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Proteasome Inhibitors Enhance TRAIL-Induced Apoptosis through the Intronic Regulation of DR5: Involvement of NF-B and Reactive Oxygen Species-Mediated p53 Activation¹

Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan

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-B elements on the DR5 first intron region were involved in proteasome inhibitor-induced DR5 expression. Both p53 small interfering RNA and NF-B inhibitor suppressed DR5 expression, strengthening the significance of p53 and NF-B in DR5 transcription. The protein stability, Ser³⁹² phosphorylation and Lys³⁷³/Lys³⁸² acetylation of p53 were enhanced by MG132. In addition to p53, IB degradation and NF-B 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-B 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Research Grant NSC-96-2320-B002-018 from the National Science Council of Taiwan.

² Address correspondence and reprint requests to Dr. Ching-Chow Chen, Department of Pharmacology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Section 1, Taipei 10018, Taiwan. E-mail address: chingchowchen{at}ntu.edu.tw

³ Abbreviations used in this paper: ROS, reactive oxygen species; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; IKK, IB kinase; DAPA, DNA affinity protein-binding assay; NAC, N-acetylcysteine; GSH, glutathione; PSI-1; proteasome inhibitor 1; CHOP, C/EBP homologous protein.

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