Role of hypoxia-related proteins in invasion of ameloblastoma cells: crosstalk between NOTCH1, hypoxia-inducible factor 1α, a disintegrin and metalloproteinase 12, and heparin-binding epidermal growth factor.

AIMS: Ameloblastoma AME is a benign tumour characterized by local invasiveness, high recurrence rates, and diverse histological patterns. The oxygen concentration is reduced in specific areas of the tumour microenvironment, which leads to intratumoral hypoxia. Crosstalk between NOTCH1, a disintegrin and metalloproteinase 12 (ADAM-12), hypoxia-inducible factor 1α (HIF-1α) and heparin-binding epidermal growth factor (HB-EGF) under hypoxic conditions has been implicated in invadopodia formation, tumour invasiveness, and metastasis development. The aim of this study was to analyse the expression of these proteins, in order to further elucidate the mechanisms underlying AME invasiveness. METHODS AND RESULTS: Twenty cases of AME, eight calcifying cystic odontogenic tumours CCOTs and 10 samples of dental follicle were used to investigate the expression of these proteins by immunohistochemistry with the primary antibodies anti-NOTCH1, anti-ADAM-12, anti-HIF-1α, and anti-HB-EGF. Immunostaining results were expressed as the percentage of stained area in images acquired in an AxioScope microscope equipped with an AxioCamHRc camera and a × 40 objective. The results showed that immunoexpression of all proteins was higher in the AME samples than in the CCOT and dental follicle samples (P < 0.05). CONCLUSIONS: AME showed an increased presence of proteins associated with tumour invasiveness, which indicates a possible role of these proteins in the biological behaviour of this tumour.

Prognostic value of the expression and localization of cell proliferation and apoptosis markers in unicystic ameloblastomas.

The aim of this study was to verify whether the expression of cell proliferation and apoptosis markers in different types of unicystic ameloblastoma (UA) is associated with the location of neoplastic cells. Immunohistochemical study with a sample of 32 cases of UA, 11 cases of conventional ameloblastoma (CAM) and ten dental follicles (DF) cases was performed. Cell proliferation was assessed using Ki-67 status, and apoptosis by caspase-3 expression. Mural UA (MUA) showed a higher immunostaining of Ki-67 (p < 0.05) and a lower immunostaining of Caspase-3 (p < 0.05) compared with luminal and intraluminal subtypes of UA and CAM. The neoplastic cells of the MUA's cystic capsule showed a higher expression of Ki-67 protein (p < 0.0001) and a lower expression of Caspase-3 (p < 0.0001) compared with the lumen. DF showed lower Ki-67 and Caspase-3 immunostaining (p < 0.05) than neoplasms. The higher immunoexpression of Ki-67 and the lower immunoexpression of Caspase-3 in MUA, in the parenchyma cells within the cystic capsule, suggest an association between the biological behaviour and location of neoplastic cells in a tumour.

Transcriptomic Response to Acidosis Reveals Its Contribution to Bone Metastasis in Breast Cancer Cells.

Bone is the most common site of metastasis in breast cancer. Metastasis is promoted by acidosis, which is associated with osteoporosis. To investigate how acidosis could promote bone metastasis, we compared differentially expressed genes (DEGs) in MDA-MB-231 cancer cells in acidosis, bone metastasis, and bone metastatic tumors. The DEGs were identified using Biojupies and GEO2R. The expression profiles were assessed with Morpheus. The overlapping DEGs between acidosis and bone metastasis were compared to the bulk of the DEGs in terms of the most important genes and enriched terms using CytoHubba and STRING. The expression of the genes in this overlap filtered by secreted proteins was assessed in the osteoporosis secretome. The analysis revealed that acidosis-associated transcriptomic changes were more similar to bone metastasis than bone metastatic tumors. Extracellular matrix (ECM) organization would be the main biological process shared between acidosis and bone metastasis. The secretome genes upregulated in acidosis, bone metastasis, and osteoporosis-associated mesenchymal stem cells are enriched for ECM organization and angiogenesis. Therefore, acidosis may be more important in the metastatic niche than in the primary tumor. Acidosis may contribute to bone metastasis by promoting ECM organization. Untreated osteoporosis could favor bone metastasis through the increased secretion of ECM organization proteins.

Expression of metallothionein in ameloblastoma. A regulatory molecule?

BACKGROUND: Ameloblastoma is a benign odontogenic tumor, exhibiting local invasiveness and high rate of recurrence. Metallothionein is a protein associated with tumorigenesis, serving as prognostic factor in different neoplasms. We are interested in mechanisms underlying ameloblastoma local invasiveness. Thus, we decided to analyze expression of metallothionein in this tumor. MATERIALS AND METHODS: An immunohistochemical evaluation of metallothionein in ameloblastoma was carried out. As control, we assessed expression of the same molecule in calcifying cystic odontogenic tumor (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium. RESULTS: We studied 12 cases of solid/multicystic ameloblastomas. Metallothionein was observed in all samples. This molecule was observed in columnar cells in the periphery and in central polyhedral cells. CCOT (four cases) also showed the presence of metallothionein. Morphometry of stained areas showed that expression of metallothionein in ameloblastoma was significantly higher compared to CCOT (P < 0.0001). CONCLUSIONS: This protein may have an impact on ameloblastoma behavior. Metallothionein would act as a zinc reservoir for important proteases related to ameloblastoma biology, such as MMPs. This protein could also display pro-mitotic and anti-apoptotic features in the tumor.

Advances in Breast Cancer Management and Extracellular Vesicle Research, a Bibliometric Analysis.

Extracellular vesicles transport variable content and have crucial functions in cell-cell communication. The role of extracellular vesicles in cancer is a current hot topic, and no bibliometric study has ever analyzed research production regarding their role in breast cancer and indicated the trends in the field. In this way, we aimed to investigate the trends in breast cancer management involved with extracellular vesicle research. Articles were retrieved from Scopus, including all the documents published concerning breast cancer and extracellular vesicles. We analyzed authors, journals, citations, affiliations, and keywords, besides other bibliometric analyses, using R Studio version 3.6.2. and VOSviewer version 1.6.0. A total of 1151 articles were retrieved, and as the main result, our analysis revealed trending topics on biomarkers of liquid biopsy, drug delivery, chemotherapy, autophagy, and microRNA. Additionally, research related to extracellular vesicles in breast cancer has been focused on diagnosis, treatment, and mechanisms of action of breast tumor-derived vesicles. Future studies are expected to explore the role of extracellular vesicles on autophagy and microRNA, besides investigating the application of extracellular vesicles from liquid biopsies for biomarkers and drug delivery, enabling the development and validation of therapeutic strategies for specific cancers.

Environmental control of mammary carcinoma cell expansion by acidification and spheroid formation in vitro.

Breast cancer is the leading cause of cancer death among women worldwide. Like other cancers, mammary carcinoma progression involves acidification of the tumor microenvironment, which is an important factor for cancer detection and treatment strategies. However, the effects of acidity on mammary carcinoma cell morphology and phenotype have not been thoroughly characterized. Here, we evaluated fundamental effects of environmental acidification on mammary carcinoma cells in standard two-dimensional cultures and three-dimensional spheroids. Acidification decreased overall mammary carcinoma cell viability, while increasing their resistance to the anthracycline doxorubicin. Environmental acidification also increased extracellular vesicle production by mammary carcinoma cells. Conditioned media containing these vesicles appeared to increase fibroblast motility. Acidification also increased mammary carcinoma cell motility when cultured with fibroblasts in spheroids. Taken together, results from this study suggest that environmental acidification induces drug resistance and extracellular vesicle production by mammary carcinoma cells that promote tumor expansion.

Metallothionein Expression and its Influence on the In Vitro Biological Behavior of Mucoepidermoid Carcinoma.

Mucoepidermoid carcinoma (MEC) is the most common tumor in the salivary glands, often presenting with recurrence and metastasis due to its high invasive capacity. Metallothionein (MT), a zinc storage protein that supplies this element for protease activity, is probably related to mucoepidermoid carcinoma behavior. This prompted us to characterize a cell line derived from mucoepidermoid carcinoma and to correlate metallothionein expression with transforming growth factor-α (TGF-α), tumor necrosis factor-α (TNF-α) and matrix metalloproteinases (MMPs). Transcriptomic analysis and cytogenetic assays were performed to detect the expression of genes of interest and cellular chromosomal alterations, respectively. MEC cells with a depleted metallothionein 2A (MT2A) gene were subjected to Western blot to correlate metallothionein expression with growth factors and MMPs. Additionally, cells with depleted MT were subjected to migration and invasion assays. The transcriptomic study revealed reads mapped to cytokeratins 19 and AE1/AE3, α-smooth muscle actin, vimentin, and fibronectin. Cytogenetic evaluation demonstrated structural and numerical alterations, including the translocation t(11;19)(q21;p13), characteristic of MEC. Metallothionein depletion was correlated with the decreased expression of TGF-α and MMP-9, while TNF-α protein levels were augmented. Migration and invasion activity were diminished after metallothionein silencing. Our findings suggest an important role of MT in MEC invasion, through the regulation of proteins involved in this process.

HIF-1α is Overexpressed in Odontogenic Keratocyst Suggesting Activation of HIF-1α and NOTCH1 Signaling Pathways.

Background: The odontogenic keratocyst (OKC) is an odontogenic cyst that shows aggressive and intriguing biological behavior. It is suggested that a hypoxic environment occurs in OKC, which led us to investigate the immunoexpression and location of hypoxia-inducible factor 1-alpha (HIF-1α) and other hypoxia-related proteins. Methods: Twenty cases of OKC were evaluated for the expression of Notch homolog 1 (NOTCH1), HIF-1α, disintegrin and metalloproteinase domain-containing protein 12 (ADAM-12), and heparin-binding epidermal growth factor-like growth factor (HBEGF) by immunohistochemistry and compared to eight control cases of calcifying odontogenic cystic (COC), orthokeratinized odontogenic cyst (OOC), and normal oral mucosa (OM) in basal and parabasal layers. Results: In OKC, all the proteins tested were expressed significantly higher in both basal (except for NOTCH1 and HBEGF in OOC) and suprabasal epithelial layers compared to controls. Looking at the epithelial layers within OKC, we observed an increased NOTCH1 and HIF-1α expression in parabasal layers. Conclusions: These results suggest that hypoxia occurs more intensively in OKC compared to COC, OM, and OOC. Hypoxia appeared to be stronger in parabasal layers as observed by higher HIF-1α expression in upper cells. Overexpression of NOTCH1, ADAM-12, and HBEGF in OKC was observed, which suggests that microenvironmental hypoxia could potentially regulate the expression of hypoxia-related proteins, and consequently, its clinical and biological behavior.

Ultrastructural and microbiological analysis of the dentin layers affected by caries lesions in primary molars treated by minimal intervention.

PURPOSE: The purpose of this in vivo study of primary teeth was to analyze the ultrastructure and microbiology of dentin layers affected by caries lesions before and after restorations with resin-modified glass ionomer. METHODS: Samples of carious dentin from primary teeth removed prior to restoration placement (baseline-0 day) were compared with samples taken after 30 and 60 days. Dentin from 8 primary molars was analyzed by scanning electron microscopy (SEM) and dentin from 22 primary molars was examined microbiologically to compare bacteria (total of viable counts, Streptococcus spp, Streptococcus mutans, Lactobacillus spp, and Actinomyces spp) before and after treatment (30 and 60 days). RESULTS: Baseline caries samples had enlarged dentinal tubules with bacteriol invasion. SEM samples after treatment suggest better tissue organization, with more compact collagen fibers arrangement and narrower dentinal tubules. The number of bacteria decreased in all samples at both 30 (98%) and 60 (96%) days, with all bacteria species showing similar trends. CONCLUSIONS: The minimal intervention approach is very effective to promote beneficial changes in the lesion environment and favorable conditions for the healing process in primary teeth.

Primary leiomyosarcoma of the mandible. A case report.

The aim of this article is to describe a case of leiomyosarcoma of the mandible with immunohistochemical analysis that was useful in making the final diagnosis. A 40-year-old woman was referred to the Stomatology Clinic of Sao Paulo Tatuape Hospital, for evaluation of a lesion on the left side of the mandible. This lesion presented a fast growth in the last 6 months. Intraoral examination showed a firm, fixed, red colored mass measuring, approximately 60-mm in diameter. No lymph nodes involvement was found. The radiographic examination showed a lytic lesion showed ill-demarcated radiolucent with facial and lingual cortical bone destruction. Microscopic examination of the mandibular lesion showed a neoplasm composed by interlacing fascicles of spindle-shaped cells. Most of the cells presented a blut-ended elongated shape. A marked cellular pleomorphism was observed, represented by cells with irregular shape and abundant eosinophilic cytoplasm. Nuclei were large, hyperchromatic, either vacuoled or cigar-shape. The cytoplasm of the cells stained red with Masson s trichrome stain. Neoplastic cells expressed vimentin, smooth-muscle actin, HHF-35 and desmin. These findings were consistent with the diagnosis of leiomyosarcoma.

Keratocystic odontogenic tumor overexpresses invadopodia-related proteins, suggesting invadopodia formation.

OBJECTIVE: Keratocystic odontogenic tumor (KOT) is an odontogenic neoplasm that shows aggressive clinical behavior and local invasiveness. Invadopodia are actin-rich cellular protrusions exhibiting proteolytic pericellular activity, thereby inducing focal invasion in neoplastic cells and increasing neoplasms aggressiveness. Thus, this study aimed to evaluate immunoexpression of invadopodia-related proteins, cortactin, MT1-MMP, Tks4, and Tks5, in KOT. STUDY DESIGN: Immunohistochemistry of 16 cases of KOT, eight cases of calcifying cystic odontogenic tumor (CCOT), and eight samples of the oral mucosa (OM) was carried out to assess the expression of the above described invadopodia-related proteins in the basal and suprabasal layer. RESULTS: KOT samples showed higher and significant immunoexpression of cortactin, MT1-MMP, TKs4, and TKs5 compared with the CCOT and OM samples. Significant expression of all these proteins was observed in the basal layer compared with the suprabasal layer in KOT. CONCLUSIONS: Overexpression of cortactin, MT1-MMP, TKs4, and TKs5 was observed in KOT compared with samples of CCOT and OM. These proteins were also overexpressed in the basal over the suprabasal layer of KOT samples. Taken together, these results suggest the participation of invadopodia-related proteins on the pathogenesis of this lesion.

The effect of laminin and its peptide SIKVAV on a human salivary gland adenoid cystic carcinoma cell line.

We have previously demonstrated that laminin modulates the expression of adhesion molecules in an adenoid cystic carcinoma cell line (CAC2 cells). We are currently studying whether laminin can induce modifications in the overall morphology of CAC2 cells. These cells were grown in a three-dimensional preparation of laminin-1. Phenotype differences were assessed by light and transmission electron microscopy. CAC2 cells grown inside laminin-1 formed ductlike and pseudocystic structures. Based on our findings we suggest that laminin is a key regulator of tubular and pseudocystic patterns of adenoid cystic carcinoma. We also analyzed the effect of a molecular domain of laminin-1, the peptide SIKVAV (Ser-Ile-Lys-Val-Ala-Val) on CAC2 cells. This peptide was chosen because it is effective in cell proliferation and differentiation, and because it has never been tested before in salivary gland neoplasms. When CAC2 cells were grown inside SIKVAV-enriched laminin-1, only pseudocystic structures were observed. Since no ductlike structures were observed in samples treated with SIKVAV, we may assume that this peptide is at least one of the molecular domains of laminin responsible for the pseudocystic pattern observed in adenoid cystic carcinoma. Function disturbing experiments strongly suggested that the integrin alpha3beta1 play a role in the effect of laminin on CAC2 cells.

Estudo morfológico de superfícies ósseas após secçäo por pontas diamantadas ou laser de érbio: YAG / Morphological evaluation of bone surfaces after sectioning with diamond points or erbium: YAG laser

O objetivo deste trabalho foi analisar morfologicamente as superfícies ósseas resultantes da secçäo por pontas diamantadas ou por laser de érbio: YAG. Cinco ratos (Rattus norvegicus albinus foram sacrificados por dose letal de fenobarbital. Após a execuçäo deste procedimento, os ossos predeterminados foram submetidos à secçäo por pontas diamantadas ou por laser de érbio: YAG em uma energia de 300 mJ por pulso e taxa de repetiçäo de 2 Hz. As amostras foram submetidas a análise em microscópio eletrônico de varredura, revelando a existência de um padräo para as secçöes obtidas com cada instrumento, sendo verificada uma superfície mais regular nas amostras seccionadas com o laser de érbio: YAG. Em aumentos da ordem de 3000 vezes, pode-se observar indícios de fusäo e seqüente solidificaçäo das superfícies seccionadas por meio do laser de érbio: YAG. Conclui-se que o laser de érbio: YAG foi eficaz na remoçäo de tecido ósseo, mas que, nos parâmetros utilizados neste estudo, foi responsável por alteraçöes morfológicas sugestivas de significativo aumento de temperatura, näo devendo ser indicado, nestas condiçöes, para a execuçäo de secçöes ósseas

Piedra negra: estudo de seu agente etiológico em microscopia eletrônica de varredura / Black piedra: a scanning electron microscopy study of its etiological agent

No presente trabalho foi realizado estudo em microscopia eletrônica de varredura do fungo Piedraia hortae, através de material obtido de lesöes no cabelo de índios do Amazonas. O exame em microscopia eletrônica revelou ascosporos poliédricos, ovóides e arredondados, arranjados isoladamente ou na forma de pseudohifas, de permeio a material extracelular densamente compactado

Cisto odontológico epitelial calcificante: estudo morfológico e imunoenzimático / Calcifying epithelial odontogenic cyst: immunohistochemical and morphologic study

Foram estudados, a nível morfológico e imunoenzimático, a secreção presente no cisto odontogênico epitelial calcificante (C.O.E.C). Para tanto, foram utilizados 16 casos de C.O.E.C. pertencentes aos arquivos do Serviço de Patologia Cirúrgica da Disciplina de Patologia Bucal da Faculdade de Odontologia da USP. Os aspectos morfológicos do C.O.E.C. foram estudados baseados na classificação proposta por CLAUSEN et alii (1981). O material foi corado pela hematoxilina e eosina (H/E), sendo parte deste material submetido ao tricrômico de Masson, P.A.S. e Papanicolaou. A análise imuno-histoquímica foi realizada pelo método da peroxidase anti-peroxidase (PAP), sendo os espécimes submetidos aos anticorpos vimentina, queratina, fibronectina e proteína S-100. A metodologia utilizada permitiu-nos concluir que as células fantasma possuem uma expressão morfológica a partir do acúmulo intracitoplasmático de queratina e que o material dentinóide se apresentou fracamente positivo ao anticorpo anti-queratina, sendo positivo ao tricrômico de Masson, sugerindo ser essa estrutura o produto final de uma interação ectomesenquimal associada

Estudo morfológico subcelular e imuno-enzimático do leiomioma vascular bucal / Morphologic subcellular and immunoenzimatic study of oral vascular leiomyoma

Foram estudados três casos de leiomioma vascular com enfoques histopatológico, imuno-enzimático e subcelular, com a finalidade de fornecer subsídios para o seu diagnóstico. Histologicamente a neoplasia caracterizou-se pela proliferaçäo de células fusiformes formando feixes entrelaçados a partir de espaços vasculares. Ao exame imuno-histoquímico as células fusiformes mostratram-se positivas para desmina e negativas para vimentina e fator VIII, caracterizando o mioblasto em replicaçäo. O estroma da lesäo, ao contrário, apresentou positividade apenas para vimentina, enquanto o fator VIII foi evidenciado somente nas células endoteliais. O estudo em M.E.T. confirmou a natureza muscular da célula neoplásica que apresentou-se bem diferenciada, com núcleo ovalado, presença de lâmina basal, às vezes descontinua, poucas mitocôndrias e cavéolas na periferia do citoplasma, além de mcirofilamentos e corpos de adensamento

Análise do crescimento de células do carcinoma adenóide cístico humano cultivadas sobre proteínas da matriz extracelular / Effect of extracellular matrix proteins on growth of a cell line derived from human adenoid cystic carcinoma

O carcinoma adenóide cístico é uma neoplasia maligna que ocorre em glândulas salivares. A matriz extracelular tem sido apontada como possível fator regulador do fenótipo final desse tumor. O objetivo deste trabalho é verificar se há modificaçäo na taxa de crescimento ou induçäo de alteraçöes fenotípicas em células cultivadas de carcinoma adenóide cístico humano (células CAC2) na presença de proteínas da matriz extracelular (colágeno I, colágeno IV e laminina). A análise das curvas de crescimento realizadas mostrou que houve maior proliferaçäo celular nas culturas crescidas sobre o colágeno I. Näo houve diferenças estatísticas entre o crescimento celular de culturas tratadas com colágeno IV e laminina e o de seus respectivos controles. Nossos resultados sugerem que o colágeno I é um fator de crescimento para a linhagem celular derivada de carcinoma adenóide cístico humano

Influência da pasta composta por Rifocort, iodofórmio e paramonoclorofenol canforado na reparação dento-alveolar: estudo morfológico em ratos / The effect of the paste composed by Rifocort, iodoform and camphorated paramonoclorophenol on alveolar wound healing: morphologic study in rats

Análise do efeito da pasta composta por Rifocort, iodofórmio e paramonoclorofenol canforado na reparação alveolar em molares de ratos. Para tanto foram utilizados 30 ratos albinos divididos em dois grupos, sendo o grupo I experimental e o grupo II o controle. Os animais foram sacrificados após 48 horas, 7, 15, 21 e 30 dias da exodontia. Após o estudo em microscópio de luz concluiu-se que a pasta acelerou o processo de reparação alveolar, quando comparado ao grupo controle, em especial aos 7 e 15 dias pós-operatórios

Carcinoma mucoepidermóide de mandíbula

Os autores apresentam um caso clínico de carcinoma mucoepidermóide de localizaçäo no interior da mandíbula, sítio este considerado raro para esta neoplasia. O tratamento empregado constou de cirurgia e de radioterapia associada. As características clínicas e anátomo-patológicas verificadas säo comparadas com as relatadas na literatura. Especial enfoque é dado a inclusäo desta hipótese do diagnóstico diferencial de lesöes intra-ósseas dos maxilares

Reaçöes da dentina cariada humana após aplicaçäo do diamino fluoreto de prata a 10 por cento: estudo através de microscópio eletrônico de varredura / Reactions of human caries dentin after using diammine silver fluoride 10 percent: microscopicy electron scanning study

Foram utilizados sete dentes permanentes de crianças, com cáries profundas e agudas. O tratamento foi realizado em duas sessöes. Na primeira, removeu-se amostras de dentina para a análise. A seguir, aplicou-se a soluçäo de diamino fluoreto de prata a 10 por cento, durante 3 minutos. O dente foi fechado com óxido de zinco eugenol do tipo II. Na segunda sessäo, após 43 a 64 dias foram recolhidas novas amostras de dentina. Os resultados evidenciaram que as amostras obtidas após a aplicaçäo do diamino fluoreto de prata a 10 por cento a cárie estava estacionária revelando maior estruturaçäo dentinária e diminuiçäo acentuada do número de microorganismos. Com estes resultados pudemos concluir que, o uso do diamino de prata a 10 por cento, provoca resposta pulpar positiva havendo alteraçöes no processo carioso, que retrocedeu da fase ativa para a estacionária