Ferroptosis-inducing agents enhance TRAIL-induced apoptosis through upregulation of death receptor 5

Ferroptosis is considered genetically and biochemically distinct from other forms of cell death. In this study, we examined whether ferroptosis shares cell death pathways with other types of cell death. When human colon cancer HCT116, CX-1, and LS174T cells were treated with ferroptotic agents such as sorafenib (SRF), erastin, and artesunate, data from immunoblot assay showed that ferroptotic agents induced endoplasmic reticulum (ER) stress and the ER stress response-mediated expression of death receptor 5 (DR5), but not death receptor 4. An increase in the level of DR5, which is activated by binding to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and initiates apoptosis, was probably responsible for synergistic apoptosis when cells were treated with ferroptotic agent in combination with TRAIL. This collateral effect was suppressed in C/EBP (CCAAT-enhancer-binding protein)-homologous protein (CHOP)-deficient mouse embryonic fibroblasts or DR5 knockdown HCT116 cells, but not in p53-deficient HCT116 cells. The results from in vitro studies suggest the involvement of the p53-independent CHOP/DR5 axis in the synergistic apoptosis during the combinatorial treatment of ferroptotic agent and TRAIL. The synergistic apoptosis and regression of tumor growth were also observed in xenograft tumors when SRF and TRAIL were administered to tumor-bearing mice.     

Ginkgetin derived from Ginkgo biloba leaves enhances the therapeutic effect of cisplatin via ferroptosis-mediated disruption of the Nrf2/HO-1 axis in EGFR wild-type non-small-cell lung cancer

BackgroundCisplatin (DDP) is the first-in-class drug for advanced and non-targetable non-small-cell lung cancer (NSCLC). A recent study indicated that DDP could slightly induce non-apoptotic cell death ferroptosis, and the cytotoxicity was promoted by ferroptosis inducer. The agents enhancing the ferroptosis may therefore increase the anticancer effect of DDP. Several lines of evidence supporting the use of phytochemicals in NSCLC therapy. Ginkgetin, a bioflavonoid derived from Ginkgo biloba leaves, showed anticancer effects on NSCLC by triggering autophagy. Ferroptosis can be triggered by autophagy, which regulates redox homeostasis. Thus, we aimed to elucidate the possible role of ferroptosis involved in the synergistic effect of ginkgetin and DDP in cancer therapy.MethodsThe promotion of DDP-induced anticancer effects by ginkgetin was examined via a cytotoxicity assay and western blot. Ferroptosis triggered by ginkgetin in DDP-treated NSCLC was observed via a lipid peroxidation assay, a labile iron pool assay, western blot, and qPCR. With ferroptosis blocking, the contribution of ferroptosis to ginkgetin + DDP-induced cytotoxicity, the Nrf2/HO-1 axis, and apoptosis were determined via a luciferase assay, immunostaining, chromatin immunoprecipitation (CHIP), and flow cytometry. The role of ferroptosis in ginkgetin + DDP-treated NSCLC cells was illustrated by the application of ferroptosis inhibitors, which was further demonstrated in a xenograft nude mouse model.ResultsGinkgetin synergized with DDP to increase cytotoxicity in NSCLC cells, which was concomitant with increased labile iron pool and lipid peroxidation. Both these processes were key characteristics of ferroptosis. The induction of ferroptosis mediated by ginkgetin was further confirmed by the decreased expression of SLC7A11 and GPX4, and a decreased GSH/GSSG ratio. Simultaneously, ginkgetin disrupted redox hemostasis in DDP-treated cells, as demonstrated by the enhanced ROS formation and inactivation of the Nrf2/HO-1 axis. Ginkgetin also enhanced DDP-induced mitochondrial membrane potential (MMP) loss and apoptosis in cultured NSCLC cells. Furthermore, blocking ferroptosis reversed the ginkgetin-induced inactivation of Nrf2/HO-1 as well as the elevation of ROS formation, MMP loss, and apoptosis in DDP-treated NSCLC cells.ConclusionThis study is the first to report that ginkgetin derived from Ginkgo biloba leaves promotes DDP-induced anticancer effects, which can be due to the induction of ferroptosis.     

