The bisphosphonate pamidronate induces apoptosis in human melanoma cells in vitro

Abstract

Pamidronate belongs to the class of nitrogen-containing bisphosphonates that are potent inhibitors of bone resorption frequently used for the treatment of osteoporosis and cancer-induced osteolysis. The inhibition of osteoclasts' growth has been suggested as the main mechanism of the inhibitory effect of pamidronate on bone metastases. Recent findings indicated that bisphosphonates also have a direct apoptotic effect on other types of tumour cells. Nitrogen-containing bisphosphonates were shown to inhibit farnesyl diphosphate synthase, thus blocking the synthesis of higher isoprenoids. By this mechanism they inactivate monomeric G-proteins of the Ras and Rho families for which prenylation is a functional requirement. On the background of the known key role of G-proteins in tumorigenesis, we investigated a possible beneficial use of pamidronate in the treatment of malignant melanoma. Our results indicate that pamidronate inhibits the cell growth and induces apoptosis in human melanoma cells in vitro. Susceptibility to pamidronate did not correlate to CD95 ligand sensitivity or p53 mutational status. Furthermore it is interesting to note that overexpression of bcl-2 did not abolish pamidronate-induced apoptosis. These data suggests that pamidronate has a direct anti-tumour effect on malignant melanoma cells, independently of the Bax/Bcl-2 level.

Copyright 2002 Cancer Research UK

Figures

Figure 1

Effect of pamidronate on melanoma cell proliferation. A375, M186, Mel2A and M221 melanoma cells were seeded at a density of 20 000 cells per cm2 and after 36 h treated with indicated concentrations of pamidronate or vehicle control for 48 h. Proliferation was measured using crystal violet staining of adherent cells. Cell growth of control cells was set as 100% and proliferation of treated cells was calculated as a percentage of control. Values represent the mean of four experiments±s.d.

Figure 2

Apoptosis of melanoma cells after pamidronate treatment. A375, M186, Mel2A and M221 melanoma cells were seeded at a density of 40 000 cells per cm2 and after 36 h treated with vehicle control or 100 μM pamidronate for 24 h. DNA-fragmentation was quantified using an enzyme-linked immunoassay detecting cytoplasmic nucleosomes. DNA-fragmentation of control cells was set as 100% and DNA-fragmentation of treated cells was calculated as a percentage of control. Values represent the mean of four experiments±s.d.

Figure 3

Apoptosis after short term exposure of A375 cells to pamidronate. A375 melanoma cells were seeded at a density of 40 000 cells per cm2 and the next day treated with vehicle or 100 μM pamidronate. Cells were either treated for 6 h with pamidronate, after which drug-containing medium was replaced with fresh culture medium, and the cells were further incubated for another 18 h or incubated with the drug containing medium for 24 h. Apoptosis was then measured as DNA-fragmentation using an enzyme-linked immunoassay detecting cytoplasmic nucleosomes. DNA-fragmentation of control cells was set as 100% and DNA-fragmentation of treated cells was calculated as a percentage of control. Values represent the mean of four experiments±s.d.

Figure 4

Effect of combination of pamidronate with DTIC. A375 melanoma cells were seeded at a density of 40 000 cells per cm2 and the next day treated with 100 μM Pamidronate, 5 μg ml−1 DTIC or both. After 24 h of incubation apoptosis was measured. DNA-fragmentation of control cells was set as 100% and DNA-fragmentation of treated cells was calculated as a percentage of control. Values represent the mean of four experiments±s.d.

Figure 5

Procaspase-3 cleavage upon pamidronate treatment in melanoma cells. A375, M186, Mel2A and M221 melanoma cells were seeded at a density of 40 000 cells per cm2 and after 36 h treated with 100 μM pamidronate (+), vehicle control (−) or 1 μg ml−1 of the CD95-agonistic monoclonal antibody CH-11 (C) as a positive control for 24 h. Whole cell lysates were prepared and 50 μg of each were separated on a 15% SDS–PAGE. After transfer to nitrocellulose the membrane was probed with anti caspase-3 antibodies recognising procaspase-3 and cleavage products forming active caspase-3 respectively. Immunocomplexes were detected using Super Signal chemiluminescence reagent.

Figure 6

Induction of apoptosis in p53-mutated and bcl-2 overexpressing melanoma cells by pamidronate. (A) The p53-mutated cell lines MeWo and SkMel23 and (B) the mock-transfected A375/pIRES and the bcl-2 overexpressing A375/mbcl-2 were seeded at a density of 40 000 cells per cm2 and after 36 h treated with vehicle control or 100 μM pamidronate for 24 h. DNA-fragmentation was quantified using an enzyme-linked immunoassay detecting cytoplasmic nucleosomes. DNA-fragmentation of control cells was set as 100% and DNA-fragmentation of treated cells was calculated as a percentage of control. Values represent the mean of four experiments±s.d.

Figure 7

Effect of isoprenoids on pamidronate-induced apoptosis in melanoma cells. A375 melanoma cells were seeded at a density of 40 000 cells per cm2 and after 36 h treated with 100 μM pamidronate in the presence of indicated concentrations of farnesol, geranylgeraniol or vehicle control for 24 h. DNA-fragmentation was quantified as cytoplasmic nucleosomes using an enzyme-linked immunoassay. DNA-fragmentation of control cells was set as 100% and DNA-fragmentation of treated cells was calculated as a percentage of control. Values represent the mean of four experiments±s.d.