Reference - Detail
|Author||Yunoki T, Tabuchi Y, Hayashi A, Kondo T.|
|Title||BAG3 protects against hyperthermic stress by modulating NF-κB and ERK activities in human retinoblastoma cells.|
|Journal||Graefes Arch. Clin. Exp. Ophthalmol.|
PURPOSE:BCL2-associated athanogene 3 (BAG3), a co-chaperone of HSP70, is a cytoprotective and anti-apoptotic protein that acts against various stresses, including heat stress. Here, we examined the effect of BAG3 on the sensitivity of human retinoblastoma cells to hyperthermia (HT).
METHODS:We examined the effects of BAG3 knockdown on the sensitivity of Y79 and WERI-Rb-1cells to HT (44 °C, 1 h) by evaluating apoptosis and cell proliferation using western blotting, real-time quantitative PCR (qPCR), flow cytometry, and a WST-8 assay kit. Furthermore, we examined the effects of activating nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK) using western blotting and real time qPCR.
RESULTS:HT induced considerable apoptosis along with the activation of caspase-3 and chromatin condensation. The sensitivity of Y79 and WERI-Rb-1 cells to HT was significantly enhanced by BAG3 knockdown. Compared to HT alone, the combination of BAG3 knockdown and HT reduced phosphorylation of the inhibitors of kappa B α (IκBα) and p65, a subunit of NF-κB, and degraded IκB kinase γ (IKKγ) during the recovery period after HT. Furthermore, BAG3 knockdown increased the HT-induced phosphorylation of ERK after HT treatment, and the ERK inhibitor U0126 significantly improved the viability of the cells treated with a combination of BAG3 knockdown and HT.
CONCLUSIONS:The silencing of BAG3 seems to enhance the effects of HT, at least in part, by maintaining HT-induced inactivity of NF-κB and the phosphorylation of ERK. These findings indicate that BAG3 may be a potential molecular target for modifying the outcomes of HT in retinoblastoma.
|MeSH||Adaptor Proteins, Signal Transducing / physiology* Apoptosis Apoptosis Regulatory Proteins / physiology* Blotting, Western Cell Line, Tumor Cell Proliferation Cell Survival Extracellular Signal-Regulated MAP Kinases / metabolism* Flow Cytometry Gene Knockdown Techniques Humans Hyperthermia, Induced* NF-kappa B / metabolism* Phosphorylation RNA, Small Interfering / genetics Real-Time Polymerase Chain Reaction Retinal Neoplasms / metabolism* Retinal Neoplasms / pathology Retinal Neoplasms / therapy Retinoblastoma / metabolism* Retinoblastoma / pathology Retinoblastoma / therapy Transfection|
|Human and Animal Cells|