T cell activation induces CuZn superoxide dismutase (SOD)-1 intracellular re-localization,production and secretion

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H₂O₂), more stable and able to freely diffuse through cell membranes. Copper–zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H₂O₂ and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H₂O₂ generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the “non-T” cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors.     

Multiplex primer-extension assay for identification of Yersinia species

A multiplex primer-extension reaction (PER) assay, was specifically designed for the identification of ten Yersinia species. The assay, directed towards the tufA (elongation factor Tu) gene, was tested on a total of 42 samples representing Yersinia species and non-Yersinia species. The primers used in the preliminary PCR, designed in highly conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 587 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of Yersinia spp. in food samples and to evaluate the potential hazard for consumers.     

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.     

Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases

Conflicting data are reported on pro- or anti-inflammatory activity of bovine lactoferrin (bLf) in different cell models as phagocytes or epithelial cell lines infected by bacteria. Here we evaluated the bLf effect on epithelial models mimicking two human pathologies characterized by inflammation and infection with specific bacterial species. Primary bronchial epithelium from a cystic fibrosis (CF) patient and differentiated intestinal epithelial cells were infected with Pseudomonas aeruginosa LESB58 isolated from a CF patient and Adherent-Invasive Escherichia coli LF82 isolated from a Crohn’s disease patient. Surprisingly, bLf significantly reduced the intracellular bacterial survival, but differently modulated the inflammatory response. These data lead us to hypothesize that bLf differentially acts depending on the epithelial model and infecting pathogen. To verify this hypothesis, we explored whether bLf could modulate ferroportin (Fpn), the only known cellular iron exporter from cells, that, by lowering the intracellular iron level, determines a non permissive environment for intracellular pathogens. Here, for the first time, we describe the bLf ability to up-regulate Fpn protein in infected epithelial models. Our data suggest that the mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells is complex and the bLf involvement in modulating cellular iron homeostasis should be taken into account.     

Quantitative Chemical Proteomics Identifies Novel Targets of the Anti-cancer Multi-kinase Inhibitor E-3810

Novel drugs are designed against specific molecular targets, but almost unavoidably they bind non-targets, which can cause additional biological effects that may result in increased activity or, more frequently, undesired toxicity. Chemical proteomics is an ideal approach for the systematic identification of drug targets and off-targets, allowing unbiased screening of candidate interactors in their natural context (tissue or cell extracts).E-3810 is a novel multi-kinase inhibitor currently in clinical trials for its anti-angiogenic and anti-tumor activity. In biochemical assays, E-3810 targets primarily vascular endothelial growth factor and fibroblast growth factor receptors. Interestingly, E-3810 appears to inhibit the growth of tumor cells with low to undetectable levels of these proteins in vitro, suggesting that additional relevant targets exist. We applied chemical proteomics to screen for E-3810 targets by immobilizing the drug on a resin and exploiting stable isotope labeling by amino acids in cell culture to design experiments that allowed the detection of novel interactors and the quantification of their dissociation constant (K_{d imm}) for the immobilized drug. In addition to the known target FGFR2 and PDGFRα, which has been described as a secondary E-3810 target based on in vitro assays, we identified six novel candidate kinase targets (DDR2, YES, LYN, CARDIAK, EPHA2, and CSBP). These kinases were validated in a biochemical assay and—in the case of the cell-surface receptor DDR2, for which activating mutations have been recently discovered in lung cancer—cellular assays.Taken together, the success of our strategy—which integrates large-scale target identification and quality-controlled target affinity measurements using quantitative mass spectrometry—in identifying novel E-3810 targets further supports the use of chemical proteomics to dissect the mechanism of action of novel drugs.The “target deconvolution” process, namely, the identification and characterization of proteins bound by a drug of interest (1), is a crucial step in drug development that allows definition of the compound selectivity and the early detection of potential side effects. Target deconvolution can be achieved by means of systematic in vitro biochemical assays measuring the ability of the drug to interact with candidate binders and, if they are enzymes, interfere with their activity. An alternative approach is chemical proteomics (chemoproteomics), which combines affinity chromatography and proteomic techniques (2, 3). Up-to-date chemical proteomics essentially consists of three main steps: (i) drug immobilization on a solid phase; (ii) drug affinity chromatography to capture drug targets in complex protein mixtures, such as cell or tissue lysates; and (iii) mass spectrometry (MS)-based¹ identification of the proteins retained by the immobilized drug (4–6).In chemical proteomics, the affinity chromatography step is typically performed under mild conditions, to allow the identification of all possible natural binders. The drawback of using mild, non-denaturing conditions is the significant number of proteins nonspecifically binding to the solid phase, which, once identified via MS, can be difficult to discern from genuine drug targets. The relatively high number of such nonspecific binders has limited the widespread use of this strategy.More recently, the development and implementation of quantitative strategies in proteomics based on the use of differentially stable isotopes to label proteomes from distinct functional states, together with significant technological and instrumental developments in the MS field concerning sensitivity and throughput, have largely allowed this limitation to be overcome. One of the most popular labeling techniques is stable isotope labeling by amino acids in cell culture (SILAC) (7). In SILAC, dividing cells are cultured in media supplemented with amino acids containing stable isotopic variants of carbon (¹²C/¹³C), nitrogen (¹⁴N/¹⁵N), or hydrogen (¹H/²H), which are incorporated into newly synthesized proteins during cell division. When extensive labeling (>98%) of cells is achieved upon the appropriate number of replications, light and heavy cells are differentially treated (e.g. exposed to drug versus vehicle), mixed in equal proportion, and subjected to proteomics analysis by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Peptides from the two functional states can be distinguished by their specific delta mass values, and their intensity ratio in MS spectra is directly proportional to the relative abundance of the corresponding proteins in the initial protein extract. Robust analysis of SILAC data is possible with dedicated software, such as MaxQuant (8). The application of SILAC strategies to interactomic studies is an efficient means of discerning specific from background binders (9). When applied to chemical proteomics, quantitative proteomics is crucial, as it offers quality filters to discern genuine drug interactors from proteins binding to the solid phase, with the use of different experimental setups (4, 5).In this study, we successfully coupled SILAC with chemical proteomics to carry out an unbiased screening of protein interactors of the anti-cancer drug E-3810, currently in Phase II clinical trials. E-3810 is a novel multi-kinase inhibitor, a class of targeted drug that comprises different molecules currently used in clinical practice (e.g. imatinib, dasatinib, sunitinib, sorafenib) (10). E-3810 exhibits both anti-tumor and anti-angiogenic properties (11). In preclinical studies, E-3810 showed broad anti-tumor activity in vivo, when used as monotherapy in a variety of human xenografts, or in conjunction with conventional chemotherapy (11, 12).Cellular vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs) are the principal targets of E-3810, as previously demonstrated by in vitro kinase assays, which showed that E-3810 inhibited VEGFR-1, -2, and -3 and FGFR-1 and -2 in the nanomolar range (11). Studies performed on several kinase inhibitors demonstrated that these molecules can elicit pleiotropic effects not easily explained by the sole inhibition of their known targets (13, 14). These effects are in most cases due to an inhibitory activity of the drug on additional kinase targets not tested in vitro that may lead to synergistic anti-cancer effects or undesirable toxicity. This could also be the case for E-3810, which was shown to inhibit in vitro additional kinase targets with high affinity, and which is able to inhibit the growth of tumor cells expressing low to undetectable levels of VEGFRs/FGFRs, suggesting that its spectrum of target inhibition has not been fully explored (11).We thus established a SILAC-based chemical proteomic platform composed of a set of affinity chromatography experiments using E-3810 immobilized on agarose resin and incubated with SILAC-labeled extract from the ovarian cancer cell line A2780. We identified proteins interacting with the resin via MS and took advantage of SILAC-based protein quantitation to discern genuine from background binders and derive quantitative information about the specific interactions. Our findings demonstrate that additional targets of E-3810 exist and that these targets may contribute to the anticancer effect of E-3810.     

SERS Properties of Gold Nanorods at Resonance with Molecular,Transverse, and Longitudinal Plasmon Excitations

The amplification of Raman signals of the heteroaromatic cation 1-(N-methylpyrid-4-yl)-2-(N-methylpyrrol-2-yl)ethylene (PEP+)) bound to Au nanorods (NRs) was investigated at different excitation wavelengths to study the effect of the laser resonance with the absorption band of the PEP+ moiety and with the two plasmon oscillation modes of the NR. Two different PEP+ derivatives, differing in the length of the alkyl chain bearing the anchoring group, were used as target molecules. Raman spectra obtained exciting at 514 or at 785 nm (i.e., exciting the transverse or the longitudinal plasmon band) present a higher intensity than that at 488 nm suggesting a higher Raman amplification when the laser excitation wavelength is resonant with one of the two plasmon modes. Moreover, considering results of Discrete Dipole Approximation (DDA) calculations of the local field generated at the NR surface when either the transverse or the longitudinal plasmon modes are excited, we deduced that the resonance condition of the 514-nm laser excitation with the absorption band of the dye strongly contributes to the amplification of the Raman signal.     

Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca²⁺ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity.     

‘Act on Oncology’ as a New Comprehensive Approach to Assess Prostate Cancer Centres – Method Description and Results of a Pilot Study

Background

Multidisciplinary care of prostate cancer is increasingly offered in specialised cancer centres. It requires the optimisation of medical and operational processes and the integration of the different medical and non-medical stakeholders.

Objective

To develop a standardised operational process assessment tool basing on the capability maturity model integration (CMMI) able to implement multidisciplinary care and improve process quality and efficiency.

Design, Setting, and Participants

Information for model development was derived from medical experts, clinical guidelines, best practice elements of renowned cancer centres, and scientific literature. Data were organised in a hierarchically structured model, consisting of 5 categories, 30 key process areas, 172 requirements, and more than 1500 criteria. Compliance with requirements was assessed through structured on-site surveys covering all relevant clinical and management processes. Comparison with best practice standards allowed to recommend improvements. ‘Act On Oncology’(AoO) was applied in a pilot study on a prostate cancer unit in Europe.

Results and Limitations

Several best practice elements such as multidisciplinary clinics or advanced organisational measures for patient scheduling were observed. Substantial opportunities were found in other areas such as centre management and infrastructure. As first improvements the evaluated centre administration described and formalised the organisation of the prostate cancer unit with defined personnel assignments and clinical activities and a formal agreement is being worked on to have structured access to First-Aid Posts.

Conclusions

In the pilot study, the AoO approach was feasible to identify opportunities for process improvements. Measures were derived that might increase the operational process quality and efficiency.     

Expression of insulin‐like growth factor‐1 receptor in metastatic uveal melanoma and implications for potential autocrine and paracrine tumor cell growth

We investigated the importance of the insulin‐like growth factor‐1 receptor (IGF‐1R) in hepatic metastases of uveal melanoma. The expression pattern of IGF‐1R in archival tissue samples of hepatic metastasis from 24 patients was analyzed by immunohistochemistry. All the samples of hepatic metastases stained positive for IGF‐1R. To investigate the biological role of IGF‐1R on the growth of metastatic uveal melanoma, a long‐term cell line obtained from a hepatic metastasis (TJU‐UM001) was evaluated. TJU‐UM001 expressed cell surface IGF‐1R (>90%) and proliferated in response to exogenous and endogenous insulin‐like growth factor‐1 (IGF‐1). Correlatively, anti‐IGF‐1R antibody completely blocked IGF‐1‐induced growth of TJU‐UM001 cells. IGF‐1 preferentially induced phosphorylation of Akt (S473) in quiescent TJU‐UM001 cells, and this was blocked by anti‐IGF‐1R antibody. This study suggests that autocrine and paracrine mechanisms underlie IGF‐1‐induced growth of metastatic uveal melanoma and underscore the potential benefit of IGF‐1 or IGF‐1R antagonism in treatment for metastatic uveal melanoma.