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1.
Asian Pac J Cancer Prev ; 13(2): 607-11, 2012.
Article in English | MEDLINE | ID: mdl-22524832

ABSTRACT

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.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Bone Marrow Cells/drug effects , Dendritic Cells/cytology , Dendritic Cells/drug effects , Fabaceae/chemistry , Leukemia, Experimental/prevention & control , Plant Extracts/pharmacology , Animals , Bone Marrow Cells/cytology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Dietary Supplements , Humans , Immunotherapy , Interleukin-6/metabolism , Leukemia, Experimental/immunology , Male , Mice , Mice, Inbred C57BL , U937 Cells
2.
Anticancer Res ; 31(5): 1647-51, 2011 May.
Article in English | MEDLINE | ID: mdl-21617222

ABSTRACT

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.


Subject(s)
Dendritic Cells/cytology , Dendritic Cells/drug effects , Hordeum/chemistry , beta-Glucans/pharmacology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Cells, Cultured , Dendritic Cells/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interleukin-6/metabolism , Mice , Molecular Weight
3.
Nutr Cancer ; 63(1): 100-8, 2011.
Article in English | MEDLINE | ID: mdl-21170811

ABSTRACT

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.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Bone Marrow Cells/drug effects , Dendritic Cells/drug effects , Mucuna , Plant Extracts/pharmacology , Animals , Bone Marrow Cells/cytology , Cell Differentiation/drug effects , Cell Survival/drug effects , Dendritic Cells/cytology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interleukin-6/biosynthesis , Male , Mice , Mice, Inbred C57BL , Superoxides/metabolism , U937 Cells
4.
Anticancer Res ; 30(2): 613-22, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20332479

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Cell Differentiation/drug effects , Dendritic Cells/drug effects , Leukemia/pathology , Ovarian Neoplasms/pathology , Plant Extracts/pharmacology , Animals , Blotting, Western , Bone Marrow Cells/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Leukemia/drug therapy , Leukemia/metabolism , Male , Mice , Mice, Inbred C57BL , Monocytes/drug effects , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Pinus/chemistry , Reactive Oxygen Species/metabolism
5.
Cancer Sci ; 100(2): 269-77, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19200258

ABSTRACT

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.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Carcinoma, Lewis Lung/blood supply , Drugs, Chinese Herbal/pharmacology , Naphthoquinones/pharmacology , Neovascularization, Pathologic/prevention & control , Animals , Apoptosis , Blotting, Western , Carcinoma, Lewis Lung/drug therapy , Cell Adhesion , Cell Movement , Cell Proliferation , Cells, Cultured , Chickens , Chorioallantoic Membrane/metabolism , Chorioallantoic Membrane/pathology , Electrophoretic Mobility Shift Assay , Female , Immunoenzyme Techniques , Immunoprecipitation , Luciferases/metabolism , Mice , Mice, Inbred C57BL , Phosphorylation , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Transfection , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism
6.
Vitam Horm ; 78: 211-26, 2008.
Article in English | MEDLINE | ID: mdl-18374196

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Membrane Potential, Mitochondrial/physiology , Neoplasms/pathology , Vitamin K 2/pharmacology , Animals , Cell Differentiation/drug effects , Cell Line, Tumor , Humans , Leukemia/pathology
7.
J Cancer Res Clin Oncol ; 134(7): 803-12, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18202854

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Cell Nucleus/metabolism , Mitochondria/metabolism , Ovarian Neoplasms/metabolism , Receptors, Steroid/biosynthesis , Receptors, Thyroid Hormone/biosynthesis , Vitamin K 2/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Female , Fluorescent Antibody Technique , Humans , Nuclear Receptor Subfamily 4, Group A, Member 1 , Ovarian Neoplasms/pathology , RNA, Small Interfering , Signal Transduction/physiology , Up-Regulation
8.
Biochim Biophys Acta ; 1782(1): 41-50, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18078828

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Deoxyuracil Nucleotides/metabolism , Lung Neoplasms/enzymology , Naphthoquinones/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrophosphatases/metabolism , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Antioxidants/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Deoxyuracil Nucleotides/genetics , Enzyme Activation/drug effects , Fluorouracil/pharmacology , Humans , Lung Neoplasms/pathology , Molecular Sequence Data , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Pyrophosphatases/chemistry , RNA, Small Interfering/genetics
9.
Biol Pharm Bull ; 30(5): 880-4, 2007 May.
Article in English | MEDLINE | ID: mdl-17473429

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Calcium/metabolism , DNA Fragmentation/drug effects , Diterpenes/pharmacology , Cytochromes c/metabolism , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , HL-60 Cells , Humans , Hydrogen-Ion Concentration , Mitochondria/metabolism , Tetradecanoylphorbol Acetate/pharmacology
10.
Anticancer Res ; 27(1A): 245-9, 2007.
Article in English | MEDLINE | ID: mdl-17352239

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Bufanolides/pharmacology , Guanine Nucleotide Exchange Factors/genetics , Apoptosis/physiology , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Guanine Nucleotide Exchange Factors/biosynthesis , HeLa Cells , Humans , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Interfering/genetics , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , Transfection
11.
J Neurosci Res ; 84(8): 1645-55, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17022039

ABSTRACT

We have found previously that pituitary adenylate cyclase-activating polypeptide (PACAP) increases the number of astrocytes generated from cultured mouse neural stem cells (NSCs) via a mechanism that is independent of the cyclic AMP/protein kinase A pathway (Ohno et al., 2005). In the present study, the signaling pathway involved in the differentiation process was further investigated. PACAP-induced differentiation was inhibited by the phospholipase C inhibitor, U73122, the protein kinase C (PKC) inhibitor, chelerythrine, and the intracellular calcium chelator, BAPTA-AM, and was mimicked by phorbol 12-myristate 13-acetate (PMA), but not by 4alpha-PMA. These results suggest that the PACAP-generated signal was mediated via the PACAP receptor, PAC1 stimulated heterotrimeric G-protein, resulting in activation of phospholipase C, followed by calcium- and phospholipid-dependent protein kinase C (cPKC). To elucidate the involvement of the different isoforms of cPKC, their gene and protein expression were examined. Embryonic NSCs expressed alpha and betaII PKC, but lacked PKCgamma. When NSCs were exposed to 2 nM PACAP, protein expression levels of the betaII isoform transiently increased two-fold before differentiation, returning to basal levels by Day 4, whereas the level of PKCalpha increased linearly up to Day 6. Overexpression of PKCbetaII with adenovirus vector synergistically enhanced differentiation in the presence of 1 nM PACAP, whereas expression of the dominant-negative mutant of PKCbetaII proved inhibitory. These results indicate that the beta isoform of PKC plays a crucial role in the PACAP-induced differentiation of mouse embryonic NSCs into astrocytes.


Subject(s)
Astrocytes/physiology , Cell Differentiation/drug effects , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , Protein Kinase C/metabolism , Stem Cells/drug effects , Adenoviridae/physiology , Analysis of Variance , Animals , Cell Count/methods , Cell Differentiation/physiology , Cells, Cultured , Drug Interactions , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/physiology , Glial Fibrillary Acidic Protein/metabolism , Intermediate Filament Proteins/metabolism , Mice , Mice, Inbred ICR , Nerve Tissue Proteins/metabolism , Nestin , Protein Isoforms/metabolism , Protein Kinase C beta , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/methods , Stem Cells/physiology , Transfection/methods
12.
Ann N Y Acad Sci ; 1070: 597-601, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16888232

ABSTRACT

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.


Subject(s)
Astrocytes/drug effects , Astrocytes/enzymology , Cell Differentiation/drug effects , Neurons/drug effects , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , Protein Kinase C/metabolism , Stem Cells/drug effects , Animals , Astrocytes/cytology , Cells, Cultured , Gene Expression Regulation, Enzymologic , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Mice, Inbred ICR , Neurons/cytology , Neurons/enzymology , Protein Kinase C/genetics , Stem Cells/cytology , Stem Cells/enzymology
13.
Apoptosis ; 11(9): 1535-43, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16763728

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Membrane Potentials/physiology , Mitochondria/drug effects , Ovarian Neoplasms/drug therapy , Superoxides/pharmacology , Vitamin K 2/pharmacology , 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt/pharmacology , Acetylcysteine/pharmacology , Cycloheximide/pharmacology , Diterpenes/pharmacology , Female , Humans , Membrane Potentials/drug effects , Mitochondria/metabolism , Ovarian Neoplasms/metabolism , Reactive Oxygen Species/pharmacology , Superoxides/metabolism , Tumor Cells, Cultured , Vitamin K 2/therapeutic use , alpha-Tocopherol/pharmacology
14.
Biochem Biophys Res Commun ; 346(2): 454-60, 2006 Jul 28.
Article in English | MEDLINE | ID: mdl-16765912

ABSTRACT

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.


Subject(s)
Cytochromes c/metabolism , Diterpenes/pharmacology , Mitochondrial Proteins/biosynthesis , Amino Acid Sequence , Apoptosis , HeLa Cells , Humans , Mitochondria/drug effects , Mitochondria/metabolism , U937 Cells
15.
Oncogene ; 24(27): 4362-9, 2005 Jun 23.
Article in English | MEDLINE | ID: mdl-15870697

ABSTRACT

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.


Subject(s)
Breast Neoplasms/genetics , Estrogen Receptor alpha/metabolism , Gene Expression Regulation, Neoplastic , Receptors, Retinoic Acid/metabolism , Animals , Benzoates/pharmacology , Breast Neoplasms/metabolism , Cell Line , Chlorocebus aethiops , Chromans/pharmacology , Estradiol/pharmacology , Estrogen Receptor Modulators/pharmacology , Estrogen Receptor alpha/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Promoter Regions, Genetic/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Retinoic Acid/antagonists & inhibitors , Receptors, Retinoic Acid/genetics , Retinoic Acid Receptor alpha , Transcriptional Activation , Up-Regulation/drug effects
16.
Regul Pept ; 126(1-2): 115-22, 2005 Mar 15.
Article in English | MEDLINE | ID: mdl-15620424

ABSTRACT

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.


Subject(s)
Astrocytes/physiology , Cell Differentiation/drug effects , Nerve Growth Factors/pharmacology , Nerve Tissue/physiology , Neuropeptides/pharmacology , Neurotransmitter Agents/pharmacology , Stem Cells/physiology , Animals , Cell Differentiation/physiology , Cells, Cultured , Mice , Nerve Tissue/cytology , Pituitary Adenylate Cyclase-Activating Polypeptide
17.
J Biol Chem ; 279(41): 42503-15, 2004 Oct 08.
Article in English | MEDLINE | ID: mdl-15292218

ABSTRACT

beta-Hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in various lines of human tumor cells. However, the way in which beta-HIVS induces apoptosis remains to be clarified. In this study, we performed cDNA array analysis and found that beta-HIVS suppressed the expression of the gene for tumor necrosis factor receptor-associated protein 1 (TRAP1), which is a member of the heat-shock family of proteins. When human leukemia HL60 cells and human lung cancer DMS114 cells were treated with beta-HIVS, the amount of TRAP1 in mitochondria decreased in a time-dependent manner during apoptosis. A similar reduction in the level of TRAP1 was also observed upon exposure of cells to VP16. Treatment of DMS114 cells with TRAP1-specific siRNA sensitized the cells to beta-HIVS-induced apoptosis. Moreover, the reduction in the level of expression of TRAP1 by TRAP1-specific siRNA enhanced the release of cytochrome c from mitochondria when DMS114 cells were treated with either beta-HIVS or VP16. The suppression of the level of TRAP1 by either beta-HIVS or VP16 was blocked by N-acetyl-cysteine, indicating the involvement of reactive oxygen species (ROS) in the regulation of the expression of TRAP1. These results suggest that suppression of the expression of TRAP1 in mitochondria might play an important role in the induction of apoptosis caused via formation of ROS.


Subject(s)
Apoptosis , HSP90 Heat-Shock Proteins/physiology , Naphthoquinones/pharmacology , Acetylcysteine/chemistry , Acetylcysteine/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Antioxidants/pharmacology , Blotting, Northern , Blotting, Western , Cell Death , Cell Line , Cell Line, Tumor , Coloring Agents/pharmacology , Cytochromes c/metabolism , Cytosol/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Etoposide/pharmacology , Gene Expression Regulation , Genetic Vectors , HL-60 Cells , Humans , K562 Cells , Mitochondria/metabolism , Mitochondria/pathology , Oligonucleotide Array Sequence Analysis , Plasmids/metabolism , RNA, Small Interfering/metabolism , Reactive Oxygen Species , Subcellular Fractions/metabolism , Time Factors , Transfection
18.
Yakugaku Zasshi ; 124(7): 371-96, 2004 Jul.
Article in Japanese | MEDLINE | ID: mdl-15235223

ABSTRACT

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.


Subject(s)
Antineoplastic Agents , Drug Design , Nootropic Agents , Taste Disorders/drug therapy , Taste/drug effects , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Cell Differentiation/drug effects , DNA Damage , Humans , Neoplasms/pathology , Nerve Tissue Proteins , Oxidation-Reduction , Signal Transduction , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Synucleins
19.
Biol Pharm Bull ; 27(5): 736-8, 2004 May.
Article in English | MEDLINE | ID: mdl-15133257

ABSTRACT

Shimizu and Tsuji established a method of preparing colloidal platinum nanoparticles, whose average size is 2 nm, by ethanol reduction of H(2)PtCl(6) in the absence of protective agents for the particles. Platinum nanoparticles have negative surface potential and are stably suspended from an electric repulsion between them. The platinum nanoparticles reduced hydrogen peroxide and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) below 0.1 ppm. It is necessary to use higher concentration of platinum nanoparticles for the reduction of 2,6-dichlorophenol indophenol (DCIP) than that of hydrogen peroxide and 2,2-diphenyl-1-picrylhydrazyl radical, because reoxidation of DCIPH(2) (reduced) by oxygen was not negligible under our experimental conditions. These results indicate that electrons on platinum nanoparticles produced by the method of Shimizu and Tsuji can reduce hydrogen peroxide, DPPH radical or DCIP transferring electrons.


Subject(s)
2,6-Dichloroindophenol/metabolism , Biphenyl Compounds/metabolism , Hydrazines/metabolism , Hydrogen Peroxide/metabolism , Nanostructures/chemistry , Platinum/pharmacology , Colloids , Dose-Response Relationship, Drug , Picrates
20.
Oncology ; 66(1): 67-75, 2004.
Article in English | MEDLINE | ID: mdl-15031601

ABSTRACT

beta-Hydroxyisovalerylshikonin (beta-HIVS) and cisplatin (CDDP) had a synergistic growth-inhibitory effect on cultured human small-cell lung carcinoma DMS114 cells, as well as on human leukemia U937 and epidermoid carcinoma A431 cells, while beta-HIVS and CDDP alone at the same respective concentrations had little effect. Growth inhibition was accompanied by induction of apoptosis, as determined by an ELISA for the detection of cell death and the TUNEL assay. Using phosphotyrosine-specific antibodies (PY20), we observed that tyrosine kinase activity in DMS114 cells was inhibited by treatment with beta-HIVS and CDDP together. The tyrosine kinase activity of isolated Src and that of isolated receptors for epidermal growth factor were also inhibited by the two agents together. The synergistic effects of the growth of DMS114 cells of beta-HIVS and CDDP were not due simply to the intracellular accumulation of CDDP or to levels of DNA adducts. Our data suggest that the synergistic effect on the growth of DMS114 cells of beta-HIVS and CDDP might be a result of the inhibition of a tyrosine kinase-dependent pathway.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Cisplatin/pharmacology , Lung Neoplasms/drug therapy , Naphthoquinones/pharmacology , Protein-Tyrosine Kinases/metabolism , Blotting, Western , Drug Synergism , Humans , In Situ Nick-End Labeling , Lung Neoplasms/enzymology , Protein-Tyrosine Kinases/drug effects , Tumor Cells, Cultured
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