Test-retest reproducibility of cerebral adenosine A(2A) receptor quantification using [C-11]preladenant

Objective To evaluate the reproducibility of cerebral adenosine A(2A) receptor (A(2A)R) quantification using [C-11]preladenant ([C-11]PLN) and PET in a test-retest study. Methods Eight healthy male volunteers were enrolled. Dynamic 90 min PET scans were performed twice at the same time of the day to avoid the effect of diurnal variation. Subjects refrained from caffeine from 12 h prior to scanning, and serum caffeine was measured before radioligand injection. Arterial blood was sampled repeatedly during scanning and the fraction of the parent compound in plasma was determined. Total distribution volume (V-T) was estimated using 1- and 2-tissue compartment models (1-TCM and 2-TCM, respectively) and Logan graphical analysis (Logan plot) (t* = 30 min). Plasma-free fraction (f(P)) of [C-11]PLN was measured and used for correction of V-T values. Distribution volume ratio (DVR) was calculated from V-T of target and reference regions and obtained by noninvasive Logan graphical reference tissue model (LGAR) (t* = 30 min). Absolute test-retest variability (aTRV), and intra-class correlation coefficient (ICC) of V-T and DVR were calculated as indexes of repeatability. Correlation between DVR and serum concentration of caffeine (a nonselective A(2A)R blocker) was analyzed by Pearson's correlation analysis. Results Regional time-activity curves were well described by 2-TCM models. Estimation of V-T by 2-TCM produced some erroneous values; therefore, the more robust Logan plot was selected as the appropriate model. Global mean aTRV was 20% for V-T and 14% for V-T/f(P) (ICC, 0.72 for V-T and 0.87 for V-T/f(P)). Global mean aTRV of DVR was 13% for Logan plot and 10% for LGAR (ICC, 0.70 for Logan plot and 0.81 for LGAR). DVR estimates using LGAR and Logan plot were in good agreement (r(2) = 0.96). Coefficients of variation for V-T, V-T/f(P), DVR (Logan plot), and DVR (LGAR) were 47%, 47%, 27%, and 18%, respectively. Despite low serum caffeine levels, significant concentration-dependent effects on [C-11]PLN binding to target regions were observed (p < 0.01). Conclusions In this study, moderate test-retest reproducibility and large inter-subject differences were observed with [C-11]PLN PET, possibly attributable to competition by baseline amount of caffeine. Analysis of plasma caffeine concentration is recommended during [C-11]PLN PET studies

Licochalcone A Potently Inhibits Tumor Necrosis Factor ␣- Induced Nuclear Factor-B Activation through the Direct Inhibition of IB Kinase Complex Activation

ABSTRACT Glycyrrhiza inflata has been used as a traditional medicine with anti-inflammatory activity; however, its mechanism has not been fully understood. Licochalcone A is a major and biogenetically characteristic chalcone isolated from G. inflata. Here, we found that licochalcone A strongly inhibited tumor necrosis (TNF)-␣-induced nuclear localization, DNA binding activity, and the transcriptional activity of nuclear factor-B (NF-B). Whereas licochalcone A had no effect on the recruitment of receptor-interacting protein 1 and IB kinase ␤ (IKK␤) to TNF receptor I by TNF-␣, it significantly inhibited TNF-␣-induced IB kinase complex (IKK) activation and inhibitor of nuclear factor-B degradation. It is interesting that we found that the cysteine residue at position 179 of IKK␤ is essential for licochalcone A-induced IKK inhibition, because licochalcone A failed to affect the kinase activity of the IKK␤ (C179A) mutant. In contrast, a structurally related compound, echinatin, failed to inhibit TNF-␣-induced IKK activation and NF-B activation, suggesting that the 1,1-dimethy-2-propenyl group in licochalcone A is important for the inhibition of NF-B. In addition, TNF-␣-induced expression of inflammatory cytokines CCL2/ monocyte chemotactic protein-1and CXCL1/KC was clearly inhibited by licochalcone A but not echinatin. Taken together, licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the inhibition of IKK activation

First clinical assessment of [ 18 F]MC225, a novel fluorine-18 labelled PET tracer for measuring functional P-glycoprotein at the blood-brain barrier

