Proteomic Analysis In The Process Of Ochratoxin A-induced Malignant Transformation In GES-1 Cells

Objective: Ochratoxin A(OTA), a mycotoxin mainly produced by ubiquitous Aspergillus and Penicillium, is one of the most abundant food-contaminating mycotoxins, particularly in cereals, grains and beans, as well as coffee, grape juice, beer and bread. OTA exhibits a wide range of toxic activities including nephrotoxicity, hepatotoxicity, neurotoxicity, immunotoxicity, teratogenecity, mutagenicity and carcinogenicity. Accordingly, OTA had been classified as “possibly carcinogenic to humans” by the International Agency for Research on Cancer since 1993.Our previous epidemiological study have showed that Zanhuang County of Hebei Province is a high incidence area of gastric carcinoma in China, where the average annual mortality rate of gastric cancer has been over 59 per 100,000 population. Our on-site investigations further confirmed that the OTA contamination in wheat was quite common in this area, and the highest content reached to 2.41 μg/kg which was significantly higher than that of provisional tolerable weekly intake(PTWI, 100 ng/kg) defined by the Joint FAO/WHO Expert Committee on Food Additives(JECFA). The high level of OTA contamination in wheat might be related to the high incidence of gastric carcinoma in rural area of China. Our previous study showed that short-term exposure OTA could induce oxidative DNA damage, cell cycle arrest and DNA repair disorder. Subsequently, we also found that long-term OTA treatment could increase the migration, invasive abilities and induce malignant transformation of GES-1 cells. However, there were no available data regarding the possible mechanism in the process of OTA-induced malignant transformation of GES-1 cells up to now.In this study, we explored the effects of key molecules in the process of OTA-induced malignant transformation in GES-1 cells. Firstly, we explored the change of protein spectrum after long-time exposure of OTA in GES-1 cells and screened out the significantly change of proteins by two-dimentional gel electrophoresis. Furthermore, on the basis of the above research results, we further explored the effect of the differential proteins in GES-1T cells by Western blot, Real-time PCR and Immunohistochemistry. The purpose of this study was to hunt the key events and possible mechanism of glycolysis pathway and to further explored the early events in OTA-induced malignant transformation in gastric epithelium cells. The study will enrich and deepen the understanding of OTA biological effect, and provide a new research field for revealing the possible mechanism of gastric carcinogenesis in the OTA-exposed populations.Methods: 1 Cell culture and treatmentGES-1 cells were routinely cultured in DMEM supplemented with 100U/ml penicillin, 100U/ml strep-tomycin, and 10% fetal bovine serum(FBS) under the condition containing 5% CO2, as well as 95% air at 37℃. GES-1 cells in logarithmic growth phase were randomly divided into control group and the experimental group. The experimental group were then treated with 2.5μm/L OTA for 72 h, once a week. Repeated this process 40 times and collected cells for further research. Our earlier research had found that long-term OTA treatment could induce malignant transformation of GES-1 cells. 2 Two-dimentional gel electrophoresisWe extracted protein samples of GES-1 cells underwent malignant transformation, separated samples by two-dimensional electrophoresis, and used coomassie brilliant blue staining to show them. Next, we scaned gels, inputed the data into the computer and analyzed results using Image Master 6.0. Then cut protein spots which were different between each other in gels out, and we got peptide mass fingerprintings using trypsin solution and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS. 3 Western BlottingThe expressions of CAPZA1, Annexin A3, GOLPH3 L and TRX at protein level in OTA-GES-1T cells were determined by Western blot. 4 Real-time PCRThe expressions of CAPZA1, Annexin A3, GOLPH3 L and TRX at m RNA level in A549 cells were determined by Real time PCR. 5 IHCThe OTA-GES-1T cells were inoculated in nude mice, and the tumor tissues were removed and fixed after 16 weeks. The expression of Annexin A3 was determined by IHC. 6 Statistical analysisThe results were presented as means ± SD. Data were analyzed using one-way analysis of variance(ANOVA) analysis. Values were considered statistically significant when P<0.05.Results: 1 Screening out differently expressed proteins related with OTA-induced malignant transformation of GES-1 cellsCompared with parallel-control cells, 58 different protein spots were found in OTA-GES-1T cells. Among them, 9 proteins’ expression levels were increased and 5 proteins’ expression levels were decreased. Using MALDI-TOF-MS, we had successfully identified 4 priotein spots, that were CAPZA1, Annexin A3, GOLPH3 L and TRX. Their functions were respectively relevant to cytoskeletal regulation, oxidation reduction and signal transduction, etc. Among them, expression levels of Annexin A3 and GOLPH3 L were significantly raised in OTA-GES-1T cells; however, that of others were conversed. 2 The expression levels of CAPZA1, Annexin A3, GOLPH3 L and TRXThe expression levels of Annexin A3 and GOLPH3 L were raised in OTA-GES-1T cells compared with the control group by Western blot and Real time PCR(P<0.05), however, the expression of others were down(P<0.05). Among them, Annexin A3 had the most significant difference.3 The expression of Annexin A3 in tumorigenic specimensAnnexin A3 were both highly expressed at protein and m RNA level in tumorigenic specimens(P<0.05). So we could got an conclusion that Annexin A3 played an important role in the progress of OTA induced malignant transformation of GES-1 cells.Conclusion: 1 We had screened out the significantly change of proteins: Annexin A3, CAPZA1, GOLPH3 L and TRX in the progress of OTA induced malignant transformation of GES-1 cells by the proteomics technology. 2 The high expression of Annexin A3 and GOLPH3 L may play an important role in the progress of OTA induced malignant transformation of GES-1 cells. 3 The low expression of CAPZA1 and TRX may play an important role in the progress of OTA induced malignant transformation of GES-1 cells. 4 The reason that long-term OTA treatment could induce malignant transformation of GES-1 cells may be relevant to the changes of cytoskeleton conformation, disorders of oxidation reduction and activations of some oncogenes.