Oncogenic circDHTKD1 promotes tumor growth and metastasis of oral squamous cell carcinoma in vitro and in vivo via upregulating miR-326-mediated GAB1

Circular RNAs (circRNAs) have been extensively elucidated with regard to their significant implications in oral squamous cell carcinoma (OSCC). This study performed the functional investigation of circRNA dehydrogenase E1 and transketolase domain containing 1 (circDHTKD1) in OSCC. RNA expression levels of different molecules were measured via quantitative real-time polymerase chain reaction (qRT-PCR). Cellular behaviors were detected by 3-(4, 5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) for cell viability, colony formation assay for clonal capacity, flow cytometry for cell apoptosis, wound healing assay for migration, and transwell assay for migration/invasion. Western blot was used for analyzing protein expression. RNA pull-down and dual-luciferase reporter assays were applied to assess the binding between targets. A xenograft tumor model was established in nude mice for in vivo experiments. Our expression analysis revealed that circDHTKD1 was upregulated in OSCC tissues and cells. circDHTKD1 knockdown was shown to impede OSCC cell growth and metastasis but motivate apoptosis. Additionally, circDHTKD1 served as a microRNA-326 (miR-326) sponge and the function of circDHTKD1 was achieved by sponging miR-326 in OSCC cells. Also, miR-326 inhibited OSCC development via targeting GRB2-associated-binding protein 1 (GAB1). circDHTKD1 could sponge miR-326 to alter GAB1 expression. Furthermore, circDHTKD1 contributed to OSCC progression in vivo via the miR-326/GAB1 axis. These data disclosed a specific circDHTKD1/miR-326/GAB1 signal axis in governing the malignant progression of OSCC, showing the considerable possibility of circDHTKD1 as a predictive and therapeutic target for clinical diagnosis and treatment of OSCC.

Glycolysis reprogramming in cancer-associated fibroblasts promotes the growth of oral cancer through the lncRNA H19/miR-675-5p/PFKFB3 signaling pathway / 国际口腔科学杂志·英文版

As an important component of the tumor microenvironment, cancer-associated fibroblasts (CAFs) secrete energy metabolites to supply energy for tumor progression. Abnormal regulation of long noncoding RNAs (lncRNAs) is thought to contribute to glucose metabolism, but the role of lncRNAs in glycolysis in oral CAFs has not been systematically examined. In the present study, by using RNA sequencing and bioinformatics analysis, we analyzed the lncRNA/mRNA profiles of normal fibroblasts (NFs) derived from normal tissues and CAFs derived from patients with oral squamous cell carcinoma (OSCC). LncRNA H19 was identified as a key lncRNA in oral CAFs and was synchronously upregulated in both oral cancer cell lines and CAFs. Using small interfering RNA (siRNA) strategies, we determined that lncRNA H19 knockdown affected proliferation, migration, and glycolysis in oral CAFs. We found that knockdown of lncRNA H19 by siRNA suppressed the MAPK signaling pathway, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and miR-675-5p. Furthermore, the lncRNA H19/miR-675-5p/PFKFB3 axis was involved in promoting the glycolysis pathway in oral CAFs, as demonstrated by a luciferase reporter system assay and treatment with a miRNA-specific inhibitor. Our study presents a new way to understand glucose metabolism in oral CAFs, theoretically providing a novel biomarker for OSCC molecular diagnosis and a new target for antitumor therapy.

Expression of cyclophilin A in oral squamous cell carcinoma and its effect on cell proliferation and invasion / 华西口腔医学杂志

OBJECTIVES@#To investigate the expression of cyclophilin A (CyPA) in oral squamous cell carcinoma (OSCC) and explore the effect of downregulating the expression of CyPA gene on the proliferation and invasion of SCC-25 cells.@*METHODS@#A total of 77 cases of patients with OSCC were selected. The expression levels of CyPA proteins in OSCC and adjacent normal tissues were evaluated. SCC-25 cells were cultured and divided into the CyPA interference sequence group, negative control group, and blank group. The expression levels of CyPA mRNA and protein in cells were detected by using real-time fluorescent quantitative polymerase chain reaction and Western blot, respectively. Cell proliferation was detected by using methyl thiazolyl tetrazolium and plate colony formation assays. Cell invasion was detected by using Transwell assay.@*RESULTS@#The positive expression rate of CyPA protein in OSCC tissues was 76.62%, which was higher than that in adjacent tissues (@*CONCLUSIONS@#The CyPA protein is highly expressed in OSCC tissues, and the downregulation of CyPA gene expression in SCC-25 cells can reduce cell proliferation and inhibit cell invasion.

circ_0005379 inhibits the progression of oral squamous cell carcinoma by regulating the miR-17-5p/acyl-CoA oxidase 1 axis / 华西口腔医学杂志

OBJECTIVES@#To investigate the effects of circ_0005379 on the proliferation, apoptosis, migration, and invasion of oral squamous cell carcinoma (OSCC) cells and its mechanism.@*METHODS@#Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of circ_0005379 and miR-17-5p in OSCC tissues and SCC15 cell lines. Western blot was used to detect the expression levels of acyl-CoA oxidase 1 (ACOX1). The circ_0005379 overexpression vector was transfected into SCC15 cells. Methyl thiazolyl tetrazolium blue staining, flow cytometry, Transwell, and Western blot were used to detect the effects of circ_0005379 overexpression on the proliferation, apoptosis, migration, and invasion of SCC15 cells and the expression of E-cadherin, β-catenin, and Snail proteins. Dual luciferase reporter assay and RNA immunoprecipitation were used to examine the regulation of circ_0005379, miR-17-5p, miR-17-5p, and ACOX1 in SCC15 cells. A nude mouse xenograft model of SCC15 cells stably overexpressing circ_0005379 was established, and the effect of circ_0005379 overexpression on the growth of xenografts in nude mice was observed.@*RESULTS@#Compared with adjacent cancer tissues, the expression levels of circ_0005379 and ACOX1 proteins in OSCC tissues were decreased (@*CONCLUSIONS@#circ_0005379 may inhibit the proliferation, migration, and invasion of OSCC cells by downregulating the expression of miR-17-5p and upregulating ACOX1, which promote apoptosis and inhibit tumor growth

Expression and mechanism of long non-coding RNA HCG22 in oral squamous cell carcinoma / 华西口腔医学杂志

OBJECTIVES@#To investigate the expression and mechanism of the long non-coding RNA (lncRNA) HCG22 in oral squamous cell carcinoma (OSCC).@*METHODS@#HCG22 levels were detected in the OSCC and adjacent tissues, OSCC cells, and normal oral keratinocytes. HCG22 expression in SCC-25 and HSC-3 cells was upregulated by transfection of the overexpressing plasmi dvector. Methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, and Transwell assay were employed to detect changes in cell proliferation, apoptosis, migration, and invasion ability, while Western blotting was used to detect the expression of epithelial-mesenchymal transformation-related proteins. The expression level of miR-650 in the cells was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and dual-luciferase reporter gene assay was applied to assess the targeting relationship between HCG22 and miR-650.@*RESULTS@#Compared with that in adjacent tissues, the expression of HCG22 significantly decreased in OSCC tissues (@*CONCLUSIONS@#HCG22 is expressed at low levels in OSCC. Upregulation of the expression of this lncRNA can inhibit the proliferation, migration, invasion, and epithelial-mesenchymal transition of OSCC cells. The mechanism of action of HCG22 may be related to its targeted regulation of miR-650.

Anti-micro rna-221 a promising genetic therapy of oral squamous cell carcinoma (scc-25)

Abstract Micro-RNA-221(miR-221) is one of oncogenic miRNAs that plays a vital role in the development and progression of oral cancers. The aim of this study is to introduce a new gene therapy for oral squamous cell carcinoma by blocking the expression of oncogenic miR-221 by its inhibitor. The present work was performed on squamous cell carcinoma cell line SCC-25 and anti-miR-221 was delivered to the cells using an ultrasound micro bubbles. Assessment of the effect of miR-221 inhibitor on SCC-25 cells was done using MTT assay, cell cycle analysis and apoptosis detection. In addition, reverse transcription-polymerase chain reaction was also used to detect the expression -miR-221 and its target genes. Using ANOVA, statistical analysis of the results showed significant inhibition of cell viability with and induction of cell apoptosis of SCC-25 cell line after transfection. Moreover, the expression of miR-221, Epidermal growth factor receptor (EGFR) and CDKNIB/p27 were downregulated without significant difference. Transfection of SCC-25 by inhibitor of miR-221 resulting in blockage of its expression leading to arresting of tumor growth. These results proved the effective role of micro-RNA inhibitors as novel therapeutic agent for oral cancers.

