The Anti-carcinogenic Effect of Echinacea purpurea and Echinacea pallida on BT-549

1. The Anti-carcinogenic Effectof Echinaceapurpurea and Echinaceapallidaon BT-549 Nicole Driggins, Dr. E. Lewis Myles, & Dr. Todd Gary Department of Biological Sciences & Center of Excellence Tennessee State University, Nashville TN

2. Medicinal plants have been used to treat different ailments for centuries. Salicin – willow tree Medicinal Plants

3. Taxol® (Paclitaxel) • In 1975 researchers discovered that taxol, a substance found in the bark of a yew tree, decreases the production of cancerous tumors. • In 1994, FDA approved the use of taxol to treat breast cancer.

4. Figure 1. Current Research

5. Figure 2. Echinacea Products

6. Fig. 3 Other Echinacea Products

7. Echinacea • Genus: Echinacea • Family: Asteraceae • Number of species: Nine • North American Plant • Medicinal plant noted for the use of stimulating the immune system.

8. E. angustifolia* E. atrorubens E. laevigata** E. pallida* E. paradoxa E. sanguinea E. simulata E. tennesseensis** E. purpurea* Species of Echinacea

9. Figure 3. Echinacea purpurea Characterized by the white flowering rays that reflex backwards.

10. Figure 4. Echinacea pallida Characterized by long, branching, flowering stems and slender purple ray flowers.

11. Figure 6. Echinacea augustifolia Characterized by the purpleflowering rays that reflex backwards.

12. Figure 7. Echinacea tennesseensis Its rays spread up and outward to form an inverted cup-shaped corolla.

13. The Greenhouse

14. Materials: Part One • Plantextract • Breast Cancer Cell Line • 50 ml Tissue Culture Flasks • Media • PBS • Control: DMSO • Six Well Culture Plate • Hemocytometer • Trypan Blue

15. Breast Cancer Cells • BT-549 cell line (isolated in 1978) • The cells are from a 72 year old Caucasian female. • Grown & maintained in the lab.

16. Growth Media • RPMI – 1640 • Developed at Roswell Park Memorial Institute in 1966 for the growth of human leukemia cells. • Demonstrated a universal use in the growth and support of several mammalian cells that are anchorage dependent.

17. PBS • Phosphate Buffered Saline • (Na2HPO4·7H2O, NaH2PO4·1H2O • NaCl) • Isotonic solution used to wash • the cancer cells. • Non-toxic to the cancer cells

18. DMSO • Dimethyl Sulfoxide • Used to dissolve components of the plant • Non-toxic to the cancer cells • Obtained from Sigma-Aldrich

19. Method • The cells are removed from the tissue flasks. • The cells are counted by using a hemocytometer. • The same number of cells are placed in each well of the culture flask, which contains media.

20. Figure 8.Six Well Cell Culture Plate Ech. 1 DMSO 1 Ech. 2 DMSO 2 Ech. 3 DMSO 3

21. Anti-Carcinogen Procedure Media + Cancer Cells + Plant Extract or Control + 4 Day Incubation + Remove and Count Viable Cells

22. Fig. 9 Growth Analysis of E. purpurea

23. Fig. 10 Growth Analysis of E. pallida

24. Figure 11 Current Research Revisited

25. Plantextract Breast Cancer Cell Line 50 ml Tissue Culture Flasks Media Hemocytometer 20 ml Tissue Culture Flasks PBS Control: DMSO Cell Culture Slides Apoptosis Kit Confocal Microscope Materials: Part Two

26. Apoptosis Assay • Expose cells to E. purpurea or E. pallida for two days • Expose cells to DMSO for two days • Expose cells to labeling dye • Observe under confocal microscope • Viable Cells = green • Necrotic Cells = red & green • Apoptotic Cells = red • Sigma-Aldrich

27. Fig. 12 Labeling Dye and Apoptosis

28. Fig. 13 Labeling Dye and Necrotic Cells

29. Figure 14.Confocal Microscope

30. Figure 15.The Mechanism of a Confocal Microscopehttp://www.physics.emory.edu/~weeks/confocal/

31. Figure 16. Necrosis

32. Figure 17.Apoptosis

33. Fig. 18 Apoptosis FADD

34. Fig. 19 Apoptosis Cont.’d FADD Pro-caspase 8

35. Fig. 20 Apoptosis Cont.’d FADD Bid Caspase 8

36. Fig. 21 Apoptosis Cont.’d Bid Bax Mitochondrion Cytochrome C

37. Fig. 22 Apoptosis Cont.’d Apaf -1 Pro-caspase 9 Cytochrome C

38. Fig. 23 Apoptosis Cont.’d Apaf-1 Caspase Cascade Caspase 9 Cytochrome C Apoptosis

39. Figure 24.Apoptosis Cont.’d Bax Mitochondrion FADD Caspase 8 Bid Apaf-1 Caspase Cascade Caspase 9 Cytochrome C Apoptosis

40. Fig. 25 Confocal Image of Cells Treated with E. purpurea

41. Fig. 26 Confocal Image of Cells Treated with DMSO

42. Fig. 27 Confocal Image of Cells Treated with E. pallida

43. Figure 28.Current Research Revisited

44. Conclusion • Further Apoptosis Analysis with E. purpurea • Further Apoptosis Analysis with DMSO • Further Apoptosis Analysis with E. pallida • Determination of E. purpurea and E. pallida component(s) that causes the effect on the breast cancer cells • Addition of a normal breast cell line and an African-American breast cancer cell line • Western Blot Analysis

45. Acknowledgements • NASA, NIH • Dr. E. Lewis Myles • Dr. Todd Gary • Dr. Johnson, Dr. Blackshear, Dr. Kahlon and Dr. Whalen • Mrs. Yvonne Myles • Dr. John Robinson, Ryan Johnson, Brad Smith • Wendel Forston, Clifton Randell, Tanisha Taylor, Alicia Cleveland, Tim Udoji, Chasity Bradley and Tamela Hunt

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