NUCKS overexpression in breast cancer

<p>Abstract</p> <p>Background</p> <p>NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that <it>NUCKS </it>is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies.</p> <p>Results</p> <p>The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas <it>in situ </it>(DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of <it>NUCKS </it>expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures.</p> <p>Conclusion</p> <p>The results show overexpression of the NUCKS protein in a number of non malignant breast lesions and cancerous tissues. In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.</p

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer

Background: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting. Results: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells. Conclusion: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton

A Water Soluble Polymer as a Working Structural Model for Humic Acids: H-binding and Spectroscopic Properties

HA occur ubiquitously in terrestrial and aquatic environments, indicating that many precursors and several formation pathways exist. Distinguishing among these pathways has been difficult because individual scientists have tended to concentrate their efforts on specific types of samples (e.g. soil, sediment or water) for which different isolation or fractionation methods are often used. Humic acids ultimately result from abiotic and biotic polymerization and polycondensation reactions involving such small precursors. The relative importance of abiotic and biotic processes in humification in soils and sediments still remains obscure even though much research has been devoted to them. For example the oxidative polymerization of phenols, which is an important precursor reaction in the formation of HA, is catalysed both by enzymes and abiotic (inorganic) catalysts. Polyphenols have been shown to be important precursors in the abiotic formation of HA. More particularly, the oxidative polymerization of polyphenols in soils is thought to be one of the major processes of formation of humic substances. According to the polyphenolic theory, humic acids are formed from simple phenols and phenolic acids via the formation of a semiquinone radical. Coupling of semiquinones originating from diphenols can lead to the formation of stable humic polymers under the catalytic action of Mn (IV) oxides. In the present case, we have focused our work on molecules bearing both carboxy and phenolic moieties as well as radical activity. In this model, protocatechuic acid (3,4-dihydroxybenzoic acid), and gallic acid (3,4,5-trihydroxybenzoic acid) are among the main low molecular weight aromatic acids formed after lignin degradation. As we have reported recently polymerization of the mono-, di-, and trihydroxyphenolic compounds may occur in alkaline media i.e. under ambient O2. i.e. under conditions where radicals are formed. In the present work, we have investigated the oxidative co-polymerization of some simple hydrobenzoic molecules which have been shown to be good models certain physicochemical properties of humic acids. As we show by controlling the pH, redox potential and the ratio of the polyphenolics, a water soluble polymer is produced that mimics certain key- physicochemical and spectroscopic properties of humic acids

Humic Acid: Aggregate, Floc, Macromolecule, Micelle, Polymer, or Supramolecule?

Two humic acids, extracted from an Alfisol forest soil and a lignite mined in Greece, were purified of ash components, size fractionated by membrane dialysis, and polarity fractionated by adsorption chromatography on XAD-8 resin to determine binding mechanisms of humic acid constituents. Humic acids, contained in 12,000 to 15,000 dialysis membranes, were successively dialyzed against 0.1 M HCl, 0.2M HF, 0.1M sodium citrate at pH 10, and 0.1 M NaOH to remove ash components and disaggregate and size fractionate with increasing pH. A part of humic acid did not permeate the dialysis membranes during the acid treatment, while the humic acid that permeated during the sodium citrate and NaOH treatments, was polarity fractionated by a decreasing pH gradient on XAD-8 resin. The resulting fractions were characterized by mass balance, elemental analyses, pH titration, and FT-IR spectrometry. Certain fractions of the lignite humic acid had characteristics very similar to molecular condensates formed by radical coupling of phenolic acid precursors of lignin. Significant differences between the composition and fractionation patterns of the soil and lignite humic acids were interpreted in terms of binding mechanisms within the humic acid precipitates

A novel bentonite humic acid composite material BephosTM as an environmental-friendly tool in phosphate and ammonium management

