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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 investigate the growth inhibitory effect of tasidine derivative Tas D1-6 on Hep3B, Hep G2 and BEL7402 hepatocellular carcinoma cells and its mechanism. Methods The effects of Tas D1-6 on the proliferation of Hep3B, Hep G2 and BEL7402 cells were detected by MTT assay and real-time cell analysis (RTCA). Cell morphology was observed by crystal violet staining after screening sensitive strains. According to Annexin V FITC / Staining assay and Hoechst 33258 staining were used to investigate the effect of Tas D1-6 on apoptosis. Western blotting was used to detect the expression of apoptosis related proteins. Flow cytometry was used to detect cell cycle changes. Results Both MTT assay and RTCA assay showed that Tas D1-6 could significantly inhibit the proliferation of Hep3B cells. The IC50 of MTD assay was (11.3 ± 1.6) μmol / L, and the concentrations of 3, 6 , 12μmol / L. Morphological observation showed that under the high concentration of Tas D1-6, the proliferation of Hep3B cells was significantly inhibited and the cell shrinkage and morphological changes were observed. The results of Annexin V FITC / PI double staining and Hoechst 33258 staining showed that Tas D1-6 could induce cell apoptosis in a dose-dependent manner. Western blotting showed that Tas D1-6 upregulated the expression of p53 and pro-apoptotic protein Bax, and down-regulated the expression of Mcl-1. Flow cytometry Detection of cell cycle showed that Tas D1-6 blocked Hep3B cells in G1 phase. Conclusion Tas D1-6 can inhibit the proliferation of Hep3B cells, and its effect on Hep3B cells is most significant. The possible mechanism is that Tas D1-6 can up-regulate the expression of p53 and pro-apoptotic protein Bax, down-regulate the expression of anti-apoptotic protein Mcl-1, The cell cycle arrest in G_1 phase, inhibit cell proliferation.