Cancer Letters

Cancer Letters

Volume 432, 28 September 2018, Pages 180-190
Cancer Letters

Original Articles
CISD2 inhibition overcomes resistance to sulfasalazine-induced ferroptotic cell death in head and neck cancer

https://doi.org/10.1016/j.canlet.2018.06.018Get rights and content

Highlights

  • SAS induced ferroptotic cell death in HNC cells at different levels.

  • CISD2 expression was associated with resistance to ferroptosis by SAS.

  • CISD2 overexpression rendered cancer cells resistant to SAS-induced ferroptosis.

  • CISD2 gene silencing reversed the resistance to SAS-induced ferroptosis.

  • PGZ sensitized HNC cells to SAS treatment in vitro and in vivo.

Abstract

Sulfasalazine has been repurposed to induce ferroptotic cancer cell death via inhibition of xc-cystine/glutamate antiporter (xCT). However, cancer cells are capable of developing mechanisms to evade cell death. Therefore, we sought to determine the molecular mechanisms underlying resistance to sulfasalazine-induced ferroptosis in head and neck cancer (HNC). The effects of sulfasalazine and pioglitazone were tested in various HNC cell lines. The effects of these drugs and inhibition and overexpression of CISD2 gene were determined by evaluating viability, cell death, lipid ROS production, mitochondrial iron, and mouse tumor xenograft models. SAS induced ferroptotic cell death in HNC at different levels. CISD2 expression showed an association between its expression and ferroptosis resistance. CISD2 overexpression conferred resistance to ferroptosis by sulfasalazine. Silencing CISD2 gene rendered resistant HNC cells susceptible to sulfasalazine-induced ferroptosis, with increased levels of lipid ROS and mitochondrial ferrous iron. Pioglitazone induced over-accumulation of mitochondrial iron and ROS and sensitized resistant HNC cells to sulfasalazine treatment in vitro and in a mouse tumor-xenograft model. CISD2 inhibition overcomes HNC resistance to ferroptotic cell death induced by sulfasalazine via increased accumulation of mitochondrial ferrous iron and lipid ROS.

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).

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