Acta Pharmacologica Sinica 2015-04-01

Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro.

Yi-ming Ma, Wei Han, Jia Li, Li-hong Hu, Yu-bo Zhou

Index: Acta Pharmacol. Sin. 36(4) , 517-27, (2015)

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Abstract

To investigate the effects of physalin B insolated from Physalis divericata on human colon cancer cells in vitro and its anticancer mechanisms.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.Physalin B inhibited the viability of HCT116 cells with an IC50 value of 1.35 μmol/L. Treatment of the cells with physalin B (2.5-10 μmol/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.Physalin B induces mito-ROS, which not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in HCT116 cells in vitro.