Curcumin impairs tumor suppressor p53 function in colon cancer cells

Philip J. Moos1,3, Kornelia Edes1, James E. Mullally2 and Frank A. Fitzpatrick1,2

1 Department of Oncological Sciences and 2 Department of Medicinal Chemistry, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT 84112, USA

3 To whom correspondence should be addressed Email: philip.moos{at}hci.utah.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Curcumin (diferuloylmethane) is being considered as a potential chemopreventive agent in humans. In vitro it inhibits transcription by NF-{kappa}B, and the activity of lipoxygenase or cyclooxygenase enzymes, which facilitate tumor progression. In vivo it is protective in rodent models of chemical carcinogenesis. Curcumin contains an {alpha},ß-unsaturated ketone, a reactive chemical substituent that is responsible for its repression of NF-{kappa}B. In compounds other than curcumin this same electrophilic moiety is associated with inactivation of the tumor suppressor, p53. Here we report that curcumin behaves analogously to these compounds. It disrupts the conformation of the p53 protein required for its serine phosphorylation, its binding to DNA, its transactivation of p53-responsive genes and p53-mediated cell cycle arrest.

Abbreviations: IR, ionizing radiation; UV, ultraviolet radiation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cancer chemoprevention is evolving rapidly (13). Currently, the National Cancer Institute is investigating nearly 40 agents in phase I–III human clinical trials (24). Curcumin (diferuloylmethane) is one of the agents in phase I clinical trials (57), pending its evaluation for the prevention of colon (4,79), breast (4,8), lung and prostate cancer (10). Enthusiasm for curcumin in cancer prevention stems from its performance in pre-clinical settings. In vitro it inhibits the activity of proteins with roles in cancer progression, e.g. NF-{kappa}B, lipoxygenase (LOX) and cyclooxygenase (COX) isoenzymes (1113). In vivo it protects against colon tumors in models of chemical carcinogenesis at high concentrations (1418). Although seldom acknowledged, curcumin has traits that may limit its clinical use. First, dietary curcumin is associated with hepatocellular adenoma and carcinomas of the small intestine in mice (19). Appearance of neoplasms in the liver and intestine is consistent with the poor bioavailability of curcumin, which enhances the exposure in these two organs (20,21). Secondly, curcumin inhibits chemotherapy induced apoptosis in vitro and in vivo and it may be deleterious to breast cancer patients (22). Thirdly, curcumin contains an {alpha},ß-unsaturated ketone, which reacts covalently with NF-{kappa}B to repress transactivation (11). A reactive ß carbon in some chemicals can also cause repression of tumor suppressor p53 by an indirect mechanism that involves the inactivation of thioredoxin reductase (23). We hypothesized that curcumin would also impair p53 and therefore, it might confer risks that are inseparable from its potential benefits. Here we report that curcumin inhibits thioredoxin reductase and the folding of wild-type p53 protein into the conformation required for its phosphorylation, its binding to DNA and its transactivation of genes that execute its tumor suppressor function. Curcumin joins a growing list of electrophilic agents that may threaten genomic integrity via their impairment of p53, rather than DNA itself.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Materials
We used DMEM and supplements (Invitrogen, Carlsbad, CA); curcumin (from turmeric, typical purity 65–70%), propidium iodide, RNase A and etoposide (Sigma, St Louis, MO); complete protease inhibitor mixture and FuGene-6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN); luciferase reporter constructs for p53 (p53-Luc, Stratagene, La Jolla, CA) and pRL-RSV (Renilla luciferase control construct, Promega, Madison, WI). We used enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ), luciferase reporter lysis buffer and reporter detection reagents (Promega). We used monoclonal antibodies directed against different p53 epitopes: Pab240 (Santa Cruz, Santa Cruz, CA), Pab1620, Ab421 (Oncogene Sciences, Boston, MA, Ab5 and Ab1) and phospho-Ser15 (Cell Signaling Technologies, Beverly, MA); polyclonal antibodies against p53 (FL-393-G, Santa Cruz), horseradish peroxidase-conjugated secondary antibodies and protein A/G PLUS-agarose (Santa Cruz Biotechnology). We used iQTM SYBER® Green Supermix reagent (Bio-Rad, Hercules, CA) for quantitative PCR.

Cell culture
We maintained RKO cells (a gift from M.Meuth, Institute for Cancer Studies, University of Sheffield, Sheffield, UK) in DMEM at 37°C in a humidified incubator with 5% CO2. We supplemented media with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin and streptomycin and 10% (v/v) FBS. RKO cells have a wild-type p53 gene.

Stop-point thioredoxin reductase assay
Thioredoxin reductase activity was measured as described (24). We incubated RKO cells for 6 h with 0–60 mM curcumin. We washed the cells with phosphate-buffered saline (PBS), lysed cells in reaction buffer through sonication, and washed the lysate by centrifugation through 10 kDa MWCO Amicon filters (Millipore, Billerica, MA) to remove unreacted curcumin, due to its strong absorbance. Samples were incubated for 30 min at 22°C and absorbance was measured at 412 nm.

Immunoprecipitation of p53
We lysed cells and incubated 200 µg of total cell lysate for 16 h at 4°C with 1 µg of either Pab240 or Pab1620, antibodies that specifically recognize p53 in its mutant or wild-type conformation, respectively (25). We visualized the amount of p53 that occurred in each conformation by western blot immunochemistry (23,26).

Phosphorylation of p53
We incubated RKO cells for 6 h with 50 mM etoposide and 0–60 mM curcumin. We lysed cells, fractionated the lysate by SDS–PAGE, transferred proteins to PVDF membranes, and measured the content of p53 phosphorylated at its serine 15 residue by western blot. In some experiments we used ultraviolet (UV) irradiation (50 J/m2) or ionizing radiation (IR: 10 Gy) instead of etoposide to damage DNA and activate p53.

p53–DNA binding
We measured p53–DNA binding activity via electrophoretic mobility shift assay. We isolated nuclei and generated protein–DNA complexes as described (27). We used the wild-type p53 consensus, 5'-AGC TGG ACA TGC CCG GGC ATG TCC-3', double-stranded oligonucleotide and a mutant oligonucleotide, 5'-AGC TGG ATC GCC CCG GGC ATG TCC-3' (Active Motif, Carlsbad, CA), to evaluate sequence specific p53–DNA binding. We incubated 25000 counts per minute (CPM) (2 x 107 CPM/µg) of [32P]oligonucleotide probe with 1 µg of nuclear extract and 2 µg of Ab421 to stabilize p53–DNA binding (28). We fractionated samples on a non-denaturing 6% TBE–polyacrylamide gel. We exposed Kodak XAR film to the gel for 8–24 h at –70°C to detect p53–DNA complexes.

p53-mediated transactivation
We transfected 105 RKO cells/well with 1 µg of p53-Luc and 50 ng of pRL-RSV in 3 µl of FuGene-6. After 48 h, we incubated cells for 6 h with vehicle (DMSO), 0–60 µM of curcumin, plus 50 µM etoposide to initiate DNA damage and a p53 response. We quantified Firefly and control Renilla luciferase activity as described (23,26).

Gene expression analysis by microarray
We used cDNA microarray analysis to monitor the expression of endogenous genes in RKO cells exposed to 50 mM etoposide versus vehicle (DMSO); 60 mM curcumin versus vehicle; or 50 mM etoposide plus 60 mM curcumin versus vehicle for 6 h as described previously (23). Etoposide causes DNA damage and initiates a p53 transcriptional response (29). Therefore, these experimental pairs can show whether curcumin represses genes transcribed by p53. We isolated total RNA using RNeasy mini kits (Qiagen, Valencia, CA) and we amplified 1 µg of RNA using Message Amp kits (Ambion, Austin, TX). In duplicate experiments we alternated the Cy3 or Cy5 fluorescent labeling on each sample (so called double dye-swap design) and we ran two microarrays for each dye-orientation. The microarray slides each contain 9600 elements (‘spots’) corresponding to 8748 independent cDNA clones from the Invitrogen 40k sequence verified human clone set, deposited in duplicate with a Lucidea robotic spotter (Amersham). We hybridized microarray slides with labeled samples, washed and quantified the Cy3 and Cy5 fluorescence at each cDNA ‘spot’ with an Axon scanner (Axon Instruments, Union City, CA) and Imagene software (Biodiscovery, El Segundo, CA). We used GeneSight software (Biodiscovery) to subtract the local background and to flag spots with no signal, high background or distorted shapes. We adjusted low expression values to a minimal raw value of 5 to avoid instability by division with values much less than 1. We obtained the signal intensity ratios for treatment/vehicle and the data were normalized using robust local-linear regression (lowess) for each print-tip. We averaged data from duplicate spots of the same cDNA clone, transformed data to log2 and removed gene elements where the variation across experiments had standard deviations of zero to remove those elements that were frequently blank. The adjusted data were analyzed using Significance Analysis of Microarrays [SAM (30)] and Spotfire software (Spotfire, Somerville, MA) for statistical and hierarchical cluster analysis. Hierarchical clustering was performed using an unweighted pair-group method with arithmetic mean with a Euclidean distance metric.

Quantitative PCR
Total RNA was collected from cells treated with etoposide, UV (50 J/m2) or IR (10 Gy). We DNAse treated our RNA on purification columns (Qiagen) and made cDNA from all samples using Superscript III (Invitrogen). RNA samples were used as PCR controls for amplification with the cDNA. We performed real-time, quantitative PCR using an iQTM Cycler (Bio-Rad) with iQTM SYBER® Green Supermix. Primers used for these experiments are listed in Table I. The number of copies of each amplicon was determined from a standard curve by measuring the threshold cycle (Ct) for each condition. The relative copy numbers were adjusted based on the expression of ß-2 microglobulin as an internal standard for each treatment condition. As the copy numbers of the different genes varied considerably, the effect of curcumin treatment is represented as percent of the stimulation control. Experiments were performed in triplicate.


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Table I. PCR primers for quantitative assessment of p53 regulated genes

 
Cell cycle analysis
Cells were plated at 3 x 104 cells/chamber in LabTech II (Nunc, Rochester, NY) 8-chamber slides and allowed to grow overnight. We incubated RKO cells for 10 h after p53 stimulation by etoposide (50 mM), UV (50 J/m2) or IR (10 Gy) with or without 60 mM curcumin. Cells were fixed in situ using ice cold 70% ethanol for 24 h. Cells were washed 2x with PBS and then stained with propidium iodide (100 µg/ml) and ribonuclease A (200 µg/ml) in PBS for 30 min. After staining, the cells were covered with 90% glycerol in PBS, coverslipped and sealed with nail polish. We collected DNA content data utilizing a Laser Scanning Cytometer (CompuCyte, Cambridge, MA) by scanning areas of ~32 mm2 for each condition that translates to assessing ~10 000 ± 2000 cells. Fluorescent events were contoured using a threshold value of 3500 relative fluorescent units and the propidium iodide signal was integrated and histograms were plotted to generate cell cycle profiles. We analyzed the cell cycle profile data using WinCyte® software. Experiments and analysis were performed in triplicate.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Previously we demonstrated that lipids with {alpha},ß-unsaturated ketones can inactivate wild-type p53 through a mechanism involving attenuation of thioredoxin reductase (26). Exposure of RKO cells, which harbor a wild-type p53 gene, to 0–60 µM curcumin attenuated thioredoxin reductase activity (Figure 1A) and prevented them from accumulating p53 protein in its mature, wild-type conformation, while enhancing their accumulation of p53 protein in a mutant conformation (Figure 1B). The conformation of p53 is a critical determinant of its response to DNA damage. Phosphorylation of p53 at serine residues near its N-terminus, e.g. Ser-15, is an early event in its activation. Tumor suppressor p53 protein that occurs in a mutant conformation is a poor substrate for oligomerization and phosphorylation (32). Accordingly, in RKO cells treated with 0–60 µM curcumin plus 50 µM etoposide, the curcumin inhibited the phosphorylation of p53 at Ser-15 in a concentration-dependent manner (Figure 1C). We obtained similar results when cells were treated with curcumin plus UV irradiation or curcumin plus IR (Figure 1C).



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Fig. 1. Curcumin impairs thioredoxin reductase activity as well as the conformation and serine phosphorylation of p53. (A) RKO cells exposed to curcumin (0–60 µM) demonstrate a dose-dependent inhibition of TrxR activity. At 60 µM, TrxR activity diminished by >75%. (B) RKO cells exposed to DMSO vehicle or curcumin were lysed and incubated with an antibody that selectively immunoprecipitates p53 protein folded into its wild-type conformation. The amount of wild-type p53 protein fell by ~80% in RKO cells treated for 6 h with 60 µM curcumin (IP 1620). Cell lysates were also incubated with an antibody that selectively immunoprecipitates p53 protein folded into a mutant conformation. The amount of conformational mutant p53 protein rose in RKO cells treated with 60 µM curcumin for 1.5–6 h (IP 240). (C) RKO cells were exposed to DMSO vehicle alone, 50 µM etoposide alone or 50 µM etoposide combined with 6, 20 and 60 µM curcumin for 6 h. The amount of phosphorylated p53 (p53-phospho-S15) rose in cells treated with 50 µM etoposide and 0 curcumin, compared with cells treated with DMSO. The amount of p53-phospho-Ser15 fell to an undetectable level in cells treated with 50 µM etoposide plus 60 µM curcumin. Results were similar when cells were irradiated with 50 J/m2 of UV light or 10 Gy of IR. Curcumin inhibited the phosphorylation of p53 on Ser15.

 
Derangement of the conformational integrity, and consequent inhibition of p53 phosphorylation manifests as a loss of DNA binding and impaired p53 transactivation. RKO cells treated with 50 µM etoposide accumulated p53 in an active conformation that binds DNA (Figure 2A, lane 2). Specificity for p53 binding was demonstrated by competitive inhibition with an excess of an oligonucleotide containing a DNA binding sequence specific for p53 (Figure 2A, lane 4), but not by an excess of oligonucleotide lacking a p53 binding sequence (Figure 2A, lane 3). Curcumin inhibited the formation of p53–[32P]oligonucleotide in nuclear lysates from RKO cells treated with etoposide (Figure 2A, lanes 5 and 6). Consistent with its inhibition of p53–DNA binding, curcumin also inhibited the transactivation of a p53-luciferase reported gene in a concentration-dependent manner (Figure 2B).



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Fig. 2. Curcumin inhibits p53:DNA binding and represses transactivation of a p53-dependent reporter gene. (A) An eletrophoretic mobility shift assay demonstrates that etoposide stimulates the ability of p53 to bind DNA (lane 2, indicated by the arrow). The specific nature of this interaction is demonstrated using excess, non-radioactive mutant (mt) oligo competitor or wild-type (wt) oligo competitor (lanes 3 and 4). When curcumin is added, it attenuates the p53–DNA binding in a dose-dependent manner (lanes 5 and 6). NS is a non-specific band found in all lanes. (B) Curcumin attenuates the activation of a Fire Fly p53 luciferase reporter construct. We used a constitutively expressed Renilla luciferase vector co-transfection as a control. The squares demonstrate the dose-dependent attenuation of etoposide-stimulated (50 µM) p53 activity in RKO cells. The circle represents the background p53 activity.

 
Microarray experiments were performed for an unbiased examination of the effects of curcumin on endogenous p53-responsive genes. We compared expression from RKO cells treated with curcumin versus vehicle, etopside versus vehicle and curcumin plus etoposide versus vehicle on cDNA microarrays. The multi-class response function of SAM was used to estimate the false discovery rate for genes that differentiate these experimental conditions. Analysis of RNA expression for 8748 distinct clones found that 186 clones displayed differential gene expression among the three treatment conditions when the mean false discovery rate was minimized (Figure 3A). A visual representation is shown in the hierarchical cluster (Figure 3B). For these genes, the effect of curcumin was indistinguishable from the effect of curcumin plus etoposide. (For a listing of the gene expression changes observed by curcumin alone, please see online Supplementary Material.) This implies that curcumin overrides the effect of etoposide. Notably, PCNA, APO-1, p21WAF1, Bcl-6 and BTG2 were prominent p53-responsive genes (3335) among the endogenously expressed genes that were induced by etoposide treatment and then suppressed when etoposide and curcumin were used in combination. An example of the raw data for APO-1 is displayed in Figure 3C.



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Fig. 3. Curcumin attenuates endogenous p53-mediated transcription: microarray experiments. We performed four microarrays on RKO cells exposed for 6 h to curcumin versus vehicle, etoposide versus vehicle and curcumin + etoposide versus vehicle. (A) SAM plot indicating that 186 genes display differential expression (triangle) when the false discover rate is minimized. (B) Hierarchical clustering of the 186 genes that displayed differential expression are shown and curcumin's attenuation of p53 is observed through the fact that the curcumin samples and the etoposide + curcumin samples are intermixed while the etoposide samples separate into a distinct cluster. Five distinct p53-regulated genes are highlighted in blue. There were two independent clones of APO-1, two independent clones of p21WAF1, two independent clones of PCNA and one clone of each BTG2 and Bcl-6. (C) Raw data from the microarrays is shown for APO-1 curcumin versus vehicle (left) etoposide versus vehicle (middle) and curcumin plus etoposide versus vehicle (right).

 
Quantitative real-time PCR also showed that curcumin inhibited the expression of these five, representative p53 target genes in 14 of 15 cases, involving different genotoxic stimuli (Figure 4). Curcumin inhibited the expression of APO-1, BCl-6, BTG2 and PCNA in RKO cells stimulated with etoposide, (Figure 4, top panel), UV irradiation (middle panel) or IR radiation (bottom, panel). Curcumin also inhibited the expression of p21WAF1 in RKO cells stimulated with UV (middle panel) or IR radiation (bottom panel), but not etoposide (top panel). The reason for the p21WAF1 signal in the etoposide treatment is not clear. We performed additional PCR experiments that yielded essentially identical results. The RNA control samples do not indicate DNA contamination of the etoposide plus curcumin sample. The results from multiple clones on the microarray are consistent with the lack of cell cycle arrest (below). Overall, separate experiments that quantified p53 transactivation with a reporter assay, DNA microarray or PCR all support the conclusion that curcumin inhibits transcription by the tumor suppressor p53.



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Fig. 4. Curcumin attenuates endogenous p53-mediated transcription: quantitative PCR experiments. Real-time quantitative PCR was used to evaluate the expression of five p53 target genes highlighted in Figure 3 (APO-1, BCL-6, BTG2, p21WAF1 and PCNA) in RKO cells exposed to three distinct stimuli, etoposide 50 µM (top), UV 50 J/m2 (middle) and IR 10 Gy (bottom). The expression for each of the five genes, when treated with the genotoxic stress plus curcumin, is plotted relative to the stimulus control. APO-1, BCL-6, BTG2 and PCNA show curcumin-mediated attenuation of expression in the presence of all genotoxic stimuli. p21WAF1 shows curcumin-mediated attenuation of expression when stimulated with UV or IR but not etoposide.

 
One common consequence of DNA damage with p53 transcriptional activation is cell cycle arrest to allow for DNA repair. Curcumin permitted cell cycle progression in the presence of DNA damaging stimuli (Figure 5). A representative profile of untreated control RKO cells is shown in Figure 5A and Table II. Etoposide treatment (Figure 5B and Table II) and UV irradiation (Figure 5C and Table II) stimulated a pronounced G1 arrest, but in combination with curcumin, cell populations continue to cycle (Figure 5 and Table II). Ionizing radiation can result in G1 and G2/M arrests and we primarily see a G2/M arrest in our experiments (Figure 5D and Table II), but in combination with curcumin, the cell population continues cycling through mitosis (Table II).



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Fig. 5. Curcumin releases cells from p53-mediated cell cycle arrest. (A) Untreated control RKO cells demonstrating the laser scanned cell cycle profile of RKO cells. Cells treated with DNA damaging agents demonstrated G1 or G2/M arrests. (B) Etoposide (50 µM) treated cell cycle profile, demonstrating a G1 arrest (in black), and with curcumin, demonstrating cells that continued to cycle (in red). (C) UV (50 J/m2) stimulated cell cycle profile, demonstrating a G1 arrest (in black), and with curcumin, demonstrating cells that continued to cycle (in red). (D) IR (10 Gy) stimulated cell cycle profile, demonstrating primarily a G2/M arrest (in black), and with curcumin, demonstrating cells that continued to cycle (in red).

 

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Table II. Cell cycle analysis

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Our results show that curcumin impairs the folding of p53 into the conformation required for its phosphorylation, its binding to DNA and its transactivation of genes that execute its tumor suppression function. These results are consistent with the observations that curcumin exhibits a modest carcinogenic risk (19) and antagonizes apoptosis induced by p53-dependent chemotherapeutic agents used for breast cancer (22). Thus, curcumin joins a growing list of moderately electrophilic agents that impair tumor suppressor p53 function (23,26,36). It is noteworthy that repression of NF-{kappa}B by curcumin also depends on its electrophilic substitutents and their chemical reactivity (11). Thus, any beneficial effect of curcumin, derived from its repression of NF-{kappa}B (4), may be inseparable from potentially harmful effects derived from its repression of p53. While the genotoxic effects described here still need to be demonstrated in vivo, we suggest that further studies are needed to determine whether curcumin is a carcinogen or a tumor promoter in animal experiments.

The proposed preventive mechanisms of curcumin all require concentrations of 10–100 µM of this agent. This level is achieved in the intestinal tissue of Min mice at efficacious dietary doses (37). Other groups (20,21) have reported comparable levels of curcumin in intestinal tissue. The concentrations of curcumin that repress p53 tranactivation in vitro are within this range. Perkins et al. (37) have estimated that a dose of 1.6 g curcumin/person/day would be sufficient for efficacy in humans. In view of its poor bioavailability it seems probable that the human gastrointestinal tract and liver would be exposed to curcumin concentrations within the range we examined if it were administered at this recommended efficacious dose.

Curcumin's advance toward prevention trials in humans relies on the same type of criteria used for ß-carotene and NSAIDs—the criteria do not assure even a neutral medical outcome. In fact, clinical experience with NSAIDs and ß-carotenes shows that inferences about cancer prevention derived from animal models or high-risk sentinel groups do not necessarily extrapolate in a straightforward manner to the general population. In the case of the NSAIDs, abundant laboratory, clinical and epidemiological data converge and affirm that their regular use reduces the risk of colon cancer (3840). There is a comparably strong rationale for the use of ß-carotene in prevention of tobacco-related cancers: (i) it has preventive effects in animals; (ii) it causes regression of oral pre-cancerous lesions in humans; and (iii) its intake in the diet, or its levels in blood, correlate inversely with the risk of developing tobacco-related cancers (41,42). Remarkably, phase III prevention trials with ß-carotene have reported a significant excess of lung cancer in smokers receiving ß-carotene alone (43) or ß-carotene plus retinyl palmitate (44). This worrisome outcome from the ß-carotene trials was not readily foreseen (4345). Thus, mechanistic studies that can inform the design of any cancer prevention trials seem desirable. We investigated curcumin in this context. In the US, one phase I study is enrolling volunteers to determine the maximum tolerated dose of curcumin for prevention of colon cancer in healthy subjects (7). In the UK (5) and Taiwan (6) two completed phase I studies in cancer patients have prompted the conclusion that larger clinical trials of curcumin are merited. However, there are several reasons, in addition to our results, to proceed cautiously in healthy human subjects. First, tolerance is a function of bioavailability—low systemic bioavailability often correlates with apparently good systemic tolerance (5,6). Secondly, curcumin has no effect on levels of DNA adducts that are used as biomarkers of DNA damage, consistent with either its low bioavailability, its imposition of an oxidative stress or both (5). Thirdly, 10% of the patients treated with curcumin in one of the phase I trials progressed to frank malignancies and yet, the investigators concluded that there was an effect of curcumin in the chemoprevention of cancer (6). Fourthly, a rarely cited carcinogenicity study (19) showed that curcumin causes: (i) hyperplasia of the mucosal epithelium in the cecum and colon of male and female rats; (ii) an increased incidence of hepatocellular adenoma in male and female mice; (iii) a significantly increased incidence of thyroid gland follicular cell hyperplasia in female mice; and (iv) small but significant increases in sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells. Our data, showing that curcumin can repress tumor suppressor p53, raise the possibility that it might have adverse effects in specific circumstances. Somasundaram et al. (22) have suggested that breast cancer patients should avoid curcumin supplementation because it may antagonize their chemotherapy. Interestingly, the MCF-7 cells they used harbor a wild-type p53 gene and the chemotherapeutic agents they used propagate apoptosis, in part, via p53 (22); however, they attribute the detrimental effect of curcumin to a separate mechanism.

Curcumin, an ingredient in the spice turmeric, represents a convergence of dietary and pharmacological tactics for chemoprevention (46). The low incidence of large and small bowel cancer in India is often attributed to natural antioxidants, such as curcumin in the diet (47). However, it is imperative to recall that beneficial effects attributed to diets are seldom reproduced by administration of a single ingredient in that diet—e.g. diets rich in ß-carotene lower the risk of tobacco-related cancers; administration of ß-carotene capsules does not (4345). Likewise, population-based evidence that attributes beneficial effects to diets or ingredients in diets must be sufficiently rigorous to exclude confounding variables and alternate hypotheses. Finally, there are established precedents showing that chemoprevention agents can lower the risk of cancer in one organ system, yet aggravate the risk in another e.g., tamoxifen is preventive in the breast, carcinogenic in the uterus (48).

Any trials using curcumin to prevent colorectal cancer in healthy human subjects should consider all relevant data that reflect on the efficacy and safety of this agent.


    Supplementary material
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Supplementary material can be found at: http://www.carcin.oupjournals.org/.


    Acknowledgments
 
We thank the reviewers for their helpful comments. We thank the Huntsman Cancer Institute Microarray Resource for help with the microarrays. Supported by USPHS Grants R01AI26730 and P01CA073992, the Dee Glenn and Ida W.Smith Chair for Cancer Research and the Huntsman Cancer Institute.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received December 2, 2003; revised March 11, 2004; accepted April 3, 2004.