benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and monodansylcadaverine

We recently reported that necroptosis contributed to compression-induced nucleus pulposus (NP) cells death. In the current study, we investigated the regulative effect of necroptosis inhibitor Necrostatin-1 on NP cells apoptosis and autophagy. Necrostatin-1, autophagy inhibitor 3-Methyladenine and apoptosis inhibitor Z-VAD-FMK were employed, and NP cells were exposed to 1.0 MPa compression for 0, 24 and 36 h. Necroptosis-associated molecules were measured by Western blot and RT-PCR. Autophagy and apoptosis levels were evaluated by Western blot and quantified by flow cytometry after monodansylcadaverine and Annexin V-FITC/propidium iodide staining, respectively. The cell viability and cell death were also examined. Furthermore, we measured mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (MPTP) and indices of oxidative stress to assess mitochondrial dysfunction. The results established that Necrostatin-1 blocked NP cells autophagy, and 3-Methyladenine had little influence on NP cells necroptosis. The Necrostatin-1+3-Methyladenine treatment exerted almost the same role as Necrostatin-1 in reducing NP cells death. Necrostatin-1 restrained NP cells apoptosis, while Z-VAD-FMK enhanced NP cells necroptosis. The Necrostatin-1+Z-VAD-FMK treatment provided more prominent role in blocking NP cells death compared with Necrostatin-1, consistent with increased MMP, reduced opening of MPTP and oxidative stress. In summary, the synergistic utilization of Necrostatin-1 and Z-VAD-FMK is a very worthwhile solution in preventing compression-mediated NP cells death, which might be largely attributed to restored mitochondrial function.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Cadaverine; Cell Death; Cell Survival; Compressive Strength; Imidazoles; Indoles; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Mitochondria; Nucleus Pulposus; Oxidative Stress; Pressure; Propidium; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Tumor necrosis factor-alpha (TNF-α) exists in two forms: secretory TNF-α (sTNF-α) and transmembrane TNF-α (tmTNF-α). Although both forms of TNF-α induce tumor cell apoptosis, tmTNF-α is able to kill tumor cells that are resistant to sTNF-α-mediated cytotoxicity, indicating their differences in signal transduction. Here, we demonstrate that internalization of TNFR1 is crucial for sTNF-α- but not for tmTNF-α-induced apoptosis. sTNF-α induces binding of tumor necrosis factor receptor type 1-associated death domain protein (TRADD) to the death domain (DD) of TNFR1 and subsequent activation of nuclear factor kappa B (NF-κB), and the formation of death-inducing signaling complexes (DISCs) in the cytoplasm after internalization. In contrast, tmTNF-α induces DISC formation on the membrane in a DD-independent manner. It leads to the binding of signal transducer and activator of transcription 1 (STAT1) to a region spanning amino acids 319-337 of TNFR1 and induces phosphorylation of serine at 727 of STAT1. The phosphorylation of STAT1 promotes its binding to TRADD, and thus recruits Fas-associated protein with DD (FADD) and caspase 8 to form DISC complexes. This STAT1-dependent signaling results in apoptosis but not NF-κB activation. STAT1-deficiency in U3A cells counteracts tmTNF-α-induced DISC formation and apoptosis. Conversely, reconstitution of STAT1 expression restores tmTNF-α-induced apoptotic signaling in the cell line. Consistently, tmTNF-α suppresses the growth of STAT1-containing HT1080 tumors, but not of STAT1-deficient U3A tumors in vivo. Our data reveal an unappreciated molecular mechanism of tmTNF-α-induced apoptosis and may provide a new clue for cancer therapy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Cadaverine; Caspase 8; Cell Line; Death Domain Receptor Signaling Adaptor Proteins; Fas-Associated Death Domain Protein; HEK293 Cells; Humans; Mice; NF-kappa B; NIH 3T3 Cells; Phosphorylation; Protein Binding; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; STAT1 Transcription Factor; TNF Receptor-Associated Death Domain Protein; Tumor Necrosis Factor-alpha

Airway epithelial cell is often the initial site of attack by pathogens, and cell death is commonly caused by internalization of

Topics: A549 Cells; Amino Acid Chloromethyl Ketones; Apoptosis; Autophagy; Bacterial Proteins; Cadaverine; Caspase 3; Cell Death; Colony Count, Microbial; Epithelial Cells; Escherichia coli; Host-Pathogen Interactions; Humans; Lectins; Membrane Proteins; Microbial Viability; Microtubule-Associated Proteins; Mycobacterium smegmatis; Mycobacterium tuberculosis; Recombinant Proteins; Virulence Factors