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Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol, which induces autophagy in cancer cells a
a
a
a
Catherine Andreadi , Robert G Britton , Ketan R Patel & Karen Brown a
Department of Cancer Studies and Molecular Medicine; University of Leicester; Leicester, UK Published online: 09 Jan 2014.
Click for updates To cite this article: Catherine Andreadi, Robert G Britton, Ketan R Patel & Karen Brown (2014) Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol, which induces autophagy in cancer cells, Autophagy, 10:3, 524-525, DOI: 10.4161/auto.27593 To link to this article: http://dx.doi.org/10.4161/auto.27593
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Autophagic Punctum
Autophagy 10:3, 524–525; March 2014; © 2014 Landes Bioscience
Catherine Andreadi, Robert G Britton, Ketan R Patel, and Karen Brown*
Downloaded by [Michigan State University] at 19:03 15 February 2015
Department of Cancer Studies and Molecular Medicine; University of Leicester; Leicester, UK
R
esveratrol 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.
Keywords: resveratrol, sulfate, autophagy, senescence, colon cancer, metabolism Abbreviations: FACS, fluorescenceactivated cell sorting Submitted: 12/04/2013 Revised: 12/17/2013 Accepted: 12/19/2013 http://dx.doi.org/10.4161/auto.27593 *Correspondence to: Karen Brown; Email: [email protected] Punctum to: Patel KR, Andreadi C, Britton RG, Horner-Glister E, Karmokar A, Sale S, Brown VA, Brenner DE, Singh R, Steward WP, et al. Sulfate metabolites provide an intracellular pool for resveratrol generation and induce autophagy with senescence. Sci Transl Med 2013; 5:ra133; PMID:24089405; http://dx.doi.org/10.1126/ scitranslmed.3005870
Resveratrol (trans-3,5,4′trihydoxystilbene), is a phytochemical found in a variety of dietary sources including mulberries, peanuts, and the skin of red grapes, hence in red wine. Numerous health benefit claims have been attributed to resveratrol based on preclinical observations, including life extension, protection against cardiovascular and neurodegenerative diseases, reducing the deleterious burden of obesity, and cancer prevention properties. Upon ingestion, resveratrol is rapidly and extensively metabolized to sulfate and glucuronide conjugates, which limits its systemic bioavailability; this has raised questions over its ability to exert efficacy in humans. We set out to confirm or refute the long held idea that resveratrol sulfates actually contribute to activity by regenerating resveratrol in vivo, which may help to rationalize
524 Autophagy
the various beneficial effects reported in animal models and allow an improved assessment of the clinical potential of this agent. To truly interrogate the effects of any chemical on humans it is crucial that clinically achievable concentrations of the compound are used in preclinical in vivo and in vitro mechanistic studies and this can only be made possible from detailed human pharmacokinetic data. It is probable that much of the published information on the anticancer effects of resveratrol may in fact be irrelevant, due to the use of excessively high concentrations. To this end, we completed trials in healthy volunteers and colorectal cancer patients and quantitatively measured the resveratrol metabolite profile in plasma and colorectal tissue, respectively, following resveratrol ingestion, defining for the first time the concentration range of metabolites that must be used to produce data relevant to humans. We then conducted a pharmacokinetic study in mice to provide the first experimental proof that the major human metabolites, resveratrol monosulfates, are absorbed after oral administration and are then hydrolyzed to afford free resveratrol in plasma and tissues, including colorectal mucosa. The resveratrol generated in this manner persists for over 6 h post dosing, providing sustained exposure, which may account for the efficacy of resveratrol described in numerous mouse models, despite apparent low bioavailability. To ascertain whether sulfate metabolites might also provide a source of resveratrol in humans, 3 human colorectal cell lines were incubated with clinically achievable
Volume 10 Issue 3
©2014 Landes Bioscience. Do not distribute.
Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol, which induces autophagy in cancer cells
Downloaded by [Michigan State University] at 19:03 15 February 2015
concentrations. The degree of sulfate uptake and subsequent intracellular conversion to resveratrol differed across the cell lines but there was a remarkable correlation with antiproliferative activity. Highest concentrations of sulfates and resveratrol were attained in HT-29, followed by HCA-7 cells, which are both derived from malignant cancers. Accordingly, the most pronounced growth inhibition was observed in HT-29 cells. In contrast, resveratrol sulfates fail to enter HCEC cells, which were originally developed from normal epithelium, and as a consequence have no effect on cell numbers. These differences may be explained by higher relative expression of specific membrane transporters in the cancer cells, including OATP1B3, which contributes to sulfate uptake in HT-29 cells based on studies with a chemical inhibitor. The antiproliferative effects of resveratrol-sulfates could not be attributed to growth arrest, apoptosis or necrosis, as assessed by FACS analysis, in either of the affected cell lines (HT-29 and HCA-7), but transmission electron microscopy of HT-29 cells treated with resveratrol-sulfates revealed the presence of autophagomes, late-stage autophagic compartments, and autolysosomes, which are diagnostic of autophagy. Further analysis showed that resveratrol-sulfates
significantly increase the conversion of soluble microtubule-associated protein 1 light chain 3 (LC3-I) to lipid-bound LC3-II, which is then recruited to the phagophore membrane and leads to the initiation of autophagy, an effect that is not observed in normal HCEC cells. Resveratrol sulfates also causes significant upregulation of CDKN1A/p21 and increased SA-β-gal staining in HT-29 cells, but not in normal HCEC cells, suggesting that the cancer cells are becoming senescent. Importantly, both of these effects are diminished when HT-29 cells are coincubated with resveratrol-sulfate and the potent steroid sulfatase inhibitor estrone 3-O-sulfamate (EMATE). EMATE significantly reduces both the intracellular concentration of resveratrol and the conversion of LC3-I to LC3-II with respect to resveratrol-sulfate treatment alone. The expression of CDKN1A is also reduced with the addition of EMATE suggesting that resveratrol itself, not the sulfate metabolites, is responsible for the induction of autophagy and senescence in these colorectal cancer cells. Surprisingly, incubation of cells with clinically attainable concentrations of resveratrol fails to induce autophagy, due to lower intracellular levels of resveratrol compared with concentrations generated via sulfate hydrolysis; this suggests resveratrol
produced indirectly may be more important for in vivo activity than the unmetabolized compound. Since first being reported as a cancer preventive compound in 1997, resveratrol has been studied in myriad systems in vivo, in vitro and more recently in clinical trials. Based on published data, resveratrol can have a protective function in both settings of autophagy, as an oncogenic or tumor suppressor mechanism in carcinogenesis. Furthermore, while the role of autophagy in cancer is paradoxical, in this system it appears to be beneficial. These results explain how the major resveratrol metabolites in humans may contribute to activity, which is important for justifying the use of resveratrol in the treatment or prevention of a variety of diseases, especially those that require systemic delivery to target tissues. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
Work supported by Cancer Research UK (C325/A6691), the Leicester Experimental Cancer Medicine Centre (C325/A15575, funded by Cancer Research UK/UK Department of Health) and NCI-N01-CN-25025.
www.landesbioscience.com Autophagy 525
©2014 Landes Bioscience. Do not distribute.
Autophagic Punctum
Autophagic Punctum
Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol, which induces autophagy in cancer cells a
a
a
a
Catherine Andreadi , Robert G Britton , Ketan R Patel & Karen Brown a
Department of Cancer Studies and Molecular Medicine; University of Leicester; Leicester, UK Published online: 09 Jan 2014.
Click for updates To cite this article: Catherine Andreadi, Robert G Britton, Ketan R Patel & Karen Brown (2014) Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol, which induces autophagy in cancer cells, Autophagy, 10:3, 524-525, DOI: 10.4161/auto.27593 To link to this article: http://dx.doi.org/10.4161/auto.27593
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Autophagic Punctum
Autophagy 10:3, 524–525; March 2014; © 2014 Landes Bioscience
Catherine Andreadi, Robert G Britton, Ketan R Patel, and Karen Brown*
Downloaded by [Michigan State University] at 19:03 15 February 2015
Department of Cancer Studies and Molecular Medicine; University of Leicester; Leicester, UK
R
esveratrol 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.
Keywords: resveratrol, sulfate, autophagy, senescence, colon cancer, metabolism Abbreviations: FACS, fluorescenceactivated cell sorting Submitted: 12/04/2013 Revised: 12/17/2013 Accepted: 12/19/2013 http://dx.doi.org/10.4161/auto.27593 *Correspondence to: Karen Brown; Email: [email protected] Punctum to: Patel KR, Andreadi C, Britton RG, Horner-Glister E, Karmokar A, Sale S, Brown VA, Brenner DE, Singh R, Steward WP, et al. Sulfate metabolites provide an intracellular pool for resveratrol generation and induce autophagy with senescence. Sci Transl Med 2013; 5:ra133; PMID:24089405; http://dx.doi.org/10.1126/ scitranslmed.3005870
Resveratrol (trans-3,5,4′trihydoxystilbene), is a phytochemical found in a variety of dietary sources including mulberries, peanuts, and the skin of red grapes, hence in red wine. Numerous health benefit claims have been attributed to resveratrol based on preclinical observations, including life extension, protection against cardiovascular and neurodegenerative diseases, reducing the deleterious burden of obesity, and cancer prevention properties. Upon ingestion, resveratrol is rapidly and extensively metabolized to sulfate and glucuronide conjugates, which limits its systemic bioavailability; this has raised questions over its ability to exert efficacy in humans. We set out to confirm or refute the long held idea that resveratrol sulfates actually contribute to activity by regenerating resveratrol in vivo, which may help to rationalize
524 Autophagy
the various beneficial effects reported in animal models and allow an improved assessment of the clinical potential of this agent. To truly interrogate the effects of any chemical on humans it is crucial that clinically achievable concentrations of the compound are used in preclinical in vivo and in vitro mechanistic studies and this can only be made possible from detailed human pharmacokinetic data. It is probable that much of the published information on the anticancer effects of resveratrol may in fact be irrelevant, due to the use of excessively high concentrations. To this end, we completed trials in healthy volunteers and colorectal cancer patients and quantitatively measured the resveratrol metabolite profile in plasma and colorectal tissue, respectively, following resveratrol ingestion, defining for the first time the concentration range of metabolites that must be used to produce data relevant to humans. We then conducted a pharmacokinetic study in mice to provide the first experimental proof that the major human metabolites, resveratrol monosulfates, are absorbed after oral administration and are then hydrolyzed to afford free resveratrol in plasma and tissues, including colorectal mucosa. The resveratrol generated in this manner persists for over 6 h post dosing, providing sustained exposure, which may account for the efficacy of resveratrol described in numerous mouse models, despite apparent low bioavailability. To ascertain whether sulfate metabolites might also provide a source of resveratrol in humans, 3 human colorectal cell lines were incubated with clinically achievable
Volume 10 Issue 3
©2014 Landes Bioscience. Do not distribute.
Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol, which induces autophagy in cancer cells
Downloaded by [Michigan State University] at 19:03 15 February 2015
concentrations. The degree of sulfate uptake and subsequent intracellular conversion to resveratrol differed across the cell lines but there was a remarkable correlation with antiproliferative activity. Highest concentrations of sulfates and resveratrol were attained in HT-29, followed by HCA-7 cells, which are both derived from malignant cancers. Accordingly, the most pronounced growth inhibition was observed in HT-29 cells. In contrast, resveratrol sulfates fail to enter HCEC cells, which were originally developed from normal epithelium, and as a consequence have no effect on cell numbers. These differences may be explained by higher relative expression of specific membrane transporters in the cancer cells, including OATP1B3, which contributes to sulfate uptake in HT-29 cells based on studies with a chemical inhibitor. The antiproliferative effects of resveratrol-sulfates could not be attributed to growth arrest, apoptosis or necrosis, as assessed by FACS analysis, in either of the affected cell lines (HT-29 and HCA-7), but transmission electron microscopy of HT-29 cells treated with resveratrol-sulfates revealed the presence of autophagomes, late-stage autophagic compartments, and autolysosomes, which are diagnostic of autophagy. Further analysis showed that resveratrol-sulfates
significantly increase the conversion of soluble microtubule-associated protein 1 light chain 3 (LC3-I) to lipid-bound LC3-II, which is then recruited to the phagophore membrane and leads to the initiation of autophagy, an effect that is not observed in normal HCEC cells. Resveratrol sulfates also causes significant upregulation of CDKN1A/p21 and increased SA-β-gal staining in HT-29 cells, but not in normal HCEC cells, suggesting that the cancer cells are becoming senescent. Importantly, both of these effects are diminished when HT-29 cells are coincubated with resveratrol-sulfate and the potent steroid sulfatase inhibitor estrone 3-O-sulfamate (EMATE). EMATE significantly reduces both the intracellular concentration of resveratrol and the conversion of LC3-I to LC3-II with respect to resveratrol-sulfate treatment alone. The expression of CDKN1A is also reduced with the addition of EMATE suggesting that resveratrol itself, not the sulfate metabolites, is responsible for the induction of autophagy and senescence in these colorectal cancer cells. Surprisingly, incubation of cells with clinically attainable concentrations of resveratrol fails to induce autophagy, due to lower intracellular levels of resveratrol compared with concentrations generated via sulfate hydrolysis; this suggests resveratrol
produced indirectly may be more important for in vivo activity than the unmetabolized compound. Since first being reported as a cancer preventive compound in 1997, resveratrol has been studied in myriad systems in vivo, in vitro and more recently in clinical trials. Based on published data, resveratrol can have a protective function in both settings of autophagy, as an oncogenic or tumor suppressor mechanism in carcinogenesis. Furthermore, while the role of autophagy in cancer is paradoxical, in this system it appears to be beneficial. These results explain how the major resveratrol metabolites in humans may contribute to activity, which is important for justifying the use of resveratrol in the treatment or prevention of a variety of diseases, especially those that require systemic delivery to target tissues. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
Work supported by Cancer Research UK (C325/A6691), the Leicester Experimental Cancer Medicine Centre (C325/A15575, funded by Cancer Research UK/UK Department of Health) and NCI-N01-CN-25025.
www.landesbioscience.com Autophagy 525
©2014 Landes Bioscience. Do not distribute.
Autophagic Punctum
Autophagic Punctum
