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1.
J Toxicol Environ Health A ; 73(21-22): 1465-76, 2010.
Article in English | MEDLINE | ID: mdl-20954073

ABSTRACT

Ceramide induces cell cycle arrest and apoptotic cell death associated with increased levels of p27(kip1). The aim of this study was to examine the effects of ceramide on p27(kip1) protein levels as a measure of cell cycle arrest and apoptosis. Results showed that ceramide increased p27(kip1) protein levels through activation of protein phosphatase 2A (PP2A) in PC-3 prostate cancer cells. Treatment of cells with the PP2A inhibitor okadaic acid or with PP2A-Cα siRNA inhibited ceramide-induced enhanced p27(kip1) protein expression and Akt dephosphorylation, and prevented Skp2 downregulation. Overexpression of constitutively active Akt attenuated ceramide-induced Skp2 downregulation and p27(kip1) upregulation. In addition, ceramide stimulated binding of the PP2A catalytic subunit PP2A-Cαß to Akt as assessed by immunoprecipitation experiments, indicating that PP2A is involved in the induction of p27(kip1) via inhibition of Akt pathway. Finally, whether PP2A can regulate p27(kip1) expression independently of Akt pathway was determined. Knockdown of PP2A-Cα with siRNA reduced p27(kip1) levels in the presence of Akt inhibitor. These data reveal that PP2A is a regulator of ceramide-induced p27(kip1) expression via Akt-dependent and Akt-independent pathways.


Subject(s)
Apoptosis/drug effects , Ceramides/toxicity , Intracellular Signaling Peptides and Proteins/metabolism , Prostatic Neoplasms/drug therapy , Protein Phosphatase 2/biosynthesis , Proto-Oncogene Proteins c-akt/biosynthesis , Cell Cycle/drug effects , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p27 , Gene Silencing , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Phosphorylation/drug effects , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA, Small Interfering/metabolism , Transfection
2.
Toxicol Lett ; 189(1): 84-9, 2009 Aug 25.
Article in English | MEDLINE | ID: mdl-19463925

ABSTRACT

Human cytochrome P450 1B1 (CYP1B1) is found mainly in extrahepatic tissues and is overexpressed in a variety of human tumors. Metabolic activation of 17beta-estradiol (E2) to 4-hydroxy E2 by CYP1B1 has been postulated to be an important factor in mammary carcinogenesis. The inhibition of recombinant human CYP1B1 by 2,2',4,6'-tetramethoxystilbene (TMS) was investigated using either the Escherichia coli membranes of recombinant human CYP1B1 coexpressed with human NADPH-P450 reductase or using purified enzyme. 2,2',4,6'-TMS showed potent and selective inhibition of ethoxyresorufin O-deethylation (EROD) activity of CYP1B1 with IC(50) values of 2 nM. 2,2',4,6'-TMS exhibited 175-fold selectivity for CYP1B1 over CYP1A1 (IC(50), 350 nM) and 85-fold selectivity for CYP1B1 over CYP1A2 (IC(50), 170 nM). However, inhibition of human NADPH-P450 reductase activity by 2,2',4,6'-TMS was negligible. The modes of inhibition by 2,2',4,6'-TMS were noncompetitive for CYP1A1 and CYP1B1. Moreover, 2,2',4,6'-TMS significantly suppressed EROD activity and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 or CYP1B1 gene expression in human tumor cells such as HepG2 and MCF-10A. Taken together, our results indicate that 2,2',4,6'-TMS is a potently selective inhibitor of human CYP1B1 as well as a suppressor of CYP1B1 expression and may be a valuable tool for determining enzyme properties of human CYP1B1.


Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Recombinant Proteins/antagonists & inhibitors , Stilbenes/pharmacology , Aryl Hydrocarbon Hydroxylases , Cell Line, Tumor , Cytochrome P-450 CYP1A1/antagonists & inhibitors , Cytochrome P-450 CYP1B1 , Cytochrome P-450 Enzyme System/genetics , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Escherichia coli/genetics , Humans , Molecular Structure , Polychlorinated Dibenzodioxins/toxicity , Recombinant Proteins/genetics , Stilbenes/chemistry
3.
Arch Pharm Res ; 31(9): 1187-94, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18816901

ABSTRACT

trans-Stilbenes induce cytochrome P450 1B1 (CYP1B1) inhibition and cell death. 2,4,3',5' tetramethoxystilbene (TMS), a synthetic trans-stilbene analog, induced apoptotic cell death in PC-3 prostate cancer cells, as evidenced by a decrease in the mitochondrial membrane potential. TMS-induced apoptosis was associated with an increase in the level of cell cycle inhibitor, p27(kip1), through reduction of Akt-mediated Skp2 expression. TMS-induced activation of protein phosphatase 2A (PP2A) inhibited Akt phosphorylation and p27(kip1) expression, indicating that PP2A is involved in the induction of p27(kip1) via Akt inhibition. These results suggest that TMS may inhibit the cell cycle through induction of p27(kip1), leading to apoptotic cell death in PC-3 prostate cancer cells.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/biosynthesis , Oncogene Protein v-akt/metabolism , Prostatic Neoplasms/drug therapy , Protein Phosphatase 2/metabolism , Stilbenes/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Flow Cytometry , Glyceraldehyde-3-Phosphate Dehydrogenases/biosynthesis , Humans , Male , Membrane Potentials/drug effects , Mitochondrial Membranes/drug effects , Oncogene Protein v-akt/genetics , Phosphorylation/drug effects , Prostatic Neoplasms/pathology , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , S-Phase Kinase-Associated Proteins/biosynthesis , S-Phase Kinase-Associated Proteins/genetics , Transfection
4.
Arch Pharm Res ; 31(4): 438-44, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18449500

ABSTRACT

trans-Stilbenes have been reported to induce cytochrome P450 1B1 (CYP1B1) inhibition and cell death, however, the molecular mechanisms of the effects are not fully understood. We report here that (1-(2-{3-[2-(2,4-dimethoxy-phenyl)-vinyl]-5-methoxy-phenoxy}ethyl)-1H-imidazole), a synthetic stilbene analog (SA) significantly suppressed TCDD-stimulated CYP1B1 mRNA expression. In HL-60 cells, SA induced apoptosis through activation of p38 MAPK and inactivation of Akt, which in turn activated Bad and mitochondrial death signaling pathway, as evidenced by Bax translocation and cytochrome c release. Expression of dominant negative p38 MAPK or constitutively active Akt significantly prevented cell death and mitochondrial Bax translocation, implicating that p38 MAPK and Akt signaling pathways play crucial roles in stilbene-induced apoptosis of HL-60 cells. These results suggest that SA induces apoptotic cell death as well as CYP1B1 inhibition and may thus be beneficial in cancer prevention.


Subject(s)
Anticarcinogenic Agents/pharmacology , Apoptosis/drug effects , Mitochondria/drug effects , Oncogene Protein v-akt/metabolism , Signal Transduction/drug effects , Stilbenes/pharmacology , bcl-2-Associated X Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Aryl Hydrocarbon Hydroxylases , Cell Survival/drug effects , Cytochrome P-450 CYP1B1 , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/genetics , Cytochromes c/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Repression , HL-60 Cells , Humans , Mitochondria/enzymology , Mitochondria/metabolism , Oncogene Protein v-akt/genetics , Polychlorinated Dibenzodioxins/pharmacology , Protein Transport , RNA, Messenger/biosynthesis , Time Factors , Transfection , bcl-Associated Death Protein/metabolism , p38 Mitogen-Activated Protein Kinases/genetics
5.
Cancer Lett ; 260(1-2): 88-95, 2008 Feb 18.
Article in English | MEDLINE | ID: mdl-18054155

ABSTRACT

Ceramide induces apoptosis through caspase activation, cytochrome c release, and Bax translocation in HL-60 cells. However, the upstream signal transduction pathways that induce Bax translocation during ceramide-mediated apoptosis have not been well defined yet. In this study, the activation of p38 mitogen-activated protein kinase (MAPK) was found to be critical for the induction of apoptosis and subcellular redistribution of Bax. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a dominant-negative p38 MAPK attenuated DNA fragmentation, caspase-3 activation, and Bax translocation in response to ceramide. Overexpression of Akt also led to suppression of Bax translocation to mitochondria during ceramide-induced apoptosis in HL-60 cells. We also provide evidence for cross-talk between p38 MAPK and Akt pathways. Expression of myr-Akt or inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 had no effect on p38 MAPK activation by ceramide as assessed by phosphorylation, while inhibition of p38 MAPK by a pharmacological inhibitor or a dominant-negative p38 inhibited Akt dephosphorylation in response to ceramide, suggesting that ceramide-induced p38 MAPK activation negatively regulates the Akt pathway.


Subject(s)
Apoptosis , Ceramides/metabolism , Leukemia, Promyelocytic, Acute/metabolism , Protein Kinase Inhibitors/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , bcl-2-Associated X Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Caspase 3/metabolism , Ceramides/pharmacology , Enzyme Activation , HL-60 Cells , Humans , Imidazoles/pharmacology , Leukemia, Promyelocytic, Acute/enzymology , Leukemia, Promyelocytic, Acute/pathology , Mitochondria/metabolism , Mutation , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Transport , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Pyridines/pharmacology , Signal Transduction/drug effects , Time Factors , Transfection , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/genetics
6.
J Toxicol Environ Health A ; 70(15-16): 1259-63, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17654243

ABSTRACT

Styrene is an important industrial chemical that is extensively used in the production of resins, rubbers and fiberglass-reinforced plastics. Exposing male rats to high doses of styrene may produce sperm abnormalities or infertility. To determine the mechanism underlying styrene-mediated toxicity in male reproductive organs, a reverse transcription-polymerase chain reaction (RT-PCR) technology was employed using annealing control primers (ACPs) to identify the differentially expressed genes following styrene treatment in isolated testis of male rats. By using 120 ACPs, a total of 6 expressed sequence tags (ESTs) of genes were differentially expressed in styrene-treated rats, as compared to untreated, which were cloned and sequenced. Of the genes analyzed, 5 genes (testis-specific expressed gene 101, protein kinase C, H+-ATPase isoform 2, peroxiredoxin 1, and aquaporin 9) were inducible and one gene expression (clusterin) was significantly suppressed by styrene. Regulation of each gene by styrene was confirmed by RT-PCR. It was shown that styrene decreased clusterin expression in a concentration-dependent manner and these effects occurred mainly in testis. Taken together, these results indicate that repression of clusterin gene expression by styrene may play an important role in styrene-mediated toxicities.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation/drug effects , Spermatozoa/drug effects , Styrene/toxicity , Testis/drug effects , Administration, Oral , Animals , Clusterin/genetics , Clusterin/metabolism , Disease Models, Animal , Expressed Sequence Tags/metabolism , Male , Mutagenicity Tests , Rats , Rats, Sprague-Dawley , Styrene/administration & dosage
7.
Arch Pharm Res ; 29(12): 1140-6, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17225464

ABSTRACT

Ceramide analogs are potential chemotherapeutic agents. We report that a ceramide analog induces apoptosis in human prostate cancer cells. The ceramide analog induced cell death through an apoptotic mechanism, which was demonstrated by DNA fragmentation, the cleavage of poly ADP ribose polymerase (PARP), and a loss of membrane asymmetry. Treating the cells with ceramide analog resulted in the release of various proapoptotic mitochondrial proteins including cytochrome c and Smac/DIBLO into the cytosol, and a decrease in the mitochondrial membrane potential. In addition, the ceramide analog decreased the phospho-Akt and phospho-Bad levels. The expression of the antiapoptotic Bcl-2 decreased slightly with increasing Bax to Bcl-2 ratio. These results suggest that the ceramide analog induces apoptosis by regulating multiple signaling pathways that involve the mitochondrial pathway.


