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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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