Research ArticlesPREVENTATIVE MEDICINE

Cancer chemoprevention: Evidence of a nonlinear dose response for the protective effects of resveratrol in humans and mice

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Science Translational Medicine  29 Jul 2015:
Vol. 7, Issue 298, pp. 298ra117
DOI: 10.1126/scitranslmed.aaa7619

Abstract

Resveratrol is widely promoted as a potential cancer chemopreventive agent, but a lack of information on the optimal dose prohibits rationally designed trials to assess efficacy. To challenge the assumption that “more is better,” we compared the pharmacokinetics and activity of a dietary dose with an intake 200 times higher. The dose-response relationship for concentrations generated and the metabolite profile of [14C]-resveratrol in colorectal tissue of cancer patients helped us to define clinically achievable levels. In ApcMin mice (a model of colorectal carcinogenesis) that received a high-fat diet, the low resveratrol dose suppressed intestinal adenoma development more potently than did the higher dose. Efficacy correlated with activation of adenosine monophosphate–activated protein kinase (AMPK) and increased expression of the senescence marker p21. Nonlinear dose responses were observed for AMPK and mechanistic target of rapamycin (mTOR) signaling in mouse adenoma cells, culminating in autophagy and senescence. In human colorectal tissues exposed to low dietary concentrations of resveratrol ex vivo, we measured enhanced AMPK phosphorylation and autophagy. The expression of the cytoprotective NAD(P)H dehydrogenase, quinone 1 (NQO1) enzyme was also increased in tissues from cancer patients participating in our [14C]-resveratrol trial. These findings warrant a revision of developmental strategies for diet-derived agents designed to achieve cancer chemoprevention.

INTRODUCTION

Chemoprevention offers enormous potential for reducing society’s cancer burden. Trials of drugs such as tamoxifen and celecoxib provide proof of principle that the prevention of cancer through pharmaceutical intervention is feasible and cost-effective (13); however, use of these agents in a preventive context is severely hampered by an increased risk of serious side effects (4, 5). Diet-derived compounds, such as resveratrol, with good safety profiles are considered to be attractive alternatives to synthetic drugs. However, despite extensive preclinical data indicating that phytochemicals and micronutrients can protect against cancer, these findings have failed to translate as successful outcomes in randomized controlled trials, and in some cases, cancer incidence increased in the intervention group (6, 7). These unexpected results have been partly attributed to a failure to identify the optimal preventive dose for clinical evaluation before embarking on large costly trials (8, 9). To date, little attention has been paid to this crucial issue, and instead, the classical drug development philosophy—more is better—has been adopted. The situation is further confounded by a lack of appreciation of clinical pharmacokinetics, with the frequent use of concentrations in mechanistic in vitro studies, which often far exceed the levels attainable in human target tissues (10).

A fundamental fact seems to have been overlooked in the development of cancer chemopreventive agents: diet-derived candidates are often identified on the basis of epidemiological findings, indicating that activity is observed with chronic intake of low concentrations of the active ingredient (11, 12). This hypothesis suggests that dietarily achievable concentrations should be a focus of interest, but virtually nothing is known about the pharmacokinetics or activity of such low levels for any of the commonly investigated cancer preventive agents. This study aims to challenge the present developmental paradigm using a model phytochemical, resveratrol, which modulates multiple pathways pertinent to colorectal carcinogenesis (13). Although resveratrol has been widely promoted as an agent worthy of clinical evaluation, current knowledge gaps—specifically, identification of the optimal dose and key molecular targets in humans—prohibit the rational design of trials that assess chemopreventive efficacy. To address these deficiencies, we compared the target-tissue distribution and activity of a low dietarily relevant dose, equivalent to the amount contained in a large glass of certain red wines (14), with an intake 200 times higher, which was previously used in phase 1 clinical trials (15, 16). Our results show that low dietary exposures not only elicit biological changes in mouse and human tissues relevant to colorectal cancer prevention, but they also have superior efficacy compared to high doses, at least when combined with a high-fat diet, and should therefore be included in future preclinical testing strategies.

RESULTS

Comparison of plasma and tissue pharmacokinetics in humans reveals a linear relationship with dose

Resveratrol plasma pharmacokinetics is reasonably well characterized at high doses, but it is unlikely that quantities exceeding 1 g can be taken chronically by healthy populations because of potential gastrointestinal symptoms (17). The standard analytical techniques used previously are not sensitive enough to perform pharmacokinetic profiling of resveratrol or its metabolites generated by doses attainable through the diet. Therefore, we used accelerator mass spectrometry (AMS) (18) in two phase 1 clinical trials to afford new insight into the distribution and metabolism of resveratrol over a clinically relevant range. Such studies necessitate administration of a trace amount [44 kilobecquerel (kBq)] of [14C]-resveratrol, diluted with unlabeled compound to provide a dose of either 5 mg or 1 g. After oral ingestion of a single dose by healthy volunteers, plasma pharmacokinetic parameters for total [14C]-resveratrol equivalents increased in a linear manner, reaching average peak concentrations of 0.6 and 137 μM for intakes of 5 mg and 1 g, respectively (Fig. 1A and table S1). Overall exposure as measured by the average area under the curve (AUC) values also differed by a factor of ~200 (5.2 and 940 μmol/L/hour). At both doses, maximal plasma concentrations were typically observed around the 1-hour time point, with more than half of the volunteers (13 of 20) exhibiting a second minor peak between ~4 and 10 hours. Circulating [14C]-labeled species were still detectable in all 20 subjects as late as 24 hours after resveratrol administration, as shown by plasma C24h values in the range of 0.05 to 0.12 and 12 to 18 μM [14C]-resveratrol equivalents in the 5 mg and 1 g groups, respectively (table S1). Metabolite profiling of two randomly selected volunteers was achieved through coupling offline high-performance liquid chromatography (HPLC) separation with AMS analysis, which enables characterization of the [14C]-labeled species on the basis of chromatographic properties. Both the dietary and pharmacological doses of resveratrol were rapidly metabolized to sulfate and glucuronide conjugates, with only a small fraction of the parent compound remaining at tmax (Fig. 1B and table S1).

Fig. 1. Quantitation of resveratrol and its metabolites in humans after a low dietarily achievable dose or high pharmacological dose.

(A and B) Healthy volunteers received a single [14C]-labeled oral dose of either 5 mg or 1.005 g resveratrol (44.5 kBq, 0.962 μSv), and plasma samples were taken over 24 hours for determination of total [14C]-resveratrol equivalents by AMS analysis. (A) Graphs show average (±SD) concentrations for 10 volunteers per group, whereas the inset represents a single participant to illustrate the second peak maximum commonly observed with resveratrol due to enterohepatic recirculation. (B) Plasma metabolite profiles determined by HPLC-AMS analysis of selected samples from one patient on each resveratrol dose, taken 1 hour after ingestion. Also included are a pre-dose plasma sample for determination of background levels of radiocarbon and an ultraviolet (UV) chromatogram from the analysis of authentic metabolite standards. Peaks designated by * were tentatively assigned on the basis of their chromatographic properties because synthetic standards were not available. (C) Levels of [14C]-resveratrol equivalents in tissues of patients with colorectal cancer that received either 5 mg (n = 8) or 1 g (n = 7) resveratrol daily for 1 week before surgery, with the last dose being [14C]-radiolabeled as described in Materials and Methods. Where possible, malignant tissue and normal colorectal mucosa and muscle were obtained for each patient. For some participants, other tissue types (fat, ovarian tumor) were also available for analysis (table S2). One patient in the high-dose group had surgery delayed by 6 days after taking [14C]-resveratrol and was excluded. Enlargements are included as insets to enable comparisons at lower concentrations. (D) Metabolite profile in colorectal mucosa and muscle tissue of a patient that received 5 mg of [14C]-resveratrol, determined by HPLC-AMS analysis. Peaks of radiocarbon in both tissue types correspond to resveratrol and its 3-sulfate metabolite, on the basis of the similarity of retention times to authentic standards, and the concentrations stated translate to micromolar, assuming that 1 g of tissue equates to 1 ml.

To ascertain whether a dietarily relevant dose of resveratrol can reach its purported target tissue, we compared the distribution in normal colorectal mucosa and its underlying muscle layer as well as in malignant tissue samples obtained from patients that received either 5 mg or 1 g of resveratrol daily for 1 week before surgery. The trial followed a window study design, taking advantage of the period between diagnosis and surgery for administering test agents, with the final dose, taken the evening before surgery, containing the [14C] tracer. Resveratrol species reached the intestinal tissue of all patients even at the dietary dose. As expected, tissue resveratrol levels decreased over time, with the highest concentrations in those participants who experienced the shortest intervals between ingestion and surgery (Fig. 1C and table S2). The [14C]-resveratrol species were still detectable, albeit at low levels (0.11 pmol/mg), in the mucosa of a patient who suffered a 6-day delay of surgery after taking the high-dose [14C] capsule. The highest concentrations were achieved in the tissue excised from the right side of the colon, and there was a tendency (in 13 of 16 patients) for lower levels of resveratrol species in the underlying muscle compared to the surface mucosal layer. Although a large degree of interindividual variability precludes direct comparisons, the difference between mucosa concentrations achieved in each patient group can be explained by the 200-fold dose discrepancy (range, 0.05 to 6.38 and 4.46 to 560 pmol [14C]-resveratrol equivalents/mg tissue for the 5-mg and 1-g dose, respectively). Concentrations attained in malignant tissue were similar to colonic mucosa, ranging from 0.04 to 7.9 pmol resveratrol equivalents/mg tissue in the 5-mg group and 3 to 376 pmol/mg in the 1-g patients. We also detected [14C]-resveratrol species in peritoneal fat, which was obtained from a proportion of patients, as well as in a primary ovarian tumor taken from a participant with secondary colorectal deposits (Fig. 1C and table S2). Metabolite profiling revealed relatively high concentrations of parent resveratrol and its 3-sulfate metabolite in both the mucosa and muscle tissue of a participant on 5 mg daily. This finding parallels our previous observations in patients who received 1 g of resveratrol (19) for 8 days before surgery and supports the gastrointestinal tract, over other internal tissues, as a potential target for resveratrol.

Low-dose resveratrol has superior cancer chemopreventive efficacy

Having demonstrated detectable parent resveratrol in colorectal tissue of patients at both the pharmacological (16, 19) and dietary doses, we examined the ability of these exposures to prevent intestinal adenomas in the ApcMin mouse, a model of hereditary colorectal cancer characterized by a mutation in codon 850 of the adenomatous polyposis coli (Apc) gene. Because resveratrol is known to protect against some age-related pathologies and early mortality associated with a high-fat diet in mice (2022), we compared the effects of resveratrol in animals maintained on a standard diet (SD) or high-fat diet (HFD) from weaning. In two independent experiments involving male and female mice, only the higher resveratrol dose had a significant effect in animals that received an SD, where it caused a small (22%) reduction in adenoma numbers but failed to influence tumor volume.

In contrast, when coadministered with an HFD, the low dose of resveratrol (0.00007% w/w, equating to ~0.07 mg/kg body weight/day) significantly reduced the adenoma number by ~40% and decreased the overall tumor burden by ~52% relative to control animals of both sexes (Fig. 2A). Although the high dose (0.0143% w/w, 14 mg/kg body weight) was also efficacious, it was consistently less potent, reducing the adenoma number by one-third and the burden by 25%. Inhibition of tumor development by resveratrol at both doses was associated with a small (6.5 to 9.3%) but significant reduction in the proportion of proliferating cells in adenomas but not in histologically normal crypts of the small intestine or colon of mice on HFD, as measured by positive Ki-67 staining (Fig. 2B and fig. S1). In contrast, resveratrol had no effect on the extent of apoptosis, as judged by cleaved caspase-3 immunostaining (fig. S1). Consistent with previous observations (22), the higher resveratrol dose was associated with significantly increased body weight in males, but only the low dose correlated with increased body weight in females, and this was specific to animals on an HFD (Fig. 2C and fig. S2). Although control male mice on an HFD had lower body weights than those on an SD, which may indicate that the HFD was less palatable, a similar effect was not apparent in females. The reasons for these observations are currently unclear, but the increase in body weight associated with resveratrol might result from a tendency for increased food consumption or less malabsorption as a consequence of the lower tumor burden in these animals (fig. S3). The finding that low-dose resveratrol was efficacious only in mice on an HFD raises the possibility that a dietarily feasible intake might protect against the tumor-promoting effects of fat without reducing body weight (Fig. 2C and fig. S2). Indeed, comparison of tumor development in ApcMin mice on a control SD or an HFD culled at the same time point (14 weeks of age) revealed a strong procarcinogenic effect of fat in this model (Fig. 2D).

Fig. 2. Low-dose resveratrol inhibits adenoma development in ApcMin mice on HFD more potently than a dose 200-fold higher.

After weaning (4 weeks of age), male and female mice were maintained on SD or HFD supplemented with resveratrol (0.00007 or 0.0143%). Unless stated otherwise, mice on the SD (16% of calories from fat) were culled at 17 weeks, whereas those on the HFD (60% of calories from fat) had to be sacrificed at 14 weeks because of the tumor-promoting effects of the diet. (A) Comparison of the number of adenomas per mouse and total adenoma volume in the small intestine of each animal. Data represent the mean ± SEM of 14 to 16 female plus 17 to 19 male mice per group. Data were modeled using regression analysis (of log-transformed data for volume), and significant treatment-related differences relative to the corresponding control diet group are shown. (B) Box plot showing the effect of resveratrol on the proliferative index in intestinal adenomas of ApcMin mice on HFD, as measured by immunohistochemical staining for nuclear Ki-67. Data represent the median percentage (plus 25th and 75th percentile) of Ki-67–positive cells per field, where six different visual fields were scored for each mouse (n = 6 males and 5 females per group). Whiskers indicate the maximum and minimum values. (C) Body weight of male ApcMin mice on SD or HFD alone or containing resveratrol. All body weight data represent the mean ± SEM of 15 to 19 mice per group and were analyzed by mixed effects linear regression. High-dose resveratrol significantly increased the body weight of mice on an SD (P < 0.05) or an HFD (P < 0.001) compared to corresponding controls; low-dose resveratrol increased the body weight of animals on an HFD only (P = 0.05). Control mice on the HFD had significantly lower body weights than those on the SD (P < 0.001). (D) Effect of a control HFD on intestinal adenoma number and total volume compared to ApcMin mice on an SD. Animals in both groups (seven to nine females plus seven to eight males) were culled at 14 weeks of age, and data illustrate the mean ± SEM.

Chemopreventive efficacy correlates with enhanced adenosine monophosphate–activated protein kinase signaling

In female mice, efficacy was highly correlated with the expression and activation of the energy regulator adenosine monophosphate–activated protein kinase (AMPK) in intestinal mucosa. Neither the AMPKα protein nor its phosphorylated form was detectable in any mice in the SD or HFD control group, but both were evident in animals that ingested 0.00007% resveratrol and, to a lesser extent, those in the high-dose group (Fig. 3, A and C). This phenomenon was closely mirrored by an increased expression and phosphorylation of the AMPK target acetyl–coenzyme A carboxylase (ACC) (Fig. 3, B and C). In contrast, a random pattern of AMPK and pAMPK expression was observed in the mucosa of the male mice (fig. S4), probably as a result of overnight starvation before culling, which was not performed in the females. Starvation was necessary to enable the measurement of metabolic parameters in the fasting state, but given the results obtained in males, this was not pursued further in the study with female mice; none of the metabolic parameters measured in males were altered by the resveratrol intervention, apart from intestinal insulin-like growth factor 1 (IGF1) which was decreased in mice given the high-dose resveratrol treatment relative to that of the controls, regardless of the fat content of the diet (P = 0.05 and 0.04 for animals on SD and HFD, respectively; fig. S5).

Fig. 3. Low-dose dietary resveratrol activates AMPK and causes senescence in intestinal mucosa of mice on an HFD.

(A to C) Expression and phosphorylation of AMPK and its downstream target ACC, together with levels of autophagy and senescence markers in tissue of female ApcMin mice maintained on an SD or HFD, with or without resveratrol. The positive control sample is Apc10.1 cells exposed to 1 μM resveratrol. Mice were culled at 17 or 14 weeks of age for the standard and high-fat groups, respectively. (C) Data represent the mean ± SEM of six mice per group on HFD with or without resveratrol. (D and E) Kinetics of AMPK activation and downstream effects in intestinal tissue of C57BL/6J wild-type male mice maintained on an HFD, which received a single gavage dose of resveratrol (2.1 μg per mouse; R) or vehicle control (C). Mice were culled after dosing at the indicated time. (D) Representative immunoblots are shown for three mice per group. (E) Data represent the mean ± SEM of four to six mice per group.

Furthermore, determination of the kinetics of AMPK activation in wild-type B57BL/6J male mice preconditioned on an HFD before receiving a single oral dose of resveratrol (2.1 μg) revealed an extremely rapid response, with increased expression and phosphorylation detected in normal intestinal mucosa just 30 min after administration (Fig. 3, D and E). This low dose was equivalent to the total estimated amount ingested over the course of a day by animals receiving 0.00007% in their diet. AMPK activation persisted for only 2 hours before declining. This observation might explain the considerable variability of pAMPK levels in resveratrol-treated mice on an HFD because tissue concentrations might be dependent on when the mouse last ate. This finding also demonstrates that the ability of dietarily relevant doses of resveratrol to induce AMPK expression and phosphorylation in vivo is not gender-specific. The mechanisms through which resveratrol specifically enhances the expression of AMPK in the mucosa of mice on an HFD remain unclear.

One of the end products of AMPK activation is autophagy, a catabolic pathway required for the quality control of proteins and organelles and the maintenance of energy homeostasis, which can also serve as a tumor-suppressing mechanism (23). We detected significantly enhanced levels of soluble microtubule-associated protein 1 light chain 3 (LC3-I) in gut mucosal tissue of resveratrol-treated mice, along with increased conversion to the lipid bound LC3-II, which is a constituent of autophagosomal membranes and a marker of autophagy initiation (Fig. 3, D and E). Up-regulated autophagy appears to be a rapid but potentially short-term response to resveratrol, because it was observed in animals that received a single dose but not in the chronically treated ApcMin mice (Fig. 3, B, D, and E). Conversely, p21 expression, a marker of senescence, was increased in ApcMin mice that ingested resveratrol with an HFD, whereas a single dose was insufficient to elevate p21 protein levels over the time frame monitored (Fig. 3, B to E). Autophagy can facilitate establishment of the senescent phenotype (24), and these data imply that autophagy precedes senescence in resveratrol-treated mice.

Apc10.1 cells derived from adenomas of ApcMin mice (25) were used to further delineate the consequences of AMPK activation using a concentration range encompassing that detected in human colorectal tissue and plasma after both resveratrol doses. As the likely target in clinical chemoprevention, these cancer precursors provide a more relevant model than malignant cancer cells for assessing activity. The antitumor effects of resveratrol observed in vivo were recapitulated in Apc10.1 cells, which displayed increased autophagy, measured as Cyto-ID Green–stained autophagic vacuoles, and elevated senescence, detected by β-galactosidase staining and p21 expression (Fig. 4, A to D). After 6 days of repeated exposure to resveratrol, in which the medium was replaced every 24 hours, the expression of AMPK remained stable, but significant concentration-dependent activation was evident from 0.01 μM resveratrol and reached a maximum at 1 μM (Fig. 4, A and B). At 10 μM, AMPK phosphorylation returned to basal levels. This bell-shaped dose response was also apparent for ACC phosphorylation, which was greatest at 1 μM, reflecting the in vivo findings that lower exposures are more effective at modulating AMPK signaling. Downstream targets of AMPK were altered in a similar manner (Fig. 4, A and B); 1 μM resveratrol caused the greatest reduction in phosphorylation of the mechanistic target of rapamycin (mTOR) and its downstream effectors 4EBP1 and p70S6K, which are involved in protein translation. These effects were independent of Akt, another mTOR regulator, because resveratrol had no effect on Akt expression or activation (fig. S6).

Fig. 4. Low, dietarily achievable concentrations of resveratrol activate AMPK signaling and cause autophagy and senescence in Apc10.1 mouse adenoma cells.

(A and B) Six days of repeated exposure to resveratrol enhances AMPK phosphorylation, inhibits mTOR signaling, and increases markers of autophagy and senescence. Representative immunoblots are shown (A). (C) Detection of Cyto-ID Green–stained autophagic vacuoles visualized by fluorescence microscopy of live cells treated repeatedly with resveratrol for 6 days. Hoechst 33342–stained nuclei are in blue, and rapamycin (500 nM) was used as a positive control. (D) Proportion of senescence-associated β-galactosidase–positive stained cells after 6 days of repeated resveratrol treatment. (E) Kinetics of AMPK activation after exposure to resveratrol for 2 hours, replacement of the medium, and further incubation without resveratrol for the times indicated. (F) AMP/ATP ratio determined by HPLC analysis. Cells were treated with resveratrol for 2 hours, medium was replaced, and incubation was continued for 4 hours. (G) Levels of intracellular reactive oxygen species visualized using an Image-iT LIVE green ROS detection kit, 1 hour after the addition of resveratrol. Nuclei were counterstained blue with Hoechst 33342, and tert-butyl hydroperoxide (100 μM) was used as a positive control. (H) Effect of NAC on resveratrol-induced AMPK activation measured after 6 hours co-incubation. All graphs illustrate the mean ± SEM of three independent experiments. Significant differences relative to control incubations are indicated: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0005, using Student’s t test.

Several different routes to AMPK activation have been demonstrated for resveratrol. However, the key studies have been performed with concentrations that exceed levels achievable in human plasma (15, 2628). Given the reported dose dependency of the mechanisms engaged (26), we sought to identify the processes involved at lower but clinically relevant concentrations. Short-term (2-hour) exposure of Apc10.1 cells to resveratrol followed by its removal (to mimic metabolic clearance in the mouse intestine) caused a transient increase in pAMPK after 4 hours, which correlated with a significant increase in the adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio, consistent with ATP synthase inhibition (Fig. 4, E and F). Co-incubation with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N′-tetraacetic acid (BAPTA) or the Ca2+/calmodulin-dependent protein kinase kinase β (CamKKβ) inhibitor STO-609 had no effect on resveratrol-induced AMPK phosphorylation (fig. S6), suggesting that inhibition of phosphodiesterase (26) does not play a role in this system. Low concentrations of resveratrol induced a detectable increase in reactive oxygen species (ROS) within 1 hour of resveratrol addition (Fig. 4G). Therefore, we investigated whether increased oxidative stress may contribute to AMPK activation, as suggested for other activators (29), including 2-deoxy-d-glucose (30). Co-incubation with the antioxidant N-acetylcysteine (NAC) (31) significantly (P < 0.05, Student’s t test) blunted resveratrol-induced phosphorylation of AMPK, identifying a role for ROS at clinically achievable concentrations (Fig. 4H).

Low-dose resveratrol exerts activity consistent with cancer chemoprevention in human colorectal tissue

Analysis of AMPK signaling in colorectal tissue of the patients who received [14C]-resveratrol revealed random expression patterns, with no apparent difference between treated and control patients (fig. S7). As with the male ApcMin mice, this lack of effect might be attributable to the fact that all patients were fasted overnight before surgery and would have been without food for differing lengths of time. To overcome this issue, we performed explant cultures of human colorectal tumors that had been isolated from three individual patients and passaged in immunocompromised nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice. The tissue response to resveratrol exposure mimicked that observed in Apc10.1 cells, with rapid AMPK activation and increased autophagy at low concentrations (0.01 to 0.1 μM) and a less pronounced or no effect at higher exposures (Fig. 5, A and B).

Fig. 5. Low concentrations of resveratrol activate AMPK and increase markers of oxidative stress in human colorectal tissues.

(A and B) Exposure to resveratrol (2 hours) increases pAMPK levels and up-regulates autophagy in primary colorectal cancer explants, as assessed by Western blotting (A) and/or immunohistochemical staining (B) in samples from three different patients. (C to E) Expression of NQO1 (C and D) and levels of protein carbonylation (E) in colorectal mucosa tissue of patients participating in the [14C]-resveratrol trial who received a dose of either 5 mg or 1 g daily for 1 week before surgery, or untreated control patients. Samples were analyzed blind, and significant differences between the control and treated groups are indicated. (C) A typical Western blot for NQO1. (D and E) Data represent the mean ± SEM of six to eight and four to six patients per group, respectively; statistical analysis was performed using Student’s t test.

Further support for the existence of a nonconventional dose response in humans was provided by the observation that both NQO1 expression and protein carbonyl concentration were significantly increased in the colorectal mucosa of patients that received 5 mg of [14C]-resveratrol compared to those taking the 1-g dose and the control group (Fig. 5, C to E). NQO1 is a cytoprotective enzyme regulated by the transcription factor Nrf-2, which is activated by oxidative stress (32), whereas quantitation of carbonyl groups provides a stable measure of amino acid oxidation (33). Together, this study suggests that lower doses of resveratrol may be more active than higher supradietary intakes in humans and that the beneficial effects of resveratrol at such doses may be mediated by its prooxidant activity and up-regulation of AMPK signaling.

DISCUSSION

The ability of low, dietarily feasible resveratrol doses to selectively prevent intestinal tumor development in ApcMin mice fed an HFD highlights several pertinent points for advancing the field of cancer chemoprevention. To date, there has been little consensus on how to determine appropriate doses of phytochemicals, vitamins, and micronutrients for translation to clinical chemoprevention studies (9). Supradietary doses have frequently been administered, even when epidemiology data suggest that dietarily achievable intakes offer protection. However, with the recent results from clinical trials such as SELECT, which involves selenium and the antioxidant vitamin E, it is gradually being recognized that complex dose-response relationships exist for diet-derived agents (34, 35). Furthermore, the lack of effect or even harm seen in trials with high-dose antioxidant supplements (7) is consistent with the idea that low levels of ROS can trigger cellular defense mechanisms and are actually protective, which is suggestive of a nonlinear dose response or hormesis (36). It has been proposed by some investigators involved in the β-carotene and retinol efficacy trial that part of the reason an increase in lung cancer was observed in smokers, rather than the anticipated reduction, was that an inappropriately high dose of β-carotene was given (7, 8). In line with these emerging concepts, our observations in multiple models of murine and human colorectal cancer provide the first direct evidence that a low-dose intake of resveratrol has greater anticancer efficacy than high doses.

Translating resveratrol doses between species is challenging but ideally should take into account a comparison of the concentrations generated systemically and in the target tissue(s), where relevant. Here, dose conversion from human to mouse was performed on the basis of body weight; if body surface area had been factored in, then the doses administered to mice would actually correspond to even lower human equivalent intakes of 0.4 and 81 mg of resveratrol per day for a 70-kg person (37). The requirement for an HFD to reveal the efficacy of resveratrol explains why a dose that was ~14-fold higher than the maximum used in mice here was necessary to produce a significant, albeit moderate (30%), reduction in adenomas in analogous studies involving ApcMin mice on an SD, whereas lower exposures were ineffectual (38, 39). This interaction with fat is in agreement with results emerging from clinical trials, in which resveratrol, at doses as low as 10 mg daily, appears to have selective activity in obese humans (40) or those with metabolic disorders, such as type 2 diabetes (41, 42). Notable is the study by Timmers et al. in which resveratrol (150 mg/day) was found to mimic the effects of calorie restriction in obese men by lowering energy expenditure and improving the metabolic profile and general health of participants (40); in contrast, a comparable dose (75 mg) had no effect in nonobese women with normal glucose tolerance (43). A large daily dose 10-fold higher than that used by Timmers et al. failed to alter any metabolic parameters, even though the study followed a similar design and also involved obese men (40, 44). These reports, together with our observation that only the low 5-mg daily dose of resveratrol increased biomarkers of oxidative stress in colorectal tissue of treated patients and the fact that maximal AMPK activation and autophagy were achieved in human explants at submicromolar concentrations, lend further credence to the reality of a nonlinear dose response in humans. Our in vivo results also emphasize a role for lifestyle and physiological factors in influencing an individual’s response to intervention, which indicates the potential importance of personalizing chemopreventive therapy.

Long-term maintenance of C57BL/6J mice, the background strain of ApcMin mice, on an HFD is a commonly used model of impaired glucose intolerance and early type 2 diabetes (45), which is a well-established risk factor for colorectal cancer in humans (46, 47). Consumption of an HFD has previously been demonstrated to cause metabolic changes in ApcMin mice, with the degree of dysregulation increasing over time (48). Here, however, there was no evidence of the diabetic phenotype in mice fed an HFD, although it is difficult to draw direct comparisons because those on HFD were culled 3 weeks earlier than animals on an SD. Resveratrol appears to counteract the tumor-promoting effects of an HFD without modulating the fasting levels of plasma biomarkers previously associated with health and survival benefits in middle-aged mice (20). Thus, at the low doses used, it is likely that localized, rather than systemic, effects in colorectal cells are responsible for the chemopreventive efficacy of resveratrol, particularly because the phenotype observed in ApcMin mice was replicated in vitro.

Although widely perceived as an antioxidant, our findings suggest that the transient prooxidant activity of resveratrol is responsible, at least in part, for the activation of AMPK at very low concentrations. Whether the increased ROS are generated through inhibition of ATP synthase (49), which may also contribute to AMPK activation in adenoma cells via an increase in the AMP/ATP ratio, remains to be determined. In considering the potential mechanisms responsible for AMPK activation in this system, it should be noted that although resveratrol has been shown, using x-ray crystallography, to be capable of binding to F1-ATPase, evidence for a direct inhibitory effect is limited to studies with subcellular fractions and requires relatively high concentrations of resveratrol (10 to 30 μM) (49, 50). Other known AMPK activators including metformin and aspirin have been shown to protect against adenoma development in ApcMin mice (51, 52). Studies with aspirin, which is rapidly hydrolyzed in vivo to salicylate, an allosteric activator of AMPK, have yielded conflicting results, but aspirin appears to require lifetime administration from the point of conception to significantly suppress intestinal tumorigenesis (51, 53). Inhibitors of mTOR signaling such as rapamycin are also efficacious in this model (54), and although it is recognized that the anticancer effects of all these compounds are likely to involve multiple modes of action, our observations reinforce the potential of targeting metabolic pathways for cancer prevention.

Countless in vitro studies have described the proapoptotic effects of resveratrol in cancer cell lines (13, 55); however, these studies necessitated the use of concentrations beyond those systemically achievable in humans (15). We found no evidence within the intestines of resveratrol-treated ApcMin mice of increased apoptosis, which would most likely have been a consequence of toxicity at high concentrations; instead, the anticancer effects of resveratrol seem to be mediated through the induction of autophagy and senescence. Autophagy is a short-term response, and senescence is the result of sustained exposure to low resveratrol concentrations. Both processes have paradoxical roles in carcinogenesis (23, 56, 57) but appear to serve as tumor-suppressing mechanisms in ApcMin mice on an HFD. It is therefore encouraging that the increased ROS levels and elevated autophagy also translated to human colorectal tissue that had been exposed to concentrations of resveratrol generated in the gastrointestinal tract of patients after a 5-mg dose (~0.01 to 0.2 μM, Fig. 1D).

In summary, we provide compelling evidence of a bell-shaped dose response for resveratrol, with low doses having greater efficacy than high doses. We demonstrated that dietarily achievable doses of resveratrol halt tumor progression in mice; this correlates with the induction of AMPK and senescence, and these effects translate to human tissue. Our findings of the bell-shaped dose response and the evidence that the tumor-preventive efficacy of resveratrol depends on diet, and possibly behaviors causing obesity and metabolic syndrome, warrant a rethinking of strategies aimed at implementing resveratrol, and potentially other diet-derived agents, for cancer chemoprevention and other therapeutic indications.

MATERIALS AND METHODS

Detailed procedures are provided in Supplementary Materials.

Study design

Two phase 1 trials were conducted that aimed to compare the pharmacokinetics, tissue distribution, and metabolism of resveratrol at a dietarily achievable and pharmacological dose in healthy volunteers and patients with colorectal cancer awaiting surgical resection.

Clinical trials

The trials were approved by the Liverpool UK Research Ethics Committee, the UK Medicines and Healthcare products Regulatory Agency, the Administration of Radioactive Substances Advisory Committee, and the Institutional Review Board at Lawrence Livermore National Laboratory (LNLL). Both trials were conducted at the University Hospitals of Leicester National Health Service (NHS) Trust.

Volunteer study

Twenty healthy volunteers were fasted overnight, and a baseline control blood sample was taken before they received a single dose of either 5 mg or 1.005 g [14C]-resveratrol (4 × 250 mg unlabeled, plus 5 mg [14C]-labeled resveratrol). Blood samples were then taken within 24 hours.

Colorectal cancer patient trial

Patients (10 per group) with resectable colorectal cancer took daily unlabeled resveratrol capsules (5 mg or 1.0 g) for 6 days and then received a final [14C]-resveratrol dose before surgery, which consisted of either 5 mg or 1.005 g [14C]-resveratrol, as mentioned above. A control group without any intervention was also included. At surgical resection, both normal and malignant tissues were sampled.

Processing of all tissue, blood, and HPLC fractions for AMS analysis was performed in a designated laboratory, free from extraneous 14C contamination, and samples were measured at the LNLL (58, 59). Pharmacokinetic parameters were modeled using WinNonlin version 5.3 software (Pharsight Corporation).

Analysis of protein carbonyls and NQO1 in human colorectal tissue

Colorectal mucosa samples from cancer patients participating in the [14C]-resveratrol trial and tissue from control untreated patients were analyzed for markers of oxidative stress. All samples were coded and randomized, and the analyses were performed blind. NQO1 expression was measured by Western blotting, and the OxiSelect spectrophotometric assay (Cell Biolabs Inc.) was used for quantifying protein carbonyls.

In vivo mouse studies

The animal experiments were performed under project licenses PPL40/2496 and 60/4370, granted to the University of Leicester by the UK Home Office. The experimental design was vetted by the University of Leicester Local Ethical Committee for Animal Experimentation and met the standards required by the UK Coordinating Committee on Cancer Research guidelines (60). Resveratrol efficacy was assessed in male and female ApcMin mice (61). After weaning, animals were randomized to one of six different diets consisting of standard or high-fat AIN-93G diet containing either 0.00007 or 0.0143% resveratrol, or the corresponding control diet. Mice on the SD were sacrificed at 17 weeks of age, and those on the HFD at 14 weeks. Only the male mice were fasted overnight before culling. The multiplicity, location, and size of intestinal adenomas were recorded (62). The kinetics of AMPK activation in intestinal mucosa tissue was examined in wild-type C57BL/6J male mice on HFD that received a single dose of resveratrol (2.1 μg) by gavage or vehicle (control) alone.

Tumor passage

Colorectal tumors were obtained from patients at the University Hospitals of Leicester NHS Trust as part of an excess tissue study (approval granted by Leicestershire, Northamptonshire, and Rutland ethics committees; Research Ethics Committee reference 09/H0402/45). Tumors were passaged in mice to provide tissue for ex vivo explant cultures. Adult male NOD/SCID (NOD/SCID NOD.CB17/JHliHsd-Prkdcscid) mice were fed on normal irradiated diet (5LF-5) for maintenance. Sections of tumors from surgical resections (~2 mm thick) were washed in media 199 containing 2% antibiotic-antimycotic (Invitrogen) before implantation. The tumor tissue was inserted into the right and/or left flank of a mouse. The mice were sacrificed and the tumor was excised before reaching the size limit designated in the animal project license. The tissue was then used immediately for explant cultures.

Immunohistochemistry

Immunohistochemistry was performed using the Novolink Polymer Detection Systems (Leica Biosystems) according to the manufacturer’s instructions. For formalin-fixed mouse tissue, the Ki-67 (ab15580, Abcam) and caspase-3 (9661, Cell Signaling Technology) antibodies were used at 1:1000 and 1:200 dilutions, respectively. For the analysis of human explant tissues, the phosphorylated AMPK antibody (2535, Cell Signaling Technology) was used at a 1:100 dilution.

Cell culture

Unless stated otherwise, Apc10.1 cells were typically treated daily with resveratrol (0.001 to 1 μM) for 6 days to mimic repeated dosing in humans and were harvested 4 hours after the last treatment. We found no evidence of resveratrol accumulation within cells using this repeat-dosing protocol. Primary antibodies were all supplied by Cell Signaling Technology, apart from those for p21 and β-actin (Santa Cruz Biotechnology). Senescence was assessed using a senescence β-galactosidase staining kit (Cell Signaling Technology), autophagy was measured using the Cyto-ID autophagy detection kit (Enzo Life Sciences), and levels of intracellular ROS were visualized using an Image-iT LIVE green ROS detection kit (Invitrogen). Analysis of AMP, adenosine diphosphate (ADP), and ATP content in cells was performed by UV-HPLC (63).

Explant cultures

Explant cultures were performed with primary colorectal cancer samples originating from three different patients after passage in mice. Tumor tissues were freshly excised from NOD/SCID mice and placed in Dulbecco’s modified Eagle’s medium (low-glucose) supplemented with 1% fetal calf serum and 2% antibiotic-antimycotic. For each concentration of resveratrol and solvent control, nine pieces of tumor (~2 mm3 each) were placed in an insert, within a single well of a six-well plate. The explants were incubated overnight (37°C, 5% CO2) and then fresh medium, supplemented with either resveratrol or vehicle control (dimethyl sulfoxide), was added. After 2 hours, the tissues were harvested for either immunohistochemistry or Western blotting (nine pieces of tissue combined).

Statistical analysis

The effect of resveratrol dose on the expression of NQO1 protein in human colorectal tissue was analyzed using Student’s t test and Mann-Whitney test. The effect of resveratrol treatment on adenoma volume and number in ApcMin mice was modeled using regression analysis (of log-transformed data for volume). Mouse body weight data over time were analyzed using mixed effects linear regression. The immunohistochemistry and in vitro data were analyzed using a Student’s t test.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/298/298ra117/DC1

Experimental procedures

Fig. S1. Effect of resveratrol on cell proliferation and apoptosis in tissues of ApcMin mice.

Fig. S2. Effect of resveratrol and HFD on the body weight of female ApcMin mice.

Fig. S3. Estimated weekly diet consumption by male and female ApcMin mice.

Fig. S4. Effect of resveratrol on the expression of AMPK in the intestinal mucosa of ApcMin mice.

Fig. S5. Effect of resveratrol on metabolic parameters in plasma and intestinal mucosa of fasted male ApcMin mice.

Fig. S6. Effects of resveratrol in Apc10.1 mouse adenoma cells.

Fig. S7. Random expression and phosphorylation of AMPK in colorectal surgical tissue obtained from patients participating in the [14C]-resveratrol trial.

Table S1. Plasma pharmacokinetic parameters for total [14C]-resveratrol equivalents in healthy volunteers that ingested a single dose of either 5 mg or 1 g [14C]-resveratrol.

Table S2. Tissue concentrations of [14C]-resveratrol species in patients who received resveratrol daily.

REFERENCES AND NOTES

  1. Acknowledgments: We thank C. De Giovanni for the Apc.10.1 cells; D. Monk and M. Dunn (Medical Physics, University Hospitals of Leicester) for help with the clinical trials. Funding: This work was supported by Cancer Research UK (C325/A13101) with assistance from the Leicester Experimental Cancer Medicine Centre (C325/A15575, Cancer Research UK/UK Department of Health). A. Kholghi was funded by a studentship from the Libyan government through Benghazi University. AMS analysis was performed at the Research Resource for Biomedical AMS Laboratory, operated at LLNL, and supported by the NIH National Centre for Research Resources, Biomedical Technology Program grant #P41RR13461. Author contributions: H.C., E.S., C.A., A.R., A. Kholghi, A. Karmokar, L.H., and K.B. designed all laboratory experiments; H.C., E.S., and A. Karmokar performed in vivo studies; A. Kholghi performed all in vitro experiments with Apc10.1 cells and analysis of mouse tissues; R.G.B. synthesized resveratrol metabolites; C.A. performed the in vitro senescence and autophagy experiments; A. Karmokar, M.J., D.J., and E.H.-G. developed the methodology and performed experiments with explant cultures; H.C. conducted the HPLC analysis; E.S. prepared the clinical samples; T.O. and M.M. conducted the AMS analysis; E.S, H.C., A. Kholghi, E.H.-G., C.A., A.R., L.H., and K.B. analyzed the data; E.S., W.P.S., A.G., and K.B. designed and/or conducted clinical trials, whereas surgical expertise was provided by A.M. and D.H.; J.W., C.G., and N.K. analyzed the tissues and interpreted NQO1 data; M.V. provided statistical input; K.W. and P.G. provided pathology support and interpretation; K.B., A.G., and W.P.S. provided funding; K.B. wrote the paper. Competing interests: The authors declare that they have no competing interests.
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