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Diets containing omega-3 fat have been associated with decreased tumor development in the colon, breast, and prostate. We assessed the effects of a diet rich in omega-3 (ω-3) fat on the development of pancreatic precancer in elastase (EL)-Kras transgenic mice and examined the effect of an ω-3 fatty acid on pancreatic cancer cells in vitro.
Two cohorts of EL-Kras mice were fed a high ω-3 fat diet (23% menhaden oil) for 8 and 11 months and compared to age-matched EL-Kras mice fed standard chow (5% fat). Pancreata from all mice were scored for incidence and frequency of precancerous lesions. Immunohistochemistry was performed for proliferating cell nuclear antigen (PCNA) to assess proliferative index in lesions of mice fed either a high ω-3 or standard diet. In vitro, the effect of the ω-3 fatty acid, docosahexaenoic acid (DHA), on two pancreatic cancer cell lines was assessed. Cancer cell proliferation was assessed with an MTT assay, cell cycle analysis was performed by flow cytometry and apoptosis was assessed with Annexin/PI staining.
The incidence, frequency, and proliferative index of pancreatic precancer in EL-Kras mice was reduced in mice fed a high ω-3 fat diet compared to mice fed a standard chow. In vitro, DHA treatment resulted in a concentration dependent decrease in proliferation through both G1/G0 cell cycle arrest and induction of apoptosis.
A high ω-3 fat diet mitigates pancreatic precancer by inhibition of cellular proliferation through induction of cell cycle arrest and apoptosis.
The importance of early cancer detection has been demonstrated in breast, prostate, and colon cancer whereby early treatment has resulted in improved survival (1-3). While there are currently no ideal diagnostic modalities to detect early pancreatic cancer, imaging techniques are evolving that have greater sensitivity to detect dysplastic changes in the pancreas (4). Therefore, identifying chemopreventive measures that could prevent the process of malignant transformation are needed. One promising area of chemopreventive investigation is the role of dietary fat in the development of cancer(5). In vivo studies in a hamster and rat models of chemically induced pancreatic cancer have shown a protective effect of dietary fish oil (ω-3 fat) supplementation(6, 7). Although there is some degree of protection from cancer growth in animals supplemented with fish oil, there is no effective tumor regression or improvement in survival demonstrated in humans with advanced pancreatic cancer receiving ω-3 supplementation (8). Nonetheless, ω-3 supplementation may still have a preventive effect without having a chemotherapeutic effect, although this has never been studied prospectively in humans or mice.
In order to examined the role of fatty acids as a chemopreventive agent, we employed a mouse model that recapitulates pancreatic precancer. In humans, the most prominent genetic event observed in both Pancreatic Intraepithelial Neoplasias (PanINs) and pancreatic cancer is a mutation in the Kras allele, which causes constitutive signaling through the ras cascade. We have previously reported the development of a transgenic mouse model which targets human mutant Kras gene expression to the pancreas using a rat elastase promoter (9). Within the pancreas of these mice, precancerous lesions develop which are histologically similar to human PanINs, thus representing an ideal in vivo model to evaluate the effect of dietary changes on precancer development. These lesions can progress to invasive disease upon loss of TSG (p16) or other gene product such as Pigment Epithelium-Derived Factor (PEDF)(10).
While some studies have evaluated the effects of different fatty acid diets on rodents with chemically induced pancreatic cancer, there have been no in vivo studies examining the effects of high ω-3 diets on mice with genetic mutations that predispose them to pancreatic precancer. In this study, we evaluated the effects of high ω-3 fatty acid- supplemented diets on mice predisposed to the development of pancreatic precancer and examined the mechanisms in vitro through which alterations in lesion development may occur.
EL-Kras transgenic mice were previously generated by microinjection of FVB/N fertilized mouse embryos and subsequent implantation into pseudopregnant females as described in a prior publication and transgenic mice developed pancreatic precancer at roughly a 50% penetrance at 12 months of age (9). PCR genotyping of EL-Kras mice has demonstrated that all males were positive for the transgene while all females were negative. EL-Kras mice were subjected to tail biopsy and PCR evaluation of genomic DNA from mouse tail digests (see below). EL-Kras male mice were also bred to C57BL/6 (B6) female mice to generate EL-Kras FVB6 F1 mice (EL-Kras F1) which developed roughly a 100% penetrance of pancreatic precancer by 6 months of age. EL-Kras F1 mice were used for this diet study.
