Butyric Acid Prodrugs Are Histone Deacetylase Inhibitors That Show Antineoplastic Activity and Radiosensitizing Capacity in the Treatment of Malignant Gliomas

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Butyric acid prodrugs are histone deacetylase inhibitors that show antineoplastic activity and radiosensitizing capacity in the treatment of malignant gliomas

Michal Entin-Meer,1 Ada Rephaeli,3 Introduction 1 4 Xiaodong Yang, Abraham Nudelman, Glioblastoma multiforme carries a poor prognosis, and Scott R. VandenBerg,2 although radiation therapy prolongs patient survival, cures and Daphne Adele Haas-Kogan1 remain discouragingly elusive. The clear need for new, biologically effective agents has propelled investigations 1 Department of Radiation Oncology, Neurological Surgery, into histone deacetylase (HDAC) inhibitors (HDACI) that and Comprehensive Cancer Center and 2Department of Pathology and Neurological Surgery, University of California at San in turn have emerged as promising anticancer therapies Francisco, San Francisco, California; 3Felsenstein Medical (1–12). HDACIs display unique antineoplastic properties Research Center, Sackler School of Medicine, Tel Aviv University, most likely through increased nuclear histone acetylation 4 Beilinson Campus, Petach Tikva, Israel; and Chemistry and modulation of gene expression. A dynamic equilibri- Department, Bar Ilan University, Ramat Gan, Israel um between histone acetyltransferase and HDAC controls levels of acetylated histones in nuclear chromatin (13). Abstract Throughregulationofgeneexpressionandthrough Histone modification has emerged as a promising approach potential additional unknown mechanisms, HDACIs play to cancer therapy. We explored the efficacy of a novel class key roles in growth arrest, differentiation, and apoptosis of of histone deacetylase inhibitors in the treatment of tumor cells (2–11, 14). Several HDACIs are in preclinical malignant gliomas. Treatment of glioma cell lines with and clinical development, including a prodrug of butyric two butyric acid derivatives, pivaloylomethyl butyrate acid [pivaloylomethyl butyrate (AN-9); refs. 12, 15], (AN-9) and butyroyloxymethyl butyrate (AN-1), induced hydroxamic acids (suberoylanilide hydroxamic acid and hyperacetylation, increased p21Cip1 expression, inhibited trichostatin A; refs. 16–19), benzamide derivatives (MS-275 proliferation, and enhanced apoptosis. Histone deacetylase and CI-994; refs. 20–23), cyclic peptides (trapoxin, apicidin, inhibitor–induced apoptosis was mediated primarily by and depsipeptide; refs. 24–27), and valproic acid (28). caspase-8. Treatment of cells with AN-1 or AN-9 for Recent studies have revealed radiosensitizing capacity of 24 hours before exposure to ;-irradiation potentiated various HDACIs, including MS-275 (29), suberoylanilide further caspase-8 activity and resultant apoptosis. Clono- hydroxamic acid (30), valproic acid (31), and trichostatin genic survival curves revealed marked reductions in cell A (30, 32), indicating potential advantages to incorporating renewal capacity of U251 MG cells exposed to combina- HDACIs into multimodality therapy for gliomas. tions of AN-1 and radiation. Preliminary in vivo experi- Butyric acid, a HDACI, possesses low efficacy in vivo due ments using human glioma cell lines grown as xenografts in to its rapid metabolism (33); to overcome this limitation, mouse flanks suggest in vivo efficacy of AN-9. The data acyloxymethyl esters of butyric acid were synthesized and suggest that novel butyric acid prodrugs provide a characterized (4, 5). Acyloxymethyl esters of butyric acid promising treatment strategy for malignant gliomas as efficiently deliver butyric acid, resulting in higher intracel- single agents and in combination with radiation therapy. lular concentrations. Among these butyric acid prodrugs, [Mol Cancer Ther 2005;4(12):1952–61] AN-9, which on hydrolysis releases butyric acid, formaldehyde, and pivalic acid, is the best characterized (Fig. 1). AN-9 has been evaluated in a phase I clinical study that revealed very limited toxicities (12). In phase II clinical Received 3/25/05; revised 8/19/05; accepted 9/21/05. trials, AN-9 improved the well-being and survival of Grant support: NIH grant PO1 NS-42927-27A2 and Brain Tumor patients with non–small cell lung carcinoma (15). Antineo- Specialized Programs of Research Excellence grant P50 CA097257 plastic activity has been shown for additional butyric acid (D.A. Haas-Kogan) and Children’s Brain Tumor Foundation. prodrugs, including butyroyloxymethyl butyrate (AN-1) The costs of publication of this article were defrayed in part by the that releases two equivalents of butyric acid and one payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to equivalent of formaldehyde (11, 34). indicate this fact. We sought to assess the antineoplastic activity of this Requests for reprints: Michal Entin-Meer, Comprehensive Cancer Center, family of HDACIs in the treatment of high-grade gliomas. University of California at San Francisco, 2340 Sutter Street, San Francisco, CA 94115. Phone: 415-476-0543; Fax: 415-502-3179. AN-1 and AN-9 exhibited cytotoxic effects against all tested E-mail: [email protected] glioma cell lines, potentiation of radiation therapy, and Copyright C 2005 American Association for Cancer Research. in vivo inhibition of tumor growth in an in vivo flank model doi:10.1158/1535-7163.MCT-05-0087 of glioblastoma multiforme.

