Epigenetic Mechanism of Growth Inhibition Induced by Phenylhexyl Isothiocyanate in Prostate Cancer Cells

ANTICANCER RESEARCH 26: 1225-1230 (2006)

ANASTASIA A. BEKLEMISHEVA1, YUQIANG FANG1, JINGYANG FENG1, XUDONG MA1,2, WEI DAI1 and JEN WEI CHIAO1

1Department of Medicine, New York Medical College, Valhalla, NY 10595, U.S.A.; 2Zhangzhon Municipal Hospital, Zhangzhon, Fujian, 363000, China

Abstract. Background: Isothiocyanates, the constituents of procarcinogens, and inducing phase 2 enzymes to remove cruciferous vegetables, may be able to prevent prostate cancer. carcinogens (3). Our laborarory has investigated the The hypothesis that they could remodel chromatins and phenethyl isothiocyanate, sulforaphane, and its major activate cell cycle inhibitors, such as p21 for growth inhibition, metabolites, and demonstrated that they induce growth arrest was tested. Materials and Methods: Prostate cancer LNCaP and apoptosis in prostate cancer cells, in culture as well as in cells were exposed to phenylhexyl isothiocyanate (PHI). The xenografted tumors in immunodeficient mice (4, 5). These status of histone acetylation and the activity of histone observations, together with reports by other investigators (6, deacetylases (HDAC) were investigated. The association of p21 7), indicated that the isothiocyanates can also act at the level with hyperacetylated histones was examined by chromatin of post-initiation progression of prostate carcinogenesis. immunoprecipitation. Results: The PHI-exposed LNCaP cells Studies indicate that one of the mechanisms is targeting of had diminished activity of HDAC 1 and 2. Global and the cell cycle machinery, with the up-regulation of cell cycle selective histone acetylation was enhanced, consistent with the inhibitors such as p21WAF1 (p21) to mediate growth arrest signs of chromatin unfolding. The hyperacetylated histones (8). The upstream mechanism that leads to the cell cycle increased accessibility to the p21 promoter for transcription, arrest, however, has not been clearly deciphered. leading to G1 arrest and apoptosis. Conclusion: PHI inhibited We have since investigated the potential effects of the the activity of HDAC and remodeled chromatins to activate isothiocyanates on chromatin remodeling for transcriptional p21 for cell cycle arrest, underlying an epigenetic mechanism regulation of the cell cycle inhibitors. In this study, the effects regulating the growth of prostate cancer cells. of a synthetic phenylhexyl isothiocyanate (PHI) on chromatin remodeling were examined, since PHI is among the most The epidemiological literature has provided modest to strong potent of the isothiocyanates in blocking carcinogen-initiated support to the hypothesis that the intake of cruciferous lung tumors (9). Chromatin remodeling is influenced by vegetables, such as broccoli, cabbages, Brussels sprouts, enzymatic modifications of amino acids at the histone tails, watercress and cauliflower, reduces prostate cancer risk (1). which cause nucleosome movement, orchestrating epigenetic These studies, however, did not identify the responsible events. Covalent modifications, such as acetylation, neutralize dietary constituents or the mechanisms of action. We have the charges on histones and provide a more open examined the role of the isothiocyanates, released from conformation with the DNA. The balance of acetylation is glucosinolates upon enzyme hydrolysis when the cruciferous regulated by pairs of opposing enzymes, the acetyl vegetables are masticated (2). Natural and synthetic transferases and deacetylases (HDAC) (10). The inhibitors isothiocyanates are potent chemopreventive agents that block of HDAC represent a new class of targeted anticancer agents carcinogen-initiated tumors in rodents. The major mechanism that mediate gene expression, such as p21, to induce growth is cytoprotection, i.e. inhibiting P450s and the metabolism of arrest and apoptosis in malignant cells (11). In this study, it was demonstrated that PHI is an inhibitor of HDAC and initiates histone modifications and chromatin remodeling to activate the inhibitor of cell cycle progression, p21, leading to Correspondence to: Jen Wei Chiao, Department of Medicine, New proliferation cessation and apoptosis in prostate cancer cells. York Medical College, Valhalla, NY 10595, U.S.A. Tel: 914-594- 4198, Fax: 914-594-3689, e-mail: [email protected] Materials and Methods

Key Words: Isothiocyanates, epigenetic, histone deacetylases, Cell culture. PHI of more than 98% purity was purchased from HDAC, p21, prostate cancer cells. LKT Labs (St. Paul, MN, USA). The PHI was prepared in 75%

0250-7005/2006 $2.00+.40 1225 ANTICANCER RESEARCH 26: 1225-1230 (2006) methanol and phosphate buffer, pH 5. The LNCaP prostate cancer the cell lysates were prepared. After sonication, the cell lysates cells, in exponential growth, were seeded at 0.3x106 per ml of (200 ÌL) in a ChIP dilution buffer were incubated with a rabbit RPMI-1640 medium supplemented with 15% heat-inactivated fetal antibody against acetylated histone H3, or with a non-specific bovine serum and antibiotics. Some cultures were exposed to PHI control Ig overnight. The mixtures were incubated with salmon at various concentrations, while the controls were supplemented sperm DNA/protein A agarose slurry for 1 h, the beads were with the methanol medium. Cell viability was determined by the washed and the bound molecules eluted. After de-cross-linking, the trypan blue exclusion method and the cell density was calculated DNA molecules in the precipitates were recovered. The p21 gene by the viable cell counts per ml. fragments TATA area (–33 to +47), down TATA area (+43 to +122) and D (+3267–+3366) were selected for amplification by Apoptosis and cell cycle phases. The cell cycle phases were PCR as described (8). determined with a Becton-Dickinson FACScan flow cytometer, according to the methods described previously (12). The cells were Results incubated on ice before the DNA was stained with propidium iodide (50 Ìg/ml). The apoptotic cells were determined by the presence of DNA strand breaks with terminal deoxynucleotidyl transferase- Induction of growth inhibition and apoptosis. To evaluate the mediated biotinylated UTP nick-end labelling (TUNEL). Detection effects of PHI on the growth of prostate cancer cells, the of apoptosis in situ was performed with a detection kit (Roche prostate cancer cells LNCaP were exposed to PHI at various Molecular Biochemicals, Indianapolis, IN, USA), according to the concentrations. Floating cells appeared in the PHI-exposed manufacturer’s directions. The apoptotic cell percentages were cultures along with a regular decrease in cell density. calculated by counting 500 cells from multiple fields. Concentration-dependent growth inhibitory effects could be Protein expression. The protein levels of the LNCaP cells were detected after 2-day PHI exposure, at a minimal determined by Western blotting, according to the standard concentration of 1 ÌM. Approximately a 17% and 23% procedure (12). Total cellular proteins were obtained with a lysis decrease in cell density was observed at 5 and 10 ÌM, buffer containing freshly prepared protease inhibitors, and the respectively (Figure 1A). Figure 1A also depicts a gradual lysates collected after centrifugation at 4ÆC. The histone proteins decrease of cell viability in these cultures. The effects of were isolated with an acid extraction procedure (13). Rabbit PHI on cell proliferation were evaluated by cell cycle phase antibodies against the acetylated histones H3, H4 or H3 lysine 14 were purchased from Upstate Biotechnology (Lake Placid, NY, progression, with the DNA frequency histogram using a USA). Monoclonal antibodies against PARP, bcl-2, caspases-8 or - flow cytometric method. After 1 day of PHI exposure, the 9, or HDAC 1 were purchased from Santa Cruz (Santa Cruz, CA, S-phase cell proportion was reduced in a concentration- USA), and antibody for p21 from Dako (Carpinteria, CA, USA). related manner, with minimal effects seen at 1 ÌM. The Densitometric analysis was performed to calculate the % alteration level of S-phase cells was reduced by about 45%, from 33% of the protein levels. to 18%, after exposure to 10 ÌM PHI. An accumulation of Histone deacetylase (HDAC) activity. To evaluate the inhibition of G1 cells was observed concomitantly, indicating a blocking HDAC activity by PHI, a cell-free assay detecting HDAC 1 and 2 of cell cycle progression from G1- to S-phase (Figure 1B). (Color de Lys, Biomol, Plymouth Meeting, PA, USA) was used Many PHI-exposed cells displayed cell shrinkage and according to the manufacturer’s protocol. A nuclear extract of HeLa condensed chromatin, characteristic of apoptosis. The cells containing HDAC 1 and 2 was incubated with PHI at various presence of apoptotic cells was detected by DNA strand concentrations for 10 min at 37ÆC. A separate test was performed breaks with the TUNEL assay. The quantity of apoptotic by adding PHI to a standard HDAC activity to evaluate any cells was concentration-dependent; a minimal amount was interference between PHI and the developers. Incubation with trichostatain A (TSA) (50 nM) was used as a positive control for detected with 1-5 ÌM, increasing to approximately 26% at inhibition. The methanol medium was used as the vehicle control. 20 ÌM (Figure 1C). Proteolytic cleavage of PARP, a For detecting HDAC activity in the LNCaP cells, cellular lysates of hallmark of apoptosis, was detected in PHI-exposed cells LNCaP cells, that had been exposed to 2 mM sodium butyrate, or (Figure 1D). A minimal reduction of PARP could be PHI, were compared to the lysate from LNCaP cells exposed to the detected at 5 ÌM PHI, but in excess of 50% at 20 ÌM. In vehicle control of PHI. The HDAC reaction was initiated by adding addition, the bcl-2 protein, an inhibitor of apoptosis, was a substrate of acetylated peptides and incubating at 37ÆC for significantly diminished by PHI at 5 ÌM or more. The 30 min, followed by adding a color developer. The absorbance of triplicate samples was determined at 405 nm with a Bio-Tec relationship of PHI to the caspases, intracellular cysteine microtiter-plate reader. The Student’s t-test was used for statistical proteases in the initiation and execution of apoptosis, was analyses with p<0.05 considered to be significant. investigated. Figure 1D depicts an increased expression of caspase-9, but the level of caspase-8 was not altered. Chromatin immunoprecipitation (ChIP) assay. The LNCaP cells were cultured for 24 h with PHI at 20 mM or with vehicle control Inhibiton of HDAC and histone modification. To investigate medium. ChIP analyses were performed according to the protocol of the ChIP assay kit (Upstate Biotechnology). Briefly, the DNA the effects of PHI on chromatin remodeling as a mechanism and histones were cross-linked by adding formaldehyde to the of growth arrest, the expression of HDAC was measured. culture to a final concentration of 1%. After incubation for 10 min, The Western blot analyses, shown in Figure 2A, revealed

1226 Beklemisheva et al: Epigenetic Mechanism of Growth Inhibition Induced by PHI

Figure 1. Growth modulation and induction of apoptosis. LNCaP cells were exposed to PHI at the indicated concentrations. Graph A shows a concentration-related decrease of cell density and viability, 2 days after exposure to PHI. The vertical bars are means±SD of 3 independent experiments. Graph B illustrates a decrease of S-phase cells, concomitant with an increase of G1 cells, as determined after 1-day exposure to PHI by flow cytometry. The values are the means±SD of 3 independent experiments. Graph C shows a concentration-related induction of apoptotic cells, determined 2 days after exposure to PHI using the TUNEL assay. The values are means and ranges of 2 independent assays. Western blots in graph D describe altered expressions of caspase-9, PARP and bcl-2; exposure to PHI for 48 h. The antibody against ß-actin was used as a loading control. that reduction of the HDAC1 level could be detected with To further evaluate whether the activity of HDAC was higher PHI concentrations, with an approximately 15% actually lowered in PHI-exposed LNCaP cells, the cellular decrease at 20 ÌM after 6 h of exposure. A similar extracts of LNCaP cells were evaluated for HDAC activity. magnitude of decrease was also observed at 48 h. Figure 2C demonstrates that LNCaP cells exposed for 6 h Experiments were then performed to determine whether to PHI had diminished HDAC activity as compared to PHI inhibited the activity of HDAC. A nuclear extract of control LNCaP cells without PHI. A statistically significant HeLa cells containing HDAC was supplemented with PHI, reduction, of approximately 25% and 43%, could be and the HDAC activity determined with a cell-free HDAC achieved, respectively, with 5 and 20 ÌM PHI. A known activity assay. The concentrations of PHI used were pre- HDAC inhibitor, sodium butyrate, used as a control showed determined so that they did not interfere with the reagents an approximately 50% reduction. The results indicated that of the assay. As demonstrated in Figure 2B, PHI inhibited PHI reduced the HDAC activity in LNCaP cells. the activity of HDAC 1 and 2 in a concentration-dependent With the finding that PHI inhibited the activity and level manner, while the vehicle control of PHI showed no effect. of HDAC, experiments were performed to examine the Statistically significant inhibition could be achieved with status of acetylation of core histones. Enhancement of 50 nM or more PHI. histone acetylation could be detected after 6-h exposure to

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Figure 3. Histone acetylation by PHI. Western blots in A: Significant concentration-related enhancement of acetylated histones H3, H4 and H3K14. B: The expression of cell cycle cdk kinase inhibitor p21 was increased after 6 h of PHI exposure; anti-actin antibody shows even protein loadings.

5 ÌM, approximately 60% and 110% increases were seen with histones H3 and H4, respectively.

Figure 2. Blocking histone deacetylase (HDAC) activity by PHI. LNCaP cells Induction of p21. The cell cycle regulators were among the were exposed to PHI at various concentrations. Graph A: Western blot most common genes to be activated as a result of HDAC describes a concentration-related decrease of HDAC1 expression after exposure to PHI for 6 h. Graph B: Concentration-related inhibition of the activities of inhibition (8, 14). To examine the relationship between HDAC 1 and 2 from nuclear extract of HeLa cells by the addition of PHI with enhanced histone acetylation and cell growth, the expression a cell-free HDAC activity assay described in "Materials and Methods". TSA of the cdk kinase inhibitor p21 was evaluated. Figure 3B was included as a control of positive inhibition. The data are the means±SD shows that exposure to PHI for 6 h resulted in a significant of 3 independent experiments normalized with the controls without PHI. induction of p21 expression. A minimal increase could be *indicates statistically significant changes. Graph C: Concentration-related reduction of the activities of HDAC 1 and 2 in the lysates from 6-h PHI- detected in cells after exposure to 1 ÌM PHI, and an exposed LNCaP cells, as compared with cells without PHI. LNCaP cells approximately 40% increase with 5 ÌM. treated with sodium butyrate were used as a control of inhibition. The vertical To further determine the relationship between p21 bar values are the means±SD of 3 independent experiments. activation and acetylated histones, the ChIP assay was performed. The DNA from the immunoprecipitates was amplified according to four primers complementary to p21 PHI, with the magnitude of enhancement increasing with gene fragments, including the promoter region. As longer exposure time. By 24 h, a clear concentration- demonstrated in Figure 4, the chromatin from cells exposed dependent enhancement of acetylation of the histones H3, to PHI clearly contained the p21 DNA after precipitation with H4 and H3 lysine 14 was demonstrated (Figure 3A). At the antibody, as compared to cells exposed to the control

1228 Beklemisheva et al: Epigenetic Mechanism of Growth Inhibition Induced by PHI

Figure 4. Association of the p21 gene with acetylated histones accumulated by PHI. Schematic representation of PCR primer sets TATA, down TATA and D for the p21 gene in the upper graph. Lower figure: Chromatin fragments from LNCaP cells, after exposure for 24 h to medium without PHI (control) or 20 ÌM PHI, were precipitated with an anti-acetylated histone H3 antibody (H3-IP), or with a non-specific Ig as controls (beads). The indicated primers for areas of the p21 gene were used for PCR amplification of the DNA isolated from the immunoprecipitated chromatin. Input: DNA extracted from equal volumes of cell lysates was used as template and the PCR product served as input.

medium without PHI (Figure 4). Immunoprecipitations using similarly to some of the HDAC inhibitors such as a non-specific Ig were employed as controls to show the suberoylanilide hydroxamic acid and trichostatin A (15). negative background (Figure 4). The p21 sequence was PHI as an inhibitor of HDAC was supported by the direct detected in the promoter areas TATA, down TATA and D as inhibition of HDAC by PHI in a cell-free assay. Additionally, compared with cells not exposed to PHI where p21 was nearly the activity of HDAC from PHI-exposed LNCaP cells was undetectable. The results showed that more p21 DNA was diminished, with the extent of reduction close to that present in the precipitates of hyperacetylated histones, mediated by a known HDAC inhibitor, sodium butyrate. The indicating the association of the p21 gene with the experimental results also demonstrated that, at high PHI hyperacetylated histones. concentrations, the HDAC level was reduced, which could also contribute to increased acetylation. However, about 5 ÌM Discussion of PHI was sufficient to inhibit the activity of HDAC in LNCaP cells (25% decrease), while it required 20 ÌM to Evidence is presented that a synthetic isothiocyanate, PHI, is reduce the HDAC expression (15% decrease). This supports capable of inducing G1 arrest and apoptosis in prostate cancer the interpretation that inhibiting HDAC activity is the primary cells. We have demonstrated that there is complex cross-talk action to induce histone acetylation. Our conclusion that PHI between chromatins and the DNA that up-regulates p21 for is an inhibitor of HDAC is in line with the observation that cell cycle arrest. The transcriptional activation of p21 is the N-acetylcysteine conjugate of sulforaphane, the metabolite revealed as cumulative, occurring as a result of HDAC of an isothiocyanate found in broccoli, is also an inhibitor of inhibition, hyperacetylation of histones and the regulation of HDAC (16). The inhibitory activity of PHI, in comparison, enzymes controlling the balance of acetylation. Parallel to was similar to that of the sulforaphane conjugate. growth arrest, apoptosis was induced with caspase-9 up- Hyperacetylation of histone allows chromatin unfolding regulation and PARP degradation, suggesting that the and more accessibility of regulators to the DNA for mediation of apoptosis is through the mitochondrial pathway, transcription activation. Hypoacetylation of histone, on the

1229 ANTICANCER RESEARCH 26: 1225-1230 (2006) other hand, is associated with the formation of 5 Chiao JW, Chung F-L, Kancherla R, Ahmed T, Mittelman A heterochromatin and gene silencing (17, 18). Our study and Conaway CC: Sulforaphane and its metabolite mediate demonstrated that PHI enhanced acetylation of histones H3, growth arrest and apoptosis in human prostate cancer cells. Int J Oncol 20: 631-636, 2002. H4 and H3 lysine 14, which is consistent with the signs of 6 Fimognari C, Nüsse M and Cesari R: Growth inhibition, cell- transcriptionally competent chromatin (19). Hyperacetylation cycle arrest and apoptosis in human T-cell leukemia by the of histones by PHI may increase accessibility of the isothiocyanate sulforaphane. Carcinogenesis 23: 581-586, 2002. transcriptional machinery in the p21 promoter, with increase 7 Srivastava SK, Xiao D, Lew KL, Hershberger P, Kokkinakis of the p21 expression as a result. The ChIP analyses clearly DM, Johnson CS, Trump DL and Singh SV: Allyl demonstrated the association of the p21 gene with the highly isothiocyanate, a constituent of cruciferous vegetables, inhibits acetylated histones. The results support the hypothesis that growth of PC-3 human prostate cancer xenografts in vivo. Carcinogenesis 24: 1665-1670, 2003. there is a relationship between growth regulatory genes, like 8 Gui CY, Ngo L, Xu WS, Richon VM and Marks PA: Histone p21, and chromatin remodeling with acetylation by PHI. deacetylase (HDAC) inhibitor activation of p21WAF1 involves The transcriptional activation of p21 and cell cycle arrest changes in promoter-associated proteins, including HDAC1. mediated by PHI implies a reversal of the aberrant epigenetic Proc Natl Acad Sci USA 101: 1241-1246, 2004. events in the prostate cancer cells. Our experimental results 9 Chung FL, Kelloff G, Steele V, Pittman B, Zang E, Jiao D, indicated that there may be aberrant epigenetic events Rigotty J Choi CI and Rivenson A: Chemopreventive efficacy associated with prostate carcinogenesis. These events may of arylalkyl isothiocyanates and N-acetylcysteine for lung tumorigenesis in Fischer rats. Cancer Res 56: 772-778, 1996. include the local chromatin architecture, the status of histone 10 Jenuwein T and Allis CD: Translating the histone code. Science modifications and the activity of regulatory enzymes, which 293: 1074-1080, 2001. could represent promising targets for intervention. 11 McLaughlin F and La Thangue NB: Histone deacetylase Our previous report (5) indicated that isothiocyanates, inhibitors open new doors in cancer therapy. Biochem Pharm such as sulforaphane, down-regulate androgen receptor 68: 1139-1144, 2004. expression as an important growth regulatory mechanism in 12 Wang L, Liu D, Ahmed T, Chung F-L, Conaway C and Chiao prostatic cells. Judged together with our present findings, JW: Targeting cell cycle machinery as a molecular mechanism of sulforaphane in prostate cancer prevention. Int J Oncol 24: the growth regulation of prostatic cancer cells by 187-192, 2004. isothiocyanates may include a two-pronged mechanism, with 13 Yoshida M, Kijima M, Akita M and Beppu T: Potent and one to up-regulate p21 to modulate cell cycle progression, specific inhibition of mammalian histone deacetylase both in and the other to suppress the androgen receptor and, vivo and in vitro by trichostatin A. J Biol Chem 265: 17174- thereby, the hormonal-driven proliferation. The dual 17179, 1990. activities could function synergistically to effectively regulate 14 Chiba T, Yokosuka O, Fukai K, Kojima H, Tada M, Arai M, the growth of prostate cancer cells. This has provided an Imazeki F and Saisho H: Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, experimental basis for using the isothiocyanates to prevent trichostation A, on human hepatoma cells. Oncology 66: 481- the development and progression of prostate cancer. 491, 2004. 15 Henderson C, Mizzau M, Paroni G, Maestro R, Schneider C Acknowledgements and Brancolini C: Role of caspases, Bid, and p53 in the apoptotic response triggered by histone deacetylase inhibitors We thank Ms Ruth Gallagher for proofreading the manuscript. trichostatin-A (TSA) and suberoylanilide hydroxamic acid This research is supported by a research grant from the US Army. (SAHA). 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Hu Mol Gene 10: 693-698, 2001. 3 Zhang Y and Talalay P: Anticarcinogenic activities of organic 19 Strahl BD and Allis CD: The language of covalent histone isothiocyanates: chemistry and mechanisms. Cancer Res (suppl) modifications. Nature 403: 41-45, 2000. 54: 1976-1981, 1994. 4 Chiao JW, Wu H, Ramaswamy G, Conaway CC, Chung FL, Wang L and Liu D: Ingestion of an isothiocyanate metabolite from cruciferous vegetables inhibits growth of human prostate cancer cell xenografts by apoptosis and cell cycle arrest. Received December 9, 2005 Carcinogenesis 25: 1403-1408, 2004. Accepted February 7, 2006

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