MicroRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally and play important roles in various biological processes. We previously identified miR-203 as a skin-and keratinocyte-specific microRNA. Moreover, miR-203 has been implicated in repressing 'stemness' in epidermal progenitors. Here, we investigate the expression of miR-203 and two of its targets, p63 and suppressor of cytokine signalling-3, during human skin morphogenesis. MiR-203 in situ hybridization was performed on sections of human foetal skin ranging from 14 to 22 weeks' gestation and adult skin. MiR-203 was barely detectable at 14 weeks. Its expression became prominent from week 17 and was most pronounced in the suprabasal layers of the epidermis, while p63 and SOCS-3 were preferentially expressed in the basal layer. Differentiation markers such as involucrin and filaggrin were expressed mainly in the suprabasal layers of epidermis, similar to miR-203. Our results support the involvement of miR-203 in skin morphogenesis.
The aim of the present investigation was to compare the pharmacokinetics of two tablet formulations of 600 mg of racemic ibuprofen obtained using enantiospecific and non-enantiospecific assays, in order to explore if chiral assays should be employed in bioequivalence studies of chiral active substances. The stereoselective assay showed that, for both formulations, there was an initial phase where (R)-ibuprofen was the predominant enantiomer followed by a final phase where (S)-ibuprofen was the predominant one. Results from both analytical methods proved that the two formulations were bioequivalent. However, the chiral bioanalytical method detected a larger difference in the eutomer than that showed by the nonchiral bioanalytical method. In conclusion, although the exposure ratios of enantiomers are near unity, the measurement of unresolved ibuprofen alone is not an adequate measure of bioequivalence since it may mask the actual difference in the eutomer exposure among formulations.
Background:Ultraviolet radiation (UVR) is the major risk factor for development of malignant melanoma. Fibroblast activation protein (FAP)-α is a serine protease expressed on the surface of activated fibroblasts, promoting tumour invasion through extracellular matrix (ECM) degradation. The signalling mechanism behind the upregulation of FAP-α is not yet completely revealed.Methods:Expression of FAP-α was analysed after UVR exposure in in vitro co-culture systems, gene expression arrays and artificial skin constructs. Cell migration and invasion was studied in relation to cathepsin activity and secretion of transforming growth factor (TGF)-β1.Results:Fibroblast activation protein-α expression was induced by UVR in melanocytes of human skin. The FAP-α expression was regulated by UVR-induced release of TGF-β1 and cathepsin inhibitors prevented such secretion. In melanoma cell culture models and in a xenograft tumour model of zebrafish embryos, FAP-α mediated ECM degradation and facilitated tumour cell dissemination.Conclusions:Our results provide evidence for a sequential reaction axis from UVR via cathepsins, TGF-β1 and FAP-α expression, promoting cancer cell dissemination and melanoma metastatic spread.
Evaluation of tubulin β-3 as a novel senescence-associated gene in melanocytic malignant transformation., Pigment Cell & Melanoma Research, 2017. 30(2) SummaryMalignant melanoma might develop from melanocytic nevi in which the growth-arrested state has been broken. We analyzed the gene expression of young and senescent human melanocytes in culture and compared the gene expression data with a dataset from nevi and melanomas. A concordant altered gene expression was identified in 84 genes when comparing the growth-arrested samples with proliferating samples. TUBB3, which encodes the microtubule protein tubulin β-3, showed a decreased expression in senescent melanocytes and nevi and was selected for further studies. Depletion of tubulin β-3 caused accumulation of cells in the G2/M phase and decreased proliferation and migration.Immunohistochemical assessment of tubulin β-3 in benign lesions revealed strong staining in the superficial part of the intradermal components, which faded with depth. In contrast, primary melanomas exhibited staining without gradient in a disordered pattern and strong staining of the invasive front. Our results describe an approach to find clinically useful diagnostic biomarkers to more precisely identify cutaneous malignant melanoma and present tubulin β-3 as a candidate marker. SignificanceThe development and progression of a melanocytic nevus into a malignant melanoma is characterized by alteration of gene expression and growth promoting signaling to break the growth-arrested state. By combining the gene expression profiles from skin lesions and cell culture models, we identify candidate biomarkers that distinguish malignant melanoma from benign melanocytic lesions. Tubulin β-3 may have a diagnostic impact in melanoma.Running title: Tubulin β-3 expression in nevi and melanomas
Melanoma is a form of cancer that develops in melanocytes. While it represents only 5% of skin malignancies, it is the most aggressive and lethal. Benign proliferation of these cells form the melanocytic nevi. The definitive diagnosis of melanocytic nevi or melanoma lesions is histopathologic. However, it is estimated that a correct diagnosis is established by means of standard skin biopsy in only 83% of the melanocytic lesions; of the remaining cases 8% and 9% are overinterpreted (false positives) and under-interpreted (false negatives), respectively. This underscores the importance of additional diagnostic tests. Since cellular senescence is considered to be a tumor suppressive mechanism, immuno-histochemistry using senescence markers has been suggested for the evaluation of difficult melanocytic lesions; however, the routinely used senescence markers lack the ability to distinguish nevi from melanoma. The general aim of this thesis is therefore to identify novel senescence markers that may aid in melanoma diagnosis.In study I, we established a cellular model with nevus-mimicking characteristics consisting in primary melanocytes that become senescent. Transcriptomic analysis allowed expanding the set of senescence-associated markers that could distinguish nevi from melanoma and identifying tubulin β-3 as a potential diagnostic marker. Depletion of tubulin β-3 and pretreatment with tubulin destabilizing drugs in melanocytes and melanoma cells induced a senescence-like phenotype in vitro. In particular, reduced migration capacity and induction of cell cycle arrest in G2/M phase of the cell cycle was demonstrated.In study II, a potential inter-cellular signaling pathway between melanoma cells and stromal fibroblasts, that might facilitate melanoma invasion, was investigated. Ultraviolet (UV) radiation was shown, both in melanoma cells and fibroblasts, to promote the release and activation of TGF-β1 and subsequent increase in expression of the serine protease FAP-α, a protein that plays role in extracellular matrix degradation and therefore facilitates the invasion of melanoma cells. Such mechanism was not functional in senescent melanocytes.In study III, it was shown that tubulin β-3 immunostaining aids in the diagnosis of nevi and melanomas. The diagnostic criterium was the tubulin β-3 gradient within the melanocytic nevi that was no longer apparent in melanoma. Different patterns of tubulin β-3 immunostaining in melanoma were described, dermoscopy-immunohistochemistry associations were found, specific dermoscopic features highlighted, and the prognostic value of this tubulin β-3 marker was examined. The progression rate in patients whose melanomas had areas with loss of tubulin β-3 was 4 times higher than in patients without this feature, although statistical significance could not be reached (p=0.06).In conclusion, transcriptomic analysis expanded the set of senescence-associated markers that could distinguish nevi from melanoma and identified tubulin β-3 as novel immunohistochemistry marker shown to have diagn...
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