新的微管抑制剂YB-13诱导HeLa细胞凋亡及其机制

目的研究吲哚3-草酰胺衍生物YB-13在体外试验中诱导宫颈癌细胞株(HeLa细胞)发生凋亡及其作用机制。方法采用MTT法、细胞生长曲线、细胞集落、荧光显微镜、DNAladder和流式细胞仪等方法进行凋亡检测,用RT-PCR方法检测凋亡过程中相关基因表达的变化;间接免疫荧光观察YB-13对细胞骨架的影响,观察YB-13对微管蛋白聚合和解聚。结果吲哚3-草酰胺衍生物YB-13在体外试验中对HeLa细胞的杀伤力强,荧光显微镜观察到了凋亡小体;DNAladder法检测到凋亡时DNA降解形成的梯带,流式细胞仪检测到了细胞凋亡峰,同时观察到细胞周期的变化。在凋亡过程中,凋亡相关基因bcl-2家族中bcl-2表达下调而bax表达上调;实验观察到YB-13能影响HeLa细胞的细胞骨架,并且YB-13可影响微管蛋白聚合和解聚。结论YB-13在体外试验中能诱导HeLa细胞发生凋亡,其作用机制可能与bax基因表达上调、bcl-2基因表达下调以及对微管蛋白的抑制作用有关。

YB-13,a novel synthetic microtubule inhibitor, induces apoptosis of HeLa cells and its mechanism

Aim To study whether the derivative of indol-3-glyoxylic acid amide induced apoptosis on human galactophore cancer HeLa cells line(HeLa cells)in vitro and the feasible mechanisms of apoptosis.Methods MTT assay,cell growth curve,colony formation of cancer cells,morphological study,DNA gel electrophoresis,flow cytometry,RT-PCR,and fluorescence microscopy were carried out.Results Potent cytotoxic effect on HeLa cells could be observed by MTT assay and cell growth curve,colony formation of cancer cells.After treatment of HeLa cells with YB-13,the apparent morphological characteristic of apoptosis was detected under the fluorescent microscopes by the staining of Hoechst 33342.Both the number of apoptosis cells and the cell circle were measured by flow cytometry.A typical "Sub-G1 peak" was also checked and YB-13 blocked the tumor cells at G2+M phase and arrested followed cell circle.RT-PCR showed that YB-13 promoted bax mRNA expression,at the same time it also inhibited bcl-2 expression in a dose-dependent manner.YB-13 can interfere with microtubule polymerization and disrupting cytoskeleton.Conclusions YB-13 shows obvious anticancer activity by inducing apoptosis and causing cell cycle arrest in a dose-dependent manner and can interfere with microtubule polymerization and disrupting cytoskeleton.