Jenny Rubin

The Origin of Multiple Molecular Forms in Urine of HNL/NGAL

BACKGROUND AND OBJECTIVES Several molecular forms of human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), a novel biomarker for acute kidney injury (AKI), have been found in urine. The origin of these different forms and the effect of antibody configuration on assay performances were investigated in this report. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS The molecular forms of HNL/NGAL from human neutrophils and present in urine obtained from cardiac surgery patients and patients with urinary tract infection (UTI), as well as secreted from HK-2 cells, were studied by Western blotting. The levels of HNL/NGAL in urine were measured by ELISAs. Kidney injury was simulated by incubation of HK-2 cells under stressful conditions. RESULTS The major molecular form of HNL/NGAL secreted by neutrophils is dimeric, whereas the major form secreted by HK-2 cells is monomeric. This was reflected by a predominance of the monomeric form in urine from patients with AKI and the dimeric form in patients with UTIs. The epitope specificities of the antibody used in the ELISAs had a profound effect on assay performance and paralleled differences of the antibodies to identify the different forms of urine HNL/NGAL. CONCLUSIONS The monomeric form is the predominant form secreted by tubular epithelial cells, and the dimeric form is the predominant form secreted by neutrophils. The development of molecular form-specific assays for HNL/NGAL may be a means to identify the origin of HNL/NGAL in urine and construct more specific tools for the diagnosis of AKI.

Eosinophil Cationic Protein (ECP) Is Processed during Secretion

The eosinophil granulocyte is an inflammatory cell involved in allergic diseases such as asthma and rhinitis. Eosinophil cationic protein (ECP) is a basic and potentially cytotoxic granule protein that is released from the eosinophil upon activation. The aim was to study secretion of molecular variants of ECP from blood eosinophils with the hypothesis that the stored noncytotoxic ECP is altered into cytotoxic species upon release from the cell. Eosinophil granulocytes were purified to >95% from venous blood from birch pollen allergic subjects, with symptoms of rhinitis, and from healthy control subjects during the birch pollen season. The cells were stimulated with IL-5, GM-CSF, or serum-opsonized Sephadex particles. Concentration of ECP in cells or supernatants was measured by means of a fluoroenzyme immunoassay, and ECP heterogeneity was studied using an affinity capture assay with the surface-enhanced laser desorption/ionization-time of flight mass spectrometry technique. Extracts of unstimulated eosinophils contained 10 major ECP variants, with molecular masses ranging from 16.1 to 17.7 kDa. Stimulation with particles mainly induced the secretion of two molecular variants at 16.1 and 16.3 kDa, while cytokine stimulation gave rise to a different secretion profile. ECP variants in the pellet extracts remained unaffected by cell activation. The modifications of secreted ECP were partly explained by differences in N-linked glycosylations. Secretion of ECP from eosinophils involves protein modification. The molecular masses of released ECP have acquired the masses of the cytotoxic species.

The Coding ECP 434(G>C) Gene Polymorphism Determines the Cytotoxicity of ECP but Has Minor Effects on Fibroblast-Mediated Gel Contraction and No Effect on RNase Activity

Eosinophil cationic protein (ECP) is a secretory protein of the eosinophil granulocyte, a cell involved in innate immunity. Functional studies have implicated ECP in numerous processes, such as tissue remodeling in allergic inflammation and cytotoxicity toward a variety of pathogens. Recent genetic studies have suggested that the ECP 434(G>C) polymorphism resulting in an arg97thr substitution would alter the function of ECP in vivo. Functional (in vitro) studies of ECP up until now have either been conducted with native preparations containing an unknown mixture of the ECP97arg and ECP97thr variants, or with recombinant proteins. Therefore, we have now for the first time extracted the native ECP97arg and ECP97thr variants from healthy blood donors and tested them functionally in vitro. Our results show that the arg97thr shift dramatically alters the cytotoxic capacity of ECP in vitro; the tested ECP97arg variants were cytotoxic toward the small-cell lung cancer cell line NCI-H69, whereas ECP97thr was noncytotoxic. RNase activity was unaffected by the arg97thr substitution. Both ECP97arg and ECP97thr stimulated fibroblast-mediated collagen gel contraction, an experimental model, which depicts wound healing, in a dose-dependent manner. In conclusion, our results demonstrate that the ECP 434(G>C) gene polymorphism affects the functional properties of native ECP, but also that there is a dissociation between different biological activities; the arg97thr substitution impairs the cytotoxic potential of ECP but less the gel contraction and not at all the RNase activity.

Predictors of histology, tissue eosinophilia and mast cell infiltration in Hodgkin's lymphoma--a population-based study.

Objective:  Classical Hodgkin’s lymphoma (HL) lesions comprise few tumour cells, surrounded by numerous inflammatory cells. Like in other malignancies, the microenvironment is presumed to be clinically important in HL; however, microenvironment predictors remain poorly characterised. The aim of this study was to investigate how selected patient characteristics and genetic factors affect HL phenotype, in particular tissue eosinophilia, mast cell counts and HL histological subtype.

Protein post-translational modification in host defense: the antimicrobial mechanism of action of human eosinophil cationic protein native forms

Knowledge on the contribution of protein glycosylation in host defense antimicrobial peptides is still scarce. We have studied here how the post‐translational modification pattern modulates the antimicrobial activity of one of the best characterized leukocyte granule proteins. The human eosinophil cationic protein (ECP), an eosinophil specific granule protein secreted during inflammation and infection, can target a wide variety of pathogens. Previous work in human eosinophil extracts identified several ECP native forms and glycosylation heterogeneity was found to contribute to the protein biological properties. In this study we analyze for the first time the antimicrobial activity of the distinct native proteins purified from healthy donor blood. Low and heavy molecular weight forms were tested on Escherichia coli cell cultures and compared with the recombinant non‐glycosylated protein. Further analysis on model membranes provided an insight towards an understanding of the protein behavior at the cytoplasmic membrane level. The results highlight the significant reduction in protein toxicity and bacteria agglutination activity for heavy glycosylated fractions. Notwithstanding, the lower glycosylated fraction mostly retains the lipopolysaccharide binding affinity together with the cytoplasmic membrane depolarization and membrane leakage activities. From structural analysis we propose that heavy glycosylation interferes with the protein self‐aggregation, hindering the cell agglutination and membrane disruption processes. The results suggest the contribution of post‐translational modifications to the antimicrobial role of ECP in host defense.

Asparagine-linked glycans determine the cytotoxic capacity of eosinophil cationic protein (ECP)

Eosinophil cationic protein (ECP) is a toxic, granule-stored protein of the eosinophil granulocyte. It is a heterogeneous protein; molecular weights can differ from 15 to 22 kDa, due to glycosylations. We purified high molecular weight ECP from blood donors with the ECP434GG (rs2073342) genotype, with the aim of examining whether removal of carbohydrates could enhance the cytotoxic capacity. The cytotoxic activity of the ECP pools was tested against the NCI-H69 cell line, before and after enzymatic deglycosylation. ECP was also analysed by SELDI-TOF MS to monitor the changes in molecular mass after deglycosylation. Five high molecular weight pools of ECP (HMW-ECP I-V) with decreasing degrees of glycosylation were tested at concentrations ranging from 0.02 to 0.6 μM. The activity ranged from EC50 of >0.6 μM to 0.04 μM; HMW-ECP II had the lowest activity and HMW-ECP V the highest. After deglycosylation with N-glycosidase F, pools HMW-ECP I-III were reduced to the same molecular weight of 15.78 kDa and acquired potent cytotoxic activities. HMW-ECP IV and V with molecular species at 16.3 and 16.1 kDa were highly cytotoxic as such and were only partially deglycosylated, with slight enhancement of the toxic properties. The results suggest the presence of several HMW-ECP molecular species with differences in their post-translational modifications and cytotoxic properties. We conclude that a fraction of native ECP is stored in a non-cytotoxic form, which can be converted into a cytotoxic form by N-deglycosylation, whereas another fraction is stored as a highly cytotoxic form carrying different post-translational modifications.

The anticancer effect of mebendazole may be due to M1 monocyte/macrophage activation via ERK1/2 and TLR8-dependent inflammasome activation

Abstract Mebendazole (MBZ), a drug commonly used for helminitic infections, has recently gained substantial attention as a repositioning candidate for cancer treatment. However, the mechanism of action behind its anticancer activity remains unclear. To address this problem, we took advantage of the curated MBZ-induced gene expression signatures in the LINCS Connectivity Map (CMap) database. The analysis revealed strong negative correlation with MEK/ERK1/2 inhibitors. Moreover, several of the most upregulated genes in response to MBZ exposure were related to monocyte/macrophage activation. The MBZ-induced gene expression signature in the promyeloblastic HL-60 cell line was strongly enriched in genes involved in monocyte/macrophage pro-inflammatory (M1) activation. This was subsequently validated using MBZ-treated THP-1 monocytoid cells that demonstrated gene expression, surface markers and cytokine release characteristic of the M1 phenotype. At high concentrations MBZ substantially induced the release of IL-1β and this was further potentiated by lipopolysaccharide (LPS). At low MBZ concentrations, cotreatment with LPS was required for MBZ-stimulated IL-1β secretion to occur. Furthermore, we show that the activation of protein kinase C, ERK1/2 and NF-kappaB were required for MBZ-induced IL-1β release. MBZ-induced IL-1β release was found to be dependent on NLRP3 inflammasome activation and to involve TLR8 stimulation. Finally, MBZ induced tumor-suppressive effects in a coculture model with differentiated THP-1 macrophages and HT29 colon cancer cells. In summary, we report that MBZ induced a pro-inflammatory (M1) phenotype of monocytoid cells, which may, at least partly, explain MBZ’s anticancer activity observed in animal tumor models and in the clinic.

Mebendazole stimulates CD14+ myeloid cells to enhance T-cell activation and tumour cell killing

Mebendazole (MBZ) was recently shown to induce a tumor suppressive M1 phenotype in THP-1 monocytes and macrophages. In the present study the immune effects of MBZ was further investigated using human peripheral blood mononuclear cells (PBMCs) co-cultured with tumour cells. The Biomap platform was used to screen for biomarkers induced from MBZ exposed co-cultures of T-cell receptor activated PBMCs, HT29 colon cancer cells and either human fibroblasts or human umbilical vein endothelial cells (HUVEC) cells. In these co-culture systems MBZ at 0.3-10 μM induced significant increases in TNFα and IFNγ indicating immune stimulation. PBMC cultures alone were subsequently tested for activation status and only in PBMCs activated by CD3/IL2 stimulation and MBZ, at a clinically achievable concentration, was able to increase PBMC clustering and release of pro-inflammatory IFNγ, TNFα, IL6 and IL1β cytokines. Moreover, when PBMC cultures were functionally tested for immune cell killing of lung cancer A549NucLightRed cells, MBZ significantly increased tumour cell apoptosis and reduced the number of surviving tumour cells. This effect was dependent on the presence of CD14 monocytes/macrophages in the co-culture. In summary, MBZ potentiated the immune stimulatory and anticancer effects of anti-CD3/IL2 activated PBMCs which could be relevant to explain the anticancer activity of MBZ observed in the clinic.

Abstract 4990: High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities

Monolayer, two-dimensional (2D) cell cultures have been a predominant in vitro model in anticancer drug discovery and high-throughput screening (HTS). However, 2D cultures of cancer cells lack numerous properties of in vivo tumors, such as tissue-like structure, cell-cell interactions and nutrient/oxygen gradients. Thus, in recent years there has been an increased interest in 3D cell cultures, such as multicellular tumors spheroids (MCTS), to address some of these limitations. Recently, we and others have applied MCTS for HTS and identified oxidative phosphorylation (OXPHOS) as a selective vulnerability of quiescent cancer cells persisting in hypoxic and nutrient-deprived milieu. However, prolonged continuous exposure to OXPHOS inhibitors is necessary for the cytotoxic effect. Thus, there is a need to identify processes that could be co-targeted for enhanced anticancer activity. Here, we present two distinct HTS approaches to identify combination partner molecules for OXPHOS inhibitors. Since we were interested in targeting non-dividing nutrient-deprived cancer cells, we used quiescent MCTS (Q-MCTS), as an in vitro model. Cells in Q-MCTS experience glucose concentrations and pH similar to those observed in deep tumor parenchyma in vivo. In our first screening approach, we have applied high-throughput gene-expression profiling to study drug effects in MCTS at a large scale. Using L1000 Gene Expression Profiling method, we generated a dataset of over 1000 drug-induced gene-expression profiles and found that co-targeting of OXPHOS and the mevalonate pathway results in selective synergistic toxicity in quiescent cancer cells. In the other approach, we screened a library of 1650 biologically active compounds, with or without addition of the FDA-approved anthelmintic agent nitazoxanide (an OXPHOS inhibitor with high drug repurposing potential). After the screen, we selected molecules that demonstrated pronounced synergy when combined with nitazoxanide, but not when used alone. Then, we validated the hits in an extensive dose-response combination experiments in Q-MCTS and chose 14 compounds that demonstrated strong synergistic interaction with nitazoxanide at broad range of concentrations. These included antifungal agents, kinase inhibitors and others. In summary, we here report on novel approaches, utilizing 3D cell cultures, to identify drug combinations targeting quiescent cancer cells. By high-throughput gene-expression profiling and large-scale combinatorial drug screens, we were able to identify drug combinations preferentially toxic to quiescent cells. This work also demonstrates how 3D cell cultures yield functional insights that are not accessible through standard 2D cultures. Citation Format: Wojciech Senkowski, Madiha Nazir, Malin Jarvius, Jenny Rubin, Johan Lengqvist, Mats G. Gustafsson, Peter Nygren, Kim Kultima, Rolf Larsson, Marten Fryknas. High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4990. doi:10.1158/1538-7445.AM2017-4990

Abstract 213: Mitochondrial inhibitors and statins: a lethal combination for metabolically stressed cancer cells

Inhibition of mitochondrial oxidative phosphorylation (OXPHOS) has recently emerged as a promising strategy for treatment of therapy-resistant cancer cells. These cells often reside within hypoxic tumor regions, where nutrient concentrations are low. Recently, OXPHOS inhibitors have been demonstrated to be toxic to quiescent, nutrient-deprived cells in multicellular tumor spheroids. Such spheroids, formed without medium exchange over the culture period, can serve as an appropriate model to mimic quiescent in vivo tumor regions. These spheroids exhibit low cell proliferation and comprise necrotic cores, contrary to commonly used spheroids cultured with frequent medium change. We here aimed to characterize how quiescent cells respond to OXPHOS inhibition and thereby identify processes that could be co-targeted for enhanced toxicity. We treated HCT116 colon cancer cell line, grown as monolayer cultures and spheroids, with a range of OXPHOS inhibitors (n = 10, including FDA-approved drugs, e.g. nitazoxanide) and other compounds (n = 14) at escalating doses and in 4 biological replicates. Then, we obtained global gene expression profiles (n = 1149, including 144 vehicle controls) of all treatment conditions using L1000 Gene Expression Profiling method. We found that upon exposure to OXPHOS inhibitors cells grown as nutrient-deprived spheroids significantly and in dose-dependent manner upregulate expression of genes involved in biosynthesis of cholesterol. This response was not observed for spheroids cultured with medium change or monolayer cell cultures. Thus, we were interested if simultaneous exposure to OXPHOS inhibitors and statins, inhibitors of mevalonate (cholesterol precursor) synthesis, would result in enhanced cytotoxic effects in quiescent, metabolically stressed cells. We here demonstrate that combination of OXPHOS inhibitors and statins results in pronounced synergistic cytotoxicity in metabolically stressed spheroids. This effect was observed for various classes of OXPHOS inhibitors and different types of statins, indicating that the observed synergy was not a result of off-target effects. This notion was further strengthened by the finding that mevalonate largely abrogated the synergistic effects. In conclusion, we here report that statins enhance the toxic effects of OXPHOS inhibitors in quiescent, metabolically stressed cells. Our results can serve as a foundation for further studies on targeting therapy-resistant and nutrient-deprived cancer cells by inhibition of OXPHOS. We also demonstrate, for the first time, that the L1000 Gene Expression Profiling can be used to study 3D cell cultures. Importantly, our findings underscore the importance of using a relevant cellular model for target discovery endeavors. Citation Format: Wojciech Senkowski, Malin Jarvius, Kim Kultima, Jenny Rubin, Mats Gustafsson, Peter Nygren, Rolf Larsson, Marten Fryknas. Mitochondrial inhibitors and statins: a lethal combination for metabolically stressed cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 213.