| Objective: Ochratoxin A (OTA) is a mycotoxin produced by ubiquitousAspergilli, mainly by Aspergillus ochraceus and also by Peniciliumverrucosum that may contaminate a broad variety of foodstuff, such as grains,vegetables, coffee, dried fruits, beer, wine and meats. It is also frequentlydetected in human blood and animal blood and tissues. OTA has been shownto be nephrotoxic, hepatotoxic, teratogenic, immunotoxic, genotoxic andcarcinogenic to animals and has been classified as a possible carcinogen tohuman by the International Agency for Research on Cancer (IARC) in1993.Studies showed that OTA induced DNA strand breaks in several cell lines,such as cultured human primary kidney cells, rat and Chinese hamster ovaryfibroblast cells. Our previous study demonstrated that OTA induced DNAdouble-strand breaks (DSBs) and evoked cell cycle arrest at G2phase inhuman gastric epithelium (GES-1) cells.Human Rad51is the key protein in homologous recombination (HR)repair pathway of DSBs with homology to the Escherichia coli protein, RecArecombinase. The molecular weight of Rad51is37kDa, and the “RAD51paralogs” encoded by the Rad51B, Rad51C, Rad51D, XRCC2and XRCC3genes participate in late stages of homologous recombination. Rad51is astrand transferase that forms a nucleoprotein filament by polymerizing ontosingle-stranded DNA at the processed DNA break. The nucleoprotein filamentpromotes DNA strand exchange with the undamaged homologous chromatid.The abnormal expression of Mammalian Rad51, which could directly affectthe function of HR, may cause chromosomal instability and even lead to tumorformation.UV irradiation, ionizing radiation (IR), or mitomycin C (MMC) couldincrease the expression of Rad51by promoting the Rad51protein to relocalize and concentrate in nuclear foci of various cells, including MCF-7breastcancer cells, nasopharyngeal carcinoma (NPC) cell lines and colonic epithelialcells. There is still some controversials on the relationship between Rad51expression and cytoxicity (such as DNA damage, etc) by different cytotoxicagents. The hepatocarcinogen aflatoxin B1(AFB1) exposure increases Rad51expression to promote gene mutation in yeast Saccharomyces cerevisiae.While at the same time, different results were also found in several otherstudies. Studies showed that some chemotherapy drugs and oxygen deficitcould reduce the expression of Rad51to enhance cytoxicity. Up to now, theeffect of OTA on the expression of Rad51and its putative role in theOTA-induced cytotoxicity in normal human cells is unclear. To explore theputative role of OTA on Rad51expression and OTA-induced cytotoxity inGES-1cells, the current study first evaluated the expression of Rad51, andthen, explored its effect on DSBs and G2phase arrest induced by OTA inGES-1cells and the putative mechanisms of Rad51abnormal expression. Thisstudy is to extend our previous study by elucidating the detailed molecularmechanism of OTA-induced DSBs repair and provide insight into the possiblerelationship between OTA exposure and gastric carcinogenesis.Methods:1Cell culture and treatmentThe cells were routinely cultured in DMEM supplemented with100U/mlpenicillin,100U/ml strep-tomycin, and10%fetal bovine serum (FBS), under5%CO2/95%air. The cells were then treated with solvent (methanol) alone orwith various concentrations of OTA for24h or with same concentrations ofOTA for6-24h. OTA was diluted in methanol and added to the culture flasksto obtain the final concentration of5,10and20μM respectively. Control cellswere incubated for the same time with methanol a concentration of0.08%(ourprevious study showed that at the concentration of0.08%, methanol showedno potential effects on the pro-liferation and cell cycle distribution of GES-1cells by MTT and Flow cytometry).2P38and ERK inhibitor treatment To Investigate the effect of MAPK Inhibitors on OTA-induced Rad51down-regulation, confluent cell cultures were preincubated for0.5h with oneof the following inhibitors before the addition of20μM OTA:10μMSB203580(p38inhibitor), or10μM PD98059(ERK inhibitor).3Transfection with small interfering RNA or Rad51vectorCells were reversely transfected with siControl or siRNA targeting p53ata concentration of100pmol respectively using Lipofectamine2000(Invitrogen)according to the manufacturer’s instructions. And24h post transfection,medium was discarded, cells were washed with PBS and subsequently treatedwith20μM OTA for24h or not. Afterwards, cells were assayed by Westernblot and Flow cytometry.Exponentially growing GES-1cells (1×105cells/well) were plated for24h. The Rad51expression vectors were then transiently transfected into GES-1cells using Lipofectamine (Invitrogen) for24h, and lastly,20μM OTA wastreated or not in these cells.4Western blot analysisAfter treated with various facors to GES-1cells, the expression of variousprotein levels in GES-1cells was determined by Western blot.5Quantitative real-time polymerase chain reaction (PCR)The effects of OTA on the expression of Rad51at mRNA level in GES-1cells in vitro in different groups were determined using Real-time PCR method.For each sample, data were normalized to the housekeeping gene humanβ-actin. Analysis was done using the comparative Ct value method.6Immunofluorescence stainingThe GES-1cells were incubated with rabbit monoclonal antibodiesFITC-labeled affinity purified anti-rabbit IgG secondary antibody, then DNAwas counterstained with DAPI. The images were captured and observed usingTCS-SP2laser scanning confocal microscope (Leica, Germany). Lastly,images were coded and stored on a computer for later analysis.7FCMThe cells were collected and washed twice by PBS, then fixed with70% ethanol and used FCM to determine the distribution of different cell cyclephases.8Immunoprecipitation（IP）Cells lysate containing120μg of total protein was incubated with5μganti-CyclinB1antibody at4°C with rotation overnight. The sample buffercontaining immunoprecipitated protein was electrophoresed on15%SDS-PAGE gel and then was transferred to PVDF membrane to detect theCdc2-CyclinB1complex.9Statistical analysisFor each protocol, at least three independent experiments were performed.Results were expressed as the mean±standard error of the mean (SEM).Statistical calculations were performed with the one way analysis of variance(ANOVA) using SPSS13.0software. Differences in measured variablesbetween experimental and control groups were assessed by Regression andpaired-samples t-test. P<0.05was considered statistically significant.Results:1OTA increases the expression of γ-H2AX protein level in a dose-and time-dependent manner in GES-1cellsIt’s known that γ-H2AX is a marker of DSBs, and its protein expressionwas determined by Western blot analysis. Exposure of GES-1cells to OTA atincreasing concentrations of5,10and20μM for24h caused distinct rise in theexpression of γ-H2AX (r=0.895, P<0.05). In order for further verification,20μM OTA was added to GES-1cells for6h,12h and24h, then we found thatthe level of γ-H2AX protein was markedly elevated since6h (r=0.926,P<0.05).2OTA suppresses the expression of Rad51mRNA and protein levels in adose-and time-dependent manner in GES-1cells2.1OTA down-regulates the expression of Rad51mRNA level in GES-1cellsThe results of real time-PCR revealed that, treated with increasingconcentrations(5μM'10μM'20μM) of OTA for24h (r=-0.909, P<0.05) ortreated with20μM OTA (a concentration that maximally inhibited Rad51) at increasing time(6h'12h'24h)（r=-0.984, P<0.05) caused significantdecrease in Rad51mRNA levels in GES-1cells.2.2OTA down-regulates the expression of Rad51protein level in GES-1cellsSimilar to Rad51at mRNA level, GES-1cells were treated with variousOTA concentrations (5,10and20μM) for24h and the expression of Rad51was determined by Western blot analysis (r=-0.952, P<0.05). Then,20μMOTA was added to GES-1cells and the Rad51protein level was examined atvarious time points (6,12and24h)(r=-0.989, P<0.05).The results showed that OTA treatment induced a time-anddose-dependent decrease in cellular Rad51expression both at protein andmRNA levels.2.3OTA induces significant decrease in the number of Rad51foci in GES-1cellsImmunofluorescence results showed that the number of Rad51foci inOTA treatment (20μM) cells was significantly decreased as compared withthat in control group. The results further confirmed that OTA coulddown-regulate Rad51in GES-1cells (P<0.05).These results above-mentioned suggest that OTA down-regulates theexpression of Rad51and then suppresses the function of HR repair pathway.3Transfection of Rad51expression vector reverses OTA-induced damage inGES-1cells3.1Effect of Rad51overexpression on OTA-induced DSBs in GES-1cellsTo verify Rad51involvement in the cytotoxicity induced by OTA, aRad51vector was transfected into GES-1cells to enforce the Rad51expression in normal control cells and OTA treated cells. The results ofWestern blot revealed that transfection of Rad51expession vector blocked theup-regulation of γ-H2AX induced by OTA treatment (P<0.05).3.2Effect of Rad51overexpression on OTA-induced G2arrest in GES-1cellsFCM analysis revealed that the transfection of Rad51expression vectorcould remitted the G2phase arrest of OTA on GES-1cells (P<0.05). The results of Western blot revealed that, compared with control vectorgroup, Rad51expression vector blocked the down-regulation of Cdc2/p-Cdc2,Cdc25C/p-Cdc25C, CyclinB1and Cdc2-CyclinB1complex induced by OTAtreatment (P<0.05). Meanwhile, the results of Immunoprecipitation revealedthat the Cdc2-CyclinB1complex was significantly increased in Rad51+OTAgroup compare with that in vector+OTA group(P<0.05).4The possible molecular mechanism of OTA down-regulates Rad51expression in GES-1cells4.1The ERK-and p38-MAPK signaling pathways are involved inOTA-induced down-regulation of Rad51GES-1cells were treated with OTA (20μM) for24h after pretreated withPD98059(a specific ERK inhibitor) or SB203580(a specific p38MAPKinhibitor) for30min, then Western blot analysis was used to determine theexpression of Rad51protein. Pharmacological inhibition of ERK or p38MAPK pathway clearly restored the levels of Rad51protein in GES-1cellstreated with OTA (P<0.05). These results indicated that the ERK-and p38MAPK signaling pathways are involved in OTA-induced down-regulation ofRad51.4.2OTA triggers Rad51down-regulation by p53in GES-1cellsProtein levels of Rad51were determined by Western blot analysis.Depletion of p53by specific siRNA transfection effectively reversedOTA-induced Rad51down-regulation in GES-1cells (P<0.05). The resulttherefore suggests that p53is involved in OTA-induced Rad51down-regulation in GES-1cells.Conclusion:1OTA induces DSBs and down-regulates the expression of Rad51in GES-1cells.2Rad51overexpression can significantly reverse OTA-induced DSBs inGES-1cells.3Rad51overexpression can relieve OTA-induced G2phase arrest in GES-1cells. 4The ERK-and p38-MAPK signaling pathways as well as p53are involvedin OTA-induced Rad51down-regulation in GES-1cells. |
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