Effect of tumor-stromal fibroblasts on the biological behavior of salivary gland pleomorphic adenoma cells in vitro / 华西口腔医学杂志

OBJECTIVES@#This study aims to investigate the effects of tumor-stromal fibroblasts (TSFs) on the proliferation, invasion, and migration of salivary gland pleomorphic adenoma (SPA) cells in vitro.@*METHODS@#Salivary gland pleomorphic adenoma cells (SPACs), TSFs, and peri-tumorous normal fibroblasts (NFs) were obtained by tissue primary culture and identified by immunocytochemical staining. The conditioned medium was obtained from TSF and NF in logarithmic phase. SPACs were cultured by conditioned medium and treated by TSF (group TSF-SPAC) and NF (group NF-SPAC). SPACs were used as the control group. The proliferation, invasion, and migration of the three groups of cells were detected by MTT, transwell, and scratch assays, respectively. The expression of vascular endothelial growth factor (VEGF) in the three groups was tested by enzyme linked immunosorbent assay (ELISA).@*RESULTS@#Immunocytochemical staining showed positive vimentin expression in NF and TSF. Results also indicated the weak positive expression of α-smooth muscle actin (SMA) and fibroblast activation protein (FAP) in TSFs and the negative expression of α-SMA and FAP in NFs. MTT assay showed that cell proliferation in the TSF-SPAC group was significantly different from that in the NF-SPAC and SPAC groups (P<0.05). Cell proliferation was not different between the NF-SPAC and SPAC groups (P>0.05). Transwell and scratch assays showed no difference in cell invasion and migration among the groups (P>0.05). ELISA showed that no significant difference in VEGF expression among the three groups (P>0.05).@*CONCLUSIONS@#TSFs may be involved in SPA biological behavior by promoting the proliferation of SPACs but has no effect on the invasion and migration of SPACs in vitro. Hence, TSF may be a new therapeutic target in SPA treatment.

Avaliação da via do c-kit no adenoma pleomórfico e carcinoma adenoide cístico das glândulas salivares: estudo da expressão das proteínas da via, das mutações e da regulação pós-transcricional do gene kit / c-Kit signaling cascade in pleomorphic adenoma and adenoid cystic carcinoma of salivary glands: evaluation of cascade proteins, mutations and post-transcriptional regulation of gene Kit

Introdução: As neoplasias das glândulas salivares têm amplo espectro histológico resultante da múltipla diferenciação celular tumoral. O adenoma pleomórfico (AP) e o carcinoma adenoide cístico (CAC) são as mais comuns neoplasias benignas e malignas provenientes do ducto intercalado, respectivamente, além de serem compostas por estruturas luminais e células mioepiteliais. Em estudo realizado previamente pelo nosso grupo, detectamos que a proteína c-kit está envolvida nos processos da morfogênese das glândulas salivares e no adenoma pleomórfico. A proteína c-Kit tem papel importante no desenvolvimento de muitos processos embrionários, incluindo a gametogênese, melanogênese e hematopoiese, e também na biologia de tumores. Sua ativação induz diversas respostas intracelulares através de cascatas de sinalização de vias como PI3K/AKT e MAPK. Em tumores da glândula salivar ainda há poucos estudos sobre as alterações do gene KIT e das proteínas relacionadas a sua via de sinalização, assim como sua regulação pós-transcricional, realizada principalmente por meio dos microRNAs. O presente estudo avaliou, em APs e CACs (a) a localização das proteínas das vias PI3K/AKT/mTOR e MAPK por meio da técnica de imunoistoquímica; (b) a expressão dos microRNAs 221 e 222, relacionados ao gene KIT (c) a associação dos achados laboratoriais com variáveis clínicas, patológicas e sobrevida. Resultados: Nos casos de AP a proteína c-Kit foi identificada em formações luminais e em raras células isoladas no parênquima tumoral. Já nos CAC, observou-se positividade na membrana das células ductais. Para a via de PI3K/AKT/mTOR, no AP, a proteína PI3K beta mostrou-se parcialmente positiva no citoplasma das células próximas à capsula tumoral, e as proteínas AKT e mTOR fosforiladas, foram expressas especialmente nas células epiteliais e em poucas células mioepiteliais. Já no CAC, a proteína PI3K beta e AKT fosforilada mostraram-se negativas na maioria dos casos, e a proteína mTOR fosforilada foi expressa no citoplasma das células epiteliais e em algumas células mioepiteliais. Para a via MAPK, as proteínas RAS, MEK-1 fosforilada e ERK 1/2 foram negativas na maioria dos AP e CAC; B-Raf e MEK-2 fosforilada foram observadas nas células luminais dos AP. Nos CAC, estruturas luminais neoplásicas foram positivas para a proteína MEK-2 fosforilada; B-Raf foi positivo nas células luminais e mioepiteliais. Além disso, os pacientes que expressaram as proteínas mTOR e MEK-2 fosforilada apresentaram sobrevida câncer-específica significativamente aumentada (p=0,040 e p=0,005, respectivamente). Na análise do microRNAs, a expressão do miR-221 foi variável nas 13 amostras analisadas, tendo baixa expressão em 30,77% dos casos, expressão normal em 38,46 e expressão aumentada em 30,77% dos casos. Já nos APs o miR-221 foi detectado em 19 amostras, sendo 36,84% com baixa expressão, 52,63% com expressão normal e expressão aumentada foi vista em 10,53% dos casos. A expressão do miR-222 foi detectada em 14 CACs, sendo que a maioria dos casos (8 casos ­ 57,1%) a expressão do miR-222 foi semelhante ao observado nas amostras não neoplásicas. Nos APs, o miR-222 foi detectado em 22 amostras, sendo 31,8% com baixa expressão, 31,8% com expressão normal e 36,4% com expressão aumentada. Conclusão: Apesar de a proteína c-Kit ser expressa em ambas as neoplasias ­ AP e CAC, sua influência sobre as vias de sinalização MAPK e PI3K/AKT/mTOR ainda permanece por ser estabelecida. Ainda, os microRNAs 221 e 222 não mostram correlação consistente com a expressão de c-Kit nos tipos tumorais estudados.

