We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9-and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G 2 /M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3␤. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G 2 /M arrest. HCT116 human colon cancer-derived Chk2 ؊/؊ cells were significantly more resistant to G 2 /M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G 2 /M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.Epidemiological studies have revealed an inverse correlation between the dietary intake of cruciferous vegetables and the risk for certain types of cancers, including prostate cancer (1-5). Laboratory studies indicate that the anticancer effect of cruciferous vegetables is caused by isothiocyanates that exist as thioglucoside conjugates (glucosinolates) in a variety of edible plants including broccoli, cabbage, watercress, and so forth (6 -9). Cruciferous vegetable-derived organic isothiocyanates are generated by hydrolytic cleavage of corresponding glucosinolates through catalytic mediation of myrosinases, which are released when the plant cells are damaged because of cutting or chewing (6 -9). Sulforaphane (SFN) 1 is one such isothiocyanate analog that has received a great deal of attention not only because it is present in high concentrations in certain varieties of broccoli but also because of its potent anticancer activity (10 -15). For example, oral administration of SFN (1-isothiocyanato-4-(methylsulfinyl)butane; CH 3 -SO-(CH 2 ) 4 -NϭCϭS)) caused a statistically significant reduction in 9,10-dimethyl-1,2-benzanthr...

Glutathione S‐transferase P1‐1 isoforms, differing in a single amino acid residue (Ile104 or Val104), have been previously identified in human placenta [Ahmad, H., Wilson, D. E., Fritz, R. R., Singh, S. V., Medh, R. D., Nagle, G. T., Awasthi, Y. C. & Kurosky, A. (1990) Arch. Biochem. Biophys. 278, 398–408]. In the present report, the enzymic properties of these two proteins are compared. [I104]glutathione S‐transferase P1‐1 has been expressed from its cDNA in Escherichia coli and purified to homogeneity by affinity chromatography; the cDNA has been mutated to replace Ile104 by Val104, and [V104]glutathione S‐transferase P1‐1 was expressed and isolated as described for [I104]glutathione S‐transferase P1‐1. The two enzymes differed in their specific activity and affinity for electrophilic substrates (KM values for 1‐chloro‐2,4‐dinitrobenzene were 0.8 mM and 3.0 mM for [I‐104]glutathione S‐transferase P1‐1 and [V‐104]glutathione S‐transferase P1‐1, respectively), but were identical in their affinity for glutathione. In addition, the two enzymes were distinguishable by their heat stability, with half‐lives at 45°C of 19 min and 51 min, respectively. The resistance to heat denaturation was differentially modulated by the presence of substrates. These data, in conjunction with molecular modeling, indicate that the residue in position 104 helps to define the geometry of the hydrophobic substrate‐binding site, and may also influence activity by interacting with residues directly involved in substrate binding.

Chronic arsenic exposure is a worldwide health problem. Although arsenic-induced cancer has been widely studied, comparatively little attention has been paid to arsenic-induced vascular disease. Epidemiological studies have shown that chronic arsenic exposure is associated with increased morbidity and mortality from cardiovascular disease. In addition, studies suggest that susceptibility to arsenic-induced vascular disease may be modified by nutritional factors in addition to genetic factors. Recently, animal models for arsenic-induced atherosclerosis and liver sinusoidal endothelial cell dysfunction have been developed. Initial studies in these models show that arsenic exposure accelerates and exacerbates atherosclerosis in apolipoprotein E-knockout mice. Microarray studies of liver mRNA and micro-RNA abundance in mice exposed in utero suggest that a permanent state of stress is induced by the arsenic exposure. Furthermore, the livers of the arsenic-exposed mice have activated pathways involved in immune responses suggesting a pro-hyperinflammatory state. Arsenic exposure of mice after weaning shows a clear dose-response in the extent of disease exacerbation. In addition, increased inflammation in arterial wall is evident. In response to arsenic-stimulated oxidative signaling, liver sinusoidal endothelium differentiates into a continuous endothelium that limits nutrient exchange and waste elimination. Data suggest that nicotinamide adenine dinucleotide phosphate oxidase-derived superoxide or its derivatives are essential second messengers in the signaling pathway for arsenic-stimulated vessel remodeling. The recent findings provide future directions for research into the cardiovascular effects of arsenic exposure.

Background-Heme oxygenase-1 (HO-1) is an inducible stress-response protein that imparts antioxidant and antiapoptotic effects. However, its pathophysiological role in cardiac remodeling and chronic heart failure (HF) is unknown. We hypothesized that induction of HO-1 in HF alleviates pathological remodeling. Methods and Results-Adult male nontransgenic and myocyte-restricted HO-1 transgenic mice underwent either sham operation or coronary ligation to induce HF. Four weeks after ligation, nontransgenic HF mice exhibited postinfarction left ventricular (LV) remodeling and dysfunction, hypertrophy, fibrosis, oxidative stress, apoptosis, and reduced capillary density, associated with a 2-fold increase in HO-1 expression in noninfarcted myocardium. Compared with nontransgenic mice, HO-1 transgenic HF mice exhibited significantly (PϽ0.05) improved postinfarction survival (94% versus 57%) and less LV dilatation (end-diastolic volume, 46Ϯ8 versus 85Ϯ32 L), mechanical dysfunction (ejection fraction, 65Ϯ9% versus 49Ϯ16%), hypertrophy (LV/tibia length 4.4Ϯ0.4 versus 5.2Ϯ0.6 mg/mm), interstitial fibrosis (11.2Ϯ3.1% versus 18.5Ϯ3.5%), and oxidative stress (3-fold reduction in tissue malondialdehyde). Moreover, myocyte-specific HO-1 overexpression in HF promoted tissue neovascularization and ameliorated myocardial p53 expression (2-fold reduction) and apoptosis. In isolated mitochondria, mitochondrial permeability transition was inhibited by HO-1 in a carbon monoxide (CO)-dependent manner and was recapitulated by the CO donor tricarbonylchloro(glycinato)ruthenium(II) (CORM-3). HO-1-derived CO also prevented H 2 O 2 -induced cardiomyocyte apoptosis and cell death. Finally, in vivo treatment with CORM-3 alleviated postinfarction LV remodeling, p53 expression, and apoptosis. Conclusions-HO-1 induction in the failing heart is an important cardioprotective adaptation that opposes pathological LV remodeling, and this effect is mediated, at least in part, by CO-dependent inhibition of mitochondrial permeability transition and apoptosis. Augmentation of HO-1 or its product, CO, may represent a novel therapeutic strategy for ameliorating HF.