Embryotrophic effects of extracellular vesicles derived from outgrowth embryos in pre- and peri-implantation embryonic development in mice.

Recently, many studies have investigated the role of extracellular vesicles (EVs) on reproductive events, including embryo development and death, oviduct-embryo crosstalk, in vitro fertilization and others. The aim of this study was to demonstrate whether outgrowth embryo-derived EVs function as bioactive molecules and regulate mouse embryonic developmental competence in vitro and implantation potential in utero. The EVs from mouse outgrowth embryos on 7.5 days postcoitum were detected and selectively isolated to evaluate the embryotrophic functions on preimplantation embryos. Developmental outcomes such as the percentage of blastocyst formation, hatching, and trophoblastic outgrowth were assessed. Furthermore, the total cell number and apoptotic index of blastocysts, which were incubated with EVs during the culture period, were evaluated by fluorescence microscopy. Implantation potential in utero was investigated following embryo transfer. The EVs from outgrowth embryo-conditioned media have rounded membrane structures that range in diameter from 20 to 225 nm. Incubation with EVs improved preimplantation embryonic development by increasing cell proliferation and decreasing apoptosis in blastocysts. Moreover, the implantation rates following embryo transfer were significantly higher in EV-supplemented embryos compared with the control. Collectively, EVs from outgrowth embryo could enhance the embryonic developmental competence and even implantation potential in mice.

Identification of differentially expressed microRNAs in outgrowth embryos compared with blastocysts and non-outgrowth embryos in mice.

Recurrent implantation failure (RIF) is one of the main causes for the repeated failure of IVF, and the major reason for RIF is thought to be a miscommunication between the embryo and uterus. However, the exact mechanism underlying embryo-uterus cross-talk is not fully understood. The aim of the present study was to identify differentially expressed microRNAs (miRNAs) among blastocysts, non-outgrowth and outgrowth embryos in mice using microarray analysis. A bioinformatics analysis was performed to predict the potential mechanisms of implantation. The miRNA expression profiles differed significantly between non-outgrowth and outgrowth embryos. In all, 3163 miRNAs were detected in blastocysts and outgrowth embryos. Of these, 10 miRNA candidates (let-7b, miR-23a, miR-27a, miR-92a, miR-183, miR-200c, miR-291a, miR-425, miR-429 and miR-652) were identified as significant differentially expressed miRNAs of outgrowth embryos by in silico analysis. The expression of the miRNA candidates was markedly changed during preimplantation embryo development. In particular, let-7b-5p, miR-200c-3p and miR-23a-3p were significantly upregulated in outgrowth embryos compared with non-outgrowth blastocysts. Overall, differentially expressed miRNAs in outgrowth embryos compared with blastocysts and non-outgrowth embryos could be involved in embryo attachment, and interaction between the embryo proper and maternal endometrium during the implantation process.

Ovarian injury during cryopreservation and transplantation in mice: a comparative study between cryoinjury and ischemic injury.

STUDY QUESTION: What is the main cause of ovarian injury during cryopreservation and transplantation in mice: cryoinjury or ischemic injury? SUMMARY ANSWER: Post-transplantation ischemia is the main cause of ovarian injury during cryopreservation and transplantation for restoring ovarian function. WHAT IS KNOWN ALREADY: During cryopreservation and the transplantation of ovaries, cryoinjury and ischemic injury inevitably occur, which has a detrimental effect on ovarian quality and reserve. STUDY DESIGN, SIZE, DURATION: A total of 80 B6D2F1 female mice were randomly allocated to 2 control and 6 experimental groups according to the presence or the absence of transplantation (n = 10/group). The control groups consisted of fresh or vitrified-warmed controls that had the whole ovary fixed without transplantation (fresh and vitri-con, respectively). The experimental groups were further divided according to the presence of vitrification (fresh or vitrified-warmed) and the transplantation period (2 [D2], 7 [D7] or 21 [D21] days). PARTICIPANTS/MATERIALS, SETTING, METHODS: In the control groups, fresh and vitrified-warmed ovaries were immediately fixed after the collection (fresh) and the vitrification-warming process (vitrification control, vitri-con), respectively. Of those experimental groups, three were auto-transplanted with fresh whole ovary (FrOT; FrOT-D2, FrOT-D7 and FrOT-D21). For the other three groups, the ovaries were harvested and stored in liquid nitrogen for 1 week after vitrification and then warmed to auto-transplant the vitrified whole ovaries (vitrified ovary [VtOT]; VtOT-D2, VtOT-D7 and VtOT-D21). After 2, 7 or 21 days of grafting, the grafts and blood sera were collected for analysis by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, CD31 immunohistochemistry and follicle-stimulating hormone enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: The vitrification-warming procedure decreased the proportion of intact follicles (Grade 1, G1) (vitri-con 50.3% versus fresh 64.2%) but there was a larger decrease due to ischemic injury after transplantation (FrOT-D2: 42.5%). The percentage of apoptotic follicles was significantly increased in the vitrified-warmed ovary group compared with the fresh control, but it increased more after transplantation without vitrification (fresh: 0.9%, vitri-con: 6.0% and FrOT-D2: 26.8%). The mean number of follicles per section and percentage of CD31-positive area significantly decreased after vitrification but decreased to a larger extent after transplantation (number of follicles, fresh: 30.3 ± 3.6, vitri-con: 20.6 ± 2.9, FrOT-D2: 17.9 ± 2.1; CD31-positive area, fresh: 10.6 ± 1.3%, vitri-con: 5.7 ± 0.9% and FrOT-D2: 4.2 ± 0.4%). Regarding the G1 follicle ratio and CD31-positive area per graft, only the FrOT groups significantly recovered with time after transplantation (G1 follicle ratio, FrOT-D2: 42.5%, FrOT-D7: 56.1% and FrOT-D21: 70.7%; CD31-positive area, FrOT-D2: 4.2 ± 0.4%, FrOT-D7: 5.4 ± 0.6% and FrOT-D21: 7.5 ± 0.8%). Although there was no significant difference between the two transplantation groups at each evaluation, the serum follicle-stimulating hormone level of both groups significantly decreased over time. LIMITATIONS AND REASONS FOR CAUTION: It is unclear how far these results can be extrapolated from mice to the human ovary. WIDER IMPLICATIONS OF THE FINDINGS: Minimizing ischemic injury should be the first priority rather than preventing cryoinjury alone, and decreasing the combination of cryoinjury and ischemic injury is necessary to improve ovarian quality after cryopreservation and transplantation. STUDY FUNDING/COMPETING INTEREST: This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055). The authors have no conflict of interest to declare.

A combination of simvastatin and methylprednisolone improves the quality of vitrified-warmed ovarian tissue after auto-transplantation.

STUDY QUESTION: Does the preoperative administration of simvastatin and methylprednisolone enhance mouse ovarian quality after auto-transplantation of vitrified-warmed ovarian tissue (OT)? SUMMARY ANSWER: Treatment with combined simvastatin and methylprednisolone enhances the quality of transplanted mouse OTs. WHAT IS KNOWN ALREADY: The prevention of ischemic injury after transplantation of OT is critical for preserving the ovarian follicles. Preoperative administration of simvastatin (a cholesterol-lowering drug) has beneficial effects on various organ transplantations. Moreover, donor treatment with simvastatin and methylprednisolone (main effects are on immune response) prevents ischemia-reperfusion injury and has a beneficial effect on allograft survival in rat cardiac allografts. STUDY DESIGN, SIZE, DURATION: A total of 232 6-week-old B6D2F1 mice were randomly distributed into fresh control, vitrified-warmed control and experimental groups (n = 10-17 per group). The experimental groups were as follows: sham control, simvastatin, methylprednisolone and co-treatment groups. In the experimental groups, the mice were administered simvastatin (5 mg/kg, orally), methylprednisolone (15 mg/kg, i.v.) or a combination of simvastatin and methylprednisolone 2 h before ovariectomy, whereas the sham control mice received normal saline. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole ovaries were removed from the mice and vitrified by two-step vitrification procedures. The vitrified ovaries were warmed 1 week later and auto-transplanted under the bilateral kidney capsules. The ovaries and blood samples were collected 2, 7 and 21 days (D) after transplantation for histological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, immunohistochemistry for CD31 and serum anti-Mullerian hormone (AMH) level estimation. Embryonic development was evaluated after IVF of oocytes obtained from the transplanted ovary. MAIN RESULTS AND THE ROLE OF CHANCE: The group that received simvastatin and methylprednisolone showed a significantly improved intact (Grade 1) follicle ratio (D2: P < 0.001, D7: P < 0.05 and D21: P < 0.001), apoptotic follicle ratio (D21: P < 0.05), CD31-positive area (D7: P < 0.05 and D21: P < 0.05) and serum AMH level (D7: P < 0.001) after transplantation when compared with the sham control. However, no difference was noted in the fertilization and blastocyst formation rates, number of total and apoptotic blastomeres per blastocyst and inner cell mass/trophectoderm ratio among the four transplantation groups. LIMITATIONS, REASONS FOR CAUTION: Although we evaluated the beneficial effects of simvastatin and methylprednisolone in the present study, we did not unravel the corresponding protective mechanisms. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that a combination of simvastatin and methylprednisolone has beneficial effects on the quality and functioning of transplanted OT. This combined treatment can potentially be applied clinically to humans and domestic animals subject to further studies.

