Elevated cyclic AMP suppresses ConA-induced MT1-MMP expression in MDA- MB-231 human breast cancer cells

We have previously reported that induction of MMP-2 activation by Concanavalin A (ConA) in MDA-MB-231 human breast cancer cells involves both transcriptional and post-transcriptional mechanisms, and that the continuous presence of ConA is required for MMP-2 activation (Yu et al. Cancer Res, 55, 3272-7, 1995). In an effort to identify signal transduction pathways which may either contribute to or modulate this mechanism, we found that three different cAMP-inducing agents, cholera toxin (CT), forskolin (FSK), and 3- isobutyl-1-methylxanthine (IBMX) partially inhibited ConA-induced MT1-MMP expression and MMP-2 activation in MDA-MB-231 cells. Combinations of CT or FSK with IBMX exhibited additive effects on reduction of MT1-MMP mRNA expression and MMP-2 activation. Agents which increase cAMP levels appeared to target transcriptional aspects of ConA induction, reducing MT1-MMP mRNA and protein in parallel with the reduced MMP-2 activation. In the absence of ConA, down-regulation of constitutive production of MT1-MMP mRNA and protein was observed, indicating that cAMP acts independently of ConA. These observations may help to elucidate factors regulating MT1-MMP expression, which may be pivotal to the elaboration of invasive machinery on the cell surface.

Analysis of unsaturated lipids by ozone-induced dissociation

Recent developments in analytical technologies have driven significant advances in lipid science. The sensitivity and selectivity of modern mass spectrometers can now provide for the detection and even quantification of many hundreds of lipids in a single analysis. In parallel, increasing evidence from structural biology suggests that a detailed knowledge of lipid molecular structure including carbon-carbon double bond position, stereochemistry and acyl chain regiochemistry is required to fully appreciate the biochemical role(s) of individual lipids. Here we review the capabilities and limitations of tandem mass spectrometry to provide this level of structural specificity in the analysis of lipids present in complex biological extracts. In particular, we focus on the capabilities of a novel technology termed ozone-induced dissociation to identify the position (s) of double bonds in unsaturated lipids and discuss its possible role in efforts to develop workflows that provide for complete structure elucidation of lipids by mass spectrometry alone: so-called top-down lipidomics. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrom. (C) 2011 Elsevier B.V. All rights reserved.

Remodeling of purinergic receptor-mediated Ca 2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.

Tensor-based item recommendation using probabilistic ranking in social tagging systems

A common problem with the use of tensor modeling in generating quality recommendations for large datasets is scalability. In this paper, we propose the Tensor-based Recommendation using Probabilistic Ranking method that generates the reconstructed tensor using block-striped parallel matrix multiplication and then probabilistically calculates the preferences of user to rank the recommended items. Empirical analysis on two real-world datasets shows that the proposed method is scalable for large tensor datasets and is able to outperform the benchmarking methods in terms of accuracy.

Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M(r) in equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M(r) zymographic activities at 50, 46 and 37 kDa M(r) are collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M(r) are increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (~2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

Synthesis and characterization of titanium sol–gels in varied gravity

Two unique test systems were designed and built to allow the effects of varied gravity (high, normal, reduced) during synthesis of titanium sol–gels to be studied. A centrifuge capable of providing high gravity environments of up to 70 g for extended periods while applying a 100 mbar vacuum and a temperature of 40–50 °C to the reaction chambers was developed. The second system was used in the QUT Microgravity Drop Tower Facility also provided the same thermal and vacuum conditions used in the centrifuge, but was required to operate autonomously during free fall. Through the use of post synthesis instrumental characterization, it was found that increased gravity levels during synthesis, had the greatest effect on the final products. Samples produced in reduced and normal gravity appeared to form amorphous gels containing very small particles with moderate surface areas. Whereas crystalline anatase (TiO2), was found to form in samples synthesized above 5 g with significant increases in crystallinity, particle size and surface area observed when samples were produced at gravity levels up to 70 g. It is proposed that for samples produced in higher gravity, an increased concentration gradient of water is forms at the bottom of the reacting film due to forced convection. The particles formed in higher gravity diffuse downward toward this excess of water, which favors the condensation reaction of remaining sol–gel precursors with the particles promoting increased particle growth. Due to the removal of downward convection in reduced gravity, particle growth due to condensation reaction processes are physically hindered hydrolysis reactions favored instead. Another significant finding from this work was that anatase could be produced at relatively low temperatures of 40–50 °C instead of the conventional method of calcination above 450 °C solely through sol–gel synthesis at higher gravity levels.

Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin induced dormant ring stages of Plasmodium falciparum

Artemisinin (ART) based combination therapy (ACT) is used as the first line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART induced ring stage dormancy and recovery has been implicated as possible cause of recrudescence; however, little is known about the characteristics of dormant parasites including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA) induced dormancy and recovery. Transcription analysis showed an immediate down regulation for 10 genes following exposure to DHA, but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, were also maintained. Additions of inhibitors for biotin acetyl CoA carbozylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively following DHA treatment. Our results demonstrate most metabolic pathways are down regulated in DHA induced dormant parasites. In contrast fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment.

Neutrophil gelatinase-associated lipocalin (NGAL) an early-screening biomarker for ovarian cancer : NGAL is associated with epidermal growth factor-induced epithelio-mesenchymal transition

The expression of neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be upregulated in ovarian cancer cells. In this study, we report that the expression of immunoreactive NGAL (irNGAL) in ovarian tumors changes with disease grade and that this change is reflected in the concentration of NGAL in peripheral blood. A total of 59 ovarian tissues including normal, benign, borderline malignant and grades 1, 2 and 3 malignant were analyzed using immunohistochemistry. irNGAL was not present in normal ovaries and the NGAL expression was weak to moderate in benign tissues. Both borderline and grade 1 tumors displayed the highest amount of NGAL expression with moderate to strong staining, whereas in grade 2 and 3 tumors, the extent of staining was significantly less (p < 0.01) and staining intensity was weak to moderate. Staining in all cases was confined to the epithelium. NGAL expression was analyzed by ELISA in 62 serum specimens from normal and different grades of cancer patients. Compared to control samples, the NGAL concentration was 2 and 2.6-fold higher in the serum of patients with benign tumors and cancer patients with grade 1 tumors (p < 0.05) and that result was consistent with the expression of NGAL performed by Western blot. NGAL expression was evaluated by Western blot in an immortalized normal ovarian cell line (IOSE29) as well as ovarian cancer cell lines. Moderate to strong expression of NGAL was observed in epithelial ovarian cancer cell lines SKOV3 and OVCA433 while no expression of NGAL was evident in normal IOSE29 and mesenchyme-like OVHS1, PEO.36 and HEY cell lines. NGAL expression was downregulated in ovarian cancer cell lines undergoing epithelio-mesenchymal transition (EMT) induced by epidermal growth factor (EGF). Down-regulation of NGAL expression correlated with the upregulation of vimentin expression, enhanced cell dispersion and downregulation of E-cadherin expression, some of the hallmarks of EMT. EGF-induced EMT phenotypes were inhibited in the presence of AG1478, an inhibitor of EGF receptor tyrosine kinase activity. These data indicate that NGAL may be a good marker to monitor changes of benign to premalignant and malignant ovarian tumors and that the molecule may be involved in the progression of epithelial ovarian malignancies.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Zymosan-induced inflammation stimulates neo-adipogenesis

Objective: To investigate the potential of inflammation to induce new adipose tissue formation in the in vivo environment. Methods and results: Using an established model of in vivo adipogenesis, a silicone chamber containing a Matrigel and fibroblast growth factor 2 (1 μg/ml) matrix was implanted into each groin of an adult male C57Bl6 mouse and vascularized with the inferior epigastric vessels. Sterile inflammation was induced in one of the two chambers by suspending Zymosan-A (ZA) (200-0.02 μg/ml) in the matrix at implantation. Adipose tissue formation was assessed at 6, 8, 12 and 24 weeks. ZA induced significant adipogenesis in an inverse dose-dependent manner (P<0.001). At 6 weeks adipose tissue formation was greatest with the lowest concentrations of ZA and least with the highest. Adipogenesis occurred both locally in the chamber containing ZA and in the ZA-free chamber in the contralateral groin of the same animal. ZA induced a systemic inflammatory response characterized by elevated serum tumour necrosis factor-α levels at early time points. Aminoguanidine (40 μg/ml) inhibited the adipogenic response to ZA-induced inflammation. Adipose tissue formed in response to ZA remained stable for 24 weeks, even when exposed to the normal tissue environment. Conclusions: These results demonstrate that inflammation can drive neo-adipogenesis in vivo. This suggests the existence of a positive feedback mechanism in obesity, whereby the state of chronic, low-grade inflammation, characteristic of the condition, may promote further adipogenesis. The mobilization and recruitment of a circulating population of adipose precursor cells is likely to be implicated in this mechanism.

Host rather than graft origin of Matrigel-induced adipose tissue in the murine tissue-engineering chamber

We have recently shown that Matrigel-filled chambers containing fibroblast growth factor-2 (FGF2) and placed around an epigastric pedicle in the mouse were highly adipogenic. Contact of this construct with pre-existing tissue or a free adipose graft was required. To further investigate the mechanisms underpinning formation of new adipose tissue, we seeded these chambers with human adipose biopsies and human adipose-derived cell populations in severe combined immunodeficient mice and assessed the origin of the resultant adipose tissue after 6 weeks using species-specific probes. The tissues were negative for human-specific vimentin labeling, suggesting that the fat originates from the murine host rather than the human graft. This was supported by the strong presence of mouse-specific Cot-1 deoxyribonucleic acid labeling, and the absence of human Cot-1 labeling in the new fat. Even chambers seeded with FGF2/Matrigel containing cultured human stromal-vascular fraction (SVF) labeled strongly only for human vimentin in cells that did not have a mature adipocyte phenotype; the newly formed fat tissue was negative for human vimentin. These findings indicate that grafts placed in the chamber have an inductive function for neo-adipogenesis, rather than supplying adipocyte-precursor cells to generate the new fat tissue, and preliminary observations implicate the SVF in producing inductive factors. This surprising finding opens the door for refinement of current adipose tissue-engineering approaches.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and β-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, β-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase- 2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial- to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4- MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen- induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen- stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.

A natural oil-based emulsion containing allantoin versus aqueous cream for managing radiation-induced skin reactions in patients with cancer : a phase III double-blind randomised controlled trial

Purpose To investigate the effects of a natural oil-based emulsion containing allantoin versus aqueous cream for preventing and managing radiation induced skin reactions (RISR). Methods and Materials A total of 174 patients were randomised and participated in the study. Patients either received Cream 1 (the natural oil-based emulsion containing allantoin) or Cream 2 (aqueous cream). Skin toxicity, pain, itching and skin-related quality of life scores were collected for up to four weeks after radiation treatment. Results Patients who received Cream 1 had a significantly lower average level of Common Toxicity Criteria at week 3 (p<0.05), but had statistically higher average levels of skin toxicity at weeks 7, 8 and 9 (all p<0.001). Similar results were observed when skin toxicity was analysed by grades. With regards to pain, patients in the Cream 2 group had a significantly higher average level of worst pain (p<0.05) and itching (p=0.046) compared to the Cream 1 group at week 3, however these differences were not observed at other weeks. In addition, there was a strong trend for Cream 2 to reduce the incidence of grade 2 or more skin toxicity in comparison to Cream 1 (p=0.056). Overall, more participants in the Cream 1 group were required to use another topical treatment at weeks 8 (p=0.049) and 9 (p=0.01). Conclusion The natural oil-based emulsion containing allantoin appears to have similar effects for managing skin toxicity compared to aqueous cream up to week 5, however, it becomes significantly less effective at later weeks into the radiation treatment and beyond treatment completion (week 6 and beyond). There were no major differences in pain, itching and skin-related quality of life. In light of these results, clinicians and patients can base their decision on costs and preferences. Overall, aqueous cream appears to be a more preferred option.

Characterisation of the micro-architecture of direct writing melt electrospun tissue engineering scaffolds using diffusion tensor and computed tomography microimaging

This article describes the first steps toward comprehensive characterization of molecular transport within scaffolds for tissue engineering. The scaffolds were fabricated using a novel melt electrospinning technique capable of constructing 3D lattices of layered polymer fibers with well - defined internal microarchitectures. The general morphology and structure order was then determined using T 2 - weighted magnetic resonance imaging and X - ray microcomputed tomography. Diffusion tensor microimaging was used to measure the time - dependent diffusivity and diffusion anisotropy within the scaffolds. The measured diffusion tensors were anisotropic and consistent with the cross - hatched geometry of the scaffolds: diffusion was least restricted in the direction perpendicular to the fiber layers. The results demonstrate that the cross - hatched scaffold structure preferentially promotes molecular transport vertically through the layers ( z - axis), with more restricted diffusion in the directions of the fiber layers ( x – y plane). Diffusivity in the x – y plane was observed to be invariant to the fiber thickness. The characteristic pore size of the fiber scaffolds can be probed by sampling the diffusion tensor at multiple diffusion times. Prospective application of diffusion tensor imaging for the real - time monitoring of tissue maturation and nutrient transport pathways within tissue engineering scaffolds is discussed.