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 mm 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.

Proteomic analysis of PTCH1+/- fibroblast lysate and conditioned culture media isolated from the skin of healthy subjects and nevoid basal cell carcinoma syndrome patients.

BACKGROUND: The pathogenesis underlying the increased predisposition to the development of basal cell carcinomas (BCCs) in the context of Gorlin-Goltz syndrome is linked to molecular mechanisms that differ from sporadic BCCs. Patients with Gorlin syndrome tend to develop multiple BCCs at an early age and present with tumors of non-sun-exposed skin. The aim of this study was to compare the proteomic profile of cultured fibroblast and fibroblast conditioned culture media of PTCH1+ and nonmutated fibroblasts. RESULTS: Proteomic analysis was performed using Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry in PTCH1+ fibroblast conditioned media isolated from not affected sun-protected skin areas of Gorlin patients and from healthy subjects. 12 protein cluster peaks, >5 kDa, had significant differences in their peak intensities between PTCH1+ and PTCH1- subject groups. We detected a strongly MMP1 overexpression in PTCH1+ fibroblasts obtained from NBCCS patients with respect to healthy donors. CONCLUSION: Protein profiles in the fibroblast conditioned media revealed statistically significant differences between two different types (missense versus nonsense) of PTCH1 mutations. These differences could be useful as signatures to identify PTCH1 gene carriers at high risk for the development of NBCCS-associated malignancies and to develop novel experimental molecular tailored therapies based on these druggable targets.

Lack of evidence for an association between seminoma and human papillomavirus infection using GP5+/GP6+ consensus primers.

Testicular germ cell tumors account for about 1% of all cancers. The incidence of these tumors is increasing and they represent the most common solid malignancies of young men aged 15-40 years with seminoma being one of the most common histotype. Pathogenesis of testicular germ cell tumors remains unknown and, although cryptorchidism is considered the main risk factor, there is evidence of an association with environmental and genetic risk factors. Human papillomaviruses (HPV) are a family of DNA viruses and represent a major risk factor for cervical cancer. In addition, they have been associated with other human non-malignant and malignant diseases, including breast and head and neck cancer. HPV sequences have been detected throughout the male lower genitourinary tract as well as in seminal fluid and an increased testicular tumorigenesis has been reported in HPV transgenic mice. Aim of this study was to evaluate the potential involvement of HPV in human testicular tumorigenesis. Real-time PCR employing GP5+/GP6+ consensus HPV primers was used to examine the presence of HPV sequences in a subset of human seminoma (n = 61) and normal testicles (n = 23). None of the specimens tested displayed the presence of HPV DNA. These findings do not support an association between HPV and human seminoma and warrant further studies to assess definitively the role of these viruses in human testicular tumorigenesis.

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

Melanoma 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.

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.

18beta-glycyrrhetinic acid and glabridin prevent oxidative DNA fragmentation in UVB-irradiated human keratinocyte cultures.

BACKGROUND: UVB radiation is the major etiological factor in the pathogenesis of skin aging and cancer development. New approaches to prevent and reverse UVB damage are needed to reduce sunlight-induced skin cancer. This study aimed to investigate a possible protective activity of liquorice root extracts glycyrrhizin (GL), 18ß-glycyrrhetinic acid (18ß-GA) and glabridin (GLB) against UVB radiation damage in human keratinocyte cultures. MATERIALS AND METHODS: The MTT test was performed to assess cell viability. DNA damage was evaluated by comet assay, whereas generation of intracellular reactive oxygen species (ROS) was measured by fluorescent 2'7'-dichlorodihydrofluorescein diacetate assay. In addition, the activation of p53, regulation of BCL-2 and PARP cleavage were analyzed by Western blot analysis. RESULTS: The treatment of human keratinocytes with 18ß-GA and GLB prevented direct and indirect DNA damage avoiding apoptosis activation. CONCLUSION: 18ß-glycyrrhetinic acid and glabridin are potent antioxidants that prevent oxidative DNA fragmentation and the activation of apoptosis-associated proteins in human keratinocytes.

Pharmacological and toxicological evaluation of a new series of thymidylate synthase inhibitors as anticancer agents.

Thymidylate synthase (TS) is responsible for catalysing the de novo biosynthesis of doexythymidine monophosphate and is a target for many anticancer drugs. A series of thymidylate synthase inhibitors (TSIs), synthesised in our laboratory, were submitted to primary anticancer screening by the National Cancer Institute (NCI). Four compounds, 3,3-bis(4-methoxyphenyl)-1H, 3H-naphtho[1,8-cd]pyran-1-one (MR7), 6-chloro-3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR21), 3,3-bis(3-fluoro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR35) and 6-bromo-3,3-bis(3-chloro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR36), passed the criteria and were automatically scheduled for evaluation against the full panel of 60 human tumour cell lines. In this study, the antiproliferative activity of the substances against SK-MEL-2 cells (from metastatic tissue) and SK-MEL-28 cells (from primary malignant melanoma cells) was investigated. Neutral Red uptake and the MTT test were performed to confirm the results of the NCI, and [3H]-thymidine incorporation was performed as a test of the proliferation rate. Our results indicated that compounds MR21 and MR36 were the most active agents and the [3H]-thymidine test was the best in predicting toxicity against melanoma cells.