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Experimental Therapeutics, Preclinical Pharmacology |
Cell Biology Program [L. M. B., Y. W., T. R. T., M. C. K., V. M. R., R. A. R., P. A. M.], Laboratory of Tumor Biology [D. B. A., B. H.], Department of Surgery [M. P. L.], Department of Pathology [M. D., C. C-C.], Genitourinary Oncology Service [H. I. S.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021; Department of Chemistry, Columbia University, New York, New York 10027 [Y. W., R. B.]; and Department of Pediatrics, Joan and Sandford I. Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10021 [M. C. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design and Results: Pyroxamide, at micromolar concentrations, induced terminal differentiation in murine erythroleukemia (MEL) cells and caused growth inhibition by cell cycle arrest and/or apoptosis in MEL, prostate carcinoma, bladder carcinoma, and neuroblastoma cells. Administration of pyroxamide (100 or 200 mg/kg/day) to nude mice at doses that caused little evident toxicity significantly suppressed the growth of s.c. CWR22 prostate cancer xenografts. Despite the potent growth-inhibitory effects of pyroxamide in this tumor model, serum prostate-specific antigen levels in control versus pyroxamide-treated mice were not significantly different. Pyroxamide is a potent inhibitor of affinity-purified HDAC1 (ID50 = 100 nM) and causes the accumulation of acetylated core histones in MEL cells cultured with the agent. Human CWR22 prostate tumor xenografts from mice treated with pyroxamide (100 or 200 mg/kg/day) showed increased levels of histone acetylation and increased expression of the cell cycle regulator p21/WAF1, compared with tumors from vehicle-treated control animals.
Conclusions: The findings suggest that pyroxamide may be a useful agent for the treatment of malignancy and that induction of p21/WAF1 in transformed cells by pyroxamide may contribute to the antitumor effects of this agent.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The hydroxamic acid-based HPCs are potent inhibitors of affinity-purified HDAC enzymes, and cause the accumulation of acetylated histones in treated cells (2) . Cocrystallization of one of these agents, SAHA, and a HDAC-like protein showed that SAHA fits into the catalytic pocket of the enzyme. The hydroxamic acid moiety binds to the Zn2+ ion at the base of the pocket, resulting in the inhibition of HDAC activity (6) . In general, histone hyperacetylation is associated with a more open conformation of chromatin and transcriptional activation, whereas deacetylation of histones is associated with transcriptional repression (7 , 8) . On the basis of the analysis of cells cultured with the HDAC inhibitor trichostatin A, <2% of expressed genes have altered patterns of expression (9) . We have previously shown that SAHA induces expression of the cyclin-dependent kinase inhibitor p21/WAF1 in transformed cells (1 , 10) . Induction of p21/WAF1 has also been reported for other HDAC inhibitors, including trichostatin A (11) , butyrate (12 , 13) , MS-27-275 (14) , trapoxin (15) and oxamflatin (16) . It has been hypothesized that these agents modulate chromatin structure in transformed cells to selectively induce the expression of genes, e.g., p21/WAF1, involved in differentiation, growth arrest, and apoptosis (17) .
Pyroxamide (suberoyl-3-aminopyridineamide hydroxamic acid; Fig. 1A
) is a new member of this group of hydroxamic acid-based compounds that are potent inhibitors of HDACs in transformed cells. Each member of this class of agents has structural variations (17)
, associated with different solubility, potency, and pharmacokinetic properties, which may affect the clinical utility of these agents. In this study, we investigated the effects of pyroxamide on the differentiation and proliferation of transformed cells in vitro and in vivo. Pyroxamide, at low micromolar concentrations, induced terminal differentiation of MEL cells and inhibited the growth of several human transformed cell types in culture. In vivo, pyroxamide inhibited the growth of a human prostate cancer xenograft grown s.c. in nude mice. Pyroxamide is a potent inhibitor of affinity-purified HDAC1. Accumulation of acetylated histones was detected in MEL cells cultured with pyroxamide and in prostate tumors removed from mice treated with pyroxamide. In addition, the tumors from the pyroxamide-treated mice had increased levels of p21/WAF1 expression compared with vehicle-treated controls. These results suggest that pyroxamide may be a useful agent for the treatment of malignancy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ref. 18
) as follows: melting point, 181°C; mass spectroscopy (CI), m/z 266 (M + 1); 1H-NMR (200 MHz, DMSO-d6, 
); 1.2–1.4 (m, 4H), 1.4–1.8 (m, 4H), 2.0 (t, 2H), 2.3 (t, 2H), 7.3 (m, 1H), 8.0 (dd, 1H), 8.2 (dd, 1H); 8.6 (s, 1H), 8.7 (s, 1H), 10.1 (s, 1H), 10.3 (s, 1H). For animal studies, pyroxamide was synthesized by a high-yield process analogous to the published synthesis of SAHA (1
, 19)
, substituting 3-aminopyridine for aniline (National Cancer Institute). For all of the studies pyroxamide was dissolved and diluted in a vehicle of DMSO.
Cell Culture.
MEL DS19/Sc9 cells, derived from 745A cells, were maintained in 
-MEM containing 10% FCS (1)
. Cultures were initiated with MEL cells in logarithmic growth phase at a density of 1–2 x 105 cells per ml. Measurements of cell density, cell viability and benzidine reactivity were performed as described previously (20
, 21)
.
T24 bladder carcinoma cells, LNCaP prostate carcinoma cells and KCN-69n neuroblastoma cells were obtained from the American Type Culture Collection. T24 cells were cultured in -MEM containing 10% FCS, LNCaP cells in RPMI 1640 containing 10% FCS, and KCN-69n cells in a 1:1 mixture of RPMI 1640 and F-12 medium containing 10% FCS. Cells were cultured in triplicate with pyroxamide (1.25–20 µM) in the appropriate growth medium for 1–4 days at 37°C. Cells were counted every 24 h using a hemocytometer, and cell viability was assessed by trypan blue exclusion. FACS analysis of cell cycle distribution was performed on untreated and pyroxamide-treated cells after 12, 24, or 48 h of culture, as described previously (22)
. Sub-G1 values were obtained by standard histogram analysis using CellQuest software (Becton Dickinson, San Jose, CA).
Histone Isolation.
Cultures of MEL DS19/Sc9 cells in logarithmic growth phase were cultured with 4 µM pyroxamide for 4, 24, or 48 h at 37°C. Cells cultured without pyroxamide were harvested at the same time points for comparison of histone acetylation levels. At the appropriate time points, cells were harvested, washed with ice-cold PBS and suspended in 1 ml of ice-cold histone lysis buffer [8.6% sucrose, 1% Triton X-100, 50 mM sodium bisulfite, 10 mM Tris-HCl (pH 6.5), and 10 mM MgCl2]. For the animal studies, tumor tissues were excised and Dounce homogenized on ice in histone lysis buffer. Histones were extracted from the lysates as described previously (2)
.
HDAC Inhibition Assays.
We used a HDAC1 enzyme assay, modified from Richon et al. (2)
. A MEL cell line expressing the epitope Flag-tagged HDAC1 was generated. HDAC1-Flag was affinity purified by immunoprecipitation using M2 anti-Flag antibody-coated agarose (Kodak, Rochester, NY), followed by elution from the agarose using the Flag peptide. [3H]acetate-labeled cellular histones were prepared from MEL cells as described previously (23)
and were used as a substrate for the HDAC activity assay. Released [3H]acetic acid was quantified by scintillation counting. For inhibition studies, the enzyme preparations were preincubated with pyroxamide (10 to 100,000 nM) for 30 min at 4°C.
Western Blotting.
Histones (2.5 µg) were loaded onto 15% polyacrylamide gels (Ready Gels; Bio-Rad, Hercules, CA) and blotted onto nitrocellulose membranes. Acetylated histones H3 and H4 were detected using 1:1000 dilutions of rabbit polyclonal antibodies (Upstate Biotechnology, Lake Placid, NY) as described previously (22)
. Acetylated histone H2A was detected using a 1:800 dilution of rabbit antisera raised against histone 2A acetylated at lysine residue 5 (Serotec, Oxford, United Kingdom), and acetylated histone H2B was detected using a 1:400 dilution of rabbit antisera raised against acetylated forms of histone H2B (Serotec). A donkey antirabbit secondary antibody (1:5000 dilution) was used to detect the primary signal (Amersham, Buckinghamshire, United Kingdom). All of the antibodies were diluted in 3% milk in PBS. The signals were visualized using the Super Signal chemiluminescence system (Pierce, Rockford, IL).
For detection of p21/WAF1 expression in tumor samples, frozen tissues were ground under liquid nitrogen and suspended in NP40 lysis buffer [50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 5 mM EDTA, and 0.5% NP40] containing protease inhibitors (Boehringer Mannheim). Lysates (25 µg) were electrophoresed through 15% polyacrylamide gels (Ready Gels; Bio-Rad) and blotted onto nitrocellulose membranes. P21/WAF1 was detected using Santa Cruz H164 rabbit polyclonal antibody at 1 µg/ml, and cdk2 (used as a loading control) was detected using 1 µg/ml Santa Cruz M2 rabbit polyclonal antibody. Both primary antibodies were diluted in 5% milk in Tris-buffered saline containing 0.05% Tween-20 and incubations were performed at 4°C overnight. The signals were detected using a donkey antirabbit secondary antibody (Amersham) and visualized with the Super Signal chemiluminescence system (Pierce).
Inoculation of Mice with the CWR22 Xenograft.
Male athymic (nu/nu) BALB/c mice were purchased from the National Cancer Institute-Frederick Cancer Center. The CWR22 human prostate cancer xenograft was kindly provided by Dr. Thomas G. Pretlow (Case Western Reserve University, Cleveland, OH; Ref. 24
). Mice were implanted with a 12.5 mg sustained-release testosterone pellet (Innovative Research of America, Sarasota, FL), 7 days before the injection of tumor cells. Tumors from seed mice were minced finely through a sterile mesh and suspended in Matrigel. An equal volume of the tumor cell suspension was injected s.c. on the right flank of each mouse, and the mice were monitored until palpable s.c. tumors (5 mm x 5 mm) were detected (7 days after tumor inoculation).
Treatment of Mice with Pyroxamide.
Mice bearing palpable CWR22 tumors of similar size (5 mm x 5 mm) were randomized into four treatment groups. Mice received daily i.p. injections of vehicle alone (DMSO) or 100, 200, or 300 mg/kg of pyroxamide, for a total treatment period of 21 days. Injection volumes were kept constant at 1 µl per g of body weight. Tumor length and width were measured twice weekly by calipers, and tumor volume was calculated using the following formula:
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After 21 days of treatment, at which time the control animals had large tumors requiring sacrifice of the animals, all of the mice were killed 6 h after the final ,injection and the tumors and spleens were removed. Tissues were flash-frozen in liquid nitrogen or fixed in formalin and embedded in paraffin.
During the treatment period, mice were weighed weekly and monitored for any overt signs of toxicity. A complete necropsy, including a complete blood count and differential, was performed on one animal from each treatment group at the end of the treatment period (21 days) by the Research Animal Resource Center of Cornell University and Memorial Sloan-Kettering Cancer Center.
Measurement of Serum PSA Levels.
Mice were bled from the lateral tail vein via tail nick on days 0 and 9 of treatment and via cardiac puncture at the time of sacrifice (day 21). PSA levels were measured in the serum using a Tandem-R radioimmunometric assay (Hybritech, San Diego, CA).
Statistical Analyses.
A permutation test was used to compare differences between groups with respect to tumor volume over time. The null hypothesis for this test is that the growth rates in two treatment groups are equal. The statistic used to test this hypothesis is the absolute difference between mean tumor volume in each treatment group summed overall of the time points. All of the possible permutations of the treatment group affiliation were computed.
PSA values between pyroxamide dose levels were compared relative to their baseline (day 0) value. A Wilcoxon rank-sum test was used to test whether the change in PSA value from baseline was different between groups, and whether there were any differences among the treatment groups in the absolute PSA values at day 21 (the end of treatment).
Immunohistochemistry.
Five-µm sections of CWR22 tumors fixed in paraffin were stained for expression of p21/WAF1 as described previously (25)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is a hydroxamic acid-based HPC that was designed in an attempt to increase solubility in aqueous solutions. To evaluate the biological activity of pyroxamide, we first determined whether pyroxamide induces terminal differentiation of MEL DS19/Sc9 cells by measuring benzidine positivity, reflecting accumulation of hemoglobin in the cells. MEL cells were cultured with pyroxamide (0.625–8 µM). Maximal positivity of 64% of the cells was achieved with 4 µM pyroxamide (Fig. 1B)
. The effective concentration for induction of differentiation caused a decrease both in cell density and in the percentage of viable cells in the culture (Fig. 1C)
.
Inhibition of HDAC by Pyroxamide.
We next investigated the activity of pyroxamide as an inhibitor of HDACs. Pyroxamide inhibited the enzymatic activity of affinity-purified HDAC1 at submicromolar concentrations (Fig. 2A)
. The ID50 value for inhibition of HDAC1 activity by pyroxamide was 
100 nM.
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. Cells cultured without the agent had low basal levels of acetylated histones at the same time points (Fig. 2B)
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Pyroxamide Inhibits the Growth of Transformed Cells in Culture.
