Abstract A40: Modification of P2X7-pAkt signaling pathway in long-term treatment with statins
Authors: Miraglia Erica , Högberg Johan , Stenius Ulla 
Published: January 2011

Abstract
The 3-hydroxy-3-methylglutaryl-CoenzymeA reductase inhibitors, statins, are widely used as safe and potent cholesterol lowering drugs in the prevention of cardiovascular diseases. Interestingly, statins exhibit also strong anti-tumor effects in several in vitro and in vivo models, and recent epidemiology indicates that statins reduce the risk of colon, lung, pancreatic and prostatic cancer [1]. However, the molecular mechanisms behind these effects of statins are not clear. We previously demonstrated that statins rapidly activate the purinergic receptor P2X7 and affect Akt signalling, which may have important anti-cancer effects [2]. P2X7 system is an important pro-apoptosis modulator in epithelial cells, and the use of P2X7 agonists has been proposed as a novel pharmacological approach for prevention and treatment of epithelial cancers [3]. The aim of the current study was to investigate the role of the P2X7-Akt axis in the anti-cancer effects of statins, using epithelial cells as a model. We focused on the long-term effects of statins because they mimic the conditions of chemopreventive strategies in vivo. By culturing A549 cells in the presence of stepwise increasing concentrations of atorvastatin, we selected five sub-populations which were resistant to different concentrations of statin (15, 30, 50, 60 and 75 µM). The expression and subcellular localization of proteins involved in the P2X7-pAkt axis were analyzed by Western Blot and Immunostaining methods. Cell proliferation and cell death were evaluated with MTT assay and Trypan Blue exclusion. The functionality of the P2X7 system was assessed with the fluorescent dye Fura-2 as increase of cytosolic free Ca2+ upon agonists (e.g. ATP and BzATP) stimulation.
Our results demonstrate that long-term treatment with statins selected cell sub-populations expressing altered signalling pathways, as compared to parental cells. Selected cells exhibited induced P2X7 expression, increased PTEN levels, enhanced PHLPP2 and an inhibited downstream pAkt signalling. In parallel to the increase of P2X7 levels, atorvastatin-selected cells were more sensitive to agonists of this receptor, in terms of rising cytosolic free Ca2+ and induction of apoptosis. Further, statin-selected cells displayed reduced cell number and increased p53 levels, suggesting DNA-damage stress. Statins significantly inhibited cell proliferation in response to cytostatic drugs. In fact, statin-selected cells were more vulnerable to etoposide- or 5-fluorouracil-elicited DNA-damage, confirming the ability of statins to sensitize cancer cells to cytostatic agents. These results may also indicate that pre-neoplastic cells, which often exhibit “DNA-damage responses,” are more vulnerable to statins via p53 stabilization.
We conclude that long term treatment with high concentrations of statins selects cells exhibiting adaptations in the P2X7-Akt signalling. These adaptations suggest that a toxic effect of statins is mediated by the P2X7-Akt axis, which selectively eliminates pre-neoplastic cells. Our results are highly novel with respect to statins anti-cancer mechanism of action and strongly support their potential use in future chemopreventive designs.
References:
1. Demierre MF, Nat Rev Cancer 2005, 5:930-42
2. Mistafa O, Biochem Biophys Res Commun 2008, 365:131-6
3. Gorodeski GI. Expert Opin Ther Targets 2009, 13:1313-32
Citation Information: Cancer Prev Res 2010;3(12 Suppl):A40.