Claudia Di Serio

Evidence for differential expression of Notch receptors and their ligands in melanocytic nevi and cutaneous malignant melanoma

The Notch signaling has been implicated in the regulation of self-renewal of adult stem cells and differentiation of precursors along a specific cell lineage, in normal embryonic development and organogenesis. There is also evidence that signaling through Notch receptors regulate cell proliferation and cell survival in several types of cancer, with opposing results depending on tissue context. No data are available in the literature concerning modulation of the expression of Notch receptors, and their ligands, in human cutaneous malignant melanoma. Here, we have investigated, for the first time, the expression of Notch-1, Notch-2, Jagged-1, Jagged-2 and Delta-like 1 proteins, by immunohistochemistry, in a series of benign and malignant human melanocytic lesions: five common melanocytic nevi, five ‘dysplastic nevi’ and 20 melanomas (five in situ, five T1–T2, five T3–T4 and five metastatic melanomas). We found that the expression of Notch-1 and Notch-2, as well as Notch ligands, was upregulated in ‘dysplastic nevi’ and melanomas as compared with common melanocytic nevi. These results indicate that the activation of Notch may represent an early event in melanocytic tumor growth and upregulation of Notch signaling may sustain tumor progression.

Renal, but not systemic, hemodynamic effects of dopamine are influenced by the severity of congestive heart failure*

Objective:To determine whether the short-term systemic and renal hemodynamic response to dopamine is influenced by clinical severity of congestive heart failure. Design:Effects of increasing doses of dopamine were assessed in patients consecutively admitted for acutely decompensated congestive heart failure. Setting:Intensive care unit. Patients:We enrolled 16 congestive heart failure patients stratified by clinical severity (New York Heart Association [NYHA] class III, n = 8; NYHA class IV, n = 8) and two additional NYHA class III patients as controls. Interventions:Measurements were carried out throughout five 20-min experimental periods: baseline, dopamine infusion at 2, 4, and 6 μg·kg−1·min−1, and recovery. Controls received a similar amount of saline. Measurements and Main Results:Systemic and renal hemodynamics were determined respectively by right cardiac catheterization and radioisotopes (iodine 131-labeled hippuran and iodine 125-labeled iothalamate clearance). The peak increase in heart rate and cardiac index occurred at a dopamine dose of 4–6 μg·kg−1·min−1. The dose-response relation was similar in NYHA classes III and IV. Improvement in effective renal plasma flow and glomerular filtration rate, peaking at 4 μg·kg−1·min−1, was more rapid and marked in NYHA class III than class IV patients, in whom the renal fraction of cardiac output failed to increase. The systemic and renal effects of dopamine were independent of age. No change occurred in controls. Conclusions:The dose of dopamine producing an optimal improvement of systemic and renal hemodynamics in congestive heart failure is higher than usually reported. A greater clinical severity of congestive heart failure impairs the renal effects of dopamine, probably through a selective loss in renal vasodilating capacity.

NOTCH SIGNALLING MODULATES HYPOXIA-INDUCED NEUROENDOCRINE DIFFERENTIATION OF HUMAN PROSTATE CANCER CELLS

Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro. Results: Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. Conclusions: Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription. Mol Cancer Res; 10(2); 230–8. ©2011 AACR.

Kynurenic acid inhibits the release of the neurotrophic fibroblast growth factor (FGF)-1 and enhances proliferation of glia cells, in vitro.

Summary1.Kynurenic (KYNA) and quinolinic (QUIN) acids are neuroactive tryptophan metabolites formed along the kynurenine pathway: the first is considered a non-competitive antagonist and the second an agonist of glutamate receptors of NMDA type. The affinity of these compounds for glutamate receptors is, however, relatively low and does not explain KYNA neuroprotective actions in models of post-ischemic brain damage.2.We evaluated KYNA effects on the release of fibroblast growth factor (FGF)-1, a potent neurotrophic cytokine. Because KYNA exhibits a neuroprotective profile in vitro and in vivo, we anticipated that it could function as an autocrine/paracrine inducer of FGF-1 release. Studies were performed in several models of FGF-1 secretion (FGF-1 transfected NIH 3T3 cells exposed to heat shock, A375 melanoma cells exposed to serum starvation, growth factor deprived human endothelial cells). To our surprise, KYNA, at low concentration, inhibited FGF-1 release in all cellular models. QUIN, a compound having opposite effects on glutamate receptors, also reduced this release, but its potency was significantly lower than that of KYNA.3.KYNA and QUIN also displayed a major stimulatory effect on the proliferation rate of mouse microglia and human glioblastoma cells, in vitro.4.Our data suggest that minor changes of local KYNA concentration may modulate FGF-1 release, cell proliferation, and ultimately tissue damage in different pathological conditions.

S100A13 is a new angiogenic marker in human melanoma

Angiogenesis is critical in melanoma progression and metastasis and relies on the synthesis and release of proangiogenic molecules such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factors (FGFs). S100A13 is a small calcium-binding protein that facilitates the release of FGF-1, the prototype of the FGF family. S100A13 is upregulated in astrocytic gliomas, in which it correlates with VEGF-A expression, microvessel density and tumor grading, and promotes a more aggressive, invasive phenotype in lung cancer-derived cell lines. To investigate the involvement of S100A13 in human cutaneous melanoma, we analyzed a series of 87 cutaneous melanocytic lesions: 14 common acquired melanocytic nevi, 14 atypical, so-called ‘dysplastic’ nevi, 45 melanomas (17 radial growth phase and 28 vertical growth phase) and 14 melanoma metastases. Main clinical and pathological features, including histotype, Breslow thickness, Clarks level and outcome were recorded. Microvessel density was determined with CD105/endoglin staining. Semiquantitative determination of S100A13, FGF-1 and VEGF-A protein expression was obtained by immunostaining. Quantification of S100A13 mRNA was achieved by real-time PCR. We found that S100A13 was expressed in melanocytic lesions; compared with benign nevi, S100A13 protein expression was significantly upregulated in melanomas (P=0.024), in which it correlated positively with the intensity of VEGF-A staining (P=0.041) and microvessel density (P=0.007). The level of expression of S100A13 mRNA also significantly increased with progression of disease, from radial growth phase (0.7±0.7) to vertical growth phase (3.6±3.1) to metastases (7.0±7.0) (P<0.001). Furthermore, S100A13 mRNA correlated positively with VEGF-A (P=0.023), TNM stage (P=0.05), risk of relapse (P=0.014) and status at follow-up (P=0.024). In conclusion, S100A13 is expressed in melanocytic lesions when the angiogenic switch occurs and it may cooperate with VEGF-A in supporting the formation of new blood vessels, favoring the shift from radial to vertical tumor growth. Therefore, S100A13 may represent a new angiogenic and prognostic marker in melanoma.

Notch3 is activated by chronic hypoxia and contributes to the progression of human prostate cancer

Prostate cancer (PC) is still the second cause of cancer‐related death among men. Although patients with metastatic presentation have an ominous outcome, the vast majority of PCs are diagnosed at an early stage. Nonetheless, even among patients with clinically localized disease the outcome may vary considerably. Other than androgen sensitivity, little is known about which other signaling pathways are deranged in aggressive, localized cancers. The elucidation of such pathways may help to develop innovative therapies aimed at specific molecular targets. We report that in a hormone‐sensitive PC cell line, LNCaP, Notch3 was activated by hypoxia and sustained cell proliferation and colony formation in soft agar. Hypoxia also modulated cellular cholesterol content and the number and size of lipid rafts, causing a coalescence of small rafts into bigger clusters; under this experimental condition, Notch3 migrated from the non‐raft into the raft compartment where it colocalized with the γ‐secretase complex. We also looked at human PC biopsies and found that expression of Notch3 positively correlated with Gleason score and with expression of carbonic anhydrase IX, a marker of hypoxia. In conclusion, hypoxia triggers the activation of Notch3, which, in turn, sustains proliferation of PC cells. Notch3 pathway represents a promising target for adjuvant therapy in patients with PC.

Excessive vasoconstriction after stress by the aging kidney: Inadequate prostaglandin modulation of increased endothelin activity

The adaptive capacity of the aging kidney to stimulation of the sympathetic nervous system, as induced by a 30-minute mental stress (MS), was assessed in 8 elderly healthy women (68 to 82 years of age) and compared with that of 8 younger women (24 to 40 years of age). The study encompassed 4 consecutive 30-minute periods (baseline, mental stress, recovery 1, and recovery 2). In the elderly subjects, baseline effective renal plasma flow (ERPF)(iodine 131-labeled hippurate clearance) was lower and glomerular filtration rate (GFR)(iodine 125-labeled iothalamate clearance) was proportionally less reduced than in the younger group; the filtration fraction (FF) was higher. The elderly group excreted more endothelin 1 (ET-1) (P < .05), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1alpha (6-keto PGF1alpha)(P < .001 for both)(radioimmunoassay). Mental stress induced similar increases in blood pressure, heart rate, and plasma catecholamines in the 2 age groups, limited to the stimulation period. In the elderly group, mental stress caused a prolonged decrease in ERPF that reached its maximum 60 minutes after mental stress (-33%, P < .05), while GFR remained constant during the whole experiment, so that FF increased. In the younger subjects, renal hemodynamic changes were limited to the mental stress period. ET-1 increased during mental stress and the first recovery period in the elderly group (+50% and +25%, P < .05) as it did in the younger group, but the elderly group differed from the younger in that vasodilating prostaglandins increased only during mental stress. In conclusion, the aging kidney reacts to adrenergic stimulation with more-pronounced and -prolonged vasoconstriction that is probably caused by a defect in prostaglandin modulation of endothelin activity. Autoregulation of GFR is maintained at the expense of increased intraglomerular pressure.

Increased renal formation of thromboxane A 2 and prostaglandin F 2α in heart failure

Renal formation of the vasoconstrictor prostaglandins thromboxane A 2 (TXA 2 ) and prostaglandin F 2α (PGF 2α ) was investigated in 25 patients with cardiac failure, divided into New York Heart Association functional classes I to IV, and in eight healthy control subjects. Plasma renin activity (PRA) and hemodynamic parameters were also investigated. Renal vasoconstrictor eicosanoid formation, measured in urinary daily excretion, was not different between patients in class I and control subjects. Class II to IV patients showed progressively increasing production of PGF 2α (F = 49.8, p < 0.001, analysis of variance) and TXA 2 (F = 37.8, p < 0.002). PGF 2α excretion peaked in class IV (+1266% vs class I, p < 0.001). Compared with class I, urinary excretion of thromboxane B 2 was +816% in class III and +1561% in class IV (both p < 0.001). PRA was significantly increased only in class IV (+1558%, p < 0.001). The current results indicate a progressive increase in renal production of vasoconstrictor eicosanoids directly related to New York Heart Association class and suggest that these prostanoids may have a role in deterioration of renal function. (Am Heart J 1997;133:94-100.)

Renal adaptation to stress: A possible role of endothelin release and prostaglandin modulation in the human subject

The aim of this study was to define the neurohumoral response associated with the renal hemodynamic perturbations induced by mental stress acting as an adrenergic stimulus. In 8 healthy women, the effects of mental stress were studied during four consecutive 30-minute periods (baseline, mental stress, recovery I, recovery II). Mental stress induced sympathetic activation as evidenced by increases in blood pressure, heart rate, and plasma norepinephrine level. Effective renal plasma flow (iodine 131-labeled hippurate clearance) decreased only during mental stress (-22%, p < 0.05 vs baseline); glomerular filtration rate (iodine 125-labeled iotalamate clearance) remained constant during the entire experiment; the filtration fraction increased significantly during mental stress and recovery I (+30% and +22%, respectively, p < 0.02 for both). Complex neuroendocrine responses were associated with the hemodynamic changes. Urinary excretion of endothelin-1 and 6-keto-PGF(1alpha) increased during mental stress (+53%, p < 0.01, and +20%, p < 0.01, respectively) and recovery I (+49% and +29%, respectively, p < 0.01 for both). Urinary cyclic guanosine monophosphate rose only during mental stress (+77%, p < 0.05), whereas excretion of PGE2 showed a stepwise increase throughout recovery I and II (+292%, p < 0.01, and +360%, p < 0.001, respectively). In conclusion, the present experiments demonstrate that renal hemodynamic response induced by mental stress is a complex reaction in which endothelin-1, prostaglandins, and presumably nitric oxide take part.

The release of fibroblast growth factor-1 from melanoma cells requires copper ions and is mediated by phosphatidylinositol 3-kinase/Akt intracellular signaling pathway.

Melanoma is a highly invasive tumor with elevated mortality rates. Progression and aggressiveness appear related to the achievement of an angiogenic phenotype. Melanoma cells express several angiogenic factors, including fibroblast growth factor (FGF)-1 and FGF-2. The autocrine production and release of FGFs and the subsequent activation of FGF receptors, have a central role in melanoma tumor progression. We demonstrated that FGF-1 is secreted from a human melanoma cell line, A375, under conditions of serum deprivation. The release of FGF-1 is inhibited by the copper chelator ammonium tetrathiomolybdate, suggesting a role of copper in the secretory pathway, and is triggered by activation of phosphatidylinositol 3-kinase (PI3K)/Akt intracellular signaling. Interestingly, overexpression or activation of Akt has been correlated with poor prognosis in melanoma patients. Our data indicate a novel role for Akt in supporting the progression of human melanomas and advocate the need for new treatments targeting PI3K/Akt signaling pathway, to control tumor development and progression.