Cung Hoa Thien Quach

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.

Resveratrol-loaded polymeric nanoparticles suppress glucose metabolism and tumor growth in vitro and in vivo.

Resveratrol (RSV) is a natural phenol with promising anti-tumor activities, but its use for in vivo cancer treatment is limited by low aqueous solubility and poor stability. In this study, we prepared RSV-loaded polyethylene glycol-polylactic acid (PEG-PLA; M.W. 5000-5000) polymer nanoparticles (NPs) for improved stability and controlled delivery, and investigated its metabolic and anti-tumor effect in vitro and in vivo. CT26 colon cancer cells displayed significantly reduced cell number to 5.6% and colony forming capacity to 6.3% of controls by 72 h treatment with 40 and 20 μM of RSV-NP, respectively. Flow cytometry and western blots demonstrated increased apoptotic cell death, and (18)F FDG uptake and reactive oxygen species was significantly reduced by RSV-NP. All of these effects were comparable to or greater in potency compared to free RSV. When RSV-NP was intravenously administered to CT26 tumor bearing mice, there was a reduction of (18)F FDG uptake on PET/CT by day 4. Longer treatment led to retardation of tumor growth accompanied by an improvement in survival compared to empty NP-injected controls. These results demonstrate that the in vitro and in vivo metabolic and anti-tumor effects of RSV is preserved by PEG-PLA NP loading, and provide an encouraging outlook on the potential of polymeric NPs as an effective method to deliver RSV for cancer therapy.

18F-FDG PET/CT Monitoring of β3 Agonist–Stimulated Brown Adipocyte Recruitment in White Adipose Tissue

There is rising interest in recruitment of brown adipocytes into white adipose tissue (WAT) as a means to augment energy expenditure for weight reduction. We thus investigated the potential of 18F-FDG uptake as an imaging biomarker that can monitor the process of WAT browning. Methods: C57BL/6 mice were treated daily with the β3 agonist CL316,243 (5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylic acid disodium salt), whereas controls received saline. 18F-FDG small-animal PET/CT was serially performed at 1 h after CL316,243 injection. After sacrifice, interscapular brown adipose tissue (BAT) and WAT depots were extracted, weighed, and measured for 18F-FDG uptake. Tissues underwent immunostaining, and UCP1 content was quantified by Western blotting. Results: PET/CT showed low 18F-FDG uptake in both BAT and inguinal WAT at baseline. BAT uptake was substantially increased by a single stimulation with CL316,243. Uptake in inguinal WAT was only modestly elevated by the first stimulation uptake but gradually increased to BAT level by prolonged stimulation. Ex vivo measurements recapitulated the PET findings, and measured 18F-FDG uptake in other WAT depots was similar to inguinal WAT. WAT browning by prolonged stimulation was confirmed by a substantial increase in uncoupling protein 1 (UCP1), cytochrome-c oxidase 4 (COX4), and PR domain containing 16 (PRDM16) staining as markers of brown adipocytes. UCP1 content, which served as a measure for extent of browning, was low in baseline inguinal WAT but linearly increased over 10 d of CL316,243 injection. Finally, image-based and ex vivo–measured 18F-FDG uptake in inguinal WAT correlated well with UCP1 content. Conclusion: 18F-FDG PET/CT has the capacity to monitor brown adipocyte recruitment into WAT depots in vivo and may thus be useful for screening the efficacy of strategies to promote WAT browning.

Annexin V imaging detects diabetes-accelerated apoptosis and monitors the efficacy of benfotiamine treatment in ischemic limbs of mice.

The role of apoptosis imaging for monitoring treatment response in ischemic limbs has not been properly explored. In this study, we investigated the ability of annexin V (AnxV) imaging to assess the efficacy of antiapoptotic treatment in ischemic limbs of diabetic mice. Normal C57BL/6 mice and streptozotocin-induced diabetic mice were subject to hindlimb ischemia. AnxV-conjugated fluorescent streptavidin probes were intravenously injected, and optical imaging was performed. Tissue apoptosis was quantified by histochemistry and Western blotting. The AnxV probes showed specific targeting to apoptotic cells on confocal microscopy and flow cytometry. Intravenous AnxV probes displayed substantially greater accumulation in ischemic limbs of diabetic mice. Benfotiamine (BFT) treatment of diabetic mice led to better perfusion recovery on laser Doppler imaging and reduced AnxV binding on optical imaging. TUNEL staining and cleaved caspase-3 Western blots confirmed accelerated apoptosis by diabetes and its suppression by BFT treatment. Furthermore, AnxV-SAv-PEcy5.5 uptake in the ischemic limbs closely correlated to cleaved caspase-3 expression. Thus, AnxV imaging may be useful for monitoring the efficacy of therapeutic agents designed to suppress ischemia-induced apoptosis.

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.

EGF receptor targeted tumor imaging with biotin-PEG-EGF linked to 99mTc-HYNIC labeled avidin and streptavidin

INTRODUCTION As direct radiolabeled peptides suffer limitations for in vivo imaging, we investigated the usefulness of radioloabeled avidin and streptavidin as cores to link peptide ligands for targeted tumor imaging. METHODS Human epidermal growth factor (EGF) was site specifically conjugated with a single PEG-biotin molecule and linked to (99m)Tc-HYNIC labeled avidin-FITC (Av) or streptavidin-Cy5.5 (Sav). Receptor targeting was verified in vitro, and in vivo pharmacokinetic and biodistribution profiles were studied in normal mice. Scintigraphic imaging was performed in MDA-MB-468 breast tumor xenografted nude mice. RESULTS Whereas both (99m)Tc-Av-EGF and (99m)Tc-Sav-EGF retained receptor-specific binding in vitro, the two probes substantially diverged in pharmacokinetic and biodistribution behavior in vivo. (99m)Tc-Av-EGF was rapidly eliminated from the circulation with a T1/2 of 4.3 min, and showed intense hepatic accumulation but poor tumor uptake (0.6%ID/gm at 4 h). (99m)Tc-Sav-EGF displayed favorable in vivo profiles of longer circulation (T1/2β, 51.5 min) and lower nonspecific uptake that resulted in higher tumor uptake (3.8 %ID/gm) and clear tumor visualization at 15 h. CONCLUSION (99m)Tc-HYNIC labeled streptavidin linked with growth factor peptides may be useful as a protein-ligand complex for targeted imaging of tumor receptors.

Mild Alkalization Acutely Triggers the Warburg Effect by Enhancing Hexokinase Activity via Voltage-Dependent Anion Channel Binding

To fully understand the glycolytic behavior of cancer cells, it is important to recognize how it is linked to pH dynamics. Here, we evaluated the acute effects of mild acidification and alkalization on cancer cell glucose uptake and glycolytic flux and investigated the role of hexokinase (HK). Cancer cells exposed to buffers with graded pH were measured for 18F-fluorodeoxyglucose (FDG) uptake, lactate production and HK activity. Subcellular localization of HK protein was assessed by western blots and confocal microscopy. The interior of T47D breast cancer cells was mildly alkalized to pH 7.5 by a buffer pH of 7.8, and this was accompanied by rapid increases of FDG uptake and lactate extrusion. This shift toward glycolytic flux led to the prompt recovery of a reversed pH gradient. In contrast, mild acidification rapidly reduced cellular FDG uptake and lactate production. Mild acidification decreased and mild alkalization increased mitochondrial HK translocation and enzyme activity. Cells transfected with specific siRNA against HK-1, HK-2 and voltage-dependent anion channel (VDAC)1 displayed significant attenuation of pH-induced changes in FDG uptake. Confocal microscopy showed increased co-localization of HK-1 and HK-2 with VDAC1 by alkaline treatment. In isolated mitochondria, acidic pH increased and alkaline pH decreased release of free HK-1 and HK-2 from the mitochondrial pellet into the supernatant. Furthermore, experiments using purified proteins showed that alkaline pH promoted co-immunoprecipitation of HK with VDAC protein. These findings demonstrate that mild alkalization is sufficient to acutely trigger cancer cell glycolytic flux through enhanced activity of HK by promoting its mitochondrial translocation and VDAC binding. This process might serve as a mechanism through which cancer cells trigger the Warburg effect to maintain a dysregulated pH.

Troglitazone Stimulates Cancer Cell Uptake of 18F-FDG by Suppressing Mitochondrial Respiration and Augments Sensitivity to Glucose Restriction

We evaluated how troglitazone influences cancer cell glucose metabolism and uptake of 18F-FDG, and we investigated its molecular mechanism and relation to the drug’s anticancer effect. Methods: Human T47D breast and HCT116 colon cancer cells that had been treated with troglitazone were measured for 18F-FDG uptake, lactate release, oxygen consumption rate, mitochondrial membrane potential, and intracellular reactive oxygen species. Viable cell content was measured by sulforhodamine-B assays. Results: Treatment with 20 μM troglitazone for 1 h acutely increased 18F-FDG uptake in multiple breast cancer cell lines, whereas HCT116 cells showed a delayed reaction. In T47D cells, the response occurred in a dose-dependent (threefold increase by 40 μΜ) manner independent of peroxisome proliferator-activated receptor-γ and was accompanied by a twofold increase of lactate production, consistent with enhanced glycolytic flux. Troglitazone-treated cells showed severe reductions of the oxygen consumption rate, indicating suppression of mitochondrial respiration, which was accompanied by significantly decreased mitochondrial membrane potential and increased concentration of reactive oxygen species. Troglitazone dose-dependently reduced T47D and HCT116 cell content, which was significantly potentiated by restriction of glucose availability. In T47D cells, cell reduction closely correlated with the magnitude of increase in relative 18F-FDG uptake (r = 0.821, P = 0.001). Conclusion: Troglitazone stimulates cancer cell uptake of 18F-FDG through a shift of metabolism toward glycolytic flux, likely as an adaptive response to impaired mitochondrial oxidative respiration.

Abstract B111: Targeting of calreticulin exposure for optical imaging of etoposide-induced immunogenic tumor cell death.

Objective: There is growing interest in reinforcing response to anticancer therapies by promoting immunogenic cell death. This mode of cell death induces surface exposure of calreticulin (CRT) in a manner that precedes apoptosis. In this study, we explored whether CRT-targeted optical imaging may have potential as a biomarker of immunogenic cell death. Methods: We used a monoclonal antibody against human CRT (FMC 75) conjugated with a phycoerythrin label as a fluorescent imaging probe for CRT. Human CT-26 colon cancer cells were used for in vitro studies, and immunogenic cell death was induced by treatment with 25 M etoposide. Targeting of the probe to CRT exposed on the surface of etoposide-treated cells was verified by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. In vivo imaging was performed in tumor bearing Balb/C nude mice xenografted by subcutaneous injection of MDA-MB-468 human breast cancer cells. Tumors approximately 1 cm in diameter were treated with 100 μM etoposide intra-tumorally injected in 100 μl PBS. Phycoerythrin labeled anti-CRT antibody was injected into the tail vein 3 hr later. Optical imaging of fluorescent signals was performed on an IVIS® Spectrum system (Xenogen) at 4 and 21 hr post-injection using an excitation filter of 570 nm and emission filter of 620 nm. Results: Confocal microscopy displayed specific binding of phycoerythrin anti-CRT antibodies to the surface of etoposide-treated CT-26 cancer cells. FACS analysis with the probe showed a right-shift of fluorescent signals in cells treated with etoposide. Hence, signal-positive cells were increased from 1.83 ± 0.1% to 5.81 ± 0.5% by 4 and 24 hr of treatment, respectively. In vivo optical imaging following intra-tumoral etoposide injection demonstrated clear tumor uptake of phycoerythrin anti-CRT antibody from 4 hr post-injection. Tumor contrast was substantially enhanced at 21 hr post-injection owing to reduction of background activity, which resulted in high tumor-to-muscle signal ratios. Conclusion: This study provides the first evidence that targeted imaging of cell surface-exposed CRT may be useful for noninvasive monitoring of immunogenic cell death during anticancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B111.