Simone Braig

Validating Metal‐Organic Framework Nanoparticles for Their Nanosafety in Diverse Biomedical Applications

&NA; Metal‐organic frameworks (MOFs) are promising platforms for the synthesis of nanoparticles for diverse medical applications. Their fundamental design principles allow for significant control of the framework architecture and pore chemistry, enabling directed functionalization for nanomedical applications. However, before applying novel nanomaterials to patients, it is imperative to understand their potential health risks. In this study, the nanosafety of different MOF nanoparticles is analyzed comprehensively for diverse medical applications. The authors first evaluate the effects of MOFs on human endothelial and mouse lung cells, which constitute a first line of defense upon systemic blood‐mediated and local lung‐specific applications of nanoparticles. Second, we validated these MOFs for multifunctional surface coatings of dental implants using human gingiva fibroblasts. Moreover, biocompatibility of MOFs is assessed for surface coating of nerve guidance tubes using human Schwann cells and rat dorsal root ganglion cultures. The main finding of this study is that the nanosafety and principal suitability of our MOF nanoparticles as novel agents for drug delivery and implant coatings strongly varies with the effector cell type. We conclude that it is therefore necessary to carefully evaluate the nanosafety of MOF nanomaterials with respect to their particular medical application and their interacting primary cell types, respectively. &NA; Metal‐organic frameworks (MOFs) are a promising platform for the synthesis of porous nanoparticles for diverse medical applications. The aim of this study is to comprehensively investigate the nanosafety of different MOF nanoparticles for distinct fields of medical applications. Data presented here suggest the need to evaluate the nanosafety of each MOF nanomaterial with respect to their particular medical application. Figure. No caption available.

Pharmacological targeting of membrane rigidity: implications on cancer cell migration and invasion

The invasive potential of cancer cells strongly depends on cellular stiffness, a physical quantity that is not only regulated by the mechanical impact of the cytoskeleton but also influenced by the membrane rigidity. To analyze the specific role of membrane rigidity in cancer progression, we treated cancer cells with the Acetyl-CoA carboxylase inhibitor Soraphen A and revealed an alteration of the phospholipidome via mass spectrometry. Migration, invasion, and cell death assays were employed to relate this alteration to functional consequences, and a decrease of migration and invasion without significant impact on cell death has been recorded. Fourier fluctuation analysis of giant plasma membrane vesicles showed that Soraphen A increases membrane rigidity of carcinoma cell membranes. Mechanical measurements of the creep deformation response of whole intact cells were performed using the optical stretcher. The increase in membrane rigidity was observed in one cell line without changing the creep deformation response indicating no restructuring of the cytoskeleton. These data indicate that the increase of membrane rigidity alone is sufficient to inhibit invasiveness of cancer cells, thus disclosing the eminent role of membrane rigidity in migratory processes.

Pretubulysin: a new option for the treatment of metastatic cancer

Tubulin-binding agents such as taxol, vincristine or vinblastine are well-established drugs in clinical treatment of metastatic cancer. However, because of their highly complex chemical structures, the synthesis and hence the supply issues are still quite challenging. Here we set on stage pretubulysin, a chemically accessible precursor of tubulysin that was identified as a potent microtubule-binding agent produced by myxobacteria. Although much simpler in chemical structure, pretubulysin abrogates proliferation and long-term survival as well as anchorage-independent growth, and also induces anoikis and apoptosis in invasive tumor cells equally potent to tubulysin. Moreover, pretubulysin posseses in vivo efficacy shown in a chicken chorioallantoic membrane (CAM) model with T24 bladder tumor cells, in a mouse xenograft model using MDA-MB-231 mammary cancer cells and finally in a model of lung metastasis induced by 4T1 mouse breast cancer cells. Pretubulysin induces cell death via the intrinsic apoptosis pathway by abrogating the expression of pivotal antiapoptotic proteins, namely Mcl-1 and Bcl-xL, and shows distinct chemosensitizing properties in combination with TRAIL in two- and three-dimensional cell culture models. Unraveling the underlying signaling pathways provides novel information: pretubulysin induces proteasomal degradation of Mcl-1 by activation of mitogen-activated protein kinase (especially JNK (c-Jun N-terminal kinase)) and phosphorylation of Mcl-1, which is then targeted by the SCFFbw7 E3 ubiquitin ligase complex for ubiquitination and degradation. In sum, we designate the microtubule-destabilizing compound pretubulysin as a highly promising novel agent for mono treatment and combinatory treatment of invasive cancer.

Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ.

Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as innovative cancer drugs. This is moreover supported by the in vivo efficacy of Chondramide triggered by abrogation of PKCɛ signaling shown in a xenograft breast cancer model.

Simplified Pretubulysin Derivatives and Their Biological Effects on Cancer Cells

Tubulin binding agents are a potent group of cancer chemotherapeutics. Most of these substances are naturally derived compounds. A novel substance class of destabilizing agents is the group of tubulysins. The tubulysins and their derivative pretubulysin have shown high efficacy in vitro and in vivo. Due to their complex chemical structures, one major bottleneck of the tubulysins is their accessibility. Biotechnological as well as chemical production is challenging, especially on larger scales. Thus, the synthesis of chemically simplified structures is needed with retained or improved biological activity. Herein is presented the biological evaluation of two pretubulysin derivatives [2-desmethylpretubulysin AU816 (1) and phenylpretubulysin JB337 (2)] in comparison to pretubulysin. Both 1 and 2 display a simplification in chemical synthesis. It was shown that both compounds exhibited potent biological activity against cancer cells. These simplified compounds inhibited tubulin polymerization in the nanomolar range. The cytotoxic effects of 1 and 2 were in a similar range, when compared with pretubulysin [IC50 (nM): pretubulysin: 0.6; 1: 10; 2: 100]. Furthermore, it was shown that cell cycle arrest is induced and migration is hampered in MDA-MB-231 breast cancer cells. In conclusion, 1 was shown to be about 10-fold more active than 2 and as potent as pretubulysin.

New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP131-18

Marine actinobacteria are drawing more and more attention as a promising source of new natural products. Here we report isolation, genome sequencing and metabolic profiling of new strain Streptomyces sp. MP131-18 isolated from marine sediment sample collected in the Trondheim Fjord, Norway. The 16S rRNA and multilocus phylogenetic analysis showed that MP131-18 belongs to the genus Streptomyces. The genome of MP131-18 isolate was sequenced, and 36 gene clusters involved in the biosynthesis of 18 different types of secondary metabolites were predicted using antiSMASH analysis. The combined genomics-metabolics profiling of the strain led to the identification of several new biologically active compounds. As a result, the family of bisindole pyrroles spiroindimicins was extended with two new members, spiroindimicins E and F. Furthermore, prediction of the biosynthetic pathway for unusual α-pyrone lagunapyrone isolated from MP131-18 resulted in foresight and identification of two new compounds of this family – lagunapyrones D and E. The diversity of identified and predicted compounds from Streptomyces sp. MP131-18 demonstrates that marine-derived actinomycetes are not only a promising source of new natural products, but also represent a valuable pool of genes for combinatorial biosynthesis of secondary metabolites.

A Small Molecule Inhibits Protein Disulfide Isomerase and Triggers the Chemosensitization of Cancer Cells

Resistance to chemotherapeutic agents represents a major challenge in cancer research. One approach to this problem is combination therapy, the application of a toxic chemotherapeutic drug together with a sensitizing compound that addresses the vulnerability of cancer cells to induce apoptosis. Here we report the discovery of a new compound class (T8) that sensitizes various cancer cells towards etoposide treatment at subtoxic concentrations. Proteomic analysis revealed protein disulfide isomerase (PDI) as the target of the T8 class. In-depth chemical and biological studies such as the synthesis of optimized compounds, molecular docking analyses, cellular imaging, and apoptosis assays confirmed the unique mode of action through reversible PDI inhibition.

The pleiotropic profile of the indirubin derivative 6BIO overcomes TRAIL resistance in cancer

TRAIL (TNFα-related apoptosis-inducing factor) has been promoted as a promising anti-cancer agent. Unfortunately many tumor cells develop resistance towards TRAIL due to numerous defects in apoptotic signaling. To handle this problem combination therapy with compounds affecting as many different anti-apoptotic targets as possible might be a feasible approach. The bromo-substituted indirubin derivative 6BIO meets this challenge: Treatment of breast cancer and bladder carcinoma cell lines with micromolar concentrations of 6BIO abrogates cellular growth and induces apoptosis. Combination of subtoxic amounts of 6BIO with ineffective doses of TRAIL completely abolishes proliferation and long-term survival of cancer cells. As shown in two-dimensional as well as three-dimensional cell culture models, 6BIO potently augments TRAIL-induced apoptosis in cancer cell lines. The potent chemosensitizing effect of 6BIO to TRAIL-mediated cell death is due to the pleiotropic inhibitory profile of 6BIO. As shown previously, 6BIO abrogates STAT3, PDK1 as well as GSK3 signaling and moreover, inhibits the expression of the anti-apoptotic Bcl-2 family members Bcl-xL and Mcl-1 on mRNA as well as on protein level, as demonstrated in this study. Moreover, the expression of cFLIP and cIAP1 is significantly downregulated in 6BIO treated cancer cell lines. In sum (subtoxic concentration of) the multi-kinase inhibitor 6BIO serves as a potent chemosensitizing agent fighting TRAIL resistant cancer cells.

Pharmacological characterization of actin-binding (-)-doliculide.

Natural compounds offer a broad spectrum of potential drug candidates against human malignancies. Several cytostatic drugs, which are in clinical use for decades, derive directly from natural sources or are synthetically optimized derivatives of natural lead structures. An eukaryote target molecule to which many natural derived anti-cancer drugs bind to is the microtubule network. Of similar importance for the cell is the actin cytoskeleton, responsible for cell movements, migration of cells and cytokinesis. Nature provides also a broad range of compounds directed against actin as intracellular target, but none of these actin-targeting compounds has ever been brought to clinical trials. One reason why actin-binding compounds have not yet been considered for further clinical investigations is that little is known about their pharmacological properties in cancer cells. Herein, we focused on the closer characterization of doliculide, an actin binding natural compound of marine origin in the breast cancer cell lines MCF7 and MDA-MB-231. We used fluorescence-recovery-after-photobleaching (FRAP) analysis to determine doliculides early effects on the actin cytoskeleton and rhodamin-phalloidin staining for long-term effects on the actin CSK. After validating the disruption of the actin network, we further investigated the functional effects of doliculide. Doliculide treatment leads to inhibition of proliferation and impairs the migratory potential. Finally, we could also show that doliculide leads to the induction of apoptosis in both cell lines. Our data for the first time provide a closer characterization of doliculide in breast cancer cells and propagate doliculide for further investigations as lead structure and potential therapeutic option as actin-targeting compound.

Targeting de novo lipogenesis as a novel approach in anti-cancer therapy

Background:Although altered membrane physiology has been discussed within the context of cancer, targeting membrane characteristics by drugs being an attractive therapeutic strategy has received little attention so far.Methods:Various acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FASN) inhibitors (like Soraphen A and Cerulenin) as well as genetic knockdown approaches were employed to study the effects of disturbed phospholipid composition on membrane properties and its functional impact on cancer progression. By using state-of-the-art methodologies such as LC-MS/MS, optical tweezers measurements of giant plasma membrane vesicles and fluorescence recovery after photobleaching analysis, membrane characteristics were examined. Confocal laser scanning microscopy, proximity ligation assays, immunoblotting as well as migration, invasion and proliferation experiments unravelled the functional relevance of membrane properties in vitro and in vivo.Results:By disturbing the deformability and lateral fluidity of cellular membranes, the dimerisation, localisation and recycling of cancer-relevant transmembrane receptors is compromised. Consequently, impaired activation of growth factor receptor signalling cascades results in abrogated tumour growth and metastasis in different in vitro and in vivo models.Conclusions:This study highlights the field of membrane properties as a promising druggable cellular target representing an innovative strategy for development of anti-cancer agents.