The Study Of Relationships Between Notch-1 Signaling Pathway And Gemcitabine Chemoresistance, And Correlations Between Notch-1 Expression And Patients' Clinicopathological Factors And Prognosis In Pancreatic Cancer

Background and Objective Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors in gastrointestinal tract, which has the characteristics of rapid progression, low resection rate and poor prognosis. The mechanism of initiation and progression of PDAC still remains unclear. Gemcitabine is current first-line chemotherapy for advanced pancreatic cancer; however, high chemoresistance becomes a major reason of low treatment response. Notch signaling pathway plays a key role in cell fate decisions and is involved in contributing to chemoresistances in multiple tumors. Besides, aberrantly activated Notch signaling has been implicated in carcinogenesis and progression of various human malignancies. This research aims to investigate the relationships between Notch-1 signaling pathway and gemcitabine chemoresistance, and to evaluate the correlations between Notch-1 expression and patients'clinicopathological factors and prognosis in PDAC.Materials and Methods Three human PDAC cell lines as AsPC-1,BxPC-3 and MIAPaCa-2 were employed for in vitro experiments. The expression differences of Notch-1 signaling pathway after gemcitabine treatment were tested by real-time PCR and Western blot. Chemosensitivity changes to gemcitabine were evaluated after applyingγ-Secretase inhibitors (GSI) and small interfering RNA transfection as down-regulators for Notch-1 pathway activity and pIRES-EGFR-NICD transfection as up-regulators. Finally, changes of apoptosis rate, caspase-3 activity and relative protein expression in apoptosis pathway were observed in gemcitabine treatment combined with Notch-1 signaling pathway inhibition compared with gemcitabine treatment alone. Moreover, expression of Notch-1 protein was examined by immunohistochemistry in 73 surgically resected specimens of PDAC and adjacent tissues. The relationships between Notch-1 expression and clinicopathological and prognostic variables of patients were further evaluated.Results Notch-1 mRNA expression was decreased and the target protein Hes-1 expression was increased after gemcitabine treatment, suggesting activation of Notch-1 signaling pathway. Inhibition of Notch-1 pathway by GSI and Notch-1 siRNA induced increased chemosensitivity to gemcitabine, which accompanied with higher apoptosis and caspase-3 activity, overexpression of Bax and decreased expression of Bcl-2 and Bcl-XL protein. Contrarily, pIRES-EGFR-NICD transfection which promoted Notch-1 pathway caused a decreased chemosensitivity to gemcitabine. Immunohistochemistry staining showed that the positive expression rate of Notch-1 in tumor tissue was significantly higher than in normal tissue. There were correlations between Notch-1 expression and distant metastasis. Kaplan-Meier analysis showed positive expression of Notch-1 had a significantly poorer prognosis. Multivariate Cox proportional hazards regression analysis confirmed that Notch-1 was an independent predictor for overall survival.Conclusions Gemcitabine chemotherapy induced Notch-1 signaling pathway activation. Inhibition of Notch-1 signaling pathway enhanced gemcitabine sensitivity by activating the apoptosis pathway through intrinsic stress pathway. Notch-1 expression predicts an unfavorable prognosis and may serve as a novel independent prognostic marker.