Meanwhile, PTH has PLX-4720 in vitro been shown to increase cell proliferation of human prostate cancer in vitro  and to promote bone metastasis in mouse xenograft model of prostate cancer . Therefore, reducing PTH secretion could potentially interrupt SHPT and be of substantial clinical benefit in prostate cancer patients. In fact, a functional CaSR was detected in human prostate cancer cells [9, 10]. However, the biological effect of calcimimetic agents on prostate cancer cells has not been evaluated. Therefore, in this study, we tested the biological effect of calcimimetic agent NPS R-568 on multiple prostate cancer cells. We surprisingly found for the first time that NPS R-568 induced apoptotic cell
death, which is dependent on the CaSR and is modulated by anti-apoptotic Bcl-xL pathway.
Materials and methods Cell Culture, Reagents and Antibodies Human prostate cancer PC-3 and LNCaP, as well as LNCaP sublines (LNCaP/Bclxl and LNCaP/LN11) were described in our previous publication . Briefly, LNCaP/Bclxl cells were established by stable transfection of LNCaP cells with a vector bearing HA-tagged selleck human bcl-xl cDNA ARN-509 research buy sequence (pcDNA3.1-Bclxl.HA). LN11 is a LNCaP cell subline that lost Bcl-xL expression, as described . Cells were maintained in a humidified atmosphere of 5% CO2, RPMI 1640 supplemented with 10% fetal bovine serum (FBS) with antibiotics (Invitrogen, Carlsbad, CA). Antibodies for PARP, caspase-3, CaSR and Actin were purchased from Santa Cruz Biotech (Santa Cruz, CA). CaSR small interference RNA (siRNA) mixture and the negative control siRNA were obtained from Santa Cruz Biotech. The calcimimetic R isomer of N-[3-[2-chlorophenyl]propyl]-[R]-α-methyl-3-methoxybenzylamine (NPS R-568) and its inactive isomer NPS S-568 were kindly provided by Amgen, Inc. (Thousand Oaks, CA). Cell Viability Analyses For MTT [3-[4,5-dimethylthazol-2-yl]-2,5-diphenyl tetrazolium-Bromide] assay, which is based on the conversion of MTT to MTT-formazan by mitochondrial enzyme, a cell growth
determination kit (Sigma Co., St Louse, MO) was utilized according to the instruction from the manufacturer. Selleck Cisplatin Briefly, cells were seeded at a density of 2 × 103 cells/well in 96-well plates in triplicates and allowed to attachment overnight. Cells were then maintained in various conditions as indicated in the figures. The MTT solution was added in an amount equal to 10% of the culture volume. After 3 h incubation, the culture media was removed and the MTT solvent was added. The plates were read at a wavelength of 570 nM. For trypan blue assay, cells were seeded in 12-well plates, and then treated with various reagents as indicated in the figures. At the end of experiments, viable cells was counted using a hemocytometer after staining with trypan blue as described in our recent publication .