The Effect Of 17-DMAG On Radiosensitization Of Hypoxic Cancer Cells

Objective:To explore the effect of 17-DMAG on Radiosensitization of human uterine cervix cancer cell line(HeLa) in chemical hypoxia induced by CoCl2, and reseach of possible mechanism. The expression of HIF-1αand HSP70 protein was detected by Western-Blotting; The effects of 17-DMAG combined with radiation on cell survival rate, cell apoptosis, and cell cycles was determined.Methods:HeLa cells were seeded in the medium containing 200μmol/L CoCl2. The expression of HIF-1αand HSP70 of HeLa cells under hypoxia after exposure to17-DMAG combined with radiation was detected by Western-Blotting. Colon forming assay is carried out to study the cells survival rate of HeLa cells under hypoxia after exposure to17-DMAG combined with radiation 17-DMAG; Apoptosis of hypoxic HeLa cells after exposure to17-DMAG combined with radiation was observed by confocal microscope, and evaluation of cell cycle phase distribution was performed using Flow Cytometer.Results:(1) Different concentrations of CoCl2 treat HeLa cells for 24 hours, expression of HIF-1αprotein increased with the concentration-dependent of CoCl2, to determine the most appropriate drug concentration with 200μmol/L on HeLa cells in chemical hypoxia; Exposure to different concentrations of 17-DMAG significantly reduced the expression of HIF-1αof HeLa cells under hypoxia, and there was a concentration-dependent decrease in the levels of HIF-1α; The expression levels of HSP70 was enhanced, and was dependent on the concentration 17-DMAG.(2) Colon forming assay showed that there was a significant difference between HeLa cells treated by different concentrations 17-DMAG and different irradiation doses(P<0.01). The survival rate of hypoxia group more than normal controls, that show in the hypoxia environment of tumor cells with radiation resistance. The Cell survival curve became smaller and linear slope increased significantly. The survival rate of all dose points was lower than the hypoxia group, and it indicated 17-DMAG had a significantly radiosensitivity effect on the HeLa cells of hypoxia environment.(3) The effect of different concentrations 17-DMAG on apoptosis: Early apoptosis rate of the normal control group, slightly higher than the group of hypoxia, but the effect of 17-DMAG combined with irradiation under hypoxia show that irradiation group of early and late apoptosis rate was significantly higher than that of hypoxia group, the reason is hypoxia can reduction the apoptosis rate induced by radiation. hypoxia drug group compare with hypoxia group show that The enhanced of apoptosis rate was in the drug and dose-dependent. We can speculate 17-DMAG can promote hypoxia-induced apoptosis in HeLa cells.(4) The effect of 17-DMAG combined with irradiation on the cell cycle distribution show that Hypoxic irradiation group G2/M phase cells was significantly increased compared with hypoxic group; 17-DMAG pretreatment had no effect on cell cycle, but 17-DMAG can inhibited G2/M arrest caused of different doses of radiation. Therefore, we can infer that 17-DMAG on the HeLa cells, the mechanism of radiosensitization may be related to reduced the cell cycle of G2/M arrest.Conclusions:(1)By CoCl2 treated cells, can induce expression of HIF-1α, 17-DMAG inhibits simulation of HIF1-α, the inhibition of HSP90 led to HSP70 overexpression of HeLa cells in the hypoxic environment.(2) 17-DMAG could significantly reduced the cell survival rate after irradiation in hypoxic environment, suggesting that: inhibition of HSP90 can increase the radiation sensitivity of hypoxic cells.(3) 17-DMAG could increase apoptosis rate that induced by the radiation in hypoxic cells, and the enhancement was in the drug concentration-dependent.(4) 17-DMAG pretreatment does not alter cell cycle phase distribution in hypoxia, but could reduce G2/M arrest induced by the radiation, and enhanced the radiosensitivity of hypoxic HeLa cells.