Cell-to-Cell Signaling Influences the Fate of Prostate Cancer Stem Cells and Their Potential to Generate More Aggressive Tumors

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44+CD24− phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications

An investor-oriented approach for regulation on equity crowdfunding

Abstract Purpose \u2013 The aim of this research is to contribute to the existing literature about the entrepreneurial conditions in crowd-based contexts by describing how different European countries regulate equity crowdfunding market in order to incentive the investments and protect investors. Design/methodology/approach \u2013 Based on a legal acts\u2019 analysis, we conduct a qualitative study comparing the crowdfunding regulation addressed to investors. In particular, we focus our analysis on the European countries with the highest concentration of crowdfunding platforms (i.e. the UK, Germany, France, Italy and Spain). Findings \u2013 The results show that some countries, such as the UK, Germany and France, present an investor- oriented approach based on non-restrictive regulation, while other countries, such as Spain and Italy, have a restrictive approach that protects investors excessively and discourages them. In particular, the case study of France shows how the introduction of unrestricted regulation can produce positive effects on the volume of crowdfunding transactions. Practical implications \u2013 The paper is addressed to investors, policymakers and intermediaries (platforms) to help the first in orienting themselves between the different crowdfunding regulations and the latter in aligning and orchestrating rules and norms. Originality/value \u2013 This is the first study that analyses the role of investor-oriented regulations in the promotion of entrepreneurship through the identification of four key factors to monitor equity crowdfunding regulations

Action of retinoic acid receptor on EGFR gene transactivation and breast cancer cell proliferation: Interplay with the estrogen receptor

In the present report, we investigated the action of retinoic acid (RA) on the transactivation of the epidermal growth factor receptor (EGFR) gene promoter. In a previous study, we showed that the estrogen receptor (ER) α activated by 17β-estradiol (E 2) increased EGFR expression by enhancing the binding of the transcription factor Sp1 to the EGFR minimal promoter in HeLa cells. Here, we demonstrate that ligand-activated RA receptor (RAR) α inhibited EGFR transactivation by competing with Sp1 for binding to the same promoter fragment in the same cell model. When RARα and ERα were coexpressed, the inhibitory effect of RA on transactivation of the EGFR promoter counteracted the enhancement induced by E 2-activated ERα and became more pronounced in the presence of ligand-free ERα. In the MCF7 breast cancer cell line, which endogenously expresses RARα and ERα, RA exerted anti-proliferative effects in the presence of ligand-free ERα. Moreover, interplay between the pathways mediated by the two receptors was observed, as RA counteracted E 2-induced cell proliferation. Our results suggest that the interference with the activity of Sp1 on the EGFR promoter could be related to the observed RA-mediated growth suppression of breast cancer cells. © 2011 Elsevier Masson SAS

Safety and immunogenicity of co-administered MF59-adjuvanted 2009 pandemic and plain 2009-10 seasonal influenza vaccines in rheumatoid arthritis patients on biologicals

Rheumatoid arthritis (RA) patients under immunosuppressive therapy are particularly susceptible to infections, mainly of the respiratory tract, thus vaccination may represent a strategy to reduce their incidence in this vulnerable population. In the 2009-10 influenza season, the safety and immunogenicity of co-administered non-adjuvanted seasonal and MF59-adjuvanted pandemic influenza vaccines were evaluated in this study in 30 RA patients under therapy with anti-tumour necrosis factor (TNF)-α agents or Abatacept and in 13 healthy controls (HC). Patients and HC underwent clinical and laboratory evaluation before (T0), 1 (T1) and 6 months (T2) after vaccinations. No severe adverse reactions, but a significant increase in total mild side effects in patients versusHC were observed. Both influenza vaccines fulfilled the three criteria of the Committee for Proprietary Medicinal Products (CPMP). Seroconversion rate for any viral strain in patients and HC was, respectively, 68 versus 45 for H1-A/Brisbane/59/07, 72 versus 81 for H3-A/Brisbane/10/07, 68 versus 54 for B/Brisbane/60/08 and 81 versus 54 for A/California/7/2009. A slight increase in activated interferon (IFN)-γ-, TNF-α- or interleukin (IL)-17A-secreting T cells at T1 compared to T0, followed by a reduction at T2 in both patients and HC, was registered. In conclusion, simultaneous administration of adjuvanted pandemic and non-adjuvanted seasonal influenza vaccines is safe and highly immunogenic. The largely overlapping results between patients and HC, in terms of antibody response and cytokine-producing T cells, may represent further evidence for vaccine safety and immunogenicity in RA patients on biological

Parallelism between the phenotype of cells held in tumors and of CSC grown <i>in vitro</i>.

<p>In particular, CSC grown in FBS-medium aimed to reproduce the injection of 1 spheroid in mice, whereas CSC grown in CM from DU145 cells allowed us to simulate the injection of 5,000 unsorted DU145 cells.</p

Analysis of CSC <i>vs</i> DU145 cells by microarray, IPA and Q-PCR analysis.

<p>(<b>A</b>) Selected 8 top biological functions found enriched within the set of transcripts modulated in CSC with respect to total DU145 cells by microarray analysis. (<b>B</b>) The 22 deregulated genes, as determined by IPA, are positioned in subcellular layouts, and relationships are marked by arrows: filled and dashed line arrows mark direct and indirect interactions, respectively. Genes in red showed increased expression in CSC, while genes in green were down-regulated. (<b>C</b>) Differences in gene expression determined by microarray analysis were confirmed by Q-PCR, choosing 7 representative genes among selected 22. Results are expressed as ratio between each target gene and the endogenous control GAPDH. Expression in CSC was compared to what was observed in DU145 cells to which a value equal to 1 was arbitrarily assigned. Values are mean ± SEM from 3 experiments. *, <i>p</i><0.05 relative to DU145 cells.</p

Structure and composition of spheroids.

<p>(<b>A</b>) Phase contrast evaluation of spheroid structure (10×). (<b>B</b>) TEM analysis showing desmosomes and adherent junctions (top and bottom, respectively) as well as the annulate lamellae (<b>C</b>) in spheroid cells. (<b>D</b>) Phase contrast (20×) of a fragment of spheroid surrounded by cells characterized by protruding blebs of empty cytoplasm, which present intact nuclei as shown by DAPI staining (40×, <b>E</b>). (<b>F</b>) Staining with trypan blue shows the presence of dead cells in spheroids (10×). Immunofluorescence staining of a section of spheroids and DU145 cells (original magnification ×200) showing the expression of E-cadherin (<b>G</b>), β-catenin (<b>H</b>) and vimentin (<b>I</b>).</p

Analysis of tumors generated in NOD/SCID mice.

<p>(<b>A</b>) Histological staining with H&E of tumors grown following the injection of 1 spheroid or 5,000 DU145 cells showing highly invasive (left panel) and scantly invasive (right) phenotype (5×). Asterisks indicate the borders between tumors and the surrounding tissues. (<b>B</b>) Representative western blots showing the expression of E-cadherin, β-catenin and vimentin in tumors. (<b>C</b>) IHC staining with anti-mouse CD31 antibody highlighting higher vessel density in spheroid-derived tumors (indicated by asterisks, left panel) compared to tumors from DU145 cells (right). (<b>D</b>) Q-PCR showing mouse CD31 expression in tumors. Mouse B2M was used as endogenous control. Values are mean ± SEM from 3 experiments. *, <i>p</i><0.05 relative to tumors generated from spheroid. (<b>E</b>) IHC with anti-CD56 antibody showing higher density of NE cells in tumors generated from spheroid than from DU145 cells (20×). (<b>F</b>) Q-PCR displaying CGA mRNA level in tumors. Results are expressed as ratios between the target gene and the endogenous control GAPDH and represent mean ± SEM from 3 independent experiments. *, <i>p</i><0.05 with respect to tumors generated from spheroid. (<b>G</b>) A representative FACS analysis showing the presence of CD44⁺CD24⁻ cells in tumors generated from the injection of 1 spheroid in mice. Tumors originating from the injection of 5,000 DU145 cells showed a comparable CD44⁺CD24⁻ cell population (data not shown).</p

Prevalence of Hepatitis B Virus Markers in Patients with Autoimmune Inflammatory Rheumatic Diseases in Italy

Chronic hepatitis B virus (HBV) infection may be reactivated by immunosuppressive drugs in patients with autoimmune inflammatory rheumatic diseases. This study evaluates HBV serum markers&rsquo; prevalence in rheumatic outpatients belonging to Spondyloarthritis, Chronic Arthritis and Connective Tissue Disease diagnostic groups in Italy. The study enrolled 302 subjects, sex ratio (M/F) 0.6, mean age &plusmn; standard deviation 57 &plusmn; 15 years, 167 (55%) of whom were candidates for immunosuppressive therapy. The Spondyloarthritis group included 146 subjects, Chronic Arthritis 75 and Connective Tissue Disease 83 (two patients had two rheumatic diseases; thus, the sum is 304 instead of 302). Ten subjects (3%) reported previous anti-HBV vaccination and tested positive for anti-HBs alone with a titer still protective (&gt;10 IU/mL). Among the remaining 292 subjects, the prevalence of positivity for HBsAg, isolated anti-HBc, anti-HBc/anti-HBs, and any HBV marker was 2%, 4%, 18%, and 24%, respectively. A total of 26/302 (9%) patients with &gamma;-globulin levels &le;0.7 g/dL were more frequently (p = 0.03455) prescribed immunosuppressive therapy, suggesting a more severe rheumatic disease. A not negligible percentage of rheumatic patients in Italy are at potential risk of HBV reactivation related to immunosuppressive therapy. Before starting treatment, subjects should be tested for HBV markers. Those resulting positive should receive treatment or prophylaxis with Nucleos (t) ides analogue (NUCs) at high barrier of resistance, or pre-emptive therapy, according to the pattern of positive markers. HB vaccination is recommended for those who were never exposed to the virus

Influence of culture conditions on the expression of lipocalin-2 and E-cadherin in CSC.

<p>Q-PCR showing changes in mRNA levels for lipocalin-2 and E-cadherin in DU145 cells, CSC contained in control spheroids, and CSC grown for 20 days in FBS-medium or CM from DU145 cells. Data represent mean ± SEM from 3 individual experiments. Asterisks indicate a significant difference (*, <i>p</i><0.05).</p