Ras-selective Lethal Small Molecule Research Articles

TCP1 expression alters the ferroptosis sensitivity of diffuse large B-cell lymphoma subtypes by stabilising ACSL4 and influences patient prognosis

Diffuse large B-cell lymphoma (DLBCL), an invasive lymphoma with substantial heterogeneity, can be mainly categorised into germinal centre B-cell-like (GCB) and non-GCB subtypes. DLBCL cells are highly susceptible to ferroptosis, which offers an effective avenue for treating recurrent and refractory DLBCL. Moreover, various heat shock proteins are involved in regulating the sensitivity of tumour cells to ferroptosis. Among these proteins, tailless complex polypeptide 1 (TCP1), a subunit of chaperonin-containing T-complex protein-1 (CCT), plays a role in tumour proliferation and survival. Therefore, we explored the role of TCP1 in different DLBCL subtypes, the sensitivity of GCB and non-GCB subtypes to the ferroptosis inducer RAS-selective lethal small molecule 3 (RSL3), and the underlying molecular mechanism. In GCB cells, TCP1 promoted RSL3-induced ferroptosis. Notably, TCP1 could bind with acyl-CoA synthetase long-chain family member 4 (ACSL4), a key enzyme regulating lipid composition and facilitating ferroptosis, to reduce its ubiquitination and degradation. This interaction activated the ACSL4/LPCAT3 signalling pathway and promoted ferroptosis in the GCB subtype. However, in the non-GCB subtype, TCP1 did not act as a positive regulator but served as a predictor of an unfavourable prognosis in patients with non-GCB. In conclusion, our results suggest that in DLBCL, high TCP1 expression enhances the sensitivity of GCB tumour cells to ferroptosis and serves as a marker of poor prognosis in patients with non-GCB DLBCL.

Chlorobenzoquinones Aggravate RSL3-Induced Ferroptosis in ROS-Dependent Manner.

Chlorobenzoquinones (CBQs) are a class of emerging water disinfection byproducts that pose significant risks to public health. In this study, we found that three CBQs (tetrachloro-1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone, and 2-chloro-1,4-benzoquinone) can significantly aggravate cell death caused by Ras-selective lethal small molecule 3 (RSL3). Further study showed that the cell death caused by CBQs, either alone or in combination with RSL3, was related to iron accumulation and GPX4 inactivation, suggesting the occurrence of ferroptosis. Furthermore, reactive oxygen species are found to play a potential key role in mediating the toxicity of CBQs in CBQs and RSL3-induced ferroptosis. These findings will be helpful in understanding the toxic mechanism of CBQs to mammalian cells.

Injectable alginate hydrogel promotes antitumor immunity through glucose oxidase and Fe3+ amplified RSL3-induced ferroptosis

Ferroptosis induced by RAS-selective lethal small molecule 3 (RSL3) can trigger anti-tumor immune responses by reversing the immunosuppressive tumor microenvironment (TME). However, it is challenging to achieve sufficient ferroptosis in the tumor via RSL3 alone. Because of the excellent reactive oxygen species (ROS) production capacity of glucose oxidase (GOx) and Fe3+, we hypothesized that GOx and Fe3+ could increase intracellular lipid peroxidation (LPO) accumulation, and strengthen RSL3-induced ferroptosis in tumor cells. Herein we designed an in-situ gelation strategy based on sodium alginate (SA) to realize localized transport and specific retention of GOx, RSL3, and Fe3+ in tumor tissues. We loaded hydrophobic RSL3 with the tannic acid (TA)/Fe3+ complexes to form nanoparticles (RTF NPs). GOx diluted in the SA solution was blended with RTF NPs to obtain a homogeneous solution. The solution could form hydrogels in the tumor site (RTFG@SA) upon injection. The retained GOx and Fe3+ amplified the induction of ferroptosis by RSL3, augmented immunogenic cell death (ICD) and promoted antitumor immunity. The RTFG@SA hydrogel presented a significant restraint of tumor growth and metastasis in the 4T1 tumor model. This hydrogel could offer an effective means of co-delivery of hydrophilic drugs, hydrophobic drugs, and metal ions.

Modified Shoutai Pill inhibited ferroptosis to alleviate recurrent pregnancy loss

Ethnopharmacological relevanceModified Shoutai Pill, also called Jianwei Shoutai Pill (JSP), is a traditional Chinese medicine prescription that has been used as an effective agent for the treatment of miscarriage. Aim of the studyTo explore the potential molecular mechanism of JSP against recurrent pregnancy loss (RPL). Materials and methodsIn vivo, CBA/J mated DBA/2 mice were used to conduct RPL model, while CBA/J mated BALB/c mice were seen as the control group. Mice were orally administered with JSP, Fer-1 (a ferroptosis inhibitor) or distilled water from day 0.5–12.5 of gestation (GD 0.5–12.5). Pregnancy outcomes were analyzed and ferroptosis related indexes of the whole implantation sites were measured on GD 12.5. In vitro, human trophoblast cell line HTR-8/SVneo was cultured and treated with RAS-selective lethal small molecule 3 (RSL3) (a ferroptosis agonist) or different concentrations of JSP. Then, ferroptosis related indexes were tested to analyze whether JSP could inhibit ferroptosis in HTR-8/SVneo cells. ResultsIn vivo consequences demonstrated that JSP or Fer-1 alleviated pregnancy outcomes including lower resorption rate and abortion rate. In addition, excessive iron accumulation and MDA level were inhibited, while GSH and GPX content were raised under JSP or Fer-1 exposure. Also, JSP or Fer-1 enhanced protein expressions of GPX4 and SLC7A11 which suppress ferroptosis, and lightened protein expression of ACSL4 which boosts ferroptosis. In vitro, JSP rescued HTR-8/SVneo cell death and migration ability that were injured by RSL3. Furthermore, JSP inhibited RSL3-induced intracellular reactive oxygen species (ROS), lipid ROS and iron deposition. ConclusionsCollectively, our findings illustrated that the mechanism of JSP in treating RPL might be related to inhibiting ferroptosis, which provided a novel insight into the application of JSP in RPL intervention.

Scutellarein alleviates chronic obstructive pulmonary disease through inhibition of ferroptosis by chelating iron and interacting with arachidonate 15-lipoxygenase.

Ferroptosis, an iron-dependent cell death characterized by lethal lipid peroxidation, is involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Therefore, ferroptosis inhibition represents an attractive strategy for COPD therapy. Herein, we identified natural flavonoid scutellarein as a potent ferroptosis inhibitor for the first time, and characterized its underlying mechanisms for inhibition of ferroptosis and COPD. In vitro, the anti-ferroptotic activity of scutellarein was investigated through CCK8, real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, flow cytometry, and transmission electron microscope (TEM). In vivo, COPD was induced by lipopolysaccharide (LPS)/cigarette smoke (CS) and assessed by changes in histopathological, inflammatory, and ferroptotic markers. The mechanisms were investigated by RNA-sequencing (RNA-seq), electrospray ionization mass spectra (ESI-MS), local surface plasmon resonance (LSPR), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and molecular dynamics. Our results showed that scutellarein significantly inhibited Ras-selective lethal small molecule (RSL)-3-induced ferroptosis and mitochondria injury in BEAS-2B cells, and ameliorated LPS/CS-induced COPD in mice. Furthermore, scutellarein also repressed RSL-3- or LPS/CS-induced lipid peroxidation, GPX4 down-regulation, and overactivation of Nrf2/HO-1 and JNK/p38 pathways. Mechanistically, scutellarein inhibited RSL-3- or LPS/CS-induced Fe2+ elevation through directly chelating Fe2+ . Moreover, scutellarein bound to the lipid peroxidizing enzyme arachidonate 15-lipoxygenase (ALOX15), which resulted in an unstable state of the catalysis-related Fe2+ chelating cluster. Additionally, ALOX15 overexpression partially abolished scutellarein-mediated anti-ferroptotic activity. Our findings revealed that scutellarein alleviated COPD by inhibiting ferroptosis via directly chelating Fe2+ and interacting with ALOX15, and also highlighted scutellarein as a candidate for the treatment of COPD and other ferroptosis-related diseases.

