Study On Radiaosensitization Of Caffeine In P53-deficient Human Lung Adenocarcinoma H1299 Cell Line And Its Mechanism

Objective To observe the G2 phase arrest after irradiation and the abrogation of radiation-induced G2 phase arrest,and the radiosensitization of p53-deficient human lung adenocarcinoma H1299 cell line by caffeine,as well as to preliminarily investigate its mechanism.Methods Human lung adenocarcinoma H1299 cell line with p53-deficient was used in this study.(1) Detecting different concentrations of caffeine on H1299 cells growth inhibition by trypan blue exclusion test and the survival rate of H1299 cells after the effect of caffeine at different time points by MTT assay. (2) The effect of caffeine on radiosensitivity was determined by clonogenic assay and was quantified by calculating the sensitive enhancement ratio(SER). (3) Flow cytometry was used to observe the cell-cycle effects of irradiation and caffeine on the abrogation of radiation-induced G2 phase arrest in H1299 cells.(4) The effect of caffeine on apoptosis in irradiated H1299 cells was determined by flow cytometry. (5) Real-time quantitative PCR was used to observe the effect of irradiation and caffeine on Cdc2 mRNA expression in H1299 cells.Results (1)Caffeine significantly inhibit the growth of H1299 cells in a concentration-dependent and time-dependent manner. (2)Caffeine significantly reduced H1299 cells cloning efficiency after radiation. 1mmol/L and 2mmol/L caffeine resulted in a radiosensitization with SER of 1.20 and 2.07 in H1299 cell. (3)Irradiation resulted in G2 phase arrest in H1299 cells in a dose-dependent manner.At 24h after irradiation of 0Gy,2Gy,4Gy and 6Gy,the proportion of H1299 cells in G2 phase is 13.800%, 20.026%, 26.929% and 49.163%. (4)Radiation-induced G2 phase arrest was abrogated by caffeine in a concentration-dependent manner. Caffeine in the concentrations of 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L and 5mmol/L reduced the proportion of cells in G2 phase from 20.026% in the irradiation alone group to15.866%, 11.271%, 3.461%, 3.281% and 3.208%. (5) In H1299 cells, caffeine was able to increase the radiation-induced apoptosis, and the percentage of apoptosis was 10.2% in irradiation alone group and 26.3% in irradiation plus caffeine group after 24h. (6)The expression of cell cycle protein Cdc2 mRNA increased with the arrest of G2 phase after irradiation in H1299 cells. Caffeine could surpress the expression of radiation-induced Cdc2 mRNA, which was in accordance with the abrogation of radiation-induced G2 phase arrest.Conclusions (1)Caffeine is able to inhibit p53-deficient human lung adenocarcinoma H1299 cell line growth. (2)Caffeine is able to potentiate the radiosensitivity of p53-deficient human lung adenocarcinoma H1299 cell line. (3) Caffeine can abrogate radiation-induced G2 phase arrest and inhibit the repair of radiation-induced DNA damage,with increasing the radiation-induced cell apoptosis. (4)Caffeine can decrease the Cdc2 mRNA expression after radiation in H1299 cells, indicating that caffeine can incresse radiosentivity by altering the cell cycle signal factors.