The Radiosensitization Of DNP And Its Mechanism

Background and Objective:DNP(2,4-dinitrophenol)was a protonophore or agent for uncoupling oxidative phosphorylation, getting protons to leak across the inner mitochondrial membrane,which makes ATP energy production less efficient. It has been once a diet pill. Tumor cell is a kind of cell with higher energy expenditure for its growth, proliferation and repair after injured by irradiation. So it potentially as a radiosensitizer for tumor cell.Method: MTT cytotoxicity assay and cell clone formation assay were used to detect the effects of different concentrations of DNP on the growth and proliferation of Hela and KB cells. And then the appropriate drug concentration 200μmol/L was selected to continue the next experiments. The cell clone formation assay was used to confirm whether DNP was a radiosensitizer for tumor radioterapy. The cell cycle distribution and mitochondrial membrane potential of Hela and KB cells were detected by flow cytometry. The p H in lysosome was detected by laser confocal microscopy with PDMPO lysosome yellow/blue fluorescent probe. The subcellular structures, APs and ALs of Hela and KB cells were observed by the transmission electron microscopy.Result: The growth and proliferation of Hela and KB cells were obviously inhibited by the DNP, and KB cell was more sensitive. The distinct radiosensitization of DNP was confirmed, the SERD0 of two kind of cells was 1.45 and 1.18 respectively. The proportion of G2/M phase increased significantly due to the G2/M phase arrest after DNP action 24 hours or combined with 4 Gy X-rayradiation. Mitochondrial membrane potential detection was also found that the proportion of cells with low mitochondrial membrane potential increased significantly when the cells were affected by DNP, irradiation with 4 Gy dose or combined effect with two factors, and the latter was the strongest. Under observed by transmission electron microscopy, the both autophagosome and autolysosome accumulate markedly in Hela and KB cell after DNP action. Compared with control cell, the blue fluorescence intensity in lysosome of KB cell was higher, which means the p H value changed to neutral intra lysosome. However, there was no change in lysosome of Hela.Conclusion: The radiosensitization of DNP was confirmed. The mechanisms may be included below. The G2/M phase arrest was induced by DNP. The mitochondrial function was inhibited by the mitochondrial membrane potential reduce.The p H value changed to neutral intra lysosome which enhanced the autophagy to process of cell death. DNP may be a potential radiasensitizer.