Nrf2 activation inhibits LXRα activity and LXRα-dependent liver steatosis by competing with FXR for p300, causing FXR activation and FXR-mediated SHP induction. Our findings provide important information on a strategy to prevent and/or treat steatosis.
Cytoprotective effects of chemopreventive agents may be attributed to the induction of antioxidant enzymes. Among these, the induction of glutamate-cysteine ligase (GCL) protects cells from oxidative injury by increasing glutathione (GSH) content. Nuclear factor erythroid-2-related factor 2 (Nrf2) transcriptionally regulates the expression of genes encoding for GCL and other cysteine-metabolizing enzymes. Despite extensive studies on the components in garlic, little information is available on organosulfur by-products made from garlic. In this study, we investigated whether ajoene, a chemically stable garlic by-product, has the ability to activate Nrf2 and induce GCL, and, if so, what is the role of activating Nrf2 in cytoprotection against oxidative stress. Immunoblottings and reporter gene assays were performed in HepG2 cells. Ajoene treatment activated Nrf2, as indicated by increased phosphorylation and nuclear accumulation of Nrf2, decreased interaction with Kelch-like ECH-associated protein-1, and decreased Nrf2 ubiquitination. Consistently, treatment of ajoene increased antioxidant response element reporter gene activity and the mRNA and protein levels of GCL subunits. Ajoene activated protein kinase C-delta (PKCdelta). Inhibition of PKCdelta activation by rottlerin abrogated its ability to activate Nrf2 and induce GCL, suggesting that ajoene promotes the Nrf2-dependent antioxidant defense system via PKCdelta activation. Consequently, ajoene prevented cell death, GSH depletion, and hydrogen peroxide production elicited by tert-butylhydroperoxide. The important role of Nrf2 in cytoprotection was verified by the reversal of ajoene's ability to protect hepatocytes in Nrf2-knockout mice. Our results demonstrate that ajoene increases PKCdelta-dependent Nrf2 activation, GCL induction, and the cellular GSH concentration, which may contribute to protecting cells from oxidative stress.
BACKGROUND AND PURPOSESauchinone, an antioxidant lignan, protects hepatocytes from iron-induced toxicity. This study investigated the protective effects of sauchinone against acetaminophen (APAP)-induced toxicity in the liver and the role of nuclear factor erythroid-2-related factor-2 (Nrf2) in this effect. EXPERIMENTAL APPROACHBlood biochemistry and histopathology were assessed in mice treated with APAP or APAP + sauchinone. The levels of mRNA and protein were measured using real-time PCR assays and immunoblottings. KEY RESULTSSauchinone ameliorated liver injury caused by a high dose of APAP. This effect was prevented by a deficiency of Nrf2. Sauchinone treatment induced modifier subunit of glutamate-cysteine ligase, NAD(P)H:quinone oxidoreductase-1 (NQO1) and heat shock protein 32 in the liver, which was abolished by Nrf2 deficiency. In a hepatocyte model, sauchinone activated Nrf2, as evidenced by the increased nuclear accumulation of Nrf2, the induction of NQO1-antioxidant response element reporter gene, and glutamate-cysteine ligase and NQO1 protein induction, which contributed to the restoration of hepatic glutathione content. Consistently, treatment of sauchinone enhanced Nrf2 phosphorylation with a reciprocal decrease in its interaction with Kelch-like ECH-associated protein-1. Intriguingly, sauchinone activated protein kinase C-d (PKCd), which led to Nrf2 phosphorylation. In addition, it increased the inhibitory phosphorylation of glycogen synthase kinase-3b (GSK3b), derepressing Nrf2 activity, which was supported by the reversal of sauchinone's activation of Nrf2 by an activated mutant of GSK3b. Moreover, phosphorylation of GSK3b by sauchinone depended on PKCd activation. CONCLUSION AND IMPLICATIONSOur results demonstrate that sauchinone protects the liver from APAP-induced toxicity by activating Nrf2, and this effect is mediated by PKCd activation, which induces inhibitory phosphorylation of GSK3b. AbbreviationsALT, alanine aminotransferase; AMPK, AMP-activated protein kinase; APAP, acetaminophen; ARE, antioxidant response element; AST, aspartate aminotransferase; GCL, glutamate-cysteine ligase; GCLM, modifier subunit of glutamate-cysteine ligase; GSH, glutathione; GSK3b, glycogen synthase kinase 3b; HSP32, heat shock protein 32; Keap1, Kelch-like ECH-associated protein-1; KO, knockout; LDH, lactate dehydrogenase; NAPQI, N-acetyl-p-benzoquinoneimine; NQO1, NAD(P)H:quinone oxidoreductase-1; Nrf2, nuclear factor erythroid-2-related factor-2; PKCd, protein kinase C d; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; WT, wild-type
Hypoxia and growth factor stimulation induce hypoxia-inducible factor-1a (HIF-1a), conferring upon cancer cells the ability to adapt to microenvironments and enhance proliferation, angiogenesis and metastasis. Hemin, an iron-binding porphyrin, has been used to treat porphyria attacks, particularly in acute intermittent porphyria. Although the anti-inflammatory and antitumor effects of hemin were reported, no information is available regarding its effect on HIF-1a. Our study investigated whether hemin and other protoporphyrin compounds have the ability to inhibit HIF-1a activity, and if so, what is the molecular basis of inhibition. Hemin treatment prevented CoCl 2 -induced HIF-1a expression. HIF-1a inhibition by hemin resulted from an increase in its facilitated ubiquitination and degradation, as shown by the experimental results using cychloheximide treatment and ubiquitination assays. Consistently, hemin repressed HIF-1a-dependent gene transactivation. Intriguingly, hemin directly impeded the binding between heat shock protein 90 (HSP90) and HIF-1a, which was reversed by the addition of an excess amount of ATP required for HSP90 activity. In addition, hemin decreased the expression of client proteins of HSP90. Thus, the inhibition of HIF-1a activity by hemin might result from its interaction with HSP90. Moreover, treatment of protoporphyrin IX, ZnPP or Co(III)PP, but not Mn(III)PP, inhibited HIF-1a induction, indicating that protoporphyrin ring in association with the nature of binding metal leads to HSP90 inhibition. In an in vivo model, hemin treatment inhibited not only the formation of new vessels but also cancer cell proliferation and migration/invasion, supporting the notion that hemin may be applied to the prevention and/or treatment of angiogenesis and/or cancer metastasis.
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