Tamoxifen and Flaxseed Alter Angiogenesis Regulators in Normal Human Breast Tissue In Vivo

The incidence of breast cancer is increasing in the Western world and there is an urgent need for studies of the mechanisms of sex steroids in order to develop novel preventive strategies. Diet modifications may be among the means for breast cancer prevention. Angiogenesis, key in tumor progression, is regulated by the balance between pro- and anti-angiogenic factors, which are controlled in the extracellular space. Sampling of these molecules at their bioactive   compartment is therefore needed. The aims of this study were to explore if tamoxifen, one of the most used anti-estrogen treatments for breast cancer affected some of the most important endogenous angiogenesis regulators, vascular endothelial growth factor (VEGF), angiogenin, and endostatin in normal breast tissue in vivo and if a diet supplementation with flaxseed had similar effects as tamoxifen in the breast. Microdialysis was used for in situ sampling of extracellular proteins in normal breast tissue of women before and after six weeks of tamoxifen treatment or before and after addition of 25 g/day of ground flaxseed to the diet or in control women. We show significant correlations between estradiol and levels of VEGF, angiogenin, and endostatin in vivo, which was verified in ex vivo breast tissue culture. Moreover, tamoxifen decreased the levels of VEGF and angiogenin in the breast whereas endostatin increased significantly. Flaxseed did not alter VEGF or angiogenin levels but similar to tamoxifen the levels of endostatin increased significantly. We conclude that one of the mechanisms of tamoxifen in normal breast tissue include tipping of the angiogenic balance into an anti-angiogenic state and that flaxseed has limited effects on the pro-angiogenic factors whereas the anti-angiogenic endostatin may be modified by diet. Further studies of diet modifications for breast cancer prevention are warranted. (Authors abstract)

Long-term exposure to sex steroids such as estradiol and/or progesterone increases the risk of breast cancer. A potent proangiogenic factor is angiogenin a 14.2 kD polypeptide member of the RNase A superfamily, which induces endothelial cell proliferation after its nuclear translocation. The nuclear translocation of angiogenin is also necessary for the angiogenic response of VEGF, and other angiogenesis regulator. Angiogenin is estrogen regulated in a similar fashion as VEGF in normal breast tissue and experimental breast cancer. Human endostatin inhibits endothelial cell proliferation and migration, and induces apoptosis in proliferating endothelial cells. Estradiol down-regulates and tamoxifen up-regulates endostatin generation in experimental breast cancer via effects on matrix metalloproteinase activity. Lignans, which are found in in flaxseed, have been associated with reduced breast cancer risk. The present study determined if a diet of flaxseed or treatment with tamoxifen affected potent angiogenic regulators in normal human breast tissue in vivo. It was shown in this study that the most potent endogenous angiogenesis inhibitor, endostatin, exhibits a negative correlation with estradiol in normal human breast tissue in situ. Tamoxifen increased the endostatin levels both in women in vivo and in cultured breast biopsies. In a similar fashion as tamoxifen, a diet of flaxseed to healthy premenopausal women increased the extracellular endostatin in breast tissue significantly. A diet of flaxseed did, however, not alter VEGF or angiogenin in the breast. A tipping of the angiogenic balance to favor a pro-angiogenic environment is a key event in the initiation, growth, and progression of tumors. In this study, tamoxifen had significant effects on the proangiogenic proteins investigated. In this study it was postulated that anti-angiogenic effects similar to that of tamoxifen would be induced by dietary flaxseed in healthy volunteers. The data did not show any effects of VEGF levels in breast tissue after the addition of flaxseed to the diet. The difference compared with previous animal data, where VEGF levels decreased by flaxseed, may be a difference of cancerous tissue versus normal tissue and the fact that there is a magnitude of difference in lignan exposure in women (1 mg/kg bwt) and mice (10 mg/kg bwt). Moreover, in animal studies, the amount of flaxseed added to the diet equates approximately 10% of the daily caloric intake while in women 25 g of ground flaxseed is less than 5% of the total calorie intake. No differences in estradiol and/or progesterone levels after flaxseed ingestion were seen in the women ruling out an effect of flaxseed on estrogen levels. This may suggest that there is no strong dose-response relationship with endostatin and lignans, or the cohort was too small to detect a possible relationship or, that other component(s) in flaxseed, not measured in our study, may modulate endostatin in breast tissue. A diet modification with flaxseed for one menstrual cycle does not seem to be equally powerful as  tamoxifen in tipping the angiogenic balance towards angiogenesis inhibition in normal breast tissue. Further studies of flaxseed for a longer duration are warranted. (Editors comments)