Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca²⁺ in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca²⁺ signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia.     

Perspectives of purinergic signaling in stem cell differentiation and tissue regeneration

Replacement of lost or dysfunctional tissues by stem cells has recently raised many investigations on therapeutic applications. Purinergic signaling has been shown to regulate proliferation, differentiation, cell death, and successful engraftment of stem cells originated from diverse origins. Adenosine triphosphate release occurs in a controlled way by exocytosis, transporters, and lysosomes or in large amounts from damaged cells, which is then subsequently degraded into adenosine. Paracrine and autocrine mechanisms induced by immune responses present critical factors for the success of stem cell therapy. While P1 receptors generally exert beneficial effects including anti-inflammatory activity, P2 receptor-mediated actions depend on the subtype of stimulated receptors and localization of tissue repair. Pro-inflammatory actions and excitatory tissue damages mainly result from P2X7 receptor activation, while other purinergic receptor subtypes participate in proliferation and differentiation, thereby providing adequate niches for stem cell engraftment and novel mechanisms for cell therapy and endogenous tissue repair. Therapeutic applications based on regulation of purinergic signaling are foreseen for kidney and heart muscle regeneration, Clara-like cell replacement for pulmonary and bronchial epithelial cells as well as for induction of neurogenesis in case of neurodegenerative diseases.     

Purinergic signaling in inflammatory cells: P2 receptor expression, functional effects, and modulation of inflammatory responses

Extracellular ATP and related nucleotides promote a wide range of pathophysiological responses via activation of cell surface purinergic P2 receptors. Almost every cell type expresses P2 receptors and/or exhibit regulated release of ATP. In this review, we focus on the purinergic receptor distribution in inflammatory cells and their implication in diverse immune responses by providing an overview of the current knowledge in the literature related to purinergic signaling in neutrophils, macrophages, dendritic cells, lymphocytes, eosinophils, and mast cells. The pathophysiological role of purinergic signaling in these cells include among others calcium mobilization, actin polymerization, chemotaxis, release of mediators, cell maturation, cytotoxicity, and cell death. We finally discuss the therapeutic potential of P2 receptor subtype selective drugs in inflammatory conditions.     

Differential expression of P2Y receptors in the rat cochlea during development

Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂, during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the driving force for sound transduction. In addition, early postnatal P2Y₂ and P2Y₄ protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca²⁺-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance of extracellular nucleotide and nucleoside signaling to hearing.     

Adenosine arrests breast cancer cell motility by A3 receptor stimulation

In neutrophils, adenosine triphosphate (ATP) release and autocrine purinergic signaling regulate coordinated cell motility during chemotaxis. Here, we studied whether similar mechanisms regulate the motility of breast cancer cells. While neutrophils and benign human mammary epithelial cells (HMEC) form a single leading edge, MDA-MB-231 breast cancer cells possess multiple leading edges enriched with A3 adenosine receptors. Compared to HMEC, MDA-MB-231 cells overexpress the ectonucleotidases ENPP1 and CD73, which convert extracellular ATP released by the cells to adenosine that stimulates A3 receptors and promotes cell migration with frequent directional changes. However, exogenous adenosine added to breast cancer cells or the A3 receptor agonist IB-MECA dose-dependently arrested cell motility by simultaneous stimulation of multiple leading edges, doubling cell surface areas and significantly reducing migration velocity by up to 75 %. We conclude that MDA-MB-231 cells, HMEC, and neutrophils differ in the purinergic signaling mechanisms that regulate their motility patterns and that the subcellular distribution of A3 adenosine receptors in MDA-MB-231 breast cancer cells contributes to dysfunctional cell motility. These findings imply that purinergic signaling mechanisms may be potential therapeutic targets to interfere with the motility of breast cancer cells in order to reduce the spread of cancer cells and the risk of metastasis.     

Signalling by extracellular nucleotides in health and disease

Nucleotides are released from all cells through regulated pathways or as a result of plasma membrane damage or cell death. Outside the cell, nucleotides act as signalling molecules triggering multiple responses via specific plasma membrane receptors of the P2 family. In the nervous system, purinergic signalling has a key function in neurotransmission. Outside the nervous system, purinergic signalling is one of the major modulators of basal tissue homeostasis, while its dysregulation contributes to the pathogenesis of various disease, including inflammation and cancer. Pre-clinical and clinical evidence shows that selective P2 agonists or antagonists are effective treatments for many pathologies, thus highlighting the relevance of extracellular nucleotides and P2 receptors as therapeutic targets.     

Purinergic receptors in gastrointestinal inflammation

Purinergic receptors comprise a family of transmembrane receptors that are activated by extracellular nucleosides and nucleotides. The two major classes of purinergic receptors, P1 and P2, are expressed widely in the gastrointestinal tract as well as immune cells. The purinergic receptors serve a variety of functions from acting as neurotransmitters, to autocoid and paracrine signaling, to cell activation and immune response. Nucleosides and nucleotide agonist of purinergic receptors are released by many cell types in response to specific physiological signals, and their levels are increased during inflammation. In the past decade, the advent of genetic knockout mice and the development of highly potent and selective agonists and antagonists for the purinergic receptors have significantly advanced the understanding of purinergic receptor signaling in health and inflammation. In fact, agonist/antagonists of purinergic receptors are emerging as therapeutic modalities to treat intestinal inflammation. In this article, the distribution of the purinergic receptors in the gastrointestinal tract and their physiological and pathophysiological role in intestinal inflammation will be reviewed.     

Mechanisms of apoptosis modulation by curcumin: Implications for cancer therapy

Cancer incidences are growing and cause millions of deaths worldwide. Cancer therapy is one of the most important challenges in medicine. Improving therapeutic outcomes from cancer therapy is necessary for increasing patients’ survival and quality of life. Adjuvant therapy using various types of antibodies or immunomodulatory agents has suggested modulating tumor response. Resistance to apoptosis is the main reason for radioresistance and chemoresistance of most of the cancers, and also one of the pivotal targets for improving cancer therapy is the modulation of apoptosis signaling pathways. Apoptosis can be induced by intrinsic or extrinsic pathways via stimulation of several targets, such as membrane receptors of tumor necrosis factor-α and transforming growth factor-β, and also mitochondria. Curcumin is a naturally derived agent that induces apoptosis in a variety of different tumor cell lines. Curcumin also activates redox reactions within cells inducing reactive oxygen species (ROS) production that leads to the upregulation of apoptosis receptors on the tumor cell membrane. Curcumin can also upregulate the expression and activity of p53 that inhibits tumor cell proliferation and increases apoptosis. Furthermore, curcumin has a potent inhibitory effect on the activity of NF-κB and COX-2, which are involved in the overexpression of antiapoptosis genes such as Bcl-2. It can also attenuate the regulation of antiapoptosis PI3K signaling and increase the expression of MAPKs to induce endogenous production of ROS. In this paper, we aimed to review the molecular mechanisms of curcumin-induced apoptosis in cancer cells. This action of curcumin could be applicable for use as an adjuvant in combination with other modalities of cancer therapy including radiotherapy and chemotherapy.     

P2Y purinergic signaling in prostate cancer: Emerging insights into pathophysiology and therapy

Despite recent advances in the treatment landscape for prostate cancer, many challenges still remain. A more profound understanding of prostate cancer pathogenesis and the underlying mechanisms is critical to developing novel therapeutics strategies. Extracellular nucleotides play a central role in the growth and progression of a variety of cancer types – almost all tumor cells and immune cells express purinergic membrane receptors for extracellular nucleotides (ATP, ADP, UTP, UDP, UDP-sugar) and their metabolic nucleoside products (e.g., adenosine). Herein we review the pathological and immunomodulatory roles of P2Y purinergic nucleotide receptors in prostate cancer and their potential as therapeutic targets to address some of the clinical limitations in prostate cancer treatment.     

Effect of P2X purinergic receptors in tumor progression and as a potential target for anti-tumor therapy

The development of tumors is a complex pathological process involving multiple factors, multiple steps, and multiple genes. Their prevention and treatment have always been a difficult problem at present. A large number of studies have proved that the tumor microenvironment plays an important role in the progression of tumors. The tumor microenvironment is the place where tumor cells depend for survival, and it plays an important role in regulating the growth, proliferation, apoptosis, migration, and invasion of tumor cells. P2X purinergic receptors, which depend on the ATP ion channel, can be activated by ATP in the tumor microenvironment, and by mediating tumor cells and related cells (such as immune cells) in the tumor microenvironment. They play an important regulatory role on the effects of the skeleton, membrane fluidity, and intracellular molecular metabolism of tumor cells. Therefore, here, we outlined the biological characteristics of P2X purinergic receptors, described the effect of tumor microenvironment on tumor progression, and discussed the effect of ATP on tumor. Moreover, we explored the role of P2X purinergic receptors in the development of tumors and anti-tumor therapy. These data indicate that P2X purinergic receptors may be used as another potential pharmacological target for tumor prevention and treatment.

     

Physiological and pathological functions of P2X7 receptor in the spinal cord

ATP-mediated signaling has widespread actions in the nervous system from neurotransmission to regulation of proliferation. In addition, ATP is released during injury and associated to immune and inflammatory responses. Still, the potential of therapeutic intervention of purinergic signaling during pathological states is only now beginning to be explored because of the large number of purinergic receptors subtypes involved, the complex and often overlapping pharmacology and because ATP has effects on every major cell type present in the CNS. In this review, we will focus on a subclass of purinergic-ligand-gated ion channels, the P2X7 receptor, its pattern of expression and its function in the spinal cord where it is abundantly expressed. We will discuss the mechanisms for P2X7R actions and the potential that manipulating the P2X7R signaling pathway may have for therapeutic intervention in pathological events, specifically in the spinal cord.     

High expression levels of pyrimidine metabolic rate–limiting enzymes are adverse prognostic factors in lung adenocarcinoma: a study based on The Cancer Genome Atlas and Gene Expression Omnibus datasets

Reprogramming of metabolism is described in many types of cancer and is associated with the clinical outcomes. However, the prognostic significance of pyrimidine metabolism signaling pathway in lung adenocarcinoma (LUAD) is unclear. Using the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets, we found that the pyrimidine metabolism signaling pathway was significantly enriched in LUAD. Compared with normal lung tissues, the pyrimidine metabolic rate–limiting enzymes were highly expressed in lung tumor tissues. The high expression levels of pyrimidine metabolic–rate limiting enzymes were associated with unfavorable prognosis. However, purinergic receptors P2RX1, P2RX7, P2RY12, P2RY13, and P2RY14 were relatively downregulated in lung cancer tissues and were associated with favorable prognosis. Moreover, we found that hypo-DNA methylation, DNA amplification, and TP53 mutation were contributing to the high expression levels of pyrimidine metabolic rate–limiting enzymes in lung cancer cells. Furthermore, combined pyrimidine metabolic rate–limiting enzymes had significant prognostic effects in LUAD. Comprehensively, the pyrimidine metabolic rate–limiting enzymes were highly expressed in bladder cancer, breast cancer, colon cancer, liver cancer, and stomach cancer. And the high expression levels of pyrimidine metabolic rate–limiting enzymes were associated with unfavorable prognosis in liver cancer. Overall, our results suggested the mRNA levels of pyrimidine metabolic rate–limiting enzymes CAD, DTYMK, RRM1, RRM2, TK1, TYMS, UCK2, NR5C2, and TK2 were predictive of lung cancer as well as other cancers.

     

New insights into purinergic receptor signaling in neuronal differentiation,neuroprotection, and brain disorders

Ionotropic P2X and metabotropic P2Y purinergic receptors are expressed in the central nervous system and participate in the synaptic process particularly associated with acetylcholine, GABA, and glutamate neurotransmission. As a result of activation, the P2 receptors promote the elevation of free intracellular calcium concentration as the main signaling pathway. Purinergic signaling is present in early stages of embryogenesis and is involved in processes of cell proliferation, migration, and differentiation. The use of new techniques such as knockout animals, in vitro models of neuronal differentiation, antisense oligonucleotides to induce downregulation of purinergic receptor gene expression, and the development of selective inhibitors for purinergic receptor subtypes contribute to the comprehension of the role of purinergic signaling during neurogenesis. In this review, we shall discuss the participation of purinergic receptors in developmental processes and in brain physiology, including neuron-glia interactions and pathophysiology.     

Purinergic interplay between erythrocytes and platelets in diabetes-associated vascular dysfunction

Cardiovascular complications in diabetes are the leading causes for high morbidity and mortality. It has been shown that alteration of purinergic signaling contributes to diabetes-associated cardiovascular complications. Red blood cells (RBCs) and platelets play a fundamental role in regulation of oxygen transport and hemostasis, respectively. Of note, these cells undergo purinergic dysfunction in diabetes. Recent studies have established a novel function of RBCs as disease mediators for the development of endothelial dysfunction in type 2 diabetes (T2D). RBC-released ATP is defective in T2D, which has implication for induction of vascular dysfunction by dysregulating purinergic signaling. Platelets are hyperactive in diabetes. ADP-mediated P2Y₁ and P2Y₁₂ receptor activation contributes to platelet aggregation and targeting P2Y receptors particularly P2Y₁₂ receptor in platelets is effective for the treatment of cardiovascular events. In contrast to other P2Y₁₂ receptor antagonists, platelet-targeting drug ticagrelor has potential to initiate purinergic signaling in RBCs for the beneficial cardiovascular outcomes. It is increasingly clear that altered vascular purinergic signaling mediated by various nucleotides and nucleoside contributes to diabetes-associated vascular dysfunction. However, the contribution of complex purinergic networks between RBCs and platelets to the vascular dysfunction in diabetes remains unclear. This study discusses the possible interplay of RBCs and platelets via the purinergic network for diabetes-associated vascular dysfunction.     

P2Y12 receptor expression is a critical determinant of functional responsiveness to ATX's MORFO domain

In the central nervous system, the formation of the myelin sheath and the differentiation of the myelinating cells, namely oligodendrocytes, are regulated by complex signaling networks that involve purinergic receptors and the extracellular matrix. However, the exact nature of the molecular interactions underlying these networks still needs to be defined. In this respect, the data presented here reveal a signaling mechanism that is characterized by an interaction between the purinergic P2Y(12) receptor and the matricellular extracellular matrix protein autotaxin (ATX), also known as ENPP2, phosphodiesterase-Iα/ATX, or lysoPLD. ATX has been previously described by us to mediate intermediate states of oligodendrocyte adhesion and to enable changes in oligodendrocyte morphology that are thought to be crucial for the formation of a fully functional myelin sheath. This functional property of ATX is mediated by ATX's modulator of oligodendrocyte remodeling and focal adhesion organization (MORFO) domain. Here, we show that the expression of the P2Y(12) receptor is necessary for ATX's MORFO domain to exert its effects on differentiating oligodendrocytes. In addition, our data demonstrate that exogenous expression of the P2Y(12) receptor can render cells responsive to the known effects of ATX's MORFO domain, and they identify Rac1 as an intracellular factor mediating the effect of ATX-MORFO-P2Y(12) signaling on the assembly of focal adhesions. Our data further support the idea that a physical interaction between ATX and the P2Y(12) receptor provides the basis for an ATX-MORFO-P2Y(12) signaling axis that is crucial for mediating cellular states of intermediate adhesion and morphological/structural plasticity.     

Purinergic receptors in the endocrine and exocrine pancreas

The pancreas is a complex gland performing both endocrine and exocrine functions. In recent years there has been increasing evidence that both endocrine and exocrine cells possess purinergic receptors, which influence processes such as insulin secretion and epithelial ion transport. Most commonly, these processes have been viewed separately. In beta cells, stimulation of P2Y(1) receptors amplifies secretion of insulin in the presence of glucose. Nucleotides released from secretory granules could also contribute to autocrine/paracrine regulation in pancreatic islets. In addition to P2Y(1) receptors, there is also evidence for other P2 and adenosine receptors in beta cells (P2Y(2), P2Y(4), P2Y(6), P2X subtypes and A(1) receptors) and in glucagon-secreting alpha cells (P2X(7), A(2) receptors). In the exocrine pancreas, acini release ATP and ATP-hydrolysing and ATP-generating enzymes. P2 receptors are prominent in pancreatic ducts, and several studies indicate that P2Y(2), P2Y(4), P2Y(11), P2X(4) and P2X(7) receptors could regulate secretion, primarily by affecting Cl(-) and K(+) channels and intracellular Ca(2+) signalling. In order to understand the physiology of the whole organ, it is necessary to consider the full complement of purinergic receptors on different cells as well as the structural and functional relation between various cells within the whole organ. In addition to the possible physiological function of purinergic receptors, this review analyses whether the receptors could be potential therapeutic targets for drug design aimed at treatment of pancreatic diseases.