Research On Apoptosis Induction Of Paeonol On Human Nasopharyngeal Carcinoma Cell Line CNE-1

Objective: To Study the proliferation inhibition effect of paeonol on human nasopharyngeal carcinoma CNE-1 cells in vitro and to explore the mechanism of apoptosis of CNE-1 induced by paeonol with molecular biology techniques.The results of research could be prepared as a basis for the treatment of nasopharyngeal carcinoma with paeonol.Method: 1 MTT method detecting the influence of paeonol on the proliferation activity of the human nasopharyngeal carcinoma CNE-1 cells: The CNE-1 cells were treated by the different doses of paeonol,and the experiment time was not the same including the 24 h,48h and 72 h.The negative control group and the positive control group were set up.At last,the morphological changes of each group were observed and the cellular proliferation inhibition rate was calculated so as to analyze the time and dose response relationship of paeonol treating the human nasopharyngeal carcinoma CNE-1 cells.2 Immunofluorescence staining detecting the apoptosis after the human nasopharyngeal carcinoma CNE-1 cells were treated with paeonol: The attached cells of the negative control group and the middle and low dose group of paeonol were cultured 24 h and 48 h.Then the apoptotic status was observed under the fluorescence microscope after the AO/EB fluorescence staining.3 Flow Cytometry detecting the cell apoptosis rate and the cell growth cycle: The attached cells of the negative control group and the middle and low dose group of paeonol were cultured 48 h.Then use the Flow Cytometry to detect the cell apoptosis rate and the cell growth cycle.4 Immunoblotting(Western Blot)measuring the expression level of the cell-associated protein: The attached cells of the negative control group and the middle and low dose group of paeonol were cultured 48 h,then the total cellular protein was extracted.The expression of the apoptotic proteins(Bax?Bcl-2)were detected by the Immunoblotting method.Results: 1 The MTT Experiment: The human nasopharyngeal carcinoma CNE-1 cells were disposed by paeonol,then the experiment data was analyzed by the researchers.Compared with the negative control group,there existed remarkable difference which has the statistical significance(P<0.001).There was dose-effect relationship between paeonol and the human nasopharyngeal carcinoma CNE-1 cells.2 The Immune Staining Experiment: The human nasopharyngeal carcinoma CNE-1 cells were disposed by paeonol,then the cells were observed under the inverted fluorescence microscope.Compared with the negative control group,there were more typical apoptosis in paeonol treatment group showing nuclear condensation,distortion,etc.And there appearing the early apoptotic and the late apoptotic cells by the different nuclear staining.3 The detection of the cell cycle and the apoptosis by the Flow Cytometry: The human nasopharyngeal carcinoma CNE-1 cells were disposed by paeonol.Compared with the negative control group,the distribution of cells in the S phase increased significantly,and the difference was statistically significant(P<0.01)after the data was statistical analyzed.What's more,the apoptosis rate of paeonol treatment group increased more significantly,and the difference had the remarkable statistically significance(P<0.001).4 The Western Blot Experiment: Compared with the negative control group,the expression of Bax had increased more significantly in paeonol treatment group,the expression of Bcl-2 is just the opposite.And the ratio of Bax/Bcl-2 was up-regulated significantly.The difference was statistically significant(P<0.01)after the data was analyzed.Conclusions: 1 Paeonol can significantly inhibit the growth of the human nasopharyngeal carcinoma CNE-1 cells.It was related to the dosage and time.2 Paeonol can significantly inhibit the growth of the human nasopharyngeal carcinoma CNE-1 cells.The mechanism may relate to both the induction of apoptosis and the cell cycle arrest,and arresting cell cycle progression in S phase may induce apoptosis.3 In the process that paeonol induce the apoptosis of the human nasopharyngeal carcinoma CNE-1 cells,The mechanism may be relate to the expression of Bax increased significantly and the expression of Bcl-2 decreased.It may be associated with mitochondria mediated apoptosis,which needed further study.