sm-164 and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Necroptosis is a form of regulated programmed cell death that is mediated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting serine/threonine protein kinase-3 (RIPK3), and mixed lineage kinase domain-like protein (MLKL); however, it is not known whether zinc finger protein 91 (ZFP91) is involved in this process. Here, we investigated ZFP91 as a potential mediator of necroptosis. Our mechanistic study demonstrates that ZFP91 promotes RIPK1-RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. ZFP91 stabilized RIPK1 to promote cell death by inducing RIPK1 de-ubiquitination. ZFP91 also significantly increased production of mitochondrial reactive oxygen species (ROS). Accumulation of ROS promoted RIPK3-independent necroptosis triggered by tumor necrosis factor (TNF). in vivo, ZFP91 knockdown alleviated TNFα-induced systemic inflammatory response syndrome (SIRS). These results provide direct evidence that ZFP91 plays an important role in the initiation of RIPK1/RIPK3-dependent necroptosis in vitro and in vivo. We discussed the potential of ZFP91 as a novel therapeutic target for necroptosis-associated diseases.

Topics: Amino Acid Chloromethyl Ketones; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Survival; Gene Expression Regulation; Humans; Mice; Protein Kinases; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Systemic Inflammatory Response Syndrome; Triazoles; Tumor Necrosis Factor-alpha; Ubiquitin-Protein Ligases

Chemoradiation is the treatment of choice for locally advanced head and neck squamous cell carcinoma (HNSCC). However, radioresistance, which contributes to local recurrence, remains a significant therapeutic problem. In this study, we characterized SM-164, a small second mitochondria-derived activator of caspase -mimetic compound that promotes degradation of cellular inhibitor of apoptosis-1(cIAP-1; also known as baculoviral IAP repeat-containing protein 2, BIRC2) and releases active caspases from the X-linked inhibitor of apoptosis inhibitory binding as a radiosensitizing agent in HNSCC cells. We found that SM-164 at nanomolar concentrations induced radiosensitization in some HNSCC cell lines in a manner dependent on intrinsic sensitivity to caspase activation and apoptosis induction. Blockage of caspase activation via short interfering RNA knockdown or a pan-caspase inhibitor, z-VAD-fmk, largely abrogated SM-164 radiosensitization. On the other hand, the resistant lines with a high level of Bcl-2 that blocks caspase activation and apoptosis induction became sensitive to radiation on Bcl-2 knockdown. Mechanistic studies revealed that SM-164 radiosensitization in sensitive cells was associated with NF-κB activation and TNFα secretion, followed by activation of caspase-8 and -9, leading to enhanced apoptosis. Finally, SM-164 also radiosensitized human tumor xenograft while causing minimal toxicity. Thus, SM-164 is a potent radiosensitizer via a mechanism involving caspase activation and holds promise for future clinical development as a novel class of radiosensitizer for the treatment of a subset of head and neck cancer patients.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Baculoviral IAP Repeat-Containing 3 Protein; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Squamous Cell; Caspase Inhibitors; Caspases; Cell Line, Tumor; Combined Modality Therapy; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; Head and Neck Neoplasms; Humans; Inhibitor of Apoptosis Proteins; Mice; Mice, Nude; Molecular Structure; Proto-Oncogene Proteins c-bcl-2; Radiation-Sensitizing Agents; Radiotherapy; RNA Interference; Triazoles; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays