Reporter PET Images Bortezomib Treatment-Mediated Suppression of Cancer Cell Proteasome Activity.

Proteasomal protein degradation is a promising target for cancer therapy. Here, we developed a positron emission tomography (PET) technique based on the sodium-iodide symporter (NIS) gene fused with the carboxyl-terminal of ornithine decarboxylase (cODC) that noninvasively images cancer cells with inhibited proteasome activity. A retroviral vector was constructed in which the murine cODC degron was fused to the human NIS gene (NIS-cODC). Transiently transduced CT26 and HT29 colon cancer cells and stably expressing CT26/NIS-cODC cells were prepared. In cancer cells transiently transduced with NIS-cODC, NIS expression and transport activity was low at baseline, but NIS protein and 125I uptake was significantly increased by inhibition of proteasome activity with bortezomib. Stable CT26/NIS-cODC cells also showed increased cytosolic and membrane NIS by bortezomib, and four different stable clones displayed bortezomib dose-dependent stimulation of 125I and 99mTc-04- uptake. Importantly, bortezomib dose-dependently suppressed survival of CT26/NIS-cODC clones in a manner that closely correlated to the magnitudes of 125I and 99mTc-04- uptake. CT26/NIS-cODC tumors of bortezomib-treated mice demonstrated greater 124I uptake on PET images and increased NIS expression on tissue staining compared to vehicle-injected animals. NIS-cODC PET imaging may allow noninvasive quantitative monitoring of proteasome activity in cancer cells treated with bortezomib.

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.

UNLABELLED: We evaluated how troglitazone influences cancer cell glucose metabolism and uptake of (18)F-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 (18)F-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 (18)F-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 (18)F-FDG uptake (r = 0.821, P = 0.001). CONCLUSION: Troglitazone stimulates cancer cell uptake of (18)F-FDG through a shift of metabolism toward glycolytic flux, likely as an adaptive response to impaired mitochondrial oxidative respiration.

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 ß3 agonist-stimulated brown adipocyte recruitment in white adipose tissue.

UNLABELLED: 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 (18)F-FDG uptake as an imaging biomarker that can monitor the process of WAT browning. METHODS: C57BL/6 mice were treated daily with the ß3 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. (18)F-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 (18)F-FDG uptake. Tissues underwent immunostaining, and UCP1 content was quantified by Western blotting. RESULTS: PET/CT showed low (18)F-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 (18)F-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 (18)F-FDG uptake in inguinal WAT correlated well with UCP1 content. CONCLUSION: (18)F-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 (99m)Tc-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 (T1/2ß, 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.