Proteasome inhibitors enhance TRAIL-induced apoptosis through the intronic regulation of DR5: involvement of NF-kappa B and reactive oxygen species-mediated p53 activation.
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-kappaB elements on the DR5 first intron region were involved in proteasome inhibitor-induced DR5 expression. Both p53 small interfering RNA and NF-kappaB inhibitor suppressed DR5 expression, strengthening the significance of p53 and NF-kappaB in DR5 transcription. The protein stability, Ser(392) phosphorylation and Lys(373)/Lys(382) acetylation of p53 were enhanced by MG132. In addition to p53, IkappaBalpha degradation and NF-kappaB 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-kappaB 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.
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Jun-Jie
JJ
Chen
Chia-Wei
CW
Chou
Yu-Fan
YF
Chang
Ching-Chow
CC
Chen
D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000276:immunology; D016166:Free Radical Scavengers / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000276:immunology; D045325:HCT116 Cells; D006801:Humans; D007106:Immune Sera / Q000502:physiology; D007438:Introns / Q000276:immunology; D007976:Leupeptins / Q000494:pharmacology; D016328:NF-kappa B / Q000502:physiology*; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D017382:Reactive Oxygen Species / Q000378:metabolism* / Q000494:pharmacology; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D020545:Respiratory Mucosa / Q000166:cytology / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000378:metabolism* / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology
D015853:Cysteine Proteinase Inhibitors; D016166:Free Radical Scavengers; D007106:Immune Sera; D007976:Leupeptins; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D016159:Tumor Suppressor Protein p53; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde