The Mechanism Of Necroptosis Of Gastric Cancer Cells Induced By Celastrol Targeting Biglycan

BackgroundChemotherapy is the most effectivecurative approach for patients with advanced gastric cancer,which is one kind of the most common cancers.However,the emergence of apoptosis resistance in chemotherapy is an important obstacle to treatment.It takes the non-apoptotic cell death such as necroptosis into accountin anti-tumor treatment.The large leucine-rich Biglycan(BGN)is highly expressed in a variety of tumors and is closely related to tumor migration and invasion.Celastrol is a triterpene alcohol compound obtained from plant of Tripterygium that induces apoptosis to produce antitumor activity.However,it has not been reported whether celastrol induce necroptosis of gastric cancer cells and whether Biglycan participates in this process.Based on the above background,we hypothesized that celastrol may induce necroptosis of gastric cancer cells by regulating the level of Biglycan.Therefore,in this study,we synthetically studied the antitumor effects of celastrol induced necroptosis on AGS and HGC-27 gastric cancer cells,and further explored the role and molecular mechanism of Biglycan in this process.Our results provide ideas and strategies for new personalized anti-tumor therapy based on Biglycan expression and necroptosis.Objective1.To study the antitumor effect of celastrol induced necroptosis in gastric cancer cell;2.To study the role of Biglycan in the cell necroptosis of gastric cancer induced by celastrol and its possible molecular mechanism.Methods1.Effect of celastrol on the necroptosis of gastric cancer cellsThe effect of celastrol on the survival rate of gastric cancer cells were analyzed by MTT assay and flow cytometry;the effect of celastrol on the necroptosis of gastric cancer cells were analyzed western blot.2.Celastrol targeted regulation of Biglycan involves programmed necroptosis in gastric cancer cellsThe expression of Biglycan in gastric cancer cells treated with celastrol were detected by Western blot,and the overexpression of lentivirus was detected by Western blot;MTT?morphological observation and flow cytometry were used to analyze the effect of Biglycan on the programmed necrosis of gastric cancer cells induced by celastrol.3.Effect of biglycan on the necroptosis of gastric cancer cells induced by celastrol and its molecular mechanismCombined with Lentiviral vector over-expression Biglycan,MTT assay,morphological,Nec-1 and NSA were used to analyze the effects of celastrol;further use of flow cytometry,western blot,immunofluorescence and other methods to analyze the effect of up regulation of Biglycan on RIP1,RIP3 and downstream signal MLKL in gastric cancer cells after exposure to celastrol,and to explore effects of celastrol on the regulation of Biglycan in the inhibition of programmed necrosis gastric cancer cells.Results1.It was found that the survival rate of gastric cancer cells AGS and HGC-27 were decreased and the necroptosis rate were increased by celastrol in a concentration-dependent manner,and the expression of necroptosis signals RIP1 and RIP3 were increased,which were through MTT,flow cytometry and western blot experiments;2.Our study found that the expression of Biglycan decreased with the increase of the treatment dose of celastrol in gastric cancer cells,Overexpression of Biglycan can block the necroptosis of gastric cancer cells induced by celastrol which were through Western blot,MTT and morphological observation experiments;3.we found that overexpression of biglycan can inhibit celastrol-induced necroptosis of gastric cancer cells,showing inhibition expression of necroptosis signal RIP1 and RIP3 and translocation of MLKL from the nucleus to the cytoplasm.Necroptosis inhibitor Nec-1 significantly antagonized the anti-tumor effect of celastrol and increased the level of biglycan at the same time,which were through flow cytometry,morphological observation,western blot,immunofluorescence and necroptosis inhibitors Nec-1 and NSA.Conclusion1.Celastrol promotes necroptosis of gastric cancer cells and exerts anti-tumor effect;2.Celastrol decreases the expression of biglycan to induce necroptosis in gastric cancer cells;3.Celastrol-induced the expression of necroptosis protein marker RIP1 and RIP3 and the translocation of MLKL were inhibited by over-expression of biglycan in gastric cancer cells.