Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release, and caspase activation.
Silymarin, a plant flavonoid, has been shown to inhibit skin carcinogenesis in mice. However, the mechanism responsible for the anti-skin carcinogenic effects of silymarin is not clearly understood. Here, we report that treatment of JB6 C141 cells (preneoplastic epidermal keratinocytes) and p53+/+ fibroblasts with silymarin and silibinin (a major constituent of silymarin) resulted in a dose-dependent inhibition of cell viability and induction of apoptosis in an identical manner. Silymarin-induced apoptosis was determined by fluorescence staining (8-64% apoptosis) and flow cytometry (12-76% apoptosis). The silymarin-induced apoptosis was primarily p53 dependent because apoptosis occurred to a much greater extent in the cells expressing wild-type p53 (p53+/+, 9-61%) than in p53-deficient cells (p53-/-, 6-20%). The induction of apoptosis in JB6 C141 cells was associated with increased expression of the tumor suppressor protein, p53, and its phosphorylation at Ser15. The constitutive expression of antiapoptotic proteins Bcl-2 and Bcl-xl were decreased after silymarin treatment, whereas the expression of the proapoptotic protein Bax was increased. There was a shift in Bax/Bcl-2 ratio in favor of apoptotic signal in silymarin-treated cells, which resulted in increased levels of cytochrome c release, apoptotic protease-activating factor-1, and cleaved caspase-3 and poly(ADP-ribose) polymerase in JB6 C141 cells. The shift in Bax/Bcl-2 ratio was more prominent in p53+/+ fibroblasts than in p53-/- cells. Silymarin-induced apoptosis was blocked by the caspase inhibitor (Z-VAD-FMK) in JB6 C141 cells which suggested the role of caspase activation in the induction of apoptosis. These observations show that silymarin-induced apoptosis is primarily p53 dependent and mediated through the activation of caspase-3.
Katiyar
Santosh K
SK
Department of Dermatology, University of Alabama at Birmingham, 1670 University Boulevard, Volker Hall 557, P.O. Box 202, Birmingham, AL 35294, USA. skatiyar@uab.edu
Roy
Anshu M
AM
Baliga
Manjeshwar S
MS
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D015536:Down-Regulation; D005347:Fibroblasts / Q000187:drug effects; D015603:Keratinocytes / Q000187:drug effects; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D000077385:Silybin; D012838:Silymarin / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
D000590:Amino Acid Chloromethyl Ketones; C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; C494827:Bax protein, mouse; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012838:Silymarin; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D000077385:Silybin; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases