2-(n-(7-nitrobenz-2-oxa-1-3-diazol-4-yl)amino)-2-deoxyglucose and Breast-Neoplasms

Metabolic reprogramming is being recognized as a fundamental hallmark of cancer, and efforts to identify drugs that can target cancer metabolism are underway. In this study, we used human breast cancer (BC) cell lines and established their invading phenotype (INV) collected from transwell inserts to compare metabolome differences and evaluate prognostic significance of the metabolome in aggressive BC invasiveness.. The invasiveness of seven human BC cell lines were compared using the transwell invasion assay. Among these, INV was collected from SUM149, which exhibited the highest invasiveness. Levels of metabolites in INV were compared with those of whole cultured SUM149 cells (WCC) using CE-TOFMS. The impact of glycolysis in INV was determined by glucose uptake assay using fluorescent derivative of glucose (2-NBDG), and significance of glycolysis, or tricarboxylic acid cycle (TCA) and electron transport chain (ETC) in the invasive process were further determined in aggressive BC cell lines, SUM149, MDA-MB-231, HCC1937, using invasion assays in the presence or absence of inhibitors of glycolysis, TCA cycle or ETC.. SUM149 INV sub-population exhibited a persistent hyperinvasive phenotype. INV were hyper-glycolytic with increased glucose (2-NBDG) uptake; diminished glucose-6-phosphate (G6P) levels but elevated pyruvate and lactate, along with higher expression of phosphorylated-pyruvate dehydrogenase (pPDH) compared to WCC. Notably, inhibiting of glycolysis with lower doses of 2-DG (1 mM), non-cytotoxic to MDA-MB-231 and HCC1937, was effective in diminishing invasiveness of aggressive BC cell lines. In contrast, 3-Nitropropionic acid (3-NA), an inhibitor of succinate dehydrogenase, the enzyme that oxidizes succinate to fumarate in TCA cycle, and functions as complex II of ETC, had no significant effect on their invasiveness, although levels of TCA metabolites or detection of mitochondrial membrane potential with JC-1 staining, indicated that INV cells originally had functional TCA cycles and membrane potential.. Hyper-glycolytic phenotype of invading cells caters to rapid energy production required for invasion while TCA cycle/ETC cater to cellular energy needs for sustenance in aggressive BC. Lower, non-cytotoxic doses of 2-DG can hamper invasion and can potentially be used as an adjuvant with other anti-cancer therapies without the usual side-effects associated with cytotoxic doses.

Topics: 4-Chloro-7-nitrobenzofurazan; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cellular Reprogramming; Citric Acid Cycle; Deoxyglucose; Female; Glucose; Glycolysis; Humans; Metabolome; Neoplasm Invasiveness

With the large number of women diagnosed and treated for breast cancer each year, the importance of studying recurrence has become evident due to most deaths from breast cancer resulting from tumor recurrence following therapy. To mitigate this, cellular and molecular pathways used by residual disease prior to recurrence must be studied. An altered metabolism has long been considered a hallmark of cancer, and several recent studies have gone further to report metabolic dysfunction and alterations as key to understanding the underlying behavior of dormant and recurrent cancer cells. Our group has used two probes, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) and tetramethyl rhodamine ethyl ester (TMRE), to image glucose uptake and mitochondrial membrane potential, respectively, to report changes in metabolism between primary tumors, regression, residual disease, and after regrowth in genetically engineered mouse (GEM)-derived mammospheres. Imaging revealed unique metabolic phenotypes across the stages of tumor development. Although primary mammospheres overexpressing Her2 maintained increased glucose uptake ("Warburg effect"), after Her2 downregulation, during regression and residual disease, mammospheres appeared to switch to oxidative phosphorylation. Interestingly, in mammospheres where Her2 overexpression was turned back on to model recurrence, glucose uptake was lowest, indicating a potential change in substrate preference following the reactivation of Her2, reeliciting growth. Our findings highlight the importance of imaging metabolic adaptions to gain insight into the fundamental behaviors of residual and recurrent disease. IMPLICATIONS: This study demonstrates these functional fluorescent probes' ability to report metabolic adaptations during primary tumor growth, regression, residual disease, and regrowth in Her2 breast tumors.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Animals, Genetically Modified; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Deoxyglucose; Female; Gene Expression Regulation, Neoplastic; Glucose; Humans; Mammary Glands, Animal; Membrane Potential, Mitochondrial; Neoplasm Recurrence, Local; Organometallic Compounds; Phenotype; Receptor, ErbB-2

