The Plant Derived 3-3′-Diindolylmethane (DIM) Behaves as CB2 Receptor Agonist in Prostate Cancer Cellular Models

3-3′-Diindolylmethane (DIM) is a biologically active dimer derived from the endogenous conversion of indole-3-carbinol (I3C), a naturally occurring glucosinolate found in many cruciferous vegetables (i.e., Brassicaceae). DIM was the first pure androgen receptor antagonist isolated from the Brassicaceae family and has been recently investigated for its potential pharmacological use in prostate cancer prevention and treatment. Interestingly, there is evidence that DIM can also interact with cannabinoid receptors. In this context, by considering the well-known involvement of the endocannabinoid system in prostate cancer, we have pharmacologically characterized the properties of DIM on both CB1 and CB2 cannabinoid receptors in two human prostate cancer cell lines: PC3 (androgen-independent/androgen receptor negative) and LNCaP (androgen-dependent). In the PC3 cell line, DIM was able to activate CB2 receptors and potentially associated apoptotic pathways. On the other hand, although DIM was also able to activate CB2 receptors in the LNCaP cell line, no apoptotic effects were observed. Our evidence confirms that DIM is a CB2 receptor ligand and, moreover, it has a potential anti-proliferative effect on androgen-independent/androgen receptor-negative prostate cancer cells.


Introduction
3-3 -Diindolylmethane (DIM) is a biologically active dimer derived from the conversion of indole-3-carbinol (I3C). I3C is produced endogenously from naturally occurring glucosinolates (GLs) contained in a wide variety of plant food substances, including members of the family Cruciferae, and particularly members of genus Brassica [1][2][3]. The amount of GLs in Brassicaceae vegetables is 206-3895 mg/kg [4]. Whether the plant tissues are crushed or cooked, an endogenous thioglucosidase (myrosinase) is activated and converts glucosinolates to indoles, principally to I3C [5]. When I3C is orally ingested, due to its chemical instability in acidic conditions, such as in the stomach environment, the compound is promptly condensed into DIM, which is the bioactive product [6], as demonstrated by pharmacokinetics studies conducted in animals [7,8] and human models [9,10]. DIM has been shown to possess hepatoprotective, antioxidant and anticancer properties [11][12][13]. In particular, the anticancer effect of DIM has been positively associated with a reduction of cancer incidence when a regular consumption of cruciferous vegetables is part of the dietary regime [14]. Specifically, DIM can induce apoptosis and can reduce proliferation and metastasis of tumor cells, as well as the inflammation process. Moreover, DIM appears to be a promising agent for the prevention of the recurrence of hormone-dependent cancers, such as prostate cancer [6]. In this context, hormone-dependent prostate cancer represents the sixth leading cause of cancer deaths in males, as well as the second most common 2 of 14 form [15]. Indeed, most prostate cancer cases are dependent on androgen at initial stages, and the inhibition of the function of androgen receptors (AR) (i.e., by using anti-androgen drugs), is currently a coadjutant therapeutical approach, along with surgery or radiation, in the treatment of this type of cancer [16]. Moreover, DIM is the first androgen receptor antagonist derived from plants with a binding affinity similar to antiandrogens, such as cyproterone acetate and bicalutamide. In addition, it can downregulate AR signaling to prevent the translocation of these receptors from the cytoplasm to the nucleus [17].
Interestingly, DIM has also demonstrated an ability to antagonize the aryl hydrocarbon receptor [18], as well as modulate cytochrome P450 1A1 activity [19]. Further evidence has also confirmed the protective role of DIM in androgen-independent prostate cancer. For example, microarray analysis conducted on PC3 cells (androgen-independent/androgen receptor negative) showed that many genes involved in the control of carcinogenesis and cell survival are modulated in their expression by indoles, including DIM [20]. Similarly, clinical trial results have demonstrated the therapeutic efficacy of DIM in the treatment of patients with high-grade prostatic intraepithelial neoplasia [21]. DIM has been demonstrated to interact with the endocannabinoid system, by stimulating both CB 1 and CB 2 cannabinoid receptors. Specifically, in an experimental model using hCB 1 /CB 2 -CHO transfected cells, DIM was able to interact as a slight inverse agonist on CB 1 receptors and as a partial agonist on CB 2 receptors [22]. Therefore, the endocannabinoid system has been proposed as a possible target for the treatment of prostate cancer, and extensive studies have demonstrated the involvement of the system in the pathology. In fact, results from preclinical studies have highlighted altered levels of endocannabinoids in patients' plasma and identified a correlation between high CB 1 /CB 2 receptor expression and a poor prognosis [23,24]. In human prostate cancer cell lines (e.g., LNCaP and PC3), higher levels of CB 1 and CB 2 receptor expressions are detected in comparison with non-cancerous prostate cells [25], plus elevated levels for the enzymes responsible for endocannabinoid degradation, suggesting a potential role of the endocannabinoid system in prostate cell proliferation [26]. In fact, a number of synthetic and endogenous agonist ligands for CB 1 and CB 2 receptors, along with inhibitors of endocannabinoid enzyme degradation, have anticancer effects [27]. In PC3 cells and in primary cultures from benign prostate hyperplasia or prostate cancer, the endocannabinoid system is responsible for decreasing cell viability, mostly through the activation of CB 1 receptors [28]. Considering the role played by the endocannabinoid system in prostate cancer, and the potential anticancer effect of DIM, the aim of this study was to characterize the ability of DIM to interact with both CB 1 and CB 2 receptors and to evaluate any effect on apoptotic processes in cellular models of prostate cancer, such as PC3 (androgen-independent human prostate cancer cells) and LNCaP (androgen-dependent human prostate cancer cell line) that naturally express the endocannabinoid system [29,30].

