橙皮素衍生物对肝癌细胞SMMC-7721的促凋亡作用

摘要

目的 以橙皮素为原型,合成了新型化合物HY-12,研究其体外对SMMC-7721细胞的促凋亡作用及机制.方法 在3个时间点(24、48、72 h)采用 MTT 比色法观察不同浓度HY-12对肝癌细胞、肝细胞增殖的影响,计算半数抑制率(IC50).根据结果选取了24 h,12.5×10-6,25×10-6,50×10-6 mol/L 3个浓度进行下述实验.通过Annexin V/PI双染,流式细胞仪检测细胞凋亡率;制备细胞爬片, Hoechst 33342染色,在荧光倒置显微镜下观察细胞凋亡的形态学改变;采用Western blot 法检测细胞凋亡相关因子Bcl-2、Bax蛋白水平的表达情况.结果 ① MTT法显示在对肝细胞活性无显著影响的前提下,HY-12在体外可抑制SMMC-7721细胞的增殖,且具有剂量-时间依赖性;② 流式细胞仪检测显示HY-12作用后,SMMC-7721细胞凋亡率增加,且呈浓度依赖性趋势;③ 经Hoechst 33342染色,药物处理后细胞核皱缩断裂呈现亮蓝色新月状;④ Western blot 结果显示药物处理可以使Bax蛋白表达上调,相反地Bcl-2蛋白表达下调.结论 橙皮素衍生物HY-12有明显抑制肝癌细胞SMMC-7721增殖作用.其机制可能是通过调节Bcl-2、Bax表达以诱导细胞凋亡.HY-12可能成为治疗肝癌的有效化疗药物.%Objective This experiment take hesperitin as the prototype, synthesize a new compound HY-12.To explore the anti-cancer activity of HY-12 on SMMC-7721 cells and preliminary discussed its mechanism.Methods MTT assay was used to detect the effect of HY-12 on cell proliferation rate and then the IC50 was calculated.According to the results, 24 h and three concentrations of HY-12 (12.5×10-6,25×10-6,50×10-6 mol/L) for the following experiments were choosen.Apoptotic ratio was measured by flow cytometer after Annexin V/PI staining.Prepared the cell-climbing slide, and then the apoptosis morphological changes were observed after Hoechst 33342 staining.The expression of Bcl-2 and Bax were measured by Western blot.Results ① HY-12 had inhibitory effect on proliferation of SMMC-7721 under the condition of no obvious significance on liver cell activity, which was time and dose dependent, and half inhibiting rate were 41.7×10-6,38.7×10-6,31.36×10-6 mol/L at three time points.② The result of flow cytometer indicated that HY-12 could increase the apoptotic ratio of SMMC-7721 cell with time and dose dependent.③ After treatment with indicated concentrations of HY-12, light blue condensed nuclei present as bicornuous were observed by staining with Hoechst 33342.④ According to Western blot, the expression of Bax were increased and conversely Bcl-2 were decreased in HY-12 treated groups.Conclusion The new compound derivate from hesperitin could inhibit the proliferation of SMMC-7721 cells in vitro, its mechanism is probably inducing cell apoptosis by regulating Bcl-2, Bax expression,indicating that HY-12 might be a candidate for the treatment of hepatocellular carcinoma.