Selenium Supplementation Blocks Angiogenic Switch in Mammary Endothelial Cells in a Thioredoxin Reductase-Dependent Manner

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802

Performing Department
VETERINARY SCIENCE

Non Technical Summary
Breast cancer is a leading cause of death in women in the United States, and a dietary deficiency of the micronutrient selenium (Se) is associated with an increase in the formation of breast cancer. Foods that contain Se causes the incorporation of Se into proteins such as thrioredoxin reductase (TrxR), which is known to combat the cellular stress caused by oxidative metabolism, and possibly block formation of new blood vessels in breast cancer (i.e. angiogenesis). The objectives of this project include determining whether Se affects the angiogenic potential of cow mammary endothelial cells directly through the involvement of TrxR or transcription factors (AP-1 and HIF-1), which regulate the activation of pro-angiogenic genes in endothelial cells.

Animal Health Component

(N/A)

Research Effort Categories

Basic

90%

Applied

(N/A)

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	5010	1010	50%
702	5010	1040	50%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
5010 - Food;

Field Of Science
1040 - Molecular biology; 1010 - Nutrition and metabolism;

Goals / Objectives
The overall objective is to determine the effect of selenium (Se) supplementation and thioredoxin reductase (TrxR) on angiogenesis of bovine mammary endothelial cells (BMEC). The first objective is to determine if blocking TrxR activity in Se deficient BMEC yields altered expression of vascular endothelial cell growth factor (VEGF) or VEGF receptors (i.e. KDR and Flt-1), and a lowered angiogenic potential. The second objective is to determine if the selenoprotein TrxR regulates the expression of VEGF or VEGF receptors in Se deficient, oxidant stressed BMEC through the transcription factors hypoxia inducing factor-1 and/or activator protein-1.

Project Methods
Oxidant stress induced by Se deficiency in BMEC will be confirmed by assaying for glutathione peroxidase activity, and the generation of reactive oxidant species via flow cytometry. The effect of Se supplementation on the expression of vascular endothelial cell growth factor (VEGF) and VEGF receptors mRNA and protein will be assessed using quantitative competitive RT-PCR and immunoblotting. TrxR activity will be blocked using two independent techniques, a chemical selective inhibitor and RNA interference. TrxR activity will be measured using NADPH-dependent reduction of DTNB to confirm the activity of these inhibitors. Once TrxR activity is blocked, expression of VEGF and VEGF receptors will be assessed using quantitative competitive RT-PCR and immunoblotting techniques. Angiogenic switch in TrxR inhibited BMEC will be assessed using 3H-thymidine incorporation assays. Blocking of the expression of VEGF receptors will be accomplished using the addition of anti-sense oligonucleotides specific for bovine VEGF receptors (i.e. KDR and Flt-1). Activation of transcription factors (activator protein-1 (AP-1) and hypoxia inducing factor-1 (HIF-1)) in response to inhibition of TrxR or VEGF receptors will be assessed by performing electrophoretic mobility shift and luciferase-driven reporter assays. Once the activation of AP-1 and HIF-1 are known in response to blocking TrxR or TrxR, the activity of AP-1 and HIF-1 will be blocked by transfecting dominant negative plasmids for each transcription factor. The effect of blocking these transcription factors on expression of VEGF and VEGF receptors will be determined, as described above.

Progress 07/01/03 to 06/30/05

Outputs
Selenium deficient (-Se) bovine mammary endothelial cells (BMEC) produce significantly more mRNA and protein for vascular endothelial cell growth factor (VEGF)165, as compared to selenium sufficient (+Se) BMEC. In addition, -Se BMEC show significantly increased migratory potential as compared to +Se BMEC. We tested whether selenium supplementation affects the expression of Flk-1 (also known as KDR) or Flt-1, which are receptor for VEGF. Selenium deficient BMEC express more basal surface associated Flk-1, whereas +Se BMEC express significantly less. Selenium supplementation does not affect the surface expression of Flt-1. Because selenium supplementation affect the expression of selenoproteins, we examined whether thioredoxin reductase (TrxR) was involved in regulating surface expression of Flk-1 or Flt-1. Inhibition of TrxR by 1-chloro-dinitrobenzene (DNCB) caused +Se BMEC to express surface levels of Flk-1, similar to that of -Se BMEC. Thus, TrxR appears, in part, to regulate the expression of Flk-1. Because DNCB may selectively inhibit other selenoproteins, a RNA interference based approach to knock-down TrxR mRNA expression is being evaluated. Stable transfected BMEC expressing RNA interference vector for TrxR are under antibiotic selection, and will be used to further determine whether TrxR is involved in Flk-1 expression.

Impacts
Results of this proposal will result in a more comprehensive understanding of the mechanism by which selenium supplementation affects the early progression of breast cancer. In addition to breast cancer, these findings may be broadly applicable to a variety of other cancers, such as prostate and colorectal, where the clinical chemo preventative effect of selenium supplementation is currently being tested.

Publications

No publications reported this period