RESUMO
The complexity of different components of tumor stroma poses huge challenges for therapies targeting the neuroblastoma (NB) microenvironment. The present study aimed to evaluate platinum-based response in IMR-32 neuroblastoma cell line cultured in monolayer (2D) and neurosphere (3D) models. For this, we evaluated mRNA expression of heat shock proteins HSPA1A, HSPB1, TRAP1, HSPA1AL, HSPD1, and DNA damage repair gene ERCC1. After treatment, residual cells were grafted on CAM (chicken chorioallantoic membrane) to evaluate the growth capability and histological paraffin sections were made to assess Ki-67 and HER-2 proteins by immunofluorescence. Our results showed that cisplatin induces mRNA downregulation of Heat Shock Proteins and ERCC1 in IMR-32 cells cultured in 2D or 3D models. In addition, the cisplatin-treatment approach increased HER-2 expression in residual IMR-32 cells grafted on the CAM. Therefore, these insights provide many advances in neuroendocrine tumor biology and knowledge about cisplatin-response in neuroblastoma.
Assuntos
Antineoplásicos , Células-Tronco Neurais , Neuroblastoma , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Proteínas de Choque Térmico HSP90 , Humanos , Recidiva Local de Neoplasia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Microambiente TumoralRESUMO
The identification of the best reference gene is a critical step to evaluate the relative change in mRNA expression of a target gene by RT-qPCR. In this work, we evaluated nineteen genes of different functional classes using Real Time Human Reference Gene Panel (Roche Applied Sciences), to identify the internal housekeeping genes (HKGs) most suitable for gene expression normalization data in human cell lines. Normal cell lines CCD-19LU (lung fibroblast), HEK-293 (epithelial cell of embryonic kidney), WI-26 VA4 (lung fibroblast), and human cancer cells, BT-549 (breast cancer), Hs 578T (breast cancer), MACL-1 (breast cancer), HeLa (cervical carcinoma), U-87 MG (glioblastoma/astrocytoma), RKO-AS45-1 (colorectal carcinoma), and TOV-21G (ovarian adenocarcinoma) were cultivated according to manufacturer's protocol. Twelve candidate reference genes were commonly expressed in five cell lines (CCD-19Lu, HEK-293, RKO-AS45-1, TOV-21G, and U-87 MG). To verify the expression stability, we used the RefFinder web tool, which integrates data from the computational programs Normfinder, BestKeeper, geNorm, and the comparative Delta-Ct method. The ACTB was the most stable reference gene to the CCD-19Lu and HEK-293 cells. The best combination of HKGs for the RKO-AS45-1 and TOV-21G cell lines were B2M/GAPDH and PBGD/B2M, respectively. For the U-87 MG cells, GAPDH and IPO8 were the most suitable HKGs. Thus, our findings showed that it is crucial to use the right HKGs to precise normalize gene expression levels in cancer studies, once a suitable HKG for one cell type cannot be to the other.
Assuntos
Adenocarcinoma , Genes Essenciais , Genes Essenciais/genética , Células HEK293 , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Padrões de ReferênciaRESUMO
The epithelial-to-mesenchymal transition (EMT) is a phenomenon during which cancer epithelial cells undergo changes in plasticity and lose cell-cell adhesion with consequent remodeling of the extracellular matrix and development of mesenchymal characteristics. Long non-coding RNAs (lncRNAs) have been described as EMT modulation markers, becoming a promising target in the development of new therapies for cancer. The present study aimed to investigate the role of everolimus at 100 nM as inductor of the EMT phenomenon in cell lines derived from human breast (BT-549), colorectal (RKO-AS45-1) and ovary (TOV-21G) cancer. The integrity of cellular junctions was monitored using an in vitro model of epithelial resistance. The results demonstrated that the EMT genes ZEB1, TWIST1 and TGFB1 were differentially expressed in cells treated with everolimus compared with in untreated cells. lncRNA HOTAIR was upregulated post-treatment only in BT-549 cells compared with in untreated cells. After treatment with everolimus, the intensity of fluorescence of P-cadherin decreased, and that of fibronectin increased in RKO-AS45-1 and TOV-21G cells compared with control cells. The transepithelial electrical resistance at the RKO-AS45-1 monolayer treated with everolimus started to decrease at 48 h. The changes in the gene expression and epithelial resistance may confirm the role of everolimus in EMT.