Mechanistic Studies Of Tetrachlorobenzoquinone-induced The DNA Damage Repair Signaling In MDA-MB-231 Cells

Tetrachlorobenzoquinone(TCBQ)is a mainly reactive metabolite of chlorophenols pentachlorophenol(PCP).PCP,as a typical representative of persistent organic pollutants(POPs),can cause carcinogenic,teratogenic,mutagenic toxicity and environmental hormone effect.The chemical properties of PCP are stable,but it can be degraded and generate tetrachlorohydroquinone(TCHQ),TCBQ by electrochemical reaction,photocatalytic reaction and metabolism in organisms and cells.The metabolic process of PCP in organisms will generate a large amount of reactive oxygen species(ROS)through oxidation-reduction cycling reaction,which can break the body’s oxidation-reduction balance,cause a series of serious human diseases such as inflammation,neurodegenerative diseases,immunodeficiency,premature aging and cancer.DNA double strands break(DSBs)is known as one of the most serious kinds of DNA damage,if it cannot timely and accurately be repaired,may cause gene mutation,chromosomes breakage and DNA damage,which may lead to the occurrence of cancer.Eukaryotic organisms protect the integrity and stability of genetic information by DNA damage response(DDR)mechanisms,which transmits the damage signals to a variety of effector proteins,and finally initiates the cell cycle arrest,DNA damage repair,programmed cell death(such as apoptosis and necrosis),senescence or other pathways.There are two main mechanisms of DNA repair when DSBs is caused by the external or internal factors: non homologous end joining(NHEJ)and homologous recombination repair(HR).HR pathway is a kind of high accuracy repair pathway and depends on homologous DNA sequence.RAD51 is one of the key proteins in the HR pathway of DSBs repair,which might find homologous sequences and carry out strand exchange.It is an essential recombinant protease in homologous recombination mediated DNA double strands repair pathway.p53,as an important component in DDR signaling network,which plays a pivotal role in DNA damage repair process.When cells are damaged,p53 is activated,starting the DNA repair pathways to allow cell survival.If cells were severely damaged and can not effectively repair,p53 induces cell apoptosis to promote cell death,thereby it reduces the generation of cancerous cells.Part Ⅰ: In the first part,MDA-MB-231 cell viability was detected by CCK-8 essay after exposure to TCBQ for different concentrations or times.The results showed that cell growth was inhibited after TCBQ exposure with concentration and time dependence.TCBQ-induced DSBs were then assessed by two different methods.DNA damage was detected by comet assay after cells were treated with 25 μM for different times.With the increasing of TCBQ dose or exposure time,olive tail moment increased significantly.It was indicating that TCBQ can lead to DNA damage.DSBs marker proteins γ-H2 AX and 53BP1,indicating cellular TCBQ responses to genotoxic stress,were detected by western blot and immunofluorescence.Immunofluorescence experiments found that γ-H2 AX and 53BP1 assembled in the nucleus was dependent on the increasing of TCBQ concentration or time.Western blot assay further demonstrated that TCBQ promotes the expression of γ-H2 AX and 53BP1,indicating that TCBQ can induce MAD-MB-231 cells DNA damage.Furthermore,the apoptosis of MDA-MB-231 cells was detected by flow cytometry.TCBQ significantly increased cell apoptosis.The cleavege of caspase-3,which was the key factor of apoptosis,was also significantly increased after TCBQ stimulation.Finally,we explored the effect of RAD51 on TCBQ-induced DSBs.RAD51 protein and mRNA expression was detected by western blot and RT-qPCR.We found that the exposure of TCBQ for 6 h slightly decreased the level of RAD51.However,a two-phase change of RAD51 was found in our time-course study.Compared with the control group,RAD51 protein and mRNA expression increased with the increasing of the TCBQ-exposure time.After reached a peak,the leve of RAD51 protein and mRNA reduced gradually,wherein higher concentration faced a faster response.We also explored the effect of TCBQ on the formation of RAD51 foci in the nucleus by immunofluorescence analysis,and the trends were similar to RAD51 protein and mRNA expression.In summary,these data indicated that TCBQ acts as a genotoxic agent,which induces DNA damage and apoptosis in