tosylphenylalanyl-chloromethyl-ketone and Necrosis

Necrosis is the main mode of cell death, which leads to multiple clinical conditions affecting hundreds of millions of people worldwide. Its molecular mechanisms are poorly understood, hampering therapeutics development. Here, we identify key proteolytic activities essential for necrosis using various biochemical approaches, enzymatic assays, medicinal chemistry, and siRNA library screening. These findings provide strategies to treat and prevent necrosis, including known medicines used for other indications, siRNAs, and establish a platform for the design of new inhibitory molecules. Indeed, inhibitors of these pathways demonstrated protective activity

Topics: Animals; Brain Injuries, Traumatic; Cell Death; Chemical and Drug Induced Liver Injury; High-Throughput Screening Assays; Humans; Mice, Inbred BALB C; Mice, Inbred C57BL; Myocardial Infarction; Necroptosis; Necrosis; Pancreatic Elastase; Protease Inhibitors; RNA, Small Interfering; U937 Cells

Superior cervical ganglion (SCG) cells from neonatal rats underwent apoptosis upon treatment with colchicine, a microtubule-disrupting agent. Western blotting and activity measurements showed that caspase-3 was indeed activated, but its peptide inhibitor (Ac-DEVD-CHO) neither suppressed nuclear fragmentation nor rescued the neurons from cell death. z-VAD-fmk, the general inhibitor of caspases, prevented nuclear fragmentation and delayed the cell death. Moreover, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), but not N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK), prevented nuclear fragmentation and provided neuronal protection as well. The combination of both z-VAD-fmk and TLCK provided a long-term neuronal protection (>4 days), whereas neither one alone could do so, suggesting that there are both caspase-dependent and -independent pathways. TLCK-sensitive serine protease is also likely to act upstream of caspase-3 in a caspase-dependent pathway. Electron microscopic observations demonstrated that z-VAD-fmk suppressed nuclear fragmentation and improved mitochondrial swelling, but failed to prevent vesicular formation, which resulted in a slowly-occurring necrosis. More importantly, TLCK effectively blocked this abundant vesicular formation along with suppressing chromatin condensation. Thus, the combination of both conferred a nearly normal morphology, which is consistent with the results of cell survival experiments. These findings clearly indicate that TLCK-sensitive serine protease plays multiple roles in caspase-dependent and -independent pathways of colchicine-induced cell death, and suggest a novel mechanism underlying a necrotic pathway involving ER swelling and vesicular formation.

Topics: Animals; Animals, Newborn; Apoptosis; Bucladesine; Caspase 3; Caspases; Cells, Cultured; Colchicine; Enzyme Inhibitors; Immunohistochemistry; Microscopy, Electron; Microtubule Proteins; Necrosis; Nervous System; Neurons; Neuroprotective Agents; Potassium; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Serine Endopeptidases; Serine Proteinase Inhibitors; Signal Transduction; Superior Cervical Ganglion; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone