Inhibiting the glutamate/cystine antiporter system x, a key antioxidant defense machinery in the CNS, could trigger a novel form of regulated necrotic cell death, ferroptosis. The underlying mechanisms of system x-dependent cell demise were elucidated using primary oligodendrocytes (OLs) treated with glutamate to block system x function. Pharmacological analysis revealed ferroptosis as a major contributing factor to glutamate-initiated OL death. A sphingolipid profile showed elevations of ceramide species and sphingosine that were preventable by inhibiting of an acid sphingomyelinase (ASM) activity. OL survival was enhanced by both downregulating ASM expression and blocking ASM activity. Glutamate-induced ASM activation seems to involve posttranscriptional mechanisms and was associated with a decreased GSH level. Further investigation of the mechanisms of OL response to glutamate revealed enhanced reactive oxygen species production, augmented lipid peroxidation, and opening of the mitochondrial permeability transition pore that were attenuated by hindering ASM. Of note, knocking down sirtuin 3, a deacetylase governing the mitochondrial antioxidant system, reduced OL survival. The data highlight the importance of the mitochondrial compartment in regulated necrotic cell death and accentuate the novel role of ASM in disturbing mitochondrial functions during OL response to glutamate toxicity, which is essential for pathobiology in stroke and traumatic brain injury.
Our results indicate that NGF-RAGE/p75 signaling may be a therapeutic target in ALS. Antioxid. Redox Signal. 28, 1587-1602.
Hepatocarcinoma is one of the most common cancer types in the U.S. with limited treatment options and poor survival rate. Recently, cancer bioenergetics emerged as a promising source of targets to develop novel anticancer agents. The identification of biomarkers is essential for screening and validation of small molecules proposed as chemotherapeutic agents. In this regard, there is a great need for high-throughput/high-content methods, which require short and reproducible sample preparation. In this project, we used the erastin-like compound X1 in the well-characterized SNU-449 hepatocarcinoma cell line. Erastin initially enhances mitochondrial function and ROS formation leading to subsequent mitochondrial dysfunction. As readout, we employed directly and indirectly labeled fluorescent antibodies to visualize and quantify expression of proliferative and antiproliferative markers Ki-67 and cytoskeleton-associated protein 4/p63 (CKAP4/p63), as well as expression and intracellular translocation of the tumor suppressor protein p53. Using the Agilent BioTek Cytation 5 cell imaging multimode reader and Agilent BioTek Gen5 microplate reader and imager software, we successfully validated these markers and the corresponding antibodies as tools to study long-term effects of xenobiotics on mitochondrial bioenergetics. The method allows for fast and reliable screening of potential drug candidates. Acknowledgement: This work was supported in part by the Chan Zuckerberg Initiative, Silicon Valley Community Foundation, and the NIH S10 OD028663 to M.G. and by the U.S. National Institutes of Health (NIH/NCI) R01 CA184456, and South Carolina Translational Research Pilot Project UL1 TR001450-SCTR to E.N.M. Citation Format: Bradley R. Larson, Monika Gooz, Eduardo Maldonado. Expression and intracellular translocation of cancer biomarkers in hepatocarcinoma cells induced by changes in mitochondrial metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1382.
Bakground: Mitochondrial membrane potential (DJ) and generation of reactive oxygen species (ROS) requires a respiratory chain fueled by the flux of metabolites into mitochondria through voltage dependent anion channels (VDAC). Free tubulin induces reversible blockage of VDAC both in vitro and in cells. Erastin, a small molecule lethal to cancer cells, antagonizes the inhibitory effect of free tubulin on VDAC and upregulates mitochondrial metabolism. Here, we hypothesized that erastin and "erastin-like" compounds open VDAC, increase mitochondrial metabolism and ROS formation, and activate JNK, which in turn cause mitochondrial dysfunction. Our AIM was to evaluate the effects of erastin/erastin-like compounds on DJ, NADH, ROS and JNK in intact cells. METHODS: DJ was assessed by confocal microscopy of tetramethylrhodamine methylester (TMRM) fluorescence and ROS by chloromethyldichlorofluorescein (cmDCF) and MitoSOX Red fluorescence. Mitochondrial NADH autofluorescence was assessed by multiphoton microscopy. Total and phosphorylated JNK was determined by Western blotting. RESULTS: In lipid bilayers, erastin antagonized tubulin inhibition on VDAC. In HepG2 human hepatoma cells, erastin increased DJ by 46% and NADH by 30%. Mitochondrial hyperpolarization plateaued after 2 h. Subsequently depolarization occurred (3-4 h), suggesting mitochondrial dysfunction. Erastin-like compounds X1 and X2, identified in a highthroughput screening, similarly caused mitochondrial hyperpolarization/ depolarization. Erastin also caused increases of DCF and Mitosox Red fluorescence beginning after~30 min and reaching a maximum after 2 h. Additionally, erastin activated JNK (maximum pJNK at 60 min). JNK activation and ROS formation both preceded mitochondrial depolarization. Conclusion: Erastin relieves tubulin-dependent inhibition of VDAC conductance, leading to mitochondrial hyperpolarization that increases ROS production with concomitant activation of the stress kinase JNK. These events, in turn, may induce the mitochondrial permeability transition, mitochondrial dysfunction and ultimately cell death. The opening of a high-conductance and long-lasting mitochondrial permeability transition pore (mPTP) induces uncoupling of respiration from ADP phosphorylation, and causes mitochondrial injury and cell death. However, low-conductance and transient openings of mPTP may limit mitochondrial calcium load and mediate mitochondrial reactive oxygen species (ROS) signaling. Transient openings of mPTP have been proposed, and could be cardioprotective, but evidence for this in cells is indirect and not thoroughly studied. To address the cellular mechanism, we measured mitochondrial [Ca] ([Ca]Mito) with Rhod-2 AM and membrane potential (Dcm) in isolated single permeablized myocytes during cyclical sarcoplasmic reticulum (SR) Ca release using 2-D confocal imaging where individual mitochondria can be seen. Rapid and transient decreases in both mitochondrial [Ca] and Dcm were observed during SR Ca release. The frequency of these candidate transient mPTP...
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