TR3的线粒体靶向参与TPA诱导乳腺癌细胞凋亡的机制

Mitochondrial Targeting of TR3 is Involved in the Mechanism of TPA-induced Apoptosis in Breast Cancer Cells

目的研究TPA对乳腺癌细胞增殖、凋亡及线粒体功能的影响,并探讨TR3在TPA调控乳腺癌细胞功能中的作用。方法采用2.5 nM和10 nM的12-O-十四烷酰佛波醇-13-乙酸酯(TPA)干预人乳腺癌细胞MCF-748 h,对照组用DMSO干预。MTT法检测细胞增殖抑制率,流式细胞仪检测细胞凋亡率、线粒体膜电位、Ca 2+水平和ROS水平,Western Blot检测细胞中caspase-9和TR3蛋白的表达,生物信息学分析TR3对乳腺癌患者总体生存率(OS)的影响。结果与对照组相比,2.5TPA组和10TPA组细胞增殖抑制率、细胞凋亡率和cleaved-caspase-9蛋白显著增加(P<0.05),pro-caspase-9蛋白表达无显著变化。2.5TPA组和10TPA组中细胞线粒体膜电位显著降低(P<0.05),Ca 2+水平和ROS水平显著增加(P<0.05)。2.5TPA组和10TPA组细胞核和细胞质(不含线粒体)中TR3蛋白的表达显著降低(P<0.05),而细胞质(含线粒体)和线粒体中TR3蛋白的表达显著增加(P<0.05)。TR3在乳腺癌患者中的表达显著降低,且TR3高表达的乳腺癌患者总体生存率显著提高(P<0.05)。结论TPA诱导乳腺癌细胞凋亡取决于TR3蛋白的表达,分子机制是促进TR3核输出并定位于线粒体,从而引起线粒体凋亡。

Objective To investigate the effects of TPA on proliferation,apoptosis and mitochondrial function of breast cancer cells,and to explore the role of TR3 in the regulation of breast cancer cell function by TPA.Methods Human breast cancer cells MCF-7 were treated with 2.5 nM and 10 nM 12-O-tetradecanoylphorbol-13-acetate(TPA)for 48 h,and the control group was treated with DMSO.Cell proliferation inhibition rate was detected by MTT assay.Apoptosis rate,mitochondrial membrane potential,Ca 2+level and ROS level were detected by flow cytometry.Western blot was used to detect the expression of caspase-9 and TR3 protein in cells.The effect of TR3 on overall survival rate(OS)of breast cancer patients were analyzed by bioinformatics methods.Results Compared with the control group,the cell proliferation inhibition rate,apoptosis rate and cleaved-caspase-9 protein expression were significantly increased in the 2.5 TPA group and the 10 TPA group(P<0.05),and the pro-caspase-9 protein expression was not significantly changed.The mitochondrial membrane potential of the cells in the 2.5TPA group and the 10TPA group was significantly decreased(P<0.05),and the Ca 2+level and ROS level were significantly increased(P<0.05).The expression of TR3 protein in the nucleus and cytoplasm(excluding mitochondria)of 2.5TPA group and 10TPA group was significantly decreased(P<0.05),while the expression of TR3 protein in cytoplasm(containing mitochondria)and mitochondria was significantly increased(P<0.05).The expression of TR3 was significantly lower in breast cancer patients,and the overall survival rate of breast cancer patients with high TR3 expression was significantly increased(P<0.05).Conclusion The apoptosis of breast cancer cells induced by TPA depends on the expression of TR3 protein.The molecular mechanism is to promote the nuclear export of TR3 and localize to mitochondria,which causes mitochondrial apoptosis.