Patient advocacy group involvement in health technology assessments: an observational study.

BACKGROUND: In some jurisdictions, patients and patient groups may be invited to provide input when Health Technology Assessment (HTA) is used to inform decisions about which medicines should be subsidised or funded. This input can help frame the evidence from a patient perspective, address uncertainties in the evidence and interpret it for the local setting. However, there is currently no evidence linking patient involvement with positive reimbursement decisions. AIM: We aimed to understand the expectations of patient involvement in the reimbursement process, especially among cancer patient advocacy groups (PAGs) in New Zealand (Aotearoa), South Korea and Taiwan. METHODS: We developed an online survey to help understand the role that cancer PAGs play in reimbursement processes and identify knowledge gaps about the processes that might impact the efforts of PAGs. The survey elicited the views of staff and patients affiliated with PAGs (n = 43) on current practices and how the assessment and reimbursement of new cancer drugs might be improved. RESULTS: There was variability in knowledge of the HTA assessment processes and in experience of being involved in them. Those with HTA experience were more likely to have confidence in the process. Those who had not been involved tended to have little awareness of, or frustration with, decision-making processes. Most identified cost, finances and economic assessments as key considerations in current processes. Some respondents had clear ideas about how their knowledge and involvement could improve processes to determine the value of new medicines. However, for many, a lack of information about the basis for decision making and opportunities to be involved was a barrier to identifying process improvement. CONCLUSIONS: HTA is implemented primarily in countries seeking to have fair and equitable processes for funding medicines. PAGs often recognise the financial challenges of funding new medicines and share the desire for procedural fairness. The connection PAGs make between patient involvement and improved access to new medicines may be based on the belief they can add information to the evidence base, help solve problems, ensure fairness through transparency and/or influence the culture towards increased access to medicines they value.

WHAT ARE HEALTH TECHNOLOGY ASSESSMENTS?: Health technologies are medicines, devices and services used to prevent, diagnose or treat medical conditions. A Health Technology Assessment (HTA) considers the value of a health technology to help governments make fair decisions about which treatments to fund or subsidise. Patients and Patient Advocacy Groups (PAGs) may be invited to provide input into this process. WHAT DID OUR STUDY LOOK AT?: We surveyed staff and patients from cancer PAGs in New Zealand (Aotearoa), South Korea and Taiwan to understand their experiences and expectations of involving patients in HTA. A total of 43 people completed our survey. WHAT WERE THE RESULTS?: Overall, 20 of the 43 surveyed people (47%) were aware of the assessment process used to decide which medicines were funded in their country. (27 of these 50 people were PAG staff members.) Patients from South Korea and New Zealand were rarely included in the assessment process. Patient involvement was more commonly reported in Taiwan. The impact of input from patients on HTA was rated low by patients in both South Korea and New Zealand and average in Taiwan. A general lack of transparency, feedback on the process, and support from assessment agencies was also reported. Many of the patients surveyed wanted to see more patient involvement in the assessment processes. WHAT IS THE MAIN TAKE-HOME MESSAGE FROM THIS STUDY?: With improved guidance and feedback from assessment agencies, PAGs and patients believe their involvement can improve decision making on treatment funding and increase fairness through transparency.

Reactive oxygen species-driven HIF1α triggers accelerated glycolysis in endothelial cells exposed to low oxygen tension.

Endothelial cells and their metabolic state regulate glucose transport into underlying tissues. Here, we show that low oxygen tension stimulates human umbilical vein endothelial cell 18F-fluorodeoxyglucose (18F-FDG) uptake and lactate production. This was accompanied by augmented hexokinase activity and membrane Glut-1, and increased accumulation of hypoxia-inducible factor-1α (HIF1α). Restoration of oxygen reversed the metabolic effect, but this was blocked by HIF1α stabilization. Hypoxia-stimulated 18F-FDG uptake was completely abrogated by silencing of HIF1α expression or by a specific inhibitor. There was a rapid and marked increase of reactive oxygen species (ROS) by hypoxia, and ROS scavenging or NADPH oxidase inhibition completely abolished hypoxia-stimulated HIF1α and 18F-FDG accumulation, placing ROS production upstream of HIF1α signaling. Hypoxia-stimulated HIF1α and 18F-FDG accumulation was blocked by the protein kinase C (PKC) inhibitor, staurosporine. The phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, blocked hypoxia-stimulated 18F-FDG uptake and attenuated hypoxia-responsive element binding of HIF1α without influencing its accumulation. Thus, ROS-driven HIF1α accumulation, along with PKC and PI3K signaling, play a key role in triggering accelerated glycolysis in endothelial cells under hypoxia, thereby contributing to 18F-FDG transport.

Oxidized low-density lipoprotein stimulates macrophage 18F-FDG uptake via hypoxia-inducible factor-1α activation through Nox2-dependent reactive oxygen species generation.

UNLABELLED: 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 oxLDL-induced 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 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 (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.

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.

Resveratrol suppresses cancer cell glucose uptake by targeting reactive oxygen species-mediated hypoxia-inducible factor-1α activation.

UNLABELLED: 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-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 (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-1α protein was markedly reduced by resveratrol, and inhibiting HIF-1α expression with cycloheximide or specific small interfering RNAs suppressed (18)F-FDG uptake. The proteosomal inhibitor MG132 partly restored HIF-1α 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-1α accumulation.

131I-MIBG targeting of neuroblastoma cells is acutely enhanced by KCl stimulation through the calcium/calmodulin-dependent kinase pathway.

The efficacy of (131)I-metaiodobenzylguanidine (MIBG) therapy relies on norepinephrine transporter (NET) function. The ionic make-up of the extracellular fluid critically controls neuronal cell activity and can also affect substrate transport. In this study, we explored the effect of treatment with elevated KCl concentration on MIBG uptake in SK-N-SH neuroblastoma cells. KCl stimulation caused a rapid increase of (131)I-MIBG uptake in a manner that was calcium-dependent and accompanied by activation of calcium/calmodulin-dependent protein kinase (CaMK)II. The effect was completely abolished by KN93, an inhibitor of CaMKI, II, and IV. STO609, a selective inhibitor of CaMK kinase required for activation of CaMKI and IV, but not CaMKII, only modestly attenuated the response. The KCl effect was also completely abrogated by ML7, a selective inhibitor of myosin light chain kinase (MLCK). This restricted form of CaMK activates myosin, which is required for vesicle trafficking. Saturation kinetic analysis revealed KCl stimulation to increase maximal transport velocity without affecting substrate affinity. In conclusion, KCl stimulation rapidly upregulates NET function through the CaMK pathway via activation of CaMKII and MLCK. These findings allow a better understanding of how NET function is acutely modulated by the ionic environment, which in turn may ultimately help improve the efficacy of (131)I-MIBG therapy.

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.

Hydrazinonicotinamide prolongs quantum dot circulation and reduces reticuloendothelial system clearance by suppressing opsonization and phagocyte engulfment.

In this study, we investigated the effects of hydrazinonicotinamide (HYNIC)-a bifunctional crosslinker widely used to (99m)Tc radiolabel protein and nanoparticles for imaging studies-on quantum dot opsonization, macrophage engulfment and in vivo kinetics. In streptavidin-coated quantum dots (SA-QDots), conjugation with HYNIC increased the net negative charge without affecting the zeta potential. Confocal microscopy and fluorescence-activated cell sorting showed HYNIC attachment to suppress SA-QDot engulfment by macrophages. Furthermore, HYNIC conjugation suppressed surface opsonization by serum protein including IgG. When intravenously injected into mice, HYNIC conjugation significantly prolonged the circulation of SA-QDots and reduced their hepatosplenic uptake. Diminished reticuloendothelial system clearance of SA-QDots and aminoPEG-QDots by HYNIC conjugation was also demonstrated by in vivo and ex vivo optical imaging. The effects of HYNIC on the opsonization, phagocytosis and in vivo kinetics of quantum dots were reversed by removal of the hydrazine component from HYNIC. Thus, surface functionalization with HYNIC can improve the in vivo kinetics of quantum dots by reducing phagocytosis via suppression of surface opsonization.

