Effect Of FNC On Proliferation And Apoptosis In Non-Small Cell Lung Cancer

Background and Objective:Lung cancer is the most common malignancy with the highest morbidity and mortality in the world.According to the latest statistics,patients with lung cancer in China account for one-third of the world^[1].Histologically,approximately 85%of all new lung cancer cases are classified as non-small cell lung cancers（NSCLC）.The traditional treatment of non-small cell lung cancer is platinum-based dual chemotherapy,but the current efficacy has reached a platform,and the clinical application was limited due to the adverse effects and toxicity,so there is an urgent need to develop new drugs to treat lung cancer.FNC（2’-deoxy-2’-β-fluoro-4’azide-nucleoside）is a novel nucleoside analog designed and synthesized by the School of Chemistry and Molecular Engineering of Zhengzhou University,which has applied for a national patent for invention,and the patent number is ZL201010506595.X.FNC showed marked antitumor activity,in addition,FNC can inhibit growth,invasion and metastasis of various lymphoma cells.The previous research was displayed that FNC had a significantly inhibitory effect on human lung cancer cells A549 in vitro,and the half inhibitory concentration was 1.22μmol·L^-1,suggesting that FNC may have a good therapeutic effect on lung cancer.In this study,non-small cell lung cancer cells,namely human H460 cells and mouse Lewis cells,were selected to study the effects of novel nucleoside compounds FNC on proliferation and apoptosis of non-small cell lung cancer in vitro and in vivo.Methods:1.The H460 cells were treated with 0.019,0.078,0.313,1.250,5.000μmol·L^-1FNC for 24 h,48 h,72 h,and the cell growth inhibition was determined by MTT assay and cisplatin was used as a positive control drug.2.H460 cells were treated with different concentrations of FNC(0.313,1.250,5.000μmol·L^-1)for 48 hour,and apoptosis rate was detected by flow cytometry（Annexin V-FITC/PI）;The apoptotic morphology was observed by acridine orange/ethidium bromide（AO/EB）double fluorescent staining.Cytochrome C released from mitochondria were detected by Western Blot.The expression of Bcl-2、Bax and Caspase-3 in H460 cells were detected by Western Blot.3.C57BL/6 mice were inoculated subcutaneously with Lewis lung cancer cells to establish a tumor-bearing mouse model and tumor-bearing mice were randomly divided into five groups:negative control group（0.9%physiological saline）,positive control group(cisplatin 2mg·kg^-1),low(2 mg·kg^-1),medium(4mg·kg^-1),high dose groups(8mg·kg^-1)of FNC.All mice were given by intraperitoneal injection,and every group was administered daily,and continuous administration was performed for 12days.At the end of trial,the mice were killed,stripped and weighed the turmors.Western Blot was used to detect the expression of Bcl-2,Bax and Caspase-3 proteins in the tumor tissue.Results:1.FNC significantly inhibited the proliferation of H460 cells,and the suppression was in time-and dose-response relationship(F_dose=222.389,F_time=405.892,F_interaction=73.271,P<0.05).The half inhibitory concentration after 24h,48h and 72h were 6.971,0.632,and 0.267μmol·L^-1.The IC₅₀ values of the positive control drug cisplatin were 39.271,8.032,3.193μmol·L^-1.2.The cell morphology changed and the apoptosis rate increased significantly after FNC treatment（P<0.05）.The cells were treated with 0.313,1.250,5.000μmol·L^-1 FNC and the apoptotic rates were（25.0±1.5）%,（42.5±2.4）%,（67.1±1.0）%.The mitochondrial cytochrome C were released due to the mitochondrial permeability increased.FNC down-regulated the expression of Bcl-2 protein in the cells and up-regulated the expression of Bax and Caspase-3 proteins in a dose-dependent manner.3.The tumor inhibition rates at low,medium,and high dose groups of FNC were 45.29%,71.75%,and 84.90%,respectively（P<0.05）.FNC can down-regulate the expression of Bcl-2 protein,up-regulate the expression of Bax and Caspase-3protein in the tumor tissue.The suppression was in time-and dose-response relationship.Conclusion:1.FNC can significantly inhibit the proliferation of NSCLC.2.FNC can induce apoptosis of NSCLC through mitochondrial apoptosis pathway.3.Induction of apoptosis is an important mechanism of FNC inhibiting the proliferation of NSCLC.