Research Article Proteomic Changes Induced by Effective Chemopreventive Ratios of n-3:n-6 Fatty Acids and Tamoxifen against MNU-Induced Mammary Cancer in the Rat Christine G. Skibinski 1 , Henry J. Thompson 4 , Arunangshu Das 1 , Andrea Manni 2 , James D. Bortner 1 , Anne Stanley 3 , Bruce A. Stanley 3 , and Karam El-Bayoumy 1 Abstract We used a proteomic approach to gain insights into the mechanisms of protection at the protein level by a high n-3:n-6 ratio in the absence and presence of Tamoxifen. Four groups were treated with 1-methyl-1- nitrosourea (MNU) and fed the following diets with varied n-3:n-6 ratios; group 1 ¼ 1:1; groups 2 and 3 ¼ 10:1 and 25:1, respectively; group 4: (25:1) plus Tamoxifen (1 mg/kg diet). The plasma from six rats/group was pooled and analyzed with the isobaric tags for relative and absolute quantitation method; 148 proteins were identified with 95% confidence by ProteinPilot 4.0. In plasma of rats fed 10:1, 25:1 n-3:n-6, and 25:1 plus Tamoxifen, the number of proteins that met our criteria (P  0.05, error factor  2) were 10, 14, and 19 proteins, respectively. Selected proteins were further validated by Western blotting. Compared to 1:1, both 10:1 and 25:1 diets upregulated vitamin D binding protein, gelsolin, and 14-3-3 sigma, reported to have tumor suppressive effects, whereas alpha-1B-glycoprotein, which has been reported to be elevated in the serum of breast cancer patients was decreased. Compared to 25:1, the 25:1 plus Tamoxifen diet down- regulated apolipoprotein E, haptoglobin, and inter-a-inhibitor H4 heavy chain. Ingenuity pathway analysis determined that the trends of specific proteins were related to lipid metabolism in the 25:1 n-3:n-6 group, whereas the 25:1 n-3:n-6 plus Tamoxifen group included proteins involved in cancer and inflammation. Our results show that several proteins were altered in a manner consistent with chemoprevention. Such proteins may serve as biomarkers to monitor efficacy of n-3 and Tamoxifen in future clinical chemopre- vention trials. Cancer Prev Res; 6(9); 979–88. Ó2013 AACR. Introduction Breast cancer is the second leading cause of cancer death in women (1). Although effective local and synthetic ther- apies are available, prevention represents the best approach to reduce breast cancer morbidity and mortality. The anti- estrogens, Tamoxifen and Raloxifene, have been shown to be effective chemopreventive agents reducing the risk of breast cancer in high-risk women by about 50% and 38%, respectively (2). However, only a very small portion of high- risk women take these drugs for chemoprevention primarily because of fear of side effects, particularly thromboembolic events (2). Furthermore, antiestrogens are only able to inhibit the development of estrogen receptor (ER) positive tumors, whereas they are totally ineffective at inhibiting ER negative ones. Clearly there is an urgent need to develop mechanistically based naturally occurring chemopreventive agents which can be used individually and in combination with antiestrogens. The combined approach may allow the use of lower doses of antiestrogens to minimize their side effects. It has been postulated that the risk of breast cancer can be influenced by certain dietary components such as the amount and type of dietary fats ingested (3–7). Among the fatty acids, omega-3 (n-3) and omega-6 (n-6) fatty acids have been suggested to decrease and increase breast cancer risk, respectively. Despite the prevailing hypothesis that the ratio of n-3:n-6 may be important for chemoprevention, the optimum ratio has not been rigorously tested in many previously published reports (7). Furthermore, several che- moprevention studies in animal models used diets that were high in the percent of dietary calories provided from fat which are not consistent with the U.S. Dietary Guidelines (8). Because both issues (% calories from fat and n-3:n-6 ratio) are problematic from the translational perspectives, this stimulated our team (4) to formulate a series of experi- ments testing the chemopreventive efficacy of purified diets modified to reflect levels of dietary fat which are currently Authors' Affiliations: Departments of 1 Biochemistry & Molecular Biology and 2 Medicine; 3 Proteomics Mass Spectrometry Core Facility, Pennsylva- nia State University College of Medicine, Hershey, Pennsylvania; and 4 Cancer Prevention Laboratory, Colorado State University, Fort Collins, Colorado Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/). Corresponding Author: Karam El-Bayoumy, Cancer Institute, Penn State University College of Medicine, 500 University Drive, PO Box, Hershey, PA 17033. Phone: 717-531-1005; Fax: 717-531-0002; E-mail: kee2@psu.edu doi: 10.1158/1940-6207.CAPR-13-0152 Ó2013 American Association for Cancer Research. Cancer Prevention Research www.aacrjournals.org 979 Research. on January 22, 2016. © 2013 American Association for Cancer cancerpreventionresearch.aacrjournals.org Downloaded from Published OnlineFirst July 23, 2013; DOI: 10.1158/1940-6207.CAPR-13-0152