Effect of a Purified Extract of Olive Mill Waste Water on Endothelial Cell Proliferation, Apoptosis, Migration and Capillary-Like Structure in vitro and in vivo.

Olive oil, a major feature of the Mediterranean diet, is an important source of phenolic compounds. Poliphenols are associated with inhibition of several pathological processes, including cancer. Soluble phenols are contained in the aqueous part of olive and are discarded during oil production in the \u2018olive mill wastewaters\u2019. Here we investigated the properties of a purified extract of olive mill wastewaters, named A009, as potential anti-angiogenic compound. While the strong anti-oxidant activity of olive derived phenolic compounds has been well characterized, little is known about their anti-angiogenic properties. We investigated effects of A009 on endothelial cell morphogenesis, proliferation, migration and apoptosis, comparing the results obtained with the activity of a well-characterized olive oil phenol, Hydroxytyrosol (HT). Further, we tested the effects of A009 and HT in an in vivo angiogenesis assay. We found that A009 exerted strong anti-angiogenic effects both in vitro and in vivo, and that the complex natural purified extract has stronger anti-angiogenic potential when compared to the same concentrations of HT in most of the assays performed. These data demonstrate that a novel purified, phenols enriched, extract with anti-angiogenic, and angiopreventive potential can be obtained from olive oil mill waste material, recovering useful products from an agricultural waste

Biomarkers of cancer angioprevention for clinical studies

With the great advances made in the treatment and prevention of infectious diseases over the last century, chronic degenerative Diseases-cardiovascular, cerebrovascular, and cancer-represent the major causes of death in the developed world. Although massive efforts and investments have been made in cancer therapy, the progress made towards reducing mortality has been more successful for cardiovascular disease than for tumours. This can be attributable largely to an active prevention approach implemented for cardiovascular disease. Cardiologists treat their patients before the overt disease becomes life threatening, performing early interventions in phenotypically healthy patients, by using several markers that predict risk. If the concept of prevention could be applied to cancer in a more extensive way, a significant number of tumours could be avoided through preventive measures. Prevention approaches range from avoiding tobacco exposure to dietary strategies to active pharmacological approaches in higher risk groups. Host targets rather than the tumour cells themselves are attractive for chemoprevention, in particular endothelial and immune cells. Angioprevention i.e. preventing cancer angiogenesis is a key concept that we introduced; yet one of the major current challenges for anti-angiogenesis in therapy and prevention is finding the right biomarkers. Here we discuss the importance of angioprevention and the potential use of VEGF, PlGF, CD31, Ang and Tie, circulating vascular cell precursors, and microRNA as potential biomarkers

Preliminary evidence on the diagnostic and molecular role of circulating soluble EGFR in non-small cell lung cancer

Assessment of biological diagnostic factors providing clinically-relevant information to guide physician decision-making are still needed for diseases with poor outcomes, such as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a promising molecule in the clinical management of NSCLC. While the EGFR transmembrane form has been extensively investigated in large clinical trials, the soluble, circulating EGFR isoform (sEGFR), which may have a potential clinical use, has rarely been considered. This study investigates the use of sEGFR as a potential diagnostic biomarker for NSCLC and also characterizes the biological function of sEGFR to clarify the molecular mechanisms involved in the course of action of this protein. Plasma sEGFR levels from a heterogeneous cohort of 37 non-advanced NSCLC patients and 54 healthy subjects were analyzed by using an enzyme-linked immunosorbent assay. The biological function of sEGFR was analyzed in vitro using NSCLC cell lines, investigating effects on cell proliferation and migration. We found that plasma sEGFR was significantly decreased in the NSCLC patient group as compared to the control group (median value: 48.6 vs. 55.6 ng/mL respectively; p = 0.0002). Moreover, we demonstrated that sEGFR inhibits growth and migration of NSCLC cells in vitro through molecular mechanisms that included perturbation of EGF/EGFR cell signaling and holoreceptor internalization. These data show that sEGFR is a potential circulating biomarker with a physiological protective role, providing a first approach to the functional role of the soluble isoform of EGFR. However, the impact of these data on daily clinical practice needs to be further investigated in larger prospective studies

Tracing Behçet's disease origins along the Silk Road: an anthropological evolutionary genetics perspective

Objective. Behçet's disease is a multifactorial vasculitis that shows its highest prevalence in geographical areas historically involved in the Silk Road, suggesting that it might have originated somewhere along these ancient trade routes. This study aims to provide a first clue towards genetic evidence for this hypothesis by testing it via an anthropological evolutionary genetics approach. Methods. Behçet's disease variation at ancestry informative mitochondrial DNA control region and haplogroup diagnostic sites was characterised in 185 disease subjects of Italian descent and set into the Eurasian mitochondrial landscape by comparison with nearly 9,000 sequences representative of diversity observable in Italy and along the main Silk Road routes. Results. Dissection of the actual genetic ancestry of disease individuals by means of population structure, spatial autocorrelation and haplogroup analyses revealed their closer relationships with some Middle Eastern and Central Asian groups settled along the Silk Road than with healthy Italians. Conclusion. These findings support the hypothesis that the Behçet's disease genetic risk has migrated to western Eurasia in parallel with ancestry components typical of Silk Road-related groups. This provided new insights that are useful to improve the understanding of disease origins and diffusion, as well as to inform future association studies aimed at properly accounting for the actual genetic ancestry of the examined Behçet's disease samples in order to minimise the detection of spurious associations and to improve the identification of genetic variants with actual clinical relevance

Effects of mir-133b and erlotinib on EGFR and EGFR pathway.

<p><b>A</b> Flow cytometry to evaluate EGFR surface expression. Expression relative to lipofectamine treated cells is shown (mean ± SEM of three independent experiments is shown). *<i>p</i><0.05 by the one sample <i>t</i> test with hypothetical value = 100. <b>B</b> Western blot assay to detect total EGFR, total ERK, GAPDH and phosphorylated ERK. Cropped blots are displayed. Full length blots are showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196350#pone.0196350.s001" target="_blank">S1 Fig</a>. Exposure time: EGFR 200 sec, GAPDH 90 sec, pERK 420 sec, total EGFR 90 sec.</p

Effects of miR-133b in combination to erlotinib on NSCLC cell lines.

<p>Cells were seeded at 1.5 x 10⁵ cells/well and cultured up to 72 hours. Data are presented as mean ± SEM of three independent experiments. <b>A</b> Cell growth relative to lipofectamine-treated cells. <b>B</b> Photographs of A549 and H1299 after 72 h treatment. Magnification 400X. <b>C</b> Cell shape measured as forward scatter (FSC) shown on the top and side scatter (SSC) shown on the bottom. Note that the axes in each diagram are displayed in relative percent scale. *<i>p</i><0.05 by the one sample <i>t</i> test with hypothetical value = 100.</p

MiRNAs expression in relation to response to erlotinib.

<p><b>A</b> Analysis of miRNA expression was investigated using TaqMan real-time quantitative PCR in NSCLC specimen from "responders" (n = 8) and "non-responders" patients (n = 24). Results are shown as normalized expression: 2^−ΔCt compared with Mann-Whitney test. <b>B</b> ROC curve analysis of miRNA levels to predict response to erlotinib. AUC = Area Under the Curve; CI = Confidence Interval; Thr = Threshold; Se = Sensitivity; Sp = Specificity.</p

Clinicopathological characteristics of NSCLC patients at diagnosis classified as responders and non-responders to erlotinib.

<p>Clinicopathological characteristics of NSCLC patients at diagnosis classified as responders and non-responders to erlotinib.</p