Ankur Karmokar

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 role of cancer stem cells in the anti-carcinogenicity of curcumin

Many cancers contain cell subpopulations that display characteristics of stem cells. These cells are characterised by their ability to self-renew, form differentiated progeny and develop resistance to chemotherapeutic strategies. Cancer stem cells may utilise many of the same signalling pathways as normal stem cells including Wnt, Notch and Hedgehog. The dietary agent curcumin exerts a plethora of anti-carcinogenic effects both in vitro and in vivo, and can also inhibit many of the signalling pathways associated with stem cell biology. Emerging evidence suggests that curcumin can exert its anti-carcinogenic activity via targeting cancer stem cells through the disruption of stem cell signalling pathways. In this review we summarise the ability of curcumin to interfere with signalling pathways Wnt, Hedgehog, Notch, Signal Transducers and Activator (STAT) and interleukin-8, and report curcumin-induced changes in function and properties of cancer stem cells. We present evidence that the effects of curcumin on cancer stem cells mediate, or contribute to, its anti-carcinogenic activity.

Dietary intake of rosmarinic acid by Apc(Min) mice, a model of colorectal carcinogenesis: levels of parent agent in the target tissue and effect on adenoma development.

SCOPE Rosmarinic acid (RA), a constituent of culinary herbs is considered to possess cancer chemopreventive properties. It has been shown to inhibit the development of cancer in preclinical models but data are conflicting and whether it can protect against gastrointestinal malignancies in vivo has not been examined. This study aimed to investigate the effect of RA on the development of intestinal adenomas in the Apc(Min) mouse model of colorectal carcinogenesis, and to correlate efficacy with levels of RA achieved in the plasma and gastrointestinal tract. METHODS AND RESULTS RA inhibited the growth of APC10.1 cells derived from Apc(Min) mouse adenomas, with an IC₅₀ of 43 μM. Consumption of dietary RA (0.3%) by Apc(Min) mice for 8 weeks post weaning decreased adenoma burden by ∼35%, but the difference from controls was not significant. Although RA significantly decreased the frequency of large adenomas, the number of small ones increased. Using a novel validated HPLC assay, average levels of RA in the plasma and intestinal mucosa of these mice were found to be 1.1 μM and 38 nmol/g, respectively. CONCLUSION Chronic consumption of RA furnished quantifiable levels of parent compound in the plasma and intestinal tract of Apc(Min) mice and may slow adenoma development.

Targeting cancer stem‐like cells using dietary‐derived agents – where are we now?

Diet has been linked to an overwhelming proportion of cancers. Current chemotherapy and targeted therapies are limited by toxicity and the development of resistance against these treatments results in cancer recurrence or progression. In vitro evidence indicates that a number of dietary-derived agents have activity against a highly tumorigenic, chemoradiotherapy resistant population of cells within a tumour. This population is associated with cancer recurrence and is therefore clinically significant. Targeting this subpopulation, termed cancer stem-like cells with dietary-derived agents provides a potentially low toxicity strategy to enhance current treatment regimens. In addition, dietary-derived compounds also provide a novel approach to cancer prevention strategies. This review focusses on selected diet-derived agents that have been shown to specifically target cancer stem-like cells using in vivo models, or in clinical trials. Furthermore, the potential limitations of these studies are discussed, and areas of research that need to be addressed to allow successful translation of dietary-derived agents to the clinical arena are highlighted.

Characterization and Propagation of Tumor Initiating Cells Derived from Colorectal Liver Metastases: Trials, Tribulations and a Cautionary Note

Tumor initiating cells (TIC) are increasingly being put forward as a potential target for intervention within colorectal cancer. Whilst characterisation and outgrowth of these cells has been extensively undertaken in primary colorectal cancers, few data are available describing characteristics within the metastatic setting. Tissue was obtained from patients undergoing surgical resection for colorectal liver metastases, and processed into single cell suspension for assessment. Tumor initiating cells from liver metastases were characterised using combinations of EPCAM, Aldehyde dehydrogenase activity, CD133 and CD26. CD133 expression was significantly lower in patients who had received chemotherapy, but this was accounted for by a decrease observed in the male patient cohort only. ALDHhigh populations were rare (0.4 and 0.3% for EPCAM+/ALDHhigh/CD133- and EPCAM+/ALDHhigh/CD133+ populations respectively) and below the limits of detection in 28% of samples. Spheroid outgrowth of metastatic tumor cells across all samples could not be readily achieved using standard spheroid-formation techniques, thus requiring further method validation to reliably propagate cells from the majority of tissues. Spheroid formation was not enhanced using additional growth factors or fibroblast co-culture, but once cells were passaged through NOD-SCID mice, spheroid formation was observed in 82% samples, accompanied by a significant increase in CD26. Order of spheroid forming ability was ALDHhigh>CD133>CD26. Samples sorted by these markers each had the ability to reform ALDHhigh, CD133 and CD26 positive populations to a similar extent, suggestive of a high degree of plasticity for each population. Ex vivo TIC models are increasingly being utilised to assess efficacy of therapeutic interventions. It is therefore essential that such investigations use well-characterised models that are able to sustain TIC populations across a large patient cohort in order that the inherent heterogeneity observed in cancer populations is maintained.

Abstract 258: Targeting Nanog in 3D explant models for the evaluation of cancer prevention agents

Background: The transcription factor Nanog is crucial for the self-renewal of cancer stem-like cells (CSCs). Nanog expression in colorectal cancer (CRC) tissue correlates with lymph node metastasis and poor prognosis. Since Nanog is not expressed in most tissues, including normal adult stem-cells, it represents a therapeutic target specific to cancer cells. Curcumin inhibits proliferation and expansion of CSCs derived from CRC and adenomas. In NOD/SCID mice bearing xenografts from patient-derived CRC CSCs, curcumin significantly inhibited tumour growth and improved survival. In addition, curcumin binds directly to Nanog recombinant protein (quantified using microscale thermophoresis). We have developed 3D in-vitro primary human explant models to further characterise the effects of curcumin on Nanog. In this work the hypothesis is tested that Nanog may be an early marker of response for CRC prevention agents. Methods: Patient-derived CRC and adenoma tissue was cubed (2x2x2mm) and treated for 24 hours with curcumin. Following treatment, explant tissues were processed for analysis by immunohistochemistry (IHC) (n=6) and flow cytometry (n=17). The effect of curcumin on CSCs (defined by expression of aldehyde dehydrogenase (ALDH) or Nanog) and differentiation (via Mucin 2 expression) was analysed using IHC. Additionally, cells expressing Nanog (Nanog+) or Nanog plus proliferation marker Ki67 (Nanog+Ki67+) were assessed using flow cytometry. Results: A range of adenoma (n=5) and Dukes stage A-C CRC (n=18) samples were studied. Following exposure to curcumin, a 30% reduction was observed in Nanog+ and Nanog+Ki67+ cells. Nanog+ cell number was decreased in a curcumin concentration-dependent fashion in 6 samples and concentration-independently in a further 8. No response was observed in 3 samples. A reduction in Nanog and ALDH with concurrent increase in differentiation was observed via IHC in one sample. Conclusion: Our data suggest Nanog is targeted by curcumin in adenoma and CRC tissues. Nanog may serve as a biomarker in clinical trials to identify individuals most amenable to treatment with curcumin alone or in combination treatment. Crucially, this will help select those who are likely to benefit from curcumin as a cancer prevention agent. Ultimately, this concept may be applicable to the evaluation of novel CRC prevention agents. Citation Format: Sam Khan, Ankur Karmokar, Zahirah Sidat, Nalini Foreman, David Moore, Jennifer Higgins, Emma Parrott, Despoina Theofanous, Dominic Hobbs, Lynne Howells, Anne Thomas, Karen Brown. Targeting Nanog in 3D explant models for the evaluation of cancer prevention agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 258.