| Ochratoxin A(OTA,C20H18ClNO6;molecular weight:403.8),mainly produced by the fungal species of Penicillium verrucosum,Aspergillus carbonarius,A.niger and A.ochraceus,is the most toxic chemical in the group of ochratoxins.It is a potent pentaketide nephrotoxin diffusely distributed in food,feed products and even bottled water.Therefore,humans could contact OTA in many different ways.Studies on toxicity and carcinogenicity of OTA in different animals have showed that kidney is the main target organ of OTA,which can be induced to form renal adenoma accompanied by urinary tract tumor.Difference in sensitivity to OTA has been found among different species and sexes.In humans,exposure to high levels of OTA in the diet has been linked with chronic renal disease(Balkan Endemic Nephropathy)and an increased incidence of urinary tract tumors.Therefore,OTA has been classified as“possibly carcinogenic to humans”by the International Agency for Research on Cancer since 1993.China is one of the countries with the highest incidence of gastric cancer.The disease burden is so high that it has always been the focus of cancer prevention in our country.According to the latest statistics from Chinese National Cancer Registration Center in 2015,there were about 679,000 new cases of gastric cancer in China.Our previous epidemiological study has showed that Zanhuang County of Hebei Province is an area in China with high incidence of gastric carcinoma,which might be closely related to the OTA contamination in the local grains.Our previous studies have also found that the low-dose(2.5?M)and long-term(40 weeks)OTA treatment on human gastric epithelial(GES-1)cells could cause malignant transformation through activating the Wnt/?-catenin pathway.These results further suggest that OTA may be closely related to the occurrence of gastric cancer.During the research progress of carcinogenic mechanism for OTA,our studies have found that OTA could induce oxidative stress to generate DNA double-strand breaks(DSBs)and simultaneously inhibit the expression of Rad51,which is one of the key proteins in homologous recombination(HR),the precise repair pathway of DSBs.Therefore,we concluded that OTA can inhibit the repair efficiency of HR.DSBs is the most serious type of DNA damage,and if its accumulative effect cannot be effectively repaired in time it will eventually lead to genome instability and oncogenesis.However,how the non-homologous terminal junction(NHEJ),another error-prone repair pathway of DSBs,responds to DSBs,and whether the low-dose long-term OTA treatment affect genome stability and energy metabolism in GES-1 cells remain to be investigated.To address these questions,this study explored the selection of DSBs repair pathways with the NHEJ pathway as a main line,as well as the effects of low-dose long-term OTA on genome instability and glycolysis pathways in GES-1cells.This study is divided into three parts:first,detect if the two repair pathways,NHEJ and HR,play roles in the repair of DSBs damage induced by OTA in GES-1 cells;second,investigate the role of PARP-1 in alternative nonhomologous end joining(alt-NHEJ)and possible molecular mechanism;and third,examine the effects of the low-dose long-term OTA treatment on the genome instability and glycolysis pathway in GES-1 cells.The results of this study provide new insights into mechanisms revealing the relationship between OTA and the occurrence of gastric cancer,and supply theoretical basis for discovery of new therapeutic targets to gastric cancer.Part one Effects of OTA on DSBs repair mechanism in GES-1 cellsObjective:To explore the selection of two repair pathways of NHEJ and HR,and the important role of Ku protein in c-NHEJ repair pathway after DSBs generation in GES-1 cells treated with OTA.Methods:1.Cell viability of GES-1 under the treatment of OTA at different concentrations and different times was detected by CCK-8 assay.2.ROS changes in GES-1cells under the OTA treatment were analyzed by flow cytometry(FCM).3.The expression of?H2AX in GES-1 cells treated with different concentrations OTA and the protein expression of key regulatory factors in HR and NHEJ pathways were detected by Western blot.4.The m RNA levels of key regulatory factors in HR and NHEJ pathways under the treatment of OTA were detected by Realtime PCR.5.The expression of H2AX,Rad51,Ku70 and Ku80 proteins in GES-1cells under the treatment of OTA was observed by using Immunofluorescence and Confocal laser scanning microscopy.6.The selection of HR and alt-NHEJ pathways,the cell viability and cell apoptosis were evaluated in GES-1 cells transiently transfected with Ku70/Ku80 siRNA by Realtime PCR,Western blot and FCM.Results:1.OTA has a significant effect on GES-1 cell viabilityResults showed that the viabilty of GES-1 cells treated with different concentrations OTA(5?M?10?M?20?M)and different times(6 h,12 h,24h and 48 h)significantly decreased in a dose and time dependent manner(P<0.05).2.OTA induced GES-1 cells ROS increase to produce DSBsFCM results showed that compared with the solvent control group,the treatment of 20?M OTA on GES-1 cells for 24 h resulted in significant ROS increase(P<0.05).Moreover,DSBs significantly increased with the increase of OTA concentration(P<0.01).It indicated that GES-1 cells produce ROS through oxidative stress and lead to DSBs generation.3.DSBs induced by OTA in GES-1 cells are mainly repaired by NHEJ pathwayThe key molecule Rad51 of the HR pathway was indeed inhibited(P<0.05),but the expression of the key molecule of c-NHEJ and alt-NHEJ pathways was increased in GES-1 cells treated with different concentrations(5?M?10?M?20?M)OTA for 24 h by Western blot,Realtime PCR and laser confocal immunofluorescence.The results showed that PARP-1,a key molecule involved in the alt-NHEH pathway,especially showed significant OTA sensitivity,and its expression was significantly increased with the increase of OTA concentration(P<0.05).4.OTA significantly increased the repair ability of alt-NHEJ and reversed the repair ability of HR in GES-1 cells transfected with Ku70/80 siRNAKu70 and Ku80 participated in DSBs repair in the form of complex as the key molecules of c-NHEJ pathway by means of Immunoprecipitation.Not only the expression of PARP-1,the key molecule of the alt-NHEJ pathway was significantly increased(P<0.05),but also the repair ability of the HR pathway was reversed and the expression of Rad51 and BRCA1/2 was significantly increased(P<0.05)in GES-1 cells co-transfected with Ku70/80siRNA after 20?M treatment for 24 h by Western blot and Realtime PCR.5.GES-1 cells co-transfected with Ku70/80 siRNA showed decreased cell activity,obvious G2/M phase arrest,and significantly increased apoptosis rateAfter c-NHEJ pathway was inhibited by co-transfection of Ku70/Ku80siRNA,it was found that the viability of GES-1 cells induced by OTA was significantly decreased(P<0.05),the proportion of G2/M phase arrest cells was increased(P<0.05),and the apoptosis rate was significantly increased(P<0.05).These indicate that c-NHEJ plays a major repair role in DSBs repair induced by OTA.Part two Role of PARP-1 in OTA-induced DSBs repair in GES-1 cellsObjective:In the previous results,it has known that PARP-1,a key molecule of the alt-NHEJ pathway,showed significant OTA sensitivity,and its expression was significantly increased with the increase of OTA concentration(P<0.05).Alt-NHEJ pathway has always been considered as an error-prone repair pathway.Whether the abnormal sensitivity of this pathway to OTA is closely related to genome instability of GES-1 cells induced by OTA need to further be explored in the progress of DSBs repair.Methods:1.The expressions of core proteins in c-NHEJ and HR pathways and the expressions of cell cycle and apoptotic molecules were detected in GES-1cells after transfection of PARP-1 siRNA.2.The relationship between the expression of PARP-1 and G2/M phase arrest was detected in GES-1 cells applying ATM inhibitor KU55933 by Western blot.3.The effect of OTA on GES-1 cell viability and mechanism of apoptosis were detected in GES-1 after transfection of PARP-1 siRNA by Realtime PCR,Western blot and immunofluorescence.4.After the biological function of PARP was completely destroyed by Olaparib,a PARP-1/2 inhibitor,the effect of Olaparib on GES-1 cells was detected by CCK-8,FCM and Western blot.5.The selection of DSBs repair pathway and its influence