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Four types of fatty acids exert differential impact on pancreatic cancer growth
ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Four types of fatty acids exert differential impact on pancreatic cancer growth Ming Yu a, Hongyi Liu b, Yijie Duan a,b, Dapeng Zhang b, Shasha Li c, Feng Wang b,d,* a
Department of Nutrition and Food Hygiene, School of Public Health, Tianjin Medical University, Tianjin 300070, China Tianjin Institute of Integrative Medicine for Acute Abdominal Diseases, Nankai Hospital, Tianjin 300100, China Graduate School, Tianjin Medical University, Tianjin 300070, China d Department of Surgery, Karolinska University Hospital, Huddinge 14186, Sweden b c
A R T I C L E
I N F O
Article history: Received 20 December 2014 Received in revised form 23 January 2015 Accepted 3 February 2015 Keywords: Pancreatic cancer High fat diet Fatty acids Orthotopic xenograft model
A B S T R A C T
Increased fatty acids (FAs) regulate pancreatic cancer progression, however, the detailed mechanism is not clear, and different forms of FAs may play diversified roles in pancreatic cancer. To elucidate the underlying mechanism, we compared the effects of four major types of FAs on pancreatic cancer growth both in cell culture and in a mouse model. HPAF pancreatic cancer cells were implanted in nude mice for 14 weeks, and the mice were fed with four different high-fat/high-energy diets (15% fat, 4 kcal/g), an iso-caloric diet (5% fat, 4 kcal/g) and a normal diet (4% fat, 3 kcal/g). The high fat diets were rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and n-6 and n-3 polyunsaturated fatty acids (n6- and n3PUFAs), respectively. While n3PUFA diet decreased tumor viability, the other high fat diets stimulated tumor viability by apparently different mechanisms. For instance, xenografts whose carriers were fed with SFA diet had marked expression of cancer-related proteins and lipid droplets. Although mice that were fed with MUFA- and n6PUFA diets had pancreatic tumors of similar size, liver metastasis occurred more frequently in those with the n6PUFA diet. In experiments in vitro, the HPAF-cell population was increased by SFAs and MUFAs, decreased by n3PUFAs and not changed by n6PUFAs. In conclusion, different fatty acids have different impact on pancreatic cancer cells. The effects of fatty acids on pancreatic cancer cells were consistent in vivo and in vitro except that n6PUFAs only had regulatory effects in vivo. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Fatty acids (FAs) are principal constituents of dietary fat, so high fat diets (HFDs) increase the intake of FAs. By structure, dietary FAs are mostly of three major types namely saturated FA (SFA), monounsaturated FA (MUFA) and n-6 polyunsaturated FA (n6PUFA) [1]. In flaxseed and in fish oil, n-3 polyunsaturated FAs (n3PUFAs) also contribute as principal FAs [1]. Previous studies have shown that increased FAs regulate pancreatic cancer [2–8]. For instance, diets rich in SFAs and n6PUFAs accelerate the process of pancreatic carcinogenesis in the animal, whereas HFDs made of fish oil and thus rich in n3PUFAs sometimes inhibit the carcinogenetic process [3–7]. To date, it remains unclear whether dietary increase in MUFAs regulates pancreatic cancer. Similarly unclear is whether a diet rich in flaxseed-derived n3PUFAs regulates pancreatic cancer, though such a diet inhibited non-pancreatic cancers in animals [9,10]. When pancreatic cancer cells were exposed to different FAs in vitro, their growth
* Corresponding author. Tel. +862227435362; fax: +862227435362. E-mail address: [email protected] (F. Wang).
was stimulated by SFAs and MUFAs, inhibited by n3PUFAs, and either stimulated or inhibited by n6PUFAs [11,12]. Mechanistically, FAs may regulate pancreatic cancer cells by different pathways. First of all, FAs are energy substrates when they are degraded by β-oxidation in the mitochondria. Thus, increased FAs may improve energy production in cancer cells [3]. In keeping with this notion, inhibitors of β-oxidation such as etomoxir inhibited tumor cells [13]. Further, medium-chain acyl-CoA dehydrogenase (MCAD) is a key regulator of β-oxidation [14], and the enzyme was increased in human pancreatic cancer cells carried by nude mice that were fed a diet rich in SFAs [3]. As a second mechanism, FAs may regulate pancreatic cancer cells by modulating hypoxiainducible factor-1 (HIF-1) expression. HIF-1 is a transcription factor composed of HIF-1α and HIF-1β subunits [15]. Because HIF-1α is degraded in the presence of oxygen, mammalian cells normally have no HIF-1. However, cancer cells frequently express HIF-1α, hence HIF-1 [16]. The proteins encoded by HIF-1 target genes include glucose transporters and growth factors such as vascular endothelial growth factors (VEGF). Thus, HIF-1 expression increases cell viability [17]. According to a recent study, n6PUFAs increase prostaglandin E2 (PGE2) in pancreatic cancer cells and thereby stimulate HIF-1α expression and increase cell viability [18]. In contrast,
http://dx.doi.org/10.1016/j.canlet.2015.02.002 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
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n3PUFAs increase prostaglandin E3 (PGE3) and thus inhibit HIF-1α expression and decrease cell viability [18]. The PUFA-induced effects may be because n6- and n3PUFAs increase cyclooxygenase-2 (Cox2) expression and thus increase prostaglandin synthesis [18–20]. Lipid droplets are a hallmark of lipid metabolism and their surface is coated by two groups of proteins namely perilipins and caveolins [21]. We previously demonstrated that one HFD rich in SFAs increased lipid droplets in pancreatic cancer cells [3]. In addition, increased caveolin-1 (cav-1) is found as a prognostic factor for pancreatic cancer [22,23]. Recently, the gene of cav-1 was added to the list of HIF-1 target genes [24]. We undertook the present study to investigate how different types of dietary FAs regulate pancreatic cancer cells. We started the study with two experiments in vitro. In the first experiment, we examined cell viability in three pancreatic-cancer cell lines (namely HPAF, MiaPaCa2, and Panc-1) when they were incubated with a fattyacid mix. In the next experiment, eight FAs (two for each FA type) were studied individually for their effects on HPAF cells. In a nudemouse experiment, we compared four HFDs that were enriched with SFAs, MUFAs, n6PUFAs and flaxseed-derived n3PUFAs, respectively. In the 14-week experiment, six groups of nude mice that carried HPAF cells were fed with these HFDs and two control diets, respectively. Tumor grafts and their carriers were examined for the effects that were derived from increased dietary fatty acids. Materials and methods Materials The human pancreatic cancer cell lines of HPAF, MiaPaCa2, and Panc-1 were bought from American Type Culture Collection (Rockville, MD, USA). Eight fatty acids namely palmitic acid, lauric acid, oleic acid, palmitoleic acid, linoleic acid, arachidonic acid (ARA), linolenic acid, and docosahexaenoic acid (DHA) as well as a solution of oleic acid (800 μg/ml) and linoleic acid (800 μg/ml) were bought from Sigma Aldridge (St. Louis, MO, USA). Six diets were prepared for an animal experiment, including four HFDs, an iso-caloric control (Iso-C) diet and a normal control (NC) diet. The HFDs and the Iso-C diet were designed and produced by Harlan Laboratories Inc. (Madison, WI, USA). The NC diet was described previously [3]. Cell proliferation assay HPAF, MiaPaCa2, and Panc-1 cells were cultured in 96-well plates using Dulbecco Modified Essential Media (DMEM) with 10% fetal bovine serum. When cells were 90% confluent, they were incubated for 24 hours in serum-free DMEM wherein an oleic acid-linoleic acid solution was diluted in different concentrations (3 μM– 100 μM). In an experimental group, media contained both fatty acids and 100 μM etomoxir. Media for control cells contained no fatty acids or etomoxir. After incubation, the cell population was determined by MTT assay as we described elsewhere [25]. In the next experiment, HPAF cells were seeded in 96-well plates and incubated in serum-containing DMEM. When cells were 90% confluent, they were incubated for 24 hours in serum-free DMEM supplemented with a fatty acid in the final concentrations of 4-, 20-, and 60 μM. Eight FAs were tested, with each type of fatty acids being represented by two FAs. In some groups, culture media also contained 100 μM etomoxir. Cells in the control group were incubated in the absence of fatty acids and etomoxir. After incubation, cell population was determined by MTT assay. Transplantation of pancreatic cancer cells in nude mice After arrival, all 60 male nude mice (5 weeks old, weighing 15–20 g) were fed with NC diet for two weeks. The composition of the NC diet was described elsewhere [3]. Mice were randomly divided into six groups (10 in each). One group was still fed with NC diet, and the other five groups were fed with four HFDs and the Iso-C diet, respectively (Tables 1 and 2). The four HFDs were made of four different oils, more than 50% of whose weight came from a single type of FA (Table 1, ref. 1). One week after the feeding was started, 3 × 106 HPAF cells were transplanted in the pancreas [3]. During the experiment, food intake and body weight were noted on a weekly basis. In the end of week 14, mice were sacrificed under anesthesia. Tumor grafts, liver, and hind-leg muscle were removed and stored at −80 °C. Effects of HFDs on hepatic and skeletal-muscle FA profiles Liver and skeletal-muscle samples (0.5–1.0 g) were homogenized in a mix of benzene (1 ml) and petroleum ether (1 ml). Homogenates were centrifuged (5000 RPM, 15 min) and supernatants mixed with 2 ml methanol (0.5 M NaOH). After a
Table 1 Six groups of nude mice and basic information of their diets. Groups
Mice-NC Mice-SFA Mice-MUFA Mice-n6PUFA Mice-n3PUFA Mice-IsoC
Diets Name
Fat content
Principal fatty acids (ref. 1)
Energy
NC SFA MUFA n6PUFA n3PUFA Iso-C
4% soybean oil 15% cocoa oil 15% olive oil 15% soybean oil 15% flaxseed oil 5% soybean oil
n6PUFAs SFAs MUFAs n6PUFAs n3PUFAs n6PUFAs
3 kcal/g 4 kcal/g 4 kcal/g 4 kcal/g 4 kcal/g 4 kcal/g
30-min incubation at 30 °C, water was added to retain water-soluble contaminants. The top layer of organic solvents was removed. Fatty acid methyl esters were separated by gas–liquid chromatography on a 30-m DB-WAX capillary column, using Agilent 7890-5975C system. Fatty acids were identified by retention time, using fatty acid standards as reference. A total of 25 FAs were identified in different samples. In later analysis, these FAs were grouped according to their types, giving SFA-, MUFA-, n6PUFA-, and n3PUFA groups. Histology Frozen tumors were cut along the longest axis (8 μm thick) and stained with hematoxylin and eosin (H&E). In a microscope (Leica DM 4000B), sections were photographed region by region, using a CCD camera (Leica DFC 500). Resulting images were merged using the LAS software. Total areas and necrotic areas were quantified in reconstituted tumor image, using the software of ImageJ (NIH, version 2.1). In other sections, intracellular lipid droplets were stained with Oil-Red-O. HIF-1α and proliferating cell nuclear antigen (PCNA) were stained by immunocytochemistry in consecutive sections, using anti-HIF-1α (Novus 100449) and anti-PCNA (Abcam, #912) antisera and a Vector kit (Burlingame, CA, USA). Western blotting Tumor samples were homogenized. Whole-cell proteins were extracted, separated in SDS gel, and transferred to PVDF membranes. The membranes were incubated with antisera against MCAD (Abcam 13677), COX-2 (ab15191), HIF-1α (Novus 100– 449), VEGF (BD555036), cav-1 (Abcam 32577), and β-actin (Abcam 8227). They were incubated with appropriate secondary antisera and then treated with chemiluminescence detection reagents. Specific blots for target proteins were recorded in an image analysis system. Statistics Data are means ± SEM. When three or more groups were involved, results were analyzed using analysis of variance (ANOVA). When fewer groups were involved, Student’s t test was used. P < 0.05 was considered significant.
Results Effects of fatty acids in vitro The mix of oleic and linoleic acids dose-dependently increased HPAF and MiaPaCa2 cells (Fig. 1). However, the effects did not exist anymore in the presence of etomoxir (Fig. 1). The same mix of fatty acids had no effects on Panc-1 cells (Fig. 1). Fig. 2 shows the effects of single FAs on HPAF cells. The eight FAs tested were derived from four FA types with each type being represented by two FAs. Control cells were incubated in the absence of FAs. MTT values seen in control cells were taken as baselines to which MTT values from the other cells were related. The baseline (100%) bars for control cells were omitted in Fig. 2. Both SFAs tested increased cell population (Fig. 2A), but the addition of etomoxir in culture media removed the stimulating effect of SFAs. Both MUFAs tested increased HPAF cells as well, and the stimulating effect was also inhibited by etomoxir (Fig. 2B). Neither of the two n6PUFAs tested induced any significant effects on HPAF cells (Fig. 2C). The growth of HPAF cells was inhibited by two n3PUFAs namely linolenic acid and DHA, and the addition of etomoxir removed the inhibiting effects of DHA but not linolenic acid (Fig. 2D).
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Table 2 Six groups of nude mice and their tumor grafts. Groups
NC Iso-C SFA MUFA n6PUFA n3PUFA a b c d e
Survivors (n)
Tumor grafts
6 8 6 7 6 9
Liver metastasis (n)
Weight (mg)
Total area (pixel, 1 × 106)
Normal area (pixel, 1 × 106)
Big necrosisa (n)
465 ± 100 356 ± 49 592 ± 100b 597 ± 96c 567 ± 100d 364 ± 60
1.3 ± 0.3 1.3 ± 0.1 1.6 ± 0.2 1.7 ± 0.3 2.3 ± 0.3c 1.6 ± 0.1
0.9 ± 0.3 0.9 ± 0.1 1.0 ± 0.2 1.5 ± 0.3e 1.7 ± 0.3c 0.9 ± 0.1
3 4 5 1 3 6
2 (33%) 3 (38%) 4 (67%) 2 (29%) 5 (83%) 2 (22%)
Necrotic areas were >25% of the tumor area. P = 0.07 vs. group-IsoC. P < 0.05 vs. groups-IsoC and n3PUFA. P = 0.08 vs. groups-IsoC and n3PUFA. P = 0.06–P = 0.09 vs. groups-IsoC and n3PUFA.
were lost in the last four weeks when they were either found dead or were sacrificed for looking sick. During the experiment, body weight and food intake were checked weekly. Results from three or four neighboring weeks were pooled, so weekly food intake and body weight gain were averaged in the periods. Mice fed with five high-energy diets showed decreases in both food
Animal survival and nutritional status
% of ctrl (0 µM) value
Two mice in group- SFA died the day after cancer-cell transplantation and were removed from the study. Forty-two out of the 58 remaining mice (72%) survived experiment, with each group having 6–9 mice (Table 2). The 16 non-survivors
150
Panc-1
MiaPaCa2
HPAF-II
* **
#
**
** **
#
**
100 50
16 9 8 15 11
16 7
8
0
6 13 25 50 50+ 100 eto
8 13 14 11 9
14 7
8
0
6 13 25 50 50+ 100 eto
7 12 13 11 9
13 7
7
0
6 13 25 50 50+ 100 eto
0 3
3
3
fatty acid concentrations (µM) Fig. 1. Effects of oleic and linoleic acids on three pancreatic cancer cells. HPAF, MiaPaCa2, and Panc-1 cells were incubated for 24 h in media containing a mix of oleic acid and linoleic acid. Final concentrations of fatty acids are shown in the figure. In a group, culture media contained both fatty acids (50 μM) and etomoxir (100 μM). Cell population was measured in MTT assay. Numbers in bars indicate the times (n) of experiments. Each time, every condition was tested in three wells of cells. *P < 0.05 and **P < 0.01, compared to control cells; #P < 0.05, compared to cells incubated with 50 μM fatty acids in the absence of etomoxir.
palmitic
100 50
9
B 150
lauric
*
#
9
9
*
#
*
9
9
8 10 9
#
*
#
9 11
% of ctrl
% of ctrl
A 150
linoleic
D 125
ARA
100 75 50
14 15
9 14
9
25
* *
11 13 12
9
9
11 13
# 9 13
9
0 4 20 20+ 60 60+ eto eto
11 13
9 13 9
% of ctrl
% of ctrl
C 125
50
palmitoleic
*
100
0 4 20 20+ 60 60+ eto eto
oleic
4 20 20+ 60 60+ eto eto
4 20 20+ 60 60+ eto eto
linolenic
DHA
100
* *
75 50
11
25
0
13 9
** ** 13 9
*
*
11 13 9 12 9
0 4
20 20+ 60 60+ eto eto
4 20 20+ 60 60+ eto eto
4
20 20+ 60 60+ eto eto
4 20 20+ 60 60+ eto eto
fatty acid concentrations (µM) Fig. 2. Effects of eight fatty acids on HPAF cells. HPAF cells were incubated for 24 h in media that contained eight fatty acids from SFA (A), MUFA (B), n6PUFA (C), and n3PUFA (D) groups. Each fatty acid was tested in different concentrations in the absence or presence of etomoxir (100 μM). Cell population was determined in MTT assay. Numbers in bars indicate experiment times. Each time, every condition was tested in three wells of cells. *P < 0.05 and **P < 0.01, compared to control cells. #P < 0.05, compared to cells incubated with the same concentration of fatty acids in the absence of etomoxir.
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Fig. 3. Food intake and body weight in nude mice. Six groups of nude mice carrying HPAF pancreatic cancer cells were fed with four high-fat diets and two control diets. The high fat diets were rich in SFAs, MUFAs, n-6PUFAs and n3PUFAs. A control diet was normal chow (NC) and the other control diet (namely Iso-C) was isocaloric to high fat diets. Food intake (A) and body weight gain (B) were noted during the experiment. Letters a, b, c, and d denote that the indicated data in group-NC were greater (P < 0.05) than data from the other groups at the same time points: a. compared to groups-MUFA and n6PUFA; b. compared to all the other groups; c. compared to groups-SFA and -n6PUFA; d. compared to groups-IsoC, -SFA and -n6PUFA.
intake (Fig. 3A) and body-weight gain (Fig. 3B), as compared to group-NC. FA profiling in tumor-carrying mice Before we investigated whether increased dietary FAs changed fatty acid profiles, we had to choose FA data from one control group as baselines. Thus, we compared liver and skeletal muscle FA profiles between group-NC and group-IsoC. The profiles were similar, except that group-IsoC had decreased n3PUFAs in liver and increased MUFAs in skeletal muscle (Fig. 4A). We decided to use group-NC data as baselines. Data from HFD groups were converted to fold increase (or decrease), using the corresponding results in group-NC as 1. In the liver, SFAs and MUFAs were not increased when the same FAs were increased in diets (Fig. 4B). However, the SFA diet made of cocoa oil increased MUFAs in the liver (Fig. 4B). Considering that MUFAs accounted for 33% (w/w) of cocoa oil [1], it is not inconceivable that cocoa oil-based diet increased MUFAs in the liver. Importantly, HFDs rich in n6PUFAs and n3PUFAs increased the same PUFAs in the liver (Fig. 4B). When mice were fed with diets rich in SFAs, MUFAs or
Fig. 4. Fatty acid profiles in the liver and skeletal muscle of nude mice. Fatty acids (FAs) were determined by chromatography in the liver and skeletal muscle. Results were aggregated in groups-SFA, -MUFA, -n6PUFA, and n3PUFA. A. Liver and skeletal muscle FA profiles were compared between groups-NC and -IsoC. B and C: To study FA profiles in four HFD groups, FA data in these groups were calculated as fold increase (or decrease), using data from group-NC as 1. FA profiles in the liver (B) and skeletal muscle (C) were compared among the four HFD groups. **P < 0.01, ***P < 0.001.
n6PUFAs, the same types of FAs were increased in skeletal muscle (Fig. 4C). When mice were fed a cocoa-oil-based diet, MUFAs were increased in skeletal muscle (Fig. 4C), as in the liver. The HFD made of flaxseed oil increased n6PUFAs but not n3PUFAs in skeletal muscle (Fig. 4C). Considering that n6PUFAs accounted for 14% of flaxseed oil, the present increase in n6PUFAs is not inconceivable. Tumor grafts Tumors from groups-SFA, -MUFA, and -n6PUFA apparently increased in weight (Table 2). This increase was statistically significant in group-MUFA and marginally so in groups-SFA and -n6PUFA (Table 2). When tumor sections were analyzed, the mean total area in group-n6PUFA was significantly bigger than that in groups-IsoC and -n3PUFA (Table 2).
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Fig. 5. Whole-section views of tumor grafts. Pancreatic tumors were stained by H&E. Each section was photographed region by region. Photos were merged to reconstitute the image of the whole section. Necrotic regions were stained in dark red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
All tumors contained different amounts of necrotic regions. Fig. 5 shows representative necrotic regions in different groups. Necrotic lesions in groups-NC, -IsoC, and -SFA (Fig. 5A–C) were relatively greater than those in group-MUFA and -n6PUFA (Fig. 5D and E, Table 2). Tumors from group-n3PUFA were small in size, but they also contained significant amounts of necrotic regions (Fig. 5F, Table 2). We defined necrotic areas bigger than 25% of the total tumor area as big necrosis. Such lesions were more frequent in groups-SFA and -n3PUFA (Table 2). When we subtracted necrotic areas from total areas, group-n6PUFA had bigger tumor areas than groups-IsoC and -n3PUFA (Table 2). Liver metastasis was the most frequent in group-n6PUFA and the least so in group-n3PUFA (Table 2). Considering that when mice were fed with diets rich in n6PUFAs and n3PUFAs, the same types of PUFAs were increased in the liver, the present results suggest that the dietinduced change in hepatic FA profiles may have effects on the likelihood of liver metastasis. When tumor sections were stained with Oil-Red-O, lipid droplets were scarce in groups-NC and -IsoC (Fig. 6A and B). In contrast, lipid droplets were numerous in tumors from groups-SFA and -MUFA (Fig. 6C and D). Lipid droplets in tumors from groups-n6 and -n3PUFA were increased moderately (Fig. 6E and F) compared to control groups. When HIF-1α and PCNA were stained in consecutive sections, they were found to be co-expressed (Fig. 7). In addition, the co-expression was the most frequent in tumors from groups-MUFA and -n6PUFA. When whole-cell proteins were extracted, pancreatic tumor grafts from group-MUFA yielded little proteins for an unknown reason. Unfortunately, these tumors were excluded from the assay. Fig. 8A shows representative results for the remaining groups. First, MCAD expression was increased in HFD groups compared to control groups (Fig. 8A). Further, COX-2, HIF-1, VEGF, and cav-1 were increased in examined HFD groups and in the Iso-C group (Fig. 8A), which suggests that the increase may depend on the high-energy contents of the diets, rather than high dietary fat. Relatively speaking, tumors from group-SFA had greater expression of COX-2, VEGF, and cav-1 than tumors from the other groups (Fig. 8A). We also compared VEGF expression in metastatic tumors that were derived from groups-n6PUFA and -n3PUFA. The tumors from group-n6PUFA had greater
VEGF expression than those from group-n3PUFA (Fig. 8B). The data suggest that dietary increases in n6- and n3-PUFAs regulated liver metastasis in opposite directions. Discussion In the present study, the five diets that were produced by Harlan Laboratories had 30% more energy than normal chow (NC). In keeping with this, mice in group-NC ate 30% more. This being the case, it appears that mice in all six groups had similar energy homeostasis. However, the mice in group-NC had greater body weight than those in the other groups. It suggests that NC diet was better utilized than were the high-energy diets. Mechanisms underlying the difference are unclear. Of note, the high-energy diets had more cellulose than NC diet (see Table 3 and ref. 3). The increased cellulose may stimulate intestinal movement and decrease nutrient adsorption. When certain types of FAs were increased in the diet, the same FAs were increased in the liver or in skeletal muscle. The dietinduced increase in hepatic n6PUFAs was associated with an increase in liver metastasis, whereas the diet-induced increase in n3PUFAs decreased liver metastasis. These suggest that dietary PUFAs may regulate hepatic metastasis by changing hepatic FA profiles. SFA-, MUFA- and n6PUFA-enriched diets increased pancreatic cancer cells in vivo. Previously, we used the same pancreatic cancer model to study the effects of a diet containing 21% fat from cocoa and found that the intake of the diet increased the weight of tumor grafts [3]. In previous studies, n6PUFAs either increased or decreased pancreatic cancer cells in vitro [11,12]. In the present study, n6PUFAs had no effects on HPAF cells in vitro, which is in contrast to the stimulating effect seen in the n6PUFA-enriched diet on HPAF cells in vivo. Reasons for the discrepancy are unclear. A tentative explanation is that the stimulating effect of the n6PUFA-diet was derived from n6PUFAs other than those tested in the present study in vitro. When fish-oil-based n3PUFA diets were used in carcinogen-treated hamsters, pancreatic carcinogenesis was inhibited in some but not all studies [6–8]. Although flaxseed-based
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Fig. 6. Lipid droplets in tumor grafts. Tumor sections were stained with Oil-Red-O to show intracellular lipid droplets. Representative sections are shown for the tumors whose carriers were fed with NC (A), Iso-C (B), SFA (C), MUFA (D), n6PUFA (E), and n3PUFA (F) diets. Arrows in A and B show lipid droplets that were scarce in tumors from the control groups.
n3PUFA diets have not been used in pancreatic cancer models, they indeed inhibited non-pancreatic cancers [9,10]. In the present study, the intake of flaxseed-based diet was associated with decreased liver metastasis and increased tumor necrosis. Previously, we showed that the diet rich in SFAs increased MCAD expression in pancreatic cancer cells [3], which suggests that SFAs may stimulate β-oxidation in pancreatic cancer cells. In the present study, we saw that the intake of diets rich in n6PUFAs and n3PUFAs increased tumoral MCAD as well. This suggests that β-oxidation is a common pathway by which different types of FAs stimulated pan-
creatic cancer cells. In vitro, etomoxir inhibited the stimulating effects of SFAs and MUFAs, which is another piece of evidence that suggests that β-oxidation is involved in FA-induced stimulation on pancreatic cancer cells. Interestingly, etomoxir also removed inhibiting effect of DHA on pancreatic cancer cells, which suggests that β-oxidation is also involved in DHA-induced inhibition of pancreatic cancer cells. In the same previous study, the intake of cocoabased HFD also increased intracellular lipid droplets, which suggests that an increase in dietary SFAs may augment lipid metabolisms in pancreatic cancer cells. In the present study, increased lipid
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Fig. 7. Co-expression of HIF-1α and PCNA in tumor graft. Consecutive sections from a tumor in group-MUFA were stained by immunohistochemistry to show HIF-1α (A) and proliferating cell nuclear antigen (PCNA) (B).
droplets were seen in tumors from all HFD groups, which suggests that an increase in any of four major types of fatty acids may augment lipid metabolisms in pancreatic cancer cells. According to a previous study, dietary n6PUFAs increased PGE2 in pancreatic cancer cells and thereby stimulated HIF-1α expression. In the present study, we found by histology that HIF-1α was co-expressed with PCNA in the same cancer cells, suggesting that HIF-1α expression was in favor of cancer-cell proliferation. In Western blotting assay, HIF-1α expression was increased consistently in examined tumors whose carriers were fed with high-energy diets. The HIF-1α expression was also associated with increased expression of two HIF-1-regulated proteins namely VEGF and cav-1. The mechanism of HIF-1α-induced regulation on pancreatic cancer cells requires further investigation. In summary, we systematically investigated four major types of dietary FAs for their possible involvement in pancreatic cancer. We found evidence that suggests that increased SFAs, MUFAs and n6PUFAs augmented lipid metabolisms so as to increase the via-
bility of pancreatic cancer cells. In contrast, increased n3PUFAs augmented lipid metabolisms so as to promote pancreatic cancer cell death. Thus, four types of FAs may exert differential impact on pancreatic cancer cells, though they all increase lipid metabolisms in the cells. It is the specific metabolic pathway for each FA type that determines how this FA type regulates pancreatic cancer cells. More studies are required to investigate these specific metabolic pathways by which different types of fatty acids regulate pancreatic cancer cells in different ways.
Acknowledgement We thank Tianjin Municipal Government (0190020104) for financial support.
Conflict of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Table 3 Ingredients in four high fat diets and an iso-caloric normal-fat dieta.
Fig. 8. Protein expression by tumor cells. MCAD, COX-2, HIF-1α, VEGF, and cav-1 expression was determined by Western blotting in pancreatic tumors (A). VEGF expression was determined by Western blotting in 5 cases of liver metastasis, 3 from group-n6PUFA and 2 from group-n3PUFA (B).
Casein L-cystine Corn starch Vegetable oilb Maltodextrin Sucrose Cellulose Mineral mix Calcium phosphate Vitamin mix Choline bitartrate Antioxidants a b
Four high fat diets
Iso-caloric control (Iso-C) diet
Harlan No. 09578-09581 (g/kg)
Harlan No. 09582 (g/kg)
200 3 248 150 150 100 100 35 1.7 10 2.5 0.03
200 3 448 50 100 100 50 35 1.7 10 2.5 0.01
All were produced by Harlan Laboratories Inc. See Table 1 for fat origins.
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[13] L.S. Pike, A.L. Smift, N.J. Croteau, D.A. Ferrick, M. Wu, Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells, Biochim. Biophys. Acta 1807 (2011) 726–734. [14] F. Ouali, F. Djouadi, C. Merlet-Benichou, J. Bastin, Dietary lipids regulate beta-oxidation enzyme gene expression in the developing rat kidney, Am. J. Physiol. 275 (1998) F777–F784. [15] G.L. Semenza, G.L. Wang, A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation, Mol. Cell. Biol. 12 (1992) 5447–5454. [16] H. Zhong, A.M. De Marzo, E. Laughner, M. Lim, D.A. Hilton, D. Zagzag, et al., Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases, Cancer Res. 59 (1999) 5830–5835. [17] J.W. Lee, S.H. Bae, J.W. Jeong, S.H. Kim, K.W. Kim, Hypoxia-inducible factor (HIF-1)alpha: its protein stability and biological functions, Exp. Mol. Med. 36 (2004) 1–12. [18] H. Funahashi, M. Satake, S. Hasan, H. Sawai, R.A. Newman, H.A. Reber, et al., Opposing effects of n-6 and n-3 polyunsaturated fatty acids on pancreatic cancer growth, Pancreas 36 (2008) 353–362. [19] A. Greenhough, H.J.M. Smartt, A.E. Moore, H.R. Roberts, A.C. Williams, C. Paraskeva, et al., The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment, Carcinogenesis 30 (2009) 377–386. [20] A. Kaidi, D. Qualtrough, A.C. Williams, C. Paraskeva, Direct transcriptional up-regulation of cyclooxygenase-2 by hypoxia-inducible factor (HIF)-1 promotes colorectal tumor cell survival and enhances HIF-1 transcriptional activity during hypoxia, Cancer Res. 66 (2006) 6683–6691. [21] D.A. Brown, Lipid droplets: Protein floating on a pool of fat, Curr. Biol. 11 (2001) R446–R449. [22] M. Suzuoki, M. Miyamoto, K. Kato, K. Hiraoka, T. Oshikiri, Y. Nakakubo, et al., Impact of caveolin-1 expression on prognosis of pancreatic ductal adenocarcinoma, Br. J. Cancer 87 (2002) 1140–1144. [23] A.K. Witkiewicz, K.H. Nguyen, A. Dasgupta, E.P. Kennedy, C.J. Yeo, M.P. Lisanti, et al., Co-expression of fatty acid synthase and caveolin-1 in pancreatic ductal adenocarcinoma: implications for tumor progression and clinical outcome, Cell Cycle 7 (2008) 3021–3025. [24] Y. Wang, O. Roche, C. Xu, E.H. Moriyama, P. Heir, J. Chung, et al., Hypoxia promotes ligand-independent EGF receptor signaling via hypoxia-inducible factor-mediated upregulation of caveolin-1, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 4892–4897. [25] H. Xiao, S. Li, D. Zhang, T. Liu, M. Yu, F. Wang, Separate and concurrent use of 2-deoxy-D-glucose and 3-bromopyruvate in pancreatic cancer cells, Oncol. Rep. 29 (2013) 329–334.
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Original Articles
Four types of fatty acids exert differential impact on pancreatic cancer growth Ming Yu a, Hongyi Liu b, Yijie Duan a,b, Dapeng Zhang b, Shasha Li c, Feng Wang b,d,* a
Department of Nutrition and Food Hygiene, School of Public Health, Tianjin Medical University, Tianjin 300070, China Tianjin Institute of Integrative Medicine for Acute Abdominal Diseases, Nankai Hospital, Tianjin 300100, China Graduate School, Tianjin Medical University, Tianjin 300070, China d Department of Surgery, Karolinska University Hospital, Huddinge 14186, Sweden b c
A R T I C L E
I N F O
Article history: Received 20 December 2014 Received in revised form 23 January 2015 Accepted 3 February 2015 Keywords: Pancreatic cancer High fat diet Fatty acids Orthotopic xenograft model
A B S T R A C T
Increased fatty acids (FAs) regulate pancreatic cancer progression, however, the detailed mechanism is not clear, and different forms of FAs may play diversified roles in pancreatic cancer. To elucidate the underlying mechanism, we compared the effects of four major types of FAs on pancreatic cancer growth both in cell culture and in a mouse model. HPAF pancreatic cancer cells were implanted in nude mice for 14 weeks, and the mice were fed with four different high-fat/high-energy diets (15% fat, 4 kcal/g), an iso-caloric diet (5% fat, 4 kcal/g) and a normal diet (4% fat, 3 kcal/g). The high fat diets were rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and n-6 and n-3 polyunsaturated fatty acids (n6- and n3PUFAs), respectively. While n3PUFA diet decreased tumor viability, the other high fat diets stimulated tumor viability by apparently different mechanisms. For instance, xenografts whose carriers were fed with SFA diet had marked expression of cancer-related proteins and lipid droplets. Although mice that were fed with MUFA- and n6PUFA diets had pancreatic tumors of similar size, liver metastasis occurred more frequently in those with the n6PUFA diet. In experiments in vitro, the HPAF-cell population was increased by SFAs and MUFAs, decreased by n3PUFAs and not changed by n6PUFAs. In conclusion, different fatty acids have different impact on pancreatic cancer cells. The effects of fatty acids on pancreatic cancer cells were consistent in vivo and in vitro except that n6PUFAs only had regulatory effects in vivo. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Fatty acids (FAs) are principal constituents of dietary fat, so high fat diets (HFDs) increase the intake of FAs. By structure, dietary FAs are mostly of three major types namely saturated FA (SFA), monounsaturated FA (MUFA) and n-6 polyunsaturated FA (n6PUFA) [1]. In flaxseed and in fish oil, n-3 polyunsaturated FAs (n3PUFAs) also contribute as principal FAs [1]. Previous studies have shown that increased FAs regulate pancreatic cancer [2–8]. For instance, diets rich in SFAs and n6PUFAs accelerate the process of pancreatic carcinogenesis in the animal, whereas HFDs made of fish oil and thus rich in n3PUFAs sometimes inhibit the carcinogenetic process [3–7]. To date, it remains unclear whether dietary increase in MUFAs regulates pancreatic cancer. Similarly unclear is whether a diet rich in flaxseed-derived n3PUFAs regulates pancreatic cancer, though such a diet inhibited non-pancreatic cancers in animals [9,10]. When pancreatic cancer cells were exposed to different FAs in vitro, their growth
* Corresponding author. Tel. +862227435362; fax: +862227435362. E-mail address: [email protected] (F. Wang).
was stimulated by SFAs and MUFAs, inhibited by n3PUFAs, and either stimulated or inhibited by n6PUFAs [11,12]. Mechanistically, FAs may regulate pancreatic cancer cells by different pathways. First of all, FAs are energy substrates when they are degraded by β-oxidation in the mitochondria. Thus, increased FAs may improve energy production in cancer cells [3]. In keeping with this notion, inhibitors of β-oxidation such as etomoxir inhibited tumor cells [13]. Further, medium-chain acyl-CoA dehydrogenase (MCAD) is a key regulator of β-oxidation [14], and the enzyme was increased in human pancreatic cancer cells carried by nude mice that were fed a diet rich in SFAs [3]. As a second mechanism, FAs may regulate pancreatic cancer cells by modulating hypoxiainducible factor-1 (HIF-1) expression. HIF-1 is a transcription factor composed of HIF-1α and HIF-1β subunits [15]. Because HIF-1α is degraded in the presence of oxygen, mammalian cells normally have no HIF-1. However, cancer cells frequently express HIF-1α, hence HIF-1 [16]. The proteins encoded by HIF-1 target genes include glucose transporters and growth factors such as vascular endothelial growth factors (VEGF). Thus, HIF-1 expression increases cell viability [17]. According to a recent study, n6PUFAs increase prostaglandin E2 (PGE2) in pancreatic cancer cells and thereby stimulate HIF-1α expression and increase cell viability [18]. In contrast,
http://dx.doi.org/10.1016/j.canlet.2015.02.002 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
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n3PUFAs increase prostaglandin E3 (PGE3) and thus inhibit HIF-1α expression and decrease cell viability [18]. The PUFA-induced effects may be because n6- and n3PUFAs increase cyclooxygenase-2 (Cox2) expression and thus increase prostaglandin synthesis [18–20]. Lipid droplets are a hallmark of lipid metabolism and their surface is coated by two groups of proteins namely perilipins and caveolins [21]. We previously demonstrated that one HFD rich in SFAs increased lipid droplets in pancreatic cancer cells [3]. In addition, increased caveolin-1 (cav-1) is found as a prognostic factor for pancreatic cancer [22,23]. Recently, the gene of cav-1 was added to the list of HIF-1 target genes [24]. We undertook the present study to investigate how different types of dietary FAs regulate pancreatic cancer cells. We started the study with two experiments in vitro. In the first experiment, we examined cell viability in three pancreatic-cancer cell lines (namely HPAF, MiaPaCa2, and Panc-1) when they were incubated with a fattyacid mix. In the next experiment, eight FAs (two for each FA type) were studied individually for their effects on HPAF cells. In a nudemouse experiment, we compared four HFDs that were enriched with SFAs, MUFAs, n6PUFAs and flaxseed-derived n3PUFAs, respectively. In the 14-week experiment, six groups of nude mice that carried HPAF cells were fed with these HFDs and two control diets, respectively. Tumor grafts and their carriers were examined for the effects that were derived from increased dietary fatty acids. Materials and methods Materials The human pancreatic cancer cell lines of HPAF, MiaPaCa2, and Panc-1 were bought from American Type Culture Collection (Rockville, MD, USA). Eight fatty acids namely palmitic acid, lauric acid, oleic acid, palmitoleic acid, linoleic acid, arachidonic acid (ARA), linolenic acid, and docosahexaenoic acid (DHA) as well as a solution of oleic acid (800 μg/ml) and linoleic acid (800 μg/ml) were bought from Sigma Aldridge (St. Louis, MO, USA). Six diets were prepared for an animal experiment, including four HFDs, an iso-caloric control (Iso-C) diet and a normal control (NC) diet. The HFDs and the Iso-C diet were designed and produced by Harlan Laboratories Inc. (Madison, WI, USA). The NC diet was described previously [3]. Cell proliferation assay HPAF, MiaPaCa2, and Panc-1 cells were cultured in 96-well plates using Dulbecco Modified Essential Media (DMEM) with 10% fetal bovine serum. When cells were 90% confluent, they were incubated for 24 hours in serum-free DMEM wherein an oleic acid-linoleic acid solution was diluted in different concentrations (3 μM– 100 μM). In an experimental group, media contained both fatty acids and 100 μM etomoxir. Media for control cells contained no fatty acids or etomoxir. After incubation, the cell population was determined by MTT assay as we described elsewhere [25]. In the next experiment, HPAF cells were seeded in 96-well plates and incubated in serum-containing DMEM. When cells were 90% confluent, they were incubated for 24 hours in serum-free DMEM supplemented with a fatty acid in the final concentrations of 4-, 20-, and 60 μM. Eight FAs were tested, with each type of fatty acids being represented by two FAs. In some groups, culture media also contained 100 μM etomoxir. Cells in the control group were incubated in the absence of fatty acids and etomoxir. After incubation, cell population was determined by MTT assay. Transplantation of pancreatic cancer cells in nude mice After arrival, all 60 male nude mice (5 weeks old, weighing 15–20 g) were fed with NC diet for two weeks. The composition of the NC diet was described elsewhere [3]. Mice were randomly divided into six groups (10 in each). One group was still fed with NC diet, and the other five groups were fed with four HFDs and the Iso-C diet, respectively (Tables 1 and 2). The four HFDs were made of four different oils, more than 50% of whose weight came from a single type of FA (Table 1, ref. 1). One week after the feeding was started, 3 × 106 HPAF cells were transplanted in the pancreas [3]. During the experiment, food intake and body weight were noted on a weekly basis. In the end of week 14, mice were sacrificed under anesthesia. Tumor grafts, liver, and hind-leg muscle were removed and stored at −80 °C. Effects of HFDs on hepatic and skeletal-muscle FA profiles Liver and skeletal-muscle samples (0.5–1.0 g) were homogenized in a mix of benzene (1 ml) and petroleum ether (1 ml). Homogenates were centrifuged (5000 RPM, 15 min) and supernatants mixed with 2 ml methanol (0.5 M NaOH). After a
Table 1 Six groups of nude mice and basic information of their diets. Groups
Mice-NC Mice-SFA Mice-MUFA Mice-n6PUFA Mice-n3PUFA Mice-IsoC
Diets Name
Fat content
Principal fatty acids (ref. 1)
Energy
NC SFA MUFA n6PUFA n3PUFA Iso-C
4% soybean oil 15% cocoa oil 15% olive oil 15% soybean oil 15% flaxseed oil 5% soybean oil
n6PUFAs SFAs MUFAs n6PUFAs n3PUFAs n6PUFAs
3 kcal/g 4 kcal/g 4 kcal/g 4 kcal/g 4 kcal/g 4 kcal/g
30-min incubation at 30 °C, water was added to retain water-soluble contaminants. The top layer of organic solvents was removed. Fatty acid methyl esters were separated by gas–liquid chromatography on a 30-m DB-WAX capillary column, using Agilent 7890-5975C system. Fatty acids were identified by retention time, using fatty acid standards as reference. A total of 25 FAs were identified in different samples. In later analysis, these FAs were grouped according to their types, giving SFA-, MUFA-, n6PUFA-, and n3PUFA groups. Histology Frozen tumors were cut along the longest axis (8 μm thick) and stained with hematoxylin and eosin (H&E). In a microscope (Leica DM 4000B), sections were photographed region by region, using a CCD camera (Leica DFC 500). Resulting images were merged using the LAS software. Total areas and necrotic areas were quantified in reconstituted tumor image, using the software of ImageJ (NIH, version 2.1). In other sections, intracellular lipid droplets were stained with Oil-Red-O. HIF-1α and proliferating cell nuclear antigen (PCNA) were stained by immunocytochemistry in consecutive sections, using anti-HIF-1α (Novus 100449) and anti-PCNA (Abcam, #912) antisera and a Vector kit (Burlingame, CA, USA). Western blotting Tumor samples were homogenized. Whole-cell proteins were extracted, separated in SDS gel, and transferred to PVDF membranes. The membranes were incubated with antisera against MCAD (Abcam 13677), COX-2 (ab15191), HIF-1α (Novus 100– 449), VEGF (BD555036), cav-1 (Abcam 32577), and β-actin (Abcam 8227). They were incubated with appropriate secondary antisera and then treated with chemiluminescence detection reagents. Specific blots for target proteins were recorded in an image analysis system. Statistics Data are means ± SEM. When three or more groups were involved, results were analyzed using analysis of variance (ANOVA). When fewer groups were involved, Student’s t test was used. P < 0.05 was considered significant.
Results Effects of fatty acids in vitro The mix of oleic and linoleic acids dose-dependently increased HPAF and MiaPaCa2 cells (Fig. 1). However, the effects did not exist anymore in the presence of etomoxir (Fig. 1). The same mix of fatty acids had no effects on Panc-1 cells (Fig. 1). Fig. 2 shows the effects of single FAs on HPAF cells. The eight FAs tested were derived from four FA types with each type being represented by two FAs. Control cells were incubated in the absence of FAs. MTT values seen in control cells were taken as baselines to which MTT values from the other cells were related. The baseline (100%) bars for control cells were omitted in Fig. 2. Both SFAs tested increased cell population (Fig. 2A), but the addition of etomoxir in culture media removed the stimulating effect of SFAs. Both MUFAs tested increased HPAF cells as well, and the stimulating effect was also inhibited by etomoxir (Fig. 2B). Neither of the two n6PUFAs tested induced any significant effects on HPAF cells (Fig. 2C). The growth of HPAF cells was inhibited by two n3PUFAs namely linolenic acid and DHA, and the addition of etomoxir removed the inhibiting effects of DHA but not linolenic acid (Fig. 2D).
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Table 2 Six groups of nude mice and their tumor grafts. Groups
NC Iso-C SFA MUFA n6PUFA n3PUFA a b c d e
Survivors (n)
Tumor grafts
6 8 6 7 6 9
Liver metastasis (n)
Weight (mg)
Total area (pixel, 1 × 106)
Normal area (pixel, 1 × 106)
Big necrosisa (n)
465 ± 100 356 ± 49 592 ± 100b 597 ± 96c 567 ± 100d 364 ± 60
1.3 ± 0.3 1.3 ± 0.1 1.6 ± 0.2 1.7 ± 0.3 2.3 ± 0.3c 1.6 ± 0.1
0.9 ± 0.3 0.9 ± 0.1 1.0 ± 0.2 1.5 ± 0.3e 1.7 ± 0.3c 0.9 ± 0.1
3 4 5 1 3 6
2 (33%) 3 (38%) 4 (67%) 2 (29%) 5 (83%) 2 (22%)
Necrotic areas were >25% of the tumor area. P = 0.07 vs. group-IsoC. P < 0.05 vs. groups-IsoC and n3PUFA. P = 0.08 vs. groups-IsoC and n3PUFA. P = 0.06–P = 0.09 vs. groups-IsoC and n3PUFA.
were lost in the last four weeks when they were either found dead or were sacrificed for looking sick. During the experiment, body weight and food intake were checked weekly. Results from three or four neighboring weeks were pooled, so weekly food intake and body weight gain were averaged in the periods. Mice fed with five high-energy diets showed decreases in both food
Animal survival and nutritional status
% of ctrl (0 µM) value
Two mice in group- SFA died the day after cancer-cell transplantation and were removed from the study. Forty-two out of the 58 remaining mice (72%) survived experiment, with each group having 6–9 mice (Table 2). The 16 non-survivors
150
Panc-1
MiaPaCa2
HPAF-II
* **
#
**
** **
#
**
100 50
16 9 8 15 11
16 7
8
0
6 13 25 50 50+ 100 eto
8 13 14 11 9
14 7
8
0
6 13 25 50 50+ 100 eto
7 12 13 11 9
13 7
7
0
6 13 25 50 50+ 100 eto
0 3
3
3
fatty acid concentrations (µM) Fig. 1. Effects of oleic and linoleic acids on three pancreatic cancer cells. HPAF, MiaPaCa2, and Panc-1 cells were incubated for 24 h in media containing a mix of oleic acid and linoleic acid. Final concentrations of fatty acids are shown in the figure. In a group, culture media contained both fatty acids (50 μM) and etomoxir (100 μM). Cell population was measured in MTT assay. Numbers in bars indicate the times (n) of experiments. Each time, every condition was tested in three wells of cells. *P < 0.05 and **P < 0.01, compared to control cells; #P < 0.05, compared to cells incubated with 50 μM fatty acids in the absence of etomoxir.
palmitic
100 50
9
B 150
lauric
*
#
9
9
*
#
*
9
9
8 10 9
#
*
#
9 11
% of ctrl
% of ctrl
A 150
linoleic
D 125
ARA
100 75 50
14 15
9 14
9
25
* *
11 13 12
9
9
11 13
# 9 13
9
0 4 20 20+ 60 60+ eto eto
11 13
9 13 9
% of ctrl
% of ctrl
C 125
50
palmitoleic
*
100
0 4 20 20+ 60 60+ eto eto
oleic
4 20 20+ 60 60+ eto eto
4 20 20+ 60 60+ eto eto
linolenic
DHA
100
* *
75 50
11
25
0
13 9
** ** 13 9
*
*
11 13 9 12 9
0 4
20 20+ 60 60+ eto eto
4 20 20+ 60 60+ eto eto
4
20 20+ 60 60+ eto eto
4 20 20+ 60 60+ eto eto
fatty acid concentrations (µM) Fig. 2. Effects of eight fatty acids on HPAF cells. HPAF cells were incubated for 24 h in media that contained eight fatty acids from SFA (A), MUFA (B), n6PUFA (C), and n3PUFA (D) groups. Each fatty acid was tested in different concentrations in the absence or presence of etomoxir (100 μM). Cell population was determined in MTT assay. Numbers in bars indicate experiment times. Each time, every condition was tested in three wells of cells. *P < 0.05 and **P < 0.01, compared to control cells. #P < 0.05, compared to cells incubated with the same concentration of fatty acids in the absence of etomoxir.
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Fig. 3. Food intake and body weight in nude mice. Six groups of nude mice carrying HPAF pancreatic cancer cells were fed with four high-fat diets and two control diets. The high fat diets were rich in SFAs, MUFAs, n-6PUFAs and n3PUFAs. A control diet was normal chow (NC) and the other control diet (namely Iso-C) was isocaloric to high fat diets. Food intake (A) and body weight gain (B) were noted during the experiment. Letters a, b, c, and d denote that the indicated data in group-NC were greater (P < 0.05) than data from the other groups at the same time points: a. compared to groups-MUFA and n6PUFA; b. compared to all the other groups; c. compared to groups-SFA and -n6PUFA; d. compared to groups-IsoC, -SFA and -n6PUFA.
intake (Fig. 3A) and body-weight gain (Fig. 3B), as compared to group-NC. FA profiling in tumor-carrying mice Before we investigated whether increased dietary FAs changed fatty acid profiles, we had to choose FA data from one control group as baselines. Thus, we compared liver and skeletal muscle FA profiles between group-NC and group-IsoC. The profiles were similar, except that group-IsoC had decreased n3PUFAs in liver and increased MUFAs in skeletal muscle (Fig. 4A). We decided to use group-NC data as baselines. Data from HFD groups were converted to fold increase (or decrease), using the corresponding results in group-NC as 1. In the liver, SFAs and MUFAs were not increased when the same FAs were increased in diets (Fig. 4B). However, the SFA diet made of cocoa oil increased MUFAs in the liver (Fig. 4B). Considering that MUFAs accounted for 33% (w/w) of cocoa oil [1], it is not inconceivable that cocoa oil-based diet increased MUFAs in the liver. Importantly, HFDs rich in n6PUFAs and n3PUFAs increased the same PUFAs in the liver (Fig. 4B). When mice were fed with diets rich in SFAs, MUFAs or
Fig. 4. Fatty acid profiles in the liver and skeletal muscle of nude mice. Fatty acids (FAs) were determined by chromatography in the liver and skeletal muscle. Results were aggregated in groups-SFA, -MUFA, -n6PUFA, and n3PUFA. A. Liver and skeletal muscle FA profiles were compared between groups-NC and -IsoC. B and C: To study FA profiles in four HFD groups, FA data in these groups were calculated as fold increase (or decrease), using data from group-NC as 1. FA profiles in the liver (B) and skeletal muscle (C) were compared among the four HFD groups. **P < 0.01, ***P < 0.001.
n6PUFAs, the same types of FAs were increased in skeletal muscle (Fig. 4C). When mice were fed a cocoa-oil-based diet, MUFAs were increased in skeletal muscle (Fig. 4C), as in the liver. The HFD made of flaxseed oil increased n6PUFAs but not n3PUFAs in skeletal muscle (Fig. 4C). Considering that n6PUFAs accounted for 14% of flaxseed oil, the present increase in n6PUFAs is not inconceivable. Tumor grafts Tumors from groups-SFA, -MUFA, and -n6PUFA apparently increased in weight (Table 2). This increase was statistically significant in group-MUFA and marginally so in groups-SFA and -n6PUFA (Table 2). When tumor sections were analyzed, the mean total area in group-n6PUFA was significantly bigger than that in groups-IsoC and -n3PUFA (Table 2).
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Fig. 5. Whole-section views of tumor grafts. Pancreatic tumors were stained by H&E. Each section was photographed region by region. Photos were merged to reconstitute the image of the whole section. Necrotic regions were stained in dark red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
All tumors contained different amounts of necrotic regions. Fig. 5 shows representative necrotic regions in different groups. Necrotic lesions in groups-NC, -IsoC, and -SFA (Fig. 5A–C) were relatively greater than those in group-MUFA and -n6PUFA (Fig. 5D and E, Table 2). Tumors from group-n3PUFA were small in size, but they also contained significant amounts of necrotic regions (Fig. 5F, Table 2). We defined necrotic areas bigger than 25% of the total tumor area as big necrosis. Such lesions were more frequent in groups-SFA and -n3PUFA (Table 2). When we subtracted necrotic areas from total areas, group-n6PUFA had bigger tumor areas than groups-IsoC and -n3PUFA (Table 2). Liver metastasis was the most frequent in group-n6PUFA and the least so in group-n3PUFA (Table 2). Considering that when mice were fed with diets rich in n6PUFAs and n3PUFAs, the same types of PUFAs were increased in the liver, the present results suggest that the dietinduced change in hepatic FA profiles may have effects on the likelihood of liver metastasis. When tumor sections were stained with Oil-Red-O, lipid droplets were scarce in groups-NC and -IsoC (Fig. 6A and B). In contrast, lipid droplets were numerous in tumors from groups-SFA and -MUFA (Fig. 6C and D). Lipid droplets in tumors from groups-n6 and -n3PUFA were increased moderately (Fig. 6E and F) compared to control groups. When HIF-1α and PCNA were stained in consecutive sections, they were found to be co-expressed (Fig. 7). In addition, the co-expression was the most frequent in tumors from groups-MUFA and -n6PUFA. When whole-cell proteins were extracted, pancreatic tumor grafts from group-MUFA yielded little proteins for an unknown reason. Unfortunately, these tumors were excluded from the assay. Fig. 8A shows representative results for the remaining groups. First, MCAD expression was increased in HFD groups compared to control groups (Fig. 8A). Further, COX-2, HIF-1, VEGF, and cav-1 were increased in examined HFD groups and in the Iso-C group (Fig. 8A), which suggests that the increase may depend on the high-energy contents of the diets, rather than high dietary fat. Relatively speaking, tumors from group-SFA had greater expression of COX-2, VEGF, and cav-1 than tumors from the other groups (Fig. 8A). We also compared VEGF expression in metastatic tumors that were derived from groups-n6PUFA and -n3PUFA. The tumors from group-n6PUFA had greater
VEGF expression than those from group-n3PUFA (Fig. 8B). The data suggest that dietary increases in n6- and n3-PUFAs regulated liver metastasis in opposite directions. Discussion In the present study, the five diets that were produced by Harlan Laboratories had 30% more energy than normal chow (NC). In keeping with this, mice in group-NC ate 30% more. This being the case, it appears that mice in all six groups had similar energy homeostasis. However, the mice in group-NC had greater body weight than those in the other groups. It suggests that NC diet was better utilized than were the high-energy diets. Mechanisms underlying the difference are unclear. Of note, the high-energy diets had more cellulose than NC diet (see Table 3 and ref. 3). The increased cellulose may stimulate intestinal movement and decrease nutrient adsorption. When certain types of FAs were increased in the diet, the same FAs were increased in the liver or in skeletal muscle. The dietinduced increase in hepatic n6PUFAs was associated with an increase in liver metastasis, whereas the diet-induced increase in n3PUFAs decreased liver metastasis. These suggest that dietary PUFAs may regulate hepatic metastasis by changing hepatic FA profiles. SFA-, MUFA- and n6PUFA-enriched diets increased pancreatic cancer cells in vivo. Previously, we used the same pancreatic cancer model to study the effects of a diet containing 21% fat from cocoa and found that the intake of the diet increased the weight of tumor grafts [3]. In previous studies, n6PUFAs either increased or decreased pancreatic cancer cells in vitro [11,12]. In the present study, n6PUFAs had no effects on HPAF cells in vitro, which is in contrast to the stimulating effect seen in the n6PUFA-enriched diet on HPAF cells in vivo. Reasons for the discrepancy are unclear. A tentative explanation is that the stimulating effect of the n6PUFA-diet was derived from n6PUFAs other than those tested in the present study in vitro. When fish-oil-based n3PUFA diets were used in carcinogen-treated hamsters, pancreatic carcinogenesis was inhibited in some but not all studies [6–8]. Although flaxseed-based
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Fig. 6. Lipid droplets in tumor grafts. Tumor sections were stained with Oil-Red-O to show intracellular lipid droplets. Representative sections are shown for the tumors whose carriers were fed with NC (A), Iso-C (B), SFA (C), MUFA (D), n6PUFA (E), and n3PUFA (F) diets. Arrows in A and B show lipid droplets that were scarce in tumors from the control groups.
n3PUFA diets have not been used in pancreatic cancer models, they indeed inhibited non-pancreatic cancers [9,10]. In the present study, the intake of flaxseed-based diet was associated with decreased liver metastasis and increased tumor necrosis. Previously, we showed that the diet rich in SFAs increased MCAD expression in pancreatic cancer cells [3], which suggests that SFAs may stimulate β-oxidation in pancreatic cancer cells. In the present study, we saw that the intake of diets rich in n6PUFAs and n3PUFAs increased tumoral MCAD as well. This suggests that β-oxidation is a common pathway by which different types of FAs stimulated pan-
creatic cancer cells. In vitro, etomoxir inhibited the stimulating effects of SFAs and MUFAs, which is another piece of evidence that suggests that β-oxidation is involved in FA-induced stimulation on pancreatic cancer cells. Interestingly, etomoxir also removed inhibiting effect of DHA on pancreatic cancer cells, which suggests that β-oxidation is also involved in DHA-induced inhibition of pancreatic cancer cells. In the same previous study, the intake of cocoabased HFD also increased intracellular lipid droplets, which suggests that an increase in dietary SFAs may augment lipid metabolisms in pancreatic cancer cells. In the present study, increased lipid
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Fig. 7. Co-expression of HIF-1α and PCNA in tumor graft. Consecutive sections from a tumor in group-MUFA were stained by immunohistochemistry to show HIF-1α (A) and proliferating cell nuclear antigen (PCNA) (B).
droplets were seen in tumors from all HFD groups, which suggests that an increase in any of four major types of fatty acids may augment lipid metabolisms in pancreatic cancer cells. According to a previous study, dietary n6PUFAs increased PGE2 in pancreatic cancer cells and thereby stimulated HIF-1α expression. In the present study, we found by histology that HIF-1α was co-expressed with PCNA in the same cancer cells, suggesting that HIF-1α expression was in favor of cancer-cell proliferation. In Western blotting assay, HIF-1α expression was increased consistently in examined tumors whose carriers were fed with high-energy diets. The HIF-1α expression was also associated with increased expression of two HIF-1-regulated proteins namely VEGF and cav-1. The mechanism of HIF-1α-induced regulation on pancreatic cancer cells requires further investigation. In summary, we systematically investigated four major types of dietary FAs for their possible involvement in pancreatic cancer. We found evidence that suggests that increased SFAs, MUFAs and n6PUFAs augmented lipid metabolisms so as to increase the via-
bility of pancreatic cancer cells. In contrast, increased n3PUFAs augmented lipid metabolisms so as to promote pancreatic cancer cell death. Thus, four types of FAs may exert differential impact on pancreatic cancer cells, though they all increase lipid metabolisms in the cells. It is the specific metabolic pathway for each FA type that determines how this FA type regulates pancreatic cancer cells. More studies are required to investigate these specific metabolic pathways by which different types of fatty acids regulate pancreatic cancer cells in different ways.
Acknowledgement We thank Tianjin Municipal Government (0190020104) for financial support.
Conflict of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Table 3 Ingredients in four high fat diets and an iso-caloric normal-fat dieta.
Fig. 8. Protein expression by tumor cells. MCAD, COX-2, HIF-1α, VEGF, and cav-1 expression was determined by Western blotting in pancreatic tumors (A). VEGF expression was determined by Western blotting in 5 cases of liver metastasis, 3 from group-n6PUFA and 2 from group-n3PUFA (B).
Casein L-cystine Corn starch Vegetable oilb Maltodextrin Sucrose Cellulose Mineral mix Calcium phosphate Vitamin mix Choline bitartrate Antioxidants a b
Four high fat diets
Iso-caloric control (Iso-C) diet
Harlan No. 09578-09581 (g/kg)
Harlan No. 09582 (g/kg)
200 3 248 150 150 100 100 35 1.7 10 2.5 0.03
200 3 448 50 100 100 50 35 1.7 10 2.5 0.01
All were produced by Harlan Laboratories Inc. See Table 1 for fat origins.
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