去甲斑蝥素对乳腺癌细胞系MCF-7^(adr)生长与凋亡的影响

Effects of norcantharidin on the growth and apoptosis of breast cancer cell line MCF-7^(adr)

目的:探讨去甲斑蝥素(NCTD)对耐阿霉素乳腺癌细胞系MCF-7adr生长及凋亡的影响,为其临床应用提供实验依据。方法:采用MTT法测定NCTD抑制效应;利用光镜及荧光显微镜观察处理后的细胞形态变化;利用流式细胞术检测细胞凋亡;利用细胞免疫化学检测增殖、凋亡相关因子bcl-2、p53的表达。结果:NCTD对乳腺癌细胞MCF-7adr的生长有明显的抑制作用,且呈剂量、时间依赖性。NCTD处理48h后,MCF-7adr细胞内ki-67阳性指数由(96.8±1.3)%下降至(18.8±1.9)%;PCNA蛋白阳性指数由(95.4±1.1)%降至(59.2±1.9)%。NCTD处理48h后,在光镜下可见MCF-7adr细胞出现发泡、皱缩,有些细胞出现碎裂,经Hoechst染色证实NCTD处理后出现典型的凋亡表现。流式细胞术显示NCTD处理48h后,出现亚二倍体峰,随剂量的增加(120、240、360、480μmol/L),凋亡率分别增加11.67%、16.51%、18.29%、20.79%。NCTD处理48h后,MCF-7adr细胞突变型p53蛋白表达率为(97.8±1.64)%,经NCTD240μmol/L处理后表达率下降为(47.2±9.8)%;Bcl-2蛋白表达率由(98.87±3.74)%下降为(17.5±1.64)%。结论:NCTD能抑制MCF-7adr细胞生长、增殖,降低ki-67阳性指数率,促进MCF-7adr细胞凋亡与凋亡相关因子bcl-2及突变型p53蛋白的表达。

Objective：To explore the effect of norcantharidin （NCTD） on the growth and apoptosis of breast cancer cell line MCF-7^adr for providing reliable evidence for clinical application. Methods：Suppressive effect of NCTD on cell line was detected by MTr assay. Morphological alterations were observed by light microscope and fluorescence microscope. Flow cytometry were used to observe the effect on cell cycle and apoptosis. The related factors of proliferation, apoptosis were determined by immunocytochemisty. Results： NCTD inhibited the growth and proliferation of MCF-7^adr cells in a dose dependent manner and a time dependent manner. NCTD reduced the positive percentage of the ki-67 positive index from （96. 8 ± 1.3）% to（ 18.8 ± 1.9）% at 48h after treatment; the percentage of PCNA was decreased from （95.4 ± 1.1）% to（59. 2 ± 1.9）%. Morphological alterations of NCTD-treated cells under a microscope were membrane blebbing, condensation of nuclear chromatin, nuclear fragmentation, and apoptotic bodies. Hoechst staining assay confirmed this. Sub-G1 peaks were increasing in a dose dependent manner at 48h after NCTD treated cells. When dose of NCTD treated cells by 120,240,360 and 480μM, The increasing apoptosis ratio was 11.67%, 16. 51%, 18.29% and 20. 79%, respectively. The express ratio of p53 protein reduced from （97. 8 ± 1.64）% to （47. 2 ± 9. 8）%. The express ration of bcl-2 protein decreased from （98. 87 ±3.74）% to （ 17.5 ± 1.64）%. Conclusion：This study shows the inhibition of NCTD on MCF-7^adr cells. After NCTD treatment, cell apoptosis is promoted. The expression of apoptosis relative factors, mutant p53 is down-regulated.