Antitumour treatment
Valproic acid as epigenetic cancer drug: Preclinical, clinical and transcriptional effects on solid tumors

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Summary

Among many anticancer drugs collectively named “targeted or molecular therapies” epigenetic drugs are clearly promising. Differently from other agents targeting a single gene product, epigenetic drugs have chromatin as their target through inhibition of histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) therefore, yet unspecific, they may act upon most or all tumor types, as deregulation of the methylation and deacetylation machinery are a common hallmark of neoplasia. In the last years, valproic acid (VPA) as emerged as a promising drug for cancer treatment. VPA has shown potent antitumor effects in a variety of in vitro and in vivo systems, and encouraging results in early clinical trials either alone or in combination with demethylating and/or cytotoxic agents. In addition, whole genome expression by microarray analysis from the primary tumors of patients treated with VPA show significant up-regulation of hundred of genes belonging to multiple pathways including ribosomal proteins, oxidative phosphorylation, MAPK signaling; focal adhesion, cell cycle, antigen processing and presentation, proteasome, apoptosis, PI3K, Wnt signaling, calcium signaling, TGF-beta signaling, and ubiquitin-mediated proteolysis among others. Despite in general, industry is not particularly interested in funding the clinical development of VPA, -at least in comparison to novel HDAC inhibitors-, existing preclinical and preliminary clinical data strongly suggest that VPA could be a drug that eventually will be used in combination therapies, either with classical cytotoxics, other molecular-targeted drugs or radiation in a number of solid tumors.

Introduction

It is well-known that cancer cells do contain multiple gene defects, namely mutations, deletions, duplications, translocations that lead to oncogene activation and lost of tumor suppressor gene function, however, defects in gene transcription primarily mediated by epigenetic mechanisms are another hallmark of the malignant phenotype that act in concern with genetic alterations for cancer development.1

Epigenetics can be defined as the study of mitotically and/or meiotically heritable changes in gene function that can not be explained by changes in DNA sequence. There are two epigenetic systems that affect animal development and fulfill the heritability criterion: DNA methylation, and the polycomb–trithorax group (Pc-G/trx) protein complexes; nevertheless, post-transductional modification of histones possess some attributes of an epigenetic process and as such are studied within this field.2 The two most widely studied epigenetic changes in cancer are DNA methylation and histone acetylation; however, the picture is much more complicated than this, with new players coming into the scene, such as the RNA interference phenomenon, proven to be implicated in transcriptional silencing through small duplex RNA molecules that recruit silencing complexes to the chromatin, and the BORIS (Brother of the Regulator of Imprinted Sites)/CTCF gene family, which is involved in epigenetic reprogramming events.3, 4

Section snippets

Histones and post-translational modifications

How double-strand DNA is packaged into the dynamic structure of chromatin is crucial for the transcriptional control process by regulating transcription factor accessibility to DNA regulatory sequences. Chromatin is constituted of nucleosomes, which are comprised of 146 base pairs of DNA wrapped around a core of two copies each of histones H2A, H2B, H3, and H4. The N-terminal tails of histones which undergo post-translational modifications such as acetylation, methylation, ubiquitination,

Valproic acid

Valproic acid was synthesized in the search for organic solvents in 1882 but was not until 1963 when Eymard uncovered its antiepileptic potential,14, 15 Although valproic acid is a small branched fatty acid, its chemical properties allow easy delivery to the organism and cells. It is slightly soluble in water and highly soluble in organic solvents and it is stable at room temperature. Since valproic acid exists in a dissociated form in water solutions containing alkali metals, it can be easily

Inhibition of HDAC by valproic acid

The finding that VPA was an effective inhibitor of HDACs surged from the observations that VPA was able to relieve transcriptional repression of a peroxisomal proliferation and activation of a glucocorticoid receptor (GR)–PPARϵ hybrid receptor and a RAR-dependent reporter gene expression system, suggesting that it acts on a common factor in gene regulation such as corepressor-associated HDACs rather than on individual transcription factors or receptors. Consistent with this finding, it was

Preclinical studies of VPA in solid tumors models

As shown in Table 2, VPA has shown potent antitumor effects in a variety of in vitro and in vivo systems, by modulating multiple pathways including cell cycle arrest, apoptosis, angiogenesis, metastasis, differentiation and senescence. These effects seem to be cell type specific which may depend also on the level of differentiation and the underlying genetic alterations. Most of preclinical and clinical data on the anticancer effects of VPA has been generated for malignant hematological

Glioma

As early as 1985, VPA was shown to inhibit the mitotic index of the rat glioma G6 cell line at an IC50 of 0.5 mM whereas its continued exposure at 1 mM concentration lead to increased cell differentiation and adhesiveness.28 The growth inhibitory effect of VPA upon C6 glioma cultured continuously in the presence of 1 mM valproate has also been reported, and was associated with an inhibition of transient alpha2,3 sialylation of a 65 kDa glycoprotein expressed maximally at 4 h into the G1 phase of the

Neuroblastoma

VPA inhibits growth and induces differentiation of human neuroblastoma cells in in vitro at concentrations that have been achieved in humans with no significant adverse effects. Treatment of UKF-NB-2 and UKF-NB-3 NB cell lines with VPA at concentrations ranging from 0.5 to 2 mM results in neuronal morphological differentiation characterized by extension of cellular processes without significant effects on cell viability. Ultrastructural features of VPA-treated cells were consistent with the

Breast

VPA strongly reduces the growth of MCF-7 breast cancer cell line at a concentration of 0.23 mM, effect that seems independent of the estrogen receptor.36 VPA, in combination with retinoic acid and a DNA methyltransferase inhibitor leads to reactivation of the silenced tumor suppressor gene, RARβ2, in human breast cancer cells and also inhibits the proliferation of receptor positive and negative MCF-7 and MDA-231 cells either alone or in combination with retinoic acid and a DNA methyltransferase

Colon

VPA has been evaluated in the colon cancer cell lines HCA-7 which have wild-type β-catenin and APC proteins and in SW620, and HT-29 which are APC mutant. While VPA induces significant level of apoptosis in HCA-7 cells, SW620 and HT-29 cells are resistant when treated at the same doses. Artificial expression of APC in HT-29 cells leads to sensitivity to VPA-induced apoptosis which is accompanied by down-regulation of survivin.40 Nevertheless, a subsequent report of VPA treatment upon these same

Prostate

VPA treatment of the prostate cancer cell line LNCaP induces apoptosis which was associated with up-regulation of the pro-apoptotic factor caspase-3, tissue inhibitor of matrix metalloproteinase-3 and insulin-like growth factor binding protein-3.44 In other models, apoptosis is related to down-regulation of bcl-2 in short-term studies whereas in chronic treatment it has observed that apoptosis is related to enhancement of FAS-dependent apoptosis, associated with over-expression in Fas and Fas

Thyroid

Poorly differentiated thyroid cancer are frequently characterized by the lost of molecular machinery for iodide uptake. Treatment with VPA to N-PA (poorly differentiated) and ARO (anaplastic) cells results in Na+/I− symporter (NIS) gene expression in the cell membrane which is accompanied by I125 uptake. In ARO cells, despite VPA increases NIS mRNA it was not followed by changes in iodide uptake.48 The effect of VPA on thyroid carcinoma cells is not limited to the restitution of iodine uptake

Hepatocarcinoma

VPA treatment for five days has shown to induce a dose-dependent decrease in cell viability in the hepatocarcinoma cell lines HepG2, HuH-7 and PLC/PRF/5 at IC50 ranging between 1.3 and 2.5 mM and induced apoptosis at 2 mM concentration. Interestingly, VPA was proven nontoxic to primary normal hepatocytes when treated for 48 h at concentrations between 1 and 2 mM.52 VPA has shown to up-regulate the expression of MHC classI chain-related molecules MICA and MICB in the surface of hepatoma cells which

Cervix

The antitumor effects of VPA have also been evaluated in cervical cancer cell lines. A study has shown that VPA at a 1 mM concentration leads to growth inhibition in HeLa, SiHa and Casky cells. Interestingly, the antitumor effect of valproate in cervical cancer may at least partially depend on an up-regulating effect on the p53 gene and in the valproate-induced hyperacetylation of p53 protein protecting it from its degradation by E6.55 Further, it has been shown that VPA increases the

Endometrial

The biological and therapeutical effects of VPA have been investigated in the endometrial cancer cells lines HEC-1B, RL95-2, KLE, AN3CA, HEC59 and Ishikawa. VPA inhibits clonal proliferation of all of the endometrial cancer cell lines tested in a dose-dependent manner with a 50% clonal growth inhibition between 0.7 mM 3.8 mM. In the HEC-IB cell line, the inhibitory effect was associated with G1–G0 arrest at 5 mM. Likewise, apoptosis is significantly increased by VPA at ranges between 1 and 5 mM.

Neuroectodermal

VPA has demonstrated growth inhibitory activity in a supratentorial primitive neuroectodermal tumor (sPNET) cell line where it induces potent growth inhibition, cell cycle arrest, apoptosis, senescence, cell differentiation and suppressed colony-forming efficiency in vitro at concentrations ranging from 0.6 and 1 mM.59

Melanoma

The effect of VPA in melanoma was tested in the melanoma cell line M14 demonstrating that VPA induces cell cycle arrest and apoptosis. At a IC50 of 2.9 mM the arrested fraction was up to 75% which was associated with up-regulation of p16, p21 and cyclin-D1 related to Rb hypo-phosphorilation. In addition VPA activated apoptosis (50%) when given alone or in combination with antitumoral agents.60

Ovarian

VPA has shown growth inhibitory activity upon a number of ovarian cancer cell lines including SK-OV-3, OVCAR-3, TOV-21G, OV-90, TOV-112D, OVCA420, OVCA429, OVCA432, and OVCA433 at concentrations that inhibit 50% clonal growth in range of 0.93–2.4 mM with variable effects on the cell cycle characterized by either G1 or G2 arrest depending on the cell line. VPA was also shown to increase from 26% to 49% the proportion of apoptotic cells at 5 mM. When tested in a xenograft model with the SK-OV-3

Teratocarcinoma

In a culture system to study the teratogenic effect of VPA, it was shown that this drug but not with its non-teratogenic-analogue, 2-isopropylpentanoic acid (IPPA), inhibits cell growth and has a cytotoxic effect. Differentiation was evaluated by immunostaining for neurofilament proteins, microtubule associated proteins-2 (MAP-2), tau and neural cell adhesion molecule, NCAM.62

Medulloblastoma

The antitumor activity of VPA has also been evaluated in medulloblastoma. In the cell lines DAOY and D283-MED, VPA at concentrations between 0.6 mM and 1 mM, induces potent growth inhibition, cell cycle arrest, apoptosis, senescence, cell differentiation and suppressed colony-forming efficiency in vitro. When evaluated in a severe combined immunodeficient mice xenografted with cell lines, daily intraperitoneal injection of VPA at 400 mg/kg for 28 days exerts a significant antitumor effect. These

Thoracic tumors (lung, esophagus, mesothelioma)

VPA at concentrations varying from 3.2 to 5 mM induces dose-dependent growth arrest and apoptosis in cultured non–small cell lung cancer cells H460 and H322; esophageal cancer (EsC) cells TE2 and TE12; and malignant pleural mesothelioma (MPM) cells H211 and H513. In addition to these effects, VPA sensitizes these cells to Apo2L/TRAIL, as indicated by a fourfold to a >20-fold reduction of Apo2L/TRAIL IC50 values in combination-treated cells. Thus, VPA (0.5–5 mM) or Apo2L/TRAIL (20 ng/ml) alone

Bladder

Acute VPA treatment (72 h) causes a dose-dependent decrease in invasion in bladder cancer cell lines T24, TCC-SUP, HT1376 but not in RT4, a noninvasive papilloma. Chronic VPA treatment (34 days), significantly inhibits growth of T24 tumor xenografts.64

EBV-related tumors

EBV is the causative agent of infectious mononucleosis and is associated with African Burkitt’s lymphomas, Hodgkin’s disease, nasopharyngeal and gastric carcinomas. It has been shown that switching the latent form of viral infection into the lytic form may induce tumor cell death. VPA treatment induces a modest increase in the level of lytic viral gene expression, but strongly enhances the ability of chemotherapeutic agents to induce lytic EBV gene expression in EBV-positive epithelial and

Carcinoid

Carcinoid tumors are neoplasms that arise from the disseminated neuroendocrine cell system of the gastrointestinal (GI) tract, lungs, and other organs. A study has shown that VPA leads to a profound dose-dependent inhibition of growth of BON gastrointestinal and H727 pulmonary human carcinoid tumor cells by arresting them at G1 phase of the cell cycle and related to increase in p21 and p27 while cyclin D1 is downregulated. These effects seems to result from cell differentiation induced by VPA

Fibrosarcoma

In a sarcoma xenograft model in nu/nu mice with HT1080 cells, treatment with VPA plus hydralazine, a DNA methylation inhibitor, added to adriamycin, it was observed that, yet in the animals treated with adriamycin with or without VPA plus hydralazine, tumors disappeared at day 21, there was a rapid regrowth starting at day 21 in the animals treated only with adriamycin, whereas animals treated with VPA plus hydralazine remained free of tumor. These results suggest that the antitumor efficacy of

Clinical studies of valproic acid

The realization that neoplastic cells exhibit aberrant gene expression has led to strategies to correct these genetic perturbations through pharmacological manipulation of the epigenome, consequently, there has been considerable effort to develop HDAC inhibitors (HDACi) as inappropriate modulation of chromatin structure by HDACs and subsequently repression of gene expression appear to be major factors in the development of cancer.

Over the last years several HDACi have been introduced into

Phase I studies

In the first phase I study published, 12 patients with untreated cervical carcinoma were studied in the window from the diagnosis to the beginning of definitive treatment with chemoradiation. These patients had a macroscopic tumor accessible for punch biopsy and the study consisted in the administration of magnesium valproate in cohorts of four patients at 20 mg/kg, 30 mg/kg and 40 mg/kg from day 1 to 5. At day 6 tumor biopsies and blood samples were taken.

All patients completed the study

Phase I–II studies

A third study with VPA combined with epirubicin was recently published. This was a phase I–II study in patients with advanced solid tumor malignancies, an ECOG performance status of 0–2, and adequate organ function. Prior anthracyclines were permitted (doxorubicin ⩽300 mg/m2 and epirubicin ⩽600 mg/m2). In this study an intravenous loading dose of VPA was followed by five oral doses administered every 12 h beginning 1 h after the loading dose, though later in the study, the intravenous loading dose

Phase II studies

A number of preclinical studies have consistently demonstrated that DNA demethylating agents and HDAC inhibitors, either alone or in combination, are able to modulate gene expression,74, 75 and that both classes of agents synergize not only the antitumor effects76, 77, 78, 79, 80 but also the extent of gene up-regulation.81, 82 We recently confirmed this synergy between hydralazine, a weak DNA methylation inhibitor83, 84, 85, 86, 87, 88 and VPA regarding global gene expression by studying colon

Transcriptome changes induced by valproate and hydralazine

The rationale for utilizing epigenetic drugs (DNA methylation and HDAC inhibitors) for cancer treatment has relied on the thought that reversing epigenetic aberrations would turn-on tumor suppressor genes and consequently exert antitumor effects. Most of this knowledge has been generated at the level of individual genes by candidate-gene approach, however, it has been proposed that an epigenomic reactivation screening strategy that combines treatment of cancer cells in vitro with DNA

Conflict of interest statement

The authors have declared that no competing interests exist.

Funding

The laboratory and clinical work of Dr. Duenas-Gonzalez’ group has been supported by CONACyT grants SALUD-2002-C01-6579, AVANCE C01-294, and by Psicofarma, S.A. de C.V., Mexico. Sponsors did not participate in data collection, analysis, and interpretation of data; writing of the paper; nor decision to submit it for publication.

Acknowledgment

Dr. Dueñas-Gonzalez is a recipient of the Catedra ICP-PUIS UNAM.

References (127)

  • C.M. Olsen et al.

    Antiepileptic drugs inhibit cell growth in the human breast cancer cell line MCF7

    Mol Cell Endocrinol

    (2004)
  • D. Zgouras et al.

    Modulation of angiogenesis-related protein synthesis by valproic acid

    Biochem Biophys Res Commun

    (2004)
  • M.F. Ziauddin et al.

    Valproic acid, an antiepileptic drug with histone deacetylase inhibitory activity, potentiates the cytotoxic effect of Apo2L/TRAIL on cultured thoracic cancer cells through mitochondria-dependent caspase activation

    Neoplasia

    (2006)
  • W.R. Parulekar et al.

    Novel endpoints and design of early clinical trials

    Ann Oncol

    (2002)
  • M. Candelaria et al.

    A phase II study of epigenetic therapy with hydralazine and magnesium valproate to overcome chemotherapy resistance in refractory solid tumors

    Ann Oncol

    (2007)
  • A. Bird

    DNA methylation patterns and epigenetic memory

    Genes Dev

    (2002)
  • H. Kawasaki et al.

    Transcriptional gene silencing by short interfering RNAs

    Curr Opin Mol Ther

    (2005)
  • A. Eberharter et al.

    Dynamic chromatin: concerted nucleosome remodelling and acetylation

    J Biol Chem

    (2005)
  • M.H. Kuo et al.

    Roles of histone acetyltransferases and deacetylases in gene regulation

    Bioessays

    (1998)
  • J. Taunton et al.

    A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p

    Science

    (1996)
  • J.E. Bolden et al.

    Anticancer activities of histone deacetylase inhibitors

    Nat Rev Drug Discov

    (2006)
  • D. Marchion et al.

    Development of histone deacetylase inhibitors for cancer treatment

    Exp Rev Anticancer Ther

    (2007)
  • B.S. Burton

    On the propyl derivatives and decomposition products of ethylacetoacetate

    Am Chem J

    (1882)
  • H. Meunier et al.

    Pharmacodynamic properties of N-dipropylacetic acid

    Therapie

    (1963)
  • W. Loscher

    Basic pharmacology of valproate: a review after 35 years of clinical use for the treatment of epilepsy

    CNS Drugs

    (2002)
  • E. Perucca

    Pharmacological and therapeutic properties of valproate: a summary after 35 years of clinical experience

    CNS Drugs

    (2002)
  • C.L. DeVane

    Pharmacokinetics, drug interactions, and tolerability of valproate

    Psychopharmacol Bull

    (2003)
  • C.U. Johannessen et al.

    Valproate: past, present, and future

    CNS Drug Rev

    (2003)
  • J.H. DiLiberti et al.

    The fetal valproate syndrome

    Am J Med Genet

    (1984)
  • R. Alsdorf et al.

    Teratogenicity of sodium valproate

    Expert Opin Drug Saf

    (2005)
  • M. Göttlicher et al.

    Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells

    EMBO J

    (2001)
  • N. Gurvich et al.

    Histone deacetylase is a target of valproic acid-mediated cellular differentiation

    Cancer Res

    (2004)
  • D.C. Marchion et al.

    Valproic acid alters chromatin structure by regulation of chromatin modulation proteins

    Cancer Res

    (2005)
  • A. Kuendgen et al.

    Valproic acid for the treatment of myeloid malignancies

    Cancer

    (2007)
  • M.M. Knüpfer et al.

    Valproic acid inhibits proliferation and changes expression of CD44 and CD56 of malignant glioma cells in vitro

    Anticancer Res

    (1998)
  • A. Chavez-Blanco et al.

    Antineoplastic effects of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in cancer cell lines

    Cancer Cell Int

    (2006)
  • Das CM, Aguilera D, Vasquez H, et al. Valproic acid induces p21 and topoisomerase-II (alpha/beta) expression and...
  • J. Cinatl et al.

    Antitumor activity of sodium valproate in cultures of human neuroblastoma cells

    Anticancer Drugs

    (1996)
  • M. Michaelis et al.

    Valproic acid and interferon-alpha synergistically inhibit neuroblastoma cell growth in vitro and in vivo

    Int J Oncol

    (2004)
  • R.A. Blaheta et al.

    Valproic acid inhibits adhesion of vincristine- and cisplatin-resistant neuroblastoma tumour cells to endothelium

    Br J Cancer

    (2007)
  • N.P. Mongan et al.

    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)
  • L. Hodges-Gallagher et al.

    Inhibition of histone deacetylase enhances the anti-proliferative action of antiestrogens on breast cancer cells and blocks tamoxifen-induced proliferation of uterine cells

    Breast Cancer Res Treat

    (2006)
  • D.C. Marchion et al.

    In vivo synergy between topoisomerase II and histone deacetylase inhibitors: predictive correlates

    Mol Cancer Ther

    (2005)
  • X. Huang et al.

    Adenomatous polyposis coli determines sensitivity to histone deacetylase inhibitor-induced apoptosis in colon cancer cells

    Cancer Res

    (2006)
  • I. Friedmann et al.

    Synergistic effects of valproic acid and mitomycin C in adenocarcinoma cell lines and fresh tumor cells of patients with colon cancer

    J Chemother

    (2006)
  • J.S. Isenberg et al.

    Modulation of angiogenesis by dithiolethione-modified NSAIDs and valproic acid

    Br J Pharmacol

    (2007)
  • P. Thelen et al.

    Expressional changes after histone deacetylase inhibition by valproic acid in LNCaP human prostate cancer cells

    Int J Oncol

    (2004)
  • A. Angelucci et al.

    Valproic acid induces apoptosis in prostate carcinoma cell lines by activation of multiple death pathways

    Anticancer Drugs

    (2006)
  • Q. Xia et al.

    Chronic administration of valproic acid inhibits prostate cancer cell growth in vitro and in vivo

    Cancer Res

    (2006)
  • S. Shabbeer et al.

    Multiple molecular pathways explain the anti-proliferative effect of valproic acid on prostate cancer cells in vitro and in vivo

    Prostate

    (2007)
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