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Department of Anatomy and Cell Biology, University Hospital of Aachen, Aachen, Germany (C.J.W., L.-O.B., T.P.); and Institute of Pharmacology (M.E.G., T.H.) and Department of Trauma Surgery (D.V.), University Hospital of Schleswig-Holstein UK-SH, Kiel, Germany
Received October 5, 2007; accepted March 10, 2008
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Abstract |
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; Butterfield et al., 2001) and by in vitro studies (Behl et al., 1994). Oxidative stress can cause cell death by damaging cardinal cellular components, such as lipids, proteins, or DNA and RNA. The brain is especially sensitive to oxidative stress because of its high concentration of readily oxidized fatty acids and high oxygen consumption. In AD, oxidative stress is suspected to be generated by the amyloid β peptide (Behl et al., 1994; Butterfield et al., 2001).
Therefore, treatment with antioxidants might theoretically act to retard spreading of neuronal damage and to improve neurological outcome. Indeed, several studies investigated whether dietary intake of antioxidants, especially vitamins, might prevent or reduce the progression of AD. Although a few of the antioxidants showed some efficacy in these trials, no answer is yet available as to whether antioxidants are truly protective in AD (Boothby and Doering, 2005).
Another way to render neuronal cells more resistant to oxidative stress is to up-regulate the endogenous protection system. During evolution, cells have developed complex mechanisms to defend from oxidative and electrophilic stress. A battery of genes encoding detoxifying and antioxidative enzymes is orchestrated on exposure to electrophiles and reactive oxygen species. This coordinated response is regulated through a cis-acting element, the antioxidant response element (ARE) within the regulatory regions of this "safeguard" gene. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and resultant binding to the ARE initiates or enhances the transcription of these genes, such as NAD(P)H:quinone oxidoreductase-1, thioredoxin reductase, glutathione peroxidase, and hemeoxygenase-1 (Jaiswal, 2004; Lee and Johnson, 2004). Like other "stress response" transcription factors (e.g., hypoxia inducible factor-1
), Nrf2 is expressed in a constitutive manner, and it subsequently degraded within minutes. An essential step in the stabilization and activation of Nrf2 is the liberation of the Nrf2 inhibitor Keap1, which binds Nrf2 and promotes its proteasomal degradation. Oxidative stress or electrophiles but also Nrf2 phosphorylation by kinases disrupt the Keap1-Nrf2 complex, leading to stabilization and activation of Nrf2 (Tong et al., 2006).
The relevance of Nrf2 in neuronal protection could be shown using transgenic techniques. Neural cells from these Nrf2 knockout mice were more vulnerable to oxidative stress compared with those from Nrf2 wild-type mice (Lee et al., 2003a,b). In addition, overexpression of Nrf2 dramatically increased the resistance of neurons to oxidative cell death (Shih et al., 2003).
In search of agents that activate Nrf2, three analytically pure kavalactones—methysticin, yangonin, and kavain—were under examination. These kavalactones are the main components of the rhizome and roots of kava (Piper methysticum G. Forst), a Piperaceae common on some Pacific Ocean islands (Bilia et al., 2004). Anthropological evidence suggests that kava has been cultivated and consumed for more than 3000 years. It is still used today by a wide range of Pacific societies for spiritual, medicinal, and recreational purposes. Kavalactones showed only moderate antioxidant activities (Wu et al., 2002), but they have well known psychopharmacological properties. The most observed effects of kava extract at low doses are mood relaxation or euphoric behavior, depending on the circumstances of ingestion, whereas higher doses cause sleepiness and skeletal muscle relaxation (Singh, 1992; Singh and Singh, 2002). Several other effects have been reported, such as antithrombotic action (Gleitz et al., 1997), anticonvulsant properties (Jamieson et al., 1989), inhibition of nuclear factor-
B (Folmer et al., 2006), and cyclooxygenases (Wu et al., 2002) as well as neuroprotection in focal cerebral ischemia in mice and rats (Backhauss and Krieglstein, 1992). No interactions with neuroreceptors have yet been found that would explain the various pharmacological effects.
Concerns have recently been raised about the safety of kava consumption. There have been several reports of rare but severe liver toxicity, including liver failure in some people who have used dietary supplements containing kava extract. In this context, Nerurkar et al. (2004) showed toxicity for the kava alkaloid pipermethystine but not for kavalactones such as methysticin and yangonin. This study suggests that pipermethystine, rather than kavalactones, is responsible for the hepatotoxic reactions to kava. On this account, we examine the kavalactones methysticin, yangonin, and kavain for their properties to activate Nrf2 and evolve neuroprotective effects.
The present study provides the first documentation of the neuroprotective properties of kavalactones from amyloid β-toxicity. We could further show that the investigated kavalactones exert their protective effects by inducing the expression of cytoprotective genes through Nrf2 activation in an ERK1/2-dependent manner.
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Materials and Methods |
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-GCS, and β-actin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Amyloid β-(1-42) was purchased from Tocris Cookson Inc. (Bristol, UK). All other chemicals were of the highest quality commercially available.
Cell Culture
The pheochromocytoma cell line PC-12 and rat astrocytoma cell line C6 (both purchased from LGC Promochem, Wesel, Germany) were grown in Dulbecco's modified Eagle's medium-Ham's F-12 (1:1) with 2 mM glutamine and N2-supplement containing putrescine, insulin-like growth factor-1, transferrin, progesterone, and selenite. PC-12 cells were differentiated for 6 days with 50 ng/ml nerve growth factor. For WST, lactate dehydrogenase (LDH), and luciferase assay, 5000 cells were plated per well on BIOCOAT Collagen I 96-well plates (VWR International, Hamburg, Germany) in 200 µl of serum-free medium.
Toxicity Assays
WST Assay. For WST assay, media were supplemented with 10 µl/well WST 2 h before spectrophotometric evaluation. Conversion of WST to formazan was measured at 450 nm by microplate spectrophotometry (model 680; Bio-Rad Laboratories, Hercules, CA). This reaction reflects the reductive capacity of the cells, which represented the viability of the cells, and it is expressed relative to the value of 100 ± S.E.M., which represented the reductive capacity of the untreated control.
LDH Assay. LDH is a stable cytoplasmic enzyme present in all cells, including neurons. It is rapidly released into the cell culture supernatant when the cell plasma membrane is damaged. Thus, the LDH level in the culture medium is a reliable biochemical index for neuronal plasma membrane damage. In this study, LDH release from the cytosol of damaged PC-12 cells into the culture medium after amyloid β-(1-42) exposure was measured using a cytotoxicity detection assay (Roche Diagnostics, Mannheim, Germany), which determines the LDH activity in the culture medium to enzymatically convert the lactate and NAD+ to pyruvate and NADH. The tetrazolium salt produced in the enzymatic reaction was then reduced to red formazan in the presence of protons, thereby allowing colorimetric detection of neuronal membrane integrity. LDH release is expressed relative to the value of 100 ± S.E.M., which represented the maximum LDH release that occurred after freezing overnight at -70°C and subsequent rapid thawing of each culture, which induced nearly complete cell damage, established from n = 8 wells per one experiment from three separate experiments.
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RNA Interference
The mammalian expression vector pGE1 (Stratagene, La Jolla, CA) was used for the stable expression of shRNA against Nrf2-mRNA in PC-12 cells. The gene-specific insert, which is specified by a 29-nucleotide sequence 5'-GTCTTCAGCATGTTACGTGATGAGGATGG-3' of the rat Nrf2, is separated by an eight-nucleotide non-complementary spacer (GAAGCTTG) from the reverse complement of the same 29-nucleotide sequence. The oligonucleotides were synthesized by Tib Molbiol. This construct was inserted into the pGE1 using BamHI and XbaI restriction sides, and it is referred to as pGE1-rNrf2. A control vector (pGE1-negative) serves as a nonsilencing control (Stratagene).
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Western Immunoblotting
HO-1 and -GCS protein levels, ERK1/2 phosphorylation in whole-cell lysates, and Nrf2 in nuclear extracts were measured by Western immunoblotting, using protocols described previously (Varoga et al., 2006). For the Nrf2 detection in the nucleus, nuclear and cytoplasmic fractions of PC-12 cells were separated with the NE-PER kit purchased from Pierce Chemical (Rockford, IL). To monitor potential artifacts in loading and transfer among samples in different lanes, the blots for phospho-ERK1/2 were stripped and reprobed with antibodies against total ERK1/2. The digitized images were quantitated with the PCBAS program (Raytest Isotopen Meßgeräte GmbH, Straubenhardt, Germany).
