This article is part of the 2019 Oncology Special Issue of Natural Medicine Journal. Read the full issue here.
Darwito D, Dharmana E, Riwanto I, et al. Effects of omega-3 supplementation on Ki-67and VEGF expression levels on clinical outcomes of locally advanced breast cancer patients treated with neoadjuvant CAF chemotherapy: a randomized controlled trial report. Asian Pac J Cancer Prev. 2019;20(3):911-916.
To determine effects of omega-3 supplementation on Ki-67 and vascular endothelial growth factor (VEGF) expression levels at the time of surgery in women with locally advanced, invasive carcinoma. Overall survival and progression-free survival were also tracked.
Randomized double-blind placebo-controlled trial
Participants and Study Intervention
Participants were women with locally advanced stage IIIB invasive ductal breast cancer (n=48) between the ages of 25 and 60. All participants received 3 cycles of neoadjuvant chemotherapy (cyclophosphamide, doxorubicin, and fluorouracil, or CAF).
Study participants were assigned to receive 1 g/day of omega-3 fatty acids from fish oil (n=24) or placebo (n=24) alongside their chemotherapy (51 days total). The contents of the placebo were not disclosed.
- Expression of Ki-67and VEGF were semi-quantified using immunohistochemistry from paraffin blocks of tumor materials obtained from mastectomy after neoadjuvant chemotherapy.
- Overall survival and disease-free survival were assessed via Kaplan-Meier curve and Cox-regression tests.
- Decreased Ki-67 expression was evident in the intervention group compared to the control group (P=0.032)
- Decreased VEGF expression was also observed in the intervention group versus the control group (P=0.041)
- VEGF was positively correlated with Ki-67 expression (P<0.001)
- Overall survival in the intervention group was significantly longer compared to the control (mean survival: 30.9 vs 25.9 weeks; P=0.048).
- Disease-free survival in the intervention group compared to the control group was also reported to be significantly longer (mean survival: 28.5 vs 23.7 weeks; P=0.044).
Overview of Omega 3s and Breast Cancer
Before discussing this study on the use of omega-3 fatty acids on the expression of Ki-67 and VEGF in locally advanced breast cancer patients undergoing neoadjuvant CAF, it is useful to consider the evidence to date supporting the use of omega 3s in breast cancer.
Women who consumed high intake ratios of marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to omega-6 arachidonic acid have been found to have a reduced risk of breast cancer compared with those with low ratios in some but not all case-control and cohort studies.1 If increasing EPA and DHA relative to arachidonic acid is effective in reducing breast cancer risk, mechanisms may include reduction in pro-inflammatory lipid derivatives, inhibition of nuclear factor-kappa B–induced cytokine production, and altered cell signaling. More interventional trials designed to assess biomarkers or cancer incidence as endpoints need to be done to definitively determine if omega-3 fatty acids may be lowering risk.
Evidence from several observational studies suggests that higher intakes of omega 3s are associated with a lower risk of breast cancer. In the prospective Singapore Chinese Health Study of 35,298 women aged 45–74 years, those in the top 3 quartiles of dietary omega-3 intake had a 26% lower risk of breast cancer after an average of 5.3 years of follow-up than those in the lowest quartile.2
Evidence from several observational studies suggests that higher intakes of omega 3s are associated with a lower risk of breast cancer.
Similarly, among 35,016 female participants aged 50–76 years in the Vitamins and Lifestyle cohort, those who reported current use of fish-oil supplements had a 32% lower risk of breast cancer after a mean of 6 years than those who did not take fish oil.3
According to a systematic review of 3 case-control studies and 5 prospective studies published between 2007 and 2011, evidence increasingly suggests that higher intakes of dietary and supplemental omega 3s are associated with a lower risk of breast cancer. Similarly, the authors of a meta-analysis of data from 21 prospective cohort studies concluded that women with the highest dietary intakes and/or tissue levels of omega-3s had a 14% lower risk of breast cancer than those with the lowest intakes and tissue levels.4 These authors also found a dose-response relationship between higher intakes of combined omega 3s and reduced breast cancer risk.
While these studies don’t speak to the effect of omega 3s on VEGF and Ki-67 expression, they suggest that omega-3 fatty acid ingestion is associated with less incident breast cancer.
