RESUMEN
BACKGROUND: The aminoisoquinoline FX-9 shows pro-apoptotic and antimitotic effects against lymphoblastic leukemia cells and prostate adenocarcinoma cells. In contrast, decreased cytotoxic effects against non-neoplastic blood cells, chondrocytes, and fibroblasts were observed. However, the actual FX-9 molecular mode of action is currently not fully understood. METHODS: In this study, microarray gene expression analysis comparing FX-9 exposed and unexposed prostate cancer cells (PC-3 representing castration-resistant prostate cancer), followed by pathway analysis and gene annotation to functional processes were performed. Immunocytochemistry staining was performed with selected targets. RESULTS: Expression analysis revealed 0.83% of 21,448 differential expressed genes (DEGs) after 6-h exposure of FX-9 and 0.68% DEGs after 12-h exposure thereof. Functional annotation showed that FX-9 primarily caused an activation of inflammatory response by non-canonical nuclear factor-kappa B (NF-κB) signaling. The 6-h samples showed activation of the cell cycle inhibitor CDKN1A which might be involved in the secondary response in 12-h samples. This secondary response predominantly consisted of cell cycle-related changes, with further activation of CDKN1A and inhibition of the transcription factor E2F1, including downstream target genes, resulting in G1-phase arrest. Matching our previous observations on cellular level senescence signaling pathways were also found enriched. To verify these results immunocytochemical staining of p21 Waf1/Cip1 (CDKN1A), E2F1 (E2F1), PAI-1 (SERPNE1), and NFkB2/NFkB p 100 (NFKB2) was performed. Increased expression of p21 Waf1/Cip1 and NFkB2/NFkB p 100 after 24-h exposure to FX-9 was shown. E2F1 and PAI-1 showed no increased expression. CONCLUSIONS: FX-9 induced G1-phase arrest of PC-3 cells through activation of the cell cycle inhibitor CDKN1A, which was initiated by an inflammatory response of noncanonical NF-κB signaling.
Asunto(s)
Antineoplásicos/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Isoquinolinas/farmacología , FN-kappa B/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Antineoplásicos/uso terapéutico , Factor de Transcripción E2F1/antagonistas & inhibidores , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Humanos , Isoquinolinas/uso terapéutico , Masculino , Persona de Mediana Edad , Células PC-3 , Inhibidor 1 de Activador Plasminogénico/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Puntos de Control de la Fase S del Ciclo Celular , Factores de Tiempo , Análisis de Matrices TisularesRESUMEN
Ultrasound-guided fine-needle aspiration (US-FNA) biopsy is a widely used minimally invasive sampling procedure for cytological diagnosis. This study investigates the feasibility of using US-FNA samples for both cytological diagnosis and whole transcriptome RNA-sequencing analysis (RNA-Seq), with the ultimate aim of improving canine prostate cancer management. The feasibility of the US-FNA procedure was evaluated intra vitam on 43 dogs. Additionally, aspirates from 31 euthanised dogs were collected for standardising the procedure. Each aspirate was separated into two subsamples: for cytology and RNA extraction. Additional prostate tissue samples served as control for RNA quantity and quality evaluation, and differential expression analysis. The US-FNA sampling procedure was feasible in 95% of dogs. RNA isolation of US-FNA samples was successfully performed using phenol-chloroform extraction. The extracted RNA of 56% of a subset of US-FNA samples met the quality requirements for RNA-Seq. Expression analysis revealed that only 153 genes were exclusively differentially expressed between non-malignant US-FNAs and tissues. Moreover, only 36 differentially expressed genes were associated with the US-FNA sampling technique and unrelated to the diagnosis. Furthermore, the gene expression profiles clearly distinguished between non-malignant and malignant samples. This proves US-FNA to be useful for molecular profiling.