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.

Combination of nitric oxide stimulation with high-dose 18F-FDG promotes apoptosis and enhances radiation therapy of endothelial cells.

INTRODUCTION: High-dose (18)F-FDG can provide targeted nuclear therapy of cancer. Endothelial cell injury is a key determinant of tumor response to radiotherapy. Here, we tested the hypothesis that activation of endothelial cell glycolytic metabolism with nitric oxide can enhance the therapeutic effect of high-dose (18)F-FDG. METHODS: Calf pulmonary artery endothelial (CPAE) cells were treated with graded doses of (18)F-FDG. Glycolysis was stimulated by 24 h of exposure to the nitric oxide donor, sodium nitroprusside (SNP). Cell viability was assessed by MTT and clonogenic assays. Apoptosis was evaluated by ELISA of cytosolic DNA fragments and Western blots of cleaved caspase-3. RESULTS: SNP stimulation (0.1 and 1 mM) augmented CPAE cell (18)F-FDG uptake to 2.6- and 4.6-fold of controls without adverse effects. Treatment with 333 µCi/ml (18)F-FDG alone reduced viable cell number to 35.4% of controls by Day 3. Combining 0.1 mM SNP stimulation significantly enhanced the killing effect, reducing cell numbers to 19.2% and 39.2% of controls by 333 and 167 µCi/ml of (18)F-FDG, respectively. (18)F-FDG also suppressed clonogenic survival to 80.8% and 43.2% of controls by 83 and 167 µCi/ml, which was again intensified by SNP to 59.7% and 21.1% of controls. The cytotoxic effect of (18)F-FDG was attributed to induction of apoptosis as shown by increased cytosolic fragmented DNA and cleaved caspase-3 levels (26.4% and 30.7% increases by 167 µCi/ml). Combining SNP stimulation significantly increased both of these levels to 1.8-fold of control cells. CONCLUSION: High-dose (18)F-FDG combined with nitric oxide-stimulated glycolysis is an effective method to inhibit endothelial cell survival and promote apoptosis. These results suggest a potential role of this strategy for targeted radiotherapy of angiogenic vasculature.

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.

UNLABELLED: 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 (99m)Tc-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: (99m)Tc-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: (99m)Tc-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.

17beta-estradiol augments 18F-FDG uptake and glycolysis of T47D breast cancer cells via membrane-initiated rapid PI3K-Akt activation.

UNLABELLED: Use of (18)F-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 (18)F-FDG uptake in responsive breast cancer cells and investigated the mediating molecular mechanisms. METHODS: T47D breast cancer cells were stimulated with 17ß-estradiol (E(2)) or bovine serum albumin (BSA)-E(2) and measured for (18)F-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: E(2) augmented T47D cell (18)F-FDG uptake in a dose- and time-dependent manner that preceded and surpassed its proliferative effect. With exposure to 10 nM E(2), protein content-corrected (18)F-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 E(2) effect was not blocked by tamoxifen and was mimicked by membrane-impermeable BSA-E(2), consistent with nongenomic membrane-initiated E(2) action. Inhibition by cycloheximide demonstrated the requirement of a new protein synthesis. Immunoblots displayed rapid phosphorylation of PI3K and Akt within minutes of E(2) treatment, and the specific PI3K inhibitors wortmannin and LY294002 abolished the ability of E(2) to elevate (18)F-FDG uptake. CONCLUSION: Estrogen augments breast cancer cell (18)F-FDG uptake by stimulating glycolysis and hexokinase activity via membrane-initiated E(2) 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 (18)F-FDG uptake as a surrogate marker of therapeutic response.

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.

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.

Fibronectin stimulates endothelial cell 18F-FDG uptake through focal adhesion kinase-mediated phosphatidylinositol 3-kinase/Akt signaling.

