Formononetin induces pyroptosis in lung cancer cells and inhibits PD-L1-mediated immune escape
Objectives To investigate the mechanism of inhibition of non-small cell lung cancer(NSCLC)A549 cells by formononetin(formononetin).Methods MTT assay,cell scratch and clone formation assays were performed to investigate the inhibitory ability of formononetin(20-100 μmol L-1)on proliferation,migration and clone formation of A549 cells.A549 cells were pre-incubated with formononetin for 24 h,and then the nuclear staining was performed by Hoechst 33342,and Dil-labeled Jurkat cells(human T-lymphocyte leukemia cells)were added to co-culture for 30 min,and the co-localization images of the two cells were captured by laser confocal microscopy.Mitochondrial membrane potential(JC-1 staining)and glutathione(GSH)of A549 cells were detected by kit method to investigate the induction effect of onondine on mitochondrial stress.The effects of formononetin on non-classical pyroptosis-associated proteins and PD-L1 proteins after mitochondrial stress in A549 cells were examined by Western blotting.Results Formononetin inhibited the proliferation,migration,and clone-forming ability of A549 cells(P＜0.01,0.001),and enhanced the recruitment and killing of T cells in the cell co-culture model(P＜0.01,0.001).Formononetin induced mitochondrial stress,induced a decrease in the mitochondrial membrane potential,lowered the level of cellular GSH(P＜0.05,0.01),and up-regulated Bax,cleaved-Caspase-3 in A549 cells,Gasdermin E-N protein expression induced cellular pyroptosis(P＜0.01,0.001),while down-regulating p-PI3K,Akt,NF-κB,PD-L1 protein expression inhibited immune escape(P＜0.05,0.001).Conclusion Formononetin has the effect of inducing focal death of NSCLC cells and blocking immune escape.The mechanism is that formononetin can enhance tumor immunity by inducing mitochondrial stress in A549 cells and promoting cellular pyroptosis to recruit T cells on the one hand;on the other hand,it inhibits the p-PI3K/Akt/NF-κB signaling axis,which in turn reduces the expression of PD-L1 protein and inhibits immune escape from A549 cells.
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