| Objective:Our aim is to study the effects of MSH2 gene expression, telomerase and NADPH oxidase on radiation sensitivity and to explore its mechanism. The study includes four aspects: First, the effect of X-ray and heavy ion irradiation on MSH2 expression in human Hepatoma Hep G2 cells and the biological effects; Second, inhibiting the expression of MSH2 increased radiation sensitivity of human Hep G2 cells exposed to heavy-ion irradiation; Third, the effect of telomerase inhibitors of h TERT gene on heavy ion radiation sensitivity of human breast cancer cells was explored. Forth, the research of the relationship between reactive oxygen species and the heavy-ion radiation sensitivity of Hep G2 was studied. Materials and Method:Human Hep G2 cells and breast cancer cells were exposed to different LET carbon-ion beams and X-rays. MTT method was used to study the tumor cell proliferation; Cloning experiment were used to detect radiation sensitivity of cells; Flow cytometry was used to evaluate cell cycle and apoptosis changes after irradiation; RT-PCR, Western Blotting, and confocal microscopy were used to detect MSH2 expression,telomerase subunits, PGC-1a and NOX family proteins. According MSH2 sequence provided by Genebank, Hep G2 cells were transfected by the synthetic oligonucleotides specific MSH2-si RNA and the expression of MSH2 was inhibited to detect MSH2 expression in Hep G2 cells and its role of radiosensitivity. Human breast cancer MCF-7 cells were treated with the telomerase inhibitor MST-312, telomerase activity was detected by using the corresponding kit, the amount of mitochondria, mitochondrial DNA copy number, mitochondrial membrane potential and cell senescence were detected.Last, human Hep G2 cells were treated with NADPH oxidase inhibitor DPI. Fluorescent probes DCFH-DA was used to detect ROS levels; laser scanning confocal microscope was used to observe the expression of NADPH family proteins, p47 phox subunits within the cell and NOX2 the distribution and abundance, and also explore heavy ion irradiation effect of NADPH oxidase in the radiation sensitive. Results:First, the human Hep G2 cell was exposed to low LET X-rays and high LET carbon-ion irradiation at the same dosage, and the cell survival decreased, but the high LET carbon-ions induced more cells death. MSH2 expression in human Hep G2 cell exposed to high LET carbon-ion irradiation increased at 2 h, reached the peak at 6 h, then decreased slowly, and nearly disappeared at 24 h. Speculated that time of MSH2 DNA repair function was at 6 h-12 h.Second, there was no significant difference of the colony formation rate between the three groups in the Hep G2 cell group, MSH2 non-silencing group and the si RNA group, but the tumor cells treated with silencing of MSH2 and irradiation increased cell radiosensitivity. The tumor cells treated silencing of MSH2 and irradiation, occurred G2/M phase arrest in a short time, then decreased, followed by increase of sub-G1. The tumor cells were treatedwith silencing of MSH2 and different dosage irradiation. It is shown that, with the time and dosage prolonged, p53 protein expression gradually increased and the above data had significant statistical significance compared the control group.Third, compared h TERT m RNA expression in human breast cancer MCF-7 cellsbefore and after irradiation, real-time PCR in m RNA level and Western Blotting at the protein levelconfirmed that h TERT were decreased and the difference was statistically significant, and the telomerase activity in in human breast cancer MCF-7 cells exposed to 4 Gy and 10 Gy irradiation was decreased. The combination of 2 μM h TERT inhibitors and 2-4 Gy irradiation significantly reduced survival rate. When the cells were treated byirradiation and MST-312, clone survival rate was 17%. The cells exposed to heavy ion irradiation can occur G2/M phase stop after exposure 24 h, and decreased after 48 h irradiation, but the level was still higher than control. With the addition MST-312, G2/M to G1/S transition; S phase was instead of G2/M phase stop and the G1/S cell mass is probably 2 times in human breast cancer MCF-7 cells treated by irraditioan and MST-312 than irradiation group alone. Heavy ion radiation can significantly affect telomerase activity, thereby inhibiting the normal function of mitochondria MCF-7, Further analysis showed that inhibition of telomerase activity and heavy ion irradiation combined treatment can be reduced by PGC-1a expression mediated by the aging of MCF-7.Forth, human hepatoma Hep G2 cells were exposed 4 Gy heavy ion irradiation after 12 h, intracellular reactive oxygen increased significantly. When the application NADPH oxidase inhibitors diiodo benzene(diphenylene iodonium, DPI), the active oxygen significantly reduced and a significant difference compared with the single heavy ion treatment group. There was no significant difference compared with DPI alone treatment group and control group 4 Gy heavy ion irradiated Hep G2 cells, p47 phox subunit significantly increased. After administration of inhibitors, p47 phox subunit significantly reduced in Hep G2 cells exposed irradiation. The difference was statistically significant Furthermore; NOX2 changes were consistent with p47 phox subunits. Thus, Most NOX protein family in tumor cells exposed to heavy ion irradiation after 1 h increased significantly, and was dose-dependent manner; there is only NOX2, NOX3 in tumor cells after 4 Gy radiation, which were decreased more after 1 Gy irradiation, indicating that NOX2 and NOX3 also played an important role in tumor cells after low dosage irradiation. Conclusion:First, MSH2 gene of tumor cells to overcome the heavy ion radiation resistance is an important factor and down regulated the expression of MSH2 can improve the sensitivity. p53 signal pathway plays an important role in this process.Second, Telomerase inhibitor MST 312 can enhance the radiation sensitivity of human breast cancer cell line.Third, Carbon-ion irradiation can enhance the radiation sensitivity of heavy ions by activating NADPH oxidase to produce ROS. |
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