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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 18, 2001 as doi:10.1096/fj.00-0675fje. |
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2
* Laboratory of Molecular Cytology, Department of Internal Medicine, University of Innsbruck, A-6020 Innsbruck, Austria;
Tyrolean Cancer Research Institute at the University of Innsbruck, A-6020 Innsbruck, Austria;
# Institute for General and Experimental Pathology, University of Innsbruck, A-6020 Innsbruck, Austria;
Centre National de la Recherche Scientifique, UMR-1599, Institute Gustave Roussy, F-94805 Villejuif, France; and
¶ Institute of Medical Chemistry and Biochemistry, University of Innsbruck, A-6020 Innsbruck, Austria
2Correspondence: Laboratory of Molecular Cytology, Department of Internal Medicine, University of Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria. E-mail: Richard.Greil{at}uibk.ac.at
SPECIFIC AIMS
The clinical application of the tumor-suppressive agent resveratrol in cancer treatment requires an exact understanding of which molecules sensitize cells to, or antagonize them against, its specific signaling cascade. Bcl-2 is frequently expressed in hematological tumors, is associated with an unfavorable prognosis in some of them, and inhibits the efficacy of cytotoxic agents. Bcl-2 functions may be diverse, but predominantly involve mitochondria. We therefore focused on the mitochondrial signaling pathways induced by resveratrol and potentially counteracted by Bcl-2.
PRINCIPAL FINDINGS
1. Resveratrol-induced apoptosis is inhibited by Bcl-2
To study the role of Bcl-2 in resveratrol-induced apoptosis, we
used subclones of the CEM-C7H2 T-ALL cell line, which stably
overexpressed Bcl-2. After resveratrol treatment of the vector control
cell line, apoptosis was induced in a time-dependent manner. Bcl-2
overexpression protected cells from resveratrol-induced apoptosis
even over the extended incubation period of 72 h. Bcl-2 has been
shown to localize to mitochondria and stabilize mitochondrial
functions, thereby suppressing the release of proapoptotic effector
molecules. In our study, a very rapid, transient breakdown of
m after addition of resveratrol and a
second, persisting decrease of m could be
observed in the vector control cell line, which was significantly
reduced in the subclone with high Bcl-2 expression levels. This drug
also had a direct effect on the permeability of the mitochondrial
membranes of isolated rat mitochondria, which was prevented by
cyclosporin A (CsA), a substance that blocks the opening of the
permeability transition pore complex.
2. Reactive oxygen species are produced after resveratrol
stimulation of C7H2 cells
Production of reactive oxygen species (ROS) can contribute to
mitochondrial damage that may facilitate the further release of ROS
into the cytoplasm. We detected intracellular ROS production after
resveratrol treatment that could already be observed 30 min after the
addition of resveratrol and steadily increased over the period of
observation. According to the inhibitory effect of Bcl-2 expression on
the breakdown of m after resveratrol
addition, Bcl-2 significantly reduced resveratrol-induced production of
ROS. To elucidate whether the increase in ROS is responsible for the
loss of m and for resveratrol-induced
apoptosis, we used the radical scavenger N-acetylcysteine (N-AC).
Pretreatment with N-AC before addition of resveratrol significantly
lowered the generation of ROS as well as the breakdown of
m, suggesting that ROS may not only develop
downstream from changes in the mitochondrial transmembrane potential,
but may also act upstream and contribute to the loss of
m. As expected, pretreatment with N-AC also
protected cells from DNA fragmentation induced by resveratrol.
3. Resveratrol-induced apoptosis does not involve cytochrome
c release
To identify whether cytochrome c release contributes to
the proapoptotic effects of resveratrol, we examined whether its
subcellular localization was changed by resveratrol treatment. Despite
the clear effect of resveratrol on the mitochondrial permeability
transition pore complex and transmembrane potential, no translocation
of cytochrome c to the cytosol could be detected at the
times analyzed (1, 4, 8, 16, and 24 h; Fig. 1
and data not shown).
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4. Resveratrol does not trigger the activation of death receptor
type II pathways
To clarify whether the effects of resveratrol on mitochondria
might be mediated by its activation of Bid, a Bcl-2 family member and
downstream target of activated caspase-8, extracts from untreated cells
or from cells treated with resveratrol were analyzed for the
unprocessed and cleaved form of Bid. No early processing of Bid could
be detected, which supports our previous findings that
resveratrol-induced apoptosis does not involve the activation of a
death receptor type II pathway.
5. Resveratrol leads to the activation of caspase-9, -2, -3,
and -6
Since cytochrome c translocation is thought to
represent a prerequisite for efficient caspase-9 activation, we
wondered whether caspase-9 is activated by resveratrol. Cell lysates
from untreated cells and from cells treated with resveratrol were
analyzed for the processed form of caspase-9. Activation of caspase-9
became detectable in the vector control cells, but not in the Bcl-2
overexpressing subclone. Furthermore, resveratrol treatment triggered
the activation of caspase-2, -3, and -6 in a Bcl-2 controlled manner.
6. DNA cleavage by resveratrol occurs downstream of mitochondrial
signaling
To elucidate whether resveratrol-induced DNA cleavage occurs
before the mitochondrial changes observed, TUNEL assays in untreated
cells or cells treated with resveratrol were performed. Significant
induction of DNA strand breaks by resveratrol followed
m changes and ROS production and was
significantly blocked in the Bcl-2 overexpressing subclone.
7. Resveratrol does not induce the translocation of
apoptosis-inducing factor (AIF)
After different proapoptotic stimuli, the loss of mitochondrial
transmembrane potential leads to the release of AIF from the
mitochondrial intermembrane space, thus representing the link between
mitochondria and nucleus in resveratrol-induced apoptosis. We prepared
nuclear and mitochondrial fractions of C7H2-VC cells and the Bcl-2
overexpressing clones and detected AIF by immunoblotting. The signal of
AIF in the mitochondrial fractions did not decrease after resveratrol
treatment of cells, suggesting that this pathway of caspase-independent
activation of nucleases is not significantly involved in
resveratrol-induced apoptosis.
CONCLUSIONS
Our present work provides evidence that the modulation of
m and generation of ROS constitute early
events of the resveratrol-induced apoptotic pathway that are not
preceded by direct activation of Bid and are not accompanied by
subcellular translocation of cytochrome c. Despite the lack
of cytochrome c release from mitochondria, caspase-2, -3,
-6, and -9 are activated in resveratrol-induced apoptosis and function
downstream of m breakdown and ROS
generation (Fig. 2
). Moreover, the proapoptotic effects of resveratrol cannot be
attributed to a direct induction of DNA damage. Mitochondrial changes
and ROS production are both inhibited by overexpression of Bcl-2, which
also blocked all further downstream effects including
phosphatidylserine exposure, caspase activation, and DNA damage.
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A protective effect of Bcl-2 against m
breakdown has been observed after treatment of cells with
glucocorticoid, ceramide, tumor necrosis factor, Fas, and p53-induced
apoptosis. Bcl-2-specific maintenance of m
might result from its capacity to prevent physical interaction of the
proapoptotic protein Bax with the adenine nucleotide translocator
protein resulting in the formation of a pore, followed by the transient
breakdown of m, and by osmotic matrix
swelling of mitochondria, both of which we observed after application
of resveratrol to intact cells and to isolated mitochondria,
respectively. These data favor the idea that resveratrol directly
interacts with mitochondrial membrane components. This hypothesis is
substantiated by data demonstrating that resveratrol inhibits
F0F1 ATPase activity by
targeting the F1 region at the inner membrane of mitochondria.
We found that in addition to its effect on mitochondrial potential,
resveratrol leads to an increase in ROS production in CEM-C7H2 cells.
Generation of ROS by the antioxidant resveratrol seems paradoxical,
particularly because it has been shown that resveratrol can decrease
the activity of complex III of the mitochondrial respiratory chain, the
site where ROS are generated. At this stage, we cannot rule out that
the antioxidant activity of resveratrol diminishes the increase in ROS
levels generated by its action on mitochondria. Such opposing effects
of either an increase or decrease of ROS by resveratrol, depending on
its concentration, have been observed in blood platelets. Since we
observed an early transient loss of m,
which was followed by a time-dependent increase in ROS and a second,
lasting decrease of m, we propose a model
where transient m breakdown causes local
generation of ROS, which in a positive feedback loop then further
reduces m. The generation of ROS by
resveratrol in CEM-C7H2 cells was also blocked by overexpression of
Bcl-2. Besides the direct interaction of Bcl-2 with mitochondrial
components, its interference with oxidative cell death has been shown
to act on at least two other levels: inhibition of free radical
production and detoxification of ROS. We conclude from our results that
Bcl-2, which inhibited the early transient and the second lasting loss
of m, might operate in two ways: by
directly antagonizing the early loss of m
and ROS production and by preventing the emergence of the feedback
loop.
We could not detect cytochrome c in the cytosolic compartment of C7H2 cells at any of the time points analyzed after addition of resveratrol. This might be explained by the formation of a pore that is too small to allow the efflux of cytochrome c. However, we cannot exclude the possibility that resveratrol triggers a very rapid release of cytochrome c, which then rapidly re-enters mitochondria.
There is agreement among scientists working in the field of apoptosis
that after its release from mitochondria, cytochrome c binds
to APAF-1 and then activates caspase-9. In addition, it has been shown
that caspase-9 activity is indispensable for the cytochrome
c-inducible activation of all other downstream caspases:
caspase-2, -3, -6, -7, -8, and -10. Our data now provide evidence that
the activation of caspase-9, -2, -3, and -6 by resveratrol does not
depend on cytochrome c release, at least at concentrations
detectable by the methods applied in this study, that it follows the
breakdown of m, and is regulated by Bcl-2.
Cytochrome c-independent release of caspase-9 from
mitochondria and its activation by cytotoxic endoribonuclease treatment
have been reported recently. Disruption of
m, ROS production, and activation of
caspase-3-like proteases in the absence of Bid activation and of
cytochrome c release has also been described in p53-induced
apoptosis in HeLa cells. From these reports and from our study, it can
be concluded that either high cytoplasmic concentrations of cytochrome
c are not indispensable for efficient activation of
caspase-9 or that caspase-9 activation in the apoptosome might be
initiated via an as yet unidentified alternative component.
It has recently been shown that resveratrol-induced apoptosis occurs only in cells expressing wild-type p53, but not in p53-deficient cells. The CEM-C7H2 cell line used in this study is heterozygous for the mutant p53 alleles R175H and R248Q, which results in absent trans-activation of known endogenous p53-responsive genes. However, functions of p53 independent of its transcriptional activity may be conserved, and this cell line has been shown to express all relevant signaling molecules for p53-mediated apoptosis. To answer the question of whether resveratrol might be used for treatment of tumors with a loss-of-function mutation in the p53 gene and whether its therapeutic application can replace sophisticated gene transfer of wild-type p53, a broader spectrum of tumor cell models needs to be analyzed.
In conclusion, elevated Bcl-2 expression of tumor cells might represent an important obstacle to the efficacy of resveratrol when applied as a single agent in tumor therapy. Combined strategies with Bcl-2-antagonizing cytotoxic agents should therefore be evaluated in future studies.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0675fje ; to cite this
article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0675fje 
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