RESUMO
Uncontrolled inflammation is a leading cause of various chronic diseases. Cinnamaldehyde (CA) is a major bioactive compound isolated from the essential oil of the leaves of Cinnamomum osmophloeum kaneh that exhibits anti-inflammatory activity; however, the use of CA is limited by its cytotoxicity. Here, we synthesized three CA derivatives and identified 4-hydroxycinnamaldehyde-galactosamine (HCAG) as a low toxicity anti-inflammatory compound in vitro (HCAG IC50 â« 1600 µM; CA IC50=40 µM) and in vivo. HCAG reduced pro-inflammatory mediator expression in LPS-activated macrophages by inhibiting MAPK and PKC-α/δ phosphorylation, decreasing ROS generation and reducing NF-κB activation. HCAG also reduced NLRP3 inflammasome-derived IL-1ß secretion by inhibiting the ATP-mediated phosphorylation of AKT and PKC-α/δ. In a mouse model of LPS-induced renal inflammation, we observed reduced albuminuria and a mild degree of glomerular proliferation, glomerular sclerosis and periglomerular inflammation in the HCAG-treated mice compared with the vehicle-treated mice. The underlying mechanisms for these renoprotective effects involved: (1) inhibited NLRP3 inflammasome activation; (2) decreased superoxide anion levels and apoptosis; and (3) suppressed activation of NF-κB and related downstream inflammatory mediators.
Assuntos
Acroleína/análogos & derivados , Anti-Inflamatórios/farmacologia , Galactosamina/análogos & derivados , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Nefrite/tratamento farmacológico , Acroleína/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Galactosamina/farmacologia , Inflamassomos/antagonistas & inibidores , Interleucina-6/metabolismo , Rim/efeitos dos fármacos , Rim/imunologia , Rim/patologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Nefrite/imunologia , Nefrite/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismoRESUMO
Ganoderma lucidum is a white rot fungus widely used as a tonic for the promotion of longevity and health. Extracts of G. lucidum have been recognized as an alternative adjuvant treatment for diabetes. Among the many biologically active constituents of G. lucidum, polysaccharides, proteoglycans, proteins and triterpenoids have been shown to have hypoglycemic effects. G. lucidum polysaccharides have been reported to have hypoglycemic activity by increasing plasma insulin levels and decreasing plasma sugar levels in mice. Protein tyrosine phosphatase 1B is a promising therapeutic target in diabetes, and G. lucidum proteoglycan can inhibit this enzyme in vitro. Moreover, G. lucidum triterpenoids were shown to have inhibitory activity on aldose reductase and α-glucosidase that can suppress postprandial hyperglycemia. In addition, a protein Ling Zhi-8 extracted from G. lucidum significantly decreased lymphocyte infiltration and increased the antibody detection of insulin in diabetic mice. This review summarizes most of the research about the hypoglycemic action effects of polysaccharides, proteoglycans, proteins and tritrerpenoids from G. lucidum as a guide for future research.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , alfa-Glucosidases/metabolismo , Aldeído Redutase/metabolismo , Animais , Masculino , Camundongos , Fitoterapia , Polissacarídeos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , ReishiRESUMO
Nowadays, depression is a serious psychological disorder that causes extreme economic loss and social problems. Previously, we discovered that the water extract of Gastrodia elata Blume (WGE) improved depressive-like behavior by influencing neurotransmitters in rats subjected to the forced swimming test. To elucidate possible mechanisms, in the present study, we performed a proteomics and bioinformatics analysis to identify the related pathways. Western blot-validated results indicated that the core protein network modulated by WGE administration was closely associated with down-regulation of the Slit-Robo pathway, which modulates neuronal cytoskeletal remodeling processes. Although Slit-Robo signaling has been well investigated in neuronal development, its relationship with depression is not fully understood. We provide a potential hint on the mechanism responsible for the antidepressive-like activity of WGE. In conclusion, we suggest that the Slit-Robo pathway and neuronal cytoskeleton remodeling are possibly one of the pathways associated with the antidepressive-like effects of WGE.