Pyroxamide (1.25–20 µM) caused dose-dependent inhibition of the growth of prostate carcinoma (LNCaP), neuroblastoma (KCN-69n), and bladder carcinoma (T24) cells in culture (Fig. 3)
, with similar efficacy in all of the cell lines. There was no induction of cell death by pyroxamide in the LNCaP or T24 cell lines (Fig. 3)
at doses of up to 20 µM. Dose-dependent induction of cell death was observed in the KCN-69n neuroblastoma cells, with up to 90% of cells dead after culture in 20 µM pyroxamide for 3 days (Fig. 3)
. FACS analysis revealed that LNCaP cells cultured with 5, 10, or 20 µM pyroxamide for 24 h arrested in the G1 phase of the cell cycle (Fig. 4A)
. T24 cells arrested in G1 after culture with 10 µM pyroxamide, but arrested in the G1 and G2-M phases after culture with 20 µM for 24 h (Fig. 4A)
. The G1 cell cycle arrest induced in the T24 and LNCaP cells by pyroxamide (10 µM) was reversible, as determined by washing out the drug from the cultures. LNCaP cells incubated with 10 µM pyroxamide for 24 h showed a reduction in S phase from 33.6% (untreated cells) to 5.4%. After the washing out of the drug and incubation of the pyroxamide-treated cells in medium without pyroxamide for an additional 24 h, the percentage of cells in S phase increased to 29.8% (data not shown). Similar results were obtained with T24 cells. There was no change in the sub-G1 fraction in either the LNCaP or the T24 cells after 24 h of culture with pyroxamide (data not shown). FACS analysis revealed an increased sub-G1 cell fraction in KCN-69n cells treated with pyroxamide (5 or 20 µM) for 12, 24, or 48 h (Fig. 4B)
, indicative of DNA fragmentation and cell death.
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. A dose of 300 mg/kg pyroxamide was lethal to all of the mice in the treatment group within 1 week.
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Necropsy analysis of at least 26 tissues and organs was performed on one mouse from each treatment group at the end of the 21-day treatment period. The animals receiving vehicle alone or 100 or 200 mg/kg/day of pyroxamide had no apparent lesions other than mild peritonitis, presumably resulting from daily i.p. injections.
Serum PSA Levels.
s.c. growth of the human CWR22 prostate cancer xenograft in nude mice is associated with an increase in the serum PSA levels in the mice that is related to the tumor volume (24)
. In the current study, mice receiving daily i.p. injections of vehicle alone showed an average 17-fold increase in serum levels of PSA over the 21-day treatment period (Fig. 5B)
. Despite having significantly lower tumor volumes than the vehicle-treated controls, mice receiving 100 or 200 mg/kg/day of pyroxamide had similar levels of serum PSA as the vehicle-treated controls at the end of the 21-day treatment period (Fig. 5B)
.
Accumulation of Acetylated Histones after in Vivo Administration of Pyroxamide.
Histones were isolated from the CWR22 tumors excised from two mice in each treatment group at the end of the 21-day treatment period, 6 h after the final injection of vehicle or pyroxamide. Tumors from mice treated with vehicle alone showed low basal levels of histone H4 acetylation (Fig. 6A)
. Tumors removed from mice treated with 100 mg/kg and, more strikingly, 200 mg/kg pyroxamide showed increased accumulation of acetylated histone H4 (Fig. 6A)
.
|
. In agreement with previous findings using other HDAC inhibitors (15
, 16
, 26) , there was no change in the levels of cdk2 protein in the tumors from mice treated with pyroxamide. Immunohistochemical staining of p21/WAF1 in the tumor tissues showed an increase in the number of tumor cells positive for p21/WAF1 in tumors from mice treated with 200 mg/kg/day pyroxamide (Fig. 6C)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have previously shown that SAHA and the related compound CBHA inhibit the activity of affinity-purified HDAC1 in vitro (2) . In the present study, we found that pyroxamide inhibits the activity of HDAC1. Pyroxamide causes the accumulation of acetylated core histones (H2A, H2B, H3, and H4) in MEL cells cultured with the agent within four h. Accumulation of acetylated histones was observed in human prostate tumor xenografts after the administration of 200 mg/kg pyroxamide by daily i.p. injection to nude mice, confirming that the HDAC inhibitory activity of pyroxamide can be detected in vivo. Consequently, the accumulation of acetylated histones may serve as a biological marker for the activity of this HDAC inhibitor.
