Cancer Prevention Research Eleostearic Acid Inhibits Breast Cancer Proliferation by Means of an Oxidation-Dependent Mechanism

Eleostearic acid (α-ESA) is a conjugated linolenic acid that makes up ∼60% of Momordica charantia (bitter melon) seed oil. Prior work found that water extract from bitter melon was able to inhibit breast cancer. Here, we investigated effects of α-ESA on both estrogen receptor (ER)–negative MDA-MB-231 (MDA-wt) and ER-positive MDA-ERα7 human breast cancer cells. We found that α-ESA inhibited proliferation of both MDA-wt and MDA-ERα7 cells, whereas conjugated linoleic acid had comparatively weak antiproliferative activity at 20 to 80 μmol/L concentrations. We also found that α-ESA (40 μmol/L) treatment led to apoptosis in the range of 70% to 90% for both cell lines, whereas conjugated linoleic acid (40 μmol/L) resulted in only 5% to 10% apoptosis, similar to results for control untreated cells. Addition of α-ESA also caused loss of mitochondrial membrane potential and translocation of apoptosis-inducing factor as well as endonuclease G from the mitochondria to the nucleus. Additionally, α-ESA caused a G2-M block in the cell cycle. We also investigated the potential for lipid peroxidation to play a role in the inhibitory action of α-ESA. We found that when the breast cancer cells were treated with α-ESA in the presence of the antioxidant α-tocotrienol (20 μmol/L), the growth inhibition and apoptosis effects of α-ESA were lost. An AMP-activated protein kinase inhibitor (Dorsomorphin) was also able to partially abrogate the effects of α-ESA, whereas a caspase inhibitor (BOC-D-FMK) did not. These results illustrate that α-ESA can block breast cancer cell proliferation and induce apoptosis through a mechanism that may be oxidation dependent. Anumber of studies indicate that total dietary fat intake may play a major role in the development and progression of breast cancer in humans (1, 2). However, the impact of different types of fat in the diet on breast cancer development has proven complex with results from previous studies supporting both positive and negative roles for specific fats. Some long chain polyunsaturated fatty acids (LC-PUFA) have been reported to have protective effects as shown by an inverse relationship between high fish consumption and breast cancer first noted over 30 years ago (3, 4). We have identified bitter melon as a plant containing a LCPUFA that seems to have potential anticancer properties. Bitter melon originated in tropical Asia, but due to its apparent health benefits, it is now grown and used medicinally in many countries and is widely available in a variety of different supplement forms. Prior work showed that multiple types of extracts from bitter melon had in vivo (5–7) and in vitro (8) anticancer activity. Of particular interest to us is eleostearic acid (α-ESA), also known as 9Z,11E,13E-octadecatrienoic acid, a LC-PUFA that makes up ∼60% of bitter melon seed oil. In vitro studies using pure α-ESA have reported anticancer activity. For example α-ESA significantly reduced viability of transformed NIH-3T3 mouse fibroblast (SV-T2) and monocytic leukemia (U-937) cells (9). In additional reports, DLD-1 colorectal adenocarcinoma cells treated with α-ESA in vitro were growth inhibited and underwent DNA laddering indicative of apoptosis (10), and both Caco-2 and HT-29 colon cancer cells had decreased viability and increased DNA fragmentation when treated with α-ESA (11). In a follow-up study using Caco-2 cells, α-ESA was again found to reduce cell viability and increase DNA fragmentation such as would be seen with apoptosis. Cell viability was maintained by addition of the antioxidant α-tocopherol in a concentration-dependent manner, suggesting that the reduction in viability is dependent on lipid peroxidation (12). The potential for α-ESA to inhibit breast cancer and possible mechanisms of action of α-ESA in breast cancer has not been addressed to this point. In this study, we investigated the anticancer effects of purified α-ESA using human breast cancer cells with and without estrogen receptor by comparison of cell proliferation and apoptosis in the presence and absence of α-ESA. We have also Authors' Affiliation: The Hormel Institute, University of Minnesota, Austin, MN Received 4/30/09; revised 8/21/09; accepted 8/24/09; published OnlineFirst 9/29/09. Grant support: The Breast Cancer Research Foundation and Hormel