2. Natural Compounds as Ferroptosis Inducers

Several natural compounds, alone or in combination, have been found to induce ferroptosis in different in vitro cancer models (Table 1).

Amentoflavone is a flavonoid mainly found in Selaginella tamariscina (P. Beauv.) Spring and in other species of Selaginella, as well as in many other plant species [55]. Amentoflavone exhibits anticancer effects in several tumor cells by inducing apoptosis, autophagy and ferroptosis, and by inhibiting cell-cycle progression [27,56,57,58,59,60,61]. In U251 and U373 glioma cell lines and in a glioma xenograft model, but not in normal human astrocytes, it triggered ferroptotic cell death by reducing GSH and ferritin heavy chain (FTH) intracellular levels, thus leading to the accumulation of lipid ROS and malondialdehyde (MDA), a PUFAs oxidation product, and subsequent cell death [27] (Table 1). Hence, amentoflavone induces ferroptosis through the rupture of iron homeostasis by reducing the intracellular levels of FTH, which is involved in the intracellular iron storage [27]. Interestingly, both in vitro and in vivo, amentoflavone induced the degradation of FTH by activating autophagy via AMPK (AMP-activated protein kinase)/mTOR (mammalian target of rapamycin)/P70S6K (phosphoprotein 70 ribosomial protein S6 kinase) signaling pathway, suggesting the induction of autophagy-dependent ferroptosis [27]. Autophagy is known as a potent ferroptosis enhancer. Ferritinophagy, in particular, degrades the iron storage protein ferritin and increases the release of free iron, leading to ferroptosis induction [62,63,64].

Two other natural compounds that trigger autophagy-dependent ferroptosis are dihydroartemisinin (DHA) and typhaneoside. DHA is a semi-synthetic derivative of artemisinin, a sesquiterpene lactone derived from Artemisia annua L. currently used as antimalarial agent, which promotes ferroptosis in glioma cells [40] and ferroptosis together with apoptosis in acute myeloid leukemia (AML) cancer cells [39] (Table 1). Typhaneoside, a flavonoid found in the extract of Pollen Typhae, triggered apoptotic and ferroptotic cell death in AML cancer cells [52] (Table 1). In particular, in AML cancer cells, as for amentoflavone, both DHA and typhaneoside induced autophagy-dependent ferroptosis [39,52] by raising the degradation of ferritin through ferritinophagy; moreover, autophagy inhibition mitigated ferroptosis induction by the two natural compounds [39,52] (Table 1). In another experimental setting, DHA did not trigger ferroptosis itself, but it sensitized resistant cancer cells to ferroptosis. In particular, in vitro [mouse embryonic fibroblasts (MEFs) and human osteosarcoma HT1080 cells] and in vivo (GPX4 iKO H292-xenografted female athymic nude-Foxn 1^nu/Foxn1⁺ mice), DHA perturbed iron homeostasis leading to an increase in intracellular iron levels, which concurred to the restoration of RSL3′s and erastin’s ability to induce ferroptosis (Table 1) [65].

Artesunate is another semi-synthetic derivative of artemisinin. It induces ferroptosis in pancreatic [34,36], ovarian [35], head and neck cancer (HNC) [33], T-cell leukemia/lymphoma (ATLL) [32], and in Burkitt’s lymphoma [31] through the modulation of different molecular targets (Table 1). One of these targets is the endoplasmic reticulum (ER). ER stress is a condition of oxidative stress and perturbations in the ER folding machinery provoked by the accumulation of unfolded/misfolded proteins. ER stress activates a signaling process, called unfolded protein response (UPR), in order to lessen ER stress and to restore ER homeostasis [66,67]. In DAUDI and CA-46 lymphoma cells, artesunate triggered ferroptosis and ER stress through the activation of ATF4 (activating transcription factor-4)-CHOP (C/EBP [CCAAT-enhancer-binding protein] homologous protein)-CHAC1 (glutathione-specific γ-glutamylcyclotransferase 1) pathway and PERK [protein kinase RNA (PKR)-like ER kinase] branch of UPR [31] (Table 1). As proposed by the authors [31], the upregulation of CHAC1 possessing a GSH degradation activity [68,69] probably contributes to artesunate-induced ferroptosis [31]. Besides, it is well known that ATF4 could be upregulated by the depletion of amino acids [70], such as that of intracellular cysteine caused by ferroptosis inducers through the system X_c⁻ inhibition. Hence, artesunate might induce ER stress in Burkitt’s lymphoma cells by altering the system X_c⁻, even if it has to be confirmed. ER stress is also involved in artesunate-induced ferroptosis in KRas mutant pancreatic cancer cells (PaTU8988 and AsPC-1) and in AsPC-1 xenografted BALB/c nude mice [34] (Table 1 and Table S2). Indeed, knockdown of glucose-regulated protein 78 (GRP78), which is considered the master regulator of the UPR signaling process [71], and the inhibition of the three UPR transducers [PERK, IRE1 (inositol requiring protein-1) and ATF6 (activating transcription factor-6] [72], enhanced artesunate-induced ferroptosis in vitro and in vivo [34] (Table 1). Of note, artesunate triggered ferroptosis in a most efficient way in pancreatic cancer cells carrying mutationally-active KRas mutations (i.e., AsPC-1) rather than in pancreatic cancer cells expressing wild type KRas (i.e., COLO-357 and BxPC-3) [36]. This outcome is not odd since KRas mutation often leads to low antioxidant ferritin and transferrin levels and increased number of transferrin receptors and may sensitize pancreatic adenocarcinomas to ferroptosis [33,73]. Still, given that KRas mutant tumors are hardly druggable, these results are quite auspicious [33,74,75].

The role of nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) in ferroptosis is still a matter of debate. Normally, Nrf2 is kept inactivated by Kelch-like ECH-associated protein 1 (Keap-1). Under increased oxidative stress conditions, Nrf2 dissociates from Keap1, translocates into the nucleus, and starts the transcription of the so-called antioxidant responsive element (ARE)-dependent genes [76,77,78]. Most of the Nrf2 target genes are involved in the maintenance of redox homeostasis [79,80], including the regulation of system X_c⁻ [81,82,83], and also in iron and heme homeostasis. They regulate heme-oxygenase 1 (HO-1), ferroportin, and light chain and heavy chain of ferritin (FTL/FTH1) [76,84,85]. In other words, Nrf2 activation could be considered a negative ferroptosis regulator since it endorses antioxidant elements and iron storage, and limits cellular ROS production [86]. Furthermore, since it has been shown that ferroptosis inducers capable of activating Nrf2 pathway promote cellular adaptation and survival and render cancer cells less sensitive to ferroptosis induction themselves [87,88,89], it could be thought that they cannot be considered good candidates for anticancer therapy. However, the activation of Nrf2 pathway could promote ferroptotic cell death. Shifting the focus from the antioxidant properties of Nrf2 effectors to their ability in increasing intracellular iron content, that evidence is not surprising. For instance, HO-1 is responsible for heme catabolism, which produces iron, monoxide, and biliverdin. Thus, it is plausible assuming that the Nrf2 antioxidant response cannot balance the strong iron production, which leads cells to ferroptosis [90]. Accordingly, Kwon et al. [90] demonstrated that hemin, the most prevalent heme metabolite originated by HO-1 catabolism, induced lipid peroxidation as a consequence of iron increase [90]. The opposite role of Nrf2 in ferroptosis seems to be cell-type specific [91], since the activation of Nrf2 pathway protected hepatocellular carcinoma cells against ferroptosis [87], while it promoted ferroptosis in neuroblastoma [54]. Taken together, those results support the hypothesis that Nrf2 could act as a double-edge sword. Even if further studies are needed to disentangle this knot, artesunate supports this hypothesis inducing different effects in different cell lines.

In HNC cells, but not in human oral keratinocytes and fibroblasts, artesunate decreased GSH intracellular levels and increased lipid ROS production and led to ferroptosis [33] (Table 1). However, in HNC cells and cisplatin-resistant HNC cells, artesunate activated the Nrf2 pathway [33] (Table 1), favoring the onset of ferroptosis resistance. As a matter of fact, Keap1 silencing decreased cancer cells’ sensitivity towards artesunate-mediated ferroptosis in both resistant and non-resistant cells, while Nrf2 silencing restored the ability of inducing ferroptosis [33]. In Panc-1 pancreatic cancer cells, induction of ferroptosis by artesunate was accompanied by an increase of HO-1 protein expression [36] (Table 1), which authors associated with the ability of artesunate to increase ROS levels, that in turn activates Nrf2-mediated antioxidant response. Hence, it could be postulated that artesunate induces ferroptosis in pancreatic cancer cells through the HO-1-mediated enhancement of intracellular labile iron (LIP) (i.e., ionic Fe complexes that are redox active). Promotion of ferroptosis by artesunate has been reported also in vivo. In Burkitt’s lymphoma xenograft model, it suppressed tumor growth by inducing lipid peroxidation [31].

Withaferin A (WA) is a naturally occurring steroidal lactone derived from Withania somnifera, a medicinal plant used in Ayurvedic medicine [92]. In a variety of cancer cells, WA showed to exhibit anticancer activity through a plethora of mechanisms, including proteasome and cell-cycle inhibition, modulation of oxidative stress, and induction of apoptosis [92]. In neuroblastoma cells, WA promoted ferroptosis through a dual mechanism: at high dose (10 μM), WA directly binds and inactivates GPX4, thus inducing canonical ferroptosis; at lower dose (1 μM), WA targets Keap1 and activates the Nrf2 pathway, leading to an excessive upregulation of HO-1 and a subsequent LIP increase [54] (Table 1). Through these two mechanisms, WA also promoted ferroptosis and eradicated neuroblastoma xenografts in BALB/c mice [54]. Of note, WA outperformed the full-blown chemotherapeutic agent etoposide both in vitro and in vivo. In vitro, WA efficiently killed a panel of high-risk and etoposide-resistant neuroblastoma cells by inducing ferroptosis [54] (Table 1). In vivo, WA intratumoral administration showed the same efficacy of etoposide in suppressing tumor growth [54]. Most importantly, in contrast to etoposide, WA treatment also repressed neuroblastoma relapse rates in four out of five mice [54]. Hence, taking into account that WA-induced cell death was associated with CD45-positive immune cells infiltration in tumor tissue [54], we could speculate that WA could have activated the immune system, thus inducing an anticancer vaccine effect. This result may itself be a further step in demonstrating that ferroptosis possesses an immunogenic nature, especially in light of the recent confirmation that ferroptosis could promote antitumor immunity [93]. The only sore point of the study, together with the small number of animals used in the experimentation, was the toxic effect of WA observed in vivo. Since upon WA-systemic injection severe weight loss–related adverse effects were detected and given the scarce water solubility of WA, authors formulated WA-encapsulated nanoparticles (WA-NPs) [54]. WA-NPs showed the same efficacy of non-encapsulated WA in vitro (Table 1) and in vivo, constraining systemic side-effects induced by WA, thus allowing systemic application and an effective tumor targeting of WA [54].