RESUMO
Desalted Salicornia europaea L. (SE) inhibits acetylcholine esterase, attenuates oxidative stress and inflammatory cytokines, and activates neurotrophic pathway. We performed 12-week, randomized, double-blind, placebo-controlled study to evaluate the efficacy of PhytoMeal(a desalted SE)-ethanol extract (PM-EE), in improving the cognitive performance in patients with subjective memory impairment. 63 participants complaining memory dysfunction without dementia (Korean Mini-Mental State Examination [K-MMSE] score ≥ 23) were assigned to PM-EE 600 mg/day or placebo. The cognitive domain of the Alzheimer's disease assessment scale-Korean version (ADAS-K) was set as the primary outcome. After 12 weeks, there was no differences in the changes in the primary outcome or the frequency of adverse events between the groups. In the subgroup analysis for the 30 subjects with mild cognitive impairment (MCI, baseline K-MMSE scores ≤ 28), PM-EE significantly improved the color-reading score of the Korean color-word stroop test (8.2 ± 25.0 vs. - 4.7 ± 13.2, P = 0.018). Our findings suggest that PM-EE is safe but might not be effective in this setting of this study. However, PM-EE may improve the frontal executive function in the patients with MCI. Further large-sized studies with longer follow-up period is warranted (trial registration number KCT0003418).
Assuntos
Doença de Alzheimer/fisiopatologia , Chenopodiaceae/química , Etanol/química , Transtornos da Memória/tratamento farmacológico , Extratos Vegetais/farmacologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Transtornos da Memória/etiologia , Transtornos da Memória/patologia , Pessoa de Meia-Idade , PrognósticoRESUMO
Hyperactivation of microglia, the resident innate immune cells of the central nervous system, exacerbates various neurodegenerative disorders, including Parkinson's disease (PD). Parkinson's disease is generally characterized by a severe loss of dopaminergic neurons in the nigrostriatal pathway, with substantial neuroinflammation and motor deficits. This was experimentally replicated in animal models, using neurotoxins, i.e., LPS (lipopolysaccharides) and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Salicornia europaea L. (SE) has been used as a dietary supplement in Korea and Europe for several years, due to its nutritional and therapeutic value. In this study, we intend to investigate the antineuroinflammatory and anti-PD-like effects of the bioactive fraction/candidate of the SE extract. Initially, we screened various fractions of SE extract using an in vitro antioxidant assay. The optimal fraction was investigated for its in vitro antineuroinflammatory potential in LPS-stimulated BV-2 microglial cells and in vivo anti-PD-like potential in MPTP-intoxicated mice. Subsequently, to identify the potential candidate responsible for the elite therapeutic potential of the optimal fraction, we conducted antioxidant activity-guided isolation and purification; the bioactive candidate was structurally characterized using nuclear magnetic resonance spectroscopy and chromatographic techniques and further investigated for its in vitro antioxidative and antineuroinflammatory potential. The results of our study indicate that SE-EA and its bioactive candidate, Irilin B, effectively alleviate the deleterious effect of microglia-mediated neuroinflammation and promote antioxidative effects. Thus, they exhibit potential as therapeutic candidates against neuroinflammatory and oxidative stress-mediated PD-like neurodegenerative complications.
Assuntos
Chenopodiaceae/química , Isoflavonas/farmacologia , Intoxicação por MPTP/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/química , Acetatos/química , Animais , Antioxidantes/metabolismo , Chenopodiaceae/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Isoflavonas/uso terapêutico , Lipopolissacarídeos/farmacologia , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/uso terapêutico , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Recently, our research team reported the anti-amnesic potential of desalted-hydroethanolic extracts of Salicornia europaea L. (SE-EE). In this study, we performed bioactivity-guided isolation and identification of Acanthoside B (Aca.B), from SE-EE, as the potential bioactive candidate and examined anti-amnesic activity with its potential mechanism of action using an in vivo model. S7-L3-3 purified from SE-EE showed enhanced in vitro acetylcholinesterase (AChE) inhibitory activity. The isolated S7-L3-3 was identified and characterized as Aca.B using varied spectral analyses, i.e., Nuclear magnetic resonance (NMR), Ultraviolet-visible (UV-Vis), and Electrospray ionization-mass spectrometry (ESI-MS). In the in vitro studies, Aca.B exhibited negligible toxicity and showed a dose-dependent nitric oxide inhibitory potential in Lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. In the in vivo studies, the oral administration of Aca.B to mice showed enhanced bioavailability and dose-dependent repression of the behavioral/cognitive impairment by regulating the cholinergic function, restoring the antioxidant status, attenuating the inflammatory cytokines/mediators and actively enriching neurotropic proteins in the hippocampal regions of the scopolamine-administered mice.
Assuntos
Amnésia/induzido quimicamente , Furanos/farmacologia , Glucosídeos/farmacologia , Inflamação/metabolismo , Lignanas/farmacologia , Receptores Colinérgicos/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Glicoproteínas de Membrana/metabolismo , Camundongos , Oxirredução , Proteínas Tirosina Quinases/metabolismo , Escopolamina/farmacologiaRESUMO
The Salicornia europaea L. (SE) plant is a halophyte that has been widely consumed as a seasoned vegetable, and it has been recently reported to counteract chronic diseases related to oxidative and inflammatory stress. In this study, we performed an initial phytochemical analysis with in vitro biochemical tests and chromatographic profiling of desalted and enzyme-digested SE ethanol extract (SE-EE). Subsequently, we evaluated the anti-neuroinflammatory and ameliorative potential of SE-EE in LPS-inflicted BV-2 microglial cells and scopolamine-induced amnesic C57/BL6N mice, respectively. SE-EE possess considerable polyphenols and flavonoids that are supposedly responsible to improve its bio-efficacy. SE-EE dose-dependently attenuated LPS-induced inflammation in BV-2 cells, significantly repressed behavioural/cognitive impairment, dose-dependently regulated the cholinergic function, suppressed oxidative stress markers, regulated inflammatory cytokines/associated proteins expression and effectively ameliorated p-CREB/BDNF levels, neurogenesis (DCX stain), neuron proliferation (Ki67 stain) in scopolamine-administered mice. Thus, SE-EE extract shows promising multifactorial disease modifying activities and can be further developed as an effective functional food, drug candidate, or supplemental therapy to treat neuroinflammatory mediated disorders.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Amnésia/tratamento farmacológico , Antioxidantes/administração & dosagem , Chenopodiaceae/química , Inflamação/tratamento farmacológico , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Amnésia/induzido quimicamente , Amnésia/genética , Amnésia/patologia , Animais , Antioxidantes/química , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Modelos Animais de Doenças , Proteína Duplacortina , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Escopolamina/toxicidadeRESUMO
CONTEXT: Salicornia europaea (Amaranthaceae) (SE) has been shown to reduce obesity, but it remains a problem as a food supplement because of its high salt content (25-35% NaCl). OBJECTIVES: This study investigated the anti-obesity effects and mechanism of action of desalted SE powder (DSP). MATERIALS AND METHODS: Sprague-Dawley rats (n = 50) were divided into a normal control group (NC), a high-fat diet (HFD)-induced obesity control group (HFD), and HFD groups co-administered DSP (250 and 500 mg/kg) or Garcinia cambogia (Clusiaceae) extract (GE, 200 mg/kg, standard control) orally each day for 12 weeks. RESULTS: The body weight was significantly reduced by co-administration of DSP (596.51 ± 19.84 kg, 4.60% and 562.08 ± 9.74 kg, 10.10%, respectively) and GE (576.00 ± 11.29 kg, 7.88%) relative to the HFD group (625.25 ± 14.02 kg) and was accompanied by reduced abdominal fat mass, and serum lipid levels, with no effects on feed intake. To find the underlying mechanism of the anti-obesity effects, trans-ferulic acid (TFA) was identified as the main ingredient and investigated with regard to whether it attenuated adipogenesity in 3T3L-1 cells. DSP-derived TFA suppressed adipocyte differentiation and accumulation of intracellular lipids. TFA also down-regulated the adipogenesis-related gene expression of sterol regulatory element-binding protein 1, peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein-α and fatty acid synthase. CONCLUSIONS: These findings suggest that DSP may be considered for use as a food supplement intent of controlling obesity through its antiobesity and antiadipogenic properties.
Assuntos
Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/uso terapêutico , Chenopodiaceae , Ácidos Cumáricos/uso terapêutico , Obesidade/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Células 3T3-L1 , Adipogenia/fisiologia , Animais , Fármacos Antiobesidade/isolamento & purificação , Fármacos Antiobesidade/farmacologia , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Ácidos Cumáricos/isolamento & purificação , Ácidos Cumáricos/farmacologia , Relação Dose-Resposta a Droga , Masculino , Camundongos , Obesidade/metabolismo , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
A Gram-stain-negative, motile-by-gliding, aerobic, non-spore-forming, rod-shaped and yellow-pigmented bacterium was isolated from Salicornia herbacea in the Yellow Sea and designated as strain MBLN091T. It belonged to the family Flavobacteriaceae. The 16S rRNA gene sequence of this isolated strain was similar to that of Flavimarina pacifica IDSW-73T with 94.8â% similarity, and with 92.3-92.8â% similarities to those of other closely related species of the genus Leeuwenhoekiella. The similarities of the RNA polymerase subunit B gene between this strain and F. pacifica KCTC 32466T and Leeuwenhoekiella marinoflava DSM 3653T were 80.5 and 80.2â%, respectively. Growth of strain MBLN091T was observed in the presence of 0.5â15.0â% (w/v) NaCl at 4â35 °C and pH 6.0-8.0, with optimal growth in the presence of 2.5â5.0â% (w/v) NaCl at 20â25 °C and pH 7.0. This isolate was able to hydrolyse gelatin. The only respiratory quinone was MK-6. The major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and two unidentified lipids. Major fatty acids of the isolate were iso-C15â:â0, summed feature 3 (C16â:â1 ω7c and/or C16â:â1 ω6c), iso-C17â:â0 3-OH and iso-C15â:â1 G. The genomic DNA G+C content was 39.6 mol%. The physiological features were closely related to F. pacifica. Therefore, strain MBLN091T is considered to represent a novel species within the genus Flavimarina, for which the name Flavimarina flava sp. nov. is proposed. The type strain is MBLN091T (=KCTC 52527T=JCM 31731T).
Assuntos
Chenopodiaceae/microbiologia , Flavobacteriaceae/classificação , Filogenia , Água do Mar/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Fosfatidiletanolaminas/química , Pigmentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
Salicornia europaea L. (SE) has been used as folk medicine for the treatment of various diseases such as obesity, diabetes, and cancer. However, its effects on atherosclerotic events in vascular smooth muscle cells (VSMCs) remain unknown. The present study explored the effects of the ethyl acetate fraction of desalted SE hot water extract (SEWEAF) on atherosclerotic responses (especially migration and proliferation) in VSMCs and vascular neointima formation. Treatment with the SEWEAF significantly suppressed the platelet-derived growth factor (PDGF)-BB-induced VSMC migration and proliferation as well the phosphorylation of mitogen-activated protein kinases (MAPKs) such as the p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2. Moreover, oral administration of the SEWEAF resulted in the attenuation of neointima formation in balloon-injured rat carotid arteries. Additionally, HPLC analysis showed that the major components in the two subfractions of the SEWEAF were five phenolic acids and four flavonols. In the SEWEAF components, for which atherosclerosis-linked responses in VSMCs have not been known, p-coumaric acid, quercetin-3-ß-d-glucoside, and isorhamnetin-3-ß-d-glucoside inhibited both PDGF-BB-induced migration and proliferation and isorhamnetin attenuated only PDGF-BB-stimulated VSMC proliferation. These results suggest that the SEWEAF may suppress PDGF-BB-induced VSMC migration by downregulating the phosphorylation of p38 MAPK and ERK1/2, thus leading to the reduction of neointimal hyperplasia during vascular remodeling. Therefore, the desalted SE extract, SEWEAF may be a potential ingredient for dietary supplements or nutraceuticals to ameliorate and/or prevent vascular remodeling-related disorders.
Assuntos
Movimento Celular/efeitos dos fármacos , Chenopodiaceae/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Neointima/enzimologia , Neointima/patologia , Extratos Vegetais/farmacologia , Acetatos/química , Animais , Becaplermina , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Flavonóis/farmacologia , Hidroxibenzoatos/farmacologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-sis/farmacologia , Ratos Sprague-Dawley , ÁguaRESUMO
High-fat and high-salt intakes are among the major risks of chronic diseases including obesity, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). Salicornia is a halophytic plant known to exert antioxidant, antidiabetic, and hypolipidemic effects, and Salicornia-extracted salt (SS) has been used as a salt substitute. In this study, the effects of SS and purified salt (PS) on the aggravation of NAFLD/NASH were compared. C57BL/6J male mice (8-wk-old) were fed a high-fat diet (HFD) for 6 mo and divided into 3 dietary groups, which were additionally fed HFD, HFD + SS, and HFD + PS for 13 wk. PS induced aggravation of NAFLD/NASH in HFD-fed mice. Although the actual salt intake was same between the PS and SS groups as 1% of the diet (extrapolated from the World Health Organization [WHO] guideline), SS induced less liver injury and hepatic steatosis compared to PS. The hepatic mRNA expressions of inflammatory cytokines and fibrosis marker were significantly lower in the SS group than the PS group. Oxidative stress is one of the major causes of inflammation in NAFLD/NASH. Results of the component analysis showed that the major polyphenols that exhibited antioxidant activity in the Salicornia water extract were ferulic acid, caffeic acid, and isorhamnetin. These results suggest that even the level of salt intake recommended by WHO can accelerate the progression of liver disease in obese individuals consuming HFD. It is proposed that SS can be a salt substitute for obese individuals who consume HFD.
