Proteasome Inhibitors Enhance TRAIL-Induced Apoptosis through the Intronic Regulation of DR5: Involvement of NF-κB and Reactive Oxygen Species-Mediated p53 Activation

Abstract

Manipulation of TRAIL receptor 2 (DR5) pathway is a promising therapeutic strategy to overcome TRAIL-resistant lung cancer cells. Preclinical studies have shown that proteasome inhibitors enhance TRAIL-induced apoptosis in lung cancer cells, but the underlying mechanism has not been fully elucidated. In this study, we demonstrated the enhancement of TRAIL-mediated apoptosis in human alveolar epithelial cells by proteasome inhibitors that up-regulate DR5 expression. This effect was blocked by DR5-neutralizing Ab. Using reporter assay, we demonstrated that the p53 and NF-κB elements on the DR5 first intron region were involved in proteasome inhibitor-induced DR5 expression. Both p53 small interfering RNA and NF-κB inhibitor suppressed DR5 expression, strengthening the significance of p53 and NF-κB in DR5 transcription. The protein stability, Ser392 phosphorylation and Lys373/Lys382 acetylation of p53 were enhanced by MG132. In addition to p53, IκBα degradation and NF-κB translocation was also observed. Moreover, the binding of p53 and p65 to the first intron of DR5 was demonstrated by DNA affinity protein-binding and chromatin immunoprecipitation assays. Intracellular reactive oxygen species (ROS) generation after MG132 treatment contributed to p53, but not p65 nuclear translocation and DNA-binding activity. ROS scavenger dramatically inhibited the apoptosis induced by proteasome inhibitors plus TRAIL. The p53-null H1299 cells were resistant to proteasome inhibitor-induced DR5 up-regulation and enhancement of TRAIL-induced apoptosis. These findings reveal that proteasome inhibitor-mediated NF-κB and ROS-dependent p53 activation are contributed to intronic regulation of DR5 transcription, and resulted in the subsequent enhancement of TRAIL-induced apoptosis in human lung cancer cells.

γ-Tocotrienol Promotes TRAIL-Induced Apoptosis through Reactive Oxygen Species/Extracellular Signal-Regulated Kinase/p53–Mediated Upregulation of Death Receptors

“…These findings corroborate the previous findings with curcumin (34), rosiglitazone (36), sulforaphane (35), proteasome inhibitors (33), and baicalein (37).…”

“…Previously, ROS have been implicated in induction of receptors by certain agents (33)(34)(35)(36)(37). Whether γ-T3 can generate ROS was examined by FACS.…”

Death receptor 5-mediated TNFR family signaling pathways modulate γ-humulene-induced apoptosis in human colorectal cancer HT29 cells

“…The transcriptional regulation of DR5 is complex and the multiple transcription factor binding sites of CHOP (CCAAT/ enhancer-binding protein-homologous protein), p53 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), are present in the DR5 upstream region (26,27). Recently, it was also reported that p53 is a key regulator activating the extrinsic pathway through the induction of genes encoding transmembrane proteins such as DR4 and DR5 (18).…”

Nucleophosmin Blocks Mitochondrial Localization of p53 and Apoptosis

“…It is well documented that increased phosphorylation of p53 at the Ser 392 position stabilizes the tetramer formation of tumor suppressor protein p53 (32). It has been shown that proteasome inhibitor MG132 increases the stability of p53 by enhancing the expression of p53 S-392 phosphorylation (33). It has also been shown that stress-dependent stabilization of p53 in the cytoplasmic pool targets p53 to locate in the mitochondria after monoubiquitylation (34).…”