Abstracts of 2nd Scientific Meeting on Bone Disease in Multiple Myeloma
Molecular mechanisms of action of bisphosphonates and anti-tumour effects in multiple myeloma
MJ Rogers, S Gordon, JC Frith, FP Coxon, J Dunford, H Sati, M Greaves, SH Ralston, MH Helfrich Dept of Medicine & Therapeutics, University of Aberdeen Medical School, Scotland, UK
Recently, important breakthroughs have been made in understanding the molecular mechanisms by which bisphosphonates inhibit bone resorption[1]. All bisphosphonate drugs, by virtue of their P-C-P backbone structure, target to calcified tissues, where they are released and internalised selectively by bone-resorbing osteoclasts. Once internalised, bisphosphonates inhibit the ability of osteoclasts to resorb bone by mechanisms that interfere with cytoskeletal organisation and formation of the ruffled border, and that cause cell death by apoptosis. The anti-resorptive potency of bisphosphonates has long been known to be influenced by the chemical and three-dimensional structure of the side chain attached to the central carbon of the P-C-P backbone, with the presence of a nitrogen-containing group (especially within a heterocyclic ring, as in risedronate and zoledronic acid) at a critical distance from the central carbon conferring increased potency. Bisphosphonates can therefore be grouped into two distinct classes on the basis of structure and potency - those that lack a nitrogen group (such as clodronate and etidronate) and those that contain a nitrogen.
Following the discovery that some nitrogen-containing bisphosphonates could inhibit the intracellular mevalonate pathway[2;3], it is now clear that this is their major route of action. The biosynthetic mevalonate pathway is responsible for the production of cholesterol and isoprenoid lipids such as farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) [Fig. 1]. Recent studies have shown that nitrogen-containing bisphosphonates are inhibitors of FPP synthase[4-6]. For a wide range of bisphosphonates, we have found a significant correlation between potency for inhibition of recombinant human FPP synthase in vitro and anti-resorptive potency in vivo, suggesting that this enzyme is the major pharmacologic target of these drugs. The most potent anti-resorptive bisphosphonates such as zoledronic acid and risedronate are very potent inhibitors of FPP synthase, with IC50 values as low as 3nM and 10nM respectively[6].
Inhibition of FPP synthase prevents the formation of FPP and its derivative GGPP. These isoprenoid lipids are necessary for the post-translational lipid modification (prenylation) of small GTPase proteins such as Ras, Rho, Rac and Rab[7]. Prenylation involves the transfer of a farnesyl or geranylgeranyl lipid group onto a cysteine residue in characteristic carboxy-terminal motifs[8], giving rise to farnesylated and geranylgeranylated proteins. Small GTPases (the majority of which are geranylgeranylated), are important components of signalling pathways that regulate a variety of cell processes important for osteoclast function, including integrin signalling, membrane ruffling, trafficking of endosomes, and apoptosis. Prenylation is required for the correct function of these proteins, since the lipid prenyl group serves to anchor the proteins in cell membranes and may also participate in protein:protein interactions[9]. Inhibition of FPP synthase and loss of prenylation of small GTPases such as Rho, Rac, cdc42 and Rab accounts for most, if not all, of the various effects on osteoclast function, including loss of the ruffled border and disruption of the actin cytoskeleton, and the induction of osteoclast apoptosis. Studies with J774 macrophages and purified osteoclasts in vitro have provided direct evidence that nitrogen-containing bisphosphonates inhibit protein prenylation, since these compounds prevent the incorporation of [14C]mevalonate into prenylated proteins (both farnesylated and geranylgeranylated proteins), whereas the bisphosphonates that lack a nitrogen in the R2 side (clodronate and etidronate) have no effect [5; 10-12]. Risedronate and zoledronic acid almost completely inhibit protein prenylation at a concentration of 10-5M, which is similar to concentrations of bisphosphonates that affect osteoclast viability in vitro and could be achieved within the osteoclast resorption lacuna[13]. Furthermore, changes to the structure of the R2 side chain or to the phosphonate groups which influence anti-resorptive potency in vivo also influence in a similar manner the ability to