Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro

Abstract

Nitrogen-containing bisphosphonates were shown to cause macrophage apoptosis by inhibiting enzymes in the biosynthetic pathway leading from mevalonate to cholesterol. This study suggests that, in osteoclasts, geranylgeranyl diphosphate, the substrate for prenylation of most GTP binding proteins, is likely to be the crucial intermediate affected by these bisphosphonates. We report that murine osteoclast formation in culture is inhibited by both lovastatin, an inhibitor of hydroxymethylglutaryl CoA reductase, and alendronate. Lovastatin effects are blocked fully by mevalonate and less effectively by geranylgeraniol whereas alendronate effects are blocked partially by mevalonate and more effectively by geranylgeraniol. Alendronate inhibition of bone resorption in mouse calvaria also is blocked by mevalonate whereas clodronate inhibition is not. Furthermore, rabbit osteoclast formation and activity also are inhibited by lovastatin and alendronate. The lovastatin effects are prevented by mevalonate or geranylgeraniol, and alendronate effects are prevented by geranylgeraniol. Farnesol and squalene are without effect. Signaling studies show that lovastatin and alendronate activate in purified osteoclasts a 34-kDa kinase. Lovastatin-mediated activation is blocked by mevalonate and geranylgeraniol whereas alendronate activation is blocked by geranylgeraniol. Together, these findings support the hypothesis that alendronate, acting directly on osteoclasts, inhibits a rate-limiting step in the cholesterol biosynthesis pathway, essential for osteoclast function. This inhibition is prevented by exogenous geranylgeraniol, probably required for prenylation of GTP binding proteins that control cytoskeletal reorganization, vesicular fusion, and apoptosis, processes involved in osteoclast activation and survival.

The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells: evidence for synergy with paclitaxel

“…These observations are consistent with previous studies that have demonstrated that bisphosphonates cause a significant reduction in osteoclast number (Hughes et al, 1995;Fisher et al, 1999). Importantly, studies in human myeloma cells have shown that nitrogen-containing bisphosphonates, including zoledronic acid, also inhibit cell proliferation (Shipman et al, 1997(Shipman et al, , 1998Aparicio et al, 1998;Derenne et al, 1999).…”

“…We have also demonstrated that zoledronic acid-induced apoptosis of MCF-7 breast cancer cells can be inhibited by addition of intermediates of the mevalonate pathway, which is consistent with observations in osteoclasts, macrophages and myeloma cells (Luckman et al, 1998;Shipman et al, 1998;Fisher et al, 1999). This pathway has been shown to be important in the posttranslational modification of small GTP-binding proteins including Ras, by the process of farnesylation, and Rho and Rac by geranylgeranylation.…”

“…*P < 0.05 compared with contro.l MCF-7 cells from zoledronic acid-induced apoptosis, whereas the addition of farnesol only partially inhibited tumour cell death. Recent observations by Fisher et al suggest that this may also be the case in osteoclasts where disruption of the cytoskeleton and loss of the osteoclast ruffled border, as well as apoptosis, are seen with bisphosphonate treatment (Fisher et al, 1999). However, this contrasts with previous studies in J774 macrophages and the JJN3 myeloma cells in which bisphosphonate-induced apoptosis could be prevented by the addition of both geranylgeraniol and farnesol.…”

“…bisphosphonates such as pamidronate, incadronate, and zoledronic acid (Hughes et al, 1995;Rogers et al, 1996;Shipman et al, 1997Shipman et al, , 1998Aparicio et al, 1998;Fisher et al, 1999). Although there were significant differences between the levels of apoptosis detected by the 2 different techniques, the relative changes induced by bisphosphonate treatment were similar.…”

Structure, function, and regulation of tartrate-resistant acid phosphatase

“…78 In the case of alendronate at least this results from the inhibition of an unidentified enzyme involved in the conversion of mevalonate to geranylgeranyldiphosphate, the substrate for prenylation of most GTP binding proteins. 33 …”