Alternation Of Akt/mTOR Singaling Pathway In Prostate Cancer Cell Lines And Tissues

Objective:(1) To investigate the effect of mTOR inhibitor Rapamycin on androgen independent prostate cancer cell line PC-3 and DU145.(2) To evaluate the alternation of Akt/mTOR singaling pathway in prostate cancer tissues and to explore its prognostic value.Methods:(1) CCK-8 assay was used to identify the effect of rapamycin on proliferation inhibition in PC-3 and DU145 cells.The cell cycle and apoptosis was detected by flow cytometry.The invasion assay was performed using 24-well Transwell paltes to analyse the invasion ability of PC-3 and DU145 cells after the treatment of rapamycin.Western blot assay was used to detect the expression of protein p-Akt,p-mTOR,4E-BP1,p-4E-BP1,p70S6K and p-p70S6K in PC3 and DU145 cell before and after rapamycin treatment.(2) This study included prostate cancer tissue samples from 182 Chinese prostate cancer patients including 104 patients who had distant metastasis at diagnosis.Archival formalin-fixed paraffin-embedded specimens from prostatectomies and prostate biopsies were obtained.For patients who underwent radical prostatectomy,the prostate biopsy specimens were not included in this study.Therefore,51 prostatectomy and 131 biopsy specimens were selected.The activation of several important molecule markers in the upstream and downstream of mTOR pathway including PTEN,Akt, mTOR,p70S6 kinase and 4E-BP1 in our Chinese prostate cancer patients were evaluated using Immunohistochemical technique.Because Akt and mTOR are kinases, we examined the phosphorylation status of them and their downstream effectors using phosphorylation-specific antibodies.Results:(1) Rapamycin 1nM-1000nM can inhibit the proliferation of PC3 cell and DU145 cell and present an obvious time/concentration dependent.Rapamycin has a better inhibitory effect on PC3 cell compared with DU145 cell.Rapamycin significantly increase the percentage of G0/G1 phase in PC3 cell and DU145 cell when employed for 24 hours at concentrations of 10nM-100nM.Transwell study found that Rapamycin can inhibit the invasion ability of PC-3 cell and DU145 cell in vitro significantly.Western blot assay showed that rapamycin didn't affect the expression of p-Akt,p-mTOR,4E-BP1,p70S6K protein in PC3 and DU145 cell.The expression level of p-4E-BP1 and p-p70S6K protein can be downregulated by rapamycin.(2) The expression level of p-Akt,p-mTOR,p-4E-BP1 and p-p70S6K all increased significantly as disease progressed from BPH to HGPIN to prostate cancer and the expression level of PTEN decreased significantly as disease progressed from BPH to HGPIN to prostate cancer.PETN expression level was significantly inversely correlated with p-Akt expression level but not with p-mTOR,p-4E-BP1 and p-p70S6K expression level in 182 prostate cancer patients,p-Akt expression level was significantly positively correlated with p-mTOR,p-p70S6K and p-4E-BP1 expression level.Prostate cancer patients with distant metastasis tended to have more positive p-Akt,p-mTOR,p-4E-BP1 and p-p70S6K expression in their tumors.However,there was no significant association between patient distant metastatic status and PTEN expression.46.2%(48/104) patients with TxNxM1 stage disease at diagnosis died of prostate cancer at the end of this study.Substage of metastatic prostate cancer and expression level of p-Akt,p-mTOR p-4E-BP1 and p-p70S6K were significant univariate predictors of death from prostate cancer,multivariate analysis showed that p-Akt expression level and substage of metastatic prostate cancer were independent predictors of cancer related death.Conclusions:(1) Rapamycin can inhibit the proliferation of PC3 and DU145 cell and present an obvious time/concentration dependent.Rapayncyn significantly increase the percentage of G0/G1 phase and induce apoptosis in PC3 and DU145 cell. Rapamycin inhibit cell proliferation and induce apoptosis by block Akt/mTOR signaling pathway.(2) Akt/mTOR signaling pathway plays an important role in the initiataion and development of prostate cancer.Akt/mTOR signaling pathway was more active in distant metastatic prostate cancer patients.The protein markers in Akt/mTOR pathway affect the cancer specific survival rate in distant metastatic prostate cancer patients.