Discovery and development of sulforaphane as a cancer chemopreventive phytochemical¹

Introduction

Phytochemicals in broad terms are a synonym of plant chemicals (phyto is Greek for plant); however, in common usage the term is more limited in scope and usually refers to plant chemicals that are bioactive and are not part of the traditional nutrients, such as vitamins and minerals. Although these compounds are generally viewed as non-essential for normal body functioning, an increasing number of them have been shown to possess disease-fighting activities, including anticarcinogenic and anticancer activities^[1–4]. In this review, we attempt to provide an overview of the discovery and development of sulforaphane (SF) as a cancer chemopre-ventive phytochemical. SF was isolated from broccoli in the early 1990s in our laboratory as an inducer of phase 2 enzymes and has since been extensively studied by numerous investigators and shows a highly promising cancer-fighting ability^[5–10].

Isolation and identification of SF

Prochaska and coworkers in the late 1980s developed a cell-culture system (known as the Prochaska assay) for the detection of inducers of phase 2 enzymes based on the induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) in murine hepatoma Hepa 1c1c7 cells grown in microtiter plates^[11,12]. These researchers then used the assay to screen organic solvent extracts of a broad collection of fruit and vegetables for activities involved in the induction of phase 2 enzymes. This was an important effort because it was recognized that the induction of phase 2 enzymes, such as NQO1 and glutathione S-transferase (GST), is an important strategy for achieving protection against carcinogenesis^[13,14] and that consumption of fruit and vegetables reduces cancer risk^[15]. They found that many extracts exhibited significant inducer activities, but the broccoli extract was one of the richest sources of inducer activity^[16]. In an attempt to identify the inducer(s) in broccoli, we subjected the extracts to multiple runs of fractionation by high performance liquid chromatography and examined each fraction for inducer activity using the Prochaska assay. We succeeded in isolating a liquid substance that was responsible for more than 80% of the total inducer activity in broccoli extracts. This substance was soon identified as SF (1-isothiocyanato-4-(methylsulfinyl)-butane) (Table 1). Approximately 9 mg of SF was isolated from the extracts representing 640 g fresh broccoli florets; however, it became clear later that the actual amount of SF in the extracts was approximately 10-fold more^[17]. Laboratory synthesis of SF provided a sufficient quantity for evaluation in animals, and SF was shown to significantly induce both NQO1 and GST in multiple organs of mice after oral dosing^[18]. Interestingly, a literature search revealed that SF had already been isolated more than 30 years earlier from hoary cress (a cruciferous weed) for its antimicrobial activity^[19,20], and a recent article reports that SF occurs in a wide variety of plants^[21]. We also became aware at this time that SF was one of a large number of naturally occurring isothiocyanates (ITC)^[22], and several ITC had previously been shown to inhibit carcinogenesis in animal experiments^[23].

Table 1 SF analogs: relation of structure to inducer activity
Full table

SF analogs: relationship between structure and inducer activity

In an attempt to ascertain the structural features of SF and in the hope of generating a more potent inducer of phase 2 enzymes, more than 40 analogs of SF were synthesized and evaluated using the Prochaska assay (see Table 1 for representative analogs)^[18,24]. SF isolated from broccoli is chiral, possessing the R configuration, but both R-SF and the synthetic (R,S)-SF show identical inducer potency. Change of the oxidation state of the sulfur atom in the methylthiol group from sulfoxide to sulfone reduced inducer activity 4-fold, and the sulfide analog was more than 10-fold less active. Moreover, if the sulfoxide group was replaced with the methylene group, the inducer activity was reduced 75-fold. However, the sulfoxide group could be replaced with a carbonyl group without losing any inducer activity. A change in the number of methylene units from 4 to 5 or 3 did not significantly affect inducer activity (results not shown), nor did the rigidity of the methylene bridge have much effect on inducer activity, as shown by the finding that the norbonyl ITC were almost equally active (Table 1). Although these findings shed new light on the importance of SF structure, we were unable to generate a more potent inducer of phase 2 enzymes than SF. Other investigators reported that converting the –N=C=S of SF to various dithiocarbamate structures (-NH-CS-SR, R representing various alkyl groups) did not generate a more potent inducer either^[25,26].

