纳米雄黄酸飞品对人乳腺癌细胞增殖、凋亡及自噬的影响

Effects of Acid Cleaning-Realgar Nanoparticles on Proliferation,Apoptosis and Autophagy of Human Breast Cancer Cell

目的探讨纳米雄黄酸飞品(Acid Cleaning-Realgar Nanoparticles,NRPP)对人乳腺癌细胞增殖、凋亡及对自噬的影响,并初步研究其可能作用机制。方法采用CCK-8法检测不同浓度(0、10、20、40、60、80、100、120μg·mL^(-1))和不同干预时间(12、24、48 h)的NRPP对人乳腺癌MCF-7、MDA-MB-231、MDAMB-435S细胞增殖的影响;荧光显微镜观察MDA-MB-435S细胞形态变化;Hoechst 33258染色法观察细胞凋亡现象;透射电子显微镜(TEM)观察细胞超微结构变化;蛋白免疫印迹法(Western blot)检测凋亡(Bcl-2和PARP)、自噬(LC3和p62)及自噬通路相关蛋白的表达水平。结果NRPP对上述3种乳腺癌细胞的增殖均有显著的抑制作用,且呈现一定的浓度和时间依赖性。在荧光显微镜下观察,随着NRPP浓度的增大,细胞数量减少,细胞间隙增大,细胞逐渐皱缩、变圆、脱落,未见凋亡小体。TEM下观察,对照组细胞的细胞器状态良好,NRPP组有明显的自噬体。Western blot结果显示Bcl-2和PARP表达量未发生明显的趋势变化;LC3Ⅱ/Ⅰ表达上调,p62表达下调,并呈一定的浓度和时间依赖性;PI3K/Akt/mTOR自噬通路蛋白表达量未发生明显的趋势变化;COXIV表达下调,并呈浓度依赖性。结论NRPP可抑制3种乳腺癌细胞的增殖,且可能通过线粒体自噬诱导MDA-MB-435S细胞死亡。

Objective To explore the effects of Acid Cleaning-Realgar Nanoparticles Processed(NRPP)on the proliferation,apoptosis and autophagy of human breast cancer cells and its possible mechanism.Methods CCK-8 method was used to detect the effects of NRPP with different concentrations(0,10,20,40,60,80,100,120μg·mL^(-1))and different time(12,24,48 h)on proliferation of human breast cancer MCF-7,MDA-MB-231 and MDA-MB-435S cells.The morphological changes of MDA-MB-435S cells were observed by fluorescence microscope.Apoptosis and the ultrastructure were observed by Hoechst 33258 staining and transmission electron microscope(TEM)respectively.Expression level of apoptosis related proteins Bcl-2 and cleaved PARP,autophagy related proteins LC3 and p62 and autophagy pathway related proteins were detected by Western blotting.Results NRPP had significant inhibitory effects on the above three kinds of human breast cancer cells with dose-dependence or time-dependence certainly.The number of cells gradually decreased,the gap became larger,cells gradually shrank,became round and fell off,and no apoptotic bodies on MDA-MB-435S cells with the increase of NRPP concentration.The control group that cells were well and autophagy lysosomes were found in the NRPP group under TEM.Western blotting showed no significant trend of Bcl-2and cleaved PARP expression,the expression of LC3Ⅱ/Ⅰwas up-regulated and p62 was down-regulated with dosedependence and time-dependence certainly.The expression of PI3K/Akt/mTOR pathway did not change significantly and the expression of COXIV was down-regulated in dose-dependence.Conclusion NRPP can inhibit the proliferation of three kinds of human breast cancer cells.It may induce MDA-MB-435S cells death through mitophagy.