Janina M. Tomm

MiR-130a, miR-203 and miR-205 jointly repress key oncogenic pathways and are downregulated in prostate carcinoma

With ∼30 000 deaths annually in the United States, prostate cancer (PCa) is a major oncologic disease. Here we show that the microRNAs miR-130a, miR-203 and miR-205 jointly interfere with the two major oncogenic pathways in prostate carcinoma and are downregulated in cancer tissue. Using transcriptomics we show that the microRNAs repress several gene products known to be overexpressed in this cancer. Argonaute 2 (AGO2) co-immunoprecipitation, reporter assays and western blot analysis demonstrate that the microRNAs directly target several components of the mitogen-activated protein kinase (MAPK) and androgen receptor (AR) signaling pathways, among those several AR coregulators and HRAS (Harvey rat sarcoma viral oncogene homolog), and repress signaling activity. Both pathways are central for the development of the primary tumor and in particular the progression to its incurable castration-resistant form. Reconstitution of the microRNAs in LNCaP PCa cells induce morphological changes, which resemble the effect of androgen deprivation, and jointly impair tumor cell growth by induction of apoptosis and cell cycle arrest. We therefore propose that these microRNAs jointly act as tumor suppressors in prostate carcinoma and might interfere with progression to castration resistance.

Combined Proteomic and Metabolomic Profiling of Serum Reveals Association of the Complement System with Obesity and Identifies Novel Markers of Body Fat Mass Changes

Obesity is associated with multiple adverse health effects and a high risk of developing metabolic and cardiovascular diseases. Therefore, there is a great need to identify circulating parameters that link changes in body fat mass with obesity. This study combines proteomic and metabolomic approaches to identify circulating molecules that discriminate healthy lean from healthy obese individuals in an exploratory study design. To correct for variations in physical activity, study participants performed a one hour exercise bout to exhaustion. Subsequently, circulating factors differing between lean and obese individuals, independent of physical activity, were identified. The DIGE approach yielded 126 differentially abundant spots representing 39 unique proteins. Differential abundance of proteins was confirmed by ELISA for antithrombin-III, clusterin, complement C3 and complement C3b, pigment epithelium-derived factor (PEDF), retinol binding protein 4 (RBP4), serum amyloid P (SAP), and vitamin-D binding protein (VDBP). Targeted serum metabolomics of 163 metabolites identified 12 metabolites significantly related to obesity. Among those, glycine (GLY), glutamine (GLN), and glycero-phosphatidylcholine 42:0 (PCaa 42:0) serum concentrations were higher, whereas PCaa 32:0, PCaa 32:1, and PCaa 40:5 were decreased in obese compared to lean individuals. The integrated bioinformatic evaluation of proteome and metabolome data yielded an improved group separation score of 2.65 in contrast to 2.02 and 2.16 for the single-type use of proteomic or metabolomics data, respectively. The identified circulating parameters were further investigated in an extended set of 30 volunteers and in the context of two intervention studies. Those included 14 obese patients who had undergone sleeve gastrectomy and 12 patients on a hypocaloric diet. For determining the long-term adaptation process the samples were taken six months after the treatment. In multivariate regression analyses, SAP, CLU, RBP4, PEDF, GLN, and C18:2 showed the strongest correlation to changes in body fat mass. The combined serum proteomic and metabolomic profiling reveals a link between the complement system and obesity and identifies both novel (C3b, CLU, VDBP, and all metabolites) and confirms previously discovered markers (PEDF, RBP4, C3, ATIII, and SAP) of body fat mass changes.

