Cholesterol depletion induces mesenchymal properties in oral squamous cell carcinoma cell line.

BACKGROUND: Cholesterol in cell membranes is crucial for cell signaling, adhesion, and migration. Membranes feature cholesterol-rich caveolae with caveolin proteins, playing roles in epithelial-mesenchymal transition and cancer progression. Despite elevated cholesterol levels in tumors, its precise function and the effects of its depletion in oral squamous cell carcinoma remain unclear. The aim of this study was to evaluate the influence of cholesterol depletion in oral squamous cell carcinoma cell line and epithelial-mesenchymal transition process. METHODS: Cholesterol depletion was induced on SCC-9 cells by methyl-ß-cyclodextrin and cell viability, proliferation, apoptosis, and colony formation capacities were evaluated. Gene and protein expressions were evaluated by reverse transcription polymerase chain reaction (RT-qPCR) and Western Blot, respectively, and cell sublocalization was assessed by immunofluorescence. RESULTS: Cholesterol depletion resulted in alteration of oral squamous cell carcinoma cell morphology at different concentrations of methyl-ß-cyclodextrin, as well as decreased cell proliferation and viability rates. Analysis of CAV1 transcript expression revealed increased gene expression in the treated SCC-9 during the 24 h period, at different concentrations of methyl-ß-cyclodextrin: 5 , 7.5, 10, and 15 mM, in relation to parental SCC-9. CAV1 protein expression was increased, with subsequent dose-dependent decrease. A statistically significant difference was observed in samples treated with 5 mM of methyl-ß-cyclodextrin (p = 0.02, Kruskal-Wallis test). The immunofluorescence assay showed lower cytoplasmic and membrane labeling intensity in the treated samples for CAV1. CONCLUSION: These findings indicate the modulation of cholesterol as a possible mechanism underlying the regulation of these molecules and activation of epithelial-mesenchymal transition in oral squamous cell carcinoma.

Influence of conditioned medium from squamous cell carcinoma of the tongue on lymphoblasts and peripheral blood mononuclear cells.

BACKGROUND: Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the oral cavity and is associated with high morbidity and mortality. Attention has been given to the role of inflammatory cells in carcinogenesis because of the ability of cancer cells to subvert the immune response. However, little is known about how molecules from neoplastic cells interact with lymphoblasts and circulating immune cells. This study aimed to understand the mechanisms by which SCC cells modulate the immune response by analyzing the influence of conditioned medium derived from SCC cell lines on immune cells. METHODS: Lymphoblastic cells (CEM) and peripheral blood mononuclear cells (PBMC) were cultured in a conditioned medium derived from squamous cell carcinoma cells (SCC9 or SCC4) and analyzed for cell viability, CD4/CD8/FOXP3 profile by flow cytometry, and chemokine levels. RESULTS: Conditioned medium derived from SCC4 and SCC9 presented higher concentrations of IL-6 and IL-8 than IL-1ß, IL-10, and IFN-γ. CEM and PBMCs when cultured with conditioned medium derived from SCC4 and SCC9 reduced IL-1ß, IL-8, and IFN-γ concentrations. Conditioned medium from SCC4 increased CD4+ population in both CEM and PBMCs, while in conditioned medium from SCC9 it occurred only in PBMCs. PBMCs when cultured with both conditioned mediums increased CD8+ /FOXP3+ cells. CEM cells when cultured with conditioned medium derived from SCC4 and SCC9 reduced. CONCLUSION: Collectively, our results suggest that the products derived from squamous cell carcinoma on inflammatory cells can promote an immunosuppressed environment by reducing cell viability, changing cytokine expression, and altering the cell immunoprofile.

Secretory carcinoma of the salivary gland is rich in lactoferrin: a possible lactational-like differentiation?

PURPOSE: It has been hypothesised that secretory carcinoma of the salivary gland (SCsg) might have a lactational-like differentiation. Therefore, we aimed to assess the immunoexpression of breast hormonal receptors and milk-related proteins in cases of SCsg and other salivary gland tumours with prominent secretory activity. METHODS: Immunohistochemistry against prolactin and growth hormone receptors, lactoferrin, human milk fat globule 1, MUC 1 and MUC4 was performed in twelve cases of SCsg and 47 other salivary gland tumours. RESULTS: Most cases of SCsg were negative for prolactin and growth hormone receptors. All cases of SCsg showed enhanced membranous-cytoplasmic staining for human milk fat globule 1, a pattern seen in other tumour groups. Only SCsg showed widespread strong staining for lactoferrin, concomitantly in the cell compartment and secretion. The other positive tumour types exhibited restricted staining. MUC1 and MUC4 showed no distinct pattern of expression. CONCLUSION: Although SCsg failed to demonstrate a complete lactational-like differentiation, lactoferrin showed a distinctive expression pattern in SCsg compared to other tumour types, which makes it a good marker to help in its differential diagnosis.

Central giant cell granulomas of the jaws stromal cells harbour mutations and have osteogenic differentiation capacity, in vivo and in vitro.

BACKGROUND: Central giant cell granulomas (CGCG) of the jaws are osteolytic lesions that may behave aggressively and respond poorly to surgery. Microscopically, in addition to giant cells, there is a mononuclear cell population composed of macrophage/monocytic cells and spindle-shaped cells of mesenchymal origin. Seventy two percent of these tumours harbour mutually exclusive TRPV4, KRAS and FGFR1 mutations. We aimed to assess the mutational status of mononuclear and giant cells and the osteogenic potential of stromal cells in vitro and in vivo. METHODS AND RESULTS: We screened CGCG for signature mutations and used laser-capture microdissection to demonstrate that the mutations are restricted to the mononuclear cells. Additionally, we established CGCG primary cell culture and observed that the cells retained the mutations throughout passages. By flow cytometry, we observed predominance of CD14- CD51- CD61- cells, consistent with the expected profile for stromal cells. Considering the mesenchymal origin of stromal cells, we assessed the osteogenic differentiation potential of CGCG cells in culture by cytochemistry (von Kossa and alizarin red staining), alkaline phosphatase (ALP) activity assay and gene expression of osteogenic markers. CGCG cells presented self-capacity to increase ALP levels in a time-dependent manner and under osteogenic induction presented increasing number of calcium deposits, and overall higher expression of osteocalcin, RUNX2, ALPL and osteopontin than cells without osteogenic induction. A patient-derived xenograft model for CGCG was established, and osteoid material deposition was observed. CONCLUSION: Collectively, the results confirm that the signature mutations are restricted to stromal cells in CGCG, and the in vitro and in vivo results support that these cells have the capacity to differentiate into osteoblasts, in line with the bone formation often observed in the stroma of these lesions.

Photodynamic therapy for squamous cell carcinoma of the head and neck: narrative review focusing on photosensitizers.

This narrative review aimed to evaluate the effectiveness of PDT in early or advanced squamous cell carcinoma of the head and neck (SCCHN). Scopus, MEDLINE/PubMed, and Embase were searched electronically following the PRISMA protocol. Quality assessment was performed according to JBI, NIH, and AMSTAR protocols. The main outcomes evaluated were treatment response, recurrence, survival, and adverse effects. A total of 49 articles met the search criteria: 43 case series, two cohort studies, two prospective before-after clinical trials, one systematic review, and one meta-analysis. Data from 2121 SCCHN patients were included. The response to PDT was variable according to the type of photosensitizer, tumor location, and tumor stage. In general, higher complete responses rated were observed in T1/T2 SCCHN, mainly with mTHPC-mediated PDT. With regard to T3/T4 or advanced SCCHN tumors, there is no compelling evidence suggesting the effectiveness of PDT. Any adverse effects reported were well tolerated by patients. The present review suggests that PDT is a promising treatment modality for early-stage SCCHN. Although there are limitations due to the low level of evidence of the included studies, we believe that the present review could help to design robust clinical trials to determine the efficacy of PDT in SCCHN.

Effects of photobiomodulation on cellular viability and cancer stem cell phenotype in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy; it has been shown that cancer stem cells (CSC) are present in OSCC and associated with tumor growth, invasion, metastasis, and therapeutic resistance. Photobiomodulation (PBM) is an alternative tool for oncologic treatment adverse effects such as oral mucositis (OM); however, controversy exists regarding the undesirable effects of PBM on tumor or CSC. This study aimed to evaluate in vitro, the effects of PBM, with the same dosimetric parameters as those used in the clinic for OM prevention and treatment, on OSCC cellular viability, as well as PBM's effect on CSC properties and its phenotype. OSCC cell lines were submitted to single or daily PBM with 3 J/cm2 and 6 J/cm2 and then the cellular viability was evaluated by MTT, NRU (neutral red uptake), and CVS (crystal violet staining). The CSC populations were evaluated by clonogenic formation assay, flow cytometry, and RT-qPCR. The single PBM with the 3 J/cm2 group was associated with increased cellular viability. Daily PBM with 3 J/cm2 and 6 J/cm2 was associated with a significant decrease in cellular viability. Additionally, daily PBM was not able to promote CSC self-renewal or the CD44high/ESAlow and CD44high/ESAhigh cellular phenotypes. Moreover, a decrease in the number of spheres and in the expression of the CSC related gene BMI1 was observed after daily PBM with 6 J/cm2. Daily PBM with 3 J/cm2 and 6 J/cm2 showed an inhibitory effect on cellular viability and was not able to promote the CSC self-renewal or phenotype.

Expression of upstream and downstream targets of mTOR pathway in seven cases of secretory carcinoma of salivary gland origin.

PURPOSE: To investigate the expression of upstream and downstream targets of mTOR signalling pathway in the secretory carcinoma of salivary gland origin (SCsg). METHODS: Seven cases of secretory carcinoma diagnosed by a combination of immunohistochemistry and/or molecular testing were retrieved from our pathology files. For comparison purposes, 27 other salivary carcinomas were selected. Immunohistochemical staining was performed against phospho-Akt, PTEN, phospho-mTOR, phospho-4E-BP, eIF4E and phospho-S6 ribosomal protein. RESULTS: With the exception of Akt, all the other proteins were present at some level in the SCsg and in other salivary carcinomas. PTEN was diffusely expressed in 57.1% of SCsg, but only in 14.8% of other salivary carcinomas. mTOR is expressed in more than half of the cases both for SCsg and other salivary tumour types. Most cases of SCsg showed negative expression for S6 ribosomal protein (71.4%) and 4E-BP1 (57.1%). For both groups evaluated, eIF4E was the most expressed protein. CONCLUSION: SCsg shows different expression patterns for the mTOR signalling molecules, but only eIF4E was highly expressed. This may suggest alternative signalling pathways other than Akt and mTOR in this group of tumours.

Differential expression of inflammatory and anti-inflammatory mediators by M1 and M2 macrophages after photobiomodulation with red or infrared lasers.

In response to stimuli in the microenvironment, macrophages adopt either the M1 or M2 phenotype to coordinate the tissue repair process. Photobiomodulation (PBM) plays an important role in the modulation of acute inflammation, including cellular influx, macrophage polarization, and the release of inflammatory mediators. The aim of the present study was to evaluate the effects of red and infrared PBM on the mRNA expression of cytokines and chemokines in macrophages polarized to the M1 and M2 phenotypes. J774 macrophages activated to induce M1 (lipopolysaccharide + interferon gamma) or M2 (interleukin-4) phenotypes were irradiated with red or infrared PBM (1 J). After 4 and 24 h, gene expression was analyzed by qPCR. PBM at 660 nm decreased the mRNA expression of CCL3, CXCL2, and TNF-α in M1 macrophages and CXCL2 in M2 macrophages 4 h after irradiation. Similarly, PBM at 780 nm decreased mRNA expression levels of CCL3 and IL-6 by M1 macrophages 24 h after irradiation. Moreover, PBM at 780 nm increased the mRNA expression of TGFß1 4 h after irradiation and decreased the expression of this gene after 24 h in M2 macrophages. Although red and infrared PBM were able to modulate and reduce M1/M2a-related markers, infrared laser irradiation promoted a temporal increase in the expression of TGFß1 in M2 macrophages. Thus, depending on the time PBM is used on injured tissue, different parameters can promote optimal results by modulating specific macrophage phenotypes.

