Wnt signalling in prostate cancer stem-like cells

The work reported in this thesis describes the characterisation of cell derived colonies from cancer cell lines and mechanisms of a key signalling pathway within these. PC3 cells were cultured at clonal density and resultant colony formation objectively described. Wnt signalling was investigated in colony types using a live [Ca2+]i assay. Exploratory work regarding ion channel gene and protein expression in 3 prostate cancer cell lines was performed. Preliminary investigation on the effects of MPRC (membrane potential regulating compounds) was performed using a cell proliferation assay. PC3 prostate cancer cells form distinct colonies with different morphologies. These different colony types can be characterised using quantitative parameters such as cell density and percentage cellularity, unlike previously employed visual only characteristics. PC3 cells form two types of colonies from a single cell. In this thesis these are termed holo/meroclones and paraclones. Cells within both colony types respond to Wnt activation, demonstrated by a response in free intracellular calcium [Ca2+]i . In addition, there is a suggestion of increased expression of, and translocation into the nucleus of the transcription factor co- activator β-catenin again in response to both Wnt 5A and Wnt 9B in both colony types. The characteristics of Wnt induced calcium release vary between different types of colonies. This is the first report of Wnt mediated [Ca2+]i release in response to both Wnt 5A and Wnt 9B in different PC3 colony types and the first report suggestive of β-catenin translocation following Wnt activation in colonies derived from stem-like cells

Planar charged-particle trajectories in multipole magnetic fields

International audienceThis paper provides a complete generalization of the classic result that the radius of curvature (?) of a charged-particle trajectory confined to the equatorial plane of a magnetic dipole is directly proportional to the cube of the particle's equatorial distance (?) from the dipole (i.e. ? ? ?3). Comparable results are derived for the radii of curvature of all possible planar charged-particle trajectories in an individual static magnetic multipole of arbitrary order m and degree n. Such trajectories arise wherever there exists a plane (or planes) such that the multipole magnetic field is locally perpendicular to this plane (or planes), everywhere apart from possibly at a set of magnetic neutral lines. Therefore planar trajectories exist in the equatorial plane of an axisymmetric (m = 0), or zonal, magnetic multipole, provided n is odd: the radius of curvature varies directly as ?n+2. This result reduces to the classic one in the case of a zonal magnetic dipole (n =1). Planar trajectories exist in 2m meridional planes in the case of the general tesseral (0 m n) magnetic multipole. These meridional planes are defined by the 2m roots of the equation cos[m(? ? ?nm)] = 0, where ?nm = (1/m) arctan (hnm/gnm); gnm and hnm denote the spherical harmonic coefficients. Equatorial planar trajectories also exist if (n ? m) is odd. The polar axis (? = 0,?) of a tesseral magnetic multipole is a magnetic neutral line if m > 1. A further 2m(n ? m) neutral lines exist at the intersections of the 2m meridional planes with the (n ? m) cones defined by the (n ? m) roots of the equation Pnm(cos ?) = 0 in the range 0 ? ?, where Pnm(cos ?) denotes the associated Legendre function. If (n ? m) is odd, one of these cones coincides with the equator and the magnetic field is then perpendicular to the equator everywhere apart from the 2m equatorial neutral lines. The radius of curvature of an equatorial trajectory is directly proportional to ?n+2 and inversely proportional to cos[m(? ? ?nm)]. Since this last expression vanishes at the 2m equatorial neutral lines, the radius of curvature becomes infinitely large as the particle approaches any one of these neutral lines. The radius of curvature of a meridional trajectory is directly proportional to rn+2, where r denotes radial distance from the multipole, and inversely proportional to Pnm(cos ?)/sin θ. Hence the radius of curvature becomes infinitely large if the particle approaches the polar magnetic neutral line (m > 1) or any one of the 2m(n ? m) neutral lines located at the intersections of the 2m meridional planes with the (n ? m) cones. Illustrative particle trajectories, derived by stepwise numerical integration of the exact equations of particle motion, are presented for low-degree (n ? 3) magnetic multipoles. These computed particle trajectories clearly demonstrate the "non-adiabatic'' scattering of charged particles at magnetic neutral lines. Brief comments are made on the different regions of phase space defined by regular and irregular trajectories

Influence of molecular imaging on patient selection for treatment intensification prior to salvage radiation therapy for prostate cancer: a post hoc analysis of the PROPS trial.

BACKGROUND: The impact of molecular imaging (MI) on patient management after biochemical recurrence (BCR) following radical prostatectomy has been described in many studies. However, it is not known if MI-induced management changes are appropriate. This study aimed to determine if androgen deprivation therapy (ADT) management plan is improved by MI in patients who are candidates for salvage radiation therapy. METHODS: Data were analyzed from the multicenter prospective PROPS trial evaluating PSMA/Choline PET in patients being considered for salvage radiotherapy (sRT) with BCR after prostatectomy. We compared the pre- and post-MI ADT management plans for each patient and cancer outcomes as predicted by the MSKCC nomogram. A higher percentage of predicted BCR associated with ADT treatment intensification after MI was considered as an improvement in a patient's management. RESULTS: Seventy-three patients with a median PSA of 0.38 ng/mL were included. In bivariate analysis, a positive finding on MI (local or metastatic) was associated with decision to use ADT with an odds ratio of 3.67 (95% CI, 1.25 to 10.71; p = 0.02). No factor included in the nomogram was associated with decision to use ADT. Also, MI improved selection of patients to receive ADT based on predicted BCR after sRT : the predicted nomogram 5-year biochemical-free survivals were 52.5% and 43.3%, (mean difference, 9.2%; 95% CI 0.8 to 17.6; p = 0.03) for sRT alone and ADT±sRT subgroups, while there was no statistically significant difference between subgroups before MI. CONCLUSIONS: PSMA and/or Choline PET/CT before sRT can potentially improve patient ADT management by directing clinicians towards more appropriate intensification

Contractile and structural properties of detrusor from children with neurogenic lower urinary tract dysfunction

Neurogenic lower urinary tract (NLUT) dysfunction in paediatric patients can arise after congenital or acquired conditions that affect bladder innervation. With some patients, urinary tract dysfunction remains and is more difficult to treat without understanding the pathophysiology. We measured in vitro detrusor smooth muscle function of samples from such bladders and any association with altered Wnt-signalling pathways that contribute to both foetal development and connective tissue deposition. A comparator group was tissue from children with normally functioning bladders. Nerve-mediated and agonist-induced contractile responses and passive stiffness were measured. Histology measured smooth muscle and connective tissue proportions, and multiplex immunohistochemistry recorded expression of protein targets associated with Wnt-signalling pathways. Detrusor from the NLUT group had reduced contractility and greater stiffness, associated with increased connective tissue content. Immunohistochemistry showed no major changes to Wnt-signalling components except down-regulation of c-Myc, a multifunctional regulator of gene transcription. NLUT is a diverse term for several diagnoses that disrupt bladder innervation. While we cannot speculate about the reasons for these pathophysiological changes, their recognition should guide research to understand their ultimate causes and develop strategies to attenuate and even reverse them. The role of changes to the Wnt-signalling pathways was minor. View Full-Tex

A Novel Docetaxel-Loaded Poly (ε-Caprolactone)/Pluronic F68 Nanoparticle Overcoming Multidrug Resistance for Breast Cancer Treatment

Multidrug resistance (MDR) in tumor cells is a significant obstacle to the success of chemotherapy in many cancers. The purpose of this research is to test the possibility of docetaxel-loaded poly (ε-caprolactone)/Pluronic F68 (PCL/Pluronic F68) nanoparticles to overcome MDR in docetaxel-resistance human breast cancer cell line. Docetaxel-loaded nanoparticles were prepared by modified solvent displacement method using commercial PCL and self-synthesized PCL/Pluronic F68, respectively. PCL/Pluronic F68 nanoparticles were found to be of spherical shape with a rough and porous surface. The nanoparticles had an average size of around 200 nm with a narrow size distribution. The in vitro drug release profile of both nanoparticle formulations showed a biphasic release pattern. There was an increased level of uptake of PCL/Pluronic F68 nanoparticles in docetaxel-resistance human breast cancer cell line, MCF-7 TAX30, when compared with PCL nanoparticles. The cytotoxicity of PCL nanoparticles was higher than commercial Taxotere®in the MCF-7 TAX30 cell culture, but the differences were not significant (p > 0.05). However, the PCL/Pluronic F68 nanoparticles achieved significantly higher level of cytotoxicity than both of PCL nanoparticles and Taxotere®(p < 0.05), indicating docetaxel-loaded PCL/Pluronic F68 nanoparticles could overcome multidrug resistance in human breast cancer cells and therefore have considerable potential for treatment of breast cancer

Chemoproteomics reveals Toll-like receptor fatty acylation

Partial funding for Open Access provided by The Ohio State University Open Access Fund.Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells. Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.This work was supported by funding from the NIH/NIAID (grant R00AI095348 to J.S.Y.), the NIH/NIGMS (R01GM087544 to HCH), and the Ohio State University Public Health Preparedness for Infectious Diseases (PHPID) program. NMC is supported by the Ohio State University Systems and Integrative Biology Training Program (NIH/NIGMS grant T32GM068412). BWZ is a fellow of the National Science Foundation Graduate Research Fellowship Program (DGE-0937362)

Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy

<p>Abstract</p> <p>Background</p> <p>Many peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge.</p> <p>Methods</p> <p>To address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) <it>in vitro</it>.</p> <p>Results</p> <p>We confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses.</p> <p>Conclusion</p> <p>Encapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.</p

PGC-1α Inhibits Oleic Acid Induced Proliferation and Migration of Rat Vascular Smooth Muscle Cells

BACKGROUND: Oleic acid (OA) stimulates vascular smooth muscle cell (VSMC) proliferation and migration. The precise mechanism is still unclear. We sought to investigate the effects of peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1 alpha (PGC-1alpha) on OA-induced VSMC proliferation and migration. PRINCIPAL FINDINGS: Oleate and palmitate, the most abundant monounsaturated fatty acid and saturated fatty acid in plasma, respectively, differently affect the mRNA and protein levels of PGC-1alpha in VSMCs. OA treatment resulted in a reduction of PGC-1alpha expression, which may be responsible for the increase in VSMC proliferation and migration caused by this fatty acid. In fact, overexpression of PGC-1alpha prevented OA-induced VSMC proliferation and migration while suppression of PGC-1alpha by siRNA enhanced the effects of OA. In contrast, palmitic acid (PA) treatment led to opposite effects. This saturated fatty acid induced PGC-1alpha expression and prevented OA-induced VSMC proliferation and migration. Mechanistic study demonstrated that the effects of PGC-1alpha on VSMC proliferation and migration result from its capacity to prevent ERK phosphorylation. CONCLUSIONS: OA and PA regulate PGC-1alpha expression in VSMCs differentially. OA stimulates VSMC proliferation and migration via suppression of PGC-1alpha expression while PA reverses the effects of OA by inducing PGC-1alpha expression. Upregulation of PGC-1alpha in VSMCs provides a potential novel strategy in preventing atherosclerosis