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Western Blot Analysis Whole-cell lysates or acid-extracted histones were solubilized in sample buffer and equal amounts of protein were separated on SDS-PAGE. Purified histones were transferred to nitrocellulose membranes and whole-cell lysates to polyvinylidene difluoride membranes (Bio-Rad Laborato- ries, Hercules, CA). Membranes were probed with primary antibody and then with horseradish peroxidase–conjugated species-specific secondary antibody (Amersham Bio- sciences, Piscataway, NJ). Band intensities, visualized by Figure 1. Chemical structures of AN-9 and AN-1. On in vivo metabolic enhanced chemiluminescence, were measured using Scion breakdown, AN-9 releases butyric acid, formaldehyde, and pivalic acid, Image software (Scion Corp., based on NIH image for whereas AN-1 releases two equivalents of butyric acid and one equivalent macintosh, Frederick, MD). of formaldehyde. HDAC ActivityAssay Triplicate samples of 25 Ag brain extract or 3 Ag cell Materials and Methods extract were loaded per well of 96-well plates and HDAC Cell Culture and Irradiation activity was assayed following the manufacturer’s instruc- U87 MG, U251 MG, and SF188 glioma cell lines as well as tions (Biomol, Plymouth Meeting, PA). HDAC activity was primary human astrocytes (35) were grown in a humidified reflected in increased fluorescence that was emitted by the j incubator at 37 C and 8% CO2 in DMEM supplemented deacetylated substrate. Each experiment was carried out with 10% fetal bovine serum (Invitrogen Life Technologies, twice starting with different sets of animals or cells. Carlsbad, CA) and 100 units/mL penicillin-100 Ag/mL Cell Proliferation streptomycin (Life Technologies). Cells were irradiated at U251 MG and U87 MG (3,000 cells per well), SF188, and room temperature in a Mark I-68 Cesium 137 irradiator (J.L. primary human astrocytes (5,000 cells per well) were Shepherd & Associates, San Fernando, CA) at a dose rate of seeded in 96-well plates. Sixteen hours later, medium was 2.26 Gy/min. aspirated, and fresh medium (100 AL) containing escalating Reagents drug doses was added. Cells were then incubated for The butyric acid derivatives AN-9 and AN-1 were pre- 48 hours followed by addition of 3-(4-5-dimethylthiazol-2- pared as described previously (7, 36). For in vitro studies, yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- compounds were solubilized in DMSO followed by dilution tetrazolium salt (MTS) reagent (Promega, Madison, WI) in medium to a final DMSO concentration of V0.2%. Caspase and further incubation for 1 to 3 hours. Light absorbance, inhibitors included the pan-caspase peptide inhibitor Z-Ala- which reflects cellular viability, was measured using a plate Asp-fluoromethylketone (Z-VAD-fmk; FK-009) and a more reader at A490 nm. caspase-8 specific inhibitor Z-Ile-Glu(OMe)-Thr-Asp(OMe) Colony Formation Assays FMK (Z-IETD-fmk; FK-012; Enzyme Systems Products, For clonogenic survival assays, exponentially growing Livermore, CA). Antibodies included rabbit anti-acetylated U251 MG cells were treated with escalating doses of AN-1 for histone H4 (Lys12), rabbit anti-histone H3 (Lys23), rabbit anti- 24 hours and harvested by trypsinization. Specified numbers total H4, rabbit anti-cleaved caspase-3, mouse anti-p21Cip1, of cells were plated in six-well plates already containing mouse anti–poly(ADP-ribose) polymerase (PARP; all from 20,000 cells per well of lethally irradiated feeder SF188 cells Cell Signaling Technology, Beverly, MA), mouse anti-Ki-67 to maximize plating efficiency. For colony formation assays (DakoCytomation California Inc., Carpinteria, CA; clone assessing interactions with radiation, specified numbers of MIB1), mouse anti-h-actin (Sigma-Aldrich, St. Louis, MO), cells were plated in medium containing AN-1 (35 Amol/L). biotinylated horse anti-mouse, and biotinylated goat anti- Following a 16-hour incubation, cells were irradiated with rabbit (Vector Laboratories, Burlingame, CA). specified doses and colony-forming efficiency was deter- Histone Acetylation mined in the continued presence of AN-1. Cultures were Three million cells per 100-mm plate were seeded, allowed then incubated for 8 days and colonies of >50 cells were to adhere for 24 hours, and then treated with HDACI or scored. Cell survival measurements were fitted to a linear control medium. At specified times following treatment, cells quadratic mathematical model using the FIT 2.5 program were collected by trypsinization, washed with PBS, and lysed (37, 38). Within each of at least two independent experiments, in 10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2,10mmol/ two to four different dilutions were made per radiation L KCl, 0.5 mmol/L DTT, and 1.5 mmol/L phenylmethylsul- dose and each dilution was plated in multiples of six. fonyl fluoride. Histones were acid extracted with H2SO4 Caspase Activity (0.2 mol/L) for 1 hour and dialyzed against 0.1 mol/L acetic Caspase activity was determined by measuring the acid for 2 hours and twice against water (2 hours each). A fluorescent emission of 7-amino-4-trifluoromethyl couma- third overnight dialysis was carried out against water rin on its cleavage from Z-IETD substrate (for caspase 8) or containing 50% glycerol. A similar procedure was carried from LEHD substrate (for caspase-9). The assay was carried out to assess in vivo histone acetylation using homogenized out following the manufacturer’s instructions (Oncogene brain tissue harvested at specified times following treatment. Science, Cambridge, MA). Two independent experiments,

