Histone deacetylase inhibitor sulforaphane: The phytochemical with vibrant activity against prostate cancer

Biomedicine & Pharmacotherapy 81 (2016) 250–257

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Review

Histone deacetylase inhibitor sulforaphane: The phytochemical with vibrant activity against prostate cancer Shabir Ahmad Ganai Department of Biotechnology, University of Kashmir, Hazratbal, Srinagar, 190006 Jammu & Kashmir, India

A R T I C L E I N F O

Article history: Received 26 December 2015 Received in revised form 10 April 2016 Accepted 10 April 2016 Keywords: HDACs TRAIL Therapeutic intervention

A B S T R A C T

Epigenetic modifications are closely involved in the patho-physiology of prostate cancer. Histone deacetylases (HDACs), the transcriptional corepressors have strong crosstalk with prostate cancer progression as they influence various genes related to tumour suppression. HDACs play a marked role in myriad of human cancers and as such are emerging as striking molecular targets for anticancer drugs and therapy. Histone deacetylase inhibitors (HDACi), the small-molecules interfering HDACs are emerging as promising chemotherapeutic agents. These inhibitors have shown multiple effects including cell growth arrest, differentiation and apoptosis in prostate cancer. The limited efficacy of HDACi as single agents in anticancer therapy has been strongly improved via novel therapeutic strategies like doublet therapy (combined therapy). More than 20HDACi have already entered into the journey of clinical trials and four have been approved by FDA against diverse cancers. This review deals with plant derived HDACi sulphoraphane (SFN; 1-isothiocyanato-4-(methylsulfinyl)-butane) and its potential role in prostate cancer therapy along with the underlying molecular mechanism being involved. The article further highlights the therapeutic strategy that can be utilized for sensitizing conventional therapy resistant cases and for acquiring the maximum therapeutic benefit from this promising inhibitor in the upcoming future. ã 2016 Elsevier Masson SAS. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distinct classes of HDACs: brief glimpse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structurally distinct groups of HDACi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Impaired expression of HDACs: trigger to prostate cancer . . . . . . . . . . . . . . . . . . Sulforaphane: its processing, bioavailabilty and metabolism . . . . . . . . . . . . . . . . Sulforaphane: the highly promising molecule for prostate cancer chemotherapy Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Aberrant gene expression plays a crucial role in tumour onset and progression. This impaired gene expression in turn is triggered by epigenetic dysregulation. Epigenetic gene silencing plays multiple roles in eukaryotes organisms including differentiation,

E-mail address: [email protected] (S.A. Ganai). http://dx.doi.org/10.1016/j.biopha.2016.04.022 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.

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development and imprinting [1]. Dysregulation of this silencing forms the etiology for plethora of diseases, notably cancer. DNA methylation, post-translational modifications of histones, nucleosome positioning and non-coding RNAs are the critical processes responsible for epigenetic gene silencing. Post-translational modifications, especially histone acetylation and deacetylation, occurring mainly on the tail residues of histone proteins are the major determinants of epigenetic gene regulation [2,3]. This dynamic post-translational modification is tightly regulated by the

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antagonistic activities of histone acetyl transferases (HATs) and histone deacetylases (HDACs). HATs deposit the acetyl moiety on the lysine residues of nucleosomal histones creating the permissive state of chromatin structure by enhancing the electrostatic repulsion between polyanionic DNA and polycationic histones resulting in transcriptional activations [4–6]. HDACs erase acetyl group from the nucleosomal histones creating the repressive chromatin structure by enhancing electrostatic attraction between DNA and histones leading to transcriptional silencing [4,7]. Recent studies have revealed more than 50 non-histone targets for HDACs as well suggesting that HDACs modulate both histone and nonhistone substrates inside a eukaryotic cell [8,9]. Many of the HDAC substrates are perform regulatory functions being involved in cell adhesion, cell division and apoptosis. For instance members of class I HDACs like HDAC1, 2 and 3 are involved in cell cycle regulation by regulating cell cycle genes like p21 [10–12]. HDAC8 participates in cellular proliferation besides regulating smooth muscle contractility [13,14]. HDAC6, the multifunctional HDAC regulates several vital processes such as cell migration, protein folding, cellular stress and immune synapse formation [15–17]. HDAC6 has been reported to destabilize androgen receptor and alpha-tubulin protein [15,18]. Moreover, HDAC6 and HDAC10 play significant roles in HSP-mediated vascular endothelial receptors (VEGFR) regulation [19]. Thus, it is quite evident that HDACs have a broad spectrum of targets and thus act as master regulators of many diseases including the cancer. Over-representation of HDACs in cancers culminates in enhanced proliferation, altered cell cycle regulation, enhanced tumour angiogenesis apart from induction of several oncogenes. Therapeutic intervention using small-molecules namely HDACi have shown encouraging results in various diseases including cancer and neurodegeneration [1,20]. Due to certain dose limited toxicities like diarrhoea, electrolytic changes, taste disturbances, fatigue, thrombocytopenia etc., encountered during the therapeutic intervention with synthetic HDACi [21,22], the current studies have begun to focus on plant derived molecules. Experimental evidences have shown that plant derived molecules like chrysin [23], pomiferin [24], and sulforaphane possess inhibitory activity against HDACs [25]. Sulforaphane shows promising activity against several cancers like pancreatic [26], colon [27] and breast [28]. The defined inhibitor exhibits considerable neuroprotective effect in various experimental paradigms [29]. Sulforaphane destabilizes HSP90 client proteins and hampers NF-B DNA binding in various pancreatic cancer models resulting in cytotoxic effect [30,31]. Sulforaphane induces death in gemcitabine-resistant pancreatic cancer models by suppressing gemicitabine-induced Notch-1 upregulation [31]. Despite its remarkable effect against transformed cells, sulforaphane shows no toxicity against normal cells which determines its clinical relevance [31]. Keeping in view the above grim facts this review focuses on plant derived HDAC inhibitor sulforaphane (Fig. 1) and its potential role in prostate cancer therapy along with the underlying molecular mechanism being involved. Moreover, the article highlights the therapeutic strategy that can be used for tackling the conventional therapy resistant cases and for enhancing the therapeutic efficacy of this promising inhibitor in the upcoming future.

