Aline Corrêa Abrahão

A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies.

Immunohistochemical expression of p53, p16 and hTERT in oral squamous cell carcinoma and potentially malignant disorders.

Oral carcinogenesis is a multi-step process. One possible step is the development of potentially malignant disorders known as leukoplakia and erytroplakia. The objective of this study was to use immunohistochemistry to analyze the patterns of expression of the cell-cycle regulatory proteins p53 and p16(INK4a) in potentially malignant disorders (PMD) of the oral mucosa (with varying degrees of dysplasia) and in oral squamous cell carcinomas (OSCC) to correlate them with the expression of telomerase (hTERT). Fifteen PMD and 30 OSCC tissue samples were analyzed. Additionally, 5 cases of oral epithelial hyperplasia (OEH) were added to analyze clinically altered mucosa presenting as histological hyperplasia without dysplasia. p53 positivity was observed in 93.3% of PMD, in 63.3% of OSCC and in 80% of OEH. Although there was no correlation between p53 expression and the grade of dysplasia, all cases with severe dysplasia presented p53 suprabasal immunoexpression. p16(INK4a) expression was observed in 26.7% of PMD, in 43.3% of OSCC and in 2 cases of OEH. The p16(INK4a) expression in OEH, PMD and OSCC was unable to differentiate non-dysplastic from dysplastic oral epithelium. hTERT positivity was observed in all samples of OEH and PMD and in 90% of OSCC. The high hTERT immunoexpression in all three lesions indicates that telomerase is present in clinically altered oral mucosa but does not differentiate hyperplastic from dysplastic oral epithelium. In PMD of the oral mucosa, the p53 immunoexpression changes according to the degree of dysplasia by mechanisms independent of p16(INK4a) and hTERT.

NFκB mediates cisplatin resistance through histone modifications in head and neck squamous cell carcinoma (HNSCC)

Cisplatin‐based chemotherapy is the standard treatment of choice for head and neck squamous cell carcinoma (HNSCC). The efficiency of platinum‐based therapies is directly influenced by the development of tumor resistance. Multiple signaling pathways have been linked to tumor resistance, including activation of nuclear factor kappa B (NFκB). We explore a novel mechanism by which NFκB drives HNSCC resistance through histone modifications. Post‐translational modification of histones alters chromatin structure, facilitating the binding of nuclear factors that mediate DNA repair, transcription, and other processes. We found that chemoresistant HNSCC cells with active NFκB signaling respond to chemotherapy by reducing nuclear BRCA1 levels and by promoting histone deacetylation (chromatin compaction). Activation of this molecular signature resulted in impaired DNA damage repair, prolonged accumulation of histone γH2AX and increased genomic instability. We found that pharmacological induction of histone acetylation using HDAC inhibitors prevented NFκB‐induced cisplatin resistance. Furthermore, silencing NFκB in HNSCC induced acetylation of tumor histones, resulting in reduced chemoresistance and increased cytotoxicity following cisplatin treatment. Collectively, these findings suggest that epigenetic modifications of HNSCC resulting from NFκB‐induced histone modifications constitute a novel molecular mechanism responsible for chemoresistance in HNSCC. Therefore, targeted inhibition of HDAC may be used as a viable therapeutic strategy for disrupting tumor resistance caused by NFκB.

Clinical, histological and immunohistochemical features of ectomesenchymal chondromyxoid tumor

OBJECTIVE Ectomesenchymal chondromyxoid tumor is a rare oral soft tissue neoplasm that should be differentiated from other neural and chondromyxoid entities. The aim of this study was to report the clinical, histological, and immunohistochemical features of 3 additional cases of this condition. METHODS Clinical data were obtained from the clinical records and all cases were evaluated through light microscopy and immunohistochemistry to cytokeratins, vimentin, S100 protein, desmin, smooth muscle actin, and glial fibrilary acidic protein. RESULTS All 3 cases affected the tongue as a long-lasting submucosal swelling and were managed through conservative surgery. They all showed myxoid and chondroid histological patterns, and vimentin, S100, and glial fibrilary acidic protein immunoexpression. CONCLUSIONS These findings reinforce the typical features of ectomesenchymal chondromyxoid tumor previously described, helping to confirm and establish the clinical, histopathological, and immunohistochemical profile of this uncommon lesion.

Different expression patterns of pAkt, NF-κB and cyclin D1 proteins during the invasion process of head and neck squamous cell carcinoma: an in vitro approach.

BACKGROUND Several signaling pathways are involved in the progression of squamous cell carcinoma. Among them, activated PI3K/Akt may result in NF-κB nuclear translocation, thus leading to the transcription of genes enrolled in cellular invasion and proliferation, such as cyclin D1. This study sought to evaluate the expression of pAkt, NF-κB and cyclin D1 proteins in head and neck squamous cell carcinoma cell lines and their respective in vitro-obtained invasive clones. METHODS Squamous cell carcinoma cell lines originating from the tongue, pharynx and the metastatic lymph node were submitted to an in vitro invasion assay to select invasive clones. All experimental groups were submitted to immunofluorescence and Western blot assays. Statistical analysis was performed through a Students t-test with a significance level of 5%. RESULTS The pAkt and NF-κB expression differed from cytoplasm and nucleus depending on the studied cell line. The invasive clone from the tongue presented a network-like structure of pAkts cytoplasmic expression. This lineage as well as the invasive clone from pharynx also showed pAkt and NF-κB nuclear transportation. Significant pAkt and NF-κB increases were observed in the tongue and pharynx invasive clones. Cyclin D1 was detected in the nucleus of all studied cells and was significantly enhanced in the invasive clones from tongue and pharynx. CONCLUSION This study suggests the participation of pAkt, NF-κB and cyclin D1 in the invasion process of head and neck squamous cell carcinoma. Moreover, cytoplasmic pAkt network-like structure was probably related to cytoskeleton changes presented during invasion.

Recurrent bilateral gingival peripheral calcifying epithelial odontogenic tumor (Pindborg tumor): A case report

Calcifying epithelial odontogenic tumor (CEOT) is an extremely rare, benign neoplasm, accounting for approximately 1% of all odontogenic tumors. Peripheral CEOTs commonly resemble oral hyperplastic or reactive lesions and are histologically similar to their intraosseous counterparts. We report an unusual case of multifocal peripheral CEOT. A 40-year-old female presented with bilateral soft, painful, erythematous, gingival swellings localized in premolar areas of the mandibular gingiva. The presumptive diagnosis was bilateral pyogenic granuloma. The masses were surgically excised under local anesthesia without bone curettage and both recurred 12 months later. Morphologic features, and histochemical and immunohistochemical tests revealed bilateral peripheral calcifying odontogenic epithelial tumor. There is no clinical or radiographic evidence of recurrence 3.5 years after excision. This multifocal phenomenon has been reported previously only for intraosseous CEOT. Gingival masses must be carefully evaluated for clinical and histologic evidence of neoplasia.

Assessment of Bone Healing in Rabbit Calvaria Grafted with Three Different Biomaterials

This study evaluated the bone regeneration process in rabbit calvaria induced by three types of biomaterials: two xenogenous, consisting of deproteinized bovine bone, while the other was alloplastic, based on biphasic calcium phosphate. Five New Zealand white rabbits weighing between 2,900 and 3,500 g were submitted to four standard 8 mm-diameter perforations at the parietal bone. Three perforations were filled with three grafts and biomaterials, two of them received bovine Bio-Oss® and Endobon® Xenograft Granules, and the other consisted of fully alloplastic Straumann® Bone Ceramic. The fourth remaining cavity was used as control with coagulum. After eight weeks, the animals were sacrificed, and the samples were prepared for morphometric and qualitative analysis. The cavities filled with alloplastic biomaterials showed higher percentages of newly formed bone (p<0.05), while the cavities with xenogenous biomaterials showed higher amount of residual graft (p<0.05). Although the results showed greater bone formation with Straumann® Bone Ceramic, further studies are required to prove which is the more effective biomaterial for bone induction process.

Clinicopathologic and immunohistochemical features of five new cases of solitary fibrous tumor of the oral cavity

OBJECTIVE To describe the clinicopathologic and immunohistochemical features of five cases of oral solitary fibrous tumor. STUDY DESIGN Clinical data were collected from charts of two oral pathology laboratories of Latin America. All cases were evaluated by conventional hematoxylin and eosin staining and an extended immunohistochemical panel comprising vimentin, CD34, CD99, bcl-2, HHF-35, smooth muscle actin, calponin, S-100 protein, h-caldesmon, and Ki-67. RESULTS The study included 1 male (20%) and 4 female (80%) patients, with a median age of 43 years. The most common affected site was the buccal mucosa (40%). Tumors were characterized by proliferation of spindled and ovoid cells in a variably vascular and collagenized stroma. All tumors were positive for vimentin, CD34, bcl-2, and CD99. Recurrence was not observed after complete surgical enucleation. CONCLUSIONS Oral solitary fibrous tumors usually appear as well-delimited submucous nodules with a firm-rubbery consistency and covered by intact mucosa. Immunoreactivity for CD34, bcl-2, and CD-99 is helpful to confirm the diagnosis.

Effects of celecoxib treatment over the AKT pathway in head and neck squamous cell carcinoma

BACKGROUND Celecoxib, a non-steroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2 (COX-2), has shown an important anticarcinogenic effect for the treatment of squamous cell carcinoma. The use of COX-2 inhibitors has effectively inhibited the growth of Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines, while a recent phase 1 trial demonstrated good response rate of cancer cells to this drug with minimal toxicity. Possible targets of celecoxib include proteins involved in cell proliferation and apoptosis control. Additionally, celecoxib antitumoral activity has been linked with a COX-2-independent event. METHODS To better understand which cellular mechanisms are targeted by celecoxib, its effects upon the Akt signaling pathway using two different HNSCC cell lines were analyzed through cell viability assay, immunofluorescence, and Western blotting. RESULTS The results showed decreased levels of Cyclin D1 and pAkt protein expression in vitro. The number of viable cells was also diminished after celecoxib treatment. CONCLUSION As Akt pathway seems to be a valuable target for the HNSCC therapy, the results presented herein confirm that celecoxib can be considered as an alternative adjuvant drug for HNSCC treatment.

Clinicopathological characteristics of tumours of the intraoral minor salivary glands in 170 Brazilian patients.

Tumours of the minor salivary glands are relatively uncommon, and publications from around the world normally include tumours of both the minor and major salivary glands, making it difficult to assess their prevalence and distribution. Our aim was to evaluate retrospectively the clinicopathological features of a series of tumours of the intraoral minor salivary glands from two universities in Rio de Janeiro, Brazil, and to compare the data with those from other epidemiological studies. A total of 170 such tumours were diagnosed from 1942 to 2012, and were selected from two university departments of oral pathology. Eighty-nine of the tumours were benign (52%). Pleomorphic adenoma (n=75) and mucoepidermoid carcinoma (n=23) were the most common benign (44%) and malignant tumours (14%), respectively. There were 104 female patients (61%) and both benign and malignant tumours affected more women than men. Significantly more tumours were in the palate (n=95, 56%; p=0.001). We conclude that these tumours had features similar to those from other studies from North and Latin America, but differ from the results presented from Asia. Further studies should be designed to highlight possible geographical and population-specific characteristics of these tumours.