Extracellular purines, purinergic receptors and tumor growth.

Virtually, all tumor cells as well as all immune cells express plasma membrane receptors for extracellular nucleosides (adenosine) and nucleotides (ATP, ADP, UTP, UDP and sugar UDP). The tumor microenvironment is characterized by an unusually high concentration of ATP and adenosine. Adenosine is a major determinant of the immunosuppressive tumor milieu. Sequential hydrolysis of extracellular ATP catalyzed by CD39 and CD73 is the main pathway for the generation of adenosine in the tumor interstitium. Extracellular ATP and adenosine mold both host and tumor responses. Depending on the specific receptor activated, extracellular purines mediate immunosuppression or immunostimulation on the host side, and growth stimulation or cytotoxicity on the tumor side. Recent progress in this field is providing the key to decode this complex scenario and to lay the basis to harness the potential benefits for therapy. Preclinical data show that targeting the adenosine-generating pathway (that is, CD73) or adenosinergic receptors (that is, A2A) relieves immunosuppresion and potently inhibits tumor growth. On the other hand, growth of experimental tumors is strongly inhibited by targeting the P2X7 ATP-selective receptor of cancer and immune cells. This review summarizes the recent data on the role played by extracellular purines (purinergic signaling) in host-tumor interaction and highlights novel therapeutic options stemming from recent advances in this field.

The P2X7 receptor is a key modulator of the PI3K/GSK3ß/VEGF signaling network: evidence in experimental neuroblastoma.

Neuroblastoma (NB) is an aggressive pediatric tumor, responsible for 15% of cancer-related deaths in childhood, lacking an effective treatment in its advanced stages. The P2X7 receptor for extracellular ATP was associated to NB cell proliferation and recently emerged as a promoter of tumor engraftment, growth and vascularization. In an effort to identify new therapeutic options for neuroblastoma, we studied the role of P2X7 receptor in NB biology. We first analyzed the effect of P2X7 activation or down-modulation of the main biochemical ways involved in NB progression: the PI3K/Akt/GSK3ß/MYCN and the HIF1α/VEGF pathways. In ACN human NB cells, P2X7 stimulation enhanced PI3K/Akt, while decreasing GSK3ß activity. In the same model, P2X7 silencing or antagonist administration reduced the activity of PI3K/Akt and increased that of GSK3ß, leading to a decrease in cellular glycogen stores. Similarly, P2X7 downmodulation caused a reduction in HIF1α levels and vascular endothelial growth factor (VEGF) secretion. Systemic administration of two different P2X7 antagonists (AZ10606120 or A740003) in nude/nude mice reduced ACN-derived tumor growth. An even stronger effect of P2X7 blockade was obtained in a syngeneic immune-competent neuroblastoma model: Neuro2A cells injected in AlbinoJ mice. Together with tumor regression, treatment with P2X7 antagonists caused downmodulation of the Akt/HIF1α axis, leading to reduced VEGF content and decreased vessel formation. Interestingly, in both experimental models, P2X7 antagonists strongly reduced the expression of the probably best-accepted oncogene in NB: MYCN. Finally, we associated P2X7 overexpression with poor prognosis in advanced-stage NB patients. Taken together, our data suggest that P2X7 receptor is an upstream regulator of the main signaling pathways involved in NB growth, metabolic activity and angiogenesis, and a promising therapeutic target for neuroblastoma treatment.

The P2X7 receptor is a key modulator of aerobic glycolysis.

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.

cAMP efflux from human trophoblast cell lines: a role for multidrug resistance protein (MRP)1 transporter.

Cyclic adenosine 3'-5'-monophosphate (cAMP) is a second messenger, which exerts an important role in the control of human first-trimester trophoblast functions. In the present study we demonstrate the existence of a mechanism that is able to extrude cAMP from trophoblast-derived cell lines, and show evidence indicating the involvement of multidrug resistance protein (MRP) 1, a transporter belonging to the ATP-binding cassette family, in cAMP egress. MRP1 is expressed in trophoblast cell lines and cAMP efflux is highly reduced by the MRP1 inhibitor, MK-571. In addition, interleukin-1beta and estrone are able to enhance MRP1 gene expression and influence extracellular cAMP concentration. The occurrence of a MRP1-dependent cAMP efflux is also shown in human first-trimester placenta explants. Extracellular cAMP could represent a source for adenosine formation, which in turn could regulate cAMP-dependent responses in placental tissue. Evidence is provided that adenosine receptor subtypes are present and functional in human trophoblast-derived cells. A role for cAMP egress mechanism in the fine modulation of the nucleotide homeostasis is therefore suggested.

Somatostatin as a regulator of first-trimester human trophoblast functions.

We have tested the hypothesis that human early trophoblast is a target for somatostatin (SRIF) regulatory actions. We report for the first time that SSTR2A and 2B transcripts and proteins are present in first-trimester human chorionic villi and the trophoblast-derived HTR-8/SVneo and JAR cells. In both cell lines, SSTR are functional since SRIF inhibits cyclic AMP pathway, stimulates arachidonic acid release and enhances cell proliferation. Moreover, in HTR-8/SVneo cells, considered a good model of first-trimester EVT, SRIF also enhances migration. An involvement of the cyclic AMP pathway in mediating SRIF effects on proliferation and migration is suggested. Our data support the idea that SRIF regulates early trophoblast functions mainly through an interaction with SSTR2.

Increased proliferation rate of lymphoid cells transfected with the P2X(7) ATP receptor.

Human leukocytes can express the P2X(7) purinergic receptor, an ionic channel gated by extracellular ATP, for which the physiological role is only partially understood. Transfection of P2X(7) cDNA into lymphoid cells that lack this receptor sustains their proliferation in serum-free medium. Increased proliferation of serum-starved P2X(7) transfectants is abolished by the P2X(7) receptor blocker oxidized ATP or by the ATP hydrolase apyrase. Both wild type and P2X(7)-transfected lymphoid cells release large amounts of ATP into the culture medium. These data suggest the operation of an ATP-based autocrine/paracrine loop that supports lymphoid cell growth in the absence of serum-derived growth factors.

Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2.

The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure-driven multiple sequence alignment with remote homologous protein of known three-dimensional (3D) structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure-driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative "oxyanion hole" involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.

Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily.

We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B'-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11+/-2 microM (mean+/-S.D., n=3) and 6610+/-880 s-1 (mean+/-S.D., n=3) respectively at 70 degreesC and pH7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (+/-)-3-bromo-5-(hydroxymethyl)-Delta2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relationship is discussed in relation to those of the homologous members of the HSL group.

Reliability of a new algorithm for automatic mode switching from DDDR to DDIR pacing mode in sinus node disease patients with chronotropic incompetence and recurrent paroxysmal atrial fibrillation.

To evaluate the safety and efficacy of a new algorithm for automatic mode switching (AMS) from DDD-DDDR to DDIR, 26 patients, 16 females and 10 males, mean age 73 +/- 6 years of age, affected by sinus node disease, chronotropic incompetence, and recurrent paroxysmal atrial fibrillation (PAF) received the Medtronic Thera DR pacemaker. The device continuously calculates, in ms, the running average of the intrinsic atrial rate (MAR) and compares the current atrial interval (CAI) with the stored MAR. When the CAI is greater than the MAR it increases by 8 ms, and when the CAI is less than the MAR, it decreases by 23 ms. When MAR < or = 330 ms (182 beats/min), tachycardia is detected and AMS is activated. All patients had clinical evaluation, 12-lead ECG, Holter monitoring, and exercise testing after implantation and every 3 months for 1 year. The results were compared with the data stored in the pacemaker memory: AMS episodes number; the histogram of the last 14 episodes; and atrial electrogram recording. Twenty-two Holter recordings in 13 patients showed PAF and in all of them AMS occurred simultaneously. AMS lasted between 10 seconds and 20 hours, and MAR ranged from 195-400 beats/min. No episode of PAF and no AMS were recorded in 39 Holter recordings in 22 patients. Appropriate AMS was confirmed in five patients by stored atrial electrogram and in nine by 12-lead ECG and pacemaker event markers. Mean atrial sensing was 2.13 +/- 1.04 mV during PAF and 3.18 +/- 1.46 mV during sinus rhythm. No PAF episode and no AMS were recorded during exercise testing. In conclusion, this new algorithm was very reliable, sensitive, and specific.

[Prognostic evaluation with invasive technics in patients with hyperkinetic ventricular arrhythmias]. / Valutazione prognostica con metodiche invasive di pazienti con aritmie ipercinetiche ventricolari.

The prognostic value of induction of ventricular tachycardia (VT) by programmed electrical stimulation (PES) was analyzed in 123 patients: 64 (Group I) with spontaneous recurrent VT and 59 (Group II) without a history of serious arrhythmias. Thirty-three patients with spontaneous VT underwent coronary and left ventricular angiography to compare electrical instability with the presence of ventricular disfunction and/or the extent of coronary artery disease (CAD). PES reproducibly induced VT in 49/64 patients with spontaneous VT (sensitivity = 77%) and in 6/59 patients without VT (specificity = 90%). Twenty-two patients (66%) had ventricular disfunction defined by an ejection fraction of less than or equal to 40% or regional wall motion abnormalities. Only 4 patients (33%) had proximal 3-vessel CAD. The mean follow-up period was 16 +/- 12 months. Eight of Group I patients died suddenly and 24 had recurrent symptomatic VT. Three of Group I patients died (1 cardiac failure, 2 non-cardiac deaths), all the survivors were free of serious arrhythmias. In Group I patients mortality was correlated with: recent anterior myocardial infarction, inducible sustained VT with PES, ejection fraction less than or equal to 0.40, ventricular ipoasynergy and or at least one coronary stenosis greater than or equal to 70%. This study suggests that inducible VT is a marker of the risk of sudden death. Electrical instability may occur independent from the etiology of cardiopathy, ventricular disfunction and extent of CAD, but these parameters are correlated to global and sudden mortality in the group of patients with spontaneous VT.