curcumin inhibits growth of saccharomyces cerevisiae through iron
TRANSCRIPT
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
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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.05163-11 EC Accepts, published online ahead of print on 9 September 2011
Minear, S., et al
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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
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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
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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
112
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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
bathopenanthrolinebisulfonic acid (BPS), bathocuprine disulphonate (BCS), 118
ferrous sulfate (Fe2SO4), copper chloride (CuCl2) and ferric chloride (FeCl3) were 119
from Sigma-Aldrich, Inc. (St. Louis, MO). BY4743 (MATa/α his3Δ1/his3Δ1 120
leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0) or BY4741 (MATa 121
his3Δ1 leu2Δ0 LYS2 met15Δ0 ura3Δ0) were used as the wild-type strains. The 122
homozygous diploid deletion collection, based on BY4743, was purchased from 123
Open Biosystems (Huntsville, AL). Cells used in growth analysis on plates were 124
grown to saturation and then plated in 5-fold serial dilutions with a starting 125
concentration of approximately 1.0x107 cells. 126
Pooled competitive growth assays 127
The experimental design is detailed in (11). In brief, yeast strains from the 128
homozygous deletion collection were pooled as described (12, 32) and stored at 129
-80°C. The pooled cells were then thawed and diluted in YPD to an optical 130
density (OD) of 0.0625 at 600 nm. This dilution ensures that 300 individual cells 131
from each deletion strain are represented at the onset of the experiment. 150 μM 132
curcumin was added to the pooled cells and cells were grown for five generations 133
(to OD600 = 2.0) in a Tecan GENios microtiter plate reader (Tecan, Durham, NC). 134
Cells were harvested and genomic DNA extracted, used as a template in PCR-135
amplification of ‘UPTAG’ and ‘DOWNTAG’ sequences that mark each gene 136
deletion. The fluorescently-tagged PCR products were hybridized to TAG3 137
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microarrays from Affymetrix (Santa Clara, CA) (11) and fluorescence intensity of 138
each pair of tags was determined. Fitness-defect scores were calculated for each 139
strain in the pool. These scores are based on a tag-specific algorithm that takes 140
into account the intensities of each tag on the experimental array and the 141
corresponding intensities on a set of control arrays, preformed using pooled-cells 142
grown without the compound of interest (control set) (12). Tag intensities were 143
log transformed, mean normalized, and the intensities for replicates averaged 144
into a single value. A mean and standard deviation were calculated for both the 145
UPTAG and DOWNTAG hybridizations. Z-scores for the UPTAG and 146
DOWNTAG for each strain were calculated by subtracting the control average 147
intensity from the experimental (curcumin treated) average intensity and dividing 148
by the standard deviation of the control-set intensities. This generates a z-score 149
value for both the UPTAG and DOWNTAG sequences which are then averaged 150
into a single fitness-defect score for the strain. 151
β-galactosidase assays 152
BY4741 cells were transformed by the lithium acetate method with a plasmid 153
bearing the PrFET3-lacZ reporter (provided by Caroline Philpott, NIDDK National 154
Institutes of Health), or a plasmid containing the PrFRE2-lacZ reporter (provided by 155
Dennis Winge, University of Utah) (28). Cells were then grown in synthetic 156
complete medium (lacking the required amino acid for plasmid selection) made 157
with YNB lacking iron and copper from BIO101 (Solon, OH) to OD600= 1.0. 158
Cultures where diluted to OD600= 0.01 into fresh low iron/copper synthetic 159
complete medium with additions of curcumin or BPS, in the presence or absence 160
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of Fe2SO4, and grown an additional 12 h. Cells were harvested and proteins 161
extracted for use in β-galactosidase activities as described in (4). The β-162
galactosidase activity reported is the maximum rate of OD415 163
change/minute/amount of protein. Two independent transformants were assayed 164
in triplicate (for a minimum of six replicates) for each condition and error bars 165
represent the standard deviation of the mean. 166
Inductively-coupled plasma atomic emission spectroscopy (ICP-AES) 167
ICP-AES was performed as described in (10). In brief, wild-type (BY4743) cells 168
were grown from OD600= 0.5 to saturation in YPD with no additions or with 169
addition of 100 μM BPS, 500 μM BCS, 25 μM curcumin, or 50 μM curcumin. 170
Cells were then harvested by filtration, washed with water, resuspended in 500 μl 171
of 30% nitric acid and digested overnight at 65°C. Cell digests were centrifuged 172
at 12,000 x g for 10 minutes and the supernatants were transferred to new tubes 173
and used in ICP-AES analysis on a Varian Vista ICP-AES (Varian Inc, Palo Alto, 174
CA, USA). Data analysis was performed as indicated in (10) for the following 175
elements: Ca, Co, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, and Zn. All element 176
measurements were corrected for differences in cell mass. This correction 177
assumes that treated and untreated cells differ only in cell mass which appears 178
to be the case as most elements assessed did not change significantly between 179
experimental and control cells (data not shown). The mean iron and copper 180
content for 5 experimental replicates is presented as a percentage relative to the 181
untreated (YPD) control cells. Error bars represent the standard error of the 182
mean. 183
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Yeast growth curves, cell morphology, and viability analysis 184
Wild-type (BY4743) cells were grown to saturation in YPD, washed and 185
inoculated at OD600=0.5 into YPD with no additives or containing 80 μM BPS, 50 186
μM curcumin, or 100 μM curcumin +/- 50 μM Fe2SO4. Three independent 187
cultures were monitored for each condition and cells were grown in a Tecan 188
GENios plate reader with orbital shaking at room temperature (between 23-27°C) 189
with OD600 readings taken every 30 min. A path-length correction factor was then 190
applied to Tecan GENios OD600 readings, and these data are reported. For bud-191
type assessments, cells were harvested after 7 h of growth, fixed in 3.7% 192
formaldehyde and bud morphology scored. The morphology of 200 cells was 193
assessed for each of three replicate cultures (for a total ‘n’ of 600 cells in 3 194
replicates). Data presented are the mean of these values, and the error bars 195
represent the standard deviation of the mean. 196
197
In addition to cell morphology, cells used in the growth curve analysis were 198
scored for viability after 7 hours of incubation with the additives indicated. This 199
analysis was performed to determine if the growth lag observed for certain 200
additions was due to cell death. 20 μl of each culture was harvested by 201
centrifugation, resuspended in 3.7% methylene blue in 100 mM phosphate 202
buffered saline (adjusted to pH 7), and scored for viability. No less than 100 cells 203
were scored per culture and cells staining dark blue were considered 204
metabolically inactive and therefore inviable. As a control in this experiment, an 205
additional sample was taken from the YPD grown cultures and heat-treated for 206
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10 min. at 70°C to kill all cells. As expected, 100% of cultures treated this way 207
stained dark blue and were unable to form colonies. Viability was further 208
confirmed for all cultures by assessing colony-forming units on YPD after 7 h of 209
incubation with the additives indicated. 210
211
212
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Results and Discussion 213 214 215 Yeast mutants with defects in iron and copper homeostasis are 216
hypersensitive to curcumin 217
218
Curcumin was tested at a range of concentrations, and found to inhibit growth of 219
wild-type yeast in a dose-dependent manner (Figure 1A). To determine the 220
mechanism by which growth was inhibited, we sought to identify mutant yeast 221
strains with increased sensitivity to curcumin. First a competitive growth assay 222
was used (11), in which a genomic collection of ~4,700 homozygous diploid 223
deletion strains (39) was grown in a single culture using medium that contained 224
curcumin. After 5 generations of growth, the relative abundance of each strain in 225
the pool was assessed by hybridization to a DNA microarray that contained 226
sequence identifiers unique to each deletion strain (see Methods). Mutants 227
whose growth is inhibited by curcumin are underrepresented in this pool. 228
Analysis of these data identified 42 mutant strains that displayed a significant 229
fitness defect during growth in curcumin-containing media (Table S1). Growth of 230
the 30 most sensitive strains was then tested individually on solid medium 231
containing 150 μM curcumin (Figure 1B and Table S1), however pho86Δ, ypk1Δ 232
and vma5Δ were excluded because they grew poorly on YPD alone. Of the 233
strains tested, 18 were hypersensitive to 150 μM curcumin in the solid-medium 234
growth assay and are hereafter referred to as curcumin hypersensitive strains 235
(Figure 1B). The degree of hypersensitivity in the initial liquid growth assay did 236
not strongly correlate with the degree of growth inhibition observed on solid 237
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medium containing curcumin. This could be due to the difference between liquid 238
and solid media growth, competitive versus clonal growth, and/or due to the 239
length of exposure to curcumin: In the competitive growth assay, cells were 240
exposed to curcumin for only 5 generations, compared to prolonged curcumin 241
exposure for 2-4 days in the solid medium growth assay (i.e. ~20 generations of 242
growth are required to form a colony). The combination of both assays allowed 243
for a more accurate assessment of hypersensitivity. 244
245
Next, the functions of genes deleted in the curcumin hypersensitive strains 246
were analyzed. The 18 mutant strains identified only 17 genes as two strains, 247
ccc2Δ and ydr269cΔ, delete overlapping open reading frames, and thus both 248
compromise Ccc2 function. Remarkably, 8 out of 17 of the genes identified 249
encode products that function in the transport and homeostasis of iron and 250
copper ions (Table 1). Specifically, genes whose products mediate iron transport 251
(FTR1 and FET3) as well as regulation of gene expression under iron limitation 252
conditions (AFT1) were identified (8, 40). Genes that encode proteins required 253
for copper transport (CTR1) and regulation of gene expression during copper 254
limitation (MAC1) were also identified (6, 23). Interestingly, these processes are 255
coupled in vivo (7), as an essential component of high affinity iron uptake, Fet3p, 256
is a copper-dependent ferro-oxidoreductase, and deletions of genes whose 257
products are required for incorporation of copper ion into Fet3 (CCC2, ATX1, 258
GEF1) are sensitive to curcumin (13, 18, 42). Other genes identified include 259
those that disrupt vacuolar acidification (VMA3, VMA13, VMA21), a process 260
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required for intracellular iron utilization (9, 37). In addition, several mutants that 261
disrupt the lipid composition of membranes, and thus have wide-ranging defects 262
in membrane function and trafficking are hypersensitive to curcumin (SAC1, 263
ERG3, LEM3). Overall, these findings suggest that strains with defects in iron 264
and/or copper homeostasis are hypersensitive to curcumin. 265
266
Differential rescue of curcumin hypersensitivity by copper and iron 267
268
To further define the mechanism by which curcumin inhibits yeast growth, we 269
tested the ability of curcumin-hypersensitive deletion strains to grow when iron or 270
copper supplements were added to the growth medium. Importantly, addition of 271
iron or copper to YPD medium at the concentrations employed had no effect on 272
cell growth in the absence of curcumin (data not shown). Addition of 50 μM iron 273
significantly improved the growth of all curcumin-sensitive strains, whereas 274
addition of a 10-fold higher amount of copper (500 μM) was able to rescue 275
growth of only a small subset of strains (fet3Δ, gef1Δ, ccc2Δ, atx1Δ and mac1Δ), 276
(Figure 1B). 277
278
We next compared the curcumin sensitivity profile to that of BPS, a known iron 279
chelator, and BCS, a known copper chelator. The 100 μM curcumin sensitivity 280
profile was strikingly similar to 80 μM BPS-sensitivity profile (Figure 1C), with 281
erg3Δ as the single exceptor showing sensitivity to curcumin and not BPS. 282
Similar experiments comparing growth on 100 μM curcumin to growth on as 283
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much as 500 μM BCS identified only three of the candidates, vma21Δ, vma13Δ 284
and cup5Δ, as BCS sensitive (data not shown). Taken together these 285
observations suggest that curcumin inhibits yeast growth primarily through 286
limiting iron availability. 287
288
Competitive growth assays similar to the one described here for curcumin 289
have been performed using 75-100 μM BPS (21). This BPS concentration range 290
results in a similar yeast sensitivity profile to that observed for 150 μM curcumin 291
(Figure 1C and data not shown), which was the curcumin concentration 292
employed in our competitive growth assays. Strikingly, 18 of 42 of our curcumin 293
sensitive candidates were also identified in the BPS-sensitivity screen (21). While 294
the curcumin-hypersenstive strains identified in this study were well-represented 295
in the BPS hypersensitivity profile, the BPS competitive growth assays described 296
by Jo et al., (21) identified approximately three-fold more sensitive strains. This 297
difference could be due to the fact that cells were exposed to BPS for 15 298
generations, as opposed to the 5 generations used in the curcumin screen. 299
Furthermore, a less stringent fitness cutoff was used when assigning BPS-300
sensitive strains. Taken together these data further support the interpretation that 301
curcumin-mediated growth inhibition is due to iron starvation. 302
303
304
305
306
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Curcumin accumulates in yeast endoplasmic-reticulum membranes 307
308
Chelators can deplete the exogenous pool of metal ions from the growth 309
medium, enter cells and deplete the intracellular reserves of metal ions, or act 310
both intra and extracellularly to alter metal ion pools. Curcumin is able to bind 311
iron in solution, demonstrating that it is an effective extracellular iron chelator (2). 312
To determine if curcumin can also traverse the yeast cell wall and plasma 313
membrane to act intracellularly, we exploited the natural fluorescent properties of 314
curcuminoids. Curcumin auto-fluorescence can be excited at ~455 nm and emits 315
at approximately 540 nm (19, 24). We treated cells containing an HDEL-Ds-Red 316
marker, which uniformly stains both cortical and peri-nuclear ER-membranes 317
(16), with either DMSO or 150 μM curcumin for 5 hours and visualized curcumin 318
fluorescence using a filter set for GFP that overlaps with the curcumin excitation 319
and emission wavelengths (Figure 2). Curcumin fluorescence co-localized with 320
the HDEL-Ds-Red marker in 69% of cells (n=103) examined, indicating that 321
curcumin not only enters yeast cells but also accumulates within the ER-322
membrane (Figure 2A). While there is clear co-localization of curcumin with the 323
ER-membrane, curcumin may also localize to additional intracellular membranes 324
accounting for the background of curcumin fluorescence that does not co-localize 325
with the ER HDEL marker (Figure 2A). These observations are consistent with 326
demonstrations of curcumin accumulation in intracellular membranes of 327
mammalian cells, including the ER (19, 24). These findings further suggest that 328
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curcumin may impact intracellular iron pools in addition to chelation of 329
extracellular iron. 330
To test whether addition of iron to the growth medium suppresses curcumin 331
sensitivity by preventing curcumin uptake into cells, cells grown in curcumin and 332
iron were examined by fluorescence microscopy. Curcumin also co-localized with 333
the ER-membrane in iron-treated cells, indicating that it still enters cells under 334
conditions of iron supplementation (Figure 2B). Curcumin fluorescence was 335
reduced in iron-treated cells. However, because iron decreases the fluorescence 336
intensity of curcumin-containing solutions in a dose-dependent manner (data not 337
shown), fluorescence per se cannot be used to compare intracellular curcumin 338
concentrations under these two conditions. Nonetheless, our findings indicate 339
that curcumin permeates yeast cells grown in the presence or absence of 340
supplemental iron, and are consistent with the hypothesis that curcumin acts 341
intracellularly to chelate iron and induce iron starvation. 342
343
Curcumin treatment results in reduced intracellular iron 344
345
We tested the effect of curcumin on cellular iron levels directly using inductively-346
coupled plasma atomic emission spectroscopy (ICP-AES). Wild-type cells were 347
treated with curcumin, BPS, or BCS (see Materials and Methods). The majority of 348
elements assessed this way did not change as a result of these treatments (data 349
not shown). As expected, the iron content of cells treated with 100 μM BPS was 350
dramatically reduced (80% less iron compared to cells grown in YPD). Curcumin 351
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also significantly decreased cellular iron levels, with cells grown in the presence 352
of 25 μM and 50 μM curcumin containing 20-40% less iron, respectively, versus 353
cells grown in YPD (Figure 3A). Treatment with BCS reduced cellular-copper 354
content by 90% of that of cells grown in YPD, however, consistent with our earlier 355
findings, curcumin-treatment did not significantly alter copper levels in 356
comparison to YPD-grown cells (Figure 3A). 357
358
The physiological relevance of this cellular iron depletion was next examined 359
by measuring the transcriptional response induced by curcumin. Under 360
conditions of iron starvation, the Aft1 transcription factor up-regulates genes 361
responsible for high-affinity iron transport (41). Two targets of Aft1-transcription 362
regulation are FET3, the high affinity iron transporter, and FRE2, a gene 363
encoding an iron reductase that functions as the rate limiting step in high-affinity 364
iron import (1, 43). Transcription of FET3 can also be induced by Mac1p, a 365
transcription factor that activates gene expression in response to copper 366
starvation (14); however, regulation of FRE2 is independent of copper starvation 367
(28). Following 50 μM curcumin treatment, FET3 and FRE2 expression increased 368
3-fold and 6-fold, respectively (Figures 3B and 3C). Because the curcumin-369
mediated induction of FRE2 and FET3 are similar, the most parsimonious 370
explanation is that curcumin predominantly induces an iron-starvation response. 371
These data suggest that growth of yeast in the presence of curcumin leads to a 372
decrease in available iron and, as a result, induces the iron-starvation 373
transcriptional response. 374
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375
Curcumin slows yeast cell cycle progression with no accompanying 376
decrease in cell viability 377
378
Next, we examined the kinetics of curcumin-induced yeast cell growth inhibition. 379
Yeast cells were grown to mid-log phase and treated with curcumin or BPS, in 380
the presence or absence of additional iron, and growth rate (i.e. doubling time) 381
monitored. As expected, addition of curcumin or BPS slowed yeast growth rate 382
(Figure 4A), with treatments of either 80 μM BPS or 150 μM curcumin increasing 383
doubling time approximately two-fold (Figure 4B). Addition of iron to the BPS- or 384
curcumin-treated cells rescued this growth inhibition, shortening the doubling 385
times to levels similar to those observed for untreated-control cells grown in YPD 386
(Figure 4A and Figure 4B). 387
388
After ~10 hours of treatment with either BPS or curcumin, at which point 389
control cells in YPD had doubled twice, cell viability and cell cycle progression 390
were assessed for the various conditions. Viability of cells grown in the presence 391
of curcumin or BPS was monitored using the vital stain methylene blue (see 392
Materials and Methods) and by assessing colony-forming units of washed cells 393
plated on YPD. 100% cell viability was observed for all cultures (data not shown) 394
at 10 hours post-treatment, consistent with the hypothesis that these compounds 395
cause a delay in cell cycle progression rather than causing cell death. To assess 396
cell cycle progression, we analyzed the morphology of yeast exposed to 397
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curcumin and BPS. In G1 phase cells are unbudded, while cells in S, G2 and M 398
phases of the cell cycle possess buds of characteristic sizes (reviewed in (25)). 399
During mid-log phase growth in YPD, 25% of cells were unbudded and 75% of 400
cells were budded (Figure 4C). Exposure to curcumin or BPS caused an 401
increase in the fraction of unbudded cells, which is commonly observed for 402
treatments that increase the length of G1 (Figure 4C). Curcumin exposure 403
caused a dose-dependent increase in the proportion of unbudded cells to 40% 404
and 45% for 100 μM and 150 μM curcumin, respectively. BPS treatment 405
increased the unbudded fraction to 55%. Taken together, these observations 406
demonstrate that curcumin treatment lengthens the time required to complete the 407
cell cycle, and suggest that the duration of G1 is increased. 408
409
To determine if iron starvation was the cause of this increased doubling 410
time, iron was added to the medium to counter the effect of curcumin on cellular 411
iron levels. Supplementation with 50 μM iron in cultures with 100 μM or 150 μM 412
curcumin or 80 μM BPS completely alleviated the growth inhibition, resulting in 413
restoration of the cell-morphology distributions to those observed for YPD grown 414
cells (Figure 4C). Thus, BPS- or curcumin-induced iron starvation increases 415
yeast doubling time and this is at least in part due to a prolonged G1 phase. 416
Treatment of yeast with other metal chelators has been reported to induce a G1 417
growth arrest that can be rescued by addition of exogenous metal ions to the 418
growth medium (22). 419
420
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Curcumin and human cultured cells 421
422
Exposure of certain human cancer cells to curcumin results in a G1 cell cycle 423
arrest (30) and treatment of human cells with other iron chelators also induces a 424
reversible G1 cell cycle arrest (3, 29). To extend the observations made in yeast, 425
we further explored the effects of curcumin on cell growth by treating human 426
osteosarcoma U2OS cells with curcumin and monitoring cell density relative to a 427
DMSO drug-vector control. The density of control cultures increased 3-fold in 428
24 h, whereas the curcumin-treated culture density did not increase (Figure 5). 429
Addition of iron to the curcumin treated-cells suppressed this growth defect and 430
allowed for population doubling in 24 hours (Figure 5). These data suggest that 431
curcumin’s effects are conserved and it inhibits growth of both yeast and human 432
cells by limiting iron availability. 433
434
An understanding of curcumin's mechanism of action on human cells has 435
been elusive, due in part to the myriad effects associated with curcumin 436
treatment (reviewed in (15)). We used yeast as a model system to explore the 437
mechanism of action of curcumin, and our results strongly suggest that curcumin 438
inhibits eukaryotic cell growth predominantly by chelating intracellular iron. Yeast 439
cells with defects in iron homeostasis are sensitive to curcumin, and curcumin 440
delays cell cycle progression in an iron-dependent manner. These findings 441
suggest that the potent chemotherapeutic effects of curcumin may be explained 442
by iron depletion of cancer cells, which inhibits their proliferation. Iron chelation 443
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may also explain the diverse physiological effects of curcumin. Many cellular 444
enzymes require iron as a co-factor and several signaling pathways are also 445
affected by iron availability. Curcumin penetrates both yeast and mammalian 446
cells and accumulates in intracellular membranes, including the ER. This 447
accumulation in intracellular compartments may selectively limit the availability of 448
essential iron co-factors, impairing specific enzymatic functions and altering 449
cellular signaling to produce the diverse physiological effects associated with 450
curcumin treatment. 451
452
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Figure Legends 453
Figure1. Yeast sensitivity to curcumin can be rescued by iron 454
supplementation and is similar to yeast sensitivity to BPS, an iron chelator. 455
(A) Yeast cells are sensitive to curcumin. Serial dilutions of WT (BY4743) cells 456
were plated on YPD +/- curcumin at the concentration indicated and grown for 2 457
days at 30°C. (B) Deletion of iron and copper homeostasis genes exacerbates 458
yeast sensitivity to curcumin. WT (BY4743) and isogenic homozygous deletion 459
strains (lacking the gene indicated) were plated on YPD +/- 150 μM curcumin, 460
500 μM Cu2SO4, and 50 μM Fe2SO4 and grown for 3 days at 30°C. (C) In parallel 461
to the panels shown in (B) the same cultures were plated on YPD +/- 100 μM 462
curcumin or 80 μM BPS and grown for 3 days at 30°C. 463
464
Figure 2. Curcumin enters yeast cells and accumulates in ER-membranes. 465
(A) Wild-type yeast cells expressing HDEL-DsRed (from strain VHY87 described 466
in (16) were treated with either DMSO (left column) or 150 μM curcumin and 467
visualized by fluorescence microscopy using the GFP filter set for curcumin and 468
the RFP filter set for the HDEL-DsRed. Co-localization of curcumin fluorescence 469
with the ER-marker was observed for 69% of cells examined (n=103). (B) Cells 470
from VHY87 expressing the HDEL-DsRed marker were treated with either 150 471
μM curcumin with or without the addition of 50 μM FeSO4 and visualized as in 472
(A). 473
474
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Figure 3. Curcumin depletes cellular iron and activates iron-starvation 475
induced transcription. (A) WT (BY4743) cells were treated with 100 μM BPS, 476
500 μM BCS or the indicated concentration of curcumin. The mean cellular iron 477
and copper levels relative to YPD untreated cells are shown (as determined by 478
ICP-AES measurement - see Materials and Methods) for a minimum of five 479
experimental replicates. Error bars represent the standard error of the mean. 480
Paired Student’s t-tests were performed between YPD control cultures and each 481
of the treatment conditions. * denotes a p-value of <0.05 and ** denotes a p-482
value of <0.0005. (B) WT (BY4741) cells containing the PrFET3-lacZ reporter were 483
grown in low Fe/Cu medium and treated for 12 hours with 100 μM Fe2SO4, 10 484
μM BPS, or the indicated concentrations of curcumin. Protein extracts were 485
prepared and β-galactosidase activity was determined for a minimum of two 486
transformants, each of which was assayed in triplicate. The mean β-487
galactosidase activity and standard deviation are reported. (C) WT (BY4741) 488
cells containing the PrFRE2-lacZ reporter were grown and assayed for β-489
galactosidase activity as in (B). The mean β-galactosidase activity and standard 490
deviation are reported. 491
492
Figure 4. Curcumin treatment causes a G1 growth arrest in yeast that is 493
alleviated by iron supplementation. (A) WT (BY4743) cells were grown 494
overnight to saturation, inoculated at OD600 = 0.5 into fresh YPD with either no 495
supplement, 100 μM curcumin, 150 μM curcumin, or 80 μM BPS +/- 50 μM 496
Fe2SO4 (indicated as + Fe) and optical density measurements were obtained 497
Minear, S., et al
23
every 30 minutes using a Tecan Genios microtiter plate reader. Addition of 498
curcumin to the growth medium results in a shift in absorbance of approximately 499
OD600 = 0.6 for 100 μM curcumin and OD600 = 1.0 for 150 μM curcumin, therefore 500
growth below these densities cannot be measured accurately for this 501
concentration of drug. The growth curves presented are the average of 502
measurements for three independent cultures. (B) The cell doubling time during 503
logarithmic growth was determined for the cultures used in (A). Values presented 504
are the average of the doubling time determined for three independent cultures 505
and error bars represent the standard error of the mean. (C) Cells were grown as 506
in (A) and at 7 hours post-inoculation (during growth curve analysis) samples 507
were taken and bud morphology was scored for three independent cultures with 508
a minimum of 200 cells counted for each. 509
510
Figure 5. Curcumin-mediated inhibition of U2OS cells is alleviated by 511
addition of iron. (A) Three independent cultures of U2OS cells were grown for 512
24 hours in DMEM, 10% FCS with addition of either DMSO (as a vehicle control), 513
100 μM FeCl3 and/or 7.5 μM curcumin (NB - This is ~20x lower concentration 514
than what we routinely used for yeast. The difference in human vs yeast cell 515
sensitivity to curcumin may be due to the yeast cell wall or the multiple drug 516
efflux pumps present in yeast cells). Cells were then trypsinized (100 μl of 0.05% 517
trypsin; 5 min RT), resuspended in DMEM and FCS, harvested by centrifugation 518
and resuspended in PBS. Cell density was determined for each culture by 519
counting three fields with a hemocytometer. Fold growth presented is relative to 520
Minear, S., et al
24
the density of the starting culture (t = 0) and error bars represent the standard 521
deviation of the mean. 522
523
Acknowledgements 524
We thank Caroline Philpott and Dennis Winge for kindly providing plasmids used 525
in this work. We thank the Environmental Measurement: Gas-Solution Analytical 526
Center at Stanford University for their assistance with the ICP-AES 527
measurments. We also acknowledge Aaron Goldman for key technical 528
assistance. M.S.C. is supported by National Institutes of Health research grant 529
GM-48728. T.P.S is supported by NIH grant GM52022. C.N. and G.G. are 530
supported by a grant from NHGRI and the Canadian Institutes of Health (84305) 531
CN and (81340) GG. 532
533
Minear, S., et al
25
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Minear, S., et al
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
FET3, FTR1, AFT1 CTR1, MAC1 ATX1, GEF1, CCC2 (YDR269c also disrupts CCC2) SAC1 GAL11 ERV14 VMA3, VMA13, VMA21 LEM3 ERG3 YSP1
Minear, S., et al., Figures
Figure 1
WTfet3ΔΔ
ydr269cΔΔsac1ΔΔgef1ΔΔysp1ΔΔccc2ΔΔctr1ΔΔerg3ΔΔatx1ΔΔ
vma21ΔΔ
WTmac1ΔΔvma13ΔΔlem3ΔΔcup5ΔΔgal11ΔΔerv14ΔΔftr1ΔΔaft1ΔΔ
A
B
YPD+ 150 μMCurcumin
+ 150 μMCurcumin+ Cu2SO4
+ 150 μMCurcumin+ Fe2SO4
+ 100 μMCurcumin
+ 80 μMBPS
C
050
100210400600
Cur
cum
in (μ
M)
Cell number
Cell number
Minear, S., et al., Figures
Figure 2
ADMSO
150 µM Curcumin
Mer
geD
sRed
(HD
EL)
FITC
(Cur
cum
in)
DIC
B
Mer
geD
sRed
(HD
EL)
FITC
(Cur
cum
in)
150 µM Curc. 150 µM Curc. + Fe
Minear, S., et al., Figures
A
Figure 3
Rel
ativ
e %
cel
lula
r ion
con
tent
Mea
n β-
gala
ctos
idas
e ac
tivity
YP
D
+ B
PS
+ B
CS
+ 25
μM
Cur
c.
+ 50
μM
Cur
c.
Low
Fe/
Cu
+ B
PS
+ 25
μM
Cur
c.
+ 50
μM
Cur
c.
+ Fe
2SO
4
Low
Fe/
Cu
+ 50
μM
Cur
c.
+ Fe
2SO
4
B C
Mea
n β-
gala
ctos
idas
e ac
tivity
0
20
40
60
80
100
120
140
!"#$ %"&$ %'&$ '()*$+,$ '()*$,-$
Cu
Fe
*
**
**
*
0
1
2
3
4
0 5 10 15 20
0
1
2
3
4
0 5 10 15 20
Minear, S., et al., Figures
Figure 4
A
YPD100 Curc150 Curc
150 Curc + Fe100 Curc + Fe
YPD80 BPS80 BPS + Fe
OD
600
OD
600
Time (hours)B
Dou
blin
g tim
e (h
ours
)
YPD
C
Budded
No Bud
Per
cent
age
of c
ell p
opul
atio
ng
pp
0%
20%
40%
60%
80%
100%
�� �� �� �� �� �� �
No
addi
tive
+ B
PS
+ B
PS
+ F
e
+ 10
0 C
urc.
+ F
e
+ 15
0 C
urc.
+ F
e
+ 15
0 C
urc.
+ 10
0 C
urc.
Minear, S., et al., Figures
Figure 5
Fold
Gro
wth
Fold
Gro
wth
t = 0
cul
ture
+ D
MS
O+
Fe+
Cur
c.+
Cur
c. +
Fe
t = 24 hrs