RESUMO
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.
Assuntos
Inibidores da Angiogênese/farmacologia , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Medicamentos de Ervas Chinesas/farmacologia , Naftoquinonas/farmacologia , Neovascularização Patológica/prevenção & controle , Animais , Apoptose , Western Blotting , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Galinhas , Membrana Corioalantoide/metabolismo , Membrana Corioalantoide/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Técnicas Imunoenzimáticas , Imunoprecipitação , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Neoplasias Ovarianas/metabolismo , Receptores de Esteroides/biossíntese , Receptores dos Hormônios Tireóideos/biossíntese , Vitamina K 2/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Imunofluorescência , Humanos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Neoplasias Ovarianas/patologia , RNA Interferente Pequeno , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Nucleotídeos de Desoxiuracil/metabolismo , Neoplasias Pulmonares/enzimologia , Naftoquinonas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Nucleotídeos de Desoxiuracil/genética , Ativação Enzimática/efeitos dos fármacos , Fluoruracila/farmacologia , Humanos , Neoplasias Pulmonares/patologia , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Pirofosfatases/química , RNA Interferente Pequeno/genéticaRESUMO
Neural development is controlled by region-specific factors that regulate cell proliferation, migration and differentiation. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts a wide range of effects on different cell types in the brain as early as the fetal stage. Here we review current knowledge concerning several aspects of PACAP expression in embryonic and neonatal neural tissue: (i) the distribution of PACAP and PACAP receptors mRNA in the developing brain; (ii) the characteristic generation of neurons, astrocytes and oligodendrocytes in brain areas where the PACAP receptor is expressed and (iii) the role of PACAP as a regulator of neural development, inducing differentiation and proliferation in association with other trophic factors or signal transduction molecules.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Modelos Biológicos , Organogênese , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismoRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Diterpenos/farmacologia , Citocromos c/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Células HL-60 , Humanos , Concentração de Íons de Hidrogênio , Mitocôndrias/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bufanolídeos/farmacologia , Fatores de Troca do Nucleotídeo Guanina/genética , Apoptose/fisiologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Células HeLa , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , TransfecçãoRESUMO
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.
Assuntos
Astrócitos/fisiologia , Diferenciação Celular/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Proteína Quinase C/metabolismo , Células-Tronco/efeitos dos fármacos , Adenoviridae/fisiologia , Análise de Variância , Animais , Contagem de Células/métodos , Diferenciação Celular/fisiologia , Células Cultivadas , Interações Medicamentosas , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/metabolismo , Nestina , Isoformas de Proteínas/metabolismo , Proteína Quinase C beta , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células-Tronco/fisiologia , Transfecção/métodosRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. PACAP prevents ischemic delayed neuronal cell death (apoptosis) in the hippocampus. PACAP inhibits the activity of the mitogen-activated protein kinase (MAPK) family, especially JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia-reperfusion, both pyramidal cells and astrocytes increased their expression of the PACAP receptor (PAC1-R). Reactive astrocytes increased their expression of PAC1-R, released interleukin-6 (IL-6) that is a proinflammatory cytokine with both differentiation and growth-promoting effects for a variety of target cell types, and thereby protected neurons from apoptosis. These results suggest that PACAP itself and PACAP-stimulated secretion of IL-6 synergistically inhibit apoptotic cell death in the hippocampus. The PAC1-R is expressed in the neuroepithelial cells from early developmental stages and in various brain regions during development. We have recently found that PACAP, at physiological concentrations, induces differentiation of mouse neural stem cells into astrocytes. Neural stem cells were prepared from the telencephalon of mouse embryos and cultured with basic fibroblast growth factor. The PAC1-R immunoreactivity was demonstrated in the neural stem cells. When neural stem cells were exposed to PACAP, about half of these cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. This phenomenon was significantly antagonized by a PAC1-R antagonist (PACAP6-38), indicating that PACAP induces differentiation of neural stem cell into astrocytes. Other our physiological studies have demonstrated that PACAP acts on PAC1-R in mouse neural stem cells and its signal is transmitted to the PAC1-R-coupled G protein Gq but not to Gs. These findings strongly suggest that PACAP plays very important roles in neuroprotection in adult brain as well as astrocyte differentiation during development.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/crescimento & desenvolvimento , Fármacos Neuroprotetores/farmacologia , Neurotransmissores/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Apoptose/efeitos dos fármacos , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Diferenciação Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Proteína Quinase C/metabolismo , Células-Tronco/efeitos dos fármacos , Animais , Astrócitos/citologia , Células Cultivadas , Regulação Enzimológica da Expressão Gênica , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Neurônios/citologia , Neurônios/enzimologia , Proteína Quinase C/genética , Células-Tronco/citologia , Células-Tronco/enzimologiaRESUMO
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.
Assuntos
Citocromos c/metabolismo , Diterpenos/farmacologia , Proteínas Mitocondriais/biossíntese , Sequência de Aminoácidos , Apoptose , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células U937RESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Superóxidos/farmacologia , Vitamina K 2/farmacologia , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Acetilcisteína/farmacologia , Cicloeximida/farmacologia , Diterpenos/farmacologia , Feminino , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Ovarianas/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Superóxidos/metabolismo , Células Tumorais Cultivadas , Vitamina K 2/uso terapêutico , alfa-Tocoferol/farmacologiaRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores do Ácido Retinoico/metabolismo , Animais , Benzoatos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular , Chlorocebus aethiops , Cromanos/farmacologia , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Ativação Transcricional , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Retinoic acid receptor (RAR) activation induces cell differentiation and may antagonize cancer progression. Cellular retinol-binding protein I (CRBP-I) functions in retinol storage and its expression is lower in human cancers than in normal cells. We hypothesized that retinol storage might be linked to RAR activation and thus that lowered CRBP-I function might impair RAR activity and cell differentiation. METHODS: Sarcoma virus 40-immortalized human mammary epithelial cells (MTSV1-7) devoid of CRBP-I were transfected with wild-type CRBP-I or CRBP-I point mutants with low RA binding affinity. The subcellular localization of CRBP-I was investigated in these cells and in wild-type or CRBP-I null mouse mammary epithelial cells (MECs), using indirect immunofluorescence and sucrose gradient fractionation. RAR activity was assessed using reporter gene assays. Acinar differentiation and in vivo tumor growth were assessed in reconstituted basement membrane and athymic mice, respectively. RESULTS: In cells expressing wild-type CRBP-I but not the CRBP-I mutants, CRBP-I was found mainly in lipid droplets, the retinol storage organelle, and this localization was associated with promotion of retinol storage by wild-type CRBP-I only. RAR activity was higher and acinar differentiation was observed in cells expressing wild-type but not mutant CRBP-I. RAR antagonist treatment blocked and chronic RA treatment mimicked, the CRBP-I induction of cell differentiation. Finally, CRBP-I suppressed tumorigenicity in athymic mice. CONCLUSIONS: Physiologic RAR activation is dependent on CRBP-I-mediated retinol storage, and CRBP-I downregulation chronically compromises RAR activity, leading to loss of cell differentiation and tumor progression.
Assuntos
Neoplasias da Mama/metabolismo , Células Epiteliais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Animais , Diferenciação Celular , Progressão da Doença , Regulação para Baixo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Glândulas Mamárias Humanas/citologia , Camundongos , Camundongos Nus , Mutação Puntual , Receptores do Ácido Retinoico/genética , Proteínas de Ligação ao Retinol/genética , Proteínas Celulares de Ligação ao Retinol , Sacarose/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Astrócitos/fisiologia , Diferenciação Celular/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Tecido Nervoso/fisiologia , Neuropeptídeos/farmacologia , Neurotransmissores/farmacologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Camundongos , Tecido Nervoso/citologia , Polipeptídeo Hipofisário Ativador de Adenilato CiclaseRESUMO
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.
Assuntos
Apoptose , Proteínas de Choque Térmico HSP90/fisiologia , Naftoquinonas/farmacologia , Acetilcisteína/química , Acetilcisteína/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/farmacologia , Northern Blotting , Western Blotting , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , Corantes/farmacologia , Citocromos c/metabolismo , Citosol/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Regulação da Expressão Gênica , Vetores Genéticos , Células HL-60 , Humanos , Células K562 , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio , Frações Subcelulares/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Ischemic delayed neuronal cell death (apoptosis) in the hippocampus is prevented by PACAP. PACAP inhibits the increasing activity of the MAP kinase family, especially on JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia-reperfusion, both pyramidal cells and astrocytes increased their expression of PACAP receptors (PAC1-Rs). The pyramidal cells degenerated but reactive astrocytes increased their expression of PAC1-R. PACAP does not only inhibit apoptotic cell death directly, but also affects astrocytes through PAC1-Rs. Interleukin-6 (IL-6), produced in astrocytes, has several effects on the prevention of brain ischemia and trauma and stimulating neuronal growth. IL-6 secretion into the CSF was markedly stimulated after PACAP infusion, suggesting that PACAP stimulates IL-6 secretion from astrocytes. We studied the effects of PACAP on the wild type and IL-6 KO mice after brain ischemia. In wild-type animals, PACAP significantly inhibited cell death, but in IL-6 KO animals, no cytoprotective effect of PACAP was seen. These results suggest that PACAP inhibits apoptotic cell death partly through IL-6. It is considered that PACAP itself and IL-6, stimulated secretion by PACAP, both synergistically inhibit the JNK/SAPK and p38 signaling pathway, thereby protecting against neuronal cell death.