Role of Omega 3s in Cancer
Omega-3 fatty acids are used in a variety of conditions for their role as a systemic anti-inflammatory.5 Chronic inflammation is a known risk factor for cancer growth and progression.6 Research has shown that omega-3 fatty acids DHA and EPA influence cancer cell proliferation, differentiation, and apoptosis while also inhibiting angiogenesis, tumor cell invasion, and metastasis.7 Omega 3s can positively influence the genetic expression related to cancer and DNA repair.1 Studies have also demonstrated the influence of omega-3 fatty acids and their ability to decrease the potent inflammatory markers interleukin-6 (IL-6),8 tumor necrosis factor (TNF)-alpha,9 and C-reactive protein.10
Expression Levels of Ki-67 and VEGF in Breast Cancer
VEGF has been recognized as playing a role in breast cancer progression.11 VEGF expression in breast cancer is well documented and is produced by both macrophages and cancer cells in breast carcinoma.12 VEGF receptor expression has also been observed on breast cancer cells.13
Ki-67 is present in all proliferating cells, and its role as a proliferation marker attracts considerable interest as a biomarker of cancer growth and treatment.14 Ki-67 is a nuclear nonhistone protein present in all active phases of cell cycle, except the resting phase (G0).15 Potential uses of Ki-67 in breast cancer include prognosis of relative responsiveness, resistance to chemotherapy or endocrine therapy, or as a dynamic biomarker of treatment efficacy in neoadjuvant therapy.16
Influence of Omega 3s on Ki-67 and VEGF
An extensive amount of evidence has shown that the omega-3 fatty acids EPA and DHA play an essential role in the regulation of inflammatory responses.17 In the current study under review, immune staining of Ki-67 and VEGF in the intervention and control groups was performed at baseline and after neoadjuvant chemotherapy plus supplementation of omega 3 or placebo.
The authors reported an expression of Ki-67 that was positively correlated with VEGF expression (P<0.001). The results of this study imply that omega-3 supplementation may be a consideration to lower VEGF and Ki-67 expression in locally advanced breast cancer. Although the mechanism of action for the omega-3 fatty acids was not investigated, local anti-inflammatory effects may have influenced expression of VEGF and Ki-67.
Effects of Omega 3s in Chemotherapy
Omega-3 fatty acids have the ability to modulate inflammatory responses and intracellular signaling; thus incorporation of omega-3 fatty acids to improve clinical outcomes during standard chemotherapy may benefit patients through regulation of cytokines and immune cell signaling.18
The authors of this study suggest the results of this trial contribute to the evidential use of omega-3 supplementation in conjunction with chemotherapy. This conclusion may carry some bias based on the failure to reach statistical significance. However, this conclusion does not seem to be an entirely false claim based on the previous body of evidence. There is a significant evidence base for the use of dietary intake of omega-3 fatty acids to improve the efficacy of chemotherapeutic agents, in vivo and in vitro and in animal and clinical studies.19,20
In a previous study, fish oil was shown to improve the effectiveness of chemotherapy agents, including 5-fluorouracil (5-FU), paclitaxel, doxorubicin, and oxoplatin.21 While the overall survival was short for both cohorts in this current study, intervention with omega-3 fatty acids was associated with both significantly longer overall survival and longer disease-free progression. Based on this and previous evidence, one could argue that there is significant benefit for the utilization of omega-3 fish oil in conjunction with chemotherapy.10,21,22
Omega-3 Dosing, Bioavailability, Quality
Standard doses of omega-3 fatty acids range from 300 mg to 2,000 mg (EPA/DHA).23 All participants in this study received standard neoadjuvant CAF chemotherapy as well as 1 g/day omega-3 fatty acids or placebo.
The study intervention was observing the effects of fish oil on VEGF and Ki-67 expression. The authors fail to mention the ratio of EPA/DHA, which could alter the effects on VEGF and Ki-67 expression. They also omit the sourcing of the fish oil, which could also impair the effectiveness of this intervention.
Dietary supplements can contain several different forms of omega 3s, including natural triglycerides, free fatty acids, ethyl esters, re-esterified triglycerides, and phospholipids.24 Triglycerides are the form that occurs naturally in fish oil, whereas ethyl esters are synthesized by removing the natural glycerol backbone of the triglycerides and replacing it with ethyl ester end caps on each fatty acid. Re-esterified triglycerides are formed by the conversion of ethyl esters back to triglycerides. Omega 3s as re-esterified triglycerides, natural triglycerides, and free fatty acids have somewhat higher bioavailability than ethyl esters, but consumption of all forms significantly increases plasma EPA and DHA levels.25
Since the authors of this study did not assess bioavailability, it might be beneficial to look at the absorption via erythrocyte levels in the blood of patients to evaluate their nutrient status prior to treatment; this would also help to define a more appropriate/optimal dose for patients.
During treatment, 3 patients in the study suffered from diarrhea, and their omega-3 supplementation was discontinued for 5-7 days. The authors did not mention whether these adverse events were from treatment intervention or neoadjuvant therapy. Safety was not assessed in this study.
Overall Survival and Progression-Free Survival
The authors of this study claim that “omega-3 fatty acid supplementation improved overall survival and progression-free survival of locally advanced breast cancer treated with CAF neoadjuvant chemotherapy and mastectomy.” The study was conducted in Indonesia, where women are often diagnosed in late stages, the staging itself is prone to missing stage IV disease due to limitations of diagnostic equipment, and there are much higher mortality rates and shorter disease-free progression in that geographic region.26
- Limitations of this study include:
- Small sample size (n=48)
- Recruitment of locally advanced stage IIIB breast cancer only
- Failure to reach statistical significance
- No stated reasons for choosing this dose of fish oil
- Source of the fish oil capsules in the intervention group is not divulged
- Single dosage (1 g of fish oil)
In future studies, we should consider a trial that includes intervention groups at incremental doses of omega 3s. Expanding a trial with multiple dose levels would likely provide a clearer evidence base as well as optimal dosing.
