Effect of glutathione-S-transferase polymorphisms on the cancer preventive potential of isothiocyanates: an epidemiological perspective

Isothiocyanates (ITCs) are widely distributed in cruciferous vegetables and are biologically active against chemical carcinogenesis due to their ability to induce phase II conjugating enzymes. Among these is the glutathione-S-transferase (GST) family of enzymes, which in turn catalyzes the metabolism of ITCs, for which it has high substrate specificity. A recent body of epidemiologic data on the inverse association between cruciferous vegetable/ITC intake and cancers of the colo-rectum, lung and breast, also support that this protective effect is greater among individuals who possess the GSTM1 or T1 null genotype, and who would be expected to accumulate higher levels of ITC at the target tissue level, a pre-requisite for their enzyme-inducing effects. The association between ITC and cancer, and its modification by GST status, is most consistent for lung cancer and appears to be strongest among current smokers. Within limits, a comparison between groups which have been stratified by GST genotype may be less susceptible to confounding by other variables, given the random assortment of genes in gametogenesis. While a more complete understanding of the overall effects on health will need to take into account other components such as indoles and anti-oxidants, the interaction between ITC intake and GST genotype may provide a firmer basis to support a biologically significant role for ITC in cruciferous vegetables.     

Low concentrations of isothiocyanates protect mesenchymal stem cells from oxidative injuries, while high concentrations exacerbate DNA damage

Isothiocyanates (ITCs) are molecules naturally present in many cruciferous vegetables (broccoli, black radish, daikon radish, and cauliflowers). Several studies suggest that cruciferous vegetable consumption may reduce cancer risk and slow the aging process. To investigate the effect of ITCs on cellular DNA damage, we evaluated the effects of two different ITCs [sulforaphane (SFN) and raphasatin (RPS)] on the biology of human mesenchymal stem cells (MSCs), which, in addition to their ability to differentiate into mesenchymal tissues, contribute to the homeostatic maintenance of many organs. The choice of SFN and RPS relies on two considerations: they are among the most popular cruciferous vegetables in the diet of western and eastern countries, respectively, and their bioactive properties may differ since they possess specific molecular moiety. Our investigation evidenced that MSCs incubated with low doses of SFN and RPS show reduced in vitro oxidative stress. Moreover, these cells are protected from oxidative damages induced by hydrogen peroxide, while no protection was evident following treatment with the UV ray of a double strand DNA damaging drug, such as doxorubicin. High concentrations of both ITCs induced cytotoxic effects in MSC cultures and further increased DNA damage induced by peroxides. In summary, our study suggests that ITCs, at low doses, may contribute to slowing the aging process related to oxidative DNA damage. Moreover, in cancer treatment, low doses of ITCs may be used as an adjuvant to reduce chemotherapy-induced oxidative stress, while high doses may synergize with anticancer drugs to promote cell DNA damage.     

Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform

Epidemiological studies have shown that consumption of cruciferous vegetables, such as, broccoli and cabbages, is associated with a reduced risk of developing cancer. This phenomenon has been attributed to specific glucosinolates among the ~30 glucosinolates that are typically present as natural products characteristic of cruciferous plants. Accordingly, there has been a strong interest to produce these compounds in microbial cell factories as it will allow production of selected beneficial glucosinolates. We have developed a versatile platform for stable expression of multi-gene pathways in the yeast, Saccharomyces cerevisiae. Introduction of the seven-step pathway of indolylglucosinolate from Arabidopsis thaliana to yeast resulted in the first successful production of glucosinolates in a microbial host. The production of indolylglucosinolate was further optimized by substituting supporting endogenous yeast activities with plant-derived enzymes. Production of indolylglucosinolate serves as a proof-of-concept for our expression platform, and provides a basis for large-scale microbial production of specific glucosinolates for the benefit of human health.     

Covalent binding to tubulin by isothiocyanates. A mechanism of cell growth arrest and apoptosis

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.     

Proteomic identification of binding targets of isothiocyanates: A perspective on techniques

Intake of cruciferous vegetable is inversely associated with the risk of several cancer types. Isothiocyanates (ITCs) are believed to be important constituents contributing to these cancer-preventive effects. Although several mechanisms, including induction of apoptosis, have been proposed for the anti-carcinogenesis activities of ITCs, detailed upstream triggering events are still not fully understood. Identification of ITC binding targets in cellular proteins is crucial for not only mechanistic studies but also future drug screening and design. In this review, we summarize recent progress in discovery of ITC protein targets from a technical perspective. The advantages and limitations of each method are discussed to facilitate future studies on target discovery of ITCs and perhaps other compounds.     

Micronucleus formation and induction of apoptosis by different isothiocyanates and a mixture of isothiocyanates in human lymphocyte cultures

Isothiocyanates (ITCs) are the main sulfur-containing metabolites found in cruciferous vegetables. There is evidence that some ITCs may act as chemopreventive agents against different tumor types and induce apoptosis and modulate cell-cycle progression of highly proliferative cancer cells. However, there are also studies reporting genotoxic or co-carcinogenic effects for some ITCs, such as benzyl ITC and phenyl ITC. Since selectivity for transformed cells and absence of genotoxicity for healthy cells are important pre-requisites for new chemopreventive agents, we investigated micronucleus formation and induction of apoptosis by 4-(methylthio)butylisothiocyanate (MTBITC), sulforaphane and a mixture of ITCs in human T-lymphocyte cultures. We demonstrate that MTBITC, sulforaphane and the mixture of ITCs did not induce micronuclei. Moreover, sulforaphane induced a dose-dependent increase in the number of apoptotic cells, which was significant at the highest concentration tested (30 microM) (41% versus 18% in the untreated samples, P<0.05). The mixture of ITCs presented a trend similar to that found for sulforaphane. In fact, the mixture of ITCs was able to induce a dose-dependent increase in the percentage of apoptotic cells, which reached a maximum value at the concentration of 13 microg/ml (46% versus 19% in control samples, P<0.05). Induction of apoptosis was not observed in cultures treated with MTBITC. Our results suggest that different ITCs can have different effects. Moreover, although the mixture of glucosinolates (GLs) used in the present study does not reflect the exact composition of broccoli, our findings demonstrate that the quantitative effects of a single, specific ITC can be significantly different from those of an ITC mixture, where other ITCs of the mixture contribute to the outcome observed.     

Determination of new biomarkers to monitor the dietary consumption of isothiocyanates

Isothiocyanates (ITCs) found in cruciferous vegetables have been associated with a reduced cancer risk in humans. We determined serum albumin adducts of allyl isothiocyanate (AITC), benzylisothiocyanate (BITC), phenylethylisothiocyanate (PEITC) and sulforaphane (SFN) in 85 healthy men from a dietary, randomized, controlled trial. After enzymatic digestion of albumin we determined the adducts of the ITCs with lysine (Lys) using liquid chromatography–tandem mass spectrometry. At the beginning of the study (and after 4 weeks) 4.7% (2.4%), 48.2% (35.3%), 5.9% (10.6%), and 24.7% (23.5%) of the samples were found positive for AITC-Lys, BITC-Lys, PEITC-Lys and SFN-Lys, respectively. This method enables the quantification of ITC adducts in albumin from large, prospective studies on diet and cancer.     

Pharmacokinetics,Tissue Distribution,and Anti-Lipogenic/Adipogenic Effects of Allyl-Isothiocyanate Metabolites

Allyl-isothiocyanate (AITC) is an organosulfur phytochemical found in abundance in common cruciferous vegetables such as mustard, wasabi, and cabbage. Although AITC is metabolized primarily through the mercapturic acid pathway, its exact pharmacokinetics remains undefined and the biological function of AITC metabolites is still largely unknown. In this study, we evaluated the inhibitory effects of AITC metabolites on lipid accumulation in vitro and elucidated the pharmacokinetics and tissue distribution of AITC metabolites in rats. We found that AITC metabolites generally conjugate with glutathione (GSH) or N-acetylcysteine (NAC) and are distributed in most organs and tissues. Pharmacokinetic analysis showed a rapid uptake and complete metabolism of AITC following oral administration to rats. Although AITC has been reported to exhibit anti-tumor activity in bladder cancer, the potential bioactivity of its metabolites has not been explored. We found that GSH-AITC and NAC-AITC effectively inhibit adipogenic differentiation of 3T3-L1 preadipocytes and suppress expression of PPAR-γ, C/EBPα, and FAS, which are up-regulated during adipogenesis. GSH-AITC and NAC-AITC also suppressed oleic acid-induced lipid accumulation and lipogenesis in hepatocytes. Our findings suggest that AITC is almost completely metabolized in the liver and rapidly excreted in urine through the mercapturic acid pathway following administration in rats. AITC metabolites may exert anti-obesity effects through suppression of adipogenesis or lipogenesis.     

Bioactive food components and cancer risk reduction

Research over the last three decades has provided convincing evidence to support the premise that diets rich in fruits and vegetables may be protective against the risk of different types of cancers. Initial evidence for protective effect of fruits and vegetables against cancer risk came from population-based case-control studies, which prompted intense research aimed at (a) identification of bioactive component(s) responsible for the anticancer effects of fruits and vegetables, (b) elucidation of the mechanisms by which bioactive food components may prevent cancer, and (c) determination of their efficacy for prevention of cancer in animal models. The bioactive components responsible for cancer chemopreventive effects of various edible plants have now been identified. For instance, anticancer effect of Allium vegetables including garlic is attributed to organosulfur compounds (e.g., diallyl trisulfide). Interestingly, unlike cancer chemotherapy drugs, many bioactive food components selectively target cancer cells. Molecular basis for selectivity of anticancer bioactive food components towards cancer cells remains elusive, but these agents appear promiscuous and target multiple signal transduction pathways to inhibit cancer cell growth in vitro and in vivo. Despite convincing observational and experimental evidence, however, limited effort has been directed towards clinical investigations to determine efficacy of bioactive food components for prevention of human cancers. This article reviews current knowledge on cancer chemopreventive effects of a few highly promising dietary constituents, including garlic-derived organosulfides, berry compounds, and cruciferous vegetable-derived isothiocyanates, and serves to illustrate complexity of the signal transduction mechanisms in cancer chemoprevention by these promising bioactive food components.     

The role of Sulforaphane in cancer chemoprevention and health benefits: a mini-review

Cancer is a multi-stage process resulting from aberrant signaling pathways driving uncontrolled proliferation of transformed cells. The development and progression of cancer from a premalignant lesion towards a metastatic tumor requires accumulation of mutations in many regulatory genes of the cell. Different chemopreventative approaches have been sought to interfere with initiation and control malignant progression. Here we present research on dietary compounds with evidence of cancer prevention activity that highlights the potential beneficial effect of a diet rich in cruciferous vegetables. The Brassica family of cruciferous vegetables such as broccoli is a rich source of glucosinolates, which are metabolized to isothiocyanate compounds. Amongst a number of related variants of isothiocyanates, sulforaphane (SFN) has surfaced as a particularly potent chemopreventive agent based on its ability to target multiple mechanisms within the cell to control carcinogenesis. Anti-inflammatory, pro-apoptotic and modulation of histones are some of the more important and known mechanisms by which SFN exerts chemoprevention. The effect of SFN on cancer stem cells is another area of interest that has been explored in recent years and may contribute to its chemopreventive properties. In this paper, we briefly review structure, pharmacology and preclinical studies highlighting chemopreventive effects of SFN.     

Role of 4-hydroxynonenal in chemopreventive activities of sulforaphane

Chemoprevention of cancer via herbal and dietary supplements is a logical approach to combating cancer and currently it is an attractive area of research investigation. Over the years, isothiocyanates, such as sulforaphane (SFN) found in cruciferous vegetables, have been advocated as chemopreventive agents, and their efficacy has been demonstrated in cell lines and animal models. In vivo studies with SFN suggest that in addition to protecting normal healthy cells from environmental carcinogens, it also exhibits cytotoxicity and apoptotic effects against various cancer cell types. Among several mechanisms for the chemopreventive activity of SFN against chemical carcinogenesis, its effect on drug-metabolizing enzymes that cause activation/neutralization of carcinogenic metabolites is well established. Recent studies suggest that SFN exerts its selective cytotoxicity to cancer cells via reactive oxygen species-mediated generation of lipid peroxidation products, particularly 4-hydroxynonenal (HNE). Against the background of the known biochemical effects of SFN on normal and cancer cells, in this article we review the underlying molecular mechanisms responsible for the overall chemopreventive effects of SFN, focusing on the role of HNE in these mechanisms, which may also contribute to its selective cytotoxicity to cancer cells.     

Induction of apoptosis in tumor cells by naturally occurring sulfur-containing compounds

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Among naturally occurring products, sulfur-containing compounds (OSCs), especially garlic compounds (GCs) and isothiocyanates (ITCs), represent two important and promising chemopreventive families because of their potent chemopreventive effects in various in vivo and in vitro models. In recent years, numerous investigations have shown that sulfur-containing compounds induce apoptosis in multiple cell lines and experimental animals. In the course of apoptosis induction by GCs and ITCs, multiple signal-transduction pathways and apoptosis intermediates are modulated. In particular, modulation of MAPKs and production of reactive oxygen species (ROS) seem to play pivotal roles in apoptosis induction by most GCs and ITCs. However, the role of P53 is still controversial. Based on present knowledge, GCs and ITCs may target not only the metabolism of carcinogens but also apoptosis signaling molecules. The effects of ITCs and GCs at multiple points of cancer development make these compounds highly promising candidates in cancer chemoprevention. However, the mechanisms of their anticancer effects are not fully understood, and further studies are required, especially to elucidate the role of cell-death receptors (the extrinsic pathway) and whether these agents induce apoptotic effects in non-tumor cells.     

3,3'-Diindolylmethane induces gastric cancer cells death via STIM1 mediated store-operated calcium entry

3,3''-Diindolylmethane (DIM), a natural phytochemicals isolated from cruciferous vegetables, has been reported to inhibit human gastric cancer cells proliferation and induce cells apoptosis as well as autophagy, but its mechanisms are still unclear. Store-operated calcium entry (SOCE) is a main Ca²⁺ influx pathway in various of cancers, which is activated by the depletion of endoplasmic reticulum (ER) Ca²⁺ store. Stromal interaction molecular 1 (STIM1) is the necessary component of SOCE. In this study, we focus on to examine the regulatory mechanism of SOCE on DIM-induced death in gastric cancer. After treating the human BGC-823 and SGC-7901 gastric cancer cells with DIM, cellular proliferation was determined by MTT, apoptosis and autophagy were detected by flow cytometry or Hoechst 33342 staining. The expression levels of related proteins were evaluated by Western blotting. Free cytosolilc Ca²⁺ level was assessed by fluorescence monitoring under a laser scanning confocal microscope. The data have shown that DIM could significantly inhibit proliferation and induce apoptosis as well as autophagy in two gastric cancer cell lines. After DIM treatment, the STIM1-mediated SOCE was activated by upregulating STIM1 and decreasing ER Ca²⁺ level. Knockdown STIM1 with siRNA or pharmacological inhibition of SOCE attenuated DIM induced apoptosis and autophagy by inhibiting p-AMPK mediated ER stress pathway. Our data highlighted that the potential of SOCE as a promising target for treating cancers. Developing effective and selective activators targeting STIM1-mediated SOCE pathway will facilitate better therapeutic sensitivity of phytochemicals acting on SOCE in gastric cancer. Moreover, more research should be performed to validate the efficacy of combination chemotherapy of anti-cancer drugs targeting SOCE for clinical application.     

Dietary organic isothiocyanates are cytotoxic in human breast cancer MCF-7 and mammary epithelial MCF-12A cell lines

Organic isothiocyanates (ITCs) are dietary components present in cruciferous vegetables. The purpose of this investigation was to examine the cytotoxicity of 1-naphthyl isothiocyanate (NITC), benzyl isothiocyanate (BITC), beta-phenethyl isothiocyanate (PEITC), and sulforaphane in human breast cancer MCF-7 and human mammary epithelium MCF-12A cell lines, as well as in a second human epithelial cell line, human kidney HK-2 cells. The cytotoxicity of NITC, BITC, PEITC, and sulforaphane, as well as the cytotoxicity of the chemotherapeutic agents daunomycin (DNM) and vinblastine (VBL), were examined in MCF-7/sensitive (wt), MCF-7/Adr (which overexpresses P-glycoprotein), MCF-12A, and HK-2 cells. Cell growth was determined by a sulforhodamine B assay. The IC50 values for DNM and VBL in MCF-7/Adr cells were 7.12 +/- 0.42 microM and 0.106 +/- 0.004 microM (mean +/- SE) following a 48-hr exposure; IC50 values for BITC, PEITC, NITC, and sulforaphane were 5.95 +/- 0.10, 7.32 +/- 0.25, 77.9 +/- 8.03, and 13.7 +/- 0.82 microM, respectively, with similar values obtained in MCF-7/wt cells. Corresponding values for BITC, PEITC, NITC, and sulforaphane in MCF-12A cells were 8.07 +/- 0.29, 7.71 +/- 0.07, 33.6 +/- 1.69, and 40.5 +/- 1.25 microM, respectively. BITC and PEITC can inhibit the growth of human breast cancer cells as well as human mammary epithelium cells at concentrations similar to those of the chemotherapeutic drug DNM. Sulforaphane and NITC exhibited higher IC50 values. The effect of these ITCs on cell growth may contribute to the cancer chemopreventive properties of ITCs by suppressing the growth of preclinical tumors, and may indicate a potential use of these compounds as chemotherapeutic agents in cancer treatment.     

3,3'-Diindolylmethane, a cruciferous vegetable derived synthetic anti-proliferative compound in thyroid disease

Considerable epidemiological evidence exists to link thyroid disease with differing patterns of dietary consumption, in particular, cruciferous vegetables. We have been studying the anti-thyroid cancer (TCa) activity of indole-3-carbinol (I3C) found in cruciferous vegetables and its acid catalyzed dimer, 3,3'-diindolylmethane (DIM). There are no studies as yet to elucidate the effect of these compounds on the altered proliferative patterns in goiter or thyroid neoplasia. In this study, we tested the anti-proliferative effects of I3C and DIM on four different thyroid cancer cell lines representative of papillary (B-CPAP and 8505-C) and follicular carcinoma of the thyroid (CGTH-W-1 and ML-1), and primary human goiter cells. Cell survival and IC50 values for I3C and DIM were calculated by the XTT assay and cell cycle distribution analysis was done by flow cytometry. DIM was found to be a better anti-proliferative agent than I3C in both papillary and follicular TCa resulting in a greater cytotoxic effect at a concentration over three fold lower than predicted by the molar ratio of DIM and I3C. The anti-proliferative activity of DIM in follicular TCa was mediated by a G1 arrest followed by induction of apoptosis. DIM also inhibited the growth of primary goiter cells by 70% compared to untreated controls. Contrary to traditional belief that cruciferous vegetables are "goitrogenic", DIM has anti-proliferative effects in glandular thyroid proliferative disease. Our preclinical studies provide a strong rationale for the clinical exploration of DIM as an adjuvant to surgery in thyroid proliferative disease.     

RLIP: An existential requirement for breast carcinogenesis

Breast cancer (BC) is the most common cancer among women worldwide. Due to its complexity in nature, effective BC treatment can encounter many challenges. The human RALBP1 gene encodes a 76-kDa splice variant protein, RLIP (ral-binding protein1, RalBP1), a stress-protective mercapturic acid pathway (MAP) transporter protein, that also plays a key role in regulating clathrin-dependent endocytosis (CDE) as a Ral effector. Growing evidence shows that targeting RLIP may be an effective strategy in cancer therapy, as RLIP is over-expressed in multiple cancers and is known to induce resistance to apoptosis and chemotherapeutic drugs. Recent studies demonstrated that RLIP is expressed in human BC tissues, as well as BC cell lines. Knockdown of RLIP resulted in apoptotic death of BC cells in vitro, and targeted inhibition and depletion of RLIP resulted in regression of BC in xenograft studies of nude mice. Signaling studies showed that RLIP depletion inhibited endocytosis and differentially regulated signaling to Akt, Myc, and ERK1/2. However, the proliferation and multi-specific transport mechanisms that promote RLIP-mediated cell death in BC are not well understood. In this review, we will discuss a missing but an essentially determining and connecting piece of the puzzle on the understanding of proliferation and transport mechanisms by focused analyses of the apoptotic, drug- and radiation-sensitivity regulated by RLIP, a stress-responsive non-ATP-binding cassette (ABC), high capacity MAP transporter, in breast cancer.     

十字花科植物中莱菔硫烷防癌机制研究进展

十字花科蔬菜中的异硫氰酸酯--莱菔硫烷(sulforaphane,SF)可以在癌症的起始、发展和增生3个阶段阻滞癌细胞的生长发育,降低癌症发生的风险,其前体物主要存在于西兰花中.本文主要对国内外近年来有关SF对化学致癌物代谢酶的影响、激活抗氧化基因活性、激活转录因子、阻滞细胞周期、诱导细胞凋亡等方面的研究进展进行综述,以探讨SF的防癌机制,为防癌抗癌食品和药物的研发提供参考.