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Neuroblastoma (NB) is the most frequently observed among extracranial pediatric solid tumors. It displays an extreme clinical heterogeneity, in particular for the presentation at diagnosis and response to treatment, often depending on cancer cell differentiation/stemness. The frequent presence of elevated hematic and urinary levels of catecholamines in patients affected by NB suggests that the dissection of adrenergic system is crucial for a better understanding of this cancer. β3-adrenoreceptor (β3-AR) is the last identified member of adrenergic receptors, involved in different tumor conditions, such as melanoma. Multiple studies have shown that the dysregulation of the bioactive lipid sphingosine 1-phosphate (S1P) metabolism and signaling is involved in many pathological diseases including cancer. However, whether S1P is crucial for NB progression and aggressiveness is still under investigation. Here we provide experimental evidence that β3-AR is expressed in NB, both human specimens and cell lines, where it is critically involved in the activation of proliferation and the regulation between stemness/differentiation, via its functional cross-talk with sphingosine kinase 2 (SK2)/S1P receptor 2 (S1P2) axis. The specific antagonism of β3-AR by SR59230A inhibits NB growth and tumor progression, by switching from stemness to cell differentiation both in vivo and in vitro through the specific blockade of SK2/S1P2 signaling.

BACKGROUND AND PURPOSE: Stress‐related catecholamines have a role in cancer and β‐adrenoceptors; specifically, β(2)‐adrenoceptors have been identified as new targets in treating melanoma. Recently, β(3)‐adrenoceptors have shown a pleiotropic effect on melanoma micro‐environment leading to cancer progression. However, the mechanisms by which β(3)‐adrenoceptors promote this progression remain poorly understood. Catecholamines affect the immune system by modulating several factors that can alter immune cell sub‐population homeostasis. Understanding the mechanisms of cancer immune‐tolerance is one of the most intriguing challenges in modern research. This study investigates the potential role of β(3)‐adrenoceptors in immune‐tolerance regulation. EXPERIMENTAL APPROACH: A mouse model of melanoma in which syngeneic B16‐F10 cells were injected in C57BL‐6 mice was used to evaluate the effect of β‐adrenoceptor blockade on the number and activity of immune cell sub‐populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with β‐blockers (propranolol and SR59230A) and specific β‐adrenoceptor siRNAs targeting β(2)‐ or β(3)‐adrenoceptors were used. KEY RESULTS: Only β(3)‐, but not β(2)‐adrenoceptors, were up‐regulated under hypoxia in peripheral blood mononuclear cells and selectively expressed in immune cell sub‐populations including Treg, MDSC, and NK. SR59230A and β(3)‐adrenoceptor siRNAs increased NK and CD8 number and cytotoxicity, while they attenuated Treg and MDSC sub‐populations in the tumour mass, blood, and spleen. SR59230A and β(3)‐adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes. CONCLUSIONS AND IMPLICATIONS: Our data suggest that β(3)‐adrenoceptors are involved in immune‐tolerance, which opens the way for new strategic therapies to overcome melanoma growth. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors—New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc

Neuroblastoma (NB) is the most frequently observed among extracranial pediatric solid tumors. It displays an extreme clinical heterogeneity, in particular for the presentation at diagnosis and response to treatment, often depending on cancer cell differentiation/stemness. The frequent presence of elevated hematic and urinary levels of catecholamines in patients affected by NB suggests that the dissection of adrenergic system is crucial for a better understanding of this cancer. β3-adrenoreceptor (β3-AR) is the last identified member of adrenergic receptors, involved in different tumor conditions, such as melanoma. Multiple studies have shown that the dysregulation of the bioactive lipid sphingosine 1-phosphate (S1P) metabolism and signaling is involved in many pathological diseases including cancer. However, whether S1P is crucial for NB progression and aggressiveness is still under investigation. Here we provide experimental evidence that β3-AR is expressed in NB, both human specimens and cell lines, where it is critically involved in the activation of proliferation and the regulation between stemness/differentiation, via its functional cross-talk with sphingosine kinase 2 (SK2)/S1P receptor 2 (S1P2) axis. The specific antagonism of β3-AR by SR59230A inhibits NB growth and tumor progression, by switching from stemness to cell differentiation both in vivo and in vitro through the specific blockade of SK2/S1P2 signaling.

Although there is an increasing evidence that cancer stem cell (CSC) niches in the tumor microenvironment (TME) plays a crucial role in sustaining solid tumors progression, several molecular players involved in this regulation still remain unknown. The role of &beta, adrenergic signaling in enhancing tumor growth through &beta, 2-adrenoreceptors (&beta, 2-ARs) has been confirmed in different cancer models, but the role played by the &beta, 3-adrenergic receptor (&beta, 3-AR) has recently emerged. Previous studies showed that &beta, 3-AR promotes cancer growth through the activation of different stromal cells in the TME, and leads to melanoma malignancy progression through inflammation, angiogenesis, and immunotolerance. Here we show that in B16 melanoma-bearing mice, the pharmacological &beta, 3-AR blockade is able to reduce the expression of CSC markers, and to induce a differentiated phenotype of hematopoietic subpopulations in TME. In particular, cytofluorimetric analysis (FACS) of the tumor mass shows that &beta, 3-AR antagonist SR59230A promotes hematopoietic differentiation as indicated by increased ratios of lymphoid/hematopoietic stem cells (HSCs) and of myeloid progenitor cells/HSCs, and increases the number of Ter119 and natural killer (NK) precursor cells, and of granulocyte precursors, indicating active hematopoiesis within the tumor tissue. Moreover, pharmacological antagonism of &beta, 3-AR induces mesenchymal stem cell (MSC) differentiation into adipocytes subtracting a potential renewal of the stem compartment by these cells. Here we demonstrate that &beta, 3-AR blockade in the TME by inducing the differentiation of different stromal cells at the expense of stemness traits could possibly have a favorable effect on the control of melanoma progression.

Matrix metalloproteinases (MMPs) play a critical role in inflammation and ulcerations in gut of Crohn׳s disease (CD) patients. Intestinal subepithelial myofibroblasts (ISEMFs) secrete MMPs in response to inflammatory stimuli. Previous data showed in CD-ISEMFs increased oxidative status. The aim of this study was to investigate the role of ISEMFs in modulating the production of MMP-3 and TIMP-1, an inhibitor of MMPs activity. A relationship among oxidative stress, activity of antioxidants and MMP-3/TIMP-1 was also studied. ISEMFs isolated from CD patient colon and human colonic cell line of myofibroblasts (18Co) were used. Oxidative state was modulated by buthionine sulfoximine, an inhibitor of glutathione (GSH) synthesis, and N-acetylcysteine (NAC), GSH precursor. An up-regulation of MMP-3 due to increased oxidative state was found in CD-ISEMFs. Stimulation by tumor necrosis factor (TNF)α increased further MMP-3 levels. On the contrary, no change in TIMP-1 production was determined. NAC treatment decreased MMP-3 production in CD-ISMEFs and removed the enhancement due to TNFα. Similar effects were observed in 18Co cells treated with curcumin, antioxidant with anti-inflammatory properties. The involvement of MAPKs on MMP-3 redox regulation was also shown. This study demonstrates the involvement of ISEMFs and high oxidative state in the increased MMP-3 production found in intestinal mucosa of CD patients. NAC and curcumin normalize MMP-3 levels mainly in TNFα stimulated cells. A modulation of MMP-3 production by NAC and curcumin due to their direct action on transcriptional factors has been also suggested. Therefore, they could have a therapeutic use for the prevention and treatment of fistulaes in CD.

Estrogen deficiency causes bone loss as a result of microdamage, oxidative stress, and osteocyte apoptosis. A relationship between oxidative stress-induced apoptosis, c-Jun N-terminal kinase (JNK) activation, and expression of factors involved in bone remodeling has been demonstrated in osteocytes. However, the molecular regulation of these events in osteocytes treated with 17β-estradiol (17β-E2) remains unexplored. The MLO-Y4 murine osteocyte-like cell line was used as a model to study starvation-induced apoptosis and ROS production during 17β-E2 treatment. Expression of glutathione S-transferase P1-1 (GSTP1-1), receptor activator kB ligand (RANKL), osteoprotegerin (OPG), sclerostin, and kinases activation were measured by western blot. In addition, the GSTP1-1/JNK association was assessed by immunoprecipitation, and GSTP1-1 involvement in the osteocyte response to 17β-E2 was detected by specific siRNA transfection. 17β-E2 prevents starvation-induced apoptosis (DNA fragmentation and caspase activation), the increase in sclerostin expression and the RANKL/OPG ratio, which are all related to JNK activation due to oxidative stress in osteocytes. This occurs through GSTP1-1 overexpression, which can inhibit JNK activation by formation of a GSTP1-1/JNK complex. No early antioxidant action of 17β-E2 has been found but the estrogen effect is similar to N-acetylcysteine which, by increasing the intracellular redox state, maintains JNK bound to GSTP1-1. Thus, the antiapoptotic and osteogenic effect of 17β-E2 in MLO-Y4 occurs by a redox-independent process involving GSTP1-1/JNK association. This study clarifies at molecular level the effect of 17β-E2 on osteocyte activity and identifies a possible role of GSTP1-1 and JNK activity in bone remodeling and repair mechanisms.

Resveratrol (RE), a polyphenolic compound present in some food and plants, is characterized by anti-inflammatory and antioxidant properties. However, it is quickly metabolized with consequent loss of its efficacy. In this study, the antioxidant effect of 2-phenyl-benzoselenophene derivatives (VD0, VD1 and VD2) was detected in intestinal myofibroblast and osteocyte cell lines in which the oxidative stress was induced by GSH depletion or starvation, respectively. In fact, the oxidative stress is involved in pathogenesis of inflammatory bowel diseases (IBD) and in increased osteoclastogenesis in osteoporosis. Our results show that these derivatives have major antioxidant power in reducing and/or restoring radical oxygen species to control values than RE itself in both cell types. Moreover, derivatives have different antioxidant capacity in myofibroblasts and in osteocytes and this can be due to different degree of oxidative stress and structural characteristics of these compounds. Some of the synthesized RE analogs have shown anti-bacterial role in IBD and anti-resorptive activity in bone pathologies related to inflammatory and osteoporotic processes. Thus, we suggest benzoselenophene derivatives as good candidates for alternative therapy and/or therapeutic support in these pathologies.

Intercellular adhesion molecule-1 (ICAM-1) is distributed and expressed on cell surface and is present in circulation as soluble form (sICAM-1). Tumor necrosis factor-alpha (TNFα) and radical oxygen species (ROS) up-regulate the expression of ICAM-1. This study demonstrates for the first time in 18 Co cells, a myofibroblast cell line derived from human colonic mucosa, an up-regulation of ICAM-1 expression and sICAM-1 release induced by oxidative stress and TNFα stimulation. The intracellular redox state was modulated by L-buthionine-S,R-sulfoximine (BSO) or N-acetylcysteine (NAC), inhibitor and precursor respectively of GSH synthesis. ROS production increases in cells treated with BSO or TNFα, and this has been related to an up-regulation of ICAM-1 expression and sICAM-1 release. The involvement of metalloproteinases in ICAM-1 release has been demonstrated. Moreover, also expression and activation of A disintegrin and metalloproteinase 17, a membrane-bound enzyme known as TNFα-converting enzyme (TACE), have been related to ROS levels. This suggests the possible involvement of TACE in the cleavage of ICAM-1 on cell surface in condition of oxidative stress. NAC down-regulates the expression and release of ICAM-1 as well as the expression and activation of TACE. However, in TNFα stimulated cells NAC treatment reduces only in part ICAM-1 expression and sICAM-1 release. Given this TNFα may also act on these events by a redox-independent mechanism.

Osteocyte apoptosis due to microdamage and/or oxidative stress is related to increased local bone turnover and resorption observed in various bone diseases. Previous data on osteoblasts and osteoclasts have linked reactive oxygen species and antioxidants to bone remodelling. This study performs a comprehensive analysis on the effect of antioxidants such as glutathione (GSH), N-acetylcysteine and lipoic acid (LA) on starvation-induced osteocyte apoptosis and on cytokines involved in bone remodelling such as the receptor activator kB ligand (RANKL), osteoprotegerin (OPG) and sclerostin. For this study, apoptosis was induced by serum starvation in a murine osteocyte-like cell line MLO-Y4; this condition mimics in part osteocyte apoptosis due to microdamage. The results show that starvation-induced apoptosis and expression of RANKL, OPG and sclerostin are redox regulated processes. All antioxidants are able to inhibit the apoptosis due to starvation. They down-regulate the expression and the release of RANKL, the expression of sclerostin and RANKL/OPG ratio, whereas they only in part up-regulate OPG expression. Antioxidants mediate their effect on starvation-induced apoptosis by JNK signalling and on cytokine expression by both JNK and ERK1/2 activities. This study shows the possible involvement of biological antioxidants such as GSH and LA on redox regulated mechanisms related to apoptosis and expression of cytokines involved in bone remodelling. Moreover, it suggests that both JNK and ERK1/2 may be useful biological targets for drugs affecting bone diseases associated with increased oxidative stress.

The early phases of embryonic development and cancer share similar strategies to improve their survival in an inhospitable environment: both proliferate in a hypoxic and catecholamine-rich context, increasing aerobic glycolysis. Recent studies show that β3-adrenergic receptor (β3-AR) is involved in tumor progression, playing an important role in metastasis. Among β-adrenergic receptors, β3-AR is the last identified member of this family, and it is involved in cancer cell survival and induction of stromal reactivity in the tumor microenvironment. β3-AR is well known as a strong activator of uncoupling protein 1 (UCP1) in brown fat tissue. Interestingly, β3-AR is strongly expressed in early embryo development and in many cancer tissues. Induction of uncoupling protein 2 (UCP2) has been related to cancer metabolic switch, leading to accelerated glycolysis and reduced mitochondrial activity. In this study, for the first time, we demonstrate that β3-AR is able to promote this metabolic shift in both cancer and embryonic stem cells, inducing specific glycolytic cytoplasmic enzymes and a sort of mitochondrial dormancy through the induction of UCP2. The β3-AR/UCP2 axis induces a strong reduction of mitochondrial activity by reducing ATP synthesis and mitochondrial reactive oxygen species (mtROS) content. These effects are reverted by SR59230A, the specific β3-AR antagonist, causing an increase in mtROS. The increased level of mtROS is neutralized by a strong antioxidant activity in embryonic stem cells, but not in cancer stem cells, where it causes a dramatic reduction in tumor cell viability. These results lead to the possibility of a selective antitumor therapeutic use of SR59230A. Notably, we demonstrate the presence of β3-AR within the mitochondrial membrane in both cell lines, leading to the control of mitochondrial dormancy.