曲古霉素A通过调控Ku70乙酰化水平对结肠癌HCT116细胞上皮间充质转化和侵袭转移的影响
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摘要
目的探讨曲古霉素A(TSA)通过调控Ku70乙酰化水平对结肠癌HCT116细胞上皮间充质转化(EMT)和侵袭转移的影响。方法体外培养HCT116细胞,采用MTT法筛选并确定TSA的最佳给药浓度;采用Western印迹(WB)检测对照组(DMSO处理组)和TSA处理组细胞中Ku70及其乙酰化水平以及EMT相关蛋白E-钙黏蛋白(cadherin)、N-钙黏蛋白、Snail和Slug的表达情况;采用Transwell-迁移和侵袭实验观察TSA介导Ku70乙酰化对HCT116细胞侵袭和转移能力的影响。结果 MTT检测结果表明TSA可明显降低HCT116细胞的活性(P<0.05);WB结果显示,TSA可明显上调HCT116细胞中acetyl-Ku70的蛋白水平并抑制其EMT,表现为上调上皮细胞标志蛋白E-钙黏蛋白的表达,并下调EMT标志蛋白N-钙黏蛋白及EMT相关转录因子Snail和Slug的表达(P<0.05);此外,Transwell-迁移和侵袭实验结果表明,TSA可明显抑制HCT116细胞的迁移和侵袭(P<0.05)。结论 TSA可能通过上调HCT116细胞中Ku70乙酰化水平,降低Ku70与DNA的亲和力,从而抑制肿瘤细胞DNA的损伤修复能力,发挥抑制结肠癌细胞EMT和迁移侵袭的作用,为临床抗结肠癌转移治疗提供新的思路和作用靶点。
Objective To investigate the effect of trichostatin A(TSA)on epithelial-mesenchymal transition(EMT),invasion and metastasis of colon cancer HCT116 cells by regulating the level of Ku70 acetylation. Methods MTT assay was used to detect the effect of TSA on cell viability of colon cancer HCT116 cells. Western blot(WB)was used to detect the expressions of Ku70,acetyl-Ku70 and EMT-related proteins E-cadherin,N-cadherin,Snail and Slug in the control(DMSO-treated)group and TSA-treated groups. Transwell-migration and invasion assays were used to observe the effect of Ku70 acetylation on invasion and metastasis abilities of colon cancer HCT116 cells after TSA treatment. Results MTT assay results showed that TSA could significantly reduce the activity of colon cancer HCT116 cells(P<0.05). WB results showed that TSA could significantly up-regulate the expression of acetyl-Ku70 and inhibit the EMT of HCT116 cells by upregulating the expression of epithelial cell marker E-cadherin and down-regulating the expression of mesenchymal cell marker N-cadherin and other EMT-related transcription factors,such as Snail and Slug(P<0.05). In addition,Transwellmigration and invasion assays results showed that TSA could also significantly inhibit the migration and invasion of colon cancer HCT116 cells by promoting Ku70 acetylation(P<0.05). Conclusion TSA may significantly reduce the affinity of Ku70 to DNA by significantlyup-regulatingtheacetylationlevelofKu70 incoloncancerHCT116 cells,therebyinhibitingthe EMT,migration and invasion of colon cancer HCT116 cells. This study can provide a new treatment strategy for clinical anti-colon cancer metastasis.
Objective To investigate the effect of trichostatin A(TSA)on epithelial-mesenchymal transition(EMT),invasion and metastasis of colon cancer HCT116 cells by regulating the level of Ku70 acetylation. Methods MTT assay was used to detect the effect of TSA on cell viability of colon cancer HCT116 cells. Western blot(WB)was used to detect the expressions of Ku70,acetyl-Ku70 and EMT-related proteins E-cadherin,N-cadherin,Snail and Slug in the control(DMSO-treated)group and TSA-treated groups. Transwell-migration and invasion assays were used to observe the effect of Ku70 acetylation on invasion and metastasis abilities of colon cancer HCT116 cells after TSA treatment. Results MTT assay results showed that TSA could significantly reduce the activity of colon cancer HCT116 cells(P<0.05). WB results showed that TSA could significantly up-regulate the expression of acetyl-Ku70 and inhibit the EMT of HCT116 cells by upregulating the expression of epithelial cell marker E-cadherin and down-regulating the expression of mesenchymal cell marker N-cadherin and other EMT-related transcription factors,such as Snail and Slug(P<0.05). In addition,Transwellmigration and invasion assays results showed that TSA could also significantly inhibit the migration and invasion of colon cancer HCT116 cells by promoting Ku70 acetylation(P<0.05). Conclusion TSA may significantly reduce the affinity of Ku70 to DNA by significantlyup-regulatingtheacetylationlevelofKu70 incoloncancerHCT116 cells,therebyinhibitingthe EMT,migration and invasion of colon cancer HCT116 cells. This study can provide a new treatment strategy for clinical anti-colon cancer metastasis.
引文
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[22]Wang JH,Lee EJ,Ji M,et al.HDAC inhibitors,trichostatin Aand valproic acid,increase E-cadherin and vimentin expression but inhibit migration and invasion of cholangiocarcinoma cells[J].Oncol Rep,2018,40(1):346-354.
