Anthocyanidins Inhibit Activator Protein 1 Activity and Cell Transformation: Structure Activity Relationship and Molecular Mechanisms
Anthocyanidins Inhibit Activator Protein 1 Activity and Cell Transformation: Structure Activity Relationship and Molecular Mechanisms
Anthocyanidins Inhibit Activator Protein 1 Activity and Cell Transformation: Structure Activity Relationship and Molecular Mechanisms
2936, 2004
DOI: 10.1093/carcin/bgg184
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D.-X.Hou et al.
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(45), and purified by HPLC (purity is 495%). All of the anthocyanidins used
were dissolved in dimethyl sulfoxide (DMSO, final concentration was 0.2%).
Antibodies were from Cell Signaling Technology (Beverly, MA). Luciferase
assay substrate was obtained from Promega (Madison, WI). Fetal
bovine serum (FBS) was from Equitech-Bio (Kerrville, TX). TPA, p-iodonitrotetrazolium violet (INT), superoxide dismutase (SOD), d-mannitol and
catalase were from Sigma (St Louis, MO).
The JB6 P mouse epidermal cell line, Cl41 (28), and its AP-1-luciferase
reporter stable transfectant P 11 cells (40) were cultured at 37 C, 5% CO2 in
EMEM containing 5% FBS, 2 mM l-glutamine and 25 mg/ml gentamicin.
Anchorage-independent transformation assay
The inhibitory effects of anthocyanidins on TPA-induced cell transformation
were investigated in the parental JB6 Cl41 cells or AP-1-luciferase stable
transfectant JB6 cells (P 11 ) (46). There is no marked difference in TPAinduced transformation between both cell lines (Hou et al., unpublished data).
Cells (1 104 ) were suspended in 2 ml of 0.38% BME agar medium over 3 ml
of 0.5% BME agar medium containing 10% FBS, 20 ng/ml TPA with or
without anthocyanidins (520 mM). The cultures were maintained in a 37 C,
5% CO2 incubator for 14 days, and the anchorage-independent colonies after
staining with INT were scored by a computerized image analyzer. The efficiency of anthocyanidin inhibition of TPA-induced cell transformation is
expressed as a percentage of the transformation frequency when the cells
were treated with TPA alone.
Luciferase assay for AP-1-dependent transactivation
AP-1-luciferase stable transfectant JB6 cells (P 11 ) (46) were used to assay
AP-1-dependent transactivation. Viable cells (2 104 ) were plated in a 48well dish for 24 h before each experiment. The cells were starved by being
cultured in 0.1% FBSEMEM for another 18 h to eliminate the influence of
FBS on AP-1 activity, and then treated with or without anthocyanidin for
30 min before they were exposed to 20 ng/ml TPA for 24 h. For antioxidant
agents, the cells were treated with SOD, catalase or d-mannitol alone or with
SOD plus anthocyanidins for 30 min, respectively, before the cells were
exposed to 20 ng/ml TPA for 24 h. The cells were extracted with lysis buffer,
and the luciferase activity was measured by a luminometer (Berthold) according to the supplier's recommendations. AP-1 activity is expressed as fold
induction relative to the control cells without TPA treatment (47,48).
Western blotting analysis
After the cells (1.5 106 ) were cultured in 10-cm dish for 24 h, the cells were
starved in serum-free for another 4 h to eliminate the influence of FBS on
MAPK activation. The cells were then treated with or without delphinidin for
30 min before they were exposed to 20 ng/ml TPA for the different times. The
harvested cells were lysed and the supernatants were boiled for 5 min. Protein
concentration was determined using dye-binding protein assay kit (Bio-Rad
Hercules, CA) as described in the manufacturer's manual. Lysate protein of 40
mg was run on 10% SDAPAGE and electrophoretically transferred to PVDF
membrane (Amershan Pharmacia Biotech, Little Chalfont, UK). After blotting,
the membrane was incubated with specific primary antibody overnight at 4 C,
and further incubated for 1 h with HRP-conjugated secondary antibody. Bound
antibodies were detected by ECL system with a Lumino Image Analyzer. The
relative amount of proteins associated with specific antibody was quantified by
using the Imager Gauge Software (Fuji Photo Film).
Statistical analyses
Differences between the treated and the control were analyzed by the Student's
t-test. A probability of P 5 0.05 was considered significant. The multiplicative
models for the interaction between two agents were used to determine the
effect of a combination of SOD with delphinidin (49,50). The additive model
predicts the effect of a combination to be equal to the effect of its constituents;
an observed effect of the combination higher than predicted by the additive
model indicates synergism (49).
Results
Anthocyanidins inhibit TPA-induced JB6 cell transformation
and AP-1 activation in a structureactivity relationship
Cells (1 104 ) were exposed to 20 ng/ml TPA in soft agar
for 14 days, 10002000 transformed colonies were induced
whereas in the solvent control group (50.1% DMSO) there is
no colony formation. TPA-induced cell transformation was
significantly inhibited by delphinidin, petunidin and cyanidin,
but not by pelargonidin, peonidin and malvidin (P 5 0.05) at
the concentration range from 5 to 20 mM. Previous studies
have suggested that AP-1 transactivation is required for TPAinduced cell transformation in mouse JB6 cells (25,40,51).
We thus tested the effects of those anthocyanidins on TPAinduced AP-1 activity by using a reporter gene assay. The
treatments of delphinidin, petunidin and cyanidin, but not
pelargonidin, peonidin and malvidin, markedly inhibited
TPA-induced AP-1 activity at the same concentration range
(Figure 2B; P 5 0.05). The inhibitory actions by delphinidin,
petunidin and cyanidin were not caused by their cytotoxicity,
because the concentration range that inhibited cell transformation and AP-1 activity did not affect cellular viability
D.-X.Hou et al.
Fig. 3. Delphinidin blocks TPA-induced ERK and JNK, but not p38
phosphorylation. (A) Delphinidin blocks TPA-induced ERK and JNK
phosphorylation. After JB6 cells were starved in serum-free medium for 4 h.
The cells were treated with the indicated concentrations of delphinidin for
30 min, and then exposed to 20 ng/ml TPA for 2 h to target ERK, and for
12 h to target JNK and p38. (B) Peonidin does not block TPA-induced
phosphorylation of ERK, JNK and p38. After JB6 cells were starved in
serum-free medium for 4 h. The cells were treated with 20 mM peonidin or
without for 30 min, and then exposed to 20 ng/ml TPA for 2 h to target ERK,
and for 12 h to target JNK and p38. Western blotting analysis of MAPKs
were performed with specific antibodies, respectively, as described in
`Materials and methods'. Histograms show the densitometric analysis of
phosphorylated protein expression normalized to total MAPK. The data
represent the mean SD of three to four separate experiments, and the
figure is a representative of those experiments each with similar results.
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Discussion
D.-X.Hou et al.
Fig. 6. Synergistic inhibition between delphinidin and SOD. JB P 11 cell culture and luciferase activity assay were done as described in Figure 2B. Each value
represents the mean SD of three to six separate experiments. (A) SOD, but not catalase or d-mannitol, inhibits TPA-induced AP-1 activity. The cells were
treated with or without SOD, catalase and d-mannitol for 30 min at the indicated concentrations before they were exposed to 20 ng/ml TPA for 24 h. P 5 0.05
versus control. (B) Delphinidin, cyanidin and petunidin, but not pelargonidin, peonidin or malvidin, show greater inhibition of TPA-induced AP-1 activity
with SOD. The cells were treated by six anthocyanidins (5 mM) with or without SOD (200 U/ml) for 30 min before they were exposed to 20 ng/ml TPA for 24 h.
P 5 0.05 versus SOD or anthocyanidin alone. (C) Delphinidin synergistically inhibits AP-1 activity with SOD. The cells were treated with the indicated
concentrations of delphinidin in the presence or absence of various doses of SOD. Results represent the mean SD of three experiments as a percentage of AP-1
activity in the presence of TPA alone. (D) Comparison between the actual AP-1 activity (shaded bars) and the expected value for additive effects (open bars) in
combinations of them.
Acknowledgements
This work was supported by grant-in-aid for scientific research (C) of the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) of
Japan (13660130), and by grant for New Kagoshima Study to D.-X. Hou.
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inhibition was observed in combinations of SOD with anthocyanins those have the ortho-dihydroxyphenyl structure
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