resveratrol: challenges in translation to the clinic · 29/10/2010 · now that 60 minutes,...
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1 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Copyright © 2010 American Association for Cancer Research
Resveratrol: challenges in translation to the clinic
A critical discussion
Lalita Subramanian, Sherry Youssef, Saswati Bhattacharya, Jason Kenealey, Arthur S.
Polans and Paul R. van Ginkel
The University of Wisconsin Carbone Cancer Center, the Department of Ophthalmology
and Visual Sciences, the UW Eye Research Institute, and the Department of
Biomolecular Chemistry, University of Wisconsin, Madison 53792
Corresponding Author: Paul van Ginkel, Department of Ophthalmology and Visual
Sciences, University of Wisconsin School of Medicine and Public Health, Room K6/454
Clinical Sciences Center, 600 Highland Avenue, Madison, WI 53792. Phone: 608-265-
5409; Fax: 608-265-6021; E-mail: prvangin@wisc.edu
Statement of Translational Relevance
Side effects from chemotherapy highlight the need for effective non-toxic anti-cancer
agents. Resveratrol has consistently proven effective at tumor inhibition in diverse
human cancer models, while remaining non-toxic. This suggests that resveratrol could
pioneer a novel class of chemotherapeutics. It already is being extensively promoted in
the unregulated nutraceutical sector despite recent controversies regarding target
interaction, bioavailability and other essential matters. However, fundamental aspects of
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Copyright © 2010 American Association for Cancer Research
resveratrol action need to be understood before it can be developed into a clinically
viable anti-cancer drug. We present a short but critical discussion of these issues and
suggest important experiments that could pave the way to the successful translation of
resveratrol to the clinic.
Abstract
Low cancer survival rates and the serious side effects often associated with current
chemotherapeutics highlight the need for new and effective non-toxic anti-cancer
agents. Since 1997 when Jang et al. first described resveratrol’s ability to inhibit
carcinogenesis, it has consistently proven effective at tumor inhibition in diverse human
cancer models. This has raised the hope that resveratrol would pioneer a novel class of
non-toxic chemotherapeutics. As a consequence of initial basic and pre-clinical studies,
resveratrol is now being extensively promoted in the unregulated nutraceutical sector.
However, some fundamental aspects of resveratrol’s action need to be understood
before it can be developed into a clinically viable anti-cancer drug. These pertain to the
key mechanism(s) by which resveratrol potentiates its anti-tumor effects. Current
research suggests that these might be through novel pathways requiring an
understanding of cellular uptake, sentinel targets and in vivo biological networks. The
metabolism of resveratrol and its bioavailablity also warrant further consideration in light
of recent in vitro and in vivo studies. Finally, we need to appreciate the sorts of
information about resveratrol that may translate between different disease entities. We
present a critical discussion of these issues and suggest important experiments that
could pave the way to the successful translation of resveratrol to the clinic.
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Now that 60 minutes, Barbara Walters, National Public Radio, Dr. Oz and a
myriad of columnists at the New York Times and other papers have offered their
comments and recommendations regarding the health benefits of resveratrol, including
its potential anti-cancer properties, perhaps it’s time for scientists to ask a few essential
questions about the popular product before considering a further pitch to the public.
In 1997 Jang et al. published their seminal paper on resveratrol’s ability to inhibit
the three major stages of carcinogenesis (1). Since then, there has been an explosion
of literature about resveratrol’s effects on cancer. Some fundamental aspects of our
understanding of resveratrol, however, are yet to be resolved. These gaps in our
knowledge must be addressed before the full potential of this non-toxic compound as an
anti-cancer drug can be realized.
I. Cellular uptake
What evidence do we have that resveratrol actually enters cancer cells?
A critical first step to delineate the mechanisms of drug action is to determine
whether resveratrol mediates its effects by cell surface receptors or intracellular targets
in tumor and other cells. Both intra and extracellular resveratrol targets have been
proposed (see section II). However, a direct binding partner has yet to be convincingly
established. Elucidation of whether resveratrol enters cells or acts at the cell surface will
shed some light on the targets on which it acts.
Resveratrol is a hydrophobic compound and currently the only compelling
evidence for entry of resveratrol into cells comes from highly specialized cell types:
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intestinal and liver cells. When a monolayer of colonic Caco-2 cells was treated in vitro
with resveratrol, a saturating dose-dependent apical to basolateral transport was
observed (2, 3). In another study, the human hepatoblastoma cell line HepG2 and
human hepatocytes, were used to compare resveratrol transport in normal and tumor
cells (4). Resveratrol uptake was detected by its intrinsic fluorescence properties as well
as by using radiolabeled drug. Concentration- and temperature-dependence of uptake
were examined and results indicated that both passive diffusion and carrier-mediated
transport might be involved.
