Studies on the synergistic effects of combined treatment of … · 2017. 12. 18. · Malignant mesothelioma (MM), is an aggressive, treatment-resistant tumor, which is increasing

Studies on the synergistic effects of combined treatment of

gamma-tocotrienol and statin on human malignant

mesothelioma cells

2014

Guligena Tuerdi

1

Contents

List of abbreviations…………………………………….…………………………….…..2

Preface………………………………………………………………………………….…...4

Chapter1 Synergistic effect of combined treatment with gamma-tocotrienol and

statin on human malignant mesothelioma cells……………………………...8

Introduction…………………………………………………………………………….8

1.1 Cytotoxic effects of a combined treatment with gamma-T3 and statin…..…..9

1.1.1 Materials and methods…………………………………………………………9

1.1.2 Results……………………………………………………………………..…….12

1.2 Combination effects of on HMGR levels…………………………………….….…16

1.2.1 Materials and methods…………………………………………………..….…16

1.2.2 Results………………………………………………………………..……....….18

1.3 Antiproliferative effect of the mevalonate pathway and downstream

factors…………………………………………………………………………….....….20

1.3.1 Materials and methods………………………………………………..….…...20

1.3.2 Results……………………………………………………………………...…....22

1.4 Effect on intrinsic apoptosis ………………………………………………..…...…27

1.4.1 Materials and methods…………………………………………….…........…27

1.4.2 Results…………………………………………………………………....…..…29

1.5 Discussion……………………………………………………………………....…..…33

1.6 Summary………………………………………………………………………...….…37

Chapter2 Involvement of endoplasmic reticulum stress with the combination

effect of gamma-tocotrienol and statin……………………………..…….38

Introduction…………………………………………………………………..….…....38

2.1 Materials and methods………………………………………………………...……40

2.2 Results……………………………………………………………………………....…42

2.3 Discussion……………………………………………………………………………..50

2.4 Summary………………………………………………………………………………53

Conclusion ……………………………………………………………………..…….……54

References……………………………………………………………………………….…57

List of publication……………………………………………………………..……….…69

Acknowledgements……………………………….………………………….....….….…70

Examiners……………………………………………………………………....………....71

2

List of abbreviations

・APS : ammonium peroxodisulfate

・ATV : atorvastatin

・ASK1: apoptosis signaling regulation kinase

・BPB : bromophenol blue

・BSA : bovine serum albumine

・CDDP : cisplatin

・CHOP : C/EBP-homologous protein

・DMSO : dimethylsulfoxide

・DTT : dithiothreitol

・D.W : diluted water

・EDTA : ethylenediamine-N,N,N’,N’-tetraacetic acid

・ER : endoplasmic reticulum

・FACS : fluorescence-activated cell sorter

・FBS : fetal bovine serum

・FPP : farnesyl pyrophosphate

・GGPP : geranylgeranyl pyrophosphate

・GRP78 : glucose-regulated protein 78

・HMGR : 3-hydroxy-3-methylglutaryl-CoA reductase

・IPP : isopentenyl pyrophosphate

・IE : immuno enhancer

・JNK : c-jun N-terminal kinase

・MAPK : mitogen-activated protein kinase

・MVL : mevalonate

・PBS : phosphate buffered saline

・PCR : polymerase chain reaction

・PI : propidium iodide

・PVDF : polyvinylidene difluoride

・PMSF : phenylmethanesulfonyl fluoride

・RPL : ribosomal protein L

・RT : reverse transcription

・SDS : sodium dodecyl sulfate

・SMV : simvastatin

・Sal : salubrinal

3

・SP600125 : JNK inhibitor

・SREBPs : sterol regulatory element binding proteins

・T3 : tocotrienol

・TBS : tris bufferd saline

・TE : Tris-HCl + EDTA

・TEMED : N,N,N’,N’-tetramethyl-ethylenediamine

・TM : tunicamycin

・UPR : unfolded protein response

4

Preface

Cancer continues to be a worldwide killer; there are more than 20 different types

of cancer. In 2008 approximately 12.7 million cancers were diagnosed [1] and in

2010 nearly 7.98 million people died [2]. Cancer can develop from almost any type of

cell in the body. Cancers as a group account for approximately 13% of all deaths

each year with the most common being: lung cancer (1.4 million deaths), stomach

cancer (740,000 deaths), liver cancer (700,000 deaths), colorectal cancer (610,000

deaths), and breast cancer (460,000 deaths) [3]. Cancer is caused by both internal

factors such as inherited mutations, hormones, and immune conditions and

environmental/acquired factors such as tobacco, diet, radiation, and infectious

organisms; This makes invasive cancer the leading cause of death in the developed

world and the second leading cause of death in the developing world [1,3].

Malignant mesothelioma (MM), is an aggressive, treatment-resistant tumor, which

is increasing in frequency throughout the world [4]. Moreover, although MM is still

a rare cancer, its incidence is increasing in many countries [5–8]. From 1994 to 2008

the 15-year magnitude of mesothelioma is estimated to be 174,300 in a group of 56

countries that report the disease, and 38,900 for the same 15-year period by

extrapolation to a group of 33 countries that do not systematically report the

disease [9]. Key changes that occur during the development of the disease include

the loss of the normal restraints on proliferation and the acquisition of resistance to

apoptosis, and asbestos, a complete carcinogen for mesothelial cells, plays a complex

role in altering both proliferation and apoptosis [10].

MM patients have a history of asbestos exposure. Asbestos refers to a family of six

mineral fibers and is classified into two subgroups: (i) the amphiboles, a group of

rod-like fibers including amosite (brown asbestos), crocidolite (blue asbestos),

anthophyllite, actinolite and tremolite; (ii) the serpentine group, consisting of

chrysotile (white asbestos) [11], which promotes difficult disease 30-40 years after

the exposure [12]. There is a direct causal relationship between asbestos exposure

and the development of mesothelioma, with an etiological fraction of 80% or more

[4]. And several mechanisms might account for this finding that the asbestos fibers

stick out from the lung surface and cause repeated cycles of scratching, damage,

inflammation, and repair in the adjacent parietal mesothelial cell layer [13].

Although the association between MM and asbestos is well established, other

http://en.wikipedia.org/wiki/Diagnosis

http://en.wikipedia.org/wiki/Cancer#cite_note-Epi11-6

http://en.wikipedia.org/wiki/Cancer#cite_note-Loz2012-146

http://en.wikipedia.org/wiki/Lung_cancer

http://en.wikipedia.org/wiki/Stomach_cancer

http://en.wikipedia.org/wiki/Stomach_cancer

http://en.wikipedia.org/wiki/Liver_cancer

http://en.wikipedia.org/wiki/Colorectal_cancer

http://en.wikipedia.org/wiki/Breast_cancer

http://en.wikipedia.org/wiki/Cancer#cite_note-WHO-147

http://en.wikipedia.org/wiki/Developed_country

http://en.wikipedia.org/wiki/Developed_country

http://en.wikipedia.org/wiki/Developing_country

http://en.wikipedia.org/wiki/Cancer#cite_note-Epi11-6

5

carcinogenic factors must be involved in MM development because only 10% of all

MM cases occur in asbestos exposed subjects [14]. Simian virus 40 (SV40), a DNA

virus, has been implicated in the development of several cancers including MM [15].

That report showed that SV40 oncogenes could lead to malignant transformation of

mesothelial cells and used an in vitro mouse model mesothelial cells which were

more susceptible to asbestos induced apoptosis than normal mesothelial cells [15].

However, the putative involvement of SV40 in the pathogenesis of MM has become

a controversial issue, and its role remains unclear and unproved [13]. One study

showed that long-term exposure (100 days) to both SV40 and asbestos, primary

peritoneal mouse mesothelioma cells become resistant to stress induced senescence

[15]. On the other hand, occur by silencing promoter of the Cdk inhibitor p21 (p21)

through p53 inactivation by SV40 proteins and/or the weaker activation of p21 by

alternative pathways [16]. A greater reduction in p21 expression will cause greater

aggression and result in a poorer prognosis of human mesothelioma [16]. Therefore,

new therapeutic approaches are urgently needed for MM patients.

The anti-folate pemetrexed was the first licensed drug in combination with

cisplatin (CDDP) for malignant mesothelioma. Results were presented at the

Society of Clinical Oncology and the European Crohn’s and Colitis Organization in

2007 [12]. Pemetrexed is a potent inhibitor of thymidylate synthase, which is

required for DNA synthesis. A multicentre phase Ⅲ study in 448 patients

compared this drug combination with CDDP alone, and showed an improvement in

overall survival of nearly 3 months with the combination, with an objective

response rate of 41% [4]. The addition of folic acid and vitamin B12 supplementation

resulted in a significant reduction in pemetrexed related toxicity. Thus, pemetrexed

and CDDP is likely to be used widely as first-line chemotherapy for MM [17].

Vitamin E administration is an attractive alternative therapy for cancer patients.

Since the discovery of this fat-soluble vitamin in 1922, many studies have focused

on the potential health benefits and therapeutic use of α-, β-, δ- and γ-tocopherol

[18,19]. Similarly, γ-tocotrienol (γ-T3) (Fig.A), another isoform of vitamin E, induces

apoptosis in a variety of cancer cell types [20,21]. Additionally, γ-T3 has gained

much interest owing to its lipid-lowering effects in cell-based studies and especially

for its ability to lower cholesterol by inhibiting 3-hydroxymethyl-3-methylglutaryl

coenzyme A reductase (HMGR) [22]. Compared to other vitamin E isomers, γ-T3

has superiorperior abilities in anticancer, neuroprotective and cholesterol-lowering

activities [23,24]. Most recently, γ-T3 has exhibited anticancer activity in numerous

human cancers, including prostate [25], breast [26], colon [27], liver [28] cancers.

6

Statins are currently some of the most widely used pharmaceutical agents in the

world [29,30] which include atorvastatin (Fig.B), simvastatin (Fig.C), lovastatin,

and pravastatin. Their main function is to inhibit the endogenous synthesis of

cholesterol [29]; in addition to that, they appear to have pleiotropic effects such as

modulation of cell growth, apoptosis, and inflammation [31]. Statins are competitive

inhibitors of HMGR, which has been found to directly block tumor cell growth both

in vitro and in vivo [32], thus making it a unique molecular target for anticancer

therapy. Furthermore, statins have been found to induce apoptosis [33]. In most

clinical studies, the debate has centered on whether the use of statins causes

cognitive decline, diabetes mellitus [34], or cancers such as prostate [33], breast [35],

colorectal [31], liver [29], and pancreatic tumors [36]. Thus statins are attractive

therapeutic compounds for life style diseases, however, high concentration of statins

would induce severe side-effect. Clinical trials with prostate, breast, colorectal,

ovarian, and lung cancer patients determined that high-dose treatment was

associated with severe myopathy with the maximum dose of lovastatin that could be

tolerated to be 25 mg/kg, which produced a peak plasma concentration of 3.9 μM

[37]. Since the dose of lovastatin required to inhibit growth and apoptosis of cancer

cells in culture ranged between 0.1 and 100 μM, toxic doses of lovastatin must be

used to effectively treat cancer patients. Moreover, to be achieved apoptosis of

cancer cells, more than several tens μM of lovastatins was required. Therefore it

limits clinical use of statins though they possess strong anticancer effects.

The endoplasmic reticulum (ER) is an essential intracellular organelle with

multiple roles including the synthesis of nascent proteins, Ca2+ storage,

glycosylation, and the trafficking of newly-synthesized membrane and secretory

proteins. Perturbations of these processes have been demonstrated to interfere with

the proper functioning of ER, thus leading to a condition defined as ER stress [38].

Inadequate supplies of glucose affect the glycosylation of secretory pathway

proteins and ATP production, both of which lead to the accumulation of unfolded

proteins in the ER, resulting in ER stress [39]. The tumor microenvironment is

characterized by poor vascularization, low oxygen supply, and nutrient deprivation

all of which are activators of ER stress. In rapidly growing cancers, unfolded protein

response (UPR) has been shown to exert an important cytoprotective role that

assists folding of newly synthesized proteins necessary for tumor growth [40]. These

integrated mechanisms, although they may reflect tumor cell specificities related to

their metabolism or proliferation ability, could represent interesting avenue for

therapeutic strategy as their inhibition/activation would prevent tumor cell

7

adaptation to environmental challenges [41].

Thus, combination chemotherapy could optimize the effectiveness of each drug by

inciting a complimentary and synergetic therapeutic response while concurrently

reducing toxic adverse side effects associated with high-dose single-agent therapy.

In this context, the present study for the first time investigates the possible

synergistic effects of γ-T3 in combination with statin in human MM cell lines. As

estimated molecular targets, we concentrated on HMGR expression and the

following signaling (common targets of γ-T3 and statins), ER stress markers

(targets of γ-T3), and intrinsic apoptotic factors; Bcl-2 family in mitochondria and

caspase 3 (common targets of γ-T3 and statins).

Fig. A structural formula of tocotrienol

Fig. B structural formula of atorvastatin Fig. C structural formula of simvastatin

R1

HO

R2

R3

O CH3

CH3CH3 CH3 CH3

R1 R2 R3

α -tocotrienol CH3 CH3 CH3

β -tocotrienol CH3 H CH3

γ -tocotrienol H CH3 CH3

δ -tocotrienol H H CH3

２

NNH

OH3C CH3

O-

OOH OHHH

F

Ca2+

3H2O

CH3H

O

H3C

H

H

O

OOH

O

CH3H3C

H3C

8

Chapter1

Synergistic effect of combined treatment with gamma-tocotrienol

and statin on human malignant mesothelioma cells

Introduction

The product of HMGR, mevalonate (MVL), is the precursor for many important

intermediates in a pathway commonly known as “mevalonate pathway”. Inhibition

of HMGR leads to block the formation of many intermediate products such as

farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which

isoprenylate various proteins, including small G-proteins such as the Ras/Rho super

family proteins [42]. Ras communicates with a number of downstream targets

involved in cell function. Two targets, the PI3K/Akt pathway and the

Raf/MEK/MAPK/ERK pathways are involved in apoptosis and cell cycle progression.

Therefore, the cancer prevention activity reported for statin is believed to be due to

their inhibition of isoprenylation of G-proteins and subsequent alteration of

downstream signaling pathways. On the other hand, the mechanisms of action by

which γ-T3 and statins suppress HMGR activity are different [43]. In most cases,

the combination of γ-T3 with statin exhibits better anticancer activity than either

agent alone [26,27,44,45]. For example, combining low dose γ-T3 and statin was

shown to inhibit the growth of highly malignant mammary epithelial cells [44]. In

addition, atorvastatin (ATV) and γ-T3 exhibited a strong synergistic effect against

human colon cancer cells and caused cell cycle arrest and apoptosis [27]. Moreover,

both γ-T3 and statin also have reported to have HMGR-independent targets. For

example, statins inhibit the protein degradation machinery, specifically the

proteasomes. Inhibition of proteasome activity is likely to be related to the effect of

statins in G1 cell cycle arrest [27]. In general, statins exert their cancer prevention

effects by regulating several disease-associated cellular events including

inflammation, immunomodulation, angiogenesis, apoptosis and proliferation [26,27].

With regard to γ-T3, it has been well-studied about its endoplasmic reticulum (ER)

stress inducing effect. Wali et al. suggested that levels of HMGR (it is an

ER-transmembrane enzyme) decreased following γ-T3 treatment but it might not be

required for γ-T3 induced apoptosis, whereas ER stress apoptotic signaling was

associated with γ-T3-induced apoptosis in mammary tumor cells [20].

9

1.1Cytotoxic effects of a combined treatment with gamma-T3

and statin

In chapter 1-1, present study is the first to demonstrate the synergetic effect of

statins (atorvastatin; ATV and simvastatin; SMV) and gamma-tocotrienol (γ-T3) on

human malignant mesothelioma (MM) at 24 and 48 h.

1.1.1 Materials and methods

[Cell lines and culture conditions]

The human MM cell lines H2052 (a sarcomatoid), H28 (an epithelioid), H2452

(an epithelioid), and MSTO-211H (a biphasic; MSTO) cells were obtained from

the American Type Culture Collection (Rockville, MD, USA) and were cultured

in RPMI-1640 medium.

MSTO and H2052 cells were grown in medium containing 10% fetal bovine

serum (FBS) (Equitech-Bio, Kerrville, TX, USA), 0.5 units/mL of penicillin,

and 1 μg/mL of streptomycin.

H2452 and H28 cells were grown in the same medium supplemented with 4.5

g/L of glucose, 1 mM sodium pyruvate, and 10 mM HEPES buffer solution

(GIBCO, Life Technologies Japan Ltd., Minato-ku, Tokyo, Japan). All cell

lines were maintained at 37 °C in a fully humidified atmosphere of 5% CO2.

[Reagents]

All cultures and reagents were purchased from Sigma Chemical Company (St.

Louis, MO, USA) unless otherwise indicated. γ-T3 was a gift from Dr. Yano T (Toyo

University). Simvastatin (SMV) was purchased from Cayman Chemical Company

(Ann Arbor, MI, USA) and atorvastatin (ATV) was purchased from LKT

Laboratories, Inc. (St. Paul, MN, USA).

1) RPMI1640 (SIGMA)

2) FBS (EQUITECH-BIO, SIGMA)

3) Penicillin-Streptomycin (Antibiotics) (GIBCO)

4) 100mM Sodium Pyrvate (GIBCO)

5) HEPES Buffer Solution (1M) (GIBCO)

6) D-Glucose (GIBCO)

10

7) Dulbecco’s PBS(－) (Nissui Pharmaceutical Co.,Ltd)

8) 0.25% Trypsin-EDTA solution (SIGMA)

9) 0.4% Trypan blue solution (SIGMA)

10) Dimethyl sulfoxide(DMSO) (Wako)

11) Ethanol(Wako)

12) T3 content 39.3% of γ-T3

→γ-T3 is 3mM Was dissolved in FBS and ethanol.

13) Simvastatin (SMV) (Cayman Chemical)

14) Atorvastatin (ATV) (LKT Laboratories, Inc)

→Was dissolved in DMSO so that the 100mM.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (DOJINDO)

→MTT was dissolved in PBS so as to have a 5 mg / ml, was prepared at the

time to 0.25 mg / ml by using (1% Antibiotics) serum-free medium.

[Cytotoxicity assay (MTT assay)]

Cytotoxicity studies were performed in 96-well plates. The cells were cultured at a

density of 3.0 × 103 cells per well in 100 μL of RPMI-1640 medium for 24 h, then

started incubation with γ-T3, statin (ATV, SMV), combination of “γ-T3 + ATV” and

“γ-T3 + SMV”. After the cells were incubated for 24 or 48 h, 100 μL of MTT

solution (500 μg/mL) was added to each well and incubated for 1 h. Formazan

crystals were dissolved in DMSO (100 μL/well). The absorbance was measured at

540 nm with a Multiskan JX microplate reader (Thermo Lab Systems, Cheshire,

UK).

[Cell cycle analysis]

Cells were plated at a density of 2.0 × 105 cells in a 60-mm dish and incubated

for 24 h prior to treatment with γ-T3, statin (ATV, SMV), or a combination of γ-T3

and statin for 24 h. Thereafter, the cells were collected and fixed in 70% ice-cold

ethanol at −30 °C. Before the analysis, the cells were treated with 200 μg/mL

RNase A and 50 μg/mL of propidium iodide for 30 min in the dark at 37 °C. The

stained cells were analyzed using flow cytometry by using a MoFlo cell sorter (Dako

Cytomation, Kyoto, Japan).

[Statistical analysis]

We performed at least three independent experiments, and all data are presented

as mean ± standard deviation (SD) or standard error of the mean (SEM).

11

Statistical analysis was performed using the Tukey–Kramer test for the comparison

of multiple groups. Values with p < 0.05 were considered statistically significant.

12

1.1.2 Results

The cytotoxic effects of γ-T3 and statin either alone or in combination on human

MM cells were examined. As shown in Fig. 1.1.1, ATV (Fig. 1.1.1a) and SMV (Fig.

1.1.1b) decreased the cell viability of human MM cells; however, each statin alone

did not exert significant effects compared to the control. Compared to γ-T3 or statin

alone, the combination treatment significantly inhibited the growth of human MM

cells. Additionally, the combination treatment effect occurred in a time-dependent

manner. To examine the mechanism of the combination effect, MSTO and H2452

cells, which were especially sensitive to the combination among four cell lines, were

selected for the experiments that follow.

DNA damage causes cell cycle arrest. Cells with irreversible damage might

undergo apoptosis, causing an accumulation of cells in sub-G1 phase [46]. As shown

in Fig. 1.1.2, almost no phase shift was observed in MSTO cells that were treated

with γ-T3 (20 μM), ATV, or SMV (6.25 μM) alone for 24 h. On the other hand, the

sub-G1 population appeared to slightly increase in the group treated with a

combination of γ-T3 and statin. This population was also higher in groups treated

with ATV or SMV for 48 h than that in untreated control cells. Moreover, there was

a 3- to 8-fold increase in sub-G1 cells in groups treated for 48 h with a combination

of γ-T3 and ATV (or SMV) than in untreated control cells.

13

(a) γ-T3+ATV

14

Fig. 1.1.1 Effects of γ-T3 given alone or in combination with statins on human

MM cells. Cells (MSTO, H2452, H2052, and H28) were seeded in 96-well plates and

treated for 24 or 48 h with γ-T3 (20 μM) alone or in combination with (a) ATV (6.25

μM) or (b) SMV (6.25 μM). Cell viability was evaluated using the MTT assay. Data

are expressed as mean ± S.D of at least three independent experiments.

Significant differences between each of the indicated groups were determined using

the Tukey–Kramer test. **p < 0.01; ***p < 0.001.

(b) γ-T3+SMV

15

(a)

Fig. 1.1.2 Effects of combination treatment with γ-T3 and statins on the cell cycle

distribution in MSTO cells. Cells were treated with γ-T3 (20 μM) alone or in

combination with ATV (6.25 μM) or SMV (6.25 μM) for (a) 24 h or (b) 48 h. Each

parameter was determined using fluorescence-activated cell sorting (FACS)

analysis. Columns represent the mean value of a relative percentage of cells in each

cell cycle phase over the time course. Data were obtained from three independent

experiments.

control γT3 ATV γT3

+ATV

γT3

+SMV

SMV

control γT3 ATV γT3

+ATV

γT3

+SMV

SMV

(b)

16

1.2 Combination effects of on HMGR levels

In chapter 1-2, demonstrate the synergetic effect of statins (ATV and SMV) and

γ-T3 on human MM cells. Involvement of Mevalonate upstream pathway HMGR

mRNA expression.



Refer to 1.2.1 Materials and methods.

[Real-time RT-PCR analysis]

Total RNA was isolated using an RNeasy® Mini Kit (Qiagen, Valencia, CA, USA),

and cDNA was synthesized using a PrimeScript RT Reagent Kit (Takara, Shiga,

Japan). Real-time reverse transcription (RT) PCR was performed using the ABI

StepOne Real-time PCR System (Applied Biosystems Japan Ltd, Tokyo, Japan)

using SYBR® Premix ExTaq™ (TaKaRa Bio Inc., Shiga, Japan) according to the

manufacturer ’ s instructions. The sequences of primers used to amplify

3-hydroxy-3-methylglutaryl CoA reductase (HMGR), and ribosomal protein L32

(RPL32) are shown in Table 1. The PCR was performed at 95 ℃ for 10 s followed by

40 cycles of 95 °C for 5 s and 60 °C for 31 s. All data were normalized to the

internal standard RPL32.

17

＜Reverse transcription (RT) reaction＞

Reagents：PrimeScript RT reagent Kit (Perfect Real Time) (TaKaRa)

Equipment：PCR Thermal Cycler (TaKaRa)

Composition：

Volume（/tube）

①5×PrimeScript Buffer (for Real Time) 2 l

②PrimeScript RT Enzyme Mix I 0.5 l

③Oligo dT Primer 0.5 l

④Random 6 mers 0.5 l

RNA template 0.25 g

⑤RNase Free dH2O

Total 10 l

＜Real-time PCR＞

Reagents：SYBR Premix Ex Taq (Perfect Real Time) (TaKaRa)

Equipment：ABI StepOne Real Time PCR System (Applied Biosystems)

Composition： Volume（/well）

HMGR / RPL

SYBR Premix Ex Taq (2×) 10 l

Primer Mix（10 M） 0.4 l

ROX Reference Dye (50×) 0.4 l

D.W 7.2l

cDNA template 2 l

Total 20 l

Table1

Sequence of oligonucleotide primers used for real-time quantitative RT-PCR.

Primer Sequence

HMGR (F) 5’-TACCATGTCAGGGGTACGTC-3’ Tm=62.3℃

(R) 5’-CAAGCCTAGAGACATAATCATC-3’ Tm=57.0℃

<Product: 153bp>

RPL32 (F) 5’-CATCTCCTTCTCGGCATCA-3’ Tm=64.0℃

(R) 5’-AACCCTGTTGTCAATGCCTC-3’ Tm=63.9℃

RPL32 was used as an internal standard.

Transcription reaction

37℃ 15 min

85℃ 5 sec

4℃ ∞

Transcription reaction

95℃ 10 sec

95℃ 5 sec 40 cycle

60℃ 31 sec

18

1.2.2 Results

The effects of single treatment of γ-T3 (20 μM)/ATV (6.25 μM)/SMV (6.25 μM) or

combined treatment with γ-T3 and one of the statins on HMGR mRNA levels were

measured using real-time PCR. As shown in Fig. 1.2.1, ATV or SMV (6.25 μM) alone

significantly increased HMGR mRNA expression approximately 1.5-fold above the

control levels, although the effect of SMV in H2452 was not significant. On the other

hand, combined treatment with γ-T3 and ATV or SMV maintained the HMGR

expression as control level.

19

Fig. 1.2.1 Effects of γ-T3 and statins on HMG-CoA reductase (HMGR) mRNA

levels in MSTO and H2452 cells. Cells were treated with γ-T3 (20 μM) given alone

or in combination with ATV (6.25 μM) or SMV (6.25 μM). The mRNA levels were

determined using real-time RT-PCR after a 24 h incubation, and each mRNA level

was normalized to the mRNA level of RPL32. Data are expressed as mean ± SEM

from three independent experiments. Significant differences between each of the

indicated groups were determined using the Tukey–Kramer test. ***p < 0.001.

20

1.3 Antiproliferative effect of the mevalonate pathway and

downstream factors

In chapter 1-3, demonstrate the synergetic effect of statins (ATV and SMV) and

γ-T3 on human MM cells. Involvement of the mevalonate pathway downstream

intermediates, including FPP, GGPP and MVL. Then investigation of downstream

factors Akt and ERK protein expression.




[Reagents and antibodies]

P44/42 MAP Kinase (137F5), Phospho-p44/42 MAPK (Thr202/Tyr204), and

Rabbit monoclonal antibodies were obtained from Cell Signaling Technology Inc.

(Danvers, MA, USA). Anti-AKT (rabbit) polyclonal and Anti-AKT pS473 (mouse)

monoclonal antibodies were purchased from Rockland Immunochemicals

(Gilbertsville, PA, USA).

[Cytotoxicity assay (MTT assay) with intermediates of mevalonate

pathway]

Cytotoxicity studies were performed in 96-well plates. The cells were cultured at a

density of 3.0 × 103 cells per well in 100 μL of RPMI-1640 medium for 24 h, then

started incubation with γ-T3, statin (ATV, SMV), combination of “γ-T3 + ATV”, and

“γ-T3 + SMV”. After the cells were incubated for 24 or 48 h, 100 μL of MTT solution

(500 μg/mL) was added to each well and incubated for 1 h. Formazan crystals were

dissolved in DMSO (100 μL/well). The absorbance was measured at 540 nm with a

Multiskan JX microplate reader (Thermo Lab Systems, Cheshire, UK). Mevalonate

(MVL) pathway is one of the main and common targets for both γ-T3 and statins.

Therefore, to examine the involvement of this pathway with cytotoxic effect induced

by γ-T3 and statins, cells were then co-treated with intermediate metabolites of

MVL pathway (these compounds promote the pathway), such as farnesyl

pyrophosphate (FPP; 5 μM), geranylgeranyl pyrophosphate (GGPP; 5 μM), or MVL

(50 μM) in addition to γ-T3 and statins. High concentrations of MVL (100, 200 μM)

21

were also examined to elucidate if the effect is concentration dependent or not.

[Western blotting analysis]

Human MM cells were cultured at a density of 1.0 × 106 cells in a 60-mm dish for

24 h. The cells were then harvested and lysed in ice-cold lysis buffer (50 mM

Tris–HCl [pH 6.5], 10% glycerol, 10% β-mercaptoethanol, 0.5 mM phenylmethane

sulfonyl fluoride [PMSF] solution, 2% sodium dodecyl sulfate [SDS] [Wako], 1 mM

sodium orthovanadate, and 1% protease inhibitor cocktail). The cells were

incubated on ice for 20 min following centrifugation for 10 min at 12,000 rpm and

4 °C. Samples containing 20 μg of protein were electrophoresed through a 10%

SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF)

membrane (Atto Corp, Tokyo, Japan). The membranes were blocked with TBS-T

(13.7 mM NaCl, 2.5 mM Tris, 0.05% Tween20 [Wako]) containing 5% skim milk

(Yukijirushi, Tokyo, Japan) for 1 h, incubated with primary antibodies for 1 h, and

incubated with secondary antibody for 1 h. The antibody dilutions were as follows:

ERK (1:1000), P-ERK and AKT (1:2000), P-AKT (1:3000), and ß-actin (1:2000). All

antibodies were diluted with TBS-T containing 0.1% skim milk. ß-actin was used as

the internal standard. Detection was accomplished using Immobilon Western

Chemiluminescent HRP Substrate (Millipore, Tokyo, Japan) and an LAS-1000plus

Image Analyzing System (Fujifilm, Tokyo, Japan).


Refer to 1.1.1 statistical analysis.

22

1.3.2 Results

To examine whether cytotoxicity induced by γ-T3 and statin was involved in the

modulatory effects of the MVL pathway, the cells were cultured with γ-T3, ATV, and

SMV in the absence (control) or presence of 5 μM FPP, 5 μM GGPP, and 50 μM MVL

Cell viability was measured using the MTT assay. FPP did not have a significant

effect on the viability of MSTO or H2452 cells. On the other hand, the addition of

GGPP and MVL increased cell viability, which was more readily apparent at 48 h

than at 24 h in MSTO cells (Fig. 1.3.1a). Additionally, the viability of cells treated

with γ-T3, ATV, and SMV was examined in the absence or presence of 50 μM, 100

μM, and 200 μM MVL (Fig. 1.3.1b). The results suggest that MVL treatment

reversed the growth inhibitory effect of the combined treatment (γ-T3 with ATV or

SMV) in both MSTO and H2452 cells.

Oncogenes promote cell cycle progression (i.e., cell proliferation) via regulation of

intracellular signaling pathways. Two major pathways that are intimately involved

in the G1-to-S transition are the RAS-activated RAF-MEK-ERK and PI3-AKT

pathways. PI3K-AKT is known to impart a strong survival signal that potentially

raises the cellular threshold for apoptosis. Therefore, AKT is frequently activated in

MM cells [47]. However, γ-T3 and statin (either alone or in combination) did not

affect AKT phosphorylation (Fig. 1.3.2a). On the other hand, γ-T3 and statin (alone

or in combination) significantly inhibited ERK phosphorylation in MSTO cells (***:

p < 0.001). No effects were noted in H2452 cells. These results suggest that γ-T3 and

statin inhibit the downstream signaling of the MVL pathway, which partially

contributes to the inhibition of cell growth (Fig. 1.3.2b).

23

(a)

24

Fig. 1.3.1 Effect of combined treatment with γ-T3 and statins on the MVL

pathway in MSTO and H2452 cells. (a) Under stimulation with FPP (5 μM), GGPP

(5 μM), and MVL (50 μM), growth inhibitory effects induced by γ-T3, ATV, and SMV

were examined in MSTO and H2452 cells. (b) The effect of MVL (50–200 μM) on the

growth inhibition induced by γ-T3, ATV, and SMV in MSTO and H2452 cells was

examined. Cell viability was determined using the MTT assay. Data are expressed

as mean ± S.D from three independent experiments. In each treatment group,

significant differences from (a) no metabolites or (b) MVL 0 μM were determined

using the Tukey–Kramer’s test. *p < 0.05; **p < 0.01; ***p < 0.001.

(b)

25

p-Akt/Akt (MSTO)

(a) Akt activation

26

Fig. 1.3.2 Effects of a combined treatment with γ-T3 and statins on the expression

levels of growth factors located downstream of the MVL pathway in MSTO and

H2452 cells. Cells were treated with γ-T3 (20 μM), ATV (6.25 μM), or SMV (6.25 μM)

given alone or in combination for 24 h. Subsequently, (a) Akt activation and (b) ERK

activation levels were examined. Protein expression levels were determined using

western blotting analysis. The bar graph shows the ratio of phosphoprotein density

to total protein density. Data are expressed as mean ± SEM of four independent

experiments. ***p < 0.001 vs. control by the Tukey–Kramer test.

(b) ERK activation

27

1.4 Effect on intrinsic apoptosis

In chapter 1-4, demonstrate the synergetic effects of intrinsic apoptosis,

unchanged expression of Bcl-2 family proteins and activation of caspase 3 on statins

(ATV and/or SMV) and γ-T3 in human MM cells.




[Reagents and antibodies]

Bax (N-20), Purified mouse anti-Bcl-2 and monoclonal anti-ß-actin were obtained

from Sigma Chemical Company (St. Louis, MO, USA). Donkey anti-rabbit IgG,

HRP-conjugate and Goat anti-mouse IgG, HRP-conjugate were purchased from

Beckam Coulter (Tokyo, Japan).




It is the same as 1.3.1 Materials and methods except for antibodies’ condition.

The antibody dilutions were as follows: The antibody dilutions were as follows:

Bax (1:1000), Bcl-2 (1:500), and ß-actin (1:2000). Bax and ß-actin were diluted with

TBS-T containing 0.1% skim milk. Bcl-2 was diluted with Immuno Enhancer (IE).

ß-actin was used as the internal standard.

[Caspase 3 activity assay]

1.0 × 106 cells were cultured in 60-mm dishes for 24 h. Subsequently, the cells

were harvested and centrifuged, and the cell pellets were washed with 2 mL of PBS.

Then the caspase 3-activity assay was performed according to the manufacturer’s

protocol (Biovision, Mountain View, CA, USA). In brief, 100 μL of cold lysis buffer

was added to the cell pellets. Subsequently, the cell pellets were incubated for 10

min on ice, and the lysate was centrifuged at 10,000 rpm for 1 min at 4 °C. The

supernatant was transferred into a micro-centrifuge tube and mixed with the DC

28

Protein Assay Reagent (Bio-Rad, Tokyo, Japan). Supernatants (50 μL) were

dispensed into a 96-well plate (100 μg of protein/well). We then added 10 μL of DTT

to 1 mL of 2 × reaction buffer and dispensed 50 μL of this mixture into each well in

addition to 5 μL of 4 mM DEVD-pNA substrate. The plate was then incubated for

2 h at 37 °C. To detect caspase 3 activity, the absorbance was measured at 405 nm

with a microplate reader.


Refer to o 1.1.1 statistical analysis.

29

1.4.2 Results

The expression of Bax, a pro-apoptotic protein, did not change after γ-T3 or statin

treatment. Similarly, the expression of Bcl-2, another cell death-associated protein,

did not change (Fig. 1.4.1). On the other hand, a combination of γ-T3 and ATV or

SMV caused a strong induction of caspase 3 activity in both in MSTO and H2452

cells (Fig. 1.4.2). Treatment with ATV alone also strongly activated caspase 3 in

MSTO cells (Fig. 1.4.2a); therefore, the effect might be mainly due to statins. The

caspase 3 inhibitor Z-DEVD-FMK significantly inhibited the reduction in MSTO

cell viability in response to ATV treatment, although the cell viability was not

reversed in H2452 cells even after treatment with Z-DEVD-FMK (Fig. 1.4.2b).

30

Fig. 1.4.1 Effects of a combined treatment with γ-T3 and statins on the expression

levels of Bcl-2 and Bax proteins in MSTO and H2452 cells. Cells were treated with

γ-T3 (20 μM), ATV (6.25 μM), or SMV (6.25 μM) given alone or in combination for 24

h. Subsequently, Bax and Bcl-2 protein expression levels were examined. Each

protein expression level was determined and quantified using western blotting.

ß-actin was used as an internal standard. The lower columns indicate the Bax/Bcl-2

ratio, which illustrates the apoptotic balance. Data are expressed as mean ± SEM

of four independent experiments.

31

(a)

32

Fig. 1.4.2 Effects of a combined treatment with γ-T3 and statins on caspase 3

activity in MSTO and H2452 cells. Cells were treated with γ-T3 (20 μM), ATV

(6.25 μM), or SMV (6.25 μM) given alone or in combination for 24 h. Subsequently,

(a) the activity of casepase-3 in MSTO and H2452 cells was examined. Caspase 3

activity was detected as absorbance at 405 nm. (b) The effect of a caspase 3

inhibitor (Z-DEVD-FMK) on apoptosis induced by ATV in MSTO and H2452 cells

was analyzed. The cells were treated with ATV (6.25 μM) in the absence or

presence of Z-DEVD-FMK (2 μM). Cell viability was measured using the MTT

assay. ***p < 0.001 vs. control; ##p < 0.05 vs. ATV by the Tukey–Kramer test.

Data are expressed as mean ± S.D of three independent experiments. Cell

viability was measured using the MTT assay. ***p < 0.001 vs. control; ##p < 0.05

vs. ATV by the Tukey–Kramer test. Data are expressed as mean ± S.D of three

independent experiments.

(b)

33

1.5 Discussion

Malignant mesothelioma cells exhibit resistance to single-agent and combination

chemotherapy strategies, therefore successful chemotherapy is a key point for the

treatment of these patients [48]. Now pemetrexed and cisplatin combination

chemotherapy is the first-line treatment in inoperable disease with a median

survival of 9 – 12.4 months [49]. Despite notable improvement using this

combination therapy, disease recurrence and progression remain a problem. On the

other hand, one previous study addressing the efficacy of γ-T3 combination

treatment with cisplatin-induced cytotoxicity in human mesothelioma H28 cells in

culture has been reported [50]. Additionally, several in vitro [27] and in vivo [51]

studies support the anticancer effect of γ-T3 alone or in combination with statin.

Cytostatic effects were observed when 20 μM of γ-T3 was combined with 6.5 μM

of ATV or 6.5 μM of SMV (Fig. 1.1.1). Previous study has shown that combining

low-dose γ-T3 and statin induced cell cycle arrest at G1 in mammary tumor, leading

to a large reduction in cyclin D1 and a marked increase in p27 expression; however,

γ-T3 or statin alone had little or no effect on the distribution of cells in different cell

cycle phases compared with the combining group [52]. On the other hand, γ-T3

inhibited human gastric adenocarcinoma (SGC-7901) cell growth at the G0/G1

phase [53]. Our results indicate that the sub-G1 population is increased by

combination treatment with γ-T3 and statins, and such an increase is an indicator

of apoptosis. No phase shift was observed in MSTO cells treated with γ-T3 (20 μM),

ATV (6.25 μM), or SMV (6.25 μM) alone for 24 and 48 h (Fig. 1.1.2). These results

suggest that the effects of γ-T3 or statin on cell cycle distribution may be dependent

on cell types. Further studies are needed to confirm which phase is a major target.

It has been reported that the synergistic effects of γ-T3 and statin appear to result

from the cooperative action of γ-T3, which down-regulates HMGR mRNA expression,

and statins, which directly inhibit HMGR activity [43]. Several studies have been

performed to determine if the antiproliferative effects of combined statin and γ-T3

treatment result solely from the inhibition of HMGR or if additional

HMGR-independent mechanisms are involved [26,43]. In the present study, a

decreasing trend of HMGR mRNA expression was observed in both MSTO and

H2452 cells treated with γ-T3; however, this decrease was not significant compared

to control in both cell lines. On the other hand, statins alone increased the mRNA

expression, which was also shown in previous reports [54,55]. This statin-induced

increase was reversed by co-treatment with γ-T3 (Fig. 1.2.1). Previous reports

34

support our results: tocotrienols have been shown to reduce HMGR activity by

triggering (1) the retention of sterol regulatory element binding proteins (SREBPs),

which are membrane-bound transcription factors that activate all of the genes

required to produce cholesterol, in the ER and (2) the degradation of HMGR by

stimulating ubiquitination or reduction in mRNA level [45,56]. Moreover, the ability

of sub-effective doses of γ-T3 to synergistically enhance the growth inhibitory effects

of statins can be explained, at least in part, on the basis of the finding that statin

inhibition of HMGR activity is associated with a compensatory feedback mechanism

that results in the up-regulation of HMGR expression [44]. Therefore, synergistic

attenuation of HMGR activity via the application of the 2 compounds might

contribute to strong growth inhibition.

Next, to investigate whether the anticancer activity of combination (γ-T3 + statin)

therapy is mediated via inhibition of the MVL pathway, the effects of exogenous

intermediates of this pathway on cell growth were examined. The inhibition of

HMGR by statins leads to reduced production of cholesterol and isoprenoids as well

as downstream intermediates, including MVL (the first product of this pathway),

FPP, and GGPP [57]. Human farnesyl pyrophosphate synthase (hFPPS) is

responsible for the catalytic elongation of dimethylallyl pyrophosphate (DMAPP) to

FPP via the successive condensation of 2 isopentenyl pyrophosphate (IPP) units,

and it controls the intracellular levels of all downstream isoprenoids, including

GGPP [58]. Moreover, the angiostatic effects of statins at high therapeutic

concentrations are lipid-independent and are reversible by supplementation with

MVL and GGPP [59]. Both FPP and GGPP are important lipid attachments

required for the post-translational modification of a variety of small GTPase

proteins such as Ras and Rho GTPases. Thus, inhibitors of the MVL pathway might

have the opportunity to inhibit the invasion of cancer cells, as was reported for

HMGR inhibitors [60]. In the present study, the inhibition of cell growth by γ-T3

and statin was reversed after the addition of MVL or GGPP in a dose-dependent

manner (Fig. 1.3.1). Therefore, the synergistic antiproliferative effects of combined

γ-T3 and statin treatment may result directly from an inhibition of HMGR activity

and subsequent suppression of MVL synthesis.

The Raf/MEK/MAPK/ERK pathway and the PI3K/AKT pathway are important

downstream targets of activated Ras. ERK and AKT are vital growth factors

involved in cell cycle promotion and apoptosis regulation. We investigated whether

there was any change in protein expression among the treated cells; however,

neither the single nor the combination treatment altered AKT expression (Fig.

35

1.3.2). On the other hand, γ-T3 and statin (alone or in combination) significantly

inhibited ERK phosphorylation in MSTO cells (Fig. 1.3.2). Therefore, the growth

inhibition observed in the present study might mainly occur via ERK signaling. It

was previously shown that γ-T3 inhibits cell growth in sympathoadrenal (SA) +

mammary cells specifically by suppressing EGF-dependent Stat and AKT mitogenic

signaling [45]. This disparity in response to γ-T3 might be derived from the

different characteristics of each cancer type.

Indeed, AKT is often activated in both primary tumor and MM cell lines [47]. Our

results also showed positive AKT phosphorylation in all the cells, which was not

affected by any treatment. MMs with a positive AKT phosphorylation status have

also been shown to be phosphorylated (activated) mammalian targets of rapamycin

(mTOR), a molecule that functions downstream of the AKT pathway [47]. Moreover,

a PTEN homozygous deletion, which was also responsible for AKT activation, was

detected in a small subset of MM cell lines [61]. Based on these observations, one of

the small molecule inhibitors of receptor tyrosine kinases (RTKs), imatinib

mesylate was used in clinical trial for MM patients; however, a clear effectiveness

was not observed [62]. This lower-than-expected level of responsiveness was

thought to be due to the intrinsic resistance of MM cells against RTK inhibitor [47].

As a result of this acquired secondary resistance, the effectiveness of inhibitors

targeted against constitutive activation of a specific RTK is thought to be abrogated

via a new mutation in the RTK gene or the activation of another RTK. Taken

together, these results indicate that mono-target therapy will be ineffective in the

near future for aggressive cancer types such as MM.

Finally, the effects on the intrinsic apoptotic pathway were investigated. Statins

initiate apoptosis by suppressing Bcl-2, increasing Bax expression, and activating

caspase 3 [35]. The propensity of a cell to undergo apoptosis is finely balanced by

anti-apoptotic and pro-apoptotic factors. The Bcl-2 family has been shown to play an

important regulatory role in apoptosis either as an activator (i.e., Bax and Bak) or

as an inhibitor (i.e., Bcl-2 and Bcl-xL). The ratio of Bcl-2 to Bax proteins is

recognized as a key factor in the regulation of the apoptotic process or cell death [63].

Additionally, because the Bcl-2 family proteins are involved in most of the apoptosis

pathways, they are attractive targets for cancer therapy [64]. Our studies have

shown that Bax and Bcl-2 were expressed in both MSTO and H2452 cells, but they

were not affected by γ-T3 and statin. These results are consistent with a previous

report indicating that γ-T3 induced a mitochondrial disruption pathway without

affecting Bax/Bcl-2 expression in human breast cancer MDA-MB-231 cells [65].

36

MM cell lines and tumors were shown to express the Bcl-2 family members Bcl-xL

and Mcl-1 [66]. The Bcl-2 family is a group of more than 20 protein-coding genes.

Among these, the Bcl-xL protein is at least as effective as Bcl-2 for preventing

apoptosis in a variety of human tumors, including MM, when challenged with

pro-apoptotic stimuli [67]. On the other hand, we found that caspase 3 was

activated in human MM cell lines by the γ-T3 and statin combination treatment

(Fig. 1.4.1). Cao et al. showed that Bcl-2/Bcl-xL inhibitors, such as 2-methoxy

antimycin A3, increased caspase 3 activation without altering the protein

expression of members of the Bcl-2 family [68]. Therefore, the expression of Bcl-2

family members should be further examined, and functional assays should be

performed.

On the other hand, extent of caspase 3 reactivity induced by statins, especially

ATV, was the most different point between MSTO and H2452 cells (Fig. 1.4.1). In

this study, H2452 cells seems to response mildly to stains compared with MSTO

cells, for example, regarding to ERK activation (Fig. 1.3.2b), casepase 3 activation

(Fig. 1.4.1). The cause of such difference between cells did not clear out in this study,

however, caspase 3 original levels might be involved with the different responses.

Soini et al. showed that there was a tendency for better prognosis of mesotheliomas

from patients with moderate or intense caspase 3 immunoreativity as compared to

cases with negative or low caspase 3 [66]. They also indicated that another famous

apoptotic inducing factor, Fas ligand was not detected in the most drug resistant

mesothelioma cell line although Fas/Fas ligand has been suggested to participate in

the apoptosis in mesothelioma cells [69]. Additionally it is suggested that cell origin

difference might be involved to each responses in MSTO and H2452; MSTO is

derived from metastatic site (lung) of mesothelioma patients while H2452 is from

primary tumor though both cell lines are biphasic type. To explore the factor which

determines drug sensitivity, levels of caspase 3 and other apoptotic markers should

be examined and be compared between cell lines.

37

1.6 Summary

Combination with γ-T3 and statin induced apoptosis.

γ-T3 was down-regulated HMGR mRNA expression.

Growth inhibiting effect by γ-T3 and statin was reversed with MVL or GGPP

in a dose-dependent.

γ-T3 and statin alone or combination inhibited ERK phosphorylation.

Extent of caspase 3 activation was induced by statin, and combination with

γ-T3 further made further stronger activation.

38

Chapter2

Involvement of endoplasmic reticulum stress with the

combination effect of gamma-tocotrienol and statin

Introduction

The endoplasmic reticulum (ER) stress is defined as accumulation of unfolded or

misfolded proteins in the ER, which induces monitored and maintained via a

coordinated adaptive program, the unfolded protein response (UPR) [70]. The

biochemistry of the ER stress and UPR pathways has been the subject of many

recent reviews [71]. The investigation of ER-nuclear signaling transduction has

three signaling pathways [72]: (1) the presence of misfolded or unfolded protein in

the organelle (unfolded protein response, UPR). (2) The overloading of the ER with

correctly folded proteins (ER-overload proteins, EOR). (3) The starvation of

cholesterol (sterol regulatory element binding protein, SREBP).

There are three branches of UPR that are detected by distinct ER stress

transducers located on the ER membrane [73]: (1) PER-like endoplasmic reticulum

kinase (PERK), (2) inositol-requiring enzyme (IRE1), and (3) activating

transcription factor 6 (ATF6). Moreover, ER stress activates a large number of genes

involved in the control of cell fate, including antiapoptotic and proapoptotic

molecules like C/EB homologous protein (CHOP), c-Jun N-terminal kinases (JNK)

or members of the BCL-2 proteins family [74].

PERK is a type 1 transmembrane protein. This activates and phosphorylates

phosphorylated α subunit of eukaryotic initiation factor 2 (eIF2α) causing a

translation arrest [75]. IRE1α is an ER transmembrane sensor that activates the

UPR to maintain the ER and cellular function. Further understanding of IRE1α

substrate preferences will reveal how IRE1α coordinates cellular homeostasis to

determine cell fate under ER stress [76]. During ER stress, Ask1 is recruited to

oligomerized IRE1α complexes containing TRAF2, activating Ask1 kinase and

causing downstream activation of JNK and P38 MAPK [38]. Release of GRP78 from

N-terminus of ATF6 triggers different mechanism of protein activation, compared

with PERK and IRE1α [38].

In chapter 2, present study is the first to demonstrate the synergetic effect of

statins and γ-T3 on ER stress in human MM cells. ER stress was evaluated by

39

expression of typical ER stress markers such as CHOP, GRP78, caspase 4

apoptosis-related cysteine protease (caspase 4) and JNK signaling. Finally

involvement of caspase family was examined as well.

40

2.1 Materials and methods


The human MM cell lines H2452 (an epithelial), and MSTO-211H (a biphasic;

MSTO) cells were obtained from the American Type Culture Collection

(Rockville, MD, USA) and were cultured in RPMI-1640 medium.

MSTO cells were grown in medium containing 10% fetal bovine serum (FBS)

(Equitech-Bio, Kerrville, TX, USA), 0.5 units/mL of penicillin, and 1 μg/mL of

streptomycin.

H2452 cells were grown in the same medium supplemented with 4.5 g/L of

glucose, 1 mM sodium pyruvate, and 10 mM HEPES buffer solution (GIBCO,

Life Technologies Japan Ltd., Minato-ku, Tokyo, Japan). All cell lines were

maintained at 37 °C in a fully humidified atmosphere of 5% CO2.

[Reagents]

All cultures and reagents were purchased from Sigma Chemical Company (St.

Louis, MO, USA) unless otherwise indicated. γ-T3 was a gift from Dr. Yano T (Toyo

University). Simvastatin (SMV) was purchased from Cayman Chemical Company

(Ann Arbor, MI, USA) and atorvastatin (ATV) was purchased from LKT

Laboratories, Inc. (St. Paul, MN, USA).


Refer to Chapter 1, 1.1.1 Materials and methods.

Use the following agents to examine ER stress induced apoptosis:

Tunicamycin (0.125 μg/ML); ER stress inducer

Salubrinal (12.5 μM); Protector from ER stress-induced apoptosis

JNK inhibitor (SP600125; 2 μM)

Caspase inhibitors (2 μM) [caspase 8 inhibitor (Z-IETD-FMK), caspase 9

inhibitor (Z-LEHD-FMK), and pan caspase inhibitor (Z-VAD-FMK)] and

caspase 4 inhibitor (Z-LEVD-FMK; 10 μM)

[Real-time RT-PCR analysis for ER stress markers]

Refer to Chapter 1, 1.2.1 Materials and methods.

The sequences of primers used to amplify CHOP, Caspase 4, GRP78, and RPL32

(internal standard) are shown in Table 2. All data were normalized to the internal

41

standard RPL32.

Table 2

Sequence of oligonucleotide primers used for real-time quantitative RT-PCR.

Primer Sequence

RPL32 (F) 5’-CATCTCCTTCTCGGCATCA-3’ Tm=64.0℃

(R) 5’-AACCCTGTTGTCAATGCCTC-3’ Tm=63.9℃

<Product: 247bp>

GRP78 (F)5’-GCTCGACTCGAATTCCAAAG-3’ Tm=63.7℃

(R) 5’-TTTGTCAGGGGTCTTTCACC-3’ Tm=63.8℃

<Product: 331bp>

CHOP (F) 5’-GAGTCATTGCCTTTCTCCTTCG-3’ Tm=66.0℃

(R) 5’-TTTGATTCTTCCTCTTCATTTCCA-3’ Tm=64.5℃

<Product: 140bp>

Caspase 4 (F) 5’-CTGAAGGACAAACCCAAGGTCA-3’ Tm=67.2℃

(R) 5’-CACTTCCAAGGATGCTGGAGAG-3’ Tm=67.1℃

<Product: 96bp>

RPL32 was used as an internal standard.


Refer to Chapter 1, 1-3-1 Materials and methods.

The antibody dilutions were shown as follows: JNK (1:1000), pJNK, and ß-actin

(1:2000). All antibodies were diluted with TBS-T containing 0.1% skim milk. ß-actin

was used as the internal standard. Detection was accomplished using Immobilon

Western Chemiluminescent HRP Substrate (Millipore, Tokyo, Japan) and an

LAS-1000plus Image Analyzing System (Fujifilm, Tokyo, Japan).


Refer to Chapter 1, 1.1.1 statistical analysis.

42

2.2 Results

GRP78, CHOP, and caspase 4 are key molecules involved in the ER

stress-associated apoptosis pathway. Their mRNA expression levels were quantified

using real-time RT-PCR after 24 h of treatment with γ-T3 and statins given alone or

in combination. As shown in Fig. 2.2.1a and Fig. 2.2.1b, the expression levels of

GRP78 and CHOP in MSTO cells significantly increased from baseline in the

presence of γ-T3. ATV or SMV alone did not affect them at all. In H2452 cells, γ-T3

also increased GRP78 and CHOP expressions though it seemed milder compared

with those in MSTO cells (Fig. 2.2.1a and 2.2.1b). Although no differences were

observed in caspase 4 expression levels in MSTO cells, treatment with γ-T3 alone

caused a significant increase in the levels in H2452 cells (Fig. 2.2.1c). The response

to γ-T3 seems also relatively weak in H2452 regarding to ER stress markers, CHOP

(Fig. 2.2.1a) and GRP78 (Fig. 2.2.1b), although caspase 4 increase by γ-T3 was only

observed in H2452 cells. Additionally we examined if caspase 4 activation involves

the combination effect or not with caspase 4 inhibitor (Fig.2.2.2) but cell viability

after γ-T3 and statin treatment were unchanged both in presence or absence the

inhibitor.

Tunicamycin (TM) is an ER glycosylation inhibitor that impairs the synthesis of

proteins in the ER which induces apoptosis. And Salubrinal (Sal) is a selective

inhibitor of eIF2α dephosphorylation that was recently developed as a protective

agent against ER stress-mediated apoptosis [77]. Sal alone did not affect cell

viability so much whereas TM alone decreased the viability to almost 30% compared

with control (no treatment). In case of the combination with TM and Sal, cell

viability had been kept around 50%, which was significantly higher than in case of

TM alone (Fig.2.2.3a). On the other hand, γ-T3 seemed to exhibit similar effect like

TM; γT3 induced-growth inhibition was recovered with Sal although there was no

significant difference between γT3 alone and γT3 was combination Sal (Fig.2.2.3b).

It was considered that the growth inhibitory effect of γ-T3 might be partly occurred

via ER stress.

Next, as shown in Fig.2.2.4 γ-T3 and statin treated samples were probed by

western blot analysis for JNK and p-JNK. γ-T3 and ATV alone did not significantly

alter either JNK or p-JNK levels. On the other hand, p-JNK/JNK ratio was

significantly increased by γ-T3 and ATV combination comparing with control (p≺

43

0.05) in human MM cells.

Then we examined whether such as JNK activation could contribute to the

growth inhibitory effect or not, using JNK kinase inhibitor, SP600125 with γ-T3 and

statins. Contrary to our expectation, our results showed that SP600125 did not

significantly alter the cell viability in cells which were treated with γ-T3, ATV alone

or combination in neither MSTO nor H2452 cells (Fig.2.2.5).

To determine which caspase are required for the induction of apoptosis by γ-T3,

ATV alone or combination, cells were treated with caspase 8 inhibitor

(Z-IETD-FMK), caspase 9 inhibitor (Z-LEHD-FMK) or pan caspase inhibitor

(Z-VAD-FMK) at a concentration of 2 μM. Any of caspase inhibitors did not block

cell growth inhibition induced by γ-T3 and statins in MSTO and H2452 cells

(Fig.2.2.6).

44

Fig. 2.2.1 Effects of combined treatment with γ-T3 and statins on the relative

mRNA levels of ER stress-associated factors in MSTO and H2452 cells. Cells were

treated with γ-T3 (20 μM), ATV (6.25 μM), or SMV (6.25 μM) given alone or in

combination for 24 h. Subsequently, (a) CHOP, (b) GRP78, and (c) caspase 4 mRNA

expression levels were analyzed. Each mRNA expression level was determined

using real-time RT-PCR and quantified. The expression levels of each gene were

normalized to the expression level of RPL32. Data are expressed as mean ± SEM

of at least three independent experiments. ***p < 0.001; **p < 0.01 vs. control by

the Tukey–Kramer test.

MSTO

H2452

(a) CHOP

(b) GRP78

(c) Caspase 4

45

Fig.2.2.2 Effects of caspase 4 inhibitor (Z-LEVD-FMK; 10 μM) treatment with

γ-T3 (20 μM) and ATV (6.25 μM) alone or combination in MSTO and H2452 cells for

24 h. Cell viabilities were determined by MTT assay. Data are expressed as mean

±S.D of at least three independent experiments.

0

20

40

60

80

100

120

control γT3 ATV γT3+ATV

Ce

ll v

iab

ilit

y (

% o

f c

on

tro

l)

MSTO

0

20

40

60

80

100

120

control γT3 ATV γT3+ ATV

Ce

ll v

iab

ilit

y (

% o

f c

on

tro

l) H2452

Caspase4 inhibitor (-)

Caspase4 inhibitor (+)

46

Fig.2.2.3 The effects of Salubrinal (Sal) (12.5 μM), Tunicamycin (TM) (0.125 μg/ml)

and γ-T3 (50 μM) given alone and in combination of each other in MSTO cells for 24

h. (a) TM induced ER stress (b) γ-T3 induced ER stress. Cell viabilities were

determined by MTT assay. Data are expressed as mean ±S.E. of at least three

independent experiments. ***: p<0.001, **: p<0.01 vs. control by Tukey-kramer’s

test.

(a)

TM induced ER stress

(b)

γ-T3 induced ER stress

47

Fig.2.2.4 Effects of combined treatment with γ-T3 (20 μM), and statins (6.25 μM)

on the relative expressions of JNK and phosphorylated-JNK (p-JNK) protein in

MSTO and H2452 cells. p-JNK/JNK ratio was normalized to control in MSTO and

H2452 cells. Each protein level was determined by western blotting. Data are

expressed as mean ± S.D of at least three independent experiments. *:p<0.05 vs.

control by Tukey-Kramer’s test.

48

Fig.2.2.5 Effects of JNK inhibitor (SP600125; 2 μM) treatment with γ-T3 and ATV

alone or combination in MSTO and H2452 cells for 24 h. Cell viabilities were

determined by MTT assay. Data are expressed as mean ±S.D of at least three

independent experiments. ***: p<0.001 vs. control by Tukey-kramer’s test.

49

Fig.2.2.6 Contribution of caspase family to cell growth inhibition of γ-T3 and ATV

in MSTO and H2452 cells. Cells were seeded in 96-well plates and treated for 48 h

with γ-T3 (20 μM), ATV (6.25 μM) alone or in combination with caspase 8 inhibitor

(Z-IETD-FMK), caspase 9 inhibitor (Z-LEHD-FMK) and pan caspase (Z-VAD-FMK)

(2 μM). Cell viability was evaluated using the MTT assay. Data are expressed as

mean ± S.D of at least three independent experiments. ***: p<0.001 vs. control, ###:

p<0.001 vs. γ-T3 or ATV Tukey-Kramer’s test.

50

2.3 Discussion

In this chapter, we examined the involvement of ER stress with the synergistic

effect of γ-T3 and statins. ER stress is another expected target of γ-T3. Until now,

several apoptotic pathways have been identified in cells that are responsive to

cytotoxic insult, including apoptosis mediated by the activation of death receptors

(extrinsic), mitochondria-dependent signaling (intrinsic), and ER-induced apoptotic

cell death. In many instances, deregulation of ER homeostasis has been correlated

with pathologic states and particularly with cancer [78]. ER stress can lead to

adaptive responses or apoptosis, both of which follow activation of the UPR [79].

The UPR regulator GRP78, an ER chaperone, induces ER stress and is widely

up-regulated in cancer [51]. Similarly, the activation of CHOP and caspase 4 is a key

step in ER stress-induced apoptosis [80]. In fact, γ-T3 induced apoptosis may be

associated with induction of early response genes and the transcriptional response

to ER stress in breast cancer cells [81]. In the present study, the observed

up-regulation of GRP78 and CHOP led us to further investigate the involvement of

ER stress in γ-T3-induced apoptosis. We found that the expression levels of GRP78

and CHOP were unchanged by treatment with ATV or SMV alone (Fig. 2.2.1a and

Fig. 2.2.1b). It indicates that statins in the present study did not affect ER stress,

and also that statins did not inhibit γ-T3 -induced ER stress which might lead the

following apoptosis. Therefore, this supports the idea that γ-T3 enhances

statin-induced apoptosis. Although caspase 4 is also a known critical mediator of

programmed cell death [82], we found that the caspase 4 level was altered only by

treatment with γ-T3 alone in H2452 cells (Fig. 2.2.1c). Result of MTT assay using

caspase 4 inhibitor also suggested it was not significant factor for growth inhibitory

effect of γ-T3 and statins in the present study (Fig. 2.2.2).

The TM inhibits protein glycosylation in the ER, there by inducing ER stress.

Apoptosis is induced by severe and prolonged ER stress [77]. Salubrinal protected

MSTO cells from cytotoxicity induced by TM, as demonstrated by increased cell

viability. A recent study has shown that phosphorylation of eIF2α protected cells

from ER stress [83]. And TM is the most commonly used pharmacological agent to

experimentally induce ER stress [84]. As shown in Fig.2.2.3b, γ-T3 induced

cytotoxicity was slightly recovered by salubrinal which suggests the ER stress has a

role in γ-T3-induced growth inhibition; however, it was not significant change.

Another assay which detects apoptosis from the different aspect, such as

flowcytometry and TUNEL assay, will support to understand the role of ER stress

51

in the present case.

The, JNK belong to the mitogen-activated protein kinase (MAPK) family. JNK

studies provided a mechanistic link between stress and growth factor [85]. Moreover,

JNK is important role in cell stress reactions and being activated by many

extracellular stress signals, JNK is also called stress activated protein kinase

(SAPK), and is involved in stress reaction and cell death [86]. Our results indicated

that p-JNK/JNK ratio was significantly increased by γ-T3 combination with ATV,

but not affected by γ-T3 or ATV single treatment (Fig.2.2.4). A recent report

suggested JNK as a mediator of simvastatin- induced growth inhibition and cell

death in breast cancer cells [87]. Another report suggested that γ-T3 induces

apoptosis through activation of JNK signaling via ER stress in breast cancer cells

[88]. Moreover, the regulation of JNK transduction pathway also has been

mentioned in tumorigenesis and metastatic potential of gastric cancer [89]. JNK is

known to be activated by ER stress through the IRE1α pathway, but the interplay

with mitochondria and the mechanism for sustained JNK activation in ER stress is

not known [90]. In response to ER stress, the kinase domain of IRE1 binds to the

TRAF2 adaptor molecule and activates the apoptosis signaling regulation kinase

(ASK1), which in turn the causes the phosphorylation and activation of JNK.