Central giant cell granuloma: A clinicopathological and immunohistochemical study of macrophages, blood vessels, lymphatic vessels and regulatory proteins.

OBJECTIVE: This study seeks to investigate immunohistochemical parameters that could distinguish non-aggressive Central giant cell granuloma (CGCG) from aggressive CGCG, two groups of lesions which differ in their clinical and radiographic features and prognosis. MATERIAL AND METHODS: 12 cases of non-aggressive CGCG and 11 cases of aggressive CGCG were investigated and associated the immunohistochemical expression of macrophages (CD68 and CD163), blood vessels (CD34 and CD105), lymphatic vessels (D2-40) and regulator proteins (p63 and Ki-67). Clinical and radiographic features were also studied. RESULTS: Associations between all proteins in non-aggressive and aggressive CGCG were not significant (p > 0.05). With respect to non-aggressive CGCG, there were no significant correlations, while in aggressive CGCG there was a significant positive correlation between CD68 and CD163 (p = 0.031), between CD34 and D2-40 proteins (p = 0.04), whereas a significant negative correlation was observed between CD105 and CD68 (p = 0.040). However, regardless of aggressiveness of CGCG, there was a significant positive correlation between CD68 and CD163 (p = 0,04). Among the clinical and immunohistochemical aspects, only the symptomatology was a significant risk factor for the occurrence of aggressive CGCG (OR = 12.00/p = 0.016). CONCLUSION: Macrophages and angiogenesis contribute to their maintenance and development of CGCG. In addition, immunohistochemistry used here was not able to differentiate their aggressiveness. However, symptomatology was proved to be a risk factor for the occurrence of aggressive CGCG. It is possible that clinical features, particularly symptomatology, represent the most appropriate parameter to attempt to distinguish GCCG.

Anticancer Activities of the Quinone-Methide Triterpenes Maytenin and 22-ß-hydroxymaytenin Obtained from Cultivated Maytenus ilicifolia Roots Associated with Down-Regulation of miRNA-27a and miR-20a/miR-17-5p.

Natural triterpenes exhibit a wide range of biological activities. Since this group of secondary metabolites is structurally diverse, effects may vary due to distinct biochemical interactions within biological systems. In this work, we investigated the anticancer-related activities of the quinone-methide triterpene maytenin and its derivative compound 22-ß-hydroxymaytenin, obtained from Maytenus ilicifolia roots cultivated in vitro. Their antiproliferative and pro-apoptotic activities were evaluated in monolayer and three-dimensional cultures of immortalized cell lines. Additionally, we investigated the toxicity of maytenin in SCID mice harboring tumors derived from a squamous cell carcinoma cell line. Both isolated molecules presented pronounced pro-apoptotic activities in four cell lines derived from head and neck squamous cell carcinomas, including a metastasis-derived cell line. The molecules also induced reactive oxygen species (ROS) and down-regulated microRNA-27a and microRNA-20a/miR-17-5p, corroborating with the literature data for triterpenoids. Intraperitoneal administration of maytenin to tumor-bearing mice did not lead to pronounced histopathological changes in kidney tissue, suggesting low nephrotoxicity. The wide-ranging activity of maytenin and 22-ß-hydroxymaytenin in head and neck cancer cells indicates that these molecules should be further explored in plant biochemistry and biotechnology for therapeutic applications.

Potential role of Hedgehog signaling pathway and myofibroblastic differentiation in central giant cell granuloma-A preliminary study.

