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학 사 학 논

Sodium-dependent bile acid

transporter expression modulates

deoxycholate-induced effects on

hepatocellular carcinoma cells

간 포암종 포주에 담즙산 달체 이

시 린산에 한 효과에 미 는 향

2013 2 월

울 학 학원

학과 내과학 공

장

Sodium-dependent bile acid

transporter expression modulates

deoxycholate-induced effects on

hepatocellular carcinoma cells

February 2013

Seoul National University

Internal Medicine

Eun Sun Jang

간 포암종 포주에 담즙산

달체 이 시 린산에

한 효과에 미 는 향

지도

이 논 학 사 학 논 출함

2012 10 월

울 학 학원

학과 내과학 공

장

장 학 사 학 논 인 함

2012 12 월

원 장 (인)

부 원장 (인)

원 (인)

원 (인)

원 (인)

Sodium-dependent bile acid

transporter expression modulates

deoxycholate-induced effects on

hepatocellular carcinoma cells

by

Eun Sun Jang, M.D.

A Thesis Submitted to the Department of Internal Medicine

in Partial Fulfillment of the Requirements

for the Degree of Doctor of Philosophy in Medicine

at the Seoul National University College of Medicine

December, 2012

Approved by Thesis Committee:

Professor Chairman

Professor Vice Chairman

Professor

Professor

Professor

i

Abstract

Sodium-dependent bile acid

transporter expression modulates

deoxycholate-induced effects on

hepatocellular carcinoma cells

Eun Sun Jang

Department of Internal Medicine

The Graduate School

Seoul National University College of Medicine

Advanced HCC has been a therapeutic challenge despite the advances

in treatment modalities. In particular, hypoxia is the main obstacle to

treat HCC because it induces aggressive growth of the tumor.

Hydrophobic bile acid (BA), such as deoxycholate (DC), is

accumulated in cholestatic patients and can induce hepatocytes

apoptosis. Therefore, DC may also be applied to treat hepatocellular

carcinoma (HCC). However, the roles of DC and its transporter are

still not established in HCC cells. Thus, this study aimed to

investigate DC-induced HCC cell growth alterations, particularly

focused on the BAT expression.

Four human HCC cell lines (Huh-BAT, Huh-7, SNU-761 and SNU-

ii

475) were used to determine whether those cells expressed BAT or not.

Huh-BAT (BAT+ HCC cells) and Huh-7 cells which do not express

BAT (BAT- HCC cells) were grown either in a normoxic or hypoxic

condition with DC. Cell viability was assessed using the MTS assay.

BAT expression and apoptotic signaling cascades activation were

examined by immunoblot analyses. Wound healing and invasion

assays were performed to evaluate migration and invasion ability of

cells, respectively. Real-time PCR analysis was performed to measure

IL-8 expression levels. Nuclear factor kappa B (NF-κB) activity was

evaluated by ELISA method.

HCC cells revealed various BAT expression levels. In BAT+ HCC

cells, DC increased apoptosis in a dose dependent manner, and BAT

mediated the DC-induced apoptosis. Moreover, the susceptibility to

DC was markedly enhanced in hypoxia, which was dependent on

enhanced ER stress following DC treatment. This enhanced ER stress

in hypoxia accelerated apoptosis of BAT+ HCC cells via JNK

activation. On the contrary, DC promoted migration and invasion of

BAT- HCC cells and induced IL-8 overexpression. Particularly, DC-

induced IL-8 overexpression was dependent on NF-

κB/cyclooxygenase-2 (COX-2) activity. Wound healing and invasion

assays revealed that IL-8 was responsible for this augmented cellular

migration and invasion of BAT- HCC cells, and these were preventable

iii

by a COX-2 inhibitor.

In summary, hydrophobic BA had different effects on HCC cells

according to their BAT expression status. It induced apoptosis of

BAT-containing HCC cells, especially in hypoxic condition. In HCC

cells without BAT, hydrophobic BA and simultaneous blockage of

COX-2 activity could markedly decrease aggressive cellular behaviors

through the inhibition of NF-κB/COX-2/IL-8 pathway. Therefore,

hydrophobic bile acid had a therapeutic potential for advanced HCC.

Keywords : deoxycholate, bile acid, hepatocellular carcinoma,

apoptosis, JNK, COX-2, IL-8

Student Number : 2011-30575

iv

Contents

Abstract ............................................................................................ ⅰ

Contents .............................................................................................. ⅳ List of figures ...................................................................................... ⅴ

List of abbreviations ........................................................................... ⅶ

1. Introduction .................................................................................... 1

2. Materials and Methods .................................................................. 4

3. Results ............................................................................................ 11

4. Discussion ..................................................................................... 19

5. References ....................................................................................... 25

국 ............................................................................................. 34

v

List of figures

Figure 1. Human hepatocellular carcinoma cells expressed various

levels of bile acid transporter

Figure 2. Deoxycholate has a different effect on the growth of human

hepatocellular carcinoma cells depending on the bile acid transporter

expression, especially in hypoxic condition

Figure 3. Deoxycholate induced apoptosis of a human hepatocellular

carcinoma cell line with bile acid transporter

Figure 4. Bile acid transporter expression affected deoxycholate-

induced apoptosis of human hepatocellular carcinoma cells

Figure 5. Hypoxia augmented mitochondrial Smac and cytochromc C

releases by deoxycholate in human hepatocellular carcinoma cells with

bile acid transporter

Figure 6. Deoxycholate enhanced endoplasmic reticulum stress

dependent apoptosis of human hepatocellular carcinoma cells with bile

acid transporter via c-Jun N-terminal kinase (JNK) activation

Figure 7. Deoxycholate induced interleukin-8 overexpression in a

human hepatocellular carcinoma cells without bile acid transporter was

dependent on cyclooxygenase-2 activity

Figure 8. Interleukin-8 is responsible for enhanced cellular migration

activity by deoxycholate of hepatocellular carcinoma cells without bile

vi

acid transporter

Figure 9. Enhanced cellular migration ability of human hepatocellular

carcinoma cells without bile acid transporter by deoxycholate is

reversed by cyclooxygenase-2 inhibition

Figure 10. Enhanced cellular invasion ability of human hepatocellular

carcinoma cells without bile acid transporter by deoxycholate is

cyclooxygenase-2 dependent

Figure 11. Neither epidermal growth factor receptor nor G-protein

coupled bile acid receptor were responsible for deoxycholate induced

interleukin-8 overexpression of human hepatocellular carcinoma cells

without bile acid transporter

Figure 12. Deoxycholate induced interleukin-8 overexpression was

reversed by gugglesterone in human hepatocellular carcinoma cells

without bile acid transporter

Figure 13. Guggulsterone inhibited deoxycholate induced nuclear

factor kappa B activation in human hepatocellular carcinoma cells

without bile acid transporter

vii

List of abbreviations HCC, hepatocellular carcinoma

TACE, transarterial chemoembolization

BA, bile acid

CDC, chenodeoxycholate

DC, deoxycholate

GCDC, glycochenodeoxycholate

BAT, bile acid transporter

NTCP, Na+-dependent taurocholate cotransporting polypeptides

mEH, microsomal epoxide hydrolase

OATP, organic anion transporting polypeptides

BSEP, bile acids export pump

EGFR, epidermal growth factor receptor

TGR5, G-protein coupled bile acid receptor

FXR, farnesoid-X-receptor

NF-κB, nuclear factor kappa B

DMEM, Dulbecco’s Modified Eagle’s Media

FBS, fetal bovine serum

CytC, cytochrome c

AIF, apoptosis inducing factor

Smac, second mitochondria-derived activator of caspases

eIF2α, eukaryotic initiation factor 2α

viii

JNK, c-Jun N-terminal Kinase

IL, interleukin

siRNA, small interfering ribonucleic acid

MTS, 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium salt

ELISA, enzyme-linked immunosorbent assay

RNA, ribonucleic acid

cDNA, complementary deoxyribonucleic acid

mRNA, messenger ribonucleic acid

PCR, polymerase chain reaction

GAPDH, Glyceraldehyde-3-phosphate dehydrogenase

SD, standard deviation

ER, endoplasmic reticulum

COX-2, cyclooxygenase-2

ERK, extracellular signal-regulated kinase

1

1. Introduction

Hepatocellular carcinoma (HCC) is a leading cause of cancer-

related mortality worldwide1. In the recent decades, unresectable

HCC has been a therapeutic challenge despite the advances in treatment

modalities. Although transarterial chemoembolization (TACE) has

been developed to control HCC with characteristic hypervascularity,

some recurred HCCs after TACE show more aggressive growth than

the original tumor resulting from hypoxic insult. Although systemic

targeted therapy with sorafenib is currently available for advanced

HCC, the survival benefit of the drug to advanced HCC patients is

unsatisfactory2.

Bile acids (BA) are synthesized from cholesterol via enzymatic

modification in hepatocytes and regulate lipid and fat metabolism in

human. It can be classified into primary and secondary BAs

according to the synthetic process, and into hydrophilic and

hydrophobic BAs according to their molecular nature. In cholestatic

liver disease, the elevated hepatic hydrophobic BA such as

chenodeoxycholate (CDC), deoxycholate (DC), lithocholate,

glycochenodeoxycholate (GCDC) trigger cellular damage and death3.

BAs circulate enterohepatic system. In the liver, BAs are

dissociated from albumin, and transported across the hepatocytes via

bile acid transporters (BATs). On the basolateral membrane of

2

hepatocytes, Na+-dependent taurocholate cotransporting polypeptides

(NTCPs) uptake most BAs into the cells, and microsomal epoxide

hydrolase (mEH) and organic anion transporting polypeptides (OATPs)

also transport BAs in Na+-dependent and independent manners. After

uptake, BAs are exported across the canalicular membrane with bile

acids export pump (BSEP) and reach intestinal lumen through bile

duct4.

During enterohepatic circulation, BAs affect carcinogenesis of

cholangiocarcinoma or colon cancer. According to previous

population based studies, an increased fecal concentration of BAs are

related with a high colorectal cancer incidence5, 6. Several

experimental studies showed that an excessive exposure to BA can

induce cellular oxidative stress and DNA damage resulted in mutant

cells7. Additionally, BA induces cellular invasiveness of

cholangiocarcinoma8. Several pathways regulated by epidermal

growth factor receptor (EGFR), G-protein coupled bile acid receptor

(TGR5), farnesoid-X-receptor (FXR) or nuclear factor kappa B (NF-

κB) have documented to be stimulated by BAs9.

Although the liver is a major organ involved in the enterohepatic

circulation of BA, the effect of BAs on hepatocellular carcinoma

(HCC) is still controversial. Some investigators documented that

CDC or GCDC induced apoptosis of HepG2, HepG2-NTCP, and Huh-7

3

cells10, 11. On the contrary, others reported that GCDC prolonged

survival of Huh-7 cells12. However, those previous in vitro studies did

not consider the differences of BAT expression among the used cells

which facilitated intracellular uptake of BAs.

There are few clinical studies which focus on role of BAT in

human HCC. Knisely et al. have reported that a familial BA export

pump deficiency which results BA accumulation in hepatocytes is

related with a HCC development in children13. According to other

clinical studies, BA uptake and BAT expression are decreased in human

HCC14. However, the role of BAT expression on the growth of human

HCC is not established so far.

Therefore, we intended in this study to evaluate the effect of

hydrophobic BA on the HCC cells according to BAT expression, and to

elucidate the molecular mechanism.

4

2. Materials and Methods

2.1. Cell lines and culture

Four human HCC cell lines were used in this study: Huh-715 which

is derived from a well-differentiated HCC, Huh-BAT9 which is Huh-7

cells stably transfected with a bile acid transporter NTCP, SNU-76116

and SNU-47517 which are derived from a poorly differentiated HCC.

Cells were grown either in Dulbecco’s Modified Eagle’s Media

(DMEM, for Huh-BAT and Huh-7 cells) or in Roswell Park Memorial

Institute-1640 (for SNU-761 and SNU-475 cells) supplemented with

10% fetal bovine serum (FBS), 100,000 U/L of penicillin, and 100

mg/L of streptomycin. According to the experiments, cells were

incubated under standard culture conditions (20% O2, and 5% CO2 at

37 °C) or hypoxic culture condition (1% O2, 5% CO2 and 94% N2, at

37 °C).

2.2. Materials and reagents

Sodium deoxycholate was purchased from Sigma-Aldrich (St.

Louis, MO, USA). Rabbit anti-caspase 7 and anti-caspase 9, anti-bax,

anti-cytochrome C (CytC), anti-apoptosis inducing factor (AIF), anti-

second mitochondria-derived activator of caspases (Smac), and mouse

anti-p’- eukaryotic initiation factor 2α (eIF2α), anti-p’-c-Jun N-terminal

Kinase (JNK), anti-NTCP were purchased from Cell Signaling

5

Technology, Inc. (Danvers, MA, USA). Rabbit anti-NTCP was

purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

SP600125 (JNK inhibitor), celecoxib (selective cyclooxygenase-2

inhibitor), AG1478 (EGFR inhibitor) and Z-guggulsterone were

obtained from Sigma-Aldrich (St. Louis, MO, USA). Celecoxib, a

selective cyclooxygenase-2 inhibitor, was obtained from Tocris

Bioscience (Bristol, UK). NTCP, interleukin (IL)-8 and TGR5 small

interfering ribonucleic acids (siRNAs) were purchased from Santa Cruz

Biotechnology Inc. (Santa Cruz, CA, USA).

2.3. Small interfering RNA transfection

Cells were seeded on a 6-well culture plate (2x105 cells/well) in 2

mL antibiotic-free medium supplemented with 10% FBS. At 60-80 %

confluence, transfection of siRNA was carried out with siRNA

Transfection Reagent mixture (Santa Cruz Biotechnology Inc., Santa

Cruz, CA, USA) according to the manufacturer’s instruction. After 6

hour incubation at 37℃, cells were supplied with normal growth

medium containing 20% FBS and antibiotics, and incubated for an

additional 18 hours. Then, the medium was aspirated and replaced

with fresh medium with 10% FBS and antibiotics. At 24 hours after

the transfection, cells were treated for further experiments.

6

2.4. Cell viability test

Cellular proliferation was measured using the Cell Titer 96 Aqueous

One Solution cell proliferation assay (Promega, Madison, WI, USA),

based on the cellular conversion of the colorimetric reagent MTS [3,4-

(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium salt] into soluble formazan by

dehydrogenase enzymes found only in metabolically active,

proliferating cells. After each scheduled treatment, 20 μL of dye

solution was added into each well of a 96-well plate. The plate was

incubated for 1 to 2 hours. After the incubation, optical densities were

recorded at 490 nm using an enzyme-linked immunosorbent assay

(ELISA) plate reader (Molecular Devices, Sunnyvale, CA, USA).

2.5. Immunoblot assay

Cells were lysed for 20 min on ice with lysis buffer (50 mM tris-

HCl [pH 7.4]; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mM

NaCl; 1 mM EDTA; 1 mM phenyl-methylsulfonyl fluoride; 1 ug/mL

aprotinin, leupeptin, and pepstatin; 1mM Na3Vo4; and 1 mM NaF)

and centrifuged for 10 min at 14,000 x g at 4℃. Samples were

resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis,

transferred to nitrocellulose membranes, and blotted with appropriate

primary antibodies. The blots were incubated with peroxidase-

7

conjugated secondary antibodies (Biosource International, Camarillo,

CA, USA). The bound antibodies were visualized using a

chemiluminescent substrate (ECL; Amersham, Arlington Heights, IL,

USA) and exposed to Kodak X-OMAT film. Images were detected

using an image analyzer (LAS-1000, Fuji Photo Film, Tokyo, Japan),

and densitometric analysis was carried out using Image Gauge software

(Fuji Photo Film, Tokyo, Japan).

2.6. Mitochondrial regulatory protein release

A mitochondria/cytosol fractionation kit (Merk Millipore, Billerica,

MA, USA) was used to isolate mitochondria from cellular fraction of

treated cells according to the manufacturer’s instruction. After

fractionation, standard western blotting methods were performed as

described above, against bax, CytC, AIF, and Smac.

2.7. Wound healing assay

For wound healing assay using the scratch method, Huh-7 cells

were grown to confluence on 6-well plates and then serum-starved

using DMEM media for 16 hours. After the serum starvation, a

scratch was introduced with a yellow pipette tip as described

elsewhere18. Cells were further incubated in various conditions and

areas re-filled by migrating cells were documented. Images of three

8

non-overlapping fields were captured, and the re-filled area was

analyzed using image analyzing software (Videotest® v4.0; Ista-

Videotest Company, St. Petersburg, Russia).

2.8. Invasion assay

In vitro invasion assay was performed using the 24-well chamber.

After transfer inserts into the wells, each inserts were coated with

matrigel (BD biosciences, Billerica, MA, USA) for 30 minutes at 37 °C.

Huh-7 cells were suspended in serum-free medium and seeded on

Matrigel-coated upper chambers (5x104 cells/chamber), and DMEM

containing 10% FBS was added in the lower chamber. After 6 hour

incubation at 37 °C, cells were treated with or without 20 μM of

celecoxib. After 1 hour of the pretreatment, cells were incubated in

the presence or absence of DC (50 μM) for 24 hours. Cells were

stained with 4 μg/mL calcein AM (BD biosciences, Billerica, MA,

USA) in HBSS at 37 °C for 90 minutes. Invasion ability was

determined as relative fluorescence units measured at an excitation of

494 nm and an emission of 517 nm using a multifunctional plate reader

(EnVision Multilabel Reader; PerkinElmer Inc., Waltham, MA, USA).

2.9. Real time PCR analysis

Total RNA was extracted from cells using AxyPrep Multisource

9

Total RNA Miniprep Kit® according to the manufacturer’s

recommendations (Axygen Biosciences, Union city, CA, USA). The

complementary deoxyribonucleic acid (cDNA) templates were

synthesized using oligo-dT random primers and Moloney Murine

Leukemia Virus (MoMLV) reverse transcriptase. Interleukin 8 (IL-8)

messenger RNA (mRNA) was quantified by real-time polymerase

chain reaction (PCR) using the following primers19: forward, 5´-GGC

AGC TTC CTG ATT TCT G-3´; reverse, 5´-CGC AGT GTG GTC

CAC TCT CA-3´. Glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) primers (Takara Bio inc., Shiga, Japan) were used as a

control for RNA integrity. Amplification was carried out via real-time

PCR (Thermal Cycler DiceTM Real Time System; Takara Bio inc.,

Shiga, Japan) using SYBR green as the fluorophore (Takara Bio inc.,

Shiga, Japan).

2.10. Nuclear factor kappa B activity assay

Huh-7 cells were plated at 5x104 cells/well in 1ml of media in a six-

well plate. After 16 hours of serum starvation, cells were treated with

or without DC (50 μM) ± Z-guggulsterone (10 μM) for 8 hours.

Nuclear fractions were extracted using Nuclear Extract Kit (Active

Motif, Shinjuku, Japan) and NF-κB p65 concentrations were

determined by TransAMTM NF-κB p65 kit (Active Motif, Shinjuku,

10

Japan) which is a sensitive ELISA-based method, according to the

manufacturer’s instruction.

2.11. Statistical analysis

All data represent at least three independent experiments and are

expressed as means ± standard deviations (SDs) unless otherwise

indicated. Statistical evaluations of numeric variables in each group

were conducted using the Mann-Whitney U test. All statistical

analyses were performed using SPSS 17.0 for Windows (SPSS Inc.,

Chicago, IL, USA). Differences with a p-value of <0.05 were

considered statistically significant.

11

3. Results

3.1. Human hepatocellular carcinoma cells expressed various levels

of sodium-dependent bile acid transporter

As shown in Figure 1, 4 human HCC cells presented various

levels of NTCP, a major BAT in human hepatocyte. In contrast with

Huh-7 cells which barely expressed NTCP regardless of DC treatment,

Huh-BAT cells showed consistent NTCP expression. Poorly

differentiated human HCC cell lines SNU-761 and SNU-475 revealed

moderate expression of NTCP. After DC treatment, the expression

level of NTCP in those 2 cell lines seemed to be decreased slightly

(Figure 1).

3.2. Deoxycholate had different cytotoxicity to hepatocellular

carcinoma cells depending on the bile acid transporter expression

To explore whether DC had different effects on HCC cells

depending on their BAT expression levels, Huh-BAT and Huh-7 cells

were treated with DC, respectively. In Huh-BAT cells, DC showed

dose-dependent cytotoxic activity (Figure 2A). As shown in Figure 3,

we confirmed that intrinsic apoptotic pathway (caspase 7 and 9) of

Huh-BAT cells was activated after DC treatment. On the contrary, DC

did not suppress growth of Huh-7 cells which do not express NTCP, a

major sodium-dependent BAT on the human hepatocyte (Figure 2B).

12

Then, SNU-761 (Figure 4A) and SNU-475 (Figure 4B) cells,

which were expressed moderate levels of NTCP, were transfected with

control or NTCP siRNA, and treated with or without DC. As a result,

DC activated caspase 7 and 9 of both cell lines transfected with control

siRNA. However, DC-induced apoptosis of those cells were

attenuated by blocking NTCP with siRNA transfection.

3.3. Hypoxia enhanced susceptibility to deoxycholate of

hepatocellular carcinoma cells with bile acid transporter

Since we have documented the apoptotic signaling pathways of

hypoxic HCC cells could be different from those of normoxic cells in

previous other studies20, we treated BAT positive cells with DC under

both normoxic and hypoxic conditions. As mentioned above,

normoxic Huh-BAT cells showed susceptibility to DC. Especially, the

cytotoxic effect of DC on Huh-BAT cells was enhanced markedly

under hypoxic condition (Figure 2A).

3.4. Hypoxia augmented mitochondrial Smac and cytochromc C

releases by deoxycholate in human hepatocellular carcinoma cells

with bile acid transporter.

Because hypoxia can change oxidative stress and resultant

mitochondrial function21 which regulates cellular apoptosis directly, we

13

evaluated whether DC affected mitochondrial apoptosis regulations in

HCC cells under normoxic and hypoxic conditions, respectively. As

shown in Figure 5, DC increased mitochondrial translocation of Bax,

and consequent cytosolic releases of CytC/Smac under normoxic

condition in Huh-BAT cells. These mitochondrial apoptosis

regulatory proteins were also increased by DC under hypoxic condition,

but they did not differ between normoxia and hypoxia. AIF release was

not increased by DC under both normoxic and hypoxic conditions.

3.5. Enhanced endoplasmic reticulum stress under hypoxic

condition accelerated apoptosis of hepatocellular carcinoma cells

with bile acid transporter via c-Jun N-terminal kinase activation

Then, we hypothesized that DC could affect endoplasmic reticulum

(ER) stress which is known to be induced under hypoxic condition22

and may mediate cellular apoptosis. Thus, immunoblot assay was

performed against p’-eIF2α to compare ER stress levels in cells treated

with or without DC. As a result, ER stress was more augmented by

DC (Figure 6A, right) and under hypoxic condition (Figure 6A,

bottom) in Huh-BAT cells compared to by control (Figure 6A, left) and

under normoxic condition (Figure 6A, top), respectively.

Since ER stress is often coupled to activation of JNK20, we

explored the effect of DC on JNK pathway in BAT positive HCC cells.

