| Objective:Cholangiocarcinoma is a common malignancy of biliary tract,which has Late clinical symptoms and is difficult to diagnosis early.And its radical surgical resection rate is low and the prognosis is poor.At present,some datas indicate that cholangiocarcinoma is not sensitive to chemical treatment,and exact cause is unclear.In recent years,with the study of the mechanism of TRAIL -induced apoptosis of tumor cells,we found that TRAIL can selectively induce tumor cell apoptosis.And we also found that some tumors are not sensitive to the treatment of TRAIL,but these tumors after the stimulation of chemotherapy are sensitive to it again.TRAIL and chemotherapeutic drugs in combination may provide a new way for the treatment of cholangiocarcinoma.Our research aims at the effect of TRAIL and 17-AAG on growth of Cholangiocarcinoma cell through culture of the Cholangiocarcinoma cell line RBE,and discussed this phenomenon preliminarily.First of all,we found that RBE cholangiocarcinoma cell line cells are resistant to the effect of TRAIL alone.After the role of different concentrations of TRAIL in RBE cells,the rate of RBE cell survival are 1.88%±0.405%,2.50%±0.525%,2.70%±1.416%,2.33%±1.098%and 2.31%±0.136% respectively.It can be concluded that RBE is resistant to TRAIL.Grouth inhibition of cell after TRAIL(100ng/ml) combined with different 17-AAG (0.1μmol,0.3μmol,0.5μmol,1μmol) for 24 hours were 14.35%±2.325%,31.01%±2.681%,38.74%±2.373%,42.11%±2.810%respectively,comparing with the same dose of 17-AAG group,the difference had marked significance.It did not cause obvious cell apoptosis in a separate group of TRAIL or 17-AAG.However,in Joint Application of the group,the sensitivity of cell changed after pre-treating by 17-AAG.Joint application of the two significantly increased the ability of TRAIL to kill cells.Then through DNA ladder detection we confirmed that 17-AAG increased RBE sensitivity of cells to TRAIL is by inducing apoptosis.We deal with the cells with 17-AAG(0.5μmol) and TRAIL (100ng/ml),then have DNA ladder detection.The results shows that clear DNA ladder can be seen when 17-AAG and TRAIL are used and less DNA ladder turn up when TRAIL are used alone.As can be seen 17-AAG can enhance TRAIL-induced apoptosis of REB cells.For further study of that,we use Western blot to detect the expression of Caspas-8 and Caspas-3 protein in TRAIL group and 17-AAG in combination with TRAIL group.The results show that TRAIL alone can not activate Caspas-8 precursor,so it can not induce cell apoptosis. However the two applications,there is obviously Caspas-8 and Caspas-3 activation with cleavage.It indicated that chemotherapy drugs in combination with TRAIL,lifted the suppression condition of Caspas-8 and restored the apoptosis signal transduction,and promoted the occurrence of apoptosis by some mechanism.In order to further explore the mechanism that 17-AAG in combination with TRAIL activated Caspas-8.We detected the expression of receptor DR5 on tumor cell surface and that of upstream regulatory protein RIP of Caspase-8 which are influenced by TRAIL combined with chemotherapy drugs.The results show that,DR5 expression of RBE cells which have 17-AAG and TRAIL co-treatment comparing with TRAIL alone treatment group and control group has not changed.The mechanism that 17-AAG changed the sensitivity of RBE cell to TRAIL is not mainly due to the up-regulation of the expression of TRAIL death receptor.We can see significant down-regulation of the expression of RIP.This shows that after pre-chemotherapy treatment,it may lift the inhibition of Caspase-8 by down-regulation of the expression of RIP and then induce apoptosis.Conclusion:(1)The 17-AAG could overcome the resistant to TRAIL in Cholangiocarcinoma cell line RBE.(2)TRAIL conbined with 17-AAG could induce the death of Cholangiocarcinoma cell line RBE by apoptosis.(3)17-AAG and TRAIL in inducing apoptosis of cholangiocarcinoma cells have significant synergies.The mechanism may lift the inhibition of Caspase-8 by down-regulation of the expression of RIP and then induce apoptosis.. |
PDF Full Text Request