Inhibition Of Liver Cancer Cell Growth Of Peroxisome Proliferator-activated Receptor γ

Human hepatocellular carcinoma (HCC) is one of the most common cancers, threatening the peoples' health severely, which characterized with high malignant grade and poor prognosis. At present, surgical resection remains the first and most effective treatment for HCC patients, though the doctors have made great progress in these decades, the prognosis is unsatisfied with a 5-years survival rate at 30～40% postoperatively. As a critical adjuvant treatment method, chemotherapy faces a problem, the experiential effective rate at only 25% about. Therefore, it is essential to find more new therapy methods.Recent researches indicate that Peroxisome proliferator-activated receptor γ (PPARγ) is therapy site for many diseases, such as obesity, type 2 diabetes mellitus, hyperlipaemia, arteriosclerosis and tumor's prevention and treatment, etc., the research deeper as the time goes on. At present, it is found that PPARγ plays an important role in fat metabolism, tissue's inflammation, cell differentiation and regulation of cell cycle.Actiation of PPARγ makes a low expression of phosphatase PP2A, and enhances its phosphorylation, which casts down the binding activation of DNA and its substrate: E2F/DP transcription factor, which is the one of the main transcription regulation factors in cell cycle. Consequently, a cascade reaction is activated by PPARγ, the cell cycle suspend at the peak of the reaction. Based on the foundations of PPARγ on cell differentiation and cell cycle regulation, some argue that whether the activated products of PPARγ can be used in tumor therapy? Some research infer that some tumor cell's proliferation are restrained after being given the activator of PPARγ, such as liposarcoma, breast cancer, prostatic csarcinoma and colonrectal carcinoma, furthermore, the activator can induce the apoptosis of lipocyte, macrophage, breast cancer cell, prostatic csarcinoma cell and non-small cell lung carcinoma cell, but the research are comparatively rare in HCC.According to the former research, it is safe to conclude that the new clues and concepts can be obtained after analysis the abnormity activation of PPARγ, and the new bio-target drug can thrust the bio-therapy effectively combining with traditional cytotoxicity drug. According to the investigating of ciglitazone—PPARγ's activator, the study probe into the growth mechanism of HCC, especially on the influence of HCC cell's proliferation and apoptosis. The scientific significances are: 1.The research explores a new strategy of tumor therapy aiming at the direct causation of chemotherapy curative effect, and gives the theory base for the research and development of new efficient and low-toxicity anticancer drugs, and improve the chemotherapy effect. 2. The research has a brilliant prospect in tumor therapy, would be used in clinical therapy effectively in the future.The research focus on the following aspects:1. Study the expression of PPARγ in HCC cell according to RT-PCR and Western-Blot techniques.2. Observe the growth change of HepG2 cell line after the effect ofPPARγ ligand------ ciglitazone according to MTT technique, andtest the cell apoptosis index by flow cytometry.3. Test the apoptosis and restraining mechanism of HepG2 cell under the induce of PPARγ, via western blot technique.4. To investigate the yole of NF-κB in the process of differential regulation of ciglitazone on the liver cancer cells HepG2.5. Establish a nude mice model of HCC, inject PPARγ ligand and chemotherapy drug into the tumor, then test the expressions of VEGF and VIII factor according to immunohistochemistry technique, and the change of caspase3, cyclin D1 and p21 protein's expression, research into the anti-tumor mechanism of PPARγ. Part 1 Expression and implication of PPARγ in hepatic cancer cellsObjective To investigate the expression of Peroxisome proliferator-activated receptor γ( PPARγ), and determine the role in hepatocellular cancernoma. Methods PPARγ in 2 strains of hepatic cancer cells and 1 strain normal hepatic liver cell were detected by RT-PCR and western-blot. Results PPARγ was detected in all the three strains cell, however PPARγ expresses significantly in 2 hepatic cancer cells than that in L02 cells. Conclusion A high expression of PPARγ in Hepatic cells suggest it play an important aim to treat HCC.Part 2 Induction of cell cycle arrest in human liver cancer by activation of PPARγI Effects of ciglitazone on proliferation and apoptosis of the hepatocellular carcinoma cell line (HepG2) in vitro Objective To investigate the effects of ciglitazone on proliferation and apoptosis of the hepatocellular carcinoma cell line (HepG2) in vitro, and elucidate the the change of cycle protein. Methods The study in vitro was carried out in the culture of HepG2 lines. Various concentrations of ciglitazone were added and incubated. Cell proliferation was detected with MTT colorimetric assay. Cell apoptosis was detected by electron microscopy and flow cytometry. Results The proliferation of HepG2 was inhibited by ciglitazone, which depending on dose and time significantly. Cells growth well on 1, 3 day at different concentrations, The HepG2 proliferation rates of groups at the end concentrations 10, 25 and 50μmol/L were respectively, significantly lower than that of normal control group(P<0.01) on 7^th day. Meanwhile, it was showed that the inducing effects of ciglitazone on HepG2 apoptosis were dose-dependent and time-dependent. The apoptosis index(AI) was detected by FCM. The HepG2 apoptosis index of groups at the end concentrations 10, 25 and 50μmol/L were 7.90%, 28.18% and 38.36% respectively, significantly higher than that of normal control group(P<0.05). Conclusions Ciglitazone could significantly inhibit HepG2 proliferation and increase the apoptosis index of HepG2 dose-dependently and time-dependently.II Mechamsin of ciglitazone on proliferation and apoptosis of the HepG2 in vitroObjective To investigate the mechamsin of ciglitazone on proliferation and apoptosis of the hepatocellular carcinoma cell line (HepG2) in vitro. Methods The study in vitro was carried out in the culture of HepG2 lines. 50μmol/L ciglitazone were added and incubated. Cyclin D1, p21 and caspase3 protein were detected by western blot. Results P21 and caspase3 significantly were higher than that of normal control group(P<0.05), but cyclinD1 expression was lower than that of normal control group. Conclusions Ciglitazone could significantly inhibit HepG2 proliferation and its mechanisms can be it can induce proliferation and inhibit apoptosis.Part 3 Influence of NF-κB on ciglitazone inducing livercancer cells HepG2 apoptosisObjective To investigate the role of NF-κB in the process of differential regulation of ciglitazone on the liver cancer cells HepG2. Methods NF-κB inhibitor was added into HepG2 cells, electrophoretic mobility shift assays(EMSA) was performed to investigate the activation of NF-κB. When ciglitazone was added, the proliferation was observed by growth curve and the differentiation of cells was observed by flow cytometry, the expression of Bcl-2 and Bax was observed by Western blot. the activity of caspase3 was found by quantitative assay Results EMSA showed PDTC could decrease the activation of NF-κB in HepG2. Ciglitazone could obviously inhibit the growth and induce the differentiation of HepG2 cells that were added with PDTC. Treated with 50μmol/ L ciglitazone (for 7day) it induced typical apoatosis. After treatment, a decrease in Bcl-2 expression in HepG2 cells was observed by Western blot analysis, and the expression of Bax, however, was up-regulated. Caspase3 activity was increased. Conclusion Inhibiting NF-κB could enhance the effects of ciglitazone on proliferative suppression and differential induction of HepG2 cells.Part 4 Inhibition of ciglitazone on tumor angiogenesisand growth in vivoI Inhibition of ciglitazone on tumor angiogenesis in nude mice transplants of hepatic carcinoma.Objective To investigate the effect of ligant of Peroxisome proliferator-activated receptor gamma (PPARγ) on tumor angiogenesis in nude mice transplants of hepatic carcinoma. Methods RT-PCR was use to detect PPAR mRNA expression in hepatic carcinoma tissuse, to detect the VIII factor and VEGF of the ciglitazone-treate nude mice transplants of hepaticcarcinoma by using SP method and western blot. Results PPAR mRNA was detected in tuomr, and ciglitazone treatment group, tumor grew slowly [207+192.9mm³, p<0.05]], The MVD and VEGF expression in ciglitazone group were lower than that of control group. Conclusion Ciglitazone treatment, tumor grow slowly, decrease of tumor MVD and VEGF. Those strongly suggest that ciglitazone maybe play an important role in angiogenesis.II Influence of ciglitazone on hepatic cancer cells HepG2 growth in vivo and its mechanismsObjective To study the effect of the ciglitazone on hepatic cancer cells HepG2 growth in vivo and its mechanisms. Methods HepG2 cells(1×10⁶ /mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group(group A, n=10), the ciglitazone treated group(group B, n=10). The expressions of cyclinD1, caspase3 and P21 were analyzed by Western blot. Results The direct injection of ciglitazone into HepG2-induced tumors was able to suppress hepatic cancer growth in nude mice. The expression of cyclinDl in group A was increased significantly than that of group B, the lever of P21 and caspase3 were decreased significantly in group A when compared with group B. Conclusions Ciglitazone could significantly inhibit HepG2 growth and its mechanism may be due to the fact that It can induce the differentiation of HepG2 and inhibit cells apoptosis. In summary,it comes the following point of views1. activator ciglitazon of peroxisome proliferator activated receptor γ( PPARγ) has high dose expression, it can be taken as a therapy site for HCC.2. Ciglitazone can restrain the growth of HCC cell, furthermore, can induce the apoptosis of the cell, and has an obviously time and dose dependence.3. Ciglitazone can restrain the proliferation of HCC cell, the mechanism of inducing apoptosis has correlation with the activity change of cell cycle protein and caspase3 protein.4. Inhibiting NF-κB could enhance the effects of ciglitazone on proliferative suppression and differential induction of HepG2 cells.5. Ciglitazone can restrain the growth of HCC cell, restrain the vascularization of the tumor maybe one of the mechanism.