Cancer prevention (chemoprevention) by using naturally occurring dietary agents has gained immense interest due to the broad safety window of these compounds. However, many of these compounds are hydrophobic and poorly soluble in water. They frequently display low bioavailability, poor systemic delivery, and low efficacy. To circumvent this problem, we explored a novel approach towards chemoprevention using nanotechnology to deliver luteolin, a natural compound present in green vegetables. We formulated water soluble polymer-encapsulated Nano-Luteolin from hydrophobic luteolin, and studied its anticancer activity against lung cancer and head and neck cancer. In vitro studies demonstrated that, like luteolin, Nano-Luteolin inhibited the growth of lung cancer cells (H292 cell line) and squamous cell carcinoma of head and neck (SCCHN) cells (Tu212 cell line). In Tu212 cells, the IC50 value of Nano-Luteolin was 4.13μM, and that of luteolin was 6.96μM. In H292 cells, the IC50 of luteolin was 15.56μM, and Nano-Luteolin was 14.96μM. In vivo studies using a tumor xenograft mouse model demonstrated that Nano-Luteolin has a significant inhibitory effect on the tumor growth of SCCHN in comparison to luteolin. Our results suggest that nanoparticle delivery of naturally occurring dietary agents like luteolin has many advantages and may have potential application in chemoprevention in clinical settings.

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 18 F-FDG uptake. Assays were performed for lactate production; hexokinase activity and intracellular ROS; and immunoblotting for hypoxia-inducible factor-1a (HIF-1a), 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 18 F-FDG uptake in Lewis lung carcinoma, HT-29 colon, and T47D breast cancer cells. Hence, 18 F-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 18 F-FDG uptake. Scavenging of ROS with N-acetyl cysteine, but not inhibition of nicotinamide adenine dinucleotide phosphate oxidase, was sufficient to suppress 18 F-FDG uptake. Conversely, ROS inducers effectively reversed the metabolic response of resveratrol. HIF-1a protein was markedly reduced by resveratrol, and inhibiting HIF-1a expression with cycloheximide or specific small interfering RNAs suppressed 18 F-FDG uptake. The proteosomal inhibitor MG132 partly restored HIF-1a level and 18 F-FDG uptake in resveratrol-treated cells. Resveratrol also inhibited Akt activation; in addition, inhibitors and small interfering RNAs against phosphoinositide 3-kinase decreased 18 F-FDG uptake. Finally, small-animal PET results showed resveratrol treatment to suppress tumor 18 F-FDG uptake in vivo. Conclusion: Resveratrol suppresses cancer cell 18 F-FDG uptake and glycolytic metabolism in a manner that depends on the capacity of resveratrol to inhibit intracellular ROS, which downregulates HIF-1a accumulation. Ther e is recently growing interest in natural products as an addition to the repertoire of agents that may be beneficial in our battle against cancer (1). Resveratrol, a natural polyphenol compound found in such fruits as grapes and berries, has particularly gained intense attention for its promising anticancer effects (2). Initially recognized for its ability to inhibit carcinogenesis at multiple stages (3,4), resveratrol has since been found to exert significant antitumor effects including inhibition of growth (5-7), induction of apoptosis (6-8), and suppression of metastatic potential (9,10).Resveratrol is known to reduce energy expenditure in vivo, mimicking the effects of caloric restriction (11). Recently, several in vitro studies have described an inhibitory effect of resveratrol ...

We developed an 89 Zr-labeled anti-programmed death ligand 1 (anti-PD-L1) immune PET that can monitor chemotherapy-mediated modulation of tumor PD-L1 expression in living subjects. Methods:Anti-PD-L1 underwent sulfohydryl moiety-specific conjugation with maleimide-deferoxamine followed by 89 Zr radiolabeling. CT26 colon cancer cells and PD-L1 overexpressing CT26/PD-L1 cells underwent binding assays, flow cytometry, and Western blotting. In vivo pharmacokinetics, biodistribution, and PET imaging was evaluated in mice. Results: 89 Zr-anti-PD-L1 synthesis was straightforward and efficient. SDS PAGE showed that reduction produced half-antibody fragments, and MALDI-TOF analysis estimated 2.18 conjugations per antibody, indicting specific conjugation at the hinge region disulfide bonds. CT26/PD-L1 cells showed 102.2 ± 6.7-fold greater 89 Zr-anti-PD-L1 binding compared to weak expressing CT26 cells. Excellent target specificity was confirmed by a drastic reduction of binding by excess cold antibody. Intravenous 89 Zr-anti-PD-L1 followed bi-exponential blood clearance. PET/CT image analysis demonstrated decreases in major organ activity over 7 days, whereas high CT26/PD-L1 tumor activity was maintained. Again, this was suppressed by excess cold antibody. Treatment of CT26 cells with gemcitabine for 24 h augmented PD-L1 protein to 592.4 ± 114.2% of controls and increased 89 Zr-anti-PD-L1 binding. This was accompanied by increased AKT activation and reduced PTEN. In CT26 tumor-bearing mice, gemcitabine treatment substantially increased tumor uptake from 1.56 ± 0.48 to 6.24 ± 0.37 %ID/g (tumor/blood ratio: 34.7). Immunoblots revealed significant increases in tumor PD-L1 and activated AKT and a decrease of PTEN. Conclusion: 89 Zr-anti-PD-L1 showed specific targeting with favorable imaging properties. Gemcitabine treatment upregulated cancer cell and tumor PD-L1 expression and increased 89 Zr-anti-PD-L1 uptake. 89 Zr-anti-PD-L1 PET may thus be useful for monitoring chemotherapy-mediated tumor PD-L1 modulation in living subjects.

For 18 F-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 18 F-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 18 F-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 18 F-FDG uptake by 24 h, which was accompanied by increased lactate production, membrane GLUT1 expression, and hexokinase activity. oxLDL-stimulated 18 F-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 oxLDLinduced ROS production and 18 F-FDG uptake. oxLDL increased Nox2 expression, and nicotinamide adenine dinucleotide phosphate oxidase inhibition totally blocked increased ROS generation and 18 F-FDG uptake by oxLDL. Finally, there was a clear ROSdependent 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 18 F-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.

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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 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. 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. Subcut aneous and visceral white adipose tissue (WAT) contributes to obesity by storing excess energy as intracellular lipid (1,2). By contrast, brown adipose tissue (BAT) can have a negative influence on weight gain by dissipating energy as heat (3,4). This thermoregulatory response is mediated through norepinephrineactivated b3 adrenergic receptor (b3AR) signaling. The exciting recent revelation that functional BAT is present not only in animals and young children but also in human adults (5,6) has fueled attempts to exploit its energy-consuming property to counter obesity (7,8). Unfortunately, however, obese patients have insufficient amounts of BAT, limiting the value of constitutive BAT for weight reduction (9).On the other hand, obese subjects have abundant amounts WAT. It has recently become known that WAT can recruit clusters of adipocytes with a brownlike phenotype. This process, called WAT browning, can be seen in mice stimulated by prolonged cold exposure or by b3AR agonists as a mimetic of cold stress (10,11). Brownlike cells recruited by WAT browning are referred to as beige (pale brown) adipocytes (12)(13)...

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