Substantial progress has been made in understanding the causal relationship between aberrant epigenetic changes and tumorigenesis.1 Dysregulated acetylation of core histones is among the epigenetic modifications that contribute to inappropriate gene expression in cancer cells. Acetylation and deacetylation of histones alter higher-order conformation of chromatin by influencing the interaction of histones with DNA.2,3 In eukaryotes, the packaging of genomic DNA into the chromatin structure provides a dynamic mechanism that allows the regulation of gene expression by controlling access of transcription factors and RNA polymerases to promoter regions (Figure 1) .
Illustration depicting the role of HDAC and histone acetyltransferase (HAT) enzymes in the regulation of transcription. Acetylation of nucleosomes by histones results in decondensed chromatin structures that facilitate transcription by allowing access of transcription factors and RNA polymerases to promoter regions. HDACi = HDAC inhibitor. Ac = Acetylation site.
Citation: American Journal of Veterinary Research 69, 7; 10.2460/ajvr.69.7.938
Histones are small, basic proteins that consist of a globular domain and an N-terminal tail that protrudes from the nucleosome. In the hypoacetylated state, nucleosomes are tightly compacted, which results in repression of transcription because transcription factors are prevented from accessing the targeted DNA. Histone acetylation of nucleosomes results in relaxed structures that permit transcription. The degree of this posttranslational modification is maintained by a dynamic balance between the activities of histone acetyltransferases and HDACs. Aberrant regulation of histone acetylation can cause inappropriate gene expression, which is a key event in carcinogenesis.2 Inhibition of HDACs induces expression of a specific subset of genes associated with inhibition of cell growth, induction of differentiation, or apoptosis in many types of tumor cells by reactivating transcription of a small number of genes.2 In particular, histone hypoacetylation represses the expression of tumor-suppressor genes. By inducing conditions that permit transcription, HDAC inhibitors effectively upregulate the expression of tumor-suppressor genes.2 Furthermore, HDAC inhibitors can modify the structure and function of many nonhistone proteins that are involved in the regulation of cell proliferation and cell death.2
The effects of epigenetic factors on the pathogenesis of cancer are of particular interest because these factors are attractive targets for pharmacologic intervention.2,3 In particular, inhibition of HDAC activity is recognized as a promising means of treating cancer patients because HDACs have been implicated in the pathogenesis of solid and hematologic malignancies.4,5 Substantial evidence from preclinical experiments suggests that HDAC inhibitors can arrest growth and induce differentiation and apoptosis in cancer cells in vitro and suppress tumor growth in mice. The effects of many structurally distinct, small-molecule HDAC inhibitors are the subject of investigation in phase I and phase II clinical trials in humans. The first of this class of drugs, SAHA (commonly known as vorinostat), has been approved for clinical use in humans.6
Histone deacetylase inhibitors differ with respect to their antitumor activity, toxicity, and stability.2 The hydroxamic acid SAHA is the prototypical inhibitor of HDACs. It can inhibit class I and class II HDACs at concentrations of approximately 50nM and can arrest the growth of various transformed cells in vitro, generally at low micromolar concentrations.6,7 Among the clinically relevant HDAC inhibitors, phenylbutyrate and other short-chain fatty acids are weak inhibitors of HDACs that are potent at millimolar concentrations in vitro.8 These compounds include a novel class of potent phenylbutyrate-based HDAC inhibitors that are active at submicromolar concentrations.8,9 One hydroxamatetethered derivative of the phenylbutyrate-derived class, OSU-HDAC42, achieves an IC50 for HDAC inhibition at low nanomolar concentrations.8,9 This compound is reportedly a more potent inhibitor of prostate cancer and hepatocellular carcinoma cell viability in vitro and tumor growth in vivo, and it is more effective at inducing apoptosis than SAHA.10,11
We are unaware of any studies in which investigators have evaluated HDAC inhibitors in canine tumors other than a case report.12 The purpose of the study reported here was to determine whether exposure of canine cancer cells to HDAC or SAHA results in increased histone acetylation and decreased cell viability and whether changes in viability involve induction of apoptosis or alterations in progression of the cell cycle.
Materials and Methods
Sample Population—Nine canine cancer cell lines were evaluated, including osteosarcoma (D17a), transitional cell carcinoma (K9TCCb), prostatic carcinoma (ACE-1c), apocrine gland carcinoma (ASAc), T-cell lymphoma (OSW13), mast cell tumor (C2d), melanoma (323610e), histiocytic sarcoma (030210f), and hemangiosarcoma (SB-HSA-2g).14–18 Cells from lines D17, OSW, C2, 030210, 323610, and SB-HSA-2 were cultured in RPMI 1640,h those from lines ACE-1 and ASA were cultured in a mixture of Dulbecco modified Eagle medium and Ham F12,h and those from line K9TCC were cultured in Dulbecco modified Eagle mediumi alone. All media were supplemented with 10% fetal bovine serumj and antimicrobialsk (penicillin [100 U/ mL] and streptomycin [0.1 mg/mL]). Cell lines were maintained in a humidified atmosphere of 5% carbon dioxide at 37°C.
Synthesis of HDAC inhibitors—Synthesis of OSUHDAC42 and SAHA was performed by our laboratory group; purities exceeded 99% as assessed by nuclear magnetic resonance spectroscopy (300 MHz).9 Both compounds were dissolved in 100% DMSOl at concentrations of 10mM to generate stock solutions, which were subsequently shielded from light and stored at −80°C. As needed, stock solutions were thawed and diluted with DMSO to provide a range of concentrations. Solutions were further diluted by mixing with the appropriate cell culture medium to generate working solutions with inhibitor concentrations ranging from 0.1 to 100μM and a constant DMSO concentration of ≤ 0.1% by volume. Fresh OSU-HDAC42 and SAHA solutions were prepared from stock solutions for each experiment.
Assessment of cell viability—Cells were seeded in 96-well plates in triplicate at a density of 5,000 to 20,000 cells/well in the aforementioned appropriate medium, which was supplemented with 10% fetal bovine serum (final volume, 200 μL/well). The number of cells plated per well was determined separately for each cell line on the basis of growth characteristics (ie, doubling time, size, and adherence). Cells were allowed to attach to the wells for 24 hours and were then exposed to various concentrations (0.25 to 10μM) of OSU-HDAC42 or SAHA for 24, 48, or 72 hours. Control cells were treated with DMSO vehicle at a concentration equal to that of drug-treated cells (final DMSO concentration, ≤ 0.1% by volume). Cell proliferation and viability were determined by use of a tetrazolium salt assay,m which quantifies the reduction of a tetrazolium salt by mitochondria in viable cells. Number of cells per well was determined on the basis of absorbance values of known numbers of cells. After 24, 48, or 72 hours of treatment with an inhibitor, 20 μL of WST-1 reagent was added to each well, and wells were then incubated for 2 to 4 hours at 37°C. After subtraction of the background value, the absorbance of the reaction product was measured at 450 nm by use of an automated microplate reader.n All treatments were evaluated in triplicate, with values expressed as mean ± SD. Each experiment was repeated at least 3 times.
Evaluation of induction of apoptosis—To assess drug-induced apoptosis among cells from the 2 cell lines most sensitive to HDAC inhibitors (OSW and C2) and in a cell line that was relatively resistant to the same compounds (K9TCC), detection and quantification of cytoplasmic nucleosomes were performed with an ELISA kito used in accordance with the manufacturer's instructions. This ELISA provides a quantitative assessment of cytoplasmic histone–associated DNA fragments (mononucleosomes or oligonucleosomes) that are generated after induced apoptosis.
