Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro
Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro
复制标题
Physalin B 不仅能抑制泛素-蛋白酶体途径，还能在体外诱导人结肠癌细胞不完全自噬反应。
DOI：
10.1038/aps.2014.157
复制
发表时间：
2015-04-01
影响因子：
8.2
通讯作者：
Zhou, Yu-bo
中科院分区：
文献类型：
--
作者：
Ma, Yi-ming;Han, Wei;Zhou, Yu-bo
目的：目的：研究从酸浆中分离得到的酸浆素B对人结肠癌细胞株HCT 116的体外抗肿瘤作用及其机制。检测细胞存活率和凋亡率，并采用Western blot分析检测相关蛋白。在稳定的GFP-LC3 HCT 116细胞中观察到自噬体。在GFP-LC 3/RFP-LAMP 1共转染的细胞中评价自噬体和溶酶体的定位。共聚焦显微镜观察微管和F-肌动蛋白微丝。结果：酸浆苦素B对HCT 116细胞有明显的抑制作用，其IC 50值为1.35 μ mol/L;用酸浆素B（2.5 - 10 μ mol/L）处理细胞可诱导细胞凋亡以及PARP和caspase-3的裂解。同时，酸浆素B处理诱导了自噬体的形成，LC 3-II和p62的积累，但降低了Beclin 1蛋白的水平。酸浆素B处理后的细胞中微管和F-肌动蛋白微丝发生明显变化，导致自噬体和溶酶体的共定位受阻。酸浆素B处理后，p38、ERK和JNK的磷酸化水平呈剂量依赖性增加，而p38抑制剂SB 202190、ERK抑制剂U0126或JNK抑制剂SP 600125可部分降低酸浆素B诱导的PARP切割和p62积累。此外，酸浆苦素B处理剂量依赖性地增加细胞中线粒体活性氧的产生，而活性氧清除剂NAC可以逆转酸浆苦素B诱导的效应，包括不完全的自噬反应，泛素化蛋白的积累，微管和F-actin的变化，p38，ERK和JNK的激活，以及细胞死亡和凋亡。酸浆苦素B诱导线粒体活性氧的产生，不仅抑制了泛素-蛋白酶体途径，而且在体外诱导了HCT 116细胞的不完全自噬反应。
Aim: To investigate the effects of physalin B insolated from Physalis divericata on human colon cancer cells in vitro and its anticancer mechanisms.Methods: Human HCT116 colon cancer cell line was tested. Cell viability and apoptosis were detected, and relevant proteins were measured using Western blot analyses. Autophagosomes were observed in stable GFP-LC3 HCT116 cells. Localization of autophagosomes and lysosomes was evaluated in GFP-LC3/RFP-LAMP1-co-transfected cells. Microtubules and F-actin microfilaments were observed with confocal microscope. Mitochondrial ROS (mito-ROS) was detected with flow cytometry in the cells stained with MitoSox dye.Results: Physalin B inhibited the viability of HCT116 cells with an IC50 value of 1.35 mu mol/L. Treatment of the cells with physalin B (2.5-10 mu mol/L) induced apoptosis and the cleavage of PARP and caspase-3. Meanwhile, physalin B treatment induced autophagosome formation, and accumulation of LC3-II and p62, but decreased Beclin 1 protein level. Marked changes of microtubules and F-actin microfilaments were observed in physalin B-treated cells, which led to the blockage of co-localization of autophagosomes and lysosomes. Physalin B treatment dose-dependently increased the phosphorylation of p38, ERK and JNK in the cells, whereas the p38 inhibitor SB202190, ERK inhibitor U0126 or JNK inhibitor SP600125 could partially reduce physalin B-induced PARP cleavage and p62 accumulation. Moreover, physalin B treatment dose-dependently increased mito-ROS production in the cells, whereas the ROS scavenger NAC could reverse physalin B-induced effects, including incomplete autophagic response, accumulation of ubiquitinated proteins, changes of microtubules and F-actin, activation of p38, ERK and JNK, as well as cell death and apoptosis.Conclusion: Physalin B induces mito-ROS, which not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in HCT116 cells in vitro.