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1.
Exp Dermatol ; 32(11): 1982-1995, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37727050

RESUMEN

The aim of this study is to examine the effects of ageing on dermal fibroblast heterogeneity based on samples obtained from the same donor. We used a dermal fibroblast lineage (named ASF-4 cell lines) isolated from the inner side of the upper arm of a healthy male donor over a 35-year period, beginning at 36 years of age. Because clonal analysis of ASF-4 cell lines demonstrated a donor age-dependent loss of proliferative capacity and acquisition of senescent traits at the single-cell level, cultured cells frozen at passage 10 at ages 36 and 72 years were subjected to single-cell RNA sequencing. Transcriptome analysis revealed an increase in senescent fibroblasts and downregulation of genes associated with extracellular matrix remodelling with ageing. In addition, two putative differentiation pathways, with one endpoint consisting of senescent fibroblasts and the other without, were speculated using a pseudo-time analysis. Knockdown of the characteristic gene of the non-senescent fibroblast cluster endpoint, EFEMP2, accelerated cellular senescence. This was also confirmed in two other normal human dermal fibroblast cell lines. The detection of a common cellular senescence-related gene from single-donor analysis is notable. This study provides new insights into the behaviour of dermal fibroblasts during skin ageing.


Asunto(s)
Fibroblastos , Piel , Humanos , Masculino , Adulto , Persona de Mediana Edad , Anciano , Células Cultivadas , Fibroblastos/metabolismo , Diferenciación Celular , Senescencia Celular , Análisis de la Célula Individual
2.
Regul Pept ; 194-195: 55-62, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25229126

RESUMEN

Galanin is a neuropeptide expressed in the central and peripheral nervous systems. Galanin is known to be biosynthesized in neural and endocrine cells, but little evidence exists for its synthesis in other cells. In this study, we explored galanin-releasing nonneural cells using radioimmunoassay, finding that some fibroblasts produced and released the galanin-like immunoreactive component (galanin-LI). The molecular weight of the galanin-LI obtained from the fibroblasts, as measured by gel filtration chromatography and Western blotting, was 14 kDa and suggested that the compound was progalanin. Peptide mass fingerprinting analysis identified the large form of galanin-LI as progalanin without its signal sequence. In addition, galanin-LI was located in the Golgi bodies and vesicle-like structures of the fibroblasts. Furthermore, the addition of brefeldin A, an inhibitor of transport from the ER, decreased the release of galanin-LI. In this study, we showed that the fibroblast, a nonneural and nonendocrine cell type, produced and released a galanin precursor, progalanin, without processing via Golgi bodies or secretory vesicles.


Asunto(s)
Fibroblastos/metabolismo , Galanina/biosíntesis , Galanina/metabolismo , Animales , Células Cultivadas , Pollos , Cricetulus , Galanina/química , Humanos , Ratones
3.
Cell Signal ; 25(9): 1731-8, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23707520

RESUMEN

Calcineurin inhibitors such as cyclosporin A (CsA) and FK506 have been used in solid organ and hematopoietic stem cell transplantations to suppress immune function. However, these immunosuppresants are associated with severe endothelial dysfunction. We investigated whether CsA and FK506 induce endothelial dysfunction using a three-dimensional culture blood vessel model, in which human umbilical vein endothelial cells form and maintain capillary-like tube and lumen structures. We found that FK506, but not CsA, induced breakdown of the tube structures and endothelial cell death. FK506 inhibited calcineurin activity, but FK506-induced tube breakdown and cell death was not suppressed by RNA interference targeting calcineurin Aα. FK506 also induced caspase activation, but caspase inhibition by zVAD(OMe)-fmk failed to suppress FK506-induced tube breakdown and cell death. FK506 induced attenuation of Akt and extracellular-regulated kinase 1/2 (ERK1/2). Furthermore, Akt inhibition by LY294002 or ERK1/2 inhibition by PD98059 induced tube breakdown and cell death. Present results suggest that FK506 induces endothelial dysfunction through attenuation of Akt and ERK1/2 independently of calcineurin inhibition and the caspase pathway.


