Combination Treatment of p53-Null HL-60 cells with Histone Deacetylase Inhibitors and Chlorambucil Augments Apoptosis and Increases BCL6 and p21 Gene Expression

Author(s): Faith A.A. Kwa*, Merrole F. Cole-Sinclair, Miroslav K. Kapuscinski.

Journal Name: Current Molecular Pharmacology

Volume 12 , Issue 1 , 2019

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Abstract:

Background: Treatment of hematological malignancies with conventional DNA-damaging drugs, such as chlorambucil (CLB), commonly results in p53-dependent chemo-resistance. Chromatin modifying agents, such as histone deacetylase inhibitors (HDACIs), sodium butyrate (NaBu) and trichostatin A (TSA), may reverse chemo-resistance by modulating the activity of chromatin remodeling enzymes and/or genes that control cell proliferation, differentiation and survival.

Objective: This study examined the potential use of HDACIs and CLB combination therapies in an in vitro chemo-resistant leukemia model.

Methods: The p53-null promyelocytic leukemia cell line, HL60, was used as an in vitro model of chemo-resistant leukemia. Drug cytotoxicity was determined by tetrazolium salt-based colorimetric assays and Annexin V/propidium iodide staining (flow cytometry). The level of mRNA expression of the chromatin modifying genes was measured by quantitative real-time PCR.

Results: Micromolar concentrations of CLB combined with either NaBu or TSA triggered synergistic cytotoxic effects in HL-60 cells (p < 0.001). The effects of the combination treatments resulted in upregulated p21 gene expression (up to 59-fold; p<0.001) that preceded an increase in BCL6 gene expression (up to 20-fold; p < 0.001). Statistically significant but smaller magnitude changes (≤ 2-fold; p <0.05) were noted in the expression of other genes studied regardless of the treatment type.

Conclusion: The combination treatment of p53-null HL-60 cells with DNA-damaging agent CLB and HDACIs NaBu and TSA triggered additive to synergistic effects on apoptosis and upregulated BCL6 and p21 expression. These findings reveal BCL6 and p21 as potential targets of chemo-resistance for the development of anti-leukemic drugs.

Keywords: Chlorambucil, chromatin, drug resistance, cyclin-dependent kinase inhibitor p21, histone deacetylase inhibitors, proto-oncogene BCL6, sodium butyrate, trichostatin A.

[1]
Eichhorst, B.; Goede, V.; Hallek, M. Treatment of elderly patients with chronic lymphocytic leukemia. Leuk. Lymphoma, 2009, 50, 171-178.
[2]
Hillmen, P.; Robak, T.; Janssens, A.; Babu, K.G.; Kloczko, J.; Grosicki, S.; Doubek, M.; Panagiotidis, P.; Kimby, E.; Schuh, A.; Pettitt, A.R.; Boyd, T.; Montillo, M.; Gupta, I.; Wright, O.; Dixon, L.; Carey, J.L.; Chang, C.; Lisby, S.; McKeown, A.; Offner, F. Chlorambucil plus ofatumumab versus chlorambucil alone in previously untreated patients with chronic lymphocytic leukaemia (COMPLEMENT 1): A randomised, multicentre, open-label phase 3 trial. Lancet, 2015, 385, 1873-1883.
[3]
Stilgenbauer, S. Prognostic markers and standard management of chronic lymphocytic leukemia. Ash Educ. Program Book, 2015, 2015(1), 368-377.
[4]
Rigg, R.A.; Aslan, J.E.; Healy, L.D.; Wallisch, M.; Thierheimer, M.L.; Loren, C.P.; Pang, J.; Hinds, M.T.; Gruber, A.; McCarty, O.J. Oral administration of Bruton’s tyrosine kinase inhibitors impairs GPVI-mediated platelet function. Am. J. Physiol. Cell Physiol., 2016, 310(5), C373-C380.
[5]
Hebb, J.; Assouline, S.; Rousseau, C.; DesJardins, P.; Caplan, S.; Egorin, M.J.; Amrein, L.; Aloyz, R.; Panasci, L. A phase I study of imatinib mesylate in combination with chlorambucil in previously treated chronic lymphocytic leukemia patients. Cancer Chemother. Pharmacol., 2011, 68(3), 643-651.
[6]
Hallek, M.; Shanafelt, T.D.; Eichhorst, B. Chronic lymphocytic leukaemia. The Lancet, 2018, 39(10129), 1524-1537.
[7]
Strati, P.; Jain, N.; O’Brien, S. Chronic Lymphocytic Leukemia: Diagnosis and Treatment. Mayo Clin. Proc., 2018, 93(5), 651-664.
[8]
Sturm, I.; Bosanquet, A.G.; Hermann, S.; Güner, D.; Dörken, B.; Daniel, P.T. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy. Cell Death Differ., 2003, 10, 477.
[9]
Bush, J.A.; Li, G. Cancer chemoresistance: The relationship between p53 and multidrug transporters. Int. J. Cancer, 2002, 98(3), 323-330.
[10]
Wattel, E.; Preudhomme, C.; Hecquet, B.; Vanrumbeke, M.; Quesnel, B.; Dervite, I.; Morel, P.; Fenaux, P. p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies. Blood, 1994, 84(9), 3148-3157.
[11]
Herman, G.; Baylin, S.B. Gene silencing in cancer in association with promoter hypermethylation. N. Engl. J. Med., 2003, 349, 2042-2054.
[12]
Carter, B.Z.; Milella, M.; Altieri, D.C.; Andreeff, M. Cytokine-regulated expression of survivin in myeloid leukemia. Blood, 2001, 97, 2784-2790.
[13]
Ma, X.; Ezzeldin, H.H.; Diasio, R.B. Histone deacetylase inhibitors: Current status and overview of recent clinical trials. Drugs, 2009, 69, 1911-1934.
[14]
Shaffer, A.L.; Yu, X.; He, Y.; Boldrick, J.; Chan, E.P.; Staudt, L.M. BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity, 2000, 13, 199-212.
[15]
Phan, R.T.; Saito, M.; Basso, K.; Niu, H.; Dalla-Favera, R. BCL6 interacts with the transcription factor Miz-1 to suppress the cyclin-dependent kinase inhibitor p21 and cell cycle arrest in germinal centre B cells. Nat. Immunol., 2005, 6(10), 1054-1060.
[16]
Fujiki, R.; Kim, M.; Sasaki, Y.; Yoshimura, K.; Hirochika, K.; Kato, S. Ligand-induced transrepression by VDR through association of WSTF with acetylated histones. EMBO J., 2005, 24, 3881-3894.
[17]
Nahi, H.; Selivanova, G.; Lehmann, S.; Mollgard, L.; Bengtzen, S.; Concha, H.; Svensson, A.; Wiman, K.G.; Merup, M.; Paul, C. Mutated and non-mutated TP53 as targets in the treatment of leukaemia. Br. J. Haematol., 2008, 141, 445-453.
[18]
Kachalaki, S.; Baradaran, B.; Majidi, J.; Yousefi, M.; Shanehbandi, D.; Mohammadinejad, S.; Mansoori, B. Reversal of chemoresistance with small interference RNA (siRNA) in etoposide resistant acute myeloid leukemia cells (HL-60). Biomed. Pharmacother., 2015, 75, 100-104.
[19]
Pepper, C.; Thomas, A.; Tucker, H. Flow cytometric assessment of three different methods for the measurement of in-vitro apoptosis. Leuk. Res., 1998, 22, 439-444.
[20]
Rieger, A.M.; Nelson, K.L.; Konowalchuk, J.D.; Barreda, D.R. Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death. J. Vis. Exp., 2011, 50, 2597.
[http://dx.doi.org/10.3791/2597]
[21]
Aubry, J-P.; Blaecke, A.; Lecoanet-Henchoz, S.; Jeannin, P.; Herbault, N.; Caron, G.; Moine, V.; Bonnefoy, J.Y. Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity. Cytometry, 1999, 37, 197-204.
[22]
Jones, L.B.; Secomb, T.W.; Dewhirst, M.W.; El-Kareh, A.W. The additive damage model: A mathematical model for cellular responses to drug combinations. J. Theor. Biol., 2014, 357, 10-20.
[23]
Abruzzo, L.V.; Lee, K.Y.; Fuller, A.; Silverman, A.; Keating, M.J.; Coombes, K.R. Validation of oligonucleotide microarray data using microfluidic low-density arrays: A new statistical method to normalize real-time RT-PCR data. Biotechniques, 2005, 38, 785-792.
[24]
Kwa, F.A.; Cole-Sinclair, M.; Kapuscinski, M. Chlorambucil-sensitive and -resistant lymphoid cells display different responses to the histone deacetylase inhibitor, sodium butyrate. Biochem. Biophys. Res. Commun., 2010, 403, 288-292.
[25]
Mullick, A.; Elias, M.; Harakidas, P.; Marcil, A.; Whiteway, M.; Ge, B.; Hudson, T.J.; Caron, A.W.; Bourget, L.; Picard, S.; Jovcevski, O.; Massie, B.; Thomas, D.Y. Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans. Infect. Immun., 2004, 72(1), 414-429.
[26]
Rigg, R.A.; Aslan, J.E.; Healy, L.D.; Wallisch, M.; Thierheimer, M.L.D.; Loren, C.P.; Pang, J.; Hinds, M.T.; Gruber, A.; McCarty, O.J.T. Oral administration of Brutons tyrosine kinase inhibitors impairs GPVI-mediated platelet function. Am. J. Physiol. Cell Physiol., 2016, 310(5), C373.
[27]
Ahmadzadeh, A.; Khodadi, E.; Shahjahani, M.; Bertacchini, J.; Vosoughi, T.; Saki, N. The Role of HDACs as Leukemia Therapy Targets using HDI. Int. J. Hematol. Oncol. Stem Cell Res., 2015, 9(4), 203-214.
[28]
Mann, B.S.; Johnson, J.R.; Cohen, M.H.; Justice, R.; Pazdur, R. FDA approval summary: Vorinostat for treatment of advanced primary cutaneous T-cell lymphoma. Oncologist, 2007, 12(10), 1247-1252.
[29]
Kwa, F.A.; Balcrczyk, A.; Licciardi, P.; El-Osta, A.; Karagiannis, T.C. Chromatin modifying agents - the cutting edge of anticancer therapy. Drug Discov. Today, 2011, 16, 543-547.
[30]
Medina, V.; Edmonds, B.; Young, G.P.; James, R.; Appleton, S.; Zalewski, P.D. Induction of caspase-3 protease activity and apoptosis by butyrate and trichostatin a (inhibitors of histone deacetylase): Dependence on protein synthesis and synergy with a mitochondrial/cytochrome c-dependent pathway. Cancer Res., 1997, 57, 3697-3707.
[31]
Wei, Z.; Gao, W.; Wu, Y.; Ni, B.; Tian, Y. Mutual interaction between BCL6 and miRNAs contributing to the pathogenesis of various cancers. Clin. Transl. Oncol., 2015, 17(11), 841-846.
[32]
Cerchietti, L.C.; Yang, S.N.; Shaknovich, R.; Hatzi, K.; Polo, J.M.; Chadburn, A.; Dowdy, S.F.; Melnick, A. A peptomimetic inhibitor of BCL6 with potent antilymphoma effects in vitro and in vivo. Blood, 2009, 113(15), 3397-3405.
[33]
Choi, J.H.; Min, N.Y.; Park, J.; Kim, J.H.; Park, S.H.; Ko, Y.J.; Kang, Y.; Moon, Y.J.; Rhee, S.; Ham, S.W.; Park, A.J.; Lee, K.H. TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116. Biochem. Biophys. Res. Commun., 2010, 39(1), 449-454.
