Several lines of evidence suggest that altered adenosine deaminase (ADA) activity, especially its ADA2 iso-enzyme, is associated with malignant breast cancer (BC) development. Triple-negative breast cancer (TNBC) is currently the most challenging BC subtype due to its metastatic potential and recurrence. Herein, we analyzed the sources of ADA iso-enzymes in TNBC by investigating the effects of cell-to-cell interactions between TNBC cells, macrophages, lymphocytes, and endothelial cells. We also examined the potential relationship between ADA activity and cancer progression in TNBC patients. In vitro analyses demonstrated that the interactions of immune and endothelial cells with MDA-MB-231 triple negative BC cells modulated their extracellular adenosine metabolism pattern. However, they caused an increase in the ADA1 activity, and did not alter ADA2 activity in cancer cells. In turn, the co-culture of MDA-MB-231 cells with THP-1 monocyte/macrophages, Jurkat cells, and human lung microvascular endothelial cells (HULEC) caused the increase in ADA2 activity on THP-1 cells and ADA1 activity on Jurkat cells and HULEC. Clinical sample analysis revealed that TNBC patients had higher plasma ADA2 activities and lower ADA1/ADA2 ratio at advanced stages of cancer development than in the initial stages, while patients with hormone receptor positive, HER2 negative (HR+HER2-), and triple positive (HR+HER2+) breast cancers at the same stages showed opposite trends. TNBC patients also demonstrated positive associations between plasma ADA2 activity and pro-tumor M2 macrophage markers, as well as between ADA1 activity and endothelial dysfunction or inflammatory parameters. The analysis of TNBC patients, at 6 and 12 months following cancer treatment, did not showed significant changes in plasma ADA activities and macrophage polarization markers, which may be the cause of their therapeutic failure. We conclude that alterations in both ADA iso-enzymes can play a role in breast cancer development and progression by the modulation of extracellular adenosine-dependent pathways. Additionally, the changes in ADA2 activity that may contribute to the differentiation of macrophages into unfavorable pro-tumor M2 phenotype deserve special attention in TNBC.

Background Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation. Methods Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients. Results Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine. Conclusions This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors.

Our recent studies identified a novel pathway of nicotinamide metabolism that involves 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) and demonstrated its endothelial cytotoxic effect. This study tested the effects of 4PYR and its metabolites in experimental models of breast cancer. Mice were divided into groups: 4T1 (injected with mammary 4T1 cancer cells), 4T1 + 4PYR (4PYR-treated 4T1 mice), and control, maintained for 2 or 21 days. Lung metastasis and endothelial function were analyzed together with blood nucleotides (including 4PYR), plasma amino acids, nicotinamide metabolites, and vascular ectoenzymes of nucleotide catabolism. 4PYR metabolism was also evaluated in cultured 4T1, MDA-MB-231, MCF-7, and T47D cells. An increase in blood 4PYR in 4T1 mice was observed at 2 days. 4PYR and its metabolites were noticed after 21 days in 4T1 only. Higher blood 4PYR was linked with more lung metastases in 4T1 + 4PYR vs. 4T1. Decreased L-arginine, higher asymmetric dimethyl-L-arginine, and higher vascular ecto-adenosine deaminase were observed in 4T1 + 4PYR vs. 4T1 and control. Vascular relaxation caused by flow-dependent endothelial activation in 4PYR-treated mice was significantly lower than in control. The permeability of 4PYR-treated endothelial cells was increased. Decreased nicotinamide but enhanced nicotinamide metabolites were noticed in 4T1 vs. control. Reduced N-methylnicotinamide and a further increase in Met2PY were observed in 4T1 + 4PYR vs. 4T1 and control. In cultured breast cancer cells, estrogen and progesterone receptor antagonists inhibited the production of 4PYR metabolites. 4PYR formation is accelerated in cancer and induces metabolic disturbances that may affect cancer progression and, especially, metastasis, probably through impaired endothelial homeostasis. 4PYR may be considered a new oncometabolite.

Huntington’s disease (HD) is a rare neurodegenerative disease that is accompanied by skeletal muscle atrophy and cardiomyopathy. Tissues affected by HD (central nervous system [CNS], skeletal muscle, and heart) are known to suffer from deteriorated cellular energy metabolism that manifests already at presymptomatic stages. This work aimed to test the effects of peroxisome proliferator-activated receptor (PPAR)-γ agonist—rosiglitazone on grip strength and heart function in an experimental HD model—on R6/1 mice and to address the mechanisms. We noted that rosiglitazone treatment lead to improvement of R6/1 mice grip strength and cardiac mechanical function. It was accompanied by an enhancement of the total adenine nucleotides pool, increased glucose oxidation, changes in mitochondrial number (indicated as increased citric synthase activity), and reduction in mitochondrial complex I activity. These metabolic changes were supported by increased total antioxidant status in HD mice injected with rosiglitazone. Correction of energy deficits with rosiglitazone was further indicated by decreased accumulation of nucleotide catabolites in HD mice serum. Thus, rosiglitazone treatment may not only delay neurodegeneration but also may ameliorate cardio- and myopathy linked to HD by improvement of cellular energetics.

BackgroundAdenosine deaminase (ADA) via two isoenzymes, ADA1 and ADA2, regulates intra- and extracellular adenosine concentrations by converting it to inosine. In the central nervous system (CNS), adenosine modulates the processes of neuroinflammation and demyelination that together play a critical role in the pathophysiology of multiple sclerosis (MS). Except for their catalytic activities, ADA isoenzymes display extra-enzymatic properties acting as an adhesion molecule or a growth factor.AimsThis study aimed to explore the distribution and activity of ADA1 and ADA2 in the plasma and the CSF of MS patients as well as in the human brain microvascular endothelial cells (HBMEC), human brain vascular pericytes and human astrocytes.Methods and resultsThe enzyme assay following reverse phase-high performance liquid chromatography (HPLC) analysis was used to detect the ADA1 and ADA2 activities and revealed an increased ratio of ADA1 to ADA2 in both the plasma and the CSF of MS patients. Plasma ADA1 activity was significantly induced in MS, while ADA2 was decreased in the CSF, but significance was not reached. The brain astrocytes, pericytes and endothelial cells revealed on their surface the activity of ADA1, with its basal level being five times higher in the endothelial cells than in the astrocytes or the pericytes. In turn, ADA2 activity was only observed in pericytes and endothelial cells. Stimulation of the cells with pro-inflammatory cytokines TNFα/IL17 for 18 h decreased intracellular nucleotide levels measured by HPLC only in pericytes. The treatment with TNFα/IL17 did not modulate cell-surface ATP and AMP hydrolysis nor adenosine deamination in pericytes or astrocytes. Whereas in endothelial cells it downregulated AMP hydrolysis and ADA2 activity and upregulated the ADA1, which reflects the ADA isoenzyme pattern observed here in the CSF of MS patients.ConclusionIn this study, we determined the impaired distribution of both ADA isoenzymes in the plasma and the CSF of patients with MS. The increased ADA1 to ADA2 ratio in the CSF and plasma may translate to unfavorable phenotype that triggers ADA1-mediated pro-inflammatory mechanisms and decreases ADA2-dependent neuroprotective and growth-promoting effects in MS.