Download metabolomic analysis of resveratrol-induced effects on

METABOLOMIC ANALYSIS OF RESVERATROL-INDUCED EFFECTS ON THE HUMAN
BREAST CANCER CELL LINES MCF-7 AND MDA-MD-231
W. Jaeger*1, A. Gruber2, B. Giessrigl3, G. Krupitza3, D. Sonntag2
1
Department of Clinical Pharmacy and Diagnostics, University of Vienna, Austria, 2 BIOCRATES Life
Sciences AG, Innsbruck, Austria; 3Institute of Clinical Pathology, Medical University of Vienna, Austria
INTRODUCTION
Breast cancer is a major cause of cancer death in
women worldwide. Evidence from epidemiological
and experimental studies indicates that natural
constituents of the diet may well act as
chemopreventive agents and inhibit mammary
carcinogenesis. One of such compounds is
resveratrol (RES), 3,4’,5-trihydroxy-trans-stilbene,
which is produced by several plants, berries,
fruits, and is mainly found in the skin of grapes
and red wine.
OH
HO
OH
The antiproliferative property of RES has been
demonstrated in vitro against breast cancer cells
due to the induction of apoptosis via downregulation of NF-kappa B, Bcl-2, inhibition of
ribonucleotide reductase, and DNA polymerase.
In addition, recent experiments have shown
significantly less tumor growth in human breast
cancer xenografts in vivo, supporting the use of
this polyphenol as a potential chemotherapeutic
agent. Although gene and protein expression
have been extensively profiled in breast cancer
cells after RES treatment, there are no data about
metabolic alterations that characterize the effects
of this compound. Most breast cancer biomarker
discovery studies are based on the qualitative or
quantitative changes of large biopolymers (e.g.
DNA, RNA, glycans and proteins). In contrast,
metabolic biomarker discovery approaches that
focus an small molecules (below 1 kDa) have
received significantly less attention, despite the
fact that the identification and quantification of
specific metabolites in tumor cells provides highresolution biochemical snapshots depicting the
functional endpoints of drug disposure (Fig.1).
There are two approaches to metabolomics, i.e.,
targeted metabolomics and metabolic profiling.
While both approaches are complementary,
targeted metabolomics, i.e., the identification and
quantification of defined sets of structurally known
and biochemically annotated metabolites, takes
advantage of our detailed understanding of most
biochemical pathways (Fig.2).
RESULTS & DISCUSSION
Sample cohorts
Fig. 1: Omics technologies used for the development of
molecular diagnostics
Metabolic profiling
(e.g. full scan LC-MS)
Targeted metabolomics
(ID / quantitation by MS/MS)
Differential pattern
information
Metabolite concentration
shifts
Identification of relevant
metabolites
Functional annotation
Resveratrol significantly reduced cell viability in
the cancer cell lines MCF-7 ZR and MDA-MB231, yielding IC50 of 68.3 ± 2.6 and 67.6 ± 4.1
µM, respectively. Cell growth inhibition was
accompanied by substantial metabolic changes,
which were dose-dependent and similar in both
cell lines. Results obtained for MCF-7 cells were
chosen to demonstrate the metabolic effects of
resveratrol on mammary carcinoma cells. The
most interesting findings were related to the
following metabolite classes:
Amino acids:
Resveratrol induced degradation or release of
amino acids (Fig. 3). Enzymatical conversion of
tryptophan to the bioactive metabolite serotonin
was stimulated, whereas methionine underwent
increased
non-enzymatical
oxidation
to
methionine-sulfoxide. Taurine, on the other hand,
was substantially released from the cells, which is
often caused by cell swelling and the occurrence
of reactive oxygen species (Lambert, 2007).
Fig. 2: Metabolic profiling versus targeted metabolomics
EFFECT OF RESVERATROL ON THE METABOLISM OF
Polyamines:
Upon treatment with resvertarol, a profound
modulation of polyamine biosynthesis could be
seen in mammary cancer cell lines (Fig. 4).
While the synthesis of putrescine from ornithine
by ornithine decarboxylase, seemed to be
inhibited, an up to 8-fold increased synthesis of
spermidine from putrescine was observed,
indicating activation of spermidine synthase.
Interestingly, synthesis of spermine from
spermidine was not stimulated, but rather
inhibited. Putrescine and spermidine are essential
for a variety of cellular processes related to signal
transduction,
growth
and
differentiation.
Therefore, resveratrol-induced changes in
polyamine metabolism could be directly linked to
cell vitality (Takao et al, 2006). Conversion of
putrescine to metabolically active spermidine and
spermine occurs early during cell proliferation,
and the activity of the two rate-limiting enzymes
of
polyamine
biosynthesis,
ornithine
decarboxylase (ODC) and S-adenosylmethionine
decarboxylase (SADMC) is increased in
proliferating cells (Milovic et al, 2000).
Resveratrol
and
the
analog
(Z)-3,5,4`trimethoxystilbene have been shown to reduce
ODC and SADMC activities by depletion of the
polyamines putrescine and spermidine, hence
exerting the cytotoxic effects by depleting the
intracellular pool of polyamines (Wolter et al,
2003; Schneider et al, 2003).
Arachidonic acid:
A
pronounced
increase
in
extracellular
arachidonic acid (AA) and its metabolite 12SHETE could be observed with high doses of
resveratrol (Fig. 5). AA is released from cell
membrane phospholipids upon activation of
phospholipase A2 and subsequently converted to
12S-HETE by the action of 12-lipoxygenase.
Increased levels of 12S-HETE may therefore
indicate oxidative stress in tumor cells under
resveratrol treatment. (Nazarewicz et al, 2007).
Furthermore,
resveratrol
also
reduced
prostaglandin E2 (PGE2) levels (from >4 to 0
pmol/mio cells), thus confirming that this
polyphenol is an inhibitor of cyclooxygenase 2
(Murias et al. 2005).
AMINO ACIDS
 Stimulation of tryptophan degradation
Tryptophanhydroxylase
Tryptophan
Serotonin
Cell culture medium
MCF-7 cells
 Release of taurine from cells
Cell swelling
ROS
Leakage
Taurine
Cell culture medium
MCF-7 cells
Cell culture medium
MCF-7 cells
 Formation of methionine-sulfoxide
ROS
OBJECTIVE
To investigate possible
metabolic alterations in
MCF-7 and MDA-MD-231
breast cancer cells after
different doses of
resveratrol treatment
focusing on the following
classes of analytes:
Methionine
Methionine sulfoxide
Fig. 3: Stimulation of serotonin synthesis, taurine release and methionine-sulfoxide formation by resveratrol
POLYAMINES
A
Arginine
 amino acids
Cell culture medium
MCF-7 cells
 biogenic amines and
polyamines
 eicosanoids and oxidized
polyunsaturated fatty
acids (PUFAs)
CONCLUSION
Arginase
Ornithine
A Methionine
Ornithine decarboxylase
B
Putrescine
Spermidine synthase
Cell culture medium
S-Adenosylmethionine
B
Decarboxylated
S-Adenosylmethionine
MCF-7 cells
Spermidine
Spermine synthase
C
Cell Culture
MCF-7 and MDA-MD-2131 cells were cultured in
phenol red-free D-MEM/F-12 (GIBCO 21041-025)
containing 10% heat inactivated FCS and 1%
P/S. For the experiment cells were seeded in
15 cm plates. After 24 h, medium was changed to
15 mL phenol red-free D-MEM/F-12 (GIBCO
21041-025) containing 10% DCC-stripped FCS
and 1% P/S. Cells were treated with resveratrol
(0 µM, 25 µM, 50 µM, 100 µM) for 72 hours
(DMSO final concentration: 0.1 %).
C
Spermine
METHODS
Cell culture medium
MCF-7 cells
Fig. 4: Modulation of polyamine synthesis by resveratrol
ARACHIDONIC ACID
REFERENCES
CELL MEMBRANE PHOSPHOLIPIDS
Phospholipase A2
Lambert, I, Am J Physiol Cell Physiol 2007;293:C390-C400
Milovic V et al, Cancer Letters 2000;154:195-200
Murias et al, Bioorganic & Medicinal Chemistry
2004;12:5571-5578
Nazarewicz R et al, Arch Biochem Biophys
2007;468,114-120
Takao K et al, Int. Journal of Biochemistry & Cell Biology
2006;38:621-628
Wolter F et al, Carcinogenesis 2003;24(3):469-474
Schneider Y et al, Int. J Cancer 2003;107(2): 189-196
Mobilization of arachidonic acid
Targeted Metabolomics
By using a high-throughput liquid chromatography-based mass spectrometry platform, we
applied
targeted
metabolomics
for
the
identification and quantitation of almost 70
analytes of 4 different classes in cell pellets and
in cell culture medium. MRM detection was
performed using a 4000 Q TRAP® tandem mass
spectrometry instrument (AB Sciex), and data
were finally exported for biochemical analysis.
12-Lipoxygenase
Cell culture medium
SADMC
The
metabolic
effects
of
resveratrol on breast cancer cell
lines could be revealed using
targeted metabolomics. Biogenic
amines and polyamines, namely
serotonin, putrescine, spermidine,
and methionine-sulf-oxide, as well
as arachidonic acid and its
metabolite
12S-HETE
were
identified as potential markers for
the pharmacological response of
resveratrol, which must be
considered in humans following
oral uptake of dietary resveratrol
or of other substances intented to
be chemopreventive agents.
Cell culture medium
Fig. 5: Release of arachidonic acid from membrane lipids by lipase A2 and increased production of 12S-HETE
ACKNOWLEDGEMENT
We would like to thank the Method Development and Contract
Research groups of BIOCRATES Life Sciences AG for their
valuable work and great effort within this project.