Proteome Screening And Expression Analysis Of Proteins Related To Radiation-induced Carcinogenesis

Carcinogenesis is the most serious late effect of ionizing radiation, but the mechanism of ionization radiation induced carcinogenesis remains to know. As the atomic power used widely and widely, the radiation effect on health becomes more and more concerned by people than before. To elucidate the mechanism of induced radiation carcinogenesis will help us efficient prevention and treatment for carcinomas. Carcinogenesis is a very complicated network regulation process. Lots of genes and proteins are involved in. Studying on a single gene or protein can not completely elucidate the mechanism of carcinogenesis. On this consideration, gene chips were widely used in cancer research. Through this technology, we can easily get the profile of gene alteration between normal and tumor tissue. But there are many conflicting consequences between the abundance of mRNA and proteins level. Because genes performed their functions through proteins, studying on the protein profile is very necessary and important in cancer research. The high-throughput proteomic technology will conduce to comprehensive understanding of carcinogenetic processes.Objective and Methods:In this study, we used 2-D protein electrophoresis technology to obtain protein alteration profile between human lung cancer cells induced by gamma-ray and normal cells. We selected some proteins which may be involved in radiation carcinogenetic process, then observe its expression level in different stage of carcinogenesis. We try to find some proteins that may play an important role in the process of carcinogenesis induced by radiation, and observed the contribution of these proteins in the different stage of carcinogenesis.We selected human bronchial epithelial cell line (BEAS-2B) as the cell model. Gamma-ray (3 times, total dose: 22Gy) induce BEAS-2B cells transforming to malignancy after 45 population doublings. The malignant level of cells was detectedby nude mice tumorigenesis test.The different expression proteins between normal and cancer cells were obtained after 2-D electrophoresis and been identified by MALDI-TOF MS technology. Four proteins (a-enolase, Glutathione peroxidase K dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 and Peroxiredoxin 1) were selected as research targets from these different expression proteins. Western blot and fluorescence quantitative PCR were used to observe the expression level of these four proteins in the different stage of carcinogenesis.Results:lxlO7 BR22P35 (P35: passage 35;R22: 22Gy y-ray), BR22P45 and BR22P5o cells were subcutaneouly injected into cervical part of nude mice. After 50 days observing, BR22P50 cells were successfully transplanted into two nude mice. Transplanted tumor tissue was identified as human bronchial epithelial cell cancer by pathology and immunohistochemistry.The sliver stained gels of 2-D electrophoresis were scanned at 256 grayscale and 8 bits degree. After analyzed by ImageMaster 2D Elite 3.10, 1102±29 spots in BNP50 cells and 1058±33 spots in BR22P50 cells were detected. Normalized gel of BNP50 cells was selected as reference gel. After Spots matching with other gels, the unstably developed difference expression spots between the four times electrophoresis was precluded from analysis. There are 82.89% and 79.58% of the spots could be matched for BNP50 and BR22P50 gels. Difference analysis showed that 59 credible protein spots were differentially expressed between BNP50 and BR22P50 cells. In these spots, fourteen protein spots were only expressed in BR22P50 cells, fifteen were only expressed in BNP50. In other 30 spots, seven spots were higher expressed and 23 spots were lower expressed in BR22P50 cells.Using MALDI-TOF MS technology, twenty-six spots were identified by PMF, including enzymes, structure proteins, cell signal proteins, binding proteins, metabolism related proteins, some unknown function proteins and ploy-peptides.Western blots results: ? The expression level of EN01 in BR22P35 (middle stage of irradiation induced carcinogenesis) and BR22P50 (advanced stage of irradiation induced carcinogenesis) cells was significantly higher than in BNP50 (normal cells), BR22P20 (earlier stage of irradiation induced carcinogenesis) and BRsPi(acute stage of radiation) cells (P<0.01). There is no statistical difference in expression of ENO1 among BNP50, B6R1 and BR22P20 cells (P>0.05). The expression level in BR22P35 cells were analogous to BR22P50 (P>0.05). ? The expression level of Prx I was found to be correlated with the radiation dose and the post-radiation time, and it was increased with the elevation of radiation dose and post-radiation time. There was significant difference the in the expression of Prx I between BR22P50 and BNP50 cells (PO.01). (3) The expression level of Dyrk2 in BNP50 and BRsPi cells was significantly higher from BR22P20, BP22P35 and BR22P50 cells (PO.01). No significant difference was found in expression between BR6P1 and BNP50 cells (P>0.05) and the same among BR22P20, BR22P35, BR22P5o cells (P>0.05). ? The expression level of GPXl in BNP50 and BR6P1 cells was significantly higher from BR22P20, BR22P35 and BP22P50 cells (PO.01), and the expression level in BR22P20 was higher than BR22P35 and BR22P50 cells (P>0.05). No statistical difference was observed in expression between BRsPi and BNP50 cells (P>0.05). The expression level in BR22P35 cells was similar to BR22P50 (P>0.05).FQ-PCR results: ? The mRNA quantities of ENO1 decreased gradually in BR22P50, BR22P35, BR22P20 cells, and the difference was statistically significant (P<0.05). There is no statistical difference between BNP50 and BRePi cells (P>0.05) and the same between BRePi and BR22P20 cells (P>0.05). ? The mRNA quantity of Prx I was increased with the increment of radiation dose and post-radiation time. There was significant difference in mRNA quantity among each groups (PO.05) except the difference between BR22P50 and BR22P35 cells (P>0.05). (3) The significant difference in mRNA quantity of Prx I was found among each groups (P<0.05) except that between BR22P50 and BR22P35 cells, and the mRNA quantity of Prx I increased with the rise of radiation dose and post-radiation time. @ The mRNAquantities of GPX1 decreased gradually in BNP50, BR^Pi, BR22P20 and BR22P35 cells. The difference was statistically significant (PO.01). No statistical difference was observed in mRNA quantities between B22R35 and B22R50 cells (P>0.05).Discussing:ENO1 is an enzyme in the glycolytic pathway catalysing the formation of phosphoenolpyruvate from 2-phosphoglycerate. The alternative translated product of ENO1, known as MBP-1, acts as a negative regulator of the c-myc oncogene, making the ENO1 gene a candidate as a tumour suppressor gene. Katarina found that the expression of EN01 was decreased in some tumor cells, and transfection of ENO1 cDNA into lp-deleted neuroblastoma cell lines causes reduced number of viable cells over time compared to a negative control and it induces apoptosis. But there are some research showed different results. Racz A. observed a higher expression of EN01 in squamous cell lung carcinoma. Zhang D. found that ENO1 was up-regulated in HER-2/neu-positive breast tumors. In this study, ENO1 was found to be up-regulated in BR22P50 and BR22P35 cells, but not in BR22P20 and BRgPi. It demonstrated that ENO1 may not be involved in the earlier stage of carcinogenesis induced by gamma-ray, and there is possibility that the enhancement of glycolytic pathway contributes to the up-regulation of ENO1. Considering the known function of ENO1 as a negative regulator of c-myc expression, c-myc may not be involved in radiation induced carcinogenetic process.Prx I is a member of Peroxiredoxin anti-oxidation protein family. Prx I is an inducible protein that it can be up-regulated by radiation oxidative stress, and the increment of Prx I is correlated with the radiation dose and the post-radiation time. Prx I was found be up-regulated in some tumors including lung, breast and liver cancer. Our research got the same results and it may be caused by the increase of free radicle and hyperoxide generated by radiation. Crowley Webe observed that Prx I can repress the apoptosis of tumor cells induced by Cisplatin. Chen MF reported that the low expression of Prx I can led to growth retardation and decreasing radiation resistance in tumor cells. All of these were demonstrated that Prx I may plays animportant role in tumor growth and anti-apoptosis of tumors.Dyrk2 is a member of Dyrk (Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase) family. The Dyrk family is distinguished from other protein kinase families by their ability to phosphorylate both Ser/Thr and Tyr substrates (dual-specificity). Several researches reported that Dyrk2 was highly expressed in lung and alimentary system adencarcinoma cells. Park GH found that the mRNA of Dyrk2 was down-regulated in human neuroblastoma cells treated by anticancer agent 8-Cl-cAMP. It implied that Dyrk2 may play a important role in carcinogenetic process. However, we got a completely contrary result that Dyrk2 was significant down-regulated in human bronchial epithelial cell cancer induced by gamma-ray in our research. Potential reasons were considered as follows: ? The up-regulation of Dyrk2 was only found in adencarcinoma and few reports was found in squamous carcinoma till now. We suspected that the expression of Dyrk2 is specific in adencarcinoma cells. (2) The factor of radiation may contribute to the down-regulation of Dyrk2. (3) Dyrk2 dos not play an important role in the carcinogenesis, and the signal pathway network negatively regulated Dyrk2 during the radiation induced carcinogenetic process.Cellular glutathione peroxidase-1 (GPXl) has been widely considered to be a major antioxidant enzyme, and GPXl is the first identified and the most abundant selenoprotein in mammals. Several researches showed that the reduction of tumor incidence, such as colon carcinoma, was correlated with the selenium level in body, and the antitumous effect of selenium may be mediated by GPXl. In recent researches, GPXl has been implicated in the development of cancer of the head and neck, lung, and breast, in part because of allelic loss at the GPXl locus. Yajun Hu reported that the allelic loss of GPXl was found both in colorectal tumor tissue and in adjacent histopathologically normal tissue. The results demonstrated that allelic loss of GPXl may be an early event in tumorigenesis. In our research, we also found that GPXl was significantly down-regulated in BR22P20 cells, and then decreased in BR22P35 and BR22P50 cells continuously. Taken together, these date indicated that GPXl plays animportant role in the initiate stage of carcinogenesis.