Biochemometry identifies suppressors of pro-inflammatory gene expression in Pterocarpus santalinus heartwood.

The heartwood extract of the Ayurvedic medicinal plant Pterocarpus santalinus L. f. has previously been shown to significantly suppress the expression of CX3CL1 and other pro-inflammatory molecules in IL-1-stimulated human endothelial cells. Here, we identify the pigment-depleted extract PSD as the most promising yet still complex source of metabolites acting as an inhibitor of CX3CL1 gene expression. For the target-oriented identification of the constituents contributing to the observed in vitro anti-inflammatory effect of PSD, the biochemometric approach ELINA (Eliciting Nature's Activities) was applied. ELINA relies on the deconvolution of complex mixtures by generating microfractions with quantitative variances of constituents over several consecutive fractions. Therefore, PSD was separated into 35 microfractions by means of flash chromatography. Their 1H NMR data and bioactivity data were correlated by heterocovariance analysis. Complemented by LC-MS-ELSD data, ELINA differentiated between constituents with positive and detrimental effects towards activity and allowed for the prioritization of compounds to be isolated in the early steps of phytochemical investigation. A hyphenated high-performance counter-current chromatographic device (HPCCC+) was employed for efficient and targeted isolation of bioactive constituents. A total of 15 metabolites were isolated, including four previously unreported constituents and nine that have never been described before from red sandalwood. Nine isolates were probed for their inhibitory effects on CX3CL1 gene expression, of which four isoflavonoids, namely pterosonin A (1), santal (6), 7,3'-dimethylorobol (12) and the previously unreported compound pterosantalin A (2), were identified as pronounced inhibitors of CX3CL1 gene expression in vitro.

Pterocarpus santalinus Selectively Inhibits a Subset of Pro-Inflammatory Genes in Interleukin-1 Stimulated Endothelial Cells.

Based on the traditional use and scientific reports on the anti-inflammatory potential of red sandalwood, i.e., the heartwood of Pterocarpus santalinus L., we investigated its activity in a model of IL-1 stimulated endothelial cells. Endothelial cells were stimulated with IL-1 with or without prior incubation with a defined sandalwoodextract (PS), and analyzed for the expression of selected pro-inflammatory genes. The activity of NF-κB, a transcription factor of central importance for inflammatory gene expression was assessed by reporter gene analysis, Western blotting of IκBα, and nuclear translocation studies. In addition, microarray studies were performed followed by verification of selected genes by qPCR and supplemented by bioinformatics analysis. Our results show that PS is able to suppress the induction of E-selectin and VCAM-1, molecules that mediate key steps in the adhesion of leukocytes to the endothelium. It also suppressed the activity of an NF-κB reporter, IκBα phosphorylation and degradation, and the nuclear translocation of NF-κB RelA. In contrast, it stimulated JNK phosphorylation indicating the activation of the JNK signaling pathway. Gene expression profiling revealed that PS inhibits only a specific subset of IL-1 induced genes, while others remain unaffected. Most strongly suppressed genes were the signal transducer TRAF1 and the chemokine CX3CL1, whereas IL-8 was an example of a non-affected gene. Notably, PS also stimulated the expression of certain genes, including ones with negative regulatory function, e.g., members of the NR4A family, the mRNA destabilizing protein TTP as well as the transcription factors ATF3 and BHLHB40. These results provide mechanistic insight into the anti-inflammatory activity of PS, and suggest that it acts through the interplay of negative and positive regulators to achieve a differential inhibition of inflammatory gene expression.

Peucedanum ostruthium Inhibits E-Selectin and VCAM-1 Expression in Endothelial Cells through Interference with NF-κB Signaling.

Twenty natural remedies traditionally used against different inflammatory diseases were probed for their potential to suppress the expression of the inflammatory markers E-selectin and VCAM-1 in a model system of IL-1 stimulated human umbilical vein endothelial cells (HUVEC). One third of the tested extracts showed in vitro inhibitory effects comparable to the positive control oxozeaenol, an inhibitor of TAK1. Among them, the extract derived from the roots and rhizomes of Peucedanum ostruthium (i.e., Radix Imperatoriae), also known as masterwort, showed a pronounced and dose-dependent inhibitory effect. Reporter gene analysis demonstrated that inhibition takes place on the transcriptional level and involves the transcription factor NF-κB. A more detailed analysis revealed that the P. ostruthium extract (PO) affected the phosphorylation, degradation, and resynthesis of IκBα, the activation of IKKs, and the nuclear translocation of the NF-κB subunit RelA. Strikingly, early effects on this pathway were less affected as compared to later ones, suggesting that PO may act on mechanism(s) that are downstream of nuclear translocation. As the majority of cognate NF-κB inhibitors affect upstream events such as IKK2, these findings could indicate the existence of targetable signaling events at later stages of NF-κB activation.

LC-ESI-FT-MSn Metabolite Profiling of Symphytum officinale L. Roots Leads to Isolation of Comfreyn A, an Unusual Arylnaphthalene Lignan.

Preparations of comfrey (Symphytum officinale L.) roots are used topically to reduce inflammation. Comfrey anti-inflammatory and analgesic properties have been proven in clinical studies. However, the bioactive compounds associated with these therapeutic activities are yet to be identified. An LC-ESI-Orbitrap-MSn metabolite profile of a hydroalcoholic extract of comfrey root guided the identification of 20 compounds, including a new arylnaphthalene lignan bearing a rare δ-lactone ring, named comfreyn A. Its structure was determined using extensive 2D NMR and ESI-MS experiments. Additionally, the occurrence of malaxinic acid, caffeic acid ethyl ester, along with the lignans ternifoliuslignan D, 3-carboxy-6,7-dihydroxy-1-(3',4'-dihydroxyphenyl) -naphthalene, globoidnan A and B, and rabdosiin was reported in S. officinale for the first time. These results helped to redefine the metabolite profile of this medicinal plant. Finally, caffeic acid ethyl ester and comfreyn A were found to significantly inhibit E-selectin expression in IL-1ß stimulated human umbilical vein endothelial cells (HUVEC), with EC values of 64 and 50 µM, respectively.

A Biochemometric Approach for the Identification of In Vitro Anti-Inflammatory Constituents in Masterwort.

Peucedanum ostruthium (L.) Koch, commonly known as masterwort, has a longstanding history as herbal remedy in the Alpine region of Austria, where the roots and rhizomes are traditionally used to treat disorders of the gastrointestinal and respiratory tract. Based on a significant NF-κB inhibitory activity of a P. ostruthium extract (PO-E), this study aimed to decipher those constituents contributing to the observed activity using a recently developed biochemometric approach named ELINA (Eliciting Nature's Activities). This -omics tool relies on a deconvolution of the multicomponent mixture, which was employed by generating microfractions with quantitative variances of constituents over several consecutive fractions. Using an optimized and single high-performance counter-current chromatographic (HPCCC) fractionation step 31 microfractions of PO-E were obtained. 1H NMR data and bioactivity data from three in vitro cell-based assays, i.e., an NF-ĸB reporter-gene assay and two NF-κB target-gene assays (addressing the endothelial adhesion molecules E-selectin and VCAM-1) were collected for all microfractions. Applying heterocovariance analyses (HetCA) and statistical total correlation spectroscopy (STOCSY), quantitative variances of 1H NMR signals of neighboring fractions and their bioactivities were correlated. This revealed distinct chemical features crucial for the observed activities. Complemented by LC-MS-CAD data this biochemometric approach differentiated between active and inactive constituents of the complex mixture, which was confirmed by NF-κB reporter-gene testing of the isolates. In this way, four furanocoumarins (imperatorin, ostruthol, saxalin, and 2'-O-acetyloxypeucedanin), one coumarin (ostruthin), and one chromone (peucenin) were identified as NF-κB inhibiting constituents of PO-E contributing to the observed NF-ĸB inhibitory activity. Additionally, this approach also enabled the disclose of synergistic effects of the PO-E metabolites imperatorin and peucenin. In sum, prior to any isolation an early identification of even minor active constituents, e.g. peucenin and saxalin, ELINA enables the targeted isolation of bioactive constituents and, thus, to effectively accelerate the NP-based drug discovery process.

Feed­back loops integrating RELA, SOX18 and FAK mediate the break­down of the lymph­endothelial barrier that is triggered by 12(S)­HETE.

Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre­metastatic niches, leading to their colonisation. 'S' stereoisomer 12S­hydroxy­5Z,8Z,10E,14Z­eicosatetraenoic acid [12(S)­HETE] is secreted by a variety of cancer cell types and has been indicated to open up these barriers. In the present study, another aspect of the endothelial unlocking mechanism was elucidated. This was achieved by investigating 12(S)­HETE­treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v­rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY­box transcription factor 18 (SOX18), prospero homeobox 1 (PROX1) and focal adhesion kinase (FAK). These key players of LEC retraction, which is a prerequisite for cancer cell transit into vasculature, were analysed using western blot analysis, reverse transcription­quantitative PCR and transfection with small interfering (si)RNA. The silencing of a combination of these signalling and executing molecules using siRNA, or pharmacological inhibition with defactinib and Bay11­7082, extended the mono­culture experiments to co­culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent­induced defect' assay. 12(S)­HETE was indicated to induce the upregulation of the RELA/SOX18 feedback loop causing the subsequent phosphorylation of FAK, which fed back to RELA/SOX18. Therefore, 12(S)­HETE was demonstrated to be associated with circuits involving RELA, SOX18 and FAK, which transduced signals causing the retraction of LECs. The FAK­inhibitor defactinib and the NF­κB inhibitor Bay11­7082 attenuated LEC retraction additively, which was similar to the suppression of FAK and PROX1 (the target of SOX18) by the transfection of respective siRNAs. FAK is an effector molecule at the distal end of a pro­metastatic signalling cascade. Therefore, targeting the endothelial­specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.

A Symphytum officinale Root Extract Exerts Anti-inflammatory Properties by Affecting Two Distinct Steps of NF-κB Signaling.

Symphytum officinale, commonly known as comfrey, constitutes a traditional medicinal plant with a long-standing therapeutic history, and preparations thereof have been widely used for the treatment of painful muscle and joint complaints, wound and bone healing, and inflammation. Today, its topical use is based on its analgesic and anti-inflammatory effects, which have been substantiated by modern clinical trials. However, the molecular basis of its action remained elusive. Here, we show that a hydroalcoholic extract of comfrey root impairs the development of a pro-inflammatory scenario in primary human endothelial cells in a dose-dependent manner. The extract, and especially its mucilage-depleted fraction, impair the interleukin-1 (IL-1) induced expression of pro-inflammatory markers including E-selectin, VCAM1, ICAM1, and COX-2. Both preparations inhibit the activation of NF-κB, a transcription factor of central importance for the expression of these and other pro-inflammatory genes. Furthermore, our biochemical studies provide evidence that comfrey inhibits NF-κB signaling at two stages: it dampens not only the activation of IKK1/2 and the subsequent IκBα degradation, but also interferes with NF-κB p65 nucleo-cytoplasmatic shuttling and transactivation. These results provide a first mechanistic insight into the mode of action of a century-old popular herbal medicine.

FOXF1 Mediates Endothelial Progenitor Functions and Regulates Vascular Sprouting.

Endothelial colony forming cells (ECFC) or late blood outgrowth endothelial cells (BOEC) have been proposed to contribute to neovascularization in humans. Exploring genes characteristic for the progenitor status of ECFC we have identified the forkhead box transcription factor FOXF1 to be selectively expressed in ECFC compared to mature endothelial cells isolated from the vessel wall. Analyzing the role of FOXF1 by gain- and loss-of-function studies we detected a strong impact of FOXF1 expression on the particularly high sprouting capabilities of endothelial progenitors. This apparently relates to the regulation of expression of several surface receptors. First, FOXF1 overexpression specifically induces the expression of Notch2 receptors and induces sprouting. Vice versa, knock-down of FOXF1 and Notch2 reduces sprouting. In addition, FOXF1 augments the expression of VEGF receptor-2 and of the arterial marker ephrin B2, whereas it downmodulates the venous marker EphB4. In line with these findings on human endothelial progenitors, we further show that knockdown of FOXF1 in the zebrafish model alters, during embryonic development, the regular formation of vasculature by sprouting. Hence, these findings support a crucial role of FOXF1 for endothelial progenitors and connected vascular sprouting as it may be relevant for tissue neovascularization. It further implicates Notch2, VEGF receptor-2, and ephrin B2 as downstream mediators of FOXF1 functions.

12(S)-HETE induces lymph endothelial cell retraction in vitro by upregulation of SOX18.

Metastasising breast cancer cells communicate with adjacent lymph endothelia, intravasate and disseminate through lymphatic routes, colonise lymph nodes and finally metastasize to distant organs. Thus, understanding and blocking intravasation may attenuate the metastatic cascade at an early step. As a trigger factor, which causes the retraction of lymph endothelial cells (LECs) and opens entry ports for tumour cell intravasation, MDA-MB231 breast cancer cells secrete the pro-metastatic arachidonic acid metabolite, 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(S)-HETE]. In the current study, treatment of LECs with 12(S)-HETE upregulated the expression of the transcription factors SRY-related HMG-box 18 (SOX18) and prospero homeobox protein 1 (PROX1), which determine endothelial development. Thus, whether they have a role in LEC retraction was determined using a validated intravasation assay, small interfering RNA mediated knockdown of gene expression, and mRNA and protein expression analyses. Specific inhibition of SOX18 or PROX1 significantly attenuated in vitro intravasation of MDA-MB231 spheroids through the LEC barrier and 12(S)-HETE-triggered signals were transduced by the high and low affinity receptors, 12(S)-HETE receptor and leukotriene B4 receptor 2. In addition, the current findings indicate that there is crosstalk between SOX18 and nuclear factor κ-light-chain-enhancer of activated B cells, which was demonstrated to contribute to MDA-MB231/lymph endothelial intravasation. The present data demonstrate that the endothelial-specific and lymph endothelial-specific transcription factors SOX18 and PROX1 contribute to LEC retraction.

Matrix Metalloproteinase-2 Impairs Homing of Intracoronary Delivered Mesenchymal Stem Cells in a Porcine Reperfused Myocardial Infarction: Comparison With Intramyocardial Cell Delivery.

BACKGROUND: Intracoronary (IC) injection of mesenchymal stem cells (MSCs) results in a prompt decrease of absolute myocardial blood flow (AMF) with late and incomplete recovery of myocardial tissue perfusion. Here, we investigated the effect of decreased AMF on oxidative stress marker matrix metalloproteinase-2 (MMP-2) and its influence on the fate and homing and paracrine character of MSCs after IC or intramyocardial cell delivery in a closed-chest reperfused myocardial infarction (MI) model in pigs. METHODS: Porcine MSCs were transiently transfected with Ad-Luc and Ad-green fluorescent protein (GFP). One week after MI, the GFP-Luc-MSCs were injected either IC (group IC, 11.00 ± 1.07 × 106) or intramyocardially (group IM, 9.88 ± 1.44 × 106). AMF was measured before, immediately after, and 24 h post GFP-Luc-MSC delivery. In vitro bioluminescence signal was used to identify tissue samples containing GFP-Luc-MSCs. Myocardial tissue MMP-2 and CXCR4 receptor expression (index of homing signal) were measured in bioluminescence positive and negative infarcted and border, and non-ischemic myocardial areas 1-day post cell transfer. At 7-day follow-up, myocardial homing (cadherin, CXCR4, and stromal derived factor-1alpha) and angiogenic [fibroblast growth factor 2 (FGF2) and VEGF] were quantified by ELISA of homogenized myocardial tissues from the bioluminescence positive and negative infarcted and border, and non-ischemic myocardium. Biodistribution of the implanted cells was quantified by using Luciferase assay and confirmed by fluorescence immunochemistry. Global left ventricular ejection fraction (LVEF) was measured at baseline and 1-month post cell therapy using magnet resonance image. RESULTS: AMF decreased immediately after IC cell delivery, while no change in tissue perfusion was found in the IM group (42.6 ± 11.7 vs. 56.9 ± 16.7 ml/min, p = 0.018). IC delivery led to a significant increase in myocardial MMP-2 64 kD expression (448 ± 88 vs. 315 ± 54 intensity × mm2, p = 0.021), and decreased expression of CXCR4 (592 ± 50 vs. 714 ± 54 pg/tissue/ml, p = 0.006), with significant exponential decay between MMP-2 and CXCR4 (r = 0.679, p < 0.001). FGF2 and VEGF of the bioluminescence infarcted and border zone of homogenized tissues were significantly elevated in the IM goups as compared to IC group. LVEF increase was significantly higher in IM group (0.8 ± 8.4 vs 5.3 ± 5.2%, p = 0.046) at the 1-month follow up. CONCLUSION: Intracoronary stem cell delivery decreased AMF, with consequent increase in myocardial expression of MMP-2 and reduced CXCR4 expression with lower level of myocardial homing and angiogenic factor release as compared to IM cell delivery.

Anti-thrombotic and pro-fibrinolytic effects of levosimendan in human endothelial cells in vitro.

AIMS: Levosimendan is an inodilator for the treatment of acute decompensated heart failure (HF). Data from clinical studies suggest that levosimendan is particularly effective in HF due to myocardial infarction. After acute revascularization, no reflow-phenomenon is a common complication that may lead to pump failure and cardiogenic shock. Our aim was to examine whether levosimendan interferes with the pro-thrombotic phenotype of activated endothelial cells in vitro. METHODS: Human heart microvascular endothelial cells (HHMEC) and human umbilical vein endothelial cells (HUVEC) were treated with interleukin-1ß (IL-1ß) (200U/mL) or thrombin (5U/mL) and co-treated with or without levosimendan (0.1-10µM) for 2-24h. In addition, flow experiments were performed. Effects on plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression and activity were measured by rt-PCR, specific ELISA and flow cytometry. RESULTS: Treatment with IL-1ß or thrombin significantly increased the expression of PAI-1 and TF in endothelial cells. Co-treatment with levosimendan strongly attenuated the effects of IL-1ß and thrombin on PAI-1 and TF mRNA by up to 50% and 45%, in a dose- and time-dependent manner. Similar results were obtained under flow conditions. Furthermore, co-treatment with levosimendan dampened the antigen production of PAI-1 and the surface expression of TF by 35% and 45%, respectively. Additionally, levosimendan diminished both TF and PAI-1 activity. CONCLUSION: Levosimendan down-regulates the expression of the pro-thrombotic and anti-fibrinolytic biomolecules TF and PAI-1 in activated human endothelial cells. Our findings may, at least in part, explain some of the beneficial effects of levosimendan after myocardial reperfusion.

Three-Dimensional Coculture Model to Analyze the Cross Talk Between Endothelial and Smooth Muscle Cells.

The response of blood vessels to physiological and pathological stimuli partly depends on the cross talk between endothelial cells (EC) lining the luminal side and smooth muscle cells (SMC) building the inner part of the vascular wall. Thus, the in vitro analysis of the pathophysiology of blood vessels requires coculture systems of EC and SMC. We have developed and validated a modified three-dimensional sandwich coculture (3D SW-CC) of EC and SMC using open µ-Slides with a thin glass bottom allowing direct imaging. The culture dish comprises an intermediate plate to minimize the meniscus resulting in homogenous cell distribution. Human umbilical artery SMC were sandwiched between coatings of rat tail collagen I. Following SMC quiescence, human umbilical vein EC were seeded on top of SMC and cultivated until confluence. By day 7, EC had formed a confluent monolayer and continuous vascular endothelial (VE)-cadherin-positive cell/cell contacts. Below, spindle-shaped SMC had formed parallel bundles and showed increased calponin expression compared to day 1. EC and SMC were interspaced by a matrix consisting of laminin, collagen IV, and perlecan. Basal messenger RNA (mRNA) expression levels of E-selectin, angiopoietin-1, calponin, and intercellular adhesion molecule 1 (ICAM-1) of the 3D SW-CC was comparable to that of a freshly isolated mouse inferior vena cava. Addition of tumor necrosis factor alpha (TNF α) to the 3D SW-CC induced E-selectin and ICAM-1 mRNA and protein induction, comparable to the EC and SMC monolayers. In contrast, the addition of activated platelets induced a significantly delayed but more pronounced activation in the 3D SW-CC compared to EC and SMC monolayers. Thus, this 3D SW-CC permits analyzing the cross talk between EC and SMC that mediate cellular quiescence as well as the response to complex activation signals.

Tissue factor is induced by interleukin-33 in human endothelial cells: a new link between coagulation and inflammation.

Tissue factor (TF) is the primary trigger of coagulation. Elevated levels of TF are found in atherosclerotic plaques, and TF leads to thrombus formation when released upon plaque rupture. Interleukin (IL)-33 was previously shown to induce angiogenesis and inflammatory activation of endothelial cells (ECs). Here, we investigated the impact of IL-33 on TF in human ECs, as a possible new link between inflammation and coagulation. IL-33 induced TF mRNA and protein in human umbilical vein ECs and coronary artery ECs. IL-33-induced TF expression was ST2- and NF-κB-dependent, but IL-1-independent. IL-33 also increased cell surface TF activity in ECs and TF activity in ECs-derived microparticles. IL-33-treated ECs reduced coagulation time of whole blood and plasma but not of factor VII-deficient plasma. In human carotid atherosclerotic plaques (n = 57), TF mRNA positively correlated with IL-33 mRNA expression (r = 0.691, p < 0.001). In this tissue, IL-33 and TF protein was detected in ECs and smooth muscle cells by immunofluorescence. Furthermore, IL-33 and TF protein co-localized at the site of clot formation within microvessels in plaques of patients with symptomatic carotid stenosis. Through induction of TF in ECs, IL-33 could enhance their thrombotic capacity and thereby might impact on thrombus formation in the setting of atherosclerosis.

Interleukin-33 stimulates GM-CSF and M-CSF production by human endothelial cells.

Interleukin (IL)-33, a member of the IL-1 family of cytokines, is involved in various inflammatory conditions targeting amongst other cells the endothelium. Besides regulating the maturation and functions of myeloid cells, granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) have been shown to play a role in such pathologies too. It was the aim of our study to investigate a possible influence of IL-33 on GM-CSF and M-CSF production by human endothelial cells. IL-33, but not IL-18 or IL-37, stimulated GM-CSF and M-CSF mRNA expression and protein production by human umbilical vein endothelial cells (HUVECs) and human coronary artery ECs (HCAECs) through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in an IL-1-independent way. This effect was inhibited by the soluble form of ST2 (sST2), which is known to act as a decoy receptor for IL-33. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor fluvastatin could also be shown to moderately reduce the IL-33-mediated effect on M-CSF, but not on GM-CSF expression. In addition, IL-33, IL-1ß, GM-CSF and M-CSF were detected in endothelial cells of human carotid atherosclerotic plaques using immunofluorescence. Upregulation of GM-CSF and M-CSF production by human endothelial cells, an effect that appears to be mediated by NF-κB and to be independent of IL-1, may be an additional mechanism through which IL-33 contributes to inflammatory activation of the vessel wall.

AHR/CYP1A1 interplay triggers lymphatic barrier breaching in breast cancer spheroids by inducing 12(S)-HETE synthesis.

A causal link between overexpression of aryl hydrocarbon receptor (AHR) and its target cytochrome P450 1A1 (CYP1A1) and metastatic outgrowth of various cancer entities has been established. Nevertheless, the mechanism how AHR/CYP1A1 support metastasis formation is still little understood. In vitro we discovered a potential mechanism facilitating tumour dissemination based on the production of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Utilising a three-dimensional lymph endothelial cell (LEC) monolayer & MDA-MB231 breast cancer cell spheroid co-culture model in combination with knock-down approach allowed elucidation of the molecular/biochemical basis of AHR/CYP1A1-induced tumour breaching through the LEC barrier. Enzyme immunoassay evidenced the potential of recombinant CYP1A1 to synthesise 12(S)-HETE in vitro and qPCR and Western blotting measured gene and protein expression in specific experimental settings. In detail, AHR induced CYP1A1 expression and 12(S)-HETE secretion in tumour spheroids, which caused LEC junction retraction thereby forming large discontinuities allowing transmigration of the tumour. This was enforced by the activating AHR ligand 6-formylindolo (3,3-b)carbazole (FICZ), or inhibited by the AHR antagonist 3,3'-diindolylmethane (DIM) as well as by siRNA against AHR and CYP1A1. AHR and NF-κB were negatively cross talking and therefore, the inhibition of AHR (but not CYP1A1) induced RELA, RELB, NFKB1, NFKB2 and the NF-κB target MMP1, which itself promotes tumour intravasation by a mechanism that is different from 12(S)-HETE. Conversely, the inhibition of NFKB2 induced AHR, CYP1A1 and 12(S)-HETE synthesis. The approved clinical drugs guanfacine and vinpocetine, which inhibit CYP1A1 and NF-κB, respectively, significantly inhibited LEC barrier breaching in vitro indicating an option to reduce metastatic dissemination.

NF-κB contributes to MMP1 expression in breast cancer spheroids causing paracrine PAR1 activation and disintegrations in the lymph endothelial barrier in vitro.

RELA, RELB, CREL, NFKB1 and NFKB2, and the upstream regulators NEMO and NIK were knocked-down in lymph endothelial cells (LECs) and in MDA-MB231 breast cancer spheroids to study the contribution of NF-κB in vascular barrier breaching. Suppression of RELA, NFKB1 and NEMO inhibited "circular chemo-repellent induced defects" (CCIDs), which form when cancer cells cross the lymphatic vasculature, by ~20-30%. Suppression of RELB, NFKB2 and NIK inhibited CCIDs by only ~10-15%. In MDA-MB231 cells RELA and NFKB1 constituted MMP1 expression, which caused the activation of PAR1 in adjacent LECs. The knock-down of MMP1 in MDA-MB231 spheroids and pharmacological inhibition of PAR1 in LECs inhibited CCID formation by ~30%. Intracellular Ca(2+) release in LECs, which was induced by recombinant MMP1, was suppressed by the PAR1 inhibitor SCH79797, thereby confirming a functional intercellular axis: RELA/NFKB1 - MMP1 (MDA-MB231) - PAR1 (LEC). Recombinant MMP1 induced PAR1-dependent phosphorylation of MLC2 and FAK in LECs, which is indicative for their activity and for directional cell migration such as observed during CCID formation. The combined knock-down of the NF-κB pathways in LECs and MDA-MB231 spheroids inhibited CCIDs significantly stronger than knock-down in either cell type alone. Also the knock-down of ICAM-1 in LECs (a NF-κB endpoint with relevance for CCID formation) and knock-down of MMP1 in MDA-MB231 augmented CCID inhibition. This evidences that in both cell types NF-κB significantly and independently contributes to tumour-mediated breaching of the lymphatic barrier. Hence, inflamed tumour tissue and/or vasculature pose an additional threat to cancer progression.

The germacranolide sesquiterpene lactone neurolenin B of the medicinal plant Neurolaena lobata (L.) R.Br. ex Cass inhibits NPM/ALK-driven cell expansion and NF-κB-driven tumour intravasation.

BACKGROUND: The t(2;5)(p23;q35) chromosomal translocation results in the expression of the fusion protein NPM/ALK that when expressed in T-lymphocytes gives rise to anaplastic large cell lymphomas (ALCL). In search of new therapy options the dichloromethane extract of the ethnomedicinal plant Neurolaena lobata (L.) R.Br. ex Cass was shown to inhibit NPM/ALK expression. PURPOSE: Therefore, we analysed whether the active principles that were recently isolated and found to inhibit inflammatory responses specifically inhibit growth of NPM/ALK+ ALCL, leukaemia and breast cancer cells, but not of normal cells, and the intravasation through the lymphendothelial barrier. METHODS: ALCL, leukaemia and breast cancer cells, and normal peripheral blood mononuclear cells (PBMCs) were treated with isolated sesquiterpene lactones and analysed for cell cycle progression, proliferation, mitochondrial activity, apoptosis, protein and mRNA expression, NF-κB and cytochrome P450 activity, 12(S)-HETE production and lymphendothelial intravasation. RESULTS: In vitro treatment of ALCL by neurolenin B suppressed NPM/ALK, JunB and PDGF-Rß expression, inhibited the growth of ALCL cells late in M phase, and induced apoptosis via caspase 3 without compromising mitochondrial activity (as a measure of general exogenic toxicity). Moreover, neurolenin B attenuated tumour spheroid intravasation probably through inhibition of NF-κB and CYP1A1. CONCLUSION: Neurolenin B specifically decreased pro-carcinogenic NPM/ALK expression in ALK+ ALCL cells and, via the inhibition of NF-kB signalling, attenuated tumour intra/extravasation into the lymphatics. Hence, neurolenin B may open new options to treat ALCL and to manage early metastatic processes to which no other therapies exist.

Alpha-catulin contributes to drug-resistance of melanoma by activating NF-κB and AP-1.

Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.

Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rß, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this early step of metastatic progression.

Levosimendan exerts anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro.

Levosimendan is a positive inotropic drug for the treatment of acute decompensated heart failure (HF). Clinical trials showed that levosimendan was particularly effective in HF due to myocardial infarction. Myocardial necrosis induces a strong inflammatory response, involving chemoattractants guiding polymorphonuclear neutrophils (PMN) into the infarcted myocardial tissue. Our aim was to examine whether levosimendan exhibits anti-inflammatory effects on human adult cardiac myocytes (HACM) and human heart microvascular endothelial cells (HHMEC). Cardiac myocytes and endothelial cells were stimulated with interleukin-1ß (IL)-1ß (200 U/ml) and treated with levosimendan (0.1-10 µM) for 2-48 hours. IL-1ß strongly induced expression of IL-6 and IL-8 in HACM and E-selectin and intercellular adhesion molecule-1 (ICAM-1) in HHMEC and human umbilical vein endothelial cells (HUVEC). Treatment with levosimendan strongly attenuated IL-1ß-induced expression of IL-6 and IL-8 in HACM as well as E-selectin and ICAM-1 in ECs. Levosimendan treatment further reduced adhesion of PMN to activated endothelial cells under both static and flow conditions by approximately 50 %. Incubation with 5-hydroxydecanoic acid, a selective blocker of mitochondrial ATP-dependent potassium channels, partly abolished the above seen anti-inflammatory effects. Additionally, levosimendan strongly diminished IL-1ß-induced reactive oxygen species and nuclear factor-κB (NF-κB) activity through inhibition of S536 phosphorylation. In conclusion, levosimendan exhibits anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro. These findings could explain, at least in part, the beneficial effects of levosimendan after myocardial infarction.