OBJECTIVEThe purpose of this study was to assess the feasibility of fluorescent 2-deoxyglucose analog, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), that could be taken up by breast cancer cells highly expressing glucose transporter 1 (GLUT-1). The purpose of this study was to clarify if a fluorescent 2-deoxyglucose analog, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), can be taken up by breast cancer cells highly expressing glucose transporter 1 (GLUT-1), and to assess whether it can be used as a targeting imaging agent.

METHODSThe expressions of GLUT-1 mRNA and protein in breast cancer MDA-MB-231 cells were detected by RT-PCR and immunohistochemistry, respectively. The difference of GLUT-1 protein expression between breast cancer MDA-MB-231 cells and MCF-7 cells was compared by Western blot. Secondly, MDA-MB-231 cells which were grown in 6-well plates were incubated with 2-NBDG, and the result of 2-NBDG uptake was analyzed by fluorescence microscopy and flow cytometry. The difference of 2-NBDG absorption in MDA-MB-231 and MCF-7 cells was compared by flow cytometry.

RESULTSThe results of RT-PCR and immunohistochemistry confirmed that MDA-MB-231 cells highly expressed GLUT-1. Furthermore, Western blot revealed that GLUT-1 expression of MDA-MB-231 cells (0.946 ± 0.007) was higher than that in the MCF-7 cells (0.833 ± 0.010). Fluorescence microscopic and flow cytometric analysis showed that 2-NBDG was uptaken rapidly by MDA-MB-231 cells. Addition of 50 mmol/L D-glucose to the media with 2-NBDG reduced its uptake by 46.0%. Moreover, flow cytometry indicated that the fluorescence intensity of MDA-MB-231 cells (25.10 ± 0.57) was higher than that of MCF-7 cells (10.12 ± 0.62) when incubated with 2-NBDG for 20 minutes.

CONCLUSIONThe preliminary data clearly demonstrate that 2-NBDG is taken up and accumulated in breast cancer cells that highly express GLUT-1, and may be used as an optical probe for glucose uptake in hypermetabolic malignant cells.

4-Chloro-7-nitrobenzofurazan ; analogs & derivatives ; pharmacokinetics ; Blotting, Western ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Deoxyglucose ; analogs & derivatives ; pharmacokinetics ; Female ; Flow Cytometry ; Glucose Transporter Type 1 ; genetics ; metabolism ; Humans ; Immunohistochemistry ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo compare the differences in uptake of 2-deoxy-D-glucose (2-DG)-conjugated nanoparticles between breast carcinoma MDA-MB-231 cells with high metabolism and breast fibroblasts with normal metabolism, and investigate the feasibility of using the coated nanoparticles as a MRI-targeted contrast agent for highly metabolic carcinoma cells.

METHODSThe γ-Fe2O3@DMSA-DG was prepared. The glucose metabolism level of both cell lines was determined. The targeting efficacy of γ-Fe2O3@DMSA-DG and γ-Fe2O3@DMSA NPs to breast carcinoma MDA-MB-231 cells and breast fibroblasts at 10 min, 30 min, 1 h and 2 h was measured with Prussian blue staining and UV colorimetric assay. MRI was performed to visualize the changes of T2WI signal intensity.

RESULTSPrussian blue staining showed more intracellular blue granules in the MDA-MB-231 cells of γ-Fe2O3@DMSA-DG NPs group than that in the γ-Fe2O3@DMSA NPs group, and the γ-Fe2O3@DMSA-DG uptake was greatly competed by free D-glucose. As revealed by UV colorimetric assay, MDA-MB-231 cells also showed that the cellular iron amount of γ-Fe2O3@DMSA-DG group was significantly higher than that of the γ-Fe2O3@DMSA group and γ-Fe2O3@DMSA-DG + D-glucose group, statistically with a significant difference between them. MRI showed that the signal intensity of γ-Fe2O3@DMSA-DG group was decrease significantly, the T2 signal intensity was decreased by 10.5%, 37.5%, 72.9%, 92.0% for 10 min, 30 min, 1 h and 2 h, respectively. In contrast, the signal intensity did not show obvious decrease in the γ-Fe2O3@DMSA-DG group, the T2 signal intensity was decreased by 8.5%, 11.4%, 32.0%, 76.7% for 10 min, 30 min, 1 h and 2 h, respectively. However, HUM-CELL-0056 cells did not produce apparent difference for positive staining in the γ-Fe2O3@DMSA-DG group, γ-Fe2O3@DMSA group and γ-Fe2O3@DMSA-DG+D-glucose group, and the signal intensity also did not produce apparent difference.

