RESUMO
Renal cell carcinoma (RCC) is the most frequent upper urinary tract cancer in humans and accounts for 80-85% of malignant renal tumors. Eker rat represents a unique animal model to study RCC since these rats develop spontaneous renal tumors and leiomyoma, which may be due to tuberous sclerosis 2 (TSC2) mutation resulting in the activation of the mammalian target of rapamycin (mTOR) pathway. This study examines the role of a lycopene-rich diet in the development of RCC in the TSC2 mutant Eker rat model. Ten-week old female Eker rats (n=90) were assigned in equal numbers to receive 0, 100 or 200mg/kg of lycopene as part of their daily diet. After 18 months the rats were sacrificed and the kidneys were removed. Immunohistochemical staining with antibodies against mTOR, phospho-S6 and EGFR were performed, as well as hematoxylin-eosin staining for histologic examination of the tumors. Tumors were counted and measured in individual kidneys. Presence of tumor decreased from 94% in control animals to 65% in the experimental group, but the difference was not statistically significant (P<0.12). However, mean numbers of renal carcinomas were statistically significantly decreased in the lycopene-treated rats (P<0.008) when compared to untreated controls. In the lycopene group, tumor numbers decreased (P<0.002) and the numbers tended to decrease linearly (P<0.003) as supplemental lycopene increased from 0 to 200. Control rats fed only basal diet had a greater length of tumors (23.98 mm) than rats fed lycopene supplement groups (12.90 mm and 11.07 mm) (P<0.05). Moreover tumor length decreased (P<0.02) and tumor length tended to decrease linearly (P<0.03) as supplemental lycopene increased from 0 to 200mg/kg. All tumors showed strong staining with antibodies against mTOR, phospho-S6 and EGFR. In conclusion, dietary supplementation with lycopene attenuates the development of renal cell cancers in the predisposed TSC2 mutant Eker rat model. These results suggest that lycopene may play a role in the prevention of RCC.
Assuntos
Anticarcinógenos/farmacologia , Carcinoma de Células Renais/prevenção & controle , Carotenoides/farmacologia , Neoplasias Renais/prevenção & controle , Mutação , Proteínas Supressoras de Tumor/genética , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Feminino , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Licopeno , Fosfoproteínas/metabolismo , Ratos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose TuberosaRESUMO
Substances in olive products contribute to improved health as suggested by epidemiological data. In this study we assessed the effects of hydroxytyrosol (HT) on inflammatory mediators, cytokines and chemokines, and identified anti-inflammatory constituents of aqueous olive extracts, I.E., olive vegetation water (OVW). Murine macrophages (RAW264.7 cells) were stimulated with lipopolysaccharide (LPS) in the absence or presence of substances; inflammatory mediators [nitric oxide (NO), prostaglandin E2 (PGE2), cytokines, interleukins, chemokines] were determined by the Griess reaction, EIA, or multiplex ELISA (Luminex technology). Expression of inflammatory genes was determined by RT-PCR. Aqueous olive extracts were fractionated by preparative HPLC and the fractions investigated for their effects on NO and PGE2 production. Results were further analyzed by principal component analysis. HT inhibited production of NO and PGE2 with an IC50 of 11.4 and 19.5 µM, respectively, reflecting strong anti-inflammatory activity. HT and OVW diminished secretion of cytokines (IL-1 α, IL-1 ß, IL-6, IL-12, TNF- α), and chemokines (CXCL10/IP-10, CCL2/MCP-1). HT and OVW concentration-dependently reduced the expression of genes of inducible nitric oxide synthase (iNOS), IL-1 α, CXCL10/IP-10, MIP-1 ß, matrix metalloproteinase-9, and prostaglandin E2 synthase (PGES). The effects of HT were partly mediated VIA the NF- κB pathway, as shown by RT-PCR analysis. HT was identified as the main bioactive compound of OVW. The data provide a molecular basis for elucidating the effects of HT on inflammatory processes. The effects of HT on NO and chemokine production point to their impact on chronic inflammatory processes in endothelium or arthritis.
