Inhibition Of The Prolyl Isomerase Pin1 Enhances The Ability Of Sorafenib To Induce Cell Death And Inhibit Tumor Growth In Hepatocellular Carcinoma

Objective:This study aimed to elucidate the role of Pin1 on Sorafenib drug resistance in liver cancer,to enhance Sorafenib efficacy by targeting on Pin1,and to provide a new approach and strategy for the treatment of hepatocellular carcinoma cells.Methods:1.Western Blot and QPCR were used to determine the effects of Sorafenib on the Pin1 exprssion in HCC cell lines.2.Lentivirus expressing Pin1 sh RNA was used to knockdown Pin1 in HCC cell lines.Then,p I/Hoechst assay was used to determine the potential of Sorafenib induced cell death in the cells with or without Pin1 knockdown.Western Blot was used to determine the expression of Pin1.3.Treat the HCC cell lines with Sorafenib and ATRA,the Pin1 inhibitor,then the Pin1 protein level was detected by Western Blot.Using p I/Hoechst staining kits and Flow cytometry technique to detect the cell death of HCC cell lines after treatement of Sorafenib combined with ATRA.The protein level was detected by Western Blot.4.Several inhibitors were used to figure out the type of cell death induced by the combination of ATRA and Sorafenib.The protein level of Caspase3/9 was detected by Western Blot.5.Animal models,Huh7 cells were inoculated subcutaneously into the nude mice.Mice with xenograft tumor were divided into 4 groups randomly:ATRA treatment,Sorafenib treatment,and combination of Sorafenib and ATRA or vehicle saline.Examine the tumor growth by measuring the tumor volume and weight and determine the expression of proteins by Western Blot.Results:1.Sorafenib treatment not only led to a does-dependent decrease in Pin1 protein levels,also m RNA level of Pin1 in all HCC cell lines examined.2.We successfully knockdowned Pin1 in HCC cell lines including Hep G2,Huh7 and sk-Hep-1.Results of p I/Hoechst staining and flow cytometry indicated that Pin1 knockdown increased the sensitivity of HCC to Sorafenib,and enhanced the ability of Sorafenib to induce cell death in multiple human HCC cells.The expression level of apototic proteins was proved to be higher in cell lines with Pin1 knockdown.3.Pin1 inhibiitor,ATRA,significantly enhanced Sorafenib induced down-regulation of Pin1 expression.p I/Hochest and flow cytometry assays showed that ATRA enhanced the ability of Sorafenib to induce cell death in human HCC cells,supported by upregulation of protein levels of Caspase3/9.4.Caspase inhibitor,z VAD,dramatically blocked cell death induced by the combination of Sorafenib and ATRA in a dose dependent manner.5.Xenograft assay showed that ATRA alone had the modest effect on tumor growth,while Sorafenib alone did not inhibit tumor growth until the late stages,the combination of ATRA with Sorafenib synergistically stopped HCC tumor growth,even leading to tumor shrinkage at the late stage.Conclusions:Our results showed that Sorafenib can downregulate the protein level and the m RNA level of Pin1.And the effect of Sorafenib induced HCC cell death was enhanced when Pin1 was inhibited.Therefore,we hypothesized that Pin1 is a key regulator of the anti-tumor effects of Sorafenib.It was further confirmed by the results showing that ATRA with Sorafenib synergistically induced HCC cell death.And it might be a kind of apotosis.Taken together,we demonstrated that Pin1 played an important role in Sorafenib induced cell death.Furthermore,development of Pin1 inhibitor is promising stategy to enhance the therapeutic efficacy of Sorafenib against HCC.