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initiated with two different sets of cells in culture, were Data Analysis

done. In each experiment, three independent measure- IC50 values of drug activity were determined by linear ments were taken for each sample. The mean F SE values regression of at least three independent dose-response were generated from the two mean values obtained for titrations. Comparisons of drug activity were done by two- each sample in the two individual experiments. sided t testsortwo-wayANOVA(MicrosoftExcel). Apoptosis Assay Survival was estimated by Kaplan-Meier curves, and Glioma cells were seeded in six-well plates (100,000 differences in survival between the groups were analyzed per well), allowed to adhere, and incubated with AN-1 or by the Cox Mantel test (Statistica, Tulsa, OK, StatSoft AN-9 for 48 hours. Adherent and nonadherent cells were Derived Comparisons, Inc., 1984-2001). collected and double-stained with Annexin V-FITC and propidium iodide according to the manufacturer’s instructions (Calbiochem, San Diego, CA). Apoptotic cells were Results Cip1 quantitated by flow cytometry using a FACSCalibur HDAC Activity and p21 Expression cytometer (Becton Dickinson, Rockville, MD). We explored whether AN-9 and AN-1 inhibit HDAC In vivo Flank Xenografts and Drug Administration activity and increase histone acetylation, as their purported Xenografts of human glioma cell lines were established mechanism of action would predict. Treatment of by s.c. inoculation of 3 Â 106 U251 MG cells into flanks of U251 MG in vitro with 100 Amol/L AN-1 or AN-9 increased BALB/c nu/nu athymic mice (Charles River Laboratories, histone H4 and H3 acetylation, with a histone H4 Wilmington, MA). Mice were monitored according to the acetylation peak of 5 to 30 hours for both AN-1 and AN-9 protocol approved by the Institutional Animal Care and Use and histone H3 acetylation peaks of 1.5 to 18 and 3 to 5 Committee. BALB/c nu/nu athymic mice were inoculated hours for AN-1 and AN-9, respectively (Fig. 2A). In vivo with 3 Â 106 U251 MG cells, and drug administration began experiments using histones purified from brains of mice when xenografts reached 30 mm3 in size. Mice were that were treated with AN-9 (100 mg/kg given orally) randomly assigned to one of two groups: mineral oil vehicle exhibited a broad peak of histone H3 and H4 acetylation alone as control or 200 mg/kg AN-9 diluted in mineral oil. 1.5 to 5 hours after drug administration followed by a Agents were given by oral gavage using 20-gauge curved decline in acetylation 18 hours after treatment. Of note, needles (Popper and Sons, New Hyde Park, NY). Oral drug acetylated histone H4 levels were still higher than control administration continued for the entire duration of the levels even at this later time point (Fig. 2B). Consistent experiment, consisting of 150 days after initiation of treat- with the observed elevation in histone acetylation, HDAC ment, unless the tumor reached 2.5 cm, at which point mice activity in extracts of U251 MG and mouse brains exhibited were