A heat-stable extract from Mucuna stimulates the differentiation of bone marrow cells into dendritic cells and induces apoptosis in cancer cells.

Pine cone extract is known to induce differentiation of human mononuclear cells into dendritic cells (DCs) and also to induce apoptosis in human cancer cells. In the present study, we screened edible plants that contain components with biological activities similar to or more potent than those of pine cone extract. We found that Mucuna (Mucuna pruviens var. utilis) contains a DC differentiation/maturation-inducing activity and a component that induces apoptosis in human cancer cell lines. Mucuna extract specifically stimulated differentiation of BM cells to immature DCs. Marked production of IL-6 was observed by sequential treatment with at least 10 µg/mL of Mucuna extract followed by LPS. The sequential treatment with Mucuna extract followed by LPS produced a much higher ratio of IL-12 to IL-6 and a lower ratio of TNF-α to IL-6 than that obtained by sequential treatment with a medicinal mushroom Phellinus linteus extract and then LPS. The DC differentiation/maturation activity and the component inducing apoptosis in cancer cells were separable by column chromatography.

A tyrosine kinase inhibitor, beta-hydroxyisovalerylshikonin, induced apoptosis in human lung cancer DMS114 cells through reduction of dUTP nucleotidohydrolase activity.

Apoptotic cell death was induced in human lung cancer DMS114 cells by treatment with beta-hydroxyisovalerylshikonin (beta-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases. Changes in phosphoprotein profiles were analyzed by two-dimensional-polyacrylamide gel electrophoresis (2D-PAGE) after the cells were treated with beta-HIVS. One spot on the 2D gel showed a marked decrease in intensity and the corresponding protein was identified by mass spectrometry as dUTP nucleotidohydrolase (dUTPase). The beta-HIVS-induced decrease of dUTPase in the phosphoprotein fraction of DMS114 cells was confirmed using immunoblotting. Treatment of the cells with beta-HIVS-induced rapid reduction of dUTPase activity. An antioxidant N-acetyl-cysteine inhibited both the reduction of phosphorylated dUTPase and the induction of apoptosis by beta-HIVS treatment of DMS114 cells. Introduction of siRNA directed against dUTPase mRNA into DMS114 cells enhanced the susceptibility of beta-HIVS-induced apoptosis. Treatment of DMS114 cells with beta-HIVS and 5-fluorouracil, a specific inhibitor of thymidylate synthase used as a chemotherapeutic drug, revealed the synergistic effects of these drugs on the inhibition of cell growth. These results suggest that dUTPase activity is one of the crucial factors involved in apoptotic cell death in lung cancer cells.

Mechanism of inhibition of tumor angiogenesis by beta-hydroxyisovalerylshikonin.

Shikonin and beta-hydroxyisovalerylshikonin (beta-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that beta-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. beta-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using beta-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. beta-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. beta-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and beta-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling.

Involvement of Tiam1 in apoptosis induced by bufalin in HeLa cells.

BACKGROUND: It has been previously demonstrated that bufalin, an active agent in the Chinese medicine chan'su, induces apoptosis in human leukemia cells by altering the expression of apoptosis-related genes, such as bcl-2 and c-myc. Tiam1 was also found to play a critical role in bufalin-induced apoptosis through the activation of the Rac1, PAK and JNK pathway in human leukemia cell lines. In the present study, the involvement of the Tiam1 gene products in bufalin-induced apoptosis in human solid tumor HeLa cells was examined. MATERIALS AND METHODS: HeLa cells were treated with 10(-8) M bufalin and apoptosis was measured by ELISA quantification of nucleosomes. Tiam1 mRNA levels were quantified by real-time PCR analysis and inhibited by transfected siRNA specific for Tiam1. RESULTS: Apoptosis was induced in HeLa cells by treatment with 10(-8) M bufalin. Expression of both Tiam1 mRNA and its protein was induced 0.5 h after the start of the bufalin treatment. Transfection of Tiam1-specific siRNA into HeLa cells markedly inhibited bufalin-induced apoptosis. CONCLUSION: Our results suggest that Tiam1 is a downstream mediator of bufalin-induced apoptosis in the human solid tumor HeLa cell line, as well as in leukemia cell lines.

Expression of estrogen receptor alpha, retinoic acid receptor alpha and cellular retinoic acid binding protein II genes is coordinately regulated in human breast cancer cells.

Human breast cancer cell lines expressing the estrogen receptor alpha (ERalpha), all-trans-retinoic acid (ATRA) receptor alpha (RARalpha) and cellular retinoic acid binding protein II (CRABPII) genes are sensitive to ATRA-mediated growth inhibition. To study the relationship among ERalpha, RARalpha and CRABPII expression, the protein levels of each member were compared in five breast cancer cell lines (T47D, MCF-7, ZR-75-1, Hs587 T and MDA-MB-231 cells) and two immortalized nontumorigenic breast epithelial cell lines (MTSV1.7 and MCF-10A). ERalpha, RARalpha and CRABPII proteins were detected in T47D, MCF-7 and ZR-75-1 cells but not in other tested cell lines. RARalpha and CRABPII proteins were either reduced or undetectable in T47D/C4:2W and MCF-7/ADR cells with lost expression of ERalpha. Estradiol increased and anti-estrogens (tamoxifen and ICI 164,384) downregulated the expression of both RARalpha and CRABPII proteins in T47D and MCF-7 cells. RARalpha antagonist Ro-41-5253 inhibited CRABPII expression, but not RARalpha expression in estradiol-treated T47D and MCF-7 cells. Suppression of ERalpha by small interfering RNA (siRNA) reduced RARalpha and CRABPII gene expression and siRNA suppression of RARalpha reduced CRABPII expression while having no effect on ERalpha in T47D cells. Transient transfection of either RARalpha or ERalpha expression vectors increased CRABPII expression in MDA-MB-231 cells but only RARalpha, not ERalpha, activated hCRABPII promoter reporter. These results indicate that there is a gene activation pathway in which ERalpha drives RARalpha transcription and RARalpha drives CRABPII transcription in ERalpha-positive human breast cancer cells.

Involvement of protein kinase C in the PACAP-induced differentiation of neural stem cells into astrocytes.

Expression of members of the conventional protein kinase C (cPKC) family in the differentiation of mouse neural stem cells (NSCs) induced by pituitary adenylate cyclase-activating polypeptide (PACAP) was investigated. In particular, expression of the alpha and beta subtypes of cPKC in NSCs was observed. In response to activation by PACAP, cPKCbeta transiently increased twofold by day 2 and returned to basal levels by day 4, suggesting that cPKCbeta might be responsible for the differentiation process.

Beta-hydroxyisovalerylshikonin induces apoptosis in human leukemia cells by inhibiting the activity of a polo-like kinase 1 (PLK1).

beta-Hydroxyisovalerylshikonin (beta-HIVS), which was isolated from the plant, Lithospermum radix, induces apoptosis in various lines of human tumor cells. To identify genes involved in beta-HIVS-induced apoptotic process, we performed cDNA array analysis and found that beta-HIVS suppresses the expression of the gene for a polo-like kinase 1 (PLK1) that is involved in control of the cell cycle. When U937 and HL60 cells were treated with 10(-6) M beta-HIVS for 0.5 h, both the amount of PLK1 itself and the kinase activity of this enzyme were decreased. By contrast, Bcr-Abl-positive K562 cells were resistant to the induction of apoptosis by beta-HIVS and this compound did not suppress the kinase activity of PLK1 in these cells. However, simultaneous treatment of K562 cells with both beta-HIVS and STI571, which selectively inhibits the protein tyrosine kinase (PTK) activity of Bcr-Abl, strongly induced apoptosis. Moreover, beta-HIVS increased the inhibitory effect of STI571 on PTK activity. Treatment of K562 cells with antisense oligodeoxynucleotides (ODNs) specific for PLK1 sensitized these cells to the beta-HIVS-induced fragmentation of DNA. These results suggest that suppression of the activity of PLK1 via inhibition of tyrosine kinase activity by beta-HIVS might play a critical role in the induction of apoptosis.

Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes.

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT-PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP(6-38), had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.

Regulation of cytosolic prostaglandin E synthase by phosphorylation.

cPGES [cytosolic PG (prostaglandin) E synthase] is constitutively expressed in various cells and can regulate COX (cyclo-oxygenase)-1-dependent immediate PGE2 generation. In the present study, we found that cPGES underwent serine phosphorylation, which was accelerated transiently after cell activation. Several lines of evidence suggest that a cPGES-activating protein kinase is CK-II (casein kinase II). Recombinant cPGES was phosphorylated directly by and associated with CK-II in vitro, resulting in marked reduction of the K m for the substrate PGH2. In activated cells, cPGES phosphorylation occurred in parallel with increased cPGES enzymic activity and PGE2 production from exogenous and endogenous arachidonic acid, and these processes were facilitated by Hsp90 (heat-shock protein 90), a molecular chaperone that formed a tertiary complex with cPGES and CK-II. Treatment of cells with inhibitors of CK-II and Hsp90 and with a dominant-negative CK-II attenuated the formation of the cPGES-CK-II-Hsp90 complex and attendant cPGES phosphorylation and activation. Mutations of either of two predicted CK-II phosphorylation sites on cPGES (Ser113 and Ser118) abrogated its phosphorylation and activation both in vitro and in vivo. Moreover, the CK-II-Hsp90-mediated activation of cPGES was ameliorated by the p38 mitogen-activated protein kinase inhibitor SB20358 or by the anti-inflammatory glucocorticoid dexamethasone. Taken together, the results of the present study have provided the first evidence that the cellular function of this eicosanoid-biosynthetic enzyme is under the control of a molecular chaperone and its client protein kinase.

Synergistic inhibitory effects of transplatin and beta-hydroxyisovalerylshikon on carcinoma A431 cells involve epidermal growth factor receptor.

Treatment of human epidermoid carcinoma A431 cells with transplatin (trans-DDP) and beta-hydroxyisovalerylshikon (beta-HIVS) together had a strong inhibitory effect on A431 cells. By contrast, trans-DDP and beta-HIVS by themselves, at the same respective concentrations, had practically no effect. The tyrosine kinase activities of v-Src and epidermal growth factor (EGFR) were inhibited by each of the two agents alone and strongly inhibited by combination. The observed synergistic inhibitory effect on the growth of A431 cells might have resulted from the inhibition of EGFR by trans-DDP, as well as by beta-HIVS.

Vitamin K(2) selectively induced apoptosis in ovarian TYK-nu and pancreatic MIA PaCa-2 cells out of eight solid tumor cell lines through a mechanism different from geranylgeraniol.

PURPOSE: In this study, we examined the effects of vitamin K(2) (menaquinone 4), which has a geranylgeranyl side chain, on various lines of cells derived from human solid tumors and compared them with the effects of geranylgeraniol (GGO). METHODS: Cell proliferation was determined with 3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium- bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT), and the induction of apoptosis was analyzed by TUNEL staining and flow cytometry as well as by measurement of DNA fragmentation, released nucleosomes and caspase-3 activity. Levels of Bcl-2, Bax and cytochrome c were determined by immunoblotting. RESULTS: GGO inhibited the growth of all eight cell lines derived from solid tumors, while vitamin K(2) selectively inhibited the proliferation of ovarian TYK-nu and pancreatic MIA PaCa-2 cancer cells, inducing apoptosis in both cell lines. Far more time was required for the induction of apoptosis in these two cell lines by vitamin K(2) than by GGO. Apoptotic signals induced in TYK-nu cells during the first 2 days that followed the addition of vitamin K(2) to the culture medium were reversible, but these signals became irreversible after 3 days of treatment with vitamin K(2). The induction of apoptosis in TYK-nu cells by vitamin K(2) was inhibited by cycloheximide and also by starvation at a low concentration of serum. Neither cycloheximide nor starvation had any effect on the induction of apoptosis by GGO. Cytochrome c was released simultaneously with the initiation of apoptosis on treatment of TYK-nu cells with vitamin K(2) or GGO. However, GGO induced the release of cytochrome c from isolated mitochondria, while vitamin K(2) did not. The amount of Bcl-2 in TYK-nu cells was reduced by vitamin K(2), but not by GGO. CONCLUSIONS: In contrast to GGO, vitamin K(2) induced apoptosis selectively in pancreatic MIA-PaCa 2 and ovarian TYK-nu cancer cells. It is suggested that de novo protein synthesis might be necessary for induction of apoptosis by vitamin K(2) but not by GGO, and thus, that vitamin K(2) and GGO might induce apoptosis by different mechanisms.

[Basic studies for the development of anticancer, antidementia, and taste modifier drugs].

We developed various types of differentiation- and apoptosis-inducing agents against tumor cells and also studied the function and structure of synucleins and taste modifiers. Differentiation- and apoptosis-inducing agents are classified into DNA-damaging agents, Na(+), K(+)-ATPase inhibitors, agents affecting the redox states of tumor cells, agents affecting signal transduction pathways, isoprenoid compounds, and ATP-noncompetitive tyrosine kinase inhibitors. These include camptothecin, etoposide, cisplatin, transplantin, bufalin, arsenic trioxide, costunolide, C(2)- ceramide, daidzein, geranylgeranylacetone, geranylgeraniol, vitamin K(2), sophoranone, and beta-hydroxyisovalerylshikonin. The mechanisms of action of these differentiation- and apoptosis-inducing agents are described. The structure and function of synucleins are also reviewed for the development of potential antidementia agents. In addition, the structures of three purified taste modifiers are described.

Stimulation of dendritic cell maturation and induction of apoptosis in leukemia cells by a heat-stable extract from azuki bean (Vigna angularis), a promising immunopotentiating food and dietary supplement for cancer prevention.

Non-toxic stimulation of dendritic cells (DCs), which are central immunomodulators, may aid the prevention of cancer. Furthermore, induction of apoptosis in cancer cells by anticancer agents contributes to the induction of DC maturation. We previously reported that extracts from Pinus parviflora Sieb. et Zucc pine cone and Mucuna seed induce differentiation of mouse bone marrow cells into mature dendritic cells and also induce apoptosis in various human cancer cell lines. In the present study, we screened 31 kinds of edible beans with biological activity similar to that of extracts from pine cone and Mucuna and found that the heat-stable extract from azuki bean (Vigna angula) stimulated differentiation of bone marrow cells into immature DCs with the greatest efficacy. The level of IL-6 produced by sequential treatment of DCs with azuki extract and lipopolysaccharide was the highest among the examined beans. Azuki extract also inhibited the growth of human leukemia U937 cells, leading to induction of apoptosis. These results suggest that azuki bean and its extract are immunopotentiating foods that can be used as a dietary supplement for cancer prevention and immunotherapy.

Barley low molecular weight ß-glucan potently induces maturation of mouse dendritic cells.

BACKGROUND: Accumulating evidence indicates that non-toxic immunostimulants with strong differentiation/maturation-inducing activity for dendritic cells (DCs) might be useful for preventing or even curing cancer. MATERIALS AND METHODS: Mouse bone marrow (BM) cells were cultured in the presence of various glucans and their differentiation/maturation-inducing activities were compared by measuring cytokines secreted in the culture medium. RESULTS: Barley-derived ß-glucan with an average molecular weight of 2 kDa (BBG-Low) remarkably stimulated the formation of mature DCs from immature mouse DCs. The amount of interleukin-6 produced by sequential treatment of BM cells with granulocyte-macrophage colony-stimulating factor and 10 µg/mL of BBG-Low was approximately 30 times higher than that obtained by a similar sequential treatment using barley ß-glucan of 40-70 kDa instead of BBG-Low. CONCLUSION: BBG-Low induces the formation of mature DCs from immature DCs and suggests that BBG-Low will be useful as a potent nontoxic immunostimulator.

Dendritic cell differentiation and tumor cell apoptosis induced by components of a poly-phenylpropanoid polysaccharide complex.

BACKGROUND: As part of the indigenous folk medicine in Japan, aqueous extracts of pine cones have been used for over a century to treat cancer and other illnesses and references to their use can be found in ancient Greek literature. However, the mechanisms by which such extracts work are largely unknown. MATERIALS AND METHODS: Murine bone marrow (BM)-derived dendritic cells (DCs) and human monocyte U937 cells were treated in vitro with an extract prepared from pine cones (termed poly-phenylpropanoid polysaccharide complex, PPC). RESULTS: The components of the PPC were separated into different molecular weight fractions with distinct biological activities. One fraction, consisting of relatively high molecular weight material, was found to induce the differentiation of murine BM cells into immature DC, as well as the maturation of immature DCs into mature DCs. A second fraction, consisting of low molecular weight material, was found to inhibit the in vitro growth of the U937 cells and two other human cancer cell lines. The inhibition of tumor cell growth was found to be associated with the generation of reactive oxygen species (ROS) and the induction of a mitochondria-dependent apoptotic pathway. CONCLUSION: The effects on dendritic cells and the inhibition of tumor growth, if they occur to a significant level in vivo, could help explain the apparent usefulness of PPC in the treatment of cancer.

Vitamin K2-mediated apoptosis in cancer cells: role of mitochondrial transmembrane potential.

Vitamin K2 induces differentiation and apoptosis in a wide array of human cancer cell lines. Vitamin K2-mediated apoptosis proceeds much more slowly than the apoptosis induced by conventional anticancer agents. Thus, it is possible to analyze the underlying mechanism in detail. In this chapter, we focus on the pro-apoptotic effects of vitamin K2 on mitochondrial physiology with particular emphasis on changes in mitochondrial membrane potential (DeltaPsim). Upon treatment of ovarian cancer TYK-nu cells with vitamin K2, superoxide is produced after two to three days, followed shortly thereafter by release of mitochondrial cytochrome c. This is accompanied by other apoptotic features such as characteristic morphological changes and DNA fragmentation by day four. Data suggest that superoxide production might cause damage to mitochondrial membranes, open permeability transition pores, and result in disruption of DeltaPsim with subsequent release of cytochrome c. Both vitamin K2-induced production of superoxide and reduction of DeltaPsim are completely inhibited by alpha-tocopherol such that cell viability is retained. Thus, we propose that the loss of DeltaPsim caused by superoxide might be the major cause of apoptosis following exposure to vitamin K2. However, other pathways may be involved since cyclosporin A failed to completely inhibit vitamin K2-induced apoptosis.

Induction of apoptosis in PA-1 ovarian cancer cells by vitamin K2 is associated with an increase in the level of TR3/Nur77 and its accumulation in mitochondria and nuclei.

PURPOSE: We examined the growth-inhibitory and apoptosis-inducing effects of vitamin K(2) (VK(2); menaquinone-4) on various lines of human ovarian cancer cells to study the mechanism of induction of apoptosis by VK(2). METHODS: Cell proliferation was determined by XTT method, and apoptotic cells were detected by Hoechst staining. TR3, also known as Nur77 and NGFI-B, was detected by immunoblotting and immunofluorescence analysis. Role of TR3 on induction of apoptosis was examined by a siRNA experiment. RESULTS AND CONCLUSIONS: We found that PA-1 cells were the most sensitive to VK(2) (IC(50) = 5.0 +/- 0.7 microM), while SK-OV-3 cells were resistant to VK(2). Immunoblotting and immunofluorescence analyses indicated that levels of TR3 were elevated in cell lysates 48 h after the start of treatment with 30 microM VK(2). In the VK(2)-treated cells, TR3 accumulated at significant levels in mitochondria, as well as in the nuclei of PA-1 cells. No similar changes were observed in SK-OV-3 cells under the same conditions. Treatment of PA-1 cells with small interfering RNA (siRNA) directed against TR3, and with cycloheximide or SP600125 (an inhibitor of c-jun N-terminal kinase; JNK), separately, inhibited the VK(2)-induced synthesis of TR3 and apoptosis. From these results, we can conclude that an increase in the synthesis of TR3 and the accumulation of TR3 in mitochondria and in nuclei might be involved in the induction of apoptosis by VK(2) and that the synthesis of TR3 might be regulated through a JNK signaling pathway.

Increase in intracellular Ca(2+) concentrations and the corresponding intracellular acidification are early steps for induction of apoptosis by geranylgeraniol in HL60 cells.

To elucidate the mechanism of induction of apoptosis by geranylgeraniol (GGO), which is a potent inducer of apoptosis in various lines of human cancer cells, we examined the role of intracellular acidification during GGO-induced apoptosis using human leukemia HL60 cells. Flow cytometry analysis revealed that apoptosis induced in human leukemia HL60 cells by GGO was associated with intracellular acidification. Both GGO-induced intracellular acidification and apoptosis as analyzed by DNA fragmentation were inhibited by phorbol myristate acetate (TPA) and O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), an intracellular Ca(2+) chelator, but not by ethyleneglycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). These results suggest that the early concentration change of intracellular Ca(2+) and the corresponding decrease in intracellular pH are required for the induction of apoptosis in HL60 cells by GGO.

A novel 21-kDa cytochrome c-releasing factor is generated upon treatment of human leukemia U937 cells with geranylgeraniol.

Geranylgeraniol (GGO) induces apoptosis in various lines of human tumor cells through a mitochondrion-dependent pathway. The present study describes identification of a 21-kDa cytochrome c-releasing factor that appears in the cytosolic fraction after treatment of human leukemia U937 cells with GGO. Incubation of isolated mitochondria with a lysate of U937 cells that had been treated with GGO resulted in the release of cytochrome c from the mitochondria. Utilizing this cell-free system, we purified a 21-kDa protein that induced the release of cytochrome c from mitochondria and appeared to be involved in the apoptosis that is induced in U937 cells by GGO. We designated this protein cytochrome c-releasing factor 21 (CRF21). Overexpression of CRF21 in HeLa cells induced the release of cytochrome c from mitochondria, with subsequent apoptosis. Our results suggest that CRF21 might play an important role in the induction of apoptosis by GGO in leukemia U937 cells.

Production of superoxide and dissipation of mitochondrial transmembrane potential by vitamin K2 trigger apoptosis in human ovarian cancer TYK-nu cells.

We reported previously that vitamin K(2) selectively induces apoptosis in human ovary cancer cells (TYK-nu cells) and pancreatic cancer cells (MIA PaCa-2 cells) through a mitochondrion-dependent pathway. In the present study, we examined the details of the mechanism of vitamin K(2)-induced apoptosis in TYK-nu cells. We found that superoxide (O(2)(*-)) was produced by TYK-nu cells between 2 and 3 days after the start of treatment with vitamin K(2), whereas it was produced within 30 min after the start of treatment with geranylgeraniol. The vitamin K(2)-induced apoptosis was inhibited by anti-oxidants, such as alpha-tocopherol, Tiron and N-acetyl-L-cysteine (NAC). Furthermore, both the production of superoxide and the induction of apoptosis by vitamin K(2) were inhibited almost completely by cycloheximide, an inhibitor of protein synthesis, suggesting that the synthesis of enzymes for the production of superoxide might be required for these processes. In parallel with the production of superoxide, the mitochondrial transmembrane potential, as measured by staining with Mitotracker Red CMXRos, dissipated during treatment of TYK-nu cells with vitamin K(2) for 3 days. The vitamin K(2)-induced depolarization of mitochondrial membranes was completely inhibited by alpha-tocopherol and, to a lesser extent, by Tiron and NAC. Since alpha-tocopherol reacts with oxygen radicals, such as superoxide, within the hydrophobic environment of the mitochondrial membrane, we postulate that vitamin K(2)-induced oxidative stress in mitochondria might damage mitochondrial membranes, with subsequent release of cytochrome c, the activation of procaspase 3 and, eventually, apoptosis.