CD44 and ALDH1 immunoexpression as prognostic indicators of invasion and metastasis in oral squamous cell carcinoma.

BACKGROUND: Tumour metastasis has been associated with cancer stem cells, a small population with stem-like cells properties, higher rate of migration and metastatic potential compared to cells from the tumour bulk. Our aim was to evaluate the immunoexpression of the putative cancer stem cell biomarkers ALDH1 and CD44 in primary tumour and corresponding metastatic lymph nodes. METHODS: Tumour tissue specimens (n = 50) and corresponding metastatic lymph nodes (n = 25) were surgically obtained from 50 patients with oral squamous cell carcinoma and submitted to immunohistochemistry. CD44 and ALDH1 were semi-quantitatively scored according to the proportion and intensity of positive cells within the invasive front and metastatic lymph nodes as a whole. A combined score was obtained by multiplying both parameters and later dichotomized into a final score classified as low (≤2) or high (>2) immunoexpression. RESULTS: ALDH1 immunoexpression and CD44 immunoexpression were detected in both tumour sites, although the means of ALDH1 (P = .0985) and CD44 (P = .4220) cells were higher in metastasis compared to primary tumours. ALDH1high was positively associated (P = .0184) with angiolymphatic invasion, while CD44high was positively associated (P = .0181) with metastasis (N+). At multivariate analysis, CD44 significantly increased the odds of lymph node metastasis, regardless of T stage (OR = 8.24; 1.64-65.64, P = .0088). CONCLUSIONS: CD44 immunoexpression was a significant predictor of lymph node metastasis, while ALDH1high immunostaining was associated with angiolymphatic invasion. Altogether, it suggests that immunoexpression of CD44 and ALDH1 links the cancer stem cell phenotype with oral squamous cell carcinoma invasion and metastasis.

Elevation in 5-FU-induced apoptosis in head and neck cancer stem cells by a combination of CDHP and GSK3ß inhibitors.

BACKGROUND: Cancer stem cells (CSCs) are involved in both tumourigenesis and in tumour recurrence after therapy. In head and neck squamous cell carcinoma (HNSCC), there are two biologically different CSC phenotypes both of which express high levels of CD44 but differ in their expression levels of epithelial-specific antigen (ESA). One phenotype is CD44(high)/ESA(high) and has epithelial features (Epi-CSCs), while the other is CD44(high) /ESA(low), has undergone epithelial to mesenchymal transition (EMT-CSCs), has mesenchymal features and is migratory (Biddle et al., 2011). CSCs are resistant to therapeutically induced apoptosis but the molecular mechanisms by which they develop apoptotic resistance remains unclear. However, glycogen synthase kinase 3ß (GSK3ß) contributes to regulation of both the self-renewal and switching of these two CSC phenotypes (Shigeishi et al., 2013). METHODS: CD44(high) /ESA(low), CD44(high) /ESA(high) and CD44(low) cells were FACS sorted from the HNSCC cell line LUC4, and 5-FU-induced apoptosis was analysed by Annexin V staining followed by flow cytometry analysis. RESULTS: CD44(high) /ESA(low) cells exhibited marked resistance to 5-FU-induced apoptosis and had high expression of dihydropyrimidine dehydrogenase (DPD). The DPD inhibitor, 5-chloro-2, 4-dihydroxypyridine (CDHP) significantly enhanced 5-FU-induced apoptosis of CD44(high)/ESA(low) cells. Inhibition of GSK3ß induced CD44(high) /ESA(low) cells to undergo mesenchymal-to-epithelial transition (MET) to CD44(high)/ESA(high) cells and pre-existing CD44(high) /ESA(high) cells to differentiate. Apoptosis induced by 5-FU was thus facilitated. Combination of both CDHP and GSK3ß inhibitors markedly enhanced 5-FU-induced apoptosis of CD44(high) /ESA(low) cells. CONCLUSIONS: Our results suggest potentially new approaches for the elimination of the therapy resistant HNSCC CSC population.

Maintenance of stem cell self-renewal in head and neck cancers requires actions of GSK3ß influenced by CD44 and RHAMM.