Assuntos
Chenopodiaceae/química , Fígado Gorduroso/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Cloreto de Sódio/efeitos adversos , Animais , Antioxidantes/administração & dosagem , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Cloreto de Sódio/metabolismoRESUMO
To develop novel mechanism-based preventive approaches for lung cancer, we examined the effect of oral consumption of a human achievable dose of pomegranate fruit extract (PFE) on growth, progression, angiogenesis, and signaling pathways in two mouse lung tumor protocols. Benzo(a)pyrene [B(a)P] and N-nitroso-tris-chloroethylurea (NTCU) were used to induce lung tumors, and PFE was given in drinking water to A/J mice. Lung tumor yield was examined on the 84th day and 140 days after B(a)P dosing and 240 days after NTCU treatment. Mice treated with PFE and exposed to B(a)P and NTCU had statistically significant lower lung tumor multiplicities than mice treated with carcinogens only. Tumor reduction was 53.9% and 61.6% in the B(a)P + PFE group at 84 and 140 days, respectively, compared with the B(a)P group. The NTCU + PFE group had 65.9% tumor reduction compared with the NTCU group at 240 days. Immunoblot analysis and immunohistochemistry were used to determine effect on cell survival pathways and markers of cellular proliferation and angiogenesis. PFE treatment caused inhibition of (a) activation of nuclear factor-kappaB and IkappaBalpha kinase, (b) degradation and phosphorylation of IkappaBalpha, (c) phosphorylation of mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase 1/2, and p38), (d) phosphatidylinositol 3-kinase (p85 and p110), (e) phosphorylation of Akt at Thr(308), (f) activation of mammalian target of rapamycin signaling, (g) phosphorylation of c-met, and (h) markers of cell proliferation (Ki-67 and proliferating cell nuclear antigen) and angiogenesis (inducible nitric oxide synthase, CD31, and vascular endothelial growth factor) in lungs of B(a)P- and NTCU-treated mice. Thus, our data show that PFE significantly inhibits lung tumorigenesis in A/J mice and merits investigation as a chemopreventive agent for human lung cancer.
Assuntos
Anticarcinógenos/farmacologia , Frutas , Neoplasias Pulmonares/tratamento farmacológico , Lythraceae , Fitoterapia/métodos , Extratos Vegetais/farmacologia , Administração Oral , Animais , Benzo(a)pireno , Processos de Crescimento Celular/efeitos dos fármacos , Feminino , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos A , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Neovascularização Patológica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina-Treonina Quinases TOR , Ureia/análogos & derivadosRESUMO
Developing novel mechanism-based chemopreventive approaches for lung cancer through the use of dietary substances which humans can accept has become an important goal. In the present study, employing normal human bronchial epithelial cells (NHBE) and human lung carcinoma A549 cells, we first compared the growth inhibitory effects of pomegranate fruit extract (PFE). Treatment of PFE (50-150 microg/ml) for 72 h was found to result in a decrease in the viability of A549 cells but had only minimal effects on NHBE cells as assessed by the MTT and Trypan blue assays. PFE treatment of A549 cells also resulted in dose-dependent arrest of cells in G0-G1 phase of the cell cycle (as assessed by DNA cell cycle analysis). We further found that PFE treatment also resulted in (i) induction of WAF1/p21 and KIP1/p27, (ii) decrease in the protein expressions of cyclins D1, D2 and E, and (iii) decrease in cyclin-dependent kinase (cdk) 2, cdk4 and cdk6 expression. The treatment of cells with PFE inhibited (i) phosphorylation of MAPK proteins, (ii) inhibition of PI3K, (iii) phosphorylation of Akt at Thr308, (iv) NF-kappaB and IKKalpha, (v) degradation and phosphorylation of IkappaBalpha, and (vi) Ki-67 and PCNA. We also found that PFE treatment to A549 cells resulted in inhibition of NF-kappaB DNA-binding activity. Oral administration of PFE (0.1 and 0.2%, wt/vol) to athymic nude mice implanted with A549 cells resulted in a significant inhibition in tumor growth. Our results provide a suggestion that PFE can be a useful chemopreventive/chemotherapeutic agent against human lung cancer.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Frutas/química , Neoplasias Pulmonares/tratamento farmacológico , Lythraceae/química , Fitoterapia , Extratos Vegetais/uso terapêutico , Transdução de Sinais , Animais , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/prevenção & controle , Masculino , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
Solar UV radiation, in particular its UVB component, is the primary cause of many adverse biological effects, the most damaging of which is skin cancer. Here, we assessed the photochemopreventive effect of delphinidin, a major anthocyanidin present in many pigmented fruits and vegetables, on UVB-mediated responses in human immortalized HaCaT keratinocytes and SKH-1 hairless mouse skin. We found that pretreatment of cells with delphinidin (1-20 microM for 24 hours) protected against UVB (15-30 mJ/cm2, 24 hours)-mediated (i) decrease in cell viability and (ii) induction of apoptosis. Furthermore, we found that pretreatment of HaCaT cells with delphinidin inhibited UVB-mediated (i) increase in lipid peroxidation; (ii) formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG); (iii) decrease in proliferating cell nuclear antigen expression; (iv) increase in poly(ADP-ribose) polymerase cleavage; (v) activation of caspases; (vi) increase in Bax; (vii) decrease in Bcl-2; (viii) upregulation of Bid and Bak; and (ix) downregulation of Bcl-xL. Topical application of delphinidin (1 mg/0.1 ml DMSO/mouse) to SKH-1 hairless mouse skin inhibited UVB-mediated apoptosis and markers of DNA damage such as cyclobutane pyrimidine dimers and 8-OHdG. Taken together our results suggest that treatment of HaCaT cells and mouse skin with delphinidin inhibited UVB-mediated oxidative stress and reduced DNA damage, thereby protecting the cells from UVB-induced apoptosis.
Assuntos
Antocianinas/farmacologia , Apoptose/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Protetores contra Radiação/farmacologia , Pele/efeitos da radiação , 8-Hidroxi-2'-Desoxiguanosina , Animais , Antioxidantes/farmacologia , Apoptose/efeitos da radiação , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Pelados , Estresse Oxidativo/efeitos da radiação , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Dímeros de Pirimidina/metabolismo , Pele/efeitos dos fármacos , Proteína X Associada a bcl-2/análiseRESUMO
We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNA-mediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment.
Assuntos
Regulação Neoplásica da Expressão Gênica , Metaloproteinase 9 da Matriz/genética , Neoplasias da Próstata/patologia , Proteínas S100/metabolismo , Transcrição Gênica , Animais , Genes Neoplásicos/genética , Humanos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Células-Tronco Neoplásicas , Neoplasias da Próstata/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Ativação Transcricional/genética , Transfecção , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Epigallocatechin 3-gallate (EGCG), the major polyphenol found in green tea, exerts antiproliferative and proapoptotic effects in many cancer cells. However, we found that among many cancer cells human lung adenocarcinoma A549 cells are markedly resistant to apoptosis induction by EGCG (even at 100 microm for 72 h). Heme oxygenase-1 (HO-1) induced by stress stimuli represents a prime cellular defense mechanism, but it may be associated with enhanced cell proliferation and chemoresistance in some cancer cells. Because we found that A549 cells constitutively overexpress HO-1 and its associated transcription factor Nrf2, we tested an hypothesis that EGCG resistance in these cells may be linked with Nrf2-mediated HO-1 overexpression. HO-1 inhibition with tin-protoporphyrin IX and silencing with RNA interference rendered cells more sensitive to apoptosis induction by EGCG and classical prooxidants. Interestingly, EGCG at high concentration (>200 microm) induced apoptosis by suppressing expression of HO-1 protein and mRNA, and this effect correlated with a decrease in both Nrf2-ARE binding and HO-1-ARE-luciferase activity, suggesting Nrf2-driven transcriptional activation of ho-1. Because we observed notably high levels of phosphorylated protein kinase C alpha and its suppression by EGCG and deferoxamine (an iron chelator), a possible mechanism involving phosphorylated protein kinase C alpha and iron in Nrf2-HO-1 activation was further investigated. Collectively, our findings suggest that Nrf2-mediated HO-1 overexpression confers resistance to apoptosis induction by EGCG; therefore, its inactivation may be a target for overcoming the resistance to chemoprevention and chemotherapy.
Assuntos
Apoptose/efeitos dos fármacos , Catequina/análogos & derivados , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antioxidantes/metabolismo , Catequina/farmacologia , Catequina/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Heme Oxigenase-1/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Proteína Quinase C-alfa/metabolismo , RNA Interferente Pequeno/genética , Elementos de Resposta , Sensibilidade e Especificidade , Ativação Transcricional/genéticaRESUMO
Induction of detoxifying phase II genes by chemopreventive agents represents a coordinated protective response against oxidative stress and neoplastic effects of carcinogens. We have earlier shown that a novel antioxidant from the bamboo leaves constituent 3-O-caffeoyl-1-methylquinic acid (MCGA3) induces heme oxygenase-1 (HO-1) and protects endothelial cells from ROS-induced endothelial injury. The purpose of this study was to elucidate the induction mechanism of HO-1 and other phase II genes by MCGA3 in human umbilical vascular endothelial cells (HUVECs). Using Northern blotting and RT-PCR, we found that treatment of HUVECs with MCGA3 increased, in a dose and time-dependent manner, steady-state mRNA levels of the selected phase II genes including HO-1, ferritin, gamma-glutamylcysteine lygase, glutathione reductase, and glutathione transferase, which were dependent on Nrf2 nuclear translocation. The observed phase II gene induction by MCGA3 was found to be associated with MCGA3-mediated cytoprotective activity, ROS-scavenging potency, and the increase in the cellular levels of both reduced (GSH) and oxidized glutathione (GSSG). Interestingly, exposure to MCGA3 resulted in a decreased ratio of GSH/GSSG, which was negatively related with mRNA level of phase II genes. By employing N-acetylcysteine and GSH biosynthetic enzyme inhibitors as well as prooxidants, hemin and H(2)O(2), we show that a decreased intracellular GSH/GSSG homeostasis, at least in part, may be involved in the MCGA3-mediated phase II gene induction and Nrf2 translocation, although the attenuation of HO-1 expression with SP 600125 supports a partial involvement of JNK signaling.
Assuntos
Antioxidantes/farmacologia , Glutationa/metabolismo , Desintoxicação Metabólica Fase II/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Quínico/análogos & derivados , Acetilcisteína/farmacologia , Transporte Ativo do Núcleo Celular , Carmustina/farmacologia , Células Cultivadas , Desidroepiandrosterona/farmacologia , Ferritinas/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Oxirredução/efeitos dos fármacos , Fosforilação , Ácido Quínico/química , Ácido Quínico/farmacologia , RNA Mensageiro/genética , Fatores de Tempo , Ativação Transcricional , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/metabolismoRESUMO
Because of unsatisfactory treatment options for prostate cancer (CaP) there is a need to develop novel preventive approaches for this malignancy. One such strategy is through chemoprevention by the use of non-toxic dietary substances and botanical products. We have shown previously that panduratin A isolated from the extract of Kaempferia pandurata (Zingiberaceae) is a strong inhibitor of cyclooxygenase-2 in RAW264.7 cells and induces apoptosis in HT-29 cells. In the present study, we provide evidence that panduratin A treatment to androgen-independent human CaP cells PC3 and DU145 result in a time and dose-dependent inhibition of cell growth with an IC50 of 13.5-14 microM and no to little effect on normal human prostate epithelial cells. To define the mechanism of these anti-proliferative effects of panduratin A, we determined its effect on critical molecular events known to regulate the cell cycle and the apoptotic machinery. Annexin V/propidium iodide staining provided the evidence for the induction of apoptosis which was further confirmed by the observation of cleavage of poly (ADP-ribose) polymerase and degradation of acinus. Panduratin A treatment to cells was found to result in inhibition of procaspases 9, 8, 6 and 3 with significant increase in the ratio of Bax:Bcl-2, suggesting the involvement of a mitochondrial-dependent apoptotic pathway. Panduratin A-mediated apoptosis was accompanied with upregulation of Fas death receptor and TNF-related apoptosis-inducing ligand (TRAIL). Furthermore, cell cycle analysis using flow cytometry showed that panduratin A treatment of cells resulted in a G2/M arrest in a dose-dependent manner. The immunoblot analysis data revealed that in both cell lines panduratin A treatment resulted in a dose-dependent (i) induction of p21WAF1/Cip1 and p27Kip1, (ii) downregulation of cdks 2, 4 and 6 and (iii) decrease in cyclins D1 and E. These findings suggest that panduratin A may be an effective chemopreventive or therapeutic agent against CaP.
Assuntos
Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Chalconas/farmacologia , Fitoterapia , Neoplasias da Próstata/patologia , Zingiberaceae/química , Androgênios/metabolismo , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Caspases/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/efeitos dos fármacos , Ciclinas/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Masculino , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Extratos Vegetais/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Proteína X Associada a bcl-2/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/efeitos dos fármacos , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
In prostate cancer, a fine balance between cell proliferation and apoptotic death is lost, resulting in increased cellular mass and tumor progression. One approach to redress this imbalance and control this malignancy is its preventive intervention through the use of dietary natural agents. Here, we investigated the growth-inhibitory effect and associated mechanisms of Lupeol, a triterpene present in fruits and vegetables, in androgen-sensitive human prostate cancer cells. Lupeol treatment resulted in significant inhibition of cell viability in a dose-dependent manner and caused apoptotic death of prostate cancer cells. Lupeol was found to induce the cleavage of poly(ADP-ribose) polymerase protein and degradation of acinus protein with a significant increase in the expression of FADD protein. Among all death receptor targets examined, Lupeol specifically caused a significant increase in the expression of Fas receptor. The small interfering RNA-mediated silencing of the Fas gene and inhibition of caspase-6, caspase-8, and caspase-9 by their specific inhibitors confirmed that Lupeol specifically activates the Fas receptor-mediated apoptotic pathway in androgen-sensitive prostate cancer cells. The treatment of cells with a combination of anti-Fas monoclonal antibody and Lupeol resulted in higher cell death compared with the additive effect of the two compounds alone, suggesting a synergistic effect. Lupeol treatment resulted in a significant inhibition in growth of tumors with concomitant reduction in prostate-specific antigen secretion in athymic nude mice implanted with CWR22Rnu1 cells. Because early clinical prostate cancer growth is an androgen-dependent response, the results of the present study suggest that Lupeol may have a potential to be an effective agent against prostate cancer.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Triterpenos/farmacologia , Receptor fas/fisiologia , Androgênios/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Apoptose/fisiologia , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Triterpenos Pentacíclicos , Poli(ADP-Ribose) Polimerases/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , Receptores Androgênicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor fas/genética , Receptor fas/imunologiaRESUMO
Pancreatic cancer is an exceptionally aggressive disease, the treatment of which has largely been unsuccessful due to higher resistance offered by pancreatic cancer cells to conventional approaches such as surgery, radiation and/or chemotherapy. The aberration of Ras oncoprotein has been linked to the induction of multiple signaling pathways and to the resistance offered by pancreatic cancer cells to apoptosis. Therefore, there is a need for development of new and effective chemotherapeutic agents which can target multiple pathways to induce responsiveness of pancreatic cancer cells to death signals. In this study, human pancreatic adenocarcinoma cells AsPC-1 were used to investigate the effect of Lupeol on cell growth and its effects on the modulation of multiple Ras-induced signaling pathways. Lupeol caused a dose-dependent inhibition of cell growth as assessed by MTT assay and induction of apoptosis as assessed by flow cytometry, fluorescence microscopy and western blotting. Lupeol treatment to cells was found to significantly reduce the expression of Ras oncoprotein and modulate the protein expression of various signaling molecules involved in PKCalpha/ODC, PI3K/Akt and MAPKs pathways along with a significant reduction in the activation of NFkappaB signaling pathway. Our data suggest that Lupeol can adopt a multi-prong strategy to target multiple signaling pathways leading to induction of apoptosis and inhibition of growth of pancreatic cancer cells. Lupeol could be a potential agent against pancreatic cancer, however, further in-depth in vivo studies are warranted.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Triterpenos/farmacologia , Proteínas ras/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Western Blotting , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Frutas/química , Humanos , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Ornitina Descarboxilase/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Triterpenos Pentacíclicos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais Cultivadas , Verduras/químicaRESUMO
