| 1.Background:Primary carcinoma of the liver is the common malignant tumor that do harm to thehuman beings' health. Epidemic disease investigation showed that Primary carcinoma of the liver and HBV and HCV chronic influence is related closely. The influence of hepatitis has high incidence of a disease in our country. The infected persons of the HCV and HBV that exceeds one hundred millions, thus the rate of incidence of liver cancer keeps high. According to statistics of Department of Health, Primary liver cancer is the second position lethal factor in all tumors, which is next only to lung cancer in city or cancer of the stomach in rural. Therefore the research of treating liver cancer is urgent. Now, surgical operation, chemotherapy, radiation therapy and biotherapy are four kinds of main way to treat it. In recent years, with progress of the pathogenesis and treatment research of liver cancer, it was known that the unusual mechanism of cell apoptosis in liver cancer occuring and treatment took an important role. From the recent research, we induce cancer cell apoptosis in much anti-cancer therapy.TRAIL, a type II transmembrane protein, is a recently identified member of the TNF family and is capable of inducing apoptosis in various transformed cells. Of the three major death ligands ?TNF, FasL, TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), was the most effective at inducing apoptosis in the cell lines. TRAIL induces apoptotic death on binding to either of two proapoptotic TRAIL receptors, TRAIL Rl (DR4) or TRAIL R2 (DR5). Normal cells are believed to be resistant to TRAIL because of expressing higher levels of TRAIL decoy receptors TRID (DcRl) or TRUNDD (DcR2) on their cell surface. TRAIL, unlike TNF and Fas ligand, has shown a relative sparing of normal cells. Given systemically, has been safe in animals. TNF-related apoptosis inducing ligand is a promising agent for development as a cancer therapeutic agent. However, the potential utility and safety of systemic administration of TRAIL has recently been questioned because of results showing sensitivity of human but not monkey; nonhuman primates or mouse hepatocytes to recombinant human TRAIL in vitro. If TRAIL is ultimately found to be toxic to human hepatocytes in vivo, it will limit the clinical use of systemic TRAIL. Many viruses could induce apoptosis on infected cells, such as RNA viruses HIV, Measles virus, Influenza virus, etc. ; but some DNA viruses could inhibit apoptosis on infected cells, including Herpesvirus, Adenovirus, Human papilloma virus, etc. 2.Objective:To study TRAIL cooperated with chemotherapeutic agents induces apoptosis in human hepatocellular carcinoma cell lines. Normal human liver cell could be protected from TRAIL-induced apoptosis with the exposure to caspase-9 inhibitor (Z-LEHD-FMK) and the effect of HBV on TRAIL inducing hepatocyte apoptosis. 3.Methods:1) Cytotoxicity Determination Assays: The cells were plated in 96-well microtiter plates (NUNCLON Surface, Denmark) with a density of 2 104 cells/well. Each plate was incubated with human recombinant TRAIL (PeproTech EC Ltd, England) of different concentration, either 50-1600ng/ml) or 200ng/ml, as well as the presence of 0. 1 g/mldoxorubicin (Bedford Laboratories, Bedford, OH). Caspase 9 inhibitor 1 (Z-LEHD-fmk) (Calbiochem, U. S. A) and Lamivudine (GlaxoSmithKline, U. S. A) were incubated for 18 hours at 37 in 5% CO2. 10% MTT, dissolved in phosphate buffered saline with a concentration of 5mg/ml, was added to the medium directly to incubate about 4 hours. The absorbance of each well was measured at 570nm with a microtiter plate reader. The mean value of three assays performance and the percentage of cell viability with a reference to the untreated controls were calculated. The percentage of cytotoxicity was calculated by the following equation.Percentage cytotoxicity =[1-(absorbance of experimental wells/absorbance of control wells)] 100%.2) Analysis of Apoptosis: Annexin V assay for apoptosis was conducted wi... |
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