Sorafenib Combined With RSL3 In Anti-hepatocellular Carcinoma And Its Preliminary Study On GSDME-mediated Pyroptosis

OBJECTIVE: This study is mainly to clarify that the combination of low-concentration sorafenib and RSL3 can enhance the killing effect of liver cancer cells.To analyze the relationship between the combined with RSL3 through gasdermin-E(GSDME)to mediated proptosis.To clarify that the combination of solareinib and RSL3 can mediates cell pyroptosis through GSDME to enhance the sensitivity of liver cancer to drugs.Providing new treatment idea and experimental basis for clinical reversal of drug resistance in liver cancer.METHODS:1.The effect of sorafenib,RSL3 and combination drugs to enhance the sensitivity of sorafenib to kill liver cancer.(1)Observe the morphological effects of sorafenib,RSL3,combined use of sorafenib and RSL3 on liver cancer cells under a microscope.(2)Cell Counting Kit-8(CCK8)detects the effects of different concentrations of sorafenib,RSL3 and their combination on the proliferation of liver cancer cells.(3)Western-blot(WB)test was used to detect the effects of sorafenib,RSL3,sorafenib combined with RSL3 on caspase-3,GSDME,GPX4 protein levels in liver cancer cells.2.Cellular level to explore the effect of GSDME expression level on sorafenib and RSL3 combined treatment which induced cell pyroptosis.(1)Construct a recombinant lentivirus GSDME-sh RNA silencing cell line and use western-blot method to detect the effect of low expression of GSDME on the expression of pyroptosis-related proteins.(2)Construct a recombinant lentiviral caspase-3-sh RNA silencing cell line,and use western-blot method to detect the effect of low expression of caspase 3on cell pyroptosis-related protein expression.3.Animal experiments explore the effect of GSDME-mediated pyroptosis on enhancing the anticancer effect of sorafenib combined with RSL3.(1)Construct human SK-Hep1 cell transplanted tumor models in BALB/c nude mice.To study the effect of sorafenib combined with RSL3 on human liver cancer cells in nude mice.(2)Construct a murine model of Hep1-6 cell transplantation tumor in BALB/c nude mice.To study the effect of sorafenib combined with RSL3 on the liver cancer cells in nude mice.(3)Build mouse-derived Hep1-6 cell transplanted tumor model in C57BL/6mice.To study the effect of sorafenib combined with RSL3 on the effect of killing liver cancer cells in mice.Immunohistochemistry was used to detect the infiltration of CD3+Tcells,CD49b+ NK cells,CD11C+ dendritic cells and F4/80+ macrophages in tumor tissues.RESULT:1.The combination of low-dose Sorafenib and RSL3 significantly enhanced the effect of killing liver cancer cells.(1)Larger dose of sorafenib can kill liver cancer cells.We used sorafenib at a concentration of 30 μmol/L on killing effect on SK-Hep1,97 L and Hep G2 hepatoma cells(p<0.05),which no killing effect on Huh7 and SMMC-7721.(2)Larger doses of RSL3 can kill liver cancer cells.RSL3 significantly inhibited the growth of SK-Hep1 and Huh7 cells at 20 μmol/L Secondly are SMMC-7721 and MHCC-97 L cells.(P<0.05).(3)The combination of low-dose sorafenib and RSL3 has a significant killing effect on liver cancer cells.Compared with the above-mentioned single drugs,the combination of sorafenib(5 μmol/L)+ RSL3(1 μmol/L)can show a more obvious effect on killing liver cancer cells.Even Hep G2 cell lines that are resistant to sorafenib and RSL3 also showed significant death and had little toxic and side effects.2.The combination of sorafenib and RSL3 group showed obvious morphological characteristics of cell pyroptosis and changes of pyroptosis-related proteins.(1)Obvious morphological characteristics of cell pyroptosis appear in the combined drug group.Microscopic observations showed that the liver cancer cells in the drug alone group grew well.The cell morphology of the combined medication group showed swollen cytoplasm,rupture of the cell membrane,which the cells showed a more obvious characteristic of cell burnout-bulb-like.T he nucleus was squeezed to the side.The cell membrane was swollen like a bulb-like.This is a difference from the cell death characteristics reported in the literature that sorafenib and RSL3 mainly induce iron death.(2)The level of pyroptosis executive protein GSDME-N increased in the combined medication group.WB detected the effect of drugs on the level of pyroptosis executive protein of liver cancer cells.WB found that an obvious GSDME-N-terminal shear band in the combination group.Sorafenib and RSL3 are both activators of iron death.So we also tested GPX4,an iron death-related indicator.We found that the iron death inhibitor protein GPX4 was significantly inhibited.3.The pyroptosis executive protein GSDME enhances the sensitivity of the combination of sorafenib and RSL3 to kill liver cancer cells.(1)After using sh RNA to silence GSDME in liver cancer cell lines,the killing effect of sorafenib combined with RSL3 on GSDME low expression liver cancer cells was significantly reduced the pyroptosis executive protein GSDME which enhances the sensitivity of the combination drug to kill liver cancer cells through pyroptosis.(2)After using sh RNA to silence caspase-3 in hepatocellular carcinoma cell lines,the effect of sorafenib combined with RSL3 on killing hepatocarcinoma cells with low expression of caspase-3 did not change significantly.It is suggested that caspase-3 may not participate in the killing effect of coke-induced cell pyroptosis.CONCLUSION: Compared with large doses of sorafenib and RSL3 in order to kill liver cancer cells.The combination of low concentration of sorafenib and RSL3 obviously exerts its killing effect on liver cancer cells.The cell morphology is mainly pyroptosis.Pyroptosis executive protein GSDME-N level increase is not obvious which not depend on the expression level of caspase-3.The combination of sorafenib and RSL3 enhances the sensitivity of sorafenib to liver cancer in vivo.It is possible that GSDME mediates cell pyroptosis and release factors induce tumor immune cell infiltration.