Fig. 1. Structure of sulforaphane.

substrates. Class II includes two subclasses namely class IIa and class IIb. Class IIa encompasses HDAC4, 5, 7, 9 while class IIb covers HDAC6 and 10 [32]. Class II HDACs exhibit tissue specific distribution, possess shuttling ability and deacetylate both histone and non-histone substrates. Class III HDACs also known as sirtuins includes sirt1-sirt7 are mechanistically distinct [33]. Class IV includes HDAC11 as the sole member and has properties of both class I and class II HDACs. Class I, II and class IV HDACs require zinc for their catalytic activity under in vitro conditions; however, there is no report till date which supports the zinc requirement in living

2. Distinct classes of HDACs: brief glimpse HDACs as aforementioned erase the acetyl group from the

e-N-acetyl lysine amino acid residues and thus modulate

chromatin architecture. This modulation creates the closed state of chromatin culminating in gene silencing [7]. HDACs have been broadly classified into four major classes viz. class I, class II, class III and class IV. Class I including HDAC1, 2, 3 and 8 are ubiquitous in distribution, usually lack shuttling ability and deacetylate histone

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Fig. 2. Detailed classification of HDACs.

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system [34]. Class IV HDACs are NAD+ dependent and are considered as non-classical HDACs (Fig. 2) [34,35]. 3. Structurally distinct groups of HDACi Histone deacetylase inhibitors (HDACi) are the emerging drug candidates tuning the histone acetylation dysregulated in disease states. Based on structural distinction they have been classified broadly into four major groups. Histone deacetylase inhibitors may be hydroxamates like SAHA; benzamide derivatives like entinostat; cyclic tetrapeptides like HC-toxin; short chain fatty acids like valproic acid and sodium butyrate (Fig. 3) [36–38]. HDAC inhibitors may be natural like Trichostatin A, sodium butyrate or synthetic like sodium phenyl butyrate, sodium valproate and so on [39]. HDACi may be pan-inhibitors (SAHA, TSA) targetting HDACs of many classes or selective inhibitors targetting HDACs of a particular class or single isoform. Selective inhibitors targetting members of single class are known as class selective (Entinostat, mocetinostat) while those targetting a single isoform are termed as isoform selective (tubacin and tubastatin) [40–43]. Most of the HDACi are reversible however few like trapoxin, depudecin and chlamydocin are irreversible inhibitors [44,45]. 4. Impaired expression of HDACs: trigger to prostate cancer HDACs are upregulated in plethora of cancers including prostate cancer. In majority of prostate cancer samples strong expression of HDAC1, 2 and 3 has been reported and the differential

expression of these HDACs in prostate cancer is believed to play a role in cancer progression [46]. Lower expression of HDAC8 has been found in benign and malignant prostate tissue compared to various non-prostatic malignancies [47]. Studies have shown the predominant localization of HDAC4 in cytoplasm of benign prostate hyperplasia cells and in primary prostate cancer cells as well compared to hormone refractory cancers where it is predominantly present in nucleus and thus may contribute to more aggressive behavior [48]. HDAC7, the class II HDAC localizes to the mitochondrial inner membrane space in prostate epithelial cells and in response to the initiation of apoptotic signalling exhibits cytoplasmic relocalization [49]. SIRT1 overexpression has been reported in androgen-refractory PC3 and DU145 cells compared to androgen-sensitive LNCaP cells suggesting the possible role of the defined HDAC in promoting cell growth and chemo-resistance [50]. Downregulation or SIRT1 activity has been reported to enhance the sensitivity of prostate cancer cells to the effects induced by androgens. Besides the androgen antagonistmediated growth suppression depends on the presence of SIRT1 [51]. 5. Sulforaphane: its processing, bioavailabilty and metabolism Sulphoraphane occurs in foods in the form of glycoside glucoraphanin which is an isothiocyanate [52,53]. The main source of sulphoraphane/glucoraphanin are Brassica oleracea commonly known as broccoli (44–171 mg/100 g dry weight) and broccoli sprouts (1153 mg/100 g dry weight) [54]. Glucoraphanin is acted

Fig. 3. Structurally distinct groups of HDAC inhibitors with typical examples.

