Study On The Effect Of Rapamycin And NVP-BEZ235 Alone Or In Combination On Proliferation And Apoptosis Of CNE-2 Cells And Its Mechanism

Objectives:To investigate the influence of rapamycin,dual PI3K/mTOR inhibitor NVP-BEZ235 and their combined treatment on the growth,proliferation,apoptosis and the main targets in PI3K/Akt/mTOR signaling pathways of nasopharyngeal carcinoma CNE-2 cell lines.To explore the effect and possible mechanism of the combined treatment of NVP-BEZ235 and rapamycin on nasopharyngeal carcinoma CNE-2 cell lines in vitro.Methods:Nasopharyngeal carcinoma CNE-2 cell lines were cultured in vitro and divided into four experimental groups: NVP-BEZ235 group;Rapamycin group;NVP-BEZ235+Rapamycin group;control group.NVP-BEZ235 group refers to CNE-2 cells co-incubated with NVP-BEZ235(concentration: 100 nmol/L);Rapamycin group refers to CNE-2 cells co-incubated with Rapamycin(concentration:100 nmol/L),while NVP-BEZ235+Rapamycin group refers to CNE-2 cells co-incubated with the mixture of NVP-BEZ235 and Rapamycin(total concentration:100 nmol/L).Cells in the control group were treated with the same amount of solvents.Specific methods include: 1.The microscopic photography was utilized to observe the morphologic change of nasopharyngeal carcinoma CNE-2 cell lines in four group.2.The viability of nasopharyngeal carcinoma CNE-2 cell lines was studied by MTT test.3.RT-PCR and Western Blot were used to detect the mRNA expression level and protein expression level of PI3 K,Akt,mTOR respectively.4.Apoptosis was detected by flow cytometry.Results:1.After 48 h treatment,the CNE-2 cells in the rapamycin or NVP-BEZ235 group appear apoptosis morphological changes.And NVP BEZ235 + rapamycin group compared with single drug groups,CNE-2 cells shows more obvious apoptosismorphological changes.2.After drug treatment,the difference in cell growth inhibition rate between CNE-2 cells in three treatment group and control group was statistically significant(P < 0.01),but the difference of cell growth inhibition rate in combined treatment group between single drug groups show no statistical significance(P > 0.05).That prove NVP-BEZ235,Rapamycin single drug group and their combination group can obviously inhibit the growth of CNE-2 cells,but the combination group has no obvious improvement compared to single drug group in cell proliferation inhibition effect.3.(1)NVP-BEZ235 single drug group reduce the mRNA and protein expression level of PI3 K and Akt in CNE-2 cells;Rapamycin single drug group show no obvious effect in the mRNA and protein expression level of PI3 K and Akt;Joint application of two drugs present a significant decrease in the mRNA and protein expression level of PI3 K and Akt compared to NVP-BEZ23 or rapamycin single drug group,there was statistically significant difference(P <0.05).(2)rapamycin and NVP-BEZ235 single drug groups were significantly reduced the expression level of mTOR m RNA and protein;and the combination group compared to the single drug group,significantly reduced the expression level of mTOR mRNA and protein in CNE-2 cells,there was statistically significant difference(P < 0.05).4.NVP-BEZ235,Rapamycin single drug group compared with control group respectively,the apoptosis rate of CNE-2 cells increased,the difference was statistically significant(P < 0.05);Joint group compared with control group,the apoptosis rate increased significantly,and the difference is statistically significant(P < 0.01);Compared with the two single drug group,apoptosis rate of CNE-2 cell increased in the combination group,and the difference is statistically significant(P < 0.05).The above prove that the combination group induced stronger apoptosis than the NVP-BEZ235 single drug or drug rapamycin single group.Conclusion:1.NVP-BEZ235 and Rapamycin block PI3K/Akt/mTOR signaling pathways,and inhibit the growth and proliferation of nasopharyngeal carcinoma CNE-2 cells,induce apoptosis in vitro;2.The combined application of Rapamycin and NVPBEZ235 level can improve the antitumor activity of nasopharyngeal carcinomaCNE-2 cells in vitro.