The human HDAC (histone deacetylase) family, a well-validated anticancer target, plays a key role in the control of gene expression through regulation of transcription. While HDACs can be subdivided into three main classes, the class I, class II and class III HDACs (sirtuins), it is presently unclear whether inhibiting multiple HDACs using pan-HDAC inhibitors, or targeting specific isoforms that show aberrant levels in tumours, will prove more effective as an anticancer strategy in the clinic. To address the above issues, we have tested a number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lys™ (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rhHDAC3 and rhHDAC8) and class II HDAC isoforms (rhHDAC4, rhHDAC6, rhHDAC7 and rhHDAC9) was determined. MS-275 was HDAC1-selective, MGCD0103 was HDAC1- and HDAC2-selective, apicidin was HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I HDACs. The hydroxamic acid-derived compounds (trichostatin A, NVP-LAQ824, panobinostat, ITF2357, vorinostat and belinostat) were potent pan-HDAC inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted in increased acetylation of histones, but only pan-HDAC inhibitor treatment resulted in increased tubulin acetylation, which is in agreement with their activity towards the HDAC6 isoform.

INTRODUCTION

HDACs (histone deacetylases) are a family of enzymes involved in the regulation of a number of cellular processes [1], including cell proliferation, apoptosis and assembly of the cytoskeleton. However, the best-characterized HDAC function is the control of gene expression through regulation of transcription [2,3]. This involves an interplay between HATs (histone acetyltransferases) and HDACs, both of which are involved in post-translational modification of histone proteins [4]. HATs and HDACs have opposing roles in acetylating and deacetylating highly conserved lysine residues on the N-terminal tail of histones, thus altering chromatin assembly and transcriptional activity [5,6]. HDACs are also involved in regulating the acetylation of a number of non-histone proteins, such as α-tubulin and the tumour suppressor p53 [79]. These results, and reports of aberrant HDAC activity in a number of tumour types [10], suggest that inhibition of HDACs represents a viable anticancer strategy [1113].

To date, 18 members of the human HDAC family have been identified. These HDACs are subdivided into three individual classes based on structural and functional similarities. The class I isoforms (HDACs 1, 2, 3 and 8) and class II (HDACs 4, 5, 6, 7, 9, 10 and 11) are Zn-dependent enzymes, whereas class III HDACs [Sir1–Sir7 (sirtuins 1–7)] are NAD+-dependent [14,15]. HDACs exist in large multiprotein complexes, and there is evidence that most, if not all, HDAC isoforms require interaction with other HDACs or proteins for optimal enzymatic activity. For example, HDAC3 activity requires an interaction with SMRT/NCoR (silencing mediator for retinoid and thyroid receptors/nuclear receptor co-repressor) [16,17], whereas suppression of HDAC4 binding by the HDAC3–SMRT/NCoR complex results in the loss of HDAC4 enzymatic activity [18]. More recently, the presence of two intramolecular or intermolecular catalytic domains was reported to be required for protein deacetylation [19].

A number of HDAC inhibitors are currently undergoing clinical trials as anticancer drugs [2022], some of which have been characterized as selective HDAC inhibitors and some as pan-HDAC inhibitors. Recently vorinostat was registered as an HDACi (HDAC inhibitor) drug (Zolinza®; Merck). In the present paper we provide a comprehensive evaluation of the potency and selectivity of various HDAC inhibitors against recombinant HDAC isoforms.

MATERIALS AND METHODS

Belinostat (6,888,027), MS-275 (6,174,905), vorinostat (5,369,108), NVP-LAQ824 (6,552,065), panobinostat (6,552,065), ITF2357 (WO 03/013493) and MGCD0103 (6,897,220) were synthesized as described in the recent U.S. or World (WO) patent applications given in parentheses. All solvents were purified before use by routine techniques. The reaction products were isolated by evaporating the solvent using a vacuum rotary evaporator. Compounds were purified using column chromatography on an Acros silica-gel 0.035–0.070-mm-particle-size column. Reversed-phase HPLC on a Varian ProStar HPLC system, equipped with a spectrophotometer, was used to check purity of the synthesized compounds. The melting point was determined with micro melting-point apparatus (Boëtius or Fisher) and the uncorrected values were used. NMR spectra, obtained on Varian WH-90/DS or Mercury 200 spectrometers, were used to confirm the chemical structure of the compounds. The chemical-shift (δ) values are presented in p.p.m. (parts per million). Elemental analyses were carried out on a Carlo Erba EA 1108 instrument [23]. Apicidin, TSA and VPA (valproic acid) were purchased from Sigma–Aldrich.

Expression of HDAC isoforms

Recombinant HDAC isoforms were produced using a baculovirus expression vector system. In addition, mouse SMRT, a co-activator of HDAC3, was also expressed. The DAD (deacetylase activating domain) of SMRT, comprising 95 amino acids (395–489 in mouse SMRT) was expressed with a translation initiation codon (methionine) added to the N-terminus.

Cloning of cDNAs

cDNAs comprising the complete coding sequences of the human HDAC isoforms and the DAD portion of mouse SMRT were isolated from a human cDNA library by PCRs using primers listed in Supplementary Table 1 at http://www.BiochemJ.org/bj/409/bj4090581add.htm. Restriction sites for either BamHI or BglII were incorporated in the sense primers, and XhoI or SalI in the antisense primers, in order to facilitate subsequent cloning. In addition, sequences coding for either FLAG or His6 tags were incorporated in the primers. The PCR amplicons were subcloned into the cloning vector pCR2.1TOPO and nucleotide sequences were determined. The cDNAs were then subcloned into BamHI and XhoI sites of appropriate expression vectors (see Supplementary Table 2 at http://www.BiochemJ.org/bj/409/bj4090581add.htm).

Generation of recombinant baculoviruses

Recombinant baculoviruses were generated using either the Bac-N-Blue™ baculovirus system or the Bac-to-Bac® baculovirus expression system according to the instructions of the manufacturer (Invitrogen; see Supplementary Table 2). Sf9 insect cells, derived from Spodoptera frugiperda (fall armyworm), were cultured in Ex-Cell™ 420 medium supplemented with 5% (v/v) foetal bovine serum and served as host cells for virus generation and/or protein production.

The Bac-N-Blue™ baculovirus system was used to produce recombinant HDAC isoforms with no fusion protein; however, FLAG tags were added to the C-termini. In this system the cDNAs were first subcloned into the transfer vector pBlueBac4.5. Sf9 cells grown in adherent format were co-transfected with the transfer vector carrying the HDAC coding sequence and Bac-N-Blue™ linearized baculovirus DNA. Recombinant viruses were collected and cloned in plaque assays. Six to ten virus clones were tested in a small-scale infection for their ability to produce recombinant HDAC and to generate low-titre virus stock. One of these virus stocks was selected for generating high-titre virus stock and large-scale infection. High-titer virus stocks were produced by infecting a 200 ml culture of Sf9 cells [(1.5–2.0)×106 cells/ml] with 200 μl of the low-titre stock. Virus-laden media were harvested when the cell viabilities decreased to 40% or below (4–6 days post infection). The titre of the viral stocks was determined using the FastPlax™ Titer Kit (Novagen).

HDAC isoforms with N-terminal fusion of GST (glutathione transferase) with or without FLAG tags were produced using the Bac-to-Bac® baculovirus expression system. The transfer vector pFastBac1 was first modified to include the GST coding sequence at the 5′ end of the multiple cloning site. The HDAC cDNAs were inserted downstream of the GST coding sequence through subcloning. Competent Escherichia coli DH10Bac cells were transformed with the recombinant transfer vector. Recombinant bacmids were isolated and verified for the presence of the cDNAs coding for HDACs by PCR, using appropriate primers. Sf9 cells were transfected with the bacmid, and the resulting recombinant viruses were tested for their ability to produce recombinant HDACs. This low-titre stock was used to generate a larger-scale and high-titre viral stock as described above.

Large-scale infections

Production cultures were performed either in two to four 3-litre shake flasks (1 litre culture volume per flask) or in 20-litre Wave Biotech LLC (Somerset, NJ, U.S.A.) bioreactors (10 litre culture volume. Flasks or wave bioreactors were seeded with Sf9 cells at 0.5×106 cells/ml. When the cell densities reached (1.5–2.0)×106 cells/ml, the cultures were inoculated with appropriate recombinant baculovirus stocks at multiplicities of infection of 3–5. At 3 days after infection, cells were harvested by centrifugation at 1200 g for 15 min at 4 °C in a Sorvall Evolution RC centrifuge. Cell pellets were stored at −80 °C until protein purification.

Purification of recombinant HDAC isoforms

Lysis of infected cell pellets

Infected cells were resuspended in ice-cold lysis buffer (50 mM sodium phosphate, 300 mM NaCl and 1% Triton-X-100, pH 8.0), using 1 ml of lysis buffer for every 2×107 cells. Complete EDTA-free protease-inhibitor cocktail tablets (Roche Applied Science) were dissolved in the lysis buffer just prior to use at a ratio of one tablet for every 50 ml of buffer. The suspension was homogenized for 1 min at a setting of 1 on a T 25 basic Ultra-Turrax® dispersing instrument (IKA Works) and incubated on ice for 45 min. Insoluble material was removed by centrifugation at 23000 g at 4 °C for 45 min in a Sorvall Evolution RC centrifuge. The clarified lysate was processed immediately using batch chromatography.

Batch-mode affinity purification using glutathione–Sepharose

GST-tagged proteins were purified using glutathione–Sepharose 4 Fast Flow (GE Healthcare) (see Supplementary Table 2). Settled Sepharose beads were washed by suspending them in a 10-fold excess of ice-cold lysis buffer, followed by centrifugation at 4 °C for 5 min at 213 g in a Sorvall Legend RT tabletop centrifuge equipped with a swinging-bucket rotor. The wash step was repeated two more times. The washed beads were suspended in lysis buffer to make a 50% (w/v) slurry. The lysate was split into 50 ml aliquots in disposable centrifuge tubes, and 1.25 ml of Sepharose slurry was added to each tube. Tubes were incubated for 1 h at 4 °C with end-over-end mixing. The charged matrix was collected by centrifugation, and the beads were washed by centrifugation three times with 40 ml of ice-cold PBS. Finally, the matrix was resuspended in PBS and transferred to a Poly-Prep disposable chromatography column (Bio-Rad Laboratories). The column was placed at 4 °C and allowed to drain until the top of the bed was dry. One-third packed bed volume of ice-cold elution buffer (50 mM Hepes/40 mM reduced L-glutathione, pH 9.0) was carefully added to the top of the bed and allowed to drain through the column. The effluent was discarded. Two packed bed volumes of elution buffer were carefully added to the top of the bed, and the effluent was collected as a single fraction. The effluent containing the HDAC was immediately divided into aliquots and frozen at −80 °C.

Batch-mode affinity purification using anti-FLAG–agarose

FLAG-tagged proteins were purified using ANTI-FLAG M2 Affinity Gel (Sigma) (Supplementary Table 2). Settled agarose beads were washed as described above, twice with ice-cold PBS, once with 0.1 M glycine, pH 2.8, and twice with PBS containing an additional 150 mM NaCl. The washed beads were suspended in PBS containing an additional 150 mM NaCl to make a 50% slurry. Batch-mode capture, washing with PBS, and transfer to a Poly-Prep chromatography column were performed as described above. The column was placed at 4 °C and allowed to drain until the top of the bed was dry. Elution buffer was prepared as follows. 3×FLAG peptide (Sigma) was dissolved at a concentration of 25 μg/ml in 0.5 M Tris/HCl/1 M NaCl, pH 7.5, and the solution was diluted 1:4 with water to make a 5 μg/ml stock which was stored at −20 °C. Elution buffer was prepared just prior to use by adding the 3×FLAG peptide stock to PBS to a final concentration of 250 ng/ml. The pre-elution and elution steps, and the division of samples into aliquots were performed as described above.

HDAC assays

The deacetylase activity of rhHDACs (recombinant human HDACs) 1, 2, 3, 4, 6, 7 and 9 was assayed with a pan-HDAC substrate [initially KI-104 (Biomol International) and subsequently I-1925 (Bachem)]. The deacetylase activity of rhHDAC8 was assayed with HDAC8-specific FDL (Fluor de Lys™) substrate (KI-178; Biomol International). The total pan-HDAC assay volume was 50 μl and all the assay components were diluted in Hepes buffer (25 mM Hepes, 137 mM NaCl, 2.7 mM KCl and 4.9 mM MgCl2, pH 8.0). The reaction was carried out in half-volume white 96-well plates (Costar 3693). In brief, the pan-HDAC assay mixture contained HDAC substrate (30 μM, 25 μl), rhHDAC isoform (0.5–3 μl, diluted to 15 μl final volume) and inhibitor (5×the required assay concentration, diluted to 10 μl final volume). Positive controls contained all the above components except the inhibitor. The negative controls contained neither enzyme nor inhibitor. In each case these were replaced with an equivalent volume of buffer. The assay components were incubated at 37 °C for 3 h. The reaction was quenched with the addition of 50 μl HDAC–FDL Developer (KI-105; Biomol) 20×stock diluted to 1:400 in Hepes buffer and containing 2 μM TSA. The plates were incubated for 25 min at room temperature (22–22 °C) to allow the fluorescence signal to develop. The fluorescence generated was monitored at wavelengths 355 nm (excitation) and 460 nm (emission).

The HDAC8-specific assay was as described for the pan-HDAC assay, except for the changes indicated. The Hepes assay buffer contained BSA (1 mg/ml). The FDL–HDAC8 substrate was 25 μM. The assay components were incubated (3 h) at room temperature and then the reaction was quenched with HDAC–FDL Developer (KI-176, Biomol) 5×stock diluted 1:110 in Hepes buffer containing BSA (1mg/ml) and 2 μM TSA. The plates were incubated (25 min) at 4 °C to allow the fluorescence signal to develop.

Cell-proliferation assays

HeLa cells were grown in monoculture and treated with serial dilutions of the HDACis. The cells were incubated for 48 h at 37 °C. The number of viable cells was assessed using Cell Proliferation Reagent WST-1 (Roche). Media only and HeLa cells without HDACis were used as negative and positive controls respectively.