Assuntos
Antidepressivos/administração & dosagem , Citoesqueleto/metabolismo , Depressão/tratamento farmacológico , Regulação para Baixo/efeitos dos fármacos , Gastrodia/química , Neurônios/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Animais , Depressão/genética , Depressão/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas RoundaboutRESUMO
Methionine adenosyltransferase (MAT) is the cellular enzyme that catalyzes the synthesis of S-adenosylmethionine (SAM), the principal biological methyl donor and a key regulator of hepatocyte proliferation, death and differentiation. Two genes, MAT1A and MAT2A, encode 2 distinct catalytic MAT isoforms. A third gene, MAT2B, encodes a MAT2A regulatory subunit. In hepatocellular carcinoma (HCC), MAT1A downregulation and MAT2A upregulation occur, known as the MAT1A:MAT2A switch. The switch is accompanied with an increasing expression of MAT2B, which results in decreased SAM levels and facilitates cancer cell growth. Berberine, an isoquinoline alkaloid isolated from many medicinal herbs such as Coptis chinensis, has a wide range of pharmacological effects including anti-cancer effects. Because drug-induced microRNAs have recently emerged as key regulators in guiding their pharmacological effects, we examined whether microRNA expression is differentially altered by berberine treatment in HCC. In this study, we used microRNA microarrays to find that the expression level of miR-21-3p (previously named miR-21*) increased after berberine treatment in the HepG2 human hepatoma cell line. To predict the putative targets of miR-21-3p, we integrated the gene expression profiles of HepG2 cells after berberine treatment by comparing with a gene list generated from sequence-based microRNA target prediction software. We then confirmed these predictions through transfection of microRNA mimics and a 3' UTR reporter assay. Our findings provide the first evidence that miR-21-3p directly reduces the expression of MAT2A and MAT2B by targeting their 3' UTRs. In addition, an overexpression of miR-21-3p increased intracellular SAM contents, which have been proven to be a growth disadvantage for hepatoma cells. The overexpression of miR-21-3p suppresses growth and induces apoptosis in HepG2 cells. Overall, our results demonstrate that miR-21-3p functions as a tumor suppressor by directly targeting both MAT2A and MAT2B, indicating its therapeutic potential in HCC.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/fisiopatologia , Metionina Adenosiltransferase/metabolismo , MicroRNAs/farmacologia , Berberina/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Primers do DNA/genética , Citometria de Fluxo , Células HEK293 , Humanos , Luciferases , MicroRNAs/genética , MicroRNAs/metabolismo , Análise em Microsséries , Mutagênese , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Wheatgrass is one of the most widely used health foods, but its functional components and mechanisms remain unexplored. Herein, wheatgrass-derived oligosaccharides (WG-PS3) were isolated and found to induce CD69 and Th1 cytokine expression in human peripheral blood mononuclear cells. In particular, WG-PS3 directly activated the purified monocytes by inducing the expression of CD69, CD80, CD86, IL-12, and TNF-α but affected NK and T cells only in the presence of monocytes. After further purification and structural analysis, maltoheptaose was identified from WG-PS3 as an immunomodulator. Maltoheptaose activated monocytes via Toll-like receptor 2 (TLR-2) signaling, as discovered by pretreatment of blocking antibodies against Toll-like receptors (TLRs) and also determined by click chemistry. This study is the first to reveal the immunostimulatory component of wheatgrass with well defined molecular structures and mechanisms.
Assuntos
Leucócitos Mononucleares/imunologia , Oligossacarídeos/imunologia , Extratos Vegetais/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/metabolismo , Triticum/química , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Células Cultivadas , Cromatografia em Gel , Citocinas/metabolismo , Expressão Gênica/imunologia , Glucanos/imunologia , Glucanos/isolamento & purificação , Humanos , Fatores Imunológicos/imunologia , Fatores Imunológicos/isolamento & purificação , Lectinas Tipo C/metabolismo , Leucócitos Mononucleares/metabolismo , Oligossacarídeos/isolamento & purificação , Extratos Vegetais/isolamento & purificaçãoRESUMO
Sweet broomweed (Scoparia dulcis) is an edible perennial medicinal herb widely distributed in tropical and subtropical regions of Asia, Africa, and the Americas. Four compounds, (2R)-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-O-ß-galactopyranoside [(2R)-HMBOA-2-O-Gal], 3,6-dimethoxy-benzoxazolin-2(3H)-one (3,6-M2BOA), 3-hydroxy-6-methoxy-2-benzoxazolinone (3-OH-MBOA), and scutellarein 7-O-ß-glucuronamide, along with eight known compounds, including two 7-methoxy-1,4-benzoxazin-3(2H)-one 3-O-hexopyranosides [(2R)-HMBOA-2-O-Glc and (2R)-HDMBOA-2-O-Glc], 6-methoxy-benzoxazolin-2(3H)-one (MBOA), acteoside, sodium scutellarin, p-coumaric acid, and two monosaccharides (fructose and glucose), were isolated from the aqueous extract of S. dulcis. Antiproliferative activities of the six benzoxazinoid compounds against the DU-145 human prostate cancer cell line were assayed, and one of these displayed an IC50 of 65.8 µg/mL.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Benzoxazinas/farmacologia , Scoparia/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Benzoxazinas/química , Benzoxazinas/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
A new biflavonol glycoside, quercetin-3-O-ß-D-glucopyranoside-(3'âO-3''')-quercetin-3-O-ß-D-galactopyranoside (9), together with eight known compounds was isolated for the first time from the leaves of Machilus zuihoensis Hayata (Lauraceae). The structure of compound 9 was elucidated by various types of spectroscopic data analysis. Analysis of the biological activity assay found that compound 9 showed significant superoxide anion scavenging activity (IC50 is 30.4 µM) and markedly suppressed LPS-induced high mobility group box 1 (HMGB-1) protein secretion in RAW264.7 cells. In addition, the HMGB-1 protein secretion was also inhibited by quercitrin (3), ethyl caffeate (6), and ethyl 3-O-caffeoylquinate (7) treatment. In the LPS-stimulated inducible nitric oxide synthase (iNOS) activation analysis, two known compounds, quercetin (1) and ethyl caffeate (6), were found to markedly suppress nitric oxide (NO) production (IC50 value, 27.6 and 42.9 µM, respectively) in RAW264.7 cells. Additionally, it was determined that ethyl caffeate (6) down-regulated mRNA expressions of iNOS, IL-1ß, and IL-10 in the LPS-treatment of RAW264.7 cells via a suppressed NF-kB pathway. These results suggested for the first time that the new compound 9 and other constituents isolated from M. zuihoensis have potential anti-inflammatory and superoxide anion scavenging effects. These constituents may be useful for treating various inflammatory diseases.
Assuntos
Anti-Inflamatórios , Sequestradores de Radicais Livres , Lauraceae/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Ácidos Cafeicos/farmacologia , Linhagem Celular , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/farmacologia , Galactosídeos/química , Proteína HMGB1/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Monossacarídeos/química , NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Quercetina/análogos & derivados , Quercetina/química , Quercetina/farmacologia , Superóxidos/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Honokiol (HNK) is a phenolic compound isolated from the bark of houpu (Magnolia officinalis), a plant widely used in traditional Chinese and Japanese medicine. While substantial evidence indicates that HNK possesses anti-inflammatory activity, its effect on dendritic cells (DCs) during the inflammatory reaction remains unclear. The present study investigates how HNK affects lipopolysaccharide (LPS)-stimulated human monocyte-derived DCs. Our experimental results show that HNK inhibits the inflammatory response of LPS-induced DCs by (1) suppressing the expression of CD11c, CD40, CD80, CD83, CD86, and MHC-II on LPS-activated DCs, (2) reducing the production of TNF-α, IL-1ß, IL-6, and IL-12p70 but increasing the production of IL-10 and TGF-ß1 by LPS-activated DCs, (3) inhibiting the LPS-induced DC-elicited allogeneic T-cell proliferation, and (4) shifting the LPS-induced DC-driven Th1 response toward a Th2 response. Further, our results show that HNK inhibits the phosphorylation levels of ERK1/2, p38, JNK1/2, IKKα, and IκBα in LPS-activated DCs. Collectively, the findings show that the anti-inflammatory actions of HNK on LPS-induced DCs are associated with the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways.