UNLABELLED: There has been recent interest in the relationship between (18)F-FDG uptake and the angiogenic activity of endothelial cells (ECs). The angiogenic process is strongly dependent on the interaction of ECs with matrix fibronectin (FN), a key regulator of EC survival, migration, and proliferation. Therefore, we investigated how FN influences EC glucose uptake and elucidated the signaling pathways that mediate this effect. METHODS: Human umbilical vein ECs were allowed to adhere to FN-coated plates and were compared with control cells for (18)F-FDG uptake, membrane GLUT1 levels, and hexokinase activity. The roles of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), and Akt were evaluated with Western blotting, small interfering RNA (siRNA), and specific inhibitors. RESULTS: FN adhesion significantly enhanced the protein-corrected (18)F-FDG uptake in HUVEC, to 2.1-, 2.7-, and 4.3-fold that in control cells by 2, 3, and 5 d, respectively. This effect was mediated by the upregulation of both membrane GLUT1 expression and hexokinase activity and was accompanied by FAK activation. Silencing of FAK signaling by siRNA completely abrogated both FN-induced FAK phosphorylation and (18)F-FDG uptake. FN also activated PI3K and Akt, well-known angiogenesis mediators, and the inhibition of either pathway totally abolished the effect of FN on (18)F-FDG uptake. Nitric oxide, a downstream Akt effector that stimulates glucose uptake, was not involved in the metabolic effect of FN. CONCLUSION: The results of this study demonstrated that an EC-FN interaction induces strong enhancement of (18)F-FDG uptake through the upregulation of GLUT1 expression and hexokinase activity. The findings also showed that the response occurs through FAK-mediated activation of PI3K and Akt, indicating a role for this pathway in modulating EC glucose metabolism.

Mitogen-activated protein kinase signaling enhances sodium iodide symporter function and efficacy of radioiodide therapy in nonthyroidal cancer cells.

UNLABELLED: 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 (125)I 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. (131)I-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 (131)I 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 anesthetic agents on cellular 123I-MIBG transport and in vivo 123I-MIBG biodistribution.

OBJECTIVES: Small animal imaging with meta-iodobenzylguanidine (MIBG) allows characterization of animal models, optimization of tumor treatment strategies, and monitoring of gene expression. Anesthetic agents, however, can affect norepinephrine (NE) transport and systemic sympathetic activity. We thus elucidated the effects of anesthetic agents on MIBG transport and biodistribution. METHODS: SK-N-SH neuroblastoma and PC-12 pheochromocytoma cells were measured for (123)I-MIBG uptake after treatment with ketamine (Ke), xylazine (Xy), Ke/Xy, or pentobarbital (Pb). NE transporters were assessed by Western blots. Normal ICR mice and PC-12 tumor-bearing mice were injected with (123)I-MIBG 10 min after anesthesia with Ke/Xy, Ke, Xy, or Pb. Plasma NE levels and MIBG biodistribution were assessed. RESULTS: Cellular (123)I-MIBG uptake was dose-dependently inhibited by Ke and Xy but not by Pb. Treatment for 2 h with 300 microM Ke, Xy, and Ke/Xy decreased uptake to 46.0 +/- 1.6, 24.8 +/- 1.5, and 18.3 +/- 1.6% of controls. This effect was completely reversed by fresh media, and there was no change in NE transporter levels. In contrast, mice anesthetized with Ke/Xy showed no decrease of MIBG uptake in target organs. Instead, uptakes and organ-to-blood ratios were increased in the heart, lung, liver, and adrenals. Plasma NE was notably reduced in the animals with corresponding decreases in blood MIBG, which partly contributed to the increase in target organ uptake. CONCLUSION: In spite of their inhibitory effect at the transporter level, Ke/Xy anesthesia is a satisfactory method for MIBG imaging that allows favorable target tissue uptake and contrast by reducing circulating NE and MIBG.

Direct targeting of tumor cell F(1)F(0) ATP-synthase by radioiodine angiostatin in vitro and in vivo.

