塔斯品碱衍生物TasD1-6对肝癌Hep3B细胞生长的抑制作用研究

Inhibition effects of taspine derivatives TasD1-6 on human liver cancer cell of Hep3B

目的考察塔斯品碱衍生物Tas D1-6对肝癌细胞株Hep3B、Hep G2和BEL7402的生长抑制作用及其作用机制。方法应用MTT法和实时细胞分析(RTCA)技术检测Tas D1-6对Hep3B、Hep G2和BEL7402细胞增殖的影响,筛选敏感株后采用结晶紫染色进行细胞形态学观察,根据Annexin V FITC/PI双染法和Hoechst 33258染色法考察Tas D1-6对细胞凋亡的影响,采用Western blotting法检测凋亡相关蛋白表达,流式细胞仪检测细胞周期变化。结果 MTT法和RTCA结果均表明Tas D1-6能明显抑制Hep3B细胞的增殖,MTT检测其半数抑制浓度(IC50)值为(11.3±1.6)μmol/L,确定后续给药浓度分别为3、6、12μmol/L;形态学观察结果表明Tas D1-6高浓度下能明显抑制Hep3B细胞增殖,且细胞皱缩,形态发生明显变化;Annexin V FITC/PI双染法和Hoechst 33258染色法均表明Tas D1-6可诱导细胞发生凋亡,且呈剂量依赖性;Western blotting结果表明Tas D1-6可上调p53和促凋亡蛋白Bax表达,并下调抑凋亡蛋白Mcl-1表达;流式细胞仪检测细胞周期表明Tas D1-6可将Hep3B细胞阻滞于G1期。结论 Tas D1-6可抑制肝癌细胞增殖,其对Hep3B细胞作用最为显著,其作用机制可能为通过上调p53和促凋亡蛋白Bax,下调抑凋亡蛋白Mcl-1诱导细胞凋亡的产生,同时将细胞周期阻滞于G_1期,抑制细胞增殖。

Objective To analyze inhibitory effect of taspine derivative Tas D1-6 on human liver cancer cells of Hep3B, HepG2, and BEL7402, and to study their preliminary mechanism of anticancer action. Methods Effect of Tas D1-6 on cell growth of Hep3 B, HepG2, and BEL7402 were determined by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide（MTT） and real-time cellular analysis（RTCA） assay. Effect of Tas D1-6 on Hep3B cell morphology was investigated with crystal violet staining. Next, Hep3B cell apoptosis was analyzed by Annexin V FITC/PI and Hoechst 33258 staining. Flow cytometry was used to determine Hep3B cell cycle. Effect of Tas D1-6 on cell apoptosis protein expression in Hep3B cell was determined by Western blotting. Results MTT and RTCA assay demonstrated that Tas D1-6 significantly inhibited the growth of Hep3B cell and the IC50 was（11.3 ± 1.6） μmol/L. Hep3B cell morphology indicated the shrinkage and obvious morphology changes. And Tas D1-6 induced the Hep3B cell apoptosis in dose-dependent manner. Cell was stopped as G1 phase by Tas D1-6. Western blotting analysis showed Tas D1-6 could upregulate the protein expression of p53 and Bax and downregulate Mcl-1 protein expression（P〈0.05）. Conclusion Taspine derivative Tas D1-6 shows better inhibitory action on the growth of human liver cancer Hep3B cell than HepG2 and BEL7402 cells. At the same time, Tas D1-6 can change cell cycle and induce cell apoptosis, which probably related to upregulation of p53 and Bax protein expression and downregulation of Mcl-1 protein expression.