Robert G. Britton

Sulfate Metabolites Provide an Intracellular Pool for Resveratrol Generation and Induce Autophagy with Senescence

Biologically active concentrations of resveratrol can be generated intracellularly after uptake of the major human sulfate metabolites by selected cells. Cheers for Resveratrol Sulfates Red wine drinkers rejoiced at the news that their chosen beverage contains the antioxidant resveratrol, a phytochemical that has health benefits and extends longevity in preclinical studies. But resveratrolߣs rapid metabolism appears to limit translation of its health benefits to humans, even if one chugs liters of red wine. Now, Patel et al. show that certain metabolites might contribute to resveratrol’s in vivo activity through metabolic regeneration of the parent compound. The authors first detected and quantitated the sulfate and glucuronide conjugates of resveratrol in plasma and tissue after resveratrol ingestion, over time, by cancer patients and healthy control subjects. A daily dose of 1 g of resveratrol gave rise to plasma concentrations of these metabolites in the micromolar range. A pharmacokinetic analysis of resveratrol-3- and 4′-O-sulfate metabolites in mice revealed that sulfate hydrolysis generated free resveratrol, a reaction also observed in human colorectal cells in culture. The addition of resveratrol-sulfate metabolites to human cancer cells in culture correlated with an increase in autophagy and senescence, effects that were blocked with a sulfatase inhibitor that decreased the amounts of intracellular resveratrol. These findings suggest that resveratrol can enter tissues as a stable sulfate-conjugate. Selected cells then generate the free parent compound, which appears to be the form responsible for biological benefits. The phytochemical resveratrol has been shown to exert numerous health benefits in preclinical studies, but its rapid metabolism and resulting poor bioavailability may limit translation of these effects to humans. Resveratrol metabolites might contribute to in vivo activity through regeneration of the parent compound. We present quantitation of sulfate and glucuronide conjugates of resveratrol in human plasma and tissue after repeated ingestion of resveratrol by volunteers and cancer patients, respectively. Subsequent pharmacokinetic characterization of a mixture of resveratrol-3-O-sulfate and resveratrol-4′-O-sulfate in mice showed that these metabolites are absorbed orally but have low bioavailabilities of ~14 and 3%, respectively. Sulfate hydrolysis in vivo liberated free resveratrol, which accounted for ~2% of the total resveratrol species present in mouse plasma. Monosulfate metabolites were also converted to the parent in human colorectal cells. The extent of cellular uptake was dependent on specific membrane transporters and dictated antiproliferative activity. Sulfate metabolites induced autophagy and senescence in human cancer cells; these effects were abrogated by inclusion of a sulfatase inhibitor, which reduced intracellular resveratrol. Together, our findings suggest that resveratrol is delivered to target tissues in a stable sulfate-conjugated form and that the parent compound is gradually regenerated in selected cells and may give rise to the beneficial effects in vivo. At doses considered to be safe in humans, resveratrol generated via this route may be of greater importance than the unmetabolized form.

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

Low-dose resveratrol prevents tumor growth in mice and in human tissues, suggesting a revision of development strategies for preventive dietary agents. Less is more From energy drinks to supplements to skin serums, resveratrol has been sold to the public for myriad health benefits, most famously in the anti-aging arena. In fact, at a posh wine bar, one might overhear a patron lamenting the small dose of resveratrol one receives in a glass of the red variety. Now, Cai et al. show that a low rather than a high dose of resveratrol prevents tumor growth in mice and alters metabolic pathways in human tissues. The authors compared the dose-response curves of a dietary dose of resveratrol and a 200-fold higher amount in mice that spontaneously develop colorectal adenomas—precursors to cancer—that were fed a standard or a high-fat diet. In the mice on the high-fat diet, low-dose resveratrol reduced intestinal tumor development much better than did the high dose. In mouse tumor cells, resveratrol efficacy was tracked with an increase in autophagy and senescence markers and activation of adenosine monophosphate (AMP)–activated protein kinase (AMPK)—an enzyme that functions in the maintenance of cellular energy homeostasis. Exposure of human colorectal cancer tissue to low concentrations of resveratrol also caused an increase in autophagy and activation of AMPK. Colorectal mucosal samples isolated from cancer patients who received a low-dose resveratrol regimen before tumor resection showed an increase in expression of the cytoprotective, oxidative stress-activated enzyme NAD(P)H dehydrogenase, quinone 1 (NQO1). These findings suggest that resveratrol operates by modulating energy balance and responding to stress. At a time when “supersizing” is popular, the nonlinear dose-response documented in the new work suggests that its time for a revision in development strategies for preventative dietary agents. 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.

Prolonged biologically active colonic tissue levels of curcumin achieved after oral administration--a clinical pilot study including assessment of patient acceptability.

Curcumin, the main constituent of turmeric, is suspected to possess cancer chemopreventive properties. Pharmacokinetic and pharmacodynamic parameters have been reported, but few data exist describing whether methodologies are suitably robust for curcuminoid detection in colonic biopsy specimens. Information on the acceptability of prolonged administration of daily curcumin is not available. This is of vital importance to implement chemoprevention strategies. This study aimed to quantify levels of curcuminoids in colorectal mucosa of patients undergoing colorectal endoscopy or surgical resection and to obtain information on the acceptability and compliance with daily curcumin. Curcumin C3 complex (2.35 g) was administered to patients once daily for 14 days before endoscopic biopsy or colonic resection. Safety and tolerance were monitored. Analysis of curcuminoids in plasma, urine, and colonic mucosa was conducted by ultraperformance liquid chromatography (UPLC)-UV with characterization by liquid chromatography/tandem mass spectrometry (LC/MS-MS). Twenty-four of 26 patients commencing curcumin completed the course. Six patients reported mild gastrointestinal adverse events. Curcuminoids were detectable in nine of 24 plasma samples, 24 of 24 urine samples, and in the colonic mucosa of all 23 biopsied participants. Mean tissue levels were 48.4 μg/g (127.8 nmol/g) of parent curcuminoids. The major conjugate, curcumin glucuronide, was detectable in 29 of 35 biopsies. High levels of topical curcumin persisted in the mucosa for up to 40 hours postadministration. Sixteen participants (67%) stated that they would take curcumin long-term should it be of proven benefit. In summary, pharmacologically active levels of curcumin were recovered from colonic mucosa. The regimen used here seems safe, and patients support its use in long-term trials. Cancer Prev Res; 6(2); 119–28. ©2012 AACR.

Flavones as Colorectal Cancer Chemopreventive Agents—Phenol-O-Methylation Enhances Efficacy

Flavonoids occur ubiquitously in plants, and some possess preclinical cancer chemopreventive activity. Little is known about molecular features that mediate chemopreventive efficacy of flavonoids. Here, three related flavones, apigenin (4′,5,7-trihydroxyflavone), tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone), and 3′,4′,5′,5,7-pentamethoxyflavone (PMF), were compared in terms of their effects on (a) adenoma development in ApcMin mice, a model of human gastrointestinal malignancies; (b) growth of APC10.1 mouse adenoma cells in vitro; and (c) prostaglandin E-2 generation in HCA-7 human-derived colorectal cancer cells in vitro. Life-long consumption of PMF with the diet at 0.2% reduced ApcMin mouse adenoma number and burden by 43% and 61%, respectively, whereas apigenin was inactive. Tricin has previously shown activity in this model. IC50 values for murine adenoma cell growth inhibition by PMF, tricin, and apigenin were 6, 13, and 18 μmol/L, respectively. In ApcMin mice that received flavones (0.2%) for 4 weeks, adenoma cell proliferation as reflected by Ki-67 staining was reduced by PMF and tricin, but not by apigenin. On incubation with HCA-7 cells for 6 hours, PMF reduced prostaglandin E-2 generation with an IC50 of 0.8 μmol/L, a fraction of the respective values reported for tricin or apigenin. In silico PMF docked into the cyclooxygenase active site with greater affinity than tricin or apigenin. The results suggest that the rank order of cancer chemopreventive efficacy in ApcMin mice is PMF > tricin > apigenin, supporting the notion that the presence of O-methyl in the flavone molecular scaffold promotes gastrointestinal cancer chemopreventive efficacy.

Direct molecular targets of resveratrol: identifying key interactions to unlock complex mechanisms.

To truly understand the mechanisms through which resveratrol exerts its biological effects, the key direct interactions between resveratrol and its target biomolecules must be identified. With an increasing number of biochemical tools to measure and quantify direct physical interactions between biomolecules, there have been around 20 proteins identified as having a specific affinity to resveratrol to date. Resveratrol has been described as a promiscuous molecule, and one would expect it to bind with numerous proteins, which would help explain why resveratrol appears to have so many health benefits and has been shown to act upon various different pathways related to a diverse range of conditions. The aim of this review is to present the direct protein targets of resveratrol that are currently known and highlight the consequences of direct binding and the methods used to identify the nature of these interactions.

Design, synthesis and biological evaluation of new tryptamine and tetrahydro-beta-carboline-based selective inhibitors of CDK4

We present the design, synthesis and biological activity of a library of substituted (biphenylcarbonyl)-tryptamine and (biphenylcarbonyl)-tetrahydro-beta-carboline compounds related to the natural product fascaplysin, as novel inhibitors of CDK4/cyclin D1. We show all these molecules, prepared using the Suzuki-Miyaura reaction, being selective inhibitors of CDK4 over CDK2. The most active compounds have a CDK4 IC(50) in the range 9-11 microM, three of them containing the para-biphenyl plus para-substituents supporting the existence of a pi-stacking pocket within the active site of CDK4.

A Short, Organocatalytic Formal Synthesis of (−)-Swainsonine and Related Alkaloids

A short synthesis of hydroxyalkyl dihydropyrroles has been developed that involves the coupling of propargylamines with α-chloroaldehydes, followed by Lindlar reduction and a one-pot epoxide formation/opening sequence. The application of this process to the synthesis of unnatural iminosugars and a formal synthesis of (-)-swainsonine is described.

Dose-Response Relationships for N7-(2-Hydroxyethyl)Guanine Induced by Low-Dose [14C]Ethylene Oxide: Evidence for a Novel Mechanism of Endogenous Adduct Formation

Ethylene oxide (EO) is widely used in the chemical industry and is also formed in humans through the metabolic oxidation of ethylene, generated during physiologic processes. EO is classified as a human carcinogen and is a direct acting alkylating agent, primarily forming N7-(2-hydroxyethyl)guanine (N7-HEG). To conduct accurate human risk assessments, it is vital to ascertain the relative contribution of endogenously versus exogenously derived DNA damage and identify the sources of background lesions. We have therefore defined in vivo dose-response relationships over a concentration range relevant to human EO exposures using a dual-isotope approach. By combining liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography-accelerator mass spectrometry analysis, both the endogenous and exogenous N7-HEG adducts were quantified in tissues of [(14)C]EO-treated rats. Levels of [(14)C]N7-HEG induced in spleen, liver, and stomach DNA increased in a linear manner from 0.002 to 4 adducts/10(8) nucleotides. More importantly, the extent of damage arising through this route was insignificant compared with the background abundance of N7-HEG naturally present. However, at the two highest doses, [(14)C]EO exposure caused a significant increase in endogenous N7-HEG formation in liver and spleen, suggesting that EO can induce physiologic pathways responsible for ethylene generation in vivo and thereby indirectly promote N7-HEG production. We present evidence for a novel mechanism of adduct formation to explain this phenomenon, involving oxidative stress and 1-aminocyclopropane-1-carboxylic acid as a potential biosynthetic precursor to ethylene in mammalian cells. Based on the proposed pathway, N7-HEG may have potential as a biomarker of cellular oxidative stress.

Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol, which induces autophagy in cancer cells

Resveratrol has many proposed health benefits, including the prevention of cancers, but its low bioavailability is considered a limiting factor in translating these effects to humans. Based on in vivo and clinical studies we have shown that resveratrol is indeed rapidly metabolized by phase II enzymes, and that resveratrol sulfates are deconjugated by steroid sulfatases to afford free resveratrol in vitro and in vivo and hence act as an intracellular reservoir for resveratrol. Further, we have demonstrated that at clinically achievable concentrations of resveratrol sulfate, parent resveratrol is regenerated within human colorectal cancer, but not normal epithelial cells, and is responsible for inducing autophagy with senescence selectively in cancer cells.

Preclinical Colorectal Cancer Chemopreventive Efficacy and p53-Modulating Activity of 3′,4′,5′-Trimethoxyflavonol, a Quercetin Analogue

Some naturally occurring flavonols, exemplified by quercetin, seem to possess experimental cancer chemopreventive efficacy. Modulation of p53 is a mechanism thought to contribute to their activity. The hypothesis was tested that a synthetic flavonol, 3′,4′,5′-trimethoxyflavonol (TMFol), can interfere with tumor development and p53 expression in two models of colorectal carcinogenesis, ApcMin mice and human-derived HCT116 adenocarcinoma–bearing nude mice. Mice received TMFol with their diet (0.2%) from weaning to week 16 in the case of ApcMin or from either day 7 before (“TMFol early”) or day 7 after (“TMFol late”) tumor inoculation in HCT116 mice. The ability of TMFol to affect tumor proliferation or apoptosis, as reflected by staining for Ki-67 or cleaved caspase-3, respectively, was studied in HCT116 tumors. TMFol tumor levels were measured by high-performance liquid chromatography. Consumption of TMFol reduced small intestinal adenoma burden in ApcMin mice by 47%, compared with control mice (P < 0.002). The TMFol early regimen approximately halved HCT116 tumor size (P < 0.05), decreased tumor proliferation, and increased apoptosis, whereas the TMFol late regimen had no significant effect when compared with controls. In tumor tissues from mice, in which TMFol reduced tumor development, p53 expression was increased 3-fold in ApcMin and 1.5-fold in HCT116 tumor–bearing mice (P = 0.02). TMFol increased p53 also in cells derived from these tumors. TMFol was detected in HCT116 tumors, but levels did not correlate with tumor burden. TMFol was not mutagenic in the Ames test. The results suggest that chemical modification of the flavonol structure may generate safe and efficacious cancer chemopreventive agents. Cancer Prev Res; 3(8); 929–39. ©2010 AACR.