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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Int J Cancer. Author manuscript; available in PMC 2013 October 15.
Published in final edited form as:
PMCID: PMC3376252
NIHMSID: NIHMS361919

Atorvastatin delays progression of pancreatic lesions to carcinoma by regulating PI3/AKT signaling in p48Cre/+.LSL-KrasG12D/+ mice

Abstract

Pancreatic cancer is the one of most common causes of cancer deaths and has the worst prognosis. Clinical observational studies suggest that statins may reduce the risk of pancreatic cancer. The chemopreventive efficacy of the statin atorvastatin (Lipitor®) and the role of the phosphatidyl-inositol 3-kinase(PI3/AKT) signaling pathway were evaluated for the progression of pancreatic intraepithelial neoplasms (PanINs) to pancreatic ductal adenocarcinoma (PDAC) in conditional p48Cre/+-LSL-KrasG12D/+ transgenic mice. Six-week old male p48Cre/+-LSL-KrasG12D/+ (20/group) mice were fed AIN-76A diets containing 0, 200, and 400 ppm atorvastatin for 35 weeks. At termination, pancreata were evaluated histopathologically for PanINs and PDAC, and for various PI3/AKT signaling markers, and inflammatory cytokines, by immunohistochemistry/immunohistoflourscence, ELISA, Western blotting and/or Reverse Transcription-PCR methods. Control diet-fed mice showed 85% incidence of PDAC; whereas, mice fed with atorvastatin showed PDAC incidence of 65 and 35% respectively (p<0.0001). Similarly, significant suppression of PanIN-3 (22.6%) was observed in mice fed 400 ppm atorvastatin. Importantly, pancreata from atorvastatin-treated mice were ~68% free from ductal lesions. Furthermore, pancreas of mice administered with atorvastatin had significantly reduced expressions levels of PCNA, p2X7, p-ERK, RhoA, cyclin D1, survivin, Akt, pAKT, β-catenin, cyclin E, cdK2, and caveolin-1. Also, atorvastatin-treated mice had shown dose-dependent suppression of inflammatory cytokines and a significant increase in tunnel-positive cells, p21 and PARP expression levels in pancreas. Atorvastatin significantly delays the progression of PanIN-1 and -2 lesions to PanIN-3 and PDAC by modulating PI3/AKT signal molecules in a preclinical model, suggesting potential clinical benefits of statins for high risk pancreatic cancer patients.

Keywords: PI3/AKT, Atorvastatin, Pancreatic cancer, p48Cre/+-LSL- KrasG12D/+ mice

Introduction

Pancreatic ductal adenocarcinoma (PDAC), a neoplastic disease with a 5 year survival rate of less than 5%, still remains one of the worst cancers. In the US alone, it is estimated that 44,030 individuals (22,050 men and 21,980 women) will be diagnosed with and 37,660 of them (19,360 men and 18,300 women) will die of cancer of the pancreas in 2011 (1). It is the fourth leading cause of cancer related death in USA, characterized by low responsiveness to conventional chemotherapies. Despite widespread knowledge and advances in the field of molecular genetics in human pancreatic cancers over the past 60 years, the identification of putative molecular targets and the development of targeted therapies have not yet translated to improved rate of overall patient survival (2). PDAC is generally believed to arise predominantly through progression of pancreatic intraepithelial neoplasia (PanIN), ranging from low-grade PanINs (termed PanIN-1A, -1B) to high-grade PanINs (termed PanIN-2, -3), to ductal adenocarcinoma. The preclinical study of PanINs has recently been made possible by the generation of genetically modified animal models, which recapitulate human PanINs on genetic and histomorphologic levels (3).

More than 95% of pancreatic cancers are characterized by K-ras mutations, the most frequent and earliest genetic event at early stages, and the accumulations of multiple additional genetic abnormalities over time (4,5). Mice harboring the conditional K-ras mutant allele (LSL-KrasG12D/+) in combination with a pancreas-specific Cre recombinase transgene (p48Cre/+) develop a full range of premalignant lesions in the pancreas, termed pancreatic intraepithelial neoplasia, before succumbing to invasive PDAC and other tumors at late ages (6,7). These mice are an excellent model that mimics human pancreatic cancer development and they are useful for understanding pathobiology and development of potential chemopreventive and therapeutic agents to suppress the progression of PanINs to PADC (6).

Statins are small molecule inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which are used widely as cholesterol lowering drugs (8). Epidemiological and experimental data supports that frequent statin use may reduce the risk of many epithelial cancers including pancreatic cancer (912). Platz et al. (11) showed a significant (<50%) risk reduction in metastatic or fatal prostate cancer among statin users. An even stronger effect was registered in a cross-sectional case-controlled study retrospectively analyzed from prospectively collected data 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% (12). During the past 10 years, the antiproliferative effects of statins were demonstrated both in in-vitro as well as in vivo studies on various cancers including hepatocellular carcinoma, lung, colorectal and pancreatic cancer (9, 1318). Also, evidence shows that statins inhibit growth of pancreatic cancer cells and sensitize them to cytostatic drugs like Gemcitabine (9, 1922). Besides their in vitro effects, statins have been shown to inhibit pancreatic tumor growth in-vivo (19,20,23).

Mutation in Kras leads to constitutive activation with subsequent stimulation of downstream signal transduction pathways. The phosphatidyl inositol 3-kinase (PI3/AKT) pathway has been implicated as one of the major targets for Kras activation and PI3/AKT has been shown to regulate cell survival, apoptosis, angiogenesis, metabolism, protein synthesis and proliferation (24). Several lines of evidence have pointed to the importance of this pathway and its downstream signaling elements in PDAC. Inhibition of PI3-K prevents ras-induced cell transformation, supporting the importance of PI3/AKT pathway as a downstream effector of the survival signal of kras activation (25). Therefore it is hypothesized that strategies leading to inactivation of PI3/AKT signaling would represent a promising approach for the prevention and treatment of pancreatic cancer.

Although several laboratory and observational studies have demonstrated potential anticancer effects of statins against different types of cancers (912), the potential chemopreventive properties and molecular mechanisms of atorvastatin action against pancreatic cancer have not been fully established using mouse models that develop PDAC in a stepwise manner similar to humans. Thus, in this present study, we evaluate the effects of atorvastatin on progression of PanINs to PDAC and assess the importance of PI3-/AKT pathway on expression of biomarkers that would be modified during progression of PanIN lesions to PDAC in a conditional p48Cre/+-LSL-KrasG12D/+ mouse model. We found that atorvastatin significantly inhibited PDAC development and, at least in part, regulated PI3/AKT pathway signals.

Materials and Methods

Animals, diets and care

All animal experiments were done in accordance with the institutional guidelines of the American Council of Animal Care. Breeder pairs of LSL-K-RASG12D/+ and p48Cre/+ in the C57BL/6 genetic background were obtained from Dr. Howard Crawford at the University of New York at Stony Brook, NY. Required quantities of activated KrasG12D/+ mice were generated as described below. Animals were housed in ventilated cages under standardized conditions (21°C, 60% humidity, 12-h light/12-dark cycle, 20 air changes/hour) in the University of Oklahoma Health Sciences Center rodent barrier facility. Semi-purified modified AIN-76A diet ingredients were purchased from Bioserv, Inc., NJ. Atorvastatin (Fig. 1A) was procured from the NCI DCP chemoprevention drug repository (Bethesda, MD). Atorvastatin (200 and 400 ppm) was premixed with small quantities of casein and then blended into the diet using a Hobart Mixer. Both control and experimental diets were prepared weekly and stored in the cold room. Mice were allowed ad libitum access to the respective diets and to automated tap water purified by reverse osmosis.

Figure 1
A. Structure of the statin atorvastatin/lipitor, B. Genotyping of p48Cre/+ and LSL-KrasG12D/+ offspring by PCR. C. Experimental design for chemopreventive efficacy evaluation of Atorvastatin in male p48Cre/+-LSL-K-RasG12D/+ mice. At 6-weeks of age, groups ...

Breeding and Genotyping analysis

LSL-KrasG12D/+ and p48Cre/+ mice were maintained in a C57BL/6 heterozygous genetic background. LSL-KrasG12D/+ and p48Cre/+ mice were bred and the offspring of male activated KrasG12D/+ (25%) were generated at required quantities. The genotype of each pup was confirmed by tail DNA extraction and PCR as described elsewhere (6). Briefly, genomic DNA was extracted from snap-frozen tail tissue samples using the mini-prep kit (Invitrogen). PCR was performed for K-ras and Cre genes using the following conditions: denaturation at 95°C for 5 minutes, followed by 35 cycles at 95°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. Oligonucleotide primer sequences used were as follows: K-ras 5'-CCTTTACAAGCGCACGCAGAG-3' sense, 5'-AGCTAGCCACCATGGCTTGAGTAAGTCTGCA-3' anti-sense; and Cre 5'-ACCGTCAGTACGTGAGATATCTT-3' sense and 5'-ACCTGAAGATGTTCGCGATTATCT-3' antisense. PCR products were separated on a 2 % agarose gel. Successful recombination yields are 550 and 210-bp products (Fig. 1B).

Bioassay: Chemopreventive Efficacy of Atorvastatin

Genotyped male p48Cre/+-LSL-KrasG12D/+ transgenic mice were used in the efficacy study. The experimental protocol is summarized in Fig. 1C. Five week-old mice were selected and randomized so that average body weights in each group were equal (n=20/group p48Cre/+-LSL-KrasG12D/+ mice and n=12 C57BL/6 wild-type mice) and were fed with AIN-76A diet for one week. At 6 weeks of age, mice were fed with control or experimental diets containing 0 ppm, 200 ppm or 400 ppm atorvastatin in the diet until termination of the study. Mice were routinely checked for signs of weight loss, any signs of toxicity or any abnormalities. Food intake and body weight of each animal were measured once weekly for the first 6 weeks and then once a month till termination. After 35 weeks (~9 months) on experimental diets, all mice were euthanized by CO2 asphyxiation and necropsied; pancreata were collected from all groups (Fig. 1D–F), weighed and snap frozen in liquid nitrogen for further analysis. Pancreata (head to tail) required for histopathologic and IHC evaluations, to identify PanIN lesions and PDAC for evaluation of various molecular markers were fixed in 10% neutral-buffered formalin.

Histologic evaluation

Formalin-fixed, paraffin-embedded tissues were sectioned (4 µm) and stained with H&E (Hematoxylin & Eosin). Twenty sections of each pancreas were histologically evaluated by a pathologist blinded to the experimental groups. PanIN lesions and carcinoma were classified according to histopathologic criteria as recommended elsewhere (3). To quantify the progression of PanIN lesions, the total number of ductal lesions and their grade were determined. Pancreatic ducts of the entire fixed specimen (head, body, and tail of the pancreatic sections) were analyzed for each animal. The relative proportion of each PanIN lesion grade to the overall number of analyzed ducts was recorded for each animal. Similarly, pancreatic carcinoma and normal appearing pancreatic tissue were evaluated for all the animals.

HMG-CoA reductase assay

Pancreata harvested from mice fed with or without atorvastatin were homogenized in ice cold buffer [100 mM sucrose, 50 mM KCl, 5 mM MDTT, 30 mM EDTA pH 7.4]. HMG-COA reductase assay was measured in duplicates for 6–8 samples per treatment group by previously published method using conversion of [14C]mevalonate to [14C]-mevalonolactone (18). Results are expressed as pmoles 14C incorporated into mevalonolactone per min per mg of total protein.

Immuno-histochemistry/Immuno-histoflourescence

The effects of atorvastatin on expression of proliferating cell nuclear antigen (PCNA), β-catenin and p21 were evaluated by immunohistochemistry (IHC) or Immunohistoflourescence (IHF). Briefly, paraffin sections were deparaffinized in xylene, rehydrated through graded ethanol solutions and washed in phosphate-buffered saline (PBS). Antigen retrieval was carried out by heating sections in 0.01 M citrate buffer (pH 6) for 30 minutes in a boiling water bath. Endogenous peroxidase activity was quenched by incubation in 3% H2O2 in PBS for 5 minutes. Nonspecific binding sites were blocked using Protein Block for 20 minutes. Sections were then incubated overnight at 4°C with 1:300 dilutions of monoclonal antibodies against PCNA, β-catenin and p21 (AbCam/Santa Cruz Biotechnology, CA). After several washes with PBS, the slides were incubated with appropriate secondary antibody for two hours and then washed and incubated with avidin-biotin complex reagent (Zymed laboratories). After rinsing with PBS, the slides were incubated with the chromogen 3, 3”-diaminobenzidine (DAB) for three minutes, then rinsed and counterstained with hematoxylin. Non-immune rabbit immunoglobulins were substituted for primary antibodies as negative controls. Slides were observed under an Olympus microscope 1X701 and digital computer images were recorded with an Olympus DP70 camera. For immunohistofluorscence (IHF), after overnight incubation with primary antibody, the slides were washed thrice with PBS for 5 min and then were incubated with secondary antibody tagged with FITC/TRITC in the dark for one hour. Slides were then washed with PBS for 5 minutes thrice in the dark room and incubated with 0.5 µg/mL DAPI for 5 min. Slides were rinsed with PBS and observed for fluorescence under FITC/ TRITC filters using an Olympus microscope IX701 and digital computer images were recorded with an Olympus DP70 camera.

Apoptosis assay

Paraffin sections of 5µ thickness mounted on slides were rehydrated, and stained using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. Briefly, slides were incubated with 3% H2O2 in PBS for 5 min, rinsed, and then incubated in TdT buffer [140 mmol/L cacodylate (pH 7.2), 30 mmol/L Tris-HCl, 1 mmol/L CoCl2] for 15 min at room temperature. TdT reaction mixture [0.2 unit/AL TdT, 2 nmol/L biotin-11-dUTP, 100 mmol/L cacohydrate, 2.5 mmol/L CoCl2, 0.1 mmol/L dithiothreitol (DTT), and 0.05 mg/mL bovine serum albumin (BSA)] was added, and the slides were incubated for an additional 30 minutes at 37 °C. After blocking with 2% BSA and incubation with avidin-biotin peroxidase complexes, the TUNEL reaction was visualized by chromogenic staining with DAB, and slides were counterstained with malachite green. Stained apoptotic epithelial cells (a minimum of 10 microscopic fields per section) were counted manually in a single-blind fashion.

Western-blot analysis of protein expression

Pancreata harvested from mice fed with or without atorvastatin were homogenized and lysed in ice cold lysis buffer (Sigma). After a brief vortexing, the lysates were separated by centrifugation at 12,000 × g for 15 minutes at 4°C, and protein concentrations were measured with the Bio-Rad Protein Assay reagent (Hercules, CA). An aliquot (50 µg protein/lane) of the total protein was separated with 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% milk powder, membranes were probed for expression of caveolin-1 (Cav-1), RhoA, PCNA, cdK2, survivin, p21, polyp(ADP-ribose) polymerase (PARP) cyclin E, AKT, pAKT, ERK, pERK, b-actin and in hybridizing solution [1:500, in TBS-Tween 20 solution] using respective primary antibodies (cell signaling/AbCam/Santa Cruz Biotechnology) and then probed with their respective HRP-conjugated secondary antibodies. Detection was performed using the SuperSignal® West Pico Chemiluminescence procedure (Pierce, Rockford, IL). The bands were captured on Ewen Parker Blue sensitive X-ray films and quantified by densitometry.

Reverse Transcription-PCR for AKT-1, P2X7, p21, β-catenin, Cyclin D1, and COX-2 (cyclo-oxygenase-2) mRNA expression

Total RNA from pancreas samples was extracted using the Totally RNA™ Kit (Ambion) as per the manufacturer's instructions. Equal quantities of DNA-free RNA were used in reverse transcription reactions for making cDNA using SuperScript™ reverse transcriptase (Invitrogen). PCR reactions were performed for AKT-1, P2X7, Cyclin D1, β-catenin, p21 and COX-2 using the following conditions. For AKT-1, denaturation at 94°C for 3 minutes was followed by 35 cycles at 94°C for 45 seconds, 60°C for 25 seconds, and 72°C for 1 minute. Oligonucleotide primer sequences used for AKT-1 were: 5'-AGCAAACAGGCTCACAGGTT-3' sense, 5'-TAAGTCCTCCCCATGTCCCT3' anti-sense. For p2X7, denaturation at 94°C for 3 minutes, was followed by 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 68°C for 1 minute. Oligonucleotide primer sequences used for p2X7 were as follows: 5'-TCTTCCGACTAGGGGACATCT-3' sense, 5'-ATGGGACCAGCTGTCTAGGTT-3' anti-sense. For β-catenin, denaturation at 94°C for 3 minutes was followed by 35 cycles at 94°C for 30 seconds, 60°C for 20 seconds, and 72°C for 45 seconds. Oligonucleotide primer sequences used for the p21 gene were as follows: 5’-CGTCAGTGCAGGAGGCCGAG-3’ sense, 5’-TCCTCAGGGTTGCCCTTGCCA-3’ antisense. For p21, denaturation at 94°C for 3 minutes was followed by 35 cycles at 94°C for 30 seconds, 60°C for 20 seconds, and 72°C for 45 seconds. Oligonucleotide primer sequences used for p21 were as follows: 5'-TCCTGGTGATGTCCGACCTG-3' sense, 5'-TCCGTTTTCGGCCCTGAG-3' anti-sense. For Cyclin D1, denaturation at 94°C for 3 minutes was followed by 35 cycles at 94°C for 30 seconds, 60°C for 20 seconds, and 72°C for 45 seconds. Oligonucleotide primer sequences used for the cyclin D1 gene were as follows: 5'-ATGGAACACCAGCTCCTGTG-3' sense, 5'-ACCTCCAGCATCCAGGTGGC-3' anti-sense. For COX-2, denaturation at 94°C for 2 minutes was followed by 35 cycles at 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute. Oligonucleotide primer sequences used for COX-2 were as follows: 5'-CCTGTGCCTGATGATTGC-3' sense, 5'-CGGTGAAACTCTGGCTAG-3' anti-sense. PCR was done using the Taq polymerase, 10 mM dNTP, and buffers from Invitrogen. The PCR products were visualized and photographed under UV illumination.

Serum Cytokines

Blood samples were collected at the time of sacrifice to measure the serum concentrations of IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, TNF-ά, and GM-CSF. In addition, serum triglycerides were determined in the non-hemolysed serum with InfinityTM Triglycerides liquid stable reagent (Thermo scientific) as per the manufacturer’s instructions. Determination of inflammatory cytokine levels in serum from KrasG12D/+ mice fed with or without atorvastatin were evaluated by Multi-Analyte ELISA (SA Biosciences) as per manufacturer’s instruction. Results are expressed as ng/mL of serum. Determination was carried out in triplicates from each sample.

Statistical analysis

The data are presented as means ± SE. Differences in body weights were analyzed by ANOVA. Statistical differences between control and treated groups were evaluated using Fisher’s exact test for PDAC incidence and unpaired t-test with Welch’s correction was used for PanINs and PDAC lesions. Differences between groups are considered significant at p<0.05.

Results

General observations

KrasG12D/+ mice fed the control and experimental diets had steady body weight gains. At the end of the experiment, wild-type and 400 ppm atorvastatin-fed KrasG12D/+ mice had slightly higher body weight gains (p<0.05) in comparison with the KrasG12D/+ mice on control diet. No significant body weight change differences were observed between the treatment groups and the control group during the course of the study (Fig 2 A). None of the animals fed the experimental diets exhibited any observable toxicity or any gross changes attributable to liver, kidney, or lung toxicity despite notable difference in the pancreatic weights as described below. We observed high PDAC incidence rates in our study compared to previous studies (7).

Figure 2
A. Effect of atorvastatin on the body weight gain (Means ± SE, N=20 or 12). No significant differences were observed between the KrasG12D/+ vs. wild-type (WT) mice treated with atorvastatin. B. Pancreas weights (Means ± SE, N=20 or 12) ...

Dietary administration of atorvastatin significantly inhibited PDAC formation and delayed the progression of PanIN lesions to PDAC in KrasG12D/+ mice

Pancreas from C57BL/6 wild-type mice fed control diet weighed about 0.28 (0.26–0.30) gms and did not differ significantly from pancreas of wild-type mice fed with atorvastatin diets (Fig. 1D–F, ,2B).2B). However, the pancreas from control diet-fed KrasG12D/+ mice weighed 1.25 (0.9–1.4) gms, almost 5-fold more than the pancreas from wild-type mice. A significant decrease in pancreas weights (>50%, p<0.007) was observed in KrasG12D/+ mice fed with the atorvastatin diet (Fig 2B). Also, there was mortality rate of ~15% (3 of 20 mice) in control group and ~5% (1 of 20 mice) from the treatment groups. KrasG12D/+ mice (control and treated) spontaneously develop pancreatic cancer arising from progression of PanINs through low-grade PanINs (1A and 1B) to high-grade PanINs (PanIN-2, -3) (Fig 2C–H). C57BL/6 wild-type mice fed with control diet or experimental diets containing atorvastatin showed no evidence of PanIN lesions or carcinoma (data not shown).

The efficacy endpoints used in this study were inhibition of PanINs and PDAC. The criterion for invasive disease incidence is groups of adenocarcinoma cells atleast 3% invading with adenomatous stroma streaming around the cells. Many of the invasive groups also invaded muscles with few animals showing tumors in lymph nodes and liver showing signs of metastases. Clinical signs of cancer progression (icterus, hemorrhagic ascites, cachexia etc.) were not noticeable at 40 weeks of disease progression in these mice. Fig 3A summarizes the efficacy of atorvastatin on PDAC incidence in KrasG12D/+ mice. Mice that were fed control diet had an incidence of 85% (percentage of mice with PDAC); whereas 200 ppm atorvastatin-fed mice showed an incidence of 65% PDAC (p=0.2, NS), while 400 ppm atorvastatin-fed mice had an incidence of 35% carcinoma as determined via histological analysis (p<0.003). Control diet-fed mice showed about 49% of the pancreas involved with invasiveness of ductal carcinoma, whereas atorvastatin-fed mice showed only 5 and 2 % invasive ductal carcinoma, with 200 and 400 ppm respectively (Fig 3B). Also, control diet-fed KrasG12D/+ mice developed, on the average, about 62 PanIN1, 51 PanIN2 and 146 PanIN3 lesions, whereas dietary atorvastatin at 200 ppm for 35 weeks caused a significant increase in PanIN 1, 2 and 3 lesions (68, 141, 227) respectively; and 400 ppm atorvastatin caused inhibition of PanIN 3 (113) lesions with a significant increase in PanIN1 and 2 (247 and 130) lesions (Fig 3C). Furthermore, mice fed 200 or 400 ppm atorvastatin exhibited 36 and 68%, respectively, normal appearing pancreatic tissue (free from the PanINs and PDAC) in comparison to control group (Fig 3D, 2G &H).

Figure 3
A. Effect of atorvastatin on the incidence (percentage of mice with carcinomas) of pancreatic ductal adenocarcinoma. B. Percentage of carcinoma spread per pancreas. C. Effect of atorvastatin on the PanINs multiplicity (Means ± SE). D. Percentage ...

Effect of atorvastatin on HMG-CoA reductase activity

Atorvastatin is a known HMG-CoA reductase inhibitor and some of its modes of action are shown by inhibition of HMG-CoA reductase activity. Radiometric HPLC analysis of pancreatic samples showed a decreased HMG-CoA reductase activity in a dose-dependent manner (24.2 and 47.5% respectively) with atorvastatin treatment compared with control diet mice (Fig 3E).

Dietary atorvastatin decreases serum triglycerides

A decrease in serum triglycerides (TGs) was observed in mice fed atorvastatin compared with control mice. Mice fed with 200 and 400 ppm atorvastatin diets showed dose-dependent suppression of serum TGs (53.6% P<0.001, 62.0% P<0.001 respectively) (Fig. 3F).

Inhibition of PCNA (Proliferating cell nuclear antigen) and up regulation of p21 expression by atorvastatin in PanINs and carcinoma of pancreas

Fig. 4A & B summarizes the effects of atorvastatin on tumor cell proliferation as measured by fraction of PCNA positive cells (labeling index). PCNA labeling index was significantly lowered by atorvastatin treatment. Qualitative microscopic examination of PCNA-stained sections showed a substantial decrease in PCNA- labeling index in the pancreas of atorvastatin-treated mice compared with pancreas from mice fed control diet. As shown in Fig 4C, minimal expression of p21 was observed in the pancreatic ductal lesions and PDAC of KrasG12D/+ mice fed with control diets. However administration of 200 and 400 ppm atorvastatin diet resulted in a significant increase in p21 protein expression levels in the pancreas. The quantification of PCNA staining showed 80 ± 4.6 (Mean ± SEM) PCNA-labeling index in control diet fed PDAC, as compared with 30.2 ±2.4 and 18 ±1.8 (Mean ± SEM) PCNA- labeling index in 200 and 400 ppm atorvastatin fed mice PDAC, accounting for a decrease in the proliferation index by ~77% (P < 0.0001) (Fig. 4B).

Figure 4
Effect of Lipitor/atorvastatin on cell proliferation and targeted markers in pancreatic tumors. Immunohistochemical analysis was performed with paraffin-embedded and micro-sectioned pancreatic tissues as described in the methods section. A. Proliferating ...

Effect of dietary administration of atorvastatin on apoptotic index

Fig. 4D summarizes the effects of atorvastatin on tumor cell apoptosis. Qualitative microscopic examination of Tunnel-stained sections showed a substantial and dose-dependent increase in tunnel-positive cells in the pancreatic tissue of atorvastatin-fed mice compared with the control diet-fed mice. The quantification of apoptotic staining showed 12.5 ± 1.3 (Mean ± SEM) tunnel-positive cells (apoptotic index) in PDAC from control, as compared with 55.20 ± 5.4 and 65.8 ± 3.3 (Mean ± SEM) tunnel-positive cells in PDAC from 200 and 400 ppm atorvastatin-fed mice, accounting for a increase in the apoptotic index of >75% (P < 0.0001) (Fig.4D & E).

Inhibition of β-catenin, Cav-1, cdK2, RhoA, PCNA, survivin, p21, Cyclin E, PARP, AKT, pAKT, ERK and pERK expression

Protein analysis demonstrated that pancreatic tissues from atorvastatin-fed mice exhibited significantly reduced expression of β-catenin (Fig. 4F), Cav-1, cdK2, RhoA, PCNA, survivin, cyclin E, AKT, pAKT, pERK, compared with pancreatic tissues from mice fed control diet (Fig 5A). In addition, Rho A and pERK, which are known to be upregulated due to the Kras mutations in various malignancies, are down-regulated in the pancreata of mice fed atorvastatin compared with those from mice on the control diet (Fig 5A, Supplementary Fig. 1). Importantly, the ratio of pAKT to AKT is significantly lower in the pancreatic tissues of high dose treatment groups compared to control. Similarly, up-regulation of ERK, p21 expression and cleaved PARP was observed in the pancreatic tumor tissues of atorvastatin-fed mice compared with control diet-fed mice (Fig. 5A, Supplementary Fig. 1).

Figure 5
A. Effect of atorvastatin on protein expression levels of caveolin-1, cdk2, RhoA, PCNA, Survivin, p21, cyclin E, PARP, AKT, pAKT, ERK and pERK expression in pancreatic ductal adenocarcinoma. Protein expression was analyzed by Western blotting as described ...

Modulation of AKT-1, P2X7, cyclin D1, β-catenin, p21 and COX-2

RT-PCR analysis demonstrated that pancreatic tissues from atorvastatin-fed mice exhibited significant up-regulation of p21 mRNA expression compared with pancreatic tumor tissues from control diet-fed mice (Fig 5B). Significant decreases in inflammatory molecules P2X-7 along with AKT-1, cyclinD1, β-catenin were observed in pancreatic tissues of atorvastatin-fed mice compared with control diet-fed mice (Fig. 4F & 5B). These results further support both the immunohistological and/or western immunoblotting observations described above for PCNA, cyclin D1 and p21.

Modulation of inflammatory cytokines by atorvastatin

Expression levels of inflammatory cytokines IL-2, IL6, IL12 and GM-CSF, which induce COX-2 or act as proinflammatory cytokines, are significantly decreased in serum from atorvastatin-treated mice (Suplementary Table. 1). Interestingly, IL-4, IL-10 and IFN-γ were increased in atorvastatin-treated mice compared with control diet-fed mice (Suplementary Table. 1).

DISCUSSION

Current therapies for managing pancreatic cancer lack efficacy. An urgent need remains for the development of novel strategies for prevention and treatment. Recent studies have demonstrated the possible anticancer activity of statins on several kinds of cancers, including pancreatic cancer (12,17, 2632). In this study, we observed high PDAC incidence in the untreated group compared to previously reported studies (7). This may due to the several reasons. The original LSL-KrasG12D/+ and p48Cre/+ (from MIT, Dr. Tyler Jacks) initially developed in B6/29SVJ genetic background, were bred into C57BL/6 background and then were inbred more than 20 times before being applied in the chemoprevention studies. Our observations show that extended breeding in C57BL/6 (p48Cre/+LSL-KrasG12D/+) genetic background lead to a significant enhancement of PDAC (faster progression from PanINs to PDAC) when compared to earlier reports (7). Most importantly, male mice but not female mice with Kras activation develop much more aggressive PDAC. Hence we used only male p48Cre/+-LSL-KrasG12D/+ mice to evaluate chemopreventive efficacy of statin.

In this study, we show that the most commonly used statin provided chemopreventive efficacy against PanINs and PDAC in KrasG12D/+ transgenic mice. The results also suggest potential antitumoral effects as evidenced by delayed progression of PanIN-1 and -2 to PanIN-3 and to PDAC in KrasG12D/+ mice fed with atorvastatin (Fig 3B & C). Atorvastatin reduces PanIN-3 and PDAC incidence and multiplicity without any observable toxicity, suggesting safe chemopreventive effects of atorvastatin on pancreatic cancer (Fig 3A & C). Safety of statins has been documented extensively in clinical trials investigating the value of statins as anticancer agents (33). Our study clearly shows that statin use for more than 9 months is associated with a 56–94% reduction of PDAC (carcinoma spread/pancreas) with a strong dose-response effect in a transgenic mouse model that mimics human pancreatic cancer progression (Fig 3B).

The exact mechanism of action that would lead to suppression of PDAC by atorvastatin is not yet fully known. Statins may inhibit the PI3/AKT signaling pathway (34,35). PI3/AKT has been implicated in the resistance of pancreatic cancer to gemcitabine (36). More than 95% of pancreatic cancers bear activating mutations in the Kras proto-oncogene, which result in loss of GTPase activity, thus leading to protracted K-ras activation. Suppression of K-ras signaling is an important factor in controlling progression of PanIN lesions to PDAC. Mechanistically, inhibition of farnesyltransferase (FTase)- and geranylgeranyltransferase I (GGTase )-mediated preneylation, which leads to the activation of K-ras, would be an ideal approach; however, application of FTase and GGTase inhibitors is limited due to extensive toxicities associated with these inhibitors in Phase I human trials. Alternatively, statins, may influence the ras activation by limiting the substrates of FTase and GGTase. Importantly, safety and toxicity of statins is not a major concern, given extensive use in cardiovascular outcomes studies (33, 37,38). In the present study, a significant dose-responsive protective effect of atorvastatin against Kras-driven progression of PanINs to PDAC in the transgenic LSL-KrasG12D/+ mice was observed. To our knowledge, few studies have been reported on chemoprevention of PanIN lesions and these have used selective COX-2 / EGFR / ACE inhibitors / capsaicin (6, 39, 40, 41). The present study further supports chemopreventive approaches targeted towards individuals at high risk for pancreatic cancer.

We have shown that atorvastatin significantly reduced several PI3/AKT signaling molecules, such as AKT, pAKT, P2X7, RhoA, pERK, Cdk2, cyclin D1, β-catenin, cyclin E, survivin, Cav-1, and COX-2. This effect was associated with inhibition of cell proliferation (decrease in PCNA and an increased p21) and induction of apoptosis (PARP and tunnel positive cells). In the present study, over-expression of Cav-1 was corroborated with proliferation markers and several down-stream signal molecules, such as RhoA, pERK and the cdK2. Cav-1 knockdown significantly reduced beta1 integrin expression and Akt phosphorylation, induced Caspase 3- and Caspase 8-dependent apoptosis, and enhanced the radiosensitivity of 3D pancreatic cancer cell cultures (42). The exact mechanism by which statins influence Cav-1 is not clearly known; however, our previous studies on colon cancer cell lines exposed to lovastatin show significantly suppressed Cav-1 localization to the membrane lipid rafts (43). Also, lovastatin-induced Cav-1 inhibition was associated with down- regulation of the PI3/AKT signaling pathway. Furthermore, clinical studies have shown that Cav-1 expression is associated with pancreatic tumor progression and poor prognosis for patient survival (44). Our results support previous observations that oncogenic Kras enhances progression of pancreatic ductal cells to a malignant phenotype through the activation of Cav-1 and the PI3/AKT signaling pathway.

Gemcitabine is the current standard chemotherapeutic drug for pancreatic cancer; but it has modest benefit. It was observed that atorvastatin increased the effectiveness of Gemcitabine and 5-Fu by decreasing AKT, supporting the hypothesis that statins may act through the PI3/AKT pathway (22). Several lines of evidence indicate that the statin-induced inhibition of pancreatic cancer cell lines was mediated by AKT via the P2X7 ATP-gated cation channel purinergic receptor that leads to pleiotropic effects (45,46). Activation of the p2X7 receptor is able to stimulate release of proinflammatory cytokines and P2X7 activation can, thus, be involved in inflammation. Chronic pancreatitis is a risk factor for development of pancreatic cancer and recent studies show increased levels of P2X7 in chronic pancreatitis and pancreatic cancers (47). An increased incidence of pancreatic cancer in patients with chronic pancreatitis has been observed in several studies (48,49). Inflammatory mediators, such as COX-2 overexpression in chronic pancreatitis, also have been linked to pancreatic carcinogenesis. Studies from our laboratory and others have shown previously that statin chemopreventive agents such as lovastatin, pitavastatin and atorvastatin prevent colon cancer by inhibiting COX-2 and thus leading to apoptotic cell death (18,50). Also, circulating Inflammatory cytokines IL-2, IL-6, IL-10, IL-12 and GMSF were observed to be significantly higher whereas reduced IL-4 and IFN-γ levels in pancreatic cancer patients compared to normal subjects (51). In the present study, we observed significant decrease in the circulating serum cytokines IL-2, IL-6, IL-12 and GMCSF upon atorvastatin treatment and substantial increase in the levels of IL-4 and IFN-γ (Supplementary Table 1). The mechanistic information on antiproliferative, antiinflammatory and proapoptotic effects of statins supports the efficacy of these agents observed in various experimental tumor models, including the present study (9,10,1518,21). Thus, it is conceivable that the development of agents such as atorvastatin, which will target proliferative signaling and inflammation pathways, is likely to have significant impact on prevention/treatment of pancreatic cancer.

In summary, this study demonstrated significant chemopreventive efficacy of dietary atorvastatin, on progression of PanIN lesions to PDAC. Inhibition of PanINs and PDAC by atorvastatin is associated with significant suppression of tumor cell proliferation and suppression of multiple signaling molecules involved in proliferation, such as AKT, pAKT, Cav-1, Rho A, pERK, cdK2, p2X7, β-catenin, COX-2, cyclin D1, E, survivin. In addition, atorvastatin caused a significant increase in apoptosis and in PARP and p21 expression. Fig 6 depicts the pictorial representation of the effects of atorvastatin on expressions of various signaling molecules based on the present study and previous studies in pancreatic cancer with treatment of atorvastatin (9,10, 1517, 21). The similarity of the KrasG12D/+ mouse model to human premalignant (PanINs) and malignant pancreatic cancer (PDAC) supports the usefulness of stains for delaying the progression of pancreatic cancer in clinical settings. Furthermore, mechanistic observations in the present study further highlight the importance of targeting Cav-1/PI3/AKT signaling for the prevention and treatment of pancreatic cancer.

Figure 6
Pictorial representation of effects of atorvastatin on expressions of various signaling molecules affected by atorvastatin treatment.

Supplementary Material

Supp Fig S1

01

Acknowledgement

We thank the University of Oklahoma Health Sciences Center Rodent Barrier Facility staff. We thank Dr. Julie Sando for editing this manuscript. We also want to thank Dr. Howard Crawford for providing K-ras mice breeder pairs. This work was in part supported by the National Cancer Institute N01- CN-53300.