The novel antioxidant 3-O-caffeoyl-one-methylquinic acid (MCGA3) is a methyl chlorogenic acid derivative isolated from bamboo leaves. MCGA3 scavenges reactive oxygen species (ROS) and inhibits lipid peroxidation and xanthine oxidase in vitro. In this study, we evaluated the cytoprotective effect of MCGA3, which occurs via heme oxygenase-1 (HO-1) induction in bovine vascular endothelial cells exposed to tert-butylhydroperoxide (tBHP). Cells treated with 1 mM tBHP (6-18 h) generated substantial ROS and concomitantly lost most intracellular lactate dehydrogenase (LDH), which then caused necrotic cell death. Of the several MCGA antioxidants and structurally related phenolic acids examined in this study, MCGA3 (0.01-0.15 mM) was found to completely block this necrosis and generation of ROS by tBHP. Surprisingly, MCGA3 by itself was found to be a potent inducer of HO-1. We observed the time- and dose-dependent induction of HO-1 mRNA and protein, which was closely associated with decreased intracellular ROS and necrosis against tBHP. Deesterified or Al-chelated MCGA3 or co-treatment with MCGA3 and actinomycin D abolished HO-1 induction and the antinecrotic effect of MCGA3. Zinc protoporphyrin IX and cycloheximide attenuated the cytoprotection afforded by MCGA3, but did not reduce HO-1 mRNA. Interestingly, N-acetylcysteine (1 mM) enhanced the HO-1 induction of MCGA3, but N-acetylcysteine itself did not induce HO-1. These results suggested that not only ortho-dihydroxyl groups but also aromatic ester and methoxyl ester moieties are necessary for full HO-1 induction and cytoprotection against toxic tBHP-derived ROS. Ferritin mRNA was also upregulated during all HO-1 induction by MCGA3, which might decrease iron and lower ROS levels. Consequently, the combined action of HO-1 and ferritin may protect cells from toxic tBHP-mediated necrosis.
Assuntos
Crioprotetores/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Ácido Quínico/farmacologia , Acetilcisteína/farmacologia , Animais , Antioxidantes/metabolismo , Bovinos , Linhagem Celular , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Ferritinas/genética , Citometria de Fluxo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1 , Hidroxibenzoatos/química , Hidroxibenzoatos/farmacologia , Estrutura Molecular , Necrose , Protoporfirinas/farmacologia , Ácido Quínico/análogos & derivados , Ácido Quínico/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , terc-Butil Hidroperóxido/toxicidadeRESUMO
Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.
Assuntos
Antígenos de Helmintos/imunologia , Via Clássica do Complemento/imunologia , Proteínas de Helminto/imunologia , Receptores de Superfície Celular/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Complemento C2/imunologia , Complemento C2/metabolismo , Antígeno de Forssman/imunologia , Cobaias , Proteínas de Helminto/genética , Hemólise/imunologia , Masculino , Dados de Sequência Molecular , Receptores de Superfície Celular/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Choque/imunologia , Relação Estrutura-AtividadeRESUMO
Bamboo, Phyllostachys edulis produces well known edible shoots in Asia. Immunostimulating anticomplementary (complement activating) substances have been recognized as a characteristic biological response modifier (BRM). In the present study, we isolated and characterized three anticomplementary beta-glucans (BS-BGA, BS-BGB, and BS-BGC) from bamboo shoots. Hot-water extraction, DEAE-Toyopearl 650M-column chromatography, amylase digestion and concanavalin A adsorption, and Sephacryl S-100 HR column chromatography were applied to isolate the beta-glucans. The average molecular masses of the beta-glucans were estimated to be from 14,500 to 85,300 Da by HPSEC-MALLS-RI. All three beta-glucans (0.1-1.0 mg/mL) activated the complement system via the alternative pathway, and could cleave human complement C3 under Ca 2+-free gelatin veronal buffered saline. Among them, the largest molecule, BS-BGA was the most potent complement activator. Methylation analysis and NMR spectroscopy were used to achieve their structural characterization. They are all water-soluble and composed mainly of backbone structures of beta-(1-->3)-glucan with beta-(1-->4)-linked side chains varying in degree of branching. BS-BGA consisted of a higher proportion of 3-linked glucopyranosyl residues and a lower degree of branching than BS-BGB and BS-BGC. In particular, BS-BGA contained a small amount of O-acetyl groups at C-6 of the 3-linked glucopyranosyl residues. These data demonstrate that the structural characteristics including molecular size, degree of branching, and O-acetyl substitution are involved, at least in part, in their different anticomplementary activities.