Análise da expressão de proto-oncogenes e genes supressores tumorais em células epiteliais orais normais e neoplásicas promovida por metabólitos de biofilmes simples e misto de Candida albicans e Staphylococcus aureus
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Data
2020-12-09
Autores
Amaya Arbeláez, Maria Isabel
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Universidade Estadual Paulista (Unesp)
Resumo
A microbiota oral desempenha um papel importante, tanto no microbioma humano como na saúde. Os micro-organismos presentes na cavidade bucal têm a capacidade de produzir metabólitos e toxinas, gerando relação disbiótica com o hospedeiro e levando ao desenvolvimento de diversas patologias. A inflamação crônica produzida pelo estímulo bacteriano, a inibição da apoptose e produção de espécies reativas de oxigênio, tem sido descritos como mecanismos de ação da microbiota oral na tumorigênese de cabeça e pescoço. Dados do nosso grupo de pesquisa evidenciam que metabólitos derivados de biofilmes simples e misto de Candida albicans e Staphylococcus aureus, são capazes de promover resposta inflamatória e morte celular de queratinócitos orais normais. Neste sentido, o objetivo deste trabalho foi analisar a expressão de proto-oncogenes e genes supressores tumorais em células epiteliais orais normais (NOK-si) e neoplásicas (SCC 25 e Detroit 562) estimuladas com metabólitos (fatores solúveis) provenientes de biofilmes simples e mistos de C. albicans e S. aureus. Para a obtenção dos fatores solúveis (FS) produzidos pelos micro-organismos, foram formados biofilmes jovens e maduros simples e mistos de C. albicans e S. aureus, com 16 e 36 horas de cultivo, respectivamente. Os FS foram coletados e incubados com as células epiteliais orais. Primeiramente, foi realizado o ensaio de viabilidade celular por AlamarBlueTM e a morfologia celular foi observada por microscopia confocal para determinar o tempo de exposição das linhagens celulares aos FS dos biofilmes. Realizou-se a análise de perfil do ciclo celular por citometria de fluxo. A análise da expressão de proto-oncogenes e genes supressores tumorais foi realizada por qRT-PCR. Em geral, a exposição aos FS de biofilmes de C. albicans e misto causou redução na viabilidade das células NOK-si e aumento na fase sub G0 do ciclo celular, com diminuição da expressão gênica em HRAS, BRAF, MEK1, AKT, mTOR,TP53, CDKN1A, CDKN1B. No entanto, nesta linhagem, os FS de biofilmes simples de S. aureus produziram uma superexpressão do gene CDKN1A e Bcl-2, seguido pela manutenção da viabilidade celular, e um aumento significativo da população G2/M. Nas células tumorais, SCC 25 e Detroit 562, os estímulos dos FS provenientes dos biofilmes, regularam positivamente proto-oncogenes como HRAS e mTOR, bem como a superexpressão de Bcl-2 e CDKN1A. As células tumorais apresentaram capacidade de sobrevivência mesmo após 24 h de estímulo com FS de 16 h e 36 h, sem mudanças significativas na progressão do ciclo celular para a linhagem SCC 25 e com uma tendência significativa de aumento de fase G2/M, para a linhagem Detroit 562. Os resultados mostraram que metabólitos provenientes de biofilmes fúngicos e bacterianos de C. albicans e S. aureus, tanto jovens como maduros, são capazes de geram alterações ligadas ao processo de tumorigênese, devido à capacidade de promover alterações na viabilidade, dinâmica do ciclo celular e na expressão de genes pro-tumorais e supressores tumorais em linhagens epiteliais orais normais e neoplásicas.
