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Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
GROWTH ARREST AND CASPASE-DEPENDENT APOPTOSIS
INDUCED BY 5,6-DIHYDROXY-2,4-DIMETHOXY-9,10-
DIHYDROPHENANTHRENE DERIVED FROM
Dioscorea membranacea PIERRE IN HUMAN
LUNG ADENOCARCINOMA A549 CELLS
BY
MISS WIPADA DUANGPROMPO
A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY PROGRAM IN
BIOCHEMISTRY AND MOLECULAR BIOLOGY
FACULTY OF MEDICINE
THAMMASAT UNIVERSITY
ACADEMIC YEAR 2016
COPYRIGHT OF THAMMASAT UNIVERSITY
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
GROWTH ARREST AND CASPASE-DEPENDENT APOPTOSIS
INDUCED BY 5,6-DIHYDROXY-2,4-DIMETHOXY-9,10-
DIHYDROPHENANTHRENE DERIVED FROM
Dioscorea membranacea PIERRE IN HUMAN
LUNG ADENOCARCINOMA A549 CELLS
BY
MISS WIPADA DUANGPROMPO
A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY PROGRAM IN
BIOCHEMISTRY AND MOLECULAR BIOLOGY
FACULTY OF MEDICINE
THAMMASAT UNIVERSITY
ACADEMIC YEAR 2016
COPYRIGHT OF THAMMASAT UNIVERSITY
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Dissertation Title GROWTH ARREST AND CASPASE-
DEPENDENT APOPTOSIS INDUCED BY
5,6-DIHYDROXY-2,4-DIMETHOXY-9,10-
DIHYDROPHENANTHRENE DERIVED
FROM Dioscorea membranacea PIERRE IN
HUMAN LUNG ADENOCARCINOMA A549
CELLS
Author Miss Wipada Duangprompo
Degree Doctor of Philosophy Program in Biochemistry
and Molecular Biology
Major Field/Faculty/University Biochemistry and Molecular Biology
Faculty of Medicine
Thammasat University
Dissertation Advisor
Dissertation Co-Advisor
Assistant Professor Pintusorn Hansakul, Ph.D.
Assistant Professor Kalaya Aree, Ph.D.
Academic Years 2016
ABSTRACT
An active compound of Dioscorea membranacea Pierre called 5,6-
dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP) has been shown to
possess the selective antiproliferative effect against human lung large cell carcinoma
COR-L23 cells. In this study, an adequate amount of HMP was isolated from D.
membranacea Pierre using column chromatography on silica gel 60 as the stationary
phase, and the column was eluted by gradient elution in increasing order of polarity.
The isolated compound was determined to be HMP by comparing its spectral data of
proton nuclear magnetic resonance (1H NMR) with those of previously isolated
compound and was further tested for cell-type-specific cytotoxicity in two main types
of lung cancer cell lines including non-small cell lung cancer (NSCLC) and small cell
lung cancer (SCLC) using the sulforhodamine B (SRB) assay. The results showed that
the validated HMP exhibited the most antiproliferative and cytotoxic effects on
adenocarcinoma cell line A549, one of the three main subtypes of NSCLC. Therefore,
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the aims of this study were to comprehensively investigate the antiproliferative and
cytotoxic effects through cell cycle arrest and apoptosis in human lung carcinoma A549
cells.
In the present study, the antiproliferative and cytotoxic effects of HMP were
analyzed by the SRB assay. Cell division, cell cycle distribution, membrane asymmetry
changes and intracellular ROS generation were each performed with different
fluorescent dyes including carboxyfluorescein succinimidyl ester (CFSE), propidium
iodide (PI), annexin V-FITC double staining and 2´,7´-dichlorofluorescein (DCF),
respectively, and they were then analyzed by flow cytometry. Cell cycle- and apoptosis-
related mRNA and proteins levels were measured by real-time PCR and western blot
analyses, respectively. The nuclear morphology of apoptotic cells stained with 4’, 6-
diamidino-2-phenylindole (DAPI) and DNA fragmentation were detected by
fluorescence microscopy and gel electrophoresis, respectively. The results showed that
HMP exerted strong antiproliferative (represented as IC50 = 9.37 µM and TGI = 54.81
µM) and cytotoxic effects (represented as LC50 = 94.01 µM) in A549 cells with the
highest selectivity index as compared with the human lung fibroblast cell line MRC-5.
Treatment of A549 cells with HMP induced a rapid arrest of cell division and halted
the cell cycle at G2/M phase through down-regulation of the expression levels of G2/M
regulatory proteins cdc25C, cdk1 and cyclin B1. Moreover, HMP treatment induced
early apoptotic cells with externalized phosphatidylserine and subsequent apoptotic
cells in the sub-G1 phase of the cell cycle and concurrent activation of caspase-3, whose
activity was completely abolished with pan-caspase inhibitor Z-VAD-fmk. Indeed, the
active form of caspase-3 was detected, and its actions were supported by the results of
cleavage of its target PARP and morphological alterations of apoptotic cell death such
as nuclear condensation, DNA fragmentation with accompanying DNA ladder
formation. In addition, HMP significantly increased the Bax/Bcl-2 mRNA and protein
ratios of proapoptotic, especially at 72 h of incubation, leading to subsequent caspase-9
activation and further indicating the induction of the intrinsic apoptotic pathway. Also,
HMP induced apoptosis via the extrinsic pathway by causing the proteolytic cleavage
of Bid. This study, furthermore, demonstrated that HMP could generate excessive
intracellular ROS, which was confirmed using ROS scavenger NAC. This inhibitor was
used to further study the involvement of ROS in HMP-induced apoptosis.
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In conclusion, this is the first molecular evidence of HMP that exerted its
anticancer actions through the induction of G2/M cell cycle arrest as well as the intrinsic
and extrinsic apoptotic pathways in A549 cells. These data support the potential role of
HMP as a cell-cycle arrest and apoptosis-inducing agent for treatment of NSCLC and
the use of D. membranacea Pierre in Thai traditional herbal remedies for cancer
treatment.
Keywords: Apoptosis, Anticancer effect, G2/M arrest, Dioscorea membranacea Pierre,
5,6-Dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene
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ACKNOWLEDGEMENTS
I would like to express my deepest and sincere gratitude to my advisor,
Assistant Professor Dr. Pintusorn Hansakul for her kindness in providing an opportunity
to be my advisor. I am also grateful for her valuable supervision, valuable suggestions,
supporting, encouragement, guidance and criticism throughout the course of my study.
I also would like to express my greatest appreciation and sincere gratitude to my co-
advisors, Assistant Professor Dr. Kalaya Aree for her valuable comments and
suggestions.
I wish to express my sincere appreciation to Associate Professor Dr. Treetip
Ratanavalachai, Dr. Saengsoon Charoenvilaisiri and Dr. Srisopa Ruangnoo for being
my external committee and for giving helpful suggestions.
I would like to thank Associate Professor Dr. Arunporn Itharat and Dr.
Pakakrong Thongdeeying, Department of Applied Thai Traditional Medicine, Faculty
of Medicine, Thammasat University for their help and suggestion on the laboratory
techniques in the part of plant extraction and HMP isolation.
I am grateful to all staffs and friends of the Faculty of Medicine, Thammasat
University, for their kind help and friendship.
My special thanks are extended to Mr. Suebkul Kanchanasuk, Mr. Worawat
Surarit, Miss Kedsara Junmakho and whom it concern to my study as I did not mention
for their kindness, help support and friendships during a time of the study.
Finally, I would like to express my sincere gratitude and appreciation to my
dear parents for their love, pushing up, cheerfulness, devoting and encouragement
throughout my life.
This research was mainly supported by National Research University Project
of Thailand, Office of the Higher Education Commission and Center of Excellence of
Applied Thai Traditional Medicine Research of Thammasat University. The authors
gratefully acknowledge the financial support provided by the Research Grants of
Thammasat University for Ph.D. students and TU Research Scholar, Contract No. 79/2558.
Miss Wipada Duangprompo
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TABLE OF CONTENTS
Page
ABSTRACT (1)
ACKNOWLEDGEMENTS (4)
LIST OF TABLES (10)
LIST OF FIGURES (11)
LIST OF ABBREVIATIONS (15)
CHAPTER 1 INTRODUCTION 1
1.1 Rational and Background 1
1.2 Aims of this study 3
1.2.1 Overall aims 3
1.2.2 Specific aims 3
1.3 Outcomes 4
CHAPTER 2 REVIEW OF LITERATURE 5
2.1 Lung cancer 5
2.1.1 Incidence and etiology 5
2.1.2 Pathology and staging of lung cancer 6
2.1.2.1 Non-small cell lung cancer (NSCLC) 6
2.1.2.2 Small cell lung cancer (SCLC) 10
2.1.3 Lung cancer treatment 11
2.1.4 Molecular and genetic aspects of lung cancer 11
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2.1.4.1 Proto-oncogenes 12
2.1.4.2 Tumor suppressor genes 13
2.2 Alteration of cell cycle in cancer cells 18
2.2.1 Normal cell cycle regulation and cell cycle checkpoint 18
2.2.1.1 Cell cycle regulation 19
(1) Regulation of G1 phase progression 22
(2) Regulation of S phase progression 24
(3) Regulation of G2 phase progression 25
(4) Regulation of M phase progression 25
2.2.1.2 Cell cycle checkpoints 26
(1) Restriction checkpoint 27
(2) Replication checkpoint 27
(3) Spindle checkpoint 27
(4) DNA damage checkpoint 28
2.2.2 Alterations of cell cycle regulation in cancer cells 28
2.2.2.1 Oncogenes 29
2.2.2.2 Deregulated tumor suppressor genes 31
2.3 Classification of cell death 33
2.3.1 Autophagy 33
2.3.2 Necrosis 35
2.3.3 Apoptosis 35
2.4 Alteration of apoptotic cell death in cancer cells 36
2.4.1 Apoptosis in normal cells 36
2.4.1.1 The mitochondrial pathway (or intrinsic pathway) 37
(1) Caspase-dependent apoptosis 38
(2) Caspase-independent apoptosis 39
2.4.1.2 The death receptor pathway (or extrinsic pathway) 39
2.4.2 Apoptosis in cancer cells 40
2.4.3 Reactive oxygen species (ROS) leading to apoptosis in cancer cells 40
2.5 Targeting for cancer treatment 43
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2.5.1 Targeting cell cycle regulators in cancer treatment 43
2.5.2 Targeting apoptosis in cancer treatment 45
2.6 Thai medicinal plants (Hua-Khao-Yen) 47
2.6.1 Dioscorea membranacea Pierre 47
2.6.1.1 General description 47
2.6.1.2 Biological activities 51
(1) Antiproliferative activity 51
(2) Anti-allergic activity 53
(3) Anti-HIV activity 54
(4) Antioxidant activity 54
(5) Anti-inflammatory activity 54
2.6.2 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene 55
CHAPTER 3 RESEARCH METHODOLOGY 56
3.1 Conceptual framework of this study 56
3.2 Extraction of Dioscorea membranacea Pierre 58
3.3 Isolation of 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydrophenanthrene 59
3.4 Cell culture 60
3.5 Growth inhibitory and cytotoxic effects 60
3.6 Cell proliferation by CFSE assay 62
3.7 Cell cycle analysis 64
3.8 Annexin-V/PI double staining assay 65
3.9 Caspase-3 activity assay 66
3.10 Real-time Quantitative PCR Analysis 67
3.11 Western blot analysis 69
3.12 Tubulin polymerization assay 70
3.13 DNA fragmentation assay 71
3.14 Nuclear staining with DAPI 72
3.15 Intracellular reactive oxygen species (ROS) measurement 73
3.16 Statistical analysis 74
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CHAPTER 4 RESULTS AND DISCUSSION 75
4.1 Extraction of Dioscorea membranacea Pierre 75
4.2 Isolation and purification of HMP 75
4.3 Antiproliferative and cytotoxic effects of HMP against 88
a panel of human lung cancer cell lines
4.4 Inhibitory effects of HMP on cell division 90
4.5 Effects of HMP on the cell cycle distribution 92
4.6 Effect of HMP on protein expression of cell cycle regulatory proteins 95
4.7 Effect of HMP on interfering microtubule formation 96
4.8 Effect of HMP on apoptosis induction in A549 cells 97
4.9 Effect of HMP on caspase-3 activity in A549 cells 100
4.10 Bax and Bcl-2 mRNA and protein expression levels 104
4.11 Effect of HMP on expression of active caspases and their targets 107
4.12 Effect of HMP on nuclear morphological changes 108
4.13 Effect of HMP on DNA fragmentation 111
4.14 Effect of HMP on the generation of intracellular ROS 112
CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 117
5.1 Antiproliferative effect of HMP in A549 cells 117
5.2 Molecular mechanism underlying antiproliferative effect of HMP 118
5.3 Cytotoxic effects of HMP in A549 cells 120
5.4 Apoptosis underlying cytotoxic effects of HMP 120
5.5 Effects of HMP on the generation of intracellular ROS and 122
the relationship between enhanced ROS and apoptosis
REFERENCES 123
APPENDICES 143
APPENDIX A GROWTH CURVE 144
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APPENDIX B STANDARD CURVE FOR PROTEIN 150
DETERMINATION
APPENDIX C HPLC CHROMATOGRAMS 151
APPENDIX D FLOW CYTOMETRIC ANALYSIS 154
APPENDIX E REAGENTS FOR LABORATORY EXPERIMENTS 155
BIOGRAPHY 160
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LIST OF TABLES
Tables Page
2.1 The TNM staging system for lung cancer 8
2.2 Cancer genes and their functions found in lung cancer 15
2.3 Cyclin/CDKs complex are activated within specific phases of 20
the cell cycle.
4.1 The percent yield of the ethanolic extract of D. membranacea Pierre 75
4.2 The percent yield of HMP-1 isolated from D. membranacea Pierre 79
4.3 The percent yield of HMP-2 isolated from D. membranacea Pierre 81
4.4 1H NMR spectral data (500 MHz) of HMP-1, HMP-2 and previously 85
isolated 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydrophenanthrene
4.5 The retention time, area under the curve and percentage area of HMP-1, 87
HMP-2 and 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene
analyzed by HPLC at wavelengths 254 and 270 nm.
4.6 Antiproliferative effects of HMP on a panel of human cell lines 89
4.7 The percentages of HMP-treated cells in each phase of cell cycle 94
4.8 The percentages of cells in the respective quadrants 99
4.9 The percentages of cells in each phase of cell cycle 102
4.10 The percentages of cells in each phase of cell cycle 116
25 µM HMP treatment for 24 h.
E-1 Recipes for resolving and stacking gels 157
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LIST OF FIGURES
Figures Page
2.1 Signal transduction pathway that promotes cell division 13
2.2 Phases of the cell cycle. 19
2.3 Changes in cyclins during the cell cycle. 20
2.4 Regulation of cdk-cyclin complex by phosphorylation and 21
dephosphorylation
2.5 A schematic representation of various changes in the activity of 23
cyclin-cdk complex during the cell cycle
2.6 The cell cycle checkpoint 26
2.7 The mechanisms that lead to the conversion of proto-oncogenes to 30
oncogenes
2.8 The mechanisms that lead to the deregulation of 32
tumor suppressor genes
2.9 Characteristics of autophagy, apoptosis and necrosis 34
2.10 A schematic representation of intrinsic and extrinsic pathways of 37
Apoptosis
2.11 JNK/p38 MAPK signaling pathways, apoptosis pathway and multiple 42
molecular targets of plant-derived agents
2.12 The characteristics of D. membranacea Pierre 48
2.13 Dioscorea membranacea Pierre (Male plant) 49
2.14 Dioscorea membranacea Pierre (Female plant) 50
2.15 Chemical structures of isolated compounds from the rhizomes of 52
D. membranacea Pierre
2.16 Chemical structures of isolated compounds from the rhizomes of 53
D. membranacea Pierre
2.17 The structure of 5,6-dihydroxy-2,4-dimethoxy-9,10- 55
dihydrophenanthrene
3.1 The physical characteristics of the rhizome of D. membranacea Pierre 58
3.2 Formation of fluorescent compound CFSE by intracellular esterase 63
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3.3 DNA content distribution during the various phases of the cell cycle 65
obtained by flow cytometric analysis
3.4 Dot plot analysis by Annexin V-FITC/PI double staining 66
3.5 DNA fragmentation analysis 72
3.6 Formation of fluorescent compound DCF by ROS and RNS 74
4.1 TLC analysis of the 14 combined fractions of D. membranacea Pierre 76
extract obtained from the first silica gel column chromatography
4.2 TLC analysis of the odd numbered fractions, ranging from 25-99, 77
eluted from the second silica gel column chromatography
4.3 TLC analysis of the 3 groups of the combined fractions eluted from 77
the second silica gel column chromatography
4.4 The schematic flow chart for isolation of clearly separated bands 78
using a TLC glass plate
4.5 TLC isolation of the combined fractions in group 2 79
4.6 TLC analysis of the odd numbered fractions, ranging from 1-51, 80
eluted from the third silica gel column chromatography
4.7 TLC analysis for checking the purity of HMP-2 in three different 81
solvent systems of varying polarity
4.8 1H NMR spectrum of HMP-1 in deuterated chloroform (CDCl3) 82
4.9 1H NMR spectrum of HMP-2 in deuterated chloroform (CDCl3) 83
4.10 1H NMR spectrum of 5, 6-dihydroxy-2, 4-dimethoxy-9, 10- 84
dihydrophenanthrene in deuterated chloroform (CDCl3)
4.11 HPLC chromatograms of HMP-1, HMP-2 and previously isolated 86
5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene
4.12 Effects of HMP on antiproliferative and cytotoxic activities 89
in A549 cells
4.13 Antiproliferative effects of HMP on A549 cells 91
4.14 Effects of HMP on cell cycle distribution in A549 cells 93
4.15 Effects of HMP on protein levels of cdc25C, cdk1 and cyclin B1 95
in A549 cells
4.16 Effect of HMP on in vitro tubulin polymerization 96
4.17 Effects of HMP on apoptotic induction in A549 cells 98
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4.18 Inhibitory effects of Z-VAD-fmk on sub-G1 populations 101
4.19 Effects of HMP on caspase-3 activity in A549 cells 103
4.20 The quantification of relative mRNA levels of Bax and Bcl-2 105
in A549 cells using Real-time PCR
4.21 Effects of HMP on protein expression of Bax and Bcl-2 in A549 cells 106
4.22 Effects of HMP on expressions of apoptotic proteins in A549 cells 108
4.23 Effects of HMP on nuclear morphological changes by DAPI staining 109
under bright-field microscopy (400x magnification)
4.24 Effects of HMP on nuclear morphological changes by DAPI staining 110
under fluorescent microscopy (400x magnification)
4.25 Effect of HMP on DNA fragmentation of A549 cells 111
4.26 Effect of HMP on ROS production in A549 cells treated with 113
25 µM HMP at different incubation times
4.27 Effect of the ROS scavenger NAC on ROS production in A549 cells 114
4.28 Inhibitory effects of NAC on sub-G1 populations 115
A-1 Growth curve of human lung carcinoma cell line A549 144
in 96-well plates
A.2 Growth curve of human lung squamous carcinoma cell line NCI-H226 145
in 96-well plates
A-3 Growth curve of human large cell lung cancer line COR-L23 146
in 96-well plates
A-4 Growth curve of human small cell lung cancer cell line NCI-H1688 147
in 96-well plates
A-5 Growth curve of human lung fibroblast cell line MRC-5 148
in 96-well plates
A-6 Growth curve of human lung carcinoma cell line A549 149
in 24-well plates
B-1 Standard curve for protein determination by Bradford’s method 150
B-2 Standard curve for protein determination by BCA Assay 150
C-1 HPLC chromatogram of HMP-1 151
C-2 HPLC chromatogram of HMP-2 152
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C-3 HPLC chromatogram of 5, 6-dihydroxy-2, 4-dimethoxy-9, 10- 153
Dihydrophenanthrene
D-1 Flow cytometric analysis of the DNA from A549 cells treated with 154
NAC alone at different concentrations (0.1, 1 and 5 mM) for 72 h.
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LIST OF ABBREVIATIONS
Symbols/Abbreviations Terms
α
β
%
µg
µl
µM
°C
APS
AUC
bp
BCA
BSA
CAD
CDCl3
Cdc25C
Cdk1 CFSE
CO2
CT
DAPI
DCF
DCFH-DA DMSO
DNase
DNA
cDNA
et al.
Alpha
Beta
Percent
Microgram
Microliter
Micromolar
Degree Celsius
Ammonium per sulfate
Area under the curve
Base pair
Bicinchoninic acid
Bovine serum albumin
Caspase-activated deoxyribonuclease
Deuterochloroform
Cell Division Cycle 25C
Cyclin-dependent kinase 1
Carboxyfluorescein succinimidyl
Carbon dioxide
Comparative threshold
4′,6-diamidino-2-phenylindole
2,7-dichlorofluorescein
2´,7´-dichlorofluorescein diacetate
Dimethyl sulphoxide
Deoxyribonuclease
Deoxyribonucleic acid
Complementary deoxyribonucleic acid
et alibi, and others
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FBS
FITC
g
h
HMP
HPLC
i.e.
IAPs
ICAD
LC50
M
mAU
mg
ml
mm
mM
mole
MOMP
mRNA
M.W.
nm
NAC
NMR
1H NMR
NSCLC
O.D.
PARP
PBS
Fetal bovine serum
Fluorescein isothiocyanate
Gram
Hour
5,6-dihydroxy-2,4-dimethoxy-9,10-
dihydrophenanthrene
High-performance liquid chromatography
id est (Latin), that is or in other words
Inhibitor apoptotic proteins
Inhibitor of caspase-activated
deoxyribonuclease
50% lethal concentration
Molar (concentration)
Milli- absorbance units
Milligram
Milliliter
Millimeter
Millimolar
Mole
Mitochondrial outer membrane
permeabilization
Messenger ribonucleic acid
Molecular weight
Nanometer
N-acetylcysteine
Nuclear magnetic resonance
Proton nuclear magnetic resonance
Non-small cell lung cancer
Optical density
Poly (ADP-ribose) polymerase
Phosphate buffer saline
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PCR
PI
PS
PVDF
ROS
RNA
Rpm
Rf
RT
RT-PCR
RQ
SCLC
SD
SDS
SDS-PAGE
SI
SRB
TBS
TBST
TCA
TLC
TGI
UV
w/w
Polymerase chain reaction
Propidium iodide
Phosphatidylserine
Polyvinylidene fluoride
Reactive oxygen species
Ribonucleic acid
Revolutions Per Minute
Retention factor
Retention time
Reverse transcription polymerase chain
reaction
Relative quantitation
Small cell lung cancer
Standard deviation
Sodium dodecyl sulfate
Sodium dodecyl sulfate polyacrylamide
gel electrophoresis
Selectivity index
Sulphorhodamine B
Tris-buffered saline
Tris-buffered saline with Tween20
Trichloroacetic acid
Thin-layer chromatography
Total growth inhibition
Ultraviolet
weight per weight
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CHAPTER 1
INTRODUCTION
1.1 Rational and Background
Lung cancer, one of the most common cancers, has become increasingly a
significant health problem in the world. The treatment options such as surgery,
radiotherapy, chemotherapy, and targeted therapy are currently being used depending
on the type and stage of lung cancer. Although chemotherapy treatment is the most
common regimen to treat patients, it has many unpleasant side effects such as bone
marrow suppression, gastrointestinal problems (nausea, vomiting, diarrhea), alopecia
(or hair loss) and others (Chun, Garrett, & Vail, 2007). Moreover, chemotherapy
resistance continues to be a major problem for lung cancer treatment. For this reason,
the search for new anticancer agents with increased safety and efficacy, and with
affordable price, is one of the most effective strategies to overcome the limitation of
currently available chemotherapeutic drugs. These new anticancer agents must exert
potent and specific cytotoxicity as well as their actions at the molecular level should be
clearly understood.
As cancer cells acquire defects in cell cycle control and apoptosis, new
promising anticancer agents should thus be able to potently block cell division via cell
cycle arrest (Choi, Lim do, & Park, 2009; Choi & Yoo, 2012) and concurrently restore
apoptosis towards normality (Wong, 2011). Over the years, induction of cell cycle
arrest and apoptosis has emerged as the major mechanisms by which active anticancer
agents act to inhibit the growth of cancer cells and eliminate them (Feng et al., 2011).
Therefore, investigating whether agents exert their cytotoxic actions through the
induction of cell cycle arrest and apoptosis appears to be a powerful strategy to obtain
effective agents for the development of chemotherapeutic drugs.
Many studies have shown that plant-derived compounds mediating cell
cycle arrest and apoptosis have increasingly attracted scientific interest, such as
curcumin (Curcuma longa) (Tan et al., 2006; Lee, Lee, & Kim, 2009; Wu et al., 2010;
Cheng et al., 2016), shikonin (Lithospermum erythrorhizon) (Wu et al., 2004; Gong &
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Li, 2011; Tian, Li, & Gao, 2015), magnolol (Magnolia officinalis) (Zhou et al.,2013;
Li et al., 2015), genistein (soybean) (Ouyang et al., 2009; Zhang et al., 2013),
resveratrol (red grape skins) (Aziz, Nihal, Fu, Jarrard, & Ahmad, 2006; Gogada et al.,
2011) and so forth. Moreover, some of them have the effects on the generation and
accumulation of intracellular reactive oxygen species (ROS). The excessive ROS
production leads to the activation of mitochondria-mediated apoptosis pathway (Gong
& Li, 2011; Singh, Zaidi, Shyam, Sharma, & Balapure, 2012; Qui et al., 2015).
Dioscorea membranacea Pierre, also called Hua-Khao-Yen-Tai in Thai, is
one of Thai medicinal plants, which has long been used to prepare Thai traditional
medicine for cancer treatment (Itharat, Singchangchai, & Ratanasuwan, 1998;
Subchareon, 1998). Previous studies have shown that the ethanolic extract of D.
membranacea Pierre and its active compounds exhibited high cytotoxic activity against
a panel of human cancer cell lines (Itharat et al., 2003; Itharat et al., 2004; Itharat et al.,
2007; Itharat, Thongdeeying, & Ruangnoo, 2014). Among these active compounds,
dioscorealide B (Saekoo, Dechsukum, Graidist, & Itharat, 2010; Saekoo, Graidist,
Leeanansaksiri, Dechsukum, & Itharat, 2010) and dioscoreanone (Hansakul, Aree,
Tanuchit, & Itharat, 2014) have been elucidated for their molecular mechanisms of
action. Itharat et al. (2014) have demonstrated that an active compound named 5,6-
dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP) exerted the selective
cytotoxic effects against human lung, breast and prostate cancer cell lines whereas it
was less toxic to the normal cell line. However, the molecular mechanisms underlying
its cytotoxic effect have not yet been studied.
Thus, this study we further investigated the antiproliferative effect of HMP
against a panel of different human lung cancer cell lines. A549 cell line, one of cell
lines displaying the most potent inhibitory effect with the highest selectivity index, was
chosen to investigate the molecular mechanisms underlying anticancer effect through
the induction of cell cycle arrest and apoptosis. In addition, the effects of HMP on the
generation and accumulation of intracellular reactive oxygen species (ROS) were
studied.
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1.2 Aims of this study
1.2.1 Overall aims
To investigate molecular mechanisms underlying the anticancer activity of
HMP in human lung adenocarcinoma cell line A549 through cell cycle arrest and
apoptosis
1.2.2 Specific aims
The aims of this study were as follows:
1.2.2.1 To isolate 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenan
threne (HMP) from Dioscorea membranacea Pierre
1.2.2.2 To determine the antiproliferative effect of HMP on different
lung cancer cell lines as compared to the normal cell line
1.2.2.3 To determine the cytotoxic effect of HMP on human lung
carcinoma cell line A549
1.2.2.4 To investigate the antiproliferative activity of HMP through
the induction of cell cycle arrest in A549 cells
1.2.2.5 To investigate the cytotoxic activity of HMP through the
induction of apoptosis in A549 cells
1.2.2.6 To investigate the intracellular reactive oxygen species (ROS)
levels in HMP-treated A549 cells and further examine whether ROS is associated with
HMP-induced apoptosis
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1.3 Outcomes
These research findings provided:
1.3.1 Knowledge on HMP-induced G2/M arrest through modulation of
specific regulatory proteins and mitotic spindle disruption in A549 cells.
1.3.2 Knowledge on HMP-induced apoptosis via caspase-dependent
pathway in A549 cells.
1.3.3 Data supporting the development of HMP as a novel anticancer
drug.
1.3.4 Data supporting the high economic value of D. membranacea
Pierre as a source of potential anticancer compounds.
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CHAPTER 2
REVIEW OF LITERATURE
2.1 Lung cancer
2.1.1 Incidence and etiology
Lung cancer is one of the most important malignancies and the most
common cause of cancer death worldwide. In 2012, data from GLOBOCAN project
produced by the International Agency for Research on Cancer (IARC) have shown that
lung cancer is responsible for more cancer-related deaths than any other types of cancer
(Torre et al., 2015). The major reason that contributes to its high mortality rate is the
fact that a large proportion of these cases are diagnosed with the advanced or metastatic
stage. The data from American Cancer Society (2015) have revealed that more than
half (57%) of lung cancer are diagnosed at a distant stage, for which the 1- and 5-year
survival is 26% and 4%, respectively. The 5-year survival for small cell lung cancer
(6%) is lower than that for non-small cell (21%). Moreover, an estimated 1.8 million
new lung cancer cases occurred in both men and women, accounting for 13% of total
cancer diagnoses. The high prevalence of lung cancer is increasingly becoming a
significant health problem in many regions of the world (Torre et al., 2015).
In Thailand during 2004-2006, lung cancer is the second most common
cancer in males after liver cancer, and the fourth in females after cervix, breast, and
liver cancer (Müller-Hermelink et al., 2004; Sriplung et al., 2005). In 2002, Vatanasapt,
Sriamporn, & Vatanasapt have reported that the cancer incidence rate appears to depend
on the geographical regions. For example, lung cancer predominates in the northern
part of Thailand whereas liver cancer, especially cholangiocarcinoma, is high in the
Northeast.
A major risk factor for developing lung cancer is tobacco consumption
because tobacco contains a complex mixture of potent carcinogens, predominantly
polycyclic aromatic hydrocarbons derived from combustion of tars. Other known risk
factors for lung cancer include gender, occupation, diet, radon exposure and passive
smoking. Interactions between risk factors, and in particular with cigarette smoking,
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may increase lung cancer risk significantly. Hereditary factors and genetic
susceptibility to lung cancer currently remain ill-defined. Recently, outdoor pollution
has also been determined to cause lung cancer (Codony-Servat, Verlicchi, & Rosell,
2016).