on cell cycle and apoptosis in GES-1 cells exposed to OTA after transient transfection of PARP-1 overexpression plasmid were detected by Western blot,Realtime PCR and FCM.Results:1.The repair capacity of c-NHEJ increased in OTA-induced cells after transfection of PARP-1 siRNA,while there was no significant change in HR repair pathwayWestern blot and laser confocal immunofluorescence were used to observe the enhanced sensitivity of GES-1 cells to OTA after transfection of PARP-1 siRNA,and the expression of?H2AX protein significantly increased(P<0.05).The expression of Ku70 and Ku80,key molecules in c-NHEJ pathway,was significantly increased(P<0.05),while the HR pathway showed no significant changes after the expression of PARP-1 was inhibited.2.OTA promoted G2/M phase arrest of GES-1 cells after transfection of PARP-1 siRNA,but had no effect on cell activity and apoptosisCombined with FCM,Western Blot and CCK-8 assay,it was found that OTA aggravated G2/M phase arrest(P<0.05)in GES-1 cells after transfection of PARP-1 siRNA,but no significant changes were observed in cell viability and apoptosis.Cells arrested at G2/M phase were not effectively repaired because H2AX increased significantly(P<0.05).3.The positive correlation between the expression of PARP-1 and G2/M phase arrest was verified by KU55933KU55933 inhibited OTA-induced G2/M phase arrest and PARP-1 protein expression also decreased(P<0.05)by Western blot.4.G2/M phase arrest in GES-1 cells treated with OTA was increased after transfection of PARP siRNA,which may inhibit apoptosis through the parthanatos pathwayWestern blot and immunofluorescence were used to analyze and observe the expression and cytoplasmic transfer of AIF,the key molecule of the parthanatos pathway,and it was found that the expression of AIF in the cytoplasm increased(P<0.05).5.Olaparib was used to completely destroy the function of PARP,and OTA increased G2/M phase arrest and apoptosis in GES-1 cellsIn order to further verify the PARP-1 influence on cell cycle in GES-1cells treated with OTA,PARP inhibitor,Olaparib,was applied and destroyed the function of PARP1/2.Olaparib relied on ATM/p53/p21 pathway activation to block the GES 1 cells in G2/M phase and the ratio of G2/M arrest increased significantly(P<0.05)with the changes of cell proliferation and apoptosis.The significance of the synergistic effect of PARP-1 and PARP-2 was illustrated by inverted microscope observation,CCK 8,Western Blot and FCM method.6.Olaparib inhibited the alt-NHEJ pathway and activated c-NHEJ pathwayRealtime PCR and Western blot showed that after the biological function of PARP was destroyed by Olaparib,the alt-NHEJ pathway was inhibited,while the c-NHEJ repair pathway was activated(P<0.05),and the HR repair pathway was not significantly changed.7.Overexpression of PAPR-1 reduced OTA-induced G2/M arrest of GES-1 cells and apoptosis rateThe increased expression of PARP-1 could reduce the rate of G2/M phase arrest(P<0.05)and reduce the apoptosis rate of cells(P<0.05)by FCM,Western blot and CCK-8 methods.8.Overexpression of PARP-1 enhanced the repair ability of alt-NHEJWestern blot was used to detect the expression of the cell key DSBs repair proteins in GES-1 cells transfected with PARP-1 overexpressed plasmid,and it was found that the increase of PARP-1 expression would enhance the repair ability of alt-NHEJ(P<0.05),while the repair ability of c-NHEJ and HR did not change significantly.Part three The effect of long-term and low-dose OTA exposure on the genome stability and energy metabolism of GES-1 cellsObjective:Low dose of OTA acts on GES-1 cells for a long time,and continuous DNA damage will damage the genome stability and cause rearrangement of energy metabolism.Therefore,we established a chronic OTA-induced cell model that was continuously poisoned with 5?M OTA for30 d,60 d and 90 d,and observed the changes in genome stability and energy metabolism.Methods:1.The morphological changes and cell proliferation in GES-1 cells treated with 5?M OTA for 30 d,60 d and 90 d were observed by using inverted microscope,CCK-8 method and Western blot.2.The effects of long-term treatment of GES-1 cells with low dose OTA on the genome stability were observed and analyzed by using the micronucleus method and chromosome karyotype analysis.3.The expression of E-cadherin and Vimentin in GES-1 cells treated with5?M OTA for 30 d,60 d and 90 d was detected by Western.4.Changes in the Wnt/?-catenin signal pathway were detected by Western blot and immunofluorescence.5.Glucose and lactic acid in cell cultures after 30 d,60 d and 90 d of 5?M OTA administration were determined by colorimetry.6.The expression of key kinases in the glycolytic pathway in GES-1 cells was detected by Realtime PCR and Western blot.7.The protein expression of PI3K/Akt pathway was detected in GES-1cells treated with 5?M OTA for 30 d,60 d and 90 d by Western blot.Results:1.The morphology and cell proliferation changed after treatment with low dose OTA for 90 dGES-1 cells were observed under an inverted microscope after treatment with 5?M OTA for 90 d,and significant changes were observed in cell morphology.Moreover,CCK-8 assay was used to observe the cell proliferation,and it was found that the cell proliferation rate started to elevate from 60 d after 5?M OTA treatment.2.Chromosome aberration and micronucleus rate were increased in GES-1 cells treated with low dose OTA for 90 dChromosome aberration rate(including bicentric,break/gap,deletion and ring formation)and micronucleus incidence rate in GES-1 treated with 5?M OTA for 30 d,60 d and 90 d were detected by chromosome karyotypic analysis and micronucleus assay,and found that the chromosome aberration rate and micronucleus incidence rate were significantly increased(P<0.05).3.Malignant transformation of GES-1 cells possibly occurred after 90 d of low-dose OTA treatmentThe expression of E-cadherin and Vimentin in GES-1 cells treated 5?M OTA for 30 d,60 d and 90 d was detected by Western blot and showed that the expression of E-cadherin was significantly decreased from 90 d(P<0.01),while the expression of Vimentin was significantly increased(P<0.05).4.OTA possibly caused malignant transformation of GES-1 cells by activating key molecules expression of Wnt/?-catenin signal pathwayThe significantly increased expressions of key molecules of Wnt/?-catenin signaling pathway,p-GSK-3?,Wnt2 and?–catenin,were detected in GES-1 cells treated 5?M OTA for 30 d,60 d and 90 d(P<0.01 or P<0.05)by Western blot.Transfer from the nucleus to the cytoplasm of?-catenin was observed by means of immunofluorescence and confocal laser observation.5.Energy metabolism of GES-1 cells was rearranged after treatment with low dose OTA for 90 dBiovision glucose and lactic acid detection kit was used to detect the consumption of glucose and lactate production in the supernatant of cell treated with 5?M OTA for 30 d,60 d and 90 d,and it was found that glucose content decreased(P<0.05)and lactic acid production increased(P<0.01),so the glycolytic pathway was preferred.6.Treatment of low dose OTA for 90 d possibly strengthened the glycolysis metabolism with the upregulated expression of HIF1?,PI3K and p-AKt proteinsThe protein expression of glycolytic pathway and several important driving molecules from GES-1 cells treated with 5?M OTA for 30 d,60 d and90 d by Realtime PCR and Western blot.The results showed that the key kinase expression of the glycolysis pathway increased(P<0.05),and the expression of HIF1?,PI3K and p-AKt proteins also increased(P<0.05).Conclusion:1.HR pathway was inhibited in GES-1 cells treated with OTA,and the expression of key molecules on c-NHEJ and alt-NHEJ pathways showed a significant increase.2.Although the expression of Ku protein complex,the key molecule of c-NHEJ pathway,didn't show sensitivity to OTA concentration,it played a vital role in DSBs repair.3.The expression of PARP-1 depended on G2/M phase arrest,and the knockdown of PARP-1 may inhibit apoptosis of GES-1 cells through activating the parthanatos pathway.4.Long-term treatment of low-dose OTA on GES-1 cells caused genome instability and significantly increased chromosome aberration and micronucleus formation.5.Long-term treatment of low-dose OTA on GES-1 cells might result in rearrangement of cellular energy metabolism with the upregulated expression of HIF1?,PI3K and p-AKt proteins. |
PDF Full Text Request