Markus Klinger

Adenosine receptors: G protein-mediated signalling and the role of accessory proteins.

Ever since the discovery of the effects of adenosine in the circulation, adenosine receptors continue to represent a promising drug target. Firstly, this is due to the fact that the receptors are expressed in a large variety of cells; in particular, the actions of adenosine (or, respectively, of the antagonistic methylxanthines) in the central nervous system, in the circulation, on immune cells and on other tissues can be beneficial in certain disorders. Secondly, there exists a large number of ligands, which have been generated by introducing several modifications in the structure of the lead compounds (adenosine and methylxanthine), some of them highly specific. Four adenosine receptor subtypes have been identified by molecular cloning; they belong to the family of G protein-coupled receptors, which transfer signals by activating heterotrimeric G proteins. It has been appreciated recently that accessory proteins impinge on the receptor/G protein interaction and thus modulate the signalling reaction. These accessory components may be thought as adaptors that redirect the signalling pathway to elicit a cell-specific response. Here, we review the recent literature on adenosine receptors and place a focus on the role of accessory proteins in the organisation of adenosine receptor signalling. These components have been involved in receptor sorting, in the control of signal amplification and in the temporal regulation of receptor activity, while the existence of others is postulated on the basis of atypical cellular reactions elicited by receptor activation.

Bevacizumab protects against sinusoidal obstruction syndrome and does not increase response rate in neoadjuvant XELOX/FOLFOX therapy of colorectal cancer liver metastases

AIM In patients suffering from colorectal cancer liver metastases, 5-fluorouracil-based chemotherapy plus oxaliplatin ensures superior response rates at the cost of hepatic injury. Knowledge about the consequences of bevacizumab on chemotherapy-induced hepatic injury and tumor response is limited. METHODS Resected liver specimens from patients of two prospective, non-randomized trials (5-fluorouracil/oxaliplatin+/-bevacizumab) were analyzed retrospectively. Hepatotoxicity to the non-tumor bearing liver was evaluated for sinusoidal obstruction syndrome, hepatic steatosis and fibrosis. Tumor response under chemotherapy was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS Bevacizumab decreased the severity of the sinusoidal obstruction syndrome. Bevacizumab had no impact on hepatic steatosis and fibrosis. The addition of bevacizumab to chemotherapy had no effect on tumor response compared to combination chemotherapy alone. CONCLUSIONS This analysis shows that bevacizumab protects against the sinusoidal obstruction syndrome and thus provides the histological explanation of the safe use of bevacizumab prior to liver resection. Furthermore, we show that bevacizumab does not improve tumor response according to RECIST.

Activation of Mitogen-activated Protein Kinase by the A2A-adenosine Receptor via a rap1-dependent and via a p21ras-dependent Pathway

The A2A-adenosine receptor, a prototypical Gs-coupled receptor, activates mitogen-activated protein (MAP) kinase in a manner independent of cAMP in primary human endothelial cells. In order to delineate signaling pathways that link the receptor to the regulation of MAP kinase, the human A2A receptor was heterologously expressed in Chinese hamster ovary (CHO) and HEK293 cells. In both cell lines, A2A agonist-mediated cAMP accumulation was accompanied by activation of the small G protein rap1. However, rap1 mediates A2A receptor-dependent activation of MAP kinase only in CHO cells, the signaling cascade being composed of Gs, adenylyl cyclase, rap1, and the p68 isoform of B-raf. This isoform was absent in HEK293 cells. Contrary to CHO cells, in HEK293 cells activation of MAP kinase by A2A agonists was not mimicked by 8-bromo-cAMP, was independent of Gαs, and was associated with activation of p21 ras . Accordingly, overexpression of the inactive S17N mutant of p21 ras and of a dominant negative version of mSos (the exchange factor of p21 ras ) blocked MAP kinase stimulation by the A2A receptor in HEK 293 but not in CHO cells. In spite of the close homology between p21 ras and rap1, the S17N mutant of rap1 was not dominant negative because (i) overexpression of rap1(S17N) failed to inhibit A2A receptor-dependent MAP kinase activation, (ii) rap1(S17N) was recovered in the active form with a GST fusion protein comprising the rap1-binding domain of ralGDS after A2A receptor activation, and (iii) A2A agonists promoted the association of rap1(S17N) with the 68-kDa isoform of B-raf in CHO cells. We conclude that the A2A receptor has the capacity two activate MAP kinase via at least two signaling pathways, which depend on two distinct small G proteins, namely p21 ras and rap1. Our observations also show that the S17N version of rap1 cannot be assumed a priori to act as a dominant negative interfering mutant.

Generation of Peptide Mimics of the Epitope Recognized by Trastuzumab on the Oncogenic Protein Her-2/neu

Immunizations with the oncogenic protein Her-2/neu elicit Abs exerting diverse biological effects--depending on epitope specificity, tumor growth may be inhibited or enhanced. Trastuzumab (herceptin) is a growth-inhibitory humanized monoclonal anti-Her-2/neu Ab, currently used for passive immunotherapy in the treatment of breast cancer. However, Ab therapies are expensive and have to be repeatedly administered for long periods of time. In contrast, active immunizations produce ongoing immune responses. Therefore, the study aims to generate peptide mimics of the epitope recognized by trastuzumab for vaccine formulation, ensuring the subsequent induction of tumor growth inhibitory Abs. We used the phage display technique to generate epitope mimics, mimotopes, complementing the screening Ab trastuzumab. Five candidate mimotopes were isolated from a constrained 10 mer library. These peptides were specifically recognized by trastuzumab, and showed distinctive mimicry with Her-2/neu in two experimental setups. Subsequently, immunogenicity of a selected mimotope was examined in BALB/c mice. Immunizations with a synthetic mimotope conjugated to tetanus toxoid resulted in Abs recognizing Her-2/neu in a blotted cell lysate as well as on the SK-BR-3 cell surface. Analogous to trastuzumab, the induced Abs caused internalization of the receptor from the cell surface to endosomal vesicles. These results indicate that the selected mimotopes are suitable for formulation of a breast cancer vaccine because the resulting Abs show similar biological features as trastuzumab.

MAP Kinase Stimulation by cAMP Does Not Require RAP1 but SRC Family Kinases

The small G protein RAP1 and the kinase B-RAF have been proposed to link elevations of cAMP to activation of ERK/mitogen-activated protein (MAP) kinase. In order to delineate signaling pathways that link receptor-generated cAMP to the activation of MAP kinase, the human A2A-adenosine receptor, a prototypical Gs-coupled receptor, was heterologously expressed in Chinese hamster ovary cells (referred as CHO-A2A cells). In CHO-A2A cells, the stimulation of the A2A-receptor resulted in an activation of RAP1 and formation of RAP1-B-RAF complexes. However, overexpression of a RAP1 GTPase-activating protein (RAP1GAP), which efficiently clamped cellular RAP1 in the inactive GDP-bound form, did not affect A2A-agonist-mediated MAP kinase stimulation. In contrast, the inhibitor of protein kinase A H89 efficiently suppressed A2A-agonist-mediated MAP kinase stimulation. Neither dynamin-dependent receptor internalization nor receptor-promoted shedding of matrix-bound growth factors accounted for A2A-receptor-dependent MAP kinase activation. PP1, an inhibitor of SRC family kinases, blunted both the A2A-receptor- and the forskolin-induced MAP kinase stimulation (IC50 = 50 nm); this was also seen in PC12 cells, which express the A2A-receptor endogenously, and in NIH3T3 fibroblasts, in which cAMP causes MAP kinase stimulation. In the corresponding murine fibroblast cell line SYF, which lacks the ubiquitously expressed SRC family kinases SRC, YES, and FYN, forskolin barely stimulated MAP kinase; this reduction was reversed in cells in which c-SRC had been reintroduced. These findings show that activation of MAP kinase by cAMP requires a SRC family kinase that lies downstream of protein kinase A. A role for RAP1, as documented for the β2-adrenergic receptor, is apparently contingent on receptor endocytosis.

The human D2 dopamine receptor synergizes with the A2A adenosine receptor to stimulate adenylyl cyclase in PC12 cells

The adenosine A2A receptor and the dopamine D2 receptor are prototypically coupled to Gs and Gi/Go, respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor–G protein coupling. We tested this model in PC12 cells which endogenously express A2A receptors. The human D2 receptor was introduced into PC12 cells by stable transfection. A2A-agonist-mediated inhibition of D2 agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D2 agonist quinpirole depended on the expression level of the D2 receptor: at low and high receptor levels, the A2A-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A2A-receptor-mediated cAMP formation was inhibited by other Gi/Go-coupled receptors that were either endogenously present (P2y12-like receptor for ADP) or stably expressed after transfection (A1 adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca2+ channels were inhibited by the endogenous P2Y receptor and by the heterologously expressed A1 receptor but not by the D2 receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D2 and A2A receptors are closely associated, but they highlight the fact that this interaction can also support synergism.

Liver resection remains a safe procedure after neoadjuvant chemotherapy including bevacizumab: a case-controlled study.

Objective:This study was conducted to analyze if the combination of Bevacizumab with standard chemotherapy increases postoperative morbidity and mortality after resection of colorectal liver metastases as compared with resection after chemotherapy alone. Parameters contributing to an increased morbidity were evaluated. Summary Background Data:Most patients referred for colorectal liver metastases are treated with neoadjuvant chemotherapy before hepatic surgery. Targeted agents like the vascular endothelial growth factor—antagonist Bevacizumab are increasingly added to standard therapy to prolong survival; however, little is known about the consequences of this policy in the perioperative period. Methods:One hundred-two patients treated between 2005 and 2009, who received neoadjuvant chemotherapy combined with Bevacizumab (CHT + B) were identified. A cohort of 112 patients treated without chemotherapy alone before resection served as the control group (CHT). Complications were graded within an established staging system and the therapeutic consequences were laid down. Uni- and multivariate analysis of factors contributing to postoperative complications in the CHT + B group was performed using a logistic regression model. Results:Postoperative complications occurred in 45 (44%, CHT + B) and 38 (34%, CHT) patients, respectively (P = 0.216). The incidence of severe complications requiring surgical or radiologic intervention or leading to organ failure was 10.8% in the CHT + B group and 7.1% in the CHT group (P = 0.350). Increased age, low serum albumin, resection of more than 3 liver segments and synchronous bowel procedures requiring an anastomosis were associated with an increased morbidity rate in the multivariate regression analysis. No patient died in either group. Conclusions:The addition of Bevacizumab to standard chemotherapy before resection of colorectal liver metastases does not seem to increase postoperative morbidity. Caution should be given to extended resections >3 liver segments and synchronous bowel anastomoses.

Simvastatin decreases free radicals formation in the human abdominal aortic aneurysm wall via NF-κB.

OBJECTIVES Statins have been reported to suppress the progression of abdominal aortic aneurysm (AAA). However, the effects of statins on inflammatory processes and free radicals generation are poorly understood. METHODS Wall samples from 51 patients (simvastatin patients, n = 34; non-statin patients, n = 17; matched by sex, age and aneurysm size) subjected to elective open AAA repair were analysed. We examined the effects of simvastatin on lipid peroxidation (4-hydroxy-trans-2-nonenal (4-HNE)), hydrogen peroxide (H(2)O(2)), tumour necrosis factor alpha (TNF-α) concentration, superoxide dismutase (SOD) and catalase (CAT) activity as well as nuclear factor kappa B (NF-κB) pathway activation in human AAA wall samples. RESULTS Treatment with simvastatin resulted in a decrease in 4-HNE and TNF-α concentration (median 4.18 μg/mg protein vs. 4.75, p = 0.012; median 10.33 pg/ml vs. 11.81, p = 0.026, respectively). CAT activity was higher in the simvastatin group (median 3.98 U ml vs. 3.19, p = 0.023). NF-κB expression was lower (p = 0.018) in the simvastatin group. However, simvastatin had little effect on H(2)O(2) concentration (p = 0.832) and SOD activity (p = 0.401). CONCLUSION Simvastatin inhibits free radicals and TNF-α generation and improves antioxidant capacity of human AAA wall tissue, possibly through the suppression of NF-κB activity. This may be one possible explanation how statins can inhibit AAA oxidative stress.

Inhibition of Xenograft Tumor Growth and Down-Regulation of ErbB Receptors by an Antibody Directed against Lewis Y Antigen

The blood group-related Lewis Y antigen is expressed on the majority of human cancers of epithelial origin with only limited expression on normal tissue. Therefore, the Lewis Y antigen represents an interesting candidate for antibody-based treatment strategies. Previous experiments showed that the humanized Lewis Y-specific monoclonal antibody, IGN311, reduced ErbB-receptor-mediated stimulation of mitogen-activated protein kinase by altering receptor recycling. Here, we tested whether binding of IGN311 to growth factor receptors is relevant also to inhibition of tumor growth in vivo. Prolonged incubation with IGN311 of human tumor cell lines, which express high levels of ErbB1 (A431) or ErbB2 (SK-BR-3), resulted in down-regulation of the receptors and inhibition of cell proliferation. IGN311 inhibited the growth of tumors derived from A431 cells xenografted in nude mice. Treatment with IGN311 was associated with a down-regulation of ErbB1 in the excised tumor tissue. Importantly, these effects of IGN311 were also mimicked by the Fab fragment of IGN311. These data indicate that tumor cell growth inhibition by IGN311 cannot solely be accounted for by invoking cellular and humoral immunological mechanisms. A direct effect on signaling via binding to Lewis Y glycosylated growth factor receptors on tumor cells is also likely to contribute to the therapeutic effect of IGN311 in vivo.

Decreased Tissue Levels of Cyclophilin A, a Cyclosporine A Target and Phospho-ERK1/2 in Simvastatin Patients with Abdominal Aortic Aneurysm

BACKGROUND Cyclophilin A (CyPA), a cyclosporine A-binding protein, influences abdominal aortic aneurysm (AAA) formation and the ERK1/2 signalling pathway in animal and in vitro studies. Statins decrease CyPA in smooth muscle cells although their influence on CyPA in human AAA is unknown. MATERIAL AND METHODS The study was performed on AAA wall-tissue samples obtained from 30 simvastatin-treated and 15 non-statin patients (2:1 case to control). The patients were matched by age, sex and AAA diameter. We investigated the gene expression of CyPA, its receptor extracellular matrix metalloproteinase inducer (EMMPRIN) by real-time RT-PCR. CyPA and EMMPRIN protein level and phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured by Western blot. RESULTS The AAA wall tissue from simvastatin-treated patients had significantly lower CyPA gene expression and protein levels (P = 0.0018, P = 0.0083, respectively). Furthermore, phosphorylation of ERK1 and ERK2 was markedly suppressed in the simvastatin group (P = 0.0002, P = 0.0027, respectively). However, simvastatin did not influence EMMPRIN gene and protein expression. CONCLUSION Simvastatin-treated patients with AAA exert lower CyPA messenger RNA (mRNA), as well as CyPA intracellular protein levels and a decreased amount of phospho-ERK1/2. Thus, the interference with signalling pathways leading to CyPA formation and ERK1/2 activation reveals a new anti-inflammatory role of statins in AAA.