Sustained inhibition of tumor growth and prolonged survival following sequential administration of doxorubicin and zoledronic acid in a breast cancer model Penelope D. Ottewell, Diane V. Lefley, Simon S. Cross, C. Alyson Evans, Robert E. Coleman and Ingunn Holen Academic Unit of Clinical Oncology, School of Medicine and Biomedical Sciences, University of Sheffield, United Kingdom Combination therapy, using agents that target the microenvironment as well as the cancer cells, is common in the treatment of advanced breast cancer. Here, we show that a 6-week course of weekly sequential administration of the cytotoxic drug doxorubicin (2 mg/kg), followed 24 hr later by the antiresorptive agent zoledronic acid (100 lg/kg), causes substantial inhibition of subcutaneous MDA-MB-436 breast tumor growth in immunocompromised mice, leading to significantly increased survival. Tumor growth did not resume following withdrawal of treatment after 6 weeks, with 60% of the animals in this group surviving for more than 160 days. In comparison, animals receiving single-agent therapy all died within 50 days. Molecular analysis of the tumors showed no effect on cell cycle or apoptosis following administration of 100 lg/kg zoledronic acid or 2 mg/kg doxorubicin alone. When doxorubicin was administered 24 hr before zoledronic acid, tumors displayed decreased expression of CYCLINS E1, B, D1 and D3 as well as CDK2, CDC2, CDK4 and CDK7, indicative of cell-cycle inhibition. Tumors from animals receiving sequential treatment also showed induction of both intrinsic- and extrinsic-apoptotic pathways, with increased expression of BAX, decreased expression of BCL-2 and activation of CASPASE 3, 8 and 9. Accumulation of the unprenylated form of RAP1a, a surrogate marker for uptake of zoledronic acid, was only detected in tumors from animals treated with doxorubicin 24 hr before zoledronic acid. Our data are the first to show a sustained antitumor effect in vivo following a limited course of sequential administration of doxorubicin followed by zoledronic acid. Patients with advanced breast cancer may receive chemother- apeutic agents to reduce tumor growth as well as antiresorp- tive drugs to control cancer-induced bone disease, but the optimal way of combining these agents remains to be estab- lished. In vivo studies from different tumor types have reported increased antitumor effects in bone when cytotoxic drugs are combined with bisphosphonate zoledronic acid (reviewed in Ref. 1 ). This has been shown by combining zole- dronic acid with UHT in breast cancer models, 2 with imati- nib mesylate or paclitaxel in a prostate cancer model 3 and with ifosfamide in an osteosarcoma model. 4 However, these studies used high doses of zoledronic acid ranging from a single injection of 250–100 lg/kg administered 2 per week. In the B02 model of breast cancer bone metastases, we have shown that a clinically relevant dose of doxorubicin (2 mg/ kg), combined with a single administration of zoledronic acid (100 lg/kg), reduces tumor growth in bone. 5 There is emerg- ing evidence that combination treatment with zoledronic acid may also affect tumors outside the skeleton. A recent clinical trial in breast cancer has reported that adding zoledronic acid to standard endocrine therapy leads to a reduction in re- currence at 5 years, including at visceral and locoregional sites. 6 In support of this, in vivo studies of subcutaneously implanted tumors have reported that zoledronic acid com- bined with cytotoxic agents may reduce tumor growth depending on dosing regimens. 1 This has been shown for combinations of zoledronic acid with paclitaxel/Gefitinib in prostate cancer 3,7 with paclitaxel in Ewing sarcoma 8 and with Imatinib in leukemia. 9 We have found that weekly treatment with 2 mg/kg doxorubicin followed 24 hr later by 100 lg/kg zoledronic acid eliminated subcutaneous breast tumor growth in mice and that sequential administration was more effective than giving the 2 drugs simultaneously. 10 How doxorubicin and zoledronic acid elicit these com- bined antitumor effects remains to be established, but the individual mechanism of action of the drugs is well estab- lished. Doxorubicin is an anthracyclin that inhibits topoisom- erase II and induces DNA double-strand breaks, 11 interferes with DNA unwinding, 12 induces differentiation and generates oxygen-free radicals. 13 Doxorubicin does not appear to induce apoptosis in breast cancer cells in vitro, but induces growth arrest and nonapoptotic cell death accompanied by Key words: doxorubicin, zoledronic acid, breast cancer, apoptosis, cell cycle Conflict of interest: R.E. Coleman: Research funding, consultancy and speakers fee from Novartis. I. Holen: Research funding and speakers fee from Novartis Grant sponsor: Breast Cancer Campaign, United Kingdom DOI: 10.1002/ijc.24756 History: Received 12 Feb 2009; Accepted 24 Jun 2009; Online 20 Jul 2009 Correspondence to: Ingunn Holen, Academic Unit of Clinical Oncology; School of Medicine and Biomedical Sciences; University of Sheffield, Beech Hill Road; Sheffield, S10 2RX, United Kingdom, Fax: þ44-0-114-271-1711, E-mail: I.Holen@sheffield.ac.uk Cancer Therapy Int. J. Cancer: 126, 522–532 (2009) V C 2009 UICC International Journal of Cancer IJC