The Research Of The Combination Treatment Of Heptocellular Carcinoma Cell Lines With TRAIL And Chemotherapeutic Drugs

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. TRAIL (Tumor necrosis factor-Related Apoptosis Inducing Ligand) is a recently identified new members of the TNF family, and has emerged as a novel therapeutic agent because it selectively induced apoptosis in tumorogenic or transformed cells over normal cells. However some studies have revealed that almost all of the Hepatocellular carcinoma (HCC) cell lines show strong resistant against TRAIL-induced cytotoxicity, therefore there is a limitation to treat Hepatocellular carcinoma using TRAIL alone unless combined with some other therapeutic agents.In this study, we found that HCC cell lines such as HepG2 and Hep3B were significantly sensitized to TRAIL-induced apoptosis by using TRAIL in combination with some chemotherapeutic agents, in particular, camptothecin, cisplatin and celecoxib, and the expression of c-FLIP and RIP might involve in this process.First of all, our results showed that HCC cell lines were resistant to TRAIL alone, after treated with 1000ng/ml TRAIL for 24h, the cell viability of Hep3B and HepG2 were 96.39%±5.66% and 96.91%±5.69%, there was no significant difference compared with control group. But pretreated the HepG2 and Hep3B cells with lower dose of chemotherapeutic agents, such as 2.5μg/ml Cisplatin, 0.3μg/ml Camptothecin and 50μmol Celecoxib could overcome the resistant to TRAIL-induced cytotoxicity by apoptosis, cell cycle analysis indicated that cell growth were inhibited at G1 phase and formed the subG1 peak before G1 cotreated with TRAIL and chemo drugs, but at S phase when treated with chemo drugs alone. Further study revealed that it also could downregulate the expression of c-FLIP and RIP, and then activate the most up-stream caspase, caspase-8, and the critical down-stream caspase, caspase-3, eventually induce cell death. The upregulatation of DR5 and FADD might also contribute to the sensitization of HCC cell lines to TRAIL-induced apoptosis.To further investigate the effect of expression of c-FLIP and RIP in HCC cell lines resistant to TRAIL, we did the analysis of TRAIL-induced DISC after treatment with chemotherapeutic agents and interfered the expression of c-FLIP and RIP by the technique of small interfering RNA, the results showed that the expression of c-FLIP and RIP were downregulated, the FADD was upregulated, and DR4 and DR5 in DISC did not change after treated by chemo drugs, therefore, the high expression of c-FLIP and RIP in DISC made the signal of apoptosis inhibite at the level of inactivation of Caspase-8. Next, we constructed the c-FLIPsiRNA and RIPsiRNA cell lines using the technique of small interfering RNA, which were sensitive to TRAIL by cell viability analysis, furthermore, cell cycle analysis indicated that the siRNA cell lines formed the subG1 peak before G1 phase, and the cell growth were inhibited at G1 phase compared with control group. In addition, compared with control group, the transduction protein of apoptosis signal, such as precaspase-8, was activated and cleavaged, and then activated the effective caspase, caspase-3, in the end, made the DFF45 occur the cleavaged bands, that were P25, P17, and P11, from this, it can be seen that the apoptosis were induced and the signal transduction were recovered after interfering the expression of c-FLIP and RIP. Another result was that the expression of p-Akt and p-IKb-α, which were the upstream important regulated proteins of NF-κB pathway, were downregulated and the expression of Akt and IKb-αwere upregulated when interfered the gene of RIP, the result indicated that the activation of NF-κB was blocked because of the inhibition of the phosphorylation of Akt and IKb-α. All of these results above confirmed that the higher expression of c-FLIP and RIP were major resistant machenism of HCC cells to TRAIL.So, co-treatment of TRAIL with chemotherapeutic agents could overcome the limitation of TRAIL alone, the Caspase-8 avtivity was inhibited by high expression of c-FLIP and RIP in DISC and the NF-κB pathway was activated because of the expression of RIP might contribute to the HCC cell lines survival. We thus concluded that TRAIL-based tumor therapy in combination with chemotherapeutic agents might be a powerful therapeutic tool in the treatment of human HCCs.