Androgen receptor (AR) is a key regulator involved in prostate cancer (PCA) growth; therefore, identification and development of novel non-toxic phytochemicals that target androgen and AR signaling remains a priority for PCA control. Several studies by us have shown both anticancer and chemopreventive effects of silymarin (crude milk thistle extract) and silibinin (major active diastereomeric fraction in silymarin) against PCA. Recently, we have isolated and purified additional silibinin stereoisomers from silymarin, and found isosilybin B as a novel potent agent against human PCA cells in culture. Here we conducted detailed mechanistic and efficacy studies with isosilybin B employing androgen-dependent human LNCaP PCA cells as a model. Treatment of cells with isosilybin B strongly decreased AR levels in both nuclear and cytosolic fractions. This effect of isosilybin B might have altered AR binding with ARE resulting in a decreased AR mediated transcriptional activity, as evidenced by a decrease in both intracellular and secreted form of PSA in isosilybin B-treated cells. To further support the finding that isosilybin B indeed targets androgen-AR pathway, cells were grown in charcoal-stripped serum and then stimulated with a synthetic androgen R1881 without or with isosilybin B treatment. In this study also, isosilybin B showed a strong anti-androgenic effect in terms of a decrease in both PSA secretion and cell growth. In the studies identifying the mechanism of AR degradation, isosilybin B treatment of cells resulted in an increased phosphorylation of Akt at Ser473 and of Mdm2 at Ser166, which might be linked with AR degradation as pre-treatment with PI3K inhibitor (LY294002) restored AR level through inhibiting phosphorylation of both Akt and Mdm2. Further studies showed that isosilybin B increases Mdm2 (an E3 ligase) binding with phospho-Akt and AR, which might be responsible for increased AR ubiquitination followed by proteosomal degradation; more studies in future are needed to support this assumption. In terms of its biological effects, isosilybin B decreased cell number and resulted in G1 arrest, which was associated with a marked decrease in the protein levels of CDK2 and CDK4 and cyclins namely D1, D3, E and A, together with a strong induction of CDK inhibitors Cip1/p21 and Kip1/p27. In additional studies assessing whether isosilybin B-mediated AR degradation plays a role in its biological activity, restoration of AR level by LY294002 pre-treatment also attenuated isosilybin B-caused cell cycle arrest, suggesting a possible association between these two events. Together, this study provides more in-depth knowledge about isosilybin B effects in androgen-dependent LNCaP cells, and suggests additional efficacy and mechanistic investigations with this agent in PCA models.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]