Inducing ferroptosis by traditional medicines: a novel approach to reverse chemoresistance in lung cancer

Lung cancer is the leading cause of global cancer-related deaths. Platinum-based chemotherapy is the first-line treatment for the most common type of lung cancer, i.e., non-small-cell lung cancer (NSCLC), but its therapeutic efficiency is limited by chemotherapeutic resistance. Therefore, it is vital to develop effective therapeutic modalities that bypass the common molecular mechanisms associated with chemotherapeutic resistance. Ferroptosis is a form of non-apoptotic regulated cell death characterized by iron-dependent lipid peroxidation (LPO). Ferroptosis is crucial for the proper therapeutic efficacy of lung cancer-associated chemotherapies. If targeted as a novel therapeutic mechanism, ferroptosis modulators present new opportunities for increasing the therapeutic efficacy of lung cancer chemotherapy. Emerging studies have revealed that the pharmacological induction of ferroptosis using natural compounds boosts the efficacy of chemotherapy in lung cancer or drug-resistant cancer. In this review, we first discuss chemotherapeutic resistance (or chemoresistance) in lung cancer and introduce the core mechanisms behind ferroptosis. Then, we comprehensively summarize the small-molecule compounds sourced from traditional medicines that may boost the anti-tumor activity of current chemotherapeutic agents and overcome chemotherapeutic resistance in NSCLC. Cumulatively, we suggest that traditional medicines with ferroptosis-related anticancer activity could serve as a starting point to overcome chemotherapeutic resistance in NSCLC by inducing ferroptosis, highlighting new potential therapeutic regimens used to overcome chemoresistance in NSCLC.


Introduction
Lung cancer is broadly classified into two types: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC).SCLC and NSCLC comprise >85% of all cases, are highly prevalent, and are very aggressive, with an estimated 2.2 million new cases and 1.8 million deaths in 2020 (Leiter et al., 2023).Globally, lung cancer is the second leading cause of cancer death after breast cancer in women and is the leading cause of cancer mortality in men (Sung et al., 2021).
Although multiple approaches including surgery, immunotherapy, targeted therapy, and radiotherapy are recommended for NSCLC patients, systemic chemotherapy is still the mainstay regimen for NSCLC, especially for advanced-stage patients.Platinum-based chemotherapy is recommended as the standard first-line regimen for patients with advanced NSCLC and is also prescribed for patients at earlier stages (Nagasaka and Gadgeel, 2018).Platinum-based chemotherapy is frequently combined with gemcitabine, pemetrexed, and vinorelbine or taxanes as first-line therapeutic regimens for NSCLC.However, the therapeutic efficacy of this regimen varies remarkably among individuals and is limited by chemoresistance (Yin et al., 2016).Therefore, understanding the novel molecular mechanism behind chemoresistance in lung cancer will be vital to develop effective therapies (Herbst et al., 2018;Lim and Ma, 2019).
Recent publications have discovered that using traditional medicines to pharmacologically induce ferroptosis holds great therapeutic potential by either boosting the efficacy of chemotherapy or overcoming chemoresistance in NSCLC.In this review, we first introduce the role of chemoresistance in lung cancer and then discuss the core mechanisms of ferroptosis.We then comprehensively summarize small-molecule compounds from traditional medicines that may boost the anti-tumor activity of chemotherapeutic agents or overcome chemotherapy drug resistance in NSCLC.Cumulatively, we suggest that the pharmacological induction of ferroptosis by traditional medicines with ferroptosis-related anticancer activity could overcome chemotherapy resistance in NSCLC, potentially producing therapeutic regimens that may overcome chemoresistance in NSCLC.

Chemoresistance in lung cancer
Chemotherapy remains a dominant treatment cornerstone for many types of cancers at different stages (El-Hussein et al., 2021).
Conventional chemotherapy remains a cornerstone in the treatment of patients with NSCLC, especially those with advanced-stage disease (Min and Lee, 2021).Platinum-based chemotherapy is still the standard treatment option and mainstay regimen for patients with SCLC (Herzog et al., 2021).However, the development of chemoresistance, i.e., resistance to chemotherapeutic agents, poses a significant challenge and obstacle to the treatment efficiency of patients with NSCLC (Min and Lee, 2021).Although most patients with SCLC initially have a good response to platinum-based chemotherapy, most patients develop chemoresistance within 1 year (Jin et al., 2023), making chemoresistance almost a universal driving factor behind patient mortality (Herzog et al., 2021).Therefore, it is necessary to understand the mechanisms underlying chemoresistance to develop efficacious chemotherapeutic approaches for lung cancer.

Iron homeostasis
Iron functions as a cofactor for iron-dependent enzymes, i.e., arachidonate lipoxygenases (ALOXs) and cytochrome P450 oxidoreductase (POR), or catalyzes the Fenton reaction to promote LPO during the process of ferroptosis (Lei et al., 2022).The overproduction of ROS and reactive nitrogen species (RNS) can directly damage lipid membranes.In an iron-catalyzed process, ROS (such as LO • or HO • ) can react with polyunsaturated fatty acid (PUFA)-containing phospholipids (PUFA-PLs) to produce lipid hydroperoxides through the Fenton reaction.The Fenton reaction is an Fe 2+ -catalyzed reaction that converts hydrogen peroxide (H 2 O 2 ) to toxic HO • , triggering LPO (Ryter et al., 2007;Dos Santos et al., 2023).In the enzymatic LPO pathway, Fe 2+ promotes the activity of iron-dependent peroxidases, in which LOXs initiate the dioxygenation of the membrane PUFA-PLs (Chen et al., 2020;David et al., 2022).

Lipid peroxidation
PUFA-PLs are the substrates for LPO during ferroptosis (Hadian and Stockwell, 2020).There are two pathways for LPO, the non-enzymatic and enzymatic LPO pathways (Hassannia et al., 2019;Chen et al., 2021;Liang et al., 2022).The non-enzymatic LPO pathway is a radical-driven chain reaction-dependent autooxidation of lipids, in which ROS initiate PUFA oxidation.The hydroxyl radical (OH • ), a highly mobile water-soluble form of ROS produced from Fenton reactions, is involved in initiating LPO (Ayala et al., 2014).One OH • first abstracts a hydrogen radical from a PUFA to produce a lipid radical (L • ), which rapidly reacts with molecular oxygen (O 2 ) to yield a lipid peroxyl radical (LOO • ).LOO • subsequently abstracts a hydrogen radical from an adjacent PUFA, producing lipid hydroperoxide (LOOH).In the presence of ferrous iron, LOOH is converted to an alkoxyl radical (LO • ), which subsequently reacts with an adjacent PUFA to initiate another lipid radical chain reaction.When the ferroptosis defense systems that keep LPO in check fail, this iron-and oxygen-catalyzed oxidation process can lead to membrane destruction and cell death (Hassannia et al., 2019).
Enzymatic LPO is mediated by the activity of ALOXs and POR in a controlled manner.Iron initiates the Fenton reaction by functioning as an essential cofactor for ALOXs and POR.In enzymatic processes, acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) catalyzes the generation of PUFA-CoAs by ligating free PUFAs with CoA to form phospholipids (Dixon et al., 2015;Doll et al., 2017).Then, acyl groups are inserted into lysophospholipids by lysophosphatidylcholine acyltransferase 3 (LPCAT3), which incorporates free PUFAs into phospholipids (PLs) to generate PUFA-PLs (Dixon et al., 2015;Kagan et al., 2017).The incorporated PUFA-PLs are then peroxidated by PORs and ALOXs by labile iron and O 2 to generate PUFA-PL hydroperoxides (PUFA-PL-OOH) or peroxidated PUFA-PLs (Hadian and Stockwell, 2020;Zou et al., 2020).Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) are the two secondary products of LPO activity, leading to the formation of membrane pores and, from such cell death, ferroptosis (Tang and Kroemer, 2020).

