| Background and objective:Lung cancer, mainly originated in the bronchial epithelium and sometimes referred to as bronchogenic lung carcinoma, is one of the most common primary malignant pulmonary tumors. According to the World Health Organization (WHO) statistics, the incidence of lung cancer each year has been more than1.3million and lung cancer has become the first killer of men and women in the global with the number of mortality from lung cancer up to1.1milion. Radiotherapy, one of the traditional treatments of patients with lung cancer, plays an important role in tumor local control and mitigating symptoms caused by metastatic lesions. The generation of tumor cell resistance to radiation, however, often leads to long-term survival of patients with lung cancer very low. Therefore, there is an urgent need to develop more effective anti-cancer modalities. Studies on radiobiology in recent years have shown that cells at different cell cycle phases have different sensitivity to radiation. Therefore, more focus on the tumor radiation sensitizer was also transferred to the regulation of cell cycle. It has now been confirmed that Ataxia Telangiectasia mutated (ATM) protein plays a crucial role in the regulation of cell cycle which is regulated by corresponding cyclin. Unmethylated CpG motifs-containing oligodeoxy-nucleotide (CpG ODN) is able to stimulate the immune system, thus enhancing the body immunity to produce a variety of anti-tumor effect. It has been reported in recent years that CpG ODN can directly inhibit the proliferation of tumor cells and induce tumor cell apoptosis. In previous experiments, we observed that CpG ODN7909with the final concentration of10μg/ml inhibited proliferation activity of human lung adenocarcinoma A549cells. Therefore, objective of this study is to further explore both the effect of CpG ODN7909on the sensitivity of human lung adenocarcinoma A549cells to radiation and potential role of ATM kinase in this process.Methods:Human lung adenocarcinoma A549cells were used as experimental subject and randomly divided into6groups followed by Control group, CpG group, X-ray group, CpG+X-ray group, ATM-siRNA+CpG+X-ray group and Control-siRNA+CpG+X-ray group. Among them, Control group was dealt without any treatment. CpG group was dealt with10μg/ml CpG ODN7909for24hours. X-ray group was treated with10Gy doses of irradiation. CpG+X-ray group was dealt with10μg/ml CpG ODN7909for24hours and then irradiated with10Gy doses of X-ray. ATM-siRNA+CpG+X-ray group and Control-siRNA+CpG+X-ray group were transf-ected with ATM-siRNA and Control-siRNA for6hours, respectively, and then were continually cultured for another18hours before CpG being administered. In colony formation assay, colony-formation numbers in each group were counted through a microscope at10days after X-ray irradiation. In both apoptosis and cell cycle experiments, cells in each group were analyzed by flow cytometry at24hours after X-ray irradiation. In Wetern-blotting experiments, samples in each group were collected at3hours after X-ray irradiation.Results:The difference of colony formation, apoptosis and cell cycle distribution between Control group and CpG group was not significante. Compared to the situation under X-ray irradiation alone, decreased cell clonogenic survival, prolonged G2/M arrest, and increased cell apoptosis were observed after the combined treatment with CpG ODN7909and X-rays, and the difference was statistically significant. Compared to ATM-siRNA+CpG+X-ray group, there were increase of colony-formation numbers and decreased numbers of apoptosis and cells at G2/M phase in CpG+X-ray group, and there was significant difference between two groups. Compared Control-siRNA+CpG+X-ray group with CpG+X-ray group, the difference was no statistical significance in terms of colony formation, apoptosis and cell cycle distribution. It was shown in Western-blotting tests that there were no changes in the expression of ATM, Chk2and p53protein in each group in addition to ATM-siRNA+CpG+X-ray group. Moreover, the phosphorylation on ATM, Chk2, and p53was not observed in both Control group and CpG group. Compared to X-ray group, there was increased phosphorylation on ATM, Chk2, and p53in CpG+X-ray group. Compared ATM-siRNA+CpG+X-ray group with Control-siRNA+CpG+X-ray group, it was shown that the expression of ATM was specifically inhibited by ATM-siRNA, while the entire transfection system had no effect on the expression of proteins such as ATM, Chk2and p53.Conclusion:CpG ODN7909might in vitro enhance the radiosensitivity of human lung adenocarcinoma A549cells via enhancing proliferation inhibition, apoptosis and G2/M phase arrest induced by X-ray irradiation, which was perhaps associated with increased phosphorylation of ATM kinase. |
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