Hannu Mönkkönen

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

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. 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. 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. 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.

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.

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.

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.

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 μg/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 μCT 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 μmol/l zoledronic acid or 500 μmol/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 μmol/l zoledronic acid or 10–2000 μmol/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.

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.

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′(β,γ‐dichloromethylene) triphosphate (AppCCl2p).

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.

The Cellular Uptake and Metabolism of Clodronate in RAW 264 Macrophages

AbstractPurpose. Non-nitrogen-containing bisphosphonates, such as clodronate (dichloromethylene bisphosphonate), appear to act as prodrugs, their active form being the AppCp-type analogues of ATP. To further elucidate this, we examined the cellular uptake of clodronate and intracellular accumulation of the metabolite of clodronate (AppCCl2p) in RAW 264 macrophages, the influence of clodronate metabolism on the intracellular ATP concentration, and the time course of clodronate metabolism and the effects of clodronate on cytokine secretion from macrophages. Methods. The cellular uptake of clodronate was measured using 14C-labeled clodronate. AppCCl2p was determined in cell extracts by using an ion-pairing HPLC-ESI-MS. The cytokine concentrations in the culture supernatants were measured with time-resolved fluoroimmunoassay. Intracellular ATP concentration was measured with a luminometer using a luciferin-luciferase assay. Results. Of the clodronate internalized by macrophages in vitro, 30-55% is metabolized to AppCCl2p, which accumulates to high intracellular concentrations during the first 12 h of exposure. This accumulation does not affect the ATP levels in the cells. The time course of metabolite appearance in the cells and the inhibition of cytokine secretion were very similar. Conclusions. These results strongly support the idea that clodronate acts as a prodrug, the active form being its intracellular AppCCl2p metabolite.