Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADPsensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Modification of cell volume and proliferative capacity of Pseudokirchneriella subcapitata cells exposed to metal stress

The impact of metals (Cd, Cr, Cu and Zn) on growth, cell volume and cell division of the freshwateralga Pseudokirchneriella subcapitata exposed over a period of 72 h was investigated. The algal cells wereexposed to three nominal concentrations of each metal: low (closed to 72 h-EC10values), intermediate(closed to 72 h-EC50values) and high (upper than 72 h-EC90values). The exposure to low metal concen-trations resulted in a decrease of cell volume. On the contrary, for the highest metal concentrations anincrease of cell volume was observed; this effect was particularly notorious for Cd and less pronouncedfor Zn. Two behaviours were found when algal cells were exposed to intermediate concentrations ofmetals: Cu(II) and Cr(VI) induced a reduction of cell volume, while Cd(II) and Zn(II) provoked an oppositeeffect. The simultaneous nucleus staining and cell image analysis, allowed distinguishing three phases inP. subcapitata cell cycle: growth of mother cell; cell division, which includes two divisions of the nucleus;and, release of four autospores. The exposure of P. subcapitata cells to the highest metal concentrationsresulted in the arrest of cell growth before the first nucleus division [for Cr(VI) and Cu(II)] or after thesecond nucleus division but before the cytokinesis (release of autospores) when exposed to Cd(II). Thedifferent impact of metals on algal cell volume and cell-cycle progression, suggests that different toxic-ity mechanisms underlie the action of different metals studied. The simultaneous nucleus staining andcell image analysis, used in the present work, can be a useful tool in the analysis of the toxicity of thepollutants, in P. subcapitata, and help in the elucidation of their different modes of action.

Effects of Environmental Organochlorine Pesticides on Human Breast Cancer: Putative Involvement on Invasive Cell Ability

Human exposure to persistent organic pollutants (POPs) is a certainty, even to long banned pesticides like o,p′-dichlorodiphenyltrichloroethane (o,p′-DDT), and its metabolites p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE), and p,p′-dichlorodiphenyldichloroethane (p,p′-DDD). POPs are known to be particularly toxic and have been associated with endocrine-disrupting effects in several mammals, including humans even at very low doses. As environmental estrogens, they could play a critical role in carcinogenesis, such as in breast cancer. With the purpose of evaluating their effect on breast cancer biology, o,p′-DDT, p,p′-DDE, and p,p′-DDD (50–1000 nM) were tested on two human breast adenocarcinoma cell lines: MCF-7 expressing estrogen receptor (ER) α and MDA-MB-231 negative for ERα, regarding cell proliferation and viability in addition to their invasive potential. Cell proliferation and viability were not equally affected by these compounds. In MCF-7 cells, the compounds were able to decrease cell proliferation and viability. On the other hand, no evident response was observed in treated MDA-MB-231 cells. Concerning the invasive potential, the less invasive cell line, MCF-7, had its invasion potential significantly induced, while the more invasive cell line MDA-MB-231, had its invasion potential dramatically reduced in the presence of the tested compounds. Altogether, the results showed that these compounds were able to modulate several cancer-related processes, namely in breast cancer cell lines, and underline the relevance of POP exposure to the risk of cancer development and progression, unraveling distinct pathways of action of these compounds on tumor cell biology.

Molecular and biochemical mechanisms involved in the toxicity of a marine cyanobacteria compound on cancer cell lines

