C.A. Evans

Sequence‐ and schedule‐dependent enhancement of zoledronic acid induced apoptosis by doxorubicin in breast and prostate cancer cells

We investigated whether the combination of zoledronic acid and doxorubicin induced apoptosis of breast and prostate cancer cell lines, and if synergistic interaction was present. We investigated whether the levels of cell death altered depending on the sequence in which the drugs were administered and the possible mechanism of action responsible for the increased cell death following combined treatments. Breast and prostate cancer cells were treated with zoledronic acid alone, doxorubicin alone, or drugs in sequence (doxorubicin before, after, or with zoledronic acid), and the levels of apoptotic death were determined by evaluation of nuclear morphology. We found that clinically relevant concentrations of doxorubicin and zoledronic acid induced sequence‐ and schedule‐dependent apoptosis of breast and prostate cancer cells. For maximal apoptosis, cells had to be pretreated for 24 hr with doxorubicin before immediate treatment with zoledronic acid for 1 hr. This observation is a characteristic feature of cell cycle phase‐specific synergistic effect. Replacing zoledronic acid with the nonnitrogen‐containing bisphosphonate clodronate did not induce increased apoptosis. Induction of apoptosis was mainly via inhibition of the mevalonate (MVA) pathway, as addition of the MVA pathway intermediary geranylgeraniol inhibited the induction of apoptosis by doxorubicin followed by zoledronic acid. In conclusion, combined treatment of breast and prostate cancer cell lines with clinically relevant doses of doxorubicin and zoledronic acid induces apoptosis in a synergistic fashion. These findings may have relevance for the clinical setting, particularly breast cancer patients receiving these drugs in the adjuvant setting.

Osteoprotegerin (OPG) produced by bone marrow stromal cells protects breast cancer cells from TRAIL-induced apoptosis

Advanced breast cancer is often associated with metastatic bone disease, causing a number of serious complications for the patients such as hypercalceamia, pain, nerve compression and fractures. The formation of bone metastases depends on complex interactions between tumour cells and the cells of the bone microenvironment, but the precise molecular mechanisms involved in the development of tumour-induced bone disease have not been identified. We have investigated the ability of bone marrow stromal cells (BMSC) isolated from breast cancer patients to generate osteoprotegerin (OPG), a molecule involved both in bone turnover and cell survival. The potential survival effects of OPG are mediated through binding to a member of the TNF super family, TNF-related Apoptosis Inducing Ligand (TRAIL), preventing association between TRAIL and its death-inducing receptors present on a number of tumour cell types. In the present report we show that bone marrow stromal cells isolated from breast cancer patients produce OPG when grown in culture. The levels of OPG present in BMSC conditioned medium is sufficient to protect breast cancer cells from undergoing TRAIL induced apoptosis. Our data suggest that bone-derived OPG may increase survival of breast cancer cells that reach the bone microenvironment as part of the metastatic process.

Mechanisms of the Synergistic Interaction between the Bisphosphonate Zoledronic Acid and the Chemotherapy Agent Paclitaxel in Breast Cancer Cells in vitro

Breast cancer patients often receive both paclitaxel and zoledronic acid as part of their treatment, and these drugs are reported to have synergistic effects on the induction of apoptosis of breast cancer cells in vitro. We have found that the synergistic interaction is drug sequence dependent, with maximal levels of apoptosis achieved when cells are treated with paclitaxel followed by zoledronic acid, as opposed to the reverse sequence or simultaneous treatment. The synergistic interaction persists at clinically relevant concentrations and incubation periods. We report that the sequential treatment is associated with cell cycle changes and depends on breast cancer cell characteristics, with hormone independence, mutated p53 status and presence of BRCA1 gene being associated with higher levels of apoptosis. Finally, we have found that the synergistic induction of apoptosis is via zoledronic acid-mediated inhibition of the mevalonate pathway.

Location matters: osteoblast and osteoclast distribution is modified by the presence and proximity to breast cancer cells in vivo

Bone metastasis is a common incurable complication of breast cancer affecting around 70% of patients with advanced disease. In order to improve outcomes for these patients, the cellular and molecular mechanisms underlying bone metastasis need to be established. The majority of studies to date have focused on end-stage disease and little is known about the events taking place following initial tumour cell colonisation of bone. Here we report the results of a longitudinal study that provides detailed analysis of the spatial and temporal relationship between bone and cancer cells during progression of bone metastasis. Tumour growth in bone was initiated by intra-cardiac inoculation of MDA-MB-231-GFP breast cancer cells in immunocompromised mice. Differentiating between areas of bone in direct contact with the tumour and areas distal to the cancer cells but within the tumour bearing bone, we performed comprehensive analyses of the number and distribution of osteoclasts and osteoblasts. Tumour colonies were detectable in bone from day 10, while reduced trabecular bone volume was apparent from day 19 onwards. Cancer-induced changes in osteoblast and osteoclast numbers differed substantially depending on whether or not the cells were in direct contact with the tumour. Compared to naïve controls, areas of bone in direct contact with the tumour had significantly reduced osteoblast but increased osteoclast numbers, whereas the reverse was found in distal areas. Our data demonstrate that tumour cells induce substantial changes in the bone microenvironment prior to the appearance of bone lesions, suggesting that early therapeutic intervention may be required to oppose the tumour-induced changes to the microenvironment und thus tumour progression.

A single administration of combination therapy inhibits breast tumour progression in bone and modifies both osteoblasts and osteoclasts

We have previously shown that repeated sequential administration of doxorubicin, followed 24 h later by zoledronic acid, inhibits tumour growth in models of established breast cancer bone metastasis. As breast cancer patients only receive zoledronic acid every 3–4 weeks, the aim of the current study was to establish the anti-tumour and bone effects of a single administration of doxorubicin/zoledronic acid combination therapy in a bone metastasis model. MDA-MB-231-GFP cells were injected i.c. in 6-week-old nude mice. On day 2, animals received PBS, doxorubicin (2 mg/kg i.v.), zoledronic acid (100 μg/kg s.c.) or doxorubicin followed 24 h later by zoledronic acid. Anti-tumour effects were assessed on days 15/23 by quantification of apoptotic and proliferating cells and changes in expression of genes implicated in apoptosis, proliferation and bone turnover. Bone effects were assessed by μCT analysis, bone histomorphometry and measurement of serum markers. A tumour-free control group was included. Combination treatment reduced bone tumour burden compared to single agent or PBS control and increased levels of tumour cell apoptosis on day 15, but this was no longer detectable on day 23. Animals receiving zoledronic acid had increased bone density, without evidence of tumour-induced lesions. Bone histomorphometry showed that zoledronic acid caused a decrease in osteoblast and osteoclast numbers and an increase in osteoclast size, in both tumour-free and tumour-bearing animals. Our data show that although zoledronic acid modifies the bone microenvironment through effects on both osteoblasts and osteoclasts, this does not result in a significant anti-tumour effect in the absence of doxorubicin.