Dexamethasone blocks the migration of the human neuroblastoma cell line SK-N-SH

Glucocorticoids (Gc) influence the differentiation of neural crestderived cells such as those composing sympathoadrenal tumors like pheochromocytomas, as well as neuroblastomas and gangliomas. In order to obtain further information on the effects of Gc on cells evolving from the neural crest, we have used the human neuroblastoma cell line SK-N-SH to analyze: 1) the presence and the binding characteristics of Gc receptors in these cells, 2) the effect of dexamethasone (Dex) on the migration of SK-N-SH cells, and 3) the effect of Dex on the organization of the cytoskeleton of SK-N-SH cells. We show that: 1) receptors that bind [ 3 H]-Dex with high affinity and high capacity (Kd of 9.6 nM, Bmax of 47 fmol/mg cytosolic protein, corresponding to 28,303 sites/cell) are present in cytosolic preparations of SK-N-SH cells, and 2) treatment with Dex (in the range of 10 nM to 1 µM) has an inhibitory effect (from 100% to 74 and 43%, respectively) on the chemotaxis of SK-N-SH cells elicited by fetal bovine serum. This inhibition is completely reversed by the Gc receptor antagonist RU486 (1 µM), and 3) as demonstrated by fluorescent phalloidin-actin detection, the effect of Dex (100 nM) on SK-NSH cell migration is accompanied by modifications of the cytoskeleton organization that appear with stress fibers. These modifications did not take place in the presence of 1 µM RU486. The present data demonstrate for the first time that Dex affects the migration of neuroblastoma cells as well as their cytoskeleton organization by interacting with specific receptors. These findings provide new insights on the mechanism(s) of action of Gc on cells originating in the neural crest

Factors released by rat type 1 astrocytes exert different effects on the proliferation of human neuroblastoma cells (SH-SY5Y) in vitro

Brain metastases derived from abdominal neuroblastoma are an uncommon complication of this tumour; however, an increase in their occurrence has recently been reported. In the present study, we have investigated the influence of factors derived from central nervous system glial cells on the proliferation of human neuroblastoma cells (SH-SY5Y) in vitro. Co-culture experiments show that a 24-h exposure to factors released by type 1 astrocytes (A1) may induce a significant decrease in [(3)H]thymidine ([(3)H]TdR) incorporation by SH-SY5Y cells. This effect was not duplicated by fresh A1-conditioned medium (A1-CM); A1-CM became active only when it was heated or frozen. In contrast to this short-lived inhibitory effect, long-term treatment (3, 6 and 9 days) with A1-CM produced a significant and dose-dependent increase in SH-SY5Y cell number. Immunoneutralisation of A1-CM with an anti-transforming growth factor-beta antibody eliminated the inhibitory effect on [(3)H]TdR uptake in SH-SY5Y cells, but did not affect the increased number of viable cells observed after long-term treatments. In conclusion, these results showed that factor(s) released by A1 may affect the proliferation/survival of a human neuroblastoma cell line in vitro inducing: (a) a short transient negative effect on DNA synthesis and (b) an overall sustained trophic action. These results are suggestive of a possible role of glial cells in the establishment of brain metastases of neuroblastomas

Antiprogestins inhibit the binding of opioids to mu-opioid receptors in nervous membrane preparations

The present study showed that the glucocorticoid/progesterone antagonists, 17 beta-hydroxy-1 1 beta-(4-dimethylamino-phenyl-1)-17-(prop-1-ynyl)estra-4,9-dien+ ++-3-one (RU486) and 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl-1)-17-(propan-3-ol)estra-4,9-dien-3-o ne (ZK 98299), inhibit the binding of labeled dihydromorphine to mu-opioid receptors present on membrane preparations derived from rat and mouse brain, as well as from human neuroblastoma cells. The inhibitory effect of RU486 was dose-dependent and linked to a decrease of the affinity of labeled dihydromorphine to the mu-opioid receptors. Kinetic experiments have shown that RU486 induces a decrease of the association rate constant (k + 1) of dihydromorphine. RU486 also proved able to dissociate the dihydromorphine-mu-opioid receptor complex, although at a rate slower than that exhibited by unlabeled dihydromorphine. Finally, the addition of NaCl (100 mM) to the incubation buffer induced a 50% decrease of the inhibitory effect of RU486. A 6-day treatment of neuroblastoma cells with RU486 eliminated the inhibitory effect morphine exerts on the intracellular accumulation of cyclic AMP induced by prostaglandin E1. These results indicate that RU-486 may interact with brain mu-opioid receptors in vitro, by decreasing the affinity of opioid ligands

Role of glucocorticoid hormones in the differentiation of the human neuroblastoma cell line SK-N-SH

It is known that glucocorticoids (Gc) are involved in the differentiation of primordial cells derived from the neural crest. Neuroblastoma, the most common extracranial solid tumor of childhood, is a tumor derived from the neural crest sympathoadrenal lineage. Previous data performed in our laboratory have demonstrated that glucocorticoid receptors (GRs) are expressed in the SK-N-SH cells, a human neuroblastoma cell line. It is known that the human GR gene generates different transcripts differing in the 5\u2019 non-translated forms of exon 1. In order to get further information on the GR gene expression and on the mechanism of action of Gc on neuroblastoma cells, experiments have been performed to study the presence of different transcripts of GR in SK-N-SH cells. The RT-PCR analysis performed in our laboratory revealed that three GR-gene derived transcripts (1A1, 1A3 and C) are present in SK-N-SH cells. In a second series of experiments, the effect(s) of the exposure to Dex for 6 days on the number and/or differentiation of SK-N-SH cells has been investigated. It has been found that Dex addition produces a dose-dependent (from 10-8 to 10-6 M) increase of the number of SK-N-SH viable cells, as evaluated by the MTT method. In addition, Dex treatment induces a trans-differentiation of the neuroblast-like component of SK-N-SH cells toward an epithelial-like phenotype. This phenotypic modification is apparently accompanied by a remodeling of cell cytoskeleton and redistribution of F-actin, formation of stress fibers and loss of ruffles and lamellipodia present in untreated cells, as indicated by a staining with fluorescent phalloidine. It has further been analyzed whether Dex could modify the chemomigratory response of SK-N-SH cells in the presence of a chemoattractant by a microchemotaxis assay. A 6-day treatment of SK-N-SH cells with different doses of Dex (10-10 M - 10-6M), significantly decreases their chemomigratory response to fetal bovine serum. In summary, the data here reported suggest that: a) three different exon1-derived transcripts (1A1, 1A3 and C) of the GR gene are present in SK-N-SH cells; b) the chronic treatment with Dex induces an increase of cell proliferation and/or survival; c) under these cirmcumstances, Dex stimulates a differentation of SK-N-SH neuroblastoma cells to a quite homogeneous epithelial-like phenotype; d) Gc may affect the organization of the actin cytoscheleton of SK-N-SH neuroblastoma cells; e) Gc may also affect in an inhibitory way the migratory activity of these cells. These results suggest that SK-N-SH cells may represent a useful tool to study the molecular mechanisms through which Gc act on the differentiation of neural crest-derived cells

New insight on the molecular aspects of glucocorticoid effects in nervous system development

Adrenal glucocorticoids (Gc) are among the most significant hormones in the mammalian organisms; these steroids may reach and penetrate all tissues where they interact with cytoplasmic/nuclear receptors, through which they exert multiple and very multifaceted actions. The effects of physiological concentrations of Gc on brain functions have not been completely clarified, even though Gc are recognized to influence behavioral responses, emotions, cognitive processes and to take part in the neuroendocrine control of body homeostasis. Developmental programming effects of Gc in animal models and humans have been proposed. Actually, prenatal stress, or exposure to high Gc levels, would somehow affect neuronal developmental events in some structure and this can lead to central nervous system's altered functions, as the impairment of neuroendocrine activities, cognitive processes, sleep and mood disorders. Interestingly, it has been observed that these abnormalities may not be limited to the first directly exposed individuals, but transmissible across generations. The establishment of animal models with localized prenatal glucocorticoid receptors deficiency led to the accumulation of data on the possible roles of these hormones on development of the central and peripheral nervous system. The most recent findings on the effects of Gc on neuroblast development, with particular attention to neuronal migration, will be presented

Dexamethasone blocks the migration of the human neuroblastoma cell line SK-N-SH

Glucocorticoids (Gc) influence the differentiation of neural crest-derived cells such as those composing sympathoadrenal tumors like pheochromocytomas, as well as neuroblastomas and gangliomas. In order to obtain further information on the effects of Gc on cells evolving from the neural crest, we have used the human neuroblastoma cell line SK-N-SH to analyze: 1) the presence and the binding characteristics of Gc receptors in these cells, 2) the effect of dexamethasone (Dex) on the migration of SK-N-SH cells, and 3) the effect of Dex on the organization of the cytoskeleton of SK-N-SH cells. We show that: 1) receptors that bind [³H]-Dex with high affinity and high capacity (Kd of 9.6 nM, Bmax of 47 fmol/mg cytosolic protein, corresponding to 28,303 sites/cell) are present in cytosolic preparations of SK-N-SH cells, and 2) treatment with Dex (in the range of 10 nM to 1 µM) has an inhibitory effect (from 100% to 74 and 43%, respectively) on the chemotaxis of SK-N-SH cells elicited by fetal bovine serum. This inhibition is completely reversed by the Gc receptor antagonist RU486 (1 µM), and 3) as demonstrated by fluorescent phalloidin-actin detection, the effect of Dex (100 nM) on SK-N-SH cell migration is accompanied by modifications of the cytoskeleton organization that appear with stress fibers. These modifications did not take place in the presence of 1 µM RU486. The present data demonstrate for the first time that Dex affects the migration of neuroblastoma cells as well as their cytoskeleton organization by interacting with specific receptors. These findings provide new insights on the mechanism(s) of action of Gc on cells originating in the neural crest

Antiproliferative effect of mifepristone ({RU}486) on human neuroblastoma cells ({SK}-N-{SH}): in vitro and in vivo studies

RU486 (mifepristone), a glucocorticoid and progesterone receptor antagonist, has been reported to exert antiproliferative effects on tumor cells. Experiments were performed to analyze the effects of RU486 on the proliferation of the human neuroblastoma, both in vitro and in vivo, using the human neuroblastoma SK-N-SH cell line. The exposure in vitro of SK-N-SH cells to RU486 revealed a dose-dependent inhibition of 3H-thymidine incorporation due to a rapid but persistent inhibition of MAPKinase activity and ERK phosphorylation. A significant decrease of SK-N-SH cell number was evident after 3, 6, and 9 days of treatment (up to 40% inhibition), without evident cell death. The inhibitory effect exerted by RU486 was not reversed by the treatment of the cells with dexamethasone or progesterone. Moreover, RU486 induced a shift in SK-N-SH cell phenotypes, with an almost complete disappearance of the neuronal-like and a prevalence of the epithelial-like cell subtypes. Finally, the treatment with RU486 of nude mice carrying a SK-N-SH cell xenograft induced a strong inhibition (up to 80%) of tumor growth. These results indicated a clear effect of RU486 on the growth of SK-N-SH neuroblastoma cells that does not seem to be mediated through the classical steroid receptors. RU486 acted mainly on the more aggressive component of the SK-N-SH cell line and its effect in vivo was achieved at a concentration already used to inhibit oocyte implantation

Effects and metabolism of steroid hormones in human neuroblastoma cells

The development of the central nervous system is influenced by sex steroids and by their metabolites. However, little information on the possible effects of steroid hormones on neuroblastoma cells is available. Human neuroblastoma cell lines have been used as a model of human neuroblasts in vitro to study the metabolism of steroid hormones; in addition, the effects of steroids and steroid antagonists on neuroblastoma cell growth have also been investigated. The results obtained show that SH-SY5Y human neuroblastoma cells may actively metabolize testosterone and progesterone to their respective 5 alpha-reduced metabolites and that differentiation of neuroblastoma cells is paralleled by a significant increase in expression of the type-1 5 alpha-reductase and of the formation of steroid metabolites. All these data are suggestive of a potential role of steroid 5 alpha-reduced metabolites in the biology of neuroblastoma cells. Studies performed to analyze the role of steroid hormones on neuroblastoma cell proliferation show that progesterone at low doses may induce minor stimulation, and at higher doses, a toxic effect on the neuroblastoma cell line SK-N-SH is seen. Moreover, the antiprogestin 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl-1)-17-(prop-1-ynyl)estra-4,9-dien+ ++-3-one (RU486) decreases the proliferation of these cells in a dose-dependent manner. The effect of RU486 is not antagonized by either progesterone or dexamethasone, a result that seems to exclude the action of RU486 via classic intracellular steroid hormone receptors