Detection and activity of MMP-2 and MMP-9 in Leishmania amazonensis and Leishmania braziliensis promastigotes.

Metalloproteinases (MMPs) are remarkable zinc-dependent endopeptidases, critical for degrading components of the extracellular matrix, thus actively influencing cell migration. Their impact on intracellular parasites, such as the enigmatic protozoan Leishmania, elicits intriguing queries. This study explores into the untapped territory of MMP-2 and MMP-9 within Leishmania spp. promastigotes. Notably, we successfully detected and quantified these MMPs, while also evaluating their activity in two distinct Leishmania species-L. amazonensis (La) and L. braziliensis (Lb)-at various growth stages and isolated from distinct clinical tegumentar disease forms. The results unveiled the presence of MMP-2 and MMP-9 in both species, albeit with distinct localization patterns. Specifically, MMP-9 exhibited significantly higher gelatinolytic activity in La when compared to Lb. Moreover, our data cleverly illustrated the presence and release of MMP-2 and MMP-9 by La and Lb promastigotes, exposing their ability to invade and migrate within a collagen matrix. This pioneering study establishes a compelling correlation between MMP-2 and MMP-9 and their potential role in the dynamics of La and Lb infection. Suggesting their potential as prognostic markers for severe leishmaniasis and promising target molecules for therapeutic interventions, this research opens new avenues for combatting this debilitating parasitic disease.

Expression of stem cell markers SALL4, LIN28A, and KLF4 in ameloblastoma.

BACKGROUND: Ameloblastoma (AME) is a benign odontogenic tumour of epithelial origin characterised by slow but aggressive growth, infiltration, and recurrence; it is capable of reaching large dimensions and invading adjacent structures. Stem cell research has proven to be significant in the sphere of tumour biology through these cells' possible involvement in the aetiopathogenesis of this tumour. METHODS: Immunohistochemistry was performed on AME, dentigerous cyst (DC), and dental follicle (DF) samples, and indirect immunofluorescence was performed on the AME-hTERT cell line to determine the expression of SALL4, LIN28A, and KLF4. RESULTS: Expression of proteins related to cellular pluripotency was higher in AME cells than in DC and DF cells. The analysis revealed that the proteins in question were mainly expressed in the parenchyma of AME tissue samples and were detected in the nuclei of AME-hTERT cells. CONCLUSIONS: Stem cells may be related to the origin and progression of AME.

Immunoexpression of stem cell markers SOX-2, NANOG AND OCT4 in ameloblastoma.

Background: Ameloblastoma (AME) is characterized by a locally invasive growth pattern. In an attempt to justify the aggressiveness of neoplasms, the investigation of the role of stem cells has gained prominence. The SOX-2, NANOG and OCT4 proteins are important stem cell biomarkers. Methodology: To verify the expression of these proteins in tissue samples of AME, dentigerous cyst (DC) and dental follicle (DF), immunohistochemistry was performed and indirect immunofluorescence were performed on the human AME (AME-hTERT) cell line. Results: Revealed expression of SOX-2, NANOG and OCT4 in the tissue samples and AME-hTERT lineage. Greater immunostaining of the studied proteins was observed in AME compared to DC and DF (p < 0.001). Conclusions: The presence of biomarkers indicates a probable role of stem cells in the genesis and progression of AME.

Downregulation of metallothionein 2A reduces migration, invasion and proliferation activities in human squamous cell carcinoma cells.

BACKGROUND: The invasive behaviour of squamous cell carcinoma (SCC), a common malignant tumour of the mouth, is a process mediated by cell proliferation, extracellular matrix proteolysis and other factors. Studies have shown a potential relationship between growth factors, metallothionein 2A (MT2A) and matrix metalloproteinase (MMP) activation in malignant tumours. The aim of this study was to downregulate MT2A in cells (Cal27) derived from human squamous cell carcinoma. METHODS: Cal27 cells with reduced MT2A were subjected to proliferation, migration and invasion assays. Immunofluorescence and western blot confirmed MT2A depletion by siRNA. Growth curve assays assessed cell proliferation. Indirect immunofluorescence analysed the expression of MT2A, MMP-2, MMP-9, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), tumour necrosis factor alpha (TNF-α) and Ki67. Zymography evaluated the effects of MT2A silencing on MMP-2 and -9 expression. Migration and invasion activities were evaluated using migration and invasion assays. RESULTS: CAL27 cells displayed MT2A, MMP-2, MMP-9, EGF, TGF-α, TNF-α and Ki67. MT2A depletion decreased MMP-9, EGF, TGF-α and Ki67 protein levels, while increasing TNF-α. CONCLUSIONS: MT2A downregulation reduced cell proliferation, migration and invasion activities. Therefore, MT2A has an important role in cell proliferation, migration and invasion in human oral SCC cells.

Key proteins of invadopodia are overexpressed in oral squamous cell carcinoma suggesting an important role of MT1-MMP in the tumoral progression.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most relevant malignant neoplasm among all head and neck tumours due to its high prevalence and unfavourable prognosis. Tumour invasion and metastasis that affect prognosis are result of a set of complex events that cells with invasive potential use to spread to other regions. These cells use several mechanisms to invade tissues, including a type of finger-like membrane protrusion called invadopodia. This study aims to investigate the immunoexpression of invaopodia related-proteins TKs5, cortactin, TKs4 and MT1-MMP in OSCC and correlate it to clinicopathological data. METHODS: An immunohistochemical evaluation of fifty cases of OSCCs and 20 cases of oral mucosa (OM) were assessed. The expression of invadopodia proteins were analysed in comparison to normal tissue (OM) and correlated to different clinical-stage and histological grade of OSCC. RESULTS: TKs5, cortactin, TKs4 and MT1-MMP were significantly overexpressed in OSCC when compared to OM (p < 0.0001). Among tumour stages, TKs5 showed a statistical difference in immunolabelling between stage I and III (p = 0.026). Cortactin immunolabelling was statistically higher in grade I than in grade II and III. No differences were seen on TKs4 expression based on tumour staging or grading. MT1-MMP was higher expressed and showed statistical difference between stages I and III and grades I compared to II and III. CONCLUSIONS: The invadopodia related-proteins were found to be overexpressed in OSCC when compared to OM, suggesting invadopodia formation and activity. Besides overexpressed in OSCC, cortactin, TKs4 and TKs5 showed no or ambiguous differences in protein expression when compared among clinical-stages or histological grades groups. Conversely, the expression of MT1-MMP increased in advanced stages and less differentiated tumours, suggesting MT1-MMP expression as a promising prognostic marker in OSCC.

Immunohistochemical analysis of ADAMTS-1, versican and pEGFR expressions in periapical granuloma and radicular cyst.

BACKGROUND: ADAMTS expression can be associated with several inflammatory processes, and has been correlated with tumorigenesis of some neoplasms, but its participation in the development of periapical lesions has not been investigated. Therefore, our objective was to verify the expression of ADAMTS-1, versican and pEGFR in Periapical Granuloma (PG) and in the Radicular Cyst (RC) since they are the most common lesions of the periapex. METHODS: 25 samples of RC and 10 of PG were used. As a control, 10 samples of inflammatory fibrous hyperplasia (IFH) and 10 of dental follicle (DF) were used. The expression of these proteins was investigated using immunohistochemistry. RESULTS: In the epithelium of RC, IFH and DF, the expression of ADAMTS-1 was greater in DF than in RC (p < .001). Versicano showed greater expression in IFH than in RC, DF than in RC (p < .001). pEGFR showed greater expression in IFH and RC than in DF (p < .01 and p < .05, respectively). In connective tissue, ADAMTS-1 expression was greater in PG and RC than in IFH and DF (p < .001). Versicano showed greater expression in PG, RC and IFH compared to DF (p < .001). In pEGFR there was a higher expression in PG when compared to RC, IFH and DF (p < .001). Greater immunostaining occurred in the RC than in the DF (p < .001). CONCLUSIONS: Our results suggest that the studied proteins may participate in the pathogenesis of PG and RC, through the interaction of these proteins, in the remodeling of the ECM (versican) by ADAMTS-1, producing bioactive fragments, which could activate EGFR, contributing to the formation, growth and maintenance of injuries.

Hypoxia and proangiogenic proteins in human ameloblastoma.

Ameloblastomas are epithelial odontogenic tumours that, although benign, are locally invasive and may exhibit aggressive behaviour. In the tumour microenvironment, the concentration of oxygen is reduced, which leads to intratumoral hypoxia. Under hypoxia, the crosstalk between the HIF-1α, MMP-2, VEGF, and VEGFR-2 proteins has been associated with hypoxia-induced angiogenesis, leading to tumour progression and increased invasiveness. This work showcases 24 ameloblastoma cases, 10 calcifying odontogenic cysts, and 9 dental follicles, used to investigate the expression of these proteins by immunohistochemistry. The anti-HIF-1α, anti-MMP-2, anti-VEGF, and anti-VEGFR-2 primary antibodies are used in this work. The results have been expressed by the mean grey value after immunostaining in images acquired with an objective of 40×. The ameloblastoma samples showed higher immunoexpression of HIF-1α, MMP-2, VEGF, and VEGFR-2 when compared to the dental follicles and calcifying odontogenic cysts. Ameloblastomas show a higher degree of expression of proteins associated with intratumoral hypoxia and proangiogenic proteins, which indicates the possible role of these proteins in the biological behaviour of this tumour.

Role of hypoxia-related proteins in adenoid cystic carcinoma invasion.

BACKGROUND: Among cancers affecting the oral cavity, adenoid cystic carcinoma (ACC) is a relatively common malignant neoplasm. It has high rates of metastasis and recurrence and is associated with significant morbidity. During the progression of ACC, the oxygen concentration is reduced in specific areas of the tumour microenvironment, leading to intratumoural hypoxia. The expression of NOTCH1, a disintegrin and metalloproteinase 12 (ADAM-12), hypoxia-inducible factor 1 alpha (HIF-1α), and heparin-binding epidermal growth factor (HB-EGF) under hypoxic conditions has been implicated in invadopodia formation, tumour invasiveness, and metastasis. The aim of this study was to analyse the expression of these proteins to elucidate the mechanisms underlying ACC invasiveness. METHODS: Fifteen ACC samples and 10 normal-looking salivary gland (SG) samples were used to investigate the expression of these proteins by immunohistochemistry. Primary antibodies against NOTCH1, ADAM-12, HIF-1α, and HB-EGF were used. RESULTS: The immunoexpression of all proteins was higher in ACC samples than in SG samples (p < 0.05). CONCLUSIONS: There was increased expression of proteins associated with hypoxia and tumour invasiveness in ACC samples, which indicates a possible role of these proteins in the biological behaviour of this tumour.

Central Giant Cell Granuloma Treated with Intralesional Corticosteroid Injections and Bisphosphonates: A Long-Term Follow-Up Case Study.

Central giant cell granuloma (CGCG) is a benign intraosseous lesion of the head and neck with potential for aggressive and locally destructive behaviour. Lesions of the maxilla tend to expand more than those of the mandible due to the thinner cortices and spongy tissue of this location. Surgical removal is the most common treatment; however, it may be disfiguring in aggressive cases, especially for lesions located in the maxilla. Alternative treatments, such as intralesional corticosteroid injections, have been performed with satisfactory results. We report a case of a 12-year-old female patient with a CGCG of the left maxilla that was treated with 40 doses of intralesional triamcinolone acetonide infiltrations combined with alendronate sodium and calcium carbonate. Clinical and imaging follow-up over 12 years demonstrates improvement in the patient's condition.

Oxidative Damage in Human Periodontal Ligament Fibroblast (hPLF) after Methylmercury Exposure.

Human exposure to mercury (Hg) is primary associated with its organic form, methylmercury (MeHg), through the ingestion of contaminated seafood. However, Hg contamination is also positively correlated with the number of dental restorations, total surface of amalgam, and organic mercury concentration in the saliva. Among the cells existing in the oral cavity, human periodontal ligament fibroblast (hPLF) cells are important cells responsible for the production of matrix and extracellular collagen, besides sustentation, renewal, repair, and tissue regeneration. In this way, the present study is aimed at investigating the potential oxidative effects caused by MeHg on hPLF. Firstly, we analyzed the cytotoxic effects of MeHg (general metabolism status, cell viability, and mercury accumulation) followed by the parameters related to oxidative stress (total antioxidant capacity, GSH levels, and DNA damage). Our results demonstrated that MeHg toxicity increased in accordance with the rise of MeHg concentration in the exposure solutions (1-7 µM) causing 100% of cell death at 7 µM MeHg exposure. The general metabolism status was firstly affected by 2 µM MeHg exposure (43.8 ± 1.7%), while a significant decrease of cell viability has arisen significantly only at 3 µM MeHg exposure (68.7 ± 1.4%). The ratio among these two analyses (named fold change) demonstrated viable hPLF with compromised cellular machinery along with the range of MeHg exposure. Subsequently, two distinct MeHg concentrations (0.3 and 3 µM) were chosen based on LC50 value (4.2 µM). hPLF exposed to these two MeHg concentrations showed an intracellular Hg accumulation as a linear-type saturation curve indicating that metal accumulated diffusively in the cells, typical for metal organic forms such as methyl. The levels of total GSH decreased 50% at exposure to 3 µM MeHg when compared to control. Finally, no alteration in the DNA integrity was observed at 0.3 µM MeHg exposure, but 3 µM MeHg caused significant damage. In conclusion, it was observed that MeHg exposure affected the general metabolism status of hPLF with no necessary decrease on the cell death. Additionally, although the oxidative imbalance in the hPLF was confirmed only at 3 µM MeHg through the increase of total GSH level and DNA damage, the lower concentration of MeHg used (0.3 µM) requires attention since the intracellular mercury accumulation may be toxic at chronic exposures.

Abnormal activation of the Akt signaling pathway in adenoid cystic carcinoma.

PURPOSE: Adenoid cystic carcinoma (ACC) is an intriguing lesion because it shows a slow growth in the beginning, but a late poor prognosis due to perineural invasion, metastasis and recurrence. This study aimed to investigate whether Akt signaling would be deregulated in adenoid cystic carcinoma and its consequence in the expression of associated proteins. METHODS: The expression of the Akt, p-Akt, NFκB, ß-catenin, cyclin D1 and COX-2 was assessed by immunohistochemistry in 10 cases of ACC, 17 cases of pleomorphic adenoma (PA), and 7 cases of normal salivary gland (NSG). RESULTS: p-Akt was overexpressed in ACC when compared to NSG. NFκB, ß-catenin, and COX-2 were overexpressed in ACC and PA when compared to NSG. Most proteins were slightly higher expressed in ACC than in PA, but they never reached significance. p-Akt expression positively correlated with NFκB, ß-catenin, cyclin D1 and COX-2 in ACC and PA, while this correlation trended to be negative in for these proteins (except for NFκB) in NSG using Person's correlation analysis, but without reaching significance. CONCLUSIONS: Our results indicate an abnormal activation of Akt signaling pathway, which can be an important regulator of tumor biology in ACC. Activated Akt correlated with the expression of NFκB, ß-catenin and COX-2, which can potentially influence cell survival in ACC.

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.

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.

EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma.

Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion, and MMP secretion of ameloblastoma-derived cells.