RESUMO
Arthrobacter sp. KFRI-F3372 is a Gram-positive bacterium with a high G + C content of 65.7%, which was isolated from Doenjang, a traditional Korean fermented soybean paste. In this report, we introduce the complete genome sequence of Arthrobacter sp. KFRI-F3372.
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The receptor tyrosine kinase c-Met is elaborated in embryogenesis, morphogenesis, metabolism, cell growth, and differentiation. JNJ38877605 (JNJ) is an inhibitor of c-Met with anti-tumor activity. The c-Met expression and its role in adipocyte differentiation are unknown. Here, we investigated the c-Met expression and phosphorylation, knockdown (KD) effects, and pharmacological inhibition of c-Met by JNJ on fat accumulation in murine preadipocyte 3T3-L1 cells. During 3T3-L1 preadipocyte differentiation, strikingly, c-Met expression at the protein and mRNA levels and the protein phosphorylation on Y1234/1235 and Y1349 is crucial for inducing its kinase catalytic activity and activating a docking site for signal transducers were increased in a time-dependent manner. Of note, JNJ treatment at 20 µM that strongly inhibits c-Met phosphorylation without altering its total expression resulted in less lipid accumulation and triglyceride (TG) content with no cytotoxicity. JNJ further reduced the expression of adipogenic regulators, including CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and perilipin A. Moreover, JNJ treatment increased cAMP-activated protein kinase (AMPK) and liver kinase B-1 (LKB-1) phosphorylation but decreased ATP levels. Significantly, KD of c-Met suppressed fat accumulation and triglyceride (TG) quantity and reduced the expression of C/EBP-α, PPAR-γ, FAS, ACC, and perilipin A. Collectively, the present results demonstrate that c-Met is a novel, highly conserved mediator of adipogenesis regulating lipid accumulation in murine adipocytes.
Assuntos
Adipogenia , Receptores Ativados por Proliferador de Peroxissomo , Animais , Camundongos , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Ácido Graxo Sintases/metabolismo , Metabolismo dos Lipídeos , Lipídeos/farmacologia , Perilipinas/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , PPAR gama/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Triglicerídeos/metabolismoRESUMO
AlaskOmega® Omega 7 500, also known as Omega7 fatty acid or 7MEGA™, is a highly concentrated palmitoleic acid (C16:1). Little is known about how 7MEGA regulates skin inflammation and wrinkle formation in cultured skin cells. The present study aimed to investigate the effects of 7MEGA on the expression of cyclooxygenase2 (COX2), matrix metallopeptidase (MMP)1/3 and type 1 procollagen, which are markers of skin inflammation and wrinkle formation, in ultraviolet B (UVB)irradiated human dermal fibroblasts (HDFs) and keratinocytes (HaCaT). No toxicity was observed upon treatment of HDFs and HaCaT cells with 0.52.5 µl/ml 7MEGA. The exposure of HaCaT cells to 10 mJ/cm2 UVB for 6 h resulted in increased protein and/or mRNA expression of COX2 and MMP3. Treatment of HaCaT cells with 2.5 µl/ml 7MEGA suppressed the UVBinduced expression of COX2 and MMP3 in these cells. In addition, treatment with 2.5 µl/ml 7MEGA attenuated the UVBinduced expression and phosphorylation levels of cFos and cJun, two components of the activator protein1 (AP1) transcription factor, in HaCaT cells. Exposure of HDFs to 60 mJ/cm2 UVB for 6 h significantly decreased the expression of type 1 procollagen protein, whereas treatment with 2.5 µl/ml 7MEGA partially reversed the effects of UVB on the expression of type 1 procollagen protein. These results demonstrated for the first time that 7MEGA regulated the expression of COX2, MMP3 and type 1 procollagen in UVBirradiated skin cells. The present study suggested that 7MEGA may serve as a novel agent against UVBinduced skin inflammation and damage.
Assuntos
Colágeno Tipo I/biossíntese , Ciclo-Oxigenase 2/biossíntese , Derme/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Queratinócitos/metabolismo , Metaloproteinase 3 da Matriz/biossíntese , Raios Ultravioleta , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , HumanosRESUMO
Excessive preadipocyte differentiation/adipogenesis is closely linked to the development of obesity. LY3009120 is a panRaf kinase inhibitor and is known for its anticancer activities. In the present study, the effect of LY3009120 on 3T3L1 cell adipogenesis was investigated. The differentiation of 3T3L1 preadipocytes into adipocytes was measured by Oil Red O staining and AdipoRed assay. Changes of cellular protein expression and phosphorylation levels in differentiating 3T3L1 preadipocytes in the absence or presence of LY3009120 were determined by western blotting analysis. Cell count assay was used to assess the cytotoxicity of LY3009120 on 3T3L1 cells. At 0.3 µM, LY3009120 markedly inhibited lipid accumulation and decreased triglyceride content in differentiating 3T3L1 cells. However, it had minimal effect on the elevated expression and phosphorylation of three Raf kinase isoforms (CRaf, ARaf, and BRaf) observed in the cells. LY3009120 reduced not only the expression of CCAAT/enhancerbinding proteinα (C/EBPα), peroxisome proliferatoractivated receptorγ (PPARγ), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and perilipin A, but also reduced the phosphorylation of signal transducer and activator of transcription3 (STAT3) in differentiating 3T3L1 cells. LY3009120 also increased the phosphorylation of adenosine 3',5'cyclic monophosphate (cAMP)activated protein kinase (AMPK), but did not affect the phosphorylation or expression of liver kinase B1 in these cells. In summary, this is the first report, to the best of our knowledge, demonstrating that LY3009120 has an antiadipogenic effect on 3T3L1 cells, which may be mediated through control of the expression and phosphorylation of C/EBPα, PPARγ, STAT3, FAS, ACC, perilipin A, and AMPK.
Assuntos
Adipogenia/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas/metabolismo , Pirimidinas/farmacologia , Quinases raf/antagonistas & inibidores , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ácido Graxo Sintases/metabolismo , Camundongos , PPAR gama/metabolismo , Perilipina-1/metabolismo , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismoRESUMO
Meridianin C is a marine natural product known for its anti-cancer activity. At present, the anti-tumour effects of meridianin C on oral squamous cell carcinoma are unknown. Here, we investigated the effect of meridianin C on the proliferation of four different human tongue cancer cells, YD-8, YD-10B, YD-38 and HSC-3. Among the cells tested, meridianin C most strongly reduced the growth of YD-10B cells; the most aggressive and tumorigenic of the cell lines tested. Strikingly, meridianin C induced a significant accumulation of macropinosomes in the YD-10B cells; confirmed by the microscopic and TEM analysis as well as the entry of FITC-dextran, which was sensitive to the macropinocytosis inhibitor amiloride. SEM data also revealed abundant long and thin membrane extensions that resemble lamellipodia on the surface of YD-10B cells treated with meridianin C, pointing out that meridianin C-induced macropinosomes was the result of macropinocytosis. In addition, meridianin C reduced cellular levels of Dickkopf-related protein-3 (DKK-3), a known negative regulator of macropinocytosis. A role for DKK-3 in regulating macropinocytosis in the YD-10B cells was confirmed by siRNA knockdown of endogenous DKK-3, which led to a partial accumulation of vacuoles and a reduction in cell proliferation, and by exogenous DKK-3 overexpression, which resulted in a considerable inhibition of the meridianin C-induced vacuole formation and decrease in cell survival. In summary, this is the first study reporting meridianin C has novel anti-proliferative effects via macropinocytosis in the highly tumorigenic YD-10B cell line and the effects are mediated in part through down-regulation of DKK-3.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Alcaloides Indólicos/farmacologia , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pinocitose/efeitos dos fármacos , Pirimidinas/farmacologia , Neoplasias da Língua/metabolismo , Neoplasias da Língua/patologia , Proteínas Adaptadoras de Transdução de Sinal , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocinas , Humanos , Alcaloides Indólicos/química , Indóis/química , Pirimidinas/química , Neoplasias da Língua/ultraestrutura , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Bacillus subtilis and B. velezensis are frequently isolated from various niches, including fermented foods, water, and soil. Within the Bacillus subtilis group, B. velezensis and B. subtilis subsp. subtilis have received significant attention as biological resources for biotechnology-associated industries. Nevertheless, radical solutions are urgently needed to identify microbes during their ecological succession to accurately confirm their action at the species or subspecies level in diverse environments, such as fermented materials. Thus, in this study, previously published genome data of the B. subtilis group were compared to exploit species- or subspecies-specific genes for use as improved qPCR targets to detect B. velezensis and B. subtilis subsp. subtilis in kimchi samples. In silico analyses of the selected genes and designed primer sequences, in conjunction with SYBR Green real-time PCR, confirmed the robustness of this newly developed assay. Consequently, this study will allow for new insights into the ontogeny and succession of B. velezensis and B. subtilis subsp. subtilis in various niches. Interestingly, in white kimchi without red pepper powder, neither B. subtilis subsp. subtilis nor B. velezensis was detected.
