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Riboflavin Trafficking in Breast Cancer
Background: Riboflavin (vitamin B2) serves as a precursor for the biosynthesis of coenzymes FMN (riboflavin 5-phosphate) and FAD (flavin adenine dinucleotide) that are crucial to oxidation-reduction reactions involved in cellular metabolic pathways. Recently, our laboratory using subcellular localization techniques coupled with studies involving pharmacological alteration of vesicular trafficking has shown riboflavin transport to follow a receptor-mediated endocytic pathway, although the exact mechanism remains elusive. The working model proposes the involvement of soluble riboflavin-binding proteins (RfBP) in sequestering riboflavin followed by internalization via cell surface receptors associated with the recruitment of clathrin. RfBP are 37 kDa estrogen-responsive phosphoglycoproteins that exhibit increased expression in rapidly dividing cells such as early and advanced breast carcinomas and the developing fetus. Consequently, the enhanced expression of serum RfBP associated with marked increase in cell proliferation has been implicated as a potential serum and tissue marker for the clinical screening of breast tumors. However, literature reports on riboflavin trafficking via the riboflavin-RfBP system in breast cancer are scarce. Objective/Hypothesis: Breast cancer is associated with an increase in receptor-mediated endocytosis of riboflavin. Specific Aims:  To measure and correlate riboflavin uptake with RfBP expression levels in established breast cancer and non-tumorigenic cell models.  To investigate the involvement of an endocytic mechanism in riboflavin trafficking in cellular models of breast cancer.  To determine tumor uptake of riboflavin in whole animals xenografted with cellular strains of breast cancer. Study Design: Aim  Transpithelial transport of [3H]-riboflavin combined with HPLC analysis of flavin content (FMN and FAD) will be determined in the estrogen-dependent (MCF-7, T-47D), estrogen-independent (MDA-MB231, SK-BR-3) breast cancer models, and normal mammary epithelia (MCF-10A). In parallel, RfBP expression levels will be quantitated by western blot analysis using polyclonal antibodies raised in rabbits directed against the homologous cRfBP. Aim  Sub-cellular distribution of a fluorescent riboflavin analog (rhodamine-riboflavin) will be determined using confocal microscopy. In addition, co-localization of the early endocytic components namely, clathrin, and Rab5 will be visualized by immunofluorescence. In order to identify the specific endocytic pathway, expression of ADP-ribosylation factors (ARFs) 1 and 6 will be detected by Northern and western blot analyses following incubation with riboflavin. In addition, the effect of pharmacological modifiers of vesicular machinery such as brefeldin A, neomycin, nocodazole, and wortmannin on riboflavin uptake will be examined. Aim  Biodistribution and tumor uptake of [125I]-riboflavin following i.v. administration in BALB/c nude mice xenografted with breast tumors will be determined by gamma counting. Potential Outcomes and Benefits of the Research: The study aims to delineate the mechanism underlying the translocation of an essential nutrient (vitamin B2) in breast cancer. Understanding this riboflavin-RfBP system will provide further insight into potential qualitative and/or quantitative markers for the detection of breast tumors and in addition, may lead to novel chemotherapeutic approaches. Targeting the tumor abundant RfBP component should provide a rational basis in the design of chemotherapeutic agents that will bind and translocate into the breast cancer cells via the vitamin transport system.
Riboflavin more commonly referred to as vitamin B2, is an essential nutrient required for normal cellular functioning that promotes growth and development. Since humans cannot make riboflavin, dietary intake of this vitamin followed by absorption in the intestine contributes to maintaining the nutritional status in humans. Riboflavin deficiency has been evidenced as a causal/risk factor for anemia, cardiovascular disease, neuro-degenerative disorders, and although not conclusively established in humans, possibly cancer. Consequently, the mechanism(s) underlying riboflavin absorption from its dietary source has generated considerable interest. The outcome of several investigative approaches from our laboratory indicates cellular riboflavin uptake to occur via a specialized process referred to as receptor-mediated endocytosis (RME). The proposed model suggests that proteins called riboflavin binding proteins (RfBP) specifically bind the vitamin and are taken up within vesicles following interaction with cell surface receptors. These RfBP are overexpressed under stimulation from the female steroid hormone estrogen in rapidly dividing cells such as breast tumors and the developing fetus, making them a suitable marker for early detection in clinical screening of breast cancer. However, the role of these overexpressed proteins in riboflavin absorption in breast cancer remains unanswered. Hence, the overall objective of this study is to investigate the process of riboflavin absorption in breast tumors. Our first goal is to correlate the levels of RfBP with cellular accumulation of riboflavin in established breast cancer and non-tumorigenic cell models. In order to quantitatively detect RfBP, an antibody that selectively recognizes RfBP will be developed in rabbits, characterized, and used. In addition, levels of intracellular riboflavin and its derivatives FMN (riboflavin 5-phosphate), and FAD (flavin adenine dinucleotide) will be measured using various analytical tools. Secondly, we propose to investigate whether cellular uptake of riboflavin occurs via RME in breast cancer cells. This will be accomplished using sensitive imaging techniques that will identify the participation of components integral to the process of RME. Furthermore, this qualitative evaluation will be supplemented with studies that assess cellular riboflavin uptake following treatment with agents that specifically interfere with RME. Together, the resulting data should provide insight into riboflavin uptake via RME in breast cancer. Lastly, we aim to study the tumor accumulation of riboflavin using an animal model. Cellular strains of breast cancer will be transplanted into immunosuppressed mice. Uptake of radiolabeled riboflavin following intravenous administration will be quantitatively measured in tumors and other tissues utilizing analytical techniques. The results at the end of this project will provide a better understanding of the riboflavin transport system in breast cancer. This will aid the design of chemotherapeutic entities that may be selectively targeted to breast tumors using this vitamin transport system.