Serum enterolactone concentrations are low in colon but not in rectal cancer patients

The dietary lignan metabolite, enterolactone, has been suggested to have anti-cancer functions, and high serum enterolactone concentrations have been associated with decreased risk of breast and prostate cancers. We hypothesized that serum enterolactone concentrations as a marker of plant-based foods are associated with decreased risk in colorectal cancer (CRC). We measured serum enterolactone glucuronide and sulfate concentrations by liquid chromatography-tandem mass spectrometry in 115 CRC patients and 76 sex- and age-matched controls and analyzed the results with respect to tumor parameters, clinical parameters, and systemic inflammatory markers. Patients with colon cancer had significant lower serum enterolactone glucuronide and sulfate concentrations than controls (glucuronide: median 3.14 nM vs. 6.32 nM, P < 0.001; sulfate: median 0.13 nM vs. 0.17 nM, P = 0.002), whereas rectal cancer patients had similar enterolactone levels as controls (glucuronide: median 5.39 nM vs. 6.32 nM, P = 0.357; sulfate: median 0.19 nM vs. 0.17 nM, P = 0.452). High serum enterolactone concentrations were associated with low tumor grade, high serum creatinine levels, and concomitant diabetes. In summary, our results suggest that serum enterolactone concentrations are decreased in colon but not in rectal cancer. Further investigations are required to assess whether this reflects an altered lignan metabolism by the colon microbiome.


Relationships between serum enterolactone concentrations and clinicopathological parameters.
Next, we analyzed the relationships between serum enterolactone concentrations and tumor and patient characteristics (Table 2). Low serum abundance of both the glucuronide and sulfate conjugated forms were associated with tumor localization, namely colon compared to the rectum (P = 0.049 and P = 0.012, respectively). Indeed, patients with rectal cancer (n = 38) had similar enterolactone concentrations to controls (glucuronide: median 5.39 nM vs. 6.32 nM, P = 0.357; sulfate: median 0.19 nM vs. 0.17 nM, P = 0.452, Table 2), whereas the patients with colon cancer (n = 77) had significantly lower concentrations of both enterolactone forms compared with control subjects (glucuronide: median 2.61 nM vs. 6.32 nM, P < 0.001; sulfate: median 0.13 nM vs. 0.17 nM, P = 0.002, Table 2). In addition, tumor differentiation was associated with serum enterolactone levels: the CRC patients with low-grade tumors had higher enterolactone levels than the patients with poorly differentiated tumors (glucuronide: P = 0.032, sulfate: P = 0.011, Table 2). Serum enterolactone levels were not associated with patient age, gender, or body mass index (BMI), tumor stage, tumor necrosis, or the the fraction of tumor cells positive for proliferation marker Ki-67 (Tables 2 and 3).

Serum enterolactone concentrations in CRC patients with concomitant diseases. We fur-
ther assessed the effect of the concomitant diseases on serum enterolactone levels in CRC patients (Table 2). Hypertension-or cholesterol-lowering medication was not associated with altered serum enterolactone levels. Interestingly, patients with diabetes had higher enterolactone glucuronide and sulfate concentrations than the patients without diabetes (Glucuronide: P = 0.028 and sulfate: P = 0.021, Table 2). Previously, increased serum CRC patients (n = 115) Healthy controls (n = 76) www.nature.com/scientificreports www.nature.com/scientificreports/ enterolactone concentrations have been shown in patients with diabetic renal disease 18 . Therefore, we assessed the relationship between serum levels of enterolactone and kidney function marker creatinine among CRC patients. We found a significant positive correlation between enterolactone glucuronide and creatinine (r = 0.200, P = 0.042, Table 3) and a tendency towards positive correlation between enterolactone sulfate and creatinine (r = 0.177, P = 0.077, Table 3).

Discussion
Dietary lignan derived enterolactone has been reported to possess anti-cancer activities and to be associated with lowered risk of prostate cancer and breast cancer. Our main finding is that colon but not rectal cancer patients have lower serum enterolactone concentrations than healthy controls. In CRC patients, low tumor grade, increased serum creatinine levels indicating impaired renal function, and concomitant diabetes were significantly associated with higher serum enterolactone concentrations. Enterolactone is metabolized from diet-derived lignans in a multistep process catalyzed by intestinal bacteria. For example, secoisolariciresinol diglucoside, which is a lignan found in high concentration in flaxseed, is converted to enterolactone in four sequential reactions catalyzed by phylogenetically and functionally distantly related anaerobic bacteria 25 . Accordingly, high urinary excretion of enterolactone has been associated with a high diversity of the gut microbial community 7 . In addition, low serum enterolactone levels have been associated with low fecal total bacteria and Lactobacillus-Enterococcus counts 26 . Thus, we hypothesize that low serum enterolactone concentrations in colon cancer patients may reflect paucity of lignan-converting bacteria in these patients, or an intracolonic environment sub-optimal to bacterial metabolism and lignan conversion. Accordingly, CRC have been linked to gut microbiota dysbiosis 27 and antibiotics are known to reduce circulating enterolactone levels 28,29 . Unfortunately, we do not have data on the frequency of antibiotics usage in our cohort subjects.
Earlier studies have reported that enterolactone exerts anti-proliferative activities on prostate and breast cancer in vivo 30,31 . However, in our CRC patient cohort, serum enterolactone abundance was not associated with tumor cell proliferation, as assessed by Ki-67 immunohistochemistry (Table 3). We further hypothesized that serum enterolactone could be altered by the presence of a systemic inflammatory response to the tumor. However, we detected no association with different cytokines or CRP. Furthermore, serum enterolactone levels were not associated with tumor stage. Currently, the prognostic classification of CRC is mainly based on tumor stage, while additional prognostic parameters could help to target the tumor with more individualized treatments 32,33 . Given the reported anti-tumor effects of enterolactone, we hypothesized that high serum enterolactone could be associated with improved patient outcome. Our cohort had a 10-year survival follow-up but no significant associations were detected between serum enterolactone levels and patient survival.
We measured serum enterolactone as glucuronide and sulfate conjugates. Consistent with earlier studies in healthy subjects 34,35 we found that glucuronide conjugate was the major circulating enterolactone metabolite in both CRC patients and controls. Enterolactone, as well as other polyphenols, are readily a mg/L, b pg/mL, c %, d μmol/L, median (IQR)  www.nature.com/scientificreports www.nature.com/scientificreports/ conjugated in the intestinal wall and in the liver 36 . Glucuronidation and sulfation are detoxification mechanisms to eliminate potentially harmful effects of enterolactone 37,38 . Tumor targeted glucuronic acid cleavage from the enterolactone-glucuronide in the tumor tissue is currently under investigation as a potential adjuvant therapy in prostate cancer 39 .
We found that CRC patients with diabetes had increased serum enterolactone levels. A previous study found elevated serum enterolactone levels in patients with diabetic nephropathy and a strong correlation between serum enterolactone and creatinine levels 18 . Accordingly, our results support the positive correlation between serum enterolactone glucuronide and creatine, suggesting that decreased urinary secretion of enterolactone may contribute to the increased serum enterolactone levels in a subset of CRC patients.
Earlier reported determinants of serum enterolactone concentrations include consumption of lignan-containing foods, constipation, smoking and BMI 34,40 . Especially, lignan-containing plant foods such as grains, fruits and vegetables are enriched with the dietary fiber, phytoestrogen, and unsaturated fatty acids. Increased dietary intake of lignans or fortification of food with the plant lignans have resulted in increased serum enterolactone levels 2,3,41 . In the Nordic population, a healthy diet includes apples and berries, roots and cabbage, rye, oats, barley, low-fat milk products, rapeseed oil, and fish 42,43 . However, in our CRC cohort detailed food intake questionnaires were not available. Nevertheless, we did not detect statistically significant association between serum enterolactone levels and the BMI of CRC patients.
Our CRC cohort was cross sectional study with a long follow-up period from a single province in Northern Finland (65° latitude). The study was limited by its sample size, comprising 115 CRC patients and 76 healthy controls. Due to cross-sectional study design, we were not able to assess, whether enterolactone reduced the risk for colon cancer 15 and/or affected colon cancer progression at early stages. Therefore, additional studies are required to further clarify the significance of the decreased serum enterolactone levels in colon cancer patients.
In conclusion, we found significantly decreased levels of enterolactone in colon cancer, but not in rectal cancer patients. Since a diverse gut microbiota plays a crucial role for an efficient conversion of plant to enterolignans, microbiome homeostasis may be disturbed in patients with colon, but not with rectal cancer, which requires further investigation. Serum enterolactone levels were not significantly associated with patient gender or age, tumor stage, systemic inflammatory markers, and survival.  44 . Serum CRP levels and serum albumin levels were measured in the laboratory of Oulu University Hospital and mGPS was calculated from serum CRP and albumin values 44,51 . Serum creatinine levels were analyzed with nuclear magnetic resonance metabolomics platform, equipped with Bruker AVANCE III 500 MHz and Bruker AVANCE III 600 MHz spectrometers (Bruker, Billerica, MA, USA) 19,52 . Enterolactone measurements. Serum enterolactone concentrations were quantified using a novel high-throughput liquid chromatography-mass spectrometry (LC-MS)/MS method by measuring the free enterolactone or intact form of either glucuronide or sulphate conjugated enterolactone, as previously described 34 . The method was validated according to the guidelines of the U.S. Food and Drug Administration (FDA) and the European Medicines Agency. Internal standards and their stability was tested previously 53 . Intrabatch accuracy, precision and recovery of enterolactone were tested at low, medium and high concentrations and the relative standard deviation did not exceed 15%. Briefly, standard curves and test serum sample were prepared using standards of (±)-enterolactone-mono-β-D-glucuronide and (±)-enterolactone monosulfate ammonium salt from ReseaChem (Burgdorf, Switzerland) and enterolactone from Plantech (Berkshire, UK). Serum samples were cleaned in solid phase extraction (SPE) hydrophilic/lipophilic balanced (HBL) 96-well plates from Waters (Torrance, CA, USA) and eluted with 50/40/10% acetonitrile (ANC)/methanol (MeOH)/H 2 O and internal standard (Glycine-1 13C) was added to the eluate. The LC-MS/MS measurements were performed on a microLC 200 series from Eksigent/AB Sciex (Redwood City, CA, USA) and QTrap 500 mass spectrometer from AB Sciex equipped with an ESI source. The microLC was equipped with a phenyl column from Eksigent/AB Sciex (Redwood City, CA, USA). The data were analysed using the Analyst software 1.6 from AB Sciex (Framingham, MA, USA).

Statistical analyses.
Statistical analyses were performed using IBM SPSS Statistics for Windows version 22.0 (IBM Corporation, Armonk, NY, USA). Normally distributed continuous variables are presented as mean (standard deviation, SD), whereas other continuous variables are presented as median (interquartile range, IQR). Correlations between two continuous variables were presented as Pearson correlation coefficients (r). Statistical significances of the differences in serum enterolactone levels between the different study groups and categorical variables were analyzed by Mann-Whitney U test or Kruskal-Wallis test. ROC analysis was used to evaluate the capacity of the serum enterolactone level to discriminate survivors from non-survivors. Cox regression models were used in the survival analyses. In all the tests P < 0.05 was considered statistically significant.  Table 4. Multivariate analysis of 120-month cancer-specific survival (CSS) and overall survival (OS) of CRC patients. Abbreviations: CI: confidence interval; HR: hazard ratio.