Efeitos da variação térmica no sistema pigmentar extracutâneo do anuro Eupemphix nattereri (Anura: Leiuperidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Morphological effects of bacterial compounds on the testes of Eupemphix nattereri (Anura)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise da expressão protéica de moléculas de adesão celular em melanomas cutâneos

Cutaneous melanoma is a malignancy caused by the uncontrolled proliferation of melanocytes. Has been increasing in incidence and mortality over the years. This type of tumor may arise in the skin or any other place where there was migration of neural crest cells. Melanocytes are located in the basal layer of the epidermis forming the epidermal melanin unit, involving a melanocyte to 36 keratinocytes. The proliferation of melanocytes depends on this relationship with the keratinocytes, gap junctions, E-cadherin and desmoglein. The basal membrane is responsible for promoting the integrity of the skin, their adhesion structures shown to be altered in several skin diseases. Some molecules that play the role of adherence in this membrane are the laminins, type IV collagen and type XVII and perlecan. Analysis of protein expression of cell adhesion molecules in melanoma by immunohistochemistry on tissue microarray (TMA). We selected paraffin blocks of patients who were diagnosed with cutaneous melanoma in the period 1995 to 2010, reaching a sample of 124 cases. All these have gone through review of preparation for subsequent histological analysis of tissue microarray (TMA). The protein expression will be analyzed by immunohistochemistry, using markers anti-laminin, anti-collagen type IV and type XVII and anti-perlecan. There was a predominance of female patients, Caucasians and southeastern Brazil. There were frequent in type of extensive superficial ulcers with Breslow depth greater than 4 mm, Clark level IV and mitotic index greater than 6 mitoses per cga. The patients, most of them were female with mean age of 57.13 years and Caucasian. The most frequent histological type was extensive superficial followed by acral lentiginous. Predominated cases very deep, with Breslow thickness greater than 4 mm, Clark IV, ulcerated and on average by 9 cga mitosis, leading to worse prognosis in this sample. It...(Complete abstract click electronic access below)

Gene and protein expression of oestrogen-beta and progesterone receptors in facial melasma and adjacent healthy skin in women

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Changes in nuclear morphology and chromatin texture of basal keratinocytes in melasma

BackgroundThe pathogenesis of melasma and the role of keratinocytes in disease development and maintenance are not completely understood. Dermal abnormalities, the expression of inflammatory mediators, growth factors, epithelial expression of melanocortin and sexual hormones receptors suggest that not only melanocytes, but entire epidermal melanin unit is involved in melasma physiopathology.ObjectivesTo compare nuclear morphological features and chromatin texture between basal keratinocytes in facial melasma and adjacent normal skin.MethodsWe took facial skin biopsies (2mm melasma and adjacent normal skin) from women processed for haematoxylin and eosin. Thirty non-overlapping basal keratinocyte nuclei were segmented and descriptors of area, highest diameter, perimeter, circularity, pixel intensity, profilometric index (Ra) and fractal dimension were extracted using ImageJ software.ResultsBasal keratinocyte nuclei from facial melasma epidermis displayed larger size, irregular shape, hyperpigmentation and chromatin heterogeneity by fractal dimension than perilesional skin.ConclusionBasal keratinocytes from facial melasma display changes in nuclear form and chromatin texture, suggesting that the phenotype differences between melasma and adjacent facial skin can result from complete epidermal melanin unit alterations, not just hypertrophic melanocytes.

Avaliação da atividade da unidade epidermo-melânica e do dano dérmico no melasma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeitos da alta temperatura na pigmentação visceral e no epitélio germinativo masculino em Physalaemus nattereri e Leptodactylus fuscus (Anura)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Potencial do uso do ácido cafeico em formulações cosméticas: atividade antioxidante, atividade despigmentante e citotoxicidade

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Potencial do uso do ácido cafeico em formulações cosméticas: atividade antioxidante, atividade despigmentante e citotoxicidade

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Proteasome inhibition and ROS generation by 4-nerolidylcatechol induces melanoma cell death

Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half-life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4-Nerolidylcatechol (4-NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4-NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl-1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4-NC is time-dependent upon the pro-apoptotic protein Noxa, which is able to bind and neutralize Mcl-1. We demonstrate the role of 4-NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4-NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.

Novel properties of melanins include promotion of DNA strand breaks, impairment of repair, and reduced ability to damage DNA after quenching of singlet oxygen

Melanins have been associated with the development of melanoma and its resistance to photodynamic therapy (PDT). Singlet molecular oxygen (102), which is produced by ultraviolet A solar radiation and the PDT system, is also involved. Here, we investigated the effects that these factors have on DNA damage and repair. Our results show that both types of melanin (eumelanin and pheomelanin) lead to DNA breakage in the absence of light irradiation and that eumelanin is more harmful than pheomelanin. Interestingly, melanins were found to bind to the minor grooves of DNA, guaranteeing close proximity to DNA and potentially causing the observed high levels of strand breaks. We also show that the interaction of melanins with DNA can impair the access of repair enzymes to lesions, contributing to the perpetuation of DNA damage. Moreover, we found that after melanins interact with 102, they exhibit a lower ability to induce DNA breakage; we propose that these effects are due to modifications of their structure. Together, our data highlight the different modes of action of the two types of melanin. Our results may have profound implications for cellular redox homeostasis, under conditions of induced melanin synthesis and irradiation with solar light. These results may also be applied to the development of protocols to sensitize melanoma cells to PDT. (c) 2012 Elsevier Inc. All rights reserved.

Generierung eines BAC-transgenen Mausstamms zur Analyse von Melanomen

Da Tumorerkrankungen ein enormes Gesundheitsproblem in der westlichen Welt darstellen, wird eine Vielzahl neuer Behandlungsstrategien entwickelt. Neuartige Tumor-Therapeutika werden jedoch üblicherweise zunächst an Tiermodellen evaluiert, bevor sie am Menschen angewandt werden.rnIn der vorliegenden Arbeit wurde ein BAC-transgenes Mausmodell generiert, welches als autochthones Melanommodell zur Anwendung kommen sollte.rnZunächst wurde dafür ein DNA-Konstrukt erzeugt. Dieses enthält die Melanom-Onkogene BrafV600E, Cdk4R24C und Mitf deren Expression durch die Tamoxifen-induzierbare Rekombinase CreERT2 kontrollierbar sein sollte. Die Verwendung des Tyrosinasepromoters sollte die melanozytenspezifische Expression der eingebrachten Gene gewährleisten. Ein weiterer Bestandteil des Konstrukts ist ein Luziferase-Gen, welches die Lokalisierung Onkogen-exprimierender Zellen durch in vivo-Biolumineszenz-Imaging erlaubt, da die Onkogen- und Luziferase-Expression durch 2A-Sequenzen gekoppelt sind.rnVor der Generierung der transgenen Tiere sollten in vitro Analysen die Funktionalität des Konstruktteils, bestehend aus den Onkogenen und der Luziferase, klären. Zu diesem Zweck wurde die Zelllinie C22 mit einem Expressionsvektor transfiziert, welcher den genannten Konstruktteil enthielt. Es konnte ein Anstieg der Braf- und Cdk4-Expression auf Protein Ebene, das Vorhandensein von Luziferase-Aktivität und die Aktivierung des MAP-Kinase-Signalwegs nachgewiesen werden. Die Funktionalität des untersuchten Konstruktteils war damit nahegelegt und die Generierung der transgenen Tiere wurde fortgesetzt.rnDie Pronukeus-Injektion resultierte schließlich in 3 Founder-Tieren, die mittels PCR und Southern Blot identifiziert wurden und die Bezeichnung „B6 tg Tyr iOnkogene“ (TyriOn) erhielten. Durch Verkreuzen der Founder-Tiere mit C57BL/6 Mäusen wurden im weiteren Verlauf 3 Linien erzeugt. Bei in vivo Biolumineszenz-Messungen zeigten Tiere der Linie D einen gewissen Grad an Hintergrund-Luziferase-Aktivität, die jedoch durch Tamoxifen-Injektionen verstärkt werden konnte. In den Folgegenerationen ging diese Tamoxifen-induzierte Verstärkung der Luziferase-Aktivität teilweise verloren. Es wurde die Vermutung angestellt, dass funktionelle und nicht-funktionelle Varianten des Transgens an unterschiedlichen Stellen im Genom von Founder D integriert hatten, und sich in den folgenden Generationen auf die Nachkommen verteilten. Die mangelnde Induzierbarkeit betroffener Tiere konnte nicht auf fehlende Integrität der Sequenz „iOnkogene“ in diesen Tieren oder auf nicht-funktionelle loxP-Stellen im Konstrukt zurückgeführt werden.rnTamoxifen-Injektionen führten in TyriOn-D Tieren im Laufe von 15 Monaten nicht zur Entwicklung von Tumoren. Ebenso wenig konnten in TyriOn-D / Cre del Tieren, welche die eingebrachten Onkogene maximal exprimieren sollten, Tumoren detektiert werden. Um zu analysieren, ob die eingebrachten Onkogene die Bildung von Tumoren begünstigen, wurden TyriOn-D Tiere mit dem Melanom-anfälligen Stamm MT/ret verkreuzt. Hierzu konnte im Rahmen dieser Arbeit noch kein Ergebnis erzielt werden. Allerdings konnte in Melanomen von TyriOn-D / MT/ret Tieren Luziferase-Aktivität bei in vivo Biolumineszenz-Messungen und CreERT2 RNA durch RT-PCR detektiert werden.rnTyriOn-D / MT/ret Tiere werden im weiteren Verlauf dieses Projektes nicht nur der Analyse der Melanomentwicklung dienen. Deren Tumore ermöglichen außerdem weitere Untersuchungen bezüglich der Funktionalität des Konstrukts, die teilweise in TyriOn Tieren keine Resultate ergaben.

MMP19 is upregulated during melanoma progression and increases invasion of melanoma cells

During the progression of cutaneous melanomas, matrix metalloproteinases (MMPs) facilitate the tumour cells to traverse the basement membrane and invade the dermis. In this study, we analysed the expression of MMP19 in the course of melanoma progression. Although MMP19 was absent in melanocytes and melanoma cells of early stages of melanoma development, its expression was strongly upregulated in the neighbouring keratinocytes that may facilitate the vertical outgrowth of melanoma cells. In contrast to early stages, MMP19 was upregulated during the vertical growth phase of melanoma and in metastases. The upregulation of MMP19 in melanoma of Clark levels IV and V correlates with that of MMP2 and also simultaneously with ceased expression of E-cadherin. To reveal whether MMP19 facilitates the invasion of melanomas, we examined adhesion and migratory capacity of selected melanoma cell lines. Melanoma cell lines with low expression of MMP19 exhibited increased adhesion to various substrates and lower migration in comparison with the cell line with higher expression of MMP19. Moreover, ectopic expression of MMP19 could restore the migratory capacity of melanoma cells with low endogenous level of MMP19. These results suggest that the increase of MMP19 expression hallmarks the progression of cutaneous melanoma and might augment melanoma growth by promoting the invasion of tumour cells.

Antagonistic TSC22D1 variants control BRAF(E600)-induced senescence

Oncogene-induced cellular senescence (OIS) is an increasingly recognized tumour suppressor mechanism that confines the outgrowth of neoplastic cells in vivo. It relies on a complex signalling network, but only few components have been identified so far. Gene-expression profiling revealed a >100-fold increase in the levels of the transcription factor and putative tumour suppressor gene TGFβ-stimulated clone 22 (TSC22D1) in BRAF(E600)-induced senescence, in both human fibroblasts and melanocytes. Only the short TSC22D1 transcript was upregulated, whereas the abundance of the large protein variant was suppressed by proteasomal degradation. The TSC22D1 protein variants, in complex with their dimerization partner TSC22 homologue gene 1 (THG1), exerted opposing functions, as selective depletion of the short form, or conversely, overexpression of the large variant, resulted in abrogation of OIS. This was accompanied by the suppression of several inflammatory factors and p15(INK4B), with TSC22D1 acting as a critical effector of C/EBPβ. Our results demonstrate that the differential regulation of antagonistic TSC22D1 variants is required for the establishment of OIS and suggest distinct contributions of TSC22 family members to the progression of BRAF(E600)-driven neoplasia.

Effectiveness of a 308-nm excimer laser in treatment of vitiligo: a review

Vitiligo is a relatively common acquired disorder, characterized by progressive loss of melanocytes from the epidermis and the epidermal appendages. The disease is associated with considerable morbidity because of a major impact on the quality of life. The treatment for vitiligo is generally unsatisfactory and challenging. There are a variety of therapeutic possibilities including topical corticosteroids, topical calcineurin inhibitors, as well as phototherapy with Psoralen plus UVA (PUVA), narrow-band UVB, and a 308-nm excimer laser and/or lamps. Furthermore, surgical methods encompass grafting and transplantation while depigmentation treatments and psychological support may also be considered. The objective is to assess the effect of the 380-nm excimer laser in the treatment of vitiligo based on the available studies and case series. We searched the relevant literature about vitiligo and excimer laser published between 1990 and 2012 using the MEDLINE database. We reviewed all relevant articles about 308-nm excimer laser and light sources assessing their efficacy in the management of vitiligo as well as their side effects. The value of combination treatment methods was also analyzed. The available studies provide strong evidence that the excimer laser represents the most effective approach to treat vitiligo compared to ordinary phototherapy. Excimer laser is relatively safe and effective for localized disease. UV-sensitive areas respond best as well as a short duration of the disease. More frequent treatments achieve better results. Compared to other treatment modalities, the excimer laser most likely constitutes the treatment of choice for localized vitiligo. Its efficacy can be further improved in combination with other therapies such as corticosteroids, pimecrolimus, or tacrolimus.