Assuntos
Anti-Inflamatórios/farmacologia , Regulação da Expressão Gênica/genética , Olea/química , Álcool Feniletílico/análogos & derivados , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Linhagem Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Dinoprostona/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Álcool Feniletílico/química , Álcool Feniletílico/isolamento & purificação , Álcool Feniletílico/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Polifenóis/química , Polifenóis/isolamento & purificação , RNA/genéticaRESUMO
Epidemiological evidence links lycopene consumption with decreased prostate cancer risk. Several signaling pathways have been identified as players in prostate cancer development. Chronic prostatitis, for example, due to infections, is a suggested risk factor for prostate cancer. Endogenous production of reactive oxygen species during inflammation may lead to oxidative DNA damage, which can be mutagenic, if unrepaired. Androgen signaling, cytokine (IL-6, IL-4) and growth factor signaling (e.g., IGF, Wnt/beta-catenin) cross-talk via PI3K/Akt, MAPK, and Jak/STAT pathways have been identified as major controllers of prostate growth. During disease progression, and after androgen ablation therapy, the remaining operational pathways are upregulated to compensate for the lost growth signal, finally resulting in androgen-independent prostate cancer. Lycopene modulates several of the aforementioned pathways, providing a promising rationale for prostate cancer risk reduction by lycopene: In many experimental setups, lycopene reduced inflammatory signals, prevented oxidative DNA damage, modulated the expression or activity of IGF axis members, of Wnt/beta-catenin and androgen signalling, and enhanced gap junctional communication. Lycopene's influence on these pathways likely contributes to the observed cell growth inhibition and apoptosis induction by lycopene. A substantial part of the lycopene effects can be explained by its antioxidant action, but other mechanisms might also be involved.
Assuntos
Anticarcinógenos/uso terapêutico , Carotenoides/uso terapêutico , Suplementos Nutricionais , Próstata/metabolismo , Neoplasias da Próstata/prevenção & controle , Antagonistas de Androgênios/uso terapêutico , Animais , Apoptose , Cateninas/antagonistas & inibidores , Cateninas/metabolismo , Ciclo Celular , Dano ao DNA , Progressão da Doença , Junções Comunicantes/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Licopeno , Masculino , Desintoxicação Metabólica Fase II , Próstata/fisiopatologia , Neoplasias da Próstata/epidemiologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Prostatite/epidemiologia , Prostatite/metabolismo , Prostatite/prevenção & controle , Risco , Transdução de Sinais , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismoRESUMO
We studied the influence of beta-carotene on the tobacco smoke carcinogen 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor development in the A/J-mouse model. The normally low beta-carotene absorption was facilitated with a diet enriched in fat and bile salt, resulting in plasma and lung tissue levels similar to humans. beta-Carotene enhanced NNK-induced early bronchial cell proliferation, however, this effect was not predictive for later tumor development. Tumor multiplicity was not significantly affected by beta-carotene, neither in carcinogen-initiated nor in uninitiated mice, and regardless of dose and time point of supplementation during tumor development. RARbeta isoform and CYP26 gene expression levels analyzed by quantitative RT-PCR were weakly, but significantly, inversely correlated and showed evidence for altered retinoid signaling and catabolism in the lungs of NNK-initiated, beta-carotene supplemented mice. However, this interaction did not translate into enhanced tumor multiplicity. These results indicate that impaired retinoid signaling is not likely a key factor in lung tumorigenesis in this mouse model.
Assuntos
Adenoma/patologia , Carcinógenos , Neoplasias Pulmonares/patologia , Pulmão/efeitos dos fármacos , Nitrosaminas , beta Caroteno/farmacologia , Adenoma/induzido quimicamente , Adenoma/metabolismo , Animais , Brônquios/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Modelos Animais de Doenças , Regulação para Baixo , Interações Medicamentosas , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Pulmão/química , Pulmão/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Isoformas de Proteínas/biossíntese , Receptores do Ácido Retinoico/biossíntese , Receptores do Ácido Retinoico/genética , Ácido Retinoico 4 HidroxilaseRESUMO
The influence of beta-carotene (BC) and its derivatives on differentiation, proliferation and apoptosis in three human acute leukemia cell lines was studied. We investigated: (i) the cellular uptake of BC, (ii) the cytotoxicity, (iii) the effect on cell cycle progression and/or apoptosis. The dose- and time-dependent pattern of cellular BC uptake in all studied cell lines was seen. We did not observe any cytotoxic effect of BC and ATRA in the chosen concentrations. There was only limited effect of BC on gene expression. The microarrray analysis of U-937 cell line exposed to BC for 72 h showed an increased expression of BAX gene. This finding was confirmed by real-time Q-PCR analysis, and supported by a flow cytometry apoptosis tests. We did not observe any influence of studied components on cellular proliferation. The induction of differentiation after incubation with ATRA in HL-60 cells was noted. The induction of cellular apoptosis by BC was seen in all studied cell lines. We demonstrated that BC used in the concentrations achievable in vivo does not affect the proliferation and differentiation process of the studied leukemic cell lines, but can influence and enhance the apoptosis by modulating the expression of the regulatory genes.
Assuntos
Apoptose/efeitos dos fármacos , beta Caroteno/farmacologia , Apoptose/genética , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia/classificação , Leucemia/tratamento farmacológico , Análise em Microsséries , Células U937 , beta Caroteno/uso terapêuticoRESUMO
Epidemiological evidence links consumption of lycopene, the red carotenoid of tomato, to reduced prostate cancer risk. We investigated the effect of lycopene in normal prostate tissue to gain insight into the mechanisms, by which lycopene can contribute to primary prostate cancer prevention. We supplemented young rats with 200 ppm lycopene for up to 8 wk, measured the uptake into individual prostate lobes, and analyzed lycopene-induced gene regulations in dorsal and lateral lobes after 8 wk of supplementation. Lycopene accumulated in all four prostate lobes over time, with all-trans lycopene being the predominant isoform. The lateral lobe showed a significantly higher total lycopene content than the other prostate lobes. Transcriptomics analysis revealed that lycopene treatment mildly but significantly reduced gene expression of androgen-metabolizing enzymes and androgen targets. Moreover, local expression of IGF-I was decreased in the lateral lobe. Lycopene also consistently reduced transcript levels of proinflammatory cytokines, immunoglobulins, and immunoglobulin receptors in the lateral lobe. This indicates that lycopene reduced inflammatory signals in the lateral prostate lobe. In summary, we show for the first time that lycopene reduced local prostatic androgen signaling, IGF-I expression, and basal inflammatory signals in normal prostate tissue. All of these mechanisms can contribute to the epidemiologically observed prostate cancer risk reduction by lycopene.
Assuntos
Biomarcadores/metabolismo , Carotenoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Próstata/efeitos dos fármacos , Próstata/metabolismo , Androgênios/metabolismo , Animais , Carotenoides/farmacocinética , Citocinas/genética , Regulação para Baixo/efeitos dos fármacos , Nível de Saúde , Imunoglobulinas/genética , Inflamação/genética , Inflamação/metabolismo , Fator de Crescimento Insulin-Like I/genética , Licopeno , Masculino , Próstata/química , Próstata/crescimento & desenvolvimento , Ratos , Receptores Fc/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVES: Endothelial cells and their progenitors play an important role in angiogenesis that is essential for organogenesis and tissue remodelling, as well as for inflammatory responses and carcinogenesis in all periods of life. In the present study, the authors concentrated on the direct effect of beta-carotene (BC) on human umbilical cord-originated endothelial progenitor cells and human umbilical vein endothelial cells. METHODS: BC uptake was measured using high-performance liquid chromatography. The effect on cell proliferation was measured based on bromodeoxyuridine incorporation. Chemotaxis was performed in a Boyden chamber. The influence on tubular-like structure formation was investigated using a three-dimensional assay in Matrigel (Becton Dickinson, USA) both in an in vitro and in vivo model. Changes in gene expression were analyzed using the microarray hybridization method. Quantitative gene expression was estimated using the real-time polymerase chain reaction method. RESULTS: It was shown that BC, in the physiological range of concentrations found in human blood, is a potent activator of chemotaxis of endothelial cells. Microarray data analysis revealed that the genes involved in cell-cell and cell-matrix adhesion, matrix reorganization and activation of chemotaxis were the most affected by BC genes in human umbilical vein endothelial cells and endothelial progenitor cells. These results were also confirmed in an in vivo angiogenesis model. CONCLUSION: BC, in the range of physiological concentrations, stimulates early steps of angiogenic activity of endothelial cells by activation of cellular migration, as well as by matrix reorganization and a decrease in cellular adhesion.
RESUMO
Ultraviolet light A (UVA) exposure is thought to cause skin aging mainly by singlet oxygen ((1)O(2))-dependent pathways. Using microarrays, we assessed whether pre-treatment with the (1)O(2) quencher beta-carotene (betaC; 1.5 microM) prevents UVA-induced gene regulation in HaCaT human keratinocytes. Downregulation of growth factor signaling, moderate induction of proinflammatory genes, upregulation of immediate early genes including apoptotic regulators and suppression of cell cycle genes were hallmarks of the UVA effect. Of the 568 UVA-regulated genes, betaC reduced the UVA effect for 143, enhanced it for 180, and did not interact with UVA for 245 genes. The different interaction modes imply that betaC/UVA interaction involved multiple mechanisms. In unirradiated keratinocytes, gene regulations suggest that betaC reduced stress signals and extracellular matrix (ECM) degradation, and promoted keratinocyte differentiation. In irradiated cells, expression profiles indicate that betaC inhibited UVA-induced ECM degradation, and enhanced UVA induction of tanning-associated protease-activated receptor 2. Combination of betaC-promoted keratinocyte differentiation with the cellular "UV response" caused synergistic induction of cell cycle arrest and apoptosis. In conclusion, betaC at physiological concentrations interacted with UVA effects in keratinocytes by mechanisms that included, but were not restricted to (1)O(2) quenching. The retinoid effect of betaC was minor, indicating that the betaC effects reported here were predominantly mediated through vitamin A-independent pathways.
Assuntos
Antioxidantes/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , beta Caroteno/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Expressão Gênica/efeitos da radiação , Humanos , Fatores Imunológicos/genética , Queratinócitos/citologia , Metaloproteinase 10 da Matriz , Metaloendopeptidases/genética , Inibidores de Proteases/metabolismo , Receptor PAR-2/genética , Tretinoína/metabolismo , Raios UltravioletaRESUMO
Epidemiological studies have consistently associated high intakes of lycopene or vitamin E with a reduced prostate cancer risk. Both compounds were tested in the MatLyLu Dunning prostate cancer model to gain insight into the in vivo action of lycopene and vitamin E. Supplementation for 4 weeks with 200 ppm lycopene, 540 ppm vitamin E, or both led to plasma levels comparable with those in humans. Both compounds also accumulated in tumor tissue. Macroscopic evaluation of the tumors by magnetic resonance imaging showed a significant increase in necrotic area in the vitamin E and the lycopene treatment groups. Microarray analysis of tumor tissues revealed that both compounds regulated local gene expression. Vitamin E reduced androgen signaling without affecting androgen metabolism. Lycopene interfered with local testosterone activation by down-regulating 5-alpha-reductase and consequently reduced steroid target genes expression (cystatin-related protein 1 and 2, prostatic spermine binding protein, prostatic steroid binding protein C1, C2 and C3 chain, probasin). In addition, lycopene down-regulated prostatic IGF-I and IL-6 expression. Based on these findings, we suggest that lycopene and vitamin E contribute to the reduction of prostate cancer by interfering with internal autocrine or paracrine loops of sex steroid hormone and growth factor activation/synthesis and signaling in the prostate.
Assuntos
Comunicação Autócrina/efeitos dos fármacos , Carotenoides/farmacologia , Modelos Animais de Doenças , Comunicação Parácrina/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Vitamina E/farmacologia , Androgênios/metabolismo , Androgênios/farmacologia , Animais , Peso Corporal , Carotenoides/administração & dosagem , Carotenoides/análise , Carotenoides/sangue , Colestenona 5 alfa-Redutase/metabolismo , Dieta , Suplementos Nutricionais , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/metabolismo , Licopeno , Imageamento por Ressonância Magnética , Masculino , Necrose , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Ratos , Vitamina E/administração & dosagem , Vitamina E/análise , Vitamina E/sangueRESUMO
UVA exposure causes skin photoaging by singlet oxygen (1)O(2)-mediated induction of, e.g., matrix metalloproteases (MMPs). We assessed whether pretreatment with beta-carotene, a (1)O(2) quencher and retinoic acid (RA) precursor, interferes with UVA-induced gene regulation. HaCaT keratinocytes were precultured with beta-carotene at physiological concentrations (0.5, 1.5, and 3.0 microM) prior to exposure to UVA from a Hönle solar simulator (270 kJ/m(2)). HaCaT cells accumulated beta-carotene in a time- and dose-dependent manner. UVA irradiation massively reduced the cellular beta-carotene content. Beta-carotene suppressed UVA-induction of MMP-1, MMP-3, and MMP-10, three major matrix metalloproteases involved in photoaging. We show that regulation by not only MMP-1, but also MMP-10, involves (1)O(2)-dependent mechanisms. Beta-carotene dose-dependently quenched (1)O(2)-mediated induction of MMP-1 and MMP-10. Thus, as in chemical solvent systems, beta-carotene quenches (1)O(2) also in living cells. Vitamin E did not cooperate with beta-carotene to further inhibit MMP induction. HaCaT cells produced weak retinoid activity from beta-carotene, as demonstrated by mild upregulation of RAR beta and activation of an RARE-dependent reporter gene. Beta-carotene did not regulate the genes encoding other RARs, RXRs, or the two beta-carotene cleavage enzymes. These results demonstrate that beta-carotene acts photoprotectively, and that this effect is mediated by (1)O(2) quenching.
Assuntos
Antioxidantes/farmacologia , Queratinócitos/enzimologia , Metaloproteinase 1 da Matriz/genética , Metaloendopeptidases/genética , Oxigênio Singlete/metabolismo , Luz Solar , Raios Ultravioleta , beta Caroteno/farmacologia , Análise de Variância , Sequência de Bases , Transporte Biológico , Linhagem Celular , Primers do DNA , Epiderme , Humanos , Queratinócitos/efeitos dos fármacos , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 10 da Matriz , Metaloendopeptidases/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Caroteno/farmacocinéticaRESUMO
OBJECTIVE: To describe the response of a child with persistent fungemia to caspofungin, a member of the echinocandin class of antifungals. DESIGN: Descriptive case report. SETTING: Pediatric intensive care unit at a university teaching hospital. PATIENT: A 3-yr-old female with persistent candidemia. INTERVENTION: After >5 wks of persistent candidemia, caspofungin was added to an antifungal regimen that included amphotericin B and flucytosine. MEASUREMENTS AND MAIN RESULTS: The addition of caspofungin resulted in rapid clearance of the candidemia. The child recovered without evidence of further fungal infection or overt toxicity. CONCLUSION: Caspofungin was administered safely in this pediatric patient and possibly contributed to her clinical improvement. Caspofungin may be considered in children with severe persistent fungal infections that are not responsive to standard therapy. More study in pediatric patients is necessary before recommending its general use.
Assuntos
Antifúngicos/uso terapêutico , Candidíase/tratamento farmacológico , Fungemia/tratamento farmacológico , Peptídeos Cíclicos , Peptídeos/uso terapêutico , Anfotericina B/uso terapêutico , Candidíase/complicações , Caspofungina , Pré-Escolar , Quimioterapia Combinada , Equinocandinas , Endocardite/etiologia , Feminino , Flucitosina/uso terapêutico , Fungemia/complicações , Humanos , LipopeptídeosRESUMO
Oxidative stress and mitochondrial dysfunction are known to play important roles in type 2 diabetes mellitus (T2DM) and insulin resistance. However, the pathology of T2DM remains complicated; in particular, the mechanisms of mitochondrial dysfunction in skeletal muscle and other insulin-sensitive tissues are as yet unclear. In the present study, we investigated the underlying mechanisms of oxidative stress and mitochondrial dysfunction by focusing on mitochondrial dynamics, including mitochondrial biogenesis and autophagy, in skeletal muscle of a nonobese diabetic animal model--the Goto-Kakizaki (GK) rat. The results showed that GK rats exhibited impaired glucose metabolism, increased oxidative stress and decreased mitochondrial function. These dysfunctions were found to be associated with induction of LC3B, Beclin1 and DRP1 (key molecules mediating the autophagy pathway), while they appeared not to affect the mitochondrial biogenesis pathway. In addition, (-)-epigallocatechin-3-gallate (EGCG) was tested as a potential autophagy-targeting nutrient, and we found that EGCG treatment improved glucose tolerance and glucose homeostasis in GK rats, and reduced oxidative stress and mitochondrial dysfunction in skeletal muscle. Amelioration of excessive muscle autophagy in GK rats through the down-regulation of the ROS-ERK/JNK-p53 pathway leads to improvement of glucose metabolism, reduction of oxidative stress and inhibition of mitochondrial loss and dysfunction. These results suggest (a) that hyperglycemia-associated oxidative stress may induce autophagy through up-regulation of the ROS-ERK/JNK-p53 pathway, which may contribute to mitochondrial loss in soleus muscle of diabetic GK rats, and (b) that EGCG may be a potential autophagy regulator useful in treatment of insulin resistance.
