Study On The Mechanism Of Oleanolic Acid - Induced Autophagic Death In Human Gastric Cancer Cells

Oleanolic acid (OA), a plant-derived pentacyclic terpenoid, is known to have hepatoprotective effects. In this study, we first found that OA effectively inhibited cell proliferation in a dose- and time-dependent manner in human gastric cancer SGC-7901, MGC-803 and BGC-823 cells according to MTT assay. Previous studies have shown that OA induces apoptosis in various types of cancer cells. However, in OA-treated human gastric cancer SGC-7901, MGC-803 and BGC-823 cells, we did not detect an increase in apoptosis (Annexin V-FITC/PI double staining and caspase-3 assay). The apoptosis inhibitor z-VAD also failed to attenuate OA-induced cell death. These results indicate that OA-induced cell death does not occur via caspase-dependent apoptosis in these cells.In contrast, OA was found to cause autophagy, rather than apoptosis, in SGC-7901, MGC-803 and BGC-823 cells. OA induced a noticeably greater amount of Beclin-1 and LC3-Ⅱ in SGC-7901, MGC-803 and BGC-823 cells after OA treatment. Confocal microscopy analysis revealed that,in contrast to the relatively homogeneous distribution in control cells, GFP-RFP-LC3 puncta accumulated in the cytoplasm of OA-treated cell. In addition, OA-treated SGC-7901, MGC-803 and BGC-823 cells also showed cytoplasmic accumulation of autophagosomes, a morphological marker of autophagy as determined by TEM. We found that inhibiting autophagy by 3-MA and knocking down the expression of Beclin-1 in SGC-7901, MGC-803 or BGC-823 cells by siRNA led to a reduction in OA-induced cell death, autophagosomes and autolysosomes. Which indicate that OA-induced cytotoxicity in SGC-7901, MGC-803 and BGC-823 cells is caused by autophagy.mTOR has been reported to be involved in the cell autophagy process, and the suppression of mTOR phosphorylation is considered to be responsible for the early triggering of autophagy, which is regulated by the PI3K/AKT, ERK/MAPK and AMPK signalling pathways. We investigated the role of mTOR in OA-induced autophagy. we found that OA treatment decreased the phosphorylation of PI3K, AKT, ERK1/2, p38 and mTOR, but increased the phosphorylation of AMPK. These data suggest that OA induces autophagy by suppressing phospho-mTOR through inhibition of the PI3K/AKT and ERK/p38 MAPK signalling pathways and activation of the AMPK signalling pathway.We found that OA-induced depolarization of the mitochondrial membrane potential (MMP) and increasing of ROS, The antioxidant NAC blocked the increase in ROS, but failed to prevent the OA-induced collapse of MMP indicated that increasing ROS generation might be a result of the OA-induced disruption of MMP, which leads to respiratory dysfunction and electron escape from the mitochondria. Depolarization of MMP which is associated with the mitochondrial permeability transition pore (MPTP), resulted in autophagic cell death. Inhibition of cyclophilin D (CypD, a component of MPTP) by cyclosporin A or siRNA blocked OA-induced cell death, autophagy and depolarization of MMP, suggesting that CypD may be the target of OA in the autophagic cell death process.In conclusion, OA induced apoptosis-independent autophagic cell death in multiple human gastric cancer cells via modulation of the expression of phospho-mTOR, which is associated with the inhibition of the PI3K/AKT and ERK/p38 MAPK signalling pathways and the activation of the AMPK signalling pathway. Our mechanistic study indicated that OA induced the depolarization of the MMP associated with the MPTP and that CypD might be specific for the OA-induced cytotoxicity. The results contribute to our understanding of OA and provide clear evidence for its promising potential in preclinical and clinical situations.