IGF-1R inhibition sensitizes breast cancer cells to ATM-Related Kinase (ATR) inhibitor and cisplatin

The complexity of the IGF-1 signalling axis is clearly a roadblock in targeting this receptor in cancer therapy. Here, we sought to identify mediators of resistance, and potential co-targets for IGF-1R inhibition. By using an siRNA functional screen with the IGF-1R tyrosine kinase inhibitor (TKI) BMS-754807 in MCF-7 cells we identified several genes encoding components of the DNA damage response (DDR) pathways as mediators of resistance to IGF-1R kinase inhibition. These included ATM and Ataxia Telangiectasia and RAD3-related kinase (ATR). We also observed a clear induction of DDR in cells that were exposed to IGF-1R TKIs (BMS-754807 and OSI-906) as indicated by accumulation of γ-H2AX, and phosphorylated Chk1. Combination of the IGF-1R/IR TKIs with an ATR kinase inhibitor VE-821 resulted in additive to synergistic cytotoxicity compared to either drug alone. In MCF-7 cells with stably acquired resistance to the IGF-1R TKI (MCF-7-R), DNA damage was also observed, and again, dual inhibition of the ATR kinase and IGF-1R/IR kinase resulted in synergistic cytotoxicity. Interestingly, dual inhibition of ATR and IGF-1R was more effective in MCF-7-R cells than parental cells. IGF-1R TKIs also potentiated the effects of cisplatin in a panel of breast cancer cell lines. Overall, our findings identify induction of DDR by IGF-1R kinase inhibition as a rationale for co-targeting the IGF-1R with ATR kinase inhibitors or cisplatin, particularly in cells with acquired resistance to TKIs.

Endothelial cell-derived Pentraxin 3 limits the vasoreparative therapeutic potential of Circulating Angiogenic Cells

AIMS: Circulating Angiogenic Cells (CACs) promote revascularization of ischemic tissues although their underlying mechanism of action and the consequences of delivering varying numbers of these cells for therapy remain unknown. This study investigates molecular mechanisms underpinning CAC modulation of blood vessel formation.

METHODS & RESULTS: CACs at low (2x10(5)cells/ml) and mid (2x10(6)cells/ml) cellular densities significantly enhanced endothelial cell (EC) tube formation in vitro, while high density CACs (2x10(7)cells/ml) significantly inhibited this angiogenic process. In vivo, Matrigel-based angiogenesis assays confirmed mid-density CACs as pro-angiogenic and high density CACs as anti-angiogenic. Secretome characterization of CAC-EC conditioned media identified pentraxin 3 (PTX3) as only present in the high density CAC-EC co-culture. Recombinant PTX3 inhibited endothelial tube formation in vitro and in vivo Importantly, our data revealed that the anti-angiogenic effect observed in high density CAC-EC co-cultures was significantly abrogated when PTX3 bioactivity was blocked using neutralizing antibodies or PTX3 siRNA in endothelial cells. We show evidence for an endothelial source of PTX3, triggered by exposure to high density CACs. In addition, we confirmed that PTX3 inhibits FGF2-mediated angiogenesis, and that the PTX3 N-terminus, containing the FGF-binding site, is responsible for such anti-angiogenic effects.

CONCLUSIONS: Endothelium, when exposed to high density CACs, releases PTX3 which markedly impairs the vascular regenerative response in an autocrine manner. Therefore, CAC density and accompanying release of angiocrine PTX3 are critical considerations when using these cells as a cell therapy for ischemic disease.

Leccinum vulpinum Watling induces DNA damage, decreases cell proliferation and induces apoptosis on the human MCF-7 breast cancer cell line

The current work aimed to study the antitumour activity of a phenolic extract of the edible mushroom Leccinum vulpinum Watling, rich essentially in hydroxybenzoic acids. In a first approach, the mushroom extract was tested against cancer cell growth by using four human tumour cell lines. Given the positive results obtained in these initial screening experiments and the evidence of some studies for an inverse relationship between mushroom consumption and breast cancer risk, a detailed study of the bioactivity of the extract was carried out on MCF-7 cells. Once the selected cell line to precede the work was the breast adenocarcinoma cell line, the human breast non-malignant cell line MCF-10A was used as control. Overall, the extract decreased cellular proliferation and induced apoptosis. Furthermore, the results also suggest that the extract causes cellular DNA damage. Data obtained highlight the potential of mushrooms as a source of biologically active compounds, particularly with antitumour activity.

Associação entre o proteassoma e o inibidor de protease BTCI : caracterização fisico-química e efeitos moleculares em células de câncer de mama (MCF-7)

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2010.

