ABSTRACT
Chronic inflammation is at least partially mediated by the chemokine-mediated attraction and by the adhesion molecule-directed binding of leukocytes to the activated endothelium. Therefore, it is therapeutically important to identify anti-inflammatory compounds able to control the interaction between leukocytes and the endothelial compartments of the micro- and macrocirculation. When testing novel drug candidates, it is, however, of the utmost importance to detect side effects, such as potential cytotoxic and barrier-disruptive activities. Indeed, minor changes in the endothelial monolayer integrity may increase the permeability of small blood vessels and capillaries, which, in extreme cases, can lead to edema development. Here, we describe the development of a high-throughput screening (HTS) platform, based on AlphaLISA technology, able to identify anti-inflammatory nontoxic natural or synthetic compounds capable of reducing tumor necrosis factor (TNF)-induced chemokine (interleukin [IL]-8) and adhesion molecule (ICAM-1) expression in human lung microvascular endothelial cells. Quantification of cell membrane-expressed ICAM-1 and of cell culture supernatant-associated levels of IL-8 was analyzed in HTS. In parallel, we monitored monolayer integrity and endothelial cell viability using the electrical cell substrate impedance sensing method. This platform allowed us to identify natural secondary metabolites from cyanobacteria, capable of reducing ICAM-1 and IL-8 levels in TNF-activated human microvascular endothelial cells in the absence of endothelial monolayer barrier disruption.
Subject(s)
Endothelial Cells/drug effects , High-Throughput Screening Assays/methods , Anti-Inflammatory Agents , Capillary Permeability/drug effects , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cyanobacteria/chemistry , Cytokines/genetics , Cytokines/metabolism , Depsipeptides/isolation & purification , Depsipeptides/pharmacology , Electric Impedance , Endothelial Cells/metabolism , Endothelial Cells/physiology , Endothelium, Vascular/drug effects , Gene Expression/drug effects , Humans , Imidazoles/pharmacology , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Kinetics , Lung/blood supply , Microvessels/cytology , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacology , Pyridines/pharmacology , Reproducibility of Results , Transcriptional Activation/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Tumor Necrosis Factor-alpha/physiologyABSTRACT
The remarkable ability of TNF, especially in combination with Interferon-gamma or melphalan, to inhibit the growth of malignant tumor cells is so far unmatched. Unfortunately, its high systemic toxicity and hepatotoxicity prevent its systemic use in cancer patients. An elegant manner to circumvent this problem is the isolated limb and liver perfusion for the treatment of melanoma, soft tissue sarcoma and liver tumors, respectively, although the latter method can lead to a reversible hepatotoxicity. In order to allow also the treatment of other cancers with TNF, new strategies have to be developed that aim at sensitizing tumor cells to TNF and at reducing its systemic and liver toxicity, without losing its antitumor efficiency. Moreover, the lectin-like domain of TNF, which is spatially distinct from the receptor binding sites, could be useful in reducing cancer treatment-related pulmonary edema formation. This review will discuss some recent developments in these areas, which can lead to a renewed interest in TNF for the systemic treatment of cancer.