DIM Is a CB 2 Receptor Agonist in Both PC3 and LNCaP Cells
When tested in PC3 cell membranes, DIM was able to stimulate [ 35 S]GTPγS binding (EC 50 : 9.28 nM) showing full agonist behavior ( Figure 1a) with a potency similar to that of the full CB 1 /CB 2 receptor agonist CP 55,940 (EC 50 : 6.14 nM) (Figure 1d). To determine whether the elicited effect of DIM was due to the activation of CB 1 or CB 2 receptors, we tested whether the effect of DIM could be antagonized with well-known CB 1 and CB 2 receptor antagonists/inverse agonists SR141716A (SR1) and SR144528 (SR2) respectively. SR1 (10 nM) produced no significant dextral shift in the log concentration-response curve ( Figure 1b). Conversely, SR2 (10 nM) did produce a significant dextral shift in the log concentration-response curve of DIM ( Figure 1c). As a positive control, the effect of CP 55,940 was assayed in the same system producing a marked dextral shift in the log concentration-response dose observed in the presence of SR2 (10 nM) in addition to a slight, but significant, dextral shift in the log concentration-response in the presence of SR1 (10 nM) (Figure 1e,f, respectively). 55,940 was assayed in the same system producing a marked dextral shift in the log concentration-response dose observed in the presence of SR2 (10 nM) in addition to a slight, but significant, dextral shift in the log concentration-response in the presence of SR1 (10 nM) (Figure 1e,f, respectively).   Figure 2c, in the presence of the CB 2 receptor antagonist SR2 (10 nM), a significant dextral shift in the log concentration-response curve of DIM was observed, whilst no significant effect was observed in presence of the CB 1 receptor antagonist SR1 (Figure 2b). Since there was no evidence of a significant interaction of DIM with CB 1 receptors, further experiments with intact cells focused on its actions through CB 2 receptors.

DIM Induces an Antiproliferative Effect through CB 2 Receptor Stimulation Only in PC3 Cells
When tested in the cell viability assay, DIM was able to induce an antiproliferative effect in PC3 cells (IC 50 : 38.93 µM) (Figure 3a). This apoptotic effect was antagonized by the CB 2 receptor antagonist SR2, with a significant dextral shift in the log concentrationresponse observed. Interestingly, while DIM also induced an antiproliferative effect in LNCaP cells, no antagonist effect with SR2 was observed (Figure 3b), indicating this was not via CB 2 receptors, so these were not considered further. To better understand the CB 2 -mediated effects, further experiments were conducted on PC3 cells.
In PC3 cells, DIM was able to inhibit FSK-induced cAMP production with a potency in the nanomolar range (IC 50 : 13.49 nM) (Figure 4b), showing a similar effect to the wellknown CB 1 /CB 2 receptor agonist CP 55,940 (IC 50 : 4.85 nM) (Figure 4a). The inhibitory effect of both DIM and CP 55, 940 on FSK-induced cAMP production, was tested in presence of the CB 2 receptor antagonist SR2. Again, as for the viability assay, when tested at the dose of 10 nM, SR2 did not induce any significant effect on FSK-induced cAMP production inhibition elicited in the presence of both CP 55,940 and DIM. However, previous studies suggest a dextral shift requires a much higher dose of antagonists in an intact cell assay compared to isolated cell membranes [31][32][33]. At the dose of 100 nM, SR2 was indeed able to significantly inhibit both CP 55,940 and DIM effects on FSK-induced cAMP production ( Figure 4a,b). In contrast, the CB 1 receptor antagonist SR1 had no effect on DIM's inhibition of FSK-induced cAMP production.

DIM Induces an Antiproliferative Effect through CB2 Receptor Stimulation Only in PC3 Cells
When tested in the cell viability assay, DIM was able to induce an antiproliferative effect in PC3 cells (IC50: 38.93 μM) ( Figure 3a). This apoptotic effect was antagonized by the CB2 receptor antagonist SR2, with a significant dextral shift in the log concentrationresponse observed. Interestingly, while DIM also induced an antiproliferative effect in LNCaP cells, no antagonist effect with SR2 was observed (Figure 3b), indicating this was not via CB2 receptors, so these were not considered further. To better understand the CB2mediated effects, further experiments were conducted on PC3 cells.

DIM Induces Changes in pAKT (Reduction) and Cleaved-CASP-3 (Increase) Levels through CB 2 Receptor Stimulation in PC3 Cell Line
Since DIM had an antiproliferative effect on PC3 cells, we tested the compound for its ability to modulate the expression of the phosphorylated form of AKT (pAKT) at doses of 50 nM and 50 µM. There was no significant reduction at 50 nM but at the higher dose of 50 µM, a strong and significant reduction of pAKT level was observed ( Given this evidence for the regulation of pAKT levels in PC3 cells, the same experimental protocol described above was used to test the ability of DIM to modulate the activity of CASP-3. Both doses of DIM, 50 nM and 50 µM, induced a significant increase of cleaved CASP-3 level in PC3 cells ( Figure 6, lanes 2 and 3) and this increase was antagonized by SR2 in a dose-related manner ( Figure 6, lanes 4-7). In PC3 cells, DIM was able to inhibit FSK-induced cAMP production with a potency in the nanomolar range (IC50: 13.49 nM) (Figure 4b), showing a similar effect to the wellknown CB1/CB2 receptor agonist CP 55,940 (IC50: 4.85 nM) (Figure 4a). The inhibitory effect of both DIM and CP 55, 940 on FSK-induced cAMP production, was tested in presence of the CB2 receptor antagonist SR2. Again, as for the viability assay, when tested at the dose of 10 nM, SR2 did not induce any significant effect on FSK-induced cAMP production inhibition elicited in the presence of both CP 55,940 and DIM. However, previous studies suggest a dextral shift requires a much higher dose of antagonists in an intact cell assay compared to isolated cell membranes [31][32][33]. At the dose of 100 nM, SR2 was indeed able to significantly inhibit both CP 55,940 and DIM effects on FSK-induced cAMP production (Figure 4a,b). In contrast, the CB1 receptor antagonist SR1 had no effect on DIM's inhibition of FSK-induced cAMP production.

DIM Induces Changes in pAKT (Reduction) and Cleaved-CASP-3 (Increase) Levels through CB2 Receptor Stimulation in PC3 Cell Line
Since DIM had an antiproliferative effect on PC3 cells, we tested the compound for its ability to modulate the expression of the phosphorylated form of AKT (pAKT) at doses of 50 nM and 50 μM. There was no significant reduction at 50 nM but at the higher dose of 50 μM, a strong and significant reduction of pAKT level was observed ( Figure 5  Given this evidence for the regulation of pAKT levels in PC3 cells, the same experimental protocol described above was used to test the ability of DIM to modulate the activity of CASP-3. Both doses of DIM, 50 nM and 50 μM, induced a significant increase of cleaved CASP-3 level in PC3 cells ( Figure 6, lanes 2 and 3) and this increase was antagonized by SR2 in a dose-related manner ( Figure 6, lanes 4-7).

Discussion
The experimental results obtained in our study have clearly demonstrated that the pharmacological effect of DIM was mediated by CB2 receptors as shown by [ 35 S]GTPγS assay in PC3 and, to a lesser extent, in LNCap cells.
Given the robust effect in PC3 cells, the CB2 receptor-mediated effect of DIM was

Discussion
The experimental results obtained in our study have clearly demonstrated that the pharmacological effect of DIM was mediated by CB 2 receptors as shown by [ 35 S]GTPγS assay in PC3 and, to a lesser extent, in LNCap cells.
Given the robust effect in PC3 cells, the CB 2 receptor-mediated effect of DIM was further investigated in this cell line. In the cAMP assay experiments, DIM inhibited FSKinduced cAMP production, inhibited pAKT and enhanced cleavage of CASP-3, all of which were robustly antagonized by the CB 2 receptor antagonist.
To our knowledge, there are very few previous studies that describe the ability of DIM to interact with CB 2 cannabinoid receptors, and these used stable CB 2 receptortransfected cellular models. Thus, this may be the first study to describe the effects in cells endogenously expressing this receptor and the associated signaling pathways. Specifically, a study conducted in 2009 by Yin et al. [22] showed that DIM was only able to partially activate CB 2 receptors when tested in a β-arrestin assay. However, our data in cancer cells in both the [ 35 S]GTPγS and cAMP assays, clearly indicate DIM actually displays full agonist activity, similar to the well-known full CB 1 /CB 2 receptor agonist CP 55,940.
The difference in CB 2 receptor properties between the two studies is likely due to the different cellular models (recombinant vs. naturally expressing cell lines) used, although it may also reflect the different assays used to characterize the pharmacological response of the CB 2 receptor to DIM. Whatever the reason, our findings suggest that for the pharmacological characterization of potential CB 2 receptor ligands, systems naturally expressing the receptor represent a more reliable model than recombinant ones when compared to in vivo models [32]. As mentioned above, when tested in both cell lines, DIM behaved as a full CB 2 receptor agonist and, at least in PC3 cells, the compound was also able to induce apoptosis through a significant reduction of the pAKT level. The latter is important since pAKT is a key kinase in multiple cellular pathways involved in oncogenic and proliferative functions that can regulate androgen receptor levels in prostate cancer cells [34].
The main factors influencing the growth and progression of prostate cancer are the androgen receptor and the PI3K/AKT pathway. Our observations, therefore, confirm previous studies showing that the activation of the CB 2 receptor was responsible for AKT protein kinase dephosphorylation in addition to the inhibition of the phosphatidylinositol-3-kinase (PI3K)-AKT pathway [35,36], but importantly, it shows that DIM induces the same effects. The glycogen synthase kinase 3 (GSK-3) and other targets are regulated by AKT phosphorylation increased by the phosphatase and tensin homolog (PTEN) tumor suppressor gene inactivation [37]. PTEN has been reported to be lost or mutated in most advanced prostate cancer cases [38] and there are numerous inhibitors in development that target various PI3K cascade nodes, including PI3K, AKT, mTOR [34]. The effect of DIM on PTEN/PI3K-AKT signaling has been reported in other cancer models [39] and our results suggest it invokes the same inhibitory activity in PC3 cells through CB 2 receptors. On the other hand, PI3K or AKT inhibition might not be enough to significantly reduce tumor size in prostate cancer, and repression of the AR axis is also necessary for maximum effectiveness [37]. In fact, several investigations showed that PI3K and AR signaling interact [40,41]. This may explain the lack of efficacy in LNCaP cell lines, which are thought to be androgen-dependent, requiring androgens for growth. In contrast, PC3 cell lines are thought to be androgen-independent, not requiring or affecting androgen for growth [42]. It is likely that LNCaP cells with a functioning androgen pathway prevent DIM from activating CB2. In LNCaP cells, the DIM-induced inhibition of cell viability was not antagonized by the CB 2 receptors antagonist, suggesting the possible involvement of other non-cannabinoid receptors in the mechanism. In fact, in LNCaP cells, DIM can inhibit cancer cell proliferation through different and overlapping mechanisms [43,44] and some cannabinoid ligands have been reported to induce apoptosis through a combination of cannabinoid receptor-independent cellular and molecular mechanisms [45]. Our results, obtained by stimulating LNCaP with DIM, show only a weak effect on cell proliferation which may be ascribed to a non-specific mechanism or to a lower expression of CB 2 receptors in this cell line [45].
In PC3 cells, activation of the CB 2 receptor can elicit an antiproliferative effect by promoting an increase in ceramide levels [46]. Ceramide generation occurs from sphingomyelin hydrolysis, with cannabinoids having a stimulatory effect in the process [47]. Indeed, increasing ceramide levels can inhibit the AKT-mammalian target of the rapamycin (mTOR) pathway and activate the initiation factors involved in autophagy regulation and endoplasmic reticulum (ER) stress response [27]. Also, increasing levels of ceramide due to ER stress enhancement can trigger the activation of the caspase cascade, leading to apoptosis as well [27]. As cysteine proteases, caspases are formed constitutively in the cells and are normally present as inactive proenzymes [48]. In this context, our results have shown that treating PC3 cells with DIM caused a significantly increased level of the active pro-apoptotic caspase-3 (CASP-3) kinase, confirming the ability of the compound to induce apoptosis in this human prostate cancer cell line [48].
The CB 2 receptor-mediated AKT dephosphorylation in PC3 cells after DIM administration may suggest a different signaling pathway is involved, one not involving the activation of PI3K, a kinase normally activated by G βγ subunits upon CB 2 receptor stimulation [49]. Indeed, the PI3K pathway is directly responsible for AKT activation and provides an intrinsic bypass mechanism able to enhance both the survival and proliferation of prostate cancer cells lacking AR signaling, making these cells resistant to androgen therapy [50]. Thus, the inhibitory role of CB 2 receptors on AKT, possibly through a mechanism independent of PI3K, may represent promising and beneficial effects of DIM that are worth further investigation, considering the lack of demonstrable efficacy in clinical studies where direct PI3K inhibitor drugs were tested. [50].
Our data suggest that PC3 cells exhibit a constitutively active tone by the endocannabinoids on CB 2 receptors which modulates the AKT pathway. This is demonstrated as the AKT phosphorylation increased significantly above the basal levels when the effect of DIM was antagonized with the highest dose of CB 2 receptor antagonist [28].
As mentioned above, in human prostate cancer cells, antiproliferative and apoptotic effects can also be mediated by ceramide accumulation and these compounds have been suggested as possible mediators of cannabinoid action, and more specifically, of the endogenous cannabinoid anandamide [27]. Consistent with this, altered levels of intracellular ceramide have been found in both LNCaP and PC3 cells [51].