UNLABELLED: Recent discoveries have identified endothelial cell-surface F(1)F(0) adenosine triphosphate (ATP) synthase as the key target for angiostatin (AST) action. As this enzyme is also present on tumor cells, we investigated whether radiolabeled AST may directly target cancer cell-surface ATP synthase in vitro and in vivo. METHODS: Cell-binding characteristics of (125)I-AST was evaluated on human umbilical vein endothelial (HUVE) and SNU-C5 colon carcinoma cells. The molecular target for binding was investigated with anti-ATP synthase antibodies and RGDyV. Biodistribution and imaging experiments were performed in mice xenografted with carcinoma and sarcoma tumors. Tumor localization of ATP synthase and exogenous AST was compared via double immunostaining. RESULTS: Both HUVE and SNU-C5 cells showed specific (125)I-AST binding that was dose-dependently inhibited by excess AST, with IC(50) values of 3.5 and 0.2 microM, respectively. Both cell types stained positive for ATP synthase and demonstrated an approximately 50% reduction in AST binding by antibodies against the alpha- and beta-subunit of the enzyme. (123)I-AST injected in mice allowed for the clear tumor visualization with significant tumor accumulation and uptake ratios. Immunostaining revealed a localization of exogenous AST to closely correlate to that of tumor-cell ATP synthase. CONCLUSIONS: AST can directly target tumor-cell ATP synthase, and AST imaging may thus be useful for monitoring tumor ATP synthase expression.

Favorable biokinetic and tumor-targeting properties of 99mTc-labeled glucosamino RGD and effect of paclitaxel therapy.

UNLABELLED: Compared with the recent advancements in radiohalogenated Arg-Gly-Asp (RGD) peptides for alpha(v)beta(3)-targeted imaging, there has been limited success with (99m)Tc-labeled RGD compounds. In this article, we describe the favorable in vivo kinetics and tumor-imaging properties of a novel (99m)Tc-RGD compound that contains a glucosamine moiety. METHODS: Glucosamino (99m)Tc-d-c(RGDfK) was prepared by incorporating (99m)Tc(CO)(3) to the glucosamino peptide precursor in high radiochemical yield. Cell-binding characteristics were tested on human endothelial cells. Mice bearing RR1022 fibrosarcoma and Lewis lung carcinoma (LLC) tumors were used for in vivo biodistribution and blocking experiments and for imaging studies. Separate LLC-bearing mice underwent antiangiogenic therapy with 0, 20, or 40 mg of paclitaxel per kilogram of body weight every 2 d. Tumor volume was serially monitored, and tumor glucosamino (99m)Tc-d-c(RGDfK) uptake and Western blots of alpha(v) integrin expression were analyzed at day 14. RESULTS: Glucosamino (99m)Tc-d-c(RGDfK) binding to endothelial cells was dose-dependently inhibited by excess RGD. Biodistribution in mice showed rapid blood clearance of glucosamino (99m)Tc-d-c(RGDfK), with substantially lower liver uptake and higher tumor uptake compared with (125)I-c(RGD(I)yV). Tumor uptake was 1.03 +/- 0.21 and 1.18 +/- 0.26 %ID/g at 1 h and 0.85 +/- 0.05 and 0.89 +/- 0.28 %ID/g at 4 h for sarcomas and carcinomas, respectively. Excess RGD blocked uptake by 76.5% and 70.2% for the respective tumors. gamma-Camera imaging allowed clear tumor visualization, with an increase of sarcoma-to-thigh count ratios from 5.5 +/- 0.7 at 1 h to 10.1 +/- 2.2 at 4 h and sustained carcinoma-to-thigh count ratios from 4 to 17 h. Pretreatment with excess cRGDyV significantly reduced tumor contrast on images. Paclitaxel therapy in LLC tumor-bearing mice significantly retarded tumor growth. This was accompanied by a corresponding reduction of tumor glucosamino (99m)Tc-d-c(RGDfK) uptake, which correlated significantly with tumor alpha(v) integrin expression levels. CONCLUSION: Glucosamino (99m)Tc-d-c(RGDfK) has favorable in vivo biokinetics and tumor-imaging properties and may be useful for noninvasive evaluation of tumor integrin expression and response to antiangiogenic therapeutics. Because of the wide accessibility of gamma-cameras and high availability and excellent imaging characteristics of (99m)Tc, glucosamino (99m)Tc-d-c(RGDfK) may be an attractive alternative to radiohalogenated RGD peptides for angiogenesis-imaging research.