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Regulation of the p53/SLC7A11/GPX4 Pathway by Gentamicin Induces Ferroptosis in HEI-OC1 Cells

Li Yingying,Xu Hui,Shi Jinping,Li Cong,Li Mengxin,Zhang Xiaoling,Xue Qing,Qiu Jingjing,Cui Limei,Sun Yan,Song Xicheng,Chen Liang

Background Gentamicin is a commonly used aminoglycoside antibiotic, with ototoxicity as a significant side effect. Ferroptosis, an iron-dependent form of cell death, has been implicated in a variety of disorders. Whether ferroptosis impacts gentamicin ototoxicity is not yet known. The current work used an in-vitro model to examine the influence of gentamicin-induced ferroptosis on cochlear hair cell damage and probable molecular biological pathways. Methods House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were treated with different concentrations of gentamicin for 24 hours, with or without ferrostatin-1 pretreatment, to observe gentamicin-induced ferroptosis. The role of p53/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling in gentamicin-induced ferroptosis was explored by pretreating cells with the p53 inhibitor pifithrin-α (PFT-α). We investigated the effect of gentamicin on cells by assessing cell viability. Cellular proteins were isolated and Western blots were performed to detect changes in the expression of p53, SLC7A11, and GPX4. Fluorescence staining was used to assess levels of reactive oxygen species. An enzymatic detection kit was used to detect glutathione, Fe, and malondialdehyde markers. Results Gentamicin reduced cell viability, glutathione content, and SLC7A11 and GPX4 protein levels, and increased levels of p53 protein, reactive oxygen species, malondialdehyde, and Fe. These effects were largely blocked by pretreatment with ferrostatin-1. Pretreatment with the p53 inhibitor PFT-α prevented the gentamicin-induced reduction in SLC7A11 and GPX4, which alleviated several features of ferroptosis including glutathione depletion, iron overload, and lipid peroxidation build-up. Conclusion Gentamicin induces ferroptosis in the HEI-OC1 cell line, and the mechanism may be related to the p53/SLC7A11/GPX4 signaling pathway.

Single fluorescent probe reveals glutathione-viscosity crosstalk in the endoplasmic reticulum during ferroptosis and extracellular vesicles in synergistic ferroptosis-apoptosis

Yan-Ping Liu,Ling-Li Wang,Jing-Yu Piao,Ming-Hua Zheng,Jing-Yi Jin

Ferroptosis is an iron-dependent cell death triggered by lipid peroxidation accumulation, representing a redox imbalance in cells. The increased knowledge of ferroptosis has led to various potential strategies to develop new cancer therapies, such as synergistic ferroptosis-apoptosis. The endoplasmic reticulum (ER) is the major hub for lipid metabolism in cells and plays a central role in lipid peroxidation. Glutathione (GSH) and viscosity levels in ER are important parameters for intracellular redox homeostasis, which remain unclear in ferroptosis and synergistic ferroptosis-apoptosis. This study developed an ER-targeting fluorescent probe (MMPY) with a dual-emission response to GSH and viscosity. Fluorescent cell imaging using MMPY revealed different GSH-viscosity crosstalk in the ER for the four ferroptosis pathways induced by erastin, RSL3, FIN56, and FINO2. GSH and viscosity changes in the ER during synergistic ferroptosis-apoptosis were also investigated. Interestingly, the new types of extracellular vesicles were observed during the synergistic process, which burdened the ER fragments with different GSH contents.

Loss of AR-regulated AFF3 contributes to prostate cancer progression and reduces ferroptosis sensitivity by downregulating ACSL4 based on single-cell sequencing analysis

Fan Aoyu,Li Yunpeng,Zhang Yunyan,Meng Wei,Pan Wei,Chen Meixi,Ma Zhongliang,Chen Wei

