Inhibition of CAL27 Oral Squamous Carcinoma Cell by Targeting Hedgehog Pathway With Vismodegib or Itraconazole.

Oral Squamous Cell Carcinoma (OSCC) presents an important challenge for the health systems worldwide. Thus, unraveling the biological mechanisms involved in OSCC pathogenesis is essential to the discovery of new drugs with anticancer potential. The Hedgehog (HH) pathway has shown promising results as a therapeutic target both in vitro and in vivo. This study aimed to investigate the effects of vismodegib and itraconazole on the expression of Hedgehog (HH) genes (PTCH1, SMO, and GLI1), cell cycle and cell death in OSCC cells. Alamar Blue assay was used to assess the cytotoxicity of vismodegib and itraconazole in a panel of oral cancer cell lines, including CAL27. The expression of HH signaling components after treatment with vismodegib and itraconazole, at concentrations of 25 or 50 µg/ml was evaluated by qPCR. Cell cycle and apoptosis were evaluated by flow cytometry after 72 h treatment with 50 µg/ml of vismodegib or itraconazole. HH signaling was activated in OSCC cell lines CAL27, SCC4, SCC9, and HSC3. Vismodegib and itraconazole significantly reduced CAL27 cell viability after 48 h of treatment. Gene expression of PTCH1, SMO, and GLI1 decreased in response to 24 h of treatment with vismodegib or itraconazole. Furthermore, CAL27 cells exhibited alterations in morphology, cell size, and cellular granularity. An increase in the DNA fragmentation was observed after treatment and both inhibitors induced apoptosis after 72 h. In conclusion, SMO inhibitors vismodegib and itraconazole demonstrably reduced the expression of HH genes in CAL27 OSCC cell line. In addition, treatment with vismodegib and itraconazole reduced cellular viability and altered the morphology of CAL27 cells, and also induced apoptosis.

Hedgehog pathway activation in oral squamous cell carcinoma: cancer-associated fibroblasts exhibit nuclear GLI-1 localization.

The purpose of this study was to evaluate the expression of Hedgehog (HH) signaling molecules (SHH and GLI-1) by cancer-associated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). Immunohistochemistry was used to detect molecular HH signaling and CAF-related protein expression, including α-SMA and S100A4, in 70 samples of human OSCC. The colocalization of α-SMA and S100A4 with SHH was also evaluated by double-staining. In vitro study was performed using primary normal oral fibroblast (NOF) and CAF through immunofluorescence and Western Blot for CAF-proteins, SHH, and GLI-1. Forty-five cases (64.28%) were positive for α-SMA exclusively in tumor stroma, and S100A4 was identified in the cytoplasm of CAFs in 94.28% (n = 66) of the cases. With respect to stromal cells, 64 (91.43%) OSCC cases were positive for SHH, and 31 were positive for GLI-1 (44.29%); positive correlations were found between SHH and α-SMA (p < 0.0001, φ = 0.51), as well as between SHH and S100A4 (p = 0.087, φ = 0.94). Protein expression of SHH and GLI-1 was observed in primary CAFs and NOFs. Although SHH was found to be localized in the cellular cytoplasm of both cell types, GLI-1 was present only in the nuclei of CAF. Our results indicate that CAFs are not only potential sources of HH ligands in tumor stroma, but may also respond to HH signaling through nuclear GLI-1 activation. We further observed that elevated SHH expression by OSCC cells was associated with higher CAF density, reinforcing the chemoattractant role played by these molecules.

The sonic hedgehog signaling pathway contributes to the development of salivary gland neoplasms regardless of perineural infiltration.

The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands whose histopathology is heterogeneous. The sonic hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC, and 20 cases of MEC. Using immunohistochemistry, SHH, GLI1, SUFU, HHIP, and STAT3 were investigated. For comparative purposes, MCM3 (cellular proliferation marker) was also included. In PA, there was high expression of cytoplasmic SHH and SUFU and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP, and STAT3 and low expression of SHH, SUFU, and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU, and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p = 0.0064), STAT3 (p = 0.0003), and MCM3 (p = 0.0257) when all tumors were compared and a higher expression in parenchyma for all tumors when stroma and parenchyma were compared (p < 0.05). These findings suggests a possible role of Hh pathway in the development and maintenance of the cytoarchitectural pattern of PA, ACC, and MEC, as well as the participation of STAT3 in the development of ACC, irrespective perineural infiltration.

Epithelioid hemangioma in the oral mucosa ­ A case report.

Introduction: Epithelioid hemangioma is an uncommon benign vasoproliferative neoplasm that usually manifests as multiple red nodules in middle-aged adults Case Outline: 52-year-old male patient presented with a one-year history of a nodular lesion in the left buccal mucosa measuring 3 cm. The clinical hypothesis was lipoma. An excisional biopsy revealed a circumscribed lesion composed of lobules of vessels with perceptible or poor lumina, associated with a prominent inflammatory infiltrate consisting of eosinophils, histiocytes and chronic inflammatory cells. The endothelial cells composing the lesion had an epithelioid morphology and contained abundant eosinophilic cytoplasm. Immunohistochemistry for CD34, factor VIII, collagen IV, alpha-smooth muscle actin, and mast cells, as well as histochemical staining with Weigert's orcein were performed. Conclusion: Vascular proliferations of soft tissues are a diverse and morphologically complex group of lesions that are difficult to diagnose. This report presents a case of oral epithelioid hemangioma, highlighting relevant morphological and immunohistochemical features that could help distinguish this condition from other neoplasms.

Accessory mental foramen: A rare anatomical variation detected by cone-beam computed tomography.

The mental foramen is a bilateral opening in the vestibular portion of the mandible through which nerve endings, such as the mental nerve, emerge. In general, the mental foramen is located between the lower premolars. This region is a common area for the placement of dental implants. It is very important to identify anatomical variations in presurgical imaging exams since damage to neurovascular bundles may have a direct influence on treatment success. In the hemimandible, the mental foramen normally appears as a single structure, but there are some rare reports on the presence and number of anatomical variations; these variations may include accessory foramina. The present report describes the presence of accessory mental foramina in the right mandible, as detected by cone-beam computed tomography before dental implant placement.

Branch of the canalis sinuosus: a rare anatomical variation--a case report.

The canalis sinuosus (CS) is a neurovascular canal, a branch of the infraorbital canal through which the anterior superior alveolar nerve passes. There are no studies or case reports of anatomical variations related to this canal. A rare case of anatomical variation in the CS is reported that was detected by cone beam computed tomography done in a 47-year-old female as a pre-operative workup before dental implants. In this case, in the region slightly medial to tooth 23, a wide accessory branch from the CS was observed, running an intraosseous course in the inferior and posterior direction up to a foramen located in the hard palate, slightly medial in relation to tooth 23. The location of this branching, as well as its neurovascular component, is important for dental implant planning because of its proximity to the upper teeth. Identification of neurovascular bundles is fundamental to avoid complications for the patient.

Estudo imunoistoquímico de componentes da Via Sonic Hedgehog, Stat3 e Mcm3 em tumores de glândula salivar / Immunohistochemical study components of Via Sonic Hedgehog, Stat3 and MCM3 in salivary gland tumors

O adenoma pleomórfico (AP), o carcinoma mucoepidermóide (CME) e o carcinoma adenóide cístico (CAC) representam tumores frequentes em glândula salivar. A via de sinalização Sonic Hedgehog (Hh) e o Transdutor de sinal e ativador da transcrição 3 (STAT3) desempenham funções importantes na proliferação celular, favorecendo o desenvolvimento tumoral e a proteína MCM3 tem sido considerada uma nova classe de marcadores de proliferação celular. Portanto, o presente trabalho propõe-se a estudar componentes da via Hh, bem como o STAT3 e o MCM3 em neoplasias de glândula salivar, na tentativa de adicionar informações sobre as características biológicas dessas neoplasias. Foram utilizados 9 casos de AP, 17 casos de CAC e 20 casos de CME e, por meio da técnica imunoistoquímica, realizou-se a detecção das seguintes proteínas: SHH, GLI1, SUFU, HHIP, STAT3 e MCM3. No AP, observou-se alta expressão citoplasmática de SHH e SUFU, e baixa expressão de STAT3 e MCM3. No CAC, observou-se alta expressão de GLI1, HHIP e STAT3 e baixa expressão de SHH, SUFU e MCM3. No CME, observou-se alta expressão de SHH, GLI1, SUFU e HHIP e baixa expressão de STAT3 e MCM3. Quando comparado entre os tipos tumorais, observou-se diferença estatisticamente significante para expressão de SHH (p=0.0064), STAT3 (p=0.0003) e MCM3 (p=0.0257)...

The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC) and the adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands. The Sonic Hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The MCM3 protein has been considered as a novel class of cell proliferation markers. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 and MCM3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC and 20 cases of MEC. Using immunohistochemistry, were investigated: SHH, GLI1, Sufu, HHIP, STAT3 and MCM3. In PA, there was high expression of cytoplasmic SHH and Sufu, and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP and STAT3 and low expression of SHH, SUFU and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p=0.0064), STAT3 (p=0.0003) and MCM3 (p=0.0257) when all tumors were compared...

Density of mast cells and microvessels in minor salivary gland tumors.

The aim of this study was to investigate the density of mast cells and microvessels in minor salivary gland tumors. Forty-one cases of minor salivary gland tumors (pleomorphic adenoma, n = 10; adenoid cystic carcinoma, n = 11; mucoepidermoid carcinoma, n = 10; and polymorphous low-grade adenocarcinoma) were investigated using immunohistochemistry for mast cell tryptase and von-Willebrand factor. Density of mast cells was higher in mucoepidermoid carcinoma; however, no differences in the number of these cells were observed between the different types of tumors (p > 0.05). The number of mast cells was higher in periparenchymal areas in all tumors, but the difference was not significant (p > 0.05). Mucoepidermoid carcinoma showed the largest number of periparenchymal mast cells, whereas pleomorphic adenomas showed the smallest number of intraparenchymal mast cells (p > 0.05). The highest microvessel density was observed in mucoepidermoid carcinomas, being this difference statistically significant when mucoepidermoid carcinoma was compared to pleomorphic adenoma (p = 0.0034) and polymorphous low-grade adenocarcinoma (p = 0.004). Microvessel density was significantly higher in adenoid cystic carcinoma when compared to pleomorphic adenoma (p = 0.0406) and polymorphous low-grade adenocarcinoma (p = 0.0123). Comparison of mast cells and microvessel densities showed no significant difference between tumors. A quantitative difference in mast cells and microvessels was observed, particularly in mucoepidermoid carcinoma, a finding supporting the aggressive behavior of malignant salivary gland tumors without myoepithelial differentiation. Further studies are needed to determine the role of mast cells in angiogenesis, as well as in the development and biological behavior of these tumors.

Queilite actínica: um estudo de 35 casos com destaque para os aspectos morfológicos / Actinic cheilitis: a study of 35 cases highlighting their morphological features

A queilite actínica (QA) é uma alteração crônica, pré-maligna, resultante da exposição solar frequente e prolongada. Afeta principalmente o lábio inferior de pessoas idosas, do sexo masculino e de pelo clara. Sabendo-se que a QA é influenciada pela radiação solar e que ao nosso conhecimento não existem dados clínicos e histológicos dessa lesão no Nordeste do Brasil, o presente estudo analisou uma série de 35 casos de QA na população dessa região brasileira. As lâminas referentes a cada caso foram coradas pela técnica da hematoxilina e eosina (HE) e analisadas por microscopia de luz, sendo descritos os aspectos morfológicos das lesões. Os dados clínicos foram obtidos a partir das fichas de solicitação do exame anatomopatológico. Dos 35 caos de nossa amostra, 60 por cento representou o sexo masculino. A idade média foi de 54,6 anos. A maioria dos pacientes era leucoderma. As lesões de QA caracterizavam-se por epitélio pavimentoso estratificado exibindo graus variáveis de queratinização. Elastose solar esteve presente em todos os casos. Quanto ao grau de displasia, 17 casos apresentavam displasia discreta, 13 eram de displasia moderada e 5 eram representados por displasia severa. Embora os casos desse estudo apresentassem displasia epitelial, estas não foram suficientemente severas para gerar uma neoplasia invasiva. Contudo, é importante que estudos dessa natureza sejam realizados em outras regiões do país para que políticas públicas sejam estabelecidas para evitar o aparecimento e malignização dessas lesões.

