Lysosomal Iron Research Articles

Changes in meat quality of Esox Lucius during postmortem storage: Based on the lysosomal-mitochondrial apoptotic pathway

In this study, we explored the correlation between the lysosome–mitochondrial apoptosis pathway and fish softening, as well as the correlation between ferritin degradation and lysosomal iron changes. The results indicated that ferritin levels gradually decreased, lysosomal iron first increased and then decreased and tended to stabilize, and lysosomal membrane stability significantly decreased (p < 0.05). Spearman's analysis suggested that an increase in lysosomal iron was associated with ferritin degradation. Lysosomal instability promoted the release of cathepsin D, thereby increasing the release of Bid and Bax, and inhibiting the expression of Bcl-2. Subsequently, caspase-9/−3 was activated. In addition, transmission electron microscopy revealed ultrastructural damage to mitochondria and cell nuclei, which are morphological features of apoptosis during post-mortem storage. Moreover, TUNEL staining confirmed the occurrence of apoptosis. We concluded that the lysosome– mitochondrial apoptosis pathway was active during the storage of Esox Lucius, in which ferritin degradation and increased lysosomal iron were key factors inducing lysosomal damage, and cathepsin D released by lysosomes was a key factor connecting lysosomes and mitochondria.

Abstract IA-01: Targeting autophagy in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most vexing problems in cancer with 5-year overall survival rates of 13%, thus there is a need for new therapeutic targets. PDAC have a distinct dependence on autophagy, a conserved eukaryotic catabolic pathway that serves to degrade proteins, other macromolecules, and organelles via the lysosome as part of a recycling process to maintain cellular homeostasis during basal and stress conditions. The unique importance of autophagy in PDAC suggests that there may be a subset of proteins that are specifically targeted by autophagy for degradation, which would promote proliferation and survival, and that these may represent new targets for therapy. Using a quantitative PDAC cellular autophagosomal proteomics approach, we identified NCOA4 as the autophagy receptor for ferritin, the cellular iron storage complex, and demonstrated that ferritin autophagy (ferritinophagy) is a highly regulated process and plays a central role in the larger context of cellular and organismal iron homeostasis. Given the reliance of PDAC, on both high levels of autophagy as well as iron, we studied the role of NCOA4 and ferritinophagy in PDAC thereby identifying NCOA4-mediated ferritinophagy and more broadly lysosomal iron metabolism as a PDAC dependency and therapeutic target. We recently determined the cryo-EM structure of the NCOA4-Ferritin complex for future drug design. To identify the role of autophagy and the lysosome in PDAC metastatic progression, we co-developed an in vivo capable lysosomal enrichment that enables capture and proteomic profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. Employing this strategy, we successfully captured lysosomal proteins from primary tumors (pancreas), as well as lung and liver metastases. Our preliminary analysis revealed an enrichment of resident lysosomal membrane and luminal proteins in the datasets, confirming successful isolation of intact lysosomes from these complex tissues. Additionally, we identified non-resident lysosomal proteins (termed cargo) that are enriched and co-evolve with advancing tumor stages. Notably, our results reveal distinct differences in the lysosomal proteome of tumor cells across different tissues. These findings emphasize the critical role of lysosomal function in tumor progression and suggest that targeting lysosomal components could be a promising therapeutic strategy for treating metastatic PDAC. Finally, given the challenging pharmacokinetic and pharmacodynamic properties and limited clinical efficacy of hydroxychloroquine as an autophagy/lysosomal inhibition strategy, we are now exploring novel autophagy inhibition strategies in PDAC to effectively target this pathway for clinical benefit. Citation Format: Joseph D. Mancias. Targeting autophagy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-01.

Lysosomal iron accumulation and subsequent lysosomes-mitochondria iron transmission mediate PFOS-induced hepatocyte ferroptosis

Perfluorooctane sulfonate (PFOS) is known as a persistent organic pollutant. A significant correlation between PFOS and liver ferroptosis has been unveiled, but the precise mechanism needs to be elucidated. In prior research, we found that PFOS treatment provoked mitochondrial iron overload. In this study, we observed a gradual increase in lysosomal iron in L-O2 cells after exposure to PFOS for 0.5–24 h. In PFOS-exposed L-O2 cells, suppressing autophagy relieved the lysosomal iron overload. Inhibiting transient receptor potential mucolipin 1 (TRPML1), a calcium efflux channel on the lysosomal membrane, led to a further rise in lysosomal iron levels and decreased mitochondrial iron overload during PFOS treatment. Suppressing VDAC1, a subtype of voltage-dependent anion-selective channels (VDACs) on the outer mitochondrial membrane, had no impact on PFOS-triggered mitochondrial iron overload, whereas restraining VDAC2/3 relieved this condition. Although silencing VDAC2 relieved PFOS-induced mitochondrial iron overload, it had no effect on PFOS-triggered lysosomal iron overload. Silencing VDAC3 alleviated PFOS-mediated mitochondrial iron overload and led to an additional increase in lysosomal iron. Therefore, we regarded VDAC3 as the specific VDACs subtype that mediated the lysosomes-mitochondria iron transfer. Additionally, in the presence of PFOS, an enhanced association between TRPML1 and VDAC3 was found in mice liver tissue and L-O2 cells. Our research unveils a novel regulatory mechanism of autophagy on the iron homeostasis and the effect of TRPML1-VDAC3 interaction on lysosomes-mitochondria iron transfer, giving an explanation of PFOS-induced ferroptosis and shedding some light on the role of classic calcium channels in iron transmission.

Erdafitinib promotes ferroptosis in human uveal melanoma by inducing ferritinophagy and lysosome biogenesis via modulating the FGFR1/mTORC1/TFEB signaling axis

Uveal melanoma (UM) is a rare yet lethal primary intraocular malignancy affecting adults. Analysis of data from The Cancer Genome Atlas (TCGA) database revealed that FGFR1 expression was increased in UM tumor tissues and was linked to aggressive behavior and a poor prognosis. This study assessed the anti-tumor effects of Erdafitinib, a selective pan-FGFR inhibitor, in both in vitro and in vivo UM models. Erdafitinib exhibited a robust anti-cancer activity in UM through inducing ferroptosis in the FGFR1-dependent manner. Transcriptomic data revealed that Erdafitinib mediated its anti-cancer effects via modulating the ferritinophagy/lysosome biogenesis. Subsequent research revealed that Erdafitinib exerted its effects by reducing the expression of FGFR1 and inhibiting the activity of mTORC1 in UM cells. Concurrently, it enhanced the dephosphorylation, nuclear translocation, and transcriptional activity of TFEB. The aggregation of TFEB in nucleus triggered FTH1-dependent ferritinophagy, leading to lysosomal activation and iron overload. Conversely, the overexpression of FGFR1 served to mitigate the effects of Erdafitinib on ferritinophagy, lysosome biogenesis, and the activation of the mTORC1/TFEB signaling pathway. In vivo experiments have convincingly shown that Erdafitinib markedly curtails tumor growth in an UM xenograft mouse model, an effect that is closely correlated with a decrease in FGFR1 expression levels. The present study is the first to demonstrate that Erdafitinib powerfully induces ferroptosis in UM by orchestrating the ferritinophagy and lysosome biogenesis via modulating the FGFR1/mTORC1/TFEB signaling. Consequently, Erdafitinib emerges as a strong candidate for clinical trial investigation, and FGFR1 emerges as a novel and promising therapeutic target in the treatment of UM.

Fluoride Induces Neurocytotoxicity by Disrupting Lysosomal Iron Metabolism and Membrane Permeability.

The detrimental effects of fluoride on neurotoxicity have been widely recorded, yet the detailed mechanisms underlying these effects remain unclear. This study explores lysosomal iron metabolism in fluoride-related neurotoxicity, with a focus on the Steap3/TRPML1 axis. Utilizing sodium fluoride (NaF)-treated human neuroblastoma (SH-SY5Y) and mouse hippocampal neuron (HT22) cell lines, our research demonstrates that NaF enhances the accumulation of ferrous ions (Fe2+) in these cells, disrupting lysosomal iron metabolism through the Steap3/TRPML1 axis. Notably, NaF exposure upregulated ACSL4 and downregulated GPX4, accompanied by reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity and increased malondialdehyde (MDA) levels. These changes indicate increased vulnerability to ferroptosis within neuronal cells. The iron chelator deferoxamine (DFO) mitigates this disruption. DFO binds to lysosomal Fe2+ and inhibits the Steap3/TRPML1 axis, restoring normal lysosomal iron metabolism, preventing lysosomal membrane permeabilization (LMP), and reducing neuronal cell ferroptosis. Our findings suggest that interference in lysosomal iron metabolism may mitigate fluoride-induced neurotoxicity, underscoring the critical role of the Steap3/TRPML1 axis in this pathological process.

Cardiomyocyte-specific deletion of the mitochondrial transporter Abcb10 causes cardiac dysfunction via lysosomal-mediated ferroptosis.

Heart function is highly dependent on mitochondria, which not only produce energy but also regulate many cellular functions. Therefore, mitochondria are important therapeutic targets in heart failure. Abcb10 is a member of the ABC transporter superfamily located in the inner mitochondrial membrane and plays an important role in haemoglobin synthesis, biliverdin transport, antioxidant stress, and stabilization of the iron transporter mitoferrin-1. However, the mechanisms underlying the impairment of mitochondrial transporters in the heart remain poorly understood. Here, we generated mice with cardiomyocyte-specific loss of Abcb10. The Abcb10 knockouts exhibited progressive worsening of cardiac fibrosis, increased cardiovascular risk markers and mitochondrial structural abnormalities, suggesting that the pathology of heart failure is related to mitochondrial dysfunction. As the mitochondrial dysfunction was observed early but mildly, other factors were considered. We then observed increased Hif1α expression, decreased NAD synthase expression, and reduced NAD+ levels, leading to lysosomal dysfunction. Analysis of ABCB10 knockdown HeLa cells revealed accumulation of Fe2+ and lipid peroxides in lysosomes, leading to ferroptosis. Lipid peroxidation was suppressed by treatment with iron chelators, suggesting that lysosomal iron accumulation is involved in ferroptosis. We also observed that Abcb10 knockout cardiomyocytes exhibited increased ROS production, iron accumulation, and lysosomal hypertrophy. Our findings suggest that Abcb10 is required for the maintenance of cardiac function and reveal a novel pathophysiology of chronic heart failure related to lysosomal function and ferroptosis.

L-serine restored lysosomal failure in cells derived from patients with BPAN reducing iron accumulation with eliminating lipofuscin

Defective mitochondria and autophagy, as well as accumulation of lipid and iron in WDR45 mutant fibroblasts, is related to beta-propeller protein-associated neurodegeneration (BPAN). In this study, we found that enlarged lysosomes in cells derived from patients with BPAN had low enzyme activity, and most of the enlarged lysosomes had an accumulation of iron and oxidized lipid. Cryo-electron tomography revealed elongated lipid accumulation, and spectrometry-based elemental analysis showed that lysosomal iron and oxygen accumulation superimposed with lipid aggregates. Lysosomal lipid aggregates superimposed with autofluorescence as free radical generator, lipofuscin. To eliminate free radical stress by iron accumulation in cells derived from patients with BPAN, we investigated the effects of the iron chelator, 2,2′-bipyridine (bipyridyl, BIP). To study whether the defects in patient-derived cells can be rescued by an iron chelator BIP, we tested whether the level of iron and reactive oxygen species (ROS) in the cells and genes related to oxidative stress were rescued BIP treatment. Although BIP treatment decreased some iron accumulation in the cytoplasm and mitochondria, the accumulation of iron in the lysosomes and levels of cellular ROS were unaffected. In addition, the change of specific RNA levels related to free radical stress in patient fibroblasts was not rescued by BIP. To alleviate free radical stress, we investigated whether l-serine can regulate abnormal structures in cells derived from patients with BPAN through the regulation of free radical stress. l-serine treatment alleviated increase of enlarged lysosomes and iron accumulation and rescued impaired lysosomal activity by reducing oxidized lipid accumulation in the lysosomes of the cells. Lamellated lipids in the lysosomes of the cells were identified as lipofuscin through correlative light and electron microscopy, and l-serine treatment reduced the increase of lipofuscin. These data suggest that l-serine reduces oxidative stress-mediated lysosomal lipid oxidation and iron accumulation by rescuing lysosomal activity.

Nebulized Therapy of Early Orthotopic Lung Cancer by Iron-Based Nanoparticles: Macrophage-Regulated Ferroptosis of Cancer Stem Cells.

Cancer stem cells (CSCs) within protumorigenic microlesions are a critical driver in the initiation and progression of early stage lung cancer, where immune cells provide an immunosuppressive niche to strengthen the CSC stemness. As the mutual interactions between CSCs and immune cells are increasingly recognized, regulating the immune cells to identify and effectively eliminate CSCs has recently become one of the most attractive therapeutic options, especially for abundant tumor-associated macrophages (TAMs). Herein, we developed a nebulized nanocatalytic medicine strategy in which iron-based nanoparticle-regulated TAMs effectively target CSC niches and trigger CSC ferroptosis in the early stage of lung cancer. Briefly, the iron-based nanoparticles can effectively accumulate in lung cancer microlesions (minimum 122 μm in diameter) through dextran-mediated TAM targeting by nebulization administration, and as a result, nanoparticle-internalized TAMs can play a predominant role of the iron factory in elevating the iron level surrounding CSC niches and destroying redox equilibrium through downregulating glucose-6-phosphate metabolite following their lysosomal degradation and iron metabolism. The altered microenvironment results in the enhanced sensitivity of CSCs to ferroptosis due to their high expression of the CD44 receptor mediating iron endocytosis. In an orthotopic mouse model of lung cancer, the initiation and progression of early lung cancer are significantly suppressed through ferroptosis-induced stemness reduction of CSCs by nebulization administration. This work presents a nebulized therapeutic strategy for early lung cancer through modulation of communications between TAMs and CSCs, which is expected to be a general approach for regulating primary microlesions and micrometastatic niches of lung cancer.

Sodium Sulfite-Triggered Hepatocyte Ferroptosis via mtROS/Lysosomal Membrane Permeabilization-Mediated Lysosome Iron Efflux.

Sodium sulfite is a widely used preservative in the food industry. Ferroptosis has been a newly discovered form of iron-dependent oxidative cell death in recent years. However, the potential connection between sodium sulfite and ferroptosis has not been explored. In our study, we observed the abnormal expression of ferroptosis marker protein in vivo, suggesting that sodium sulfite caused ferroptosis in vivo. Next, our study revealed that sodium sulfite caused the overproduction of mitochondrial reactive oxygen species (mtROS) in the AML-12 cells. It is well established that reactive oxygen species (ROS) can induce lysosomal membrane permeabilization. After lysosomal membrane permeabilization occurs, the outflow of Fe2+ in lysosomes triggers the Fenton reaction and subsequently results in the increase of intracellular ROS level, which is closely related to ferroptosis. As speculated, acridine orange (AO) staining and LysoTracker red staining showed that sodium sulfite-induced lysosomal membrane permeabilization could be alleviated by mtROS scavenger TEMPO. In addition, TEMPO, lysosomal stabilizer mannose, and lysosomal iron chelator deferoxamine (DFO) inhibited sodium sulfite-induced ferroptosis. Overall, the results showed that sodium sulfite induced lysosomal iron efflux through the mtROS-lysosomal membrane permeabilization pathway and eventually led to ferroptosis. Our study might provide a new mechanism for the hepatotoxicity of sodium sulfite and a theoretical basis for the risk assessment of sodium sulfite as a food additive.

Ferritinophagy-mediated apoptosis and paraptosis induction involved MAPK and PI3K/AKT pathway in mechanism of an iron chelator

Melanoma cells were more resistant to ferroptosis with still poor therapy outcomes. Sensitizing melanoma cell to the ferroptosis inducer was a crucial strategy for treatment of melanoma. In the present study, 2,2′-di-pyridylketone hydrazone dithiocarbamate s-butyric acid (DpdtbA) displayed superior inhibitory activity than ferroptosis inducer Erastin in melanoma cells, which prompt us to explore the underlying mechanism. The analyses from flow cytometry and Western blot showed that the growth inhibition of DpdtbA against SK-MEL-28 and A375 cells involved apoptosis induction and G1 phase arrest. Surprisingly, the cytoplasmic vacuoles were found upon the treatment; transmission electron microscopy and endoplasmic reticulum (ER) staining revealed that the cytoplasmic vacuoles were in ER; while down-regulation of alix and requirement of protein synthesis suggested there was an occurrence of paraptosis. However, both NAC and 3-MA could significantly attenuate the cytoplasmic vacuolization and growth inhibition, hinting that both ROS and autophagy involved the paraptosis induction. The additional evidence revealed that there was an occurrence of continuous ferritinophagy, which was responsible for the ROS production. Downregulation of NCOA4 clearly attenuated the apoptosis and paraptosis induction. In addition, activation of MAPK involved regulation of paraptosis, but only ERK and JNK had role in the formation of cytoplasmic vacuoles and growth inhibition. Furthermore, a ROS dependent regulation of PI3K/AKT pathway was also involved. Taken together, our result firstly demonstrated that a continuous ferritinophagy contributed to the apoptosis and paraptosis induction, highlighting that the lysosomal labile iron pool had a crucial role in control of melanoma cell fate.

Improving lysosomal ferroptosis with NMN administration protects against heart failure.

Myocardial mitochondria are primary sites of myocardial energy metabolism. Mitochondrial disorders are associated with various cardiac diseases. We previously showed that mice with cardiomyocyte-specific knockout of the mitochondrial translation factor p32 developed heart failure from dilated cardiomyopathy. Mitochondrial translation defects cause not only mitochondrial dysfunction but also decreased nicotinamide adenine dinucleotide (NAD+) levels, leading to impaired lysosomal acidification and autophagy. In this study, we investigated whether nicotinamide mononucleotide (NMN) administration, which compensates for decreased NAD+ levels, improves heart failure because of mitochondrial dysfunction. NMN administration reduced damaged lysosomes and improved autophagy, thereby reducing heart failure and extending the lifespan in p32cKO mice. We found that lysosomal damage due to mitochondrial dysfunction induced ferroptosis, involving the accumulation of iron in lysosomes and lipid peroxide. The ameliorative effects of NMN supplementation were found to strongly affect lysosomal function rather than mitochondrial function, particularly lysosome-mediated ferroptosis. NMN supplementation can improve lysosomal, rather than mitochondrial, function and prevent chronic heart failure.

Chronic cadmium exposure triggered ferroptosis by perturbing the STEAP3-mediated glutathione redox balance linked to altered metabolomic signatures in humans

Cadmium (Cd), a predominant environmental pollutant, is a canonical toxicant that acts on the kidneys. However, the nephrotoxic effect and underlying mechanism activated by chronic exposure to Cd remain unclear. In the present study, male mice (C57BL/6J, 8 weeks) were treated with 0.6 mg/L cadmium chloride (CdCl2) administered orally for 6 months, and tubular epithelial cells (TCMK-1 cells) were treated with low-dose (1, 2, and 3 μM) CdCl2 for 72 h (h). Our study results revealed that environmental Cd exposure triggered ferroptosis and renal dysfunction. Spatially resolved metabolomics enabled delineation of metabolic profiles and visualization of the disruption to glutathione homeostasis related to ferroptosis in mouse kidneys. Multiomics analysis revealed that chronic Cd exposure induced glutathione redox imbalance that depended on STEAP3-driven lysosomal iron overload. In particular, glutathione metabolic reprogramming linked to ferroptosis emerged as a metabolic hallmark in the blood of Cd-exposed workers. In conclusion, this study provides the first evidence indicating that chronic Cd exposure triggers ferroptosis and renal dysfunction that depend on STEAP3-mediated glutathione redox imbalance, greatly increasing our understanding of the metabolic reprogramming induced by Cd exposure in the kidneys and providing novel clues linking chronic Cd exposure to nephrotoxicity.

TFEB regulates cellular labile iron and prevents ferroptosis in a TfR1-dependent manner