Resumo Micro-RNA-221 (miR-221) é um dos miRNAs oncogênicos que desempenham um papel vital no desenvolvimento e progressão de carcinomas orais. O objetivo deste estudo é apresentar uma nova terapia gênica para o carcinoma epidermóide oral por meio do bloqueio da expressão do miR-221 oncogênico por seu inibidor. O presente trabalho foi realizado na linhagem de células de carcinoma de células escamosas SCC-25 e o anti-miR-221 foi administrado às células usando micro-bolhas de ultrassom. A avaliação do efeito do inibidor miR-221 em células SCC-25 foi feita usando ensaio de MTT, análise do ciclo celular e detecção de apoptose. Além disso, a reação em cadeia da polimerase com transcrição reversa também foi usada para detectar a expressão -miR-221 e seus genes-alvo. Usando ANOVA, a análise estatística dos resultados mostrou inibição significativa da viabilidade celular e indução da apoptose celular da linhagem celular SCC-25 após a transfecção. Além disso, a expressão de miR-221, receptor do fator de crescimento epidérmico (EGFR) e CDKNIB/p27 foram regulados para baixo sem diferença significativa. A transfecção de SCC-25 por inibidor de miR-221 resultou no bloqueio de sua expressão, levando à interrupção do crescimento do tumor. Esses resultados comprovaram o papel eficaz dos inibidores de micro-RNA como novo agente terapêutico para carcinomas orais.

The effect of Maras powder and smoking on the microRNA deregulation of oral mucosa

Abstract Objective This study aimed to investigate the effects of Maras powder (a type of smokeless tobacco obtained from Nicotiana rustica Linn and mixed with the ashes of wood, especially from oak, walnut or grapevine) on the microRNA (miRNA) deregulation of oral mucosa, and it compares these effects with those of smoking. Methodology Oral mucosal samples were collected from 74 patients, consisting of 16 nonusers, 26 smokers, and 32 Maras powder users. Genes associated with oral cancer were selected and 90 microRNAs targeting these genes were identified. MicroRNA were isolated and purified using the microRNA isolation kit. MicroRNA were expressed using Fluidigm RT-PCR. Results A positive correlation between the duration of Maras powder use with miR-31 expression levels, and a negative correlation between the Maras powder chewing time and miR-372 expression levels was found. In addition, there is a negative correlation between the amount of Maras powder consumed and expression levels of miR-375, miR-378a, miR-145, and miR-10b; moreover, another negative correlation is observed between the number of cigarettes consumed and the expression levels of miR-23a, miR-23b, miR-203a, miR-200b, and miR-375. However, miR-200b and miR-92a levels were downregulated significantly more in Maras powder users when compared with smokers and nonusers (p<0.05). Conclusion The results show both chewing Maras powder and smoking have an effect on deregulation of miR-200b and miR-92a expressions. This leads to the belief that assessing the expression of these two miRNAs is a promising noninvasive method of analysis, especially in mutagen exposures. Finally, large-scale and high-throughput studies may help to identify an extensive miRNA expression profile associated with tobacco use and improve the understanding of oral malignancies.

MicroRNA model that can predict the prognosis of oral squamous cell carcinoma based on bioinformatics analysis / 华西口腔医学杂志

OBJECTIVE@#The microRNA (miRNA) prognostic model can predict the prognosis of patients with oral squamous cell carcinoma (OSCC) on the basis of bioinformatics. Moreover, it can accurately group OSCC patients to improve targeted treatment.@*METHODS@#We downloaded the miRNA and mRNA expression profile and clinical data of OSCC from The Cancer Genome Atlas (TCGA). The risk score model of miRNA was screened and established by univariate and multivariate Cox regression models. The performance of this prognostic model was tested by receiver operating characteristic (ROC) curves and area under the curve (AUC). The target genes of six miRNAs were predicted and intersected with differential mRNA for enrichment analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway and gene ontology (GO) enrichment analysis. A protein protein interaction network (PPI) was constructed to screen hub genes.@*RESULTS@#By using univariate and multivariate Cox regression analyses, the prognostic risk model was obtained. The AUC of the ROC curve for predicting 5-year survival in the training group, test group, and whole cohort were 0.757, 0.673, and 0.724, respectively. Furthermore, univariate Cox regression and multivariate Cox regression considering other clinical factors showed that the six-miRNAs signature could serve as an independent prognostic factor (P<0.001). The top 10 hub genes in the PPI network screened by intersecting target genes include CCNB1, EGF, KIF23, MCM10, ITGAV, MELK, PLK4, ADCY2, CENPF, and TRIP13. EGF and ADCY2 were associated with survival prognosis (P<0.05).@*CONCLUSIONS@#The six-miRNAs signature could efficiently function as a novel and independent prognostic model for OSCC patients, which may be a new method to guide the accurate targeting treatment of OSCC.

Comparative evaluation of micronuclei in saudi smokers and non-smokers without any visible oral lesions: a pilot study / Evaluación comparativa de micronúcleos en fumadores y no fumadores sauditas sin lesiones orales visibles: un estudio piloto

Objectives: A systematic review was conducted to evaluate effectiveness and safety of beta carotenes for the treatment of oral leukoplakia regarding clinical resolution and prevention of malignant transformation. Material and Methods: The systematic search was conducted in three electronic databases and the study's selection was performed according to pre-set eligibility criteria. Four studies evaluating the efficacy of beta carotenes in oral leukoplakia compared to placebo were included in the review; three of which were assigned for quantitative analysis. Data were extracted, tabulated, quality assessed and statistically analyzed. Results: The meta-analysis revealed that when comparing clinical resolution the beta carotene group favored was favored compared to placebo, with statistically significant difference. However, a meta-analysis comparing beta carotene and placebo groups regarding malignant transformation as a primary outcome failed to show any significant benefit. Furthermore, results showed evidence of beta carotene safety. Conclusion: the overall quality of evidence about efficacy of beta carotene in oral leukoplakia treatment was not high. However, given the obvious safety of this agent, data suggests it could have a promising effect in clinical improvement of oral leukoplakia lesions. However, no evidence supporting its benefits in reducing risk of malignant transformation in these lesions was found. Therefore, further long term, well designed randomized clinical trials are highly recommended.

Introducción: el cáncer oral es un problema grave con alta mortalidad y morbilidad, a pesar de la disponibilidad de los mejores tratamientos. Uno de los factores más importantes para una mortalidad tan alta es su diagnóstico tardío. La mejor manera de enfrentar un problema de este tipo es evitar su aparición creando conciencia entre la población y tenendo un diagnóstico más temprano. El cáncer oral es una enfermedad multifactorial, donde el daño genómico tiene un papel. Se ha demostrado que los micronúcleos (MNi) son un biomarcador importante y en este estudio se utilizó como una herramienta para crear conciencia sobre el riesgo de cáncer oral. Objetivo: evaluar y comparar la frecuencia de MNi en fumadores sin ninguna lesión oral visible (Grupo I) y no fumadores sanos (Grupo II). Materiales y métodos: se obtuvieron citoestimuladores de fumadores sauditas (n = 15, Grupo I) sin ninguna lesión oral visible y no fumadores sanos (n = 15, control, Grupo II) y se tiñeron con hemotoxilina y eosina para evaluar la frecuencia de MNi y las observaciones fueron sometidas a análisis estadístico utilizando la prueba t de Student. Resultados: La frecuencia media de MNi en el Grupo I fue significativamente mayor (p<0.05) que en el Grupo II. El estudio ayuda a educar, motivar y crear conciencia, alentando así a los pacientes a dejar de fumar, y evitando así el cáncer oral antes de su inicio.

Susceptibilidad genética frente al cáncer bucal por infección del virus del papiloma humano / Genetic susceptibility to oral cancer due to human papillomavirus infection

Resumen Los carcinógenos ambientales y el virus del papiloma humano (VPH) son los principales responsables del cáncer bucal. Los factores de susceptibilidad en el genoma humano desempeñan un papel modulador del riesgo, sin embargo, no todos los individuos expuestos a los carcinógenos padecen cáncer. El objetivo de la presente revisión es describir los principales factores de susceptibilidad genética para cáncer bucal en individuos con infección por VPH. Se realizó una búsqueda sistemática en tres bases de datos en inglés; solo siete artículos cumplieron con los criterios de selección. Se registraron polimorfismos genéticos en tres categorías relacionados con el VPH y que participan en la oncogénesis. Se identificaron tres artículos relativos a la desregulación de los mecanismos de control del ciclo celular, uno relativo a mutaciones en la vía de la apoptosis y tres a polimorfismos en genes de respuesta inflamatoria e inmune. La asociación entre polimorfismos para el desarrollo de cáncer bucal y VPH es evidente, aunque continúa en estudio ya que no siempre el patrón de oncogénesis de las neoplasias bucales está relacionado con el VPH, sino con otros factores ambientales o epigenéticos.

Abstract Environmental carcinogens and human papillomavirus (HPV) are the main responsible factors for oral cancer. Susceptibility factors in the human genome play a risk-modulating role; however not all individuals exposed to these carcinogens suffer from cancer. The purpose of the present review is to describe the main factors of genetic susceptibility to oral cancer due to HPV infection. A systematic search was carried out in three databases in English, with only 7 articles meeting the selection criteria. Genetic polymorphisms are shown in three categories, which are related to HPV and participate in oncogenesis. Three articles related to deregulation of cell cycle control mechanisms were identified, as well as one referring to mutations in the apoptosis pathway and three about polymorphisms in inflammatory and immune response genes. The association of polymorphisms for the development of oral cancer by HPV is evident, although it remains under study. Oral neoplasms’ oncogenesis pattern is not always associated with HPV, but with other environmental or epigenetic factors.

