NOX5-S Knockdown Inhibits Esophageal Adenocarcinoma Cell Progression by Inactivating the PI3K/AKT/NF-κB Pathway

Abstract

Background: Esophageal adenocarcinoma (EAC), the major form of esophageal cancer, has the high incidence worldwide and patients with EAC have poor prognosis due to limited treatment. It is critical to develop a comprehensive understanding of progression of EAC cells for developing more effective and targeted therapies. Since the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 5 (NOX5)-S has been associated with the progression of EAC, we aimed to explore the effect of NOX5-S on the EAC cell progression and to elucidate the mechanisms underlying this effect. Methods: The differential expression of NOX5-S in human normal esophageal epithelial cells Het1A and human EAC cells OE19 was evaluated using western blot and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The cells were transfected with pCMV-tag5a-NOX5-S (overexpressing plasmids (OE)-NOX5-S), si-NOX5-S, and blank control (OE-NC) to assess the effects of NOX5-S on cell proliferation, migration, and apoptosis using cell counting kit-8, colony formation, transwell, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assays. The western blot was used for evaluating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor-kappaB (NF-κB) axis activation, the Recilisib was used for PI3K/AKT activation and the Prostratin was used for NF-κB pathway activation. The induced alterations of cell proliferation, migration and apoptosis were measured. Results: The expression of NOX5-S was higher in EAC OE19 cells (p < 0.001). Transfection of small interfering (si)-RNA against si-NOX5-S successfully decreased the NOX5-S expression (p < 0.001), and transfection of NOX5-S overexpressing plasmids (OE)-NOX5-S successful increased the NOX5-S expression (p < 0.001). Increased/decreased NOX5-S expression promoted/inhibited EAC cell proliferation (p < 0.001) and migration (p < 0.001), induced less/more EAC cell apoptosis (p < 0.001), and increased/decreased all levels of p-PI3K/PI3K, p-AKT/AKT and p-NF-κB/NF-κB (p < 0.001). Recilisib elevated p-AKT/AKT, p-NF-κB/NF-κB and p-PI3K/PI3K in NOX5-S knockdown cells (p < 0.001) and Prostratin increased p-NF-κB/NF-κB (p < 0.001) in NOX5-S knockdown cells. Both Recilisib and Prostratin reversely increased the proliferation (p < 0.001) and migration (p < 0.001), and reversely decreased apoptosis (p < 0.001) in EAC cells. Conclusions: The NOX5-S knockdown suppresses the progression of EAC cells by inactivating the PI3K/AKT/NF-κB pathway. Knockdown of NOX5-S and inactivation of the PI3K/AKT and NF-κB signaling pathways could be potential strategies for treating EAC.