Indução de morte celular em células de adenocarcinoma cervical humano (HeLa) pela lectina da esponja Cinachyrella apion (CaL)

Cancer is a term used to represent a set of more than 100 diseases, including malignant tumors from different locations. The malignancies are the second leading cause of death in the population, representing approximately 17% of deaths of known cause. Strategies that induce differentiation have had limited success in the treatment of established cancers. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated due to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death via apoptosis in tumor cells. The antiproliferative activity of CaL was tested against cell lines, with the highest inhibition of tumor growth for HeLa, reducing cell growth at a dose dependent manner, with a concentration of 10 μg/mL. The hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. The results showed the lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase, with accumulation of cells of approximately 57% in this phase, and acting as both dependent and/or independent of caspases pathway. These results suggest that CaL has the potential to be used as drug treatment against cancer.

A Catalytically Inactive Lys49 PLA2 Isoform from Bothrops jararacussu venom that Stimulates Insulin Secretion in Pancreatic Beta Cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A soy-based product fermented by Enterococcus faecium and Lactobacillus helveticus inhibits the development of murine breast adenocarcinoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on Ca-45 efflux, cytoplasmic Ca2+ concentration [Ca2+](i), and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the Ca-45 efflux from isolated islets in a dose-dependant manner. Tetracaine did not affect the increase in Ca-45 efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+](i) in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 mu M D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 mu M thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in Ca-45 efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.

Dexamethasone-induced insulin resistance is associated with increased connexin 36 mRNA and protein expression in pancreatic rat islets

Augmented glucose-stimulated insulin secretion (GSIS) is an adaptive mechanism exhibited by pancreatic islets from insulin-resistant animal models. Gap junction proteins have been proposed to contribute to islet function. As such, we investigated the expression of connexin 36 (Cx36), connexin 43 (Cx43), and the glucose transporter Glut2 at mRNA and protein levels in pancreatic islets of dexamethasone (DEX)-induced insulin-resistant rats. Study rats received daily injections of DEX (1 mg/kg body mass, i.p.) for 5 days, whereas control rats (CTL) received saline solution. DEX rats exhibited peripheral insulin resistance, as indicated by the significant postabsorptive insulin levels and by the constant rate for glucose disappearance (K-ITT). GSIS was significantly higher in DEX islets (1.8-fold in 16.7 mmol/L glucose vs. CTL, p < 0.05). A significant increase of 2.25-fold in islet area was observed in DEX vs. CTL islets (p < 0.05). Cx36 mRNA expression was significantly augmented, Cx43 diminished, and Glut2 mRNA was unaltered in islets of DEX vs. CTL (p < 0.05). Cx36 protein expression was 1.6-fold higher than that of CTL islets (p < 0.05). Glut2 protein expression was unaltered and Cx43 was not detected at the protein level. We conclude that DEX-induced insulin resistance is accompanied by increased GSIS and this may be associated with increase of Cx36 protein expression.

Low protein diet confers resistance to the inhibitory effects of interleukin1 beta on insulin secretion in pancreatic islets

High protein content in the diet during childhood and adolescence has been associated to the onset insulin-dependent diabetes mellitus. We investigated the effect of interleukin-1 beta (IL-I beta) on insulin secretion, glucose metabolism, and nitrite formation by islets isolated from rats fed with normal protein (NP, 17%) or low protein (LP, 6%) after weaning. Pretreatment of islets with IL-1 beta for 1 h or 34 h inhibited the insulin secretion induced by glucose in both groups, but it was less marked in LP than in NP group. Islets from LP rats exhibited a decreased IL-1 beta -induced nitric oxide (NO) production, lower inhibition of D-[(UC)-C-14]-glucose oxidation to (CO2)-C-14, and less pronounced effect of IL-1 beta on alpha -ketoisocaproic acid-induced insulin secretion than NP islets. However, when the islets were stimulated by high concentrations of K+ the inhibitory effect of IL-1 beta on insulin secretion was not different between groups. In conclusion, protein restriction protects beta -cells of the deleterious effect of IL-1 beta, apparently, by decreasing NO production. The lower NO generation in islets from protein deprived rats may be due to increased free fatty acids oxidation and consequent alteration in Ca2+ homeostasis. (C) 2001 Elsevier B.V. All rights reserved.

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dexamethasone treatment in vivo counteracts the functional pancreatic islet alterations caused by malnourishment in rats

The effects of dexamethasone (Dex) on the metabolic parameters, peripheral insulin, and glucose sensitivity in vivo as well as on islet function ex vivo of rats submitted to low-protein diet were analyzed. Dexamethasone (1.0 mg/kg body weight) was administered intraperitoneally daily to adult Wistar rats fed on a normal-protein diet or low-protein diet (LPD) for 5 days, whereas control rats fed on a normal-protein diet or low-protein diet (LP) received saline alone. At the end of the experimental period, LP rats showed a significant reduction in serum insulin, total serum protein, and serum albumin levels compared with rats fed on a normal-protein diet (P < .05). All these parameters tended to be normalized in LPD rats (P < .05); furthermore, these rats exhibited increased serum glucose and nonesterified fatty acid levels compared with LP rats (P < .05). Rats submitted to the low-protein diet demonstrated normal peripheral glucose sensitivity and improved peripheral insulin sensitivity, which was reversed by Dex treatment. A reduced area of islets from LP rats was partially recovered in LPD rats (P < .05). At 16.7 mmol/L glucose, insulin secretion from LPD islets was also partially recovered and was significantly higher than that from LP islets (P < .05). In conclusion, induction of insulin resistance by Dex treatment reverses most of the metabolic alterations in rats submitted to a low-protein diet. In addition, several islet functions were also improved by Dex, confirming the plasticity of pancreatic islets in adverse conditions. (C) 2008 Elsevier B.V. All rights reserved.

Insulin signaling proteins in pancreatic islets of insulin-resistant rats induced by glucocorticoid

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Increased pancreatic islet mass is accompanied by activation of the insulin receptor substrate-2/serine-threonine kinase pathway and augmented cyclin D(2) protein levels in insulin-resistant rats

It is well known that glucocorticoids induce peripheral insulin resistance in rodents and humans. Here, we investigated the structural and ultrastructural modifications, as well as the proteins involved in beta-cell function and proliferation, in islets from insulin-resistant rats. Adult male Wistar rats were made insulin resistant by daily administration of dexamethasone (DEX; 1mg/kg, i.p.) for five consecutive days, whilst control (CTL) rats received saline alone. Structure analyses showed a marked hypertrophy of DEX islets with an increase of 1.7-fold in islet mass and of 1.6-fold in islet density compared with CTL islets (P < 0.05). Ultrastructural evaluation of islets revealed an increased amount of secreting organelles, such as endoplasmic reticulum and Golgi apparatus in DEX islets. Mitotic figures were observed in DEX islets at structural and ultrastructural levels. Beta-cell proliferation, evaluated at the immunohistochemical level using anti-PCNA (proliferating cell nuclear antigen), showed an increase in pancreatic beta-cell proliferation of 6.4-fold in DEX islets compared with CTL islets (P < 0.0001). Increases in insulin receptor substrate-2 (IRS-2), phosphorylated-serine-threonine kinase AKT (p-AKT), cyclin D(2) and a decrease in retinoblastoma protein (pRb) levels were observed in DEX islets compared with CTL islets (P < 0.05). Therefore, during the development of insulin resistance, the endocrine pancreas adapts itself increasing beta-cell mass and proliferation, resulting in an amelioration of the functions. The potential mechanisms that underlie these events involve the activation of the IRS-2/AKT pathway and activation of the cell cycle, mediated by cyclin D(2). These adaptations permit the maintenance of glycaemia at near-physiological ranges.

Endurance training stimulates growth and survival pathways and the redox balance in rat pancreatic islets

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin - An immunohistochemical survey of 476 primary and metastatic carcinomas

CDX2 is a recently cloned homeobox gene that encodes an intestine-specific transcription factor, expressed in the nuclei of epithelial cells throughout the intestine, from duodenum to rectum. While expression of CDX2 protein in primary and metastatic colorectal carcinomas has been previously documented, neither the sensitivity nor the specificity of CDX2 expression, as determined by immunohistochemistry, for colorectal adenocarcinoma has been determined. We performed an immunohistochemical survey of 476 tumors with a monoclonal antibody, CDX2-88, including 89 tumors from the colon and duodenum and 95 tumors from other gastrointestinal sites, including the esophagus, stomach, pancreatobiliary system, gastrointestinal carcinoids, and liver. CDX2 was expressed uniformly (that is, in 76-100% of tumor cells) in all but one of the evaluated colorectal and duodenal tumors. High-level expression of CDX2 was also found, however, in mucinous ovarian carcinomas and adenocarcinomas primary to the urinary bladder of which 64% and 100% were positive, respectively. Gastric, gastroesophageal, and pancreatic adenocarcinomas and cholangiocarcinomas all showed similar, heterogeneous patterns of CDX2 expression. Most tumors in each group showed CDX2 expression by a minority of cells, whereas a substantial minority of cases in each group was completely negative and a smaller minority was uniformly positive. Gastrointestinal carcinoids gave similarly varied results, but the majority (58%) was negative. Hepatocellular carcinomas showed no expression of CDX2. Only very rare examples of carcinomas of the genitourinary and gynecologic tracts, breast, lung, and head and neck showed significant levels of CDX2 expression. In this study of primary and metastatic epithelial tumors, uniform CDX2 expression is demonstrated to be an exquisitely sensitive and highly, but incompletely, specific marker of intestinal adenocarcinomas. Compared with villin, a previously described marker of GI adenocarcinomas, CDX2 demonstrated superior sensitivity and comparable specificity. CDX2 expression can be seen, however, in selected non-GI adenocarcinomas such as mucinous ovarian carcinomas and adenocarcinomas of the urinary bladder.

Genotyping of AR and PSA polymorphisms in a patient with Klinefelter syndrome, non-Hodgkin lymphoma, and adenocarcinoma of the prostate

It has been hypothesized that the AR (androgen receptor) gene binds the two PSA (prostate-specific antigen) alleles with differing affinities and may differentially influence prostate cancer risk. In this article, we report a case of adenocarcinoma of the prostate in a 56-year-old man with Klinefelter syndrome (47,XXY) and non-Hodgkin lymphoma, as well as the AR and PSA genotype. AR and PSA gene polymorphisms were analyzed by polymerase chain reaction-based methods using DNA from peripheral white blood cells and the prostate cancer. We determined the methylation status of the AR gene on the X chromosome. The patient presents with the AG genotype for the ARE-I (androgen response element) region of the PSA gene. We detect the presence of two short AR alleles with 19 and 11CAG repeats each. Unmethylated alleles were demonstrated for both. The shorter allele was inactive in more than 60% of total DNA in both control blood and prostate cancer cells. The presence of short AR alleles and the G allele of the PSA gene may contribute to the development of prostate cancer in a 47,XXY patient. (C) 2004 Elsevier B.V. All rights reserved.

Polysaccharide fraction of Agaricus brasiliensis avoids tumor-induced IL-10 production and changes the microenvironment of subcutaneous Ehrlich adenocarcinoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)