Gamma-linolenic acid inhibits both tumour cell cycle progression and angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb, p53 and p27 protein expression

Background: Gamma-linolenic acid is a known inhibitor of tumour cell proliferation and migration in both in vitro and in vivo conditions. The aim of the present study was to determine the mechanisms by which gamma-linolenic acid (GLA) osmotic pump infusion alters glioma cell proliferation, and whether it affects cell cycle control and angiogenesis in the C6 glioma in vivo. Methods: Established C6 rat gliomas were treated for 14 days with 5 mM GLA in CSF or CSF alone. Tumour size was estimated, microvessel density (MVD) counted and protein and mRNA expression measured by immunohistochemistry, western blotting and RT-PCR. Results: GLA caused a significant decrease in tumour size (75 +/- 8.8%) and reduced MVD by 44 +/- 5.4%. These changes were associated with reduced expression of vascular endothelial growth factor (VEGF) (71 +/- 16%) and the VEGF receptor Flt1 (57 +/- 5.8%) but not Flk1. Expression of ERK1/2 was also reduced by 27 +/- 7.7% and 31 +/- 8.7% respectively. mRNA expression of matrix metalloproteinase-2 (MMP2) was reduced by 35 +/- 6.8% and zymography showed MMP2 proteolytic activity was reduced by 32 +/- 8.5%. GLA altered the expression of several proteins involved in cell cycle control. pRb protein expression was decreased (62 +/- 18%) while E2F1 remained unchanged. Cyclin D1 protein expression was increased by 42 +/- 12% in the presence of GLA. The cyclin dependent kinase inhibitors p21 and p27 responded differently to GLA, p27 expression was increased (27 +/- 7.3%) while p21 remained unchanged. The expression of p53 was increased (44 +/- 16%) by GLA. Finally, the BrdU incorporation studies found a significant inhibition (32 +/- 11%) of BrdU incorporation into the tumour in vivo. Conclusion: Overall the findings reported in the present study lend further support to the potential of GLA as an inhibitor of glioma cell proliferation in vivo and show it has direct effects upon cell cycle control and angiogenesis. These effects involve changes in protein expression of VEGF, Flt1, ERK1, ERK2, MMP2, Cyclin D1, pRb, p53 and p27. Combination therapy using drugs with other, complementary targets and GLA could lead to gains in treatment efficacy in this notoriously difficult to treat tumour.

Gamma-linolenic Acid Alters Ku80, E2F1, and Bax Expression and Induces Micronucleus Formation in C6 Glioma Cells In Vitro

Gamma-linolenic acid (GLA) is an inhibitor of tumor cell proliferation in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of 150 mu M GLA on the expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 in C6 rat glioma cells. The Ku proteins were chosen as previous studies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation. The fact that GLA exposure is known to enhance the efficacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other molecules such as roscovitine and flavopiridol. GLA altered the mRNA expression of E2F1, cyclin D1, and bax, but no changes were found for bcl2, Ku70, and Ku80. Alterations in protein expression were observed for bax, Ku80, and E2F1. The 45% decrease in E2F1 expression was proportional to decreased cell proliferation (44%). Morphological analysis found a 25% decrease in mitotic activity in the GLA-treated cells, which was accompanied by a 49% decrease in S-phase by FACS analysis. A 39% increase in the number of micronuclei detected by Hoechst fluorescence points to GLA`s effects on cell division even at concentrations that do not produce significant increases in apoptosis. Most important was the finding that Ku80 expression, a critical protein involved in DNA repair as a heterodimer with Ku70, was decreased by 71%. It is probable that reduced Ku80 is responsible for the increase in micronucleus formation in GLA-treated cells in a similar manner to that found in Ku80 null cells exposed to radiation. The decreased expression of Ku80 and E2F1 could make cells more susceptible to radiotherapy and chemotherapy. (C) 2009 IUBMB

Cigarette smoke exposure severely reduces peripheral insulin sensitivity without changing GLUT4 expression in oxidative muscle of Wistar rats

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

Time course of training-induced microcirculatory changes and of vegf expression in skeletal muscles of spontaneously hypertensive female rats

Exercise-induced vessel changes modulate arterial pressure (AP) in male spontaneously hypertensive rats (SHR). Vascular endothelial growth factor (VEGF) is important for angiogenesis of skeletal muscle. The present study evaluated the time course of VEGF and angiogenesis after short- and long-term exercise training of female SHR and Wistar Kyoto (WKY) rats, 8-9 weeks (200-250 g). Rats were allocated to daily training or remained sedentary for 3 days (N = 23) or 13 weeks (N = 23). After training, the carotid artery was catheterized for AP measurements. Locomotor (tibialis anterior and gracilis) and non-locomotor skeletal muscles (temporalis) were harvested and prepared for histologic and protein expression analyses. Training increased treadmill performance by all groups (SHR = 28%, WKY = 64%, 3 days) and (SHR = 141%, WKY = 122%, 13 weeks). SHR had higher values of AP than WKY (174 ± 4 vs 111 ± 2 mmHg) that were not altered by training. Three days of running increased VEGF expression (SHR = 28%, WKY = 36%) simultaneously with an increase in capillary-to-fiber ratio in gracilis muscle (SHR = 19%, WKY = 15%). In contrast, 13 weeks of training increased gracilis capillary-to-fiber ratio (SHR = 18%, WKY = 19%), without simultaneous changes in VEGF expression. Training did not change VEGF expression and capillarity of temporalis muscle. We conclude that training stimulates time- and tissue-dependent VEGF protein expression, independent of pressure levels. VEGF triggers angiogenesis in locomotor skeletal muscle shortly after the exercise starts, but is not involved in the maintenance of capillarity after long-term exercise in female rats.

Vascular endothelial growth factor (VEGF) and VEGF-receptor expression in placenta of hyperglycemic pregnant women

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

Absence of chemopreventive influence of propolis on the rat liver altered foci development

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

Expression of interferon-gamma, interferon-alpha and related genes in individuals with Down syndrome and periodontitis

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

IGFR-I expression and structural analysis of the hard palatine mucosa in an ethanol-drinking rat strain (UChA and UChB)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Interleukin 4 induces the expression of inducible nitric oxide synthase in eosinophils

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Low triioothyronine (T-3) or reverse triioothyronine (rT(3)) syndrome modifies gene expression in rats with congestive heart failure

Heart failure (NF) is frequently associated with euthyroid sicksyndrome (low T-3 and elevated rT(3)). We investigated if altered thyroid hormone in HF could affect expression of the TH receptor (TR alpha 1), and alpha and beta myosin heavy chains (alpha-MHC beta-MHC). HF was provoked in rats by aortic stenosis. We showed that rT(3) generated front liver and kidney deiodination significantly increased and T-3 decreased in HE; there was significantly higher TR alpha 1 expression, no alpha-MHC expression, but beta-MHC expression. Changes in TR alpha 1 could be compensating for low T-3 from HF.

Expression and Function of Fibroblast Growth Factor 18 in the Ovarian Follicle in Cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Altered Glucose Homeostasis and Hepatic Function in Obese Mice Deficient for Both Kinin Receptor Genes

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

Gene expression in placentation of farm animals: An overview of gene function during development

Eutherian mammals share a common ancestor that evolved into two main placental types, i.e., hemotrophic (e.g., human and mouse) and histiotrophic (e.g., farm animals), which differ in invasiveness. Pregnancies initiated with assisted reproductive techniques (ART) in farm animals are at increased risk of failure; these losses were associated with placental defects, perhaps due to altered gene expression. Developmentally regulated genes in the placenta seem highly phylogenetically conserved, whereas those expressed later in pregnancy are more species-specific. To elucidate differences between hemotrophic and epitheliochorial placentae, gene expression data were compiled from microarray studies of bovine placental tissues at various stages of pregnancy. Moreover, an in silico subtractive library was constructed based on homology of bovine genes to the database of zebrafish - a nonplacental vertebrate. In addition, the list of placental preferentially expressed genes for the human and mouse were collected using bioinformatics tools (Tissue-specific Gene Expression and Regulation [TiGER] - for humans, and tissue-specific genes database (TiSGeD) - for mice and humans). Humans, mice, and cattle shared 93 genes expressed in their placentae. Most of these were related to immune function (based on analysis of gene ontology). Cattle and women shared expression of 23 genes, mostly related to hormonal activity, whereas mice and women shared 16 genes (primarily sexual differentiation and glycoprotein biology). Because the number of genes expressed by the placentae of both cattle and mice were similar (based on cluster analysis), we concluded that both cattle and mice were suitable models to study the biology of the human placenta. (C) 2011 Elsevier B.V. All rights reserved.

Effects of BST and high energy diet on gene expression in mammary parenchyma of dairy heifers

The objective of this study was to determine the effects of dietary energy and recombinant bovine somatotropin (bST) injection to identify genes that might control mammogenesis. Total RNA was extracted from the parenchymal tissue of 32 heifers randomly assigned to one of four treatments: two diets (a standard diet and a high energy, high protein diet), each with or without bST. To perform microarray experiments, RNA samples were pooled (2 animals/pool) before reverse transcription and labeling with Cy3 or Cy5. A 4-node loop design was used to examine the differential gene expression among treatments using a bovine-specific cDNA micro array (National Bovine Functional Genomics Consortium Library, NBFGC) containing 18,263 unique expressed sequence tags (EST). Significance levels of differential gene expression among treatments were assessed using a mixed model approach. Injection of bST altered the expression of 12 % of the genes on NBFGC slide related to tissue development, whereas 6% were altered by diet. Administration of bST increases the expression of genes positively related to cell proliferation and mammary parenchyma to a greater extent than a high energy diet. © 2013 Sociedade Brasileira de Zootecnia.

Detection of cytokeratins in ghost cells of calcifying cystic odontogenic tumor indicates an altered keratinization and hair follicle differentiation for their development

Calcifying cystic odontogenic tumors (CCOTs) are benign cystic lesions of odontogenic origin characterized by an ameloblastoma-like epithelium and the presence of a group of cells named ghost cells. The pattern of cytokeratin (Ck) expression on these lesions remains unclear and needs to be clarified. To this end, the expression of Ck6, Ck13, Ck14, Ck18, and Ck19 in the epithelium lining of 7 cases of CCOTs was evaluated by immunohistochemistry. For this, the epithelium lining was divided into 3 distinct regions: basal layer, suprabasal layer, and the compartment composed of ghost cells. In this study, 6 cases (85.7%) were classified as type 1 and 1 (14.3%) as type 4. All cases were negative for Ck13 and Ck18, despite the epithelial layer, as well as in the ghost cells. Ck6 was only positive in the ghost cells. Positivity for Ck14 and Ck19 was found in the basal and suprabasal layers, including the ghost cells. The results showing positivity for Ck14 and Ck19 in all of the analyzed cases reinforce CCOT as being of odontogenic origin, and the restricted expression of Ck6 in the ghost cells may be indicative that these cells suffer an altered differentiation into hair follicles in CCOTs. © 2013 Elsevier Inc. All rights reserved.