Elsevier

Biochemical Pharmacology

Volume 78, Issue 9, 1 November 2009, Pages 1115-1126
Biochemical Pharmacology

Statins inhibit Akt/PKB signaling via P2X7 receptor in pancreatic cancer cells

https://doi.org/10.1016/j.bcp.2009.06.016Get rights and content

Abstract

Cholesterol-lowering statins have been shown to inhibit growth of pancreatic cancer cells 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. We found that atorvastatin decreased constitutive- and insulin-induced pAkt in Panc-1 and MIA PaCa-2 cells. Statins also inhibited pAkt in combination with gemcitabine- and 5-fluorouracil, and sensitized cells to gemcitabine- and 5-fluorouracil-induced apoptosis and inhibition of cell proliferation. In line with our previous data, it was found that the P2X7-purinergic receptor mediated the effects of statins in Panc-1 and MIA PaCa-2 cells. Thus, experiments employing P2X7 siRNA and inhibitors supported an involvement of P2X7. 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 NFκB and Raf/MEK pathways. As shown previously, these latter effects can be explained by an inhibited protein prenylation. Our data suggest that statins primarily target a functional P2X7-Akt signaling in pancreatic cancer cells. By targeting the P2X7-Akt axis, statins can sensitize pancreatic cancer cells to chemotherapeutic drugs. Our data are also in line with a role for P2X7 in the chemopreventive effect of statins on pancreatic cancer.

Graphical abstract

Summary of statin-induced effects in pancreatic cancer cell lines.

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Introduction

Pancreatic cancer is resistant to conventional radio- and chemotherapy and is associated with poor prognosis. Improvement of therapies targeting specific signaling pathways in this tumor type is thus urgently needed. 3-Hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, statins, have anticancer effects in different in vitro and in vivo models [1], [2]. Several studies show that statins inhibit growth of pancreatic cancer cell lines in vitro and sensitize them for cytostatic drugs [3], [4], [5], [6]. Gemcitabine, which is presently used for the treatment of pancreatic cancer, is one of these drugs. Thus, fluvastatin was shown to enhance the antiproliferatory effect of gemcitabine in MIA PaCa-2 pancreatic cancer cells [5]. Besides their in vitro effects, statins have been shown to inhibit pancreatic tumor growth in vivo[3], [5], [7]. In a recent study performed on nude mice it was shown that different statins decreased the tumor growth of transplanted Capan-2 cells [7]. This effect was explained by an inhibited cholesterol synthesis and an inhibited prenylation of signaling proteins such as Ras.

Statin-induced anticancer effects on pancreatic cancer progression are supported by epidemiological data [6], [8], [9], [10]. A recent study indicate a dramatic (<50%) risk reduction in metastatic or fatal prostate cancer among statin users [9]. An even stronger effect was registered in a case-control study, involving about half a million veterans. In this study it was found that four years on statins reduced the risk of pancreatic cancer by 80% [10]. Together these data indicate that statins strongly affect pancreatic cancer cells and preneoplastic cells in pancreas. However, mechanisms for these anticancer effects are still not well characterized.

There are three major signaling pathways commonly altered in pancreatic cancers [11]. These include phosphatidylinositol 3-kinase- (PI3K/Akt), nuclear factor kappa-B- (NF-κB), and mitogen-activated protein kinase (MAPK) pathways [12], [13], [14]. We have previously shown that statins inhibit one of these pathways, PI3K/Akt signaling, in different cell lines [15], [16]. Thus, incubation with statins decrease the levels of phosphorylated Akt in the cytoplasm and the nucleus and sensitized HepG2 and A549 cells to cytostatic drugs [16].

The Akt pathway is one of the major anti-apoptotic factors in cells. Akt is activated by growth factors and cellular stress and is commonly overexpressed in pancreatic cancer, but is also often induced by cytostatic treatment [12], [17], [18]. In addition, PI3K/Akt has been implicated in the resistance of pancreatic cancer to gemcitabine [19]. Therefore, Akt is an attractive target for cancer therapy and in particular for pancreatic cancer.

Our recent data show that the statin-induced inhibition of nuclear pAkt is mediated via the P2X7-purinergic receptor in A549 lung cancer cells. Furthermore, we found evidence that P2X7 receptors, when activated by its natural ligand ATP, can regulate nuclear pAkt in epithelial cells [20]. P2X7 is expressed in pancreatic cells and is activated by extracellular purinergic nucleotides such as ATP. P2X7 receptor activation might stimulate pleiotropic cellular effects including the release of pro and inhibitory inflammatory cytokines [21], [22]. Increased levels of P2X7 were recently detected in chronic pancreatitis and pancreas cancer indicating a possible involvement of P2X7 receptors in pancreatic cancer development [23].

In the present study, we have investigated the effects of atorvastatin on pancreatic cancer cell lines. We report that pharmacologically relevant concentrations of statins decrease the levels of pAkt in Panc-1 and MIA PaCa-2 cells and sensitize them to gemcitabine and 5-Fu. We also report that these effects are mediated by P2X7. On the other hand, in Capan-2 cells, with low expression of P2X7, statins did not affect pAkt levels and did not sensitize them to cytostatic drugs.

Section snippets

Cell culture

Human pancreatic cell lines, Panc-1, Capan-2 and MIA PaCa-2, were obtained from the American Type Culture Collection, ATCC (Manassas, VA, USA). Capan-2 is well- to moderately well-differentiated ductal adenocarcinoma cell line expressing wt p53, while MIA PaCa-2 and Panc-1 are poorly differentiated ductal adenocarcinoma cell lines with mutant p53. Panc-1 and MIA PaCa-2 cells were maintained in Dulbecco's modified Eagle's medium (ATCC) while Capan-2 cells were maintained in McCoy's 5A Modified

Statins decrease pAkt levels and inhibit cell proliferation in Panc-1 cells

The PI3K/Akt pathway has been implicated in the resistance of pancreatic cancer to cytostatic drugs [19] and we have recently shown that statins readily inhibit Akt in cancer cells [16]. Therefore, the effect of atorvastatin on the constitutive level of phosphorylated Akt (pAkt) was examined in Panc-1 cells. We found that incubation of cells with atorvastatin (1 μM for 1 or 24 h) decreased a high constitutive level of Akt phosphorylation at residue Ser473 (pAkt Ser473) (Fig. 1A and B). This

Discussion

Our data show that statins in pharmacologically relevant concentrations inhibit phosphorylation of Akt in Panc-1 and MIA PaCa-2 pancreatic cancer cells. This effect was associated with inhibition of cell proliferation and induction of apoptosis. In addition, statin increased the effect of cytostatic drugs commonly used to treat pancreatic cancer. We also present data indicating that these effects are mediated by the P2X7 receptor.

The Akt pathway is commonly altered in pancreatic cancer [12]. We

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