Assuntos
Autofagia/efeitos dos fármacos , Catequina/análogos & derivados , Diabetes Mellitus Tipo 2/fisiopatologia , Hipoglicemiantes/farmacologia , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Glicemia , Catequina/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Jejum , Resistência à Insulina , Masculino , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Ratos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
The Nrf2-Keap1 pathway is believed to be a critical regulator of the phase II defense system against oxidative stress. By activation of Nrf2, cytoprotective genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO-1) and γ-glutamyl-cysteine ligase (GCL) are induced. GCL-induced glutathione (GSH) production is believed to affect redox signaling, cell proliferation and death. We here report that tert-butyl hydroperoxide (t-BHP)-induced GSH reduction led to mitochondrial membrane potential loss and apoptosis in cultured human retinal pigment epithelial cells from the ARPE-19 cell line. Hydroxytyrosol (HT), a natural phytochemical from olive leaves and oil, was found to induce phase II enzymes and GSH, thus protect t-BHP-induced mitochondrial dysfunction and apoptosis. Depletion of GSH by buthionine-[S,R]-sulfoximine enhanced t-BHP toxicity and abolished HT protection. Overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss. Meanwhile, HT-induced GSH enhancement and induction of Nrf2 target gene (GCLc, GCLm, HO-1, NQO-1) messenger RNA (mRNA) were inhibited by Nrf2 knockdown, suggesting that HT increases GSH through Nrf2 activation. In addition, we found that HT was able to activate the PI3/Akt and mTOR/p70S6-kinase pathways, both of which contribute to survival signaling in stressed cells. However, the effect of HT was not inhibited by the PI3K inhibitor LY294002. Rather, c-Jun N-terminal kinase (JNK) activation was found to induce p62/SQSTM1 expression, which is involved in Nrf2 activation. Our study demonstrates that Nrf2 activation induced by the JNK pathway plays an essential role in the mechanism behind HT's strengthening of the antiapoptotic actions of the endogenous antioxidant system.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Glutationa/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Álcool Feniletílico/análogos & derivados , Epitélio Pigmentado da Retina/efeitos dos fármacos , Antioxidantes/farmacologia , Linhagem Celular , Humanos , Álcool Feniletílico/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Proteína Sequestossoma-1 , Transdução de SinaisRESUMO
Physical exercise is considered to exert a positive effect on health, whereas strenuous or excessive exercise (Exe) causes fatigue and damage to muscle and immune functions. The underlying molecular mechanisms are still unclear. We designed a protocol to mimic Exe and explore the ensuing cellular damage and involvement of mitochondrial dynamics. We found that Exe was prone to decrease endurance capacity and induce damage to renal function and the immune system. Muscle atrophy markers atrogin-1 and MuRF1 mRNA were increased by Exe, accompanied by increased autophagy and mitochondrial fission in skeletal muscle. Exe caused a decrease in PGC-1α and complex I expression; it also activated JNK and Erk1/2 pathways and consequently induced p53, p21, and MnSOD expression in skeletal muscle. The involvement of oxidant-induced autophagy and mitochondrial dysfunction was confirmed in C2C12 myoblasts. Hydroxytyrosol (HT), a natural olive polyphenol, efficiently enhanced endurance capacity and prevented Exe-induced renal and immune system damage. Also, HT treatment inhibited both the Exe-induced increase in autophagy and mitochondrial fission and the decrease in PGC-1α expression. In addition, HT enhanced mitochondrial fusion and mitochondrial complex I and II activities in muscle of Exe rats. These results demonstrate that Exe-induced fatigue and damage to muscle and immune functions may be mediated via the regulation of mitochondrial dynamic remodeling, including the downregulation of mitochondrial biogenesis and upregulation of autophagy. HT supplementation may regulate mitochondrial dynamic remodeling and enhance antioxidant defenses and thus improve exercise capacity under Exe conditions.
Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Músculo Esquelético/imunologia , Álcool Feniletílico/análogos & derivados , Condicionamento Físico Animal , Animais , Masculino , Camundongos , Modelos Biológicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Álcool Feniletílico/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: To our knowledge, there is no direct information on lycopene metabolism in humans. OBJECTIVE: The objective of this study was to quantify the long-term human bioavailability of lycopene in plasma and skin after a single dose of (14)C-lycopene and to profile the metabolites formed. DESIGN: We preselected 2 male subjects as lycopene absorbers and gave them an oral dose of 10 mg synthetic lycopene combined with ≈6 µg [6,6',7,7'-(14)C]lycopene (≈30,000 Bq; 92% trans lycopene). The appearance of (14)C in plasma, plasma triacylglycerol-rich lipoprotein (TRL) fraction, urine, expired breath carbon dioxide, and skin biopsies was measured over 42 d. The (14)C in lycopene-isomer fractions from plasma and TRL fraction was measured to assess the isomerization of lycopene in vivo. RESULTS: We quantified (14)C from (14)C-lycopene in plasma, the plasma TRL fraction, expired carbon dioxide, urine, and skin. The time to maximum concentration (t(max)) of total (14)C-lycopene in plasma was 6 h, and the elimination half-life (t(1/2)) was 5 d, which were different from the t(max) and t(1/2) of unlabeled lycopene (0.5 and 48 d, respectively). (14)C-Lycopene was extensively isomerized after dosing as a 92% all-trans isomer at dosing but changed to 50% trans, 38% 5 cis, 1% 9 cis, and 11% other cis isomers after 24 h. A similar pattern of isomerization was seen in plasma TRL fractions. CONCLUSIONS: Lycopene was extensively isomerized after dosing and rapidly metabolized into polar metabolites excreted into urine with the rapid peak of (14)CO(2) after dosing, which implies that ß-oxidation was involved in the lycopene metabolism. Lycopene or its metabolites were detected in skin for up to 42 d.
Assuntos
Carotenoides/farmacocinética , Pele/metabolismo , Adulto , Disponibilidade Biológica , Biópsia , Testes Respiratórios , Dióxido de Carbono/metabolismo , Isótopos de Carbono/metabolismo , Carotenoides/sangue , Carotenoides/metabolismo , Humanos , Isomerismo , Lipoproteínas/sangue , Licopeno , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Triglicerídeos/sangue , UrináliseRESUMO
Hydroxytyrosol (HT) in extra-virgin olive oil is considered one of the most important polyphenolic compounds responsible for the health benefits of the Mediterranean diet for lowering incidence of cardiovascular disease, the most common and most serious complication of diabetes. We propose that HT may prevent these diseases by a stimulation of mitochondrial biogenesis that leads to enhancement of mitochondrial function and cellular defense systems. In the present study, we investigated effects of HT that stimulate mitochondrial biogenesis and promote mitochondrial function in 3T3-L1 adipocytes. HT over the concentration range of 0.1-10 micromol/L stimulated the promoter transcriptional activation and protein expression of peroxisome proliferator-activated receptor (PPAR) coactivator 1 alpha (PPARGC1 alpha, the central factor for mitochondrial biogenesis) and its downstream targets; these included nuclear respiration factors 1 and 2 and mitochondrial transcription factor A, which leads to an increase in mitochondrial DNA (mtDNA) and in the number of mitochondria. Knockdown of Ppargc1 alpha by siRNA blocked HT's stimulating effect on Complex I expression and mtDNA copy number. The HT treatment resulted in an enhancement of mitochondrial function, including an increase in activity and protein expression of Mitochondrial Complexes I, II, III and V; increased oxygen consumption; and a decrease in free fatty acid contents in the adipocytes. The mechanistic study of the PPARGC1 alpha activation signaling pathway demonstrated that HT is an activator of 5'AMP-activated protein kinase and also up-regulates gene expression of PPAR alpha, CPT-1 and PPAR gamma. These data suggest that HT is able to promote mitochondrial function by stimulating mitochondrial biogenesis.
Assuntos
Adipócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Álcool Feniletílico/análogos & derivados , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/enzimologia , Adipócitos/fisiologia , Animais , Doenças Cardiovasculares/dietoterapia , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/prevenção & controle , Respiração Celular/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos não Esterificados/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Mitocôndrias/enzimologia , Mitocôndrias/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fatores Nucleares Respiratórios/genética , Fatores Nucleares Respiratórios/metabolismo , Concentração Osmolar , Fosforilação Oxidativa/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Álcool Feniletílico/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Studies in this laboratory have previously shown that hydroxytyrosol, the major antioxidant polyphenol in olives, protects ARPE-19 human retinal pigment epithelial cells from oxidative damage induced by acrolein, an environmental toxin and endogenous end product of lipid oxidation, that occurs at increased levels in age-related macular degeneration lesions. A proposed mechanism for this is that protection by hydroxytyrosol against oxidative stress is conferred by the simultaneous activation of two critically important pathways, viz., induction of phase II detoxifying enzymes and stimulation of mitochondrial biogenesis. Cultured ARPE-19 cells were pretreated with hydroxytyrosol and challenged with acrolein. The protective effects of hydroxytyrosol on key factors of mitochondrial biogenesis and phase II detoxifying enzyme systems were examined. Hydroxytyrosol treatment simultaneously protected against acrolein-induced inhibition of nuclear factor-E2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor coactivator 1 alpha (PPARGC1α) in ARPE-19 cells. The activation of Nrf2 led to activation of phase II detoxifying enzymes, including γ-glutamyl-cysteinyl-ligase, NADPH (nicotinamide adenine dinucleotide phosphate)-quinone-oxidoreductase 1, heme-oxygenase-1, superoxide dismutase, peroxiredoxin and thioredoxin as well as other antioxidant enzymes, while the activation of PPARGC1α led to increased protein expression of mitochondrial transcription factor A, uncoupling protein 2 and mitochondrial complexes. These results suggest that hydroxytyrosol is a potent inducer of phase II detoxifying enzymes and an enhancer of mitochondrial biogenesis. Dietary supplementation of hydroxytyrosol may contribute to eye health by preventing the degeneration of retinal pigment epithelial cells induced by oxidative stress.
Assuntos
Acroleína/efeitos adversos , Antioxidantes/farmacocinética , Desintoxicação Metabólica Fase II , Estresse Oxidativo , Álcool Feniletílico/análogos & derivados , Epitélio Pigmentado Ocular/citologia , Acroleína/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Suplementos Nutricionais , Flavonoides/metabolismo , Humanos , Canais Iônicos/metabolismo , Degeneração Macular/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , PPAR alfa/metabolismo , Fenóis/metabolismo , Álcool Feniletílico/farmacocinética , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/metabolismo , Polifenóis , Fatores de Transcrição/metabolismo , Proteína Desacopladora 2RESUMO
Angiogenesis is a process of new blood vessel formation from pre-existing ones. The most important steps in angiogenesis include detachment, proliferation, migration, homing and differentiation of vascular wall cells, which are mainly endothelial cells and their progenitors. The study focused on the effect of beta-carotene (BC) supplementation (12,000 mg/kg) in the diet on angiogenesis in Balb/c mice. Female Balb/c mice were fed for 5 weeks with two different diets: with BC or without BC supplementation. After 4 weeks of feeding, Balb/c mice were injected subcutaneously with two matrigel plugs with or without basic fibroblast growth factor (bFGF). Six days later, the animals were killed, and the matrigel plugs were used for immunohistochemical staining with CD31 antibody and for gene expression analysis. Microarray and Real-Time PCR data showed down-regulation of genes involved in proliferation and up-regulation of genes encoding inhibitors of apoptosis, proteins regulating cell adhesion, matrix-degrading enzymes and proteins involved in the VEGF pathway. The results of this study demonstrated that BC proangiogenic activity (with or without bFGF) in vivo seemed to be more significantly associated with cells' protection from apoptosis and their stimulation of chemotaxis/homing than cell proliferation.
RESUMO
Insulin resistance is an important feature of type 2 diabetes and obesity. The underlying mechanisms of insulin resistance are still unclear and may involve pathological changes in multiple tissues. Mitochondrial dysfunction, including mitochondrial loss and over-production of oxidants, has been suggested to be involved in the development of insulin resistance. Increasing evidence suggests that targeting mitochondria to protect mitochondrial function as a unique measure, i.e. mitochondrial medicine, could prevent and ameliorate various diseases associated with mitochondrial dysfunction. In this review, we have summarized recent progress in pharmaceutical and nutritional studies of drugs and nutrients to targeting mitochondria by stimulating mitochondrial metabolism (biogenesis and degradation) to improve mitochondrial function and decrease oxidative stress for preventing and ameliorating insulin resistance. We have focused on nutrients from natural sources to stimulating mitochondrial biogenesis in cellular systems and in animal models. The in vitro and in vivo studies, especially our own work on the effects and mechanisms of mitochondrial targeting nutrients or their combinations, may help us to understand the importance and mechanisms of mitochondrial biogenesis in insulin resistance, and provide hope for developing mitochondria-targeting agents for preventing and treating insulin resistance in type 2 diabetes and obesity.
Assuntos
Diabetes Mellitus/metabolismo , Suplementos Nutricionais , Sistemas de Liberação de Medicamentos/métodos , Resistência à Insulina/fisiologia , Mitocôndrias/metabolismo , Obesidade/metabolismo , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/prevenção & controle , Humanos , Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/tratamento farmacológico , Modelos Biológicos , Obesidade/tratamento farmacológico , Obesidade/prevenção & controle , Estresse Oxidativo/efeitos dos fármacosRESUMO
The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 x 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells.