The Histone Deacetylase Inhibitor Trichostatin A Sensitizes Human Renal Carcinoma Cells to TRAIL-Induced Apoptosis through Down-Regulation of c-FLIPL

  • Han, Min Ho (Department of Biochemistry, College of Oriental Medicine, Dong-Eui University) ;
  • Park, Cheol (Department of Molecular Biology, College of Natural Sciences, Dongeui University) ;
  • Kwon, Taek Kyu (Department of Immunology, School of Medicine, Keimyung University) ;
  • Kim, Gi-Young (Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University) ;
  • Kim, Wun-Jae (Department of Urology, College of Medicine, Chungbuk National University) ;
  • Hong, Sang Hoon (Department of Internal Medicine, College of Oriental Medicine, Dong-Eui University) ;
  • Yoo, Young Hyun (Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Mitochondria Hub Regulation Center) ;
  • Choi, Yung Hyun (Department of Biochemistry, College of Oriental Medicine, Dong-Eui University)
  • Received : 2014.08.11
  • Accepted : 2014.10.07
  • Published : 2015.01.01


Histone acetylation plays a critical role in the regulation of transcription by altering the structure of chromatin, and it may influence the resistance of some tumor cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by regulating the gene expression of components of the TRAIL signaling pathway. In this study, we investigated the effects and molecular mechanisms of trichostatin A (TSA), a histone deacetylase inhibitor, in sensitizing TRAIL-induced apoptosis in Caki human renal carcinoma cells. Our results indicate that nontoxic concentrations of TSA substantially enhance TRAIL-induced apoptosis compared with treatment with either agent alone. Cotreatment with TSA and TRAIL effectively induced cleavage of Bid and loss of mitochondrial membrane potential (MMP), which was associated with the activation of caspases (-3, -8, and -9) and degradation of poly (ADP-ribose) polymerase (PARP), contributing toward the sensitization to TRAIL. Combined treatment with TSA and TRAIL significantly reduced the levels of the cellular Fas-associated death domain (FADD)-like interleukin-$1{\beta}$-converting enzyme (FLICE) inhibitory protein (c-FLIP), whereas those of death receptor (DR) 4, DR5, and FADD remained unchanged. The synergistic effect of TAS and TRAIL was perfectly attenuated in c-$FLIP_L$-overexpressing Caki cells. Taken together, the present study demonstrates that down-regulation of c-FLIP contributes to TSA-facilitated TRAIL-induced apoptosis, amplifying the death receptor, as well as mitochondria-mediated apoptotic signaling pathways.



Supported by : National Research Foundation of Korea (NRF)


  1. Abdulghani, J. and El-Deiry, W. S. (2010) TRAIL receptor signaling and therapeutics. Expert Opin. Ther. Targets 14, 1091-1108.
  2. Ather, M. H., Masood, N. and Siddiqui, T. (2010) Current management of advanced and metastatic renal cell carcinoma. Urol. J. 7, 1-9.
  3. Barneda-Zahonero, B. and Parra, M. (2012) Histone deacetylases and cancer. Mol. Oncol. 6, 579-589.
  4. Benayoun, B., Baghdiguian, S., Lajmanovich, A., Bartoli, M., Daniele, N., Gicquel, E., Bourg, N., Raynaud, F., Pasquier, M. A., Suel, L., Lochmuller, H., Lefranc, G. and Richard, I. (2008) NF-kappaB-dependent expression of the antiapoptotic factor c-FLIP is regulated by calpain 3, the protein involved in limb-girdle muscular dystrophy type 2A. FASEB J. 22, 1521-1529.
  5. Billen, L. P., Shamas-Din, A. and Andrews, D. W. (2008) Bid: a Bax-like BH3 protein. Oncogene 27, S93-104.
  6. Brugarolas, J. (2007) Renal-cell carcinoma--molecular pathways and therapies. N. Engl. J. Med. 356, 185-187.
  7. Carew, J. S., Giles, F. J. and Nawrocki, S. T. (2008) Histone deacetylase inhibitors: mechanisms of cell death and promise in combination cancer therapy. Cancer Lett. 269, 7-17.
