Effect And Molecular Mechanism Of How Cepharanthine Works On Human Nasopharyngeal Carcinoma Cell Lines CNE-2and 5-8F

OBJECTIVE:To investigate whether cepharanthine can inhibit the growth of human nasopharyngeal carcinoma CNE-2 and 5-8F cells in a concentration-and time-dependent manner,and to explore its mechanism.METHODS:(1)Cell proliferation and toxicity assay kit(CCK-8)was used to measure the cytotoxicity of cepharanthine on CNE-2 and 5-8F cells: cepharanthine at different concentrations(1.25 mg/L,2.5 mg/L,5 mg/L,10 mg/L,20 mg/L)acted on CNE-2 and 5-8F cells at different times(24 h,48 h,72 h),and then the absorbance was measured by enzyme standard instrument,and the inhibition rate of cepharanthine on CNE-2 and 5-8F cells was calculated.(2)Flow cytometry(FCM)was used to measure the cell cycle and apoptosis rate of CNE-2 and 5-8F cells.(3)Real-time fluorescence quantitative PCR(qRT-PCR)was used to measure the expression of apoptosis-related proteins in CNE-2 and5-8F cells.The results showed that half inhibitory concentration(IC50concentration)was selected as the medium concentration,and three concentrations of Stephania alkaloids were set up to treat CNE-2,5-8F cells for24 h,48h and 72 h,respectively.The expression of each gene in the three groups was measureed;(4)Use western blot to measure the expression of three proteins.RESULTS:(1)The results of CCK-8 showed that the IC50 values of cepharanthine for CNE-2 cells at 24,48 and 72 hours were 16.0737 mg/L,9.8561 mg/L and5.61277 mg/L,respectively;the IC50 values for 5-8F at 24,48 and 72 hours were 27.5491 mg/L,19.5802 mg/L and 10.9068 mg/L,respectively.It shows time and concentration dependence;(2)The results of FCM cycle showed that:With the increase of drug concentration,the proportion of G2 phase cells of CNE-2 increased from 14.20+0.23 at 0 mg/L to 64.86+0.29 at 20 mg/L,and the proportion of G2 phase cells of 5-8F increased from 10.31+1.25 at 0 mg/L to53.90+1.43 at 20 mg/L(P<0.01).The apoptotic rate of FCM cells showed that with the increase of drug concentration,the apoptotic rate of CNE-2 cells increased from 6.26+1.43 at 0 mg/L.The difference was significant(P < 0.01).Figure 2-2-2 showed that the apoptotic rate of 5-8F cells increased from5.12+0.30 at 0 mg/L to 33.07+0.72 at 20 mg/L with the increase of drug concentration(P<0.01).(3)The results of qRT-PCR showed that the expression of Bcl-2 and Cyclin D1 decreased with the increase of concentration and time,and the expression of Bax increased with the increase of drug concentration and time;(4)Western blot showed that the expression of Bcl-2 and Cyclin D1 increased with drug concentration.The expression of Bax protein increased with the increase of drug concentration and the prolongation of action time.CONCLUSION:(1)Cepharanthine can inhibit the proliferation of human nasopharyngeal carcinoma CNE-2 and 5-8F cells,block cell cycle at G2 phase and induceapoptosis of CNE-2 and 5-8F cells;(2)Cepharanthine may affect cell cycle and induce apoptosis by down-regulating the expression of Bcl-2 and Cyclin D 1 in CNE-2 and 5-8F cells.