Detrimental effect of cancer preventive phytochemicals silymarin, genistein and epigallocatechin 3-gallate on epigenetic events in human prostate carcinoma DU145 cells.
BACKGROUND
Targeting epigenetic events associated with autonomous growth of advanced prostate cancer (PCA) is a practical approach for its control, prevention, and treatment. Recently we showed that treatment of prostate carcinoma DU145 cells with cancer preventive flavonoid silymarin at 100-200 microM doses inhibits erbB1-Shc mitogenic signaling and modulates cell cycle regulators leading to a G1 arrest and inhibition of cell growth and anchorage-independent colony formation. Here, we asked the question whether these important findings could be extended to other cancer preventive flavonoids and isoflavones such as epigallocatechin 3-gallate (EGCG) and genistein.


METHODS
DU145 cells were treated with similar doses (100-200 microM) of silymarin, genistein or EGCG, cell lysates prepared, and levels of activated signaling molecules (erbB1-Shc-ERK1/2) and cell cycle regulators (CDKIs, CDKs, and cyclins) analyzed employing immunoprecipitation and/or immunoblotting techniques. Cell growth studies were done by cell counting during 5 days of treatment with these agents, and cell death was determined by Trypan blue staining.


RESULTS
Treatment of cells with silymarin, genistein or EGCG at 100-200 microM resulted in a complete inhibition of TGFalpha-caused activation of erbB1 followed by a moderate to strong inhibition (10-90%) of Shc activation without an alteration in their protein levels. Silymarin and genistein, but not EGCG, also inhibited (10% to complete) ERK1/2 activation suggesting that these agents impair erbB1-Shc-ERK1/2 signaling in DU145 cells. In other studies, silymarin, genistein or EGCG caused a strong induction of Cip1/p21 (up to 2.4-fold) and Kip1/p27 (up to 150-fold), and a strong decrease in CDK4 (40-90%) but had moderate effect on CDK2, and cyclins D1 and E. An enhanced level of CDKIs also led to an increase in their binding to CDK4 and CDK2. Treatment of cells with silymarin, genistein or EGCG also resulted in 50-80% cell growth inhibition at lower doses, and complete inhibition at higher doses. In contrast to silymarin, higher doses of genistein showed cytotoxic effect causing 30-40% cell death. A more profound cytotoxic effect was observed with EGCG accounting for 50% cell death at lower doses and complete loss of viability at higher doses.


CONCLUSIONS
These results suggest that similar to silymarin, genistein and EGCG also inhibit mitogenic signaling pathway(s) and alter cell cycle regulators, albeit at different levels, leading to growth inhibition and death of advanced and androgen-independent prostate carcinoma cells. More studies are, therefore, needed with these agents to explore their anti-carcinogenic potential against human prostate cancer.
Bhatia
N
N
Center for Cancer Causation and Prevention, AMC Cancer Research Center, 1600 Pierce Street, Denver, CO 80214, USA.
Agarwal
R
R
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D016923:Cell Death / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D066246:ErbB Receptors / Q000502:physiology; D019833:Genistein / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D010936:Plant Extracts / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology / Q000517:prevention & control*; D011485:Protein Binding; D011506:Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D055765:Shc Signaling Adaptor Proteins; D012838:Silymarin / Q000494:pharmacology*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016211:Transforming Growth Factor alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D016588:Anticarcinogenic Agents; D010936:Plant Extracts; D011506:Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D012838:Silymarin; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016211:Transforming Growth Factor alpha; D002392:Catechin; C045651:epigallocatechin gallate; D019833:Genistein; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases