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The SLC25A20 gene encodes carnitine-acylcarnitine translocase (CACT), facilitating the transport of long-chain acylcarnitine required for energy production via β-oxidation into the mitochondria. Loss-of-function mutations in this gene lead to CACT deficiency, a rare autosomal recessive disorder of fatty acid metabolism characterized by severe symptoms including cardiomyopathy, hepatic dysfunction, rhabdomyolysis, hypoketotic hypoglycemia, and hyperammonemia, often resulting in neonatal mortality. Here, we utilized CRISPR/Cas9 gene editing to isolate slc25a20 mutant zebrafish. Homozygous mutants displayed significant lethality, with the majority succumbing before reaching maturity. However, we identified a notably rare homozygous individual that survived into adulthood, prompting a histological examination. Firstly, we observed adipose tissue accumulation at various sites in the homozygous mutant. The mutant heart exhibited hypertrophy, along with degenerated myocardial and muscle cells containing numerous eosinophilic nuclei. Additionally, we found no large oil droplet vacuoles in the mutant liver; however, the hepatocytes displayed numerous small vacuoles resembling lipid droplets. Iron deposition was evident in the spleen and parts of the liver. Overall, our slc25a20 zebrafish mutant displayed tissue pathologies analogous to human CACT deficiency, suggesting its potential as a pathological model contributing to the elucidation of pathogenesis and the improvement/development of therapies for CACT deficiency.

Abstract SARS-CoV-2 infection causes severe pulmonary manifestations, with poorly understood mechanisms and limited treatment options. Hyperferritinemia and disrupted lung iron homeostasis in COVID-19 patients imply that ferroptosis, an iron-dependent cell death, may occur. Immunostaining and lipidomic analysis in COVID-19 lung autopsies reveal increases in ferroptosis markers, including transferrin receptor 1 and malondialdehyde accumulation in fatal cases. COVID-19 lungs display dysregulation of lipids involved in metabolism and ferroptosis. We find increased ferritin light chain associated with severe COVID-19 lung pathology. Iron overload promotes ferroptosis in both primary cells and cancerous lung epithelial cells. In addition, ferroptosis markers strongly correlate with lung injury severity in a COVID-19 lung disease model using male Syrian hamsters. These results reveal a role for ferroptosis in COVID-19 pulmonary disease; pharmacological ferroptosis inhibition may serve as an adjuvant therapy to prevent lung damage during SARS-CoV-2 infection.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells’ susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with – or caused by – ferroptosis.

Summary: Extracellular vesicles (EVs) are complex structures that transport various DNA, RNA, and protein. Recently, new EV secretion mechanisms have been identified through the iron regulatory system in mammalian cells. We revealed that ferroptosis increases EV secretion, which is named ferroptosis-dependent EVs (FedEVs). Here, we describe a step-by-step procedure to isolate GFP-expressing FedEVs for in vitro analysis. The FedEVs are further analyzed by imaging and flow cytometry analysis.For complete details on the use and execution of this protocol, please refer to Ito et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus that is causally associated with several malignancies. In addition to latent factors, lytic replication contributes to cancer development. In this study, we examined whether the lytic gene BNRF1, which is conserved among gamma-herpesviruses, has an important role in lymphomagenesis. We found that lymphoblastoid cell lines (LCLs) established by BNRF1-knockout EBV exhibited remarkably lower pathogenicity in a mice xenograft model than LCLs produced by wild-type EBV (LCLs-WT). RNA-seq analyses revealed that BNRF1 elicited the expression of interferon-inducible protein 27 (IFI27), which promotes cell proliferation. IFI27 knockdown in LCLs-WT resulted in excessive production of reactive oxygen species, leading to cell death and significantly decreased their pathogenicity in vivo. We also confirmed that IFI27 was upregulated during primary infection in B-cells. Our findings revealed that BNRF1 promoted robust proliferation of the B-cells that were transformed by EBV latent infection via IFI27 upregulation both in vitro and in vivo.

Abstract Organoids are a three-dimensional (3D) culture system that simulate actual organs. Therefore, tumor organoids are expected to predict precise response to chemotherapy in patients. However, to date, few studies have studied the drug responses in organoids of malignant mesothelioma (MM). The poor prognosis of MM emphasizes the importance of establishing a protocol for generating MM-organoid for research and clinical use. Here, we established murine MM organoids from p53 +/- or wild-type C57BL/6 strain by intraperitoneal injection either with crocidolite or carbon nanotube. Established MM-organoids proliferated in Matrigel as spheroids. Subcutaneous injection assays revealed that the MM-organoids mimicked actual tissue architecture and maintained the original histological features of the primary MM. RNA sequencing and pathway analyses revealed that the significant expressional differences between the 2D- and 3D-culture systems were observed in receptor tyrosine kinases, including IGF1R and EGFR, glycosylation and cholesterol/steroid metabolism. MM-organoids exhibited a more sensitive response to cisplatin through stable plasma membrane localization of a major cisplatin transporter, copper transporter 1/Slc31A1 (Ctr1) in comparison to 2D-cultures, presumably through glycosylation and lipidation. The Matrigel culture system facilitated the localization of CTR1 on the plasma membrane, which simulated the original MMs and the subcutaneous xenografts. These results suggest that the newly developed protocol for MM-organoids is useful to study strategies to overcome chemotherapy resistance to cisplatin.

Research on nonthermal plasma (NTP) devices first began almost five decades ago. NTP devices discharge electrons, positive ions, ultraviolet light, reactive oxygen species (ROS) and reactive nitrogen species (RNS) at near-physiological temperatures. Advances in plasma science have enabled the manipulation of ROS/RNS irradiation for medical applications. During preclinical stages and in human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral, and biofilm-related infections, wound healing, and cancer therapy. Previously, tetrachloroaurate (III) increased NTP-induced oxidative stress that was attenuated by reduced and oxidized glutathione, indicating that the presence of interactions between metal ions and biomolecules may modulate biological effects. In this study, using 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) as a spin probe in electron paramagnetic resonance (EPR), we observed that the tetrachloroaurate-induced M4PO-X spin adduct was significantly suppressed by ascorbate and α-tocopherol, while dehydroascorbate (DHA) and Trolox were ineffective. Tetrachloroaurate-induced lipid peroxidation, which was measured by 2-thiobarbituric acid-reactive substances (TBARS) in combination with NTP exposure, was suppressed by ascorbate, α-tocopherol and Trolox, while DHA was ineffective. Furthermore, N-acetyl-l-cysteine and dithiothreitol efficiently suppressed tetrachloroaurate-induced M4PO-X spin adduct and lipid peroxidation. LC‒MS/MS analyzes identified hexanal that was significantly elevated by NTP exposure and/or tetrachloroaurate. However, 25 and 250 μM ascorbate did not significantly suppress the formation of aldehydes, such as acetaldehyde, hexanal, nonanal, nonenal, and 4‑hydroxy-2-nonenal. Further studies are warranted to elucidate the redox reactions between metal ions, including gold (III), and biomolecules to expand the possibility of NTP application in medicine and agriculture.

Toxoplasmosis is a major infectious disease, affecting approximately one-third of the world's population; its main clinical manifestation, ocular toxoplasmosis (OT), is a severe sight-threatening disease. Nevertheless, the diagnosis of OT is based on clinical findings, which needs improvement, even with biochemical tests, such as polymerase chain reaction and antibody detections. Furthermore, the efficacy of OT-targeted treatment is limited; thus, additional measures for diagnosis and treatments are needed. Here, we for the first time report a significantly reduced iron concentration in the vitreous humor (VH) of human patients infected with OT. To obtain further insights into molecular mechanisms, we established a mouse model of T. gondii infection, in which intravitreally injected tracer 57Fe, was accumulated in the neurosensory retina. T. gondii-infected eyes showed increased lipid peroxidation, reduction of glutathione peroxidase-4 expression and mitochondrial deformity in the photoreceptor as cristae loss. These findings strongly suggest the involvement of ferroptotic process in the photoreceptor of OT. In addition, deferiprone, an FDA-approved iron chelator, reduced the iron uptake but also ameliorated toxoplasma-induced retinochoroiditis by reducing retinal inflammation. In conclusion, the iron levels in the VH could serve as diagnostic markers and iron chelators as potential treatments for OT.

Abstract Cancer is the primary cause of human mortality in Japan since 1981. Although numerous novel therapies have been developed and applied in clinics, the number of deaths from cancer is still increasing worldwide. It is time to consider the strategy of cancer prevention more seriously. Here we propose a hypothesis that cancer can be side effects of long time-use of iron and oxygen and that carcinogenesis is an evolution-like cellular events to obtain “iron addiction with ferroptosis-resistance” where genes and environment interact each other. Among the recognized genetic risk factors for carcinogenesis, we here focus on BRCA1 tumor suppressor gene and how environmental factors, including daily life exposure and diets, may impact toward carcinogenesis under BRCA1 haploinsufficiency. Although mice models of BRCA1 mutants have not been successful for decades in generating phenotype mimicking the human counterparts, a rat model of BRCA1 mutant was recently established that reasonably mimics the human phenotype. Two distinct categories of oxidative stress, one by radiation and one by iron-catalyzed Fenton reaction, promoted carcinogenesis in Brca1 rat mutants. Furthermore, mitochondrial damage followed by alteration of iron metabolism finally resulted in ferroptosis-resistance of target cells in carcinogenesis. These suggest a possibility that cancer prevention by active pharmacological intervention may be possible for BRCA1 mutants to increase the quality of their life rather than preventive mastectomy and/or oophorectomy.

Abstract Diagnostic markers of malignant mesothelioma (MM) have been extensively investigated. Immunohistochemistry (IHC) markers, such as calretinin, have been used for pathologic diagnosis. However, more diagnostic markers are required to improve the specificity and sensitivity of pathologic diagnosis. This study proposed two proteins as diagnostic markers for epithelioid MM. One is RhoA, an MM mutation-susceptible locus-derived protein, and another is vigilin, a lung small cell carcinoma marker. IHC was performed using 93 MM (epithelioid, 71 cases; sarcomatoid, 13 cases; and biphasic, 9 cases), 64 lung adenocarcinoma (LAC), 60 lung squamous cell carcinoma (LSC), and 14 normal mesothelial (NM) tissues. The majority of epithelioid MM cases were positive for both RhoA and vigilin, whereas both IHCs showed lower stainability in biphasic and sarcomatoid MM. Besides, both IHCs showed significantly higher stainability for RhoA and vigilin in epithelioid MM than in LAC and LSC (p 0.05). In the differential diagnosis of MM from lung cancer, the accuracy and specificity of RhoA, vigilin, and calretinin staining were almost equivalent. Further, H-score test showed that there was no significant difference between RhoA versus calretinin and vigilin versus calretinin in IHC positivity in epithelioid MM (p > 0.05). In conclusion, RhoA and vigilin may be candidates for immunohistochemical markers for epithelioid MM.

Ras mutations induce cell competition and cellular senescence to inhibit the proliferation of oncogenic mutated cells. Here the authors demonstrate that cellular senescence inhibits cell competition-induced elimination of oncogenic cells through HGF signalling.

Thioredoxin, encoded by Txn1, acts as a critical antioxidant in the defense against oxidative stress by regulating the dithiol/disulfide balance of interacting proteins. The role of thioredoxin in the central nervous system (CNS) is largely unknown. A phenotype-driven study of N-ethyl-N-nitrosourea-mutated rats with wild-running seizures revealed the importance of Txn1 mutations in CNS degeneration. Genetic mapping identified Txn1-F54L in the epileptic rats. The insulin-reducing activity of Txn1-F54L was approximately one-third of that of the wild-type (WT). Bilateral symmetrical vacuolar degeneration in the midbrain, mainly in the thalamus and the inferior colliculus, was observed in the Txn1-F54L rats. The lesions displayed neuronal and oligodendrocytic cell death. Neurons in Txn1-F54L rats showed morphological changes in the mitochondria. Vacuolar degeneration peaked at five weeks of age, and spontaneous repair began at seven weeks. The TUNEL assay showed that fibroblasts derived from homozygotes were susceptible to cell death under oxidative stress. In five-week-old WT rats, energy metabolism in the thalamus was significantly higher than that in the cerebral cortex. In conclusion, in juvenile rats, Txn1 seems to play an essential role in reducing oxidative stress in the midbrains with high energy metabolism.

Yogosawa et al find that mice lacking the Dyrk2 kinase display congenital malformations in multiple organs and die from respiratory failure associated with disrupted lung mesenchyme and reduced expression of Foxf1. Using an ex vivo lung culture system, they rescue the expression of Foxf1 and its target by restoring Shh signalling, altogether providing insights into Dyrk2 function.

Abstract Low-temperature plasma is being widely used in the various fields of life science, such as medicine and agriculture. Plasma-activated solutions have been proposed as potential cancer therapeutic reagents. We previously reported that plasma-activated Ringer’s lactate solution exhibited selective cancer-killing effects, and that the plasma-treated L-sodium lactate in the solution was an anti-tumor factor; however, the components that are generated through the interactions between plasma and L-sodium lactate and the components responsible for the selective killing of cancer cells remain unidentified. In this study, we quantified several major chemical products, such as pyruvate, formate, and acetate, in plasma-activated L-sodium lactate solution by nuclear magnetic resonance analysis. We further identified novel chemical products, such as glyoxylate and 2,3-dimethyltartrate, in the solution by direct infusion-electrospray ionization with tandem mass spectrometry analysis. We found that 2,3-dimethyltartrate exhibited cytotoxic effects in glioblastoma cells, but not in normal astrocytes. These findings shed light on the identities of the components that are responsible for the selective cytotoxic effect of plasma-activated solutions on cancer cells, and provide useful data for the potential development of cancer treatments using plasma-activated L-sodium lactate solution.

Germline-mutation in BRCA1 tumor suppressor gene is an established risk for carcinogenesis not only in females but also in males. Deficiency in the repair of DNA double-strand breaks is hypothesized as a responsible mechanism for carcinogenesis. However, supporting data is insufficient both in the mutation spectra of cancers in the patients with BRCA1 germline-mutation and in murine knockout/knock-in models of Brca1 haploinsufficiency. Furthermore, information on the driving force toward carcinogenesis in BRCA1 mutation carriers is lacking. Here we applied Fenton reaction-based renal carcinogenesis to a rat heterozygously knockout model of BRCA1 haploinsufficiency (mutant [MUT] model; L63X/+). Rat MUT model revealed significant promotion of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA). Array-based comparative genome hybridization of the RCCs identified significant increase in chromosomal amplification, syntenic to those in breast cancers of BRCA1 mutation carriers, including c-Myc, in comparison to those in the wild-type. Subacute-phase analysis of the kidney after repeated Fe-NTA treatment in the MUT model revealed dysregulated iron metabolism with mitochondrial malfunction assessed by expression microarray and electron microscopy, leading to renal tubular proliferation with iron overload. In conclusion, we for the first time demonstrate that biallelic wild-type BRCA1 provides more robust protection for mitochondrial metabolism under iron-catalyzed oxidative stress, preventing the emergence of neoplastic cells with chromosomal amplification. Our results suggest that oxidative stress via excess iron is a major driving force for carcinogenesis in BRCA1 haploinsufficiency, which can be a target for cancer prevention and therapeutics.

Tertiary lymphoid tissues (TLTs) facilitate local T and B cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here, we identified TNF superfamily CD153/CD30 signaling between 2 unique age-dependent lymphocyte subpopulations, CD153+PD-1+CD4+ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL-21 and IFN-γ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis, and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153/CD30 signaling in TLT formation and propose targeting the CD153/CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.