Culturing and Treatment of Cells
Human prostate cancer cell lines, LNCaP and PC3, were obtained from the European Collection of Animal Cell Cultures. These were cultured in RPMI 1640 medium (Merck Life Science Ltd, Gillingham, UK) containing 10% (vol:vol) fetal bovine serum and 1% (vol:vol) Penicillin-Streptomycin Solution (10,000 U/mL penicillin and 10 mg/mL streptomycin in 0.9% sodium chloride (Merck Life Science Ltd., Gillingham, UK).

Membrane Preparation
Binding assays with [ 35 S]GTPγS were performed with PC3 or LNCaP cell membranes [53,54]. Both PC3 and LNCaP cells were removed from flasks by scraping and then frozen as a pellet at −20 • C until required [55]. Before use in a radioligand-binding assay, cells were defrosted, diluted in Tris buffer (50 mM Tris-HCl, 50 mM Tris-Base) and homogenized. Protein assays were performed using a Bio-Rad Dc kit (Bio-Rad©, Watford, UK).

[ 35 S]GTPγS-Binding Assays
The measurement of ligand-stimulated [ 35 S]GTPγS binding to cannabinoid CB 1 or CB 2 receptors was adapted from methods described previously [55,56]. The assays were carried out with GTPγS-binding buffer (50 mM Tris-HCl, 50 mM Tris-Base, 5 mM MgCl 2 , 1 mM ethylenediaminetetraacetic acid, 100 mM NaCl, 1 mM dithiothreitol and 0.1% bovine serum albumin) in the presence of [ 35 S]GTPγS and GDP, in a final volume of 500 µL. Binding was initiated by the addition of [ 35 S]GTPγS to the wells. Non-specific binding was measured in the presence of 30 µM GTPγS. The drugs were incubated in the assay for 60 min at 30 • C. The reaction was terminated by a rapid vacuum filtration method using Tris-binding buffer, as described previously [32], and the radioactivity was quantified by liquid scintillation spectrometry. In all the [ 35 S]GTPγS-binding assays, we used 0.1 nM [ 35 S]GTPγS, 30 µM GDP and a protein concentration of 40 µg per well for PC3 and LNCaP cell membranes. Compounds were stored at −20 • C in DMSO.

