Recent studies from our group and others showed that Bcl-xL is a major cellular survival

factor in castration-resistant prostate cancers [11, 13–15]. Therefore, we evaluated if Bcl-xL modulates R-568-induced apoptosis. Two previously confirmed EPZ015938 ic50 LNCaP sublines, LNCaP/Bclxl (Bcl-xL overexpression) and LNCaP/LN11 (Bcl-xL null) described in our recent publication [11], were used in a trypan blue exclusion assay. Compared to the parental LNCaP cells, enforced Bcl-xL expression abolished R-568-induced cell death in LNCaP/Bclxl cells while loss of Bcl-xL expression significantly increased R-568-induced cell death in LNCaP/LN11 cells [Fig 4A]. Consistently, caspase-3 processing and PARP cleavage were also dramatically attenuated due to altered levels of Bcl-xL expression in response to R-568 treatment [Fig 4B]. These data further confirmed that R-568-induced

cytotoxicity is due to mitochondria-related mechanism in prostate cancer cells. LY2603618 Figure 4 R-568-induced apoptosis is attenuated by altered Bcl-xL expression in prostate cancer cells. A LNCaP cells and its two sublines, LNCaP/Bclxl and LNCaP/LN11, were seeded in 12-well plates and treated with R-568 at the indicated doses for 48 h. The control cells received no treatment. Cells were harvested at the end of experiment and stained in 0.4% trypan blue solution. The dead (blue) cells were counted and the average of death rate in each well was presented. Data represent three different experiments. The asterisk indicates a significant difference (P < 0.05) between R-568 treatment and the control. B LNCaP/Bclxl and LNCaP/LN11 cells were treated with R-568 at indicated doses for 24 h and then harvested for protein extraction. Equal amounts of cellular proteins were subjected to Western blot assay to assess caspase-3 processing and PARP Grape seed extract cleavage. Primary antibodies used are indicated on the left side. Actin blot served as the protein loading control. Data

represent two different experiments. Discussion The primary goal of this study was to determine the biological effect of the Foretinib calcimimetic NPS R-568 on prostate cancer cells. Using two commonly used prostate cancer cell lines, AR-positive LNCaP and AR-negative PC-3, we demonstrated that R-568 reduced cell viability of both cell lines in a dose- and time-dependent manner. R-568-induced cell death is an apoptotic response through a mitochondria-related mechanism and CaSR is essential for R-568-induced cell death. These data provided the preliminary evidence that the calcimimetic R-568 might be useful as adjunctive therapeutic agent for advanced prostate cancers although further pre-clinical testing is desirable. Currently, limited information is available for calcimimetic NPS R-568-induced apoptosis in mammalian cells.