Studies On The Regulatory Mechanisms Of Hepatocyte Growth Factor Receptor Inhibitor SU11274 And Oridonin-Induced Human Non-small Cell Lung Cancer A549 Cell Death

Apoptosis and autophagy are two major forms of programmed cell death. The study showed that c-Met, receptor of hepatocyte growth factor, is overexpressed on A549 cell membrane, and the expression of c-Met is HGF-independent. According to the above phenomenon, we treated A549 cells with SU11274, a small molecular inhibitor of c-Met. To further investigate the anti-tumor activity of oridonin and SU11274, we demonstrated the molecular mechanisms in which oridonin and SU11274 induced A549 cell apoptosis and autophagy in the study.We found that treatment of A549 cells with SU11274 led to autophagic cell death. The expression of c-Met was suppressed and then ERK and p53 were activated after SU11274 treatment. SU11274 augmented Bcl-2 phosphorylation, decreased Bcl-2 expression, and then induced Beclin-1 release. Moreover, activated ERK could induce release of Beclin-1 by enhancing Bcl-2 phosphorylation and down-regulating Bcl-2 expression.It has been widely reported that reactive oxygen species (ROS) generation act as an important factor in expression of c-Met, therefore, we investigated the possible roles of ROS in SU11274-treated cell autophagy. We demonstrated that the exposure of A549 cells to SU11274 led to a rapid increase in ROS generation and ROS level reached a plateau level after 24 h. There is a negative feedback loop between ROS induction and SU11274-induced autophagy. Further studies indicated that ROS promoted the expression of c-Met and its downstream factors including PI3K-Akt and Grb2/SOS-Ras-p38 signalling pathways. SU11274 mediated protective ROS generation in a short time; however, it finally induced A549 autophagic cell death.We further found that oridonin treatment resulted in autophagy and apoptosis in A549 cells. Inhibition of c-Met increased oridonin-induced A549 cell autophagy and apoptosis. Inhibition of c-Met increased oridonin-induced Bax transition, cytochrome c release and ERK-p53 activation, indicating that c-Met plays an anti-apoptotic role in oridonin-induced A549 cell apoptosis. We further investigated the role of c-Met in oridonin-induced A549 cell autophagy, and discovered that c-Met inhibitor increased oridonin-induced autophagy, followed by the punctuate distribution of MDC staining and GFP-LC3, as well as Beclin-1 activation and conversion from LC3-I to LC3-Ⅱ. Subsequently, we found that inhibition of autophagy by 3-MA or siRNA against Beclin-1 and Atg5 also inhibited oridonin-induced cell apoptosis. Apoptosis and autophagy acted in synergy to mediate cell death in oridonin-treated A549 cells.c-Met have been found to be overexpressed in a variety of human lung cancer tissues, and c-Met/HGF signaling plays a key role in growth, invasion, metastasis and angiogenesis. Thus HGF/c-Met will be an attractive target for therapeutic inhibition in NSCLC clinical trials. So, we further investigated the molecular mechanisms mediating HGF-induced protection against oridonin-induced apoptosis in A549 cells. Oridonin induced decrease in Bcl-2/Bax ratio, activation of caspase-3, down-regulation of iPLA2 expression and decrease in PGE2 production, while these processes were reversed by HGF, suggesting that HGF played an anti-apoptotic role in oridonin-induced A549 cell death. HGF-induced protective effect was partially attributed to the activation of NF-κB and cyclooxygenase 2 (COX-2). Then the activated COX-2 could prevent cells from initiating the apoptotic response by promoting PGE2 release. Activation of NF-κB-COX-2 by HGF-treatment triggered the increase in Bcl-2/Bax ratio, inhibition of procaspase-3 cleavage, promotion of iPLA2 expression and augmentation of PGE2 release, leading to antagonizing oridonin-induced cell death in A549 cells. iPLA2, downstream effector of caspase-3, also participated in these processes.In conclusion, these findings indicated that c-Met, receptor of hepatocyte growth factor, is overexpressed on A549 cell membrane. SU11274, a small molecule inhibitor of c-Met, induce A549 autophagy through inhibiting overexpression of c-Met and activating ERK-p53 signalling pathway. And meanwhile, SU11274 induced generationg of ROS, ROS mediated activation of PI3K-Akt and Grb2/SOS-Ras-p38 signalling pathways and negative regulated A549 cell autophagy. Using SU11274 or c-Met siRNA can promote oridonin-induced apoptosis and autophagy in A549 cells, in the process, ERK-p53 signalling pathway was upregulated. And HGF could inhibit oridonin-induced apoptosis through activating NF-κB-COX-2 pathway.