Evaluation of histone acetylation—Histone acetylation in cells from all 9 canine cancer cell lines was assessed by use of western blot analysis. Lysates of cells treated with various concentrations of OSU-HDAC42, SAHA, or DMSO vehicle were collected after 48 hours of treatment. Cells were lysed in mammalian protein extraction reagentp unless otherwise stated. To avoid degradation as a result of dephosphorylation of proteins in samples of lysates, a protease inhibitor solutionq (1%) and phosphatase inhibitorsr,s (1mM) were added to the mixture. Lysates were washed with ice-cold PBS solution and resuspended in lysis buffer containing 20mM Tris-HCl (pH, 8.0), 137mM NaCl, 1mM CaCl2, 10% glycerol, 1% NP 40, 0.5% deoxycholate, 0.1% SDS, 100μM 4-(2-aminoethyl)-benzenesulfonyl fluoride, leu peptin (10 μg/mL), and aprotinin (10 μg/mL). Soluble fractions of cell lysates were collected after centrifugation (10,621 Xg for 5 minutes), quantification, and boiling for 10 minutes. Equivalent amounts of proteins (25 μg) from each lysate were resolved on precast 10% polyacrylamide gelst and electrophoretically transferred to nitrocellulose membranes.u Transblotted membranes were washed 3 times with TBST. Membranes were blocked by the addition of 5% skim milk in TBST and incubation for 1 hour at 20°C; probed with primary antibodies against acetylated histone 3,v acetylated histone 4,w caspase 3,x and B-actiny at 1:1,000 dilutions in 5% blocking reagentz in TBST; and incubated overnight at 4°C. Membranes were then washed 3 times in TBST for 15 minutes, treated with goat anti-rabbit IgG conjugated to horseradish peroxidaseaa at 1:15,000 in 2.5% milk in TBST for 1 hour, and washed 3 times. Antigen and antibody reactions were detected by use of chemiluminescence reagent.bb To evaluate whether histone acetylation had occurred, gels were visually inspected for differences in band intensity.
Evaluation of effects on cell cycle—To determine whether OSU-HDAC42 or SAHA affected progression of the cell cycle, cells from lines OSW, C2, and K9TCC were seeded onto 6-well plates at a density of 2.0 × 105 cells/well. Cells were incubated for 24 hours, after which the medium was replaced with medium containing DMSO vehicle (control) or 1 or 10μM OSUHDAC42 or SAHA. After 24 hours of exposure, cells were washed with 0.1% glucose in PBS solution and removed from the plates with 0.1% trypsin-EDTA. Cells were collected in 500 μL of ice-cold PBS solution and then fixed in 7 mL of ice-cold 70% ethanol. Cell suspensions were incubated at 4°C overnight. Fixed cells were washed 2 times in 5 mL of PBS solution and suspended in 500 μL of PBS solution containing RNase A (100 μg/mL) and propidium iodide (50 μg/mL). Flow cytometric analysis was performed by use of a multipurpose flow cytometercc and analysis software.dd Samples of 10,000 cells each were assayed in duplicate. Accumulation of cells in the subG1 phase is an indicator of DNA fragmentation and apoptosis and was used to quantify cell death.
Statistical analysis—Statistical analyses comparing mean optical densities between control (DMSO) and treated wells for each drug concentration were performed by use of 1-way ANOVA. Pairwise multiple comparisons were performed by use of the Dunnett procedure; other multiple comparisons were performed by use of a Bonferroni correction. All analyses were performed by use of statistical software.ee Values of P < 0.05 were considered significant.
Results
Assessment of cell viability—Viability of cells from 9 cancer cell lines was significantly (P < 0.01 to P < 0.05) inhibited in a dose-dependent manner after exposure to OSU-HDAC42 or SAHA (Figure 2) . For adherent cells from lines that were sensitive to HDAC inhibitors, macroscopic and microscopic examination revealed cellular fragmentation and loss of attachment of cells to the cell culture plate. In contrast, no evidence of toxic effects was detected for vehicle-treated cells. After 48 hours, the IC50 for OSU-HDAC42 or SAHA was reached in cells from 6 and 4 cell lines, respectively, at concentrations that ranged from 0.4 to 1.3μM for OSUHDAC42 and 0.6 to 4.8μM for SAHA. The lowest IC50s of OSU-HDAC42 and SAHA were detected in cells from the OSW line (0.6μM and 1.1μM, respectively) and the C2 line (0.4μM and 1.5μM, respectively). In the hemangiosarcoma cell line (SB-HSA-2), no significant differences between cell viability in vehicle-treated cells and inhibitor-treated cells were detected for SAHA, and significant but small differences in cell viability were detected in OSU-HDAC42–treated cells at the concentrations evaluated (≤ 10μM). Although significant differences in cell viability were detected in the treated melanoma cell line (32360), the differences were small and varied little with dose. Cell lines SB-HSA-2 and 32360 were considered relatively resistant. Inhibitor concentrations > 10μM were not evaluated because concentrations in blood > 10μM may not be achievable in vivo.
Mean ± SD time- and dose-dependent effects of various concentrations of OSU-HDAC42 and SAHA on viability of cells from 9 canine cancer cell lines (transitional cell carcinoma [line K9TCC; A], apocrine gland adenocarcinoma [line ASA; B], prostate adenocarcinoma [line ACE-1; C], osteosarcoma [line D17; D], melanoma [line 323610; E], mast cell tumor [line C2; F], histiocytic sarcoma [line 030210; G], T-cell lymphoma [line OSW; H], and hemangiosarcoma [line SB-HSA-2; I]) representative from 3 separate experiments. Cells were exposed to OSU-HDAC42 or SAHA in 96-well plates at indicated concentrations for 24 (white bars), 48 (gray bars), or 72 (black bars) hours, and cell viability was assessed by a tetrazolium salt assay. Toxic effects were not detected (ie, cell viability was approx 100%) in control cells treated with DMSO vehicle at concentrations equal to those of drug-treated cells. *Significant (P < 0.01) difference between results for treated cells and respective control cells.
Citation: American Journal of Veterinary Research 69, 7; 10.2460/ajvr.69.7.938
Evaluation of histone acetylation—Exposure to OSU-HDAC42 and SAHA resulted in a dose-dependent increase in H3 and H4 histone acetylation in cells from all cell lines (Figure 3) . Histone acetylation was increased in inhibitor sensitive (OSW and C2) and inhibitor resistant (323610) cells.
Representative immunoblots from western blot analysis of the dose-dependent effects of 1 or 10μM of OSU-HDAC42 or SAHA on acetylation of histone H3 (AcH3), histone H4 (AcH4), and actin achieved after 48 hours in cells from 9 canine cancer cell lines (transitional cell carcinoma [line K9TCC; A], apocrine gland adenocarcinoma [line ASA; B], prostate adenocarcinoma [line ACE-1; C], osteosarcoma [line D17; D], melanoma [line 323610; E], mast cell tumor [line C2; F], histiocytic sarcoma [line 030210; G], T-cell lymphoma [line OSW; H], and hemangiosarcoma [line SB-HSA2; I]). Cells cultured in DMSO vehicle served as controls. Exposure to OSU-HDAC42 and SAHA resulted in a significant dose-dependent increase in H3 and H4 histone acetylation in all cell lines.
Citation: American Journal of Veterinary Research 69, 7; 10.2460/ajvr.69.7.938
Evaluation of induction of apoptosis—Both HDAC inhibitors (OSUHDAC42 and SAHA) induced a significant (P < 0.01) dose-dependent increase in cytoplasmic nucleosomes in cells from 2 inhibitor-sensitive cell lines (OSW and C2) and 1 inhibitor-resistant cell line (323610; Figure 4). Cleaved caspase 3 was detected in cells from the same 3 cell lines by use of western blot analysis. Moreover, OSU-HDAC42 was deemed the more potent of the 2 inhibitors because caspase-3 cleavage was detected after cells were treated with 1μM OSUHDAC42, whereas caspase-3 cleavage was detected only after cells were treated with 10μM SAHA.
Dose-dependent effects of OSU-HDAC42 and SAHA on apoptosis in cells from 3 canine cancer cell lines. A–Mean ± SD amount of fragmentation of cytoplasmic nucleosomal DNA in T-cell lymphoma (line OSW; left), mast cell tumor (line C2; middle), and transitional cell carcinoma (line K9TCC; right) cells treated with OSU-HDAC42 (black bars) or SAHA (white bars) at the indicated concentrations for 48 hours; results are representative of 2 experiments. Cells treated with DMSO vehicle were used as control cells. Fragmentation of cytoplasmic nucleosomal DNA was detected by use of an ELISA. Notice that the scale on the y-axis differs between cell lines. †Significant (P < 0.001) difference between results for cells treated with 1μM and 10μM of OSU-HDAC42 or SAHA. B–Representative immunoblots from western blot analyses of the cleavage of caspase 3 in OSW (left), C2 (middle), and K9TCC (right) cells treated with DMSO alone or OSU-HDAC42 or SAHA at the indicated concentrations for 48 hours. Results represent intact caspase 3 (top), cleaved caspase 3 (middle), and actin as a control compound (bottom). Both HDAC inhibitors induced an increase in cytoplasmic nucleosomes in treated cells. Caspase-3 cleavage occurred at a logarithm lower drug concentration in OSU-HDAC42 treated cells. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 69, 7; 10.2460/ajvr.69.7.938
Evaluation of effects on cell cycle—Cells from lines OSW and C2 underwent apoptosis after treatment with OSU-HDAC42 or SAHA, as detected by an accumulation of cells in the subG1 phase (Figure 5) . This effect was detected at a lower concentration of OSU-HDAC42 (1 logarithm lower than for SAHA). Cells from the transitional cell carcinoma (K9TCC) cell line were relatively resistant to HDAC inhibition, with little evidence of cell death detected after treatment with either drug at 24 hours. The HDAC inhibition did not result in arrest of the cell cycle in OSW or C2 lines, but it did induce endoreplication (DNA replication without cell division) in the K9TCC line.
Representative histograms of results of flow cytometric analyses of the proportion of cells in the subG1 phase from 3 canine cancer cell lines (T-cell lymphoma [line OSW; A, D, G, J, and M]; mast cell tumor [line C2; B, E, H, K, and N]; and transitional cell carcinoma [line K9TCC; C, F, I, L, and O]) after treatment with DMSO vehicle (A, B, and C), 1μM OSU-HDAC42 (D, E, and F), 1μM SAHA (G, H, and I), 10μM OSU-HDAC42 (J, K, and L), and 10μM SAHA (M, N, and O) for 24 hours. Cells were collected, washed, rendered permeable, and stained with propidium iodide immediately prior to analysis. The M1 region indicates the subG1 population.
Citation: American Journal of Veterinary Research 69, 7; 10.2460/ajvr.69.7.938
Discussion
Epigenetics involves the study of factors that influence gene expression. Changes in gene expression, particularly the inactivation of tumor-suppressor genes and consequent termination of protein expression, can destabilize the cell cycle, prevent apoptosis, and lead to uncontrolled cell proliferation, which can lead to tumor formation. Two important forms of epigenetic gene regulation are DNA methylation and histone acetylation. Both processes modify gene expression without changing the DNA sequence. Antiproliferative effects of HDAC inhibitors have been reported6,7,19,20 in cells from various human cancer cell lines, which suggests that HDAC inhibitors could be useful agents for the treatment of patients with many different types of cancer. Although SAHA has been approved for the treatment of humans with cutaneous T-cell lymphoma,6 many other HDAC inhibitors are in various stages of development and evaluation in human medicine.
In the study reported here, the in vitro effect of 2 HDAC inhibitors, OSU-HDAC42 and SAHA, on cells from a panel of canine cancer cell lines was investigated. First, our intent was to determine whether HDAC inhibitors may be useful in the treatment of dogs with cancer. Because SAHA is commercially available for clinical use in humans, it may become available for extralabel use in canine patients with cancer. Future options for extralabel use may arise as additional HDAC inhibitors, such as OSU-HDAC42 and others, become commercially available. Second, we intended to determine whether the difference in potency between OSUHDAC42 and SAHA that has been detected in cells from human cancer cell lines also exists in cells from canine cancer cell lines. Third, we believed that if canine cancer cells were sensitive to HDAC inhibitors, then treatment of naturally developing tumors in dogs may be an ideal method for evaluation of new HDAC inhibitors prior to clinical trials in humans.
We determined the in vitro efficacy of SAHA and OSU-HDAC42. The effects of both drugs on cells from canine cancer cell lines are similar to their effects on cells from human cancer cell lines; in both types of cells, the IC50s of sensitive cells are in the low micromolar range. Similar to the reported effects of HDAC inhibitors on cells from human cancer cell lines,6–9 the HDAC inhibitors evaluated in our study reduced the viability and proliferation of cells and induced apoptosis. Both HDAC inhibitors induced acetylation of cellular histones; however, histone acetylation did not always correlate with reduction of cell viability and proliferation or induction of apoptosis. Although histones are a primary site of action for HDAC inhibitors, the antitumor effects of these inhibitors may also be attributed to modulation of the acetylation state of nonhistone proteins such as nuclear factor κB, STAT3, p53, and heat shock protein-90.2 Thus, differences in the protein substrates that are modified in cells from various cell lines or tumors and differences between the effects of various HDAC inhibitors on nonhistone protein substrates likely contribute substantially to the differences in antitumor effects detected for various HDAC inhibitors and tumors.
In the study reported here, cells from certain canine cancer cell lines responded to 2 HDAC inhibitors; however, OSU-HDAC42 was generally more potent than SAHA. Increases in histone acetylation were detected after treatment of cells with a 10-fold lower concentration of OSU-HDAC42 than of SAHA. Similarly, induction of apoptosis and decreased cell viability were detected at lower concentrations of OSU-HDAC42, compared with effects for SAHA. More intensive investigation of the effects of histone acetylation on gene expression and modification of nonhistone protein substrates is required to determine whether the effects of OSU-HDAC42 differ from those of SAHA in other ways. Certainly, differences in toxic effects in vivo would be relevant to the clinical usefulness of various HDAC inhibitors.
To our knowledge, the only reported clinical use of an HDAC inhibitor in a dog with cancer is a case report12 in which investigators describe the use of SAHA to treat a dog with hemangiosarcoma after splenectomy. In that dog, treatment with SAHA (3 mg/kg, PO, q 24 h) was apparently tolerated well. Although the dog survived for > 1,000 days after splenectomy while receiving SAHA, the role SAHA may have had in the clinical outcome is uncertain.
Our study of canine cancer cell lines provided evidence that HDAC inhibitors are a potentially promising new class of anticancer drugs to treat dogs with cancer. Furthermore, the concentrations of drug required to increase histone acetylation, decrease proliferation, and induce apoptosis are probably achievable in vivo. In phase I studies21,22 of SAHA in humans, SAHA was safely administered by use of IV and various orally administered dosage protocols. Plasma concentrations of SAHA > 1μM were achieved via both routes of administration. In rats, the maximum plasma concentration achieved with 1 dose of OSU-HDAC42 (50 mg/kg, PO) was 7.1μM.ff The panel of canine cancer cell lines used in the study reported here represented 9 histologically distinct types of cancer; an IC50 of ≤ 10μM was achieved in cells from 6 and 4 of those lines after treatment with OSU-HDAC42 and SAHA, respectively. Cells from T-cell lymphoma and mast cell tumor lines were the most sensitive to both drugs. Interestingly, SAHA was initially approved to treat humans with cutaneous T-cell lymphoma.6 In a phase II clinical trial of orally administered SAHA for refractory cutaneous T-cell lymphoma, 8 of 35 patients previously treated with other chemotherapeutic regimens achieved a partial response.23 Similarly, in a phase I trial of another HDAC inhibitor, depsipeptide (FK228), responses were detected in patients with T-cell lymphoma.24 Both drugs induce histone acetylation, cell cycle arrest, and apoptosis in cells from human T-cell lymphoma lines; however, the specific mechanism of the sensitivity of cells to HDAC inhibitors is unclear.25,26 Mast cell proliferation is also affected by HDAC inhibition. The HDAC inhibitor trichostatin A increases acetylation of histones, reduces cell viability, and induces apoptosis in cells from the p815 mouse mastocytoma line.27 In our study, similar effects of increased histone acetylation, reduced cell viability, and induction of apoptosis were detected in cells from canine T-cell lymphoma and mast cell tumor lines treated with OSU-HDAC42 and SAHA.
In the study reported here, as in most studies of the effects of HDAC inhibitors in humans and mice, histone acetylation did not correlate with antitumor responses. A dose-dependent increase in histone acetylation was detected in cells from all lines, regardless of whether there was a concurrent decrease in cell viability and increase in apoptosis. It is important to mention that because each tumor type was represented by only 1 cell line in our study, general conclusions regarding the sensitivity of various tumor types to OSU-HDAC42 and SAHA cannot be made; however, these results suggested that additional investigation of HDAC inhibitors in canine lymphoma and mast cell tumors is warranted. Finally, our study provided in vitro evidence that HDAC inhibitors induced nuclear histone acetylation, decreased cell proliferation, and induced apoptosis in cells from canine cancer cell lines, similar to their effects on cells from human cancer cell lines, which suggests that naturally developing cancers in dogs may be a good method for the preclinical evaluation of HDAC inhibitors in humans.
ABBREVIATIONS
| DMSO | Dimethyl sulfoxide |
| HDAC | Histone deacetylase |
| IC50 | Concentration at which 50% inhibition of cell viability was achieved |
| OSU-HDAC42 | S(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)benzamide |
| SAHA | Suberoylanilide hydroxamic acid |
| TBST | 0.05% Tween-20 in tris-buffered saline solution |
American Type Culture Collection, Manassas, Va.
Provided by Dr. Deborah Knapp, Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, Ind.
Provided by Dr. Thomas Rosol, Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio.
Provided by Dr. Warren Gold, Cardiovascular Research Institute, University of California, San Francisco, Calif.
Provided by Dr. Michael Kent, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Calif.
Provided by Dr. Cheryl London, Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio.
Provided by Dr. Jaime Modiano, University of Minnesota Cancer Center, University of Minnesota, Minneapolis, Minn.
Gibco, Invitrogen, Carlsbad, Calif.
DMEM, GIBCO, Invitrogen, Carlsbad, Calif.
Gemini, West Sacramento, Calif.
Penstrep, GIBCO, Invitrogen, Carlsbad, Calif.
Fisher Scientific, Fair Lawn, NJ.
WST-1 assay, Roche Diagnostics, Indianapolis, Ind.
Spectra Max M2 plate reader, Molecular Devices, Sunnyvale, Calif.
Cell Death Detection ELISA, Roche Diagnostics, Indianapolis, Ind.
M-PER, Pierce Biotechnology, Rockford, Ill.
Halt protease inhibitor cocktail, Pierce Biotechnology, Rockford, Ill.
Sodium vanadate, Sigma Chemical Co, St Louis, Mo.
Sodium fluoride, Sigma Chemical Co, St Louis, Mo.
GeneMate 10% express gels, ISC-Bioexpress, Keysville, Utah.
Biotrace NT, Pall Corp, Pensacola, Fla.
Anti-acetylated histone 3 (H3) rabbit polyclonal antibody, Upstate Biotechnology, Temecula, Calif.
Anti-acetylated histone 4 (H4) rabbit polyclonal antibody, Upstate Biotechnology, Temecula, Calif.
Anti-caspase 3 rabbit polyclonal antibody, Cell Signaling Technology Inc, Beverly, Mass.
Anti-actin rabbit polyclonal antibody, Sigma Chemical Co, St Louis, Mo.
Blotto, Bio-Rad Laboratories, Hercules, Calif.
Goat anti-rabbit IgG conjugated to horseradish peroxidase, Cell Signaling Technology Inc, Beverly, Mass.
ECL reagent, Perkin-Elmer, Boston, Mass.
BD FACSCalibur, Becton-Dickinson, San Jose, Calif.
Cell Quest Pro software, Becton Dickinson Biosciences, San Jose, Calif.
GraphPad Prism, version 4.0, GraphPad Software Inc, San Diego, Calif.
Chan KK, College of Pharmacy, The Ohio State University, Columbus, Ohio: Personal communication, 2007.
References
- 2.↑
Lin HY, Chen CS, Lin SP, et al. Targeting histone deacetylase in cancer therapy. Med Res Rev 2006;26:397–413.
- 3.
Conley BA, Wright JJ, Kummar S. Targeting epigenetic abnormalities with histone deacetylase inhibitors. Cancer 2006;107:832–840.
- 4.
Marks P, Rifkind RA, Richon VM, et al. Histone deacetylases and cancer: causes and therapies. Nat Rev Cancer 2001;1:194–202.
- 5.
Cress WD, Seto E. Histone deacetylases, transcriptional control, and cancer. J Cell Physiol 2000;184:1–16.
- 6.↑
Marks PA, Breslow R. Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug. Nat Biotechnol 2007;25:84–90.
- 7.
Marks PA. Discovery and development of SAHA as an anticancer agent. Oncogene 2007;26:1351–1356.
