Study On The Mechanism By Which The Interaction Of XRCC5/p300 Promotes The Growth Of Colorectal Cancer By Upregulating Cyclooxygeanse-2

Colorectal cancer(CRC)is a common carcinoma in digestive tract;CRC has become the third most commonly diagnosed cancer in males and the second one in females worldwide.In males,about 750,000 new CRC cases are diganosed and about370,000 deaths can be atributed to CRC annully,CRC has been listed as the fourth cause of death from cancer.In females,the annually diagnosed new CRC cases can reach 610,000,CRC causes 320,000 deaths annully,CRC is the third cause of death from cancer.As CRC being a serious threat on human health,health institutions in various countries are paying great attention to this disease.To date,only early detection and curative colorectal operation have the possibility to cure CRC.However,a great amount of CRC patients are found with remote metastasis at first hospital visit and diagnosed with advanced stage CRC,a curative resection of CRC is not possible for these patients.Local radiotherapy,systemic chemotherapy,traditional Chinese medicine and molecule-targeted drugs are the mainstays for the treatment of advanced stage CRC.However,local radiotherapy and systemic chemotherapy have limited treating effect but great side effect.The combination therapy of cytotoxic drugs including 5-fluorouracil,irinotecan,leucovorin,oxaliplatin and capecitabine is the first line chemotherapy for advanced CRC with metastasis.During the combination chemotherapy for CRC,many CRC cases develop drug resistance,which causes treatment failure.With intensive studies on signaling in tumors development,various molecular treatment targets for CRC are found.Consequently,molecule-targeted drugs are developed for advanced stage CRC treatment.Bevacizumab(vascular endothelial growth factor A inhibitor,VEGF-A inhibitor)and cetuximab(epidermal growth factor receptor inhibitor,EGFR inhibitor)have been used to treat advanced stage CRC successfully.Because of multiple pathways involved in tumor genesis,when one pathway is inhibited,other compensatory pathways would be enhanced.So it is not rare that some CRC patients also develop drug resistance to bevacizumab and cetuximab.So search for new molecular targets to treat advanced stage CRC is of great significance for improving the prognosis of CRC patients clinically.Cyclooxygenase(COX)is the rate-limiting enzyme in prostaglandins(PG)synthesis.COX convert arachidonic acid to PGG2,PGG2 is converted to PGH2 subsequently,PGH2 is then converted to physiologically active PGs(PGE2,PGD2 and thromboxane A2)which are of great importance in inflammation,stress and digestive tract mucosa protection processes.Three COX isoforms(COX-1,COX-2 and COX3)exist naturally in humans.COX-1 as a constitutive enzyme exists in vessels,stomach and kidney,the function of COX-1 includes mucosa protection in stomach,blood flow regulation and platelet aggregation regulation.COX-3 is a splice variant of COX-1whose function is not clearly elucidated to date.COX-2 is the most studied COX isoform,COX-2 can be induced to be overexpressed during tissue damage,inflammation and cell malignant transformation.Previous studies show that over 50%tumor tissues overexpress COX-2,which indicates COX-2 having great importance in tumor genesis and COX-2 being a tumor promoting factor.COX-2 has the potential to become the target for tumor treatment.PGE2 as the product of COX-2 transduces cellular signals into target cells via G protein coupled receptor(GPCR)family(EP1,EP2,EP3,EP4 and peroxisome proliferator-activated receptors)on cellular membrane.After coupled with EP4,PGE2 activates phosphatidylinositol 3-kinase(PI3K)and protein kinase B(PKB)pathways to inhibit apoptosis and promote proliferation of target cells.PGE2 can also activate mitogen-activated protein kinase(MAPK)signaling pathway to promote tumorigenesis.Moreover,PGE2 induces the expression of Bcl-2 and increases the activity of nuclear factor kappa B(NF-?B)to inhibit apoptosis.COX-2 can also increase the expression of vascular endothelial growth factor(VEGF),which has the ability to promote angiogenesis of tumors.Furthermore,COX-2 can regulate NOTCH/WNT via EP4 to promote cancer stem cell formation.The immunological characteristic of tumor microenvironment is the shift from a Th1 predominant immune response to a Th2 predominant one.Tumor necrosis factor-?(TNF-?),interferon-?(IFN-?)and interleukin-2(IL-2)can increase Th1 cell proliferation.Correspondingly,interleukin-4(IL-4),interleukin-6(IL-6)and interleukin-10(IL-10)can promote Th2 cell proliferation.Previous studies show that COX-2 decreases the expression of TNF-?,IFN-? and IL-2,and increases the expression of IL-4,IL-6 and IL-10.So COX-2 can facilitate immune response shift in tumor microenvironment,which make tumor cells evade from host immunity.In summary,COX-2 promotes tumorigenesis through increasing cell proliferation,inhibiting apoptosis,promoting cancer stem cell formation and evading from host immunity,COX-2 is an important treatment target for tumor treatment.X-ray repair cross-complementing protein 5(XRCC5)also called Ku80 is encoded by XRCC5 gene located in 2q33-34.XRCC5 encoded 732 aminoacids;its molecular weight is about 86 kDa.XRCC5 and XRCC6 form a XRCC5/XRCC6 heterodimer which is a DNA-dependent protein kinase complex(DNA-PK).Previous studies have proved that XRCC5 participates in repair and recombination of DNA double strand breaks,which is of great importance in maintaining stability of the whole genome and chromosomes.XRCC5/XRCC6 heterodimer binds the ends of breaked DNA double strands to accomplish DNA non homologous end joining repair.Studies also indicate that the function of XRCC5 is not limited in DNA double strand breaks repairs;XRCC5is also an adherence factor participating in cell adherence,migration and invasion of tumors.XRCC5 has been proved to be overexpressed in various tumor tissues(including CRC),which implies that XRCC5 is also a tumor promoting factor.Previous studies have proved that XRCC5 can increase COX-2 expressions via synergistic interaction with CBP to promote cell proliferation in lung cancer cells.However,the interaction between XRCC5 and COX-2 in CRC development is not clear.In the present study,streptavidin-agarose pulldown assay was employed to search and identify novel transcription factors,which was able to bind COX-2 promotors specifically and regulate COX-2 expressions in the level of gene transcriptions in CRC cell lines.During this essay,we found that XRCC5 was able to bind COX-2 promotors to increase COX-2 gene transcription and up-regulate COX-2 expression in protein level.Through down-regulating and up-regulating XRCC5,we further evaluated the positive effect of XRCC5 on COX-2 regulation in CRC cell lines.Moreover,we employed cell proliferation essay in CRC cell lines and the experiment of tumorformation in nude mice to prove that XRCC5 could promote CRC growth through increasing COX-2 expressions both in vitro and in vivo.In order to further elucidate the mechanism of XRCC5 acting on COX-2 promotors,we used co-immunoprecipitation to identify co-activators of XRCC5 and found that transcription co-activator p300 had the ability to bind XRCC5.Further cell experiments also proved that p300 acetylated XRCC5 to regulate COX-2 transcriptions.Our study provided important theoretical and experimental foundations for the utilization of XRCC5 as a potential therapeutic target in CRC treatment.Part I XRCC5 as the specific COX-2 promotor binding protein regulates COX-2 expression in CRC cell linesObject: To identify novel transcription factors binding COX-2 promotors with streptavidin-agarose pulldown assay,and to evaluate the effect of the identified transcription factor on regulating COX-2 expression in CRC cell lines.Methods: In order to identify novel transcription factors binding COX-2 promotors,we designed a 478bp(-30 to-508)double strand DNA probe labeled with biotin.In four CRC cell lines(RKO,LoVo,DLD-1 and SW480),we separated COX-2 promotor binding protein sediment with streptavidin-agarose pulldown assay.After elution of the separated COX-2 promotor binding protein sediment,SDS-PAGE and silver staining were used to observe COX-2 promotor binding protein bands.The potential eligible protein band was then enzymolyzed.Mass spectral analysis and protein database comparison were utilized subsequently to identify the target protein.Reverse transcription PCR(RT-PCR)and Western blot were used to assess the expression of identified protein in four CRC cell lines.We used siRNA to knockdown the identified protein expression in LoVo cell line.COX-2 promoter luciferase essay was utilized to assess the binding ability of the identified protein on the promoter of COX-2 and the inhibitory effect of down-regulation of the identified protein on COX-2 promoter activation.We also used LPS to induce COX-2 expression to evaluate whether COX-2inducer could attenuate the above inhibitory effect on COX-2 pomoter activation.In LoVo and RKO cell lines,immunofluorescence localization experiment was employed to evaluate whether this protein was located in nucleus and acted as a potential transcription factor.We used si RNA to knockdown and plasmid transfection toup-regulate this protein to evaluate its regulating effect on COX-2 protein expression which was detected with Western blot.Results:1.After streptavidin-agarose pulldown assay,COX-2 promotor binding protein sediment was separated successfully.SDS-PAGE with silver staining showed one protein band with a molecular weight of about 90 kD.Mass spectral analysis and protein database comparison of this protein proved it to be XRCC5.2.XRCC5 and COX-2 were highly expressed in four CRC cell lines in both transcription level and protein level.Moreover,the expression of XRCC5 and COX-2were positively correlated to some extent.3.In LoVo cell line,down-regulation of XRCC5 decreased COX-2 promoter activation,COX-2 inducer LPS was able to attenuate the inhibitory effect of down-regulation of XRCC5 on COX-2 promoter activation.In LoVo and RKO cell lines,immunofluorescence localization experiment proved XRCC5 to be located in nucleus.In the protein level,down-regulation of XRCC5 decreased COX-2 protein expression,and overexpression of XRCC5 increased COX-2 protein expression.Conclusions: XRCC5 is a specific binding protein for COX-2 promotor,XRCC5 can activate COX-2 promoter and increase the transcription of COX-2.XRCC5 is highly expressed in four CRC cell lines in both transcription level and protein level.XRCC5 protein is able to regulate COX-2 expression and this regulation is a positive one.Part II Transcriptional co-activator p300 acetylates XRCC5 to regulate COX-2 expression in CRC cell linesObject: To evaluate the interaction between XRCC5 and transcriptional co-activator p300 with co-immunoprecipitation method,to assess whether p300 could acetylate XRCC5 to regulate COX-2 expression with p300 plasmid transfection and p300 acetylation inhibitor C646 in CRC cell lines.Methods: Nuclear proteins were exacted,co-immunoprecipitation with XRCC5 antibody was then used to separate immune complex,Western blot was used subsequently to detect whether P300 antibody could bind to the separated immune complex.Correspondingly,co-immunoprecipitation with p300 antibody was used toseparate immune complex in nuclear proteins,Western blot was used to detect whether XRCC5 antibody could bind to the separated immune complex.Immunofluorescence localization experiment was used to evaluate whether XRCC5 and p300 were localized in the same position in nucleus.Western blot was used to detect the expression of acetylated XRCC5 in RKO,LoVo and SW480 CRC cell lines.p300 overexpression plasmid was used to up-regulate p300 expression and increase its acetylization function,and p300 acetylization inhibitor C646 was used to inhibit p300 acetylization function.After that,we used Western blot to evaluated the regulation effect of p300 on XRCC5.Furthermore,we used anti-acetyl antibody to perform co-immunoprecipitation.Western blot was used to detect the expression of CRCC5 and acetylated XRCC5 in the separated immune complex.Through this way,we evaluated whether p300 had the ability to acetylate XRCC5.Furthermore,we up-regulated p300 expression with p300 overexpression with plasmid transfection and inhibited p300 acetylization function with C646 to evaluate the effect of p300 acetylization function on COX-2 expression.Results:1.After co-immunoprecipitation with XRCC5 antibody,Western blot with p300 antibody detected p300 protein in the separated immune complex.Correspondingly,after co-immunoprecipitation with p300 antibody,Western blot with XRCC5 antibody detected XRCC5 protein in the separated immune complex.2.Immunofluorescence localization experiment indicated that XRCC5 and p300 were both located in nucleus,and their locations were identical.3.Western blot also proved that high expression of acetylated XRCC5 existed in RKO,LoVo and SW480 CRC cell lines.Transfection with p300 overexpression plasmid to up-regulate p300 expression was able to improve the acetyliztion level of XRCC5 but not XRCC5 expression,and p300 acetylization inhibitor C646 decreased the acetyliztion level of XRCC5.Moreover,transfection with p300 overexpression plasmid to up-regulate p300 expression increased the expression of COX-2,and p300 acetylization inhibitor C646 decreased the expression of COX-2.Conclusions: XRCC5 and transcriptional co-activator p300 interact in the nucleus of CRC cells.p300 acetylates XRCC5 to promote the expression of COX-2 in CRC cells.Part III XRCC5 and p300 synergistically regulate COX-2 to promote CRC growthObject: To evaluate the effect of XRCC5 regualting COX-2 to promote CRC growth with cell proliferation experiment and tumor formation experiment in nude mice.To verify the synergistic effect of XRCC5 and p300 on COX-2 expression to promote CRC growth.Methods: In LoVo and RKO cell lines,we used siRNA to knockdown XRCC5 expression,MTS method was employed to assess cell viability,clone formation essay was used to evaluate cell clonablity,and microscopic observation was used to assess the morphology of cells.Moreover,XRCC5 overexpression plasmid transfection was used to up-regulate the expression of XRCC5,MTS method was used to assess viability of CRC cells.In LoVo and RKO cell lines,COX-2 inhibitor celecoxib was used to inhibit COX-2 expression to evaluate the effect of COX-2 inhibition on the proliferation of CRC cells.Furthermore,we used XRCC5 overexpression plasmid to up-regulate XRCC5 expression,and COX-2 inhibitor celecoxib was used to down-regulate COX-2expression at the same time to evaluate the attenuating effect of COX-2 inhibitor on the promoting effect of upregulation of XRCC5 on CRC cell proliferation.In the tumor formation experiment in nude mice,we used siRNA to knockdown XRCC5 expression to evaluate its effect on tumor proliferation in vivo.We also used the COX-2 inducer LPS to further evaluate whether the inhibitory effect of XRCC5 siRNA on tumor proliferation could be attenuated by COX-2 inducer in nude mice.We up-regulated p300 expression to assess its promoting effect on CRC cell proliferation.To further evaluate the synergistic effect of p300 and XRCC5 on CRC cell proliferation,we up-regulated p300 expression and down-regulated XRCC5 expression simultaneously to evaluate whether the promoting effect of p300 on cell proliferation could be attenuated by low XRCC5 expression.Correspondingly,we used p300 acetylization inhibitor to inhibit p300 acetylization function and up-regulated XRCC5 expression simultaneously to evaluate whether the promoting effect of XRCC5 on cell proliferation could be attenuated by p300 acetylization inhibitor.Results1.MTS essay showed that compared with controls,down-regulation of XRCC5 expression inhibited CRC cell viability significantly,and XRCC5 overexpressionenhanced CRC cell viability.2.Microscopic observation indicated that CRC cells with down-regulated XRCC5 expression had weaker attachment ability and more cell fragments compared with controls.Clone formation essay also showed that CRC cells with down-regulated XRCC5 expression had lower clone formation ability compared with controls.3.XRCC5 overexpression promote CRC cell viablility,and COX-2 inhibitor celecoxib attenuated the above promoting effect of XRCC5 on CRC cell viability.4.Tumor formation experiment in nude mice showed that down-regulation of XRCC5 inhibited tumor growth,and COX-2 inducer LPS attenuated the inhibitory effect of down-regulation of XRCC5 on tumor growth.5.Overexpression of p300 or XRCC5 increased proliferation of CRC cells.Down-regulation of XRCC5 expression inhibited the promoting effect of p300 on CRC cell proliferation.p300 acetylization inhibitor attenuated the promoting effect of XRCC5 on CRC cell proliferation.Conclusions: p300 regulates the level of XRCC5 acetylization.p300 acetylates XRCC5,and acetylated XRCC5 promotes CRC growth via increaseing COX-2expression.