Nrf2 inhibition reverses the resistance of cisplatin-resistant head and neck cancer cells to artesunate-induced ferroptosis

Abstract

Artesunate, an anti-malarial drug, has been repurposed as an anticancer drug due to its induction of cell death via reactive oxygen species (ROS) production. However, the molecular mechanisms regulating cancer cell death and the resistance of cells to artesunate remain unclear. We investigated the molecular mechanisms behind the antitumor effects of artesunate and an approach to overcome artesunate resistance in head and neck cancer (HNC). The effects of artesunate and trigonelline were tested in different HNC cell lines, including three cisplatin-resistant HNC cell lines. The effects of these drugs as well as the inhibition of Keap1, Nrf2, and HO-1 were assessed by cell viability, cell death, glutathione (GSH) and ROS production, protein expression, and mouse tumor xenograft models. Artesunate selectively killed HNC cells but not normal cells. The artesunate sensitivity was relatively low in cisplatin-resistant HNC cells. Artesunate induced ferroptosis in HNC cells by decreasing cellular GSH levels and increasing lipid ROS levels. This effect was blocked by co-incubation with ferrostatin-1 and a trolox pretreatment. Artesunate activated the Nrf2–antioxidant response element (ARE) pathway in HNC cells, which contributed to ferroptosis resistance. The silencing of Keap1, a negative regulator of Nrf2, decreased artesunate sensitivity in HNC cells. Nrf2 genetic silencing or trigonelline reversed the ferroptosis resistance of Keap1-silenced and cisplatin-resistant HNC cells to artesunate in vitro and in vivo. Nrf2–ARE pathway activation contributes to the artesunate resistance of HNC cells, and inhibition of this pathway abolishes ferroptosis-resistant HNC.Condensed abstractOur results show the effectiveness and molecular mechanism of artesunate treatment on head and neck cancer (HNC). Artesunate selectively killed HNC cells but not normal cells by inducing an iron-dependent, ROS-accumulated ferroptosis. However, this effect may be suboptimal in some cisplatin-resistant HNCs because of Nrf2–antioxidant response element (ARE) pathway activation. Inhibition of the Nrf2–ARE pathway increased artesunate sensitivity and reversed the ferroptosis resistance in resistant HNC cells.

Ferroptosis is programmed by the coordinated regulation of glutathione and iron metabolism by BACH1

“…Even though NRF2 increases the intracellular glutathione amount, it only weakly protects cells from ferroptosis (Cao et al, 2019). Other reports have shown that NRF2 can inhibit ferroptosis (Fan et al, 2017, Roh et al, 2017, Sun et al, 2016). Therefore, ferroptosis execution may depend on the initial amounts and kinetics of the induction or reduction of these transcription factors.…”

Markers of oxidative‐nitrosative stress induced by artesunate are followed by clastogenic and aneugenic effects and apoptosis in human lymphocytes

“…ARS has been found to induce ROS production and accumulation in different cancer cell lines, including the generation of O 2 − (Greenshields et al, ; Li et al, ; Roh et al, ; Wu, Zhang, Li, Yang, & Zhao, ; Yang et al, ). In contrast, ARS‐induced ROS production in control normal cells appears to be dependent on the cell type, once elevated ROS content was verified in certain kinds of cells whereas reduction or no alteration was observed in other cell types (Beccafico et al, ; Cheng et al, ).…”

“…In contrast, ARS‐induced ROS production in control normal cells appears to be dependent on the cell type, once elevated ROS content was verified in certain kinds of cells whereas reduction or no alteration was observed in other cell types (Beccafico et al, ; Cheng et al, ). This could explain, at least in part, the absence or low susceptibility of normal cells to ARS toxicity compared with neoplastic cells (Beccafico et al, ; Du et al, ; Holien et al, ; Jiang et al, ; Jing & Hui, ; Pang et al, ; Roh et al, ; Xu et al, ). Interestingly, we have previously observed that human lymphocytes were highly sensitive to ARS incubation displaying a significant increase in the number of apoptotic and necrotic cells (Mota et al, ).…”

“…ARS induced cell death of different cell types in vitro, including the Chinese hamster ovary cell line, human vascular endothelial cells and human lymphocytes (Cheng et al, ; Li et al, ; Mota et al, ). In addition, a number of cancer cell lines were observed to respond to ARS by decreasing cell viability and increasing cell death (Greenshields, Shepherd, & Hoskin, ; Li et al, ; Pang, Qin, Wu, Wang, & Chen, ; Roh, Kim, Jang, & Shin, ). Similarly, incubation with ARS led to oxidative DNA damage, induction of DNA double‐strand breaks and increase of micronuclei (MN) frequency in both cancer cell lines and normal cells, including human lymphocytes (Aquino et al, ; Aquino, Ferreira, & Luis, ; Berdelle, Nikolova, Quiros, Efferth, & Kaina, ; Li et al, ; Mota et al, ; Wang et al, ).…”

“…Likewise, both caspase-dependent and caspaseindependent mechanisms of apoptotic cell death were observed to happen in cancer cell lines subjected to ARS treatment (Greenshields et al, 2017;Ilamathi & Sivaramakrishnan, 2017;Li et al, 2017;Papanikolaou et al, 2014). Additionally, ARS triggers cell death mechanisms other than apoptosis, such as necrosis, autophagy, oncosis and ferroptosis, which might be coupled with elevated ROS production (Berdelle et al, 2011;Chen et al, 2014;Du, Zhang, Ma, & Ji, 2010;Eling, Reuter, Hazin, & Hamacher-brady, 2015;Greenshields et al, 2017;Roh et al, 2017).…”