Chaetocin Induces Apoptosis In Gastric Cancer Cells Through Bid/AIF Pathway Independent Of ROS Production

Gastric cancer is one of the most serious malignancies that threaten human health.With the development of science and technology,we have a deep understanding of the occurrence,development and treatment of gastric cancer.However,the biological diversity,drug resistance,invasion and metastasis of tumors make the treatment of gastric cancer still difficult for humans to overcome.Therefore,seeking and exploring new drugs for treating gastric cancer has become the goal of many researchers.Chaetocin is a natural metabolite isolated from Chaetomium fungi and belongs to the group of thiodiketopyrazine complexes.Studies have shown that chaetocin is a specific inhibitor of the eukaryotic histone methyltransferases Su(VAR)3-9 and G9a.In addation,chaetocin also has protective effects against virus,cancers and cardiovascular disease,therefore,more and more attentions are paid to chaetocin.Although chaetocin is found to have anti-tumor effects in a variety of tumors such as lung cancer,liver cancer,colon cancer,etc.,it is not clear whether it is also effective for gastric cancer.This article aims to investigate the role of chaetocin in inducing apoptosis of gastric cancer cells and the related mechanisms.In this paper,we selected gastric cancer cell lines AGS and HGC-27 and NCI-N87 as the research object.First,MTT assay,PI staining combined flow cytometry,and Hoechst33342 staining were used to illustrate that chaetocin could induce apoptosis in gastric cells in dose-and time-dependent manners and the IC50 consentrations were 120 nM,400 nM,880 nM,respectively.To further clarify the potential molecular mechanism of apoptosis induced by chaetocin,we used western blotting to detecte apoptosis-related proteins,and the results showed chaetocin could activate the caspase-dependent apoptotic pathways.However,pan-caspase inhibitor Z-VAD-FMK only partially suppressed the cell apoptosis,suggesting that chaetocin could also induced caspase-independent apoptosis in gastric cancer cells.Next,we first used lentivirus-mediated RNA interference technology to knock down endogenous AIF expression in gastric cancer cells.MTT assay and flow cytometry found that knockdown of AIF greatly attenuated chaetocin-induced apoptosis.Immunofluorescence and nucleoplasmin isolation experiments further demonstrated that chaetocin could induce the translocation of AIF from the mitochondria into the nucleus in gastric cancer cells;moreever,pretreatment cells with BI-6C9,an inhibitor of the AIF upstream protein BID,effectively prevented the mitochondria-nuclear translocation of AIF and inhibited the cell death induced by chaetocin,thus indicating that chaetocin promotes AIF translocation into the nucleus through BID protein to induce caspase-independent apoptosis in gastric cancer cells.In addition,although chaetocin did not induce ROS release,it still caused a decrease in mitochondrial membrane potential,which might be related to the changed of apoptosis-related proteins Bcl-2 and BAX that controling mitochondrial membrane channel switching.Autophagy often plays an antagonistic role in drug-induced cell death.We found that chaetocin could also induce autophagy in gastric cancer cells by western blotting and acridine orange staining;morever co-treatment with autophagy inhibitor CQ could significantly enhance the apoptosis induced by chaetocin in gastric cancer cells.Therefore,our study may provide theoretical basis for the combination use of chaetocin with autophagy inhibitors to enhance the therapeutic effect of gastric cancer.The above studies systematically elucidated the role of chaetocin in inducing apoptosis of gastric cancer cells and the related molecular mechanisms.In particular,it was proposed that chaetocin can promote caspase-independent apoptosis signaling pathway through BID-mediated nuclear translocation of AIF.At the same time,we unexpectedly found that chaetocin induced apoptosis of gastric cancer cells does not depend on the release of ROS,This phenomenon is extremely rare in the process of drug-induced apoptosis.The specific mechanisms,advantages and disadvantages still need further exploration.These findings have enriched the antitumor effect of chaetocin and laid a good theoretical foundation for its clinical application.