Luisa Benassi

Ultraviolet B radiation induces activation of neutral and acidic sphingomyelinases and ceramide generation in cultured normal human keratinocytes

The sphingomyelin pathway is an ubiquitous, evolutionary conserved signaling system which transduces an extracellular signal into the cell. During the past few years increasing evidence has shown that the sphingolipid ceramide may play a role as a second messenger in intracellular signal transduction. The ceramide generation via sphingomyelinase (SMase) is followed by three major cellular responses: cell growth arrest, induction of cell differentiation and/or induction of programmed cell death or apoptosis. The aim of this study is to investigate whether activation of SMases and generation of ceramide can be induced by UVB radiation in normal human keratinocytes. The present data show that exposure to UVB radiation results in rapid generation of ceramide. The ceramide accumulation starts 15 min after UV exposure and progressively increases up to 24 h. In vitro measurement of SMase activity following exposure to UVB evidences an activation of both neutral and acidic SMases. Moreover, UVB induces apoptosis in normal human keratinocytes as shown by TUNEL technique and FACS analysis. These data indicate that UVB induced ceramide generation and activation of both neutral and acidic SMases, suggesting that sphingolipids metabolism may be involved in the UVB signaling pathway.

Ceramide 2 (N‐acetyl sphingosine) is associated with reduction in Bcl‐2 protein levels by Western blotting and with apoptosis in cultured human keratinocytes

Background  Ceramides produced by sphingomyelin hydrolysis activate a cycle that is followed by three different major cellular responses: downregulation of cell proliferation, induction of cell differentiation and apoptosis. In the skin, the generation of intracellular ceramide may also provide a link between an extracellular signal and the induction of the apoptosis programme for the elimination of damaged cells. Objectives We investigated the effect of ceramides capable of entering cells on cultured keratinocytes. Methods Human keratinocytes from neonatal skin were cultured in serum‐free medium with or without increasing concentrations of ceramide 2 (CER‐2; N‐acetyl sphingosine) (5, 10, 20 and 40 µmol L−1). Proliferative effects were studied either by cell counts or by 3H‐thymidine incorporation and flow cytometric analysis. Apoptosis was studied by TUNEL staining and Western blot analysis of Bcl‐2 protein. Results Cell counts and DNA synthesis were reduced in a dose‐dependent manner following CER‐2 treatment. TUNEL staining showed CER‐2‐induced apoptosis at 48, 72 and 96 h. Western blot analysis showed that CER‐2 induces downregulation of Bcl‐2 at 24–96 h. Conclusions These results demonstrate that CER‐2 inhibits cell proliferation and induces apoptosis, possibly via a Bcl‐2‐dependent mechanism.

Functionalized gold nanoparticles for topical delivery of methotrexate for the possible treatment of psoriasis.

Gold nanoparticles (AuNPs) represent an effective choice for topical drug delivery systems thanks to their small size, general non-toxicity, ease of functionalization and high surface to volume ratio. Even if systemic, methotrexate still plays an important role in psoriasis treatment: its topical use shows insufficient percutaneus penetration owing to limited passive diffusion, high molecular weight and dissociation at physiological pH. The aim of our study was to design a new drug delivery nanocarrier for Methotrexate and to improve its solubility, stability and biodistribution. AuNPs were on purpose prepared with a hydrophilic stabilizing layer, in order to improve the colloidal stability in water. Water-soluble gold nanoparticles functionalized by sodium 3-mercapto-1-propansulfonate (Au-3MPS) were prepared and loaded with methotrexate (MTX). The loading efficiency of MTX on Au-3MPS was assessed in the range 70-80%, with a fast release (80% in one hour). The release was studied up to 24h reaching the value of 95%. The Au-3MPS@MTX conjugate was fully characterized by spectroscopic techniques (UV-vis, FTIR) and DLS. Preliminary toxicity tests in the presence of keratinocytes monolayers allowed to assess that the used Au-3MPS are not toxic. The conjugate was then topically used on C57BL/6 mouse normal skin in order to trace the absorption behavior. STEM images clearly revealed the distribution of gold nanoparticles inside the cells. In vitro studies showed that Methotrexate conjugated with Au-3MPS is much more efficient than Methotrexate alone. Moreover, DL50, based on MTT analysis, is 20 folds reduced at 48 h, by the presence of nanoparticles conjugation. UV-vis spectra for in vivo tracing of the conjugate on bare mouse skin after 24h of application, show increased delivery of Methotrexate in the epidermis and dermis using Au-3MPS@MTX conjugate, compared to MTX alone. Moreover we observed absence of the Au-3MPS in the dermis and in the epidermis, suggesting that these layers of the skin do not retain the nanoparticles. Based on our data, we found that the novel Au-3MPS@MTX conjugate is an effective non-toxic carrier for the satisfactory percutaneous absorption of Methotrexate and could help in possible topical treatment of psoriasis.

Brooke-Spiegler syndrome: Report of two cases not associated with a mutation in the CYLD and PTCH tumor-suppressor genes

Brooke‐Spiegler syndrome represents an autosomal dominant disease characterized by the occurrence of multiple cylindromas, trichoepitheliomas and (sporadically) spiroadenomas. Patients with Brooke‐Spiegler syndrome are also at risk of developing tumors of the major and minor salivary glands. Patients with Brooke‐Spiegler syndrome have various mutations in the CYLD gene, a tumor‐suppressor gene located on chromosome 16q. To date, 68 unique CYLD mutations have been identified. We describe two families with Brooke‐Spiegler syndrome, one with familial cylindromatosis and one with multiple familial trichoepithelioma, which showed wide inter‐family phenotypic variability. Analysis of germline mutations of the CYLD and PTCH genes was performed using peripheral blood. In addition, formalin‐fixed paraffin‐embedded tumor samples were analyzed for PTCH somatic mutations and cylindroma cell cultures were obtained directly from patients for further growth and analysis. Clinically, the major features of Brooke‐Spiegler syndrome include the presence of heterogeneous skin tumors and wide inter‐ and intra‐familial phenotypic variability. Histopathologically, both cylindromas and trichoepitheliomas were found in affected individuals. Mutations or loss of heterozygosity was not found in CYLD and PTCH genes. In CYLD and PTCH mutation‐negative patients, other genes may be affected and further studies are needed to clarify whether these patients may be affected by de novo germline mutations.

Melanocytes--a novel tool to study mitochondrial dysfunction in Duchenne muscular dystrophy.

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal–epidermal junction. In addition the full‐length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments. J. Cell. Physiol. 228: 1323–1331, 2013.

In vitro testing of tensides employing monolayer cultures: a comparison with results of patch tests on human volunteers

Evaluation of the irritant potential of new products or ingredients prior to human testing is generally performed in vivo on animals. However, according to the 6th amendment and following updates of the European Community directive on cosmetic products (93/35/EEC), animal testing will be banned when suitable substitutes will be available. To know whether in vitro tests for assessment of skin irritancy provide results approaching human conditions, comparisons have to be made between data deriving from in vitro tests and skin response in humans. The aim of our study was to assess the validity of the monolayer culture system of normal human keratinocytes as a model for the evaluation of the irritant effects of detergents, by comparing in vitro cell culture data to in vivo acute skin irritancy effects of cocamidopropyl betaine (CAPB), an amphoteric compound, Tween 20 (TW20) (polysorbate 20) and Tween 80 (TW80) (polysorbate 80), representing nonionic compounds, applied to the skin of 24 healthy volunteers at a concentration similar to that employed in commercial products. As parameters for cytotoxicity, cell proliferation, cell membrane integrity and cell metabolism were assessed by cell counts, thymidine incorporation, MTT conversion, and Neutral Red uptake. In order to increase the sensitivity of the in vivo evaluation, bioengineering methods for assessment of the effects of test products on the skin were employed. Whereas all 4 in vitro methods ranked the tensides according to their toxicity in the following order: CAPB>SLS>TW20>TW80, both in vivo methods agreed in identifying SLS as the most irritating substance. Moreover, as compared with the irritation potential on human skin, all 4 in vitro tests overestimated the toxicity of CAPB. This suggests that the keratinocyte monolayer cell culture technique cannot directly replace in vivo methods, and that data obtained by this method should be interpreted cautiously.

Stem cell properties in cell cultures from different stage of melanoma progression.

Cutaneous melanoma is an extremely heterogenous human cancer. The most aggressive melanoma may contain deregulated cells with undifferentiated/stem cell-like phenotype. A critical mechanism by which melanoma cells enhance their invasive capacity is the dissolution of the intercellular adhesion and the acquisition of mesenchymal features as a part of an epithelial-to-mesenchymal transition. The aim of this study was to clarify the role of a stem cell-like population in human melanomas by means of melanocytic cell culture analysis obtained from distinct histotypes of primary and metastatic malignant melanoma. Patients with advanced melanoma >2 cm in diameter and/or >300 mm2 surface were enrolled. The melanoma cells were isolated from skin biopsies of lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and metastatic melanoma. The colony forming unit assay and alkaline phosphatase stain were evaluated. Cells were subsequently cultured and maintained in different media to evaluate their ability to differentiate into osteogenic and adipogenic lineages. Immunohistochemistry and flow cytometry analysis were performed to evaluate antigenic markers CD90, CD73, CD105, CD146, CD20, CD166, and Nestin. This study confirms that melanoma can include heterogenous cell populations with the ability both to self-renew and to a give rise to differentiated progeny. Melanoma cells displayed intratumoral heterogeneity and dynamic antigen phenotypes. Histologically, transitions from normal skin to melanoma were associated with a gradual increase in the expression of CD146, CD20, CD133, Nestin, and CD73. These molecular profiles could be further analyzed and, in the future, used for the development of novel biomolecular targeted-therapy approaches.

Decrease in toxic potential of mixed tensides maintained below the critical micelle concentration: an in vitro study.

Sodium lauryl sulphate (SLS) is an anionic tenside widely utilized in commercial topical preparations that may cause skin irritation. It has been shown that the barrier damage caused by SLS in vivo is lower when SLS is used in combination with other tensides which are able to reduce the critical micelle concentration (CMC). The aim of our study was to evaluate if the cytotoxic effect of SLS is reduced by the association with different tensides also at concentrations well below the CMC. Normal human keratinocytes from plastic surgery were grown in serum-free medium. At subconfluency, the cells were treated with SLS at a dose of 0.0025% in combination with cocamidopropyl betaine, Tween 20 and Tween 80 at the minimum toxic dose. Following tenside treatment, the culture medium was changed, and after 24 h the cells were collected for 3H-thymidine incorporation, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and neutral red (NR) uptake. The cytotoxic effect on normal human keratinocytes, as evaluated by 3H-thymidine incorporation, MTT assay and NR uptake, was significantly decreased by the combination with all the tested tensides. The correlation between cytotoxicity and physical properties was also studied by a conductimetric assay to investigate the mechanism involved in this toxicity reduction.

Nerve Growth Factor Receptor and Neurochemical Markers in Human Oral Mucosa: An Immunohistochemical Study

BACKGROUND The innervation of the oral mucosa has so far been studied mainly by histochemical and ultrastructural techniques. Only few studies have investigated the presence of neural proteins and neurotransmitters in human gingival mucosa. OBJECTIVE The purpose of the present study was to evaluate the presence and distribution of neural structural and transmitter proteins in different areas of normal human oral mucosa. METHOD Indirect immunofluorescence was employed on specimens taken from different mucosal regions (gingiva, lips, gums, palate). Both structural (low-affinity nerve growth factor receptor, NGFr; protein gene product 9.5, PGP 9.5) and neuropeptide markers (substance P; calcitonin gene-related peptide; vasoactive intestinal peptide, neuropeptide Y) were used. RESULTS NGFr and PGP 9.5 intensely labelled both nerve fibres and selected epithelial cells, while neuropeptide immunoreactivity was scarcely expressed and exclusively localized in nerve fibres. CONCLUSIONS Similarly in the distribution pattern and neurochemistry between oral and cutaneous innervation is apparent. Expression of NGFr could be relevant to the trophism of both the oral innervation and epithelium.

Biological evaluation of MR36, a novel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines

SummaryMelanoma is one of the most common cancers, and its incidence has continued to increase over the past few decades. Chemotherapy resistance and related defects in apoptotic signaling are critical for the high mortality of melanoma. Effective drugs are lacking because apoptosis regulation in this tumor type is not well understood. The folate pathway has been considered an interesting target for anticancer therapies, and approaches targeting this pathway have recently been extended to melanoma treatment. In this study, the intracellular apoptosis signaling pathways of two melanoma cells lines (SK-MEL-2 and SK-MEL-28) were investigated after treatment with a new experimental antifolate substance (MR36) that targets thymidylate synthase. In both melanoma cell lines, apoptosis induction was triggered by a p53-independent mechanism. MR36-induced apoptosis was associated with a loss of both mitochondrial membrane potential and caspase-3 activation. Induction of cell cycle arrest by MR36 was associated with changes in the expression of key cell cycle regulators, such as p21 and cyclin D1, and the hypophosphorylation of pRb. In addition, Fas signaling was also analyzed. These findings suggest that, unlike classical antifolates, MR36 exerted an inhibitory effect on both the enzymatic function and expression of thymidylate synthase, thereby inducing apoptosis through the activation of the extrinsic and intrinsic pathways in the melanoma cell lines. MR36 showed a different mechanism of action from the known antifolates (Nolatrexed and Pemetrexed) that resulted in higher anticancer activity. Therefore, MR36 should be included as a potential new therapeutic treatment in melanoma research.