Abstract
Polyphyllin VII (PP7), a pennogenyl saponin isolated from Rhizoma Paridis, exhibited strong anticancer activities in various cancer types. Previous studies found that PP7 induced apoptotic cell death in human hepatoblastoma cancer (HepG2) cells. In the present study, we investigated whether PP7 could induce autophagy and its role in PP7-induced cell death, and elucidated its mechanisms. PP7 induced a robust autophagy in HepG2 cells as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, formation of punctate LC3-positive structures, and autophagic vacuoles tested by western blot analysis or InCell 2000 confocal microscope. Inhibition of autophagy by treating cells with autophagy inhibitor (chloroquine) abolished the cell death caused by PP7, indicating that PP7 induced an autophagic cell death in HepG2 cells. C-Jun N-terminal kinase (JNK) was activated after treatment with PP7 and pretreatment with SP600125, a JNK inhibitor, reversed PP7-induced autophagy and cell death, suggesting that JNK plays a critical role in autophagy caused by PP7. Furthermore, our study demonstrated that PP7 increased the phosphorylation of AMPK and Bcl-2, and inhibited the phosphorylation of PI3K, AKT and mTOR, suggesting their roles in the PP7-induced autophagy. This is the first report that PP7 induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy.
Highlights
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide
Primary antibodies against light chain 3 (LC3)-I, LC3-II, Beclin-1, sequestosome-1 (SQSTM1, P62), p53, caspase-3, phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K (P-PI3K/p85 at Tyr458), protein kinase B (AKT), P-AKT at Ser473, Jun N-terminal kinase (JNK), P-JNK at Thr183/ Tyr185, mammalian target of rapamycin (mTOR), P-mTOR at Ser2448, adenosine monophosphate-activated protein kinase (AMPK), P-AMPK at Thr172, Bcl-2, P-Bcl-2 at Ser87, and GAPDH, and secondly antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) or Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA)
The results showed that Polyphyllin VII (PP7) significantly inhibited the growth of HepG2 cells in a dosedependent manner, with a 50% inhibitory concentration value of 1.32 ± 0.04 μM after PP7 treatment for 24 h
Summary
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. The major therapeutic strategy for HCC, is only effective for 15% to 25% early stage of HCC patients. Most HCC patients of late-stage die within a few months after diagnosis due to poor response to chemotherapy and radiotherapy [1]. Polyphyllin VII Induces an Autophagic Cell Death in HepG2 Cells. Co-author Zhinan Mei is employed by College of Pharmacy at South Central University for Nationalities, which provided support in the form of salary for author ZM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript
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