The objective of the present study was to examine the feasibility of using a novel modified bentonite as adsorbent for phosphate and ammonium removal from natural eutrophic waters. The uptake of phosphate and ammonium in adsorption kinetics A critical parameter was the initial phosphate concentration aiming to represent a eutrophic natural ecosystem. Moreover, its adsorption capacity and efficiency as a restoration tool was compared with a number of materials tested on the removal of phosphate and ammonium from aqueous solutions. Bephos™ is capable for simultaneous adsorption of both phosphorus and ammonia and this is an important advantage against other materials used to restore eutrophic water bodies. Adsorption kinetics showed that more than 90% and 70% removal of phosphate and ammonium respectively from water within 30 min. Bephos™ is potent for remediation of phosphate and ammonium at low concentrations that occur in natural water ecosystems

An Investigation into the Possibility of Reusing Wastewater and Sludge on Soil Enriched with Humic Acid of Young Growing Seedlings of Cupressus Arizonica Greene

The influence of Humic acids (HA) in combination to Treated Municipal Waste Water (TMWW) and biological sludge (S) reuse was studied, for the characteristics of growth and metal accumulation in the forestal species Cupressus Arizonica Greene and soil characteristics. The experiment was held at the forestal seedbed of Agrinio at an experimental spacing of random groups containing 12 combinations of TMWW, S and HA plus control, in 4 replicates, meaning 12x4. The findings were: The interventions affected statistically significant the micronutrients capacity of Cu, Fe, Zn, and Mn of soil, leaves and roots. Humic acids raised significantly the availability of Cu and Zn micronutrients in the soil, but did not affect their concentrations in leaves and roots. The higher concentration of Cu and Zn in the leaves was found in the absence of humics with the intervention of E1 (control), while in the presence of humics with the intervention of E11 (Control+TMWW+200mg/kg HA). In the roots the higher concentration of Cu and Zn was achieved in the absence of humics with the intervention of E1 (control), while in the presence of humics with the intervention of E2 (Control+200mg/kg HA). Closing, in the soil, the highest concentration of Cu and Zn was observed in the absence of humics with the intervention of E10 (control+S), while in the presence of humics with the intervention of E8 (TMWW+S+200mg/kg HA)

Determination of Antioxidant Charge and Total Phenolic Content in Greek aromatic plants by EPR/UV-Vis Spectroscopy

Aromatic plants and herbs are among the more important sources of phenolic compounds, which have been reported to show strong antioxidant activity. In the present work the total phenolic content and the antioxidant charge of five aromatic plants, Origanum vulgare L., Rosmarinus officinalis L., Ocimum basilicum L., Salvia officinalis L. and Achillea millefolium L., biologically grown in Western Greece, was determined by UV-vis and Electron Paramagnetic Resonance (EPR) spectroscopy. Dried plant leaves extracted in 50% methanol/ 48,5% H2O/ 1,5% formic acid solution were measured by UV-vis spectroscopy for the determination of total phenolics. Quantitative analysis has been done by using Gallic acid as standard solution. In addition the dry matter of plant leaves was measured at 77Kelvin by EPR spectroscopy. The EPR spectra of all samples contained stable radical signal with g-values 2.0046-49 and a linewidth of 3-5Gauss. These are characteristic of phenolic or quinone-type radicals with the unpaired electron partially on the Oxygen atom of the phenolic ring. Quantitative analysis of the radicals has been done using DPPH [1mm3 2x1013 spins] as reference. The calculations have been done per dry mass of the material. The present results reveal significant differences of radical concentration between samples of different origin by EPR spectra. The higher radical content was measured in Origanum vulgare L. and the lower in Ocimum basilicum L. plant leaves respectively. In addition a significant difference in total phenolics content was determined between plants. The higher concentration was found in Origanum vulgare L. and the lower in Achillea millefolium L. plant leaves respectively. It is concluded that a fraction of the total phenolics accumulation corresponds to the redox active antioxidant charge in plant leaves