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upon by Myrosinase [55] to form an unstable intermediate and ultimate conversion into Sulphoraphane depends upon the Epithiospecifier protein (ESP) which if active will result in formation of sulphoraphane nitrile (5-methylsulfinylpentane nitrile) lacking anticancer activity and if less active results in the formation of sulphoraphane via an alternative pathway [56]. Experiments have shown that heating of broccoli enhances sulphoraphane absorption as heating causes denaturation of (ESP) while as excessive heating diminishes the absorption of sulphoraphane by deactivating Myrosinase [57,58]. Sulphoraphane is mainly absorbed in jejunum [59] and reports have shown the metabolites of sulphoraphane appear in urine soon after its consumption [60]. Reports have shown that bioavailability of serum nearly equal to 74% [59]. Sulphoraphane has the ability to cross blood brain barrier and is thus able to show neurological effect [61]. Single doses of 200 mmol broccoli sprouts ITC (isothiocyanate) preparation given to human subjects showed peak concentrations between 0.943 and 2.27 mmol/L one hour post-feeding and showed half-life times of 1.77
0.13 h [62]. Sulforaphane inhibited survival in PC-3 cells in a concentration-dependent fashion and showed an IC50 of 40 mM against the defined model [63]. However, the IC50 varies from one type of cancer to another. Studies with human subjects have deciphered that raw broccoli is quickly absorbed compared to cooked broccoli and both forms show close excretion half-lives (2.6 and 2.4 h) respectively [64]. Sulforaphane is metabolized through the mercapturic acid pathway involving conjugation with glutathione. Glucoraphanin present in broccoli is converted into sulforaphane by myrosinase-producing bacteria in the human gut. Sulforaphane thus formed is passively transported to intestinal lumen and is conjugated with glutathione with the help of glutathione S-transferases. Further metabolism is assisted by g-glutamyltranspeptidase (GTP), cysteinyl-glycinease (GCase) and N-acetyltransferase (NAT) respectively. The conjugates are transported into system circulation by active transport where the mercapturic acid (SF-NAC) and its precursors are excreted in urine [59,65]. The stepwise mechanism of glucoraphanin and sulforaphane metabolism is summarized in Fig. 4. 6. Sulforaphane: the highly promising molecule for prostate cancer chemotherapy Prostate cancer is regarded as the second leading cause of cancer death in America, the first being lung cancer [66]. Most of the cases are diagnosed in men aged 65 (6 out of 10) and is rare below the age of 40. Although androgen ablation therapy at initial stage benefits patients with metastatic prostate cancer, most patients die of hormone-refractory prostate cancer within few years emphasizing the desperate need of novel therapeutic strategy against the defined cancer [1]. Emerging evidences have revealed the role of epigenetic enzymes namely HDACs in prostate and other cancer [1,5]. Aberrant expression of class IIb HDAC (HDAC6) has been reported in prostate cancer signalling providing a novel target for epigenetic inhibitors [18]. As aforementioned, HDAC6 is over-represented in prostate cancer and the anticancer activity of Sulphoraphane is mainly due to its ability to restrain HDACs [25]. Sulphoraphane acts as chemoprotective in xenograft models of prostate cancer [63]. Sulphoraphane induced apoptosis in PC-3 human prostate cancer cells by activating caspases 3, 9 and 8. Such cells showed Bax upregulation and downregulation of antiapoptotic Bcl-2. Last but not least this antiproliferative and apoptosis was accompanied by poly (ADP-ribose) polymerase (PARP) cleavage [63]. Epidemiological studies have shown that high consumption of cruciferous vegetables decreases the risk of prostate cancer. Androgen receptor is the central player in prostate cancer signalling. Heat shock protein 90 (HSP90) is a cellular chaperone that stabilizes androgen receptor [18]. HDAC6

Fig. 4. Glucoraphanin is converted into Sulforaphane (SF) by gut microflora. Sulforaphane is passively absorbed and is then conjugated with glutathione with the help of glutathione S-transferases (GSTs) to form (SF-GST). The defined conjugate is sequentially metabolized by a series of enzymes like g-glutamyltranspeptidase (GTP), cysteinyl-glycinease (GCase) and N-acetyltransferase (NAT).

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Table 1 Distinct molecular players modulated by sulforaphane in exerting cytotoxic effect against prostate cancer. Drug/combination

Cancer type

Gene/mRNA/Protein upregulated/activated

Gene/mRNA/Protein/downregulate/inhibited

Sulforaphane

Prostate cancer Sulforaphane Prostate cancer Sulforaphane Prostate cancer Sulforaphane + TRAIL Prostate cancer Sulforaphane + TRAIL Prostate cancer Sulforaphane Prostate cancer Sulforaphane Prostate cancer

Reference

HDAC6 caspases 3, 9 and 8, Bax P21, Bak, Bax, Bim, NOXA, caspase-3 and caspase-9, TRAIL-R1/DR4 and TRAIL-R2/DR5, FOXO3a

Nrf2, NQO-1, Cytochrome c, Fas, caspase-8

deacetylates HSP90 which augments its association with AR thereby stabilizing the latter. Sulphoraphane destabilizes AR by hyperacetylating HSP90 via restraining HDAC6 (Table 1). This enhances the proteasomal degradation of AR and thereby attenuates prostate cancer signalling [18]. An important factor in determining safety and clinical relevance is the ability of a drug to show cytotoxic effect against cancer cells without affecting normal ones. Therapeutic intervention using sulforaphane (15 mM) has been reported to show selective induction of cell cycle arrest and apoptosis in benign hyperplasia epithelial cells (BPH1), androgen dependent prostate cancer epithelial cells (LNCaP), androgen-independent prostate cancer epithelial cells (PC3) and neutrality towards normal (PrEC) prostate epithelial cells [67]. Sulforaphane treatment resulted in selective decrease in HDAC activity, class I and class II HDAC protein levels. The defined inhibitor enhanced the acetylation status of histone H3 at the p21 promotor resulting in p21 induction and increased tubulin acetylation in prostate cancer cells (Table 1). Further studies have revealed that HDAC6 overexpression rescues the cytotoxicity induced by sulforaphane. Sulforaphane based therapeutic intervention resulted only in transient reduction of HDAC activity without affecting the other end points tested, in normal PrEC [67]. Speaking in few words, sulforaphane treatment shows differential effects on cell proliferation, HDAC activity and downstream targets in normal and cancer cell models [67]. Sulforaphane has been found to enhance the antiproliferative and proapoptotic effects of TNF-related apoptosis-inducing ligand (TRAIL) in prostate cancer cell models. Sulforaphane was found to have cytotoxic effect both against androgen-independent PC-3 cells and androgen-dependent LNCap cells. Combined treatment of sulforaphane augmented the cytotoxic effect of TRAIL in PC-3 models and sensitized the TRAIL-resistant LNCaP cells to TRAIL [68]. Sulforaphane induced apoptosis was accompanied with increase in production of reactive oxygen species (ROS), decrease in mitochondrial membrane potential, inhibited expression of antiapoptotic Bcl-2, Mcl-1, and Bcl-XL, upregulated proapoptotic Bak, Bax, Bim, and NOXA in the predefined model. Increased activation of caspase-3 and caspase-9 has been reported upon the individual treatment of sulforaphane and TRAIL in PC-3 cells [68]. Pretreatment of this cell models with sulforaphane followed by TRAIL treatment showed marked increase in caspase-3 and caspase-9 activities. Further the combined treatment has been found to be more effective in dropping mitochondrial membrane potential compared to individual treatment involving either of the drugs. Increased expression of TRAIL-R1/DR4 and TRAIL-R2/DR5 receptors with no marked increase in expression of decoy The conjugates enter into systemic circulation via active transport where the merapturic acid (SF-NAC) and its conjugates are excretion products.