MicroRNA-21 targets tumor suppressor genes ANP32A and SMARCA4

MicroRNA-21 (miR-21) is a key regulator of oncogenic processes. It is significantly elevated in the majority of human tumors and functionally linked to cellular proliferation, survival and migration. In this study, we used two experimental-based strategies to search for novel miR-21 targets. On the one hand, we performed a proteomic approach using two-dimensional differential gel electrophoresis (2D-DIGE) to identify proteins suppressed upon enhanced miR-21 expression in LNCaP human prostate carcinoma cells. The tumor suppressor acidic nuclear phosphoprotein 32 family, member A (ANP32A) (alias pp32 or LANP) emerged as the most strongly downregulated protein. On the other hand, we applied a mathematical approach to select correlated gene sets that are negatively correlated with primary-miR-21 (pri-miR-21) expression in published transcriptome data from 114 B-cell lymphoma cases. Among these candidates, we found tumor suppressor SMARCA4 (alias BRG1) together with the already validated miR-21 target, PDCD4. ANP32A and SMARCA4, which are both involved in chromatin remodeling processes, were confirmed as direct miR-21 targets by immunoblot analysis and reporter gene assays. Furthermore, knock down of ANP32A mimicked the effect of enforced miR-21 expression by enhancing LNCaP cell viability, whereas overexpression of ANP32A in the presence of high miR-21 levels abrogated the miR-21-mediated effect. In A172 glioblastoma cells, enhanced ANP32A expression compensated for the effects of anti-miR-21 treatment on cell viability and apoptosis. In addition, miR-21 expression clearly increased the invasiveness of LNCaP cells, an effect also seen in part upon downregulation of ANP32A. In conclusion, these results suggest that downregulation of ANP32A contributes to the oncogenic function of miR-21.

Chlorinated Benzenes Cause Concomitantly Oxidative Stress and Induction of Apoptotic Markers in Lung Epithelial Cells (A549) at Nonacute Toxic Concentrations

In industrialized countries, people spend more time indoors and are therefore increasingly exposed to volatile organic compounds that are emitted at working places and from consumer products, paintings, and furniture, with chlorobenzene (CB) and 1,2-dichlorobenzene (DCB) being representatives of the halogenated arenes. To unravel the molecular effects of low concentrations typical for indoor and occupational exposure, we exposed human lung epithelial cells to CB and DCB and analyzed the effects on the proteome level by 2-D DIGE, where 860 protein spots were detected. A set of 25 and 30 proteins were found to be significantly altered due to exposure to environmentally relevant concentrations of 10(-2) g/m(3) of CB or 10(-3) g/m(3) of DCB (2.2 and 0.17 ppm), respectively. The most enriched pathways were cell death signaling, oxidative stress response, protein quality control, and metabolism. The involvement of oxidative stress was validated by ROS measurement. Among the regulated proteins, 28, for example, voltage-dependent anion-selective channel protein 2, PDCD6IP protein, heat shock protein beta-1, proliferating cell nuclear antigen, nucleophosmin, seryl-tRNA synthetase, prohibitin, and protein arginine N-methyltransferase 1, could be correlated with the molecular pathway of cell death signaling. Caspase 3 activation by cleavage was confirmed for both CB and DCB by immunoblotting. Treatment with CB or DCB also caused differential protein phosphorylation, for example, at the proteins HNRNP C1/C2, serine-threonine receptor associated protein, and transaldolase 1. Compared to previous results, where cells were exposed to styrene, for the chlorinated aromatic substances besides oxidative stress, apoptosis was found as the predominant cellular response mechanism.

Influence of agglomeration and specific lung lining lipid/protein interaction on short-term inhalation toxicity.

Abstract Lung lining fluid is the first biological barrier nanoparticles (NPs) encounter during inhalation. As previous inhalation studies revealed considerable differences between surface functionalized NPs with respect to deposition and toxicity, our aim was to investigate the influence of lipid and/or protein binding on these processes. Thus, we analyzed a set of surface functionalized NPs including different SiO2 and ZrO2 in pure phospholipids, CuroSurfTM and purified native porcine pulmonary surfactant (nS). Lipid binding was surprisingly low for pure phospholipids and only few NPs attracted a minimal lipid corona. Additional presence of hydrophobic surfactant protein (SP) B in CuroSurfTM promoted lipid binding to NPs functionalized with Amino or PEG residues. The presence of the hydrophilic SP A in nS facilitated lipid binding to all NPs. In line with this the degree of lipid and protein affinities for different surface functionalized SiO2 NPs in nS followed the same order (SiO2 Phosphate ∼ unmodified SiO2 < SiO2 PEG < SiO2 Amino NPs). Agglomeration and biomolecule interaction of NPs in nS was mainly influenced by surface charge and hydrophobicity. Toxicological differences as observed in short-term inhalation studies (STIS) were mainly influenced by the core composition and/or surface reactivity of NPs. However, agglomeration in lipid media and lipid/protein affinity appeared to play a modulatory role on short-term inhalation toxicity. For instance, lipophilic NPs like ZrO2, which are interacting with nS to a higher extent, exhibited a far higher lung burden than their hydrophilic counterparts, which deserves further attention to predict or model effects of respirable NPs.

Subtoxic and toxic concentrations of benzene and toluene induce Nrf2‐mediated antioxidative stress response and affect the central carbon metabolism in lung epithelial cells A549

Since people in industrialized countries spend most of their time indoors, the effects of indoor contaminants such as volatile organic compounds become more and more relevant. Benzene and toluene are among the most abundant compounds in the highly heterogeneous group of indoor volatile organic compounds. In order to understand their effects on lung epithelial cells (A549) representing lungs first line of defense, we chose a global proteome and a targeted metabolome approach in order to detect adverse outcome pathways caused by exposure to benzene and toluene. Using a DIGE approach, 93 of 469 detected protein spots were found to be differentially expressed after exposure to benzene, and 79 of these spots were identified by MS. Pathway analysis revealed an enrichment of proteins involved in Nrf2‐mediated and oxidative stress response glycolysis/gluconeogenesis. The occurrence of oxidative stress at nonacute toxic concentrations of benzene and toluene was confirmed by the upregulation of the stress related proteins NQO1 and SOD1. The changes in metabolism were validated by ion chromatography MS/MS analysis revealing significant changes of glucose‐6‐phosphate, fructose‐6‐phosphate, 3‐phosphoglycerate, and NADPH. The molecular alterations identified as a result of benzene and toluene exposure demonstrate the detrimental effect of nonacute toxic concentrations on lung epithelial cells. The data provided here will allow for a targeted validation in in vivo models.

Benzo[a]pyrene affects Jurkat T cells in the activated state via the antioxidant response element dependent Nrf2 pathway leading to decreased IL-2 secretion and redirecting glutamine metabolism

There is a clear evidence that environmental pollutants, such as benzo[a]pyrene (B[a]P), can have detrimental effects on the immune system, whereas the underlying mechanisms still remain elusive. Jurkat T cells share many properties with native T lymphocytes and therefore are an appropriate model to analyze the effects of environmental pollutants on T cells and their activation. Since environmental compounds frequently occur at low, not acute toxic concentrations, we analyzed the effects of two subtoxic concentrations, 50nM and 5μM, on non- and activated cells. B[a]P interferes directly with the stimulation process as proven by an altered IL-2 secretion. Furthermore, B[a]P exposure results in significant proteomic changes as shown by DIGE analysis. Pathway analysis revealed an involvement of the AhR independent Nrf2 pathway in the altered processes observed in unstimulated and stimulated cells. A participation of the Nrf2 pathway in the change of IL-2 secretion was confirmed by exposing cells to the Nrf2 activator tBHQ. tBHQ and 5μM B[a]P caused similar alterations of IL-2 secretion and glutamine/glutamate metabolism. Moreover, the proteome changes in unstimulated cells point towards a modified regulation of the cytoskeleton and cellular stress response, which was proven by western blotting. Additionally, there is a strong evidence for alterations in metabolic pathways caused by B[a]P exposure in stimulated cells. Especially the glutamine/glutamate metabolism was indicated by proteome pathway analysis and validated by metabolite measurements. The detrimental effects were slightly enhanced in stimulated cells, suggesting that stimulated cells are more vulnerable to the environmental pollutant model compound B[a]P.

Evaluation of the tolerability and immunogenicity of ultraviolet C-irradiated autologous platelets in a dog model

BACKGROUND: The THERAFLEX ultraviolet (UV) platelets (PLTs) pathogen reduction system for PLT concentrates (PCs) operates using ultraviolet C (UVC) light at a wavelength of 254 nm. UVC treatment can potentially alter proteins, which may affect drug tolerance in humans and influence the immunogenicity of blood products. This preclinical study in beagle dogs was designed to evaluate the safety pharmacology of UVC‐irradiated PCs after intravenous administration and to determine whether they are capable of eliciting humoral responses to PLTs and plasma proteins.

Proteomics analysis of dendritic cell activation by contact allergens reveals possible biomarkers regulated by Nrf2

Allergic contact dermatitis is a widespread disease with high clinical relevance affecting approximately 20% of the general population. Typically, contact allergens are low molecular weight electrophilic compounds which can activate the Keap1/Nrf2 pathway. We performed a proteomics study to reveal possible biomarkers for dendritic cell (DC) activation by contact allergens and to further elucidate the role of Keap1/Nrf2 signaling in this process. We used bone marrow derived dendritic cells (BMDCs) of wild-type (nrf2+/+) and Nrf2 knockout (nrf2-/-) mice and studied their response against the model contact sensitizers 2,4-dinitrochlorobenzene (DNCB), cinnamaldehyde (CA) and nickel(II) sulfate by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS). Sodium dodecyl sulfate (SDS, 100μM) served as irritant control. While treatment with nickel(II) sulfate and SDS had only little effects, CA and DNCB led to significant changes in protein expression. We found 18 and 30 protein spots up-regulated in wild-type cells treated with 50 and 100μM CA, respectively. For 5 and 10μM DNCB, 32 and 37 spots were up-regulated, respectively. Almost all of these proteins were not differentially expressed in nrf2-/- BMDCs, indicating an Nrf2-dependent regulation. Among them proteins were detected which are involved in oxidative stress and heat shock responses, as well as in signal transduction or basic cellular pathways. The applied approach allowed us to differentiate between Nrf2-dependent and Nrf2-independent cellular biomarkers differentially regulated upon allergen-induced DC activation. The data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization.

Occupational IgE-mediated softwood allergy: characterization of the causative allergen.

Allergic reactions to wood dust allergens are rare, and only few in vitro diagnostic tools and information about relevant allergens are available. To differentiate between protein-based allergy and probably clinically silent glycogenic sensitization, it is helpful to characterize the relevant protein allergens and specify IgE binding. The current case report deals with the occupational softwood allergy of a carpenter exposed to different wood dusts. Skin tests and IgE tests against wood were performed with specifically tailored ImmunoCAPs and cross-reactive carbohydrate determinants. Potential allergens were identified by IgE blots and tandem mass spectrometry. The clinical relevance was verified by challenge tests. Specific IgE to softwood (spruce, pine and larch wood), beech wood, natural rubber latex (NRL) and horseradish peroxidase (HRP) were detected. Allergens in spruce wood, the dominant allergen source, were identified as peroxidases. Softwood were the strongest inhibitors. HRP reduced IgE binding to softwood to <50%, indicating predominantly proteinogenic epitopes, whereas IgE binding to NRL and beech wood was reduced to >50% by HRP, indicating predominantly glycogenic IgE epitopes. Skin and challenge tests underlined that softwoods were the source of sensitization. For the polysensitized patient, a clinically relevant softwood allergy was diagnosed, not only by challenge tests but also with specifically tailored in vitro tools.