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. 2023 Sep 27;14(9):637.
doi: 10.1038/s41419-023-06153-9.

Dehydroascorbic acid sensitizes cancer cells to system xc- inhibition-induced ferroptosis by promoting lipid droplet peroxidation

Luciano Ferrada  1 , María José Barahona  2   3 , Matías Vera  2 , Brent R Stockwell  4   5 , Francisco Nualart  2   3
Affiliations

Dehydroascorbic acid sensitizes cancer cells to system xc- inhibition-induced ferroptosis by promoting lipid droplet peroxidation

Luciano Ferrada et al. Cell Death Dis. .

Abstract

Since the discovery of ferroptosis, it has been postulated that this type of cell death could be utilized in treatments for cancer. Unfortunately, several highly aggressive tumor models are resistant to the pharmacological induction of ferroptosis. However, with the use of combined therapies, it is possible to recover sensitivity to ferroptosis in certain cellular models. Here, we discovered that co-treatment with the metabolically stable ferroptosis inducer imidazole ketone erastin (IKE) and the oxidized form of vitamin C, dehydroascorbic acid (DHAA), is a powerful therapy that induces ferroptosis in tumor cells previously resistant to IKE-induced ferroptosis. We determined that DHAA and IKE + DHAA delocalize and deplete GPX4 in tumor cells, specifically inducing lipid droplet peroxidation, which leads to ferroptosis. Moreover, in vivo, IKE + DHAA has high efficacy with regard to the eradication of highly aggressive tumors such as glioblastomas. Thus, the use of IKE + DHAA could be an effective and safe therapy for the eradication of difficult-to-treat cancers.

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Conflict of interest statement

BRS is an inventor on patents and patent applications involving ferroptosis; co-founded and serves as a consultant to ProJenX, Inc. and Exarta Therapeutics; holds equity in Sonata Therapeutics; serves as a consultant to Weatherwax Biotechnologies Corporation and Akin Gump Strauss Hauer & Feld LLP; and receives sponsored research support from Sumitomo Dainippon Pharma Oncology. The other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. IKE + DHAA treatment induces ferroptosis in GBM and PCa models.
a IncuCyte dose-response curve analysis showing that GBM cells exhibit resistance to IKE-induced ferroptosis. b IncuCyte dose-response curve analysis showing that GBM cells can be sensitized to death by IKE + DHAA co-treatment. c, d Dose-response curve of the effect of IKE plus DHAA co-treatment on cell death PCa models determined by IncuCyte. e, f Incucyte time-plot showing that IKE + DHAA induces ferroptosis in GBM and PCa. g Quantification of GSH levels. h BSO + DHAA fails to induce ferroptosis in GBM and PCa. i Cyst(e)ine depletion and DHAA co-treatment recapitulates the effects of IKE + DHAA to induce ferroptosis. j IncuCyte analysis showing that pharmacological doses of AA, but not DHAA, induce cell death in 22Rv1 cells. k Pharmacological doses of AA induce cell death by production of H2O2, but not by ferroptosis. l IncuCyte analysis showing that IKE + DHAA mediated death induction does not involve necroptosis, apoptosis or H2O2 production. In panels k and l the compounds were used at the following concentrations: Fer 5 μM; Trolox 100 μM; Catalase 200 U/mL; Nec-1 30 μM, Nec-1s 10 μM and Z-VAD-FMK 30 μM. Data are presented as mean ± SEM, from at least three independent biological replicates. ***P < 0.001; **P < 0.01, *P < 0.05, and n.s., not significant (P > 0.05).
Fig. 2
Fig. 2. IKE + DHAA induces tumor death by ferroptosis in 3D models.
a, b Death kinetics assays in 3D spheroids of U87 and 22Rv1 cells determined by IncuCyte and incorporation of Sytox green. c, d IKE + DHAA induces ferroptotic death in 3D models of GBM and PCa. e, f Treatment with IKE, DHAA (2 mM) or IKE + DHAA does not have toxic effects on primary neurospheres. Data are presented as mean ± SEM, from at least three independent biological replicates.
Fig. 3
Fig. 3. IKE + DHAA induces ferroptosis by peroxidation of lipid droplets.
a, b, c Determination of mitochondrial ROS by FACS in U87 and 22Rv1 cells. d, e Determination of total ROS by FACS in U87 and 22Rv1 cells. f, g Determination of lipid ROS by FACS in U87 and 22Rv1. h Super-resolution live-cell microscopy using bodipy C-11 for lipid staining (green, oxidized lipids; red, reduced lipids), cellmask as a membrane marker (white) and Hoechst as a nuclear marker (blue), where it is observed that IKE + DHAA does not induce lipid peroxidation at the plasma membrane. i Super-resolution live-cell microscopy using bodipy C-11 for oxidized lipid staining (green), LipidSpot 610 for lipid droplet labeling (magenta) and Hoechst as nuclear marker (blue), where DHAA and IKE+ are observed DHAA induces lipid droplet peroxidation, which is completely inhibited by blocking DGAT1i. j, k IncuCyte analysis showing that inhibition of lipid droplet biosynthesis driven by DGAT1 blockade prevents IKE + DHAA-induced ferroptosis (n = 3 independent biological replicates). l Representative photomicrographs of j and k. m Validation of Dgat1 knockdown by qrt-PCR in 22Rv1 cells. n Dgat1 knockdown inhibits IKE + DHAA-induced. o, p IncuCyte analysis showing that inhibition of lipid droplet biosynthesis driven by DGAT1 blockade does not prevent ferroptosis in IKE-sensitive cells. In all cases, DHAA was used at a concentration of 1 mM and IKE was used at 5 µM for U87 cells and 2 µM for 22Rv1 cells. Data are presented as mean ± SEM, from at least three independent biological replicates. ***P < 0.001; **P < 0.01, *P < 0.05; and n.s., not significant (P > 0.05).
Fig. 4
Fig. 4. IKE + DHAA induces depletion and delocalization of GPX4 from lipid droplets.
a Western blot analysis of GPX4 abundance. b Quantification of GPX4 abundance in U87 cells. c qRT-PCR of GPX4 mRNA levels in U87 and 22Rv1 cells at 7 and 5 h post-treatment, respectively. d, e IncuCyte analysis showing that GPX4 overexpression inhibits IKE + DHAA-induced ferroptosis. f Lightning super-resolution microscopy showing GPX4 delocalization in lipid droplets in 22Rv1. g Lightning super-resolution microscopy showing GPX4 delocalization and inaccessibility in lipid droplets in U87. h 3D reconstruction of the large lipid droplet and GPX4 in U87 cells. i, j 3D live cell microscopy showing that large lipid droplets are specifically peroxidized in U87 and 22Rv1 cells. Data are presented as mean ± SEM, from at least three independent biological replicates. ***P < 0.001, **P < 0.01, *P < 0.05, and n.s., not significant (P > 0.05).
Fig. 5
Fig. 5. Intratumorally injection of IKE + DHAA induces GBM eradication.
a Scheme of the tumor induction and treatment protocol. b Histological analysis by H&E of the effect of IKE and IKE + DHAA at 7 days post treatment. c Tumor area quantification. n = 7–12 mice per condition. Each point represents a brain slice from a different analyzed animal. d H&E analysis of third ventricle invasion. e Quantification of tumor invasion in the anteroposterior axis. n = 5 mice per condition. f Quantification of GBM cell invasion of the third ventricle. n = 5 mice per condition g Confocal microscopy analysis of GBM cells in vivo. h Super-resolution analysis for the identification of ferroptotic cells (3F3-FMA stain, green) and lipid peroxidation (MDA stain, red). Data are presented as mean ± SEM. ***P < 0.01, **P < 0,01, *P < 0.05; and n.s., not significant (P > 0.05).
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