Anke J. Roelofs

Molecular mechanisms of action of bisphosphonates: current status.

Purpose: Bisphosphonates are currently the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor-associated osteolysis and hypercalcemia. These compounds have high affinity for calcium ions and therefore target bone mineral, where they are internalized by bone-resorbing osteoclasts and inhibit osteoclast function. Experimental Design: This article reviews the pharmacology of bisphosphonates and the relationship between chemical structure and antiresorptive potency. We also describe new insights into their intracellular molecular mechanisms of action, methods for assessing the effects of bisphosphonates on protein prenylation, and their potential as direct antitumor agents. Results: Nitrogen-containing bisphosphonates act intracellularly by inhibiting farnesyl diphosphate synthase, an enzyme of the mevalonate pathway, thereby preventing prenylation of small GTPase signaling proteins required for normal cellular function. Inhibition of farnesyl diphosphate synthase also seems to account for their antitumor effects observed in vitro and for the activation of γ,δ T cells, a feature of the acute-phase response to bisphosphonate treatment in humans. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that seems to involve primarily the formation of cytotoxic metabolites in osteoclasts. Conclusions: Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect osteoclasts in vivo but could also have direct effects on other cell types, such as tumor cells. After >30 years of clinical use, their molecular mechanisms of action on osteoclasts are finally becoming clear but their exact antitumor properties remain to be clarified.

Peripheral blood monocytes are responsible for γδ T cell activation induced by zoledronic acid through accumulation of IPP/DMAPP

Nitrogen‐containing bisphosphonates indirectly activate Vγ9Vδ2 T cells through inhibition of farnesyl pyrophosphate synthase and intracellular accumulation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but the cells responsible for Vγ9Vδ2 T cell activation through IPP/DMAPP accumulation are unknown. Treatment of human peripheral blood mononuclear cells (PBMCs) with a pharmacologically relevant concentration of zoledronic acid induced accumulation of IPP/DMAPP selectively in monocytes, which correlated with efficient drug uptake by these cells. Furthermore, zoledronic acid‐pulsed monocytes triggered activation of γδ T cells in a cell contact‐dependent manner. These observations identify monocytes as the cell type directly affected by bisphosphonates responsible for Vγ9Vδ2 T cell activation.

Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells

Bisphosphonates (BPs) target bone due to their high affinity for calcium ions. During osteoclastic resorption, these drugs are released from the acidified bone surface and taken up by osteoclasts, where they act by inhibiting the prenylation of small GTPases essential for osteoclast function. However, it remains unclear exactly how osteoclasts internalise BPs from bone and whether other cells in the bone microenvironment can also take up BPs from the bone surface. We have investigated this using a novel fluorescently-labelled alendronate analogue (FL-ALN), and by examining changes in protein prenylation following treatment of cells with risedronate (RIS). Confocal microscopic analysis showed that FL-ALN was efficiently internalised from solution or from the surface of dentine by resorbing osteoclasts into intracellular vesicles. Accordingly, unprenylated Rap1A accumulated to the same extent whether osteoclasts were cultured on RIS-coated dentine or with RIS in solution. By contrast, J774 macrophages internalised FL-ALN and RIS from solution, but took up comparatively little from dentine, due to their inability to resorb the mineral. Calvarial osteoblasts and MCF-7 tumour cells internalised even less FL-ALN and RIS, both from solution and from the surface of dentine. Accordingly, the viability of J774 and MCF-7 cells was drastically reduced when cultured with RIS in solution, but not when cultured on dentine pre-coated with RIS. However, when J774 macrophages were co-cultured with rabbit osteoclasts, J774 cells that were adjacent to resorbing osteoclasts frequently internalised more FL-ALN than J774 cells more distant from osteoclasts. This was possibly a result of increased availability of BP to these J774 cells due to transcytosis through osteoclasts, since FL-ALN partially co-localised with trancytosed, resorbed matrix protein within osteoclasts. In addition, J774 cells occupying resorption pits internalised more FL-ALN than those on unresorbed surfaces. These data demonstrate that osteoclasts are able to take up large amounts of BP, due to their ability to release the BP from the dentine surface during resorption. By contrast, non-resorbing cells take up only small amounts of BP that becomes available due to natural desorption from the dentine surface. However, BP uptake by non-resorbing cells can be increased when cultured in the presence of resorbing osteoclasts.

Fluorescent risedronate analogues reveal bisphosphonate uptake by bone marrow monocytes and localization around osteocytes in vivo.

Bisphosphonates are effective antiresorptive agents owing to their bone‐targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non‐osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein‐labeled risedronate (FAM‐RIS) and Alexa Fluor 647–labeled risedronate (AF647‐RIS), were used to address this question. Twenty‐four hours after injection into 3‐month‐old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM‐RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14high bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14+ cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone‐resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo.

Bisphosphonates: Molecular Mechanisms of Action and Effects on Bone Cells, Monocytes and Macrophages

Bisphosphonates are widely used in the treatment of diseases involving excessive bone resorption, such as osteoporosis, cancer-associated bone disease, and Pagets disease of bone. They target to the skeleton due to their calcium-chelating properties, where they primarily act by inhibiting osteoclast-mediated bone resorption. The simple bisphosphonates, clodronate, etidronate and tiludronate, are intracellularly metabolised to cytotoxic ATP analogues, while the more potent, nitrogen-containing bisphosphonates act by inhibiting the enzyme FPP synthase, thereby preventing the prenylation of small GTPases that are necessary for the normal function and survival of osteoclasts. In recent years, these concepts have been refined, with an increased understanding of the exact mode of inhibition of FPP synthase and the consequences of inhibiting this enzyme. Recent studies further suggest that the R2 side chain, as well as determining the potency for inhibiting the target enzyme FPP synthase, also influences bone mineral binding, which may influence distribution within bone and duration of action. While bisphosphonates primarily affect the function of resorbing osteoclasts, it is becoming increasingly clear that bisphosphonates may also target the osteocyte network and prevent osteocyte apoptosis, which could contribute to their anti-fracture effects. Furthermore, increasing evidence implicates monocytes and macrophages as direct targets of bisphosphonate action, which may explain the acute phase response and the anti-tumour activity in certain animal models. Bone mineral affinity is likely to influence the extent of any such effects of these agents on non-osteoclast cells. While alternative anti-resorptive therapeutics are becoming available for clinical use, bisphosphonates currently remain the principle drugs used to treat excessive bone resorption.

A role for L-α-lysophosphatidylinositol and GPR55 in the modulation of migration, orientation and polarization of human breast cancer cells

Background and purpose:  Increased circulating levels of L‐α‐lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G‐protein‐coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.

The bisphosphonate zoledronic acid has antimyeloma activity in vivo by inhibition of protein prenylation

Nitrogen‐containing bisphosphonates (N‐BPs) are effective antiosteolytic agents in patients with multiple myeloma. Preclinical studies have also demonstrated that these agents have direct antitumor effects in vitro and can reduce tumor burden in a variety of animal models, although it is not clear whether such effects are caused by direct actions on tumor cells or by inhibition of bone resorption. N‐BPs prevent bone destruction in myeloma by inhibiting the enzyme farnesyl pyrophosphate synthase in osteoclasts, thereby preventing the prenylation of small GTPase signaling proteins. In this study, utilizing a plasmacytoma xenograft model without complicating skeletal lesions, treatment with zoledronic acid (ZOL) led to significant prolongation of survival in severe combined immunodeficiency mice inoculated with human INA‐6 plasma cells. Following treatment with a clinically relevant dose of ZOL, histological analysis of INA‐6 tumors from the peritoneal cavity revealed extensive areas of apoptosis associated with poly (ADP‐ribose) polymerase cleavage. Furthermore, Western blot analysis of tumor homogenates demonstrated the accumulation of unprenylated Rap1A, indicative of the uptake of ZOL by nonskeletal tumors and inhibition of farnesyl pyrophosphate synthase. These studies provide, for the first time, clear evidence that N‐BPs have direct antitumor effects in plasma cell tumors in vivo and this is executed by a molecular mechanism similar to that observed in osteoclasts.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower‐affinity analogues deoxy‐risedronate and 3‐PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower‐affinity compounds showed preferential binding to resorption lacunae, whereas the highest‐affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower‐affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high‐ and low‐affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that “recycling” had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton.

Cell-based Approaches to Joint Surface Repair : A Research Perspective

Summary Repair of lesions of the articular cartilage lining the joints remains a major clinical challenge. Surgical interventions include osteochondral autograft transfer and microfracture. They can provide some relief of symptoms to patients, but generally fail to durably repair the cartilage. Autologous chondrocyte implantation has thus far shown the most promise for the durable repair of cartilage, with long-term follow-up studies indicating improved structural and functional outcomes. However, disadvantages of this technique include the need for additional surgery, availability of sufficient chondrocytes for implantation, and maintenance of their phenotype during culture-expansion. Mesenchymal stem cells offer an attractive alternative cell-source for cartilage repair, due to their ease of isolation and amenability to ex vivo expansion while retaining stem cell properties. Preclinical and clinical studies have demonstrated the potential of mesenchymal stem cells to promote articular cartilage repair, but have also highlighted several key challenges. Most notably, the quality and durability of the repair tissue, its resistance to endochondral ossification, and its effective integration with the surrounding host tissue. In addition, challenges exist related to the heterogeneity of mesenchymal stem cell preparations and their quality-control, as well as optimising the delivery method. Finally, as our knowledge of the cellular and molecular mechanisms underlying articular cartilage repair increases, promising studies are emerging employing bioactive scaffolds or therapeutics that elicit an effective tissue repair response through activation and mobilisation of endogenous stem and progenitor cells.

The bisphosphonate zoledronic acid decreases tumor growth in bone in mice with defective osteoclasts

Bisphosphonates (BPs), bone targeted drugs that disrupt osteoclast function, are routinely used to treat complications of bone metastasis. Studies in preclinical models of cancer have shown that BPs reduce skeletal tumor burden and increase survival. Similarly, we observed in the present study that administration of the Nitrogen-containing BP (N-BP), zoledronic acid (ZA) to osteolytic tumor-bearing Tax+ mice beginning at 6 months of age led to resolution of radiographic skeletal lesions. N-BPs inhibit farnesyl diphosphate (FPP) synthase, thereby inhibiting protein prenylation and causing cellular toxicity. We found that ZA decreased Tax+ tumor and B16 melanoma viability and caused the accumulation of unprenylated Rap1a proteins in vitro. However, it is presently unclear whether N-BPs exert anti-tumor effects in bone independent of inhibition of osteoclast (OC) function in vivo. Therefore, we evaluated the impact of treatment with ZA on B16 melanoma bone tumor burden in irradiated mice transplanted with splenic cells from src(-/-) mice, which have non-functioning OCs. OC-defective mice treated with ZA demonstrated a significant 88% decrease in tumor growth in bone compared to vehicle-treated OC-defective mice. These data support an osteoclast-independent role for N-BP therapy in bone metastasis.