Mechanism Of Apoptosis And Autophagy In HVJ-E Induced Cell Death In A549 And A549/DDP

Lung cancer is one of the most common malignant tumor worldwide, its incidence and mortality have increasing trend. Lung cancer is a multifactorial disease, cigarette smoking is one of the main factors causing lung cancer.At present, the treament of lung cancer is mainly treated by operation, chemotherapy and radiotherapy, but the survival rate is not high in general. So, we need to seek a new approach to the treament of lung cancer. Studies have shown that inactivated Sendai virus (Hemaglutinating virus of Japan Envelope, HVJ-E) not only can be used as a carrier to deliver anticancer drugs,but also be able to activate multiple anti-tumor immunity. In this study, we found that HVJ-E can directly induce human lung adenocarcinoma cell (A549), cisplatin resistant human lung adenocarcinoma cell (A549/DDP) death, to explore its anti-tumor mechanism.Firstly, A549 and A549/DDP cells were treated with HVJ-E and RIG-I, IFN-βwere observed by Western blot and ELISA. The results showed that RIG-I was activated, HVJ-E can induce IFN-P production and was dose-dependent. The results indicated that HVJ-E fused effectively with cells and the broken viral genome were recognized by RIG-I, which resulted in an increasing amount of IFN-β production in A549 and A549/DDP cells with the increament of HVJ-E.Secondly, the effects of HVJ-E on the prooliferation and viability of A549 and A549/DDP cells were examined. Treatment of these cells with HVJ-E, we observed growth arrest, shrinkage and collapse. Apoptotic chromatin condensation and nuclear fragmention were readily observed by Hoechst 33258 staining.Cell inhibition and apoptosis detected by CCK-8 and FACS assay indicated that the proliferation of treated cells was inhibited,and the apoptosis of the cells was dose-dependent by HVJ-E administration. It showed that the HVJ-E can directly cause A549 and A549/DDP cells apoptosis.Thirdly, we assayed p53,Bcl-2/Bax, Caspase pathway related proteins by Western blot, the cleavage of caspase-8, caspase-9, caspase-3, PARP can be detected with the increasing of HVJ-E concentration. To further confirm the apoptotic induction by HVJ-E is caspase pathway dependent, cells were pretreated with Caspase inhibitor Z-VAD-FMK, and than the cleavage of caspase-3, PARP were markedly suppressed, cell apoptosis was also inhibited significantly. The experiment proved that Caspase pathway is involved in HVJ-E induced A549 and A549/DDP cells apoptosis.Then, to investigate the regulation of the MAPK pathways in HVJ-E treated A549 and A549/DDP cells, the p-p38, p-JNK, p-ERK can be detected by Western blot. To further investigate the role of MAPK pathway,specific inhibitors, SB203580, SP600125 and PD98059, targeting p38, JNK and ERK, respectively, were added to cells 30 minutes prior to HVJ-E. The Western blot results showed that the phosphorylated forms were all significantly inhibited. The CCK-8 results showed significant inhibition of cytotoxicity by HVJ-E. All the above results showed MAPK pathway plays an important role in HVJ-E induced A549 and A549/DDP cells apoptosis.Finally, we investigated the role of autophagy in HVJ-E induced A549 and A549/DDP cells apoptosis. We used GFP-LC3 plasmid and Lipofectamine 2000 transfect cells, we set negative control, HVJ-E and Rapa three groups. After 4 hours, we observed autophagy under inverted fluorescence microscope. We found a lot of autophagic vacuole in Rapa group, and a few in HVJ-E group. The Western blot results showed the expression of p-mTOR and p-P70S6K were increased with the increase of HVJ-E concentration. It appears that autophagy is involved in the HVJ-E promote cell death. We used Rapa and CQ respectively pre-stimulated cells. Western blot assay found that in A549 cells,maximun amount of cleaved caspase-3 and cleaved PARP were in CQ group, while in A549/DDP cells, they were in Rapa group. The CCK-8 results showed the CQ group had the highest mortality rate in A549 cells, the Rapa group had the highest mortality rate in A549/DDP cells. So, for the A549 cell apoptosis, inhibit autophagy can promote HVJ-E induced, but for A549/DDP cell apoptosis, activa tautophagy can promote HVJ-E induced.