GEFT-Rac1/Cdc42 Inhibits Rhabdomyosarcoma Cell Pyroptosis By Affecting The Caspase-3/GSDME Pathway

Objective: To investigate the effect of GEFT-Rac1/Cdc42 on the pyroptosis of rhabdomyosarcoma cells by regulating the caspase-3/GSDME pathway.Methods:(1)Western blot and q RT-PCR experiments were used to detect the expression of pyroptosis-related molecules GSDME and caspase-3 in normal skeletal muscle cells and rhabdomyosarcoma cells.(2)The cells were treated with dactinomycin,and the morphology of pyroptosis was observed by microscopy.The types of cell death were analyzed by flow cytometry,and the changes of pyroptosis-related molecules were detected by LDH release assay,Western blot,and immunofluorescence assay.(3)GSDME was knocked down using small interfering RNA,and then the relationship between GSDME and dactinomycin-induced pyroptosis was studied by light microscopy observation,LDH release assay,Western blot,and flow cytometry.The effects of GSDME knockdown on the biological behavior of rhabdomyosarcoma cells were investigated by Ed U,plate clone,Transwell,and AO/EB double staining experiments.(4)GEFT was overexpressed and knocked down in rhabdomyosarcoma cells,and the effects on pyroptosis were detected by light microscopy observation,LDH release assay,and Western blot.Moreover,the effect of GEFT expression on cell biological behavior was detected by Ed U,plate cloning,and Transwell experiments.Results:(1)The expression levels of pyroptosis-related molecules(GSDME,caspase-3)were lower in RMS cells.(2)Compared with the control group,after dactinomycin treatment,microscopic observation revealed that rhabdomyosarcoma cells blistered,swelled,and ruptured.The release of LDH in the cell supernatant increased.Flow cytometry showed the pyroptosis rate of RMS cells.Western blot results showed caspase-3activation(cleaved caspase-3),and GSDME cleavage(GSDME-N)were enhanced.Immunofluorescence colocalization experiments showed that dactinomycin cleaved GSDME in the nucleus by inducing the nuclear translocation of caspase-3,and then GSDME-N was released into the cytoplasm.(3)After GSDME knockdown,dactinomycin-induced GSDME-N terminus was reduced,whereas cleaved PARP was increased.In addition,pyroptosis was transformed into apoptosis.The proliferation,invasion,and migration of rhabdomyosarcoma cells did not change significantly.(4)After GEFT overexpression,the activities of Rac1 and Cdc42 were increased.Dactinomycin-induced cleaved caspase-3 and GSDME-N terminus were significantly reduced,and the proliferation,invasion,and migration abilities of cells were enhanced.After knocking down GEFT,the opposite results were observed.Conclusions:(1)The chemotherapeutic drug dactinomycin induces pyroptosis in rhabdomyosarcoma cells through the caspase-3/GSDME pathway.Dactinomycin-induced pyroptosis can be converted into apoptosis after GSDME knockdown.(2)GEFT can affect caspase-3 by regulating Rac1/Cdc42,which in turn affects dactinomycin-induced GSDME-dependent pyroptosis.