Low-dose zoledronate for the treatment of bone metastasis secondary to prostate cancer

Abstract

Background: Bisphosphonates (BPs) including zoledronate (zol) have become standard care for bone metastases as they effectively inhibit tumor-induced osteolysis and associated pain. Several studies have also suggested that zol has direct anti-tumor activity. Systemic administration at high doses is the current approach to deliver zol, yet it has been associated with debilitating side effects. Local therapeutic delivery offers the ability to administer much lower total dosage, while at the same time maintaining sustained high-local drug concentration directly at the target treatment site. Here, we aimed to assess effects of lower doses of zol on bone metastases over a longer time.

Methods: Prostate cancer cell line LAPC4 and prostate-induced bone metastasis cells were treated with zol at 1, 3 and 10 µM for 7 days. Following treatment, cell proliferation was assessed using Almarblue^®, Vybrant MTT^®, and Live/Dead^® viability/cytotoxicity assays. Additionally, cell migration and invasion were carried out using Falcon™ cell culture inserts and Cultrex^® 3D spheroid cell invasion assays respectively.

Results: We show that treatment with 3-10 µM zol over 7-days significantly decreased cell proliferation in both the prostate cancer cell line LAPC4 and cells from spine metastases secondary to prostate cancer. Using the same low-dose and longer time course for treatment, we demonstrate that 10 µM zol also significantly inhibits tumor cell migration and 3D-cell growth/invasion.

Conclusions: This project harnesses the potential of using zol at low doses for longer treatment periods, which may be a viable treatment modality when coupled with biomaterials or biodevices for local delivery.

Keywords: Bone metastases secondary to prostate; Cellular assays; Direct in vitro treatment; Low doses; Zoledronate.

Fig. 1

Cell proliferation using alamarblue^® assay of LAPC4 cells treated with vehicle (PBS1x) or zol 10 to 1000 µM for 7 days in 1% serum conditions. The histogram represents the ratio of drug-treated cells divided by vehicle-treated cells (PBS1x). Results are the mean ± SD of three independent experiments, p < 0.05

Fig. 2

Cell proliferation using alamarblue^® assay (a) and Vybrant MTT^® assay (b) of LAPC4 cells treated with vehicle (PBS1x) or zoledronate 1 µM, 3 µM, 10 µM and 100 µM for 7 days in 1% serum conditions. The histograms in (a) and (b) represent the ratio of drug-treated cells divided by vehicle-treated cells (PBS1x) in three independent experiments for all variables except for 100 µM that was done as a single experiment in triplicate. c Representative photos of Live/Dead assay carried out on LAPC4 following vehicle or zol treatment at different concentrations. Live cells are in green and dead cells are in red. d Percentage of viable cells [number of live cells/(number of live cells + number of dead cells) * 100] and e ratio of live cells or dead cells in vehicle or zol-treated conditions, Results are mean ± SD, p < 0.05. Scale bar 250 µm

Fig. 3

Cell proliferation using alamarblue^® assay (a) and Vybrant MTT^® assay (b) of prostate-induced bone metastasis cells treated with vehicle (PBS1x) or zol 1 µM, 3 µM and 10 µM for 7 days in 1% serum conditions. The histograms in (a) and (b) represent the ratio of drug-treated cells divided by vehicle-treated cells (PBS1x). c representative photos of Live/Dead assay carried out on LAPC4 following vehicle or zol treatment at different concentrations. Live cells are in green and dead cells are designated in red arrow heads. d Percentage of viable cells [number of live cells/(number of live cells + number of dead cells) * 100] and e ratio of live cells or dead cells in vehicle or zol-treated conditions, Results are mean ± SD of three independent experiments, p < 0.05. Scale bar 100 µm

Fig. 4

Migration (Falcon™ insert assay) of LAPC4 (a and b) and prostate-induced bone metastasis cells (c and d) treated with vehicle (PBS1x) or zol 1 µM, 3 µM and 10 µM for 7 days in 1% serum conditions. Representative images of LAPC4 (a) and prostate-induced bone metastasis patient cells (c) cells from vehicle or zol-treated conditions. The histograms represent the ratio of drug-treated cells divided by vehicle-treated cells (PBS1x) for LAPC4 (b) and prostate-induced bone metastasis cells (d). Results are the mean ± SD of three independent experiments, p < 0.05. Scale bar 100 µm

Fig. 5

Spheroid growth (a and b) and spheroid invasion (c and d) of LAPC4 cells treated with vehicle (PBS1x) or Zol 1 µM, 3 µM and 10 µM for 7 and 14 days in 1% serum conditions. Representative brightfield images of LAPC4 spheroids growth (a) and invasion (c) from vehicle or zol-treated conditions. Histograms for growth (b) and invasion (d) showing the mean ± SD of three independent experiments, p < 0.05. Each condition (drug-treated cells or vehicle-treated cells) on each day was normalized to day 0 and then all normalized conditions were normalized to vehicle (PBS1x)

Fig. 6

Spheroid growth (a and b) and spheroid Invasion (c and d) assays of prostate-induced bone metastasis cells treated with vehicle (PBS1x) or zol 10 µM for 7 days in 1% serum conditions. Representative brightfield images of spheroids growth (a) and invasion (c) of prostate-induced bone metastasis cells from vehicle or zol-treated conditions. Histograms for spheroids growth (b) and invasion (d) showing the mean ± SD of three independent experiments, p < 0.05. Each condition (drug-treated cells or vehicle-treated cells) on each day was normalized to day 0 and then all normalized conditions were again normalized to vehicle (PBS1x)