Glycolytic pathway candidate markers in the prognosis of oral squamous cell carcinoma: a systematic review with meta-analysis

Molecular changes that affect mitochondrial glycolysis have been associated with the maintenance of tumor cells. Some metabolic factors have already been described as predictors of disease severity and outcomes. This systematic review was conducted to answer the question: Is the glycolytic pathway correlated with the prognosis of oral squamous cell carcinoma (OSCC)? A search strategy was developed to retrieve studies in English from PubMed, Scopus, and ISI Web of Science using keywords related to squamous cell carcinoma, survival, and glycolytic pathway, with no restriction of publication date. The search retrieved 1273 publications. After the titles and abstracts were analyzed, 27 studies met inclusion criteria. Studies were divided into groups according to two subtopics, glycolytic pathways and diagnosis, which describe the glycolytic profile of OSCC tumors. Several components of tumor energy metabolism found in this review are important predictors of survival of patients with OSCC.

Anticancer effects of 6-shogaol via the AKT signaling pathway in oral squamous cell carcinoma

Abstract Objective Oral squamous cell carcinoma (OSCC) is one of the common type of cancer that leads to death; and is becoming a global concern. Due to the lack of efficient chemotherapeutic agents for patients with oral cancer, the prognosis remains poor. 6-shogaol, a bioactive compound of ginger, has a broad spectrum of bioactivities and has been widely used to relieve many diseases. However, its effects on human oral cancer have not yet been fully evaluated. In our study, we investigated the anticancer effects of 6-shogaol on the proliferation, migration, invasion, apoptosis, and underlying mechanisms within human OSCC cell lines. Methodology We investigated the effect of 6-shogaol on the growth of OSCC cells by cell viability and soft agar colony formation assay. Migration and invasion assays were conducted to confirm the effect 6-shogaol on OSCC cell metastasis. Apoptosis was detected by flow cytometry and the underlying mechanism on the antigrowth effect of 6-shogaol in OSCC cells was assessed using western blotting. Results In our results, 6-shogaol not only suppressed proliferation and anchorage-independent cell growth in OSCC cells, but also induced apoptosis by regulating the apoptosis-associated factors such as p53, Bax, Bcl-2, and cleaved caspase-3. Migration and invasion of OSCC cells were inhibited following the regulation of E-cadherin and N-cadherin by 6-shogaol. Additionally, 6-shogaol treatment significantly inhibited the PI3K/AKT signaling pathway. Conclusion Therefore, our results may provide critical evidence that 6-shogaol can be a potential new therapeutic candidate for oral cancer.

Immunohistochemistry analysis of checkpoint kinase 2 in oral squamous cell carcinoma

Background: Oral squamous cell carcinoma (OSCC) is the most frequently occurring malignant tumor of the head and neck region. Chk2 (Checkpoint kinase 2) is considered a tumor suppressor gene that acts on the cellular response to DNA damage. However, the role of Chk2 in OSCC prognosis is not yet fully understood. The objective of this study was to evaluate Chk2 immunoexpression in OSCC and to elucidate the association between its expression and clinicopathological parameters of prognostic importance, including overall survival, disease-free survival, and metastasis-free survival. Methods: Chk2 expression was analyzed in 101 samples from patients with OSCC using immunohistochemistry. We stratified the patients into high expression (> 66% of cells positive for Chk2) and low expression (< 66%) groups. Results: Chk2 showed high expression in 57.43% of OSCC. In our study, the expression of Chk2 did not correlate with any of the prognostic parameters evaluated. There was no difference between overall survival, metastasis-free survival, and disease-free survival according to Chk2 expression. Conclusion: Despite the great importance of Chk2 in the development of different types of cancer, our findings do not favor Chk2 as a prognostic marker in oral squamous cell carcinoma.

