千金藤素对乳腺癌细胞MDA-MB-231自噬降解的影响

目的 探讨千金藤素对乳腺癌细胞MDA-MB-231自噬的影响及其机制.方法 激光共聚焦显微镜观察千金藤素对转染了EGFP-LC3、tfLC3(mRFP-EGFP-LC3)质粒的MDA-MB-231细胞自噬体形成及自噬流变化的影响;Western blot检测自噬相关蛋白LC3B、p62以及Beclin1的表达水平.激光共聚焦显微镜观察千金藤素、Bafilomycin A1、Rapamycin分别对转染了EGFP-LC3、tfLC3(mRFP-EGFP-LC3)质粒的MDA-MB-231细胞自噬体形成及自噬流变化的影响;Western blot检测溶酶体相关膜蛋白LAMP1、LAMP2的表达.结果 千金藤素处理后细胞中的自噬体(EGFP-LC3荧光团)数量明显多于对照组(30.40 ±6.42)vs(5.00±2.12),P＜0.01],自噬相关蛋白LC3B-Ⅱ、p62的表达也呈量效和时效增多.转染ffLC3质粒后,千金藤素组和Bafilomycin A1组红绿荧光重叠呈现较多的黄色荧光团,而Rapamycin组出现较多的红色荧光团.千金藤素联用Bafilomycin A1并未进一步升高p62和LC3B-Ⅱ蛋白的表达水平,但联用Rapamycin明显升高了LC3B-Ⅱ蛋白的表达水平,同时,逆转了Rapamycin导致的p62蛋白表达水平减少.千金藤素既不影响对溶酶体的染色,也不减少LAMP1、LAMP2蛋白的表达水平,但影响了mRFP-LC3和mGFP-LAMP1的共定位.结论 千金藤素通过阻断自噬体与溶酶体的融合抑制MDA-MB-231细胞自噬体降解.

Cepharanthine inhibits autophagic degradation in breast cancer MDA-MB-231 cells

Objective To investigate the effect of cepharanthine on autophagy in breast cancer MDA-MB-231 cells.Methods Confocal laser scanning microscopy was used to observe the formation of autophagosome and autophagic flux in breast cancer MDA-MB-231 cells after the transfection with EGFP-LC3 or tfLC3 plasmids.Western blot analysis was used to detect the expression of autophagy related proteins,including LC3B,p62 and Beclin1.Confocal laser scanning microscopy was used to observe the LysoTracker staining and the co-localization of autophagosome (mRFP-LC3) and lysosome (mGFP-LAMP1).Western blot analysis was also used to detect the expression of lysosomal-associated membrane proteins LAMP1 and LAMP2.Results The number of EGFP-LC3 puncta in the cells treated with cepharanthine was significantly greater than that in control cells (30.40 ± 6.42 vs 5.00 ± 2.12,P ＜ 0.01),and the expression levels of LC3B-Ⅱ and p62 were increased in a dose-and time-dependent manner.Exposure to cepharanthine and bafilomycin A1 caused pronounced formation of LC3 puncta that displayed both green and red fluorescence intensity producing a yellow overlay after transfected with tfLC3.In contrast,the cells exposed to rapamycin led to the production of large amounts of red-only puncta.Cepharanthine combined with bafilomycin A1 did not show any significant increase in the accumulation of LC3B-Ⅱ and p62.In contrast,treatment with rapamycin resulted in a slight increase in the levels of LC3 B-Ⅱ that were further enhanced by cepharanthine treatment.Furthermore,treatment with rapamycin resulted in decreased p62 levels that were markedly reversed by cepharanthine treatment.Further studies indicated that cepharanthine did not affect lysosomal staining nor decrease the expression of LAMP1 and LAMP2,but it affected the colocalization of mRFP-LC3 and mEGFP-LAMP1.Conclusion Cepharanthine inhibits autophagic degradation in breast cancer MDA-MB-231 cells through blocking autophagosome-lysosome fusion.