The Experimental Study Targeting On APE1/Ref-1 In Tumor Radiotherapy

With the development of radiation oncology, the radiotherapy plays an important role in the tumor treatment, and has become one of the most commonly approaches. Some tumors can be cured by the radiotherapy, but the efficacy of radiotherapy on some other advanced tumors is not satisfied, meanwhile, the incidence of radiation-induced injuries is high. In order to solve the problem of radioresistance of tumor cells, improve the cure rate of radiotherapy and control the radiation-induced injury, the clinical doctors and scientists have sought out a lot of new methods. In all these research, the radiotherapy combined with gene therapy against tumor, also known as tumor gene radiotherapy, were considered as a promising stategy for future tumor treatment.The apurinic/aprimidinic endonuclease/redox factor-1(APE1/Ref-1) is a bifunctional biomacromolecule. APE1/Ref-1 plays a vital role in DNA damage repair and oxidation reduction or redox signal transmission process. The abnormal expression, distribution and functional changes of APE1/Ref-1 are not only associated with anti-apoptosis and tumor progression, moreover, the experimental modification of its activity could change the response of cells and organism to DNA damage agents. Wild-type p53 plays a key role in cell cycle control and apoptosis, and the mutation of p53 gene is correlated with the carcinogenesis and progression of tumor. Based on these results and conclusion, we proposed that APE1/Ref-1 might be a molecular target in tumor therapy and prevention of radiation-induced injuries. In order to prove the assumption, we examined the inhibitory effect of chimeric adenoviral vector Ad5/F35 carrying human APE1/Ref-1siRNA and recombinant adenovirus carrying wild-type p53 combined with radiotherapy on human hepatocellular carcinoma cell proliferation in vivo. We also explored the action mechanism of Ad5/F35-APE1/Ref-1siRNA combined with rAd-p53 on radiosensitivity of human hepatocellular carcinoma cells. Then we detected the expression of APE1/Ref-1 in radiation-induced lung injury by immunohistochemistry and western blot. Mice lung tissues were transferred by the plasmid pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 via the tail vein by injection to overexpress APE1/Ref-1, and we investigated the protective effect of APE1/Ref-1 on radiation-induced lung injury in mice model. The study will present a new strategy for the combination of radiotherapy and gene therapy against tumor and the control of radiation-induced injury.Objective1. To investigate the effects of Ad5/F35-APE1/Ref-1siRNA on radiosensitivity of human hepatocellular carcinoma and its synergistic effect with rAd-p53 in nude mice model.2. To investigate the expression of APE1/Ref-1 in mice model of radiation-induced lung injury and explore its correlation with radiation-induced lung injury.3. To observe the lung tissues of mice transferred by the plasmid pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 injected via tail vein and investigate the protective effect of APE1/Ref-1 on radiation-induced lung injury in mice model.Materials and Methods1. Study of the effects of Ad5/F35-APE1/Ref-1siRNA and rAd-p53 on radiosensitivity of human hepatocellular carcinoma cells in tumor-bearing nude mice model: Nude mice hepatocarcinoma xenograft model was established with SMMC-7721 human hepatocellular carcinoma cell line. When the tumor volumes reached 40~50mm3 on the seventh to tenth day after inoculation of SMMC-7721 cells, 28 mice were randomly divided into seven groups: control group, the radiotherapy group, Ad5/F35- APE1/Ref-1siRNA group, Ad5/F35-APE1/Ref-1siRNA plus radiotherapy group, rAd-p53 group, rAd-p53 plus radiotherapy group and Ad5/F35-APE1/Ref-1siRNA combined with rAd-p53 plus radiotherapy group. The tumor volumes were measured and the tumor growth curves were drawn. The histology of specimens was examined by HE stain. The expression of Ki-67 protein was detected by immunohistochemistry. Apoptosis index was detected by TUNEL technique.2. Expression of APE1/Ref-1 and its significance in mice model of radiation-induced lung injuryThe mice model of radiation-induced lung injury was established. The histology of specimens was examined by HE stain. The expression of APE1/Ref-1 in the lung tissues of mice of radiation lung injury was detected by immunohistochemistry and western blot methods. We also explored the correlation of APE1/Ref-1 with the radiation-induced lung injury. The study will present a new strategy for the control of radiation-induced injury by gene therapy.3. To investigate the protective effect of APE1/Ref-1 over expression in mice model of radiation-induced lung injury:The mice omni-thorax irradiation pulmonary model was established. We observed the lung tissues of mice transferred by the plasmid pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 injected via the tail vein and investigated the protective effect of APE1/Ref-1 over-expression in mice model of radiation-induced lung injury. The histology and collagen of lungs was examined by HE stain and Masson-trichrome separately, the expression of APE1/Ref-1 protein was detected by immunohistochemistry and western blot. Enzyme linked-immuno-sorbent assay (ELISA) was applied to detect the TGF-β1 in serum.Results1. Study of the effects of Ad5/F35-APE1/Ref-1siRNA and rAd-p53 on radiosensitivity of human hepatocellular carcinoma cells in tumor-bearing nude mice model:Ad5/F35-APE1/Ref-1siRNA suppressed significantly the expression of APE1/Ref-1 protein, and rAd-p53 enhanced significantly the expression of p53 protein in vivo. The tumor growth inhibition ratio, PI and AI in Ad5/F35-APE1/Ref-1siRNA combined with rAd-p53 plus radiotherapy group were significantly different, compared with the control group and other therapy groups (p<0.01).2. Expression of APE1/Ref-1 and its significance in mice model of radiation-induced lung injuryThe high expression of APE1/Ref-1 was detected in nuclei of epithelial cells and endothelial cells in normal mice lung tissues. During the process of radiation-induced lung injury in mice model, the changes of three types of APE1/Ref-1 positive staining were observed; the expression of APE1/Ref-1 protein was detected in nuclear, cytoplasmic and mixed type. The expression of APE1/Ref-1 protein began to increase 1d after irradiation, reached its peak value on 3d, it was 2.2-fold compared with control group, and then decreased at 7d~56d. The expression of APE1/Ref-1 protein was significantly increased after 3d post-irradiation, and then was significantly decreased at 56d, and was 0.2-fold compared with control group.3. To investigate the protective effect of APE1/Ref-1 over expression in mice model of radiation-induced lung injury:After transfection of pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 in vivo, the expression of APE1/Ref-1 protein in epithelial cells and endothelial cells of mice lung tissue was increased. It was 2.3-fold and 2.8-fold compared with lung tissue epithelial cells and endothelial cells of untransfected mice, separately. After single irradiation at omni-thorax of the mice with 20 Gy of 8 MV X irradiation, the pathological study showed marked lung injury in the radiation group while only slight hyperemia, hemorrhage, exudation and the forming of collagen in prevention and treatment groups by HE stain and Masson-trichrome. The levels of TGF-β1 in the serum increased 7d after irradiation, and then increased at 14d~56d, it reached its peak value at 56d, there was significant difference comparaed with the control group(P<0.01), and also reduced in two prevention and treatment groups and the radiation group(p<0.05). However, there was no significant difference between two prevention and treatment groups (p>0.05).Conclusion1. Ad5/F35-APE1/Ref-1siRNA and rAd-p53 combined with radiotherapy can significantly inhibit the growth and enhance radiosensitivity of human hepatocellular carcinoma cells in tumor-bearing nude mice model. Therefore, the gene therapy of Ad5/F35-APE1siRNA combined with rAd-p53 may be a promising approach to treat human hepatocellular carcinoma in the future.2. Irradiation can up-regulate the expression of APE1/Ref-1 protein, while APE1/Ref-1 has the characteristic translocation from nucleus to cytoplasma. The expression of APE1/Ref-1 protein was increased at first and then decreased, which suggests that the APE1/Ref-1 may play a very important role in the development of radiation-induced lung injury. 3. After transfection of pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 in vivo, the expression of APE1/Ref-1 protein in epithelial cells and endothelial cells of mice lung tissue was increased.4. APE1/Ref-1 could show protective effects on the radiation-induced lung injury and the mechanism may be through repairing DNA lesions, blocking the production of reactive oxygen species and inhibiting the activity of redox regulation, decreasing the expression of TGF-β1, reducing the forming of collagen so as to ameliorate the radiation-induced lung injury.