Bone Abstracts

Nitrogen-containing bisphosphonates (N-BPs) are used to treat osteolytic bone metastases. N-BPs have been previously shown to enter macrophages via macropinocytosis, but the mechanisms how they are taken up by breast cancer cells are so far incompletely known. In breast cancer primary tumours N-BPs have been shown, by other researchers, to be bound to micro-calcifications present in the tumour stroma. In our study we have characterized how different N-BP formulations, free, calcium-bound and liposome encapsulated are internalized to MCF-7 and T47D breast cancer cells.

All of the three different N-BP formulations were shown to utilize the various dynamin dependent endocytosis routes under which the T47D cells used more the clathrin-dependent route and MCF-7 cells a caveolin and clathrin independent route. In both of the cell lines the uptake of ¹⁴C-zoledronic acid was enhanced by calcium chelation of the drug (MCF7: free drug 50 c/s per mg protein vs [¹⁴C]-Zol-Ca²⁺2157 c/s per mg protein, P<0.001; T47D: 36.6 c/s per mg protein vs 3368.2 c/s per mg protein, P<0.001). Cytotoxicity was also enhanced by calcium chelation (IC₅₀, MCF7 cells: free drug, 2.3×10⁻⁴ M vs calcium bound, 4.5×10⁻⁴ M, P<0.05; T47D cells: 8.97×10⁻⁴ M vs 7.2×10⁻⁵ M, P<0.001,respectively) and liposome encapsulation of the drug (MCF-7: 8×10⁻⁷ M, T47D: 6.9×10⁻⁶, P<0.001 for both of the cells lines when compared to the free drug). Liposome encapsulation favoured the use of the dynamin and caveolin dependent endocytosis route. Moreover, liposome encapsulation prolonged the drug effect in cells as monitored by the intracellular formation of isopentenyl pyrophosphate (IPP) and it AMP conjugate ApppI. At 72 h post treatment ApppI was still detected and the IPP concentration was highest in the cells treated with the liposomal drug. These findings suggest that breast cancer cells use different uptake mechanisms for N-BPs than macrophages, which is relevant when cell-specific targeting strategies for N-BPs are planned.