Microenvironment and microvascular wall modulate cancer cell progression

Tumour progression is a complex process that frequently brings to cancer metastasis, the first cause of poor prognosis of cancer affected patients. Metastasis are generated by cells escaped from a primary mass and able to enter in the circulation, survive and proliferate in a new, distant site of the organism. To reach all these goal, many different phenomena had occur within both the cancer cells and the surrounding microenvironment. In the first part of this thesis, the focus was pointed on the metastatic potential of a leiomyosarcoma cell model. The studied cancer cells demonstrated a strong invasive capacity of the ECM in vitro, principally by production of matrix metalloproteinases 2 and 9, and robust pro-angiogenic activity in the chick CAM model, that facilitate its dissemination through same chick embryo internal organs. This study, with the title “MMPs and angiogenesis affect the metastatic potential of a human vulvar leiomyosarcoma cell line”, is presented in the published form. In the second part of this work, the emphasis was given to the microvascular element of the tumour microenvironment and specifically to the perivascular pericytes. These are intriguing cells due to their uncertain involvement in the biology of cancer. It is not clear how pericytes change within the tumour microenvironment and which is their contribute during the tumour dissemination. After the characterization of the chosen pericytic cell model, an in vitro study of the interaction between pericytes and different cancer cell lines where performed. Indirect and direct cell-cell interaction as well as movement of cancer cells in presence of pericytes conditioned media was analysed, in order to investigate the reciprocal influence of pericytes and tumour cells in the context of cancer progression.

Transcription of liver X receptor is down-regulated by 15-deoxy-Δ12,14-prostaglandin J2 through oxidative stress in human neutrophils

Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. They play important roles in controlling cholesterol homeostasis and as regulators of inflammatory gene expression and innate immunity, by blunting the induction of classical pro-inflammatory genes. However, opposite data have also been reported on the consequences of LXR activation by oxysterols, resulting in the specific production of potent pro-inflammatory cytokines and reactive oxygen species (ROS). The effect of the inflammatory state on the expression of LXRs has not been studied in human cells, and constitutes the main aim of the present work. Our data show that when human neutrophils are triggered with synthetic ligands, the synthesis of LXRα mRNA became activated together with transcription of the LXR target genes ABCA1, ABCG1 and SREBP1c. An inflammatory mediator, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), hindered T0901317-promoted induction of LXRα mRNA expression together with transcription of its target genes in both neutrophils and human macrophages. This down-regulatory effect was dependent on the release of reactive oxygen species elicited by 15dPGJ2, since it was enhanced by pro-oxidant treatment and reversed by antioxidants, and was also mediated by ERK1/2 activation. Present data also support that the 15dPGJ2-induced serine phosphorylation of the LXRα molecule is mediated by ERK1/2. These results allow to postulate that down-regulation of LXR cellular levels by pro-inflammatory stimuli might be involved in the development of different vascular diseases, such as atherosclerosis.

Platelet-activating factor downregulates the expression of liver X receptor-α and its target genes in human neutrophils

Liver X receptors (LXRs) are ligand-activated members of the nuclear receptor superfamily that regulate the expression of genes involved in lipid metabolism and inflammation, although their role in inflammation and immunity is less well known. It has been reported that oxysterols/LXRs may act as anti-inflammatory molecules, although opposite actions have also been reported. In this study, we investigated the effect of platelet-activating factor (PAF), a proinflammatory molecule, on LXRα signalling in human neutrophils. We found that PAF exerted an inhibitory effect on mRNA expression of TO901317-induced LXRα, ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, and sterol response element binding protein 1c. This negative action was mediated by the PAF receptor, and was dependent on the release of reactive oxygen species elicited by PAF, as it was enhanced by pro-oxidant treatment and reversed by antioxidants. Current data also support the idea that PAF induces phosphorylation of the LXRα molecule in an extracellular signal-regulated kinase 1/2-mediated fashion. These results suggest that a possible mechanism by which PAF exerts its proinflammatory effect is through the downregulation of LXRα and its related genes, which supports the notion that LXRα ligands exert a modulatory role in the neutrophil-mediated inflammatory response.

Oleic acid modulates mRNA expression of liver X receptor (LXR) and its target genes ABCA1 and SREBP1c in human neutrophils

Purpose: Regulation of liver X receptors (LXRs) is essential for cholesterol homeostasis and inflammation. The present study was conducted to determine whether oleic acid (OA) could regulate mRNA expression of LXRα and LXRα-regulated genes and to assess the potential promotion of oxidative stress by OA in neutrophils. Methods: Human neutrophils were treated with OA at different doses and LXR target gene expression, oxidative stress production, lipid efflux and inflammation state were analyzed. Results: We describe that mRNA synthesis of both LXRα and ABCA1 (a reverse cholesterol transporter) was induced by OA in human neutrophils. This fatty acid enhanced the effects of LXR ligands on ABCA1 and LXR expression, but it decreased the mRNA levels of sterol regulatory element-binding protein 1c (a transcription factor that regulates the synthesis of triglycerides). Although OA elicited a slight oxidative stress in the short term (15–30 min) in neutrophils, it is unlikely that this is relevant for the modulation of transcription in our experimental conditions, which involve longer incubation time (i.e., 6 h). Of physiological importance is our finding that OA depresses intracellular lipid levels and that markers of inflammation, such as ERK1/2 and p38 mitogen-activated protein kinase phosphorylation, were decreased by OA treatment. In addition, 200 μM OA reduced the migration of human neutrophils, another marker of the inflammatory state. However, OA did not affect lipid peroxidation induced by pro-oxidant agents. Conclusions: This work presents for the first time evidence that human neutrophils are highly sensitive to OA and provides novel data in support of a protective role of this monounsaturated acid against the activation of neutrophils during inflammation.

Ascorbic acid induces an oxidative stress in CCRF cells activating the transcription factors AP-1 and NFRB

We have previously tested the effects of high dose AA supplements on human volunteers in terms of reducing DNA damage, as a possible mechanism of the vitamin’s proposed protective effect against cancer and detected a transient, pro-oxidant effect at high doses (500 mg/day). Herein, we present evidence of a pro-oxidant effect of the vitamin when added to CCRF cells at extracellular concentrations which mimic those present in human serum in vivo (50–150AM). The activation of the transcription factor AP-1 was optimal at 100 AM AA following 3h exposure at 37jC. A minimum dose of 50 AM of AA activated NFnB but there appeared to be no dose-dependent effect. Increases of 2–3 fold were observed for both transcription factors when cells were exposed to 100 AM AA for 3h, comparing well with the pro-oxidant effect of H2O2 at similar concentrations. In parallel experiments the activation of AP-1 (binding to DNA) was potentiated when cells were pre-incubated with AA prior to exposure with H2O2. Cycloheximide pretreatment (10 Ag/ml for 15min) caused a 50% inhibition of AP-1 binding to DNA suggesting that it was due to a combination of increasing the binding of pre-existing Fos and Jun and an increase in their de novo synthesis. Cellular localisation was confirmed by immunocytochemistry using antibodies specific for c-Fos and c-Jun proteins. These results suggest that extracellular AA can elicit an intracellular stress response resulting in the activation of the oxidative stress-responsive transcription factors AP-1 and NFnB. These transcription factors are involved in the induction of genes associated with an oxidative stress response, cell cycle arrest and DNA repair confirmed by our cDNA microarray analysis (Affymetrix). This may explain the abilty for AA to appear to inhibit 8-oxodG, yet simultaneously generate another oxidative stress biomarker, 8-oxo-dA. These results suggest a completely novel DNA repair action for AA. Whether this action is relevant to our in vivo findings will be the subject of our future research.

Deoxycytidine glyoxal:Lesion induction and evidence of repair following vitamin C supplementation in vivo

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p = .001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p = .0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected. © 2003 Elsevier Science Inc.

The effect of complexes on combustion

The twin goals of low and efficient fuel use and minimum emissions are increasingly being addressed by research in both the motor and the catalyst industries of the world. This study was designed to attempt to investigate these goals. For diesel engine vehicles, this can be achieved by improving the efficiency of the fuel combustion in the combustion chamber. By having a suitable oxidation catalyst in the fuel one would expect the efficiency of the fuel combustion to be increased and fewer partial oxidation products to be formed. Also by placing a catalyst converter in the exhaust system partial oxidation products may be converted to more desirable final products. Finally, in this research the net catalytic effect of using an additive treated fuel and a blank ceramic monolith to trap the metal in the exhaust gases for potential use as catalytic converter was investigated. Suitable metal additives must yield a stable solution in the fuel tank. That is, they should not react with the air, water and rust that are always present. The research was targeted on the synthesis of hydrocarbon-soluble complexes that might exhibit unusually slow rates of ligand substitution. For materials containing metal ions, these properties are best met by using multi-dentate ligands that form neutral complexes. Metal complexes have been synthesised using acetylacetone derivatives, schiff base ligands and macrocyclic polyamine ligands, as potential pro-oxidant additives. Their thermal stabilities were also investigated using a differential thermal analysis instrument. The complexes were then investigated as potential additives for use in diesel fuel. The tests were conducted under controlled conditions using a diesel combustion bomb simulating the combustion process in the D.I. diesel engine, a test bed engine, and a vehicle engine.

Time controlled photo-oxidation of polyolefins by polymer bound additives

A variety of iron compounds containing vinyl or thiol functional groups (used as photoactivators) have been synthesised and some of these were successfully bound to both polyethylene and polypropylene backbones during processing in the presence of peroxide and interlinking agent. Concentrates (masterbatches) of the photoactivators in PP and PE were prepared and the pro-oxidant effect of the diluted masterbatches in absence and presence of an antioxidant was evaluated. An antioxidant photoactivator (FeDNC ) was found to sensitise the photoactivity of pro-oxidants (Metone A / Metone M) whereas an antioxidant (ZnDNC) was found to stabilise the polymer (PP and PE) containing both of these combinations. It was observed that the lower concentration of FeDNC sensitises the stability of the polymer containing very small concentration of NiDNC whereas higher concentration of FeDNC stabilises the polymer (LDPE) containing same amount of NiDNC compared to FeDNC alone. The photostability of unstabilised PP containing FeAc could be varied by varying the concentration of ZnDEC. Both the induction period and the UV - life time of the polymer increased by increasing concentration of ZnDEC. It is suggested that ligand exchange reaction may take place between FeAc and ZnDNC. A polymer bound UV stabiliser (HAEB) and a thermal stabiliser (DBBA) were used with a non extractable photoactivator (FeAc) in PP. Small concentrations of the stabilisers (HAEB and DBBA) in combination with the photoactivator (FeAc) sensitise the polymer. The antioxidant present in commercial polymer (LDPE and PP) was found to be of a hindered phenol type, which was found to antagonise with ZnDNC when used in combination with the photoactivators.

Effects of oxidative stress and neuroprotection in apoptosis in neuronal cell models

