Characterization of antitumoral and antiangiogenic responses induced by treatment with bisphosphonates in breast cancer cell line

Orientador: Karina Gottardello ZecchinDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O comprometimento ósseo em pacientes com câncer é responsável por significativas morbidade e redução da qualidade de vida, principalmente quando se trata de câncer de mama, no qual o esqueleto é o principal sítio de desenvolvimento das metástases. Bisfosfonatos (BPs) contendo ou não nitrogênio são os principais agentes usados no combate à perda óssea como na osteoporose, uma vez que induzem apoptose de osteoclastos. Recentemente foram demonstradas ações antitumoral e antiangiogência dos BPs, in vitro e in vivo. Desse modo, o objetivo deste trabalho foi analisar e comparar os efeitos antitumorais e antiangiogênicos de BP que não contêm nitrogênio (clodronato, CLO) e BP contendo nitrogênio (ácido zoledrônico, ZOL), em linhagem celular derivada de câncer de mama, MCF-7, e em culturas de células endoteliais. Os resultados mostram que ZOL reduziu a proliferação das MCF-7 de maneira dose-dependente, com aumento do número de células nas fases G0/G1, sem alterar a viabilidade. Paralemamente, CLO não mostrou nenhum efeito sobre as células tumorais. O tratamento de células endoteliais de coelho, RAEC, com ZOL ou CLO reduziu a proliferação celular derivada, sem alterar sua viabilidade. Células endoteliais humanas HUVEC mostraram 15% de redução da viabilidade quando tratadas com ZOL, enquanto CLO não mostrou efeitos citotóxicos. A incubação de células HUVEC com meio condicionado por células MCF-7 tratadas com ZOL culminou na redução da proliferação das células endoteliais e na redução da formação de estruturas semelhantes a vasos, in vitro. Esse mesmo meio condicionado pelas células tumorais tratadas com ZOL resultou em menor proliferação de células provenientes de aorta murina, em ensaio ex vivo. Ensaios de imunoabsorbância revelaram diminuição nos níveis de VEGFA total nesse meio condicionado, assim como aumento da quantidade da isoforma antiangiogênica VEGFA165b, após tratamento das MCF-7 com ZOL. Por último, o tratamento das células MCF-7 com ZOL modulou a expressão de VEGFA total e de suas isoformas. Em conjunto, estes achados mostram que ZOL exibe ação antitumoral e antiangiogênica em células de câncer de mama e em células endoteliais. Tais dados contribuem para um maior esclarecimento sobre os mecanismos de ação dos BPs, permitindo buscas por melhorias no uso desses agentes como coadjuvantes na quimioterapia, a fim de minimizar a morbidade decorrente de metástases ósseas em tumores malignosAbstract: Patients with cancer frequently develop bone metastasis, resulting in considerable morbidity and affecting quality of life. This is particularly important considering breast cancer, in which skeletal is the main site for metastasis. Non-nitrogen-containing and nitrogen-containing bisphosphonates (BPs) are the mainly agents to treat bone loss due to osteoporosis, Paget disease, bone metastasis, multiple myeloma, hypercalcemia and osteogenesis imperfecta. BPs impair bone loss by inducing apoptosis in osteoclasts. Recently it was shown anti-tumoral and anti-angiogenic effects of BPs, in vitro and in vivo. The present study aim to analyze and compare the anti-angiogenic pathway of the non-nitrogen-containing BP, clodronate (CLO), and the nitrogen-containing BP, zoledronic acid (ZOL), using estrogen receptor-positive breast cancer cell line, MCF-7, and endothelial cells. ZOL treatment reduced MCF-7 cell proliferation and induced cell cycle arrest in a dose-dependent manner, while CLO did not affect these cells. Treatment with ZOL or CLO also reduced RAEC cells proliferation, without changes in cell viability. HUVEC cells showed reduced viability after incubation with ZOL, but not with CLO. The conditioned medium by MCF-7 ZOL-treated cells reduced the formation of blood vessels in vitro and proliferation of HUVEC cells, together with lower endothelial cells proliferation derived from mouse aortic rings. Enzyme-linked immunoabsorbent assay showed reduction of ~25% in VEGFA levels, and increased amount of VEGFA165b isoform in the conditioned medium by ZOL-treated MCF-7 cells. The nitrogen-containing BP also modulated the expression of mRNAs for VEGF in MCF-7 treated cells. In all assays, CLO showed less anti-angiogenic properties when compared with ZOL. Data provided from this study amplify the knowledge of BPs actions, contributing for new approaches of bone metastases in malignant tumorsMestradoEstomatologiaMestre em Estomatopatologi

Osseous oral hyaline ring granuloma mimicking a mandible tumor in a child with congenital agenesis of the corpus callosum

Hyaline ring granuloma (HRG) of the oral cavity is an uncommon disorder considered to be a foreign-body reaction resulting from implantation of food vegetable particles. Microscopically, it is characterized by the presence of structures of hyaline rings in an inflamed fibrous tissue background, which contains multinucleated giant cells. We present the case of a 4-year-old boy diagnosed with a mandible osseous HRG, which showed clinical and tomographic aspects suggestive of an aggressive bone tumor. The patient underwent surgical exploration and histopathologic analysis showed fragments composed predominantly of widespread dense connective tissue with an acute and chronic inflammatory infiltrate containing multinucleated giant cells and scattered areas of eosinophilic material associated with hyaline rings, strongly suggestive of vegetable particles. The eosinophilic material was positive for periodic acid-Schiff (PAS) and resistant to diastase digestion. These features led to diagnosis of osseous HRG. Scanning electron microscopy (SEM) analysis was performed for illustrative purposes and the multiple structures resembling vegetable particles were characterized in more detail. Although rare, this case highlights the importance of the clinician’s awareness regarding the existence of an osseous counterpart of HRG

Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma

Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers

Educomunicação e suas áreas de intervenção: Novos paradigmas para o diálogo intercultural

oai:omp.abpeducom.org.br:publicationFormat/1O material aqui divulgado representa, em essência, a contribuição do VII Encontro Brasileiro de Educomunicação ao V Global MIL Week, da UNESCO, ocorrido na ECA/USP, entre 3 e 5 de novembro de 2016. Estamos diante de um conjunto de 104 papers executivos, com uma média de entre 7 e 10 páginas, cada um. Com este rico e abundante material, chegamos ao sétimo e-book publicado pela ABPEducom, em seus seis primeiros anos de existência. A especificidade desta obra é a de trazer as “Áreas de Intervenção” do campo da Educomunicação, colocando-as a serviço de uma meta essencial ao agir educomunicativo: o diálogo intercultural, trabalhado na linha do tema geral do evento internacional: Media and Information Literacy: New Paradigms for Intercultural Dialogue

