The Effect Of GP130 Small Molecule Inhibitor-SC144 On The Apoptosis Of Hepatocellular Carcinoma Cells

Back ground: Primary liver cancer is one of the most common digestive system malignant tumors in the world.For advanced hepatocellular carcinoma,sorafenib is the only targeted drug currently approved for clinical use.However,the multiple drug resistance is a serious factor affecting the efficacy of sorafenib.Therefore,the research and development of new targets is very important.In recent years,the relationship between GP130 and tumor proliferation,metastasis and apoptosis has attracted much attention.More and more studies focus on GP130.As a small molecule inhibitor of GP130 with oral activity,in vitro and in vivo study of SC144 in colorectal cancer,ovarian cancer,inhibition of tumor cell proliferation and promote the apoptosis have been confirmed,but the study of hepatocellular carcinoma is not clear.Therefore,in this experiment,the experimental model of human hepatocyte L-02,human hepatocellular cell PLC/PRF/5 and human hepatocellular cell MHHC-97 H was used to study the effect of SC144 on the apoptosis of hepatocellular cells and to determine the effective drug concentration,and to provide a favorable basis for the research and development of the target for the treatment of primary liver cancer.Therefore,this experiment in human liver cell L02 and human liver cancer cell lines PLC/PRF/5 and MHHC-97 H in vitro model to investigate the effect of SC144 on apoptosis of cancer cells and to determine the effective drug concentration and try to provide a favorable basis for the development of primary liver cancer target research.Objectives: 1.We investigate the difference in expression of GP130 in human liver cell L-02,human hepatocellular cell PLC/PRF/5,and human hepatocellular cell MHHC-97 H.2.We explore the effective concentration of the GP130 small molecule inhibitor SC144,which can effectively promote the apoptosis of hepatocellular cells and reduce the effect on normal cells as much as possible.3.We try to explore the mechanism of SC144 to promote the apoptosis of hepatocellular cells.4.We will provide strong in vitro evidence for the development of new targets for the treatment of liver cancer.Methods: 1.We detected the difference of GP130 m RNA expression levels in human liver cell L-02,human hepatocellular cell PLC/PRF/5 and human hepatocellular cell MHHC-97 H by real-time PCR.2.We used SC144 drugs with different concentration(0 ?mol/L?1 ?mol/L?5 ?mol/L?10 ?mol/L)treatment of L-02 cell and PLC/PRF/5 cell and MHHC-97 H cell in different time,through the absorbance detection experiment CCK8 cell proliferation difference,so that the cell survival rate differences,explore the different effects of SC144 on cell proliferation.3.We compared the number of apoptotic phenomenon in L-02 cell,PLC/PRF/5 cell and MHHC-97 H cell after 48 hours of intervention with different concentrations of SC144 drugs by Hoechst 33258 fluorescence staining,so as to prove the difference in apoptosis promoting effect of SC144 drugs on each group.We detected the difference of early apoptosis rate,late apoptosis rate and total apoptosis rate after 48 hours of intervention of different concentrations of SC144 drugs on L-02 cell,PLC/PRF/5 cell and MHHC-97 H cell by flow cytometry.4.We detected the difference of the expression level of three apoptotic proteins between Caspase-3,Bax and Bcl-2 in the experimental group after 48 hours of intervention of SC144 drugs on L-02 cell,PLC/PRF/5 cell and MHHC-97 H cell by WB,trying to explain the mechanism of SC144 promoting the apoptosis of hepatocellular cells.Results: 1.Real-time PCR results showed that the expression level of GP130 m RNA in human hepatocellular cell PLC/PRF/5 and human hepatocellular cell MHHC-97 H was significantly higher than that in normal cell L-02.2.CCK8 cell proliferation test showed that different concentrations of SC144 drugs intervened in L-02 cells until 48 hours,and no obvious proliferation inhibition was displayed.PLC/PRF/5 cells and MHHC-97 H cells were interfered with different concentrations of PLC/PRF/5 cells and MHHC-97 H cells,respectively,with the increase of drug concentration,and the results were statistically significant difference;3.Hoechst 33258 fluorescence staining was used to observe the effects of different concentrations of SC144 drugs on L-02 cells,PLC/PRF/5 cells and MHHC-97 H cells for 48 hours.With the increase of drug concentration,there was no significant increase in the formation of apoptotic phenomenon in the L-02 of human hepatocytes,but in human hepatocellular cells PLC/PRF/5 and MHHC-97 H,the drug concentration increased with the increase of drug concentration.When the flow cytometry was used to detect different concentrations of SC144 in L-02 cells,PLC/PRF/5 cells and MHHC-97 H cells for 48 hours,the apoptosis rate,the late apoptosis rate and the total apoptosis rate in human hepatocyte L-02 were not significantly increased with the increase of drug concentration,but in human hepatocellular cell PLC/PRF/5 and MHHC-97 H,with the increase of drug concentration,the early apoptosis rate,the late apoptosis rate and the total apoptosis rate increased significantly.4.5 ?mol/L SC144 drugs intervented for L-02 cells,PLC/PRF/5 cells and MHHC-97 H cells for 48 hours,We detected the difference in expression of Caspase-3,Bax,and Bcl-2 of apoptosis related proteins in each group by WB.Compared with the control group,the expression of Bax increased only in human hepatocellular cell MHHC-97 H,with statistical difference,but no significant difference was found in the rest.Conclusion: 1.The expression of GP130 gene is highly expressed in the hepatocellular cells.2.SC144 can effectively promote the apoptosis of hepatocellular cells.3.Cell membrane receptor GP130 is a potential target for the treatment of liver cancer.