RESUMO
Clinically, serum level of folate has been negatively correlated to the stage and progression of liver cancer. Nevertheless, the functional consequence of folate deficiency (FD) in malignancy has not been fully investigated. Human hepatocellular carcinoma (HCC) cells (as study model) and other cancer types such as lung and glioma were cultured under folate deficient (FD) and folate complete (FD) conditions. Molecular characterization including intracellular ROS/RNS (reactive oxygen/nitrogen species), viability, colony formation, cancer stem-like cell (CSC) phenotype analyses were performed. In vivo tumorigenesis under FD and FC conditions were also examined. FD induced a significant increase in ROS and RNS, suppressing proliferative ability but inducing metastatic potential. Mesenchymal markers such as Snail, ZEB2, and Vimentin were significantly up-regulated while E-cadherin down-regulated. Importantly, CSC markers such as Oct4, ß-catenin, CD133 were induced while PRRX1 decreased under FD condition. Furthermore, FD-conditioned HCC cells showed a decreased miR-22 level, leading to the increased expression of its target genes including HDAC4, ZEB2 and Oct4. Finally, xenograft mouse model demonstrated that FD diet promoted tumorigenesis and metastasis as compared to their FC counterparts. Our data provides rationales for the consideration of folate supplement as a metastasis preventive measure.
Assuntos
Transição Epitelial-Mesenquimal , Deficiência de Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Deficiência de Ácido Fólico/genética , Deficiência de Ácido Fólico/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Estresse Nitrosativo , Estresse Oxidativo , Fenótipo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Despite a plethora of literature has documented that osteoarthritis (OA) is veritably associated with oxidative stress-mediated chondrocyte death and matrix degradation, yet the possible involvement of synoviocyte abnormality as causative factor of OA has not been thoroughly investigated. For this reason, we conduct the current studies to insight into how synoviocytes could respond to an episode of folate-deprived (FD) condition. First, when HIG-82 synoviocytes were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature mediated through FD-evoked overproduction of reactive oxygen species (ROS) and drastically released of cytosolic calcium (Ca2+) concentrations. Next, we uncovered that FD-evoked ROS overproduction could only be strongly suppressed by either mitochondrial complex II inhibitors (TTFA and carboxin) or NADPH oxidase (NOX) inhibitors (AEBSF and apocynin), but not by mitochondrial complex I inhibitor (rotenone) and mitochondrial complex III inhibitor (antimycin A). Interestingly, this selective inhibition of FD-evoked ROS by mitochondrial complex II and NOX inhibitors was found to correlate excellently with the suppression of cytosolic Ca2+ release and reduced the magnitude of the apoptotic TUNEL-positive cells. Taken together, we present the first evidence here that FD-triggered ROS overproduction in synoviocytes is originated from mitochondrial complex II and NOX. Both elevated ROS in tandem with cytosolic Ca2+ overload serve as final arbitrators for apoptotic lethality of synoviocytes cultivated under FD condition. Thus, folate supplementation may be beneficial to patients with OA.
Assuntos
Cálcio/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Deficiência de Ácido Fólico/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carboxina/farmacologia , Linhagem Celular , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Ácido Fólico/metabolismo , Células HeLa , Humanos , NADPH Oxidases/antagonistas & inibidores , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Coelhos , Rotenona/farmacologia , Sulfonas/farmacologia , Tenoiltrifluoracetona/farmacologiaRESUMO
Patients with hepatocellular carcinoma (HCC) are prone to folate deficiency (FD). Here we showed that, in cell line-specific manner, FD caused resistance to FD-induced oxidative stress and multi-drug resistance (MDR). This resistance was due to upregulation of glucose-regulated protein 78 (GRP78) and Survivin. Using siRNA and Epigallocatechin gallate (EGCG), we found that GRP78 and Survivin cooperatively conferred MDR by decreasing FD-induced ROS generation. Our data showed that FD increases GRP78 and Survivin, which serve as ROS inhibitors, causing MDR in HCC. We suggest that folate supplementation may enhance the efficacy of chemotherapy.
Assuntos
Ácido Fólico/farmacologia , Proteínas de Choque Térmico/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Catequina/análogos & derivados , Catequina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Chaperona BiP do Retículo Endoplasmático , Ácido Fólico/metabolismo , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Proteínas Inibidoras de Apoptose/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Oxirredução , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , SurvivinaRESUMO
BACKGROUND: Glucose-regulated protein 78 (GRP78) plays an important role in the therapeutic treatment and progression of cancer. However, little is known about the effect of GRP78 expression to curcumin in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: In this study, we generated GRP78 knockdown cells (GRP78KD) by a short interfering RNA (siRNA) technique. The antiproliferation effects of curcumin were determined by MTT assay, TUNEL assay, and cell cycle determination. RESULTS: We found that GRP78KD cells were more resistant to curcumin treatment compared with the parental cells in MTT assay. The apoptosis cell population was increased in scrambled-siRNA cells treated with curcumin compared with GRP78KD cells in cell cycle distribution and TUNEL assays. Finally, we found that knocking down GRP78 causes resistance to curcumin treatment through the suppression of caspase-3 and caspase-8 expression levels. CONCLUSIONS: We conclude that the expression level of GRP78 may contribute to the therapeutic effect of curcumin on HCC cells.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Curcumina/farmacologia , Proteínas de Choque Térmico/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Citometria de Fluxo , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
BACKGROUND/PURPOSE: Betel quid (BQ) chewing is a popular oral masticatory activity, and there are approximately 600 million BQ chewers worldwide. Although chewing BQ has been linked to the patho-genesis of oral cancer, leukoplakia, and oral submucous fibrosis. The question whether the mixed constituents present in areca nut, which may exert cytotoxic effects on red blood cells (RBCs), has never been addressed. METHODS: Heparinized blood specimens were obtained with informed consent from healthy laboratory personnel. RBCs were separated with the standard procedure and adjusted to 10% hematocrit with PBS. Various concentrations of areca nut extract (ANE; 100-800 microg/mL) were added to these RBC preparations and incubated at 37 degrees C for 4 hours. Two portions (0.4 mL each) of the incubated RBCs were then used for measuring osmotic deformability index and for observing RBC morphology with scanning electron microscopy. The remaining RBCs were used for determining membrane sulfhydryl groups and protein profiles by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Blood incubated with various concentrations of ANE showed concentration-dependent decreases in osmotic deformability index and membrane sulfhydryl groups. Membrane protein profiles revealed a significant loss of the band 3 fraction, with the concomitant appearance of several new protein bands in the electropheretogram. Finally, drastic morphological changes of ANE-treated RBCs were observed. CONCLUSION: We suggest that to assure the quality of transfusion, the blood donated by a habitual BQ chewer should be used with caution because of its possible contamination with areca nut ingredients that may be cytotoxic to RBCs.
