An important link between cancer and inflammation involves cyclooxygenase-2 (COX-2), the inducible form of cyclooxygenase, which is overexpressed in multiple malignancies (Wang et al., 2007). In the COX pathway, the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) is released from cellular membranes and then enzymatically converted into various bioactive lipid molecules, including prostaglandin E2 (PGE2). PGE2 promotes carcinogenesis through stimulating cellular proliferation, inhibiting apoptosis, promoting angiogenesis, and/or enhancing invasiveness (Wang and Dubois, 2006). Upregulation of COX-2 occurs in 50% of colon adenomas and 85% of colon cancers and is considered a key and early oncogenic event in colorectal carcinogenesis (Gupta and Dubois, 2001).
Eicosapentaenoic acid (EPA), a n-3 long-chain (LC) PUFA found in fish oil, is converted into eicosanoids through the same enzymatic pathways as AA, but produces series-3 prostanoids that have less inflammatory actions because of lower receptor affinities when compared with AA-derived series-2 prostanoids (Calder, 2002; Wada et al., 2007). In addition, EPA reduces tissue levels of AA through directly displacing AA from the sn2 position (second carbon) in the glycerol backbone in cell phospholipid membranes and inhibits COX enzymes (Garg et al., 1988; Ferretti et al., 1991; Blair et al., 1993; Smith, 2005). Several animal and human studies have demonstrated that increased dietary intake of n-3 LCPUFAs are associated with reduced tissue levels of AA and reduced PGE2 production (Boudreau et al., 1991; Ferretti et al., 1991; Blair et al., 1993; Wander et al., 1997; Broughton and Wade, 2002; Trebble et al., 2003).
Tissues stores of AA come from both exogenous (i.e. diet) and endogenous sources (the conversion of linoleic acid to AA). Multiple genome-wide association studies (GWAS) have reported associations between single nucleotide polymorphisms (SNPs) in enzymes responsible for AA biosynthesis pathway and tissue levels of AA. Variants in a single gene, FADS1 (which encodes Δ5-desaturase enzyme), have been found to explain up to 28% of the additive variance in tissue levels of AA. Specifically, homozygotes for the T allele in the rs174537 FADS1 SNP have lower fatty acid desaturase activity and subsequently lower tissues levels of AA (Schaeffer et al., 2006; Rzehak et al., 2009). A recent GWAS study of loci associated with CRC risk found that SNPs in FADS1 are associated with colorectal cancer (CRC) risk (Zhang et al., 2014).
We conducted a randomized, double-blind, controlled trial of marine-derived n-3 LC-PUFA (fish oil) supplementation compared with olive oil supplementation on the markers of colorectal proliferation and apoptosis in patients with known previous history of colorectal adenomas. Because of the hypothesis that genetically determined tissue levels of AA might be associated with an increased risk for CRC, participants were stratified based on rs174537 FADS1 genotype. In this report, we describe the results of the intervention on the secondary outcomes of the levels of urinary and rectal eicosanoids.
Participants and methods
Participants were recruited from individuals who were identified as colorectal adenoma cases within the Tennessee Colorectal Polyp Study or were identified through electronic medical record reviews to have undergone a colonoscopy at Vanderbilt University Medical Center and diagnosed with an adenoma. Eligible participants were between the ages of 40 and 80 years, had a past medical history of one or more adenomas, and had a known genotype for the rs174535 SNP of FADS1. Participants were excluded if they had a previous resected CRC, congestive heart failure or coronary artery disease, inflammatory bowel disease, any cancer (except nonmelanoma skin cancer), advanced kidney disease, cirrhosis, were pregnant or breast feeding, using fish oil supplements or anticoagulants, or allergic to fish products.
The Fatty Acid Desaturase Activity, Fish Oil, and Colorectal Cancer Prevention study was approved by the Vanderbilt University Medical Center Institutional Review Board and registered at ClinicalTrials.gov (NCT01661764).
Participants were randomized into either three capsules of the fish oil-sourced supplement Lovaza, each containing 465 mg of EPA and 375 mg docosahexaenoic acid (DHA) (a total daily dose of 1395 mg EPA plus 1125 mg DHA) or three capsules of an olive oil supplement with each capsule contained 1000 mg of olive oil. Olive oil was chosen as the control arm as previous studies have found no effect on colonic eicosanoid production (Hillier et al., 1991). Both capsules were similar in size, shape, and appearance. The rationale for this dose is that it was similar to US clinical trials that have successfully lowered the indices of rectal mucosal proliferation and is well tolerated without any reported adverse effects (Bartoli et al., 1993; Anti et al., 1994). Medications were dispensed by the Vanderbilt University Investigational Drug Service.
Randomization, allocation, adherence, and blinding
Randomization was performed according to a permuted block randomization scheme stratified on FADS1 rs174537 genotypes (GG, GT, and TT). Randomization proceeded within these three strata with a block size of balancing interval, varying randomly according to the outcome of a computer-generated random number. The randomization code was kept by the Investigational Drug Service. Participants and study personnel were unaware of the randomization list or treatment assignment. To assess the study blinding, participants were asked at the end of the study which treatment they thought they had received. Adherence was assessed by weekly telephone calls, pill counts, and red blood cell n-3 LC-PUFA measurements.
Study participation lasted 24 weeks with three in-person visits: at baseline, 12 weeks, and 24 weeks. At each visit, participants arrived after an overnight fast, and 20 ml of blood and 30 ml of spot urine were collected. Eight rectal epithelial biopsies were collected at baseline and at 24 weeks. Participants did not undergo a bowel preparation and samples were obtained using an anoscope and a 2.4 mm×160 cm Captura biopsy forceps (Cook Medical LLC, Bloomington, Indiana, USA). Biopsies were taken circumferentially ~8 cm from the anal verge using biopsy forceps. Two samples were placed in formalin and six were snap frozen in liquid nitrogen.
Red blood cell phospholipid membrane determination
Total lipids were extracted from 200 μl of double-washed packed red blood cells (RBCs) using the method described by Folch et al. (1957). Phospholipids were isolated using thin-layer chromatography on Silica Gel 60A plates and fatty acids, methylated using BF3/methanol (Morrison and Smith, 1964). The methylated fatty acids were analyzed by gas chromatography using an HP 7890A gas chromatograph equipped with flame ionization detectors and a capillary column (SP2380, 0.25 mm×30 mm, 0.20 µm film; Supelco, Bellefonte, Pennsylvania, USA). Helium was used as a carrier gas. Fatty acid methyl esters were identified by comparing the retention times to those of known standards. The inclusion of the internal standard, dipentadecanoyl phosphatidylcholine (C15:0), permitted quantitation of the amount of phospholipid in the sample. Fatty acid values are presented as percentage of total RBC membrane phospholipid fatty acid content. The lowest level of detection for individuals’ fatty acids is less than 0.5% of the total profile.
Rectal epithelial cell eicosanoid measurement
Tissue was added to 5 ml of ice-cold methanol containing indomethacin, to inhibit COX activation, and 1.0 ng of the internal standard [2H4]-PGE2 (Cayman Chemicals, Ann Arbor, Michigan, USA). The sample was then extracted and analyzed using gas chromatography/negative-ion chemical ionization mass spectrometry as described previously (Liu et al., 2012; Boutaud et al., 2016). The interday variability for each assay was less than 10%. The precision for each assay was ±5%, whereas the accuracy for each assay was 95%.
Urinary prostaglandin E2 metabolite determination
To quantify endogenous PGE2 production, the level of urinary PGE2 metabolite PGE-M (11 α-hydroxy-9,15-dioxo-2,3,4,5-tetranorprostane-1,20-dioic acid) was measured using a previously described liquid chromatography/tandem mass spectrometric method (Mohebati et al., 2013). In our laboratory the coefficient of variation for samples analyzed in multiple batches was 7.2%.
The primary outcome of the study was changes in the rectal cell markers of proliferation and apoptosis with the secondary outcomes being changes in the eicosanoid production. The primary outcomes are completing analyses and pathologic review, and this paper reports our secondary outcomes. Sample-size estimates were derived using previously described clinical trial information using similar primary outcomes and doses of fish oil supplements. Courtney et al. (2007) found a change in the mean±SD level of Ki67 labeling of −5.86±±8.8 for individuals allocated to the fish oil supplement group (n=14) and 3.62±8.0 for the placebo group (n=14) (Cheng et al., 2003). With a sample size of 22 per group, each genotype would provide at least 90% of power to detect 1-SD (8.4) difference in ΔKi67 between the two groups within each genotype with type I error rate of 5%. Assuming ~10% drop-out rate, a total of 150 patients (25 patients/group per genotype×2 groups×3 genotypes=150) would provide the power to detect a clinically significant difference between the intervention and placebo groups.
Baseline characteristics stratified by treatment allocation were compared using the Wilcoxon rank order sum test for continuous variables and the χ2 for categorical variables. We compared RBC membrane percentages of PUFAs at baseline stratified by genotype using the Wilcoxon rank order sum test. Urinary PGE-M and rectal eicosanoids were right skewed and log transformed for analyses. We present medians and interquartile ranges for the ease of interpretation. We used repeated-measures linear regression to determine the effect of the intervention on urinary PGE-M levels. Models were adjusted for genotype and sample batch. For rectal eicosanoids, we used general linear regression and analysis of covariance to adjust for baseline rectal eicosanoid levels, genotype, and batch number. We conducted a secondary analysis stratifying the analysis by aspirin/NSAID use (regular use, no regular use), baseline RBC membrane n-3 LC-PUFA content (below median, above median), baseline RBC membrane AA content (below median, above median), and FADS genotype (G/G, G/T, T/T). We calculated Spearman’s partial correlation coefficients between urinary PGE-M and rectal PGE2 adjusting for age, sex, aspirin/NSAID use, genotype, and study allocation. All analyses were conducted using SAS v9.4 (SAS Institute Inc., Cary, North Carolina, USA).
Three-hundred and seventy-nine potential participants were assessed for eligibility, and 141 were randomized to either fish oil (n=70) or olive oil (n=71) (Fig. 1). Sixty-two (89%) participants allocated to fish oil and 64 (90%) participants allocated to olive oil completed the study. Reasons for discontinuation are presented in Fig. 1.
We were unable to obtain pill/bottle returns for 17 (13%) participants. Of the participants who did return unused pills or empty bottle, 92% (100/109) reported greater than 80% compliance. In the fish oil arm 33% of participants (20/61) believed they were taking fish oil, 28% (17/61) believed they were taking olive oil, and 39% (24/61) were unsure of their treatment allocation. In the olive oil arm, 11% (7/64) reported they were taking fish oil, 33% (21/64) reported they were taking olive oil, and 56% (36/64) were unsure of their treatment allocation.
There was no difference in age, sex, or baseline RBC membrane fatty acids between the two groups (Table 1). As expected, RBC membrane AA content was lower in individuals who were homozygous for the T allele of the FADS gene (Table 2). In addition, the RBC membrane content of linoleic acid, a precursor of endogenously synthesized AA, was greater in individuals who had the T allele.
Fish oil supplementation reduced urinary PGE-M in individuals allocated to the fish oil arm compared with the olive oil arm (P=0.03) (Fig. 2). The reduction in urinary PGE-M was greater at the 3-month measurement with a waning of the effect at the 6-month measurement period, which was no longer statistically significant. We found no evidence of an interaction between fish oil supplementation and urinary PGE-M based on the FADS genotype (Pinteraction=0.39). We also found no evidence of effect modification between n-3 LC-PUFA supplementation and urinary PGE-M based on the NSAID use (Pinteraction=0.64).
Overall, we found no changes in the rectal mucosal levels of PGE2 after 6 months of supplemental fish oil (Fig. 3). Rectal PGE2 did decrease over the course of the study in the fish oil arm compared with the olive oil arm; however, the difference was not statistically significant. We also compared the baseline and the 24-month changes in rectal PGE3, PGD2, and PGD3. For each measured rectal eicosanoid, there was a decrease from the baseline in the fish oil arm, but the difference was not statistically significant (Fig. 3). Similar to the urinary PGE-M, we found no evidence of an interaction between fish oil supplementation and rectal eicosanoids by genotype (data not shown).
We did find a statistically significant interaction between NSAID use and fish oil supplementation (Pinteraction=0.008). In participants who did not use regular NSAIDs over the course of the study (n=57, fish oil group=23, olive oil group=34), supplemental fish oil reduced rectal PGE2 levels from 3.10±0.32 (log mean±SE) at baseline to 2.54±0.28 at 6 months (18% reduction) compared with a change from 2.80±0.23 to 3.09±0.19 in participants allocated to placebo (10% increase) (P=0.04). In contrast, participants who did use regular NSAIDs (n=84, fish oil group=47, olive oil group=37), supplemental fish oil did not significantly reduce rectal PGE2 levels compared with placebo; levels went from 2.34±0.22 at baseline to 1.84±0.19 at 6 months compared with a change from 1.61±0.38 to 1.41±0.36 in participants allocated to placebo (P=0.19). We found no evidence of interaction with other rectal eicosanoids (data not shown).
We found a small but statistically significant positive correlation with an increase in the urinary PGE-M being correlated with an increase in the rectal PGE2 levels (adjusted correlation coefficient=0.25; 95% confidence interval: 0.06, 0.42; P=0.01).
Over the course of the study, RBC membrane percentages of n-3 LC-PUFA increased by 76% compared with 6% for individuals allocated to olive oil (Fig. 4). Similarly, individuals allocated to the fish oil arm had a statistically significant decrease in the RBC membrane AA content.
We found in a randomized controlled trial that fish oil reduced urinary PGE-M levels compared with olive oil. This effect was strongest during the first 3 months of the study, but we found no effect at 6 months. Changes in the rectal mucosa PGE2 were smaller and significant only in individuals not using NSAIDs. We also found that the FADS1 polymorphism rs174537 did not interact with fish oil intake, even though this variant did influence AA levels. Finally, rectal mucosa PGE2 and urinary PGE-M had only a negligible correlation.
PGE-M, the major urinary metabolite of PGE2, is an established marker of systemic PGE2 (Sterz, Scherer et al., 2012; Wang and DuBois, 2013). Several case–control studies have found higher levels of PGE-M in patients with high-risk adenomas or known CRC than in controls (Cai et al., 2006; Johnson et al., 2006; Shrubsole et al., 2012; Bezawada et al., 2014; Davenport et al., 2016). A recent meta-analysis concluded that PGE-M levels are strongly associated with an increased risk for multiple small tubular adenoma, advanced adenoma, and CRC, and that PGE-M is the most promising urinary biomarker for the CRC risk assessment and screening (Altobelli et al., 2016).
PGE-M may also be an important marker to identify the subsets of patients who would benefit the most from interventions targeted toward reducing inflammation. A nested case–control study found that regular NSAID use was associated with a reduction in adenoma risk in patients with high baseline PGE-M, but not in those with low PGE-M (Bezawada et al., 2014). This suggests that PGE-M could help with both evaluating the risk for CRC and assessing the response to interventions; it has the potential to become a ‘risk-response’ biomarker, similar to the role the lipid panel plays in the field of cardiovascular medicine in helping assess the risk as well as evaluating the response to statin therapy (Colbert Maresso et al., 2014). Our finding that fish oil supplementation significantly decreased urinary PGE-M levels in our study population – which was by design a higher risk group in that all participants had known history of adenomas – further supports the potential use of urinary PGE-M as a response marker. However, one concern was the relatively weak correlation between rectal PGE2 and urinary PGE-M seen in our study. Currently underway is the seAFOod polyp prevention trial, a large randomized controlled trial to examine whether EPA, alone or in combination with aspirin, prevents colorectal adenomas (Hull et al., 2013).
Several interventional studies have examined the effect of fish oil on the production of eicosanoids and other inflammatory markers, with mixed but overall promising results. In 40 healthy male volunteers, supplementation of 15 g/day of fish oil for 10 weeks led to a 14% reduction in urinary PGE-M compared with 15 g/day of placebo oil (Ferretti et al., 1991). In a cross-over feeding trial, 10 healthy male volunteers had a 24% reduction in PGE-M when fed a salmon-rich diet versus a placebo diet with identical macronutrient breakdown (Ferretti et al., 1991). Another cross-over trial found that rectal PGE2 levels were significantly lower after 4 weeks of fish oil supplementation compared with corn oil supplementation (435.5 vs. 671.5 pg/mg wet tissue, P<0.05). A third cross-over trial found that the urinary excretion of thromboxane B2, a metabolite of platelet activator thromboxane A2, which is also derived from AA, was significantly reduced with a salmon diet compared with a reference diet (Prakash et al., 1994). A study examining varying doses of fish oil supplementation found that PGE2 in peripheral mononuclear cells was decreased significantly for all doses of fish oil (0.3, 1, and 2 g of EPA plus DHA), with a trend toward a dose response (Trebble et al., 2003).
Several meta-analyses have been published on this topic (Jiang et al., 2016). Five of the included studies reported PGE2 changes in response to fish oil supplementation; only two of these showed reduction in PGE2 (Andrade et al., 2007; Tartibian et al. 2011). However, of the three studies that did not show a significant reduction in PGE2, one involved healthy participants (Rees et al., 2006) and the other two included very limited patient populations, namely patients on hemodialysis (Peck et al., 1996) and patients with rheumatoid arthritis (Park et al., 2013).
In our study, fish oil only statistically significantly lowered rectal mucosa PGE2 levels in nonusers but not in the users of aspirin/NSAIDs. Of note, the baseline PGE2 levels were lower in the aspirin/NSAID users than in nonusers. After fish oil supplementation for 24 weeks, rectal mucosa PGE2 levels in the nonusers of aspirin/NSAID approached but did not reach the baseline levels in aspirin/NSAID users. This suggests that rectal PGE2 is reduced with fish oil supplementation, but this effect is not seen with NSAID use. Intriguingly, our baseline rectal PGE2 levels were lower in aspirin/NSAID users who were allocated to olive oil supplementation. This difference was not statistically significant (P=0.14), but may have impacted our stratified results. As this was a secondary analysis and participants were not randomized based on NSAID use, this difference in baseline rectal PGE2 status may be related to confounding factors. We found no differences in the groups based on age, BMI, sex, smoking status, or educational level (data not shown); however, unmeasured confounders may have also influenced the results.
Strengths of the study included the randomized, controlled design. We had good compliance with the study medication as determined by pill counts and changes in RBC membrane PUFAs. Our participant demographics are similar to patient populations that would likely benefit the most from improved colon cancer prevention strategies – namely, middle-aged and older patients who have risk factors for CRC. The study has some weaknesses. Most notably, the results we report are of a secondary outcome of our study, and our study might have been underpowered to detect an overall effect on rectal PGE2. We did not collect rectal biopsies at 12 weeks and cannot report whether rectal PGE2 levels change similarly to urinary PGE-M, with an initial strong decrease followed by a waning effect. Another weakness was our choice of FADS polymorphism to stratify the participant. While this polymorphism is associated with endogenous AA production, it has not been associated with CRC in GWAS studies. Our choice of dose for n-3 LC-PUFA supplementation could have also impacted the results, as limited data exists to determine whether a dose–response effect exists between n-3 LC-PUFA supplementation and eicosanoid production. Finally, the control arm included olive oil supplementation, which may have had an impact on eicosanoid production.
This study showed that supplemental fish oil reduced urinary PGE-M levels; however, this reduction was seen at 3 months and not at 6 months. It would be important to replicate our findings as this could indicate that studies of a shorter duration might suggest a stronger eicosanoid modifying the effect of fish oil than might exist over longer periods of time. Additionally, we found a very weak correlation between urinary PGE-M levels and rectal PGE2 levels suggesting that generalized PGE2 production only weakly represents colonic production. Finally, our results suggest that fish oil is more effective in reducing rectal PGE2 in individuals not using NSAIDs. These results might be better delineated after the results of the seAFOod Polyp Prevention Trial are reported.
This study was supported through the National Institute of Health grants R01CA160938, R01CA143288, P50CA95103, and R01CA97386. Surveys and sample collection, processing, and preparation for this study were conducted by the Survey and Biospecimen Shared Resource, which is supported in part by P30CA068485. Fatty acid analyses were performed by the Vanderbilt Diabetes Research and Training Core Lipid Core lab supported by DK20593. The study was supported in part by the Vanderbilt CTSA grant UL1TR002243. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.
Conflicts of interest
There are no conflicts of interest.
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Keywords:Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
colonic neoplasms; controlled clinical trials; randomized; eicosanoids; fish oil; omega-3 fatty acids; prostaglandin E2