Assuntos
Bacillus/fisiologia , Fermentação , Alimentos Fermentados/microbiologia , Microbiologia de Alimentos , Bacillus/classificação , Bacillus/genética , Bacillus subtilis , Evolução Biológica , Genoma Bacteriano , Genômica/métodos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Lactobacillus plantarum is one of the most extensively studied Lactobacillus species because of its presence in a variety of environmental niches, versatility, and metabolic capabilities, resulting in the use of this organism in many industrial applications. However, although extensive effort has been invested in screening this species from a variety of habitats, a reliable and accurate method for studying the succession and ontogeny of this organism in complex ecosystems is still required to confirm the activity of L. plantarum at the subspecies level. Therefore, in this study, novel subspecies-specific genes for the quantitative detection of two L. plantarum subspecies were identified by comparative genomic analysis. The specificity of primer sets for selected genes specific to each targeted microbe was confirmed in kimchi samples. Interestingly, in all the kimchi samples at 4 °C, the presence of L. plantarum subsp. argentoratensis was not observed. Hence, we found that low temperatures markedly affected the ontogeny of L. plantarum subsp. argentoratensis during kimchi fermentation. Subsequently, this touchstone method will offer new insight and metrics to understand the ontogeny and succession of L. plantarum subsp. plantarum and L. plantarum subsp. argentoratensis in various niches.
Assuntos
Lactobacillus plantarum/genética , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ecossistema , Ontologia Genética , Genes Bacterianos , Genômica , Lactobacillus plantarum/classificação , Lactobacillus plantarum/isolamento & purificação , Fenótipo , RNA Ribossômico 16S/genéticaRESUMO
The proviral integration moloney murine leukaemia virus (Pim) kinases, consisting of Pim-1, Pim-2 and Pim-3, are involved in the control of cell growth, metabolism and differentiation. Pim kinases are emerging as important mediators of adipocyte differentiation. AZD1208 is a pan-Pim kinase inhibitor and is known for its anti-cancer activity. In this study, we investigated the effect of AZD1208 on adipogenesis and lipolysis in 3T3-L1 cells, a murine preadipocyte cell line. AZD1208 markedly suppressed lipid accumulation and reduced triglyceride contents in differentiating 3T3-L1 cells, suggesting the drug's anti-adipogenic effect. On mechanistic levels, AZD1208 reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and perilipin A but also the phosphorylation of signal transducer and activator of transcription-3 (STAT-3) in differentiating 3T3-L1 cells. Remarkably, AZD1208 increased cAMP-activated protein kinase (AMPK) and LKB-1 phosphorylation while decreased intracellular ATP contents in differentiating 3T3-L1 cells. Furthermore, in differentiated 3T3-L1 adipocytes, AZD1208 also partially promoted lipolysis and enhanced the phosphorylation of hormone-sensitive lipase (HSL), a key lipolytic enzyme, indicating the drug's HSL-dependent lipolysis. In summary, the findings show that AZD1208 has anti-adipogenic and lipolytic effects on 3T3-L1 adipocytes. These effects are mediated by the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, ACC, perilipin A, STAT-3, AMPK and HSL.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Lipólise/efeitos dos fármacos , Tiazolidinas/farmacologia , Células 3T3-L1 , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , PPAR gama/genética , Perilipina-1/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Receptor fas/genéticaRESUMO
Tanshinone IIA is a diterpene quinone isolated from the roots of Salviamiltiorrhiza bunge that has traditionally been used in China for the treatment of cardiovascular and cerebrovascular disorders. Although there is recent evidence showing that tanshinone IIA has an anti-obesity effect, its underlying mechanism of anti-obesity effect is poorly understood. Here, we investigated the effect of tanshinone IIA on lipid accumulation in 3T3-L1 preadipocytes and zebrafish. Notably, tanshinone IIA at 10 µM concentration greatly reduced lipid accumulation and triglyceride (TG) contents during 3T3-L1 preadipocyte differentiation, suggesting its anti-adipogenic effect. On mechanistic levels, tanshinone IIA reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3/5 (STAT-3/5) in differentiating 3T3-L1 cells. In addition, tanshinone IIA strongly inhibited leptin and resistin mRNA expression in differentiating 3T3-L1 cells. Importantly, the tanshinone IIA's lipid-reducing effect was also seen in zebrafish. In sum, these findings demonstrate that tanshinone IIA has anti-adipogenic effects on 3T3-L1 cells and zebrafish, and its anti-adipogenic effect on 3T3-L1 cells is largely attributable to the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3/5.
Assuntos
Abietanos/farmacologia , Adipogenia/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/genética , Animais , Biomarcadores , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipólise , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Peixe-ZebraRESUMO
Sulforaphane (SFN) is a dietary isothiocyanate abundantly available in cruciferous vegetables and has been shown to possess anti-inflammatory and immunomodulatory activities. Chemokines are important mediators of inflammation and immune responses due to their ability to recruit and activate macrophages and leukocytes. To date, little is known about the SFN-mediated regulation of chemokine expression in pancreatic ß-cells. In this study, we investigated the inhibitory effects and mechanisms of SFN on the interferon-γ (IFN-γ)-induced expression of a subset of chemokines, including monokine induced by IFN-γ (MIG), IFN-inducible protein of 10 kDa (IP-10) and IFN-inducible Tcell alpha chemoattractant (I-TAC), in INS1 cells, a rat pancreatic ß-cell line. Notably, IFN-γ treatment led to an increase in the mRNA expression levels of MIG, IP-10 and I-TAC in the INS1 cells. However, SFN strongly blocked the mRNA expressions of MIG, IP-10 and I-TAC induced by IFN-γ in INS1 cells. On the mechanistic level, SFN significanlty decreased not only the mRNA expression levels of interferon regulatory factor-1 (IRF-1), but also the phosphorylation levels of signal transducer and activator of transcription-1 (STAT-1) and protein kinase B (PKB) which were induced by IFN-γ in the INS1 cells. Pharmacological inhibition experiments further revealed that treatment with JAK inhibitor I weakly inhibited the IFN-γ-induced expression of IP-10, whereas it strongly suppressed the IFN-γ-induced expression of MIG and I-TAC in the INS1 cells. Moreover, treatment with LY294002, a PI3K/PKB inhibitor, was able to slightly repress IFNγinduced expressions of MIG and I-TAC, but not IP-10, in INS1 cells. Importantly, the IFN-γ-induced increase in the expression levels of MIG, IP-10 and I-TAC in the INS-1 cells was strongly inhibited by SFN, but not by other natural substances, such as curcumin, sanguinarine, resveratrol, triptolide and epigallocatechin gallate (EGCG), suggesting the specificity of SFN in downregulating the levels of these chemokines. To the best of our knowledge, these results collectively demonstrate for the first time that SFN strongly inhibits the IFN-γ-induced expression of MIG, IP-10 and I-TAC in INS1 cells and this inhibition is, at least in part, mediated through the reduced expression and phosphorylation levels of IRF-1, STAT-1 and PKB.
Assuntos
Quimiocina CXCL10/biossíntese , Quimiocina CXCL11/biossíntese , Quimiocina CXCL9/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Fator Regulador 1 de Interferon/biossíntese , Interferon gama/farmacologia , Isotiocianatos/farmacologia , Proteínas Proto-Oncogênicas c-akt/biossíntese , Fator de Transcrição STAT1/biossíntese , Animais , Linhagem Celular Transformada , Células Secretoras de Insulina/citologia , Ratos , SulfóxidosRESUMO
The proviral integration site for moloney murine leukemia virus (Pim) kinases, consisting of Pim-1, Pim-2 and Pim-3, belongs to a family of serine/threonine kinases that are involved in controlling cell growth and differentiation. Pim kinases are emerging as important mediators of adipocyte differentiation. SGI-1776, an inhibitor of Pim kinases, is widely used to assess the physiological roles of Pim kinases, particularly cell functions. In the present study, we examined the effects of SGI-1776 on adipogenesis. The antiadipogenic effect of SGI1776 was measured by Oil Red O staining and AdipoRed assays. The effect of SGI1776 on the growth of 3T3L1 adipocytes was determined by cell count analysis. The effects of SGI1776 on the protein and mRNA expression of adipogenesis-related proteins and adipokines in 3T3L1 adipocytes were also evaluated by western blot analysis and RTPCR, respectively. Notably, SGI-1776 markedly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. On a mechanistic level, SGI-1776 inhibited not only the expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ) and fatty acid synthase (FAS), but also the phosphorylation of signal transducer and activator of transcription-3 (STAT-3). Moreover, SGI-1776 decreased the expression of adipokines, including the expression of leptin and regulated on activation, normal T cell expressed and secreted (RANTES) during adipocyte differentiation. These findings demonstrate that SGI-1776 inhibits adipogenesis by downregulating the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS and STAT-3.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Piridazinas/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , PPAR gama/genética , Fator de Transcrição STAT3/genéticaRESUMO
Differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. Excessive adipogenesis, however, is largely linked to the development of obesity. Herein we investigated a library of 53 novel chemicals, generated from a number of polyphenolic natural compounds, on adipogenesis. Strikingly, among the chemicals tested, KMU-3, a derivative of gallic acid, strongly suppressed lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. On mechanistic levels, KMU-3 inhibited expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) during adipocyte differentiation. Moreover, KMU-3 reduced expressions of adipokines, including retinol binding protein-4 (RBP-4), leptin, and regulated on activation, normal T cell expressed and secreted (RANTES) during adipocyte differentiation. Of further note, KMU-3 rapidly blocked the phosphorylation of signal transducer and activator of transcription-3 (STAT-3) during the early stage of adipogenesis. Importantly, pharmacological inhibition studies revealed that AG490, a JAK-2/STAT-3 inhibitor suppressed adipogenesis and STAT-3 phosphorylation, implying that early blockage of STAT-3 activity is crucial for the KMU-3-mediated anti-adipogenesis. These findings demonstrate firstly that KMU-3 inhibits adipogenesis by down-regulating STAT-3, PPAR-γ, C/EBP-α, and FAS. This work shows that KMU-3 is an inhibitor of adipogenesis and thus may have therapeutic potential against obesity.
Assuntos
Adipogenia/efeitos dos fármacos , Benzamidas/farmacologia , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Benzamidas/síntese química , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Quimiocina CCL5/genética , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Ácido Gálico/síntese química , Leptina/genética , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Receptor fas/genéticaRESUMO
Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a-7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases' inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of these meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-α, PPAR-γ, FAS, STAT-3 and STAT-5.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Alcaloides Indólicos/farmacologia , Leptina/biossíntese , Metabolismo dos Lipídeos/fisiologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , CamundongosRESUMO
Hypoxia-inducible factor-1 (HIF-1) is a tumor angiogenic transcription factor composed of an α and ß subunit. We investigated the effect of glucosamine hydrochloride (GS-HCl) on the expression of HIF-1α and HIF-1ß in serumtreated YD-8 human tongue cancer cells. While long-term (24 h) treatment with GS-HCl strongly repressed the expression of HIF-1α and HIF-1ß at both the protein and mRNA levels, short-term (4 h) GS-HCl treatment inhibited HIF-1α at the protein level. Short-term GS-HCl treatment also decreased phosphorylation of p70S6K and S6, translation-related proteins. However, the results of subsequent pharmacological inhibition and protein stability analyses indicated that HIF-1α protein downregulation induced by short-term GS-HCl treatment was not through modulation of the mTOR/p70S6K/S6 signaling pathways, the 26S proteasomal and lysosomal activities and HIF-1α protein stability. Importantly, our further analyses identified that HIF-1α protein downregulation induced by short-term GS-HCl treatment was blunted by exogenous administration of the citric acid cycle metabolites citrate and 2-oxoglutarate, but not the glycolytic end byproducts pyruvate and lactate. These findings demonstrate firstly that short-term GS treatment selectively downregulates HIF-1α at the protein level in YD-8 cells via interference of production of the citric acid cycle metabolites. It is proposed that short-term GS-HCl exposure may be applied for the treatment of oral tumors with high expression of HIF-1α.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Glucosamina/administração & dosagem , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias da Língua/metabolismo , Linhagem Celular Tumoral , Ácido Cítrico/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Gluconatos/farmacologia , Glucosamina/farmacologia , Humanos , Técnicas In Vitro , Ácido Láctico/farmacologia , Leupeptinas/farmacologia , Lisossomos/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ácido Pirúvico/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Evidence suggests that solar ultraviolet B (UVB) radiation inhibits growth and/or induces apoptosis of human skin cells. However, mechanisms underlying the UVB-induced anti-survival and pro-apoptotic effects on human skin cells remain unclear. In this study, we investigated the effect of UVB radiation on survival and apoptosis of HaCaT human keratinocytes and determined possible molecular, cellular and signaling mechanisms including cross-regulation, which are responsible for the UVB's anti-survival and/or pro-apoptotic effects. The results showed that UVB radiation at 400 mJ/cm² for 8 h largely decreased cell survival and induced DNA fragmentation, an index of apoptosis, in HaCaT human keratinocytes. On a mechanistic level, UVB radiation triggered the activation of caspase-9, cleavage of poly(ADP-ribose) polymerase, and downregulation of myeloid cell leukemia-1 (Mcl-1), human inhibitor of apoptosis protein-1 (HIAP-1), X-linked IAP (XIAP), and protein kinase B (PKB), but did not affect the expression of B-cell lymphoma-2 in HaCaT cells. UVB radiation also upregulated the expression of glucose-regulated protein 78 (GRP78), an endoplasmic reticulum (ER) stress marker, in HaCaT cells. Of note, results of pharmacological inhibition studies have demonstrated that pretreatment with z-VAD-fmk, a pan-caspase inhibitor strongly attenuated UVB-induced apoptosis, the activation of caspase-9, downregulation of Mcl-1, XIAP and PKB (but not HIAP-1), and upregulation of GRP78, while pretreatment with GF109203 or GO6983, pan-PKC inhibitors, substantially blocked the UVB-induced reduction of cell survival, activation of caspase-9, downregulation of HIAP-1, XIAP, and PKB (but not Mcl-1), and GRP78 upregulation in HaCaT cells. Collectively, these results demonstrated that UVB has strong anti-survival and pro-apoptotic effects on HaCaT cells and the effects were largely mediated via the activation of caspase-9 and protein kinase Cs, which subsequently downregulated PKB, XIAP, HIAP-1 and Mcl-1, and triggered ER stress.
Assuntos
Caspase 9/biossíntese , Proteínas Inibidoras de Apoptose/biossíntese , Queratinócitos/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Animais , Caspase 9/genética , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Proteínas de Choque Térmico/biossíntese , Humanos , Proteínas Inibidoras de Apoptose/genética , Queratinócitos/efeitos da radiação , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína Quinase C/biossíntese , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ubiquitina-Proteína Ligases , Raios Ultravioleta , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
Excessive and/or long-term glucocorticoid therapy reduces ß-cell mass and induces hyperglycemia, which contribute to the development of steroidinduced diabetes. Ceriporia (C.) lacerata is one of the whiterot fungi and has been used in bioremediations, such as lignocellulose degradation, in nature. The pharmacologic effect of C. lacerata on steroid-induced ß-cell toxicity is not known. In this study, we evaluated the effect of a crude extract from a submerged cultivation of C. lacerata on the survival and apoptosis of INS-1 rat insulin-secreting cells exposed to dexamethasone (Dex), a synthetic diabetogenic glucocorticoid. Treatment with the C. lacerata crude extract (CLCE) largely blocked the Dex-induced reduction in survival and apoptosis of INS-1 cells. Moreover, CLCE treatment inhibited Dex-induced protein kinase B (PKB) dephosphorylation without affecting Dex-induced extracellular signal-regulated protein kinase-1/2 dephosphorylation and MKP-1 upregulation. Importantly, the protective effect of CLCE on Dex-induced cytotoxicity in INS-1 cells was attenuated by LY294002, an inhibitor of PI3K/PKB. CLCE treatment, however, did not protect the INS-1 cells from the cytotoxic effects triggered by other insults, such as interleukin-1ß (an inflammatory cytokine), streptozotocin (a diabetogenic drug), thapsigargin (a calcium mobilizing agent), and tunicamycin (an ER stress inducer). Collectively, these findings demonstrate for the first time the ability of CLCE to specifically protect INS-1 cells from Dex-induced cytotoxicity through the modulation of the PI3K/PKB pathway. It is suggested that CLCE may be applied for the prevention and/or treatment of steroid diabetes in which reduction of ß-cell survival and induction of ß-cell apoptosis play pathogenic roles.
Assuntos
Basidiomycota/química , Misturas Complexas/farmacologia , Dexametasona/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Fosfatase 1 de Especificidade Dupla/metabolismo , Interleucina-1beta/toxicidade , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Morfolinas/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Estreptozocina/toxicidade , Tapsigargina/toxicidade , Tunicamicina/toxicidadeRESUMO
Platelet-derived growth factor (PDGF)-D is suggested to be a key factor in the development of several renal pathologies, including mesangioproliferative glomerulonephritis. Cyclooxygenase (COX)-2 is a protein involved in the biosynthesis of inflammatory prostaglandins. In this study, we investigated the effect of PDGF-D on the regulation of COX-2 expression in rat mesangial cells (RMCs). Treatment with PDGF-D induced COX-2 at both the protein and mRNA levels in RMCs, suggesting that the PDGF-D-mediated induction of COX-2 is due to COX-2 transcriptional upregulation. PDGF-D treatment also led to a rapid but transient activation of PKB and extracellular signal regulated kinase (ERK)-1/2. Activities of JNK-1/2 and p38 MAPK, however, were not influenced by PDGF-D in RMCs. Markedly, pharmacological inhibition studies showed that pretreatment with LY294002 (a PI3K/PKB inhibitor) or GF109203X (a pan-PKC inhibitor) suppressed the PDGF-D-induced expression of COX-2 protein and mRNA, while pretreatment with PD98059 (an ERK-1/2 inhibitor) or PP1 (an Src inhibitor) had no effect on it. These findings collectively demonstrate for the first time that PDGF-D induces COX-2 by transcriptional upregulation in RMCs and the induction is largely related to PI3K/PKB and PKCs activities.
Assuntos
Ciclo-Oxigenase 2/genética , Linfocinas/metabolismo , Células Mesangiais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Ciclo-Oxigenase 2/metabolismo , Ativação Enzimática , Células Mesangiais/citologia , Células Mesangiais/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , RNA Mensageiro/genética , Ratos , Transdução de Sinais , Ativação Transcricional , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Evidence suggests that mistletoe extract has the potential to be used as an anticancer agent. Abnobaviscum F® is a European mistletoe extract from the host tree Fraxinus. We investigated the effect of Abnobaviscum F on the growth and survival of different leukemia cell lines. Abnobaviscum F treatment strongly reduced survival and induced apoptosis of K562 (human myeloid leukemia), RPMI-8226 (human plasmacytoma) and L1210 (murine lymphocytic leukemia) cells in culture. Using K562 cells to further investigate the mechanism of action of Abnobaviscum F, we showed that Abnobaviscum F-induced cell death was associated with the activation of caspase-9, JNK-1/2 and p38 MAPK, as well as with the downregulation of Mcl-1, and inhibition of ERK-1/2 and PKB phosphorylation. Moreover, Abnobaviscum F treatment led to both a reduction of cellular glutathione (GSH) and the induction of ER stress (GRP78 and CHOP induction and eIF-2α phosphorylation). By contrast, Abnobaviscum F did not impact the expression of the DR4 and DR5 death receptors. The Abnobaviscum F-induced apoptosis of K562 cells was blocked by pretreatment with either GSH, z-VAD-fmk or SP600125. Our results, therefore, show that Abnobaviscum F induces apoptosis of K562 cells through the activation of the intrinsic caspase pathway, the phosphorylation of JNK-1, the reduction of cellular GSH, and the induction of ER stress.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia Linfoide/patologia , Leucemia Mieloide/metabolismo , Extratos Vegetais/farmacologia , Viscum album/química , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Antracenos/farmacologia , Caspase 9/biossíntese , Caspase 9/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Glutationa/metabolismo , Glutationa/farmacologia , Proteínas de Choque Térmico/biossíntese , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células K562 , Leucemia Linfoide/metabolismo , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fosforilação/efeitos dos fármacos , Plasmocitoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Fator de Transcrição CHOP/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
High exposure to cadmium is a risk factor for many neuronal diseases. Overexpression of cyclooxygenase (COX)-2 is linked to many neuroinflammatory and neoplastic diseases. We, herein, investigated the effect of cadmium on the expression of COX-2 in C6 rat glioma cells. Treatment with cadmium sulfate (cadmium) increased the expression of COX-2 mRNA. Remarkably, cadmium treatment further increased expression of not only the N-glycosylated COX-2 protein of 72 kDa but also the unglycosylated COX-2 of 66 kDa, as assessed by the unglycosylated COX-2 induced by tunicamycin or glucosamine, known inhibitors of COX-2 N-glycosylation. Of note, when translation was blocked in the presence of cycloheximide (CHX), levels of both N-glycosylated and unglycosylated COX-2 proteins induced by cadmium rapidly declined but the decline was prevented by MG132, a 26S proteasomal inhibitor. However, in the absence of CHX, cadmium induced and maintained expression of the unglycosylated COX-2 proteins. Pharmacological inhibition studies importantly demonstrated that the cadmium-mediated COX-2 transcriptional upregulation in C6 cells was not shown by exogenous glutathione (GSH) supplementation or treatment with inhibitors of extracellular signal-regulated protein kinase-1/2 (ERK-1/2), p38 MAPK and c-Jun N-terminal protein kinase-1/2 (JNK-1/2), respectively. Expression of COX-2 was not noted in C6 cells exposed to other heavy metals (cobalt or manganese). These results demonstrate that cadmium specifically induces expression of COX-2 through both transcriptional and co-translational (N-glycosylation) regulation in C6 cells in which the cadmium-induced COX-2 transcriptional upregulation is closely related to oxidative stress-dependent activation of the family of MAPKs and the cadmium-induced expression of both N-glycosylated and unglycosylated COX-2 proteins is proteasome- and translation-dependent.
Assuntos
Cádmio/metabolismo , Ciclo-Oxigenase 2/genética , Poluentes Ambientais/metabolismo , Glioma/genética , Regulação para Cima , Animais , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glicosilação , Sistema de Sinalização das MAP Quinases , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , RNA Mensageiro/genética , Ratos , Ativação TranscricionalRESUMO
Evidence suggests anti-tumor activities of glucosamine-hydrochloride (GS-HCl). In the present study, we investigated anti-proliferative, growth suppressive and/or pro-apoptotic effects of GS-HCl on YD-8 human oral squamous cell carcinoma (OSCC) cells. Fundamentally, treatment with GS-HCl strongly inhibited proliferation and induced apoptosis in YD-8 cells, as determined by MTS and DNA fragmentation analyses. Of further note, as measured by Western analyses, GS-HCl treatment led to activation of caspase-3, cytosolic accumulation of cytochrome c, down-regulation of Mcl-1 and HIF-1α, up-regulation of GRP78, an indicator of ER stress, and generation of ROS in YD-8 cells. Importantly, results of pharmacological inhibition studies showed that treatment with z-VAD-fmk, a pan-caspase inhibitor, but not with vitamin E, an anti-oxidant strongly blocked the GS-HCl-induced apoptosis in YD-8 cells. Analyses of additional cell culture works further revealed that GS-HCl had a strong growth suppressive effect on not only YD-8 but also YD-10B and YD-38, two other human OSCC cell lines. These findings collectively demonstrate that GS-HCl has anti-proliferative, anti-survival, and pro-apoptotic effects on YD-8 cells and the effects appear to be mediated via mechanisms associated with the mitochondrial-dependent activation of caspases, down-regulation of Mcl-1, and induction of ER stress. Considering HIF-1α as a tumor angiogenic transcription factor, the ability of GS-HCl to down-regulate HIF-1α in YD-8 cells may further support its anti-cancer property. It is thus suggested that GS-HCl may be used as a potential anti-cancer drug against human OSCC.