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

Abstract

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.

Figure 1

Identification of IPP and ApppI in rabbit osteoclasts in vivo. Vitronectin receptor-positive osteoclasts were isolated by immunomagnetic bead separation from rabbit bone marrow 24 h after single injection with 100 µg·kg⁻¹ zoledronic acid (ZOL). Acetonitrile cell extracts were then analysed by HPLC-ESI-MS. Selective reaction monitoring chromatogram of osteoclast extract isolated from a saline injected rabbit (A, B), extract isolated from zoledronic acid-treated rabbit (C, D), untreated rabbit osteoclast extract with added 37 nmol·mg⁻¹ protein IPP (E) and 11 nmol·mg⁻¹ protein ApppI (F). The detection limits for IPP and ApppI are 2 pmol·mg⁻¹ protein and 0.2 pmol·mg⁻¹ protein respectively. No IPP or ApppI were detected in the chromatogram of osteoclast extracts generated from saline injected rabbits. The chromatograms are drawn on the same scale. ApppI, triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester; ESI, electrospray ionization; HPLC, high pressure liquid chromatography; IPP, isopentenyl pyrophosphate; MS, mass spectrometry.

Figure 3

IPP/ApppI concentrations (pmol·mg⁻¹ protein) in each osteoclast fraction from zoledronic acid (ZOL)-treated rabbits. Animals were injected subcutaneously with 100 µg·kg⁻¹ or 1 mg·kg⁻¹ zoledronic acid in PBS. The osteoclasts were isolated by immunomagnetic bead separation 24 or 48 h after injection. The molar amounts of IPP/ApppI in acetonitrile cell extracts were determined by HPLC-ESI-MS. ApppI, triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester; ESI, electrospray ionization; HPLC, high pressure liquid chromatography; IPP, isopentenyl pyrophosphate; MS, mass spectrometry; PBS, phosphate-buffered saline.

Figure 2

Mass spectrometric identification of IPP and ApppI. MS/MS spectra of IPP from the peak of Figure 1C (A) and Figure 1E (B), MS/MS spectra of ApppI from the peak of Figure 1D (C) and Figure 1F (D). m/z = mass-to-charge ratio. ApppI, triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester; IPP, isopentenyl pyrophosphate; MS, mass spectrometry.

Figure 4

Effect of 1-100 µmol·L⁻¹ zoledronic acid (ZOL) on IPP/ApppI formation (A), and the cell viability (B) of confluent MCF-7 cells after 24 h, assessed by mass spectrometry and MTT test respectively. (mean ± SEM, n= 9).***P < 0.001 compared with control. ApppI, triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester; IPP, isopentenyl pyrophosphate; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide.

Figure 5

Zoledronic acid (ZOL)-induced IPP/ApppI formation (A), accumulation of AppCCl₂p (B), the cellular uptake of zoledronic acid (C) and clodronate (CLOD; D), in confluent MCF-7 cells after a pulse exposure to BPs. The MCF-7 cells were treated with 25 µmol·L⁻¹ ZOL or 500 µmol·L⁻¹ CLOD for 1 h, and the samples were collected 0–48 h after drug removal. The intracellular concentration of BPs was determined by comparing the radioactivity of medium, washes and cell extracts relative to amount of protein in the cell extract. The molar amounts of IPP/ApppI or AppCCl₂p were determined in acetonitrile cell extracts by using HPLC-ESI-MS (mean ± SEM, n= 4–8). ApppI, triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester; AppCCl₂p, adenosine 5′(β,γ-dichloromethylene) triphosphate; BP, bisphosphonate; ESI, electrospray ionization; HPLC, high pressure liquid chromatography; IPP, isopentenyl pyrophosphate; MS, mass spectrometry.

Figure 6

Zoledronic acid(ZOL)-induced IPP/ApppI formation (A), accumulation of AppCCl₂p (B), the cellular uptake of zoledronic acid (C) and clodronate (CLOD; D), in confluent MCF-7 cells. The cells were treated with 25 µmol·L⁻¹ ZOL or 500 µmol·L⁻¹ CLOD continuously for 1–48 h. The intracellular concentration of BPs was determined by comparing the radioactivity of medium, washes and cell extracts relative to amount of protein in the cell extract. The molar amount of IPP/ApppI or AppCCl₂p were determined in acetonitrile cell extracts by using HPLC-ESI-MS (mean ± SEM, n= 4–8). ApppI, triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester; AppCCl₂p, adenosine 5′(β,γ-dichloromethylene) triphosphate; BP, bisphosphonate; ESI, electrospray ionization; HPLC, high pressure liquid chromatography; IPP, isopentenyl pyrophosphate; MS, mass spectrometry.