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Background

Valproic acid, a widely used anticonvulsant drug, is a potent teratogen resulting in various congenital abnormalities. However, the mechanisms underlying valproic acid induced teratogenesis are nor clear. Recent studies indicate that histone deacetylase is a direct target of valproic acid.

Methods

In the present study, we have used histological analysis and RT-PCR assays to examine the cardiac abnormalities in mice treated with sodium valproate (NaVP) and determined the effects of NaVP on histone deacetylase activity and the expression of heart development-related genes in mouse myocardial cells.

Results

The experimental data show that NaVP can induce cardiac abnormalities in fetal mice in a dose-dependent manner. NaVP causes a dose-dependent inhibition of hitone deacetylase (HDAC) activity in mouse myocardial cells. However, the expression levels of HDAC (both HDAC1 and HDAC2) are not significantly changed in fetal mouse hearts after administration of NaVP in pregnant mice. The transcriptional levels of other heart development-related genes, such as CHF1, Tbx5 and MEF2, are significantly increased in fetal mouse hearts treated with NaVP.

Conclusions

The study indicates that administration of NaVP in pregnant mice can result in various cardiac abnormalities in fetal hearts, which is associated with an inhibition of histone deacetylase without altering the transcription of this enzyme.

Retinoic acid derivatives have shown their greatest benefit in acute promyelocytic leukemia, but have also demonstrated pre-clinical anti-cancer effects in some solid tumors. Histone deacetylase inhibitors, by upregulating gene expression, are able to limit cancer cell proliferation and induce apoptosis. The combination of all-trans retinoic acid (ATRA) and the histone deacetylase inhibitor valproic acid has been previously studied in hematologic malignancies. We conducted a Phase I two-step dose escalation trial of the liposomal ATRA analog ATRA-IV and divalproex sodium (Depakote®) in nine patients with advanced solid tumors refractory to prior therapy. Side effects attributed to therapy had a severity ≤grade 2 and included skin toxicity and thrombocytopenia. The best disease response seen was disease stabilization in one patient. Expression of cellular retinoic acid binding protein-2 in peripheral blood mononuclear cells was detected as a marker of drug effect. The maximum tolerated dose (MTD) of both drugs in combination could not be established due to early closure of the trial resulting from a halt in the commercial availability of ATRA-IV.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by cognitive impairments that progress to dementia and death. The earliest symptoms of AD present as a relatively pure deficit in memory retrieval. Therefore, drug treatments that intervene in the early stages of AD by rescuing memory deficits could be promising therapies to slow, or even reverse progression of the disease. In this study, we tested the potential of systemic histone deacetylase inhibitor (HDACi) treatment to rescue cognitive deficits in a mouse model of AD. APPswe/PS1dE9 mice showed pronounced contextual memory impairments beginning at 6 months of age. Chronic HDACi injections (2–3 weeks) did not alter contextual memory formation in normal mice, but had profound effects in transgenic animals. Injections of sodium valproate, sodium butyrate, or vorinostat (suberoylanilide hydroxamic acid; Zolinza®) completely restored contextual memory in these mutant mice. Further behavioral testing of the HDACi-treated transgenic mice showed that the newly consolidated memories were stably maintained over a 2-week period. Measurement of the HDAC isoform selectivity profile of sodium valproate, sodium butyrate, and vorinostat revealed the common inhibition of class I HDACs (HDAC1, 2, 3, 8) with little effect on the class IIa HDAC family members (HDAC4, 5, 7, 9) and inhibition of HDAC6 only by vorinostat. These preclinical results indicate that targeted inhibition of class I HDAC isoforms is a promising avenue for treating the cognitive deficits associated with early stage AD.

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 μg/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.

