Purinergic system in cancer stem cells.

Accumulating evidence supports the idea that cancer stem cells (CSCs) are those with the capacity to initiate tumors, generate phenotypical diversity, sustain growth, confer drug resistance, and orchestrate the spread of tumor cells. It is still controversial whether CSCs originate from normal stem cells residing in the tissue or cancer cells from the tumor bulk that have dedifferentiated to acquire stem-like characteristics. Although CSCs have been pointed out as key drivers in cancer, knowledge regarding their physiology is still blurry; thus, research focusing on CSCs is essential to designing novel and more effective therapeutics. The purinergic system has emerged as an important autocrine-paracrine messenger system with a prominent role at multiple levels of the tumor microenvironment, where it regulates cellular aspects of the tumors themselves and the stromal and immune systems. Recent findings have shown that purinergic signaling also participates in regulating the CSC phenotype. Here, we discuss updated information regarding CSCs in the purinergic system and present evidence supporting the idea that elements of the purinergic system expressed by this subpopulation of the tumor represent attractive pharmacological targets for proposing innovative anti-cancer therapies.

Transactivation of receptor tyrosine kinases by purinergic P2Y and adenosine receptors.

Transactivation of receptor tyrosine kinases (RTK) is a crosstalk mechanism exhibited by G-protein-coupled receptors (GPCR) to activate signaling pathways classically associated with growth factors. The discovery of RTK transactivation was a breakthrough in signal transduction that contributed to developing current concepts in intracellular signaling. RTK transactivation links GPCR signaling to important cellular processes, such as cell proliferation and differentiation, and explains the functional diversity of these receptors. Purinergic (P2Y and adenosine) receptors belong to class A of GPCR; in the present work, we systematically review the experimental evidence showing that purinergic receptors have the ability to transactivate RTK in multiple tissues and physiopathological conditions resulting in the modulation of cellular physiology. Of particular relevance, the crosstalk between purinergic receptors and epidermal growth factor receptor is a redundant pathway that participates in multiple pathophysiological processes. Specific and detailed knowledge of purinergic receptor-regulated pathways advances our understanding of the complexity of GPCR signal transduction and opens the way for pharmacologic intervention in the pathological context.

Autocrine and paracrine purinergic signaling in the most lethal types of cancer.

Cancer comprises a collection of diseases that occur in almost any tissue and it is characterized by an abnormal and uncontrolled cell growth that results in tumor formation and propagation to other tissues, causing tissue and organ malfunction and death. Despite the undeniable improvement in cancer diagnostics and therapy, there is an urgent need for new therapeutic and preventive strategies with improved efficacy and fewer side effects. In this context, purinergic signaling emerges as an interesting candidate as a cancer biomarker or therapeutic target. There is abundant evidence that tumor cells have significant changes in the expression of purinergic receptors, which comprise the G-protein coupled P2Y and AdoR families of receptors and the ligand-gated ion channel P2X receptors. Tumor cells also exhibit changes in the expression of nucleotidases and other enzymes involved in nucleotide metabolism, and the concentrations of extracellular nucleotides are significantly higher than those observed in normal cells. In this review, we will focus on the potential role of purinergic signaling in the ten most lethal cancers (lung, breast, colorectal, liver, stomach, prostate, cervical, esophagus, pancreas, and ovary), which together are responsible for more than 5 million annual deaths.

Purinergic signaling in hepatic disease.

Extracellular purines (ATP and adenosine) are ubiquitous intercellular messengers. During tissular damage, they function as damage-associated molecular patterns (DAMPs). In this context, purines announce tissue alterations to initiate a reparative response that involve the formation of the inflammasome complex and the recruitment of specialized cells of the immune system. The present review focuses on the role of the purinergic system in liver damage, mainly during the onset and development of fibrosis. After hepatocellular injury, extracellular ATP promotes a signaling cascade that ameliorates tissue alterations to restore the hepatic function. However, if cellular damage becomes chronic, ATP orchestrates an aberrant reparative process that results in severe liver diseases such as fibrosis and cirrhosis. ATP and adenosine, their receptors, and extracellular ectonucleotidases are mediators of unique processes that will be reviewed in detail.

Cellular Migration Ability Is Modulated by Extracellular Purines in Ovarian Carcinoma SKOV-3 Cells.

Extracellular nucleotides and nucleosides have emerged as important elements regulating tissue homeostasis. Acting through specific receptors, have the ability to control gene expression patterns to direct cellular fate. We observed that SKOV-3 cells express the ectonucleotidases: ectonucleotide pyrophosphatase 1 (ENPP1), ecto-5'-nucleotidase (NT5E), and liver alkaline phosphatase (ALPL). Strikingly, in pulse and chase experiments supplemented with ATP, SKOV-3 cells exhibited low catabolic efficiency in the conversion of ADP into AMP, but they were efficient in converting AMP into adenosine. Since these cells release ATP, we proposed that the conversion of ADP into AMP is a regulatory node associated with the migratory ability and the mesenchymal characteristics shown by SKOV-3 cells under basal conditions. The landscape of gene expression profiles of SKOV-3 cell cultures treated with apyrase or adenosine demonstrated similarities (e.g., decrease FGF16 transcript) and differences (e.g., the negative regulation of Wnt 2, and 10B by adenosine). Thus, in SKOV-3 we analyzed the migratory ability and the expression of epithelium to mesenchymal transition (EMT) markers in response to apyrase. Apyrase-treatment favored the epithelial-like phenotype, as revealed by the re-location of E-cadherin to the cell to cell junctions. Pharmacological approaches strongly suggested that the effect of Apyrase involved the accumulation of extracellular adenosine; this notion was strengthened when the incubation of the SKOV-3 cell with α,ß-methylene ADP (CD73 inhibitor) or adenosine deaminase was sufficient to abolish the effect of apyrase on cell migration. Overall, adenosine signaling is a fine tune mechanism in the control of cell phenotype in cancer. J. Cell. Biochem. 118: 4468-4478, 2017. © 2017 Wiley Periodicals, Inc.

Nucleotides and nucleoside signaling in the regulation of the epithelium to mesenchymal transition (EMT).

The epithelium-mesenchymal transition (EMT) is an important process of cell plasticity, consisting in the loss of epithelial identity and the gain of mesenchymal characteristics through the coordinated activity of a highly regulated informational program. Although it was originally described in the embryonic development, an important body of information supports its role in pathology, mainly in cancerous and fibrotic processes. The purinergic system of inter-cellular communication, mainly based in ATP and adenosine acting throughout their specific receptors, has emerged as a potent regulator of the EMT in several pathological entities. In this context, cellular signaling associated to purines is opening the understanding of a new element in the complex regulatory network of this phenotypical differentiation process. In this review, we have summarized recent information about the role of ATP and adenosine in EMT, as a growing field with high therapeutic potential.

The P2RY2 Receptor Induces Carcinoma Cell Migration and EMT Through Cross-Talk With Epidermal Growth Factor Receptor.

Extracellular nucleotides are signaling elements present in the tumor microenvironment; however, their role in tumor growth is not completely understood. In the present study, we asked whether nucleotides regulate cell migration in ovarian carcinoma-derived cells. We observed that 100 µM UTP induced migration in SKOV-3 cells (1.57 ± 0.08 fold over basal), and RT-PCR showed expression of transcripts for the P2RY2 and P2RY4 receptors. Knockdown of P2RY2 expression in SKOV-3 cells (P2RY2-KD) abolished the UTP-induced migration. The mechanism activated by UTP to induce migration involves transactivation of the epidermal growth factor receptor (EGFR) since we observed that the EGFR kinase inhibitor AG1478 and the PI3K inhibitor Wortmannin inhibit this response (to 0.76 ± 0.23 and 0.46 ± 0.14 relative to the control, respectively). In agreement with these observations, UTP was able to modify the phosphorylation state of the EGFR; likewise, the induction of ERK1/2 phosphorylation promoted by UTP was abolished by a 30-60 min treatment with AG1478. Our data also suggested that the enhanced cell migration involves the epithelium to mesenchymal transition (EMT) process, since a 12 h stimulation of SKOV-3 cells with 100 µM UTP showed an increase in vimentin and SNAIL protein levels (459.8 ± 132.4% over basal for SNAIL). Interestingly, treatment with apyrase (10 U/mL) reduces the migration of control cells and induces a considerable enrichment of E-cadherin in the cell-cell contacts, favoring an epithelial phenotype and strongly suggesting that the nucleotides released by tumor cells and acting through the P2RY2 receptor are potential regulators of invasiveness.

Differential expression of the P2X7 receptor in ovarian surface epithelium during the oestrous cycle in the mouse.

Purinergic signalling has been proposed as an intraovarian regulatory mechanism. Of the receptors responsible for purinergic transmission, the P2X7 receptor is an ATP-gated cationic channel that displays a broad spectrum of cellular functions ranging from apoptosis to cell proliferation and tumourigenesis. In the present study, we investigated the functional expression of P2X7 receptors in ovarian surface epithelium (OSE). P2X7 protein was detected in the OSE layer of the mouse, both in situ and in primary cultures. In cultures, 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) activation of P2X7 receptors increased [Ca(2+)]i and induced apoptosis. The functionality of the P2X7 receptor was investigated in situ by intrabursal injection of BzATP on each day of the oestrous cycle and evaluation of apoptosis 24h using the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end-labelling (TUNEL) assay. Maximum effects of BzATP were observed during pro-oestrus, with the effects being blocked by A438079, a specific P2X7 receptor antagonist. Immunofluorescence staining for P2X7 protein revealed more robust expression during pro-oestrus and in OSE regions behind the antral follicles, strongly supporting the notion that the differences in apoptosis can be explained by increased receptor expression, which is regulated during the oestrous cycle. Finally, P2X7 receptor expression was detected in the OSE layer of human ovaries, with receptor expression maintained in human ovaries diagnosed with cancer, as well as in the human ovarian carcinoma SKOV3 cell line.

Vascular alpha-1D-adrenoceptors are overexpressed in aorta of the aryl hydrocarbon receptor null mouse: role of increased angiotensin II.

1 The hypothesis that alpha(1D)-adrenoceptors may mediate the pro-hypertensive actions of angiotensin II (Ang II) was tested in isolated aorta (alpha(1D)-adrenoceptor bearing tissue) of the aryl hydrocarbon receptor null mouse (AhR(-/-)), which shows increased levels of Ang II, cardiac hypertrophy and hypertension. 2 The effect of captopril (an angiotensin converting enzyme inhibitor) on both blood pressure and aortic alpha(1D)-adrenoceptor expression and function in mice were determined. 3 Basal blood pressure was higher in AhR(-/-) mice, while captopril therapy decreased it to wild-type (WT) values. 4 Aortas of adult WT and AhR(-/-) mice were stimulated by phenylephrine or noradrenaline to induce contraction; the maximal effect was higher in AhR(-/-) mice, without a significant change in pEC(50). 5 PA(2) values for the selective alpha(1D)-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazynil]ethyl]-8-azaspiro [4.5]decane-7,9-dione) were 9.19 and 8.94 for WT and AhR(-/-), respectively; while Schild slopes were not different from 1. 6 PCR experiments showed c. 77% increase in AhR(-/-)alpha(1D)-adrenoceptors cDNA compared with WT mice; while western blot analysis demonstrated c. 88% increase in alpha(1D)-adrenoceptor protein in AhR(-/-) mice. 7 Captopril therapy decreased alpha(1D)-adrenoceptor-induced contraction and protein in AhR(-/-) mice to WT levels. 8 These data support the hypothesis that under conditions where Ang II is elevated, vascular alpha(1D)-adrenoceptors are increased, and further suggest that both Ang II and vascular alpha(1D)-adrenoceptors could be related in the onset of hypertension.

Phosphorylation and desensitization of alpha1d-adrenergic receptors.

In rat-1 fibroblasts stably expressing rat alpha(1d)-adrenoceptors, noradrenaline and PMA markedly decreased alpha(1d)-adrenoceptor function (noradrenaline-elicited increases in calcium in whole cells and [(35)S]guanosine 5'-[gamma-thio]triphosphate binding in membranes), suggesting homologous and heterologous desensitizations. Photoaffinity labelling, Western blotting and immunoprecipitation identified alpha(1d)-adrenoceptors as a broad band of 70-80 kDa. alpha(1d)-Adrenoceptors were phosphorylated in the basal state and noradrenaline and PMA increased it. The effect of noradrenaline was concentration-dependent (EC(50) 75 nM), rapid (maximum at 1 min) and transient. Phorbol ester-induced phosphorylation was concentration-dependent (EC(50) 25 nM), slightly slower (maximum at 5 min) and stable for at least 60 min. Inhibitors of protein kinase C decreased the effect of phorbol esters but not that of noradrenaline. Evidence of cross-talk of alpha(1d)-adrenoceptors with receptors endogenously expressed in rat-1 fibroblasts was given by the ability of endothelin, lysophosphatidic acid and bradykinin to induce alpha(1d)-adrenoceptor phosphorylation. In summary, it is shown for the first time here that alpha(1d)-adrenoceptors are phosphoproteins and that receptor phosphorylation is increased by the natural ligand, noradrenaline, by direct activation of protein kinase C and via cross-talk with other receptors endogenously expressed in rat-1 fibroblasts. Receptor phosphorylation has functional repercussions.