Asunto(s)
Células Endoteliales/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inmunosupresores/farmacología , Transducción de Señal/efectos de los fármacos , Tacrolimus/farmacología , Calcineurina/metabolismo , Inhibidores de la Calcineurina , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Ciclosporina/farmacología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo
4.
Mol Nutr Food Res ; 55(11): 1730-4, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21936049

RESUMEN

Angiogenesis is a promising target for cancer prevention and treatment. This study aimed to determine the antiangiogenic effects of melinjo (Gnetum gnemon L.) seed extract and its resveratrol derivative components, such as gnetin C (GC), gnetin L (GL), gnemonoside A (GMA), gnemonoside C (GMC), and gnemonoside D (GMD). An ethanol extract of melinjo seeds (EEMS) and the two gnetins markedly inhibited the proliferation and tube formation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor and basic fibroblast growth factor. The inhibitory effects of GC and GL were much stronger than those of resveratrol. GMC and GMD inhibited only proliferation, whereas GMA had almost no effect on the two endothelial cell functions. The EEMS and GC also reduced the cell viability of tube-forming HUVEC, with accompanying ERK1/2 inactivation, and suppressed the migration of HUVEC. Furthermore, dietary intake of EEMS significantly inhibited tumor angiogenesis in a mouse dorsal air sac assay. In conclusion, we found that the EEMS and its resveratrol derivatives, particularly GC, suppress multiple angiogenesis-related endothelial cell functions and/or tumor angiogenesis, indicating that the melinjo seeds and the natural resveratrol derivatives may be useful for cancer prevention and treatment.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos Fitogénicos/farmacología , Descubrimiento de Drogas , Gnetum/química , Neovascularización Patológica/tratamiento farmacológico , Extractos Vegetales/farmacología , Estilbenos/farmacología , Inhibidores de la Angiogénesis/química , Animales , Antineoplásicos Fitogénicos/química , Bioensayo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Glucósidos/química , Glucósidos/farmacología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Ratones , Ratones Endogámicos ICR , Extractos Vegetales/química , Resveratrol , Semillas/química , Estilbenos/química
5.
Artículo en Inglés | MEDLINE | ID: mdl-19351710

RESUMEN

We recently reported that propolis suppresses tumor-induced angiogenesis through tube formation inhibition and apoptosis induction in endothelial cells. However, molecular mechanisms underlying such angiogenesis suppression by propolis have not been fully elucidated. The aim of this study was to investigate the effects of ethanol extract of Brazilian propolis (EEBP) on two major survival signals, extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, and to elucidate whether changes in these signals were actually involved in antiangiogenic effects of the propolis. Detection by western blotting revealed that EEBP suppressed phosphorylation of ERK1/2, but not that of Akt. Pharmacological inhibition by U0126 demonstrated that ERK1/2 inactivation alone was enough to inhibit tube formation and induce apoptosis. It was also shown that EEBP and U0126 similarly induced activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) and lamin A/C, all of which are molecular markers of apoptosis. These results indicate that inhibition of survival signal ERK1/2, and subsequent induction of apoptosis, is a critical mechanism of angiogenesis suppression by EEBP.

6.
J Nutr ; 140(1): 1-6, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19889811

RESUMEN

We previously reported that indole-3-carbinol (I3C), found in cruciferous vegetables, suppresses angiogenesis in vivo and in vitro. However, the underlying molecular mechanisms still remain unclear. Antiangiogenic effects of its major metabolite, 3,3'-diindolylmethane (DIM), also have not been fully elucidated. In this study, we investigated the effects of these indoles on angiogenesis and tested a hypothesis that I3C and DIM inhibit angiogenesis and induce apoptosis by affecting angiogenic signal transduction in human umbilical vein endothelial cells (HUVEC). We found that I3C and DIM at 25 micromol/L significantly inhibited tube formation and only DIM induced a significant increase in apoptosis in tube-forming HUVEC. DIM showed a stronger antiangiogenic activity than I3C. At the molecular level, I3C and DIM markedly inactivated extracellular signal-regulated kinase 1/2 (ERK1/2) and the inhibitory effect of DIM was significantly greater than that of I3C. DIM treatment also resulted in activation of the caspase pathway and inactivation of Akt, whereas I3C did not affect them. These results indicate that I3C and DIM had a differential potential in the regulation of the 2 principal survival signals, ERK1/2 and Akt, in endothelial cells. We also demonstrated that pharmacological inhibition of ERK1/2 and/or Akt was enough to inhibit tube formation and induce caspase-dependent apoptosis in tube-forming HUVEC. We conclude that both I3C and DIM inhibit angiogenesis at least in part via inactivation of ERK1/2 and that inactivation of Akt by DIM is responsible for its stronger antiangiogenic effects than those of I3C.


Asunto(s)
Células Endoteliales/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Indoles/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Apoptosis/efectos de los fármacos , Butadienos/farmacología , Caspasas/metabolismo , Cromonas/farmacología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/genética , Humanos , Morfolinas/farmacología , Nitrilos/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal
7.
Biochem Biophys Res Commun ; 388(2): 328-32, 2009 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-19665004

RESUMEN

A novel telomerase-associated protein was isolated from porcine testis. The 115-kDa protein, purified with telomerase activity, was molecular cloned using human cDNA library, and identified as MOV10. The expression levels of both MOV10 mRNA and MOV10 protein in cancer cells were 2-3 times higher than that of the normal cells, and MOV10 mRNA was highly expressed in human testis and ovary. The anti-MOV10 antibody precipitated the telomerase activity from cancer cell extracts, and inhibited the telomerase activity in vitro. Sf9-expressed MOV10 protein bound to G-rich strand of both single- and double-stranded telomere-sequenced DNA, but not to single C-rich strand. ChIP assay showed the binding of MOV10 to telomere region in vivo. These data suggest that MOV10 is involved in the progression of telomerase-catalyzing reaction via the interaction of telomerase protein and telomere DNA.


Asunto(s)
ARN Helicasas/metabolismo , Telomerasa/metabolismo , Telómero/enzimología , Animales , Clonación Molecular , ADN/metabolismo , Biblioteca de Genes , Células HeLa , Humanos , Masculino , ARN Helicasas/genética , Porcinos , Testículo/enzimología
8.
Hum Cell ; 22(2): 31-7, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19385097

RESUMEN

The relationship between cellular aging and aging of entire organisms has been studied extensively.The findings are confusing, however, and no clear relationships have been demonstrated.The conflicting data may be due to individual differences among the donors of the studied cells.It is crucial to identify the changes in cellular properties that are the result of the aging process.Here, we used human dermal fibroblast cell lines established from a single donor at different ages to assess the influence of ultraviolet A (UVA) on cellular aging. These cell lines have the same genetic background and were obtained from a restricted body region. The results indicated that cellular aging was accelerated by UVA irradiation in a donor age-dependent manner. The ratio of lifespan shortening increased with donor age. Increased donor age not only decreased cell division, but also increased the growth arrest response to UVA irradiation. The characteristics of the cultured cells reflected the age-related changes in dermal fibroblasts.


Asunto(s)
Envejecimiento/fisiología , Senescencia Celular/efectos de la radiación , Dermis/citología , Fibroblastos/efectos de la radiación , Rayos Ultravioleta , Adulto , Ciclo Celular/efectos de la radiación , Línea Celular , Relación Dosis-Respuesta en la Radiación , Fibroblastos/patología , Humanos , Persona de Mediana Edad
9.
Hum Cell ; 22(2): 38-42, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19385098

RESUMEN

Human fibroblasts, which have a finite lifespan in cultures, have been widely used as a model system for cellular aging, and frequently used as one model of human aging. But whether cellular aging contributes to organismal aging has been controversial. To reinvestigate this question, we cultured human fibroblasts from the skin of one individual volunteer collected at different ages. Over a period of 27 years (donor age 36 years to 62 years), we obtained skin cells four times at appropriate intervals, and established eight fibroblast lines. These human fibroblasts have presented evidence for a correlation between donor age and proliferative lifespan in vitro. This result parallels the fact that telomeric DNA size cultured fibroblasts decrease with the increase in donor age. These cell lines had a normal diploid human chromosome constitution and will be useful in studies of human biology including aging.


