Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg-1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.

Macrocyclic peptides as allosteric inhibitors of nicotinamide N-methyltransferase (NNMT).

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide (MNA) using S-adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides an indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In the search for structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC50 values as low as 229 nM. The peptides were also found to downregulate MNA production in cellular assays. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT.

Esterase-Sensitive Prodrugs of a Potent Bisubstrate Inhibitor of Nicotinamide N-Methyltransferase (NNMT) Display Cellular Activity.

A recently discovered bisubstrate inhibitor of Nicotinamide N-methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC50 value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into strong cellular activity. This prodrug strategy relies on the temporary protection of the amine and carboxylic acid moieties of the highly polar amino acid side chain present in the bisubstrate inhibitor. The modification of the carboxylic acid into a range of esters in the absence or presence of a trimethyl-lock (TML) amine protecting group yielded a range of candidate prodrugs. Based on the stability in an aqueous buffer, and the confirmed esterase-dependent conversion to the parent compound, the isopropyl ester was selected as the preferred acid prodrug. The isopropyl ester and isopropyl ester-TML prodrugs exhibit improved cell permeability, which also translates to significantly enhanced cellular activity as established using assays designed to measure the enzymatic activity of NNMT in live cells.

Exogenous Vitamins K Exert Anti-Inflammatory Effects Dissociated from Their Role as Substrates for Synthesis of Endogenous MK-4 in Murine Macrophages Cell Line.

Vitamins K exert a range of activities that extend far beyond coagulation and include anti-inflammatory effects, but the mechanisms involved in anti-inflammatory action remain unclear. In the present study, we showed that various forms of exogenous vitamins-K1, K3, K2 (MK-4, MK-5, MK-6 and MK-7)-regulated a wide scope of inflammatory pathways in murine macrophages in vitro, including NOS-2, COX-2, cytokines and MMPs. Moreover, we demonstrated for the first time that macrophages are able to synthesise endogenous MK-4 on their own. Vitamins with shorter isoprenoid chains-K1, K3 and MK-5-exhibited stronger anti-inflammatory potential than vitamins with longer isoprenoid chains (MK-6 and MK-7) and simultaneously were preferably used as a substrate for MK-4 endogenous production. Most interesting, atorvastatin pretreatment inhibited endogenous MK-4 production but had no impact on the anti-inflammatory activity of vitamins K. In summary, our results demonstrate that macrophages are able to synthesise endogenous MK-4 using exogenous vitamins K, and statin inhibits this process. However, the anti-inflammatory effect of exogenous vitamins K was independent of endogenous MK-4 synthesis.

Nicotinamide N-methyltransferase in endothelium protects against oxidant stress-induced endothelial injury.

Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC, HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by NNMT inhibitors (5-amino-1-methylquinoline - 5MQ and 6-methoxynicotinamide - JBSF-88) or by shRNA-mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and presence of menadione. Our results revealed that all studied endothelial lines express relatively high levels of functionally active NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endothelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1. Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role that safeguards endothelial cell viability under oxidant stress insult.

Unexpected effects of long-term treatment with acetylsalicylic acid on late phase of pulmonary metastasis in murine model of orthotopic breast cancer.

Long-term administration of acetylsalicylic acid (ASA) was effective in prevention of colorectal cancer, whereas the efficacy of this compound in other cancer types, including breast cancer, has been less convincingly documented. Indeed, the antimetastatic effect of low-dose ASA was observed only in the early intravascular phase of metastasis of breast cancer. In the present work, we characterized the effects of long-term treatment with ASA on the late phase of pulmonary metastasis in a mouse orthotopic 4T1 breast cancer model. Mice were treated with ASA at a dose of 12 mg·kg-1 of body weight daily starting one week prior to inoculation of 4T1 breast cancer cells, and the treatment was continued throughout progression of the disease. ASA administration decreased platelet TXB2 production in ex vivo assays but did not change thrombin-induced platelet reactivity. Although the number of metastases in the lungs remained unchanged in ASA-treated mice, infiltration of inflammatory cells was increased concomitantly with higher G-CSF and serotonin concentrations in the lungs. Pulmonary NO production was compromised compared to control 4T1 mice. ASA treatment also evoked an increase in platelet and granulocyte counts and decreased systemic NO bioavailability along with increased markers of systemic oxidant stress such as higher GSSG/lower GSH concentrations in RBC. Analysis of eicosanoids in stirred blood demonstrated that administration of ASA at a dose of 12 mg·kg-1 to cancer-bearing mice had an effect beyond inhibition of platelet COX-1, suggesting long-term treatment with low-dose aspirin is not a selective murine platelet COX-1/TXA2 pathway inhibitor in cancer-bearing mice. In summary, quite surprisingly, long-term treatment with low-dose ASA administered until the advanced phase of breast cancer in a murine orthotopic model of 4T1 breast cancer negatively affected the phenotype of the disease.

Alterations in arginine and energy metabolism, structural and signalling lipids in metastatic breast cancer in mice detected in plasma by targeted metabolomics and lipidomics.

BACKGROUND: The early detection of metastasis based on biomarkers in plasma may improve cancer prognosis and guide treatment. The aim of this work was to characterize alterations in metabolites of the arginine pathway, energy metabolism, and structural and signalling lipids in plasma in the early and late stages of murine breast cancer metastasis. METHODS: Mice were orthotopically inoculated with 4T1 metastatic breast cancer cells, and plasma was analysed along the pulmonary metastasis progression using LC-MS/MS-based targeted metabolomics and lipidomics. RESULTS: Based on primary tumour growth and pulmonary metastases, 1-2 weeks after 4T1 cancer cell inoculation was defined as an early metastatic stage, and 3-4 weeks after 4T1 cancer cell inoculation was defined as a late metastatic stage. Early metastasis was featured in plasma by a shift of L-arginine metabolism towards arginase (increased ornithine/arginine ratio) and polyamine synthesis (increased putrescine). Late metastasis was reflected in plasma by further progression of changes in the arginine pathway with an additional increase in asymmetric dimethylarginine plasma concentration, as well as by a profound energy metabolism reprogramming towards glycolysis, an accelerated pentose phosphate pathway and a concomitant decrease in tricarboxylic cycle rate ("Warburg effect"). The late but not the early phase of metastasis was also characterized by a different lipid profile pattern in plasma, including a decrease in total phosphatidylcholines, a decrease in diester-bound phospholipid fraction and an increase in lysophospholipids associated with an increase in total sphingomyelins. CONCLUSIONS: The early phase of metastasis in murine 4T1 metastatic breast cancer was associated with plasma metabolome changes characteristic of arginase activation and polyamine synthesis. The late metastasis was reflected in plasma not only by the alterations in arginine pathways but also by a shift towards glycolysis and the pentose pathway, remodelling of structural lipids and activation of lipid signalling, all of which coincided with metastasis progression.