Impact Of DTPA-DG On Hexokinase Phosphorylation And A GLUT-specific Blocking Agent

Objective: 1. To assess the role of diethylenetriaminepentaac-eti acid-2-deoxy-D-glucose ( DTPA-DG ) and 2-deoxy-D-glucose (DG) in hexokinase (HK) phosphate metabolism, to determine the mechanism of cellular uptake of 99mTc-diethylenetriaminepentaacetic acid 2-deoxy-D-glucose (99mTc-DTPA-DG). 2. To explore the relevant mechanisms of the specific inhibitor, cytochalasin B (CB), of glucose transporter. Methods: 1. The HK phosphorylation experiments were divided into four groups: DG, DG+DTPA-DG, DTPA-DG, and control (distilled water). High-performance liquid chromatography (HPLC) was used to detect the content of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) at 0.5, 1, 2, and 2.5 hours. The HK inhibition experiments were divided into three groups: DG, DG+DTPA-DG, and control. Six-phosphate glucose dehydrogenase (G6PD) coupling colorimetry was used to detect HK activity of DTPA-DG and DG in red blood cells. 2. Nasopharyngeal carcinoma cells were plated into 24-well culture plates, and divided into treatment and control groups (normal saline [NS]). The drugs used in this study included CB, 5% glucose, 10% glucose, and diethyl sulfoxide at 3 concentrations (50μg/ml, 100μg/ml, and 150μg/ml). After 2 hours, 99mTc-DTPA-DG (0.1 ml 37 MBq/L) was added to each well. The cells were incubated for another 2 hours, digested and collected, then the rate of uptake of 99mTc-DTPA-DG was determined using aγ-counter. Results: 1. There was a significant difference between the consumed ATP and the generated ADP in all four groups in the HK phosphorylation experiments (P<0.05). Furthermore, at a substrate concentration of 100μg/ml the reaction was most thorough at 2 hours. There were differences in the activity of HK in three drug concentrations in the HK inhibition experiments. HK activity was the highest with a substrate concentration of 100μg/ml. 2. After different concentrations of CB reacted for 2 hours, the 99mTc-DTPA-DG uptake rates were determined. There was a significant difference between the concentration at 100μg/ml group and the other groups (P<0.05). A CB concentration of 100μg/ml was optimal for blocking Glut. At the same concentration of 100μg/ml and reacted for 2 hours, the 99mTc-DTPA-DG uptake rates were significant different between five different drugs, CB is a specific blockers of Glut. Conclusion: 1. DTPA-DG and DG are able to be phosphorylated. Through the phosphorylation, 99mTc-DTPA-DG can enter cells, therefore it can be used as an imaging agent for glucose metabolism. 2. CB has a similar functional mechanism to glucose and may specifically block Glut. Moreover, CB is a better specific blocker of Glut than glucose.