Ferroptosis defense mechanisms
Cellular antioxidant systems constitute the ferroptosis defense systems, which directly neutralize lipid peroxides (Gu et al., 2023).Five major ferroptosis defense systems exist with specific subcellular localizations.

FSP1-CoQH 2 system
Ubiquinone (coenzyme Q 10 or CoQ 10 ), a component of mitochondria and diverse membranes, works as a second endogenous mechanism to inhibit LPO and ferroptosis.Ferroptosis suppressor protein 1 (FSP1) localizes to the plasma membrane and was first discovered to operate independently of GPX4 to halt ferroptosis (Bersuker et al., 2019;Doll et al., 2019), which reduces ubiquinone CoQ 10 to regenerate CoQ 10 -H 2 (CoQ 10 ubiquinol), acting as a NAD(P)H-dependent oxidoreductase.This traps LOO • , thereby suppressing ferroptosis by inhibiting LPO.FSP1 halts ferroptosis by repairing damage to the plasma membrane and by activating the endosomal sorting complex required for transport III (ESCRT-III) complex (Dai et al., 2020;Pedrera et al., 2021).

DHODH-CoQH 2 system
The dihydroorotate dehydrogenase (DHODH)dihydroubiquione (CoQH 2 ) system is the third ferroptosis defense system independent of GPX4, which detoxifies mitochondrial lipid peroxides compensating for GPX4 loss (Mao et al., 2021).In the inner mitochondrial membrane, DHODH, originally discovered to be involved in pyrimidine synthesis, reduces CoQ 10 to CoQH 2 , thereby reducing mitochondrial CoQ 10 , analogous to the function of FSP1 in the extramitochondrial membranes (Mao et al., 2021).Once GPX4 is acutely inactivated, DHODH-mediated flux is significantly increased to promote the generation of CoQH 2 , which neutralizes LPO and halts ferroptosis that originates from the mitochondria (Mao et al., 2021).

SC5D-7-DHC axis
The lathosterol oxidase (SC5D)-7-dehydrocholesterol (7-DHC) axis is a newly identified inhibitor of ferroptosis, discovered by Freitas et al. (2024), Li et al. (2024), Freitas et al. (2024), Li et al. (2024), and Li et al. (2024), who both reported a previously unknown natural inhibitor of ferroptosis, i.e., 7-DHC.Synthesized in the endoplasmic reticulum, 7-DHC is found on the cell membrane and mitochondria.It is generated in the cholesterol synthesis pathway, which includes the intermediates of zymosterol/lathosterol and the enzymes EBP, SC5D, and DHCR7.When radicals attack phospholipids, the lipid is oxidized, and it fragments.Here, 7-DHC absorbs radicals and inhibits lipid peroxidation in both the plasma membrane and mitochondria by diverting the peroxidation pathway from phospholipids, thus mitigating ferroptosis.

Reversing chemotherapy resistance by inducing ferroptosis in NSCLC
New reports suggest that small-molecule drugs may function as ferroptosis-inducing bioactive compounds, enhancing chemotoxicity toward cancers (Yin et al., 2022;Xing et al., 2023;Li et al., 2024).Small-molecule drugs are organic compounds that impact cellular activity, which, due to their low molecular weight, can provide high cellular permeability.Small-molecule drugs are generally derived from two major practices: isolation from natural products or rational design to target proteins with a known function (Ibarrola-Villava et al., 2018;Niu et al., 2023).Emerging ferroptosisinducing bioactive compounds (Figure 2) could boost the antitumor activity of ferroptosis induced by chemotherapeutic agents, overcoming chemotherapeutic drug resistance in NSCLC (Figure 3).Table 1 lists some natural compounds that induce ferroptosis to overcome chemoresistance in NSCLC.