euthanized in accordance with the protocol approved peak inhibition 3 to 5 hours following treatment with either by the Institutional Animal Care and Use Committee. AN-1 or AN-9, with gradual recovery thereafter (Fig. 2C). H&E Staining and Immunohistochemical Analysis of The data show that these butyric acid–releasing prodrugs Ki-67 and Cleaved Caspase-3 efficiently cross the blood-brain barrier and effectively Formalin-fixed, paraffin-embedded sections, each 5 Am increase acetylation, prompting us to investigate their thick, were cut from tumors excised from euthanized mice. utility in the treatment of malignant gliomas. For H&E staining, deparaffinized slides were stained with Previous studies have indicated that increased expression hematoxylin for 5 minutes, washed with acetalcohol (differ- of the cyclin-dependent kinase inhibitor p21Cip1 is a entiation solution) for 30 seconds, and then stained with eosin hallmark of HDACI activity (2). Elevation in p21Cip1 for 2 minutes (all of which from Sigma). For immunohisto- expression is associated with cell cycle arrest, induction chemistry procedures, peroxidase activity was quenched of differentiation, and/or apoptosis (8), all cellular with 1% H2O2 in PBS. For Ki-67 staining, sections were then responses induced by HDACI treatment. Consistent with sequentially incubated with 5% FCS, mouse anti-Ki-67 (1:100 these roles of p21Cip1, a dose-dependent increase in p21Cip1 dilution; 4jC overnight), biotinylated horse anti-mouse, and expression in all glioma cell lines was observed following finally streptavidin-horseradish peroxidase (1:200 dilution; AN-1 and AN-9 treatment. As predicted by their p53 status, Zymed Laboratories, S. San Francisco, CA) for 30 minutes. basal p21Cip1 expression levels were high in primary For cleaved caspase-3 staining, sections were stained astrocytes and U87 MG that express wild-type p53 and with rabbit anti-cleaved caspase-3 primary antibody (1:200 low in U251 MG and SF188 that harbor p53 mutations dilution; room temperature for 1 hour), biotinylated goat (Fig. 3A). anti-rabbit secondary antibody (1:200 dilution; 30 minutes), Effect of Butyric Acid Prodrugs on Proliferation and and avidin-biotin complex (1:100 dilution; Vectastain ABC Colony Formation of Gliomas and Primary Astrocytes kit, Vector Laboratories) for 30 minutes. Staining was We investigated the biological consequences of AN-1 and visualized with 3,3V-diaminobenzidine tetrahydrochloride AN-9 treatment, focusing first on cellular viability and at 500 mg/mL in PBS for 5 minutes at room temperature, proliferation. To establish drug concentrations resulting in and slides were counterstained with hematoxylin. Slides 50% growth inhibition (IC50), MTS assays were done 48 were examined using an Olympus (Melville, NY) BX41 light hours after drug treatment. IC50 values were calculated by microscope with UPlanFl objectives. Images were obtained linear regression and averages of three to five independent with a DP70 single CCD cooled 1.5-megapixel camera. experiments are shown in Table 1. Both AN-1 and AN-9

