Inhibitory Effect Of Mangiferin On Nasopharyngeal Carcinoma Cells2(CNE2) And Possible Mechanism

ObjectiveMangiferin is one of the natural flavonoid compounds.The report showed mangiferin can inhibit the proliferation of nasopharyngeal carcinoma cells,but the mechanism is not yet clear.To study the biology behavior and mechanism of human nasopharyngeal carcinoma CNE2 cells treated by different concentration of mangiferin and different time,in vitro cell culture.To observe the effect of mangiferin on cell proliferation through the cell toxicity test,to detect the cell cycle distribution and apoptosis by flow cytometry.Using biology method to test the change of apoptosis related gene Bcl-2 and Bax,in mRNA and protein expression level.To provide experimental base for clinical application in treatment of nasopharyngeal carcinoma(NPC)by mangiferin.Method(1)Consulting relevant literature and the purification method of mangiferin provided by the clinical trials center of guangxi national hospital,crush the leaves of the mangiferin,and do the extraction,separation and purification of mangiferin by using ethanol diacolation method.Identify the purity of mangiferin by appearance observation,microscopic examination,Chemical identification,Ultraviolet spectrum scan and High performance capillary electrophoresis method.Prepare dimethyl sulfoxide(DMSO)solution of different concentration for the following cell experiment.(2)CNE2 is provided by ENT laboratory experiment center of Guangxi m edical university.Conventional cell culture method,in vitro,by using RPMI1640 culture medium added 10%fetal bovine serum.When CNE 2 cell grows to 70-80%fusion,add culture medium containing differen t concentration of mangiferin,compared to blank control without mangi ferin.Observe the change of Cell morphology and cell number by Inv erted microscope in different time points.Apply CCK-8 cell proliferatio n-toxicity detection kit to detect the inhibition rate of CNE2.(3)To culture CNE2 cell in vitro by conventional method,when CNE2 ce 11 grows to 70-80%fusion,add culture medium containing different co ncentration of mangiferin,three days latter:①To observe cell apoptosis situation by fluorescence microscope.②Using Flow cytometry to anal yse the change in the cell cycle and apoptosis rate.(4)To culture CNE2 cell in vitro by conventional method,when CNE2 cell grows to 70-80%fusion,add culture medium containing different concentration of mangiferin,three days latter:①collect cell,extract the RNA and reverse transcription into cDNA,using Real-time PCR to detect the change of apoptosis related gene Bcl-2 and Bax in mRNA expression level.②collect cell,extract the protein,using Western blot to detect the Bcl-2 and Bax protein,involving the change of protein content.ResultThe purity of mangiferin extracted from the local characteristic resources in guangxi Mango tree leaves is nearly up to 97.4%,close to the Sigma level.The source of mangiferin is accessible,and extraction cost is relatively low.To test the proliferation of CNE2 deader with mangiferin by using CCK-8,the results show that mangiferin has influence on the growth of CNE2,inhibits the proliferation of CNE2.When the concentration of mangiferin is 200 μmol/L,five days later,the inhibition rate is 55.9%(P<0.05).The effect shows a dose-effect relationship.Observed by microscope,we can find the number of the cell does not increase,the forming of colonies is inhibited,light transmittance decrease,Cell becomes round and whole outline is not clear,Cells fall off from the bottle wall.The higher concentration of mangiferin results in more obvious performance.To detect cell cycle distribution in CNE2 synchronized by flow cytometry.Finding that mangiferin can interfere with the growth of CNE2 cell cycle,make the stagnation of cells in the G2-M.Cell apoptosis occurs at the same time,the effect is obvious after 3 days and concentration of mangiferin as 200 μmol/L,especially,the apoptosis is 44.1%,the G2/M value is 14.6%.Using AnnexinV-FITC/PI apoptosis detection kit and fluorescence microscope,show that the number of cell membranes showed green fluorescence is more,that is cell in early apoptosis.But the number of cells appear red fluorescence are very few,indicate that death or in the terminal stage of apoptosis cells are less.The two above experiments both show a dose-effect relationship.Via RT-,real-time fluorescent PCR,to detect expression of Bcl-2 and Bax in gene level in CNE2 synchronized,at the same time,using Western Blot to detect protein expression of Bcl-2 and Bax.Results show that mangiferin can decrease expression of Bcl-2,and increase expression of Bax relatively.This also presented dose-effect relationship.Proving that mangiferin induce apoptosis of nasopharyngeal carcinoma CNE2 cells occurring in a certain extent.Conclusion(1)Mangiferin can inhibit the growth of nasopharyngeal carcinoma CNE2 cells in dose and time related.(2)Mangiferin on nasopharyngeal carcinoma CNE2 cell growth inhibition induce CNE2 stagnating in G2/M phase and occurring early apoptosis,the mechanism may be through the can decrease the expression of Bcl-2 protein levels,and relative increase protein expression of Bax.