Objective: 5-(1-(2-[18F]fluoroethoxy))-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalen ([18F]MC225) is a selective substrate for P-glycoprotein (P-gp), possessing suitable properties for measuring overexpression of P-gp in the brain. This is the first-in-human study to examine safety, radiation dosimetry and P-gp function at the blood-brain barrier (BBB) of [18F]MC225 in healthy subjects. Methods: [18F]MC225 biodistribution and dosimetry were determined in 3 healthy male subjects, using serial 2 h and intermittent 4 and 6 h whole-body PET scans acquired after [18F]MC225 injection. Dynamic [18F]MC225 brain PET (90 min) was obtained in 5 healthy male subjects. Arterial blood was sampled at various time intervals during scanning and the fraction of unchanged [18F]MC225 in plasma was determined. T1-weighted MRI was performed for anatomical coregistration. Total distribution volume (VT) was estimated using 1- and 2-tissue-compartment models (1-TCM and 2-TCM, respectively). VT was also estimated using the Logan graphical method (Logan plot) (t* = 20 min). Surrogate parameters without blood sampling (area-under the curve [AUC] of regional time-activity curves [TACs] and negative slope of calculated TACs) were compared with the VT values. Results: No serious adverse events occurred throughout the study period. Although biodistribution implied hepatobiliary excretion, secretion of radioactivity from liver to small intestine through the gallbladder was very slow. Total renal excreted radioactivity recovered during 6 h after injection was 0.9). AUCs of TACs were positively correlated with VT (2-TCM) values (r2: AUC0-60 min = 0.61, AUC0-30 min = 0.62, AUC30-60 min = 0.59, p < 0.0001). Negative slope of SUV TACs was negatively correlated with VT (2-TCM) values (r2 = 0.53, p < 0.0001). Conclusions: This initial evaluation indicated that [18F]MC225 is a suitable and safe PET tracer for measuring P-gp function at the BBB. Keywords: Blood–Brain barrier; Dosimetry; First-in-human; P-glycoprotein; Positron emission tomography

EBP2, a novel NPM‐ALK‐interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53

The oncogenic fusion protein nucleophosmin‐anaplastic lymphoma kinase (NPM‐ALK), found in anaplastic large‐cell lymphoma (ALCL), localizes to the cytosol, nucleoplasm, and nucleolus. However, the relationship between its localization and transforming activity remains unclear. We herein demonstrated that NPM‐ALK localized to the nucleolus by binding to nucleophosmin 1 (NPM1), a nucleolar protein that exhibits shuttling activity between the nucleolus and cytoplasm, in a manner that was dependent on its kinase activity. In the nucleolus, NPM‐ALK interacted with Epstein–Barr virus nuclear antigen 1‐binding protein 2 (EBP2), which is involved in rRNA biosynthesis. Moreover, enforced expression of NPM‐ALK induced tyrosine phosphorylation of EBP2. Knockdown of EBP2 promoted the activation of the tumor suppressor p53, leading to G0/G1‐phase cell cycle arrest in Ba/F3 cells transformed by NPM‐ALK and ALCL patient‐derived Ki‐JK cells, but not ALCL patient‐derived SUDH‐L1 cells harboring p53 gene mutation. In Ba/F3 cells transformed by NPM‐ALK and Ki‐JK cells, p53 activation induced by knockdown of EBP2 was significantly inhibited by Akt inhibitor GDC‐0068, mTORC1 inhibitor rapamycin, and knockdown of Raptor, an essential component of mTORC1. These results suggest that the knockdown of EBP2 triggered p53 activation through the Akt‐mTORC1 pathway in NPM‐ALK‐positive cells. Collectively, the present results revealed the critical repressive mechanism of p53 activity by EBP2 and provide a novel therapeutic strategy for the treatment of ALCL

Soluble form of the ST2 gene product exhibits growth promoting activity in NIH-3T3 cells

The ST2 gene is induced in murine fibroblast cells at the start of cell proliferation. Although IL-33 has been identified as a ligand for one of the two major gene products of ST2 – namely, the transmembrane receptor form ST2L – prompting immunological research on inflammation, the roles of the ST2 gene products in cell proliferation remain to be elucidated. Using a cell proliferation assay system with NIH-3T3 cells, a normal murine fibroblast cell line, we found that treatment with recombinant ST2 caused an acceleration of cell proliferation, suggesting that ST2 acts in an autocrine/paracrine fashion. Strikingly, shRNA-induced knockdown of both ST2 gene products, ST2 and ST2L, reduced cell proliferation. This effect was effectively canceled by the expression of shRNA-resistant ST2, but not shRNA-resistant ST2L. The novel enhancement of cell proliferation by ST2 appears to involve positive feedback. Since the ST2 level is increased in various diseases involving inflammation, future investigations into the role of ST2 gene products in relation to various diseases, including malignancies, may be warranted

Treatment with ODC inhibitor, DFMO, significantly inhibited tumorigenesis induced by JAK2 (V617F).

<p>Ba/F3 cells were infected with empty virus (-) and retrovirus encoding JAK2 V617F mutant and EpoR. Transduced Ba/F3 cells were s.c. injected into nude mice (1×10⁷ cells/mice) and then given either standard water (H₂O) or water containing 1% DFMO for 10 days. (A) Nude mice were photographed 16 days post-inoculation. Arrows indicate tumors in nude mice. (B, C) Sixteen days post-inoculation, three mice were sacrificed. Morphological changes of the spleen and liver are shown in the photograph. The weights of the tumor, liver, and spleen were measured and plotted in the graph. * and ** indicate significant differences p<0.05 and p<0.01, respectively. (D) Sixteen days after inoculation, sections of the liver were stained with H&E (magnification: ×100). (E) Mouse survival was monitored daily for 30 days post-inoculation (n = 10).</p

c-Myc T58A mutant conferred growth-factor independence on Ba/F3 cells.

<p>(A) Schematic diagram of wild-type (c-Myc), T58A and In373 mutants of c-Myc. (B-H) Ba/F3 cells were infected with empty virus (−) or retrovirus encoding wild-type c-Myc (c-Myc) or two c-Myc mutants (T58A, In373). (B) Transduced cells were incubated without IL-3 (2 ng/mL) for 6 hr and whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody or anti-β-actin antibody. (C, D) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 12 hr. (C) ODC mRNA was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (D) Whole cell lysates were immunoblotted (IB) with anti-ODC antibody or anti-β-actin antibody. (E) Viable transduced Ba/F3 cells and VF/EpoR cells were counted in the presence and absence of IL-3 (2 ng/mL) for 3 days. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (F) Transduced Ba/F3 cells were left untreated or stimulated with IL-3 (2 ng/mL) for 24 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (G) Transduced cells were cultured without IL-3 (2 ng/mL) for 24 hr. Cells were then fixed, treated with propidium iodide (PI) and subjected to FACS analysis. (H) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 24 hr. DNA was isolated from cells and subjected to agarose gel electrophoresis.</p

JAK2 (V617F) induced expression of c-Myc through STAT5 activation.

<p>(A–E) Ba/F3 cell lines were infected with empty virus (−), retroviruses encoding wild-type JAK2 c-HA (WT), JAK2 mutant c-HA (V617F) and EpoR c-Flag as indicated in each figure. (A) WT/EpoR cells and VF/EpoR cells were cultured without Epo for 12 hr, and then total RNAs were prepared from each cell line. The enhancement of gene expression induced by JAK2 (V617F) was determined by DNA array. The ratio of the altered gene expressions in VF/EpoR cells was calculated by dividing the amount of each gene in VF/EpoR cells by their amount in WT/EpoR cells. (B, C) A series of Ba/F3 cells expressing the described genes was cultured untreated or stimulated with Epo (5 U/mL) for 12 hr. (D, E) A series of Ba/F3 cells expressing the indicated genes were cultured in the absence of Epo, and treated with DMSO (0.1%) or AG490 (30 µM) for 12 hr. (F) HEL cells were treated with DMSO (0.1%) or AG490 (10, 20, 30 µM) for 24 hr. (G, H) VF/EpoR cells were infected with retrovirus harboring shRNA against firefly luciferase (control) or STAT5. (I, J) Ba/F3 cells were infected with empty virus (−), retrovirus encoding wild-type STAT5 (WT) or the constitutively active mutant of STAT5 (1*6). The cells were cultured without Epo for 12 hr. (B, E, H, J) The mRNA expression of c-Myc and ODC was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three experiments. (C, D, F, G, I) Whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody, anti-ODC antibody, anti-HA antibody, anti-EpoR antibody, anti-phospho-JAK2 antibody (Y1007/1008), anti-phospho-STAT5 antibody (Y694), anti-STAT5 antibody or anti-β-actin antibody.</p