When monitoring response to cancer therapy, it is important to differentiate changes in glucose tracer uptake caused by altered delivery versus a true metabolic shift. Here, we propose an optical imaging method to quantify glucose uptake and correct for in vivo delivery effects. Glucose uptake was measured using a fluorescent D-glucose derivative 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-D-glucose (2-NBDG) in mice implanted with dorsal skin flap window chambers. Additionally, vascular oxygenation (SO2) was calculated using only endogenous hemoglobin contrast. Results showed that the delivery factor proposed for correction, "RD", reported on red blood cell velocity and injected 2-NBDG dose. Delivery-corrected 2-NBDG uptake (2-NBDG60/RD) inversely correlated with blood glucose in normal tissue, indicating sensitivity to glucose demand. We further applied our method in metastatic 4T1 and nonmetastatic 4T07 murine mammary adenocarcinomas. The ratio 2-NBDG60/RD was increased in 4T1 tumors relative to 4T07 tumors yet average SO2 was comparable, suggesting a shift toward a "Warburgian" (aerobic glycolysis) metabolism in the metastatic 4T1 line. In heterogeneous regions of both 4T1 and 4T07, 2-NBDG60/RD increased slightly but significantly as vascular oxygenation decreased, indicative of the Pasteur effect in both tumors. These data demonstrate the utility of delivery-corrected 2-NBDG and vascular oxygenation imaging for differentiating metabolic phenotypes in vivo.

Topics: 4-Chloro-7-nitrobenzofurazan; Adenocarcinoma; Animals; Blood Glucose; Breast Neoplasms; Cell Line, Tumor; Deoxyglucose; Female; Fluorescent Dyes; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Optical Imaging; Oxygen Consumption

The relationship between estrogen and some types of breast cancer has been clearly established. However, although several studies have demonstrated the relationship between estrogen and glucose uptake via phosphatidylinositol 3-kinase (PI3K)/Akt in other tissues, not too much is known about the possible cross talk between them for development and maintenance of breast cancer. This study was designed to test the rapid effects of 17β-estradiol (E2) or its membrane-impermeable form conjugated with BSA (E2BSA) on glucose uptake in a positive estrogen receptor (ER) breast cancer cell line, through the possible relationship between key components of the PI3K/Akt signaling pathway and acute steroid treatment. MCF-7 human breast cancer cells were cultured in standard conditions. Then 10 nM E2 or E2BSA conjugated were administered before obtaining the cell lysates. To study the glucose uptake, the glucose fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose was used. We report an ER-dependent activation of some of the key steps of the PI3K/Akt signaling pathway cascade that leads cells to improve some mechanisms that finally increase glucose uptake capacity. Our data suggest that both E2 and E2BSA enhance the entrance of the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, and also activates PI3K/Akt signaling pathway, leading to translocation of glucose transporter 4 to the plasma membrane in an ERα-dependent manner. E2 enhances ER-dependent rapid signaling triggered, partially in the plasma membrane, allowing ERα-positive MCF-7 breast cancer cells to increase glucose uptake, which could be essential to meet the energy demands of the high rate of proliferation.

Topics: 4-Chloro-7-nitrobenzofurazan; Blotting, Western; Breast Neoplasms; Cell Membrane; Deoxyglucose; Estradiol; Estrogen Receptor alpha; Estrogens; Female; Glucose; Glucose Transporter Type 4; Humans; MCF-7 Cells; Phosphatidylinositol 3-Kinase; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-akt; Serine; Signal Transduction

We demonstrate an optical strategy using intravital microscopy of dorsal skin flap window chamber models to image glucose uptake and vascular oxygenation in vivo. Glucose uptake was imaged using a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). SO2 was imaged using the differential absorption properties of oxygenated [HbO2] and deoxygenated hemoglobin [dHb]. This study was carried out on two sibling murine mammary adenocarcinoma lines, 4T1 and 4T07. 2-NBDG uptake in the 4T1 tumors was lowest when rates of delivery and clearance were lowest, indicating perfusion-limited uptake in poorly oxygenated tumor regions. For increasing rates of delivery that were still lower than the glucose consumption rate (as measured in vitro), both 2-NBDG uptake and the clearance rate from the tumor increased. When the rate of delivery of 2-NBDG exceeded the glucose consumption rate, 2-NBDG uptake decreased with any further increase in rate of delivery, but the clearance rate continued to increase. This inflection point was not observed in the 4T07 tumors due to an absence of low delivery rates close to the glucose consumption rate. In the 4T07 tumors, 2-NBDG uptake increased with increasing rates of delivery at low rates of clearance. Our results demonstrate that 2-NBDG uptake in tumors is influenced by the rates of delivery and clearance of the tracer. The rates of delivery and clearance are, in turn, dependent on vascular oxygenation of the tumors. Knowledge of the kinetics of tracer uptake as well as vascular oxygenation is essential to make an informed assessment of glucose demand of a tumor.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Breast Neoplasms; Cell Line, Tumor; Deoxyglucose; Disease Models, Animal; Female; Fluorescent Dyes; Hypoxia; Kinetics; Mice; Neoplasm Metastasis; Oxygen Consumption; Regional Blood Flow; Sulfur Dioxide; Time Factors

To determine use of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) as a tracer for detection of hypermetabolic circulating tumor cells (CTC) by fluorescence imaging.. Human breast cancer cells were implanted in the mammary gland fat pad of athymic mice to establish orthotopic human breast cancer xenografts as a mouse model of circulating breast cancer cells. Near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DeoxyGlucosone 750 (2-DG 750) was conducted to assess glucose metabolism of xenograft tumors. Following incubation with fluorescent 2-NBDG, circulating breast cancer cells in the blood samples collected from the tumor-bearing mice were collected by magnetic separation, followed by fluorescence imaging for 2-NBDG uptake by circulating breast cancer cells, and correlation of the number of hypermetabolic circulating breast cancer cells with tumor size at the time when the blood samples were collected.. Human breast cancer xenograft tumors derived from MDA-MB-231, BT474, or SKBR-3 cells were visualized on near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DG 750. Hypermetabolic circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in the blood samples from tumor-bearing mice and visualized by fluorescence imaging, but not in the blood samples from normal control mice. The number of hypermetabolic circulating breast cancer cells increased along with growth of xenograft tumors, with the number of hypermetabolic circulating breast cancer cells detected in the mice bearing MDA-MB231 xenografts larger than those in the mice bearing BT474 or SKBR-3 xenograft tumors.. Circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in mice bearing human breast cancer xenograft tumors by fluorescence imaging, suggesting clinical use of 2-NBDG as a tracer for fluorescence imaging of hypermetabolic circulating breast cancer cells.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Biological Transport; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Deoxyglucose; Feasibility Studies; Female; Glucose; Humans; Mice; Molecular Imaging; Neoplastic Cells, Circulating; Spectrometry, Fluorescence

This study quantifies uptake of a fluorescent glucose analog, (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-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. Expressions of glucose transporter (GLUT 1) and hexokinase (HK I), which phosphorylates 2-NBDG, were measured via western blot in two normal mammary epithelial and eight breast cancer cell lines of varying biological subtype. Fluorescence intensity of each cell line labeled with 100 lM 2-NBDG for 20 min or unlabeled control was quantified. A subset of cancer cells was treated with anticancer and endocrine therapies, and 2-NBDG fluorescence changes were measured. Expression of GLUT 1 was necessary for uptake of 2-NBDG, as demonstrated by lack of 2-NBDG uptake in normal human mammary epithelial cells (HMECs). GLUT 1 expression and 2-NBDG uptake was ubiquitous among all breast cancer lines. Reduction and stimulation of 2-NBDG uptake was demonstrated by perturbation with anticancer agents, lonidamine (LND), and a-cyano-hydroxycinnamate (a-Cinn), respectively. LND directly inhibits HK and significantly reduced 2-NBDG fluorescence in a subset of two breast cancer cell lines. Conversely, when cells were treated with a-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. Results indicate that 2-NBDG uptake can be used to measure changes in glycolysis and has potential for use in early drug development.

Topics: 4-Chloro-7-nitrobenzofurazan; Antineoplastic Agents, Hormonal; Blotting, Western; Breast; Breast Neoplasms; Cells, Cultured; Deoxyglucose; Female; Glucose; Glucose Transporter Type 1; Hexokinase; Humans; Phosphorylation; Receptors, Estrogen; Tamoxifen; Treatment Outcome

The 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.. The 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.. The 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.. The 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.

Topics: 4-Chloro-7-nitrobenzofurazan; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Deoxyglucose; Female; Flow Cytometry; Glucose Transporter Type 1; Humans; Immunohistochemistry; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger