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

IntroductionProstate Cancer

The second most common cancer among American menAmerican Cancer Society estimated for 2002: 30,200 men would die 189,000 new casesDifficult to test possible treatments on human subjects

IntroductionProstate Cancer Cell Line TRAMP-C1

Dr. Norman Greenberg, Baylor College of MedicineTRAMP-C1: in vitro cell cultureTransgenic Adenocarcinoma Mouse ProstateGenetically manipulated C57BL/6 miceProstate cancer after pubertyTumors: elevated p53

IntroductionProstate Cancer Cell Line LNCaP

LNCaP = human50-year-old man1977AggregateSlow-growing (DT = 60 h)

IntroductionMilk Thistle

Silybum marianum (SM)Traditional herbal therapy: hepatitis, cirrhosis, mushroom & alcohol poisoning, psoriasisReadily available as commercial product Silybum marianum (Milk

Thistle)2003 Nature Conservancy

IntroductionMilk Thistle

SM inhibits cancer cell growth in vitroSilymarin blocks NF-kappa B activation by TNF reduces effects of azoxymethane in

colons of F344 ratsSilibinin inhibits rat H-7, I-8, I-26 inhibits human PC-3, DU145

IntroductionTUNEL Reaction

Roche Applied Science 2000

TdT adding fluorescein labeled nucleotides to DNA strand breaks

Anti-fluorescein-antibody conjugated with peroxidase

Substrate for peroxidase

IntroductionHypothesis

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

Materials and MethodsCell line maintenance

Cells grown on surface of sterile plastic flasks or plates in liquid growth mediumExperimental plates contained approx. 5000 cellsCells maintained in humidified incubator at 37°C and 5% CO2

Materials and MethodsPreparation of Herbal Extract

Dissolved commercial milk thistle extract in waterFiltered suspensionFreeze-dried filtrateDetermined exact weight of SMRehydrated SM (known concentration)Filter-sterilized solution

Materials and MethodsDetermination of LD50

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

Materials and MethodsTUNEL Assay Protocol

Cells incubated with 0.8 mg/ml SM for 2 and 8 hrsCells fixed with paraformaldehydeNucleases blocked w/ H2O2 in methanolCells permeabilized w/ Triton X-100TUNEL reaction performedCells stained by oxidized substrate observed under light microscope

ResultsBest dosage (LD50) was 0.8 mg/mlSM induced apoptosis in both TRAMP-C1 and LNCaP

ResultsTRAMP-C1

LNCaP

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

ResultsTRAMP-C1

LNCaP

Apoptotic nuclei

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

ResultsTRAMP-C1

LNCaP

Apoptotic nuclei

Necrotic nuclei Photos: Brian Y. Y. Wong,

Ph.D.

ResultsTRAMP-C1

LNCaP

Apoptotic nuclei

Necrotic nuclei Photos: Brian Y. Y. Wong,

Ph.D.

Unstained nuclei

ResultsApoptosis was indicated at both incubation timesGreater number of cells were apoptotic than necroticEffects of SM were time-dependent

Results

Results

ConclusionsSM kills prostate cancer cells in vitro by apoptosisOptimal incubation time with SM for TRAMP-C1 = 2 hrsOptimal time for LNCaP = at least 8 hrsSM has potentially chemopreventive properties against prostate cancer

AcknowledgmentsMy primary advisor for this project was Dr. Brian WongCell lines were a gift from Dr. Norman GreenbergPhotographs were provided by the Marketing Dept. at Union CollegeStudent research travel award was provided by the Nebraska Academy of SciencesSupport for research was provided by the Union Scholars Program

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