Iron overload and accumulation are linked to various diseases like PD, AD, and neurodegeneration with brain iron accumulation (NBIA) [35]. Given the wide range of adverse effects associated with current iron chelators, exogenous antioxidants such as NAC, flavonoids, carotenoids, and diterpenes have been tested against iron overload [19, 36]. Taking into account the promising antioxidant activity and favorable pharmacokinetic and adverse effect profiles of dithiolethiones [37], we present their effects against iron-induced cytotoxicity and ferroptosis in this study.
Astrocytes have gained a spotlight as models of iron overload-induced oxidative stress [38]. These cells serve as iron stores and gatekeepers for the transport of iron and other toxic substances in the brain [39]. Therefore, any toxic damage to astrocytes is associated with neurodegeneration [40]. In our study, we used U-87 MG cells which are astrocytoma cells. The FAC salt was used to induce iron toxicity owing to its readily water-soluble property and widespread use in past iron-related studies [5].
While D3T and ACDT have previously shown cytoprotection against various toxicants, to our knowledge, this is the first study depicting their effects against iron-induced toxicity and associated cell death pathways. A 50% cytotoxicity with 10 mM FAC at 24 hours [41] and 5 mM FAC at 48 hours [42] were previously reported in SH-SY5Y neuroblastoma cells. The requirement of higher concentration of FAC (15 mM) in our study to produce similar of cell death is likely due to the cell line itself, as astrocytes can store large amounts of iron and are more resistant to toxicity compared to neurons [43]. Here, we chose the 50 µM concentration for both our test dithiolethiones based on the results from previous reports. ACDT at 50 µM concentration exerted significant neuroprotection against 6-OHDA [30] and Mn-induced cytotoxicity [31] in SH-SY5Y cells. Similarly, Li et al., showed that 50 µM D3T protected against ultra-violet radiation-induced toxicity in retinal cells [44]. We included NAC as a concentration-matched standard comparator. This dual antioxidant directly neutralizes free radicals and indirectly acts a precursor to GSH [45]. Importantly, all three compounds demonstrated an excellent counteractivity against iron overload-induced cell death (Fig. 1b and Fig. 1c). A structural comparison of D3T and ACDT with NAC reveals that dithiolethiones are at an advantage wherein they possess three sulfur groups while NAC has only one. This may help partly explain the stronger antioxidant effect observed with these compounds in some of our reported experiments.
FAC-induced oxidative stress (ROS generation and lipid peroxidation) is the principal mechanism of iron toxicity [46]. The successful mitigation of ROS production (Fig. 2b) and lipid peroxidation (Fig. 2c) by D3T and ACDT are key findings. These effects are explained by the established mechanism of a Nrf2-driven upregulation of the cellular antioxidant machinery by dithiolethiones [47]. A 4 hour FAC exposure for the ROS assay was chosen because ROS levels peaked significantly at this time point (Fig. 2a) and this is in line with the previous FAC-related studies [48]. The significant increase in intracellular GSH observed with exposure to FAC (Fig. 2d) was consistent with a past study [49] and likely due to the cellular response to FAC-induced oxidative insult. Exposure to D3T alone for 24 hours has previously been reported to increase GSH levels in SH-SY5Y cells [23] and cardiomyocytes [50]. Likewise, Betharia et al. showed that a 24 hour treatment with ACDT increases intracellular GSH in SH-SY5Y cells [30]. In our study, the FAC-induced increase in GSH levels was successfully reduced by D3T and ACDT (Fig. 2d). We hypothesize that pretreatment with dithiolethiones may have preemptively bolstered the GSH stores via Nrf2 activation thereby preventing FAC-induced oxidative stress and reducing the need for further rise in GSH production. Interestingly, this effect of dithiolethiones was similar to that observed with the well-established antioxidant NAC which increases GSH levels by acting as a source of cysteine, an amino acid required for GSH biosynthesis.
The labile iron pool is strictly regulated by various mechanisms including the Nrf2-mediated transcriptional upregulation of ferritin and ferroportin (FPN1) expression [51]. Both these proteins reduce cellular iron wherein ferritin is an intracellular iron storage protein that stores excess iron in a redox inactive form [52], while FPN1 is the iron exporter that exports iron from the cell into the plasma [53]. Incubation with ferric iron has been reported to increase ferritin expression in Caco-2 cells [54] and astrocytes [55] as a potential cellular defense mechanism against iron overload which is in line with our results (Fig. 2e). Remarkably, pretreatment with D3T and ACDT but not NAC, further increased ferritin expression against FAC. In explanation, dithiolethione-mediated activation of the Nrf2-ferritin heavy chain (Fth1)-ARE pathway can induce ferritin expression beyond normal in response to oxidative stress [56]. In our study, in contrast to the results from Tangudu and group [57], FAC did not alter FPN1 levels (Fig. 2f). However, hepcidin levels were unchanged in response to FAC in astrocytes [58]. Since hepcidin regulates FPN1 expression, the absence of FPN1 upregulation in our study is justified.
