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

Hyemi Lee,Bo-Hwa Choi,Eun-Taex Oh,Heon Joo Park,Min-Jeong Song,Byung Uk Lim,Moon-Taek Park,Chulhee Kim,Jeonghun Lee,Mikhail V Blagosklonny

doi:10.1371/journal.pone.0021533

Abstract

Backgroundβ-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.Methodology/Principal Findingsβ-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.Conclusions/SignificanceOur 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.

Highlights

Introduction b-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione; b-lap), a naturally occurring quinone obtained from the bark of the Lapacho tree (Tabebuia vellanedae), has been reported to exert a variety of pharmacological activities, such as anti-bacterial, -fungal, -trypanocidal, and -cancer [1,2]
The futile cycling between the oxidized and reduced forms of b-lap caused by NAD(P)H quinone oxidoreductase 1 (NQO1) leads to progressive depletion of NADH and NAD(P)H, which in turn induces a massive release of Ca2+ from Endoplasmic Reticulum (ER) to cytosol, causing activation of calpain known as a Ca2+ dependent proteinase and subsequent apoptosis [1,2,5]
As the activation of caspase plays a key role in the induction of apoptotic cell death [17], we investigated whether caspase activities are required for b-lap-induced apoptotic cell death in NQO1+-MDA-MB-231 cells using a broad-spectrum caspase inhibitor, z-VAD-fmk

Summary

Introduction

Introduction b-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione; b-lap), a naturally occurring quinone obtained from the bark of the Lapacho tree (Tabebuia vellanedae), has been reported to exert a variety of pharmacological activities, such as anti-bacterial, -fungal, -trypanocidal, and -cancer [1,2]. We demonstrate here that JNK activation due to ER stress significantly contributes to mitochondria-mediated cell death caused by b-lap treatment. Subcellular fractionation showed that the treatment of NQO1+-MDA-MB-231 cells with b-lap dramatically induced the translocation of AIF and Endo G to the nucleus (Fig. 2A).

Results

Conclusion