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 37C
  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

23
References

• Baylor College of Medicine. 2003. TRAMP-C1,
  TRAMP-C2 and TRAMP-C3 Novel epithelial cell
  lines derived from urine prostate cancer. Online
  at www.research.bcm.tmc.edu
• Center for Disease Control (CDC). 2002.
  Accessed 30 Mar. 2003. Online at www.cdc.gov
• Dhanalakshmi, S. R. P. Singh C. Agarwal and R.
  Agarwal. 2002. Silibinin inhibits constitutive
  and TNF"-induced activation of NF-6B and
  sensitizes human prostate carcinoma DU145 cells
  to TNF"-induced apoptosis. Oncogene
  21(11)1759-67.
• Fraschini, F G. Demartini and D. Esposti.
  2002. Pharmacology of silymarin. Clinical Drug
  Investigation 22(1)51-65.
• Golsby, R. A. T. J. Kindt and B. A. Osborne.
  2000. Kuby Immunology, 4th ed. New York W. H.
  Freeman.
• Greenberg, N. M. F. DeMayo M. J. Finegold D.
  Medina W. D. Tilley J. O. Aspinall G. R.
  Cunha A. A. Donjacour R. J. Matusik and J. M.
  Rosen. Prostate cancer in a transgenic mouse.
  Proceedings of the National Academy of Sciences
  USA 923439-43.
• Guide to Apoptotic Pathways. Manheim Boehringer.
  
• Iannotta, Beatrice. 2003. LNCaP. Biotech.
  Online at www.biotech.ist
• In situ cell death detection kit, POD. 2001.
  Instruction Manual. Roche Applied Science.
• Kohno, H. T. Tanaka K. Kawabata Y. Hirose S.
  Sugie H. Tsuda and H. Mori. 2002. Silymarin,
  a naturally occuring polyphenolic antioxidant
  flavonoid, inhibits azoxymethane-induced colon
  carcinogenesis in male F344 rats. Intl Jrnl of
  Cancer 101(5)461-8.
• Manna, S. K. A. Mukhopadhyay N. T. Van and B.
  B. Aggarwal. 1999. Silymarin suppresses
  TNF-induced activation of NF-6B, c-Jun N-terminal
  kinase, and apoptosis. Journal of Immunology
  163(12)6800-9.
• Piqueras, B. P. D. Autran and G. Gorochov.
  1996. Detection of apoptosis at the single-cell
  level by direct incorporation of fluorescein-dUTP
  in DNA strand breaks. Biotechniques
  20(4)634-640.

24
References

• Roche Applied Science. 2000. Apoptosis special
  interest site In situ cell death detection kit,
  POD. Accessed on 23 Apr. 2003. Online at
  www.roche-applied-science.com
• Saller, R. R. Meier and R. Brignoli. 2001.
  The use of silymarin in the treatment of liver
  diseases. Drugs 61(14)2035-63.
• Shimizu, Ichiro. 2000. Sho-saiko-to Japanese
  herbal medicine for protection against hepatic
  fibrosis and carcinoma. Journal of
  Gastroenterology Hepatology 15(3)84-90.
• Singh, R. P. S. Dhanalakshmi A. K. Tyagi D. C.
  Chan C. Agarwal and R. Agarwal. 2002. Dietary
  feeding of silibinin inhibits advance human
  prostate carcinoma growth in athymic nude mice
  and increases plasma insulin-like growth
  factor-binding protein-3 levels. Cancer Research
  62(11)3063-9.
• Tyagi, A. N. Bhatia M. S. Condon M. C.
  Bosland C. Agarwal and R. Agarwal. 2002.
  Antiproliferative and apoptotic effects of
  silibinin in rat prostate cancer cells. Prostate
  53(3)211-7.
• Tyagi, A. K. R. P. Singh C. Agarwal D. C.
  Chan R. Agarwal. 2002. Silibinin strongly
  synergizes human prostate carcinoma DU145 cells
  to doxorubicin-induced growth inhibition, G2-M
  arrest, and apoptosis. Clinical Cancer Research
  8(11)3512-9.
• Wildland Invasive Species Team. 2003. Nature
  Conservancy. Accessed 1 Apr. 2003. Online at
  www.tncweeds.ucdavis.edu
• Wong, B. Y. Y. 1992. Modulation of rat hepatic
  S9-dependent mutagenesis, DNA binding, and
  metabolism of aflatoxin B1 and benzoapyrene by
  four Chinese medicinal herbs. Dissertation for
  Degree of Doctor of Philosophy in Biology.
• Zi, X. J. Zhang R. Agarwal and M. Pollak.
  2000. Silibinin up-regulates insulin-like growth
  factor-binding protein 3 expression and inhibits
  proliferation of androgen-independent prostate
  cancer cells. Cancer Research 60(20)5617-20.

25
Questions?