Cell Technology Topics: PI3K/Akt/mTOR Signaling Pathway

OBJECTIVE: To investigate the molecular mechanisms of PI3K/Akt/mTOR signaling pathway activity and biological behavior in HepG2 and Hep3B cell lines by specifically blocking PI3K and mTOR.

METHODS: PI3K (p110α subunit), PTEN, pAkt (S473, T308) were detected by immunoblotting (Western blot) on cultured HepG2, Hep3B human hepatoma cell line and human normal liver cell line QSG-7701. And p-mTOR (S2448) expression; HepG2 and Hep3B cells were incubated with PI3K inhibitor LY294002 (50 μmol/ml) and mTOR inhibitor Rapamycin (RAPA, 50 nmol/ml), respectively, and cells were detected by MTT colorimetry. The cell proliferation and apoptosis were detected by flow cytometry. The expression changes of pAkt (S473, T308) and p-mTOR (S2448) were detected by Western blot.

Results: PTEN was not expressed in HepG2 and Hep3B cells, and was highly expressed in QSG-7701 cells. The expression of pAkt and p-mTOR in HepG2 and Hep3B cells was significantly higher than that in QSG-7701 cells. Both LY294002 and RAPA were present. Dose-time dependent inhibition of HepG2 and Hep3B cell growth. After 24 hours of saturation effect of LY294002 and RAPA, HepG2 and Hep3B cells showed significant G0/G1 phase arrest, and the proportion of cells in S phase was significantly lower than that of the control group (P<0.01). HepG2 in the two administration groups The apoptosis rate of cells and Hep3B cells was significantly increased compared with the control group (P<0.01). The apoptosis rate of HepG2 cells in the two administration groups was significantly higher than that of Hep3B cells (P<0.01 or P<0.05), and the HepG2 cells were The apoptotic rate was significantly higher in the RAPA-administered group than in the LY294002-administered group (P<0.01), but the apoptosis rate of Hep3B cells was not significantly different between the two groups. After 48 hours of saturation effect concentration of LY294002, the expression levels of pAkt (T308, S473) and p-mTOR (S2448) in HepG2 and Hep3B cells were significantly lower than those in the control group (P<0.01), and the saturation effect concentration of RAPA was 48. After an hour, the expression levels of P-mTOR (S2448) in HepG2 and Hep3B cells were significantly lower than those in the control group (P<0.01), while the expression levels of pAkt (T308, S473) were significantly higher than those in the control group (P< 0.01).

Conclusion: (1) HepG2 and Hep3B cells have constitutive activation of PI3K/Akt/mTOR signaling pathway; (2) LY294002 and RAPA can effectively block PI3K/Akt/mTOR signaling pathway in HepG2 and Hep3B cells, inhibiting the growth and proliferation of HCC cells Induces cell cycle arrest and promotes apoptosis, and the blocking effect is related to the expression of P53 in HCC cells; (3) HepG2 cells are more sensitive to PI3K/Akt/mTOR signaling pathway blockers within a certain concentration range. Hep3B cells, RAPA partial blocking effect on PI3K/Akt /mTOR signaling pathway is better than LY294002.

OBJECTIVE: To observe the effects of doxorubicin (DOX) alone or in combination with RAPA on the biological behavior of HepG2 and Hep3B cells and the activity of PI3K/Akt/mTOR signaling pathway, and to explore the role of mTOR inhibitors in enhancing the efficacy of HCC chemotherapy drugs. mechanism.

METHODS: HepG2 and Hep3B cells in logarithmic growth phase were cultured at different concentrations of DOX alone or in combination with RAPA (20 nmol/ml) for 48 h or 24 h. MTT assay was used to determine the proliferation inhibition of HepG2 and Hep3B cells. The apoptosis was detected by flow cytometry. The expression changes of P-p70S6K (T389), P-4E-BP1 (S65) and pS6 (S235/236) were detected by Western blot.

RESULTS: DOX inhibited the proliferation of HepG2 and Hep3B cells in a concentration-dependent manner in a concentration range of 0.156-2.5 mg/L. The relative survival rate of Hep3B cells was significantly greater than that of HepG2 cells in the concentration range of 0.625-10 mg/L. P<0.01); Compared with DOX alone, DOX (0.156-2.5 mg/L) combined with RAPA significantly reduced the survival rate of HepG2 and Hep3B cells (P<0.01 or P<0.05), DOX (0.156-10 mg/ L) In combination with RAPA, the survival rate of Hep3B cells was significantly higher than that of HepG2 cells (P<0.01). DOX (0.156～10mg/L) could induce apoptosis of HepG2 and Hep3B cells alone or in combination with RAPA for 24h, and the apoptosis was increased with the increase of DOX concentration. Compared with DOX alone, DOX (1.25～10mg) /L) Combined with RAPA, the apoptosis rate of HepG2 cells was significantly increased (P<0.01 or P<0.05), DOX (2.5-10 mg/L) was combined with RAPA, and the apoptosis rate of Hep3B cells was significantly increased ( P<0.05); DOX (5.0～10mg/L) alone, the apoptosis rate of HepG2 cells was significantly higher than that of Hep3B cells (P<0.05), DOX (2.5～10 mg/L) combined with RAPA, HepG2 cells withered The mortality rate was significantly greater than that of Hep3B cells (P<0.01, respectively). RAPA (20nmol/L), DOX (2.5mg /L) and their combination can reduce the expression of P-p70S6K (T389), P-4E-BP1 (S65) and pS6 (S235/236) in HepG2 or Hep3B cells. And the effect of DOX combined with RAPA is most obvious.

Conclusion: (1) DOX can inhibit the growth and proliferation of HepG2 and Hep3B cells, promote apoptosis and down-regulate the activation of PI3K/Akt/mTOR signaling pathway; (2) DOX combined with RAPA can significantly inhibit the proliferation of HepG2 and Hep3B cells. Promotes apoptosis, down-regulates the activation of PI3K/Akt/mTOR signaling pathway, and exhibits synergistic effects within a certain concentration range; (3) Sensitivity of HepG2 cells to DOX alone or in combination with RAPA within a certain concentration range Significantly higher than Hep3B cells, may be related to the difference in expression of p53 in HCC cells.

OBJECTIVE: To analyze the relationship between the activation level of PI3K/Akt/mTOR signaling pathway and the expression of p53 and clinicopathological parameters of HCC in human HCC tissues, and to evaluate the role of PI3K/Akt/mTOR signaling pathway in HCC diagnosis and prognosis evaluation.

Methods: 76 cases of HCC tissue samples were classified by clinical pathological staging. The expressions of p53, pAkt, PTEN, p-mTOR and pS6 in HCC tissues and paracancerous tissues were detected by immunohistochemistry. The pathological characteristics of these indicators in different HCC were analyzed. Positive expression rate under conditions and compared with normal liver tissue.

Results: p53, pAkt, PTEN, p-mTOR and pS6 were expressed in different degrees in human HCC tissues, HCC tissues p53 (60.5%, 46/76), pAkt (92.1%, 70/76), p-mTOR (84.2 The positive expression ratios of %, 64/76) and pS6 (88.2%, 67/76) were higher than those of adjacent liver tissues (10.5%, 8/76, 63.2%, 48/76, 46.1%, 35/76, 52.6, respectively). %, 40/76) and normal liver tissue (0%, 55.0%, 11/20, 50.0%, 10/20, 45.0%, 9/20, respectively) were significantly elevated (P<0.01), while PTEN (65.8) The positive expression rate of %, 50/76) was significantly lower than that of adjacent liver tissues (89.5%, 68/76) and normal liver tissues (85.0%, 17/20) (P<0.05); p53 positively expressed HCC tissue pAkt, The positive expression rate of p-mTOR and pS6 was significantly higher than that of p53 negatively expressed tissues (P<0.01), while the positive expression rate of PTEN was significantly lower than that of p53 negatively expressed tissues (P<0.01). The positive expression rate of p53, pAkt, p-mTOR and pS6 in HCC tissues with poor differentiation, vascular invasion and high TNM stage was significantly higher than that of corresponding HCC tissues with better differentiation, no vascular invasion and low TNM stage (P <0.01 or P<0.05); and the positive expression rate of PTEN was significantly decreased (P<0.01 or P<0.05, respectively).

Conclusions: (1) Activation of PI3K/Akt/mTOR signaling pathway in human HCC tissues; (2) Activation of PI3K/Akt/mTOR signaling pathway in human HCC tissues may be correlated with p53 expression; (3) Human HCC tissues The degree of activation of PI3K/Akt/mTOR signaling pathway is related to the clinicopathological features of HCC. The higher the degree of activation, the lower the degree of differentiation, the more vascular invasion and the higher TNM stage.