Histone acetylation and histone deacetylase activity of magnesium valproate in tumor and peripheral blood of patients with cervical cancer. A phase I study

Alma Chavez-Blanco, Blanca Segura-Pacheco, Enrique Perez-Cardenas, Lucia Taja-Chayeb, Lucely Cetina, Myrna Candelaria, David Cantu, Aurora Gonzalez-Fierro, Patricia Garcia-Lopez, Pilar Zambrano, Carlos Perez-Plasencia, Gustavo Cabrera, Catalina Trejo-Becerril, Enrique Angeles, Alfonso Duenas-Gonzalez, Alma Chavez-Blanco, Blanca Segura-Pacheco, Enrique Perez-Cardenas, Lucia Taja-Chayeb, Lucely Cetina, Myrna Candelaria, David Cantu, Aurora Gonzalez-Fierro, Patricia Garcia-Lopez, Pilar Zambrano, Carlos Perez-Plasencia, Gustavo Cabrera, Catalina Trejo-Becerril, Enrique Angeles, Alfonso Duenas-Gonzalez

Abstract

Background: The development of cancer has been associated with epigenetic alterations such as aberrant histone deacetylase (HDAC) activity. It was recently reported that valproic acid is an effective inhibitor of histone deacetylases and as such induces tumor cell differentiation, apoptosis, or growth arrest.

Methods: Twelve newly diagnosed patients with cervical cancer were treated with magnesium valproate after a baseline tumor biopsy and blood sampling at the following dose levels (four patients each): 20 mg/kg; 30 mg/kg, or 40 mg/kg for 5 days via oral route. At day 6, tumor and blood sampling were repeated and the study protocol ended. Tumor acetylation of H3 and H4 histones and HDAC activity were evaluated by Western blot and colorimetric HDAC assay respectively. Blood levels of valproic acid were determined at day 6 once the steady-state was reached. Toxicity of treatment was evaluated at the end of study period.

Results: All patients completed the study medication. Mean daily dose for all patients was 1,890 mg. Corresponding means for the doses 20-, 30-, and 40-mg/kg were 1245, 2000, and 2425 mg, respectively. Depressed level of consciousness grade 2 was registered in nine patients. Ten patients were evaluated for H3 and H4 acetylation and HDAC activity. After treatment, we observed hyperacetylation of H3 and H4 in the tumors of nine and seven patients, respectively, whereas six patients demonstrated hyperacetylation of both histones. Serum levels of valproic acid ranged from 73.6-170.49 microg/mL. Tumor deacetylase activity decreased in eight patients (80%), whereas two had either no change or a mild increase. There was a statistically significant difference between pre and post-treatment values of HDAC activity (mean, 0.36 vs. 0.21, two-tailed t test p < 0.0264). There was no correlation between H3 and H4 tumor hyperacetylation with serum levels of valproic acid.

Conclusion: Magnesium valproate at a dose between 20 and 40 mg/kg inhibits deacetylase activity and hyperacetylates histones in tumor tissues.

Figures

Figure 1
Figure 1
Western blots for anti-acetylated H3 and H4 histones pre- and post-treatment in patients receiving a dose of 20 mg/kg. Patients 2 and 4 are missed due to insufficient sample. Positive and negative controls are HeLa cells treated or not with trichostatinA. Loading control are gels stained with coomassie blue. Graphs in the inferior panel are HDAC activity expressed as optical densities (ODs), in the same scale as positive and negative controls that are also HeLa cells extracts with and without trichostatinA treatment. At the bottom are values of valproic acid in serum.
Figure 2
Figure 2
Western blots for anti-acetylated H3 and H4 histones pre- and post-treatment in patients receiving a dose of 30 mg/kg. Positive and negative controls are HeLa cells treated or not with trichostatin A. Loading control are gels stained with coomassie blue. Graphs in the inferior panel are HDAC activity expressed as ODs, in the same scale as positive and negative controls that are also HeLa cells extracts with and without trichostatinA treatment. At the bottom are values of valproic acid in serum.
Figure 3
Figure 3
Western blots for anti-acetylated H3 and H4 histones pre- and post-treatment in patients receiving a dose of 40 mg/kg. Positive and negative controls are HeLa cells treated or not with trichostatin A. Loading control are gels stained with coomassie blue. Graphs in the inferior panel are HDAC activity expressed as ODs, in the same scale as the positive and negative controls that are also HeLa cells extracts with and without trichostatin A treatment. At the bottom are values of valproic acid in serum.
Figure 4
Figure 4
Western blots for anti-acetylated H3 and H4 histones pre- and post-treatment in PBMN cells of patients 1, 2, 5, and 8. Patient 1 and 2 received a dose of 20 mg/kg, whereas patients 5 and 8 each received a dose of 30 mg/kg. Positive and negative controls are HeLa cells treated or not with thrichostatin A. Loading control are gels stained with Coomassie blue.

