Exploring cell death mechanisms in spheroid cultures using a novel application of the RIP3-caspase3-assay.

This study explores the application of the RIP3-caspase3-assay in heterogeneous spheroid cultures to analyze cell death pathways, emphasizing the nuanced roles of apoptosis and necroptosis. By employing directly conjugated monoclonal antibodies, we provide detailed insights into the complex mechanisms of cell death. Our findings demonstrate the assay's capability to differentiate between RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, marking a significant advancement in organoid research. Additionally, we investigate the effects of TNFα on isolated intestinal epithelial cells, revealing a concentration-dependent response and an adaptive or threshold reaction to TNFα-induced stress. The results indicate a preference for RIP1-independent cell death pathways upon TNFα stimulation, with a notable increase in apoptosis and a secondary role of necroptosis. Our research underscores the importance of the RIP3-caspase3-assay in understanding cell death mechanisms in organoid cultures, offering valuable insights for disease modeling and the development of targeted therapies. The assay's adaptability and robustness in spheroid cultures enhances its potential as a tool in personalized medicine and translational research.

Pulmonary delivery and tissue distribution of aerosolized antisense 2'-O-Methyl RNA containing nanoplexes in the isolated perfused and ventilated rat lung.

Pulmonary delivery of drugs, particularly in the treatment of lung cancer, is an attractive strategy for future targeted therapy. In this context, inhalation of nanoplexes might offer a new mode for drug delivery in gene therapy. However, limited data are currently available demonstrating pulmonary delivery, cellular uptake as well as local tolerability in lung tissue. The aim of this study was to elucidate the pulmonary delivery, tissue distribution and local tolerability of aerosolized chitosan-coated poly(lactide-co-glycolide) based nanoplexes containing antisense 2'-O-Methyl RNA (OMR). Therefore, an aerosol of OMR-nanoplexes or OMR alone was administered intra-tracheally using the model of the isolated perfused and ventilated rat lung. Localization of OMR in rat lung tissue was examined by immunohistochemistry. Administration of the OMR-nanoplex formulation resulted in significantly higher cellular OMR uptake of the respiratory epithelium in contrast to the administration of OMR alone, indicating that drug administration via aerosolized nanoplexes is able to target lung tissue. No prominent changes in lung physiology parameters were observed following inhalation, suggesting good local tolerability of OMR-nanoplex formulation.

Telomerase as an emerging target to fight cancer--opportunities and challenges for nanomedicine.

Telomerase as an enzyme is responsible for the renewal of the chromosomal ends, the so-called telomeres. By preventing them from shortening with each cell cycle, telomerase is able to inhibit cellular senescence and apoptosis. Telomerase activity, which is detectable in the majority of cancer cells, allows them to maintain their proliferative capacity. The thus obtained immortality of those cells again is a key to their malignancy. Based on these discoveries, it is obvious that telomerase inhibitors would represent an innovative approach to fight cancer, and a variety of such candidate molecules are currently in the pipeline. Telomerase inhibitors largely fall in two classes of compounds: small synthetic molecules and nucleotide-based biologicals. For several candidates, some proof of concept studies have been demonstrated, either on cell cultures or in animal models. But the same studies also revealed that inefficient delivery is largely limiting the translational step into the clinic. The most appealing feature of telomerase inhibitors, which distinguishes them from conventional anticancer drugs, is probably seen in their intrinsic non-toxicity to normal cells. Nevertheless, efficient delivery to the target cells, i.e. to the tumor, is still required. Here, some well-known biopharmaceutical problems such as insufficient solubility, permeability or even metabolic stability are frequently encountered. To address these challenges, there is a clear need for adequate delivery technologies, for example by using nanomedicines, that would allow to overcome their biopharmaceutical shortcomings and to warrant a sufficient bioavailability at the target side. This review first briefly explains the concept of telomerase and telomerase inhibition in cancer therapy. It secondly aims to provide an overview of the different currently known telomerase inhibitors. Finally, the biopharmaceutical limitations of these molecules are discussed as well as the possibilities to overcome those limits by novel drug carrier systems and formulation approaches.

Down-regulation of E-cadherin gene expression by collagen type I and type III in pancreatic cancer cell lines.

E-cadherin-mediated cell-cell adhesion is reduced in epithelial tumors, which is thought to be a prerequisite to acquire invasive properties. We observed that several pancreatic carcinoma cell lines with high metastatic potential expressed normal levels of E-cadherin and possessed functional E-cadherin/catenin adhesion complexes. When the cell lines PANC-1, BxPC-3, and PaTu8988s were cultured either on type I or type III collagen, E-cadherin gene expression was repressed, and E-cadherin and catenin protein concentrations were reduced. In contrast, growth on fibronectin and collagen type IV had no influence. Collagen type I- or type III-dependent reduction of E-cadherin expression led to decreased cell-cell adhesion, increased proliferation, and migratory activity as well as morphological transformation. Overexpression of activated c-Src in PANC-1 cells mimicked collagen-induced E-cadherin down-regulation and changed the elevated cell proliferation and migration. Conversely, treatment of cells with the Src-inhibitors PP1 or herbimycin A resulted in complete suppression of collagen type I-induced E-cadherin decrease. Our data demonstrate that specific collagens are able to promote metastatic behavior by down-regulation of E-cadherin gene expression in a Src-kinase-dependent manner. This points toward a novel mechanism for substrate-dependent signaling and underlines the significance of extracellular matrix environment for tumor growth and invasiveness.