Potential markers and epigenetic relatioship in primary and metastatic melanomas

Orientadores: Karina Gottardello Zecchin, Edgard GranerTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O desenvolvimento do Melanoma tem sido relacionado à exposição à radiação ultravioleta, mas uma série de alterações genéticas e epigenéticas também parecem estar envolvidas em sua patogênese. Estudos prévios mostram que a inibição da enzima ácido graxo sintase (FASN - fatty acid synthase) reduz a proliferação de células de melanoma através de alterações nas proteínas envolvidas na transição das fases G0/G1 para S do ciclo celular. Recentemente, verificou-se que a perda do marcador epigenético 5-hidroximetilcitosina (5-hmC) regulado pela família TET (ten-eleven translocation), resulta em aumento dos níveis da proteína do filamento intermediário, nestina, associada a progressão tumoral e pior prognóstico em melanomas. Este estudo apresenta dois principais objetivos: (i) avaliar a expressão de FASN, proteínas do ciclo celular, CD34 (marcador vascular) e Ki67 (índice proliferativo) em melanomas cutâneos primários (MCP) e melanomas metastáticos (MM), correlacionando-os com dados clínicos, histopatológicos e taxas sobrevida; (ii) avaliar a regulação epigenética de nestina através da via TET2/5-hmC, e correlacioná-los com a progressão e prognóstico desta neoplasia. MCP e MM foram organizados em microarranjos teciduais e submetidos a reações de imunoistoquímica (IHQ) contra FASN, Cdk-4, p16Ink4a, p21WAF1/Cip1, p27Kip1, Rb, CD34 e Ki-67. IHQ e imunofluorescencia (IF) contra nestina e 5-hmC foram realizadas, avaliando-se a expressão de nestina com a perda de 5-hmC nos melanoma selecionados. Além disso, observamos os efeitos de 5-hmC sob nestina, através do aumento de TET2 em melanomas in vitro e pelo sequenciamento gênico por imunoprecipitação DNA metilado (hMeDIP-seq) de 5-hmC em nevos e melanomas. A sobrevida global (SG) dos casos de MCP foi significativa para fatores prognósticos como índice mitótico, estágio clínico e, discretamente, espessura de Breslow; também foi relevante para uma expressão aumentada de Cdk-4, FASN e CD34. E uma correlação significantemente positiva foi encontrada entre FASN e CD34. A SG dos MM mostrou-se reduzida diante do aumento de Cdk-4, p21WAF1/Cip1, p27Kip1, Rb e foi marginalmente significativa para p16 Ink4a. FASN, p21 WAF1/Cip1 e Rb apresentaram maiores valores nos casos de MM, quando comparados aos primários. IHQ e IF revelaram importante correlação negativa na expressão de nestin e 5-hmC. Relevante redução na expressão de nestina em melanomas com a reintrodução de TET2 in vitro, restabelecendo 5-hmC nessas células, foi encontrada. Em conjunto, estes resultados sugerem que FASN, p16Ink4a, Cdk-4 e CD34 são possíveis fatores prognóstico na progressão do melanoma. FASN mostrou correlação positiva com CD34, sugerindo que a expressão de FASN está associada à neoformação vascular em melanomas. A regulação epigenética de nestina na progressão tumoral pode estar relacionada aos efeitos de TET2 mediados por 5-hmC. Mais estudos são indicados para definir a progressão tumoral associada a perda de 5hmC, incluindo aquelas relacionadas a células-tronco cancerígenas no melanomaAbstract: Melanoma development has been associated with ultraviolet radiation exposure, but a variety of genetic and epigenetic changes might be also involved in its pathogenesis. Previous studies has shown that fatty acid synthase enzyme (FASN) inhibition reduces melanoma cells proliferation through changes in the proteins involved in the transition cell cycle phases G0 / G1 to S. Recently, it has been verified that the loss of the epigenetic marker 5-hydroxymethylcytosine (5-hmC), regulated by the TET family (ten-eleven translocation), results in a higher expression of nestin, an intermediate filament protein, involved in tumor progression and melanoma virulence. The aims of this work were: (i) evaluate the expression of the proteins FASN, cell cycle proteins, CD34 (a vascular marker), Ki-67 (a proliferation cell marker) in primary cutaneous melanoma (PCM) and metastatic melanoma (MM) and correspond these results with clinicopathological parameters and survival rates of the patients; (ii) access the epigenetic regulation of nestin through TET/5-hmC pathway, correlating it with progression and prognosis of the melanoma patients. PCM and MM were organized in tissue microarrays and submitted to immunohistochemical reactions (IHC) against FASN, Cdk-4, p16Ink4a, p21WAF1/Cip1, p27Kip1, Rb, CD34 and Ki-67. IHC and immunofluorescence (IF) against nestin and 5-hmC were also realized. The impact of 5-hmC in nestin expression by overexpression of TET2 in melanomas in vitro and through genome-wide mapping using Hydroxymethylated DNA Immunoprecipitation Sequencing (hMeDIP-seq) of 5-hmC in nevi and melanomas was also evaluated. For PCM, the overall survivor (OS) was significantly lower in the presence of elevated prognosis factors such as mitotic index, clinical stage and marginally significant to Breslow. It was also found relevancy for higher expression of Cdk-4, FASN and CD34; and a positive correlation was observed between FASN and CD34. The OS for MM was reduced in the presence of high levels of Cdk-4, p21WAF1/Cip1, p27Kip1, Rb and slightly to p16 Ink4a. Expressive increased values of FASN, p21WAF1/Cip1 and Rb were found in MM compared to the reciprocals PCM. A negative correlation of nestin expression and 5hmC was observed. We created a reduction in nestin expression in melanoma by reintroduction of TET2 in vitro restoring 5-hmC. Hence, our results suggest that FASN, p16 Ink4a, Cdk-4 and CD34 are possible critical prognostic factors in melanoma progression. Besides, FASN expression shown a positive correlation with CD34, suggesting that of FASN expression is related with angiogenesis in melanoma. Epigenetic regulation of nestin expression in melanoma tumorigenic growth may relate in part to effects of TET2-mediated 5-hmC. Further studies are indicated to define additional virulence pathways affected by loss of 5-hmC, including those related to melanoma stem cell functionDoutoradoPatologiaDoutora em Estomatopatologia2012/14008-0FAPESPCAPE