Alpha-hederin induces the apoptosis of oral cancer SCC-25 cells by regulating PI3K/Akt/mTOR signaling pathway

BACKGROUND: Oral cancer is one of the common malignant tumors of the head and neck. However, current treatments have numerous side effects, and drugs from natural sources may have better therapeutic potential. This research investigated the induction of apoptosis by α-hederin (α-HN), a constituent of Pulsatilla chinensis (Bunge) Regel, in the oral cancer cell line SCC-25 and its underlying mechanism. RESULTS: SCC-25 cells were treated with 50, 100, and 200 µmol/L α-HN. Cell proliferation; extent of apoptosis; activities of caspases-3, 8, and 9; and the expression of Bcl-2, Bax, phosphorylated (p)-phosphoinositide 3-kinase (PI3K), p-Akt, and p-mammalian target of rapamycin (mTOR) proteins were determined using the 3-(4,5)-2-thiazole-(2,5)-diphenyl tetrazolium bromide, flow cytometry, caspase activity detection kits, and western blot assays, respectively. The results showed that the proliferation of SCC-25 cells in the α-HN-treated groups decreased significantly, and the inhibitory effect was time and concentration dependent. Compared with cells in the control group, the extent of apoptosis increased significantly, caspase-3 and -9 activities were significantly enhanced, and the Bcl-2 level was lowered and the Bax level was elevated significantly in SCC-25 cells treated with α-HN for 48 h (P b 0.05). The expression of p-PI3K, p-Akt, and p-mTOR was also significantly lower in SCC-25 cells treated with α-HN than that in the control group (P b 0.05). CONCLUSION: These results indicate that α-HN can inhibit proliferation and induce apoptosis of SCC-25 cells and may exert these effects by inhibiting the PI3K/Akt/mTOR signaling pathway.

Inmunoexpresión de E-cadherina y vimentina en mucosa oral normal, displasia epitelial oral y carcinoma oral de células escamosas / Immunoexpression of E-cadherin and vimentin in normal oral mucosa, oral epithelial displasia and oral squamous cell carcinoma

El objetivo fue evaluar la inmunoexpresión de E-cadherina y Vimentina en mucosa oral normal (MON), displasia epitelial oral (DEO) y carcinoma oral de células escamosas (COCE). Se realizó un estudio descriptivo de una serie de casos analizandolos mediante técnica de inmunohistoquímica contra E-cadherina y Vimentina 16 muestras de MON, 16 de DEO y 19 de COCE. La inmunotinción fue evaluada cualitativamente considerando extensión e intensidad para E-cadherina e intensidad para Vimentina. El análisis de la extensión e intensidad de la inmunotinción de E-cadherina y Vimentina según diagnóstico reveló una asociación estadísticamente significativa (p<0,001). Siendo la expresión de E-cadherina más alta en MON, seguido por DEO y más baja en COCE, inversamente a lo que se observó con Vimentina. El presente estudio reveló la subregulación del marcador molecular E-cadherina junto con la expresión aberrante por parte de células epiteliales del marcador mesenquimal Vimentina en muestras de MON, DEO y COCE.

The aim was to evaluate the expression of E-cadherin and Vimentin in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), in comparison with normal oral mucosa (NOM) in a descriptive case study using immunohistochemistry. A total of fifty-one (N=51) histological samples were included; as follows: n = 16 (NOM), n = 16 (OED) and n = 19 (OSCC). All samples were analyzed using immunohistochemistry against the expression of E-cadherin and Vimentin. Immunostaining was qualitatively evaluated by extent and intensity of its expression for E-cadherin and intensity for Vimentin. Extension and intensity analysis of E-cadherin and Vimentin immunostaining according to group revealed a statistically significant association (r<0.001). E-cadherin expression was found to be highest in NOM followed by OED and lowest in OSCC, inverse to what was observed with Vimentin. The present study revealed the down regulation of the molecular marker E-cadherin, suggestive of reduction in dysplastic cells on comparison to NOM cells, and aberrant expression of the mesenchymal marker Vimentin by epithelial cells in samples of NOM, OED and OSCC; questioning their value as a prognostic marker.

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.

Difference in EGFR expression and mean vascular density in normal oral mucosa, oral epithelial dysplasia and oral squamous cell carcinoma

To evaluate the expression of the epidermal growth factor receptor (EGFR) and mean vascular density (MVD) in normal oral mucosa (NOM), oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Material and methods: Descriptive case study. Nineteen histological samples diagnosed with NOM, 18 diagnosed with OED, and 19 with OSCC, were analyzed with immunohistochemistry against EGFR and CD31. EGFR expression was evaluated by extent and intensity of its expression in normal, dysplastic and neoplastic epithelium. MVD was determined through the detection of blood vessels by antibodies against CD31. Results: Extension of EGFR expression was highest in OSCC followed by OED and lowest in NOM, resulting in significant different between the degrees of extension (p<0.001). Intensity of EGFR was similar in NOM, OED and OSCC, without differences in its expression (p=0.533). Differences in MVD were found between NOM and OSCC groups (p<0.01), and between OED and OSCC groups (p<0.01), with no differences between NOM and OED groups (p=0.91). MVD was 21.17 +/- 4.98 in NOM, 23.40 +/- 5.77 in OED and 33.92 +/- 8.39 in OSCC. Conclusion: EGFR is expressed in normal, dysplastic or neoplastic oral epithelium. However, the extent of its expression is greater as malignancy increases. MVD varies according to the diagnosis.

Expression of E-cadherin and involucrin in leukoplakia and oral cancer: an immunocytochemical and immunohistochemical study

Abstract To assess the immunocytochemical and immunohistochemical correlation of adhesion (E-cadherin) and cell differentiation (involucrin) molecules in oral leukoplakia and oral squamous cell carcinoma. Cytological samples and biopsies were obtained from male and female patients aged over 30 years with oral leukoplakia (n = 30) and oral squamous cell carcinoma (n = 22). Cell scrapings and the biopsy were performed at the site of the lesion and histological slides were prepared for the immunocytochemical analysis of exfoliated oral mucosal cells and for the immunohistochemical analysis of biopsy tissues using E-cadherin and involucrin. Spearman’s correlation and kappa coefficients were used to assess the correlation and level of agreement between the techniques. Immunostaining for E-cadherin and involucrin by both techniques was similar in the superficial layers of the histological sections compared with cell scrapings. However, there was no statistical correlation and agreement regarding the immunocytochemical and immunohistochemical expression of E-cadherin and involucrin in oral leukoplakia (R = 0.01, p = 0.958) (Kappa = 0.017, p = 0.92) or in oral squamous cell carcinoma (R = 0.26, p = 0.206) (Kappa = 0.36, p = 0.07). The immunoexpression of E-cadherin and involucrin in tissues is consistent with the expression patterns observed in exfoliated oral mucosal cells, despite the lack of a statistically significant correlation. There is an association of the histopathological characteristics of leukoplakia with the expression E-cadherin and of the microscopic aspects of oral squamous cell carcinoma with immunohistochemical expression of involucrin.

Ki-67 protein predicts survival in oral squamous carcinoma cells: an immunohistochemical study

Abstract The aim of this study was to identify the expression of Ki-67 and MCM3 in oral squamous cell carcinoma (OSCC) as well as to address the correlation with patient survival and clinical features. Samples were collected from 51 patients with OSCC who presented for follow-up. Immunohistochemical expression of Ki-67 and MCM3 in all groups was performed. The scoring system was previous published by Tsurutani in 2005. We used Kappa index to evaluate observers agreement degree. The associations between protein expression and clinical variables were examined for statistical significance using the chi-squared test. The overall survival rates were estimated by the Kaplan-Meier method and the relationship between protein expression and survival was compared using the log-rank test (p < 0.05). The overall survival time for a patient with positive immunostaining for Ki-67 is shorter than for a patient with negative immunostaining, (log-rank test, p = 0.00882). Patients with tumor size T3 and T4 showed a statistically significant relationship with Ki-67 immunoexpression (log-rank test, p = 0.0174). The relationship between Ki-67 expression and the relation between age, gender, smoking, tumor site, lymph node metastasis and disease stage was not significant. The examiners agreement degree by Kappa presented p value < 0.05. There was not a significant correlation when we evaluated MCM3 expression regarding clinical characteristics and survival rate. From these results, the present study suggests that positive Ki-67 expression found in OSCC patients may contribute to predict the survival in OSCC samples, as well as the relation between the protein and the tumor size.

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.

Dysregulation and detection methods of EGFR in oral cancer: a narrative review / Desregulación y métodos de detección del EGFR en cáncer oral: revisión narrativa

Abstract: epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein, with an intracellular domain and tyrosine kinase function (TK) involved in cell proliferation. Dysfunctions in EGFR signaling pathways have been associated with oral malignant tumors such as oral squamous cell carcinoma (OSCC). Dysfunctions of EGFR may result from: increased EGF ligand; EGFR overexpression and copy number gain of the EGFR gene (EGFR CNG); EGFR mutations; failure in the downregulation of EGFR; and EGFR crosstalk. Of these alterations, overexpression of EGFR is by far the most studied dysfunction in OSCC. Clinicians should identify possible alterations of EGFR in the oral mucosa of patients, as EGFR can act as a biomarker for the diagnosis and prognosis of OSCC. Currently, there are several methods and techniques for detecting EGFR. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR), are used to identify overexpression of EGFR, EGFR CNG and EGFR mutations, respectively. Detection of EGFR as a biomarker is key to identify any oral malignant transformation. Consequently, it becomes imperative to implement a non-invasive and inexpensive method of early diagnosis for OSCC in clinical practice.

Resumen: el receptor del factor de crecimiento epidérmico (EGFR) es una glicoproteína transmembrana, con un dominio intracelular y función tirosina quinasa (TK) que participa en la proliferación celular. Las fallas en las vías de señalización del EGFR se han asociado con la formación de tumores malignos orales como el carcinoma oral de células escamosas (COCE). El incorrecto funcionamiento del EGFR puede producirse por: aumento del ligando EGF; sobreexpresion del EGFR y ganancia en el número de copias del gen EGFR (GNC EGFR); mutaciones del EGFR; falla en la regulación negativa del EGFR; y diafonía del EGFR. De las alteraciones mencionadas, la sobreexpresion de EGFR es por lejos la disfunción más estudiada en COCE. Para el clínico es importante poder identificar las posibles alteraciones del EGFR en la mucosa oral del paciente, esto debido a que el EGFR puede actuar como un biomarcador de diagnóstico y pronóstico para COCE. En la actualidad existen diversos métodos para detectar el EGFR. La inmunohistoquímica (IHC), la hibridación fluorescente in situ (FISH) y la reacción en cadena de la polimerasa (PCR), son técnicas utilizadas para identificar la sobreexpresion del EGFR, GNC EGFR y mutaciones del EGFR, respectivamente. La necesidad de detección de estas alteraciones se debe a la transcendencia del EGFR como biomarcador de transformación maligna. Lo anterior, hace necesario implementar un método de diagnóstico precoz para COCE que sea no invasivo y de bajo costo para la práctica clínica.

TGF-beta alterations in oral squamous cell carcinoma: narrative review / Alteraciones de TGF-beta en el carcinoma oral de células escamosas: revisión narrativa

The transforming growth factor beta (TGF-beta) is a cytokine that plays crucial roles in the regulation of angiogenesis, immune response, proliferation, migration and apoptosis of cells. In addition, it can inhibit cell progression and stimulate apoptosis in early stages of cancer. TGF-beta is a multifunctional homodimeric protein secreted by various cell lines, which have three different isoforms: TGF-beta1, TGF-beta2 and TGF-beta3. In normal conditions, TGF-beta1 activates some tumor suppressor cell signaling pathways that inhibit proliferation and are involved in cell migration, differentiation and apoptosis. However, in more advanced stages of cancer, when TGF-beta1 is altered, it acts as a promoter of tumorigenesis and may cause: 1) increased TGF-beta1, 2) overexpression of TGF-beta1 receptors (TbetaR), 3) TbetaR mutations, and 4) downregulation of TGF-beta receptor. In oral squamous cell carcinoma, the path is altered especially at the level of transmembrane receptors, with the TbetaR-II and TbetaR-III subtypes being the most affected. However, there is little information on the prognostic role it plays in the various types of cancers. It is important to study the signaling pathways of TGF-beta in order to develop techniques that may help detect their alterations and restore their normal operation. The objective of this review is to describe the alterations of TGF-beta in oral squamous cell carcinoma.

