Effect And Molecular Mechanism Of HDAC Inhibitor Romidepsin In Cholangiocarcinoma

BackgroundIn the hepatobiliary system,cholangiocarcinoma is a kind of malignant tumor disease with high degree of deterioration.At present,radical resection is the main treatment.But the disease is occult in the early stage of the disease,so that some patients have been in the middle or late stage of the disease when diagnosed,thus missing the best opportunity for surgical treatment,resulting in poor prognosis.For patients with advanced or unresectable cholangiocarcinoma,chemotherapy is the main treatment.For example,gemcitabine combined with cisplatin as the first-line chemotherapy can benefit some patients,but it only increases the median survival time of these patients by about 3 months.At present,there is no specific drug for cholangiocarcinoma in clinic.With the deepening of cholangiocarcinoma related research,the key to improve the therapeutic effect of cholangiocarcinoma is to find new therapeutic drugs and diagnosis and treatment targets.Histone and DNA modifications affect the epigenetics of tumors and eventually lead to carcinogenesis.Histone can be covalently modified,in which deacetylation and acetylation have corresponding effects.The degree of acetylation is related to the biological effect of chromatin core histone.Histone deacetylase and histone acetyltransferase are the key proteases to maintain deacetylation and acetylation.When pathogenic factors cause the enhanced catalysis of histone deacetylase,histone deacetylation is obvious,resulting in the blocking of DNA transcription process,which will affect the relevant molecular signaling pathways and increase the incidence of tumor.Histone deacetylase inhibitor(HDACi)can inhibit the activity of deacetylase,destroy the dynamic balance between histone acetyltransferase and histone deacetylase,lead to histone acetylation,then activate the expression of tumor suppressor gene and inhibit the formation of tumor.HDACis is a kind of targeted therapeutic drugs with anti-tumor effect and the current clinical attention to this kind of drugs is increasing.More and more scholars carry out research around HDACis,hoping to get more extensive application in the next clinical.Romidepsin is a new type of HDACi,which was officially approved by FDA in November 2009.Initially,it was mainly used for the treatment of hematological tumors.In recent years,with the deepening of research,it has been reported that Romidepsin also has biological effects on solid tumors.In digestive tract tumors,it has been reported that Romidepsin has anti-tumor effect on gastric cancer,liver cancer,etc.,but there is no relevant research report on Romidepsin in cholangiocarcinoma before this project.Under the background of poor therapeutic effect and high mortality of cholangiocarcinoma in China,in order to improve the prognosis of patients with cholangiocarcinoma and prolong the overall survival of patients with cholangiocarcinoma,it is necessary to explore new targeted drugs for cholangiocarcinoma.This project aims to study the anti cholangiocarcinoma effect and related mechanism of Romidepsin.The solution of this scientific problem may provide a new strategy for the clinical treatment of cholangiocarcinoma,and provide a new direction and choice for the comprehensive treatment of cholangiocarcinoma.Objectives1.To research the effect of Romidepsin on proliferation inhibition and apoptosis induction of cholangiocarcinoma cells.2.To research the potential molecular mechanism of Romidepsin on proliferation inhibition and apoptosis induction of cholangiocarcinoma cells.3.To research the inhibitory effect of Romidepsin on cholangiocarcinoma cells in vivo.Methods1.The effects of different concentrations of Romidepsin on the viability of cholangiocarcinoma cell lines CCLP-1 and HCCC-9810 were detected by CCK-8 assay.2.The effects of different concentrations of Romidepsin on cell cycle changes of cholangiocarcinoma cell lines CCLP-1 and HCCC-9810 were detected by flow cytometry.3.The effects of different concentrations of Romidepsin on cell cycle related proteins CyclinB1 and p-cdc2 in cholangiocarcinoma cell lines CCLP-1 and HCCC-9810 were detected by Western blot.4.The effects of different concentrations of Romidepsin on apoptosis of cholangiocarcinoma cell lines CCLP-1 and HCCC-9810 were detected by flow cytometry.5.The effects of different concentrations of Romidepsin on apoptosis related proteins Cleaved-caspase-3 and Cleaved-PARP in cholangiocarcinoma cell lines CCLP-1 and HCCC-9810 were detected by Western blot.6.To establish subcutaneous cholangiocarcinoma model in nude mice and to evaluate the effect of Romidepsin in vivo.The expression of Ki67 protein in nude mice was detected by immunohistochemistry and to evaluate the inhibitory effect of Romidepsin on cholangiocarcinoma cell proliferation in vivo.Results1.CCK-8 assay was used to detect cell viability.In the cholangiocarcinoma cell lines of CCLP-1 and HCCC-9810,the activity of cholangiocarcinoma cells decreased with the increasing concentration of Romidepsin.Similarly,with the gradual prolongation of its action time,the cell viability also decreased.2.Cell cycle was detected by flow cytometry.Compared with the control group,in CCLP-1 cells treated with Romidepsin(5 nM,10 nM),the number of cells in G2/M phase increased significantly,while the number of cells in S phase decreased(P<0.01).In HCCC-9810 cells treated with Romidepsin(2.5 nM,5 nM,10 nM),the number of cells in G2/M phase was significantly increased,and the number of cells in S phase was also decreased(P<0.05).3.Cyclins were detected by Western blot.After treatment with Romidepsin(5 nM,10 nM)for 48 h,the expression level of cyclin B1,which promotes the progression of G2/M phase,decreased significantly in CCLP-1 and HCCC-9810.After treatment with Romidepsin(2.5 nM,5 nM,10 nM)for 48 h,the expression level of cyclin related protein p-cdc2,which inhibits the progression of G2/M phase,was significantly increased(P<0.05).4.Apoptosis was detected by flow cytometry.In cholangiocarcinoma cell lines CCLP-1 and HCCC-9810,the percentage of apoptotic cells increased significantly after treatment with Romidepsin(2.5 nM,5 nM,10 nM)(P<0.05).5.Western blot was used to detect apoptosis related proteins.Cholangiocarcinoma cell lines CCLP-1 and HCCC-9810 were treated with 2.5 nM,5 nM and 10 nM Romidepsin respectively.After 48 h,the expression of PARP did not change significantly(P>0.05),but the expression of cleaved-caspase-3 and cleaved-PARP increased significantly(P<0.05).6.Subcutaneous tumorigenesis in nude mice.Compared with the control group,the volume and weight of tumor in Romidepsin(0.5 mg/kg)group were significantly reduced(P<0.05).The weight of nude mice in each experimental group increased steadily,but there was no significant difference(P>0.05).Compared with the control group,the expression level of Ki67 protein in Romidepsin(0.5 mg 1 kg)group was significantly decreased(P<0.05).Conclusion1.Romidepsin can effectively inhibit the proliferation of cholangiocarcinoma cell lines CCLP-1 and HCCC-9810 in vitro in a time and concentration dependent manner.2.Romidepsin can induce G2/M phase arrest of cholangiocarcinoma cell lines CCLP-1 and HCCC-9810 by inhibiting CyclinB1 and promoting the expression of p-cdc2 in a concentration dependent manner.3.Romidepsin can promote the expression of apoptosis-related proteins Cleaved-caspase-3 and Cleaved-PARP,then induce apoptosis of cholangiocarcinoma cell lines CCLP-1 and HCCC-9810 in a concentration dependent manner.4.Romidepsin can effectively inhibit the growth of transplanted tumor in nude mice and the proliferation of cholangiocarcinoma cells in vivo.Romidepsin can effectively inhibit the growth of transplanted tumor in nude mice and the proliferation of cholangiocarcinoma cells in vivo,so it has anti-cholangiocarcinoma effect.