In vivo phosphoantigen levels in bisphosphonate-treated human breast tumors trigger Vγ9Vδ2 T-cell antitumor cytotoxicity through ICAM-1 engagement.

PURPOSE: Nitrogen-containing bisphosphonates (N-BP) such as zoledronate and risedronate exhibit antitumor effects. They block the activity of farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway, leading to intracellular accumulation of mevalonate metabolites (IPP/ApppI), which are recognized as tumor phosphoantigens by Vγ9Vδ2 T cells. However, mechanisms responsible for Vγ9Vδ2 T-cell recognition of N-BP-treated tumors producing IPP/ApppI remain unclear. EXPERIMENTAL DESIGN: The effects of N-BPs on Vγ9Vδ2 T-cell expansion and anticancer activity were evaluated in vitro and in animal models of human breast cancers. The modalities of recognition of breast tumors by Vγ9Vδ2 T cells in N-BP-treated animals were also examined. RESULTS: We found a strong correlation between Vγ9Vδ2 T-cell anticancer activity and intracellular accumulation of IPP/ApppI in risedronate-treated breast cancer cells in vitro. In addition, following risedronate treatment of immunodeficient mice bearing human breast tumors, human Vγ9Vδ2 T cells infiltrated and inhibited growth of tumors that produced high IPP/ApppI levels but not those expressing low IPP/ApppI levels. The combination of doxorubicin with a N-BP improved, however, Vγ9Vδ2 T-cell cytotoxicity against breast tumors expressing low IPP/ApppI levels. Moreover, Vγ9Vδ2 T-cell cytotoxicity in mice treated with risedronate or zoledronate did not only depend on IPP/ApppI accumulation in tumors but also on expression of tumor cell surface receptor intercellular adhesion molecule-1 (ICAM-1), which triggered the recognition of N-BP-treated breast cancer cells by Vγ9Vδ2 T cells in vivo. CONCLUSION: These findings suggest that N-BPs can have an adjuvant role in cancer therapy by activating Vγ9Vδ2 T-cell cytotoxicity in patients with breast cancer that produces high IPP/ApppI levels after N-BP treatment.

Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset.

Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.

High phosphoantigen levels in bisphosphonate-treated human breast tumors promote Vgamma9Vdelta2 T-cell chemotaxis and cytotoxicity in vivo.

The nitrogen-containing bisphosphonate zoledronic acid (ZOL), a potent inhibitor of farnesyl pyrophosphate synthase, blocks the mevalonate pathway, leading to intracellular accumulation of isopentenyl pyrophosphate/triphosphoric acid I-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (IPP/ApppI) mevalonate metabolites. IPP/ApppI accumulation in ZOL-treated cancer cells may be recognized by Vγ9Vδ2 T cells as tumor phosphoantigens in vitro. However, the significance of these findings in vivo remains largely unknown. In this study, we investigated the correlation between the anticancer activities of Vγ9Vδ2 T cells and the intracellular IPP/ApppI levels in ZOL-treated breast cancer cells in vitro and in vivo. We found marked differences in IPP/ApppI production among different human breast cancer cell lines post-ZOL treatment. Coculture with purified human Vγ9Vδ2 T cells led to IPP/ApppI-dependent near-complete killing of ZOL-treated breast cancer cells. In ZOL-treated mice bearing subcutaneous breast cancer xenografts, Vγ9Vδ2 T cells infiltrated and inhibited growth of tumors that produced high IPP/ApppI levels, but not those expressing low IPP/ApppI levels. Moreover, IPP/ApppI not only accumulated in cancer cells but it was also secreted, promoting Vγ9Vδ2 T-cell chemotaxis to the tumor. Without Vγ9Vδ2 T-cell expansion, ZOL did not inhibit tumor growth. These findings suggest that cancers-producing high IPP/ApppI levels after ZOL treatment are most likely to benefit from Vγ9Vδ2 T-cell-mediated immunotherapy.

Correlation between time-dependent inhibition of human farnesyl pyrophosphate synthase and blockade of mevalonate pathway by nitrogen-containing bisphosphonates in cultured cells.

A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (K(isom)) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated K(isom) values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway. As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the K(isom) calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.

Nitrogen-containing bisphosphonates can inhibit angiogenesis in vivo without the involvement of farnesyl pyrophosphate synthase.

Nitrogen-containing bisphosphonates (N-BPs) are widely used to block bone destruction associated with bone metastasis because they are effective inhibitors of osteoclast-mediated bone resorption. More specifically, once internalized by osteoclasts, N-BPs block the activity of farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway. In addition to their antiresorptive activity, preclinical evidence shows that N-BPs have antiangiogenic properties. However, the exact reasons for which N-BPs inhibit angiogenesis remain largely unknown. Using different angiogenesis models, we examined here the effects of zoledronate, risedronate and three structural analogs of risedronate (NE-58025, NE-58051 and NE-10790) with lower potencies to inhibit FPPS activity. Risedronate and zoledronate were much more potent than NE-compounds at inhibiting both endothelial cell proliferation in vitro and vessel sprouting in the chicken egg chorioallantoic membrane (CAM) assay. In addition, only risedronate and zoledronate inhibited the revascularization of the prostate gland in testosterone-stimulated castrated rats. Moreover, as opposed to NE-compounds, risedronate and zoledronate induced intracellular accumulation of isopentenyl pyrophosphate (IPP) in endothelial cells by blocking the activity of the IPP-consuming enzyme FPPS. Thus, these results indicated that N-BPs inhibited angiogenesis in a FPPS-dependent manner. However, drug concentrations used to inhibit angiogenesis, both in vitro and in the CAM and prostate gland assays, were high. In contrast, a low concentration of risedronate (1 µM) was sufficient to inhibit blood vessel formation in the ex vivo rat aortic ring assay. Moreover, NE-58025 (which had a 7-fold lower potency than risedronate to inhibit FPPS activity) was as effective as risedronate to reduce angiogenesis in the rat aortic ring assay. In conclusion, our results suggest that low concentrations of N-BPs inhibit angiogenesis in a FPPS-independent manner, whereas higher drug concentrations were required to inhibit FPPS activity in vivo.

Aminobisphosphonate-pretreated dendritic cells trigger successful Vgamma9Vdelta2 T cell amplification for immunotherapy in advanced cancer patients.

Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vgamma9Vdelta2 T lymphocytes represents an attractive strategy. Indeed, Vgamma9Vdelta2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vgamma9Vdelta2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vgamma9Vdelta2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vgamma9Vdelta2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vgamma9Vdelta2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vgamma9Vdelta2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vgamma9Vdelta2 T cells as measured by IFN-gamma production. Moreover, we demonstrated that the cytotoxic level of Vgamma9Vdelta2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vgamma9Vdelta2 T cells leads eligible for Vgamma9Vdelta2 T cell adoptive immunotherapy the HCC and mCRC patients.

Activation of γδ T cells by bisphosphonates.

After decades of successful clinical use, the exact molecular mechanisms by which the anti-resorptive bisphosphonate drugs (BPs) exert their effects are now being revealed. In addition to their anti-resorptive effects, it is now apparent that nitrogen-containing BPs (N-BPs) have immunomodulatory properties. Specifically, these drugs activate immune cells called gamma, delta T lymphocytes. In this chapter we discuss the mechanism of gamma, delta T cell activation by N-BPs and propose that N-BPs may provide a safe and effective means for manipulating gamma,delta T cell activity in future immunotherapeutic approaches.

Zoledronic acid repolarizes tumour-associated macrophages and inhibits mammary carcinogenesis by targeting the mevalonate pathway.

It is unknown whether zoledronic acid (ZA) at clinically relevant doses is active against tumours not located in bone. Mice transgenic for the activated ErbB-2 oncogene were treated with a cumulative number of doses equivalent to that recommended in human beings. A significant increase in tumour-free and overall survival was observed in mice treated with ZA. At clinically compatible concentrations, ZA modulated the mevalonate pathway and affected protein prenylation in both tumour cells and macrophages. A marked reduction in the number of tumour-associated macrophages was paralleled by a significant decrease in tumour vascularization. The local production of vascular endothelial growth factor and interleukin-10 was drastically down-regulated in favour of interferon-γ production. Peritoneal macrophages and tumour-associated macrophages of ZA-treated mice recovered a full M1 antitumoral phenotype, as shown by nuclear translocation of nuclear factor kB, inducible nitric oxide synthase expression and nitric oxide production. These data indicate that clinically achievable doses of ZA inhibit spontaneous mammary cancerogenesis by targeting the local microenvironment, as shown by a decreased tumour vascularization, a reduced number of tumour-associated macrophages and their reverted polarization from M2 to M1 phenotype.

Mevalonate pathway intermediates downregulate zoledronic acid-induced isopentenyl pyrophosphate and ATP analog formation in human breast cancer cells.

Increasing evidence is accumulating that zoledronic acid (ZOL), a nitrogen-containing bisphosphonate (N-BP), is able to affect tumor cells by inhibiting the enzyme farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway (MVP). The consequent accumulation of unprenylated proteins is believed to largely account for the cytotoxic effects of ZOL. FPPS inhibition leads also to the accumulation of isopentenyl pyrophosphate (IPP) and the apoptotic ATP analog, ApppI, but the role of this mechanism in the cytotoxic action of bisphosphonates is less clear. Since treatment with MVP intermediates has been shown to overcome N-BP-induced apoptosis via rescuing protein prenylation, our aim here was to determine their mechanism of action on ZOL-induced IPP/ApppI accumulation. Interestingly, the results revealed that ZOL-induced IPP/ApppI accumulation in MCF-7 cells were decreased by farnesol, and almost completely blocked by geranylgeraniol and geranylpyrophosphate. The functionality of the regulatory enzymes of IPP and ApppI, IPP isomerase and aminoacyl-tRNA-synthase, respectively, or protein levels of FPPS were not affected by the treatments. However, the protein levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and unprenylated Rap1A were observed to be strongly downregulated by geranylgeraniol and geranylpyrophosphate. This study represents a novel insight into the mechanism of action of MVP intermediates on the regulation of MVP after FPPS inhibition. The data implies that in addition to the previously reported effects on rescuing protein prenylation, MVP intermediates can preserve cell activity by inhibiting the accumulation of IPP/ApppI via HMGR downregulation. This supports the hypothesis that IPP/ApppI formation is a significant mechanism in the anticancer action of ZOL.

Analysis of endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid chromatography-electrospray ionization mass spectrometry.

Nitrogen-containing bisphosphonates (N-BPs) are shown to inhibit a key enzyme of intracellular mevalonate pathway, FPP synthase, leading to intracellular accumulation of pathway metabolites isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In our previous studies we have shown that a new type of ATP analog, ApppI (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester), is also formed in addition to IPP and DMAPP accumulation. ApppI has cytotoxic effects leading to direct apoptosis of various cancer cells. In this study we present a validated method based on ion-pair LC-MS(2) for the analysis of isomeric mevalonate pathway metabolites and ATP analogs in cell culture samples. Limit of quantitation for IPP and DMAPP was 0.030microM (1.35fmol on-column) and for ApppI and ApppD 0.020microM (0.9fmol on-column). Acceptable accuracies and precision were also obtained for quality control samples in low and high concentrations of the calibration curve. In addition, we present a new method for quantitation of each coeluting isomer utilizing the peak intensity ratios of two characteristic fragment ions of each compound. For IPP and DMAPP, fragment ions m/z 177 and m/z 159 in the MS(2) were monitored, whereas for ATP analogs, ApppI and ApppD (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-2-enyl) ester), the same fragments in the MS(3) spectra were followed. IPP and DMAPP accumulation as well as ApppI and ApppD formation was demonstrated using MCF-7 breast cancer cells. Cells were treated with 25muM zoledronic acid (an N-BP) for 24h, conditions found to induce significant production of the metabolites. We found that the total amount of IPP and DMAPP was 2.4nmol/mg of protein and amount of ApppI and ApppD was 1.1nmol/mg protein. Relative portions of the isomers were approximately 1:4 IPP:DMAPP and 3:7 ApppI:ApppD. Untreated control samples did not contain IPP, DMAPP, ApppI or ApppD.