A piece of mouse tail was digested with proteinase K (Fermentas) solution in GNT Buffer (50mM KCl, 1.5mM MgCl2, 10mM Tris-HCl, 0.01% gelatin, 0.45% NP40, and 0.45% Tween-20 pH=8.5). The digested, heat-inactivated supernatant was used as a DNA template in the following 25 ul PCR reactions: 1 uM of each primer (EL-Kras 1f primer 5’ 3’ and 2r primer 5’ 3’) in a solution with 1× PCR Buffer, 1.5mM MgCl2, 1 uM dNTPs, and 0.2 − 0.4 U Taq polymerase (Fermentas or Promega) was used in a standard PCR reaction. The 25 ul samples were incubated at 94°C for 3−5 minutes followed by 35 cycles of 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute followed by 5 minutes at 72°C and an indefinite soak at 4−10°C.
Groups of mice were administered ad libitum either a standard chow (~5% fat) or a diet with 23% menhaden oil (ω-3). The high omega 3 fatdiet has a 1:2.5 omega-3:omega-6 polyunsaturated fatty acid ratio while the standard diet has a 1:9.6 omega-3:omega-6 polyunsaturated fatty acid ratio (For further diet details see Table 1). Five EL-Kras F1 mice were fed a 23% menhaden oil diet and sacrificed at 8 months of age while six EL-Kras F1 mice were fed 23% menhaden oil and sacrificed at 11 months of age. Mice on the high ω-3 diet were compared to an equal number of age-matched EL-Kras F1 mice fed the standard chow. Following euthanasia, mouse pancreatic tissue was fixed in 10% buffered formalin overnight. Tissue blocks were sectioned at 4−6 um and placed on positively charged slides. Following removal of paraffin by xylenes and subsequent hydration, the tissue was stained with H&E and then dehydrated to xylenes and mounted with a cover slip. H&E stained tissue was scored for the presence of ductal carcinoma in situ (pancreatic precancer). Several parameters were noted including incidence (number of mice with lesions per number of mice with genotype) and frequency (number of lesions per random section/per mouse).
After deparafinization, the slides were heated in the microwave in Dako antigen retrieval solution [(S1699), Dako (Carpinteria, CA)] and then washed in Dako wash buffer (S3006). Sections were blocked with 0.5% BSA in PBS for 30 minutes at room temperature followed by Dako peroxidase block for 30 minutes. Primary anti-PCNA (Proliferating Cell Nuclear Antigen) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was placed on sections at a concentration of 1:1000 at room temperature for 1 hour. Sections were then treated with Dako secondary antibody (anti-mouse IgG) HRP-linked (Dako Envision Kit K4008) for 30 minutes at room temperature and developed with DAB reagent for 30 sec. Counterstaining was performed with Gills-2 hematoxylin. Immunohistochemistry for PCNA was independently evaluated manually by two investigators (P.G. and M.B.), verified by ImageJ software (National Institute of Heatlh, Bethesda MD.), and calculated as percentage of positive nuclei per lesion.
Human pancreatic cancer cell lines BxPC-3 and AsPC-1 were obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C incubator in a humidified atmosphere of 5% CO2. Fatty acid-free bovine serum albumin (BSA, sigma Chemical) was prepared as a 125 μmol/L solution in DMEM. A purified ω-3 fat, Docosahexaenoic acid (DHA), purchased as a sodium salt (Sigma Chemical), was solubilized to 600 μmol/L stocks in the BSA medium. BSA was used as a carrier protein for DHA and maintained at a constant ratio of 4.8:1 for all experiments based on previously published in vitro ω-3 culture models(11). Cell growth was measured using the 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma) colorimetric dye reduction method. Pancreatic cancer cells were seeded at a density of 1 × 104 cells for both ASPC-1 and BXPC-3 per well in 96-well plates in complete cell culture medium containing 0.5% FBS. After 24 hours, culture medium was then replaced with serum-free medium together with indicated reagents. After 48 hours, 10 μL MTT with 5 mg/mL concentration was added to the medium and cultured for another 2 h. Then, the medium was discarded and 200 μL DMSO was added into each well. The plate was then put on a shaker for 10 min. The absorbencies of each well were read at a wavelength of 570 nm using a spectrophotometric plate reader (Bio-Tek Instruments). Proliferation assays were repeated in triplicate for each cell line.
AsPC-1 and BxPC-3 cells were grown to 50% confluence in T25-cm2 flasks and then incubated in Dubelco's Modified Eagles Medium (DMEM) with 0.5% FBS overnight. Cells were treated with either DHA (40μM) or BSA (40μM) for 48 hours and then harvested with a trypsin-EDTA solution to produce a single-cell suspension and were then washed twice with PBS. The cells were suspended in 0.5mL PBS, fixed in 70% ethanol at 4°C, centrifuged at ×300g for 10 minutes and then washed with PBS. After resuspension in 1ml PBS, the cells were incubated with 5μL Rnase I (10mg/ml) at 37°C for half an hour. Then, 150μL of propidium iodide (400g/mL, Sigma (St. Louis, MO) was added and the cells were shaken for 2 hours in the dark at room temperature. The samples were analyzed by flow cytometry.
