Tumor Biology

, Volume 36, Issue 12, pp 9347–9356 | Cite as

Suberoylanilide hydroxamic acid enhances chemosensitivity to 5-fluorouracil in hepatocellular carcinoma via inhibition of thymidylate synthase

  • Bo Liao
  • Huifang Liang
  • Jin Chen
  • Qiumeng Liu
  • Bixiang Zhang
  • Xiaoping Chen
Research Article


Hepatocellular carcinoma (HCC) is associated with a high rate of mortality worldwide. Here, we investigated the effect of combination treatment with suberoylanilide hydroxamic acid (SAHA) and 5-fluorouracil (5-FU) on HCC cells. HepG2, SMMC7721, and BEL7402 cells were treated with SAHA and/or 5-FU and subjected to cell viability, colony formation, and soft agarose assays; cell cycle, apoptosis, and reverse transcription-PCR analyses; western blotting; immunohistochemistry; and xenograft tumorigenicity assay. SAHA and 5-FU inhibited the proliferation of the three cell lines, and combination treatment with SAHA and 5-FU resulted in a combination index <1 and a dose-reduction index value >1, indicating a synergistic effect. Co-treatment with SAHA and 5-FU caused G0/G1 phase arrest and induced caspase-dependent apoptosis, inhibiting tumorigenicity in vitro and in vivo. 5-FU upregulated thymidylate synthase, whereas SAHA downregulated its expression. Our results indicate that SAHA and 5-FU act synergistically to inhibit cell growth and tumorigenicity in HCC via the induction of cell-cycle arrest and apoptosis through a mechanism involving the inhibition of thymidylate synthase, suggesting that combination treatment with 5-FU and SAHA may be beneficial for the treatment of inoperable HCC.


Hepatocellular carcinoma Suberoylanilide hydroxamic acid 5-fluorouracil Combination Thymidylate synthase 



This work was supported by the State Key Project on Infectious Diseases of China (Grant Nos. 2012ZX10002016-004, 2012ZX10002010-001-004), the National Natural Science Foundation of China (Nos. 81372495 and 81372327) and Hubei Province for the Clinical Medicine Research Centre of Hepatic Surgery (2014BKB0892012DCA130032013BCB026). The authors thank Arian Laurence for his assistance in revising the manuscript.

Conflicts of interest



  1. 1.
    Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90.CrossRefPubMedGoogle Scholar
  2. 2.
    Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. Hepatology. 2011;53(3):1020–2.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet. 2012;379(9822):1245–55.CrossRefPubMedGoogle Scholar
  4. 4.
    El-Serag HB. Hepatocellular carcinoma. N Engl J Med. 2011;365(12):1118–27.CrossRefPubMedGoogle Scholar
  5. 5.
    Forner A, Reig ME, de Lope CR, Bruix J. Current strategy for staging and treatment: the BCLC update and future prospects. Semin Liver Dis. 2010;30(1):61–74.CrossRefPubMedGoogle Scholar
  6. 6.
    Llovet JM, Real MI, Montana X, Planas R, Coll S, Aponte J, et al. Arterial embolisation or chemoembolisation versus symptomatic treatment in patients with unresectable hepatocellular carcinoma: a randomised controlled trial. Lancet. 2002;359(9319):1734–9.CrossRefPubMedGoogle Scholar
  7. 7.
    Duschinsky R, Pleven E, Oberhansli W. Synthesis of 5-fluoropyrimidine metabolites. Acta Unio Int Contra Cancrum. 1960;16:599–604.PubMedGoogle Scholar
  8. 8.
    Suzuki M, Tsukagoshi S, Saga Y, Ohwada M, Sato I. Enhanced expression of thymidylate synthase may be of prognostic importance in advanced cervical cancer. Oncology. 1999;57(1):50–4.CrossRefPubMedGoogle Scholar
  9. 9.
    Shintani Y, Ohta M, Hirabayashi H, Tanaka H, Iuchi K, Nakagawa K, et al. New prognostic indicator for non-small-cell lung cancer, quantitation of thymidylate synthase by real-time reverse transcription polymerase chain reaction. Int J Cancer. 2003;104(6):790–5.CrossRefPubMedGoogle Scholar
  10. 10.
    Pestalozzi BC, Peterson HF, Gelber RD, Goldhirsch A, Gusterson BA, Trihia H, et al. Prognostic importance of thymidylate synthase expression in early breast cancer. J Clin Oncol. 1997;15(5):1923–31.CrossRefPubMedGoogle Scholar
  11. 11.
    Hagiwara K, Kochi M, Fujii M, Song K, Tamegai H, Watanabe M, et al. Radiochemotherapy for esophageal squamous cell carcinoma in elderly patients. Hepatogastroenterology. 2014;61(134):1617–22.PubMedGoogle Scholar
  12. 12.
    Johnston PG, Lenz HJ, Leichman CG, Danenberg KD, Allegra CJ, Danenberg PV, et al. Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors. Cancer Res. 1995;55(7):1407–12.PubMedGoogle Scholar
  13. 13.
    Suzuki R, Kang Y, Li X, Roife D, Zhang R, Fleming JB. Genistein potentiates the antitumor effect of 5-fluorouracil by inducing apoptosis and autophagy in human pancreatic cancer cells. Anticancer Res. 2014;34(9):4685–92.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Mizutani Y, Wada H, Yoshida O, Fukushima M, Nonomura M, Nakao M, et al. Significance of thymidylate synthase activity in renal cell carcinoma. Clin Cancer Res. 2003;9(4):1453–60.PubMedGoogle Scholar
  15. 15.
    Leichman L, Leichman CG. Therapy of disseminated colorectal cancer: the emerging role of intratumoral molecular biology. Eur J Surg Suppl. 2001;586:43–8.CrossRefGoogle Scholar
  16. 16.
    Lakshmaiah KC, Jacob LA, Aparna S, Lokanatha D, Saldanha SC. Epigenetic therapy of cancer with histone deacetylase inhibitors. J Cancer Res Ther. 2014;10(3):469–78.PubMedGoogle Scholar
  17. 17.
    Bose P, Dai Y, Grant S. Histone deacetylase inhibitor (HDACI) mechanisms of action: emerging insights. Pharmacol Ther. 2014;143(3):323–36.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Anestopoulos I, Voulgaridou GP, Georgakilas AG, Franco R, Pappa A, Panayiotidis MI. Epigenetic therapy as a novel approach in hepatocellular carcinoma. Pharmacol Ther. 2015;145C:103–19.CrossRefGoogle Scholar
  19. 19.
    Jafary H, Ahmadian S, Soleimani M. The enhanced apoptosis and antiproliferative response to combined treatment with valproate and nicotinamide in MCF-7 breast cancer cells. Tumour Biol. 2014;35(3):2701–10.CrossRefPubMedGoogle Scholar
  20. 20.
    Duvic M, Talpur R, Ni X, Zhang C, Hazarika P, Kelly C, et al. Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory cutaneous T-cell lymphoma (CTCL). Blood. 2007;109(1):31–9.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Olsen EA, Kim YH, Kuzel TM, Pacheco TR, Foss FM, Parker S, et al. Phase IIb multicenter trial of vorinostat in patients with persistent, progressive, or treatment refractory cutaneous T-cell lymphoma. J Clin Oncol. 2007;25(21):3109–15.CrossRefPubMedGoogle Scholar
  22. 22.
    Lane AA, Chabner BA. Histone deacetylase inhibitors in cancer therapy. J Clin Oncol. 2009;27(32):5459–68.CrossRefPubMedGoogle Scholar
  23. 23.
    Lee MJ, Kim YS, Kummar S, Giaccone G, Trepel JB. Histone deacetylase inhibitors in cancer therapy. Curr Opin Oncol. 2008;20(6):639–49.CrossRefPubMedGoogle Scholar
  24. 24.
    Bots M, Johnstone RW. Rational combinations using HDAC inhibitors. Clin Cancer Res. 2009;15(12):3970–7.CrossRefPubMedGoogle Scholar
  25. 25.
    Lee JH, Park JH, Jung Y, Kim JH, Jong HS, Kim TY, et al. Histone deacetylase inhibitor enhances 5-fluorouracil cytotoxicity by down-regulating thymidylate synthase in human cancer cells. Mol Cancer Ther. 2006;5(12):3085–95.CrossRefPubMedGoogle Scholar
  26. 26.
    Kim MJ, Lee JS, Park SE, Yi HJ, Jeong IG, Kang JS, Yun J, Lee JY, Ro S, Choi EK, Hwang JJ, Kim CS. Combination treatment of renal cell carcinoma with Belinostat and 5-fluorouracil: a role for oxidative stress induced DNA damage and HSP90 regulated thymidine synthase. J Urol. 2014.Google Scholar
  27. 27.
    Noro R, Miyanaga A, Minegishi Y, Okano T, Seike M, Soeno C, et al. Histone deacetylase inhibitor enhances sensitivity of non-small-cell lung cancer cells to 5-FU/S-1 via down-regulation of thymidylate synthase expression and up-regulation of p21(waf1/cip1) expression. Cancer Sci. 2010;101(6):1424–30.CrossRefPubMedGoogle Scholar
  28. 28.
    Fazzone W, Wilson PM, Labonte MJ, Lenz HJ, Ladner RD. Histone deacetylase inhibitors suppress thymidylate synthase gene expression and synergize with the fluoropyrimidines in colon cancer cells. Int J Cancer. 2009;125(2):463–73.CrossRefPubMedGoogle Scholar
  29. 29.
    Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006;58(3):621–81.CrossRefPubMedGoogle Scholar
  30. 30.
    Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010;70(2):440–6.CrossRefPubMedGoogle Scholar
  31. 31.
    Chen L, Zhang W, Zhou QD, Yang HQ, Liang HF, Zhang BX, et al. HSCs play a distinct role in different phases of oval cell-mediated liver regeneration. Cell Biochem Funct. 2012;30(7):588–96.CrossRefPubMedGoogle Scholar
  32. 32.
    Ding ZY, Jin GN, Wang W, Chen WX, Wu YH, Ai X, et al. Reduced expression of transcriptional intermediary factor 1 gamma promotes metastasis and indicates poor prognosis of hepatocellular carcinoma. Hepatology. 2014;60(5):1620–36.CrossRefPubMedGoogle Scholar
  33. 33.
    Zhang B, Halder SK, Kashikar ND, Cho YJ, Datta A, Gorden DL, et al. Antimetastatic role of Smad4 signaling in colorectal cancer. Gastroenterology. 2010;138(3):969–80.e1–3.CrossRefPubMedGoogle Scholar
  34. 34.
    Wei S, Xiong M, Zhan DQ, Liang BY, Wang YY, Gutmann DH, et al. Ku80 functions as a tumor suppressor in hepatocellular carcinoma by inducing S-phase arrest through a p53-dependent pathway. Carcinogenesis. 2012;33(3):538–47.CrossRefPubMedGoogle Scholar
  35. 35.
    Chen L, Zhang W, Liang HF, Zhou QF, Ding ZY, Yang HQ, et al. Activin A induces growth arrest through a SMAD- dependent pathway in hepatic progenitor cells. Cell Commun Signal. 2014;12:18.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Zhang B, Chen X, Bae S, Singh K, Washington MK, Datta PK. Loss of Smad4 in colorectal cancer induces resistance to 5-fluorouracil through activating Akt pathway. Br J Cancer. 2014;110(4):946–57.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Lachenmayer A, Toffanin S, Cabellos L, Alsinet C, Hoshida Y, Villanueva A, et al. Combination therapy for hepatocellular carcinoma: additive preclinical efficacy of the HDAC inhibitor panobinostat with sorafenib. J Hepatol. 2012;56(6):1343–50.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Lee YH, Seo D, Choi KJ, Andersen JB, Won MA, Kitade M, et al. Antitumor effects in hepatocarcinoma of isoform-selective inhibition of HDAC2. Cancer Res. 2014;74(17):4752–61.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Fan J, Lou B, Chen W, Zhang J, Lin S, Lv FF, et al. Down-regulation of HDAC5 inhibits growth of human hepatocellular carcinoma by induction of apoptosis and cell cycle arrest. Tumour Biol. 2014;35(11):11523–32.CrossRefPubMedGoogle Scholar
  40. 40.
    Carlisi D, Vassallo B, Lauricella M, Emanuele S, D’Anneo A, Di Leonardo E, et al. Histone deacetylase inhibitors induce in human hepatoma HepG2 cells acetylation of p53 and histones in correlation with apoptotic effects. Int J Oncol. 2008;32(1):177–84.PubMedGoogle Scholar
  41. 41.
    Ocker M, Alajati A, Ganslmayer M, Zopf S, Luders M, Neureiter D, et al. The histone-deacetylase inhibitor SAHA potentiates proapoptotic effects of 5-fluorouracil and irinotecan in hepatoma cells. J Cancer Res Clin Oncol. 2005;131(6):385–94.CrossRefPubMedGoogle Scholar
  42. 42.
    Liu YL, Yang PM, Shun CT, Wu MS, Weng JR, Chen CC. Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma. Autophagy. 2010;6(8):1057–65.CrossRefPubMedGoogle Scholar
  43. 43.
    Kramer OH, Mahboobi S, Sellmer A. Drugging the HDAC6-HSP90 interplay in malignant cells. Trends Pharmacol Sci. 2014;35(10):501–9.CrossRefPubMedGoogle Scholar
  44. 44.
    Aoyagi S, Archer TK. Modulating molecular chaperone Hsp90 functions through reversible acetylation. Trends Cell Biol. 2005;15(11):565–7.CrossRefPubMedGoogle Scholar
  45. 45.
    Bali P, Pranpat M, Bradner J, Balasis M, Fiskus W, Guo F, et al. Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors. J Biol Chem. 2005;280(29):26729–34.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  1. 1.Hepatic Surgery Centre, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina

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