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


METHODS
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.


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
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.
Department of Pharmaceutics, University of Kuopio, Yliopistonranta 1 B, P.O. Box 1627, Kuopio FIN-70211, Finland. Johanna.Raikkonen@uku.fi
Johanna
J
Räikkönen
Julie C
JC
Crockett
Michael J
MJ
Rogers
Hannu
H
Mönkkönen
Seppo
S
Auriola
Jukka
J
Mönkkönen
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D050071:Bone Density Conservation Agents / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004002:Clodronic Acid / Q000378:metabolism; D004164:Diphosphonates / Q000008:administration & dosage / Q000494:pharmacology*; D045782:Hemiterpenes / Q000096:biosynthesis*; D006801:Humans; D007093:Imidazoles / Q000008:administration & dosage / Q000494:pharmacology*; D066298:In Vitro Techniques; D009943:Organophosphorus Compounds; D010010:Osteoclasts / Q000187:drug effects* / Q000378:metabolism; D011817:Rabbits; D000077211:Zoledronic Acid
D050071:Bone Density Conservation Agents; D004164:Diphosphonates; D045782:Hemiterpenes; D007093:Imidazoles; D009943:Organophosphorus Compounds; C543712:triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester; D004002:Clodronic Acid; C004809:isopentenyl pyrophosphate; D000077211:Zoledronic Acid; C056864:adenosine-5'-O-(beta,gamma-dichloromethane)triphosphate; D000255:Adenosine Triphosphate