Dynamic interplay between breast cancer cells and normal endothelium mediates the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells

Background Breast cancer\u2013endothelium interactions provide regulatory signals facilitating tumor progression. The endothelial cells have so far been mainly viewed in the context of tumor perfusion and relatively little is known regarding the effects of such paracrine interactions on the expression of extracellular matrix (ECM), proteasome activity and properties of endothelial cells. Methods To address the effects of breast cancer cell (BCC) lines MDA-MB-231 and MCF-7 on the endothelial cells, two cell culture models were utilized; one involves endothelial cell culture in the presence of BCCs-derived conditioned media (CM) and the other co-culture of both cell populations in a Transwell system. Real-time PCR was utilized to evaluate gene expression, an immunofluorescence assay for proteasome activity, and functional assays (migration, adhesion and invasion) and immunofluorescence microscopy for cell integrity and properties. Results BCC-CM decreases the cell migration of HUVEC. Adhesion and invasion of BCCs are favored by HUVEC and HUVEC-CM. HA levels and the expression of CD44 and HA synthase-2 by HUVEC are substantially upregulated in both cell culture approaches. Adhesion molecules, ICAM-1 and VCAM-1, are also highly upregulated, whereas MT1-MMP and MMP-2 expressions are significantly downregulated in both culture systems. Notably, the expression and activity of the proteasome \u3b25 subunit are increased, especially by the action of MDA-MB-231-CM on HUVEC. Conclusions and general significance BCCs significantly alter the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells. Deep understanding of such paracrine interactions will help to design novel drugs targeting breast cancer at the ECM level. This article is part of a Special Issue entitled Matrix-mediated cell behavior and properties

NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis

Preparation and toxicological assessment of functionalized carbon nanotube-polymer hybrids.

Novel Carbon Nanotube-Polymer Hybrids were synthesized as potential materials for the development of membranes for water treatment applications in the field of Membrane Bioreactors (MBRs). Due to the toxicological concerns regarding the use of nanomaterials in water treatment as well as the rising demand for safe drinking water to protect public health, we studied the functionalization of MWCNTs and Thin-MWCNTs as to control their properties and increase their ability of embedment into porous anisotropic polymeric membranes. Following the growth of the hydrophilic monomer on the surface of the properly functionalized CNTs, that act as initiator for the controlled radical polymerization (ATRP) of sodium styrene sulfonate (SSNa), the antimicrobial quaternized phosphonium and ammonium salts were attached on CNTs-g-PSSNa through non-covalent bonding. In another approach the covalent attachment of quaternized ammonium polymeric moieties of acrylic acid-vinyl benzyl chloride copolymers with N,N-dimethylhexadecylamine (P(AA12-co-VBCHAM)) on functionalized CNTs has also been attempted. Finally, the toxicological assessment in terms of cell viability and cell morphological changes revealed that surface characteristics play a major role in the biological response of functionalized CNTs

Schematic illustration of the functionalization of CNTs-OH with P(AA12-co-VBCHAM).

<p>Schematic illustration of the functionalization of CNTs-OH with P(AA12-co-VBCHAM).</p

Schematic illustration of the functionalization of CNTs with SSNa moieties (A) and the introduction of quaternized ammonium groups (B).

<p>Schematic illustration of the functionalization of CNTs with SSNa moieties (A) and the introduction of quaternized ammonium groups (B).</p

¹Η-NMR spectra of the copolymer P(AA12-co-VBC) (A) and the quaternized copolymer P(AA12-co-VBCHAM) (B) and FT-IR spectra of the above copolymers (C).

<p>¹Η-NMR spectra of the copolymer P(AA12-co-VBC) (A) and the quaternized copolymer P(AA12-co-VBCHAM) (B) and FT-IR spectra of the above copolymers (C).</p

TGA curves of pristine Thin-MWCNTs, Thin-MWCNTs-ΟH, Thin-MWCNTs-g-P(ΑΑ12-co-VBCHAM) and the quaternized P(ΑΑ12-co-VBCHAM).

<p>TGA curves of pristine Thin-MWCNTs, Thin-MWCNTs-ΟH, Thin-MWCNTs-g-P(ΑΑ12-co-VBCHAM) and the quaternized P(ΑΑ12-co-VBCHAM).</p

TGA curves of pristine Thin-MWCNTs and Thin-MWCNTs-g-PSSNa, as well as Τhin-MWCNTs-g-PSSAmC₁₆ (A), and Τhin MWCNTs-g-PSSPhC₁₆ (B)_.

<p>TGA curves of the copolymers of PSSNa and PSSPhC₁₆ are also shown.</p

Effects of pristine and hydroxyl decorated CNTs on lung fibroblasts cells proliferation for a 24 h incubation period.

<p>(<b>A</b>) <b><i>i</i></b><i>.</i> Pristine MWCNTs and <b><i>ii</i></b><i>.</i> MWCNTs-OH and (<b>B</b>) <b><i>i</i></b>. Pristine Thin-MWCNTs and <b><i>ii</i></b><i>.</i> Thin-MWCNTs-OH. A range of concentrations from 0.125 µg/mL to 25 µg/mL was assayed. The results are expressed as mean ±SD of three separate experiments in triplicate. Statistically significant differences were evaluated using the ANOVA test. Statistically significant differences among the 1% PF-127 treated and control cells are shown by (#) (p≤0.05). Statistically significant differences among the CNTs-treated and 1% PF-127 are shown by (*) (p≤0.05), (**) (p≤0.01) and (***) (p≤0.001).</p