Less is more for cancer chemoprevention: evidence of a non-linear dose response for the protective effects of resveratrol in humans and mice

Resveratrol is widely promoted as a potential cancer chemopreventive agent, but a lack of information on the optimal dose prohibits rationally designed trials assessing 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 and metabolite profile of [C]-resveratrol in colorectal tissue of patients helped define clinically achievable concentrations. In Apc mice receiving a high-fat diet the low dose suppressed intestinal adenoma development more potently than the higher dose. Efficacy correlated with increased AMP-activated protein kinase (AMPK) activation and the senescence marker p21. Non-linear dose responses were observed for AMPK and mTOR signalling in adenoma cells, culminating in autophagy and senescence. In human tissues low dietary exposures caused enhanced AMPK phosphorylation, autophagy and expression of the cytoprotective enzyme NQO1. These findings warrant revision of developmental strategies for diet-derived agents for cancer chemoprevention. Introduction Chemoprevention offers enormous potential for reducing the burden of cancer in society. 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 (1-3); however, use of these agents in this context is severely hampered by an increased risk of serious side effects (4,5). Diet-derived compounds are considered an attractive alternative to synthetic drugs for prevention of malignancies in healthy populations, with those that are consumed regularly by humans likely to have a good safety profile. However, despite extensive preclinical data indicating that phytochemicals and micronutrients can protect against cancer, these findings have failed to translate into successful outcomes in randomised controlled trials, and in some cases cancer incidence has actually 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 classic drug development philosophy has been adopted, that in terms of dosage, more is better. The situation is further confounded by a lack of appreciation of clinical pharmacokinetics, with the frequent use of concentrations in mechanistic in vitro studies that 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, in that diet-derived candidates are often identified on the basis of epidemiological observations indicating activity at low, chronic intake (11,12). This would suggest that dietary 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 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 assessing chemopreventive efficacy. To address these deficiencies we compared the target tissue distribution and activity of a low dietary relevant dose, equivalent to the amount contained in a large glass of certain red wines (14) with an intake 200-times higher that has previously been used in phase I 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 chemoprevention, but they 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 Comparative plasma and tissue pharmacokinetics in humans Resveratrol plasma pharmacokinetics are reasonably well characterised at high doses, but it is unlikely that quantities exceeding 1g can be taken chronically by healthy populations due to potential gastrointestinal symptoms (17). The standard analytical techniques previously utilised are not sensitive enough to perform pharmacokinetic profiling of resveratrol or its metabolites generated by doses attainable through the diet. Therefore, we employed accelerator mass spectrometry (18) in two 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 kBq) of [C]-resveratrol, diluted with unlabelled compound to provide a dose of either 5mg or 1g. Following oral ingestion of a single dose by healthy volunteers plasma pharmacokinetic parameters for total [C]-resveratrol equivalents increased in a linear manner, reaching average peak concentrations of 0.6 and 137 μmol/L for intakes of 5mg and 1g, respectively (Fig. 1A, Table S1). Overall exposure as measured by the average area under the curve values (AUC) also differed by a factor of ~200 (5.2 and 940 μmol/L/h). At both doses, maximal plasma concentrations were typically observed around the 1h time point, with over half the volunteers (13/20) also exhibiting a second minor peak between ~4-10h. Importantly, circulating [C]-labelled species were still detectable in all twenty subjects as late as 24h after resveratrol administration, as shown by C24h values in the range 0.05-0.12 and 12-18 μmol/L [C]-resveratrol equivalents in the 5mg and 1g groups, respectively (Table S1). Metabolite profiling of two randomly selected volunteers was achieved through coupling off-line HPLC separation with AMS analysis, which enables characterisation of the [C]-labelled species based on chromatographic properties. Accordingly, both the dietary and pharmacological doses of resveratrol were found to be rapidly metabolised to sulfate and glucuronide conjugates with only a small fraction of parent compound remaining at tmax (Fig. 1B and Table S1). To ascertain whether a dietary-relevant dose of resveratrol can reach its purported target tissue we compared the distribution in normal colorectal mucosa and underlying muscle layer as well as malignant tissue samples obtained from patients that received either 5mg or 1g resveratrol daily for one week. 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 [C]-tracer. Resveratrol species reached the intestinal tissue of all patients, even at the dietary dose. As might be expected, levels decreased over time, with highest concentrations in those participants that experienced the shortest intervals between ingestion and surgery (Fig. 1C, Table S2). Surprisingly, [C]-resveratrol species were still detectable, albeit at low levels (0.11 pmol/mg), in the mucosa of a patient that suffered a six day delay of surgery, after taking the high dose [C]-capsule. Greatest concentrations were achieved in tissue excised from the right-side of the colon, and there was a tendency (in 13/16 patients) for lower levels in the muscle compared to the surface mucosal layer. Although the large degree of inter-individual variability precludes direct comparisons, the difference between mucosa concentrations achieved in each patient group can be explained by the 200-fold dose discrepancy (range 0.05-6.38 and 4.46-560 pmol [C]-resveratrol equivalents/mg tissue, for the 5mg and 1g dose, respectively). Concentrations attained in malignant tissue were similar to colonic mucosa, ranging from 0.04-7.9 pmol resveratrol equivalents/mg tissue in the 5mg group and 3-376 pmol/mg in the 1g patients. We also detected [C]-resveratrol species in peritoneal fat, which was obtained from a proportion of patients, as well as a primary ovarian tumour taken from a participant with secondary colorectal deposits (Fig. 1C and Table S2). Importantly, metabolite profiling revealed relatively high concentrations of parent resveratrol and its 3-sulfate, in both the mucosa and muscle tissue of a participant on 5mg daily. This finding parallels our previous observations in patients receiving 1g resveratrol (19) and supports the gastrointestinal tract, over other internal tissues, as a potential target for resveratrol. Superior cancer chemopreventive efficacy of low dose resveratrol 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 Apc mouse, a model of hereditary colorectal cancer characterised by a mutation in codon 850 of the adenomatous polyposis coli (Apc) gene. Since resveratrol is known to protect against age-related pathologies and early mortality associated with high fat in mice (20-22), we compared the effects of resveratrol in animals maintained on a standard (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 receiving SD, where it caused a small (22%) reduction in adenoma number but failed to influence tumour volume. In contrast, when co-administered with a HFD the low dose of resveratrol (0.00007% w/w, equating to ~0.07 mg/kg body weight per day) significantly reduced adenoma number by ~40% and decreased the overall burden by ~52% relative to control animals of both gender (Fig. 2A). Surprisingly, although the high dose (0.0143% w/w; 14 mg/kg body weight) was also efficacious, it was consistently less potent, reducing adenoma number by one third, and burden by 25%. Inhibition of tumour development by resveratrol at both doses was associated with a small (6.5-9.3%) significant reduction in the proportion of proliferating cells in adenomas, but not histologically normal crypts of the small intestine or colon, in mice on the HFD, as measured by positive Ki-67 staining (Fig. 2B and Fig. S1). In contrast, resveratrol had no effect on the extent of apoptosis, adjudged by cleaved caspase-3 immunostaining (Fig. S1). Consistent with previous observations (22), the higher resveratrol dose was associated with significantly increased bodyweight in males, but in females only the low dose correlated with increased bodyweight and this was specific to animals on HFD (Fig. 2C and Fig. S2). Although control male mice on HFD had lower bodyweights than those on SD, which may indicate the HFD was less palatable, a similar effect was not apparent in females. Whilst the reasons are currently unclear, the increase in bodyweight associated with resveratrol may potentially be due to less malabsorption as a consequence of lower tumour burden in these animals or a tendency for increased food consumption (Fig. S3). Given that low dose resveratrol was only efficacious in mice on HFD, this raises the possibility that dietary feasible intakes may protect against the tumour promoting effects of fat, without reducing body weight (Fig. 2C and Fig. S2). Indeed, comparison of tumour development in Apc mice on control SD and HFD, culled at the same time point (14 weeks of age) revealed a strong pro-carcinogenic effect of fat in this model (Fig. 2D). Importance of AMPK signalling in the chemopreventive effects of resveratrol In female mice, efficacy was highly correlated with the expression and activation of the energy regulator AMPK in intestinal mucosa. Neither AMPKα protein nor its phosphorylated form were detectable in any mice on the SD or in the high fat control group, but both were evident in animals that ingested 0.00007% resveratrol, and to a lesser extent those on the high dose (Fig. 3A,C). This phenomenon was closely mirrored by an increased expression and phosphorylation of the AMPK target acetyl-CoA carboxylase (ACC) (Fig 3B,C). In contrast, a random pattern of AMPK and pAMPK expression was observed in mucosa of the male mice (Fig. S4), probably due to overnight starvation prior to culling, which was not performed in the females; this was necessary to enable measurement of metabolic parameters in the fasting state, none of which were altered by intervention, apart from intestinal IGF1, which was decreased by the high dose resveratrol regardless of fat content (Fig. S5). Furthermore, determination of the kinetics of AMPK activation in wild-type B57BL/6J male mice preconditioned on a HFD before receiving a single oral dose of resveratrol (2.1μg), revealed an extremely rapid response with increased expression and phosphorylation detected in normal intestinal mucosa just 30 min after administration (Fig. 3D,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 2h before declining. This may explain the considerable variability of pAMPK levels in resveratrol-treated mice on HFD, as tissue concentrations may be dependent on when the mouse last ate. This finding also demonstrates that the ability of dietary-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 a HFD are currently unclear. One of the end products of AMPK activation is autophagy, a catabolic pathway required for the quality control of proteins/organelles and maintenance of energy homeostasis, which can also serve as a tumour suppressing mechanism (23). We detected significantly enhanced levels of soluble microtubule-associated protein 1 light chain 3 (LC3-I) in mucosa tissue of resveratroltreated mice, along with increased conversion to the lipid bound LC3-II, which is a constituent of autophagosomal membranes and marker of autophagy initiation (Fig. 3D,E). Upregulated autophagy appears to be a rapid but potentially short-term response to resveratrol since it was observed in animals that received the single dose but not in the chronically treated Apc mice (Fig. 3B,D,E). Conversely, p21 expression, a marker of senescence, was increased in Apc mice that ingested resveratrol with a HFD, whilst a single dose was insufficient to elevate p21 protein levels over the time frame monitored (Fig. 3B-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 Apc 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 following 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 anti-tumour 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 βgalactosidase staining and p21 expression (Fig. 4A-D). After 6 days’ repeated exposure to resveratrol, where the medium was replaced every 24h, the expression of AMPK remained stable but significant concentration-dependent activation was evident from 0.01μM and reached a maximum at 1μM (Fig. 4A,B). At 10μM, AMPK phosphorylation returned to basal levels. This bell-shaped dose response was also apparent for ACC phosphorylation, which was greatest at 1μM, reflecting the in vivo findings that lower exposures are more effective. Downstream targets of AMPK were altered in a similar manner (Fig. 4A,B); 1μM resveratrol caused the greatest reduction in phosphorylation of the mechanistic target of rapamycin (mTOR), and its downstream effectors 4EBP1 and S6K, which are involved in protein translation. These effects were independent of Akt, another mTOR regulator, since resveratrol had no effect on Akt expression and activation (Fig. S6). 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 (15,26-28). 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 (2h) exposure of Apc10.1 cells to resveratrol followed by removal, to mimic the process of metabolic clearance in the mouse intestine, caused a transient increase in pAMPK after 4h, which correlated with a significant increase in the AMP/ATP ratio, consistent with ATP synthase inhibition (Fig. 4E-F). Co-incubation with the calcium chelator BAPTA or the CamKKβ inhibitor STO-609 had no effect (Fig. S6), suggesting that inhibition of phosphodiesterase (26) does not play a role in this system. Intriguingly, low concentrations of resveratrol induced a detectable increase in reactive oxygen species (ROS) within 1h (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 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 signalling in colorectal tissue of the patients that received [C]-resveratrol revealed random expression patterns, with no apparent difference between treated and control patients (Fig. S7). As with the male Apc mice, this lack of effect may be due to the fact all patients were fasted overnight prior to surgery and would have been without food for differing lengths of time. To overcome this issue, we performed explant cultures of human colorectal tumours isolated from three individual patients and passaged in immuno-compromised NODSCID mice. Notably, the response to resveratrol exposure mimicked that observed in Apc10.1 cells, with rapid AMPK activation and increased autophagy at low concentrations (0.01-0.1μM) and a less pronounced or no effect with higher exposures (Fig. 5A-B). Further support for the existence of a non-conventional dose response in humans is provided by the observation that both NQO1 expression and protein carbonyl concentration were significantly increased in the colorectal mucosa of patients that received 5mg [C]-resveratrol compared to those taking the 1g dose and the control group (Fig. 5C-E). NQO1 is a cytoprotective enzyme regulated by the transcription factor Nrf-2, which is activated by oxidative stress (32), whilst quantitation of carbonyl groups provides a stable measure of amino acid oxidation (33). Taken together, this study suggests that lower doses of resveratrol may be more active than higher supra-dietary intakes in humans and that the beneficial effects of resveratrol at such doses may be mediated by its pro-oxidant activity and upregulation of AMPK signalling. Discussion The ability of low, dietary feasible resveratrol doses to selectively prevent intestinal tumour development in Apc mice fed a HFD highlights several pertinent points important for advancing the field of 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). Supra-dietary doses have frequently been administered, even when epidemiology data suggest it is dietary achievable intakes that offer protection. However, with recent results from trials such as SELECT, involving selenium and the antioxidant vitamin E, it is gradually being recognized that complex dose-response relationships exist for dietary-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 non-linear 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 because 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 low intakes of resveratrol have 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. In this study, dose conversion from human to mouse was performed based on 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 81mg of resveratrol a day for a 70kg person (37). The requirement for a HFD to reveal the efficacy of resveratrol explains why a dose ~14-fold higher than the maximal used here in mice was necessary to produce a significant, albeit moderate (30%), reduction in adenomas in analogous studies involving Apc mice on a SD, whilst lower exposures were ineffectual (38,39). This interaction with fat is in agreement with results emerging from clinical trials, where resveratrol at doses as low as 10mg daily, appears to have selective activity in obese humans (40) or those with metabolic disorders, such as type 2 diabetes mellitus (41,42). Of particular note is the study by Timmers et al. in which resveratrol (150mg per 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 (75mg) had no effect in non-obese women with normal glucose tolerance (43). Interestingly, 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 5mg 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, lends further credence to the reality of a non-linear 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 personalising chemopreventive therapy. Long-term maintenance of C57BL/6J mice, the background strain of Apc mice, on a 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 a HFD has previously been demonstrated to cause metabolic changes in Apc mice, with the degree of dysregulation increasing over time (48). However, in the present study there was no evidence of the diabetic phenotype in mice on HFD, although it is difficult to draw direct comparisons because those on HFD were culled 3 weeks earlier than animals on SD. Resveratrol appears to counteract the tumour-promoting effects of a HFD without modulating the fasting levels of plasma biomarkers previously associated with health and survival benefits in middle aged mice (20). Therefore, at the low doses used, it is likely that localised rather than systemic effects in colorectal cells are responsible for the chemopreventive efficacy of resveratrol, particularly as the phenotype observed in Apc mice was replicated in vitro. Although widely perceived as an antioxidant our findings suggest that the transient pro-oxidant activity of resveratrol is responsible, at least in part, for activation of AMPK at very low concentrations. Whether the increased ROS involved 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 whilst resveratrol has been shown capable of binding to F1-ATPase using X-ray crystallography, evidence for a direct inhibitory effect is limited to studies with subcellular fractions and require relatively high concentrations of resveratrol (10-30μM) (49,50). Other known AMPK activators including metformin and aspirin have been shown to protect against adenoma development in Apc mice (51,52). Studies with aspirin, which is rapidly hydrolysed in vivo to salicylate, an allosteric activator of AMPK, have yielded conflicting results but it appears to require lifetime administration from the point of conception to significantly suppress intestinal tumorigenesis (51,53). Inhibitors of mTOR signalling such as rapamycin are also efficacious in this model (54), and whilst it is recognized that the anticancer effects of all these compounds are likely to involve multiple modes of action, these observations reinforce the potential of targeting metabolic pathways for cancer prevention. Countless in vitro studies have described pro-apoptotic effects of resveratrol in cancer cell lines (13,55), however these necessitated the use of concentrations beyond those systemically achievable in humans (15). We found no evidence of increased apoptosis, which is likely to be a consequence of toxicity at high concentrations, within the intestines of treated Apc mice; 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 the result of sustained exposure to low concentrations. Both processes have paradoxical roles in carcinogenesis (23,56,57), but appear to serve as tumour supressing mechanisms in Apc mice on a HFD. It is therefore encouraging that the increased ROS stimulus and elevated autophagy also translate to human colorectal tissue exposed to concentrations of resveratrol generated in muscle and mucosa after a 5mg dose (~0.01-0.2 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 dietary achievable doses of resveratrol halt tumour progression in mice; this correlates with the induction of AMPK and senescence and these effects translate to human tissue. Moreover, we unveiled how the tumour preventive efficacy of resveratrol is dependent on animal diet and, probably, human lifestyle, particularly behaviours that cause obesity and metabolic syndrome. This work warrants a deep 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 Supplemental Experimental Procedures. Clinical Trials The trials were approved by the Liverpool UK Research Ethics Committee, the UK Medicines and Healthcare products Regulatory Authority, the Administration of Radioactive Substances Advisory Committee (ARSAC) and the Institutional Review Board at Lawrence Livermore National Laboratory. Both trials were conducted at The University Hospitals of Leicester 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 5mg [C]-resveratrol or 1.005g [C]-resveratrol (4 x 250mg, plus 5mg [C]-resveratrol). Blood samples were then taken over 24h. Colorectal cancer patient trial: Patients (10 per group) with resectable colorectal cancer took daily unlabelled resveratrol capsules (5mg or 1.0g) for 6 days and then received a final [C]resveratrol dose prior to surgery which consisted of either 5mg or 1.005g [C]-resveratrol, as above. A control group without any intervention was also included. At surgical resection, both normal and malignant tissue was sampled. Processing of all tissue, blood and HPLC fractions for AMS analysis was performed in a designated laboratory, free from extraneous C-contamination and samples were measured at Lawrence Livermore National Laboratory (58,59). Pharmacokinetic parameters were modelled using WinNonlin Version 5.3 software (Pharsight Corporation, Mountain View, California, US). Analysis of protein carbonyls and NQO1 in human colorectal tissue Colorectal mucosa samples from cancer patients participating in the [C]-resveratrol trial, and tissue from control untreated patients were analysed for markers of oxidative stress. All samples were coded and randomised and the analyses were performed blind. NQO1 expression was measured by Western blotting and the OxiSelectTM 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 Leicester University by the UK Home Office. The experimental design was vetted by the Leicester University Local Ethical Committee for Animal Experimentation and met the standards required by the UKCCCR guidelines (60). Resveratrol efficacy was assessed in male and female Apc mice (61). After weaning, animals were randomised to one of 6 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. Male mice only were fasted overnight prior to 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) only. Tumour passaging: Colorectal tumours 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 committee, REC reference 09/H0402/45). Tumours 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 tumours from surgical resections (~2mm thick) were washed in media 199 containing 2% Antibiotic-Antimycotic (Invitrogen) prior to implantation. The tumour tissue was inserted into the right and/or left flank of a mouse. Mice were sacrificed and the tumour excised before reaching the size limit designated in the animal project licence. Tissue was then used immediately for explant cultures. Immunohistochemistry Immunohistochemistry was performed using the Novolink Polymer Detection system (Leica Biosystems, Newcastle, UK) according to the manufacturer’s instructions. For formalin fixed mouse tissue the anti-Ki-67(ab15580, Abcam) and Caspase-3 (9661, Cell Signalling) were used at 1:1000 and 1:200 dilutions, respectively. For analysis of human explant tissues the phosphorylated AMPK antibody (2535, Cell Signalling) was used at a 1:100 dilution. Cell culture Unless stated otherwise, Apc10.1 cells were typically treated daily with resveratrol (0.001-1 μM) for six days to mimic repeated dosing in humans and harvested 4h 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 Signalling Tech., apart from those for p21 and βactin (Santa Cruz Biotechnologies). Senescence was assessed using a senescence βgalactosidase staining kit (Cell Signalling Tech.), autophagy was measured using the Cyto-ID Autophagy detection kit (Enzo Life Sciences, Switzerland) and levels of intracellular ROS were visualised using an Image-iTTM LIVE green ROS detection kit (Invitrogen). Analysis of AMP, 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. Tumour tissues were freshly excised from NOD/SCID mice and placed in DMEM (low glucose) media supplemented with 1% fetal calf serum and 2% antibiotic-antimycotic. For each concentration of resveratrol and the solvent control, nine pieces of tumour (~2mm each) were placed in an insert, within a single well of a six well plate. The explants were incubated overnight (37°C, 5% CO2) then fresh media, supplemented with either resveratrol or vehicle control (DMSO), was added. After 2h the tissues were harvested for either immunohistochemistry or Western blotting (9 pieces of tissue combined). Statistical analysis The effect of resveratrol dose on the expression of NQO1 protein in human colorectal tissue was analysed using a T-test and Mann Whitney test. The effect of resveratrol treatment on adenoma volume and number in Apc mice was modelled using regression analysis (of logtransformed data for volume). Mouse body weight data over time were analysed using mixed effects linear regression. The immunohistochemistry and in vitro data were analysed using a Ttest. Supplementary Materials Materials and Methods Fig. S1. Effect of resveratrol on cell proliferation and apoptosis in tissues of Apc mice. Fig. S2. Effect of resveratrol and a high-fat diet on body weight of female Apc mice. Fig. S3. Estimated weekly diet consumption by male and female Apc mice. Fig. S4. Effect of resveratrol on the expression of AMPK in the intestinal mucosa of Apc mice. Fig. S5. Effect of resveratrol on metabolic parameters in plasma and intestinal mucosa of fasted male Apc 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 [C]-resveratrol trial. Table S1. Plasma pharmacokinetic parameters for total [C]-resveratrol equivalents in healthy volunteers that ingested a single dose of either 5 mg or 1 g [C]-resveratrol. Table S2. 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Chemopreventive efficacy and pharmacokinetics of curcumin in the min/+ mouse, a model of familial adenomatous polyposis. Cancer Epidemiol. Biomarkers Prev. 11, 535-540 (2002). 63) Stocchi, V., Cucchiarini, L., Magnani, M., Chiarantini, L., Palma, P. and Crescentini, G. Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells. Anal. Biochem. 146, 118-124 (1985). Acknowledgements We thank Carla De Giovanni for the Apc.10.1 cells. We are grateful to David Monk and Mike Dunn (Medical Physics, University Hospitals of Leicester) for help with the clinical trials. 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., A.Kholghi., C.A., R.G.B., E. H-G., A.Karmokar, M.J., L.H., A.R. and K.B. designed and/or performed all laboratory experiments; H.C., E.S. and A.Karmokar performed in vivo studies; 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, while surgical expertise was provided by A.M. and D.H.; J.W., C.G. and N.K. analysed 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. Figure 1. Comparison of the plasma pharmacokinetics and target tissue distribution of [C]resveratrol and its metabolites in humans following a low dietary achievable dose or high pharmacological dose. (A-B) Healthy volunteers received a single [C]-labelled oral dose of either 5 mg or 1.005 g resveratrol (44.5 kBq, 0.962 μSv) and plasma samples were taken over 24 h for determination of total [C]-resveratrol equivalents by AMS analysis. (A) Graphs show average (± SD) concentrations for 10 volunteers per group, whilst the inset represents a single participant to illustrate the second peak maxima 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 h after ingestion. Also included are a pre-dose plasma sample for determination of background levels of radiocarbon and a UV chromatogram from the analysis of authentic metabolite standards. Peaks designated by * were tentatively assigned on the basis of their chromatographic properties, since synthetic standards were not available. (C) Levels of [C]-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 prior to surgery, with the last dose being [C]-radiolabelled as described in the 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 tumour) were also available for analysis (Supplementary Table 2). One patient in the high dose group had surgery delayed by 6 days after taking [C]-resveratrol and has been 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 [C]-resveratrol, determined by HPLC-AMS analysis. Peaks of radiocarbon in both tissue types correspond to resveratrol and its 3-sulfate, based on similarity of retention times to authentic standards, and the concentrations stated translate to μM, assuming 1g of tissue equates to 1mL. Figure 2. Low dose resveratrol inhibits adenoma development in Apc mice on HFD more potently than a dose 200-fold higher. Male and female mice were maintained on SD or HFD from weaning (4 weeks of age) 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 whilst those on the HFD (60% of calories from fat) had to be killed at 14 weeks due to the tumour promoting effects of the latter. (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 14-16 female plus 17-19 male mice per group. 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 Apc mice on HFD, as measured by immunohistochemical staining for nuclear Ki-67. Data represent the median percentage (plus 25 and 75 percentile) of Ki-67 positive cells per field, where 6 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 Apc mice on SD or HFD alone or containing resveratrol. Data represent the mean ± SEM of 15-19 mice per group. High dose resveratrol significantly increased the body weight of mice on SD (p<0.05) and HFD (p<0.001) compared to corresponding controls; low dose resveratrol increased the body weight of animals on HFD only (p=0.05). Control mice on the HFD had significantly lower bodyweights than those on the standard diet (P<0.001). (D) Effect of a control HFD on intestinal adenoma number and total volume compared to Apc mice on a SD. Animals in both groups (7-9 females plus 7-8 males) were culled at 14 weeks of age and data illustrate the mean ± SEM. Figure 3. Low dose dietary resveratrol activates AMPK and causes senescence in intestinal mucosa of mice on HFD. (A-C) Expression and phosphorylation of AMPK and its downstream target ACC, together with levels of autophagy and senescence markers in tissue of female Apc mice maintained on 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 6 mice per group on HFD with or without resveratrol. (D-E) Kinetics of AMPK activation and downstream effects in intestinal tissue of C57BL/6J wild-type male mice maintained on HFD which received a single gavage dose of resveratrol (2.1 μg per mouse; R) or vehicle control (C). Mice were culled post-dosing at the indicated time. (D) Representative immunoblots are shown for 3 mice per group. (E) Data represent the mean ± SEM of 4-6 mice per group. Figure 4. Low, dietary achievable concentrations of resveratrol activate AMPK signalling and cause autophagy and senescence in Apc10.1 mouse adenoma cells. (A-B) Six days of repeated exposure to resveratrol enhances AMPK phosphorylation, inhibits mTOR signalling and increases markers of autophagy and senescence. Representative immunoblots are shown (A). (C) Detection of Cyto-ID Green-stained autophagic vacuoles visualised 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 following exposure to resveratrol for 2 h, replacement of the media and further incubation without resveratrol for the times indicated. (f) AMP/ATP ratio determined by HPLC analysis. Cells were treated with resveratrol for 2 h, media was replaced and incubation continued for 4 h. (G) Levels of intracellular reactive oxygen species visualised using an Image-iT LIVE green ROS detection kit, 1 h after 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 h co-incubation. All graphs illustrate the mean ± SEM of three independent experiments. Significant differences relative to control incubations are indicated by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0005). Figure 5. Low concentrations of resveratrol activate AMPK and increase markers of oxidative stress in human colorectal tissues. Exposure to resveratrol (2h) increases pAMPK levels and upregulates autophagy in primary colorectal cancer explants, as assessed by Western blotting (A) and/or immunohistochemical staining (B) in samples from three different patients. Expression of NQO1 (C-D) and levels of protein carbonylation (E) in colorectal mucosa tissue of patients participating in the [C]resveratrol trial who received a dose of either 5 mg or 1 g daily for one week prior to surgery, or untreated control patients. Samples were analysed blind, and significant differences between control and treated groups are indicated. (C) A typical Western blot for NQO1. (D) Data represent the mean ± SEM of 6-8 patients per group. (e) Data show the mean ± SEM of 4-6 patients per group.