inhibit FPP synthase and prevent protein prenylation[6; 14]. Importantly, the effects of nitrogen-containing bisphosphonates on osteoclasts can be overcome by addition of components of the mevalonate pathway, which bypass the inhibition of FPP synthase and restore protein prenylation. In particular, geranylgeraniol (a cell-permeable form of GGPP), prevents inhibition of osteoclast formation, the reduction in osteoclast number and inhibition of resorption by nitrogen-containing bisphosphonates in vitro[15;16]. Since geranylgeraniol can be used as a substrate for protein geranylgeranylation, and since farnesol (a cell-permeable form of FPP) has little protective effect[15;16], this strongly suggests that the inhibitory effect of nitrogen-containing bisphosphonates on osteoclast formation and osteoclast function is due to loss of geranylgeranylated proteins rather than loss of farnesylated proteins. This is supported by the evidence that a selective inhibitor of protein geranylgeranylation (GGTI-298) mimicks the effects of bisphosphonates on osteoclast formation, function and survival in vitro, whereas an inhibitor of farnesylation (FTI-277) has little effect on osteoclasts[12]. The signaling pathways involving geranylgeranylated small GTPases such as Rho, Rac, cdc42 and Rab that are affected by bisphosphonates and that lead to loss of osteoclast function, caspase activation and apoptosis remain to be determined. However, taken together, these recent observations all provide compelling evidence that inhibition of protein prenylation (especially protein geranylgeranylation) is the major molecular mechanism by which nitrogen-containing bisphosphonates inhibit bone resorption.
Fig. 1. Schematic representation of the mevalonate pathway and the site of inhibition by nitrogen-containing bisphosphonates
Since the bisphosphonates that lack a nitrogen group do not inhibit FPP synthase, affect the mevalonate pathway or inhibit protein prenylation[2; 6; 10-11; 15-17] these drugs must have a different molecular mechanism of action to that of the nitrogen-containing bisphosphonates. In osteoclasts, macrophages and other cell types in vitro, clodronate, etidronate and tiludronate can be metabolically incorporated by aminoacyl-tRNA synthetases into non-hydrolysable, methylene-containing (AppCp-type) analogues of adenosine triphosphate (ATP), containing the P-C-P moiety in place of the b,g P-O-P moiety[11; 18;19]. However, the nitrogen-containing bisphosphonates with larger, bulkier side chains are not metabolised. The identity of the AppCp-type bisphosphonate metabolites has recently been confirmed using electrospray mass spectrometry[11]. We have recently shown that osteoclasts, purified from clodronate-treated rabbits using immunomagnetic beads, also contain the metabolite of clodronate (AppCCl2p)[20]. Owing to the non-hydrolysable nature of the AppCp-type metabolites, their accumulation in osteoclasts in vivo is likely to inhibit numerous intracellular metabolic enzymes, thus having detrimental effects on cell function and survival. In accord we have found that chemically-synthesised AppCCl2p mimicks the effect of clodronate, causing osteoclast apoptosis and inhibiting bone resorption in vitro. This confirms that AppCp-type bisphosphonate metabolites are cytotoxic and suggests that this class of bisphosphonates act as prodrugs, being converted to cytotoxic metabolites following intracellular uptake by osteoclasts in vivo.
Given that the mevalonate pathway and aminoacyl-tRNA synthetases are ubiquitous, it is not surprising that bisphosphonates can affect most cell types, including tumour cells, in vitro. For example, we have shown that nitrogen-containing bisphosphonates can cause apoptosis of human myeloma cells in vitro by inhibiting protein prenylation[21]. However, it remains unclear whether bisphosphonates could directly affect cells other than osteoclasts in vivo, and whether this could account for the apparent anti-tumour effects of bisphosphonates and increased survival observed in some animal models and clinical studies. Using FACS analysis of bone marrow aspirates taken before and after a single infusion of pamidronate, we have observed a significant increase in myeloma cell apoptosis in vivo following pamidronate treatment, suggesting that bisphosphonates may have modest anti-tumour effects in multiple myeloma. Additional studies are required to determine whether these effects are direct or indirect and could be exploited further.
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