Christoph P. Bagowski

Retinoic Acid Receptor Antagonists Inhibit miR-10a Expression and Block Metastatic Behavior of Pancreatic Cancer

BACKGROUND & AIMS The infiltrating ductal adenocarcinoma of the pancreas is among the most lethal of all solid malignancies, largely owing to a high frequency of early metastasis. We identified microRNA-10a (miR-10a) as an important mediator of metastasis formation in pancreatic tumor cells and investigated the upstream and downstream regulatory mechanisms of miR-10a. METHODS Northern blot analysis revealed increased expression levels of miR-10a in metastatic pancreatic adenocarcinoma. The role of miR-10a was analyzed by Morpholino and short interfering RNA transfection of pancreatic carcinoma cell lines and resected specimens of human pancreatic carcinoma. Metastatic behavior of primary pancreatic tumors and cancer cell lines was tested in xenotransplantation experiments in zebrafish embryos. RESULTS We show that miR-10a expression promotes metastatic behavior of pancreatic tumor cells and that repression of miR-10a is sufficient to inhibit invasion and metastasis formation. We further show that miR-10a is a retinoid acid target and that retinoic acid receptor antagonists effectively repress miR-10a expression and completely block metastasis. This antimetastatic activity can be prevented by specific knockdown of HOX genes, HOXB1 and HOXB3. Interestingly, suppression of HOXB1 and HOXB3 in pancreatic cancer cells is sufficient to promote metastasis formation. CONCLUSIONS These findings suggest that miR-10a is a key mediator of metastatic behavior in pancreatic cancer, which regulates metastasis via suppression of HOXB1 and HOXB3. Inhibition of miR-10a expression (with retinoic acid receptor antagonists) or function (with specific inhibitors) is a promising starting point for antimetastatic therapies.

Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model.

BackgroundAberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour.MethodsWe fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish.ResultsIn the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature.ConclusionOur results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen.

Iridium Complex with Antiangiogenic Properties

Substitutionally inert metal complexes are promising emerging scaffolds for targeting enzyme active sites. Over the last several years, our research group has demonstrated that inert ruthenium(II) complexes can serve as highly selective nanomolar and even picomolar inhibitors of protein kinases. Octahedral metal coordination geometries in particular offer new gateways to design rigid, globular molecules with defined shapes that can fill protein pockets such as enzyme active sites in a unique fashion (Figure 1). However, the

On the Biological Properties of Alkynyl Phosphine Gold(I) Complexes

Gold complexes have a long tradition in the treatment of the symptoms of rheumatoid arthritis. 2] Therapeutically used drugs include mainly gold(I) thiolates (e.g. aurothiomalate and auranofin), which belong to the group of diseasemodifying antirheumatic drugs (DMARDs) that are used to slow down or stop the progression of this severe and disabling rheumatic disorder. Interestingly, in vitro studies on cultured tumor cells have also indicated the considerable potential of this class of metallodrugs for tumor chemotherapy, and thioredoxin reductase is one of the enzymes identified as a critical target. Intensified research on the development of gold antitumor drugs has led to many active species such as gold(I) complexes with phosphine, thiolate, chloride, and carbene ligands as well as gold(III) derivatives. 10–12] However, a major issue in the development of new bioactive gold complexes is the preparation of complexes that show suitable stability under physiological conditions. Gold complexes with alkynyl ligands, which are widely used because of their catalytic and luminescent properties, might display reasonably stable coordinative bonds. In fact, recent initial reports on the bioactivity of alkynyl gold complexes indicate that this type of organometallic complex offers opportunities for the development of new chemotherapeutics against cancer and infectious diseases. Despite these prospectives, only three studies on the biological potential of alkynyl gold complexes have been reported so far. Here, we present the outcome of a pilot study aimed at establishing the biological profile of alkynyl phosphine gold(I) complexes. Our study shows that the critical target enzyme thioredoxin reductase can be efficiently and selectively inhibited and that cysteine and selenocysteine residues are presumably the sites of molecular interaction with the enzyme. Moreover, we quantified the cellular uptake of the complexes, established their effects on tumor cell metabolism and mitochondrial respiration, and investigated their antiangiogenic properties in zebrafish embryos. A series of six alkynyl gold(I) complexes (1–6, see Figure 1) was prepared by reacting the respective alkynes with chloro(triphenylphosphine)gold(I). The structures were confirmed by H, C, P NMR, and IR spectroscopy and

Modulation of the Biological Properties of Aspirin by Formation of a Bioorganometallic Derivative

Despite recent advances in modern tumor therapy the development of effective drugs remain a challenge for medicinal chemists. The demand for innovative agents triggers interest in novel chemical strategies and new concepts for modern drug design. The vast majority of drugs used to date are purely “organic” compounds. However, stimulated by the tremendous success of the inorganic compound cisplatin in modern tumor therapy, interest in the development of other metal complexes has been rapidly growing. Bioorganometallic chemistry is a novel emerging field in medicinal chemistry, which aims at probing the biological (and therapeutic) potential of organometallic compounds. As a result of their different coordination geometries, chemical properties, and reactivities, metal complexes offer a wide spectrum of functional groups more or less unexplored in modern drug design and development. The hexacarbonyldicobalt moiety Co2(CO)6 bound to an alkyne, is one such functional group, for which promising results on medical applications have been reported. For example, Co2(CO)6 derivatives of antiepileptic drugs (e.g. carbamazepine) were used as diagnostic tools in the so-called carbonyl metallo immuno assay (CMIA), and complexes with fructopyranose, nucleoside, and neuropeptide ligands displayed interesting bioactivities. We have recently reported on alkyne hexacarbonyldicobalt species with promising antiproliferative properties. Interestingly, the cell growth inhibitory activity of the complexes depended strongly on the chemical structure of the alkyne ligand. Weakly active and inactive derivatives showed that the cobalt cluster does not cause general (unspecific) cytotoxic effects. In further studies the Co2(CO)6 complex of the aspirin (o-acetylsalicylic acid, ASS) derivative prop-2-ynyl-2-acetoxybenzoate (Co-ASS) emerged as a lead compound for this class of antiproliferative agents.

Transcriptome analysis of the response to chronic constant hypoxia in zebrafish hearts

Insufficient blood supply during acute infarction and chronic ischemia leads to tissue hypoxia which can significantly alter gene expression patterns in the heart. In contrast to most mammals, some teleost fishes are able to adapt to extremely low oxygen levels. We describe here that chronic constant hypoxia (CCH) leads to a smaller ventricular outflow tract, reduced lacunae within the central ventricular cavity and around the trabeculae and an increase in the number of cardiac myocyte nuclei per area in the hearts of two teleost species, zebrafish (Danio rerio) and cichlids (Haplochromis piceatus). In order to identify the molecular basis for the adaptations to CCH, we profiled the gene expression changes in the hearts of adult zebrafish. We have analyzed over 15,000 different transcripts and found 376 differentially regulated genes, of which 260 genes showed increased and 116 genes decreased expression levels. Two notch receptors (notch-2 and notch-3) as well as regulatory genes linked to cell proliferation were transcriptionally upregulated in hypoxic hearts. We observed a simultaneous increase in expression of IGF-2 and IGFbp1 and upregulation of several genes important for the protection against reactive oxygen species (ROS). We have identified here many novel genes involved in the response to CCH in the heart, which may have potential clinical implications in the future.

Naphthalimide gold(I) phosphine complexes as anticancer metallodrugs

Gold(I) phosphine complexes exhibit promising properties for anticancer drug development. Here we report on a series of gold(I) phosphine complexes containing a naphthalimide ligand. Strong antiproliferative effects were observed in MCF-7 breast cancer cells as well as in HT-29 colon carcinoma cells. The cellular and nuclear gold levels were increased compared to analogues, in which the naphthalimide ligand was replaced by a chloro ligand. Compound 4a was selected for more detailed biochemical and biological studies, which revealed solvent dependent fluorescence emission, uptake of the compound into the organelles of tumor cells as well as antiangiogenic effects concerning angiogenesis and tumor-induced angiogenesis in vivo. Antiangiogenic properties of 4a were observed in two different zebrafish angiogenesis models, including a tumor-cell induced neovascularization assay.

Molecular evolution of the MAGUK family in metazoan genomes

BackgroundDevelopment, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.ResultsOur results indicate that at least four of the seven MAGUK subfamilies were present in early metazoan lineages, such as Porifera. We employed domain sequence and structure based methods to infer a model for the evolutionary history of the MAGUKs. Notably, the phylogenetic trees for the guanylate kinase (GK)-, the PDZ- and the SH3-domains all suggested a matching evolutionary model which was further supported by molecular modeling of the 3D structures of different GK domains. We found no MAGUK in plants, fungi or other unicellular organisms, which suggests that the MAGUK core structure originated early in metazoan history.ConclusionIn summary, we have characterized here the molecular and structural evolution of the large MAGUK family. Using the MAGUKs as an example, our results show that it is possible to derive a highly supported evolutionary model for important multidomain families by analyzing encoded protein domains. It further suggests that larger superfamilies encoded in the different genomes can be analyzed in a similar manner.

LIMK1 and LIMK2 are important for metastatic behavior and tumor cell-induced angiogenesis of pancreatic cancer cells.

The two LIM kinases (LIMKs) LIMK1 and LIMK2 are members of the PDZ/LIM family. These serine/threonine protein kinases are involved in actin cytoskeleton reorganization through phosphorylation and inactivation of ADF/cofilin. Different subcellular localizations of LIMK1 and LIMK2 suggest different functions. LIMK1 is implicated in microtubule disassembly in endothelial- and cancer cells, whereas LIMK2 plays a role in cell cycle progression. To compare the role of the two LIMKs in cancer-related processes, we used a cell-based in vitro migration assay, as well as two zebrafish xenograft assays. We analyzed here the metastatic behavior and tumor cell-induced neovascularization of pancreatic cancer cells in which both LIMK genes were silenced by siRNAs. Both LIMK1 and LIMK2 single knock down led to a reduction of invasion and metastatic behavior in the zebrafish xenograft metastasis assay. Interestingly, the double knock down completely blocked invasion and formation of micrometastasis in vivo. Moreover, in the zebrafish xenograft angiogenesis assay, we observed a reduction of pancreatic cancer cell-induced angiogenesis for both the LIMK1 and LIMK2 knockdowns. Our results demonstrate similar functions for the two LIMKs in pancreatic cancer cells and suggest an important role for both LIMK1 and LIMK2 in tumor progression and metastasis formation.

Insights into the molecular evolution of the PDZ/LIM family and identification of a novel conserved protein motif.

The PDZ and LIM domain-containing protein family is encoded by a diverse group of genes whose phylogeny has currently not been analyzed. In mammals, ten genes are found that encode both a PDZ- and one or several LIM-domains. These genes are: ALP, RIL, Elfin (CLP36), Mystique, Enigma (LMP-1), Enigma homologue (ENH), ZASP (Cypher, Oracle), LMO7 and the two LIM domain kinases (LIMK1 and LIMK2). As conventional alignment and phylogenetic procedures of full-length sequences fell short of elucidating the evolutionary history of these genes, we started to analyze the PDZ and LIM domain sequences themselves. Using information from most sequenced eukaryotic lineages, our phylogenetic analysis is based on full-length cDNA-, EST-derived- and genomic- PDZ and LIM domain sequences of over 25 species, ranging from yeast to humans. Plant and protozoan homologs were not found. Our phylogenetic analysis identifies a number of domain duplication and rearrangement events, and shows a single convergent event during evolution of the PDZ/LIM family. Further, we describe the separation of the ALP and Enigma subfamilies in lower vertebrates and identify a novel consensus motif, which we call ‘ALP-like motif’ (AM). This motif is highly-conserved between ALP subfamily proteins of diverse organisms. We used here a combinatorial approach to define the relation of the PDZ and LIM domain encoding genes and to reconstruct their phylogeny. This analysis allowed us to classify the PDZ/LIM family and to suggest a meaningful model for the molecular evolution of the diverse gene architectures found in this multi-domain family.