Chemotherapeutic Paclitaxel And Cisplatin Differentially Induce Pyroptosis In A549 Lung Cancer Cells Via Caspase-3/GSDME Activation

Aim:Paclitaxel and cisplatin are two first-line chemotherapy drugs for treatment of non-small-cell lung cancer(NSCLC)and a wide range of other malignancies.It has been reported that two chemotherapeutic agents could induce apoptosis in lung cancers via different action mechanisms.Recent researches reveal that GSDME upon chemotherapy drug stimulation can be cleaved by activated caspase-3 to generate its N-terminal fragment(GSDME-NT),which executes pyroptosis by perforating the plasma membrane.GSDME is expressed in many human lung cancers including A549 cells.However,it remains unclear whether they can induce GSDME-mediated secondary necrosis/pyroptosis in A549 lung cancer cells.Thus we aimed to investigate the mechanism of paclitaxel and cisplatin induces secondary necrosis/pyroptosis in A549 cells to provide experimental evidence for reducing the adverse effects and optimizing clinical chemotherapeutical scheme.Methods:(1)The cytotoxicity of these drugs was detected by using WST-1 assay to calculate the 50% cell inhibition(IC50)of cell viability.(2)The pattern and characteristic morphology of cell death after treated by paclitaxel and cisplatin in cells were determined by Annexin V/7-AAD,fixable viability dye(FVD)and PI/Hoechst 33342 staining.(3)The mitochondrial membrane potential was determined by mitochondrial membrane potential assay kit with JC-1.(4)The expression levels of cleaved caspase-3,GSDME-NT,PARP and other proteins in cell lysates were evaluated by Western blotting.(5)It was investigated whether blockade of caspase-3 activation,by pre-treatment of the cells with its specific inhibitor Ac-DEVD-CHO(DEVD),attenuated the secondary necrosis/pyroptosis induced by paclitaxel or cisplatin.(6)It was explored whether GSDME knockdown by small interfering RNA(si RNA)could attenuate the secondary necrosis/pyroptosis in cells induced by paclitaxel and cisplatin.Result:(1)Paclitaxel did not reach the IC50 of cell viability while the IC50 value of cisplatin was about 25 ?M after 48 h-treatment.(2)Both paclitaxel and cisplatin evidently induced apoptosis and secondary necrosis/pyroptosis in cells while cisplatin induced higher levels of secondary necrosis/pyroptosis than paclitaxel.Upon same treatment concentrations and time lengths,paclitaxel-induced cells got round,underwent cell shrinkage and showed blebbing with the cell membrane integrity being retained.After cisplatin treatment,many cells also displayed cell shrinkage and blebbing but their cell membrane integrity was damaged,and the PI-positive cells(lytic cell death)among them had large bubbles blowing from the plasma membrane,which has been regarded as a typical characteristic of secondary necrosis/pyroptosis.Increased paclitaxel concentrations(up to 240 ?M considering its solubility)did not increase the number of PI-positive cells,and more typical pyroptotic cells were observed at 48 h.(3)Paclitaxel significantly decreased the mitochondrial membrane potential(??m)in A549 cells,but cisplatin did not significantly induce ??m reduction.(4)Both paclitaxel and cisplatin induced apoptotic initiator caspase-8/-9,executioner caspase-3/-7 and their substrate PARP were activation in turn;GSDME was cleaved into GSDME-NT fragment by paclitaxel and cisplatin which was correlated with the activation levels of caspase-3 and the secondary necrocis/pyroptosis of A549 cells.However,cisplatin showed higher ability in inducing the activation of these apoptotic or pyroptotic markers,in comparison with the same concentrations and times of paclitaxel.(5)Caspase-3 specifc inhibitor(Ac-DEVD-CHO)suppressed cisplatin-induced GSDME-NT generation and concurrently reduced the secondary necrosis/pyroptosis.(6)GSDME knockdown signifcantly inhibited cisplatin-but not paclitaxelinduced secondary necrosis/pyroptosis.Conclusion:Paclitaxel and cisplatin could induce secondary necrosis/pyroptosis in A549 cells via caspase-3/GSDME activation while cisplatin induced higher levels of secondary necrosis/pyroptosis than paclitaxel,suggesting that cisplatin may provide additional advantages in the treatment of lung cancers with high levels of GSDME expression.