Nitrogen-containing bisphosphonates inhibit cell cycle progression in human melanoma cells

Abstract

Cutaneous melanoma is one of the highly malignant human tumours, due to its tendency to generate early metastases and its resistance to classical chemotherapy. We recently demonstrated that pamidronate, a nitrogen-containing bisphosphonate, has an antiproliferative and proapoptotic effect on different melanoma cell lines. In the present study, we compared the in vitro effects of three different bisphosphonates on human melanoma cell lines and we demonstrated that the two nitrogen-containing bisphosphonates pamidronate and zoledronate inhibited the proliferation of melanoma cells and induced apoptosis in a dose- and time-dependent manner. Moreover, cell cycle progression was altered, the two compounds causing accumulation of the cells in the S phase of the cycle. In contrast, the nonaminobisphosphonate clodronate had no effect on melanoma cells. These findings suggest a direct antitumoural effect of bisphosphonates on melanoma cells in vitro and further support the hypothesis of different intracellular mechanisms of action for nitrogen-containing and nonaminobisphosphonates. Our data indicate that nitrogen-containing bisphosphonates may be a useful novel therapeutic class for treatment and/or prevention of melanoma metastases.

Figure 1

Effects of bisphosphonates on melanoma cell proliferation. A375 (A) and M186 (B) melanoma cells were incubated for 24 h with the indicated doses of pamidronate (squares) or zoledronate (circles). Cell proliferation was evaluated using the crystal violet technique. Four independent experiments were performed in quadruplicate with similar results. One representative experiment is shown. Results are given as % of controls±s.d. (n=4) (^*P<0.05; ^**P<0.01).

Figure 2

Effect of bisphosphonates on the induction of apoptosis in melanoma cells. Preconfluent A375 (A) and M186 (B) melanoma cells were treated with the indicated concentrations of zoledronate (white columns), or pamidronate (grey columns) for 24 h. (C) A375 (light grey columns) and M186 (hatched columns) cells were treated for 24 h with clodronate in the indicated concentrations. DNA fragmentation was measured using the ‘Cell death detection ELISA^PLUS’ as described under Materials and methods. Four independent experiments were performed in quadruplicate, with similar results. One representative experiment is shown. Results are given as % of controls±s.d. (n=4) (^*P<0.05; ^**P<0.01).

Figure 3

Nitrogen-containing bisphosphonates induce caspase-3/7 activity in melanoma cells. Preconfluent A 375 (A) and M186 (B) cells were treated with vehicle control (white bars), 100 μM pamidronate (grey columns) or 100 μM zoledronate (black columns) for 24 h. For specific activation of caspase-3, cells were pretreated with the respective caspase-3 inhibitor 1 h prior to stimulation and then treated for 24 h with 100 μM pamidronate or zoledronate in combination with the inhibitor (square bars). Caspase-3/7 activity was determined with the Apo-One™ homogeneous caspase-3/7 assay as described under Materials and methods. Three independent experiments were performed in quadruplicate, with similar results. One representative experiment is shown. Results are given as % of control±s.d. (n=4) (^*P<0.05; ^**P<0.01).

Figure 4

Time dependency of bisphosphonate-induced apoptosis in melanoma cells. Preconfluent A375 melanoma cells were treated with the indicated concentrations of zoledronate (grey columns) or pamidronate (black columns), or vehicle for 6, 12 and 24 h. At each time point, DNA fragmentation was measured using the ‘Cell death detection ELISA^PLUS’ as described under Materials and methods. Three independent experiments were performed in quadruplicate, with similar results. One representative experiment is shown. Results are given as % of controls±s.d. (n=4) (^*P<0.05; ^**P<0.01).

Figure 5

Distribution in various phases of the cell cycle of cultured melanoma cells after bisphosphonate treatment. A375 cells were treated with (A) pamidronate, (B) zoledronate or (C) clodronate at the indicated concentrations for 24 h. DNA content of cells was determined by propidium iodide staining using FACS analysis, and the distribution of cells in G0/G1 (diamonds), S (squares) and G2/M (triangles) phases of the cell cycle is shown. Results are given as the percentage of cells in G0/G1, S and G2/M phases as determined by ModFitLT software (version 2.0). Values represent the mean of three independent experiments±s.d. (n=3) (^*P<0.05).