The Research Of Radiosensitization By RAD001 For The Different PIK3CA Gene And KRAS Gene Type Human NSCLC Cells

Objective Study the role of mTOR inhibitor RAD001 in the radiotherapy for NSCLC, then discuss the relevance among this effect, PIK3CA gene and KRAS gene.Methods To detect the 50% inhibition concentration(IC50) of RAD001 in the human NSCLC cells NCI-H661 cells (PIK3CA and KRAS gene wild type) and NCI-H460 cells (PIK3CA and KRAS gene mutation) by MTT in vitro. To determine the concentration for the radiosensitization. Using RAD001 in these two cells for 24h or X-ray only, then, the cells were irradiated by X-ray with 0, 2, 4, 6, 8Gy.To calculate the surviving fraction of two cells respectively. To fit the curves by multi-target single-hit model, then calculate D0, Dq, SF2 of two cells. To observe the changes of theγ-H2AX protein of the radiation alone group and the RAD001 + irradiated group by immunofluorescence after 4Gy X-ray exposed at 0m, 30m, 60m, 4h, 12h, 24h. To detect the p-p70S6K protein and theγ-H2AX protein by western blot.Results The 50% inhibition concentration(IC50) of RAD001 in two cells respectively is NCI-H661 (23.101±0.056) nM and NCI-H460 (61.894±0.049) nM(p﹤0.05); The D0, Dq, SF2 of NCI-H661 in the radiation alone group and the RAD001 + irradiated group respectively is 2.244,1.927,0.712 and 1.596,1.369,0.548,SER is 1.406. The D0, Dq, SF2 of NCI-H460 in the radiation alone group and the RAD001 + irradiated group respectively is 2.412,2.894,0.851 and 2.359,1.848,0.706,SER is 1.022;The immunofluorescence shows that the residues ofγ-H2AX foci in NCI-H661 cells in the RAD001 + irradiated group become higher than anther group at 24h.But the similar phenomenon does not appear in the NCI-H460 cells. The p-p70S6K proteins of both two cells reduce after using RAD001 for 24h. The western blot shows that theγ-H2AX protein of NCI-H661 cells in the RAD001 + irradiated group is obviously higher at 24h than anther group. The summit ofγ-H2AX protein of NCI-H460 cells in the RAD001 + irradiated group is at 30m.Conclusions RAD001 has significantly radiosensitization for the human NSCLC cells. This function is related to the state of PIK3CA gene and KRAS gene. The radiosensitization is notable in PIK3CA gene and KRAS gene wild type cells than the mutation ones.