11.11. Novel information on the molecular mechanisms of action of bisphosphonates

Public examination of a doctoral dissertation in the field of Biopharmacy

Doctoral candidate: MSc (Pharm) Johanna Räikkönen

Time and venue: 11.11.2011 at 12 noon, Medistudia ML2, Kuopio Campus

Language of the public examination: English

Language of the doctoral dissertation: English

This study explored the molecular mechanisms of action of bisphosphonates (BPs). BPs are currently among the most used and effective class of drugs developed for the treatment of metabolic bone disorders that are associated with increased bone destruction, such as osteoporosis and tumor-induced osteolysis. Moreover, there is now extensive preclinical evidence available that BPs could have the ability to act directly on tumor cells of various origins. However, the underlying mechanisms are still unclear.

Non-nitrogen-containing BPs (non-N-BPs), such as clodronate, are metabolised into cytotoxic ATP analogs capable of inducing apoptosis in cells, while nitrogen-containing BPs (N-BPs), such as zoledronic acid, act by inhibiting the farnesyl pyrophosphate synthase (FPPS) enzyme of the mevalonate pathway causing a concomitant decrease in the formation of the isoprenoid lipids required for normal cellular function. Moreover, a new possible mechanism of action of N-BPs has been revealed, a finding which may account for the observed effects of N-BPs. N-BPs induce the formation of a novel endogenous ATP analog ApppI as a consequence of the inhibition of the FPPS enzyme, and the subsequent accumulation of isopentenyl pyrophosphate (IPP). ApppI is a pharmacologically active compound capable of directly triggering apoptosis.

This study aimed to characterize the role of the intracellular accumulation of IPP/ApppI in the mechanism of action of N-BPs. The detailed aims were: (1) to investigate BP-induced IPP accumulation and ATP analog formation in various cancer cell lines, (2) to obtain detailed data, such as time-course and dose-dependence, on N-BP-induced IPP/ApppI formation in cancer cells, (3) to explore N-BP-induced ApppI formation in rabbit osteoclasts, in vivo, (4) to identify the specific intermediates and enzymes in the mevalonate pathway involved in regulating the BP-induced IPP/ApppI formation in cancer cells.

This study provided the first conclusive evidence that the pro-apoptotic ApppI is formed in osteoclasts in vivo, the pharmacological target cells for BPs, even after a single clinically relevant dose of N-BP. This result is of considerable importance, as it establishes the biological significance of this molecule. Furthermore, it was shown for the first time, that BPs induce the accumulation of cytotoxic ATP analogs, ApppI and AppCCl2p, in several types of cancer cells. The metabolites accumulated in cells in a time- and dose-dependent manner. However, the accumulation of these metabolites was remarkably cell line dependent suggesting that BPs may have distinct antitumor effects dependent upon the extent of accumulation of cytotoxic compounds within the cells. In addition to cancer cell type, the intracellular amounts of IPP and ApppI were shown to be dependent on the FPPS binding characteristics of the N-BP. N-BP-induced IPP/ApppI levels in cells can also be downregulated by the mevalonate pathway intermediates, isoprenoids. This represents a novel insight into the mechanism of action of isoprenoids on the regulation of the mevalonate pathway after inhibition by N-BPs, and supports the role of ApppI in mediating N-BP-induced apoptosis.

In conclusion, this study provided further evidence that BP-induced IPP and the subsequent ApppI accumulation are important metabolic events underlying the molecular mechanisms of action of BPs. Furthermore, the mass spectrometric IPP/ApppI analysis was demonstrated to be a useful and sensitive tool for investigating the intracellular action of BPs and other mevalonate pathway inhibitor candidates under development. The introduction of novel markers such as IPP and ApppI as surrogates of N-BP efficacy might be also useful both in preclinical and clinical research to better understand the anticancer activities of N-BPs.

The doctoral dissertation of MSc (Pharm) Johanna Räikkönen, entitled Bisphosphonate-induced IPP/Appp1 accumulation in cells will be examined at the Faculty of Health Sciences. The opponent in the public examination will be Professor Graham Russell, University of Sheffield Medical School, and the custos will be Professor Jukka Mönkkönen, University of Eastern Finland. The public examination will be held in English.

Photo available for download at http://uef.fi/vaitoskuvat

For further information, please contact MSc Johanna Räikkönen, johanna.raikkonen@uef.fi, tel. 040-355 3465

Publishing year: 2011

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