The Function And Molecular Mechanism Of Matrine-induced Autophagy

Background:Matrine, one of the main components extracted from Sophora flavescens, has exhibited potent pharmacological effects on some tumors. However, the mechanism underlying this process remains elusive. Aim:This study is to investigate the function and molecular mechanism of autophagy promoted by matrine in hepatic cancer cells and liver oval cells.The first part:the function and molecular mechanism of matrine-induced autophagy in WB-F344cellsMaterials and methods:We established an in vitro model of liver stem cells using the WB-F344cell line under matrine treatment. Apoptosis was monitored in matrine-treated WB-F344cells through flow cytometry analysis. Autophagy was detected in matrine-treated WB-F344cells through confocal microscopy, a western blot, and transmission electron microscopy (TEM). The signal transduction in apoptosis and autophagy activation was detected in WB-F344cells through immunoblotting analysis and Reverse transcription polymerase chain reaction (PCR).Results:Initially, the significant increase of MDC positive cells and LC3-II amount, a specific marker for detecting autophagy, was observed in matrine-treated WB-F344cells, showing that autophagy was stimulated by matrine, and then transmission electron microscopy (TEM) confirmed the observations. Meanwhile, a marked elevation of apoptosis was detected in WB-F344cells after exposure to matrine at0.4mg/ml and0.8mg/ml in a dose-dependent manner. A decrease of Bcl-2mRNA expression but an increase of Bax mRNA expression was demonstrated in matrine-treated oval cells, and led to a down-regulation of the Bcl-2/Bax ratio, confirming the molecular marker for determining the extent of apoptosis. Subsequently,3-methyladenine (3-MA), a specific inhibitor of autophagy, enhanced matrine-induced apoptosis in WB-F344cells. Next, we explored the possible molecular mechanism of matrine-induced autophagy in WB-F344cells. Matrine treatment attenuated β-catenin degradation, then activation of β-catenin induced an increase of LC3-Ⅱ amount, and reversed the Bcl-2/Bax ratio under matrine treatment, whereas inhibition of β-catenin by RNA interference (RNAi) induced a decrease of LC3-Ⅱamount and the Bcl-2/Bax ratio in WB-F344cells, suggesting that β-catenin is linked to the increased autophagy and apoptosis by matrine. Furthermore, β-catenin was significantly down-regulated by Rapamycin, a foregone chemical agonist of autophagy, and up-regulated by3-MA, indicating that β-catenin protein levels is reversely regulated by autophagy.Conclusion:These results demonstrate that autophagy is stimulated by matrine in liver stem cells, and autophagy inhibition enhances matrine-induced apoptosis. Additionally, β-catenin is involved in matrine-induced autophagy and apoptosis, whereas p-catenin is negatively regulated by autophagy as feedback. We conclude that β-catenin may be a crosstalk of autophagy and apoptosis. The second part:the function and molecular mechanism of matrine-induced autophagy in human hepatocellular carcinoma cellsMaterials and methods:The cultured human hepatocellular carcinoma cell line (HepG2) and redifferentiation of human hepatoma cells (SMMC-7721) were treated with matrine. The signal transduction in autophagy activation was detected in HepG2cells. A gene expression profile was used in matrine-treated HepG2cells.Results:Matrine stimulated autophagy in human hepatoma cells, which is mammalian Target of Rapamycin (mTOR)-dependent manner in SMMC-7721cells, but mTOR-independent manner in HepG2cells. Then, chemical inhibition of autophagy significantly aggravated apoptosis in matrine-treated HepG2cells. Next, in HepG2cells, autophagy induction by matrine is regulated by p53inactivation through AMP-activated protein kinase (AMPK) signaling transduction, then AMPK suppression switches autophagy to apoptosis. The p53protein isoforms-p53β, p53γ,△133p53, and△133p53γ, due to alternative splicing of intron9, are implicated in the p53-mediated autophagy. Furthermore, the interferon (IFN)-induciable genes, such as interferon alpha-inducible protein27(IFI27) and interferon induced transmembrane protein1(IFITM1), are involved in matrine-induced autophagy and as downstream effectors of p53.Conclusions:These results show that matrine induces autophagy in human hepatoma cells with novel mechanism, and autophagy inhibition enhances matrine-induced apoptosis. That is, p53/AMPK and p53/IFN signaling transduction are implicated in matrine-induced autophagy. P53isoforms, for the first time, are relative to the promotion of autophagy. Thus target of autophagy is the potential of matrine in liver cancer therapy in potentiating tumor cell death.