Author : Christine Skibinski
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (943 download)
Book Synopsis Preclinical Investigations Into the Role of Omega-3 Fatty Acids for Breast Cancer Prevention by : Christine Skibinski
Download or read book Preclinical Investigations Into the Role of Omega-3 Fatty Acids for Breast Cancer Prevention written by Christine Skibinski and published by . This book was released on 2015 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: As discussed in Chapter 1, Breast cancer is the second leading cause of cancer death in women in the United States, with about 2 million women at high risk for developing the disease. A current strategy, approved by the FDA, for breast cancer prevention is the daily administration of selective estrogen receptor modulators(SERMS), tamoxifen and raloxifene. These SERMS have proven to be effective at reducing breast cancer incidence in women that are at high risk by 50% and 38%, respectively. However, these agents are poorly accepted as oral chemopreventives even by women at high risk for breast cancer because of concerns of side effects which include thromboembolic events and an increase in endometrial cancers. Furthermore, both agents are ineffective against the more aggressive estrogen receptor negative tumors. A series of experiments have been conducted in our laboratories to test the hypothesis that chemoprevention can be improved by combining SERMS with agents with different mechanisms of action. Such an approach can allow the use of low doses of SERMS and thus reduce their side effects. Literature data provide some support of the protective effects of omega-3 fatty acids against the development of several cancers, including breast cancer. However, the results remain inconsistent which could be due to confounding variables. These confounding variables which have been reported by our group include omega-3:omega-6(n-3:n-6) ratio and caloric intake. A previous study conducted in our laboratories showed that high ratios of omega-3:omega-6 fatty acids(25:1 n-3:n-6) are required to inhibit mammary carcinogenesis in the rat and such high ratios of omega-3:omega-6 fatty acids potentiated the chemopreventive efficacy of tamoxifen. Studies conducted in Chapter 2 were aimed to test the hypothesis that by using a proteomics approach, novel proteins can be identified that can provide insights into the molecular mechanism by which high ratios of omega-3:omega-6 fatty acids inhibit mammary carcinogenesis. We further hypothesize that proteins identified in a minimally invasive fashion can be used for early detection and to monitor the efficacy of the chemopreventive agents.We used an isobaric Tagging for Relative and Absolute Quantitation (iTRAQ) method to provide insights into the mechanism, at the protein level, responsible for the chemopreventive action of the high omega-3:omega-6 fatty acid ratios in the absence and presence of tamoxifen in the 1-methyl-1-nitrosourea(MNU)-induced mammary tumor model in the rat; selective proteins were further validated by western blotting. Compared to control (n-3:n-6, 1:1) diet, both 10:1 and 25:1 n-3:n-6 diets upregulated plasma 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 n-3:n-6, the 25:1 n-3:n-6 plus tamoxifen diet downregulated apolipoprotein E, haptoglobin, and inter-alpha-inhibitor H4 heavy chain. Ingenuity Pathway Analysis (IPA) 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. Collectively, our results show that several proteins were altered in a manner consistent with chemoprevention. The Western diet is composed of a 15:1 n-6:n-3 ratio and would be impractical for women to consume high omega-3:omega-6 ratios on a daily basis. The chemopreventive efficacy of the high omega-3:omega-6 ratios suggests that a component of omega-3 fatty acids(eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)) or one of its metabolites which is present in small amounts could account for the chemopreventive effects observed in the rat. Furthermore, considering the superior efficacy of DHA over EPA, in preclinical mammary carcinogenesis models as reported in the literature provided a strong rationale to focus our studies on DHA. However, the highly unsaturated structure of DHA makes it susceptible to oxidation by aqueous medium, therefore a liposomal formulation of DHA would be ideal for oral administration, which is considered an appropriate route for chemoprevention studies. In Chapter 3, we hypothesize that an ether lipid based nanoliposome delivery system of DHA would protect DHA from oxidation and fluctuation in pH of the gastrointestinal system, resulting in an ideal oral delivery system. Oral delivery is the appropriate route for chemoprevention studies. We further hypothesize that liposomal DHA will be more efficacious than free DHA at providing an anti-cancer effect in vitro, specifically in both estrogen receptor positive and estrogen receptor negative human breast cancer cell lines. Our results indicated that our liposomal formulation of DHA(size: 136nm, charge: -1.3mV) protected DHA from oxidation and acidic pH, as determined by dynamic light scattering. Our liposome also had a 60-80% encapsulation efficiency as determined by LC-MS/MS. Furthermore, liposomal DHA was found to be more efficacious at reducing cell viability, and increasing apoptosis in both MCF-7 and MDA-MB-231 cell lines. In addition, western blot analysis revealed that liposomal DHA was more efficacious at altering protein markers (P-AKT, P-S6, p21, cleaved PARP, BCL-2) of cell viability, the cell cycle, and apoptosis in human breast cancer cell lines in a manner consistent with chemoprevention. We decided in Chapter 4 to test our hypothesis that the oral administration of our acid stable liposome formulation of DHA would protect DHA from oxidation and acidic pH and thus increasing the bioavailability of DHA and its metabolites in the serum and at the mammary fat pad. To test our hypothesis, it was crucial to initially demonstrate whether our oral liposomal formulation was capable of delivering DHA and its metabolites to the circulation and mammary fat pad. Female rats (21-day old) were gavaged with liposomal DHA and sacrificed at 1, 3, and 6 hours and tissues and serum were collected at each time point. Fatty acids were extracted with a solution ethyl acetate/hexane and methylated using a solution of acetyl chloride, butylated hydroxytoluene, and methanol. Detection and quantification of DHA (expressed as % of administered dose) were achieved using Gas Chromatography Flame Ionization Detection(GC-FID), and liquid chromatography tandem mass spectroscopy(LC-MS/MS). The lipoxygenase (LOX) metabolites of DHA, known to play a role in anti-inflammation, were quantified using LC MS/MS. Following the oral administration of liposomal DHA, a significantly higher amount of DHA was found in the mammary fat pad (15-20%), 2-3 fold higher, than in the serum (4.5-6%). The LOX metabolites 4-hydroxy DHA, 14-hydroxy DHA, and 17-hydroxy DHA were also detected in both the serum (0.14%) and mammary fat pad (0.45%). In summary, we demonstrated that high omega-3 fatty acids ratios(25:1) with and without tamoxifen were able to alter plasma proteins in a manner consistent with chemoprevention. Furthermore, we demonstrated for the first time the successful formulation of liposomal DHA for oral delivery of DHA. Our formulation protected DHA from acidic pH and oxidation and in vitro and in vivo results provided experimental data demonstrating the potential utility of liposomal DHA in the prevention of breast cancer initially in preclinical models and ultimately in the clinic. Future studies (detailed in Chapter 5) will be aimed at comparing the pharmacokinetics, pharmacodynamics and chemopreventive efficacy of liposomal DHA and free DHA in the rat.
