Preventive effects of zoledronic acid on bone metastasis in mice injected with human breast cancer cells

Abstract

Bisphosphonates are used routinely to reduce bone-related events in breast cancer patients with bone metastasis. We evaluated the effects of zoledronic acid, a third generation, nitrogen-containing bisphosphonate, to prevent bone metastasis in breast cancer. Zoledronic acid or vehicle alone was administered to nude mice either simultaneously or after intracardiac injection of human breast cancer MDA-MB-231 cells. Nude mice treated with zoledronic acid at early time points showed a lower incidence of bone metastases than did vehicle-treated nude mice, but these differences were not statistically significant. Only 37.5% of mice treated with zoledronic acid at the time of tumor cell inoculation developed bone metastases compared to over 51.8% of mice receiving vehicle alone (P = 0.304). Cell count of apoptosis confirmed by immunohistochemical staining in metastatic bone tissue significantly increased in the zoledronic acid-treated groups compared to non-treated group (1,018.3 vs 282.0; P = 0.046). However, metastatic tumor cells, which invade soft tissue around the bone, did not show extensive apoptosis; there were no differences between the zoledronic acid-treated and control groups. These results suggest that zoledronic acid increases apoptosis of metastatic breast tumor cells in the bone and could therefore reduce metastatic tumor burden. These results support the use of zoledronic acid to reduce the incidence of bone metastasis in breast cancer.

Keywords: Bone Metastasis; Breast Neoplasms; Prevention; Zoledronic Acid.

Fig. 1

Experimental schedule of zoledronic acid administration.

Fig. 2

Metastatic activities visualized using bioluminescent imaging in the control group (A; dorsal side, B; ventral side). First and third mice show hot spots on their ventral sides, confirmed as rib metastases.

Fig. 3

Incidence of bone metastases in each group. Differences between group 1 and control (P = 0.304), between group 2 and control (P = 0.747), and between group 3 and control (P = 0.447) were not statistically significant. Additionally, the differences between each test group were not statistically significant.

Fig. 4

Immunohistochemical staining results for apoptosis in metastatic tumor cells in the bone (× 400). ApopTag peroxidase in situ results of metastatic tumor tissues of bone in test (A) and control (B) groups. Many apoptotic tumor cells were positively stained in the test groups.

Fig. 5

Immunohistochemical staining results for apoptosis in metastatic tumor cells outside the bone (× 400). ApopTag peroxidase in situ results of metastatic tumor tissues outside the bone in the test (A) and control (B) groups. No differences were seen between the groups.

Fig. 6

Number of apoptotic tumor cells inside and outside of the bone in each group. Apoptosis of metastatic cells increased on average by 1,018.3 in the zoledronic acid-treated groups (zol) and on average by 282.0 in the control groups (con), indicating a significant increase in apoptosis in the zoledronic acid-treated groups (P = 0.046). On the other hand, apoptosis of cells metastasized to the outside of the bone was 173.6 in the zoledronic acid-treated groups and 217.3 in the control groups, suggesting no increase in any of the groups and no differences between groups (P = 0.86).