Prevalence of HPV and EBV infection andtheir relationship with the p53 and PCNA expression in oral carcinoma patients

Introduction: Infection caused by potentially oncogenic viruses, such as HPV and EBV, favors the role of certain oncoproteins that can induce dysplasias and malignant lesions. Objective: To evaluate the prevalence of HPV and EBV and their relation with the expression of p53 and PCNA in patients with oral carcinoma. Methodology: Twenty-seven oral squamous cell carcinomas (OSCC) were evaluated; DNA extraction was conducted using the QIAamp DNA mini kit; viral detection was obtained using the INNO-LiPA kit for HPV, and nested PCR was used for EBV. The evaluation of molecular markers was performed through immunohistochemical staining. Results: The mean age of the patients was 60.55 +/- 13.94 years, and 52 percent of these were female. Of the patients, 59 percent were tobacco users and 63 percent were alcohol consumers. HPV was detected in 70 percent of the patients with the predominance of genotype 16 (60 percent). As for EBV infection, it was observed in 59 percent of cases. p53 and PCNA immunopositivity corresponded to 44 percent and 59 percent, respectively. The tongue was the anatomical location with highest positivity for both viruses as well as for the expression of molecular markers. The 48 percent of the cases presented infection by both viruses. Conclusion: HPV and EBV infection together with the expression of p53 and PCNA were more frequently observed in advanced stages of the disease, suggesting a more relevant role in the progression than in tumor genesis.

Polymorphisms in methylenetetrahydrofolate reductase and cystathionine beta-synthase in oral cancer - a case-control study in southeastern Brazilians / Polimorfismos em metilenotetrahidrofolato redutase, cistationina beta-sintase no câncer de boca - um estudo de caso-controle no Sudeste brasileiro

ABSTRACT INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a serious public health problem, due to its high mortality rate and worldwide rising incidence. OSCC susceptibility is mediated by interactions between genetic and environmental factors. Studies suggest that genetic variants encoding enzymes involved in folate metabolism may modulate OSCC risk by altering DNA synthesis/repair and methylation process. OBJECTIVE: The goals of this study were to evaluate the association of three genotypic polymorphism (MTHFR C677T, MTHFR A1298C and CBS 844ins68) and oral cancer risk in southeastern Brazilians and evaluate the interactions between polymorphisms and clinical histopathological parameters. METHODS: This case-control study included 101 cases and 102 controls in the state of Espírito Santo, Brazil. MTHFR genotyping was done by PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) and CBS genotyping by PCR (polymerase chain reaction) analysis. RESULTS: MTHFR C677T polymorphism was associated with lymph node involvement. Genotype CT + TT acted as a protective factor. MTHFR A1298C AC + CC genotype was associated with tumor differentiation, and possibly with a better prognosis. In risk analysis, no correlation was observed between genotypes and OSCC. CONCLUSION: We concluded that MTHFR C677T, MTHFR A1298C and CBS 844ins68 polymorphisms were not associated with OSCC risk in southeastern Brazilians; however, we suggest a prognosis effect associated with MTHFR C677T and A1298C polymorphisms in OSCC.

Resumo Introdução: O carcinoma espinocelular oral (CECO) trata-se de um importante problema de saúde pública, devido à elevada taxa de mortalidade e incidência crescente em todo o mundo. A susceptibilidade ao CECO é mediada por interações entre fatores genéticos e ambientais. Estudos sugerem que as variantes genéticas que codificam as enzimas envolvidas no metabolismo do folato podem modular o risco de CECO, alterando a síntese/reparação do DNA e o processo de metilação. Objetivo: Os objetivos deste estudo foram avaliar a associação de três polimorfismos genotípicos (MTHFR C677T, MTHFR A1298C e CBS 844ins68) e o risco de câncer oral em brasileiros da região Sudeste, e avaliar as interações entre polimorfismos e parâmetros clínico-histopatológicos. Método: Este estudo de caso-controle incluiu 101 casos e 102 controles no estado do Espírito Santo, Brasil. A genotipagem do polimorfismo MTHFR foi realizada por PCR-RFLP (Reação de Polimerase em Cadeia - Polimorfismo no Comprimento de Fragmento de Restrição) e a do CBS por análise da PCR (Reação de Polimerase em Cadeia). Resultados: O polimorfismo MTHFR C677T foi associado ao envolvimento de gânglios linfáticos. O genótipo CT + TT atuou como um fator protetor. O genótipo MTHFR A1298C AC + CC foi associado à diferenciação do tumor e, possivelmente, a um prognóstico melhor. Na análise de risco, a correlação entre os genótipos e o CECO não foi observada. Conclusão: Concluímos que os polimorfismos MTHFR C677T, MTHFR A1298C e CBS 844ins68 não estão associados ao risco de CECO nos brasileiros da região Sudeste; no entanto, sugerimos um efeito prognóstico associado aos polimorfismos MTHFR C677T e A1298C em CECO.

Salivary Biomarkers for Oral Squamous Cell Carcinoma: An Overview.

Oral Cancer encompasses an important faction of neoplasms of head and neck. More than 90% of oral cancers are oral squamous cell carcinomas. Ideally, this fatal disease is detected through a comprehensive clinical examination by the oral health care professionals. Regardless of the fact that the oral cavity is easily accessible, most oral cancers are typically detected at an advanced stage. This has attributed to lower survival rate. Saliva, an aqueous biological fluid is in direct contact with the oral cancer lesion. Hence, the abnormal DNA, RNA, protein molecules released by the malignant cells can be easily obtained from saliva. Saliva, being a noninvasive diagnostic aid can be an alternative to serum for early detection, monitoring post therapy status, prognosis of oral cancer patients. This article aims to provide a brief overview of various salivary biomarkers and their implications in oral cancer.

Effect of Super-Oxidized Water, Sodium Hypochlorite and EDTA on Dentin Microhardness

The present study aimed to evaluate the influence of the following irrigating solutions on the microhardness of root canal dentin: 2% sodium hypochlorite (2NaOCl), 5% sodium hypochlorite (5NaOCl), super-oxidized water (400 ppm Sterilox - Sx) and 17% EDTA (E). Eighty roots from bovine incisors were randomly divided into 8 groups (n=10): 2NaOCl, 5NaOCl, Sx, and 2NaOCl + E, 5NaOCl + E, Sx + E (associated with E as final irrigant for 5 min), E solely and distilled water (dH2O) as the negative control. Root canal preparation was performed by hand instruments, using one of the irrigation protocols for 30 min. Then, 5 mm of the cervical root third were cut out from each sample and subjected to the Vickers microhardness test, at two points, one at approximately 500-1000 µm from the root canal lumen (distance 1), and the other at approximately 500-1000 µm from the external root surface (distance 2). Data were analyzed by Wilcoxon and Kruskal-Wallis tests at 5% significance level. Microhardness values at distance 1 were significantly lower than those at distance 2 for all groups, except 5NaOCl and 5NaOCl + E groups (p>0.05). EDTA showed the lowest microhardness values. However, no statistically significant difference was detected among groups at distance 1 and EDTA was significantly different only from Sx at distance 2. In conclusion, all tested solutions showed lower microhardness at the most superficial root canal dentin layer compared to the one found near the external root surface, except 5NaOCl and 5NaOCl + E; EDTA promoted lower microhardness values in comparison to Sterilox at this site.

O presente estudo teve como objetivo avaliar a influência das seguintes soluções irrigadoras na microdureza da dentina do canal radicular: hipoclorito de sódio a 2% (NaOCl2), hipoclorito de sódio a 5% (NaOCl5), água superoxidada (Sterilox(r) 400 ppm - Sx) e EDTA a 17% (E). Oitenta raízes de incisivos bovinos foram divididas aleatoriamente em 8 grupos (n=10): NaOCl2, NaOCl5, Sx e NaOCl2 + E, NaOCl5 + E, Sx + E (associados ao E como irrigante final por 5 min), E isolado e água destilada (H2Od), como controle negativo. O preparo dos canais radiculares foi realizado com instrumentos manuais, usando um dos protocolos de irrigação por 30 min. A seguir, 5 mm do terço cervical de cada amostra foram cortados perpendicularmente e submetidos ao teste de microdureza de Vickers, em dois pontos, um aproximadamente 500-1000 µm da luz do canal radicular (distância 1), e o outro aproximadamente 500-1000 µm da superfície externa da raiz (distância 2). Os dados foram analisados pelos testes de Wilcoxon e Kruskal-Wallis com um nível de significância de 5%. Os valores de microdureza na distância 1 foram significativamente menores do que na distância 2 para todos os grupos, exceto NaOCl5 e NaOCl5 +E (p>0,05). O EDTA mostrou os menores valores de microdureza. No entanto, não foi detectada diferença estatisticamente significativa entre os grupos na distância 1 e o EDTA foi significativamente diferente apenas do Sx na distância 2. Pode-se concluir que todas as soluções testadas mostraram menor microdureza na camada de dentina mais superficial do canal radicular em comparação aos valores encontrados próximo à superfície radicular externa, exceto NaOCl5 e NaOCl5 + E; o EDTA promoveu menor microdureza em comparação ao Sterilox(r) neste ponto.

Genetic polymorphisms in the Cytochrome P450 family and squamous cell carcinoma of the oral cavity, pharynx and larynx / Polimorfismos genéticos da família Citocromo P450 e carcinoma de células escamosas de cavidade oral, faringe e laringe

Objective: To analyze the genetic polymorphisms of the cytochrome P450 family and their relationship with squamous cell carcinoma of the oral cavity, pharynx and larynx. Methods: We present a narrative literature review, conducted in Pubmed, Lilacs and Cochrane Databases of articles published in the last five years correlating genetic polymorphisms of the cytochrome P450 family and cancer risk in different populations worldwide. Results: We initially found 65 articles and, after selection criteria, 20 case-control studies with various populations worldwide were eligible. The most studied polymorphisms were those of CYP2E1 and CYP1A1 subfamilies. There is little about the other subfamilies. The association found between polymorphisms and cancer risk amounted to a countless number of variables, amongst them: population, selection methods, racial factors and different modes of exposure to carcinogens, genotyping methods, and nomenclature of the polymorphisms. Conclusion: so far, there is no proven link between genetic polymorphisms of cytochrome P450 family and squamous cell carcinoma of the oral cavity, pharynx and larynx relationship. .

Objetivo: analisar os polimorfismos genéticos da família Citocromo P450 e sua relação com o carcinoma de células escamosas de cavidade oral, faringe e laringe. Métodos: por meio de uma Revisão Narrativa de literatura, realizada nas principais bases de dados Pubmed, Lilacs, e Cochrane Database, de artigos publicados nos últimos cinco anos, correlacionando polimorfismos genéticos da família citocromo P450 e risco de câncer nas diversas populações mundiais. Resultados: foram encontrados inicialmente 65 artigos, que, após critérios de seleção, tornaram elegíveis 20 artigos do tipo caso-controle em diversas populações mundiais. Os polimorfismos mais estudados foram os das subfamílias CYP1A1 e CYP2E1. Pouco existe sobre as demais subfamílias. A associação entre os polimorfismos encontrados e risco de câncer sofreu um incontável número de variáveis, entre elas, população estudada, métodos de seleção, fatores raciais e diferentes modos de exposição aos carcinógenos, métodos de genotipagem, e nomenclatura dos polimorfismos. Conclusão: até o momento, não existe relação comprovada entre os polimorfismos genéticos da família Citocromo P450 e o carcinoma de células escamosas de cavidade oral, faringe e laringe. .

Assessing extent of single stranded DNA damage in oral mucosal cells of patients with oral squamous cell carcinoma and its correlation with TNM staging.

Context: This study was carried out on the assumption that oral mucosal cells might show DNA damage in oral squamous cell carcinoma (OSCC). Aims: To evaluate the extent of DNA damage in oral smears of patients with OSCC and determine correlation if any of the extent of DNA damage to TNM staging of oral cancer. Settings and design: A randomized controlled study at a regional cancer centre was designed for this project. Smears were taken from lesion proper of 30 patients with OSCC and from the buccal mucosa of 30 normal healthy volunteers. Materials and methods: Collected cells were centrifuged and single-cell gel electrophoresis (SCGE) assay was performed. DNA damage was visualized under a fluorescent microscope. Statistical analysis used : Mean DNA damage levels of both the groups were measured and statistically analyzed with students' test. The extent of DNA damage was correlated with the TNM stages by employing the one way ANOVA 'F' technique. Results: High statistical significance (P < 0.0001) was found in DNA damage levels between control and study groups. A stepwise increase in DNA damage levels with high statistical significance (P < 0.005) was also found between all the TNM stages. Conclusions: Statistically significant increased DNA damage levels in OSCC patients and their correlation to clinical staging suggest that comet assay may be used effectively to assess the prognosis of OSCC.

Padronização da análise de mutações no gene TP53 em carcinomas de células escamosas de boca em material parafinado / Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues

Introduction: The tumor protein p53 gene (TP53) is a constant target of investigation in cancer pathogenesis. Analysis by immunohistochemistry provides limited data about p53 in oral carcinogenesis, and TP53 sequencing can contribute to this analysis. However, obtaining high-quality and contamination-free deoxyribonucleic acid (DNA) for a proper amplification can be a difficult task when using paraffin-embedded tissues. Objective: Standardize DNA extraction, polymerase chain reaction (PCR) amplification and DNA sequencing techniques for TP53 mutation analysis. Material and methods: Thirty-nine cases of oral squamous cell carcinoma (OSCC) were selected from the Pathology Division of Instituto Nacional de Câncer (Inca). The DNA extraction method used was the QIAamp® DNA minikit® system. After DNA quantification by spectrophotometry, 250 ng of genetic material obtained from TP53 gene were amplified by PCR for exon 2 and by nested PCR for exon 6. Out of the total sample, 11 cases were selected for exon 2 sequencing. Results: The DNA samples presented mean concentration of 119.74 ± 88.86 ng/µl (28.9-556.4) and purity of 1.69 ± 0.18 (1-1.9). Thirty-three (84.6%) samples were amplified for exon 2, and all samples for exon 6 (39/100%). Readable sequencing data were obtained in 10 (90.9%) cases. Conclusion: Optimization of conditions for TP53 sequencing was obtained, and this will facilitate the analysis of mutations in paraffin-embedded tissues, allowing molecular retrospective studies...

