南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (02): 162-.doi: 10.12122/j.issn.1673-4254.2019.02.06

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自噬相关基因3过表达促进乳腺癌细胞自噬并抑制盐霉素诱导的细胞凋亡

李芳，黄果，彭平，刘瑶，李双辉，刘罗根，章运生   

  • 出版日期:2019-02-20 发布日期:2019-02-20

Overexpression of autophagy-related gene 3 promotes autophagy and inhibits salinomycin-induced apoptosis in breast cancer MCF-7 cells

  • Online:2019-02-20 Published:2019-02-20

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摘要： 目的研究人自噬相关基因3（ATG3）过表达对乳腺癌细胞自噬及盐霉素诱导细胞凋亡的影响和机制。方法采用慢病毒 的方法，构建了稳定过表达ATG3的乳腺癌MCF-7细胞株；通过Western blotting、免疫荧光和透射电镜等方法分析了ATG3过表 达对乳腺癌MCF-7细胞自噬的影响；并用AKT/mTOR的激动剂（SC79和MHY1485）分析AKT/mTOR信号通路对ATG3过表 达促进细胞自噬的影响；通过Western blotting和流式细胞术，同时结合盐霉素和自噬抑制剂（3-MA），分析了自噬对ATG3过表 达抑制盐霉素诱导细胞凋亡的影响。结果成功构建稳定高表达ATG3的MCF-7细胞株。ATG3能够促进MCF-7细胞自噬，并 且抑制AKT/mTOR信号通路。ATG3 明显抑制了盐霉素诱导的细胞凋亡（P<0.01），且ATG3 过表达组中促凋亡分子cleavedcaspase 3（P<0.01）和Bax表达明显减少（P<0.05），抗凋亡蛋白Bcl-2表达增多（P<0.05）。自噬的阻滞能够削弱ATG3对盐霉素 诱导细胞凋亡的抑制作用。结论ATG3可能通过抑制AKT/mTOR信号通路诱导乳腺癌MCF-7细胞自噬，并通过细胞自噬减 少盐霉素诱导的细胞凋亡。综上提示诱发细胞自噬可能是乳腺癌细胞耐药的机制之一。

Abstract: Objective To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms. Methods We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor). Results In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis (P<0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 (P<0.01) and Bax (P<0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 (P<0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis. Conclusion ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.