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Hyperpigmentation disorders causing emotional distress require the topical use of depigmenting agents of natural origin. In this study, the anti-melanogenic effects of the Lilium lancifolium root extract (LRE) were investigated in B16F10 cells. Consequently, a non-cytotoxic concentration of the extract reduced intracellular melanin content and tyrosinase activity in a dose-dependent manner, correlating with the diminished expression of core melanogenic enzymes within cells. LRE treatment also inhibited cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)/microphthalmia-associated transcription factor signaling, which regulates the expression of tyrosinase-related genes. Upon examining these findings from a molecular mechanism perspective, LRE treatment suppressed the phosphorylation of protein kinase A (PKA), p38, and extracellular signal-related kinase (ERK), which are upstream regulators of CREB. In addition, L-phenylalanine and regaloside A, specifically identified within the LRE using liquid chromatography-mass spectrometry, exhibited inhibitory effects on melanin production. Collectively, these results imply that LRE potentially suppresses cAMP-mediated melanogenesis by downregulating PKA/CREB and mitogen-activated protein kinase (MAPK)/CREB signaling pathways. Therefore, it can be employed as a novel therapeutic ingredient of natural origin to ameliorate hyperpigmentation disorders.
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The aim of the present study was to investigate the potential inhibitory effects of nodakenin, a coumarin glucoside derivative from the root extract of Angelica gigas Nakai (AGN), on melanogenesis and its underlying mechanisms in B16F10 melanoma cells. The inhibitory effects of nodakenin on melanogenesis were evaluated by determining melanin contents and tyrosinase activity in α -melanocyte stimulating hormone (α-MSH)-treated B16F10 melanoma cells. The mechanisms associated with the anti-pigmentation effect of nodakenin were investigated by quantitative real-time PCR and immunoblotting analysis. Using the UVB-irradiated conditioned media culture system and UVB-irradiated co-cultivation system of HaCaT keratinocytes and B16F10 melanoma cells mimicking in vivo melanin biosynthesis, the effect of nodakenin on melanin production was evaluated. Melanin content analysis showed that nodakenin decreased cellular melanin biosynthesis in α-MSH-treated B16F10 cells. Immunoblotting revealed that CREB phosphorylation, MITF, a mastering transcription factor of melanogenesis and its downstream genes tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2 were downregulated by nodakenin in a dose-dependent manner. Interestingly, nodakenin did not affect the phosphorylation of PKA and p38 MAPK but the phosphorylation of ERK1/2 and MSK1. In addition, the inhibition of melanin accumulation by nodakenin in the UVB-irradiated conditioned media culture system and UVB-irradiated co-cultivation system of HaCaT and B16F10 cells suggests that nodakenin has potential as an anti-pigmentation activity. These data suggest that nodakenin inhibits the melanogenesis in B16F10 cells by interfering the ERK/ MSK1/CREB axis and thus preventing MITF expression.
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Melanoma Experimental , Melanoma , Animales , alfa-MSH , Línea Celular Tumoral , Cumarinas/farmacología , Medios de Cultivo Condicionados/farmacología , Glucósidos/farmacología , Sistema de Señalización de MAP Quinasas , Melaninas , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/metabolismo , Transducción de Señal , RatonesRESUMEN
Background: Melatonin, a neurohormone, maybe involved in physiological processes, such as antioxidation, anti-inflammation, and hair growth. In the present study, we investigated the effects of melatonin on proliferation and intracellular signaling in DP cells using a three-dimensional (3D) spheroid culture system that mimics the in vivo hair follicle system. Methods: DP cells were incubated in monolayer (2D) and 3D spheroid culture systems. The expression levels of melatonin receptors in DP cells were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. The effect of melatonin on the hair-inductive property of DP cells was analyzed using a WST-1-based proliferation assay, determination of DP spheroid size, expression analysis of DP signature genes, and determination of ß-catenin stabilization in DP cells. The AKT/GSK3ß/ß-catenin signaling pathway associated with melatonin-induced ß-catenin stabilization in DP cells was investigated by analyzing changes in upstream regulator proteins, including AKT, GSK3ß, and their phosphorylated forms. Results: The expression levels of the melatonin receptors were higher in human DP cells than in human epidermal keratinocytes and human dermal fibroblast cells. Comparing the expression level according to the human DP cell culture condition, melatonin receptor expression was upregulated in the 3D culture system compared to the traditional two-dimensional monolayer culture system. Cell viability analysis showed that melatonin concentrations up to 1 mM did not affect cell viability. Moreover, melatonin increased the diameter of DP cell 3D spheroids in a dose-dependent manner. Immunoblotting and qRT-PCR analysis revealed that melatonin upregulated the expression of hair growth-related genes, including alkaline phosphatase, bone morphogenetic protein 2, versican, and wingless-int 5A, in a melatonin receptor-dependent manner. Cell fractionation analysis showed that melatonin increased the nuclear localization of ß-catenin. This result correlated with the increased transcriptional activation of T-cell factor/lymphoid enhancer factor-responsive luciferase induced by melatonin treatment. Interestingly, melatonin induced the phosphorylation of protein kinase B/AKT at serine 473 residue and GSK-3ß at serine 9 residue. To determine whether AKT phosphorylation at serine 473 induced ß-catenin nuclear translocation through GSK3ß phosphorylation at serine 9, the PI3K/AKT inhibitor LY294002 was cotreated with melatonin. Immunoblotting showed that LY294002 inhibited melatonin-induced phosphorylation of GSK3ß at serine 9 residue and ß-catenin activation. Conclusion: Collectively, this report suggests that melatonin promotes growth properties by activating the AKT/GSK3ß/ß-catenin signaling pathway through melatonin receptors.
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Melatonina , Proteínas Proto-Oncogénicas c-akt , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Melatonina/farmacología , Receptores de Melatonina , Células Cultivadas , beta Catenina/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
Diarylpropionitrile (DPN), a selective agonist for estrogen receptor ß (ERß), has been reported to regulate various hormonal responses through activation of ERß in tissues including the mammary gland and brain. However, the effect of DPN on melanogenesis independent of ERß has not been studied. The aim of this study is to examine the possibility of anti-melanogenic effect of DPN and its underlying mechanism. Melanin contents and cellular tyrosinase activity assay indicated that DPN inhibited melanin biosynthesis in alpha-melanocyte stimulating hormone-stimulated B16F10 melanoma cell line. However, DPN had no direct influence on in vitro tyrosinase catalytic activity. On the other hand, 17ß-estradiol had no effect on inhibition of melanogenesis, suggesting that the DPN-mediated suppression of melanin production was not related with estrogen signaling pathway. Immunoblotting analysis showed that DPN down-regulated the expression of microphthalmia-associated transcription factor (MITF), a central transcription factor of melanogenesis and its down-stream genes including tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. Also, DPN attenuated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). Additionally, DPN suppressed the melanin synthesis in UVB-irradiated HaCaT conditioned media culture system suggesting that DPN has potential as an anti-melanogenic activity in physiological conditions. Collectively, our data show that DPN inhibits melanogenesis via downregulation of PKA/CREB/MITF signaling pathway.
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In this study, the protective functions of bacteria-free conditioned media from Bifidobacterium and Lactobacillus species against ultraviolet radiation-induced skin ageing and associated cellular damage were investigated. The effects of ultraviolet radiation-induced reactive oxygen species production were suppressed by all conditioned media; particularly, the loss of cell viability and downregulation of collagen gene expression were significantly reversed by the conditioned media from B. longum and B. lactis. Further exa mination of potential anti-pigmentation effects revealed that the B. lactis-derived conditioned media significantly inhibited tyrosinase activity and alpha-melanocyte-stimulating hormone-induced melanin production in human epidermal melanocytes. Further, the conditioned media suppressed the phosphorylation of extracellular signal- related kinase, which functions as an upstream regulator of melanogenesis. Therefore, B. lactis-derived conditioned media can potentially protect against cellular damage involved in skin-ageing processes.
