CD73 mitigates ZEB1 expression in papillary thyroid carcinoma.

BACKGROUND: ZEB1, a core transcription factor involved in epithelial-mesenchymal transition (EMT), is associated with aggressive cancer cell behavior, treatment resistance, and poor prognosis across various tumor types. Similarly, the expression and activity of CD73, an ectonucleotidase implicated in adenosine generation, is an important marker of tumor malignancy. Growing evidence suggests that EMT and the adenosinergic pathway are intricately linked and play a pivotal role in cancer development. Therefore, this study focuses on exploring the correlations between CD73 and ZEB1, considering their impact on tumor progression. METHODS: We employed CRISPR/Cas9 technology to silence CD73 expression in cell lines derived from papillary thyroid carcinoma. These same cells underwent lentiviral transduction of a reporter of ZEB1 non-coding RNA regulation. We conducted studies on cell migration using scratch assays and analyses of cellular speed and polarity. Additionally, we examined ZEB1 reporter expression through flow cytometry and immunocytochemistry, complemented by Western blot analysis for protein quantification. For further insights, we applied gene signatures representing different EMT states in an RNA-seq expression analysis of papillary thyroid carcinoma samples from The Cancer Genome Atlas. RESULTS: Silencing CD73 expression led to a reduction in ZEB1 non-coding RNA regulation reporter expression in a papillary thyroid carcinoma-derived cell line. Additionally, it also mitigated ZEB1 protein expression. Moreover, the expression of CD73 and ZEB1 was correlated with alterations in cell morphology characteristics crucial for cell migration, promoting an increase in cell polarity index and cell migration speed. RNA-seq analysis revealed higher expression of NT5E (CD73) in samples with BRAF mutations, accompanied by a prevalence of partial-EMT/hybrid state signature expression. CONCLUSIONS: Collectively, our findings suggest an association between CD73 expression and/or activity and the post-transcriptional regulation of ZEB1 by non-coding RNA, indicating a reduction in its absence. Further investigations are warranted to elucidate the relationship between CD73 and ZEB1, with the potential for targeting them as therapeutic alternatives for cancer treatment in the near future.

The crossroads of adenosinergic pathway and epithelial-mesenchymal plasticity in cancer.

Epithelial-mesenchymal transition (EMT) is a key mechanism related to tumor progression, invasion, metastasis, resistance to therapy and poor prognosis in several types of cancer. However, targeting EMT or partial-EMT, as well as the molecules involved in this process, has remained a challenge. Recently, the CD73 enzyme, which hydrolyzes AMP to produce adenosine (ADO), has been linked to the EMT process. This relationship is not only due to the production of the immunosuppressant ADO but also to its role as a receptor for extracellular matrix proteins, being involved in cell adhesion and migration. This article reviews the crosstalk between the adenosinergic pathway and the EMT program and the impact of this interrelation on cancer development and progression. An in silico analysis of RNAseq datasets showed that several tumor types have a significant correlation between an EMT score and NT5E (CD73) and ENTPD1 (CD39) expressions, with the strongest correlations being in prostate adenocarcinoma. Furthermore, it is evident that the cooperation between EMT and the adenosinergic pathway in tumor progression is context and tumor-dependent. The increased knowledge about this topic will help broaden the view to explore new treatments and therapies for different types of cancer.

Epidermal melanocytes metabolize extracellular nucleotides by purinergic enzymes.

The human epidermal melanocyte (hEM) are melanin-producing cells that provide skin pigmentation and protection against ultraviolet radiation. Although purinergic signaling is involved in skin biology and pathology, the presence of NTPDase members, as well as the rate of nucleotides degradation by melanocytes were not described yet. Therefore, in this study, we analyzed the expression of ectonucleotidases in hEM derived from discarded foreskin of male patients. The expression of purinergic enzymes was confirmed by mRNA and flow cytometry. Among the ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5´-nucleotidase were the ectoenzymes with higher expressions. The hydrolysis rate for ATP, ADP, and AMP was low in comparison to other primary cells already investigated. The amount of ATP in the culture medium was increased after a scratch wound and decreased to basal levels in 48 h, while the NTPDase1 and P2X7 expressions increased. Therefore, it is possible to suggest that after cell injury, the ATP released by hEM into the extracellular space will be hydrolyzed by ectonucleotidases as the NTPDase1 that will control the levels of nucleotides in the skin micro-environment.

A Systematic Review of the Role of Purinergic Signalling Pathway in the Treatment of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global health concern. Three years since its origin, despite the approval of vaccines and specific treatments against this new coronavirus, there are still high rates of infection, hospitalization, and mortality in some countries. COVID-19 is characterised by a high inflammatory state and coagulation disturbances that may be linked to purinergic signalling molecules such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine (ADO), and purinergic receptors (P1 and P2). These nucleotides/nucleosides play important roles in cellular processes, such as immunomodulation, blood clot formation, and vasodilation, which are affected during SARS-CoV-2 infection. Therefore, drugs targeting this purinergic pathway, currently used for other pathologies, are being evaluated in preclinical and clinical trials for COVID-19. In this review, we focus on the potential of these drugs to control the release, degradation, and reuptake of these extracellular nucleotides and nucleosides to treat COVID-19. Drugs targeting the P1 receptors could have therapeutic efficacy due to their capacity to modulate the cytokine storm and the immune response. Those acting in P2X7, which is linked to NLRP3 inflammasome activation, are also valuable candidates as they can reduce the release of pro-inflammatory cytokines. However, according to the available preclinical and clinical data, the most promising medications to be used for COVID-19 treatment are those that modulate platelets behaviour and blood coagulation factors, mainly through the P2Y12 receptor.

The implications of the purinergic signaling throughout pregnancy.

Purinergic signaling is a necessary mechanism to trigger or even amplify cell communication. Its ligands, notably adenosine triphosphate (ATP) and adenosine, modulate specific membrane-bound receptors in virtually all human cells. Regardless of the stage of the pregnancy, cellular communication between maternal, placental, and fetal cells is the paramount mechanism to sustain its optimal status. In this review, we describe the crucial role of purinergic signaling on the regulation of the maternal-fetal trophic exchanges, immune control, and endocrine exchanges throughout pregnancy. The nature of the modulation of both ATP and adenosine on the embryo-maternal interface, going through placental invasion until birth delivery depends on the general maternal-fetal health state and consequently on the selective activation of their specific receptors. In addition, an increasing number of studies have been demonstrating the pivotal role of ATP and adenosine in modulating deleterious effects of suboptimal conditions of pregnancy. Here, we discuss the role of purinergic signaling on the balance that coordinates the embryo-maternal exchanges and a promising therapeutic venue in the context of pregnancy disorders.

The adenosinergic pathway in mesenchymal stem cell fate and functions.

Mesenchymal stem cells (MSCs) play an important role in tissue homeostasis and damage repair through their ability to differentiate into cells of different tissues, trophic support, and immunomodulation. These properties made them attractive for clinical applications in regenerative medicine, immune disorders, and cell transplantation. However, despite multiple preclinical and clinical studies demonstrating beneficial effects of MSCs, their native identity and mechanisms of action remain inconclusive. Since its discovery, the CD73/ecto-5'-nucleotidase is known as a classic marker for MSCs, but its role goes far beyond a phenotypic characterization antigen. CD73 contributes to adenosine production, therefore, is an essential component of purinergic signaling, a pathway composed of different nucleotides and nucleosides, which concentrations are finely regulated by the ectoenzymes and receptors. Thus, purinergic signaling controls pathophysiological functions such as proliferation, migration, cell fate, and immune responses. Despite the remarkable progress already achieved in considering adenosinergic pathway as a therapeutic target in different pathologies, its role is not fully explored in the context of the therapeutic functions of MSCs. Therefore, in this review, we provide an overview of the role of CD73 and adenosine-mediated signaling in the functions ascribed to MSCs, such as homing and proliferation, cell differentiation, and immunomodulation. Additionally, we will discuss the pathophysiological role of MSCs, via CD73 and adenosine, in different diseases, as well as in tumor development and progression. A better understanding of the adenosinergic pathway in the regulation of MSCs functions will help to provide improved therapeutic strategies applicable in regenerative medicine.

ENTPD5: identification of splicing variants and their impact on cancer survival.

NTPDase5 is a nucleotidase of the endoplasmic reticulum that plays an important role in proteostasis as a regulator of protein N-glycosylation. This enzyme was first identified in hamster as a proto-oncogene activated upon a single nucleotide deletion that causes a frameshift leading to a truncated protein. Truncated NTPDase5 proteins were detected in human samples, but an oncogene was never identified. Searching for transcript variants in the GenBank database and using TCGA data, we discovered that splice variants could originate truncated human NTPDase5 proteins. We identified three main splicing events in the ENTPD5 gene: alternative acceptors, exon skipping, and alternative terminators. The analysis of impact of splicing events in cancers showed that skipping of exon 11-the event that leads to truncated proteins similar in size to the hamster oncogene-does not affect the hazard ratio of most tumors and was, in fact, a protective factor in the only two cancer studies where it was significant. We also identified four main patterns of impact of ENTPD5 in cancer and a potential variant-specific regulation by miR-215. Our findings shed light on a two-decade uncertainty about the origin of truncated NTPDase5 and contribute to the characterization of its impacts in cancer.

A three-dimensional microenvironment alters CD73 expression in cervical cancer.

Stem-like cells (CSCs) have a tumour-initiating capacity and play critical role in tumour metastasis, relapse and resistance to therapy. The ectoenzyme CD73, encoded by the NT5E gene, which catalyses the hydrolysis of AMP into adenosine, has been associated to an immunosuppressive tumour microenvironment, tumour cell adhesion and migration. Therefore, we investigated the expression and activity of CD73 in sphere-forming cells from cervical cancer in comparison to monolayer cells in vitro. In addition, in silico analysis was performed to determine the expression of CD73 and other members of purinergic signalling in CSC-like population derived from different tumour types in comparison to monolayer cells. CD73 protein expression levels and functionality in SiHa cells were analysed by flow cytometry and enzymatic assay, respectively. In silico investigation was performed through the analysis of seven datasets from different tumour types using GEO database. In vitro analysis showed a decreased CD73 protein expression and enzymatic activity in cervical spheres, when compared to monolayers. In addition, when sphere-derived cells are re-plated as monolayer culture, the CD73 expression and activity are restored. Supporting the in vitro results, in silico analysis showed that three-dimensional spheres derived from cervical, thyroid and breast cancer presented decreased expression of CD73, when compared to their adherent counterparts. The decreased expression of CD73 in sphere-derived cells or CSC-enriched population reinforce its important role in cell adhesion, tumour spreading ability and metastasis, suggesting CD73 as potential target to be further investigated in cervical cancer.

Xanthan gum-based hydrogel containing nanocapsules for cutaneous diphenyl diselenide delivery in melanoma therapy.

The aim of this study was to further evaluate the antitumoral effect of (PhSe)2-loaded polymeric nanocapsules (NC (PhSe)2) against a resistant melanoma cell line (SK-Mel-103) and develop a xanthan gum-based hydrogel intending the NC (PhSe)2 cutaneous application. For the in vitro evaluation, cells were incubated with free (PhSe)2 or NC (PhSe)2 (0.7-200 µM) and after 48 h the MTT assay, propidium iodide uptake (necrosis marker) and nitrite levels were assessed. The hydrogels were developed by thickening of the NC (PhSe)2 suspension or (PhSe)2 solution with xanthan gum and characterized in terms of average diameter, polydispersity index, pH, drug content, spreadability, rheological profiles and in vitro permeation in human skin. The results showed that NC (PhSe)2 provided a superior antitumoral effect in comparison to free (PhSe)2 (IC50 value of 47.43 µM and 65.05 µM, respectively) and increased the nitrite content. Both compound forms induced propidium iodide uptake, suggesting a necrosis-related pathway could be involved in the cytotoxic action of (PhSe)2. All hydrogels showed pH values around 7, drug content close to the theoretical values (5 mg/g) and mean diameter in the nanometric range. Besides, formulations were classified as non-Newtonian flow with pseudoplastic behavior and suitable spreadability factor. Skin permeation studies revealed that the compound content was higher for the nano-based hydrogel in the dermis layer, demonstrating its superior permeation, achieved by the compound encapsulation. It is the first report on an adequate formulation development for cutaneous application of NC (PhSe)2 that could be used as an adjuvant treatment in melanoma therapy.

Imatinib mesylate affects extracellular ATP catabolism and expression of NTPDases in a chronic myeloid leukemia cell line.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists of treatment with imatinib mesylate, which selectively inhibits the BCR-ABL protein by competing for its ATP-binding site. Adenine nucleotide signaling is modulated by the ectonucleotidases and this pathway is related to tumorigenic processes. Considering the relationship between ATP and cancer, we aimed to evaluate the influence of imatinib mesylate on the expressions and functions of the NTPDase and ecto-5'-nucleotidase (CD73) enzymes in imatinib-sensitive and -resistant K-562 cell lines. mRNA analysis showed that K-562 cells express all ENTPDs and NT5E. However, when treated with imatinib mesylate for 24 h, the expression of ENTPD1, -2, -3 and -5 increased, leading to a higher nucleotides hydrolysis rate. HPLC analysis identified increased ATP degradation in cells after 24 h of treatment, with consequent ADP and AMP formation, corroborating the increase in gene and protein expression of ectonucleotidases as observed in previous results. On the other hand, we observed that imatinib-resistant K-562 cells presented a decrease in nucleotide hydrolysis and expressions of ENTPD1 and -5. These results suggest an involvement of imatinib in modulating ectonucleotidases in CML that will need further investigation. Since these ectonucleotidases have important catalytic activities in the tumor microenvironment, their modulation in CML cells may represent an important therapeutic approach to regulate levels of extracellular adenine nucleotides.

Dipyridamole impairs autophagic flux and exerts antiproliferative activity on prostate cancer cells.

Autophagy is a cellular bulk degradation process used as an alternative source of energy and metabolites and implicated in various diseases. Inefficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy making its modulation valuable as a therapeutic strategy for cancer treatment, especially in combination with chemotherapy. Dipyridamole (DIP) is a vasodilator and antithrombotic drug. Its major effects involve the block of nucleoside uptake and phosphodiestesase inhibition, leading to increased levels of intracellular cAMP. Here we report that DIP increases autophagic markers due to autophagic flux blockage, resembling autophagosome maturation and/or closure impairment. Treatment with DIP results in an increased number of autophagosomes and autolysosomes and impairs degradation of SQSTM1/p62. As blockage of autophagic flux decreases the recycling of cellular components, DIP reduced the intracellular ATP levels in cancer cells. Autophagic flux blockage was neither through inhibition of lysosome function nor blockage of nucleoside uptake, but could be prevented by treatment with a PKA inhibitor, suggesting that autophagic flux failure mediated by DIP results from increased intracellular levels of cAMP. Treatment with DIP presented antiproliferative effects in vitro alone and in combination with chemotherapy drugs. Collectively, these data demonstrate that DIP can impair autophagic degradation, by preventing the normal autophagosome maturation, and might be useful in combination anticancer therapy.

Characterization of soluble CD39 (SolCD39/NTPDase1) from PiggyBac nonviral system as a tool to control the nucleotides level.

Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

Purinergic Signaling in Glioma Progression.

Among the pathological alterations that give tumor cells invasive potential, purinergic signaling is emerging as an important component. Studies performed in in vitro, in vivo and ex vivo glioma models indicate that alterations in the purinergic signaling are involved in the progression of these tumors. Gliomas have low expression of all E-NTPDases, when compared to astrocytes in culture. Nucleotides induce glioma proliferation and ATP, although potentially neurotoxic, does not evoke cytotoxic action on the majority of glioma cells in culture. The importance of extracellular ATP for glioma pathobiology was confirmed by the reduction in glioma tumor size by apyrase, which degrades extracellular ATP to AMP, and the striking increase in tumor size by over-expression of an ecto-enzyme that degrades ATP to ADP, suggesting the effect of extracellular ATP on the tumor growth depends on the nucleotide produced by its degradation. The participation of purinergic receptors on glioma progression, particularly P2X7, is involved in the resistance to ATP-induced cell death. Although more studies are necessary, the purinergic signaling, including ectonucleotidases and receptors, may be considered as future target for glioma pharmacological or gene therapy.

Physical exercise and human adipose-derived mesenchymal stem cells ameliorate motor disturbances in a male rat model of Parkinson's disease.

Parkinson's disease (PD) is a disabling and highly costly neurodegenerative condition with worldwide prevalence. Despite advances in treatments that slow progression and minimize locomotor impairments, its clinical management is still a challenge. Previous preclinical studies, using mesenchymal stem cell (MSC) transplantation and isolated physical exercise (EX), reported beneficial results for treatment of PD. Therefore, this experimental randomized study aimed to elucidate the therapeutic potential of combined therapy using adipose-derived human MSCs (ADSCs) grafted into the striatum in conjunction with aerobic treadmill training, specifically in terms of locomotor performance in a unilateral PD rat model induced by 6-hydroxydopamine (6-OHDA). Forty-one male Wistar rats were categorized into five groups in accordance with the type of treatment to which they were subjected (Sham, 6-OHDA - injury, 6-OHDA + exercise, 6-OHDA + cells, and 6-OHDA + combined). Subsequently, dopaminergic depletion was assessed by the methylphenidate challenge and the specified therapeutic intervention was conducted in each group. The foot fault task was performed at the end of the experiment to serve as an assessment of motor skills. The results showed that despite disturbances in motor balance and coordination, locomotor dysfunction was ameliorated in all treatment categories in comparison to the injury group (sign test, p < 0.001, effect size: 0.71). The exercise alone and combined groups were the categories that exhibited the best recovery in terms of movement performance (p < 0.001). Overall, this study confirms that exercise is a powerful option to improve motor function and a promising adjuvant intervention for stem cell transplantation in the treatment of PD motor symptoms. OPEN PRACTICES: This article has been awarded Open Data. All materials and data are publicly accessible at https://figshare.com/s/18a543c101a17a1d5560. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.

Analysis of NTPDase2 in the cell membrane using fluorescence recovery after photobleaching (FRAP).

NTPDase2, a member of the CD39/NTPDase family, is an ecto-nucleotidase anchored to the plasma membrane by two transmembrane domains, with a catalytic site facing the extracellular space and preferentially hydrolyzing nucleoside triphosphates. While NTPDase2 is expressed in many cell types, its unique functionality, mobility and dynamics at the cell membrane remain unexplored. We therefore constructed a recombinant NTPDase2 linked to the yellow fluorescent protein (EYFP) to investigate its dynamics by confocal microscopy. The present study shows that the expression of EYFP-NTPDase2 in different cell lines does not affect its proliferation, migration and adhesion to extracellular matrices (ECM). Moreover, in human embryonic kidney cells 293 (HEK293) grown on collagen type I and fibronectin, EYFP-NTPDase2 fluorescence is greater in free plasma membrane regions than in cell-cell contacts, in comparison with cells grown on other substrates. Differences in the time required for fluorescence recovery after photobleaching (FRAP) in free membrane regions and cell-cell contacts indicate that the mobility of EYFP-NTPDase2 depends on the matrix to which the cells are attached. © 2018 International Society for Advancement of Cytometry.

Cervical cancer stem-like cells: systematic review and identification of reference genes for gene expression.

Cervical cancer is the fourth most common cancer affecting women worldwide. Among many factors, the presence of cancer stem cells, a subpopulation of cells inside the tumor, has been associated with a worse prognosis. Considering the importance of gene expression studies to understand the biology of cervical cancer stem cells (CCSC), this work identifies stable reference genes for cervical cancer cell lines SiHa, HeLa, and ME180 as well as their respective cancer stem-like cells. A literature review was performed to identify validated reference genes currently used to normalize RT-qPCR data in cervical cancer cell lines. Then, cell lines were cultured in regular monolayer or in a condition that favors tumor sphere formation. RT-qPCR was performed using five reference genes: ACTB, B2M, GAPDH, HPRT1, and TBP. Stability was assessed to validate the selected genes as suitable reference genes. The evaluation validated B2M, GAPDH, HPRT1, and TBP in these experimental conditions. Among them, GAPDH and TBP presented the lowest variability according to the analysis by Normfinder, Bestkeeper, and ΔCq methods, being therefore the most adequate genes to normalize the combination of all samples. These results suggest that B2M, GAPDH, HPRT1, and TBP are suitable reference genes to normalize RT-qPCR data of established cervical cancer cell lines SiHa, HeLa, and ME180 as well as their derived cancer stem-like cells. Indeed, GAPDH and TBP seem to be the most convenient choices for studying gene expression in these cells in monolayers or spheres.

The Epithelial-to-Mesenchymal Transition-Like Process in Glioblastoma: An Updated Systematic Review and In Silico Investigation.

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer due to its highly invasive nature that impedes the surgical removal of all tumor cells, making relapse inevitable. However, the mechanisms used by glioma cells to invade the surrounding tissue are still unclear. In this context, epithelial-to-mesenchymal transition (EMT) has emerged as a key regulator of this invasive state and although the real relevance of this program in malignant glioma is still controversial, it has been strongly associated with GBM malignancy. EMT is a very complex process regulated by several families of transcriptional factors through many signaling pathways that form a network that allows cancer cells to acquire invasive properties and penetrate the neighboring stroma, resulting in the formation of an advantageous microenvironment for cancer progression and metastasis. In this systematic review, we focus on the molecular mechanisms of EMT including EMT-factors, drug resistance, miRNA, and new therapeutic strategies. In addition, we address controversial questions about mesenchymal shift in GBMs with a bioinformatics analysis to show that in terms of epithelial and mesenchymal phenotype, the majority of GBMs samples analyzed have a profile more mesenchymal than epithelial. If induced, this phenotype can be shifted toward an even more mesenchymal phenotype in an EMT-like process in glioma cells. A better understanding of the molecular regulation of the EMT during tumor spreading will help to provide potential therapeutic interventions to target this program when treating GBM.

Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105+ , CD44+ , CD14- , CD34- , CD45- , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc.

Modulation of Autophagy by Calcium Signalosome in Human Disease.

Autophagy is a catabolic process that is largely regulated by extracellular and intracellular signaling pathways that are central to cellular metabolism and growth. Mounting evidence has shown that ion channels and transporters are important for basal autophagy functioning and influence autophagy to handle stressful situations. Besides its role in cell proliferation and apoptosis, intracellular Ca(2+) is widely recognized as a key regulator of autophagy, acting through the modulation of pathways such as the mechanistic target of rapamycin complex 1, calcium/calmodulin-dependent protein kinase kinase 2, and protein kinase C. Proper spatiotemporal Ca(2+) availability, coupled with a controlled ionic flow among the extracellular milieu, storage compartments, and the cytosol, is critical in determining the role played by Ca(2+) on autophagy and on cell fate. The crosstalk between Ca(2+) and autophagy has a central role in cellular homeostasis and survival during several physiologic and pathologic conditions. Here we review the main findings concerning the mechanisms and roles of Ca(2+)-modulated autophagy, focusing on human disorders ranging from cancer to neurologic diseases and immunity. By identifying mechanisms, players, and pathways that either induce or suppress autophagy, new promising approaches for preventing and treating human disorders emerge, including those based on the modulation of Ca(2+)-mediated autophagy.

Analysis of the safety of mesenchymal stromal cells secretome for glioblastoma treatment.

BACKGROUND AIMS: The purpose of this study was to investigate whether the secretome of human adipose-derived stem cells (hADSC) affects human glioblastoma (GBM) cancer stem cell (CSC) subpopulation or has any influence on drug resistance and cell migration, evaluating the safety of hADSCs for novel cancer therapies. METHODS: hADSCs were maintained in contact with fresh culture medium to produce hADSCs conditioned medium (CM). GBM U87 cells were cultured with CM and sphere formation, expression of genes related to resistance and CSCs-MGMT, OCT4, SOX2, NOTCH1, MSI1-and protein expression of OCT4 and Nanog were analyzed. The influence of hADSC CM on GBM resistance to temozolomide (TMZ) was evaluated by measuring cumulative population doubling and hADSC CM influence on tumor cell migration was analyzed using transwell assay. RESULTS: hADSC CM did not alter CSC-related features such as sphere-forming capacity and expression of genes related to CSC. hADSC CM treatment alone did not change proliferation rate of U87 cells and, most important, did not alter the response of tumor cells to TMZ. However, hADSC CM secretome increased the migration capacity of glioblastoma cells. DISCUSSION: hADSC CM neither induced an enrichment of CSCs in U87 cells population nor interfered in the response to TMZ in culture. Nevertheless, paracrine factors released by hADSCs were able to modulate glioblastoma cells migration. These findings provide novel information regarding the safety of using hADSCs against cancer and highlight the importance of considering hADSC-tumor cells interactions in tumor microenvironment in the design of novel cell therapies.