The Role And Mechanism Of PI3K/AKT/mTOR Signaling Pathway In Cisplatin-based Chemotherapy In Epithelial Ovarian Cancer

Epithelial ovarian cancer is the leading killer among gynecological malignancies. About 67% of patients in epithelial ovarian cancer initially present with advanced stage, missing good chance for operation. Then chemotherapy becomes one of the important treatments. Unfortunately, parallel to high efficacy in the treatment, the development of resistance is a major obstacle. Previous studies indicated that the PI3K/AKT pathway played a pivotal role in many human malignances, including ovarian cancer. Increased activation of AKT was noted in ovarian cancer, which exerted anti-apoptotic effect, antagonized cell cycle arrest, modulated angiogenesis, and mediated mRNA translation through mTOR signaling. However, a limited amount of information was available regarding the function of the PI3K/AKT pathway in the context of cisplatin-induced apoptosis in ovarian cancer.This study was scheduled to analyze the expression of PI3K/AKT in epithelial ovarian cancer and examine the involvement of the PI3K/AKT pathway in human ovarian cancer cell line SKOV3 in the forms of monolayer and three-dimensional cell cultures, as models of tumor cells in peritoneal dissemination of ovarian cancer. Furthermore, comparison between parental and drug-resistant cells would be taken to explore important clues for successful cisplatin therapy. The identification of the PI3K/AKT signaling responsible for cisplatin-based chemotherapy may provide more targets for combination therapy aimed to circumvent drug-resistance.Part I Expression and clinicopathologic meaning of PI3K-p85α,AKT in serous epithelial ovarian carcinomaObjective To explore the expression and significance of phosphatidylinositol-3 kinase-p85αand AKT at protein and mRNA levels in serous epithelial ovarian carcinoma.Methods The expression of PI3K-p85αand AKT at protein and mRNA levels were evaluated by western blot and RT-PCR in normal ovarian epithelium(group N, n=20), benign serous ovarian tumors(group Bl , n=15), borderline serous ovarian tumors(group B2, n=15) and serous epithelial ovarian carcinoma(group E, n=24).The relevant clinical pathological parameters were analyzed. Results The tendency of two methods was coincidence on the whole. There was no positive expression of PI3K p85αprotein in group N, but 13% in group B2, 83% in group E. PI3K-p85αmRNA was 0.167±0.092 in group N, 0.389±0.095 in group B1, 0.576±0.037 in group B2 and 1.008±0.223 in group E. There was significant difference between group N, group B1, group B2, and group E(P<0.05). There was significant difference between p85αexpression and tumor differentiation degree and clinicalopathological staging (P <0.05, P <0.05). Overactivation of AKT was detected in group E, compared with group N, group B1, and group B2(P<0.05). Three isoforms of AKT mRNA were detected in each group. However, AKT1, AKT2, and AKT3 mRNA were overexpressed in group E compared with group N, group B1, and group B2(P<0.05). Significant differences were noted between AKT activation and tumor differentiation degree and clinicopathological staging (P <0.05, P <0.05).Conclusions Overactivation of PI3K/AKT signaling existed in serous epithelial ovarian carcinoma. The alteration of PI3K-p85αand AKT were correlated with clinicopathological staging and tumor differentiation degree.Part II Effects of AKT activation on cell proliferation, apoptosis, migration, and invasion in epithelial ovarian cancer cellsObjective To explore the effects of activation of AKT on cell growth, cell cycle, cell invasive, cell migration, cell clone formation and cell apoptosis, and to explore related mechanism.Methods Western blot was used to detecte the activation of AKT by IGF-1 stimulation. Recombinant plasmids carrying the full-length AKT1 cDNA or AKT1 siRNA were constructed. Plasmids were transfected into SKOV3 cells. Western blot and RT-PCR were used to detect the transfection efficiency. Flow cyctometry was used to detect cell early apoptosis rate and cell cycle progression. CCK-8 and clone formation assay were used to examine cell proliferation. Transwell-Matrigel assay was used to analyse cell invasion. Wound healing assay was used to analyse cell migration. RT-PCR was used to explore the expression of several cell migration related moleculars at mRNA level.Results Western blot detected the intact activation of AKT in SKOV3 cells by IGF-1 stimulation on time dependent manner. A plasmid (pEF-1α-AKT1) containing full length of AKT1 cDNA and specific AKT1-targeted shRNA expression plasmids were constructed successfully, which could effectively upregulate or downregulate the activation of AKT after transfected into SKOV3 cells. Compared with untranfected cells or non-target shRNA transfected cells, down-regulation AKT1 inhibited cell growth and clone formation, decreased cell migration and invasion, and reduced cell population in the S phase(P <0.05), but induced cell apoptosis. Additionally, down-regulation AKT1 decreased the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level(P<0.05). On the other hand, up-regulation AKT1 increased cell migration, proliferation, and invasion(P<0.05). Up-regulation AKT1 increased the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level(P <0.05).Conclusions AKT played an important role in cell growth and suvivial in epithelial ovarian cancer, and might impact cell migration and invasion by regulating the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level.Part III Role and mechanism of AKT/mTOR signaling in cisplatin-based chemotherapy in epithelial ovarian cancer cellsObjective To investigate the role and mechanism of AKT/mTOR signaling in cisplatin-based chemotherapy in epithelial ovarian cancer cells.Methods Cisplatin was administrated at different concerntrations and different times. Western blot was used to examine the activation of AKT and mTOR in SKOV3 and ES-2 cells. SKOV3/DDP monolayer cells and SKOV3/MCA (multicellular aggregates) were constructed as chemoresistant models. Western blot was used to detect the expression of PI3K/AKT signaling in both models comparing with SKOV3 cells. The role and mechanism of AKT siRNA in different models before and after cisplatin treatment were detected by MTT assay. Western blot was used to check the expression of survivin and multidrug resistant protein before or after AKT siRNA transfection.Results Cisplatin increased the activation of AKT and mTOR dramatically on dose-dependent and time-dependent manners in SKOV3 and ES-2 cells. SKOV3/DDP monolayer cells and SKOV3/MCA were constructed and proved as cisplatin-resistant models. Elevated activation of AKT/mTOR signaling was observed in SKOV3/DDP cells and SKOV3/MCA(P<0.05). AKT siRNA could attenuate cisplatin resistance dominantly(P<0.05) and decreased the protein expression of survivin and P-gp.Conclusions Cisplatin could play an anti-apoptotic role by inducing the activation of AKT and mTOR in epithelial ovarian cancer cells. The AKT/mTOR/survivin signaling was involved in cisplatin-based chemoresistance in epithelial ovarian cancer. AKT siRNA could enhaced the effects of cisplatin-based chemotherapy by decreasing the expression of survivin and P-gp.Part IV Enhanced effects of Triciribine or Rapamycin on cisplatin-based chemotherapy in epithelial ovarian cancer cellsObjective To explore the effects of Triciribine or Rapamycin singly or combined with cisplatin on cell proliferation and apoptosis in SKOV3 and ES-2 cells.Methods Western blot detected the optical concerntration of Ticiribine and Rapamycin in cells. Methyl thiazolyl tetrazolium and flow cytometry were used to detect the growth rate and apoptosis rate after cisplatin-based chemotherapy, Triciribine or Rapamycin treatment or combination treatment, respectively.Results (1) The activation of AKT was dramatically inhibited after exposured to 10μmol/L Triciribine under normal culture condition(P <0.05). Triciribine enhanced the sensitivity of cisplatin-based chemotherapy in SKOV3 and ES-2 cells. (2) The activation of mTOR was dramatically inhibited after exposured to 100nmol/L Rapamycin under normal culture condition(P <0.05). Rapamycin enhanced the sensitivity of cisplatin-based chemotherapy in SKOV3 and ES-2 cells.Conclusions Both Triciribine and Rapamycin could enhanced cisplatin-based chemotherapy in epithelial ovarian cancer cells by inhibiting corresponding taget.