Assuntos
Areca/toxicidade , Doadores de Sangue , Deformação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Nozes/química , Extratos Vegetais/química , Areca/metabolismo , Transfusão de Sangue , Eletroforese em Gel de Poliacrilamida , Humanos , Mastigação , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Varredura , Nozes/efeitos adversos , Nozes/metabolismo , Extratos Vegetais/efeitos adversos , Dodecilsulfato de SódioRESUMO
(-)-Anonaine has been shown to have some anticancer activities, but the mechanisms of (-)-anonaine inducing cell death of human cancer cells is not fully understood. We investigated the mechanisms of apoptosis induced by (-)-anonaine in human HeLa cancer cells. Treatment with (-)-anonaine induces dose-dependent DNA damage that is correlated with increased intracellular nitric oxide, reactive oxygen species, glutathione depletion, disruptive mitochondrial transmembrane potential, activation of caspase 3, 7, 8, and 9, and poly ADP ribose polymerase cleavage. Our data indicate that (-)-anonaine up-regulated the expression of Bax and p53 proteins in HeLa cancer cells. The apoptosis and expression of Bax induced by (-)-anonaine could be inhibited when the HeLa cells were pretreated with Boc-Asp(OMe)-fmk, which is a broad caspases inhibitor. There was no obvious DNA damage in the (-)-anonaine-treated Madin-Darby canine kidney and Vero cell lines. Both Madin-Darby canine kidney and Vero cell lines are kidney epithelial cellular morphology. These results suggest that (-)-anonaine might be considered a potent compound for chemotherapy against cervical cancer or a health food supplement for cancer chemoprevention.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Aporfinas/farmacologia , Caspases/fisiologia , Dioxóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/fisiologia , Proteínas Reguladoras de Apoptose/biossíntese , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citometria de Fluxo , Glutationa/biossíntese , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Potenciais da Membrana/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Óxido Nítrico/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Espécies Reativas de Oxigênio/metabolismo , Sais de Tetrazólio , TiazóisRESUMO
The protective effects of a freeze-dried extracts of vegetables and fruits (BauYuan; BY) on the hydroxyl radical-mediated DNA strand breakages and the structural integrity of human red blood cells (RBCs) were investigated. First, the supercoiled plasmid (pEGFP-C1) DNA was subjected to oxidation damage by an ascorbate-fortified Fenton reaction and the protective effects were analyzed by agarose gel electrophoresis. In the absence of BY extracts, exposure of the high-throughput .OH-generating system (Fe2+ concentration >1.0 microM) caused a complete fragmentation of DNA. Supplementation of BY extract (1 mg/mL) to the plasmid DNA prior to the exposure could prevent it significantly. In contrast, as the plasmid exposed to a low-grade .OH-generating system (Fe2+<0.1 microM), the BY extract (1 mg/mL) provided an almost complete protection. Next, the cell deformabilities were measured to assess the protection effects of various BY extracts on human erythrocytes exposed to the oxidative insults. We found that both the aqueous extract and the organic solvent-derived extracts could strongly protect human RBCs from the reactive oxygen species (ROS)-mediated decrease in the deformability indices. The results implicated that the BY extracts could effectively protect the cell membrane integrity via scavenging ROS which enabling RBCs to maintain a balance of water content and surface area to prevent the drop of cell deformability.
Assuntos
Dano ao DNA , Liofilização , Frutas , Radical Hidroxila , Verduras , Animais , DNA/química , Deformação Eritrocítica , Eritrócitos/metabolismo , Radicais Livres , Humanos , Ferro/química , Camundongos , Células NIH 3T3 , Concentração Osmolar , Estresse Oxidativo , Oxigênio/metabolismoRESUMO
Chemoprevention by the use of naturally occurring substances is becoming a promising strategy to prevent cancer. In this study, the effects of isoobtusilactone A, a novel constituent isolated from the leaves of Cinnamomum kotoense, on the proliferation of human hepatoma Hep G2 cells were studied. Under our experimental conditions, isoobtusilactone A was found to elicit a concentration-dependent growth impediment (IC(50)=37.5 microM). The demise of these cells induced by isoobtusilactone A was apoptotic in nature, exhibiting a concentration-dependent increase in sub-G(1) fraction and DNA fragmentation. Subcellular fractionation analysis further revealed that Bax translocation to mitochondria resulted in a rapid release of cytochrome c, followed by activation of caspase 3 and PARP cleavage, and finally cell death. Isoobtusilactone A-treated cells also displayed transient increase of ROS during the earlier stage of the experiment, followed by the disruption of mitochondrial transmembrane potential (DeltaPsi(m)). The presence of a ROS scavenger (N-acetyl-L-cysteine) and an inhibitor of NADPH oxidase (diphenyleneiodonium chloride) blocked ROS production and the subsequent apoptotic cell death. In addition, in order to investigate the acute toxicity of isoobtusilactone A, groups of 5-6-week old Sprague-Dawley rats were subjected to oral administration of 350, or 700 mg/kg bw isoobtusilactone A four times each week for two weeks. There was no significant difference between control animals and treated animals with respect to the body weight gain, the body weight ratio of liver, spleen and kidney, haematological and clinical chemistry parameters. Taken together, our data suggest that ROS generated through the activation of NADPH oxidase plays an essential role in apoptosis induced by isoobtusilactone A, and the dosages of isoobtusilactone A tested in this study did not cause animal toxicity.
