ABSTRACT
ABSTRACT Background: Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p 0.001) or indirectly (p 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.
ABSTRACT
Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.(AU)
Subject(s)
Animals , Peptides , Bothrops , Crotalid Venoms/isolation & purification , Lectins, C-Type/isolation & purification , Neuroblastoma , Neutrophils , In Vitro TechniquesABSTRACT
Abstract The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.
Resumo As propriedades antitumorais de extratos de glândulas salivares de carrapatos ou proteínas recombinantes foram relatadas recentemente, mas pouco se sabe sobre as propriedades antitumorais dos componentes secretados da saliva. O objetivo deste estudo foi investigar o efeito in vitro da saliva bruta do carrapato duro Amblyomma sculptum sobre as linhagens celulares de neuroblastoma. Células SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32 e CHLA-20 foram suscetíveis à saliva, com redução de 80% na sua viabilidade em comparação com controles não tratados, como demonstrado pelo ensaio de Azul de Metileno. Investigações posteriores utilizando CHLA-20 revelaram apoptose, com aproximadamente 30% de células positivas para anexina-V, e G0/G1 (> 60%) após tratamento com saliva. O potencial de membrana mitocondrial (Δψm) foi reduzido significativamente (p <0,05), e o citoesqueleto de actina foi desestruturado, como indicado pela microscopia de fluorescência. A viabilidade do fibroblasto humano (células HFF-1), usado como controle não tumoral, diminuiu em aproximadamente 40%. No entanto, não foram observadas alterações na progressão do ciclo celular, morfologia e Δψm nestas células. O presente trabalho fornece novas perspectivas para a caracterização das moléculas presentes na saliva e suas propriedades antitumorais.
Subject(s)
Animals , Saliva/chemistry , Biological Products/pharmacology , Cytoskeleton/drug effects , Ixodidae/chemistry , Arthropod Proteins/pharmacology , Neuroblastoma/pathology , Antineoplastic Agents/pharmacology , Biological Products/isolation & purification , Cell Survival/drug effects , Apoptosis/drug effects , Cell Line, Tumor , Arthropod Proteins/isolation & purification , Antineoplastic Agents/isolation & purificationABSTRACT
ABSTRACT INTRODUCTION: According to the cancer stem-cell theory, tumors originate from a small population of cancer stem cells, which lose the mechanism of self-regulation and begin to differentiate and proliferate indefinitely. The CD44+/CD24- phenotype may be considered a stem-cell marker in breast cancer. OBJECTIVE: To evaluate the correlation between CD44+/CD24- phenotype and different molecular subtypes of breast cancer in invasive ductal carcinoma samples. METHODS: The expression of CD44, CD44v6, and CD24 markers was investigated in 133 cases of invasive mammary carcinoma with immunohistochemistry. CD44+/CD24- phenotype was identified and correlated with the molecular subtypes and classical prognostic factors such as age, histological grade, tumor size, and lymph node status. RESULTS: Eighteen (14%) cases were positive for CD44+/CD24- (CD44+/CD24- or CD44v6+/CD24-) phenotype; among these, 11.1%, 27.8%, 38.9%, and 22.2% were luminal, luminal B-human epidermal growth factor receptor 2 (HER2), HER2, and triple-negative subtype, respectively. CD44+/ CD24- phenotype was more common in HER2 subgroup (p = 0.0197). CONCLUSION: CD44+/CD24- phenotype was correlated with molecular subtypes of breast cancer. The highest expression of CD44+/CD24- phenotype was reported in patients with HER2+ disease, a molecular subtype associated with more aggressive behavior and worse prognosis.
RESUMO INTRODUÇÃO: De acordo com a teoria das células-tronco tumorais, os tumores são originários de uma pequena população de células-tronco que perdem o mecanismo de autorregulação e começam a se diferenciar e proliferar indefinidamente. O fenótipo CD44+/CD24- pode ser considerado um marcador de células-tronco tumorais no câncer de mama. OBJETIVO: Avaliar a correlação entre o fenótipo CD44+/CD24- e os diferentes subtipos moleculares do câncer de mama em amostras de carcinoma ductal invasor. MÉTODOS: A expressão dos marcadores CD44, CD44v6 e CD24 foi investigada em 133 casos de carcinoma mamário invasor por meio de imuno-histoquímica. O fenótipo CD44+/CD24- foi identificado e correlacionado com os subtipos moleculares e os fatores prognósticos clássicos, como idade, grau histológico, tamanho do tumor e status do linfonodo. RESULTADOS: Dezoito (14%) casos foram positivos para o fenótipo CD44+/CD24- (CD44+/CD24- ou CD44v6+/CD24-), sendo 11, 1%, 27, 8%, 38, 9% e 22, 2% dos subtipos luminal, luminal B-human epidermal growth factor receptor 2 (HER2), HER2 e triplo negativo, respectivamente. O fenótipo CD44+/CD24- foi mais comum no subgrupo HER2 (p = 0, 0197). CONCLUSÃO: O fenótipo CD44+/CD24- foi correlacionado com os subtipos moleculares do câncer de mama. A maior expressão do fenótipo CD44+/CD24- foi encontrada em pacientes com doença HER2+, subtipo molecular associado a um comportamento mais agressivo e a um pior prognóstico.