Mechanism Of Silencing Of Notch-1Enhances The Chemosensitivity Of Docetaxel To Prostate Cancer PC-3Cells

Prostate cancer is a common malignant tumor and prostate cancer has the highest incidence rate among males in the Europe and America. In our country the incidence rate of prostate cancer is also increasing year by year. Endocrine therapy is the main treatment for prostate cancer, but most progressive prostate cancer patients who receive hormone treatment exhibit tumor progression and require chemotherapy within18-24months, at which point their condition is termed castration-resistant prostate cancer (CRPC), also known as androgen-independent prostate cancer. CRPC treatment is docetaxel-based chemotherapy. Docetaxel is a semi-synthetic paclitaxel derivatives and it can form stable non-functional microtubules by strengthening of tubulin polymerization and inhibiting microtubule depolymerization, destroy the mitosis of tumor cells, and arrest the cells at the G2/M phase, eventually leading to tumor cell apoptosis. Docetaxel is the first drug approved by Food and Drug Administration (FDA) for the treatment of CRPC and docetaxel-based combination therapy is the standard treatment for CRPC.Despite the efficacy of docetaxel-based chemotherapy, tumor progression occurs following a median of6.3months, resulting in treatment failure. At present, studies on resistance to chemotherapy are mainly concentrated in paclitaxel resistance and many scholars have conducted in-depth analysis mechanisms of drug resistance in lung cancer and ovarian cancer. These studies found that over-expression of α-and β-tubulin, mutations of tubulin and post-translational modification of microtubule protein could lead to drug resistance by antagonizing paclitaxel microtubule-stabilizing effect, reducing the sensitivity of tumor cells to paclitaxel, and reducing microtubule polymerization rate. Meanwhile, TRAG-3and P-glycoprotein expressions are elevated may also result in paclitaxel resistance.Notch is an transmembrane receptor protein family. The precise regulation of the Notch signaling pathway is essential for the normal development of most organizations. Aberrant regulation of Notch signaling leads to abnormal tissue development and the occurrence of tumors and other diseases. There are a number of studies have shown that inhibition of Notch-1expression in tumor cells increased apoptosis, inhibited proliferation and invasion. The anti-tumor effect of docetaxel is reflected in the regulation of proliferation and apoptosis of tumor cells through multiple signaling pathways. Whether docetaxel has cross-talk with the Notch signaling pathway has not been reported. If inhibition of Notch pathway can increase anti-tumor effect of docetaxel, it may become an effective therapeutic target for prostate cancer.Chapter Ⅰ:Screening of Notch-1siRNA and the biological behavior of PC-3cells caused by the silencing of Notch-1Objective:To synthesis and screen effective Notch-1siRNA sequence and to research the alteration of biological behavior of PC-3cell caused by the silencing of Notch-1.Methods:Three pairs of sequences siRNA against Notch-1were transfected into PC-3cells and real time RT-PCR and Western blot were used to detect Notch-1mRNA and protein expression. The proliferation of siRNA-transfected PC-3cells were examined by MTT. The apoptosis and cell cycle distribution of PC-3cells were examined by flow cytometry. The invasion of the tumor cells were studied by Transwell assay.Results:siRNA-6150with the best efficiency was selected for subsequent experiments. Compared with that of control group and the negative control group, the apoptosis rate increased3.3times in Notch-1siRNA-transfected PC-3cells. Compared to control treatment, the silencing of Notch-1induced a significant increase PC-3cells in S arrest. PC-3cells to the the membrane of the Transwell chamber decreased after transfection of Notch-1siRNA.Conclusions:Silencing Notch-1can inhibit proliferation and promote apoptosis, cell cycle arrest in S phase. Silencing of Notch-1can reduce the invasion ability of PC-3cells. Chapter II:Mechanism of synergistic effects of silencing of Notch-1and docetaxel on proliferation and apoptosis in PC-3cellsObjective:To study on whether silencing of Notch-1could enhance the induction of apoptosis and the inhibition of proliferation in PC-3cells by docetaxel.Methods:The proliferation of PC-3cells treated by different concentrations of docetaxel at different times were detected by MTT assay. Notch-1siRNA was transfected into PC-3prostate cancer cells before IC30docetaxel treatment. The proliferation and apoptosis of PC-3cells were examined in the presence or absence of docetaxel by MTT and flow cytometry. The expression of Bcl-2and Bax in PC-3cells were detected by real-time RT-PCR and Western blot.Results:The results demonstrated that silencing the Notch-1gene effectively inhibits proliferation and induces apoptosis in PC-3cells. In addition, docetaxel treatment results in decreased proliferation and increased apoptosis in the Notch-1-silenced cells compared to the control PC-3cells. Docetaxel treatment was also accompanied by an upregulation of Bax and a downregulation of Bcl-2.Conclusions:Silencing of Notch-1down-regulates the anti-apoptotic protein Bcl-2, and up-regulates the pro-apoptotic protein Bax, which ultimately results in increased sensitivity of PC-3cells to docetaxel. Chapter III:Mechanism of synergistic effects of silencing of Notch-1and docetaxel on cell cycle in PC-3cells Objective:Aimed to investigate the effects of Notch-1silencing on the sensitivity of prostate cancer cells to docetaxel treatment.Methods:The proliferation of PC-3cells treated by different concentrations of docetaxel after silencing Notch-1were detected by MTT assay. Notch-1siRNA was transfected into PC-3prostate cancer cells before IC30docetaxel treatment. The cell cycle distribution of PC-3cells were examined in the presence or absence of docetaxel by MTT and flow cytometry. The expression of p21wafl/cipl and Akt as well as the activation of Akt in PC-3cells were detected by real-time RT-PCR and Western blot.Results:The inhibition rate significantly increased in the interference group than that of control group and negative control group. The combination of silencing of Notch-1and docetaxel caused G2/M cycle arrest, accompanied by up-regulation of p21wafl/cipl and down-regulation of Akt and p-Akt.Conclusion:Silencing of Notch-1promoted docetaxel induced cell growth inhibition and cell cycle arrest in PC-3cells. In addition, these effects were associated with increased p2lwafl/cipl expression and decreased Akt expression and activation in PC-3cells.