14

As shown in Figure 6B, DC activated JNK in Huh-BAT cells time

dependently. Then, ER stress and apoptosis of BAT positive HCC

cells were evaluated after JNK inhibitor treatment with or without DC

(Figure 6C). As a result, solitary JNK inhibitor treatment augmented

ER stress in Huh-BAT cells, but activation of caspase 7 was not

observed (Figure 6C). With DC treatment, JNK inhibition also

augmented ER stress but decreased the apoptosis of Huh-BAT cells.

Consequently, DC induced ER stress was responsible to the apoptosis,

and JNK was a downstream mediator of ER stress-dependent apoptosis

in BAT positive human HCC cells .

3.6. Interleukin-8 was responsible for enhanced cellular migration

activity by deoxycholate of hepatocellular carcinoma cells without

bile acid transporter

As mentioned above, DC did not reveal significant cytotoxic

effect on Huh-7 cells which does not express BAT (Figure 2B). Since

IL-8 has documented to correlate with the tumorigenicity and

metastasis of tumor23, we measured IL-8 expression in Huh-7 cells

which showed resistance to DC treatment. As shown in Figure 7, DC

stimulated IL-8 expression of Huh-7 cells (Figure 7). Thus, we

performed wound healing assay using Huh-7 cells transfected with

control or IL-8 siRNA to confirm the function of IL-8 in BAT negative

15

HCC cells. As a result, DC augmented migration of control siRNA-

transfected Huh-7 cells (Figure 8, solid line with diamond vs. short

broken line with triangle). However, the augmented migration activity

by DC was not observed in those transfected with IL-8 siRNA (Figure

8, long broken line with square vs. dot and broken line with circle).

According to these results, IL-8 was responsible for enhanced cellular

migration ability of BAT negative HCC cells after DC treatment.

3.7. Overexpression of interleukin-8 by deoxycholate is dependent

to cyclooxygenase-2 activation

In human malignancies including HCC, cyclooxygenase-2 (COX-

2) has been reported to affect many mechanisms involved in a

stimulation of cell growth or invasion24, 25. To elucidate whether DC-

induced IL-8 overexpression in Huh-7 cells was dependent on COX-2

activity, IL-8 mRNA levels were measured by real time PCR after a

selective COX-2 inhibitor celecoxib treatment on Huh-7 cells. As

shown in Figure 7, the overexpression of IL-8 after DC treatment was

restored by COX-2 inhibition. As a result of wound healing assay,

celecoxib reversed the enhanced migration of BAT negative human

HCC cells after DC treatment (Figure 9). Collectively, enhanced

cellular migration of BAT negative HCC cells by DC was related to the

COX-2 dependent IL-8 overexpression.

16

3.8. Enhanced cellular invasion ability of human hepatocellular

carcinoma cells without bile acid transporter by deoxycholate was

cyclooxygenase-2 dependent

Since DC augmented cellular migration of Huh-7, we evaluated

the change of invasion ability of those cells after DC treatment.

According to our invasion assay using fluorescent dye, the number of

invaded Huh-7 cells increased after DC treatment (Figure 10).

Similarly with the migration, the enhanced invasion of Huh-7 cells after

DC treatment was reversed by COX-2 inhibitor (Figure 10). Taken

together, DC induced aggressive cellular behavior of human HCC cells

without BAT via COX-2 dependent IL-8 overexpression.

3.9. Neither epidermal growth factor receptor nor G-protein

coupled bile acid receptor were responsible for deoxycholate

induced interleukin-8 overexpression of human hepatocellular

carcinoma cells without bile acid transporter

Since bile acids can elicit their effects on cells via several receptor-

mediated alteration of gene transcription without its intracellular

transportation26-28, we evaluated that the blockage of several

documented bile acid receptors on the cell surface was responsible for

DC-induced IL-8 overexpression in Huh-7 cells. As shown in Figure

17

11A, EGFR inhibitor could not reverse the IL-8 overexpression induced

by DC treatment on Huh-7 cells. In addition, TGR5 blockage with

siRNA could not affect the DC-induced IL-8 overexpression (Figure

11B).

3.10. Deoxycholate induced interleukin-8 overexpression was

mediated by nuclear factor kappa B in human hepatocellular

carcinoma cells without bile acid transporter

Then, we treated Huh-7 cells with guggulsterone, which suppresses

broad bile acid-induced signals via multiple nuclear receptors29. As a

result, BAT negative cells treated with DC and guggulsterone showed

significantly lower IL-8 mRNA expression levels compared to those

treated with DC alone (Figure 12). As a next step, we measured NF-

κB p65 activities in Huh-7 cells treated with or without DC, in the

presence or absence guggulsterone, because COX-2 transcription can

be activated by NF-κB30, 31 and guggulsterone has documented to

modulate NF-κB activity32. As shown in Figure 13, BAT negative

cells treated with DC showed markedly increased NF-κB p65

concentration, but a combined guggulsterone and DC treatment

reversed the increased activity. Therefore, we thought that DC

activated NF-κB/COX-2/IL-8 pathway and consequent

migration/invasion of human HCC cells without BAT.

18

4. Discussion

In the present study, we demonstrated that DC, a hydrophobic BA,

showed different cytotoxicity according to sodium-dependent BAT

expression on the HCC cells. HCC cells with BAT were susceptible

to hydrophobic BA, especially in hypoxic condition, and it was related

to the JNK dependent ER stress enhancement. HCC cells without

BAT were resistant to hydrophobic BA and rather revealed augmented

cellular migration/invasion activities. The increased aggressiveness of

HCC cells with BAT was related to COX-2 dependent IL-8

overexpression through NF-κB activation. To our best knowledge,

this is the first study to investigate the importance of sodium-dependent

BAT expression level on the human HCC cells.

DC induced ER stress and resultant JNK-dependent apoptosis of

HCC cells with BAT (figure 6). The cytotoxic effects of hydrophobic

BAs are documented in normal hepatocytes. Previous studies showed

that both mitochondrial dysfunction via oxidative stress and nitric oxide

dependent non-mitochondrial pathway were responsible to the BA

induced apoptosis of hepatocytes33, 34. Recently, ER stress is also

suggested as a major process of cholestasis induced apoptosis of

hepatocyte35. Many studies have documented that ER stress induces

cellular apoptosis36, 37, and DC activates several gene promoters that

respond to ER stress37. Moreover, it has been reported that ER stress

19

can directly trigger the JNK cascade through IRE1 activation38.

Regarding HCC cells, hydrophobic BAs induce upregulation of death

receptor 5/TRAIL-receptor 2 expressions and consequent apoptosis of

BAT positive HCC cells, and it is dependent on the JNK activation39, 40.

In the same context, we demonstrated that JNK inhibition potentiated

ER stress triggered by DC due to blocking of downstream signalling of

unfolded protein response, but attenuated apoptosis of DC treated cell

(Figure 6).

Another notable finding of the present study was that the cytotoxic

effect of DC on HCC cells with BAT was augmented in hypoxia

(Figure 2A). Because the current clinical practice to treat unresectable

HCC is based on the TACE or sorafenib, the hypoxic status from

insufficient vascularization of the tumor is a major obstacle to the HCC

treatment41. Moreover, hypoxia tends to stimulate the growth of HCC

via hexokinase II22 or Notch homolog 142 expressions. Therefore,

anti-cancer therapy with hydrophobic bile acids against HCC with BAT

could be a promising strategy to control hypoxic HCCs.

Nevertheless, BAT systems on hepatocytes are not only

constitutively expressed but also regulated at transcriptional and post-

transcriptional levels43. The expression of BAT on HCC cells have

been studied with respect to the usefulness of drug-BA conjugates as

anti-tumor agents. In 5 surgically resected HCC tissues, NTCP

20

proteins are present on the cell surface, but the level of NTCP

expression is reduced to 56% compared to the surrounding non-tumor

liver tissue44. Moreover, in vivo study showed that HCC can be

developed spontaneously in the absence of BAT45. Thus, we designed

this study to compare the effect of BAT on the HCC cells using Huh-

BAT and Huh-7, which are based on the same cell line (Huh-7) except

their BAT expression level.

In contrast to HCC cells with BAT, those without BAT were not

susceptible to the hydrophobic BA treatment. Instead, DC treatment

stimulated migration and invasion abilities of those cells that meant

potential aggressive behavior. Although the carcinogenic effects of

hydrophobic BAs in cholangiocarcinoma9 and colon cancer7 are

documented, it has not been fully studied in HCCs which arise within

the liver, a major involving system of enterohepatic circulation of BAs.

A previous study revealed that hydrophobic BAs enhance Mcl-1

expression through activation of the EGFR signaling pathways

participated in carcinogenesis of cholangiocytes28. Although

hydrophobic BA can stimulate Mcl-1 phosphorylation and subsequent

extracellular signal-regulated kinase (ERK) 1/2 activation, resulting

chemoresistance in Huh-7 cells12, we could not succeed to confirm

Mcl-1 phosphorylation nor ERK1/2 activation (data not shown).

Alternatively, we found that the increased migration activity was

21

related with IL-8 overexpression, which was not dependent to EGFR

but to COX-2 pathway. A high levels of IL-8 are relevant to poor

prognosis of HCC46, 47, and it has been recently determined that IL-8 is

associated with the chemosensitivity of cancer cells48.

COX-2 is overexpressed in several cancer cells, and the inhibition

of the enzyme reduced cancer development, especially colon cancer30,

49. The enzyme also promotes HCC cell growth, and the inhibition of

COX-2 is anticipated as a anticancer mechanism50. According to our

results, COX-2 inhibitor reversed the DC effect on the HCC cells

without BAT which are the cellular migration stimulation and the

overexpression of IL-8, a well-known mediator of invasion and

metastasis of cancer. Interestingly, the inhibitory effect of celecoxib

combined with DC on the cellular migration was more remarkable

compared with those of single celecoxib (Figure 9). This finding

supported the feasibility of combined treatment with COX-2 inhibitor

and bile acid as an anti-cancer drug in HCC.

Unfortunately, the precise mechanism of DC induced intracellular

signaling in HCC cells without BAT did not fully elucidated in the

present study. Inhibitions of several well-known bile acid receptors,

such as EGFR and TGR5, could not suppress the DC-induced IL-8

overexpression in Huh-7 cells as shown in Figure 11. However,

hydrophobic bile acid was thought to bind to nuclear receptors27, 51

22

directly and owing to its lipophilicity. To inhibit broad bile acid

related signals caused by DC, we used guggulsterone in this study.

Guggulsterone, which is a plant steroid from Commiphora mukul, can

lower serum cholesterol29 by bile acid metabolism regulation via FXR

antagonism and BSEP enhancement52. Additionally, several other bile

acid related signals such as NF-κB32, 53 and constitutive causal-related

homeobox 254 are affected by guggulsterone according to recent studies.

Similar with several previous reports32, 41, guggulsterone attenuated

DC-induced NF-κB/COX-2/IL-8 pathway activation on HCC cells

without BAT (Figure 12 and 13). Since FXR antagonizes NF-κB in

HepG2 cells and primary hepatocytes55, the reversal of IL-8

overexpression in Huh-7 cells after guggulsterone treatment might not

be mediated by FXR. Further studies will be needed to detect another

intracellular bile acid binding sites except FXR.

In summary, hydrophobic BA deoxycholate had different effects on

HCC cells according to their BAT expression status. It induced

apoptosis of BAT-containing HCC cells via ER stress-related JNK

activation, especially in hypoxic condition. On the contrary, HCC

cells without BAT were resistant to hydrophobic BA. Hydrophobic

BA paradoxically stimulated cellular migration and invasion in HCC

cells without BAT via COX-2 dependent IL-8 overexpression.

However, hydrophobic BA and simultaneous blockage of COX-2

23

activity could markedly decrease aggressive cellular behaviors.

Therefore, hydrophobic bile acid had a therapeutic potential for

advanced HCC.

24

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33

시 린산 등 소 담즙산 담즙 체가 생하는 경우

상 간 포 자멸사를 증가시키는 것 알 있다. 그러나

소 담즙산이 간 포암종 장 사멸에 미 는 향 잘

있지 않다. 또한, 상 간 포에 는 담즙산 달체가

담즙산 포내 이동 매개하지만, 간 포암종 포에 이러한

담즙산 달체 그 향에 한 연구는 거 이루어진

가 없다. 라 본 연구에 는 소 담즙산이 간 포암종

포에 미 는 향 담즙산 달체 여부에 라 알아보고자

하 다.

본 연구에 는 담즙산 달체 평가하 하여 4

개 인간 간 포암 포주, 즉 Huh-BAT, Huh-7, SNU-761

SNU-475 를 사용하 다. 특히, 주요 담즙산 달체인 Na+-

taurocholate cotransporting polypeptide (NTCP) 이 거

없는 Huh-7 과 NTCP 를 모 하는 Huh-BAT 에 소

담즙산 시 린산 처리하 며, 산소 상태 상

산소농도 상태에 각각 포를 양하 다. 포 증식 MTS

실험 통하여 하 고, 면역탁본법 이용하여 포자멸사

경 , NTCP 도를 인하 다. 상처 검사 침습

검사를 이용하여 포 이동 침습능 변 를 인하 다.

인 루킨-8 도를 실시간 역 사 합연쇄 실험

이용하여 평가하 다. Nuclear factor kappa B (NF-κB)

도는 ELISA 하 다.

간 포암종 포주들 담즙산 달체인 NTCP 를 다양한

도 하는 것 나타났다. 담즙산 달체를 잘 하는

간 포암종 포주에 는 시 린산에 하여 간 포암종

자멸사가 용량 존 증가하 며, 이는 NTCP 를 통해

34

매개 었다. 또한 이러한 시 린산 포독 특히 산소

상태에 있는 간 포암종 포에 하 며, 이는 포질 망

스트 스 증가에 른 JNK 과 이 있었다. 한편, 담즙산

달체를 거 하지 않는 간 포암종 포주에 는

시 린산이 포 이동과 침습 증가시 며 이는

인 루킨-8 에 해 매개 었다. 시 린산 NF-κB 를

통하여 cyclooxygenase (COX)-2 를 도하고, 그

결과 인 루킨-8 증가시키는 것 나타났다. 특히

소 담즙산 COX-2 억 동시에 처리하 ,

간 포암종 포 이동과 침습 도는 각 단독 요법에 해 욱

감소하는 것 나타났다.

요약하면, 소 담즙산인 시 린산 담즙산 달체

에 라 간 포암종 포에 다른 효과를 나타냈다.

담즙산 달체를 하는 간 포암종 포주에 는 소

담즙산에 해 포사멸이 진 었다. 한편, 담즙산 달체를

하지 않는 포주에 는 COX-2 억 소 담즙산

동시에 여하 NF-κB/COX-2/IL-8 신 경 를

차단하여 포 이동과 침습 억 하 다. 라 소

담즙산 진행 간 포암종 료 용할 있 리라

하며, 특히 산소 상태 간 포암종 료에 보다 효과 일

것 생각 다.

주요어 : 담즙산, 시 린산, 간 포암종, 담즙산 달체,

포질 망 스트 스, 인 루킨-8, cyclooxygenase-2

학번 : 2011-30575

Figure 1. Human hepatocellular carcinoma cells expressed various

levels of sodium-dependent bile acid transporter. Huh-7, Huh-BAT,

SNU-761, and SNU-475 cells were harvested at 30 minutes after

treatment with deoxycholate (200 μM, DC) or not (0 μM, control).

Cell lysates were resolved by SDS-PAGE and immunoblot analysis

performed for anti-NTCP.

Figure 2. Deoxycholate has a different effect on the growth of

human hepatocellular carcinoma cells depending on the bile acid

transporter expression, especially in hypoxic condition. Huh-BAT

(A) and Huh-7 (B) cells were cultured for 8 hours in the presence of

deoxycholate at various concentration after serum starvation for 16

hours, under stardard normoxic culture condition (20% O2 and 5% CO2,

at 37 °C) or hypoxic culture condition (1% O2, 5% CO2 and 94% N2, at

37°C). Cell viability (MTT) assay was performed according to the

manufacturer’s instruction. Data were expressed as mean ± SD of

relative number of viable cells as compared to that of the time 0 in

normoxic condition. * p=0.029, vs. time 0 in normoxia. ** p=0.05,

vs. time 0 in normoxia.

(A)

(B)

Figure 3. Deoxycholate induced apoptosis of a human

hepatocellular carcinoma cell line with bile acid transporter. Huh-

BAT cells were harvested the each time points after treatment with

deoxycholate (100 μM, DC) or not (0 μM, control). Cell lysates were

resolved by SDS-PAGE and immunoblot analysis performed for

casepase 7 and 9. Immunoblot analysis using anti-β-actin was

performed as a control for protein loading.

Figure 4. Bile acid transporter expression affected deoxycholate-

induced apoptosis of human hepatocellular carcinoma cells. SNU-

761 (A) or SNU-475 (B) cells were transfected with control or Na+-

dependent taurocholate cotransporting polypeptides (NTCP) siRNA for

24 hours. Cells were harvested at each time points after deoxycholate

(200 μM, DC) or distilled water (control) treatment. Cell lysates were

resolved by SDS-PAGE and immunoblot analysis performed for

casepase 7 and 9.

(A)

(B)

Figure 5. Hypoxia augmented mitochondrial Smac and cytochromc

C releases by deoxycholate in human hepatocellular carcinoma

cells with bile acid transporter. Huh-BAT cells were cultured with or

without deoxycholate (100 μM), under stardard normoxic condition (20%

O2 and 5% CO2, at 37 °C) or hypoxic condition (1% O2, 5% CO2 and

94% N2, at 37°C). Harvested cells in each time points were processed

using a Mitochondria/Cytosol Fractionation Kit. Immunoblot analysis

was performed with the respective fractions (mitochondria and cytosol)

for bax, cytochrome C, second mitochondria-derived activator of

caspases (Smac), and apoptosis inducing factor (AIF).

Figure 6. Deoxycholate enhanced endoplasmic reticulum stress

dependent apoptosis of human hepatocellular carcinoma cells with

bile acid transporter via c-Jun N-terminal kinase (JNK) activation.

(A) Huh-BAT cells were cultured with or without deoxycholate (100

μM), under stardard normoxic condition (20% O2 and 5% CO2, at

37 °C) or hypoxic condition (1% O2, 5% CO2 and 94% N2, at 37°C).

(B) Huh-BAT cells were treated with 100 μM of deoxycholate (DC) or

without it (control). (C) Huh-BAT cells were grown at variable

treatment conditions, with or without deoxycholate (100 μM), and with

or without JNK inhibitor (10 μM). Cells were harvested at the each

time points after variable treatment and immunoblot analyses

performed for phospho-eIF2α, phospho-JNK and caspase-7.

Immunoblot analysis using anti-β-actin was performed as a control for

protein loading.

(A)

(B)

(C)

Figure 7. Deoxycholate induced interleukin-8 overexpression in a

human hepatocellular carcinoma cells without bile acid transporter

was dependent on cyclooxygenase-2 activity. After serum starvation

for 16 hours, Huh-7 cells were treated with or without 50 μM of

deoxycholate, in the presence or absence of Cox-2 inhibitor (celecoxib,

20 μM). Quantitative real-time PCR for IL-8 was carried out using

total RNAs extracted from Huh-7 cells after 8 hours of each treatment.

Results were expressed as a relative ratio of product mRNA expression

level to the housekeeping gene expression level from the same RNA

sample, assuming a control of 1. Columns, mean; bars, SD. *,

p=0.025 versus control.

Figure 8. Interleukin-8 is responsible for enhanced cellular

migration activity by deoxycholate of hepatocellular carcinoma

cells without bile acid transporter. Huh-7 cells were transfected

with control or interleukin (IL)-8 siRNA for 24 hours. After serum

starvation for 16 hours, wound healing assay was performed. At 0, 24,

and 48 hours after deoxycholate (50 μM, DC) or with distilled water

(control) treatment, wound healing areas were measured in 3 non-

overlapping fields.

Figure 9. Enhanced cellular migration ability of human

hepatocellular carcinoma cells without bile acid transporter by

deoxycholate is reversed by cyclooxygenase-2 inhibition. After

serum starvation for 16 hours, Huh-7 cells were treated with or without

celecoxib (20 μM) for wound healing assay. After 1 hour of the

pretreatment, cells were cultures in the presence or absence of

deoxycholate (50 μM) for 0, 6, 24, and 48 hours. Cell-free areas at

each time points were measured in 3 non-overlapping fields.

Figure 10. Enhanced cellular invasion ability of human

hepatocellular carcinoma cells without bile acid transporter by

deoxycholate is cyclooxygenase-2 dependent. Huh-7 cells were

seeded on Matrigel-coated upper chambers (5x104 cells/chamber), and

DMEM containing 10% FBS was added in the lower chambers of 24-

well plate. After 6 hour incubation, cells were treated with or without

20 μM of celecoxib. After 1 hour of the pretreatment, cells were

incubated in the presence or absence of deoxycholate (DC, 50 μM) for

24 hours. Invasion ability was determined as relative fluorescence

units measured in a fluorescence plate reader using BD Calcein AM

Fluorescent dye. Columns, mean; bars, SD. *, p=0.05 versus cells

treated with DC without celecoxib.

*

Figure 11. Neither epidermal growth factor receptor nor G-protein

coupled bile acid receptor were responsible for deoxycholate

induced interleukin-8 overexpression of human hepatocellular

carcinoma cells without bile acid transporter. (A) After serum

starvation for 16 hours, Huh-7 cells were treated with or without 50 μM

of deoxycholate, in the presence or absence of either 10 μM of the

EGFR inhibitor. (B) Huh-7 cells were transfected with control or

TGR5 siRNA for 24 hours. After serum starvation for 16 hours,

transfected cells were treated with or without 50 μM of deoxycholate

(DC). Quantitative real-time PCR for IL-8 was carried out using total

RNAs extracted from Huh-7 cells after 6 hours of DC treatment.

Results were expressed as a relative ratio of product mRNA expression

level to the housekeeping gene expression level from the same RNA

sample, assuming a control of 1. Columns, mean; bars, SD. *,

p=0.025 versus control; **, p=0.039 versus cells without DC treatment.

(A)

(B)

Figure 12. Deoxycholate induced interleukin-8 overexpression was

reversed by gugglesterone in human hepatocellular carcinoma cells

without bile acid transporter. After serum starvation for 16 hours,

Huh-7 cells were treated with or without 50 μM of deoxycholate (DC),

in the presence or absence of either 10 μM of gugglesterone.

Quantitative real-time PCR for IL-8 was carried out using total RNAs

extracted from Huh-7 cells after 8 hours of each treatment. Results

were expressed as a relative ratio of product mRNA expression level to

the housekeeping gene expression level from the same RNA sample,

assuming a control of 1. Columns, mean; bars, SD. *, p=0.05

versus control; **, p=0.05 vs. cells treated with DC.

Figure 13. Guggulsterone inhibited deoxycholate induced nuclear

factor kappa B activation in human hepatocellular carcinoma cells

without bile acid transporter. After 16 hours of serum starvation,

Huh-7 cells were treated with or without 50 μM of deoxycholate (DC),

in the presence or absence of either 10 μM of gugglesterone. NFκB

concentrations in nuclear extracts from each treated cells were

measured using ELISA. Columns, mean; bars, SD. *, p=0.05

versus control; **, p=0.05 vs. cells treated with DC.