References

    1. Verma M, Maruvada P, Srivastava S. Epigenetics and cancer. Crit Rev Clin Lab Sci. 2004;41:585–607. doi: 10.1080/10408360490516922.
    1. Rountree MR, Bachman KE, Baylin SB. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nature Genet. 2000;25:269–277. doi: 10.1038/77023.
    1. Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature. 1998;393:386–389. doi: 10.1038/30764.
    1. Shao Y, Gao Z, Marks PA, Jiang X. Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci U S A. 2004;101:18030–18035. doi: 10.1073/pnas.0408345102.
    1. Michaelis M, Suhan T, Cinatl J, Driever PH, Cinatl J., Jr Valproic acid and interferon-alpha synergistically inhibit neuroblastoma cell growth in vitro and in vivo. Int J Oncol. 2004;25:1795–1799.
    1. Cinatl J, Jr, Kotchetkov R, Blaheta R, Driever PH, Vogel JU, Cinatl J. Induction of differentiation and suppression of malignant phenotype of human neuroblastoma BE(2)-C cells by valproic acid: enhancement by combination with interferon-alpha. Int J Oncol. 2002;20:97–106.
    1. Kawagoe R, Kawagoe H, Sano K. Valproic acid induces apoptosis in human leukemia cells by stimulating both caspase-dependent and -independent apoptotic signaling pathways. Leuk Res. 2002;26:495–502. doi: 10.1016/S0145-2126(01)00151-5.
    1. Knupfer MM, Pulzer F, Schindler I, Hernaiz Driever P, Knupfer H, Keller E. Different effects of valproic acid on proliferation and migration of malignant glioma cells in vitro. Anticancer Res. 2001;21:347–351.
    1. Marks PA, Richon VM, Rifkind RA. Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. J Natl Cancer Inst. 2000;92:1210–1215. doi: 10.1093/jnci/92.15.1210.
    1. Kim MS, Blake M, Baek JH, Kohlhagen G, Pommier Y, Carrier F. Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Res. 2003;63:7291–7300.
    1. Castro-Galache MD, Ferragut JA, Barbera VM, Martin-Orozco E, Gonzalez-Ros JM, Garcia-Morales P, Saceda M. Susceptibility of multidrug resistance tumor cells to apoptosis induction by histone deacetylase inhibitors. Int J Cancer. 2003;104:579–586. doi: 10.1002/ijc.10998.
    1. Camphausen K, Scott T, Sproull M, Tofilon PJ. Enhancement of xenograft tumor radiosensitivity by the histone deacetylase inhibitor MS-275 and correlation with histone hyperacetylation. Clin Cancer Res. 2004;10:6066–6071.
    1. Kim JH, Shin JH, Kim IH. Susceptibility and radiosensitization of human glioblastoma cells to trichostatin A, a histone deacetylase inhibitor. Int J Radiat Oncol Biol Phys. 2004;59:1174–1180. doi: 10.1016/j.ijrobp.2004.03.001.
    1. Zhang Y, Jung M, Dritschilo A, Jung M. Enhancement of radiation sensitivity of human squamous carcinoma cells by histone deacetylase inhibitors. Radiat Res. 2004;161:667–674.
    1. Loscher W. Basic pharmacology of valproate: a review after 35 years of clinical use for the treatment of epilepsy. CNS Drugs. 2002;16:669–669.
    1. Perucca E. Pharmacological and therapeutic properties of valproate: a summary after 35 years of clinical experience. CNS Drugs. 2002;16:695–714.
    1. Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem. 2001;276:36734–36741. doi: 10.1074/jbc.M101287200.
    1. Kramer OH, Zhu P, Ostendorff HP, Golebiewski M, Tiefenbach J, Peters MA, Brill B, Groner B, Bach I, Heinzel T, Gottlicher M. The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2. EMBO J. 2003;22:3411–3420. doi: 10.1093/emboj/cdg315.
    1. Vigushin DM, Coombes RC. Targeted histone deacetylase inhibition for cancer therapy. Curr Cancer Drug Targets. 2004;4:205–218. doi: 10.2174/1568009043481560.
    1. Shi H, Wei SH, Leu YW, Rahmatpanah F, Liu JC, Yan PS, Nephew KP, Huang TH. Triple analysis of the cancer epigenome: an integrated microarray system for assessing gene expression, DNA methylation, and histone acetylation. Cancer Res. 2003;63:2164–2171.
    1. Peart MJ, Smyth GK, van Laar RK, Bowtell DD, Richon VM, Marks PA, Holloway AJ, Johnstone RW. Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors. Proc Natl Acad Sci U S A. 2005;102:3697–3702. doi: 10.1073/pnas.0500369102.
    1. Mei S, Ho AD, Mahlknecht U. Role of histone deacetylase inhibitors in the treatment of cancer (Review) Int J Oncol. 2004;25:1509–1519.
    1. Chapman A, Keane PE, Meldrum BS, Simiand J, Vernieres JC. Mechanism of anticonvulsant action of valproate. Prog Neruobiol. 1982;28:963–964.
    1. Gurvich N, Tsygankova OM, Meinkoth JL, Klein PS. Histone deacetylase is a target of valproic acid-mediated cellular differentiation. Cancer Res. 2004;64:1079–1086.
    1. Driever PH, Knüpfer MM, Cinatl J, Wolff JFA. Valproic acid for the treatment of pediatric malignant glioma. Klin Pediatr. 1999;211:323–328.
    1. Olsen CM, Meussen-Elholm ETM, Roste LS, Tauboll E. Antiepileptic drugs inhibit cell growth in the human breast cancer cell line MCF7. Mol Cell Endocrinol. 2004;213:173–179. doi: 10.1016/j.mce.2003.10.032.
    1. Davis R, Peters DH, McTavish D. Valproic acid: A reappraisal of its pharmacological properties and clinical efficacy in epilepsy. Drugs. 1994;47:332–372.
    1. Parulekar WR, Eisenhauer EA. Novel endpoints and design of early clinical trials. Ann Oncol. 2002;13:139–43.
    1. Hunsberger S, Rubinstein LV, Dancey J, Korn EL. Dose escalation trial designs based on a molecularly targeted endpoint. Stat Med. 2005;24:2171–2181. doi: 10.1002/sim.2102.
    1. Parulekar WR, Eisenhauer EA. Phase I trial design for solid tumor studies of targeted, non-cytotoxic agents: theory and practice. J Natl Cancer Inst. 2004;96:990–997.
    1. Korn EL. Nontoxicity endpoints in phase I trial designs for targeted, non-cytotoxic agents. J Natl Cancer Inst. 2004;96:977–978.
    1. Piekarz RL, Robey R, Sandor V, Bakke S, Wilson WH, Dahmoush L, Kingma DM, Turner ML, Altemus R, Bates SE. Inhibitor of histone deacetylation, depsipeptide (FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report. Blood. 2001;98:2865–2868. doi: 10.1182/blood.V98.9.2865.
    1. Sandor V, Bakke S, Robey R, Kang MH, Blagaskonny M, Brooks R, Piekarz R, Tucker E, Figg WD, Chan KK, Goldspiel B, Sausville E, Balcerzak SP, Bates SE. Phase I trial of the histone deacetylase inhibitor, depsipeptide (FR90 NSC 630176), in patients with refractory neoplasms. Clin Cancer Res. 1228;8:718–728.
    1. Kelly WK, Richon VM, O'Connor O, Curley T, MacGregor-Curtelli B, Tong W, Klang M, Schwartz L, Richardson S, Rosa E, Drobnjak M, Cordon-Cordo C, Chiao JH, Rifkind R, Marks PA, Scher H. Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously. Clin Cancer Res. 2003;9:3578–3588.
    1. Miljkovic B, Pokrajac M, Varagic V, Levic Z. Single dose and steady state pharmacokinetics of valproic acid in adult epileptic patients. Int J Clin Pharmacol Res. 1991;11:137–141.
    1. Bruni J, Wilder BJ, Willmore LJ, Perchalski RJ, Villarreal HJ. Steady-state kinetics of valproic acid in epileptic patients. Clin Pharmacol Ther. 1978;24:324–332.
    1. Tisdale JE, Tsuyuki RT, Oles KS, Penry JK. Relationship between serum concentration and dose of valproic acid during monotherapy in adult outpatients. Ther Drug Monit. 1992;14:416–423.
    1. Atmaca A, Maurer A, Heinzel T, Gotlicher M, Neumann A, Al-Batran SE, Martin E, Bartshc I, Knuth A, Jaegen E. A dose-scalating phase I study with valproic acid (VPA) in patients (pts) with advanced cancer. Procc ASCO. 2004;23 (abstract 3169).
    1. Garcia-Manero G, Kantarjian H, Sanchez-Gonzalez B, Verstovsek S, Ravandi F, Ryttling M, Cortes J, Yang H, Fiorentino J, Rosner G, Issa J. Results of a Phase I/II Study of the Combination of 5-aza-2-deoxycytidine and 'valproic acid in patients with acute myeloid leukemia and myelodysplastic syndrome. Procc ASCO. 2005;24 (abstract 6544).
    1. Munster PN, Marchion DC, Bicaku E, Sullivan P, Beam C, Mahany J, Lush R, Sullivan DM, Daud A. Phase I trial of the histone deacetylase Inhibitor, valproic acid and the topoisomerase II inhibitor, epirubicin: A clinical and translational study. Procc ASCO. 2005;24 (abstract 3084).
    1. Spiller HA, Krenzelok EP, Klein-Schwartz W, Winter ML, Weber JA, Sollee DR, Bangh SA, Griffith JR. Multicenter case series of valproic acid ingestion: serum concentrations and toxicity. J Toxicol Clin Toxicol. 2000;38:7557–7560. doi: 10.1081/CLT-100102388.
    1. Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet. 1999;21:103–107. doi: 10.1038/5047.
    1. Zhu WG, Otterson GA. The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells. Curr Med Chem Anti-Cancer Agents. 2003;3:187–199. doi: 10.2174/1568011033482440.
    1. Mongan NP, Gudas LJ. Valproic acid, in combination with all-trans retinoic acid and 5-aza-2'-deoxycytidine, restores expression of silenced RARbeta2 in breast cancer cells. Mol Cancer Ther. 2005;4:477–486.
    1. Segura-Pacheco B, Trejo-Becerril C, Perez-Cardenas E, Taja-Chayeb L, Mariscal I, Chavez A, Acuna C, Salazar AM, Lizano M, Duenas-Gonzalez A. Reactivation of tumor suppressor genes by the cardiovascular drugs hydralazine and procainamide and their potential use in cancer therapy. Clin Cancer Res. 2003;9:1596–603.
    1. Angeles EE, Vazquez-Valadez VH, Vasquez-Valadez O, Velazquez-Sanchez AM, Ramirez A, Martinez L, Diaz-Barriga S, Romero-Rojas A, Cabrera G, Lopez-Castañares R, Duenas-Gonzalez A. Computational studies of 1-hydrazinophthalazine (Hydralazine) as antineoplasic agent. Docking studies on methyltransferase. Letters Drug Design Discovery. 2005;4:282–286. doi: 10.2174/1570180054038413.
    1. Zambrano P, Segura-Pacheco B, Perez-Cardenas E, Cetina L, Revilla-Vazquez A, Taja-Chayeb L, Chavez-Blanco A, Angeles E, Cabrera G, Sandoval K, Trejo-Becerril C, Chanona-Vilchis J, Duenas-Gonzalez A. A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes. BMC Cancer. 2005;5:44. doi: 10.1186/1471-2407-5-44.
    1. Zhu WG, Lakshmanan RR, Beal MD, Otterson GA. DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors. Cancer Res. 2001;61:1327–1333.
    1. Jolley ME. Fluorescence polarization immunoassay for determination of therapeutic drug levels in human plasma. J Anal Tox. 1981;5:236–240.

Source: PubMed

赞助者和合作者

医疗条件

药物干预

3
订阅