Assuntos
Alcanos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Lactonas/farmacologia , Neoplasias Hepáticas/patologia , Mitocôndrias Hepáticas/patologia , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Administração Oral , Alcanos/uso terapêutico , Animais , Antineoplásicos Fitogênicos/toxicidade , Carcinoma Hepatocelular/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cinnamomum/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lactonas/uso terapêutico , Neoplasias Hepáticas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
Mahlavu cells, poorly differentiated and p53 mutants of a human hepatoma subline, are known to be highly refractory to a number of chemotherapeutic agents and radiotherapy due to their high expressions of multidrug resistance gene-1 (MDR-1) and Bcl-2 proteins. Thus, it is desirable to search for an alternative strategy for effective eradication of this type of cancer cells. We present evidence here for the first time that 6-shogaol (6-SG), an alkanone isolated from the rhizomes of ginger, can effectively induce apoptotic cell death of Mahlavu cells via an oxidative stress-mediated caspase-dependent mechanism. The cascade of events in 6-SG-induced apoptosis of these cells involved an initial overproduction of reactive oxygen species (ROS) followed by a severe depletion of intracellular glutathione (GSH) contents. Both events consequently entailed a significant drop in mitochondrial transmembrane potential (DeltaPsim), which ultimately activated the activities of caspases 3/7 resulting in the DNA fragmentation. Interestingly, we also found that N-acetylcysteine (NAC), an antioxidant and a precursor of GSH biosynthesis, could offer a near complete protection of apoptotic cell death exerted by 6-SG. Similarly, exogenously added GSH could also provide protection with an equal efficacy. However, it was paradoxical that both Boc-Asp(OMe)-fmk (a broad caspases inhibitor) and cyclosporin A (an mitochondrial permeability transition opening inhibitor) could only partially protect these cells from 6-SG-induced apoptosis. Taking these data into consideration, it is obvious that GSH depletion is the major contributing factor in arbitrating 6-SG-induced apoptosis of Mahlavu cells. In conclusion, we provide here a novel modality that can help to eradicate a p53 mutant of human hepatoma cells by using a natural consistent isolated form of ginger. These data also provide evidence to reaffirm the notion that consumption of certain foodstuffs can be beneficial to health because some of the constituents contained in them may be anticarcinogenic.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Caspases/metabolismo , Catecóis/farmacologia , Neoplasias Hepáticas/patologia , Estresse Oxidativo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Zingiber officinale/química , Humanos , Mutação , Extratos Vegetais , Raízes de Plantas/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
We report here the development of a probe-based ultraweak chemiluminescence (uwCL) method capable of detecting a panel of four oxygen-derived free radicals (ODFRs) including superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radical (*OH), and peroxyl radical (ROO*) using different probes specific for these radicals performed by the same uwCL analyzer. The selected radical-generating systems and their corresponding uwCL-probing emitters were validated. These ODFR-detecting systems were subsequently utilized by us to assess the radical-scavenging ability (RSA) of a variety of extracts and purified constituents derived from foods and herbal preparations. Our approach for assessing RSA for these constituents is based on the suppression of uwCL generated by each ODFR, and the degrees of inhibition have been shown to be dose-dependent. For this reason, the estimation of IC50 for each testing compound can be obtained from the curve constructed based on the percent of inhibitions of uwCL versus the concentrations of the compound tested. To illustrate the practical applications of our devised methodology, data for comparative studies of RSA activities of fermented extracts of Cordeceps sinensis, purified methylgallate isolated from Toona sinesis, resveratrol purified from grape seeds, plus epimedin C from the aerial part of the Epimedium plant (yinyanghuo) are to be presented.
Assuntos
Análise de Alimentos , Sequestradores de Radicais Livres/análise , Medições Luminescentes/métodos , Extratos Vegetais/química , Espécies Reativas de Oxigênio/análise , Fermentação , Sequestradores de Radicais Livres/química , Meliaceae/química , Espécies Reativas de Oxigênio/químicaRESUMO
Actinodaphnine, extracted from Cinnamomum insularimontanum (Lauraceae), possesses cytotoxicity in some cancers, but the mechanism by which actinodaphnine induces apoptosis in human hepatoma cells remains poorly understood. In this study, we investigated the mechanisms of apoptosis induced by actinodaphnine in human hepatoma Mahlavu cells. Treatment with actinodaphnine dose-dependently induced apoptosis in Mahlavu cells that correlated with increased intracellular nitric oxide (NO) and reactive oxygen species (ROS), disruptive mitochondrial transmembrane potential (DeltaPsi(m)), and activation of caspase 3/7. Our data also demonstrated that actinodaphnine down-regulated activity of nuclear factor kappaB (NF-kappaB). The apoptotic response to actinodaphnine was markedly decreased in Mahlavu cells pretreated with dexsamethasone, a NO inhibitor, N-acetylcysteine (NAC), an antioxidant, and Boc-Asp(OMe)-fmk, a broad caspases inhibitor. These results suggested that actinodaphnine-induced apoptosis is initially mediated through the NO and/or ROS increase and caspases-dependent pathway. In conclusion, our results indicate that an increase of ROS and/or NO is the initial essential event that results in the decrease of DeltaPsi(m) and the activation of caspases that commits the cells to the apoptotic pathway in actinodaphnine-treated hepatoma Mahlavu cells.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Dioxolanos/farmacologia , NF-kappa B/efeitos dos fármacos , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Dano ao DNA , Relação Dose-Resposta a Droga , Regulação para Baixo , Medicamentos de Ervas Chinesas/farmacologia , Ativação Enzimática , Humanos , Marcação In Situ das Extremidades Cortadas , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Células Tumorais CultivadasRESUMO
Annoglabayin (1), a novel Annona dimeric kaurane diterpenoid, has been isolated from Annona glabra, and its structure was determined on the basis of spectroscopic analysis. Annoglabayin (1) contains a unique carbon bridge between two nor-ent-kaurane monomeric units. The dose-response of 2 in Hep G2 cells indicated that 2 increased DNA damage. In addition, our results showed that 2 induced a noticeable decrease in mitochondrial transmembrane potential during treatment. These results indicate that 2 produces apoptotic events in Hep G2 cells, through inducing changes in mitochondria.
Assuntos
Annona/química , Diterpenos do Tipo Caurano/isolamento & purificação , Plantas Medicinais/química , Apoptose/efeitos dos fármacos , Diterpenos do Tipo Caurano/química , Diterpenos do Tipo Caurano/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Taiwan , Células Tumorais CultivadasRESUMO
Casuarinin has been shown to be an antioxidant in acellular experiments. This study was designed to assess the ability of casuarinin, extracted from Terminalia arjuna, to protect cultured Madin-Darby canine kidney (MDCK) cells against H2O2-mediated oxidative stress. A comparison with trolox, a hydrosoluble vitamin E analogue was performed. MDCK cells were pretreated with casuarinin or trolox for 1 h, then exposed to H2O2. After incubation with 0.8 mM H2O2 for 1 h, casuarinin caused a decrease in intracellular peroxide production as shown by dichlorofluorescein (DCF) fluorescence in a concentration-dependent manner. After 3 h exposure to 8 mM H2O2, the percentage of intracellular glutathione (GSH)-negative cells was reduced in the casuarinin-treated group. Addition of 32mM H2O2 to MDCK cells for 3 h induced an increase in the percentage of cells containing 8-oxoguanine but the level of such cells declined in casuarinin-treated cells. These results show that casuarinin is more effective against H2O2-induced oxidative damage than trolox. The data suggest that casuarinin attenuates H2O2-induced oxidative stress, decreases DNA oxidative damage and prevents the depletion of intracellular GSH in MDCK cells.