Impact Of Pharmacist Interventions On Drug-related Problems And Laboratory Markers In Outpatients With Human Immunodeficiency Virus Infection.

Substantial complexity has been introduced into treatment regimens for patients with human immunodeficiency virus (HIV) infection. Many drug-related problems (DRPs) are detected in these patients, such as low adherence, therapeutic inefficacy, and safety issues. We evaluated the impact of pharmacist interventions on CD4+ T-lymphocyte count, HIV viral load, and DRPs in patients with HIV infection. In this 18-month prospective controlled study, 90 outpatients were selected by convenience sampling from the Hospital Dia-University of Campinas Teaching Hospital (Brazil). Forty-five patients comprised the pharmacist intervention group and 45 the control group; all patients had HIV infection with or without acquired immunodeficiency syndrome. Pharmaceutical appointments were conducted based on the Pharmacotherapy Workup method, although DRPs and pharmacist intervention classifications were modified for applicability to institutional service limitations and research requirements. Pharmacist interventions were performed immediately after detection of DRPs. The main outcome measures were DRPs, CD4+ T-lymphocyte count, and HIV viral load. After pharmacist intervention, DRPs decreased from 5.2 (95% confidence interval [CI] =4.1-6.2) to 4.2 (95% CI =3.3-5.1) per patient (P=0.043). A total of 122 pharmacist interventions were proposed, with an average of 2.7 interventions per patient. All the pharmacist interventions were accepted by physicians, and among patients, the interventions were well accepted during the appointments, but compliance with the interventions was not measured. A statistically significant increase in CD4+ T-lymphocyte count in the intervention group was found (260.7 cells/mm(3) [95% CI =175.8-345.6] to 312.0 cells/mm(3) [95% CI =23.5-40.6], P=0.015), which was not observed in the control group. There was no statistical difference between the groups regarding HIV viral load. This study suggests that pharmacist interventions in patients with HIV infection can cause an increase in CD4+ T-lymphocyte counts and a decrease in DRPs, demonstrating the importance of an optimal pharmaceutical care plan.

Synthesis, characterization, and bactericidal properties of composites based on crosslinked resins containing silver

Two different commercial crosslinked resins (Amberlite GT73 and Amberlite IRC748) were employed for anchoring silver. The -SH and -N(CH2COOH)2 groups, respectively, present on these resins were used for Ag+ chelation from an aqueous solution. The Ag+ ions were reduced with three different reductants: hydrazine, hydroxylamine, and formaldehyde (under an alkaline pH). The produced composites were characterized with thermogravimetry/differential thermogravimetry and scanning electron microscopy combined with a backscattered scanning electron detector. Energy-dispersive X-ray spectroscopy coupled to scanning electron microscopy allowed the observation of submicrometer particles of silver, and chemical microanalysis of emitted X-rays revealed the presence of metal on the internal and external surfaces of the composite microspheres. The amount of incorporated silver was determined by titration. The antibacterial activity of the silver/resin composites was determined toward 10(3)-10(7) cells/mL dilutions of the auxotrophic AB1157 Escherichia coli strain; the networks containing anchored submicrometer silver particles were completely bactericidal within a few minutes because of the combined action of silver and functional groups of the resins. (c) 2007 Wiley Periodicals, Inc.

Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular localization following heat shock

The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42 degreesC for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37 degreesC, those exposed to heat stress (42 degreesC for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate. hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism

Context: Necdin activates GNRH gene expression and is fundamental for the development, migration, and axonal extension of murine GNRH neurons. In humans, necdin plays a potential role in the hypogonadotropic hypogonadism phenotype in patients with Prader-Willi syndrome. Aim: To investigate necdin gene (NDN) variants in patients with isolated hypogonadotropic hypogonadism (IHH). Patients and methods: We studied 160 Brazilian patients with IHH, which includes 92 with Kallmann syndrome and 68 with normosmic IHH. Genomic DNA was extracted and the single NDN exon was amplified and sequenced. To measure GNRH transcriptional activity, luciferase reporter plasmids containing GNRH regulatory regions were transiently transfected into GT1-7 cells in the presence and absence of overexpressed wild-type or mutant necdin. Results: A heterozygous variant of necdin, p.V318A, was identified in a 23-year-old male with Kallmann syndrome. The p.V318A was also present in affected aunt and his father and was absent in 100 Brazilian control subjects. Previous FGFR1 gene analysis revealed a missense mutation (p.P366L) in this family. Functional studies revealed a minor difference in the activation of GNRH transcription by mutant protein compared with wild type in that a significant impairment of the necdin protein activity threshold was observed. Conclusion: A rare variant of necdin (p.V318A) was described in a family with Kallmann syndrome associated with a FGFR1 mutation. Familial segregation and in vitro analysis suggested that this non-synonymous variant did not have a direct causative role in the hypogonadism phenotype. NDN mutations are not a frequent cause of congenital IHH.

