Introduction

Breast cancer (BRC) is the most frequent cancer in women, with an incidence of 2.1 million new cases worldwide in 20201. Adjuvant systemic therapies, such as chemotherapy, are still the golden standard in BRC therapy. In the past 35 years, the efficacy of chemotherapy has significantly increased, resulting in reduced morbidity and improved survival rate of cancer patients2. Despite the evident beneficial effects3, the treatments are related to complications and induce different side effects. Acute reversible side effects include alopecia, nausea, vomiting, fatigue, and myelosuppression, whereas long-term potentially irreversible side effects include neuropathy, cardiomyopathy, and acute leukemia4. Many of them are mediated by proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-a) and interleukin 6 (IL-6)5, often found in high concentration in patients long-term after cancer treatment6. Additionally, chemotherapy significantly alters lipid metabolism, reducing the HDL-cholesterol and apoA1 and increasing apoB concentrations, as well as impairing nutritional status, leading to increased risk for the development of cardiovascular diseases7,8. Therefore, the routes for ameliorating side effects of chemotherapy with natural hypolipemic and anti-inflammatory agents during and/or after the treatment are increasingly the focus of numerous studies9,10.

The favorable effects of fish oil, rich in docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) on clinical outcomes in patients receiving chemotherapy, have been documented in several studies11. EPA is the main precursor for the synthesis of anti-inflammatory cytokines. The consumption of EPA and DHA in BRC patients may lead to several beneficial outcomes, including maintenance of optimal body weight and nutritional status, reduction in proinflammatory cytokines, a decrease of fatigue and pain, improvement of the quality of life, as well as overall better recovery of the metastatic BRC patients12,13. Moreover, both in vitro and animal studies indicate the synergistic action of DHA and EPA with chemo- and radiotherapy in destroying tumor cells, reducing metastases, and inhibiting angiogenesis14. Regardless of these findings, there is no consensus on the dose and the duration of administration of fish oil, and n-3 polyunsaturated fatty acids (PUFA) are not a part of the multimodal therapeutic approach in BRC patients.

Apart from n-3 PUFA, levels of gamma-linolenic acid (GLA, 18:3n-6) in plasma lipids of BRC patients were also found to be extremely low, as compared with healthy individuals15. Although GLA belongs to n-6 PUFA, the latter mainly having pro-inflammatory potential, it exhibits anti-inflammatory properties through its conversion to dihommo-gamma-linolenic acid (DGLA) and its further conversion to anti-inflammatory prostaglandin PGE116. As a rich source of GLA, evening primrose oil (EPO) alone or in combination with tamoxifen, exerted anti-cancer effects in BRC cell lines. Accordingly, it has been suggested that in combination with tamoxifen (endocrine therapy), EPO may improve the efficacy of therapy and reduce side effects in BRC patients17. However, there is a gap in knowledge on the potential benefit of EPO intake in BRC patients undergoing chemotherapy.

We hypothesized that fish oil combined with EPO may decrease systemic inflammation and improve the nutritional status and lipid metabolism of BRC patients undergoing chemotherapy. The benefit of simultaneous supplementation with anti-inflammatory n-3 PUFAs and GLA is supported by the results of our previous studies, showing low plasma levels of n-3 PUFA and GLA in BRC patients15, and on the beneficial effects of this combination in patients with rheumatoid arthritis18. Thus, the objective of the present study was to evaluate whether fish oil (rich in EPA and DHA), and evening primrose oil (rich in GLA) supplementation influence concentration of cytokine (IL-6, IL-8, IL-10, and TNF-alpha), nutritional status/body composition, and fatty acids profile in BRC patients receiving adjuvant chemotherapy.

Results

GC analysis of fish oil and evening primrose oil capsules content is presented in Table 1. Results showed that fish oil contained 35.09% EPA and 22.68% DHA per tablet, and the rest were mixture of other PUFA, MUFA and SFA. EPO contained more than 80% linoleic and gamma-linolenic acids and the rest were MUFA and SFA. These results were expected and confirmed the label.

