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Journal of Digestive Diseases 2014; 15; 684–693
doi: 10.1111/1751-2980.12191
Original article
Ursodeoxycholic acid induces apoptosis of hepatocellular carcinoma cells in vitro Lei ZHU,*1 Lu Juan SHAN,† Yue Jian LIU,† Dan CHEN,‡ Xiao Guang XIAO§ & Yan LI* *Department of Gastroenterology, Shengjing Hospital, China Medical University, Shenyang, †Central Laboratory, ‡Department of Clinical Pathology, and §Clinical Laboratory of Gene Diagnosis, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning Province, China
OBJECTIVE: Ursodeoxycholic acid (UDCA) is widely used to treat chronic liver diseases, and its cytoprotective effect on normal hepatocytes has been shown. This study aimed to investigate the apoptotic effects of UDCA on hepatocellular carcinoma (HCC) cells and the underlying molecular events in vitro. METHODS: HCC cells were treated by UDCA at different doses and periods of time to assess cell morphology, viability, apoptosis and gene expression using methyl thiazolyl tetrazolium (MTT), Annexin V/propidium iodide (PI) stain, transferase dUTP nick end labeling (TUNEL), enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and quantitative reverse transcription polymerase chain reaction, respectively. KEY WORDS:
CONCLUSIONS: The induction of HCC cell apoptosis by UDCA was dose-dependent and timedependent and was mediated by the regulation of Bax to Bcl-2 ratio, the expressions of Smac and Livin, and caspase-3 expression and activity.
apoptosis, gene expression, hepatocellular carcinoma, ursodeoxycholic acid.
Correspondence to: Yan LI, Department of Gastroenterology, Shengjing Hospital, China Medical University, 36 Sanhao Street, Shenyang, Liaoning Province 110004, China. Email: [email protected] 1
Current address: Department of Gastroenterology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning Province 116011, China. Conflict of interest: None. © 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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RESULTS: UDCA treatment reduced cell viability but induced HCC cell apoptosis in dose-dependent and time-dependent manners. UDCA arrested HepG2 cells at phase S of the cell cycle. At the gene levels, UDCA downregulated Bcl-2 and second mitochondria-derived activator of caspase (Smac) protein expressions, but upregulated Bax and Livin proteins in HCC cells. At the highest concentration, UDCA inhibited Livin mRNA expression but increased Smac and caspase-3 mRNA expressions as well as the activity of caspase-3 in HCC cells.
INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common solid tumors worldwide. According to the cancer statistics, a total of 748 300 new cases and 695 900 cancer-related deaths were estimated to occur in 2008.1 Approximately half of these cases and deaths have been estimated to occur in China because of a high prevalence of hepatitis B (HBV) and C virus (HCV) infection. The development of HCC might be attributed to a long-term effect of multiple factors at
Journal of Digestive Diseases 2014; 15; 684–693 the molecular level, one of which might be the imbalance of cell apoptosis and anti-apoptosis at the disease onset and during the progression of this disease. Thus, regulation of tumor cell apoptosis could be a strategy in the prevention of and treatment for HCC. Ursodeoxycholic acid (UDCA) is one of the secondary bile acids, and is widely used to treat chronic hepatobiliary diseases such as primary biliary cirrhosis (PBC) and cholelithiasis in the clinical setting. UDCA can reduce both the secretion of cholesterol into the bile and bile absorption from the intestines. Therefore, UDCA is used to dissolve cholesterol gallstones, while it is also known to have direct cytoprotective effects on hepatocytes.2 The mechanisms of action for UDCA are complicated, including the regulation of cellular signaling and the protection of hepatocytes against apoptosis.3 However, previous studies have demonstrated that UDCA is able to induce the apoptosis of human hepatoblastoma cells and the addition of UDCA to HCC cell culture leads to cell apoptosis in a dose-dependent manner.4,5 Thus, UDCA might play an important role in the chemoprevention of human cancers6–8 although its pro-apoptotic action remains to be clarified. UDCA-induced cell apoptosis occurs via the p53-independent pathway in human breast cancer cells, cell cycle regulation and an increase in the ratio of Bax to Bcl-2 in HCC cells.9,10 Another study showed that UDCA induced colon cancer cell apoptosis by suppressing cyclooxygenase-2 (COX-2) upregulation.11 In this study, we aimed to further evaluate the effects of UDCA on HCC cell apoptosis in vitro and explore the underlying molecular events playing in this progression. MATERIALS AND METHODS Cell culture Human HCC cell line HepG2, kindly provided by Professor Lu Juan SHAN from the Central Laboratory, The First Affiliated Hospital of Dalian Medical University (Dalian, Liaoning Province, China), were cultured in the RPMI1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 U/mL penicillin G (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL streptomycin (Sigma-Aldrich) at 37°C in an incubator (5% CO2; Thermo Fisher Scientific Inc., Waltham, MA, USA).
UDCA induction of HCC cell apoptosis
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UDCA UDCA was dissolved in dimethyl sulphoxide (DMSO) (both from Sigma-Aldrich) to achieve the final concentrations of 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively. UDCA with varied doses was used to treat the HCC cells for up to 72 h in order to identify its effect on cell proliferation and apoptosis as well as cell cycle. Cells treated with UDCA at 0.25, 0.50 and 0.75 mmol/L were further investigated to determine the expressions of apoptosis-associated factors. Methyl thiazolyl tetrazolium (MTT) assay for cell viability The cells at the logarithmic phase were seeded in 96-well plates at a density of 6 × 103 cells/well and incubated for 24 h. UDCA was then added to the medium and the cells were cultured for up to 72 h. After the 72-h culture, 20 μL of MTT based Cell Growth Determination Kit (Sigma-Aldrich) was added to each well and the cells were incubated for additional 4 h. After the growth medium was removed, 100 μL DMSO was added to each well and the optical density (OD) was measured at 570 nm using a spectrophotometer (UNICO, Shanghai, China). The experiments were performed in quintuplicate and repeated at least twice. The inhibitory rate (IR) of cell viability was calculated using the following formula: IR = (1 − OD570 of treated group [mean value]/OD570 of control [mean value]) × 100%. Flow cytometry (FCM) for cell cycle and apoptosis After the cells were treated with UDCA, cell apoptosis was assessed using an Annexin V/propidium iodide (PI) Flow Cytometric Assay with an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Briefly, HepG2 cells were gently trypsinized and washed twice with phosphate buffer saline (PBS), and 1 × 105 cells were then collected after centrifugated at 800 ×g for 5 min and resuspended in 500 μL of binding buffer. Annexin V-FITC of 2 μL 5 μL of PI were added, followed by cell incubation for 5 min at room temperature in the dark. To evaluate the cell cycle, the cells were further incubated with 5 μL of RNase and 100 μg/mL of PI at the room temperature in the dark for 30 min. Both cell samples were then analyzed by a flow cytometer (BD FACSCalibur). The cells without UDCA treatment were used as negative control. The experiments were performed in triplicate and repeated once.
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) To evaluate the late-stage HepG2 cell apoptosis induced by UDCA, TUNEL analysis was performed using a One-Step TUNEL Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer’s instructions. Briefly, after having been treated with UDCA at the final concentrations of 0.25, 0.50 and 0.75 mmol/L for 24 h, the HepG2 cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min and incubated in PBS with 0.1% Triton X-100 (Sigma-Aldrich) in an ice bath for 2 min. The TUNEL detection reagents were added and the cells were then analyzed by FCM. The cells without UDCA treatment was used as the negative control. The experiments were performed in triplicate and repeated once. Meanwhile, the HepG2 cells were seeded on coverslips in 6-well plates. Similarly, after treated with UDCA for 24 h, the coverslips were washed once with PBS, fixed with 4% paraformaldehyde for 30 min and then incubated in PBS with 0.1% Triton X-100 (Sigma-Aldrich) in an ice bath for 2 min for the addition of the TUNEL detection reagents. The apoptotic cells were observed and quantified under fluorescent microscopy. Immunocytochemistry HepG2 cells were seeded on coverslips in 6-well plates and incubated for 24 h, and were then treated with UDCA at the final concentrations of 0.25, 0.50 and 0.75 mmol/L for 48 h. The cells on the coverslips were fixed with 4% paraformaldehyde for 30 min and washed with PBS and were then treated with different primary antibodies, including the Mouse Anti-Human Bax monoclonal antibody (mAb) or the Rabbit AntiHuman Bcl-2 mAb (both from Maxim, Fuzhou, Fujian Province, China), Livin (D61D1) XP® Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) and anti-second mitochondria-derived activator of caspase (Smac)/Diablo Mouse mAb (Cell Signaling Technology), overnight at 4°C. On the next day, the coverslips were washed with PBS and further incubated with a MaxVisionTM Kit (Maxim) according to the manufacturer’s instructions. The cells were then stained with diaminobenzidine and counterstained with hematoxylin and eosin (HE). The coverslips were mounted with neutral gum and evaluated under microscope independently by two pathologists. The expressions of Livin, Smac, Bax and Bcl-2 were regareded as brown stain in the cells. The labeling index was calculated as the number of positive cells divided by the total number of cells on three coverslips.
Journal of Digestive Diseases 2014; 15; 684–693 FCM analysis of Livin and Smac expressions The HepG2 cells were treated with UDCA at final concentrations of 0.25, 0.50 or 0.75 mmol/L for 48 h, and were then gently trypsinized, transferred to a centrifuge tube and washed twice with PBS. Altogether 1 × 105 cells were collected after centrifugated at 800 ×g for 3 min and resuspended in 1 mL PBS. After having been fixed with 70% ethanol overnight at 4°C and washed twice with PBS, the cells were incubated in 100 μL Livin (D61D1) XP® Rabbit mAb or antiSmac/Diablo Mouse mAb (both from Cell Signaling Technology) at 4°C overnight. The next day, the cells were washed with PBS and the supernatants were discarded. Fluorescent labeled goat anti-mouse or goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) of 100 μL was added, and the samples were incubated in the dark at room temperature for 30 min before FCM. Cells treated with DMSO without UDCA were regarded as the negative control. The experiments were performed in triplicate and repeated once. Enzyme linked immunosorbent assay (ELISA) for caspase-3 activity To detect caspase-3 activity in cells treated with UDCA, the caspase-3 activity colorimetric assay kit (Beyotime Institute of Biotechnology) was used according to the manufacturer’s instructions. In brief, after having been treated with UDCA at the concentrations of 0.25, 0.50 and 0.75 mmol/L for 24 h or 48 h, HepG2 cells were added to a cell lysis solution, incubated on ice for 15 min and centrifugated at 10 000 ×g for 10 min at 4°C. The supernatant was then collected and added in the reaction buffer and coupling substrate, followed by incubation at 37°C in a water bath for 1 h. The absorbance values of the samples were determined using a spectrophotometer (UNICO) at a wavelength of 405 nm. The values were then calculated as the relative activity of caspase-3. The experiment was performed in triplicate and repeated once. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of Livin, Smac and caspase-3 mRNA levels Total cellular RNA was isolated from the cells after treated with UDCA for 24 or 48 h using Trizol (Invitrogen, Carlsbad, CA, USA) and were then reversely transcribed into cDNA using a PrimeScriptTM Reverse Transcription (RT) Reagent Kit (TaKaRa Biotechnology [Dalian] Co., Ltd., Dalian, Liaoning
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2014; 15; 684–693 Table 1.
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Primers used for quantitative reverse transcription polymerase chain reaction
Gene
Genbank symbol
Sequences
Length (bp)
Livin
NM_022161.2
104
Smac
NM_019887.4
Caspase-3
NM_004346.3
β-actin
NM_001101.3
Forward: 5′-TTCTATGACTGGCCGCTGACTG-3′ Reverse: 5′-TAGCAGAAGAAGCACCTCACCTTGT-3′ Forward: 5′-CTGTCGCGCAGCGTAACTTC-3′ Reverse: 5′-GGTTACTCCAAAGCCAATCGTCA-3′ Forward: 5′-GACTCTGGAATATCCCTGGACAACA-3′ Reverse: 5′-CTGAGGTTTGCTGCATCGACA-3′ Forward: 5′-TGGCACCCAGCACAATGAA-3′ Reverse: 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′
Province, China) according to the manufacturers’ instructions. Quantitative polymerase chain reaction (qPCR) was then performed using a SYBR® Premix Ex Taq™ Kit (TaKaRa Biotechnology [Dalian] Co., Ltd.) according to the manufacturer’s instructions. The sequences of the primers are shown in Table 1. The total reaction system was 20 μL and a two-step PCR reaction was performed under the following conditions: the first cycle of denaturation at 95°C for 30 s, 40 cycles at 95°C for 5 s and at 60°C for 20 s, and then a melting curve analysis at 95°C for 0 s, at 65°C for 15 s, and at 95°C for 0 s. The data were summarized as the percentages of control by normalized to the β-actin mRNA levels.
(a)
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138 143 186
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Statistical analysis Statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The values were expressed as mean ± standard deviation. A one-way analysis of variance (ANOVA) was used to determine the differences among all groups, and the Student– Newman–Keuls test was performed to compare all pairs of means following one-way ANOVA. P ≤ 0.05 was considered statistically significant.
Figure 1. The changes of HepG2 cell morphology after ursodeoxycholic acid (UDCA) treatment. (a) HepG2 cells without UDCA treatment. HepG2 cells treated with (b) 0.25 mmol/L, (c) 0.50 mmol/L, (d) 0.75 mmol/L and (e) 1.0 mmol/L UDCA, respectively.
RESULTS HepG2 cell morphology after UDCA treatment HepG2 cells became deformed and smaller after treated with UDCA compared with the control. The higher the concentration of UDCA, the more obvious the morphological changes of HepG2 cells. With a UDCA concentration of 1.0 mmol/L, in particular, some adherent cells were suspended from the bottom of the culture flask and the number of the remaining adherent cells was decreased (Fig. 1).
Reduction of HepG2 cell viability after UDCA treatment After having been treated with UDCA, the cell viability of HepG2 cells were significantly reduced in a dose-dependent manner (Fig. 2). Similarly, the longer the duration that HepG2 cells were treated with UDCA at the given concentrations, the lower the cell viability, indicating a time-dependent manner in the reduction of cell viability after UDCA treatment (Fig. 2).
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Figure 2. Methyl thiazolyl tetrazolium (MTT) analysis on the reduction of cell viability after ursodeoxycholic acid (UDCA) treatment at different time points and different concentrations of UDCA. , 24 h; , 48 h; , 72 h. PI
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Cell cycle distribution was analyzed by FCM (Fig. 5). Compared with the control cells, UDCA treatment arrested cells at the S phase of the cell cycle at both 24 h and 48 h. And UDCA treatment also showed a dose-dependent arrest of tumor cells at the S phase of the cell cycle. After having been treated with 0.75 mmol/L UDCA for 48 h, the percentage of HepG2 cells arrested at the S phase reached 90.87%.
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Figure 3. Cell apoptosis after ursodeoxycholic acid (UDCA) treatment detected by Annexin V/propidium iodide (PI) assay. (a) 0.25 mmol/L, (b) 0.5 mmol/L and (c) 0.75 mmol/L UDCA for 24 h. (d) 0.25 mmol/L, (e) 0.5 mmol/L and (f) 0.75 mmol/L UDCA for 48 h. Apoptotic rate (%)
Cell cycle arrested at S phase after UDCA treatment
102 101
Induction of cell apoptosis after UDCA treatment Apoptosis of HepG2 cells induced by UDCA at different concentrations were detected by Annexin V/PI stain (Figs. 3 and 4). The control cells showed an apoptotic rate of 0.36% ± 0.45% and 0.93% ± 0.68%, respectively, after treated with UDCA for 24 h and 48 h, whereas the cells induced by UDCA at the doses from 0.25 mmol/L to 0.75 mmol/L showed steadily increased apoptotic rates. Specifically, at a concentration of 0.75 mmol/L, the apoptotic rate was 10.03% ± 2.46% for 24 h and 23.95% ± 3.21% for 48 h (P < 0.05 compared with control). UDCA-induced HepG2 cell apoptosis was both dose-dependent and time-dependent. Furthermore, the TUNEL analysis also showed that with the increase of UDCA concentration, HepG2 cells showed more late-stage cell apoptosis, that is, the apoptotic rate of UDCA-treated cells was 8.82% ± 0.28% (0.25 mmol/L UDCA), 20.06% ± 1.44% (0.50 mmol/L UDCA) and 26.78% ± 1.63% (0.75 mmol/L UDCA), respectively, which was higher than that of the control cells (0.02% ± 0.005%) (P < 0.05).
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40 30 20 10 0 Control
0.25 0.50 UDCA concentration (mmol/L)
0.75
Figure 4. Annexin V/propidium iodide (PI) analysis for hepatocellular carcinoma cell apoptosis after treated with ursodeoxycholic acid (UDCA). , 24 h; , 48 h.
Changes in Livin, Smac, Bax and Bcl-2 protein expressions and Livin, Smac and caspase-3 mRNA expressions after UDCA treatment Livin, Smac, Bax and Bcl-2 protein expressions after UDCA treatment were assessed using
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Additionally, after treated with UDCA at the concentrations of 0.25, 0.50 and 0.75 mmol/L for 24 h or 48 h, qRT-PCT showed that Livin mRNA expression was decreased, whereas Smac and caspase-3 mRNA expressions were increased (Table 3).
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Smac proteins were higher than those in the control (Fig. 6 and Table 2). Furthermore, FCM also showed that the labeling rates of Livin and Smac proteins were 8.51% ± 0.77% and 0.54% ± 0.25%, respectively, in control cells, whereas those of Livin protein were decreased (4.01% ± 0.66% for 0.25 mmol/L UDCA, 1.20% ± 0.31% for 0.50 mmol/L UDCA and 0.84% ± 0.12% for 0.75 mmol/L UDCA, respectively) and of Smac protein were increased (1.55% ± 0.07% for 0.25 mmol/L UDCA, 1.81% ± 0.03% for 0.50 mmol/L UDCA and 3.54% ± 0.95% for 0.75 mmol/L UDCA, respectively) after UDCA treatment (Fig. 7).
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Figure 5. Cell cycle distribution of HepG2 cells by flow cytometry at 24h after treated with (a–d) 0, 0.25, 0.50 and 0.75 mmol/L ursodeoxycholic acid (UDCA), respectively, or at 48 h after treated with (e–h) 0, 0.25, 0.50 and 0.75 mmol/L UDCA, respectively. The part of oblique graticule presents as the distribution of HepG2 cells arrested in phase S. , Dip G1; , Dip G2; , Dip S.
immunohistochemistry. After treated with DMSO or UDCA at the concentrations of 0.25, 0.50 and 0.75 mmol/L for 48 h, the accumulation of Livin, Smac, Bax and Bcl-2 protein expressions in the HepG2 cells was indicated by brown color in the cytoplasm. Compared with the control cells, the expressions of Bcl-2 and Livin in HepG2 cells were decreased in a dose-dependent manner. In contrast, those of Bax and
Subsequently, we also detected the relative activity of caspase-3 protein using ELISA. Without UDCA treatment, the absorbance value of the cells was 0.01257 ± 0.0004 at 24 h and 0.02150 ± 0.0054 at 48 h. After the concentrations of UDCA was increased from 0.25 mmol/L to 0.75 mmol/L, the absorbance values were increased steadily (0.25 mmol/L UDCA: 0.019047 ± 0.0006 at 24 h and 0.025748 ± 0.0066 at 48 h; 0.50 mmol/L UDCA: 0.025202 ± 0.0003 at 24 h and 0.027636 ± 0.0069 at 48 h; and 0.75 mmol/L UDCA: 0.027738 ± 0.0004 at 24 h and 0.044247 ± 0.0057 at 48 h).
DISCUSSION In the current study, we demonstrated that UDCA reduced HCC cell viability and induced its apoptosis in both dose-dependent and time-dependent manners. However, UDCA had no effect on the viability of human liver Chang cells (normal human liver cells; data not shown). After treated with different concentrations of UDCA, HepG2 cells were arrested at the S phase of the cell cycle. Two different apoptosis assays (i.e. Annexin V/PI stain and TUNEL) revealed that UDCA treatment at different doses for 24 and 48 h induced both early-stage and late-stage apoptosis of HepG2 cells. At the gene level, UDCA treatment led to reduced expression of Bcl-2 and Livin proteins but increased expressions of Smac and Bax. Caspase-3
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Journal of Digestive Diseases 2014; 15; 684–693
Control
0.25 mmol/L UDCA
0.50 mmol/L UDCA
0.75 mmol/L UDCA
Bcl-2
Bax
Livin
Smac
Figure 6. Expressions of Bcl-2, Bax, Livin and Smac proteins in HepG2 cells after ursodeoxycholic acid (UDCA) treatment. The cells are grown and treated with (0.25 mmol/L, 0.5 mmol/L or 0.75 mmol/L) or without UDCA for up to 48 h and then subjected to immunocytochemistry.
Table 2. Expression of Bcl-2, Bax, Livin and Smac proteins in HepG2 cells after treated with ursodeoxycholic acid (UDCA) for 48 h UDCA
Bcl-2 Bax Livin Smac
Control
0.25 mmol/L
0.5 mmol/L
0.75 mmol/L
75.8 ± 2.1 13.1 ± 1.3 65.9 ± 0.7 10.4 ± 1.8
59.4 ± 3.5* 19.7 ± 0.9* 47.7 ± 1.1** 36.2 ± 2.6**
38.7 ± 1.2** 43.4 ± 2.2** 41.0 ± 2.3** 60.0 ± 3.1**
22.4 ± 2.4** 49.0 ± 2.0** 14.5 ± 2.3** 71.5 ± 1.6**
*P < 0.05 and **P < 0.01 compared with control.
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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3.63 31.07
2.49 27.98
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Figure 7.
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Fluorescent labeling rates of (a) Livin and (b) Smac proteins by flow cytometry.
Table 3. Quantitative reverse transcription polymerase chain reaction detection of Livin, Smac and caspase-3 mRNA expressions relative to β-actin UDCA
Livin Smac Caspase-3
24 h 48 h 24 h 48 h 24 h 48 h
Control
0.25 mmol/L
0.50 mmol/L
0.75 mmol/L
2.5250 ± 0.2419 15.9990 ± 3.1751 0.0591 ± 0.0166 0.3135 ± 0.0295 0.2581 ± 0.0335 0.2099 ± 0.0042
1.8705 ± 0.1168* 1.8862 ± 0.0532** 0.2705 ± 0.0350** 9.8468 ± 1.5805** 0.6790 ± 0.0309** 1.2415 ± 0.0358**
0.8634 ± 0.1045** 0.1948 ± 0.0871** 0.6318 ± 0.0575** 28.2446 ± 1.5832** 0.8370 ± 0.0512** 1.4619 ± 0.1121**
0.6819 ± 0.0399** 0.0311 ± 0.0056** 0.8152 ± 0.1067** 74.9187 ± 13.2296** 1.4599 ± 0.2644** 1.9807 ± 0.1715**
*P < 0.05, **P < 0.01 compared with control. UDCA, ursodeoxycholic acid.
