Research paper The cell cycle arrest and the anti-invasive effects of nitrogen-containing bisphosphonates are not mediated by DBF4 in breast cancer cells Mahdieh Mansouri a , Seyed Abbas Mirzaei a, b , Hermann Lage b , Seyyedeh Soghra Mousavi a , Fatemeh Elahian a, b, * a Department of Pharmaceutical Biotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Iran b Institute of Pathology, Charité Campus Mitte, University Medicine Berlin, Berlin, Germany article info Article history: Received 19 July 2013 Accepted 11 November 2013 Available online 26 November 2013 Keywords: Bisphosphonates Cell cycle DBF4 Invasion assay Risedronate Zoledronate abstract Recent work has shown that a DBF4 analog in yeast may be a target of nitrogen-containing bisphosphonates. DBF4 is an essential protein kinase required for DNA replication from primary eu- karyotes to humans and appears to play a critical role in the S-phase checkpoint. It is also required for cell migration and cell surface adhesion. The effects of Pamidronate, risedronate, or zoledronate on cell viability and DBF4 expression were measured via MTT assays and western blotting. In addition, FACS cell cycle analyses and invasion assays were conducted in cells in the presence of nitrogen-containing bisphosphonates to identify any correlations between DBF4 expression and S-phase arrest or anti- invasive effects of the bisphosphonates. Zoledronate transiently down-regulated DBF4 expression in all three cell lines in the first 24 h of the experiment, but after 72 h, DBF4 expression returned to the control levels in all treated cells. Following treatment of the tumor cells with the bisphosphonates, the number of cells in S-phase was increased. Pamidronate and zoledronate showed anti-invasive effects in BT20 cells. The anti-invasive effects of pamidronate, risedronate and zoledronate appeared after 48 h of exposure. In MDA-MB231 cells a reduction of invasiveness was only observed after 72 h of the pamidronate exposure. We finally concluded that the anti-invasive and cell cycle arrest-inducing effects of nitrogen-containing bisphosphonates are not DBF4 mediated, and other mediators are therefore needed to explain the observed complex behaviors. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Bisphosphonates are a series of synthetic compounds that are widely used in the treatment of bone metabolic diseases associated with high bone resorption, including destructive arthropathy, fibrous dysplasia, heterotopic ossification, osteogenesis imperfecta, and Paget’s disease [1]. Bisphosphonates are chemically and enzymatically stable and resemble pyrophosphate analogs char- acterized by a PeCeP bond [2]. The germinal carbon in this moiety is covalently bonded to two different lateral chains that are usually referred to as the R1 and R2 groups. R1 and R2 confer a pronounced binding affinity for hydroxyapatite and antiresorptive properties in bone, respectively. Based on their molecular structure and mech- anism of action, two distinct types of bisphosphonates have been defined. Non-nitrogen-containing bisphosphonates (non-NBPs), such as clodronate and etidronate, are metabolized to form potentially cytotoxic analogs of ATP that accumulate in cells and deactivate osteoclasts, leading to cell death [1]. In contrast, nitrogen-containing bisphosphonates (NBPs), such as alendronate, ibandronate, pamidronate, risedronate, and zoledronate, inhibit intracellular farnesyl diphosphate synthase (FPPS) in the mevalo- nate pathway, which results in decreased levels of farnesyl diphosphate and geranylgeranyl diphosphate and inhibition of the prenylation of GTP-binding proteins (such as Ras, Rho, and Rac), which is considered to represent the main mechanism of osteoclast activity inhibition and apoptosis promotion [3,4]. Although these bioactive agents are only FDA approved for the treatment of bone metabolic diseases, they have recently gained attention due to their strong apoptosis induction effect in human myeloma cells [5], immunomodulation and stimulation of cytotoxic T-cells targeting tumor cells [4], inhibition of tumor angiogenesis [3], inhibition of the adhesion of invasive cancer cells to bone matrices [6], inhibition of cell invasion and proliferation, and * Corresponding author. School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran. Tel.: þ98 241 4273636; fax: þ98 241 4273639. E-mail addresses: elahian@zums.ac.ir, dr.elahian@yahoo.com (F. Elahian). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.11.010 Biochimie 99 (2014) 71e76