Effect Of PFDA-induced DNA Damage In Gastric Cancer And Its Mecha Nism

Gastric cancer is one of the most common malignant tumors,and accounts for about 10%of all invasive cancers worldwide.It also is probably the second leading cause of cancer deaths.In recent years,the impact of environmental pollution on human health has become a concern of governments and the public around the world.It has been confirmed that 75 to 80%of malignant tumors are associated with carcinogens in the environment,and environmental pollution is also positively correlated with the occurrence of gastric cancer;The mechanism of environmental pollution promoting gastric cancer is not fully understood.Perfluorinated chemicals(PFCs)have long been used in the manufacture of cookware such as non-stick pans,fire-fighting foams and many other industrial products.Now PFCs can be detected globally in the environment,wildlife and humans.In Asian oceanic waters,the level of detected PFCs can reach 17.8-192ng/L.Because PFCs are physically and chemically stable,they are highly persistent and these compounds are not biodegradable in the body and thus bioaccumulated.Studies have shown that some PFCs may have a half-life in water of more than 40 years.Unfortunately,there are no extensive regulations or guidelines on PFCs emissions and disposal in many parts of the world,mainly due to the lack of toxicity data on perfluorinated chemicals.Hence there is an urgent need to understand more deeply the environmental and ecological impacts of these pollutants and the associated risks.Perfluorodecanoic acid(PFDA),also a perfluorinated compound(PFCs),has been reported to cause toxic effects in rats causing loss of appetite and severe weight loss,bradycardia,hypothermia and decreased serum thyroid hormone levels The reported cellular and physiological effects of PFDA include reproductive endocrine and hepatotoxicity as well as lipid metabolism disorders.Genotoxicity refers to the destruction of a genetic material,such as chromosome or DNA,by a physical or chemical substance.Genotoxicity can lead to serious biological dysfunction or even disease,the action of which usually takes a long time to occur The ability of compounds to interfere with DNA integrity and gene expression renders them potentially mutagenic and even carcinogenic.It has been reported that PFCs can induce disturbances of DNA metabolism homeostasis.Other studies have reported that gene expression of important biological functions such as energy expenditure and reproduction may be affected by exposure to PFCs.Other reports confirm that PFCs suppress the expression of inflammation and immune-related genes.However,little is known of the destructive effect of PFCs on DNA damage and repair,as well as how it acts.The purpose of the current study was to investigate the mechanism of PFDA in DNA damage and repair.The results showed that PFDA treatment increased the number of DSBs in gastric cells.Through microarray and expression regulation analysis,we found that the mechanism of the DSBs production might be due to the inhibition of DNA repair by PFDA,particularly suppressed the non-homologous end-joining mechanism.NHEJ impairment usually triggers cell death,but the experimental results showed that PFDA inhibited the expression of P53 and thus protects cells from apoptosis.This article discovers for the first time that PFCs induces an increase in intracellular DSBs and the mechanism is through inhibition of the NHEJ major repair factor XRCC4 and regulation of XRCC4 expression by TCF4.PART I:THE ROLE OF PFDA IN THE DNA DAMAGE OFGASTRIC CANCERObjectives:Western-blot assay was used to detect the change of DNA damage index yH2AX after PFDAtreatment in vivo and vitro and detect the degree of DNA damage by DNA end-ligation detection.Method:1.Experiments were performed using gastric cancer cell lines AGS and SGC.10%fetal bovine serum containing F12 and 1640 DMEM with 10%fetal bovine serum were used,respectively.2.PFDA was added to AGS and SGC cell culture medium for 72h,the expression of DNA damage index y-H2AX was detected by western-blot.3.The mice were divided into a control group and an experimental group,and the water containing the DMSO in the control group and the water containing the PFDA and DMSO in the experimental group were fed separately.Changes in γ-H2AX expression in gastric tissue of mice were measured by western-blot 15 days later.4.DNA end-ligation detection was used to further investigate whether the intracellular DNA repair mechanism was impaired after PFDA treatment.plasmid pUC19 fragment digested with EcoRI and BamHI was used.These fragments were incubated with different treated nuclear proteins(six-well plates,50 mmol/LPFDA 0.2.4.6 ul per well)and the DNA repair efficiency was compared by QPCR assays.Result:1.The expression of DNA damage index γ-H2AX was significantly increased at the protein level after treated with PFDA in AGS and SGC cells.2.In vivo experiments,DNA damage index y-H2AX expression was significantly increased at the protein level in the PFDA-treated mouse gastric tissue.3.The results showed that the PFDA treatment group(4 ul)showed a significant reduction in DSBs repair ability compared with the blank control group.Conclusion:1.In vivo and vitro,PFDA treatment caused DNA damage in gastric cancer cells.2.PFDA treatment decreased the ability of NHEJ in gastric cancer cells.PARTH:THE EFFECT OF PFDA ON CELL APOPTOSIS AFTER DNA DAMAGE AND ITS MECHANISMObjectives:In the experiment,DNA damage of gastric cancer cells treated by PFDA was found,but no obvious apoptosis was found.The cell apoptosis arrested was evaluated by the method of cell apoptosis analysis and western-blot.Method1.Cell apoptosis analysis was performed as instructed by the Annexin V-FITC conjugate manual.Briefly,5×105 AGS cells in a well of 6-well plates were treated with certain concentration of PFDA and control DMSO as well as P53 plasmid administration,incubating for 72 h before the cells were digested and harvested by centrifugation.After harvested the cells and washed in cold phosphate-buffered saline(PBS),then the cells were centrifuged and discard the supernatants,Add 400ul of Annexin-binding buffer to the cells and mix.Add 5 μl of Annexin V-FITC to resolve the suspension cells.In the cell suspension,mix and incubate for 15 min at 2-8 ° C in the dark.Then add the suspension with 10μl PI and mix gently at 2-8 ℃ in the dark for 5 min.Be sure to perform cytometry flow assay within 1 hour.2.AGS and SGC were cultured with PFDA,then Western-blot assay was performed to assess the expression of P53.3.P53 overexpression plasmid were added to the cell line to further detect the relationship between apoptosis and P53 expression.4.Gene sequencing of P53was performed after PFDA treated in AGS cells.Result:1.PFDA inhibited the expression of P53 significantly in AGS and SGC cells.2.PFDA treated AGS and SGC cells were supplemented with P53 up-regulation plasmid(1,2,4,8 μg P53 plasmid per well)significantly increased apoptosis.3.The P53 gene sequence of PFDA treated AGS cells was aligned with the P53 gene sequence of the NCBI database,and the P53 gene was not mutated.Conclusions:1.PFDA induces a decrease in the expression of P53 protein in gastric cancer cells.2.PFDA reduces apoptosis by inhibiting P53 expression.thereby preventing cells after DNA injury from apoptosis.PARTⅢ:THE EFFECT OF PFDA ON RADIOTHERAPY AND CHEMOTHERAPYObjectives:In vitro the PFDA treated gastric cancer cells were cultured with classic chemotherapy drugs and radiotherapy methods to study whether the PFDA can influence the effect of chemotherapy drugs and radiotherapy.The cell survival rate was measured by CCK8 to evaluate the effect of PFDA on gastric cancer.Method:1.RadiotherapyCells with 40%to 50%confluence in 6-well plates were treated with PFDA for 3 days and then received radiation therapy with different prescription dosages(such as 6 MeV photons),the cells without PFDA treatment as a control.The planned target volume(PTV)encompassed all six wells with a margin of approximately 1.0 cm.After radiotherapy,the cells were incubated with trypsin/EDTA to release the wells from the 6-well plates,and then plated in a 96 well after counting.10 μL of CCK8 was added to each well,and incubated at 37℃ for 2 hours.The optical density(OD)at 450 nm was measured by a microplate reader The relative percentage of cell viability was calculated using the following formula:(OD of the experimental group/OD of the control group)×100%.2.ChemotherapyCells with 40%to 50%confluence in 6-well plates were treated with PFDA for 3 days and then exposed to different concentrations of cisplatin and 5-Fluorouracil(5-FU)at 37 ℃ for 48 h,the cells without PFDA treatment as a control.The following cell viability assay was same as the Radiotherapy described above.Results:1.The survival of cells in the PFDA group treated with CDDP at a concentration of 1 μgg/ml;10 μg/ml;100 μg/ml was significantly higher than that of the blank control group treated with the same concentration of CDDP.2.The survival of cells in the PFDA group treated with the concentration of 0.1 μg/ml;1μg/ml;10 μg/ml;100 μg/ml 5-FU was not significantly different from that of the blank control group treated with 5-FU.3.The experimental results showed that there was no significant difference in the number of cells in the PFDA group treated with radiotherapy(16,20,40Gray)and the blank control group of the same dose of radiation.Conclusions:1.PFDA-caused a decrease in sensitivity to CDDP chemotherapy after DNA damage in gastric cancer cells.2.PFDA had no significant effect on the sensitivity to 5-FU chemotherapy.in gastric cancer cells.3.PFDA has no significant effect on radiotherapy in gastric cancer cells.PART IV:MECHANISM OF HOW PFDA INDUCE DNA DAMAGE AND INHIBIT NHEJObjectives:The gene sequencing method was used to detect the changes of major factors related to DNA damage repair in gastric cancer cells treated with PFDA.RT-PCR and western-blot assays were used to detect the repair factor of DNA after DNA damage in gastric cancer cells.Furthermore,the mechanism of how PFDA causes DNA damage was also studied.Method:1.Experiments were performed using gastric cancer cell lines AGS and SGC.10%fetal bovine serum containing F12 and 1640 DMEM with 10%fetal bovine serum were used,respectively.2.The cells were incubated in six-well culture plates at 37℃ with 5%CO2 for 24h.The liquid supernatant was then discarded;1800μl fresh F12 or DMEM 1640 without FBS and 60 multiplicity of infection(MOI)of Ad-XRCC4 or null adenoviruses was added into each well.After 4 hours,200μl FBS was added into each well.The blank control group cells were cultured with F12 or 1640 and added 10%FBS.Then the cells were cultured at 37℃with 5%C02 for 48h.3.Si-RNA transfection:After culturing the cells in a six-well plate for 24 hours,the transfect reagent lipotamine 3000,5 μl,RNA oligo 50 nM,5 μl 250 μl opti-MEM were mixed and allowed to stand for 10-15 min to be added to each well,and the cells were cultured at 37℃.Change the liquid after 6-8h.Expression of mRNA or protein levels was detected at 36-72h.4.RT-PCR was performed to assess the mRNA level of the main NHEJ repair factor including xrcc4,xrcc5,xrcc6,DNA-pk,DNA-ligase4 xlf the mRNA level and the Wnt signal factor β-catenin was assessed at the mRNA level.5.Total cellular protein was isolated from AGS cells and SGC cells treated with PFDA,XRCC4 adenovirus and si-XRCC4 using RIPA buffer,and Western-blot assay was performed to assess XRCC4 and DNA-ligase4 expression,and XRCC4 and y-H2AX expression treated with si-TCF4.6.DNA end joining activity assay was performed to detect the change of DNA Damage repair ability.Specifically,nucleoprotein was extracted by Nucleoprotein Extraction Kit and PUC19 plasmid was extracted with the TIANprep Rapid Mini Plasmid Extraction Kit Then the PUC19 plasmid was digested by EcoR1 and BamHI,the digestion products were used for electrophoresis and the large fragment was harvested.Then a 20μl system containing the following items was created:50mM triethanolamine(pH7.5),60mM potassium acetate,50μM dNTP,2mM ATP,1mM DTT and 0.1mg/ml bovine Serum albumin.6μg of nucleoprotein were incubated in at 37°C for 5min before added into the 20μl system that containing 20ng PUC19 plasmid large fragment.The reaction system without nucleoprotein was used as a negative control.The reaction system was incubated at 18℃ for 24 hours.7.Microarray analysis:The microarray chip consisted of 27 326 probes for different human cDNAs(Capitalbio Company,Beijing,China),in which the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase was served as internal control.The cDNAs extracted from PFDA-treated AGS cells were labeled with Cy3,whereas the cDNAs from the control DMSO-treated AGS cells were labeled with Cy5.The labeled cDNAs were then hybridized with microarray chip under standard conditions according to manufacturer’s instructions.Changes in mRNA expression in response to PFDA stimulation were assayed in DNA microarrays.Two fold up or down regulation were set as cutoff values,and changes in gene expression were analyzed using the Database for Annotation,Visualization,and Integrated Discovery(DAVID).8.CCK8 assay was performed to assess the viability of the PFDA-treated group and the XRCC4 adenovirus group cells.Result:1.Microarray analysis observed the NHEJ major repair factor XRCC4 was the most down-regulated gene in response to PFDA treatment,which was 43%of the control group.Moreover,the expression of the major gene of homologous recombination repairs(HR)Rad51,BRCAl and BRCA2 is also reduced after PFDA-treated.2.The results of NHEJ major factor mRNA assay after PFDA treatment showed that the expression levels of xrcc4,xrcc5,xrcc6,DNA-pk and DNA-ligase4 mRNA were significantly down-regulated compared with the control group.3.Western-blot analysis of AGS cells treated with PFDA showed that the expression levels of NHEJ major repair factors XRCC4 and Lig4 were significantly lower than those of the blank control group.In addition,XRCC4 and Lig4 expression was reduced to 23.1%and 54.7%,respectively,in SGC cells treated with PFDA.4.AGS cells were treated with PFDA and compared with the control group.XRCC4 gradually decreased at the protein level with time,and gradually became obvious after 48 hours.This time-dependent reduction was also observed in SGC cells.5.The AGS cells were treated with PFDA for 24-96h and the XRCC4 adenovirus was added during the period.Compared with the XRCC4 adenovirus alone,the expression level of XRCC4 was decreased by PFDA treatment and gradually decreased with time.This time-dependent reduction was also observed in SGC cells.6.Detection of NHEJ repair ability:group:1.Negative control group(plasmid pUC19 fragment digested with EcoR1 and BamH1 without added nucleoprotein);2.blank control group 3.DMSO group;3,PFDA group;4.control Si-RNA group;5.xrcc4 si-RNA group;6.empty adenovirus group;7.xrcc4 adenovirus group,the up regulation of XRCC4 express the NHEJ repair ability was increased.7.Adenovirus and siRNA were used to up-regulate and down-regulate the expression of XRCC4,further verifying the change of protein level,the expression of XRCC4 protein was up-regulated after up-regulation of XRCC4 gene.Validation was performed in AGS cells and SGC cells,respectively.8.Accordingly,γ-H2AX levels were significantly increased after PFDA treatment in AGS cells,but decreased in cells overexpressing XRCC4,down-regulating XRCC4 y-H2AX expression up-regulated and verified in SGC cells.9.After PFDA treatment and PFDA treatment with addition of XRCC4 adenovirus,the CCK8 assay showed that the survival of cells in the XRCC4 adenovirus group was significantly higher than that in the PFDA-only group in AGS gastric cells.10.The expression of TCF4 was down-regulated by TCF4 siRNA transfected in AGS cells;and AGS cells treated with PFDA showed that TFC4 expression was down-regulated.We also found that XRCC4 expression was down-regulated after TCF down-regulation and y-H2AX expression was up-regulated.And the results were verified in the SGC cells.11.The microarray and the results of qPCR showed that β-catenin increased by 1.36 times compared with the control group after treatment with PFDA.Conclusion:1.PFDA mainly reduces the repair ability of DSBs by inhibiting NHEJ activity.2.PFDA inhibits NHEJ function mainly by down-regulating XRCC4.3.PFDA regulates XRCC4 expression via the TCF4 signaling pathway.