Study Of The Pathogenic Role And Targeted Therapy Of PI3K/Akt/mTOR Signaling Pathway In Hepatocellular Carcinoma

Objective:The purpose of this study is to investigate the molecular mechanisms associated with PI3K/Akt/mTOR pathway in hepatocellular carcinoma (HCC).Method:We firstly observed the expression of PI3K (subunit of p110α), PTEN, pAkt (S473, T308) and p-mTOR (S2448) in HepG2, Hep3B and QSG-7701 by Western Blot analysis; And then, the cell proliferation, cell cycle, apoptosis and expression of pAkt (S473, T308) and p-mTOR (S2448) were determined in HepG2 and Hep3B after incubation with rapamycin (50nmol/ml), the mTOR inhibitor and LY294002 (50 umol/ml), the PI3K inhibitor.Result:There was absence or low of PTEN expression in HepG2 and Hep3B, but it is highly expressed in QSG-7701; The pAkt and p-mTOR levels were significantly higher in HepG2 and Hep3B than in QSG-7701; LY294002 and RAPA inhibit the proliferation of HepG2 and Hep3B in both dose-and time-dependent manner; HepG2 and Hep3B in G0/G1 phase were both inhibited remarkably after treated by saturation concentration of LY294002 and Rapamycin for 24 hours, while the proportion of cells in S phase is largely less than the control group (P<0.01), and the apoptotic rates of HepG2 and Hep3B in both group are significantly higher than that of the control group (P<0.01), the apoptotic rate of HepG2 is obvious higher than that of Hep3B in the both group(P<0.01 or 0.05), and the apoptotic rate of HepG2 treated by saturation concentration of RAPA is obvious higher than that of the HepG2 treated by saturation concentration of LY294002 (P<0.01),but the apoptotic rates of Hep3B between the both group have no significant difference. The expressions of pAkt (T308, S473) and p-mTOR (S2448) in HepG2 and Hep3B both down-regulated significantly compared with the control group (P<0.01) after saturation concentration of LY294002 treatment for 48 hours, while the results of p-mTOR (S2448) expression in both kinds of cells which treated with RAPA for 48 hours were almost the same, but the expression of pAkt (T308, S473) was up-regulated (P<0.01).Conclusion:(1) there is a constitutive activation of PI3K/Akt/mTOR pathway in HCC cells of HepG2 and Hep3B; (2) LY294002 and RAPA could effectively block PI3K/Akt/mTOR pathway in HepG2 and Hep3B, inhibit the growth of HCC cells, induce cell cycle arrest and promote apoptosis, while the effect of blocking is probably related to the expression of P53 in HCC cells; (3) Within certain concentration, HepG2 is more sensitive to inhibitors of PI3K/Akt/mTOR pathway than Hep3B, while RAPA has a more effective impact in partly blocking PI3K/Akt/mTOR pathway than LY294002.Objective:The purpose of this study is to observe the influences of Doxorubicin (DOX) alone or DOX combined with RAPA on the biological actions and the functions of PI3K/Akt/mTOR pathway in HepG2 and Hep3B, and explore the mechanism of mTOR inhibitor which enhancing the effect of chemotherapy on HCC.Method:HepG2 and Hep3B in logarithmic phase were treated with DOX in different concentration alone or with different concentration of DOX combined with RAPA (20 nmol/ml) for 24 or 48 hours respectively. The growth inhibition of HepG2 and Hep3B were assessed by MTT; the apoptosis of both cells were analysed by flow cytometry; and expression changes of P-p20S6K (T389), P-4E-BP1 (S65) and pS6 (S235/236) were assayed by Western blot.Result:Within the concentration of 0.156 to 2.5 mg/L, the proliferation of HepG2 and Hep3B were inhibited by DOX in a dose-dependent manner; within the concentration of 0.625 to 10 mg/L the relative survival rate of Hep3B were higher than HepG2 (P<0.01), the survival rate was decreased significantly when HepG2 was treated with DOX (0.312-5mg/L) plus RAPA compared with DOX alone (P＜0.01 