Effects And Mechanism On Biological Behavior And Mechanisms Of Nasopharyngeal Carcinoma Cells In Vitro Of Theaflavin

Background:Nasopharyngeal carcinoma?NPC?has a high degree of malignancy and rapid progression,which is a common malignant tumor of the neck in the central and southern parts of China.Radiotherapy combined with chemotherapy is the main treatment for nasopharyngeal carcinoma.The local control rate is over 80%,but recurrence and distant metastasis after radiotherapy are two major problems in clinical treatment.Therefore,finding anti-tumor drugs with good curative effect,small side effects and capable of addressing the above problems is a clinical problem to be solved urgently in clinical practice.Theaflavin?TF?,which are abundant in black tea,are one of the products of tea fermentation.Studies have shown that theaflavin can inhibit the proliferation of various tumor cells and induce their apoptosis,which is an ideal anti-tumor Chinese medicine monomers.In the screening of traditional Chinese medicines for nasopharyngeal carcinoma,we found that theaflavin also have inhibitory effect on nasopharyngeal carcinoma,but there is no research on the effects of theaflavin on nasopharyngeal carcinoma and its molecular mechanisms.Therefore,the study will explore the mechanism of anti-Nasopharyngeal carcinoma effect in vitro of theaflavin.Objective:To investigate the anti-cancer effects of theaflavin on human nasopharyngeal carcinoma cells in vitro,and make a exploration of the cell proliferation,apoptosis,invasion and migration,as well as the underlying molecular mechanism.Methods:Human nasopharyngeal carcinoma CNE1 and CNE2 cells were treated with different concentrations of theaflavin.Firstly,the CNE1 and CNE2cells growth inhibition were detected at 24,48,72h by CCK-8 assay,and the cell inhibition rate were calculated.The clonality of CNE1 and CNE2 cells were measured by cell plate clone formation assay,and the invasion and migration of CNE1 and CNE2 cells were observed by Transwell assay.The cell migration assay was used to detect the migration of CNE1 and CNE2 cells.The apoptosis of CNE1 and CNE2 cells were observed by Hoechst 33258 staining,and the rate of apoptosis and cycle change were detected by flow cytometry.The mRNA expression level of CNE1 and CNE2 cells'proliferation-and apoptosis-relevant genes were detected by real-time fluorescent quantitative PCR.The expression of apoptosis-related proteins were detected by Western Blot.Results:Our results of the experimental study showed that,with the increase of the concentration of theaflavin and the prolongation of the action time,CCK-8assay showed that it significantly inhibited CNE1 and CNE2 cells growth in vitro in a dose-and time-dependent manner.Cell plate cloning experiments showed that theaflavin could significantly reduced the colony forming ability of CNE1and CNE2 cells.Cell scratch assay and Traswell assay showed that theaflavin can inhibit the migration and invasion of CNE1 and CNE2 cells.Mechanismly,the anti-cancer effect to CNE1 and CNE2 cells by theaflavin were involved into cell apoptotic process,which were proved by nuclear pyknosis after Hoechst33258staining.After the treatment of CNE1 and CNE2 cells by theaflavin,the apoptotic rate increased with the increase of drug concentration accordingly by cell flowmetry.And the cycle results showed that CNE1 cells were arrested in S and G₂/M phases,and CNE2 cells were arrested in G₂/M phase.Real-time quantitative PCR was used to detect the proliferation and expression of apoptosis-related genes in CNE1 and CNE2 cells.It was found that theaflavin can up-regulate the expression of apoptosis-related genes bax,caspase-3 in CNE1 and CNE2 cells,down-regulate the expression of bcl-2.The expression of p21 and p53 were up-regulated,the expression of CDK1 and CDK2 were downregulated.Western blot results showed that CNE1 and CNE2 cells up-regulated the expression of apoptosis-related proteins caspase-3 and bax,and down-regulated bcl-2.Conclusions:1.Theaflavin could up-regulate p53,causing the activation of p21,which in turn inhibits the expression of CDK1 and CDK2,arresting nasopharyngeal carcinoma cells in S and G₂/M phases,and inhibiting the proliferation of nasopharyngeal cells.2.Theaflavin could induce the apoptosis of nasopharyngeal carcinoma cells via up-regulate the expression of bax and caspase-3,and down-regulate the expression of bcl-2.3.Theaflavin also could inhibit the invasion and migration of nasopharyngeal carcinoma cells.