Study On The Mechanism Of Endothermic Stress Induced By Matrine And Its Related Pharmacodynamics

Matrine is an active compound extracted from the root of a traditional Chinese herb, Sophora flavescens Ait. It has been reported that MAT has been widely used in the treatment of viral hepatitis, hepatic fibrosis, cancer, cardiac arrhythmia and skin diseases in China. Although much research has been investigated into the mechanisms responsible for the pharmacological effects of MAT, there are still lots of key questions that still need to be answered. In the present study, in order to guide rational application and improve the therapeutic efficacy of MAT in clinic, we have further studied the mechanisms underlying its pharmacological effects.We initially assessed the cytotoxic effects of MAT on different cell lines, MTT assay results showed that the human hepatic carcinoma cell line HepG2were more sensitive to MAT while the human normal hepatic L02cells were resistant to MAT. Then HepG2and the human breast adenocarcinoma cell line MCF-7were selected to carry out the subsequent studies. MAT induced apoptosis via activation of caspase-3and caspase-9, generation of ROS and disruption of mitochondria membrane potential in HepG2and MCF-7cells. We here confirmed that although MAT induced futures of autophagy, it finally inhibited the late stage of autophagy by suppressing the maturation of the lysosomal protease cathepsin D. The action of MAT on autophagy is similar to autophagy inhibitor Chloroquine (CQ). The autophagy inhibitor3-methyladenine (3-MA) or silencing of ATG7failed to suppress MAT-induced cytoplasmic vacuoles, suggesting that they were distinct from the typic autophagic vacuoles. The H+-ATPase inhibitor Bafilomycin Al (Baf Al) suppressed MAT-induced cytoplasmic vacuoles, indicating that their formation is correlated with H+-ATPase.For the first time, we reported that MAT induced endoplasmic reticulum. Endoplasmic reticulum stress markers78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) were upregulated upon MAT treatment, suggesting the activation of ER stress. Three ER stress sensors, PERK (protein kidnase-like endoplasmic reticulum kinase), IRE1(inositol-requiring enzyme1), and ATF6(activating transcription factor6) were all activated by MAT treatment. The endoplasmic reticulum4-Phenylbutyric acid (4-PBA) attenuated the induction of GRP78and CHOP, futher confirming that ER stress was triggered upon MAT treatment. Using4-PBA to suppress ER stress, we found that the cytoxicity of MAT was greatly enhanced. Moreover, knockdown of CHOP of RNA interference attenuated MAT-induced apoptosis, indicating that ER stress is involved in MAT-induced apoptosis.According to the current knowledge about MAT, there is still lack of studies aiming to investigate the cellular factors that contribute to MAT sensitivity. In clinic, MAT has been mainly used in the treatment of liver diseases, such as viral hepatitis, hepatic fibrosis. We observed that the expression levels of GRP78were lower in liver cells compared with cells from other organs by immunoblotting analysis. Therefore, we infer that the cellular expression levels of GRP78were associated with the pharmacological effects of MAT. The expression levels of GRP78and p53were correlated with MAT sensitivity in cancer cells, cells expressing higher levels of GRP78or p53were more resistant to MAT, and vice versa. Moreover, knockdown of GRP78or p53by RNA interference potentiated the cytotoxicity of MAT in HepG2and MCF-7cells, further indicating that the expression levels of GRP78and p53contribute to MAT sensitivity.The mechanisms underlying the cell cycle arrest of MAT and its action on cell senescence were also investigated here. MAT arrested cell cycle at Gl phase via induction of p21and p27expression as well as downregulation of Cyclin D1. In addition, MAT can’t induce the increase in β-galactosidase-positive cells; therefore cell cycle arrest induced by MAT can’t trigger cell senescence.The above studies show that MAT induces ER stress which contributes to apoptosis and MAT sensitivity. Moreover, MAT suppresses the late stage of autophagy and cytoplasmic vacuoles induced by MAT are distinct from MAT-induced autophagic vacuoles. Our studies provide strong evidence for the basic research and clinic application of MAT in the future.