The E-NTPDase Family of Ectonucleotidases: Structure Function Relationships and Pathophysiological Significance

Ectonucleotidases are ectoenzymes that hydrolyze extracellular nucleotides to the respective nucleosides. Within the past decade, ectonucleotidases belonging to several enzyme families have been discovered, cloned and characterized. In this article, we specifically address the cell surface-located members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) family (NTPDase1,2,3, and 8). The molecular identification of individual NTPDase subtypes, genetic engineering, mutational analyses, and the generation of subtype-specific antibodies have resulted in considerable insights into enzyme structure and function. These advances also allow definition of physiological and patho-physiological implications of NTPDases in a considerable variety of tissues. Biological actions of NTPDases are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides and consequent effects on P2-receptor signaling. It further appears that the spatial and temporal expression of NTPDases by various cell types within the vasculature, the nervous tissues and other tissues impacts on several patho-physiological processes. Examples include acute effects on cellular metabolism, adhesion, activation and migration with other protracted impacts upon developmental responses, inclusive of cellular proliferation, differentiation and apoptosis, as seen with atherosclerosis, degenerative neurological diseases and immune rejection of transplanted organs and cells. Future clinical applications are expected to involve the development of new therapeutic strategies for transplantation and various inflammatory cardiovascular, gastrointestinal and neurological diseases

CD73 promotes proliferation and migration of human cervical cancer cells independent of its enzyme activity

100 ΟM Adenosine treatment did not change the expression of EGFR, VEGF and Akt. (JPG 335 kb

Syntenin-1 is a promoter and prognostic marker of head and neck squamous cell carcinoma invasion and metastasis.

Metastasis represents a key factor associated with poor prognosis of head and neck squamous cell carcinoma (HNSC). However, the underlying molecular mechanisms remain largely unknown. In this study, our liquid chromatography with tandem mass spectrometry analysis revealed a number of significantly differentially expressed membrane/membrane-associated proteins between high invasive UM1 and low invasive UM2 cells. One of the identified membrane proteins, Syntenin-1, was remarkably up-regulated in HNSC tissues and cell lines when compared to the controls, and also over-expressed in recurrent HNSC and high invasive UM1 cells. Syntenin-1 over-expression was found to be significantly associated with lymph node metastasis and disease recurrence. HNSC patients with higher syntenin-1 expression had significantly poorer long term overall survival and similar results were found in many other types of cancers based on analysis of The Cancer Genome Atlas data. Finally, knockdown of syntenin-1 inhibited the proliferation, migration and invasion of HNSC cells, and opposite findings were observed when syntenin-1 was over-expressed. Collectively, our studies indicate that syntenin-1 promotes invasion and progression of HNSC. It may serve as a valuable biomarker for lymph node metastasis or a potential target for therapeutic intervention in HNSC

A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment

Within tumors, some areas are less oxygenated than others. Since their home ground is under chronic hypoxia, tumor cells adapt to this condition by activating aerobic glycolysis; however, this hypoxic environment is very harsh for incoming immune cells. Deprivation of oxygen limits availability of energy sources and induces accumulation of extracellular adenosine in tumors. Extracellular adenosine, upon binding with adenosine receptors on the surface of various immune cells, suppresses pro-inflammatory activities. In addition, signaling through adenosine receptors upregulates a number of anti-inflammatory molecules and immunoregulatory cells, leading to the establishment of a long-lasting immunosuppressive environment. Thus, due to hypoxia and adenosine, tumors can discourage anti-tumor immune responses no matter how the response was induced, whether it was spontaneous or artificially introduced with a therapeutic intention. Preclinical studies have shown the significance of adenosine in tumor survival strategy by demonstrating tumor regression after inactivation of adenosine receptors, inhibition of adenosine-producing enzymes or reversal of tissue hypoxia. These promising results indicate a potential use of the inhibitors of the hypoxia-adenosine pathway for cancer immunotherapy

5'-nucleotidases, nucleosides and their distribution in the brain: pathological and therapeutic implications

Elements of the nucleoside system (nucleoside levels, 5'-nucleotidases (5'NTs) and other nucleoside metabolic enzymes, nucleoside transporters and nucleoside receptors) are unevenly distributed in the brain, suggesting that nucleosides have region-specific functions in the human brain. Indeed, adenosine (Ado) and non-Ado nucleosides, such as guanosine (Guo), inosine (Ino) and uridine (Urd), modulate both physiological and pathophysiological processes in the brain, such as sleep, pain, memory, depression, schizophrenia, epilepsy, Huntington's disease, Alzheimer's disease and Parkinson's disease. Interactions have been demonstrated in the nucleoside system between nucleoside levels and the activities of nucleoside metabolic enzymes, nucleoside transporters and Ado receptors in the human brain. Alterations in the nucleoside system may induce pathological changes, resulting in central nervous system (CNS) diseases. Moreover, several CNS diseases such as epilepsy may be treated by modulation of the nucleoside system, which is best achieved by modulating 5'NTs, as 'NTs exhibit numerous functions in the CNS, including intracellular and extracellular formation of nucleosides, termination of nucleoside triphosphate signaling, cell adhesion, synaptogenesis and cell proliferation. Thus, modulation of 5'NT activity may be a promising new therapeutic tool for treating several CNS diseases. The present article describes the regionally different activities of the nucleoside system, demonstrates the associations between these activities and 5'NT activity and discusses the therapeutic implications of these associations

CD73 a novel marker for the diagnosis of benign and malignant salivary gland tumors

Ecto-5?-nucleotidase (CD73) plays an important role in the development of several types of cancer; however, its prognostic significance in salivary gland tumors remains unknown. The current study was conducted to investigate the expression of CD73 in such tumors. In this retrospective study, immunohistochemical expression of CD73 was evaluated in 25 pleomorphic adenomas, 20 mucoepidermoid carcinomas and 20 adenoid cystic carcinomas using the Envision technique. Labeling indices of CD73 expression were calculated and compared between lesions. Immunohistochemical analysis demonstrated that the CD73 expression was significantly higher in salivary gland tumors than in normal salivary gland tissue (p0.05). The findings suggest that CD73 can be an independent and useful biomarker for predicting the clinical behavior of salivary gland tumors

Studies of overcoming acquired resistance : molecular mechanisms and development of novel drugs

Chemotherapeutic agents have become widely applied for treatment of various types of malignancies. Drug resistance unfortunately remains as a major obstacle for the effectiveness of chemotherapy. Cancer drug resistance includes two broad categories: intrinsic and acquired. In this thesis I have examined the problem of acquired drug resistance and have aimed to develop novel approaches to overcome acquired resistance. Clofarabine is a second-generation nucleoside analogue which has been employed primarily for the treatment of hematological malignancies. In paper I, we found that clofarabine inhibited [14C]-thymidine uptake, presumably by decreasing DNA synthesis. Clofarabine was also found to induce apoptosis of a solid tumor cell line, a finding which may open new fields of application of this drug. In paper II, 5’-nucleotidases, a family of enzymes known to confer resistance to nucleoside analogues, were found to be expressed at various levels in samples from CLL patients. Moreover, degradation of fludarabine monophosphate was found to be associated with CN2 activity, and degradation of cladribine monophosphate was associated with CN1 activity. This result helps to explain why some leukemic patients may show resistance to different nucleoside analogues. In paper III, we screened the RPMI 8226 myeloma cell line and its multidrug resistant subline 8226/Dox40 for the response to 3,000 chemically diverse compounds. We found one compound, designated VLX40, which showed strong cytotoxicity to the drug resistant cells. VLX40 was found to be cytotoxic to myeloid and lymphoid leukemia cells. Mechanistic studies showed that VLX40 is a novel microtubule inhibitor. The efficacy of the compound may potentially be improved leading to the development of novel tubulin active agents that are insensitive to common mechanisms of cancer drug resistance. b-AP15 is a novel small molecule inhibitor of the ubiquitin-proteasome system. b- AP15 inhibits the USP14/UCHL5 deubiquitinases of the 19S proteasome and shows anti-tumor activity in a number of tumor models. In paper IV, we examined the anti- multiple myeloma activity of b-AP15. We found that b-AP15 has significant efficacy in multiple myeloma (MM) disease models, including cells resistant to the proteasome inhibitor bortezomib. The finding provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in MM. In paper V, we show that despite the fact that b-AP15 is a reversible enzyme inhibitor, it induces rapid commitment to apoptosis/cell death. We show that the compound is rapidly taken up and enriched in cells, findings that explain the difference in potency of b-AP15 in biochemical and cellular assays. Based on the effective anti-cancer activity of b-AP15, we performed a lead optimization procedure aimed to identify efficient b-AP15 analogues with improved solubility. In paper VI, we identified the analogue VLX1570 which has similar biochemical activity as b-AP15. VLX1570 has strong antineoplastic activity in multiple myeloma cells and is capable of overcoming bortezomib resistance. We conclude that VLX1570 is a promising candidate for the clinical management of multiple myeloma