tearate has been found to inhibit invasion, inhibit proliferation and induce apoptosis of breast cancer and other cells, as well as induce cytotoxicity in a variety of normal and malignant cell lines (9
). This body of work indicates the possibility for stearate to function as an anti-metastasic agent in vivo
. This is the first study to demonstrate that stearate does inhibit breast cancer metastasis in vivo
and does so by approximately 50%. Interestingly stearate also inhibits the primary tumor size and weight by approximately 50% although stearate's inhibition of metastasis was independent of primary tumor size. While this does not necessarily rule out a relationship between the effects of stearate on the primary tumor and decreased metastasis it certainly increases the possibility that stearate effects another part of the metastatic cascade. In vitro
, stearate induced apoptosis of the MDA-MB-435 breast cancer cells in a dose dependent manner (). As 50 μM can be considered a high-normal stearate concentration in the plasma of humans, this suggests that physiological concentrations of stearate are sufficient to induce apoptosis of breast cancer cells. This may partially explain the decrease of tumor size and metastases seen in the stearate-fed mice compared to the low fat and high safflower fed mice. However, because the primary tumors have similar growth patterns up to 3 weeks post-injection, if stearate is affecting the viability of the cancer cells, it is most likely not acting through an initial selection of sub-populations of cells that are sensitive to the apoptotic effects of stearate.
The three week delay in dietary stearate decreasing primary tumor volumes may suggest changes in the tumor microenvironment such as an inhibition of angiogenesis. Compounds derived from arachidonic acid and those derived from EPA appear to have opposing roles in the cell. Studies have found that linoleic acid enhanced the development of tumors in animals that were injected in the mammary fat pad with MDA-MB-435 or MDA-MB-231 breast cancer cells. To date, the mechanism underlying this increase is unknown. However, it has been hypothesized that the increase in linoleic acid causes an increase in arachidonic acid, thereby increasing prostaglandin synthesis (Reviewed in 20, 21). Consistent with this hypothesis, treatment of animals on a high linoleic acid diet with indomethacin, a cyclooxygenase inhibitor, inhibited prostaglandin synthesis and linoleic acid induced metastasis (22
). Omega-3 fatty acids such as EPA, and DHA have been shown to inhibit tumor angiogensis and breast cancer cell metastasis, perhaps due to a difference in lipid metabolism or an inhibition of arachidonic-derived eicosanoids (7
). To date, the role of saturated fatty acids on prostaglandin synthesis has not been investigated in breast cancer tumors. However, in vitro
, 20 μM stearate is sufficient to almost completely inhibit cyclooxgenase-1 activity and partially inhibit cyclooxygenase-2 activity (26
). Furthermore, stearate has been shown to induce apoptosis of human endothelial cells, although the 200-300 μM concentrations tested are within the pathophysiological range (27
). These results indicate stearate may inhibit angiogenesis by inhibiting prostaglandin synthesis or affecting vessel viability in the mammary fat pad tumors, and, therefore, may cause a decrease in primary tumor size and inhibit metastasis.
Stearate could also be modulating the immune response of nude mice to the tumors, thereby accounting for the variation observed in the stearate (B) subgroups. These animals have been shown previous to display a large amount of heterogeneity concerning specific T-cell subpopulations (29
). This suggests that although the mice are an inbred population, the immune response to the tumor may vary between mice. Interestingly, macrophages, cells known to be modulated by fatty acids, have been extensively studied for their cancer promoting or cancer inhibiting effects. Two lineages of macrophages have been uncovered that effect tumors in opposing manners. The M1 lineage is generally associated with tumor suppression and resistance whereas the M2 linage is associated with tumor promotion and angiogenesis (Reviewed in 30). Although much work has been done analyzing the effect of n-3 and n-6 fatty acids in regard to immune response, little is known concerning the saturated fatty acids. Stearate has been demonstrated previously to induce apoptosis of the murine macrophage cell line J774 (31
). Dietary stearate has been associated with a decrease in natural killer T-cells in the spleen of mice, whereas palmititate, a 16-carbon saturated fatty acid has been associated with an increase in activity (32
). NK cells are known to stimulate M2 macrophages through the release of certain cytokines (30
). These results suggest that dietary stearate may inhibit the differentiation of monocytes into M2 macrophages, thereby inhibiting tumor progression. These data also suggest that the saturated fatty acids have differing effects in vivo
. Future studies are necessary to the cause of the decrease in tumor size.
Interestingly, the animals in the stearate (B) group displayed a range in terms of tumor growth, but not macroscopic metastases. The tumors in the B-i and B-ii subgroups reached a size comparable to the low fat and safflower groups although an additional 2-3 weeks of tumor growth were necessary for this to occur (9 weeks post-injection for the low fat and safflower animals versus 11 and 12 weeks post injection for B-i and B-ii, respectively). The tumors in the B-iii group reached a maximum volume approximately 6 weeks after cancer cell injection. The underlying cause of this plateau is unknown although stearate could be inhibiting proliferation or inducing apoptosis of the cancer cells. Stearate has been shown previously to inhibit epidermal growth factor dependent proliferation in the Hs578t human breast cancer cells and we present evidence here that the MDA-MB-435 breast cancer cells are sensitive to stearate induced apoptosis ((9
); ). Furthermore, as cancer cell masses are generally accepted as a heterogeneous population of neoplastic, there could be a selection occurring for stearate-insensitive cells. Further studies are necessary to test these hypotheses.
Given the complexity of dietary studies, the effect seen with the high stearate diet may be due to a decrease in linoleate, the predominate fatty acid in safflower oil. Safflower oil is composed of 77% linoleic acid. The stearate diet was 3% safflower oil, meaning the diet was 2.3% linoleic acid as compared to the 15.5% in the safflower diet. Using the same mammary fat pad injection model with the MDA-MB-435 breast cancer cells, Rose et al. reported that significantly more macroscopic lung metastases in animals fed a diet composed of 12% linoleic acid than animals fed a diet with 2% linoleic acid, although no difference was observed in the growth rate of the primary tumor. (4
). These results would argue our effect may, at least in part, be due to a decrease in linoleic acid. However, corn oil is 56% linoleic acid, meaning that our low fat diet was composed of 2.8% linoleic acid. Given no significant difference was observed between the low fat (2.8% linoleic acid) and safflower (15.5% linoleic acid) diets in terms of tumor volume or lung metastases, the effects seen with the stearate diet are most likely not due to a decrease in linoleic acid. Also, there was a significant reduction in primary tumor and metastasis seen with the stearate diet compared to the low fat diet that contained comparable amounts of linoleic acid.
In summary, dietary stearate inhibited the growth of MDA-MB-435 human breast cancer cells in the mammary fat pad model system and partially reduced metastatic burden in the lung. The inhibition of metastasis was independent of the size of the primary tumor as animals that developed larger tumors also had an inhibition of metastasis. Physiological concentrations of stearate where also sufficient to induce apoptosis in vitro, providing a potential mechanism observed in vivo. There is also evidence in the literature to support other potential in vivo mechanisms, including inhibition of angiogenesis and modulation of the immune system, and these are on going areas of investigation in our laboratory. The degree of inhibition of metastasis by dietary stearate indicates that it may be a potential adjuvant therapeutic strategy for breast cancer patients to increase the suppression of metastatic disease.