Differential expression of galectin-3, beta-catenin, and cyclin D1 in adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma of salivary glands

Background: Galectin-3 has been implicated in tumor progression of some malignancies as thyroid, prostate, and salivary gland tumors. Recently, it has been suggested that this protein may be an important mediator of the beta-catenin/Wnt pathway. Moreover, nuclear galectin-3 expression has been implicated in cell proliferation, promoting cyclin D1 activation. Thus, the present study aimed to correlate galectin-3 expression with beta-catenin and cyclin D1 expressions in adenoid cystic carcinoma (ACC) and in polymorphous low-grade adenocarcinoma (PLGA). Methods: Fifteen formalin-fixed paraffin-embedded cases of each tumor were retrieved from the files of the Surgical Oral Pathology Service at the University of Sao Paulo and the proteins were analyzed by immunohistochemistry. Results: Adenoid cystic carcinoma showed galectin-3 immunostaining mainly in the nuclei, while PLGA revealed a positive mostly cytoplasmic reaction to galectin-3 in the largest part of tumor cells. Both tumors showed intense cytoplasmic/nuclear staining for beta-catenin in majority of cases. Cyclin D1 immunoreactivity was not detected in 14/15 PLGA and showed specific nuclear staining in 10/15 cases of ACC in more than 5% of the neoplastic cells. Cyclin D1 expression was correlated with cytoplasmic and nuclear galectin-3 expression in ACC (P < 0.05). Conclusions: These results suggest that in ACC galectin-3 may play a role in cellular proliferation through cyclin D1 activation. In addition, nuclear expression of galectin-3 in ACC may be related to a more aggressive behavior of this lesion. Although beta-catenin seems to play a role in carcinogenesis in both lesions, it seems that it does not bind to galectin-3 for cyclin D1 stimulation.

WNT-5A, but not matrix metalloproteinase 3 or beta-catenin protein, expression is related to early stages of lip carcinogenesis

Background: Oncogenic Wnt/beta-catenin signaling occurs in numerous types of cancers, but little is known about the role of the Wnt protein family member, WNT-5A, in lip carcinogenesis. The aim of this study was to investigate WNT-5A, beta-catenin, and matrix metalloproteinase (MMP)-3 protein expression in actinic cheilitis (AC), and lip squamous cell carcinoma (LSCC). Methods: Twenty-one cases of AC, and fifty-one cases of LSCC were analyzed, with normal lip mucosa used as a control. Qualitative and semi-quantitative analyses of WNT-5A, beta-catenin, and MMP-3 immunostaining pattern and cellular distribution were performed. Results: WNT-5A was observed in more than 50% of the cells, scattered in all layers of AC, in contrast to the absence of immunostaining in normal lip mucosa. AC presented a higher level of WNT-5A expression than LSCC (P = 0.0289, Fisher test), while MMP-3 immunoexpression was statistically more significant in LSCC than in AC (P = 0.0285, Fisher test). Immunolabeling of beta-catenin protein was differentially distributed between samples; the majority of AC cases (61.90%) demonstrated a membranous-cytoplasmic pattern, while a considerable number of LSCC cases (29.41%) revealed a cytoplasmic pattern, instead of the usual membranous pattern. Conclusions: The present results suggest that WNT-5A may be an important marker during initial events of AC malignant transformation, in which non-canonical and canonical Wnt/beta-catenin signaling pathways could be involved. Additionally, WNT-5A might recruit other events in LSCC, such as MMP-3 protein synthesis, as its presence is increased in established malignant processes without beta-catenin dependency.

CASPASE EXPRESSION IN ORAL SQUAMOUS CELL CARCINOMA

Background. Apoptosis is a genetically programmed form of cell death, of which caspases are the central components. Methods. By tissue microarray of 229 cases of oral squamous cell carcinoma (OSCC), we analyzed the immunoexpression of caspases 3, 6, 7, 8, 9, and 10. Results. All proteins that we examined were expressed in primary OSCC samples. Caspases 8 and 9 were prominently expressed, and caspases 3, 6, 7, and 10 were occasionally expressed. Disease-free survival differed significantly between caspase 7 high-expressing and low-expressing patients, and our multivariate analysis suggested that expression of caspase 7 is an independent prognostic factor for patients with OSCC. Conclusion. This study suggests that caspases regulate the tumorigenesis of OSCC and that caspase 7 expression is a predictor of locoregional recurrence of OSCC. (C) 2010 Wiley Periodicals, Inc. Head Neck 33: 1191-1198, 2011

Expression of proteins in the extracellular matrix of pulp tissue in human primary teeth during physiologic root resorption

Objectives: To analyze the expression of tenascin, fibronectin, collagens I and III, osteonectin, and bone morphogenetic protein 4 (BMP4) in the extracellular matrix of pulp tissue in primary teeth during physiologic root resorption. Method and Materials: Eighteen teeth were decalcified and equally distributed into 3 groups (group I, teeth with two-thirds root length; group II, teeth with one-third root length; and group III, teeth lacking the root). Results: Immunohistochemical analysis showed that all the proteins were expressed. Tenascin, collagen I, and osteonectin showed strong and broad reactivity in group I, with weaker and rare reactivity in groups II and III. The expression of fibronectin, collagen III, and BMP4 did not vary with root resorption phase. Conclusion: The expression of tenascin, collagen I, and osteonectin was reduced in the extracellular matrix and odontoblasts during root resorption. This fact may be related to the decreasing pulp response to damage and treatment during the progression of root resorption. (Quintessence Int 2009; 40: 553-558)

Substance P regulates the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in cultured human gingival fibroblasts

Background and Objective: Substance P may play a role in the pathogenesis of periodontal disease; however, its mechanisms of modulation are not clear. This study evaluated the effect of two concentrations of Substance P on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cultured human gingival fibroblasts. Materials and Methods: Fibroblasts were stimulated for 48 h with 10(-4) or 10(-9) m Substance P; untreated fibroblasts served as controls. The expression of MMP-1, -2, -3, -7 and -11 and of TIMP-1 and -2 was evaluated using real-time polymerase chain reaction and western blotting. Resulsts: There was a significant, concentration-dependent stimulatory effect of Substance P on MMP-1, -2, -3 and -7 and TIMP-2 gene expression (p < 0.05), and a probable effect on MMP-11 (p = 0.06). At the higher concentration (10(-4) m Substance P), MMP-1, -2, -3, -7 and -11 and TIMP-2 showed the greatest up-regulation; at the lower concentration (10(-9) (M) Substance P), MMP-1, -3 and -7 and TIMP-2 exhibited diminished up-regulation, with MMP-2 and -11 showing down-regulation (p < 0.05). Expression of TIMP-1 was not affected by Substance P (p > 0.05). Western blotting confirmed that Substance P up-regulated MMP-1, -2, -3 and -11 and TIMP-2. MMP-1, -3 and -11 and TIMP-2 showed greater up-regulation at the higher Substance P concentration and diminished up-regulation at the lower concentration. MMP-2 was up-regulated to a similar degree at both Substance P concentrations. Conclusion: In gingival fibroblast cells, Substance P at the higher concentration (10(-4) m) induced greater up-regulation of MMP-1, -3 and -11 and TIMP-2 expression, but at the lower concentration (10(-9) (M)) induced diminished up-regulation, which may represent a mechanism for modulating periodontal breakdown.

STAT3 expression in salivary gland tumours

The aim of the present study was to evaluate the signal transducer and activator of transcription (STAT3) expression, which is constitutively active in different types of malignant tumours, in salivary gland tumours. Fifty biopsies of salivary gland tumours (9 pleomorphic adenomas, 12 adenoid cystic carcinomas, 7 epithelial-myoepithelial carcinomas, 10 polymorphous low-grade adenocarcinomas and 12 mucoepidermoid carcinomas) and 10 normal. salivary glands were immunohistochemically labeled for STAT3 and Phospho-STAT3 (STAT3P). The labeled sections were quatitatively and quantitatively evaluated. The results showed that, in normal salivary gland, STAT3 was expressed in cytoplasm and STAT3P in nuclei of all tissue cells, except in large mucous acinar cells for which both antibodies were negative. In pleomorphic adenoma, the expression was the same as in normal glands. In malignant tumours, there were variations in the expression of these antibodies. The most important one was the presence of STAT3 in the nuclei of the malignant tumour cells, most evident in the cribriform-type of adenoid cystic carcinoma. Both toss and variation of STAT3P expression were also observed. The presence of STAT3 in the nuclei of malignant salivary gland tumours may represent an important event in oncogenesis probably contributing to tumour cell proliferation white blocking apoptosis. However, further investigation will. be necessary to support this hypothesis. (c) 2007 Pubtished by Elsevier Ltd.

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Enhanced programmed death 1 (PD-1) and PD-1 ligand (PD-L1) expression in patients with actinic cheilitis and oral squamous cell carcinoma

PD-1 and PD-L1 can be involved in tumor escape, and little is known about the role of these molecules in oral tumors or pre-malignant lesions. In the present study, we investigated the expression of PD-1 and PD-L1 in the blood and lesion samples of patients with actinic cheilitis (AC) and oral squamous cell carcinoma (OSCC). Our results showed that lymphocytes from peripheral blood and tissue samples exhibited high expression of PD-1 in both groups analyzed. Patients with AC presented higher percentage as well as the absolute numbers of CD4(+)PD-1(+) and CD8(+)PD-1(+) lymphocytes in peripheral blood mononuclear cells (PBMC) than healthy individuals, while patients with OSCC presented an increased frequency of CD8(+)PD1(+) in PBMC when compared with controls. On the other hand, increased frequency of CD4(+) and CD8(+) T cells expressing PD-1(+) accumulate in samples from OSCC, and the expression of PD-L1 was intense in OSCC and moderate in AC lesion sites. Lower levels of IFN-gamma and higher levels of TGF-beta were detected in OSCC samples. Our data demonstrate that PD-1 and PD-L1 molecules are present in blood and samples of AC and OSCC patients. Further studies are required to understand the significance of PD-1 and PD-L1 in oral tumors microenvironment.

The broad effects of the functional IL-10 promoter-592 polymorphism: modulation of IL-10, TIMP-3, and OPG expression and their association with periodontal disease outcome

Periodontal diseases are infectious diseases, in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. It occurs through the generation of metalloproteinases and the activation of bone resorption mechanisms. Anti-inflammatory cytokines such as IL-10 seem to attenuate periodontal tissue destruction through the induction of tissue inhibitors of metalloproteinases (TIMPs) and the inhibitor of osteoclastogenesis osteoprotegerin (OPG). A high individual variation in levels of IL-10 mRNA is verified in periodontitis patients, which is possibly determined by genetic polymorphisms. In this study, the IL-10 promoter -592C/A single nucleotide polymorphism ( SNP), which is associated with a decrease in IL-10 production, was analyzed by RFLP in 116 chronic periodontitis (CP) patients and 173 control (C) subjects, and the IL-10, TIMPs, and OPG mRNA expression levels in diseased gingival tissues were determined by real-time-PCR. The IL-10-592 SNP CA (P=0.0012/OR=2.4/CI:1.4-4.1), AA (P=0.0458/OR=2.3/CI:1.1-4.9), and CA+AA (P=0.0006/OR=2.4/CI: 1.4-3.4) genotypes and the allele A (P=0.0036/OR=1.7/CI:1.2-2.4) were found to be significantly more prevalent in the CP group when compared with control subjects. Both CA and AA genotypes were associated with lower levels of IL-10, TIMP-3, and OPG mRNA expression in diseased periodontal tissues and were also associated with disease severity as mean pocket depth. Taken together, the results presented here demonstrate that IL10-592 SNP is functional in CP, being associated with lower levels of IL-10 mRNA expression, which is supposed to consequently decrease the expression of the downstream genes TIMP-3 and OPG, and influence periodontal disease outcome. J. Leukoc. Biol. 84: 1565-1573; 2008.

Expression of placental glutathione S-transferase in rat tongue mucosa exposed to cigarette smoke

Glutatione S-transferases (GSTs) are a family of enzymes involved in detoxification of xenobiotics. Placental GST, known as GST-P, has been detected in tissues following exposure to carcinogenic agents being regarded a reliable biomarker of exposure and susceptibility in early phases of carcinogenesis. The aim of this study was to investigate the expressivity of GST-P positive foci in the rat tongue mucosa exposed to cigarette smoke by means of immunohistochemistry. A total of twelve male Wistar rats were distributed into two groups: negative control and experimental group exposed to cigarette smoke during 75 days. After experimental period, no histopathological changes in the tongue mucosa were evidenced in the negative control and the experimental group. However, a total of five GST-P positive foci were detected in two out of six animals exposed to cigarrette smoke. None control animals were noticed GST-P positive foci. These data indicate that expression of GST-P may reflect the carcinogenic effect of cigarette smoke as well as the genetic susceptibility of animals in relation to continuous carcinogens exposure.

Expression analysis of matrix metalloproteinase-9 in epithelialized and nonepithelialized apical periodontitis lesions

Objective. The objective of this study was to determine the expression of matrix metalloproteinase-9 (MMP-9) in apical periodontitis lesions. Study design. Nineteen epithelialized and 18 nonepithelialized apical periodontitis lesions were collected after periapical surgery. After histological processing, serial sectioning, H&E staining, and microscopic analysis, 10 epithelialized and 10 nonepithelialized lesions were selected for immunohistochemical analysis for MMP-9 and CD 68. At least one third of each specimen collected was frozen at -70 degrees C for further mRNA isolation and reverse transcription into cDNA for real-time-PCR procedures. Geometric averaging of multiple housekeeping genes normalized MMP-9 mRNA expression level. Results. Polymorphonuclear neutrophils, macrophages and lymphocytes presented MMP-9 positive immunostaining in both types of lesions. When present, epithelial cells were also stained. The number and the ratio of MMP-9(+)/total cells were greater in nonepithelialized than epithelialized lesions (P = .0001) presenting a positive correlation to CD68(+)/total cells (P = .045). Both types of lesions presented increased MMP-9 expression (P < .0001) when compared to healthy periapical ligaments. However, no significant differences were observed for MMP-9 mRNA expression between ephithelized and nonephithelized lesions. Conclusion. The present data suggest the participation of several inflammatory cells, mainly CD68(+) cells, in the MMP-9 expression in apical periodontitis lesions. MMP-9 could be actively enrolled in the extracellular matrix degradation in apical periodontitis lesions. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 107: 127-132)

Differential patterns of receptor activator of nuclear factor kappa B ligand/osteoprotegerin expression in human periapical granulomas: Possible association with progressive or stable nature of the lesions

Receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) are expressed in apical periodontitis, suggesting a role for these molecules during lesion development. However, the profiles of RANKL/OPG expression in periapical lesions remain unknown. In this study we investigated the patterns of RANKL and OPG mRNA expression by real-time polymerase chain reaction in human periapical granulomas (N = 44) and compared them with sites presenting characteristic bone resorbing activity: healthy (n = 14) and orthodontically stretched and compressed periodontal ligament (n = 26), healthy gingiva (n = 24), chronic gingivitis (n = 32), and chronic periodontitis (n = 34) samples. Both RANKL and OPG mRNA expression was higher in periapical granulomas when compared with healthy periodontal ligament. Distinct patterns of RANKL and OPG expression ratio were found in the granulomas and in different physiologic and pathologic conditions, with characteristic bone resorption activity potentially being indicative of the stable or progressive nature of the lesions. Lesions with radiographic image smaller than 5 mm showed higher RANKL/OPG expression than images greater than 5 mm. Periapical granulomas presented heterogeneous patterns of RANKL and OPG expression, ranging from samples with RANKL/OPG ratio similar to that seen in sites with minimal or absent bone resorption to samples with RANKL/OPG expression pattern comparable with active bone resorption sites.

Substance P and NK-1R expression in oral precancerous epithelium

The objectives of this study were to investigate the presence and distribution of substance P and neurokinin 1 receptor in oral premalignant epithelium and their relation with the presence of dysplasia, and to analyze whether the expression of substance P can be considered an early oncogenic event in oral carcinogenesis. Substance P and neurokinin I receptor expression was immunohistochemically studied in 83 oral carcinomas and adjacent nontumor epithelia. The presence and degree of epithelial dysplasia was assessed according to WHO criteria. The nuclear, cytoplasmic, and membrane expression of substance P and the cytoplasmic and membrane expression of neurokinin 1 receptor were assessed in tumor and adjacent non-tumor epithelium. Nuclear and cytoplasmic expression of substance P in non-tumor epithelium was significantly associated with the presence of epithelial dysplasia (p<0.001) and carcinoma in. situ (p=0.021). Nuclear, cytoplasmic, and membrane expressions of substance P in non-tumor epithelium were significantly (p<0.001) associated with its expression in the corresponding tumor. These findings suggest that substance P plays a role in early oral carcinogenesis by promoting the proliferation and growth of premalignant fields.

Expression of bone resorption regulators (RANK, RANKL, and OPG) in odontogenic tumors

Objective. To investigate the expression of bone resorption regulators (receptor activator of nuclear factor kappa B [RANK], RANK ligand [RANKL], and osteoprotegerin [OPG]) in calcifying cystic odontogenic tumor (CCOT), adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), odontogenic myxoma (OM), and ameloblastic fibroma (AF). Study design. The expression of these mediators was evaluated by means of immunohistochemistry. Results. All specimens demonstrated positive immunoreactivity to RANK, RANKL, and OPG. The quantification of these mediators in epithelium revealed a similar pattern of expression for RANKL and OPG in CCOT, AOT, CEOT, and AF. With regard to stromal/mesenchymal cells, the majority of AOT and CCOT cases showed a higher content of OPG than RANKL, whereas CEOT, OM, and especially AF had a tendency to present a greater content of RANKL than OPG. Conclusion. Our data indicate that the CCOT, AOT, CEOT, OM, and AF cell constituents express key regulators of bone metabolism that might locally modulate tumor-associated bone resorption. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:548-55)

Expression of matrix metalloproteinases-2 and-9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.