Subject(s)
Apoptosis/drug effects , Ceramides/pharmacology , Prostatic Neoplasms/drug therapy , Blotting, Western , Cell Proliferation/drug effects , Cytochromes c/metabolism , DNA Fragmentation/drug effects , Genes, bcl-2 , Humans , Male , Membrane Potentials/drug effects , Mitochondria/physiology , Neoplasm Proteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Prostatic Neoplasms/pathology , Signal Transduction/drug effects , Signal Transduction/physiology , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism
8.
Drug Metab Dispos ; 33(12): 1771-6, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16120791

ABSTRACT

We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a synthetic trans-stilbene analog, is one of the most potently selective inhibitors of recombinant human cytochrome P450 1B1 (CYP1B1) in vitro. In the present studies, the effects of TMS on CYP1B1 expression were investigated in human cancer cells. TMS significantly inhibited CYP1-mediated 7-ethoxyresorufin O-deethylation activity in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced MCF-7 cells or lung microsomes of Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene. TCDD-stimulated CYP1B1 protein and mRNA expression was significantly suppressed by TMS in a concentration-dependent manner in MCF-7, MCF-10A, and HL-60 cells. Whereas TMS down-regulated TCDD-induced CYP1B1 gene expression, the levels of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNA expression were not changed by TMS treatment. In human cancer cells, TMS induced apoptotic cell death, and the cytotoxic effects of TMS were significant when the cells were incubated with TCDD. CYP1B1 was able to convert TMS to a metabolite(s) when incubated with NADPH. Metabolic activation of TMS by CYP1B1 induced by TCDD may mediate cellular toxicity of TMS in human cancer cells because the sensitivity to TMS in MCF-7 cells treated with TCDD was more significant than in HL-60 cells treated with TCDD. Taken together, our results indicate that TMS acts as a strong modulator of CYP1B1 gene expression as well as a potent selective inhibitor in vitro. The ability of TMS to induce apoptotic cell death in tumor cells, as well as CYP1B1 inhibition, may contribute to its usefulness for cancer chemoprevention.


Subject(s)
Anticarcinogenic Agents/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Gene Expression Regulation, Enzymologic/drug effects , Plant Extracts/pharmacology , Stilbenes/pharmacology , Apoptosis/drug effects , Aryl Hydrocarbon Hydroxylases , Cell Line, Tumor , Cytochrome P-450 CYP1B1 , Cytochrome P-450 Enzyme System/genetics , Dose-Response Relationship, Drug , Humans , Plant Extracts/metabolism , Polychlorinated Dibenzodioxins/pharmacology , Stilbenes/metabolism
9.
Arch Pharm Res ; 28(1): 87-92, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15742814

ABSTRACT

The cyclin-dependent kinase inhibitor p27kip1 (p27) has been implicated in the regulation of cell cycle and apoptosis. Recently, we have demonstrated that ceramide induces apoptotic cell death associated with increase in the level of p27 in HL-60 cells. In the present study, we showed that overexpression of p27 increases ceramide-induced apoptotic cell death in HL-60 cells. Furthermore, overexpression of p27 accelerated DNA fragmentation, PARP cleavage and cytochrome c release induced by ceramide. In addition, ceramide induced Bax expression independent of p27. These findings indicate that enhanced effect on apoptosis by p27 is associated with mitochondrial signaling which involves cytochrome c release.


Subject(s)
Apoptosis/drug effects , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Ceramides/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Apoptosis/physiology , Cyclin-Dependent Kinase Inhibitor p27 , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , HL-60 Cells , Humans
10.
J Toxicol Environ Health A ; 67(23-24): 1987-2000, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15513897

ABSTRACT

Previously it was reported that various hydroxystilbene compounds strongly inhibit human cytochrome P-450 1 enzymes and were postulated as candidate chemopreventive agents. In this study, the inhibitory potential of P-450 1 enzyme activities by 3,5,3,4,5-pentamethoxystilbene (PMS), a synthetic stilbene compound, was evaluated with the Escherichia coli (E. coil) membranes of recombinant human cytochrome P-450 1A1, 1A2, or 1B1 coexpressed with human NADPH-P-450 reductase. PMS produced a significant inhibition of ethoxyresorufin O-deethylation (EROD) activities with IC50 values of 0.14, 934, and 3.2 M for 1A1, 1A2, and 1B1, respectively. PMS did not significantly inhibit EROD activities in human liver microsomes. To elucidate the mechanism of inhibition by PMS, kinetic studies were performed. Analysis of the mode of inhibition indicated a mixed-type inhibition of P-450 1A1. The inhibition of P-450 1A1-mediated EROD activity by PMS was not irreversible-mechanism based. The loss of EROD activity of P-450 1A1 with PMS was blocked by trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol. Moreover, PMS significantly suppressed P-450 1A1-mediated EROD activity and P-450 1A1 gene expression in HepG2 cells induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Taken together, the results suggested that PMS is a potent and selective inhibitor of human P-450 1A1 and may be considered for use as a cancer chemopreventive agent in humans.


Subject(s)
Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP1A1/pharmacology , Enzyme Inhibitors/pharmacology , Stilbenes/pharmacology , Carcinoma, Hepatocellular/pathology , Cell Membrane , Escherichia coli , Gene Expression Regulation/drug effects , Humans , Liver Neoplasms/pathology , Microsomes, Liver/enzymology , Tumor Cells, Cultured
11.
Arch Pharm Res ; 27(2): 199-205, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15022723

ABSTRACT

Cytochrome P450 (P450) 1 enzymes such as P450 1A1, 1A2, and 1B1 are known to be involved in the oxidative metabolism of various procarcinogens and are regarded as important target enzymes for cancer chemoprevention. Previously, several hydroxystilbene compounds were reported to inhibit P450 1 enzymes and were rated as candidate chemopreventive agents. In this study, we investigated the inhibitory effect of 2-[2-(3,5-dimethoxyphenyl)vinyl]-thiophene (DMPVT), produced from the chemical modification of oxyresveratrol, on the activities of P450 1 enzymes. The inhibitory potential by DMPVT on the P450 1 enzyme activity was evaluated with the Escherichia coli membranes of the recombinant human cytochrome P450 1A1, 1A2, or 1B1 coexpressed with human NADPH-P450 reductase. DMPVT significantly inhibited ethoxyresorufin O-deethylation (EROD) activities with IC50 values of 61, 11, and 2 nM for 1A1, 1A2, and 1B1, respectively. The EROD activity in DMBA-treated rat lung microsomes was also significantly inhibited by DMPVT in a dose-dependent manner. The modes of inhibition by DMPVT were non-competitive for all three P450 enzymes. The inhibition of P450 1B1-mediated EROD activity by DMPVT did not show the irreversible mechanism-based effect. The loss of EROD activity in P450 1B1 with DMPVT incubation was not blocked by treatment with the trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol. Taken together, the results suggested DMPVT to be a strong noncompetitive inhibitor of human P450 1 enzymes that should be considered as a good candidate for a cancer chemopreventive agent in humans.


Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Thiophenes/chemical synthesis , Thiophenes/pharmacology , Animals , Cytochrome P-450 CYP1A1/metabolism , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Humans , In Vitro Techniques , Isoenzymes/antagonists & inhibitors , Kinetics , Lung/drug effects , Lung/enzymology , Microsomes/drug effects , Microsomes/enzymology , NADP/metabolism , Rats , Recombinant Proteins/antagonists & inhibitors
12.
FEBS Lett ; 555(2): 217-22, 2003 Dec 04.
Article in English | MEDLINE | ID: mdl-14644418

ABSTRACT

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.


Subject(s)
Apoptosis/physiology , Enzyme Inhibitors/pharmacology , Mitochondria/metabolism , Naphthoquinones/pharmacology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , cdc25 Phosphatases/antagonists & inhibitors , Amino Acid Chloromethyl Ketones/pharmacology , Apoptosis/drug effects , BH3 Interacting Domain Death Agonist Protein , Carrier Proteins/metabolism , Caspase Inhibitors , Caspases/metabolism , Cytochromes c/metabolism , Enzyme Activation/drug effects , HL-60 Cells , Humans , Immunoblotting , Naphthoquinones/antagonists & inhibitors , Protein Serine-Threonine Kinases/pharmacology , Proto-Oncogene Proteins/pharmacology , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction , bcl-2-Associated X Protein , bcl-Associated Death Protein
13.
Arch Pharm Res ; 26(5): 405-10, 2003 May.
Article in English | MEDLINE | ID: mdl-12785737

ABSTRACT

Vitamin K-related analogs induce growth inhibition in various cancer cell lines. A naphthoquinone analog, termed 2,3-dichloro-5, 8-dihydroxy-1,4-naphthoquinone (DDN), induces apoptosis in human promyeloid leukemic HL-60 cells, and shows antitumor activity in vivo. Following treatment with DDN, evidence of apoptosis, including DNA fragmentation and cleavage of poly ADP ribose polymerase (PARP), was observed. DDN induced an upregulation of proapoptotic Bax protein, and Bid cleavage. Antiapoptotic Bcl-2 protein levels were not changed by DDN, but the expression of Bcl-xL was decreased. In addition, DDN reduced the mass of solid tumor in the Sarcoma 180 tumor-bearing mouse model. These results indicate that DDN exerts antitumor activity, which appears to be related to the induction of apoptosis by regulating Bcl-2 family proteins.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Naphthoquinones/pharmacology , Animals , Antineoplastic Agents/therapeutic use , Blotting, Western , Cell Line, Tumor , Cell Survival/drug effects , DNA Fragmentation/drug effects , Humans , Mice , Mice, Inbred ICR , Naphthoquinones/therapeutic use , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma 180/drug therapy , Sarcoma 180/pathology
14.
Biochem Biophys Res Commun ; 298(5): 687-92, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12419308

ABSTRACT

Cytochrome P450 3A4 (CYP3A4) enzyme is responsible for the metabolic activation and inactivation of the majority of clinically used drugs in human liver and intestines. Recent studies have increasingly implicated various inflammatory stimuli to cause changes in the activities and expression levels of CYPs. However, the underlying mechanisms are largely unknown. In the present study, our studies investigated the effects of ceramide on CYP3A4 expression in human colon carcinoma HT-29 cells. Treatment with the cell-permeable ceramide analog C(6)-ceramide to the cells significantly decreased the expression of CYP3A4. By contrast, C(6)-dihydroceramide, a biologically inactive analog of C(6)-ceramide, did not affect CYP3A4 expression. We found that bacterial sphingomyelinase (SMase) and tumor necrosis factor-alpha (TNF), which are known to increase intracellular ceramide levels, also markedly suppressed the synthesis of CYP3A4. To elucidate whether nitric oxide (NO) participates in suppression of CYP3A4 expression by ceramide, the effects of NO modulators were determined. Treatment with N(G)-monomethyl-L-arginine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), was able to protect ceramide-dependent CYP3A4 suppression. In contrast, the addition of S-nitroso-N-acetylpenicillamine, a NO donor, to HT-29 cells reduced CYP3A4 expression. The addition of iNOS antisense oligonucleotide prevented ceramide-mediated induction of iNOS expression and restored CYP3A4 expression. Wortmannin which is known to inhibit phosphatidylinositol 3-kinase (PI3-K) blocked CYP3A4 suppression by ceramide. Taken together, our results demonstrate that ceramide-mediated suppression of CYP3A4 is due to production of NO, which might result from activation of PI3-K.


Subject(s)
Ceramides/pharmacology , Cytochrome P-450 Enzyme System/genetics , Androstadienes/pharmacology , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/biosynthesis , Enzyme Inhibitors/pharmacology , Gene Expression/drug effects , Humans , Nitric Oxide/biosynthesis , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Oligodeoxyribonucleotides, Antisense/pharmacology , Phosphoinositide-3 Kinase Inhibitors , S-Nitroso-N-Acetylpenicillamine/pharmacology , Sphingomyelin Phosphodiesterase/pharmacology , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacology , Wortmannin , omega-N-Methylarginine/pharmacology
15.
FEBS Lett ; 522(1-3): 161-7, 2002 Jul 03.
Article in English | MEDLINE | ID: mdl-12095638

ABSTRACT

Recently, p73 was identified as a structural and functional homolog of p53. The p73 protein activates the transcription of genes downstream of p53 and induces apoptosis when overexpressed in several cell lines, similar to the tumor suppressor p53. However, the extracellular stimuli and molecular mechanisms regulating p73 activity remain to be elucidated. In this paper, we present evidence that the naphthoquinone analog, 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (NA), is a novel apoptotic stimulus that induces p73beta expression. Treatment with NA induced the expression of p73beta mRNA and protein and its downstream genes, p21 and bax, in HeLa cells. Similar results were obtained in MCF7 cells (p53(+/+), p73(+/+)). In the MCF7 cells, p53 protein level was rather decreased by NA treatment. Overexpression of p73beta led to the apoptosis of HeLa cells and enhancement of NA-induced cell death. Expression of p73beta was mediated by E2F-1, which was activated via release from pRB after exposure of cells to NA. We additionally observed that overexpression of pRB inhibited NA-induced apoptosis. These results imply that p53-independent p73beta-dependent p21 expression is involved in NA-induced apoptosis of HeLa cells.


Subject(s)
Apoptosis , Cell Cycle Proteins , DNA-Binding Proteins/genetics , Gene Expression , Genes, Tumor Suppressor , Naphthoquinones/pharmacology , Nuclear Proteins/genetics , Transcription Factors/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , E2F Transcription Factors , E2F1 Transcription Factor , HeLa Cells , Humans , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Transcriptional Activation , Tumor Cells, Cultured , Tumor Protein p73 , Tumor Suppressor Proteins , bcl-2-Associated X Protein
16.
Planta Med ; 68(2): 123-7, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11859461

ABSTRACT

3, 4'-Dimethoxy-5-hydroxystilbene (DMHS) is a hydroxystilbene compound obtained by methylation and acid hydrolysis of piceid (resveratrol-3-O-glucoside) from Polygonum cuspidatum. Herein, we report that DMHS induces programmed cell death or apoptosis in human promyelocytic leukemic HL-60 cells. We found that treatment of HL-60 cells with DMHS suppressed the cell growth in a concentration-dependent manner with an IC50 value of 25 microM. DMHS increased internucleosomal DNA fragmentation in a time-dependent manner. The cell death by DMHS was partially prevented by the caspase inhibitor, zVAD-fmk. DMHS caused activation of caspases such as caspase-3, -8, and -9. Immunoblot experiments revealed that DMHS-induced apoptosis was associated with the induction of Bax expression. The release of cytochrome c from mitochondria into the cytosol was increased in response to DMHS. Taken together, our present results indicated that DMHS leads to apoptotic cell death in HL-60 cells through increased Bax expression and release of cytochrome c into cytosol and may be considered as a good candidate for a cancer chemopreventive agent in humans.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Glucosides/pharmacology , HL-60 Cells/drug effects , Polygonum , Proto-Oncogene Proteins c-bcl-2 , Stilbenes/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Blotting, Western , Caspase 3 , Caspase 8 , Caspase 9 , Caspase Inhibitors , Caspases/metabolism , Cell Division/drug effects , Cytochrome c Group/metabolism , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Gene Expression/drug effects , Glucosides/chemistry , HL-60 Cells/metabolism , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Plant Extracts/chemistry , Plant Extracts/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Signal Transduction , Stilbenes/chemistry , Stilbenes/isolation & purification , bcl-2-Associated X Protein
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