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Probióticos , Rayos Ultravioleta , Humanos , Medios de Cultivo Condicionados/farmacología , Medios de Cultivo Condicionados/metabolismo , Rayos Ultravioleta/efectos adversos , Piel , Envejecimiento , Probióticos/farmacologíaRESUMEN
BACKGROUND: Salvianolic acid B (SAB) is one of the main active ingredients of Salvia Miltiorrhiza. It has significant skin anti-aging, whitening, and sun protection properties. AIMS: The study aimed at studying the mechanism underlying the effect of salvianolic acid Bon collagen synthesis, which has good anti-aging efficacy and modulates microcirculation. METHODS: This study employed available public databases, bioinformatics methodologies, and the inverse docking approach to explore the effectiveness of SAB in the regulating collagen synthesis, and then used an human dermal fibroblast (HDF)- Human dermal microvascular endothelial cell (HDMEC) in vitro model to validate the predicted mechanism of SAB in influencing collagen synthesis. RESULTS: The results showed that NO production in SAB-treated HDMEC-conditioned medium was increased compared to that in control media, and the same tendency was also observed for growth factor production. SAB also upregulated HDMEC cellular eNOS and VEGF. When SAB-treated HDMEC conditioned medium was transferred to HDFs, the expression of collagen I, collagen III, and elastin in HDFs was upregulated and MMP-1 was downregulated. CONCLUSIONS: The results show that SAB regulates collagen through the HDMEC-HDF pathway. Furthermore, the mechanisms might be closely related to the microcirculation factors NO and VEGF.
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Fibroblastos , Factor A de Crecimiento Endotelial Vascular , Benzofuranos , Células Cultivadas , Colágeno/metabolismo , Medios de Cultivo Condicionados/metabolismo , Células Endoteliales , Humanos , Piel , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Ginsenoside Rg4 is a rare ginsenoside that is naturally found in ginseng, and exhibits a wide range of biological activities including antioxidant and anti-inflammatory properties in several cell types. The purpose of this study was to use an in vivo model of hair follicle (HF)-mimic based on a human dermal papilla (DP) spheroid system prepared by three-dimensional (3D) culture and to investigate the effect of Rg4 on the hair-inductive properties of DP cells. Treatment of the DP spheroids with Rg4 (20 to 50 µg/ml) significantly increased the viability and size of the DP spheres in a dose-dependent manner. Rg4 also increased the mRNA and protein expression of DP signature genes that are related to hair growth including ALP, BMP2, and VCAN in the DP spheres. Analysis of the signaling molecules and luciferase reporter assays further revealed that Rg4 induces the activation of phosphoinositide 3-kinase (PI3K)/AKT and the inhibitory phosphorylation of GSK3ß, which activates the WNT/ß-catenin signaling pathway. These results correlated with not only the increased nuclear translocation of ß-catenin following the treatment of the DP spheres with Rg4 but also the significant elevation of mRNA expression of the downstream target genes of the WNT/ß-catenin pathway including WNT5A, ß-catenin, and LEF1. In conclusion, these results demonstrated that ginsenoside Rg4 promotes the hair-inductive properties of DP cells by activating the AKT/GSK3ß/ß-catenin signaling pathway in DP spheres, suggesting that Rg4 could be a potential natural therapy for hair growth.
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Dermis/efectos de los fármacos , Ginsenósidos/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Cabello/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , beta Catenina/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Dermis/citología , Dermis/metabolismo , Cabello/crecimiento & desarrollo , Folículo Piloso/citología , Folículo Piloso/efectos de los fármacos , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Esferoides Celulares , Proteínas Wnt/metabolismoRESUMEN
Developing dermatitis therapeutics has been faced with challenges including adverse effects of topical steroid and high cost of new developing drugs. Here, we found the expression levels of dopamine receptor D2 is higher in skin biopsies of dermatitis patients and an oxazolone-induced animal model of dermatitis. We used perphenazine, an FDA-approved dopamine receptor antagonist to determine the therapeutic effect. Two different animal models including 12-o-tetradecanoylphorbol-13-acetate (TPA) and oxazolone (OXA)-induced dermatitis were employed. TPA and OXA-mediated ear swelling was attenuated by perphenazine. Moreover, perphenazine inhibited infiltrated mast cells into lesion area. We found levels of serum IgE, histamine and cytokines are decreased in mice cotreated with perphenazine and OXA compared to OXA-treated mice. Overall, this is a first study showing that the FDA-approved, anti-psychotic drug, perphenazine, alleviates animal models of dermatitis.
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Dermatitis Alérgica por Contacto/tratamiento farmacológico , Antagonistas de Dopamina/uso terapéutico , Perfenazina/uso terapéutico , Animales , Citocinas/metabolismo , Dermatitis Alérgica por Contacto/etiología , Antagonistas de Dopamina/farmacología , Inmunoglobulina G/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Oxazolona/toxicidad , Perfenazina/farmacología , Acetato de Tetradecanoilforbol/toxicidad , Células Th2/efectos de los fármacos , Células Th2/inmunologíaRESUMEN
Metformin, a potent AMPK activator is the most commonly used drug for diabetes. According to recent reports, metformin lowers the risk of diabetic complications and inflammatory diseases. We found the expression levels of AMPK subunits including PRKAA1, PRKAA2, PRKAB1 and PRKAB2 are decreased in skin biopsies of dermatitis patients from multiple datasets. Interestingly, metformin treatment ameliorates dermatitis symptom in animal model of dermatitis using O-tetradecanoylphorbol-13-acetate (TPA). Especially, the levels of epidermis and dermis thickness were decreased by metformin. We found NFκB activity as well as of gene expression associated with collagen synthesis are attenuated by metformin treatment. These results suggest that metformin treatment alleviates animal model of dermatitis.
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Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Dermatitis/tratamiento farmacológico , Activadores de Enzimas/farmacología , Metformina/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Colágeno/metabolismo , Dermatitis/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Piel/efectos de los fármacos , Piel/patología , Acetato de TetradecanoilforbolRESUMEN
Cellular metabolism is one of the crucial factors to regulate epigenetic landscape in various cells including immune cells, embryonic stem cells and hair follicle stem cells. Dermal papilla cells (DP) interact with epithelial stem cells to orchestrate hair formation. Here we show that active DP exhibit robust aerobic glycolysis. We observed decrease of signature genes associated with hair induction by DP in presence of low glucose (2 mM) and glycolysis inhibitors. Moreover, hair shaft elongation was attenuated by glycolysis inhibitors. Interestingly, excessive glucose is able to increase the expression of hair inductive genes and elongation of hair shaft. We also observed glycolysis-mediated histone acetylation is increased and chemical inhibition of acetyltransferase reduces expression of the signature genes associated with hair induction in active DP. These results suggest that glucose metabolism is required for expression of signature genes associated with hair induction. This finding may be beneficial for establishing and maintaining of active DP to generate hair follicle in vitro.
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Dermis/metabolismo , Glucosa/metabolismo , Folículo Piloso/metabolismo , Histonas/metabolismo , Acetilación , Animales , Western Blotting , Supervivencia Celular/fisiología , Carbohidratos de la Dieta/metabolismo , Femenino , Glucólisis/fisiología , Metaanálisis como Asunto , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Nintedanib, a receptor tyrosine kinase (RTK) inhibitor has been developed as therapeutics for idiopathic pulmonary fibrosis and non-small lung cancer. We found that the expression levels of RTK, especially VEGFR1 is increased in skin biopsies of dermatitis patients from multiple independent datasets. Moreover, VEGFR1 is highly expressed by infiltrated cells in dermis from oxazolone (OXA) treated mice. Interestingly, nintedanib alleviates dermatitis symptom in OXA-induced animal model. Especially, levels of epidermis thickness, infiltrated immune cells including mast cells and eosinophils were decreased from mice cotreated with nintedanib and OXA compared with OXA treated mice. Moreover, serum IgE and Th2 cytokines including IL-4 and IL-13 were decreased by nintedanib treatment. These results suggest an evidence that nintedanib alleviates animal model of dermatitis.
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Dermatitis/tratamiento farmacológico , Dermatitis/metabolismo , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Biomarcadores , Biopsia , Línea Celular , Supervivencia Celular , Dermatitis/etiología , Dermatitis/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Ratones , Oxazolona/efectos adversos , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Glucose metabolism is a key metabolic pathway that orchestrates cellular homeostasis by generating ATP, nucleotides, and amino acids. Abnormal glucose signaling has been found in many diseases including cancers and inflammatory diseases. According to recent report, glycolysis contributes to pathogenesis of psoriasis and ablation of Glut1 attenuates animal models of psoriasis. While we were screening a molecular target for atopic dermatitis, we found the levels of glucose transporters including Glut1 (SLC2a1) and Glut3 (SLC2a3) are highly expressed in skin biopsies of dermatitis patients from multiple datasets. We demonstrated that administration of 2-deoxy-d-glucose (2DG) ameliorates animal models of 12-o-tetradecanoylphorbol-13-acetate (TPA) and oxazolone induced dermatitis using morphological and histological analysis. These results suggest that inhibition of glucose metabolism ameliorates dermatitis in animal models.
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HDAC2, one of the class I histone deacetylase regulates epigenetic landscape through histone modification. Because HDAC2 is overexpressed in many cancers, cancer therapeutics against HDAC2 have been developed. Here we show novel mechanism of HDAC2 regulation by E3 ligase RCHY1. We found inverse correlation RCHY1 and HDAC2 levels in tumor tissue from six independent dataset using meta-analysis. Ectopic expression of RCHY1 decreased the level of HDAC2 from cancer cells including p53 wildtype, mutant and null cells. In addition, HDAC2 was increased by RCHY1 knockdown. RCHY1 directly interacts with HDAC2. Ectopic expression of wild type but not RING mutant RCHY1 increased HDAC2 levels. These data provide an evidence that RCHY1 negatively regulates HDAC2.
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Histona Desacetilasa 2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células Cultivadas , Histona Desacetilasa 2/genética , Humanos , Ratones , Ratones DesnudosRESUMEN
Loliolide is one of the most ubiquitous monoterpenoid compounds found in algae, and its potential therapeutic effect on various dermatological conditions via agent-induced biological functions, including anti-oxidative and anti-apoptotic properties, was demonstrated. Here, we investigated the effects of loliolide on hair growth in dermal papilla (DP) cells, the main components regulating hair growth and loss conditions. For this purpose, we used a threedimensional (3D) DP spheroid model that mimics the in vivo hair follicle system. Biochemical assays showed that low doses of loliolide increased the viability and size of 3D DP spheroids in a dose-dependent manner. This result correlated with increases in expression levels of hair growth-related autocrine factors including VEGF, IGF-1, and KGF. Immunoblotting and luciferase-reporter assays further revealed that loliolide induced AKT phosphorylation, and this effect led to stabilization of ß-catenin, which plays a crucial role in the hair-inductive properties of DP cells. Further experiments showed that loliolide increased the expression levels of the DP signature genes, ALP, BMP2, VCAN, and HEY1. Furthermore, conditioned media from loliolide-treated DP spheroids significantly enhanced proliferation and the expression of hair growth regulatory genes in keratinocytes. These results suggested that loliolide could function in the hair growth inductivity of DP cells via the AKT/ ß-catenin signaling pathways.
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Benzofuranos/farmacología , Dermis/efectos de los fármacos , Folículo Piloso/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , beta Catenina/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Dermis/citología , Células HEK293 , Folículo Piloso/crecimiento & desarrollo , Humanos , Queratinocitos/efectos de los fármacos , Monoterpenos/farmacología , Fosforilación/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
HDAC3, one of the class I histone deacetylase modulates epigenetic landscape through histone modification. HDAC3 also interacts with non-histone proteins including p53 for deacetylation. Moreover, HDAC3 serves as a transcriptional repressor, interacting with NCor1/SMRT complex. Although HDAC3 plays a critical role for cellular homeostasis, regulatory mechanism of HDAC3 have been poorly understood. Here we report a novel regulatory mechanism of HDAC3 about its monoubiquitination and stabilization by Mdm2. HDAC3 levels were increased by ectopic expression of Mdm2 and decreased by Mdm2 ablation in various cell lines. We found that Mdm2 directly interacts with HDAC3 and induces HDAC3 protein levels without alteration of mRNA levels. Ectopic expression of wild type but not RING mutant of Mdm2 increased HDAC3 monoubiquitination. In addition, MdmX is beneficial for mdm2-mediated HDAC3 regulation. Ablation of Mdm2 and Mdm2/MdmX decreased cell migration along with the decrease of HDAC3 levels. These data provide an evidence that Mdm2 positively regulates HDAC3 monoubiquitination and stability.
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Histona Desacetilasas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Histona Desacetilasas/química , Humanos , Ratones , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , UbiquitinaciónRESUMEN
Chemoresistance is one of most critical clinical problems encountered when treating patients with ovarian cancer, due to the fact that the disease is usually diagnosed at advanced stages. Metformin is used as a firstline drug for the treatment of type 2 diabetes; however, drug repositioning studies have revealed its antitumor effects, mainly mediated through AMPactivated protein kinase (AMPK) activation and AKT/mammalian target of rapamycin (mTOR) pathway inhibition in various types of cancer, including drugresistant cancer cells. The current study revealed that the novel antitumor mechanism of metformin is mediated by regulation of mitochondrial E3 ubiquitin protein ligase 1 (MUL1) expression that negatively regulates AKT. The results demonstrated that metformin decreased the expression of AKT protein levels via MUL1 E3 ligase. In addition, metformin increased both mRNA and protein levels of MUL1 and promoted degradation of AKT in a proteasomedependent manner. Silencing MUL1 expression suppressed the metforminmediated AKT degradation and its downstream effects. Cell cycle analysis and a clonogenic assay demonstrated that knockdown of MUL1 significantly diminished the antitumor effects of metformin. Together, these data indicate that MUL1 regulates metforminmediated AKT degradation and the antitumor effects of metformin in chemoresistant ovarian cancer cell lines.
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Resistencia a Antineoplásicos/efectos de los fármacos , Metformina/administración & dosificación , Neoplasias Ováricas/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Reposicionamiento de Medicamentos , Femenino , Humanos , Metformina/farmacología , Ratones , Neoplasias Ováricas/enzimología , Proteolisis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Progesterone receptor membrane component1 (PGRMC1) is a heme-binding protein involved in cancers and Alzheimer's disease. PGRMC1 consists of a short N-terminal extracellular or luminal domain, a single membrane-spanning domain, and a long cytoplasmic domain. Previously, we generated two monoclonal antibodies (MAbs) 108-B6 and 4A68 that recognize cell surface-expressed PGRMC1 (csPGRMC1) on human pluripotent stem cells and some cancer cells. In this study, flow cytometric analysis found that an anti-PGRMC1 antibody recognizing the N-terminus of PGRMC1 could not bind to csPGRMC1 on cancer cells, and 108-B6 and 4A68 binding to csPGRMC1 was inhibited by trypsin treatment, suggesting that the epitopes of 108-B6 and 4A68 are trypsin-sensitive. To examine the epitope specificity of 108-B6 and 4A68, glutathione-S-transferase (GST)-fused PGRMC1 mutants were screened to identify the epitopes targeted by the antibodies. The result showed that 108-B6 and 4A68 recognized C-terminal residues 183-195 and 171-182, respectively, of PGRMC1, where trypsin-sensitive sites are located. A polyclonal anti-PGRMC1 antibody raised against the C-terminus of PGRMC1 could also recognized csPGRMC1 in a trypsin-sensitive manner, suggesting that the C-terminus of csPGRMC1 is exposed on the cell surface. This finding reveals that csPGRMC1 has a non-conventional plasma membrane topology, which is different from that of intracellular PGRMC1.
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Anticuerpos Monoclonales/inmunología , Membrana Celular/metabolismo , Mapeo Epitopo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Receptores de Progesterona/inmunología , Receptores de Progesterona/metabolismo , Línea Celular , Humanos , Espacio Intracelular/metabolismo , Proteínas de la Membrana/química , Receptores de Progesterona/químicaRESUMEN
Skin photoaging, which is mainly induced by ultraviolet B (UVB) radiation, is prevented by the application of UV-protective agents. The microalga Nannochloropsis oceanica (N. oceanica) has been primarily reported as a potential biofuel; however, in this study, we investigated whether N. oceanica extracts exerted photoprotective effects against UVB-irradiated human dermal fibroblasts (HDFs) and which single component was responsible for the protective effect of the extracts. Two extracts-pigment and nonpigment-were prepared from N. oceanica biomass. WST-1 assay and expression analysis of interleukin genes showed that the pigment extracts were not significantly cytotoxic to HDFs. Further experiments revealed that treatment with the pigment extract upregulated the expression of collagen genes and significantly blocked UVB-induced damage such as decreased cell viability and increased ROS production. Next, to investigate the pigment composition of the extracts, HPLC analysis was conducted and violaxanthin was identified as the major pigment. The UVB photoprotective effect of the pigment extracts was confirmed in violaxanthin-treated HDFs. In addition, violaxanthin significantly attenuated UVB-induced G1 phase arrest, senescence-associated ß-galactosidase activation, p16 and p21 upregulation, ERK phosphorylation and the downregulation of ECM molecules in HDFs. Therefore, we concluded that violaxanthin was a potential antiphotoaging agent.
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Algas Marinas/química , Piel/efectos de los fármacos , Piel/efectos de la radiación , Rayos Ultravioleta , Biomasa , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/efectos de la radiación , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Humanos , Piel/citología , Xantófilas/farmacologíaRESUMEN
Abnormal melanin synthesis results in several hyperpigmentary disorders such as freckles, melanoderma, age spots, and other related conditions. In this study, we investigated the antimelanogenic effects of an extract from the microalgae Chlamydomonas reinhardtii (CE) and potential mechanisms responsible for its inhibitory effect in B16F10, normal human epidermal melanocyte cells, and human skin-equivalent models. The CE extract showed significant dose-dependent inhibitory effects on α-melanocyte-stimulating, hormone-induced melanin synthesis in cells. Additionally, the CE extract exhibited suppressive effects on the mRNA and protein expression of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. The CE extract also inhibited the phosphorylation of protein kinase A and extracellular signal-related kinase, which function as upstream regulators of melanogenesis. Using a three-dimensional, reconstructed pigmented epidermis model, the CE-mediated, anti-pigmentation effects were confirmed by Fontana-Masson staining and melanin content assays. Taken together, CE extract can be used as an anti-pigmentation agent.
Asunto(s)
Chlamydomonas reinhardtii/química , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Pigmentación/efectos de los fármacos , Extractos Vegetales/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Descubrimiento de Drogas , Células Epidérmicas/efectos de los fármacos , Células Epidérmicas/patología , Epidermis/efectos de los fármacos , Epidermis/patología , Quinasas MAP Reguladas por Señal Extracelular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Oxidorreductasas Intramoleculares/metabolismo , Melaninas/metabolismo , Melanoma , Glicoproteínas de Membrana/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/metabolismo , Oxidorreductasas/metabolismo , Fosforilación , ARN Mensajero/efectos de los fármacos , Piel , alfa-MSH/metabolismoRESUMEN
Arctiin, a lignin isolated from Arctium lappa, exhibits a variety of biological effects, including antiviral, antiinflammatory, and antiproliferative actions, in mammalian cells. In a previous study, arctiin was demonstrated to induce procollagen type I synthesis and exhibited protective effects against ultraviolet B (UVB) radiation in normal human dermal fibroblasts (nHDFs). However, the underlying molecular mechanism of arctiinmediated collagen synthesis remains unknown. In the present study, the mechanism for increased expression of collagen type 1α 1 chain (COL1A1) mRNA in arctiininduced nHDFs was identified. The expression of microRNA378b (miR378b), downregulated by arctiin, was correlated with the expression of sirtuin6 (SIRT6) mRNA, a regulator of COL1A1 mRNA. Furthermore, it was revealed that arctiin protected the UVB radiationmediated decrease in COL1A1 mRNA expression, through the miR378b/SIRT6 signaling pathway. In conclusion, these results suggest that arctiin regulates COL1A1 through the miR378bSIRT6 axis.