erastin and Necrosis

Necroptosis and ferroptosis are two distinct necrotic cell death modalities with no known common molecular mechanisms. Necroptosis is activated by ligands of death receptors such as tumor necrosis factor-α (TNF-α) under caspase-deficient conditions, whereas ferroptosis is mediated by the accumulation of lipid peroxides upon the depletion/or inhibition of glutathione peroxidase 4 (GPX4). The molecular mechanism that mediates the execution of ferroptosis remains unclear. In this study, we identified 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO), a compound known to inhibit heat shock protein 90 (HSP90), as an inhibitor of necroptosis that could also inhibit ferroptosis. We found that HSP90 defined a common regulatory nodal between necroptosis and ferroptosis. We showed that inhibition of HSP90 by CDDO blocked necroptosis by inhibiting the activation of RIPK1 kinase. Furthermore, we showed that the activation of ferroptosis by erastin increased the levels of lysosome-associated membrane protein 2a to promote chaperone-mediated autophagy (CMA), which, in turn, promoted the degradation of GPX4. Importantly, inhibition of CMA stabilized GPX4 and reduced ferroptosis. Our results suggest that activation of CMA is involved in the execution of ferroptosis.

Topics: Apoptosis; Autophagy; Caspases; Cell Death; Glutathione Peroxidase; HSP90 Heat-Shock Proteins; Humans; Iron; Ligands; Lipid Peroxides; Lysosomal-Associated Membrane Protein 2; Molecular Chaperones; Necrosis; Phospholipid Hydroperoxide Glutathione Peroxidase; Piperazines; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Various types of cell death, including apoptosis, necrosis, necroptosis, and ferroptosis, are induced in renal tubular epithelial cells following exposure to environmental stresses and toxicants such as osmotic stress, ischemia/reperfusion injury, cisplatin, and cadmium. This is known to cause renal dysfunction, but the cellular events preceding stress-induced cell death in renal tubules are not fully elucidated. The activin receptor-like kinase (ALK) 4/5, also known as activin-transforming growth factor (TGF) β receptor, is involved in stress-induced renal injury. We, therefore, studied the role of ALK4/5 signaling in HK-2 human proximal tubular epithelial cell death induced by cisplatin, cadmium, hyperosmotic stress inducer, sorbitol, and the ferroptosis activator, erastin. We found that ALK4/5 signaling is involved in cadmium- and erastin-induced cell death, but not sorbitol- or cisplatin-induced apoptotic cell death. Cadmium exposure elevated the level of phosphorylated Smad3, and treatment with the ALK4/5 kinase inhibitors, SB431542 or SB505124, suppressed cadmium-induced HK-2 cell death. Cadmium-induced cell death was attenuated by siRNA-mediated ALK4 or Smad3 silencing, or by treatment with SIS3, a selective inhibitor of TGFβ1-dependent Smad3 phosphorylation. Furthermore, ALK4/5 signaling activated Akt signaling to promote cadmium-induced HK-2 cell death. In contrast, siRNA-mediated Inhibin-bA silencing or treatment with TGFβ1 or activin A had little effect on cadmium-induced HK-2 cell death. On the other hand, treatment with SB431542 or SB505124 attenuated erastin-induced ferroptosis by hyperactivating Nrf2 signaling in HK-2 cells. These results suggest that blockade of ALK4/5 signaling protects against cadmium- and erastin-induced HK-2 cell death via Akt and Nrf2 signaling pathways, respectively.

Topics: Activin Receptors, Type I; Activins; Apoptosis; Cadmium; Cell Death; Cell Line; Cisplatin; Epithelial Cells; Ferroptosis; Glutathione; Humans; Kidney Tubules, Proximal; Necroptosis; Necrosis; NF-E2-Related Factor 2; Piperazines; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type I; RNA Interference; RNA, Small Interfering; Signal Transduction; Sorbitol; Transforming Growth Factor beta1

System x

Topics: Cell Death; Cell Line, Tumor; Cisplatin; Glutathione; Humans; Necrosis; Neoplasms; Piperazines

Cell death and its recently discovered regulated form ferroptosis are characterized by distinct morphological, electrophysiological, and pharmacological features. In particular ferroptosis can be induced by experimental compounds and clinical drugs (i.e., erastin, sulfasalazine, sorafenib, and artesunate) in various cell types and cancer cells. Pharmacologically, this cell death process can be inhibited by iron chelators and lipid peroxidation inhibitors. Relevance of this specific cell death form has been found in different pathological conditions such as cancer, neurotoxicity, neurodegeneration, and ischemia. Distinguishing cell viability and cell death is essential for experimental and clinical applications and a key component in flow cytometry experiments. Dead cells can compromise the integrity of the data by nonspecific binding of antibodies and dyes. Therefore it is essential that dead cells are robustly and reproducibly identified and characterized by means of cytometry application. Here we describe a procedure to detect and quantify cell death and its specific form ferroptosis based on standard flow cytometry techniques.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Dactinomycin; Flow Cytometry; Humans; Indicators and Reagents; Iron; Necrosis; Niacinamide; Phenylurea Compounds; Piperazines; Propidium; Rats; Sorafenib

Ferroptosis is a recently identified form of regulated necrosis that can be experimentally induced in cancer cells with the chemical inducer erastin. Recently, we identified sorafenib, an inhibitor of oncogenic kinases, as an inducer of ferroptosis in hepatocellular carcinoma cells. Whether sorafenib is able to exert its ferroptotic activity in cancer cells originating from other tissues is presently unclear.. We compared the levels of ferroptosis induced by sorafenib with those induced by the reference compound erastin in a panel of ten human cell lines originating from various tissues.. Sorafenib induced ferroptosis in different cancer cell lines. We found a positive correlation between the ferroptotic potency of sorafenib and erastin. Compared to other kinase inhibitors, sorafenib is the only drug that displays ferroptotic efficacy.. The findings establish sorafenib as the first clinically-approved anticancer drug that can induce ferroptosis.

Topics: Apoptosis; Biomarkers, Tumor; Blotting, Western; Cell Proliferation; Humans; L-Lactate Dehydrogenase; Necrosis; Neoplasms; Niacinamide; Phenylurea Compounds; Piperazines; Protein Kinase Inhibitors; Sorafenib; Tumor Cells, Cultured