Klaus Zanger

Combined cytotoxic and anti-invasive properties of redox-active nanoparticles in tumor–stroma interactions

Tumor-stroma interaction plays an important role in tumor progression. Myofibroblasts, pivotal for tumor progression, populate the microecosystem of reactive stroma. The formation of myofibroblasts is mediated by tumor derived transforming growth factor β1 (TGFβ1) which initiates a reactive oxygen species cell type dependent expression of alpha-smooth muscle actin, a biomarker for myofibroblastic cells. Myofibroblasts express and secrete proinvasive factors significantly increasing the invasive capacity of tumor cells via paracrine mechanisms. Although antioxidants prevent myofibroblast formation, the same antioxidants increase the aggressive behavior of the tumor cells. In this study, the question was addressed of whether redox-active polymer-coated cerium oxide nanoparticles (CNP, nanoceria) affect myofibroblast formation, cell toxicity, and tumor invasion. Herein, nanoceria downregulate both the expression of alpha-smooth muscle actin positive myofibroblastic cells and the invasion of tumor cells. Furthermore, concentrations of nanoceria being non-toxic for normal (stromal) cells show a cytotoxic effect on squamous tumor cells. The treatment with redox-active CNP may form the basis for protection of stromal cells from the dominating influence of tumor cells in tumor-stroma interaction, thus being a promising strategy for chemoprevention of tumor invasion.

Downregulation of tumor growth and invasion by redox-active nanoparticles.

AIMS Melanoma is the most aggressive type of malignant skin cancer derived from uncontrolled proliferation of melanocytes. Melanoma cells possess a high potential to metastasize, and the prognosis for advanced melanoma is rather poor due to its strong resistance to conventional chemotherapeutics. Nanomaterials are at the cutting edge of the rapidly developing area of nanomedicine. The potential of nanoparticles for use as carrier in cancer drug delivery is infinite with novel applications constantly being tested. The noncarrier use of cerium oxide nanoparticles (CNPs) is a novel and promising approach, as those particles per se show an anticancer activity via their oxygen vacancy-mediated chemical reactivity. RESULTS In this study, the question was addressed of whether the use of CNPs might be a valuable tool to counteract the invasive capacity and metastasis of melanoma cells in the future. Therefore, the effect of those nanoparticles on human melanoma cells was investigated in vitro and in vivo. Concentrations of polymer-coated CNPs being nontoxic for stromal cells showed a cytotoxic, proapoptotic, and anti-invasive capacity on melanoma cells. In vivo xenograft studies with immunodeficient nude mice showed a decrease of tumor weight and volume after treatment with CNPs. INNOVATION In summary, the redox-active CNPs have selective pro-oxidative and antioxidative properties, and this study is the first to show that CNPs prevent tumor growth in vivo. CONCLUSION The application of redox-active CNPs may form the basis of new paradigms in the treatment and prevention of cancers.

Stromal interaction molecule 1 (STIM1) is involved in the regulation of mitochondrial shape and bioenergetics and plays a role in oxidative stress

Background: Store-operated Ca2+ entry is regulated by the sensor STIM1 and the channel ORAI1. Results: Deficiency alters mitochondrial shape and increases mitochondrial activity resulting in increased susceptibility to oxidative stress and cell death by nuclear translocation of apoptosis-inducing factor. Conclusion: Store-operated Ca2+ entry regulates mitochondrial function and vulnerability. Significance: STIM1 plays a role in oxidative stress by regulating mitochondrial function. Calcium ions are involved in a plethora of cellular functions including cell death and mitochondrial energy metabolism. Store-operated Ca2+ entry over the plasma membrane is activated by depletion of intracellular Ca2+ stores and is mediated by the sensor STIM1 and the channel ORAI1. We compared cell death susceptibility to oxidative stress in STIM1 knock-out and ORAI1 knockdown mouse embryonic fibroblasts and in knock-out cells with reconstituted wild type and dominant active STIM1. We show that STIM1 and ORAI1 deficiency renders cells more susceptible to oxidative stress, which can be rescued by STIM1 and ORAI1 overexpression. STIM1 knock-out mitochondria are tubular, have a higher Ca2+ concentration, and are metabolically more active, resulting in constitutive oxidative stress causing increased nuclear translocation of the antioxidant transcription factor NRF2 triggered by increased phosphorylation of the translation initiation factor eIF2α and the protein kinase-like endoplasmic reticulum kinase PERK. This leads to increased transcription of antioxidant genes and a high basal glutathione in STIM1 knock-out cells, which is, however, more rapidly expended upon additional stress, resulting in increased release and nuclear translocation of apoptosis-inducing factor with subsequent cell death. Our data suggest that store-operated Ca2+ entry and STIM1 are involved in the regulation of mitochondrial shape and bioenergetics and play a role in oxidative stress.

Limitations of the Colloidal Silica Method in Mapping the Endothelial Plasma Membrane Proteome of the Mouse Heart

The endothelial cell (EC) membrane is an important interface, which plays a crucial role in signal transduction. Our aim was to selectively purify luminal EC membrane proteins from the coronary vasculature of the isolated perfused mouse heart and analyze its composition with mass spectrometry (MS). To specifically label coronary ECs in the intact heart, the colloidal silica method was applied, which is based on the binding of positively charged colloidal silica to the surface of EC membranes. Transmission electron microscopy revealed the specific labeling of ECs of macro and microvessels. Two different methods of tissue homogenization (Teflon pestle and ultra blade) together with density centrifugation were used for membrane protein enrichment. Enrichment and purity was controlled by Western blot analysis using the EC-specific protein caveolin 1 and various intracellular marker proteins. The ultra blade method resulted in a tenfold enrichment of caveolin 1, while there was negligible contamination as judged by Western blot. However, protein yield was low and required pooling of ten hearts for MS. When enriched endothelial membrane proteins were digested with trypsin and analyzed by LC-MS, a total of 56 proteins could be identified, of which only 12 were membrane proteins. We conclude that coronary endothelial membranes can be conveniently labeled with colloidal silica. However, due to the ionic nature of interaction of colloidal silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity.

Mycoplasma salivarium detected in a microbial community with Candida glabrata in the biofilm of an occluded biliary stent.

Mycoplasma salivarium, preferentially an inhabitant of the human oral cavity, has rarely been found in other locations associated with disease. We describe here, for what is believed to be the first time, the detection of M. salivarium, together with Candida glabrata, in an occluded biliary stent of an icteric, cholestatic patient.

Nephrocytes of harvestmen, Leiobunum limbatum and L. rotundum.

Nephrocytes are said to be able to take up substances from the hemolymph. In Opiliones, which were examined electron microscopically three different types of nephrocytes were found. Numerous large nephrocytes lie clustered between the muscles in the anterior region of the body. Smaller nephrocytes occur scattered throughout the opilionid body, often affixed to tracheoles. The third group, pericardial cells, are always attached to the heart wall by connective ligaments. All nephrocytes are surrounded by a thick basement membrane and their plasma membrane forms pedicels. Junctional complexes link the adjacent pedicels to form diaphragm-like slit-membranes, which form the entrance to an extracellular compartment. The cytoplasm of the nephrocytes contains many pinocytotic vesicles and tubular elements. Different types of large electron-dense and electron-lucent vesicles can be distinguished. Occasionally a large bundle of unmyelinated nerve fibers is enclosed by a pericardial cell. Morphological differences between the types of nephrocytes are described and the ultrastructural characteristics of the nephrocytes of harvestmen are compared with those of other arthropods. Functional aspects are discussed with respect to their ultrafiltration structures.

Effect of Fe3O4 Nanoparticles on Skin Tumor Cells and Dermal Fibroblasts.

Iron oxide (Fe3O4) nanoparticles have been used in many biomedical approaches. The toxicity of Fe3O4 nanoparticles on mammalian cells was published recently. Though, little is known about the viability of human cells after treatment with Fe3O4 nanoparticles. Herein, we examined the toxicity, production of reactive oxygen species, and invasive capacity after treatment of human dermal fibroblasts (HDF) and cells of the squamous tumor cell line (SCL-1) with Fe3O4 nanoparticles. These nanoparticles had an average size of 65 nm. Fe3O4 nanoparticles induced oxidative stress via generation of reactive oxygen species (ROS) and subsequent initiation of lipid peroxidation. Furthermore, the question was addressed of whether Fe3O4 nanoparticles affect myofibroblast formation, known to be involved in tumor invasion. Herein, Fe3O4 nanoparticles prevent the expression alpha-smooth muscle actin and therefore decrease the number of myofibroblastic cells. Moreover, our data show in vitro that concentrations of Fe3O4 nanoparticles, which are nontoxic for normal cells, partially reveal a ROS-triggered cytotoxic but also a pro-invasive effect on the fraction of squamous cancer cells surviving the treatment with Fe3O4 nanoparticles. The data herein show that the Fe3O4 nanoparticles appear not to be adequate for use in therapeutic approaches against cancer cells, in contrast to recently published data with cerium oxide nanoparticles.

Mechanical properties of the skin of Rana esculenta (Anura, Amphibia) with some notes on structures related to them

Summary The mechanical properties of the skin of the semiaquatic anuran Rana esculenta were investigated, using a technical tensiometer. Stress-strain curves and the histology of dorsal and ventral skin indicate the significance of collagen fibers in skin mechanics. Histological stains recommended for elastin fibers in paraffin sections, such as van Gieson in combination with Orcein, color a variety of structures, but only a few found within the perforating bundles are typical elastic fibers as revealed by electron microscopy. This small number of fibers does not play an important role in the mechanical properties. Angles between the collagen fibers in the dermal stratum compactum range from 50° to 70°. Mechanical parameters such as thickness, extension load, maximal tensile strength, modulus of elasticity and breaking energy show considerable variability within the specimens investigated, but are within the range of values measured for other lower vertebrates.

Immunocytochemical localization of lysozyme in the nephrocytes of the harvestman, Leiobunum rotundum

Nephrocytes are cells involved in the metabolism of hemolymph components and are characterized by peripheral finger-like projections bordering a labyrinthine channel system. The antibacterial protein lysozyme was localized in anterior and pericardial nephrocytes of the harvestman, Leiobunum rotundum, by biochemical and immuno-gold postembedding-labelling techniques. Lysozyme-activity was demonstrated in experimental animals, challenged by Micrococcus luteus Gram positive bacteria. With SDS-electrophoresis, lysozyme was proved to be present in nephrocytes adjoining the heart, before studies at the electron microscopical level were carried out. The opilionid lysozyme was found to have a molecular weight of 14,000 Da, thus belonging to the c-type lysozyme which is represented by hen-egg-white lysozyme. In untreated animals the presence of lysozyme could not be proved by electrophoresis. With immunogold labelling, however, lysozyme could be clearly localized in the vesicles of nephrocytes of untreated animals. Additional control experiments were carried out which revealed that the nephrocytes of L. rotundum in vivo are able to take up native lysozyme from an exterior medium. Therefore, the problem needs to be solved if nephrocyte vesicles transport lysozyme into or out of the cell.