Jin-Young Paik

Resveratrol Suppresses Cancer Cell Glucose Uptake by Targeting Reactive Oxygen Species–Mediated Hypoxia-Inducible Factor-1α Activation

Resveratrol is gaining attention for its anticancer effects and is also recognized for its antioxidant properties and influence on glucose metabolism. Augmented reactive oxygen species (ROS) and high glycolytic flux are common characteristics of malignant cells. We thus evaluated the effect of resveratrol on cancer cell glucose metabolism and investigated the role of ROS in the response. Methods: Cancer cells were measured for cell content and 18F-FDG uptake. Assays were performed for lactate production; hexokinase activity and intracellular ROS; and immunoblotting for hypoxia-inducible factor-1α (HIF-1α), Akt, mammalian target of rapamycin, and glucose transporter type 1 (Glut-1). Animal studies were performed with small-animal PET imaging of Lewis lung carcinoma tumor–bearing mice. Results: Resveratrol mildly decreased cell content and more pronouncedly suppressed 18F-FDG uptake in Lewis lung carcinoma, HT-29 colon, and T47D breast cancer cells. Hence, 18F-FDG uptake normalized to cell content was reduced to less than half of controls by 24-h exposure to resveratrol. This reduction was attributed to reduced glycolytic flux and Glut-1 expression. Resveratrol also decreased intracellular ROS in patterns that closely paralleled 18F-FDG uptake. Scavenging of ROS with N-acetyl cysteine, but not inhibition of nicotinamide adenine dinucleotide phosphate oxidase, was sufficient to suppress 18F-FDG uptake. Conversely, ROS inducers effectively reversed the metabolic response of resveratrol. HIF-1α protein was markedly reduced by resveratrol, and inhibiting HIF-1α expression with cycloheximide or specific small interfering RNAs suppressed 18F-FDG uptake. The proteosomal inhibitor MG132 partly restored HIF-1α level and 18F-FDG uptake in resveratrol-treated cells. Resveratrol also inhibited Akt activation; in addition, inhibitors and small interfering RNAs against phosphoinositide 3-kinase decreased 18F-FDG uptake. Finally, small-animal PET results showed resveratrol treatment to suppress tumor 18F-FDG uptake in vivo. Conclusion: Resveratrol suppresses cancer cell 18F-FDG uptake and glycolytic metabolism in a manner that depends on the capacity of resveratrol to inhibit intracellular ROS, which downregulates HIF-1α accumulation.

Oxidized Low-Density Lipoprotein Stimulates Macrophage 18F-FDG Uptake via Hypoxia-Inducible Factor-1α Activation Through Nox2-Dependent Reactive Oxygen Species Generation

For 18F-FDG PET to be widely used to monitor atherosclerosis progression and therapeutic response, it is crucial to better understand how macrophage glucose metabolism is influenced by the atherosclerotic microenvironment and to elucidate the molecular mechanisms of this response. Oxidized low-density lipoprotein (oxLDL) is a key player in atherosclerotic inflammation that promotes macrophage recruitment, activation, and foam cell formation. We thus explored the effect of oxLDL on macrophage 18F-FDG uptake and investigated the underlying molecular mechanism including the roles of hypoxia-inducible factor-1α (HIF-1α) and reactive oxygen species (ROS). Methods: RAW264.7 macrophages were stimulated with native LDL, oxLDL, or lipopolysaccharide. Cells were assessed for 18F-FDG uptake, lactate production, membrane glucose transporter 1 (GLUT1) expression, and hexokinase activity. ROS generation, Nox expression, and HIF-1α activity were also measured. Results: oxLDL (20 μg/mL) induced a 17.5 ± 1.7-fold increase in macrophage 18F-FDG uptake by 24 h, which was accompanied by increased lactate production, membrane GLUT1 expression, and hexokinase activity. oxLDL-stimulated 18F-FDG uptake was completely blocked by inhibitors of Src or phosphoinositide 3-kinase. ROS generation was increased to 262.4% ± 17.9% of controls by oxLDL, and N-acetyl-l-cysteine completely abrogated both oxLDL-induced ROS production and 18F-FDG uptake. oxLDL increased Nox2 expression, and nicotinamide adenine dinucleotide phosphate oxidase inhibition totally blocked increased ROS generation and 18F-FDG uptake by oxLDL. Finally, there was a clear ROS-dependent increase of HIF-1α accumulation by oxLDL, and silencing of HIF-1α completely abolished the metabolic effect of oxLDL. Conclusion: oxLDL is a strong stimulator of macrophage 18F-FDG uptake and glycolysis through upregulation of GLUT1 and hexokinase. This metabolic response is mediated by Nox2-dependent ROS generation that promotes HIF-1α activation.

Use of insulin to improve [18f]fluorodeoxyglucose labelling and retention for in vivo positron emission tomography imaging of monocyte trafficking

While 18F-FDG labelling of monocytes would allow in vivo trafficking with positron emission tomography (PET), present methods suffer from poor retention of radioactivity. We investigated the feasibility of utilizing insulin for improved [18F]fluorodeoxyglucose (18F-FDG) labelling. Separated human monocytes and lymphocytes were labelled with 18F-FDG with or without 3 h insulin pre-incubation. Insulin had no effect on lymphocyte labelling (21.4±0.8% vs 20.8±1.1% efficiency, P = NS). However, for monocytes, insulin pre-incubation led to a 169±9% increase in labelling efficiency (19.3±4.1 vs 32.5±1.8, P<0.05), without significant effects on cell activation or viability. Moreover, while only 57.7±4.8% and 40.4±5.6% of the 18F-FDG was retained at 1 and 3 h for controls, the retention rate increased to 91.6±2.1% (P = 0.01) and 86.5±1.9% (P<0.01) after insulin pre-incubation. Improved 18F-FDG retention was accompanied by a 70.3±7.4% decrease in glucose-6-phosphatase activity (P = 0.02). PET imaging of rats showing hepatic ischaemia-reperfusion injury demonstrated higher liver uptake for monocytes labelled after insulin treatment. Thus, insulin improves monocytic 18F-FDG uptake and retention, and may provide a feasible labelling method for PET imaging.

17β-Estradiol Augments 18F-FDG Uptake and Glycolysis of T47D Breast Cancer Cells via Membrane-Initiated Rapid PI3K–Akt Activation

Use of 18F-FDG uptake as a surrogate marker of therapeutic response requires the recognition of biologic factors that influence cancer cell glucose metabolism. Estrogen is a potent stimulator of breast cancer proliferation, a process that requires sufficient energy, which is likely met by increased glycolysis. We thus explored the effect of estrogen on 18F-FDG uptake in responsive breast cancer cells and investigated the mediating molecular mechanisms. Methods: T47D breast cancer cells were stimulated with 17β-estradiol (E2) or bovine serum albumin (BSA)–E2 and measured for 18F-FDG uptake, lactate release, and mitochondrial hexokinase activity. The effects of antiestrogens, cycloheximide, and major protein kinase inhibitors were investigated. Immunoblots were performed for membrane glucose transporter type 1, phosphorylated phosphatidylinositol 3-kinase (PI3K), and Akt. Results: E2 augmented T47D cell 18F-FDG uptake in a dose- and time-dependent manner that preceded and surpassed its proliferative effect. With exposure to 10 nM E2, protein content–corrected 18F-FDG uptake reached 172.7% ± 6.6% and 294.4% ± 9.5% of controls by 24 and 48 h, respectively. Lactate release reached 110.9% ± 7.3% and 145.2% ± 10.5% of controls at 24 and 48 h, and mitochondrial hexokinase activity increased to 187.1% ± 31.6% at 24 h. Membrane glucose transporter type 1 expression was unaltered. The effect was absent in estrogen receptor (ER)–negative breast cancer cells and was abrogated by ICI182780, indicating ER dependence. The E2 effect was not blocked by tamoxifen and was mimicked by membrane-impermeable BSA-E2, consistent with nongenomic membrane-initiated E2 action. Inhibition by cycloheximide demonstrated the requirement of a new protein synthesis. Immunoblots displayed rapid phosphorylation of PI3K and Akt within minutes of E2 treatment, and the specific PI3K inhibitors wortmannin and LY294002 abolished the ability of E2 to elevate 18F-FDG uptake. Conclusion: Estrogen augments breast cancer cell 18F-FDG uptake by stimulating glycolysis and hexokinase activity via membrane-initiated E2 action that activates the PI3K–Akt pathway. These findings yield important insight into our understanding of the biology of breast cancer metabolism and may have potential implications for 18F-FDG uptake as a surrogate marker of therapeutic response.

99mTc-Hydrazinonicotinamide Epidermal Growth Factor–Polyethylene Glycol–Quantum Dot Imaging Allows Quantification of Breast Cancer Epidermal Growth Factor Receptor Expression and Monitors Receptor Downregulation in Response to Cetuximab Therapy

Therapy of cancer, including basallike breast tumors, that targets the epidermal growth factor receptor (EGFR) would greatly benefit from noninvasive methods that can quantitatively monitor receptor status and treatment response. Methods: Here, we investigated the potential of a novel technique based on streptavidin cadmium selenide/zinc sulfide quantum dots (Qdots) multiplexed with polyethylene glycol (PEG), epidermal growth factor (EGF), and 99mTc-hydrazinonicotinamide. In vitro binding affinity and specificity were evaluated in cultured cells. Biodistribution studies and in vivo imaging were performed in murine breast tumor xenografts of basallike phenotype MDA-MB-468 cells and EGFR-negative cells. Results: 99mTc-hydrazinonicotinamide EGF-PEG-Qdot showed specific and high-affinity EGFR targeting on confocal microscopy, immunoblotting, and binding assays. When intravenously injected, MDA-MB-468 tumors were visualized with high contrast by both optical and scintigraphic imaging. Scintigraphic image–based quantification correctly discriminated high–EGFR-expressing MDA-MB-468 tumors from other tumors, and image-based tumor uptake closely correlated to EGFR content. Importantly, serial imaging of MDA-MB-468 tumors responding to cetuximab therapy could detect a significant reduction of tumor uptake that was paralleled by downregulation of EGFR expression. Furthermore, high baseline uptake predicted good response to cetuximab therapy. Conclusion: 99mTc-hydrazinonicotinamide EGF-PEG-Qdot provides EGFR-targeted imaging of breast tumors and may allow noninvasive monitoring of EGFR status in living subjects before and after targeted therapies.

α2-Adrenergic agonists including xylazine and dexmedetomidine inhibit norepinephrine transporter function in SK-N-SH cells.

α2-Adrenergic agonists simulate norepinephrine (NE) action on α2 receptors of sympathetic neurons to mediate feedback inhibition of NE release. These agents are used as valuable adjuncts for management of hypertension and for anesthesia. Their action, equivalent to NE on α2 adrenergic receptors, raises the question whether α2 agonists may also target NE transporters (NETs), another major control mechanism for noradrenergic neurotransmission. We thus investigated the effect of α2 agonists on transport of the NE analog, (131)I-metaiodobenzylguanidine (MIBG). Results from this investigation showed that xylazine and dexmedetomidine dose-dependently blocked [(3)H]nisoxetine binding in neuron-like SK-N-SH cells. Furthermore, the agents acutely suppressed cellular MIBG uptake in a dose-dependent manner. This effect was uninfluenced by the α2 antagonist yohimbine, but was completely reversed by drug removal. There was no change in membrane NET density by the agents. Moreover, saturation analysis showed that xylazine and dexmedetomidine significantly increased Km without affecting Vmax, indicating competitive inhibition of MIBG transport. Thus, the α2 adrenergic agonists xylazine and dexmedetomidine, acutely suppress NET function through competitive inhibition of substrate transport, likely by direct interaction on a region that over-laps with the nisoxetine binding site.

Mitogen-Activated Protein Kinase Signaling Enhances Sodium Iodide Symporter Function and Efficacy of Radioiodide Therapy in Nonthyroidal Cancer Cells

Although the success of sodium/iodide symporter (NIS) gene–based cancer therapy is critically dependent on the level of radioiodide accumulation attained, recent evidence indicates that successful therapy relies not solely on NIS amount but also crucially on its functional activity. In this study, we investigated the role of kinase-linked signaling on the regulation of NIS function in cancer cells. Methods: T47D human breast cancer and PC-12 rat pheochromocytoma cells were transduced with the human NIS genes via an adenoviral vector. Cells were measured for 125I uptake, and the effects of activation or inhibition of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathways were evaluated. Membrane localization of NIS was evaluated by biotinylation-immunoblotting and confocal microscopy. 131I-mediated cancer cell killing was evaluated by clonogenic assays. Results: NIS function was acutely reduced by short stimulation with the PKC activator phorbol 12-myristate 13-acetate and increased by its inhibition with staurosporine or prolonged phorbol 12-myristate 13-acetate exposure. Surprisingly, epidermal growth factor (EGF) caused a strong dose-dependent augmentation of radioiodide transport, accompanied by extracellular signal-regulated kinase (ERK)-1/2 activation. Both effects were completely abrogated by specific MAP kinase kinase (MEK) inhibitors, which also reduced basal NIS function. Hence, radioiodide uptake levels could differ 24-fold, depending on ERK activity. Biotinylation-immunoblotting and confocal microscopy revealed that EGF increases plasma membrane–localized NIS without affecting total cellular levels. EGF stimulation was sufficient to enhance the killing effect of 131I on the cancer cells. Conclusion: Thus, PKC and ERK signaling play important roles in the regulation of NIS function, and control of these signaling pathways may help enhance the efficacy of radioiodide cancer therapy.

Effects of theophylline on radioiodide uptake in MCF-7 breast cancer and NIS gene-transduced SNU-C5 colon cancer cells.

BACKGROUND We investigated whether theophylline has the potential to increase radioiodide uptake in nonthyroidal cancer cells. MATERIALS AND METHODS MCF-7 cells that express endogenous sodium/iodide symporter (NIS) and SNU-C5 cells adenovirally transduced with the human NIS gene (SNU-C5/NIS) were treated with 10(-7)-2x10(-4) mol/L theophylline for 24 hours before incubation with (125)I, and then, radioiodide uptake and retention were measured. NIS expression was assessed by immunohistochemistry and Western blot analysis, using an antihuman NIS monoclonal antibody. RESULTS Theophylline at 10(-6)-2x10(-4) mol/L significantly and dose dependently augmented radioiodide uptake in MCF-7 cells and at 10(-6)-10(-5) mol/L in SNU-C5/NIS cells, without affecting radioiodide efflux. Abrogation by KClO(4)(-) demonstrated that the effect of theophylline occurred through specific iodide transport. Immunohistochemistry revealed dose-dependent increases of NIS staining in MCF-7 and SNU-C5/NIS cells by 10(-6)-10(-4) and 10(-6)-10(-5) mol/L theophylline, respectively. Western blot analysis demonstrated similar findings, showing increased expression of NIS on the membrane of SNU-C5/NIS and MCF-7 cells by theophylline treatment. CONCLUSIONS Theophylline can augment radioiodide uptake in breast cancer cells and NIS gene-transduced cancer cells through the upregulation of NIS expression. Therefore, further investigations are warranted to explore the potential utility of this phenomenon for enhancing radioiodide-based imaging and therapies of NIS gene-transduced cancer cells.

Quantification of early adipose-derived stem cell survival: comparison between sodium iodide symporter and enhanced green fluorescence protein imaging

OBJECTIVE Strategies to overcome the problem of extensive early stem cell loss following transplantation requires a method to quantitatively assess their efficacy. This study compared the ability of sodium/iodide symporter (NIS) and enhanced green fluorescent protein (EGFP) imaging to monitor the effectiveness of treatments to enhance early stem cell survival. METHODS Human adipose-derived stem cells (ADSCs) transduced with an adenoviral vector to express both NIS and EGFP were mixed with culture media (control), matrigel (matrigel group) or pro-survival cocktail (PSC group), and 5×10(6) cells were injected into thigh muscles of C57BL/6 mice. Animals underwent serial optical imaging and (99m)TcO(4)(-) scintigraphy. Image-based EGFP fluorescence and (99m)TcO(4)(-) uptake was measured by region-of-interest analysis, and extracted tissues were measured for (99m)Tc activity. Fluorescent intensity measured from homogenized muscle tissue was used as reference for actual amount of viable ADSCs. RESULTS ADSCs were efficiently transduced to express EGFP and NIS without affecting proliferative capacity. The absence of significant apoptosis was confirmed by annexin V FACS analysis and Western blots for activated caspase-3. Both fluorescence optical imaging and (99m)TcO(4)(-) scintigraphy visualized implanted cells in living mice for up to 5days. However, optical imaging displayed large variations in fluorescence intensity, and thus failed to detect difference in cell survival between groups or its change over time. In comparison, (99m)TcO(4)(-) scintigraphy provided more reliable assessment of within-in group donor cell content as well as its temporal change. As a result, NIS imaging was able to discern beneficial effects of matrigel and pro-survival cocktail treatment on early ADSC survival, and provided quantitative measurements that correlated to actual donor cell content within implanted tissue. CONCLUSION NIS reporter imaging may be useful for noninvasively assessing the efficacies of strategies designed to improve early survival of transplanted stem cells.

Trypsinization severely perturbs radioiodide transport via membrane Na/I symporter proteolysis: implications for reporter gene imaging

INTRODUCTION Cell preparation procedures injurious to Na/I symporters (NIS) could deter their usefulness for reporter gene assays and in vivo cell imaging. In this study, we investigated the effects of cell collection by trypsinization on radioiodide transport and in vivo cell imaging results. METHODS The influence of trypsinization procedures on (125)I transport was evaluated using Huh-7/NIS hepatoma cells. The effects of graded concentrations of trypsin and EDTA were assessed on Huh-7/NIS and A431/NIS lung cancer cells. Trypsin-induced NIS proteolysis was investigated by immunoblots of plasma membrane prepared from adenovirus-infected mouse liver tissue. (99m)Tc-O(4)(-) scintigraphy was performed in Balb/C nude mice at 1 and 4 h following administration of Huh-7/NIS cells collected with and without trypsin. RESULTS (125)I Transport ability of Huh-7/NIS cells was severely impaired within minutes of standard trypsinization and further deteriorated up to 24 h after termination of treatment. This perturbation was caused by trypsin, which dose- and time-dependently induced substantial reductions of (125)I uptake in Huh-7/NIS and A431/NIS cells. Immunoblot analysis revealed significant dose- and time-dependent losses of membrane NIS protein by trypsin. NIS proteolysis was completely blocked by soybean trypsin inhibitor, and partial protection was offered by the substrates iodide and perchlorate. On (99m)Tc-O(4)(-) scintigraphy of mice, cells prepared by trypsinization were poorly visualized, whereas those collected with a nonenzymatic method showed significantly better uptake and contrast. CONCLUSION Trypsinization leads to serious perturbations in iodide accumulating capacity through tryptic degradation of membrane NIS protein. Hence, NIS-based reporter assays and in vivo cell imaging studies may benefit from better-optimized cell cultivation and harvesting procedures.