Table 1 Fatty acid profile in fish and evening primrose oil, Mean ± SD.
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Participant's characteristics

Of the 60 patients screened for entry into the trial, 28 did not meet the criteria for randomization (Fig. 1). Of 32 eligible patients, 16 were randomly assigned to the intervention group and 16 to placebo. One patient dropped out due to discontinuation of the treatment (in the placebo group) and two did not show up at the post-treatment measurements (in the intervention group), thus, N = 29 women completed the study. As presented in Table 2 there were no inter- or intra- groups differences regarding examined nutritional status during the study.

Figure 1
figure 1

Disposition of the study participants.

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Table 2 Characteristics and relevant parameters of the breast cancer patients on adjuvant chemotherapy at baseline and after 12 weeks (end of the study), Mean ± SD.
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Biochemical and hematological parameters in intervention and placebo groups

Table 3 presents values of hematological parameters, at baseline and after 12 weeks. The count of leukocytes and erythrocytes, and the level of hemoglobin significantly decreased throughout the intervention period in both groups, without inter-group variabilities. Regarding biochemical parameters, all remained stable in both groups during the study (Supplementary Table S1).

Table 3 Hematological parameters of the breast cancer patients on adjuvant chemotherapy at baseline and after 12 weeks (end of the study), Mean ± SD.
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Fatty acid profile and estimated activity of desaturase and elongase in intervention and placebo groups

The proportions of fatty acid changed in both groups. As expected, the supplementation with fish oil and EPO led to significant changes in FA profiles in the intervention group, resulting in increased plasma levels of n-3 PUFA (docosapentaenoic acid (22:5n-3), DHA, and total n-3PUFA), followed by a decreased palmitoleic and oleic acid as well as the n-6/n-3PUFA ratio (p < 0.01) (Table 4). Interestingly, no differences in GLA concentration were found, but the concentration of docosatetraenoic (DTA, 22:4n-6) acid, as the last product of GLA metabolism, was increased after the supplementation period. Some changes were also detected in the placebo group. Namely, the level of DGLA (20:3n-6) was significantly higher at the end of the study (Table 4).

Table 4 Plasma fatty acids profile from breast cancer patients on adjuvant chemotherapy at the start and the end of the study.
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Comparisons between groups at the end of the study, adjusted for baseline values, showed significantly higher levels, of DPA, DHA, n-3 PUFA, and total PUFA, in the intervention group. Additionally, lower levels of palmitoleic and oleic acids, DGLA, as well as an n-6/n-3 PUFA ratio (Table 4), as well as the lower estimated activity of SCD-16 were also noted in the intervention group after 12 weeks of treatment (Supplementary Table S2).

Levels of cytokines in intervention and placebo groups

The concentrations of inflammatory markers IL-8, IL-10, and TNF-alpha remained unchanged, while the concentration of IL-6 significantly decreased in both groups during chemotherapy (Table 5). Additionally, the intergroup comparisons at the end of the study adjusted for baseline levels revealed a significantly (p < 0.01) lower concentration of IL-6 in the intervention group compared to the control group.

Table 5 The cytokines concentration in breast cancer patients on adjuvant chemotherapy at the start and the end of the study.
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Discussion

This randomized double-blind controlled study investigated the effects of supplementation with an anti-inflammatory combination of n-3 and n-6 PUFA vs. placebo on body composition, lipid metabolism, and inflammatory response in breast cancer patients receiving the adjuvant chemotherapy during the period of 12 weeks.

Although chemotherapy commonly leads to changes in weight, BMI, waist circumference, and body fat percentage19,20, in this study there was no change in these parameters during the first 12 weeks of adjuvant chemotherapy in either of the groups. Freedman et al. have shown that although some women with BRC did not experience significant changes in weight or BMI after chemotherapy, they had unfavorable changes in body composition (decreased fat free mass and increased percentage of body fat)21. Although gains in weight and/or waist circumference are positively associated with baseline values of these parameters22, premenopausal women display a significantly higher risk for body fat percentage gains compared to postmenopausal women20, during chemotherapy. Maintenance of all anthropometric parameters and body composition in all participants of our study could probably be explained by the fact that only post-menopausal women were included. Moreover, body weight changes also depend on other variables such as the type and length of chemotherapy23, decreased physical fitness, anxiety, depression and appetite change24. Further, the literature data have shown the most prominent positive effects of supplements in cancer patients with cachexia25. However, all the patients included in this study had BRC at early stages, and the effects of supplements are not significant, but the follow-up of these patients could show if the nutritional intervention at early stages prevents nutritional decline in the future. This is important since it will show if the supplementation at early stages should be included in the BRC therapy.

Since leukopenia and neutropenia are common side effects of many cytotoxic agents26, often leading to delayed cycles of chemotherapy in cancer patients, we also followed changes in hematological parameters during this study. Although n-3 PUFA has a proliferative effect on lymphocytes27, and improves white and red blood cell counts in mice under chemotherapeutics28, only a few human studies showed a beneficial effect of PUFA supplementation on hematological parameters during chemotherapy29. In our study, fish oil and EPO supplementation did not prevent the reduction in hematological parameters during chemotherapy in BRC patients grade IIa–IIIa, which is in line with the results of other studies29.

Biochemical parameters were stable during the 12 weeks of the study in both groups. Although some other studies have shown unfavorable change in lipid profile during chemotherapy7, this was not a case in the present study.

The n-3 PUFA and GLA supplementation during chemotherapy in our study participants significantly increased DPA, DHA, total n-3 PUFA, and DTA levels, and decreased the n-6/n-3 PUFA ratio in the intervention group. The optimal n-6/n-3 PUFA ratio is important not only for reducing the BRC risk30 but also for decreasing the invasive potential of tumor cells31,32. Moreover, DHA can positively influence the effectiveness of chemo- and radiotherapy in BRC patients33. More precisely, DHA from food or supplements is quickly incorporated into all phospholipids, including those of tumor tissues, thus making the tissue more flexible and permeable to antitumor agents. Importantly, these supplements do not increase the susceptibility of healthy tissues to the drugs, e.g., to exposed toxicity of chemotherapeutics33,34. Therefore, the observed increase in DHA probably reflected the increase in DHA in the tumor tissue and could increase the effectiveness of chemotherapy in patients with BRC.

In the placebo group participants who remained on the standard dietary intake, only the DGLA concentration significantly increased after 12 weeks of the study, while it remained the same in the intervention group. DGLA is a precursor of arachidonic acid (AA), but also anti-inflammatory PGE1 and thromboxane A1, and could inhibit the synthesis of pro-inflammatory leukotrienes LTB4, LTC4, and LTD4. The higher concentrations of DGLA in the placebo group may indicate reduced conversion to AA and subsequent anti-inflammatory molecules. As the estimated activity of ∆5 desaturase, which catalyzes the conversion of DGLA to AA, did not change significantly during the study in both groups, the increased level of DGLA may be due to the chemotherapy-reduced conversion to anti-inflammatory molecules, further adversely affecting BRC patients and leading to inflammation.

Unlike the other members of the n-6 PUFA family, GLA exhibits anti-inflammatory, vasodilating, and anti-aggregation effects. In addition, GLA has a cytotoxic effect on tumor cells35, inhibits angiogenesis36, and stimulates apoptosis of breast, pancreatic, colon, and brain cancer cells37,38. In vitro studies have shown that GLA improves the effectiveness and reduces the side effects of antitumor therapy, and the response time to the therapy39. Despite the promising results, few interventional studies have addressed GLA supplementation in BRC. To the best of our knowledge, this is the first study that examined the effect of simultaneous supplementation with fish oil and EPO in women with breast cancer on adjuvant chemotherapy. In our intervention group the level of DTA (adrenic acid), as the final product of GLA conversion, was significantly increased after the supplementation, suggesting that DTA could inhibit AA release and formation of the LTB440, leading to the decrease of inflammation in vivo. All the observed changes in PUFA have shifted the FA profiles in the intervention group towards anti-inflammatory profiles. It is worth to mention that although EPO contains 72% of linoleic acid (18:2 n-6), its effect in the supplemented group is not exerted, since the amount of LA in EPO is not significant in comparison to the dietary intake of this fatty acid.