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Figure 2. Western blot analysis of histones purified from (A) U251 MG cells treated with AN-1 or AN-9 (100 Amol/L) and (B) brain extracts of BALB/c nu/nu athymic mice treated orally with AN-9 (100 mg/kg). Purified histones (6 Ag/well) were analyzed by Western blot using anti-acetylated H4 (Lys12), anti-acetylated H3 (Lys23), and anti-total H4 to verify equal protein loading. Columns, ratios of H3 and H4 acetylation in treated versus untreated samples normalized to total H4. Each Western analysis was repeated thrice and a representative blot is shown. C, HDAC activity in U251 MG cells and in brain lysates of BALB/c nu/nu athymic mice treated with AN-1 or AN-9. Y axes, ratios of HDAC activity in treated samples versus untreated controls of two independent experiments; bars, SE. displayed inhibitory activity in all three glioma cell lines detect Annexin V-FITC and propidium iodide staining. tested as well as in human primary astrocytes. AN-1 was Following 48 hours of drug treatment, adherent and somewhat more potent than AN-9, reflected in more nonadherent cells were pooled and viable cells were pronounced decreases in MTS values following AN-1 identified as those stained with neither Annexin V-FITC treatment (P < 0.05; two-sided t test). nor propidium iodide. Percentages of viable cells are Clonogenic survival assays confirmed the sensitivity of plotted in Fig. 3B and show that primary astrocytes U251 MG to AN-1 treatment (Table 1). As expected, the underwent little to no apoptosis following treatment, IC50 value for clonogenic survival (39 Amol/L) was lower whereas all three glioma cell lines displayed significant than the corresponding value generated by MTS assay (118 apoptotic responses. Whereas MTS assays did not show Amol/L) because sustained clonogenic capacity is a more substantial differences among the cell lines, flow cytometry rigorous measure of cell survival than persistent prolifer- revealed significant differences in susceptibility to AN-9- ation measured by MTS. induced apoptosis. Primary human astrocytes displayed Decreases in MTS values may reflect cell death, growth only 20% cell death at 200 Amol/L AN-9, indicating that arrest, or a combination thereof. To determine the inhibition observed in MTS assays reflected mostly growth mechanism of AN-9- and AN-1-induced cytotoxicity, arrest. After identical treatments, virtually no SF188 cells cellular viability was quantitated using flow cytometry to remained viable, indicating that in this pediatric glioma cell

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line AN-9-induced decreases in MTS values reflected astrocytes and cell death in glioma cell lines, reflecting chiefly cell death. U87 MG and U251 MG displayed an some selective toxicity of AN-9 for malignant cells. Of note, intermediate susceptibility to AN-9-induced cell death, 49% different mechanisms contribute to the efficacy of AN-9 in and 58% cell death at 200 Amol/L AN-9, respectively. The various glioma cell lines, for example, predominantly above data indicate that following AN-9 treatment the apoptosis in SF188 and a combination of apoptosis and dominant cellular response is growth arrest in normal growth arrest in U251 MG and U87 MG.

Figure 3. Induction of cell death by acyloxymethyl esters of butyric acid. A, PARP cleavage and p21Cip1 expression. Glioma cells and primary astrocytes were treated with escalating doses of AN-1 for 24 h. Cell lysates (20 Ag) were subjected to Western blot analysis using p21cip1 and PARP antibodies as well as h-actin as loading control. B, primary astrocytes and glioma cells were treated with AN-9 for 48 h. Adherent and nonadherent cells were pooled, double stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry. Columns, mean of three independent experiments; bars, SE. C, activities of caspase-8 and caspase-9 in U251 MG and SF188 treated with AN-1 for designated periods of time. Treatment with tumor necrosis factor (TNF; 20 ng/mL) and cycloheximide (CHX;30Ag/mL) was used as a positive control for caspase-8 activity. UV irradiation (400 J) was used as a positive control for caspase-9 activity. Y axes, ratios of activity in treated samples versus untreated controls. C, untreated controls. Columns, mean of two independent experiments; bars, SE. D, rescue of U251 MG and SF188 cells from apoptosis by 200 Amol/L Z-VAD or Z-IETD was evaluated by MTS assays. Y axis, percent proliferation in treated samples versus untreated controls. Columns, mean of three independent experiments; bars, SE.

