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.