[34]
Parker, B.S.; Cutts, S.M.; Nudelman, A.; Rephaeli, A.; Phillips, D.R.; Sukumar, S. Mitoxantrone mediates demethylation and reexpression of Cyclin D2, estrogen receptor and 14.3.3 sigma in breast cancer cells. Cancer Biol. Ther., 2003, 2(3), 259-263.
[35]
Kato, S.; Fujiki, R.; Kim, M.S.; Kitagawa, H. Ligand-induced transrepressive function of VDR requires a chromatin remodelling complex, WINAC. J. Steroid Biochem. Mol. Biol., 2007, 103, 372-380.
[36]
Daniel, C.; Schroder, O.; Zahn, N.; Gaschott, T.; Steinhilber, D.; Stein, J.M. The TGF beta/Smad 3-signaling pathway is involved in butyrate-mediated vitamin D receptor (VDR)-expression. J. Cell. Biochem., 2007, 102(6), 1420-1431.
[37]
Ting, H.J.; Bao, B.Y.; Reeder, J.E.; Messing, E.M.; Lee, Y.F. Increased expression of corepressors in aggressive androgen-independent prostate cancer cells results in loss of 1 alpha,25-dihydroxyvitamin D-3 responsiveness. Mol. Cancer Res., 2007, 5(9), 967-980.
[38]
Secchiero, P.; di Iasio, M.G.; Gonelli, A.; Barbarotto, E.; Melloni, E.; Tiribelli, M.; Chiaruttini, C.; Zauli, G. Differential gene expression induction by TRAIL in B chronic lymphocytic leukemia (B-CLL) cells showing high versus low levels of zap-70. J. Cell. Physiol., 2007, 21(1), 229-236.
[39]
Park, D.G. The changes of expression of survivin by butyrate in HCT116 colon cancer cells. J. Korean Surg. Soc., 2009, 77(5), 297-305.
[40]
Sah, N.K.; Munshi, A.; Hobbs, M.; Carter, B.Z.; Andreeff, M.; Meyn, R.E. Effect of downregulation of survivin expression on radiosensitivity of human epidermoid carcinoma cells. Int. J. Radiat. Oncol. Biol. Phys., 2006, 66(3), 852-859.
[41]
Saldanha, S.; Kala, R.; Tollefsbol, T. Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate. Exp. Cell Res., 2014, 324(1), 40-53.
[42]
Ge, Z.; Wang, H.; Parthum, M.R.; Hoffman, C.S.; Annunziato, A.T. Nuclear Hat1p complex (NuB4) components participate in DNA repair-linked chromatin reassembly. J. Biol. Chem., 2011, 286, 16790-16799.
[43]
Lemercier, C.; Brocard, M.; Puvion-Dutilleul, F.; Kao, H.; Albagli, O.; Khochbin, S. Class II histone deacetylases are directly recruited by BCL6 transcriptional repressor. J. Biol. Chem., 2002, 277, 22045-22052.
[44]
Zhang, H.; Okada, S.; Hatano, M.; Okabe, S.; Tokuhisa, T. A new functional domain of BCL6 family that recruits histone deacetylases. Biochim. Biophys. Acta, 2001, 540, 188-200.
[45]
Mottet, D.; Castronovo, V. Histone deacetylases: Target enzymes for cancer therapy. Clin. Exp. Metastasis, 2008, 25, 183-189.
[46]
Saito, M.; Novak, U.; Piovan, E.; Basso, K.; Sumazin, P.; Schneider, C.; Crespo, M.; Shen, Q.; Bhagat, G.; Califano, A. BCL6 suppression of BCL2 via Miz1 and its disruption in diffuse large B cell lymphoma. Proc. Natl. Acad. Sci. USA, 2009, 106(27), 11294-11299.
[47]
Heider, U.; Rademacher, J.; Lamottke, B.; Mieth, M.; Moebs, M.; von Metzler, I.; Assaf, C.; Sezer, O. Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteosome inhibitor bortezomib in cutaneous T cell lymphoma. Eur. J. Haematol., 2009, 82, 440-449.
[48]
Zhou, J.X.; Lee, C.H.; Qi, C.F.; Wang, H.; Naghashfar, Z.; Abbasi, S.; Morse, III, H.C. IFN Regulatory Factor 8 regulates MDM2 in germinal center B cells. J. Immunol., 2009, 183, 3188-3194.


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Article Details

VOLUME: 12
ISSUE: 1
Year: 2019
Page: [72 - 81]
Pages: 10
DOI: 10.2174/1874467211666181010161836
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