Novel tubulin antagonist pretubulysin displays antivascular properties in vitro and in vivo.

OBJECTIVE: Pretubulysin (PT) is a novel, synthetically accessible myxobacterial compound that acts as a tubulin-depolymerizing agent and inhibits cancer cell growth in vitro and in vivo. Moreover, PT was found to attenuate tumor angiogenesis. Here, we hypothesized that PT could exert antivascular activities on existing tumor vessels. APPROACH AND RESULTS: We aimed to characterize the antivascular effects of PT and to elucidate the underlying mechanisms in endothelial cells. In vitro, PT rapidly induced endothelial hyperpermeability and a concentration-dependent disassembly of established endothelial tubes on Matrigel and in an ex vivo aortic ring model. It disrupted endothelial cell junctions and triggered F-actin stress fiber formation and cell contraction by the RhoA/Rho-associated protein kinase pathway without causing cell death. In vivo, using a hamster dorsal skinfold chamber preparation, PT significantly decreased blood flow and vessel diameter in hamster A-Mel-3 amelanotic melanoma tumors but not in the neighboring healthy tissue. In a second tumor model using mice with subcutaneous murine B16 melanoma tumors, a single dose of PT (10 mg/kg) caused a shut down of tumor blood flow and a strong central tumor cell necrosis within 24 hours. Repeated PT administration significantly decelerates tumor growth and seems to be well tolerated. CONCLUSIONS: In summary, we could show for the first time that the antitumor effect of PT is, at least in part, mediated via its antivascular activities on existing tumor vessels.

Chemo- and thermosensory responsiveness of Grueneberg ganglion neurons relies on cyclic guanosine monophosphate signaling elements.

Neurons of the Grueneberg ganglion (GG) in the anterior nasal region of mouse pups respond to cool temperatures and to a small set of odorants. While the thermosensory reactivity appears to be mediated by elements of a cyclic guanosine monophosphate (cGMP) cascade, the molecular mechanisms underlying the odor-induced responses are unclear. Since odor-responsive GG cells are endowed with elements of a cGMP pathway, specifically the transmembrane guanylyl cyclase subtype GC-G and the cyclic nucleotide-gated ion channel CNGA3, the possibility was explored whether these cGMP signaling elements may also be involved in chemosensory GG responses. Experiments with transgenic mice deficient for GC-G or CNGA3 revealed that GG responsiveness to given odorants was significantly diminished in these knockout animals. These findings suggest that a cGMP cascade may be important for both olfactory and thermosensory signaling in the GG. However, in contrast to the thermosensory reactivity, which did not decline over time, the chemosensory response underwent adaptation upon extended stimulation, suggesting that the two transduction processes only partially overlap.

Exploring Cytotoxic mRNAs as a Novel Class of Anti-cancer Biotherapeutics.

New treatments to overcome the obstacles of conventional anti-cancer therapy are a permanent subject of investigation. One promising approach is the application of toxins linked to cell-specific ligands, so-called immunotoxins. Another attractive option is the employment of toxin-encoding plasmids. However, immunotoxins cause hepatoxicity, and DNA therapeutics, among other disadvantages, bear the risk of insertional mutagenesis. As an alternative, this study examined chemically modified mRNAs coding for diphtheria toxin, subtilase cytotoxin, and abrin-a for their ability to reduce cancer cell growth both in vitro and in vivo. The plant toxin abrin-a was the most promising candidate among the three tested toxins and was further investigated. Its expression was demonstrated by western blot. Experiments with firefly luciferase in reticulocyte lysates and co-transfection experiments with EGFP demonstrated the capability of abrin-a to inhibit protein synthesis. Its cytotoxic effect was quantified employing viability assays and propidium iodide staining. By studying caspase-3/7 activation, Annexin V-binding, and chromatin condensation with Hoechst33258 staining, apoptotic cell death could be confirmed. In mice, repeated intratumoral injections of complexed abrin-a mRNA resulted in a significant reduction (89%) of KB tumor size compared to a non-translatable control mRNA.

Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA.

Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2) protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form. For this purpose, we advanced the latest RNA technology for liver- and lung-targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA) leads to robust translation of fully matured, membrane-anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles and in lipid nanoparticles led to liver- and lung-targeted translation of significant amounts of ACE2 protein, respectively. In summary, we provide evidence that RNA transcript therapy (RTT) is a promising approach for ACE2-based treatment of liver and lung fibrosis to be tested in fibrotic disease models.