Ferroptosis is a form of regulated cell necrosis, as a consequence of Fe(II)-dependent lipid peroxidation. Although ferroptosis has been linked to cancer cell death, neurodegeneration and reperfusion injury, physiological roles of ferroptosis have not been elucidated to date mostly due to the lack of appropriate methodologies. Here, we show that 4-hydroxy-2-nonenal (HNE)-modified proteins detected by a HNEJ-1 mouse monoclonal antibody is a robust immunohistochemical technology to locate ferroptosis in tissues in combination with morphological nuclear information, based on various models of ferroptosis, including erastin-induced cysteine-deprivation, conditional Gpx4 knockout and Fe(II)-dependent renal tubular injury, as well as other types of regulated cell death. Specificity of HNEJ-1 with ferroptosis was endorsed by non-selective identification of HNE-modified proteins in an Fe(II)-dependent renal tubular injury model. We further comprehensively searched for signs of ferroptosis in different developmental stages of Fischer-344 rats from E9.5–2.5 years of age. We observed that there was a significant age-dependent increase in ferroptosis in the kidney, spleen, liver, ovary, uterus, cerebellum and bone marrow, which was accompanied by iron accumulation. Not only phagocytic cells but also parenchymal cells were affected. Epidermal ferroptosis in ageing SAMP8 mice was significantly promoted by high-fat or carbohydrate-restricted diets. During embryogenesis of Fischer-344 rats, we found ferroptosis in nucleated erythrocytes at E13.5, which disappeared in enucleated erythrocytes at E18.5. Administration of a ferroptosis inhibitor, liproxstatin-1, significantly delayed erythrocyte enucleation. Therefore, our results demonstrate for the first time the involvement of ferroptosis in physiological processes, such as embryonic erythropoiesis and aging, suggesting the evolutionally acquired mechanism and the inevitable side effects, respectively.

Asbestos-associated diseases remain a social burden worldwide. Our previous studies identified asbestos-induced iron-rich milieu for mesothelial cells with ceaseless macrophage ferroptosis. However, molecular mechanisms how this mutagenic milieu influences mesothelial cells have not been elucidated yet. Here, we propose a novel mechanism that extracellular vesicles (EVs) mediate asbestos-associated mutagenic factors to mesothelial cells. In a mice model of intraperitoneal crocidolite injection, mutagenic milieu highly expressed CD63, an exosomal marker. We then used a GFP-CD63 labeled THP-1 macrophage model exposed to crocidolite/iron, which generated EVs under ferroptotic process. We observed that MeT-5A mesothelial cells can receive and internalize these EVs. Furthermore, we comprehensively analyzed the ferroptosis-dependent EVs (FedEVs) for transported proteins and identified ferritin heavy/light chains as major components. Therefore, we inferred that FedEVs transport iron from ferroptotic macrophages to mesothelial cells. RNA sequencing revealed that the mesothelial cells receiving higher amounts of the FedEVs were mitotic, especially at the S and G2/M phases, by the use of Fucci mesothelial cells. Nuclear 8-hydroxy-2′-deoxyguanosine and γ-H2AX were significantly increased in the recipient mesothelial cells after exposure to FedEVs. Collectively, we here demonstrate a novel mechanism that FedEVs act as a key mutagenic mediator by transporting iron, which contribute to asbestos-induced mesothelial carcinogenesis.

Non-thermal plasma (NTP), an engineered technology to generate reactive species, induces ferroptosis and/or apoptosis specifically in various-type cancer cells. NTP-activated Ringer's lactate (PAL) is another modality for cancer therapy at preclinical stage. Here we found that PAL induces selective ferroptosis of malignant mesothelioma (MM) cells, where non-targeted metabolome screening identified upregulated citrulline-nitric oxide (.NO) cycle as a PAL target. .NO probe detected biphasic peaks transiently at PAL exposure with time-dependent increase, which was responsible for inducible . NO synthase (iNOS) overexpression through NF-κB activation. .NO and lipid peroxidation occupied lysosomes as a major compartment with increased TFEB expression. Not only ferrostatin-1 but inhibitors for . NO and/or iNOS could suppress this ferroptosis. PAL-induced ferroptosis accompanied autophagic process in the early phase, as demonstrated by an increase in essential amino acids, LC3B-II, p62 and LAMP1, transforming into the later phase with boosted lipid peroxidation. Therefore, .NO-mediated lysosomal impairment is central in PAL-induced ferroptosis.

Summary: Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.

Inosine triphosphate pyrophosphatase (ITPA) hydrolyzes inosine triphosphate (ITP) and other deaminated purine nucleotides to the corresponding nucleoside monophosphates. In humans, ITPA deficiency causes severe encephalopathy with epileptic seizure, microcephaly, and developmental retardation. In this study, we established neural stem cell–specific Itpa–conditional KO mice (Itpa-cKO mice) to clarify the effects of ITPA deficiency on the neural system. The Itpa-cKO mice showed growth retardation and died within 3 weeks of birth. We did not observe any microcephaly in the Itpa-cKO mice, although the female Itpa-cKO mice did show adrenal hypoplasia. The Itpa-cKO mice showed limb-clasping upon tail suspension and spontaneous and/or audiogenic seizure. Whole-cell patch-clamp recordings from entorhinal cortex neurons in brain slices revealed a depolarized resting membrane potential, increased firing, and frequent spontaneous miniature excitatory postsynaptic current and miniature inhibitory postsynaptic current in the Itpa-cKO mice compared with ITPA-proficient controls. Accumulated ITP or its metabolites, such as cyclic inosine monophosphates, or RNA containing inosines may cause membrane depolarization and hyperexcitability in neurons and induce the phenotype of ITPA-deficient mice, including seizure.

Ovarian endometriosis (OE) provides women of reproductive age with not only severe menstrual pain but also infertility and an increased risk for ovarian carcinogenesis. Whereas peritoneal endometriosis models have been developed with syngeneic implantation of minced uterine tissue and oncogenic K-ras allele with conditional Pten deletion within ovarian surface epithelium generated preneoplastic endometrial glandular morphology, followed by endometrioid adenocarcinoma, there has been no mouse model of OE similar to human counterparts, applicable to preclinical studies. Here we for the first time established a murine OE model that reveals infertility, and evaluated the involvement of iron catalyzed oxidative stress in the pathogenesis. Minced uterine tissue from female mice was implanted on ovarian surface of syngeneic mice after bursectomy to induce OE. Ectopic growth of endometrium was observed in association with ovary 4 weeks after implantation in 85.7% (12/14) of the operated mice with our protocol. Endometriotic lesions involved intestine, pancreas and peritoneal wall. Fibrosis around the ovary was prominent and increased time-dependently in the OE group. Iron accumulation was significantly increased in the OE group, leading to oxidative stress in each stage of the follicles as evaluated by 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2′-deoxyguanosine. Expression of follicle stimulating hormone receptor in the follicles revealed a significant decrease during pre-antral, antral and pre-ovulatory phases in the OE group. Finally, the number of pups was significantly reduced in the OE group in comparison to the controls. This model affords an opportunity to evaluate agents or procedures to counteract ovarian endometriosis in the preclinical settings.

Asbestos is still a social burden worldwide as a carcinogen causing malignant mesothelioma. Whereas recent studies suggest that local iron reduction is a preventive strategy against carcinogenesis, little is known regarding the cellular and molecular mechanisms surrounding excess iron. Here by differentially using high-risk and low-risk asbestos fibers (crocidolite and anthophyllite, respectively), we identified asbestos-induced mutagenic milieu for mesothelial cells. Rat and cell experiments revealed that phagocytosis of asbestos by macrophages results in their distinctive necrotic death; initially lysosome-depenent cell death and later ferroptosis, which increase intra- and extra-cellular catalytic Fe(II). DNA damage in mesothelial cells, as assessed by 8-hydroxy-2′-deoxyguanosine and γ-H2AX, increased after crocidolite exposure during regeneration accompanied by β-catenin activation. Conversely, β-catenin overexpression in mesothelial cells induced higher intracellular catalytic Fe(II) with increased G2/M cell-cycle fraction, when p16INK4A genomic loci localized more peripherally in the nucleus. Mesothelial cells after challenge of H2O2 under β-catenin overexpression presented low p16INK4A expression with a high incidence of deletion in p16INK4A locus. Thus, crocidolite generated catalytic Fe(II)-rich mutagenic environment for mesothelial cells by necrotizing macrophages with lysosomal cell death and ferroptosis. These results suggest novel molecular strategies to prevent mesothelial carcinogenesis after asbestos exposure.

A 62-year-old previously healthy male who was a welder/smoker/drinker was admitted to Kani Tono Hospital for severe hypoxemia (Day 0). Initial physical and radiological examinations suggested an acute exacerbation of chronic obstructive pulmonary disease. However, respiratory failure developed rapidly, and he died on Day + 4. Aspergillus fumigatus was identified after his death, and he was diagnosed with invasive pulmonary aspergillosis. The clinical and pathological features are precisely described with pathogenetic considerations. Keywords: Invasive pulmonary aspergillosis, Aspergillus fumigatus, Immunocompetent host, Smoker, Welder, Alcoholism

Abstract Non-thermal atmospheric pressure plasma has been proposed as a new therapeutic tool for cancer treatment. Recently, plasma-activated medium (PAM) has been widely studied in various cancer types. However, there are only few reports demonstrating the anti-tumour effects of PAM in an animal model reflecting pathological conditions and the accompanying mechanism. Here we investigated the inhibitory effect of PAM on the metastasis of ovarian cancer ES2 cells in vitro and in vivo. We demonstrated that ES2 cell migration, invasion and adhesion were suppressed by PAM at a certain PAM dilution ratio, whereas cell viability remained unaffected. In an in vivo mouse model of intraperitoneal metastasis, PAM inhibited peritoneal dissemination of ES2 cells, resulting in prolonged survival. Moreover, we assessed the molecular mechanism and found that MMP-9 was decreased by PAM. On further investigation, we also found that PAM prevented the activation of the MAPK pathway by inhibiting the phosphorylation of JNK1/2 and p38 MAPK. These findings indicate that PAM inhibits the metastasis of ovarian cancer cells through reduction of MMP-9 secretion, which is critical for cancer cell motility. Our findings suggest that PAM intraperitoneal therapy may be a promising treatment option for ovarian cancer.

Abstract Estradiol (E2) and the oestrogen receptor-alpha (ERα) signalling pathway play pivotal roles in the proliferative activity of breast cancer cells. Recent findings show that the brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signalling modulation in breast cancer cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using the ERα activity-regulator synthetic peptide (ERAP: 165–177 amino acids), derived from α-helical BIG3 sequence, resulted in a significant anti-tumour effect. However, the duration of this effect was very short for viable clinical application. We developed the chemically modified ERAP using stapling methods (stapledERAP) to improve the duration of its antitumour effects. The stapledERAP specifically inhibited the BIG3-PHB2 interaction and exhibited long-lasting suppressive activity. Its intracellular localization without the membrane-permeable polyarginine sequence was possible via the formation of a stable α-helix structure by stapling. Tumour bearing-mice treated daily or weekly with stapledERAP effectively prevented the BIG3-PHB2 interaction, leading to complete regression of E2-dependent tumours in vivo. Most importantly, combination of stapledERAP with tamoxifen, fulvestrant, and everolimus caused synergistic inhibitory effects on growth of breast cancer cells. Our findings suggested that the stapled ERAP may be a promising anti-tumour drug to suppress luminal-type breast cancer growth.

Hypoxia and acidity provide microenvironment for selection under evolutionary pressure and proliferation in cancer cells. Carbonic anhydrases (CAs) are a superfamily of metalloenzymes present in all life kingdoms, equilibrating the reactions among CO2, bicarbonate and H+. CA9, a membrane-associated α-CA, has been a drug target for various cancers. Whereas iron is essential not only for cancer cells but also for all the lives on earth, little is known on the association among hypoxia, iron metabolism, extracellular acidity and redox regulation. Malignant mesothelioma (MM), an aggressive tumor with poor prognosis, is an intriguing model in that asbestos-associated pathogenesis includes excess iron environment during carcinogenesis. Re-analysis of rat asbestos-induced MM model revealed an inverse association between high CA9 expression and survival. Here we used human MMs to identify the molecular events surrounding CA9 from the viewpoint of iron metabolism. CA9 expression was significantly higher in MM cells than in MeT-5A mesothelial cells, which was further amplified under hypoxia (1%O2) with increased catalytic Fe(II). CA9 suppression by inhibitors (S4 and U104) decreased viability and migration of MM cells, accompanied by overexpression of TFRC, IREB1/2 and FPN1(SLC40A1) and by downregulation of FTH/FTL. This expressional pattern was similar to that of erastin-induced ferroptosis in the same cells. Furthermore, we observed mitochondrial fission and enhanced autophagy with increased catalytic Fe(II) in both mitochondria and lysosomes after CA9 inhibition, accompanied by increased peroxides, mitochondrial O2− and lipid peroxidation. The eventual cell death was significantly inhibited by deferoxamine, ferrostatin-1 and Z-VAD-FMK, suggesting a mixed cell death of ferroptosis and apoptosis. Therefore, CA9 plays a role in equilibrating among hypoxia, iron metabolism and redox regulation in MM cells. Keywords: Carbonic anhydrase, Malignant mesothelioma, Iron metabolism, Tumor biology, Apoptosis, Ferroptosis, Catalytic Fe(II)

Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

Clear cell adenocarcinoma of the ovary (OCC) is a chemo-resistant tumor with a relatively poor prognosis and is frequently associated with endometriosis. Although it is assumed that oxidative stress plays some role in the malignant transformation of this tumor, the characteristic molecular events leading to carcinogenesis remain unknown. In this study, an array-based comparative genomic hybridization (CGH) analysis revealed Met gene amplification in 4/13 OCC primary tumors and 2/8 OCC cell lines. Amplification of the AKT2 gene, which is a downstream component of the Met/PI3K signaling pathway, was also observed in 5/21 samples by array-based CGH analysis. In one patient, both the Met and AKT2 genes were amplified. These findings were confirmed using fluorescence in situ hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemistry. In total, 73 OCC cases were evaluated using real-time quantitative PCR; 37.0% demonstrated Met gene amplification (>4 copies), and 8.2% had AKT2 amplification. Furthermore, stage 1 and 2 patients with Met gene amplification had significantly worse survival than patients without Met gene amplification (p

Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC) after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.

The human lens is composed of a monolayer of lens epithelial cells (LECs) and elongated fibers that align tightly but are separated by the plasma membrane. The integrity of the lens plasma membrane is crucial for maintaining lens cellular structure, homeostasis, and transparency. Glutathione peroxidase 4 (GPX4), a selenoenzyme, plays a critical role in protecting against lipid peroxidation. This study aims to elucidate the role of GPX4 in lens plasma membrane stability during lens development using in vitro, ex vivo, and in vivo systems. Our findings reveal that GPX4 deficiency triggers lens epithelial apoptosis-independent but ferroptosis-mediated cell death. Blocking lens GPX4 activity during ex vivo culture induces lens opacification, LEC death, and disruption of lens fiber cell arrangement. Deletion of lens-specific Gpx4 results in significant unsaturated phospholipid loss and an increase in oxidized phospholipids. Consequently, lenses with Gpx4 deficiency exhibit massive disruption of lens fiber cell structure, significant loss of LECs via ferroptosis, and formation of newborn cataracts. Remarkably, administering the lipid peroxidation inhibitor, liproxstatin-1, to pregnant mothers at embryonic days 9.5 significantly prevents lipid peroxidation, LEC death, and lens developmental defects. Our study unveils the crucial role of GPX4 in lens development and transparency, and also provides a successful intervention approach to prevent lens developmental defects through lipid peroxidation inhibition. [ABSTRACT FROM AUTHOR]