Experimental Study On The Synergistic Inhibition Of Hepatoma Cell Lines With Histone Deacetylase Inhibitor And 5-FU

The Primary Hepatocellular carcinoma is one of the most common malignant cancers, because of the lower excision ratio during operation, the higher mobidity and mortality, and the resistance to chemotherapy and ratiotherapy, the therapeutic options are very limited.5-FU remains one of the main anticancer agents used to a large number species of cancers , including hepatocellular carcinoma (HCC).However some studies have revealed that almost all of the hepatocellular carcinoma (HCC) cell lines show strong resistant against 5-FU-induced cytotoxicity, therefore there is a limitation to treat Hepatocellular carcinoma using 5-FU alone unless combined with some other therapeutic agents.Many studies have certified that tumor cells have the abnormality of gene phenotype states, the modify of acetylation and deacetylation is most major way that modify gene phenotype.Such sytle paritipate in the development and occurrence of many tumors.The mechanism that HDACIs cure tumor is a hotpoint that interven the gene phenotype modify.Summarize previous studies , we sum up several mechanism of HDACI:induce the differentiation of tumor; change the cycle of tumor through impacting some gene related to the cell cycle; induce the apoptosis of tumor; recover the sensitivity of drug-resistant tumor cells to drugs, and so on. In this study, we found that HCC cell lines such as HepG2 and Hep3B were significantly sensitized to inhibit and apoptosis by using 5-FU in combination with some HDACI such as MS-275 and the expression of some protein such as Bcl-2, p21, CyclinD1 might involve in this process. Moreover ,the expression of protein TS was very important in this process.Frist, we screen the optimal concentration of MS-275 in HCC cell line.We found that 0.5μmol/L and higher concentration MS-275 have obviously suppression effect to three kinds of HCC lines.The 1.0μmol/L and higher concentration MS-275 can obviously induce apoptosis. We get the optimal drug concentration of MS-275 which suppression rate is obvious but apoptosis rate is not.At the same time,we found that MS-275 showed significant growth effect on HCC cell lines effect on which were time and dose-dependent and were correlated with the p53 expression level. The growth inhibition effects of 5-FU on 3 HCC cell lines HepG2,Hep3B and Huh-7 were detected by MTT assay. 5-FU showed significant growth effect on HCC cell lines. The number of IC50 were correlated with the p53 expression level.We found the drug combination of MS-275 and 5-FU can increase the inhibition rate of two kinds of HCC cell, compared with MS-275 or 5-FU only, such two drugs are both concentration and time dependent style. We also found out that better synergistical effect would be get when HCC cell was exposed to MS-275 firstly in our test.MS-275 and 5-FU can both arrest such two kinds of HCC cells in G1 stage to suppress the proliferation of cell.the drug combination of MS-275 and 5-FU can arrest cycle in G1 stage obviously compared with MS-275 and 5-FU only(P<0.05). From the detection of gene and protein we found that 5-FU and MS-275 all enhanced p21 protein expression and suppressed the expression of CyclinD1 in Hep3B cells,group M→F increased more p21 expression level and suppressed the expression of CyclinD1 than group F and M.All of these maybe related with the proliferation of cell in G1 stage. Through FCM we found that the percentage of apoptosis increased after co administrated 5-FU with MS-275 in HepG2 and Hep3B cell line.These maybe related with down regulation in the expression level of Bcl-2. The expression level of Bax did not change.We also researched the role of TS in the combination of MS-275 and 5-FU. Result displayed that MS-275 can down-regulate TS expression and thus sensitize resistant cancer cells to 5-FU. We believe that MS-275 regulates TS expression in two ways—by indirect transcriptional repression of the TS gene and by the proteasomal degradation of TS protein.In this study, we found that HCC cell lines were significantly sensitized by using 5-FU in combination with HDACI such as MS-275, the expression of TS and the other protein relating cell cycle and apoptosis might involve in this process.We thus concluded that 5-FU-based tumor therapy in combination with HDACI agents might be a powerful therapeutic tool in the treatment of human HCCs.