If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
* This work was supported by National Institutes of Health Grants CA-66134 and ES/HL-09249-01 and awards from the American Cancer Society and the Council for Tobacco Research. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ These authors contributed equally to this work. ¶ Supported by National Institutes of Health Grants HL-57940 and M01-RR-00080.
A variety of environmental stresses stimulate the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEKK) > stress-activated protein kinase (SAPK)-ERK kinase (SEK) > SAPK/c-Jun NH2-terminal kinase (JNK) stress-activated protein kinase cascade and coordinately activate the transcription factor NFκB. Mechanisms of stress activation upstream of MEKK1 have not been precisely determined. Redox mechanisms involving sulfhydryls are likely because N-acetyl-cysteine at millimolar concentrations blocks stress signals. Because intracellular sulfhydryl concentrations can be regulated through redox cycling involving reactive quinones (
), we tested the ability of quinone reductase inhibitors to alter stress signaling. Several quinone reductases are inhibited by dicoumarol, a coumarin derivative. Dicoumarol prevented SAPK activation in vivo by chemical cell stressors and also prevented SAPK activation induced by expression of the tumor necrosis factor α (TNFα) receptor-associated protein TRAF2 but not by expression of truncated active MEKK1. Other coumarin derivatives failed to block SAPK activation, but other inhibitors of quinone reductases, particularly menadione, similarly blocked SAPK activation. Cells deficient in a major quinone reductase, NQO1, displayed hypersensitivity to dicoumarol stress inhibition, whereas SAPK in cells reconstituted with the NQO1 gene displayed relative dicoumarol resistance. Consistent with the proposed role of overlapping upstream signaling cascades in activation of NFκB, dicoumarol also blocked NFκB activation in primary macrophages stimulated with either lipopolysaccharide or TNFα. In addition, dicoumarol strongly potentiated TNFα-induced apoptosis in HeLa cells, probably by blocking the anti-apoptotic effect of NFκB. The ability of dicoumarol to simultaneously inhibit SAPK and NFκB activation and to potentiate apoptotic cell death suggests that SAPK is not an obligate participant in apoptosis. Dicoumarol, currently in clinical use as an oral anticoagulant, represents a potential therapeutic inhibitor of the SAPK and NFκB response.
). Reactive oxygen species are implicated in activation of stress kinase pathways in response to TNFα and UV irradiation. Reducing agents such as N-acetyl-cysteine at millimolar concentrations can block stress signals (
), 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. 188.8.131.52) (
). 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 NFκB signaling. In addition, dicoumarol potentiates the apoptotic effect of TNFα, probably by preventing anti-apoptotic events dependent upon NFκB.
MATERIALS AND METHODS
SAPK Activation Assays
Human embryonic kidney 293 cells, or other cells as indicated, were seeded at 105 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 (
) using rabbit anti-holo-SAPK-β1 (p54) antiserum and glutathioneS-transferase-Jun (5–79) as substrate. Anti-MAPK C-terminal peptide polyclonal serum was obtained from Michael Dunn (Milwaukee, WI). Following gel electrophoresis, proteins were transferred to polyvinylidene difluoride membrane and labeled proteins detected and quantified using a Packard Instant Imager.
Plasmid encoding the catalytic fragment of MEKK under a CMV promoter have been described (
); these were transfected into recipient cells using calcium phosphate. The SAPK-β1 (p54) allele expressed as a glutathioneS-transferase fusion protein using the pEBG vector was obtained from James Woodgett (Toronto, Ontario).
Human pulmonary alveolar macrophages were isolated by bronchoalveolar lavage of healthy adults. Nuclear extracts were prepared either immediately upon isolation or after 30 min of incubation in medium with or without LPS or TNFα. DNA binding of NFκB in nuclear extracts was determined using the radiolabeled oligonucleotide 5′-CTAGTAGCGGAAAGTCCCTTG-3′. Polyclonal antisera detecting p65 and p50 amino termini was a gift from Nancy Rice, NCI, Frederick, MD.
To determine whether quinone reductase inhibitors affect transmission of stress signals, we pretreated cultures of human embryonic kidney 293 cells with dicoumarol before stimulation with hyperosmotic sorbitol or anisomycin. Dicoumarol alone did not affect SAPK but completely blocked activation by osmotic shock and anisomycin in 293 cells (Fig. 1A) and in Jurkat and resting peripheral T lymphocytes (data not shown and see below). Dicoumarol also blocked SAPK activation by UV irradiation and ceramide induced by treatment with sphingomyelinase (Fig.1B). Warfarin and coumarin, agents that inhibit the vitamin K cycle like dicoumarol, did not inhibit SAPK activation (see TableI), suggesting that vitamin K metabolism is unrelated to stress signaling.
Table IInhibition of SAPK by respiratory and redox-active chemicals
Residual SAPK Activity (±S.D.)
0.08 ± 0.01
0.94 ± 0.06
Dicoumarol 300 μm + warfarin
Warfarin (18 h)
Warfarin (18 h)
0.94 ± 0.1
1.10 ± 0.12
0.87 ± 0.25
1.06 ± 0.15
0.73 ± 0.12
1.07 ± 0.17
1.01 ± 0.03
0.96 ± 0.06
0.99 ± 0.27
0.97 ± 0.16
1.24 ± 0.41
BAPTA 1 mm + EGTA 2 mm
1.14 ± 0.42
0.96 ± 0.09
1.03 ± 0.01
1.04 ± 0.04
0.73 ± 0.02
0.73 ± 0.01
Assay of hyperosmotic sorbitol stimulated SAPK in 293 cells treated with the indicated inhibitors. Except as noted, inhibitor pretreatment preceded stimulation with sorbitol by 10 min and were assayed for SAPK activity as described above 20 min following stimulation. Agents are grouped together under broad categories but typically have several known inhibitory roles. Inhibitory agents were compared with controls treated with the appropriate solvent. Averages and standard deviations were derived from assays in separate experiments.
Inhibition of SAPK activation by dicoumarol was dose-dependent (Fig. 2), with an IC50 between 19 and 38 μm in 293 cells. Dicoumarol blocked SAPK activation in response to expression of TRAF2, a TNFα receptor interacting protein that activates SAPK (
) (Fig. 3A). In contrast, dicoumarol was unable to block SAPK activation in response to expression of truncated active MEKK1, indicating that the point of dicoumarol inhibition of SAPK activation lies downstream of TRAF2 but upstream of MEKK1 in a poorly characterized segment of the stress-signaling cascade.
In contrast to its ability to inhibit activation of SAPK by agents such as sorbitol and anisomycin, dicoumarol failed to inhibit activation of SAPK in response to heat shock (Fig. 3B), which is believed to proceed by an alternate pathway (
). The specificity of dicoumarol inhibition for stress signaling was demonstrated by its failure to inhibit activation of MAPK in CV1 cells in response to TPA (Fig.3C). However dicoumarol did block MAPK activation resulting from osmotic shock, which likely proceeds via upstream pathways that overlap the SAPK signaling pathway, including the involvement of MEKK1 (
). Although these cells are still capable of activating SAPK in response to hyperosmotic sorbitol, they have a reduced IC50 for dicoumarol inhibition of SAPK activation, about 5 μm (Fig.4A). Reconstitution of NQO1 by stable expression of the NQO1 gene (
) reduced the sensitivity of these cells to dicoumarol (Fig. 4B). Thus, inhibition of SAPK activation by dicoumarol can be partly abrogated by expression of NQO1, genetically supporting a role for NQO1 and other quinone reductases in stress signaling.
We tested whether other metabolic toxins and redox inhibitors might be able to inhibit SAPK activation (Table I). No inhibition of SAPK activation was seen with coumarin or warfarin anticoagulants, inhibitors of mitochondrial oxidative phosphorylation, or extracellular superoxide dismutase or catalase, which reduce intracellular levels of superoxide and hydrogen peroxide, respectively. The vitamin K-related quinone menadione blocked SAPK activation with an IC50approximately the same as dicoumarol. Notably, menadione is an in vitro substrate of quinone reductases, and competitively inhibits these enzymes in vivo. Two other quinone-related compounds, hydroquinone and butylated hydroxyanisole, inhibited SAPK at lower efficiency, requiring very high concentrations to see a significant inhibition. Thus dicoumarol and menadione, followed by other quinones, were most effective at blocking SAPK activation. These inhibitor studies suggest a role for quinone reduction, rather than events such as oxidative phosphorylation, in stress signaling.
These data suggest that dicoumarol inhibition of SAPK signaling may be related to a change in the state of quinone reduction in the cell. Specifically, inhibition of quinone reductases by dicoumarol would result in a decrease in the levels of reduced quinones in the cell. We examined whether replacing the reduced quinones would restore the ability of the cells to signal to SAPK (Fig.5). Although dicoumarol completely inhibited activation of SAPK in response to sorbitol (lane 4), the addition of increasing amounts of hydroquinone restored the SAPK activation in a dose-responsive manner (lanes 5–8). Treating the cells with hydroquinone alone had no effect on SAPK activity (lanes 9–12). These data suggest that the inhibition of SAPK activation by dicoumarol is due in large part to the decrease in reduced quinones that results from the inhibition of quinone reductases, supporting a role for quinone reduction in establishing an intracellular environment that is permissive for stress signaling.
We examined consequences of interfering with stress signaling using dicoumarol. Many stress stimuli, for example TNFα and LPS, activate both SAPK and the NFκB transcription factor. Recent findings have suggested that the upstream pathways leading to NFκB activation may significantly overlap with those for SAPK activation, including a role for MEKK1 in both signaling cascades (
). Disruption of TNFα-induced gene expression by cycloheximide or actinomycin D synergizes with TNFα-induced apoptosis. Like cycloheximide, dicoumarol also potentiated apoptosis of HeLa cells treated with TNFα (Fig. 6B). This synergism is likely a result of the inhibition of NFκB activation by dicoumarol and points to possible therapeutic uses for dicoumarol in synergism with this or other apoptosis-inducing agents. Importantly, the simultaneous inhibition of SAPK/JNK activation and potentiation of apoptosis demonstrates that SAPK/JNK activation is not a required event in the apoptotic response.