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Results |
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). Therefore, the viability of PC-12 and C6 cells was measured by the use of WST assay after incubation with kavalactones and curcumin in different concentrations for 24 h. These studies reveal a significant cytotoxicity in PC-12 as well as C6 cells beginning at 5 µM for curcumin. The kavalactones showed no cytotoxic effects up to 100 µM in PC-12 and C6 cells (data not shown). Induction of ARE-Mediated Reporter Activity by Kavalactones. To investigate the efficacy of kavalactones to activate the cis-acting element ARE, we carried out a dual-luciferase reporter gene assay with the ARE of the rat NQO1-gene. ARE activation was determined in a dose-response assay up to 50 µM for kavalactones in PC-12 and C6 cells for 12-h incubation time. Kavalactones (0.5 µM) activate the luciferase gene expression 3-fold in PC-12 cells (Fig. 1A) and 2-fold in C6 cells (Fig. 1B). The activation reached a plateau at 5 µM with a 7-fold induction over control in PC-12 cells (Fig. 1A) and 6-fold in C6 cells (Fig. 1B). Higher concentrations of kavalactones yielded no further increase in the luciferase gene expression. Therefore, 5 µM kavalactones were used in all further experiments. DMSO used as vehicle in a concentration of 0.25% showed no significant effect on ARE activation (Fig. 1, A and B).
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-GCS Expression. PC-12 cells were exposed to methysticin, yangonin, or kavain at the final concentration of 5 µM to examine its effect on Nrf2 protein stability over time via Western blot of the nuclear fraction. As shown in Fig. 2A, treatment with kavalactones caused a significant time-dependent increase in Nrf2 protein stabilization in nuclear extracts. We measured Nrf2 induction within 30 min, pictured by occurrence of Nrf2 in the nuclear fraction. The induction is still measurable after 24 h. As loading control, we reprobed the blots for Nrf1, a transcription factor with constant expression. The density of both bands, Nrf2 and Nrf1, were measured and the ratio of Nrf2/Nrf1 was calculated. The median of three independent experiments is shown in Fig. 2B.
To further confirm that kavalactones are activators of the Nrf2-ARE system, we studied the effect of kavalactones on the expression of two well known Nrf2 target genes, HO-1 and -GCS, via Western blot of the whole extract. Both HO-1 and -GCS were up-regulated over time by incubation of PC-12 cells with 5 µM kavalactones (Fig. 3, A-D). As loading control, we reprobed the blots for β-actin. The densities of both bands were measured, and the ratio was calculated. The medians of three independent experiments are shown in Fig. 3, B and D.
Kavalactone-Mediated ARE Activation Was ERK1/2-Dependent. To address the role of individual MAPK pathways in ARE gene regulation by kavalactones, we examined the effects of various kinase inhibitors. We observed the activation of ERK1/2 to be a prerequisite for the activation of Nrf2 by all kavalactones investigated because kavalactone-mediated Nrf2 activation is exclusively inhibited by the MEK1 inhibitor PD98059 at 20 µM as well as the MEK1/2 inhibitor U0126 (10 µM) in PC-12 cells (Fig. 4A). The c-Jun NH2-terminal kinase inhibitor SP600125 at 2 µM, the inhibitor of p38-MAPK SB203580 at 5 µM, and wortmannin at 1 µM, an inhibitor of phosphoinositol-3-kinase, did not diminish the Nrf2 activation in PC-12 cells (Fig. 4A).
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Kavalactone-Mediated Nrf2 Activation Was Not Dependent on Oxidative Stress Production or Glutathione Depletion. To exclude the possibility that kavalactones activate Nrf2 via production of oxidative stress or glutathione depletion, PC-12 cells were pretreated with the antioxidants ascorbic acid at 50 µM, Trolox at 50 µM, and NAC at 5 µM, and GSH-MEE at 2 mM. None of these antioxidants had an inhibitory effect of the kavalactone-induced Nrf2 activation, providing evidence that activation is not dependent on oxidative stress production or glutathione depletion. Although DTT by itself induced a small increase in ARE activation (Haridas et al., 2004), pretreatment of cells with 10 µM DDT almost completely blocked the effects of incubations with 5 µM kavalactones for 24 h in PC-12 cells. Unlike NAC and GSH-MEE, DTT seems to have a mechanism of protein thiol reduction independent of GSH (Rafeiro et al., 1994), but the exact mechanism has not been shown. These observations suggest a protection of critical thiol groups of Keap1 by DTT, whereas replenishment of GSH content via NAC and GSH-MEE does not. Moreover, treatment of PC-12 cells for 24 h with 50 µM ascorbic acid down-regulates Nrf2 activation significantly compared with control cells (Fig. 5).
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Nrf2 Activation Conferred Cytoprotection. We further investigated a causal relationship between Nrf2 activation and cytoprotection mediated by kavalactones. Therefore, we designed a shRNA against mRNA coding for rat Nrf2 (pGE1-rNrf2), and we produced a PC-12 cell line (PC-12-shNrf2) by stable transfection of this shRNA construct. In a NQO1-ARE luciferase assay, PC-12-shNrf2 cells are not responsible for ARE-dependent reporter gene induction mediated by kavalactones, demonstrating the efficacy of pGE1-rNrf2 (Fig. 7A). We used these PC-12-shNrf2 cells for cytoprotection assays against Aβ-(1-42) toxicity conferred by kavalactones. As shown in Fig. 7, B and C, kavalactones are not capable to protect PC-12-shNrf2 cells from Aβ-(1-42) toxicity (Fig. 7, B and C). Furthermore, PC-shNrf2 cells are more vulnerable to Aβ-(1-42) toxicity. After incubation with 10 µM Aβ-(1-42) the cell viability of PC-12-shNrf2 cells declined by 75% (Fig. 7B) versus the 30% decline, which was found with naive PC-12 cells (Fig. 6A). PC-12-shNrf2 cells consistently showed a greater increase of released LDH activity after Aβ-(1-42) treatment than naive PC-12 cells, because PC-12-shNrf2 showed an 18% increase from 17% (control) to 35% [Aβ-(1-42)-treated] (Fig. 7C), whereas naive PC-12 showed an 10% increase from 13% (control) to 23% [Aβ-(1-42)-treated] (Fig. 6B). Data in percentage of maximum LDH activity equal 100%. The higher vulnerability of PC-12-shNrf2 cells emphasizes the important role of Nrf2 in the defense from Aβ-(1-42) toxicity.
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Discussion |
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Kavalactones Activate Nrf2 at Nontoxic Concentration in Vitro. The first criterion was low toxicity of the tested substances. In a viability assay, the kavalactones methysticin, yangonin, and kavain showed no toxicity to differentiated PC-12 and C6 cells up to 100 µM (Fig. 1). These results are consistent with the literature describing kava-kava and particularly purified kavalactones as virtually nontoxic substances (Clouatre, 2004; Nerurkar et al., 2004; Sorrentino et al., 2006).
Next, we examined the potential of kavalactones to activate Nrf2 in neural PC-12 and glial C6 cells. We used glial in addition to neural cells because Murphy et al. (2001) showed that within the brain the protection against oxidants is mainly supported by astrocytes. We established a dual luciferase assay with an ARE of the rat NQO-1 gene transfected in PC-12 and C6 cells. In these systems, exposure of kavalactones activated the Nrf2-ARE system dose-dependently (Fig. 2, A and B). The induction of the ARE system could be confirmed by Western blot showing the time-dependent stabilization of Nrf2 in the nucleus (Fig. 3, A and B) and upregulation of the Nrf2-target genes -GCS and HO-1 in PC-12 cells (Fig. 3, C-F).
In contrast to most other Nrf2 activators like flavonoides, sulforaphane, or curcumin, which are already toxic at low concentration, kavalactones showed no toxicity in our assays in vitro, and they are thereby able to activate Nrf2 at extremely low concentrations.
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). To examine whether MAPKs are involved in the signal transduction of kavalactones, we tested various MAPK inhibitors with respect to Nrf2 activation. From all tested inhibitors, only the MEK1 inhibitor PD98059 and the MEK1/2 inhibitor U0126 showed an inhibitory effect on the Nrf2 activation (Fig. 4A). This identifies ERK1/2 activation to be a prerequisite for Nrf2 activation by kavalactones. However, Nrf2 might not directly be a substrate of ERK1/2. Instead, it is discussed that ERK1/2 phosphorylates the nuclear transcription coactivator CREB-binding protein, and that CREB-binding protein enhances Nrf2 transcriptional response (Katoh et al., 2001; Shen et al., 2004). In addition, we stimulated PC-12 cells with kavalactones, and we analyzed the phosphorylation status of ERK1/2 to back-reference the activation status of the kinases. All three tested kavalactones showed a strong but transient activation of ERK1/2 after 30 min up to 1 h, followed by a period of weaker activation for a minimum 6 h. After 24 h, the phosphorylation status renormalized similar to control (Fig. 4B).
ERK1/2 are traditionally viewed as the survival factors of the MAPK family. In contrast, there are suggestions that ERK1/2 activation may also be associated with neuronal cell death in various neurodegeneration models, especially if occurring coincidentally with oxidative stress (Zhu et al., 2004). However, activation of ERK1/2 in situations devoid of prevailing oxidative stress, as may be the case with kavalactones, could be neuroprotective (see below; Culmsee et al., 2005).
Kavalactone-Mediated Nrf2 Activation Is Not Dependent on Oxidative Stress Production or Glutathione Depletion. The Keap1-Nrf2 complex serves as a cytoplasmic sensor enabling the cell to respond to electrophiles and oxidative stress. Many Nrf2 activators, also those considered to act as protective substances such as curcumin, sulforaphane, flavonoids, or epigallocatechin gallate, activate Nrf2 via oxidative stress (Gong et al., 2004; Jakubíková et al., 2006; Lee-Hilz et al., 2006; Wu et al., 2006). The protective effect of these substances may by based upon a phenomenon referred to as preconditioning, an effect well established for hypoxia via hypoxia inducible factor-1 or hyperthermia via heat shock transcription factor 1.
To test whether kavalactones also activate Nrf2 via oxidative stress, PC-12 cells were pretreated with the various antioxidants. But none of the tested antioxidants had an inhibitory effect on kavalactone-induced Nrf2 activation (Fig. 4), suggesting another mechanism of Nrf2 activation than oxidative stress production. To address whether kavalactones activate Nrf2 via decline of the GSH/oxidized glutathione ratio, we pretreated the cells with GSH monoethyl ester. This enhancement of the GSH pool did not decrease the Nrf2 activation, giving evidence that glutathione depletion is not the way kavalactones activate Nrf2. It is noteworthy that treatment of PC-12 cells with 50 µM ascorbic acid but not Trolox down-regulates the Nrf2 activation significantly compared with control cells (Fig. 4), indicating an adaptive effect on the reduced oxidative burden of the cells.
We further tested the effect of DTT, a reagent commonly used in biochemical studies as an agent to prevent the oxidation of thiol groups and for reducing disulfides to dithiols. Although DTT by itself induced a small increase in ARE activation, pretreatment of cells with DTT almost completely blocked the effects of kavalactones in PC-12 cells (Fig. 4). Because of the ,β-unsaturated carbonyl group present in its lactone ring (Fig. 9), kavalactones may act as a Michael reaction acceptor and readily interact with critical cellular nucleophiles, such as cysteine thiol groups in proteins such as Keap1, but the exact mechanism has not been shown. Therefore, the inhibition of kavalactone-induced Nrf2 activation by DTT might suggest that kavalactones regulate critical redox-sensitive thiol groups of Keap1. Whether kavalactones interact directly with cysteine residues of Keap1 has to be elucidated in further studies.
According to these results, we presumed that kavalactones do not activate Nrf2 via oxidative stress production or glutathione depletion. This may explain the low toxicity of kavalactones, compared with other Nrf2 activating substances. This is of great interest, particularly in reference to treatment of neurodegenerative conditions, in which an additional increase of oxidative stress could have destructive consequences.
Kavalactones Protect against Amyloid β-(1-42) Toxicity. Our hypothesis was that Nrf2 activation with a nontoxic substance would render neuronal cells more resistant to Aβ-(1-42)-induced oxidative stress and toxicity. To test this hypothesis, we pretreated differentiated PC-12 cells with kavalactones for 16 h to allow a complete up-regulation of genes encoding detoxifying and antioxidative enzymes, and we confronted them with Aβ-(1-42). Indeed, kavalactone-pretreated cells were more resistant to Aβ-(1-42) toxicity than untreated cells as shown in cell viability and cytotoxicity assays (Fig. 6, A and B). Furthermore, the protective doses tested showed a dose-response relationship and correlation with those of Nrf2 induction (Fig. 1).
A neuroprotective effect of kava extract and its constituents kavain, dihydrokavain, methysticin, dihydromethysticin, and yangonin on ischemic brain damage in mice and rats was first shown by Backhauss and Krieglstein (1992). The molecular mechanisms of these effects were not further elucidated. Because the production of reactive oxygen species has been implicated in reperfusion injury after cerebral ischemia, it is likely that Nrf2 also plays a role in cerebral ischemia (Love, 1999). Indeed, Zhao et al. (2006) give the first evidence that this hypothesis holds true.
To elucidate the role of Nrf2 in the protective effect of kavalactones, we used a PC-12 cell line (PC-12-shNrf2) that carried a stable transfected shRNA against Nrf2-mRNA. This cell line was no longer able to activate the Nrf2-ARE system (Fig. 7A). We used shRNA technology rather than dominant-negative Nrf2 overexpression, because shRNA against Nrf2 knocks down solely Nrf2, whereas dominant-negative Nrf2 blocks the binding to ARE by competitive inhibition and thereby blocks all factors with affinity to ARE.
In these Nrf2 deficient PC-12 cells, kavalactones are no longer protective against Aβ-(1-42) toxicity (Fig. 7, B and C), supporting the pivotal function of Nrf2 in cytoprotection mediated by kavalactones; in addition, Nrf2-deficient cells are more vulnerable to Aβ-(1-42) toxicity.
We further examined whether the ERK1/2 activation is required for the protective effects of kavalactones by treating ERK1/2 inhibitor-preincubated PC-12 cells with kavalactones and testing these cells for their Aβ-(1-42) vulnerability. In fact, the ERK1/2 inhibitor abolished the protective effects of kavalactones in toxicity assays (Fig. 8, A and B). Supposing that kavalactone-mediated ERK1/2 activation occurred also in PC-12-shNrf2 cells, ERK1/2 activation without Nrf2 induction would not be sufficient for cytoprotection (Fig. 7, B and C).
According to these results, we propose that kavalactones activate Nrf2 and thereby elevate cytoprotective gene expression as exemplified by -GCS and HO-1 up-regulation. Other Nrf2 target genes that were not under examination here surely contribute to the described cytoprotection.
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Conclusions |
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). Consequently efforts to identify and promote new therapeutic strategies for patients with AD are still of great interest. We found that a beneficial effect can be induced in neuronal as well as glial cells via kavalactones. Thus, kavalactones are able to activate ERK1/2 and Nrf2 at nontoxic concentrations and thereby mediate an up-regulation of a battery of genes encoding detoxifying and antioxidative enzymes, effective in protecting neurons against amyloid β-(1-42) toxicity in vitro. If studies using kavalactones in an in vivo model of Alzheimer's disease prove this beneficial effect, the use of kavalactones might be considered as an adjunct therapeutic strategy to combat neural demise in Alzheimer's disease and other oxidative stress-related diseases.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: AD, Alzheimer's disease; Nrf2, nuclear factor erythroid 2-related factor 2; ARE, antioxidant response element; DDT, dithiothreitol; NAC, N-acetylcysteine; GSH, glutathione; GSH-GEE, glutathione monoethyl ester; PD98059, 2'-amino-3'-methoxyflavone; SP600125, anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; LDH, lactate dehydrogenase; WST, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; p-, phospho-; ERK, extracellular signal-regulated kinase; HO-1, heme oxygenase-1; -GCS, -glutamylcysteine synthetase; DMSO, dimethyl sulfoxide; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; Aβ, amyloid β-peptide; ANOVA, analysis of variance.
Address correspondence to: Dr. Christoph J. Wruck, Department of Anatomy and Cell Biology, RWTH Aachen, Wendlingweg 2, 52074 Aachen, Germany. E-mail: cwruck{at}ukaachen.de
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
Balogun E, Hoque M, Gong P, Killeen E, Green CJ, Foresti R, Alam J, and Motterlini R (2003) Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem J 371: 887-895.[CrossRef][Medline]
Behl C, Davis JB, Lesley R, and Schubert D (1994) Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77: 817-827.[CrossRef][Medline]
Bilia AR, Scalise L, Bergonzi MC, and Vincieri FF (2004) Analysis of kavalactones from Piper methysticum (kava-kava). J Chromatogr B Analyt Technol Biomed Life Sci 812: 203-214.[Medline]
Boothby LA and Doering PL (2005) Vitamin C and vitamin E for Alzheimer's disease. Ann Pharmacother 39: 2073-2080.
Butterfield DA, Drake J, Pocernich C, and Castegna A (2001) Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid beta-peptide. Trends Mol Med 7: 548-554.[CrossRef][Medline]
Clouatre DL (2004) Kava kava: examining new reports of toxicity. Toxicol Lett 150: 85-96.[CrossRef][Medline]
Culmsee C, Gerling N, Landshamer S, Rickerts B, Duchstein HJ, Umezawa K, Klumpp S, and Krieglstein J (2005) Nitric oxide donors induce neurotrophin-like survival signaling and protect neurons against apoptosis. Mol Pharmacol 68: 1006-1017.
Folmer F, Blasius R, Morceau F, Tabudravu J, Dicato M, Jaspars M, and Diederich M (2006) Inhibition of TNFalpha-induced activation of nuclear factor kappaB by kava (Piper methysticum) derivatives. Biochem Pharmacol 71: 1206-1218.[CrossRef][Medline]
Gleitz J, Beile A, Wilkens P, Ameri A, and Peters T (1997) Antithrombotic action of the kava pyrone (+)-kavain prepared from Piper methysticum on human platelets. Planta Med 63: 27-30.[Medline]
Gong P, Hu B, and Cederbaum AI (2004) Diallyl sulfide induces heme oxygenase-1 through MAPK pathway. Arch Biochem Biophys 432: 252-260.[CrossRef][Medline]
Götz ME, Kunig G, Riederer P, and Youdim MB (1994) Oxidative stress: free radical production in neural degeneration. Pharmacol Ther 63: 37-122.[CrossRef][Medline]
Haridas V, Hanausek M, Nishimura G, Soehnge H, Gaikwad A, Narog M, Spears E, Zoltaszek R, Walaszek Z, and Gutterman JU (2004) Triterpenoid electrophiles (avicins) activate the innate stress response by redox regulation of a gene battery. J Clin Invest 113: 65-73.[CrossRef][Medline]
Jaiswal AK (2004) Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med 36: 1199-1207.[CrossRef][Medline]
Jakubíková J, Sedlak J, Bod'o J, and Bao Y (2006) Effect of isothiocyanates on nuclear accumulation of NF-kappaB, Nrf2, and thioredoxin in caco-2 cells. J Agric Food Chem 54: 1656-1662.[CrossRef][Medline]
Jamieson DD, Duffield PH, Cheng D, and Duffield AM (1989) Comparison of the central nervous system activity of the aqueous and lipid extract of kava (Piper methysticum). Arch Int Pharmacodyn Ther 301: 66-80.[Medline]
Katoh Y, Itoh K, Yoshida E, Miyagishi M, Fukamizu A, and Yamamoto M (2001) Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription. Genes Cells 6: 857-868.[Abstract]
Klafki HW, Staufenbiel M, Kornhuber J, and Wiltfang J (2006) Therapeutic approaches to Alzheimer's disease. Brain 129: 2840-2855.
Lee-Hilz YY, Boerboom AM, Westphal AH, Berkel WJ, Aarts JM, and Rietjens IM (2006) Pro-oxidant activity of flavonoids induces EpRE-mediated gene expression. Chem Res Toxicol 19: 1499-1505.[CrossRef][Medline]
Lee JM, Calkins MJ, Chan K, Kan YW, and Johnson JA (2003a) Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis. J Biol Chem 278: 12029-12038.