Abstract 4777: Exploring ferroptosis pathway for the development of therapeutic strategy in liposarcoma

Abstract Background: Liposarcoma (LPS) is one of the most common soft tissue sarcoma (STS) subtypes. Both well-differentiated LPS (WDLPS) and dedifferentiated LPS (DDLPS) have 12q13-15 amplification. Mouse double minute 2 homolog (MDM2) is one of the most important oncogenes within this region. Ferroptosis is a unique type of necrotic cell death featured accumulation of lipid-based reactive oxygen species (ROS) derived from the oxidative modification of phospholipid membranes. Cysteine metabolism is the critical part of ferroptosis regulation. Cystine-glutamate antiporter (system xc-; xCT), encoded by two subcomponents, the solute carrier family 7 member 11 (SLC7A11) and SLC3A2, is responsible for cystine supply from extra-cellular environment. Imported cystine is then reduced to cysteine, a key component of tripeptide glutathione (GSH). Glutathione peroxidase 4 (GPX4) requires GSH to repair lipid peroxidation and prevent ferroptosis. Inactivation of GPX4, either through cystine deprivation due to xCT inhibitor erastin, or by GPX4 inhibitor Ras-selective lethal small molecule 3 (RSL3), will lead to accumulation of lipid-based ROS, and eventually ferroptosis cell death. The tumor suppressor p53 (TP53) could suppress the expression of SLC7A11 and induce ferroptosis. TP53 could also indirectly suppress 3-hydroxy-3-methyl-glutaryl (HMG)-coenzyme A (CoA) reductase (HMGCR) activity, a key pathway for generation of biomolecules with anti-ferroptosis property. MDM2, the key oncogene in DDLPS, and homolog MDM4 could facilitate ferroptotic death in TP53-dependent or -independent mechanisms. Methods: We explored public domain database to identify possible aberrations of ferroptosis-related genes in DDLPS. We then examined the sensitivity of DDLPS cell lines to possible ferroptosis-inducing agents. Furthermore, we used nutlin-3, a MDM2 inhibitor, to modulate the MDM2 expression in DDLPS cell line, and explored the potential synergistic effect of nutlin-3 with ferroptosis-inducing agents. Finally, we used immunoblotting study to reveal the potential mechanism responsible to possible synergistic effect. Results: Expression level of GPX4 is significantly lower in DDLPS than adipose tissue and WDLPS, and DPP4 was general higher in LPS than adipose tissue. HMGCR, SLC7A11 and SLC3A2 were mostly up-regulated in LPS in comparison with benign counterpart. Both DDLPS cell lines showed sensitivity to erastin and RSL3. In addition, nutlin-3 could exert synergistic ferroptosis-inducing effect and cytotoxicity with erastin and RSL3 in sequential manner. Furthermore, nultin-3 treatment could upregulate SLC3A2 with altered cystine/glutamate exchange in DDLPS cell lines, indicating possible mechanism responsible for nultin-3 resistance and the synergistic effect of nultin-3 with erastin or RSL3. Conclusion: Our study showed that modulation of ferroptosis is a potential treatment strategy in DDLPS. Citation Format: Chueh-Chuan Yen, San-Chi Chen, Chih-Hsueh Chen, Jir-You Wang, Chao-Ming Chen, Po-Kuei Wu, Muh-Hwa Yang. Exploring ferroptosis pathway for the development of therapeutic strategy in liposarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4777.

Application of singlet oxygen-activatable nanocarriers to boost X-ray-induced photodynamic therapy and cascaded ferroptosis for breast cancer treatment.

Ferroptosis has appealing antitumor potential that is mainly based on the accumulation of lipid peroxide to a lethal level. The cytotoxic singlet oxygen (1O2) generated from nanoscale X-ray-induced photodynamic therapy (X-PDT) may facilitate glutathione (GSH) depletion and further activate ferroptosis. To realize combined X-PDT and ferroptosis, a nanocarrier (D-NPVR) was engineered with a hyperbranched copolymer with 1O2-sensitive linkers, where both the photosensitizer (verteporfin) and ferroptosis inducer RAS-selective lethal small molecule 3 (RSL3) were encapsulated. Upon X-ray radiation, D-NPVR could produce a large amount of 1O2 for apoptosis. Subsequently, 1O2 triggered D-NP dissociation by cleavage of 1,2-bis(2-hydroxyethylthio)ethylene bonds to boost payload release and decrease levels of intracellular GSH via thiol oxidation. Liberated RSL3 is a covalent inhibitor for glutathione peroxide 4 (GPX4), which is responsible for detoxifying lipid peroxides to lipid alcohols with GSH assistance, and both 1O2-induced GSH depletion and GPX4 inactivation thereby produced ferroptotic cell death. Tumor growth inhibition in murine 4T1 tumor-bearing mice demonstrated that D-NPVR produced pronounced therapeutic efficiency where ferroptosis induction was supported by the GPX4 content and expression. This study highlights the contribution of 1O2-sensitive nanocarriers for promoting the potency of combined X-PDT and ferroptosis.

The endosomal sorting complex required for transport repairs the membrane to delay cell death.

The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in the repair of damaged plasma membranes with puncta form and removes pores from the plasma membrane in regulated cell death, apoptosis, necroptosis, pyroptosis, ferroptosis, and autophagy. ESCRT-I overexpression and ESCRT-III-associated charged multivesicular body protein (CHMP) 4B participate in apoptosis, and the ESCRT-1 protein TSG 101 maintains low levels of ALIX and ALG-2 and prevents predisposition to apoptosis. The ESCRT-III components CHMP2A and CHMP4B are recruited to broken membrane bubble sites with the requirement of extracellular Ca2+, remove membrane vesicles from cells, and delay the time required for active MLKL to mediate necroptosis, thus preserving cell survival. CHMP4B disturbed pyroptosis by recruiting around the plasma membrane neck to remove the GSDMD pores and preserve plasma membrane integrity depending on Ca2+ influx. The accumulation of the ESCRT-III subunits CHMP5 and CHMP6 in the plasma membrane is increased by the classical ferroptosis activators erastin-1 and ras-selective lethal small molecule 3 (RSL3) upon cytosolic calcium influx and repairs the ferroptotic plasma membrane. ESCRT-III- and VPS4-induced macroautophagy, ESCRT-0-initiated microautophagy. ESCRT-I, ESCRT-II, ESCRT-III, ALIX, and VPS4A are recruited to damaged lysosomes and precede lysophagy, indicating that ESCRT is a potential target to overcome drug resistance during tumor therapy.

Cytoglobin Silencing Promotes Melanoma Malignancy but Sensitizes for Ferroptosis and Pyroptosis Therapy Response.

Despite recent advances in melanoma treatment, there are still patients that either do not respond or develop resistance. This unresponsiveness and/or acquired resistance to therapy could be explained by the fact that some melanoma cells reside in a dedifferentiated state. Interestingly, this dedifferentiated state is associated with greater sensitivity to ferroptosis, a lipid peroxidation-reliant, iron-dependent form of cell death. Cytoglobin (CYGB) is an iron hexacoordinated globin that is highly enriched in melanocytes and frequently downregulated during melanomagenesis. In this study, we investigated the potential effect of CYGB on the cellular sensitivity towards (1S, 3R)-RAS-selective lethal small molecule (RSL3)-mediated ferroptosis in the G361 melanoma cells with abundant endogenous expression. Our findings show that an increased basal ROS level and higher degree of lipid peroxidation upon RSL3 treatment contribute to the increased sensitivity of CYGB knockdown G361 cells to ferroptosis. Furthermore, transcriptome analysis demonstrates the enrichment of multiple cancer malignancy pathways upon CYGB knockdown, supporting a tumor-suppressive role for CYGB. Remarkably, CYGB knockdown also triggers activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and subsequent induction of pyroptosis target genes. Altogether, we show that silencing of CYGB expression modulates cancer therapy sensitivity via regulation of ferroptosis and pyroptosis cell death signaling pathways.

Berberine protects cardiac cells against ferroptosis.

ABSTRACTObjectives:Cardiovascular diseases are one of the primary causes of death. Cardiomyocyte loss is a significant feature of cardiac injury. Ferroptosis is iron-dependent cell death, which occurs due to excess iron and reactive oxygen species (ROS) accumulation causing lipid peroxidation, and subsequent cell death. Ferroptosis has been confirmed to mediate ischemia/reperfusion-induced cardiomyopathy and chemotherapy-induced cardiotoxicity. Berberine (BBR) has been proven to protect the heart from cardiomyopathies, including cardiac hypertrophy, heart failure, myocardial infarction, and arrhythmias. It protects cardiomyocytes from apoptosis and autophagy. However, the relation between BBR and ferroptosis is still unknown. This study aimed to confirm if BBR reduces cardiac cell loss via inhibiting ferroptosis.Materials and Methods:We used erastin and Ras-selective lethal small molecule 3 (RSL3) to establish a ferroptosis model in an H9c2 cardiomyoblast cell line and rat neonatal cardiomyocytes to prove that BBR has a protective effect on cardiac cells via inhibiting ferroptosis.Results:In H9c2 cardiomyoblasts, the results showed that BBR reduced erastin and RSL3-induced cell viability loss. Moreover, BBR decreased ROS accumulation and lipid peroxidation in cells induced with ferroptosis. Furthermore, quantitative polymerase chain reaction results showed that Ptgs2 mRNA was reduced in BBR-treated cells. In rat neonatal cardiomyocytes, BBR reduced RSL3-induced loss of cell viability.Conclusion:These results indicated that BBR inhibited ferroptosis via reducing ROS generation and reducing lipid peroxidation in erastin and RSL3-treated cardiac cells.

Ferroptotic stress promotes macrophages against intracellular bacteria.

Rational: Intracellular bacterial survival is a major factor causing chronic or recurrent infection, leading to the failure of both host defense and/or antibiotic treatment. However, the elimination of intracellular bacteria is challenging as they are protected from antibiotics and host immune attack. Recent studies have indicated that iron helps macrophages against intracellular bacteria, contradictory to traditional “nutritional immunity”, in which iron is considered a key nutrient for bacterial survival in host cells. However, how iron facilitates intracellular bacterial death has not been fully clarified. In this study, we found that ferroptotic stress can help macrophages suppress intracellular bacteria by reversing the importation of ferrous iron into bacterial vacuoles via ferroportin and thereby inducing in situ ferroptosis-like bacterial death.Methods: A macrophage model of bacterial invasion was established to monitor dynamic changes in ferroptotic hallmarks, including ferrous iron and lipid peroxidation. Ferroptosis inducers and inhibitors were added to the model to evaluate the relationship between ferroptotic stress and intracellular bacterial survival. We then determined the spatiotemporal distributions of ferroportin, ferrous iron, and lipid peroxidation in macrophages and intracellular bacteria. A bacterial infection mouse model was established to evaluate the therapeutic effects of drugs that regulate ferroptotic stress.Results: Ferrous iron and lipid peroxidation increased sharply in the early stage of bacterial infection in the macrophages, then decreased to normal levels in the late stage of infection. The addition of ferroptosis inducers (ras-selective lethal small molecule 3, sulfasalazine, and acetaminophen) in macrophages promoted intracellular bacterial suppression. Further studies revealed that ferrous iron could be delivered to the intracellular bacterial compartment via inward ferroportin transportation, where ferrous iron induced ferroptosis-like death of bacteria. In addition, ferroptotic stress declined to normal levels in the late stage of infection by regulating iron-related pathways in the macrophages. Importantly, we found that enhancing ferroptotic stress with a ferroptosis inducer (sulfasalazine) successfully suppressed bacteria in the mouse infection models.Conclusions: Our study suggests that the spatiotemporal response to ferroptosis stress is an efficient pathway for macrophage defense against bacterial invasion, and targeting ferroptosis may achieve therapeutic targets for infectious diseases challenged by intracellular pathogens.

Ginkgolide B protects against cognitive impairment in senescence-accelerated P8 mice by mitigating oxidative stress, inflammation and ferroptosis

Alzheimer's disease (AD) is a destructive neurodegenerative disease that currently has no effective treatment option available. Ginkgolide B (GB) is a terpene lactone derivative of Ginkgo biloba that possesses neuroprotective effects in various diseases. The aim of the present study was to investigate whether GB protects against cognitive impairment in AD by mitigating oxidative stress, inflammation and ferroptosis using senescence-accelerated P8 (SAMP8) mice. The results showed that GB improved the cognitive dysfunction of SAMP8 mice in the Morris water maze and novel object recognition test, which was associated with the attenuation of oxidative stress, inflammation and nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4 (GPX4) pathway-mediated ferroptosis. Furthermore, Ras-selective lethal small molecule 3, a GPX4 inhibitor and ferroptosis inducer, compromised GB-induced cognitive performance in SAMP8 mice. These findings suggested that GB alleviated AD-induced cognitive defects by mitigating oxidative stress, neuroinflammation and ferroptosis, and that the inhibition of ferroptosis is required for GB to have beneficial effects in AD.

Characterization of Novel Diphenylamine Compounds as Ferroptosis Inhibitors.

Ferroptosis is a form of oxidative cell death that is increasingly recognized as a key mechanism not only in neurodegeneration but also in regulated cell death, causing disease in other tissues. In neurons, major hallmarks of ferroptosis involve the accumulation of lipid reactive oxygen species (ROS) and impairment of mitochondrial morphology and function. Compounds that interfere with ferroptosis could provide novel treatment options for neurodegenerative disorders and other diseases involving ferroptosis. In the present study, we developed new compounds by refining structural elements of the BH3 interacting-domain death agonist inhibitor BI-6c9, which was previously demonstrated to block ferroptosis signaling at the level of mitochondria. Here, we inserted an antioxidative diphenylamine (DPA) structure to the BI-6c9 structure. These DPA compounds were then tested in models of erastin, and Ras-selective lethal small molecule 3 induced ferroptosis in neuronal HT22 cells. The DPA compounds showed an increased protective potency against ferroptotic cell death compared with the scaffold molecule BI-6c9. Moreover, hallmarks of ferroptosis such as lipid, cytosolic, and mitochondrial ROS formation were abrogated in a concentration- and time-dependent manner. Additionally, mitochondrial parameters such as mitochondrial morphology, mitochondrial membrane potential, and mitochondrial respiration were preserved by the DPA compounds, supporting the conclusion that lipid ROS toxicity and mitochondrial impairment are closely related in ferroptosis. Our findings confirm that the DPA compounds are very effective agents in preventing ferroptotic cell death by blocking ROS production and, in particular, via mitochondrial protection. SIGNIFICANCE STATEMENT: Preventing neuronal cells from different forms of oxidative cell death was previously described as a promising strategy for treatment against several neurodegenerative diseases. This study reports novel compounds based on a diphenylamine structure that strongly protects neuronal HT22 cells from ferroptotic cell death upon erastin and Ras-selective lethal small molecule 3 induction by preventing the development of different reactive oxygen species and by protecting mitochondria from ferroptotic impairments.

Positive and Negative Regulation of Ferroptosis and Its Role in Maintaining Metabolic and Redox Homeostasis.

Ferroptosis is a recently recognized regulated form of cell death characterized by accumulation of lipid-based reactive oxygen species (ROS), particularly lipid hydroperoxides and loss of activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4). This iron-dependent form of cell death is morphologically, biochemically, and also genetically discrete from other regulated cell death processes, which include autophagy, apoptosis, necrosis, and necroptosis. Ferroptosis is defined by three hallmarks, defined as the loss of lipid peroxide repair capacity by GPX4, the bioavailability of redox-active iron, and oxidation of polyunsaturated fatty acid- (PUFA-) containing phospholipids. Experimentally, it can be induced by many compounds (e.g., erastin, Ras-selective lethal small-molecule 3, and buthionine sulfoximine) and also can be pharmacologically inhibited by iron chelators (e.g., deferoxamine and deferoxamine mesylate) and lipid peroxidation inhibitors (e.g., ferrostatin and liproxstatin). The sensitivity of a cell towards ferroptotic cell death is tightly associated with the metabolism of amino acid, iron, and polyunsaturated fatty acid metabolism, and also with the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis sensitivity is also governed by many regulatory proteins, which also link ferroptosis to the function of key tumour suppressor pathways. In this review, we highlight the discovery of ferroptosis, the mechanism of ferroptosis regulation, and its association with other cellular metabolic processes.

Neuroprotective Effects of the Anti-cancer Drug Lapatinib Against Epileptic Seizures via Suppressing Glutathione Peroxidase 4-Dependent Ferroptosis.

Epilepsy is a complex neurological disorder characterized by recurrent and unprovoked seizures. Neuronal death process is implicated in the development of repetitive epileptic seizures. Therefore, cell death can be harnessed for ceasing seizures and epileptogenesis. Oxidative stress is regarded as a contributing factor of neuronal death activation and there is compelling evidence supporting antioxidants hold promise in abrogating seizure-related cell modality. Lapatinib, a well-known anti-cancer drug, has been traditionally reported to exert anti-tumor effect via modulating oxidative stress and a recent work illustrates the improvement of encephalomyelitis in rodent models after lapatinib treatment. However, whether lapatinib is beneficial for inhibiting neuronal death and epileptic seizure remains unknown. Here, we found that lapatinib remarkably prevented kainic acid (KA)-epileptic seizures in mice and ferroptosis, a newly defined cell death which is associated with oxidative stress, was involved in the neuroprotection of lapatinib. In the ferroptotic cell death model, lapatinib exerted neuroprotection via restoring glutathione peroxidase 4 (GPX4). Treatment with GPX4 inhibitor ras-selective lethal small molecule 3 (RSL3) abrogated its anti-ferroptotic potential. In a mouse model of KA-triggered seizure, it was also validated that lapatinib blocked GPX4-dependent ferroptosis. It is concluded that lapatinib has neuroprotective potential against epileptic seizures via suppressing GPX4-mediated ferroptosis.

HCAR1/MCT1 Regulates Tumor Ferroptosis through the Lactate-Mediated AMPK-SCD1 Activity and Its Therapeutic Implications

Ferroptosis is a recently discovered form of programed cell death caused by the metabolically regulated lipid peroxidation and holds promise for cancer treatment, but its regulatory mechanisms remain elusive. In this study, we observe that lactate-rich liver cancer cells exhibit enhanced resistance to the ferroptotic damage induced by common ferroptosis inducers such as Ras-selective lethalsmall molecule3 (RSL3) and Erastin and that the monocarboxylate transporter 1 (MCT1)-mediated lactate uptake could promote ATP production in hepatocellular carcinoma (HCC) cells and deactivate the energy sensor AMP-activated protein kinase (AMPK), leading to the upregulation of sterol regulatory element-binding protein 1 (SREBP1) and the downstream stearoyl-coenzyme A (CoA) desaturase-1 (SCD1) to enhance the production of anti-ferroptosis monounsaturated fatty acids. Additionally, blocking the lactate uptake via hydroxycarboxylic acid receptor 1 (HCAR1)/MCT1 inhibition promotes ferroptosis by activating the AMPK to downregulate SCD1, which may synergize with its acyl-coenzyme A synthetase 4 (ACSL4)-promoting effect to amplify the ferroptotic susceptibility. Invitro and invivo evidence confirms that lactate regulates the ferroptosis of HCC cells and highlights its translational potential as a therapeutic target for ferroptosis-based tumor treatment.

Electron-Accepting Micelles Deplete Reduced Nicotinamide Adenine Dinucleotide Phosphate and Impair Two Antioxidant Cascades for Ferroptosis-Induced Tumor Eradication.

Ferroptotic antitumor therapy has been compromised by various intracellular antioxidants, particularly glutathione and thioredoxin. Both are cofactors of glutathione peroxide 4 (GPX4) that act against oxidative stress via catalyzing the reduction of lipid peroxides. It was postulated that tailored polymer micelles could enhance ferroptotic antitumor efficacy via diminishing glutathione and thioredoxin under hypoxia. The aim was to engineer hypoxia-responsive micelles for selective enhancement of ferroptotic cell death in solid tumor. The polymer contains hydrophilic poly(ethylene glycol) (PEG) that is linked by azobenzene linker with nitroimidazole-conjugated polypeptide. The tailored polymer could self-assemble into nanoscale micelles to encapsulate RAS-selective lethal small molecule 3, a covalent GPX4 inhibitor. Under hypoxia, the azobenzene moiety enabled PEG shedding and enhanced micelles uptake in 4T1 cells. Likewise, the nitroimidazole moiety was reduced by the overexpressed nitroreductase with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as the cofactor, resulting in transient depletion of NADPH. This impaired both the glutathione and thioredoxin redox cycle, leading to diminished intracellular glutathione and thioredoxin. The selective potency of ferroptotic micelles in depleting NADPH, glutathione and thioredoxin was further verified in vivo in the 4T1 tumor xenograft mice model. This work highlights the role of hypoxia-responsive polymers in enhancing the potency of ferroptotic inducers against solid tumors without additional side effects to healthy organs.