OBJECTIVE: This study investigated components of the Hedgehog (HH) signaling pathway (SHH, GLI1), cyclin D1, and smooth muscle actin (SMA) in central giant cell granulomas (CGCG). The relationship between these proteins and myofibroblasts was also studied. MATERIAL AND METHODS: Twelve cases of non-aggressive CGCG and 11 cases of aggressive CGCG were studied using immunohistochemistry for SHH, GLI1, Cyclin D1, and SMA. RESULTS: Associations between all proteins in non-aggressive and aggressive CGCG were not significant (P > .05). All cases of CGCG showed significantly higher expression of SMA compared with the other proteins (P < .01). A positive correlation (P = .04) was only observed between SHH and GLI1 for all cases of CGCG. Furthermore, a positive correlation between SHH and GLI1 in non-aggressive CGCG (P = .04) and between GLI1 and cyclin D1 in aggressive CGCG (P = .03) were observed. There was also a negative correlation between the expression of SHH and SMA in non-aggressive CGCG (P = .031). CONCLUSIONS: This study provided insights into the activation of the HH signaling pathway in CGCG. In addition, the activation of this pathway (SHH and GLI1) might play some role in the differentiation of stromal myofibroblasts, although these markers including Cyclin D1 and SMA do not indicate aggressiveness of the CGCG. Furthermore, this myofibroblastic differentiation process would occur at the expense of maturation of these lesions.

Photobiomodulation is associated with a decrease in cell viability and migration in oral squamous cell carcinoma.

The treatment of squamous cell carcinoma (SCC) involves surgery, chemotherapy, and/or radiotherapy, which can cause mucositis (inflammation of the oral mucosa that causes considerable pain and can compromise the continuity of oncological treatment). Photobiomodulation (PBM) has been successfully used in the treatment of mucositis, but doubts arise regarding the use of laser for areas in which tumor cells may remain. In this study, the effect of PBM on the viability, mitochondrial activity, proliferation, apoptosis, and migration of cells derived from oral SCC was evaluated. SCC9 cells were irradiated with laser (660 and 780 nm, using 11 dosimetric parameters) and submitted to mitochondrial and caspase 3 activity tests after 1 and 3 days. Based on the results, cell viability (neutral red assay), proliferation (BrdU assay), and migration (scratch-wound assay) were evaluated using only the dosimetric parameters recommended for mucositis. Non-irradiated cells served as the control. The experiments were performed in triplicate. The 11 parameters diminished mitochondrial activity and induced tumor cell apoptosis. Using the parameters recommended for mucositis, irradiation with 780 nm (70 mW, 4 J/cm2) proved to be the safest and led to a reduction in cell viability, the induction of apoptosis, and a reduction in the migration capacity of the tumor cells.

PAI-1, CAIX, and VEGFA expressions as prognosis markers in oral squamous cell carcinoma.

BACKGROUND: In oral squamous cell carcinoma (OSCC), the HIF-1 complex promotes the expression of genes involved in specific mechanisms of cell survival under hypoxic conditions, such as plasminogen activator inhibitor-1 (PAI-1), carbonic anhydrase 9 (CAIX), and vascular endothelial growth factor A (VEGFA). The study aimed to investigate the presence and prognostic value of PAI-1, CAIX, and VEGFA in OSCC. MATERIALS AND METHODS: Immunohistochemistry was used to analyze the expressions of these proteins in 52 tumoral tissue samples of patients with OSCC, surgically treated and followed by a minimum of 24 months after surgery. The correlations between protein expressions and clinicopathological parameters and prognosis were analyzed. RESULTS: Positive PAI-1 membrane expression was significantly associated with local disease relapse (P = .027). Multivariate analysis revealed that the positive PAI-1 membrane expression is an independent marker for local disease relapse, with approximately 14-fold increased risk when compared to negative expression (OR = 14.49; CI = 1.40-150.01, P = .025). Strong PAI-1 cytoplasmic expression was significantly associated with the less differentiation grade (P = .027). Strong CAIX membrane expression was significantly associated with local disease-free survival (P = .038). Positive CAIX cytoplasmic expression was significantly associated with lymph node affected (P = .025) and with disease-specific survival (P = .022). Multivariate analysis revealed that the positive CAIX cytoplasmic expression is an independent risk factor for disease-related death, increasing their risk approximately 3-fold when compared to negative expression (HR = 2.84; CI = 1.02-7.87, P = .045). Positive VEGFA cytoplasmic expression was significantly associated with less differentiation grade (P = .035). CONCLUSION: Our results suggest a potential role for these expressions profiles as tumor prognostic markers in OSCC patients.

Embryonic stem cells markers Oct4 and Nanog correlate with perineural invasion in human salivary gland mucoepidermoid carcinoma.

BACKGROUND: Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy and is successfully treated by surgery and radiation. However, some patients have recurrent tumours and in these cases, few treatments options are available. Cancer stem cells (CSC) have been observed and isolated from different solid tumours based on the expression of stem cell markers. These cells are associated with tumour initiation, progression as well as treatment resistance. In this study, the expression of stem cell markers CD44, Bmi1, Oct4 and Nanog was evaluated in non-neoplastic salivary tissue and in MEC. METHODS: Twenty-eight samples of MEC and their corresponding non-neoplastic salivary tissue were examined by immunohistochemistry and the stem cell markers expression was correlated with histological and clinical parameters. RESULTS: CD44 was expressed in the membrane of serous and mucous acini as well as in the ductal cells in normal gland tissue. Bmi1, Oct4 and Nanog were mainly expressed in ductal structures. In MEC, CD44 and Bmi1 showed strong expression in all types of neoplastic cells and both markers revealed intense expression in tumour invasive front. Oct4 and Nanog protein expression was associated with desmoplasia and perineural invasion. Only Oct4 positive tumours were associated with dissociative growth pattern and committed margins. CONCLUSION: The stem cell markers CD44, Bmi1, Oct4 and Nanog are frequently expressed in MEC in relation to normal salivary gland and Oct4 and Nanog expression may contribute to aggressiveness and worst prognosis in MEC patients.

Methylation status of homeobox genes in common human cancers.

Approximately 300 homeobox loci were identified in the euchromatic regions of the human genome, of which 235 are probable functional genes and 65 are likely pseudogenes. Many of these genes play important roles in embryonic development and cell differentiation. Dysregulation of homeobox gene expression is a frequent occurrence in cancer. Accumulating evidence suggests that as genetics disorders, epigenetic modifications alter the expression of oncogenes and tumor suppressor genes driving tumorigenesis and perhaps play a more central role in the evolution and progression of this disease. Here, we described the current knowledge regarding homeobox gene DNA methylation in human cancer and describe its relevance in the diagnosis, therapeutic response and prognosis of different types of human cancers.

Fatal hepatocellular carcinoma presenting with oral metastasis in a patient with synchronic primary malignancies of prostate and liver.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most frequent type of liver cancer and its occurrence in the oral cavity as a metastatic neoplasm is a rare event. We describe a fatal case of HCC with oral metastasis in a patient firstly diagnosed with prostatic and hepatic carcinomas. The histopathological examination revealed a hepatocyte-like tumour cells arranged in organoid structures as well as positivity to cytokeratin 8 and Hep Par 1. The present findings highlight the importance of a complete medical evaluation of the patient to identify possible oral repercussions of primary diseases.

Comparative effects of low-level laser therapy pre- and post-injury on mRNA expression of MyoD, myogenin, and IL-6 during the skeletal muscle repair.

This study analyzed the effect of pre-injury and post-injury irradiation with low-level laser therapy (LLLT) on the mRNA expression of myogenic regulatory factors and interleukin 6 (IL-6) during the skeletal muscle repair. Male rats were divided into six groups: control group, sham group, LLLT group, injury group; pre-injury LLLT group, and post-injury LLLT group. LLLT was performed with a diode laser (wavelength 780 nm; output power 40 mW' and total energy 3.2 J). Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior (TA) muscle. After euthanasia, the TA muscle was removed for the isolation of total RNA and analysis of MyoD, myogenin, and IL-6 using real-time quantitative PCR. Significant increases were found in the expression of MyoD mRNA at 3 and 7 days as well as the expression of myogenin mRNA at 14 days in the post-injury LLLT group in comparison to injury group. A significant reduction was found in the expression of IL-6 mRNA at 3 and 7 days in the pre-injury LLLT and post-injury LLLT groups. A significant increase in IL-6 mRNA was found at 14 days in the post-injury LLLT group in comparison to the injury group. LLLT administered following muscle injury modulates the mRNA expression of MyoD and myogenin. Moreover, the both forms of LLLT administration were able to modulate the mRNA expression of IL-6 during the muscle repair process.

Light-emitting diode therapy increases collagen deposition during the repair process of skeletal muscle.

This study analyzed the effects of light-emitting diode (LED) therapy on the morphology of muscle tissue as well as collagen remodeling and matrix metalloproteinase 2 (MMP-2) activity in the skeletal muscle of rats following acute injury. Wistar rats were divided into four groups: (1) control, (2) sham, (3) untreated cryoinjury, and (4) cryoinjury treated with LED. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior muscle. For treatment, the LED equipment (wavelength 850 nm, output power 30 mW, and total energy 3.2 J) was used daily. The study periods were 1, 3, and 7 days after cryoinjury. Morphological aspects were evaluated through hematoxylin-eosin staining. The amount of collagen fibers was evaluated using Picro Sirius Red staining under polarized light. The gelatinase activity of MMP-2 was evaluated using zymography. The results showed significant reductions in inflammatory infiltrate after 3 days and an increased number of immature muscle fibers after 7 days. Furthermore, treatment induced a reduction in the gelatinolytic activity of MMP-2 after 1, 3, and 7 days in comparison to the untreated injury groups and increased the collagen deposition after 3 and 7 days in the treated groups. LED therapy at 850 nm induced a significant reduction in inflammation, decreased MMP-2 activity, and increased the amount of immature muscle and collagen fibers during the muscle repair process following acute injury.

DNA methyltransferase immunohistochemical expression in odontogenic tumours.

BACKGROUND: Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalysed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyses the expression of DNMTs in odontogenic tumours. METHODS: Formalin-fixed and paraffin-embedded tissue samples of 20 ameloblastomas, 10 calcifying cystic odontogenic tumours, 10 calcifying epithelial tumours, 10 adenomatoid odontogenic tumours, 10 keratocystic odontogenic tumours, five ameloblastic fibromas, two ameloblastic fibro-odontomas, four central odontogenic fibromas, seven peripheral odontogenic fibromas and 10 odontogenic myxomas were included. Immunohistochemical expression of DNMT1, 3A and 3B was assessed using a semi-quantitative analysis, and also a correlation with p21, p27 and E-cadherin immunoexpression was made. RESULTS: DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. DNMT1 expression was directly correlated with p27 expression in ameloblastomas. CONCLUSION: The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.

Low-level laser irradiation modulates cell viability and creatine kinase activity in C2C12 muscle cells during the differentiation process.

Low-level laser irradiation (LLLI) is increasingly used to treat musculoskeletal disorders, with satisfactory results described in the literature. Skeletal muscle satellite cells play a key role in muscle regeneration. The aim of the present study was to evaluate the effect of LLLI on cell viability, creatine kinase (CK) activity, and the expression of myogenic regulatory factors in C2C12 myoblasts during the differentiation process. C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2% horse serum and submitted to irradiation with GaAlAs diode laser (wavelength, 780 nm; output power, 10 mW; energy density, 5 J/cm2). Cell viability and the expression of myogenic regulatory factors were assessed 24, 48, and 72 h after irradiation by 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) assay and quantitative real-time polymerase chain reaction (RT-qPCR), respectively. CK activity was analyzed at 24 and 72 h. An increase in cell viability was found in the laser group in comparison to the control group at all evaluation times. CK activity was significantly increased in the laser group at 72 h. Myogenin messenger RNA (mRNA) demonstrated a tendency toward an increase in the laser group, but the difference in comparison to the control group was non-significant. In conclusion, LLLI was able to modulate cell viability and CK activity in C2C12 myoblasts during the differentiation process.