Original ArticlesCISD2 inhibition overcomes resistance to sulfasalazine-induced ferroptotic cell death in head and neck cancer
Introduction
Sulfasalazine (SAS) has been commonly used to treat inflammatory arthritis and bowel diseases, including rheumatoid arthritis, ulcerative colitis and Crohn's disease [1]. It was repurposed for inducing the death of therapy-resistant cancer cells, either as a monotherapy or in combination with other chemotherapeutic drugs or radiotherapy [[2], [3], [4]]. SAS exhibits anti-inflammatory and anti-cancer effects by inhibiting the system xc− -cystine/glutamate antiporter (xCT) [5]. xCT exchanges extracellular cystine for intracellular glutamate, as a source for the major cellular antioxidant, glutathione (GSH) [6]. SAS-induced cystine depletion leads to a marked reduction in cellular GSH and a marked inhibition of in vivo tumor growth without severe toxicity [7]. The SAS-induced inhibition of xCT sensitizes cancer cells to ferroptosis, a recently recognized form of iron-dependent, non-apoptotic cell death [8].
Ferroptosis is a novel form of regulated cell death that occurs via iron accumulation and lipid peroxidation, distinct from apoptosis, necroptosis, and autophagic cell death [9]. The key molecules related to ferroptosis include xCT and glutathione peroxidase (GPX4), an essential regulator of ferroptosis that suppresses lipid peroxidation [6,10]. Inhibition of xCT and GPX4 may eradicate cancer cells resistant to conventional chemotherapy or radiotherapy [9]. Inhibition of xCT induces GSH depletion by blocking cystine uptake and sensitizes cancer cells to chemotherapeutic agents [11,12]. GPX4 inhibition also sensitizes mesenchymal therapy-tolerant, persistent cancer cells to ferroptotic cancer cell death [13].
Nutrient-deprivation autophagy factor-1 (NAF-1) is a member of iron-sulfur (FeS) protein family, encoded by CDGSH iron sulfur domain 2 (CISD2) gene [14]. The protein functions to transfer its 2Fe-2S cluster to an apo-acceptor protein and iron to mitochondria [15]. The NEET proteins mitoNEET (encoded by CISD1) and NAF-1 interact together by transferring 2F2-2 S cluster to maintain the levels of iron in the mitochondria [16]. The NEET proteins are required for cell proliferation and resistance to oxidative stress [17]. Overexpression of NAF-1 or mitoNEET is associated with aggressive phenotypes and clinical outcomes of various human cancers, and silencing of its expression inhibits tumor proliferation [[17], [18], [19], [20]]. The mitoNEET was recently reported as a negative regulator of ferroptotic cancer cell death that protects against mitochondrial lipid peroxidation [21]. In addition, the 2Fe-2S cluster biosynthetic enzyme NFS1 also confers the protection of lung cancer cells from ferroptotic cell death to oxidative damage under high oxygen environment [22].
Resistance to xCT inhibition allows therapy-resistant cancer cells to evade cell death including ferroptosis. NAF-1, as an interacting protein with mitoNEET [16], might be also involved in the mechanisms of resistance to ferroptotic cancer cell death. NAF-1 is localized to the outer mitochondrial membrane and endoplasmic reticulum (ER) where may serve as the cytoplasmic place of a selective cargo receptor for ferritinophagy highly enriched with NCOA4 [23]. Therefore, NAF-1 might be implicated in ferroptosis by the modulation of iron-sulfur cluster and free iron as previously reported in other key proteins, such as mitoNEET and NFS1 [21,22]. Further understanding of the mechanisms underlying resistance to ferroptosis would facilitate the implementation of new approaches to overcome cancer resistance. Therefore, this study sought to investigate the molecular mechanism of resistance to SAS-induced ferroptosis in head and neck cancer (HNC). CISD2 expression was related to the resistance to SAS therapy and CISD2 inhibition rendered resistant HNC susceptible to SAS-induced ferroptotic cancer cell death in vitro and in vivo.