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Table 1. Effects of AN-1 and AN-9 on cell proliferation and Radiation has a proven role in the treatment of gliomas and colony formation we therefore sought to investigate the combined effects of ionizing radiation and HDACIs. To optimize this combined Cell type AN-1 AN-9 treatment, preliminary MTS assays were done to explore (IC , Amol/L) (IC , Amol/L) 50 50 several treatment sequences. Pretreatment of cells with AN- MTS proliferation assay 9 followed by irradiation 24 hours later maximally enhanced Primary astrocytes 147 F 16 219 F 10 growth inhibition (data not shown). Other sequencing U251 MG 118 F 13 177 F 4 approaches, such as irradiation before AN-9 or concurrent SF188 94 F 5 141 F 8 treatments, did not enhance growth inhibition. U87 MG 88 F 10 134 F 7 To extend these analyses, gliomas and primary astrocytes Colony formation assay were treated with AN-9 or AN-1 for 24 hours before U251 MG (plating efficiency, 60%) 39 F 4 Not done irradiation and subjected to analysis by flow cytometry 48 hours later. Figure 4A and Table 2 show that combined HDACI and radiation treatments resulted in at least To further assess apoptosis after HDACI treatment, additive cell death in all cell lines tested. In SF188, AN-9 PARP cleavage was evaluated by Western blot analysis. (100 Amol/L) caused 4% cell death, 3 Gy alone resulted in The data (Fig. 3A) are consistent with those obtained by 16% mortality, but combination thereof enhanced cell death flow cytometry (Fig. 3B) and show that PARP cleavage was to 51% (Table 2). most prominent in SF188, somewhat less apparent in U251 To confirm the potentiation of radiation by HDACIs, MG, and absent in U87 MG and primary astrocytes. These clonogenic survival assays were done by pretreating U251 data complement the flow cytometry results in showing the MG with AN-1 (35 Amol/L) followed 24 hours later by range of HDACI-induced susceptibility to apoptosis seen in irradiation. Cell survival curves shown in Fig. 4B confirmed gliomas. Our observation that primary astrocytes express the efficacy and potentiation of combinatorial treatments exceedingly low levels of PARP proform is consistent with with HDACI and radiation. previously published reports (39). To examine the mechanism of cytotoxic potentiation Mechanism of HDACI-Induced Apoptosis in Gliomas of HDACI and radiation, caspase-8 activity assays were The mechanism of AN-1-induced apoptosis was investi- repeated, this time with AN-1 and radiation as single agents gated by asking which caspase pathways were activated in and in combination (Fig. 4C). In SF188, AN-1 (75 Amol/L) in gliomas following HDACI treatment. By using caspase- combination with irradiation (2 Gy) significantly enhanced specific fluorescent substrates, the activities of caspase- the apoptotic response compared with each treatment alone 8 and caspase-9 were measured following treatment with after 6 and 24 hours (P < 0.05, two-way ANOVA) but not 100 Amol/L AN-1 (Fig. 3C). In SF188, AN-1 treatment after 48 hours (P = 0.6). In U251 MG, such potentiation be- induced activities of both caspase-8 and caspase-9, suggest- tween AN-1 (75 Amol/L) and irradiation (3 Gy) was sta- ing that both death receptor– and mitochondria-mediated tistically significant only when assayed 48 hours (P < 0.03) pathways of apoptosis triggered cell death. In U251 MG, but not 6 or 24 hours after irradiation (P = 0.6 and 0.5, res- however, AN-1 exposure induced only caspase-8 activity, pectively). Potentiation of irradiation by HDACIs occurred indicating that only the death receptor–mediated pathway at earlier time points in SF188 than in U251 MG, consistent executed apoptosis in this cell line. Levels of caspase- with higher sensitivities of SF188 to both modalities. The 8 activity were lower in treated U251 MG than in treated data suggest that caspase-8 activity is res-ponsible at least SF188 cells, consistent with higher susceptibility to apop- in part for the potentiation of cytotoxicity documented with tosis of SF188 exposed to HDACIs. combinations of HDACIs and radiotherapy. We further examined the roles of caspases in HDACI- In vivo Efficacy of AN-9 induced apoptosis by using a pan-caspase inhibitor, Z- In view of the encouraging in vitro data, we sought to VAD, and a more specific caspase-8 inhibitor, Z-IETD. expand our studies to test the in vivo efficacy of AN-9 in Incubation with Z-VAD partially overcame AN-1-induced mice bearing flank xenografts of human glioma cell lines. We inhibition of cell proliferation (50-60%) for both U251 MG concentrated our efforts on AN-9, rather than on AN-1, and SF188. In U251 MG, Z-IETD mimicked the protection because completed and ongoing clinical trials are testing afforded by Z-VAD, consistent with the dominant role AN-9, and a maximum tolerated dose already exists for this of caspase-8 in AN-1-induced apoptosis of U251 MG drug, thus paving the way for clinical studies of AN-9 (Fig. 3D). In SF188, Z-IETD enhanced cell proliferation in glioma patients. In a preliminary experiment, BALB/c by only 32% compared with 53% seen with Z-VAD, nu/nu athymic mice were inoculated with 3 Â 106 U251 MG reflecting the dual roles of both caspase pathways in cells, and drug administration began when xenografts HDACI-induced apoptosis of this cell line. reached 30 mm3 in size. Mice were randomly assigned CombinationTreatment of Radiation and Butyric Acid to one of two groups: mineral oil vehicle as control or Prodrugs 200 mg/kg AN-9 diluted in the same vehicle. Agents were AN-1 and AN-9 exhibited promising antineoplastic given by oral gavage thrice weekly for a complete 150-day activities as single agents in vitro, an important finding course or until tumors reached 2.5 cm, at which point mice given the global resistance of gliomas to anticancer agents. were euthanized.

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The survival curves of the treated mice in this prelimi- exponentially with no apparent growth delay. Survival nary experiment are shown in Fig. 5A. All tumors in the analysis revealed a trend toward longer survival in mice control group (8 of 8) grew exponentially, showing no receiving AN-9 (P = 0.1, Mantel-Cox comparison test). evidence of regression. In three of seven mice receiving Of note, no constitutional signs or toxicities were observed 200 mg/kg AN-9, tumors did not grow beyond 150 mm3 in any of these mice for the entire duration of the for the entire duration of the experiment. Flank tumors in experiment. Specifically, no changes were observed in the remaining four mice receiving 200 mg/kg grew any animal’s weight or activity and no pathologic signs

Figure 4. Effects of concurrent treatment with radiation and HDACIs. A, flow cytometry analysis of SF188 cells treated with AN-1 or AN-9 (100 Amol/L) and irradiation (3 Gy) as indicated. Percent viable cells for each sample are shown. B, colony formation assay of U251 MG treated with AN-1 (35 Amol/L) and irradiation. Clonogenic survival curves were generated after normalizing to cell killing by AN-1 alone. C, caspase-8 activities in U251 MG and SF188 treated with AN-1 (75 Amol/L), radiation (3 Gy for U251 MG and 2 Gy for SF 188), or combinations thereof. Results are presented as ratios of treated samples versus untreated controls. Points, mean of samples obtained from two independent experiments; bars, SE.

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Table 2. Effects of combined butyric acid–releasing HDACIs Of the three surviving mice, one tumor shrunk and was and irradiation on glioma cell viability no longer detectable following treatment, precluding further biochemical tissue analyses. Tumors excised from Treatment % Mortality the other two surviving animals following euthanization SF188 were assessed for morphology by H&E staining, for 3Gy 16 apoptosis by immunohistochemistry staining for the 100 Amol/L AN-1 50 cleaved form of caspase-3, and for cellular proliferation 100 Amol/L AN-1 + 3 Gy 92.5* by immunohistochemistry staining for Ki-67. 100 Amol/L AN-9 4 H&E staining revealed that tumors from control animals 100 Amol/L AN-9 + 3 Gy 51* were highly cellular neoplasms with marked cellular U251 MG pleomorphism, variable necrosis, and stromal desmoplasia. 6 Gy 13.1 In contrast, tumors from the AN-9-treated animals were A 100 mol/L AN-1 6.8 more heterogeneous; desmoplastic and less cellular zones 100 Amol/L AN-1 + 6 Gy 19.5* adjacent to adipose tissue were admixed with cellular areas 100 Amol/L AN-9 11 100 Amol/L AN-9 + 6 Gy 27.7* (Fig. 5B, top). In addition, tumors excised from the animals U87 MG that were treated with AN-9 displayed a dramatic 10 Gy 0 reductionintheproportionof proliferating cells as 100 Amol/L AN-9 15 reflected by significantly reduced Ki-67 antigen staining 100 Amol/L AN-9 + 6 Gy 20.0* (Fig. 5B, middle). Apoptotic cell populations in both control Primary human astrocytes and drug-treated animals were low, with only a marginal 6Gy 0 increase in cleaved caspase-3 staining in treated animals 200 Amol/L AN-9 20 (Fig. 5B, bottom). Our in vivo immunohistochemistry results A 200 mol/L AN-9 + 6 Gy 21.1* indicate that, after oral administration of AN-9, growth arrest plays a key role in the cytotoxicity induced by these *Cellular viability for combined treatment modalities are shown in bold. novel HDACIs. were documented. To further assess any potential toxicity, gross and microscopic examinations were done on livers and Discussion kidneys isolated from representative euthanized mice. The promise of HDACIs for cancer treatment, as single These organs displayed normal architecture with no agents and in combination with standard therapies, is observed toxicities, except for rare calcifications observed supported by in vitro assays, preclinical experiments, and in the kidney, most consistent with the age of the mice rather clinical studies. Recent studies show that butyric acid than treatment effects. prodrugs that undergo intracellular esterase-catalyzed

Figure 5. In vivo effects of AN-9. A, survival of nude mice treated with AN-9. BALB/c nu/nu athymic mice bearing 30 mm3 flank tumors were randomly assigned to one of two treatment groups: control vehicle or 200 mg/kg AN-9. The agents were given orally thrice weekly and tumor sizes were recorded. B, analyses of tumor sections from control (left) and AN-9-treated (right) BALB/c nu/nu athymic mice bearing xenografts of U251 MG. Top, H&E staining; middle, Ki-67 staining; bottom, caspase-3 staining.

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hydrolysis are orally bioavailable, reduce tumor growth, Preliminary in vivo experiments reported herein suggest and inhibit metastases in xenograft models of prostate that in vivo efficacy of AN-9 is coupled with a safe toxicity cancer (34). Furthermore, AN-9 in intralipid formulation profile. The human nuclear antigen Ki-67 reflects cell (Pivanex) increases survival and improves the well-being of proliferation because it is present in the nucleus during patients in phase II clinical studies (12, 15). late G1,S,G2, and M phases (44). Reduced Ki-67 staining in In this study, we have shown that, whereas the dominant tumor sections from AN-9-treated animals indicates that effects of AN-1 and AN-9 on normal astrocytes are chiefly growth arrest plays a key role in the antineoplastic effects cell cycle arrest, malignant gliomas display marked of these novel HDACIs in vivo.Ofnote,significant apoptotic responses. Of note, prodrug hydrolysis produces variability in responses of individual animals is evident not only butyric acid but also one equivalent of formalde- among AN-9-treated mice. This unexpected variation hyde, which has also been shown to induce apoptosis and among syngeneic mice may be due to differences among inhibit proliferation of cancer cells (40). Thus, both butyric flank tumors in subpopulations of U251 MG that formed acid and formaldehyde have antineoplastic activity, the tumors or to differences in proximity to blood supply and former acid by inhibiting HDAC and the latter aldehyde by other extracellular components. an unknown mechanism. Our plans for in vivo experiments include comparisons of As prodrugs of HDACIs, AN-1 and AN-9 augment the oral and i.v. modes of drug delivery. Although the ease of acetylation of H3 and H4 in glioma cell lines and in murine oral administration offers obvious benefits for patients, the brain extracts. Consistent with the induction of histone maximum tolerated dose is already established for i.v. acetylation are decreased HDAC activity and increased given Pivanex, allowing a more expeditious, direct route to p21Cip1 expression in vitro and in vivo. p21Cip1 elevation is clinical use. In addition, the treatment regimen that proves shown in all tested cell lines, including those with mutant most effective in the flank model will be further tested in an p53, in which basal p53 levels are predictably much lower orthotopic intracranial model (45, 46). Furthermore, be- than in wild-type p53-expressing cells. These data are cause in vitro data presented herein indicate that butyric consistent with previous reports documenting p53-inde- acid–delivering HDACIs enhance the efficacy of ionizing pendent increases in p21Cip1 (8). radiation, combinations of AN-9 and clinically relevant We explored the mechanism of HDACI-induced cytotox- radiation doses will be the focus of our next study. icity in glioma cell lines and found that caspase-regulated Preliminary in vivo data reported herein suggest prom- pathways of apoptosis, both receptor-mediated (caspase-8) ising efficacy of AN-9 in the treatment of gliomas. Our and mitochondria-mediated (caspase-9) components, par- in vivo results, coupled with additional preclinical studies ticipate in HDACI-induced cell death. However, experi- of acyloxymethyl esters of butyric acid (34), show exceed- ments using caspase inhibitors suggest that additional ingly low toxicities of these novel agents and set them apart caspase-independent mechanisms of cell death may come from other HDACIs, such as suberoylanilide hydroxamic to bear as well. Such caspase-independent mechanisms of acid and MS-275 (19, 23). Thus, the promise of butyric acid cell death may include nucleases, nuclease activators, and prodrugs for cancer treatment in general and for highly serine proteases (41). morbid radioresistant malignant gliomas in particular, rests Radiosensitizing functions have been attributed to on their low toxicity, oral bioavailability, ability to cross the butyric acid, the hydrolytic derivative of AN-1 and AN- blood-brain barrier, and potential synergy with chemother- 9, two decades ago (42, 43). The prominent role of apy and radiation. Indeed, the above shown differential radiation therapy in standard treatments of many neo- effects of the butyric acid prodrugs AN-1 and AN-9 on plasms has focused attention on radiosensitizing effects high-grade glioma cell lines versus their normal cellular of various HDACIs. Our data indicate that butyric acid– counterparts, primary human astrocytes, shed light on their delivering prodrugs may serve as effective radiosensitiz- potential clinical use for glioma therapy. We have shown ing agents. Further in vivo experiments are now required in vitro and in vivo efficacy of novel butyric acid–delivering to confirm enhanced effects of combined treatments prodrugs in the treatment of gliomas, and given their compared with each modality alone. 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Michal Entin-Meer, Ada Rephaeli, Xiaodong Yang, et al.

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