Assuntos
Antioxidantes/farmacologia , Taninos Hidrolisáveis/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Terminalia , Animais , Antioxidantes/administração & dosagem , Antioxidantes/uso terapêutico , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Dano ao DNA , Cães , Relação Dose-Resposta a Droga , Citometria de Fluxo , Glutationa/metabolismo , Peróxido de Hidrogênio , Taninos Hidrolisáveis/administração & dosagem , Taninos Hidrolisáveis/uso terapêutico , Casca de Planta , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Espécies Reativas de Oxigênio/metabolismoRESUMO
Previous reports have suggested that high-dose L-arginine could be used in diabetic patients as a prophylactic blocker for the initial glycation reaction of proteins by methylglyoxal (MG), a reactive dicarbonyl compound of glucose metabolism. Here, we present several lines of evidence to substantiate that this prophylactic intervention may be inappropriate and should be used with care. First, we demonstrated that when various concentrations of L-arginine (2.0-8.0 mM) were added to a fixed concentration of MG (1.56 microM) in a buffered lucigenin solution, dose-dependent generation of superoxide anion (O(-)(2))-mediated ultraweak chemiluminescence (uwCL) occurs. The suppression of uwCL generation by exogenously added superoxide dismutase further substantiated that the interaction between MG and L-arginine generated O(-)(2). This phenomenon can also be demonstrated in a serum-based system. Furthermore, when a fixed concentration of L-arginine (8.0 mM) was added exogenously to a group of sera obtained from either diabetic patients (n = 10) or their matched nondiabetic controls (n = 10), a marked discrepancy in the generation of O(-)(2)-mediated uwCL could be demonstrated (12,534 +/- 3,147 vs. 950 +/- 350 counts; p < 0.001). Taken together, this evidence demonstrates that the appropriateness of using high-dose L-arginine for prophylactic measures in diabetic patients may be questioned, because the inhibition of the glycation reaction between MG and proteins by high-dose L-arginine unexpectedly produces plethoric O(-)(2) as a by-product, which may subsequently aggravate the preexisting oxidative stress status of diabetic patients.
Assuntos
Arginina/efeitos adversos , Arginina/farmacologia , Diabetes Mellitus/tratamento farmacológico , Produtos Finais de Glicação Avançada/metabolismo , Aldeído Pirúvico/farmacologia , Diabetes Mellitus/sangue , Relação Dose-Resposta a Droga , Humanos , Valores de Referência , Superóxidos/sangueRESUMO
Shikonin has been demonstrated to exhibit anti-cancer activity, but the underlying mechanisms are poorly understood. In this report, we showed that the administration of shikonin could result in the induction of apoptotic cell death of human hepatoma cell line, SK-Hep-1. As evident by the flow-cytometric studies, shikonin has the capability of generating increased amounts of intracellular reactive oxygen species (ROS) during the early stage of this apoptotic process (ca. one-hour), and subsequently accompanied by the dissipation of mitochondrial transmembrane potential (deltapsi (m)) at 3 hours. Further studies indicated that this apoptotic process could effectively be protected by the pretreatment of shikonin-treated cells with glutathione (GSH) and N-acetylcysteine (NAC), a precursor of GSH, but not by cyclosporin A (CyA), an inhibitor of mitochondrial permeability transition (MPT) pore. These data further proved that ROS-mediated oxidative stress was the pivotal element involved in the induction of apoptosis of SK-Hep-1 cells. Taken together, we suggest that shikonin-induced apoptosis of SK-Hep-1 cells proceeds by an oxidative stress-mediated pathway.