The above cell types, however, have specialized uptake mechanisms. The
intestinal epithelium is adapted for absorption of nutrients from food. A study of drug
transport using a Caco-2 monolayer may indicate transepithelial transport of a drug, but
does not necessarily imply that other cells have similar uptake mechanisms. Similarly,
one of the main functions of hepatocytes is to extract protein-bound drugs and
metabolites from circulation. Consequently, hepatocytes are equipped with many
different transport proteins in the basolateral membrane. The only other evidence of
possible resveratrol uptake has been shown indirectly through sulfotransferase 1A1
activity associated with two breast cancer cell lines resulting in the conversion of
exogenously added resveratrol to resveratrol 3-O-sulfate (5). These studies have not
been replicated in other tumor cells.
If resveratrol cannot be shown to efficiently enter all tumor cells, it may be more
likely that its anti-tumor effect is the result of binding to an extracellular target.
Therefore, further studies are necessary to directly address cellular uptake in cancer
cells. New technology, such as two-photon microscopy, may be particularly appropriate
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for detecting the real-time uptake of unmodified resveratrol and its metabolites by taking
advantage of their intrinsic fluorescence under conditions of minimal photo-bleaching.
Results of such studies will indicate the validity of intracellular targets identified thus far
and aid in the identification of new sentinel targets.
II. Targets — fast and slow
What are the direct targets of resveratrol and can we differentiate binding events
from indirect effects of resveratrol?
The wealth of resveratrol literature is in part a result of the myriad of pathways
and molecules that are altered in response to resveratrol treatment (reviewed in 6).
Some of these effects may be cell type specific, tissue type specific or depend on
whether a cell is transformed. Through parametric analysis of gene set enrichment
(PAGE analysis), resveratrol has been found to cause a significant alteration in 127
pathways (7). Certain effects of drug treatment are measurable in a matter of seconds
(8), whereas others only become apparent after hours or even days. This then brings up
the question which of these effects is a direct result of resveratrol binding and which are
downstream effects of the initial target interaction.
Several molecular targets have been postulated as mediators of resveratrol’s
anti-cancer effects (7). Approaches such as a phage-display screen using biotinylated
resveratrol identified several candidates (9). However, the direct binding of resveratrol
to these targets in a cellular context has yet to be convincingly demonstrated.
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An example of a direct binding target identified through column chromatography
is quinone reductase 2 (10). The binding constant for NQO2 was in a physiologically
relevant range. However, not all tumor cells have measurable levels of this protein
despite having similar sensitivity to the drug (11). Therefore, binding to this protein may
not be part of a general mechanism for resveratrol anti-tumor action.
Other targets reported include DNA polymerase α, PKC, PKD, COX2,
ribonucleotide reductase, and F0/F1 ATPase/ATP synthase based on alterations of
enzymatic activity. Only F0/F1 ATPase/ATP synthase however has been shown to
interact directly with resveratrol. An additional complication in identifying and validating
such targets is that binding and enzymatic experiments in vitro or in cells in culture may
not reflect in vivo conditions. Since levels of unmetabolized resveratrol in vivo don’t
exceed the low micromolar range (11), it is possible that some observed target binding
and modulations of pathways are only induced because of exposure of isolated
enzymes or cells to constant and high drug concentrations in vitro.
A more recent group of potential targets, the sirtuins, has been the subject of
intense research as mediators of resveratrol’s life extending activity as well as anti-
tumor activity. The direct binding of resveratrol to sirtuins has not been shown. Early
work showing direct resveratrol activation of purified SIRT1 using a fluorescently
modified substrate for sirtuin was shown to be an artifact of the assay (12). Additionally,
the effects of resveratrol on sirtuins in cells appear to be measureable only after several
hours. This is suggestive of downstream or indirect effects, if at all. More recent studies
also conflict with the original observation that resveratrol has an effect on Sir2 activity in
vivo (13) or an effect on the lifespan of yeast (13), Drosophila, or C. elegans (14).
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Extracellular receptors for resveratrol such as insulin growth factor receptor
(IGFR) and integrin αVß3 have been proposed. The IGFR study compared downstream
activity using known ligands (15). No direct binding was shown in this study. The αVß3
study relied on competition assays using a known ligand and binding studies using
membrane fractions (16). Radiolabelled resveratrol was used to suggest direct binding
to the ß3 subunit of the integrin. However, given its properties, resveratrol is likely to
associate with membranes readily, making it difficult to assess the specificity of these
binding studies. Even though further work is needed to establish that resveratrol binds
to these extracellular receptors, these studies suggest that resveratrol could activate
several extracellular targets.
One of the very early in vitro events observed in tumor cells following resveratrol
addition is an increase in intracellular calcium and can be measured within seconds
after drug treatment (8). Calcium signaling (Fig. 1) may be a shared early response
underlying the non-toxic feature of different anti-cancer natural products (17). If so, it
may be possible to define a novel calcium-based mechanistic model for the
development of a new generation of non-toxic chemotherapeutics using resveratrol as a
model compound. However, the sentinel targets involved in calcium activation are yet to
be identified. Further, we have to recognize that some binding targets for resveratrol
activate early events while others might potentiate later events.
In spite of important progress, the binding targets of resveratrol that mediate its
anti-cancer activity remain unknown. The relevant targets mediating effects in vitro
versus in vivo might differ, and there appear to be several targets within a single cell
type as well as differences between cell types. To better understand the mechanisms
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through which resveratrol mediates its anti-cancer properties, studies need to better
reflect conditions in vivo at drug levels that lead to inhibition of tumor growth.
III. Resveratrol Actions
Is resveratrol anti-proliferative, pro-apoptotic and anti-angiogenic?
In vitro studies of resveratrol using a variety of tumor cell lines have shown the
drug to be anti-proliferative as well as pro-apoptotic, through the activation of many
different pathways. Resveratrol inhibits cell cycle progression by modulating major cell
cycle regulators, arresting cells at the G1/S, S or G2/M phase and by inhibiting DNA
synthesis. These effects, however, have not been demonstrated in vivo. The
mechanisms causing tumor inhibition therefore are unclear even though tumor inhibition
is observed in many different animal models.
Alternatively, it is well established that angiogenesis is critical to tumor
progression. Resveratrol has been reported to have an anti-angiogenic effect in certain
tumor models as indicated by decreased vessel density in treated tumors (18-21).
However, an observed decrease in microvessel density cannot differentiate between
cause and effect. Either the inhibition of tumor growth results in fewer blood vessels or
an anti-angiogenic effect causes decreased tumor outgrowth due to a lack of nutrients
and oxygen. The tumor-inhibitory effects in vivo could be due either to direct effects of
resveratrol on tumor cell growth or indirect effects on angiogenesis. An initial study in a
mouse model of Lewis Lung Carcinoma indicated a resveratrol effect on
neovascularization (19). However, more in vivo studies are needed to show a direct
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effect of resveratrol on blood vessel formation as the mechanism for tumor inhibition
using cell-specific reporter systems.
In contrast to tumor inhibition, higher local doses of resveratrol result in massive
tumor cell death and tumor regression as indicated by TUNEL staining and histology
(11, 22). Thus the reported pro-apoptotic effects of resveratrol may only be relevant
when local concentrations of resveratrol more closely match the higher concentrations
studied in vitro. Therefore, strategies to improve the bioavailability of resveratrol either
systemically or locally will provide more potent anti-cancer effects.
IV. Bioavailability
How do we reconcile the anti-cancer effects of resveratrol determined from in
vitro studies with in vivo measurements of resveratrol bioavailability?
Preclinical studies using resveratrol in animal models of different human cancers
indicate that resveratrol has potent anti-tumor properties. However, measurements of
serum levels of unmetabolized resveratrol suggest that its bioavailability is very low after
oral or intraperitoneal administration of the drug. This is mainly due to poor absorption
and ready metabolism to glucuronidated and sulfated compounds, followed by rapid
clearance of these metabolites. Serum levels of unmetabolized resveratrol peak in the
sub to low micromolar range (<3 µM) within minutes of oral drug administration, and
decrease rapidly thereafter (11, 23-26). One implication of these findings is that the
direct activation of cellular targets by resveratrol needs to reflect this transient
occurrence of both resveratrol and the metabolites. In addition, no accumulation of
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resveratrol in tumors or normal tissues has been found after extended treatment
regimens (11). These data contrast sharply with in vitro studies wherein sustained
concentrations of tens of micromolars are required to obtain substantive anti-tumor
effects. This apparent conundrum was discussed elegantly by Gescher and Steward
(27). These observations may suggest one of two different things: 1) the mechanisms of
action of resveratrol are different in vivo and in vitro; or 2) the in vitro conditions for
growing cells somehow alter the sensitivity to resveratrol. In the first case, the anti-
angiogenic activity of resveratrol could indirectly inhibit tumor growth. Such properties of
the tumor environment are not recapitulated in vitro. In the second case, growth of
tumor cells on a two dimensional surface, the presence of high concentrations of growth
factors, high O2 levels and lack of inhibitory cell-cell contacts with surrounding normal
cells could decrease the sensitivity of cells to resveratrol. These optimized growth
conditions for tumor cells in culture may necessitate higher drug concentrations in vitro.
