A WARD NUMBER : W 81 XWH - 08 - 1 - 0240 TITLE : Multidisciplinary Biomarkers of Early Mammary

Purpose: This study quantifies uptake of a fluorescent glucose analog, (2-(N-(7-nitrobenz-2oxa-1,3diazol-4-yl)amino)-2-deoxyglucose), (2-NBDG) in a large panel of breast cancer cells and demonstrates potential to monitor changes in glycolysis caused by anticancer and endocrine therapies. Methods: Expressions of glucose transporter (GLUT 1) and hexokinase (HK I), which phosphorylates 2NBDG, were measured via Western blot in 2 normal mammary epithelial and 8 breast cancer cell lines of varying biological subtype. Fluorescence intensity of each cell line labeled with 100 μM 2-NBDG for 20 minutes or unlabeled control was quantified. A subset of cancer cells was treated with anticancer and endocrine therapies and 2-NBDG fluorescence changes were measured. Results: Expression of GLUT 1 was necessary for uptake of 2-NBDG, as demonstrated by lack of 2NBDG uptake in normal mammary epithelial cells (HMECs). GLUT 1 expression and 2-NBDG uptake was ubiquitous amongst all breast cancer lines. Reduction and stimulation of 2-NBDG uptake was demonstrated by perturbation with anticancer agents, lonidamine (LND) and α-cyanohydroxycinnamate (α-Cinn), respectively. LND directly inhibits HK and significantly reduced 2-NBDG fluorescence in a subset of 2 breast cancer cell lines. Conversely, when cells were treated with α-Cinn, a drug used to increase glycolysis, 2-NBDG uptake was increased. Furthermore, tamoxifen (tam), a common endocrine therapy, was administered to estrogen receptor positive and negative (ER+/-) breast cells and demonstrated a decreased 2-NBDG uptake in ER+ cells, reflecting a decrease in glycolysis. Conclusions: Results indicate 2-NBDG uptake can be used to measure changes in glycolysis and has potential for use in early drug development. DEPARTMENT OF BIOMEDICAL ENGINEERING TELEPHONE: (919) 660-5131 PRATT SCHOOL OF ENGINEERING Fax: (919) 684-4488 ROOM 136, HUDSON HALL BOX 90281 Nirmala Ramanujam, Ph.D. Associate Professor Phone (919) 660-5307, Email: [email protected] March 26, 2010 Marc E. Lippman, Editor-in-Chief Breast Cancer Research and Treatment