However, JNK signaling elicits many cellular responses besides cell death and can

even promote cell survival in specific circumstances [91].

SP600125 is a specific, small molecule inhibition of JNK that prevents the

phosphorylation of c-Jun and blocks the expression of proinflammatory cytokines

and subsequent apoptosis cell death [92]. Hence, SP600125 treatment significantly

induced apoptosis consistent with the increasing levels of phosphorylated

anti-apoptotic proteins like Bcl-2 and Bcl-xL (un-activated form) in U937 leukemia

cells [93]. In recent report shown that downstream of JNK activates caspases via

the translation of Bax to mitochondria from the cytosol. The translocated Bax

induces an increase of mitochondrial membrane permeabilization and cytochrome c

release [94]. In the recent report provided further details for the action mechanism

of SP600125 on the ER and mitochondrial functions [95]. Considering these reports,

the action of SP600125 used in our study should be revised carefully. There is still a

possibility that results would be changing by the concentrations or timing of

SP600125 when it was treated with γ-T3 (Fig.2.2.5). Further studies are needed

that concentrate on more efficient upstream signals leading to the JNK activation

as well and this notion remains to be tested.

Caspase are the final effectors of both extrinsic and intrinsic apoptosis,

52

therefore it is expected that interfering with their function impairs these pathway

leading to a survival advantage for cancer cells and indeed caspase alterations are

not rare in a variety of tumors [96]. It has been demonstrated those two pathways

through which the caspase family proteases can be activated: one is the death

signal-induced, death receptor-mediated pathway; the other is the stress-induced,

mitochondrion-mediated pathway [97]. Typically, the death receptor-initiated

pathway involved activation of initiator caspase 8, which in turn activates caspase 3

[97]. In addition, caspase 3 and caspase 9 as well as caspase generated truncated

Bid that triggers cytochome c release [98]. There is a report that when the caspase

12 activity was inhibited by Z-ATAD-FMK, the mitochondria dependent activation

of caspase 9 and caspase 3 was not provoked to a sufficient level required for

subsequent activation of caspase 8 in Jurkat T cells [99]. On the other hand, in all

MM cell lines, Z-VAD-FMK 10 µM resulted no significant effect on

sorafenib-mediated cell killing whereas the caspase inhibitor did potently inhibit

the cytotoxicity of TRAIL in those cell lines sensitive to TRAIL [100]. Therefore,

they didn’t significantly affect the synergistic cell growth inhibition of γ-T3 and

statin that needed to explore the molecular mechanisms of ER stress.

53

2.4 Summary

γ-T3 increased ER stress markers; GRP78 and CHOP but not caspase 4.

γ-T3 induced growth inhibition was slightly blocked by salubrinal, a protector

of ER stress related apoptosis in MSTO cells.

γ-T3 and statin (ATV) combination up-regulated JNK activation but it had

almost no effect on their synergistic growth inhibitory effect.

Caspase 8 and caspase 9 inhibitors did not significantly change cell

cytotoxicity induced by γ-T3 and statin (ATV, SMV).

54

Conclusion

In this study, we analyzed that treatment effect of γ-T3 and statin exhibited a

synergistic effect against human malignant mesothelioma cells in vitro.

In Chapter1, synergistic effect of combined treatment with gamma-tocotrienol and

statin on human malignant mesothelioma cells was revealed.

γ-T3 and statin exhibited a synergistic effect against human MM cells by

complimentary and synergetic mechanism; inhibition of the MVL pathway

including HMGR and the following MAPK signaling (induced by both γ-T3 and

statin), and caspase 3 activation (induced by statin). Whether caspase 3 activation

was derived from MVL pathway or not had not been confirmed yet. In this study,

stains were used at 6.25 μM when combined with γ-T3. It still seems a little bit high

concentration in consideration of previous clinical tolerance of statin [37], however,

there is a possibility that lower than 6.25 μM concentration of statins combined

with high concentration of γ-T3 (more than 20 μM) could work effectively since γ-T3

is assumed to be safe. Moreover, our combination use of γ-T3 and statin has shown

not only cytostatic effect but also cytotoxicity, as shown sub-G1 increase and caspase

3 activation.

In Chapter2, it was revealed that endoplasmic reticulum stress seemed to be

involved with the combination effect of γ-T3 and statin.

Our work found that the expression levels of GRP78 and CHOP were

unchanged by treatment with ATV or SMV alone (Fig. 2.2.1a and b). It indicates

that statins in the present study did not affect ER stress, and also that statins did

not inhibit γ-T3 induced ER stress which might lead the following apoptosis. Then

we used ER stress factors such as JNK and caspase family members, caspase 8 and

caspase 9 which are reported to be related with ER stress, although they didn’t

significantly affect the synergistic cell growth inhibition of γ-T3 and statin.

Therefore, the downstream apoptotic pathway of ER stress mediated cell death is

still unclear.

Taken together, current research shows that γ-T3 synergistically enhances the

anti-proliferative effect of statins on MM cells via mevalonate pathway, and

promotes apoptosis via ER stress signal and intrinsic apoptosis pathway, in which

55

one the vital inducer could be caspase 3 (Fig. D). Overall, the combination of γ-T3

and statins could be useful for MM therapy and functions in a complementary style.

These findings suggest that a combination treatment may cause significant

inhibition of cancer cell growth, which will help to overcome the resistance of MM to

current chemotherapies. The distinct molecular targets of this combined therapy

should be explored in further detail.

56

Fig. D Mechanism scheme of “γ-T3 + statins”. Bold arrows indicate each effects

induced by γ-T3, statins, and γ-T3 + statins, respectively which have shown in this

study.

Caspase 9

Caspase 8

57

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http://www.ncbi.nlm.nih.gov/pubmed?term=Katz%20SI%5BAuthor%5D&cauthor=true&cauthor_uid=20038816

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhou%20L%5BAuthor%5D&cauthor=true&cauthor_uid=20038816

http://www.ncbi.nlm.nih.gov/pubmed?term=Chao%20G%5BAuthor%5D&cauthor=true&cauthor_uid=20038816

http://www.ncbi.nlm.nih.gov/pubmed?term=Smith%20CD%5BAuthor%5D&cauthor=true&cauthor_uid=20038816

http://www.ncbi.nlm.nih.gov/pubmed?term=Ferrara%20T%5BAuthor%5D&cauthor=true&cauthor_uid=20038816

http://www.ncbi.nlm.nih.gov/pubmed?term=Wang%20W%5BAuthor%5D&cauthor=true&cauthor_uid=20038816

http://www.ncbi.nlm.nih.gov/pubmed?term=Dicker%20DT%5BAuthor%5D&cauthor=true&cauthor_uid=20038816

http://www.ncbi.nlm.nih.gov/pubmed?term=El-Deiry%20WS%5BAuthor%5D&cauthor=true&cauthor_uid=20038816


69

List of publication

Guligena Tuerdi, Saki Ichinomiya, Hiromi Sato, Sana Siddig, Eriko Suwa, Hiroki Iwata,

Tomohiro Yano, Koichi Ueno. Synergistic effect of combined treatment with

gamma-tocotrienol and statin on human malignant mesothelioma cells. Cancer Letters,

2013, 339(1), 116-127.

70

Acknowledgements

First and foremost, I would like to express my sincere gratitude and appreciation

to my supervisor, Professor Koichi Ueno for his continues guidance and support

during my studies. Many thanks for his continuous discussions and encouragement.

Many thanks for his efforts in reviewing and correcting this thesis manuscript.

Second, I would like my teacher, Assistant Professor Hiromi Sato whose useful

suggestions, incisive comments and constructive criticism have contributed greatly

to the completion of this thesis. She devotes a considerable portion of her time to

reading my manuscripts. Her tremendous assistance in developing the framework

for analysis and in having gone through the draft versions of this thesis several

times as well as her great care in life deserve more thanks than I can find words to

express.

After that, I am also greatly indebted to all my teachers who have helped me

directly and indirectly in my studies. Any progress that I have made is the result of

their profound concern and selfless devotion. Among them the following require

mentioning: Associate Professor Katsunori Yamaura, Professor Toshihiko Toida,

and Professor Rena Kasimu.

Then, I would like to thank deeply to Department of Geriatric Pharmacology and

Therapeutics laboratory mates and friends companionship and assistance especially

Ph.D. Eriko Suwa.

Last but not least, I would like to deepest thanks go to my beloved family for their

encouragement and support which have been rendered to me throughout my study

in Japan. They have reminded me that what I do would been important. When I am

feeling down or stressed out their listening to me helped me a lot. My parents are

my proud of my life.

71

Examiners

This thesis for the doctorate in pharmaceutical sciences was examined by the

following referees authorized by the Graduated School of Pharmaceutical Sciences

Chiba University, Japan.

Professor of Chiba University (Graduate School of Pharmaceutical Sciences)

Nobunori Sato, Ph.D. (Pharm. Sci) ~Chief examiner~


Hiroyuki Takano, M.D. & Ph.D.


Yuko Sekine, Ph.D. (Pharm. Sci)

Documents

Studies on the synergistic effects of combined treatment of … · 2017. 12. 18. · Malignant mesothelioma (MM), is an aggressive, treatment-resistant tumor, which is increasing