- 8.↑
Lu Q, Yang YT, Chen CS, et al. Zn2+-chelating motif-tethered short-chain fatty acids as a novel class of histone deacetylase inhibitors. J Med Chem 2004;47:467–474.
- 9.↑
Lu Q, Wang DS, Chen CS, et al. Structure-based optimization of phenylbutyrate-derived histone deacetylase inhibitors. J Med Chem 2005;48:5530–5535.
- 10.
Kulp SK, Chen CS, Wang DS, et al. Antitumor effects of a novel phenylbutyrate-based histone deacetylase inhibitor, (S)-HDAC42, in prostate cancer. Clin Cancer Res 2006;12:5199–5206.
- 11.
Lu YS, Kashida Y, Kulp SK, et al. Efficacy of OSU-HDAC42, a novel histone deacetylase inhibitor, in murine models of hepatocellular carcinoma. Hepatology 2007;46:1119–1130.
- 12.↑
Cohen LA, Powers B, Amin S, et al. Treatment of canine haemangiosarcoma with suberoylanilide hydroamic acid, a histone deacetylase inhibitor. Vet Comp Oncol 2004;2:243–248.
- 13.↑
Kisseberth WC, Nadella MV, Breen M, et al. A novel canine lymphoma cell line: a translational and comparative model for lymphoma research. Leuk Res. 2007;31:1709–1720.
- 14.
LeRoy BE, Thudi NK, Nadella MV, et al. New bone formation and osteolysis by a metastatic, highly invasive canine prostate carcinoma xenograft. Prostate 2006;66:1213–1222.
- 15.
Knapp DW, Chan TC, Kuczek T, et al. Evaluation of in vitro cytotoxicity of nonsteroidal anti-inflammatory drugs against canine tumor cells. Am J Vet Res 1995;56:801–805.
- 16.
Forsberg LS, Lazarus SC, Seno N, et al. Dog mastocytoma proteoglycans: occurrence of heparin and oversulfated chondroitin sulfates, containing trisulfated disaccharides, in three cell lines. Biochim Biophys Acta 1988;967:416–428.
- 17.
Shelly S, Chien MB, Yip B, et al. Exon 15 BRAF mutations are uncommon in canine oral malignant melanomas. Mamm Genome 2005;16:211–217.
- 18.
Liao AT, McMahon M, London C. Characterization, expression, and function of c-Met in canine spontaneous cancers. Vet Comp Oncol 2005;3:61–72.
- 19.
Papeleu P, Vanhaecke T, Elaut G, et al. Differential effects of histone deacetylase inhibitors in tumor and normal cells—what is the toxicological relevance? Crit Rev Toxicol 2005;35:363–378.
- 20.
Garcia-Manero G, Issa JP. Histone deacetylase inhibitors: a review of their clinical status as antineoplastic agents. Cancer Invest 2005;23:635–642.
- 21.
Kelly WK, Richon VM, O'Connor O, et al. Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously. Clin Cancer Res 2003;9:3578–3588.
- 22.
Kelly WK, O'Connor OA, Krug LM, et al. Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer. J Clin Oncol 2005;23:3923–3931.
- 23.↑
Duvic M, Talpur R, Ni X, et al. Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory cutaneous T-cell lymphoma (CTCL). Blood 2007;109:31–39.
- 24.↑
Piekarz RL, Robey R, Sandor V, et al. Inhibitor of histone deacetylation, depsipeptide (FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report. Blood 2001;98:2865–2868.
- 25.
Zhang C, Richon V, Ni X, et al. Selective induction of apoptosis by histone deacetylase inhibitor SAHA in cutaneous T-cell lymphoma cells: relevance to mechanism of therapeutic action. J Invest Dermatol 2005;125:1045–1052.
- 26.
Piekarz RL, Robey RW, Zhan Z, et al. T-cell lymphoma as a model for the use of histone deacetylase inhibitors in cancer therapy: impact of depsipeptide on molecular markers, therapeutic targets, and mechanisms of resistance. Blood 2004;103:4636–4643.
- 27.↑
Yee SB, Kim MS, Baek SJ, et al. Trichostatin A induces apoptosis of p815 mastocytoma cells in histone acetylation- and mitochondria-dependent fashion. Int J Oncol 2004;25:1431–1436.