Introduction: Salivary gland tumors present broad histological spectrum resulting from multiple tumor cell differentiation. Pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) are the commonest benign and malignant salivary gland neoplasms originated from the intercalated duct region, respectively, and are composed by luminal structures and myoepithelial cells. In a previous study we detected that protein c-kit is involved in the process of salivary gland morphogenesis and PA. c-Kit protein is important during embryogenesis, including gametogenesis, melanogeneis and hematopoiesis as well as in tumorigenesis. Its activation induces various intracellular responses through pathways such as MAPK and PI3K/AKT/mTOR signaling cascades. In salivary gland neoplasms, only a few reports have shown that alterations in KIT gene are present and proteins related to its signaling pathway as well as its post-transcriptional regulation. This study has aimed at evaluating in PA and ACC: (a) the proteins location of PI3K/AKT/mTOR and MAPK pathways using immunohistochemistry (IHC); (b) expression of miR-221 and miR-222, related to KIT gene; and (c) the association of these findings with clinical, pathological and survival data of patients. Results: In PA c-kit was positive in isolated luminal cells; in ACC, neoplastic luminal structures were positive for c-Kit. In PA, PI3K beta protein was shown to be partially positive in the cytoplasm of cells near the tumor capsule and phosphor AKT and phospho mTOR, are specifically expressed in epithelial cells and in a few myoepithelial. In ACC, PI3K and phosphor AKT protein showed to be negative in most of cases. Phospho mTOR protein was expressed in the cytoplasm of epithelial cells and some myoepithelial cells. In MAPK pathway, Ras, ERK1/2 and phosphor MEK-1 proteins were negative in most PAs and CACs; B-Raf and phospho MEK-2 were detected in luminal cells of PA. In ACC neoplastic luminal structures were positive for phospho MEK-2; B-Raf was also positive in myoepithelial and epithelial cells. In addition, cases with expressed phospho-mTOR and phosphor MEK-2 proteins were significantly associated with higher cancer-specific survival (p = 0.040 and p = 0.005, respectively). Moreover, expression of miR-221 was detected in 13 CAC samples and 19 PA samples. In CAC, expression of miR-221 was downregulated in 30,77% of the samples, upregulated in 30,77% samples, and normal in 38,46% samples. In PA, miR-221 expression was downregulated in 36,84% samples, upregulated in 10,53% samples, and normal in 52,63% samples. Expression of miR-222 was detected in 14 CAC samples and 22 PA samples. In the majority of CAC samples, the expression of miR-222 was similar to that observed in non-neoplastic samples. In PA samples, expression of miR-222 was downregulated in 31,8% samples, upregulated in 36,4% samples, and normal in 31,8% samples. Conclusion: Although c-Kit expression is detected in PA and ACC, its influence on the MAPK e PI3K/AKT/mTOR signaling cascades remains to be established. miR-221 e -222 did not show a robust correlation with c-Kit expression in the tumors studied.