Zoledronic acid induces formation of a pro-apoptotic ATP analogue and isopentenyl pyrophosphate in osteoclasts in vivo and in MCF-7 cells in vitro.

BACKGROUND AND PURPOSE: Bisphosphonates (BPs) are highly effective inhibitors of bone resorption. Nitrogen-containing bisphosphonates (N-BPs), such as zoledronic acid, induce the formation of a novel ATP analogue (1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester triphosphoric acid; ApppI), as a consequence of the inhibition of farnesyl pyrophosphate synthase and the accumulation of isopentenyl pyrophosphate (IPP). ApppI induces apoptosis, as do comparable metabolites of non-nitrogen-containing bisphosphonates (non-N-BPs). In order to further evaluate a pharmacological role for ApppI, we obtained more detailed data on IPP/ApppI formation in vivo and in vitro. Additionally, zoledronic acid-induced ApppI formation from IPP was compared with the metabolism of clodronate (a non-N-BP) to adenosine 5'(beta,gamma-dichloromethylene) triphosphate (AppCCl2p). EXPERIMENTAL APPROACH: After giving zoledronic acid in vivo to rabbits, IPP/ApppI formation and accumulation was assessed in isolated osteoclasts. The formation of ApppI from IPP was compared with the metabolism of clodronate in MCF-7 cells in vitro. IPP/ApppI and AppCCl2p levels in cell extracts were analysed by mass spectrometry. KEY RESULTS: Isopentenyl pyrophosphate/ApppI were formed in osteoclasts in vivo, after a single, clinically relevant dose of zoledronic acid. Furthermore, exposure of MCF-7 cells in vitro to zoledronic acid at varying times and concentrations induced time- and dose-dependent accumulation of IPP/ApppI. One hour pulse treatment was sufficient to cause IPP accumulation and subsequent ApppI formation, or the metabolism of clodronate into AppCCl2p. CONCLUSIONS AND IMPLICATIONS: This study provided the first conclusive evidence that pro-apoptotic ApppI is a biologically significant molecule, and demonstrated that IPP/ApppI analysis is a sensitive tool for investigating pathways involved in BP action.

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

Nitrogen-containing bisphosphonates indirectly activate Vgamma9Vdelta2 T cells through inhibition of farnesyl pyrophosphate synthase and intracellular accumulation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but the cells responsible for Vgamma9Vdelta2 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 gammadelta T cells in a cell contact-dependent manner. These observations identify monocytes as the cell type directly affected by bisphosphonates responsible for Vgamma9Vdelta2 T cell activation.

Antitumor effects of doxorubicin followed by zoledronic acid in a mouse model of breast cancer.

BACKGROUND: The potent antiresorptive drug zoledronic acid (Zol) enhances the antitumor effects of chemotherapy agents in vitro. We investigated the effects of clinically achievable doses of doxorubicin (Dox) and Zol, given alone, in sequence, and in combination, on the growth of established breast tumors in vivo. METHODS: Female MF1 nude mice were inoculated subcutaneously with 5 x 10(5) human breast cancer MDA-MB-436 cells that stably expressed green fluorescent protein (ie, MDA-G8 cells). Beginning on day 7 after tumor cell injection, the mice were injected weekly for 6 weeks with saline, Dox (2 mg/kg body weight via intravenous injection), Zol (100 microg/kg body weight via intraperitoneal injection), Dox plus Zol, Zol followed 24 hours later by Dox, or Dox followed 24 hours later by Zol (n = 8-9 mice per group). The effects of treatment on tumor growth were determined by measuring tumor volume; on tumor cell apoptosis and proliferation by immunohistochemistry using antibodies for caspase-3 and Ki-67, respectively; and on bone by microcomputed tomography and bone histomorphometry. All P values are two-sided. RESULTS: Treatment with Dox or Zol alone or Zol followed 24 hours later by Dox did not statistically significantly decrease final tumor volume compared with saline. Mice treated with Dox plus Zol had statistically significantly smaller final tumor volumes than those treated with Dox alone (mean = 122 mm(3) vs 328 mm(3), difference = 206 mm(3), 95% confidence interval [CI] = 78 to 335 mm(3), P < .001), with Zol alone (122 mm(3) vs 447 mm(3), difference = 325 mm(3), 95% CI = 197 to 454 mm(3), P < .001), or with Zol followed 24 hours later by Dox (122 mm(3) vs 418 mm(3), difference = 296 mm(3), 95% CI = 168 to 426 mm(3), P < .001). Treatment with Dox followed 24 hours later by Zol almost completely abolished tumor growth. Tumors from mice that were treated with Dox followed by Zol had more caspase-3-positive cells than tumors from mice treated with saline (mean number of caspase-3-positive cells per square millimeter: 605.0 vs 82.19, difference = 522.8, 95% CI = 488.2 to 557.4, P < .001), with Zol alone (605.0 vs 98.44, difference = 506.6, 95% CI = 472.0 to 541.2, P < .001), or with Zol followed by Dox (605.0 vs 103.1, difference = 501.9, 95% CI = 467.3 to 536.5, P < .001). The treatment-induced increase in the number of caspase-3-positive cells was mirrored by a decrease in the number of tumor cells positive for the proliferation marker Ki-67. No evidence of bone disease was detected in any of the treatment groups following microcomputed tomography and histological analysis of bone. CONCLUSION: Sequential treatment with Dox followed by Zol elicited substantial antitumor effects in subcutaneous breast tumors in vivo, in the absence of bone disease.

Differential effect of doxorubicin and zoledronic acid on intraosseous versus extraosseous breast tumor growth in vivo.

PURPOSE: Breast cancer patients with bone metastases are commonly treated with chemotherapeutic agents such as doxorubicin and zoledronic acid to control their bone disease. Sequential administration of doxorubicin followed by zoledronic acid has been shown to increase tumor cell apoptosis in vitro. We have therefore investigated the antitumor effects of clinically relevant doses of these drugs in a mouse model of breast cancer bone metastasis. EXPERIMENTAL DESIGN: MDA-MB-231/BO2 cells were injected via the tail vein into athymic mice. Tumor-induced osteolytic lesions were detected in all animals following X-ray analysis 18 days after tumor cell inoculation (day 18). Mice were administered saline, 100 microg/kg zoledronic acid, 2 mg/kg doxorubicin, doxorubicin and zoledronic acid simultaneously, or doxorubicin followed 24 h later by zoledronic acid. Doxorubicin-treated animals received a second injection on day 25. Tumor growth in the marrow cavity and on the outside surface of the bone was measured as well as tumor cell apoptosis and proliferation. The effects of treatments on bone were evaluated following X-ray and muCT analysis. RESULTS: Sequential treatment with doxorubicin followed by zoledronic acid caused decreased intraosseous tumor burden, which was accompanied by increased levels of tumor cell apoptosis and decreased levels of proliferation, whereas extraosseous parts of the same tumors were unaffected. Administration of zoledronic acid, alone or in combination with doxorubicin, resulted in significantly smaller tumor-induced osteolytic lesions compared with control or doxorubicin-treated animals. CONCLUSIONS: This is the first study to show that sequential treatment with clinically relevant doses of doxorubicin, followed 24 h later by zoledronic acid, reduces intraosseous but not extraosseous growth of BO2 breast tumors. Our results suggest that breast cancer patients with metastatic bone disease may benefit from sequential treatment using doxorubicin and zoledronic acid.

Bisphosphonate-induced ATP analog formation and its effect on inhibition of cancer cell growth.

Bisphosphonates (BPs) are effective inhibitors of tumor-induced bone resorption. Recent studies have demonstrated that BPs inhibit growth, attachment and invasion of cancer cells in culture and promote apoptosis. The mechanisms responsible for the observed anti-tumor effects of BPs are beginning to be elucidated. Recently, we reported that nitrogen-containing bisphosphonates (N-BPs) induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of farnesyl diphosphate synthase in the mevalonate pathway. Similar to AppCp-type metabolites of non-N-BPs, ApppI is able to induce apoptosis. This study investigated BP-induced ATP analog formation and its effect on cancer cell growth. To evaluate zoledronic acid (a N-BP)-induced ApppI accumulation, inhibition of protein prenylation and clodronate (a non-N-BP) metabolism to AppCCl2p, MCF-7 and MDA-MB-436 breast cancer cells, MCF-10A nonmalignant breast cells, PC-3 prostate cancer cells, MG-63 osteosarcoma cells, RPMI-8226, and NCI-H929 myeloma cells were treated with 25 micromol/l zoledronic acid or 500 micromol/l clodronate for 24 h. The inhibition of cell growth by zoledronic acid and clodronate was studied in MCF-7, MDA-MB-436, and RPMI-8226 cells by exposing the cells with 1-100 micromol/l zoledronic acid or 10-2000 micromol/l clodronate for 72 h. Marked differences in zoledronic acid-induced ApppI formation and clodronate metabolism between the cancer cell lines were observed. The production of cytotoxic ATP analogs in tumor cells after BP treatment is likely to depend on the activity of enzymes, such as farnesyl diphosphate synthase or aminoacyl-tRNA synthetases, responsible for ATP analog formation. Additionally, the potency of clodronate to inhibit cancer cell growth corresponds to ATP analog formation.

Zoledronic acid-induced IPP/ApppI production in vivo.

Bisphosphonates are currently the most important class of anti-resorptive drugs used for the treatment of diseases involving excess bone resorption. Recently we discovered a new mechanism of action for bisphosphonates. Previously it has been shown that nitrogen-containing bisphosphonates (N-BPs) are not metabolized. However, our studies revealed that N-BPs induce formation of a novel pro-apoptotic ATP analog (ApppI), as a consequence of the inhibition of FPP synthase in the mevalonate pathway, and the subsequent accumulation of isopentenyl pyrophosphate (IPP) in vitro. The primary aim of the current study was to determine whether zoledronic acid (a N-BP) induces IPP/ApppI formation in vivo. Mass spectrometry was used to identify whether in vivo administration of zoledronic acid-induced IPP/ApppI production by mouse peritoneal macrophages or bone marrow cells. IPP/ApppI could be detected in extracts from peritoneal macrophages isolated from zoledronic acid-treated animals. Increasing IPP/ApppI accumulation was determined up to 7 days after drug injection, indicating prolonged FPP synthase inhibition by zoledronic acid. Importantly, this is the first report of in vivo production of ApppI, supporting the biological significance of this molecule.

Bisphosphonates in cancer therapy.

Bisphosphonates are the standard of care in the treatment of malignant bone diseases, because of their ability to inhibit osteoclast-mediated bone destruction. We review here preclinical evidence that bisphosphonates also exert direct antitumour effects and antiangiogenic properties. Furthermore, we describe new insights on how bisphosphonates may act synergistically in combination with antineoplastic drugs or gammadelta T cells to exhibit antitumour activity. These findings reveal new exciting possibilities to fully exploit the antitumour potential of bisphosphonates in the clinical practice.

A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates.

1. Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. On the basis of their molecular mechanism of action, bisphosphonates can be divided into two pharmacological classes; nitrogen-containing (N-BPs) and non-nitrogen-containing bisphosphonates (non-N-BP). Both classes induce apoptosis but they evoke it differently; N-BPs by inhibiting the intracellular mevalonate pathway and protein isoprenylation, and non-N-BPs via cytotoxic ATP analog-type metabolites. N-BPs are not metabolized to ATP analogs, but we report here that these bisphosphonates can induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of the mevalonate pathway in cells. We also investigated whether ApppI is involved in the apoptosis induced by N-BPs. 2. Mass spectrometry and NMR were used to identify ApppI in N-BP treated osteoclasts, macrophages and glioma cells. The potency of different bisphosphonates to promote ApppI production was tested in J774 macrophages. The effects of ApppI on ADP/ATP translocase in isolated mitochondria and its capability to induce apoptosis in osteoclasts were also studied. 3. ApppI production correlated well with the capacity of N-BPs to inhibit mevalonate pathway. ApppI inhibited the mitochondrial ADP/ATP translocase and caused apoptosis in osteoclasts. 4. In conclusion, these findings provide the basis for a new mechanism of action for N-BPs. Some of these very potent bisphosphonates, such as zoledronic acid, represent a third class of bisphosphonates that can act both via the inhibition of the mevalonate pathway and by the blockade of mitochondrial ADP/ATP translocase, which is known to be involved in the induction of apoptosis.

In vitro comparison of clodronate, pamidronate and zoledronic acid effects on rat osteoclasts and human stem cell-derived osteoblasts.

In the present study we compared the first generation non-nitrogen-containing bisphosphonate, clodronate with second and third generation nitrogen-containing bisphosphonates, pamidronate and zoledronic acid in dynamic rat osteoclast resorption and apoptosis assays and in human mesenchymal stem cell-derived osteoblast assay. We found that due to high bisphosphonate-bone binding affinity, bone surface exposure to clodronate for 3 min. had maximal resorption inhibition. The mechanism of action of both clodronate and zoledronic acid involved osteoclast apoptosis, whereas pamidronate had only minor apoptotic effect at dosages, which readily inhibited resorption. Zoledronic acid was not metabolised into an intracellular ATP-analogue in vitro in contrast to clodronate. All bisphosphonates had a dose-dependent inhibitory effect on the human bone marrow mesenchymal stem cell (hMSC)-derived osteoblast calcium deposition. None of the compounds had inhibitory effect on hMSC differentiation. Zoledronic acid was the most potent of all three bisphosphonates in terms of both apoptosis induction and resorption inhibition. Zoledronic acid efficacy might thus use its capacity to trigger osteoclast apoptosis in an unknown, but similar manner to that of the non-nitrogen-containing bisphosphonates. It appears that zoledronic acid has properties of both bisphosphonate classes and could well be the first member of a new class of bisphosphonates, by definition.

Cellular uptake and metabolism of clodronate and its derivatives in Caco-2 cells: a possible correlation with bisphosphonate-induced gastrointestinal side-effects.

PURPOSE: To investigate possible reasons for the low frequency of GI side-effects of clodronate, even though clodronate is known to be metabolised into a cytotoxic nucleotide analogue (AppCCl(2)p) by many cell types. The effects of some lipophilic prodrugs of clodronate were also studied. METHODS: The effects of clodronate and its lipophilic derivatives on the proliferation and viability of Caco-2 cells were examined using an MTT assay. The intracellular uptake of 14C-clodronate and the accumulation of a clodronate metabolite (AppCCl(2)p) in Caco-2 cells were evaluated using ion-pairing HPLC-ESI-MS. RESULTS: Clodronate had little effect on growth of proliferating, or the viability of confluent, Caco-2 cells. The uptake of clodronate by Caco-2 cells was only about 0.04% of total clodronate. The potentially cytotoxic clodronate metabolite, AppCCl(2)p, was detected in Caco-2 cell extracts after 3 h of exposure. Dianhydride- and triPOM-clodronate were metabolised to AppCCl(2)p more efficiently and also affected the viability of Caco-2 cells more than clodronate. CONCLUSIONS: Clodronate appears to be metabolised into a cytotoxic ATP-analogue (AppCCl(2)p) by any cell type capable of internalising the drug. However, the cytotoxicity depends on the degree of uptake of clodronate. Due to the very low initial uptake of clodronate by epithelial Caco-2 cells, they do not accumulate sufficient intracellular concentrations of AppCCl(2)p to affect cell function. This explains the low frequency of gastrointestinal side-effects caused by oral clodronate therapy.