Transcript 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
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
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
LNCaP = human
50-year-old man
1977
Aggregate
Slow-growing (DT = 60 h)
Introduction
Milk 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 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
TdT adding fluorescein
labeled nucleotides to
DNA strand breaks
Anti-fluoresceinantibody
conjugated with
peroxidase
Substrate for
peroxidase
Roche Applied Science 2000
Introduction
Hypothesis
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
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
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 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
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
Best dosage (LD50) was 0.8 mg/ml
SM induced apoptosis in both TRAMPC1 and LNCaP
Results
TRAMP-C1
LNCaP
Photos: Brian Y. Y. Wong, Ph.D.
Results
TRAMP-C1
LNCaP
Apoptotic nuclei
Photos: Brian Y. Y. Wong, Ph.D.
Results
TRAMP-C1
LNCaP
Apoptotic nuclei
Necrotic nuclei
Photos: Brian Y. Y. Wong, Ph.D.
Results
Unstained nuclei
TRAMP-C1
LNCaP
Apoptotic nuclei
Necrotic nuclei
Photos: Brian Y. Y. Wong, Ph.D.
Results
Apoptosis was indicated at both
incubation times
Greater number of cells were apoptotic
than necrotic
Effects of SM were time-dependent
Results
Results
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
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
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 azoxymethaneinduced 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.
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.
Questions?