The Research Of The DR4 And DR5 Expressions In Hepatocellular Carcinoma Apoptosis Induced By TRAIL

Objective: The Primary Hepatocellular carcinoma is one of the most common malignant cancers, which is difficult to diagnose and develop quickly. The morbidity of this disease is going to rise year by year. Operation remains the major management for this disease, and others contain ablation therapy, interventional therapy, chemotherapy, ratiotherapy, biotherapy etc. But the exist therapy is still unsatisfactory. 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 tumor 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, but were significantly sensitized to TRAIL-induced apoptosis by using TRAIL in combination with some chemotherapeutic agents, in particular, camptothecin, cisplatin and celecoxib, therefore there is a new therapeutics to treat Hepatocellular carcinoma using TRAIL combined with some other therapeutic agents.Our research aims at the effect of TRAIL and cisplatin on grouth of hepatoma carcinoma cell through culture of the hepatoma carcinoma cell (HCC) line HepG2, and discuss the expresstion of TRAIL receptors DR4, DR5 on the HepG2.Methods:1. Expression of DR4 and DR5 in 24 hepatoma tissues and 20 nomal liver tissues by using immunohistochemistry.2. Cell culture: Refrigerated HepG2 cells were anabiosised and were incubated in 37℃, 5%CO2 and saturated degree of humidity culture box. Cells were digested and subcultured with 0.25% steapsin.3. Cell proferation: HepG2 cells were seeded in 96-well culture plates at a density of 0.8-1.0×105cells/ml (100 l/well) in culture medium. After having stayed over in 24h, the cells were divided into 3 groups and their concentration of the drugs were as follows:①with TRAIL (10ng/ml, 30ng/ml, 100ng/ml, 300ng/ml, 1000ng/ml) for 24 hours.②with cisplatin (1.25 g/ml, 2.5 g/ml, 5 g/ml, 10 g/ml, 20 g/ml) for 24 hours.③with TRAIL(100ng/ml) and cisplatin(1.25 g/ml, 2.5 g/ml, 5 g/ml, 10 g/ml, 20 g/ml)collectively for 24 hours. After a time, 10 l MTT (5mg/ml) was added in every well for 4h at 37℃. Then added 200 l DMSO into each well. Then the absorbance of cells in each well was detected with ELISA Reader when wavelength was 490nm. Each assay was performed 3 times independently.4. Detect the cell cycle by flow cytometry: HepG2 cells were seeded in 96-well culture plates at a density of 0.8-1.0×105 cells/ml (100 l/well) in culture medium. After having stayed over in 24h, the cells were divided into TRAIL group (with TRAIL 100ng/ml), cisplatin group (with CIS 1.25 g/ml, 2.5 g/ml, 5 g/ml, 10 g/ml, 20 g/ml) and combine group (with TRAIL 100ng/ml and CIS 1.25 g/ml, 2.5 g/ml, 5 g/ml, 10 g/ml, 20 g/ml). After 24 hours the cell were collected to detect the cell cycle by using flow cytometry. Each assay was performed 3 times independently.5. Detect the expressions of TRAIL receptor DR4 and DR5: HepG2 cells were incubated in 37℃, 5%CO2 and saturated degree of humidity culture box. After having stayed over in 24h, the cells were divided into TRAIL group (with TRAIL 100ng/ml) and combine group (with TRAIL 100ng/ml and CIS 2.5 g/ml). Added monoclonal antibody into the liquid and after 24 hours the cell were collected to detect the expression of TRAIL receptors DR4 and DR5 by using flow cytometry. Each assay was performed 3 times independently.Results:1. The expression of DR4 and DR5 were observed in both cell membrane and cytoplasm. The expression rate of the DR4 and DR5 had no statistical significance in both the hepatoma tissues and the nomal liver tissues.2. Inhibitory rate of HepG2 cell after TRAIL treatment had no statistical significance with the control group (P>0.05). Grouth inhibition of cell after TRAIL combined with cisplatin were respectively 64.49%±1.94%,74.15%±1.64%,78.90%±0.97%, 92.31%±0.35%, comparing with the same dose of cisplatin group, the difference had marked significance.3. Cell cycle analysis indicated that cell growth were not inhibited with TRAIL but less inhibited with cisplatin group alone. But pretreated the HepG2 cells with lower dose of cisplatin (2.5μg/ml) 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. The expression rate of subG1 was 74.16%±0.28%, there was marked significant difference compared with cisplatin group alone (10.63%±0.43%).4. The positive expression of DR5 treated with TRAIL conbined with cisplatin were higher than treated with TRAIL alone (P<0.05), but the expression of DR4 of the two group had no significance.Conclusion: 1. The cisplatin could overcome the resistant to TRAIL-induced cytotoxicity in HepG2 cell. 2. TRAIL conbined with cisplatin could induce cell death by apoptosis. 3. The resistance against TRAIL-induced cytotoxicity of HepG2 relactanted with the low expression of TRAIL receptors. 4. Cisplatin enhanced the ability of apoptosis induction of TRAIL to HepG2 cells by upregualation of DR5.