The beneficial effects of polyethylene glycol-superoxide dismutase on ovarian tissue culture and transplantation.

PURPOSE: Reducing the ischemic damage from free radicals that is inflicted on ovarian tissue is critical for successful ovarian tissue transplantation. Polyethylene glycol-superoxide dismutase (PEG-SOD) is mimetic of superoxide dismutase (SOD) and powerful free radical scavenger acts by reducing superoxide anions. The objective of study was to evaluate effects of PEG-SOD on mouse ovarian tissues in in vitro culture and in autotransplantation. METHODS: Ovaries were collected and randomly divided into four groups that received different doses of PEG-SOD. To assess effects of PEG-SOD on in vitro cultures, four different doses of PEG-SOD were applied to in vitro culture media during in vitro culturing following ovarian tissue vitrification and warming. To evaluate effects of PEG-SOD on ovarian tissue transplantation, four different doses of PEG-SOD were applied for 2, 7, and 21 days to mice following vitrified-warmed mouse ovarian tissue autotransplantation. RESULTS: The percentage of primordial follicles was maintained at the highest dose of PEG-SOD for 2 h in vitro, and there was a significant decrease in the percentage of apoptotic follicles at 2 h, but not at later time points. The highest dose of PEG-SOD also maintained primordial, primary, and secondary follicles 2 days post-transplantation, but only primordial follicles were maintained up to 21 days after transplantation. CONCLUSIONS: PEG-SOD is protective mainly toward primordial follicles only for a short interval in vitro, presumably via antioxidant effects. PEG-SOD may be a promising additive for preserving ovarian tissue integrity, at least for primordial follicles, up to 21 days post-transplantation.

Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified-warmed ovarian tissue survival.

STUDY QUESTION: What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? SUMMARY ANSWER: The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. WHAT IS KNOWN ALREADY: Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified-warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. STUDY DESIGN, SIZE, DURATION: A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. PARTICIPANTS/MATERIALS, SETTING, METHODS: The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of α-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified-warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. MAIN RESULTS AND THE ROLE OF CHANCE: The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4-58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6-92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. LIMITATIONS, REASONS FOR CAUTION: The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. WIDER IMPLICATIONS OF THE FINDINGS: We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

Cholesterol-ferroptosis nexus: Unveiling novel cancer therapeutic avenues.

Ferroptosis, a novel form of regulated cell death characterized by iron-mediated lipid peroxidation, holds immense potential in cancer therapeutics due to its role in tumor progression and resistance. This review predominantly explores the intricate relationship between ferroptosis and cholesterol metabolism pathways, mainly focusing on the cholesterol biosynthesis pathway. This review highlights the therapeutic implications of targeting cholesterol metabolism pathways for cancer treatment by delving into the mechanisms underlying ferroptosis regulation. Strategies such as inhibiting HMG-CoA reductase and suppressing squalene synthesis offer promising avenues for inducing ferroptosis in cancer cells. Moreover, insights into targeting the 7-dehydrocholesterol pathway provide novel perspectives on modulating ferroptosis susceptibility and managing ferroptosis-associated diseases. Understanding the interplay between ferroptosis and cholesterol metabolism pathways underscores the potential of lipid metabolism modulation as an innovative therapeutic approach in cancer treatment.

NAD(P)H-quinone oxidoreductase 1 induces complicated effects on mitochondrial dysfunction and ferroptosis in an expression level-dependent manner.

NAD(P)H-quinone oxidoreductase 1 (NQO1) is an essential redox enzyme responsible for redox balance and energy metabolism. Despite of its importance, the brain contains high capacity of polyunsaturated fatty acids and maintains low levels of NQO1 expression. In this study, we examined how levels of NQO1 expression affects cell survival in response to toxic insults causing mitochondrial dysfunction and ferroptosis, and whether NQO1 has a potential as a biomarker in different stressed conditions. Following treatment with rotenone, overexpressed NQO1 in SH-SY5Y cells improved cell survival by reducing mitochondrial reductive stress via increased NAD+ supply without mitochondrial biogenesis. However, NQO1 overexpression boosted lipid peroxidation following treatment with RSL3 and erastin. A lipid droplet staining assay showed increased lipid droplets in cells overexpressing NQO1. In contrast, NQO1 knockdown protected cells against ferroptosis by increasing GPX4, xCT, and the GSH/GSSG system. Also, NQO1 knockdown showed lower iron contents and lipid droplets than non-transfectants and cells overexpressing NQO1, even though it could not attenuate cell death when exposed to rotenone. In summary, our study suggests that different NQO1 levels may have advantages and disadvantages depending on the surrounding environments. Thus, regulating NQO1 expression could be a potential supplementary tool when treating neuronal diseases.

Effect of nicotinamide mononucleotide on osteogenesis in MC3T3-E1 cells against inflammation-induced by lipopolysaccharide.

OBJECTIVE: Nicotinamide mononucleotide (NMN) is extensively utilized as an anti-aging agent and possesses anti-inflammatory properties. Lipopolysaccharide (LPS) activates Toll-like receptor 4, a process modulated by intracellular signaling pathways such as the Wnt/ß-catenin pathway. This study investigated the impact of NMN on osteogenesis in the presence of LPS. METHODS: To elucidate the role of NMN in osteogenesis in the context of Gram-negative bacterial infection after LPS treatment, we cultured a mouse pre-osteoblast cell line (MC3T3-E1) and subsequently incubated it with NMN and/or LPS. We then evaluated osteogenic activity by measuring alkaline phosphatase activity, assessing gene expression and protein levels, and performing Alizarin Red S staining and immunocytochemistry. RESULTS: MC3T3-E1 cells underwent successful differentiation into osteoblasts following treatment with osteogenic induction medium. LPS diminished features related to osteogenic differentiation, which were subsequently partially reversed by treatment with NMN. The restorative effects of NMN on LPS-exposed MC3T3-E1 cells were further substantiated by elucidating the role of Wnt/ß-catenin signaling, as confirmed through immunocytochemistry. CONCLUSION: This study showed that infection with Gram-negative bacteria disrupted the osteogenic differentiation of MC3T3-E1 cells. This adverse effect was partially reversed by administering a high-dose of NMN. Drawing on these results, we propose that NMN could serve as a viable therapeutic strategy to preserve bone homeostasis in elderly and immunocompromised patients.

Pioneering the future of cancer therapy: Deciphering the p53-ferroptosis nexus for precision medicine.

