Investigating the molecular mechanism of pamidronate‐induced in vitro cytotoxicity in canine osteosarcoma cells

Introduction: Pamidronate is used to treat metastatic bone lesions in human cancer patients. By directly inhibiting the mevalonate pathway, pamidronate disrupts GTPase‐binding protein prenylation, resulting in osteoclast apoptosis. Pamidronate has been demonstrated to exert dose‐ and time‐dependent cytotoxic effects in several canine malignant osteoblastic cell lines. However, the exact cytotoxic mechanism remains speculative. The purpose of this study was to investigate and characterize the molecular mechanism of pamidronate‐induced cytotoxicity in canine malignant osteoblasts. Methods: The involvement of farnesyl or geranylgeranyl pyrophosphate synthase inhibition was evaluated by performing rescue experiments with cells incubated for 48 hours with cytotoxic concentrations of pamidronate (50 and 100 μM), with or without farnesol (FOH) and geranylgeraniol (GGOH). Cell lysates were fluorometrically‐assessed for caspase‐3‐like activity (R&D Systems) following incubation with varying concentrations of pamidronate (control, 50 and 100 μM) for 48 hours. The expression of a prenylated GTPase protein, Rap1 A, was qualitatively evaluated with immunoblotting using a polyclonal goat antibody (Santa Cruz Biotechnology) in cells incubated for 48 hours with varying concentrations of pamidronate (0, 1, 10, 50, and 100 μM). Results: In cells treated with lower cytotoxic concentrations of pamidronate (50 μM) for 48 hours, the addition of GGOH, but not FOH, was able to diminish the degree cytotoxicity, p ≤ 0.05. However, cells incubated with higher cytotoxic concentrations of pamidronate (100 μM), neither GGOH nor FOH were able to reduce the level of pamidronate‐induced cytotoxicity. Caspase‐3‐like activity directly correlated with the degree of pamidronate‐induced cytotoxicity. Cells treated with pamidronate at 50 μM and 100 μM increased caspase‐3‐like activity by 5.3 and 7.1‐fold, respectively over untreated controls. The detection of Rap1A by immunoblotting requires further optimization. Conclusions: Pamidronate‐induced cytotoxicity in canine osteosarcoma cells may share a similar mechanism as has been demonstrated for osteoclasts. Inhibition of the mevalonate pathway appears to contribute to the observed cytotoxicity at lower doses of pamidronate. In addition, caspase‐3‐like activity contributes to the apoptotic process induced by pamidronate in malignant canine osteoblasts.