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Journal ： Cancer Prev Res; 3(1) January 2010

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

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Journal ： Cancer Prev Res; 3(1) January 2010

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  1. 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

  2. Outline • Introduction • Materials and Methods • Results • Discussion • Conclusions

  3. 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

  4. Exocarp color ：ranging from yellow to purple, with pink and red most common.

  5. Aril color : ranging from white to red. • Polyphenol：antioxidant activity.

  6. 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.

  7. Content • Ellagic acid (EA) ： • 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

  8. 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

  9. 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

  10. MCF-7aro cells Aromatase Testosterone Estrogens Antiaronatase? + Breast cancer cells proliferation

  11. Materials and Methods Materials and Methods

  12. High performance liquid chromatography (HPLC) Placental microsome assay Aromatase “in-cell” assay Cell proliferation assay Kinetic determinations

  13. Results

  14. 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.

  15. Results-1 Chemical structures of test compounds

  16. Materials and Methods • All solvents were high performance liquid chromatography (HPLC). • Apanel of 10 ET-derived compounds.

  17. Chemical structures of test compounds

  18. Chemical structures of test compounds

  19. Results-2 Inhibition of aromatase by ET-derived compounds

  20. Materials and Methods • Placental 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

  21. 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.

  22. 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

  23. 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.

  24. 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.

  25. Results-3 Aromatase inhibition in MCF-7aro cells treated with ET-derived compounds.

  26. 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.

  27. Materials and Methods • Aromatase“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.

  28. 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).

  29. 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

  30. 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.

  31. Results-4 Antiproliferative activity of ET-derived compounds in MCF-7aro cells

  32. Materials and Methods • Cell proliferation assay • MCF-7aro cells were plated in 96-well plates in MEM overnight. • Phenol red–free MEM with 10% cdFBS for 24 h.

  33. 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

  34. In the testosterone-induced proliferation assay 48hr P ≤ 0.01 • TFM, testosterone-free medium • T, testosterone

  35. 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.

  36. In the estrogen-induced proliferation assay 48hr P ≤ 0.01 • EFM, estrogen-free medium • E2, estrogen

  37. 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.

  38. 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.

  39. Results-5 Kinetic analysis of aromatase inhibition with UB

  40. Materials and Methods • Placental microsome assay. • UB concentrations：0, 23.5, 47 μmol/L. • 3H-androstenedione concentrations：0-200 nmol/L.

  41. 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

  42. 直接作圖 雙倒數圖形(double-reciprocal plot)

  43. Aromatase inhibition kinetic profile by UB compound 47μmol/L 23.5μmol/L Vmax 0μmol/L (Control) • Slope=Km/Vmax(1+[I]/Ki)就可以得到三個斜率值        Km

  44. 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

  45. 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.

  46. Discussion

  47. Conclusions

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