A Study On The Role And Mechanism Of Notch1 As Signal In Ovarian Carcinoma

Background Ovarian cancer is the second most common gynecologic cancer among women and the first leading cause of death from gynecologic malignancy worldwide. Metastasis beyond the ovary is found in 75% of patients at the time of diagnosis due to the absence of symptoms in early stages and lack of a reliable method for early detection, resulting in a high mortality rate despite optimal surgery and aggressive chemotherapy [1, 2]. This necessitates a better understanding of the molecular events of ovarian cancer progression, which may play an important role in early diagnosis and early therapy.Notch signaling is an evolutionarily conserved pathway that regulates critical cell fate decisions[3]. Notch ligands and receptors are type I membrane proteins that regulate cell fate during cell-cell contact [3, 4]. In mammals, the Notch family consists of four receptors (Notch1-Notch4) and five ligands (Jagged-1, Jagged-2, Delta-like-1, Delta-like-3 and Delta-like-4) [5]. Receptor–ligand interaction between two neighboring cells leads toγ-secretase–mediated proteolytic release of the Notch intracellular domain (NICD [6]. NICD then translocates into the nucleus, in which it interacts with the transcriptional cofactor CBF1 and transactivates gene targets such as those in the Hes and Hey families [7, 8]. Notch signaling and its gene targets control variety of processes, involving cell fate specification, differentiation, proliferation, and survival [3].Abnormal Notch signaling has been documented in many cancers and has been associated with tumorigenesis[9, 10]. Notch1 can function as a tumor oncogene or a suppressor, depending on the cell type and context. For instance, it acts as a tumor suppressor in murine skin tumors and non-small cell lung cancer[11, 12], but it acts as a tumor oncogene in many other types of cancers, such as renal cancer, pancreatic cancer, breast cancer and prostate carcinomas[13]. Some reports show that Notch3 and its target gene Pbx1 may play an important role in the development of ovarian cancer[14, 15]. Notch1 is considered as a tumor oncogene in ovarian cancer and a higher Notchl protein expression is found in ovarian carcinoma cell lines[16, 17]. These reports hint that Notch signaling might play important roles in ovarian cancer. The relationship between Notch siganling and ovarial carcinoma is a new field of study,and it also may be a new gene therapy for ovarial carcinoma.Purpose(1) To study the significance of Notch1 expression in ovarial carcinoma transformation and progression and its relationship with clinical pathology characteristics.(2)To determine the expression of Notch1 and hes1 in ovarian carcinoma cell lines and to screen target cells for further study at Notch1 in ovarian carcinomas. (3)To determine the expression of Notch1 and hes1 in A2780 cells after the inhibition of Notch signaling byγ-secretase inhibitor(DAPT) and study the biologic behavior of A2780 cells for providing experiment data for further study. (4) To determine the influence of cisplatin resistance by down-regulation of Notch signaling byγ-secretase inhibitor(DAPT).Methods(1) using Immunohistochemistry, real-time polymerase chain reaction and Western Blot examine the expression of Notch1 in 109 ovarian cancer tissues, 65 patient-matched opposite side normal ovarian tissues and 48 normal ovarian tissues. They are analyzed by Chi-square test of SPSS14.0.(2)using real-time polymerase chain reaction and Western Blot examine the expressions of Notch1 and hes1 in ovarian carcinoma cell lines A2780, SKOV3, HO-8910, HO-8910PM and IOSE 144 and using MTT assay, Flow cytometry, ELISA, colony-forming assay to examine biologic behavior of ovarian cancer cells after the inhibition of Notch signaling byγ-secretase inhibitor(DAPT).(3) using real-time polymerase chain reaction and Western Blot examine the expressions of hes1 after the treatment of DAPT on ovarian cancer cisplatin-resistant cells A2780/CP70 and using MTT assay, Flow cytometry, ELISA, colony-forming assay to examine biologic behavior of A2780/CP70 after DAPT-cisplatin combination on A2780/CP70 cells.Results:(1)The expression of Notch1 in ovarian cancers was correlated with differentiation status and FIGO stage(P﹤0.05), not with age, family history, tumor location, ascites, adjacent tissue infiltration, lymphaden metachoresis and pathology subtypes(P>0.05).(2) Notch1 and hes1 are highly expressed in ovarian cancer cells A2780.(3) DAPT could down-regulate the expression of Notch1 and hes1 in a time-dependent and dose-dependent manner.(4) Down-regulation of Notch1 expression by DAPT inhibited A2780 cell growth and induced cell cycle G1 arrest. (5) Down-regulation of Notch1 expression by DAPT induced apoptosis in A2780 cells.(6) Notch signaling and its target gene hes1 are downregulated by DAPT in A2780/CP70 cells.(7) DAPT potentiates cisplatin-induced growth inhibition in a drug sequence–dependent manner.(8) DAPT-cisplatin combination arrests cisplatin-resistant A2780/CP70 cells in G2 phase.(9) DAPT enhances cisplatin-induced apoptosis in A2780/CP70 cells.Conclusion:(1) Notch1 expression was increased with decreasing levels of differentiation, as well as with increasing FIGO stage, that means the higher malignancy of the tumor is relevant to the higher Notch1 expression levels.(2) Notch1 and hes1 are expressed in lots of ovarian cancer cell lines and highly expressed in ovarian cancer cells A2780. So, we could choose A2780 to finish the subsequent studies.(3) DAPT could down-regulate the expression of Notch1 and hes1 in a time-dependent and dose-dependent manner and also inhibited A2780 cell growth and induced cell cycle G1 arrest and induced A2780 cells apoptosis.γ-secretase inhibitor(DAPT) may be a potential target of new therapeutic investigation in ovarian cancer.(4) DAPT could sensitize the cisplatin-resistant ovarian cancer A2780/CP70 cells to cisplatin induced cytotoxicity and potentiates cisplatin-induced growth inhibition and also enhances cisplatin-induced apoptosis in A2780/CP70 cells. DAPT-cisplatin combination may be a target for the development of novel therapies for ovarian cancer.