Reports of the relevant serum levels of resveratrol are further complicated by
recent studies and unpublished observations indicating that resveratrol metabolites do
not inhibit tumor cell growth (28, 29) and Subramanian unpublished. This suggests that
glucuronidation and sulfation not only target the drug for removal from the body, but
also mitigate its anti-tumor activity. Minor conversion back to resveratrol by local
sulfatases, for example, would not compensate for this rapid decrease in activity. To
increase the potency of resveratrol treatment, it will be necessary to increase the
amount of unmetabolized compound at the tumor site. A major obstacle to attaining this
goal systemically is the low solubility of the drug. Future research should be directed
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towards new formulations and analogs that increase its bioavailability and delay its
metabolism (Fig. 2).
The bioavailability conundrum also brings up the issue of resveratrol’s ability to
differentiate between normal cells and cancerous cells in animal models. At low
concentrations where there is no evidence of cell death either in normal or tumor tissue,
there is nevertheless an anti-tumor effect of the drug leading to tumor inhibition. At
higher local concentrations where there is significant evidence for cell death in tumor
tissue, the surrounding normal tissue remains unaffected (11). This discrimination
between normal and tumor cells could arise due to differences in: 1) the cellular uptake
of resveratrol; 2) the expression of cellular targets or 3) a differential ability to modify
resveratrol into less active forms. Studies of these processes are necessary to
understand the non-toxic nature of resveratrol.
V. Consistencies between disease processes
Can the findings from resveratrol research done in the realm of cancer be
generalized to cardioprotection / life prolongation / diabetes prevention / and vice
versa?
Resveratrol has a wide variety of pharmacological activities making its study
relevant to several diseases such as cancer, cardiovascular and neurodegenerative
diseases and diabetes. However, it is not always clear how the same drug causes
different or even opposite effects in different cell types and in different diseases. For
example, resveratrol causes cell death in tumor cells but is effective as an anti-apoptotic
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or protective agent in nerve cells (30). Similarly, resveratrol is anti-angiogenic in certain
tumor models yet pro-angiogenic in the case of myocardial infarction (31, 32).
Resveratrol increases mitochondrial health in muscle cells while it induces
mitochondria-mediated apoptosis in tumor cells (33). How might this occur? While most
direct targets of resveratrol may be expressed in a variety of different cell types albeit at
different levels, intracellular signaling networks will be different depending on the cell
type and cell status (non-dividing, dividing, tumorigenic) leading to different outcomes
following initial target interaction.
Epidemiologic studies suggest a correlation between diabetes and cancer risk.
Resveratrol has been shown to exhibit anti-diabetic effects. A recent report of the use of
the anti-diabetic drug metformin in cancer treatment suggests that resveratrol’s anti-
diabetic effects may be relevant to its anti-tumor effects as well (32, 34). Therefore,
findings of resveratrol action will be of potential cross-disease importance. As different
molecular pathways are elucidated in greater detail and are incorporated into larger
networks, it will become increasingly apparent how addition of a drug like resveratrol
can still be effective in very disparate diseases.
Many of these diseases are prevalent simultaneously, particularly in elderly
patients. Based on current literature, it would appear that resveratrol could ameliorate
secondary conditions even as it is used to treat a primary disease. However, it is
necessary to carry out comprehensive clinical trials on both the preventive potential as
well as curative efficacy of resveratrol in different diseases before such claims can be
validated.
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VI. Market Claims
What can we tell the public?
Resveratrol has great potential as an anti-cancer drug either as primary or as
adjuvant therapy. One of its major benefits is a lack of toxicity at doses needed for its
anti-tumor effect. Toxicity remains one of the major concerns with current
chemotherapies, and resveratrol therefore may allow for lowering of the doses of toxic
compounds while maintaining or enhancing the anti-tumor efficacy. Furthermore, there
have been no reports of acquired resistance to resveratrol during treatment.
At this time no clinical trials have been completed to indicate that resveratrol will
be effective as an anti-cancer treatment in humans. All the studies to date have been
performed in tissue culture models or animal models. These studies show a strong anti-
tumor effect of resveratrol with oral dosing and the potential for even higher efficacy if
doses can be further increased by overcoming delivery and solubility obstacles.
However, as has been shown with numerous other potential drugs, results from animal
studies do not necessarily extrapolate to humans. Therefore great caution has to be
taken not to raise expectations of success in using this compound as an anti-cancer
agent before clinical trials have been successfully conducted. Even as these trials
proceed, the concerns raised in previous sections should be adequately addressed in
order to maximize resveratrol’s potential as a non-toxic anti-cancer drug. Currently, one
trial is underway in colon cancer patients with trials for a number of other types of
cancer proposed. Ultimately, based on the results of the clinical trials, the FDA alone
has the authority to approve the use of resveratrol in cancer therapy in the United
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States. In addition, its antioxidant effects currently being tested in other clinical trials,
may lead to its use as an approved chemopreventive agent or in combination with other
anti-cancer drugs. Again, the non-toxic aspects of resveratrol also make it a likely
candidate for neoadjuvant or adjuvant treatment.
Nevertheless, resveratrol is already available as a nutritional supplement.
Because nutraceuticals are more loosely regulated by the FDA, much stronger claims
regarding resveratrol’s benefits to human health and disease are touted by this industry.
Since those afflicted with cancer are desperate for a cure and are distrustful of current
chemotherapy, many turn to nutraceutical products drawn by the promise of a
successful ‘natural’ treatment. Unfortunately many of these claims regarding resveratrol,
go well beyond current research results leading to false hopes in patients. These
expectations are increased further by reports in newspapers and on television
promoting resveratrol’s anti-cancer and anti-aging properties along with a host of other
health benefits. All these claims are yet to be scientifically proven in humans. It is not
surprising that companies with a commercial interest in resveratrol may oversell its
qualities in order to promote their product. However, it is important that researchers
provide accurate information through peer-reviewed publications and to the public via
news outlets and other avenues indicating the limitations of the studies performed to
date. As results of human trials become known, we will know whether the initial
promises can be fulfilled.
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Acknowledgements
We thank Chue Vang for excellent technical assistance. This work was supported by
funds from the American Institute for Cancer Research (PvG), the Mandelbaum
Therapeutics Initiative (ASP) and the Retina Research Foundation (ASP is the M.D.
Matthews RRF Professor).
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Figure Legends
Fig 1: Resveratrol-Induced Ca2+ -Mediated Apoptosis . Resveratrol activates the
inositol triphosphate receptor (IP3R) either through an extracellular receptor or through
an intracellular target resulting in release of ER calcium reflected in a rapid rise in
intracellular calcium ([Ca2+]i). The subsequent calcium uptake by the mitochondria
causes mitochondrial membrane depolarization (Ψm), increase in reactive oxygen
species production (ROS), release of cytochrome c and smac/diablo activating the
intrinsic apoptotic pathway via caspases-9 and -3. Mitochondrial calciuminduced-
calcium-release (mCICR) results in the activation of calpain that then cleaves various
substrates including calcium exchangers and pumps like PMCA1 leading to additional
elevation of intracellular calcium further stimulating cell death.
Fig 2: Translation of Resveratrol to the Clinic. Successful translation of resveratrol
to an approved chemotherapy drug will necessitate new, more soluble formulations to
improve resveratrol bioavailability and delay metabolism to achieve significant
improvements in anti-tumor efficacy.
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© 2010 American Association for Cancer Research
Cal
pai
n
Resveratrol
OH
OH
HO
OH
OH
HO
DNAfragmentation
IP3R
Extracellularreceptor pumps/exchangers
Ca2+
mCICR
Cal
pai
n
IP3Ca2+
Ca2+
Ca2+ Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
XIA
PApaf-1Procaspase
-9Procaspase
-3
Caspase-9
Caspase-3
Ca2+
Ca2+
Cytoc
Smac/diablo
m
Ca2+
Ca2+
Ca2+Ca2+
Ca2+ Ca2+
[Ca2+]i
ROS
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© 2010 American Association for Cancer ResearchC R h
Existingformulations
Newformulation
Low solubilityand fast resveratrol
metabolism
High solubility andslower resveratrol
metabolism
Low resveratrolbioavailability
Unmetabolizedresveratrol
High resveratrolbioavailability
Unmetabolizedresveratrol
MetabolitesMetabolites
Tumor inhibition Tumor regression
InactiveInactive
IV, local
Resveratrol
OH
OH
HO
Diet Supplement Pills
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Published OnlineFirst November 2, 2010.Clin Cancer Res Lalita Subramanian, Sherry Youssef, Saswati Bhattacharya, et al. discussionResveratrol: challenges in translation to the clinic - A critical
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