Reversing chemotherapeutic resistance in lung cancer-inducing ferroptosis with natural products
One of the major active components of Dendrobii caulis and phytoestrogen is erianin, which has anti-tumor, anti-diabetic retinopathy, anti-inflammatory, antibacterial, and anti-psoriasis effects (Li et al., 2023).Erianin significantly attenuates lung cancer stemness and enhances sensitivity of lung cancer cells to 5-FU (Lin et al., 2020).The ferroptosis inhibitor Fer-1 attenuates the erianin-mediated inhibition of sphere formation in lung cancer cells, suggesting that erianin inhibits lung cancer stemness by facilitating ferroptosis (Lin et al., 2020).
Isoorientin is a natural C-glucosyl flavone that has multiple pharmacological activities, including anti-inflammatory, robust antioxidant, and anti-tumor activities (Li et al., 2020;Xu et al., 2020;Ziqubu et al., 2020;Liu et al., 2021;Cui et al., 2023).Previous experiments have shown that it promotes apoptosis by the ROSmediated MAPK/STAT3/NF-κB signaling pathway in A549 lung cancer cells (Xu et al., 2020).Further study has revealed that isoorientin overcomes drug resistance by inducing ferroptosis via the sirtuin 6 (SIRT6)/nuclear factor-erythroid factor 2-related factor 2 (Nrf2)/GPX4 signaling pathway in lung cancer (Feng et al., 2023).Isoorientin boosts the anti-tumor activity of cisplatin, as evidenced by significantly decreasing the viability of drug-resistant cells, a notable decrease in glutathione concentration, and a substantial increase in intracellular iron, MDA, and ROS production in vitro and in vivo.Mechanistically, isoorientin overcomes drug resistance by downregulating SIRT6/Nrf2/GPX4 in lung cancer cells (Feng et al., 2023).
As a natural bioactive juglone derivative, 2-methoxy-6-acetyl-7methyljuglone (2-methoxystypandrone, MAM) has anticancer, antiinflammatory, antimicrobial, antioxidant, and anti-HIV properties (Khalil et al., 2022).MAM inhibits cancer progression by promoting apoptosis, necroptosis, and deregulation signaling pathways in colon cancer cells, glioblastoma, lung cancer, and breast cancer (Sun et al., 2016;Sun et al., 2017;Sun et al., 2019;Yu et al., 2020).Anticancer activity against lung cancer was corroborated by other studies, which reported that MAM induces significant cell death in cisplatin-and AZD9291-resistant lung cancer cells, being completely reversed by NQO1 siRNA, NQO1 inhibitors, or iron chelators (Yu et al., 2023).Mechanistically, MAM triggers ROS generation by binding to activate NQO1, increasing LIP and LPO.MAM suppresses tumor growth in a tumor xenograft zebrafish model.These studies suggest that MAM induces ferroptosis by activating NQO1 in drug-resistant NSCLC cells, highlighting a novel therapeutic regimen to overcome drug resistance via inducing NQO1-mediated ferroptosis in NSCLC (Yu et al., 2023).
As a natural bioactive molecule very abundant in aromatic and medicinal plants (AMPs), α-hederin has various pharmacological Chemical structures of small-molecule compounds from traditional medicines inducing ferroptosis in lung cancer.
The natural borneol obtained from the fresh branches and leaves of Cinnamomum camphora (L.), J. Presl d-borneol has refreshing and awakening effects and is usually used for treating cerebrovascular and cardiovascular diseases.Borneol has antiinflammatory, penetration-promoting, and sedative, analgesic, and antibacterial properties.Borneol can also boost the antitumor effects of chemotherapeutic drugs in NSCLC, human esophageal squamous cell carcinoma, gliomas, and hepatocellular carcinoma (Chen et al., 2015;Meng et al., 2018;Cao et al., 2019;Li et al., 2022).Further study has revealed that d-borneol exerts anticancer activity in cisplatin-resistant NSCLC cells by inducing ferroptosis (Li et al., 2022).d-Borneol enhances tumor-inhibiting effects of cisplatin by promoting ferroptosis, as evidenced by the increased production of ROS and MDA and decreased expression of GSH, Trx, SOD, and heme oxygenase-1.Mechanistically, the combination of d-borneol and cisplatin induces ferroptosis by facilitating nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and modulating intracellular iron ion transport via decreasing PCBP2 and increasing PRNP (Li et al., 2022).
Dihydroartemisinin (DHA), an active derivative of artemisinin originally developed in China, is the first-line treatment for malaria (Dai et al., 2021).DHA has anticancer activity by boosting the efficacy of chemotherapy, targeted therapy, and even radiotherapy in a wide range of cancer types (Li et al., 2021).Recent studies have suggested that DHA boosts the efficacy of targeted therapy and immunotherapy by inducing ferroptosis in lung cancer cells (Li et al., 2022;Han et al., 2023;Lai et al., 2023).DHA facilitates chlorin e6-induced photodynamic therapy by inducing ferroptosis, inhibiting GPX4, and enhancing ROS in lung cancer cells (Han et al., 2022).

Reversing chemotherapy resistance through inducing ferroptosis by smallmolecular drugs in lung cancer
The utilization of ferroptosis-modulating small molecules or compounds is a new novel strategy to enhance chemotherapy outcomes (Yin et al., 2022), potentially acting as a vector to treat chemotherapeutic resistance (Koeberle et al., 2023).Functioning as a type 3 ferroptosis inducer, the ferroptosis-inducing agent 56 (FIN56) promotes ferroptosis by facilitating the autophagydependent protein degradation of GPX4 (Shimada et al., 2016;Sun et al., 2021).FIN56 combined with cisplatin increases cellular ROS levels, decreases antioxidant gene expression, and boosts the cisplatin cytotoxic effect in the A549 cell line, indicating that inducing ferroptosis is a promising strategy in cisplatin-resistant cancer cells (Golbashirzadeh et al., 2023).
Initially identified as a small-molecule compound that selectively kills tumor cells, erastin is an inducer of ferroptosis by modulating system XC − , p53, and the voltage-dependent anion channel (VDAC).Erastin can increase tumor sensitivity to chemotherapy and radiotherapy, highlighting a promising potential in cancer therapy (Zhao et al., 2020).Erastin and sorafenib induce ferroptosis in CDDP-resistant N5CP NSCLC cells, as evidenced by the accumulation of intracellular lipid ROS.Erastin and sorafenib, alone or in combination with CDDP, inhibit the growth of N5CP cells in vivo (Li et al., 2020).
An analog of erastin, PRLX93936, has demonstrated synergistic effects against NSCLC cells.The combination of PRLX93936 and cisplatin induces ferroptosis, as evidenced by the increased production of ROS, LPO, and Fe 2+ .Mechanistically, the cotreatment of PRLX93936 with cisplatin induces ferroptosis by inhibiting Nrf2-dependant GPX4 (Liang et al., 2021).

