Endoplasmic Reticulum Stress-Induced JNK Activation Is a Critical Event Leading to Mitochondria-Mediated Cell Death Caused by β-Lapachone Treatment

β-lapachone (β-lap) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although β-lap has been reported to induce apoptosis in various cancer types in an NQO1-dependent manner, the signaling pathways by which β-lap causes apoptosis are poorly understood.β-lap-induced apoptosis and related molecular signaling pathways in NQO1-negative and NQO1-overexpressing MDA-MB-231 cells were investigated. Pharmacological inhibitors or siRNAs against factors involved in β-lap-induced apoptosis were used to clarify the roles played by such factors in β-lap-activated apoptotic signaling pathways. β-lap leads to clonogenic cell death and apoptosis in an NQO1- dependent manner. Treatment of NQO1-overexpressing MDA-MB-231 cells with β-lap causes rapid disruption of mitochondrial membrane potential, nuclear translocation of AIF and Endo G from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNAs targeting AIF and Endo G effectively attenuate β-lap-induced clonogenic and apoptotic cell death. Moreover, β-lap induces cleavage of Bax, which accumulates in mitochondria, coinciding with the observed changes in mitochondria membrane potential. Pretreatment with Salubrinal (Sal), an endoplasmic reticulum (ER) stress inhibitor, efficiently attenuates JNK activation caused by β-lap, and subsequent mitochondria-mediated cell death. In addition, β-lap-induced generation and mitochondrial translocation of cleaved Bax are efficiently blocked by JNK inhibition.Our results indicate that β-lap triggers induction of endoplasmic reticulum (ER) stress, thereby leading to JNK activation and mitochondria-mediated apoptosis. The signaling pathways that we revealed in this study may significantly contribute to an improvement of NQO1-directed tumor therapies

NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract

Background: We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-kappa B is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-kappa B in mediating cell survival in response to cigarette smoke exposure in HBECs. Methods: Both the pharmacologic inhibitor of NF-kappa B, curcumin, and RNA interference targeting p65 were used to block NF-kappa B signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay. Results: Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-kappa B -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-kappa B by the pharmacologic inhibitor curcumin (20 mu M) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium. Conclusion: The current study demonstrates that CSE activates NF-kappa B and up-regulates Bcl-XL through NF-kappa B activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-kappa B regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000260432600001&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Respiratory SystemSCI(E)28ARTICLEnull

Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-κB-Dependent Transcription of Specific Genes after Genotoxic Stress

The NF-κB pathway is involved in immune and inflammation responses, proliferation, differentiation and cell death or survival. It is activated by many external stimuli including genotoxic stress. DNA double-strand breaks activate NF-κB in an ATM-dependent manner. In this manuscript, a direct interaction between p65(RelA) and the N-terminal extremity of ATM is reported. We also report that only one of the five potential ATM-(S/T)Q target sites present in p65, namely Ser547, is specifically phosphorylated by ATM in vitro. A comparative transcriptomic analysis performed in HEK-293 cells expressing either wild-type HA-p65 or a non-phosphorylatable mutant HA-p65S547A identified several differentially transcribed genes after an etoposide treatment (e.g. IL8, A20, SELE). The transcription of these genes is increased in cells expressing the mutant. Substitution of Ser547 to alanine does not affect p65 binding abilities on the κB site of the IL8 promoter but reduces p65 interaction with HDAC1. Cells expressing p65S547A have a higher level of histone H3 acetylated on Lys9 at the IL8 promoter, which is in agreement with the higher gene induction observed. These results indicate that ATM regulates a sub-set of NF-κB dependent genes after a genotoxic stress by direct phosphorylation of p65