Patrick Thurner

Human Anti-Macrophage Migration Inhibitory Factor Antibodies Inhibit Growth of Human Prostate Cancer Cells In Vitro and In Vivo

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biologic properties in immunology and oncology. Circulating MIF levels are elevated in several types of human cancer including prostate cancer. MIF is released presumably by both stromal and tumor cells and enhances malignant growth and metastasis by diverse mechanisms, such as stimulating tumor cell proliferation, suppressing apoptotic death, facilitating invasion of the extracellular matrix, and promoting angiogenesis. Recently described fully human anti-MIF antibodies were tested in vitro and in vivo for their ability to influence growth rate and invasion of the human PC3 prostate cancer cell line. In vitro, the selected candidate antibodies BaxG03, BaxB01, and BaxM159 reduced cell growth and viability by inhibiting MIF-induced phosphorylation of the central kinases p44/42 mitogen-activated protein kinase [extracellular signal–regulated kinase-1 and -2 (ERK1/2)] and protein kinase B (AKT). Incubation of cells in the presence of the antibodies also promoted activation of caspase-3/7. The antibodies furthermore inhibited MIF-promoted invasion and chemotaxis as transmigration through Matrigel along a MIF gradient was impaired. In vivo, pharmacokinetic parameters (half-life, volume of distribution, and bioavailability) of the antibodies were determined and a proof-of-concept was obtained in a PC3-xenograft mouse model. Treatment with human anti-MIF antibodies blunted xenograft tumor growth in a dose-dependent manner. We therefore conclude that the anti-MIF antibodies described neutralize some of the key tumor-promoting activities of MIF and thus limit tumor growth in vivo. Mol Cancer Ther; 12(7); 1223–34. ©2013 AACR.

Recruitment of a cytoplasmic chaperone relay by the A2A-adenosine receptor.

Background: The A2A receptor is known to accumulate in the endoplasmic reticulum. Results: Mass spectrometry identified molecular chaperones (HSP90 and HSP70) bound to the A2A receptor. Conclusion: Sequential recruitment of chaperones to the cytosolic face of the A2A receptor is consistent with a heat-shock protein relay assisting folding. Significance: The observations are consistent with a chaperone/COPII exchange model, where heat-shock proteins bound to the receptor preclude its premature ER export. The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.

A Two-state Model for the Diffusion of the A2A Adenosine Receptor in Hippocampal Neurons: AGONIST-INDUCED SWITCH TO SLOW MOBILITY IS MODIFIED BY SYNAPSE-ASSOCIATED PROTEIN 102 (SAP102)*

Background: Agonist activation slows diffusion of the A2A receptor in the lipid bilayer. Results: In hippocampal neurons, the agonist-induced decrease in mobility was accounted for by both the hydrophobic receptor core and its extended C terminus, which recruited SAP102. Conclusion: The observations are consistent with two diffusion states of the A2A receptor in neurons. Significance: SAP102 regulates access of the A2A receptor to a compartment with restricted mobility. The A2A receptor is a class A/rhodopsin-like G protein-coupled receptor. Coupling to its cognate protein, Gs, occurs via restricted collision coupling and is contingent on the presence of cholesterol. Agonist activation slows diffusion of the A2A adenosine receptor in the lipid bilayer. We explored the contribution of the hydrophobic core and of the extended C terminus by examining diffusion of quantum dot-labeled receptor variants in dissociated hippocampal neurons. Single particle tracking of the A2A receptor(1–311), which lacks the last 101 residues, revealed that agonist-induced confinement was abolished and that the agonist-induced decrease in diffusivity was reduced substantially. A fragment comprising the SH3 domain and the guanylate kinase domain of synapse-associated protein 102 (SAP102) was identified as a candidate interactor that bound to the A2A receptor C terminus. Complex formation between the A2A receptor and SAP102 was verified by coimmunoprecipitation and by tracking its impact on receptor diffusion. An analysis of all trajectories by a hidden Markov model was consistent with two diffusion states where agonist activation reduced the transition between the two states and, thus, promoted the accumulation of the A2A receptor in the compartment with slow mobility. Overexpression of SAP102 precluded the access of the A2A receptor to a compartment with restricted mobility. In contrast, a mutated A2A receptor (with 383DVELL387 replaced by RVRAA) was insensitive to the action of SAP102. These observations show that the hydrophobic core per se does not fully account for the agonist-promoted change in mobility of the A2A receptor. The extended carboxyl terminus allows for regulatory input by scaffolding molecules such as SAP102.

Mechanism of hERG channel block by the psychoactive indole alkaloid ibogaine

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go–related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells. Currents through hERG channels were blocked regardless of whether ibogaine was applied via the extracellular or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during activation of the channels and was not observed for resting channels. With increasing depolarizations, ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, whereas inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, these findings show that ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.

Respiratory motion artifacts during arterial phase imaging with gadoxetic acid: Can the injection protocol minimize this drawback?

To determine which of three gadoxetic acid injection techniques best reduced the contrast‐related arterial‐phase motion artifacts.

Reengineering the collision coupling and diffusion mode of the A2A-adenosine receptor: palmitoylation in helix 8 relieves confinement

Background: The A2A receptor engages Gs by restricted collision coupling and lacks a palmitoyl moiety in its C terminus. Results: Engineering palmitoylated cysteine into the C terminus relieved restricted collision coupling and resulted in accelerated diffusion of the agonist-liganded A2A receptor. Conclusion: Restricted collision coupling arises from limits imposed on receptor diffusion. Significance: Agonist induced confinement of the A2A receptor in a structure consistent with a lipid raft. The A2A-adenosine receptor undergoes restricted collision coupling with its cognate G protein Gs and lacks a palmitoylation site at the end of helix 8 in its intracellular C terminus. We explored the hypothesis that there was a causal link between the absence of a palmitoyl moiety and restricted collision coupling by introducing a palmitoylation site. The resulting mutant A2A-R309C receptor underwent palmitoylation as verified by both mass spectrometry and metabolic labeling. In contrast to the wild type A2A receptor, the concentration-response curve for agonist-induced cAMP accumulation was shifted to the left with increasing expression levels of A2A-R309C receptor, an observation consistent with collision coupling. Single particle tracking of quantum dot-labeled receptors confirmed that wild type and mutant A2A receptor differed in diffusivity and diffusion mode; agonist activation resulted in a decline in mean square displacement of both receptors, but the drop was substantially more pronounced for the wild type receptor. In addition, in the agonist-bound state, the wild type receptor was frequently subject to confinement events (estimated radius 110 nm). These were rarely seen with the palmitoylated A2A-R309C receptor, the preferred diffusion mode of which was a random walk in both the basal and the agonist-activated state. Taken together, the observations link restricted collision coupling to diffusion limits imposed by the absence of a palmitoyl moiety in the C terminus of the A2A receptor. The experiments allowed for visualizing local confinement of an agonist-activated G protein-coupled receptor in an area consistent with the dimensions of a lipid raft.

Selective serotonin reuptake inhibitors induce cell death via the unfolded protein response

Selective serotonin reuptake inhibitors (SSRIs) have been observed to drive programmed cell death in Burkitt lymphoma cells. Further studies, however, showed that SSRIs induce apoptosis with little, if any, appreciable selectivity. Actually, the selectivity appears to be so low that SSRIs can kill protozoa such as Trichomonas vaginalis. Although the serotonin transporter SERT was initially considered as SSRI target in inducing programmed cell death, this concept has been rejected repeatedly. N-acetylated versions of SSRIs, which are not capable of inhibiting serotonin reuptake anymore, were shown to kill cells in concentration ranges comparable to those of their non-acetylated original versions. Our working hypothesis is that SSRIs induce cell death via activation of the endoplasmic reticulum stress response/unfolded protein response (UPR). To address this hypothesis, we performed a luciferase reporter assay (in HEK293 and HeLa cells), in which the firefly luciferase gene is under the control of the promoter for glucose-regulated protein of 78 kD (GRP78), an endoplasmic reticulum (ER) chaperone mainly expressed during the UPR. Several SSRIs (paroxetine, fluvoxamine, fluoxetine and citalopram) and their N-acetylated versions induce GRP78 expression with steep concentration-response curves, comparable to those obtained for killing cells. SSRIs also trigger activation of caspase 3/7, as observed with a caspase 3/7-dependent fluorescent substrate, and expression of the endoplasmic reticulum stress protein C/EBP-homologous protein (CHOP-10). Our results suggest that SSRIs induce cell death via the UPR, and further experiments are designed to strengthen our hypothesis. We will also investigate, if this effect is conserved throughout species (e.g. in Trichomonas vaginalis).

Differentiation of Intrahepatic Cholangiocellular Carcinoma from Hepatocellular Carcinoma in the Cirrhotic Liver Using Contrast-enhanced MR Imaging

RATIONALE AND OBJECTIVES This study aimed to investigate the potential of contrast-enhanced magnetic resonance imaging features to differentiate between mass-forming intrahepatic cholangiocellular carcinoma (ICC) and hepatocellular carcinoma (HCC) in cirrhotic livers. MATERIALS AND METHODS This study, performed between 2001 and 2013, included 64 baseline magnetic resonance imaging examinations with pathohistologically proven liver cirrhosis, presenting with either ICC (n = 32) or HCC (n = 32) tumors. To distinguish ICC form HCC tumors, 20 qualitative single-lesion descriptors were evaluated by two readers, in consensus, and statistically classified using the chi-square automatic interaction detection (CHAID) methodology. Diagnostic performance was assessed by a receiver operating characteristic analysis. RESULTS The CHAID algorithm identified three independent categorical lesion descriptors, including (1) liver capsular retraction; (2) progressive or persistent enhancement pattern or wash-out on the T1-weighted delayed phase; and (3) signal intensity appearance on T2-weighted images that could help to reliably differentiate ICC from HCC, which resulted in an AUC of 0.807, and a sensitivity and specificity of 68.8 and 90.6 (95% confidence interval 75.0-98.0), respectively. CONCLUSIONS The proposed CHAID algorithm provides a simple and robust step-by-step classification tool for a reliable and solid differentiation between ICC and HCC tumors in cirrhotic livers.

Restricted collision coupling of the adenosine A2A receptor is due to its agonist-induced confinement in the membrane

Background The A2A adenosine receptor is of interest because of several reasons. (i) It is a frequently blocked pharmacological target, because it is the site of action of caffeine. (ii) It has a long C-terminus that provides a docking site for several proteins, which direct the fate of the receptor from its synthesis to its lysosomal degradation. (iii) The A2A receptor can only promote activation of a limited number of available Gs molecules. This coupling mode was termed restricted collision coupling. (iv) Most G protein-coupled receptors carry one or several cysteine residues in their C-terminus which is subject to palmitoylation to anchor and stabilize the amphipathic helix 8; the A2A receptor lacks this palmitoylation site. We explored the hypothesis that there is a causal link between the absence of a palmitoyl moiety and restricted collision coupling.

Tracking the A2A adenosine receptor

Background The A2A adenosine receptor has become a drug target in the treatment of Parkinson’s disease, psychotic behavior and dementia. In addition, targeted deletion of this receptor in mice leads to hypertension, increased platelet aggregation, male aggressiveness and decreased susceptibility to ischemic brain damage. The potential clinical relevance of this receptor is obvious. The A2A adenosine receptor, a prototypical GPCR, is known to signal via restricted collision coupling with Gs. In addition, it is able to stimulate MAP kinase/ERK in a Gs-independent way but dependent on the lipid microenvironment of the membrane. Hence, we characterized the mobility and the targeting of the A2A receptor in nerve cells.