| Objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in our country, which is frequently associated with high lever of malignancy, poor prognosis and high recurrence rate after operation. But its exact moleculaor mechanisms of hepatocarcinogenesis is not clarified. There is not effetive preventive measure to it yet. so it is imperisive to explore the mechanism of hepatocarcinogenesis and find a new preventive measure.Peroxisome proliferator activated receptor is a member of the NHR super family,it named after its character of being activiated by proxisome perliferator. To date , PPARs subfamily has defined as PPARα, PPARβand PPARγ. Three PPAR isoforms differ in their struture, function, tissue distribution and ligand specificity. Several lines of evidence indicate that PPARγplays an important role in regulating adipocyte and glucose metablism, adipocyte differentiation, regulating monocyte differentiation and maturity,inducing differentiation and apoptosis of tumor cell, inhabiting carcinoangeinogenesis, involving in anti-inflammatory reactions and anti-arthrosclerosis and etc. Recent data showed that ligands for PPARγoverexpressed in many cancer tissue such as colon cancer, breast cancer, prostate cancer, gastric cancer , liver cancer invovling in cancer cell perliferation, differentiation, apoptosis and carcinogenesis.The synthetic drug thiazolidinediones such as troglitazone, ciglitazone was used widely in patients with insulin resistant diabetes mellitus has been known as a relatively high affinity ligand for PPARγ. Recent data showed that PPARγactivation by its selective ligands inhibits inflammatory responses in macrophages, angiogenesis, atherosclerosis, and cancer-cell growth.Apoptosis is an important part of the cyll cycle, it is important process that regulate cell growth and maintain homeostasis, which is a self-killing phenomenon that is modefied by gene encode and undergone positive biochemical process; that is to say apoptosis is positive programmed cell death which is strictly controlled by multigene. p27kip1, a cyclin-dependent kinase (cdk) inhibitor, regulates progression from G1- to S-phase of the cell cycle by binding an inhibiting cyclin/cdks . p27kip1 is a negative regulator that reduced growth rate and increased apoptosis.S-phase kinase associated protein 2 (Skp2), a member of an F-box family, is the substrate-recognition subunit of the SCFSkp2 ubiquitin ligase complex.Skp2 has been implicated in ubiquitin-mediated degradation of the cyclin-dependent kinase (CDK) inhibitor p27kip1, and positively regulates the G1/S transition. Over-expression of Skp2 has been observed in various types of human tumors. However, little is known about the mechanism of Skp2 over-expression in cancer cells or the nature of its contribution to the malignant phenotype.The aim of this expriment is to explore the effects of peroxisome proliferator-activated receptor-γ(PPAR-γ) on the growth of human hepatocellular carcinoma cell line SMMC-7721 and detect the change of apoptosis related protein p27kip1 and Skp2. It will provide a theoretical foundation in clarifying the mechanism of troglitazone inducing the HCC apoptosis and exploring new therapy to HCC.Methods: Human hepatocellular carcinoma cell line SMMC-7721 was grown on a tissue culture plastic dish in RPMI 1640 containing 10% fetal bovine serum with essential amino acid, 100 units/ml penicillin, and 100μg/ml streptomycin and was maintained in a CO2 incubator at 37°C in a humidified atmosphere containing 5% CO2 and 95% humidity.The expression of PPAR-γligand mRNA is detected by reverse transcription polymerase chain reaction (RT-PCR). For concentration-dependent experiments, media containing various concentrations of troglitazone (0,0.1, 1, 10, and 100μM) were used. For time course experiments, media containing 10μM of troglitazone were used, and the cells were harvested daily for 3 days. Changes of cell cycle and apoptosis rate after treatment with troglitazone were detected by FCM .The expression of p27kip1 and Skp2 were detected by westen-blot.Results: 1 RT-PCR showed that PPARγmRNA is expresse in human hepatocellular carcinoma cell line SMMC-7721.2 FCM showed that the percentage of cells in G0/G1 phase increased ( P<0.05)as the percentage of cells in S phase decreases lightly( P < 0.05) and G2/M phase decreased significantly( P<0.05) in cultures treated with troglitazone. The inhibitory effect on cell cycle progression resulted from an arrest in G0/ G1 of the cell cycle( P<0.05). The rate of apoptosis increased in a dose-dependent and time-dependent manner and it ranges from 5.15% to 17.36%( P<0.05).3 Werten-blot showed that troglitazone can decrease the expression of apoptosis related protein Skp2 and increase the expression of p27kip1 in human hepatoma cell line SMMC-7721 in a dose-dependence and time-dependent manner( P<0.05).Conclutions: 1 Human hepatoma cell line SMMC-7721 express the PPARγligant,which located in 360 bp.2 Troglitazone increase the percentage of cells in G0/G1 phase and decrease the percentage of cells in S phase in human hepatocellular carcinoma cell line SMMC-7721 in a dose-dependence and time-dependent manner, moreover, it increases rate of apoptosis in a dose-dependence and time-dependent manner, which slows down growth .3 Troglitazone can decrease the expression of apoptosis related protein Skp2 and increase the expression of p27kip1 in human hepatocellular carcinoma cell line SMMC-7721 in a dose-dependence and time-dependent manner. Which sugests that Skp2 and p27kip1 may take part in the process of human hepatoma cell line SMMC-7721 cell growth arrest and apoptosis. It is suggest that troglitazone may be a potential molecular therapeutic target for Hepatocellular carcinoma.
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