| Primary carcinoma of the liver is a common malignant tumor in China. Its incidence gradually increases with high malignancy, rapid progression, poor curative effect, and its mortality ranking the third among patients with carcinoma in the world. It is often accompanied with hepatic cirrhosis and propagates within liver at the early stage. Because of the lack of specific symptoms and signs, it is difficult to be detected early. Surgical resection can be operated in the early state of liver cancer, however many terminal patients will lose the opportunity of surgical therapy. As to the late state of liver cancer, there is not a therapeutic breakthrough. The average survival of patients with HCC is only 3 to 6 months and 3-year survival is less than 13% at present. In recent years, there is a continuing increase in the incidence of primary hepatic carcinoma. Therefore, it has become a hot point to search for effective but low toxic drugs which can suppress human hepatoma and improve the life quality of patients from natural plants. It is very important to investigate on the effective drugs and available therapy of HCC further.The disorder of signal transduction pathways is closely related to the occurrence, development of cancers. Phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway is activated in many malignancies such as breast, colorectal, and ovarian cancer. The activated pathway can facilitate tumor cell proliferating, inhibit tumor cell apoptosis.As an intracellular second messenger, PI3K is related to cell signaling. This enzyme is composed of two molecules: an 85-kilodalton regulatory protein and a 110-kilodalton catalytic subunit. The p85 subunit is equipped with two SH2 domains and one SH3 domain. Activation of the growth factor receptors causes translocation to the membrane and activation of PI3K, bringing the enzyme to its substrates.Akt, also known as protein kinase B (PKB), is a serine/threonine kinase downstream target of PI3K. Akt is an important molecule of PI3K/Akt signal transduction pathways, mainly activated by PI3K. Activation of Akt is in that phosphorylation of two sites: Thr-308 and Ser-473, one in the activation domain and the other in the COOH-terminal hydrophobic motif, and both of them are necessary for full activity. It has been established that activation of Akt plays an important role in celluar survival and anti-apoptotic by phosphorylating its substrates at serine or threonine residues.Several studies showed that abnormal expression and activity of PI3K, Akt in hepatoma cells can promote their proliferation and inhibit their apoptosis. Therefore, blocking or inhibiting PI3K/Akt signal transduction pathway can lead to inhibition of proliferation and induction of apoptosis in hepatoma cells.Genistein, is the primary reconstituent of the isoflavones rich in soybeans, which maybe the main active component which has the anticancer effect. Genistein has been found to inhibit the cell proliferations of many tumor cells. The mechanism may be related to cell cycle arrest, cell apoptosis induction and the accommodation of signal transduction pathways.It has been shown that genistein can affect the ERK5 MAPK and ERK1/2MAPK signaling transduction pathways and inhibit the proliferation of breast cancer cells MDA-MB-231 cells. Up to now few studies have addressed the effects of genistein on PI3K/Akt signal transduction pathways on hepatoma cells. Hence, the human hepatoma cells SMMC-7721 were used as the vitro model to investigate the effects of genistein on PI3K/Akt signal transduction pathways and the role of PI3K/Akt signal pathways in apoptosis induced by genistein so as to provid a new clue for the study of molecular mechanism of genistein on tumor cells.Objective: To investigate the effects of genistein on PI3K/Akt signaling transduction pathway of human hepatoma cells SMMC-7721 and the role of PI3K/Akt signal pathways in apoptosis induced by genistein.Methods: SMMC-7721 cells were cultured in vitro. Western blotting was performed to detect the expressions of Akt, p-Akt and PI3K afer the genistein treatment. The SMMC-7721 cell apoptosis was detected by flow cytometry.Results:①The effect of genistein on the expression of Akt protein in human hepatoma SMMC-7721 cells. Western blot assay: After SMMC-7721 cell exposured to 50μmol·L-1 genistein for 24 h, 48 h and 72h, the levels of Akt protein expression respectively were 0.99±0.01, 0.86±0.08 and 0.81±0.12 , compared with that of 0h group (P>0.05). No difference was found after SMMC-7721 cell exposured to 5, 25, 50μmol·L-1 genistein for 48h compared with that of control group(0.99±0.13), (P>0.05) and the levels were 0.68±0.04, 0.67±0.00 and 0.67±0.01, respectively. Western blot analysis results show that the Akt expression of was not affected by genistein.②The inhibition role of genistein on the phosphorylation of Akt in SMMC-7721 cell. The level of p-Akt protein expression by Western blot in 25,50μmol·L-1 genistein group was significantly lower than that of control (1.07±0.06), (P<0.01), and the levels were 0.72±0.10 and 0.55±0.09, respectively. There was significantly difference between the protein expression of 25μmol·L-1 genistein group and 50μmol·L-1 genistein group (P<0.05). But there was no difference between that of 5μmol·L-1 genistein group (0.94±0.01) and control group (P>0.05). After SMMC-7721 cell exposured to genistein, the levels of p-Akt protein expression respectively were 0.71±0.02, 0.55±0.03, and 0.47±0.02 for 24 h group, 48 h group and 72 h group compared with that of 0 h group (0.99±0.04), P<0.01.③ The effect of genistein on the expression of PI3K-p85 protein in human hepatoma SMMC-7721 cells. Western blotting assay: no difference was found after SMMC-7721 cell exposured to 5, 25, 50μmol·L-1 genistein for 48h compared with that of control group (0.99±0.13), (P>0.05) and the levels were 0.97±0.04, 1.07±0.07 and 0.93±0.05, respectively. After SMMC-7721 cell exposured to 50μmol·L-1 genistein, the levels of PI3K-p85 protein expression respectively were 0.91±0.02, 0.90±0.05 and 0.87±0.04 for 24 h, 48 h and 72h group compared with that of 0 h group(P>0.05). Western blot analysis results show that the PI3K-p85 expression of was not affected by genistein.④The effect of genistein on the apoptosis of SMMC-7721 cells: as flow cytometry indicated that the ratios of cell apoptosis increased treated with 5-50μmol·L-1 genistein at 48 hours. Compared with control group, the apoptosis ratios had significant difference after SMMC-7721 cells exposured to 25,50μmol·L-1 genistein for 48h, and the apoptosis ratios were 5.52%±0.53% vs 2.48%±0.35%, 9.44%±0.51% vs 2.48%±0.35% respectively, (P<0.01). There was statistic difference between the cells of 25μmol·L-1 genistein group and 50μmol·L-1 genistein group, (P<0.01). But there was no difference between 5μmol·L-1 genistein group and control group,(P>0.05).⑤Correlation analysis showed that there was a negative correlation between Akt phosphorylation and apoptosis in SMMC-7721 cells(r=-0.919, P<0.01).Conclusion: genistein can inhibit the phosphorylation of Akt in SMMC-7721 cells in vitro, and genistein-induced SMMC-7721 cells apoptosis is mediated through inhihition of P13K/AKt signaling pathway.
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