Silybum marianum Induces Apoptosis in Mouse (TRAMP-C1) and Human (LNCaP) Cancer Cells

1. Silybum marianum Induces Apoptosis in Mouse (TRAMP-C1) and Human (LNCaP) Cancer Cells Peter R. McHenry, H. H. L. Wong, Union College, Lincoln, NE; N. M. Greenberg, Baylor College of Medicine, Houston, TX; B. Y. Y. Wong, Union College, Lincoln, NE

2. IntroductionProstate Cancer • The second most common cancer among American men • American Cancer Society estimated for 2002: • 30,200 men would die • 189,000 new cases • Difficult to test possible treatments on human subjects

3. IntroductionProstate Cancer Cell Line TRAMP-C1 • Dr. Norman Greenberg, Baylor College of Medicine • TRAMP-C1: in vitro cell culture • Transgenic Adenocarcinoma Mouse Prostate • Genetically manipulated C57BL/6 mice • Prostate cancer after puberty • Tumors: elevated p53

4. IntroductionProstate Cancer Cell Line LNCaP • LNCaP = human • 50-year-old man • 1977 • Aggregate • Slow-growing (DT = 60 h)

5. IntroductionMilk Thistle • Silybum marianum (SM) • Traditional herbal therapy: hepatitis, cirrhosis, mushroom & alcohol poisoning, psoriasis • Readily available as commercial product Silybum marianum (Milk Thistle) 2003 Nature Conservancy

6. IntroductionMilk Thistle • SM inhibits cancer cell growth in vitro • Silymarin • blocks NF-kappa B activation by TNF • reduces effects of azoxymethane in colons of F344 rats • Silibinin • inhibits rat H-7, I-8, I-26 • inhibits human PC-3, DU145

7. IntroductionTUNEL Reaction Anti-fluorescein-antibody conjugated with peroxidase TdT adding fluorescein labeled nucleotides to DNA strand breaks Substrate for peroxidase Roche Applied Science 2000

8. IntroductionHypothesis • We sought to determine the effects of an aqueous extract from the achenes of SM on TRAMP-C1 and LNCaP cells • We hypothesized that SM would trigger apoptosis in these prostate cancer cells

9. Materials and MethodsCell line maintenance • Cells grown on surface of sterile plastic flasks or plates in liquid growth medium • Experimental plates contained approx. 5000 cells • Cells maintained in humidified incubator at 37°C and 5% CO2

10. Materials and MethodsPreparation of Herbal Extract • Dissolved commercial milk thistle extract in water • Filtered suspension • Freeze-dried filtrate • Determined exact weight of SM • Rehydrated SM (known concentration) • Filter-sterilized solution

11. Materials and MethodsDetermination of LD50 • LD50 = 50% lethal dose • Treated each plate (approx. 5000 cells) with different doses of SM for 24 hrs • Fixed, stained plates and counted surviving cell colonies • Plotted data on graph and interpolated point at which only 50% of cells survived

12. Materials and MethodsTUNEL Assay Protocol • Cells incubated with 0.8 mg/ml SM for 2 and 8 hrs • Cells fixed with paraformaldehyde • Nucleases blocked w/ H2O2 in methanol • Cells permeabilized w/ Triton X-100 • TUNEL reaction performed • Cells stained by oxidized substrate observed under light microscope

13. Results • Best dosage (LD50) was 0.8 mg/ml • SM induced apoptosis in both TRAMP-C1 and LNCaP

14. Results TRAMP-C1 LNCaP Photos: Brian Y. Y. Wong, Ph.D.

15. Results TRAMP-C1 LNCaP Apoptotic nuclei Photos: Brian Y. Y. Wong, Ph.D.

16. Results TRAMP-C1 LNCaP Apoptotic nuclei Necrotic nuclei Photos: Brian Y. Y. Wong, Ph.D.

17. Results Unstained nuclei TRAMP-C1 LNCaP Apoptotic nuclei Necrotic nuclei Photos: Brian Y. Y. Wong, Ph.D.

18. Results • Apoptosis was indicated at both incubation times • Greater number of cells were apoptotic than necrotic • Effects of SM were time-dependent

19. Results

20. Results

21. Conclusions • SM kills prostate cancer cells in vitro by apoptosis • Optimal incubation time with SM for TRAMP-C1 = 2 hrs • Optimal time for LNCaP = at least 8 hrs • SM has potentially chemopreventive properties against prostate cancer

22. Acknowledgments • My primary advisor for this project was Dr. Brian Wong • Cell lines were a gift from Dr. Norman Greenberg • Photographs were provided by the Marketing Dept. at Union College • Student research travel award was provided by the Nebraska Academy of Sciences • Support for research was provided by the Union Scholars Program

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