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Page 2 of 13 Suominen et al. J Cancer Metastasis Treat 2019;5:14 I http://dx.doi.org/10.20517/2394-4722.2018.64

combination group.

Conclusion: No additive effects in combination of DOX and ZOL were observed in this aggressive model of breast
cancer bone metastasis.

Keywords: Combination treatment, breast cancer, bone metastasis, MDA-MB-231(SA), doxorubicin, zoledronic acid

INTRODUCTION
Despite major advances in the treatment of primary breast cancer within the past decades, treatment of
advanced breast cancer with bone metastases is still palliative in nature, as no curative treatments are
available. Inhibitors of bone resorption, such as zoledronic acid (ZOL) or denosumab are used as adjuvant
treatment in breast cancer patients with bone metastases, often simultaneously with chemotherapeutic agents
[1,2]
such as doxorubicin . In these patients bisphosphonates decrease bone pain and skeletal-related events, but
[3]
do not increase the overall survival . However, before the formation of bone metastases, bisphosphonate
treatment in combination with standard chemotherapy may prevent the occurrence of bone metastases in
[4]
postmenopausal, early stage breast cancer patients . Many preclinical studies in established experimental
breast cancer bone metastasis models have reported direct anti-tumor effects of bisphosphonates alone, such
[5-7]
as increase of apoptosis in intraosseous tumors and improved survival , but we have reported no effects on
[8]
survival in a model of established bone metastases .

Bisphosphonates seek and incorporate into the hydroxyapatite in bone matrix. Therefore, it has remained
an unanswered question how tumor cells in soft tissues can be exposed to effective concentrations of
bisphosphonates. It is also possible that what was believed to be a direct anti-tumor effect is indeed, indirect.
It has been shown that macrophages take up bisphosphonates in cytotoxic quantities, and tumor growth in
soft tissues can be inhibited due to the lack of tumor-associated macrophages [9,10] .

Some exploratory preclinical studies investigating the effects of combinations of aminobisphosphonates and
chemotherapeutic agents have suggested additional benefits. Sequential treatment with doxorubicin (DOX)
followed by a single dose of ZOL 24 h later was reported to decrease tumor volume and proliferation and to
increase apoptosis as well as mouse survival with established bone metastases with MDA-MB231/BO2 breast
[11]
cancer cells . In addition, a single cycle of sequential administration of DOX and ZOL in a very early phase,
2 days after cancer cell inoculation, was reported to decrease intraosseus tumor area in an intracardiac
[12]
mouse model using MDA-MB231 cells with green fluorescent protein (GFP) . Three additional studies by
the same group with MDA-MB436 breast cancer cells showed synergistic effects on tumor volume reduction
with the weekly administration of DOX and ZOL in combination, both on bone metastasis and primary
tumor [13-15] . In these studies, the combination treatment reduced the intraosseous tumor area and increased
survival compared to DOX or ZOL monotherapy. The mode-of-action of the combination is not understood,
[14]
but increased uptake of ZOL by tumor cells was observed with the sequential combination . Because of the
varying results of anti-tumor effects of bisphosphonates, we felt a need to investigate these effects in a fairly
aggressive and widely used model of breast cancer bone metastases, utilizing the human MDA-MB231(SA)
[13]
GFP cells in nude mice. The treatment schedule was otherwise the same as used by Ottewell et al. , but the
treatment was started 3 days earlier to accommodate the more aggressive model.

METHODS
Compounds and cell line
DOX (Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in saline (0.9% NaCl, Baxter, Deerfield, IL,
USA) to concentration of 0.5 mg/mL. ZOL (Zometa®, Novartis Pharma GmbH, Basel, Switzerland) infusion