Ubiquitinated PCNA Drives USP1 Synthetic Lethality in Cancer.

CRISPR Cas9-based screening is a powerful approach for identifying and characterizing novel drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacologic inhibition of USP1 leads to decreased DNA synthesis concomitant with S-phase-specific DNA damage. Genome-wide CRISPR-Cas9 screens identify RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition is associated with reduced PCNA protein levels. Ectopic expression of WT or ubiquitin-dead K164R PCNA reverses USP1 inhibitor sensitivity. Our results show, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to selected BRCA1/2 WT cancer cell lines enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant tumors. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to posttranslational modifications of PCNA. Taken together, USP1 inhibition may represent a novel therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.

VRK1 Is a Synthetic-Lethal Target in VRK2-Deficient Glioblastoma.

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic-lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2-M arrest and DNA damage. The VRK1-VRK2 synthetic-lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line-derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. SIGNIFICANCE: A paralog synthetic-lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate.

Oxygen sensing is central to metazoan biology and has implications for human disease. Mammalian cells express multiple oxygen-dependent enzymes called 2-oxoglutarate (OG)-dependent dioxygenases (2-OGDDs), but they vary in their oxygen affinities and hence their ability to sense oxygen. The 2-OGDD histone demethylases control histone methylation. Hypoxia increases histone methylation, but whether this reflects direct effects on histone demethylases or indirect effects caused by the hypoxic induction of the HIF (hypoxia-inducible factor) transcription factor or the 2-OG antagonist 2-hydroxyglutarate (2-HG) is unclear. Here, we report that hypoxia promotes histone methylation in a HIF- and 2-HG-independent manner. We found that the H3K27 histone demethylase KDM6A/UTX, but not its paralog KDM6B, is oxygen sensitive. KDM6A loss, like hypoxia, prevented H3K27 demethylation and blocked cellular differentiation. Restoring H3K27 methylation homeostasis in hypoxic cells reversed these effects. Thus, oxygen directly affects chromatin regulators to control cell fate.

Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions.

Neural crest cells migrate throughout the embryo, but how cells move in a directed and collective manner has remained unclear. Here, we perform the first single-cell transcriptome analysis of cranial neural crest cell migration at three progressive stages in chick and identify and establish hierarchical relationships between cell position and time-specific transcriptional signatures. We determine a novel transcriptional signature of the most invasive neural crest Trailblazer cells that is consistent during migration and enriched for approximately 900 genes. Knockdown of several Trailblazer genes shows significant but modest changes to total distance migrated. However, in vivo expression analysis by RNAscope and immunohistochemistry reveals some salt and pepper patterns that include strong individual Trailblazer gene expression in cells within other subregions of the migratory stream. These data provide new insights into the molecular diversity and dynamics within a neural crest cell migratory stream that underlie complex directed and collective cell behaviors.

An Organoruthenium Anticancer Agent Shows Unexpected Target Selectivity For Plectin.

Organometallic metal(arene) anticancer agents require ligand exchange for their anticancer activity and this is generally believed to confer low selectivity for potential cellular targets. However, using an integrated proteomics-based target-response profiling approach as a potent hypothesis-generating procedure, we found an unexpected target selectivity of a ruthenium(arene) pyridinecarbothioamide (plecstatin) for plectin, a scaffold protein and cytolinker, which was validated in a plectin knock-out model in vitro. Plectin targeting shows potential as a strategy to inhibit tumor invasiveness as shown in cultured tumor spheroids while oral administration of plecstatin-1 to mice reduces tumor growth more efficiently in the invasive B16 melanoma than in the CT26 colon tumor model.

The metalation of hen egg white lysozyme impacts protein stability as shown by ion mobility mass spectrometry, differential scanning calorimetry, and X-ray crystallography.

Metalation of hen egg white lysozyme (HEWL) with organometallics was studied with physicochemical methods in solid state, solution and the gas phase. While metalation did not affect the crystal structure of HEWL significantly, protein destabilisation was detected in gas phase and solution.

Absence of PD-L1 on tumor cells is associated with reduced MHC I expression and PD-L1 expression increases in recurrent serous ovarian cancer.

Immune-evasion and immune checkpoints are promising new therapeutic targets for several cancer entities. In ovarian cancer, the clinical role of programmed cell death receptor ligand 1 (PD-L1) expression as mechanism to escape immune recognition has not been clarified yet. We analyzed PD-L1 expression of primary ovarian and peritoneal tumor tissues together with several other parameters (whole transcriptomes of isolated tumor cells, local and systemic immune cells, systemic cytokines and metabolites) and compared PD-L1 expression between primary tumor and tumor recurrences. All expressed major histocompatibility complex (MHC) I genes were negatively correlated to PD-L1 abundances on tumor tissues, indicating two mutually exclusive immune-evasion mechanisms in ovarian cancer: either down-regulation of T-cell mediated immunity by PD-L1 expression or silencing of self-antigen presentation by down-regulation of the MHC I complex. In our cohort and in most of published evidences in ovarian cancer, low PD-L1 expression is associated with unfavorable outcome. Differences in immune cell populations, cytokines, and metabolites strengthen this picture and suggest the existence of concurrent pathways for progression of this disease. Furthermore, recurrences showed significantly increased PD-L1 expression compared to the primary tumors, supporting trials of checkpoint inhibition in the recurrent setting.

Combined Proteome and Eicosanoid Profiling Approach for Revealing Implications of Human Fibroblasts in Chronic Inflammation.

During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation.

Response Profiling Using Shotgun Proteomics Enables Global Metallodrug Mechanisms of Action To Be Established.

Response profiling using shotgun proteomics for establishing global metallodrug mechanisms of action in two colon carcinoma cell lines, HCT116 and SW480, has been applied and evaluated with the clinically approved arsenic trioxide. Surprisingly, the complete established mechanism of action of arsenic trioxide was observed by protein regulations in SW480, but not HCT116 cells. Comparing the basal protein expression in the two cell lines revealed an 80 % convergence of protein identification, but with significant expression differences, which in turn seem to affect the extent of protein regulation. A clear-cut redox response was observed in SW480 cells upon treatment with arsenic, but hardly in HCT116 cells. Response profiling was then used to investigate four anti-cancer metallodrugs (KP46, KP772, KP1339 and KP1537). Proteome alterations were mapped to selected functional groups, including DNA repair, endocytosis, protection from oxidative stress, protection from endoplasmatic reticulum (ER) stress, cell adhesion and mitochondrial function. The present data suggest that knowledge of the mechanism of action of anti-cancer metallodrugs and improved patient stratification strategies are imperative for the design of clinical studies.

Functionalization of Ruthenium(II)(η6 -p-cymene)(3-hydroxy-2-pyridone) Complexes with (Thio)Morpholine: Synthesis and Bioanalytical Studies.

Hydroxypyr(id)ones constitute an emerging platform for the design of drug molecules, owing to their favorable biocompatibility and toxicity profiles. Herein, [RuII (η6 -p-cymene)] complexes with 3-hydroxy-2-pyridinone functionalized with morpholine and thiomorpholine, as a means often used in medicinal chemistry to alter the physicochemical properties of drug compounds, are reported. The compounds underwent hydrolysis of the Ru-Cl bond and the aqua species were stable for up to 48 h in aqueous solution, as observed by 1 H NMR spectroscopy and ESI-MS. The compounds formed adducts with amino acids and proteins through cleavage of the pyridinone ligand. Binding experiments to the nucleosome core particle by means of X-ray crystallography revealed similar reactivity and exclusive binding to histidine moieties of the histone proteins. Preliminary cyclin-dependent kinase 2 (CDK2)/cyclin A kinase inhibitory studies revealed promising activity similar to that of structurally related organometallic compounds.

Drosophila poised enhancers are generated during tissue patterning with the help of repression.

Histone modifications are frequently used as markers for enhancer states, but how to interpret enhancer states in the context of embryonic development is not clear. The poised enhancer signature, involving H3K4me1 and low levels of H3K27ac, has been reported to mark inactive enhancers that are poised for future activation. However, future activation is not always observed, and alternative reasons for the widespread occurrence of this enhancer signature have not been investigated. By analyzing enhancers during dorsal-ventral (DV) axis formation in the Drosophila embryo, we find that the poised enhancer signature is specifically generated during patterning in the tissue where the enhancers are not induced, including at enhancers that are known to be repressed by a transcriptional repressor. These results suggest that, rather than serving exclusively as an intermediate step before future activation, the poised enhancer state may be a mark for spatial regulation during tissue patterning. We discuss the possibility that the poised enhancer state is more generally the result of repression by transcriptional repressors.

A 12-week intervention with nonivamide, a TRPV1 agonist, prevents a dietary-induced body fat gain and increases peripheral serotonin in moderately overweight subjects.

SCOPE: A bolus administration of 0.15 mg nonivamide has previously been demonstrated to reduce energy intake in moderately overweight men. This 12-week intervention investigated whether a daily consumption of nonivamide in a protein-based product formulation promotes a reduction in body weight in healthy overweight subjects and affects outcome measures associated with mechanisms regulating food intake, e.g. plasma concentrations of (an)orexigenic hormones, energy substrates as well as changes in fecal microbiota. METHODS AND RESULTS: Nineteen overweight subjects were randomly assigned to either a control (C) or a nonivamide (NV) group. Changes in the body composition and plasma concentrations of satiating hormones were determined at fasting and 15, 30, 60, 90, and 120 min after a glucose load. Participants were instructed to consume 0.15 mg nonivamide per day in 450 mL of a milk shake additionally to their habitual diet. After treatment, a group difference in body fat mass change (-0.61 ± 0.36% in NV and +1.36 ± 0.38% in C) and an increase in postprandial plasma serotonin were demonstrated. Plasma metabolome and fecal microbiome read outs were not affected. CONCLUSIONS: A daily intake of 0.15 mg nonivamide helps to support to maintain a healthy body composition.

Evaluation of inflammation-related signaling events covering phosphorylation and nuclear translocation of proteins based on mass spectrometry data.

Peripheral blood mononuclear cells are important players in immune regulation relying on a complex network of signaling pathways. In this study, we evaluated the power of label-free quantitative shotgun proteomics regarding the comprehensive characterization of signaling pathways in such primary cells by studying regulation of protein abundance, post-translational modifications and nuclear translocation events. The effects of inflammatory stimulation and the treatment of stimulated cells with dexamethasone were investigated. Therefore, a previously published dataset accessible via ProteomeXchange consisting of 6901 identified protein groups was re-evaluated. These data enabled us to comprehensively map the c-JUN, ERK5 and NF-κB signaling cascade in a semi-quantitative fashion. Without the application of any enrichment, 3775 highly confident phosphopeptides derived from 1249 proteins including 66 kinases were identified. Efficient subcellular fractionation and subsequent comparative analysis identified previously unrecognized inflammation-associated nuclear translocation events of proteins such as histone-modifying proteins, zinc finger proteins as well as transcription factors. Profound effects of inflammatory stimulation and dexamethasone treatment on histone H3 and ZFP161 localization represent novel findings and were verified by immunofluorescence. In conclusion, we demonstrate that multiple regulatory events resulting from the activity of signaling pathways can be determined out of untargeted shotgun proteomics data. SIGNIFICANCE: Relevant functional events such as phosphorylation and nuclear translocation of proteins were extracted from high-resolution mass spectrometry data and provided additional biological information contained in shotgun proteomics data.

Multi-omics Analysis of Serum Samples Demonstrates Reprogramming of Organ Functions Via Systemic Calcium Mobilization and Platelet Activation in Metastatic Melanoma.

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia.

Integrative Systemic and Local Metabolomics with Impact on Survival in High-Grade Serous Ovarian Cancer.

Purpose: Cancer metabolism is characterized by alterations including aerobic glycolysis, oxidative phosphorylation, and need of fuels and building blocks.Experimental Design: Targeted metabolomics of preoperative and follow-up sera, ascites, and tumor tissues, RNA sequencing of isolated tumor cells, local and systemic chemokine, and local immune cell infiltration data from up to 65 high-grade serous ovarian cancer patients and 62 healthy controls were correlated to overall survival and integrated in a Systems Medicine manner.Results: Forty-three mainly (poly)unsaturated glycerophospholipids and four essential amino acids (citrulline) were significantly reduced in patients with short compared with long survival and healthy controls. The glycerophospholipid fingerprint is identical to the fingerprint from isolated (very) low-density lipoproteins (vLDL), indicating that the source of glycerophospholipids consumed by tumors is (v)LDL. A glycerophospholipid-score (HR, 0.46; P = 0.007) and a 100-gene signature (HR, 0.65; P = 0.004) confirmed the independent impact on survival in training (n = 65) and validation (n = 165) cohorts. High concentrations of LDLs and glycerophospholipids were independently predictors for favorable survival. Patients with low glycerophospholipids presented with more systemic inflammation (C-reactive protein and fibrinogen negatively and albumin positively correlated) but less adaptive immune cell tumor infiltration (lower tumor and immune cell PD-L1 expression), less oxygenic respiration and increased triglyceride biosynthesis in tumor cells, and lower histone expressions, correlating with higher numbers of expressed genes and more transcriptional noise, a putative neo-pluripotent tumor cell phenotype.Conclusions: Low serum phospholipids and essential amino acids are correlated with worse outcome in ovarian cancer, accompanied by a specific tumor cell phenotype. Clin Cancer Res; 23(8); 2081-92. ©2016 AACR.

Role of the immune system in the peritoneal tumor spread of high grade serous ovarian cancer.

The immune system plays a critical role in cancer progression and overall survival. Still, it is unclear if differences in the immune response are associated with different patterns of tumor spread apparent in high grade serous ovarian cancer patients and previously described by us. In this study we aimed to assess the role of the immune system in miliary (widespread, millet-sized lesions) and non-miliary (bigger, exophytically growing implants) tumor spread. To achieve this we comprehensively analyzed tumor tissues, blood, and ascites from 41 patients using immunofluorescence, flow cytometry, RNA sequencing, multiplexed immunoassays, and immunohistochemistry. Results showed that inflammation markers were systemically higher in miliary. In contrast, in non-miliary lymphocyte and monocyte/macrophage infiltration into the ascites was higher as well as the levels of PD-1 expression in tumor associated cytotoxic T-lymphocytes and PD-L1 expression in tumor cells. Furthermore, in ascites of miliary patients more epithelial tumor cells were present compared to non-miliary, possibly due to the active down-regulation of anti-tumor responses by B-cells and regulatory T-cells. Summarizing, adaptive immune responses prevailed in patients with non-miliary spread, whereas in patients with miliary spread a higher involvement of the innate immune system was apparent while adaptive responses were counteracted by immune suppressive cells and factors.

Coffee consumption modulates inflammatory processes in an individual fashion.

SCOPE: Anti-inflammatory effects of coffee consumption have been reported to be caused by caffeine and adenosine receptor signaling. However, contradictory effects have been observed. Many kinds of chronic diseases are linked to inflammation; therefore a profound understanding of potential effects of coffee consumption is desirable. METHODS AND RESULTS: We performed ex vivo experiments with eight individuals investigating peripheral blood mononuclear cells isolated from venous blood before and after coffee consumption, as well as in vitro experiments applying caffeine on isolated cells. After in vitro inflammatory stimulation of the cells, released cytokines, chemokines, and eicosanoids were determined and quantified using targeted mass spectrometric methods. Remarkably, the release of inflammation mediators IL6, IL8, GROA, CXCL2, CXCL5 as well as PGA2, PGD2, prostaglandin E2 (PGE2), LTC4, LTE4, and 15S-HETE was significantly affected after coffee consumption. While in several individuals coffee consumption or caffeine treatment caused significant downregulation of most inflammation mediators, in other healthy individuals exactly the opposite effects were observed. CONCLUSION: Ruling out age, sex, coffee consumption habits, the metabolic kinetics of caffeine in blood and the individual amount of regulatory T cells or CD39 expression as predictive parameters, we demonstrated here that coffee consumption may have significant pro- or anti-inflammatory effects in an individual fashion.

Mass Spectrometry Uncovers Molecular Reactivities of Coordination and Organometallic Gold(III) Drug Candidates in Competitive Experiments That Correlate with Their Biological Effects.

The reactivity of three cytotoxic organometallic gold(III) complexes with cyclometalated C,N,N and C,N ligands (either six- or five-membered metallacycles), as well as that of two representative gold(III) complexes with N-donor ligands, with biological nucleophiles has been studied by ESI-MS on ion trap and time-of-flight instruments. Specifically, the gold compounds were reacted with mixtures of nucleophiles containing l-histidine (imine), l-methionine (thioether), l-cysteine (thiol), l-glutamic acid (carboxylic acid), methylseleno-l-cysteine (selenoether), and in situ generated seleno-l-cysteine (selenol) to judge the preference of the gold compounds for binding to selenium-containing amino acid residues. Moreover, the gold compounds' reactivity was studied with proteins and nucleic acid building blocks. These experiments revealed profound differences between the coordination and organometallic families and even within the family of organometallics, which allowed insights to be gained into the compounds mechanisms of action. In particular, interactions with seleno-l-cysteine appear to reflect well the compounds' inhibition properties of the seleno-enzyme thioredoxin reductase and to a certain extent their antiproliferative effects in vitro. Therefore, mass spectrometry is successfully applied for linking the molecular reactivity and target preferences of metal-based drug candidates to their biological effects. Finally, this experimental setup is applicable to any other metallodrug that undergoes ligand substitution reactions and/or redox changes as part of its mechanism of action.

Rhodium(I) N-Heterocyclic Carbene Bioorganometallics as in Vitro Antiproliferative Agents with Distinct Effects on Cellular Signaling.

Organometallics with N-heterocyclic carbene (NHC) ligands have triggered major interest in inorganic medicinal chemistry. Complexes of the type Rh(I)(NHC)(COD)X (where X is Cl or I, COD is cyclooctadiene, and NHC is a dimethylbenzimidazolylidene) represent a promising type of new metallodrugs that have been explored by advanced biomedical methods only recently. In this work, we have synthesized and characterized several complexes of this type. As observed by mass spectrometry, these complexes remained stable over at least 3 h in aqueous solution, after which hydrolysis of the halido ligands occurred and release of the NHC ligand was evident. Effects against mitochondria and general cell tumor metabolism were noted at higher concentrations, whereas phosphorylation of HSP27, p38, ERK1/2, FAK, and p70S6K was induced substantially already at lower exposure levels. Regarding the antiproliferative activity in tumor cells, a clear preference for iodido over chlorido secondary ligands was noted, as well as effects of the substituents of the NHC ligand.

Proteomic and Metabolomic Analyses Reveal Contrasting Anti-Inflammatory Effects of an Extract of Mucor Racemosus Secondary Metabolites Compared to Dexamethasone.

Classical drug assays are often confined to single molecules and targeting single pathways. However, it is also desirable to investigate the effects of complex mixtures on complex systems such as living cells including the natural multitude of signalling pathways. Evidence based on herbal medicine has motivated us to investigate potential beneficial health effects of Mucor racemosus (M rac) extracts. Secondary metabolites of M rac were collected using a good-manufacturing process (GMP) approved production line and a validated manufacturing process, in order to obtain a stable product termed SyCircue (National Drug Code USA: 10424-102). Toxicological studies confirmed that this product does not contain mycotoxins and is non-genotoxic. Potential effects on inflammatory processes were investigated by treating stimulated cells with M rac extracts and the effects were compared to the standard anti-inflammatory drug dexamethasone on the levels of the proteome and metabolome. Using 2D-PAGE, slight anti-inflammatory effects were observed in primary white blood mononuclear cells, which were more pronounced in primary human umbilical vein endothelial cells (HUVECs). Proteome profiling based on nLC-MS/MS analysis of tryptic digests revealed inhibitory effects of M rac extracts on pro-inflammatory cytoplasmic mediators and secreted cytokines and chemokines in these endothelial cells. This finding was confirmed using targeted proteomics, here treatment of stimulated cells with M rac extracts down-regulated the secretion of IL-6, IL-8, CXCL5 and GROA significantly. Finally, the modulating effects of M rac on HUVECs were also confirmed on the level of the metabolome. Several metabolites displayed significant concentration changes upon treatment of inflammatory activated HUVECs with the M rac extract, including spermine and lysophosphatidylcholine acyl C18:0 and sphingomyelin C26:1, while the bulk of measured metabolites remained unaffected. Interestingly, the effects of M rac treatment on lipids were orthogonal to the effect of dexamethasone underlining differences in the overall mode of action.