MAD-MB-231 cells.Besides,we found that TCBQ affect the expression of DNA repair protein RAD51.Taken together,these results indicated cell attempt to repair TCBQ induced DNA damage through the upregulation of RAD51 at the first place;however,the decay of RAD51 suggested that the rescue probably was abolished,cell apoptosis increased.Part Ⅱ: The genotoxicity of TCBQ was confirmed in the first part,however the regulatory mechanism of TCBQ on RAD51 need further analysis.In this part of the experiments,we examined the possibility of post-translational control of RAD51 by inquiring whether TCBQ promotes RAD51 proteasome-mediated degradation firstly.The ubiquitination experiment showed that TCBQ had no effect on the degradation of RAD51 protein by ubiquitin-proteasome system.Next,we determined whether TCBQ affected RAD51 at the translational or transcriptional level.CHX was added to block protein biosynthesis and ActD was used to block mRNA synthesis.CHX cotreatment with TCBQ did not affect the stability of RAD51 protein,but ActD has a great influence on the stability of mRNA.This may explain that the holistic lower level of remaining RAD51 protein with TCBQ cotreatmen.These data indicated that TCBQ-induced regulates RAD51 mRNA rather than protein stability,which accounted for the decrease of RAD51.Based on the above results,we further studied the upstream signaling pathways that affect RAD51 expression.TCBQ upregulated p53 and p38 MAPK signaling pathways in both dose-and time-dependent manners.The expression of RAD51 was detected by adding the specific blocking agent of p53 or p38 MAPK,only p53 inhibitor caused a strong inhibitory effect on TCBQ-elicited RAD51 protein level.Moreover,CCK-8 analysis indicated that pharmacological inhibition of p53 clearly restored the cell viability.The results showed that TCBQ could activate these two pathways and promote the expression of related proteins,while p53 pathway was involved in the down-regulation of RAD51 by TCBQ.p53 has been found to interact with RAD51 and inhibit its activity during DNA repair.Therefore,it is interesting to investigate how TCBQ interferes with the binding of RAD51 with p53.IP experiment showed that p53 bound to RAD51 after TCBQ treatment.p53/RAD51 interaction reached the maximum after TCBQ treatment for 3 h,and then decreased with the decrease of RAD51 protein.To further verify the effect of p53 on RAD51,knockdown of p53 by p53 siRNA significantly reversed the downregulation of RAD51 and the upregulation of γ-H2 AX and cleaved caspase-3 in TCBQ treated cells.We also found that p53 knockdown reverses TCBQ-induced formation of RAD51 foci.SCGE assay demonstrated that p53 siRNA significantly decreased OTM.Moreover,p53 siRNA treatment increased cell viability in TCBQ-treated cells.Overall,these results indicated that p53 signaling pathway was essential for the regulation of RAD51 expression upon TCBQ stimulation.These results suggested that TCBQ-treatment activated the p53 signaling pathway,and participate in the regulation of RAD51 expression.It is confirmed that p53 has a dual role in mediating apoptosis and regulating the repair of DNA damage.Part III: To study the contribution of RAD51 gene on TCBQ-induced cytotoxicity,RAD51 expression was first modulated by pEGFP-N1-RAD51 vector and was knocked down using specific siRNA.Transfection with RAD51 siRNA significantly suppressed RAD51 protein levels and western blotting showed an increase in total RAD51 protein in overexpressing MDA-MB-231 cells.However,RAD51 siRNA and overexpressing did not affect TCBQ-induced p53 activation,which suggested p53 function as the upstream molecule of RAD51.RAD51 depletion increased γ-H2 AX and cleaved caspase-3 protein levels.However,RAD51 overexpression decreased the expressions of γ-H2 AX and cleaved caspase-3.Consistently,RAD51 overexpression corresponded to a decrease in OTM.On the contrary,transfection of RAD51 siRNA significantly enhanced TCBQ-induced DNA damage.In addition,cells containing RAD51 overexpression were more resistant to TCBQ than cells containing the respective parental controls;suppression of RAD51 by corresponding siRNA significantly enhanced the cytotoxicity caused by TCBQ.According to the results,TCBQ can inhibit the homologous recombination repair by inhibiting the expression of RAD51.Thus,it appears that the RAD51 plays an important role in the resistance to TCBQ-induced cytotoxicity.