Inverse baseline expression pattern of the NEP/neuropeptides and NFκB/proteasome pathways in androgen-dependent and androgen-independent prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Castration-resistance in prostate cancer (PC) is a critical event hallmarking a switch to a more aggressive phenotype. Neuroendocrine differentiation and upregulation of NFκB transcriptional activity are two mechanisms that have been independently linked to this process.</p> <p>Methods</p> <p>We investigated these two pathways together using <it>in vitro </it>models of androgen-dependent (AD) and androgen-independent (AI) PC. We measured cellular levels, activity and surface expression of Neutral Endopeptidase (NEP), levels of secreted Endothelin-1 (ET-1), levels, sub-cellular localisation and DNA binding ability of NFκB, and proteasomal activity in human native PC cell lines (LnCaP and PC-3) modelling AD and AI states.</p> <p>Results</p> <p>At baseline, AD cells were found to have high NEP expression and activity and low secreted ET-1. In contrast, they exhibited a low-level activation of the NFκB pathway associated with comparatively low 20S proteasome activity. The AI cells showed the exact mirror image, namely increased proteasomal activity resulting in a canonical pathway-mediated NFκB activation, and minimal NEP activity with increased levels of secreted ET-1.</p> <p>Conclusions</p> <p>Our results seem to support evidence for divergent patterns of expression of the NFκB/proteasome pathway with relation to components of the NEP/neuropeptide axis in PC cells of different level of androgen dependence. NEP and ET-1 are inversely and directly related to an activated state of the NFκB/proteasome pathway, respectively. A combination therapy targeting both pathways may ultimately prove to be of benefit in clinical practice.</p

Expression of Neutral Endopeptidase, Endothelin-1, and Nuclear Factor Kappa B in Prostate Cancer: Interrelations and Associations with Prostate-Specific Antigen Recurrence after Radical Prostatectomy

Objective. To study the impact of the neutral endopeptidase (NEP)/neuropeptides (NPs) axis and nuclear factor kappa B (NFκB) as predictors of prostate-specific antigen (PSA) recurrence after radical prostatectomy (RP). Patients and Methods. 70 patients with early-stage PC were treated with RP and their tumor samples were evaluated for expression of NEP, endothelin-1 (ET-1) and NFκB (p65). Time to PSA recurrence was correlated with the examined parameters and combined with preoperative PSA level, Gleason score, pathological TNM (pT) stage, and surgical margin (SM) assessment. Results and Limitations. Membranous expression of NEP (P < 0.001), cytoplasmic ET-1 (P = 0.002), and cytoplasmic NFκB (P < 0.001) were correlated with time to PSA relapse. NEP was associated with ET-1 (P < 0.001) and NFκB (P < 0.001). ET-1 was also correlated with NFκB (P < 0.001). NEP expression (P = 0.017), pT stage (P = 0.013), and SMs (P = 0.036) were independent predictors of time to PSA recurrence. Conclusions. There seems to be a clinical model of NEP/NPs and NFκB pathways interconnection, with their constituents following inverse patterns of expression in accordance with their biological roles and molecular interrelations

Survival and New Prognosticators in Metastatic Seminoma: Results From the IGCCCG-Update Consortium

PURPOSE The classification of the International Germ-Cell Cancer Collaborative Group (IGCCCG) has been a major advance in the management of germ-cell tumors, but relies on data of only 660 patients with seminoma treated between 1975 and 1990. We re-evaluated this classification in a database from a large international consortium. MATERIALS AND METHODS Data on 2,451 men with metastatic seminoma treated with cisplatin- and etoposide-based first-line chemotherapy between 1990 and 2013 were collected from 30 institutions or collaborative groups in Australia, Europe, and North America. Clinical trial and registry data were included. Primary end points were progression-free survival (PFS) and overall survival (OS) calculated from day 1 of treatment. Variables at initial presentation were evaluated for their prognostic impact. Results were validated in an independent validation set of 764 additional patients. RESULTS Compared with the initial IGCCCG classification, in our modern series, 5-year PFS improved from 82% to 89% (95% CI, 87 to 90) and 5-year OS from 86% to 95% (95% CI, 94 to 96) in good prognosis, and from 67% to 79% (95% CI, 70 to 85) and 72% to 88% (95% CI, 80 to 93) in intermediate prognosis patients. Lactate dehydrogenase (LDH) proved to be an additional adverse prognostic factor. Good prognosis patients with LDH above 2.5× upper limit of normal had a 3-year PFS of 80% (95% CI, 75 to 84) and a 3-year OS of 92% (95% CI, 88 to 95) versus 92% (95% CI, 90 to 94) and 97% (95% CI, 96 to 98) in the group with lower LDH. CONCLUSION PFS and OS in metastatic seminoma significantly improved in our modern series compared with the original data. The original IGCCCG classification retains its relevance, but can be further refined by adding LDH at a cutoff of 2.5× upper limit of normal as an additional adverse prognostic factor

Proteomics challenging medicine

The study of the genome dictated that the whole is more than the sum of its parts; genes do not function independently but in networks. The impact of this understanding was explosive. It acknowledged the concept that this interplay of genes is at least equally significant to their individual functions, and indicated that genome alone cannot describe the whole of an organism, not without the study of the systems of gene products. Proteomics and a range of other "-omics" sciences were thus created, uterly overwhelming in their magnitude and complexity. The development of proteomics would have been impossible without the technological benefits of genome research, despite the fact that great differences exist between proteomic and genomic analysis. High-throughput technologies evolved generating a huge amount of data in dire need of interpretation. A pivotal role for bioinformatics emerged, with this science becoming able of producing primary biological knowledge. Biology was transformed into a big science and systems biology was born. All these act as a pressure generating force towards the evolution of medicine not only in terms of clinical applications but, more importantly, as a way of thinking. The classical qualitative approach previously pacing the hypothesis-driven investigations is gradually abandoned and quantitative approaches necessitate discovery-driven ways towards clinical diagnosis. Medicine clearly ought to be prepared to adapt to this revolution. Copyright © Hellenic Society of Haematology

Neuropeptide-inducible upregulation of proteasome activity precedes nuclear factor kappa B activation in androgen-independent prostate cancer cells.

ABSTRACT: BACKGROUND: Upregulation of nuclear factor kappa B (NFκB) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NFκB, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro. METHODS: We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment. RESULTS: Neuropeptide (NP) stimulation induced nuclear translocation of NFκB in a dose-dependent manner in AI cells, also evident as reduced total inhibitor κB (IκB) levels and increased DNA binding in EMSA. These effects were preceded by increased 20 S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NFκB, IκB kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA. CONCLUSIONS: Our results support evidence for a direct mechanistic connection between the NPs and NFκB/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state

Magnetic nanoparticles in medical diagnostic applications: Synthesis, characterization and proteins conjugation

Background: Magnetic nanoparticles (NPs) used in biomedical applications should be discrete with small particle sizes, narrow size distribution and superparamagnetic. NPs can be tailored to target, through chemical bonds, specific organs, cells, or even molecular markers of different diseases in vivo, with suitable surface chemistry modification. Methods: Nanoparticles are synthesized by a low cost coprecipitation reaction of ferrous and ferric salts with alkaline solution. The characteristics of the NPs are modified by varying the addition rate of the alkaline solution. NPs surface is silica coated using a modified Stöbe method. The conversion of the surface hydroxyl groups into amino-groups is performed by two different alkoxysilanes and the silanization reaction is conducted either in Methanol – Glycerol environment at elevated temperature, or in water at room temperature. The surface amine groups of the NPs are further converted, either to aldehyde groups by glutaraldehyde, or to carboxyl groups using glutaric anhydride. Bovine Serum Albumin and Vena human natural immunoglobulin are used in order to study the protein conjugation capacity of the functionalized NPs. The amount of protein attached to the nanoparticles is determined by UV–Vis spectroscopy of the supernatant. Conjugation of synthesized nanoparticles to protein BSA is examined by FTIR spectroscopy. SDS-PAGE electrophoresis followed by protein immunoblotting is used to test the effect of nano-conjugation to the antibodies. Results: Superparamagnetic Fe3O4 nanoparticles with saturation magnetization 60emu/g, a mean diameter 8-12 nm and BET surface areas between 100-250 m2/gr are obtained with total time of addition of the base between 1-5 minutes. They are coated with a thin and nearly uniform silica (SiO2) layer with thickness 1-2 nm. The most appropriate source for surface functionalization with amino groups is 3-aminopropyltriethoxysilane (APTES), while the two silanization methods used, proved to be equally efficient. NPs with surface aldehyde groups display better conjugation capacity than NPs functionalized with carboxyl groups. The FTIR spectra of the protein conjugated NPs samples, contain the two main peaks, at 1529 cm-1 and 1661 cm-1, attributed to the amide bond of the protein, which confirms the conjugation of the protein to the NPs. During a SDS-PAGE electrophoresis -protein immunoblotting experiment, the antibodies, after being conjugated to the nanoparticles, are selectively attached to their antigen, which indicates of lack of significant conformation changes secondary to the conjugation process. Conclusion: The conjugation capacity of the optimized nanoparticles is higher for Ig antibody than for BSA protein, under similar reaction conditions. The conjugational efficacy and conformational stability and the effect on electrophoretic mobility of the antibodies conjugated to the nanoparticles are verified by protein immunoblotting. © 2016 Bentham Science Publishers