Immunoblotting

Cells grown in monolayer culture were treated with compounds as indicated in the Figure legends. Cells were harvested in 2×Tris/glycine/SDS sample buffer (Invitrogen) supplemented with 10 mM dithiothreitol (Sigma). Cell lysates were boiled for 10 min, resolved on SDS/4–20% (w/v) gradient polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). Immunoblotting was performed using standard procedures and the following primary antibodies: anti-[acetylated (lys40) α-tubulin] (T6793; Sigma), anti-[acetylated (Lys9) histone H3] (9671; Cell Signaling Technology), anti-(acetylated lysine) (Ac-K-103; 9681; Cell Signaling Technology), anti-p53 [a mixture of antibodies from Santa Cruz Biotechnology (sc-126) and Calbiochem (OP03)] and anti-actin (A5060; Sigma). After incubation with the appropriate horseradish-peroxidase-conjugated secondary antibodies, ECL® (enhanced chemiluminescence; GE Healthcare) was used for detection.

RESULTS

Cloning and expression

Eight full-length rhHDACs were expressed in insect Sf9 cells using a baculoviral expression system. The purified rhHDACs consisted of four class I enzymes (FLAG–HDAC1, GST–HDAC2–FLAG, FLAG–HDAC3+FLAG–SMRT and GST-HDAC8) and four class II enzymes (GST–HDAC4–His6, His6–HDAC6, HDAC7–FLAG and GST–HDAC9–FLAG).

HDAC assay

Various hydroxamate and non-hydroxamate-based HDACis (Table 1) were tested against purified rhHDAC proteins using an FDL assay and EC50 values were determined (Table 2). Of the ten inhibitors included in this study, the hydroxamic acid derivatives proved to be the most potent HDAC inhibitors, showing activity on both class I and class II HDAC isoforms. The most potent compounds, TSA, NVP-LAQ824 and panobinostat, exhibited EC50 values in the low nM range against rhHDACs 1, 2, 3, 4 and 9, and were 5–30-fold more potent than vorinostat and belinostat. TSA was also >10-fold more potent than vorinostat and belinostat as an inhibitor of rhHDACs 6 and 7, and was approximately equipotent to belinostat as an inhibitor of rhHDAC 8. NVP-LAQ824 and panobinostat were somewhat more potent as inhibitors of rhHDACs 6 and 7 compared with vorinostat and belinostat, and were equipotent as inhibitors of rhHDAC 8 compared with belinostat. Belinostat was approximately equipotent as an inhibitor of all rhHDAC isoforms tested. Vorinostat exhibited a similar profile, although it appeared to be slightly less effective as an inhibitor of rhHDAC8. ITF2357 proved to be equipotent to vorinostat and belinostat in the rhHDAC1, 2, 3, 4, 6 and 7 assays.

Table 1
HDACis
Compound Structure Chemical class Development status 
Belinostat 
graphic
 
Hydroxamate Phase II 
Vorinostat 
graphic
 
Hydroxamate Launched 
MS-275 
graphic
 
Benzamide Phase II 
NVP-LAQ824 
graphic
 
Hydroxamate Phase I 
Panobinostat 
graphic
 
Hydroxamate Phase II 
TSA 
graphic
 
Hydroxamate Preclinical 
MGCD0103 
graphic
 
Benzamide Phase II 
Apicidin 
graphic
 
Cyclic tetrapeptide Preclinical 
ITF2357 
graphic
 
Hydroxamate Phase I 
VPA 
graphic
 
Short-chain fatty acid Phase II 
Compound Structure Chemical class Development status 
Belinostat 
graphic
 
Hydroxamate Phase II 
Vorinostat 
graphic
 
Hydroxamate Launched 
MS-275 
graphic
 
Benzamide Phase II 
NVP-LAQ824 
graphic
 
Hydroxamate Phase I 
Panobinostat 
graphic
 
Hydroxamate Phase II 
TSA 
graphic
 
Hydroxamate Preclinical 
MGCD0103 
graphic
 
Benzamide Phase II 
Apicidin 
graphic
 
Cyclic tetrapeptide Preclinical 
ITF2357 
graphic
 
Hydroxamate Phase I 
VPA 
graphic
 
Short-chain fatty acid Phase II 
Table 2
Inhibition of rhHDAC isoforms in the in vitro HDAC biochemical assay

Each compound was assayed in triplicate per plate. EC50 values were determined from the average of a minimum of two plates. Results are means±S.E.M.

  EC50 (nM) 
Inhibitor rhHDAC… 
TSA  2±0 3±0 4±1 6±2 3±1 5±2 456±59 6±5 
NVP-LAQ824  5±1 5±1 12±1 23±18 7±3 18±4 162±44 6±5 
Panobinostat  3±0 3±0 4±1 12±5 61±1 14±7 248±11 3±2 
ITF2357  28±8 56±13 21±3 52±5 27±16 163±8 ND ND 
Belinostat  41±6 125±21 30±0 115±16 82±19 67±22 216±43 128±46 
Vorinostat  68±14 164±45 48±17 101±31 90±26 104±35 1524±463 107±21 
MS-275  181±62 1155±134 2311±803 >10000 >10000 >10000 >10000 505±37 
MGCD0103  34±17 34±8 998±431 >10000 >10000 >10000 >10000 ND 
Apicidin  >10000 120±28 43±7 >10000 >10000 >10000 575±111 >10000 
VPA  1584000±302 624 3068000±0 3071000±0 ND >10000 >10000 7442000±2 740000 >10000 
  EC50 (nM) 
Inhibitor rhHDAC… 
TSA  2±0 3±0 4±1 6±2 3±1 5±2 456±59 6±5 
NVP-LAQ824  5±1 5±1 12±1 23±18 7±3 18±4 162±44 6±5 
Panobinostat  3±0 3±0 4±1 12±5 61±1 14±7 248±11 3±2 
ITF2357  28±8 56±13 21±3 52±5 27±16 163±8 ND ND 
Belinostat  41±6 125±21 30±0 115±16 82±19 67±22 216±43 128±46 
Vorinostat  68±14 164±45 48±17 101±31 90±26 104±35 1524±463 107±21 
MS-275  181±62 1155±134 2311±803 >10000 >10000 >10000 >10000 505±37 
MGCD0103  34±17 34±8 998±431 >10000 >10000 >10000 >10000 ND 
Apicidin  >10000 120±28 43±7 >10000 >10000 >10000 575±111 >10000 
VPA  1584000±302 624 3068000±0 3071000±0 ND >10000 >10000 7442000±2 740000 >10000 

MS-275 exhibited EC50 values in the nM range against rhHDAC 1. It was significantly less potent (EC50 values in the low μM range) against rhHDACs 2, 3 and 9. MS-275 did not inhibit rhHDACs 4, 6, 7 and 8. Similarly, MGCD0103 exhibited EC50 values in the low nM range against rhHDACs 1 and 2. However, it was much less potent against rhHDAC3, with an EC50 value of ∼1 μM, and did not inhibit the activity of rhHDAC8 or class II HDACs.

Apicidin inhibited rhHDACs 2 and 3 in the nanomolar range. It also inhibited rhHDAC8, but with lower potency (EC50 of 575 nM), and had no inhibitory effect on rhHDAC1 and class II rhHDACs. VPA inhibited rhHDACs 1, 2, 3 and 8 in the millimolar range, but exhibited little activity in the class II rhHDAC assays; hence potency was several orders of magnitude lower compared with that of the other compounds tested. Those compounds which gave incomplete inhibition of rhHDAC isoforms at 100 μM were given a value of >100000.

In order to make a clear comparison of the potency of the inhibitors towards rhHDACs, log EC50 values were plotted for all compounds with reference to the two major HDAC classes (Figures 1A and 1B). Of the class I HDACs, rhHDAC8 was an outlier, as most compounds tested were less potent against this isoform than against rhHDACs 1, 2 and 3 (Figure 1A). MS-275, MGCD0103, apicidin and VPA had little activity on class II isoforms, thus confirming that these compounds are class-I-selective (Figure 1B).

Inhibitory activity of the indicated HDACis on class I and class II rhHDAC isoforms

Figure 1
Inhibitory activity of the indicated HDACis on class I and class II rhHDAC isoforms

Inhibitory activity of the indicated HDACis on class I (rhHDACs 1, 2, 3 and 8; A) and class II (rhHDACs 4, 6, 7 and 9; B) isoforms was determined in a cell-free system using the FDL assay and plotted as log EC50 for the purposes of comparison.

Figure 1
Inhibitory activity of the indicated HDACis on class I and class II rhHDAC isoforms

Inhibitory activity of the indicated HDACis on class I (rhHDACs 1, 2, 3 and 8; A) and class II (rhHDACs 4, 6, 7 and 9; B) isoforms was determined in a cell-free system using the FDL assay and plotted as log EC50 for the purposes of comparison.

Cell-proliferation assay

Ten hydroxamate and non-hydroxamate HDACis (Table 3) were tested in cell-proliferation assays in HeLa cells. NVP-LAQ824, panobinostat, TSA and ITF2357 exhibited the highest potency, with EC50 values of <1 μM. MGCD0103, apicidin and belinostat had EC50 values of <2 μM. Vorinostat and MS-275 had EC50 values of 3.2 and 3.9 μM respectively. VPA had an EC50 of 7.3 mM. Belinostat was found to be 1.7-fold more potent than vorinostat in the HeLa-cell-proliferation assay.

Table 3
Inhibition of HeLa cell proliferation by HDACis

Each compound was assayed in triplicate per plate. EC50 values were determined from the average of a minimum of two plates. Results are means±S.E.M.

Inhibitor EC50 (μM) 
TSA 0.187±0.095 
NVP-LAQ824 <0.04 
Panobinostat 0.106±0.040 
ITF2357 0.408±0.183 
Belinostat 1.921±0.289 
Vorinostat 3.245±1.175 
MS-275 3.919±1.370 
MGCD0103 1.030±0.294 
Apicidin 1.550±0.919 
VPA 7275±247 
Inhibitor EC50 (μM) 
TSA 0.187±0.095 
NVP-LAQ824 <0.04 
Panobinostat 0.106±0.040 
ITF2357 0.408±0.183 
Belinostat 1.921±0.289 
Vorinostat 3.245±1.175 
MS-275 3.919±1.370 
MGCD0103 1.030±0.294 
Apicidin 1.550±0.919 
VPA 7275±247 

Determination of acetylation of levels of proteins after HDACi treatment

The lysates of HeLa cells exposed to HDACis at 2×EC50 for 4 and 24 h were examined by immunoblotting with anti-acetyl-lysine antibody. Both the pan-HDAC and the isoform-specific inhibitors induced the acetylation of low-molecular-mass proteins consistent with the size of histones H3 and H4 (17 and 11 kDa respectively) in the lysates of cells treated with inhibitor for 24 h. However, the lysates from cells exposed to MS-275 and MGCD0103 for 4 h showed a lower level of acetylation of the two low-molecular-mass proteins (Figure 2A) compared with lysates from cells treated with the same compounds for 24 h (Figure 2B). In addition, the pan-HDAC inhibitors induced the acetylation of a high-molecular-mass protein the size of which is consistent with that of tubulin (Figures 2A and 2B).

Determination of acetylation of levels of proteins after HDACi treatment

Figure 2
Determination of acetylation of levels of proteins after HDACi treatment

HeLa cells were exposed to the indicated HDACis at a concentration of 2×EC50 (as determined in a cell-viability assay on HeLa cells) for 4 and 24 h, after which time total cell lysates were prepared and examined by immunoblotting under reducing conditions with an antibody to acetyl-lysine. The higher-molecular-mass protein (∼50 kDa) is probably α-tubulin, and the lower-molecular-mass proteins (∼10–15 kDa) are probably histones. The blots were re-probed with actin to confirm equal loading

Figure 2
Determination of acetylation of levels of proteins after HDACi treatment

HeLa cells were exposed to the indicated HDACis at a concentration of 2×EC50 (as determined in a cell-viability assay on HeLa cells) for 4 and 24 h, after which time total cell lysates were prepared and examined by immunoblotting under reducing conditions with an antibody to acetyl-lysine. The higher-molecular-mass protein (∼50 kDa) is probably α-tubulin, and the lower-molecular-mass proteins (∼10–15 kDa) are probably histones. The blots were re-probed with actin to confirm equal loading

HeLa cells were exposed to the HDACis at 8-fold their EC50 for 24 h, and the whole-cell lysates were examined by immunoblotting for acetyltubulin and acetylhistone H3. The results (Figure 3) show that, compared with the control lysate, the pan-HDAC inhibitors (belinostat, vorinostat, TSA, NVP-LAQ824, panobinostat and ITF2357) induced a strong acetylation of tubulin and histone H3, whereas the isoform-specific inhibitors induced strong acetylation of histone H3, but weak, or no, acetylation of tubulin.

Acetylation of tubulin and histone H3 using HDACis at 8-fold their EC50 for 24 h

Figure 3
Acetylation of tubulin and histone H3 using HDACis at 8-fold their EC50 for 24 h

HeLa cells were exposed to the indicated HDACis at a concentration of 8×EC50 (as determined in a cell-viability assay on HeLa cells) for 24 h, after which time total cell lysates were prepared and examined by immunoblotting under reducing conditions with antibodies to acetyl-α-tubulin or acetylhistone H3. The blots were reprobed with actin to confirm equal loading.

Figure 3
Acetylation of tubulin and histone H3 using HDACis at 8-fold their EC50 for 24 h

HeLa cells were exposed to the indicated HDACis at a concentration of 8×EC50 (as determined in a cell-viability assay on HeLa cells) for 24 h, after which time total cell lysates were prepared and examined by immunoblotting under reducing conditions with antibodies to acetyl-α-tubulin or acetylhistone H3. The blots were reprobed with actin to confirm equal loading.

HCC827 (human lung cancer) and DU145 (human prostate carcinoma) cells were treated for 24 h with belinostat, MS-275 and MGCD0103 at four different concentrations (0.125×, 0.25×, 0.5× and 1×EC50). The lysates from these cells were examined by immunoblotting with an anti-p53 antibody. The results for both cell lines showed that belinostat decreased the expression of p53 in a dose-dependent manner, whereas the HDAC-isoform-specific compounds MS-275 and MGCD0103 did so only weakly, if at all (Figures 4A and 4B).

Expression of p53 in HCC827 and DU145 cells

Figure 4
Expression of p53 in HCC827 and DU145 cells

HCC827 and DU145 cells were exposed to the indicated HDACis at concentrations of 0.125–1×EC50 (as determined in a cell-viability assay on HeLa cells) for 24 h, after which time total cell lysates were prepared and examined by immunoblotting under reducing conditions with an antibody to p53. The blots were re-probed with actin to confirm equal loading.

Figure 4
Expression of p53 in HCC827 and DU145 cells

HCC827 and DU145 cells were exposed to the indicated HDACis at concentrations of 0.125–1×EC50 (as determined in a cell-viability assay on HeLa cells) for 24 h, after which time total cell lysates were prepared and examined by immunoblotting under reducing conditions with an antibody to p53. The blots were re-probed with actin to confirm equal loading.

DISCUSSION

Recently published findings indicate that both class I and class II HDACs are aberrantly expressed in human cancers. HDAC1 is up-regulated in prostate cancer [24] and gastric cancer [25], HDAC2 is up-regulated in gastric cancer [26], HDAC3 is up-regulated in lung cancer [27] and there is elevated expression of HDAC6 in oral squamous cell carcinoma [28]. HDAC8 has also recently been implicated in contributing to tumorigenesis by regulating telomerase activity via its interaction with hEST1B (human ever-shorter telomeres 1B) [29].

The main objective of the present study was to compare the potency and specificity of a number of structurally diverse small-molecule compounds as inhibitors of class I or II HDAC isoforms. A number of these inhibitors, including MGCD0103, vorinostat, panobinostat, VPA, MS-275, ITF2357 and belinostat, are currently undergoing Phase I, II and III clinical trials as anticancer drugs. A further aim was to determine the mode of action of these inhibitors, in particular their effect on the induction of acetylation of specific proteins in cells.

Purified class I and class II HDACs were obtained to address isoform specificity. Four class I and four class II active rhHDAC isoforms were expressed and purified, and although co-purification of a small number of endogenous proteins was noted in the preparations, these were always minor components compared with the recombinant enzyme. The exception was rhHDAC3 preparations, which were all inactive without the presence of the SMRT protein. As a result of this requirement, rhHDAC3 was co-expressed with SMRT protein, resulting in a highly active enzyme.

Our findings suggest that hydroxamic acid-derived compounds such as TSA, NVP-LAQ824, panobinostat, ITF2357, vorinostat and belinostat act as potent pan-HDAC isoform inhibitors. A notable observation was the similarity between belinostat and vorinostat in the biochemical isoform assays; both compounds exhibit similar EC50 values in all but the HDAC8 assay. Although vorinostat and belinostat appear similar in biochemical assays, belinostat is ∼ 1.7–7-fold more potent as an inhibitor of cell proliferation in a number of cancer cell lines [Table 3; N. Khan (TopoTarget), unpublished work; M. Jeffers (CuraGen), unpublished work; National Cancer Institute data (http://dtp.nci.nih.gov; Belinostat (NSC# 726630), Vorinostat (NSC# 701852)].

NVP-LAQ824 was more potent than TSA or panobinostat in the HeLa growth-inhibition assay (Table 3). The reason for this is unknown, and it is somewhat surprising, given the similarity in the enhanced induction of H3/H4 acetylation (Figures 2A, 2B and 3) and the relative equality of these three compounds in biochemical assays of HDAC inhibition (Table 2). However, it should be pointed out that the biochemical assay is performed in a cell-free system, whereas the growth-inhibition assay is performed on intact cells. Potential reasons for the observed enhanced growth-inhibitory activity of NVP-LAQ824, relative to TSA or panobinostat, include: (1) greater cellular uptake or retention of NVP-LAQ824; (2) superior access of NVP-LAQ824 to intracellular targets following uptake; (3) longer half-life of NVP-LAQ824 in intact cells; and (4) greater off-target effects of NVP-LAQ824.

In contrast with the hydroxamates, the benzamide compounds MS-275 and MGCD0103 appeared to be HDAC-class-I-specific, as reported previously [3033]. Our findings for MS-275 inhibition of HDACs 1, 3 and 8 agree with published data [30].

The small-chain-fatty-acid compound VPA has been shown to inhibit HDACs [34] and to induce selectively proteosomal degradation of HDAC2 [35]. VPA has been used, both as monotherapy and in combination with a number of drugs, in the treatment of cancer [3638]. Our results indicate that VPA inhibited class I HDACs 1, 2, 3 and 8 in the millimolar range, but it was ineffective against HDACs 6, 7 and 9.

When comparing the class I HDACs, the EC50 values for rhHDAC8 were consistently higher for all HDACis tested, compared with the other rhHDAC isoforms. This phenomenon may be explained by recent data on HDAC8, which shows that HDAC8 affinity for vorinostat is influenced by the identity of the bivalent ion present in the catalytic centre. The Km values with Fe(II) and Co(II) were shown to be 5-fold lower than with Zn(II). These findings indicate that HDAC8 requires Fe(II), rather than Zn(II), as the bivalent ion for catalytic activity, [39].

To determine possible modes of action of the inhibitors, the change in lysine acetylation of key proteins in HeLa cells was established. Inhibition of the HDACs should result in increased acetylation of specific (Nε-acetyl-lysine) residues on histones. To confirm this hypothesis, HeLa cells were treated with the HDACis for 4 and 24 h; controls were treated with DMSO only. The cell extracts were immunoblotted with anti-acetyl-lysine antibody. Compared with the control extracts, the inhibitor-treated cell extracts showed increased acetylation of two low-molecular-mass proteins, which corresponded in size to histones H3 and H4. Cells which had been treated with MS-275 and MGCD0103 for 4 h showed a significantly lower level of acetylation of these proteins compared with pan-inhibitor-treated cells. This difference was not apparent after 24 h inhibitor treatment. A possible explanation for this finding is that MS-275 and MGCD0103 may display slower kinetics than the other drugs. These findings indicate that both pan-HDAC and class-I-specific inhibitors bring about similar changes to the level of acetylation of lysine residues on histones H3 and H4.

Interestingly, class II HDACs, particularly HDAC6, appear to be important in a number of key cellular processes. Our results showed that treatment of HeLa cells with HDACis, followed by immunoblotting of the cell extracts with anti-(acetylated tubulin) antibody resulted in increased tubulin acetylation in extracts treated with the pan-HDAC inhibitors; this was not observed with the class-I-specific inhibitors. There is evidence that HDAC6 destabilizes the microtubule assembly by deacetylating tubulin via its tubulin deacetylase domain [7,40,41]. HDAC6 also plays a role in aggresome function [42]. Furthermore, there is evidence that inhibition of HDAC6 results in increased acetylation of HSP90 (heat-shock protein 90) and disruption of the chaperone association with its client proteins [43]. p53 is a negative regulator of cell growth. It is expressed in the mutant form in a high proportion of tumour-cell types. Mutant p53 is known to be a ‘client’ protein of HSP90 [44].

In order to determine whether there were any marked differences in p53 levels in cells treated with pan-HDAC and class-I-specific inhibitors, the lysates from DU145 cells and HCC827 cells were immunoblotted with an anti-p53 antibody, following 24 h treatment with HDACis. DU145 cells are known to express mutant p53 and HCC827 cells are likely to express p53. Treatment of cells with belinostat showed a dose-dependent decrease in p53 levels in the lysates of both cell types. This depletion of mutant p53 was not observed in the lysates from cells treated with the class-I HDAC inhibitors MS-275 and MGCD0103. These findings collectively emphasize the importance of HDAC6 and suggest that it could also be an important anticancer target. If this hypothesis proves to be correct, a pan-HDAC inhibitor may be more effective in the clinic than a class-I-selective inhibitor.

One theoretical consideration is whether inhibition of specific HDACs by selective inhibitors will prove to be less toxic in the clinic than pan-inhibition of both class I and II HDACs. Evidence available from early-phase clinical trials suggests that toxicities associated with pan-HDAC inhibitors, such as belinostat and vorinostat [4547], are no more severe than those observed in patients treated with class-I-selective inhibitors, such as MS-275 and MGCD0103 [4850]. The most viable conclusion may therefore be that, until further evidence highlighting the benefits of class-I-selective inhibitors becomes available, pan-HDAC inhibitors may be advantageous, since they inhibit important class II HDAC isoforms, such as HDAC6, without demonstrating increased toxicity. However, the ultimate factor(s) deciding between a pan-HDAC or class-selective inhibitor may depend on the cancer type and/or the chemotherapeutic combination being tested.

Abbreviations

     
  • DAD

    deacetylase activating domain

  •  
  • FDL

    Fluor de Lys™

  •  
  • GST

    glutathione transferase

  •  
  • HAT

    histone acetyltransferase

  •  
  • HDAC

    histone deacetylase

  •  
  • HDACi

    HDAC inhibitor

  •  
  • HSP90

    heat-shock protein 90

  •  
  • NCoR

    nuclear receptor co-repressor

  •  
  • rh

    recombinant human

  •  
  • SMRT

    silencing mediator for retinoid and thyroid receptors

  •  
  • TSA

    trichostatin A

  •  
  • VPA

    valproic acid

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Supplementary data