AsPC-1 and BxPC-3 cells were grown to 50% confluence in T25-cm2 flasks and then incubated in serum DMEM with 0.5% FBS overnight. Cells were treated with either DHA (40μM) or BSA (40μM) for 48 hours and then harvested with a trypsin-EDTA solution to produce a single-cell suspension and were then washed twice with PBS. Annexin V-FITC and PI analysis (BD Biosciences kit 556547) was performed according to manufacture protocol (BD Biosciences, San Jose, CA) and samples were analyzed by flow cytometry. Cell treatement with DHA and Annexin-PI staining was repeated in triplicate for each cell line.
Data were analyzed by ANOVA, followed by Dunnett's Multiple Comparison Test as appropriate for post-hoc testing and paired t-tests where appropriate. The analyses were performed with the Prism software package (Graphpad, San Diego, CA). Data were expressed as mean ± SEM.
In order to assess the phenotypic effect of the ω-3 fat diet on pancreatic carcinogenesis, five ELKras F1 transgenic mice were fed a high ω-3 fat diet and sacrificed at eight months while six EL-Kras F1 mice were fed a high ω-3 fat diet and sacrificed at eleven months. Each cohort of mice was compared to an equal number of age matched EL-Kras F1 mice fed a standard chow. On histological examination, all of the EL-Kras F1 mice on a standard diet developed precancerous pancreatic lesions (representative lesion shown in Figure 1, panel A) while only 3 of 5 eight-month and 3 of 6 eleven-month old mice on the ω-3 fat diet demonstrated precancer on histological examination.(Figure 1, panels C and D) The histology of the precancerous lesions in mice fed the high omega-3 fat diet was identical to that of mice fed the standard diet, however, there was a tendency towards smaller lesions in mice fed the high omega-3 diet. The frequency of precancerous lesions in eight month and eleven month old ω-3 diet mice were 1.0 +/− 0.55 and 2.2 +/− 1.28 lesions per mouse respectively while the eight and eleven month old standard diet mice had a frequency of 4.8 +/− 0.37 and 9.5 +/− 2.25 lesions per mouse respectively.[(p<0.0001 and p<0.01 for the 8 month and 11 month groups respectively) Figure 1, panels E and F]. There was no statistically significant difference in weights between ω -3 fed mice compared to age-matched mice on standard chow (Data not shown).
Due to evidence that high fat diets may affect the rate of pancreatic cancer proliferation in vitro (12-15), we performed immunohistochemistry for PCNA, a nuclear marker expressed by proliferating cells, on the precancerous lesions to assess whether the diets affect pancreatic precancer proliferation rates in vivo. All of the EL-Kras F1 mice with precancer on either a standard or high ω-3 fat diet were immunohistochemically stained with PCNA and the lesions were scored for percentage of positive nuclei per lesion for each mouse examined.(Representative IHC demonstrated in Figure 2, panels A and B) In mice fed the high ω-3 fat diet, 7.0% of precancer cells stained positive for PCNA compared to 24.3% in age matched mice fed the standard diet.[(p<0.05) Figure 2 Panel C]
In order to implicate the ω-3 fat as the causative agent for the alterations in pancreatic precancer demonstrated in vivo, we evaluated the effect of an ω-3 fat, DHA, on pancreatic cancer cells in vitro. In both BxPC-3 and AsPC-1 pancreatic cancer cells, we demonstrated a concentration dependent decrease in cellular proliferation in cancer cells treated with DHA compared to untreated cells at 48 hours.(Figure 3, panels A and B) In both cell lines, a statistically significant decrease in proliferation was obtained at a concentration of 40μm DHA and above (p<0.05) and thus a concentration of 40 μm DHA was used for the following in vitro experiments.
After demonstrating a decrease in proliferative rate in pancreatic cancer cells treated with ω-3 fat, we sought to further elucidate the mechanism of this decrease by performing cell cycle analysis and flow cytometry for apoptosis in cells treated with DHA. In both AsPC-1 and BxPC-3 cells, we demonstrated an increase in G1 arrest in cells treated with 40 μm DHA compared to controls.[(78.5% vs. 69.5% and 89.8% vs 84.4% respectively) Figure 4, panels A and B] We next performed flow cytometry with Annexin and PI staining to assess apoptosis rates in AsPC-1 and BxPC-3 cells treated with 40 μm DHA compared to controls. In both AsPC-1 and BxPC-3 cells, there was a statistically significant induction in apoptosis following a 48 hour treatment with DHA.[(17.6% vs. 9.7% and 30.2% vs. 21.6%, p<0.05 and p<0.01 respectively) Figure 5, panels A and B]
There is increasing evidence that dietary intake of ω-3 fatty acids suppress progression of breast, prostate and colon cancers.(16-19) However, there is limited understanding of how dietary fatty acids may affect the process of pancreatic carcinogenesis and cancer progression. Decreased tumor growth rates in BOP-induced pancreatic cancer were observed in hamsters fed a high ω-3 fat diet.(20) To date, all of the in vivo studies regarding the effect of different polyunsaturated fatty acid (PUFA) diets during pancreatic carcinogenesis have utilized carcinogen-induced(7, 20-23) and xenograft(24, 25) models that develop invasive pancreatic cancer. The goal of this study was to assess the possible chemopreventive role of a diet rich in ω-3 fat during the development of precancer in vivo and determine if this chemopreventive effect was generated by a similar mechanism to that observed in pancreatic cancer cell lines following treatment with an ω-3 fatty acid. To our knowledge, this is the first study to evaluate the effects of an ω-3 fat diet on transgenic mice predisposed to the development of pancreatic precancer.
The most striking finding in this study was the significantly decreased incidence and frequency of pancreatic precancer in EL-Kras F1 mice fed a high ω-3 fat diet versus those fed a standard diet. This change occurred early in the eight-month old mice and persisted in the eleven-month old mice. In addition, the cellular proliferation rate in precancerous lesions was significantly reduced in the ω-3 fatty acid group compared to the standard diet group. Since the Kras mutation is targeted to the acinar compartment, the ω -3 diet likely plays a role in both retarding the transformation process, which leads to the initial formation of pancreatic precancer and then dampering the proliferative ability of the precancerous lesions once they develop. Previous findings in prostate (26), mammary (27), skin (28), and colon(29) in a precancerous setting demonstrated that administration of high ω-3 diets generated an environment that suppressed tumor establishment. Our results extend this idea into pancreatic precancer development and support previously identified in vivo effects of high fat diets during pancreatic carcinogenesis but were generated in a precancerous setting in the pancreas. The suppressive nature of an ω-3 fat diet on EL-Kras mice was even more pronounced than those observed in BOP-induced pancreatic cancer in hamsters(20) since some EL-Kras F1 ω-3 fed mice do not develop precancer.
Two issues remain regarding the phenotypic effects of high PUFA diets on the development of pancreatic precancer in vivo: (1) other potential variables that may contribute to these changes and (2) the mechanisms responsible for cellular/tissue abnormalities leading to precancer. When examining the differences between the high omega-3 and standard diets (see Table 1), the ω-3 fat diet had a higher calorie content per gram than the standard diet. The only means to assess lower fat composition in an isocaloric setting would be to compensate calories from fat with calories from sugar, which changes the whole dynamic of this study from an evaluation of a high dietary omega-3 fat to the source of caloric intake. Even in a proposed isocaloric setting, total calorie intake would be difficult to maintain unless each mouse consumes the same amount of isocaloric diet. Nevertheless, EL-Kras F1 mice fed a high ω-3 fat diet had no significant difference in body weights compared to mice fed a control diet. Moreover, our in vitro data demontrating a lower proliferation rate in pancreatic cancer cells treated with DHA supports our in vivo findings and implicates the ω-3 fat as the main factor associated the proliferative changes and precancer inhibition. Furthermore, we demonstrated that DHA affects proliferation through both induction of G1 arrest and apoptosis.
In conclusion, we demonstrated for the first time, that a diet high in ω-3 fat may mitigate the development of pancreatic precancer. Omega-3 fats appear to have this effect by inhibiting pancreatic lesion proliferation through induction of G1 cell cycle arrest and apoptosis. More work is needed to further elucidate the mechanisms of action of ω-3 fatty acids and is the focus of our next study. The present study serves as provocative preclinical data that dietary intake of omega-3 fat may mitigate the development of pancreatic precancer or cancer in high-risk pancreatic cancer kindreds. Likely, the most effective method to alter a patient's intake of omega-3 fat would be in the form of vitamin supplementation (fish oil, flax seed oil etc.). Ultimately, a high ω-3 fat diet may hold promise as a means of impeding the development and progression of pancreatic precancer.
We gratefully acknowledge Dr. Diane Birt for her assistance with diet formulations. We appreciate the assistance of Meg Bowden for helping procure non-transgenic control tissue. We kindly acknowledge the consultation of Dr. Eric Sandgren regarding background strain issues with EL-Kras transgenic mice.
Financial Support: This study was funded in part by a National Institute of Health (N.I.H.) R21 (P.G. 5-R21-CA123041-02).
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