Iron overload-induced oxidative stress can cause DNA damage and trigger various cell death pathways such as apoptosis, necrosis, autophagy, and the more recently identified ferroptosis [59]. Activation of caspases and the pro-apoptotic protein Bax are reported in past studies in response to FAC exposure [60]. Since we did not observe activation of caspases (supplementary Fig. 1), we evaluated the possibility of a lesser explored necrotic cell death by FAC. Similar to studies in rat hepatocytes [61] and in bone marrow cells [62], our results indicate FAC-induced necrosis for the first time in U-87 MG cells (supplementary Fig. 2). The successful counteraction of this form of cell death by dithiolethiones can be attributed to the alleviation of ROS and MDA-induced oxidative stress by the Nrf2-mediated upregulation of GSH and ferritin levels. It is important to point out that while the effects of dithiolethiones matched those of NAC in ROS, GSH, and MDA experiments, their effects on ferritin upregulation and protection against cytotoxicity were significantly higher.
While reports on iron overload-induced necrosis are sparse, recent research has expanded to other forms of iron-induced cell death such as ferroptosis. First coined in 2012, ferroptosis is an iron-dependent, non-apoptotic cell death characterized by increased lipid peroxidation [63]. The specific ferroptosis inhibitor ferrostatin-1 is a lipophilic antioxidant which specifically inhibits ferroptosis by preventing lipid peroxidation and lacks an effect on other forms of cell death [64]. FAC-induced cell death (Fig. 3b), ROS production (Fig. 3c), and lipid peroxidation (Fig. 3d) were counteracted by ferrostatin-1, thus collectively providing the first evidence that FAC induces ferroptosis in U-87 MG cells. This is in line with a previous study where 10 µM ferrostatin-1 counteracted the lipid peroxidation and ferroptosis against FAC in primary hepatocytes [12]. Additionally, FAC-induced changes in the morphology of U-87 MG cells (Fig. 3a) were similar to those observed in three different colorectal cancer cells exposed to the ferroptosis inducer RSL3 [65].
The Nrf2 transcription factor is involved in inhibiting ferroptosis by regulating the expression of various ferroptosis-related proteins [66]. As dithiolethiones are activators of the Nrf2 pathway, we tested their potential role in inhibiting ferroptosis against the canonical ferroptosis inducers erastin and RSL3 using ferrostatin-1 as a standard comparator. Notably, ours is the first study to test the effects of erastin and RSL3 in U-87 MG cells. Erastin causes ferroptosis by inhibiting the transmembrane cystine/glutamate antiporter (xCT). This transporter normally allows the uptake of cystine into the cells which is then reduced to cysteine, a molecule indispensable in GSH synthesis. Therefore, inhibition of xCT leads to a decrease in cysteine levels and consequent intracellular GSH depletion, increased lipid peroxidation, and resulting ferroptosis [67]. Our results conform with previous reports of erastin-induced downregulation of xCT expression and increased lipid peroxidation in primary cortical neurons [68], and GSH depletion in human cervical adenocarcinoma cells [67] and HT-1080 human fibrosarcoma cells [69]. Moreover, increase in the xCT expression by D3T and ACDT against erastin (Fig. 4b) is one of our valuable findings. This effect is likely via activation of the Nrf2 pathway which is reported to control the expression of xCT antiporter [70]. Notably, the dithiolethiones also inhibited erastin-induced cell death (Fig. 4a), GSH depletion (Fig. 4c), and lipid peroxidation (Fig. 4d) providing the first evidence that they serve as ferroptosis inhibitors similar to ferrostatin-1. As Nrf2 target antioxidants are involved in upregulation of intracellular antioxidants and prevention of lipid peroxidation, the protective role of D3T and ACDT against ferroptosis is justified.
RSL3 is reported to directly inhibit the GPX4 enzyme by covalently binding to the catalytic selenocysteine moiety of GPX4 [71]. This creates an imbalance between lipid peroxidation and intracellular antioxidant capacity thus leading to ferroptosis. A study in cortical neurons showed that the exposure to RSL3 decreased GPX4 protein expression [72]. In line with a previous study [73], we found RSL3-induced lipid peroxidation (Fig. 5b) and cytotoxicity (Fig. 5a) to be successfully counteracted by dithiolethiones likely via their impact on the Nrf2 pathway. Furthermore, since the GPX4 enzyme is a Nrf2 target antioxidant, dithiolethiones have been reported to upregulate GPX4 expression [74]. Surprisingly, we saw no change in GPX4 levels with D3T, ACDT, or even ferrostatin-1 in our study (Fig. 5c). A possible explanation could be the difference in duration of exposure. A study in human primary cardiomyocytes has reported that a 48 hour exposure to 50 µM of D3T induced GPX expression [75]. Additional studies are needed to reconcile this discrepancy. A schematic representation of the effects of these dithiolethiones against iron-induced toxicity and ferroptosis is shown below (Fig. 6).