RESUMO
BACKGROUND: The search for biomarkers in Parkinson's disease (PD) is crucial to identify the disease early and monitor the effectiveness of neuroprotective therapies. We aim to assess whether a gene signature could be detected in blood from early/mild PD patients that could support the diagnosis of early PD, focusing on genes found particularly altered in the substantia nigra of sporadic PD. RESULTS: The transcriptional expression of seven selected genes was examined in blood samples from 62 early stage PD patients and 64 healthy age-matched controls. Stepwise multivariate logistic regression analysis identified five genes as optimal predictors of PD: p19 S-phase kinase-associated protein 1A (odds ratio [OR] 0.73; 95% confidence interval [CI] 0.60-0.90), huntingtin interacting protein-2 (OR 1.32; CI 1.08-1.61), aldehyde dehydrogenase family 1 subfamily A1 (OR 0.86; 95% CI 0.75-0.99), 19 S proteasomal protein PSMC4 (OR 0.73; 95% CI 0.60-0.89) and heat shock 70-kDa protein 8 (OR 1.39; 95% CI 1.14-1.70). At a 0.5 cut-off the gene panel yielded a sensitivity and specificity in detecting PD of 90.3 and 89.1 respectively and the area under the receiving operating curve (ROC AUC) was 0.96. The performance of the five-gene classifier on the de novo PD individuals alone composing the early PD cohort (n = 38), resulted in a similar ROC with an AUC of 0.95, indicating the stability of the model and also, that patient medication had no significant effect on the predictive probability (PP) of the classifier for PD risk. The predictive ability of the model was validated in an independent cohort of 30 patients at advanced stage of PD, classifying correctly all cases as PD (100% sensitivity). Notably, the nominal average value of the PP for PD (0.95 (SD = 0.09)) in this cohort was higher than that of the early PD group (0.83 (SD = 0.22)), suggesting a potential for the model to assess disease severity. Lastly, the gene panel fully discriminated between PD and Alzheimer's disease (n = 29). CONCLUSIONS: The findings provide evidence on the ability of a five-gene panel to diagnose early/mild PD, with a possible diagnostic value for detection of asymptomatic PD before overt expression of the disorder.
Assuntos
Doença de Parkinson/sangue , Doença de Parkinson/genética , Idoso , Doença de Alzheimer/sangue , Biomarcadores/sangue , Feminino , Expressão Gênica , Marcadores Genéticos , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/diagnóstico , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: To evaluate the intestinal response to the induction of diabetes and to oral insulin (OI) administration in a rat. METHODS: Male Sprague-Dawley rats were divided into four experimental groups: control rats, CONTR-INS rats that were treated with OI given in drinking water for 7 days, diabetic rats that were injected with one dose of streptozotocin, and diabetic rats treated with OI. Intestinal structural changes, enterocyte proliferation and enterocyte apoptosis, bax and bcl-2 mRNA and protein levels, insulin receptor expression and ERK protein levels were determined at sacrifice. A one-way ANOVA for comparison, followed by Tukey's test for pair-wise comparison, were used for statistical analysis. RESULTS: Induction of diabetes resulted in a significant increase in bowel and mucosal weight (P < 0.05), mucosal protein (P < 0.05), villus height and crypt depth in jejunum and ileum (P < 0.05), and mucosal DNA in ileum (P < 0.05) (vs. control animals). Diabetes also enhances ERK-induced cell proliferation (P < 0.05) and concomitant bax/bcl-2 induced cell apoptosis (P < 0.05). Treatment of diabetic rats with OI resulted in a significant decrease in jejunal protein content (P < 0.05), jejunal and ileal villus height (P < 0.05), and jejunal crypt depth (P < 0.05), as well as an inhibition of ERK-related cell proliferation in ileum (P < 0.05). Expression of insulin receptor was down-regulated following OI administration in both control and diabetic animals. CONCLUSIONS: Experimental STZ-induced diabetes causes intestinal mucosal growth and enhances enterocyte turnover in a rat model. OI administration diminishes diabetes-accelerated cell turnover and diabetes-induced mucosal hyperplasia.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Insulina/administração & dosagem , Insulina/uso terapêutico , Mucosa Intestinal/patologia , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Glicemia , Western Blotting , Proteína C-Reativa/metabolismo , Proliferação de Células , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Lipídeos/sangue , Masculino , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Redução de Peso , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
INTRODUCTION: Transforming growth factor beta (TGF-ß) has been shown to affect epithelial cell differentiation and proliferation through epithelial-mesenchymal and epithelial-immune cell interaction. In the present study, we evaluated the effect of TGF-ß2-enriched polymeric diet (Modulen) on enterocyte turnover in a rat model of short bowel syndrome (SBS). METHODS: Male rats were divided into four groups: Sham rats and Sham-TGF-ß rats underwent bowel transection, and were treated with TGF-ß from the 4th postoperative day, SBS rats underwent a 75% bowel resection, and SBS-TGF-ß rats underwent bowel resection and were treated with TGF-ß-enriched diet similar to Group B. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined on day 15. Real-time PCR was used to determine Bax and Bcl-2 mRNA expression. RESULTS: Treatment of SBS animals with TGF-ß2 supplemented diet led to a significant decrease (vs. SBS rats) in bowel weight in ileum (18%, P < 0.05), mucosal DNA content in jejunum (threefold decrease, P < 0.05) and ileum (2.5-fold decrease, P < 0.05), and mucosal protein in jejunum (twofold decrease, P < 0.05) compared to SBS-untreated animals (Group B). Treatment with TGF-ß resulted in a mild decrease in enterocyte proliferation in jejunum (25%, P < 0.05) and ileum (18%, P < 0.05). A decreased cell apoptosis in the SBS-TGF-ß group was accompanied by a decreased Bax and increased Bcl-2 mRNA expression. CONCLUSIONS: In a rat model of SBS, dietary TGF-ß inhibits intestinal adaptation. Decreased enterocyte proliferation is responsible for this effect.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Suplementos Nutricionais , Intestino Delgado/cirurgia , Apoio Nutricional , Síndrome do Intestino Curto/dietoterapia , Fator de Crescimento Transformador beta/administração & dosagem , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Procedimentos Cirúrgicos do Sistema Digestório/efeitos adversos , Modelos Animais de Doenças , Enterócitos/patologia , Intestino Delgado/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Síndrome do Intestino Curto/metabolismo , Síndrome do Intestino Curto/patologiaRESUMO
BACKGROUND: The purpose of the present study was to evaluate the effects of ω-3 fatty acids (Omegaven) on early intestinal adaptation in rats with short bowel syndrome (SBS). METHODS: Male Sprague-Dawley rats were randomly assigned to 1 of 4 groups: sham rats underwent bowel transection; SBS rats underwent 75% bowel resection; SBS-O ω-3 rats underwent bowel resection and were treated with oral Omegaven given by gavage; and SBS-I ω-3 rats underwent bowel resection and were treated with Omegaven given intraperitoneally. Rats were killed on day 14. Parameters of intestinal adaptation (bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depths, cell proliferation and apoptosis) were determined at time of death. Real-time polymerase chain reaction was used to determine the level of Bax and Bcl-2 messenger RNA (mRNA). Statistical analysis was performed using Kruskal-Wallis test followed by post hoc test, with P < .05 considered statistically significant. RESULTS: Oral ω-3 supplementation did not significantly change intestinal regrowth. In contrast, parenteral ω-3 in rats that underwent resection resulted in higher bowel and mucosal weights, mucosal DNA and protein in ileum, villus height in ileum, crypt depth in jejunum and ileum, and greater rates of cell proliferation in jejunum and ileum compared with SBS animals. The initial decreased levels of apoptosis corresponded with the early decrease in Bax and increase in Bcl-2 mRNA levels. CONCLUSIONS: Parenteral but not enteral Omegaven augments and accelerates structural bowel adaptation in a rat model of SBS. Increased cell proliferation and decreased apoptosis reflect increased cell turnover in Omegaven-treated animals.
Assuntos
Ácidos Graxos Ômega-3/uso terapêutico , Intestino Delgado/efeitos dos fármacos , Nutrição Parenteral , Síndrome do Intestino Curto/terapia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Suplementos Nutricionais , Procedimentos Cirúrgicos do Sistema Digestório , Nutrição Enteral , Ácidos Graxos Ômega-3/administração & dosagem , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Mucosa Intestinal/cirurgia , Intestino Delgado/fisiopatologia , Intestino Delgado/cirurgia , Masculino , Tamanho do Órgão , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome do Intestino Curto/patologia , Síndrome do Intestino Curto/cirurgiaRESUMO
BACKGROUND: Pleiotropic (lipid lowering-independent) effects of statins are attributed to their antiinflammatory, antioxidant, and/or vascular actions. Extensive studies in various experimental models have established that pretreatment with simvastatin significantly protects heart and kidney injured by ischemia-reperfusion (IR). The purpose of the present study was to examine the effect of simvastatin on intestinal recovery and enterocyte turnover after intestinal IR injury in rats. METHODS: Male Sprague-Dawley rats were divided into three experimental groups: (1) sham rats underwent laparotomy, (2) IR-rats underwent occlusion of both superior mesenteric artery and portal vein for 30 min followed by 48 h of reperfusion, and (3) IR-SIM rats underwent IR and were treated with oral simvastatin (10 mg/kg) given by gavage immediately before and 24 h after operation. Intestinal structural changes, Park's injury score, enterocyte proliferation and enterocyte apoptosis were determined 24 h following IR. A non-parametric Kruskal-Wallis ANOVA test was used for statistical analysis with P less than 0.05 considered statistically significant. RESULTS: Treatment with simvastatin resulted in a significant increase in bowel and mucosal weight in ileum, villus height and crypt depth in jejunum and ileum compared to IR animals. IR-SIM rats had also a significantly lower intestinal injury score as well as lower apoptotic index in jejunum and ileum compared to IR animals. CONCLUSIONS: Treatment with simvastatin prevents gut mucosal damage and inhibits programmed cell death following intestinal IR in a rat.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Enteropatias/tratamento farmacológico , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Sinvastatina/administração & dosagem , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Enterócitos/efeitos dos fármacos , Enterócitos/patologia , Enteropatias/patologia , Enteropatias/fisiopatologia , Intestinos/irrigação sanguínea , Intestinos/efeitos dos fármacos , Intestinos/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Resultado do TratamentoRESUMO
PURPOSE: Among factors promoting intestinal adaptation after bowel resection, dietary fatty acids have a special role. The purpose of the present study was to evaluate the effects of palmitic acid (PA) on early intestinal adaptation in rats with short bowel syndrome (SBS). MATERIALS: Male Sprague-Dawley rats underwent either a bowel transection with re-anastomosis (sham rats) or 75% small bowel resection (SBS rats). Animals were randomly assigned to one of four groups: sham rats fed normal chow (sham-NC); SBS rats fed NC (SBS-NC), SBS rats fed high palmitic acid diet (SBS-HPA), and SBS rats fed low palmitic acid diet (SBS-LPA). Rats were sacrificed on day 14. Parameters of intestinal adaptation, overall bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depth, cell proliferation and apoptosis were determined at sacrifice. RT-PCR and Western blotting were used to determine the level of bax and bcl-2 mRNA and protein (parameters of apoptosis), and ERK protein levels (parameter of proliferation). Statistical analysis was performed using Kruskal-Wallis test followed by post hoc test for multiple comparisons with P values of less than 0.05 considered statistically significant. RESULTS: SBS-HFD rats demonstrated higher bowel and mucosal weight, mucosal DNA and protein in ileum, while deprivation of PA (SBS-LPA) inhibited intestinal re-growth both in jejunum and ileum compared to SBS-NC rats. A significant up-regulation of ERK protein coincided with increased cell proliferation in SBS-HFD rats (vs. SBS-NC). Also, the initial decreased levels of apoptosis corresponded with the early decrease in bax and increase in bcl-2 at both mRNA and protein levels. CONCLUSION: Early exposure to HPA both augments and accelerates structural bowel adaptation in a rat model of SBS. Increased cell proliferation and decreased cell apoptosis may be responsible for this effect. Deprivation of PA in the diet inhibits intestinal re-growth.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Intestino Delgado/efeitos dos fármacos , Ácido Palmítico/farmacologia , Síndrome do Intestino Curto/tratamento farmacológico , Adaptação Fisiológica , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dieta , Modelos Animais de Doenças , Enterócitos/efeitos dos fármacos , Enterócitos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Intestino Delgado/fisiologia , Masculino , Ácido Palmítico/administração & dosagem , Ratos , Ratos Sprague-Dawley , Síndrome do Intestino Curto/etiologiaRESUMO
PURPOSE: Recent evidence suggests that transforming growth factor-alpha (TGF-alpha) enhances enterocyte proliferation and exerts a gut trophic effect. The purpose of the present study was to evaluate the effect of TGF-alpha on enterocyte proliferation and intestinal recovery following methotrexate (MTX)-induced intestinal mucositis in rats and in Caco-2 cells. METHODS: Nonpretreated Caco-2 cells and those pretreated with MTX were incubated with increasing concentrations of TGF-alpha. Cell proliferation was determined by FACS cytometry. Adult rats were divided into three groups: control rats treated with vehicle, MTX rats treated with one dose (20 microg/kg) of MTX given intraperitoneally, and MTX-TGF-alpha rats treated with one dose of MTX followed by two doses of TGF-alpha (75 microg/kg a day). Three days after MTX injection, rats were sacrificed. Intestinal mucosal damage (Park's score), mucosal structural changes, and enterocyte proliferation were measured at sacrifice. Western blotting was used to determine the level of extracellular signal-related kinase (ERK) protein, a marker of cell proliferation. A nonparametric Kruskal-Wallis ANOVA test was used for statistical analysis with P value less than 0.05 considered statistically significant. RESULTS: The in vitro experiment demonstrated that treatment with TGF-alpha of Caco-2 cells resulted in a significant stimulation of cell proliferation in a dose-dependent manner. The in vivo experiment showed that treatment with TGF-alpha resulted in a significant increase in bowel and mucosal weight, DNA and protein content in jejunum and ileum, villus height in jejunum and ileum, crypt depth in ileum, and increased cell proliferation in jejunum and ileum compared to the MTX group. MTX-TGF-alpha rats also had a significantly lower intestinal injury score in ileum when compared to MTX animals. The increase in levels of cell proliferation in MTX-TGF-alpha rats corresponded with the increase in ERK protein levels in intestinal mucosa. CONCLUSION: Treatment with TGF-alpha prevents mucosal injury, enhances ERK-induced enterocyte proliferation, and improves intestinal recovery following MTX-induced intestinal mucositis in rats. These findings correlated with the observation that TGF-alpha also caused a significant stimulation of cell proliferation in a Caco-2 cell culture model treated with MTX. These observations may have significant implications for the treatment of patients on chemotherapy who develop severe mucositis.
Assuntos
Enterócitos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Mucosite/tratamento farmacológico , Fator de Crescimento Transformador alfa/farmacologia , Animais , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Enterócitos/fisiologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Masculino , Metotrexato , Mucosite/induzido quimicamente , Noxas , Ratos , Ratos Sprague-Dawley , Recuperação de Função FisiológicaRESUMO
The purpose of the present study was to evaluate the effect of transforming-growth factor-alpha (TGF-alpha) on enterocyte apoptosis following methotrexate (MTX) induced intestinal mucositis in a rat and in Caco-2 cells. Non-pretreated and pretreated with MTX Caco-2 cells were incubated with increasing concentrations of TGF-alpha. Cell apoptosis was determined by FACS cytometry. Adult rats were divided into four groups: Control, Control-TGF-alpha, MTX, and MTX- TGF-alpha rats. Three days later rats were sacrificed. Enterocyte apoptosis were measured at sacrifice. RT-PCR and Western Blotting was used to determine the level of Bax and Bcl-2 mRNA and protein. Real time PCR was used to measure epidermal growth factor receptor (EGFr) expression along the villus-crypt axis. The in vitro experiment has shown that treatment with TGF-alpha of Caco-2 cells results in a significant inhibition of cell apoptosis in a dose-dependent manner. In vivo experiment, a decreased levels of apoptosis in MTX- TGF-alpha rats corresponded with the decrease in Bax and with the increase in Bcl-2 at both mRNA and protein levels. The inhibiting effect of TGF-alpha on enterocyte apoptosis was strongly correlated with EGFr expression along the villus-crypt axis. In conclusion, treatment with TGF-alpha inhibits enterocyte apoptosis following MTX- injury in the rat.
Assuntos
Apoptose , Enterócitos/patologia , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , Mucosa Intestinal/patologia , Metotrexato/farmacologia , Fator de Crescimento Transformador alfa/metabolismo , Animais , Células CACO-2 , Enterócitos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína X Associada a bcl-2/metabolismoRESUMO
A central event of systemic inflammation and septic organ injury is infiltration of tissues with polymorphonuclear neutrophils, likely modulated by the integrity of the extracellular matrix underlying the vascular endothelium. In the present study, the effect of matrix-modifying endoglycosidase (heparanase) on endotoxin (LPS)-induced inflammatory lung injury was investigated in rats. Animals were treated with heparanase or LPS or pretreated with heparanase before LPS injection, and acute lung injury was verified histologically and characterized by analysis of bronchoalveolar lavage fluids. Pretreatment with heparanase attenuated the mortality of animals and preserved the histological structure of the lungs. Furthermore, polymorphonuclear neutrophil accumulation and activation, analyzed by myeloperoxidase release and reactive oxygen species production associated with lung injury, were significantly reduced upon heparanase pretreatment. In addition, heparanase pretreatment elevated the IL-10 levels in the pulmonary compartment. Moreover, results from in vitro experiments have identified monocyte-derived IL-10 as an important mediator used by heparanase to suppress inflammatory reactions. The protective effect of heparanase may indicate a novel therapeutic strategy for sepsis.
Assuntos
Endotoxinas/toxicidade , Glucuronidase/uso terapêutico , Pulmão/patologia , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/tratamento farmacológico , Animais , Linhagem Celular , Sinergismo Farmacológico , Humanos , Lipopolissacarídeos/toxicidade , Pulmão/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Doxorubicin (DOX) is a widely used chemotherapeutic drug for human hepatocellular carcinoma (HCC). A major limitation to its effectiveness is the development of multidrug resistance of cancer cells. In clinical trials, patients with advanced HCC were treated with high-dose acetaminophen (HAAP) in an effort to improve the antitumor activity of chemotherapeutics. In this study, we investigated the effect of concomitant treatment of DOX and HAAP on hepatoma-derived HepG2 cells. Viability, cell cycle distribution, and ultrastructure were examined. Unexpectedly, HAAP, when added to DOX-exposed cells, increased cell viability, released cell cycle arrest, and decreased apoptosis. To elucidate the mechanisms by which HAAP reduces the DOX lethal effect to HepG2 cells, we investigated the multidrug resistance P-glycoprotein (P-gp) and p44/42-mitogen-activated protein kinase (MAPK) pathways. The P-gp function was enhanced by DOX and HAAP, and it was further stimulated during combined treatment, leading to decreased DOX retention. Verapamil (VRP), when added to DOX + HAAP exposure, increased DOX accumulation and restored DOX-induced toxicity. The increased phospho-p44/42-MAPK level in DOX-exposed cells was inhibited by HAAP. In addition, suppression of p44/42 activation by the p44/42-MAPK inhibitor 2'-amino-3'-methoxyflavone (PD98059) blocked DOX-induced apoptosis. These findings suggest that the antagonistic effect of concomitant DOX + HAAP treatment occurs as a result of interactive stimulation of P-gp, generating decreased intracellular drug concentrations. Furthermore, inhibition of the p44/42-MAPK phosphorylation by HAAP could abolish the DOX-induced cell death pathway. Thus, combined treatment by DOX + HAAP, intended to improve chemotherapeutic efficacy, could have an opposite effect facilitating cancer cell survival.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Acetaminofen/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Carcinoma Hepatocelular/patologia , Morte Celular , Linhagem Celular Tumoral , Interações Medicamentosas , Humanos , Sistema de Sinalização das MAP QuinasesRESUMO
Acetaminophen overdose causes severe hepatic failure. Although the mechanisms of acetaminophen hepatotoxicity have been well investigated, little is known about the involvement of the P-glycoprotein in acetaminophen transport and toxicity. P-Glycoprotein is a membrane efflux pump, playing a significant role in regulating absorption, excretion, and tissue distribution of many drugs. To evaluate the contribution of P-glycoprotein transporter in the course of acetaminophen-induced toxicity, HepG2 and Hep3B cells with different P-glycoprotein expression and activity, were treated by acetaminophen (1-10 mM) for different time periods, with or without the P-glycoprotein inhibitor verapamil. P-Glycoprotein activity was determined by rhodamine 123 efflux assay and western blot analysis. To assess the acetaminophen-induced toxicity and effect of verapamil, we investigated cellular redox status, phosphatidylserine externalization, nuclear fragmentation and ultrastructural changes. Verapamil markedly enhanced acetaminophen-induced oxidative damage and cell death. Moreover, verapamil revealed acetaminophen toxicity even at subtoxic levels. High acetaminophen concentrations increased P-glycoprotein activity and content in both HepG2 and Hep3B cells. These observations suggest the involvement of P-glycoprotein in acetaminophen transport. Notwithstanding the differences of the investigated hepatoma cell lines in P-glycoprotein function, acetaminophen-induced toxicity was similar, possibly due to different functions of drug-metabolizing systems. We conclude that acetaminophen is a P-glycoprotein substrate and P-glycoprotein is involved in acetaminophen transport and toxicity in HepG2 and Hep3B cells. This study establishes the fact that acetaminophen can modulate P-glycoprotein in tumour cells, suggesting that its routine use in cancer patients in combination with anticancer drugs, may influence the result of chemotherapy.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Acetaminofen/toxicidade , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Resistência a Medicamentos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Acetaminofen/metabolismo , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica , Humanos , Estresse Oxidativo/efeitos dos fármacos , Verapamil/farmacologiaRESUMO
Heparanase is an endo-beta-D-glucuronidase involved in cleavage of heparan sulfate moieties and hence participates in extracellular matrix (ECM) degradation and remodeling. Traditionally, heparanase activity was correlated with the metastatic potential of a variety of tumor-derived cell types. Cloning of the heparanase gene indicated that heparanase expression is up-regulated in a variety of primary human tumors. In some cases, heparanase up-regulation correlated with increased tumor vascularity, an angiogenic feature that could be recapitulated in a number of in vitro and in vivo models. The mechanism by which heparanase enhances angiogenic responses is not entirely clear but is thought to be mediated primarily by release of ECM-resident angiogenic growth factors such as basic fibroblast growth factor and vascular endothelial growth factor (VEGF). Here, we examined the possibility that heparanase directly participates in VEGF gene regulation. We provide evidence that heparanase overexpression in human embryonic kidney 293, MDA-MB-435 human breast carcinoma, and rat C6 glioma cells resulted in a 3- to 6-fold increase in VEGF protein and mRNA levels, which correlated with elevation of p38 phosphorylation. Moreover, heparanase down-regulation in B16 mouse melanoma cells by a specific siRNA vector was accompanied by a decrease in VEGF and p38 phosphorylation levels, suggesting that VEGF gene expression is regulated by endogenous heparanase. Interestingly, a specific p38 inhibitor did not attenuate VEGF up-regulation by heparanase whereas Src inhibitors completely abrogated this effect. These results indicate, for the first time, that heparanase is actively involved in the regulation of VEGF gene expression, mediated by activation of Src family members.
Assuntos
Glucuronidase/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Glioma/enzimologia , Glioma/genética , Glioma/metabolismo , Glucuronidase/biossíntese , Glucuronidase/genética , Humanos , Neovascularização Patológica/enzimologia , Fosforilação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Transfecção , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.
Assuntos
Glucuronidase/metabolismo , Proteoglicanas de Heparan Sulfato/fisiologia , Heparina/farmacologia , Heparitina Sulfato/farmacologia , Animais , Transporte Biológico , Neoplasias da Mama , Células CHO , Linhagem Celular Tumoral , Cricetinae , Glioma , Proteoglicanas de Heparan Sulfato/farmacologia , Humanos , Cinética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Heparanase is a heparan sulfate degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Heparanase is synthesized as a 65 kDa non-active precursor that subsequently undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. The protease responsible for heparanase processing is currently unknown, as is the sub-cellular processing site. In this study, we characterize an antibody (733) that preferentially recognizes the active 50 kDa heparanase form as compared to the non-active 65 kDa heparanase precursor. We have utilized this and other anti-heparanase antibodies to study the cellular localization of the latent 65 kDa and active 50 kDa heparanase forms during uptake and processing of exogenously added heparanase. Interestingly, not only the processed 50 kDa, but also the 65 kDa heparanase precursor was localized to perinuclear vesicles, suggesting that heparanase processing occurs in lysosomes. Indeed, heparanase processing was completely inhibited by chloroquine and bafilomycin A1, inhibitors of lysosome proteases. Similarly, processing of membrane-targeted heparanase was also chloroquine-sensitive, further ruling out the plasma membrane as the heparanase processing site. Finally, we provide evidence that antibody 733 partially neutralizes the enzymatic activity of heparanase, suggesting that the N-terminal region of the molecule is involved in assuming an active conformation. Monoclonal antibodies directed to this region are likely to provide specific heparanase inhibitors and hence assist in resolving heparanase functions under normal and pathological conditions.
Assuntos
Glucuronidase/metabolismo , Lisossomos/enzimologia , Processamento de Proteína Pós-Traducional , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Células CHO , Linhagem Celular Tumoral , Cloroquina/farmacologia , Cricetinae , Ativação Enzimática/efeitos dos fármacos , Glucuronidase/antagonistas & inibidores , Glucuronidase/química , Glucuronidase/genética , Humanos , Hidrólise/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Inibidores de Proteases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico , TransfecçãoRESUMO
Heparanase is an endo-beta-glucuronidase responsible for the cleavage of heparan sulfate, participating in extracellular matrix degradation and remodeling. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase up-regulation was detected in essentially all human tumors examined, correlating, in some cases, with poor postoperative survival and increased tumor vascularity. The role of heparanase in primary tumor progression is, however, poorly understood. Here, we overexpressed the human heparanase gene in a human glioma cell line, U87. We found that heparanase overexpression induces cell invasion, as might be expected. Surprisingly, elevated heparanase expression levels correlated with decreased proliferation rates and increased cell spreading and formation of a tight monolayer rather than large cell aggregates. This phenotypic appearance was accompanied by beta1-integrin activation, FAK and Akt phosphorylation, and Rac activation. In a xenograft tumor model, relatively moderate heparanase expression levels significantly enhanced tumor development and tumor vascularity, whereas high heparanase expression levels inhibited tumor growth. These results indicate that heparanase activates signal transduction pathways and, depending on its expression levels, may modulate tumor progression.
Assuntos
Glioma/enzimologia , Glioma/patologia , Glucuronidase/fisiologia , Animais , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Galinhas , Glioma/genética , Glucuronidase/biossíntese , Glucuronidase/genética , Humanos , Integrina beta1/metabolismo , Integrina beta1/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Transfecção , Transplante HeterólogoRESUMO
Estrogen has a protective effect in adverse circulatory conditions such as trauma-hemorrhage and ischemia-reperfusion. In the present investigation uncontrolled hemorrhagic shock induced by massive splenic injury (MSI) was studied in male and female rats during the proestrus and metestrus phase of the reproductive cycle. The animals were anesthetized and randomly divided into four groups: group 1 (n = 8) were sham operated female animals; in group 2 (n = 8) MSI was induced in male animals; in group 3 (n = 8) MSI was induced in female animals in proestrus; in group 4 (n = 8) MSI was induced in female animals in metestrus. Plasma 17beta-estradiol was 112.8 +/- 16.7 pg/mL in group 3 and 44.8 +/- 16.7 pg/mL in group 4 (P < 0.04). After MSI the mean arterial pressure dropped in group 2 from 135.6 +/- 3.9 to 47.6 +/- 8.8 mmHg (P < 0.001) in 60 min. Total blood loss in 4 h was 29.6 +/- 3.5% of blood volume and mean survival time 161.3 +/- 30.6 min. In group 3 total blood loss in 4 h was 24.4 +/- 5.7% of blood volume and mean survival time 240 +/- 0 min (P < 0.03 compared with group 2). In group 4, total blood loss in 4 h was 29.6 +/- 3.9% of blood volume and mean survival time was 112.3 +/- 28.7 min (P < 0.001 compared with group 3). These results indicate that female rats in the proestrus phase of the reproductive cycle better maintain hemodynamic stability and survival in uncontrolled hemorrhagic shock than female animals in metestrus or males. This difference is attributed to factors other than blood loss.
Assuntos
Ciclo Estral/sangue , Proestro/sangue , Choque Hemorrágico/mortalidade , Ruptura Esplênica/complicações , Animais , Pressão Sanguínea , Estradiol/sangue , Feminino , Frequência Cardíaca , Hematócrito , Ácido Láctico/sangue , Masculino , Metestro/sangue , Hemorragia Pós-Operatória , Ratos , Ratos Sprague-Dawley , Choque Hemorrágico/etiologia , Taxa de SobrevidaRESUMO
We have previously observed that bolus fluid resuscitation in uncontrolled hemorrhagic shock induced by solid organ injury leads to increased blood loss and mortality. In the present investigation, we studied the effect of continuous fluid resuscitation on the hemodynamic response and survival following massive splenic injury (MSI) in rats. The animals were randomized into 11 groups: group 1, sham-operated; group 2, MSI untreated; group 3, MSI treated with 17.5 mL/kg/h of Ringers lactate (RL) solution (RL-17.5); group 4, MSI treated with 35 mL/kg/h RL (RL-35); group 5, MSI treated with 70 mL/kg/h RL (RL-70); group 6, MSI treated with 7.5 mL/kg/h of 7.5% NaCl (HTS-7.5); group 7, MSI treated with 15 mL/kg/h of 7.5% NaCl (HTS-15); group S, MSI treated with 30 mL/kg/h of 7.5% NaCl (HTS-30); group 9, MSI treated with 7.5 mL/kg/h 6% hydroxyethyl starch (HES-7.5); group 10, MSI treated with 15 mL/kg/h 6% hydroxyethyl starch (HES-15); and group 11, MSI treated with 30 mL/kg/h 6% hydroxyethyl starch (HES-30). MSI in untreated group 2 was followed by a fall of mean arterial pressure (MAP) to 50.1 +/- 6.7 mmHg (P < 0.001) in 15 min. Mean survival time (MST) was 99.5 +/- 16.6 min, and total blood loss (TBL) was 37.8% +/- 2.6% of blood volume. Fluid treatment with increasing volumes of RL in groups 3, 4, and 5 was followed by a gradual increase in TBL compared with untreated animals, and MST remained unchanged. Increasing volumes of HTS infusion in groups 6, 7, and 8 was also followed by incease in TBL, but MST remained unchanged except for an increase to 123.0 +/- 20.5 min (P < 0.05) in group 6. Increasing volumes of HES in groups 9, 10, and 11 was also followed by increase in TBL, but MST remained unchanged. In conclusion, continuous infusion of LR, HTS, and HES following massive splenic injury resulted in a significant increase in intra-abdominal bleeding, but survival time in the first hour following injury remained unchanged in contrast to bolus fluid infusion, which increases early mortality.
Assuntos
Hidratação/métodos , Choque Hemorrágico/complicações , Choque Hemorrágico/terapia , Baço/lesões , Animais , Modelos Animais de Doenças , Hemodinâmica , Soluções Isotônicas/uso terapêutico , Masculino , Substitutos do Plasma/uso terapêutico , Ratos , Ratos Sprague-Dawley , Lactato de Ringer , Choque Hemorrágico/patologia , Baço/patologia , Análise de SobrevidaRESUMO
OBJECTIVE: To examine the effect of hypertonic saline on bacterial translocation and the number and function of natural killer and T cells in controlled hemorrhagic shock. DESIGN: Randomized controlled study. Duration of follow-up was 24 hrs. SETTING: University research laboratory. SUBJECTS: Adult male Sprague-Dawley rats, weighing 310-390 g. INTERVENTIONS: Controlled hemorrhagic shock was induced by blood withdrawal (mean arterial pressure, 30-40 mm Hg) and maintained for 30 mins. The animals were randomly divided into three groups: group 1 (n = 10) was sham-operated, group 2 (n = 10) was untreated, and group 3 was treated with 5 mL/kg hypertonic saline (n = 10). The rats were killed after 24 hrs. MEASUREMENTS AND MAIN RESULTS: Infusion of hypertonic saline in group 3 was followed by reduced bacterial translocation rate (5.0 +/- 2.2% vs. 18.3 +/- 5.3%, p <.033). The total mass of bacteria isolated from hypertonic saline-treated animals with bacterial translocation was 7.8- to 10.4-fold less than that from untreated rats. Controlled hemorrhagic shock resulted in a low percentage of CD4+ cells in blood (35.2 +/- 3.9%, p <.05) and lymph nodes (44.4 +/- 4.5%, p <.05) and depressed CD4 expression on blood (82 +/- 13 arbitrary units, p <.005) and lymph node (168 +/- 24 arbitrary units, p <.03) cells. A compensatory mobilization of NKR-P1+ cells from lymph nodes (8.6 +/- 2.3%, p <.05) to blood (21.2 +/- 5.2%, p <.01) with down-regulated NKR-P1 expression on blood cells (59 +/- 10 arbitrary units, p <.005) was observed. Natural killer cell-mediated cytotoxicity was decreased (67.9 +/- 9.7%, p <.05). Hypertonic saline treatment greatly stimulated CD4 expression on blood (419 +/- 113 arbitrary units, p <.005) and lymph node (553 +/- 115 arbitrary units, p <.03) cells. Also, normalization of NKR-P1 expression (160 +/- 19 arbitrary units, p <.005) and restoration of natural killer cell-mediated cytotoxicity to near normal values (88.6 +/- 7.4%, p <.05) were demonstrated. CONCLUSION: Controlled hemorrhagic shock was accompanied by CD4+ cells suppression and excessive recruitment of natural killer cells with abnormally low NKR-P1 expression and suppressed cytolytic activity into circulation. Infusion of hypertonic saline reversed these changes and reduced bacterial translocation.
Assuntos
Translocação Bacteriana/fisiologia , Células Matadoras Naturais/imunologia , Solução Salina Hipertônica/uso terapêutico , Choque Hemorrágico/tratamento farmacológico , Choque Hemorrágico/imunologia , Linfócitos T/imunologia , Animais , Contagem de Linfócito CD4 , Linfonodos , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Choque Hemorrágico/microbiologiaRESUMO
BACKGROUND: Using a standardized massive splenic injury (MSI) model of uncontrolled hemorrhagic shock, we studied the effect of continuous fluid resuscitation and splenectomy on the hemodynamic response and survival in rats. METHODS: The animals were randomized into seven groups: group 1 (n = 8), sham-operated; group 2 (n = 8), MSI untreated; group 3 (n = 8), MSI treated with 7.5 mL/kg/h of 7.5% NaCl (hypertonic saline [HTS]) for 1 hour; group 4 (n = 8), MSI treated with 7.5 mL/kg/h hydroxyethyl starch (HES-7.5) for 1 hour; group 5 (n = 8) MSI treated with 35 mL/kg/h Ringer's lactate (RL) solution (RL-35) for 1 hour; group 6 (n = 8) MSI treated with 70 mL/kg/h RL for 1 hour (RL-70); and group 7 (n = 8), MSI treated with 105 mL/kg/h RL for 1 hour (RL-105). In all MSI groups, splenectomy was performed 45 minutes after injury. RESULTS: MSI in untreated group 2 resulted in a fall of mean arterial pressure from 105.9 +/- 10.7 mm Hg to 27.0 +/- 6.7 mm Hg (p < 0.001) in 60 minutes. Mean survival time after splenectomy in this group was 160.7 +/- 29.7 minutes, and total blood loss was 34.8 +/- 4.7% of blood volume. Continuous HTS infusion with splenectomy in group 3 was followed by a total blood loss of 38.7 +/- 4.4% and mean survival time was 176.5 +/- 23.2 minutes. HES-7.5 infusion and splenectomy was followed by a total blood loss of 39.6 +/- 2.5% and survival time was 171.6 +/- 19.5 minutes. Continuous infusion of increasing volumes of RL in groups 5, 6, and 7 was followed by increase in blood loss to 29.0 +/- 4.1%, 50.2 +/- 3.1% (p < 0.001), and 62.7 +/- 7.1% (p < 0.002) of total blood volume, respectively. Mean survival time in groups 5, 6, and 7 was 233.5 +/- 6.5 minutes (p < 0.04), 207.6 +/- 17.0 minutes (p < 0.05), and 158 +/- 26 minutes, respectively. CONCLUSION: Continuous infusion of large-volume RL and splenectomy after massive splenic injury resulted in a significant increase in intra-abdominal bleeding and shortened survival time compared with small-volume RL infusion.