Identification of edible plants or plant extracts as carriers of SF

Subsequent studies in our laboratory showed that SF was derived largely, if not entirely, from glucoraphanin, a glucosinolate (β-thioglucoside N-hydroxysulfate) (Figure 1), and that the conversion occurred during the preparation of broccoli extracts^[17]. This is not unexpected, however, because ITC are known to be synthesized and stored as gluco-sinolates in plants and are released when damage to plant tissues occurs. The conversion is catalyzed by myrosinase (thioglucoside glucohydrolase), an enzyme that coexists with, but is physically separated from, glucosinolates in normal plants^[22]. Glucosinolates, including glucoraphanin, which escape the plant myrosinase, can be partially (up to 45%) hydrolyzed in the intestinal tract because the enteric microflora are known to possess myrosinase activity^[27–30]. How-ever, the following observations indicate that it is extremely difficult to estimate human exposure to dietary SF: (1) our studies revealed that SF yield in different samples of broccoli (both frozen and fresh) sold in supermarkets might differ by as much as 9-fold, and this difference is unrelated to their physical appearance or whether grown under conventional or organic conditions^[17], thus, making it impossible to know how much glucoraphanin is present in a particular vegetable without actually measuring it; (2) different cooking conditions are likely to exacerbate variations in SF yield, as steaming or boiling vegetables will reduce the conversion of glucoraphanin to SF by inactivating myrosinase and destroying SF (SF is heat-labile)^[31]; (3) certain plants including broccoli possess epithiospecifier protein (ESP), which binds to and converts the intermediate of glucoraphanin hydrolysis (a thiohydroximate-O-sulfonate) to a nitrile at the expense of SF (Figure 1), but mild heating of broccoli (60–70 °C) inactivated ESP and preserved myrosinase and increased SF yield 3–7-fold^[32]; and (4) although glucoraphanin not hydrolyzed by vegetable myrosinase could be converted to SF in vivo by the enzyme in the enteric microflora, the growth condition of the microflora significantly affects the hydrolysis^[28] and glucosinolate hydrolysis in humans appears to differ by as much as 44-fold^[33].

Figure 1 Enzymatic hydrolysis of glucoraphanin. ESP, epithio-specifier protein.

Interestingly, our research also suggests that all gluco-sinolates in mature broccoli might already have been synthesized in the seeds. Thus, there was approximately 15-fold more glucoraphanin in 3-d-old broccoli sprouts (cv Saga) than in the florets of mature cultiva^[17]. In addition to gluco-raphanin, broccoli sprouts also contain two minor gluco-sinolates that give rise to two ITC (erucin and iberin) that closely resemble SF in both chemical structure and bioactivity^[18,34]. Although Faulkner and coworkers report that glucoraphanin content in mature broccoli could be increased 10-fold by crossing broccoli cultivars with selected wild taxa of the Brassica oleracea^[35], exploitation of broccoli sprouts may offer an advantage. Investigations have revealed that although indole glucosinolates (4-hydroxyglucobrassin, glucobrassicin and neoglucobrassicin) comprised 68% of the total in mature broccoli (cv Saga), this proportion fell to 3% in the sprouts^[17]. Similar results were obtained in sprouts grown from other varieties of broccoli seeds^[36]. Hydrolysis of indole glucosinolates by myrosinase yields highly unstable ITC that spontaneously decompose to compounds such as indole-3-carbinol, indole-3-acetonitrile and 3,3’-diindolylmethane, which may have undesired bioactivi-ties^[37,38].

We further demonstrated that aqueous extracts of broccoli sprouts were an excellent vehicle for delivering the chemopreventive activity of SF. Feeding rats with broccoli sprout extracts in which the glucosinolates either remained intact or were fully converted to ITC resulted in marked inhibition of mammary tumor development in 7,12-dimethylbenz(a)anthracene-treated female Sprague–Dawley rats^[17], and the chemoprevention efficacy of the extracts was comparable to that of pure SF at similar dose levels^[39]. The anticar-cinogenic activity of glucosinolate-containing extracts is likely to result from the conversion of the glucosinolates to ITC in vivo, as studies have shown that intact glucoraphanin does not possess significant chemopreventive activity^[17,34], and blocking the conversion of glucosinolates to ITC in broccoli sprout extracts abolishes the chemopreventive activity of the extracts^[34]. The chemopreventive activity of broccoli sprout extracts has also been demonstrated in other studies^[40,41].

Chemopreventive mechanism of SF: more than the induction of phase 2 enzymes

Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2 target genes Although SF was isolated from broccoli on the basis of NQO1 induction in cultured Hepa 1c1c7 cells (the Prochaska assay), subsequent studies have revealed that it was capable of inducing a large number of phase 2 genes, including epoxide hydrolase^[42,43], ferri-tin^[43], glutamate cysteine synthetase^[42–44], glutathione peroxidase^[43,45], glutathione reductase^[43,45], GST^[18,42], heme oxygenase-1^[43,45,46], thioredoxin and thioredoxin reductase^[43,47,48] and UDP-glucuronosyltransferase 1A^[49,50] in cultured cells or rodent tissues in vivo. Thus, SF may significantly streng-then cytoprotection because these genes are involved in various aspects of cellular defense against carcinogens and other toxicities. Extensive mechanistic studies have shown that the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2-anti-oxidant response element (ARE) signaling pathway is primarily responsible for the coordinate response of these genes to SF. Studies show that the phase 2 genes carry in their 5’-flanking region one or more cis-acting DNA regulatory elements, known as ARE, and activation of ARE leads to coordinate induction of these genes^[51]. Nrf2 is the key ARE activator, which is normally bound by its repressor Keap1 in the cytoplasm and targeted for proteosomal degradation, but dissociates from the latter in response to an inducer or other signals. Free Nrf2 translocates to the nucleus, complexes with other nuclear factors (eg small Maf) and binds to ARE to activate the transcription of the downstream gene^[52]. SF was shown to activate Nrf2 by directly reacting with the sulfhydryl groups of critical cysteine residues of Keap1^[53], although a recent study found that modifying specific cysteines of Keap1 might be insufficient for Nrf2 activation^[54] and other studies implicated the mitogen-activated protein kinase pathway in Nrf2 activation by SF^[55,56]. Nrf2 knockout rendered phase 2 genes largely unresponsive to SF^[42,43] and two mouse studies have shown that Nrf2 knockout not only increased the susceptibility of the animals to chemical carcinogenesis but also abolished the ability of SF to inhibit carcinogenesis^[57,58]. Interestingly, gene-array studies revealed that SF also upregulated a large number of non-phase-2 genes, and the response of some of these genes to SF also depended on the Keap1-Nrf2-ARE pathway^[42,43,59].

Modulation of cytochrome P-450 enzymes In addition to inducing phase 2 enzymes, several studies have also shown that SF modulates certain cytochrome P-450 (CYP) enzymes (phase 1 enzymes). CYP enzymes are important for normal metabolic processing of numerous endogenous and exogenous compounds, but may also activate certain carcinogens. For example, CYP2E1 causes the activation of carcinogens such as N-nitrosodimethylamine^[60,61] and CYP1A2 activates 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) ^[62]. SF was shown to inhibit the catalytic activity of a number of CYP enzymes, including CYP1A1,1A2, 2B1/2, 2E1 and 3A4^{[60,61,63–66]}, and to downregulate CYP3A4 in hepatocytes^[64]. However, feeding rats with SF elevated CYP1A2 expression^[65] and feeding rats with glucoraphanin (the SF precursor) elevated CYP1A1, 1A2, 2B1/2, 2C11 and 3A1 in the lungs^[67]. Hence, it remains unclear if CYP enzymes are relevant targets in SF chemo-prevention.

Induction of apoptosis and inhibition of proliferation Induction of apoptosis and inhibition of proliferation are important mechanisms for the inhibition of carcinogenesis and cancer growth. In addition to acting as an inducer of phase 2 genes, numerous studies have also documented the ability of SF to induce apoptosis and cell cycle arrest in cancer cell lines derived from bladder^[68,69], blood^[70,71], brain^[72], breast^[73], colon^[74,75], ovary^[76], pancreas^[77], prost-ate^[78,79] and skin^[80], indicating that this activity is not cell specific. SF also inhibited the growth of human cancer xenografts in mice in vivo and tumor tissues of SF-treated mice showed increased apoptosis^[77,79]. SF has been shown to activate several programmed cell death mechanisms, including mitochondria-mediated apoptosis^[74,81], death-receptor-mediated apoptosis^[82–84] and autophagic cell death^[85], and to arrest cells in G1 phase^[69,78,86], S phase^[68] and/or G2/M phase^{[68,77,87,88]}, depending on the cell line under study. Moreover, these and other studies have shown that these actions of SF are associated with the modulation of many regulators of cell death and cell cycle, including activation of mitogen-activated protein kinases, modulation of Bcl-2 family proteins, damage of mitochondria and release of apoptogenic factors from mitochondria, activation of caspases, modulation of cyclins and cdks, downregulation of Cdc25C, upregulation of p21, inhibition of histone deacetylase and tubulin polymerization^{[10,68,79,81,88–93]}. The anticancer activity of SF does not depend on p53 because SF induced apoptosis in wild-type p53-, mutated p53- and p53 knockout fibroblasts^[94], induced autophagy in both human prostate cancer PC-3 cells (p53-deficient) and LNCaP cells (p53-normal) ^[85], and induced G1 arrest in human colon cancer HT-29 cells in a p53-independent manner^[95]. SF may also potentiate other anticancer agents because it has been shown to enhance the efficacy of doxorubicin and reverse doxorubicin-resistant phenotype in mouse fibroblasts with p53 mutation^[96,97].

Inhibition of angiogenesis and metastasis More recent studies demonstrate that SF is also capable of inhibiting angiogenesis and metastasis. Using immortalized human microvascular endothelial HMEC-1 cells, SF was shown to potently reduce in vitro formation of microcapillaries, suppress capillary-like tube formation on basement membrane matrix and inhibit cell migration^[98]. These effects were not due to inhibition of cell proliferation, but were associated with transcriptional downregulation of factors important for tumor angiogenesis and metastasis, including vascular endothelial growth factor (VEGF) and its receptor KDR/flk-1, hypoxia-inducible factor-1α (Hif-1α), c-Myc and matrix metalloproteinase (MMP)-2. SF also inhibited the proliferation and tubular formation on matrigel of human umbilical vein endothelial cells in vitro^[99], and was responsible for inhibition of MMP-9 activity and invasiveness of human breast cancer MDA-MB-231 cells by broccoli extracts^[100]. Both MMP-2 and MMP-9 play an important role in cancer cell invasion^[101]. Inhibition of angiogenesis and metastasis by SF was also demonstrated in vivo. Intravenous administration of non-toxic doses of SF inhibited endothelial cell response to VEGF in a subcutaneous VEGF-impregnated matrigel plug mouse model^[102]. Moreover, although intravenous injection of B16F-10 melanoma cells into C57BL/6 mice led to formation of lung tumor nodules, SF administered intraperitoneally at very low dose (0.5 mg/kg body weight) markedly inhibited lung tumor nodule formation^[103]. The potent inhibitory effect of SF observed in this model did not appear to result from a cytotoxic effect of SF on B16F-10 cells, but was associated with inhibition of MMP activation.

Other mechanisms of SF that may also contribute to its anticarcinogenic and anticancer activity SF treatment significantly enhanced natural killer (NK) cell activity and antibody-dependent cellular cytotoxicity in both normal and Ehrlich ascites tumor-bearing mice, which was accompanied by increased proliferation of bone marrow cells, splenocytes and thymocytes, as well as increased production of inter-leukin-2 and interferon-γ^[104]. Treatment of Raw 264.7 murine macrophages with SF resulted in the inhibition of lipopoly-saccharide (LPS)-induced secretion of pro-inflammatory and procarcinogenic signaling molecules, including nitric oxide, prostaglandin E₂ and tumor necrosis factor-α, and nuclear factor-kappa B (NF-κB) was shown to be the molecular target of SF^[105]. SF also inhibited diesel-extract-induced production of pro-inflammation cytokins in primary human bronchial epithelial cells^[106]. Further studies in human prostate cancer PC-3 cells showed that suppression of NF-κB and NF-κB-regulated gene expression involved inhibition of IkB kinases (IKK) and IκBα as well as inhibition of nuclear translocation of p65^[107]. O⁶-methylguanine-DNA methyltrans-ferase (MGMT) is a DNA repair protein that protects the genome against the mutagenic action of alkylating carcino-gens, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-one (NNK) and nitrosamines. Treatment of human medulloblastoma UW228 cells and human colon carcinoma HT29 cells with SF significantly increased MGMT activity^[108]. Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine biosynthesis, and increased expression of ODC is linked to tumor promotion. SF was shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC activity in mouse epidermal ME308 cells^[109]. SF was also shown to significantly induce the expression of multidrug-resistance-associated protein 2 (MRP2) in primary hepatocytes and Caco-2 cells^[75,110], although it did not impact on MRP1 and P-glycoprotein^[111]. Increased expression of MRP2 may increase cellular protection against toxic chemicals. More-over, the antimicrobial activity of SF has long been recognized^[20] and SF was recently shown to be effective against Helicobacter pylori, a significant risk factor for gastric cancer^[57,112].

Preclinical and clinical evaluation of the in vivo efficacy of SF

Preclinical A variety of rodent models have been used to assess the in vivo efficacy of SF, some of which have been mentioned above. SF was shown to significantly inhibit tumor development induced by a number of chemical carcinogens in several rodent organ sites, including colon^[113], lungs^[114], mammary^[39], pancreas^[115], skin^[58,116] and stomach^[57], and SF was effective when it was given either in the carcinogen initiation phase or in the promotion phase. SF is believed to be responsible for the inhibition of ultraviolet (UV)-induced skin carcinogenesis by broccoli sprout extracts, which were applied topically after the completion of UV treatment (during the post-initiation phase) ^[117]. Some of these studies also highlight the importance of phase 2 induction in enabling the chemopreventive activity of SF, especially in the initiation phase, as induction of phase 2 genes in target tissues has been detected in SF-treated animals^[26,117], and knockout of Nrf2 abolished the induction of phase 2 genes by SF and abrogated the chemopreventive activity of SF^[57,58]. However, other studies show the importance of other chemopreventive mechanisms of SF. For example, SF administered to A/J mice after the completion of a combined treatment of benzo(a)pyrene and NNK inhibited malignant progression of adenoma to adenocarcinoma in the lungs with a corresponding increase in apoptotic cells and a decrease in proliferating cell nuclear antigen expression^[114]. SF supplemented in the diet significantly inhibited the formation of intestinal polyps in ApcMin/+ mice^[118]. Tumors in these mice occur spontaneously because of a mutation of the tumor suppressor adenomatous polyposis coli (APC) gene^[119]. Analysis of polyp tissues from SF-treated mice did not indicate induction of phase 2 genes, but instead showed increased apoptosis and decreased proliferation. Further study of the polyp tissues using a microarray technique showed that SF treatment caused upregulation of multiple pro-apoptotic genes and downregulation of multiple pro-survival genes^[120]. The anticancer activity of SF was further demonstrated in experiments where SF dosed either orally or intraperitoneally significantly inhibited the growth of subcutaneous xenografts of human prostate cancer PC-3 cells and human pancreatic cancer PANC-1 cells in mice^[77,79], and inhibited lung tumor formation from intravenous injection of B16F-10 melanoma cells in mice^[103].

Clinical To the best of our knowledge, SF in pure form has not yet been investigated in humans. However, the discovery of broccoli sprouts as an exceptionally rich source of SF has provided an alternative to examine its potential impact in humans. A placebo-controlled, double-blind, randomized phase 1 study of broccoli sprout extracts, containing either glucosinolate (mainly glucoraphanin) or ITC (mainly SF), showed that the extracts were well tolerated and caused no significant adverse effects when the extracts were administered orally at 8-h intervals for 7 d at doses of 25 and 100 µmol glucosinolate or 25 µmol ITC^[121]. In another randomized and placebo-controlled study involving 200 healthy adults, nightly consumption of hot water infusions of broccoli sprouts containing 400 µmol glucoraphanin (656 µmol total glucosinolate) for 2 weeks was also well tolerated and showed no adverse effects^[33]. Topical application of SF as high as 340 nmol in the form of broccoli sprout extracts to the center of a 1-cm-diameter circle of skin in humans caused no adverse reactions, but NQO1 activity in the skin tissues was elevated 1.5-fold and 4.5-fold after application of 150 nmol SF once or three times, respectively, (at 24 h-intervals) ^[40].

Metabolism and disposition of SF

Animal and human studies Many lines of evidence indicate that SF is rapidly metabolized through the mercapturic acid pathway: initial conjugation with glutathione (GSH) promoted by GST gives rise to the corresponding conjugate, which undergoes sequential enzymatic modifications to form cysteinylglycine, cysteine and N-acetylcysteine (NAC) conjugates, which are disposed in urine (Figure 2). Approximately 72% of a single oral dose of SF was recovered in the urine as NAC conjugates in rats in 24 h^[122], but only about 1% of the dose was detected in the second 24-h urine sample^[41], indicating that urinary elimination occurs almost entirely within 24 h after SF dosing. Similar changes were seen in humans because 58.3±2.8% and 77.9±6.4% of a single dose of approximately 200 µmol ITC (mainly SF) contained in broccoli sprout extracts was recovered in the urine as SF equivalents in 8 h and 72 h, respectively, although the levels of free SF and individual metabolite were not determined^[123]. These results also show that the bioavailability of SF is extremely high and inter-individual variation of SF absorption and metabolism is small. Moreover, the urinary SF elimination pattern was not significantly altered even after repeated SF dosing (oral broccoli sprout extracts containing 25 µmol ITC at 8-h intervals for 7 d) ^[121]. The rapid urinary elimination of SF is closely correlated with its rapid absorption^[124] and short plasma half-life because plasma concentrations of SF equivalents peaked (0.94–2.27 µmol/L) 1 h after feeding the extracts in the afore-described human experiment (single dose of approximately 200 µmol ITC) and declined with first-order kinetics (half-life 1.77±0.13 h). Similar results were seen in other studies in which human subjects were given a single dose of mature broccoli soup^[125,126]. These studies also revealed that free SF and its cysteine conjugate were more abundant than the other conjugates in the plasma and that significant quantities of free SF and cysteine conjugate were present in the urine in addition to the NAC conjugate. It is important to note that the thiol conjugates of SF as well as those of other ITC serve as carriers of ITC^[127], and SF-NAC has been shown to exhibit equally if not more potent chemopreventive activities in comparison with SF^{[78,114,128,129]}.

Figure 2 Metabolism of SF through the mercapturic acid pathway. The chemical structure of SF is abbreviated as R–N=C=S, where R represents CH₃-SO-(CH₂)₄. SF first reacts with glutathione to give rise to a glutathione–SF conjugate, which is promoted by glutathione S-transferase (GST). The conjugate undergoes further enzymatic modification as shown, first by γ-glutamyltranspeptidase (γ-GT) to form the cysteinylglycine–SF conjugate, then by cysteinylglycinase (CG) to form the cysteine–SF conjugate and finally by N-acetyltransferase (AT) to form the N-acetylcysteine–SF conjugate.

Cell culture studies Studies in cultured cells in our laboratory have provided an explanation for the rapid absorption and elimination of SF observed in vivo. We have shown that SF as well as other ITC are rapidly accumulated in cells, but that the accumulated ITC equivalents are rapidly exported^[130–134] (Figure 3). ITC appear to penetrate cells by diffusion, but the ITC upon entering the cells is rapidly conjugated with intracellular thiols. GSH, which is the most abundant intracellular thiol, is the major driving force for ITC accumulation, and cellular GST enhances ITC accumulation by promoting the conjugation reaction. Not surprisingly, ITC that are already conjugated with thiols are unable to accumulate in cells^[131]. It has been shown that peak intracellular ITC accumulation is achieved within 0.5–3 h after exposure, reaching 100–200-fold over the extracellular ITC concentration, and the peak intracellular ITC accumulation levels can reach the millimolar concentration range. However, intracellularly accumulated GSH conjugates of ITC, perhaps other conjugates as well, were also exported rapidly, and this appears to be mediated at least, in part, by membrane transporter MRP1^[133,134]. For example, the half-life of the accumulated SF equivalent in human prostate cancer LNCaP cells was only about 1 h. Thus, continuous intracellular accumulation seems to be possible only if there is a continuous presence of ITC in the extracellular space to allow continuous cellular uptake of ITC to offset the rapid export of accumulated ITC conjugates.

Figure 3 Cellular accumulation and export of SF and its chemopreventive mechanism. The chemical structure of SF is abbreviated as R–N=C=S, where R represents CH₃-SO-(CH₂)₄. Cdc25C, cell division cycle 25C; HDAC, histone deacetylase; Keap1, Kelch-like ECH-associated protein 1; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MGMT, O⁶-methylguanine-DNA methyltransferase; MRP2, multidrug-resistance-associated protein 2; NF-κB, nuclear factor-kappa B; Nrf2, nuclear factor erythroid 2-related factor 2; ODC, ornithine decarboxylase; VEGF, vascular endothelial growth factor; X-SH, X stands for the side chain of a sulfhydryl molecule.

Conclusion and future perspectives

Since SF was reported to be the principle inducer of phase 2 enzymes in broccoli in 1992, extensive studies of this compound have followed, which reveal that SF is a highly promising agent for cancer prevention and perhaps also useful in cancer therapy. A summary of its cellular uptake and molecular mechanisms is provided in Figure 3. Given the widespread interest in SF, our understanding about its mechanism as well as its bioactivity will undoubtedly become more sophisticated. Broccoli sprout extracts are an excellent vehicle for SF delivery and have allowed for human evaluation of SF in the absence of the approved use of pure SF. In fact, we are aware that more human trials with this substance are either ongoing or are to be initiated in the near future. These studies will not only address the utility of broccoli sprout extracts for cancer prevention/treatment in humans, but will also provide critical information as to whether the investigation of pure SF in humans is warranted. In addition, it is important to note that a few studies have shown the ability of SF to enhance the efficacy of another anticancer agent. More investigations of this ability of SF should be emphasized to determine whether SF can be used in combination therapy.

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