| Objective: Primary liver cancer(PLC) is a kind of the high malignant degree and low cure rate of clinical common malignant tumor, which incidence rate ranks sixth and mortality rate ranks third in malignant tumors of the world. The main clinical treatment of primary liver cancer includes operation excision, chemotherapy, radiotherapy, transcatheter arterial chemoembolization, molecular targeted therapy, Chinese medicine treatment. The clinical symptoms of early hepatocellular carcinoma(HCC) are not obvious, so most patients have been in advanced stage of HCC when they go to a doctor, who lost the chance of operation. With the development of precise radiotherapy for liver cancer, radiation therapy has become one of the main methods for the advanced hepatocellular carcinoma. Radiotherapy is equal to ionizing radiation, the cells will occur DNA injury when cells are exposed to the radiation. If the injury can not be promptly and accurately repaired, cells will be induced apoptosis. However, tumor cells have efficient repair mechanisms. The ionizing radiation can lead to DNA-double strand breaks(DSBs), there are two main ways in eukaryotes to repair the DSBs: non homologous end joining(NHEJ) and homologous recombination(HR). The DSBs induced by IR are repaired through non homologous end joining pathway. Thus, inhibition of repair pathway will promote the apoptosis of tumor cells, which can enhance the effect of radiation therapy. DNA dependent protein kinase(DNA-PK) comlex is a important factor for NHEJ. DNA-PKcs which is the catalytic subunit of the DNA-PK is the key protein among the complex. As far as today, there are various DNA-PK inhibitors which have been used in the tumor radiosensitizer experimental study. Synthetic small molecule compound NU7441 is a specific inhibitor of DNA-PK, which has shown radiosensitizing effect on the breast cancer, lung cancer, leukemia, prostate cancer cells radiotherapy study. NU7026 is a another inhibitor of DNA-PK. So far, there is no report about the relationship between NU7441 and hepatocellular carcinoma both here and abroad. This experiment will use the Hep G2 cells as the study object. To explore the radiosensitizing effect of NU7441 on the hepatocellular carcinoma Hep G2 cells and the mechanism of this effect by CCK-8, Western blot, confocal immunofluorescence, single cell gel electrophoresis and flow cytometry, which can provide the new idea for the development and application of radiotherapy sensitizer on liver cancer.Methods:1 The CCK-8 test was used to evaluate the effect of NU7441 on the proliferation of Hep G2 cells at different concentratons(0.1μmol/L, 1μmol/L, 5μmol/L, 10μmol/L) and times(12h, 24 h, 48 h, 72h).2 DNA-PKcs expression in normal liver cells LO2, liver cancer cells Hep G2 and the Hep G2 cells after 4Gy 60Coγ ray irradiation. The influence of NU7441 on the Ku70, Ku80, DNA-PKcs, p DNA-PKcs(S2056) proteins expression of the Hep G2 cells after 4Gy 60Coγ ray irradiation. The influence of NU7441 on the p DNA-PKcs(S2056) protein expression compared with the NU7026 which was the other radiosensitizer. All the above proteins were detected by Western blot.3 Through the neutral single cell gel electrophoresis and confocal immunefluorescence assay to evaluate the DNA repair on Hep G2 cells irradiated by 4Gy 60Coγ ray after different times with or without NU7441.4 The study was divided into four groups: the control group(Control), the irradiation group(IR), the NU7441 group(NU7441), and the NU7441 Combined with IR(NU7441+IR). The proportion at each phase of cell cycle after 12 h treatment was detected by flow cytometry(FCM).5 Statistical software 17.0 was used to analyse the experimental data. The experiment results indicated with mean±deviation. Two independent samples were analysed with t-test. Several independent samples were analysed with One-Way ANOVA and two groups were analysed with SNK. P<0.05 represent the statistical difference.Results:1 CCK-8 proliferation assay demonstrated that NU7441 had a inhibitory effect on the proliferation of Hep G2 cells in a time-dependent and dose-dependent manner. NU7441 had a obvious inhibitory effect at the concentration of 5μmol/L and above it.2 The expression of protein DNA-PKcs gradually increased in LO2 cells, Hep G2 cells and Hep G2 cells treated with 4Gy 60Coγ ray radiation. Although the proteins Ku70, Ku80 and DNA-PKcs in Hep G2 cells treated with 4Gy 60Coγ ray radiation had no changes compared with the irradiated group without NU7441, the expression lever of phosphorylated DNA-PKcs(S2056) declined significantly in NU7441 group and the activity of DNA-PKcs was inhibited by NU7441. Compare with NU7026 group, the expression lever of phosphorylated DNA-PKcs(S2056) reduced obviously and the activity of DNA-PKcs was inhibited significantly in NU7441 group. All the above proteins lever were determined by Western blot analysis.3 Confocal immunofluorescence results showed that the number of γH2AX foci increased firstly and then decreased in a certain time after the Hep G2 cells were irradiated. The same result was observed for cells treated with NU7441, whereas the decreased rate of the number of γH2AX foci was slower than the irradiated alone group(P<0.05).4 DNA damages were induced after exposure to 4Gy 60Coγ ray radiation on Hep G2 cells, which were detected by the neutral single cell gel electrophoresis. The tailmoment of comet cells for irradiated group were higher than the untreated group(P<0.05). The repair rate of irradiated group in the presence of NU7441 was slower than the irradiated alone group after 4 hours restore, which was indicated by the tailmonment value and the tailmoment was larger in the combined group than the radiation-treated group(P<0.05).5 The cell cycle distribution was evaluated by the flow cytometry. Comparing with untreated control group, the proportion of cells at G2/M phase were obviously higher in the IR group(P<0.05) while the NU7441 group had no statistical differences(P>0.05). The percentage of cells at G2/M phase were increased in NU7441 combined with IR when compared with IR group and the apoptosis rate was higher in combination group than the control group(P<0.05).Conclusion:NU7441 has a inhibitory effect on the proliferation of Hep G2 cells and this effect has time-dependent and dose-dependent. NU7441 can restrain DNA repair in irradiated Hep G2 cells by inhibiting the DNA-PKcs activity. The NU7441 can enhance the radiosensitization of Hep G2 cells by increasing the G2/M phase arrest of cell cycle and inducing the apoptosis.Thus, NU7441 has a potential radiosensitive effect. |
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