Asunto(s)
Envejecimiento/fisiología , Proliferación Celular , Fibroblastos/citología , Piel/citología , Adulto , Línea Celular , ADN , Diploidia , Humanos , Cariotipificación , Masculino , Persona de Mediana Edad , Telómero/genética
10.
Nagoya J Med Sci ; 71(1-2): 11-8, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19358471

RESUMEN

A mouse jejunum, when incubated in vitro in Ussing chambers, was found to exhibit morphological deterioration of the villi with denudation of the epithelia (J Nutr Sci Vitaminol, 51: 406, 2005). Our study examined the involvement of apoptosis in an intestinal injury model by a DNA ladder assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Electrophoresis of mucosal DNA revealed ladders, indicating the occurrence of DNA fragmentation. Cells with TUNEL-positive nuclei were detected among the villus epithelial cells (enterocytes), whereas they are rarely seen among crypt epithelial cells. These features were evident within 1 h after the start of incubation. Apoptotic death of the enterocytes was thus involved in the destruction of villi when incubated in Ussing chambers.


Asunto(s)
Apoptosis , Fragmentación del ADN , Enterocitos/patología , Mucosa Intestinal/patología , Yeyuno/patología , Animales , Animales no Consanguíneos , Cámaras de Difusión de Cultivos , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , Masculino , Ratones
11.
Mol Nutr Food Res ; 53(5): 643-51, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19065585

RESUMEN

Propolis possesses various physiological activities. In this study, we examined the antiangiogenic and antioxidant activities of various components from propolis: acacetin, apigenin, artepillin C, caffeic acid phenethyl ester, chrysin, p-coumaric acid, galangin, kaempferol, pinocembrin, and quercetin. The effects of these components were tested on in vitro models of angiogenesis, tube formation and growth of human umbilical vein endothelial cells (HUVECs). Furthermore, these components were evaluated for their antioxidant activities by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging and ferric reducing/antioxidant power (FRAP) assays. Two propolis components, caffeic acid phenethyl ester, and quercetin, possessed strong inhibitory effects on tube formation and on endothelial cell proliferation and, coincidentally, showed strong antioxidant activity. Artepillin C, galangin, and kaempferol also possessed strong antiangiogenic and antioxidant activities to a slightly less degree. In contrast, acacetin, apigenin, and pinocembrin possessed a considerable degree of antiangiogenic activities, although they showed very low antioxidant activities. From these results, we propose that components from propolis such as artepillin C, caffeic acid phenethyl ester, galangin, kaempferol, and quercetin might represent a new class of dietary-derived antioxidative compounds with antiangiogenic activities. These propolis components may have the potential to be developed into pharmaceutical drugs for the treatment of angiogenesis-dependent human diseases such as tumors.


Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antioxidantes/farmacología , Própolis/química , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Humanos , Especies Reactivas de Oxígeno/metabolismo
12.
Exp Cell Res ; 315(2): 327-35, 2009 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-19022247

RESUMEN

We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.


Asunto(s)
Apoptosis/fisiología , Caspasa 3/metabolismo , Caspasa 6/metabolismo , Caspasa 7/metabolismo , Células Endoteliales/metabolismo , Actinas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 9/metabolismo , Inhibidores de Caspasas , Hipoxia de la Célula/fisiología , Línea Celular , Núcleo Celular/efectos de los fármacos , Cromatina/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Roturas del ADN/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Humanos , Imidazoles/farmacología , Lamina Tipo A/metabolismo , Modelos Biológicos , Neovascularización Fisiológica/efectos de los fármacos , Oligopéptidos/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología
13.
Phytother Res ; 23(3): 423-7, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19003952

