Drug-induced senescence bystander proliferation in prostate cancer cells in vitro and in vivo

Senescence is a distinct cellular response induced by DNA-damaging agents and other sublethal stressors and may provide novel benefits in cancer therapy. However, in an ageing model, senescent fibroblasts were found to stimulate the proliferation of cocultured cells. To address whether senescence induction in cancer cells using chemotherapy induces similar effects, we used GFP-labelled prostate cancer cell lines and monitored their proliferation in the presence of proliferating or doxorubicin-induced senescent cancer cells in vitro and in vivo. Here, we show that the presence of senescent cancer cells increased the proliferation of cocultured cells in vitro through paracrine signalling factors, but this proliferative effect was significantly less than that seen with senescent fibroblasts. In vivo, senescent cancer cells failed to increase the establishment, growth or proliferation of LNCaP and DU145 xenografts in nude mice. Senescent cells persisted as long as 5 weeks in tumours. Our results demonstrate that although drug-induced senescent cancer cells stimulate the proliferation of bystander cells in vitro, this does not significantly alter the growth of tumours in vivo. Coupled with clinical observations, these data suggest that the proliferative bystander effects of senescent cancer cells are negligible and support the further development of senescence induction as therapy

Establishment of Protein Delivery Systems Targeting Podocytes

Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes

PM2.5 metal exposures and nocturnal heart rate variability: a panel study of boilermaker construction workers

<p>Abstract</p> <p>Background</p> <p>To better understand the mechanism(s) of particulate matter (PM) associated cardiovascular effects, research priorities include identifying the responsible PM characteristics. Evidence suggests that metals play a role in the cardiotoxicity of fine PM (PM_2.5) and in exposure-related decreases in heart rate variability (HRV). We examined the association between daytime exposure to the metal content of PM_2.5and night HRV in a panel study of boilermaker construction workers exposed to metal-rich welding fumes.</p> <p>Methods</p> <p>Twenty-six male workers were monitored by ambulatory electrocardiogram (ECG) on a workday while exposed to welding fume and a non-workday (baseline). From the ECG, rMSSD (square root of the mean squared differences of successive intervals) was summarized over the night (0:00–7:00). Workday, gravimetric PM_2.5samples were analyzed by x-ray fluorescence to determine metal content. We used linear mixed effects models to assess the associations between night rMSSD and PM_2.5metal exposures both with and without adjustment for total PM_2.5. Matched ECG measurements from the non-workday were used to control for individual cardiac risk factors and models were also adjusted for smoking status. To address collinearity between PM_2.5and metal content, we used a two-step approach that treated the residuals from linear regression models of each metal on PM_2.5as surrogates for the differential effects of metal exposures in models for night rMSSD.</p> <p>Results</p> <p>The median PM_2.5exposure was 650 μg/m³; median metal exposures for iron, manganese, aluminum, copper, zinc, chromium, lead, and nickel ranged from 226 μg/m³to non-detectable. We found inverse linear associations in exposure-response models with increased metal exposures associated with decreased night rMSSD. A statistically significant association for manganese was observed, with a decline of 0.130 msec (95% CI: -0.162, -0.098) in night rMSSD for every 1 μg/m³increase in manganese. However, even after adjusting for individual metals, increases in total PM_2.5exposures were associated with declines in night rMSSD.</p> <p>Conclusion</p> <p>These results support the cardiotoxicity of PM_2.5metal exposures, specifically manganese. However the metal component alone did not account for the observed declines in night HRV. Therefore, results suggest the importance of other PM elemental components.</p

Characterization of a Novel Binding Protein for Fortilin/TCTP — Component of a Defense Mechanism against Viral Infection in Penaeus monodon

The Fortilin (also known as TCTP) in Penaeus monodon (PmFortilin) and Fortilin Binding Protein 1 (FBP1) have recently been shown to interact and to offer protection against the widespread White Spot Syndrome Virus infection. However, the mechanism is yet unknown. We investigated this interaction in detail by a number of in silico and in vitro analyses, including prediction of a binding site between PmFortilin/FBP1 and docking simulations. The basis of the modeling analyses was well-conserved PmFortilin orthologs, containing a Ca2+-binding domain at residues 76–110 representing a section of the helical domain, the translationally controlled tumor protein signature 1 and 2 (TCTP_1, TCTP_2) at residues 45–55 and 123–145, respectively. We found the pairs Cys59 and Cys76 formed a disulfide bond in the C-terminus of FBP1, which is a common structural feature in many exported proteins and the “x–G–K–K” pattern of the amidation site at the end of the C-terminus. This coincided with our previous work, where we found the “x–P–P–x” patterns of an antiviral peptide also to be located in the C-terminus of FBP1. The combined bioinformatics and in vitro results indicate that FBP1 is a transmembrane protein and FBP1 interact with N-terminal region of PmFortilin

Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice

James Ervasti and colleagues show that injection of a truncated form of utrophin transduced all tissues examined, integrated with members of the dystrophin complex, and reduced serum levels of creatine kinase in a mouse model of muscular dystrophy

Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid β Protein

Alzheimer's disease (AD) is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of β-amyloid (Aβ) proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Aβ proteins due to their inefficient clearance at the blood-brain-barrier (BBB), places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Aβ proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT) mouse brain slices treated with fluorescein labeled Aβ40 (F-Aβ40) demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Aβ proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH) neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Aβ40 or F-Aβ42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Aβ40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Aβ40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Aβ proteins and help explain the vulnerability of cortical and hippocampal neurons to Aβ toxicity

In Vivo Imaging of HIF-Active Tumors by an Oxygen-Dependent Degradation Protein Probe with an Interchangeable Labeling System

Hypoxia-inducible factor (HIF) functions as a master transcriptional regulator for adaptation to hypoxia by inducing adaptive changes in gene expression for regulation of proliferation, angiogenesis, apoptosis and energy metabolism. Cancers with high expression of the alpha subunit of HIF (HIFα) are often malignant and treatment-resistant. Therefore, the development of a molecular probe that can detect HIF activity has great potential value for monitoring tumor hypoxia. HIF prolyl hydroxylases (HPHDs) act as oxygen sensors that regulate the fate of HIFα protein through its oxygen-dependent degradation (ODD) domain. We constructed a recombinant protein PTD-ODD-HaloTag (POH) that is under the same ODD regulation as HIFα and contains protein transduction domain (PTD) and an interchangeable labeling system. Administration of near-infrared fluorescently labeled POH (POH-N) to mouse models of cancers allowed successful monitoring of HIF-active regions. Immunohistochemical analysis for intratumoral localization of POH probe revealed its specificity to HIF-active cells. Furthermore, lack of the PTD domain or a point mutation in the ODD domain abrogated the specificity of POH-N to HIF-active cells. Overall results indicate that POH is a practical probe specific to HIF-active cell in cancers and suggest its large potential for imaging and targeting of HIF-related diseases

Selective sigma-2 ligands preferentially bind to pancreatic adenocarcinomas: applications in diagnostic imaging and therapy

<p>Abstract</p> <p>Background</p> <p>Resistance to modern adjuvant treatment is in part due to the failure of programmed cell death. Therefore the molecules that execute the apoptotic program are potential targets for the development of anti-cancer therapeutics. The sigma-2 receptor has been found to be over-expressed in some types of malignant tumors, and, recently, small molecule ligands to the sigma-2 receptor were found to induce cancer cell apoptosis.</p> <p>Results</p> <p>The sigma-2 receptor was expressed at high levels in both human and murine pancreas cancer cell lines, with minimal or limited expression in normal tissues, including: brain, kidney, liver, lung, pancreas and spleen. Micro-PET imaging was used to demonstrate that the sigma-2 receptor was preferentially expressed in tumor as opposed to normal tissues in pancreas tumor allograft-bearing mice. Two structurally distinct sigma-2 receptor ligands, SV119 and WC26, were found to induce apoptosis to mice and human pancreatic cancer cells <it>in vitro </it>and <it>in vivo</it>. Sigma-2 receptor ligands induced apoptosis in a dose dependent fashion in all pancreatic cell lines tested. At the highest dose tested (10 μM), all sigma-2 receptor ligands induced 10–20% apoptosis in all pancreatic cancer cell lines tested (p < 0.05). In pancreas tumor allograft-bearing mice, a single bolus dose of WC26 caused approximately 50% apoptosis in the tumor compared to no appreciable apoptosis in tumor-bearing, vehicle-injected control animals (p < 0.0001). WC26 significantly slowed tumor growth after a 5 day treatment compared to vehicle-injected control animals (p < 0.0001) and blood chemistry panels suggested that there is minimal peripheral toxicity.</p> <p>Conclusion</p> <p>We demonstrate a novel therapeutic strategy that induces a significant increase in pancreas cancer cell death. This strategy highlights a new potential target for the treatment of pancreas cancer, which has little in the way of effective treatments.</p

Module M1 of Zebrafish Neuroglobin Acts as a Structural and Functional Protein Building Block for a Cell-Membrane-Penetrating Activity

Neuroglobin (Ngb) is a recently discovered vertebrate globin that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection during oxidative stress that occurs, for example, during ischemia and reperfusion. Recently, we found that zebrafish, but not human, Ngb can translocate into cells. Moreover, we demonstrated that a chimeric ZHHH Ngb protein, in which the module M1 of human Ngb is replaced by the corresponding region of zebrafish Ngb, can penetrate cell membranes and protect cells against oxidative stress-induced cell death, suggesting that module M1 of zebrafish Ngb is important for protein transduction. Furthermore, we recently showed that Lys7, Lys9, Lys21, and Lys23 in module M1 of zebrafish Ngb are crucial for protein transduction activity. In the present study, we have investigated whether module M1 of zebrafish Ngb can be used as a building block to create novel cell-membrane-penetrating folded proteins. First, we engineered a chimeric myoglobin (Mb), in which module M1 of zebrafish Ngb was fused to the N-terminus of full-length human Mb, and investigated its functional and structural properties. Our results showed that this chimeric Mb protein is stable and forms almost the same heme environment and α-helical structure as human wild-type Mb. In addition, we demonstrated that chimeric Mb has a cell-membrane-penetrating activity similar to zebrafish Ngb. Moreover, we found that glycosaminoglycan is crucial for the cell-membrane-penetrating activity of chimeric Mb as well as that of zebrafish Ngb. These results enable us to conclude that such module substitutions will facilitate the design and production of novel functional proteins