Evaluation of the molecular mechanisms for the sequence-dependent, synergistic anti-tumour effects of doxorubicin and zoledronic acid in breast cancer.
Authors: Ottewell PD , Lefley DV , Coleman RE , Holen I 
Published: December 2010

Abstract
Abstract #2151
Background: Recent reports suggest that zoledronic acid (Zometa®) may have anti-tumour effects outside the skeleton. We have previously shown that administration of doxorubicin (dox) 24h prior to zoledronic acid (zol) is critical for the inhibition of growth of established breast tumours implanted subcutaneously and intra-osseously. Inhibition of tumour growth was associated with reduced tumour cell proliferation and increased apoptosis in vivo. This is the first report of the potential molecular mechanisms by which doxorubicin and zoledronic acid exert their sequence-specific anti-tumour effects.&#x2028; Materials and methods: MDA-MB-436-GFP cells were inoculated into the flank (n=10/group) or tibia (n=8/group) of female MF1 nude mice. Mice were treated 1x per week for 6 weeks with saline, 2mg/kg dox, 100μg/kg zol, dox and zol simultaneously, dox followed 24h later by zol, or zol followed 24h later by dox. Animals were sacrificed 24h following the final treatment and tumour RNA prepared. Biotin labelled tumour RNA from each treatment group was hybridised to a GEArray cell cycle and an apoptosis pathway specific microarray (n=3 tumours/array). Changes in gene expression of 2 fold or greater were confirmed by qPCR and Western blotting.&#x2028; Results: Sequential treatment with dox 24h prior to zol reduced sub-cutaneous and intra-tibial tumour volume. Molecular analysis of these tumours showed equivalent effects on cell cycle and apoptosis related genes compared with tumours treated with dox or zol alone, sequential administration caused a cell cycle block at G2, G2/M, G1 and G1/S, with an increased expression of p53 and p21 accompanied by a reduction in cyclins E1, B, D1 and D3 as well as their related cyclin dependent kinases CDK2, CDC2, CDK4 and CDK7. Tumours treated sequentially with dox followed by zol also showed an induction in both extrinsic and intrinsic apoptotic pathways, with increased expression of Bax, decreased expression of Bcl-2 and an increase in caspase 8, caspase 9 and caspase 3 cleavage. In sub-cutaneous tumours administration of 100g/μkg zol showed no effect on tumour cell cycle or apoptosis related genes. 2mg/kg dox caused a cell cycle block at G1-S with a reduction in expression of cyclin E/CDK2, whereas apoptosis-related genes were unaffected. Treatment of intra-tibial tumours with 100μg/kg zol resulted in a reduction in tumour cell proliferation as well as reduced expression of cyclins D1 and D3. No alterations in cell cycle or apoptosis related gene expression were detected in intra-tibial tumours treated with 2mg/kg dox.&#x2028; Conclusions: This is the first report showing that sequential treatment of both subcutaneous and intra-tibial breast tumours in vivo with doxorubicin followed by zoledronic acid induces changes in expression of a number of specific genes and proteins associated with regulation of the cell cycle and apoptosis, which were unaffected by treatment with the single agents. Our data suggest that breast cancer patients may benefit from receiving zoledronic acid 24h after administration of anthracycline therapy.
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2151.