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Pomegranate Ellagitannin-Derived
Compounds Exhibit Antiproliferative
and Antiaromatase Activity in
Breast Cancer Cells In vitro
Journal ：Cancer Prev Res; 3(1) January 2010
Author ：Lynn S. Adams, Yanjun Zhang, Navindra P. Seeram, David Heber, and
Shiuan Chen
南台科技大學
生物科技研究所 碩一甲班
speaker ： 許秀娟 N99H0001
Date ： 2011. 05. 08
指導教授：鄭伯智老師 林宏榮老師
Outline
• Introduction
• Materials and
Methods
• Results
• Discussion
• Conclusions
Pomegranate(石榴)
• Punica granatum L.
• Is a fruit-bearing deciduous shrubor small tree
growing between five and eight meters tall.
• Is mostly native to the Iranian Plateau and the
Himalayas in Northern India.
From Wikipedia
• Exocarp color ：ranging from yellow to purple,
with pink and red most common.
• Aril color : ranging from white to red.
• Polyphenol：antioxidant activity.
Content
• Ellagitannins (ET) ：
(1) Inhibit estrogen-responsive breast cancer.
J Nutr Biochem 2005;16:360–7
(2) Inhibit inflammatory cell signaling.
J Agric Food Chem 2006;54: 980–5
(3) Antiatherosclerotic properties.
Content
• Ellagic acid (EA) ：
(1) ETs hydrolyzed to EA.
(2) Has antiproliferative and antioxidant
properties.
Seeram NP, Adams LS, Henning SM, et al. (June 2005). Nutr. Biochem. 16 (6): 360–7
(3) Metabolized by human colonic microflora
to urolithin A (UA) and B (UB).
J Nutr 2006;136:2481–5.
Eur J Nutr 2004;43:205–20.
ur J Clin Nutr 2006; 60:245–53.
Agric Food Chem 2006;54:8956–61
Content
• Urolithin A and B：
(1) Inhibit the growth of human prostate and
breast cancer cell lines.
J Agric Food Chem 2007;55:7732–7
(2) UA and UB exhibit both estrogenic and
antiestrogenic effects in MCF-7 human breast
cancer cells and therefore may have selective
ER modulator.
J Agric Food Chem 2006;54:1611–20
Aromatase
• Converts androgen (testosterone) to estrogens.
J Nutr 2001;131:3288–93
• Estrogen stimulates the proliferation of breast cancer
cells.
• And the growth of estrogen-responsive tumors.
• Plays a key role in breast carcinogenesis.
• Aromatase inhibitors reduce serum estradiol levels in
healthy female volunteers and in animal studies.
J. Steroid Biochem. Mol. Biol. 37: 335–341
MCF-7aro cells
Testosterone
Aromatase
Antiaronatase?
Estrogens
+
Breast cancer cells
proliferation
Materials and Methods
Materials and Methods
High performance liquid
chromatography (HPLC)
Placental
microsome
assay
Aromatase
“in-cell”
assay
Cell
proliferation
assay
Kinetic
determinations
Results
• Chemical structures of test compounds.
• Inhibition of aromatase by ET-derived
compounds.
• Aromatase inhibition in MCF-7aro cells treated
with ET-derived compounds.
• Antiproliferative activity of ET-derived
compounds in MCF-7aro cells.
• Kinetic analysis of aromatase inhibition with UB.
Results-1
Chemical structures of
test compounds
Materials and Methods
фAll solvents were high performance liquid
chromatography (HPLC).
• A panel of 10 ET-derived compounds.
Chemical structures of test compounds
Chemical structures of test compounds
Results-2
Inhibition of aromatase by
ET-derived compounds
Materials and Methods
фPlacental microsome aromatase assay.
• The assay mixture consisted of (500 μL)：
(1)20 μg placental microsome
(2)100 nmol/L (3H)-androstenedione (substrate)
(3)10 μmol/L progesterone
(4)1 g/L bovine serum albumin
(5)67 mmol/L potassium phosphate buffer (pH 7.4)
(6)12 mmol/L NADPH
J Nutr 2001;131:3288–93
• Controls：
(1)blank
(2)positive control (water)
(3)vehicle control (DMSO)
• Compound concentration：47 μmol/L
• The reaction was run for 20 min at 22°C.
• The enzymatic activity was terminated with 5%
trichloroacetic acid.
• The supernatant was extracted (The supernatant
containing the aromatase product).
• The suspension was centrifuged(700 g for 10
min) and an collected.
• Counted for radioactivity (Aromatase activity).
J Nutr 2001;131:3288–93
Inhibition of aromatase by urolithins
20min
P ≤ 0.01
• The aromatase activity of vehicle-treated microsomes was set at 100%.
• Concentration ：47 μmol/L, the compounds showed varying potency.
Summary
• MUB, UBS, UB, AUB, UA, and MUA
significantly inhibited aromatase activity.
• AMUA, DMUA, GA and EA showed no
significant antiaromatase activity.
• These results revealed which metabolites of
pomegranate ingestion show antiaromatase
activity in vitro.
Results-3
Aromatase inhibition in MCF-7aro
cells treated with ET-derived
compounds.
Cell culture
• MCF-7aro cell：
The ER-positive aromatase-overexpressing MCF-7 cell line;
contains a functional ER.
• Medium：
(1)Eagle's MEM (Invitrogen)
(2)supplemented with 10% fetal bovine serum (FBS)
(3)selection antibiotic G418 (500 μg/mL; UBS Corporation).
• incubation：
37°C with 5% CO2，maintained in the linear phase of
growth.
Materials and Methods
ФAromatase“in-cell” assay
• MCF-7aro cells were cultured in six-well plates
in MEM overnight.
• phenol red–free MEM with 10% charcoal
dextran–treated FBS (cdFBS) for 24 h.
• Fresh cdFBS medium alone.
• Add DMSO control or the test compounds.
• Added plus 0.1 mmol/L (3H)-androstenedione
and 500 nmol/L progesterone for 3 h.
• Suspension was centrifuged (700 × g for 7 min)
• Collected and counted for radioactivity
(Aromatase activity).
In cell inhibitory effect of urolithin compounds on aromatase
in MCF-7aro cells
3hr
P ≤ 0.05
P ≤ 0.01
• compound： 0, 2.35, and 4.7 μmol/L
Summary
• UB significantly inhibited aromatase activity at
2.35μmol/L and 4.7μmol/L.
• GA significantly inhibited aromatase activity at
4.7μmol/L.
• This result shows that although a number of the
other test compounds were active in the microsomal
assay.
• UB was the most active in the cell-based assay.
Results-4
Antiproliferative activity of
ET-derived compounds in
MCF-7aro cells
Materials and Methods
ФCell proliferation assay
• MCF-7aro cells were plated in 96-well plates in
MEM overnight.
• Phenol red–free MEM with 10% cdFBS for 24 h.
Materials and Methods
• Cells were treated：
(1) 100μL cdFBS MEM alone ＋test samples
(2) 100μL cdFBS MEM＋1 nmol/L testosterone
(3) 100μL cdFBS MEM ＋1 nmol/L testosterone ＋test
samples
• Testosterone or estradiol(1 nmol/L) and incubated for 48-h
• Read on an Orion Microplate Luminometer
In the testosterone-induced proliferation assay
48hr
P ≤ 0.01
• TFM, testosterone-free medium
• T, testosterone
Summary
A、Testosterone induced proliferation assay：
• UB had the highest antiproliferative activity at
all test concentrations.
• GA had the second highest activity.
• UBS, UA and MUB showed a mild response.
In the estrogen-induced proliferation assay
48hr
P ≤ 0.01
• EFM, estrogen-free medium
• E2, estrogen
Summary
B、Estrogen-induced proliferation assay：
• UBS was the most potent inhibitor.
• GA again the second most potent inhibitor.
• UA, MUB, and UB all showed a mild response.
• UB only showing a significant inhibition at the
highest dose.
Summary
• UB likely inhibits the proliferation of MCF-7aro
cells primarily through aromatase inhibition.
• Other test compounds may affect an aromataseindependent mechanism：
(1) direct antagonism of ER signaling
(2) combination of aromatase dependent and
aromatase-independent mechanisms.
Results-5
Kinetic analysis of
aromatase inhibition
with UB
Materials and Methods
ф Placental microsome assay.
• UB concentrations：0, 23.5, 47 μmol/L.
• 3H-androstenedione concentrations：0-200
nmol/L.
動力學實驗操作
• 先取固定量的酵素－E
• 加入各種不同濃度的基質－S（X軸）
• 在一定時間內測生成物量－V0（Y軸）
• （ X，Y ）做圖得雙曲線之一股推漸進點－Vmax
• 當Y＝1/2 Vmax 時求其x（即S）即得－Km
直接作圖
雙倒數圖形(double-reciprocal plot)
Aromatase inhibition kinetic profile by UB compound
47μmol/L
23.5μmol/L
Vmax
Km
0μmol/L
(Control)
• Slope=Km/Vmax(1+[I]/Ki)就可以得到三個斜率值
Aromatase inhibition kinetic profile by UB compound
• 再以三個斜率值去作圖得到B圖進而推出Ki值為5umol/L.
• Ki：抑制物的抑制常數
Summary
• The Lineweaver-Burk plot shows that the
presence of UB increased the Km value and
slope with no change in the Vmax.
• This indicates that UB is a competitive inhibitor
with respect to the substrate (androstenedione).
• An approximate Kιvalue of 5μmol/L was
determined from the secondary plot.
Discussion
Conclusions
MCF-7aro cells
Testosterone
Aromatase
Estrogens
Breast cancer cells
proliferation
• These studies bring us closer to an
understanding of how the ET metabolites
resulting from pomegranate ingestion can
affect the growth of estrogen-responsive
breast cancer cells.
• The results of these analyses suggest that
pomegranate intake may be a viable strategy
for the chemoprevention of breast cancer.
Feature
• Future studies to examine the synergistic
effects of these compounds.
• Future studies into the fate of these
compounds and their effects on the cell
cycle and cell signaling will be carried out
to elucidate their mechanisms of action.