The TP53 gene, encoding the p53 protein, has been a focal point of research since its 1979 discovery, playing a crucial role in tumor suppression. Ferroptosis, a distinct form of cell death characterized by lipid peroxide accumulation, has gained prominence since its recognition in 2012. Recent studies have unveiled an intriguing connection between p53 and ferroptosis, with implications for cancer therapy. Recent research underscores p53 as a novel target for cancer therapy, influencing key metabolic processes in ferroptosis. Notably, p53 represses the expression of the cystine-glutamate antiporter SLC7A11, supporting p53-mediated tumor growth suppression. Furthermore, under metabolic stress, p53 mitigates ferroptosis sensitivity, aiding cancer cells in coping and delaying cell death. This dynamic interplay between p53 and ferroptosis has far-reaching implications for various diseases, particularly cancer. This review provides a comprehensive overview of ferroptosis in cancer cells, elucidating p53's role in regulating ferroptosis, and explores the potential of targeting p53 to induce ferroptosis for cancer therapy. Understanding this complex relationship between p53 and ferroptosis offers a promising avenue for developing innovative cancer treatments.

The Interplay between Intracellular Iron Homeostasis and Neuroinflammation in Neurodegenerative Diseases.

Iron is essential for life. Many enzymes require iron for appropriate function. However, dysregulation of intracellular iron homeostasis produces excessive reactive oxygen species (ROS) via the Fenton reaction and causes devastating effects on cells, leading to ferroptosis, an iron-dependent cell death. In order to protect against harmful effects, the intracellular system regulates cellular iron levels through iron regulatory mechanisms, including hepcidin-ferroportin, divalent metal transporter 1 (DMT1)-transferrin, and ferritin-nuclear receptor coactivator 4 (NCOA4). During iron deficiency, DMT1-transferrin and ferritin-NCOA4 systems increase intracellular iron levels via endosomes and ferritinophagy, respectively. In contrast, repleting extracellular iron promotes cellular iron absorption through the hepcidin-ferroportin axis. These processes are regulated by the iron-regulatory protein (IRP)/iron-responsive element (IRE) system and nuclear factor erythroid 2-related factor 2 (Nrf2). Meanwhile, excessive ROS also promotes neuroinflammation by activating the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). NF-κB forms inflammasomes, inhibits silent information regulator 2-related enzyme 1 (SIRT1), and induces pro-inflammatory cytokines (IL-6, TNF-α, and IL-1ß). Furthermore, 4-hydroxy-2,3-trans-nonenal (4-HNE), the end-product of ferroptosis, promotes the inflammatory response by producing amyloid-beta (Aß) fibrils and neurofibrillary tangles in Alzheimer's disease, and alpha-synuclein aggregation in Parkinson's disease. This interplay shows that intracellular iron homeostasis is vital to maintain inflammatory homeostasis. Here, we review the role of iron homeostasis in inflammation based on recent findings.

Epigenetic modulation of ferroptosis in cancer: Identifying epigenetic targets for novel anticancer therapy.

Ferroptosis is a newly recognized form of oxidative-regulated cell death resulting from iron-mediated lipid peroxidation accumulation. Radical-trapping antioxidant systems can eliminate these oxidized lipids and prevent disrupting the integrity of cell membranes. Epigenetic modifications can regulate ferroptosis by altering gene expression or cell phenotype without permanent sequence changes. These mechanisms include DNA methylation, histone modifications, RNA modifications, and noncoding RNAs. Epigenetic alterations in cancer can control the expression of ferroptosis regulators or related pathways, leading to changes in cell sensitivity to ferroptosis inducers or cancer progression. Epigenetic alterations in cancer are influenced by a wide range of cancer hallmarks, contributing to therapeutic resistance. Targeting epigenetic alterations is a promising approach to overcoming cancer resilience. However, the exact mechanisms involved in different types of cancer remain unresolved. Discovering more ferroptosis-associated epigenetic targets and interventions can help overcome current barriers in anticancer therapy. Many papers on epigenetic modifications of ferroptosis have been continuously published, making it essential to summarize the current state-of-the-art in the epigenetic regulation of ferroptosis in human cancer.

Unleashing Ferroptosis in Human Cancers: Targeting Ferroptosis Suppressor Protein 1 for Overcoming Therapy Resistance.