CONCLUSIONSγ-Fe2O3@DMSA-DG has good targeting ability to highly metabolic breast carcinoma (MDA-MB-231) cells. It is feasible to serve as a specific MRI-targeted contrast agent for highly metabolic carcinoma cells, and deserves further studies in vivo.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Colorimetry ; methods ; Contrast Media ; pharmacokinetics ; Deoxyglucose ; chemistry ; pharmacokinetics ; Female ; Ferric Compounds ; chemistry ; pharmacokinetics ; Fibroblasts ; cytology ; metabolism ; Glucose ; metabolism ; Humans ; Iron ; metabolism ; Magnetic Resonance Imaging ; methods ; Nanoconjugates ; chemistry ; Particle Size ; Succimer ; chemistry ; pharmacokinetics

OBJECTIVETo evaluate the role of 2-deoxy-D-glucose (2-DG) modified supermagnetic iron oxide nanoparticles (SPIO) (γ-Fe2O3@DMSA-DG NPs) in tumor detection as a magnetic resonance imaging (MRI) contrast agent.

METHODSγ-Fe2O3@DMSA-DG NPs was prepared. The degree of A549 cells targeted absorption of γ-Fe2O3@DMSA-DG NPs was detected by Prussian blue staining, colorimetric assay, T2W and multi-echo sequence MRI. γ-Fe2O3@DMSA NPs was used as a control agent, and free D-glucose as a competitive inhibitor. Human lung adenocarcinoma A549 xenograft tumor was prepared in nude mice. Sterile aqueous suspension of γ-Fe2O3@DMSA NPs or γ-Fe2O3@DMSA-DG NPs was injected into the tail vein of nude mice. Before and 6, 12, 24, 48 h after injection, MRI imaging of the mice was performed. T2 signal intensity of the tumor, brain, liver and thigh skeletal muscles, and T2 values of the tumors were measured.

RESULTSThe average diameter of the particles was about 10 nm, and there were no significant differences between the diameters of γ-Fe2O3@DMSA NPs and γ- Fe2O3@DMSA-DG NPs. The IR spectra showed the C-N retractable vibration peak at γ-Fe2O3@DMSA-DG NPs surface, indicating that 2-DG was conjugated to the γ-Fe2O3@DMSA NPs. The Prussian blue staining, colorimetric assay, MRI T2 signal intensity and T2 values revealed that γ-Fe2O3@DMSA-DG NPs were significantly more absorbed by A549 cells at growth peak than γ-Fe2O3@DMSA NPs, and the absorption of γ-Fe2O3@DMSA-DG NP was inhibited by free D-glucose. The results of in vivo examination showed that before and at 6, 12, 24, 48 h after injection of γ-Fe2O3@DMSA-DG NPs, the mean T2 signal intensities of the tumors were (326.00 ± 16.26)s, (276.40 ± 5.13)s, (268.40 ± 30.58)s, (240.40 ± 25.93)s, (262.20 ± 30.04)s, respectively, and the T2 values of the tumors were (735.80 ± 20.93) ms, (645.80 ± 69.58) ms, (615.00 ± 124.61) ms, (570.60 ± 67.78) ms, and (537.80 ± 105.29) ms, respectively. However, before and at 6, 12, 24, 48 h after injection of γ-Fe2O3@DMSA NPs, the mean T2 signal intensities of the tumors were (335.60 ± 4.93)s, (290.80 ± 5.93)s, (273.40 ± 15.08)s, (327.40 ± 16.65)s, and (313.20 ± 20.45)s, respectively, and T2 values were (686.00 ± 21.44)ms, (617.80 ± 69.93)ms, (645.20 ± 85.89)ms, (669.40 ± 13.72)ms, and (608.80 ± 61.90)ms, respectively. The T2 signal intensity and T2 value of the tumors were not declined generally after injection. The liver T2 signal intensity was decreased after injection of both γ-Fe2O3@DMSA-DG NPs and γ-Fe2O3@DMSA NPs, and T2 signal intensity of the brain and muscle did not show significant changes.

CONCLUSIONSγ-Fe2O3@DMSA-DG NPs has an ability to target glucose receptors overexpressed in tumors, and may serve as a MRI contrast agent for tumor detection.

Adenocarcinoma ; diagnosis ; metabolism ; pathology ; Animals ; Cell Line, Tumor ; Colorimetry ; Contrast Media ; chemistry ; pharmacokinetics ; Deoxyglucose ; chemistry ; pharmacokinetics ; Ferric Compounds ; chemistry ; pharmacokinetics ; Humans ; Image Enhancement ; Lung Neoplasms ; diagnosis ; metabolism ; pathology ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Particle Size

It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 micrometer for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.
Acetylglucosamine/*analogs & derivatives/metabolism/pharmacology ; Adipocytes/*metabolism ; Animals ; Deoxyglucose/pharmacokinetics ; Glycosylation ; Immunoprecipitation ; *Insulin Resistance ; Male ; Monosaccharide Transport Proteins/metabolism ; Oximes/*pharmacology ; Phenylcarbamates/*pharmacology ; Phosphoproteins/*metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases/*metabolism ; Proto-Oncogene Proteins/*metabolism ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Subcellular Fractions/metabolism ; beta-N-Acetylhexosaminidase/antagonists & inhibitors