Cancer cells adapt to their inherently increased oxidative stress through activation of the glutathione (GSH) and thioredoxin (TXN) systems. Inhibition of both of these systems effectively kills cancer cells, but such broad inhibition of antioxidant activity also kills normal cells, which is highly unwanted in a clinical setting. We therefore evaluated targeting of the TXN pathway alone and, more specifically, selective inhibition of the cytosolic selenocysteine-containing enzyme TXN reductase 1 (TXNRD1). TXNRD1 inhibitors were discovered in a large screening effort and displayed increased specificity compared to pan-TXNRD inhibitors, such as auranofin, that also inhibit the mitochondrial enzyme TXNRD2 and additional targets. For our lead compounds, TXNRD1 inhibition correlated with cancer cell cytotoxicity, and inhibitor-triggered conversion of TXNRD1 from an antioxidant to a pro-oxidant enzyme correlated with corresponding increases in cellular production of HO In mice, the most specific TXNRD1 inhibitor, here described as TXNRD1 inhibitor 1 (TRi-1), impaired growth and viability of human tumor xenografts and syngeneic mouse tumors while having little mitochondrial toxicity and being better tolerated than auranofin. These results display the therapeutic anticancer potential of irreversibly targeting cytosolic TXNRD1 using small molecules and present potent and selective TXNRD1 inhibitors. Given the pronounced up-regulation of TXNRD1 in several metastatic malignancies, it seems worthwhile to further explore the potential benefit of specific irreversible TXNRD1 inhibitors for anticancer therapy.

A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. Methods: A HER2-binding Affibody molecule, Z HER2:342 , was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either 11 C or 68 Ga, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the 11 C-labeled Affibody molecule. Results: Both the 11 C-and 68 Ga-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4-5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (.5-fold) for the 11 C-labeled protein, and its overall absorbed dose was considerably lower. 11 C-Z HER2:342 showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P 5 0.002), comparable to uptake in tumors with low HER2 expression. Conclusion: To our knowledge, the Sel-tagging technique is the first that enables sitespecific 11 C-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, 11 C-labeled Sel-tagged Z HER2:342 can successfully be used for rapid and repeated PET studies of HER2 expression in tumors. Overexpressi on of the human epidermal growth factor receptor 2 (HER2), found in about 20%-30% of breast cancers, is associated with poor prognosis, and HER2 is the molecular target for effective but expensive monoclonal antibody drugs such as trastuzumab (Herceptin; Roche) (1). Determination of the expression levels of HER2 is therefore important for predicting prognosis and for patient stratification for treatment. Currently, expression levels are mainly determined using biopsies for molecular assays such as in situ hybridization (2). These procedures are, however, invasive, are difficult to perform repeatedly, and risk missing overexpressing tissue due to sampling errors. Therefore, the radionuclide-based in vivo molecular imaging of HER2, primarily using polypeptide probes, is rapidly expanding, aiming for personalized medicine with image-and-treat strategies (3-6). Speed, sensitivity, and specificity are necessary for implementation in clinical routines.Comparing nuclear medicine imaging techniques, PET has better spatial resolution, higher sensitivity, and more accurate quantification than SPECT. With recent radiolabeling advances, the feasibility of using polypeptides...

Background In vivo imaging using Annexin A5-based radioligands is a powerful technique for visualizing massive cell death, but has been less successful in monitoring the modest cell death typically seen in solid tumors after chemotherapy. Here we combined dynamic positron emission tomography (PET) imaging using Annexin A5 with a serum-based apoptosis marker, for improved sensitivity and specificity in assessment of chemotherapy-induced cell death in a solid tumor model.Methodology/Principal FindingsModest cell death was induced by doxorubicin in a mouse xenograft model with human FaDu head and neck cancer cells. PET imaging was based on 11C-labeled Sel-tagged Annexin A5 ([11C]-AnxA5-ST) and a size-matched control. 2-deoxy-2-[18F]fluoro-D-glucose ([18F]-FDG) was utilized as a tracer of tissue metabolism. Serum biomarkers for cell death were ccK18 and K18 (M30 Apoptosense® and M65). Apoptosis in tissue sections was verified ex vivo for validation. Both PET imaging using [11C]-AnxA5-ST and serum ccK18/K18 levels revealed treatment-induced cell death, with ccK18 displaying the highest detection sensitivity. [18F]-FDG uptake was not affected by this treatment in this tumor model. [11C]-AnxA5-ST gave robust imaging readouts at one hour and its short half-life made it possible to perform paired scans in the same animal in one imaging session.Conclusions/SignificanceThe combined use of dynamic PET with [11C]-AnxA5-ST, showing specific increases in tumor binding potential upon therapy, with ccK18/K18 serum measurements, as highly sensitive markers for cell death, enabled effective assessment of modest therapy-induced cell death in this mouse xenograft model of solid human tumors.

Background: Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors.