Synergistic effects of cytotoxic drugs and antiresorptive agents in vitro and in vivo

Breast cancer patients commonly receive a combination of different therapies; however, our understanding of how such combined treatments work is incomplete. In an attempt to optimize treatment strategies we have focused on determining how anticancer agents can be combined in order to induce maximum levels of tumour cell death. The antiresorptive agent zoledronic acid (zol) (Novartis Pharma, Basel, Switzerland) and the chemotherapeutic agent doxorubicin (dox) (Parmachemie BV, Haarlem, The Netherlands) have been shown to synergistically increase apoptosis in breast cancer cells in vitro. In order to determine whether sequential treatment with dox and zol could have potential clinical relevance and to determine the cellular mechanisms responsible for this synergy, we have further investigated combination treatments in vitro and in vivo.

To enable visualization of intratibial tumours, MDA MB 436 breast cancer cells were stably transfected with GFP (MDA GFP 2 cells). Following sequential treatment with dox and zol, levels of MDA GFP 2 apoptosis were assessed by microscopic analysis following Hoechst and propidium iodide (PI) staining and by flow cytometry after annexin and PI staining. For in vivo dose–response studies, MDA GFP 2 cells were inoculated subcutaneously into the right flanks of female MF1 nude mice (n = 8). Mice were administered 2.5, 3, 30 or 150 μM zol intraperitoneally, or 2, 4 or 8 mg/kg dox intravenously. Combination studies were carried out against subcutaneous (n = 16) and intratibial (n = 8) MDA GFP 2 xenografts using a dosing regime of 2 mg/kg dox and/or 2.5 M zol once per week for 6 weeks, with zol being administered 24 hours after dox. The tumour volume was measured once per week for 6 weeks and mice were sacrificed 24 hours following final treatment.

In vitro sequential treatment with dox then zol synergistically increased apoptosis in MDA GFP 2 cells. In vivo combination treatment with dox then zol resulted in a significant reduction of tumour growth compared with control mice or mice treated with dox or zol alone.