Introdução: O gene TP53 (proteína tumoral p53) é alvo constante de investigação na patogênese do câncer. A imuno-histoquímica fornece dados limitados na análise de p53 no processo da carcinogênese bucal e o sequenciamento de TP53 pode contribuir nessa investigação. Contudo, a obtenção de ácido desoxirribonucleico (DNA) com qualidade para amplificação e livre de contaminação pode constituir uma tarefa difícil na utilização de material parafinado. Objetivo: Padronizar as técnicas de extração de DNA, amplificação por reação em cadeia da polimerase (PCR) e sequenciamento para a análise de mutações em TP53. Material e métodos: Foram selecionados 39 casos de carcinomas de células escamosas bucal da Divisão de Patologia do Instituto Nacional de Câncer (Inca). O DNA foi extraído utilizando o sistema comercial QIAamp® DNA minikit®. Após quantificação do DNA por espectrofotometria, 250 ng de amostra foram amplificados pela técnica de PCR para o éxon 2 e por nested PCR para o éxon 6 do gene TP53. Da amostra total, 11 casos foram selecionados para a padronização da reação de sequenciamento do éxon 2. Resultados: As amostras de DNA apresentaram concentração média de 119,74 ng/µl ± 88,86 (28,9-556,4 ng/µl) e pureza de 1,69 ± 0,18 (1-1,9). Do total das amostras analisadas, 33 (84,6%) foram amplificadas para o éxon 2, e todas (39/100%), para o éxon 6. No sequenciamento do éxon 2 obtiveram-se sequências passíveis de leitura em 10 (90,9%) casos. Conclusão: A otimização das condições para o sequenciamento de TP53 foi obtida, o que facilitará a análise de mutações em tecidos parafinados, permitindo...

Genes and oral cancer.

Oral cancers have been one of the leading causes of deaths particularly in the developing countries. Prime reason for this high mortality and morbidity is attributed to the delay in diagnosis and prompt treatment. Relentless research in the field of oncology has led to the advent of novel procedures for the early detection of oral cancers. Molecular biology is highly promising in this regard. It is a procedure that detects alterations at a molecular level much before they are seen under a microscope and much before clinical changes occur. Molecular studies serve as the basis by which we will eventually be able not only to augment clinical assessment and classification of oral lesions but also predict malignant potential of oral lesions, thus reducing the incidence and increasing the scope for early diagnosis and treatment of oral cancers. However, making such sophisticated tools available for the common man in developing countries is one of the most important challenges faced today.

Role of protein kinases and Kirsten Ras pathways in oral squamous cell carcinoma and poorly differentiated oral squamous cell carcinoma in Punjab [Pakistan] population

Oral Squamous Cell Carcinoma [OSCC] develops by accumulation of multiple genetic alterations, influenced by the patient's genetic predisposition as well as by environmental influences, that includes pan, chaalyia, tobacco, alcohol, chronic inflammation, and viral infection. This study was carried out to understand the molecular alterations which contribute to the development of OSCC in Pakistani population. The study was conducted on a sample of 53 patients collected from different hospitals of Lahore. Results of this study indicate that Akt levels shows higher expression with increase in grades. The value of Akt1 in well differentiated grade 1 tissue is of the order of 0.09+0.00, Akt2 is 0.04+0.00 and Akt3 is 0.02+0.00 while in poorly differentiated tissues the values of Akt 1, Akt and Akt3 are 0.22+0.09, 0.13+0.03 and 0.06+0.01 respectively. kRas is an oncogene which is highly elevated in both grades [well differentiated: 0.09+0.02 and poorly differentiated: 0.13+0.02] in all samples of OSCC. In conclusion, our data demonstrated that Akt isoforms and kRas significantly control the cancer transition pathway. It is seen that Akt 1expression rises from 2.5- fold in well differentiated tissues to 3.5-fold in poorly differentiated tissues. The Akt-2 on the other hand shows only 0.5-fold increases from normal tissue in grade 1 tissues, but rises to 4-fold in grade-3 tissue. On the other hand there was no change in Akt-3 as compared to normal in grade I tissues yet, 3-fold increase has been recorded in grade III tissue. The oncogene K-RaS shows consistent increase of the order of 2.5-fold in grade I and 3-fold in grade III. This information combined with histopathological reports can further improve our understanding of the prognosis of oral squamous cell carcinoma

Radiation up-regulates the expression of VEGF in a canine oral melanoma cell line

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.