El factor de crecimiento transformante beta (TGF-beta) es una citocina que cumple funciones fundamentales en la regulación de la angiogénesis, respuesta inmune, proliferación, migración y apoptosis celular. Además, puede inhibir la progresión celular y estimular la apoptosis en etapas tempranas del cáncer. El TGF-beta es una proteína homodimérica multifuncional secretada por diversas líneas celulares, que presentan 3 isoformas: TGF-beta1, TGF-beta2 y TGF-beta3. En condiciones normales TGF-beta1 activa a algunas vías de señalización celular supresoras de tumores que inhiben la proliferación, y participan en la migración, diferenciación y apoptosis. Sin embargo, cuando esta se ve alterada, en etapas más avanzadas del cáncer actúa como promotor de la tumorogénesis, pudiendo producir: 1) aumento del TGF-beta1, 2) sobre expresión de los receptores del TGF-beta1 (TbetaR), 3) mutaciones de TbetaR, y 4) falla en la regulación negativa de TbetaR. En el carcinoma oral de células escamosas, la vía se ve alterada especialmente a nivel de sus receptores transmembranales, siendo los subtipos TbetaR-II y TbetaR-III los más afectados. Sin embargo, es escasa la información sobre el rol pronóstico que juega en los diversos tipos de cánceres. Es importante estudiar las vías de señalización de TGF-beta para desarrollar técnicas que detecten sus alteraciones y restauren el funcionamiento del sistema. El objetivo de esta revisión es describir las alteraciones de TGF-beta en carcinoma oral de células escamosas.

Expression of Ki-67 and P16 INK4a in chemically-induced perioral squamous cell carcinomas in mice / Expressão KI-67 e P16 INK4a em carcinomas espinocelulares periorais quimicamente induzidos em camundongos

ABSTRACT Objective: to evaluate the influence of Ki-67 and P16INK4a proteins immunohistochemical expressions on the clinical and morphological parameters of perioral squamous cell carcinoma induced with 9,10-dimethyl-1,2-benzanthracene (DMBA) in mice. Methods: we topically induced the lesions in the oral commissure of ten Swiss mice for 20 weeks, determining the time to tumors onset and the average tumor volume up to 26 weeks. In histopathological analysis, the variables studied were histological malignancy grade and the immunohistochemical expression of Ki-67 and P16INK4a proteins. The correlation between variables was determined by application of the Spearman correlation test. Results: the mean time to onset of perioral lesions was 21.1 ± 2.13 weeks; mean tumor volume was 555.91 ± 205.52 mm3. Of the induced tumors, 80% were classified as low score and 20% high score. There was diffuse positivity for Ki-67 in 100% of lesions - Proliferation Index (PI) of 50.1 ± 18.0. There was a strong direct correlation between Ki-67 immunoreactivity and tumor volume (R = 0.702) and a low correlation with the malignancy score (R = 0.486). The P16INK4a protein expression was heterogeneous, showing a weak correlation with tumor volume (R = 0.334). There was no correlation between the immunohistochemical expression of the two proteins studied. Conclusion: in an experimental model of DMBA-induced perioral carcinogenesis, tumor progression was associated with the tumor proliferative fraction (Ki-67 positive cells) and with tumor histological grading, but not with P16INK4a expression.

RESUMO Objetivo: avaliar a influência da expressão imuno-histoquímica das proteínas Ki-67 e p16INK4a sobre parâmetros clínico-morfológicos em carcinomas espinocelulares periorais quimicamente induzidos com 9,10-dimetil-1,2-benzantraceno (DMBA) em modelo murino. Métodos: as lesões foram induzidas topicamente na comissura labial de dez camundongos Swiss durante 20 semanas, sendo determinado o momento de surgimento dos tumores e volume tumoral médio até 26 semanas. Na análise histopatológica, as variáveis estudadas foram gradação histológica de malignidade tumoral e expressão imuno-histoquímica das proteínas Ki-67 e p16INK4a. A correlação entre as variáveis estudadas foi determinada pela aplicação do teste de correlação de Spearman. Resultados: o tempo médio de surgimento das lesões periorais foi 21,1±2,13 semanas. Volume tumoral médio foi de 555,91±205,52mm3. Dos tumores produzidos, 80% foram classificados como de baixo escore e 20%, alto escore. Evidenciou-se positividade difusa para Ki-67 em 100% das lesões - índice de marcação (PI) de 50,1±18,0. Verificou-se correlação direta forte entre a imunoexpressão do Ki-67 e o volume tumoral (R=0,702) e fraca correlação com o escore de malignidade (R=0,486). A expressão da proteína p16INK4a foi heterogênea, mostrando fraca correlação com o volume tumoral (R=0,334). Não houve correlação entre a expressão imuno-histoquímica das duas proteínas estudadas. Conclusão: Em modelo experimental de carcinogênese perioral DMBA-induzida, a progressão tumoral está associada à fração proliferativa do tumor (células ki-67 positivas) e com a gradação histológica tumoral, porém não com a expressão da p16INK4a.

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.

p53 immunoprofiling of potentially malignant oral disorders: A case series analysis.

Context: p53 tumor suppressor gene which is a frequent target for mutations in a high percentage of oral cancer is regarded as an early event in carcinogenesis. Aim: The role of p53 was assessed in potentially malignant oral disorders (PMOD) to ascertain its prognostic significance. Settings and Design: Retrospective case series analysis was carried out on 30 paraffin-embedded tissue blocks of confirmed oral leukoplakia with dysplasia. Materials and Methods: 10 cases of each of mild, moderate, and severe dysplasia were immunohistochemically analyzed for p53 expression. The intensity of staining, intracellular localization, and basal and/or suprabasal distribution were assessed. Statistics: The intensity of p53 staining and its distribution were analyzed by the Chi-square test. The intracellular localization of p53 in different grades of dysplasia was subjected to one way ANOVA. P<0.05 was considered significant. Results: 21/30 cases of epithelial dysplasia were positive for p53 immunopositivity. Intensity of p53 expression was strong in 12 cases and weak in 9 cases (P<0.05). p53 positivity was confined to basal cells in mild dysplasia, while severe dysplasia showed both basal and suprabasal staining (P<0.05). Nuclear and cytoplasmic staining between and within the groups were F=9.027 and F=6.465 respectively with high significance noted between mild dysplasia and severe dysplasia. Conclusions: Increased p53 expressivity and greater cellular localization with increase in the severity of dysplasia indicated a direct association between the degree of epithelial dysplasia and p53 accretion, which occurs as an early event in oral carcinogenesis.

Fas receptor (CD95) & Fas ligand (CD178) expression in patients with tobacco-related intraoral squamous cell carcinoma.

Background & objectives: Fas receptor and Fas Ligand (FasL) system has been implicated in the resistance to apoptosis, insensitivity to chemotherapy and in providing immune privileged status to most of the tumours. However, no reports are available on Fas and FasL expression in patients with tobacco-related oral carcinoma. Therefore, the present study was undertaken to observe Fas and FasL expression and their correlation with clinicopathological features as well as cell cycle parameters. Methods: Immunohistochemistry for Fas, FasL and DNA flow cytometry for cell cycle parameters was successfully done on 41 paraffin embedded tumour and 10 normal samples. The results were evaluated for possible association of Fas and FasL with clinicopathological features and cell cycle parameters. Results: Weak Fas expression was observed on the cell membrane only in 2 of 41 (5%) oral tumours while FasL immunoreactivity was seen in 26 of 41 (63.4%) tumours. In contrast, all ten normal oral tissues exhibited strong cytoplasmic and membrane Fas receptor immunoreactivity but absence of FasL staining. Older patients, greater tumour size and lymph node positivity were found to be associated with high expression of FasL. Significantly higher (P<0.01) expression of FasL was observed in oral tumours with aggressive DNA pattern like aneuploidy and high S-phase fraction. Interpretation & conclusions: Downregulation of Fas receptor and up-regulation of Fas ligand appear to be an important feature of tobacco-related intraoral carcinoma. Association of FasL expression with advanced clinical stage and aggressive DNA pattern suggests that the Fas and FasL system may be used as an important prognostic variable in patients with tobacco-related intraoral squamous cell carcinoma.