We explored in a phase I/II clinical trial the combination of valproic acid (VPA), a clinically available histone deacetylase inhibitor, with standard chemoimmunotherapy in patients with advanced melanoma, to evaluate its clinical activity, to correlate the clinical response with the biological activity of VPA and to assess toxicity. Patients were treated initially with VPA alone for 6 weeks. The inhibition of the target in non-tumour peripheral blood cells (taken as a potential surrogate marker) was measured periodically, and valproate dosing adjusted with the attempt to reach a measurable inhibition. After the treatment with valproate alone, dacarbazine plus interferon-α was started in combination with valproate. Twenty-nine eligible patients started taking valproate and 18 received chemoimmunotherapy and are assessable for response. We observed one complete response, two partial remissions and three disease stabilisations lasting longer than 24 weeks. With the higher valproate dosages needed to reach a measurable inhibition of the target, we observed an increase of side effects in those patients who received chemoimmunotherapy. The combination of VPA and chemoimmunotherapy did not produce results overtly superior to standard therapy in patients with advanced melanoma and toxicity was not negligible, casting some doubts on the clinical use of VPA in this setting (at least in the administration schedule adopted).

Histone deacetylase (HDAC) inhibitors can radiosensitize cancer cells. Radiation is critical in high-risk neuroblastoma treatment, and combinations of HDAC inhibitor vorinostat and radiation are proposed for neuroblastoma trials. Therefore, we investigated radiosensitizing effects of vorinostat in neuroblastoma. Treatment of neuroblastoma cell lines decreased cell viability and resulted in additive effects with radiation. In a murine metastatic neuroblastoma in vivo model vorinostat and radiation combinations decreased tumor volumes compared to single modality. DNA repair enzyme Ku-86 was reduced in several neuroblastoma cells treated with vorinostat. Thus, vorinostat potentiates anti-neoplastic effects of radiation in neuroblastoma possibly due to down-regulation of DNA repair enzyme Ku-86.

Histone deacetylases (HDACs) can regulate expression of tumor suppressor genes and activities of transcriptional factors involved in both cancer initiation and progression through alteration of either DNA or the structural components of chromatin. Recently, the role of gene repression through modulation such as acetylation in cancer patients has been clinically validated with several inhibitors of HDACs. One of the HDAC inhibitors, vorinostat, has been approved by FDA for treating cutaneous T-cell lymphoma (CTCL) for patients with progressive, persistent, or recurrent disease on or following two systemic therapies. Other inhibitors, for example, FK228, PXD101, PCI-24781, ITF2357, MGCD0103, MS-275, valproic acid and LBH589 have also demonstrated therapeutic potential as monotherapy or combination with other anti-tumor drugs in CTCL and other malignancies. At least 80 clinical trials are underway, testing more than eleven different HDAC inhibitory agents including both hematological and solid malignancies. This review focuses on recent development in clinical trials testing HDAC inhibitors as anti-tumor agents.

Suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza®) is the lead compound of a new class of histone deacetylase (HDAC) inhibitors used as anticancer drugs, which have been shown to affect multiple proteins associated with gene expression, cell proliferation and migration. Studies have also demonstrated the essential role of the hydroxamate moiety of SAHA in HDAC inhibition. The ability of SAHA and its structural analog Trichostatin A (TSA) in generating NO upon oxidation was tested directly by spin trapping of NO using Electron Paramagnetic Resonance (EPR) spectroscopy and also indirectly via the determination of nitrite using the Griess assay. H2O2/metmyoglobin was used to oxidize SAHA and TSA. These studies demonstrate for the first time, the release of NO from SAHA and its structural analog TSA. We tested the protective effects of SAHA, TSA and valproic acid (VPA) in mammalian Chinese hamster V79 cells exposed to a bolus H2O2 for 1 hour and monitoring the clonogenic cell survival. Both SAHA and TSA afforded significant cytoprotection when co-incubated with H2O2 whereas VPA was ineffective. These studies provide evidence for the release of NO by hydroxamate containing HDAC inhibitors and their antioxidant effects. Such roles may be an added advantage of this class of HDAC agents which are used for epigenetic therapies in cancer.

Vorinostat, an oral histone deacetylase inhibitor with anti-tumor activity, is in clinical trials for hematological and solid tumors that metastasize and compromise bone structure. Consequently, there is a requirement to establish the effects of vorinostat on tumor growth within bone. Breast (MDA-231) and prostate (PC3) cancer cells were injected into tibias of SCID/NCr mice and the effects of vorinostat on tumor growth and osteolytic disease were assessed by radiography, μCT, histological and molecular analyses. Vorinostat-treated and control mice without tumors were also examined. Tumor growth in bone was reduced ~33% by vorinostat with inhibited osteolysis in the first few weeks of the experiment; however, osteolysis became more severe in both the vehicle and vorinostat-treated groups. Vorinostat increased the expression of tumor-derived factors promoting bone resorption, including PTHrP, IL-8 and osteopontin. After four weeks of vorinostat therapy the non-tumor bearing contra-lateral femurs as well as limbs from vorinostat-treated tumor-free SCID mice, showed significant bone loss (50% volume density of controls). Thus, our studies indicate that vorinostat effectively inhibits tumor growth in bone, but has a negative systemic effect reducing normal trabecular bone mass. Vorinostat treatment reduces tumor growth in bone and accompanying osteolytic disease as a result of decreased tumor burden in bone. However, vorinostat can promote osteopenia throughout the skeleton independent of tumor cell activity.

Background

Histone deacetylases (HDACs) are enzymes that modulate gene expression and cellular processes by deacetylating histones and non-histone proteins. While small molecule inhibitors of HDAC activity (HDACi) are used clinically in the treatment of cancer, pre-clinical treatment models suggest they also exert neuroprotective effects and stimulate neurogenesis in neuropathological conditions. However, the direct effects of HDACi on cell cycle progression and proliferation, two properties required for continued neurogenesis, have not been fully characterized in adult neural stem cells (NSCs). In this study, we examined the effects of two broad class I and class II HDACi on adult mouse NSCs, the hydroxamate-based HDACi suberoylanilide hydroxamic acid (vorinostat, SAHA) and the short chain fatty acid HDACi sodium butyrate.

Results

We show that both HDACi suppress the formation of neurospheres by adult mouse NSCs grown in proliferation culture conditions in vitro. DNA synthesis is significantly inhibited in adult mouse NSCs exposed to either SAHA or sodium butyrate and inhibition is associated with an arrest in the G1 phase of the cell cycle. HDACi exposure also resulted in transcriptional changes in adult mouse NSCs. Cdk inhibitor genes p21 and p27 transcript levels are increased and associated with elevated H3K9 acetylation levels at proximal promoter regions of p21 and p27. mRNA levels for notch effector Hes genes and Spry-box stem cell transcription factors are downregulated, whereas pro-neural transcription factors Neurog1 and Neurod1 are upregulated. Lastly, we show HDAC inhibition under proliferation culture conditions leads to long-term changes in cell fate in adult mouse NSCs induced to differentiate in vitro.

Conclusion

SAHA and sodium butyrate directly regulate cdk inhibitor transcription to control cell cycle progression in adult mouse NSCs. HDAC inhibition results in G1 arrest in adult mouse NSCs and transcriptional changes associated with activation of neuronal lineage commitment programs and a reduction of stem/progenitor state. Changes in differentiated cell state in adult mouse NSCs treated with HDACi under proliferation culture conditions suggests an intrinsic relationship between multipotency, cell cycle progression and HDAC activity in these cells.

Background

In eukaryotic cells, the genomic DNA is packed with histones to form the nucleosome and chromatin structure. Reversible acetylation of the histone tails plays an important role in the control of specific gene expression. Mounting evidence has established that histone deacetylase inhibitors selectively induce cellular differentiation, growth arrest and apoptosis in variety of cancer cells, making them a promising class of anticancer drugs. However, the molecular mechanisms of the anti-cancer effects of these inhibitors have yet to be understood.

Results

Here, we report that a key determinant for the susceptibility of cancer cells to histone deacetylase inhibitors is their ability to maintain cellular Akt activity in response to the treatment. Also known as protein kinase B, Akt is an essential pro-survival factor in cell proliferation and is often deregulated during tumorigenesis. We show that histone deacetylase inhibitors, such as valproic acid and butyrate, impede Akt1 and Akt2 expression, which leads to Akt deactivation and apoptotic cell death. In addition, valproic acid and butyrate induce apoptosis through the caspase-dependent pathway. The activity of caspase-9 is robustly activated upon valproic acid or butyrate treatment. Constitutively active Akt is able to block the caspase activation and rescues cells from butyrate-induced apoptotic cell death.

Conclusion

Our study demonstrates that although the primary target of histone deacetylase inhibitors is transcription, it is the capacity of cells to maintain cellular survival networks that determines their fate of survival.

Prostaglandin E2 (PGE2) promotes cancer progression by modulating proliferation, apoptosis, angiogenesis and the immune response. Enzymatic degradation of PGE2 involves the NAD+–dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Recent reports have shown a marked diminution of 15-PGDH expression in colorectal carcinomas. We report here that treatment of colorectal cancer (CRC) cells with histone deacetylase (HDAC) inhibitors, including sodium butyrate (NaB) and valproic acid (VPA), induces 15-PGDH expression. Additionally, we demonstrate that pre-treatment of CRC cells with HDAC inhibitors can block EGF or Snail-mediated transcriptional repression of 15-PGDH. We demonstrate an interaction between Snail and HDAC2 and the binding of HDAC2 to the 15-PGDH promoter. In vivo we observe increased Hdac2 expression in Apc-deficient mouse adenomas, which inversely correlated with loss of 15-Pgdh expression. Finally, in human colon cancers, elevated HDAC expression correlated with down-regulation of 15-PGDH. These data suggest that class I histone deacetylases, specifically HDAC2, and the transcriptional repressor Snail, play an central role in the suppression 15-PGDH expression. These results also provide a COX2-independent mechanism to explain increased PGE2 levels that contribute to progression of colorectal cancer.

Despite the availability of several Food and Drug Administration-approved drugs, advanced inoperable colorectal cancer remains incurable. In this study, we focused on the development of combined molecular targeted therapies against colon cancer by testing the efficacy of the combination of the histone deacetylase inhibitor vorinostat with the proteasome inhibitor bortezomib to determine if this resulted in synergistic antitumor effects against colorectal cancer. The effects of the histone deacetylase inhibitor vorinostat in combination with the proteasome inhibitor bortezomib on the growth of two colorectal cancer cell lines were assessed with regard to proliferation, cell cycle arrest, and apoptosis. Treatment with the combination of vorinostat and bortezomib resulted in a synergistic decrease in proliferation of both colorectal cancer cell lines compared with treatment with single agents alone. This inhibition was associated with a synergistic increase in apoptosis as measured by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase. In addition, we observed an increase in the proapoptotic protein BIM and in the number of cells arrested in the G2-M phase of the cell cycle. Although p21 levels were significantly increased, short hairpin RNA knockdown of p21 did not lead to changes in proliferation in response to the combination of drugs, indicating that although p21 is a target of these drugs, it is not required to mediate their antiproliferative effects. These data indicate that combination treatment with vorinostat and bortezomib result in synergistic antiproliferative and proapoptotic effects against colon cancer cell lines, providing a rational basis for the clinical use of this combination for the treatment of colorectal cancer.

Background

Among the epigenetic alterations occurring in cancer, DNA hypermethylation and histone hypoacetylation are the focus of intense research because their pharmacological inhibition has shown to produce antineoplastic activity in a variety of experimental models. The objective of this study was to evaluate the combined antineoplastic effect of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in a panel of cancer cell lines.

Results

Hydralazine showed no growth inhibitory effect on cervical, colon, breast, sarcoma, glioma, and head & neck cancer cell lines when used alone. On the contrary, valproic acid showed a strong growth inhibitory effect that is potentiated by hydralazine in some cell lines. Individually, hydralazine and valproic acid displayed distinctive effects upon global gene over-expression but the number of genes over-expressed increased when cells were treated with the combination. Treatment of HeLa cells with hydralazine and valproic acid lead to an increase in the cytotoxicity of gemcitabine, cisplatin and adriamycin. A higher antitumor effect of adriamycin was observed in mice xenografted with human fibrosarcoma cells when the animals were co-treated with hydralazine and valproic acid.