The oral microbiota plays an important role both in the human microbiome and in health. The microorganisms present in the oral cavity are able to produce metabolites and toxins, generating a dysbiotic relationship with the host and leading to the development of different pathologies. Thus, the aim of this study, was to analyze the expression of proto-oncogenes and tumor suppressor genes in oral epithelial cells, normal (NOK-si), and neoplastic (SCC 25 and Detroit 562) stimulated with metabolites (soluble factors) from single and dual biofilms of Candida albicans and Staphylococcus aureus. To obtain the soluble factors (SF), biofilms from C. albicans and S. aureus of 16 h and 36 h were formed. The SF were collected and incubated with the oral epithelial cells. Cell viability assay was performed by AlamarBlueTM. The cell morphology was observed by confocal microscopy to determine the exposure time of the cell lines to the biofilms SF. Cell cycle profile analysis was performed by flow cytometry. Proto-oncogenes and tumor suppressor genes expression were analyzed by qRT-PCR. In general, exposure to C. albicans and dual SF, caused a reduction in NOK-si cell viability and an increased sub G0 phase of the cell cycle, with a downregulation of HRAS, BRAF, MEK1, AKT, mTOR, TP53, CDKN1A and CDKN1B genes. However, in this cell line, S. aureus SF induced an overexpression of CDKN1A and Bcl2 genes, followed by the cell viability maintenance and a significant increase in the G2/M population. For tumoral cells, SCC 25 and Detroit 562, the stimuli of biofilms SF upregulated oncogenes such as HRAS and MTOR, as well as Bcl2 and CDKN1A overexpression. These cells could survive even after 24 h of stimuli from both SF (young and older) without significant changes in cell cycle progression for SCC 25 and a significant tendency to increase G2/M phase for Detroit 562. These results point that metabolites from fungal and bacterial biofilms of C. albicans and S. aureus, can disrupt the homeostasis of normal and neoplastic oral epithelial cells altering proto-oncogenes and tumor suppressor genes expression, remarkably CDKN1A, Bcl2, HRAS, and mTOR genes, causing disturbance on cell viability in cells with normal phenotype (NOK-si), and a pro-survival stimuli mainly in neoplastic cell lines (SCC 25 and Detroit 562). In addition, disturbances in cell cycle profile were showed.
The oral microbiota plays an important role both in the human microbiome and in health. The microorganisms present in the oral cavity are able to produce metabolites and toxins, generating a dysbiotic relationship with the host and leading to the development of different pathologies. Thus, the aim of this study, was to analyze the expression of proto-oncogenes and tumor suppressor genes in oral epithelial cells, normal (NOK-si), and neoplastic (SCC 25 and Detroit 562) stimulated with metabolites (soluble factors) from single and dual biofilms of Candida albicans and Staphylococcus aureus. To obtain the soluble factors (SF), biofilms from C. albicans and S. aureus of 16 h and 36 h were formed. The SF were collected and incubated with the oral epithelial cells. Cell viability assay was performed by AlamarBlueTM. The cell morphology was observed by confocal microscopy to determine the exposure time of the cell lines to the biofilms SF. Cell cycle profile analysis was performed by flow cytometry. Proto-oncogenes and tumor suppressor genes expression were analyzed by qRT-PCR. In general, exposure to C. albicans and dual SF, caused a reduction in NOK-si cell viability and an increased sub G0 phase of the cell cycle, with a downregulation of HRAS, BRAF, MEK1, AKT, mTOR, TP53, CDKN1A and CDKN1B genes. However, in this cell line, S. aureus SF induced an overexpression of CDKN1A and Bcl2 genes, followed by the cell viability maintenance and a significant increase in the G2/M population. For tumoral cells, SCC 25 and Detroit 562, the stimuli of biofilms SF upregulated oncogenes such as HRAS and MTOR, as well as Bcl2 and CDKN1A overexpression. These cells could survive even after 24 h of stimuli from both SF (young and older) without significant changes in cell cycle progression for SCC 25 and a significant tendency to increase G2/M phase for Detroit 562. These results point that metabolites from fungal and bacterial biofilms of C. albicans and S. aureus, can disrupt the homeostasis of normal and neoplastic oral epithelial cells altering proto-oncogenes and tumor suppressor genes expression, remarkably CDKN1A, Bcl2, HRAS, and mTOR genes, causing disturbance on cell viability in cells with normal phenotype (NOK-si), and a pro-survival stimuli mainly in neoplastic cell lines (SCC 25 and Detroit 562). In addition, disturbances in cell cycle profile were showed.
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Palavras-chave
Candida albicans, Staphylococcus aureus, Biofilmes, Câncer oral, Metabólitos, Candida albicans, Staphylococcus aureus, Biofilms, Oral cancer, Metabolites.