It is clear there is a significant evidence base for the use of omega-3 fatty acids in breast cancer. This study, while not statistically significant, supports the extensive positive data for the use of omega-3 supplementation during standard chemotherapy and clinical outcomes. Future studies should include the assessment of optimal dosing, quality of supplementation, safety, and bioavailability assessments.
- Fabian CJ, Kimler BF, Hursting SD. Omega-3 fatty acids for breast cancer prevention and survivorship. Breast Cancer Res. 2015;17:62.
- Gago-Dominguez M, Yuan JM, Sun CL, Lee HP, Yu MC. Opposing effects of dietary n-3 and n-6 fatty acids on mammary carcinogenesis: The Singapore Chinese Health Study. Br J Cancer. 2003;89(9):1686-1692.
- Brasky TM, Lampe JW, Potter JD, Patterson RE, White E. Specialty supplements and breast cancer risk in the VITamins And Lifestyle (VITAL) Cohort. Cancer Epidemiol Biomarkers Prev. 2010;19(7):1696-1708.
- Zheng JS, Hu XJ, Zhao YM, Yang J, Li D. Intake of fish and marine n-3 polyunsaturated fatty acids and risk of breast cancer: meta-analysis of data from 21 independent prospective cohort studies. BMJ. 2013;346:f3706.
- Ellulu MS, Khaza'ai H, Abed Y, Rahmat A, Ismail P, Ranneh Y. Role of fish oil in human health and possible mechanism to reduce the inflammation. Inflammopharmacology. 2015;23(2-3):79-89.
- Diakos CI, Charles KA, McMillan DC, Clarke SJ. Cancer-related inflammation and treatment effectiveness. Lancet Oncol. 2014;15(11):e493-e503.
- Merendino N, Costantini L, Manzi L, Molinari R, D'Eliseo D, Velotti F. Dietary ω -3 polyunsaturated fatty acid DHA: a potential adjuvant in the treatment of cancer. Biomed Res Int. 2013;2013:310186.
- Weiss G, Meyer F, Matthies B, Pross M, Koenig W, Lippert H. Immunomodulation by perioperative administration of n-3 fatty acids. Br J Nutr. 2002;87(Suppl 1):S89-S94.
- Zhao Y, Joshi-Barve S, Barve S, Chen LH. Eicosapentaenoic acid prevents LPS-induced TNF-alpha expression by preventing NF-kappaB activation. J Am Coll Nutr. 2004;23(1):71-88.
- Chagas TR, Borges DS, de Oliveira PF, et al. Oral fish oil positively influences nutritional-inflammatory risk in patients with haematological malignancies during chemotherapy with an impact on long-term survival: a randomised clinical trial. J Hum Nutr Diet. 2017;30(6):681-692.
- Duffy AM, Bouchier-Hayes DJ, Harmey JH. Vascular Endothelial Growth Factor (VEGF) and Its Role in Non-Endothelial Cells: Autocrine Signalling by VEGF. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013. Available from: https://www.ncbi.nlm.nih.gov/books/NBK6482/
- Lewis JS, Landers RJ, Underwood JC, Harris AL, Lewis CE. Expression of vascular endothelial growth factor by macrophages is up-regulated in poorly vascularized areas of breast carcinomas. J Pathol. 2000;192(2):150-158.
- Speirs V, Atkin SL. Production of VEGF and expression of the VEGF receptors Flt-1 and KDR in primary cultures of epithelial and stromal cells derived from breast tumours. Br J Cancer. 1999;80(5-6):898-903.
- Soliman NA, Yussif SM. Ki-67 as a prognostic marker according to breast cancer molecular subtype. Cancer Biol Med. 2016;13(4):496-504.
- Nishimura R, Osako T, Okumura Y, Hayashi M, Toyozumi Y, Arima N. Ki-67 as a prognostic marker according to breast cancer subtype and a predictor of recurrence time in primary breast cancer. Exp Ther Med. 2010;1(5):747-754.
- Dowsett M, Nielsen TO, A’Hern R, et al. Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group. J Natl Cancer Inst. 2011;103(22):1656-1664.
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- Corsetto PA, Colombo I, Kopecka J, Rizzo AM, Riganti C. ω-3 long chain polyunsaturated fatty acids as sensitizing agents and multidrug resistance revertants in cancer therapy. Int J Mol Sci. 2017;18(12):E2770.
- Lee JY, Sim TB, Lee JE, Na HK. Chemopreventive and chemotherapeutic effects of fish oil derived omega-3 polyunsaturated fatty acids on colon carcinogenesis. Clin Nutr Res. 2017;6(3):147-160.
- Murphy RA, Mourtzakis M, Chu QS, Baracos VE, Reiman T, Mazurak VC. Supplementation with fish oil increases first-line chemotherapy efficacy in patients with advanced nonsmall cell lung cancer. Cancer. 2011;117(16):3774-3780.
- Shirai Y, Okugawa Y, Hishida A, et al. Fish oil-enriched nutrition combined with systemic chemotherapy for gastrointestinal cancer patients with cancer cachexia. Sci Rep. 2017;7(1):4826.
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