  8. Crowder, R. N. and El-Deiry, W. S. (2012) Caspase-8 regulation of TRAIL-mediated cell death. Exp. Oncol. 34, 160-164.
  9. Fulda, S. (2012) Histone deacetylase (HDAC) inhibitors and regulation of TRAIL-induced apoptosis. Exp. Cell Res. 318, 1208-1212.
  10. Gimenez-Bonafe, P., Tortosa, A. and Perez-Tomas, R. (2009) Overcoming drug resistance by enhancing apoptosis of tumor cells. Curr. Cancer Drug Targets 9, 320-340.
  11. Inman, B. A., Harrison, M. R. and George, D. J. (2013) Novel immunotherapeutic strategies in development for renal cell carcinoma. Eur. Urol. 63, 881-889.
  12. Hwang, E. Y., Jeong, M. S., Park, S. Y. and Jang, S. B. (2014) Evidence of complex formation between FADD and c-FLIP death effector domains for the death inducing signaling complex. BMB Rep. 47, 488-493.
  13. Jang, J. H., Lee, T. J., Yang, E. S., Min, do. S., Kim, Y. H., Kim, S. H., Choi, Y. H., Park, J. W., Choi, K. S. and Kwon, T. K. (2010) Compound C sensitizes Caki renal cancer cells to TRAIL-induced apoptosis through reactive oxygen species-mediated down-regulation of c-FLIPL and Mcl-1. Exp. Cell Res. 316, 2194-2203.
  14. Jin, C. Y., Park, C., Cheong, J., Choi, B. T., Lee, T. H., Lee, J. D., Lee, W. H., Kim, G. Y., Ryu, C. H. and Choi, Y. H. (2007) Genistein sensitizes TRAIL-resistant human gastric adenocarcinoma AGS cells through activation of caspase-3. Cancer Lett. 257, 56-64.
  15. Jin, C. Y., Park, C., Hwang, H. J., Kim, G. Y., Choi, B. T., Kim, W. J. and Choi, Y. H. (2011) Naringenin up-regulates the expression of death receptor 5 and enhances TRAIL-induced apoptosis in human lung cancer A549 cells. Mol. Nutr. Food Res. 55, 300-309.
  16. Jonsson, G., Paulie, S. and Grandien, A. (2003) High level of cFLIP correlates with resistance to death receptor-induced apoptosis in bladder carcinoma cells. Anticancer Res. 23, 1213-1218.
  17. Kantari, C. and Walczak, H. (2011) Caspase-8 and bid: caught in the act between death receptors and mitochondria. Biochim. Biophys. Acta 1813, 558-563.
  18. Kelly, M. M., Hoel, B. D. and Voelkel-Johnson, C. (2002) Doxorubicin pretreatment sensitizes prostate cancer cell lines to TRAIL induced apoptosis which correlates with the loss of c-FLIP expression. Cancer Biol. Ther. 1, 520-527.
  19. Kouraklis, G. and Theocharis, S. (2002) Histone deacetylase inhibitors and anticancer therapy. Curr. Med. Chem. Anticancer Agents 2, 477-484.
  20. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G. and Earnshaw, W. C. (1994) Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371, 346-347.
  21. Lee, S. J., Noh, H. J., Sung, E. G., Song, I. H., Kim, J. Y., Kwon, T. K. and Lee, T. J. (2011) Berberine sensitizes TRAIL-induced apoptosis through proteasome-mediated downregulation of c-FLIP and Mcl-1 proteins. Int. J. Oncol. 38, 485-492.
  22. Li, Z. and Gao, Q. (2013) Induction of apoptosis in HT-29 cells by quercetin through mitochondria-mediated apoptotic pathway. Animal Cells Syst. 17, 147-153
  23. Macher-Goeppinger, S., Aulmann, S., Tagscherer, K. E., Wagener, N., Haferkamp, A., Penzel, R., Brauckhoff, A., Hohenfellner, M., Sykora, J., Walczak, H., The, B. T., Autschbach, F., Herpel, E., Schirmacher, P. and Roth, W. (2009) Prognostic value of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors in renal cell cancer. Clin. Cancer Res. 15, 650-659.
