Nitrogen Containing Bisphosphonates Induce Apoptosis and Inhibit the Mevalonate Pathway, Impairing Ras Membrane Localization in Prostate Cancer Cells

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Abstract
Purpose: Metastasis to bone is an important cause of morbidity in advanced prostate cancer. Despite the typically sclerotic nature of prostatic bone metastases osteolysis has a significant role in the pathogenesis of this disease. The nitrogen containing bisphosphonates (N-BPs), such as pamidronate and zoledronic acid, have greatly enhanced potency for inhibiting bone resorption and inducing apoptosis in osteoclasts. We investigated the effects of N-BPs on prostate cancer cells. Materials and Methods: Cell viability was determined with an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeyhoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) dye reduction assay. Cell cycle analysis, DNA fragmentation and caspase 3 activity were assessed using flow cytometry. Ras, Bcl-2 and Bax were quantified by Western blotting. Results: Pamidronate and zoledronic acid decreased cell viability in the 3 human cell lines DU145, PC3 and LNCaP. These effects were associated with changes in cell cycle distribution, induction of DNA fragmentation and a decrease in the Bcl-2-to-Bax ratio, which are features of apoptotic cell death. Pre-incubation with caspase inhibitors attenuated the effects of zoledronic acid and caspase 3 activity was demonstrated in treated DU145 cells. Zoledronic acid induced loss of cell viability in DU145 cells was prevented by co-treatment with farnesol, suggesting that N-BPs cause inhibition of the mevalonate pathway and Ras prenylation. A decrease in active, membrane bound Ras in zoledronic acid treated DU145 cells was shown by Western blot analysis. Conclusions: N-BPs induce apoptosis in prostate cancer via a caspase dependent mechanism. They have effects on protein prenylation via inhibition of the mevalonate pathway and impair membrane localization of Ras in prostate cancer cells.