The Cooperative Anti-Neoplastic Activity of Polyphenolic Phytochemicals on Human T-Cell Acute Lymphoblastic Leukemia Cell Line MOLT-4 In Vitro.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting pediatric patients. ALL treatment regimens with cytostatics manifest substantial toxicity and have reached the maximum of well-tolerated doses. One potential approach for improving treatment efficiency could be supplementation of the current regimen with naturally occurring phytochemicals with anti-cancer properties. Nutraceuticals such as quercetin, curcumin, resveratrol, and genistein have been studied in anti-cancer therapy, but their application is limited by their low bioavailability. However, their cooperative activity could potentially increase their efficiency at low, bioavailable doses. We studied their cooperative effect on the viability of a human ALL MOLT-4 cell line in vitro at the concentration considered to be in the bioavailable range in vivo. To analyze their potential side effect on the viability of non-tumor cells, we evaluated their toxicity on a normal human foreskin fibroblast cell line (BJ). In both cell lines, we also measured specific indicators of cell death, changes in cell membrane permeability (CMP), and mitochondrial membrane potential (MMP). Even at a low bioavailable concentration, genistein and curcumin decreased MOLT-4 viability, and their combination had a significant interactive effect. While resveratrol and quercetin did not affect MOLT-4 viability, together they enhanced the effect of the genistein/curcumin mix, significantly inhibiting MOLT-4 population growth in vitro. Moreover, the analyzed phytochemicals and their combinations did not affect the BJ cell line. In both cell lines, they induced a decrease in MMP and correlating CMP changes, but in non-tumor cells, both metabolic activity and cell membrane continuity were restored in time. (4) Conclusions: The results indicate that the interactive activity of analyzed phytochemicals can induce an anti-cancer effect on ALL cells without a significant effect on non-tumor cells. It implies that the application of the combinations of phytochemicals an anti-cancer treatment supplement could be worth further investigation regardless of their low bioavailability.

4-Pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR)-A Novel Oncometabolite Modulating Cancer-Endothelial Interactions in Breast Cancer Metastasis.

The accumulation of specific metabolic intermediates is known to promote cancer progression. We analyzed the role of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR), a nucleotide metabolite that accumulates in the blood of cancer patients, using the 4T1 murine in vivo breast cancer model, and cultured cancer (4T1) and endothelial cells (ECs) for in vitro studies. In vivo studies demonstrated that 4PYR facilitated lung metastasis without affecting primary tumor growth. In vitro studies demonstrated that 4PYR affected extracellular adenine nucleotide metabolism and the intracellular energy status in ECs, shifting catabolite patterns toward the accumulation of extracellular inosine, and leading to the increased permeability of lung ECs. These changes prevailed over the direct effect of 4PYR on 4T1 cells that reduced their invasive potential through 4PYR-induced modulation of the CD73-adenosine axis. We conclude that 4PYR is an oncometabolite that affects later stages of the metastatic cascade by acting specifically through the regulation of EC permeability and metabolic controls of inflammation.

Inhibition of LPS-stimulated ecto-adenosine deaminase attenuates endothelial cell activation.

Vascular inflammation is an important factor in the pathophysiology of cardiovascular diseases, such as atherosclerosis. Changes in the extracellular nucleotide and in particular adenosine catabolism may alter a chronic inflammation and endothelial activation. This study aimed to evaluate the relation between vascular ecto-adenosine deaminase (eADA) activity and endothelial activation in humans and to analyze the effects of LPS-mediated inflammation on this activity as well as mechanisms of its increase. Moreover, we investigated a therapeutic potential of ADA inhibition by deoxycofromycin (dCF) for endothelial activation. We demonstrated a positive correlation of vascular eADA activity and ADA1 mRNA expression with endothelial activation parameters in humans with atherosclerosis. The activation of vascular eADA was also observed under LPS stimulation in vivo along with endothelial activation, an increase in markers of inflammation and alterations in the lipid profile of a rat model. Ex vivo and in vitro studies on human specimen demonstrated that at an early stage of vascular pathology, eADA activity originated from activated endothelial cells, while at later stages also from an inflammatory infiltrate. We proposed that LPS-stimulated increase in endothelial adenosine deaminase activity could be a result of IL-6/JAK/STAT pathway activation, since the lack of IL-6 in mice was associated with lower vascular and plasma eADA activities. Furthermore, the inhibitors of JAK/STAT pathway decreased LPS-stimulated adenosine deaminase activity in endothelial cells. We demonstrated that cell surface eADA activity could be additionally regulated by transcytosis pathways, as exocytosis inhibitors including lipid raft inhibitor, methyl-ß-cyclodextrin decreased LPS-induced eADA activity. This suggests that cholesterol-dependent protein externalization mediated by lipid rafts could be an important factor in the eADA increase. Moreover, endocytosis inhibitors and exocytosis activators increased this activity on the cell surface. Furthermore, the inhibition of adenosine deaminase in endothelial cells in vitro attenuated LPS-mediated IL-6 release and soluble ICAM-1 and VCAM-1 concentration in the incubation medium through the restoration of the extracellular adenosine pool and adenosine receptor-dependent pathways. This study demonstrated that the vascular endothelial eADA activity remains under control of inflammatory mediators acting through JAK/STAT pathway that could be further modified by dyslipidemic-dependent exocytosis and transcytosis pathways. Inhibition of eADA blocked endothelial activation suggesting a crucial role of this enzyme in the control of vascular inflammation. This supports the concept of eADA targeted vascular protection therapy.

Adenosine deaminase inhibition suppresses progression of 4T1 murine breast cancer by adenosine receptor-dependent mechanisms.

The activity of a cell-surface ecto-adenosine deaminase (eADA) is markedly increased in the endothelial activation and vascular inflammation leading to decreased adenosine concentration and alterations in adenosine signalling. Depending on the specific pathway activated, extracellular purines mediate host cell response or regulate growth and cytotoxicity on tumour cells. The aim of this study was to test the effects of adenosine deaminase inhibition by 2'deoxycoformycin (dCF) on the breast cancer development. dCF treatment decreased a tumour growth and a final tumour mass in female BALB/c mice injected orthotopically with 4T1 cancer cells. dCF also counteracted cancer-induced endothelial dysfunction in orthotopic and intravenous 4T1 mouse breast cancer models. In turn, this low dCF dose had a minor effect on immune stimulation exerted by 4T1 cell implantation. In vitro studies revealed that dCF suppressed migration and invasion of 4T1 cells via A2a and A3 adenosine receptor activation as well as 4T1 cell adhesion and transmigration through the endothelial cell layer via A2a receptor stimulation. Similar effects of dCF were observed in human breast cancer cells. Moreover, dCF improved a barrier function of endothelial cells decreasing its permeability. This study highlights beneficial effects of adenosine deaminase inhibition on breast cancer development. The inhibition of adenosine deaminase activity by dCF reduced tumour size that was closely related to the decreased aggressiveness of tumour cells by adenosine receptor-dependent mechanisms and endothelial protection.

Knock-out of CD73 delays the onset of HR-negative breast cancer by reprogramming lipid metabolism and is associated with increased tumor mutational burden.

