A Study On The Effect Of γ-secretase Inhibitors On The Biological Behaviors Of Human Ovarian Cancer Cells And Related Mechanisms

Ovarian cancer is a malignant tumor occurred in ovarian tissue, accounting for about 15% of gynecologic malignancies, second only to cervical cancer and its mortality rate is ranked first. Occult ovarian cancer onset, early difficult to find, easy to transfer, and poor prognosis features, so 5-year survival in patients with ovarian cancer, a lower, becoming a serious threat to lives of patients with gynecologic tumors malignancy. Therefore, to study the mechanism of the occurrence of ovarian cancer, to find new research targets for cancer gene therapy, to provide new ideas are undoubtedly of great significance.Notch signaling pathway as an ancient and conserved signaling pathway, which by affecting apoptosis, proliferation, differentiation and other pleiotropic effects of signaling pathways regulating development of multicellular animals, the shape and form. The current study confirmed its occurrence not only in the organization have an important role in the development process, and the occurrence and development of tumor has a close relationship, a number of solid tumors in humans(such as cervical cancer, endometrial cancer, kidney cancer, lung cancer, breast cancer, pleuralmesothelioma, and salivary gland tumors), and hematological tumors have abnormal expression. Studies have shown that activation of Notch1 and its downstream gene HES1 expression in ovarian adenocarcinoma was significantly higher than in ovarian adenomas and normal ovarian tissues, indicating that Notch1 is related to the occurrence of ovarian cancer.γ-secretase enzymes, including a Presenilin dimmer, Nicastrin, presenilin enhancer-2 (PEN-2) and other components of the multi-enzyme complex. They cut APP, Notch, E-cadherin, ErB-4 receptor tyrosine kinase such as membrane proteins, and studies have shown thatγ-secretase inhibitors inhibit the growth of a variety of cells. In this study, real-time polymerase chain reaction was used to detect the relative expression of Notch1 mRNA in different ovarian cancer cell lines. Thenγ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl) ]-S-phenylglycine t-butylester (DAPT) was used to block Notch signaling pathway in human ovarian cancer cells. After suppression Notch1 protein, we found that Hes1 protein expression was down- regulated, and investigated the cell proliferation inhibition, decreased adhesion, inva- sion and matastasis byγ-secretase inhibitor in A2780 in vitro, and presume that the Notch1 protein may contribute to the biological behaviors of human ovarian cancer cells by down regulating its downstream gene HES1.As a result, we offered the expe- riment evidence that the inhibition of Notch signaling pathway byγ-secretase inhi-bitor can inhibit the the biological behaviors of human ovarian cancer cells. Therefore,γ-secretase inhibitor is a potential new choice for the treatment of human ovarian cancer.【Objectives】 1. To screen the highest expression of Notch1 gene in human ovarian cancer cell lines.2. To study the effect ofγ-secretase inhibitors on ovarian cancer cell growth, proliferation, adhesion, invasion and migration activity in vitro, and its effects on cell cycle distribution, and to explore its mechanism.【Methods and Result】Two sections were included in our study.PartⅠ: The effects ofγ-secretase inhibitors on human ovarian cancer cell growth and proliferationMethods :1. Real-time PCR was used to screen the highest expression of Notch1 gene in human ovarian cancer cell lines.2. MTT test and cell clone test were used to detect the cell growth and proliferation.3. Cell cycle distribution was detected by flow cytometry.Results:1. In A2780 cells, the relative expression levels of Notch1 in the highest, 45.68.2. The result of MTT showed thatγ-secretase inhibitors can inhibit the growth of A2780 cell, and its effect were time-and dose-dependent.3. Cloning experiment presumed thatγ-secretase inhibitors can inhibit the proliferation of A2780 cells, and the cell colony formation rate decreased (p <0.001).4. We found thatγ-secretase inhibitors can make A2780 cells have a longer G1 phase period with time-dependent manner (p <0.001).PartⅡ: The effects ofγ-secretase inhibitors on human ovarian cancer cell adhesion, invasion and migration activity.Methods :1. Real-time RT-PCR was used to detect the levels of Notch1 and HES1 mRNA expression in A2780 cells before and after treated withγ-secretase inhibitor.2. Western blot test was used to detect Notch1 and HES1 expression at the protein level.3. MTT assay was sued to find the changes of cell adhesion ability after the treatment of DAPT.4. Cell invasion and metastasis of A2780 were observed by Transwell experiment.5. Immunofluorescence test was used for the detection of HES1 in human ovarian cancer cells in the location and expression.Results:1. After treated with DAPT, the expression of Notch1 and HES1 at gene and protein levels were inhibited with a time-dependent manner, and the difference was statistically significant (p <0.001).2. We found that the adhesion, invasion and metastasis activity of A2780 cell decreased with a time-dependent manner (p <0.05).3. Immunofluorescence test showed that HES1 of human ovarian cancer cells express mainly nuclear, but also express a small amount of cytoplasm. But after treated with DAPT, the expression of HES1 of human ovarian cancer cells decreased, and is mainly concentrated in the nuclear, and the decline is in a time -dependence.【Conclusion】1.γ-secretase inhibitors can inhibit cell growth and proliferation of A2780. 2.By inhibition of Notch signaling pathway,the expression of Notch1 was downregulated and the major stagnation of A2780 cells was in the G1 cell cycle phase, suggested that Notch signaling pathway have an important role in ovarian cancer cell cycle regulation..3. Inhibit the expression of Notch1 gene can inhibit the adhesion, invasion and metastasis activity of ovarian cancer cell A2780, indicated Notch1 play an important role in human ovarian cancer cell adhesion, invasion and metastasis process, and its mechanism may be related to the fall of the HES1.4.γ-secretase inhibitors can inhibit the expression of HES1 in human ovarian cancer cells, and change its localization.5. Notch signaling pathway can be used as an effective target for treatment of ovarian cancer,γ-secretase inhibitors may become a new drug treatment for ovarian cancer...