Effect Of Ailanthone On Cell Proliferation,Cell Cycle And Apoptosis Of Human Nasopharyngeal Carcinoma CNE-2 Cells

Objective:To study the effects of ailanthone on the cell proliferation,cell cycle distribution and apoptosis of human nasopharyngeal carcinoma CNE-2cells in vitro,and the potential anti-tumor mechanism was preliminarily discussed,providing a new candidate drug for the clinical treatment of nasopharyngeal carcinoma.Methods:?1?The MTT assay was used to detect the inhibitory effect of ailanthone on the growth of human nasopharyngeal carcinoma CNE-2 cells in vitro:The human poorly differentiated nasopharyngeal carcinoma CNE-2 cells were cultured in vitro,treated with different concentrations of ailanthone?0,0.2,0.4,0.8,1.6 and 3.2?M?for 24h,48h,and 72h.Then the optical density?OD?of different concentration gradients and different time gradients were measured,and the cell survival rate and the half-inhibitory concentration?IC50?at different times?24h,48h and 72h?were calculated based on the OD value,respectively.?2?Hoechst 33342 fluorescence staining was used to observe the effect of ailanthone on the apoptotic morphology of human nasopharyngeal carcinoma CNE-2 cells:According to the results of MTT assay,the appropriate drug concentrations?0,1.6,3.2 and 6.4?M?were used to treat CNE-2 cells for 48h.After fluorescent staining with Hoechst 33342,the changes of apoptotic morphology of CNE-2 cells were observed by the fluorescent inverted microscope and recorded by photographing.?3?Flow Cytometry?FCM?analysis was used to detect the effects of different concentrations of ailanthone on the cell cycle distribution and apoptosis of human nasopharyngeal carcinoma CNE-2 cells:The cells were treated with the above drug concentrations?0,1.6,3.2 and 6.4?M?for 48h,and stained by Annexin V-FITC/PI double staining and PI single staining,then the cell apoptosis rate and cell cycle distribution of different concentrations were detected by flow cytometry.?4?Western Blot analysis was used to detect the effect of ailanthone on the expression of apoptosis-related proteins in human nasopharyngeal carcinoma CNE-2 cells:After treating with the above drug concentrations?0,1.6,3.2 and6.4?M?for 48h,the total protein was extracted,and the expressions of apoptosis-related proteins Bcl-2,Bax and caspase-3 were detected by western blot,then the results were analyzed by Image Lab software.Results:?1?The results of MTT assay showed that after treatment with different concentrations of ailanthone,the cell viability decreased with the increase of ailanthone concentration and the prolongation of the action time,in concentration-and time-dependent manners,and the IC₅₀?24,48 and 72h?were?0.66±0.07??M,?0.37±0.05??M and?0.23±0.01??M,respectively.?2?Hoechst 33342 fluorescence staining results showed that the nasopharyngeal carcinoma CNE-2 cells in the control group were stained with light blue uniform fluorescence,while the cells in the group of ailanthone treatment were stained with strong blue fluorescent with the typical apoptotic morphology,and the number of apoptosis increased with the increase of drug concentration.?3?Flow cytometry?after Annexin V-FITC/PI double staining and PI single staining?results showed that after treatment with different concentrations of ailanthone for 48h,both the apoptosis rate and the proportion of G₂/M phase cells increase with the increase of drug concentration compared with the control group.?4?Western Blot analysis showed that the expression of Bax and caspase-3increased and the expression of Bcl-2 protein decreased compared with the control group in human nasopharyngeal carcinoma CNE-2 cells after treatment with different concentrations of ailanthone for 48h.Conclusion:Ailanthone has a significant inhibitory effect on the growth of human nasopharyngeal carcinoma CNE-2 cells in concentration-and time-dependent manners,and exerts its anti-tumor effect by inducing apoptosis and cell cycle arrest.The mechanism may be related to that ailanthone has an effect on up-regulating the protein expression levels of Bax and caspase-3,and down-regulating the expression level of Bcl-2,which may be involved in the activation of mitochondrial-mediated apoptosis pathway,the specific anti-tumor mechanism needs a further investigation.In conclusion,as a natural active extract,ailanthone shows significant antitumor activity against nasopharyngeal carcinoma cells,and may become a potential candidate drug for the treatment of nasopharyngeal carcinoma.