OBJECTIVE: CD73 (ecto-5'-nucleotidase, NT5E), a cell-surface enzyme converting 5'-AMP to adenosine, is crucial for cancer progression. However, its role in the tumorigenesis process remains mostly obscure. We aimed to demonstrate CD73's role in breast cancer (BC) tumorigenesis through metabolic rewiring of fatty acid metabolism, a process recently indicated to be regulated by BC major prognostic markers, hormone receptors (HR) for estrogen (ER), and progesterone (PR). METHODS: A murine model of chemically induced mammary gland tumorigenesis was applied to analyze CD73 knock-out (KO)-induced changes at the transcriptome (RNA-seq), proteome (IHC, WB), and lipidome (GC-EI-MS) levels. CD73 KO-induced changes were correlated with scRNA-seq and bulk RNA-seq data for human breast tissues and BCs from public collections and confirmed at the proteome level with IHC or WB analysis of BC tissue microarrays and cell lines. RESULTS: CD73 KO delayed the onset of HR/PR-negative mammary tumors in a murine model. This delay correlated with increased expression of genes related to biosynthesis and ß-oxidation of fatty acids (FAs) in the CD73 KO group at the initiation stage. STRING analysis based on RNA-seq data indicated an interplay between CD73 KO, up-regulated expression of PR-coding gene, and DEGs involved in FA metabolism, with PPARγ, a main regulator of FA synthesis, as a main connective node. In epithelial cells of mammary glands, PPARγ expression correlated with CD73 at the RNA level. With cancer progression, CD73 KO increased the levels of PUFAn3/6 (polyunsaturated omega 3/6 FAs), known ligands of PPARγ and target for lipid peroxidation, which may lead to oxidative DNA damage. It correlated with the downregulation of genes involved in cellular stress response (Mlh1, Gsta3), PR- or CD73-dependent changes in the intracellular ROS levels and expression or activation of proteins involved in DNA repair or oxidative stress response in mammary tumor or human BC cell lines, increased tumor mutational burden (TMB) and genomic instability markers in CD73 low HR-negative human BCs, and the prolonged onset of tumors in the CD73 KO HR/PR-negative group. CONCLUSIONS: CD73 has a significant role in tumorigenesis driving the reprogramming of lipid metabolism through the regulatory loop with PR and PPARγ in epithelial cells of mammary glands. Low CD73 expression/CD73 KO might enhance mutational burden by disrupting this regulatory loop, delaying the onset of HR-negative tumors. Our results support combining therapy targeting the CD73-adenosine axis and tumor lipidome against HR-negative tumors, especially at their earliest developmental stage.

U937 variant cells as a model of apoptosis without cell disintegration.

The variant cell line U937V was originally identified by a higher sensitivity to the cytocidal action of tumor necrosis factor alpha (TNFα) than that of its reference cell line, U937. We noticed that a typical morphological feature of dying U937V cells was the lack of cellular disintegration, which contrasts to the formation of apoptotic bodies seen with dying U937 cells. We found that both TNFα, which induces the extrinsic apoptotic pathway, and etoposide (VP-16), which induces the intrinsic apoptotic pathway, stimulated U937V cell death without cell disintegration. In spite of the distinct morphological differences between the U937 and U937V cells, the basic molecular events of apoptosis, such as internucleosomal DNA degradation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, caspase activation and cytochrome c release, were evident in both cell types when stimulated with both types of apoptosis inducer. In the U937V cells, we noted an accelerated release of cytochrome c, an accelerated decrease in mitochondrial membrane potential, and a more pronounced generation of reactive oxygen species compared to the reference cells. We propose that the U937 and U937V cell lines could serve as excellent comparison models for studies on the mechanisms regulating the processes of cellular disintegration during apoptosis, such as blebbing (zeiosis) and apoptotic body formation.

Chemometric evaluation of urinary steroid hormone levels as potential biomarkers of neuroendocrine tumors.

Neuroendocrine tumors (NETs) are uncommon tumors which can secrete specific hormone products such as peptides, biogenic amines and hormones. So far, the diagnosis of NETs has been difficult because most NET markers are not specific for a given tumor and none of the NET markers can be used to fulfil the criteria of high specificity and high sensitivity for the screening procedure. However, by combining the measurements of different NET markers, they become highly sensitive and specific diagnostic tests. The aim of the work was to identify whether urinary steroid hormones can be identified as potential new biomarkers of NETs, which could be used as prognostic and clinical course monitoring factors. Thus, a rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection has been developed for the determination of cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone in human urine. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification were 0.5 and 1 ng mL-1 for each steroid hormone, respectively. Linearity was confirmed within a range of 1-300 ng mL-1 with a correlation coefficient greater than 0.9995 for all analytes. The described method was successfully applied for the quantification of six endogenous steroid levels in human urine. Studies were performed on 20 healthy volunteers and 19 patients with NETs. Next, for better understanding of tumor biology in NETs and for checking whether steroid hormones can be used as potential biomarkers of NETs, a chemometric analysis of urinary steroid hormone levels in both data sets was performed.

