Statin-induced cellular effects mediated by the P2X7 receptor

Detta är en avhandling från Stockholm : Karolinska Institutet, Institute of Enviromental Medicine

Sammanfattning: Cholesterol-lowering statins have been shown to inhibit growth of different cancer cells both in vitro and in vivo. Epidemiological studies also indicate a chemopreventive effect of statins. We have investigated the effect of statins on Akt/protein kinase B signaling and their sensitizing effect of cytostatic drugs. It was found that insulin- and cytostatic drug-induced Akt phosphorylations and nuclear translocation was inhibited by pravastatin and atorvastatin in HepG2, A549 and H1299 cells in an mTOR dependent manner. Statins also induced mTOR dependent phosphorylations of insulin receptor substrate 1 (IRS-1). In p53 wild-type cells (HepG2 and A549) pretreatment with statins did not sensitized cells to etoposide in concentrations which induced p53 stabilization. In line with our previous data, statins were found to attenuate the etoposide-induced p53 response. We also show that in a p53-deficient cell line (H1299) pretreatment with atorvastatin sensitized cells to etoposide, doxorubicin and 5-fluorouracil and increased the level of apoptosis. Taken together we clearly demonstrate, that an mTOR dependent, statin-induced inhibition of Akt phosphorylation and nuclear localization sensitize cells to cytostatic drugs. However, this effect can be counteracted in p53 competent cells by the ability of statins to destabilize p53. Further we address the question of possible anti-cancer effects of statins. We tested the possibility that statin-induced effects on nuclear pAkt are mediated by the P2X7 receptor. We show that low concentration of atorvastatin decreased the level of insulin-induced phosphorylated Akt in the nucleus. This effect was seen within minutes and was inhibited by P2X7 inhibitors. Our results reveal that statins via the P2X7 receptor modulate insulin-induced Akt signaling in epithelial cells. Furthermore, our data indicate that P2X7 regulate nuclear pAkt in epithelial cells. We also invastigated the effect of statins in pancreatic cancer cell lines. We found that atorvastatin decreased constitutive- and insulin-induced pAkt in Panc-1 and MIA PaCa-2 cells. Statins also inhibited pAkt in synergy with gemcitabine- and 5 fluorouracil, and sensitized cells to apoptosis and inhibited cell proliferation. It was also found that the P2X7-purinergic receptor mediated the effects of statins in Panc-1 and MIA PaCa-2 cells. Interestingly, in Capan-2 cells, which expressed P2X7 in low levels, statins did not reduce pAkt levels nor did statins sensitize them to cytostatic drugs. However, statin inhibited the growth of Capan-2 cells and this correlated to inhibition of NFkB and Raf/MEK pathways. These effect might be explained by an inhibited protein prenylation. Finally, we investigated the mechanism underlying the rapid inhibition of nuclear Akt. A role of PTEN as a negative regulator was suggested. Simultaneous activation of PTEN, PHLPP1 and 2, and calcineurin was detected in the time frame where pAkt is inhibited and abolished from nucleus. A model for how PTEN and PHLPP control Akt signaling has been suggests: PTEN prevents activation by removing the second messenger, PIP3, that activates Akt, and PHLPP inactivates Akt by direct dephosphorylation. Our data indicate, for the first time, that PTEN and PHLPP were activated simultaneously and bound the same substrate. Apparently both phosphatases participated in the down-regulation of nuclear pAkt in a rapid and coordinated action. Overall, these studies yield a novel mechanism by which Akt acts upon statin stimulation.

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