HMGB1 regulates ferroptosis through Nrf2 pathway in mesangial cells in response to high glucose

Ferroptosis, a novel type of programmed cell death, is involved in inflammation and oxidation of various human diseases, including diabetic kidney disease. The present study explored the role of high-mobility group box-1 (HMGB1) on the regulation of ferroptosis in mesangial cells in response to high glucose. Compared with healthy control, levels of serum ferritin, lactate dehydrogenase (LDH), reactive oxygen species (ROS), malonaldehyde (MDA), and HMGB1 were significantly elevated in diabetic nephropathy (DN) patients, accompanied with deregulated ferroptosis-related molecules, including long-chain acyl-CoA synthetase 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (PTGS2), NADPH oxidase 1 (NOX1), and glutathione peroxidase 4 (GPX4). In vitro assay revealed that erastin and high glucose both induced ferroptosis in mesangial cells. Suppression of HMGB1 restored cellular proliferation, prevented ROS and LDH generation, decreased ACSL4, PTGS2, and NOX1, and increased GPX4 levels in mesangial cells. Furthermore, nuclear factor E2-related factor 2 (Nrf2) was decreased in DN patients and high glucose-mediated translocation of HMGB1 in mesangial cells. Knockdown of HMGB1 suppressed high glucose-induced activation of TLR4/NF-κB axis and promoted Nrf2 expression as well as its downstream targets including HO-1, NQO-1, GCLC, and GCLM. Collectively, these findings suggest that HMGB1 regulates glucose-induced ferroptosis via Nrf2 pathway in mesangial cells.     

Acetaminophen sensitizing erastin-induced ferroptosis via modulation of Nrf2/heme oxygenase-1 signaling pathway in non-small-cell lung cancer

Growing evidence confirms that ferroptosis plays an important role in tumor growth inhibition. However, some non-small-cell lung cancer (NSCLC) cell lines are less sensitive to erastin-induced ferroptotic cell death. Elucidating the mechanism of resistance of cancer cells to erastin-induced ferroptosis and increasing the sensitivity of cancer cells to erastin need to be addressed. In our experiment, erastin and acetaminophen (APAP) cotreatment inhibited NSCLC cell viability and promoted ferroptosis and apoptosis, accompanied with attenuation of glutathione and ectopic increases in lipid peroxides. Erastin and APAP promoted NSCLC cell death by regulating nucleus translocation of nuclear factor erythroid 2-related factor 2 (Nrf2); and the ferroptosis induced by erastin and APAP was abrogated by bardoxolone methyl (BM) with less generation of reactive oxygen species and malondialdehyde. As a downstream gene of Nrf2, heme oxygenase-1 expression decreased significantly with the cotreatment of erastin and APAP, which could be rescued by BM. In vivo experiment showed that the combination of erastin and APAP had a synergic therapeutic effect on xenograft of lung cancer. In short, the present study develops a new effective treatment for NSCLC by synergizing erastin and APAP to induce ferroptosis.     

Transferrin-based radiolabeled probe predicts the sensitivity of human renal cancer cell lines to ferroptosis inducer erastin

Ferroptosis induction has been recognized as a novel cancer therapeutic strategy. To effectively apply ferroptosis-targeting cancer therapy to individual patients, a diagnostic indicator for selecting this therapeutic strategy from a number of molecular targeting drugs is needed. However, to date, methods that can predict the efficacy of ferroptosis-targeting treatment have not been established yet. In this study, we focused on the iron metabolic pathway to develop a nuclear imaging technique for diagnosing the susceptibility of cancer cells to ferroptosis. As a nuclear probe, human transferrin (Tf) was labeled with Gallium-68 (⁶⁸Ga) using 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as a chelator (⁶⁸Ga-NOTA-Tf). Western blot assay and clonogenic survival assay with human renal cancer cell lines A498 and 786-O revealed that the protein expression level of transferrin receptor1 (TfR1) and sensitivity to a ferroptosis inducer, erastin, were correlated. A cellular uptake assay with ⁶⁸Ga-NOTA-Tf revealed that the cancer cells sensitive to erastin highly internalized the ⁶⁸Ga-NOTA-Tf. Furthermore, treatment with the TfR1 inhibitor ferristatin II reduced the cellular uptake of ⁶⁸Ga-NOTA-Tf, indicating that the intracellular uptake of the probe was mediated by TfR1. These results suggest that ⁶⁸Ga-NOTA-Tf can be useful in predicting the sensitivity of cancer cells to ferroptosis inducers.     

Hsp90 induces Acsl4-dependent glioma ferroptosis via dephosphorylating Ser637 at Drp1

Ferroptosis is a newly identified form of regulated cell death (RCD) characterized by the iron-dependent lipid reactive oxygen species (ROS) accumulation, but its mechanism in gliomas remains elusive. Acyl–coenzyme A (CoA) synthetase long-chain family member 4 (Acsl4), a pivotal enzyme in the regulation of lipid biosynthesis, benefits the initiation of ferroptosis, but its role in gliomas needs further clarification. Erastin, a classic inducer of ferroptosis, has recently been found to regulate lipid peroxidation by regulating Acsl4 other than glutathione peroxidase 4 (GPX4) in ferroptosis. In this study, we demonstrated that heat shock protein 90 (Hsp90) and dynamin-related protein 1 (Drp1) actively regulated and stabilized Acsl4 expression in erastin-induced ferroptosis in gliomas. Hsp90 overexpression and calcineurin (CN)–mediated Drp1 dephosphorylation at serine 637 (Ser637) promoted ferroptosis by altering mitochondrial morphology and increasing Acsl4-mediated lipid peroxidation. Importantly, promotion of the Hsp90–Acsl4 pathway augmented anticancer activity of erastin in vitro and in vivo. Our discovery reveals a novel and efficient approach to ferroptosis-mediated glioma therapy. Subject terms: Drug development, Drug discovery     

Ferroptosis: an iron-dependent form of nonapoptotic cell death

Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.     

CIRBP promotes ferroptosis by interacting with ELAVL1 and activating ferritinophagy during renal ischaemia-reperfusion injury

Renal ischaemia-reperfusion (IR) is a major cause of acute kidney injury (AKI). Cold-inducible RNA-binding protein (CIRBP) may contribute to AKI because its deficiency protects against renal IR injury in a mechanism believed to involve ferroptosis. We aimed to investigate whether ferroptosis is associated with CIRBP-mediated renal damage. The differential expression of CIRBP was examined in tubular epithelial (HK2) cells during hypoxia-reoxygenation (HR) or in response to erastin, an inducer of ferroptosis. CIRBP expression was increased in response to HR or erastin in HK2 cells but the silencing of CIRBP inhibited HR and erastin-induced ferroptosis together with ferritinophagy. We discovered an interaction between CIRBP and ELAVL1 using STRING software, which was verified through co-immunoprecipitation and fluorescence colocalization assays. We found that ELAVL1 is a critical regulator in the activation of ferritinophagy and the promotion of ferroptosis. HR or erastin also induced the expression of ELAVL1. An autophagy inhibitor (hydroxychloroquine) or si-ELAVL1 transfection reversed CIRBP-enhanced ferritinophagy activation and ferroptosis in HK2 cells under HR. Injection of anti-CIRBP antibody into a mouse model of IR inhibited ferroptosis and decreased renal IR injury in vivo. In summary, our results provide evidence that ferritinophagy-mediated ferroptosis could be responsible for CIRBP-enhanced renal IR injury.     

Valosin-containing protein (VCP) promotes the growth,invasion, and metastasis of colorectal cancer through activation of STAT3 signaling

Valosin-containing protein (VCP) was previously shown to exhibit high expression in colorectal cancer (CRC) tissues as compared with that in normal tissues; however, the role of VCP in human CRC cells has remained to be elucidated. Two colorectal cancer cell lines HCT116 and RKO were used in the experiment. We introduced lentiviral constructs expressing VCP to infect RKO cells and lenti-shRNA targeting VCP into HCT116 cells, respectively. Cell proliferation, invasion, apoptosis, and cell cycle arrest were subsequently examined by MTT assay, transwell chamber assay, flow cytometry, and western blot analysis, respectively. Furthermore, a subcutaneous tumor mouse model and lung metastasis model was used to investigate the effects of VCP on the growth and metastasis of CRC cells in vivo. VCP knockdown was shown to inhibit cell proliferation, chemoresistance and invasion, and induce apoptosis in the HCT116 CRC cells, whereas VCP over-expression suppressed apoptosis and chemoresponse, promoted proliferation and invasion of the RKO CRC cells. In addition, in the subcutaneous tumor and lung metastasis mouse model, VCP knockdown in HCT116 cells suppressed carcinogenesis and metastasis in vivo. The findings of the present study indicated that VCP is very important for the proliferation and metastasis of CRC; therefore, targeting VCP and its downstream targets may represent novel therapies for the treatment of CRC.

     

Shuganning injection,a traditional Chinese patent medicine,induces ferroptosis and suppresses tumor growth in triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC), lacking targeted therapies currently, is susceptible to ferroptosis, a recently defined form of cell death.PurposeTo evaluate the anticancer activity of Shuganning injection (SGNI), a traditional Chinese patent medicine, on TNBC cells; To elucidate the mechanism of SGNI induced ferroptosis.MethodsThe anticancer activity of SGNI was examined via in vitro cell proliferation assays and in vivo xenograft growth assay. Ferroptosis was determined by flow-cytometric analysis of lipid ROS, labile iron pool measurement, and propidium iodide exclusion assay. The dependency on heme oxygenase 1 (HO-1) of SGNI induced ferroptosis was confirmed by genetic knockdown and pharmacological inhibition of the protein.ResultsSGNI selectively inhibited the proliferation of TNBC cells compared to non-TNBC breast cancer cells and normal cells. The cell death induced by SGNI in TNBC cells showed distinct morphology from apoptosis and could not be rescued by the pan-caspase inhibitor Z-VAD(OMe)-FMK. On the other hand, SGNI induced cell death was blocked by the lipid ROS scavengers ferrostatin-1 and liproxstatin-1, the acyl-CoA synthetase long chain family member 4 inhibitor rosiglitazone, and the iron chelators 1,10-phenanthroline and deferoxamine. These data indicated that SGNI induced a ferroptotic cell death of TNBC cells. Mechanistically, SGNI induced ferroptosis was dependent on HO-1, which promotes intracellular labile iron pool accumulation, and was alleviated by HO-1 knockdown and inhibition by tin protoporphyrin IX. In line with the in vitro data, SGNI significantly inhibited the xenograft growth of TNBC cell line MD-MB-231 in nude mice.ConclusionCollectively, our study elaborates on a promising regimen for TNBC treatment through induction of ferroptosis by SGNI, a traditional Chinese patent medicine currently available in the clinic, which merits further investigation.     

Chemotoxicity-induced exosomal lncFERO regulates ferroptosis and stemness in gastric cancer stem cells

Cancer stem cells (CSCs) are an important cause of tumor recurrence and drug resistance. As a new type of cell death that relies on iron ions and is strictly regulated by intracellular and extracellular signals, the role of ferroptosis in tumor stem cells deserves extensive attention. Mass spectrum was applied to screen for ferroptosis-related proteins in gastric cancer (GC). Sphere-formation assay was used to estimate the stemness of gastric cancer stem cells (GCSCs). Exosomal lnc-ENDOG-1:1 (lncFERO) was isolated by ultracentrifugation. Ferroptosis was induced by erastin and was assessed by detecting lipid ROS, mitochondrial membrane potential, and cell death. Furthermore, a series of functional in vitro and in vivo experiments were conducted to evaluate the effects of lncFERO on regulating ferroptosis and chemosensitivity in GCSCs. Here, we showed that stearoyl-CoA-desaturase (SCD1) played a key role in regulating lipid metabolism and ferroptosis in GCSCs. Importantly, exosomal lncFERO (exo-lncFERO) derived from GC cells was demonstrated to promote SCD1 expression by directly interacting with SCD1 mRNA and recruiting heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which resulted in the dysregulation of PUFA levels and the suppression of ferroptosis in GCSCs. Moreover, we found that hnRNPA1 was also involved in lncFERO packing into exosomes in GC cells, and both in vitro and in vivo data suggested that chemotoxicity induced lncFERO secretion from GC cells by upregulating hnRNPA1 expression, leading to enhanced stemness and acquired chemo-resistance. All these data suggest that GC cells derived exo-lncFERO controls GCSC tumorigenic properties through suppressing ferroptosis, and targeting exo-lncFERO/hnRNPA1/SCD1 axis combined with chemotherapy could be a promising CSC-based strategy for the treatment of GC.Subject terms: Stem-cell research, Gastric cancer     

Molecular mechanisms of ferroptosis and its role in cancer therapy

Ferroptosis is a newly defined programmed cell death process with the hallmark of the accumulation of iron‐dependent lipid peroxides. The term was first coined in 2012 by the Stockwell Lab, who described a unique type of cell death induced by the small molecules erastin or RSL3. Ferroptosis is distinct from other already established programmed cell death and has unique morphological and bioenergetic features. The physiological role of ferroptosis during development has not been well characterized. However, ferroptosis shows great potentials during the cancer therapy. Great progress has been made in exploring the mechanisms of ferroptosis. In this review, we focus on the molecular mechanisms of ferroptosis, the small molecules functioning in ferroptosis initiation and ferroptosis sensitivity in different cancers. We are also concerned with the new arising questions in this particular research area that remains unanswered.     

Therapeutic potential of melatonin in the intervertebral disc degeneration through inhibiting the ferroptosis of nucleus pulpous cells

Ferroptosis, a novel type of cell death mediated by the iron-dependent lipid peroxidation, contributes to the pathogenesis of the intervertebral disc degeneration (IDD). Increasing evidence demonstrated that melatonin (MLT) displayed the therapeutic potential to prevent the development of IDD. Current mechanistic study aims to explore whether the downregulation of ferroptosis contributes to the therapeutic capability of MLT in IDD. Current studies demonstrated that conditioned medium (CM) from the lipopolysaccharide (LPS)-stimulated macrophages caused a series of changes about IDD, including increased intracellular oxidative stress (increased reactive oxygen species and malondialdehyde levels, but decreased glutathione levels), upregulated expression of inflammation-associated factors (IL-1β, COX-2 and iNOS), increased expression of key matrix catabolic molecules (MMP-13, ADAMTS4 and ADAMTS5), reduced the expression of major matrix anabolic molecules (COL2A1 and ACAN), and increased ferroptosis (downregulated GPX4 and SLC7A11 levels, but upregulated ACSL4 and LPCAT3 levels) in nucleus pulposus (NP) cells. MLT could alleviate CM-induced NP cell injury in a dose-dependent manner. Moreover, the data substantiated that intercellular iron overload was involved in CM-induced ferroptosis in NP cells, and MLT treatment alleviated intercellular iron overload and protected NP cells against ferroptosis, and those protective effects of MLT in NP cells further attenuated with erastin and enhanced with ferrostatin-1(Fer-1). This study demonstrated that CM from the LPS-stimulated RAW264.7 macrophages promoted the NP cell injury. MLT alleviated the CM-induced NP cell injury partly through inhibiting ferroptosis. The findings support the role of ferroptosis in the pathogenesis of IDD, and suggest that MLT may serve as a potential therapeutic approach for clinical treatment of IDD.     

Liproxstatin-1 attenuates unilateral ureteral obstruction-induced renal fibrosis by inhibiting renal tubular epithelial cells ferroptosis

Renal fibrosis is a common pathological process that occurs with diverse etiologies in chronic kidney disease. However, its regulatory mechanisms have not yet been fully elucidated. Ferroptosis is a form of non-apoptotic regulated cell death driven by iron-dependent lipid peroxidation. It is currently unknown whether ferroptosis is initiated during unilateral ureteral obstruction (UUO)-induced renal fibrosis and its role has not been determined. In this study, we demonstrated that ureteral obstruction induced ferroptosis in renal tubular epithelial cells (TECs) in vivo. The ferroptosis inhibitor liproxstatin-1 (Lip-1) reduced iron deposition, cell death, lipid peroxidation, and inhibited the downregulation of GPX4 expression induced by UUO, ultimately inhibiting ferroptosis in TECs. We found that Lip-1 significantly attenuated UUO-induced morphological and pathological changes and collagen deposition of renal fibrosis in mice. In addition, Lip-1 attenuated the expression of profibrotic factors in the UUO model. In vitro, we used RSL3 treatment and knocked down of GPX4 level by RNAi in HK2 cells to induce ferroptosis. Our results indicated HK2 cells secreted various profibrotic factors during ferroptosis. Lip-1 was able to inhibit ferroptosis and thereby inhibit the secretion of the profibrotic factors during the process. Incubation of kidney fibroblasts with culture medium from RSL3-induced HK2 cells promoted fibroblast proliferation and activation, whereas Lip-1 impeded the profibrotic effects. Our study found that Lip-1 may relieve renal fibrosis by inhibiting ferroptosis in TECs. Mechanistically, Lip-1 could reduce the activation of surrounding fibroblasts by inhibiting the paracrine of profibrotic factors in HK2 cells. Lip-1 may potentially be used as a therapeutic approach for the treatment of UUO-induced renal fibrosis.Subject terms: Cell death, RNAi, Urinary tract obstruction