Lee JM and Johnson JA (2004) An important role of Nrf2-ARE pathway in the cellular defense mechanism. J Biochem Mol Biol 37: 139-143.[Medline]
Lee JM, Shih AY, Murphy TH, and Johnson JA (2003b) NF-E2-related factor-2 mediates neuroprotection against mitochondrial complex I inhibitors and increased concentrations of intracellular calcium in primary cortical neurons. J Biol Chem 278: 37948-37956.
Love S (1999) Oxidative stress in brain ischemia. Brain Pathol 9: 119-131.[Medline]
Murphy TH, Yu J, Ng R, Johnson DA, Shen H, Honey CR, and Johnson JA (2001) Preferential expression of antioxidant response element mediated gene expression in astrocytes. J Neurochem 76: 1670-1678.[CrossRef][Medline]
Nerurkar PV, Dragull K, and Tang CS (2004) In vitro toxicity of kava alkaloid, pipermethystine, in HepG2 cells compared to kavalactones. Toxicol Sci 79: 106-111.
Owuor ED and Kong AN (2002) Antioxidants and oxidants regulated signal transduction pathways. Biochem Pharmacol 64: 765-770.[CrossRef][Medline]
Rafeiro E, Barr SG, Harrison JJ, and Racz WJ (1994) Effects of N-acetylcysteine and dithiothreitol on glutathione and protein thiol replenishment during acetaminophen-induced toxicity in isolated mouse hepatocytes. Toxicology 93: 209-224.[CrossRef][Medline]
Shen G, Hebbar V, Nair S, Xu C, Li W, Lin W, Keum YS, Han J, Gallo MA, and Kong AN (2004) Regulation of Nrf2 transactivation domain activity. The differential effects of mitogen-activated protein kinase cascades and synergistic stimulatory effect of Raf and CREB-binding protein. J Biol Chem 279: 23052-23060.
Shih AY, Johnson DA, Wong G, Kraft AD, Jiang L, Erb H, Johnson JA, and Murphy TH (2003) Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J Neurosci 23: 3394-3406.
Singh YN (1992) Kava: an overview. J Ethnopharmacol 37: 13-45.[CrossRef][Medline]
Singh YN and Singh NN (2002) Therapeutic potential of kava in the treatment of anxiety disorders. CNS Drugs 16: 731-743.[CrossRef][Medline]
Sorrentino L, Capasso A, and Schmidt M (2006) Safety of ethanolic kava extract: results of a study of chronic toxicity in rats. Phytomedicine 13: 542-549.[CrossRef][Medline]
Tong KI, Katoh Y, Kusunoki H, Itoh K, Tanaka T, and Yamamoto M (2006) Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model. Mol Cell Biol 26: 2887-2900.
Varoga D, Paulsen F, Mentlein R, Fay J, Kurz B, Schutz R, Wruck C, Goldring MB, and Pufe T (2006) TLR-2-mediated induction of vascular endothelial growth factor (VEGF) in cartilage in septic joint disease. J Pathol 210: 315-324.[CrossRef][Medline]
Wu CC, Hsu MC, Hsieh CW, Lin JB, Lai PH, and Wung BS (2006) Upregulation of heme oxygenase-1 by Epigallocatechin-3-gallate via the phosphatidylinositol 3-kinase/Akt and ERK pathways. Life Sci 78: 2889-2897.[CrossRef][Medline]
Wu D, Yu L, Nair MG, DeWitt DL, and Ramsewak RS (2002) Cyclooxygenase enzyme inhibitory compounds with antioxidant activities from Piper methysticum (kava kava) roots. Phytomedicine 9: 41-47.[CrossRef][Medline]
Zhao J, Kobori N, Aronowski J, and Dash PK (2006) Sulforaphane reduces infarct volume following focal cerebral ischemia in rodents. Neurosci Lett 393: 108-112.[CrossRef][Medline]
Zhu X, Raina AK, Perry G, and Smith MA (2004) Alzheimer's disease: the two-hit hypothesis. Lancet Neurol 3: 219-226.[CrossRef][Medline]
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, significant differences of Nrf2-shRNA-transfected cells versus control shRNA-transfected cells, stimulated with the same kavalactone. *, significant differences of shRNA-Nrf2-transfected cells versus control-shRNA transfected cells, stimulated with the same kavalactone. #, significant difference versus kavalactone-stimulated cells.