Conclusion

Hydralazine and valproic acid, two widely used drugs for cardiovascular and neurological conditions respectively have promising antineoplastic effects when used concurrently and may increase the antitumor efficacy of current cytotoxic agents.

OBJECTIVES

To demonstrate that human smooth muscle cells derived from neurogenic bladders produce more collagen in vitro than smooth muscle cells derived from normal bladders, and that epigenetic therapy may normalize this increased collagen production.

METHODS

Human smooth muscle cells from normal (n = 3) and neurogenic bladders (n = 3) were cultured in normal culture media and at different concentrations of the histone deacetylase inhibitors trichostatin A, valproic acid, and the DNA methylation inhibitor 5-azacytidine (5-aza). Collagen type I and III gene expression was measured using real-time quantitative reverse transcription-polymerase chain reaction after varying doses of drug exposure. Cell viability was measured using trypan blue.

RESULTS

The smooth muscle cells from neurogenic bladders released significantly more collagen than the normal bladder cells (mean 4.1 vs 1.8 μg/mL in control media) when grown in normal conditions. Treatment with trichostatin A at 50 ng/mL decreased the collagen level in cells from neurogenic bladders to almost normal levels (2.1 μg/mL). In addition, valproic acid treatment decreased collagen types I and III gene expression relative to controls, with maximal effect at 300 mg/mL. These treatments had little effect on cell viability.

CONCLUSIONS

Histone deacetylase inhibitors decreased collagen production of smooth muscle cells from neurogenic bladders in vitro. These agents may be a means of effectively preventing bladder fibrosis in patients with this condition.

A correlation between components of the insulin-like growth factor (IGF) system and endometrial cancer risk has been shown in recent studies. The antitumor action of vorinostat, a histone deacetylase inhibitor, involves changes in the expression of specific genes via acetylation of histones and transcription factors. The aim of this study was to establish whether vorinostat can modify the expression of specific genes related to the IGF-I receptor (IGF-IR) signaling pathway and revert the transformed phenotype. Human endometrioid (Type I, Ishikawa) and uterine serous papillary (Type II, USPC-2) endometrial cancer cell lines were treated with vorinostat in the presence or absence of IGF-I. Vorinostat increased IGF-IR phosphorylation, produced acetylation of histone H3, up-regulated pTEN and p21 expression, and reduced p53 and cyclin D1 levels in Ishikawa cells. Vorinostat up-regulated IGF-IR and p21 expression, produced acetylation of histone H3, and down-regulated the expression of total AKT, pTEN and cyclin D1 in USPC-2 cells. Of interest, IGF-IR activation was associated with a major elevation in IGF-IR promoter activity. In addition, vorinostat treatment induced apoptosis in both cell lines and abolished the anti-apoptotic activity of IGF-I both in the absence or presence of a humanized monoclonal IGF-IR antibody, MK-0646. Finally, vorinostat treatment led to a significant decrease in proliferation and colony forming capability in both cell lines. In summary, our studies demonstrate that vorinostat exhibits a potent apoptotic and anti-proliferative effect in both Type I and II endometrial cancer cells, thus suggesting that endometrial cancer may be therapeutically targeted by vorinostat.

Background

The histone deacetylase inhibitor vorinostat is a candidate radiosensitizer in locally advanced rectal cancer (LARC). Radiosensitivity is critically influenced by hypoxia; hence, it is important to evaluate the efficacy of potential radiosensitizers under variable tissue oxygenation. Since fluoropyrimidine-based chemoradiotherapy (CRT) is the only clinically validated regimen in LARC, efficacy in combination with this established regimen should be assessed in preclinical models before a candidate drug enters clinical trials.

Methods

Radiosensitization by vorinostat under hypoxia was studied in four colorectal carcinoma cell lines and in one colorectal carcinoma xenograft model by analysis of clonogenic survival and tumor growth delay, respectively. Radiosensitizing effects of vorinostat in combination with capecitabine were assessed by evaluation of tumor growth delay in two colorectal carcinoma xenografts models.

Results

Under hypoxia, radiosensitization by vorinostat was demonstrated in vitro in terms of decreased clonogenicity and in vivo as inhibition of tumor growth. Adding vorinostat to capecitabine-based CRT increased radiosensitivity of xenografts in terms of inhibited tumor growth.

Conclusions

Vorinostat sensitized colorectal carcinoma cells to radiation under hypoxia in vitro and in vivo and improved therapeutic efficacy in combination with capecitabine-based CRT in vivo. The results encourage implementation of vorinostat into CRT in LARC trials.

Background

Uterine sarcomas are very rare malignancies with no approved chemotherapy protocols. Histone deacetylase (HDAC) inhibitors belong to the most promising groups of compounds for molecular targeting therapy. Here, we described the antitumor effects of suberoylanilide hydroxamic acid (SAHA; vorinostat) on MES-SA uterine sarcoma cells in vitro and in vivo. We investigated effects of vorinostat on growth and colony forming ability by using uterine sarcoma MES-SA cells. We analyzed the influence of vorinostat on expression of different HDACs, p21WAF1 and activation of apoptosis. Finally, we examined the antitumor effects of vorinostat on uterine sarcoma in vivo.

Results

Vorinostat efficiently suppressed MES-SA cell growth at a low dosage (3 μM) already after 24 hours treatment. Decrease of cell survival was even more pronounced after prolonged treatment and reached 9% and 2% after 48 and 72 hours of treatment, respectively. Colony forming capability of MES-SA cells treated with 3 μM vorinostat for 24 and 48 hours was significantly diminished and blocked after 72 hours. HDACs class I (HDAC2 and 3) as well as class II (HDAC7) were preferentially affected by this treatment. Vorinostat significantly increased p21WAF1 expression and apoptosis. Nude mice injected with 5 × 106 MES-SA cells were treated for 21 days with vorinostat (50 mg/kg/day) and, in comparison to placebo group, a tumor growth reduction of more than 50% was observed. Results obtained by light- and electron-microscopy suggested pronounced activation of apoptosis in tumors isolated from vorinostat-treated mice.

Conclusions

Our data strongly indicate the high therapeutic potential of vorinostat in uterine sarcomas.

Bladder cancer is one of the most common malignancies and causes hundreds of thousands of deaths worldwide each year. Bladder cancer is strongly associated with exposure to environmental carcinogens. It is believed that DNA damage generated by environmental carcinogens and their metabolites causes development of bladder cancer. Nucleotide excision repair (NER) is the major DNA repair pathway for repairing bulk DNA damage generated by most environmental carcinogens, and XPC is a DNA damage recognition protein required for initiation of the NER process. Recent studies demonstrate reduced levels of XPC protein in tumors for a majority of bladder cancer patients. In this work we investigated the role of histone deacetylases (HDACs) in XPC gene silencing and bladder cancer development. The results of our HDAC inhibition study revealed that the treatment of HTB4 and HTB9 bladder cancer cells with the HDAC inhibitor valproic acid (VPA) caused an increase in transcription of the XPC gene in these cells. The results of our chromatin immunoprecipitation (ChIP) studies indicated that the VPA treatment caused increased binding of both CREB1 and Sp1 transcription factors at the promoter region of the XPC gene for both HTB4 and HTB9 cells. The results of our immunohistochemistry (IHC) staining studies further revealed a strong correlation between the over-expression of HDAC4 and increased bladder cancer occurrence (p < 0.001) as well as a marginal significance of increasing incidence of HDAC4 positivity seen with an increase in severity of bladder cancer (p = 0.08). In addition, the results of our caspase 3 activation studies demonstrated that prior treatment with VPA increased the anticancer drug cisplatin-induced activation of caspase 3 in both HTB4 and HTB9 cells. All of these results suggest that the HDACs negatively regulate transcription of the XPC gene in bladder cancer cells and contribute to the severity of bladder tumors.

Purpose

Although microarray technology has been widely adopted by the scientific community, analysis of the ensuing data remains challenging. In this paper we present our experience with a complex design microarray experiment on resistance mechanisms of histone deacetylase inhibitors (HDACIs).

Experimental design

To improve our understanding of the underlying mechanism of HDACI resistance in prostate cancer (PCa) cells, we designed a novel “multiple loop, double cube” cDNA microarray experiment. In the experiment of 22 arrays, DU145 and PC3 cells were treated with two different HDACIs (vorinostat, and Valproic Acid, VPA) and incubation periods (48 and 96 hours). Pre-processing included exploratory analyses of the quality of the arrays and intensity-dependent within-array loess normalization. An ANOVA model was used for inference. The results were validated by western blot analysis of known treatment targets.

Results

Treatment of PC3 and DU145 cells with HDACIs caused 2.8–10% (p<.001) differential expression across conditions. 51–73% of these genes were up-regulated and 28–49% down-regulated. The extent of differential expression was associated with: cell line (DU145>PC3), HDACI (VPA≥vorinostat) and duration of treatment (96h>48h). We identified known and new treatment targets involved in cell cycle and apoptosis.

Conclusion

A multiple loop, double cube microarray design can be used to identify HDACI induced changes in gene expression, possibly related to drug resistance.

Background

Traumatic brain injury (TBI) initiates a complex series of neurochemical and signaling changes that lead to pathological events including neuronal hyperactivity, excessive glutamate release, inflammation, increased blood-brain barrier (BBB) permeability and cerebral edema, altered gene expression, and neuronal dysfunction. It is believed that a drug combination, or a single drug acting on multiple targets, may be an effective strategy to treat TBI. Valproate, a widely used antiepileptic drug, has a number of targets including GABA transaminase, voltage-gated sodium channels, glycogen synthase kinase (GSK)-3, and histone deacetylases (HDACs), and therefore may attenuate a number of TBI-associated pathologies.

Methodology/Principal Findings

Using a rodent model of TBI, we tested if post-injury administration of valproate can decrease BBB permeability, reduce neural damage and improve cognitive outcome. Dose-response studies revealed that systemic administration of 400 mg/kg (i.p.), but not 15, 30, 60 or 100 mg/kg, increases histone H3 and H4 acetylation, and reduces GSK-3 activity, in the hippocampus. Thirty min post-injury administration of 400 mg/kg valproate improved BBB integrity as indicated by a reduction in Evans Blue dye extravasation. Consistent with its dose response to inhibit GSK-3 and HDACs, valproate at 400 mg/kg, but not 100 mg/kg, reduced TBI-associated hippocampal dendritic damage, lessened cortical contusion volume, and improved motor function and spatial memory. These behavioral improvements were not observed when SAHA (suberoylanilide hydroxamic acid), a selective HDAC inhibitor, was administered.

Conclusion/Significance

Our findings indicate that valproate given soon after TBI can be neuroprotective. As clinically proven interventions that can be used to minimize the damage following TBI are not currently available, the findings from this report support the further testing of valproate as an acute therapeutic strategy.

Valproate (VPA) is a well-characterized histone deacetylase inhibitor with anti-neoplastic properties. We analyzed the growth blocking effects and the molecular mode of action of this compound in colorectal cancer cells in vitro and in vivo. Caco-2, SW-480, CX-1 or WIDR cell lines were exposed to VPA (0.25–2 mM) for various time periods. Cell growth, cell cycle progression and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide dye reduction assay and flow cytometry. Cell cycle- and apoptosis-regulating proteins and histone acetylation were assessed by Western blotting. In vivo tumor growth and regulating protein expression under VPA were investigated in a subcutaneous xenograft tumor model. VPA inhibited the growth of all cell lines with cell cycle arrest paralleled by the up-regulation of H3 and H4 acetylation. In vivo tumor growth was substantially depressed by VPA (200 mg/kg bw). Cell cycle proteins (cdk1, cdk2, cdk4, cyclin D, cyclin E, p19, p21 and p27) were differentially altered by VPA. Predominantly cdk1 was decreased and p27 was up-regulated in all models. Apoptosis-related proteins were altered in vivo with up-regulation of bax and down-regulation of bcl-2. VPA exerts anti-neoplastic activity in colorectal tumor cell lines in vitro and in vivo by altering cell cycle regulation.

