DJ-1 Protein In The Regulation Of Lung Cancer Microenvironment And Its Relationship With Cancer-Prevention By EGCG

OBJECTIVEBefore the formation of lung cancer experienced a series of morphological and molecular changes, Bronchoalveolar lavage from normal epithelial cells turn into various morphological features of lung cancer is a complex and lengthy multi-stage multi-step carcinogenesis,and Bronchial epithelial cell proliferation/differentiation control disorders are among the root causes. In the cancer-causing agents such as benzo (a) pyrene compounds for lung cancer process to promote tumor growth, These normal cells, such as the lung bronchial epithelial cells, From quantitative to qualitative change is imperceptible to the time of malignant transformation. Studies show that the polyphenols and other antioxidants could antagonize the effect. People dedicated to finding prevention strategies and excellent chemical substances, interfere with metabolism of carcinogens, do not make it into a complete carcinogen activation or inhibition,prevention of carcinogen DNA binding and cell, Inhibiting cancer or precancerous lesions evolving into its reversal to normal cells. In the previous studies, study confirmed 3,4-benzo (a) pyrene in oil injection percutaneous pulmonary rat lung tumor model building is a simple and reliable animal model of lung cancer method comfirmed, one type of tumor pathology mainly in non-small cell lung cancer. In our study, By unpaired two-dimensional gel electrophoresis to obtain tumors in rats 3,4-Benzo (a) pyrene injection into the tumor adjacent lung tissue and adjacent lung tissue in rats differentially expressed protein profiles, To screening in lung tissue microenvironment and 3,4-benzo (a) pyrene induced lung cancer associated with primary protein points. These proteins include the DJ-1, including nine particle, We believe that with 3,4-benzo (a) pyrene in lung tumors after injection of immature rats inhibited the formation of lung cancer-related proteins. Tumor microenvironment suggest DJ-1 protein can serve as a target for cancer chemoprevention, Chemoprevention against cancer-causing factors in the early control, micro-environment through a variety of cells and cytokines affect tumor phenotype intervention. Many targets the tumor microenvironment, Selecting the DJ-1 as a molecular target and find its place cells is the first step, Understand the micro-environment, changes in the level of protein molecules on tumor cells, To verify the target protein and localization of cells and with green tea (polyphenols) the relationship between lung cancer prevention.METHODS AND RESULTSThe study was carried out in three parts.In the first part,80 female SD rats were randomized into two groups(each group has 40 rats).40 rats(group A)were treated with 3,4-benzopyrene-corn oil(2mg 3,4-benzopyrene,dissolved in 0.2mL corn oil);meanwhile 40 rats(group B)were treated with corn oil only.The injection were given in every 2 weeks for 8 weeks through left middle-chest percutaneous puncture.All rats received deionized water as their sole source of drinking fluid.After 32 weeks following the first injection,the rats were killed.In group A,the rats those developed lung tumors were separated into group C and the others were separated into group D.Portions of tumor-adjacent lung tissues(group C)and 3,4-benzopyrene injection-adjacent lung tissues(group D) which might be associated with the carcinogenesis process of lung cancer. Verified by immunohistochemistry in lung tissue of lung cancer proteins inhibit the expression levels of DJ-1 and the main place cells. DJ-1 targeted siRNA transfection of human bronchial epithelial cells, from the mRNA level and protein level of DJ-1 after transfection reduced the effect of gene expression, detected by the DJ-1 HBE cells transfected with targeted siRNA on cell proliferation and apoptosis. In the second part, the establishment of human bronchial epithelial cells and lung adenocarcinoma cells were co-cultured model. Divided into three groups, group A of tumor cell culture as a separate group or the control group, group B untreated epithelial cells and lung adenocarcinoma cells were cultured group that controls, group C transfected with the siRNA targeting DJ-1 and lung epithelial cells after co-culture group of cancer cells that the experimental group. Proliferation of cell growth curve of each group and the level of cell cycle and apoptosis in three groups of nude mice experiments. In the third part, construction of 3,4-benzo (a) pyrene in oil percutaneous injection of rat lung tumor model of lung,60 female SD rats were randomized into two groups(each group has 30 rats),one for green tea(group A),and the other for control(group B).All rats were treated with 3,4-benzopyrene-corn oil(as described in Experment 1).16 weeks, rats were killed and protein from the mRNA levels were compared in rats of benzo (a) pyrene injected next to the lung tissue of DJ-1 gene expression differences. With the active ingredients of green tea (EGCG) treatment of bronchial epithelial cells, mRNA and protein levels from the determination of epithelial cells in DJ-1 gene expression in time and dose-response relationship. Successfully constructed and 3,4-benzo (a) pyrene in oil percutaneous injection of rat lung tumor model of lung, immunohistochemistry showed that DJ-1 was mainly expressed in bronchial epithelial cells, and the expression of significant differences between groups, immature lung tumor tissue levels of tumor was significantly lower than that. Location benzo (a) pyrene in the process of promoting lung cancer, lung tissue micro-environment in the anti-cancer effect of bronchial epithelial cells in DJ-1 protein target. DJ-1 HBE cells transfected with siRNA targeting, effective from the mRNA level and reduced levels of DJ-1 protein expression, a certain concentration of HBE cells transfected with the value-added and no significant effect on apoptosis. The second part of the cell experiments, group C, lung cancer cell proliferation was inhibited, the level of cell cycle arrest and apoptosis increased over the previous 2 groups. Nude mice in each group experiment indicated, group C and group A of tumor formation time and tumor volume differences, group C, the average tumor size of less than group A of indices. In the third part, group A of benzo (a) pyrene injected next to the lung tissue levels of DJ-1 gene mRNA and protein levels were significantly lower than group B. Active ingredients of green tea (EGCG) effectively reduced bronchial epithelial cells the expression of DJ-1 protein levels, and a dose and time effect.CONCLUSIONBenzo (a) pyrene in rat lungs injected construct during the primary lung tumor, DJ-1 protein in the bronchial epithelial cells may be inhibited in lung tissue micro-environment of the target protein of lung cancer. Reduced bronchial epithelial cells of the DJ-1 protein expression, On the co-cultured lung cancer cell proliferation and tumorigenesis was inhibited. Epithelial cells, the protein level may be involved in anti-tumor promotion agents (tea polyphenols) against cancer-promoting agent of lung cancer prevention.