Cells sorted from head and neck cancers on the basis of their high expression of CD44 have high potency for tumor initiation. These cells are also involved in epithelial to mesenchymal transition (EMT) and we have previously reported that cancer stem cells (CSCs) exist as two biologically distinct phenotypes. Both phenotypes are CD44(high) but one is also ESA(high) and maintains epithelial characteristics, the other is ESA(low) , has mesenchymal characteristics and is migratory. Examining CD44-regulated signal pathways in these cells we show that CD44, and also RHAMM, act to inhibit phosphorylation of glycogen synthase kinase 3ß (GSK3ß). We show that inhibitory phosphorylation reduces the formation of both "tumor spheres" and "holoclone" colonies, functional indicators of stemness. GSK3ß inhibition also reduces the expression of stem cell markers such as Oct4, Sox2, and Nanog and upregulates expression of the differentiation markers Calgranulin B and Involucrin in the CD44(high) /ESA(high) cell fraction. Transition of CSCs out of EMT and back to the epithelial CSC phenotype is induced by GSK3ß knockdown. These results indicate that GSK3ß plays a central role in determining and maintaining the phenotypes and behavior of CSCs in vitro and are likely to be involved in controlling the growth and spread of tumors in vivo.

Calcifying Cystic Odontogenic Tumour: immunohistochemical expression of matrix metalloproteinases, their inhibitors (TIMPs and RECK) and inducer (EMMPRIN).

BACKGROUND: Calcifying cyst odontogenic tumour (CCOT) is a rare benign cystic neoplasm of odontogenic origin. MMPs are responsible for extracellular matrix remodelling and, together their inhibitors and inducer, determinate the level of its turnover in pathological processes, leading to an auspicious microenvironment for tumour development. Thus, our goal was to evaluate matrix metalloproteinases (MMPs-2, -7, -9 and -14), their inhibitors (TIMPs-2, -3, -4 and RECK) and its inductor (EMMPRIN) expression in CCOT. MATERIALS AND METHODS: We used 18 cases of CCOT submitted to immunolocalization of the target proteins and analysed in both neoplastic odontogenic epithelial and stromal compartments. RESULTS: All molecules evaluated were expressed in both compartments in CCOT. In epithelial layer, immunostaining for MMPs, TIMPs, RECK and EMMPRIN was found in basal, suprabasal spindle and stellate cells surrounding ghost cells and ghost cells themselves, except for MMP-9 and TIMP-2 which were only expressed by ghost cells. In stromal compartment, extracellular matrix, mesenchymal (MC) and endothelial cells (EC) were positive for MMP-2, -7, TIMP-3 and -4, while MMP-9, TIMP-2 and RECK were positive only in MC and MMP-14 only in EC. Statistical significance difference was found between both compartments for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001), being more expressed in epithelium than in stroma. Positive correlation between both stromal EMMPRIN and RECK expression was found (R = 0.661, P = 0.003). CONCLUSIONS: We concluded that these proteins/enzymes are differentially expressed in both epithelium and stroma of CCOT, suggesting an imbalance between MMPs and their inducer/inhibitors may contribute on the tumour behaviour.

Differential expression of Cadherins switch and Caveolin-2 during stages of oral carcinogenesis.

Background: Oral squamous cell carcinoma (OSCC) accounts for 90% of oral malignancies, which may be preceded by oral potentially malignant disorders (OPMDs). Cancer progression involves the downregulation of epithelial markers (E-cadherin) and the upregulation of mesenchymal markers (N-cadherin), which together characterise the epithelial-mesenchymal transition (EMT). Furthermore, caveolin can act on cell adhesion and migration events that regulate the expression of the E-cadherin/α-ß-catenin complex, thus favouring aggressive biological behaviour. This study aimed to analyse the immunoexpression of E-cadherin, N-cadherin and caveolin-2 at different stages of oral carcinogenesis to identify reliable biomarkers to predict malignant potential. Methods: Expressions of E-cadherin and N-cadherin in 14 normal oral mucosae (NOM), 14 OPMD and 33 OSCC specimens were evaluated using immunohistochemistry. Clinicopathological parameters were also assessed. Results: E-cadherin immunoexpression was significantly reduced during the progression of oral carcinogenesis (P = 0.0018). N-cadherin immunoexpression did not show any statistical differences between these groups. However, a representative number of N-cadherin-positive OSCC cases did not express E-cadherin. The expression of caveolin-2 increased significantly with the progression of the disease, from NOM to OSCC (P value: 0.0028). Conclusion: These findings indicate that cadherin switch and caveolin-2 immunoexpression may be regulatory events in oral carcinogenesis.

AT1 receptor antagonism promotes bone loss attenuation in experimental periodontitis, blocks inflammatory mediators, and upregulates antioxidant enzymes and bone formation markers.

BACKGROUND: The initiation and progression of periodontitis might involve a local renin-angiotensin system in periodontal tissue. This study hypothesized that Losartan treatment could promote protection to rats submitted to experimental periodontitis (EP) by attenuating alveolar bone loss due to reduction in inflammatory cytokines, better reactive oxidant species regulation and maintenance of the balance between bone formation and resorption factors. METHODS: One hundred and thirty rats were submitted to EP with a silk suture thread (4.0) placed around the lower right first molar for 1, 3, 7, and 14 consecutive days. The study comprised four groups: G1-control without EP; G2-animals with EP treated with water; G3-Losartan-treated animals (treatment started at the same day of EP induction), and G4-animals previously treated with Losartan for 30 days followed by induction of EP and continuity of treatment. RESULTS: G2 rats had greater bone loss volume, increased number, and thickness and decreased separation of trabeculae. On the other hand, G4 animals showed significant improvements in these parameters. Histological analysis revealed that EP favors inflammatory cell infiltration and junctional epithelium, cementum with alveolar bone crest destruction, but animals pretreated with Losartan (G4) did not show these features. Although the G3 animals did not demonstrate the improvements detected in G4, mRNA expression results were similar. In mandibular tissue, EP promoted mRNA increases for ACE, AT1 receptor, and inflammatory mediators as well as decreases for antioxidant enzymes. However, Losartan treatments attenuated these responses in addition to promoting an increase in bone formation markers and transcription factors. CONCLUSION: AT1 receptor modulates EP progression.

Aggressive case of cherubism: 17-year follow-up.

Cherubism is an autosomal dominant disorder in which the normal bone is replaced by cellular fibrous and immature bone, resulting in painless symmetrical enlargement of the jaws. An aggressive case of cherubism with extensive swelling on several facial bones in a 19-year-old boy is reported. The disorder was diagnosed 15 years ago, but the patient has not been submitted to any type of surgery so far. The highlights of this case are the great proportion of the lesions, the enormous functional and emotional disturbances brought about by these lesions, and the difficulty to choose the most appropriate age and form of treatment.

Immunolocalization of dentin matrix protein-1 in human primary teeth treated with different pulp capping materials.

The aim of this study was to evaluate the immunolocalization of dentin matrix protein (DMP)-1 in human primary teeth treated with different pulp capping materials. Twenty-five primary molars were divided into the following groups: formocresol (FC), calcium hydroxide (CH), mineral trioxide aggregate (MTA), corticosteroid/antibiotic solution + CH (O + CH), and Portland cement (PC), and all received conventional pulpotomy treatment. The teeth at the regular exfoliation period were extracted for histological analysis and immunolocalization of DMP-1. Statistical analysis was performed using the χ(2) test (p < 0.05). Histological analysis revealed statistically significant differences in the comparison among the groups through the use of a score system regarding the presence of hard tissue barrier, odontoblastic layer, and internal resorption, but not regarding pulp calcification. Immunohistochemical analysis showed immunostaining for DMP-1 in groups CH, MTA, O + CH, and PC. Internal resorption was observed in the groups FC and CH. MTA and PC showed pulp repair without inflammation and with the presence of hard tissue barrier. DMP-1 immunostaining was higher for MTA and PC, confirming the reparative and bioinductive capacity of these materials.

Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue.

The initiation or progression of periodontitis might involve a local renin-angiotensin system (RAS) in periodontal tissue. The aim of this study was to further characterize the local RAS in human and rat periodontal tissues between healthy and periodontally-affected tissue. Components of the RAS were investigated using in vitro, ex vivo and in vivo experiments involving both human and Wistar rat periodontium. Although not upregulated when challenged with P. gingivalis-lipopolysaccharide, human gingival and periodontal ligament fibroblasts expressed RAS components. Likewise, healthy and inflamed human gingiva expressed RAS components, some of which were shown to be functional, yet no differences in expression were found between healthy and diseased gingiva. However, in inflamed tissue the immunoreactivity was greater for the AT1R compared to AT2R in fibroblasts. When compared to healthy tissue, ACE activity was increased in human gingiva from volunteers with gingivitis. Human-gingiva homogenates generated Ang II, Ang 1-9 and Ang 1-7 when incubated with precursors. In gingiva homogenates, Ang II formation from Ang I was nearly abolished only when captopril and chymostatin were combined. Ang 1-7 formation was significantly greater when human gingiva homogenates were incubated with chymostatin alone compared to incubation without any inhibitor, only captopril, or captopril and chymostatin. In rat gingiva, RAS components were also found; their expression was not different between healthy and experimentally induced periodontitis (EP) groups. However, renin inhibition (aliskiren) and an AT1R antagonist (losartan) significantly blocked EP-alveolar-bone loss in rats. Collectively, these data are consistent with the hypothesis that a local RAS system is not only present but is also functional in both human and rat periodontal tissue. Furthermore, blocking AT1R and renin can significantly prevent periodontal bone loss induced by EP in rats.

Biocompatibility of Portland cement combined with different radiopacifying agents.

The aim of this study was to evaluate the response of rat subcutaneous tissue to Portland cement combined with two different radiopacifying agents, iodoform (CHI3) and zirconium oxide (ZrO2). These materials were placed in polyethylene tubes and implanted into the dorsal connective tissue of Wistar rats for 7 and 15 days. The specimens were then stained with hematoxylin and eosin, and inflammatory reaction parameters were evaluated by light microscopy. The intensity of the inflammatory response to the sealants was analyzed by two blind calibrated observers throughout the experimental period. Histological analysis showed that all the materials caused a moderated inflammatory reaction at 7 days, which then diminished with time. At 15 days, the inflammatory reaction was almost absent, and fibroblasts and collagen fibers were observed indicating normal tissue healing. The degrees of the inflammatory reaction on different days throughout the experimental period were compared using the non-parametric Kruskal-Wallis test. Statistical analysis demonstrated no significant differences amongst the groups, and Portland cement associated with radiopacifying agents gave satisfactory results. Therefore, Portland cement used in combination with radiopacifying agents can be considered a biocompatible material. Although our results are very encouraging, further studies are needed in order to establish safe clinical indications for Portland cement combined with radiopacifying agents.

CC chemokine ligand 3 and receptors 1 and 5 gene expression in recurrent aphthous stomatitis.

OBJECTIVE: The aim of this study was to investigate the local and systemic expression of CC-chemokine ligand 3 (CCL3) and its receptors (CCR1 and CCR5) in tissue samples and peripheral blood mononuclear cells of recurrent aphthous stomatitis (RAS) patients. STUDY DESIGN: This case-control study enrolled 29 patients presenting severe RAS manifestations and 20 non-RAS patients proportionally matched by sex and age. Total RNA was extracted from biopsy specimens and peripheral blood mononuclear cells for quatitative reverse-transcription polymerase chain reaction. The data obtained by relative quantification were evaluated by the 2(-ΔΔCt) method, normalized by the expression of an endogenous control, and analyzed by Student t test. RESULTS: The results demonstrated overexpression in RAS tissue samples of all of the chemokines evaluated compared with healthy oral mucosa, whereas the blood samples showed only CCR1 overexpression in RAS patients. CONCLUSIONS: These findings suggest that the increased expression of CCL3, CCR1, and CCR5 may influence the immune response in RAS by T(H)1 cytokine polarization.