Gibbs et al. [18] Bcl-2 Singh et al. [63] HDAC2 and HDAC3, HDAC4, HDAC6 Clarke et al. [67] Bcl-2, Mcl-1, and Bcl-XL, VEGF, MT1-MMP, MMP-2, Shankar MMP-7, and MMP-9 et al. [68] NF-kB, Oct-3/4, HNF-3b, PDX-1, Otx2, TP63, GSC, Labsch et al. Snail, VEGF R2 and HCG [76] HDAC1, 4, 5 and 7, DNMT1 and DNMT3a Zhang et al. [79] GSH Singh et al. [81]

receptors DcR1 and DcR2 has been reported on therapeutic intervention with sulforaphane [68]. These results clearly suggest that up-modulation of death receptors DR4 and/or DR5 may be one of the mechanisms elevating the cytotoxic effect of TRAIL in PC-3 cells and sensitizing the LNCaP cells (TRAIL-resistant) to TRAIL. The combined therapeutic regimen proved to be more effective in inhibiting markers of angiogenesis and metastasis apart from activating FOXO3a transcription factor compared to either of the agent in singlet therapy (Table 1) [68]. NF-kB is constitutively expressed in prostate cancer cells and may facilitate cell growth and proliferation by regulating the expression profile of ample number of genes including c-myc, cyclin D1, IL-6, Bcl-2, and Bcl-XL. Moreover, NF-kB positively regulates the expression of genes having significant role in angiogenesis (IL-6, IL-8, and VEGF), invasion and metastasis (MMP-7 and MMP-9) [69]. Sulforaphane and TRAIL either alone or in combination inhibit the activation of NF-kB activity which results in suppression of its target gene products including VEGF, Bcl-2, Bcl-XL, cyclin D1, MT1-MMP, MMP2, MMP-9, COX-2, IL-6, and IL-8 [68,70]. As the combined therapeutic regimen is more effective in causing NF-kB inactivation that is why the defined strategy potentially inhibits the markers of angiogenesis and metastasis compared to monotherapy. The defined strategy involving sulforaphane and TRAIL showed superior growth inhibitory effect on orthotopically implanted prostate tumour in nude mice models compared to monotherapy involving either drug alone. Immunohistochemistry examination of tumour tissues has shown significant reduction in VEGF-positive cells upon the combined treatment compared to singlet therapy [68]. Enhanced expression of MMP is associated with elevated metastatic potential of tumour cells [71,72]. Treatment of tumour-bearing nude mice with either sulforaphane or TRAIL culminated in inhibition of MT1-MMP, MMP-2, MMP-7, and MMP-9 expression compared to control. Combined treatment of sulforaphane and TRAIL showed enhanced efficacy in inhibiting MT1-MMP, MMP-2, MMP-7, and MMP-9 expression compared to individual drug treatment (Table 1) [68]. These findings clearly suggest the beneficial effect of combined therapy in prostate cancer progression. Hormone deprivation is the most common treatment for prostate cancer, however during its progression the malignant cells lose their androgen receptors, subsequent hormone dependence culminating in therapeutic unresponsiveness and formation of androgen-independent prostate cancer (AIPC) which progresses and metastasizes, with no effective therapeutic strategy at present [73]. Cancer stem cells (CSC’s) mediate tumour formation, progression, metastasis without responding to chemo or radiotherapy [74]. Recent studies have shown that sulforaphane sensitizes prostate cancer cells to TRAIL- induced apoptosis. TRAIL

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shows selective toxicity towards malignant cells and is thus considered as a promising anticancer agent [75]. Studies with TRAIL on AIPC cell lines with CSC features (PC-3, DU145) and primary prostate CSC’s have shown minor efficacy of TRAIL in restraining clonogenicity, tumour engraftment and growth, spheroid formation apart from CSC signalling [76]. This minor efficacy of TRAIL has been attributed to induction of NF-kB activity. Therapeutic intervention with sulforaphane showed better efficacy than TRAIL in eliminating CSC features. The combined therapy involving TRAIL and sulforaphane completely restrained TRAILinduced NF-kB activity culminating in almost complete elimination of CSCs [76]. Therapeutic intervention with sulforaphane has been reported to show marked reduction in the amount of Nanog, Sox2, E-cadherin, GATA-4, HNF-3b, SOX17, Otx2, TP63, Snail, VEGF R2 and HCG. The doublet therapy involving sulforaphane and TRAIL in combination showed further reduction in the levels of Oct-3/4, HNF-3b, PDX-1, Otx2, TP63, GSC, Snail, VEGF R2 and HCG (Table 1) [76]. Thus it is quite evident that the doublet therapeutic regimen shows substantial reduction in the levels of proteins playing a critical role in self-renewal, differentiation, cell migration, EMT and tumourigenesis [68]. Mounting evidences suggest the involvement of epigenetic alteration during the development and progression of prostate cancer. Nrf2, a critical regulator of cellular antioxidant defense systems has been found to be silenced via epigenetic mechanism during tumourigenesis in vivo TRAMP (transgenic adenocarcinoma of the mouse prostate) mice [77,78] and in vitro TRAMP C1 cells [79,80]. Therapeutic intervention with sulforaphane enhanced the expressions of Nrf2 and its downstream target NQO-1 both at mRNA and protein level. Besides the protein levels of DNMT1 and DNMT3a showed marked reduction upon the pharmacological intervention of defined inhibitor [79]. Apart from this sulforaphane treatment culminated in attenuation of protein expression levels of certain HDACs like HDAC1, 4, 5 and 7 but enhanced the levels of acetylated histone H3 (Ac-H3), an active chromatin signature. Studies have revealed that sulforaphane regulates Nrf20 s CpGs demethylation and reactivation in TRAMP C1 cell models. In nutshell, sulforaphane shows therapeutic effect in part by epigenetic modulation of Nrf2 gene resulting in induction of its downstream anti-oxidative stress pathway (Table 1) [79]. Studies with human prostate cancer cell models (PC-3 and DU145) have shown that sulforaphane induced apoptotic signal arises from reactive oxygen species (ROS). Therapeutic intervention involving growth-suppressive concentrations of sulforaphane in PC-3 model has been found to induce ROS production accompanied by disruption of mitochondrial membrane potential, release of cytochrome c from mitochondria to cytoplasm and subsequent apoptosis [81]. The defined effects were markedly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. Besides, the ROS generation induced by sulforaphane substantially attenuated on previous treatment with electron transport chain complex I inhibitors including diphenyleneiodonium chloride and rotenone. Pharmacological intervention with sulforaphane has been found to cause a rapid and marked depletion in GSH levels (Table 1). Taken together, these observations suggest a probable non-mitochondrial component involving GSH depletion as well as mitochondrial component, responsible for sulforaphane induced ROS generation. SFN induced cell death has shown marked reduction upon ectopic expression of Bcl-xL in PC-3 cell model; however, similar expression of Bcl-2 does not reproduce the defined results [81]. Further, therapeutic intervention involving sulforaphane culminated in enhanced level of Fas, activation of caspase-8 apart from Bid cleavage. Speaking concisely, sulforaphane induced apoptosis is initiated by ROS generation and involves both intrinsic and extrinsic apoptotic caspase cascades [81].