Assuntos
Apoptose/genética , Isquemia Encefálica/prevenção & controle , Proteínas Quinases JNK Ativadas por Mitógeno , Neuropeptídeos/fisiologia , Animais , Astrócitos/metabolismo , Isquemia Encefálica/patologia , Hipocampo/citologia , Interleucina-6/metabolismo , Interleucina-6/fisiologia , MAP Quinase Quinase 4 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Ratos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/metabolismo , Transdução de Sinais/genética , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: The cellular retinol binding protein I gene (CRBP) is downregulated in a subset of human breast cancers and in MMTV-Myc induced mouse mammary tumors. Functional studies suggest that CRBP downregulation contributes to breast tumor progression. What is the mechanism underlying CRBP downregulation in cancer? Here we investigated the hypothesis that CRBP is epigenetically silenced through DNA hypermethylation in human and mouse breast cancer. RESULTS: Bisulfite sequencing of CRBP in a panel of 6 human breast cancer cell lines demonstrated that, as a rule, CRBP hypermethylation is closely and inversely related to CRBP expression and identified one exception to this rule. Treatment with 5-azacytidine, a DNA methyltransferase inhibitor, led to CRBP reexpression, supporting the hypothesis that CRBP hypermethylation is a proximal cause of CRBP silencing. In some cells CRBP reexpression was potentiated by co-treatment with retinoic acid, an inducer of CRBP, and trichostatin A, a histone deacetylase inhibitor. Southern blot analysis of a small panel of human breast cancer specimens identified one case characterized by extensive CRBP hypermethylation, in association with undetectable CRBP mRNA and protein. Bisulfite sequencing of CRBP in MMTV-Myc and MMTV-Neu/NT mammary tumor cell lines extended the rule of CRBP hypermethylation and silencing (both seen in MMTV-Myc but not MMTV-Neu/NT cells) from human to mouse breast cancer and suggested that CRBP hypermethylation is an oncogene-specific event. CONCLUSION: CRBP hypermethylation appears to be an evolutionarily conserved and principal mechanism of CRBP silencing in breast cancer. Based on the analysis of transgenic mouse mammary tumor cells, we hypothesize that CRBP silencing in human breast cancer may be associated with a specific oncogenic signature.
Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Proteínas de Ligação ao Retinol/genética , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Evolução Molecular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Oncogenes/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Proteínas Celulares de Ligação ao Retinol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tretinoína/farmacologiaRESUMO
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.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Naftoquinonas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Western Blotting , Sinergismo Farmacológico , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Pulmonares/enzimologia , Proteínas Tirosina Quinases/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
We generated transgenic mice that express an enhanced green fluorescent protein (EGFP) under the control of the mouse glial fibrillary acidic protein (GFAP) promoter. In one of the transgenic lines, the green fluorescence of EGFP was undetectable in almost all of the brain regions, including the neocortex, in untreated animals. However, when reactive astrogliosis was induced by cortical stab wounding, the strong fluorescence of EGFP was observed around the needle track but was not found in the corresponding area of the contralateral hemisphere. The EGFP-expressing cells had the morphological features of reactive astrocytes such as thick processes. The EGFP-expressing cells were found to overlap with the astroglial marker GFAP, but not with the microglial marker CD11b or the neuronal marker NeuN. Furthermore, there were some EGFP-expressing cells that expressed vimentin-like immunoreactivity, the specific marker for reactive astrocytes. These results strongly suggest that the EGFP-expressing cells are reactive astrocytes, but not resting astrocytes. Using these transgenic mice, immunostaining for the PAC1 receptor (PAC1-R) was performed. PAC1-R, which is a pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor, binds PACAP, which is known to have a wide variety of functions. An immunohistochemical study revealed the localization of PAC1-R in reactive astrocytes visualized with EGFP around the needle track at 5 days postsurgery.
Assuntos
Astrócitos/metabolismo , Lesões Encefálicas/metabolismo , Córtex Cerebral/lesões , Córtex Cerebral/metabolismo , Gliose/metabolismo , Neuropeptídeos/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Animais , Astrócitos/citologia , Biomarcadores , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Córtex Cerebral/fisiopatologia , Proteína Glial Fibrilar Ácida/genética , Gliose/patologia , Gliose/fisiopatologia , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Regiões Promotoras Genéticas/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Transgenes/genética , Regulação para Cima/genéticaRESUMO
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.