Recombinant Newcastle Disease Virus(rL-RVG) Triggers Autophagy And Apoptosis In Gastric Carcinoma Cells By Inducing ER Stress

Tissue hypoxia, endoplasmic reticulum calcium loss, viral infections and certain calcium ion balance, protein glycosylation, the endoplasmic reticulum and Golgi vesicle transport will interfere with ER function, leading to unfolded protein or error protein accumulation, called the unfolded response(UPR), determines survival or dying cells.NDV(Newcastle disease virus, DNV) as an avian paramyxovirus, has been used as an oncolytic virus for many years, with its high selectivity for tumor cells and high replication in tumor cells, it is considered as the next generation of new anti-cancer drugs.This study observes that apoptosis, autophagy and endoplasmic reticulum stress induced by recombinant Newcastle disease virus(r L-RVG) in human gastric cancer,explores the relationship of endoplasmic reticulum stress, autophagy and apoptosis.These findings will provide molecular bases for developing r L-RVG into a drug candidate for the treatment of gastric carcinoma. Related research is as follows:1. r L-RVG induced an ER stress response in SGC and HGC cells.After gastric cancer cells infected with r L-RVG or NDV, firstly, we detected endoplasmic reticulum-associated protein GRP78 and CHOP by Western Blot, and found that the expression of GRP78 and CHOP increased significantly. Further, we observed that the expression of GRP78 and CHOP protein was markedly upregulated in the r L-RVG-or NDV-infected group by immunofluorescence, revealing r L-RVG induces endoplasmic reticulum stress in gastric tumor cells.2. r L-RVG induced the activation of IRE1 signaling pathway After gastric cancer cells infected with r L-RVG or NDV, we detected the expression of IRE1 pathway proteins, XBP-1 protein by Western Blot and found the presence of XBP-1 m RNA splice variants by semi-quantitative PCR, revealing r L-RVG induces the activation of IRE1 signaling pathway.3. r L-RVG induced the activation of ATF6 signaling pathway After gastric cancer cells infected with r L-RVG or NDV, we detected the expression of ATF6 pathway proteins, p50-ATF6 protein by Western Blot. And found that the expression of p50-ATF6 protein increased significantly, which reveals r L-RVG induces the activation of ATF6 signaling pathway.4. r L-RVG induced the activation of PERK signaling pathway After gastric cancer cells infected with r L-RVG or NDV, we detected the expression of PERK pathway proteins by Western Blot and found that expression of p-e IF2α protein increased significantly, but no significant change in total e IF2α. These results show that r L-RVG induces the activation of PERK signaling pathway.5. r L-RVG triggered autophagy and apoptosis in gastric carcinoma cells by inducing ER stress After pretreated with 4PBA for 2h, the gastric cancer cells were infected with r L-RVG for 24 h, then detect endoplasmic reticulum stress, autophagy and apoptosis-related proteins by Western Blot. And found not only endoplasmic reticulum stress-related proteins but also autophagy and apoptosis related proteins decreased. Further flow cytometry showed the rate of apoptosis decreased significantly pretreated with 4PBA. Then interfered CHOP gene with CHOP-si RNA in gastric cancer cells and then detected autophagy and apoptosis protein expression after infected with the virus for 24 h. The results showed autophagy and apoptosis protein was decreased significantly. These demonstrated that r L-RVG triggers autophagy and apoptosis in gastric carcinoma cells by inducing ER stress.6. The autophagy induced by r L-RVG was mediated by the IRE1/JNK/beclin-1signaling pathway.After gastric cancer cells infected with r L-RVG or NDV, we found the expression of p-JNK proteins increased by Western Blot, but no significant change in total JNK. After pretreated with special JNK inhibitor(SP60015) for 2h, the gastric cancer cells were infected with r L-RVG for 24 h, then detect endoplasmic reticulum stress, autophagy and apoptosis-related proteins by Western Blot. The results showed that autophagy and apoptosis protein was decreased significantly. These dates demonstrate that autophagy induced by r L-RVG is mediated by the IRE1/JNK/beclin-1 signaling pathway.7. Autophagy played a role in ER stress-mediated apoptosis.After pretreated with special autophagy inhibitor(3MA) for 12 h, the gastric cancer cells were infected with r L-RVG for 24 h, then detected endoplasmic reticulum stress and apoptosis-related proteins by Western Blot. The results showed that autophagy mediates ER stress-mediated apoptosis.