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Phase II study of sulforaphane rich extracts was found to be safe with absence of Grade 3 adverse events. Twenty patients who had recurrent prostate cancer were treated with 200 mmoles/day of sulforaphane-rich extracts for a maximum duration of 20 weeks and the proportion of patients with 50% PSA declines (the primary end point) was determined [82]. Only one subject showed 50% PSA decline, seven patients experienced PSA decline less than 50%. A substantial lengthening of the on-treatment PSA doubling time (PSADT) has been reported in comparison to pretreatment PSADT (for pre-treatment 6.1 months versus 9.6 months on-treatment). The absence of grade 3 adverse events and the effects on PSADT modulation emphasizes the need of further higher dose studies to delineate the role of sulforaphane as a treatment agent [82]. A double-blinded, randomized, placebo-controlled multicenter trial of sulforaphane was performed in 78 patients (mean age 69
6 years) with increasing PSA levels after radical prostatectomy. The study involved stabilized free sulforaphane daily oral administration (60 mg) for 6 months (M0 to M6) followed by two months without treatment (M6 to M8). The sulforaphane group showed 86% longer PSA doubling time than the placebo. PSA increases >20% at M6 were markedly higher in the placebo group than in the sulforaphane group (71.8% and 44.4% respectively). Sulforaphane showed very good compliance and tolerance and the effects of defined inhibitor were eminent within three months of intervention [83]. 7. Conclusions Aberrant expression of HDACs alters acetylation homeostasis triggering various diseases including cancer. Tuning the dysregulated acetylation with HDACi is emerging as a promising strategy for treating cancer and neurodegeneration. Recent reports have shown that phytochemical namely sulforaphane has HDAC inhibitory activity which has been validated in diverse cancer models. Thus, this review focussed on the therapeutic effect of sulforaphane in case of prostate cancer. The defined inhibitor has shown highly promising activity in various prostate cancer models both in vitro and in vivo. Sulforaphane has been found to induce cytotoxic effect in prostate cancer models by modulating multiple pathways (pleiotropic effect). In certain cases sulforaphane restrained HDACs, while in other cases it enhanced ROS production, Nrf2 expression, inhibited DNMT1, DNMT3a, and elevated caspase activation, for bringing therapeutic effect. Sulforaphane induced cytotoxic effect has been found to be selective as no alterations were seen in normal prostate epithelial cell model thereby determining its safety and clinical relevance. Although, therapeutic intervention involving sulforaphane as a single agent (singlet therapy) showed cytotoxic effect against prostate cancer models, the doublet therapeutic regimen (combined therapy) showed maximum efficacy. Sulforaphane in combination with TRAIL has shown enhanced cytotoxic effect even in therapeutically challenging (TRAIL resistant) prostate cancer models suggesting the ability of sulforaphane to break TRAIL resistance. Recently approved HDAC inhibitor panobinostat has shown maximum efficacy when used in triplet combination against multiple myeloma. However, triplet combination studies of sulforaphane which may prove even more fruitful in prostate cancer chemotherapy are still unmet emphasizing the desperate need of further research in the defined arena. The absence of grade 3 adverse events apart from the modulation of PSADT during phase II clinical trials warrants the need of further higher dose studies to elucidate the role of sulforaphane as a prevention agent. Thus, it tempts me to speculate that sulforaphane is the highly promising molecule with vibrant activity against prostate cancer and should

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be used in combinatorial therapy for achieving maximum therapeutic benefit. Conflict of interest The authors report no conflict of interest. Acknowledgement Financial support from the DST-SERB (File No. YSS/2015/ 001267), Government of India is gratefully acknowledged. References [1] A. Abbas, S. Gupta, The role of histone deacetylases in prostate cancer, Epigenetics 3 (2008) 300–309. [2] S. Minucci, P.G. Pelicci, Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer, Nat. Rev. Cancer 6 (2006) 38–51. [3] D. Takai, P.A. Jones, Comprehensive analysis of CpG islands in human chromosomes 21 and 22, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 3740–3745. [4] X.J. Yang, E. Seto, HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention, Oncogene 26 (2007) 5310–5318. [5] S.A. Ganai, Panobinostat: the small molecule metalloenzyme inhibitor with marvelous anticancer activity, Curr. Top. Med. Chem. 16 (2016) 427–434. [6] N. Korolev, A. Allahverdi, A.P. Lyubartsev, L. Nordenskiold, The polyelectrolyte properties of chromatin, Soft Matter 8 (2012) 9322–9333. [7] S. Ganai, Strategy for enhancing the therapeutic efficacy of histone deacetylase inhibitor dacinostat: the novel paradigm to tackle monotonous cancer chemoresistance, Arch. Pharm. Res. (2015) 1–11. [8] B.N. Singh, G. Zhang, Y.L. Hwa, J. Li, S.C. Dowdy, S.-W. Jiang, Nonhistone protein acetylation as cancer therapy targets, Expert Rev. Anticancer Ther. 10 (2010) 935–954. [9] S.A. Ganai, Histone deacetylase inhibitor pracinostat in doublet therapy: a unique strategy to improve therapeutic efficacy and to tackle herculean cancer chemoresistance, Pharm. Biol. (2016) 1–10. [10] C.Y. Gui, L. Ngo, W.S. Xu, V.M. Richon, P.A. Marks, Histone deacetylase (HDAC) inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 1241–1246. [11] G. Lagger, A. Doetzlhofer, B. Schuettengruber, E. Haidweger, E. Simboeck, J. Tischler, et al., The tumor suppressor p53 and histone deacetylase 1 are antagonistic regulators of the cyclin-dependent kinase inhibitor p21/WAF1/ CIP1 gene, Mol. Cell. Biol. 23 (2003) 2669–2679. [12] A.J. Wilson, D.S. Byun, N. Popova, L.B. Murray, K. L'Italien, Y. Sowa, et al., Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer, J. Biol. Chem. 281 (2006) 13548–13558. [13] D. Waltregny, W. Glenisson, S.L. Tran, B.J. North, E. Verdin, A. Colige, et al., Histone deacetylase HDAC8 associates with smooth muscle alpha-actin and is essential for smooth muscle cell contractility, FASEB J. 19 (2005) 966–968. [14] A. Vannini, C. Volpari, G. Filocamo, E.C. Casavola, M. Brunetti, D. Renzoni, et al., Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 15064–15069. [15] C. Hubbert, A. Guardiola, R. Shao, Y. Kawaguchi, A. Ito, A. Nixon, et al., HDAC6 is a microtubule-associated deacetylase, Nature 417 (2002) 455–458. [16] A. Valenzuela-Fernandez, J.R. Cabrero, J.M. Serrador, F. Sanchez-Madrid, HDAC6: a key regulator of cytoskeleton, cell migration and cell–cell interactions, Trends Cell Biol. 18 (2008) 291–297. [17] P. Matthias, M. Yoshida, S. Khochbin, HDAC6 a new cellular stress surveillance factor, Cell Cycle 7 (2008) 7–10. [18] A. Gibbs, J. Schwartzman, V. Deng, J. Alumkal, Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 16663–16668. [19] J.H. Park, S.H. Kim, M.C. Choi, J. Lee, D.Y. Oh, S.A. Im, et al., Class II histone deacetylases play pivotal roles in heat shock protein 90-mediated proteasomal degradation of vascular endothelial growth factor receptors, Biochem. Biophys. Res. Commun. 368 (2008) 318–322. [20] S.A. Ganai, M. Ramadoss, V. Mahadevan, Histone Deacetylase (HDAC) Inhibitors  emerging roles in neuronal memory, learning, synaptic plasticity and neural regeneration, Curr. Neuropharmacol. 14 (2016) 55–71. [21] O. Bruserud, C. Stapnes, E. Ersvaer, B.T. Gjertsen, A. Ryningen, Histone deacetylase inhibitors in cancer treatment: a review of the clinical toxicity and the modulation of gene expression in cancer cell, Curr. Pharm. Biotechnol. 8 (2007) 388–400. [22] S.A. Ganai, Novel approaches towards designing of isoform-selective inhibitors against class II histone deacetylases: the acute requirement for targetted anticancer therapy, Curr. Top. Med. Chem. (2016). [23] M. Pal-Bhadra, M.J. Ramaiah, T.L. Reddy, A. Krishnan, S.N. Pushpavalli, K.S. Babu, et al., Plant HDAC inhibitor chrysin arrest cell growth and induce p21WAF1 by altering chromatin of STAT response element in A375 cells, BMC Cancer 12 (2012) 180.

[24] I.H. Son, I.M. Chung, S.I. Lee, H.D. Yang, H.I. Moon, Pomiferin, histone deacetylase inhibitor isolated from the fruits of Maclura pomifera, Bioorg. Med. Chem. Lett. 17 (2007) 4753–4755. [25] M.C. Myzak, P.A. Karplus, F.L. Chung, R.H. Dashwood, A novel mechanism of chemoprotection by sulforaphane: inhibition of histone deacetylase, Cancer Res. 64 (2004) 5767–5774. [26] T. Forster, V. Rausch, Y. Zhang, O. Isayev, K. Heilmann, F. Schoensiegel, et al., Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication, Oncotarget 5 (2014) 1621–1634. [27] H. Zeng, O.N. Trujillo, M.P. Moyer, J.H. Botnen, Prolonged sulforaphane treatment activates survival signaling in nontumorigenic NCM460 colon cells but apoptotic signaling in tumorigenic HCT116 colon cells, Nutr. Cancer 63 (2011) 248–255. [28] Y. Li, T. Zhang, H. Korkaya, S. Liu, H.F. Lee, B. Newman, et al., Sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells, Clin. Cancer Res. 16 (2010) 2580–2590. [29] A. Tarozzi, C. Angeloni, M. Malaguti, F. Morroni, S. Hrelia, P. Hrelia, Sulforaphane as a potential protective phytochemical against neurodegenerative diseases, Oxid. Med. Cell. Longev. 2013 (2013) 10. [30] Y. Li, G.E. Karagoz, Y.H. Seo, T. Zhang, Y. Jiang, Y. Yu, et al., Sulforaphane inhibits pancreatic cancer through disrupting Hsp90-p50(Cdc37) complex and direct interactions with amino acids residues of Hsp90, J. Nutr. Biochem. 23 (2012) 1617–1626. [31] G. Kallifatidis, S. Labsch, V. Rausch, J. Mattern, J. Gladkich, G. Moldenhauer, et al., Sulforaphane increases drug-mediated cytotoxicity toward cancer stemlike cells of pancreas and prostate, Mol. Ther. 19 (2011) 188–195. [32] O. Witt, H.E. Deubzer, T. Milde, I. Oehme, HDAC family: what are the cancer relevant targets, Cancer Lett. 277 (2009) 8–21. [33] W. Fischle, V. Kiermer, F. Dequiedt, E. Verdin, The emerging role of class II histone deacetylases, Biochem. Cell Biol. 79 (2001) 337–348. [34] M. Mottamal, S. Zheng, T.L. Huang, G. Wang, Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents, Molecules 20 (2015) 3898–3941. [35] S. Ropero, M. Esteller, The role of histone deacetylases (HDACs) in human cancer, Mol. Oncol. 1 (2007) 19–25. [36] J.E. Bolden, M.J. Peart, R.W. Johnstone, Anticancer activities of histone deacetylase inhibitors, Nat. Rev. Drug Discov. 5 (2006) 769–784. [37] K. Ververis, A. Hiong, T.C. Karagiannis, P.V. Licciardi, Histone deacetylase inhibitors (HDACIs): multitargeted anticancer agents, Biologics 7 (2013) 47– 60. [38] S.A. Ganai, HDAC inhibitors entinostat and suberoylanilide hydroxamic acid (SAHA): the ray of hope for cancer therapy, Mol. Life Sci. (2015) 1–16. [39] A. Mai, S. Massa, I. Cerbara, S. Valente, R. Ragno, P. Bottoni, et al., 3-(4-Aroyl-1methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides as a new class of synthetic histone deacetylase inhibitors 2. Effect of pyrrole-C2 and/or -C4 substitutions on biological activity, J. Med. Chem. 47 (2004) 1098–1099. [40] A.V. Bieliauskas, M.K.H. Pflum, Isoform-selective histone deacetylase inhibitors, Chem. Soc. Rev. 37 (2008) 1402–1413. [41] N. Khan, M. Jeffers, S. Kumar, C. Hackett, F. Boldog, N. Khramtsov, et al., Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors, Biochem. J. 409 (2008) 581–589. [42] S.A. Ganai, K. Shanmugam, V. Mahadevan, Energy-optimised pharmacophore approach to identify potential hotspots during inhibition of Class II HDAC isoforms, J. Biomol. Struct. Dyn. 33 (2015) 374–387. [43] S. Ganai, In silico approaches towards safe targeting of class I histone deacetylases. I, Mol. Life Sci. (2015) 1–9. [44] M. Kijima, M. Yoshida, K. Sugita, S. Horinouchi, T. Beppu, Trapoxin, an antitumor cyclic tetrapeptide, is an irreversible inhibitor of mammalian histone deacetylase, J. Biol. Chem. 268 (1993) 22429–22435. [45] M.P. Bhuiyan, T. Kato, T. Okauchi, N. Nishino, S. Maeda, T.G. Nishino, et al., Chlamydocin analogs bearing carbonyl group as possible ligand toward zinc atom in histone deacetylases, Bioorg. Med. Chem. 14 (2006) 3438–3446. [46] K. Halkidou, L. Gaughan, S. Cook, H.Y. Leung, D.E. Neal, C.N. Robson, Upregulation and nuclear recruitment of HDAC1 in hormone refractory prostate cancer, Prostate 59 (2004) 177–189. [47] M. Nakagawa, Y. Oda, T. Eguchi, S. Aishima, T. Yao, F. Hosoi, et al., Expression profile of class I histone deacetylases in human cancer tissues, Oncol. Rep. 18 (2007) 769–774. [48] K. Halkidou, S. Cook, H.Y. Leung, D.E. Neal, C.N. Robson, Nuclear accumulation of histone deacetylase 4 (HDAC4) coincides with the loss of androgen sensitivity in hormone refractory cancer of the prostate, Eur. Urol. 45 (2004) 382–389. [49] R.E. Bakin, M.O. Jung, Cytoplasmic sequestration of HDAC7 from mitochondrial and nuclear compartments upon initiation of apoptosis, J. Biol. Chem. 279 (2004) 51218–51225. [50] K. Kojima, R. Ohhashi, Y. Fujita, N. Hamada, Y. Akao, Y. Nozawa, et al., A role for SIRT1 in cell growth and chemoresistance in prostate cancer PC3 and DU145 cells, Biochem. Biophys. Res. Commun. 373 (2008) 423–428. [51] Y. Dai, D. Ngo, L.W. Forman, D.C. Qin, J. Jacob, D.V. Faller, Sirtuin 1 is required for antagonist-induced transcriptional repression of androgen-responsive genes by the androgen receptor, Mol. Endocrinol. 21 (2007) 1807–1821. [52] E.H. Jeffery, A.F. Brown, A.C. Kurilich, A.S. Keck, N. Matusheski, B.P. Klein, et al., Variation in content of bioactive components in broccoli, J. Food Compost. Anal. 16 (2003) 323–330.

S.A. Ganai / Biomedicine & Pharmacotherapy 81 (2016) 250–257 [53] P. Perocco, G. Bronzetti, D. Canistro, L. Valgimigli, A. Sapone, A. Affatato, et al., Glucoraphanin, the bioprecursor of the widely extolled chemopreventive agent sulforaphane found in broccoli, induces phase-I xenobiotic metabolizing enzymes and increases free radical generation in rat liver, Mutation Res 595 (2006) 125–136. [54] K. Nakagawa, T. Umeda, O. Higuchi, T. Tsuzuki, T. Suzuki, T. Miyazawa, Evaporative light-scattering analysis of sulforaphane in broccoli samples: quality of broccoli products regarding sulforaphane contents, J. Agric. Food Chem. 54 (2006) 2479–2483. [55] O.P. Thangstad, P. Winge, H. Husebye, A. Bones, The myrosinase (thioglucoside glucohydrolase) gene family in Brassicaceae, Plant Mol. Biol. 23 (1993) 511– 524. [56] N.V. Matusheski, R. Swarup, J.A. Juvik, R. Mithen, M. Bennett, E.H. Jeffery, Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. italica) inhibits formation of the anticancer agent sulforaphane, J. Agric. Food Chem. 54 (2006) 2069–2076. [57] N.V. Matusheski, J.A. Juvik, E.H. Jeffery, Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli, Phytochemistry 65 (2004) 1273–1281. [58] S. Saha, W. Hollands, B. Teucher, P.W. Needs, A. Narbad, C.A. Ortori, et al., Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli, Mol. Nutr. Food Res. 56 (2012) 1906–1916. [59] N. Petri, C. Tannergren, B. Holst, F.A. Mellon, Y. Bao, G.W. Plumb, et al., Absorption/metabolism of sulforaphane and quercetin, and regulation of phase II enzymes, in human jejunum in vivo, Drug Metab. Dispos. 31 (2003) 805–813. [60] C.C. Conaway, S.M. Getahun, L.L. Liebes, D.J. Pusateri, D.K. Topham, M. BoteroOmary, et al., Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli, Nutr. Cancer 38 (2000) 168–178. [61] A. Jazwa, A.I. Rojo, N.G. Innamorato, M. Hesse, J. Fernández-Ruiz, A. Cuadrado, Pharmacological targeting of the transcription factor Nrf2 at the basal ganglia provides disease modifying therapy for experimental parkinsonism, Antioxid. Redox Signal. 14 (2011) 2347–2360. [62] L. Ye, A.T. Dinkova-Kostova, K.L. Wade, Y. Zhang, T.A. Shapiro, P. Talalay, Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans, Clin. Chim. Acta 316 (2002) 43–53. [63] A.V. Singh, D. Xiao, K.L. Lew, R. Dhir, S.V. Singh, Sulforaphane induces caspasemediated apoptosis in cultured PC-3 human prostate cancer cells and retards growth of PC-3 xenografts in vivo, Carcinogenesis 25 (2004) 83–90. [64] M. Vermeulen, I.W. Klopping-Ketelaars, R. van den Berg, W.H. Vaes, Bioavailability and kinetics of sulforaphane in humans after consumption of cooked versus raw broccoli, J. Agric. Food Chem. 56 (2008) 10505–10509. [65] P.A. Egner, J.G. Chen, J.B. Wang, Y. Wu, Y. Sun, J.H. Lu, et al., Bioavailability of Sulforaphane from two broccoli sprout beverages: results of a short-term, cross-over clinical trial in Qidong, China, Cancer Prev. Res. (Phila) 4 (2011) 384– 395.

257

[66] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer Statistics, 2014, 64, CA, 2014, pp. 9–29. [67] J.D. Clarke, A. Hsu, Z. Yu, R.H. Dashwood, E. Ho, Differential effects of sulforaphane on histone deacetylases, cell cycle arrest and apoptosis in normal prostate cells versus hyperplastic and cancerous prostate cells, Mol. Nutr. Food Res. 55 (2011) 999–1009. [68] S. Shankar, S. Ganapathy, R.K. Srivastava, Sulforaphane enhances the therapeutic potential of TRAIL in prostate cancer orthotopic model through regulation of apoptosis, metastasis, and angiogenesis, Clin. Cancer Res. 14 (2008) 6855–6866. [69] M. Karin, Nuclear factor-kappaB in cancer development and progression, Nature 441 (2006) 431–436. [70] M. Karin, NF-kappaB and cancer: mechanisms and targets, Mol. Carcinog. 45 (2006) 355–361. [71] P. Mehlen, A. Puisieux, Metastasis: a question of life or death, Nat. Rev. Cancer 6 (2006) 449–458. [72] E.I. Deryugina, J.P. Quigley, Matrix metalloproteinases and tumor metastasis, Cancer Metastasis Rev. 25 (2006) 9–34. [73] B.J. Feldman, D. Feldman, The development of androgen-independent prostate cancer, Nat. Rev. Cancer 1 (2001) 34–45. [74] A. Abbott, Cancer: the root of the problem, Nature 442 (2006) 742–743. [75] D.W. Stuckey, K. Shah, TRAIL on trial: preclinical advances in cancer therapy, Trends Mol. Med. 19 (2013) 685–694. [76] S. Labsch, L.I. Liu, N. Bauer, Y. Zhang, E.W.A. Aleksandrowicz, J. Gladkich, et al., Sulforaphane and TRAIL induce a synergistic elimination of advanced prostate cancer stem-like cells, Int. J. Oncol. 44 (2014) 1470–1480. [77] A. Barve, T.O. Khor, S. Nair, K. Reuhl, N. Suh, B. Reddy, et al., Gamma-tocopherolenriched mixed tocopherol diet inhibits prostate carcinogenesis in TRAMP mice, Int. J. Cancer 124 (2009) 1693–1699. [78] A. Barve, T.O. Khor, K. Reuhl, B. Reddy, H. Newmark, A.N. Kong, Mixed tocotrienols inhibit prostate carcinogenesis in TRAMP mice, Nutr. Cancer 62 (2010) 789–794. [79] C. Zhang, Z.-Y. Su, T.O. Khor, L. Shu, A.-N.T. Kong, Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation, Biochem. Pharmacol. 85 (2013) 1398–1404. [80] A.A. Hurwitz, B.A. Foster, J.P. Allison, N.M. Greenberg, E.D. Kwon, The TRAMP mouse as a model for prostate cancer, Curr. Protoc. Immunol. 20 (2001) Unit 20.5. [81] S.V. Singh, S.K. Srivastava, S. Choi, K.L. Lew, J. Antosiewicz, D. Xiao, et al., Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species, J. Biol. Chem. 280 (2005) 19911–19924. [82] J.J. Alumkal, R. Slottke, J. Schwartzman, G. Cherala, M. Munar, J.N. Graff, et al., A phase II study of sulforaphane-rich broccoli sprout extracts in men with recurrent prostate cancer, Invest. New Drugs 33 (2015) 480–489. [83] B.G. Cipolla, E. Mandron, J.M. Lefort, Y. Coadou, E. Della Negra, L. Corbel, et al., Effect of sulforaphane in men with biochemical recurrence after radical prostatectomy, Cancer Prev. Res. 8 (2015) 712–719.