Ferroptosis, a novel pharmacological mechanism of anti-cancer drugs.

Ferroptosis, a form of regulated cell death, is initiated by oxidative perturbations of the intracellular microenvironment, which is under the constitutive control of glutathione peroxidase 4 (GPX4). Ferrous iron (Fe2+) accumulation and lipid peroxidation are critical events in the induction of ferroptosis, which is inhibited by iron chelators and lipophilic antioxidants. Ferroptosis terminates in mitochondrial dysfunction and toxic lipid peroxidation. It plays a vital role in inhibiting cancer growth and proliferation. It can be induced in cancer cells, and certain normal cells, by experimental compounds (e.g., erastin, Ras-selective lethal small molecule 3) or clinical drugs. The purpose of this review is to summarize the various drugs (e.g., sulfasalazine, lanperisone, sorafenib, fenugreek (trigonelline), acetaminophen, cisplatin, artesunate, combination of siramesine and lapatinib, ferumoxytol, and salinomycin (ironomycin)) that could induce ferroptosis in cancer cells and provide an overview of current knowledge regarding the mechanisms underlying ferroptosis. In future, we anticipate the development of more ferroptosis-inducing drugs, and the availability of such drugs for the clinical treatment of cancer.

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3ε.

Ras-selective lethal small molecule 3 (RSL3), a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating the glutathione peroxidase glutathione peroxidase 4 (GPx4). Here, we report the purification of the protein indispensable for GPx4 inactivation by RSL3. Mass spectrometric analysis identified 14-3-3 isoforms as candidates, and recombinant human 14-3-3ε confirms the identification. The function of 14-3-3ε is redox-regulated. Moreover, overexpression or silencing of the gene coding for 14-3-3ε consistently controls the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein operating in cell signaling further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives.

Abstract 2322: Ferroptosis induced by erastin in RAS mutant ovarian cancer cells

Abstract Although apoptosis has been thought as the principal mechanism of programed cell death in chemotherapy, other forms of non-apoptotic death also has been noted. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of cell death termed ferroptosis. This mechanism is expected to induce eradication of chemotherapeutic (e.g. platinum) resistant cancer cells. In this study we demonstrate this new form of cell death in ovarian cancer cells. The clear cell epithelial ovarian cancer cell lines, TOV-21G (KRAS_G13C) and KOC-7C (RAS wild type) were used in this study. Erastin dose-dependently inhibited growth in TOV-21G cells, but not KOC-7C cells. Treatment of TOV-21G cells with erastin resulted in increase of ROS. Iron chelator deferoxamine (DFO) rescued growth inhibition induced by erastin in TOV-21G cells. Although cisplatin inhibited growth in TOV-21G cells, this inhibition was not rescued by DFO. The GSH level was reduced by erastin, but this reduction was not rescued by DFO. In addition, the basal GSH level was higher and the basal ROS level was lower in KOC-7C cells than those in TOV-21G cells. Erastin induced ferroptosis by reducing the GSH level in RAS mutant ovarian cancer cells. Although DFO rescued growth inhibition induced by erastin, the pathway was not on System Xc-. The sensitivity to erastin might depends on the basal level of GSH in cells. Ferroptosis had great potential to become a new approach in anti-tumor therapies in ovarian cancer. Citation Format: Motoki Takenaka, Noriko Suzuki, Minako Mori, Hitomi Aoki, Tasuku Hirayama, Hideko Nagasawa, Ken-ichirou Morishige. Ferroptosis induced by erastin in RAS mutant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2322.