The Mechanisms Of Cordycepin Suppresses Cell Proliferation And Migration By Targeting DEK In Cholangiocarcinoma Via Akt And Erk1/2 Signaling Pathway

Objective:To investigate the molecular mechanisms of cordycepin on cholangiocarcinoma cell proliferation and migration inhibition.Materials and methods:Cholangiocarcinoma cells were treated with cordycepin in different concentrations,MTT assay were used to illustrate its inhibition effect on cell viability and the ability of cell colony formation were detected by colony formation assay.Hoechst33342 dye were used to detect the changes of apoptotic bodies in the cell,and Annexin V/PI double staining were applied to show the ratio of cell apoptosis after cordycepin treatment by using flow cytometry.Wound healing assay and Transwell assay were used to analyze the impact of cordycepin on cancer cell metastasis.Immunofluorescence assay were used to evaluate the expression level of E-Cadherin and Vimentin.Apoptosis associated proteins,EMT markers,key members of Akt,Erk1/2 signaling pathway and DEK were detected by western blot and the expression level were analyzed.Capacities of cholangiocarcinoma viability,colony formation and migration were detected after DEK knockdown.RT-PCR were used to analyze the mRNA expression level of DEK in both normal and various cancer cell lines.DEK expression level were demonstrated using immunohistochemical staining.In vivo study,E-Cadherin,Vimentin,p-Akt,p-Erk1/2 and DEK were also analyzed by immunohistochemical staining,the inhibition effect of cordycepin on cholangiocarcinoma were detected.Results:1.Treatment of cordycepin using 0?M,25 ?M,50 ?M,100?M,200 ?M on HuCCT1 cancer cell line and using 0 ?M,37.5 ?M,75 ?M,150?M,300 ?M on CCKS1 cancer cell line,MTT assay were used to analyzed the viability of both cell line at each 24 h,48 h,72 h time points.Compared with control group,cordycepin showed significant inhibition effects on cell viability in a dose-time dependent manner(P<0.05).2.Treatment with different concentrations of cordycepin on cholangiocarcinoma cells by colony formation assay,compared with control group,the ability of colony formation of both cell lines were significantly inhibited in a dose dependent manner(P<0.05).3.Cordycepin concentration 100?M were used to treat HuCCTl and 150?M on CCKS1 for 48 h before Hoechst33342 staining.The result showed that compared with control group,the bright blue nuclei increased significantly.4.Annexin V/PI double staining analyzed using flow cytometry were performed to determine the ratio of apoptosis after cordycepin treatment.Compared with control group the ratio of apoptosis were significantly upregulated(P<0.05).5.Wound healing assay showed that after treatment for 12 h on both cell lines using different concentrations of cordycepin,compared with control group,the transverse migration distances were significantly shortened,and both showed a dose-dependent manner(P<0.05).6.Transwell assay were also performed after cordycepin treatment for 12 h.Compared with control group,the vertical migration ability were significantly inhibited on both cell lines(P<0.05).7.Immunofluorescence staining showed that after cordycepin treatment for 24 h on both cell lines.The expression level of E-Cadherin were upregulated while Vimentin were downregulated,compared with control.8.Western blot showed that,compared with control group,the expression level of apoptosis associated proteins,Cleaved caspase 9,Cleaved caspase 3 and Cleaved PARP were upregulated.EMT markers E-Cadherin were upregulated,and Vimentin,Snail,Slug,MMP9 were downregulated.The expression of Akt singling pathway associated proteins,p-Akt and p-4EBP1 were inhibited.Key members of Erk1/2 signaling pathway,p-Erkl/2 were downregulated.No significant changes in all the total proteins.The expression of DEK were significantly inhibited.9.Immunohistochemical staining showed that DEK was overexpressed in cholangicarcinoma,compared with the periment tumor.10.RT-PCR results also demonstrated that compared with human normal biliary epithelial cells,the mRNA level of DEK were much higher in cancer cell lines.11.DEK knockdown using siRNA significantly inhibited the viability,the ability of colony formation and migration of HuCCT1(P<0.05).12.DEK knock down also inhibited the expression of p-Akt and p-Erk1/2.No significant changes were detected after the treatment using Akt inhibitor Akti 1/2 and Erk inhibitor GDC-0994.13.In vivo,compared with control group,treatment of cordycepin significantly reduced the gowth of xenograft tumors.The reduction in relative tumor volume was observed from 9 days after administration of cordycepin(P<0.05).No significant changes were detected in body weight.14.Tumor tissue harvested form these animals were processed for IHC staining,compared with control group,cordycepin treatment significantly reduced the expression of E-cadherin,Vimentin,p-Akt,p-Erkl/2 and DEK.Conclusion:1.Cordycepin inhibits cancer cell proliferation and migration by apoptosis induction and EMT progression attenuation in cholangiocarcinoma.2.Cordycepin inhibits cancer cell proliferation and migration through Akt and Erkl/2 signaling pathway reduction in cholangiocarcinoma.3.Cordycepin inhibits cancer cell proliferation and migration by targeting DEK via Akt and Erkl/2 signaling pathway in cholangiocarcinoma.