Another beneficial effect of the supplementation, in this study, is decreased stearoyl CoA desaturase (SCD-16) activity and reduced levels of oleic and palmitoleic acid, since the carcinogenesis is closely related to the increased activity of enzymes involved in the biosynthesis of monounsaturated fatty acid (MUFA). Namely, the overexpression of SCD, which is responsible for the conversion of saturated fatty acids into MUFA, has been confirmed in HER2 + BRC41 and leads to increased MUFA, mostly oleic acid, in cell membranes. Oleic acid stimulates carcinogenesis by promoting the growth and migration of cells, especially those with high metastatic potential, as confirmed in HGC-27 gastric cancer and MDA-MB-231 BRC cell lines42.

In many inflammatory diseases, fish oil supplements are used as complementary therapy as they reduced the production of TNF-alpha, IL-1β, and IL-6 by mononuclear cells43,44,45. In this study, the IL-6 concentration dropped after 12 weeks of chemotherapy in both groups, but the decrease was much more prominent in the intervention group. This result demonstrates strong anti-inflammatory potential of the fish oil- EPO combination in BRC patients on chemotherapy. The changes in IL-10, IL-8, and TNF-a were not significant, possibly due to high inter-individual variabilities. However, we cannot rule out the possibility that there were modifications of these cytokines at local levels, e.g., in the tumor tissue, although they were not sufficient to modify the systemic levels. Similar findings have been reported in newly diagnosed BRC46. Nevertheless, this study has some limitations. First, this study was conducted at a single center, affecting generalizability. Second, the study population was relatively small.

In conclusion, supplementation with fish oil and EPO of BRC patients simultaneously with chemotherapy for 12 weeks, led to significant changes in fatty acid profiles of plasma lipids and lowered levels of IL-6, suggesting a beneficial effect on lipid metabolism and inflammatory response. Additional clinical trials with a higher number of participants and long-term supplementation are required to confirm the present results.

Method

Study population

This parallel, randomized double-blind placebo-controlled study included postmenopausal women with BRC, who have started adjuvant chemotherapy by anthracycline models. Of the 60 patients screened between February 2019 and May 2021 for entry into the trial, 28 did not meet the criteria for randomization. The patients (N = 32) between 45 and 70 years of age, were recruited, from the Military Medical Academy of Belgrade, Serbia and were equally randomly assigned to be given either EPO and FO (N = 16) or mineral oil (N = 16). One of the patients in the placebo group dropped out due to discontinuation of the treatment, and two did not show up at the post-treatment measurements (in the intervention group). A total of N = 29 patients remained in the study. Eligible patients were postmenopausal women with histopathological diagnosis of breast cancer, stage IIa-IIIa, with ER+ and/or PR+ breast cancer, and human epidermal growth factor receptor 2 (HER2) negative. Exclusion criteria were a metastatic disease, HER2+ cancer, previous stroke or heart attack, presence of significant neurological deficit and consciousness disorder, presence of other malignancies, thyroid disease, allergies to fish or EPO, use of statins, fish oil, and other supplements potentially influencing lipid metabolism. The study protocol was approved by the Ethical Committee of the hospital Military Medical Academy (MMA) and study was conducted according to the Declaration of Helsinki and the principles of Good Clinical Practice. Participants provided written informed consent at the time of recruitment. During the data analysis, the personal information of the patients was not identifiable. The present study is adhered to the CONSORT guidelines.

Study design

This trial was a randomized 1:1 double-blind, placebo-controlled, parallel-group study conducted at the MMA and the Institute for Medical Research in Belgrade. The trial was registered at Clinical trials, NCT03516253 (Date of registration 04/05/2018). Random allocation was done by computer-generated permuted blocks of 4. The statistician who was not involved in the study generated a random allocation sequence, while the investigators enrolled participants, and assigned treatments. The patients were randomly assigned in a 1:1 to receive either supplements or a placebo. All capsules were identical in appearance, they were prepacked in bottles and consecutively numbered for each woman according to the randomization schedule. Patients and researchers were unaware of assignments to supplements and placebo groups until the statistical analysis was completed. The supplementation period started at the same time as chemotherapy. During 12 weeks of intervention, patients consumed two omega-3 Cardio capsules and three evening primrose oil capsules per day (Natural Wealth®, New York, USA), providing a total of 1000 mg EPA + DHA and 351 mg GLA. Compliance was assessed by the empty bottles that the participants returned at the end of the 12-week intervention period. Outcomes. The primary outcome was a change in nutritional status and level of interleukins in women with BRC during the study. The secondary outcomes were changes in hematological and biochemical parameters, fatty acid profile, in both groups during the study. Sample size. The sample size calculation was performed based on a study by Bougnox et al., which assessed the effect of DHA in BRC patients during chemotherapy47. Assuming a hypothesis that up to 5% of placebo group would have a positive response in fatty acid, inflammatory or nutritional parameters, we calculated that a minimum sample of 14 subjects in each group would allow the detection of differences caused by supplementation with n-3 PUFA, with a 80% power and 5% significance level.

Anthropometry and biochemical assays

Anthropometric measurements (weight, height, and calculated BMI) were taken at baseline and at the end of the study, via standardized methods. Blood samples for all analyses were obtained in the morning, after an overnight fast, at the beginning and at the end of the intervention. Fasting serum glucose and lipids (triglycerides (TG), total cholesterol, high-density lipoprotein (HDL)-cholesterol low-density lipoprotein (LDL)-cholesterol) concentrations and hematological parameters were measured on the same day the samples were collected using clinical chemistry automated analyzer (Dimension RxL Max and ABX MICROS 60) and commercial Roche and ABX diagnostics kits, according to the manufacturer’s instruction. Blood collected in the tubes with EDTA was centrifuged to separate plasma and red blood cells and plasma was stored at − 80 °C until the determination of FA profiles.

Analysis of the fatty acid profile of plasma lipids

Fatty acids from total plasma lipids were isolated as previously described47,48. Fatty acids were directly trans-esterified with 3 N HCl in methanol, at 85 °C for 60 min. Fatty acid methyl esters were analyzed by gas chromatograph SHIMADZU 2014 which was equipped with capillary column RESTEK Rtx 2330. The temperature program was 140–210 °C for 3°/min. Individual FA was identified by comparison with retention time of FA methyl esters commercial standards PUFA-2 (Supelco, Inc., Bellefonte, Pennsylvania, USA). Methyl esters from oils were prepared in the same way. Oils were analyzed in triplicate. The results are presented as a percentage of the total FA.

Measurement of the plasma levels of cytokines

The cytokine concentrations in the plasma were determined using commercial ELISA assays (Human IL-6 DUO SET ELISA development kit and Human TNF-/TNFSF1A DUO SET ELISA development kit; R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s protocol. IL-8 and IL-10 were determined with ELABSCIENCE kits (Elabscience Biotechnology Inc Houston, Texas, USA) as per producer instructions.

Statistical analysis

Statistical data analysis was performed by using SPSS 22.0 program. The Shapiro–Wilk test was used to check the distribution of variables. Descriptive statistics (mean, median, standard deviation, interquartile range) were used to describe examined parameters. Comparisons of values at the end of the study to baseline values were computed by two-factor analysis of variance for repeated measurements (2way RM-ANOVA) with an appropriate posthoc test. Time and treatment factors were analysed using ANCOVA where covariance is baseline values, Mann Whitney's U test and Wilcoxon's test were applied for non-parametric data. Significance was accepted at p < 0.05.