Zerumbone (ZER), a sesquiterpene from the edible plantZingiber zerumbet Smith, has recently been found to suppress tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus activation in a potent manner. In the present study, we evaluated the antiinflammatory and chemopreventive potentials of ZER in a variety of cell culture experiments. ZER effectively suppressed TPA-induced superoxide anion generation from both NADPH oxidase in dimethylsulfoxide-differentiated HL-60 human acute promyelocytic leukemia cells and xanthine oxidase in AS52 Chinese hamster ovary cells. The combined lipopolysaccharide-and interferon-γ-stimulated protein expressions of inducible nitric oxide synthase and cyclooxygenase (COX)-2, together with the release of tumor necrosis factor-α, in RAW 264.7 mouse macrophages were also markedly diminished. These suppressive events were accompanied with a combined decrease in the medium concentrations of nitrite and prostaglandin E 2 , while the expression level of COX-1 was unchanged. ZER inhibited the proliferation of human colonic adenocarcinoma cell lines (LS174T, LS180, COLO205, and COLO320DM) in a dose-dependent manner, while the growth of normal human dermal (2F0-C25) and colon (CCD-18 Co) fibroblasts was less affected. It also induced apoptosis in COLO205 cells, as detected by dysfunction of the mitochondria transmembrane, Annexin V-detected translocation of phosphatidylserine, and chromatin condensation. Intriguingly, α-humulene, a structural analog lacking only the carbonyl Abbreviations: AP, allopurinol; COX, cyclooxygenase; DMEM, Dulbecco's modified Eagle medium; DMSO, dimethylsulfoxide; DPI, diphenyleneiodonium; EBV, Epstein-Barr virus; FBS, fetal bovine serum; FITC, fluorescence isothiocyanate; HUM, α-humulene; IFN, interferon; iNOS, inducible NO; synthase; IR, inhibitory rate; LPS, lipopolysaccharide; MEM, minimum essential medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-ΦB, nuclear factor-kappaB; NO, nitric oxide; NO 2 -, nitrite; O 2 -, superoxide anion; PBS, phosphate-buffered saline; PG, prostaglandin; PI, propiodium iodide; PS, phosphatidylserine; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; XO, xanthine oxidase; ZER, zerumbone.© Oxford University Press 795 group in ZER, was virtually inactive in all experiments conducted, indicating that the α,β-unsaturated carbonyl group in ZER may play some pivotal roles in interactions with unidentified target molecule(s). Taken together, our results indicate that ZER is a food phytochemical that has distinct potentials for use in anti-inflammation, chemoprevention, and chemotherapy strategies.

In the present study, we studied the molecular mechanism underlying cell death induced by a cancer chemoprotective compound benzyl isothiocyanate (BITC). The cytotoxic effect of BITC was examined in rat liver epithelial RL34 cells. Apoptosis was induced when the cells were treated with 20 M BITC, characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane and caspase-3 activation, whereas no caspase activation and propidium iodide incorporation into cell were detected with 50 M BITC that induced necrosis. The mitochondrial death pathway was suggested to be involved in BITC-induced apoptosis because the treatment of cells with BITC-induced caspase-9-dependent apoptosis and mitochondrial transmembrane potential (⌬⌿m) alteration. We demonstrated here for the first time that BITC directly modifies mitochondrial functions, including inhibition of respiration, mitochondrial swelling, and release of cytochrome c. Moreover, glutathione depletion by diethyl maleate significantly accelerated BITC-triggered apoptosis, suggesting the involvement of a redox-dependent mechanism. This was also implicated by the observations that intracellular accumulation of reactive oxygen species, including superoxide (O 2 . ) and hydroperoxides (HPOs), was indeed detected in the cells treated with BITC and that the intracellular HPO level was significantly attenuated by pretreatment with N-acetylcysteine. The treatment with a pharmacological scavenger of O 2 . , Tiron, also diminished the HPO formation by ϳ80%, suggesting that most of the HPOs were H 2 O 2 derived from the dismutation of O 2 . . These results suggest that BITC induces apoptosis through a mitochondial redox-sensitive mechanism.

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