Distribution of mast cells in benign odontogenic tumors.

The aim of this study was to investigate the presence of mast cells in a series of odontogenic tumors. Forty-five cases of odontogenic tumors were investigated using immunohistochemistry for mast cell triptase, and differences between groups were statistically evaluated. Mast cells were present in 96% of odontogenic tumors. Mast cells present in solid ameloblastoma were observed in the tumor stroma surrounding more solid and follicular epithelial islands, with or without squamous metaplasia. The odontogenic mixoma showed few mast cells. In odontogenic tumors with a cystic structure, the mast cells were distributed throughout all areas of the lesions, mainly in keratocystic odontogenic tumor. In addition, the total density of mast cells between all odontogenic tumors showed no significant difference (p > 0.05). A greater mast cells distribution was found in keratocystic odontogenic tumor in relation to adenomatoid odontogenic tumor (p < 0.01), and when the unicystic ameloblastoma and keratocistic odontogenic tumor were compared to the odontogenic myxoma (p < 0.05). Syndrome keratocystic odontogenic tumor showed a higher mean of mast cells when compared with the other tumors of the sample. Mast cells values presented by syndrome keratocystic odontogenic tumor were significantly greater than those of the sporadic keratocystic odontogenic tumor that were not associated with the syndrome (p = 0.03). Mast cells are probably one of the major components of the stromal scaffold in odontogenic tumors. We found significant differences of mast cells between syndrome nonsyndrome keratocystic odontogenic tumors, although their distribution did not seem to have any influence on the biologic behavior of benign odontogenic tumors.

Orthokeratinized odontogenic cyst: a report of two cases in the mandible / Cisto odontogênico ortoqueratinizado: relato de dois casos em mandíbula

PURPOSE: To describe the behavior clinical and pathological features of two cases of odontogenic cyst orthokeratinized. CASE DESCRIPTION: In case 1, a 17-year-old woman presented hard and painless swelling in the left posterior mandible. Radiographically, a radiolucent area with a radiopaque was observed. A clinical diagnosis of ameloblastoma or odontogenic keratocystic tumor was established. The histopathological examination revealed an orthokeratinized odontogenic cyst. In case 2, a 23-year-old woman presented a radiolucent lesion surrounded by radiopaque halo symmetrically distributed on both partially erupted lower third molars. A clinical diagnosis of bilateral dentigerous cyst (DC) was rended. The histopathological examination showed bilateral orthokeratinized odontogenic cyst. CONCLUSION: Orthokeratinized odontogenic cyst should be considered in the differential diagnosis of lesions occurring in the jawbones associated with an impacted tooth, particularly those cases simulating dentigerous cyst. In addition, we observed the importance of radiographs taken prior to orthodontic treatment as an important tool in the diagnosis of oral pathologies. Performing routine radiograph is of high clinical value, especially before orthodontic treatment.

OBJETIVO: Descrever os aspectos clinicopatológicos de dois casos de cisto odontogênico ortoqueratinizado e a compará-los a relatos anteriores. RELATO DO CASO: No caso 1, uma mulher de 17 anos de idade, apresentou edema endurecido e indolor na mandíbula posterior esquerda. Radiograficamente, uma área radiolúcida com uma radiopacidade foi observada. O diagnóstico clínico de ameloblastoma ou tumor odontogênico ceratocístico foi estabelecido. O exame histopatológico revelou um cisto odontogênico ortoqueratinizado. No caso 2, uma mulher de 23 anos apresentou duas lesões radiolúcidas circundadas por halos radiopacos simetricamente distribuídas, associada aos terceiros molares inferiores irrompidos. Um diagnóstico clínico de cisto dentígero bilateral (DC) foi estabelecido. O exame histopatológico mostrou ser um cisto odontogênico ortoqueratinizado. CONCLUSÃO: Cisto odontogênico ortoqueratinizado deve ser considerado no diagnóstico diferencial das lesões que ocorrem na mandíbula associado com dentes impactados, especialmente aqueles casos que simulem o cisto dentígero. Além disso, observamos a importância de tomadas radiográficas antes do tratamento ortodôntico no diagnóstico de patologias bucais.