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

Testosterone Aromatase Estrogens

Breast cancer cells proliferation

Antiaronatase? +

MCF-7aro cells

Materials and Methods

Materials and Methods

High performance liquid chromatography (HPLC)

Placental microsome

assay

Aromatase “in-cell” assay

Cell proliferation

assay

Kineticdeterminations

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 MethodsфPlacental 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

• The aromatase activity of vehicle-treated microsomes was set at 100%.

• Concentration ： 47 μmol/L, the compounds showed varying potency.

P ≤ 0.01

20min

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

• compound ： 0, 2.35, and 4.7 μmol/L

P ≤ 0.05

P ≤ 0.01

3hr

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

• TFM, testosterone-free medium• T, testosterone

P ≤ 0.01

48hr

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

• EFM, estrogen-free medium• E2, estrogen

P ≤ 0.01

48hr

SummaryB 、 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 aromatase-independent 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

雙倒數圖形 (double-reciprocal plot)直接作圖

Aromatase inhibition kinetic profile by UB compound

• Slope=Km/Vmax(1+[I]/Ki) 就可以得到三個斜率值

0μmol/L(Control)

23.5μmol/L

47μmol/L

Km

Vmax

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

Testosterone Aromatase Estrogens

Breast cancer cells proliferation

MCF-7aro cells

•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.