2.1.2 Pathology and staging of lung cancer
According to pathological type, lung cancer can be divided into two
histological groups: non-small cell lung cancer (NSCLC) and small cell lung cancer
(SCLC). This broad stratification reflects fundamental differences in tumor biology and
clinical behavior as well as underlies current treatment strategies.
2.1.2.1 Non-small cell lung cancer (NSCLC)
NSCLC is the most common type of lung cancer, which accounts for
80% of the cases, and it usually grows and spreads more slowly than SCLC. NSCLC
can be divided into three major groups: adenocarcinoma, squamous cell carcinoma and
large-cell lung carcinoma based on morphological and immunohistochemical (IHC)
features (Rekhtman, Ang, Sima, Travis, & Moreira, 2011; Travis & Rekhtman, 2011;
Kadota et al., 2015).
- Adenocarcinoma is the most common histologic subtype of lung
cancer and accounts for about 50 % of NSCLC and 38 % of newly diagnosed lung
cancers. It usually originates in the periphery of the lung (outer part of the lung). The
adenocarcinoma is defined by the World Health Organization (WHO) as a malignant
epithelial tumor with glandular differentiation or mucin production, showing acinar,
papillary, bronchioloalveolar or solid with mucin growth patterns or a mixture of these
patterns (Müller-Hermelink et al., 2004). However, the adenocarcinoma can present
diverse histological patterns, which can be intermixed in the same tumor including
lepidic, acinar, papillary, micropapillary, and solid patterns. Therefore, pneumocyte
marker expression like napsin A or thyroid transcription factor 1 (TTF1) is useful in the
identification of the adenocarcinoma in challenging cases (Travis et al., 2015). For
example, solid patterns of adenocarcinoma can be confused with squamous cell
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carcinoma or large-cell lung carcinoma; the mucin production and immunohistochemical
expression of TTF-1 or napsin A can help in such diagnosis (Rodriguez-Canales, Parra-
Cuentas, & Wistuba, 2016).
- Squamous cell carcinoma represents for nearly 20 % of all lung
cancers, and it is usually found in a central location, arising in a main or lobar bronchus.
The squamous cell carcinoma is defined as a malignant epithelial tumor showing
keratinization and/or intercellular bridges that arise from the bronchial epithelium
(Müller-Hermelink et al., 2004). However, some squamous cell carcinoma may not
show such morphological features. Immunohistochemical tests including markers of
squamous cell differentiation such as p40 or p63 and cytokeratins 5/6 may be useful in
the identification of squamous cell carcinoma in difficult cases (Travis et al., 2015). For
example, a distinct entity is the basaloid squamous cell carcinoma, a poorly
differentiated malignant tumor without morphological features of squamous cell
differentiation which can be confused with small-cell lung carcinoma, but it is
characteristically positive for immunomarkers of squamous cell differentiation
including p40, p63, and cytokeratins 5/6, while TTF-1 is negative (Rodriguez-Canales
et al., 2016).
- Large-cell lung carcinoma accounts for about 3 % of all lung
cancers. The large-cell lung carcinoma is defined as undifferentiated non-small cell
carcinoma that lacks the cytologic and architectural features of small cell carcinoma
and glandular or squamous differentiation (Müller-Hermelink et al., 2004). Based on
immunohistochemistry, large-cell lung carcinoma may be positive for cytokeratins 5/6
but they are negative for TTF-1 and p40 (Travis et al., 2015; Rodriguez-Canales et al.,
2016).
The lung cancer staging system provides useful prognostic information
for patients and structures treatment plans for physicians. According to the International
Association for the Study of Lung Cancer (IASLC), the latest revision of tumor–node–
metastasis (TNM) staging, presented in the 7th edition of American Joint Committee on
Cancer (AJCC) is shown in Table 2.1.
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Table 2.1 The TNM staging system for lung cancer (Modified from Kalemkerian,
2011)
Anatomic stage/
prognostic groups Tumor (T) lymph nodes (N) Metastasis (M)
Occult Carcinoma TX N0 M0
Stage 0 Tis N0 M0
Stage IA T1a N0 M0
T1b N0 M0
Stage IB T2a N0 M0
Stage IIA T2b N0 M0
T1a N1 M0
T1b N1 M0
T2a N1 M0
Stage IIB T2b N1 M0
T3 N0 M0
Stage IIIA T1a N2 M0
T1b N2 M0
T2a N2 M0
T2b N2 M0
T3 N1 M0
T3 N2 M0
T4 N0 M0
T4 N1 M0
Stage IIIB T1a N3 M0
T1b N3 M0
T2a N3 M0
T2b N3 M0
T3 N3 M0
T4 N2 M0
T4 N3 M0
Stage IV Any T Any N M1a
Any T Any N M1b
Note: TNM descriptions for staging lung cancer
Primary Tumor (T)
TX Primary tumor cannot be assessed, or tumor proven by the presence of malignant
cells in sputum or bronchial washings but not visualized by imaging or
bronchoscopy
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T0 No evidence of primary tumor
Tis Carcinoma in situ
T1 Tumor 3 cm or less in greatest dimension, surrounded by lung or visceral pleura,
without bronchoscopic evidence of invasion more proximal than the lobar
bronchus (for example, not in the main bronchus)
T1a Tumor 2 cm or less in greatest dimension
T1b Tumor more than 2 cm but 3 cm or less in greatest dimension
T2 Tumor more than 3 cm but 7 cm or less or tumor with any of the following
features (T2 tumors with these features are classified T2a if 5 cm or less): involves
main bronchus, 2 cm or more distal to the carina; invades visceral pleura (PL1 or
PL2); associated with atelectasis or obstructive pneumonitis that extends to the
hilar region but does not involve the entire lung
T2a Tumor more than 3 cm but 5 cm or less in greatest dimension
T2b Tumor more than 5 cm but 7 cm or less in greatest dimension
T3 Tumor more than 7 cm or one that directly invades any of the following: parietal
pleural (PL3), chest wall (including superior sulcus tumors), diaphragm, phrenic
nerve, mediastinal pleura, parietal pericardium; or tumor in the main bronchus
less than 2 cm distal to the carina1 but without involvement of the carina; or
associated atelectasis or obstructive pneumonitis of the entire lung or separate
tumor nodule(s) in the same lobe
T4 Tumor of any size that invades any of the following: mediastinum, heart, great
vessels, trachea, recurrent laryngeal nerve, esophagus, vertebral body, carina,
separate tumor nodule(s) in a different ipsilateral lobe
Regional lymph nodes (N)
NX Regional lymph nodes cannot be assessed
N0 No regional lymph node metastases
N1 Metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and
intrapulmonary nodes, including involvement by direct extension
N2 Metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s)
N3 Metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or
contralateral scalene, or supraclavicular lymph node(s)
Distant Metastasis (M)
M0 No distant metastasis
M1 Distant metastasis
M1a Separate tumor nodule(s) in a contralateral lobe, tumor with pleural
nodules or malignant pleural (or pericardial) effusion
M1b Distant metastasis (in extrathoracic organs)
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2.1.2.2 Small cell lung cancer (SCLC)
SCLC is a malignant epithelial tumor consisting of small cells with
almost no visible cytoplasm, ill-defined cell borders, finely granular nuclear chromatin,
and absent or inconspicuous nucleoli. The cells are round, oval, or spindle-shaped.
Nuclear molding is prominent. Necrosis is typically extensive and the mitotic count is
high (Müller-Hermelink et al., 2004). SCLC accounts for about 20% of all lung cancers,
and it is considered to be the most aggressive form of lung cancer that has a high
propensity for metastases and a poor prognosis. Comparable to other lung cancers,
SCLC has the highest association with tobacco smoking and almost never arising in the
absence of smoking history (Pesch et al., 2012). In addition, this tumor is now generally
considered as a neuroendocrine carcinoma (with small and large cell variants), and
immunohistochemical studies have consistently demonstrated characteristic
biomarkers, including calcitonin, gastrin-releasing peptide, L-dopa decarboxylase,
chromogranin, synaptophysin, and neuron-specific enolase. However, the precise cell
of origin for lung cancer is controversial, and a mosaic of cellular elements (including
NSCLC) is common in tumors with otherwise predominantly small-cell histology
(Macdonald, Ford, & Casson, 2004).
According to the Veterans’ Administration Lung Study Group
(VALSG) system, SCLC is generally divided into two stages, limited and extensive.
Limited disease (LD) is defined as a tumor that is confined to one hemithorax and
associated regional lymph nodes whereas extensive disease (ED) is defined as tumor
outside the confines of limited stage disease including patients with malignant
pericardial and pleural effusion (Bernhardt & Jalal, 2016). Recently, the IASLC has
proposed the revised TNM staging system, presented in the 7th edition of American
Joint Committee on Cancer (AJCC), which is integrated into the classification of SCLC
(Kalemkerian, 2011). For example, LD constitutes approximately 35-40 % of patients
and includes TNM stages I through III, and ED includes patients of TNM IV. The TNM
staging seems more accurate than the limited versus extensive stage in determining
prognosis, especially, in the earlier stages of the disease (Bernhardt & Jalal, 2016).
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2.1.3 Lung cancer treatment
There are several types of lung cancer treatments such as surgery,
radiotherapy, chemotherapy and targeted therapy, either alone or in combination. In
order to eliminate abnormal cells, these treatments are selected based on the histological
types, stages of lung cancer, the patient’s general condition (Dobbelstein & Moll,
2014). For patients with SCLC, the standard treatment of limited disease includes
combination chemotherapy and radiotherapy. Also, surgery may play a role in TNM
stages I and II. In extensive disease, chemotherapy alone is the standard treatment.
However, despite the fact that patients initially have a good response to standard
treatment the vast majority relapse, with a 1-year survival rate of 40%, and 5-year
survival under 5% (Codony-Servat et al., 2016). These indicate that advances in the
treatment of SCLC remain non-satisfactory nowadays, and novel therapies are needed
to improve survival in this disease. For patients with NSCLC, the treatment options
such as surgery, radiotherapy, chemotherapy, targeted therapy or a combination of these
treatments are currently being used depending on the stage of cancer. In the early stage,
chemotherapy is often used as an adjuvant treatment, which is given after surgery or
radiation therapy to kill any remaining cancer cells (Domont, Soria, & Le Chevalier,
2005; Visbal, Leighl, Feld, & Shepherd, 2005). Chemotherapy is also used as
neoadjuvant therapy, which is given before surgery or radiation therapy to shrink tumor
(Choong & Vokes, 2005; Salvà & Felip, 2013). For later stage of cancers when surgery
is no longer an option, chemotherapy is often administered with simultaneous radiation
therapy. In addition, chemotherapy is used to treat recurrent cancer that comes back
after treatment or metastatic cancer that has spread to other parts of the body.
2.1.4 Molecular and genetic aspects of lung cancer
Lung carcinogenesis, like the development of other cancers, is a multistep
process involving the progressive accumulation of genetic and epigenetic alterations
that ultimately transform normal cells into neoplastic cells. Then, the neoplastic cells
can be benign tumors (non-cancerous) or malignant tumors (cancerous), which have
more aggressive characteristics than benign tumors. The common characteristics of
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malignant cells that make them different from other normal cells include 1) self-
sufficiency of growth signals; 2) lack of sensitivity to anti-growth signals; 3) evasion
of apoptosis; 4) limitless replicative potential; 5) sustained angiogenesis; and 6) tissue
invasion and metastasis (Hanahan & Weinberg, 2011). Specific molecular alterations
that drive malignant progression involve mutations in genes that regulate cell
proliferation (Larsen & Minna, 2011). There are two broad classes of cancer-relevant
genes: proto-oncogenes and tumor suppressor genes.
2.1.4.1 Proto-oncogenes
In normal cells, proto-oncogenes are genes that control cell growth and
code for the proteins that provide a signal for stimulating cell division. Such proteins
can be classified into six groups based on functional properties as follows: 1) growth
factors (e.g. PDGF or EGF molecules); 2) growth factor receptors (e.g. PDGF receptor
or EGF receptor); 3) plasma membrane G proteins (e.g. Ras); 4) intracellular protein
kinases (e.g. Raf, MEK, MAPK); 5) transcription factors (e.g. Fos, Jun and Myc); and
6) cell cycle or cell death regulators (e.g. cyclin, cdk, bcl-2, Mdm2) (Kleinsmith, 2006),
as shown in Figure 2.1.
In cancer cells, the genetic changes found gain-of-function mutations
in proto-oncogenes. These changes cause proto-oncogenes to become oncogenes,
which produce a mutated protein that interferes cell proliferation, thereby leading to
uncontrollable cell division seen in cancer cells.
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Figure 2.1 Signal transduction pathway that promotes cell division. (Modified from
Kleinsmith, 2006)
2.1.4.2 Tumor suppressor genes
In contrast to proto-oncogenes, tumor suppressor genes code for
negative regulator proteins that help prevent uncontrollable cell growth and promote
DNA repair and cell cycle checkpoint activation. Their normal functions are generally
to inhibit proliferation in response to certain signals such as DNA damage. The signal
is removed when the cell is fully equipped to proliferate. Tumor suppressor genes are
also broadly divided into two classes: gatekeeper genes and caretaker genes.
- Gatekeeper genes directly regulate cell growth by either inhibiting
cell proliferation or promoting apoptosis. Examples of gatekeeper genes include RB
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involved in restriction point control; APC involved in Wnt signaling and p53 involved
in DNA damage response.
- Caretaker genes do not directly regulate cell growth. Instead,
inactivation of caretaker genes leads to genetic instability that indirectly promotes
proliferation by causing an increased rate of mutation. The genes that encode proteins
involved in DNA repair are classic examples of caretaker genes, such as ATM involved
in DNA damage response; BRCA1 and BRCA2 involved in double-strand break repair;
MLH1 and MSH2 involved in DNA mismatch repair; and XP-A involved in the
nucleotide excision repair pathway (Kleinsmith, 2006).
In cancer cells, the majority of genetic changes found loss-of-function
mutations in tumor suppressor genes. Such mutations contribute to the development of
cancer by inactivating the growth inhibitory function.
As illustrated in Table 2.2, the gene families implicated in lung
carcinogenesis include dominant oncogenes and tumor suppressor genes (Larsen &
Minna, 2011).
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Table 2.2 Cancer genes and their functions found in lung cancer. (Modified from
Weber, 2007)
Genes Functions Mutation Common cancer type
1. Oncogenes
1.1 Growth factors
EGF Epidermal growth factor,
expressed in submaxillary
gland, targets epithelial/
mesenchymal/glial cells
Overexpression Breast cancer, lung
cancer
TGF-α Transforming growth
factor, expressed in
platelets targets epithelial/
mesenchymal/glial cells
Overexpression Breast
adenocarcinoma, lung
cancer
1.2 Growth factor receptors
ERBB Part of epidermal growth
factor receptor, receptor
protein tyrosine kinase
Point mutation
Amplification
Glioblastoma, breast
cancer, bladder cancer,
squamous cell lung
cancer, lung
adenocarcinoma,
head and neck cancer,
colon cancer
1.3 Signal transduction molecules associated with growth factor receptors
- Protein kinases
ERBB2
(Neu /Her-2)
Receptor protein tyrosine
kinase
Amplification,
point mutation
Neuroblastoma,
glioma, breast
adenocarcinoma,
ovarian cancer,
lung adenocarcinoma,
salivary gland cancer
- GTP-binding proteins
K-ras Guanine nucleotide-binding
protein GTPase
Point mutation
Translocation
Lung cancer, ovarian
cancer, colon cancer,
pancreas cancer
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Genes Functions Mutation Common cancer type
- Transcription factors
c-myc Acts together with MAX,
sensitizes cells to CD95-
mediated apoptosis
Chromosomal
translocation,
Insertional
mutagenesis
Burkitt lymphoma,
leukemia, breast
cancer, stomach
cancer, lung cancer
L-myc Acts together with MAX Amplification Lung cancer
E2F
Dimer with DP1 initiates
transcription of S phase
genes
Point mutation
Lung cancer, breast
cancer
2. Tumor suppressor genes
2.1 Gatekeeper genes
(1) Signal transduction molecules associated with growth factor receptors
- Blockage of cyclin-cdk activity
CDKN1A
(waf1/cip1)
(p21CIP1)
Binding to and inhibition of
cdk2 and cdk4, activated by
p53, inhibits DNA synthesis
when complexed with
PCNA,
transcription induced by
STAT1
Leukemia, lung cancer
CDKN2A
(mts1)
Cyclin-dependent kinase
inhibitor 2A Multiple
Tumor Suppressor 1
Melanoma, lung
cancer,
medulloblastoma,
CDKN2B
(mts2)
(p15INK4b)
Inhibitor of cyclin-
dependent kinases
Acute lymphoblastic
leukemia, lung cancer,
melanoma, glioma
PPP2R1B
β form of the serine/
threonine Protein
Phosphatase 2A, down-
regulates MAP kinase
cascade, inhibits nuclear
telomerase activity
Lung cancer, colon
cancer
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Genes Functions Mutation Cancers
- Transcription factors
p53
Transcription factor,
stimulates transcription of
p21, cell cycle regulator, is
phosphorylated by CDK and
Casein Kinase, induces
apoptosis via transport of
CD95 from the Golgi
complex
Osteosarcoma, breast
cancer, brain tumor,
Li-Fraumeni
syndrome, pancreas
carcinoma, small cell
lung cancer
Rb1 Negative regulation of
transcription factors E2F-
DP1, cell cycle regulation,
activity regulated by
phosphorylation (low in
G0/G1, high in G1/S)
Retinoblastoma,
osteosarcoma, small
cell lung cancer,
bladder cancer,
cervical carcinoma,
breast cancer, prostate
cancer
BRG1 Component of the SWI–SNF
chromatin remodeling
complex, inhibition of
proliferation through
interaction with RB
Prostate cancer, breast
cancer, lung cancer
(2) Function not grouped
S100 A2 Nuclear calcium-binding
protein
Breast cancer, lung
cancer
HIC-1 Located on chromosome
17p13.3, frequently
hypermethylated in cancer
Medulloblastoma,
lung cancer, colon
cancer
2.2 Caretaker genes
(1) Signal transduction molecules associated with growth factor receptors
- Inactivation of G-protein-GTP signal
RASSF1A RAS association domain
family 1 isoform A protein.
The encoded protein was
found to interact with DNA
repair protein XPA
Medulloblastoma,
nasopharyngeal
cancer, lung cancer
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2.2 Alteration of cell cycle in cancer cells
2.2.1 Normal cell cycle regulation and cell cycle checkpoint
In multicellular organisms, cells divide into two daughter cells for growth
and replacement of dead cells. In normal cell division, a cell is stimulated by growth
factors to enter the cell cycle. The binding of a growth factor to its corresponding
receptors triggers a multistep cascade in which a series of signal transduction proteins
relay the signal throughout the cell. These signal transduction proteins are encoded by
proto-oncogenes and function to regulate cell growth and division (Kleinsmith, 2006).
The cell capable of undergoing division passes through the cell cycle, which is broadly
divided into three stages: interphase, mitosis, and cytokinesis. Interphase is the period
of cellular growth and DNA synthesis and is subdivided into three phases called G1
phase, S phase, and G2 phase. Mitosis or nuclear division is a continuous process and
is divided into five phases, namely prophase, prometaphase, metaphase, anaphase, and
telophase. These divided phases are based on progress made to a specific point in the
overall nuclear division. Cytokinesis or cytoplasmic division is the last stage that ends
with the separation into two daughter cells (Chandar & Viselli, 2012), as shown in
Figure 2.2. In the absence of growth factors, cells become quiescent, and cell division
is restrained by tumor suppressor proteins such as cdk inhibitors (CKIs), Rb, and so
forth.
Also, the cell-division cycle is strictly controlled by checkpoints located at
each phase of the cell cycle to verify whether the cells are ready to progress to the next
phase. The cell cycle checkpoints can be divided into four phases: restriction
checkpoint, DNA damage checkpoint, replication checkpoint, and spindle checkpoint
(Kleinsmith, 2006). These checkpoints are considered to be safety measures for the cell,
preventing the control system from dictating the start of another cell cycle event before
the previous one has finished, or before any damage to the cell has been properly
repaired.
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Figure 2.2 Phases of the cell cycle. The cell cycle can be divided into three stages:
interphase, mitosis and cytokinesis. (Modified from Huber et al., 2013)
2.2.1.1 Cell cyle regulation
The cell cycle progression is strictly regulated by key regulatory proteins
known as cyclin-dependent kinases (cdks). Cdks are protein kinases, a class of enzymes
that regulate the activity of targeted protein molecules by catalyzing their
phosphorylation. However, cdks only exhibit protein kinase activity when they are
bound to their regulatory subunits, the cyclins. Each cdk is paired with a specific cyclin,
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and the cyclins are made and degraded during specific points in the cell cycle (Figure
2.3 and Table 2.3). Thus, cell cycle progression is controlled by several Cdk-cyclin
complexes as follows: cdk4/6-cyclin D for G1 progression, cdk2-cyclin E for the G1-S
transition, cdk2-cyclin A for S-phase progression, and cdk1-cyclin B for entry into M-
phase (Nguyen & Jameson, 1998).
Table 2.3 Cyclin/CDKs complex are activated within specific phases of the cell cycle.
CDKs Cyclins Cell cycle phases
Cdk4 Cyclin D G1 phase
Cdk6 Cyclin D G1 phase
Cdk2 Cyclin E G1/S transition
Cdk2 Cyclin A S phase and G2 phase
Cdk1 (cdc2) Cyclin A G2/M phase
Cdk1 (cdc2) Cyclin B Mitosis
Figure 2.3 Changes in cyclins during the cell cycle. The levels of different cyclins are
depicted schematically. (Modified from Nguyen & Jameson, 1998)
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Cyclin-cdk complexes are regulated in several ways, such as
phosphorylation and dephosphorylation, inhibitory proteins, proteolysis, as well as
subcellular localization.
One way to control the activity of cdk-cyclin complexes is phosphorylation.
The activity of the various cdk-cyclin complexes is controlled by reactions in which
cdk molecules are altered by phosphorylation and dephosphorylation. For example, at
the G2-to-M transition, the cdk1-cyclinB complex is initially inactive because of the
inhibitory phosphorylation of the cdk molecule by inhibiting kinase Wee1 on a
conserved tyrosine residue (Tyr15) or on an adjacent threonine residue (Thr14).
Although an activating phosphate group is added to a threonine residue (Thr161) by
cdk-activating kinase (CAK), the cdk remains inactive as long as the inhibitory
phosphate groups are present. The last step in the activation sequence is the removal of
the inhibiting phosphate by a specific enzyme called a protein phosphatase cdc25
(Gould & Forsburg, 2015), as shown in Figure 2.4.
Figure 2.4 Regulation of cdk1-cyclin complex by phosphorylation and dephosphorylation.
(Modified from Gould & Forsburg, 2015)
Conversely, whenever the cell cycle is in unfavorable conditions for
progression to the next phase, cdk inhibitors (CKIs) also regulate cdks to halt cell cycle
progression. Two classes of CKIs based on their structure and cdk specificity are
recognized. First, INK4 family members (e.g. p15 INK4b, p16INK4a, p18 INK4c and p19 INK4d)
specifically inhibit cdk4/6-cyclin D activity by binding to either cdk4 and cdk6, thereby
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preventing association between cyclin D and its catalytic partner (Lim & Kaldis, 2013).
The other one is CIP/KIP family members (e.g. p21CIP1, p27KIP1 and p57 KIP2) that bind
and strongly block cdk-cyclin complexes (Lim & Kaldis, 2013). Most CKIs identified
so far act during G1 and/or S phase and block cell cycle progression until conditions
allow them to be overcome. For example, p16 CKIs present in G1 block the cell cycle
until enough G1 cyclins are synthesized to displace them from the G1 cdks.
In addition, the control of subcellular localization of cdk–cyclins and their
regulators is essential for proper cell-cycle coordination. One of the best-understood
examples is the regulation of cyclin B localization during interphase. During interphase,
cyclin B shuttles between the nucleus and the cytoplasm because constitutive nuclear
import is counteracted by rapid nuclear export, resulting that it is mainly located in the
cytoplasm (Hagting, Jackman, Simpson, & Pines, 1999). Just before the onset of
mitosis, cyclin B is phosphorylated in the cytoplasmic retention sequence (CRS),
leading to inactivation of nuclear export signals. Therefore, cyclin B accumulates in the
nucleus. It is possible that this type of regulation also serves to bring cdk complexes
into contact with their substrates (Gould & Forsburg, 2015). The periodic availability
of cyclins is a key mechanism of regulating the catalytic activity of cdk subunits.
Cyclins accumulate at certain periods of the cell cycle to activate their cdk partners. At
cell cycle transition points, cyclins become highly unstable and cyclin destruction
irreversibly compels the cell cycle forward. The abrupt instability of cyclins is due to
activation of ubiquitin ligases that target cyclins for proteasome-mediated degradation.
(1) Regulation of G1 phase progression
When a cell receives the proper signals that trigger the process of
cell division e.g. growth factors and cytokines, these signals lead to increased
expression of genes encoding proteins that regulate cell cycle progression through the
G1 phase. Cyclin D and cyclin E are two major classes of G1 cyclins. The cyclins,
however, have no effect on G1-S transition unless they form a complex with their cdk
partners as follows: cyclin D binds mainly to cdk4 and cdk6 whereas cyclin E binds to
cdk2. In early- to mid-G1 phase, cdk4/6-cyclin D complex hypophosphorylates the
retinoblastoma tumor suppressor protein (pRb) in which the hypophosphorylated pRb
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also forms a complex with E2F family of transcription factors, resulting in inhibition
of the expression of genes required for entry into S phase, including cyclin E and cyclin
A. After progression through the cyclin D-dependent portion of the cell cycle, cyclin E
becomes activated by forming a complex with cdk2. The cdk2-cyclin E complex
triggers hyperphosphorylation of pRb, leading to the liberation of E2F to initiate the
transcription of genes needed for DNA replication (e.g. cdc6, ORC1 and the
minichromosome maintenance (MCM) proteins) and the progression into S phase (e.g.
cyclin E, cyclin A, cdk1 and cdc25A) (Xu, Sheppard, Peng, Yee, & Piwnica-Worms,
1994; Neganova & Lako, 2008; Foster, Yellen, Xu, & Saqcena, 2010). Moreover, the
activities of cyclin D- and cyclin E-dependent kinases are linked through members of
the CIP/KIP family of CKIs, including p21CIP1, p27KIP1 and p57 KIP2. These CKIs control
cell proliferation by binding to cyclin and cdk to block entry into S phase (Sherr &
McCormick, 2002) (Figure 2.5).
Figure 2.5 A schematic representation of various changes in the activity of cyclin-cdk
complex during the cell cycle. (Modified from Nguyen & Jameson, 1998)
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(2) Regulation of S phase progression
Once cells enter S phase and begin DNA replication, cyclin E that
binds to cdk2 is rapidly degraded via the ubiquitin-dependent pathway, resulting in the
reduction of its kinase activity (Nguyen & Jameson, 1998; Hwang & Clurman, 2005).
However, the continued hyperphosphorylation of pRb allows the transcription of cyclin
A and cyclin B that required for subsequent phases of the cell cycle. Cyclin A has roles
in S phase progression, and it can form complex with either cdk2 or cdk1 under different
circumstances (Pagano, Pepperkok, Verde, Ansorge, & Draetta, 1992). In fact, although
cyclin A is synthesized and associated with cdk2 during the late G1 phase, its activity
is negatively regulated by inhibitory phosphorylation of cdk2 and also by the
association of CKIs, p21CIP1 and p27KIP1. Thus, the removal of inhibitory phosphates
from a cdk2 subunit of cdk2-cyclinA complex by the cdc25A phosphatases and the
degradation of CKIs by ubiquitin ligases are required for initiation of S phase.
Cdk2-cyclinA complex is required for the initiation of DNA
replication by the disassembly of pre-replication complex through the phosphorylation
of cdc6 proteins. Briefly, at the onset of S phase, a prereplication complex is formed on
DNA replication origins by the assembly of several factors, such as origin recognition
complex (ORC), cdc6, cdt1 and minichromosome maintenance (MCM) complex.
Several processes are responsible for activation of such prereplication complex. First,
cdk2-cyclinA complex phosphorylates cdc6. Phosphorylation is an inhibitory
modification of cdc6, and inactivated cdc6 gets ubiquitinated and degrades in
proteosome. Then, cdt1 becomes inhibited by geminin, which is an inhibitor of cdt1.
With cdc6 and cdt1 no longer bond, MCM protein can unwind the double-stranded
DNA, and DNA replication begins (Marín-García, 2011). In fact, although cyclin A is
synthesized and associated with cdk2 during the late G1 phase, its activity is negatively
regulated by inhibitory phosphorylation of cdk2 and also by the association with CKIs,
p21CIP1 and p27KIP1. Thus, the removal of inhibitory phosphates from cdk2 subunit of
cdk2-cyclin A complex by the cdc25A phosphatases and the degradation of CKIs by
ubiquitin ligases are required for initiation of S phase. Moreover, the cdk2-cyclin A
complex directly binds and phosphorylates E2F, thereby preventing the binding of E2F
to DNA and turning off the Gl/S phase genes that are no longer required once the DNA
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replication has begun. The inactivation of E2F helps ensure cell cycle progression into
S phase and prevents reversion back to G1 phase (Nguyen & Jameson, 1998; Xu et al.,
1994). In late S phase, cyclin A also associates with cdk1 in which the cdk1-cyclin A
complex drives the transition between S phase and G2 phase (Pagano et al., 1992).
(3) Regulation of G2 phase progression
During G2 phase, the period between DNA synthesis and mitosis,
cdk1-cyclin A and cdk1-cyclin B complexes play an important role in G2 phase. The
accumulation of active cdk1-cyclin B, also known as maturation promoting factor
(MPF), is strictly dependent on cdk1-cyclin A activity. Briefly, the cdk1-cyclin A
complex phosphorylates cdh1, which is one of the substrate adaptor protein of the
anaphase-promoting complex (APC) that is an ubiquitin E3-ligase complex, leading to
preventing cdh1 from targeting cyclin B to the anaphase promoting complex (APC) for
ubiquitination and degradation. Active cdk1-cyclin B thus accumulates in the
cytoplasm, where it is thought to prepare structural components of the cell for the
upcoming cell division. The activity of cdk1-cyclin B complex is also controlled by
regulation of the nuclear transport of cyclin B. And the activity of cdk1 is regulated
positively by the phosphatase cdc25, which dephosphorylates tyrosine 14 and threonine
15, and negatively by the kinases Wee-1 and Myt-1, which phosphorylate these
residues. Myt-1 is cytoplasmic and phosphorylates threonine 14, while Wee-1 is
nuclear and phosphorylates tyrosine 15. Cdc25 is activated at the end of G2, leading to
permitting the cell to enter M phase (Weber, 2007).
(4) Regulation of M phase progression
In M phase, the activity of cdk1-cyclin B complex reorganizes the
microtubules and microfilaments, and phosphorylates proteins in the nuclear lamina,
resulting in the nuclear envelope breakdown, chromosome condensation, mitotic
spindle formation and fragmentation of the Golgi complex and endoplasmic reticulum.
Just before the breakdown of the nuclear membrane, the cdk1-cyclin B complex
translocates to the nucleus to target further substrates, including those that control the
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shutdown of RNA polymerase III-mediated transcription. Finally, cyclin B and cyclin
A are rapidly degraded by the APC before the end of mitosis (Skaar & DeCaprio, 2006).
The APC is inactivated by the accumulation of G1 phase cdks (Nasmyth, 1996). The
mutual inhibition between APC and cdks explains how cells suppress mitotic cdk
activity during G1 and then establish a period with elevated kinase activity from S phase
until anaphase (Weber, 2007).
2.2.1.2 Cell cycle checkpoints
The cell cycle transition, which passes from one phase to another, is
regulated by checkpoints consisting of the restriction checkpoint, DNA damage
checkpoint, replication checkpoint, and spindle checkpoint (Kleinsmith, 2006), as
shown in Figure 2.6. These checkpoints monitor conditions within the cell and
transiently halt the cell cycle at various points for correction and repair (Elledge, 1996).
If cells cannot repair such damage, they are eliminated through apoptosis.
Figure 2.6 The cell cycle checkpoint. (Modified from Kleinsmith, 2006)
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(1) Restriction checkpoint
Restriction checkpoint marks a key phase in the cell cycle, which
decides whether or not to proceed to mitosis. The cell cycle progression cannot begin
until the appropriate cellular growth has occurred during G1. Therefore, growth factors
are necessary to promote passage through the restriction point, which occurs in the late
G1 phase leading to S phase. Normally, retinoblastoma protein (pRb) functions as a
tumor suppressor to halt the cell cycle in the resting or G1 phase, by binding to a
transcription factor E2F. This restriction point is inactivated by cdk4/6-cyclin D
phosphorylation of pRb, with subsequent release of E2F that directs the synthesis of
proteins, allowing the cell cycle to proceed. Loss of restriction point control occurs in
many cancers and deregulates progression through the cell cycle.
(2) Replication checkpoint
Replication checkpoint is important for the integrity of the
genome. This checkpoint monitors the DNA replication during S phase to ensure that
DNA damage has been repaired or that DNA synthesis has been completed prior to
proceeding into M phase. For an entry into M phase, cdc25 removes inhibitory
phosphorylations from cdk1 to promote its activity. However, when the checkpoint is
engaged in response to DNA damage or incomplete DNA synthesis, cdc25 becomes
phosphorylated and is degraded, leading to no removal of the inhibitory
phosphorylations of cdk1. Thus, cdk1 remains inactive, thus preventing progression
through mitosis.
(3) Spindle checkpoint
Spindle checkpoint acts between the metaphase and anaphase
stages of mitosis. At the end of metaphase, the two sets of chromosomes are normally
lined up at the center of the mitotic spindle, and the anaphase-promoting complex
(APC) becomes active, triggering the onset of anaphase. Before chromosome
movement begins at the onset, the spindle checkpoint is invoked to make certain that
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chromosomes are all properly attached to the mitotic spindle. If chromosomes are not
properly attached, a Mad-Bub protein complex is formed and subsequently inhibits the
APC by blocking its essential activators. For this reason, the cell cycle is temporarily
halted to allow the chromosomes to become attached properly and completely to the
spindle.
(4) DNA damage checkpoint
DNA damage checkpoint monitors DNA damage and halts the cell
cycle including late G1, S, and late G2 by inhibiting different cdk-cyclin complexes. In
this checkpoint, activated p53 protein plays a central role in which its accumulation in
response to DNA damage increases transcription of its target genes including p21. This
leads to inhibition of cdk-cyclin complexes in G1 and G2 phases and subsequent cell
cycle arrest, thus giving the cells time to repair DNA damage. Therefore, DNA damage
can be avoided before division to limit heritable mutation. If the damage cannot be
repaired, p53 may also trigger cell death by apoptosis.
2.2.2 Alterations of cell cycle regulation in cancer cells
In cancer cells, the accumulation of genetic alterations that involve cell
division leads to an unrestrained cell proliferation. A special subset of cancer-relevant
genes is represented by oncogenes and deregulated tumor suppressor genes. Activation
of proto-oncogenes to become oncogenes and/or inactivation of tumor suppressor genes
causes carcinogenesis. These genes encode proteins controlling cell growth,
proliferation and survival (e.g. growth factors, growth factor receptors, signal-
transduction proteins, transcription factors, pro- or anti-apoptotic proteins, cell-cycle
control proteins, and DNA repair proteins) and play roles in cancer induction (Lodish
et al., 2000).
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2.2.2.1 Oncogenes
Oncogenes are mutated forms of the normal genes, proto-oncogenes.
The oncogenes contribute to converting a normal cell into a cancer cell, as these genes
encode proteins involved in growth signal transduction pathways, including growth
factor, growth factor receptors, proteins involved in signal transduction and nuclear
regulatory proteins (transcription factors) to be overactive. For example, the v-sis
oncogene of simian sarcoma virus, which encodes a growth factor homologous to
PDGF-β causes cells to overproduce growth factors, leading to stimulating cells to grow
(Fleming, Matsui, Molloy, Robbins, & Aaronson, 1989). Some oncogenes produce
either aberrant receptor proteins that release stimulatory signals into the cytoplasm even
when no growth factors are present in the environment or increased amount of receptor
proteins that results in increased signaling via the Ras-MAPK pathway, driving cellular
proliferation. For instance, the HER2/neu gene, which encodes transmembrane
receptors for growth factors, including EGFR, HER2, HER3, and HER4 (Burstein,
2005). Also, several proteins, which are encoded by oncogenes have their effect at the
cell membrane (e.g. the ras oncogene encodes guanine nucleotide-binding proteins
(G proteins), whereas some oncogenes act in the nucleus by binding to DNA. For
example, the myc oncogene encodes a transcription factor.
These oncogenes act as dominant genes because a mutation in only one
copy of the gene is sufficient carcinogenesis (Hunt & Dacic, 2008; Larsen & Minna,
2011). There are three main mechanisms that lead to the conversion of proto-oncogenes
to oncogenes, including mutations, gene amplification, and chromosomal
rearrangements as shown in Figure 2.7.
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Figure 2.7 The mechanisms that lead to the conversion of proto-oncogenes to oncogenes.
(Modified from Lieberman, Marks, & Peet, 2012)
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2.2.2.2 Deregulated tumor suppressor genes
Tumor suppressor genes are genes that are important in preventing
carcinogenesis. There are two major groups of tumor suppressor genes: gatekeeper
genes and caretaker genes (Kinzler & Vogelstein, 1997). Gatekeeper genes are
responsible for controlling or inhibiting cell proliferation by regulating the cell cycle
whereas caretaker genes are responsible for processes that ensure the integrity of the
genome by repairing DNA damage (Morris & Chan, 2015). However, the mutations of
these genes cause a loss or reduction in its function, leading to the induction of cancer.
For example, p53 is a well-known transcription factor that plays a crucial role in the
response of the cell to stress. The p53 is most often identified as a gatekeeper gene,
since it is directly involved in cell cycle regulation and cellular proliferation. Also, p53
is identified as a caretaker genes, as it involved in DNA repair mechanisms (Soussi &
Wiman, 2015). The mutations of this gene can grant cells with additive growth and
survival advantages, such as increased proliferation, evasion of apoptosis, and
chemoresistance (Rivlin, Brosh, Oren, & Rotter, 2011).
Tumor suppressor genes are recessive as they contribute to the
development of cancer when both copies of the gene are inactivated (Figure 2.8).
Genetic mechanisms driving to the loss of function of tumor suppressor genes can arise
at the nucleotide or chromosome level. These involve point mutation and losses of
genetic material by interstitial deletion, by the loss of arm or whole chromosome, by
unbalanced translocations, or passages in the homozygosity by mitotic recombination
or chromosome loss followed by reduplication (isodisomy) (Pierron, 2015).
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Figure 2.8 The mechanisms that lead to the deregulation of tumor suppressor genes.
(Modified from Alberts et al., 2014)
In addition, cell cycle deregulation associated with cancer occurs through
mutation of genes encoding cdks, cyclins, cdk-activating enzymes, CKIs, cdk
substrates, and checkpoint proteins at different levels of the cell cycle (Vermeulen, Van
Bockstaele, & Berneman, 2003). Changes in levels and activity of these cell cycle
regulators result in uncontrolled cell division. For example, increased levels/activity of
positive regulators, e.g. cdks and cyclins, as well as decreased levels/activity of
inhibitors of the cell cycle, e.g. CKI, can promote cancer. Furthermore, cancer cells
often lose the cycle checkpoint integrity as a result of inactivation of CKIs and/or
overexpression of cdks and cyclins. Checkpoint dysfunction contributes unregulated
cell growth.
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2.3 Classification of cell death
Cell death is a crucial process which plays an important role in controlling
development, homeostasis, and immune regulation of multicellular organisms. It also
protects the organism overall by removing all cells damaged by disease, aging,
infection, genetic mutation, and exposure to toxic agents (Saikumar & Venkatachalam,
2009). According to recommendations of the Nomenclature Committee on Cell Death
(NCCD) 2009, cell death can be classified according to its morphological appearance
e.g. apoptosis, necrosis and autophagy (Figure 2.9), enzymological criteria (with and
without the involvement of nucleases or of distinct classes of proteases, such as
caspases, calpains, cathepsins and transglutaminases), functional aspects (programmed
or accidental, physiological or pathological) or immunological characteristics
(immunogenic or non-immunogenic) (Kroemer et al., 2009).
2.3.1 Autophagy
Autophagy is a self-degradative physiological process that removes
unnecessary or dysfunctional cellular components through the actions of lysosomes.
Also, it is important for balancing sources of energy at critical times during
development and in response to nutrient starvation or other stresses (Parlato &
Mastroberardino, 2015). The specific morphological features of autophagy are defined
especially by transmission electron microscopy as follows: lack of chromatin
condensation and the presence of massive vacuolization of the cytoplasm. These
vacuoles, by definition, are two-membraned and contain degenerating cytoplasmic
organelles or cytosol. And the autophagic cell death has little or no association with
phagocytes (Kroemer et al., 2009).
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Figure 2.9 Characteristics of autophagy, apoptosis and necrosis. (Modified from Tan,
Lu, Ji, & Mao, 2014)
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2.3.2 Necrosis
Necrosis is a form of cell injury which results in the premature death of cells
in living tissue by autolysis, and it is defined as accidental cell death due to the actions
of external factors (e.g. infection, toxins, or trauma) on the cells or tissues that result in
a pathological process. The common features of necrosis are associated with cell
swelling, the rapid loss of membrane integrity and leak their intracellular components,
some of which serve as danger signals that stimulate inflammation (Rock & Kono,
2008). In contrast to apoptosis, necrotic cell death does not fragment into discrete.
2.3.3 Apoptosis
Apoptosis (or programmed cell death) is a mode of cell death that occurs to
remove unwanted cells, improperly functioning cells and injured cells without
damaging neighboring cells or inducing inflammation. Kerr, Wyllie, & Currie (1972)
have described the specific morphological features of cells undergoing apoptosis. The
features include chromatin aggregation, nuclear and cytoplasmic condensation, the
formation of membrane-bound vesicles (apoptotic bodies) which contain ribosomes,
morphologically intact mitochondria and nuclear material. Then, the apoptotic bodies
are rapidly recognized and phagocytized by either macrophages or adjacent epithelial
cells in which this phagocytic removal of apoptotic cells does not elicit an inflammatory
response (Travis et al., 2015). Also, the NCCD (2009) has formulated that apoptotic cell
death can occur with or without caspase activation (Kroemer et al., 2009).
Among three types of cell death, apoptosis is the most extensively
characterized mechanism for cancer cell killing as apoptosis is required to remove
abnormal cells without harming cells. Moreover, apoptosis has emerged as the major
mechanism by which anticancer agents act to eliminate cancer cells (Feng et al., 2011).
And the mechanisms and pathways of apoptosis are described in the topic 2.4.
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2.4 Alteration of apoptotic cell death in cancer cells
2.4.1 Apoptosis in normal cells
Apoptosis or programmed cell death is a mode of cell death in normal
homeostasis to maintain cell populations in tissues during development and aging. It
also occurs as a defense mechanism such as in immune reactions or when cells are
damaged by diseases or toxic agents (Norbury & Hickson, 2001). There are two major
pathways of apoptotic cell death namely the death receptor pathway (extrinsic pathway)
and the mitochondrial pathway (intrinsic pathway) (Xu & Shi, 2007), as shown in
Figure 2.10. These two pathways are involved with several proteins especially a group
of caspase enzymes. Therefore, caspases are central components of the regulatory
mechanisms of the apoptotic pathway because they play a central role as both initiators
(caspases 8 and 9) and executioners (caspases 3, 6 and 7) (Ghavami et al., 2009), as
shown in Figure 2.10. Caspase-3 is the most common executioner among executioner
caspases. These caspases cause the disassembly of the genome by activating the
caspase-activated DNase (CAD), which preexisted in cells as an inactive complex with
the inhibitor of caspase-activated DNase (ICAD). The executioner caspases then cleave
ICAD, thus allowing CAD to degrade chromosomal DNA into oligonucleosomal
fragments. They also disable the normal DNA repair process by directly inactivating
two key proteins involved in maintaining genomic integrity, poly (ADP-ribose)
polymerase (PARP) and DNA-dependent protein kinase (DNA-PK). In addition, the
effector caspases cause nuclear shrinking and budding to form apoptotic bodies, as well
as the structural disassembly of the cell through the direct proteolysis of the
cytoskeleton and nuclear scaffold (Hsieh & Nguyen, 2005).
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Figure 2.10 A schematic representation of intrinsic and extrinsic pathways of apoptosis.
(Modified from Ghavami et al., 2009)
2.4.1.1 The mitochondrial pathway (or intrinsic pathway)
The mitochondrial pathway or the intrinsic pathway is initiated in
response to DNA damage and involved with two major groups of Bcl-2 family proteins
based on their function (Green, 2015). The first group is antiapoptotic (also called pro-
survival) members that have four BH domains such as Bcl-2, Bcl-xL, Mcl-1, etc. The
second group is proapoptotic (or anti-survival) members, which are subdivided into two
groups: the proapoptotic proteins containing three BH domains (e.g. Bax and Bak) and
the proapoptotic BH3-only proteins carrying a single BH3 domain (e.g. Bim, Bad, Bid
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and others). Different BH3-only proteins can elicit apoptosis by either inactivating pro-
survival functions of Bcl-2 and Bcl-xL or directly stimulating Bax and Bak. In cells that
are in healthy equilibrium, these proteins are located in the mitochondrial
intermembrane space, and antiapoptotic proteins (e.g. Bcl-2, Bcl-xL, Mcl-1) inhibits
proapoptotic functions of Bax/Bak, thus providing a pro-survival signal as the default
setting in the mitochondria.
In intrinsic pathway, p53, a tumor suppressor protein, is activated in
the cytosol in response to irreparable DNA damage and translocates to the nucleus
where it activates transcription of many proapoptotic proteins while simultaneously
repressing transcription of antiapoptotic proteins. As a result, the balance between pro-
and anti-apoptotic proteins shifts to favor proapoptotic proteins. In parallel, high
concentrations of p53 also translocate to mitochondria and bind to antiapoptotic
proteins, resulting in deactivated functions of these proteins (Strayer & Rubin, 2014).
Mitochondrial outer membrane permeabilization (MOMP) is a key
feature of the intrinsic pathway. During apoptosis, DNA damage–activated p53 triggers
increased levels of proapoptotic proteins, particularly BH3-only proteins. These
proteins inactivate anti-apoptotic proteins and also directly stimulate Bax and Bak,
which then oligomerize and form the pores, resulting in disruption of the outer
membranes of the mitochondria. Then, soluble proteins normally found in the space
between the inner and outer mitochondrial membranes are released, including
cytochrome c, apoptosis-inducing factor (AIF), endonuclease G (endoG), Smac/Diablo
and HtrA2/Omi. These mitochondrial proteins activate either caspase-dependent or
-independent cell death pathways (Saelens et al., 2004).
(1) Caspase-dependent apoptosis
In caspase-dependent apoptosis, following Bax/Bak forming pores
in the outer membrane, cytochrome c is released and then interacts with Apaf-1 to begin
the formation of the apoptosome. This apoptosome is a multiprotein complex
comprising Apaf-1, cytochrome c, and caspase-9, which functions to activate caspase-3
downstream of mitochondria in response to apoptotic signals In addition, Smac/Diablo
and HtrA2/Omi activate apoptosis by neutralizing the inhibitory activity of inhibitor
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apoptotic proteins (IAPs) that inhibit caspases, leading to elevated activities of
executioner caspases-3, -6 and -7 (Chai et al., 2000; Du, Fang, Li, Li, & Wang, 2000).
The downstream caspases induce cleavage of protein kinases, cytoskeletal proteins,
DNA repair proteins, and inhibitory subunits of endonuclease as previously mentioned
(Kalimuthu & Se-Kwon, 2013).
(2) Caspase-independent apoptosis
In caspase-independent apoptosis, the soluble proteins including
AIF and endoG released from mitochondrial intermembrane space induce apoptosis in
a manner independent of caspase activities. AIF directly translocates to the nucleus and
triggers chromatin collapse and digestion into high molecular weight fragments. EndoG
also translocates to the nucleus where it cleaves nuclear chromatin to produce
oligonucleosomal DNA fragments (Bajt, Cover, Lemasters, & Jaeschke, 2006). In
addition, the use of caspase inhibitors, which block caspase-dependent apoptotic cell
death, cannot rescue these cells from caspase-independent apoptosis.
2.4.1.2 The death receptor pathway (or extrinsic pathway)
This pathway is triggered when specific death ligands of the TNF
family (e.g. TNF, Fas-ligand, TRAIL) engage their receptors (e.g. TNFR, Fas (also
called CD95 or APO-1), TRAIL-R1 and -R2 (also called DR4 and DR5), respectively
(Green, 2015). At the cell surface, death receptors become activated upon binding their
ligands. As a result, the cytoplasmic tails of these receptors bind the death domains of
docking proteins, to form a death-inducing signaling complex (DISC), leading to
subsequent stimulation of downstream procaspases-8 and -10, to become active forms.
In turn, these caspases activate executioner caspases-3, -6 and -7.
The extrinsic (death receptor) pathway intersects the intrinsic
(mitochondrial) pathway via caspase-8, which cleaves a cytoplasmic protein, Bid.
However, Bid is inactive unless it is proteolytically cleaved. Truncated Bid (tBid)
translocates to mitochondria and activates Bax and Bak to cause MOMP and apoptosis
via mitochondrial pathway (Ghavami et al., 2009; Green, 2015).
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2.4.2 Apoptosis in cancer cells
Insufficient apoptosis can lead to the development of cancer. Indeed, the
mutated cells that escape the apoptotic control typically ignore normal cellular signals
and become more proliferative than normal, allowing them to change to neoplastic cells
and even malignant cells rapidly. These cells can acquire reduction in apoptosis or
resistance to apoptosis through the mechanisms as follows: 1) impaired death receptor
signaling, e.g. reduced expression of death receptors or signals as well as expression of
decoy receptor without death domain, 2) defects or mutations in p53, 3) reduced
expression and function of caspases, 4) increased expression of IAPs and 5) disrupted
balance of pro-apoptotic and anti-apoptotic proteins, e.g. overexpression of anti-
apoptotic proteins or/and underexpression of pro-apoptotic proteins (Wong, 2011).
2.4.3 Reactive oxygen species (ROS) leading to apoptosis in cancer cells
Reactive oxygen species (ROS) are chemically reactive molecules that
contain oxygen, and they are broadly classified into two groups: free radicals and
nonradicals. The free radicals, including superoxide anion (O2•−), hydroxyl radical
(•OH), hydroperoxyl radical (HO2•−), peroxyl radical (ROO•) and alkoxyl radical (RO•),
are molecules that possess one or more unpaired electrons in their outer orbital (Islam
& Shekhar, 2015). Therefore, they are highly reactive, as they can either donate an
electron to or accept an electron from other molecules to achieve stability (Lobo, Patil,
Phatak, & Chandra, 2010). The nonradicals, including hydrogen peroxide (H2O2),
hypochlorous acid (HOCl), hypobromous acid (HOBr), ozone (O3) and singlet oxygen
(1O2), are molecules that do not have unpaired electron(s) but are chemically reactive
to generate free radicals under certain conditions with or without enzymatic catalysis
(Trachootham, Alexandre, & Huang, 2009; Shi, Zhang, Zheng, & Pan, 2012). ROS are
generated by either endogenous or exogenous sources. The former sources include
oxidases, peroxidases and oxygenases. In the cells, these intracellular enzymes bind O2
and transfer single electrons to it via a metal. Before the reaction is complete, such
reaction may accidentally release free radical intermediates. The latter sources include
environmental pollution, radiation, cigarette smoking, certain foods and drugs
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(Bhattacharyya, Chattopadhyay, Mitra, & Crowe, 2014). Normally, ROS are
effectively neutralized by the potent antioxidant system. However, when ROS are
produced beyond the antioxidant capacity of the cells, they promote genotoxic damage
and thereby cancer progression (Al-Khayal et al., 2017). Nevertheless, exogenous
administration of ROS, especially via the use of chemotherapeutic drugs, leading to
apoptotic induction has become a potential mechanism of action in eliminating cancer
cells (Ahn et al., 2014).
Several studies have shown that various anticancer drugs e.g. doxorubicin,
azidothymidine (AZT), cisplatin (Deavall, Martin, Horner, & Roberts, 2012) produced
ROS at excessive levels, resulting in irreparable DNA damage, subsequently leading to
apoptotic cell death. Moreover, recent studies have revealed that some of the plant
natural compounds e.g. resveratrol and curcumin (Singh et al., 2012), neferine
(Poornima, Quency, & Padma, 2013), capsaicin (Bu et al., 2015) as well as isoliensinine
(Zhang et al., 2015) induce apoptosis through ROS-mediated c-Jun N-terminal protein
kinase (JNK) and p38 MAPK pathways. These two kinases are stress-activated protein
kinases (MAPK). Both JNK and p38 MAPK are then activated through apoptosis
signal-regulating kinase-1 (Ask-1), whose activity is regulated by its interaction with
thioredoxin, which is a redox-regulated protein (Saitoh et al., 1998). Under normal
conditions, thioredoxin directly binds to the N-terminal noncatalytic region of Ask-1,
resulting in inhibiting kinase activity. However, in response to ROS, the oxidized form
of thioredoxin dissociates from Ask-1, thus allowing Ask-1 activation (Saitoh et al.,
1998; Liu & Min, 2002). Ask-1 signaling activates downstream MAPK kinases that
promote activation of JNK and p38 MAPK signaling pathways (Figure 2.11). Activated
JNK readily translocates to mitochondria to phosphorylate a tumor suppressor protein
p53, which activate proapoptotic proteins, such as members of the BH3-only subgroup
of the Bcl-2 family (e.g., Bid and Bim), or suppress the activity of antiapoptotic Bcl-2
and Bcl-xL proteins It is also associated with the overexpression of proapoptotic Bax,
leading to formation of Bax homodimers resulting in MOMP, the release of cytochrome
c from the inner mitochondrial membrane, apoptosome formation and finally induction
of apoptosis (Zhang, Humphreys, Sahu, Shi, & Srivastava, 2008; Liou & Storz, 2010).
However, in normal cells, levels of p53 are usually kept low by its association with a
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protein called Mdm2, which binds p53 and transports it from the nucleus to the cytosol
for proteolytic degradation by the proteasome (Shadfan, Lopez-Pajares, & Yuan, 2012).
Figure 2.11 JNK/p38 MAPK signaling pathways, apoptosis pathway, and multiple
molecular targets of plant-derived agents.
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2.5 Targeting for cancer treatment
2.5.1 Targeting cell cycle regulators in cancer treatment
The knowledge of the molecular mechanisms of cell cycle regulation is
important to control the aberrant proliferation including DNA replication and accurate
segregation of chromosomes to daughter cells, which are characteristically aberrant in
cancer cells (Ganem, Storchova, & Pellman, 2007). Many anticancer agents such as
flavopiridol (Shapiro & Harper, 1999), rifampicin (Zhuang et al., 2011), paclitaxel
(Choi & Yoo, 2012) can slow down cell division by inducing cell cycle arrest in the
G0/G1, S, or G2/M phases (Hsiao et al., 2014). They can target cdks, which are required
at different phases of the cell cycle, and inhibit the function of cdk by directly binding
the catalytic cdk subunit or indirectly targeting regulatory pathways governing cdk
activity (Schwartz & Shah, 2005). In addition, several studies have revealed that many
anticancer agents and plant-derived compounds exerted their cytotoxic effect through
arresting of the cell cycle in each phase, depending on their selectivity, as follows:
- Compounds that have been shown to arrest G0/G1 phase. For
example, curcumin derived from turmeric (Mukhopadhyay et al., 2002); tangeretin
derived from the peel of citrus fruits (Pan, Chen, Lin-Shiau, Ho, & Lin, 2002); honokiol
derived from Magnolia officinalis/grandiflora (Hahm & Singh, 2007); lycorine, a
natural alkaloid extracted from Amaryllidaceae (Li et al., 2012) and glycyrrhetinic acid
derived from glycyrrhiza (Zhu et al., 2015) have been shown to modulate the activities
of several key G1 regulatory proteins via down-regulation of cdks (e.g. cdk4, cdk6) and
cyclins (e.g. cyclin D, cyclin E) but up-regulation of the tumor suppressor protein p53.
- Compounds that have been shown to arrest S phase. For
example, crude seed extract of celery (Apium graveolens L) has been shown to down-
regulate cdk2 and cyclin A (Gao et al., 2011). Besides altering expression levels of
regulatory proteins in S phase, some anticancer agents are also incorporated directly
into DNA or RNA, and disrupt DNA synthesis during S phase, such as 5-fluorouracil,
Cytarabine, and Methotrexate, which inhibits thymidylate synthase, DNA polymerase
and dihydrofolate reductase, respectively (Payne & Miles, 2008).
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- Compounds that have been shown to arrest G2/M phase. For
example, jaceosidin, a flavonoid present in plants of genus Artemisia (Khan, Rasul, Yi,
Zhong, & Ma, 2011); myricetin, a naturally occurring flavonol widely presented in
fruits, vegetables, tea, berries and red wine (Zhang, Zou, Xu, Shen, & Li, 2011); and
curcumin (Cheng et al., 2016) have been shown to down-regulate the expression levels
of regulatory proteins of G2/M phase such as cyclin B1 and cdk1 but up-regulate p53
and p21. Moreover, Ouyang et al., (2009) have shown that genistein, a major
isoflavonoid, can increase the phosphorylation and activation of checkpoint kinases
(Chk1 and Chk2), which results in the phosphorylation and inactivation of phosphatases
(cdc25C and cdc25A), and thereby the phosphorylation and inactivation of cdc2, which
arrests cells at G2/M phase. Besides altering expression levels of regulatory proteins in
G2/M phase, some anticancer agents can bind to tubulin and lead to disrupting the
spindle apparatus of the microtubules required for chromosome segregation in M phase
(Stanton, Gernert, Nettles, & Aneja, 2011). The microtubules consist of -tubulin and
ß-tubulin and carry out polymerization and depolymerization. Altering a dynamic
balance between polymerization and depolymerization is a target for cancer drug
development (Shin et al., 2008). Certain compounds can inhibit or halt cell cycle by
interfering with microtubules during mitosis or M phase. Examples of drugs in this
category are vinca alkaloids and taxanes, which interrupt cell division by agitation of
microtubule dynamics. Vinca alkaloid (e.g. vinblastine and vincristine) binds to the
unpolymerized tubulin molecules and prevents them from polymerizing into a growing
microtubule. Taxol binds to tubulin within assembled microtubules and prevents
disassembly (Castedo et al., 2004; Mollinedo & Gajate, 2003).
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2.5.2 Targeting apoptosis in cancer treatment
The discovery of various types of defects (e.g. impaired death receptor
signaling, defects or mutations in p53, reduced expression and function of caspases,
increased expression of IAPs and disrupted the balance of proapoptotic and
antiapoptotic proteins) in the apoptotic pathways becomes an interesting target for
cancer treatment (Wong, 2011). Anticancer agents including plant-derived compounds
can restore the apoptotic signaling pathways towards normality to eliminate cancer cells
acquiring these defects, as follows:
- Some plant compounds have been reported to induce the
extrinsic apoptotic pathway by up-regulation of death receptors, e.g. Fas/CD95, DR4
and DR5 or their corresponding ligands, e.g. Fas ligand, TRAIL-R1, and -R2,
respectively. For example, triterpenediol, which comprises of isomeric mixture of 3α,
24-dihydroxyurs-12-ene and 3α, 24-dihydroxyolean-12-ene from Boswellia serrate
(Bhushan et al., 2007); γ-humulene derived from Emilia sonchifolia (Lan et al., 2011)
and curcumin (Jung et al., 2005) stimulate the clustering of DR4/DR5, TNF-R1 and
increasing of associated FADD protein levels, leading to caspase-8 and caspase-3
activation.
- Some plant compounds can restore the intrinsic apoptotic
pathway by altering the balance of pro- and antiapoptotic proteins. For example, butein,
a polyphenol derived from Dalbergia odorifera (Kim et al., 2001); curcumin derived
from Curcuma longa and the root extract of Salvia miltiorrhiza (Duval, Moreno-
Cuevas, Gonzalez-Garza, Rodriguez-Montalvo, & Cruz-Vega, 2014) as well as the
hexane extract of the leaves from Ferulago angulata (Karimian et al., 2014) have been
shown to decrease the levels of antiapoptotic proteins (e.g. Bcl-2, Bcl-xL) while it
increased the levels of proapoptotic proteins (e.g. Bax and Bak). These proapoptotic
proteins directly form pores in the mitochondrial outer membrane leading to the release
of cytochrome c. Cytochrome c induced apoptosis by activation of caspase 9 and 3,
respectively. In addition, some plant compounds such as flavokawain C, found in Kava
(Piper methysticum Forst), caused disruption of mitochondrial membrane potential,
leading to the release of AIF, Smac/DIABLO and cytochrome c from the mitochondria
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(Phang, Karsani, Sethi, & Abd Malek, 2016). These proteins then activate various
caspases (-3, -8, -9) and subsequent PARP cleavage, leading to apoptotic cell death.
Furthermore, the action of several plant-derived compounds through high
levels of ROS production is an important mechanism to eliminate cancer cells. This
excessive ROS formation can induce oxidative stress, which affects DNA damage and
leads to cell death. Briefly, in response to DNA damage, p53 is stabilized and then acts
to regulate the expression of stress response genes in DNA repair, cell-cycle arrest, and
apoptosis, in order to suppress cancer cell proliferation (Circu & Aw, 2010). Recently,
some of plant natural compounds such as curcumin (an active ingredient of turmeric),
triterpenediol (an isomeric mixture of 3α, 24-dihydroxyurs-12-ene and 3α, 24-
dihydroxyolean-12-ene from Boswellia serrate), shikonin (a naphthoquinone isolated
from Lithospermum erythrorhizon), and grape seed extract (a complex mixture of
polyphenols known as proanthocyanidins) have been shown to induce cancer cell death
through massive ROS generation, leading to an induction of intrinsic (including
caspase-dependent and caspase-independent) and extrinsic pathways of apoptosis
(Derry, Raina, Agarwal, & Agarwal, 2013; Bhushan et al., 2007; Wu et al., 2010; Gong
& Li, 2011).
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2.6 Thai medicinal plants (Hua-Khao-Yen)
Thai medicinal plants locally known as “Hua-Khao-Yen” have widely been
used to prepare Thai traditional medicine. Five species of Hua-Khao-Yen including
Dioscorea membranacea Pierre, Dioscorea birmanica Prain ex Burkill, Smilax
corbularia Kunth, Smilax glabra Roxb and Pygmaeopremna herbacea Roxb have been
extensively used by Thai folk doctors for the treatment of cancers, AIDS, septicemia,
inflammation and lymphatic diseases. The preparation of plant extracts for treating such
diseases in Thai traditional medicine is usually made by boiling in water or by soaking
in alcohol (Pongbunrod 1976; Tungtrongjit 1978). Among these species, D.
membranacea Pierre has been the most widely used to prepare Thai traditional anti-
cancer medicine. Itharat et al. (2004) have reported that its aqueous and ethanolic
extracts were more cytotoxic against cancer cell lines than other species.
2.6.1 Dioscorea membranacea Pierre
2.6.1.1 General description
Dioscorea membranacea Pierre, also called Hua-Khao-Yen-Tai, is a
member of the Dioscoreaceae family. This plant is scattered in open areas of mixed
deciduous forests to lower montane evergreen forests, often on limestone; 50-800 m,
and its distribution in Thailand, Vietnam, Laos, and Myanmar (Wilkin, 2009). The
general characteristics were described by Wilkin and Thapyai (Thapyai, 2004; Wilkin,
2009) as following: Climber to 10 m. rhizomes are 4-8 long by 1-5 cm wide, branching
and spreading, dark brown, shallowly horizontally buried, periderm hard but lacking
rigid, spine-like roots. Stems are 2-5 mm in diameter, twining to the left, unarmed.
Leaves simple, alternate, blades ovate to broadly so, membranous to thinly chartaceous
and usually translucent when dried, 7–9(–11)-veined, base shallowly to deeply cordate
apex acuminate, margins usually deeply 3-lobed, sometimes shallowly so or entire
(reproductive shoots only) or with additional lobes (towards stem base); petioles 2–13
cm long; cataphylls and bulbils absent; lateral nodal spines usually present on either
side of each node, sometimes absent (especially on distal shoots) or broken off in
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specimen preparation, curved, hard and rather brittle, to 4 mm long. Inflorescences
spicate, pendent, axes sometimes finely tuberculate, tepals inserted on a cup-shaped
torus, erect, apices not recurved, fused for 1/2 to 2/3 of their length. Male inflorescences
simple or compound, compound inflorescences 1(–2) per axil, simple/partial
inflorescences (Pl. 17C) 1–2(–3) per axil, peduncles 2.2–25 mm long, axes 4–33.5 cm
long, with an apparently sessile cymule of (1–)2–3 flowers at each node; female
inflorescences 1(–2) per axil, simple. Male flowers are 0.8–1.4 mm in diam. at anthesis,
outer tepals 1.9–2.3 by 0.6–1, narrowly obovate to obovate-oblong, inner tepals 1.9–
2.2 by 0.6–0.8 mm, obovate to obovate-oblong, stamens 6. Capsules 18–21 by 25–40
mm. Seeds 3–6 by 5–7 mm, ovoid-lenticular; wings 17–18 by 15–17 mm, extending all
around seed margin. The characteristics of D. membranacea Pierre are shown in Figure
2.12, 2.13 and 2.14.
Figure 2.12 The characteristics of D. membranacea Pierre. (Original picture)
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Figure 2.13 D. membranacea Pierre (Male plant). (Thapyai, 2004)
Note: A. Male fluorescence; B. primary bract; C. partial male inflorescence, showing
cymose pattern; D. cymular bract; E-I. male flower; E. l-section stamens and pistillode;
F. floral bract; G. bracteole; H. outer tepal; showing stamen adnation, I. inner tepal with
stamen adnation; J. rhizome.
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Figure 2.14 D. membranacea Pierre (Female plant). (Thapyai, 2004)
Note: K. inflorescence; L–Q. female flower; L. side view; M. l-section (excluded ovary)
showing staminodes, style and stigmas; N. floral bract; O. bracteole; P–Q. outer and
inner tepal respectively, with staminode adnation; R. infructescence; I. mature capsule,
l-section showing seed position; K. seed.
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2.6.1.2 Biological activities
Dioscorea membranacea Pierre has shown numerous biological
activities such as anticancer activity (Itharat et al., 2004; Itharat et al., 2014), anti-
allergic activity (Tewtrakul & Itharat, 2006), anti-HIV activity (Tewtrakul, Itharat, &
Rattanasuwan, 2006), antioxidant activity (Itharat et al., 2007) and anti-inflammatory
activity (Tewtrakul & Itharat, 2007).
(1) Antiproliferative activity
The previous study has shown that the crude ethanolic extract of
D. membranacea Pierre exhibited high cytotoxic activity against lung, breast and colon
cancer cell lines (COR-L23, MCF-7 and LS-174T, respectively) whereas its water
extracts exhibited high cytotoxic activity against breast and colon cell lines. Both the
extracts had no cytotoxic effects against keratinocyte normal cell line (SVK-14) using
the SRB assay (Itharat et al., 2004). As shown in Figure 2.15, nine compounds were
isolated by bioassay-guided fractionation from the ethanolic extract of rhizomes of
D. membranacea Pierre (Itharat 2002; Itharat et al., 2003). They include two
naphthofuranoxepins (e.g. dioscorealide A and dioscorealide B), one phenanthraquinone
(e.g. 1,4-phenanthraquinone or dioscoreanone), three steroids (e.g. β-sitosterol,
stigmasterol and β-sitosterol-3-O-β-D-glucopyranoside), three steroid sapogenins (e.g.
diosgenin 3-O-α-L-glucopyranosyl (1→2)- β-D-glucopyranoside, diosgenin 3-O-β-D-
glucopyranosyl (1→3)-β-D-glucopyranoside and diosgenin). Among these compounds,
dioscorealides B, dioscoreanone and diosgenin 3-O-α-L-rhamnopyranosyl (1→2)-β-D-
glucopyranoside exerted cytotoxic activity against lung, breast and colon cancer cell
lines (Itharat et al., 2003). Moreover, dioscorealide B (Saekoo, Dechsukum et al. 2010;
Saekoo, Graidist et al. 2010) and dioscoreanone (Hansakul et al., 2014) were studied
on the molecular mechanism underlying the anticancer activity in cancer cell lines
through induction of apoptosis.
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Figure 2.15 Chemical structures of isolated compounds from the rhizomes of
D. membranacea Pierre. (Itharat 2002; Itharat et al., 2003)
dioscorealide A (1) dioscorealide B (2) dioscoreanone (3)
β-sitosterol (4) stigmasterol (5)
diosgenin 3-O-β-D-glucopyranosyl
(1→3)- β-D-glucopyranoside (6)
β-sitosterol-3-O-β-D-glucopyranoside (7)
diosgenin (9) diosgenin 3-O-α-L-glucopyranosyl
(1→2)- β-D-glucopyranoside (8)
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In 2014, three dihydrophenanthrene compounds were subsequently
isolated from the ethanolic extract of rhizomes of D. membranacea Pierre including 5,6-
dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene, 5-hydroxy-2,4,6-trimethoxy-9,10-
dihydrophenanthrene and 5,6,2-trihydroxy-3,4-methoxy-9,10-dihydrophenanthrene
(Figure 2.16). One of them was 5,6-dihydroxy-2,4 dimethoxy-9,10-dihydrophenanthrene
that showed higher selective cytotoxicity against lung, breast and prostate cancer cell
lines (COR-L23, MCF-7 and PC3 cell lines, respectively) than other compounds using
SRB assay (Itharat et al., 2014).
Figure 2.16 Chemical structures of isolated compounds from the rhizomes of
D. membranacea Pierre. (1) 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene,
(2) 5-hydroxy-2,4,6-trimethoxy-9,10-dihydrophenanthrene and (3) 5,6,2-trihydroxy-
3,4-methoxy-9,10-dihydrophenanthrene. (Itharat et al., 2014)
(2) Anti-allergic activity
D. membranacea Pierre has been used in Thai traditional medicine
for treatment of allergy and allergy-related diseases as claimed by Thai folk doctors.
The previous study has shown that the ethanolic extract of D. membranacea Pierre
exhibited potent inhibitory activity against β -hexosaminidase release as a marker of
degranulation in rat basophilic leukemia mast cells (RBL-2H3). In addition, four
compounds isolated from this crude ethanolic extract including dioscorealide A,
dioscorealide B, dioscoreanone and diosgenin are suggested to be the active ingredients
of this plant as anti-allergic agents (Tewtrakul and Itharat 2006).
1
3
4a 5
7 8a
9
10a
10
4b 1
3
4a 5
7 8a
9
10a
10
4b 1
3
4a 5
7 8a
9
10a
10
4b
(1) (2) (3)
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(3) Anti-HIV activity
Traditional doctors have used D. membranacea Pierre in various
traditional Thai herbal remedies for treating HIV-infected persons. In 2006, the
ethanolic and water extracts of five species of Hua-Khao-Yen were investigated for
their inhibitory effects against HIV-1 protease (HIV-PR) and HIV-1 integrase (HIV-1
IN) (Tewtrakul, Itharat, & Rattanasuwan, 2006). The results showed the ethanolic
extract of S. corbularia exerted the most potent activity against HIV-1 IN whereas that
of D. membranacea Pierre possessed HIV-1 PR inhibitory effect. This data suggest the
combined usage of both plants in AIDS treatment.
(4) Antioxidant activity
Among five species of Hua-Khao-Yen, the ethanolic extract of D.
membranacea Pierre rhizomes possessed highest antioxidant activity using the lipid
peroxidation of liposomes assay (Itharat, 2010). In addition, an active compound
isolated from the ethanolic extract of this plants such as dioscoreanone also showed the
highest antioxidant activity using DPPH assay (Itharat et al., 2007).
(5) Anti-inflammatory activity
The ethanolic extract of D. membranacea Pierre rhizomes has
shown the anti-inflammatory activity in the inhibition of lipopolysaccahride (LPS)-
induced nitric oxide production in RAW264.7 cell lines. In addition, three active
ingredients of D. membranacea Pierre such as diosgenyl-3-O-α-L-rhamnopyranosyl
(1→2)-β-D-glucopyranoside, dioscoreanone and dioscorealide B are also active
principles for NO inhibitory activity, and only dioscoreanone showed potent inhibitory
effect on TNF-α release (Tewtrakul & Itharat, 2007). The anti-inflammatory activity of
the aqueous and ethanolic extracts from the rhizomes of D. membranacea Pierre was
also studied in in vivo experiments using carrageenin-induced paw edema in rats. The
results demonstrated that oral administration of both extracts at the dose of 1600 mg/kg
significantly decreased the paw edema induced by carrageenin in rats, indicating their
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anti-inflammatory activity (Reanmongkol, 2007). Such data support the use of D.
membranacea Pierre by Thai folk doctors for treatment of the inflammatory diseases.
2.6.2 5,6-Dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene
An active compound, 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophe
nanthrene, is isolated from the ethanolic extract of D. membranacea Pierre rhizomes
(Itharat et al., 2014), as shown in Figure 2.13. It is white to pale yellow solid. The
molecular formula of this compound is C16H16O4, and its molecular weight (M.W.) is
272.1049 g/mol. The chemical structure was shown in Figure 2.17. Moreover, in 2014,
Itharat et al. have demonstrated that this compound exhibited the highest cytotoxicity
activity on human large cell lung carcinoma cell line COR-L23, human breast cancer
cell line MCF-7 and human prostate cancer cell line PC-3 but less cytotoxicity activity
on the human lung fibroblast cell line MRC-5. However, the molecular mechanisms
underlying its cytotoxic effect have not yet been studied.
Figure 2.17 The structure of 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene.
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CHAPTER 3
RESEARCH METHODOLOGY
3.1 Conceptual framework of this study
Isolation of HMP
- Column Chromatography
- Thin-Layer Chromatography (TLC)
Structural analysis of HMP
- Nuclear magnetic resonance (NMR)
Determination of antiproliferative effects of HMP against cell lines
- Sulforhodamine B (SRB) assay
Normal cell lines Non-small cell lung cancer Small cell lung cancer
- A549
- NCI-H226
- COR-L23
- NCI-H1688 - MRC-5
Extraction of D. membranacea Pierre rhizomes
- Maceration in 95% ethanol
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The conceptual framework of this study (cont.)
Bax, Bcl-2
Caspase3, PARP
Bid, Caspase9
Bax, Bcl-2
Caspase 3 activity Kit
Z-VAD fmk
Estimation of cell proliferation dynamics
- CellTrace™ CFSE Cell Proliferation Kit
Assessment of growth inhibitory and cytotoxic effects
- Sulforhodamine B (SRB) assay
Determination of apoptotic cell death
Flow cytofluorometric analysis of cell cycle distribution
- Staining with Propidium Iodide (PI)
Cell cycle arrest (G2/M arrest) Apoptotic cell death (sub-G1)
Detection of PS on external
leaflet membrane of early
apoptotic cells
- Annexin V-FITC/PI
staining
Protein analysis (Western blotting)
- - cdc25, cdk1, cyclinB1
Mitotic spindle disruption
- Tubulin polymerization assay Kit
Apoptosis morphology
- DAPI Staining
Enzyme activity analysis RNA analysis
Protein analysis
NAC
ROS generation
NAC
Study on the anticancer effects of HMP on A549 cell line
General caspase inhibitor test
- Z-VAD fmk
DNA fragmentation
DNA ladder
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3.2 Extraction of Dioscorea membranacea Pierre
Dioscorea membranacea Pierre (DM) collected from Phetchabun province,
Thailand was used in this study. Authentication of plant materials was carried out at the
Herbarium of the Department of Forestry, Bangkok, Thailand, where the herbarium
vouchers are deposited (SKP A062041305). Assoc Prof. Dr. Arunporn Itharat,
Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat
University kindly provided it. In the extraction procedure, the rhizomes of D.
membranacea Pierre were cleaned, cut into small pieces and dried at 50 ºC (Figure 3.1).
Dried plant material (204.8 g) was macerated in 95% ethanol for 3 days at room
temperature. The extract was filtered through filter paper, and the supernatant was
evaporated to dryness by a rotary evaporator. Maceration of the residue was repeated
two times. The percent yield of the ethanolic extract of D. membranacea Pierre was
calculated.
Figure 3.1 The physical characteristics of the rhizome of D. membranacea Pierre.
(Original picture)
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3.3 Isolation of 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydrophenanthrene
Isolation procedures of an active ingredient of D. membranacea Pierre
namely 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydrophenanthrene (HMP) were kindly
supplied by Dr. Srisopa Ruangnoo, Department of Applied Thai Traditional Medicine,
Faculty of Medicine, Thammasat University. Briefly, the crude ethanolic extract of D.
membranacea Pierre was subjected to column chromatography on silica gel 60 (0.040-
0.063 mm) (Merck, Germany) as the stationary phase. The column was eluted by
gradient elution in increasing order of polarity. Fractions were collected in a 15-ml tube,
analyzed by thin layer chromatography (TLC) using aluminium sheets coated with
silica gel 60 (Merck, Germany) and visualized with acidic anisaldehyde spray. The
fractions containing similar spots were combined and evaporated to dryness under
reduced pressure. The combined fractions containing HMP were further separated by
additional silica gel column chromatography and/or by silica gel TLC glass plates,
followed by silica gel TLC aluminum sheets for analysis. The combined fractions or
scraped bands that contained one spot corresponding to 5, 6-dihydroxy-2, 4-dimethoxy-
9, 10-dihydrophenanthrene was checked for its purity using silica gel TLC aluminium
sheets 60 in three different solvent systems of varying polarity and High-performance
liquid chromatography (HPLC) technique using an Agilent Technologies 1200 Series
HPLC system (Agilent Technologies, USA). For HPLC analysis, 1 mg/ml of the
compound was achieved on a C18 reversed-phase HPLC column (250 x 4.60 mm 5
micron) (Phenomenex, USA) using water (eluent A) and acetonitrile (eluent B) as
mobile phase with the following gradient: 70-55% A at 0-10 min, 55% A for 5 min, 55-
30% A at 15-30 min, 30% A for 5 min, 30-70% A at 35-37 min and 70% A for 8 min.
The flow rate was 1 ml/min, and the injection volume was 10 μl. The ultraviolet (UV)
detector was used to detect the peak area of such compound with the fixed wavelength
at 254 and 270 nm.
Moreover, the chemical structure of the isolated compound was sent to
Bioresources Research Laboratory, National Center for Genetic Engineering and
Biotechnology (BIOTEC), Pathumthani, Thailand for the nuclear magnetic resonance
(NMR) analysis. In this study, such compound was determined to be 5,6-dihydroxy-
2,4-dimethoxy-9,10-dihydrophenanthrene by comparing its spectral data of proton
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nuclear magnetic resonance (1H NMR) with those of previously isolated compound
(Itharat et al., 2014). The stock solution of 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-
dihydrophenanthrene was prepared in DMSO at a concentration of 10,000 µM. In all
experiments, the final concentration of DMSO was kept below 0.5%.
3.4 Cell culture
Four cell lines used in this study were obtained from American Type
Culture Collection (ATCC, USA) e.g. 2 subtypes of human non-small cell lung cancer
(NSCLC); human lung carcinoma cell line A549 and human lung squamous carcinoma
cell line NCI-H226 as well as human small cell lung cancer cell line NCI-H1688 and
from European Collection of Cell Cultures (ECACC, UK) e.g. 1 subtype of NSCLC;
human large cell lung cancer line COR-L23. One normal cell line was obtained from
CLS-cell line service (CLS; Eppelheim, Germany) e.g. human lung fibroblast cell line
MRC-5.
The NSCLC such as A549, NCI-H226 and COR-L23 were cultured in
RPMI-1640 supplemented with 10% fetal bovine serum (FBS). The SCLC in the form
of NCI-H1688 was cultured in modified RPMI-1640 medium (2 mM L-glutamine, 10
mM HEPES, 1 mM sodium pyruvate, 4,500 mg/L glucose, 2,000 mg/L sodium
bicarbonate and 10% FBS. MRC-5 cell line was cultured in DMEM : Ham’s F12
medium supplemented with 10% FBS.
3.5 Growth inhibitory and cytotoxic effects
Growth inhibitory and cytotoxic effects of HMP were measured by
sulforhodamine B (SRB) assay. Its principle is the measurement of the cellular protein
content of living cells, based on the ability of SRB to bind to basic amino acid residues
that are fixed to the culture plate bottoms under mild acidic condition. The cell numbers
are estimated indirectly by staining total cellular protein content of each well with SRB
dye and the protein-bound dye is extracted from cells under mildly basic condition. The
optical density (O.D.) is measured using a microplate reader (Voigt, 2005).
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In this experiment, the optimal cell number for seeding in a 96-well plate
was determined using SRB assay because it is critical to ensure exponential cell growth
for the entire duration of the assay. Briefly, The various numbers of cells were seeded
in a 2-fold serial dilutions of cells, ranging from 800-12,800 cells/well, in 100 µl
medium and incubated overnight at 37 ºC with 5% CO2 to allow cell attachment prior
to adding 100 µl of complete medium and further incubated for 0, 24, 48, 72 and 96 h.
At each of the indicated time points, the cells were fixed with 100 µl of 10%
trichloroacetic acid (TCA) at least 1 h to overnight at 4 ºC, washed with distilled water
and stained with 50 µl of 0.4% (w/v) sulforhodamine B (SRB) for 30 min in the dark.
The excess dye was removed by washing with 1% (v/v) acetic acid and then dried at
room temperature for 24 h in the dark. The protein-bound dye was extracted from cells
with 100 µl of 10 mM Tris, pH 10. The optical density was measured at 570 nm using
a microplate reader (Bio Tex, USA). The absorbance values obtained from microplate
reader were plotted versus incubation times, indicating the growth rate of each cell type.
According to the exponential cell growth for the entire assay period and O.D. 1.5-2.0
at the end of the 72-h assay time, the optimal cell numbers of each cancer cell line were
showed in Appendix A.
For measurement the antiproliferative and cytotoxic effects of HMP, 100
l of cells was seeded at the indicated numbers in the bracket (A549, NCI-H226 and
COR-L23 = 3.2 x 103 cells/well as well as NCI-H1688 and MRC-5 = 12.8 x 103
cells/well) in 96-well plates. On the following day, 100 l of HMP at final
concentrations of 3.125, 6.25, 12.5 and 25µM was added to the tested wells while 100
l of complete media was added to the control wells. These cells were further incubated
at 37°C with 5% CO2 for 72 h, fixed with 100 μl of 10% TCA, washed with distilled
water and stained with 50 µl of 0.4% SRB for 30 min in the dark. The excess dye was
removed by washing with 1% acetic acid and then dried at room temperature for 24 h
in the dark. The protein-bound dye was extracted from cells with 100 µl of 10 mM Tris,
pH 10. The optical density was measured at 492 nm using a microplate reader (Bio Tex,
USA). The percentages of cell survival for each concentration are calculated using the
following formula:
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% cell survival = [(T - T0)/(C - T0)] × 100, T ≥ T0
or = [(T - T0)/ T0] × 100, T < T0
T = Average O.D. of cells treated with HMP for 72 h
T0 = Average O.D. at 0 h
C = Average O.D of cells treated with only media for 72 h
Based on the formula, the percentage of cell survival can be greater than zero, zero or
less than zero. A dose-response curve is obtained by plotting the percent cell survival
versus HMP concentrations. The concentrations of HMP required for 50% growth
inhibition (IC50), total growth inhibition (TGI), and 50% loss of cells (lethal
concentration, LC50) relative to the untreated cells are obtained by interpolating from a
dose-response cubic spline curve using GraphPad Prism 4.0 Software (GraphPad
Software, Inc., USA).
In addition, the selectivity index (SI), indicating the safety of HMP for
anticancer therapy was calculated by obtaining the ratio of IC50 of non-cancerous cell
lines and cancerous cell lines (Prayong, Barusrux, & Weerapreeyakul, 2008).
3.6 Cell proliferation by CFSE assay
Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) is a cell-
tracking dye used to label cells for examining their proliferative activity. Briefly,
CFDA-SE is colorless and non-fluorescent that diffuses passively into cells. Within the
cells, intracellular esterase cleaves its acetate groups to yield fluorescent
carboxyfluorescein succinimidyl ester (CFSE). The succinimidyl ester group reacts
with intracellular amines, thus forming fluorescent conjugates that are well retained in
the cell (Wang, Duan, Liu, Fang, & Tan, 2005) (Figure 3.2). A profile of sequential
halving of CFSE fluorescence intensity with each generation can be monitored,
allowing the visualization of the number of rounds of cell division. Inhibition of cell
division by any substance can thus be traced through changes in the CFSE profile.
The proliferative activity of A549 cells treated with or without HMP was
determined by carboxyfluorescein-succinimidyl ester (CFSE) (BD Bioscience, USA).
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According to the manufacturer’s instructions, A549 cells were labeled with 10 mM
CFSE in the dark at 37ºC with 5% CO2 for 20 min, washed two times using phosphate
buffered saline (PBS) with 10% FBS, resuspended with RPMI medium to plate at a
density of 3.2 x 104 cells/well in 24-well plates. Following overnight incubation, these
cells were incubated with 50µM HMP for 24, 48 and 72 h whereas control cells were
incubated with media only. For parent cells, non-treated cells were immediately
analyzed. These stained cells were detected using FACSCalibur flow cytometer
(Becton Dickinson, USA) and analyzed for numbers of cell division, proliferation
index, and precursor frequency with ModFit LT 3.2 program (Verity Software House,
USA). Proliferation index is the sum of the cells in all generations divided by the
number of original parent cells. Precursor frequency is defined as the fraction of the
parent population that proliferated in response to HMP treatment.
Figure 3.2 Formation of fluorescent compound CFSE by intracellular esterase.
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3.7 Cell cycle analysis
The analysis of cell cycle distribution was performed by flow cytometry to
distinguish cells in different phases of the cell cycle. Before analysis, the cells were
permeabilised and treated with propidium iodide (PI), which is a fluorescent dye that
stains DNA quantitatively. In this analysis, the fluorescence intensity of stained cells at
a certain wavelength, therefore, correlates with the amount of DNA that the cells
contain. Four distinct phases can be recognized in a proliferating cell population. For
instance, cells in the G1 and S phases containing one copy of DNA, therefore, have 1x
fluorescence intensity whereas G2 and M phases with two copies of DNA have 2x
fluorescence intensity (Figure 3.3). In addition, apoptotic cells can be observed as a
hypodiploid or sub-G1 peak in DNA histogram.
In this study, A549 cells were plated at a density of 3.2 x 104 cells/well in
24-well plates and then incubated with 25 M and 50 µM HMP for 24, 48 and 72 h at
37°C with 5% CO2. Control cells were incubated with media only. After treatment,
these cells were collected by trypsinization, fixed gently (drop by drop) in 7 ml of 80%
ethanol, and then stored at -20ºC overnight. Then, cells were washed with PBS and
stained with 0.5 ml PI/RNase staining buffer (BD bioscience, USA) for 30 min at room
temperature in the dark. These stained cells were determined using a FACSCalibur flow
cytometer (Becton Dickinson, USA) and analyzed for cell cycle phases with ModFit
LT 3.2 Software (Verity Software House, USA). Moreover, the cell cycle distribution
was determined in HMP-treated cells after pretreatment for 6 h with 1.56 to 50 M of
the pan-caspase inhibitor Z-VAD-fmk.
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Figure 3.3 DNA content distribution during the various phases of the cell cycle
obtained by flow cytometric analysis.
3.8 Annexin-V/PI double staining assay
The discrimination between intact and apoptotic cells was monitored by
annexin V-FITC (apoptotic cell marker) and propidium iodide (PI) (death cell marker)
double staining using flow cytometer. In live cells, phospholipid phosphatidylserine
(PS) is found in the inner membrane leaflet and translocates to the external membrane
leaflet in early apoptotic cells (Vermes, Haanen, Steffens-Nakken, & Reutelingsperger,
1995), these early apoptotic cells can be identified by the Annexin V-FITC staining,
which binds specifically to this externalized PS. Moreover, double staining with
propidium iodide (PI) differentiates early apoptotic cells with the intact membrane
(annexin V+/PI-) from late apoptotic/necrotic cells with leaky membranes (annexin
V+/PI+). Flow cytometric analysis was performed to quantify these cell populations.
The represented scatter dot plots demonstrate viable cells located in lower left quadrant
(annexin V-/PI-), early apoptotic cells in the lower right (annexin V+/PI-), late
apoptotic/necrotic cells, in the upper right (annexin V+/PI+), as shown in Figure 3.4.
In the present study, A549 cells were seeded and treated with HMP as
described in cell cycle analysis. After treatment, cells were trypsinized, washed with
cold PBS and then monitored by double staining with Annexin V-FITC Apoptosis
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Detection Kit I (BD Biosciences, USA) and propidium iodide (PI) (Invitrogen, USA)
using flow cytometry. Briefly, the collected cells were resuspended in 100 µl of binding
buffer containing 5 µl of Annexin V-FITC and 5 µl of PI, incubated for 20 min at room
temperature in the dark and then determined using flow cytometer (Becton Dickinson,
USA) and analyzed with CellQuest Software.
Figure 3.4 Dot plot analysis by Annexin V-FITC/PI double staining.
3.9 Caspase-3 activity assay
The activity of caspase-3 was detected using the CaspACETM Assay
System, Colorimetric Kit (Promega, USA). The kit comprises a colorimetric substrate
N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline (Ac-DEVD-pNA) and a cell-permeable pan-
caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-
fmk) for measuring caspase-3 activity. Its principle is that caspase-3 specifically
cleaves at the C-terminal side of the aspartate residue of the amino acid sequence
DEVD (Asp-Glu-Val-Asp), resulting that the chromophore p-nitroaniline (pNA) is
released from the substrate. Free pNA produces a yellow color that is monitored by a
spectrophotometer at 405 nm. The amount of yellow color produced upon cleavage is
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proportional to the amount of caspase-3 activity present in the sample. However, the
finding that the inhibition of the increased caspase-3 activity by using pan-caspase
inhibitor Z-VAD-fmk, which irreversibly binds to and blocks the cleavage site of the
caspases could confirm such increased activity. In this assay, the comparison of the
absorbance of pNA from treatment sample, in the absence of inhibitor and in the
presence of inhibitor, with an un-induced control allows determination of the fold
inhibit and increase in caspase-3 activity, respectively.
A549 cells were incubated with or without 25 µM HMP for 24, 48 and 72
hrs at 37°C with 5% CO2. According to the manufacturer’s protocol, after treatment,
cells were collected, lysed in the cold lysis buffer by freeze-thaw procedure and
incubated on ice for 20 min. The cell lysates were centrifuged at 15,000 x g for 20 min
at 4ºC and the supernatant fraction was collected. The protein content in the supernatant
was then determined by Bradford’s method.
In a 96-well plate, cell extracts with an equal amount of 80 g of total
protein were added to each reaction containing caspase assay buffer and specific
colorimetric substrate (Ac-DEVD-pNA) for caspase-3, gently mixed, incubated at 37°C
for 4 h and measured at 405 nm by a microplate reader (Bio Tex, USA). Moreover,
caspase-3 activity was measured in cell extracts treated with 25 µM HMP after
pretreatment for 6 h with 50 μM Z-VAD-fmk.
3.10 Real-time Quantitative PCR Analysis
The Bcl-2 family proteins such as proapoptotic Bax and antiapoptotic Bcl-2
proteins have been reported to regulate mitochondrial outer membrane
permeabilization (MOMP), which triggers apoptotic pathways (Suen, Norris, & Youle,
2008). To determine relative mRNA expression levels of these proteins, real-time PCR
was performed using TagMan® Gene Expression Assay.
A549 cells were treated with or without 25 μM HMP for 24, 48 and 72 h at
37°C with 5% CO2. After treatment, RNA was extracted from these cells using total RNA
extraction kit (Real Biotech Corporation, Taiwan) according to the manufacturer’s
protocol. Briefly, cell pellets were lysed with 400 µl of RB buffer, mixed and incubated
at room temperature for 5 min. The sample mixture was placed in the Filter column and
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centrifuged at 13,000 rpm for 2 min. The RNA-containing supernatant, which was passed
through this column, was collected and further added 400 µl of 70% ethanol prior to
transferring into the RB column. Such column was centrifuged at 13,000 rpm for 2 min.
The filtrate was removed whereas the column was kept. Next, this column, which
contains RNA binding to its glass fiber matrix was washed with 400 µl of R-W1 buffer
and 600 µl of R-Wash buffer, respectively, by centrifugation at 13,000 rpm for 1 min.
Such column was further centrifuged at 13,000 rpm for 3 min to dry the column matrix.
After that, RNA was eluted from this column by adding 35-50 µl of RNase-free water,
centrifugation at 13,000 for 1 min. The purity and quantification of RNA were measured
using a NanoDrop™ 2000 Spectrophotometer (Thermo Scientific, USA).
Two hundred and fifty nanograms of total RNA were converted to single-
stranded cDNA using High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems, USA) according to the manufacturer’s protocol. Briefly, one reaction
requires 25 µl, as following: 2.5 µl of 10x RT buffer, 1 µl of 25x dNTP, 2.5 µl of 10x
Random primers, 1.25 µl of Reverse transcriptase, 5.25 µl of RNase-free water and 12.5
µl of RNA sample (20 ng/µl). The reaction mixture was performed on a GeneAmp PCR
system 2700 thermocycler (Applied Biosystems, USA) as follows: 25°C for 10 min,
37°C for 120 min, 85°C for 5 min and 4°C hold.
Thirty nanograms of cDNA were used for real-time PCR amplification to
determine the mRNA expression levels of target genes using an Applied Biosystems
(ABI) StepOne™ and StepOne Plus™ Real-Time PCR System (The Applied
Biosystems, USA). The quantitative PCR was performed in duplicate using EXPRESS
qPCR Supermix, Universal (Invitrogen, USA) and commercially available primer/probe
sets, which are pre-designed FAM™ dye- labeled TaqMan® MGB (minor groove
binder) probe and primer sets (inventoried Taqman® Gene Expression Assays)
(Thermo Scientific, USA) for human Bcl-2, Bax, and GAPDH. Briefly, The total
reaction volume was 20 µl, as following: 10 µl of 2x qPCR, 0.4 µl of Rox dye, 1 µl of
20x primer/probe set, 6 µl of RNase-free water and 3 µl of cDNA (10 ng/µl). The
thermal cycling parameters are one cycle of 50°C for 2 min, 95°C for 10 min and 40
amplification cycles of 95°C for 15 sec and 60°C for 1 min. The relative quantification
of gene expression was performed using the comparative threshold (CT) method and
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determined by relative quantitation (RQ) value according to the 2-∆∆CT method. The
following equation was used to calculate RQ value.
∆CT treated group = CT of the target gene – CT of the endogenous gene (GAPDH)
∆∆CT = ∆CT of the treated group- ∆CT of the control group
Relative quantitation (RQ) value = 2 -∆∆CT
To compare fold changes in expression levels between the control group
and treated groups, CT values of all targets in the treated group were normalized to those
of GADPH as a house keeping gene.
3.11 Western blot analysis
Western blotting is an important technique used to identify specific proteins
from a mixture of proteins. The technique uses three elements to accomplish this task:
1) separation by size, 2) transfer to a solid support and 3) marking target protein using
a proper primary and secondary antibody to visualize (Mahmood & Yang, 2012).
A549 cells were treated with or without 25 μM HMP at different time
periods at 37°C with 5% CO2. After treatment, A549 cells were harvested and washed
with PBS. The cell pellets were lysed in 50-100 µl of RIPA buffer (Pierce, USA)
containing protease inhibitors cocktail (EMD Millipore, USA). The protein contents
were measured using BCA Protein Assay Kit (Pierce, USA). Forty micrograms of the
protein samples mixed with Laemmli sample buffer (Bio-Rad, USA) and 3 µl of
prestained molecular weight marker (Kaleidoscope; Bio-Rad, USA) were separated by
SDS-PAGE (7.5% or 12%) at 100 V of constant voltage until the dye front reached the
bottom of the gel. Following electrophoresis, the proteins were transferred onto
polyvinylidene fluoride (PVDF) membrane at 300 mA of constant current/slab for 3 h
(12 % gel) or 9 h (7.5% gel). After protein transfer, the protein bands on PVDF
membrane were rapidly stained using Ponceau S Staining Solution and washed with
Tris-buffered saline (TBS) until clean or colorless. Next, nonspecific sites on the
membrane were blocked using Odyssey blocking buffer (LI-COR Biosciences) in TBS
(1:1) for 1 h at room temperature. After blocking, the membrane was incubated with
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specific primary antibody [anti-cdc25C (Santa Cruz Biotechnology,USA), anti-
cyclinB1 and anti-Bid (EMD Millipore, Germany), anti-caspase9 (Upstate
Biotechnologies, Charlottesville, VA), cdk1, Bax, Bcl-2, caspase3, β-actin (Cell
Signaling Technology, USA), poly(ADP-ribose) polymerase (PARP) (BD Biosciences,
USA)] at room temperature overnight, and washed with Tris-buffered saline with
Tween20 (TBST) for 15 min (3 times). Finally, the membrane was incubated with the
fluorescently-labeled secondary antibody (LI-COR Biosciences) for 1 h at room
temperature and then washed with TBST for 15 min (3 times) and washed with TBS
for 10 min. Detection of each protein was performed using Odyssey Infrared Imaging
System, Western Blot Analysis (LI-COR Biosciences, USA).
3.12 Tubulin polymerization assay
Tubulin polymerization assay is used to determine the disruption of
microtubule formation, either by inhibiting polymerization or by preventing
depolymerization of tubulin, resulting in the cell-cycle arrest and cell death. The effect
of HMP on tubulin polymerization was analyzed using in vitro Tubulin Polymerization
Assay Kit (≥ 99% pure bovine tubulin), Catalog No. 17-10194 (EMD Millipore, UK).
The method determines light that is scattered by microtubules to an extent that is
proportional to the concentration of microtubule polymer. The resulting polymerization
curve is a representative of the three phases of microtubule polymerization, namely
nucleation, elongation, and steady state phases.
According to the manufacturer’s protocol, polymerization reactions were
performed in 96-well plate half area, which is a UV transparent plate. The
polymerization reactions occur in 70 µl final volumes, which contain 60 µl of 60 µM
tubulin in 1x PB-GTP, 9 μL of 1x PB-GTP solution and 1 μL of the test substance,
including 1,750 µM HMP, 700 µM nocodazole, 700 µM paclitaxel and 1x PB-GTP
solution, which was used as a control. The polymerization of tubulin was monitored by
measuring the turbidity variation (light scattering) every 15 seconds at 350 nm during
60 minutes using a microplate reader (Bio Tex, USA).
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3.13 DNA fragmentation assay
DNA fragmentation assay was used to determine apoptotic cell death in
A549 cells because apoptotic endonucleases, Caspase-Activated DNase; CAD, in these
cells can cleave chromosomal DNA at internucleosomal linker sites, resulting in the
formation of ladder pattern at about 180-base pair intervals in agarose gel
electrophoresis (Figure 3.5).
A549 cells were treated with or without 25 μM HMP for 48 h at 37°C with
5% CO2. After treatment, the formation of DNA fragments was detected using
Apoptotic DNA Ladder Detection Kit (ab66090) (Abcam, USA). According to the
manufacturer’s protocol, the cells were lysed with TE lysis buffer and extracted using
5 μl of Enzyme A solution incubated at 37°C for 10 min as well as 5 μl of Enzyme B
solution incubated at 50°C for 30 min. Next, 5 μl of ammonium acetate solution was
added to the reaction mixture and the DNA was then precipitated using 50 μl of
isopropanol for 30 min at -20°C. The DNA pellets were centrifuged at 10,000 rpm for
10 min, washed with 1 ml of 70% ethanol, centrifuged at 10,000 rpm for 10 min,
removed trace ethanol and air dry for 30 min at room temperature. The resulting pellets
were dissolved in 30 μl of DNA suspension buffer. The extracted DNA was separated
by electrophoresis on 1.5% agarose gel at 130 V of constant voltage. The resulting DNA
fragments were stained with ethidium bromide for 30 min, washed with distilled water
for 15 min and visualized by UV transilluminator and recorded by gel document (Alpha
Innotech, USA).
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Figure 3.5 DNA fragmentation analysis. Internucleosomal DNA cleavage, a hallmark
of apoptosis, is demonstrated by a characteristic “laddering” pattern on gel
electrophoresis.
3.14 Nuclear staining with DAPI
For the assessment of apoptosis, the morphological changes of nuclei were
visualized following DNA staining by the fluorescent dye, 4’,6-diamidino-2-
phenylindole (DAPI) (Sigma, USA). This dye is a blue fluorescent nucleic acid stain
that preferentially stains double-stranded DNA (dsDNA).
In this study, A549 cells were seeded at a density of 3.2 x 104 cells/well
into 4-well cell culture slides (SPL Life Sciences, Korea). After being seeded on culture
slides overnight, cells were treated with or without 25 and 50 µM HMP for 24, 48 and
72h. The cells were washed with PBS and fixed with 80% ethanol for 30 min at room
temperature. After fixation, the cells were washed 3 times with PBS and then stained
with 1 µg/ml of DAPI in PBS for 45 min at room temperature in the dark. The cells
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were washed 3 times with PBS, and stained nuclei were visualized using a fluorescence
microscope within 45 min (Nikon Eclipse Ci, Japan).
3.15 Intracellular reactive oxygen species (ROS) measurement
Intracellular reactive oxygen species (ROS) level was determined using
2´,7´-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA is a non-fluorescent dye
that diffuses passively into cells. Inside cells, the cellular esterases cleave the acetate
esters to non-fluorescent 2´,7´-dichlorofluorescin (DCFH). In the presence of ROS,
DCFH is rapidly oxidized to a fluorescent molecule 2´,7´-dichlorofluorescein (DCF)
(Figure 3.6). The fluorescence intensity can be evaluated quantitatively using flow
cytometric analysis (Dikalov, Griendling, & Harrison, 2007).
In this experiment, A549 cells either untreated or treated with 25, 50 and
100 μM HMP for 3, 6, 12, 24 h were incubated with 20 µM DCFH-DA for 30 min at
37 C in 5% CO2, washed, and resuspended in phosphate-buffered saline (PBS). Before
being analyzed by FACSCalibur flow cytometer (Becton Dickinson, USA), the cells
will be shortly stained with PI for live/dead cell discrimination. The median
fluorescence intensity will be quantitated by CellQuest software (Becton-Dickinson,
USA) analysis of the recorded histograms. Moreover, the inhibition of ROS generation
was performed by pretreatment N-acetylcysteine (NAC), widely known as ROS
scavengers, for 1 h prior to 25 µM HMP treatment for 12 h.
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Figure 3.6 Formation of fluorescent compound DCF by ROS and RNS.
3.16 Statistical analysis
Data are presented as mean ± SD for the indicated number of independent
experiments. Statistical differences between the control and treated groups were
analyzed using Independent-Samples T Test. Statistical differences between treatment
groups were analyzed by one-way analysis of variance (ANOVA) followed by post hoc
analysis. P value < 0.05 was considered statistically significant (SPSS 20 for
Windows).
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CHAPTER 4
RESULTS AND DISCUSSION
4.1 Extraction of Dioscorea membranacea Pierre
The percent yield of the ethanolic extract of D. membranacea Pierre was
3.57 as shown in Table 4.1.
Table 4.1 The percent yield of the ethanolic extract of D. membranacea Pierre.
Dried plant material (g) Crude extract (g) % (w/w) Yield
204.80 7.31 3.57
4.2 Isolation and purification of HMP
The crude ethanolic extract of D. membranacea Pierre (7.31 g) was
subjected to silica gel column chromatography and eluted by gradient elution in order
of increasing polarity [hexane–chloroform (9:1) 1L, hexane–chloroform (6:4) 1L,
hexane–chloroform (2:8) 1L, chloroform 500 ml, chloroform–methanol (9:1) 500 ml,
chloroform–methanol (1:1) 500 ml and methanol 500 ml, respectively]. Four hundred
and forty-two fractions (10 ml each) were collected in a 15-ml tube, and the odd
numbered fractions were spotted on TLC aluminium sheets precoated with silica gel
60. Spots on the TLC sheets were then detected under UV light and visualized with
acidic anisaldehyde spray. The fractions containing similar spots were combined, and
14 combined fractions were obtained and then spotted on a TCL aluminium sheet along
with 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene, a previously isolated
compound (Figure 4.1).
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Figure 4.1 TLC analysis of the 14 combined fractions of D. membranacea Pierre
extract obtained from the first silica gel column chromatography. (Lane 1: fractions 1-
87, lane 2: fractions 88-117, lane 3: fractions 118-143, lane 4: fractions 144-167, lane
5: fractions 168-185, lane 6: fractions 186-207, lane 7: fractions 208-217, lane 8:
fractions 218-223, lane 9: fractions 224-245, lane 10: fractions 246-279, lane 11:
fractions 280-291, lane 12: fractions 292-317, lane 13: fractions 318-331, lane 14: fractions
332-422, and lane 15: 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydrophenanthrene)
As the combined fractions 5 to 7 contained 5, 6-dihydroxy-2, 4-dimethoxy-
9, 10-dihydrophenanthrene, these fractions were then pooled, evaporated to dryness and
weighed. Six hundred and forty milligrams was obtained from these pooled fractions,
dissolved in hexane–chloroform (1:9) and subjected to the second silica gel column
chromatography. The column was eluted by gradient elution in order of increasing
polarity [hexane–chloroform (1:9) 1.5 L, chloroform 500 ml, chloroform–methanol
(1:1) 500 ml and methanol 500 ml, respectively]. Two hundred and seventy-two
fractions (3 ml each) were collected in a 15-ml tube, and the odd numbered fractions
were spotted on TLC aluminum sheets. Spots on the TLC sheets were then detected
under UV light and visualized with acidic anisaldehyde spray along with 5,6-
dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene. In Figure 4.2, TLC analysis
showed three groups of the fractions containing 5,6-dihydroxy-2,4-dimethoxy-9,10-
dihydrophenanthrene based on the characteristic features of chromatographic bands.
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Figure 4.2 TLC analysis of the odd numbered fractions, ranging from 25-99, eluted
from the second silica gel column chromatography.
The fractions in each group were combined and spotted on a TCL
aluminium sheet (group 1: fractions 38-52, group 2: fractions 53-61 and group 3:
fractions 62-83) along with 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydrophenanthrene
(Figure 4.3).
Figure 4.3 TLC analysis of the 3 groups of the combined fractions eluted from the
second silica gel column chromatography. (lane 1: group 1, lane 2: group 2, lane 3: 5,
6-dihydroxy-2, 4-dimethoxy-9, 10-dihydrophenanthrene and lane 4: group 3)
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The group 3 (lane 4) showed two clearly separated spots, and the upper spot
has the same retention factor (Rf) as the spot of 5,6-dihydroxy-2,4-dimethoxy-9,10-
dihydrophenanthrene (lane 3). The combined fractions in this group were then
evaporated to dryness and weighed. Then, 40 milligrams was obtained and further
isolated on a TLC glass plate coated with silica gel 60 (Merck, Germany) using ethyl
acetate-hexane (1:1) as the mobile phase. The plate was visualized under UV light, and
two bands were detected and marked using a pencil. The upper band was scraped from
a TLC plate, eluted, evaporated to dryness and weighed (Figure 4.4). Twelve point six
milligrams of an isolated compound was obtained and called HMP-1. The percent yield
was 0.172 % (w/w) (Table 4.2).
Figure 4.4 The schematic flow chart for isolation of clearly separated bands using a
TLC glass plate.
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Table 4.2 The percent yield of HMP-1 isolated from D. membranacea Pierre extract.
Crude extract (g) HMP-1 (mg) % (w/w) Yield
7.31 12.6 0.172
As the combined fractions in group 2 had several bands, all of which were
not well separated, the group 2 was spotted on a TLC aluminium sheet in different
solvent systems. Among these systems, ethyl acetate–hexane (4:6) is appropriate
because more clearly separated bands were observed (Figure 4.5).
Figure 4.5 TLC isolation of the combined fractions in group 2. (lane 1: one drop of
group 2, lane 2: 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydrophenanthrene and lane 3:
two drops of group 2)
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Next, the combined fractions in group 2 were evaporated to dryness and
weighed. Eighty milligrams was obtained, further dissolved in ethyl acetate–hexane
(4:6) and subjected to the third silica gel column chromatography. The column was
eluted by gradient elution in order of increasing polarity [ethyl acetate–hexane (4:6)
400 ml, ethyl acetate–hexane (6:4) 100 ml, ethyl acetate–hexane (8:2) 100 ml, ethyl
acetate 100 ml and ethyl acetate–methanol (2:8) 100 ml, respectively]. Fifty-one
fractions (3 ml each) were collected in a 15-ml tube, and the odd numbered fractions
were spotted on TLC aluminum sheets. Spots on the TLC sheet were then detected
under UV light and visualized with acidic anisaldehyde spray along with 5,6-
dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene. In Figure 4.6, fractions 15-23
containing 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene were pooled,
evaporated to dryness and weighed. Forty-eight point seven milligrams was spotted on
TLC glass plates coated with silica gel 60 and partitioned in chloroform–hexane (9:1)
twice. The plate was visualized under UV light, and two bands were detected and
marked using a pencil. The lower band that are the desired compound was scraped from
a TLC plate, eluted, evaporated to dryness and weighed (data not shown). Thirty-seven
point five milligrams of an isolated compound was obtained and called HMP-2. The
percent yield was 0.513 % (w/w) (Table 4.3).
Figure 4.6 TLC analysis of the odd numbered fractions, ranging from 1-51, eluted from
the third silica gel column chromatography.
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Table 4.3 The percent yield of HMP-2 isolated from D. membranacea Pierre extract.
Crude extract (g) HMP isolation (mg) % (w/w) Yield
7.31 37.5 0.513
The purity of compounds can be tested by running in three different solvent
systems. A pure compound should appear as a single spot whereas an impure compound
has two or more spots in a single lane. In this study, the purity of HMP-1 and HMP-2
was checked by separating them on a TLC aluminium sheet in three different solvent
systems of varying polarity [ethyl acetate–hexane (4:6), chloroform–methanol (9.8:0.2)
and chloroform–hexane (8:2)]. The results revealed a single spot in the systems as
shown in Figure 4.7, indicating that HMP-1 and HMP-2 are pure compounds.
Figure 4.7 TLC analysis for checking the purity of HMP-2 in three different solvent
systems of varying polarity. (lane 1: HMP-2 and lane 2: 5, 6-dihydroxy-2, 4-dimethoxy-
9, 10-dihydrophenanthrene)
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1H NMR spectrum of HMP-1 and HMP-2 was further analyzed as shown
in Figures 4.8 and 4.9, respectively. These spectra were compared with the spectrum of
previously isolated 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (Figure
4.10), and showed the same pattern (Table 4.4). These data strongly support that HMP-
1 and HMP-2 were 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene.
In addition, the purity of HMP-1 and HMP-2 was assessed using the HPLC
technique. The chromatograms demonstrated that the retention time (RT) of a major
peak in HMP-1 and in HMP-2 was similar to that of the previously isolated compound,
indicating that the two isolated compounds were 5,6-dihydroxy-2,4-dimethoxy-9,10-
dihydrophenanthrene (Figure 4.11 and Appendix C). The purity of a compound is the
ratio of the area under the main peak to the total area under all peaks. The results showed
that the purity of HMP-1 and HMP-2 was greater than 94% (Table 4.5).
Figure 4.8 1H NMR spectrum of HMP-1 in deuterated chloroform (CDCl3).
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Figure 4.9 1H NMR spectrum of HMP-2 in deuterated chloroform (CDCl3).
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Figure 4.10 1H NMR spectrum of 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydro
phenanthrene in deuterated chloroform (CDCl3).
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Table 4.4 1H NMR spectral data (500 MHz) of HMP-1, HMP-2 and previously
isolated 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydrophenanthrene.
Position
1H (multi, J in Hz)
HMP-1
HMP-2
previously isolated 5, 6-
dihydroxy-2, 4-
dimethoxy-9, 10-
dihydrophenanthrene
(Itharat et al., 2014)
1
2
3
4
4a
4b
5
6
7
8
8a
9
10
10a
5-OH
6-OH
2-OMe
4-Ome
6.61 (d, 2.2)
6.56 (d, 2.4)
-
-
-
-
-
6.84 (d, 7.9)
6.77 (d, 7.9)
-
2.64 (m)
2.71 (m)
-
8.25 (s)
5.99 (s)
3.86 (s)
3.99 (s)
6.61 (d, 2.4)
6.56 (d, 2.4)
-
-
-
-
-
6.83 (d, 7.9)
6.76 (d, 8.2)
-
2.63 (m)
2.71(m)
-
8.25 (s)
5.99 (s)
3.85 (s)
3.98 (s)
6.64 (d, 2.5)
6.57 (d, 2.5)
-
-
-
-
-
6.84 (d, 8.0)
6.77 (d, 8.0)
-
2.64 (m)
2.70 (m)
-
8.26 (s)
6.02 (s)
3.87 (s)
3.98 (s)
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Figure 4.11 HPLC chromatograms of HMP-1, HMP-2 and previously isolated 5,6-
dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene.
Sig = 254 nm
Sig = 270 nm
HMP-1
HMP-1
Sig = 254 nm
Sig = 270 nm
HMP-2
HMP-2
Sig = 254 nm
Sig = 270 nm
5, 6-dihydroxy-2, 4-dimethoxy-
9, 10-dihydrophenanthrene
5, 6-dihydroxy-2, 4-dimethoxy-
9, 10-dihydrophenanthrene
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Table 4.5 The retention time, area under the curve and percentage area of HMP-1,
HMP-2 and previously isolated 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene
analyzed by HPLC at wavelengths 254 and 270 nm.
Compounds Wavelength
(nm)
Retention
time (RT)
(min)
Area under the
curve (AUC)
(mAU*s)
Percentage
area
(%)
HMP-1
254 23.462 13,689.4 95.36
270 23.462 30,924.9 97.77
HMP-2
254 22.722 15,039.3 94.07
270 22.722 32,605.3 96.55
previously
isolated 5, 6-
dihydroxy-2, 4-
dimethoxy-9,
10-dihydro
phenanthrene
254 23.492 6,810.1 98.08
270 23.492 16,256.3 99.21
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4.3 Antiproliferative and cytotoxic effects of HMP against a panel of human lung
cancer cell lines
The antiproliferative effect of HMP was examined in four human lung
cancer cell lines i.e. A549, COR-L23, NCI-H226 and NCI-H1688 as well as a normal
cell line i.e. MRC-5 using SRB assay. The results demonstrated that HMP induced a
dose-dependent inhibition of growth in these cell lines (data not shown). As illustrated
in Table 4.6, HMP showed marked growth inhibitory effects on four different lung
cancer cell lines with the mean IC50 values ranging from 9.22 to 15.99 M, as compared
to a normal cell line i.e. MRC-5 (>100 M). Moreover, HMP was the most effective
against A549 cell line and also showed the highest selectivity index (SI > 10.85) toward
this cell line relative to MRC-5. Thus, we further investigated antiproliferative and
cytotoxic activities of HMP in A549 cell line through 3 parameters including IC50, TGI
and LC50. The results demonstrated that HMP exhibited growth inhibition (represented
as IC50 and TGI values) and cell death induction (represented as LC50 values), as shown
in Figure 4.12. Paclitaxel was also used as a positive control as previously described
(Hansakul et al., 2014).
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Table 4.6 Antiproliferative effects of HMP on a panel of human cell lines.
Type of cell lines IC50 ± S.D. (μM) SI
A549 9.22 ± 1.26 * > 10.85
NCI-H226 10.38 ± 0.19 * > 9.63
COR-L23 15.99 ± 1.11 * > 6.25
NCI-H1688 15.78 ± 1.31 * > 6.34
MRC-5 > 100
The data are expressed as the mean ± SD (n 3). Each experiment was performed in
triplicate. (IC50 = 50% growth inhibition, SI = selective index)
* Statistical significance (P < 0.05) versus MRC-5 cells
Figure 4.12 Effects of HMP on antiproliferative and cytotoxic activities in A549 cells.
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4.4 Inhibitory effects of HMP on cell division
The antiproliferative activity of HMP in A549 cells was further studied
using CFSE assay that monitors the number of cell divisions over time. With each round
of cell division, the relative CFSE fluorescence intensity decreases by half. Fifty µM
HMP was chosen in this study as nearly 100% growth inhibition was observed as
described in the previous results. Using ModFit LT 3.2 program, flow cytometric
analysis of CFSE-labelled cells showed that HMP-treated cells for 24, 48 and 72 h were
mainly arrested in the second round as early as 24 h (Generation 2 depicted as an orange
color, Figure 4.13A). In contrast, untreated cells proceeded through many cycles of the
division with increased incubation times. Besides the number of cell divisions, the
software provides proliferation index and precursor frequency. As for the proliferation
index, which indicates the fold-expansion of the overall culture (Roederer, 2011), the
index values of HMP-treated cells for 24, 48 and 72 h slightly increased, ranging from
1.5 to 3. On the contrary, these of untreated cells (control) considerably elevated and
reached the maximum value of approximate 16 at 72 h (Figure 4.13B). For the precursor
frequency, which defines the fraction of the parent population that divided in response
to HMP (Roederer, 2011), these values of treated cells increased slightly over
incubation periods, thus indicating that the majority of parent cells did not divide.
Conversely, untreated parent cells divided vigorously, resulting in substantial increases
in the precursor frequency over time (Figure 4.13C). Altogether, these data indicated
that HMP was effective in inhibiting cell division.
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Figure 4.13 Antiproliferative effects of HMP on A549 cells. (A) Representative
profiles of the sequential halving of CFSE fluorescence intensity of the parent cells at
0 h incubation (blue peak), untreated and 50 µM HMP-treated cells at 24, 48 and 72 h.
(B-C) Bar charts representing the proliferation index and precursor frequency. The data
are expressed as the mean ± SD (n 3). * P < 0.05 versus control at equal incubation
times.
B C
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4.5 Effects of HMP on the cell cycle distribution
As HMP was effective in inhibiting cell division, we next determined the
effect of HMP on cell cycle distribution by performing cell cycle analysis. Flow
cytometry was used to analyze DNA contents in cell cycles of A549 cells, which were
treated with various concentrations of HMP e.g. 100 µM (nearly the LC50 value), 50
µM (nearly the TGI value) and 25 µM (the half TGI value) for 24, 48 and 72 h. As
illustrated in Figure 4.14 and Table 4.7, flow cytometric analysis of the DNA from
HMP-treated cells displayed a substantial increase in the percentage of cells in G2/M
phase at 24, 48 and 72 h as compared to the control at equal incubation periods.
However, the percentage of cells in G2/M phase was inversely proportional to
incubation time. As these G2/M phase cells decreased between 48-h and 72-h incubation
periods, the marked increase in the sub-G1 peak representing apoptotic cells was also
observed in a time-dependent manner. For each incubation period, the data also
revealed that cells treated with increasing concentrations of HMP caused the decreased
percentages of cells in G2/M phase, along with the dramatically increased percentages
in G1 phase. Such data indicated that exposure to the high concentrations of HMP
resulted more DNA degradation than that the low concentrations did, leading to the
reduction in fluorescence intensity of nuclei from G2/M to G1 phase in a dose-dependent
manner. All concentrations used in this study could markedly block the cell cycle at the
G2/M phase and subsequently induced apoptosis in A549 cells. Therefore, only 25 μM
HMP was selected for further studies on molecular aspects.
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Figure 4.14 Effects of HMP on cell cycle distribution in A549 cells. Cell cycle analysis
of untreated and treated cells with 25, 50 and 100 µM for 24, 48 and 72 h was performed
using ModFit LT 3.2 program. Percentages of cells in G0/G1, S, G2/M and sub-G1
phases are represented as the mean of three independent experiments.
* P < 0.05 versus control at equal incubation periods.
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Table 4.7 The percentages of HMP-treated cells in each phase of cell cycle.
Time Samples
% non-apoptosis cells % apoptosis
cells
G1 S G2/M Sub-G1
24 h
Control 66.66 ± 3.09 10.07 ± 0.55 22.44 ± 1.83 1.25 ± 0.38
25 µM HMP 9.11 ± 0.94* 2.12 ± 0.16* 79.44 ± 4.24* 7.01 ± 1.94*
50 µM HMP 13.55 ± 0.45* 2.26 ± 0.31* 75.74 ± 0.39* 8.56 ± 0.18*
100 µM HMP 36.53 ± 2.22* 5.66 ± 1.09* 48.94 ± 1.59* 9.07 ± 2.87*
48 h
Control 71.67 ± 3.81 10.40 ± 1.58 20.50 ± 2.54 0.89 ± 0.14
25 µM HMP 12.04 ± 1.52* 7.16 ± 1.91* 55.60 ± 2.40* 26.51 ± 2.33*
50 µM HMP 15.76 ± 1.28* 7.07 ± 0.78* 48.29 ± 4.71* 29.31 ± 2.96*
100 µM HMP 33.78 ± 0.90* 8.12 ± 0.16 35.19 ± 0.46* 23.36 ± 1.23*
72 h
Control 74.99 ± 2.07 7.96 ± 1.05 15.26 ± 1.69 1.52 ± 0.56
25 µM HMP 13.30 ± 1.41* 10.27 ± 2.87 43.65 ± 4.07* 30.50 ± 3.55*
50 µM HMP 15.61 ± 1.30* 9.03 ± 1.80 44.49 ± 2.81* 32.90 ± 6.92*
100 µM HMP 33.95 ± 2.67* 9.27 ± 0.44 33.13 ± 3.79* 24.13 ± 4.25*
The data are expressed as the mean ± SD (n 3).
* Statistical significance (P <0.05) versus control at equal incubation periods.
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4.6 Effect of HMP on protein expression of cell cycle regulatory proteins
As HMP arrested the cells in the G2/M phase, we next examined whether it
manifested cell cycle distribution and subsequent inhibition of cell growth through
G2/M regulatory proteins. Therefore, the effect of HMP on the expression of the G2/M
regulatory proteins cdc25C, cdk1 and cyclin B1 was monitored by Western blot
analysis. A549 cells were either untreated or treated with 25 µM HMP for 12, 24, 48
and 72 h. As shown in Figure 4.15, the results demonstrated the basal expression levels
of constitutive cdc25C, cdk1 and cyclin B1 in untreated cells. Also, HMP treatment
induced down-regulation of cdc25C and cdk1 protein levels in a time-dependent
fashion. Interestingly, elevated levels of cyclin B1 protein were detected in A549 cells
after 12 and 24 h of HMP treatment and markedly decreased at 48 and 72 h. β-actin
levels served as an internal control and were unaffected under these conditions. These
results revealed that HMP decreased the expression levels of cdc25C, cdk1 and cyclin
B1, all of which are involved in G2 to M phase progression, thereby leading to the G2/M
arrest.
Figure 4.15 Effects of HMP on protein levels of cdc25C, cdk1 and cyclin B1 in A549
cells. The cell cycle regulatory proteins of these cells either untreated or treated with
25 µM HMP for 12, 24, 48 and 72 h were analyzed by Western blotting. Data are
representatives of three independent experiments.
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4.7 Effect of HMP on interfering microtubule formation
In addition to the alteration of the G2/M regulatory proteins, we further
investigated whether HMP could inhibit microtubule assembly in vitro using tubulin
polymerization assay kit in a cell-free system. The effect of 25 μM HMP on tubulin
polymerization was monitored every 15 seconds at 350 nm using a microplate reader,
as shown in Figure 4.16. For comparison, parallel experiments were conducted with
paclitaxel (a microtubule stabilizer), nocodazole (a microtubule depolymerizer), and
untreated tubulin as a control. The results demonstrate that 25 μM HMP, which was
shown to markedly block cell cycle at G2/M phase in A549 cells, showed very similar
tubulin polymerization to the control, indicating that HMP did not disrupt in vitro
polymerization of tubulin into microtubules.
Figure 4.16 Effect of HMP on in vitro tubulin polymerization. Sixty µM tubulin
concentration was incubated with polymerization buffer in a 96-well half area plate at
37°C in the absence (control) and the presence of 25 µM HMP, 10 µM paclitaxel and
10 µM nocodazole. Data are representatives of four independent experiments.
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4.8 Effect of HMP on apoptosis induction in A549 cells
As cell cycle analysis revealed the “sub-G1 peak” that represents apoptotic
cells in HMP-treated cells, annexin V/PI staining and flow cytometry were performed
to quantify early apoptotic cells. In these cells, phosphatidylserine (PS) is translocated
from the inner to the outer membrane leaflet and specifically binds to annexinV.
Moreover, double staining with PI differentiates early apoptotic cells with intact
membranes from late apoptotic/necrotic cells with leaky membranes. The represented
scatter dot plots demonstrate viable cells located in lower left quadrant (annexin V–/PI–),
early apoptotic cells in the lower right (annexin V+/PI–), late apoptotic/necrotic cells,
in the upper right (annexin V+/PI+).
In the present study, A549 cells were treated with or without various
concentrations of HMP for 24, 48 and 72 h. The results indicated that HMP induced a
decrease in the percentage of viable cells with a concomitant increase in the percentage
of early and late apoptotic cells in a time- and dose-dependent manner (Figure 4.17 and
Table 4.8). Therefore, such data strongly suggest a critical role of HMP in stimulating
apoptosis in A549 cells.
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Figure 4.17 Effects of HMP on apoptotic induction in A549 cells. Apoptotic profiles
of untreated and treated cells with 25, 50 and 100 µM HMP for 24, 48 and 72 h were
performed using CellQuest Software. The percentages of cells in the respective
quadrants i.e. LL: Viable cells, LR: Early apoptotic cells, UR: Late apoptotic cells, UL:
Dead cells are indicated as the mean of three independent experiments. * P < 0.05
versus control at equal incubation periods.
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Table 4.8 The percentages of cells in the respective quadrants. (i.e. LL: viable cells,
LR: early apoptotic cells, UR: late apoptotic cells, UL: dead cells)
Time samples
LL LR UR UL
% live cells
% early
apoptotic
cells
% late
apoptotic
cells
% dead
cells
24 h
Control 92.74 ± 2.17 3.13 ± 0.71 3.73 ± 1.39 0.41 ± 0.32
25 µM HMP 83.01 ± 4.08* 14.09 ± 2.15* 3.66 ± 1.01 0.81 ± 0.46
50 µM HMP 77.17 ± 5.19* 13.25 ± 2.02* 6.21 ± 2.44 1.44 ± 0.88
100 µM HMP 81.08 ± 1.36* 14.25 ± 1.68* 6.23 ± 2.67 1.21 ± 0.98
48 h
Control 91.76 ± 1.68 3.64 ± 1.23 4.06 ± 1.13 0.55 ± 0.18
25 µM HMP 74.19 ± 9.10* 26.26 ± 8.00* 5.54 ± 1.73 1.02 ± 0.59
50 µM HMP 60.83 ± 4.93* 28.37 ± 7.81* 9.21 ± 0.65* 1.85 ± 0.82
100 µM HMP 56.48 ± 7.50* 24.81 ± 2.32* 27.12 ± 4.62* 2.35 ± 0.53*
72 h
Control 93.22 ± 0.86 2.21 ± 0.82 3.29 ± 0.97 1.27 ± 0.47
25 µM HMP 65.47 ± 9.59* 25.24 ± 4.89* 10.41 ± 0.91* 3.41 ± 1.44*
50 µM HMP 54.51 ± 5.93* 25.12 ± 5.76* 15.95 ± 2.51* 3.97 ± 1.06*
100 µM HMP 39.74 ± 8.16* 21.05 ± 4.95* 48.36 ± 6.36* 4.65 ± 2.00*
The data are expressed as the mean ± SD (n 3). * Statistical significance (P <0.05)
versus control at equal incubation periods.
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4.9 Effect of HMP on caspase-3 activity in A549 cells
Next, the involvement of caspase activation was examined because caspase
enzymes, a family of cysteine proteases, are central components of the machinery for
apoptosis (Earnshaw, Martins, & Kaufmann, 1999). A549 cells were pretreated with
the pan-caspase inhibitor, Z-VAD-fmk, at different concentrations (1.56, 6.25, 25 and
50 μM) for 6 h prior to 25 µM HMP treatment for an additional 72 h and the percentage
of cells in each phase of the cell cycle was shown in Figure 4.18A and Table 4.9. In this
study, Z-VAD-fmk-pretreated cells displayed a significant decrease in sub-G1
populations as compared with unpretreated cells. Moreover, the increased percentage
of apoptotic cells with the induction of HMP treatment was markedly inhibited by Z-
VAD-fmk in a concentration-dependent manner (Figure 4.18B). These results indicated
that HMP-induced apoptosis in A549 cells was dependent on the activation of caspase
enzymes.
As caspase-3 is the primary effector (executioner) caspase responsible for
much of the cellular degradation during apoptosis, we then evaluated changes of its
activity in 25 µM HMP-treated cells for 24, 48 and 72 h using the CaspACE™ Assay
System (Promega, USA). Following HMP treatment, the relative activity of caspase-3
was not significantly different from the control at 24 h, but was significantly increased
at 48 and 72 h. Its highest level was detected at 48 h (Figure 4.19A). In addition,
pretreatment with 50 µM Z-VAD-fmk 6 h prior to HMP treatment for 48 h completely
suppressed the highest relative activity of caspase-3 (Figure 4.19B), thus ascertaining
its involvement in apoptosis.
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Figure 4.18 Inhibitory effects of Z-VAD-fmk on sub-G1 populations. (A) Representative
profiles of flow cytometric analysis. (B) Bar graphs representing the percentage of sub-
G1 peaks with the percent inhibition presented below. The data are expressed as the
mean ± SD (n 3). * P < 0.05 versus HMP-treated cells.
A
B
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Table 4.9 The percentages of cells in each phase of cell cycle. The cells were pretreated
with Z-VAD-fmk at different concentrations for 6 h prior to 25 µM HMP treatment for
72 h.
Time Samples % non-apoptosis cells
%apoptosis
cells
G1 S G2/M Sub-G1
72 h
Control 67.98 ± 1.19 23.04 ± 1.63 8.98 ± 0.98 0.13 ± 0.17
25 µM HMP 3.25 ± 1.33 37.12 ± 4.48 59.63 ± 5.37 18.46 ± 3.75
25 µM HMP 3.91 ± 0.01 20.65 ± 1.62 75.45 ± 1.61 14.06 ± 3.21
1.56 µM Z-VAD fmk
25 µM HMP 3.53 ± 0.33 22.78 ± 0.74 73.70 ± 1.07 13.11 ± 2.98
6.25 µM Z-VAD fmk
25 µM HMP 3.83 ± 0.65 13.01 ± 2.38 83.16 ± 1.73 9.47 ± 2.62
25 µM Z-VAD fmk
25 µM HMP 3.71 ± 1.02 11.97 ± 3.43 84.33 ± 2.64 8.21 ± 1.20
50 µM Z-VAD fmk
The data are expressed as the mean ± SD (n 3).
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Figure 4.19 Effects of HMP on caspase-3 activity in A549 cells. (A) The relative
caspase-3 activity of 25 µM HMP-treated cells for 24, 48 and 72 h. * (P <0.05) versus
untreated cells at equal incubation periods using Independent-samples t-test. For the
treated cell groups, bars with different lowercase letters are significantly different at p
<0.05 using One-way ANOVA. (B) The inhibition of the relative caspase-3 activity of
cells pre-incubated with 50 µM Z-VAD-fmk for 6 h prior to HMP treatment for 48 h as
compared to cells treated with HMP alone. * P < 0.05 versus HMP alone.
A
B
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4.10 Bax and Bcl-2 mRNA and protein expression levels
The significant cell cycle arrest followed by apoptosis in HMP treatment
provide a powerful hint that the intrinsic mitochondrial pathway was likely activated.
The pathway is initiated by non-receptor-mediated intracellular signals, leading to an
increase in pro-apoptotic proteins e.g. Bax relative to antiapoptotic proteins e.g. Bcl-2
and subsequent mitochondrial outer membrane permeabilization (MOMP). This event
is regulated by the balance between the Bcl-2 family proteins that shift for pro-apoptotic
proteins (Bender and Martinou, 2013). Therefore, mRNA and protein levels of pro-
apoptotic Bax and antiapoptotic Bcl-2 were determined in A549 cells treated with or
without HMP.
The results showed that treatment with HMP significantly increased Bax
mRNA (Figure 4.20) and protein levels (Figure 4.21) relative to control in almost all
incubation periods. Although significant increases relative to control were also seen in
Bcl-2 expression, Bcl-2 mRNA levels were significantly lower than those of Bax at
48 h and 72 h (Figure 4.20), and Bcl-2 protein level was notably lower than that of Bax
at 72 h (Figure 4.21). Due to higher levels of Bax relative to Bcl-2, it appeared that
apoptotic activity of Bax may be more involved in HMP-stimulated intrinsic apoptosis
than the antiapoptotic activity of Bcl-2. Moreover, the results showed more than 2-fold
increases in Bax versus Bcl-2 mRNA levels, but not in their protein levels. This made
us speculate whether high levels of Bax protein may be more affected by rapid
degradation due to cell death. Altogether, these data indicated that HMP activated
apoptosis through the intrinsic mitochondrial pathway by increasing Bax expression
over Bcl-2.
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Figure 4.20 The quantification of relative mRNA levels of Bax and Bcl-2 in A549 cells
using Real-time PCR. The cells were treated with or without 25 µM HMP for 24, 48
and 72 h. Data are expressed as mean ± SD (n = 3). * P < 0.05 versus control at equal
incubation times. # P < 0.05 versus Bax at equal incubation times.
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Figure 4.21 Effects of HMP on protein expression of Bax and Bcl-2 in A549 cells. The
cells were treated with or without 25 µM HMP for 24, 48 and 72 h. (A) Protein levels
of Bax and Bcl-2 using Western blotting. (B) Bar graphs representing the relative band
intensities of Bax and Bcl-2. Data are representatives of three independent experiments.
β-actin was used as a loading control. * P < 0.05 versus control at equal incubation
times. # P < 0.05 versus Bax at equal incubation times.
A
B
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4.11 Effect of HMP on expression of active caspases and their targets
As the previous experiment showed the highest caspase-3 activity at 48 h,
we further investigated the expression of the active form of caspase-3 and its well-
established substrate, PARP, using western blotting analysis at the same time. The
results showed that HMP induced the cleavage of procaspase-3 (35 kDa) into the active
form (19 and 17 kDa), as shown in Figure 4.22A. Similarly, the reduction of pro PARP
(113 kDa) was accompanied by the increase of cleaved PARP (89 kDa), as shown in
Figure 4.22B, indicating the action of cleaved caspase-3. These results confirmed that
HMP indeed induced apoptosis via the caspase-dependent apoptotic pathway.
Since HMP activated apoptosis through the intrinsic mitochondrial
pathway, leading to mitochondrial outer membrane permeabilization (MOMP), we
further studied the expression of cleaved caspase-9 as the crucial initiator caspase of
this pathway. Our results showed that HMP treatment for 48 h reduced expression of
procaspase-9, and its cleaved form was generated (89 kDa), as compared to the control
(Figure 4.22C). These data further confirmed that HMP induced apoptosis through
activation of the intrinsic pathway. However, caspase-8, the predominant initiator
caspase in the extrinsic pathway, also induces MOMP through the cleavage of cytosolic
Bid to truncated Bid (tBid). The tBid then translocates to mitochondria, leading to
MOMP, thus connecting the extrinsic pathway to the intrinsic mitochondrial pathway.
For these reasons, we then investigated whether HMP induced apoptosis via the
extrinsic pathway by testing the levels of Bid protein. The results showed that Bid in
HMP-treated cells was significantly lower than that in the control, indicating that HMP
triggered apoptosis via the extrinsic pathway.
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Figure 4.22 Effects of HMP on expressions of apoptotic proteins in A549 cells. Cells
were treated with 25 µM HMP for 48 h. Control cells were treated with media only.
The cellular proteins of these cells were analyzed by Western blot. Levels of caspase-3
and cleaved caspase-3 (A), PARP and cleaved PARP (B) as well as caspase-9 and Bid
(C) are shown. β-actin was used as a loading control. The immunoblots shown are
representatives of three independent experiments.
4.12 Effect of HMP on nuclear morphological changes
To determine the nuclear morphological changes during apoptosis, DAPI
staining was performed. As shown in Figure 4.23 and 4.24, HMP-treated cells exhibited
chromatin condensation and nuclear fragmentation as compared to control. Such
characteristic features can be seen in apoptotic cells. It is noteworthy that the chromatin
condensation and nuclear fragmentation decreased with prolonged incubation periods
because of the possibility of losing floating cells (dead cell) in wash steps.
A B C
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Figure 4.23 Effects of HMP on nuclear morphological changes by DAPI staining under
Bright-field microscopy (400x magnification); chromatin condensation (red arrows),
chromatin fragmentation (green arrows). Data are representatives of three independent
experiments.
Control 25 µM HMP
24 h
48 h
72 h
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Figure 4.24 Effects of HMP on nuclear morphological changes by DAPI staining under
Fluorescent microscopy (400x magnification); chromatin condensation (red arrows),
chromatin fragmentation (green arrows). Data are representatives of three independent
experiments.
Control 25 µM HMP
24 h
48 h
72 h
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4.13 Effect of HMP on DNA fragmentation
To confirm HMP-induced apoptosis, we also investigated DNA
fragmentation, which is one of the biochemical hallmarks of apoptosis. The DNA
fragmentation is associated with apoptotic endonucleases, which cleave chromosomal
DNA at internucleosomal linker sites, thereby resulting in the formation of ladder
pattern at about 180-base pair intervals in agarose gel electrophoresis. In the present
study, genomic DNA of A549 cells treated either with or without 25 µM HMP for 48
h was extracted using Apoptotic DNA Ladder Detection Kit and analyzed by 1.5%
agarose gel electrophoresis. As illustrated in Figure 4.25, the DNA fragmentation was
clearly observed in only HMP-treated cells, indicating that HMP indeed induced
apoptosis in A549 cells.
Figure 4.25 Effect of HMP on DNA fragmentation of A549 cells. DNA fragments of
cells treated with 25 µM HMP for 48 h were analyzed by 1.5% agarose gel
electrophoresis. Data are representatives of three independent experiments.
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4.14 Effect of HMP on the generation of intracellular ROS
Many studies have shown that natural plant compounds can produce high
levels of ROS in cancer cells, leading to apoptosis (Gong & Li, 2011; Jo et al., 2012;
Singh et al., 2012). Therefore, we further investigated whether HMP-induced apoptosis
was associated with the increase of ROS generation in A549 cells. The cells treated
either with or without 25 µM HMP at different incubation times were performed using
DCF assay and analyzed by flow cytometry. The results revealed that HMP could
generate ROS in treated cells. As illustrated in Figure 4.26, the ROS accumulation
reached the highest level at 12 h and then slowly decreased after 24 h. To confirm ROS
generation, cells were pretreated with the ROS scavenger NAC at 0.1, 1 and 5 mM for
1 h prior to the addition of 25 µM HMP and further incubation for 12 h. The results
showed that NAC completely inhibited the highest level of ROS in a dose-dependent
manner (Figure 4.27). The results showed that NAC completely inhibited the highest
level of ROS in a dose-dependent manner. This results indicated that HMP induced the
increased ROS generation.
We next investigated whether ROS generation induced by HMP was
directly associated with apoptotic cell death. The changes in sub-G1 populations were
thus determined in the cells, which were pretreated with NAC at different
concentrations (0.1, 1 and 5 mM) for 1 h prior to 25 µM HMP treatment for 24 h. As
shown in Figure 4.28A, 4.28B and Table 4.10, there were no statistically significant
changes in sub-G1 populations of cells that were pretreated with or without NAC. These
data indicate that the increased ROS levels by HMP did not influence cell apoptosis.
We also demonstrated that NAC alone did not induce apoptotic cell death. Flow
cytometric analysis of the DNA from cells treated with NAC alone at different
concentrations for 72 h displayed very similar cell cycle distribution to the control
(Appendix D).
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Figure 4.26 Effect of HMP on ROS production in A549 cells treated with 25 µM HMP
at different incubation times. Data are representatives of three independent
experiments.
Control
25 µM HMP 3 h 6 h 12 h
24 h 48 h 72 h
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Figure 4.27 Effect of the ROS scavenger NAC on ROS production in A549 cells.
(Upper row) Cells were pretreated with NAC at 0.1, 1 and 5 mM for 1 h before the
addition of 25 µM HMP for 12 h relative to 25 µM HMP-treated cells. (Lower row)
Cells were treated with only NAC at different concentrations for 13 h. Data are
representatives of three independent experiments.
Control Control Control Control
25 µM HMP 25 µM HMP 25 µM HMP 25 µM HMP
0.1 mM NAC 1 mM NAC 5 mM NAC + + +
Control Control Control 0.1 mM NAC 1 mM NAC 5 mM NAC
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Figure 4.28 Inhibitory effects of NAC on sub-G1 populations. A549 cells were
pretreated with NAC at different concentration (0.1, 1 and 5 mM) for 1 h prior to 25
µM HMP treatment for 24 h. (A) Representative profiles of flow cytometric analysis.
(B) Bar graphs representing the percentage of sub-G1 peaks with the percent inhibition
presented below. Data are expressed as the mean ± SD (n ≥ 3).
A
B
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Table 4.10 The percentages of cells in each phase of cell cycle. The cells were
pretreated with NAC at different concentrations for 6 h prior to 25 µM HMP
treatment for 24 h.
Time Samples % non-apoptosis cells
%apoptosis
cells
G1 S G2/M Sub-G1
24 h
Control 58.80 ± 1.06 30.23 ± 0.85 10.97 ± 1.57 0.51 ± 0.44
25 µM HMP 10.04 ± 3.98 7.63 ± 2.85 82.33 ± 1.13 6.01 ± 2.33
25 µM HMP 10.74 ± 4.06 8.47 ± 2.23 80.79 ± 4.37 6.07 ± 1.93
0.1 mM NAC
25 µM HMP 9.87 ± 3.60 8.38 ± 2.75 81.75 ± 1.98 6.89 ± 1.08
1 mM NAC
25 µM HMP 10.49 ± 3.59 6.77 ± 3.77 82.74 ± 1.52 7.39 ± 1.33
5 mM NAC
The data are expressed as the mean ± SD (n 3).
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CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
The search for plant-derived compounds has been considered to be an
interesting subject in generating new anticancer agents with high safety and high
efficacy. Exploring the precise molecular mechanisms involved in their actions has
become an important approach for preclinical evaluation of anticancer agents and
subsequent development of anticancer drugs. For this reason, the screening of
anticancer agents through induction of cell cycle arrest and apoptosis appears to be a
powerful strategy for discovery of potent anticancer agents (Li et al., 2016). An active
compound called 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP)
derived from the rhizome ethanolic extract of D. membranacea Pierre had the highest
selectivity index against human lung large cell carcinoma COR-L23 cells using the
SRB assay (Itharat et al., 2014). In this study, effects of HMP on growth inhibition were
further tested in a panel of human lung cancer cell lines representing NSCLC and SCLC
compared to the well-defined human MRC-5 fibroblast line. The effects of HMP on
growth inhibition and cell death through induction of cell cycle arrest and apoptosis
was comprehensively investigated in the most responsive cell line, A549 human lung
carcinoma cell line.
5.1 Antiproliferative effect of HMP in A549 cells
Antiproliferative effect of the active compounds is investigated by IC50
values. The lower the IC50 values, the more potent a compound is. According to
National Cancer Institute (NCI) plant screening program, pure compounds with IC50
values of 4 μg/ml or less are considered to confer significant in vitro cytotoxic activity
(Phang, Malek, & Ibrahim, 2013). Also, SI values are used to define the specificity of
the compound for cancer cells. The higher the SI values, the more selective in killing
cancer cells a compound is, as opposed to normal cells. Furthermore, SI values greater
than 3.0 are considered to be significant (Mahavorasirikul, Viyanant, Chaijaroenkul,
Itharat, & Na-Bangchang, 2010). In this study, HMP exerted strong and selective
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antiproliferative activity against the A549 cell line among other lung cancer cell types
because it had an IC50 value of 9.22 µM (or 2.51 µg/ml) for this cell line. This value
was below 4 μg/ml, the cut-off value in which any compound with this value or less is
considered to be potent. HMP also had the highest SI value of > 10.85 for the A549 cell
line versus the MRC-5 cell line. These data indicated the potential use of HMP as a
promising anticancer agent. Thus, we chose the A549 cell line to investigate the
molecular mechanism underlying antiproliferative and cytotoxic activities of HMP.
5.2 Molecular mechanism underlying antiproliferative effect of HMP
This study showed that HMP possessed strong antiproliferative activity
against A549 cells by inhibiting cell cycle arrest at G2/M phase. In this G2/M phase
transition, specific regulatory proteins, such as cdc25C, cdk1, and cyclin B1 play an
important role as follows; the cdc25C removes the inhibitory phosphates present on
cdk1, thus rendering cdk1-cyclin B1 complex active and ultimately accelerating the
transition from G2 into mitosis (M) phase (Sanchez, McElroy,& Spector, 2003;
Potapova, Daum, Byrd, & Gorbsky, 2009). Our results demonstrated that HMP
treatment downregulated the expression levels of these proteins. Such decreased levels
of these proteins could affect their activity. Indeed, decreased activity of cdc25C due to
its decreased protein levels leads to unremoved inhibitory phosphorylation of cdk1,
causing accumulation of an inactive cdk1-cyclin B1 complex (Singh et al., 2004). The
decreased proteins are possibly caused by either their enhanced degradation through
ubiquitin/proteasome pathway or their suppressed mRNA synthesis (Shabbeer et al.,
2013). The first postulate could be proved by the restoration of these decreased
regulatory proteins after pretreatment with proteasome inhibitors. The latter postulate
could be determined by detection of decreased mRNA levels of the corresponding
proteins. However, the reduction of cdc25C, cdk1, and cyclin B1 protein levels does
not exclusively cause G2/M arrest. For example, Singh et al. (2004) have shown that
sulforaphane (SFN)-induced decline in cdc25 protein levels was nearly fully blocked
in the presence of proteasome inhibitor lactacystin. Such cell cycle arrest, however,
turned out not to be significantly affected upon such restoration of cdc25C protein
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levels using this inhibitor. Cytoplasmic translocation of cdc25C appeared to be the main
mechanism of SFN-induced cell cycle arrest.
Besides the decreased levels of these regulatory proteins, there are several
other mechanisms that cause G2/M phase arrest such as the increased levels of tumor
suppressor protein p53 and cdk inhibitory (CKI) p21 (Charrier-Savournin et al., 2004),
the inhibition of mitogen‐activated protein kinases (MAPK)/extracellular signal-
regulated kinases (ERK) signaling pathway (Yin et al., 2014), as well as the disruption
of microtubule assembly as well as the disruption of microtubule assembly (Chang, Yu,
Wu, Wang, & Liu, 2011). An example of a plant-derived compound that induced
excessive G2/M phase arrest is diallyl disulfide, a natural organosulfur compound
isolated from garlic. It caused a decline in protein expression levels of cyclin B1, cdc2,
p-cdc2, cdc25C and an increase in mRNA levels of p53 and p21. Also, this compound
inhibited cell proliferation in human esophageal squamous carcinoma ECA-109 cells
through the MEK-ERK signaling pathway (Yin et al., 2014). Therefore, it is possible
that the molecular mechanisms underlying the antiproliferative effect of HMP were
likely similar to those of diallyl disulfide in that more than one mechanism is involved.
Further studies are required to elucidate. In this study, the effect of HMP on the
disruption of microtubule assembly that caused G2/M arrest was also investigated. The
results turned out that HMP at 25 μM had no effect on microtubule assembly in a cell-
free system. However, its effect may be different in cell-based systems if HMP
indirectly disrupts the polymerization of microtubules in cells by affecting some
microtubule-regulatory proteins (Duangmano, Sae-Lim, Suksamrarn, Domann, &
Patmasiriwat, 2012). Additional research is necessary to fully define the mechanisms.
Given markedly increased G2/M arrest, there may be multiple molecular mechanisms
involved in the control of G2/M-phase progression.
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5.3 Cytotoxic effects of HMP in A549 cells
Besides the antiproliferative effect, HMP also exerted the cytotoxic effect
against A549 cells by presenting the cell survival curves with parameter LC50, which
displays a significant net loss and the increased sub-G1 peak after HMP treatment. In
addition, such effect was also related to the induction of apoptotic cell death by the
presence of early apoptotic cells (annexinV+/PI–). Briefly, one of the biochemical
hallmarks of apoptosis is the translocation of PS to the outer plasma membrane where
annexin-V-FITC binds specifically to PS. Moreover, double staining with PI
differentiates early apoptotic cells with intact membrane from late apoptotic/necrotic
cells with leaky membranes and healthy cells (Vermes et al., 1995). Furthermore, HMP
treatment also presented the characteristic features of apoptosis, such as chromatin
condensation and nuclear fragmentation by DAPI staining as well as the presence of
DNA ladder by agarose gel electrophoresis.
5.4 Apoptosis underlying cytotoxic effects of HMP
In apoptosis, caspases are synthesized as inactive procaspases that need to
be proteolytically processed to generate the active enzymes in response to apoptotic
signals (Steller, 1998). There are two main apoptotic pathways including intrinsic and
extrinsic pathways. Both pathways finally converge on caspase-3, which can cleave
many key cellular proteins such as the inhibitor of caspase-activated DNase (ICAD),
PARP and other structural proteins, causing nuclear shrinking, budding to form
apoptotic bodies, cytoskeletal proteolysis, etc. (Porter & Janicke, 1999; Elmore, 2007).
In this study, our results revealed that the use of Z-VAD-fmk was efficient in blocking
apoptotic cell death in HMP-treated A549 cells, indicating that HMP induced apoptosis
through activation of caspases. In addition, we first demonstrated the effect of HMP on
caspase-3 activation through detection of both caspase-3 activity and its active
(cleaved) form. Following HMP treatment, the cleavage of PARP and the presence of
nuclear condensation, DNA fragmentation, and DNA apoptotic ladder were also
detected. These are considered as indicative of functional caspase-3 activation.
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It is well established that p53 is a sensor of cellular stress and is a critical
activator of the intrinsic apoptosis pathway (Haupt, Berger, Goldberg, & Haupt, 2003).
Once activated in the cytosol, p53 translocates to the nucleus where it activates
transcription of many proapoptotic proteins of the Bcl-2 family while simultaneously
repressing that of antiapoptotic proteins (Chumakov, 2007). As a result, proapoptotic
proteins subsequently oligomerize and form the pores, leading to the disrupted
mitochondrial outer membrane, cytochrome c release into the cytoplasm, the
apoptosome formation, and subsequent activation of caspase-9 (Zou et al.,
2003). The present study clearly showed that HMP could achieve the intrinsic apoptosis
pathway through activation of procaspase-9. The finding that levels of proapoptotic Bax
protein, one of the downstream targets of p53, were increased through its upregulated
mRNA levels, additionally supports the involvement of p53 in the intrinsic pathway.
The presence of wild-type p53 in A549 cells could increase such possibility.
Unexpectedly, however, increased expression levels of Bcl-2 were detected in HMP
treatment. This implies that Bcl-2 was likely not a key protein in HMP-mediated
apoptosis. It is possible that other antiapoptotic proteins i.e. Bcl-xL may be more
involved in this action (Zhang & Rosdahl, 2006). Therefore, further studies are needed.
The extrinsic apoptotic pathway is triggered when specific death ligands
engage their receptors on the plasma membrane, leading to the activation of initiator
caspase-8 (Elmore, 2007). Subsequently, active caspase-8 can directly cleave and
activate caspase-3, or it can alternatively cleave its downstream target Bid. This
truncated Bid can then activate proapoptotic Bax and Bak proteins directly as well as
suppress the anti-apoptotic proteins at the mitochondria, causing MOMP and
propagating the intrinsic mitochondrial pathway (Kantari & Walczak, 2011). However,
it is noteworthy to mention that the extrinsic death signals cannot directly elevate the
mRNA levels of Bax and Bak. In this study, the cleavage of Bid was detected and
therefore, provided additional insight into the extrinsic apoptosis pathway mediated by
HMP.
In summary, for the first time, this study demonstrated that HMP exerted
anticancer activity through the induction of G2/M cell cycle arrest and apoptosis in
A549 cells. Similarly, several well-known plant-derived compounds such as curcumin
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(Cheng et al., 2016) and baicalein (Mu et al., 2016) have been revealed to mediate cell
cycle arrest and apoptosis in recent years.
5.5 Effects of HMP on the generation of intracellular ROS and the relationship
between enhanced ROS and apoptosis
In addition, recent studies have shown that many plant-derived compounds
can produce excessive ROS leading to the induction of apoptosis in certain cancer cell
lines e.g., capsaicin (Ito et al., 2014), curcumin (Chang, Xing, & Yu, 2014), Plumbagin
(Tian et al., 2012), and so forth. The relationship between enhanced ROS and apoptosis
is that ROS can stimulate proapoptotic signaling molecules such as apoptosis signal-
regulating kinase 1 (ASK1), c-Jun-NH2-kinase (JNK), and p38 (Benhar, Dalyot,
Engelberg, & Levitzki, 2001; Tobiume et al., 2001), which then activate the p53 protein
pathway or engage the mitochondrial apoptotic cascade (Alexandre, Batteux, & Nicco,
2012), leading to apoptotic cell death. Our results showed that HMP induced ROS
production in a dose-dependent manner, and increased intracellular ROS levels were
completely abolished by the ROS scavenger, NAC, indicating that HMP indeed
induced elevation of ROS levels in A549 cells. However, the pretreatment with NAC
failed to inhibit the induction of apoptosis, indicating that this increased ROS was not
likely involved in HMP-mediated apoptosis.
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REFERENCES
Books and Book Articles
Alberts, B., Johnson, A., Lewis, J., Morgan, D., Raff, M., Roberts, K., & Walter, P.
(2014). Molecular Biology of the Cell, Sixth Edition. Taylor & Francis Group.
Alexandre, J., Batteux, F., & Nicco, C. (2012). Oxidative Stress. In Schwab, M. (Ed.),
Encyclopedia of Cancer (pp. 2730-2733). Heidelberg: Springer.
Chandar, N., & Viselli, S. (2010). In Harvey, R.A. (series editor), Cell and Molecular
Biology. Philadelphia: Wolters Kluwer/ Lippincott Williams & Wilkins.
Chun, R., Garrett, L. D., & Vail, D. M. (2007). Chapter 11 - Cancer Chemotherapy. In
Withrow, S.J., & Vail, D.M. (Eds.), Withrow & MacEwen's Small Animal
Clinical Oncology (Fourth Edition) (pp. 163-192). Saint Louis: W.B. Saunders.
Gould, K. L., Forsburg, S.L. (2015). Cell cycle regulation. In Plopper G., Sharp, D., &
Sikorski, E. (Eds.), Lewin’s CELLS, Third Edition (pp. 685-724).
Massachusetts: Jones & Bartlett Learning.
Green, D.R. (2015). Apoptosis. In Plopper G., Sharp, D., & Sikorski, E. (Eds.), Lewin’s
CELLS, Third Edition (pp. 725-749). Massachusetts: Jones & Bartlett Learning.
Hunt, J. L., & Dacic, S. (2008). Applications of Molecular Tests in Anatomic
Pathology. In Zander, D.S., Popper, H. H., Jagirdar, J., Haque, A. K., Cagle P.
T., & Barrios R. (Eds.), Molecular Pathology of Lung Diseases (pp. 78-82).
New York: Springer.
Islam, M. M. T., & Shekhar, H. U. (2015). Impact of Oxidative Stress on Human
Health. In Rani, V., & Yadav, U. C. S. (Eds.), Free Radicals in Human Health
and Disease (pp. 59-73). New Delhi: Springer.
Kleinsmith, L. J. (2006). Principles of Cancer Biology. San Francisco: Pearson
Benjamin Cummings.
Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E. S., Baehrecke, E.
H., . . . Melino, G. (2009). Classification of cell death: recommendations of the
Nomenclature Committee on Cell Death 2009. Cell Death Differ, 16(1), 3-11.
DOI: 10.1038/cdd.2008.150
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
124
Lieberman, M. A., Marks, A., & Peet, A. (2013). Marks' Basic Medical Biochemistry :
a clinical approach, Fourth Edition. London: Wolters Kluwer/ Lippincott
Williams & Wilkins.
Lim, S., & Kaldis, P. (2013). Cdks, cyclins and CKIs: roles beyond cell cycle
regulation. Development, 140(15), 3079-3093. DOI: 10.1242/dev.091744
Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D., & Darnell, J.
(2000). Molecular Cell Biology, Fourth Edition. New York: W. H. Freeman
Macdonald, F., Ford, C., & Casson, A. (2004). Molecular Biology of Cancer. London:
Taylor & Francis.
Marín-García, J. (2011). Cell-Cycle Signaling, Epigenetics, and Nuclear Function.
Signaling in the Heart (pp. 21-30). New York: Springer.
Müller-Hermelink, H. K., Engel, P., Kuo, T., Ströbel, P., Marx, A., Harris, N., . . .
Harris, C. (2004). Pathology & genetics, tumours of the lung, pleura, thymus
and heart. In Travis, W.D., Brambilla, E., Müller-Hermelink, H.K., & Harris,
C.C. (Eds.), World Health Organization classification of tumors (pp. 146-147).
Lyon: IARC Press.
Nguyen, L. Q., & Jameson, J. L. (1998). The Cell Cycle. In Jameson, J.L. (Ed.),
Principles of Molecular Medicine (pp. 65-72). New Jersey: Humana Press.
Payne, S., & Miles, D. (2008). Mechanisms of anticancer drugs. In Gleeson, M. (Ed.),
Scott-Brown's Otorhinolaryngology: Head and Neck Surgery, Seventh Edition
(pp. 34-46). London: Hodder Arnold.
Pierron, G. (2015). Basis for Molecular Genetics in Cancer. In Tourneau, C.L., &
Kamal, M. (Eds.), Pan-cancer Integrative Molecular Portrait Towards a New
Paradigm in Precision Medicine (pp. 15-30). Cham: Springer International
Publishing.
Saikumar, P., & Venkatachalam, M. A. (2009). Apoptosis and Cell Death. In Allen T.
C., & Cagle P. T. (Eds.), Basic Concepts of Molecular Pathology (pp. 29-40).
Massachusetts: Springer.
Skaar, J. R., & DeCaprio, J. A. (2006). Fundamental Aspects of the Cell Cycle and
Signal Transduction. In Chang A.E., Ganz P.A., Hayes D.F., Kinsella T.J., Pass
H.I., Schiller J.H., Stone R.M., & Strecher V.J. (Eds.), Oncology: An Evidence-
Based Approach (pp. 207-213). New York: Springer.
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
125
Strayer, D.S., & Rubin, E. (2015). Cell Adaptation, Injury and Death. In Strayer, D. S.,
Rubin, E., & Saffitz, J. E. (Eds.). Rubin's Pathology: Clinicopathologic
Foundations of Medicine, Seventh Edition. London: Wolters Kluwer/ Lippincott
Williams & Wilkins.
Subchareon P. (1998). Handbook of Anticancer: Thai Traditional Medicine. In New
Concept for Treated Cancer. (p.3). Bangkok: Thai Traditional Medicine
Institute.
Weber, G.F. (2007). Cell Division and Survival. In Molecular Mechanisms of Cancer
(pp. 45-191). Dordrecht: Springer.
Articles
Ahn, H. J., Kim, K. I., Hoan, N. N., Kim, C. H., Moon, E., Choi, K. S., . . . Lee, J. S.
(2014). Targeting cancer cells with reactive oxygen and nitrogen species
generated by atmospheric-pressure air plasma. PLoS One, 9(1), e86173. DOI:
10.1371/journal.pone.0086173
Al-Khayal, K., Alafeefy, A., Vaali-Mohammed, M.A., Mahmood, A., Zubaidi, A., Al-
Obeed, O., . . . Ahmad, R. (2017). Novel derivative of aminobenzenesulfonamide
(3c) induces apoptosis in colorectal cancer cells through ROS generation and
inhibits cell migration. BMC Cancer, 17, 4. DOI: 10.1186/s12885-016-3005-7
Aziz, M. H., Nihal, M., Fu, V. X., Jarrard, D. F., & Ahmad, N. (2006). Resveratrol-
caused apoptosis of human prostate carcinoma LNCaP cells is mediated via
modulation of phosphatidylinositol 3'-kinase/Akt pathway and Bcl-2 family
proteins. Mol Cancer Ther, 5(5), 1335-1341. DOI: 10.1158/1535-7163.MCT-
05-0526
Bajt, M. L., Cover, C., Lemasters, J. J., & Jaeschke, H. (2006). Nuclear translocation
of endonuclease G and apoptosis-inducing factor during acetaminophen-
induced liver cell injury. Toxicol Sci, 94(1), 217-225. DOI:
10.1093/toxsci/kfl077
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
126
Benhar, M., Dalyot, I., Engelberg, D., & Levitzki, A. (2001). Enhanced ROS
production in oncogenically transformed cells potentiates c-Jun N-terminal
kinase and p38 mitogen-activated protein kinase activation and sensitization to
genotoxic stress. Mol Cell Biol, 21(20), 6913-6926. DOI:
10.1128/MCB.21.20.6913-6926.2001
Bernhardt, E. B., & Jalal, S. I. (2016). Small Cell Lung Cancer. Cancer Treat Res, 170,
301-322. DOI: 10.1007/978-3-319-40389-2_14
Bhattacharyya, A., Chattopadhyay, R., Mitra, S., & Crowe, S. E. (2014). Oxidative
Stress: An Essential Factor in the Pathogenesis of Gastrointestinal Mucosal
Diseases. Physiol Rev, 94(2), 329-354. DOI: 10.1152/physrev.00040.2012
Bhushan, S., Kumar, A., Malik, F., Andotra, S. S., Sethi, V. K., Kaur, I. P., . . . Singh,
J. (2007). A triterpenediol from Boswellia serrata induces apoptosis through
both the intrinsic and extrinsic apoptotic pathways in human leukemia HL-60
cells. Apoptosis, 12(10), 1911-1926. DOI: 10.1007/s10495-007-0105-5
Bu, H. Q., Cai, K., Shen, F., Bao, X. D., Xu, Y., Yu, F., . . . Cui, J. H. (2015). Induction
of apoptosis by capsaicin in hepatocellular cancer cell line SMMC-7721 is
mediated through ROS generation and activation of JNK and p38 MAPK
pathways. Neoplasma, 62(4), 582-591. DOI: 10.4149/neo_2015_070
Burstein, H. J. (2005). The distinctive nature of HER2-positive breast cancers. N Engl
J Med, 353(16), 1652-1654. DOI: 10.1056/NEJMp058197
Castedo, M., Perfettini, J. L., Roumier, T., Andreau, K., Medema, R., & Kroemer, G.
(2004). Cell death by mitotic catastrophe: a molecular definition. Oncogene,
23(16), 2825-2837. DOI: 10.1038/sj.onc.1207528
Chai, J., Du, C., Wu, J. W., Kyin, S., Wang, X., & Shi, Y. (2000). Structural and
biochemical basis of apoptotic activation by Smac/DIABLO. Nature,
406(6798), 855-862. DOI: 10.1038/35022514
Chang, C. H., Yu, F. Y., Wu, T. S., Wang, L. T., & Liu, B. H. (2011). Mycotoxin
citrinin induced cell cycle G2/M arrest and numerical chromosomal aberration
associated with disruption of microtubule formation in human cells. Toxicol Sci,
119(1), 84-92. DOI: 10.1093/toxsci/kfq309
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
127
Chang, Z., Xing, J., & Yu, X. (2014). Curcumin induces osteosarcoma MG63 cells
apoptosis via ROS/Cyto-C/Caspase-3 pathway. Tumour Biol, 35(1), 753-758.
doi: 10.1007/s13277-013-1102-7
Charrier-Savournin, F. B., Chateau, M. T., Gire, V., Sedivy, J., Piette, J., & Dulic, V.
(2004). p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to
genotoxic stress. Mol Biol Cell, 15(9), 3965-3976. DOI: 10.1091/mbc.E03-12-
0871
Cheng, C., Jiao, J. T., Qian, Y., Guo, X. Y., Huang, J., Dai, M. C., . . . Shao, J. F. (2016).
Curcumin induces G2/M arrest and triggers apoptosis via FoxO1 signaling in
U87 human glioma cells. Mol Med Rep, 13(5), 3763-3770. DOI:
10.3892/mmr.2016.5037
Choi, H. J., Lim do, Y., & Park, J. H. (2009). Induction of G1 and G2/M cell cycle
arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon
cancer cells. BMC Gastroenterol, 9, 39. DOI: 10.1186/1471-230X-9-39
Choi, Y. H., & Yoo, Y. H. (2012). Taxol-induced growth arrest and apoptosis is
associated with the upregulation of the Cdk inhibitor, p21WAF1/CIP1, in
human breast cancer cells. Oncol Rep, 28(6), 2163-2169. DOI:
10.3892/or.2012.2060
Choong, N. W., & Vokes, E. E. (2005). Adjuvant and neoadjuvant therapy for early-
stage non-small-cell lung cancer. Clin Lung Cancer, 7 Suppl 3, S98-104.
Chumakov, P. M. (2007). Versatile functions of p53 protein in multicellular organisms.
Biochemistry (Mosc), 72(13), 1399-1421.
Circu, M. L., & Aw, T. Y. (2010). Reactive oxygen species, cellular redox systems, and
apoptosis. Free Radic Biol Med, 48(6), 749-762. DOI:
10.1016/j.freeradbiomed.2009.12.022
Codony-Servat, J., Verlicchi, A., & Rosell, R. (2016). Cancer stem cells in small cell
lung cancer. Translational Lung Cancer Research, 5(1), 16-25. DOI:
10.3978/j.issn.2218-6751.2016.01.01
Deavall, D. G., Martin, E. A., Horner, J. M., & Roberts, R. (2012). Drug-induced
oxidative stress and toxicity. J Toxicol, 2012, 645460. DOI:
10.1155/2012/645460
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
128
Derry, M., Raina, K., Agarwal, R., & Agarwal, C. (2013). Differential effects of grape
seed extract against human colorectal cancer cell lines: the intricate role of death
receptors and mitochondria. Cancer Lett, 334(1), 69-78. DOI:
10.1016/j.canlet.2012.12.015
Dikalov, S., Griendling, K. K., & Harrison, D. G. (2007). Measurement of reactive
oxygen species in cardiovascular studies. Hypertension, 49(4), 717-727. DOI:
10.1161/01.HYP.0000258594.87211.6b
Dobbelstein, M., & Moll, U. (2014). Targeting tumour-supportive cellular machineries
in anticancer drug development. Nat Rev Drug Discov, 13(3), 179-196. DOI:
10.1038/nrd4201
Domont, J., Soria, J. C., & Le Chevalier, T. (2005). Adjuvant chemotherapy in early-
stage non-small cell lung cancer. Semin Oncol, 32(3), 279-283.
Du, C., Fang, M., Li, Y., Li, L., & Wang, X. (2000). Smac, a mitochondrial protein that
promotes cytochrome c-dependent caspase activation by eliminating IAP
inhibition. Cell, 102(1), 33-42.
Duval, F., Moreno-Cuevas, J. E., Gonzalez-Garza, M. T., Rodriguez-Montalvo, C., &
Cruz-Vega, D. E. (2014). Liver fibrosis and protection mechanisms action of
medicinal plants targeting apoptosis of hepatocytes and hepatic stellate cells.
Adv Pharmacol Sci, 2014, 373295. DOI: 10.1155/2014/373295
Duangmano, S., Sae-Lim, P., Suksamrarn, A., Domann, F. E., & Patmasiriwat, P.
(2012). Cucurbitacin B inhibits human breast cancer cell proliferation through
disruption of microtubule polymerization and nucleophosmin/B23
translocation. BMC Complement Altern Med, 12, 185. DOI: 10.1186/1472-
6882-12-185
Earnshaw, W. C., Martins, L. M., & Kaufmann, S. H. (1999). Mammalian caspases:
structure, activation, substrates, and functions during apoptosis. Annu Rev
Biochem, 68, 383-424. DOI: 10.1146/annurev.biochem.68.1.383
Elledge, S. J. (1996). Cell cycle checkpoints: preventing an identity crisis. Science,
274(5293), 1664-1672.
Elmore, S. (2007). Apoptosis: a review of programmed cell death. Toxicol Pathol,
35(4), 495-516. DOI: 10.1080/01926230701320337
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
129
Feng, Y., Wang, N., Zhu, M., Feng, Y., Li, H., & Tsao, S. (2011). Recent progress on
anticancer candidates in patents of herbal medicinal products. Recent Pat Food
Nutr Agric, 3(1), 30-48.
Fleming, T. P., Matsui, T., Molloy, C. J., Robbins, K. C., & Aaronson, S. A. (1989).
Autocrine mechanism for v-sis transformation requires cell surface
localization of internally activated growth factor receptors. Proc Natl Acad Sci
U S A, 86(20), 8063-8067.
Foster, D. A., Yellen, P., Xu, L., & Saqcena, M. (2010). Regulation of G1 Cell Cycle
Progression: Distinguishing the Restriction Point from a Nutrient-Sensing Cell
Growth Checkpoint(s). Genes Cancer, 1(11), 1124-1131. DOI:
10.1177/1947601910392989
Ganem, N. J., Storchova, Z., & Pellman, D. (2007). Tetraploidy, aneuploidy and cancer.
Curr Opin Genet Dev, 17(2), 157-162. DOI: 10.1016/j.gde.2007.02.011
Gao, L. L., Feng, L., Yao, S. T., Jiao, P., Qin, S. C., Zhang, W., . . . Li, F. R. (2011).
Molecular mechanisms of celery seed extract induced apoptosis via S phase cell
cycle arrest in the BGC-823 human stomach cancer cell line. Asian Pac J
Cancer Prev, 12(10), 2601-2606.
Ghavami, S., Hashemi, M., Ande, S. R., Yeganeh, B., Xiao, W., Eshraghi, M., . . . Los,
M. (2009). Apoptosis and cancer: mutations within caspase genes. J Med Genet,
46(8), 497.
Gogada, R., Prabhu, V., Amadori, M., Scott, R., Hashmi, S., & Chandra, D. (2011).
Resveratrol induces p53-independent, X-linked inhibitor of apoptosis protein
(XIAP)-mediated Bax protein oligomerization on mitochondria to initiate
cytochrome c release and caspase activation. J Biol Chem, 286(33), 28749-
28760. DOI: 10.1074/jbc.M110.202440
Gong, K., & Li, W. (2011). Shikonin, a Chinese plant-derived naphthoquinone, induces
apoptosis in hepatocellular carcinoma cells through reactive oxygen species: A
potential new treatment for hepatocellular carcinoma. Free Radic Biol Med,
51(12), 2259-2271. DOI: 10.1016/j.freeradbiomed.2011.09.018
Hagting, A., Jackman, M., Simpson, K., & Pines, J. (1999). Translocation of cyclin B1
to the nucleus at prophase requires a phosphorylation-dependent nuclear import
signal. Curr Biol, 9(13), 680-689.
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
130
Hahm, E. R., & Singh, S. V. (2007). Honokiol causes G0-G1 phase cell cycle arrest in
human prostate cancer cells in association with suppression of retinoblastoma
protein level/phosphorylation and inhibition of E2F1 transcriptional activity.
Mol Cancer Ther, 6(10), 2686-2695. DOI: 10.1158/1535-7163.MCT-07-0217
Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: the next generation. Cell,
144(5), 646-674. DOI: 10.1016/j.cell.2011.02.013
Hansakul, P., Aree, K., Tanuchit, S., & Itharat, A. (2014). Growth arrest and apoptosis
via caspase activation of dioscoreanone in human non-small-cell lung cancer
A549 cells. BMC Complement Altern Med, 14, 413. DOI: 10.1186/1472-6882-
14-413
Haupt, S., Berger, M., Goldberg, Z., & Haupt, Y. (2003). Apoptosis - the p53 network.
J Cell Sci, 116(Pt 20), 4077-4085. DOI: 10.1242/jcs.00739
Hsiao, C. J., Hsiao, G., Chen, W. L., Wang, S. W., Chiang, C. P., Liu, L. Y., . . . Chung,
C. L. (2014). Cephalochromin induces G0/G1 cell cycle arrest and apoptosis in
A549 human non-small-cell lung cancer cells by inflicting mitochondrial
disruption. J Nat Prod, 77(4), 758-765. DOI: 10.1021/np400517g
Hsieh, M. H., & Nguyen, H. T. (2005). Molecular mechanism of apoptosis induced by
mechanical forces. Int Rev Cytol, 245, 45-90. DOI: 10.1016/S0074-
7696(05)45003-2
Hwang, H. C., & Clurman, B. E. (2005). Cyclin E in normal and neoplastic cell cycles.
Oncogene, 24(17), 2776-2786. DOI: 10.1038/sj.onc.1208613
Huber, F., Schnauss, J., Ronicke, S., Rauch, P., Muller, K., Futterer, C., & Kas, J.
(2013). Emergent complexity of the cytoskeleton: from single filaments to
tissue. Adv Phys, 62(1), 1-112. DOI: 10.1080/00018732.2013.771509
Itharat, A. (2010). Comparative biological activities of five Thai medicinal plants called
Hua-Khao-Yen. Thai J Pharmacol, 32(1), 327-331.
Itharat, A., Plubrukarn, A., Kongsaeree, P., Bui, T., Keawpradub, N., & Houghton, P.
J. (2003). Dioscorealides and dioscoreanone, novel cytotoxic
naphthofuranoxepins, and 1,4-phenanthraquinone from Dioscorea
membranacea Pierre. Org Lett, 5(16), 2879-2882. DOI: 10.1021/ol034926y
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
131
Itharat, A., Houghton, P. J., Eno-Amooquaye, E., Burke, P. J., Sampson, J. H., &
Raman, A. (2004). In vitro cytotoxic activity of Thai medicinal plants used
traditionally to treat cancer. J Ethnopharmacol, 90(1), 33-38.
Itharat, A., Plubrukan, A., Kaewpradub, N., Chuchom, T., Ratanasuwan, P., Houghton,
P.J. (2007). Selective cytotoxicity and antioxidant effects of compounds from
Dioscorea membranacea rhizomes. Nat Prod Commun 2, 643-648.
Itharat, A., Thongdeeying, P., & Ruangnoo, S. (2014). Isolation and characterization of
a new cytotoxic dihydrophenanthrene from Dioscorea membranacea rhizomes
and its activity against five human cancer cell lines. J Ethnopharmacol, 156,
130-134. DOI: 10.1016/j.jep.2014.08.009
Ito, K., Nakazato, T., Yamato, K., Miyakawa, Y., Yamada, T., Hozumi, N., . . . Kizaki,
M. (2004). Induction of apoptosis in leukemic cells by homovanillic acid
derivative, capsaicin, through oxidative stress: implication of phosphorylation
of p53 at Ser-15 residue by reactive oxygen species. Cancer Res, 64(3), 1071-
1078.
Jo, J. R., Park, J. S., Park, Y. K., Chae, Y. Z., Lee, G. H., Park, G. Y., & Jang, B. C.
(2012). Pinus densiflora leaf essential oil induces apoptosis via ROS generation
and activation of caspases in YD-8 human oral cancer cells. Int J Oncol, 40(4),
1238-1245. DOI: 10.3892/ijo.2011.1263
Jung, E. M., Lim, J. H., Lee, T. J., Park, J. W., Choi, K. S., & Kwon, T. K. (2005).
Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL)-induced apoptosis through reactive oxygen species-mediated
upregulation of death receptor 5 (DR5). Carcinogenesis, 26(11), 1905-1913.
DOI: 10.1093/carcin/bgi167
Kadota, K., Nitadori, J., Rekhtman, N., Jones, D. R., Adusumilli, P. S., & Travis, W.
D. (2015). Reevaluation and reclassification of resected lung carcinomas
originally diagnosed as squamous cell carcinoma using immunohistochemical
analysis. Am J Surg Pathol, 39(9), 1170-1180. DOI:
10.1097/PAS.0000000000000439
Kalemkerian, G. P. (2011). Staging and imaging of small cell lung cancer. Cancer
Imaging, 11(1), 253-258. DOI: 10.1102/1470-7330.2011.0036
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
132
Kalimuthu, S., & Se-Kwon, K. (2013). Cell survival and apoptosis signaling as
therapeutic target for cancer: marine bioactive compounds. Int J Mol Sci, 14(2),
2334-2354. DOI: 10.3390/ijms14022334
Kantari, C., & Walczak, H. (2011). Caspase-8 and bid: caught in the act between death
receptors and mitochondria. Biochim Biophys Acta, 1813(4), 558-563. DOI:
10.1016/j.bbamcr.2011.01.026
Karimian, H., Moghadamtousi, S. Z., Fadaeinasab, M., Golbabapour, S., Razavi, M.,
Hajrezaie, M., . . . Noordin, M. I. (2014). Ferulago angulata activates intrinsic
pathway of apoptosis in MCF-7 cells associated with G1 cell cycle arrest via
involvement of p21/p27. Drug Design, Development and Therapy, 8, 1481-
1497. DOI: 10.2147/DDDT.S68818
Kerr, J. F., Wyllie, A. H., & Currie, A. R. (1972). Apoptosis: a basic biological
phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer,
26(4), 239-257.
Khan, M., Rasul, A., Yi, F., Zhong, L., & Ma, T. (2011). Jaceosidin induces p53-
dependent G2/M phase arrest in U87 glioblastoma cells. Asian Pac J Cancer
Prev, 12(12), 3235-3238.
Kim, N. Y., Pae, H. O., Oh, G. S., Kang, T. H., Kim, Y. C., Rhew, H. Y., & Chung, H.
T. (2001). Butein, a plant polyphenol, induces apoptosis concomitant with
increased caspase-3 activity, decreased Bcl-2 expression and increased Bax
expression in HL-60 cells. Pharmacol Toxicol, 88(5), 261-266.
Kinzler, K. W., & Vogelstein, B. (1997). Cancer-susceptibility genes. Gatekeepers and
caretakers. Nature, 386(6627), 761, 763. DOI: 10.1038/386761a0
Lan, Y. H., Wu, Y. C., Wu, K. W., Chung, J. G., Lu, C. C., Chen, Y. L., . . . Yang, J. S.
(2011). Death receptor 5-mediated TNFR family signaling pathways modulate
gamma-humulene-induced apoptosis in human colorectal cancer HT29 cells.
Oncol Rep, 25(2), 419-424. DOI: 10.3892/or.2010.1087
Lobo, V., Patil, A., Phatak, A., & Chandra, N. (2010). Free radicals, antioxidants and
functional foods: Impact on human health. Pharmacogn Rev, 4(8), 118-126.
DOI: 10.4103/0973-7847.70902
Larsen, J. E., & Minna, J. D. (2011). Molecular biology of lung cancer: clinical
implications. Clin Chest Med, 32(4), 703-740. DOI: 10.1016/j.ccm.2011.08.003
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
133
Lee, D. S., Lee, M. K., & Kim, J. H. (2009). Curcumin induces cell cycle arrest and
apoptosis in human osteosarcoma (HOS) cells. Anticancer Res, 29(12), 5039-
5044.
Li, L., Dai, H. J., Ye, M., Wang, S. L., Xiao, X. J., Zheng, J., . . . Liu, J. (2012). Lycorine
induces cell-cycle arrest in the G0/G1 phase in K562 cells via HDAC inhibition.
Cancer Cell Int, 12(1), 49. DOI: 10.1186/1475-2867-12-49
Li, M., Zhang, F., Wang, X., Wu, X., Zhang, B., Zhang, N., . . . Liu, Y. (2015).
Magnolol inhibits growth of gallbladder cancer cells through the p53 pathway.
Cancer Sci, 106(10), 1341-1350. DOI: 10.1111/cas.12762
Li, Y. B., Yan, X., Li, R. D., Liu, P., Sun, S. Q., Wang, X., . . . Li, R. T. (2016).
Discovery of novel heteroarylmethylcarbamodithioates as potent anticancer
agents: Synthesis, structure-activity relationship analysis and biological
evaluation. Eur J Med Chem, 112, 217-230. DOI:
10.1016/j.ejmech.2016.02.015
Liou, G. Y., & Storz, P. (2010). Reactive oxygen species in cancer. Free Radic Res,
44(5), 479-496. DOI: 10.3109/10715761003667554
Liu, Y., & Min, W. (2002). Thioredoxin promotes ASK1 ubiquitination and
degradation to inhibit ASK1-mediated apoptosis in a redox activity-independent
manner. Circ Res, 90(12), 1259-1266.
Mahavorasirikul, W., Viyanant, V., Chaijaroenkul, W., Itharat, A., & Na-Bangchang,
K. (2010). Cytotoxic activity of Thai medicinal plants against human
cholangiocarcinoma, laryngeal and hepatocarcinoma cells in vitro. BMC
Complement Altern Med, 10, 55. DOI: 10.1186/1472-6882-10-55
Mahmood, T., & Yang, P. C. (2012). Western blot: technique, theory, and trouble
shooting. N Am J Med Sci, 4(9), 429-434. doi: 10.4103/1947-2714.100998
Mollinedo, F., & Gajate, C. (2003). Microtubules, microtubule-interfering agents and
apoptosis. Apoptosis, 8(5), 413-450.
Morris, L. G. T., & Chan, T. A. (2015). Therapeutic Targeting of Tumor Suppressor
Genes. Cancer, 121(9), 1357-1368. DOI: 10.1002/cncr.29140
Mu, J., Liu, T., Jiang, L., Wu, X., Cao, Y., Li, M., . . . Xu, H. (2016). The Traditional
Chinese Medicine Baicalein Potently Inhibits Gastric Cancer Cells. J Cancer,
7(4), 453-461. DOI: 10.7150/jca.13548
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
134
Mukhopadhyay, A., Banerjee, S., Stafford, L. J., Xia, C., Liu, M., & Aggarwal, B. B.
(2002). Curcumin-induced suppression of cell proliferation correlates with
down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma
protein phosphorylation. Oncogene, 21(57), 8852-8861. DOI:
10.1038/sj.onc.1206048
Nasmyth, K. (1996). Viewpoint: putting the cell cycle in order. Science, 274(5293),
1643-1645.
Neganova, I., & Lako, M. (2008). G1 to S phase cell cycle transition in somatic and
embryonic stem cells. J Anat, 213(1), 30-44. DOI: 10.1111/j.1469-
7580.2008.00931.x
Norbury, C. J., & Hickson, I. D. (2001). Cellular responses to DNA damage. Annu Rev
Pharmacol Toxicol, 41, 367-401. DOI: 10.1146/annurev.pharmtox.41.1.367
Ouyang, G., Yao, L., Ruan, K., Song, G., Mao, Y., & Bao, S. (2009). Genistein induces
G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via
activation of DNA damage checkpoint pathways. Cell Biol Int, 33(12), 1237-
1244. DOI: 10.1016/j.cellbi.2009.08.011
Pagano, M., Pepperkok, R., Verde, F., Ansorge, W., & Draetta, G. (1992). Cyclin A is
required at two points in the human cell cycle. EMBO J, 11(3), 961-971.
Pan, M. H., Chen, W. J., Lin-Shiau, S. Y., Ho, C. T., & Lin, J. K. (2002). Tangeretin
induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and
4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal
carcinoma cells. Carcinogenesis, 23(10), 1677-1684.
Parlato, R., & Mastroberardino, P. G. (2015). Editorial: Neuronal Self-Defense:
Compensatory Mechanisms in Neurodegenerative Disorders. Front Cell
Neurosci, 9, 499. DOI: 10.3389/fncel.2015.00499
Pesch, B., Kendzia, B., Gustavsson, P., Jockel, K. H., Johnen, G., Pohlabeln, H., . . .
Bruning, T. (2012). Cigarette smoking and lung cancer--relative risk estimates
for the major histological types from a pooled analysis of case-control studies.
Int J Cancer, 131(5), 1210-1219. DOI: 10.1002/ijc.27339
Phang, C. W., Malek, S. N., & Ibrahim, H. (2013). Antioxidant potential, cytotoxic
activity and total phenolic content of Alpinia pahangensis rhizomes. BMC
Complement Altern Med, 13, 243. DOI: 10.1186/1472-6882-13-243
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
135
Phang, C. W., Karsani, S. A., Sethi, G., & Abd Malek, S. N. (2016). Flavokawain C
Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic
Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in
HCT 116 Human Colon Carcinoma Cells. PLoS One, 11(2), e0148775. doi:
10.1371/journal.pone.0148775
Poornima, P., Quency, R. S., & Padma, V. V. (2013). Neferine induces reactive oxygen
species mediated intrinsic pathway of apoptosis in HepG2 cells. Food Chem,
136(2), 659-667. DOI: 10.1016/j.foodchem.2012.07.112
Potapova, T. A., Daum, J. R., Byrd, K. S., & Gorbsky, G. J. (2009). Fine tuning the cell
cycle: activation of the Cdk1 inhibitory phosphorylation pathway during mitotic
exit. Mol Biol Cell, 20(6), 1737-1748. DOI: 10.1091/mbc.E08-07-0771
Porter, A. G., & Janicke, R. U. (1999). Emerging roles of caspase-3 in apoptosis. Cell
Death Differ, 6(2), 99-104. DOI: 10.1038/sj.cdd.4400476
Prayong, P., Barusrux, S., & Weerapreeyakul, N. (2008). Cytotoxic activity screening
of some indigenous Thai plants. Fitoterapia, 79(7-8), 598-601. DOI:
10.1016/j.fitote.2008.06.007
Qiu, M., Chen, L., Tan, G., Ke, L., Zhang, S., Chen, H., & Liu, J. (2015). A reactive
oxygen species activation mechanism contributes to JS-K-induced apoptosis in
human bladder cancer cells. Sci Rep, 5, 15104. DOI: 10.1038/srep15104
Rekhtman, N., Ang, D. C., Sima, C. S., Travis, W. D., & Moreira, A. L. (2011).
Immunohistochemical algorithm for differentiation of lung adenocarcinoma
and squamous cell carcinoma based on large series of whole-tissue sections with
validation in small specimens. Mod Pathol, 24(10), 1348-1359. DOI:
10.1038/modpathol.2011.92
Reanmongkol, W., Itharat, A., Bouking, P. (2007). Investigation of the anti-
inflammatory, analgesic and antipyretic activities of the extracts from the
rhizome of Dioscorea membranacea Pierre in experimental animals.
Songklanakarin Journal of Science and Technology, 29(1): 49-57.
Rivlin, N., Brosh, R., Oren, M., & Rotter, V. (2011). Mutations in the p53 Tumor
Suppressor Gene: Important Milestones at the Various Steps of Tumorigenesis.
Genes Cancer, 2(4), 466-474. DOI: 10.1177/1947601911408889
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
136
Rock, K. L., & Kono, H. (2008). The inflammatory response to cell death. Annual review
of pathology, 3, 99-126. DOI: 10.1146/annurev.pathmechdis.3.121806.151456
Roederer, M. (2011). Interpretation of cellular proliferation data: avoid the panglossian.
Cytometry A, 79(2), 95-101. DOI: 10.1002/cyto.a.21010
Rodriguez-Canales, J., Parra-Cuentas, E., & Wistuba, II. (2016). Diagnosis and
Molecular Classification of Lung Cancer. Cancer Treat Res, 170, 25-46. DOI:
10.1007/978-3-319-40389-2_2
Saelens, X., Festjens, N., Vande Walle, L., van Gurp, M., van Loo, G., &
Vandenabeele, P. (2004). Toxic proteins released from mitochondria in cell
death. Oncogene, 23(16), 2861-2874. DOI: 10.1038/sj.onc.1207523
Sanchez, V., McElroy, A. K., & Spector, D. H. (2003). Mechanisms governing
maintenance of Cdk1/cyclin B1 kinase activity in cells infected with human
cytomegalovirus. J Virol, 77(24), 13214-13224.
Saekoo, J., Dechsukum, C., Graidist, P., & Itharat, A. (2010). Cytotoxic effect and its
mechanism of dioscorealide B from Dioscorea membranacea against breast
cancer cells. J Med Assoc Thai, 93 Suppl 7, S277-282.
Saekoo, J., Graidist, P., Leeanansaksiri, W., Dechsukum, C., & Itharat, A. (2010).
Dioscorealide B from the traditional Thai medicine Hua-Khao-Yen induces
apoptosis in MCF-7 human breast cancer cells via modulation of Bax, Bak and
Bcl-2 protein expression. Nat Prod Commun, 5(12), 1921-1926.
Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., . . . Ichijo, H.
(1998). Mammalian thioredoxin is a direct inhibitor of apoptosis signal-
regulating kinase (ASK) 1. EMBO J, 17(9), 2596-2606. DOI:
10.1093/emboj/17.9.2596
Salvà, F., & Felip, E. (2013). Neoadjuvant chemotherapy in early-stage non-small cell
lung cancer. Translational Lung Cancer Research, 2(5), 398-402.
Schwartz, G. K., & Shah, M. A. (2005). Targeting the cell cycle: a new approach to
cancer therapy. J Clin Oncol, 23(36), 9408-9421. DOI:
10.1200/JCO.2005.01.5594
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
137
Shabbeer, S., Omer, D., Berneman, D., Weitzman, O., Alpaugh, A., Pietraszkiewicz,
A., . . . Yarden, R. I. (2013). BRCA1 targets G2/M cell cycle proteins for
ubiquitination and proteasomal degradation. Oncogene, 32(42), 5005-5016.
DOI: 10.1038/onc.2012.522
Shadfan, M., Lopez-Pajares, V., & Yuan, Z.-M. (2012). MDM2 and MDMX: Alone
and together in regulation of p53. Transl Cancer Res, 1(2), 88-89.
Shapiro, G. I., & Harper, J. W. (1999). Anticancer drug targets: cell cycle and
checkpoint control. J Clin Invest, 104(12), 1645-1653. DOI: 10.1172/JCI9054
Sherr, C. J., & McCormick, F. (2002). The RB and p53 pathways in cancer. Cancer
Cell, 2(2), 103-112.
Shi, X., Zhang, Y., Zheng, J., & Pan, J. (2012). Reactive oxygen species in cancer stem
cells. Antioxid Redox Signal, 16(11), 1215-1228. DOI: 10.1089/ars.2012.4529
Shin, J. W., Son, J. Y., Kang, J. K., Han, S. H., Cho, C. K., & Son, C. G. (2008).
Trichosanthes kirilowii tuber extract induces G2/M phase arrest via inhibition
of tubulin polymerization in HepG2 cells. J Ethnopharmacol, 115(2), 209-216.
DOI: 10.1016/j.jep.2007.09.030
Singh, S. V., Herman-Antosiewicz, A., Singh, A. V., Lew, K. L., Srivastava, S. K.,
Kamath, R., . . . Baskaran, R. (2004). Sulforaphane-induced G2/M phase cell
cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell
division cycle 25C. J Biol Chem, 279(24), 25813-25822. DOI:
10.1074/jbc.M313538200
Singh, N., Zaidi, D., Shyam, H., Sharma, R., & Balapure, A. K. (2012). Polyphenols
sensitization potentiates susceptibility of MCF-7 and MDA MB-231 cells to
Centchroman. PLoS One, 7(6), e37736. DOI: 10.1371/journal.pone.0037736
Soussi, T., & Wiman, K. G. (2015). TP53: an oncogene in disguise. Cell Death Differ,
22(8), 1239-1249. DOI: 10.1038/cdd.2015.53
Sriplung, H., Sontipong, S., Martin, N., Wiangnon, S., Vootiprux, V., Cheirsilpa, A., .
. . Khuhaprema, T. (2005). Cancer incidence in Thailand, 1995-1997. Asian Pac
J Cancer Prev, 6(3), 276-281.
Stanton, R. A., Gernert, K. M., Nettles, J. H., & Aneja, R. (2011). Drugs That Target
Dynamic Microtubules: A New Molecular Perspective. Med Res Rev, 31(3),
443-481. DOI: 10.1002/med.20242
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
138
Steller, H. (1998). Artificial death switches: induction of apoptosis by chemically
induced caspase multimerization. Proc Natl Acad Sci U S A, 95(10), 5421-5422.
Suen, D. F., Norris, K. L., & Youle, R. J. (2008). Mitochondrial dynamics and
apoptosis. Genes Dev, 22(12), 1577-1590. DOI: 10.1101/gad.1658508
Tan, C. P., Lu, Y. Y., Ji, L. N., & Mao, Z. W. (2014). Metallomics insights into the
programmed cell death induced by metal-based anticancer compounds.
Metallomics, 6(5), 978-995. DOI: 10.1039/c3mt00225j
Tan, T. W., Tsai, H. R., Lu, H. F., Lin, H. L., Tsou, M. F., Lin, Y. T., . . . Chung, J. G.
(2006). Curcumin-induced cell cycle arrest and apoptosis in human acute
promyelocytic leukemia HL-60 cells via MMP changes and caspase-3
activation. Anticancer Res, 26(6B), 4361-4371.
Tewtrakul, S., & Itharat, A. (2006). Anti-allergic substances from the rhizomes of
Dioscorea membranacea. Bioorg Med Chem, 14(24), 8707-8711. DOI:
10.1016/j.bmc.2006.08.012
Tewtrakul, S., & Itharat, A. (2007). Nitric oxide inhibitory substances from the
rhizomes of Dioscorea membranacea. J Ethnopharmacol, 109(3), 412-416. DOI
10.1016/j.jep.2006.08.009
Tewtrakul, S., Itharat, A., & Rattanasuwan, P. (2006). Anti-HIV-1 protease- and HIV-
1 integrase activities of Thai medicinal plants known as Hua-Khao-Yen. J
Ethnopharmacol, 105(1-2), 312-315. DOI: 10.1016/j.jep.2005.11.021
Tian, L., Yin, D., Ren, Y., Gong, C., Chen, A., & Guo, F. J. (2012). Plumbagin induces
apoptosis via the p53 pathway and generation of reactive oxygen species in
human osteosarcoma cells. Mol Med Rep, 5(1), 126-132. DOI:
10.3892/mmr.2011.624
Tian, R., Li, Y., & Gao, M. (2015). Shikonin causes cell-cycle arrest and induces
apoptosis by regulating the EGFR-NF-kappaB signalling pathway in human
epidermoid carcinoma A431 cells. Biosci Rep, 35(2). DOI:
10.1042/BSR20150002
Tobiume, K., Matsuzawa, A., Takahashi, T., Nishitoh, H., Morita, K., Takeda, K., . . .
Ichijo, H. (2001). ASK1 is required for sustained activations of JNK/p38 MAP
kinases and apoptosis. EMBO Rep, 2(3), 222-228. DOI: 10.1093/embo-
reports/kve046
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
139
Torre, L. A., Bray, F., Siegel, R. L., Ferlay, J., Lortet-Tieulent, J., & Jemal, A. (2015).
Global cancer statistics, 2012. CA: A Cancer Journal for Clinicians, 65(2), 87-
108. DOI: 10.3322/caac.21262
Trachootham, D., Alexandre, J., & Huang, P. (2009). Targeting cancer cells by ROS-
mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov,
8(7), 579-591. DOI: 10.1038/nrd2803
Travis, W. D., & Rekhtman, N. (2011). Pathological diagnosis and classification of
lung cancer in small biopsies and cytology: strategic management of tissue for
molecular testing. Semin Respir Crit Care Med, 32(1), 22-31. DOI: 10.1055/s-
0031-1272866
Travis, W. D., Brambilla, E., Nicholson, A. G., Yatabe, Y., Austin, J. H., Beasley, M.
B., . . . Panel, W. H. O. (2015). The 2015 World Health Organization
Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic
Advances Since the 2004 Classification. J Thorac Oncol, 10(9), 1243-1260.
DOI: 10.1097/JTO.0000000000000630
Vatanasapt, V., Sriamporn, S., & Vatanasapt, P. (2002). Cancer control in Thailand.
Jpn J Clin Oncol, 32 Suppl, S82-91.
Vermes, I., Haanen, C., Steffens-Nakken, H., & Reutelingsperger, C. (1995). A novel
assay for apoptosis. Flow cytometric detection of phosphatidylserine expression
on early apoptotic cells using fluorescein labelled Annexin V. J Immunol
Methods, 184(1), 39-51.
Vermeulen, K., Van Bockstaele, D. R., & Berneman, Z. N. (2003). The cell cycle: a
review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif,
36(3), 131-149.
Visbal, A. L., Leighl, N. B., Feld, R., & Shepherd, F. A. (2005). Adjuvant
Chemotherapy for Early-Stage Non-small Cell Lung Cancer. Chest, 128(4),
2933-2943. DOI: 10.1378/chest.128.4.2933
Voigt, W. (2005). Sulforhodamine B assay and chemosensitivity. Methods Mol Med,
110, 39-48. DOI: 10.1385/1-59259-869-2:039
Wang, X. Q., Duan, X. M., Liu, L. H., Fang, Y. Q., & Tan, Y. (2005).
Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell
labeling. Acta Biochim Biophys Sin (Shanghai), 37(6), 379-385.
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
140
Wong, R. S. (2011). Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin
Cancer Res, 30, 87. DOI: 10.1186/1756-9966-30-87
Wu, S. H., Hang, L. W., Yang, J. S., Chen, H. Y., Lin, H. Y., Chiang, J. H., . . . Chung,
J. G. (2010). Curcumin induces apoptosis in human non-small cell lung cancer
NCI-H460 cells through ER stress and caspase cascade- and mitochondria-
dependent pathways. Anticancer Res, 30(6), 2125-2133.
Wu, Z., Wu, L., Li, L., Tashiro, S., Onodera, S., & Ikejima, T. (2004). p53-mediated
cell cycle arrest and apoptosis induced by shikonin via a caspase-9-dependent
mechanism in human malignant melanoma A375-S2 cells. J Pharmacol Sci,
94(2), 166-176.
Xu, G., & Shi, Y. (2007). Apoptosis signaling pathways and lymphocyte homeostasis.
Cell Res, 17(9), 759-771. DOI: 10.1038/cr.2007.52
Xu, M., Sheppard, K. A., Peng, C. Y., Yee, A. S., & Piwnica-Worms, H. (1994). Cyclin
A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-
1/DP-1 by phosphorylation. Mol Cell Biol, 14(12), 8420-8431.
Yin, X., Zhang, R., Feng, C., Zhang, J., Liu, D., Xu, K., . . . Ma, H. (2014). Diallyl
disulfide induces G2/M arrest and promotes apoptosis through the p53/p21 and
MEK-ERK pathways in human esophageal squamous cell carcinoma. Oncol
Rep, 32(4), 1748-1756. DOI: 10.3892/or.2014.3361
Zhang, H., & Rosdahl, I. (2006). Bcl-xL and bcl-2 proteins in melanoma progression
and UVB-induced apoptosis. Int J Oncol, 28(3), 661-666.
Zhang, R., Humphreys, I., Sahu, R. P., Shi, Y., & Srivastava, S. K. (2008). In vitro and
in vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated
through ROS generation and mitochondrial death pathway. Apoptosis, 13(12),
1465-1478. DOI: 10.1007/s10495-008-0278-6
Zhang, X., Wang, X., Wu, T., Li, B., Liu, T., Wang, R., . . . Shao, C. (2015).
Isoliensinine induces apoptosis in triple-negative human breast cancer cells
through ROS generation and p38 MAPK/JNK activation. Sci Rep, 5, 12579.
DOI: 10.1038/srep12579
Zhang, X. H., Zou, Z. Q., Xu, C. W., Shen, Y. Z., & Li, D. (2011). Myricetin induces
G2/M phase arrest in HepG2 cells by inhibiting the activity of the cyclin B/Cdc2
complex. Mol Med Rep, 4(2), 273-277. DOI: 10.3892/mmr.2011.417
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
141
Zhang, Z., Wang, C. Z., Du, G. J., Qi, L. W., Calway, T., He, T. C., . . . Yuan, C. S.
(2013). Genistein induces G2/M cell cycle arrest and apoptosis via ATM/p53-
dependent pathway in human colon cancer cells. Int J Oncol, 43(1), 289-296.
DOI: 10.3892/ijo.2013.1946
Zhou, Y., Bi, Y., Yang, C., Yang, J., Jiang, Y., Meng, F., . . . Yang, H. (2013). Magnolol
induces apoptosis in MCF-7 human breast cancer cells through G2/M phase
arrest and caspase-independent pathway. Pharmazie, 68(9), 755-762.
Zhu, J., Chen, M., Chen, N., Ma, A., Zhu, C., Zhao, R., . . . Zhang, X. (2015).
Glycyrrhetinic acid induces G1 phase cell cycle arrest in human nonsmall cell
lung cancer cells through endoplasmic reticulum stress pathway. Int J Oncol,
46(3), 981-988. DOI: 10.3892/ijo.2015.2819
Zhuang, W., Jia, Z., Feng, H., Chen, J., Wang, H., Guo, Y., & Meng, C. (2011). The
mechanism of the G0/G1 cell cycle phase arrest induced by activation of PXR in
human cells. Biomed Pharmacother, 65(7), 467-473. DOI:
10.1016/j.biopha.2011.04.014
Zou, H., Yang, R., Hao, J., Wang, J., Sun, C., Fesik, S. W., . . . Armstrong, R. C. (2003).
Regulation of the Apaf-1/caspase-9 apoptosome by caspase-3 and XIAP. J Biol
Chem, 278(10), 8091-8098. DOI: 10.1074/jbc.M204783200
Electronic Media
American Cancer Society. Cancer facts and figures 2015. Retrieved November 20,
2015 http://www.cancer.org/acs/groups/content/@editorial/documents/document/
acspc044552.pdf
Other Materials
Itharat, A., Singchangchai, P., Ratanasuwan, P. (1998). Wisdom of Southern Thai
Traditional Doctors (p.126). Songkla: Prince of Songkla University.
Itharat, A. (2002). Studies on bioactivity fo five Thai medical plants called Hua-Khao-
Yen (Doctoral dissertation), King's College London, London, United Kingdom.
Pongbunrod, S. (1976). Mai Thiet Mueang Thai Kasembanakit (pp.120-122). Bangkok.
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
142
Thapyai, C. (2004). Taxonomic revision of dioscoreaceae in Thailand (Doctoral
dissertation), Kasetsart University, Bangkok, Thailand.
Tungtrongjit, K. (1978). Pramuan Supphakun Ya Thai (pp.107-108). Bangkok.
Wilkin, P., Thapyai, C. (2009). Dioscoreaceae, Flora of Thailand.
Ref. code: 25595411301012PXORef. code: 25595411301012PXORef. code: 25595411301012PXO
143
APPENDICES
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APPENDIX A
GROWTH CURVE
Figure A-1 Growth curve of human lung carcinoma cell line A549 in 96-well plates.
The optimal cell numbers of this cell line were 3,200 cells/well. Data are expressed as
mean (n ≥ 3). Each experiment was performed in triplicate.
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
0 24 48 72 96
O.D
. 5
70
nm
Time (hours)
Growth curve (A549), 96 wells
12,800 cells/well
6,400 cells/well
3,200 cells/well
1,600 cells/well
800 cells/well
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Figure A-2 Growth curve of human lung squamous carcinoma cell line NCI-H226 in
96-well plates. The optimal cell numbers of this cell line were 3,200 cells/well. Data
are expressed as mean (n ≥ 3). Each experiment was performed in triplicate.
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
0 24 48 72 96
O.D
. 5
70
nm
Time (hours)
Growth curve (NCI-H226), 96-well plate
12,800 cells/well
6,400 cells/well
3,200 cells/well
1,600 cells/well
800 cells/well
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Figure A-3 Growth curve of human large cell lung cancer line COR-L23 in 96-well
plates. The optimal cell numbers of this cell line were 3,200 cells/well. Data are
expressed as mean (n ≥ 3). Each experiment was performed in triplicate.
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
0 24 48 72 96
O.D
. 5
70
nm
Time (hours)
Growth curve (COR-L23), 96-well plate
12,800 cells/well
6,400 cells/well
3,200 cells/well
1,600 cells/well
800 cells/well
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Figure A-4 Growth curve of human small cell lung cancer cell line NCI-H1688 in 96-
well plates. The optimal cell numbers of this cell line were 6,400 cells/well. Data are
expressed as mean (n ≥ 3). Each experiment was performed in triplicate.
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
0 24 48 72 96
O.D
. 5
70
nm
Time (hours)
Growth curve (NCI-H1688), 96-well plate
12,800 cells/well
6,400 cells/well
3,200 cells/well
25,600 cells/well
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Figure A-5 Growth curve of human lung fibroblast cell line MRC-5 in 96-well plates.
The optimal cell numbers of this cell line were 12,800 cells/well. Data are expressed as
mean (n ≥ 3). Each experiment was performed in triplicate.
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
0 24 48 72 96
O.D
. 5
70
nm
Time (hours)
Growth curve (MRC-5), 96-well plate
12,800 cells/well
6,400 cells/well
3,200 cells/well
1,600 cells/well
800 cells/well
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Figure A-6 Growth curve of human lung carcinoma cell line A549 in 24-well plates.
The optimal cell numbers of this cell line were 32,000 cells/well. Data are expressed as
mean (n ≥ 3). Each experiment was performed in triplicate.
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
0 24 48 72 96
O.D
. 5
70
nm
Time (hours)
Growth curve (A549), 24-well plate
128,000 cells/well
64,000 cells/well
32,000 cells/well
16,000 cells/well
256,000 cells/well
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APPENDIX B
STANDARD CURVE FOR PROTEIN DETERMINATION
Figure B-1 Standard curve for protein determination by Bradford’s method.
Figure B-2 Standard curve for protein determination by BCA Assay.
y = 0.9325x + 0.0962
R² = 0.9928
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0.000 0.250 0.500 0.750 1.000 1.250
O.D
. 5
95
nm
BSA concentration (mg/ml)
BSA standard curve by Bradford's method
y = 1.1836x + 0.0715
R² = 0.9984
0.000
0.500
1.000
1.500
2.000
2.500
3.000
0.000 0.500 1.000 1.500 2.000 2.500
O.D
. 5
62 n
m
BSA concentration (mg/ml)
BSA standard curve by BCA assay
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APPENDIX C
HPLC CHROMATOGRAMS
Figure C-1 HPLC chromatogram of HMP-1.
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Figure C-2 HPLC chromatogram of HMP-2.
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Figure C-3 HPLC chromatogram of 5, 6-dihydroxy-2, 4-dimethoxy-9, 10-dihydro
phenanthrene.
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APPENDIX D
FLOW CYTOMETRIC ANALYSIS
Figure D-1 Flow cytometric analysis of the DNA from A549 cells treated with NAC
alone at different concentrations (0.1, 1 and 5 mM) for 72 h. Data are representatives
of three independent experiments.
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APPENDIX E
REAGENTS FOR LABORATORY EXPERIMENTS
1. Reagents for cell culture
RPMI medium 1640
Dissolve 10.4 g of RPMI medium 1640 powder (Biochrom, Germany) and
2.0 g of sodium bicarbonate in distilled water to a final volume of 1,000 ml.
The medium was adjusted pH to 7.4 with 1 M HCl (hydrochloric acid) and
was then filtered through 0.22 micron of filter. The complete RPMI 1640
medium was mixed with 10% heat-inactivated FBS and was stored at 4°C.
Phosphate buffer saline (PBS) Solution
Dissolve 9.55 g of PBS powder without Ca2+, Mg2+ (Biochrom, Germany)
in 1,000 ml distilled water. PBS solution was then filtered through 0.22
micron of filter and stored at 4°C.
Trypsin-EDTA Solution
Dissolve 10 ml Trypsin (1:250)/EDTA (0.5/0.2 %) in 10x PBS without
Ca2+, Mg2+ (Biochrom, Germany) in 90 ml 1x PBS solution.
2. Reagents for SRB assay
1% glacial acetic acid
Glacial acetic acid 10.00 ml
Distilled water 990.00 ml
0.4% (w/v) Sulforhodamine B (SRB) solution
Dissolve 0.4 g of sulforhodamine B dye to a final volume of 100 ml with
1% glacial acetic acid and keep at room temperature.
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10% TCA (trichloroacetic acid)
Dissolve 10 g of trichloroacetic acid to a final volume of 100 ml with
distilled water and keep at 4°C.
Tris-base [10 mM, pH 10]
Tris 605.50 mg
Distilled water 400.00 ml
Adjust pH into 10.0, then add distilled water to a final volume 500 ml and
keep at room temperature.
3. Reagents for SDS-PAGE preparation
5x running buffer (1L)
0.25 M Tris 15.15 g
1.92 M glycine 72.00 g
1% SDS 5.00 g
Adjust to a final volume of 1,000 ml with deionized water and keep at 4ºC.
1x running buffer (working solution)
5x running buffer 200.00 ml
Distilled water 800.00 ml
4x stacking gel buffer [0.5M tris-Hcl, pH 6.8, 100ml]
Tris 6.055 g
Deionized water 80.00 ml
Adjust pH into 6.8 and add deionized water to a final volume 100 ml
4x separating (resolving) gel buffer [1.5M tris-Hcl, pH 8.8, 200ml]
Tris 36.33 g
Deionized water 150.00 ml
Adjust pH 8.8 and add deionized water to a final volume 200ml
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10% APS (ammonium per sulfate)
Ammonium per sulfate 10.00 g
Deionized water 100.00 ml
10% SDS (Sodium dodecyl sulfate)
SDS 10.00 g
Deionized water 100.00 ml
Table E-1 Recipes for resolving and stacking gels
Reagents Resolving gel
Stacking
gel
12% 7.5% 4%
30% Acrylamide/Bis Solution,
29:1 (Bio-Rad) 4.0 ml 2.5 ml 660 µl
4x resolving buffer 2.5 ml 2.5 ml -
4x stacking buffer - - 1.26 ml
10% SDS 100 µl 100 µl 50 µl
Deionized water 3.35 ml 4.85 ml 3 ml
10% APS 50 µl 50 µl 25 µl
TEMED, (Bio-Rad) 5 µl 5 µl 5 µl
Total 10 ml 10 ml 5 ml
4. Reagents for Western blotting
5x TBS (Tris-Buffered Saline) solution
200 mM Tris 12.10 g
1.5 mM NaCl 40.30 g
Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with distilled
water. Keep at 4ºC.
1x TBS solution
5x TBS 200.00 ml
Distilled water 800.00 ml
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TBST (Tris-Buffered Saline Tween-20) solution
5x TBS 200.00 ml
Distilled water 800.00 ml
0.1% Tween 20 1.00 ml
1x transfer blotting buffer (Prepare fresh buffer)
Tris 3.00 g
Glycine 14.40 g
Distilled water 800.00 ml
Methanol 200.00 ml
Destaining solution
Methanol 200.00 ml
Glacial acetic acid 100.00 ml
H2O 700.00 ml
Keep at room temperature.
Stripping solution [2.5 mM glycine, 1-2% SDS]
Glycine 0.9 g
SDS 10.00 g
Adjust pH to 2.0 with 1 M HCl and make volume up to 500 ml with
distilled water. Keep at 4ºC.
5. Reagents of DAPI staining
20 mg/ml DAPI solution (Stock solution)
Dissolve 1 mg of DAPI (Sigma, Cat.No. 9542) in 50 µl distilled water.
1 µg/ml DAPI solution
DAPI staining solution (Stock 20 mg/ml) 1.00 µl
Distilled water 20.00 ml
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80% Ethanol
Absolute ethanol 80.00 ml
Distilled water 20.00 ml
6. Reagents for DNA electrophoresis and staining solution
5x TBE buffer
Tris 53.90 g
Boric acid 27.50 g
EDTA 3.70 g
Add distilled water to a final volume of 1,000 ml
1x TBE buffer
10x TBE buffer 200.00 ml
Distilled water 800.00 ml
1.5% agarose gel
Agarose gel 1.50 g
1x TBE buffer 100.00 ml
DNA Staining solution
Ethidium bromide (10 mg/ml) 10.00 µ1
Distilled water 100.00 ml
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BIOGRAPHY
Name Miss Wipada Duangprompo
Date of Birth January 20, 1983
Educational Attainment
2004: Bachelor of Science (Medical Technology)
Faculty of Associated Medical Science
Khon Kaen University, Thailand
2007: Master of Science (Parasitology)
Department of Parasitology
Faculty of Medicine
Khon Kaen University, Thailand
Scholarship 2013: Research Grants of Thammasat University
for Ph.D. students
2015: TU Research Scholar, Contract No.79/2558
Publications
1. Duangprompo W, Aree K, Itharat A, Hansakul P. Effects of 5,6-dihydroxy-
2,4-dimethoxy-9,10-dihydrophenanthrene on G2/M cell cycle arrest and
apoptosis in human lung carcinoma A549 cell. The American Journal of
Chinese Medicine. 2016; 44 (7):1473-1490.
Oral/Poster presentation
1. Duangprompo W, Hansakul P. Antiproliferative and Apoptotic Effects of a
Novel 9, 10-Dihydrophenanthrene Isolated from Dioscorea membranacea in
the Human Non-Small Cell Lung Cancer cell line A549. The 18th World
Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for
Health and Wellness" December 1-3, 2014: Ubon Ratchathani, Thailand.
(Oral presentation).
2. Duangprompo W, Aree K, Itharat A, Hansakul P. Anticancer Effects of a
Novel 9, 10-Dihydrophenanthrene Isolated from Dioscorea membranacea
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161
Pierre in A549 cells. The 74th Annual Meeting of the Japanese Cancer
Association. October 8 –10, 2015: Nagoya Congress Center, Japan. (Poster
presentation).
3. Duangprompo W, Aree K, Itharat A, Hansakul P. Antiproliferative activity
of 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP) derived
from Dioscorea membranacea Pierre against A549 cells. The 5th
International Biochemistry and Molecular Biology Conference. May 26-27,
2016. B.P: Samila Beach Hotel, Songkhla, Thailand. (Poster presentation).