Autophagy is a major clearance pathway for misfolded α-synuclein which promotes ferroptosis through NCOA4-mediated ferritin degradation. The regulation of these two processes to achieve improved neuroprotection in Parkinson's disease (PD) must be elucidated. Transcription factor EB (TFEB) is a master regulator of both autophagy and lysosome biogenesis, and lysosomes are important cellular iron storage organelles; however, the role of TFEB in ferroptosis and iron metabolism remains unclear. In this study, TFEB overexpression promoted the clearance of misfolded α-synuclein and prevented ferroptosis and iron overload. TFEB overexpression up-regulated transferrin receptor 1 (TfR1) synthesis and increased the localization of TfR1 in the lysosome, facilitating lysosomal iron import and transient lysosomal iron storage. TFEB overexpression increased the levels of cellular iron-safe storage proteins (both ferritin light and heavy chains). These functions in iron metabolism maintain the cellular labile iron at a low level and electrical activity, even under iron overload conditions. Notably, lower levels of cellular labile iron and the upregulation of ferritin light and heavy chains were reversed after TfR1 knockdown in cells overexpressing TFEB, indicating that TFEB regulates cellular labile iron and suppresses ferroptosis in a TfR1 dependent manner. Taken together, this evidence of the regulation of iron metabolism enriches our understanding of the function of TFEB. In addition, TFEB overexpression protects against ferroptosis and iron overload and provides a new direction and perspective for autophagy regulation in PD.

Self-oriented ferritin nanocages mitigate iron overload-induced oxidative stress for acute kidney injury

Iron overload-induced oxidative injury of renal tubular epithelial cells plays crucial roles in the pathogenesis of ischemia–reperfusion acute kidney injury (IR-AKI). Curcumin is a polyphenolic compound that can effectively scavenge free radicals, but its poor water solubility and renal distribution restrict its application. Although nanotechnology helps to improve this situation by enabling solubility and renal accumulation, the headstream of oxidative injury, iron overload, has not been reverted. Ferritin (Fer) is a well-known iron storage protein with a favorable particle size for renal filtration that can potently transport excess iron into its core to prevent further oxidative damage. In this regard, ferritin nanocages loaded with curcumin (FNC/Cur) were prepared. It is worth noting that ferritin nanocage-based proteins showed a significant self-orientation capability into injured kidneys at 4 h. After kidney aggregation, glomerular filtration and renal tubular reabsorption, FNC/Cur finally internalized by the target cells (renal tubular epithelial cells) under the mediation of transferrin receptor-1 (TfR1). Once internalized, FNC/Cur released curcumin to reduce excess reactive oxygen species (ROS) with the help of lysosomal acidic environment and protease-rich environment. At the same time, the dissembled FNC or their released heavy chain are able to bind lysosomal redox-active iron for a period of time, thereby temporarily protect against iron-mediated oxidation. As a result, FNC/Cur represents a potential kidney-targeted nanoplatform for synergistic treatment of IR-AKI.

Lung microhaemorrhage drives oxidative/inflammatory damage in α1-antitrypsin deficiency.

Animal models using intratracheal instillation show that elastase, unopposed by α1-antitrypsin (AAT), causes alveolar damage and haemorrhage associated with emphysematous changes. The aim of the present study was to characterise any relationship between alveolar haemorrhage and human AAT deficiency (AATD) using bronchoalveolar lavage (BAL) and lung explant samples from AATD subjects. BAL samples (17 patients, 15 controls) were evaluated for free haem (iron protoporphyrin IX) and total iron concentrations. Alveolar macrophage activation patterns were assessed using RNA sequencing and validated in vitro using haem-stimulated, monocyte-derived macrophages. Lung explants (seven patients, four controls) were assessed for iron sequestration protein expression patterns using Prussian blue stain and ferritin immunohistochemistry, as well as ferritin iron imaging and elemental analysis by transmission electron microscopy. Tissue oxidative damage was assessed using 8-hydroxy-2'-deoxyguanosine immunohistochemistry. BAL collected from AATD patients showed significantly elevated free haem and total iron concentrations. Alveolar and interstitial macrophages in AATD explants showed elevated iron and ferritin accumulation in large lysosomes packed by iron oxide cores with degraded ferritin protein cages. BAL macrophage RNA sequencing showed innate pro-inflammatory activation, replicated in vitro by haemin exposure, which also triggered reactive oxygen species generation. AATD explants showed massive oxidative DNA damage in both lung epithelial cells and macrophages. BAL and tissue markers of alveolar haemorrhage, together with molecular and cellular evidence of macrophage innate pro-inflammatory activation and oxidative damage, are consistent with free haem stimulation. Overall, this initial study provides evidence for a pathogenetic role of elastase-induced alveolar haemorrhage in AATD emphysema.

Promotion of ferroptosis in head and neck cancer with divalent metal transporter 1 inhibition or salinomycin

Divalent metal transporter 1 (DMT1) inhibitors can selectively kill iron-addicted cancer stem cells by causing lysosomal iron overload, but their role in head and neck cancer (HNC) is unknown. We examined the role of DMT1 inhibition or salinomycin in promoting ferroptosis by lysosomal iron targeting in HNC cells. RNA interference was performed by transfection of siRNA targeting DMT1 or scrambled control siRNA in HNC cell lines. Cell death and viability, lipid peroxidation, iron contents, and molecular expression were compared between the DMT1 silencing or salinomycin group and the control. DMT1 silencing markedly accelerated cell death induced by the ferroptosis inducers. DMT1 silencing marked increases in the labile iron pool, intracellular ferrous and total iron contents, and lipid peroxidation. DMT1 silencing revealed molecular changes in iron starvation response, resulting in increases in TFRC, and decreases in FTH1. Salinomycin treatment also showed similar results to the above DMT1 silencing. DMT1 silencing or salinomycin can promote ferroptosis in HNC cells, suggesting a novel strategy for killing iron-avid cancer cells.

Iron-induced cytotoxicity mediated by endolysosomal TRPML1 channels is reverted by TFEB

Increased brain iron content has been consistently reported in sporadic Parkinson’s disease (PD) patients, and an increase in cytosolic free iron is known to cause oxidative stress and cell death. However, whether iron also accumulates in susceptible brain areas in humans or in mouse models of familial PD remains unknown. In addition, whilst the lysosome functions as a critical intracellular iron storage organelle, little is known about the mechanisms underlying lysosomal iron release and how this process is influenced by lysosome biogenesis and/or lysosomal exocytosis. Here, we report an increase in brain iron content also in PD patients due to the common G2019S-LRRK2 mutation as compared to healthy age-matched controls, whilst differences in iron content are not observed in G2019S-LRRK2 knockin as compared to control mice. Chemically triggering iron overload in cultured cells causes cytotoxicity via the endolysosomal release of iron which is mediated by TRPML1. TFEB expression reverts the iron overload-associated cytotoxicity by causing lysosomal exocytosis, which is dependent on a TRPML1-mediated increase in cytosolic calcium levels. Therefore, approaches aimed at increasing TFEB levels, or pharmacological TRPML1 activation in conjunction with iron chelation may prove beneficial against cell death associated with iron overload conditions such as those associated with PD.

Boosting ROS-Mediated Lysosomal Membrane Permeabilization for Cancer Ferroptosis Therapy.

Due to the deficient catalase, abundant reduced iron and low acidic environment in lysosomes, inducing lysosomal membrane permeabilization (LMP) through Fenton reaction-based reactive oxygen species (ROS) generation recently attracts increasing attention in cancer therapy. However, the lysosomal membranes are protected by highly glycosylated membrane proteins and several endolysosomal damage-response mechanisms can rapidly repair the injured lysosomes. To produce sufficient ROS and cause complete lysosomal membranes rupture, a lysosome-targeted ROS inducer, N-(3-Aminopropyl) morpholine grafted cross-linked lipoic acid vesicles with vitamin C-loading (VC@N3AM cLAVs), is developed. VC@N3AM cLAVs efficiently accumulate in lysosomes and convert into two redox couples LA/DHLA (dihydrolipoic acid, reduced form of LA) and VC/DHA (dehydroascorbic acid, oxidized form of VC) by the lysosomal glutathione, which can not only produce a large amount of H2 O2 by pro-oxidant action but also accelerate iron transformation through the cyclic redox reactions between each other and cause the efficient conversion of the generated H2 O2 into highly toxic •OH. Both in vitro and in vivo experiments demonstrate that VC@N3AM cLAVs can effectively enhance ROS production and boost LMP, finally initiation irreversible death of tumor cells via ferroptosis pathway, thus representing a potential anticancer drug for cancer therapy.

Bioimaging of labile lysosomal iron through naphthalimide-based fluorescent probe

Lysosomal labile iron detection is immensely important as it is related to various diseases like Alzheimer's disease, Huntington's disease, Parkinson's disease, and cell apoptosis like ferroptosis. The fluorescent-based detection methods are preferred due to their sensitive, non-invasive, and spatial-temporal detection in biological samples. However, this remains a great challenge due to the lysosomal compartment being acidic alters the photophysical properties of the probe. Herein, we have rationally designed and synthesized multi-component naphthalimide-based fluorescent marker with preferred optical properties and bio-compatibility for selective detection of labile iron present in the lysosomal compartment. The synthesized probe was characterized structurally and optically by NMR, mass spectrometry, UV–visible, and fluorescence spectroscopy. The developed probe with an appropriate linking strategy turns out to be tolerant to fluorescence alternation in lysosomal pH. The probe exhibits great selectivity and high sensitivity for Fe(III) with limit of detection of 0.44 μM and is also able to detect Fenton-type reactions. Further, the probe has been successfully applied for lysosomal imaging and detecting labile Fe(III) present in the lysosomal lumen of the live cells.

Targeting Iron Homeostasis As a Therapeutic Strategy in Multiple Myeloma

Multiple myeloma (MM) is a hematological cancer characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). Despite the survival improvement provided by current treatments, the majority of patients relapse and eventually become resistant to all treatments. Drug resistance and relapse are the main current challenges in the treatment of MM and development of new therapeutic interventions is a pressing need (Pinto et al., 2020 Cancers). Abnormal iron homeostasis is implicated in tumorigenesis and the progression of several cancers. Ironomycin is a small synthetic derivative of salinomycin that induces DNA damage and non-apoptotic cell death by sequestering iron in lysosomes (Mai et al., 2017 Nat Chem). Preclinical studies in Acute Myeloid Leukemia (Garciaz et al., 2022 Cancer Discovery) and Diffuse Large B-Cell Lymphoma (Devin et al., 2022 Can Res) have revealed the therapeutic interest of ironomycin in treating hematological malignancies. Here, we have evaluated the effect of ironomycin on MM cells, alone and in combination with standard anti-myeloma treatments. A list of 63 genes related to iron metabolism in cancer was defined. Using Maxstat R function, we showed that 6 genes out of the 63 investigated have a prognostic value in a cohort of newly diagnosed MM patients (n=345). Based on these prognostic genes, we created a gene expression profile (GEP)-based risk score as the sum of the beta coefficients weighted by ±1 according to the patient signal above or below the probe set Maxstat value as previously reported (Herviou et al., 2018 Clinical Epigenetics). This "Iron Score” (IS) was significantly associated with high-risk myeloma (OS and EFS) in 3 independent cohorts of newly diagnosed MM patients (TT2 cohort (N=345), HM cohort (N=206) and TT3 cohort (N=158)), as well as in a cohort of 188 patients at relapse treated with bortezomib monotherapy (Mulligan cohort N=188). GSEA analysis revealed that high-risk MM patients defined by iron score are significantly enriched in genes involved in cell cycle, TFRC targets and hypoxia (p < 0.001). We analyzed the therapeutic interest of ironomycin in 19 human myeloma cell lines (HMCLs). Ironomycin inhibits MM cell growth at nanomolar concentrations compared to other iron chelators. Ironomycin caused a significant cell growth inhibition mediated by cell cycle arrest and triggering of apoptosis, without significant ferroptosis induction. Moreover, cell proliferation arrest correlated with increased DNA damage and caspases activation, and a metabolic swift towards a glycolytic or quiescent metabolism. Iron could also play a role in transcriptional regulation of cancer cells mediated by epigenetic reprogramming. Iron dependent modifications of histone or DNA methylation have been shown to play a significant role in solid cancers. Ironomycin also induced a downregulation of major oncogenes of MM cells including MMSET, c-MYC and CCND1. Furthermore, ironomycin treatment resulted in an epigenetic dysregulation with H3K27ac, H3K4me3 and H3K9me3 downregulation. With major importance, we validated the therapeutic interest of ironomycin in primary MM cells from patients (n=5) without significant toxicity for non-tumor cells from the microenvironment. Furthermore, ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment. We tested the therapeutic interest to combine ironomycin with conventional drugs used in MM treatment. The combination of ironomycin with IMiDs (lenalidomide and pomalidomide) had a synergistic effect on cell growth arrest and the activation of apoptotic mechanisms, associated with a marked reduction of several targets of IMiDs including Cereblon (CRBN), MYC, IRF4, IKZF1 and IKZF3. Our data demonstrate that targeting iron homeostasis using ironomycin could be of therapeutic interest in high-risk MM defined by the IS, especially in combination with IMiDs.