Conclusion and future perspectives
In conclusion, this review summarized the novel role of ferroptosis in lung cancer and provides an overview on how pro-ferroptotic molecules may be used to overcome chemotherapeutic resistance.During the past decade, ferroptosis has attracted considerable interest in lung cancer research for its anti-tumor activity, which is thought to boost the efficacy of chemotherapy.In this review, we comprehensively summarized the small-molecule compounds from traditional medicines that may boost the anti-tumor activity of chemotherapeutic agents or overcome chemotherapy drug resistance in NSCLC, both of which serve as starting points to develop ferroptosisrelated anticancer drugs for NSCLC.Small-molecule compounds that induce ferroptosis have specific targets.Erastin targets system Xc − to prevent cysteine import, which causes GSH depletion.RSL3 is a covalent inhibitor of GPX4 that causes the accumulation of lipid peroxides.In contrast to classical small molecules, traditional medicines have the advantage of polypharmacology.For example, ginkgetin regulates Nrf2, SLC7A11, and GPX4 at the same time.
The FDA has approved some ferroptosis-targeting smallmolecule compounds for the evaluation of clinical trials with NSCLC patients (Li et al., 2024).However, research on ferroptosis is an emerging field still in its infancy.Significanr research is needed to bridge the gap from where we are to where we need to be in order to provide satisfactory biological outcomes.First, research on the role of ferroptosis in NSCLC is still ongoing, and the specific functions of ferroptosis remain ambiguous, hence warranting further investigation.As such, inevitable challenges still remain before the practical application of these treatment modalities.Second, the epigenetic modification of ferroptosis in cancer is an emerging field.The epigenetic modification of A549/Cis, Cis-resistant A549 cells; compound 23, 11β-hydroxy-ent-16-kaurene-15-one; DFO, deferoxamine; DHA, dihydroartemisinin; FTH, ferritin heavy chain; H1299/Cis, Cis-resistant H1299 cells; LIP, labile iron pool; Lip-1, liproxstatin-1; MAM, 2-methoxy-6-acetyl-7-methyljuglone; NCOA4, nuclear receptor coactivator 4; NVP-231, CERK inhibitor; Nrf2, nuclear factorerythroid factor 2-related factor 2; NSCLC, non-small-cell lung cancer; PDT, photodynamic therapy; TfR, transferrin receptor that imports iron from the extracellular environment into cells; VDAC, voltage-dependent anion channel.
ferroptosis is identified in NSCLC, which, when dysregulated, can be feasibly targeted by small-molecule compounds.However, the practical application of these treatment modalities in NSCLC still has a long way to go.Third, many key components of the ferroptosis pathway, i.e., the principal proteins and enzymes engaged in the induction and inhibition of ferroptosis are transcriptionally controlled by Nrf2 49−53 .More research is needed to discover new mechanisms that regulate ferroptosis and the role of Nrf2 in inhibiting ferroptosis, which will repurpose old drugs, i.e., Nrf2 inhibitors, as ferroptosis inducers to kill NSCLC.Nrf2 inhibitors may then be an optimal approach to treat NSCLC, but this requires further investigation.Fourth, the identification of biomarkers for ferroptosis sensitivity or resistance is urgently needed for accurately predicting the efficiency of inducing ferroptosis.Fifth, an assessment of the safety and potential toxicity of the small-molecule compounds that induce ferroptosis should be considered as this is a crucial consideration for potential therapeutic agents.
In conclusion, ferroptosis has been identified as a critical RCD triggered by ferroptosis-inducing bioactive compounds in NSCLC.Thus, the small-molecule compounds from traditional medicines hold great potential in NSCLC therapy, especially when combined with conventional chemotherapy by boosting the anti-tumor activity of chemotherapeutic agents or overcoming chemotherapy drug resistance in NSCLC.Natural-product ferroptosis-inducing small molecules may serve as an excellent starting point for the further development of ferroptosis-related anticancer drugs to overcome NSCLC chemotherapeutic resistance.

FIGURE 3
FIGURE 3Mechanisms of ferroptosis-inducing small-molecule compounds that may boost the anti-tumor activity of chemotherapeutic agents or overcome chemotherapy drug resistance in lung cancer.

TABLE 1
Small-molecule compounds as inducers of ferroptosis to overcome drug resistance in NSCLC.Hydroxy-ent-16-kaurene-15-one, is one of the ent-kaurane diterpenoids from Chinese liverworts Jungermannia tetragona Lindenb and has strong anti-tumor activity in several cancer cell lines.Compound 23 induces both apoptosis and ferroptosis by increasing cellular ROS levels in HepG2 cells.Compound 23 increases the sensitivity of cisplatin-resistant A549/CDDP cancer cells by inducing ferroptosis and apoptosis, suggesting that ent-kaurane derivatives overcome chemoresistance to cisplatin by inducing ferroptosis

TABLE 1 (
Continued) Small-molecule compounds as inducers of ferroptosis to overcome drug resistance in NSCLC.