7-bromoindirubin-3--oxime and Necrosis

Cells undergoing necrosis release endogenous danger signals that possess proinflammatory potential. In this study we show that mature IL-1beta and IL-18 are released by necrotic cells but not by apoptotic cells. We identify 7-bromoindirubin-3'-oxime, an indirubin oxime derivative that induces necrosis, as a potent inducer of caspase-1 activation and release of mature IL-1beta and IL-18. Inflammasome activation was triggered by other necrosis-inducing treatments but was not observed in response to apoptosis-inducing stimuli. Necrosis-induced inflammasome activation was mediated by the NLRP3 and ASC molecules. Release of IL-18 and IL-1beta in response to necrosis-inducing stimuli was observed in THP-1 macrophages and the MSTO-211H human mesothelioma cell line independently of LPS priming. Using the in vivo model of naphthalene-induced airway epithelial cell injury, we showed that necrosis activates the ASC inflammasome in vivo. Our study identifies a new mechanism through which necrosis generates proinflammatory molecules that contributes to the sterile inflammatory response.

Topics: Animals; Carrier Proteins; Cell Line; Epithelial Cells; Humans; Indoles; Inflammation; Interleukin-18; Interleukin-1beta; Macrophages; Mesothelioma; Mice; Mice, Knockout; Necrosis; NLR Family, Pyrin Domain-Containing 3 Protein; Oximes; Respiratory System

The new 7-bromoindirubin-3'-oxime (7BIO) compound induces caspase-independent cell death in all cell lines tested to date. Irrespective of the cell line, a 25 microM treatment for 24 h is lethal for the entire cell population. In SH-SY5Y and Jurkat cells, 7BIO (25 microM) was found to collapse the mitochondrial transmembrane potential (DeltaPsi m) at only 2-3 h of treatment. Concomitantly mitochondria swelled, cristae disrupted and, after 9 h, external cell membranes ruptured. In addition, endoplasmic reticulum dilated and, unexpectedly, the acute cytoplasmic destruction yielded isolated nuclei with preserved morphology and DNA integrity. Furthermore, the process was independent of both Bax and Bak, since cell viability and DeltaPsi m decayed indistinguishably in double Bax-/-Bak-/- mouse embryonic fibroblasts (MEFs) and their wild type counterparts. Pharmacological inhibition of the mitochondrial permeability transition pore (MPTP) did not prevent 7BIO-induced DeltaPsi m loss in none of the aforementioned cell lines. Caspase-independent inducers of cell death like AIF (Apoptosis Inducing Factor), cathepsins and calpains were not involved. Only the chemical inhibitors of serine proteases and, particularly, AEBSF afforded a significant protection thus suggesting a process regulated by this type of enzymes. As far as we know, these features are quite unique once taken together. Therefore, we propose 7BIO is triggering a specific type of necrotic cell death. Finally, the cytotoxicity of 7BIO on apoptosis-resistant cells like double Bax-/-Bak-/- MEFs seems of great interest envisaging cancer therapy.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line; DNA; Flow Cytometry; Humans; Indoles; Mice; Microscopy, Electron; Necrosis; Oximes; Serine Endopeptidases