A Study On The Role And Mechanism Of Notch As Signal And Gamma-secretase Inhibitor In Non-small Cell Lung Cancer

Background:With the increase of incidence and mortality of lung cancer,lung cancer is considered to be one of the most common cancers,and it has been one of the highest mortality of malignant tumors in our country and the world.In 2016,224,390 new cases were diagnosed with lung cancer and 158,080 were died.There were more than half of patients with lung cancer who either have advaced or metastatic disease at the time of diagnosis,due to the absence of symptoms in early stages and lack of a reliable method for early detection.at the same time,there was no effective treatment for the advanced lung cancer,which resulting in a high mortality of lung cancer.although the further study of the pathogenesis of lung cancer,the 5-year survival rate is still low.There were two main ways to treat the advanced lung cancer including platinum-based traditional chemotherapy and molecular targeted therapy,chemotherapy drugs and targeted drugs liable to occur drug resistance,so how to early detection of lung cancer and how to avoid chemoresistance is especially important for patients with lung cancer.Abnormal expression of Notch signal protein has been documented in many cancers and has been associated with tumorigenesis.Many human solid tumors,such as lung cancer,breast cancer,cervical cancer,tumor of salivary gland and blood system cancers were detected abnormal expression of notch singal protein.In Notch receptors,the more frequently-used Notchs are Notch1 and Notch3,particularly the latter,Notch1 function depends on the tumor cell type and context,For instance,it acts as a tumor suppressor in small cell lung cancer,but it acts as a tumor oncogene in many other types of cancers,such as renal cancer,pancreatic cancer and breast cancer.About Notch3,some eports showed that Notch3 may play an important role in the development of ovarian cancer,cholangiocarcinoma and urothelial carcinoma and so on,and found that over expression of Notch3 is associated with urothelial cancer chemoresistance.The relationship between Notch signaling and non-small cell lung carcinoma need to be further study,and it may be bring a new method for the treatment of lung cancer.Objective:(1)To assess the expression of Notch1 、 Notch3 、 Jagged-1(JAG-1)and Deltalike4(Delta-4)in NSCLC tissues and adjacent non-cancerous normal lung tissues.(2)To assess the correlation between over-expression of Notch1,Notch3,JAG-1,Delta4 and clinical pathological parameters and the prognosis of NSCLC.(3)By detecting expression of Notch3 protein and proliferation capacity of H1299 cells and A549 cells after induction by gemcitabine,to demonstrate whether Notch3 protein is associated with gemcitabine chemoresistance in NSCLC.(4)By detecting expression of NICD3,Bcl-2 and Bax protein when gamma secretase inhibitors DAPT blocks the Notch signal pathway,and detecting the biological behavior such as proliferation,apoptosis on H1299 cells and A549 cells or on H1299 cells and A549 cells after induction by gemcitabine,to study whether gamma secretase inhibitor DAPT can activate apoptosis pathway to kill tumor cells,and through this way to improve the chemosensitivity of gemcitabine.Methods:(1)we designed a prospective study with five years of follow-up to evaluate Notch1、Notch3、JAG-1 and Delta-4 expression in NSCLC tissues and adjacent non-cancerous normal lung tissues from 101 patients with surgical treatment by immunohistochemistry.(2)H1299 cells and A549 cells were incubated.(3)Western blot was used to detect the expression of Notch3,NICD3,Bcl-2 and Bax in H1299 cells and A549 cells.(4)MTT assay and Cell counting were used to detect proliferation of H1299 cells and A549 cells after induced by DAPT alone or synergistic gemcitabine compared with gemcitabine alone.(5)Flow cytometry was used to detect apoptosis of H1299 cells and A549 cells after induced by DAPT alone or synergistic gemcitabine compared with gemcitabine alone.Results:(1)The expression of Notch1,Notch3,JAG-1 and Delta-4 was significantly higher in tumor tissues than in adjacent non-cancerous lung tissues in 101 NSCLC.Moreover,Notch1 and Notch3 over coexpression was significantly correlated with TNM stage,lymph node metastasis and grading of tumors,and Delta-4 was correlated with TNM stage.(2)Kaplan–Meiersurvival analysis showed that the overall survival duration in patients was negatively correlated with the expression of Notch3 in NSCLC.mltivariate analysis further demonstrated that Notch3 protein expression levels was related to the prognosis of patients in non-small cell lung cancer.(3)Notch3 protein was significantly overexpressed in two cell lines and also was increased by gemcitabine induced,indicating activation of the Notch3 signaling pathway.Treated with DAPT and gemcitabine,the cell proliferation was significantly decreased at 24 h,48h and 72 h compared with adding gemcitabine alone.Similarly,through cell count of two cell lines,DAPT synergistic gemcitabine significantly reduced the number of two kinds of cells,compared with treated with gemcitabine alone,Indicating DAPT can improve the sensitivity of gemcitabine to the two kinds of cells.(4)DAPT significantly reduced NICD3 protein expression levels,and at the same time,DAPT associated with down-regulation of Bcl-2 expression and up-regulation of Bax xpression,And the Bcl-2 and Bax protein expression related to DAPT concentration.DAPT regulated the expression of apoptotic proteins and thus affected the effect of gemcitabine on two cell lines.Conclusions:(1)Overexpression of Notch1,Notch3,JAG-1 and Delta-4 were detected in NSCLC tissues,Level of Notch1 and Notch3 coexpression were significant correlated with NSCLC progression and unfavorable prognosis.Additionally,we have shown that Notch3 expression is an independent prognostic biomarker of overall survival for NSCLC.(2)Through regulating apoptosis pathway,gamma secretase inhibitors DAPT can change gemcitabine chemoresistance to H1299 cells and A4549 cells which was induced by gemcitabin.This approach indicated that co-mbined DAPT and gemcitabine would be likely to apply to treat patients who would be resistant to gemcitabine or recurrence in NSCLC.In addition,DAPT alone also can promote apoptosis of H1299 cells and A549 cells,which show that DAPT can inhibit tumor cells apoptosis,and maybe to apply in NSCLC treatment in the future.