Immunohistochemical expression of p53, p16 and hTERT in oral squamous cell carcinoma and potentially malignant disorders

Oral carcinogenesis is a multi-step process. One possible step is the development of potentially malignant disorders known as leukoplakia and erytroplakia. The objective of this study was to use immunohistochemistry to analyze the patterns of expression of the cell-cycle regulatory proteins p53 and p16INK4a in potentially malignant disorders (PMD) of the oral mucosa (with varying degrees of dysplasia) and in oral squamous cell carcinomas (OSCC) to correlate them with the expression of telomerase (hTERT). Fifteen PMD and 30 OSCC tissue samples were analyzed. Additionally, 5 cases of oral epithelial hyperplasia (OEH) were added to analyze clinically altered mucosa presenting as histological hyperplasia without dysplasia. p53 positivity was observed in 93.3 percent of PMD, in 63.3 percent of OSCC and in 80 percent of OEH. Although there was no correlation between p53 expression and the grade of dysplasia, all cases with severe dysplasia presented p53 suprabasal immunoexpression. p16INK4a expression was observed in 26.7 percent of PMD, in 43.3 percent of OSCC and in 2 cases of OEH. The p16INK4a expression in OEH, PMD and OSCC was unable to differentiate non-dysplastic from dysplastic oral epithelium. hTERT positivity was observed in all samples of OEH and PMD and in 90 percent of OSCC. The high hTERT immunoexpression in all three lesions indicates that telomerase is present in clinically altered oral mucosa but does not differentiate hyperplastic from dysplastic oral epithelium. In PMD of the oral mucosa, the p53 immunoexpression changes according to the degree of dysplasia by mechanisms independent of p16INK4a and hTERT.

A comparative study of p53 expression in hyperplastic, dysplastic epithelium and oral squamous cell carcinoma

Aim: To study oral hyperplastic epithelium, dysplastic epithelium and squamous cell carcinoma to determine (1) the prevalence of p53 protein immunoreactivity, (2) number of p53 positive cells, and (3) the area of localization of p53 protein immunoreactivity. Methods: Two contiguous sections from 30 tissue specimens (10 each from oral hyperplastic epithelium, dysplastic epithelium and squamous cell carcinoma) were subjected to hematoxylin and eosin (H/E) staining forhistopathological diagnosis and immunohistochemical (IHC) staining for demonstration of p53. p53 positivity was looked for in each IHC stained slide and the number of positive cells amongst 1,000 epithelial cells were recorded. The localization of these p53 positive cells within the strata (i.e.basal/suprabasal, spinous and superficial layers) of epithelium between 3 groups, and also with ineach group according to histological grades was recorded. Results: Higher p53 positive cell counts were demonstrated in oral squamous cell carcinoma compared to hyperplastic and dysplastic tissues. The expression of p53 in epithelial hyperkeratosis was mainly localized to basal epithelialcells whereas in epithelial dysplasia, it was predominantly localized to spinous epithelial cells. Conclusions: Qualitatively p53 is not a specific marker for malignancy of oral epithelium. However the quantitative analysis of p53 positive cells and their localization in oral epithelium is of importance as a marker for oral squamous cell carcinoma.

Death-associated protein kinase is underexpressed in high-grade oral squamous cell carcinoma / La expresión de la proteína quinasa asociada a muerte celular (DAP quinasa) está reducida en carcinoma de células escamosas oral de alto grado

An immunohistochemical analysis of 40 cases of oral squamous cell carcinoma was performed to evaluate the relationship between the expression pattern of death-associated protein kinase (DAPk) positive cells with the histological malignancy grading of these lesions. According to our results, eleven cases (27.5 percent) were high-grade malignancy tumours and 29 (72.5 percent) were low-grade ones. We found that 92.86 percent of the low-grade tumours were positive to anti-DAP kinase antibody whereas only 7.14 percent of the high-grade tumours presented positivity, and this difference was statistically significant (p<0.01). Sixteen (55.2 percent) of the low-grade carcinomas exhibited moderate immunoreactivity whereas ten cases (34.5 percent) showed weak staining and three cases (10.3 percent) were negative tumours. Immunostaining was lacking in nine (81.8 percent) of the high-grade carcinomas and "weak" in the two (18.2 percent) remaining cases. Thus, DAPk expression is significantly decreased in high-grade oral carcinomas, and evidences indicate that it might be related to the severity of cytological atypia.

Fue realizado un análisis inmunohistoquímico de 40 casos de carcinoma oral de células escamosas para evaluar la relación entre el patrón de expresión de la proteína quinasa (DAPK) asociada a la muerte celular y la clasificación histológica de malignidad de estas lesiones. Según nuestros resultados, 11 casos (27,5 por ciento) eran tumores de alto grado de malignidad y 29 (72,5 por ciento) de bajo grado. Se encontró que 92,86 por ciento de los tumores de bajo grado de malignidad fueron eran positivos para anticuerpos anti-DAP-quinasa, mientras que sólo 7,14 por ciento de los tumores de alto grado presentaron positividad, esta diferencia fue estadísticamente significativa (p <0,01). En 16 casos (55,2 por ciento) los carcinomas de bajo grado de malignidad mostraron inmunorreactividad moderada mientras que 10 casos (34,5 por ciento) mostraron una tinción débil y 3 casos (10,3 por ciento) fueron negativos. La inmunotinción estuvo ausente en 9 (81,8 por ciento) de los carcinomas de alto grado y "débil" grado de malignidad en los 2 (18,2 por ciento) casos restantes. Así, la expresión DAPK se redujo significativamente en los carcinomas orales de alto grado y las evidencias indican que podría estar relacionado con la severidad de la atipia citológica.

Molecular analysis of oral squamous cell carcinoma: A tissue microarray study.

Background : An intriguing aspect of Oral Squamous Cell Carcinomas (OSCC) is its behavioral disparity. Among patients who present with the similar clinicopathological features, some have a better prognosis than others. Identification of molecular alterations responsible for this may contribute to a greater understanding of tumor behavior. Tissue microarray (TMA) approach is a high throughput technology that enables analysis of multiple molecular targets simultaneously without causing any morphological alteration to tissue specimens. Aim and Objective : To assess the tumor behavior based on the expression of p53, Bcl-2 and E-cadherin using TMA technology. Settings and Design : This was a case series analysis using tissue microarray technology. Materials and Methods : Formalin-fixed Paraffin-embedded (FFPE) tissue blocks of histological proven cases of OSCC (n = 30) were retrieved from the department archives. Tissue microarray blocks were constructed; 4 ΅m thick sections were cut and immunostained for p53, Bcl-2 and E-cadherin. Stastistical Analysis : Mean (SD) was used to summarize age, frequencies with percentages was used to summarize categorical variable and Chi-square test was used to find association between histopathology evaluation and expression of Bcl-2, p53, E-cadherin. Results and Conclusion : Bcl-2 was the most frequently expressed biomarker. The expression of Bcl-2 was inversely related to the degree of differentiation (P = 0.005). The follow-up data showed that 63.6% of the cases that were positive for both Bcl-2 and E-cadherin were disease-free following treatment. Tissue microarray technology is a promising way to analyse multiple biomarkers simultaneously. The molecular data obtained from TMA will enhance diagnosis, provide better prognostication and will improve cancer treatment for individual patients.