High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity

Toxicogenomics, based on the temporal effects of drugs on gene expression, is able to predict toxic effects earlier than traditional technologies by analyzing changes in genomic biomarkers that could precede subsequent protein translation and initiation of histological organ damage. In the present study our objective was to extend in vivo toxicogenomic screening from analyzing one or a few tissues to multiple organs, including heart, kidney, brain, liver and spleen. Nanocapillary quantitative real-time PCR (QRT-PCR) was used in the study, due to its higher throughput, sensitivity and reproducibility, and larger dynamic range compared to DNA microarray technologies. Based on previous data, 56 gene markers were selected coding for proteins with different functions, such as proteins for acute phase response, inflammation, oxidative stress, metabolic processes, heat-shock response, cell cycle/apoptosis regulation and enzymes which are involved in detoxification. Some of the marker genes are specific to certain organs, and some of them are general indicators of toxicity in multiple organs. Utility of the nanocapillary QRT-PCR platform was demonstrated by screening different references, as well as discovery of drug-like compounds for their gene expression profiles in different organs of treated mice in an acute experiment. For each compound, 896 QRT-PCR were done: four organs were used from each of the treated four animals to monitor the relative expression of 56 genes. Based on expression data of the discovery gene set of toxicology biomarkers the cardio- and nephrotoxicity of doxorubicin and sulfasalazin, the hepato- and nephrotoxicity of rotenone, dihydrocoumarin and aniline, and the liver toxicity of 2,4-diaminotoluene could be confirmed. The acute heart and kidney toxicity of the active metabolite SN-38 from its less toxic prodrug, irinotecan could be differentiated, and two novel gene markers for hormone replacement therapy were identified, namely fabp4 and pparg, which were down-regulated by estradiol treatment

Pro-Tumoral Inflammatory Myeloid Cells as Emerging Therapeutic Targets

Since the observation of Virchow, it has long been known that the tumor microenvironment constitutes the soil for the infiltration of inflammatory cells and for the release of inflammatory mediators. Under certain circumstances, inflammation remains unresolved and promotes cancer development. Here, we review some of these indisputable experimental and clinical evidences of cancer related smouldering inflammation. The most common myeloid infiltrate in solid tumors is composed of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). These cells promote tumor growth by several mechanisms, including their inherent immunosuppressive activity, promotion of neoangiogenesis, mediation of epithelial-mesenchymal transition and alteration of cellular metabolism. The pro-tumoral functions of TAMs and MDSCs are further enhanced by their cross-talk offering a myriad of potential anti-cancer therapeutic targets. We highlight these main pro-tumoral mechanisms of myeloid cells and give a general overview of their phenotypical and functional diversity, offering examples of possible therapeutic targets. Pharmacological targeting of inflammatory cells and molecular mediators may result in therapies improving patient condition and prognosis. Here, we review experimental and clinical findings on cancer-related inflammation with a major focus on creating an inventory of current small molecule-based therapeutic interventions targeting cancer-related inflammatory cells: TAMs and MDSCs

Inflammation and Cancer: Extra- and Intracellular Determinants of Tumor-Associated Macrophages as Tumor Promoters

One of the hallmarks of cancer-related inflammation is the recruitment of monocyte-macrophage lineage cells to the tumor microenvironment. These tumor infiltrating myeloid cells are educated by the tumor milieu, rich in cancer cells and stroma components, to exert functions such as promotion of tumor growth, immunosuppression, angiogenesis, and cancer cell dissemination. Our review highlights the ontogenetic diversity of tumor-associated macrophages (TAMs) and describes their main phenotypic markers. We cover fundamental molecular players in the tumor microenvironment including extra- (CCL2, CSF-1, CXCL12, IL-4, IL-13, semaphorins, WNT5A, and WNT7B) and intracellular signals. We discuss how these factors converge on intracellular determinants (STAT3, STAT6, STAT1, NF-κB, RORC1, and HIF-1α) of cell functions and drive the recruitment and polarization of TAMs. Since microRNAs (miRNAs) modulate macrophage polarization key miRNAs (miR-146a, miR-155, miR-125a, miR-511, and miR-223) are also discussed in the context of the inflammatory myeloid tumor compartment. Accumulating evidence suggests that high TAM infiltration correlates with disease progression and overall poor survival of cancer patients. Identification of molecular targets to develop new therapeutic interventions targeting these harmful tumor infiltrating myeloid cells is emerging nowadays

Evaluación de actividades antitumorales de fitofármacos aislados de plantas nativas de Argentina: Caracterización in vitro y análisis de su expresión génica

En el marco del proyecto, se llevaron a cabo tres misiones (dos desde Argentina y una desde Hungría). La parte argentina mostró a los científicos húngaros la identificación botánica, los métodos de extracción y protocolos de análisis, que se utilizaron para la extracción de ingredientes activos de diferentes partes de plantas argentinas. Así, se prepararon cuarenta y dos extractos a partir de seis plantas usando siete solventes con distinta polaridad. La contraparte húngara realizó ensayos bioguiados a partir de los extractos a fin de obtener formas purificadas parcial o totalmente de dichos ingredientes con actividades biológicas. Para evaluar las mismas, en el laboratorio húngaro con la participación activa del científico visitante de Argentina, diferentes en ensayos celulares fueron empleados para determinar capacidad inmunomoduladora (por luminiscencia de células de melanoma transfectadas para detectar activación de NF-kB), antineoplásica (por colorimetría en líneas tumorales humanas de mama, pulmón y melanoma para evaluar pérdida de viabilidad) y/o citoprotectora antioxidante (por RT-CES en líneas celulares nerviosas y de corazón para evaluar electrónicamente cambios citológicos en tiempo real). Finalmente, se considera como exitosa la colaboración bilateral, ya que ha permitido identificar dos especies vegetales con potencial antitumoral y citoprotector según la polaridad de extracción empleada, analizando actualmente la estructura exacta de algunos compuestos activos aislados de los extractos.Fil: Soria, Elio Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Eynard, Aldo Renato. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Cantero, Juan Jose. Provincia de Córdoba. Ministerio de Ciencia y Técnica; ArgentinaFil: Puskas, Laszlo. Avidin Ltd.; HungríaFil: Nagy, Lajos Istvan. Avidin Ltd.; HungríaFil: Hackler, Laszlo. Avidin Ltd.; HungríaFil: Kitajka, Klara. University Of Szeged; Hungrí

Effects of dietary omega-3 polyunsaturated fatty acids on brain gene expression

Polyunsaturated fatty acids (PUFA) are essential structural components of the central nervous system. Their role in controlling learning and memory has been well documented. A nutrigenomic approach with high-density microarrays was used to reveal brain gene-expression changes in response to different PUFA-enriched diets in rats. In aged rats fed throughout life with PUFA-enriched diets, genes with altered expressions included transthyretin, &alpha;-synuclein, and calmodulins, which play important roles in synaptic&nbsp; plasticity and learning. The effect of perinatal omega-3 PUFA supply on gene expression later in life also was studied. Several genes showed similar changes in expression in rats fed omega-3-deficient diets in the perinatal period, regardless of whether they or their mothers were fed omega-3 PUFA-sufficient diets after giving birth. In this experiment, among the down-regulated genes were a kainate glutamate receptor and a DEAD-box polypeptide. Among the up-regulated genes were a chemokine-like factor, a tumor necrosis factor receptor, and cytochrome c. The possible involvement of the genes with altered expression attributable to different diets in different brain regions in young and aged rats and the possible mode of regulatory action of PUFA also are discussed. We conclude that PUFA-enriched diets lead to significant changes in expression of several genes in the central nervous tissue, and these effects appear to be mainly independent of their effects on membrane composition. The direct effects of PUFA on transcriptional modulators, the downstream developmentally and tissue-specifically activated elements might be one of the clues to understanding the beneficial effects of the omega-3 PUFA on the nervous system.<br /

Hypothalamic gene expression in Omega-3 PUFA-deficient male rats before, and following, development of hypertension

Dietary deficiency of &omega;-3 fatty acids (&omega;-3 DEF) produces hypertension in later life. This study examined the effect of &omega;-3 DEF on blood pressure and hypothalamic gene expression in young rats, before the development of hypertension, and in older rats following the onset of hypertension. Animals were fed experimental diets that were deficient in &omega;-3 fatty acids, sufficient in short-chain &omega;-3 fatty acids or sufficient in short- and long-chain &omega;-3 fatty acids, from the prenatal period until 10 or 36 weeks-of-age. There was no difference in blood pressure between groups at 10 weeks-of-age; however, at 36 weeks-of-age &omega;-3 DEF animals were hypertensive in relation to sufficient groups. At 10 weeks, expression of angiotensin-II1A receptors and dopamine D3 receptors were significantly increased in the hypothalamic tissue of &omega;-3 DEF animals. In contrast, at 36 weeks, &alpha;2a and &beta;1 adrenergic receptor expression was significantly reduced in the &omega;-3 DEF group. Brain docosahexaenoic acid was significantly lower in &omega;-3 DEF group compared with sufficient groups. This study demonstrates that dietary &omega;-3 DEF causes changes both in the expression of key genes involved in central blood pressure regulation and in blood pressure. The data may indicate that hypertension resulting from &omega;-3 DEF is mediated by the central adrenergic system.<br /