Drugs targeting adenosine signaling pathways: A current view.

Adenosine is an endogenous nucleoside that regulates many physiological and pathological processes. It is derived from either the intracellular or extracellular dephosphorylation of adenosine triphosphate and interacts with cell-surface G-protein-coupled receptors. Adenosine plays a substantial role in protecting against cell damage in areas of increased tissue metabolism and preventing organ dysfunction in pathological states. Targeting adenosine metabolism and receptor signaling may be an effective therapeutic approach for human diseases, including cardiovascular and central nervous system disorders, rheumatoid arthritis, asthma, renal diseases, and cancer. Several lines of evidence have shown that many drugs exert their beneficial effects by modulating adenosine signaling pathways but this knowledge urgently needs to be summarized, and most importantly, actualized. The present review collects pharmaceuticals and pharmacological or diagnostic tools that target adenosine signaling in their primary or secondary mode of action. We overviewed FDA-approved drugs as well as those currently being studied in clinical trials. Among them are already used in clinic A2A adenosine receptor modulators like istradefylline or regadenoson, but also plenty of anti-platelet, anti-inflammatory, or immunosuppressive, and anti-cancer drugs. On the other hand, we investigated dozens of specific adenosine pathway regulators that are tested in clinical trials to treat human infectious and noninfectious diseases. In conclusion, targeting purinergic signaling represents a great therapeutic challenge. The actual knowledge of the involvement of adenosinergic signaling as part of the mechanism of action of old drugs has open a path not only for drug-repurposing but also for new therapeutic strategies.

Differences in Extracellular NAD+ and NMN Metabolism on the Surface of Vascular Endothelial Cells.

The disruption of the metabolism of extracellular NAD+ and NMN may affect related signaling cascades and pathologies, such as cardiovascular or respiratory system diseases. We aimed to study NAD+ and NMN hydrolysis on surface endothelial cells of diverse origins and with genetically modified nucleotide catabolism pathways. We tested lung endothelial cells isolated from C57BL/6 J wild-type (WT) and C57BL/6 J CD73 knockout (CD73 KO) mice, the transfected porcine iliac artery endothelial cell line (PIEC) with the human E5NT gene for CD73 (PIEC CD73), and a mock-transfected control (PIEC MOCK), as well as HMEC-1 and H5V cells. Substrate conversion into the product was followed by high-performance liquid chromatography (HPLC). We showed profound differences in extracellular NAD+ and NMN metabolism related to the vessel origin, species diversity, and type of culture. We also confirmed the involvement of CD38 and CD73 in NAD+ and NMN cleavage.

In Silico Designed Gain-of-Function Variants of Complement C2 Support Cytocidal Activity of Anticancer Monoclonal Antibodies.

The molecular target for the classical complement pathway (CP) is defined by surface-bound immunoglobulins. Therefore, numerous anticancer monoclonal antibodies (mAbs) exploit the CP as their effector mechanism. Conversely, the alternative complement pathway (AP) is spontaneously induced on the host and microbial surfaces, but complement inhibitors on host cells prevent its downstream processing. Gain-of-function (GoF) mutations in the AP components that oppose physiological regulation directly predispose carriers to autoimmune/inflammatory diseases. Based on the homology between AP and CP components, we modified the CP component C2 so that it emulates the known pathogenic mutations in the AP component, factor B. By using tumor cell lines and patient-derived leukemic cells along with a set of clinically approved immunotherapeutics, we showed that the supplementation of serum with recombinant GoF C2 variants not only enhances the cytocidal effect of type I anti-CD20 mAbs rituximab and ofatumumab, but also lowers the threshold of mAbs necessary for the efficient lysis of tumor cells and efficiently exploits the leftovers of the drug accumulated in patients' sera after the previous infusion. Moreover, we demonstrate that GoF C2 acts in concert with other therapeutic mAbs, such as type II anti-CD20, anti-CD22, and anti-CD38 specimens, for overcoming cancer cells resistance to complement attack.

An unusual nicotinamide derivative, 4-pyridone-3-carboxamide ribonucleoside (4PYR), is a novel endothelial toxin and oncometabolite.

Our recent studies identified a novel pathway of nicotinamide metabolism that involves 4-pyridone-3-carboxamide-1-ß-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.

Defective apoptosis of U937 cells induced by benzyl isothiocyanate (BITC).

Isothiocyanates precursors (ITCs), including benzyl isothiocyanate (BITC), are considered as cancer chemopreventive agents. ITC derivatives were tested in clinical trials (NCT00005883, NCT01265953, NCT01790204) and preclinical studies aimed to inhibit tumor growth and modulation of their microenvironment. Although efficacy of ITCs was demonstrated with several leukemic cell lines, the final steps of BITC-induced apoptosis were not completely elucidated in the literature. Therefore, we focused on morphological and biochemical events occurring upon treatment of U937 leukemia cells with BITC. Micromolar concentrations of BITC induced cytotoxicity in U937 cells, with major features resembling the hallmarks of apoptosis: phosphatidylserine exposure, low mitochondrial membrane potential, and presence of PARP cleavage by caspases. Disassembly to apoptotic bodies, a final step of classic apoptosis, was not observed. While tracing the signalling pathways, our results showed increased levels of BAG-1 and PUMA proteins, but in contrast to other models of ITCs-induced apoptosis, downregulation of Mcl-1 protein was not noticed. Additionally, BITC-induced dying U937 cells released lower levels of chemoattractants, such as IL-8 and MCP-1, when compared to cells undergoing classical apoptosis. This may disrupt clearance of cell debris by macrophages in vivo (efferocytosis), and in turn affect the inflammatory response. In summary, BITC inhibits tumor growth which makes it a good candidate for supporting cancer treatment. However, atypical apoptosis of leukemic U937 cells induced with BITC may affect the ability of phagocytes to effectively scavenge cellular debris, which poses a question of BITC effectiveness as a chemopreventive agent for leukemias.

Distribution of ectonucleotidases in the rodent brain revisited.

Nucleotides comprise a major class of signaling molecules in the nervous system. They can be released from nerve cells, glial cells, and vascular cells where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and also adenosine is controlled and modulated by cell-surface-located enzymes (ectonucleotidases) that hydrolyze the nucleotide to the respective nucleoside. Extracellular hydrolysis of nucleotide ligands involves a considerable number of enzymes with differing catalytic properties differentially affecting the nucleotide signaling pathway. It is therefore important to investigate which type of ectonucleotidase(s) contributes to the control of nucleotide signaling in distinct cellular and physiological settings. By using a classical enzyme histochemical approach and employing various substrates, inhibitors, and knockout animals, we provide, for the first time, a comparative analysis of the overall distribution of catalytic activities reflecting four ectonucleotidase families: ecto-5'-nucleotidase, alkaline phosphatases, ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), and ectonucleotide pyrophyphatases/phosphodiesterases (E-NPPs). We place into perspective the earlier literature and provide novel evidence for a parenchymal localization of tissue non-specific alkaline phosphatase, E-NPPs, and E-NTPDases in the mouse brain. In addition, we specify the location of ectonucleotidases within the brain vasculature. Most notably, brain vessels do not express ecto-5'-nucleotidase. The preponderance of individual enzymes differs considerably between brain locations. The contribution of all types of ectonucleotidases thus needs to be considered in physiological and pharmacological studies of purinergic signaling in the brain.

The effects of pro- and anti-atherosclerotic factors on intracellular nucleotide concentration in murine endothelial cells.

Endothelial cell activation and dysfunction could lead to endothelial injury that is an important factor in the development of vascular diseases. Vascular injury is strongly associated with disturbed endothelial cell energetics and pyridine nucleotide pool. This study aimed to evaluate the effects of inflammatory stimuli (IL-6, LPS), uric acid, hyperglycemia, fatty acids, flavonoids, statins and nonsteroidal anti-inflammatory drugs on cellular concentration of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and nicotinamide adenine dinucleotide (NAD+) in cultured endothelial cells. Murine-immortalized heart endothelial cells (H5V cells) were treated with different concentrations of pro- and anti-atherosclerotic factors and intracellular concentration of nucleotides were measured using high performance liquid chromatography. Intracellular ATP concentration in H5V cells was not changed by inflammatory stimuli (IL-6 and LPS), uric acid, glucose, atorvastatin, acetylsalicylic acid, monounsaturated and polyunsaturated fatty acids. Only high concentration of palmitic acid (1 mM) and kaempferol (>0.1 mM) decreased intracellular ATP concentration. The concentration of intracellular ADP has not been altered by any of tested compounds. In turn, intracellular NAD+ pool was modified only by polyunsaturated fatty acids and atorvastatin. Linoleic acid, docosahexaenoic acid and atorvastatin increased cellular NAD+ concentration. Tested compounds have a small influence on murine endothelial cell energetics, but polyunsaturated fatty acids and atorvastatin increased intracellular NAD+ concentration that could be an important protective mechanism against endothelial cell injury.

Anti-tumor action of tumor necrosis factor against Bomirski Ab melanoma in hamsters.

INTRODUCTION: Tumor necrosis factor (TNF) is a cytokine able to exert anti-tumor activity in various models and modes of applications. However, the exact mechanism mediating the in vivo anti-tumor effect of TNF has not yet been clarified. MATERIALS AND METHODS: The effects of intratumoral injection of rat TNF into hamsters bearing Bomirski Ab amelanotic melanoma, a fast growing tumor of high metastatic potential, were tested. Subcutaneous injections of the anti-angiogenic compound TNP-470 allowed analysis of its influence on the effects of TNF administration. RESULTS: TNF application resulted in a significant inhibition of tumor growth and changes in metastasis pattern. Accelerated hemorrhagic necrosis was also observed, indicating the effect of the cytokine on tumor vessels. Moreover, the synergistic anti-tumor effect of TNF and anti-angiogenic agent TNP-470 suggested a cooperative activity of both substances on tumor vasculature. Microscopically, the effect of TNF injections was expressed by an increase in the amount of tumor cells with nuclear pyknosis and karryorrhexis. In vitro assays indicated a direct cytotoxic effect of TNF against Ab melanoma cells, most probably as an outcome of apoptosis. Intratumoral application of TNF also caused some modulation of cytokine response in melanoma-bearing hamsters as evidenced by increased levels of IL-6 in blood serum. CONCLUSIONS: This study established Bomirski Ab melanoma as a useful model for complex analysis of the anti-tumor activity of TNF.

Targeted disruption of cd73/ecto-5'-nucleotidase alters thromboregulation and augments vascular inflammatory response.

To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5'-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (-14%; P<0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl3) was significantly reduced by 20% in cd73-/- mice (P<0.05). Bleeding time after tail tip resection tended to be shorter in cd73-/- mice (-35%). In vivo platelet cAMP levels were 0.96+/-0.46 in WT versus 0.68+/-0.27 pmol/106 cells in cd73-/- mice (P<0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 micromol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73-/- compared with WT littermates (WT 98% versus cd73-/- 245%; P<0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73-/- mice (P<0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo.

Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth, Neovascularization, Angiogenesis and Macrophage Infiltration.

CD73 (ecto-5'-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A1, A2A, and A3 in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,ß-methylene 5'-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A1AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A3AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in recently proposed anti-CD73 cancer therapy.

Gel electrophoretic separation of proteins from cultured neuroendocrine tumor cell lines.

Neuroendocrine tumors (NET) often develop asymptomatically and are detected at a late stage. Currently, there exist certain markers of NET that occur only in the advanced stages of the disease. Still, there is need to develop markers specific of the early stage of cancer development. Nevertheless, biomarkers are mostly low­abundant proteins and require separation from complex protein mixtures, which remains a major challenge. The goal of the present study was to optimize one­dimensional­polyacrylamide gel electrophoresis (1D­PAGE) for separation and comparison of protein composition from neuroendocrine tumor samples. 1D­PAGE was optimized by modification of the gel concentration and by comparison of different gel staining protocols. In addition, several steps prior to electrophoresis were carried out to purify and preliminarily reduce the complexity of the sample. The results of these optimization steps indicated that use of an albumin removal kit can considerably decrease the amount of albumin in the samples, thereby allowing to detect proteins of low abundance. Optimal separation of the sample was obtained using a 12% polyacrylamide gel. Furthermore, the use of silver staining allowed detection of proteins at nanogram levels, whereas for Coomassie Brilliant Blue staining, the detection limit was 10 times higher. Optimization of the sample preparation workflow and parameters of the electrophoretic separation allowed to reduce the complexity of the studied material and facilitated further identification of proteins of low abundance in the sample. This study demonstrated that analysis of the secreted proteome of NET cells by 1D­PAGE is a simple and suitable tool for the identification of potential NET protein biomarkers.

CD73 on B16F10 melanoma cells in CD73-deficient mice promotes tumor growth, angiogenesis, neovascularization, macrophage infiltration and metastasis.

Ecto-5'-nucleotidase (CD73), an enzyme providing interstitial adenosine, mediates diverse physiological and pathological responses. In tumor progression, it has primarily an immunosuppressive role but is also thought to regulate neovascularization. However, the latter role is still in debate. When B16F10 melanoma was subcutaneously injected into CD73 knockout mice, changes in the tumor vasculature were not always observed. However, we demonstrated earlier that the growth and vascularization of B16F10 melanoma in CD73 knockout mice depend on the low presence of CD73 on tumor cells. To further analyze the role of CD73 on tumor growth and vascularization, we compared the changes in B16F10 melanoma subcutaneously injected into right flank of wild-type mice, CD73 knockout mice lacking host CD73 only, and CD73 knockout mice with tumor cell CD73 either inhibited with AOPCP (adenosine α,ß-methylene 5'-diphosphate) or permanently knocked down through genetic modification. We report here that both inhibition and knockdown of tumor CD73 further inhibited tumor growth compared to host CD73 knockout alone. MAP-kinase signaling pathway activation also decreased more strongly in the stable knockdown. There was a significant reduction in the angiogenic activation of blood microvessels as observed by decreased anti-VEGFR2 staining. Stable CD73 knockdown also reduced endothelial cell proliferation as measured by anti-CD105 staining. However, only chemical inhibition with AOPCP significantly augmented the reduction in intratumoral microvessel density induced by host CD73 knockout. Such reduction was not observed when tumor CD73 was knocked down due to the much slower tumor growth and decreased oxygen demand as indicated by the low expression of Bad, a hypoxia marker. Decreased CD73 activity also led to the decreased expression of angiogenic factors, including VEGF and bFGF that was only partially reversed by hypoxia in tumors treated with AOPCP. Both inhibition and knockdown of tumor CD73 significantly decreased tumor macrophage infiltration and induced microenvironment changes, thereby influencing MI or MII macrophage polarization. Additionally, tumor cell CD73 is important in metastasis formation through adenosine-independent attachment to endothelium. We conclude that even low tumor cell CD73 expression has an undeniable role in melanoma progression, including the regulation of many aspects of angiogenesis. CD73 is thus a viable target in anti-angiogenic melanoma therapy.

The effect of angiogenesis inhibitor TNP-470 on the blood vessels of the lungs, kidneys and livers of treated hamsters.

The growth of solid tumours and their metastases is dependent on the development of new blood vessels (angiogenesis). Therefore angiogenesis inhibitors are potential antitumour drugs. In our previous studies it was found that the angiogenesis inhibitor TNP-470 given to transplantable melanoma-bearing hamsters can decrease the rate of the tumour growth, although the survival time of the animals treated was not significantly affected. It was found finally that TNP-470 given in the vicinity of the growing tumour can cause complete remission of the melanoma in hamsters treated in this way. To check what side-effects could be evoked by such treatment, an examination of the morphology of the blood vessels of the lungs, kidneys and livers of the treated animals was carried out. It was found that the angiogenesis inhibitor applied did not cause any changes which could be observed by light and electron microscopes in the structure of the examined blood vessels of the treated animals.