The enhanced genomic 6 mA metabolism contributes to the proliferation and migration of TSCC cells / 国际口腔科学杂志·英文版

In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N6-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.

Immunoexpression of cell proliferation markers in oral squamous cell carcinoma / Inmunoexpresiones de marcadores de proliferación celular en carcinoma de células escamosas orales

This study aimed to assess the immunoexpression of cell proliferation markers (Ki-67 and Mcm-2) in oral tongue cancer, correlating it with patients' age and prognostic indicators. Sample was composed of 22 cases under 40 years and 22 over 50 years of age. Clinical staging and histological grade of malignancy were obtained. Cell proliferation was evaluated through labeling indices. Statistical analysis was performed (p<0.05 for statistical significance). Most young patients were stages III/IV (n=13/65 %) and most older patients were stages I/II (n=11/61.1 %) (p>0.05). Mean Ki-67-LI in young and older patients was 42.4 % and 44.15 %, respectively (p>0.05). Mean Mcm-2-LI was higher in older (63.6 %) than in young patients (55.75 %) (p<0.05). We found that young patients presented more aggressive lesions in comparison to older patients, however Mcm-2 expression was significantly higher in older than in young patients. SCC of tongue can be more aggressive in young patients, and this may not be related to cell proliferation. Our findings for Mcm-2 LI and Ki-67 LI suggests that Mcm-2 could be a more sensitive marker for cell proliferation.

Este estudio tuvo como objetivo evaluar la inmunoexpresión de marcadores de proliferación celular (Ki-67 y Mcm-2) en el cáncer de lengua oral, correlacionándolo con la edad de los pacientes y los indicadores pronósticos. La muestra estuvo compuesta por 22 personas menores de 40 años y 22 personas mayores de 50 años. Se identificaron los estadios clínicos y el grado histológico de malignidad. La proliferación celular se evaluó mediante índices de marcado. Se realizó análisis estadístico (p <0,05 para significación estadística). La mayoría de los pacientes jóvenes eran estadios III / IV (n = 13/65 %) y la mayoría de los pacientes mayores eran estadios I / II (n = 11 / 61,1 %) (p> 0,05). La media de Ki-67-LI en pacientes jóvenes y mayores fue 42,4% y 44,15%, respectivamente (p> 0,05). La media de Mcm-2-LI fue mayor en pacientes mayores (63,6 %) que en pacientes jóvenes (55,75 %) (p <0,05). Se encontró que los pacientes jóvenes presentaron lesiones más agresivas en comparación con los pacientes mayores, sin embargo la expresión de Mcm-2 fue significativamente mayor en pacientes mayores que en pacientes jóvenes. SCC de la lengua puede ser más agresivo en pacientes jóvenes, y esto no puede estar relacionado con la proliferación celular. Nuestros hallazgos para Mcm-2 LI y Ki-67 LI sugieren que Mcm-2 podría ser un marcador más sensible para la proliferación celular.

Correlation of β-catenin expresssion and metastasis in tongue squamous cell carcinoma / Correlação da expressão da β-catenina e presença de metástases em carcinoma de células escamosas de língua

PURPOSE: It has been reported that the oral squamous cell carcinoma (OSCC) in tongue shows a more infiltrative profile, aggressive clinical course and poor prognosis, which may be related to a higher metastatic potencial. The aim of the present study was to assess the expression of β-catenin in OSCC of the tongue and its correlation with tumor metastasis. METHODS: Twenty four cases were selected and divided in two groups: metastatic group (n=12) and non-metastatic group (n=12). A semi-quantitative analysis of the β-catenin expression was performed in the invasive tumor front and cases were graded as follows: negative (score 0), positive (score +), and strongly positive (score ++). RESULTS: It was detected that 33 percent, 50 percent and 17 percent of the cases in metastatic group were scored 0, + and ++, respectively, and the non-metastatic group showed that 42 percent were scored "0", 33 percent scored + and 25 percent scored ++. Statistical analysis showed no diference between the studied groups. CONCLUSIONS: Based on these results, it can be concluded that the immunoexpression of β-catenin does not represent a valuable tool to predict metastatic potencial of OSCC in tongue.

OBJETIVO: O carcinoma de células escamosas (CCE) quando presente em língua exibe aspecto mais infiltrativo, curso clínico agressivo e prognóstico desfavorável. Tais características podem associar-se com maior potencial metastático. O presente trabalho teve como meta investigar a expressão de β-catenina em CCE de língua e sua relação com o desenvolvimento de metástases. MÉTODOS: Foram selecionados 24 casos de CCE em língua e divididos em dois grupos: grupo metastático (n=12) e grupo sem metástase (n=12). Realizou-se uma análise semi-quantitativa da imunoexpressão da β-catenina no fronte invasivo tumoral e sendo considerada como negativa, positiva e fortemente positiva, atribuindo-se os escores, "0", "+" e "++", respectivamente. RESULTADOS: A expressão de β-catenina apresentou, no grupo metastático, escore "0" em 33 por cento dos casos, escore "+" em 50 por cento dos casos e escore "++" em 17 por cento dos casos; e, no grupo sem metástase, escore "0" em 42 por cento dos casos, escore "+" em 33 por cento dos casos e escore "++" em 25 por cento dos casos. A análise estatística não demonstrou nenhuma diferença signficativa entre a expressão da proteína nos dois grupos. CONCLUSÃO: Diante destes resultados, concluiu-se que a expressão da β-catenina não constitui um método eficaz, isoladamente, para predizer o potencial metastático do CCE em língua.

Ki-67 and p53 correlation prognostic value in squamous cell carcinomas of the oral cavity and tongue / Valor prognóstico da correlação do Ki-67 e p53 em carcinomas epidermoides da cavidade oral e língua

Epidermoid carcinomas represent from 90 percent to 95 percent of oral cavity malignant neoplasias, making up 13,470 cases/year. AIMS: To correlate p53 and Ki-67 expressions in mouth and tongue carcinomas with lymph node status, gender, histological grade, tumor volume and pathological stage. MATERIALS AND METHODS: We carried out a retrospective study of 28 cases of mouth and tongue epidermoid carcinomas. They were submitted to immunohistochemical study in order to check the expression of p53 and Ki-67 antibodies and statistically compare them in terms of lymph node status, gender, histological grade, tumor volume and pathological staging. RESULTS: The individually analyzed p53 proved to have statistical significance (p<0.05) when compared to tumor volume (p=0.029). Despite a strong tendency, the p53/tumor volume relation was not significant. When p53 + Ki67 were analyzed, tumor volume had p < 0.05 (p = 0.029). DISCUSSION: Literature shows that the expression of p53 and Ki-67 is related to the presence of metastasis to lymph nodes and a worse prognosis. CONCLUSION: In oral cavity and tongue epidermoid carcinomas, p53 and Ki-67 are related to larger tumors, metastasis to lymph nodes and very likely to a worse prognosis.

O carcinoma epidermoide representa 90 por cento a 95 por cento das neoplasias malignas da cavidade oral, responsável por 13.470 casos/ano. OBJETIVOS: Correlacionar a expressão do p53 e Ki-67 nos carcinomas epidermoides de cavidade oral e língua com o estado linfonodal, sexo, grau histológico, volume tumoral e estadiamento patológico. MATERIAIS E MÉTODOS: Foi realizado estudo retrospectivo de 28 casos de carcinomas epidermoides da cavidade oral e língua. Estes foram submetidos à técnica de imunoistoquímica para verificar a expressão dos anticorpos p53 e Ki-67 e compará-los estatisticamente quanto ao status linfonodal, sexo, grau histológico, volume tumoral e estadiamento patológico. RESULTADOS: O p53 analisado individualmente mostrou significância estatística (p<0,05) quando comparado com o volume tumoral (p=0,029). Apesar de uma forte tendência, a relação de p53 com estado linfonodal não foi significativa. Quando o p53 + Ki67 foram analisados, o volume tumoral mostrou p < 0,05 (p = 0,029). DISCUSSÃO: A literatura mostra que a expressão dos marcadores p53 e Ki-67 está relacionada com presença de metástases para linfonodos e pior prognóstico. CONCLUSÃO: Nos carcinomas epidermoides da cavidade oral e língua o p53 e o Ki-67 estão relacionados a tumores de maior volume, metástase para linfonodos e possivelmente um pior prognóstico.

Effects of hypoxia inducible factor-1alpha (HIF-1alpha) on the growth & adhesion in tongue squamous cell carcinoma cells.

BACKGROUND & OBJECTIVES: Hypoxia-inducible factor-1 alpha (HIF-1alpha) is a central transcriptional regulator of hypoxic response. Suppression of HIF-1alpha is important for exploring hypoxia-induced pathophysiological events. This study was carried out to analyze the hypoxia-induced changes of biological characteristics in the human tongue squamous cell carcinoma cell line Tca8113 and evaluate the effects of HIF-1alpha on the phenotype of the tongue squamous cell carcinoma. METHODS: HIF-1alpha gene was silenced with synthesized short interfering ribonucleic acids (siRNA). HIF-1alpha expression was measured on mRNA level by real-time reverse transcription (RT)-PCR and protein level by Western blot and immunofluorescence. The cell cycle and apoptosis of Tca8113 cells were analyzed by FACS. The proliferation and adhesion of Tca8113 cells were determined by MTT colorimetric assay. RESULTS: Tca8113 could survive and showed a more aggressive phenotype under hypoxic condition. Exposure to hypoxia induced a prolonged elevation of HIF-1alpha protein and transfection of siRNA targeting HIF-1alpha (siRNA(HIF-1alpha)) reduced HIF-1alpha synthesis as measured on mRNA and protein level. Under normoxic or hypoxic conditions, treatment of Tca8113 cells with siRNA(HIF-1alpha) induced cell apoptosis and inhibited the growth and adhesion. INTERPRETATION & CONCLUSION: siRNA(HIF-1alpha) could attenuate the tolerance against hypoxia in Tca8113 cells and inhibit their aggressive potential. Interfering with HIF-1alpha pathways by siRNA strategy may provide a therapeutic target for human tongue squamous cell carcinomas.

Immunohistochemical expression of E-cadherin and CD44v6 in squamous cell carcinomas of the lower lip and tongue

This study analyzed the immunohistochemical expression of E-cadherin and CD44v6 in 15 squamous cell carcinomas (SCCs) of lower lip and 15 SCCs of tongue in order to verify a possible association between these proteins and the anatomic location of the lesion, nodal metastasis and histological grading of malignancy. The pattern of expression and number of immunopositive cells were evaluated. The results were analyzed with the Fisher's exact test, Mann-Whitney test and Spearman's Correlation Coefficient (r). using the SPSS software 10.0 for Windows. Statistical significance was set at 5 percent determined for a p-value<0.05 for all tests. There was no significant difference (p>0.05) in the pattern of expression and number of immunopositive cells for E-cadherin and CD44v6, regarding the anatomical location and nodal metastasis. For the histological grading, low score SCCs showed higher immunopositivity for E-cadherin and CD44v6, both for the pattern of expression and number of immunopositive cells (p<0.05). There was a negative correlation between the total score of malignancy and the pattern of expression and number of immunopositive cells for E-cadherin and CD44v6 (p<0.05). In conclusion, SCCs of the lower lip and tongue did not reveal significant differences in the expression of E-cadherin and CD44v6. The expression of these adhesion molecules revealed association only with tumor histological grading of malignancy. Therefore, these results suggest that E-cadherin and CD44v6 may not help elucidating the differences between the biological behavior of SCCs of the lower lip and tongue.

Este estudo analisou a expressão imuno-histoquímica de E-caderina e CD44v6 em 15 carcinomas de células escamosas (CCEs) de lábio inferior e em 15 CCEs de língua, com o intuito de identificar possíveis associações entre a expressão destas proteínas e a localização anatômica da lesão, ocorrência de metástase nodal e gradação histológica de malignidade. Foram avaliados o padrão de expressão e o número de células imunopositivas. Os resultados foram analisados pelo teste exato de Fisher, teste de Mann-Withney e coeficiente de correlação de Spearman (r), utilizando o software SPSS 10.0 para Windows. Para todos os testes, a significância estatística foi determinada em 5 por cento, para um valor de p<0,05. Os resultados revelaram não haver diferença significativa no padrão de expressão e na quantidade de células imunopositivas para E-caderina e CD44v6 em relação à localização anatômica e metástase nodal (p>0,05). Para a gradação histológica de malignidade, os CCEs de baixo escore revelaram maior imunopositividade para E-caderina e CD44v6, tanto para o padrão de expressão quanto para o número de células imunopositivas (p<0,05). Observou-se correlação negativa entre o escore total de malignidade e o padrão de expressão e a quantidade de células imunopositivas para E-caderina e CD44v6 (p<0,05). Em conclusão, CCEs de lábio inferior e língua não revelaram diferenças significativas na expressão de E-caderina e CD44v6. A expressão destas moléculas de adesão revelou associação apenas com gradação histológica de malignidade dos CCEs. Dessa forma, os resultados sugerem que E-caderina e CD44v6 podem não ser capazes de elucidar as diferenças existentes no comportamento biológico de CCEs de lábio inferior e língua.

Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells

Phosphatidylserine (PS) exposure occurs during the cell death program and fluorescein-labeled lactadherin permits the detection of PS exposure earlier than annexin V in suspended cell lines. Adherent cell lines were studied for this apoptosis-associated phenomenon to determine if PS probing methods are reliable because specific membrane damage may occur during harvesting. Apoptosis was induced in the human tongue squamous carcinoma cell line (Tca8113) and the adenoid cystic carcinoma cell line (ACC-2) by arsenic trioxide. Cells were harvested with a modified procedure and labeled with lactadherin and/or annexin V. PS exposure was localized by confocal microscopy and apoptosis was quantified by flow cytometry. The detachment procedure without trypsinization did not induce cell damage. In competition binding experiments, phospholipid vesicles competed for more than 95 and 90 percent of lactadherin but only about 75 and 70 percent of annexin V binding to Tca8113 and ACC-2 cells. These data indicate that PS exposure occurs in three stages during the cell death program and that fluorescein-labeled lactadherin permitted the detection of early PS exposure. A similar pattern of PS exposure has been observed in two malignant cell lines with different adherence, suggesting that this pattern of PS exposure is common in adherent cells. Both lactadherin and annexin V could be used in adherent Tca8113 and ACC-2 cell lines when an appropriate harvesting procedure was used. Lactadherin is more sensitive than annexin V for the detection of PS exposure as the physical structure of PS in these blebs and condensed apoptotic cell surface may be more conducive to binding lactadherin than annexin V.

Expressão da proteína nm23 em carcinoma de células escamosas de língua metastático e não-metastático / nm23 protein expression in metastatic and non-metastatic tongue squamous cell carcinoma

O carcinoma de células escamosas oral (CCEO) exibe prognóstico desfavorável em decorrência da capacidade de invasão aos tecidos vizinhos e elevada incidência de metástases. OBJETIVO: O presente trabalho objetiva analisar a expressão imunohistoquímica da proteína nm23 em CCEs de língua metastáticos e não-metastáticos. METODOLOGIA: A técnica da imunohistoquímica para a proteína nm23-h1 foi realizada em 35 casos de CCE de língua com metástase em 15 casos. Atribuiu-se escore 0, para ausência de marcação; 1, marcação focal e 2 para marcação difusa. RESULTADOS: Observou-se marcação focal para a proteína nm23 em 9 casos, difusa em 15, e ausência de marcação em 11 espécimes. O teste exato de Fischer foi aplicado, não havendo diferença estatisticamente significativa para positividade desta proteína nos casos metastáticos e não-metastáticos (p=0.365), apesar de que em 66.7 por cento dos casos com metástase não houve marcação. CONCLUSÕES: A presença da proteína nm23 não esteve relacionada de forma positiva aos casos de CCE de língua sem metástase. Dessa forma, vários outros fatores inerentes à célula neoplásica e ao hospedeiro podem estar relacionados aos mecanismos supressores do processo metastático nesta entidade.

Oral squamous cells carcinoma (OSCC) shows unfavorable prognosis due to its invasion potential around the neighboring tissues and the elevated incidence of metastasis. AIM: the present paper aims at analyzing the immunohistochemical expression of the nm23 protein in metastatic and non-metastatic SCCs of tongue. METHODS: the immuno-expression to the nm23-hl protein was diagnosed in 35 tongue SCC (15 of which exhibiting metastasis). Nm23-hl immuno-scores were assigned as follow: score 0 = absent, 1 = focal and 2 = diffuse expression. RESULTS: The Fisher's exact test was performed and there was no statistical difference between the nm23-hl immuno-scores and the tongue SCCs studied cases (p=0.365), although 66.7 percent of metastatic cases presented negative nm23-hl expression. CONCLUSIONS: Protein nm23 was not associated with a positiveness for tongue SCC without metastasis. Thus, several others factors inherent to host and malignancy can be associated with the mechanisms that suppress the metastatic process in this disease.

Lingual schwannoma.

Schwannomas or neurilemmomas are benign, slow growing, usually solitary and encapsulated tumor, originating from Schwann cells of the nerve sheath. Intraoral schwannoma accounts for 1% of head and neck region and are commonly seen at the base region of tongue. Most of the few such reports in the literature, have described schwannomas that occurred in the tongue. In this article, we report a rare case of lingual schwannoma involving the anterior of tongue, in a young individual, in whom the lesion was completely excised via an intra oral approach.

Heterogeneous nuclear ribonuclear protein C is increased in the celecoxib-induced growth inhibition of human oral squamous cell carcinoma

Celecoxib is a selective inhibitor of cyclooxygenase-2 (COX-2) that is a critical factor in carcinogenesis, but precise mechanism of its action remains to be elucidated. Here we evaluated the inhibitory effect of celecoxib on cell growth of human oral squamous cell carcinoma (OSCC) YD-10B, which was established to be used as in vitro OSCC model, and identified celecoxib-regulated protein by proteomics techniques. Celecoxib (IC50=37 micrometer) inhibited the growth of YD-10B cells with the decrease of COX-2 protein expression. Its inhibition could be linked in the arrest of G1 phase with increased levels of p(27)protein, a specific CDK inhibitor. Using proteomics, the 10- to 20-fold increase of heterogeneous nuclear ribonuclear protein C (hnRNP C), which has been suggested to be related with the translation of p(27)mRNA, was observed in celecoxib-treated YD-10B cells. In summary, celecoxib has a potential to induce the protein expression of hnRNP C and its increase subsequently induce the translation of p(27)mRNA, which trigger the inhibition of cell growth via p(27)-regulated cell cycle arrest in YD-10B cells. In addition, YD-10B cells could be useful to study the pathological mechanism of OSCC.