Peripheral blood lymphocytes undergo DNA synthesis in response to the mitogen PHA. Pretreatment of human peripheral blood lymphocytes with hypertonic NaCl stimulated SAPK and blocked DNA synthesis in response to PHA (Table II). Pretreatment of peripheral blood lymphocytes with dicoumarol before hypertonic shock prevented SAPK activation and restored PHA-stimulated DNA synthesis. Thus, inhibition of PHA-induced cell cycle entry in response to hypertonic shock is a specific stress-induced effect rather than a nonspecific toxic effect. In future experiments, dicoumarol should similarly be useful for identifying other biologic consequences of stress signaling.
Table IIDicoumarol reverses osmotic shock blockade of PHA activation of peripheral blood mononuclear cells
Incorporation of 3H-thymidine mean cpm + S.D.
SAPK activity (Imager cpm)
597 ± 80
25,158 ± 2,165
422 ± 18
12,120 ± 992
Dicoumarol + Hypertonic NaCl
344 ± 37
24,847 ± 1,813
Resting peripheral blood mononuclear cells were pre-treated either with hypertonic saline (100 mm NaCl) for 30 min or with 300 μm dicoumarol for 10 min followed by 30 min in hypertonic saline. Lymphocytes were washed to remove stimulators and replaced in medium containing PHA. DNA synthesis during a period 24–48 h following PHA exposure was monitored by incorporation of 3H-thymidine, and the results of triplicate samples are shown. SAPK activity was assayed as in Fig. 1. Dicoumarol blocks both the osmotic shock activation of SAPK and prevents the reduction of PHA activation observed following osmotic shock. Similar results were observed in two other experiments.
Our data demonstrate an obligate role for a dicoumarol-inhibitable activity in stress signaling at a point upstream of MEKK1, and downstream of osmotic shock, LPS, anisomycin, TNFα, and the TNFα receptor-associated protein TRAF2 (Fig. 7). In contrast, this dicoumarol sensitive activity appears dispensable for activation of SAPK by heat shock, which is thought to proceed via a distinct pathway. Our data supports the hypothesis that heat shock acts via an alternate pathway which does not share the apparent dependence on quinone reductase function.
Quinone reductases likely provide an intracellular environment permissive to stress signaling rather than themselves being activated during the signaling. Inhibition of these quinone reductases by dicoumarol would interfere with this environment, and inhibit the ability of the cells to transmit stress signals. In support of this, replacing reduced quinones that are lost as a result of dicoumarol inhibition of quinone reductases restores the ability of the cells to signal to SAPK. These data suggest a role for quinone reduction in generating this permissive intracellular environment. Interestingly, very high concentrations of hydroquinone inhibit activation of SAPK in response to sorbitol, suggesting that there is a tightly regulated balance, where either too high or low levels of reduced quinones are detrimental to stress signal transmission.
Cells lacking NQO1 display increased sensitivity to dicoumarol treatment. The observation that these cells continue to signal to SAPK supports our model in which NQO1 is involved in establishing an intracellular environment permissive for stress signaling, rather than fulfilling an obligate role in a linear pathway. In the absence of NQO1, other less abundant dicoumarol-sensitive quinone reductases assume responsibility for establishing the homeostatic quinone balance. When these NQO1 deficient cells are treated with dicoumarol, this balance is more easily upset, as reflected in the decreased IC50 for SAPK signaling when compared with the cells that have been reconstituted by stable transfection of NQO1.
Inhibition of stress signaling by dicoumarol synergizes with TNF-α to promote apoptosis, suggesting a role for quinone reductases in the regulation of apoptotic events. Other evidence similarly supports this theory. First, using the SAGE technique, it was shown that expression of the apoptotic regulator p53 strongly activated expression of a quinone reductase among several other redox enzymes (
). Dicoumarol or similar quinone reductase inhibitors might serve clinically to inhibit stress and inflammatory processes. Dicoumarol has been used as an oral anticoagulant in patients for many years. Although it is a poison, the known toxic effects of its administration in animals are related solely to anticoagulation, which can be reversed by simultaneous administration of vitamin K. Inhibition of SAPK and NFκB by dicoumarol or related compounds might thus prove doubly valuable; first for identifying the function of stress signals in experimental situations, and also for translation into therapeutic utility in the clinical setting.
We thank R. Michael Sramkoski for assistance with flow cytometry at the Case Western Reserve University Cancer Center Core Facility, and Minhong Yan, Tim Shannon, and Edwina Lerner for helpful discussions.