In vitro and in silico validation of CA3 and FHL1 downregulation in oral cancer.

BACKGROUND: Aberrant methylation is a frequent event in oral cancer. METHODS: In order to better characterize these alterations, a search for genes downregulated by aberrant methylation in oral squamous cell carcinoma (OSCC) was conducted through the mining of ORESTES dataset. Findings were further validated in OSCC cell lines and patients' samples and confirmed using TCGA data. Differentially expressed genes were identified in ORESTES libraries and validated in vitro using RT-PCR in HNSCC cell-lines and OSCC tumor samples. Further confirmation of these results was performed using mRNA expression and methylation data from The Cancer Genome Atlas (TCGA) data. RESULTS: From the set of genes selected for validation, CA3 and FHL1 were downregulated in 60% (12/20) and 75% (15/20) of OSCC samples, respectively, and in HNSCC cell lines. The treatment of cell lines JHU-13 and FaDu with the demethylating agent 5'-aza-dC was efficient in restoring CA3 and FHL1 expression. TCGA expression and methylation data on OSCC confirms the downregulation of these genes in OSCC samples and also suggests that expression of CA3 and FHL1 is probably regulated by methylation. The downregulation of CA3 and FHL1 observed in silico was validated in HNSCC cell lines and OSCC samples, showing the feasibility of integrating different datasets to select differentially expressed genes in silico. CONCLUSIONS: These results showed that the downregulation of CA3 and FHL1 data observed in the ORESTES libraries was validated in HNSCC cell lines and OSCC samples and in a large cohort of samples from the TCGA database. Moreover, it suggests that expression of CA3 and FHL1 could probably be regulated by methylation having an important role the oral carcinogenesis.

Oral Mucositis in Cancer and Potential Use of Omega-3 Free Fatty Acids in Its Management: A Review.

Oral mucositis (OM) is a painful condition caused by chemotherapeutic or radiotherapeutic cancer treatments, occurring in patients with different tumour characteristics and locations. OM greatly impacts a patient's quality of life and cancer recovery. Current OM management strategies are not providing sufficient prevention and treatment; new approaches to injury management are needed. Studies on the benefit of omega-3 free fatty acids (FFA) in human health have increased significantly in recent years. FFA properties have been studied extensively, including their potential therapeutic use in inflammatory conditions. However, omega-3 FFA's use as a supplementary treatment for OM has not been clinically tested. Preliminary evidence suggests that utilising FFA to manage OM could be a useful strategy for lesion management, assisting with healthy oral mucosa recovery. This review will describe the incidence, risk factors, biology of OM and the current treatment strategies, leading to a discussion of the utility of omega-3 FFA as a novel therapeutic agent for OM.

Claudin-7 down-regulation is an important feature in oral squamous cell carcinoma.

AIMS: Claudins, a large family of essential tight junction (TJ) proteins, are abnormally regulated in human carcinomas, especially claudin-7. The aim of this study was to investigate claudin-7 expression and alterations in oral squamous cell carcinoma (OSCC). METHODS AND RESULTS: Expression of claudin-7 was analysed in 132 cases of OSCC organized in a tissue microarray. Claudin-7 mRNA transcript was evaluated using real-time polymerase chain reaction and the methylation status of the promoter was also assessed. Claudin-7 was negative in 58.3% of the cases. Loss of claudin-7 protein expression was associated with recurrence (P = 0.019), tumour size (P = 0.014), clinical stage of OSCC (P = 0.055) and disease-free survival (P = 0.015). Down-regulation of the claudin-7 mRNA transcripts was observed in 78% of the cases, in accordance with immunoexpression. Analysis of the methylation status of the promoter region of claudin-7 revealed that treatment of O28 cells (that did not express claudin-7 mRNA transcripts) with 5-Aza-2'-Deoxycytidine (5-Aza-dC) led to the re-expression of claudin-7 mRNA transcript. CONCLUSION: Loss of claudin-7 expression is associated with important subcellular processes in OSCC with impact on clinical parameters.

Three photobiomodulation protocols in the prevention/treatment of radiotherapy-induced oral mucositis.

PURPOSE: To compare three Photobiomodulation protocols to prevent/treat oral mucositis associated to radiotherapy. METHODS: Seventy-three patients with cancer in oral cavity, oropharynx, and nasopharynx, who underwent RT with dose in facial fields equal or higher than 6000 cGy were randomized into three groups (mean RT dose = 66 cGy ±4.9). Protocols of Group 1 was 660 nm, 15 mW, 3.8 J/cm2, Group 2 660 nm, 25 mW, 6.3 J/cm2 both starting on the first day of radiotherapy, and group 3 660 nm, 15 mW, 3.8 J/cm2 for therapeutic purpose. The patients of group 1 and 2 were irradiated at 40 points daily covering non-keratinizing oral mucosa. The spot size (probe's tip surface size) was 0.040 cm2 for all groups. Oral mucositis was evaluated according to both WHO and NCI scales, and pain related to oral mucositis was scored using the VAS. RESULTS: Patients from group 1 presented with grade II oral mucositis later than groups 2 and 3 (p < 0.001). Moreover, groups 2 and 3 also presented with a mean higher of oral mucositis grade than group 1, p < 0.001. Pain scores were lower in group 1 (p = 0.002). CONCLUSIONS: The Photobiomodulation used in Group 1 was more effective than the protocols used in groups 2 and 3 in controlling the grade II oral mucositis intensity, and mean pain scores.

Efeitos da fotobiomodulação na imunidade de pacientes com mucosite oral / Effects of photobiomodulation in the immunity of patients with oral mucositis

A mucosite oral é a toxicidade aguda mais comum que acomete os pacientes submetidos à radioterapia em região de cabeça e pescoço. Das diversas formas de controle e tratamento desta lesão, a terapia com laser de baixa intensidade tem demonstrado, clinicamente, ser eficaz no controle da doença. Porém, não há um completo entendimento de como esta modalidade terapêutica atua na estimulação do sistema imunológico destes pacientes. Assim, o objetivo deste estudo foi investigar os mecanismos moleculares envolvidos na ação da laserterapia como tratamento da lesão no que se refere aos aspectos imunológicos. Para isso, foi realizado RT-PCR em tempo real (TaqMan® Gene Expression Assays), utilizando um painel de genes relacionados com o sistema imunológico, onde o nível de expressão gênica foi avaliado em 60 amostras de saliva coletadas de 23 pacientes portadores de câncer de boca, orofaringe e nasofaringe tratados por radioterapia exclusiva ou radioterapia associada à quimioterapia. Estes pacientes foram divididos em 3 grupos com diferentes intensidades de laser e modalidades terapêuticas: Grupo A preventivo (Potência de 15 mW e densidade de energia de 3,8 J/ cm²) com 8 pacientes e 23 amostras, Grupo B preventivo (Potência de 25 mW e densidade de energia de 6,3 J/ cm²) com 8 pacientes e 22 amostras e o Grupo C-controle- curativo (Potência de 15 mW e densidade de energia de 3,8 J/ cm²) com 7 pacientes e 15 amostras. Os resultados obtidos, através da análise de 52 genes, demonstraram que 42 genes apresentaram alteração de expressão ao longo da evolução da mucosite oral, sendo possível identificar que os genes CCL3, CCR7, SYK foram relacionados com a instalação e progressão da doença. Além disso, através das diferentes análises de expressão entre diferentes grupos estudados foi possível inferir que a modulação da expressão dos genes CCL2, CCL3, IL-1A, MIF, NFKB2, TNF podem dar respaldo molecular aos melhores resultados clínicos previamente observados na laserterapia preventiva, com potência de 15 mW e densidade de energia de 3,8 J/ cm². Assim, este trabalho gerou dados moleculares iniciais sobre os mecanismos da imunoregulação que estão envolvidos com a instalação e progressão da mucosite e com a fotobiomodulação usada no tratamento das lesões.

Oral mucositis is the most common acute toxicity affecting patients undergoing radiotherapy in head and neck. Of the different ways of controlling and treating this lesion, low intensity laser therapy has shown clinically effectiveness in controlling the disease. However, it is not fully understood how this therapeutic modality acts in stimulating the immune system of these patients. Therefore, the objective of this study was to investigate the molecular mechanisms involved in the action of laser therapy for the treatment of this lesion regarding the immunological aspects. For this purpose, real time RT-PCR (TaqMan Gene Expression Assays) was performed, using a panel of genes related to immune system, in which the level of gene expression was evaluated in 60 saliva samples collected from 23 patients harboring mouth, oropharynx and nasopharynx cancer treated by radiotherapy alone or radiotherapy combined with chemotherapy. These patients were divided into 3 groups with different laser intensities and treatment modalities: Group A preventive (power of 15 mW and energy density of 3.8 J / cm²) with 8 patients and 23 samples, preventive Group B (25 mW Power and energy density of 6.3 J / cm²) with 8 patients and 22 samples and Group C curative (15 mW of power and energy density of 3.8 J / cm²) with 7 patients and 15 samples. The results obtained, through the analysis of the 52 genes, showed that the expression of 42 genes was changed throughout the evolution of oral mucositis. It was possible to identify that the genes CCL3, CCR7, SYK were related to the onset and progression of the disease. Moreover, through different expression analyzes among the different groups studied it was possible to infer that gene expression modulation of CCL2, CCL3, IL-1A, MIF, NFKB2, TNF can give molecular backing to the best clinical results previously observed in preventive laser therapy, with power of 15 mW and energy density of 3.8 J / cm². Thus, this study has generated initial molecular data on the mechanisms of immunoregulation involved in the onset and progression of mucositis and with photobiomodulation being used as treatment for these lesions. the photobiomodulation used in treatment of injuries.

Identificação de genes hiperexpressos em carcinomas epidermóides de cavidade oral / Identification of overexpression genes in ora! squamous cells carcinomas

O carcinoma epidermóide de cavidade oral é uma das maiores causa de morbidade e mortalidade no mundo, sendo a sexta neoplasia mais comum. Mundialmente são diagnosticados a cada ano 500.000 novos casos de câncer da cavidade oral e da faringe. Apesar dos avanços no tratamento destas neoplasias, alguns pacientes apresentam evoluções distintas no curso da doença e evoluem com metástases locoregionais e óbito. As diferenças na evolução clínica de tumores morfologicamente iguais e em um mesmo estágio clínico estão relacionadas a diversas vias de carcinogênese que são responsáveis pelo progresso dos carcinomas epidermóides, sendo assim, estes tumores podem apresentar comportamentos distintos devido a diversos padrões de expressão gênica. A análise do perfil de expressão gênica dos tumores em relação ao tecido normal poderá identificar novos potenciais marcadores de diagnóstico, prognóstico e no desenvolvimento de alvos terapêuticos. O objetivo desta pesquisa foi identificar por meio da busca em bancos de dados públicos de bibliotecas de ORESTES provenientes de cabeça de pescoço, genes que apresentavam expressão diferencial em tecidos normais e neoplásicos. Posteriormente, estas diferenças foram confirmadas por RT-PCR em tempo real através da análise dos níveis de expressão gênica em amostras de carcinomas epidermóides de cavidade oral. Foram identificados 109 genes diferencialmente expressos através desta análise, sendo que destes, 40 genes apresentavam hiperexpressão quando comparados os tecidos tumorais com os normais. Estes genes tiveram sua hiperexpressão confirmada através dos dados de EST's, sendo então selecionados 10 genes (ALDOA, AHSA1, ARHGAP1, HDAC1, KRT5, KRT6A, POLQ, PABPC1, RPL19 e RPL23) onde foi levado em consideração o nível de expressão dos transcritos, comparação entre regiões cromossômicas conhecidas como regiões de ganho descritas na literatura, o processo biológico e função celular em que o gene está envolvido, e a disponibilidade de anticorpos comerciais, para terem sua expressão diferencial avaliada em quarenta e oito amostras de carcinomas epidermóides de cavidade oral e em vinte e cinco amostras de tecidos normais de mucosa de cavidade oral. Este trabalho permitiu demonstrar que a estratégia de escolha de genes a partir a análise das bibliotecas de ORESTES foi adequada, pois genes selecionados para validação apresentaram aumento de expressão nas amostras tumorais analisadas. A quantificação da expressão desses genes revelou que três deles (ALDOA, AHSA1 e POLQ) apresentaram-se com aumento da expressão de pelo menos duas vezes em mais de 40% das amostras testadas, em relação à média dos valores de Ct das amostras de tecidos normais. O aumento de expressão destes genes nas amostras tumorais pode indicar uma associação destes com o processo de carcinogênese, porém estudos mais amplos devem ser realizados a fim de elucidar e confirmar os achados.

Oral squamous cells carcinoma is one of the most reasons of morbidity and mortality in the world, being the sixth most common malignancy. Annually, about 500000 new oral and pharyngeal cancers have been diagnosed in the world. Although the advances in treatment of these neoplasias, some patients present distinct evolutions in the course of the illness and evolve with metastases locoregional and death. The differences in clinical evolution of match morfological tumors and showing same clinical stage are related to several ways of carcinogenesis that are responsible for the squamous cells carcinomas progress, thus, these tumors may play distinct behaviors due several standards of gene expression. The tumors gene expression analysis may identify new potentials marking of diagnosis, prognostic and in the development of therapeutical targets. The aim of this research was to identify genes that are differentially expressed in normal and tumors tissues in public databases of head and neck ORESTES libraries. Later, these differences had been confirmed by quantitative real time PCR through the analysis of the gene expression levels in samples of oral squamous cells carcinomas. This first analysis had been identified a set of 109 distinguishing express genes, for a subset of 40 genes were overexpressed when comparing the tumor tissues with the normal ones. These genes had had its overexpression confirmed through the database of EST' s, and we selected 10 (ALDOA, AHSA1, ARHGAP1, HDAC1, KRT5, KRT6A, POLQ, PABPC1, RPL19 and RPL23) best genes candidates for further validation in forty-eight samples of oral squamous cells carcinomas and in twenty-five normal oral samples. We demonstrate that the strategy of genes choice through the ORESTES libraries analysis was adjusted, therefore genes selected for validation had conferred increase of expression in the tumors samples. The quantification of these genes expression revealed that three of them (ALDOA, AHSA1 and POLQ) had been presented with increase of the expression at least two times in more than 40% of tested samples, in relation to average Ct's values of the normal tissues samples. The genes overexpression in the tumors samples can indicate an association of these with the process of carcinogenesis, however ampler studies must be carried to elucidate and confirm the findings.