Footnotes

No potential conflicts of interest

References

1. American Cancer Society. Cancer Facts and Figures. Atlanta (GA): 2011.
2. Eckel F, Schneider G, Schmid RM. Pancreatic cancer: a review of recent advances. Expert Opin Investig Drugs. 2006;15:1395–1410. [PubMed]
3. Hruban RH, Adsay NV, Albores-Saavedra J, Anver MR, Biankin AV, Boivin GP, Furth EE, Furukawa T, Klein A, Klimstra DS, Kloppel G, Lauwers GY, et al. Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations. Cancer Res. 2006;66:95–106. [PubMed]
4. Sakorafas GH, Tsiotou AG, Tsiotos GG. Molecular biology of pancreatic cancer; oncogenes, tumor suppressor genes, growth factors, and their receptors from a clinical perspective. Cancer Treatment Rev. 2000;26:29–52. [PubMed]
5. Ren YX, Xu GM, Li ZS, Song YG. Detection of point mutation in K-ras oncogene at codon 12 in pancreatic diseases. World J Gastroenterol. 2004;10:881–884. [PubMed]
6. Mohammed A, Janakiram NB, Li Q, Madka V, Ely M, Lightfoot S, Crawford H, Steele VE, Rao CV. EGFR inhibitor gefitinib prevents progression of pancreatic lesions to carcinoma in a conditional LSL-K-rasG12D transgenic mice model. Cancer Prev Res. 2010;3:1417–1426. [PMC free article] [PubMed]
7. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, Ross S, Conrads TP, Veenstra TD, Hitt BA, Kawaguchi Y, Johann D, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell. 2003;4:437–450. [PubMed]
8. Demierre MF, Peter D R Higgins, Stephen B Gruber, Hawk E, Lippman SM. Statins and cancer prevention. Nat Rev Cancer. 2005;5:930–942. [PubMed]
9. Kusama T, Mukai M, Iwasaki T, Tatsuta M, Matsumoto Y, Akedo H, Inoue M, Nakamura H. 3-Hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors reduce human pancreatic cancer cell invasion and metastasis. Gastroenterology. 2002;122:308–317. [PubMed]
10. Kaminski R, Kozar K, Kopec M, Basak G, Skierski JS, Koronkiewicz M, Jakobisiak M, GolAb J. Discussion on 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors reduce human pancreatic cancer cell invasion and metastasis. Gastroenterology. 2002;123:1747–1748. [PubMed]
11. Platz EA, Leitzmann MF, Visvanathan K, Rimm EB, Stampfer MJ, Willett WC, Giovannucci E. Statin drugs and risk of advanced prostate cancer. J Natl Cancer Inst. 2006;98:1819–1825. [PubMed]
12. Khurana V, Sheth A, Caldito G, Barkin JS. Statins reduce the risk of pancreatic cancer in humans: a case-control study of half a million veterans. Pancreas. 2007;34:260–265. [PubMed]
13. Kawata S, Nagase T, Yamasaki E, Ishiguro H, Matsuzawa Y. Modulation of the mevalonate pathway and cell growth by pravastatin and dlimonene in a human hepatoma cell line (Hep G2) Br J Cancer. 1994;69:1015–1020. [PMC free article] [PubMed]
14. Feleszko W, Jakobisiak M. Lovastatin augments apoptosis induced by chemotherapeutic agents in colon cancer cells. Clin Cancer Res. 2000;6:1198–1199. [PubMed]
15. Sumi S, Beauchamp RD, Townsend CM, Uchida T, Murakami M, Rajaraman S, Ishizuka J, Thompson JC. Inhibition of pancreatic adenocarcinoma cell growth by lovastatin. Gastroenterology. 1992;103:982–989. [PubMed]
16. Mikulski SM, Viera A, Darzynkiewicz Z, Shogen K. Synergism between a novel amphibian oocyte ribonuclease and lovastatin in inducing cytostatic and cytotoxic effects in human lung and pancreatic carcinoma cell lines. Br J Cancer. 1992;66:304–310. [PMC free article] [PubMed]
17. Kusama T, Mukai M, Iwasaki T, Tatsuta M, Matsumoto Y, Akedo H, Nakamura H. Inhibition of epidermal growth factor-induced RhoA translocation and invasion of human pancreatic cancer cells by 3- hydroxyl-3-methylglutaryl-coenzyme a reductase inhibitors. Cancer Res. 2001;61:4885–4891. [PubMed]
18. Swamy MV, Patlolla JM, Steele VE, Kopelovich L, Reddy BS, Rao CV. Chemoprevention of familial adenomatous polyposis by low doses of atorvastatin and celecoxib given individually and in combination to APCMin mice. Cancer Res. 2006;66:7370–7377. [PubMed]
19. Issat T, Nowis D, Legat M, Makowski M, Klejman MP, Urbanski J, Skierski J, Koronkiewicz M, Stoklosa T, Brezezinska A, Bil J, Gietka J, et al. Potentiated antitumor effects of the combination treatment with statins and pamidronate in vitro and in vivo. Int J Oncol. 2007;30:1413–1425. [PubMed]
20. Yao CJ, Lai GM, Chan CF, Cheng AL, Yang YY, Chuang SE. Dramatic synergistic anticancer effect of clinically achievable doses of lovastatin and troglitazone. Int J Cancer. 2006;118:773–779. [PubMed]
21. Bocci G, Fioravanti A, Orlandi P, Bernardini N, Collecchi P, Del Tacca M, Danesi R. Fluvastatin synergistically enhances the antiproliferative effect of gemcitabine in human pancreatic cancer MIAPaCa-2 cells. Br J Cancer. 2005;93:319–330. [PMC free article] [PubMed]
22. Mistafa O, Stenius U. Statins inhibit AKT/PKB signaling via P2X7 receptor in pancreatic cancer cells. Biochem Pharmacol. 2009;78(11):15–26. [PubMed]
23. Gbelcov H, Len M, Zelenka J, Knejzlık Z, Dvorákov G, Zadinov M, Pouckova P, Kudla M, Balaz P, Ruml T, Vitek L. Differences in antitumor effects of various statins on human pancreatic cancer. Int J Cancer. 2008;122:1214–1221. [PubMed]
24. Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, Depinho RA. Genetics and biology of pancreatic ductal adenocarcinoma. Genes & Dev. 2006;20:18–49. [PubMed]
25. Krasilnikov MA. Phosphatidylinositol-3 kinase dependent pathways: the role in control of cell growth, survival, and malignant transformation. Biochemistry. 2000;65:59–67. [PubMed]
26. Wong WW, Dimitroulakos J, Minden MD, Penn LZ. HMG-CoA reductase inhibitors and the malignant cell: the statin family of drugs as triggers of tumor-specific apoptosis. Leukemia. 2002;16:508–519. [PubMed]
27. Kusama T, Mukai M, Ayaki M, Imamura F, Tatsuta M, Matsumoto Y, Nakamura H, Inoue M. Inhibition of lysophosphatidic acid-induced RhoA activation and tumor cell invasion by 3-hydroxyl-3-methylglutaryl-coenzyme A reductase inhibitors. Int J Oncol. 2003;23:1173–1178. [PubMed]
28. Paragh G, Kertai P, Kovacs P, Paragh G, Jr, Fulop P, Foris G. HMG CoA reductase inhibitor fluvastatin arrests the development of implanted hepatocarcinoma in rats. Anticancer Res. 2003;23:3949–3954. [PubMed]
29. Denoyelle C, Vasse M, Korner M, Mishal Z, Ganne F, Vannier JP, Soria J, Soria C. Cerivastatin, an inhibitor of HMG-CoA reductase, inhibits the signaling pathways involved in the invasiveness and metastatic properties of highly invasive breast cancer cell lines: an in vitro study. Carcinogenesis. 2001;22:1139–1148. [PubMed]
30. Chan KK, Oza AM, Siu LL. The statins as anticancer agents. Clin Cancer Res. 2003;9:10–19. [PubMed]
31. Wong WW, Tan MM, Xia Z, Dimitroulakos J, Minden MD, Penn LZ. Cerivastatin triggers tumor-specific apoptosis with higher efficacy than lovastatin. Clin Cancer Res. 2001;7:2067–2075. [PubMed]
32. Horiguchi A, Sumitomo M, Asakuma J, Asano T, Asano T, Hayakawa M. 3-Hydroxy-3-methylglutaryl-coenzyme a reductase inhibitor, fluvastatin, as a novel agent for prophylaxis of renal cancer metastasis. Clin Cancer Res. 2004;10:86–55. [PubMed]
33. Kawata S, Yamasaki E, Nagase T, Inui Y, Ito N, Matsuda Y, Inada M, Tamura S, Noda S, Imai Y, Matsuzawa Y. Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomized controlled trial. Br J Cancer. 2001;84:886–891. [PMC free article] [PubMed]
34. Pääjärvi G, Roudier E, Crisby M, Högberg J, Stenius U. HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage. FASEB J. 2005;19:476–478. [PubMed]
35. Roudier E, Mistafa O, Stenius U. Statins induce mammalian target of rapamycin (mTOR)-mediated inhibition of Akt signaling and sensitize p53-deficient cells to cytostatic drugs. Mol Cancer Ther. 2006;5:2706–2715. [PubMed]
36. Ng SSW, Tsao MS, Chow S, Hedley DW. Inhibition of phosphatidylinositide 3-kinase enhances gemcitabine-induced apoptosis in human pancreatic cancer cells. Cancer Res. 2000;60:5451–5455. [PubMed]
37. Pedersen TR, Wilhelmsen L, Faergeman O, Strandberg TE, Thorgeirsson G, Troedsson L, Kristianson J, Berg K, Cook TJ, Haghfelt T, Kjekshus J, Miettinen T, et al. Follow-up study of patients randomized in the Scandinavian simvastatin survival study (4S) of cholesterol lowering. Am J Cardiol. 2000;86:257–262. [PubMed]
38. Blais L, Desgagne A, LeLorier J. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and the risk of cancer: a nested case-control study. Arch Intern Med. 2000;160:2363–2368. [PubMed]
39. Funahashi H, Satake M, Dawson D, Huynh NA, Reber HA, Hines OJ, Eibl G. Delayed Progression of Pancreatic Intraepithelial Neoplasia in a Conditional KrasG12D/+ Mouse Model by a Selective Cyclooxygenase-2 Inhibitor. Cancer Res. 2007;67(15):7068–7071. [PubMed]
40. Fendrich V, Chen NM, Neef M, Waldmann J, Buchholz M, Feldmann G, Slater EP, Maitra A, Bartsch DK. The angiotensin-1-converting enzyme inhibitor enalapril and aspirin delay progression of pancreatic intraepithelial neoplasia and cancer formation in a genetically engineered mouse model of pancreatic cancer. Gut. 2010;59:630–637. [PubMed]
41. Bai H, Li H, Zhang W, Matkowskyj KA, Liao J, Srivastava SK, Yang GY. Inhibition of chronic pancreatitis and pancreatic intraepithelial neoplasia (PanIN) by capsaicin in LSL-KrasG12D/Pdx1-Cre mice. Carcinogenesis. 2011;32:1689–1696. [PMC free article] [PubMed]
42. Hehlgans S, Eke I, Haase M, Baretton G, Cordes N. Caveolin-1 mediated radioresistance of 3D grown pancreatic cancer cells. Radiother Oncol. 2009;92:363–370. [PubMed]
43. Guruswamy S, Rao CV. Synergistic effects of lovastatin and celecoxib on caveolin-1 and its downstream signaling molecules: Implications for colon cancer prevention. Int J Oncol. 2009;35:1037–1043. [PubMed]
44. Suzuoki M, Miyamoto M, Kato K, Hiraoka K, Oshikiri T, Nakakubo Y, Fukunaga A, Shichinohe T, Sginohara T, Itoh T, Kondo S, Katoh H. Impact of caveolin-1 expression on prognosis of pancreatic ductal adenocarcinoma. Br J Cancer. 2002;87:1140–1144. [PMC free article] [PubMed]
45. Heeneman S, Donners MM, Bai L, Daemen MJ. Drug-induced immunomodulation to affect the development and progression of atherosclerosis: a new opportunity? Expert Rev Cardiovas Ther. 2007;5:345–364. [PubMed]
46. Wassmann S, Laufs U, Muller K, Konkol C, Ahlbory K, Baumer AT, Linz W, Bohm M, Nickenig G. Cellular antioxidant effects of atorvastatin in vitro and in vivo. Arterioscler Thromb Vasc Biol. 2002;22:300–305. [PubMed]
47. Kunzli BM, Berberat PO, Giese T, Csizmadia E, Kaczmarek E, Baker C, Halaceli I, Buchler MW, Friess H, Robson SC. Upregulation of CD39/NTPDases and P2 receptors in human pancreatic disease. Am J Gastrointest Liver Physiol. 2007;292:223–230. [PubMed]
48. Otsuki M. Chronic pancreatitis in Japan: epidemiology, prognosis, diagnostic criteria, and future problems. J Gastroenterol. 2003;38:315–326. [PubMed]
49. Maisonnneuve P, Lowenfels AB. Chronic pancreatitis and pancreatic cancer. Dig Dis. 2002;20:32–37. [PubMed]
50. Shimizu YM, Shirakami Y, Sakai H, Kubota M, Hata K, Hirose Y, Tsurumi H, Tanaka T, Moriwaki H. Pitavastatin inhibits azoxymethane-induced colonic preneoplastic lesions in C57BL/KsJ-db/db obese mice. Cancer Sci. 2010;101:1701–1707. [PubMed]
51. Gabitass RF, Annels NE, Stocken DD, Pandha HA, Middleton GW. Elevated myeloid-derived suppressor cells in pancreatic, esophageal and gastric cancer are an independent prognostic factor and are associated with significant elevation of the Th2 cytokine interleukin-13. Cancer Immunol Immunother. 2011;60 1490-30. [PMC free article] [PubMed]