Pulp obliteration in a patient with sclerodermatous chronic graft-versus-host disease

Dental pulp calcification is a common finding associated with localized dental trauma, genetic disorders, and systemic inflammatory diseases. Chronic graft-versus-host disease (cGVHD) is a frequent complication after allogeneic hematopoietic cell transplantation (allo-HCT) characterized by immune-mediated injury to the skin, mouth, eyes, liver, and other tissues, resulting in significant disability and reduced quality of life. We report a patient with sclerodermatous cGVHD who presented with general pulp calcification in all teeth 5 years after allo-HCT. A review of full mouth dental radiographs obtained just before allo-HCT revealed normal-appearing pulp chambers. Based on prior reports of generalized pulp calcification associated with progressive systemic sclerosis, we hypothesized that the etiology was likely related to the presence of cGVHD with associated vascular and fibrotic tissue changes within the pulp vasculature. Clinicians should consider cGVHD in the differential diagnosis of generalized pulp calcification42467868

BMP Signaling Promotes Neural Crest Identity and Accelerates Melanoma Onset

Currently, little is known about specific factors and pathways that act to initially promote neural crest identity during tumor initiation. Recently, GDF6-activated BMP signaling was shown to regulate differentiation and survival both in melanomas and during melanocyte development. Together, these results have implicated BMP signaling as a key regulator of neural crest and melanocyte programs in normal and pathologic states of the melanocyte lineage. However, it is unknown whether BMP signaling plays a role early in melanoma initiation and progression

Reactive Tonsillar Enlargement Showing Strong 18f-fdg Uptake During The Follow-up Of Follicular Lymphoma.

The 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) has become a standard procedure for the diagnosis, staging, and restaging in lymphoma patients. However, a relative high rate of false-positive results has been reported. We report a case of a 40-year-old man with a previous history of a nodal follicular lymphoma, stage IVA, treated with R-CHOP, which showed strong 18F-FDG uptake in the Waldeyer's tonsillar ring during his follow-up, being considered highly suspicious of relapsed lymphoma. A surgical removal of the palatine tonsils and adenoids was performed, which showed reactive follicular hyperplasia. Furthermore, bone marrow biopsy revealed absence of neoplasia. The patient is still in follow-up with no signs of recurrent lymphoma. This case illustrates that, despite the high sensitivity for the detection of recurrent lymphoma, 18F-FDG uptake should be interpreted with great caution and confirmatory studies should be performed before any therapy.7258-6

Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma

Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers