Tumor Biology

, Volume 37, Issue 1, pp 963–970 | Cite as

Knockdown of Rad9A enhanced DNA damage induced by trichostatin A in esophageal cancer cells

Original Article


Histone deacetylase (HDAC) inhibitors have recently emerged as a new class of anticancer agents. As a classical HDAC inhibitor, trichostatin A (TSA) has been shown to possess many anticancer activities such as induction of cell cycle arrest, promotion of cell death, and enhancement of radiosensitity. In our previous work, we found that TSA treatment induced Rad9 gene expression, which suggested that Rad9 might play a role in TSA-induced biological effects. As Rad9 is involved in maintaining genomic integrity, we further analyzed the DNA damage induced by TSA and combined with Rad9 knockdown in esophageal cancer cells (ESCCs). Our results showed that TSA treatment alone induced significantly DNA damage in ESCC cells. Simultaneously, TSA also induced Rad9 gene expression both at transcriptional and translational levels in EC109 cells, but not in KYSE150 cells. Further, the induction of Rad9 by TSA was accompanied with increased level of histone H3K9 acetylation in Rad9 promoter region. To understand the role of Rad9 in TSA-induced DNA damage, Rad9 gene expression was efficiently knocked down by small interfering RNA (siRNA), which led to enhanced DNA damage and cell death induced by TSA. Our data suggested that Rad9 plays an important role in DNA damage, which is related to the biological effects of TSA.


TSA Esophageal cancer DNA damage Rad9A Apoptosis 



This work was supported by grants from the National Natural Science Foundation of China (No. 81272497).

Conflicts of interest


Supplementary material

13277_2015_3879_MOESM1_ESM.docx (165 kb)
ESM 1 (DOCX 165 kb)


  1. 1.
    Gaur P, Kim MP, Dunkin BJ. Esophageal cancer: recent advances in screening, targeted therapy, and management. J Carcinog. 2014;13:11.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov. 2014;13(9):673–91.CrossRefPubMedGoogle Scholar
  3. 3.
    Sachweh MC, Drummond CJ, Higgins M, Campbell J, Laín S. Incompatible effects of p53 and HDAC inhibition on p21 expression and cell cycle progression. Cell Death Dis. 2013;4, e533.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Chatterjee N, Wang WL, Conklin T, Chittur S, Tenniswood M. Histone deacetylase inhibitors modulate miRNA and mRNA expression, block metaphase, and induce apoptosis in inflammatory breast cancer cells. Cancer Biol Ther. 2013;14(7):658–71.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Yang H, Zhong Y, Xie H, Lai X, Xu M, Nie Y, et al. Induction of the liver cancer-down-regulated long noncoding RNA uc002mbe.2 mediates trichostatin- induced apoptosis of liver cancer cells. Biochem Pharmacol. 2013;85(12):1761–9.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Felisbino MB, Gatti MS, Mello ML. Changes in chromatin structure in NIH 3T3 cells induced by valproic acid and trichostatin A. J Cell Biochem. 2014;115(11):1937–47.PubMedGoogle Scholar
  7. 7.
    Sharma S, Taliyan R, Ramagiri S. Histone deacetylase inhibitor, trichostatin A, improves learning and memory in high-fat diet-induced cognitive deficits in mice. J Mol Neurosci. 2014 Nov 14.Google Scholar
  8. 8.
    Ye Q, Li Y, Jiang H, Xiong J, Xu J, Qin H, et al. Prevention of pulmonary fibrosis via trichostatin A (TSA) in bleomycin induced rats. Sarcoidosis Vasc Diffuse Lung Dis. 2014;31(3):219–26.PubMedGoogle Scholar
  9. 9.
    He G, Wang Y, Pang X, Zhang B. Inhibition of autophagy induced by TSA sensitizes colon cancer cell to radiation. Tumour Biol. 2014;35(2):1003–11.CrossRefPubMedGoogle Scholar
  10. 10.
    Barazzuol L, Jeynes JC, Merchant MJ, Wéra AC, Barry M, Kirkby KJ, et al. Radiosensitisation of glioblastoma cells using a histone deacetylase inhibitor (SAHA) comparing carbon ions with X-rays. Int J Radiat Biol. 2014;21(1):1–19.Google Scholar
  11. 11.
    Xiao W, Graham PH, Hao J, Chang L, Ni J, Power CA, et al. Combination therapy with the histone deacetylase inhibitor LBH589 and radiation is an effective regimen for prostate cancer cells. PLoS One. 2013;8(8), e74253.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Di Bernardo G, Alessio N, Dell’Aversana C, Casale F, Teti D, Cipollaro M, et al. Impact of histone deacetylase inhibitors SAHA and MS-275 on DNA repair pathways in human mesenchymal stem cells. J Cell Physiol. 2010;225(2):537–44.CrossRefPubMedGoogle Scholar
  13. 13.
    Geng L, Cuneo KC, Fu A, Tu T, Atadja PW, Hallahan DE. Histone deacetylase (HDAC) inhibitor LBH589 increases duration of gamma-H2AX foci and confines HDAC4 to the cytoplasm in irradiated non-small cell lung cancer. Cancer Res. 2006;66(23):11298–304.CrossRefPubMedGoogle Scholar
  14. 14.
    Zhang Y, Adachi M, Zou H, Hareyama M, Imai K, Shinomura Y. Histone deacetylase inhibitors enhance phosphorylation of histone H2AX after ionizing radiation. Int J Radiat Oncol Biol Phys. 2006;65(3):859–66.CrossRefPubMedGoogle Scholar
  15. 15.
    Yaneva M, Li H, Marple T, Hasty P. Non-homologous end joining, but not homologous recombination, enables survival for cells exposed to a histone deacetylase inhibitor. Nucleic Acids Res. 2005;33(16):5320–30.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Liu Y, He G, Wang Y, Guan X, Pang X, Zhang B. MCM-2 is a therapeutic target of trichostatin A in colon cancer cells. Toxicol Lett. 2013;221(1):23–30.CrossRefPubMedGoogle Scholar
  17. 17.
    Wang G, Tong X, Weng S, Zhou H. Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation. Cell Cycle. 2012;11(20):3792–800.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    He W, Ma X, Yang X, Zhao Y, Qiu J, Hang H. A role for the arginine methylation of Rad9 in checkpoint control and cellular sensitivity to DNA damage. Nucleic Acids Res. 2011;39(11):4719–27.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hopkins KM, Auerbach W, Wang XY, Hande MP, Hang H, Wolgemuth DJ, et al. Deletion of mouse rad9 causes abnormal cellular responses to DNA damage, genomic instability, and embryonic lethality. Mol Cell Biol. 2004;24(16):7235–48.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Yin Y, Zhu A, Jin YJ, Liu YX, Zhang X, Hopkins KM, et al. Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21. Proc Natl Acad Sci U S A. 2004;101(24):8864–9.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Broustas CG, Lieberman HB. Contributions of Rad9 to tumorigenesis. J Cell Biochem. 2012;113(3):742–51.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Li ZH, Zhang XB, Han XQ, Feng CR, Wang FS, Wang PG, et al. Antitumor effects of a novel histone deacetylase inhibitor NK-HDAC-1 on breast cancer. Oncol Rep. 2013;30(1):499–505.PubMedGoogle Scholar
  23. 23.
    Karagiannis TC, Harikrishnan KN, El-Osta A. Disparity of histone deacetylase inhibition on repair of radiation-induced DNA damage on euchromatin and constitutive heterochromatin compartments. Oncogene. 2007;26(27):3963–71.CrossRefPubMedGoogle Scholar
  24. 24.
    Zhang B, Wang Y, Pang X. Enhanced radiosensitivity of EC109 cells by inhibition of HDAC1 expression. Med Oncol. 2012;29(1):340–8.CrossRefPubMedGoogle Scholar
  25. 25.
    Jang SM, Kang EJ, Kim JW, Kim CH, An JH, Choi KH. Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA. Biochem Biophys Res Commun. 2013;438(2):445–51.CrossRefPubMedGoogle Scholar
  26. 26.
    Zhao ZN, Bai JX, Zhou Q, Yan B, Qin WW, Jia LT, et al. TSA suppresses miR-106b-93-25 cluster expression through downregulation of MYC and inhibits proliferation and induces apoptosis in human EMC. PLoS One. 2012;7(9), e45133.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Yao J, Qian CJ, Ye B, Zhang X, Liang Y. ERK inhibition enhances TSA-induced gastric cancer cell apoptosis via NF-κB-dependent and Notch-independent mechanism. Life Sci. 2012;91(5–6):186–93.CrossRefPubMedGoogle Scholar
  28. 28.
    Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273(10):5858–68.CrossRefPubMedGoogle Scholar
  29. 29.
    Tessarz P, Kouzarides T. Histone core modifications regulating nucleosome structure and dynamics. Nat Rev Mol Cell Biol. 2014;15(11):703–8.CrossRefPubMedGoogle Scholar
  30. 30.
    Carafa V, Nebbioso A, Altucci L. Histone deacetylase inhibitors: recent insights from basic to clinical knowledge & patenting of anti-cancer actions. Recent Pat Anticancer Drug Discov. 2011;6(1):131–45.CrossRefPubMedGoogle Scholar
  31. 31.
    Ho AS, Turcan S, Chan TA. Epigenetic therapy: use of agents targeting deacetylation and methylation in cancer management. Onco Targets Ther. 2013;6:223–32.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Smith S, Fox J, Mejia M, Ruangpradit W, Saberi A, Kim S, et al. Histone deacetylase inhibitors selectively target homology dependent DNA repair defective cells and elevate non-homologous endjoining activity. PLoS One. 2014;9(1), e87203.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Ververis K, Rodd AL, Tang MM, El-Osta A, Karagiannis TC. Histone deacetylase inhibitors augment doxorubicin-induced DNA damage in cardiomyocytes. Cell Mol Life Sci. 2011;68(24):4101–14.CrossRefPubMedGoogle Scholar
  34. 34.
    Zhang Y, Carr T, Dimtchev A, Zaer N, Dritschilo A, Jung M. Attenuated DNA damage repair by trichostatin A through BRCA1 suppression. Radiat Res. 2007;168(1):115–24.CrossRefPubMedGoogle Scholar
  35. 35.
    Lee MC, Lopez-Diaz FJ, Khan SY, Tariq MA, Dayn Y, Vaske CJ, et al. Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing. Proc Natl Acad Sci U S A. 2014;111(44):E4726–35.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Broustas CG, Lieberman HB. RAD9 enhances radioresistance of human prostate cancer cells through regulation of ITGB1 protein levels. Prostate. 2014;74(14):1359–70.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Dong Q, Sharma S, Liu H, Chen L, Gu B, Sun X, et al. HDAC inhibitors reverse acquired radio resistance of KYSE-150R esophageal carcinoma cells by modulating Bmi-1 expression. Toxicol Lett. 2014;224(1):121–9.CrossRefPubMedGoogle Scholar
  38. 38.
    Cai RL, Yan-Neale Y, Cueto MA, Xu H, Cohen D. HDAC1, a histone deacetylase, forms a complex with Hus1 and Rad9, two G2/M checkpoint Rad proteins. J Biol Chem. 2000;275(36):27909–16.PubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Xueli Pang
    • 1
  • Gang He
    • 2
  • Chao Luo
    • 2
  • Yan Wang
    • 2
  • Bo Zhang
    • 2
  1. 1.Department of Oncology, Southwest HospitalThird Military Medical UniversityChongqingChina
  2. 2.Department of Medical Genetics, College of Basic MedicineThird Military Medical UniversityChongqingChina

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