雷公藤红素促进人类乳腺癌MDA-MB-453细胞HER2蛋白降解及诱导凋亡的机制

Celastrol Degradates HER2 Protein and Induces Cell Apoptosis in MDA-MB-453 Cells

【目的】研究雷公藤红素促进人类乳腺癌MDA-MB-453细胞HER2蛋白降解及诱导凋亡的机制。【方法】MTT法检测雷公藤红素对MDA-MB-453的细胞毒作用;Hoechst 33258染色荧光显微镜观察细胞凋亡形态学改变;PI单染流式细胞术检测细胞凋亡率;Western Blot检测细胞凋亡相关蛋白变化;免疫荧光检测HER2蛋白在细胞中的定位变化。【结果】雷公藤红素对MDA-MB-453有强大的细胞毒作用,IC50为(5.22±0.29)μmol/L;该化合物可浓度依赖性地诱导MDA-MB-453细胞发生凋亡,出现典型的凋亡小体、sub-G1改变及细胞凋亡相关蛋白Caspase-3、Parp的裂解活化。值得一提的是,雷公藤红素作用后,Western Blot检测到HER2蛋白明显降低,并且原先主要表达在细胞膜上的HER2蛋白发生了异位。【结论】雷公藤红素浓度依赖性地诱导MDA-MB-453细胞发生凋亡,并且促进HER2蛋白降解,这可能与改变了HER2蛋白在细胞中的定位有关系。

[ Objective ] To investigate the mechanism of celastrol degradating HER2 protein and inducing cell apoptosis in MDA- MB-453 cells. [ Methods ] The cytotoxicity of celastrol to MDA-MB-453 cells was measured by MTT assay. Apoptotic morphology was observed after Hoechst 33258 staining. Sub-G1 DNA peak was analyzed by flow cytometry to quantify the degree of apoptosis. Changes of apoptotic related proteins were analyzed by Western blot. Subcellular distribution of HER2 was observed under fluorescence microscopy by immunofluorescence assay. [ Results] Celastrol exhibited potent cytotoxicity in HER2-overexpressing MDA-MB-453 cells, and IC50 was （5.22± 0.29）μmol/L. After MDA-MB-453 cells were treated with different concentrations of celastrol for 24 h, typical apoptotic bodies, increasing sub-G1 DNA peak and activation of Caspase-3 and Parp were detected in a dose-dependent manner. It was noteworthy that immunofluorescence study with anti-HER2 antibody showed that celastrol disturbed the subcellular distribution of HER2, with decreased location in the plasma membrane. [Conclusion] Celastrol induced MDA-MB-453 cancer cells apoptosis in a dose-dependent manner. Subcellular redistribution of HER2 maybe involved in HER2 protein degradation by celastrol.