Heiko Lemcke

Novel derivatives of 1,3,4-oxadiazoles are potent mitostatic agents featuring strong microtubule depolymerizing activity in the sea urchin embryo and cell culture assays.

A series of novel 1,3,4-oxadiazole derivatives based on structural and electronic overlap with combretastatins have been designed and synthesized. Initially, we tested all new compounds in vivo using the phenotypic sea urchin embryo assay to yield a number of agents with anti-proliferative, anti-mitotic, and microtubule destabilizing activities. The experimental data led to identification of 1,3,4-oxadiazole derivatives with isothiazole (5-8) and phenyl (9-12) pharmacophores featuring activity profiles comparable to that of combretastatins, podophyllotoxin and nocodazole. Cytotoxic effects of the two lead molecules, namely 6 and 12, were further confirmed and evaluated by conventional assays with the A549 human cancer cell line including cell proliferation, cell cycle arrest at the G2/M phase, cellular microtubule distribution, and finally in vitro microtubule assembly with purified tubulin. The modeling results using 3D similarity (ROCS) and docking (FRED) correlated well with the observed activity of the molecules. Docking data suggested that the most potent molecules are likely to target the colchicine binding site.

Ca2+-mediated Mitochondrial Reactive Oxygen Species Metabolism Augments Wnt/β-Catenin Pathway Activation to Facilitate Cell Differentiation

Background: Dissociation of the Wnt/β-catenin pathway effector Dishevelled from its complex with nucleoredoxin is a redox-sensitive process, yet the ROS sources remain elusive. Results: Mitochondrial Ca2+ influx stimulates endogenous ROS production and mediates Wnt/β-catenin pathway activity. Conclusion: Ca2+-mediated ROS production modulates the signaling efficiency of the Wnt/β-catenin pathway. Significance: Metabolic states influence fundamental and developmental signaling to drive cell differentiation. Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/β-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/β-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca2+ from the endoplasmic reticulum stores. Ca2+ subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca2+ uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/β-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca2+-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/β-catenin signaling output.

Gap junctional shuttling of miRNA — A novel pathway of intercellular gene regulation and its prospects in clinical application

The gap junctional exchange of small molecules between adjacent cells is crucial for maintaining tissue homeostasis and for a large number of cellular processes, including differentiation and proliferation. miRNAs represent a novel class of signalling molecules capable of crossing gap junction (GJ) channels, thereby directly affecting gene expression in the recipient cell. Here, we give an overview about the current knowledge on the biological significance of miRNA shuttling in different cell types (e.g. stem cells, cardiac cells, macrophages), which indicates the GJ-dependent transfer of miRNA as a general mechanism for intercellular gene regulation. Notably, shuttling via GJs is superior to exosome-mediated intercellular transfer regarding specificity and efficiency. We further elucidate this mechanism as a promising approach for miRNA delivery in clinical applications. Using a cell-based gap junctional dependent system, in vivo delivery of therapeutic miRNAs might become more efficient compared to systemic delivery methods. We will discuss the advantages of such a delivery system and the challenges that have to be overcome for its successful application in miRNA therapy.

Synthesis and SAR requirements of adamantane–colchicine conjugates with both microtubule depolymerizing and tubulin clustering activities

A series of analogues of conjugate 1, combining an adamantane-based paclitaxel (taxol) mimetic with colchicine was synthesized and tested for cytotoxicity in a cell-based assay with the human lung carcinoma cell line A549. The most active compounds (10 EC(50) 2 ± 1.0 nM, 23 EC(50) 6 ± 1.4 nM, 26 EC(50) 5 ± 1.8 nM, 28 EC(50) 11 ± 1.7 nM, 30 EC(50) 4.8 ± 0.5 nM) were found to interfere with the microtubule dynamics in an interesting manner. Treatment of the cells with these compounds promoted disassembly of microtubules followed by the formation of stable tubulin clusters. Structure-activity relationships for the analogues of 23 revealed the sensitivity of both cytotoxicity and tubulin clustering ability to the linker length. The presence of adamantane (or another bulky hydrophobic and non-aromatic moiety) in 23 was found to play an important role in the formation of tubulin clusters. Structural requirements for optimal activity have been partially explained by molecular modeling.

Design, synthesis, and bioactivity of putative tubulin ligands with adamantane core

Several adamantane-based taxol mimetics were synthesized and found to be cytotoxic at micromolar concentrations and to cause tubulin aggregation. The extent of the aggregation is maximal for N-benzoyl-(2R,3S)-phenylisoseryloxyadamantane (5) and is very sensitive to the structural modifications. A hybrid compound (15), combining adamantane-based taxol mimetic with colchicine was synthesized and found to possess both microtubule depolymerizing and microtubule bundling activities in A549 human lung carcinoma cells.

Spatio-temporal Model of Endogenous ROS and Raft-Dependent WNT/Beta-Catenin Signaling Driving Cell Fate Commitment in Human Neural Progenitor Cells

Canonical WNT/β-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/β-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear β-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of β-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/β-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream β-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/β-catenin signal or as amplifier during continuous auto-/parcrine WNT/β-catenin signaling. In addition we provide the first stochastic computational model of WNT/β-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent β-catenin activation. The model’s predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the model. Thus, our results provide both new insights and means to further our understanding of canonical WNT/β-catenin signaling and the role of ROS as intracellular signaling mediator.

Unusual Tubulin-Clustering Ability of Specifically C7-Modified Colchicine Analogues

Highly cytotoxic C7-modified colchicine analogues, exemplified by tubuloclustin, promote microtubule disassembly followed by the formation of very stable tubulin clusters, both in vitro and in cells. The proposed mechanism of action of tubuloclustin and its analogues, beyond that of colchicine, includes additional specific interactions with the α-tubulin subunit.

Involvement of connexin43 in the EGF/EGFR signalling during self-renewal and differentiation of neural progenitor cells

Neural progenitor cells (NPCs) are sensitive to epidermal growth factor (EGF), which is essential for their self-renewal. Recently we showed that high level of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) are also required to maintain NPCs in a proliferative state. In this study the connection between EGF/EGFR signalling and Cx43 expression was investigated during proliferation and differentiation of cultured ReNcell VM197 human NPCs. We found that EGF, but not basic fibroblast growth factor (bFGF), strongly stimulated both Cx43 expression and GJIC in proliferating cells. This stimulatory effect was blocked by AG1478, a specific inhibitor for EGFR kinase. Notably, knockdown of Cx43 strongly inhibited the cell proliferation promoted by EGF/EGFR signalling. High sensitivity to EGF was still maintained in differentiated NPCs. Administration of EGF to differentiating cells led to a pronounced increase (9-fold) of Cx43 expression and a re-induction of proliferation. This strong impact of EGF was found to correlate with a surprisingly massive 60-fold up-regulation of EGFR expression in differentiated cells. Our data argue for a mutual regulation between Cx43 expression and EGF/EGFR signalling during self-renewal and differentiation of NPCs.

Neuronal differentiation requires a biphasic modulation of gap junctional intercellular communication caused by dynamic changes of connexin43 expression

It was suggested that gap junctional intercellular communication (GJIC) and connexin (Cx) proteins play a crucial role in cell proliferation and differentiation. However, the mechanisms of cell coupling in regulating cell fate during embryonic development are poorly understood. To study the role of GJIC in proliferation and differentiation, we used a human neural progenitor cell line derived from the ventral mesencephalon. Fluorescence recovery after photobleaching (FRAP) showed that dye coupling was extensive in proliferating cells but diminished after the induction of differentiation, as indicated by a 2.5‐fold increase of the half‐time of fluorescence recovery. Notably, recovery half‐time decreased strongly (five‐fold) in the later stage of differentiation. Western blot analysis revealed a similar time‐dependent expression profile of Cx43, acting as the main gap junction‐forming protein. Interestingly, large amounts of cytoplasmic Cx43 were retained mainly in the Golgi network during proliferation but decreased when differentiation was induced. Furthermore, down‐regulation of Cx43 by small interfering RNA reduced functional cell coupling, which in turn resulted in a 50% decrease of both the proliferation rate and neuronal differentiation. Our findings suggest a dual function of Cx43 and GJIC in the neural development of ReNcell VM197 human progenitor cells. GJIC accompanied by high Cx43 expression is necessary (1) to maintain cells in a proliferative state and (2) to complete neuronal differentiation, including the establishment of a neural network. However, uncoupling of cells is crucial in the early stage of differentiation during cell fate commitment.

Non-viral magnetic engineering of endothelial cells with microRNA and plasmid-DNA—An optimized targeting approach

Genetic modulation of angiogenesis is a powerful tool for the treatment of multiple disorders. Here, we describe a strategy to produce modified endothelial cells, which can be efficiently magnetically guided. First, we defined optimal transfection conditions with both plasmid and microRNA, using a polyethyleneimine/magnetic nanoparticle-based vector (PEI/MNP), previously designed in our group. Further, two approaches were assessed in vitro: direct vector guidance and magnetic targeting of transfected cells. Due to its higher efficiency, including simulated dynamic conditions, production of miR/PEI/MNP-modified magnetically responsive cells was selected for further detailed investigation. In particular, we have studied internalization of transfection complexes, functional capacities and intercellular communication of engineered cells and delivery of therapeutic miR. Moreover, we demonstrated that 104 miRNA/PEI/MNP-modified magnetically responsive cells loaded with 0.37pg iron/cell are detectable with MRI. Taken together, our in vitro findings show that PEI/MNP is highly promising as a multifunctional tool for magnetically guided angiogenesis regulation.