Melanoma and the Nervous System – Novel Pathways Mediated by Neurotrophins and Their Receptors

Le neurotrofine sono una famiglia di proteine sintetizzate e prodotte da molte cellule cutanee, mentre i loro recettori sono espressi nel melanoma. Le neurotrofine stimolano la proliferazione e la migrazione delle cellule di melanoma

CD271 downregulation promotes melanoma progression and invasion in 3-dimensional models and in zebrafish

CD271 is a neurotrophin receptor variably expressed in melanoma. While contradictory data are reported on its role as a marker of tumor initiating cells, little is known on its function in tumor progression. CD271 expression was higher in spheroids derived from freshly isolated cells of primary melanomas and in primary WM115 and WM793-B cell lines, while it decreased during progression to advanced stages in cells isolated from metastatic melanomas and in metastatic WM266-4 and 1205Lu cell lines. Moreover, CD271 was scarcely detected in the highly invasive spheroids (SKMEL28 and 1205Lu). CD271, originally expressed in the epidermis of skin reconstructs, disappeared when melanoma started to invade the dermis. SKMEL8 CD271(-) cells showed greater proliferation and invasiveness in vitro, and were associated with a higher number of metastases in zebrafish, as compared to CD271(+) cells. CD271 silencing in WM115 induced a more aggressive phenotype in vitro and in vivo. On the contrary, CD271 overexpression in SKMEL28 cells reduced invasion in vitro, and CD271 overexpressing 1205Lu cells was associated with a lower percentage of metastases in zebrafish. A reduced cell-cell adhesion was also observed in absence of CD271. Taken together, these results indicate that CD271 loss is critical for melanoma progression and metastasis

Apremilast Normalizes Gene Expression of Inflammatory Mediators in Human Keratinocytes and Reduces Antigen-Induced Atopic Dermatitis in Mice

Apremilast, an oral phosphodiesterase (PDE) 4 inhibitor, has demonstrated efficacy in psoriasis, while its efficacy in atopic dermatitis (AD) was found to be modest. AD is a chronic inflammatory skin disease associated with activation of T helper (Th) 2 and Th17 immunity and a compromised epidermal barrier

A previously unreported function of beta1B integrin isoform in caspase-8-dependent integrin-mediated keratinocyte death

Integrins regulate adhesive cell-matrix interactions and mediate survival signals. On the other hand, unligated or free cytoplasmic fragments of integrins induce apoptosis in many cell types (integrin-mediated death). We have previously shown that b1 integrins expression protects keratinocyte stem cells from anoikis, while the role of the b1B integrin isoform has never been clarified. Here we report that suspended keratinocytes undergo apoptosis via the activation of caspase-8, independently of Fas/Fas Ligand system. Indeed, anti-b1 integrin neutralizing antibodies induced apoptosis in short-hairpin-RNA-Fas-Associated-Death-Domain treated cells. Moreover, before and during suspension, caspase-8 directly associated with b1 integrin, that in turn internalized and progressively degraded, shedding the cytoplasmic domain. b1B was expressed only in the cytoplasm in a perinuclear fashion and remained unaltered during suspension. At 24 hrs, as b1A located close to the nucleus, b1B co-localized with b1A and co-immunoprecipitated with caspase-8. Caspase-8 was activated earlier in b1B integrin transfected keratinocytes, and these cells underwent a higher rate of apoptosis than mock cells. By contrast, caspase-8 was not activated in siRNA b1B transfected cells. These results indicate that when b1A is unligated, b1B is responsible for “integrin-mediated death” in human keratinocytes

Self-Assembled Lipid Nanoparticles for Oral Delivery of Heparin-Coated Iron Oxide Nanoparticles for Theranostic Purposes

Recently, solid lipid nanoparticles (SLNs) have attracted increasing attention owing to their potential as an oral delivery system, promoting intestinal absorption in the lymphatic circulation which plays a role in disseminating metastatic cancer cells and infectious agents throughout the body. SLN features can be exploited for the oral delivery of theranostics. Therefore, the aim of this work was to design and characterise self-assembled lipid nanoparticles (SALNs) to encapsulate and stabilise iron oxide nanoparticles non-covalently coated with heparin (Fe@hepa) as a model of a theranostic tool. SALNs were characterised for physico-chemical properties (particle size, surface charge, encapsulation efficiency, in vitro stability, and heparin leakage), as well as in vitro cytotoxicity by methyl thiazole tetrazolium (MTT) assay and cell internalisation in CaCo-2, a cell line model used as an indirect indication of intestinal lymphatic absorption. SALNs of about 180 nm, which are stable in suspension and have a high encapsulation efficiency (>90%) were obtained. SALNs were able to stabilise the heparin coating of Fe@hepa, which are typically unstable in physiological environments. Moreover, SALNs-Fe@hepa showed no cytotoxicity, although their ability to be internalised into CaCo-2 cells was highlighted by confocal microscopy analysis. Therefore, the results indicated that SALNs can be considered as a promising tool to orally deliver theranostic Fe@hepa into the lymphatic circulation, although further in vivo studies are needed to comprehend further potential applications

Characterization of natural clays from italian deposits with focus on elemental composition and exchange estimated by edx analysis: potential pharmaceutical and cosmetic uses

Purification processes performed on natural clays to select specific clay minerals are complex and expensive and can lead to over-exploitation of some deposits. The present study aimed to examine physicochemical (mineralogy, morphology, size, surface charge, chemical composition, cation exchange capacity [CEC], and pH) and hydration (swelling, wettability, water sorption, and rheological behavior) properties of three native clays from Italian deposits for potential pharmaceutical and cosmetic uses due to the presence of phyllosilicate minerals. Particular emphasis was placed on energy dispersive X-ray (EDX) microanalysis coupled with the ‘cesium method’ to assay clay elemental composition and CEC. One bentonite of volcanic origin (BNT) and two kaolins, one of hydrothermal origin (K-H) and another of lacustrine-fluvial origin (K-L), were evaluated in comparison with a commercial, purified bentonite. The CEC assay revealed the complete substitution of exchangeable cations (Na+ and Ca2+) by Cs+ in BNT samples and CEC values consistent with those of typical smectites (100.64 7.33 meq/100 g). For kaolins, partial substitution of Na+ cations occurred only in the K-L samples because of the interstratified mineral component which has small CEC values (11.13 5.46 meq/100 g for the K-H sample and 14.75 6.58 meq/100 g for the K-L sample). The degree of isomorphous substitution of Al3+ by Mg2+ affected the hydration properties of BNT in terms of swelling, water sorption, and rheology, whereas both of the poorly expandable kaolins exhibited significant water-adsorption properties. The EDX microanalysis has proved to be of considerable interest in terms of providing more information about clay properties in comparison with other commonly used methods and to identify the role played by both chemical and mineralogical composition of natural clays for their appropriate use in pharmaceutical and cosmetic fields

E-FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro.

Epidermal fatty acid-binding protein (E-FABP) is a lipid carrier, originally discovered in human epidermis. We show that E-FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E-FABP induces overexpression of K10 and involucrin. On the other hand, E-FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF-κB and JNK signalling pathways. E-FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E-FABP as compared to the same population in normal epidermis. E-FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high-calcium conditions, E-FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E-FABP plays an important role in keratinocyte differentiation. Moreover, E-FABP modulates differentiation in psoriatic keratinocytes

Expression of nuclear survivin in normal skin and squamous cell carcinoma: a possible role in tumor invasion

Background: Survivin is detected in few adult normal cells and it is highly expressed in cancer. Nuclear survivin facilitates cell cycle entry, while the mitochondrial pool protects cells from apoptosis. Survivin is overexpressed in keratinocyte stem cells (KSC) and protects them from apoptosis. Methods: As KSC are at the origin of squamous cell carcinoma (SCC), we evaluated survivin expression in normal and cancerous skin in vivo by immunohistochemistry and western blotting. HaCaT cells overexpressing survivin and wound-healing assay are used. Anova and Student-T tests are used for statistical analysis. Results: Survivin is localized both in the cytoplasm and in the nucleus of normal adult and young keratinocytes. Nuclear survivin is detected in one every 10/11 basal keratinocytes. When present in suprabasal cells, nuclear survivin is co-expressed with K10, but not with K15 or p75-neurotrophin-receptor (p75NTR), a transit amplifying cell marker. Nuclear, but not cytoplasmic survivin expression dramatically increases in actinic keratosis and in SCC in situ, as compared to normal epidermis, and it is highest in poorly differentiated SCC. In SCC tumors, nuclear survivin-positive cells are mainly K10/p75NTR-negative and K15-positive. In poorly differentiated tumors, survivin mostly localizes in the deep infiltrating areas. When overexpressed in keratinocytes, survivin increases cell migration. Conclusion: High survivin expression and the subcellular localization of survivin correlate with keratinocyte differentiation and are associated with undifferentiated and more invasive SCC phenotype

Role of neurotrophins on dermal fibroblast survival and differentiation

Neurotrophins (NTs) belong to a family of growth factors that play a critical role in the control of skin homeostasis. NTs act through the low-affinity receptor p75NTR and the high-affinity receptors TrkA, TrkB and TrkC. Here we show that dermal fibroblasts (DF) and myofibroblasts (DM) synthesize and secrete all NTs and express NT receptors. NTs induce differentiation of DF into DM, as shown by the expression of \u3b1-SMA protein. The Trk inhibitor K252a, TrkA/Fc, TrkB/Fc or TrkC/Fc chimera prevents DF and DM proliferation. In addition, p75NTR siRNA inhibits DF proliferation, indicating that both NT receptors mediate DF proliferation induced by endogenous NTs. Autocrine NTs also induce DF migration through p75NTR and Trk, as either silencing of p75NTR or Trk/Fc chimeras prevent this effect, in absence of exogenous NTs. Finally, NGF or BDNF statistically increase the tensile strength in a dose dependent manner, as measured in a collagen gel through the GlaSbox device. Taken together, these results indicate that NTs exert a critical role on fibroblast and could be involved in tissue remodelling and wound healin