Crucifer-derived isothiocyanates: chemopreventive activity and mechanism of action.
Authors: Yuesheng Zhang
Journal: Cancer Research (2006)

Proc Amer Assoc Cancer Res, Volume 47, 2006

SY18-01 

Many cruciferous vegetables are rich sources of glucosinolates (β-thioglucoside N -hydroxysulfates, GS). While there is little evidence to show that GS themselves possess cancer-preventive activities, they are precursors of isothiocyanates (ITC), many of which have been repeatedly shown to inhibit cancer development in various animal models. In fact, several recent epidemiological studies reported an inverse association between ITC intake and the risk of cancer in the lung, breast and colon. GS are converted to ITC when vegetable tissues are damaged, as occurs in chewing or cooking, and the conversion is catalyzed by myrosinase, an enzyme that coexists with but is physically separated from GS in normal plants. Hydrolysis of GS by myrosinase generates thiohydroximate O -sulfonates, and these unstable intermediates then rearrange to form ITC or other products, depending on the chemical structure of the intermediate and/or the reaction condition. It is therefore important to note that how vegetables are prepared for human consumption can significantly affect the yield of ITC. For example, cooking vegetables at high temperature (>80 oC) likely inactivates myrosinase and inhibits GS hydrolysis, whereas vegetables that contain epithiospecifier protein (ESP) may yield very low ITC levels even at subdued temperature. ESP, which is more heat-labile than myrosinase, binds to and converts thiohydroximate O -sulfonates to nitriles or cyanoepithioalkanes at the expense of ITC. Nevertheless, GS that escape myrosinase in vegetables may be converted at least in part to ITC in the intestinal tract, as the intestinal microflora also possess myrosinase activity.

Analytical methods are available for quantifying the levels of GS and ITC in vegetable extracts and for determination of human exposure to dietary ITC. Liquid chromatography- and mass spectrometry-based methods have been used to measure individual compounds, whereas the novel cyclocondensation assay (reaction with 1,2-benzenedithiol) detects the total amount of ITC and their metabolites. Because urine is the principal disposal route for ITC metabolites, the cyclocondensation assay has been found to be an excellent tool for determining human exposure to total dietary ITC. Indeed, several epidemiological studies showing an inverse association between ITC intake and cancer risk, as mentioned above, were conducted using this simple assay.

Most studies on the cancer-preventive activities of crucifer-derived ITC have focused on those that occur abundantly in common cruciferous vegetables and are frequently consumed by humans, including allyl ITC, benzyl ITC, phenethyl ITC and sulforaphane. Numerous studies have shown that these ITC inhibit both the formation of cancer cells (anticarcinogenic activity) and the survival and proliferation of existing cancer cells (anticancer activity). Such activities with each compound have been demonstrated in multiple organ sites of rodents. Considerable information on the molecular basis for both the anticarcinogenic and anticancer effects of ITC is available. It is now clear that ITC can target cancer in multiple directions, including inhibition of carcinogen-activating enzymes, induction of carcinogen-detoxifying enzymes, induction of apoptosis and arrest of cell cycle progression, as well as other mechanisms. Molecular targets and modulation of signaling pathways by ITC will be discussed. It should be emphasized that ITC are dichotomous modulators of oxidative stress. While ITC transcriptionally stimulate many antioxidative enzymes and nonenzymatic proteins, leading to enhanced protection against oxidative stressors, they also directly alkylate and deplete cellular thiols, damage mitochondria, and elevate reactive oxygen species, leading to cellular stress. These paradoxical effects appear to occur in tandem: exposure of cells to ITC rapidly leads to an acute increase in stress, which is followed by a delayed but lasting increase in cellular protection against oxidants and carcinogens. Ironically, although ITC-induced stress may lead to oxidative damage, it has become increasingly clear that much of the chemopreventive activity of ITC stems from the response of cells to the stress induced by these compounds.

All ITC are characterized by the presence of an -N=C=S group, whose central carbon often is highly electrophilic. The biological activity of ITC appears to be primarily mediated through reaction of this carbon atom with cellular nucleophilic targets, whereas the side chains of ITC may play secondary roles, e.g., affecting the reactivity of the -N=C=S group and controlling the lipophilicity of the molecule. The -N=C=S group also governs the metabolism of ITC, which are metabolized in vivo principally by the mercapturic acid pathway. Initial conjugation of ITC through the -N=C=S group with the cysteine thiol of glutathione (GSH) gives rise to dithiocarbamate conjugates, which then undergo further enzymatic modifications to form sequentially the cysteinylglycine-, cysteine-, N -acetylcysteine-conjugates, which are excreted in the urine. Intracellular GSH is also the major driving force for rapid accumulation of ITC in cells, which is critical for ITC to exert their chemopreventive effects. The ITC conjugation reaction with GSH takes place spontaneously at a considerable rate, but it can be further enhanced by glutathione transferase (GST). Several epidemiological studies have reported that the inverse association between ITC intake and cancer risk was particularly strong among GSTM1- and/or GSTT1-null individuals. However, the hypothesis that lack of GST reduces ITC excretion and therefore enhances tissue exposure to ITC, which was proposed by some investigators, has not been sufficiently tested. Interestingly, many studies have reported that the dithiocarbamate metabolites possess similar cancer chemopreventive activity to their parent ITC. However, available evidence strongly suggests that the intact metabolites are largely unable to enter cells and the biological activity of these metabolites depend on their dissociation back to ITC, which are then taken to cells. In summary, dietary ITC are highly promising cancer chemopreventive agents, capable of inhibiting both cancer cell formation and survival. Much has been learned about the underlying chemopreventive mechanisms. Analytical methods have been developed to allow detection of ITC in vegetable extracts and assessment of human exposure to dietary ITC.