How do bisphosphonates inhibit bone metastasis in vivo?

Abstract

Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and have demonstrated clinical utility in the treatment of patients with osteolytic bone metastases. They also exhibit direct antitumor activity in vitro and can reduce skeletal tumor burden and inhibit the formation of bone metastases in vivo. However, whether such effects are caused by a direct action of bisphosphonates on tumor cells or indirectly through inhibition of bone resorption remains unclear. To address this question, we used here a structural analog of the bisphosphonate risedronate, NE-58051, which has a bone mineral affinity similar to that of risedronate, but a 3000-fold lower bone antiresorptive activity. In vitro, risedronate and NE-58051 inhibited proliferation of breast cancer and melanoma cell lines. In vivo, risedronate and NE-58051 did not inhibit the growth of subcutaneous B02 breast tumor xenografts or the formation of B16F10 melanoma lung metastasis. In contrast to NE-58051, risedronate did inhibit B02 breast cancer bone metastasis formation by reducing both bone destruction and skeletal tumor burden, indicating that the antitumor effect of bisphosphonates is achieved mainly through inhibition of osteoclast-mediated bone resorption.

Figure 1

Effects of risedronate and NE-58051 on proliferation of breast and melanoma cells in vitro. (A) Chemical structures of risedronate and NE-58051. (B) Dose-dependent inhibition of cancer cell proliferation after a 6-day treatment with 0.001 to 10 mM risedronate or NE-58051, as measured by MTT proliferation assay. Results are the mean of three independent experiments. (C) Effects of the mevalonate pathway intermediates FOH and GGOH on the inhibition of B02-GFP/βGal cell proliferation induced by risedronate or NE-58051. B02-GFP/βGal cells were treated with a bisphosphonate at its IC₅₀ concentration with or without adding 10 µM FOH or GGOH, respectively. Results are expressed as the mean OD_570nm ± SD of one experiment. *P < .001 when compared with the cells treated only with the corresponding drug, using an unpaired Student's t test.

Figure 2

Effects of risedronate and NE-58051 on bone metastasis formation. (A) Treatment protocol for bisphosphonates in a mouse model of human B02 breast cancer cell metastasis to bone. B02-GFP/βGal cells were inoculated intravenously into 4-week-old female BALB/c nude mice on day 0 (D0). Bisphosphonates were administered daily by subcutaneous injection beginning on D0. Mice were killed 35 days after tumor cell inoculation (D35). (B) Representative radiographic images of hind limbs on day 35 after cell inoculation from mice bearing B02-GFP/βGal bone metastases treated with risedronate or NE-58051. Arrows indicate osteolytic lesions. (C) Micro-CT reconstruction of hind limbs obtained from different mice on day 35. Representative images for each group are shown. (D) Micrographs of Goldner trichrome-stained sections of tibial metaphysis. For histologic tissue sections, bone is stained green, whereas bone marrow and tumor cells (asterisk) are stained red. (E) Fluorescence analyses of B02-GFP/βGal tumor cells residing in bone. Arrows indicate fluorescent tumor cells. The images shown are examples that best illustrate the effects of the treatments.

Figure 3

Measurement of CTX-I levels in the bone marrow from mice treated with risedronate, NE-58051, or vehicle only. Bone marrow from metastatic and nonmetastatic legs was flushed, and CTX-I levels were measured by a competitive ELISA using an antibody raised against an eight-amino acid sequence of type I collagen C-telopeptide. Bars indicate the average value. *P < .05.

Figure 4

(A) Effect of risedronate and NE-58051 on growth of subcutaneous breast cancer xenografts in nude mice. B02-GFP/βGal cells were inoculated subcutaneously into the right flank of animals. Animals received daily doses of 150 µg/kg risedronate or NE-58051 from day 35, when tumors became palpable, until the end of the protocol (D60). Control animals were treated with the vehicle only. None of the drugs inhibited the growth of B02 breast cancer cells in vivo. Results are the mean ± SD of five animals per group. (B) Effect of NE-58051 on lung metastasis formation. B16-F10 melanoma cells were injected intravenously to C57BL/6 mice. NE-58051 (at a dosage of 350 µg/kg per day) was administered to animals by subcutaneous injection beginning on the day of tumor cell inoculation (D0), and continuing until the end of the protocol (D14). Control animals (placebo) were treated with the vehicle only. Visible pigmented melanoma lung metastases were enumerated. Inset: images shown are examples that best illustrate metastatic lungs in each group. NE-58051 did not inhibit B16-F10 lung metastasis. Results are the mean ± SD of five animals per group.