Ferroptosis, a recently identified form of regulated cell death characterized by the iron-dependent accumulation of lethal lipid peroxidation, has gained increasing attention in cancer therapy. Ferroptosis suppressor protein 1 (FSP1), an NAD(P)H-ubiquinone oxidoreductase that reduces ubiquinone to ubiquinol, has emerged as a critical player in the regulation of ferroptosis. FSP1 operates independently of the canonical system xc-/glutathione peroxidase 4 pathway, making it a promising target for inducing ferroptosis in cancer cells and overcoming ferroptosis resistance. This review provides a comprehensive overview of FSP1 and ferroptosis, emphasizing the importance of FSP1 modulation and its potential as a therapeutic target in cancer treatment. We also discuss recent progress in developing FSP1 inhibitors and their implications for cancer therapy. Despite the challenges associated with targeting FSP1, advances in this field may provide a strong foundation for developing innovative and effective treatments for cancer and other diseases.

Targeting Iron-Sulfur Clusters in Cancer: Opportunities and Challenges for Ferroptosis-Based Therapy.

Iron dysregulation is a hallmark of cancer, characterized by an overexpression of genes involved in iron metabolism and iron-sulfur cluster (ISC) biogenesis. Dysregulated iron homeostasis increases intracellular labile iron, which may lead to the formation of excess cytotoxic radicals and make it vulnerable to various types of regulated cell death, including ferroptosis. The inhibition of ISC synthesis triggers the iron starvation response, increasing lipid peroxidation and ferroptosis in cancer cells treated with oxidative stress-inducing agents. Various methods, such as redox operations, iron chelation, and iron replacement with redox-inert metals, can destabilize or limit ISC formation and function, providing potential therapeutic strategies for cancer treatment. Targeting ISCs to induce ferroptosis represents a promising approach in cancer therapy. This review summarizes the state-of-the-art overview of iron metabolism and ferroptosis in cancer cells, the role of ISC modulation in ferroptosis, and the potential of targeting ISCs for ferroptosis induction in cancer therapy. Further research is necessary to develop and validate these strategies in clinical trials for various cancers, which may ultimately lead to the development of novel and effective treatments for cancer patients.

Altered iron metabolism as a target for ferroptosis induction in head and neck cancer.

Iron is a mineral micronutrient essential for survival and vital functions in many biological processes in living organisms. Iron plays a crucial role as a cofactor of iron-sulfur clusters in energy metabolism and biosynthesis by binding with enzymes and transferring electrons to targets. Iron can also impair cellular functions by damaging organelles and nucleic acids by producing free radicals from redox cycling. Iron-catalyzed reaction products can induce active-site mutations in tumorigenesis and cancer progression. However, the boosted pro-oxidant iron form may contribute to cytotoxicity by increasing soluble radicals and highly reactive oxygen species via the Fenton reaction. An increased redox-active labile iron pool is required for tumor growth and metastasis, but the increased cytotoxic lipid radicals also lead to regulated cell death, such as ferroptosis. Therefore, this may be a major target for selectively killing cancer cells. This review intends to understand altered iron metabolism in cancers and discuss iron-related molecular regulators highly associated with iron-induced cytotoxic radical production and ferroptosis induction, focusing on head and neck cancer.

Ferroptosis induction via targeting metabolic alterations in head and neck cancer.

Ferroptosis is a newly regulated cell death induced by the accumulation of iron-mediated lipid peroxidation. The alteration of cancer metabolism may contribute to proliferation, metastasis, and treatment resistance in human cancers, implicating the sensitivity to ferroptosis induction. Altered metabolism in cancer cells regulates oxidative stresses and changes metabolism intermediates, contributing to their deregulated growth and proliferation. Cancer metabolic changes toward the elevation of cellular free iron and polyunsaturated fatty acids sensitize cancer cells to lipid peroxidation toxicity tightly linked to ferroptosis. The altered metabolism in cancers can be served as a promising target to reverse cancer therapeutic resistance by ferroptosis induction to selectively kill cancer cells while sparing normal cells. The role of mitochondria and lipid metabolism in inducing ferroptosis in head and neck cancer (HNC) has been elucidated in previous studies. Ferroptosis is receiving attention in cancer research as treating cancers altering cellular metabolism and refractory from conventional therapies. More in-depth studies are needed to develop highly therapeutic drugs and practical methods to induce ferroptosis in diverse cancer cells and tumor microenvironments effectively. Therefore, this review intends to understand the altered metabolism and find new therapeutic possibilities using ferroptosis in HNC.

Targeting GPX4 in human cancer: Implications of ferroptosis induction for tackling cancer resilience.

Cancer metabolic alterations have been emphasized to protect cancer cells from cell death. The metabolic reprogramming toward a mesenchymal state makes cancer cells resistant to therapy but vulnerable to ferroptosis induction. Ferroptosis is a new form of regulated cell death based on the iron-dependent accumulation of excessive lipid peroxidation. Glutathione peroxidase 4 (GPX4) is the core regulator of ferroptosis by detoxifying cellular lipid peroxidation using glutathione as a cofactor. GPX4 synthesis requires selenium incorporation into the selenoprotein through isopentenylation and selenocysteine tRNA maturation. GPX4 synthesis and expression can be regulated by multiple levels of its transcription, translation, posttranslational modifications, and epigenetic modifications. Targeting GPX4 in cancer may be a promising strategy for effectively inducing ferroptosis and killing therapy-resistant cancer. Several pharmacological therapeutics targeting GPX4 have been developed constantly to activate ferroptosis induction in cancer. The potential therapeutic index of GPX4 inhibitors remains to be tested with thorough examinations of their safety and adverse effects in vivo and clinical trials. Many papers have been published continuously in recent years, requiring state-of-the-art updates in targeting GPX4 in cancer. Herein, we summarize targeting the GPX4 pathway in human cancer, which leads to implications of ferroptosis induction for tackling cancer resilience.

Epithelial-mesenchymal plasticity: Implications for ferroptosis vulnerability and cancer therapy.

Cancers polarized to a mesenchymal or poorly differentiated state can often evade cell death induced by conventional therapies. The epithelial-mesenchymal transition is involved in lipid metabolism and increases polyunsaturated fatty acid levels in cancer cells, contributing to chemo- and radio-resistance. Altered metabolism in cancer enables invasion and metastasis but is prone to lipid peroxidation under oxidative stress. Cancers with mesenchymal rather than epithelial signatures are highly vulnerable to ferroptosis. Therapy-resistant persister cancer cells show a high mesenchymal cell state and dependence on the lipid peroxidase pathway, which can respond more sensitively to ferroptosis inducers. Cancer cells may survive under specific metabolic and oxidative stress conditions, and targeting this unique defense system can selectively kill only cancer cells. Therefore, this article summarizes the core regulatory mechanisms of ferroptosis in cancer, the relationship between ferroptosis and epithelial-mesenchymal plasticity, and the implications of epithelial-mesenchymal transition for ferroptosis-based cancer therapy.

Targeting Nrf2 for ferroptosis-based therapy: Implications for overcoming ferroptosis evasion and therapy resistance in cancer.

Ferroptosis is a newly discovered form of programmed cell death caused by redox-active iron-mediated lipid peroxidation. Ferroptosis exhibits a unique morphological phenotype resulting from oxidative damage to membrane lipids. Ferroptosis induction has been shown to be effective in treating human cancers that rely on lipid peroxidation repair pathways. Nuclear factor erythroid 2-related factor 2 (Nrf2) can control the regulatory pathways of ferroptosis, which involve genes associated with glutathione biosynthesis, antioxidant responses, and lipid and iron metabolism. Resistant cancer cells often utilize Nrf2 stabilization by Keap1 inactivation or other somatic alterations in the genes from the Nrf2 pathway, which can confer resistance to ferroptosis induction and other therapies. However, pharmacological inactivation of the Nrf2 pathway can sensitize cancer cells to ferroptosis induction. Inducing lipid peroxidation and ferroptosis through regulating the Nrf2 pathway is a promising strategy for enhancing the anticancer effects of chemotherapy and radiation therapy in therapy-resistant human cancers. Despite promising preliminary studies, clinical trials in human cancer therapy have not yet been realized. A deeper understanding of their exact processes and efficacies in various cancers remains unsolved. Therefore, this article aims to summarize the regulatory mechanisms of ferroptosis, their modulation by Nrf2, and the potential of targeting Nrf2 for ferroptosis-based cancer therapy.

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.