Radio Sensitizing Effect Of Double PI3K/mTOR Inhibitor NVPBEZ235 On Hela Cells Of Cervical Cancer

Objective:In this study,human cervical cancer Hela cells were selected as the research object.The radiosensitization of NVPBEZ235,a double PI3K/m TOR inhibitor,on cervical cancer Hela cells was studied by radiation irradiation.Methods:1.Detection of antitumor effect of NVPBEZ235 in vitro by MTT.Hela cells were cultured in vitro,and the proliferation inhibition of NVPBEZ235 was detected by MTT method,and a half inhibitory concentration of IC50(n M)was calculated.The NVPBEZ235 at different doses(0,6.25,12.5,25,100nmol/L)was treated at different time(24 h,48 h,72 h)to treat Hela cells,and the effect of aging and dose effect on the proliferation of tumor cells was measured.2.Detection of radiosensitization of NVPBEZ235 by clone forming method.The effects of NVPBEZ235 and NVPBEZ235 combined with IR on the colony forming ability of tumor cells were detected by clone formation assay after treated with NVPBEZ235 for 24 h.3.Detection of tumor cell cycle by flow cytometry.The cells were treated with NVPBEZ235 for 24 h,combined with IR,and the effect of flow cytometry on the cell cycle distribution was analyzed.4.Detection of nuclear staining by Hoechst staining.Effects of NVPBEZ235 and NVPBEZ235 combined with IR on Hela cell nucleus under optical microscope observation.5.Detection of apoptosis in tumor cells by Annexin-FITC Cell apoptosis kit.Flow cytometry was used to detect the time and proportion of apoptosis induced by NVPBEZ235 and NVPBEZ235 combined with IR by Annexin-FITC cell apoptosis kit.Results:1.In vitro antitumor experiments showed that NVPBEZ235 had obvious inhibitory activity on Hela cells.The time dose dependent.MTT results showed that the survival rate of the cells decreased with the prolongation of time and the increase of drug concentration.According to calculation,the values of IC50 were 24 h:328.8 nmol/L,48 h:225 nmol/L,72 h:122.48 nmol/L respectively.2.Cloning assay showed that NVPBEZ235 could inhibit the colony formation of Hela cells.The number of clones formed in different treatment groups was analyzed and compared.It was found that the survival fraction of Hela cells was significantly lower than that of simple IR group,which indicated that the drug had radiosensitization effect on cervical cancer Hela cells.3.The effect of flow cytometry on NVPBEZ235 and radiation(6Gy)alone had little effect on the number of G0 / G1 phase,but combined action could significantly increase the proportion of G2 / M phase cells,and the cells were blocked in the G2 / M phase sensitive to radiation.4.The apoptotic cells were observed under inverted fluorescence microscope and the white apoptotic nuclei were observed in each treatment group by Hoechst staining.5.Annexin-FITC cell apoptosis kit was used to detect the time and proportion of apoptosis induced by the intervention.The apoptosis of cervical cancer Hela cells treated with NVPBEZ235 and IR 6 Gy was significantly higher than that of the single drug and IR group.The apoptotic rate of Hela cells in each group was significantly higher than that in control group.Hela and in combination group(48.85% and 52.51%,respectively),which was significantly higher than that in control group(21.5%)and IR group(46.99%).Conclusion: 1.In vitro,NVPBEZ235 can inhibit the growth and proliferation of human cervical cancer Hela cells in a time-dose dependent manner.2.The combined action of NVPBEZ235 and IR can block the cell cycle in G2 / M phase in vitro,and the two have synergistic effect.3.The radiosensitization effect of NVPBEZ235 combined with IR group was higher than that of IR group.4.NVPBEZ235 and IR could induce apoptosis of Hela cells.