石蒜碱调控线粒体氧化损伤介导人乳腺癌细胞自噬及凋亡作用机制

Mechanism of lycorine on autophagy and apoptosis in human breast cancer cell through mitochondrial oxidative damage

目的研究石蒜碱介导人乳腺癌MDA-MB-231细胞自噬及凋亡的作用机制。方法采用CCK-8法检测石蒜碱对MDA-MB-231细胞增殖的影响;倒置显微镜、荧光显微镜和激光共聚焦扫描显微镜(laser scanning confocal microscopy,LSCM)观察细胞形态;细胞集落实验检测细胞克隆形成能力;划痕实验检测细胞迁移能力;流式细胞仪检测细胞凋亡率、细胞内活性氧(reactive oxygen species,ROS)水平、线粒体膜电位;LSCM检测线粒体膜通透性转换孔(mitochondrial permeability transition pore,MPTP)开放情况;Western blotting检测线粒体自噬相关蛋白[线粒体内膜转位酶(translocase of inner membrane,TIM)、线粒体外膜转位酶(translocase of outer membrane,TOM)、E3泛素连接酶(E3 ubiquitin protein ligase,PARKIN)、PTEN诱导的激酶1(PTEN induced putative kinase 1,PINK1)、微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3-B)、p62]和凋亡相关蛋白[半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、细胞色素C(cytochrome-C,Cyt-C)]的表达。结果石蒜碱对MDA-MB-231细胞具有生长抑制作用,其半数抑制浓度(half inhibitory concentration,IC50)值为6.21μmol/L;石蒜碱能够改变细胞形态,部分细胞出现凋亡迹象;石蒜碱能够显著抑制细胞的增殖和迁移能力(P<0.01);与对照组比较,随着石蒜碱给药浓度的增加,细胞凋亡率随之升高(P<0.01),ROS水平升高(P<0.01),线粒体膜电位下降(P<0.01),MPTP开放(P<0.01);石蒜碱给药48 h后,细胞内TIM、TOM、p62和Bcl-2蛋白表达量下调(P<0.01),PARKIN、PINK1、LC3-B、Caspase-3、Bax和Cyt-C蛋白表达量上调(P<0.01),且呈浓度相关性。结论石蒜碱对MDA-MB-231细胞具有生长抑制作用,并可通过调控线粒体氧化损伤介导MDA-MB-231细胞的自噬及凋亡。

Objective To investigate the mechanism of lycorine on autophagy and apoptosis in human breast cancer MDA-MB-231 cell.Methods The effect of lycorine on MDA-MB-231 cell proliferation was detected by CCK-8;Cell morphology was observed by inversion microscopy,fluorescence microscopy and laser scanning confocal microscopy(LSCM);The cell clone formation ability was detected by cell colony assay;The cell migration ability was examined by scratch assay;The apoptosis rate,intracellular reactive oxygen species(ROS)level and mitochondrial membrane potential were determined by flow cytometry;The opening of mitochondrial membrane permeability transition pore (MPTP) was detected by LSCM;The expressions of mitochondrial autophagy-related proteins [translocase of inner membrane (TIM), translocase of outer membrane (TOM), E3 ubiquitin protein ligase (PARKIN), PTEN induced putative kinase 1 (PINK1), microtubule-associated protein light chain 3 (LC3-B), p62], and apoptosis-related proteins [cystein-asparate protease-3 (Caspase-3), Bcl-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), cytochrome-C (Cyt-C)] were detected by Western blotting. Results Lycorine had a proliferative inhibitory effect on MDA-MB-231 cells, with a half inhibitory concentration (IC50) value of 6.21 μmol/L;Lycorine could change cell morphology, and some cells showed signs of apoptosis;Lycorine could significantly inhibit the proliferation and migration capacity of MDA-MB-231 cells (P < 0.01);Compared with control group, with the increase of lycorine administration concentration, cell apoptosis rate was increased (P < 0.01), ROS level was increased (P < 0.01), mitochondrial membrane potential was decreased (P < 0.01), and MPTP was open (P < 0.01). After 48 h treatment of lycorine, the expressions of TIM, TOM, p62 and Bcl-2 protein were down-regulated (P < 0.01), and the expressions of PARKIN, PINK1, LC3-B, Caspase-3, Bax and Cyt-C were up-regulated (P < 0.01) in a concentration-dependent manner. Conclusion Lycorine has growth inhibition effect on MDA-MB-231cells, and induces autophagy and apoptosis of MDA-MB-231 cells through the regulation of mitochondrial oxidative damage.