The Significance Of RIP, C-FLIP Gene Expression And The Role In TRAIL-induced Apoptosis In Hepatic Carcinoma

Chapter One The significance of RIP, c-FLIP gene expression in hepatic carcinomaObjective:Receptor-interacting protein (RIP) is the effector to play an important role in the signaling pathways of tumor necrosis factor (TNF), and plays an important role in cell proliferation and apoptosis. Celluar FLICE-like inhibitory protein (c-FLIP) is an important inhibitor of apoptosis proteins.This chapter aim is to investigate the significance of RIP, c-FLIP gene expression in hepatic carcinoma.Methods:80cases of hepatic carcinoma were selected from January2006to December2009in General Surgery of The Third Xiangya Hospital and The Xiangya Hospital. The tissues of hepatic carcinoma were used for the study, and then40normal liver tissues of intrahepatic bile duct stones patients were taken as the control group. Immunohistochemistry was employed to measure the related expression of c-FLIP and RIP in the two kinds tissues. Then the correlations between expression of c-FLIP and RIP in hepatic carcinoma tissue and pathological grading of the tumor, clinical staging of patients and metastasis were investigated. Results:The expression of RIP and c-FLIP located in the cell cytoplasm, and the positive expression rates of hepatic carcinoma experimental group were78.8%and85.0%. While the positive expression rates in the control group were12.5%and17.5%respectively. The expression of the two cytokines was significantly associated with recurrence and distant metastasis, but not other factors such as tumor size and clinical stage. The expression of RIP and c-FLIP in hepatic carcinoma was significantly positively related.Conclusions:1. The expression of c-FLIP and RIP is significantly higher in tissue of hepatic carcinoma than those in normal liver tissue.2. The expression of c-FLIP and RIP in the tissue of hepatic carcinoma is significantly correlated with clincopathological indexes of the tumor.3. The c-FLIP and RIP might participate in cell apoptosis, occurrence and development of hepatic carcinoma together. Chapter Two The role in TRAIL-induced apoptosis in hepatic carcinomaObjective:Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces apoptosis by the death receptor pathway is an important mechanism of natural anti-tumor. The resistance of TRAIL is an important factor of liver cancer with poor prognosis. Chemotherapy drugs could reverse tolerance of the liver cancer cells HepG2and Hep3B on TRAIL-induced apoptosis, and its mechanism is lowered the expression of RIP and c-FLIP in the death-inducing signaling complex (DISC) of TRAIL signal conduction. We build the RIP and c-FLIP gene silencing in hepatic carcinoma cell model to study the impact of TRAIL resistance by RIP and c-FLIP expression.Methods:RIP and/or c-FLIP gene silencing subclone cell model of HepG2and Hep3B cell lines were established by SiRNA technology. Following studies were taken. DISC analysis after TRAIL-induced apoptosis to learn apoptotic signal conduction was blocked at the DISC level or not. Application of c-FLIP, RIP siRNA, the roles of c-FLIP and RIP were investigated in the inhibition of caspase-8activity.Results: After HepG2and Hep3B cell lines processed by100ng/ml TRAIL (treatment concentration) at different time, the degree of activation of Caspase-8/9/3reduced without the cleavage of DFF45. There were no significant effects on the expressions of DR4and DR5in DISC by mitomycin and doxorubicin (chemotherapy drug). In the TRAIL alone application group, the expressions of c-FLIP and REP were significantly strong, and the expression of FADD was low. After the regulation of chemotherapeutic drugs, the expressions of c-FLIP and RIP turned down, while the expression of FADD turned up. After the interference of gene expression of c-FLIP and RIP, the killer cells ability of TRAIL gradually increased with the increasing concentration of TRAIL. Moreover, the ability was achieved by inducing apoptosis. Compared with the control group, cell cycle analysis showed that the percentage of subGl turned higher significantly in siRNA group, and the cells were in G1phase growth retardation.Conclusions:1. The inhibited Caspase-8activity is main reason of TRAIL tolerance in hepatic carcinoma cell lines HepG2and Hep3B.2. The high expression of c-FLIP and RIP in the DISC are the main reason of the hepatic carcinoma cell lines HepG2and Hep3B TRAIL tolerance.3. The downregulation of c-FLIP and RIP chould lift the Caspase-8 inhibition of state and promote the apoptotic signal transduction. Chapter Three The effect of RIP and c-FLIP adjusting on TRAIL-induced apoptosis of hepatic carcinomaObjective:Cancer cells exhibit increased glycolysis and depend on this metabolic pathway for ATP production. This metabolic feature has evoked much interest in development of glycolytic inhibitors as potential anticancer agents.2-deoxy-D-glucose (2-DG) is a synthetic glucose analog that is phosphorylated by hexokinase upon transport into cells, but cannot be fully metabolized. The phosphorylation of2-DG accumulates in cells and interferes with glycolysis primarily by inhibition of phosphorylation of glucose by hexokinase, thus causing a depletion of ATP.2-DG can also cause inhibition of protein glycosylation that induces endoplasmic reticulum (ER) stress and gives rise to activation of the unfolded protein response (UPR). The purpose of this chapter is to explore the relationship between2-DG and RIP&c-FLIP, and then explore influence of TRAIL-induced cell apoptosis in hepatic carcinoma by the regulation of RIP and c-FLIP.Methods:Through the cell viability of analysis, changes in mitochondrial membrane potential and the cell apoptosis determined by PI stained, the effect and mechanism of apoptosis of hepatoma cells were studied by 2-DG Through quantitative RT-PCR, Western blot analysis and siRNA technology, the regulations of RIP and c-FLIP by2-DG were researched, and influence of TRAIL-induced cell apoptosis in hepatic carcinoma by the regulation of RIP and c-FLIP was studied.Results:2-DG alone did not induce significant apoptosis, however, it can inhibit cell differentiation; the combination of2-DG and TRAIL enhanced TRAIL-induced anti-differentiation and apoptosis. when the changes in JC-1fluorescent probe labeling of the mitochondrial membrane and mitochondrial membrane damaged, JC-1’s staining showed green fluorescence, normal cells showed red fluorescence. When liver cancer cells in combination with2-DG and TRAIL and mitochondrial film damaged significantly, JC-1’s staining showed green fluorescence. Caspase inhibitors (z-VAD-fmk) could inhibit HepG2cells and Hep3B cell lines without TRAIL-induced apoptosis. There was a statistically significant difference (P<0.05) in the condition of TRAIL-induced apoptosis by caspase inhibitors with or without2-DG Through Quantitative RT-PCR, the expression level of RIP and c-FLIP continued to decrease after the function of2-DG The down regulation of RIP and c-FLIP had a high correlation with2-DG during siRNA. The down regulation of RIP and c-FLIP was the key to increase the sensitivity of TRAIL-induced apoptosis of hepatoma cells during siRNA. Conclusions:1. The down regulations of RIP and c-FLIP by2-DG increase TRAIL-induced apoptosis of hepatoma cells.2.2-DG is dependent on caspase increasing TRAIL-induced apoptosis.