| Objective:To uncover the expression pattern of GSDMD in colorectal cancer and it correlation to related cliniopathological parameters.To investigate the function and mechanism of subcellular localization of GSDMD in the tumorigenesis and progression of colorectal cancer.Methods:Expression pattern of GSDMD was analyzed by IHC(Immunohistochemistry)in tissue chip of colorectal cancer paired with paratumor tissue,and statistics were carried out on correlation between subcellular localization of GSDMD and related cliniopathological markers.CCK-8 was applied to detect the growth of GSDMD stably expressing cell lines in vitro and subcutaneous tumorigenesis was carried out to observe the effects of GSDMD on cell growth in vivo.Subcellular localization of GSDMD when treated with hypoxia was observed by IF(immunofluorescence).Apoptosis of GSDMD overexpressing cell lines was detected by FCM(flow cytometry)after treatment of Oxaliplatin.Also the cleavage of GSDMD was detected after treatment of Oxaliplatin.Mass Spectra was applied to identify the interacting proteins of GSDMD,and then got verified by co-IP(co-immunoprecipitation).Subcellular localization of GSDMD and PARP-1 with treatment of Oxaliplatin was observed by IF.The effects of GSDMD on function of PARP-1 were detected by western blot to show the PAR to its substrates.IF was applied to detect the effects of GSDMD on the γ-H2 AX Foci formation,a marker of DNA Damage,after treatment of Oxaliplatin.The interacting domain of PARP-1 with GSDMD was identified by truncation co-IP,and the results got verified by co-IP and FCM to detect apoptosis with cells treated with PARP inhibitor Olaparib.IF was used to observe the effects of various chemotherapeutic drugs and ATM/ATR inhibitors on nuclear translocation of GSDMD and the results got verified by nuclear-cytosolic fractionation and FCM to detect apoptosis with treatment of ATM inhibitor Ku-55933.The expression of p-ATM(Ser1981)was observed by IHC of the above colorectal cancer tissue chip and its correlation with nuclear localized GSDMD was analyzed by statistics.MGsdmd stably expressing MC38 cell line was used for subcutaneous tumorigenesis in C57BL/6 mice treated with Oxaliplatin.Furthermore,volume measurement of tumor masses and expression of GSDMD,Ki-67 and γ-H2 AX shown by IHC were then analyzed.Results:Based on IHC results of colorectal cancer tissue chip,GSDMD is lower expressed in colorectal cancer to paratumor tissue and negative nuclear expression of GSDMD predicts a worse clinical outcomes and the positive shows a favorable outcome according to statistics,while no significant statistic differences of overall survival are observed when grouped by GSDMD whole expression level of cancer cells.GSDMD has no obvious effects of cancer cells growth in vitro measured by CCK8,while GSDMD inhibits cancer cells growth of subcutaneous injection in nude mice in vivo.Hypoxia results in a moderate translocation of GSDMD into nucleus observed by IF.As is revealed by FCM,GSDMD overexpression promotes apoptosis induced by 5-Fu and Oxaliplatin.Cell viability measured by CCK8 shows that GSDMD overexpression inhibits cell viability,and GSDMD knockdown maintains cell viability when treated with Oxaliplatin.GSDMD in cancer cells cannot be cleaved when treated with Oxaliplatin shown by western blot with positive control in THP-1 cell.According to results of mass spectra,GSDMD interacts with PARP-1,and then the interaction gets verified by semi-endogenous and endogenous co-IP.Semi-endogenous and endogenous IF shows that GSDMD predominantly localized at cytoplasm at steady state,while it translocates into nucleus after stimulation of Oxaliplatin to form co-distribution with PARP-1.GSDMD robustly inhibits the function of PARP-1,poly(ADP-ribose)ation(PAR)to its substrates revealed by smear bands of western blot,and also the formation of γ-H2 AX Foci shown by IF.Truncation co-IP demonstrates that GSDMD interacts with CA Domain of PARP-1,and the interaction is depressed by PARP inhibitor Olaparib.FCM shows that Olaparib robustly sensitizes apoptosis induced by Oxaliplatin in GSDMD knockdown cells in comparison to the Vector control ones.GSDMD translocation can be induced by various DNA damage related reagents,and ATM kinase inhibitor Ku-55933 inhibits this process,as shown by IF and nuclear-cytosolic fractionation.FCM reveals that Ku-55933 can inhibit apoptosis in GSDMD overexpression cells induced by Oxaliplatin.According to statistics on IHC results of colorectal cancer tissue chip,p-ATM expression(Ser1981)shows a positive correlation to the nuclear localization of GSDMD.IF shows that the same translocation behaviors as human GSDMD in MC38 m Gsdmd stably expressing cells.MGsdmd robustly inhibits volume of tumor masses of subcutaneous injection in C57BL/6 mice in vivo when treated with Oxaliplatin.IHC of tumor masses shows nuclear translocation of GSDMD,decreased percentage of positive staining of Ki-67 and increased percentage of positive staining of γ-H2 AX in the internal hypoxic zone of m Gsdmd overexpression without treatment of Oxaliplatin,and also in the external high-concentration zone with treatment of Oxaliplatin.As is observed,it forms a wider DNA Damage Ring of γ-H2 AX in m Gsdmd overexpressing MC38 cells in comparison to the Vector control ones.Conclusions:1.GSDMD is lower expressed in colorectal cancer to paired paratumor tissue,and negative nuclear localization indicates poor clinical outcomes.2.Full-length GSDMD,in subcellular localization dependent manner beyond pyroptosis,translocates into nucleus to block CA Domain of PARP-1 to promote apoptosis through inhibiting DNA Danage Repair,thus to sensitize chemotherapy in colorectal cancer.3.Translocation of GSDMD was shown activation of DNA Damage Response related ATM kinase dependent,thus to form positive feedback loop of “DNA Damage—p-ATM—GSDMD Translocation—PARP-1 blockage—DNA Damage& Apoptosis” signaling pathway. |
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