The PC12 and SH-SY5Y cell models have been proposed as potentially realistic models to investigate neuronal cell toxicity. The effects of oxidative stress (OS) caused by both H2O2 and Aβ on both cell models were assessed by several methods. Cell toxicity was quantitated by measuring cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) viability assay, an indicator of the integrity of the electron transfer chain (ETC), and cell morphology by fluorescence and video microscopy, both of which showed OS to cause decreased viability and changes in morphology. Levels of intracellular peroxide production, and changes in glutathione and carbonyl levels were also assessed, which showed OS to cause increases in intracellular peroxide production, glutathione and carbonyl levels. Differentiated SH-SY5y cells were also employed and observed to exhibit the greatest sensitivity to toxicity. The neurotrophic factor, nerve growth factor (NGF) was shown to cause protection against OS. Cells pre-treated with NGF showed higher viability after OS, generally less apoptotic morphology, recorded less apoptotic nucleiods, generally lower levels of intracellular peroxides and changes in gene expression. The neutrophic factor, brain derived growth factor (BDNF) and ascorbic acid (AA) were also investigated. BDNF showed no specific neuroprotection, however the preliminary data does warrant further investigation. AA showed a 'janus face' showing either anti-oxidant action and neuroprotection or pro-oxidant action depending on the situation. Results showed that the toxic effects of compounds such as Aβ and H2O2 are cell type dependent, and that OS alters glutathione metabolism in neuronal cells. Following toxic insult, glutathione levels are depleted to low levels. It is herein suggested that this lowering triggers an adaptive response causing alterations in glutathione metabolism as assessed by evaluation of glutathione mRNA biosynthetic enzyme expression and the subsequent increase in glutathione peroxidase (GPX) levels.

Photochemical degradation of polystyrene

In this work the oxidative degradation of pure polystyrene, polybutadiene and butadiene-modified polystyrene (normally called high impact polystyrene or HIPS) have been studied using a variety of physical and chemical techniques. The changes in dynamic-mechanical properties occurring during the ultra-violet light accelerated weathering of these polymers were followed by a visco-elastometric technique (Rheovibron) in the solid phase over a wide temperature range. Selective cross-linking of the polybutadiene in high-impact polystyrene caused the depression of the low temperature damping peak (tan d) with a corresponding sharp peak in tan d at ambient temperature accompanied by an integral rise in complex modulus. During the same period of photoxidation, the hydroperoxide concentration and gel content increased rapidly, reaching a maximum before decomposing photolytically with the destruction of unsaturation and with the formation of stable oxidation products. Infra-red spectroscopy showed the formation of carbonyl and hydroxyl groups. a,ß-unsaturated carbonyl was also identified and was formed by decomposition of both allylic hydroperoxide and initial peroxidic gel by ß-scission of the graft between polybutadiene and polystyrene. With further photoxidation a more stable ether gel was formed involving the destruction of the conjugating double bond of a,ß-unsaturated carbonyl. Addition of saturated and unsaturated ketones which are potential sensitisers of photoxidation to high-impact polystyrene and polybutadiene failed to photo-initiate the oxygen absorption of the polymers. A prior thermal oxidative treatment on the other hand eliminated the auto- accelerating stage leading to linear kinetics as the concentration of thermally-produced hydroperoxide approached a maximum. Antioxidants which act by destroying hydroperoxide lengthened the induction period to rapid oxygen absorption, whilst a phenolic antioxidant behaved as a weak photo-activator initially and a retarder later. Prior photolysis of high-impact polystyrene photo-activated the unsaturated component and caused similar changes in dynamic-mechanical properties to those found during photoxidation although at a much lower rate. Polybutadiene behaves as a photo-pro-oxidant for the destruction of polystyrene in high-impact polystyrene.

The behaviour of antioxidants in polyolefins under conditions of U.V. exposure

The effects of antioxidants and stabilizers on the oxidative degradation of polyolefins (low density polyethylene [LDPE] and polypropylene [PPJ have been studied after subjecting to prior high temperature processing treatments. The changes in the both chemical and physical properties of unstabilized polymers occurring during processing were found to be strongly dependent on the amount of oxygen present in the mixer. Subsequent thermal and photo-oxidation showed very similar characteristics and the chromophore primarily responsible for:both thermo and photooxidative degradation of unstabilized polymers was found to be hydroperoxide formed during processing. Removal of hydroperoxide by heat treatment in an inert atmosphere although increasing ketonic carbonyl concentration, markedly decreased the rate of photo-oxidation, introducing an induction period similar to that of an unprocessed sample. It was concluded that hydroperoxides are the most important initiators in normally processed polymers during the early stages of photo-oxidation. Antioxidants such as metal dithiocarbamates which act by destroying peroxides into non-radica1 products were found to be efficient melt stabilizers for polyolefins and effective UV stabilizers during the initial photo-oxidation stage, whilst a phenolic antioxidant, n-octadecyl-3-(3',5'-di-terbutyl 4'hydroxypheny1) propionate (Irganox 1076) retarded photo-oxidation rate in the later stages. A typical 'UV absorber' 2-hydroxy-4-octyloxy-benzophenone (HOBP) has a minor thermal antioxidant action but retarded photo-oxidation at all stages. A substituated piperidine derivative, Bis [2.2.6.6-tetramethylpiperidlnyl-4] sebacate (Tinuvin 770) behaved as an pro-oxidant during thermal oxidation of polyolefins but was an effective stabilizer against UV light. The UV absorber, HOBP synergised effectively with both peroxide decomposing antioxidants (metal dithiocarbamates) and a chain-breaking antioxidant (Irganox 1076) during photo-oxidation of the poymers studed whereas the combined effect was additive during thermal oxidation. By contrast, the peroxide decornposers and chain-breaking antioxidant (Irganox 1076) which were effective synergists during thermal oxidation of LDPE· were antagonistic during photo-oxidation. The mechanisms of these processes are discussed.

Protection of human erythrocytes against oxidative stress by berries

Berries contain several bioactive compounds that can protect against oxidative stress. In this study we evaluated the protective effect of different sequential extracts (ethyl acetate, ethanol and water) of seven berry species: bilberry (Vaccinium myrtillus), blackcurrant (Ribes nigrum), elderberry (Sambucus nigra), lingonberry (Vaccinium vitis-idaea), rose hips (Rosa sp.), sea buckthorn (Hippohae rhamnoides) and strawberry (Fragaria × ananassa). The protective effect was tested on human erythrocytes and the antioxidant capacity was also evaluated in vitro by the FRAP assay. In the erythrocyte assay all sea buckthorn extracts were superior in antioxidant effect to other berry extracts. The ethyl acetate extract of bilberries, and the ethanol and water extracts of blackcurrants, also protected the erythrocytes from oxidation. In contrast, water extracts of rose hips, bilberries and strawberries had a pro-oxidant effect on erythrocytes. The water extract of rose hips was superior to the other berry extracts in the FRAP assay. Thus, the results of the erythrocyte assay did not correlate with the results of the FRAP assay, but provided additional insights into the potential protective effects of berry extracts against oxidative stress. © 2012 - IOS Press and the authors. All rights reserved.

The role of redox signaling in the molecular mechanism of tamoxifen resistance in breast cancer.

The emergence of tamoxifen or aromatase inhibitor resistance is a major problem in the treatment of breast cancer. The molecular signaling mechanism of antiestrogen resistance is not clear. Understanding the mechanisms by which resistance to these agents arise could have major clinical implications for preventing or circumventing it. Therefore, in this dissertation we have investigated the molecular mechanisms underlying antiestrogen resistance by studying the contributions of reactive oxygen species (ROS)-induced redox signaling pathways in antiestrogen resistant breast cancer cells. Our hypothesis is that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of oxidative stress. The hypothesis of this dissertation was tested in an in vitro 2-D cell culture model employing state of the art biochemical and molecular techniques, including gene overexpression, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and anchorage-independent cell growth assays. We observed that tamoxifen (TAM) acts like both an oxidant and an antioxidant. Exposure of tamoxifen resistant LCC2 cell to TAM or 17 beta-estradiol (E2) induced the formation of reactive oxidant species (ROS). The formation of E2-induced ROS was inhibited by co-treatment with TAM, similar to cells pretreated with antioxidants. In LCC2 cells, treatments with either E2 or TAM were capable of inducing cell proliferation which was then inhibited by biological and chemical antioxidants. Exposure of LCC2 cells to tamoxifen resulted in a decrease in p27 expression. The LCC2 cells exposed to TAM showed an increase in p27 phosphorylation on T157 and T187. Conversely, antioxidant treatment showed an increase in p27 expression and a decrease in p27 phosphorylation on T157 and T187 in TAM exposed cells which were similar to the effects of Fulvestrant. In line with previous studies, we showed an increase in the binding of cyclin E-Cdk2 and in the level of p27 in TAM exposed cells that overexpressed biological antioxidants. Together these findings highly suggest that lowering the oxidant state of antiestrogen resistant LCC2 cells, increases LCC2 susceptibility to tamoxifen via the cyclin dependent kinase inhibitor p27.

Perfil imunológico associado à susceptibilidade, resistência e cura na infecção por Leishmania ( Leishmania ) infantum

Visceral Leishmaniasis (VL) is endemic in Brazil and the northeast region had the highest incidence of the disease , despite, in the last 30 years, it has spread to all geographic regions of the country. Leishmania infantum is the m ain etiological agent of VL in Latin America, Europe and North Africa. However, not all infected individuals develop the disease; in fact, the majority present spontaneous re solution of infection without symptoms. The evaluation of the immunological profil e has been mostly conducted stimulating, with Leishmania spp. antigen, peripheral blood mononuclear cells isolated from subjects with VL. These studies showed that VL patients had an inhibition of both, lymphocyte proliferation and proinflammatory response to Leishmania spp. antigen. Our study aimed to evaluate the immune response in active LV, cured post treatment and asymptomatic infection. To reach this aim, we analyzed immunophenotypic features related to activation, Treg and memory lymphocytes, by flow cytometry, as well as, evaluation of cytokine production, in ex vivo or in whole blood culture. In active VL volunteers, a longitu dinal study was conducted with reassessment at 4 and 14 months after clinical cure. The control group included individuals th at live d in endemic region and were either Positive Control, consisting of individuals with positive anti - L eishmania spp. serology and/or positive PCR for Leishmania  spp. and Negative Control composed by individuals with negative anti - Leishmania antibodie s serology and negative PCR for Leishmania . During VL, CD4 lymphocytes showed greater activation and memory profile s and were the major source of cytokines in culture when compared to CD8 lymphocytes , and these were not Leishmania specific. There were act ivated lymphocytes during VL (CD4 + CD69 + :4.9%) when compared to control groups, Positive (CD4 + CD69 + :1.96%, p=0.0045) and Negative (CD4 + CD69 + :1.35%, p=0.006), on the other hand, this was non - specific activation. The lymphocyte activation profile remain ed el evated even 14 months post treatmen t. A fter clinical cure , the activation was Leishmania specific (CD4 + CD25 + absence of SLA: 8.4%, and presence of SLA: 10.7% p=0.0279). CD8 + CD25 + lymphocytes were able to produce Leishmania specific IFN - γ in both, Positive Controls (absence of SLA 5.2% and presence of SLA: 9.5%, p=0.0391) and Cured 4 month (absence of SLA: 3.9%; presence of SLA: 10.7% p=0.0098). Whole blood culture cells, of VL patients, were able to produce IFN - γ, by SLA stimulation (absence of SLA: 28.0 pg ∕mL, and presence: 44.3 pg∕mL p=0.0020) as well as recovered groups (absence of SLA 2.3 pg∕mL and presence of SLA 139.8 pg∕mL, p=0.0005). However, the high level of IL - 10 seem ed to inhibit pro - inflammatory activity of IFN - γ and TNF - α during symptomatic dis ease . Unlike other pro - inflammatory cytokines, active VL group d id not produce Leishmania specific IL - 2 (absence of SLA 2.4 pg∕mL and presence of SLA: 2.6 pg∕mL). Based on these data we conclude that the restoration of lymphocyte activation and decreased i n IL - 10 Leishmania specific production were related to a protective immune profile.

Alternative splicing takes control of cytokine signaling

Thesis (Ph.D.)--University of Washington, 2016-08