RESUMEN

There are mainly three types of propolis whose major anticancer ingredients are entirely different: (1) CAPE (caffeic acid phenethyl ester)-based propolis in Europe, Far East and New Zealand, (2) artepillin C (ARC)-based Brazilian green propolis and (3) Brazilian red propolis. It was shown previously that NF (neurofibromatosis)-associated tumors require the kinase PAK1 for their growth, and CAPE-based propolis extracts such as Bio 30 suppress completely the growth of NF tumors in vivo by blocking PAK1 signaling. Also it was demonstrated that ARC suppresses angiogenesis, suggesting the possibility that ARC also blocks oncogenic PAK1 signaling. Here it is shown for the first time that both ARC and green propolis extract (GPE) indeed block the PAK1 signaling selectively, without affecting another kinase known as AKT. Furthermore, it was confirmed that ARC as well as GPE suppress almost completely the growth of human NF tumor xenografts in mice, as does Bio 30. These results suggest that both CAPE-based and ARC-based propolis extracts are natural anti-PAK1 remedies and could be among the first effective NF therapeutics available on the market. Since more than 70% of human cancers such as breast and prostate cancers require the kinase PAK1 for their growth, it is quite possible that GPE could be potentially useful for the treatment of these cancers, as is Bio 30.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Fenilpropionatos/farmacología , Própolis/farmacología , Quinasas p21 Activadas/metabolismo , Animales , Ácidos Cafeicos/farmacología , Línea Celular Tumoral , Femenino , Ratones , Ratones Desnudos , Neurofibromatosis/tratamiento farmacológico , Alcohol Feniletílico/análogos & derivados , Alcohol Feniletílico/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto
14.
Biosci Biotechnol Biochem ; 72(9): 2436-40, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18776674
15.
Hum Cell ; 21(3): 70-8, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18667023

RESUMEN

Plasma membranes are essential components of living cells, and phospholipids are major components of cellular membranes. Here, we used liquid chromatography/mass spectrometry to investigate changes in the membrane phospholipid content that occur in association with aging. Our results indicate that the levels of a particular species of phosphatidylcholine comprised of stearic acid and arachidonic acid increased with age. To determine the reason for the increased levels of this particular phosphatidylcholine, we examined the effect of highly unsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, on cellular aging. Applied arachidonic acid was incorporated into phosphatidylcholine molecules, but neither arachidonic acid nor other related unsaturated fatty acids had any effect. We conclude that increased levels of this distinctive phosphatidylcholine are a result of in vitro senescence.


Asunto(s)
Envejecimiento/metabolismo , Senescencia Celular/efectos de los fármacos , Dermis/citología , Fibroblastos/metabolismo , Fosfatidilcolinas/metabolismo , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Células Cultivadas , Cromatografía Liquida , Ácido Eicosapentaenoico/metabolismo , Ácido Eicosapentaenoico/farmacología , Humanos , Espectrometría de Masas
16.
Biosci Biotechnol Biochem ; 72(8): 2243-6, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18685185

RESUMEN

Indole-3-carbinol (I3C) has been reported to exert anticancer activity in vivo. However, its anticancer mechanism has not been fully elucidated. In this study, we demonstrate that I3C suppressed tumor-induced angiogenesis and tube formation of endothelial cells. I3C also induced apoptosis in endothelial cells by activating the caspase cascade. We propose that I3C exerts its anticancer effect through the induction of endothelial apoptosis.


Asunto(s)
Apoptosis/efectos de los fármacos , Indoles/farmacología , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica , Sacos Aéreos/efectos de los fármacos , Animales , Caspasas/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Indoles/química , Ratones
17.
Proteomics ; 8(14): 2897-906, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18655027

RESUMEN

We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.


Asunto(s)
Células Endoteliales/patología , Proteínas de Choque Térmico/metabolismo , Hipoxia/patología , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Proteómica , Venas Umbilicales/citología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Capilares/enzimología , Capilares/patología , Células Cultivadas , Células Endoteliales/enzimología , Proteínas de Choque Térmico HSP27 , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/deficiencia , Proteínas de Choque Térmico/genética , Humanos , Hipoxia/enzimología , Hipoxia/fisiopatología , Chaperonas Moleculares , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteoma/metabolismo , Venas Umbilicales/enzimología , Venas Umbilicales/patología
18.
Cancer Lett ; 252(2): 235-43, 2007 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-17343983