activity was also induced by UDCA. All the abovementioned results indicated that UDCA played an important role in the cell apoptosis of HCC and could be used to treat or prevent against HCC. Hepatocarcinogenesis, like most other human cancers, is a multifactor-induced process. The activation of oncogenes and the silencing of tumor suppressor genes could lead to the clonal expansion of the affected hepatocytes. Moreover, this clonal expansion can also be caused by a selective decrease in the apoptosis rate in premalignant hepatocytes. Thus, the dysregulation of cell proliferation and apoptosis plays an important role in the pathogenesis and progression of HCC. As a proto-oncogene, Bcl-2 inhibits cell apoptosis rather than promoting proliferation, whereas Bax is a pro-apoptotic member of the Bcl-2
family proteins. The overexpression of Bax can induce cell apoptosis by the suppression of Bcl-2 activity.12 Furthermore, the ratio of Bax to Bcl-2 is more vital than Bax or Bcl-2 alone in drug-induced cell survival or apoptosis.13 Both positive and negative apoptotic regulators of these two Bcl-2 family members play a key role in controlling the apoptotic threshold.14 In addition, as a novel member of the inhibitor of the apoptotic protein (IAP) family, Livin was identified in 2000, and has been found to be expressed mainly in the nuclei and in a filamentous pattern throughout the cytoplasm.15 Livin can inhibit caspase activity by binding to caspase-3 and caspase7.16 In contrast, Smac is also known as a direct IAP binding protein with a low isoelectric point. Smac protein is expressed in the mitochondrial intermembrane space, interacts with IAP and
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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prevents them from inhibiting caspases after apoptotic stress. However, during the induction of apoptosis, Smac protein is released into the cytosol together with cytochrome c.17 As a pro-apoptotic protein, Smac binds and inhibits IAP in the cytoplasm. Smac can also bind to Livin protein, while Livin protein acts as a sink for Smac. The overexpression of Bcl-2 can prevent Smac release from the mitochondria, thereby inhibiting cell apoptosis.18 A previous study has shown that the expression of Smac protein in various human cancers was altered compared with the corresponding non-cancer tissues.19 To date, Smac is known as a critical factor in the development of some tumors. In the current study, we assessed the expressions of these proteins or mRNA after UDCA treatment and found that UDCA did modulate their expressions in HCC cells to induce cell apoptosis. Indeed, clinically, UDCA is an important drug for treating chronic liver diseases that acts as an antiapoptotic agent and protects hepatocytes against apoptosis induced by different stimuli.20–23 It has been reported that UDCA prevents cytochrome c release from the mitochondria20 and reduces Bcl-2 expression in biliary epithelial cells of PBC.24 However, several other studies have revealed that UDCA administration can inhibit the proliferation of carcinoma cells4,9,25 and induce HCC cell apoptosis in a concentrationdependent manner.5 This selective effect of UDCA on tumor cells compared with normal cells makes it possible for prophylaxis and treatment for HCC associated with chronic liver diseases. Moreover, it has been demonstrated that UDCA might prevent HCC development from HCV infection-associated liver cirrhosis.26 Thus, the effects of UDCA need to be investigated, although our current study has shown that UDCA could increase the ratio of Bax to Bcl-2 and the expression of Livin and Smac. But it is to be hoped that, besides its cytoprotective effect on normal hepatocytes, the pro-apoptotic effect of UDCA on HCC cells may make it possible to use it for the prevention against the occurrence of HCC derived from viral hepatitis-associated liver disease, especially in the case of clarified mechanisms. In conclusion, besides its cytoprotective effect on hepatocytes, UDCA can induce the apoptosis of HCC cells. This pro-apoptotic effect is dose-dependent and time-dependent. At the gene level, UDCA can modulate the expressions of Bax, Bcl-2, Smac, Livin and caspase-3 in HCC cells. Thus, our further studies will focus on the effects of UDCA on the treatment and prevention of HCC in vivo.
Journal of Digestive Diseases 2014; 15; 684–693 ACKNOWLEDGMENTS We would like to thank Dr Zhi Gang SUN from Dalian Medical University for the assistance with the immunohistochemistry and Professor Shi Jun LI of Dalian Medical University for an expert review of the experiment design. REFERENCES 1 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90. 2 Lazaridis KN, Gores GJ, Lindor KD. Ursodeoxycholic acid ‘mechanisms of action and clinical use in hepatobiliary disorders’. J Hepatol 2001; 35: 134–46. 3 Guicciardi ME, Gores GJ. Ursodeoxycholic acid cytoprotection: dancing with death receptors and survival pathways. Hepatology 2002; 35: 971–3. 4 Oyama K, Shiota G, Ito H, Murawaki Y, Kawasaki H. Reduction of hepatocarcinogenesis by ursodeoxycholic acid in rats. Carcinogenesis 2002; 23: 885–92. 5 Tsagarakis NJ, Drygiannakis I, Batistakis AG, Kolios G, Kouroumalis EA. A concentration-dependent effect of ursodeoxycholate on apoptosis and caspases activities of HepG2 hepatocellular carcinoma cells. Eur J Pharmacol 2010; 640: 1–7. 6 Pardi DS, Loftus EV Jr, Kremers WK, Keach J, Lindor KD. Ursodeoxycholic acid as a chemopreventive agent in patients with ulcerative colitis and primary sclerosing cholangitis. Gastroenterology 2003; 124: 889–93. 7 Alberts DS, Martínez ME, Hess LM et al.; Phoenix and Tucson Gastroenterologist Networks. Phase III trial of ursodeoxycholic acid to prevent colorectal adenoma recurrence. J Natl Cancer Inst 2005; 97: 846–53. 8 Thompson PA, Wertheim BC, Roe DJ et al. Gender modifies the effect of ursodeoxycholic acid in a randomized controlled trial in colorectal adenoma patients. Cancer Prev Res (Phila) 2009; 2: 1023–30. 9 Im EO, Choi YH, Paik KJ et al. Novel bile acid derivatives induce apoptosis via a p53-independent pathway in human breast carcinoma cells. Cancer Lett 2001; 163: 83–93. 10 Liu H, Qin CY, Han GQ, Xu HW, Meng M, Yang Z. Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines. World J Gastroenterol 2007; 13: 1652–8. 11 Khare S, Mustafi R, Cerda S et al. Ursodeoxycholic acid suppresses Cox-2 expression in colon cancer: roles of Ras, p38, and CCAAT/enhancer-binding protein. Nutr Cancer 2008; 60: 389–400. 12 Sorenson CM. Bcl-2 family members and disease. Biochim Biophys Acta 2004; 1644: 169–77. 13 Salomons GS, Brady HJ, Verwijs-Janssen M. The Baxα: Bcl-2 ratio modulates the response to dexamethasone in leukaemic cells and is highly variable in childhood acute leukaemia. Int J Cancer 1997; 71: 959–65. 14 Opferman JT, Korsmeyer SJ. Apoptosis in the development and maintenance of the immune system. Nat Immunol 2003; 4: 410–5. 15 Lin JH, Deng G, Huang Q, Morser J. KIAP, a novel member of the inhibitor of apoptosis protein family. Biochem Biophys Res Commun 2000; 279: 820–31. 16 Kasof GM, Gomes BC. Livin, a novel inhibitor of apoptosis protein family member. J Biol Chem 2001; 276: 3238–46.
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2014; 15; 684–693 17 Verhagen AM, Ekert PG, Pakusch M et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000; 102: 43–53. 18 Adrain C, Creagh EM, Martin SJ. Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. EMBO J 2001; 20: 6627–36. 19 Yoo NJ, Kim HS, Kim SY et al. Immunohistochemical analysis of Smac/DIABLO expression in human carcinomas and sarcomas. APMIS 2003; 111: 382–8. 20 Rodrigues CM, Ma X, Linehan-Stieers C, Fan G, Kren BT, Steer CJ. Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation. Cell Death Differ 1999; 6: 842–54. 21 Azzaroli F, Mehal W, Soroka CJ et al. Ursodeoxycholic acid diminishes Fas-ligand-induced apoptosis in mouse hepatocytes. Hepatology 2002; 36: 49–54.
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22 Arisawa S, Ishida K, Kameyama N et al. Ursodeoxycholic acid induces glutathione synthesis through activation of PI3K/Akt pathway in HepG2 cells. Biochem Pharmacol 2009; 77: 858–66. 23 Im E, Akare S, Powell A, Martinez JD. Ursodeoxycholic acid can suppress deoxycholic acid-induced apoptosis by stimulating Akt/PKB-dependent survival signaling. Nutr Cancer 2005; 51: 110–6. 24 Koga H, Sakisaka S, Ohishi M, Sata M, Tanikawa K. Nuclear DNA fragmentation and expression of Bcl-2 in primary biliary cirrhosis. Hepatology 1997; 25: 1077–84. 25 Wali RK, Stoiber D, Nguyen L et al. Ursodeoxycholic acid inhibits the initiation and postinitiation phases of azoxymethane-induced colonic tumor development. Cancer Epidemiol Biomarkers Prev 2002; 11: 1316–21. 26 Tarao K, Fujiyama S, Ohkawa S et al. Ursodiol use is possibly associated with lower incidence of hepatocellular carcinoma in hepatitis C virus-associated liver cirrhosis. Cancer Epidemiol Biomarkers Prev 2005; 14: 164–9.
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Journal of Digestive Diseases 2014; 15; 684–693
doi: 10.1111/1751-2980.12191
Original article
Ursodeoxycholic acid induces apoptosis of hepatocellular carcinoma cells in vitro Lei ZHU,*1 Lu Juan SHAN,† Yue Jian LIU,† Dan CHEN,‡ Xiao Guang XIAO§ & Yan LI* *Department of Gastroenterology, Shengjing Hospital, China Medical University, Shenyang, †Central Laboratory, ‡Department of Clinical Pathology, and §Clinical Laboratory of Gene Diagnosis, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning Province, China
OBJECTIVE: Ursodeoxycholic acid (UDCA) is widely used to treat chronic liver diseases, and its cytoprotective effect on normal hepatocytes has been shown. This study aimed to investigate the apoptotic effects of UDCA on hepatocellular carcinoma (HCC) cells and the underlying molecular events in vitro. METHODS: HCC cells were treated by UDCA at different doses and periods of time to assess cell morphology, viability, apoptosis and gene expression using methyl thiazolyl tetrazolium (MTT), Annexin V/propidium iodide (PI) stain, transferase dUTP nick end labeling (TUNEL), enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and quantitative reverse transcription polymerase chain reaction, respectively. KEY WORDS:
CONCLUSIONS: The induction of HCC cell apoptosis by UDCA was dose-dependent and timedependent and was mediated by the regulation of Bax to Bcl-2 ratio, the expressions of Smac and Livin, and caspase-3 expression and activity.
apoptosis, gene expression, hepatocellular carcinoma, ursodeoxycholic acid.
Correspondence to: Yan LI, Department of Gastroenterology, Shengjing Hospital, China Medical University, 36 Sanhao Street, Shenyang, Liaoning Province 110004, China. Email: [email protected] 1
Current address: Department of Gastroenterology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning Province 116011, China. Conflict of interest: None. © 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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RESULTS: UDCA treatment reduced cell viability but induced HCC cell apoptosis in dose-dependent and time-dependent manners. UDCA arrested HepG2 cells at phase S of the cell cycle. At the gene levels, UDCA downregulated Bcl-2 and second mitochondria-derived activator of caspase (Smac) protein expressions, but upregulated Bax and Livin proteins in HCC cells. At the highest concentration, UDCA inhibited Livin mRNA expression but increased Smac and caspase-3 mRNA expressions as well as the activity of caspase-3 in HCC cells.
INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common solid tumors worldwide. According to the cancer statistics, a total of 748 300 new cases and 695 900 cancer-related deaths were estimated to occur in 2008.1 Approximately half of these cases and deaths have been estimated to occur in China because of a high prevalence of hepatitis B (HBV) and C virus (HCV) infection. The development of HCC might be attributed to a long-term effect of multiple factors at
Journal of Digestive Diseases 2014; 15; 684–693 the molecular level, one of which might be the imbalance of cell apoptosis and anti-apoptosis at the disease onset and during the progression of this disease. Thus, regulation of tumor cell apoptosis could be a strategy in the prevention of and treatment for HCC. Ursodeoxycholic acid (UDCA) is one of the secondary bile acids, and is widely used to treat chronic hepatobiliary diseases such as primary biliary cirrhosis (PBC) and cholelithiasis in the clinical setting. UDCA can reduce both the secretion of cholesterol into the bile and bile absorption from the intestines. Therefore, UDCA is used to dissolve cholesterol gallstones, while it is also known to have direct cytoprotective effects on hepatocytes.2 The mechanisms of action for UDCA are complicated, including the regulation of cellular signaling and the protection of hepatocytes against apoptosis.3 However, previous studies have demonstrated that UDCA is able to induce the apoptosis of human hepatoblastoma cells and the addition of UDCA to HCC cell culture leads to cell apoptosis in a dose-dependent manner.4,5 Thus, UDCA might play an important role in the chemoprevention of human cancers6–8 although its pro-apoptotic action remains to be clarified. UDCA-induced cell apoptosis occurs via the p53-independent pathway in human breast cancer cells, cell cycle regulation and an increase in the ratio of Bax to Bcl-2 in HCC cells.9,10 Another study showed that UDCA induced colon cancer cell apoptosis by suppressing cyclooxygenase-2 (COX-2) upregulation.11 In this study, we aimed to further evaluate the effects of UDCA on HCC cell apoptosis in vitro and explore the underlying molecular events playing in this progression. MATERIALS AND METHODS Cell culture Human HCC cell line HepG2, kindly provided by Professor Lu Juan SHAN from the Central Laboratory, The First Affiliated Hospital of Dalian Medical University (Dalian, Liaoning Province, China), were cultured in the RPMI1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 U/mL penicillin G (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL streptomycin (Sigma-Aldrich) at 37°C in an incubator (5% CO2; Thermo Fisher Scientific Inc., Waltham, MA, USA).
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UDCA UDCA was dissolved in dimethyl sulphoxide (DMSO) (both from Sigma-Aldrich) to achieve the final concentrations of 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively. UDCA with varied doses was used to treat the HCC cells for up to 72 h in order to identify its effect on cell proliferation and apoptosis as well as cell cycle. Cells treated with UDCA at 0.25, 0.50 and 0.75 mmol/L were further investigated to determine the expressions of apoptosis-associated factors. Methyl thiazolyl tetrazolium (MTT) assay for cell viability The cells at the logarithmic phase were seeded in 96-well plates at a density of 6 × 103 cells/well and incubated for 24 h. UDCA was then added to the medium and the cells were cultured for up to 72 h. After the 72-h culture, 20 μL of MTT based Cell Growth Determination Kit (Sigma-Aldrich) was added to each well and the cells were incubated for additional 4 h. After the growth medium was removed, 100 μL DMSO was added to each well and the optical density (OD) was measured at 570 nm using a spectrophotometer (UNICO, Shanghai, China). The experiments were performed in quintuplicate and repeated at least twice. The inhibitory rate (IR) of cell viability was calculated using the following formula: IR = (1 − OD570 of treated group [mean value]/OD570 of control [mean value]) × 100%. Flow cytometry (FCM) for cell cycle and apoptosis After the cells were treated with UDCA, cell apoptosis was assessed using an Annexin V/propidium iodide (PI) Flow Cytometric Assay with an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Briefly, HepG2 cells were gently trypsinized and washed twice with phosphate buffer saline (PBS), and 1 × 105 cells were then collected after centrifugated at 800 ×g for 5 min and resuspended in 500 μL of binding buffer. Annexin V-FITC of 2 μL 5 μL of PI were added, followed by cell incubation for 5 min at room temperature in the dark. To evaluate the cell cycle, the cells were further incubated with 5 μL of RNase and 100 μg/mL of PI at the room temperature in the dark for 30 min. Both cell samples were then analyzed by a flow cytometer (BD FACSCalibur). The cells without UDCA treatment were used as negative control. The experiments were performed in triplicate and repeated once.
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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) To evaluate the late-stage HepG2 cell apoptosis induced by UDCA, TUNEL analysis was performed using a One-Step TUNEL Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer’s instructions. Briefly, after having been treated with UDCA at the final concentrations of 0.25, 0.50 and 0.75 mmol/L for 24 h, the HepG2 cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min and incubated in PBS with 0.1% Triton X-100 (Sigma-Aldrich) in an ice bath for 2 min. The TUNEL detection reagents were added and the cells were then analyzed by FCM. The cells without UDCA treatment was used as the negative control. The experiments were performed in triplicate and repeated once. Meanwhile, the HepG2 cells were seeded on coverslips in 6-well plates. Similarly, after treated with UDCA for 24 h, the coverslips were washed once with PBS, fixed with 4% paraformaldehyde for 30 min and then incubated in PBS with 0.1% Triton X-100 (Sigma-Aldrich) in an ice bath for 2 min for the addition of the TUNEL detection reagents. The apoptotic cells were observed and quantified under fluorescent microscopy. Immunocytochemistry HepG2 cells were seeded on coverslips in 6-well plates and incubated for 24 h, and were then treated with UDCA at the final concentrations of 0.25, 0.50 and 0.75 mmol/L for 48 h. The cells on the coverslips were fixed with 4% paraformaldehyde for 30 min and washed with PBS and were then treated with different primary antibodies, including the Mouse Anti-Human Bax monoclonal antibody (mAb) or the Rabbit AntiHuman Bcl-2 mAb (both from Maxim, Fuzhou, Fujian Province, China), Livin (D61D1) XP® Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) and anti-second mitochondria-derived activator of caspase (Smac)/Diablo Mouse mAb (Cell Signaling Technology), overnight at 4°C. On the next day, the coverslips were washed with PBS and further incubated with a MaxVisionTM Kit (Maxim) according to the manufacturer’s instructions. The cells were then stained with diaminobenzidine and counterstained with hematoxylin and eosin (HE). The coverslips were mounted with neutral gum and evaluated under microscope independently by two pathologists. The expressions of Livin, Smac, Bax and Bcl-2 were regareded as brown stain in the cells. The labeling index was calculated as the number of positive cells divided by the total number of cells on three coverslips.
Journal of Digestive Diseases 2014; 15; 684–693 FCM analysis of Livin and Smac expressions The HepG2 cells were treated with UDCA at final concentrations of 0.25, 0.50 or 0.75 mmol/L for 48 h, and were then gently trypsinized, transferred to a centrifuge tube and washed twice with PBS. Altogether 1 × 105 cells were collected after centrifugated at 800 ×g for 3 min and resuspended in 1 mL PBS. After having been fixed with 70% ethanol overnight at 4°C and washed twice with PBS, the cells were incubated in 100 μL Livin (D61D1) XP® Rabbit mAb or antiSmac/Diablo Mouse mAb (both from Cell Signaling Technology) at 4°C overnight. The next day, the cells were washed with PBS and the supernatants were discarded. Fluorescent labeled goat anti-mouse or goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) of 100 μL was added, and the samples were incubated in the dark at room temperature for 30 min before FCM. Cells treated with DMSO without UDCA were regarded as the negative control. The experiments were performed in triplicate and repeated once. Enzyme linked immunosorbent assay (ELISA) for caspase-3 activity To detect caspase-3 activity in cells treated with UDCA, the caspase-3 activity colorimetric assay kit (Beyotime Institute of Biotechnology) was used according to the manufacturer’s instructions. In brief, after having been treated with UDCA at the concentrations of 0.25, 0.50 and 0.75 mmol/L for 24 h or 48 h, HepG2 cells were added to a cell lysis solution, incubated on ice for 15 min and centrifugated at 10 000 ×g for 10 min at 4°C. The supernatant was then collected and added in the reaction buffer and coupling substrate, followed by incubation at 37°C in a water bath for 1 h. The absorbance values of the samples were determined using a spectrophotometer (UNICO) at a wavelength of 405 nm. The values were then calculated as the relative activity of caspase-3. The experiment was performed in triplicate and repeated once. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of Livin, Smac and caspase-3 mRNA levels Total cellular RNA was isolated from the cells after treated with UDCA for 24 or 48 h using Trizol (Invitrogen, Carlsbad, CA, USA) and were then reversely transcribed into cDNA using a PrimeScriptTM Reverse Transcription (RT) Reagent Kit (TaKaRa Biotechnology [Dalian] Co., Ltd., Dalian, Liaoning
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Primers used for quantitative reverse transcription polymerase chain reaction
Gene
Genbank symbol
Sequences
Length (bp)
Livin
NM_022161.2
104
Smac
NM_019887.4
Caspase-3
NM_004346.3
β-actin
NM_001101.3
Forward: 5′-TTCTATGACTGGCCGCTGACTG-3′ Reverse: 5′-TAGCAGAAGAAGCACCTCACCTTGT-3′ Forward: 5′-CTGTCGCGCAGCGTAACTTC-3′ Reverse: 5′-GGTTACTCCAAAGCCAATCGTCA-3′ Forward: 5′-GACTCTGGAATATCCCTGGACAACA-3′ Reverse: 5′-CTGAGGTTTGCTGCATCGACA-3′ Forward: 5′-TGGCACCCAGCACAATGAA-3′ Reverse: 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′
Province, China) according to the manufacturers’ instructions. Quantitative polymerase chain reaction (qPCR) was then performed using a SYBR® Premix Ex Taq™ Kit (TaKaRa Biotechnology [Dalian] Co., Ltd.) according to the manufacturer’s instructions. The sequences of the primers are shown in Table 1. The total reaction system was 20 μL and a two-step PCR reaction was performed under the following conditions: the first cycle of denaturation at 95°C for 30 s, 40 cycles at 95°C for 5 s and at 60°C for 20 s, and then a melting curve analysis at 95°C for 0 s, at 65°C for 15 s, and at 95°C for 0 s. The data were summarized as the percentages of control by normalized to the β-actin mRNA levels.
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138 143 186
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Statistical analysis Statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The values were expressed as mean ± standard deviation. A one-way analysis of variance (ANOVA) was used to determine the differences among all groups, and the Student– Newman–Keuls test was performed to compare all pairs of means following one-way ANOVA. P ≤ 0.05 was considered statistically significant.
Figure 1. The changes of HepG2 cell morphology after ursodeoxycholic acid (UDCA) treatment. (a) HepG2 cells without UDCA treatment. HepG2 cells treated with (b) 0.25 mmol/L, (c) 0.50 mmol/L, (d) 0.75 mmol/L and (e) 1.0 mmol/L UDCA, respectively.
RESULTS HepG2 cell morphology after UDCA treatment HepG2 cells became deformed and smaller after treated with UDCA compared with the control. The higher the concentration of UDCA, the more obvious the morphological changes of HepG2 cells. With a UDCA concentration of 1.0 mmol/L, in particular, some adherent cells were suspended from the bottom of the culture flask and the number of the remaining adherent cells was decreased (Fig. 1).
Reduction of HepG2 cell viability after UDCA treatment After having been treated with UDCA, the cell viability of HepG2 cells were significantly reduced in a dose-dependent manner (Fig. 2). Similarly, the longer the duration that HepG2 cells were treated with UDCA at the given concentrations, the lower the cell viability, indicating a time-dependent manner in the reduction of cell viability after UDCA treatment (Fig. 2).
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Cell cycle distribution was analyzed by FCM (Fig. 5). Compared with the control cells, UDCA treatment arrested cells at the S phase of the cell cycle at both 24 h and 48 h. And UDCA treatment also showed a dose-dependent arrest of tumor cells at the S phase of the cell cycle. After having been treated with 0.75 mmol/L UDCA for 48 h, the percentage of HepG2 cells arrested at the S phase reached 90.87%.
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Figure 3. Cell apoptosis after ursodeoxycholic acid (UDCA) treatment detected by Annexin V/propidium iodide (PI) assay. (a) 0.25 mmol/L, (b) 0.5 mmol/L and (c) 0.75 mmol/L UDCA for 24 h. (d) 0.25 mmol/L, (e) 0.5 mmol/L and (f) 0.75 mmol/L UDCA for 48 h. Apoptotic rate (%)
Cell cycle arrested at S phase after UDCA treatment
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Induction of cell apoptosis after UDCA treatment Apoptosis of HepG2 cells induced by UDCA at different concentrations were detected by Annexin V/PI stain (Figs. 3 and 4). The control cells showed an apoptotic rate of 0.36% ± 0.45% and 0.93% ± 0.68%, respectively, after treated with UDCA for 24 h and 48 h, whereas the cells induced by UDCA at the doses from 0.25 mmol/L to 0.75 mmol/L showed steadily increased apoptotic rates. Specifically, at a concentration of 0.75 mmol/L, the apoptotic rate was 10.03% ± 2.46% for 24 h and 23.95% ± 3.21% for 48 h (P < 0.05 compared with control). UDCA-induced HepG2 cell apoptosis was both dose-dependent and time-dependent. Furthermore, the TUNEL analysis also showed that with the increase of UDCA concentration, HepG2 cells showed more late-stage cell apoptosis, that is, the apoptotic rate of UDCA-treated cells was 8.82% ± 0.28% (0.25 mmol/L UDCA), 20.06% ± 1.44% (0.50 mmol/L UDCA) and 26.78% ± 1.63% (0.75 mmol/L UDCA), respectively, which was higher than that of the control cells (0.02% ± 0.005%) (P < 0.05).
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Figure 4. Annexin V/propidium iodide (PI) analysis for hepatocellular carcinoma cell apoptosis after treated with ursodeoxycholic acid (UDCA). , 24 h; , 48 h.
Changes in Livin, Smac, Bax and Bcl-2 protein expressions and Livin, Smac and caspase-3 mRNA expressions after UDCA treatment Livin, Smac, Bax and Bcl-2 protein expressions after UDCA treatment were assessed using
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Additionally, after treated with UDCA at the concentrations of 0.25, 0.50 and 0.75 mmol/L for 24 h or 48 h, qRT-PCT showed that Livin mRNA expression was decreased, whereas Smac and caspase-3 mRNA expressions were increased (Table 3).
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Smac proteins were higher than those in the control (Fig. 6 and Table 2). Furthermore, FCM also showed that the labeling rates of Livin and Smac proteins were 8.51% ± 0.77% and 0.54% ± 0.25%, respectively, in control cells, whereas those of Livin protein were decreased (4.01% ± 0.66% for 0.25 mmol/L UDCA, 1.20% ± 0.31% for 0.50 mmol/L UDCA and 0.84% ± 0.12% for 0.75 mmol/L UDCA, respectively) and of Smac protein were increased (1.55% ± 0.07% for 0.25 mmol/L UDCA, 1.81% ± 0.03% for 0.50 mmol/L UDCA and 3.54% ± 0.95% for 0.75 mmol/L UDCA, respectively) after UDCA treatment (Fig. 7).
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Figure 5. Cell cycle distribution of HepG2 cells by flow cytometry at 24h after treated with (a–d) 0, 0.25, 0.50 and 0.75 mmol/L ursodeoxycholic acid (UDCA), respectively, or at 48 h after treated with (e–h) 0, 0.25, 0.50 and 0.75 mmol/L UDCA, respectively. The part of oblique graticule presents as the distribution of HepG2 cells arrested in phase S. , Dip G1; , Dip G2; , Dip S.
immunohistochemistry. After treated with DMSO or UDCA at the concentrations of 0.25, 0.50 and 0.75 mmol/L for 48 h, the accumulation of Livin, Smac, Bax and Bcl-2 protein expressions in the HepG2 cells was indicated by brown color in the cytoplasm. Compared with the control cells, the expressions of Bcl-2 and Livin in HepG2 cells were decreased in a dose-dependent manner. In contrast, those of Bax and
Subsequently, we also detected the relative activity of caspase-3 protein using ELISA. Without UDCA treatment, the absorbance value of the cells was 0.01257 ± 0.0004 at 24 h and 0.02150 ± 0.0054 at 48 h. After the concentrations of UDCA was increased from 0.25 mmol/L to 0.75 mmol/L, the absorbance values were increased steadily (0.25 mmol/L UDCA: 0.019047 ± 0.0006 at 24 h and 0.025748 ± 0.0066 at 48 h; 0.50 mmol/L UDCA: 0.025202 ± 0.0003 at 24 h and 0.027636 ± 0.0069 at 48 h; and 0.75 mmol/L UDCA: 0.027738 ± 0.0004 at 24 h and 0.044247 ± 0.0057 at 48 h).
DISCUSSION In the current study, we demonstrated that UDCA reduced HCC cell viability and induced its apoptosis in both dose-dependent and time-dependent manners. However, UDCA had no effect on the viability of human liver Chang cells (normal human liver cells; data not shown). After treated with different concentrations of UDCA, HepG2 cells were arrested at the S phase of the cell cycle. Two different apoptosis assays (i.e. Annexin V/PI stain and TUNEL) revealed that UDCA treatment at different doses for 24 and 48 h induced both early-stage and late-stage apoptosis of HepG2 cells. At the gene level, UDCA treatment led to reduced expression of Bcl-2 and Livin proteins but increased expressions of Smac and Bax. Caspase-3
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Journal of Digestive Diseases 2014; 15; 684–693
Control
0.25 mmol/L UDCA
0.50 mmol/L UDCA
0.75 mmol/L UDCA
Bcl-2
Bax
Livin
Smac
Figure 6. Expressions of Bcl-2, Bax, Livin and Smac proteins in HepG2 cells after ursodeoxycholic acid (UDCA) treatment. The cells are grown and treated with (0.25 mmol/L, 0.5 mmol/L or 0.75 mmol/L) or without UDCA for up to 48 h and then subjected to immunocytochemistry.
Table 2. Expression of Bcl-2, Bax, Livin and Smac proteins in HepG2 cells after treated with ursodeoxycholic acid (UDCA) for 48 h UDCA
Bcl-2 Bax Livin Smac
Control
0.25 mmol/L
0.5 mmol/L
0.75 mmol/L
75.8 ± 2.1 13.1 ± 1.3 65.9 ± 0.7 10.4 ± 1.8
59.4 ± 3.5* 19.7 ± 0.9* 47.7 ± 1.1** 36.2 ± 2.6**
38.7 ± 1.2** 43.4 ± 2.2** 41.0 ± 2.3** 60.0 ± 3.1**
22.4 ± 2.4** 49.0 ± 2.0** 14.5 ± 2.3** 71.5 ± 1.6**
*P < 0.05 and **P < 0.01 compared with control.
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
Journal of Digestive Diseases 2014; 15; 684–693
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(a) 200
Counts
160 M1
120 80
Marker
Left, Right
Events
% Gated
% Total
Mean
Geo Mean
40
All M1
1, 9647 34, 5623
9505 71
100.00 0.75
95.05 0.71
8.44 45.77
6.51 43.25
0 100
(b)
101
102
103
104
200
Counts
160 M1
120 80 40
Marker
Left, Right
Events
% Gated
% Total
Mean
Geo Mean
All M1
1, 9647 18, 3051
9568 188
100.00 1.96
95.68 1.88
3.63 31.07
2.49 27.98
0 100
Figure 7.
101
102
103
104
Fluorescent labeling rates of (a) Livin and (b) Smac proteins by flow cytometry.
Table 3. Quantitative reverse transcription polymerase chain reaction detection of Livin, Smac and caspase-3 mRNA expressions relative to β-actin UDCA
Livin Smac Caspase-3
24 h 48 h 24 h 48 h 24 h 48 h
Control
0.25 mmol/L
0.50 mmol/L
0.75 mmol/L
2.5250 ± 0.2419 15.9990 ± 3.1751 0.0591 ± 0.0166 0.3135 ± 0.0295 0.2581 ± 0.0335 0.2099 ± 0.0042
1.8705 ± 0.1168* 1.8862 ± 0.0532** 0.2705 ± 0.0350** 9.8468 ± 1.5805** 0.6790 ± 0.0309** 1.2415 ± 0.0358**
0.8634 ± 0.1045** 0.1948 ± 0.0871** 0.6318 ± 0.0575** 28.2446 ± 1.5832** 0.8370 ± 0.0512** 1.4619 ± 0.1121**
0.6819 ± 0.0399** 0.0311 ± 0.0056** 0.8152 ± 0.1067** 74.9187 ± 13.2296** 1.4599 ± 0.2644** 1.9807 ± 0.1715**
*P < 0.05, **P < 0.01 compared with control. UDCA, ursodeoxycholic acid.
activity was also induced by UDCA. All the abovementioned results indicated that UDCA played an important role in the cell apoptosis of HCC and could be used to treat or prevent against HCC. Hepatocarcinogenesis, like most other human cancers, is a multifactor-induced process. The activation of oncogenes and the silencing of tumor suppressor genes could lead to the clonal expansion of the affected hepatocytes. Moreover, this clonal expansion can also be caused by a selective decrease in the apoptosis rate in premalignant hepatocytes. Thus, the dysregulation of cell proliferation and apoptosis plays an important role in the pathogenesis and progression of HCC. As a proto-oncogene, Bcl-2 inhibits cell apoptosis rather than promoting proliferation, whereas Bax is a pro-apoptotic member of the Bcl-2
family proteins. The overexpression of Bax can induce cell apoptosis by the suppression of Bcl-2 activity.12 Furthermore, the ratio of Bax to Bcl-2 is more vital than Bax or Bcl-2 alone in drug-induced cell survival or apoptosis.13 Both positive and negative apoptotic regulators of these two Bcl-2 family members play a key role in controlling the apoptotic threshold.14 In addition, as a novel member of the inhibitor of the apoptotic protein (IAP) family, Livin was identified in 2000, and has been found to be expressed mainly in the nuclei and in a filamentous pattern throughout the cytoplasm.15 Livin can inhibit caspase activity by binding to caspase-3 and caspase7.16 In contrast, Smac is also known as a direct IAP binding protein with a low isoelectric point. Smac protein is expressed in the mitochondrial intermembrane space, interacts with IAP and
© 2014 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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prevents them from inhibiting caspases after apoptotic stress. However, during the induction of apoptosis, Smac protein is released into the cytosol together with cytochrome c.17 As a pro-apoptotic protein, Smac binds and inhibits IAP in the cytoplasm. Smac can also bind to Livin protein, while Livin protein acts as a sink for Smac. The overexpression of Bcl-2 can prevent Smac release from the mitochondria, thereby inhibiting cell apoptosis.18 A previous study has shown that the expression of Smac protein in various human cancers was altered compared with the corresponding non-cancer tissues.19 To date, Smac is known as a critical factor in the development of some tumors. In the current study, we assessed the expressions of these proteins or mRNA after UDCA treatment and found that UDCA did modulate their expressions in HCC cells to induce cell apoptosis. Indeed, clinically, UDCA is an important drug for treating chronic liver diseases that acts as an antiapoptotic agent and protects hepatocytes against apoptosis induced by different stimuli.20–23 It has been reported that UDCA prevents cytochrome c release from the mitochondria20 and reduces Bcl-2 expression in biliary epithelial cells of PBC.24 However, several other studies have revealed that UDCA administration can inhibit the proliferation of carcinoma cells4,9,25 and induce HCC cell apoptosis in a concentrationdependent manner.5 This selective effect of UDCA on tumor cells compared with normal cells makes it possible for prophylaxis and treatment for HCC associated with chronic liver diseases. Moreover, it has been demonstrated that UDCA might prevent HCC development from HCV infection-associated liver cirrhosis.26 Thus, the effects of UDCA need to be investigated, although our current study has shown that UDCA could increase the ratio of Bax to Bcl-2 and the expression of Livin and Smac. But it is to be hoped that, besides its cytoprotective effect on normal hepatocytes, the pro-apoptotic effect of UDCA on HCC cells may make it possible to use it for the prevention against the occurrence of HCC derived from viral hepatitis-associated liver disease, especially in the case of clarified mechanisms. In conclusion, besides its cytoprotective effect on hepatocytes, UDCA can induce the apoptosis of HCC cells. This pro-apoptotic effect is dose-dependent and time-dependent. At the gene level, UDCA can modulate the expressions of Bax, Bcl-2, Smac, Livin and caspase-3 in HCC cells. Thus, our further studies will focus on the effects of UDCA on the treatment and prevention of HCC in vivo.
Journal of Digestive Diseases 2014; 15; 684–693 ACKNOWLEDGMENTS We would like to thank Dr Zhi Gang SUN from Dalian Medical University for the assistance with the immunohistochemistry and Professor Shi Jun LI of Dalian Medical University for an expert review of the experiment design. REFERENCES 1 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90. 2 Lazaridis KN, Gores GJ, Lindor KD. Ursodeoxycholic acid ‘mechanisms of action and clinical use in hepatobiliary disorders’. J Hepatol 2001; 35: 134–46. 3 Guicciardi ME, Gores GJ. Ursodeoxycholic acid cytoprotection: dancing with death receptors and survival pathways. Hepatology 2002; 35: 971–3. 4 Oyama K, Shiota G, Ito H, Murawaki Y, Kawasaki H. Reduction of hepatocarcinogenesis by ursodeoxycholic acid in rats. Carcinogenesis 2002; 23: 885–92. 5 Tsagarakis NJ, Drygiannakis I, Batistakis AG, Kolios G, Kouroumalis EA. A concentration-dependent effect of ursodeoxycholate on apoptosis and caspases activities of HepG2 hepatocellular carcinoma cells. Eur J Pharmacol 2010; 640: 1–7. 6 Pardi DS, Loftus EV Jr, Kremers WK, Keach J, Lindor KD. Ursodeoxycholic acid as a chemopreventive agent in patients with ulcerative colitis and primary sclerosing cholangitis. Gastroenterology 2003; 124: 889–93. 7 Alberts DS, Martínez ME, Hess LM et al.; Phoenix and Tucson Gastroenterologist Networks. Phase III trial of ursodeoxycholic acid to prevent colorectal adenoma recurrence. J Natl Cancer Inst 2005; 97: 846–53. 8 Thompson PA, Wertheim BC, Roe DJ et al. Gender modifies the effect of ursodeoxycholic acid in a randomized controlled trial in colorectal adenoma patients. Cancer Prev Res (Phila) 2009; 2: 1023–30. 9 Im EO, Choi YH, Paik KJ et al. Novel bile acid derivatives induce apoptosis via a p53-independent pathway in human breast carcinoma cells. Cancer Lett 2001; 163: 83–93. 10 Liu H, Qin CY, Han GQ, Xu HW, Meng M, Yang Z. Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines. World J Gastroenterol 2007; 13: 1652–8. 11 Khare S, Mustafi R, Cerda S et al. Ursodeoxycholic acid suppresses Cox-2 expression in colon cancer: roles of Ras, p38, and CCAAT/enhancer-binding protein. Nutr Cancer 2008; 60: 389–400. 12 Sorenson CM. Bcl-2 family members and disease. Biochim Biophys Acta 2004; 1644: 169–77. 13 Salomons GS, Brady HJ, Verwijs-Janssen M. The Baxα: Bcl-2 ratio modulates the response to dexamethasone in leukaemic cells and is highly variable in childhood acute leukaemia. Int J Cancer 1997; 71: 959–65. 14 Opferman JT, Korsmeyer SJ. Apoptosis in the development and maintenance of the immune system. Nat Immunol 2003; 4: 410–5. 15 Lin JH, Deng G, Huang Q, Morser J. KIAP, a novel member of the inhibitor of apoptosis protein family. Biochem Biophys Res Commun 2000; 279: 820–31. 16 Kasof GM, Gomes BC. Livin, a novel inhibitor of apoptosis protein family member. J Biol Chem 2001; 276: 3238–46.
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