Inmunohistochemical expression of PCNA, p53 and bcl-2 in pleomorphic adenomas / Expresión inmunohistoquímica de PCNA, p53 y bcl-2 en adenomas pleomórficos

The aim of the study was to determine the immunohistochemical expression of the PCNA, p53 and bcl-2 proteins in pleomorphic adenomas. Nineteen specimens of pleomorphic adenomas were selected for analysis by the streptavidin-biotin-peroxidase method with antibodies againstp53, PCNA and bcl-2 proteins. It was observed weak labeling for p53 in 12 cases (63.1 percent) andforPCNA in 8 (42.1 percent). With respect to the bcl-2 labeling index, o no expression of this protein was detected in 12 cases, corresponding to 63.1 percent of the sample. Based on these findings, it was concluded that p53 and PCNA can favour the proliferative activity of pleomorphic adenomas, whereas bcl-2 probably does not effectively participate in the pathogenesis of this tumor.

El objetivo del estudio fue determinar la expresión inmmunohistoquímica de las proteínas PCNA, p53 y bcl-2 en adenomas pleomórficos. Fueron seleccionados 19 especímenes de adenomas pleomórficos para análisis a través del método de la estreptavidina-biotina-peroxidasa con anticuerpos contra las proteínas p53, PCNA y bcl-2. Fue observada leve marcación para p53 en 12 casos (63,1 por ciento) y para PCNA en 8 (42,1 por ciento). Con relación al índice de marcación para bcl-2, ono fue detectada en 12 casos (63,1 por ciento) expresión de esta proteína. En base a los resultados, se concluyó que las proteínas p53 y PCNA pueden favorecer la actividad proliferativa de adenomas pleomórficos, y por otro lado, la bcl-2 probablemente ono participaría efectivamente de la patogenia de este tumor.

Immunohistochemical evaluation of p27 (kip1) in pleomorphic adenomas and adenoid cystic carcinomas of the minor salivary glands.

BACKGROUND: p27(kip1), a universal cyclin-dependent kinase inhibitor, is a useful marker for predicting clinical aggressiveness with various human tumors. In this study, p27 expression was investigated in pleomorphic adenomas (PAs) and adenoid cystic carcinomas (ACCs) of minor salivary glands to evaluate its utility for differentiation purposes. At the same time, the correlation between p27 and ACC grading was evaluated. MATERIALS AND METHODS: Clinicopathological features of 22 patients (11 ACCs, 11 PAs), including age, sex and size of tumor were obtained from medical records. Immunohistochemical staining with p27(kip1) was performed for each specimen and p27 labelling indices were determined with a computer-assisted image-analyzing system (CAS 200). Pearson's correlation coefficient, Spearman's correlation coefficient, Students t-test, Kruskal-Wallis test and ANOVA were applied for statistical analyses using SPSS 11.5. RESULTS: p27 LIs for all PAs were above 25% whereas for ACCs they were under 25% (except one case). p27 expression (LI and intensity) was significantly lower in ACCs than PAs. The correlation between p27 expression and ACC grading was not significant. CONCLUSION: Overall, these findings suggest that reduced expression of p27 might be correlated with the development of ACC and could be an indicator of malignant behavior.

Estudo imuno-histoquímico do carcinoma adenóide cístico de glândula salivar menor / Immunohistochemical study of minor salivary gland's adenoid cystic carcinoma

A expressäo de um painel de anticorpos, constituídos pelas citoqueratinas 7, 8, 10, 13, 14, 18 e 19, vimentina e HHF-35, foi estudada em 21 casos de carcinomas adenóide císticos de glândula salivar menor, de diferentes sub-tipos histológicos, utilizando-se a técnica da imunohistoquímica, pelo método da estreptpavidina-biotina. Os resultados mostraram que nos tumores cribriformes, as células luminas das estruturas ductais foram positivas para as citoqueratinas 7, 8, 14, 18 e 19 e negativas para a vimentina e HHF-35, enquanto as células externas dos ductos, as células que revestiam os espaços pseudo-císticos e as células periféricas dos cilindros foram negativas para as citoqueratinas testadas e positivas para a vimentina e HHF-35. Nos tumores tubulares, enquanto as células internas dos ductos mostraram-se positivas para as citoqueratinas 7, 8, 14, 18 e 19 e negativas para vimentina e HHF-35, as células periféricas das estruturas ductais, apresentaram-se sempre positivas para a vimentina e HHF-35 e ocasionalmente positivas para a citoqueratina 14. Nas neoplasias do subtipo sólido, observou-se positividade esporádica das células, especialmente para as citoqueratinas 7 e 14, bem como para a vimentina. Os nossos achados mostraram que o carcinoma adenóide cístico, de um modo geral, mimetiza, em seu perfil imuno-histoquímico, o segmento de ducto intercalado da glândula salivar normal, apresentando variaçöes que sugerem diferentes graus de diferenciaçäo para os seus três subtipos histológicos. Os tumores tubulares representam a variante mais diferenciada, os tumores sólidos, o subtipo menos diferenciado, e os tumores cribiformes, neoplasias de um grau intermediário de diferenciaçäo entre aquele visto nas outras duas variantes histológicas