The present study addresses a fundamental question of causality by examining the dose response effect of n-3 PUFA on tumour outcomes in a pre-clinical model of breast cancer. Epidemiological studies to date only provide correlative evidence in support of a beneficial effect of n-3 PUFA in reducing the risk of developing breast cancer . Thus, a major gap remaining is demonstrating a cause-effect relationship in human or preclinical models . To the best of our knowledge a fundamental dose-dependent study of n-3 PUFA in breast cancer has not been previously reported. Thus, it is unknown whether effects at higher doses plateau or potentially cause harm. The results of this study provide evidence that the intake of n-3 PUFA, in a dose dependent manner results in a corresponding increase in n-3 PUFA in tumour phospholipids associated with a reduction in tumour outcomes.A key attribute of cause-effect relationships is demonstrating dose dependency. Multiple parameters in this study suggest a beneficial dose-dependent relationship across the 0%, 3% and 9% (w/w) levels of n-3 PUFA and tumour outcomes. On average, tumour latency was delayed by 2% and 9% in mice fed 3% and 9% n-3 PUFA diets, respectively (Figure 1). On average, the majority of mice were tumour free longer throughout the later time points in the 3% and 9% n-3 PUFA fed groups when compared to the 0% n-3 PUFA fed group (Figure 2). Tumour multiplicity was significantly (p < 0.05) decreased at the highest dose of 9% n-3 PUFA (Figure 3). Tumour volume was significantly (p < 0.05) and dose-dependently decreased at all levels of treatment (Figure 4).
The current study showed a greater reduction in final tumour volume in mice fed a 9% n-3 PUFA diet in comparison to findings of previous studies in MMTV-neu mice [14, 15]. Yee et al. (2005) fed mice either an 11% (w/w) corn oil diet (enriched in n-6 PUFA) or a 10% (w/w) menhaden fish oil diet beginning at 7–8 weeks of age for the duration of a 61 week study . The study utilized the MMTV-neu mouse model, a less aggressive model than the one utilized in the current study. These investigators showed that a diet supplying 22.5% of total energy (10% w/w) from menhaden oil resulted in a 30% reduction in tumour volume . In contrast, a similar amount of energy provided by lifelong exposure to a 9% n-3 PUFA diet in the present study resulted in a 70% reduction in final tumour volume. These observations suggest a potential benefit of long term exposure to n-3 PUFA from conception through in utero development, lactation, weaning, puberty and adulthood confers added protection against mammary tumour development. This is supported by previous studies demonstrating that the timing of exposure may influence future cancer risk [11, 23].
In regards to physiological relevance, the 9% n-3 PUFA diet provided mice with 22% of total calories from fat, but of greater relevance this diet provides 6.7% of the total daily energy requirements from EPA and DHA, or the equivalent of 11 g/day in humans (assuming the average human female consumes 2000 kcal/day). In humans the consumption of 267 g of fish per day will provide approximately 11 g of EPA and DHA, or alternatively the equivalent of 3 servings of an average 85 g salmon steak . While the intake of 11 g of EPA and DHA per day may seem unrealistic, a study of 48 women demonstrated that intake of up to 9 capsules of marine based n-3 PUFA, providing up to 7.56 g of DHA + EPA a day, for 6 months was well tolerated by participants . The prescribed dosages produced similar levels of compliance among participants and a dose response accumulation of EPA and DHA was observed in serum and breast adipose tissue . In contrast, the equivalent quantity of EPA and DHA from the 3% n-3 PUFA diet is achievable in humans through the consumption of 3.4 g of EPA/DHA a day. Alternatively, the balance of n-6 to n-3 PUFA is another way to characterize the physiological relevance of the diets used in the present study. In Australia, the ratio of n-6 to n-3 PUFA in an individual’s diet ranges from 2 to 80, which is similar to the variance of this ratio used in the present study .
The anti-tumourigenic properties of n-3 PUFA are largely related to their ability to incorporate into the plasma membrane of target tissues and alter membrane-protein and subsequently protein-protein interactions . The observed dose-dependent effects of a reduced n6:n-3 PUFA dietary intake were associated with a dose-dependent change in the fatty acid profile reflected in a decreased n-6:n-3 PUFA ratio, driven by increases in n-3 PUFA, mainly EPA, DPA and DHA, and a concomitant decrease in the n-6 PUFA, AA. These direct measurements importantly confirm that the bioactive components of the diet, n-3 and n-6 PUFA, are targeted to the mammary gland and tumour of interest, which coincided with reductions in tumour burden observed in the mice. Marine-based oils contain multiple n-3 PUFA, but the field has focused primarily on the study of EPA and DHA. However, the present study observed significant and appreciable incorporation of another n-3 PUFA, docosapentaenoic acid (DPA, 22:5 n-3) (Table 2). The biological role of DPA is not known, but one previous study noted a similar observation . Thus, DPA deserves more attention in future studies regarding cancer. This is highly relevant given that DHA was only preferentially incorporated into tumour phospholipids but not into mammary gland phospholipids (Tables 1 and 2). While speculative, these observations suggest differential role of individual n-3 PUFA along the cancer trajectory. Further research is required to better define the interplay between individual and combinations of n-3 PUFA on tumour outcomes.
The present study did not examine mechanisms of action, but the change in membrane fatty acid composition is important as n-6 and n-3 PUFA compete for the same eicosanoid enzymes. The observed anti-tumourigenic effects of the diets utilized in this study may be due to a dose-dependent increase in n-3 PUFA consumption, a dose-dependent reduction in n-6 PUFA intake or a combination of the two with a dose-dependent reduction in the n-6:n-3 PUFA dietary ratio. Prostaglandins, leukotrienes and thromboxanes derived from AA promote inflammation and cellular proliferation; however, when they are derived from n-3 PUFA they promote apoptosis and inhibit cell proliferation . Additionally, n-3 PUFA have been found to elevate pro-apoptotic factors including caspases 9 and 3, Bcl2, and reactive oxygen species, while also reducing DNA synthesis, cellular proliferation and the integration of growth factor receptors into the plasma membrane [18, 19]. Changes in membrane composition were observed in both mammary and tumour tissue, but it is likely that the change in tumour composition is most relevant in explaining the anti-tumourigenic effects of n-3 PUFA. The control diet utilized in this study contained 10% (w/w) safflower oil which is abundant in LA and scarce in other n-6 PUFA. The n-6 and n-3 families of fatty acids refer to many structurally similar molecules which may have different biological effects. Thus, in this study, we can only conclude that in comparison to an LA rich diet, a fish oil diet rich in EPA and DHA is protective against tumour development.
Pubertal onset has been linked to breast cancer as earlier menarches place females at a greater risk for breast cancer . In the present study, the onset of puberty in the mice was significantly (p < 0.05) delayed by ~3 days in mice fed either the 3% or 9% n-3 PUFA diets as compared to 0% n-3 PUFA fed mice. Mechanistically, n-3 PUFA may influence circulating estrogen levels, a key regulator of puberty . This observation suggests further investigation on the role of n-3 PUFA in regulating hormonal influences on puberty and cancer. Furthermore, this observation supports growing evidence that critical periods of early development such as puberty have important influences on future cancer risk [23, 30, 31].
In conclusion, the present study contributes importantly to addressing an important gap in our fundamental understanding of the causal role for n-3 PUFA in breast cancer prevention.