Breast cancer very commonly metastasizes to bone, often with devastating consequences, including pain, pathological fractures, hypercalcaemia and nerve compression syndromes. Bisphosphonates are very effective in the management of bone secondaries from breast cancer. Recent clinical studies have also suggested that adjuvant oral clodronate in patients with primary breast cancer may confer a survival benefit thereby raising the possibility that bisphosphonates have an anti-tumour effect. The aim of this PhD was to determine whether bisphosphonates have an anti-tumour effect on human breast cancer cells.
The potent third generation bisphosphonate, zoledronic acid, was found to reduce cell number and increase apoptosis, mediated by inhibition of the mevalonate pathway, in human breast cancer cells in vitro. The effects on cell apoptosis were shown to be synergistic when zoledronic acid was combined with a chemotherapeutic agent, paclitaxel. To extend the investigation to the in vivo setting, we developed a dual fluorescence labelling technique to isolate apoptotic breast cancer cells from the bone marrow of patients with breast cancer undergoing treatment with intravenous pamidronate or zoledronic acid. The technique was effective at detecting breast cancer cells but studies were inconclusive in determining the effect of intravenous bisphosphonate treatment on breast cancer cell apoptosis in vivo. These studies are ongoing. Bisphosphonates may also alter the bone microenvironment by inhibiting 1 osteoclast-mediated bone resorption and interfering with the release of cytokines and growth factors. To complete these studies, we measured the levels of a panel of cytokines and growth factors in patient serum and bone marrow before and three days after intravenous bisphosphonate treatment and also compared pretreatment levels with those in a baseline group with primary breast cancer. TGFj3-1 was significantly elevated in bone marrow plasma from patients with advanced breast cancer than in patients with primary breast cancer, along with serum IL-6 and sIL-6R. In the advanced breast cancer group, bisphosphonate treatment resulted in significantly lowered levels of serum FGF-2 and bone marrow VEGF.
The work presented in this thesis demonstrates that the bisphosphonate zoledronic acid does have anti-tumour effects on breast cancer cells in vitro, both alone and in synergy with paclitaxel, to induce apoptosis. This raises the possibility of in vivo anti-tumour effects in breast cancer. The dual fluorescence labelling technique made it possible to detect breast cancer cells in human bone marrow but the studies regarding the in vivo induction of apoptosis in breast cancer cells following intravenous bisphosphonate treatment were inconclusive. There were significant differences in bone-derived growth factors between patients with primary breast cancer and secondary bone metastases. There was also a significant reduction in certain cytokines and growth factors in the bone marrow and serum after intravenous bisphosphonate treatment, suggesting that bisphosphonates may also exert an indirect effect on the bone marrow microenvironment. Further in vivo studies in patients with breast cancer are required to confirm both the direct anti-tumour effects and indirect effects.