Expression of GLUT1 gene in breast cancer cell lines MCF-7 and MDA-MB-231.
ABSTRACT Overexpression of membrane glucose transporters belonging to GLUT family, is a common feature of different malignancies. It has been found that the level of expression of some members of this large family correlates with invasiveness of some malignant tumors. GLUT1 is an example of the most often studied and best known members of GLUT receptors. We attempted to compare the expression level of GLUT1 gene in two breast cancer cell lines: hormone-positive MCF-7 and hormone-resistant, less differentiated and more aggressive MDA-MB-231.
A multiplex PCR (after RT) was performed in order to semiquantiatively compare differences in the expression of GLUT1 in both cell lines.
We found a difference in mRNA expression of GLUT1 in two cell lines. Densitometric optical analysis of bands resulted in the following results: in MCF-7 for GLUT1: 0.624; and in MDA-MB-231 0.875.
In our studies we showed differences in GLUT1 receptor mRNA expression in two breast cancer cell lines with higher expression in MDA-MB-231. The results show that invasiveness of cancer cells may be to some extent associated with the expression of glucose transporters, including GLUT1.
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ABSTRACT: Several clinical studies have shown low or no expression of GLUT1 in breast cancer patients, which may account for the low clinical specificity and sensitivity of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) used in positron emission tomography (PET). Therefore, it has been proposed that other tumor characteristics such as the high expression of GLUT2 and GLUT5 in many breast tumors could be used to develop alternative strategies to detect breast cancer. Here we have studied the in vitro and in vivo radiopharmacological profile of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) as a potential PET radiotracer to image GLUT5 expression in breast cancers. Uptake of 6-[(18)F]FDF was studied in murine EMT-6 and human MCF-7 breast cancer cells over 60 min and compared to [(18)F]FDG. Biodistribution of 6-[(18)F]FDF was determined in BALB/c mice. Tumor uptake was studied with dynamic small animal PET in EMT-6 tumor-bearing BALB/c mice and human xenograft MCF-7 tumor-bearing NIH-III mice in comparison to [(18)F]FDG. 6-[(18)F]FDF metabolism was investigated in mouse blood and urine. 6-[(18)F]FDF is taken up by EMT-6 and MCF-7 breast tumor cells independent of extracellular glucose levels but dependent on the extracellular concentration of fructose. After 60 min, 30±4% (n=9) and 12±1% (n=7) ID/mg protein 6-[(18)F]FDF was found in EMT-6 and MCF-7 cells, respectively. 6-deoxy-6-fluoro-d-fructose had a 10-fold higher potency than fructose to inhibit 6-[(18)F]FDF uptake into EMT-6 cells. Biodistribution in normal mice revealed radioactivity uptake in bone and brain. Radioactivity was accumulated in EMT-6 tumors reaching 3.65±0.30% ID/g (n=3) at 5 min post injection and decreasing to 1.75±0.03% ID/g (n=3) at 120 min post injection. Dynamic small animal PET showed significantly lower radioactivity uptake after 15 min post injection in MCF-7 tumors [standard uptake value (SUV)=0.76±0.05; n=3] compared to EMT-6 tumors (SUV=1.23±0.09; n=3). Interestingly, [(18)F]FDG uptake was significantly different in MCF-7 tumors (SUV(15 min) 0.74±0.12 to SUV(120 min) 0.80±0.15; n=3) versus EMT-6 tumors (SUV(15 min) 1.01±0.33 to SUV(120 min) 1.80±0.25; n=3). 6-[(18)F]FDF was shown to be a substrate for recombinant human ketohexokinase, and it was metabolized rapidly in vivo. Based on the GLUT5 specific transport and phosphorylation by ketohexokinase, 6-[(18)F]FDF may represent a novel radiotracer for PET imaging of GLUT5 and ketohexokinase-expressing tumors.Nuclear Medicine and Biology 05/2011; 38(4):461-75. · 2.52 Impact Factor
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ABSTRACT: Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS-30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D-[2-(3)H-glucose] as physiological substrate. Kinetic analysis revealed two components at low- and high-glucose concentrations in both tumor cells. At low glucose and 37 degrees C, the V(max) was 55 +/- 20 and 17.2 +/- 6 nmol (min x mg protein)(-1), whereas the K(m) was 0.52 +/- 0.7 and 9.3 +/- 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC(50) = 0.44 +/- 0.1; K(i) = 0.3 +/- 0.1 microM) and phloretin (IC(50) = 8.7 microM) in AS-30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS-30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS-30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS-30D carcinoma and HeLa cells. The much lower GLUT V(max) and catalytic efficiency (V(max)/K(m)) values in comparison to those of G6P-sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target.Journal of Cellular Physiology 09/2009; 221(3):552-9. · 4.22 Impact Factor
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ABSTRACT: Various pre-clinical and clinical studies have linked diabetes and breast cancer, but, little is known regarding the molecular mechanism involved. The present study was aimed to investigate the effect of high glucose and insulin in breast cancer cells (MCF-7; non-invasive, hormone dependent and MDA-MB-231; invasive, hormone independent). In contrast to MCF-7 cells, high glucose augmented proliferation of MDA-MB-231 cells as observed by MTT and BrdU assay. High glucose condition led to increased expression of Cyclin D1, de-phosphorylation of p38 and increased phosphorylation of ERK in MDA-MB-231 cells and not in MCF-7 cells. Interestingly, we observed increased phosphorylation of GSK-3β, NF-κB and ERα only in MCF-7 cells, highlighting their role as potential targets in preventing progression of breast cancer under high glucose and insulin condition. Furthermore, insulin treatment under high glucose condition resulted in increased histone H3 phosphorylation and de-acetylation only in MDA-MB-231 cells. Taken together, we provide first evidence that high glucose and insulin promotes proliferation of MDA-MB-231 cells, by differential alteration of GSK-3β, NF-κB, ERα expression and histone H3 modifications, which may directly or indirectly modulate the expression of genes involved in its proliferation.Journal of Molecular Endocrinology 05/2013; · 3.58 Impact Factor