G9a-targeted chaetocin induces pyroptosis of gastric cancer cells

Objective:To evaluate the effect of chaetocin on pyroptosis of gastric cancer cells and its underlying mechanisms.Methods:The proliferation of gastric cancer cells was detected by trypan blue staining.Flow cytometry and Hoechst/propidium iodide double staining were used to detect apoptosis and pyroptosis.Cellular ultrastructure was observed by transmission electron microscopy.The levels of p-mixed lineage kinase domain-like(MLKL),gasdermin-D(GSDMD),gasdermin E(GSDME),N-GSDMD,and N-GSDME proteins were detected by Western blotting.In addition,lactate dehydrogenase(LDH)release assay was used to verify pyroptosis induced by chaetocin,and caspase 3 inhibition test and siRNA interference test were conducted to investigate pyroptosis mechanisms.Results:Chaetocin at concentrations of 200 nmol/L to 600 nmol/L inhibited the proliferation of AGS,HGC27,MKN28,and SGC7901 gastric cancer cells in a dose-dependent and time-dependent manner by inducing apoptosis and pyroptosis.Significant ultrastructure changes,such as chromatin condensation,vacuolization,disrupted mitochondrial cristae,and increased nuclear occupancy,were observed after treatment with chaetocin in SGC7901 cells.Chaetocin at a concentration of 400 nmol/L significantly increased the number of pyroptotic cells,LDH release,and the ratio of N-GSDME/GSDME(P<0.01),which were reversed by Z-DEVD-FMK.In addition,chaetocin did not affect the expression of GSDMD.G9a silencing abolished the effect of chaetocin on the expression levels of GSDME and N-GSDME and LDH release(P>0.05).Conclusions:In addition to inducing apoptosis,chaetocin inhibits gastric cancer cells by inducing pyroptosis via the caspase 3/GSDME pathway.G9a was the target of chaetocin to induce pyroptosis of gastric cancer cells.