Necrostatin-1 Prevents Ferroptosis in a RIPK1- and IDO-Independent Manner in Hepatocellular Carcinoma

Abstract

Ferroptosis is caused by the iron-mediated accumulation of lipid peroxidation, which is distinct from apoptosis and necroptosis. Necrostatin-1 inhibits receptor-interacting serine/threonine-protein kinase 1 (RIPK1) to initiate necroptosis; it also inhibits indoleamine 2,3-dioxygenase (IDO) to regulate tumor immunity. However, few studies have examined the off-target effect of necrostatin-1 on the ferroptosis pathway. The present study examined whether necrostatin-1 could interrupt ferroptosis induced by system xc- inhibitors (sulfasalazine and erastin) and a glutathione peroxidase 4 inhibitor (RSL3) in Huh7 and SK-HEP-1 cells. Necrostatin-1 completely prevented decreases in cell viability induced by sulfasalazine and erastin; it partially blunted decreases in cell viability induced by RSL3. Necrostatin-1, ferrostatin-1, and deferoxamine repressed sulfasalazine-provoked membrane permeabilization, as detected by 7-aminoactinomycin D staining and lipid peroxidation measured using a C11-BODIPY probe. However, other RIPK1 inhibitors (necrostatin-1s and GSK2982772) and an IDO inhibitor (1-methyl-D-tryptophan) did not recover the decrease in cell viability induced by sulfasalazine. Necrostatin-1 potentiated sulfasalazine-induced expression of xCT, a catalytic subunit of system xc- in these cells. These results demonstrated that necrostatin-1 blocked ferroptosis through a mechanism independent from RIPK1 and IDO inhibition in Huh7 and SK-HEP-1 cells, indicating that its antioxidant activity should be considered when using necrostatin-1 as a RIPK1 inhibitor.

Acute lymphoblastic leukemia necessitates GSH-dependent ferroptosis defenses to overcome FSP1-epigenetic silencing

“…2 D and E). Interestingly, Nec-1 also prevented RSL3 toxicity, in agreement with a recent report showing that Nec-1 prevents ferroptosis independently of RIPK1 [ 33 ]. On the other hand, QVD mildly reverted L-BSO toxicity in Jurkat cells, suggesting at least in part cell death is executed by apoptosis in Jurkat cells upon GSH depletion.…”

Extracellular Concentration of L-Cystine Determines the Sensitivity to System xc- Inhibitors

“…2B ). The effect of extracellular L-cystine on the sensitivity to sulfasalazine and erastin was also tested in SK-HEP-1 cells which are sensitive to ferroptosis ( Yuk et al ., 2021 ). SK-HEP-1 cells showed significantly higher percentage of PI-positive cell fraction in media containing 83 µM L-cystine than 200 µM L-cystine.…”

“…This study investigated the effects of two concentrations of L-cystine representing physiological condition and routine culture medium on ferroptosis induced by sulfasalazine, erastin, and RSL3. Ferroptosis was determined by cell viability, membrane permeabilization, and lipid peroxidation in Huh6, Huh7, and SK-HEP-1 hepatocellular carcinoma cell lines which were susceptible to ferroptotic cell death in our previous study ( Kim et al ., 2019 ; Yuk et al ., 2021 ).…”

Perspectives and mechanisms for targeting ferroptosis in the treatment of hepatocellular carcinoma

“…By maintaining the protein stability, nuclear localization and transcriptional activity of the transcriptional activator factor (ATF4), HCC cells inhibit SOR-induced ferroptosis ( Gao et al, 2021 ). In addition, Necrostatin-1 inhibits System xc − mediated ferroptosis in Huh7 and SK-HEP-1 cells probably by inducing xCT expression ( Yuk et al, 2021 ).…”

“…-MnMSN@SFB) Depletion of HSG, suppression of System xc -Tang H et al (2019); Tang H et al (2020) HKUST-1 Integrates cyclooxygenase 2 (COX-2), which depletes GSH and inhibits GPX4 activity; induces PINK1/Parkin-mediated mitochondrial autophagy Gu et al (2011); Tian et al (2022) MIL-101(Fe)@sor NPs Increased lipid peroxidation and MDA levels, decreased GSH and GPX4 Liu et al Induces the expression of SLC7A11 and inhibits ferroptosis Gao et al (2021)Necrostatin-1 Induces the expression of SLC7A11 and inhibits ferroptosisYuk et al (2021) …”