  24. Matsuda, F., Inoue, N., Goto, Y., Maeda, A., Cheng, Y., Sakamaki, K. and Manabe, N. (2008) cFLIP regulates death receptor-mediated apoptosis in an ovarian granulosa cell line by inhibiting procaspase- 8 cleavage. J. Reprod. Dev. 54, 314-320.
  25. Min, K. J., Seo, B. R., Bae, Y. C., Yoo, Y. H. and Kwon, T. K. (2014) Antipsychotic agent thioridazine sensitizes renal carcinoma Caki cells to TRAIL-induced apoptosis through reactive oxygen speciesmediated inhibition of Akt signaling and downregulation of Mcl-1 and c-FLIP(L). Cell Death Dis. 5, e1063.
  26. Mizutani, Y., Nakanishi, H., Yoshida, O., Fukushima, M., Bonavida, B. and Miki, T. (2002) Potentiation of the sensitivity of renal cell carcinoma cells to TRAIL-mediated apoptosis by subtoxic concentrations of 5-fluorouracil. Eur. J. Cancer 38, 167-176.
  27. Monneret, C. (2005) Histone deacetylase inhibitors. Eur. J. Med. Chem. 40, 1-13.
  28. Muhlethaler-Mottet, A., Flahaut, M., Bourloud, K. B., Auderset, K., Meier, R., Joseph, J. M. and Gross, N. (2006) Histone deacetylase inhibitors strongly sensitise neuroblastoma cells to TRAIL-induced apoptosis by a caspases-dependent increase of the pro- to antiapoptotic proteins ratio. BMC Cancer 6, 214.
  29. Murtaza, I., Saleem, M., Adhami, V. M., Hafeez, B. B. and Mukhtar, H. (2009) Suppression of cFLIP by lupeol, a dietary triterpene, is sufficient to overcome resistance to TRAIL-mediated apoptosis in chemoresistant human pancreatic cancer cells. Cancer Res. 69, 1156-1165.
  30. O'Kane, H. F., Watson, C. J., Johnston, S. R., Petak, I., Watson, R. W. and Williamson, K. E. (2006) Targeting death receptors in bladder, prostate and renal cancer. J. Urol. 175, 432-438.
  31. Palacios, C., Yerbes, R. and Lopez-Rivas, A. (2006) Flavopiridol induces cellular FLICE-inhibitory protein degradation by the proteasome and promotes TRAIL-induced early signaling and apoptosis in breast tumor cells. Cancer Res. 66, 8858-8869.
  32. Park, C., Jin, C. Y., Hwang, H. J., Kim, G. Y., Jung, J. H., Kim, W. J., Yoo, Y. H. and Choi, Y. H. (2012) J7, a methyl jasmonate derivative, enhances TRAIL-mediated apoptosis through up-regulation of reactive oxygen species generation in human hepatoma HepG2 cells. Toxicol. In Vitro 26, 86-93.
  33. Rae, C., Langa, S., Tucker, S. J. and MacEwan, D. J. (2007) Elevated NF-kappaB responses and FLIP levels in leukemic but not normal lymphocytes: reduction by salicylate allows TNF-induced apoptosis. Proc. Natl. Acad. Sci. U.S.A. 104, 12790-12795.
  34. Safa, A. R. and Pollok, K. E. (2011) Targeting the anti-apoptotic protein c-FLIP for cancer therapy. Cancers (Basel) 3, 1639-1671.
  35. Seo, O. W., Kim, J. H., Lee, K. S., Lee, K. S., Kim, J. H., Won, M. H., Ha, K. S., Kwon, Y. G. and Kim, Y. M. (2012) Kurarinone promotes TRAIL-induced apoptosis by inhibiting NF-${\kappa}B$-dependent cFLIP expression in HeLa cells. Exp. Mol. Med. 44, 653-664.
  36. Son, Y. G., Kim, E. H., Kim, J. Y., Kim, S. U., Kwon, T. K., Yoon, A. R., Yun, C. O. and Choi, K. S. (2007) Silibinin sensitizes human glioma cells to TRAIL-mediated apoptosis via DR5 up-regulation and down-regulation of c-FLIP and survivin. Cancer Res. 67, 8274-8284.
  37. Sonnemann, J., Gange, J., Kumar, K. S., Muller, C., Bader, P. and Beck, J. F. (2005) Histone deacetylase inhibitors interact synergistically with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis in carcinoma cell lines. Invest. New Drugs 23, 99-109.
  38. Srivastava, R. K. (2001) TRAIL/Apo-2L: mechanisms and clinical applications in cancer. Neoplasia 3, 535-546.
  39. Staehler, M., Rohrmann, K., Haseke, N., Stief, C. G. and Siebels, M. (2005) Targeted agents for the treatment of advanced renal cell carcinoma. Curr. Drug Targets 6, 835-846.
  40. Teraishi, F,, Kagawa, S., Watanabe, T., Tango, Y., Kawashima, T., Umeoka, T., Nisizaki, M., Tanaka, N. and Fujiwara, T. (2005) ZD1839 (Gefitinib, 'Iressa'), an epidermal growth factor receptortyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma. FEBS Lett. 579, 4069-4075.
  41. Thomas, L. R., Johnson, R. L., Reed, J. C. and Thorburn, A. (2004) The C-terminal tails of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas receptors have opposing functions in Fas-associated death domain (FADD) recruitment and can regulate agonist-specific mechanisms of receptor activation. J. Biol. Chem. 279, 52479-52486.
  42. Van Geelen, C. M., de Vries, E. G. and de Jong, S. (2004) Lessons from TRAIL-resistance mechanisms in colorectal cancer cells: paving the road to patient-tailored therapy. Drug Resist. Updat. 7, 345-358.
  43. Voelkel-Johnson, C. (2011) TRAIL-mediated signaling in prostate, bladder and renal cancer. Nat. Rev. Urol. 8, 417-427.
  44. Woo, S. M., Min, K. J. and Kwon, T. K. (2012) Calyculin A causes sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by ROS-mediated down-regulation of cellular FLICE-inhibiting protein (c-FLIP) and by enhancing death receptor 4 mRNA stabilization. Apoptosis 17, 1223-1234.
  45. Yang, X. J. and Seto, E. (2007) HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention. Oncogene 26, 5310-5318.
  46. Yoon, M. J., Kang, Y. J., Kim, I. Y., Kim, E. H., Lee, J. A., Lim, J. H., Kwon, T. K. and Choi, K. S. (2013) Monensin, a polyether ionophore antibiotic, overcomes TRAIL resistance in glioma cells via endoplasmic reticulum stress, DR5 upregulation and c-FLIP downregulation. Carcinogenesis 34, 1918-1928.
  47. Yoshida, M., Matsuyama, A., Komatsu, Y. and Nishino, N. (2003) From discovery to the coming generation of histone deacetylase inhibitors. Curr. Med. Chem. 10, 2351-2358.
  48. Zhang, X., Jin, T. G., Yang, H., DeWolf, W. C., Khosravi-Far, R. and Olumi, A. F. (2004) Persistent c-FLIP(L) expression is necessary and sufficient to maintain resistance to tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in prostate cancer. Cancer Res. 64, 7086-7091.

Cited by

  1. Ethanolic extract of Descurainia sophia seeds sensitizes A549 human lung cancer cells to TRAIL cytotoxicity by upregulating death receptors vol.16, pp.1, 2016,
  2. Trichostatin A potentiates TRAIL-induced antitumor effects via inhibition of ERK/FOXM1 pathway in gastric cancer vol.37, pp.8, 2016,
  3. Effects of Sodium Butyrate, a Histone Deacetylase Inhibitor, on TRAIL-mediated Apoptosis in Human Bladder Cancer Cells vol.26, pp.4, 2016,
  4. Trichostatin A Enhances the Apoptotic Potential of Palladium Nanoparticles in Human Cervical Cancer Cells vol.17, pp.8, 2016,
  5. Marine Drugs Regulating Apoptosis Induced by Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) vol.13, pp.11, 2015,
  6. Characterization of the aminopyridine derivative KRC-180 as a JAK2 inhibitor vol.14, pp.2, 2017,
  7. Preclinical trial of the multi-targeted lenvatinib in combination with cellular immunotherapy for treatment of renal cell carcinoma vol.14, pp.4, 2017,
  8. Droxinostat sensitizes human colon cancer cells to apoptotic cell death via induction of oxidative stress vol.23, pp.1, 2018,