Catherine Andreadi

Predicting the physiological relevance of in vitro cancer preventive activities of phytochemicals.

AbstractThere is growing interest in the ability of phytochemicals to prevent chronic diseases, such as cancer and heart disease. However, some of these agents have poor bioavailability and many of the in-depth studies into their mechanisms of action have been carried out in vitro using doses which are unachievable in humans. In order to optimize the design of chemopreventive treatment, it is important to determine which of the many reported mechanisms of action are clinically relevant. In this review we consider the physiologically achievable doses for a few of the best studied agents (indole-3-carbinol, diindolylmethane, curcumin, epigallocatechin-3-gallate and resveratrol) and summarize the data derived from studies using these low concentrations in cell culture. We then cite examples of in vitro effects which have been observed in vivo. Finally, the ability of agent combinations to act synergistically or antagonistically is considered. We conclude that each of the compounds shows an encouraging range of activities in vitro at concentrations which are likely to be physiologically relevant. There are also many examples of in vivo studies which validate in vitro observations. An important consideration is that combinations of agents can result in significant activity at concentrations where any single agent is inactive. Thus, for each of the compounds reviewed here, in vitro studies have provided useful insights into their mechanisms of action in humans. However, data are lacking on the full range of activities at low doses in vitro and the benefits or otherwise of combinations in vivo.

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.

The intermediate-activity (L597V)BRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway

(L597V)BRAF mutations are acquired somatically in human cancer samples and are frequently coincident with RAS mutations. Germline (L597V)BRAF mutations are also found in several autosomal dominant developmental conditions known as RASopathies, raising the important question of how the same mutation can contribute to both pathologies. Using a conditional knock-in mouse model, we show that endogenous expression of (L597V)Braf leads to approximately twofold elevated Braf kinase activity and weak activation of the Mek/Erk pathway. This is associated with induction of RASopathy hallmarks including cardiac abnormalities and facial dysmorphia but is not sufficient for tumor formation. We combined (L597V)Braf with (G12D)Kras and found that (L597V)Braf modified (G12D)Kras oncogenesis such that fibroblast transformation and lung tumor development were more reminiscent of that driven by the high-activity (V600E)Braf mutant. Mek/Erk activation levels were comparable with those driven by (V600E)Braf in the double-mutant cells, and the gene expression signature was more similar to that induced by (V600E)Braf than (G12D)Kras. However, unlike (V600E)Braf, Mek/Erk pathway activation was mediated by both Craf and Braf, and ATP-competitive RAF inhibitors induced paradoxical Mek/Erk pathway activation. Our data show that weak activation of the Mek/Erk pathway underpins RASopathies, but in cancer, (L597V)Braf epistatically modifies the transforming effects of driver oncogenes.

Mouse models for BRAF-induced cancers

Oncogenic mutations in the BRAF gene are detected in approximately 7% of human cancer samples with a particularly high frequency of mutation in malignant melanomas. Over 40 different missense BRAF mutations have been found, but the vast majority (>90%) represent a single nucleotide change resulting in a valine-->glutamate mutation at residue 600 ((V600E)BRAF). In cells cultured in vitro, (V600E)BRAF is able to stimulate endogenous MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and ERK phosphorylation leading to an increase in cell proliferation, cell survival, transformation, tumorigenicity, invasion and vascular development. Many of these hallmarks of cancer can be reversed by treatment of cells with siRNA (small interfering RNA) to BRAF or by inhibiting MEK, indicating that BRAF and MEK are attractive therapeutic targets in cancer samples with BRAF mutations. In order to fully understand the role of oncogenic BRAF in cancer development in vivo as well as to test the in vivo efficacy of anti-BRAF or anti-MEK therapies, GEMMs (genetically engineered mouse models) have been generated in which expression of oncogenic BRaf is conditionally dependent on the Cre recombinase. The delivery/activation of the Cre recombinase can be regulated in both a temporal and spatial manner and therefore these mouse models can be used to recapitulate the somatic mutation of BRAF that occurs in different tissues in the development of human cancer. The data so far obtained following Cre-mediated activation in haemopoietic tissue and the lung indicate that (V600E)BRAF mutation can drive tumour initiation and that its primary effect is to induce high levels of cyclin D1-mediated cell proliferation. However, hallmarks of OIS (oncogene-induced senescence) are evident that restrain further development of the tumour.

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.

Regulation of MEK/ERK pathway output by subcellular localization of B-Raf.

The strength and duration of intracellular signalling pathway activation is a key determinant of the biological outcome of cells in response to extracellular cues. This has been particularly elucidated for the Ras/Raf/MEK [mitogen-activated growth factor/ERK (extracellular-signal-regulated kinase) kinase]/ERK signalling pathway with a number of studies in fibroblasts showing that sustained ERK signalling is a requirement for S-phase entry, whereas transient ERK signalling does not have this capability. A major unanswered question, however, is how a cell can sustain ERK activation, particularly when ERK-specific phosphatases are transcriptionally up-regulated by the pathway itself. A major point of ERK regulation is at the level of Raf, and, to sustain ERK activation in the presence of ERK phosphatases, sustained Raf activation is a requirement. Three Raf proteins exist in mammals, and the activity of all three is induced following growth factor stimulation of cells, but only B-Raf activity is maintained at later time points. This observation points to B-Raf as a regulator of sustained ERK activation. In the present review, we consider evidence for a link between B-Raf and sustained ERK activation, focusing on a potential role for the subcellular localization of B-Raf in this key physiological event.

Abstract 1246: Development and validation of a global proteomics approach for identifying novel binding partners of resveratrol

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The dietary constituent resveratrol (trans-3,5,4′-trihydroxystilbene) has received a great deal of scientific attention and publicity due to its broad ranging pharmacological effects in preclinical systems, including cancer chemoprevention in a variety of rodent carcinogenesis models. It has been hypothesized that resveratrol exerts its effects by interacting directly with specific proteins such as cyclooxygenase 2 (COX2) and NAD(P)H dehydrogenase quinone 2 (NQO2). Nevertheless the key targets which may mediate its anticancer activity in humans have not yet been identified. An affinity purification (pull-down) assay coupled with global LC-MS/MS analysis was developed to identify proteins that bind directly to resveratrol. Resveratrol was synthetically linked via one of its phenol moieties to agarose beads using a short amine linker, generating two resveratrol bead species. Resveratrol beads were incubated with cell lysate from human-derived colon adenocarcinoma (HCA7) or adenoma cells (AAC1) and then washed to remove unbound proteins. Resveratrol-bound proteins were eluted from the beads by heat denaturation in SDS buffer. Control beads end-capped with ethanolamine were used to identify non-specific protein binding. Proteins were eluted from the beads and identified in trypsinised gel slices by PAGE coupled with either LC-MS/MS to identify all proteins bound to resveratrol or immunoblotting to identify known target proteins. More than 30 proteins were identified by LC-MS/MS that were reproducibly ‘pulled’ from cell lysates by resveratrol beads. The proteins identified fall into five main groups, including enzymes and proteins associated with nuclear transport, apoptosis, DNA repair, prostaglandin and fatty acid synthesis. Following MS/MS, 30 proteins were confirmed as selective binding partners of resveratrol by Western blotting, with 97% agreement between the two detection methods. Among the proteins identified were COX2, NQO2, fatty acid synthase (FAS) and Exportin 1 (CRM1). The first two are known resveratrol targets, which supports the validity of the method. Resveratrol at clinically achievable concentrations (0.001-10 μM) did not change the expression of the proteins identified in the pull-down assay. It is conceivable that resveratrol modulates the function of the proteins to which it binds. To test this hypothesis the effects of resveratrol on FAS activity and on Exportin 1 and its cargoes is currently under investigation. In conclusion, the protein pull-down assay described here may help discover mechanism-related protein biomarkers that can be used to monitor the efficacy of multi-targeted cancer chemopreventive diet constituents in individuals in chemoprevention trials. Citation Format: Christina J. Kovoor, Robert G. Britton, Emma Horner-Glister, Catherine Andreadi, Raj Singh, Rebekah Jukes-Jones, Kelvin Cain, William P. Steward, Andreas Gescher, Karen Brown. Development and validation of a global proteomics approach for identifying novel binding partners of resveratrol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1246. doi:10.1158/1538-7445.AM2014-1246