β-Lapachone Significantly Increases the Effect of Ionizing Radiation to Cause Mitochondrial Apoptosis via JNK Activation in Cancer Cells

Abstract

Backgroundβ-lapachone (β-lap), has been known to cause NQO1-dependnet death in cancer cells and sensitize cancer cells to ionizing radiation (IR). We investigated the mechanisms underlying the radiosensitization caused by β-lap.Methodology/Principal Findingsβ-lap enhanced the effect of IR to cause clonogenic cells in NQO1+-MDA-MB-231 cells but not in NQO1−-MDA-MB-231 cells. β-lap caused apoptosis only in NQO1+ cells and not in NQO1− cells and it markedly increased IR-induced apoptosis only in NQO1+ cells. Combined treatment of NQO1+ cells induced ROS generation, triggered ER stress and stimulated activation of ERK and JNK. Inhibition of ROS generation by NAC effectively attenuated the activation of ERK and JNK, induction of ER stress, and subsequent apoptosis. Importantly, inhibition of ERK abolished ROS generation and ER stress, whereas inhibition of JNK did not, indicating that positive feedback regulation between ERK activation and ROS generation triggers ER stress in response to combined treatment. Furthermore, prevention of ER stress completely blocked combination treatment-induced JNK activation and subsequent apoptotic cell death. In addition, combined treatment efficiently induced the mitochondrial translocation of cleaved Bax, disrupted mitochondrial membrane potential, and the nuclear translocation of AIF, all of which were efficiently blocked by a JNK inhibitor. Caspases 3, 8 and 9 were activated by combined treatment but inhibition of these caspases did not abolish apoptosis indicating caspase activation played a minor role in the induction of apoptosis.Conclusions/Significanceβ-lap causes NQO1-dependent radiosensitization of cancer cells. When NQO1+ cells are treated with combination of IR and β-lap, positive feedback regulation between ERK and ROS leads to ER stress causing JNK activation and mitochondrial translocation of cleaved Bax. The resultant decrease in mitochondrial membrane leads to translocation of AIF and apoptosis.

PARP1-mediated necrosis is dependent on parallel JNK and Ca2+/calpain pathways

“…Similar results were obtained in MEFs completely deficient in CypD (Fig. 6F,G The translocation of AIF from the mitochondrial inter-membrane space to the nucleus is thought to be the pivotal event in PARP1-induced necrotic death (Xu et al, 2006;Moubarak et al, 2007;Artus et al, 2010;Chiu et al, 2011;Park et al, 2011). Immunocytochemistry revealed that both b-Lapachone and MNNG induced the nuclear translocation of AIF (Fig.…”

“…A more recent study has also implicated RIP1 and JNK as being part of this process (Chiu et al, 2011). Similarly, JNK activation has been reported as being crucial for b-Lapachone-induced cell death (Shiah et al, 1999;Park et al, 2011), although whether RIP1 is also involved in b-Lapachone's effects has never been tested until the present study. Despite this, we were unable to recapitulate these data in 3T3-transformed Ripk1 2/2 MEFs.…”

“…It was originally thought that necrosis induced by PARP1 hyperactivation was simply due to metabolic catastrophe, where the overactive PARP1 used up the supply of NAD + in the cell, and subsequently ATP (van Wijk and Hageman, 2005). However, it is now appreciated that PARP1-induced necrosis follows more specific molecular pathways (Xu et al, 2006;Moubarak et al, 2007;Artus et al, 2010;Dong et al, 2010;Chiu et al, 2011;Park et al, 2011).…”

Tumor-Selective, Futile Redox Cycle-Induced Bystander Effects Elicited by NQO1 Bioactivatable Radiosensitizing Drugs in Triple-Negative Breast Cancers

“…Importantly, the majority of reported cellular effects in blap-treated cancer cells are the result of dramatic NAD + /ATP losses and reactive oxygen species (ROS) formation [reviewed in Bentle et al (5)]. Downstream, l-calpain and JNK activation (30,38) in b-lap-exposed NQO1-proficient (NQO1 + ) cancer cells amplify lethality. l-calpain activation results in specific atypical PARP1 and p53 proteolyses (33) that culminate in potent AIF-endonuclease G-mediated DNA fragmentation detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) + staining (6,34).…”

Mechanisms of Radiation Toxicity in Transformed and Non-Transformed Cells

“…During ER stress, IRE1 can recruit TRAF2 to the plasma membrane, which leads to the activation of the JNK pathway and subsequent cell death via caspase-12 activation [121]. Other studies demonstrated that JNK activation downstream of ER stress leads to Bax activation, MOMP, and activation of caspase 3 [122]. …”