RESUMEN

Propolis, a resinous substance collected by honeybees from various plant sources, possesses various physiological activities such as antitumor effects. We have previously shown that propolis of Brazilian origin was composed mainly of artepillin C and that its constituents were quite different from those of propolis of European origin. In this report, we examined an antiangiogenic effects of Brazilian propolis and investigated whether artepillin C was responsible for such effects. In an in vivo angiogenesis assay using ICR mice, we found that the ethanol extract of Brazilian propolis (EEBP) significantly reduced the number of newly formed vessels. EEBP also showed antiangiogenic effects in an in vitro tube formation assay. When compared with other constituents of EEBP, only artepillin C was found to significantly inhibit the tube formation of HUVECs in a concentration-dependent manner (3.13-50microg/ml). In addition, artepillin C significantly suppressed the proliferation of HUVECs in a concentration-dependent manner (3.13-50microg/ml). Furthermore, artepillin C significantly reduced the number of newly formed vessels in an in vivo angiogenesis assay. Judging from its antiangiogenic activity in vitro and in vivo, we concluded that artepillin C at least in part is responsible for the antiangiogenic activity of EEBP in vivo. Artepillin C may prove useful in the development of agents and foods with therapeutic or preventive activity against tumor angiogenesis.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Neoplasias/irrigación sanguínea , Neovascularización Patológica/prevención & control , Fenilpropionatos/farmacología , Própolis/química , Animales , Brasil , Células Cultivadas , Endotelio Vascular/citología , Humanos , Ratones
19.
J Cell Physiol ; 211(3): 673-81, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17373651

RESUMEN

In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown.


Asunto(s)
Apoptosis/fisiología , Caspasa 3/metabolismo , Endotelio Vascular/citología , Hipoxia/metabolismo , Hipoxia/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Capilares/metabolismo , Capilares/patología , Inhibidores de Caspasas , Núcleo Celular/patología , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/farmacología , Técnicas In Vitro , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Piridinas/farmacología , Transducción de Señal/fisiología , Venas Umbilicales/citología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores
20.
Cell Signal ; 19(6): 1121-31, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17303382

RESUMEN

We recently reported that hypoxia induces chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, to tube-forming HUVECs in an in vitro blood vessel model by activating p38 MAPK. In this report, we further examined what role p38 plays and how it is activated during hypoxia-induced apoptosis. First, in order to confirm that p38 can indeed induce apoptosis, the cells were treated with anisomycin, a p38 activator, during normoxia. The activator treatment induced apoptosis and activation of p38 and caspase-3 in a very short time, which indicated that p38 activation alone was sufficient to trigger apoptosis in tube-forming HUVECs. We then observed hypoxia-induced changes in intracellular signals, ERK1/2 and Akt. ERK1/2 inactivation was shown to occur prior to p38 activation and caspase-3 cleavage during hypoxia. On the other hand, anisomycin had no inhibitory effect on ERK1/2 activation during normoxia. It was also shown that the amount of Akt protein slightly decreased by either hypoxia or anisomycin treatment. We then investigated how these two survival signals, ERK1/2 and Akt, are involved in p38 activation by using MEK inhibitor U0126 and PI3K inhibitor LY294002. When tube-forming HUVECs were treated with U0126 or LY294002 during normoxia, the two inhibitors were able to induce apoptosis and activation of p38 and caspase-3 in a relatively short time. U0126 was able to inhibit ERK1/2 activation, but had almost no effect on Akt activation. In contrast, LY294002 was able to inhibit Akt activation, but had very little effect on ERK1/2 activation. These results indicate that ERK1/2 inactivation, rather than Akt decrease, is responsible for hypoxia-induced p38 activation. Taken together, our results strongly suggest that hypoxia-induced apoptosis is regulated through signal transduction in which inactivation of ERK1/2 leads to activation of p38, which then triggers caspase cascade as an execution mechanism of apoptosis.


Asunto(s)
Apoptosis , Células Endoteliales/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neovascularización Fisiológica , Venas Umbilicales/citología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Capilares/enzimología , Caspasa 3/metabolismo , Hipoxia de la Célula , Supervivencia Celular , Células Cultivadas , Células Endoteliales/citología , Activación Enzimática , Humanos , Modelos Biológicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Venas Umbilicales/enzimología
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