Yuesheng Zhang

Chemoprotection against cancer by phase 2 enzyme induction.

Mammalian cells have evolved elaborate mechanisms for protection against the toxic and neoplastic effects of electrophilic metabolites of carcinogens and reactive oxygen species. Phase 2 enzymes (e.g. glutathione transferase, NAD(P)H:quinone reductase, UDP-glucuronosyltransferases) and high intracellular levels of glutathione play a prominent role in providing such protection. Phase 2 enzymes are transcriptionally induced by low concentrations of a wide variety of chemical agents and such induction blocks chemical carcinogenesis. The inducers belong to many chemical classes including phenolic antioxidants. Michael reaction acceptors, isothiocyanates, 1,2-dithiole-3-thiones, trivalent arsenicals, HgCl2 and organomercurials, hydroperoxides, and vicinal dimercaptans. Induction by all classes of inducers involves the antioxidant/electrophile response element (ARE/EpRE). Inducers are widely, but unequally, distributed among edible plants. Search for such inducer activity in broccoli led to the isolation of sulforaphane, an isothiocyanate that is a very potent Phase 2 enzyme inducer and blocks mammary tumor formation in rats.

Consumption of Raw Cruciferous Vegetables is Inversely Associated with Bladder Cancer Risk

Cruciferous vegetables contain isothiocyanates, which show potent chemopreventive activity against bladder cancer in both in vitro and in vivo studies. However, previous epidemiologic studies investigating cruciferous vegetable intake and bladder cancer risk have been inconsistent. Cooking can substantially reduce or destroy isothiocyanates, and could account for study inconsistencies. In this hospital-based case-control study involving 275 individuals with incident, primary bladder cancer and 825 individuals without cancer, we examined the usual prediagnostic intake of raw and cooked cruciferous vegetables in relation to bladder cancer risk. Odds ratios (OR) and 95% confidence intervals (CI) were estimated with unconditional logistic regression, adjusting for smoking and other bladder cancer risk factors. We observed a strong and statistically significant inverse association between bladder cancer risk and raw cruciferous vegetable intake (adjusted OR for highest versus lowest category = 0.64; 95% CI, 0.42-0.97), with a significant trend (P = 0.003); there were no significant associations for fruit, total vegetables, or total cruciferous vegetables. The associations observed for total raw crucifers were also observed for individual raw crucifers. The inverse association remained significant among current and heavy smokers with three or more servings per month of raw cruciferous vegetables (adjusted ORs, 0.46 and 0.60; 95% CI, 0.23-0.93 and 0.38-0.93, respectively). These data suggest that cruciferous vegetables, when consumed raw, may reduce the risk of bladder cancer, an effect consistent with the role of dietary isothiocyanates as chemopreventive agents against bladder cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(4):938–44)

Inhibition of Urinary Bladder Carcinogenesis by Broccoli Sprouts

Isothiocyanates are a well-known class of cancer chemopreventive agents, and broccoli sprouts are a rich source of several isothiocyanates. We report herein that dietary administration to rats of a freeze-dried aqueous extract of broccoli sprouts significantly and dose-dependently inhibited bladder cancer development induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. The incidence, multiplicity, size, and progression of bladder cancer were all inhibited by the extract, while the extract itself caused no histologic changes in the bladder. Moreover, inhibition of bladder carcinogenesis by the extract was associated with significant induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1 in the bladder, enzymes that are important protectants against oxidants and carcinogens. Isothiocyanates are metabolized to dithiocarbamates in vivo, but dithiocarbamates readily dissociate to isothiocyanates. We found that >70% of the isothiocyanates present in the extract were excreted in the urine as isothiocyanate equivalents (isothiocyanates + dithiocarbamates) in 12 h after a single p.o. dose, indicating high bioavailability and rapid urinary excretion. In addition, the concentrations of isothiocyanate equivalents in the urine of extract-treated rats were 2 to 3 orders of magnitude higher than those in plasma, indicating that the bladder epithelium, the major site of bladder cancer development, is most exposed to p.o. dosed isothiocyanate. Indeed, tissue levels of isothiocyanate equivalents in the bladder were significantly higher than in the liver. In conclusion, broccoli sprout extract is a highly promising substance for bladder cancer prevention and the isothiocyanates in the extract are selectively delivered to the bladder epithelium through urinary excretion.

Mechanism of chemical activation of Nrf2.

NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation.

The molecular basis that unifies the metabolism, cellular uptake and chemopreventive activities of dietary isothiocyanates

Organic isothiocyanates (ITCs), which are characterized by the presence of an -N=C=S group, are among the most extensively studied cancer chemopreventive agents and show highly promising chemopreventive activities. Numerous studies have shown that ITCs can inhibit both carcinogenesis and cancer growth in a variety of animal models. Many cruciferous vegetables, which are commonly consumed by humans, are rich sources of these compounds. Of particular interest are their high bioavailability, their shared metabolic profile and their ability to target a wide array of cancer-related cellular proteins. This review is focused on discussing the molecular basis of these intriguing properties of ITCs, with a particular emphasis on the concept that cellular uptake and metabolism of ITCs and at least some of their major chemopreventive activities are all initiated through direct reaction of the carbon atom of the -N=C=S group of the ITCs with cysteine sulfhydryl groups of glutathione (GSH) and of proteins. This knowledge deepens our understanding about the biological activities of ITCs and may facilitate further research and development of these compounds for cancer prevention and treatment.

Dithiolethiones for cancer chemoprevention: where do we stand?

Dithiolethiones are a well-known class of cancer chemopreventive agents; the key mechanism of action of dithiolethiones involves activation of Nrf2 signaling and induction of phase II enzymes. In the past, attention has been focused mainly on 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz), which showed ability as a wide-spectrum inhibitor of chemical carcinogenesis in preclinical models. However, clinical trials of oltipraz have shown questionable efficacy, and at the high doses employed in such studies, significant side effects were observed. Dithiolethiones that are markedly more effective and potent than oltipraz in both induction of phase II enzymes and inhibition of chemical carcinogenesis in preclinical studies have been identified, and these compounds have shown pronounced organ specificity in vivo. Further investigation of these compounds may lead to development of effective and safe agents for cancer prevention in humans. [Mol Cancer Ther 2008;7(11):3470–9]

Intake of Cruciferous Vegetables Modifies Bladder Cancer Survival

Background: Intake of cruciferous vegetables, a rich source of dietary isothiocyanates, has been inversely associated with risk of bladder cancer. Due to the potent antiproliferative effects of dietary isothiocyanates on bladder cancer in in vitro and in vivo models, cruciferous vegetable intake may also play a role in survival among patients with bladder cancer. Methods: Using information obtained from the Roswell Park Cancer Institute Tumor Registry, patient medical records, and routinely collected questionnaire data, we examined potential associations between intake of cruciferous vegetables and survival among bladder cancer patients. As cooking can substantially reduce or destroy isothiocyanates, consumption of raw versus cooked cruciferous vegetables was examined separately. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox proportional hazard models. Results: A total of 239 bladder cancer patients were included in the study. After an average of 8 years of follow-up, 179 deaths occurred, with 101 deaths attributable to bladder cancer. After adjustment for other prognostic factors, a strong and significant inverse association was observed between bladder cancer mortality and broccoli intake, in particular raw broccoli intake (≥1 versus <1 serving per month; HR for overall death, 0.57; 95% CI, 0.39-0.83; HR for disease-specific death, 0.43; 95% CI, 0.25-0.74). There were no significant associations for total vegetables, total fruits, or other individual cruciferous vegetables. Conclusions: Considering the strong preclinical evidence, intake of broccoli may improve bladder cancer survival. Impact: Further prospective investigation is warranted to confirm the potential role of cruciferous vegetables in bladder cancer prognosis. Cancer Epidemiol Biomarkers Prev; 19(7); 1806–11. ©2010 AACR.

Potent activation of mitochondria-mediated apoptosis and arrest in S and M phases of cancer cells by a broccoli sprout extract

We have previously shown that broccoli sprouts are a rich source of chemopreventive isothiocyanates, which potently induce carcinogen-detoxifying enzymes and inhibit the development of mammary and skin tumors in rodents. However, the principal isothiocyanate present in broccoli sprout extracts, sulforaphane, not only induces carcinogen-detoxifying enzymes but also activates apoptosis and blocks cell cycle progression. In this article, we show that an aqueous extract of broccoli sprouts potently inhibits the growth of human bladder carcinoma cells in culture and that this inhibition is almost exclusively due to the isothiocyanates. Isothiocyanates are present in broccoli sprouts as their glucosinolate precursors and blocking their conversion to isothiocyanates abolishes the antiproliferative activity of the extract. Moreover, the potency of isothiocyanates in the extract in inhibiting cancer cell growth was almost identical to that of synthetic sulforaphane, as judged by their IC50 values (6.6 versus 6.8 μmol/L), suggesting that other isothiocyanates in the extract may be biologically similar to sulforaphane and that nonisothiocyanate substances in the extract may not interfere with the antiproliferative activity of the isothiocyanates. Further study showed that the isothiocyanate extract of broccoli sprouts activated the mitochondria-mediated apoptosis pathway and halted cells in S and M phases. Cell cycle arrest was associated with down-regulation of Cdc25C and disruption of mitotic spindles. These data show that broccoli sprout isothiocyanate extract is a highly promising substance for cancer prevention/treatment and that its antiproliferative activity is exclusively derived from isothiocyanates. [Mol Cancer Ther 2006;5(4):935–44]

The principal urinary metabolites of dietary isothiocyanates, N-acetylcysteine conjugates, elicit the same anti-proliferative response as their parent compounds in human bladder cancer cells.

Isothiocyanates (ITCs) are a class of well-known cancerpreventive phytochemicals, but are primarily disposed of and concentrated in the urine as N-acetylcysteine conjugates (NAC-ITCs) in vivo. Because human urinary bladder cancers occur almost exclusively in the bladder epithelium, which is directly exposed to the urine stored in the bladder, we undertook to examine the anti-cancer activity of NAC-ITCs in cultured human bladder cancer cells. In this paper, we report that the NAC conjugates of four naturally occurring ITCs, including allyl ITC, benzyl ITC (BITC), phenethyl ITC and sulforaphane, potently inhibited the growth of cells derived from both low-grade superficial and high-grade invasive human bladder cancers and drug-resistant bladder cancer cells. Moreover, the growth-inhibitory potencies were similar between the conjugates and their parent compounds. Further study of NAC-BITC and BITC as model compounds showed that both compounds accumulated in cells predominantly as the glutathione conjugate of BITC, but the accumulation of the former was slower. Moreover, both compounds also demonstrated the same anti-proliferative mechanisms: causing the cleavage of the same set of caspases (caspase−3, −8 and −9) in apoptosis induction, arresting cells in the same phases (S and G2/M) and targeting the same cell cycle regulator (Cdc25C), although a longer treatment time or slightly higher doses were needed for NAC-BITC to achieve the same effect as BITC, presumably due to slower cellular uptake of NAC-BITC. These data show that the NAC-ITCs are biologically similar to their parent compounds and are highly effective against human bladder cancer cells.

Cruciferous vegetable intake is inversely associated with lung cancer risk among smokers: a case-control study

BackgroundInverse associations between cruciferous vegetable intake and lung cancer risk have been consistently reported. However, associations within smoking status subgroups have not been consistently addressed.MethodsWe conducted a hospital-based case-control study with lung cancer cases and controls matched on smoking status, and further adjusted for smoking status, duration, and intensity in the multivariate models. A total of 948 cases and 1743 controls were included in the analysis.ResultsInverse linear trends were observed between intake of fruits, total vegetables, and cruciferous vegetables and risk of lung cancer (ORs ranged from 0.53-0.70, with P for trend < 0.05). Interestingly, significant associations were observed for intake of fruits and total vegetables with lung cancer among never smokers. Conversely, significant inverse associations with cruciferous vegetable intake were observed primarily among smokers, in particular former smokers, although significant interactions were not detected between smoking and intake of any food group. Of four lung cancer histological subtypes, significant inverse associations were observed primarily among patients with squamous or small cell carcinoma - the two subtypes more strongly associated with heavy smoking.ConclusionsOur findings are consistent with the smoking-related carcinogen-modulating effect of isothiocyanates, a group of phytochemicals uniquely present in cruciferous vegetables. Our data support consumption of a diet rich in cruciferous vegetables may reduce the risk of lung cancer among smokers.