In recent years marine biotechnology has revealed a crucial role in the future of bioindustry. Among the many marine resources, cyanobacteria have shown great potential in the production of bioactive compounds with diverse applicability. The pharmacological potential of these organisms has been one of the most explored areas in particular its antibacterial, antifungal and anticancer potential. This work was based on the assessment of potential anticancer compound E13010 F 5.4 isolated from marine cyanobacteria strain Synechocystis salina LEGE 06099. Thus the aim of this work was to explore molecular and biochemical mechanisms underlying the bioactivity detected in human cancer cells, specifically in lines RKO colon carcinoma and HT-29. The isolation of the compound was performed from biomass obtained by large-scale culture. To obtain the compound fractionation was carried and confirmation and isolation performed by Nuclear Magnetic Resonance (NMR), Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Cell viability assays were performed based on reduction of 3- (4,5-dimetiltiaziol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to assess the cytotoxic potential of the compound. From the battery of cell lines RKO (colon carcinoma), HT-29 (colorectal adenocarcinoma), MG-63 (osteosarcoma) and T47D (breast carcinoma) the cell lines RKO and HT-29 were selected for elucidation of mechanisms of cytotoxicity. For the elucidation of the mechanisms involved in cytotoxicity the cell lines RKO and HT29 were exposed to the compound. A genomic approach based in the mRNA expression of genes involved in apoptosis and cell cycle by Real-Time PCR and a proteomic approach based on the separation of proteins by two-dimensional electrophoresis (2DGE) was performed. For mRNA expression were selected the genes RPL8, HPRT1, VDAC, SHMT2, CCNE, CCNB1, P21CIP, BCL-2 and BAD and for proteomics isoelectric focussing between 3 – 10 and molecular weight of 19 – 117 kDa separated by polyacrylamide gels (2DGE). The MTT results confirmed the reduction of the cell viability. The RT-PCR results for the expression of genes studied were not yet fully elucidative. For the cell line RKO there was a significant reduction in the expression of the gene P21CIP, and a tendency for reduction in the BAD gene expression and for increased expression of gene CCNB1, pointing to an effort for cell proliferation. In HT-29 cell line, there was a tendency for increase in the expression of P21CIP and BAD, which may explain the reduction in cell viability. The 2DGE results indicate proteomic patterns with differentially altered spots in the treated and control cells with both qualitative and quantitative differences, and differences in response between the RKO and HT-29 cell lines.

Expanding our therapeutic options: β-blockers for colon cancer?

Supported by U. Porto/Santander Totta (IJUP) (PP-IJUP2011-320)

Evaluation of the role of glutathione in the lead-induced toxicity in saccharomyces cerevisiae

The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3 h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (l-glutamic acid, l-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability.

Obesidade e cancro da próstata: Avaliação do efeito dos adipócitos nas células RM1 de carcinoma da próstata

O cancro da próstata é o segundo cancro mais frequente e a sexta causa de morte mundial por cancro no sexo masculino. A obesidade tem sido associada ao aumento da incidência e mortalidade por cancro, com alguma controvérsia. As alterações nas expressões de adipocinas associadas à obesidade têm sido um dos diversos mecanismos propostos para explicar a associação entre a obesidade e o cancro da próstata, nomeadamente na promoção do desenvolvimento e progressão celular do tumor. O objetivo deste trabalho é avaliar o efeito dos fatores produzidos pelos pré-adipócitos e os adipócitos na proliferação, migração e invasão das células de carcinoma da próstata independentes dos androgénios. As células RM1 foram cultivadas na presença de diferentes concentrações de insulina e leptina, bem como em meio condicionado (MC) de pré-adipócitos e adipócitos e co-cultivadas em sistema de transwells, com as mesmas células. A proliferação celular das RM1 foi avaliada recorrendo a contagem celular em camara de Neubauer e em citometro de fluxo, e aos ensaios metabólicos alamar blue e XTT. Efetuou-se um ensaio de migração por dano nas células RM1 na presença dos meios condicionados. A invasão das células foi avaliada recorrendo a um sistema de transwells, com membrana de matrigel, quando cultivadas com pré-adipócitos e adipócitos. A insulina aumentou significativamente a proliferação celular, ao contrário da leptina que não teve efeito. O meio condicionado dos pré-adipócitos aumentou ligeiramente a proliferação, enquanto meio condicionado dos adipócitos de 1 e 2 dias aumentou significativamente a proliferação das células RM1 (p<0.01), quando avaliada por XTT. Na câmara de Neubauer não se verificaram diferenças significativas na proliferação celular. Relativamente à migração celular, observou-se um aumento significativo da migração das células RM1 cultivadas com meio condicionado de adipócitos (MCA) e pré-adipócitos (MCPA) em comparação com o controlo (p<0.01). Observou-se um aumento significativo da invasão de células RM1 cultivadas com adipócitos e pré-adipócitos (p <0.05). Os adipócitos aumentaram significativamente a proliferação das células RM1 em co-cultura (p<0.01). Em conclusão, as células RM1 parecem ser influenciadas por fatores secretados pelos adipócitos, capazes de aumentar a sua capacidade de proliferar, invadir e migrar.

Electrochemical immunosensor for the analysis of the breast cancer biomarker HER2 ECD

Human epidermal growth factor receptor 2 (HER2) is a breast cancer biomarker that plays a major role in promoting breast cancer cell proliferation and malignant growth. The extracellular domain (ECD) of HER2 can be shed into the blood stream and its concentration is measurable in the serum fraction of blood. In this work an electrochemical immunosensor for the analysis of HER2 ECD in human serum samples was developed. To achieve this goal a screen-printed carbon electrode, modified with gold nanoparticles, was used as transducer surface. A sandwich immunoassay, using two monoclonal antibodies, was employed and the detection of the antibody–antigen interaction was performed through the analysis of an enzymatic reaction product by linear sweep voltammetry. Using the optimized experimental conditions the calibration curve (ip vs. log[HER2 ECD]) was established between 15 and 100 ng/mL and a limit of detection (LOD) of 4.4 ng/mL was achieved. These results indicate that the developed immunosensor could be a promising tool in breast cancer diagnostics, patient follow-up and monitoring of metastatic breast cancer since it allows quantification in a useful concentration range and has an LOD below the established cut-off value (15 ng/mL).

Saccharomyces cerevisiae Mutants Affected in Vacuole Assembly or Vacuolar H+-ATPase are Hypersensitive to Lead (Pb) Toxicity

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16D), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16D mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H?-ATPase (VATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1D or vma2D) or membrane domain (vph1D or vma3D) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.

Adipocyte Secreted Factors Enhance Aggressiveness of Prostate Carcinoma Cells

Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

FAS -670A>G genetic polymorphism Is associated with Treatment Resistant Depression

Background Hippocampal neurogenesis has been suggested as a downstream event of antidepressants (AD) mechanism of action and might explain the lag time between AD administration and the therapeutic effect. Despite the widespread use of AD in the context of Major Depressive Disorder (MDD) there are no reliable biomarkers of treatment response phenotypes, and a significant proportion of patients display Treatment Resistant Depression (TRD). Fas/FasL system is one of the best-known death-receptor mediated cell signaling systems and is recognized to regulate cell proliferation and tumor cell growth. Recently this pathway has been described to be involved in neurogenesis and neuroplasticity. Methods Since FAS -670A>G and FASL -844T>C functional polymorphisms never been evaluated in the context of depression and antidepressant therapy, we genotyped FAS -670A>G and FASL -844T>C in a subset of 80 MDD patients to evaluate their role in antidepressant treatment response phenotypes. Results We found that the presence of FAS -670G allele was associated with antidepressant bad prognosis (relapse or TRD: OR=6.200; 95% CI: [1.875–20.499]; p=0.001), and we observed that patients carrying this allele have a higher risk to develop TRD (OR=10.895; 95% CI: [1.362–87.135]; p=0.008).Moreover, multivariate analysis adjusted to potentials confounders showed that patients carrying G allele have higher risk of early relapse (HR=3.827; 95% CI: [1.072–13.659]; p=0.039). FAS mRNA levels were down-regulated among G carriers, whose genotypes were more common in TRD patients. No association was found between FASL-844T>C genetic polymorphism and any treatment phenotypes. Limitations Small sample size. Patients used antidepressants with different mechanisms of action. Conclusion To the best of our knowledge this is the first study to evaluate the role of FAS functional polymorphism in the outcome of antidepressant therapy. This preliminary report associates FAS -670A>G genetic polymorphism with Treatment Resistant Depression and with time to relapse. The current results may possibly be given to the recent recognized role of Fas in neurogenesis and/or neuroplasticity.