Effect of zoledronic acid exposure on Ki-67 expression and proliferation in MCF7 cells resistant to apoptosis induction.

Abstract

e21129 Background: Bisphosphonates (BP) and the RANK-ligand antibody denosumab (D) are used for the treatment of bone metastases. Low to high concentrations of zoledronic acid (ZA) can induce tumor cell apoptosis in vitro via inhibition of the mevalonate pathway and/or accumulation of the ATP analog ApppI. Clinical studies indicate a benefit in tumor relapse and survival with the supportive treatment of ER-positive breast cancer using zoledronate (ZA), but the molecular mechanisms involved are under debate. Methods: MDA-MB-231 and MCF-7 breast cancer cells were treated for 3 h (pulse treatment) and 72 h (permanent treatment) with 5 – 100 µM ibandronate (IBN), alendronate (ALN), risedronate (RIS) and ZA and 1 – 100 ng/ml D for comparison. Apoptosis and proliferation was determined after 72 h. Rescue experiments for the BP effects were done using geranylgeranyl-pyrophosphate (GGPP) and atorvastatin (Ator). Microarray hybridizations were performed to identify target genes in MCF-7. Results: Permanent and pulse exposure to ZA induced apoptosis in MDA-MB-231 and inhibited proliferation in MCF-7 without affecting apoptosis. While IBN, ALN and RIS were inferior to ZA in apoptosis induction in MDA-MB-231, they were equipotent in proliferation inhibition in MCF-7. GGPP rescued ZA effects in MDA-MB-231 but not the antiproliferative effects in MCF-7, while Ator rescued the latter. qPCR and immunocytochemistry identified KLF2, KLF4, KLF6 and Ki-67 as target genes of ZA in MCF-7. RANKL did not induce proliferation in MCF-7 cells and D did not affect the untreated or RANKL pretreated cells in terms of proliferation and apoptosis. Conclusions: In summary we show here direct effects of ZA and other BP on cell proliferation and expression of tumor relevant genes in MCF-7 cells, which are relatively resistant to ZA-induced apoptosis in comparison with MDA-MB-231. Ator but not GGPP rescued these effects, possibly indicating that these effects are rather due to the accumulation of ATP analogs than to the inhibition of protein prenylation. D had no effects under these basal conditions but future experiments should address the effects in the context of estrogens and gestagens and also in coculture with bone cells.