HDAC inhibitors are currently receiving considerable attention as antitumor agents, because of their ability to induce cell cycle arrest and/or cell death in a wide range of transformed cells in vitro and in vivo (17 , 30 , 31) . Hydroxamic acid-based HDAC inhibitors, including SAHA, CBHA, ABHA, azelaic-1-hydroxamate-9-anilide (AAHA), oxamflatin, and trichostatin A, are active at inhibiting transformed cell growth in vitro at nanomolar to low micromolar concentrations (1 , 5 , 16 , 19 , 22 , 32 , 33) . Trichostatin A, the most potent of these compounds on a molar basis for the inhibition of HDAC and transformed cell growth in vitro, has not yet been shown to have appreciable tumor suppressive activity in vivo (34) . Oxamflatin, ABHA, and SAHA have been shown to have significant tumor-suppressive activity in vivo (3 , 5 , 16 , 34) . Pyroxamide was designed and shown to be more soluble in aqueous solutions than is SAHA. Here we show that pyroxamide is approximately equivalent in potency to SAHA and CBHA for the inhibition of partially purified HDAC, and it inhibits the proliferation of transformed cells in vitro and in vivo.
The cyclin-dependent kinase inhibitor p21/WAF1 has been identified as a gene induced in transformed cells by inhibitors of HDACs, including SAHA and CBHA (1) . Induction of p21/WAF1 occurs in cells lacking functional p53, and two Sp1 binding sites in the promoter of the p21/WAF1 gene are required for induction of p21/WAF1 expression by these agents (11 , 12) . Sp1 has been shown to repress transcription of the murine thymidine kinase gene promoter by interaction with HDAC1 (35) . Expression of p21/WAF1 in transformed cells after treatment with HDAC inhibitors is preceded by localized hyperacetylation of histones in the chromatin region containing the p21/WAF1 gene (10 , 15) . Overexpression of HDAC1 can reduce the level of induction of p21/WAF1 by HDAC inhibitors (13) . These findings suggest that these agents act directly to induce hyperacetylation and thereby alter chromatin structure in the region of the p21/WAF1 gene. Prevention of p21/WAF1 induction, by deletion of the p21/WAF1 gene in colon carcinoma cells, resulted in no inhibition of growth rate of the cells in response to trichostatin A or butyrate, compared with cells containing a wild-type p21/WAF1 gene (13) . We found that pyroxamide induces G1 arrest in transformed prostate and bladder cells in culture. Taken together, findings to date indicate that induction of p21/WAF1 is a likely mechanism by which HDAC inhibitors arrest the growth of transformed cells. In the present study, we show that the administration of pyroxamide to mice bearing CWR22 human prostate tumors, at a dose that induces hyperacetylation of histones (200 mg/kg/day), causes an increase in the expression of p21/WAF1, which suggests that p21/WAF1 contributes to the antitumor effects of these agents administered in vivo. Induction of p21/WAF1 by HDAC inhibitors, in addition to the accumulation of acetylated histones, may be useful intermediate biomarkers for the activity of this class of agents in clinical studies as anticancer agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from the Burke Foundation (to H. I. S. and L. M. B.), CaPCURE (to D. B. A. and H. I. S.), the PepsiCo Foundation (to D. B. A., H. I. S., M. D., and C. C. C.), the American Cancer Society (to D. B. A.), the Eleanor and Paul Stephens Foundation (to D. B. A.), the Japan Foundation for Promotion of Cancer Research, and the DeWitt Wallace Fund for Memorial Sloan-Kettering Cancer Center, and by NIH Grants CA-DK-47650 (to C. C. C.) and CA-0974823 (to R. A. R. and P. A. M.). D. B. A. was supported by a CaPCURE Young Investigator Award. 
2 Present Address: Eitan, Pearl, Latzer and Cohen-Zedek Omega Center, Advanced Technology Center, Haifa, Israel 31905.
3 Present Address: Cedars-Sinai Medical Center, Los Angeles, CA 90048.
4 Present Address: Hoffman-LaRoche Inc., Nutley, NJ 07110-1199.
5 To whom requests for reprints should be addressed, at Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-6573; Fax: (212) 639-2861; E-mail: v-richon{at}ski.mskcc.org
6 The abbreviations used are: HPC, hybrid polar compound; ABHA, azelaic bishydroxamic acid; CBHA, m-carboxycinnamic acid bishydroxamate; HDAC, histone deacetylase; MEL, murine erythroleukemia; PSA, prostate-specific antigen; SAHA, suberoylanilide hydroxamic acid; FACS, fluorescence-activated cell sorting.
Received 10/19/00; revised 1/ 8/01; accepted 1/11/01.
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, control (0 µM); 
, 1.25 µM; 
, 2.5 µM; 
, 5 µM; 
, 10 µM; 
, 20 µM. Cells were counted at the indicated times using a hemocytometer and cell viability was assessed by trypan blue exclusion. Left panels, the number of viable cells present in the cultures; right panels, the number of dead cells as a percentage of the total number of cells in the culture. Data are presented as the mean ± SD of triplicate values from a typical experiment.