[23]Zhang K,Peng T,Yan Q,et al.Jiedu sangen decoction inhibits migration and invasion of colon cancer SW480 cells via suppressing epithelial mesenchymal transition[J].Evid Based Complement Alternat Med,2018,7(29):1455-1462.
[2]Goossens S,Vandamme N,Van Vlierberghe P,et al.EMT transcription factors in cancer development re-evaluated:beyond EMT and MET[J].Biochim Biophys Acta Rev Cancer,2017,1868(2):584-591.
[3]Chanut P,Britton S,Coates J,et al.Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks[J].Nat Commun,2016,19(7):632-639.
[4]Pucci S,Polidoro C,Joubert A,et al.Ku70,Ku80,and sClusterin:a cluster of predicting factors for response to neoadjuvant chemoradiation therapy in patients with locally advanced rectal cancer[J].Int J Radiat Oncol,2017,97(2):381-388.
[5]Singh A,Singh N,Behera D,et al.Role of polymorphic XRCC6(Ku70)/XRCC7(DNA-PKcs)genes towards susceptibility and prognosis of lung cancer patients undergoing platinum based doublet chemotherapy[J].Mol Biol Rep,2018,45(3):253-261.
[6]Arai H,Wada R,Ishino K,et al.Expression of DNA damage response proteins in gastric cancer:comprehensive protein profiling and histological analysis[J].Int J Oncol,2018,52(3):978-988.
[7]Wang Z,Lin H,Hua F,et al.Repairing DNA damage by XRCC6/KU70 reverses TLR4-deficiency-worsened HCC development via restoring senescence and autophagic flux[J].Autophagy,2013,9(6):925-927.
[8]Huang Y,Shao Q,Luo X,et al.Poly(ADP-ribose)polymerase-1 promotes recruitment of meiotic recombination-11 to chromatin and DNA double-strand break repair in Ku70-deficient breast cancer cells[J].FASEB J,2018:j201800092R.
[9]Ho CK,Kornaga EN,Klimowicz AC,et al.Expression of DNAdamage response proteins in cervical cancer patients treated with radical chemoradiotherapy[J].Gynecol Oncol,2017,145(1):176-184.
[10]Kopra K,Tong-Ochoa N,Laine M,et al.Homogeneous peptidebreak assay for luminescent detection of enzymatic protein posttranslational modification activity utilizing charged peptides[J].Anal Chim Acta,2019,1055(16):126-132.
[11]Al EA,Arbon D,Jeeves M,et al.Ku70 N-terminal lysines acetylation/deacetylation is required for radiation-induced DNA-double strand breaks repair[J].Neoplasma,2018,65(5):708-719.
[12]Gong P,Li K,Li Y,et al.HDAC and Ku70 axis-an effective target for apoptosis induction by a new 2-cyano-3-oxo-1,9-dien glycyrrhetinic acid analogue[J].Cell Death Dis,2018,9(6):623.
[13]Taieb J,Andre T,Auclin E.Refining adjuvant therapy for nonmetastatic colon cancer,new standards and perspectives[J].Cancer Treat Rev,2019,75(28):1-11.
[14]Haase G,Gavert N,Brabletz T,et al.The wnt target gene L1 in colon cancer invasion and metastasis[J].Cancers(Basel),2016,8(5):7361-7368.
[15]Zan H,Tat C,Qiu Z,et al.Rad52 competes with Ku70/Ku86for binding to S-region DSB ends to modulate antibody classswitch DNA recombination[J].Nat Commun,2017,8(93):14244-14250.
[16]van den Boom J,Wolf M,Weimann L,et al.VCP/p97 extracts sterically trapped Ku70/80 rings from DNA in double-strand break repair[J].Mol Cell,2016,64(1):189-198.
[17]Mukherjee S,Chakraborty P,Saha P.Phosphorylation of Ku70subunit by cell cycle kinases modulates the replication related function of Ku heterodimer[J].Nucleic Acids Res,2016,44(16):7755-7765.
[18]Czuba LC,Hillgren KM,Swaan PW.Post-translational modifications of transporters[J].Pharmacol Ther,2018,192(46):88-99.
[19]Jiang W,Crowe JL,Liu X,et al.Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and end-ligation during nonhomologous end-joining[J].Mol Cell,2015,58(1):172-185.
[20]Difilippantonio MJ,Zhu J,Chen HT,et al.DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation[J].Nature,2000,404(6777):510-514.
[21]Franke FC,Muller J,Abal M,et al.The tumor suppressor SASH1 interacts with the signal adaptor CRKL to inhibit epithelial-mesenchymal transition and metastasis in colorectal cancer[J].Cell Mol Gastroenterol Hepatol,2019,7(1):33-53.
[22]Wang JH,Lee EJ,Ji M,et al.HDAC inhibitors,trichostatin Aand valproic acid,increase E-cadherin and vimentin expression but inhibit migration and invasion of cholangiocarcinoma cells[J].Oncol Rep,2018,40(1):346-354.
[23]Zhang K,Peng T,Yan Q,et al.Jiedu sangen decoction inhibits migration and invasion of colon cancer SW480 cells via suppressing epithelial mesenchymal transition[J].Evid Based Complement Alternat Med,2018,7(29):1455-1462.