silybum marianum induces apoptosis in mouse tramp c1 and human lncap cancer cells
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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. Introduction Prostate Cancer.

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silybum marianum induces apoptosis in mouse tramp c1 and human lncap cancer cells

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

introduction prostate cancer
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
introduction prostate cancer cell line tramp c1
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
introduction prostate cancer cell line lncap
IntroductionProstate Cancer Cell Line LNCaP
  • LNCaP = human
  • 50-year-old man
  • 1977
  • Aggregate
  • Slow-growing (DT = 60 h)
introduction milk thistle
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

introduction milk thistle6
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
introduction tunel reaction
IntroductionTUNEL Reaction

Anti-fluorescein-antibody conjugated with peroxidase

TdT adding fluorescein labeled nucleotides to DNA strand breaks

Substrate for peroxidase

Roche Applied Science 2000

introduction hypothesis
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
materials and methods cell line maintenance
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
materials and methods preparation of herbal extract
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
materials and methods determination of ld 50
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
materials and methods tunel assay protocol
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
results
Results
  • Best dosage (LD50) was 0.8 mg/ml
  • SM induced apoptosis in both TRAMP-C1 and LNCaP
results14
Results

TRAMP-C1

LNCaP

Photos: Brian Y. Y. Wong, Ph.D.

results15
Results

TRAMP-C1

LNCaP

Apoptotic nuclei

Photos: Brian Y. Y. Wong, Ph.D.

results16
Results

TRAMP-C1

LNCaP

Apoptotic nuclei

Necrotic nuclei

Photos: Brian Y. Y. Wong, Ph.D.

results17
Results

Unstained nuclei

TRAMP-C1

LNCaP

Apoptotic nuclei

Necrotic nuclei

Photos: Brian Y. Y. Wong, Ph.D.

results18
Results
  • Apoptosis was indicated at both incubation times
  • Greater number of cells were apoptotic than necrotic
  • Effects of SM were time-dependent
conclusions
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
acknowledgments
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
references
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 92:3439-43.

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.

references24
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 benzo[a]pyrene 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.

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