Antitumour treatmentValproic acid as epigenetic cancer drug: Preclinical, clinical and transcriptional effects on 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.
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