Characterisation of the recently cloned rat noradrenaline transporter - Comparison with bovine and human transporters

The aims of this study were to characterize the recently cloned rat norepinephrine transporter (NET) in more detail and in particular to study possible species differences in its pharmacological properties compared with the human and bovine NETs. The study was carried out by measuring the uptake of [3H]norepinephrine in COS-7 cells expressing the NET after transient transfection with rat, human, or bovine NET cDNA. There were small but significant differences between the rat NET and the human or bovine NETs with respect to the affinities of sodium ions (greater for rat than for bovine) of the substrates norepinephrine, epinephrine, and 1-methyl-4-phenylpyridinium (greater for human than for rat), and of the inhibitor cocaine (greater for human and bovine than for rat), whereas the affinities of dopamine and of most inhibitors, including tricyclic antidepressants, showed no species differences. The fact that the affinities for some substrates, cocaine and sodium ions exhibited small but significant interspecies differences among the rat, human, and bovine NETs suggests that ligand recognition, the translocation process, and sodium ion dependence are influenced differentially by just a few amino acid exchanges in the primary sequences of the transporters. On the other hand, the lack of any major differences in the pharmacological properties of the rat, human, and bovine NETs in this study suggests that data obtained in previous studies on rat tissues and bovine cells can be extrapolated, in all except the most quantitative analyses, to the properties of the human NET.

Bone Marrow Mononuclear Cell Therapy Led to Alveolar-Capillary Membrane Repair, Improving Lung Mechanics in Endotoxin-Induced Acute Lung Injury

The aim of this study was to test the hypothesis that bone marrow mononuclear cell (BMDMC) therapy led an improvement in lung mechanics and histology in endotoxin-induced lung injury. Twenty-four C57BL/6 mice were randomly divided into four groups (n = 6 each). In the acute lung injur;y (ALI) group, Escherichia coli lipopolysaccharide (LPS) was instilled intratracheally (40 mu g, IT), and control (C) mice received saline (0.05 ml, IT). One hour after the administration of saline or LPS, BMDMC (2 x 10(7) cells) was intravenously injected. At day 28, animals were anesthetized and lung mechanics [static elastance (E(st)), resistive (Delta P(1)), and viscoelastic (Delta P(2)) pressures] and histology (light and electron microscopy) were analyzed. Immunogold electron microscopy was used to evaluate if multinucleate cells were type II epithelial cells. BMDMC therapy prevented endotoxin-induced lung inflammation, alveolar collapse, and interstitial edema. In addition, BMDMC administration led to epithelial and endothelial repair with multinucleated type II pneumocytes. These histological changes yielded a reduction in lung E(st), Delta P(1), and Delta P(2) compared to ALI. In the present experimental ALI model, the administration of BMDMC yielded a reduction in the inflammatory process and a repair of epithelium and endothelium, reducing the amount of alveolar collapse, thus leading to an improvement in lung mechanics.

Pulmonary periarterial inflammation in fatal asthma

P>Background To date, little information has been available about pulmonary artery pathology in asthma. The pulmonary artery supplies the distal parts of the lungs and likely represents a site of immunological reaction in allergic inflammation. The objective of this study was to describe the inflammatory cell phenotype of pulmonary artery adventitial inflammation in lung tissue from patients who died of asthma. Methods We quantified the different inflammatory cell types in the periarterial region of small pulmonary arteries in lung tissue from 22 patients who died of asthma [fatal asthma (FA)] and 10 control subjects. Using immunohistochemistry and image analysis, we quantified the cell density for T lymphocytes (CD3, CD4, CD8), B lymphocytes (CD20), eosinophils, mast cells (chymase and tryptase), and neutrophils in the adventitial layer of pulmonary arteries with a diameter smaller than 500 mu m. Results Our data (median/interquartile range) demonstrated increased cell density of mast cells [FA=271.8 (148.7) cells/mmcells/mmcells/mmcells/mmcells/mmcells/mmcells predominated over tryptase (MC(T)) mast cells in the perivascular arterial space in both asthma patients and controls [MC(CT)/(MC(CT)+MC(T))=0.91 (0-1) in FA and 0.75 (0-1) in controls, P=0.86]. Conclusions Our results show that the adventitial layer of the pulmonary artery participates in the inflammatory process in FA, demonstrating increased infiltration of mast cells, eosinophils, and neutrophils, but not of T and B lymphocytes.

Systemic chemotherapy-induced microsatellite instability in the mononuclear cell fraction of women with breast cancer can be reproduced in vitro and abrogated by amifostine

Objectives Microsatellite instability (MSI) induction by alkylating agent-based chemotherapy (ACHT) may underlie both tumor resistance to chemotherapy and secondary leukaemias in cancer patients. We investigated if ACHT could induce MSI in tumor-derived plasma-circulating DNA (pfDNA) and in normal peripheral blood mononuclear (PBMN) cells. We also evaluated if amifostine could interfere with this process in an in-vitro model. Methods MSI was determined in pfDNA, PBMN cells and urine cell-free DNA (ufDNA) of 33 breast cancer patients before and after ACHT. MCF-7 cells and PBMN from normal donors were exposed in vitro to melphalan, with or without amifostine. Results We observed at least one MSI event in PBMN cells, pfDNA or ufDNA of 87, 80 and 80% of patients, respectively. In vitro, melphalan induced MSI in both MCF-7 and normal PBMN cells. In PBMN cells, ACHT-induced MSI occurred together with a significant decrease in the expression of the DNA mismatch repair gene hMSH2. Amifostine decreased hMSH2 expression and also prevented MSI induction only in normal PBMN cells. Conclusions ACHT induced MSI in PBMN cells and in tumour-derived pfDNA. Because of its protective effect against ACHT induction of MSI in normal PBMN cells in vitro, amifostine may be a potential agent for preventing secondary leukaemias in patients exposed to ACHT.

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher`s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

Tyrosine residue 271 of the norepinephrine transporter is an important determinant of its pharmacology

The aim was to examine the functional importance in the norepinephrine transporter (NET) of (i) the phenylalanine residue at position 531 in transmembrane domain (TMD) 11 by mutating it to tyrosine in the rat (rF531Y) and human (hF531Y) NETs and (ii) the highly conserved tyrosine residues at positions 249 in TMD 4 of human NET (hNET) (mutated to alanine: hY249A) and 271 in TMD 5, by mutating to alanine (hY271A), phenylalanine (hY271F) and histidine (hY271H). The effects of the mutations on NET function were for uptake of the substrates, examined by expressing the mutant and wildtype NETs in COS-7 cells and measuring the K-m and V-max for uptake of the substrates, [H-3]norepinephrine, [H-3]MPP+ and [H-3]dopamine, the K-D and B-max for [H-3]nisoxetine binding and the K-i of the inhibitors, nisoxetine, desipramine and cocaine, for inhibition of [H-3]norepinephrine uptake. The K-m values of the substrates were lower for the mutants at amino acid 271 than hNET and unaffected for the other mutants, and each mutant had a significantly lower than NET for substrate uptake. The mutations at position 271 caused an increase in the K-i or K-D values of nisoxetine, desipramine and cocaine, but there were no effects for the other mutations. Hence, the 271 tyrosine residue in TMD 5 is an important determinant of NET function, with the mutants showing an increase in the apparent affinities of substrates and a decrease in the apparent affinities of inhibitors, but the 249 tyrosine and 531 phenylalanine residues do not have a major role in determining NET function. (C) 2001 Elsevier Science B.V. All rights reserved.

Effects of short- and long-term exposure to c-AMP and c-GMP on the noradrenaline transporter

The effects of short- and long-term exposure of cells to elevated cyclic adenosine monophosphate (c-AMP), using dibutyryl-c-AMP, 8-bromo-c-AMP, cholera toxin or forskolin, or cyclic guanosine monophosphate (c-GMP), using dibutyryl-c-GMP or 8-bromo-c-GMP, on the activity and expression of the noradrenaline transporter (NAT) were examined. Short- or long-term c-GMP elevation had no effects on H-3-noradrenaline uptake by rat PC12 phaeochromocytoma cells or human SK-N-SH-SY5Y neuroblastoma cells. Short-term c-AMP elevation (for 17 min experiment duration) caused a decrease in H-3-noradrenaline uptake by PC12 cells, but had no effects on SK-N-SH-SY5Y cells or COS-7 cells transfected with human or rat NAT cDNA. c-AMP did not affect H-3-nisoxetine binding to PC12 cells. Long-term (24 h) exposure to elevated c-AMP levels caused a decrease in H-3-noradrenaline uptake and NAT mRNA in PC12 cells, but had no effects on SK-N-SH-SY5Y cells and caused a small increase in H-3-noradrenaline uptake in COS-7 cells heterologously expressing rat or human NAT. Hence, c-AMP, but not c-GMP, causes a cell type-dependent reduction in NAT activity after short-term exposure and a reduction in NAT expression after long-term exposure. (C) 2001 Elsevier Science Ltd. All rights reserved.