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EFFECT OF POTASSIUM KOETJAPATE, A
DRIVATIVE OF KOETJAPIC ACID
ISOLATED FROM SANDORICUM KOETJAPE
MERR. ON HUMAN COLORECTAL
CANCER.
by
SEYEDEH FATEMEH JAFARI
Thesis submitted in fulfillment of the requirements
for the degree of
Doctor of Philosophy
July 2018
brought to you by COREView metadata, citation and similar papers at core.ac.uk
provided by Repository@USM
DEDICATION
This thesis is dedicated to
the angels of my life, my parents,
Seyedeh Pouran Hashemi
and
Seyed Sadegh Jafari,
for their sincere love, valuable
encouragement and support
throughout my life.
ii
ACKNOWLEDGEMENT
First and Foremost, praise is to Allah, the Almighty, the most
merciful for giving me opportunity, knowledge, patient and strength to
undertake this research study.
I would like to express my special thanks to my research
supervisor, Assoc. Prof. Dr. Amin Malik Shah Abdul Majid for guiding and
supporting me during my research and my sincere thanks and gratitude to
Dr. Mohamed Khadeer Ahamed Basheer and Dr. Muhammad Asif, without
their help and incredible support throughout my research, this experimental
project would have never been successful. I would also like to express my
appreciation to my co-supervisor, Prof. Dr. Habibah A. Wahab for her kind
support.
I am deeply thankful to IPS (USM) for financial support during my
candidature (fellowshipP-FD-0082/11 R), School of Pharmaceutical
Sciences and EMAN Testing and Research Laboratory, USM, for providing
lab facilities.
Special thanks to my colleagues and friends, Dr. Muhammad Adnan
Iqbal, Dr. Fouad Saleih R. Al-Suede, Dr Zeyad D. Nassar, Hussein Bahri,
Soheila Farahani, Mohammad Ali Sarvghadi, Dr. Aman Malik Shah Abdul
Majid, Dr Syed Haroon Khalid, Dr. Loiy Elsir Ahmed Hassan, Armaghan
Shafaei, Shamsuddin Sultan Khan, Yasser Tabana, Norshirin Idris,
Sa’adiah Mohd Yusoff , Shazmin Kithur Mohamed, Hussein M Baharetha,
Mansoureh Nazari, Elham Farsi and Saad Sabbar Dahham for their kind
support and sincere help during my research.
iii
I owe my greatest appreciation to my family at Iran without their
continuous support, love and patience I would have never been able to
complete this project.
iv
TABLE OF CONTENT
ACKNOWLEDGEMENT ...................................................................... ii
TABLE OF CONTENT ........................................................................ iv
LIST OF TABLES ................................................................................ x
LIST OF FIGURES ............................................................................. xi
LIST OF ABBREVIATION ............................................................. xviii
ABSTRAK ........................................................................................xxii
ABSTRACT xxv
CHAPTER 1 - INTRODUCTION
1.1 Cancer ...................................................................................... 1
1.1.1 Colorectal carcinoma .................................................... 3
1.2 Major Signaling Pathways involving in Colon Cancer: ................ 6
1.2.1 Wnt/β-catenin Signaling Pathway: ................................. 6
1.2.2 Notch Signaling Pathway ............................................... 7
1.2.3 P53 Signaling Pathway .................................................. 8
1.2.4 Cell Cycle (pRB/ E2F) Signaling Pathway ...................... 9
1.2.5 NF-кB Signaling Pathway............................................ 10
1.2.6 Myc/Max Signaling Pathway ....................................... 11
1.2.7 Hypoxia pathway ........................................................ 12
1.2.8 MAPK Signaling Pathways .......................................... 14
1.3 Apoptosis ............................................................................... 16
1.3.1 Apoptotic Pathways .................................................... 17
1.3.1(a) Extrinsic Apoptotic Pathway ........................ 18
v
1.3.1(b) The Intrinsic Pathway of Apoptosis .............. 19
1.3.1(c) Perforin/granzyme Pathway .......................... 20
1.3.1(d) Execution Pathway ...................................... 23
1.3.2 Caspase Family ........................................................... 24
1.3.2(a) Caspase-3, -6, -7 .......................................... 25
1.3.2(b) Caspase-8 .................................................... 26
1.3.3 Apoptosis and Colon Cancer ........................................ 26
1.4 Cancer Cell Metastasis ............................................................ 29
1.5 Angiogenesis .......................................................................... 30
1.5.1 Mechanisms of Angiogenesis ....................................... 31
1.5.2 Vascular Endothelial Growth Factor (VEGF) ................ 35
1.5.3 Angiogenetic Inhibitors ............................................... 36
1.6 Anticancer Potential of Natural Products Isolated from Plants ... 38
1.7 Sandoricum koetjape ............................................................... 40
1.7.1 Characteristics of Sandoricum koetjape ........................ 40
1.7.2 Origin and Distribution ............................................... 41
1.7.3 Botanical Description: ................................................. 42
1.7.4 Traditional Medicinal Uses of S.koetjape ..................... 43
1.7.5 Phytochemistry and Pharmacological Activities of
S.koetjape ................................................................... 44
1.8 Triterpenoids as Potential Angiogenic and Cancer Inhibitors ..... 46
1.9 Pharmacological Activities of Koetjapic acid and Other Active
Triterpenoids from S.koetjape’s Bark Extracts .......................... 49
1.10 Solubility Enhanced Formulations ............................................ 51
1.11 Pharmacokinetic and Bioavailability study ............................... 52
1.12 Problem Statement and Objectives of Current Project ................ 53
vi
CHAPTER 2 - MATERIAL AND METHODS
2.1 Chemical and Reagents .................................................................. 57
2.2 Plant material ......................................................................... 61
2.3 Extraction and purification of koetjapic acid ............................ 61
2.4 Formation of potassium koetjapate (KKA) ................................ 63
2.5 Preparations of Solid Dispersions of Koetjapic Acid by Kneading
Method ................................................................................... 65
2.6 Chemical Characterization ....................................................... 65
2.6.1 Solubility studies ........................................................ 65
2.6.2 Differential scanning calorimetry studies ..................... 66
2.6.3 Fourier transform infrared spectroscopy studies ............ 66
2.6.4 HPLC analysis ............................................................ 66
2.7 Pharmacokinetics and Bioavailability of potassium koetjapate
(KKA) in Sprague Dawley Rats: .............................................. 67
2.8 In Vitro Anticancer studies: ..................................................... 69
2.8.1 Culture conditions and maintenance of Cell lines ......... 69
2.8.2 Harvesting and Counting of Cells ................................ 70
2.8.3 Assessment of Antiproliferative Effect of KKA by MTT
Assay ......................................................................... 71
2.9 Cell Death Studies .................................................................. 71
2.9.1 Determination of Nuclear Condensation Using Hoechst
33342 Stain ................................................................ 71
2.9.2 Detection of Mitochondrial Membrane Potential Using
Rhodamine 123 Stain .................................................. 72
2.9.3 Caspase Induction Assay ............................................. 72
2.9.4 Human Apoptosis Protein Profiler Array ...................... 73
2.9.5 Transmission Electron Microscope (TEM) .................... 75
2.10 Antimetastatic Studies ............................................................. 76
vii
2.10.1 Cell Invasion Assay ...................................................... 76
2.10.2 Migration Assay ........................................................... 77
2.10.3 Colony Formation Assay ............................................... 77
2.11 HCT 116 Hanging Drop Assay ................................................. 78
2.12 Transcription Factors Controlling 10 Major Cancer Signaling .... 79
2.13 Acute Toxicity Study In Rat .................................................... 80
2.14 In vivo Antitumor Studies ........................................................ 81
2.15 Anti-Angiogenic Studies ......................................................... 83
2.15.1 Assessment of Cell Viability by MTT Assay................... 83
2.15.2 EA.hy926 Cell Migration .............................................. 83
2.15.3 EA.hy926 Cell Invasion ................................................ 84
2.15.4 Tube Formation Assay .................................................. 84
2.15.5 Ex vivo Rat Aortic Ring Assay ....................................... 85
2.15.6 Assessment of VEGF Levels in HCT 116 Cells ............... 86
2.15.7 In vivo Matrigel Plug Assay .......................................... 86
2.16 Statistical Analysis ................................................................. 87
CHAPTER 3 - RESULTS AND DISCUSSION
3.1 Solubility Studies.................................................................... 88
3.2 Chemical Characterization of KKA .......................................... 89
3.2.1 FT-IR Spectroscopic Characterization of KKA ............. 89
3.2.2 FT-IR Spectroscopic Characterization of Solid Dispersions
of ............................................................................... 92
3.2.3 HPLC Analysis and Optimization of KA and KKA ........ 94
3.2.4 Differential Scanning Calorimetry ............................... 96
3.2.5 Characterization by FT-NMR spectroscopy................... 98
viii
3.3 Pharmacokinetics and Bioavailability of KKA in Sprague Dawley
Rats ....................................................................................... 99
3.4 In vitro Anticancer Activities ................................................ 107
3.4.1 Antiproliferative Effect of Koetjapic Acid Formulations on
Cell Proliferation ...................................................... 107
3.4.2 Cell Death Studies .................................................... 111
3.4.2(a) KKA Induces Morphological Modifications and
Nuclear Condensation in HCT 116 Cells ..... 111
3.4.2(b) KKA Reduces Mitochondrial Membrane
Potential in HCT 116 Cells ........................ 113
3.4.2(c) KKA Increase Caspase-8, -9 and -3/7 Levels in
HCT 116 ................................................... 116
3.4.2(d) Observation of Ultra-Structural Apoptotic
Morphology in HCT Cells Treated by KKA
Using a Transmission Electromicroscope
(TEM) ...................................................... 118
3.4.2(e) KKA Regulate the Expression of Multiple
Proteins in the Apoptotic Pathways ............ 119
3.5 Anti-metastatic Studies ......................................................... 122
3.5.1 KKA Inhibits Colon Cancer Cell Invasion .................. 122
3.5.2 KKA Inhibits Colon Cancer Cell Migration ................ 124
3.5.3 Inhibitory effect of KKA on clonogenicity of HCT 116
cells ......................................................................... 126
3.6 Potassium Koetjapate Inhibits Tumour Aggregation Property In
............................................................................................ 129
3.7 The Modulatory Effects of KKA on Carcinogenesis Signalling 132
3.8 LD50 Value of KKA in SD Rats .............................................. 133
3.9 Antitumor Activity of Potassium Koetjapate ........................... 134
3.10 Anti-Angiogenic Effect of KKA ............................................. 143
3.10.1 Effect of KKA on Cell Viability of EA.hy926
Cell Line .................................................................. 143
ix
3.10.2 KKA Prevent Blood Vessels Outgrowth in Rat Aortic
Ring Assay ............................................................... 145
3.10.3 KKA Inhibit EA.hy926 Wound Closure ...................... 147
3.10.4 KA Inhibits Differentiation of EA.hy926 cells on
Matrigel Matrix ........................................................ 149
3.10.5 Inhibition of Vascular Endothelial Growth Factor (VEGF)
Release .................................................................... 150
3.10.6 KKA Prevents Matrigel Induced Vasculature in Nude
Mice ........................................................................ 151
3.11 Discussion ............................................................................ 155
3.11.1 Develop formulation of KA with improved solubility
,characterization and assess its pharmacokinetic profile.
................................................................................ 155
3.11.2 Antiproliferative Effect of Koetjapic Acid Formulations on
Cell Proliferation ...................................................... 156
3.11.3 Pro-apoptotic and Anti-metastatic effects elicited by
Potassium koetjapate ................................................. 159
3.11.4 The Modulatory Effects of KKA on Carcinogenesis
Signalling Pathways at Transcriptional Level ............. 167
3.11.5 Preliminary safety profile and Antitumor Activity of
Potassium Koetjapate ................................................ 171
3.11.6 Anti-Angiogenic Effect of KKA ................................... 173
3.12 Summary .............................................................................. 176
CHAPTER 4 - GENERAL CONCLUSION
4.1 Conclusion ........................................................................... 178
4.2 Future Studies ....................................................................... 181
REFERENCES ................................................................................ 182
APPENDICES
LIST Of PUBLICATIONS
x
LIST OF TABLES
Page
Table 2.1 Chemical and Reagents
56
Table 2.2 Equipments and Apparatus
58
Table 3.1 Solubility Values Of Pure Koetjapic Acid,
Potassium Koetjapate (KKA) And The Solid
Dispersions Prepared With Various Carriers Via
Kneading Method In Water At 25°C
86
Table 3.2 The Selectivity Index (SI) Which Represents
IC50 For Normal Cell Line/IC50 For Cancerous
Cell Line After 48 Hours Of Treatment
95
Table 3.3 Effect Of KKA On The Expression Of Proteins
Involved In Apoptosis
96
Table 3.4 Subcutaneous Tumor Volumes In Different
Treatment Groups
97
Table 3.5 Changes In Body Weight Detected In Different
Treatment Groups
99
Table 3.6 Pharmacokinetic parameters of KKA in rat
plasma after oral administration) (n=6)
100
Table 3.7 The selectivity index (SI) which represents IC50
for normal cell line/IC50 for cancerous cell line
after 48 hours of treatment.
101
Table 3.8 Effect of KKA on the expression of proteins
involved in apoptosis
113
Table 3.9 Subcutaneous tumor volumes in different
treatment groups
128
Table 3.10 Changes in body weight detected in different
treatment groups
128
xi
LIST OF FIGURES
Page
Figure 1.1 Adenoma-Carcinoma Sequence, Adapted from
Qiagen (website)
6
Figure 1.2 Schematic Illustration Of Apoptosis. The Three
Pathways Of Apoptosis I.E. Extrinsic, Intrinsic
And Perforin/Granzyme Pathways. Adopted From
(Elmore, 2007)
19
Figure 1.3 TRAIL Death-Receptor Pathway Of Apoptosis
23
Figure 1.4 Steps Involved in Angiogenesis Adopted By
(Welti, Loges, Dimmeler, & Carmeliet, 2013).
35
Figure 1.5 Pictures of S.Koetjape Merr. Leaves, Flowers,
Fruits And Bark
44
Figure 1.6 Chemical Structure of Some Terpenoids Isolated
From The Bark
46
Figure 1.7 Chemical Structures of ursolic Acid And
oleanolic Acid
49
Figure 1.8 Chemical structure of synthetic triterpenoids with
strong antiangiogenic activity. Top: CDDO;
middle: CDDO-Me; bottom: CDDO-Im
50
Figure 2.1 Flow Sheet Diagram for Re-Crystallization
(Purification) of The Ka
61
Figure 2.2 Conversion of Koetjapic Acid Into Salt of
Potassium Koetjapate
85
Figure 3.1 Conversion of Koetjapic Acid Into Salt of
Potassium Koetjapate
88
Figure 3.2 FT-IR Spectra (Overlay) of Koetjapic Acid and
KKA
88
Figure 3.3 A Chemical Structures of Polyvinylpyrrolidone
and Koetjapic Acid(KA) B: FT-IR Spectra
(Overlay) of Koetjapic Acid and Solid
Dispersions of KA: PVP
89
Figure 3.4 HPLC Chromatograms Showing The Peaks of
Koetjapic Acid (a) and Potassium Koetjapate (b)
91
xii
Figure 3.5 The Thermograms of The Tested Samples
93
Figure 3.6 Chromatograms of blank rat plasma (A), and rat
plasma spiked with 10 µg/ml of KKA (B)
94
Figure 3.7 Calibration curve of KKA
94
Figure 3.8 Chromatograms of KKA in rat plasma at 2 hours
after oral administration of 50 mg/kg KKA (A);
rat plasma at 1 hour after intravenous
administration of 50 mg/kg KKA (B)
97
Figure 3.9 Mean plasma concentration vs. time profiles
(mean± S.E.M, n=6) of KKA after intravenous
administration at 50 mg/kg in rat plasma
98
Figure 3.10 Mean plasma concentration vs. time profiles
(mean± S.E.M, n=6) of KKA after oral
administration at 50 mg/kg in rat plasma
98
Figure 3.11 Photomicrographic images of HCT 116 cells
taken under converted phase-contrast microscope
at ×200 magnification using a digital camera at
48h.
102
Figure 3.12 Images of HT 29 Cells Taken Under an Inverted
Phase-Contrast Microscope at ×200
Magnification Using a Digital Camera At 48h.
103
Figure
3.13(a)
The Photomicrographs Depict the Images of HCT
116 Cells with Hoechst 33342 Stain Taken at 6
and 18 H after the Treatment with KKA. The
Arrows Indicate the Clear Signs of Nuclear
Condensation Including the Half-Moon
(Crescent)-Shaped Apoptotic Nuclei. The
Arrowheads Indicate the Apoptotic Cells,
Chromatin Dissolution, Breakdown and
Fragmentation.
105
Figure
3.13(b)
Graphical Representation of Percentage of
Apoptotic Indices. The Apoptotic Index for Each
Test Group Was Expressed as A Percentage of
The Ratio of Apoptotic Cells Number to The Total
Cell Number in 10 Different Fields. Values are
Presented as Mean ± Sd (N = 10), *Represents P
< 0.01 and **Represents.
106
xiii
Figure
3.14(a)
The Photomicrographs Demonstrate the Efficacy
of KKA in Disruption of The Mitochondrial
Membrane Potential. The Mitochondrial
Membrane Potential in HCT 116 Cells was
Evaluated by Visualizing the Uptake of The
Lipophilic Cation Dye Rhodamine 123 Into
Mitochondria. The Results Showed That the
Fluorescence Signal Decreased Drastically With
Respect to The Decrease of Mitochondrial
Membrane Potential Due to The Treatment With
KKA
107
Figure
3.14(b)
Graphical Representation of Percentage of
Apoptotic Indices.The Apoptotic Index for Each
Test Group Was Expressed as A Percentage of
The Ratio Of Unstained Cells Number to The
Total Cell Number in 10 Different Fields. Values
are Presented as Mean ± SD (N = 10), *Represents
P < 0.01 And **Representsp < 0.005.
108
Figure
3.15(a)
Induction of Caspase Activity in HCT 116 Cells
Treated with KKA and Betulinic Acid (5 Μm).
The Cells were Stained With The Green
Fluorescent FAM-VADFMK Dye After 8h Of
Treatment. The Photomicrograph Shows The
Fluorescence-Emitting Cells. The Images Were
Taken By EVOS Fluorescence Microscope At 20×
Magnification.
109
Figure
3.15(b)
Graphical Representation of The Per Cent
Induction of Caspase Activity in Representative
Group of Cells. Values are Presented as Mean ±
SD (N = 10), *Represents P < 0.01
And**Represents P < 0.005.
110
Figure 3.16 Ultrastructural Micrographs by TEM Reveal The
Apoptotic Property of KKA. Ultra-Structural
Micrograph of HCT After 24 H Treatment With
3.5 and 7 µm/Ml of the KKA. A Indicates
Untreated Cell That Received Distilled Water; It
Showed Normal Cell Morphology With Intact
Cell Membrane. The Treated Cells With 3.5 and 7
µm/Ml of The KKA Showed Related Apoptotic
Morphological Changes Such as Chromatin
Condensation, Fragmentation, Blebbing of Cell
Membrane and Formation Apoptotic Bodies (B-
E). Photos Were Taken at 1600x Magnification
(Scale Bar 5 µm).
111
xiv
Figure 3.17 Effect of KKA Treatment on The Expression of
Multiple Proteins Involved In The Cell Death
Cascade. Heatmap Represents Signal Intensities
of Each Protein in A Control and KKA Treatment
Group. Red Band in Cluster Diagram Shows Up-
Regulation While Green Band Indicate Down-
Regulation of Protein. Values Indicated Are Mean
± SD of Two Independent Experiments (N = 4 For
Each Protein). * = P < 0.05, ** = P < 0.01 And
*** = P < 0.001 Respectively
112
Figure 3.18 Photomicrographic Illustration of The Anti-
Invasive Activity KKA Against HCT 116 Cells.
The Cells Were Counted and The Results Are
Reported as The Average Percentage of Three
Independent Experiments (N = 3) in Term of
Invasion Inhibition Compared to The Negative
Control (Distilled Water). Values Shown are
Mean ± SD. *** Indicates P < 0.001: Significant
Difference of Invasion Inhibition at Different
Concentrations (2.5(A) And 5(C) µg/Ml) And
Positive Control 5-FU(D) Compared To The
Negative Control , Distilled Water,(B).
114
Figure 3.19 Wounds (Arrows) Of HCT Cells Treated With
The Concentration Of 2.5 And 5 µm/Ml Of KKA
And 5 µm/Ml Of 5-FU. At Zero, 6 And 18 H, And
Negative Control (Distilled Water). Both
Concentration Of KKA Show Significant
Inhibition At 6 And 18 H Compared To The
Negative Control (Distilled Water) Which Shows
Complete Wound Closure At 18 H. Photos Were
Taken At 4x Magnification (Scale Bar 1000 µm).
116
Figure 3.20 Percentage Of Inhibiting Migration Of HCT Cells
After 6h And 18 H Treatment KKA Prevented The
Migration Of HCT Cells Significantly At Two
Selected Doses Compared To The Negative
Control (Distilled Water). (*** P < 0.001).
Results Are Means ± SD Of Three Experiments.
117
Figure 3.21 (a) Effect of koetjapic acid, potassium koetjapate
and betulinic acid on colony formation of HCT
116 cells. The picture clearly depicts the dose -
dependent inhibition of HCT 116 colonies.
(b) Graphical representation depicts percentage
of plating efficiency of representative test groups.
Plating efficiency was determined by the percent
ratio of number of colonies developed to the
number of cells initially seeded.
119
xv
(c) Graphical representation illustrates the
percentage of surviving fraction obtained after
the treatment with koetjapic acid, potassium
koetjapate and betulinic acid. The percent
surviving fraction of HCT 116 colonies were
decreased with increasing concentration of
potassium koetjapate.
Figure 3.22 The Comparative Effects Of Potassium
Koetjapate On in vitro HCT 116 Tumour In
Hanging Drop Assay
122
Figure 3.23 Treatment With KKA (7 Μm/Ml) Significantly
Altered The Expression Of Multiple Cell
Signalling Pathways In HCT 116 Cells.
Significant Decrease In The Activity Of Notch,
Wnt, Hypoxia, MAPK/ERC And MAPK /JNK,
And Significant Up-Regulation Of Transcription
Factor For Cell Cycle (Prb-E2F) Pathway Was
Observed. While No Significant Changes
Detected In C-Myc,, P53, NF-Kb, And TGF-Β
Pathways. Error Bars Indicate Standard
Deviations From Mean. *P < 0.05, **P < 0.01 And
***P < 0.001
123
Figure 3.24 In Vivo Antitumor Efficacy Of KKA Determined
Using A Human Tumor Xenograft Model In
Athymic Nude Mice Bearing HCT-116 Tumors At
Day 21 Post- Inoculation.
127
Figure 3.25 Graphical Illustration Of Antitumor Effect Of
KKA In An Ectopic Xenograft Colon Cancer
Model. Values Shown Are Mean ± SD (N = = 5 –
6 Per Group). Tumor Size In The Treated Group
Was Compared To That In Negative Control
Group. *P Values < 0.05 Show Significant
Inhibition Of Tumor Growth.
129
Figure 3.28 Effect Of KKA And KA On Cell Viability Of
EA.Hy926 Cell Line. Values Exposed Are Mean
± SD Of Three Independent Experiments (n = 3)
132
Figure 3.29 Antiangiogenesis Effect Of Of KKA In The Rat
Aortic Ring Model. A = NC B = 12 µg/Ml, C = 25
µg/Ml, D = 50 µg/Ml, E = Betulinic Acid 20
µg/Ml F=100 µg/Ml Of KKA
133
xvi
Figure 3.30 Average Lengths Of Blood Vessels In KKA
Treatment And Control Groups Values Shown
Are Mean ± SD Of Three Independent
Experiments (n = 3). * = p < 0.05, ** = p < 0.01
And *** = P < 0.001 Show Significant Different
In Growth Inhibition Activity Of KKA And PC
(BA, 20 µg/Ml) Treated Groups With Negative
Control (Distilled Water).
134
Figure 3.31 Micrograph Illustration Of Anti -Migratory Effect
Of KKA Against EA.Hy926 Cells. Photos Were
Taken At 4 × Magnification
135
Figure 3.32 Graphical Representation Of The Dose And Time-
Dependent Inhibitory Effect Of KKA On
Migration Of EA.Hy926 Base On The Average
Distance Traveled By The Cells . ± SD (N = 3). *
= P < 0.05
136
Figure 3.33 Images of EA.hy926 Matrigel tube formation
assay. KA inhibits matrigel tube formation.
EA.hy926 were treated with (A) 10 µm/ml ,KKA
(B) 30 µm/ml , KA (C) 30 µm/ml ,KKA (D)
negative control(distilled water).
137
Figure 3.34 The Dose response relationship of KA on tube
formation assay, Values shown are mean ± SD of
three independent experiments (n = 3). * = p <
0.05, ** = p < 0.01 and *** = p < 0.001 show
significant activity of KKA and KA treated
groups on EA.hy926 tub formation inhibition
,compare to negative control (distilled water).
137
Figure 3.35 Effect Of KKA On The Release Of VEGF-A From
Human Colon Cancer HCT 116 Cells. Significant
Decrease In The Secretion Of VEGF Was
Detected In All The Treatment Groups. Values
Shown Are Mean ± SD Of Three Independent
Experiments (N = 3). Ns = * = P < 0.05, ** = P <
0.001 And *** = P < 0.001 Respectively.
138
xvii
Figure 3.36 In Vivo Anti-Angiogenic Activities Of KKA
Determined Using A Human Tumor Xenograft
Model In Athymic Nude Mice Bearing HCT-116
Tumors At Day 21 Post-Inoculation. KKA At
Different Doses Of 50 (B And B), 100 (C and c)
And 200 Mg/Kg (E and e)) Strongly Inhibited
Vascularization In Matrigel Plugs Implanted In
Nude Mice In Compare To Negative Control
Treated Group (A and a). Comparable With
Imatinib As PC At The Dose Of 100 Mg/Kg. (D
and d).
140
Figure 3.37 H&E Stained Cross-Sections Taken From
Matrigel Plugs Implanted Subcutaneously In
Representative Groups Of Animals. Arrows
Indicate Prompt And Well-Developed Blood
Vessels In The Control Group(A) In Compare
With Less Blood Vessels In Treated Groups: B
(KKA, Dose100 Mg/Kg) C: (PC, Imatinib 100
Mg/Kg), D: (KKA, 200 Mg/Kg).
141
Figure 3.38 Graphical Representation Of The Effect Of KKA
On The Mean Blood Vessel Count In Matrigel
Sections. (*** = P < 0.001, N = 6, Values Are
Mean ± SEM Of 10 Low Power Microscopic
Fields).
141
xviii
LIST OF ABBREVIATION
APC gene Adenomatous polyposis coli gene
LDH Lactate dehydrogenase
AIF Apoptosis inducing factor
Apaf-1 Apoptotic protease activating factor 1
BAG-1
BAK
Bax
Bcl-2 family anti-apoptotic protein
Bcl-2 homologous antagonist killer
Bcl-2 Associated-X Protein
Bcl-2
Bid
Bim
CAM
B-cell lymphoma-2
BH3 interacting-domain death agonist
Bcl-2-interacting mediator of cell death
Chick embryo chorioallantoic membrane
CC Column chromatography
CCRF-CEM
CDDO
CDDO-Me
CDDO-Im
Human lymphoblast leukemia cell line
2-cyano-3,12-dioxoolean-1,9-dien-28-oate
2-cyano-3,12-dioxoolean-1,9-dien-28-oate
methyl
2-cyano-3,12-dioxoolean-1,9-dien-28-oic
imidazolide
cFLIP Cellular FLICE-like inhibitory protein
cIAP Cellular inhibitor of apoptosis
CL-6
Cox2
Cholangiocarcinoma cells
Cyclooxygenase-2
CRC
CTL
Colorectal carcinoma
cytotoxic T lymphocytes
Cyt c
DCC
Cytochrome c
Deleted in Colorectal Carcinoma
DCFH-DA 2′,7′-Dichlorofluorescein diacetate
DcRs Decoy receptors
xix
DISC Death inducing signalling complex
DIABLO Direct inhibitor of apoptosis-binding protein
with low pI
DMSO
DNA
Dimethyl sulfoxide
Deoxyribonucleic acid
DPPH
DSC
EC
2,2 Diphenyl-1-picrylhydrazyl
Differentialtial scanning calorimetry
Endothelial cell nmr
ECM Extracellular matrix
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
ERK ½
EMT
EMSA
Extracellular-signal-regulated kinases
Epithelial–mesenchymal transition
Electrophoretic mobility shift assay
FADD Fas-associated death domains
Fas (CD95/Apo1) First apoptotic signal/ Cluster of differentiation
95/ Apoptosis antigen 1
FasL Fas Ligand
FBS Foetal bovine serum
FLIP FLICE like inhibitory protein
FRAP
FT-NMR
Ferric reducing antioxidant power
Fourier transform-nuclear magnetic resonance
GC-MS Gas chromatography mass spectrometer
Hep-2 Human laryngeal cancer cell line
HIF-1α Hypoxia inducible factor-1α
HREs Hypoxia response DNA elements
HSP Heat shock proteins
IAPs
IGF2
IGFBP-2
Inhibitors of apoptosis proteins
Insulin-like growth factor 2 receptor
Insulin like growth factor binding protein 2
xx
IGFBP-6 Insulin like growth factor binding protein 6
IκΒ Inhibitory κβ
K562
KA
KKA
Human erythroleukemia cells
Koetjapic acid
Potassium koetjape
KB Oral carcinoma cells
LD50
LEF1
Lethal dose to kill 50% of animals
Lymphoid enhancing factor-1
MAPK Mitogen activated protein kinases
MCF-7 Breast adenocarcinoma cell line
MMP Matixmetalloproteases
MOH Ministry of health
MOMP Mitochondrial outer membrane potential
MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide
MVD Microvessels density
NCR National cancer registry
NF-κB
NMR
Nuclear factor-κB
Nuclear magnetic resonance
NuMA Nuclear protein
PBS Phosphate buffer saline
PIGF Placental growth factor
pRb Retinoblastoma protein
PS
PVP
Phosphatidylserine
Polyvinylpyrrolidone
Smac
Rpm
RPMI-1640
Second mitochondria-derived activator of
caspases
Revolutions per minute
Roswell Park Memorial Institute-1640
ROS Reactive oxygen species
xxi
SD Sprague Dawley
SI
SIMPs
Selectivity index
Soluble intermembrane proteins
TCF T-cell factor
TGF
TGI
Transforming growth factor
Tumor growth inhibition
TLC Thin layer chromatography
TNF-R Tumour necrosis factor cell surface death
receptors
TRAIL Tumor necrosis factor-related apoptosis inducing
ligand
TRAILR1 (DR4) Death receptors 4
TRAILR2 (DR5) Death receptors 5
TS
USM
Tumor spheroid
Universiti Sains Malaysia
VEGF Vascular endothelial growth factor
VEGFR
WNT
VEGF receptors
Wingless integrated
xIAP X-linked inhibitor of apoptosis
xxii
KESAN KALIUM KOETJAPAT, SATU TERBITAN ASID
KOETJAPIK YANG DIASINGKAN DARIPADA SANDORICUM
KOETJAPE MERR. KE ATAS KANSER KOLOREKTAL MANUSIA.
ABSTRAK
Dalam kajian ini, usaha dilakukan untuk menambahbaik pelarutan
air acid koetjapik (KA) dan mengkaji efikasi anti - kanser kolonnya,
menggunakan metod-metod ‘in vitro’ dan ‘in vivo’. Garam kalium KA
iaitu, kalium koetjapat disediakan melalui metod separa -sintetik. Aktiviti-
aktiviti antikanser kalium koetjapat dibandingkan dengan aktiviti sebatian
asal iaitu, KA. Asai viabiliti sel MTT digunakan untuk mendapatkan dan
membandingkan nilai IC50 kedua-dua sebatian. Kesan-kesan pro-apoptotik
kalium koetjapat dinilaikan dengan menggunakan asai kaspas -kaspas (3/7,
8 dan 9), pewarna fluresen Hoechst 33342 dan Rhodamine 123. ‘Profiler
array’ protiom apoptosis manusia digunakan untuk mengenalpasti sasaran
protin yang bertanggungjawab bagi induksi apoptosis. Tambahan lagi,
kesan antitumor in vitro kalium koetjapat telah dikaji menggunakan asai
‘titik tergantung’. Tiga dos kalium koetjapat (25, 50, dan 100 mg/kg berat
badan) telah dikaji dalam model mencit bogel ‘athymic’ untuk mengkaji
efikasi in vivo anti-tumor kalium koetjapat. Dalam kajian ini pelbagai
formulasi KA telah disediakan. Kajian pelarutan menunjukkan bahawa
derivatif KA iaitu, kalium koetjapat, mempunyai pelarutan air lebih baik
daripada dispersi pepejal KA. Kajian antikanser in vitro menunjukkan
bahawa kalium koetjapat mempunyai aktiviti sitotoksik lebih baik daripada
KA dan kompleks dispersi pepejalnya, terhadap titisan sel HCT 116. asai
xxiii
pewarna fluresen menunjukkan bahawa kalium koetjapat mempunyai sifa t
menginduksi apoptosis. Ia menginduksi kondensasi kromatin dan
menurunkan potensi membran mitokondria secara kebergantungan dos.
Tambahan lagi, ia menaikkan tahap kaspas dalam sel -sel HCT 116.
Keputusan protin apoptosis ‘array’ menunjukkan bahawa kalium koetjapat
mempengaruhi aktiviti beberapa protin. Ia menurun-aturkan ekspresi
beberapa protin anti-apoptotik dan regulator negatif apoptosis termasuk
Bcl-2, HSP60, HSP90 dan IGF-1 dalam sel-sel HCT 116 dengan penaik-
aturan protin-protin TRAILR-1 dan TRAILR-2, CD40, IGFBP-6, p27,
kaspas 3 dan kaspas 8. Tambahan lagi, kalium koetjapat menunjukkan
kesan antimetastatik terhadap sel HCT 116 dalam asai -asai in vitro.
Keputusan-keputusan ini mungkin disebabkan oleh penurun-aturan laluan-
laluan signal Wnt, Notch, Hypoxia, MAPK/ERC dan MAPK/JNK dalam
sel-sel HCT 116, bersama dengan penaik-aturan faktor transkripsi untuk
laluan-laluan putaran sel (pRb-E2F). KKA juga menghalang proses
angiogenesis in vitro dengan menghalang proses -proses penembusan,
penghijrahan dan pembentukan dan pembentukan tiub sel -sel endothelium
. Kajian toksisiti akut menunjukkan bahawa kalium koetjapat mempunyai
LD50 lebih daripada 2000 mg/kg dalam tikus betina SD. Keputusan kajian
tumor ‘spheroid’ menunjukkan bahawa kalium koetjapat mempunyai
kebergantungan dos potensi antitumor dan data ini berkait dengan
keputusan kajian tumor in vivo. Kalium koetjapat menunjukkan perencatan
poten pembiakan tumor (68.15%, 82.35% dan 92.76%, pada 25, 50 dan
100mg/kg, secara berurutan). Keseluruhannya, keputusan kajian ini
xxiv
menunjukkan bahawa kalium koetjapat mempunyai aktiviti anti -kanser
terhadap kanser kolorektal.
xxv
EFFECT OF POTASSIUM KOETJAPATE, A DRIVATIVE OF
KOETJAPIC ACID ISOLATED FROM SANDORICUM KOETJAPE
MERR. ON HUMAN COLORECTAL CANCER.
ABSTRACT
In the present study an attempt was made to enhance the aqueous
solubility of KA and to study its anti -colon cancer efficacy using in vitro
and in vivo methods. Potassium salt of KA i.e., potassium koetjapate was
prepared by semi-synthetic method. Anticancer activities of potassium
koetjapate were compared with the native compound i.e., KA. MTT cell
viability assay was used to obtain and compare the IC 50 values of both the
compounds. Pro-apoptotic effects of potassium koetjapate were assessed
using caspases (3/7, 8 and 9), Hoechst 33342 and Rhodamine 123
fluorescent staining assays. Human apoptosis proteome profiler array was
used to identify the protein targets responsible for the induction of
apoptosis. Furthermore, in vitro antitumor effects of potassium koetjapate
were studied using hanging drop assay. Three doses of potassium
koetjapate (25, 50, and 100 mg/kg body weight) were tested in athymic
nude mice model to study the in vivo anti-tumorigenic efficacy of
potassium koetjapate. In this study, various formulations of KA were
prepared. Solubility studies revealed that resultant KA derivative i.e.
potassium koetjapate had better aqueous solubility than the solid
dispersions of KA. In vitro anticancer studies revealed that potassium
koetjapate has better cytotoxic activity than KA and its solid dispersion
complex towards HCT 116 cell line. Fluorescent staining assays showed
xxvi
that potassium koetjapate has apoptosis-inducing nature. It induced
chromatin condensation and decreased mitochondrial membrane potential
in a dose-dependent manner. Furthermore, it increased the levels of
caspases in HCT 116 cells. The results on apoptosis protein array show
that potassium koetjapate modulated the activity of multiple proteins. It
down-regulates the expression of multiple anti-apoptotic proteins and
negative regulators of apoptosis including Bcl -2, HSP60, HSP90 and IGF-
1 in HCT 116 cells with concomitant up-regulation of TRAILR-1 and
TRAILR-2, CD40, IGFBP-6, p27, Caspase 3 and caspase 8 proteins.
Furthermore, potassium koetjapate showed antimetastatic effect towards
HCT 116 cells in a series of in vitro assays. These results are probably due
to down regulation of Wnt, Notch, Hypoxia, MAPK/ERC and MAPK/JNK
signalling pathways in HCT 116 cells coupled with the up-regulation of
transcription factor for cell cycle (pRb-E2F) pathways. Moreover, KKA
inhibited angiogenesis in vitro by stopping endothelial cells
neovascularization, migration, tube formation and VEGF release. Acute
toxicity studies reveal that potassium koetjapate has LD50 more than 2000
mg/kg in female SD rats. Results of spheroid tumor studies show that
potassium koetjapate has dose-dependent antitumor potential and this data
correlates with the outcomes of the in vivo tumor studies. Potassium
koetjapate showed potent inhibition of tumor growth (68.15%,82.35 % and
92.76% at 25, 50 and 100mg/kg, respectively). Altogether, outcome of
present study shows that potassium koetjapate has good anti -cancer activity
towards colorectal cancer.
1
CHAPTER 1
INTRODUCTION
1.1 Cancer
A major global health issue, cancer is described as unrestrained
growth of cells leading to the invasion of local tissues and tumor metastasis
(1). Cancers are categorized by the type of cells from which the tumors
originate including carcinoma, sarcoma, blastoma, germ cell tumor,
lymphoma, leukemia and adenoma (benign tumor of glandular origin),
adenocarcinoma (malignant adenoma). Cancers de rived from epithelial
cells that include common cancers such as most types of breast, prostate,
lung and colorectal cancers are subsumed under carcinoma while sarcoma
cancer types occurring in connective tissues such as fat, bone, nerve, and
cartilage. Conditions such as leukemia and lymphoma start off from
hematopoietic whereas germ cell tumors which are mostly existing in the
testicle or the ovary result from pluripotent cells. Blastoma cancers
originate from immature “precursor” cells or embryonic tissue (2). In 2008,
lung, breast and colorectal cancers were reported to be the three most
commonly diagnosed cancers, while in case of cancer -associated
mortalities worldwide, lung, stomach, and liver cancers were found to be
the most common cancer types (3) Presently more than 200 kinds of cancer
have been recognized which are the second major cause of death
worldwide, overtaken only by heart disease(4). In 2015, cancers of breast,
colorectal, lung and prostate were estimated to be the most common causes
of cancer-related mortalities (5). According a recent report, cancer is now
2
the leading cause of death in 21 states in the USA, cancer is currently the
primary cause of death, due to exceptionally large reductions in deaths
from heart diseases. Although cancer-related death rate has decreased by
23% since 1991, or more than 1.7 mill ion deaths were prevented up to 2012,
death rates are increasing for cancers of the pancreas, liver, and uterine
corpus . In developing countries, there are more new cases than what is
documented in the developed countries (5,600,000 vs. 7,100,000 cases
respectively(6) . Cancer was found to be the third most common reason for
deaths overtaken by cardiovascular and septicaemia diseases in Malaysia
according to the Malaysian National Cancer Registry report in 2007.
According to another report, three most commonly diagnosed cancers in
Malaysia were breast, cervical and colorectal cancers followed by bone
marrow, lung, lymph node and liver cancers respectively while five most
commonly diagnosed cancers in the male population were lung, colorectal,
nasopharynx, prostate and lymphoma respectively. Whereas in the female
population, cancers of breast, colorectal, cervix, ovary and lung were at
the top list (7). A range of factors may result in an increase in the risk of
cancer. The causative factors involved in the progress of cancer include
smoking, obesity, exposure to chemicals, oxidative stress, and radiation as
external factors while inherited mutations (hyper-activation of oncogenes,
and inhibition of tumor-suppressing genes), metabolic deregulations,
hormone imbalance, and dysfunction of immune system are considered as
the main internal factors(8) . Surgery, chemotherapy and radiotherapy are
the most common treatments of cancer nowadays (9).
3
1.1.1 Colorectal carcinoma
Among various types of cancer, colorectal cancer (CRC), a
malignant tumor of large intest ine, ranks third in the world as a lethal and
metastatic carcinoma while the incidence and death rates of CRC has
decreased by around 3% per year in both men and women from 2003 up to
the end of 2012(5). Still, CRC is a significant cause of mortality in both
men and women worldwide. According to a survey, every year more than
945 000 people develop colon cancer out of which around 492 000 patients
die (10). in the Malaysian Peninsula, CRC is the most prevalent type of
cancer in men and the third most common cancer in women According to
the National Cancer Registry Report 2003-2005. The Age-Standardized
Rate (ASR) was highest among Chinese men, in whom it was more than
two times that of Indian and Malay men. Chinese women also had an ASR,
which was more than twice that of Indian and Malay women. Hypertension,
obesity, abnormal blood lipids, and high fasting blood glucose are
considered as main metabolic risk factors for colorectal cancer (11) . The
growth in colorectal tumor is due to the mutational activation of oncogenes
coupled with the mutational inactivation of tumor suppressor genes . Hence,
it is described as a multi-step disease that converts normal epithelial cells
of the colon into invasive carcinoma (Figure 1.1). An ordered series of
events, recognized as the “Adenoma -Carcinoma Sequence”, causes the
development of colorectal neoplasms. There must be at least four mutated
genes to form malignant tumors affected by few further chan ges leading to
benign tumorigenesis. The histopathologic changes occur due to genetic as
well as environmental factors. The most important environmental factors
4
include pathogen invasion, toxins, generation of ROS (Reactive Oxygen
Species) and stress conditions. Currently, the inherited and somatic genetic
deficiencies contributing to the development of colorectal carcinogenesis
have been discovered such as sustainable changes in
COX2 (Cyclooxygenase-2), KRas, Ctnn-Beta (Catenin-Beta), APC
(AdenomatousPolyposisColi),SMAD4 (Smaand MAD (MothersAgainstDe
capentaplegic), p53, TGF-BetaR2 (Transforming Growth Factor-Beta
Receptor-Type II), BAX (Bcl2 Associated-X Protein), E2F4 (E2F
Transcription Factor-4) and MMR (Mismatch Repair) genes
like, MSH2 (MutS Homolog-2), MSH3 (MutS Homolog-3), MSH6 (MutS
Homolog-6), MLH1 (MutL Homolog-1) and MMP (Matrix
Metalloproteinase)-1/2/7/9/11/12/14, loss of the 18q21 gene and
microsatellites instability (12-14). At the beginning, when the normal
colonic epithelia (Stage-0) is subject to unfavorable conditions such as
pathogenic invasions, APC and Ctnn-Beta mutations, toxins and generation
of ROS; it is changed into Dysplastic ACF (Aberrant Crypt Foci) or
Dysplastic Adenoma or Dysplast ic Epithelia (Stage-I) (15). An increase in
microsatellite repeat sequences along with KRas and COX2 mutations play
a major role in transforming Dysplastic epithelia to early adenoma phase
(Stage-II). KRas mutations occur in 39% of human colorectal cancers such
as KRas mutations combined with MSH2 (deletion) mutations DCC (point
mutation in 70% of colon cancers) and MLH1 (substitution, deletion and
hypermethylation of CpG sites), which lead to the transition fr om early
adenoma to late adenoma (Stage-III). Lastly, transitions from late adenoma
to increasingly tumor metastasis through colorectal carcinoma (Stage -IV))
5
comprise the less frequently targeted genes like BAX via frameshift
mutations which happen in over 50% of colon adenocarcinomas along with
deletion in E2F4, MSH3 and MSH6 fallowed by gene modifications of p53
and TGF-BetaR2, MMP1/MMP2/MMP7/MMP9/MMP11/MMP12 (over-
expression) and SMAD4 mutations (lack of alleles on chromosomes 17 and
18 in polyploid colorectal tumors). Particularly, point mutation on p53
gene is the main reason for causing 50% of colorectal cancers whereas
SMAD4 mutations (aneuploid/polyploid) are linked with metastatic
carcinoma (16, 17).
6
Figure 1.1: Adenoma-Carcinoma Sequence, Adapted from Qiagen (18)
1.2 Major Signaling Pathways involving in Colon Cancer:
1.2.1 Wnt/β-catenin Signaling Pathway:
The Wnt signal transduction pathway is an old pathway that is
evolutionarily conserved and has a key role in the development of the
embryo such as cell migration, cell polarity, along with a major role in cell
to cell signaling (19, 20). Mutations in the Wnt pathway have a fundamental
7
role in cancer development, especially in colon cancer. The Wnts consist of
a large family of nineteen glycoproteins in humans. So far, major signaling
branches which are downstream of the Fz receptor have been identified such
as canonical or Wnt/β-catenin dependent pathway and the non-canonical or
β-catenin independent pathway. The Planar Cell Polarity and the Wnt/Ca2+
pathways are two branches of Wnt/β-catenin dependent pathway which are
being actively dissected at the molecular and biochemical levels (21).
Around 90% of all colon cancer cases are related to the APC tumor
suppressor gene mutations, which trigger Wnt/β -catenin accumulation (22).
β-catenin and TCF/LEF transcription factors combine and consequently
boost the expression of c-Myc, cyclin D1 and MMP genes involved in
carcinogenesis and tumor angiogenesis(23) . Therefore, a possible objective
in curing different types of cancer, i.e. colon cancer, can be the down -
regulation of the Wnt pathway.
1.2.2 Notch Signaling Pathway
Notch signaling pathway can promote or suppress cell proliferation,
differentiation, death, and fate specification. This pathway significantly
interferes in some physiological programs such as apoptosis, adhesion,
migration and angiogenesis throughout adult tissue renewal leading to the
development of the organism. Because of its essential role in many
important processes, abnormal gain or loss of pathway components has
been directly linked to multiple human diseases including cancer (24, 25).
Notch can be an oncogene or a tumor suppressor gene based on factors such
as the timing, cell type, signal strength, and the n ormal function of certain
8
tissues (26) . Various types of cancer including colon, melanoma, renal,
breast, pancreas, and lung cancers have Notch pathway signaling (27, 28).
Some studies have shown strong relations between Notch and Wnt
pathways in colon cancer which strengthen the hypothesis that Notch
signaling might be in a downstream of Wnt (29-31). Thus, Notch signaling
pathway can be used in combination with Wnt inhibitors as a potential
treatment of colorectal carcinoma.
1.2.3 P53 Signaling Pathway
The P53 is a nuclear protein, which is known as “the guardian of the
genome” because of its role in the detection of genetic damage and in
triggering the genetic repair mechanisms(32). It can also trigger apoptosis
in case of irreparable DNA damage. When P53 is mutated, it will not be
able to perform its function as frequency (>50%) of mutation in the P53
gene is the most in human cancer. This indicates that the P53 tumor
suppressor gene has an a key role in cancer prevention is played by the P53
tumor suppressor gene through the cell cycle arrest mechanism which may
result in inducing apoptosis (33). Mutations in the P53 suppressor gene
are common in all cancers, which usually occur in the central DNA-binding
core domain. Mutations mostly hamper the protein’s ability to attach to its
target DNA strands, thus preventing transcriptional activation of the genes.
The P53 protein controls cell death using different mechanisms that result
in the regulation of genes entangled in both the extrinsic and intrinsic
pathways of apoptosis either via transcriptional -independent or
transcriptional-dependent mechanisms (34). Therefore, increasing the
9
amount of P53 could be a promising strategy in the treatment of cancers
with lower side effects. Recently, small molecules such as small peptides
have been used to reactivate the suppressed wild -type P53 or to return the
mutant into a wild-type P53 (35).
1.2.4 Cell Cycle (pRB/ E2F) Signaling Pathway
Retinoblastoma (Rb) is a malignancy of the developing retina, which
happens in children and is considered to be the most common malignancy
of the eye in children (36). The retinoblastoma tumor suppressor (pRB)
plays an important role in cell cycle processes and apoptosis. The pRB gene
is mutated in around 50% of all human tumors . Moreover, genes encoding
upstream regulators of pRB have been found to be mutated in the remaining
50% of all human tumors. About 60% of affected individuals have
unilateral Rb while around 40% have bilateral Rb Heritable retinoblastoma
is an autosomal dominant vulnerability to Rb RB1 was the first tumor
suppressor gene cloned. Recently, it has been reported that pRB can bind
with a series of transcription factors such as E2F and form dimmers tha t
control the expression of several downstream effector genes involved in
cell cycle control, mitosis, DNA repair and apoptosis (37). It appears that
RB1 interacts with more than 100 cell proteins resulting in regulation of
the critical G1 to S-phase transition in the cell cycle. It has also been found
that the Rb/E2F complex has the main role in maintaining G1 arrest in
connection with the p21/p27 family of cdk inhibitors (38).
10
1.2.5 NF-кB Signaling Pathway
The nuclear factor κB enhancer binding protein (NF -κB) family of
transcription factors, control the expression of a large group of genes
engaged in diverse cellular processes including the inflammation,
immunity, migration, adhesion, cell growth, apoptosis and cell survival.
Deregulated NF-κB has been linked to a variety of human diseases,
particularly cancers. The oncogenic role of NF-κB seems to be mediated
through its anti-apoptotic function, particularly through induction of Bcl -
XL. The NF- κB family consists of five DNA binding proteins as follows:
c-Rel, NF- κB 1/p50 NF- κB2/p52, RelA (p65) and RelB, which function
as various homodimers and heterodimers. A highly conserved 300 -
aminoacid-long N-terminal Rel homology domain (RHD) which is a
common domain in all five NF- κB proteins functions to dimerization, DNA
binding, interaction with the inhibitors of NF- κB as well as nuclear
translocation (39, 40). It was reported in several studies that NF-кB target
over 200 different genes such as Myc, Rel, and Cyclin D1-4 which are
engaged in cell cycle regulation, Bcl -2, Bcl-Xl, A1/Bf-1 which function in
the apoptosis process, VEGF gene which has a critical role in angiogenesis
process leading to the belief in the oncogenic potential of “normal” NF -kB
(39, 41, 42). It was also found that NF-kB signaling system has an
important role in bridging inflammation and cancer (39). Some new studies
reported on the identification of cancer-associated mutations in upstream
components of the IkB kinase -NF-kB (IKK-NF-kB) signaling system that
can result in cell autonomous activation of NF-kB in multiple myeloma
(43, 44) . Recently, Kojima and his group have reported that LPS increases
11
COX-2 expression in a certain colon carcinoma cell line through NF -kB
which is continuously activated in colorectal carcinoma tissue samples.
They also demonstrated that NF-kB is constitutively activated in colorectal
carcinoma tissues by using an electrophoretic mobility shift assay (EMSA)
andimmuno-histochemical staining. In addition, they found that NF-kB
activation is closely related to cancer progression(45) . New recent studies
suggest that activation of NF-κB appears to perform an essential role in
cancer development as it was reported that NF-κB is obviously activated
in 50% of CRC patients and those with colitis associated tumors and later
it has been established by mouse model studies that NF-κB functions
criticality in the development of Colitis -associated cancer (CAC) (40).
Recently researchers tried to synthesize compounds that target this
pathway such as cinnamaldehyde which was reported as an apoptosis
inducer agent acting via mitochondrial pathway, and hence it has been
found as a potent NF-кB pathway inhibitor. The essential roles taken by
this pathway in apoptosis inhibition and tumor maintenance suggest that
inhibitors of the pathway would be effective anti -cancer agents(45-47)
1.2.6 Myc/Max Signaling Pathway
Numerous studies demonstrated that a mutated version of Myc,
which is constitutively expressed, leads to the unregulated expression of
many genes, which result in diverse cancers. Myc has been reported to be
hyper-activated in 70% of all human cancer cases including colon, breast
and lung cancers while it acts as an angiogenesis switch as well (48, 49).
On the other hand, Myc/Max heterodimers induce intracellular transduction
12
pathways which are critical for induction of apoptosis (49) . It has been
found that Myc is activated via various mitogenic signals such as
MAPK/ERK pathway(50). A notable tumor shrinking in transgenic mice
was achieved by suppressing of the Myc/Max which resulted in targeting
this pathway(51). Another study demonstrated that c-Myc maintains
embryonal rhabdomyosarcoma (ERMS) transformed phenotype and radio -
resistance by safeguarding cancer cells from radiation-induced apoptosis
and DNA damage, while stimulating DNA repair induced by radiation. The
findings suggest that c-Myc targeting can be an effective treatment in
cancer therapy (48).
1.2.7 Hypoxia pathway
Aerobic energy metabolism processes such as oxidative
phosphorylation needs oxygen in eukaryotic cell while low oxygen
environments activate the hypoxia signaling pathways. Hypoxia signaling
dysfunction normally happens in conditions such as tumor angiogenesis
and chronic inflammation. Solid tumors frequently consist of hypoxic
regions. The cells in the core of tumor which is located too far away from
blood vessels become hypoxic. Being related to higher invasion risk and
metastasis, intratumoral hypoxia is more robust to chemotherapy and
radiation resulted in more patient mortality (52). As a transcription factor,
hypoxia inducible factor (HIF) causes most of the response in hypoxia
pathways. HIF-1 complex is a heterodimers protein comprising of an
exclusive subunit that is tightly expressed (HIF-1α) having 3 isoforms of
HIF-1α, HIF-2α, and HIF-3α and a regular constitutively-expressed beta
13
subunit (HIF-1β). The presence of oxygen provokes prolylhydroxylases to
hydroxylate HIF, resulting in the polyubiquitination and degradation of
HIF while under low oxygen conditions, prolylhydroxylase inhibition leads
HIF to accumulate which in its turn activates transcription of about 100
genes encoding proteins that mediate some major biological processes such
as angiogenesis, wound healing, invasion and metastasis, human
metabolism, chondrocyte survival in bone growth plates, autophagy and
cell death. Increased HIF-1α or HIF-2α levels are found in human colon,
breast, prostate and lung carcinomas, and are linked with increased patient
mortality. Some experimental data shows that hampering HIF-1 signaling
blocks tumor growth in mouse models(53, 54). Research has indicated that
in normal oxygen pressure, NF-κB (nuclear factor κB) directly modulates
HIF-1α expression. Investigation of siRNA (sma ll interfering RNA) as for
individual NF-κB members displayed differential effects on HIF -1α mRNA
levels, implying that NF-κB can control expression of normal HIF -1α
VEGF-A and Ang-2 genes which are associated with extreme tumor
angiogenesis and metastasis(52, 54). Another study has shown that HIF-1α
and its downstream target miR-210 is provoked by hypoxia blocking Bcl-2
expression and increasing autophagy, hence this triggers resistance to
radiotherapy in colon cancer cells. (55). The critical role of HIF in
regulating the expression of multiple genes involved in tumor metabolism
and angiogenesis makes it a potential target in cancer therapy. Hence,
greater anticancer effects may be achieved by obstruction of HIF -1.
Therefore, HIF inhibitors including the Klugine, Betulonic acid, phenethyl
14
isothiocyanate, taxol, and Acriflavine are still investigated for their anti -
cancer properties (53, 56).
1.2.8 MAPK Signaling Pathways
MAPK pathway is a term used for referring to a three kinases module
activated by phosphorylating one another sequentially as a reaction to
different types of stimuli including neurotransmitters, cellular stress,
cytokines, growth factors and cell adherencestimuli including
neurotransmitters, cellular stress, cytokines, growth factors and cell
adherence(57). This pathway uses one of the most generic singling designs
discovered in biological signal transductions. Mitogen -activated protein
kinases (MAPKs) are serine/threonine-specific protein kinases that were
broadly used during evolution in many physiological processes such as
gene expression, mitosis, growth control, cellular adaptation to chemical,
physical stress, metabolism, motility, cell differentiation and survival,
inflammation and apoptosis in all eukaryotic cells. There are 14 MAPKs in
mammals, which have been divided into seven groups. Conventional
MAPKs consist of the extracellular signal regulated kinases 1/2 (ERK1/2),
p38 isoforms (α, β, γ, and δ), c-Jun amino (N)-terminal kinases1/2/3
(JNK1/2/3), and ERK5(58) . The MAPK/JNKs and MAPK/ERKs are
involved in growth factor signaling, regulation of mitosis, migration and
apoptosis while MAPK/p38 play an important role in inflammation (59). It
was found that de-regulation of the above pathway resulted in various
diseases such as cancer, immunological disorders, degenerative and
inflammatory syndromes. As reported, MAPKs down regulate over 170
15
tumor suppressor genes including Tob1, JunD and Ddit3, which suppress
cell growth and proliferation (60). Phosphorylation activates the MAPK
pathway through a MAPKK/MKK (MAPK kinase), and that is
phosphorylated by a MAPKKK/MKKK (MAPKK kinase). It was also found
that MAPKs are disabled by several phosphatases including a preserved
family of phosphatases named MAP kinase phosphatases (MKPs). These
types of enzymes can hydrolyze the phosphate from phosphotyrosine and
the phosphothreonine remains. The deletion of each phosphate group
extremely reduces MAPK activity essentially giving up signaling. It has
been found that some types of tyrosine phosphatases take part in
inactivating MAP kinases including phosphatases such as STEP, HePTP,
and PTPRR in mammals (61, 62).
Most inducers of the MAPK pathway begin signaling via activating
receptors in the cell membrane, which result in activation of MAPKKK
typical through a small GTPase. There is a large number of known MAPK
agents including mostly of protein kinases, transcription factors, and
cytoskeletal proteins. When MAPK is activated it can transfer from the
cytoplasm to the nucleus, in which it controls gene transcription via
impacting the structure of chromatin and transforming transcription factors
activity (57, 62). It was reported that the activation of MAPK/ERK pathway
provoked cell cycle arrest and apoptosis and hence could be an effective
therapeutic target of different types of cancer such as osteosarcoma and
pancreatic cancer therefore serious attempts were made to produce
inhibitors of the ERK and JNK pathways and examine them in clinical trials
(58, 63).
16
1.3 Apoptosis
The apoptosis process was first described in 1972 as a distinct form
of cell death in term of morphology, whereas some characteristics of this
phenomenon were explained earlier (64). So far apoptosis has been
accepted as the most important type of genetically determined or
“programmed” death of cells that is involved in cell disposal. Nevertheless,
there are other types of programmed cell death which are also defined or
will be discovered in the future (65, 66). Generally, organisms having
multi-cells use two major methods for cell disposal: necrosis and apoptosis.
Necrosis can be caused due to the breaking apart of the plasmatic
membrane as a result of the formation of a swelling process. In contrast, in
apoptosis, chromatin is condensed followed by fragmentation and forming
apoptotic remains quickly engulfed by the macrophages hence this process
does not induce any inflammatory reaction (67). Apoptosis can be activated
by a variety of stimuli such as changes in the concentration of growth
factors, ionizing radiations, heat shock and other cellular stress, infection
by bacterial or viral particles, genetic mutations and damage of DNA (64,
67). Generally, cleavage of proteins, caspase cascade activation, changes
in cellular bioenergetics and membrane potential along with expression of
cell surface proteins which cause the early detection of apoptotic cells
followed by DNA fragmentation are considered as the main features of
apoptosis. Mitochondria perform a main role in mediating apoptosis by
releasing pro-apoptotic proteins such as cytochrome c, Smac, Omi and AIF
into the cytosol. Excessive apoptosis can result in the progress of acquired
immune deficiency syndrome (AIDS), renal damage, neurodegenerative
17
disorders and cardiac ischemia. On the other hand, reduced ap optosis can
result in development of cancer and autoimmune diseases (68, 69) .
1.3.1 Apoptotic Pathways
Apoptosis may be caused in mammals through three pathways based
on apoptosis regulators and the location where they act: the first pathway
is extrinsic pathway, started by the ligation of death receptors before the
activation of caspase-8 and processing extracellular death-inducing; the
second pathway is called intrinsic pathway, started by cell stress before the
activation of caspase-9, and the third type of pathway is called
Granzyme/Perforin pathway, which can trigger members of the caspase
family by processing of caspase zymogens. Nevertheless, granzyme A
functions in a caspase-independent way. Ultimately, the apoptosis
pathways lead to the execution pathway ending in cytomorphological
features of apoptosis suc cell h as chromatin condensation, shrinka ge,
cytoplasmic blebs formation prior to phagocytosing the apoptotic bodies
(64). A summarized schematic image of apoptosis pathways is illustrated
in Figure 1.2.
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Figure 1.2: Schematic illustration of apoptosis. The three pathways of
apoptosis i.e. extrinsic, intrinsic and perforin/granzyme pathways.
Adopted from (64).
1.3.1(a) Extrinsic Apoptotic Pathway
The extrinsic pathway or death receptor-mediated pathway is
triggered by binding of extra- cellular ligands with a family of tumor
necrosis factor death receptors which are located in the cell membrane. Fas
(fibroblast associated antigen) and tumour necrosis factor receptor (TNF -
R) are considered as typical death receptors which are involved in this
pathway. These receptors contain an extracellular cysteine -rich site in
order to bind the ligand, and an intracellular site for signal conduction.
FasL/FasR, TNF-alpha/TNFR1, Apo2L/DR4/DR5 and Apo3L/DR3 are
considered as the most prominent ligands and their corresponding receptors
19
of this pathway. Binding of Fas ligand to FADD (Fas-associating protein
with death domain) and the binding of TNF ligand to TNF receptor lead to
the binding of the adapter protein TRADD with complex of FADD and
RIP(64, 70) . Subsequently FADD binds to procaspase-8 through
dimerization of the death effector domain leading to formation of death -
inducing signaling complex (DISC). Next, DISC activates downstream
caspases 3 or other executioner caspases resulting in destruction of cellular
targets and apoptosis. Moreover, in certain cell types, BH3 -only protein
(Bid) is activated by caspase-8 resulting in truncated Bid (tBid).
Subsequently, tBid moves to the mitochondria and activates cytochrome c
release leading to activation of caspase-9 and caspase-3(67). The whole
process is described in Figure 1.3.
1.3.1(b) The Intrinsic Pathway of Apoptosis
The intrinsic pathway of apoptosis is initiated via non-receptor
mediated mitochondrial-stimuli that act directly on targets inside the cell
by producing intracellular signals which can be either positive or negative.
The lack of certain growth factors, cytokines and hormones create negative
signals followed by prevention of apoptosis. On the contrary, loss of
apoptotic suppression leads to activation of apoptosis via positive signals
such as radiation, oxidative stress, hypoxia, viral infections, and toxi ns
which result in Bax/Bak insertion into mitochondrial membrane. The
change in mitochondrial transmembrane by Bax/Bak is followed by loss of
the mitochondrial transmembrane potential and releasing of pro -apoptotic
proteins such as Cytochrome c into the cytosol from the intermembrane
20
space(64, 71) . Moreover, BH3-only proteins, such as Bid and Bim are
involved in homo-oligomerisation of Bax or Bak which induce their pro -
apoptotic function. Subsequently, Cytochrome c binds to the Apaf1 and
(d)ATP causing the connection of pro-caspase-9 to the complex and
forming the Apoptosome. Activated caspase-9 in turn induces caspase-3
and triggers proteolytic cascade. In contrast, anti -apoptotic Bcl-2 family
members, for example Bcl-2 and Bcl-XL, inhibit cytochrome c release,
probably via inhibition of Bax and Bak. Furthermore, mitochondria release
many other polypeptides such as AIF, endonuclease G, second
mitochondrial activator of caspases (Smac/Diablo) which can promote
caspase activation via suppressing the inhibitory effects of anti -apoptosis
proteins (IAPs) while AIF and Endo G create DNA damage and
condensation(67, 72). In case of apoptosis initiation via chemotherapeutic
agents, the mitochondrial pathway is more important than the death -
receptor pathway for example caspase 9–deficient cells and Apaf-1–
negative thymocytes are resistant to chemotherapeutic agents, however
they can be stimulated into apoptosis via Fas, TRAIL, or TNF (73).
1.3.1(c) Perforin/granzyme Pathway
Granzyme B (Gzm B) is a caspase-like serine protease that is
released by cytotoxic T lymphocytes (CTL) and natural ki ller (NK) cells
to kill virus-infected and tumor cells. Therefore, granzyme B plays a
significant role in human pathologies such as anti -viral immunity and
tumor immune surveillance. The serine proteases granzyme A and
granzyme B are the most important component within the granules which
21
have been examined in recent years (64, 74). Although caspase 3 was the
first substrate to be identified for Gzm B, other reports have shown that
Gzm B can stimulate several members of the caspase family of cysteine
proteases by proteolytic processing of their substrates (75) . Gzm B is able
to cleave proteins at critical aspartate residues, thereby stimulating pro -
caspase-10 which cleaves ICAD (Inhibitor of Caspase Activated DNAse).
Additionally, Gzm B uses the mitochondrial pathway to improve the death
signal through specific cleavage of Bid where this protein stimulates the
release of mitochondrial cytochrome c into the cytosol (76, 77) . Goping
and her colleagues also have shown that Gzm B can directly stimulate
caspase-3 resulting in the release of pro-apoptotic proteins that suppress
caspase inhibition and direct induction of the execution phase of ap optosis
which suggest that both the mitochondrial pathway and direct stimulation
of caspase-3 are essential for granzyme B-induced killing (75). Other
research indicates that death receptors and caspases do not play any role in
the apoptosis of activated T helper 2 cells induced by T cell receptors
because obstructing their ligands does not have any effect on apoptosis. In
contrast, adapter proteins with death domains, Fas -Fas ligand interaction,
and caspases are involved in apoptosis and regulating cytotoxic T helper 1
cells whereas granzyme B does not have any impact. Also granzyme A has
a major role in cytotoxic apoptosis induced by T cells and stimulation of
pathways that are independent from caspases. Granzyme A causes DNA
disintegration using DNAse NM23-H1. The nucleosome assemblage
protein SET inhibits the NM23-H1 gene. Granzyme A protease cuts the
SET complex and, therefore prevents the inhibition of NM23-H1, causing
22
apoptosis via degradation of DNA;; hence inactivation of this complex by
granzyme A probably results in apoptosis through hindering the DNA
maintenance and chromatin structure stability (64, 78).
Figure 1.3: TRAIL death-receptor pathway of apoptosis.
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1.3.1(d) Execution Pathway
Execution Pathway is considered the final pathway of apoptosis as
the extrinsic and intrinsic pathways both terminate at the execution phase.
Caspase-3, caspase-6, and caspase-7 function as effector or “executioner”
caspases, cleaving various substrates such as cytokeratins, PARP, the
nuclear protein NuMA and the plasma membrane cytoskeletal protein alpha
fodrin resulted in degradation of nuclear material and cytoskeletal proteins
that are followed by the major morphological and biochemical changes in
apoptotic cells(79). Execution caspases activation in the signaling cascade
is the apoptotic commitment point in which the cell kills itself (80) .
Caspase-3, as the main executioner caspase, is activated by any of the
initiator caspases (caspase-8, caspase-9, or caspase-10). Subsequently
caspase-3 cleaves ICAD (endonuclease CAD mixed with its inhibitor) to
release CAD which causes degradation of chromosomal DNA followed by
chromatin condensation. Moreover, caspase-3 causes cytoskeleton
rearrangement followed by disintegration of cells and formation of
apoptotic bodies(64). Screening the translation yields of small
complementary DNA pools recognized that gelsolin was a vital substrate
for caspase-3 while in Fas-activated cells it was cleaved in vivo in a
caspase-dependent manner. Caspase-3 cleaves gelsolin and its fragments
which cause cleavage of actin filaments in a calcium independent way.
Additionally, expression of the gelsolin cleavage yield in multiple cells
resulted in cell contraction and separation from the plate before
fragmentation of DNA and apoptosis induction, therefore cleaved gelsolin
is considered as an important physiological effector of morphologic change
24
during apoptosis (81) . Endonuclease-mediated DNA fragmentation and
apoptotic body formation are the final characteristic morphological
features of apoptosis. The findings show that the presence of
phosphotidylserine on the outer membrane leaflet, surface of apoptotic
cells, permits their early uptake and disposal with no release of cellular
constituents resulting in no inflammatory response (82).
1.3.2 Caspase Family
Caspases are intracellular proteases which are involved in
programmed cell death, inflammation, cell proliferation, survival and
differentiation. Activation of caspases is generated by a spec ific stimulus
through a conserved mechanism. After activation, caspases perform
proteolysis of downstream substrates to trigger a cascade of events that are
terminated in the desired biological response(83). In fact, some caspases
are vital for apoptosis but some are not required. Although aspartate at P1
position is generally necessary for all caspase substrates, different caspases
demonstrate different substrate preferences. Some caspases such as
caspase-8 and -10 have long pro-domains along with special motif like
DED while caspase-1, -2, -4, -5, -9, - 11 and -12 use domains (CARD)
which allow their responses to other proteins leading to signaling
pathways(67) . Caspases were divided into two simple groups: “apoptotic”
and “pro-inflammatory”, which was a helpful classification for several
years. However, most apoptotic members (caspase-2, -3, -6, -7, -8, -9, and
-10) are attributed to at least one non-apoptotic role. Likewise, “non -
apoptotic” candidates such as caspase -1, -4, and -5 are expected to