or P<0.05), the survival rate of Hep3B was also decreased after exposure to DOX (2.5-10mg/L) plus RAPA (P＜0.01 or P＜0.05), and the survival rate of Hep3B was largely higher than HepG2 after the treatment with DOX (0.312-5mg/L) plus RAPA (P＜0.01 or P＜0.05). Treat cell HepG2 and Hep3B with DOX(0.156-10mg/L) alone or DOX combined with RAPA for 24 hours could both induce apoptosis in a dose-dependent manner, DOX (1.25-10mg/L) combined with RAPA induces an obvious increase in apoptotic HepG2 cell number compared with DOX alone (P＜0.01 or P＜0.05), DOX (2.5-10mg/L) combined with RAPA induces a significant increase in apoptotic Hep3B cell number compared with DOX alone (P＜0.05), DOX alone causes more apoptotic cell number in cell HepG2 than cell Hep3B(P＜0.05), while DOX (2.5-10mg/L) combined with RAPA induces higher apoptosis rate in cell HepG2 than cell Hep3B (P＜0.01). RAPA (20nmol/ml) alone, DOX (2.5mg/L) alone and both of them could all induce the decreased expression of P-p20S6K (T389), P-4E-BP1(S65) and pS6 (S235/236) in HepG2 and Hep3B, and this effect was most obvious when DOX and RAPA combined.Conclusion:(1) DOX could inhibit cell proliferation, promote apoptosis, and reduce the activation of PI3K/Akt/MTOR pathway in HepG2 and Hep3B; (2) RAPA could enhance the sensitivity of HepG2 and Hep3B to DOX, which evidenced by higher inhibition of cell proliferation, apoptosis rate, and the activation of PI3K/Akt/MTOR pathway; (3) HepG2 appears more sensitive to DOX alone or DOX plus RAPA than Hep3B within certain concentration, which might relate to the differential expression of p53 in HCC cells.Objective:The purpose of this study is to analysis the activation level of PI3K/Akt/MTOR pathway and the expression of p53 in human HCC tissue, and also analysis the connection between them and clinical pathological parameters, and then assesses the value of PI3K/Akt/MTOR pathway in HCC diagnose and prognosis.Method:After classing the pathological stage and grade of HCC tissues of 76 cases, detecting the expression of p53, pAkt, PTEN, p-mTOR and pS6 in HCC tissue and adjacent tissue by immunohistochemistry, and analysis the positive expression frequency of all above indicators in different pathological condition of HCC, while also compare them with the normal hepatic tissue.Result:There were different levels of expression of p53, pAkt, PTEN, p-mTOR and pS6 in human HCC tissue, and the positive expression rates of p53 (60.5%,46/76), pAkt (92.1%,70/76), p-mTOR (84.2%,64/76) and pS6 (88.2%,67/76) in HCC tissue were significant higher than the expression rate in adjacent tissue (10.5%,8/76, 63.2%,48/76,46.1%,35/76,52.6%,40/76) and the rate in normal hepatic tissue (0%,55.0%,11/20,50.0%,10/20,45.0%,9/20) (P<0.01), while the positive expression rate of PTEN (65.8%,50/76) was lower than adjacent tissue (89.%,68/76) and normal hepatic tissue (85.0%,17/20) (P<0.05). The positive expression frequency of pAkt, p-mTOR and pS6 is higher in p53 positive HCC tissue than negative one, but the positive expression frequency of PTEN is greatly lower than the frequency of p53 negative tissue(P<0.01). HCC tissue with lower differentiation, vascular invasion and high stage of TNM demonstrated higher positive expression frequency of p53, pAkt, p-mTOR and pS6 compared with HCC tissue well differentiated, without vascular invasion and low stage of TNM (P＜0.01 or P<0.05).Conclusion:(1) It does exist the activation of PI3K/Akt/MTOR pathway in human HCC; (2) The level of activation of PI3K/Akt/MTOR pathway in human HCC may be related to the expression of p53; (3) The level of activation of PI3K/Akt/mTOR pathway is related to the clinical pathological stage of HCC, the higher activation of HCC cells, appeared to be the poorer differentiation, more vascular invasion and higher stage of TNM.