| Background and Objective:Hepatocellular carcinoma(HCC)is one of the most common malignant neoplasms worldwide and one of the leading causes of malignancy-related death in China.Surgery is the most effective option, unfortunately,the majority of patients with HCC are not amenable to surgery at diagnosis.After curative resection,the most common problem is recurrence. The complex treatments are adopted to improve the curative effects of the liver carcinoma.Presently,one of the main approaches to treat HCC is cytotoxic chemotherapy.Therefore,it is necessary to develop new drugs to treat HCC.Bile acids are polar derivatives of cholesterol essential for the absorption of dietary lipids and regulate the transcription of genes that control cholesterol homeostasis. Depending on the nature of chemical structures,different bile acids exhibit distinct biological effects.The primary bile acids chenodeoxycholic acid(CDCA)and cholic acid(CA)are the end products of hepatic cholesterol metabolites.Once they enter the colon,they are actted upon by anaerobic bacteria to produce secondary bile acids. CDCA is modified into lithocholic acid(LCA),and CA is modified into deoxycholic acid(DCA).Recently,it has been reported that synthetic chenodeoxycholic acid derivatives {N-[(3α,5β,7α)-3,7-dihydroxy-24-oxocholan-24-yl]L-phenyl alanine benzyl ester)(HS-1199)and(N-[(3α,5β,7α)-3,7-dihydroxy-24-oxocholan-24-yl] β-alanine benzyl ester}(HS-1200)induced apoptosis in several human cancer cells, including human breast carcinoma cells,human prostate cancer cells,human osteosarcoma cells,and human cervical carcinoma cells.Between the two synthetic chenodeoxycholic acid derivatives,HS-1200 showed stronger activity than HS-1199. However,the apoptotic effect of HS-1200 on human hepatoma cells was rarely reported.In the present study,we are to investigate whether HS-1200 has anti-cancer effects on human hepatoma cells and cytoxicity on normal hepatic cells and to explore the molecular mechanisms.Furthermore,we are to use BALB/c immunodeficient mice bearing s.c.xenografts of BEL7402 human hepatoma cells and to evaluate the effects of HS-1200 on tumor growth.Methods:1.The hepatoma cell line BEL7402 was treated with different concentrations of chenodeoxycholic acid(CDCA)derivative HS-1200 at different time.The cell viability of hepatoma cells was examined by 3-(4,5-dimethylthiazol-2, 1)-2,5 diphanytetrazolium bromide assay.Apoptosis of hepatoma cell line BEL7402 was detected by flow cytometry,chromatin staining with Hoechst 33258,and DNA fragmentation.The protein levels of Bcl-2,Bax,cytochrome c,caspase-3,caspase-8,caspase-9 and PARP were determined by Western blot analysis.The caspase-8 specific inhibitor z-IETDfmk and caspase-9 specific inhibitor z-LEHDfmk were used to assess the role of caspase-8 and caspase-9 in apoptosis.Alterations in mitochondrial membrane potential(△m?)were analyzed by flow cytometry using the△m?-sensitive dye,JC-1(5,5',6,6' tetrachloro-1,1',3,3' tetraethylben-zimidazolcarbocyanine iodide.2.BEL-7402 cells were cultured at 37℃,5%CO2 humid conditions.The culture medium was DMEM with 10%FBS.The cells were digested by 0.25% trypsin.A single cell suspension of BEL-7402 cells with the concentration of 5.0×106/ml was prepared for injecting.The 0.2ml cells were injected s.c.into the left flank of nude mice under the aseptic condition.The mice were raised at the aseptic constant temperature,constant humidity environment and were observed every day. The tumor size was measured every after the tumor was appeared.When the tumors were established well in mice,i.p.injection of different concentrations of HS-1200 (20mg/kg,40mg/kg,60mg/kg)once a day for 5 times.Then tumor size and weight were measured to evaluate the tumor growth.Molecular biomarkers representing proliferation and apoptosis were determined.Furthermore,western blot analysis was employed to investigate the related molecular mechanisms.Results 1.MTT assay showed that chenodeoxycholic acid(CDCA)derivative HS-1200 inhibited the proliferation of BEL7402 cells and its effect was dose-and time-dependent.BEL7402 cells were treated with chenodeoxycholic acid(CDCA) derivative HS-1200 at 40μM for 24h,48h and 72h,the inhibitory rates were (29.25±0.54)%,(38.54±1.28)%and(50.29±1.81)%,respectively;Incubation with chenodeoxycholic acid(CDCA)derivative HS-1200 at 80μM for 24h,48h and 72h, the inhibitory rates were(55.26±1.37)%,(62.49±1.42)%and(71.34±2.04)%, respectively.Statistical analysis of the data showed statistically significant differences between the treatment and control group and between the each other treatment group (p＜0.05).FCM showed that the apoptosis rates of BEL7402 incubation with HS-1200 at 40μM,60μM and 80μM for 24h were(11.42±0.54)%,(21.36±0.28)%and (42.25±0.52)%,respectively;the apoptosis rate of the control group was(1.09±0.05) %.After treated with HS-1200 at 60μM for 24h,48h and 72h,the apoptosis rates were(21.36±0.28)%,(29.82±0.31)%and(37.15±0.45)%.Statistical analysis of the apoptosis rates showed statistically significant differences between the treatment group and control group(p＜0.05).After treatment of BEL7402 cells with HS-1200, typical DNA laddering was clearly visible in ethidium bromide-stained gels.At the morphological study,the control cells displayed intact nuclear structure,while cells treated with HS-1200 displayed chromatin condensation,formation of apoptotic bodies,and fragmented nucleus.Western blot analysis showed that chenodeoxycholic acid(CDCA)derivative HS-1200 up-regulated the protein levels of Bax,cytochrome c,PARP,cleaved-casepase-9 and cleaved-casepase-3 but down-regulated the protein level of Bcl-2.HS-1200 significantly decreased the mitochondrial membrane potential of BEL7402 cells.2.HS-1200 significantly reduced the growth of BEL7402 xenografts in mice. The tumor size and weight were significantly different between the treatment and control group.The mean±SE for the percentage of apoptosis cells in control groups was(4.6±1.6)%,whereas(9.3±2.1)%,(13.3±3.2)%,and(17.5±4.3)%of apoptotic cells were detected in tumors from different concentrations of HS-1200.The mean±SE for percentage of proliferative cells in tumors from the control,HS-1200 (20mg/kg),HS-1200(40mg/kg),and HS-1200(60mg/kg)was(22.5±3.6)%,(18.9±2.7)%,(14.7±2.0)%,(9.7±1.5)%,respectively.Western blot analysis showed that expression of cleaved-caspase-3,cleaved-caspase-9,cytochrome c,Apaf-1 and Bax was up-regulated,however,the expression of Bcl-2 was down-regulated.Conclusions 1.Chenodeoxycholic acid(CDCA)derivative HS-1200 significantly inhibited viability of BEL7402 cell line,the growth inhibitory effect of HS-1200 was demonstrated to be derived from induction of apoptosis.However, HS-1200 did not inhibit viability and induce apoptosis of normal human hepatic cell line.2.The mechanism of HS-1200-induced apoptosis was related to decreasing the mitochondral membrane potential and up-regulating the protein level of Bax but down-regulating the protein level of Bcl-2.The increased ratio of Bax to Bcl-2 might contribute to the permeabilization of the OMM.OMM became permeable to intermembrane space proteins such as cytochrome c.Once released,cytochrome c promoted the activation of pro-caspase 9.The activation of caspase-9 resulted in activation of caspase-3,which functioned as downstream effectors of the cell death program.Furthermore,the specific caspase-8 inhibitor z-IETDfmk did not affect the HS-1200-mediated apoptosis,so the HS-1200 induced apoptosis of BEL7402 cells through a mitochondrial pathway.3.HS-1200 significantly retarded the growth of BEL7402 xenografts,which was related to the inhibition of cell proliferation and induction of apoptosis by HS-1200. HS-1200-induced apoptosis was also through a mitochondrial pathway.4.Taken together,the synthetic chenodeoxycholic acid derivative HS-1200 may be a potential chemotherapeutic agent for treat HCC. |
