Quinone Reductase Inhibitors Block SAPK/JNK and NFκB Pathways and Potentiate Apoptosis

Abstract

The MEKK1 protein kinase (2) is a proximate activator of the stress-activated protein kinase (SAPK, 1 alternatively known as JNK) stress-signaling pathway (3). A variety of environmental stresses, including inflammatory cytokines, hyperosmotic shock, and UV light, stimulate the cascade (4). MEKK1 is implicated in apoptotic cell death (5, 6), but perhaps surprisingly MEKK1 activates NFB pathways (7, 8) that induce expression of genes to counteract the apoptotic death response (9 -11).Stress activators upstream of MEKK1 remain obscure. The small G-proteins Rac and Rho activate stress signaling (12, 13), but their placement in relation to MEKK1 is unclear. TNF␣ and the TNF␣ receptor-associated effector protein TRAF2 activate the SAPK pathway (14, 15). Reactive oxygen species are implicated in activation of stress kinase pathways in response to TNF␣ and UV irradiation. Reducing agents such as N-acetylcysteine at millimolar concentrations can block stress signals (15), suggesting that redox mechanisms are required for an undefined and probably early event in transmission of stress signals.Intracellular thiols can be consumed by a process termed "redox cycling" in which reactive quinones catalyze the oxidation of sulfhydryls to disulfides. In the cell, reactive quinones are regulated by the enzymatic activity NAD(P)H dehydrogenase (formerly DT-diaphorase) (E.C. 1.6.99.2) (16), representing a family of cytoplasmic flavo-enzymes responsible for two-electron reduction of quinones (17), using NADPH or NADH as electron donor. NAD(P)H dehydrogenase, also termed "quinone reductase," is inhibited by dicoumarol, a coumarin derivative (18). Like other coumarins, dicoumarol is used clinically to inhibit blood coagulation processes dependent on vitamin K, a biological quinone.Here we report evidence that inhibitors of quinone reductases can inhibit SAPK and NFB signaling. In addition, dicoumarol potentiates the apoptotic effect of TNF␣, probably by preventing anti-apoptotic events dependent upon NFB. MATERIALS AND METHODSSAPK Activation Assays-Human embryonic kidney 293 cells, or other cells as indicated, were seeded at 10 5 cells/35-mm plastic dish and transferred to serum-free medium 18 h before stimulation as indicated in the figure legends. 20 min following stimulation with 400 mM sorbitol (or as indicated) cells were assayed for SAPK/JNK activity as described (3) using rabbit anti-holo-SAPK-␤1 (p54) antiserum and glutathione

Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells

“…We show that treatment with dicumarol, an inhibitor of NQOI, sensitized HeLa cells to Fas killing. This result is in agreement with earlier findings concerning the effect of dicumarol on apoptosis: it strongly potentiates TNF-a-induced apoptosis in HeLa cells (Cross et al, 1999); it abrogates the suppression of apoptosis by IGF-I in FL5.12 cells overexpressing wide-type (wt) insulin-like growth factor I receptor (IGF-IR) and it induces apoptosis in MCF-7 cells (Krause et al, 2001). Contradictory are the reports that overexpression of NQOI in MCF-7 sensitizes cells to TNF-a (Siemankowski et al, 2000) and that dicumarol blocks wt p53-mediated apoptosis in girradiated normal thymocytes and in Ml myeloid leukemic cells that overexpress wt p53 (Asher et al, 2001).…”

“…It is possible that in HeLa cells the basal levels of NQOI activity mediate a dicumarol sensitive, survival pathway. This pathway could be mediated by the survival factor NFkB as dicumarol inhibits NFkB activation in primary macrophages (Cross et al, 1999). Another potential mediator of this survival pathway is the mitogen-activated protein kinase (MAPK) as it was reported that dicumarol blocks MAPK activation in HeLa cells (Cross et al, 1999) and inhibition of the MAPK cascade sensitizes HeLa cells to Fas-induced apoptosis (Holmstrom et al, 1999).…”

“…This pathway could be mediated by the survival factor NFkB as dicumarol inhibits NFkB activation in primary macrophages (Cross et al, 1999). Another potential mediator of this survival pathway is the mitogen-activated protein kinase (MAPK) as it was reported that dicumarol blocks MAPK activation in HeLa cells (Cross et al, 1999) and inhibition of the MAPK cascade sensitizes HeLa cells to Fas-induced apoptosis (Holmstrom et al, 1999). Interestingly, extracellular-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) have been implicated in ARE activation in a complex network that suggest ARE specificity, inducer specificity and cell type/tissue specificity (Yu et al, 1999(Yu et al, , 2000a.…”

A novel arsenical has antitumor activity toward As2O3-resistant and MRP1/ABCC1-overexpressing cell lines

“…The JNK inhibitor SP600125 has previously been shown to markedly inhibit JNK activation by various agents. 33 A 48-h treatment of NB4 cells with 5 mM SP600125 inhibits the As 2 O 3 -mediated induction of c-jun expression, which is JNK-dependent (data not shown), demonstrating that 5 mM is an effective inhibitory dose. We treated NB4 cells with the combination of As 2 O 3 or darinaparsin (0.5 and 1 mM) and SP600125 (5 mM) to determine whether inhibition of JNK activity repressed As 2 O 3 or darinaparsin-induced growth Darinaparsin is more potent than As 2 O 3 in vitro Z Diaz et al inhibition.…”

Nuclear Factor-κB Activation: A Question of Life or Death

“…The quinone reductase inhibitors dicoumarol and menadione block SAPK/JNK and NF-κB, and thereby potentiate apoptosis (Cross et al,1999). Javelaud and Besancon demonstrated that the repression of JNK activation by NF-κB is involved in the anti-apoptotic effect of this transcription factor in TNF α-treated Ewing sarcoma cells (Javelaud and Besancon, 2001).…”