Assuntos
Compostos de Bifenilo/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/imunologia , Inflamação/patologia , Lignanas/farmacologia , Lipopolissacarídeos/farmacologia , Monócitos/citologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/enzimologia , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fenótipo , Células Th1/citologia , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th2/citologia , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. Tumor cell differentiation ends cancer cells' immortality, thus stopping cell growth and proliferation. In our previous study, we found that fucose-containing polysaccharide fraction F3 extracted from Ganoderma lucidum can bring about cytokine secretion and cell death in human leukemia THP-1 cells. This prompted us to further investigate on how F3 induces the differentiation in human leukemia cells. We integrated time-course microarray analysis and network modeling to study the F3-induced effects on THP-1 cells. In addition, we determined the differentiation effect using Liu's staining, nitroblue tetrazolium (NBT) reduction assay, flow cytometer, western blotting and Q-PCR. We also examined the modulation and regulation by F3 during the differentiation process. Dynamic gene expression profiles showed that cell differentiation was induced in F3-treated THP-1 cells. Furthermore, F3-treated THP-1 cells exhibited enhanced macrophage differentiation, as demonstrated by changes in cell adherence, cell cycle arrest, NBT reduction and expression of differentiation markers including CD11b, CD14, CD68, matrix metalloproteinase-9 and myeloperoxidase. In addition, caspase cleavage and p53 activation were found to be significantly enhanced in F3-treated THP-1 cells. We unraveled the role of caspases and p53 in F3-induced THP-1 cells differentiation into macrophages. Our results provide a molecular explanation for the differentiation effect of F3 on human leukemia THP-1 cells and offer a prospect for a potential leukemia differentiation therapy.
RESUMO
Chronic neurodegenerative disorders are having an increasing impact on public health as human longevity increases. Parkinson's disease (PD) is a degenerative disorder of the central nervous system and is characterized by motor system disorders resulting in loss of dopamine-producing brain cells. Pueraria thomsonii Benth. (Fabaceae) is an herbal medicine that has traditionally been used as an antipyretic agent. In the present study, the active constituents, daidzein and genistein, were isolated from P. thomsonii. Both compounds exhibited neurocytoprotective effects against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in nerve growth factor (NGF)-differentiated PC12 cells. Neither daidzein nor genistein affected 6-OHDA-induced cellular reactive oxygen species (ROS) generation according to flow cytometric analysis. Rather, they inhibited caspase-8 and partially inhibited caspase-3 activation, providing a protective mechanism against 6-OHDA-induced cytotoxicity in NGF-differentiated PC12 cells. The present results imply that daidzein and genistein may be useful in the development of future strategies for the treatment of PD.
Assuntos
Genisteína/farmacologia , Isoflavonas/farmacologia , Fator de Crescimento Neural/metabolismo , Oxidopamina/farmacologia , Pueraria/química , Animais , Inibidores de Caspase , Citometria de Fluxo , Genisteína/química , Genisteína/isolamento & purificação , Isoflavonas/química , Isoflavonas/isolamento & purificação , Estrutura Molecular , Fator de Crescimento Neural/efeitos dos fármacos , Células PC12 , Ratos , TaiwanRESUMO
Beta-amyloid (A beta) plays a key role in the pathogenesis of Alzheimer's disease (AD) by inducing neurotoxicity and cell death mainly through production of reactive oxygen species (ROS). Garcinia mangostana L. (mangosteen) has been recognized as a major source of natural antioxidants that could decrease ROS. However, its role in protection of A beta-induced cytotoxicity and apoptosis in neuronal cells remains unclear. We therefore examined such a protective effect of mangosteen extract (ME) by evaluating cell viability using MTT test, ROS level, caspase-3 activity, and cellular proteome. Treating SK-N-SH cells with 5-20 microM A beta((1-42)) for 24 h caused morphologically cytotoxic changes, decreased cell viability and increased ROS level, whereas preincubation with 50-400 microg/mL ME 30 min before the induction by A beta((1-42)) successfully prevented such cytotoxic effects in a dose-dependent manner (completely at 400 microg/mL). The A beta-induced increase in caspase-3 activity was also preventable by 400 microg/mL ME. Proteomic analysis using 2-D gel electrophoresis (n = 5 gels/group) followed by mass spectrometry revealed 63 proteins whose levels were significantly altered by A beta((1-42)) induction. Interestingly, changes in 10 proteins were successfully prevented by the ME pretreatment. In summary, we report herein the significant protective effects of ME against A beta-induced cytotoxicity, increased ROS, and increased caspase activity in SK-N-SH cells. Moreover, proteomic analysis revealed some proteins that might be responsible for these protective effects by ME. Further characterizations of these proteins may lead to identification of novel therapeutic targets for successful prevention and/or decreasing the severity of AD.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Garcinia mangostana/química , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proteoma/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Análise de Variância , Western Blotting , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Humanos , Espécies Reativas de Oxigênio/metabolismo , Estatísticas não Paramétricas , beta Carioferinas/metabolismoRESUMO
A platform for screening drugs for their ability to protect neuronal cells against cytotoxicity was developed. Nerve growth factor (NGF) differentiates PC12 cells into nerves, and these differentiated PC12 cells enter apoptosis when challenged with 6-hydroxydopamine (6-OHDA). A screening spectrophotometer was used to assay cytotoxicity in these cells; pretreatment with test samples allowed identification of compounds that protected against this neuronal cytotoxicity. The 95% ethanol extract of Phoenix hanceana Naudin var. formosana Beccari. (PH) showed potential neuroprotective activity in these assays. The PH ethanol extract was further fractionated by sequential partitioning with n-hexane, ethyl acetate (EtOAc), n-butanol (n-BuOH), and water. Subsequent rounds of assaying resulted in the isolation of ten constituents, and their structures were characterized by various spectroscopic techniques and identified by comparison with previous data as: isoorientin (1), isovitexin (2), veronicastroside (3), luteolin-7-O-beta-d-glucopyranoside (4), isoquercitrin (5), tricin-7-neohesperidoside (6), tricin-7-O-beta-d-gluco-pyranoside (7), (+)-catechin (8), (-)-epicatechin (9), and orientin 7-O-beta-d-glucopyranoside (10). Among these compounds, isovitexin (2), luteolin-7-O-beta-d-glucopyranoside (4) and (+)-catechin (8) showed significant neuroprotective activity in cell viability (WST-8 reduction), anti-apoptosis (Annexin V-FITC/propidium iodide double-labeled flow cytometry), and cellular ROS scavenging assays (besides isovitexin (2)), as well as a decreased caspase-8 activity in 6-OHDA-induced PC12 cells. Hence, isovitexin (2), luteolin-7-O-beta-d-glucopyranoside (4), and (+)-catechin (8) protected PC12 cells from 6-OHDA-induced apoptotic neurotoxicity.
Assuntos
Glucosídeos/isolamento & purificação , Glucosídeos/farmacologia , Magnoliopsida/química , Fármacos Neuroprotetores/isolamento & purificação , Fármacos Neuroprotetores/farmacologia , Oxidopamina/farmacologia , Plantas Medicinais/química , Animais , Apigenina/química , Apigenina/isolamento & purificação , Apigenina/farmacologia , Apoptose/efeitos dos fármacos , Catequina/química , Catequina/isolamento & purificação , Catequina/farmacologia , Glucosídeos/química , Luteolina/química , Luteolina/isolamento & purificação , Luteolina/farmacologia , Estrutura Molecular , Fármacos Neuroprotetores/química , Células PC12 , Quercetina/análogos & derivados , Quercetina/química , Quercetina/isolamento & purificação , Quercetina/farmacologia , Ratos , Estereoisomerismo , TaiwanRESUMO
Procedures for cytomic screening were developed for identifying compounds with immuno-modulating properties from the crude extracts of natural products. Human peripheral blood mononuclear cells (hPB-MNCs) were first cultured with different natural crude extracts for 12 hours in culture media. By analyzing the expression of early activation CD69 marker, the potential immuno-activating properties of ethanol extracts of Calocedrus formosana were observed. By the double staining of antibodies recognizing CD69 and specific cell type markers, the increase of CD69 expressions was observed in CD3 and CD14 cell populations. To examine the immuno-activating properties in CD3 T cells and CD14 monocytes, the extracts were further purified. From NMR and mass spectra, sugiol was identified as a pure functional compound, and its immuno-enhancing activities were confirmed by CD69 expressions in the affected cell populations. Furthermore, to clarify the sugiol-affected subpopulations in CD3 T cells, CD3 T cell activation in association with increase in CD8 cytotoxic T cells subpopulation was observed. To address the effect of sugiol on each isolated cell population, we found that the expression of CD69, CD80, and CD86 increased in CD14 monocytes upon exposure to sugiol, whereas for CD3 T cells, sugiol failed to induce the expressions of CD69 and CD25. However, T cell activation by co-culturing monocytes and T cells suggests that the sugiol activation of T cells in hPB-MNCs involved the accessory mechanisms of sugiol-primed monocytes. Therefore, cytomic screening as a multiple-parameter screening strategy reveals the plasticity for immuno-functional studies, leading to the applications to discover new drugs of specific immuno-modulating activities.
Assuntos
Cupressaceae/química , Avaliação Pré-Clínica de Medicamentos , Imunossupressores/isolamento & purificação , Imunossupressores/farmacologia , Antígenos CD/imunologia , Células Cultivadas , Técnicas de Cocultura , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologiaRESUMO
This report describes the synthesis of four novel paclitaxel based prodrugs with glycan conjugation (1-4). Glycans were conjugated using an ester or ether bond as the linker between 2'-paclitaxel and the 2'-glucose or glucuronic acid moiety. These prodrugs showed good water solubility and selective cytotoxicity against cancer cell lines, but showed reduced toxicity toward normal cell lines and cancer cell lines with low expression levels of GLUTs. The ester conjugated prodrug 1 showed the most cytotoxicity among the prodrugs examined and could be transported into cells via GLUTs. Fluorescent and confocal microscopy demonstrated that targeted cells exhibited morphological changes in tubulin and chromosomal alterations that were similar to those observed with paclitaxel treatment. Therefore, these glycan-based prodrugs may be good drug candidates for cancer therapy, and the glycan conjugation approach is an alternative method to enhance the targeted delivery of other drugs to cancer cells that overexpress GLUTs.
Assuntos
Sistemas de Liberação de Medicamentos , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Paclitaxel/farmacologia , Polissacarídeos/química , Pró-Fármacos/farmacologia , Animais , Células CHO , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Estrutura Molecular , Paclitaxel/química , Paclitaxel/metabolismo , Pró-Fármacos/química , Pró-Fármacos/metabolismo , EstereoisomerismoRESUMO
Calcium oxalate monohydrate (COM) is the major crystalline component found in kidney stones and its adhesion to renal tubular cells provokes tubular injury, which in turn enhances COM crystal adhesion. However, COM-induced toxic effects in these tubular cells remain largely unknown. We performed a proteomics study to characterize changes in the cellular proteome in MDCK distal renal tubular cells after an exposure to high-dose (1000 microg/mL) COM crystals for 48 h, at which percentage of cell death was significantly increased. Proteins were extracted from MDCK cells cultured with COM-containing or COM-free medium ( n = 5 individual flasks per group), resolved in individual 2-D gels, and stained with SYPRO Ruby fluorescence dye. Quantitative and statistical analyses revealed 53 proteins whose abundance levels were altered (25 were increased, whereas other 28 were decreased) by COM-induced toxicity. Among these, 50 were successfully identified by quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and/or tandem MS (MS/MS) analyses. The proteomic data were clearly confirmed by 2-D Western blot analysis. While three chaperones (GRP78, Orp150 and Hsp60) were increased, other proteins involved in protein biosynthesis, ATP synthesis, cell cycle regulator, cellular structure, and signal transduction were decreased. These data provide some novel mechanistic insights into the molecular mechanisms of COM crystal-induced tubular toxicity.
Assuntos
Oxalato de Cálcio/toxicidade , Túbulos Renais Distais/efeitos dos fármacos , Proteoma/análise , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/química , Cães , Relação Dose-Resposta a Droga , Túbulos Renais Distais/citologia , Túbulos Renais Distais/metabolismo , Mapeamento de Peptídeos , Proteoma/metabolismo , Proteômica/métodos , Fatores de TempoRESUMO
BACKGROUND: Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-alpha. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells. RESULTS: Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach. CONCLUSION: Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.
Assuntos
Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Polissacarídeos/farmacologia , Reishi/química , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Medicamentos de Ervas Chinesas/química , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Humanos , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/metabolismo , Leucemia Monocítica Aguda/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Reação em Cadeia da Polimerase , Polissacarídeos/química , Receptores de Morte Celular/genética , Receptores de Morte Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Xanthine oxidase (XO) is a key enzyme which can catalyze xanthine to uric acid causing hyperuricemia in humans. By using the fractionation technique and inhibitory activity assay, an active compound that prevents XO from reacting with xanthine was isolated from wheat leaf. It was identified by the Mass and NMR as 6-aminopurine (adenine). A structure-activity study based on 6-aminopurine was conducted. The inhibition of XO activity by 6-aminopurine (IC(50)=10.89+/-0.13 microM) and its analogues was compared with that by allopurinol (IC(50)=7.82+/-0.12 microM). Among these analogues, 2-chloro-6(methylamino)purine (IC(50)=10.19+/-0.10 microM) and 4-aminopyrazolo[3,4-d] pyrimidine (IC(50)=30.26+/-0.23 microM) were found to be potent inhibitors of XO. Kinetics study showed that 2-chloro-6(methylamino)purine is non-competitive, while 4-aminopyrazolo[3,4-d]pyrimidine is competitive against XO.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Purinas/síntese química , Purinas/farmacologia , Xantina Oxidase/antagonistas & inibidores , Alopurinol/síntese química , Alopurinol/farmacologia , Cromatografia Líquida de Alta Pressão , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Folhas de Planta/química , Purinas/isolamento & purificação , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Triticum/químicaRESUMO
A fucose-containing glycoprotein fraction which stimulates spleen cell proliferation and cytokine expression has been identified from the water-soluble extract of Ganoderma lucidum. Proteomic analysis of mouse spleen cells treated with this glycoprotein fraction showed approximately 50% change of the proteome. Further studies on the activities of this glycoprotein fraction through selective proteolysis and glycosidic cleavage indicate that a fucose containing polysaccharide fraction is responsible for stimulating the expression of cytokines, especially IL-1, IL-2 and INF-gamma.