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

Abstract

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.

Figure 1

Mevalonate pathway. The compounds (italicized) used in this study and the affected enzymes are indicated in the boxes. Biosynthesis of ApppI results from inhibition of FPP synthase by N-BPs and consequent accumulation of IPP.

Figure 2

Identification of ApppI as an endogenous metabolic product in osteoclasts treated with zoledronic acid. Before to culture, bovine bone slices were coated with N-BP by rinsing with 2 mM free zoledronic acid for 1 min. Control bone slices were rinsed with 0.9% saline. Selective reaction monitoring chromatograms of the extract from untreated cells (a), extract from osteoclasts treated with zoledronic acid for 24 h (b), untreated cell extract spiked with 0.2 μM ApppI (c), MS/MS spectrum from the peak of Figure 2b (d), MS/MS spectrum from the peak of Figure 2c (e). The chromatograms are drawn on the same scale.

Figure 3

Mass spectrometric identification of ApppI. Full scan negative-ion ESI mass spectrum (a) and MS/MS spectrum of ApppI (b).

Figure 4

The efficiency of ApppI production with different bisphosphonates. J774 macrophage cells were treated with 30 μM free bisphosphonate for 24 h. The molar amount of ApppI was determined in cell extracts by using HPLC-ESI-MS as described under Methods. clod=clodronate; alend=alendronate; iband=ibandronate; rised=risedronate; zoled=zoledronic acid. The columns and error bars represent means±s.d., n=6.

Figure 5

Zoledronic acid and risedronate dependence of ApppI production. J774 macrophage cells were treated with 10–100 μM free zoledronic acid (a) or risedronate (b) for 24 h. ApppI analysis was performed as in Figure 4. The values represent means±s.d., n=4–6. R=0.94 for zoledronic acid and R=0.89 for risedronate.

Figure 6

The effect of lovastatin on the accumulation of IPP and the production of ApppI induced by free zoledronic acid after 24 h treatment in J774 macrophages (a), IPP accumulation correlates with the ApppI production (b). The molar amounts of IPP and ApppI were determined in cell extracts by using HPLC-ESI-MS as described under Methods. Mean±s.d., n=7–9. ^***P<0.001 compared with controls.

Figure 7

The metabolism of non-N-BP to AppCp-metabolite and the formation of ApppI induced by N-BPs is catalyzed by aminoacyl-tRNA synthetases.

Figure 8

Effect of clodronate on the ApppI production evoked by risedronate (a), and risedronate on the metabolism of clodronate to AppCCl₂p (b) in J774 macrophages. BPs were in free form. The molar amounts of ApppI and AppCCl₂p were determined in cell extracts by using HPLC-ESI-MS as described under Methods. Mean±s.d., n=4–6. ^*P<0.05 compared with controls.

Figure 9

ApppI dose dependently inhibits ADP/ATP translocation in rat liver mitochondria. Reaction time 60 s and ¹⁴C-ATP concentration 150 μM. The ADP/ATP translocator activity was assayed in the ‘forward' direction as described by Paulson & Shug (1984). The symbols and error bars represent means±s.d., n=2.

Figure 10

Induction of apoptosis in osteoclasts by zoledronic acid (ZOL), ApppI, clodronate (CLOD) or AppCCl₂p. The cells were cultured on bone slices for 24 h in the presence of nontreated liposomes (CTR), 0.1 μM to 1 μM liposome-encapsulated ZOL, ApppI, AppCCl₂p or 1 μM to 10 μM liposome-encapsulated CLOD. The number of apoptotic osteoclasts was determined as described under Methods. Mean±s.e.m., n=6. ^***P<0.001 compared with controls.