The Src inhibitor AZD0530 blocks invasion and may act as a radiosensitizer in lung cancer cells

BACKGROUND: With the emergence of Src inhibitors in clinical trials, improved knowledge of the molecular responses of cancer cells to these agents is warranted. This will facilitate the development of tests to identify patients who may benefit from these agents, allow drug activity to be monitored and rationalize the combination of these agents with other treatment modalities. METHODS: This study evaluated the molecular and functional effects of Src inhibitor AZD0530 in human lung cancer cells, by Western blotting and reverse transcription-polymerase chain reaction, and by assays for cell viability, migration, and invasion. RESULTS: Src was activated in four of five cell lines tested and the level corresponded with the invasive potential and the histologic subtype. Clinically relevant, submicromolar concentrations of AZD0530 blocked Src and focal adhesion kinase, resulting in significant inhibition of cell migration and Matrigel invasion. Reactivation of STAT3 and up-regulation of JAK indicated a potential mechanism of resistance. AZD0530 gave a potent and sustained blockage of AKT and enhanced the sensitivity to irradiation. CONCLUSIONS: The results indicated that AZD0530, aside from being a potent inhibitor of tumor cell invasion which could translate to inhibition of disease progression in the clinic, may also lower resistance of lung cancer cells to pro-apoptotic signals.

Cutting edge: Natalizumab blocks adhesion but not initial contact of human T cells to the blood-brain barrier in vivo in an animal model of multiple sclerosis

The humanized anti-alpha(4) integrin Ab Natalizumab is an effective treatment for relapsing-remitting multiple sclerosis. Natalizumab is thought to exert its therapeutic efficacy by blocking the alpha(4) integrin-mediated binding of circulating immune cells to the blood-brain barrier (BBB). As alpha(4) integrins control other immunological processes, natalizumab may, however, execute its beneficial effects elsewhere. By means of intravital microscopy we demonstrate that natalizumab specifically inhibits the firm adhesion but not the rolling or capture of human T cells on the inflamed BBB in mice with acute experimental autoimmune encephalomyelitis (EAE). The efficiency of natalizumab to block T cell adhesion to the inflamed BBB was found to be more effective in EAE than in acute systemic TNF-alpha-induced inflammation. Our data demonstrate that alpha(4) integrin-mediated adhesion of human T cells to the inflamed BBB during EAE is efficiently blocked by natalizumab and thus provide the first direct in vivo proof of concept of this therapy in multiple sclerosis.

Brain electric microstates and momentary conscious mind states as building blocks of spontaneous thinking: I. Visual imagery and abstract thoughts

Prompted reports of recall of spontaneous, conscious experiences were collected in a no-input, no-task, no-response paradigm (30 random prompts to each of 13 healthy volunteers). The mentation reports were classified into visual imagery and abstract thought. Spontaneous 19-channel brain electric activity (EEG) was continuously recorded, viewed as series of momentary spatial distributions (maps) of the brain electric field and segmented into microstates, i.e. into time segments characterized by quasi-stable landscapes of potential distribution maps which showed varying durations in the sub-second range. Microstate segmentation used a data-driven strategy. Different microstates, i.e. different brain electric landscapes must have been generated by activity of different neural assemblies and therefore are hypothesized to constitute different functions. The two types of reported experiences were associated with significantly different microstates (mean duration 121 ms) immediately preceding the prompts; these microstates showed, across subjects, for abstract thought (compared to visual imagery) a shift of the electric gravity center to the left and a clockwise rotation of the field axis. Contrariwise, the microstates 2 s before the prompt did not differ between the two types of experiences. The results support the hypothesis that different microstates of the brain as recognized in its electric field implement different conscious, reportable mind states, i.e. different classes (types) of thoughts (mentations); thus, the microstates might be candidates for the `atoms of thought'.

Directed Metalation Cascade To Access Highly Functionalized Thieno2,3-fbenzofuran and Exploration as Building Blocks for Organic Electronics

A tandem directed metalation has been successfully applied to the preparation of thieno2,3-fbenzofuran-4,8-dione, providing an efficient and facile approach to symmetrically and unsymmetrically functionalize the thieno2,3-fbenzofuran core at the 2,6 positions as well as to introduce the electron-withdrawing or -donating groups (EWG or EDG) at its 4,8 positions. The presence of various functional groups makes late-stage derivatization attainable.

Inhibition of Deubiquitinase Activity and Ubiquitination of Jak2 Blocks Cytokine Signaling and Induces Tumor Cell Apoptosis

The Jak-stat pathway is critical for cellular proliferation and is commonly found to be deregulated in many solid tumors as well as hematological malignancies. Such findings have spurred the development of novel therapeutic agents that specifically inhibit Jak2 kinase, thereby suppressing tumor cell growth. Tyrphostin AG490, the first described Jak2 inhibitor, displays poor pharmacology and requires high concentrations for anti-tumor activities. Our research group screened a small library of AG490 structural analogues and identified WP1130 as a potent inhibitor of Jak2 signaling. However, unlike AG490, WP1130 did not directly inhibit Jak2 kinase activity. Our results show that WP1130 induces rapid ubiquitination and subsequent re-localization of Jak2 into signaling incompetent aggresomes. In addition to Jak2, WP1130 also induces accumulation of other ubiquitinated proteins without inhibiting 20S proteasome activity. Further analysis of the mechanism of action of WP1130 revealed that WP1130 acts as a partly selective DUB inhibitor. It specifically inhibits the deubiquitinase activity of USP9x, USP5, USP14 and UCH37. WP1130 mediated inhibition of tumor-associated DUBs resulted in down-regulation of anti-apoptotic and up-regulation of pro-apoptotic proteins, such as MCL-1 and p53 respectively. Our results demonstrate that chemical modification of a previously described Jak2 inhibitor results in the unexpected discovery of a novel compound which acts as a DUB inhibitor, suppressing Jak-Stat signaling by a novel mechanism.

Up-Scaling Sustainable Pro-Poor Energy Solutions: Addressing Stumbling Blocks

The session aims at analyzing efforts in up-scaling cleaner and more efficient energy solutions for poor people in developing countries by addressing the following questions: What are factors along the whole value chain and in the institutional, social, but also environmental space that enable up-scaling of improved pro-poor technologies? Are there differences between energy carriers or in different contexts? What are most promising entry points for up-scaling?

Constitutive notch signaling in adult transgenic mice inhibits bFGF-induced angiogenesis and blocks ovarian follicle development

Notch signaling is important in angiogenesis during embryonic development. However, the embryonic lethal phenotypes of knock-out and transgenic mice have precluded studies of the role of Notch post-natally. To develop a mouse model that would bypass the embryonic lethal phenotype and investigate the possible role of Notch signaling in adult vessel growth, we developed transgenic mice with Cre-conditional expression of the constitutively active intracellular domain of Notch1 (IC-Notch1). Double transgenic IC-Notch1/Tie2-Cre embryos with endothelial specific IC-Notch1 expression died at embryonic day 9.5. They displayed collapsed and leaky blood vessels and defects in angiogenesis development. A tetracycline-inducible system was used to express Cre recombinase postnatally in endothelial cells. In adult mice, IC-Notch1 expression inhibited bFGF-induced neovascularization and female mice lacked mature ovarian follicles, which may reflect the block in bFGF-induced angiogenesis required for follicle growth. Our results demonstrate that Notch signaling is important for both embryonic and adult angiogenesis and indicate that the Notch signaling pathway may be a useful target for angiogenic therapies.

Pestiviral E(rns) blocks TLR-3-dependent IFN synthesis by LL37 complexed RNA

The ribonuclease activity of the soluble glycoprotein E(rns) of pestiviruses represents a unique mechanism to circumvent the host's innate immune system by blocking interferon type-I synthesis in response to extracellularly added single- (ss) and double-stranded (ds) RNA. However, the reason why pestiviruses encode a ribonuclease in addition to the abundant serum RNases remained elusive. Here, we show that the 5' UTR and NS5B regions of various strains of the RNA genome of the pestivirus bovine viral diarrhea virus (BVDV) are resistant to serum RNases and are potent TLR-3 agonists. Inhibitory activity of E(rns) was restricted to cleavable RNA products, and did not extend to the synthetic TLR-7/8 agonist R-848. RNA complexed with the antimicrobial peptide LL37 was protected from degradation by E(rns)in vitro but was fully inhibited by E(rns) in its ability to induce IFN in cell cultures, suggesting that the viral protein is mainly active in cleaving RNA in an intracellular compartment. We propose that secreted E(rns) represents a potent IFN antagonist, which degrades viral RNA that is resistant to the ubiquitous host RNases in the extracellular space. Thus, the viral RNase prevents its own pathogen-associated molecular pattern (PAMP) to inadvertently activate the IFN response that might break innate immunotolerance required for persistent pestivirus infections.

The Exploitation of Versatile Building Blocks for the Self-Assembly of Novel Molecular Magnets

Using molecular building blocks to self-assemble lattices supporting long-range magnetic order is currently an active area of solid-state chemistry. Consequently, it is the realm of supramolecular chemistry that synthetic chemists are turning to in order to develop techniques for the synthesis of structurally well-defined supramolecular materials. In recent years we have investigated the versatility and usefulness of two classes of molecular building blocks, namely, tris-oxalato transition-metal (M. Pilkington and S. Decurtins, in “Magnetoscience—From Molecules to Materials,” Wiley–VCH, 2000), and octacyanometalate complexes (Pilkington and Decurtins, Chimia 54, 593 (2001)), for applications in the field of molecule-based magnets. Anionic, tris-chelated oxalato building blocks are able to build up two-dimensional honeycomb-layered structural motifs as well as three-dimensional decagon frameworks. The discrimination between the crystallization of the two- or three-dimensional structures relies on the choice of the templating counterions (Decurtins, Chimia 52, 539 (1998); Decurtins et al. Mol. Cryst. Liq. Cryst. 273, 167 (1995); New J. Chem. 117 (1998)). These structural types display a range of ferro, ferri, and antiferromagnetic properties (Pilkington and Decurtins, in “Magnetoscience—From Molecules to Materials”). Octacyanometalate building blocks self-assemble to afford two new classes of cyano-bridged compounds namely, molecular clusters and extended three dimensional networks (J. Larionova et al., Angew. Chem. Int. Ed. 39, 1605 (2000); Pilkington et al., in preparation). The molecular cluster with a MnII9MoV6 core has the highest ground state spin value, S=51/2, reported to-date (Larionova et al., Angew. Chem. Int. Ed. 39, 1605 (2000)). In the high-temperature regime, the magnetic properties are characterized by ferromagnetic intracluster coupling. In the magnetic range below 44 K, the magnetic cluster signature is lost as possibly a bulk behavior starts to emerge. The three-dimensional networks exhibit both paramagnetic and ferromagnetic behavior, since the magnetic properties of these materials directly reflect the electronic configuration of the metal ion incorporated into the octacyanometalate building blocks (Pilkington et al., in preparation). For both the oxalate- and cyanide-bridged materials, we are able to manipulate the magnetic properties of the supramolecular assemblies by tuning the electronic configurations of the metal ions incorporated into the appropriate molecular building blocks (Pilkington and Decurtins, in “Magnetoscience—From Molecules to Materials,” Chimia 54, 593 (2000)).