All Title Author
Keywords Abstract

PLOS ONE  2013 

Estrogen Receptor and PI3K/Akt Signaling Pathway Involvement in S-(-)Equol-Induced Activation of Nrf2/ARE in Endothelial Cells

DOI: 10.1371/journal.pone.0079075

Full-Text   Cite this paper   Add to My Lib


S-(-)equol, a natural product of the isoflavone daidzein, has been reported to offer cytoprotective effects with respect to the cardiovascular system, but how this occurs is unclear. Interestingly, S-(-)equol is produced by the human gut, suggesting a role in physiological processes. We report that treatment of human umbilical vein endothelial cells and EA.hy926 cells with S-(-)equol induces ARE-luciferase reporter gene activity that is dose and time dependent. S-(-)equol (10–250 nM) increases nuclear factor-erythroid 2-related factor 2 (Nrf2) as well as gene products of Nrf2 target genes heme oxygenase-1 (HO-1) and NAD(P)H (nicotinamide-adenine-dinucleotide-phosp?hate)quinone oxidoreductase 1 (NQO1). Endothelial cells transfected with an HA-Nrf2 expression plasmid had elevated HA-Nrf2, HO-1, and NQO1 in response to S-(-)equol exposure. S-(-)equol treatment affected Nrf2 mRNA only slightly but significantly increased HO-1 and NQO1 mRNA. The pretreatment of cells with specific ER inhibitors or PI3K/Akt (ICI182,780 and LY294002) increased Nrf2, HO-1, and NQO1 protein, impaired nuclear translocation of HA-Nrf2, and decreased ARE-luciferase activity. Identical experiments were conducted with daidzein, which had effects similar to S-(-)equol. In addition, DPN treatment (an ERβ agonist) induced the ARE-luciferase reporter gene, promoting Nrf2 nuclear translocation. Cell pretreatment with an ERβ antagonist (PHTPP) impaired S-(-)equol-induced Nrf2 activation. Pre-incubation of cells followed by co-treatment with S-(-)equol significantly improved cell survival in response to H2O2 or tBHP and reduced apoptotic and TUNEL-positively-stained cells. Notably, the ability of S-(-)equol to protect against H2O2-induced cell apoptosis was attenuated in cells transfected with an siRNA against Nrf2. Thus, beneficial effects of S-(-)equol with respect to cytoprotective antioxidant gene activation may represent a novel strategy to prevent and treat cardiovascular diseases.


[1]  Siow RC, Li FY, Rowlands DJ, de Winter P, Mann GE (2007) Cardiovascular targets for estrogens and phytoestrogens: transcriptional regulation of nitric oxide synthase and antioxidant defense genes. Free Radic Biol Med 42: 909–925.
[2]  Setchell KD, Clerici C (2010) Equol: pharmacokinetics and biological actions. J Nutr 140: 1363S–368S.
[3]  Setchell KD, Clerici C (2010) Equol: history, chemistry, and formation. J Nutr 140: 1355S–1362S.
[4]  Jackson RL, Greiwe JS, Schwen RJ (2011) Emerging evidence of the health benefits of S-equol, an estrogen receptor beta agonist. Nutr Rev 69: 432–448.
[5]  Clerici C, Setchell KD, Battezzati PM, Pirro M, Giuliano V, et al. (2007) Pasta naturally enriched with isoflavone aglycons from soy germ reduces serum lipids and improves markers of cardiovascular risk. J Nutr 137: 2270–2278.
[6]  McMahon M, Itoh K, Yamamoto M, Hayes JD (2003) Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J Biol Chem 278: 21592–21600.
[7]  Mann GE, Bonacasa B, Ishii T, Siow RC (2009) Targeting the redox sensitive Nrf2-Keap1 defense pathway in cardiovascular disease: protection afforded by dietary isoflavones. Curr Opin Pharmacol 9: 139–145.
[8]  Anwar AA, Li FY, Leake DS, Ishii T, Mann GE, et al. (2005) Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2. Free Radic Biol Med 39: 227–236.
[9]  Froyen EB, Steinberg FM (2011) Soy isoflavones increase quinone reductase in hepa-1c1c7 cells via estrogen receptor beta and nuclear factor erythroid 2-related factor 2 binding to the antioxidant response element. J Nutr Biochem. 22: 843–848.
[10]  Joy S, Siow RC, Rowlands DJ, Becker M, Wyatt AW, et al. (2006) The isoflavone Equol mediates rapid vascular relaxation: Ca2+-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt phosphorylation in human endothelial cells. J Biol Chem 281: 27335–27345.
[11]  Hwang J, Sevanian A, Hodis HN, Ursini F (2000) Synergistic inhibition of LDL oxidation by phytoestrogens and ascorbic acid. Free Radic Biol Med 29: 79–89.
[12]  Hwang J, Wang J, Morazzoni P, Hodis HN, Sevanian A (2003) The phytoestrogen equol increases nitric oxide availability by inhibiting superoxide production: an antioxidant mechanism for cell-mediated LDL modification. Free Radic Biol Med 34: 1271–1282.
[13]  Kamiyama M, Kishimoto Y, Tani M, Utsunomiya K, Kondo K (2009) Effects of equol on oxidized low-density lipoprotein-induced apoptosis in endothelial cells. J Atheroscler Thromb 16: 239–249.
[14]  Siow RC, Mann GE (2010) Dietary isoflavones and vascular protection: activation of cellular antioxidant defenses by SERMs or hormesis? Mol Aspects Med 31: 468–477.
[15]  Wondrak GT, Cabello CM, Villeneuve NF, Zhang S, Ley S, et al. (2008) Cinnamoyl-based Nrf2-activators targeting human skin cell photo-oxidative stress. Free Radic Biol Med 45: 385–395.
[16]  Zhang DD (2006) Mechanistic studies of the Nrf2-Keap1 signaling pathway. Drug Metab Rev 38: 769–789.
[17]  Cheng C, Wang X, Weakley SM, Kougias P, Lin PH, et al. (2010) The soybean isoflavonoid equol blocks ritonavir-induced endothelial dysfunction in porcine pulmonary arteries and human pulmonary artery endothelial cells. J Nutr 140: 12–17.
[18]  Gimenez I, Lou M, Vargas F, Alvarez-Guerra M, Mayoral JA, et al. (1997) Renal and vascular actions of equol in the rat. J Hypertens 15: 1303–1308.
[19]  Chin-Dusting JP FL, Lewis TV, Piekarska A, Nestel PJ, Husband A (2001) The vascular avtivity of some isoflavone metabolities: implications for a cardioprotective role. Br J Pharmacol 133: 595–605.
[20]  Walker HA, Dean TS, Sanders TA, Jackson G, Ritter JM, et al. (2001) The phytoestrogen genistein produces acute nitric oxide-dependent dilation of human forearm vasculature with similar potency to 17beta-estradiol. Circulation 103: 258–262.
[21]  Mahn K, Borras C, Knock GA, Taylor P, Khan IY, et al. (2005) Dietary soy isoflavone induced increases in antioxidant and eNOS gene expression lead to improved endothelial function and reduced blood pressure in vivo. FASEB J 19: 1755–1757.
[22]  Jackman KA, Woodman OL, Chrissobolis S, Sobey CG (2007) Vasorelaxant and antioxidant activity of the isoflavone metabolite equol in carotid and cerebral arteries. Brain Res 1141: 99–107.
[23]  Tormala R, Appt S, Clarkson TB, Groop PH, Ronnback M, et al. (2008) Equol production capability is associated with favorable vascular function in postmenopausal women using tibolone; no effect with soy supplementation. Atherosclerosis 198: 174–178.
[24]  Tormala RM, Appt S, Clarkson TB, Tikkanen MJ, Ylikorkala O, et al. (2007) Individual differences in equol production capability modulate blood pressure in tibolone-treated postmenopausal women: lack of effect of soy supplementation. Climacteric 10: 471–479.
[25]  Du Y, Villeneuve NF, Wang XJ, Sun Z, Chen W, et al. (2008) Oridonin confers protection against arsenic-induced toxicity through activation of the Nrf2-mediated defensive response. Environ Health Perspect 116: 1154–1161.
[26]  Zhang T, Wang F, Xu HX, Yi L, Qin Y, et al. Activation of nuclear factor erythroid 2-related factor 2 and PPARgamma plays a role in the genistein-mediated attenuation of oxidative stress-induced endothelial cell injury. Br J Nutr: 1–13.
[27]  Blair RM, Appt SE, Franke AA, Clarkson TB (2003) Treatment with antibiotics reduces plasma equol concentration in cynomolgus monkeys (Macaca fascicularis). J Nutr 133: 2262–2267.
[28]  Yuan JP, Wang JH, Liu X (2007) Metabolism of dietary soy isoflavones to equol by human intestinal microflora–implications for health. Mol Nutr Food Res 51: 765–781.
[29]  Adlercreutz H, Yamada T, Wahala K, Watanabe S (1999) Maternal and neonatal phytoestrogens in Japanese women during birth. Am J Obstet Gynecol 180: 737–743.
[30]  Setchell KD, Cassidy A (1999) Dietary isoflavones: biological effects and relevance to human health. J Nutr 129: 758S–767S.
[31]  Adlercreutz H, Mazur W (1997) Phyto-oestrogens and Western diseases. Ann Med 29: 95–120.
[32]  Nishinaka T, Ichijo Y, Ito M, Kimura M, Katsuyama M, et al. (2007) Curcumin activates human glutathione S-transferase P1 expression through antioxidant response element. Toxicol Lett 170: 238–247.
[33]  Wiegand H, Wagner AE, Boesch-Saadatmandi C, Kruse HP, Kulling S, et al. (2009) Effect of dietary genistein on Phase II and antioxidant enzymes in rat liver. Cancer Genomics Proteomics 6: 85–92.
[34]  Hernandez-Montes E, Pollard SE, Vauzour D, Jofre-Montseny L, Rota C, et al. (2006) Activation of glutathione peroxidase via Nrf1 mediates genistein`s protection against oxidative endothelial cell injury. Biochem Biophys Res Commun 346: 851–859.
[35]  Russell KS HM, Sinha D, Clerisme E, Bender JR (2000) Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling. Proc Natl Acad Sci U S A 23: 5930–5935.
[36]  Toth B, Scholz C, Saadat G, Geller A, Schulze S, et al. (2009) Estrogen receptor modulators and estrogen receptor beta immunolabelling in human umbilical vein endothelial cells. Acta Histochem 111: 508–519.
[37]  Xu SZ, Zhong W, Ghavideldarestani M, Saurabh R, Lindow SW, et al. (2009) Multiple mechanisms of soy isoflavones against oxidative stress-induced endothelium injury. Free Radic Biol Med 47: 167–175.
[38]  Duncan AM, Merz-Demlow BE, Xu X, Phipps WR, Kurzer MS (2000) Premenopausal equol excretors show plasma hormone profiles associated with lowered risk of breast cancer. Cancer Epidemiol Biomarkers Prev 9: 581–586.
[39]  Montano MM, Jaiswal AK, Katzenellenbogen BS (1998) Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor-alpha and estrogen receptor-beta. J Biol Chem 273: 25443–25449.
[40]  Yao Y, Brodie AMH, Davidson NE, Kensler TW, Zhou Q (2010) Inhibition of estrogen signaling activates the NRF2 pathway in breast cancer. Breast Cancer Res Treat 124: 585–591.
[41]  Zhou W Lo SC, Liu JH, Hannink M, Lubahn DB (2007) ERRbeta: a potent inhibitor of Nrf2 transcriptional activity. Mol Cell Endocrinol 278 52–62.
[42]  Iwasaki K MY, Haneda M, Uchida K, Nakao A, Kobayashi T (2010) Significance of HLA class I antibody-induced antioxidant gene expression for endothelial cell protection against complement attack. Biochem Biophys Res Commun 391: 1210–1215.
[43]  Langston W, Circu ML, Aw TY (2008) Insulin stimulation of gamma-glutamylcysteine ligase catalytic subunit expression increases endothelial GSH during oxidative stress: influence of low glucose. Free Radic Biol Med 45: 1591–1599.
[44]  Park SS, Kim YN, Jeon YK, Kim YA, Kim JE, et al. (2005) Genistein-induced apoptosis via Akt signaling pathway in anaplastic large-cell lymphoma. Cancer Chemother Pharmacol 56: 271–278.
[45]  Heiss EH, Schachner D, Werner ER, Dirsch VM (2009) Active NF-E2-related factor (Nrf2) contributes to keep endothelial NO synthase (eNOS) in the coupled state: role of reactive oxygen species (ROS), eNOS, and heme oxygenase (HO-1) levels. J Biol Chem 284: 31579–31586.
[46]  Rowlands DJ, Chappel S, Siow RC, Mann GE (2011) Equol-stimulated mitochondrial reactive oxygen species activate endothelial nitric oxide synthase and redox signaling in endothelial cells: roles for F-actin and GPR30. Hypertension 57: 833–840.
[47]  Haynes MP, Sinha D, Russell KS, Collinge M, Fulton D, et al. (2000) Membrane Estrogen Receptor Engagement Activates Endothelial Nitric Oxide Synthase via the PI3-Kinase-Akt Pathway in Human Endothelial Cells. Circ Res 87: 677–682.
[48]  Chung JE, Kim SY, Jo HH, Hwang SJ, Chae B, et al. (2008) Antioxidant effects of equol on bovine aortic endothelial cells. Biochem Biophys Res Commun 375: 420–424.
[49]  Arora A, Nair MG, Strasburg GM (1998) Antioxidant activities of isoflavones and their biological metabolites in a liposomal system. Arch Biochem Biophys 356: 133–141.
[50]  Mitchell JH, Gardner PT, McPhail DB, Morrice PC, Collins AR, et al. (1998) Antioxidant efficacy of phytoestrogens in chemical and biological model systems. Arch Biochem Biophys 360: 142–148.
[51]  Choi EJ (2009) Evaluation of equol function on anti- or prooxidant status in vivo. J Food Sci 74: H65–71.
[52]  Setchell KD, Zhao X, Jha P, Heubi JE, Brown NM (2009) The pharmacokinetic behavior of the soy isoflavone metabolite S-(-)equol and its diastereoisomer R-(+)equol in healthy adults determined by using stable-isotope-labeled tracers. Am J Clin Nutr 90: 1029–1037.
[53]  Setchell KD, Zhao X, Shoaf SE, Ragland K (2009) The pharmacokinetics of S-(-)equol administered as SE5-OH tablets to healthy postmenopausal women. J Nutr 139: 2037–2043.
[54]  Yee S, Burdock GA, Kurata Y, Enomoto Y, Narumi K, et al. (2008) Acute and subchronic toxicity and genotoxicity of SE5-OH, an equol-rich product produced by Lactococcus garvieae. Food Chem Toxicol 46: 2713–2720.


comments powered by Disqus