Prostate cancer (PCa) is one of the most common cancers affecting the health of men worldwide. Castration-resistant prostate cancer (CRPC), the advanced and refractory phase of prostate cancer, has multiple mechanisms of resistance to androgen deprivation therapy (ADT) such as AR mutations, aberrant androgen synthase, and abnormal expression of AR-related genes. Based on the research of the AR pathway, new drugs for the treatment of CRPC have been developed in clinical practice, such as Abiraterone and enzalutamide. However, many areas in this pathway are still worth exploring. In this study, single-cell sequencing analysis was utilized to scrutinize significant genes in the androgen receptor (AR) pathway related to CRPC. Our analysis of single-cell sequencing combined with bulk-cell sequencing revealed a substantial downregulation of AR-regulated AFF3 in CRPC. Overexpression of AFF3 restricted the proliferation and migration of prostate cancer cells whilst also increasing their sensitivity towards enzalutamide, while knockdown of AFF3 had the opposite effect. To elucidate the mechanism of tumor inhibition by AFF3, we applied GSVA and GSEA to investigate the metabolic pathways related to AFF3 and revealed that AFF3 had an impact on fatty acids metabolism and ferroptosis through the regulation of ACSL4 protein expression. Based on correlation analysis and flow cytometry, we can speculate that AFF3 can impact the sensitivity of the CRPC cell lines to the ferroptosis inducer (RSL3) by regulating ACSL4. Therefore, our findings may provide new insights into the mechanisms of drug resistance in CRPC, and AFF3 may serve as a novel prognostic biomarker in prostate cancer.

Trim65 mitigates doxorubicin-induced myocardial injury by reducing ferroptosis

Xiaofeng Ma,Wang chen,Zhihao Hu,Like Xie,Zhaobing Li,Huan Liu,Zili Li,Zhentao Jiang,Jiangwei Huang,Changrong Jiang,Ke Huang,Sujun Xiao

Background and aims The anthracycline chemotherapeutic agent doxorubicin (DOX) is widely used to treat cancer. However, DOX carries a high risk of adverse effects such as cardiovascular events. The aim of this study was to explore the role of Trim65 in DOX-induced cardiotoxicity (DIC) and its underlying mechanisms. Methods and Results Cellular studies were performed by exposing H9c2 cells (rat cardiomyocytes) to DOX. H9c2 cells were infected with lentivirus encoding negative control (LV-NC) or Trim65 (LV-Trim65). C57BL/6J mice were exposed to adeno-associated virus 9 (AAV9) containing cTnT promoter-encoded Trim65 sequence (AAV-Trim65) or AAV9 negative control sequence (AAV-NC) via tail vein injection. Furthermore, the following analysis were performed: cell viability, intracellular ROS production and lipid peroxidation. Echocardiography was used to measure the heart function of the mice. qPCR and western blotting were used to assess the expression of Trim65, p53, SLC7A11, and GPX4. According to our study, Trim65 expression was significantly downregulated in DIC. Overexpression of Trim65 exhibited considerable protection against to DIC, confirmed by both in vitro and in vivo experiments. In DOX-treated mice, mitochondria deformation was observed in the heart, as well as a high level of lipid peroxidation (signs of ferroptosis) and iron content, which were mitigated by overexpression of Trim65. Mechanistically, our study confirmed that DOX reduced the Trim65-mediated ubiquitination of p53, ultimately inhibiting the degration of p53. We also found that Trim65 mitigated DOX-induced ferroptosis via p53. Conclusion Collectively, our data demonstrated that Trim65 inhibits ferroptosis by degrading p53, thereby alleviating DIC. Therefore, Trim65 could be a promising target for the treatment of DIC.

Sarcosine sensitizes lung adenocarcinoma to chemotherapy by dual activation of ferroptosis via PDK4/PDHA1 signaling and NMDAR-mediated iron export

Shan Guangyao,Bian Yunyi,Ren Shencheng,Hu Zhengyang,Pan Binyang,Zeng Dejun,Zheng Zhaolin,Fan Hong,Bi Guoshu,Yao Guangyu,Zhan Cheng

Background Ferroptosis, a regulated cell death driven by iron-dependent lipid peroxidation, is associated with chemoresistance in lung adenocarcinoma (LUAD). This study aims to investigate the role of sarcosine in ferroptosis and its underlying mechanisms.Methods An RSL3-induced ferroptosis model was used to screen a library of 889 human endogenous metabolites and metabolomic profiling was harnessed to identify metabolites associated with ferroptosis. Cell viability, lipid-reactive oxygen species (ROS), ferrous iron, malondialdehyde (MDA), and mitochondrial integrity were assessed to evaluate sarcosine’s effects on ferroptosis. Metabolic fate was studied using 15N-labeled sarcosine. Next, we used untargeted metabolomic profiling and next-generation sequencing to dissect metabolic and transcriptomic changes upon sarcosine supplementation. The effects of sarcosine on ferroptosis and chemotherapy were further validated in patient-derived organoids (PDOs), xenograft models, and LUAD tissues.Results Sarcosine emerged as a potent ferroptosis inducer in the metabolic library screening, which was further confirmed via cell viability, lipid-ROS, ferrous iron, and MDA measurements. Metabolic flux analysis showed limited conversion of sarcosine to other metabolites in LUAD cells, while untargeted metabolomic profiling and seahorse assays indicated a metabolic shift from glycolysis to oxidative phosphorylation. Sarcosine enhanced pyruvate dehydrogenase activity to generate more ROS by interacting with PDK4, reducing PDHA1 phosphorylation. As a co-activator of N-methyl-D-aspartate receptor (NMDAR), sarcosine also exerted its pro-ferroptosis effect via regulating ferrous export through the NMDAR/MXD3/SLC40A1 axis. Given the significance of ferroptosis in chemotherapy, we validated that sarcosine enhanced the sensitization of cisplatin by promoting ferroptosis in LUAD cells, PDOs, and xenograft models.Conclusion Sarcosine promotes ferroptosis and enhances chemosensitivity, suggesting its potential as a therapeutic agent in treating LUAD.

Non-canonical role of UCKL1 on ferroptosis defence in colorectal cancer

Weili Wu,Yingying Zhao,Baifu Qin,Xin Jiang,Chuyue Wang,Rong Hu,Rui Ma,Mong-Hong Lee,Huanliang Liu,Kai Li,Ping Yuan

An ATPase-Mimicking MXene nanozyme pharmacologically breaks the ironclad defense system for ferroptosis cancer therapy

Huan Wang,Xinchen Liu,Xiangyu Yan,Yong Du,Fang Pu,Jinsong Ren,Xiaogang Qu

Anticancer nanomedicines used for ferroptosis therapy generally rely on the direct delivery of Fenton catalysts to drive lipid peroxidation in cancer cells. However, the therapeutic efficacy is limited by the ferroptosis resistance caused by the intracellular anti-ferroptotic signals. Herein, we report the intrinsic ATPase-mimicking activity of a vanadium carbide MXene nanozyme (PVCMs) to pharmacologically modulate the nuclear factor erythroid 2-related factor 2 (Nrf2) program, which is the master anti-ferroptotic mediator in the ironclad defense system in triple-negative breast cancer (TNBC) cells. The PVCMs perform high ATPase-like activity that can effectively and selectively catalyze the dephosphorylation of ATP to generate ADP. Through a cascade mechanism initiated by falling energy status, PVCMs can powerfully hinder the Nrf2 program to selectively drive ferroptosis in TNBC cells in response to PVCMs-induced glutathione depletion. This study provides a paradigm for the use of pharmacologically active nanozymes to moderate specific cellular signals and elicit desirable pharmacological activities for therapeutic applications.

Inhibition of neuronal ferroptosis in the acute phase of intracerebral hemorrhage shows long-term cerebroprotective effects

Bin Chen,Zhenghong Chen,Mingjian Liu,Xiaorong Gao,Yijun Cheng,Yongxu Wei,Zhebao Wu,Derong Cui,Hanbing Shang

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke because it has few viable therapeutic options to intervene against primary or second brain injury. Recently, evidence has suggested that ferroptosis, a nonapoptotic form of cell death, is involved in ICH. In this study, we examined whether ICH-induced neuron death is partly ferroptotic in humans and assessed its temporal and spatial characteristics in mice. Furthermore, the ferroptosis inhibitor ferrostatin-1 (Fer-1) was used to examine the role of ferroptosis after ICH. Fold changes in ferroptosis-related gene expression, intracellular iron levels, malondialdehyde (MDA) levels, and both protein levels and cellular localization of cyclooxygenase-2 (COX-2) were measured to monitor ferroptosis. Transmission electron microscopy (TEM) was also performed to examine the ultrastructure of cells after ICH. We found that the expression level of prostaglandin-endoperoxide synthase (PTGS2) was increased in both in vitro and in vivo ICH models; by comparison, expression level of RPL8 was increased in human brain tissue. In mice, iron and MDA levels were significantly increased 3 h after ICH; COX-2 levels were increased at 12 h after ICH and peaked at 3 days after ICH; COX-2 colocalized with NeuN (a neuronal biomarker); and TEM showed that shrunken mitochondria were found at 3 h, 3 days, and 7 days after ICH. Moreover, ICH-induced neurological deficits, memory impairment and brain atrophy were reduced by Fer-1 treatment. Our results demonstrated that neuronal ferroptosis occurs during the acute phase of ICH in brain areas distant from the hematoma and that inhibition of ferroptosis by Fer-1 exerted a long-term cerebroprotective effect.

Comparative effects of 6PPD and 6PPD-Quinone at environmentally relevant concentrations on hepatotoxicity, glucolipid metabolism and ferroptotic response in adult zebrafish

Zhe Wang,Mingjun Zhu,Yao Hu,Jiaxi Liu,Xiaoyu Ma,Hong Zhou

The antioxidant 6PPD and its oxidized product 6PPD-Quinone (6PPDQ) have attracted considerable attention due to their various acute toxicities to aquatic organisms. However, the chronic toxicity of two compounds in aquatic animals is still unknown. Here, adult zebrafish were exposed to 6PPD and 6PPDQ at environmentally relevant concentrations (20 μg/L) for 28 days, and histological analysis showed that 6PPD caused more severe hepatic vacuolization than 6PPDQ. Meanwhile, 6PPD induced more serious lipid accumulation and a higher increase in triglyceride and total cholesterol levels than 6PPDQ, suggesting higher hepatotoxicity of 6PPD. Furthermore, transcriptomic analysis revealed that both compounds disturbed glucolipid metabolism to different degrees by altering the expression of different peroxisome proliferator-activated receptors (PPARs), in which 6PPD inhibited gene expressions in glucolipid metabolism possibly by PPARα, PPARβ and RXR, while 6PPDQ disrupted the expressions of partial genes in similar pathways probably via PPARγ. Additionally, 6PPD but not 6PPDQ increased Fe 2+ content, decreased the protein levels of ferroportin 1, ferritin and glutathione peroxidase 4, accompanied with the increase of malondialdehyde level and the decrease of glutathione content, suggesting ferroptotic response by 6PPD. Overall, our data deepened the understanding of 6PPD- and 6PPDQ-induced hepatotoxicity association with glucolipid metabolism disorders and ferroptotic responses.

Multiple Strikes Achieve Remarkable Tumor-Inhibition Efficiency via Multi-mechanism Combination

Hanchun Yao,Pan Qiao,Zhihui Zhu,Fangfang Sun,Huijuan Zhou,Meilin Geng,Bin Du

Breast cancer treatment has been challenging all the time because cancer cells have multiple signaling pathways; so, breast cancer still remains a threat to the lives and health of many patients. While common single drug therapies inhibit only one pathway, the combination of multiple mechanisms offers the potential to simultaneously suppress multiple targets and pathways to kill cancer cells more effectively. It is reported that autophagy caused by autophagy inducers and apoptosis caused by some chemotherapeutic drugs can promote ferroptosis to some extent; herein, we combined these three pathways and constructed a multifunctional dual-responsive release nanosystem of Rap@mFe3O4–DOX–HA that achieved the ferroptosis–autophagy–apoptosis synergistic effect for cancer treatment. Mesoporous Fe3O4 (mFe3O4) was set as the carrier and can also release Fe ions for ferroptosis, the autophagy inducer rapamycin (Rap) was wrapped in the carrier to trigger autophagy, and the chemotherapeutic drug doxorubicin (DOX) was used as the apoptosis inducer. At the tumor site, the prepared Rap@mFe3O4–DOX–HA nanoparticles split and released DOX/Rap in response to H+/GSH. From in vivo and in vitro studies, it was found that Rap@mFe3O4–DOX–HA nanoparticles effectively inhibited the migration of 4T1 cells, furthermore, they struck cancer cells through multiple pathways and greatly improved the anti-tumor effect. Therefore, the strategy of multi-mechanism combination achieved a therapeutic effect of 1 + 1 > 2.

CircSCD1 inhibits ferroptosis in breast Cancer through stabilizing SCD1 protein via deubiquitinase OTUB1

Wu Zhiwei,Zhang Fan,Yang Kai,He Wenfei

Breast cancer is the leading cause of cancer-related death in women worldwide, and its developmental mechanisms involve complex factors. Recent studies have shown that ferroptosis is closely related to the occurrence and progression of breast cancer. However, the role of circular RNAs (circRNAs) in regulating ferroptosis in breast cancer remains unclear. In this study, we investigated the regulatory role of circSCD1 (hsa_circ_0019512) in breast cancer. We examined the expression of circSCD1 in breast cancer cell lines and explored its impact on cell viability and colony formation. We also evaluated the involvement of circSCD1 in ferroptosis by measuring the levels of malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), and intracellular iron. In vivo xenograft experiments were performed to confirm the role of circSCD1 in promoting tumor growth and inhibiting ferroptosis.Furthermore, we investigated the mechanism by which circSCD1 regulates SCD1 protein stability through ubiquitination and identified the interaction between circSCD1 and the deubiquitinase OTUB1. Our results showed that circSCD1 was upregulated in breast cancer cell lines and promoted cell viability and colony formation. Knockdown of circSCD1 increased MDA and ROS levels, decreased GSH levels, and enhanced ferroptosis in breast cancer cells. In vivo, circSCD1 knockdown significantly reduced tumor size and weight, while its overexpression enhanced tumor growth. Mechanistically, circSCD1 interacted with OTUB1 to inhibit the ubiquitination and degradation of SCD1 protein, thereby stabilizing its expression. Rescue experiments demonstrated that SCD1 overexpression partially reversed the effects of circSCD1 knockdown on cell proliferation and ferroptosis. Our findings suggest that circSCD1 plays a crucial role in promoting breast cancer cell growth and inhibiting ferroptosis by regulating SCD1 protein stability. Targeting the circSCD1/OTUB1/SCD1 axis may provide a potential therapeutic strategy for breast cancer treatment.

GCLC desuccinylation regulated by oxidative stress protects human cancer cells from ferroptosis

Chen Zixiang,Niu Kaifeng,Li Mengge,Deng Yuchun,Zhang Ji,Wei Di,Wang Jiaqi,Zhao Yongliang

Tumor cells evolve strong antioxidant capacities to counteract the abnormal high level of reactive oxygen species (ROS) in the tumor microenvironment. Glutamate-cysteine ligase catalyzing subunit (GCLC) for synthesis of antioxidant glutathione (GSH) represents the key enzyme to maintain redox homeostasis of tumor cells, however, whether its activity is regulated by posttranslational modifications, such as succinylation, remains to be clarified. Here, we demonstrate the existence of succinylation modification on GCLC by in vitro and in vivo assays. NAD-dependent deacetylase Sirtuin-2 (SIRT2) serves as the desuccinylase and catalyzes GCLC desuccinylation at sites of K38, K126, and K326. Specifically, GCLC directly interacts with SIRT2, which can be substantially enhanced upon ROS treatment. This strengthened association results in GCLC desuccinylation and activation, consequently promoting GSH synthesis and rendering cancer cells resistant to ferroptosis induction. Depletion of SIRT2 decreases total GSH level and meanwhile increases the cellular susceptibility to ferroptosis, which can mostly be rescued by introducing wild-type GCLC, but not its 3K-E mutant. We further demonstrated that histone acetyltransferase P300 serves as the succinyltransferase of GCLC, and their association is remarkably decreased after ROS treatment. Thus, SIRT2-regulated GCLC succinylation represents an essential signaling axis for cancer cells to maintain their redox balance in coping with oxidative stress-induced ferroptosis.