Peroxisome proliferator-activated receptor γ (PPARγ) might not be permissive to ligand activation in prostate cancer cells. Association of PPARγ with repressing factors or posttranslational modifications in PPARγ protein could explain the lack of effect of PPARγ ligands in a recent randomized clinical trial. Using cells and prostate cancer xenograft mouse models, we demonstrate in this study that a combination treatment using the PPARγ agonist pioglitazone and the histone deacetylase inhibitor valproic acid is more efficient at inhibiting prostate tumor growth than each individual therapy. We show that the combination treatment impairs the bone-invasive potential of prostate cancer cells in mice. In addition, we demonstrate that expression of E-cadherin, a protein involved in the control of cell migration and invasion, is highly up-regulated in the presence of valproic acid and pioglitazone. We show that E-cadherin expression responds only to the combination treatment and not to single PPARγ agonists, defining a new class of PPARγ target genes. These results open up new therapeutic perspectives in the treatment of prostate cancer.

Valproic Acid (VPA), a histone deacetylase inhibitor, has been demonstrated to cause a marked decrease in proliferation of prostate cancer (PCa) cells in vitro and a significant reduction in tumor volume in vivo. The goal of this study is to better understand the VPA-induced growth inhibition in vivo, by studying expression of various markers in PCa xenografts. Methods. For in vitro experiments, PCa cells were treated with 0, 0.6, and 1.2 mM VPA for 14 days. For in vivo models, experimental animals received 0.4% VPA in drinking water for 35 days. Tissue microarray was generated using cell pellets and excised xenografts. Results. VPA treatment causes cell cycle arrest in PCa cells in vivo, as determined by increase in p21 and p27 and decrease in cyclin D1 expression. Increased expression of cytokeratin18 was also seen in xenografts. LNCaP xenografts in treated animals had reduced androgen receptor (AR) expression. While decreased proliferation was found in vitro, increase in apoptosis was found to be the reason for decreased tumor growth in vivo. Also, an anti-angiogenic effect was observed after VPA treatment. Conclusion. VPA inhibits tumor growth by multiple mechanisms including cell cycle arrest, induction of differentiation, and inhibition of growth of tumor vasculature.

Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutic drugs. Their clinical utility in oncology stems from their intrinsic cytotoxic properties and combinatorial effects with other conventional cancer therapies. To date, the histone deacetylase inhibitors suberoylanilide hydroxamic acid (Vorinostat, Zolinza®) and depsipeptide (Romidepsin, Istodax®) have been approved by the US Food and Drug Administration for the treatment of refractory cutaneous T-cell lymphoma. Further, there are currently over 100 clinical trials involving the use of histone deacetylase inhibitors in a wide range of solid and hematological malignancies. The therapeutic potential of histone deacetylase inhibitors has also been investigated for numerous other diseases. For example, the cytotoxic properties of histone deacetylase inhibitors are currently being harnessed as a potential treatment for malaria, whereas the efficacy of these compounds for HIV relies on de-silencing latent virus. The anti-inflammatory properties of histone deacetylase inhibitors are the predominant mechanisms for other diseases, such as hepatitis, systemic lupus erythematosus and a wide range of neurodegenerative conditions. Additionally, histone deacetylase inhibitors have been shown to be efficacious in animal models of cardiac hypertrophy and asthma. Broad-spectrum histone deacetylase inhibitors are clinically available and have been used almost exclusively in preclinical systems to date. However, it is emerging that class- or isoform-specific compounds, which are becoming more readily available, may be more efficacious particularly for non-oncological applications. The aim of this review is to provide an overview of the effects and clinical potential of histone deacetylase inhibitors in various diseases. Apart from applications in oncology, the discussion is focused on the potential efficacy of histone deacetylase inhibitors for the treatment of neurodegenerative diseases, cardiac hypertrophy and asthma.