Abstract 5661: Silibinin inhibits advanced human prostate carcinoma-induced osteoclastogenesis
Authors: Gangar Subhash Chander , Deep Gagan , Agarwal Rajesh 
Published: February 2011

Despite significant progress, prostate cancer (PCA) remains second leading cause of cancer-related deaths in US. Advanced PCA cells metastasize preferentially to the bone in approximately 90% cases resulting in severe pain, cachexia, bone fractures, bone marrow replacement, and ultimately death of the patient. Increased osteoclastic activity plays a critical role in establishing metastatic PCA growth in bone suggesting that targeting aberrant osteoclastogenesis would alleviate the suffering of PCA patients and bone metastasis-associated mortality. Numerous studies by us have established silibinin (SB), non-toxic phytochemical from Silybum marianum seeds, as a potent anti-cancer agent against different malignancies including PCA; however, the effect of SB on the interaction between PCA and bone cells remained un-known. Accordingly, here we evaluated the efficacy of SB against PCA (PC-3, PC3MM2 and C4-2B) cells conditioned medium (CM)-induced osteoclastogenesis in RAW264.7 cells (pre-osteoclastic macrophages). PCA cells were treated with DMSO or SB; thereafter, fresh medium containing 0.5% FBS was added and collected after 12h and labeled as CCM (from DMSO treated PCA cells) or SBCM (from SB-treated PCA cells). RAW264.7 cells were treated with CCM or SBCM (volume normalized with respective PCA cell numbers) in the presence of 5 ng/ml RANKL; RAW264.7 cells in RPMI1640 (0.5% FBS) supplemented with 100 or 5 ng/ml of RANKL served as positive or negative controls, respectively. In each case, media were replaced after 48h and after 5 days, RAW264.7 cells were stained for tartrate resistant acid phosphatase (TRACP) activity (a measure of osteoclastic activity). Our results showed that CCM enhances osteoclastic activity and the number of differentiated osteoclasts compared to negative control (p&lt;0.001). However, osteoclastic activity and differentiated osteoclast numbers were significantly lesser in samples that were supplemented with SBCM (p&lt;0.001). Moreover, exogenous SB supplementation to CCM or regular medium of RAW264.7 cells containing 100 ng/ml RANKL, completely abolished osteoclastic activity and differentiated osteoclast numbers (p&lt;0.001). In mechanistic studies, treatment of serum starved RAW264.7 cells with 100 ng/ml RANKL or CCM (from PC3MM2 cells) increased the expression of phospho-IkBα (Ser-32), phospho-IKKα/β (Ser176/180) and phospho-ERK1/2(Thr202/Tyr204); however a decreased phosphorylation of these molecules was observed following SBCM treatment of starved RAW264.7 cells. In other studies, SB treatment increased the expression of osteoprotegerin (decoy receptor of RANKL), a well known inhibitor of osteoclastogenesis, in PC3MM2 and C4-2B cells. Collectively, our present findings provide novel insights for the inhibition of PCA cell-induced osteoclastogenesis by silibinin and suggest its clinical use for preventing skeletal complications in PCA patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5661.