Inhibition of the JNK signalling pathway enhances proteasome inhibitor-induced apoptosis of kidney cancer cells by suppression of BAG3 expression.
ABSTRACT Proteasome inhibitors represent a novel class of anti-tumour agents that have clinical efficacy against haematological and solid cancers. The anti-apoptotic protein BAG3 is a member of the Bcl-2-associated athanogene family. We have previously shown that BAG3 is up-regulated after exposure to proteasome inhibitors and that inhibition of BAG3 sensitized cells to apoptosis induced by proteasome inhibition. However, the mechanisms by which proteasome inhibition induced BAG3 expression remained unclear and the present experiments were designed to elucidate these mechanisms.
Effects of the proteasome inhibitor MG132 on activation of mitogenic signalling pathways were evaluated in kidney cancer cells (A498, Caki1, Caki2), with Western blotting. Specific inhibitors against individual mitogenic signalling pathways, real-time reverse transcription-polymerase chain reaction and luciferase reporter assays were used to investigate the roles of mitogenic signalling pathways in BAG3 induction after proteasome inhibition. Cell death was evaluated using Annexin V/propidium iodide staining and subsequent FACS.
MG132 activated several key mitogenic signalling pathways including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activities. Induction of BAG3 by MG132 was inhibited by blocking JNK, but not ERK1/2 and p38 MAPK signalling pathways. In addition, SP600125 and dominant-negative JNK1 suppressed BAG3 promoter-driven reporter gene expression. Furthermore, activation of the JNK pathway induced BAG in kidney cancer cells after treatment with MG132.
Our results suggested that the JNK pathway was associated with the protective response against proteasome inhibition, by mediating induction of BAG3.
Full-textDOI: · Available from: Xin Meng, Feb 07, 2015
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ABSTRACT: 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). 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. 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. 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.Albrecht von Graæes Archiv für Ophthalmologie 12/2014; 253(3). DOI:10.1007/s00417-014-2874-1 · 2.33 Impact Factor
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