The Role Of PTEN And P-Akt In Resveratrol Affecting Human Hepatoma Cells On Proliferation And Apoptosis

Primary hepatic carcinoma (PHC) is still one of malignant tumors that threaten people's health and life. Surgical resection can be operated in the early state of liver cancer, however many terminal patients will lose the opportunity of surgical therapy. Up to date, most chemotherapeutics are celltoxical drugs, which have very large toxicity. Therefore, the current study focuses on looking for noncelltoxical and higher therapeutic drugs from natural materials in the world. Domestic and foreign investigators have proven that Resveratrol possess anticancer effect in most tumors including gastric carcinoma, colon carcinoma, prostates carcinoma, hepatic carcinoma, which indicate enormous prospect in therapy carcinoma.Resveratrol (Res) is a polyphenol synthesized by plants and is present in dietary items such as grapes and peanuts. The last research indicated that Resveratrol, as atoxic and natural material, can inhibit growth of tumor cell by stagnanting cell cycle and inducing cell to apoptosis.PTEN gene is the first discovered anti-oncogene which possesses activity of phosphorylase. It play an important role in controlling signal transduction pathway of cell , including controlling normal cell growth and inhibiting tumor cell genesis, growth and metastasis. The mechanisms of PTEN mainly includes as follows: First, it can promote activity of BAD and forkhead and inhibit activity of NF-κB by resist Akt signal transduction pathways; Second , it can activate forkhead to control the cell cycle by resist activity of Akt; Third , it is indicated by lots of researches that PTEN can electively inhibit cell gathering and migrating which are carried out by integral protein; Final , it can inhibit generous form of newborn vessel which are induce by PI3K and Akt.Akt, as a proto-oncogene, play an important role in cell survival. Akt name after v-Akt which is virus-oncogene resulting mouse in leukemia because they have same source. Akt is important molecule of PI3K/Akt signal transduction pathways, who persistently activating tightly relate to tumor generating. PI3K can induce phosphorylation of Akt, and activated Akt can induce phosphorylation of MDM2, P21, cyclinD2, FXHR, TSC2, Foxo and bcl-2 to facilitate tumor cell proliferating, Invading, migrating and vessel forming, inhibit tumor cell apoptosis and chemotheraphy effect on tumor cell.Up to now, many studies have addressed about the effects of resveratrol on proliferation and apoptosis in human hepatoma cells, but it has not been reported that the role of PTEN and p-Akt in resveratrol affecting human hepatoma cells on proliferation and apoptosis. Hence, it is essential to investigate role of PTEN and p-Akt in resveratrol affecting human hepatoma cells on proliferation and apoptosis through experiment in vitro in future.Objective: To investigate role of PTEN and p-Akt in resveratrol affecting human hepatoma cells on proliferation and apoptosis.Methods: SMMC-7721 cells were cultured in vitro. MTT assay and colony-forming test were given to detect resveratrol the effects on apoptosis in human hepatoma cell line SMMC-7721; Flow cytometry (FCM) and TUNEL staining were given to detect the effects of resveratrol on apoptosis in human hepatoma cell line SMMC-7721; Immunohistochemical method was given to detect the expression of PTEN and p-Akt. Reverse transcription polymerase chain reaction were given to detect the mRNA of PTEN of hepatoma cell line SMMC-7721 before and after the resveratrol treatment.Results:1 The effect of resveratrol on the proliferation of hepatoma cell line SMMC-7721 cellsMTT assay: Contrasted to control group, at 12th hour, there was no significantly inhibition of cell growth except 200μmol·l-1 resveratrol group. But from 24th hour to 48th hour, resveratrol inhibited SMMC-7721 cell growth in dose and time-dependent manner.Colony-forming testing: Contrasted to control group, 25μmol·l-1 resveratrol could not inhibit the proliferation of hepatoma cell at 24th hour, but 50μmol·l-1,100μmol·l-1 and 200μmol·l-1 resveratrol could do this; 50μmol·l-1, 100μmol·l-1, 200μmol·l-1 resveratrol can inhibit the proliferation of hepatoma cell in a dose-dependent manner, moreover the variance contrasted to control group is significance.2 The effect of resveratrol on the apoptosis of hepatoma cell line SMMC-7721 cellsFlow cytometry (FCM) and TUNEL staining: Contrasted to control group, 25μmol·l-1 resveratrol could not facilitate the apoptosis of hepatoma cell at 24th hour, but 50μmol·l-1,100μmol·l-1 and 200μmol·l-1 resveratrol could do this; 50μmol·l-1, 100μmol·l-1, 200μmol·l-1 resveratrol can inhibit the proliferation of hepatoma cell in a dose-dependent manner, moreover the variance contrasted to control group is significance.3 The effect of resveratrol on the expressions of PTEN protein in human hepatoma SMMC-7721 cells. The PTEN protein express mainly in cytoplasm. The results of immunohistochemical indicate that after treated by 50μmol·l-1,100μmol·l-1, 200μmol·l-1 resveratrol at 24th hour, the protein expression of p-Akt can be up-regulated compared with control groups (P <0.05).4 The effect of resveratrol on the expressions of p-Akt protein in human hepatoma SMMC-7721 cells. The p-Akt protein express mainly in cytoplasm. The results of immunohistochemical indicate that after treated by 50μmol·l-1,100μmol·l-1, 200μmol·l-1 resveratrol at 24th hour, the protein expression of p-Akt can be down-regulated compared with control groups (P <0.05).5 The effect of resveratrol on the transcription of PTEN in human hepatoma SMMC-7721 cells detected by RT-PCR. PTEN andβ-actin presented special gene strap in 290bp and 500bp position. The level of mRNA transcription of PTEN could be up-regulated significantly in cells treated by 50μmol·l-1, 100μmol·l-1, 200μmol·l-1 resveratrol at 24th hour compared with control groups(P<0.05), in dose-dependent manner.Conclusion: resveratrol can inhibit proliferation and induce apoptosis of human hepatoma SMMC-7721 cells in vitro, which relate to up-regulating transcription of PTEN, up-regulating expression of PTEN and inhibiting phosphorylation of Akt.