Cyclic AMP Assay
The assays were performed using the HitHunter ® cAMP assay kit (Eurofins DiscoverX Products, Celle-L'Evescault, France) according to the vendor's protocol and previous studies [32,57]. Briefly, cells were detached using cell dissociation buffer, counted and seeded at 2 × 10 4 cells per well in 100 mL of complete medium onto white 96-well plates and incubated at 37 • C and 5% CO 2 for approximately 24 h before running the experiment. The assays and the drug dilutions were performed in a 1:1 mixture of DMEM and Ham's F12 medium without phenol red, containing 10 mM of rolipram and forskolin (FSK; 7b-acetoxy-8,13-epoxy-1a,6b,9 atrihydroxylabd-14-en-11-one). Before running the assay, the medium was discarded, and cells were washed with D-MEM/F-12 medium. Then, cells were treated with the assigned drugs (30 µL per well) and incubated for 30 min at 37 • C and 5% CO 2 . Finally, cAMP standards and the appropriate mixture of kit components were added (as described by the manufacturer, Eurofins DiscoverX Products, Celle-L'Evescault, France). Plates were incubated overnight at room temperature in the dark. Chemiluminescent signals were detected on a Synergy HT Multi-Mode Microplate Reader (ThermoFisher Scientific, Loughborough, UK).

Cell Viability Assay
A standard 3-(4,-5-dimethylthiazol-2-yl)-2,-5-diphenyl tetrazolium bromide (MTT) dye reduction assay was used to assess compounds. Briefly, cells were plated in a flatbottomed 96-well plate at seeding densities of 6 × 10 3 cells per well for LNCaP cells and 5 × 10 3 cells per well for PC3 cells. Cells were treated the following day with the appropriate agents for 24 h. Following the incubation period, the MTT solution (5 mg/mL in phosphate-buffered saline) was added and incubated for 4 h. The contents of the wells were removed and replaced with 200 µL DMSO to dissolve the MTT formazan crystals. The plates were immediately read at 570 nm in a multi-well plate reader (DynaTech MR5000; Dynex Technologies Ltd., Chantilly, VA, USA).

Data Analysis
Values for half of the maximal effective concentration required to elicit a response (EC 50 ), or for the half maximal inhibitory concentration (IC 50 ) required to inhibit a response, as well as for maximal effect (E max ), standard error of the mean (S.E.M.) and 95% confidence limits of EC 50 and IC 50 , have been calculated by nonlinear regression analysis using the equation for a sigmoid concentration-response curve (GraphPad Prism v5.0, San Diego, CA, USA). Paired Student's t-test or one-way ANOVA analysis of variance (with Tukey's post hoc analysis) was used where appropriate for cell viability and western blotting data. A value of p < 0.05 was taken as being significant.

Conclusions
Bringing together our results, we can conclude that DIM is a CB 2 receptor ligand with a potential anti-prostate cancer effect, and differing from classical cannabinoids, is without any psychotropic activity. In particular, DIM can induce a significant anti-apoptotic activity in the androgen-independent human prostate cancer cell line PC3, a cellular model that mimics prostatic small cell neuroendocrine carcinoma (SCNC), an extremely aggressive form of prostate cancer resistant to standard hormonal therapy [58,59]. According to reports, unlike LNCaP cells, PC3 cells do not express AR and their proliferation is androgen-independent, much like SCNC [58]. A more consistent body of evidence is required to confirm our findings and better understand the biological activity of DIM. The promising results obtained in this study represent a step forward in the understanding of the mechanism(s) underlying the DIM anticancer effect and encourage further exploration into the beneficial effect of this compound for the treatment of those forms of prostate cancer insensitive to the androgen treatment.
Based on our results, we have estimated that the concentrations at which DIM is showing an effect on cell viability could be obtained in vivo with an equivalent daily intake of about 60 mg of the compound. However, such daily intakes cannot be achieved by the ingestion of Brassica vegetables alone, therefore, administration in form of supplements may be required [60].