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1.
Int J Mol Med ; 35(5): 1388-94, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25738334

RESUMO

The aim of the present study was to determine the mechanisms through which 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol (20GPPD) promotes the production of hyaluronic acid (HA) in human keratinocytes. 20GPPD is the primary bioactive metabolite of Rb1, a major ginsenoside found in ginseng (Panax ginseng). We sought to elucidate the underlying mechanisms behind the 20GPPD-induced production of HA. We found that 20GPPD induced an increase in HA production by elevating hyaluronan synthase 2 (HAS2) expression in human keratinocytes. The phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was also enhanced by 20GPPD in a dose-dependent manner. The pharmacological inhibition of ERK (using U0126) or Akt (using LY294002) suppressed the 20GPPD-induced expression of HAS2, whereas treatment with an epidermal growth factor receptor (EGFR) inhibitor (AG1478) or an intracellular Ca2+ chelator (BAPTA/AM) did not exert any observable effects. The increased Src phosphorylation was also confirmed following treatment with 20GPPD in the human keratinocytes. Following pre-treatment with the Src inhibitor, PP2, both HA production and HAS2 expression were attenuated. Furthermore, the 20GPPD-enhanced ERK and Akt signaling decreased following treatment with PP2. Taken together, our results suggest that Src kinase plays a critical role in the 20GPPD-induced production of HA by acting as an upstream modulator of ERK and Akt activity in human keratinocytes.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ácido Hialurônico/biossíntese , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sapogeninas/farmacologia , Quinases da Família src/metabolismo , Células Cultivadas , Ginsenosídeos/metabolismo , Humanos , Fosforilação/efeitos dos fármacos
2.
Exp Dermatol ; 23(11): 819-24, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25181017

RESUMO

Compound K (CK) is one of the major metabolites of ginsenosides exhibiting a variety of pharmacological properties such as anti-ageing, anti-oxidation and anti-inflammatory activities. However, the protective efficacy of CK in abnormal skin conditions with inflammatory responses was not examined. Here, we investigated the effects of CK on matrix metalloproteinase-1 (MMP-1) and type I procollagen production in tumor necrosis factor-α (TNF-α)-stimulated human skin fibroblasts HS68 cells and human skin equivalents. We found that CK suppressed MMP-1 secretion and increased the level of reduced type I procollagen secretion, caused by the inhibition of extracellular signal-regulated kinase (ERK) activation, but not p38 and c-Jun N-terminal kinase (JNK) activation in TNF-α-stimulated HS68 cells. Then, we focused on the involvement of the c-Src and epidermal growth factor receptor (EGFR) as upstream signalling molecules for ERK activation by TNF-α in HS68 cells. CK suppressed the phosphorylation of c-Src/EGFR by TNF-α, which led to the inactivation of downstream signalling molecules including AKT and MEK. In addition, CK suppressed AP-1 (c-jun and c-fos) phosphorylation as downstream transcription factors of active ERK for MMP-1 expression in TNFα-stimulated HS68 cells. These results showed novel mechanisms by which CK inhibits TNF-α-induced MMP-1 expression through the inactivation of c-Src/EGFR-dependent ERK/AP-1 signalling pathway, resulting in the inhibition of collagen degradation in human fibroblast cells. Therefore, CK may be a promising protective agent for the treatment of inflammatory skin conditions such as skin ageing and atopic dermatitis.


Assuntos
Fibroblastos/metabolismo , Ginsenosídeos/química , Metaloproteinase 1 da Matriz/metabolismo , Pele/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Sobrevivência Celular , Células Cultivadas , Colágeno/química , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/metabolismo , Humanos , Inflamação , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Envelhecimento da Pele , Dermatopatias/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
3.
PLoS One ; 9(8): e104305, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25137374

RESUMO

Studies have shown that a major metabolite of the red ginseng ginsenoside Rb1, called 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol (GPD), exhibits anticancer properties. However, the chemotherapeutic effects and molecular mechanisms behind GPD action in human melanoma have not been previously investigated. Here we report the anticancer activity of GPD and its mechanism of action in melanoma cells. GPD, but not its parent compound Rb1, inhibited melanoma cell proliferation in a dose-dependent manner. Further investigation revealed that GPD treatment achieved this inhibition through the induction of autophagy and apoptosis, while Rb1 failed to show significant effect at the same concentrations. The inhibitory effect of GPD appears to be mediated through the induction of AMPK and the subsequent attenuation of mTOR phosphorylation. In addition, GPD activated c-Jun by inducing JNK phosphorylation. Our findings suggest that GPD suppresses melanoma growth by inducing autophagic cell death and apoptosis via AMPK/JNK pathway activation. GPD therefore has the potential to be developed as a chemotherapeutic agent for the treatment of human melanoma.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Ginsenosídeos/farmacologia , Melanócitos/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ginsenosídeos/isolamento & purificação , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Melanócitos/metabolismo , Melanócitos/patologia , Panax/química , Fosforilação/efeitos dos fármacos , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
4.
J Cell Biochem ; 115(10): 1702-11, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24821673

RESUMO

Various health effects have been attributed to the ginsenoside metabolite 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol (GPD); however, its effect on ultraviolet (UV)-induced matrix metalloproteinase (MMP)-1 expression and the mechanism underlying this effect are unknown. We examined the inhibitory effect of GPD on UV-induced MMP-1 expression and its mechanisms in human dermal fibroblasts (HDFs). GPD attenuated UV-induced MMP-1 expression in HDFs and suppressed the UV-induced phosphorylation of mammalian target of rapamycin (mTOR) and p70(S6K) without inhibiting the activity of phosphatidylinositol 3-kinase and Akt, which are well-known upstream kinases of mTOR. GPD augmented the phosphorylation of liver kinase B1 (LKB1) and adenosine monophosphate-activated protein kinase (AMPK), which are inhibitors of mTOR, to a greater extent than UV treatment alone. Similar to GPD, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranosyl 5'-monophosphate (AICAR), an activator of AMPK, augmented UV-induced AMPK phosphorylation to a greater extent than UV treatment alone, resulting in the inhibition of MMP-1 expression. AICAR also decreased the phosphorylation of mTOR and p70(S6K). However, compound C, an antagonist of AMPK, increased MMP-1 expression. In HDF cells with AMPK knock-down using shRNA, MMP-1 expression was increased. These results indicate that AMPK activation plays a key role in MMP-1 suppression. Additionally, the cAMP-dependent protein kinase (PKA) inhibitor, H-89, antagonized GPD-mediated MMP-1 suppression via the inhibition of LKB1. Our results suggest that the suppressive activity of GPD on UV-induced MMP-1 expression is due to the activation of AMPK as a downstream of the PKA-LKB1 pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/biossíntese , Ginsenosídeos/farmacologia , Metaloproteinase 1 da Matriz/biossíntese , Serina-Treonina Quinases TOR/antagonistas & inibidores , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibroblastos , Humanos , Isoquinolinas/farmacologia , Oxazinas/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Ribonucleotídeos/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Sulfonamidas/farmacologia , Raios Ultravioleta
5.
Artigo em Inglês | MEDLINE | ID: mdl-24324516

RESUMO

Atopic dermatitis is an inflammatory and chronically relapsing skin disorder that commonly occurs in children; the number of atopic dermatitis patients is increasing. The cause and mechanism of atopic dermatitis have not been defined clearly, although many studies are ongoing. Epidemiological studies suggest that soybean and its isoflavones have immunoregulatory activities. Here, we report that 7,3',4'-trihydroxyisoflavone (7,3',4'-THIF), a major metabolite of daidzin, effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), tumor necrosis factor (TNF)- α , and interleukin (IL)-6 production in RAW 264.7 cells, and also reduced ß -hexosaminidase secretion in RBL-2H3 cells. Moreover, 7,3',4'-THIF significantly reduced scratching time, transepidermal water loss, and mast cell infiltration. It also decreased protease-activated receptor (PAR)-2 and IL-4 expression and increased filaggrin expression in skin lesions of NC/Nga mice. These results suggest that 7,3',4'-THIF improves Dermatophagoides farina body extract-induced atopic dermatitis in NC/Nga mice.

6.
Biochem Biophys Res Commun ; 442(3-4): 165-70, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24269817

RESUMO

Autophagy is a cellular degradation process for cellular aggregates and unneeded cellular compartments including damaged mitochondria, ER, and peroxisomes. Melanosome is cellular organelle that is the cellular site of generation, storage and transports of melanin in melanocytes. Despite potential importance of autophagy, the role of autophagy in melanogenesis and melanosome autophagy are largely unknown. In here, we identified 3'-hydroxydaidzein (3'-ODI) as an autophagy inducer from a phytochemical library screening. Treatment with 3'-ODI significantly reduced α-MSH-mediated melanogenesis but efficiently increased autophagy both in melanoma cells and melanocytes. Furthermore, inhibition of autophagy significantly reduced the anti-melanogenic effects of 3'-ODI in α-MSH-stimulated melanoma cells. Taken together, these results suggest that autophagy mediates anti-melanogenic activity of 3'-ODI.


Assuntos
Autofagia/efeitos dos fármacos , Isoflavonas/farmacologia , Melaninas/antagonistas & inibidores , Melanócitos/efeitos dos fármacos , Melanossomas/efeitos dos fármacos , Animais , Autofagia/genética , Proteína 5 Relacionada à Autofagia , Linhagem Celular Tumoral , Melaninas/biossíntese , Melanócitos/metabolismo , Melanossomas/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Interferência de RNA , alfa-MSH/farmacologia
7.
Biochem Pharmacol ; 86(10): 1468-75, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24021351

RESUMO

Rutin is a well-known flavonoid that exists in various natural sources. Accumulative studies have represented the biological effects of rutin, such as anti-oxidative and anti-inflammatory effects. However, the underlying mechanisms of rutin and its direct targets are not understood. We investigated whether rutin reduced B[a]PDE-induced-COX-2 expression. The transactivation of AP-1 and NF-κB were inhibited by rutin. Rutin also attenuated B[a]PDE-induced Raf/MEK/ERK and Akt activation, but had no effect on the phosphorylation of EGFR. An in vitro kinase assay revealed rutin suppressed EGFR kinase activity. We also confirmed direct binding between rutin and EGFR, and found that the binding was regressed by ATP. The EGFR inhibitor also inhibited the B[a]PDE-induced MEK/ERK and Akt signaling pathways and subsequently, suppressed COX-2 expression and promoter activity, in addition to suppressing the transactivation of AP-1 and NF-κB. In EGFR(-/-)mouse embryonic fibroblast cells, B[a]PDE-induced COX-2 expression was also diminished. Collectively, rutin inhibits B[a]PDE-induced COX-2 expression by suppressing the Raf/MEK/ERK and Akt signaling pathways. EGFR appeared to be the direct target of rutin.


Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/toxicidade , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Poluentes Ambientais/toxicidade , Receptores ErbB/metabolismo , Rutina/farmacologia , Animais , Linhagem Celular , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Quinases raf/metabolismo
8.
Biochem Pharmacol ; 86(7): 896-903, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23948065

RESUMO

Solar UV (sUV) is an important environmental carcinogen. Recent studies have shown that sUV is associated with numerous human skin disorders, such as wrinkle formation and inflammation. In this study, we found that the isoflavone, biochanin A, inhibited the expression of sUV-induced COX-2, which is a well-characterized sUV-induced enzyme, in both human HaCaT keratinocytes and JB6 P+ mouse skin epidermal cells. Several studies have demonstrated the beneficial effects of biochanin A. However, its direct molecular target is unknown. We found that biochanin A inhibited sUV-induced phosphorylation of MKK4/JNK/c-Jun and MKK3/6/p38/MSK1. Mixed-lineage kinase 3 (MLK3) is an upstream kinase of MKK4 and MKK3/6. Thus, we evaluated the effect of biochanin A on MLK3. We found that sUV-induced MLK3 phosphorylation was not affected, whereas MLK3 kinase activity was significantly suppressed by biochanin A. Furthermore, direct binding of biochanin A in the MLK3 ATP-binding pocket was detected using pull-down assays. Computer modeling supported our observation that MLK3 is a novel target of biochanin A. These results suggest that biochanin A exerts chemopreventive effects by suppressing sUV-induced COX-2 expression mediated through MLK3 inhibition.


Assuntos
Ciclo-Oxigenase 2/metabolismo , Genisteína/farmacologia , Queratinócitos/efeitos dos fármacos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Animais , Sítios de Ligação , Células Cultivadas , Inibidores de Ciclo-Oxigenase 2/farmacologia , Genisteína/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , MAP Quinase Quinase Quinases/química , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Conformação Proteica , Transdução de Sinais/efeitos dos fármacos , Pele/citologia , Pele/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Raios Ultravioleta , MAP Quinase Quinase Quinase 11 Ativada por Mitógeno
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