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Minear, S., et al
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Curcumin inhibits growth of Saccharomyces cerevisiae 1 through iron chelation 2 3 4 Steven Minear1,3†, Allyson F. O'Donnell1,4†, Anna Ballew1,5, Guri Giaever2,6, 5 Corey Nislow2,7, Tim Stearns1, 2 and Martha S. Cyert1* 6 7 8 1Department of Biology, Stanford University, Stanford, CA 94305-5020, USA. 9 10 2Department of Genetics, Stanford University School of Medicine, Stanford, CA 11 94304, USA. 12 13 14 Current Addresses: 15 16 3Surgery Department, Stanford University School of Medicine, Stanford, CA, 17 USA. 18 19 4Department of Molecular and Cell Biology, University of California at Berkeley, 20 Berkeley, CA 94720-3202, USA. 21 22 5School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 23 USA. 24 25 6Department of Pharmaceutical Sciences and Molecular Genetics, Donnelly 26 Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada. 27 28 7Banting and Best Department of Medical Research and Donnelly Center, 29 University of Toronto, Toronto, Ontario M5S 3E1, Canada 30 31 †These authors contributed equally to this work. 32 33 *Corresponding author - Martha S. Cyert 34
371 Serra Mall 35 Department of Biology 36 Stanford University 37 Stanford, CA 94305-5020 38 Phone: 650 723-9970 39 Fax: 650 724-9945 40 Email: [email protected] 41
42 43 Current Word Count – 4722 (26, 019 characters without spaces) 44
Abstract (limit 250 words) 46 47 Curcumin, a polyphenol derived from turmeric, is an ancient therapeutic, used in 48
India for centuries to treat a wide array of ailments. Interest in curcumin has 49
increased recently, with on-going clinical trials exploring curcumin as an anti-50
cancer therapy and as a protectant against neurodegenerative diseases. In vitro, 51
curcumin chelates metal ions. However, curcumin’s mechanism of action on 52
mammalian cells remains unclear, although diverse physiological effects have 53
been documented for this compound. These studies use yeast as a model 54
eukaryotic system to dissect the biological activity of curcumin. We find that yeast 55
mutants deleted for genes required for iron and copper homeostasis are hyper-56
sensitive to curcumin and that iron supplementation rescues this sensitivity. 57
Curcumin penetrates yeast cells, concentrates in the ER-membranes, and 58
reduces the intracellular iron pool. Curcumin-treated, iron-starved cultures are 59
enriched in unbudded cells, suggesting that the G1 phase of the cell cycle is 60
lengthened. A delay in cell cycle progression could, in part, explain the anti-61
tumorigenic properties associated with curcumin. We also demonstrate that 62
curcumin causes a growth lag in cultured human cells that is remediated by 63
addition of exogenous iron. These findings suggest that curcumin-induced iron 64
starvation is conserved from yeast to humans and underlies its medicinal 65
properties. 66
67 Keywords 68 69 Curcumin, metal ion homeostasis, iron chelation 70 71
Minear, S., et al
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Introduction 72
Curcumin is the major chemical component of turmeric, a dietary spice made 73
from the root of the Curcuma longa Linn plant and used extensively in traditional 74
Indian medicine (38). Curcumin is a potent bioactive compound that is used to 75
treat cancer (5, 35), atherosclerosis (33), and neurodegenerative diseases such 76
as Alzheimer’s (26, 45) and Parkinson’s (44) as well as to promote wound 77
healing (15, 36). Curcumin is particularly appealing as a therapeutic agent 78
because of its extremely low toxicity. Many biological activities have been 79
ascribed to curcumin. For example, curcumin suppresses inflammatory 80
responses in cultured cells and in animals and also exhibits anti-oxidant 81
properties. Furthermore, curcumin’s ability to inhibit tumorogenesis, and 82
proliferation of a wide variety of cancerous cells, has been well documented. 83
Curcumin is a polyphenol and complexes readily with a number of different metal 84
ions. In aqueous solutions of neutral pH, curcumin is an effective chelator of 85
Fe(III) (2). Curcumin is also lipophilic, and readily crosses membranes (19) and 86
therefore may also chelate metal ions intracellularly. How these chemical 87
properties contribute to curcumin’s biological activities, however, is not 88
understood. 89
90
Identifying relevant in vivo targets of small molecules is technically 91
challenging. Recently, several genetic and genomic approaches have been 92
developed that use the simple eukaryote, Saccharomyces cerevisiae, or budding 93
yeast, to study the mechanism of drug action (17, 27, 31). One such method, 94
Minear, S., et al
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termed homozygous profiling, uses a comprehensive collection of 4,700 95
homozygous diploid deletion yeast strains, each bearing a deletion of a single 96
non-essential gene (39), to examine growth in the presence of a bioactive 97
compound (12). Mutant strains that display increased sensitivity to the compound 98
are identified, and the identity of the genes deleted in these hypersensitive 99
strains is used to infer the biological effects of the compound. 100
101
We carried out such a screen to identify yeast mutants whose growth is 102
strongly inhibited by curcumin. The results of this study indicate that curcumin 103
antagonizes yeast growth by chelating iron. Furthermore, iron supplementation 104
alleviates the growth inhibitory effect of curcumin on both yeast and cultured 105
human cells, suggesting a common mechanism. Previous studies established 106
that curcumin treatment causes mouse cells and tissues to display iron depletion 107
characteristics (20). The findings presented here also indicate that curcumin 108
chelates iron in vivo and suggest that iron chelation may underlie many of 109
curcumin’s therapeutic activities. 110
111
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Materials and Methods 113 114 Strains, plasmids and growth conditions 115 116 Yeast growth medium and basic methods were as described in (34). Curcumin, 117
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Table 1. Biologic Process Gene Iron ion import / iron homeostasis Copper ion import / copper homeostasis Copper and iron homeostasis Others: Cell signaling RNA Pol II-mediated transcription Vesicle-associated secretion Vacuolar acidification Phospholipid translocation Ergosterol biosynthesis Programmed cell death