ERK And P38MAPK Signaling Pathways Are Involved In Ochratoxin A-induced G2Phase Arrest In Human Gastric Epithelium Cells

Objective: Ochratoxin A （OTA） is a naturally occurring mycotoxinproduced by several species of Aspergillus and Penicillium. Thecontamination of OTA is frequently seen in a variety of animal chow andhuman food, especially in cereals, wine, coffee, spices, beer, poultry, and pork.Kidney is the primary target organ of OTA. OTA is implicated as a possiblecontributor to human Balkan endemic nephropathy （BEN） and associatedurothelial tumors and has been classified as a possible human carcinogen（group2B） by International Agency for Research on Cancer. In addition to itsnephrotoxicity, animal studies suggest that OTA be a liver toxin, an immunesuppressant and a potent teratogen. A prolonged ingestion of OTAcontaminated foodstuff also provokes hyperplasia of the forestomachepithelium and induces fibroadenoma of breast in F344/N rats. As aconsequence of its widespread occurrence in food, the human population iscontinuously exposed to OTA.Accumulating evidence has indicated that the early toxic effect of somemycotoxins （such as deoxynivalenol, zearalenone and fumonisin B1） wasleading to apoptosis, proliferation suppression and cell-cycle arrest. Ourprevious study demonstrated that OTA evoked cell cycle arrest at G2phase inGES-1cells via down-regulation the expression of cell cycle-related proteins（Cdc25C, Cdc2and CyclinB1） and the phosphorylation status of Cdc25C andCdc2. OTA also exerts its cytotoxic effect by inducing G2/M cell cycle arrestin Chinese hamster lung fibroblast V79cells and African green monkeykidney CV-1cells in vitro.Mitogen-activated protein kinase （MAPK） is an important intracellularsignal transduction system and participates in a series of physiological and pathological processes, including cell growth, differentiation and apoptosis.The most prominent members of MAPKs family are c-Jun-N-terminal kinase（JNK）, p38kinase and extracellular-regulated protein kinase （ERK）. Theactivation of JNK, ERK and p38MAPK signaling pathways was involved inthe cell-cycle arrest. In a study with Deoxynivalenol （DON）, Yang found thatDON-mediated G2/M cell cycle arrest was associated with enhanced p₂1levelvia ERK1/2MAP kinase signaling pathway. However, virtually no data isavailable involving the putative mechanism of OTA-induced G2arrest onhuman gastric epithelium cells.Considering the critical role of MAPK in cell-cycle progress, wehypothesized that OTA induced G2arrest may be through the modulation ofthe MAPK pathways. The putative role of MAPK pathways in OTA-inducedG2arrest was investigated in the present study. We aimed to revealed theputative mechanisms of G2arrest induced by ochratoxin A in human gastricepithelium cells in vitro.Methods:1Cell culture and treatmentThe cells were routinely cultured in DMEM supplemented with100U/mlpenicillin,100U/ml streptomycin, and10%fetal bovine serum （FBS）. GES-1cells in logarithmic growth phase were randomly divided into four groups. Thecells were then treated with solvent （methanol） alone or with variousconcentrations of OTA for24h. OTA was diluted in methanol and added to theculture flasks to obtain the final concentration of5,10and20μM respectively.Control cells were incubated for the same time with methanol at aconcentration of0.08%（n=3）.2Treatment in GES-1cells with p38and ERK inhibitorsTo investigate the effect of p38and ERK inhibitors on OTA-induced cellcycle arrest, confluent cell culture were preincubated for30min with10μMSB203580（p38inhibitor）, or10μM PD98059（ERK inhibitor）. Then the cellswere treated with20μM OTA for24hours.3siRNA transfection Cells were reversely transfected with Negtive control siRNA （NC siRNA）or siRNA targeting p₃8and ERK at a concentration of100pmol respectivelyusing Lipofectamine2000（Invitrogen） according to the manufacturer’sinstructions. And24h post transfection, cells were washed with PBS andsubsequently treated with20μM OTA for24h. Afterwards, cells wereharvested and assayed by Western blot and flow cytometry.4FCMAfter treated with OTA for24h, the cells were collected and washedtwice by PBS, then fixed with70%ethanol to determine the distribution ofdifferent cell cycle phases.5Western blotAfter treated with OTA for24h, the expression of JNK/Phospho-JNK,ERK/Phospho-ERK, p₃8/Phospho-p₃8, CyclinB1, Cdc2/Phospho-Cdc2andCdc25C/Phospho-Cdc25C at protein level in GES-1cells was determined byWestern blot.6Immunoprecipitation（IP）Cells lysate containing120μg of total protein was incubated with1μganti-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.7Statistical analysisThe results were presented as means±SD. Data were analyzed usingone-way analysis of variance （ANOVA） analysis. Values were consideredstatistically significant when P<0.05.Results:1Effects of OTA on JNK, ERK and p₃8signaling pathways in GES-1cells1.1OTA increases phosphorylation levels of ERK and p₃8, but not JNK inGES-1cellsThe results of Western blot showed that exposure of GES-1cells to OTAat increasing concentrations of5,10and20μM for24h caused significant increase in p38and ERK phosphorylation levels （P<0.05）. While, there wasno difference in the expression of phosphorylatd JNK following OTAtreatment （P>0.05）. At the same time, unphosphorylated forms of eachMAPKs at protein levels remained unaltered after OTA exposure.1.2p38and ERK inhibitors attenuated phosphorylation levels of ERK and p₃8increased by OTA in GES-1cellsOur results indicated that, the phosphorylation level of ERK in PD98059+OTA treatment group was significantly decreased as compared with that in20μM OTA group （P<0.05）. Similarly, OTA-mediated up-regulation of p₃8phosphorylation was also decreased by SB203580pretreatment （P<0.05）.These results suggested that OTA could activate ERK and p38but not JNKin GES-1cells in vitro.2Effect of ERK inhibition on OTA-induced G2arrest in GES-1cells2.1Effect of ERK specific inhibitors （PD98059） on OTA-induced G2arrest inGES-1cellsThe results from FCM analysis showed that, the proportion of cells inG0/G1phase in PD98059pretreatment group was significantly increasedcompared with that in OTA treatment group, while the proportion of cells inG2/M phase was significantly decreased than latter （P<0.05）. The resultindicated that PD98059pretreatment could partly inhibit G2arrest induced byOTA in GES-1cells.2.2Effect of ERK siRNA transfaction on OTA-induced G2arrest in GES-1cellsFCM analysis revealed that, there was no significant difference in cellcycle distribution between NC siRNA group and solvent control group（P>0.05）. Depletion of ERK by specific siRNA transfaction effectivelyprevent OTA-induced G2arrest in GES-1cells by increasing the proportion ofcells in G0/G1phase and decreasing that in G2/M phase （P<0.05）. This resultcomfirmed that OTA-induced G2arrest be abolished by ERK siRNAtransfaction.Taken together, the results indicated that ERK signaling pathway was involved in OTA-induced G2arrest in GES-1cells.3Effect of p38inhibition on OTA-induced G2arrest in GES-1cells3.1Effect of p38specific inhibitors （SB203580） on OTA-induced G2arrest inGES-1cellsOur results indicated that, consistent with PD98059, SB203580pretreatment also decreased the proportion of cells in G2/M phase ascompared with that in OTA20μM alone group （P<0.05）, suggesting that p₃8specific inhibitor, SB203580, could block OTA-induced G2arrest.3.2Effect of p38siRNA transfaction on OTA-induced G2arrest in GES-1cellsThe result of FCM analysis showed that, no significant difference wasfound in cell cycle distribution between NC siRNA group and solvent controlgroup （P>0.05）. p38siRNA transfaction could attenuated the G2arrest ofOTA on GES-1cells （P<0.05）.Thus, the above result confirmed that p38signaling pathway wasinvolved in G2arrest in GES-1cells induced by OTA.4Effect of ERK inhibition on OTA-downregulated G2phase related proteinsin GES-1cells4.1Effect of ERK specific inhibitors （PD98059） on G2phase related proteinsin GES-1cellsWestern blot results showed that, the expression of G2/M regulatoryproteins （Cdc25C, Cdc2and CyclinB1） and the phosphorylation status ofCdc25C and Cdc2were significantly up-regulated in PD98059-pretreatedOTA20μM-treated group as compared with20μM OTA alone treatmentgroup （P<0.05）.The results of IP revealed that, pretreatment of GES-1cells withPD98059also significantly increased the Cdc2-CyclinB1complex ascompared with that in OTA20μM alone group （P<0.05）.4.2Effect of ERK siRNA transfaction on G2phase related proteins in GES-1cellsThe results of Western blot revealed that, ERK siRNA blocked the down-regulation of Cdc2/p-Cdc2, Cdc25C/p-Cdc25C, CyclinB1andCdc2-CyclinB1complex induced by OTA treatment （P<0.05）. These proteinsshowed no significant difference between NC siRNA group and solvantcontrol group （P>0.05）.The result of IP showed that, there was no difference of Cdc2-CyclinB1complex when NC siRNA group compared with that of solvent control group（P>0.05）, however, compared with OTA group, the Cdc2-CyclinB1complexwas significantly increased when transfacted with ERK siRNA （P<0.05）.These dates indicated that ERK signaling pathway was involved inOTA-induced G2arrest in GES-1cells by modulating G2phase relatedproteins.5Effect of p38inhibition on G2phase related proteins in GES-1cells5.1Effect of p38specific inhibitors （SB203580） on G2phase related proteinsin GES-1cellsWestern blot results showed that, the expression levels of Cdc2/p-Cdc2,Cdc25C/p-Cdc25C and CyclinB1were all significantly increased inSB203580pretreatment group as compared with OTA alone treatment group（P<0.05）.In addition, the results of IP indicated that combination of SB203580andOTA treatment increased the Cdc2-CyclinB1complex as compared with thatin OTA group （P<0.05）.5.2Effect of p38siRNA transfaction on G2phase related proteins in GES-1cellsWestern blot results showed that compared with that in OTA treatmentgroup, the expression of Cdc2/p-Cdc2, Cdc25C/p-Cdc25C, and CyclinB1protein in p38siRNA+OTA treatment group was significantly decreased（P<0.05）, and there was no significant difference of these proteins betweenNC siRNA group and solvent control group （P>0.05）.The result of IP showed that no difference was found in theCdc2-CyclinB1complex beween NC siRNA group and solvent control group（P>0.05）. p38siRNA blocked the down-regulation of Cdc2-CyclinB1 complex induced by OTA treatment （P<0.05）.Our results above suggest that p38signaling pathway is involved inOTA-induced G2arrest in GES-1cells by modulating G2phase relatedproteins.Conclusion:1OTA could activate ERK and p38MAPK signaling pathway, but not JNKin GES-1cells.2Inhibition of ERK and p38MAPK signaling pathway could attenuateOTA-induced G2arrest in GES-1cells.3Inhibition of ERK and p38MAPK signaling pathway could preventOTA-induced down-regulation of Cdc2/p-Cdc2, Cdc25C/p-Cdc25C,cyclinB1and Cdc2-CyclinB1complex.4ERK and p38MAPK signaling pathways are involved in OTA-induced G2arrest in GES-1cells by up-regulating G2phase related proteins.