Section snippets
Cell culture and reagents
The HNC cell line (AMC-HN2–11) established in our hospital [24] and the SNU cell lines (SNU-1041, -1066, and -1076) that were purchased from the Korea Cell Line Bank (Seoul, Republic of Korea) were employed in this study. All cell lines were derived from the head and neck, e.g. the larynx, hypopharynx, oral cavity, and nasal cavity. The cell lines were authenticated via short tandem repeat-based DNA fingerprinting and multiplex polymerase chain reaction (PCR). The cells were cultured in Eagle's
SAS induces ferroptotic cell death in HNC at different levels
SAS decreased the viability of HNC cells in a dose-dependent manner, with HN9 and HN11 cells showing the highest sensitivity and HN10 cells showing the lowest (P < 0.01) (Fig. 1A–B). The IC50 of SAS significantly differed between HN10 and HN11 cell lines that were chosen for the following experiments (0.95 vs 0.17 mM, P < 0.01). Increased PI staining was observed in HN11 cells but no significant change was noted in HN10 cells when treated with 0.5 mM SAS (P < 0.01) (Fig. 1C). However, GSH
Discussion
Cancer chemotherapy is increasingly used as an organ-preserving treatment strategy for HNC [30,31]. Platinum drugs and molecular targeted agents are commonly associated with acquired resistance and increased toxicity, which together contribute to poor treatment outcome [32]. SAS, an old anti-inflammatory drug, was repurposed to induce ferroptotic cancer cell death by inhibition of xCT [5,7,9]. Recently, inhibition of xCT was found to sensitize therapy-resistant cancer cells to ferroptotic cell
Conflicts of interests
The authors declare no conflict of interest.
Acknowledgements
This study was supported by a grant (no. 2015R1A2A1A15054540) from the Basic Science Research Program through the National Research Foundation of Korea (NRF), Ministry of Science and ICT, Seoul, Republic of Korea (J.-L. Roh).
References (40)
- et al.
Ferroptosis: an iron-dependent form of nonapoptotic cell death
Cell
(2012) - et al.
Regulation of ferroptotic cancer cell death by GPX4
Cell
(2014) - et al.
CISD1 inhibits ferroptosis by protection against mitochondrial lipid peroxidation
Biochem. Biophys. Res. Commun.
(2016) - et al.
CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth
Canc. Cell
(2011) - et al.
Pioglitazone, an anti-diabetic drug requires sustained MAPK activation for its anti-tumor activity in MCF7 breast cancer cells, independent of PPAR-gamma pathway
Pharmacol. Rep.
(2016) - et al.
Pathway analysis of genome-wide association study on serum prostate-specific antigen levels
Gene
(2014) - et al.
Management of patients with inflammatory bowel disease and spondyloarthritis
Expet Rev. Clin. Pharmacol.
(2017) - et al.
Drug repurposing: sulfasalazine sensitizes gliomas to gamma knife radiosurgery by blocking cystine uptake through system Xc-, leading to glutathione depletion
Oncogene
(2015) - et al.
Potential use of the anti-inflammatory drug, sulfasalazine, for targeted therapy of pancreatic cancer
Curr. Oncol.
(2010) - et al.
Phase 1 study of sulfasalazine and cisplatin for patients with CD44v-positive gastric cancer refractory to cisplatin (EPOC1407)
Gastric Canc. Official J. Int. Gastric Canc. Assoc. Japanese Gastric Canc. Assoc.
(2017)
The xc- cystine/glutamate antiporter as a potential therapeutic target for small-cell lung cancer: use of sulfasalazine
Canc. Chemother. Pharmacol.
Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis
Elife
Sulfasalazine-induced cystine starvation: potential use for prostate cancer therapy
Prostate
Ferroptosis: process and function
Cell Death Differ.
xCT inhibition depletes CD44v-expressing tumor cells that are resistant to EGFR-targeted therapy in head and neck squamous cell carcinoma
Canc. Res.
Inhibiting the system xC(-)/glutathione axis selectively targets cancers with mutant-p53 accumulation
Nat. Commun.
Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition
Nature
Nutrient-deprivation autophagy factor-1 (NAF-1): biochemical properties of a novel cellular target for anti-diabetic drugs
PLoS One
A role for the CISD2 gene in lifespan control and human disease
Ann. N. Y. Acad. Sci.
Interactions between mitoNEET and NAF-1 in cells
PLoS One
Cited by (186)
Ferroptosis mechanisms and its novel potential therapeutic targets for DLBCL
2024, Biomedicine and PharmacotherapyDefect engineering to tailor structure-activity relationship in biodegradable nanozymes for tumor therapy by dual-channel death strategies
2024, Journal of Controlled ReleaseMagnetic nanoparticles for ferroptosis cancer therapy with diagnostic imaging
2024, Bioactive MaterialsUnderstanding sorafenib-induced ferroptosis and resistance mechanisms: Implications for cancer therapy
2023, European Journal of PharmacologyNon-coding RNA-mediated modulation of ferroptosis in cardiovascular diseases
2023, Biomedicine and Pharmacotherapy