Análise da acetilação de histona H3 e sua contribuição para a instabilidade genômica na leucoplasia oral e leucoplasia verrucosa proliferativa
Data
2020-02-19
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Universidade Estadual Paulista (Unesp)
Resumo
As desordens potencialmente malignas orais (DPMOs) são apresentações clínicas que apresentam risco aumentado para o desenvolvimento de câncer na cavidade oral, seja ela uma lesão precursora definida ou mucosa clinicamente normal. Dentre as principais DPMOs, a leucoplasia oral (LO) se apresenta como uma placa branca não raspável de risco questionável, podendo ser homogênea ou não homogênea, cuja taxa de transformação maligna varia de 0,2% até 17,5%. Além disso, a LO apresenta os mesmos fatores de riscos que são observados no desenvolvimento de carcinoma espinocelular (CEC) oral, o qual é a principal neoplasia maligna que afeta a cavidade oral. Outra DPMO importante é a leucoplasia verrucosa proliferativa (LVP), a qual também se apresenta como uma placa branca não raspável, porém multifocal e possui um comportamento persistente e progressivo para malignidade, com taxa de transformação maligna de 70% até 100% dos casos. Diferente da LO, os fatores de risco como tabaco, álcool e noz de areca não parecem estar associados com o desenvolvimento da LVP. Adicionalmente, a LVP apresenta resposta inadequada a todas as modalidades de tratamento, sofre rápida disseminação pelos sítios orais e muitas vezes demonstra recorrência. Os graus de displasias identificados nas DPMOs são utilizados atualmente como preditores de risco para transformação maligna, no entanto diversos estudos têm mostrado que este não é um preditor fiel. Estudos recentes sugerem ainda que o infiltrado inflamatório associado as DPMOs pode estar associado a sua etiologia e comportamento clínico. Além disso, recentemente, tem-se estudado as alterações epigenéticas envolvendo histonas e metilação de DNA em diferentes tipos de câncer, bem como em algumas DPMOs a fim de descobrir alvos para medidas terapêuticas efetivas. Dentre os diversos fatores indutores de alterações epigenéticas, a inflamação crônica tem sido apontada como um destes fatores. Assim, o presente estudo teve como objetivos específicos: (1) comparar os níveis de acetilação de histona H3, o acúmulo de danos no DNA e os níveis de metilação global de DNA entre LO e LVP; (2) avaliar se os níveis de acetilação de histona H3 em linhagens celulares displásicas e tumorais sofrem alterações pelo contato com as células mononucleares do sangue periférico (PBMCs); e (3) avaliar se tais alterações são acompanhadas de alterações na proliferação celular e transição epitélio-mesenquimal (TEM). Para alcançar estes objetivos, os níveis de acetilação de histona H3, dano de DNA e metilação foram avaliados por imumofluorescência. Adicionalmente, ensaios de co-cultura celular com PBMCs e linhagens de queratinócitos orais (NOK-SI, DOK, SCC-25 e Detroit 562) foram realizados para avaliar a influência dos PBMCs na aquisição de vantagens para a progressão tumoral. Os resultados da imunofluorescência mostraram que ambas as DPMOs estavam hipoacetiladas em relação ao controle. Contudo, quando realizada uma comparação somente entre LO e LVP, observamos que o grupo LVP está mais hiperacetilado do que LO. Foram observados também que os níveis de dano de DNA e metilação estavam aumentados no grupo LO em relação a LVP. Os experimentos com cultura de células revelaram que o contato dos queratinócitos orais displásicos e normais com os PBMCs, foi capaz de favorecer a TEM. Nossos resultados revelaram dois pontos importantes: 1) ambas as DPMOs apresentam perfil epigenético distintos; 2) o contato direto de PMBCs com queratinócitos orais normais, displásicos e tumorais parece ser importante em eventos iniciais da tumorigênese como a TEM.
Oral potentially malignant disorders (OPMDs) are clinical presentations that present an increased risk of cancer development in the oral cavity, whether in a clinically definable precursor lesion or clinically normal oral mucosa. Among the main OPMDs, oral leukoplakia (OL) presents itself as a non-scratchable white plaque of questionable risk, which can be homogeneous or non-homogeneous, whose malignant transformation rate varies from 0.2% to 17.5%. In addition, OL has the same risk factors that are observed in the development of oral squamous cell carcinoma (OSCC), which is the main malignant neoplasm affecting the oral cavity. Another important OPMD is proliferative verrucous leukoplakia (PVL), which also presents itself as a white, non-scratchable, but multifocal plaque and has a persistent and progressive behavior for malignancy, with a malignant transformation rate of 70% to 100% of the cases. Unlike OL, risk factors such as tobacco, alcohol and areca nut do not appear to be associated with the development of PVL. Additionally, PVL has a poor response to all treatment modalities, suffers rapid dissemination through oral sites and often shows recurrence. The degree of epithelial dysplasia identified in OPMDs is currently used as a risk predictor for malignant transformation, however several studies have shown that it is not a reliable predictor. Recent studies also suggest that the inflammatory infiltrate associated with OPMD may be due to its etiology and clinical comportment. Furthermore, recently, epigenetic changes involving histones and DNA methylation in different types of cancer have been studied, as well as in some OPMDs in order to discover targets for effective therapeutic measures. Among the several factors inducing epigenetic changes, chronic inflammation has been identified as one of these factors. Thus, the present study had specific objectives: (1) to compare the levels of histone H3 acetylation, the accumulation of DNA damage and the levels of global DNA methylation between OL and PVL; (2) to evaluate whether the levels of histone H3 acetylation in dysplastic and tumor cell lines are altered by the contact with peripheral blood mononuclear cells (PBMCs); and (3) assess whether such changes are accompanied by changes in cell proliferation and epithelial-mesenchymal transition (EMT). To achieve these goals, the levels of histone H3 acetylation, DNA damage and methylation were assessed by immunofluorescence. In addition, cell co-culture assays with PBMCs and oral keratinocyte lines (NOK-SI, DOK, SCC-25 and Detroit 562) were performed to assess the influence of PBMCs on the acquisition of advantages for tumor progression. The results of the immunofluorescence showed that both OPMDs were hypoacetylated compared to the control. However, when comparing only OL and PVL, we observed that the PVL group was more hyperacetylated than OL. It was also observed that the levels of DNA damage and methylation were increased in the OL group in relation to PVL. The experiments with cell culture revealed that the contact of dysplastic and normal oral keratinocytes with PBMCs was able to favor EMT. Our results revealed two main points: 1) both OPMDs have different epigenetic profiles; 2) the direct contact of PMBCs with normal, dysplastic and tumoral oral keratinocytes seems to be important in early events of tumorigenesis such as EMT.
Oral potentially malignant disorders (OPMDs) are clinical presentations that present an increased risk of cancer development in the oral cavity, whether in a clinically definable precursor lesion or clinically normal oral mucosa. Among the main OPMDs, oral leukoplakia (OL) presents itself as a non-scratchable white plaque of questionable risk, which can be homogeneous or non-homogeneous, whose malignant transformation rate varies from 0.2% to 17.5%. In addition, OL has the same risk factors that are observed in the development of oral squamous cell carcinoma (OSCC), which is the main malignant neoplasm affecting the oral cavity. Another important OPMD is proliferative verrucous leukoplakia (PVL), which also presents itself as a white, non-scratchable, but multifocal plaque and has a persistent and progressive behavior for malignancy, with a malignant transformation rate of 70% to 100% of the cases. Unlike OL, risk factors such as tobacco, alcohol and areca nut do not appear to be associated with the development of PVL. Additionally, PVL has a poor response to all treatment modalities, suffers rapid dissemination through oral sites and often shows recurrence. The degree of epithelial dysplasia identified in OPMDs is currently used as a risk predictor for malignant transformation, however several studies have shown that it is not a reliable predictor. Recent studies also suggest that the inflammatory infiltrate associated with OPMD may be due to its etiology and clinical comportment. Furthermore, recently, epigenetic changes involving histones and DNA methylation in different types of cancer have been studied, as well as in some OPMDs in order to discover targets for effective therapeutic measures. Among the several factors inducing epigenetic changes, chronic inflammation has been identified as one of these factors. Thus, the present study had specific objectives: (1) to compare the levels of histone H3 acetylation, the accumulation of DNA damage and the levels of global DNA methylation between OL and PVL; (2) to evaluate whether the levels of histone H3 acetylation in dysplastic and tumor cell lines are altered by the contact with peripheral blood mononuclear cells (PBMCs); and (3) assess whether such changes are accompanied by changes in cell proliferation and epithelial-mesenchymal transition (EMT). To achieve these goals, the levels of histone H3 acetylation, DNA damage and methylation were assessed by immunofluorescence. In addition, cell co-culture assays with PBMCs and oral keratinocyte lines (NOK-SI, DOK, SCC-25 and Detroit 562) were performed to assess the influence of PBMCs on the acquisition of advantages for tumor progression. The results of the immunofluorescence showed that both OPMDs were hypoacetylated compared to the control. However, when comparing only OL and PVL, we observed that the PVL group was more hyperacetylated than OL. It was also observed that the levels of DNA damage and methylation were increased in the OL group in relation to PVL. The experiments with cell culture revealed that the contact of dysplastic and normal oral keratinocytes with PBMCs was able to favor EMT. Our results revealed two main points: 1) both OPMDs have different epigenetic profiles; 2) the direct contact of PMBCs with normal, dysplastic and tumoral oral keratinocytes seems to be important in early events of tumorigenesis such as EMT.
Descrição
Palavras-chave
Leucoplasia oral, Histonas, Acetilação, Oral leukoplakia, Histones, Acetylation