鹅脱氧胆酸衍生物HS-1200对肝肿瘤细胞株凋亡及增殖的影响

Effects of inducing apoptosis and inhibiting proliferation on hepatoma cell line by chenodeoxycholic acid derivative HS-1200

目的探讨鹅脱氧胆酸衍生物HS-1200诱导肝肿瘤细胞株凋亡及抑制增殖的作用及机制。方法分别用40、60及80μM的HS-1200作用于肝肿瘤细胞株BEL7402,于作用后12、24及36 h采用MTT检测细胞活性,流式细胞术、DNA梯状条带、荧光显微镜检测细胞凋亡,Western-blot法检测bcl-2、bax、细胞色素C及caspase-3的表达。结果HS-1200可有效地抑制BEL7402生长,其作用随药物浓度提高和作用时间延长而增强,呈一定的剂量、时间依赖性;FCM结果表明,随作用浓度和作用时间延长,凋亡率显著增加,有显著性差异(P<0.05);Ho-echst33258染色结果显示细胞呈凋亡形态学改变,凝胶电泳显示典型的凋亡梯形条带,Western blot结果显示为HS-1200可提升bax、细胞色素C及caspase-3的表达,降低bcl-2的蛋白表达水平。结论HS-1200对BEL7402有显著的抑制增殖及诱导凋亡的作用,其机理可能为提升bax、细胞色素C及caspase-3的表达,降低bcl-2的表达;HS-1200可能是一种有效的治疗肝癌的化疗药物。

[ Objective] To investigate effects of inducing apoptosis and inhibiting proliferation on hepatoma cell line by chenodeoxycholic acid（CDCA） derivative HS-1200, and to observe mechanisms of the effects. [ Methods ] The hepatoma cell line BEL7402 was treated with different concentrations（40,60 and 80 μmol） of CDCA derivative HS-1200 at different time. At 12,24 and 36 h after treatment, the cell viability of hepatoma cells was examined by MTT assay. Apeptosis of hepatoma cell line BEL7402 was detected by flow cytometry, DNA ladder, and fluorescence microscope. The protein levels of Bcl-2, Bax, cytochrome C and casepase-3 were determined by Western blot analysis. [ Results ] HS-1200 inhibited the proliferation of BEL7402 cells and its effect was dose- and time-dependent. FCM analysis of the apeptosis rates showed significant differences between the treatment group and control group（P 〈0. 05）. Typical DNA laddering was clearly visible in ethidium bromide-stained gels. At the morphological study, cells treated with HS-1200 displayed characteristic nuclear features of apeptosis. Western blot analysis showed that HS-1200 up-regulated the protein levels of bax, cytochrome C and casepase-3 but down-regulated the protein level of bcl-2. [ Conclusion] HS-1200 can inhibit proliferation and induce apeptosis of BEL7402 cell line by up-regulating the protein levels of Bax, cytochrome C and casepase-3 but down-regulating the protein level of Bcl-2. Therefore HS-1200 might be a promising chemotherapeutic agent for treating hepatocellular carcinoma.