Targeting BMAL1 reverses drug resistance of acute myeloid leukemia cells and promotes ferroptosis through HMGB1-GPX4 signaling pathway.

PURPOSE: Acute myeloid leukemia (AML) is a refractory hematologic malignancy that poses a serious threat to human health. Exploring alternative therapeutic strategies capable of inducing alternative modes of cell death, such as ferroptosis, holds great promise as a viable and effective intervention. METHODS: We analyzed online database data and collected clinical samples to verify the expression and function of BMAL1 in AML. We conducted experiments on AML cell proliferation, cell cycle, ferroptosis, and chemotherapy resistance by overexpressing/knocking down BMAL1 and using assays such as MDA detection and BODIPY 581/591 C11 staining. We validated the transcriptional regulation of HMGB1 by BMAL1 through ChIP assay, luciferase assay, RNA level detection, and western blotting. Finally, we confirmed the results of our cell experiments at the animal level. RESULTS: BMAL1 up-regulation is an observed phenomenon in AML patients. Furthermore, there existed a strong correlation between elevated levels of BMAL1 expression and inferior prognosis in individuals with AML. We found that knocking down BMAL1 inhibited AML cell growth by blocking the cell cycle. Conversely, overexpressing BMAL1 promoted AML cell proliferation. Moreover, our research results revealed that BMAL1 inhibited ferroptosis in AML cells through BMAL1-HMGB1-GPX4 pathway. Finally, knocking down BMAL1 can enhance the efficacy of certain first-line cancer therapeutic drugs, including venetoclax, dasatinib, and sorafenib. CONCLUSION: Our research results suggest that BMAL1 plays a crucial regulatory role in AML cell proliferation, drug resistance, and ferroptosis. BMAL1 could be a potential important therapeutic target for AML.

PLAG1 interacts with GPX4 to conquer vulnerability to sorafenib induced ferroptosis through a PVT1/miR-195-5p axis-dependent manner in hepatocellular carcinoma.

BACKGROUND: Sorafenib is a standard first-line treatment for advanced hepatocellular carcinoma (HCC), yet its effectiveness is often constrained. Emerging studies reveal that sorafenib triggers ferroptosis, an iron-dependent regulated cell death (RCD) mechanism characterized by lipid peroxidation. Our findings isolate the principal target responsible for ferroptosis in HCC cells and outline an approach to potentially augment sorafenib's therapeutic impact on HCC. METHODS: We investigated the gene expression alterations following sgRNA-mediated knockdown induced by erastin and sorafenib in HCC cells using CRISPR screening-based bioinformatics analysis. Gene set enrichment analysis (GSEA) and the "GDCRNATools" package facilitated the correlation studies. We employed tissue microarrays and cDNA microarrays for validation. Ubiquitination assay, Chromatin immunoprecipitation (ChIP) assay, RNA immunoprecipitation (RIP) assay, and dual-luciferase reporter assay were utilized to delineate the specific mechanisms underlying ferroptosis in HCC cells. RESULTS: Our study has revealed that pleiomorphic adenoma gene 1 (PLAG1), a gene implicated in pleomorphic adenoma, confers resistance to ferroptosis in HCC cells treated with sorafenib. Sorafenib leads to the opposite trend of protein and mRNA levels of PLAG1, which is not caused by affecting the stability or ubiquitination of PLAG1 protein, but by the regulation of PLAG1 at the transcriptional level by its upstream competitive endogenous long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1). Data from 139 HCC patients showed a significant positive correlation between PLAG1 and GPX4 levels in tumor samples, and PLAG1 is instrumental in redox homeostasis by driving the expression of glutathione peroxidase 4 (GPX4), the enzyme that reduces lipid peroxides (LPOs), which further leads to ferroptosis inhibition. CONCLUSIONS: Ferroptosis is a promising target for cancer therapy, especially for patients resistant to standard chemotherapy or immunotherapy. Our findings indicate that PLAG1 holds therapeutic promise and may enhance the efficacy of sorafenib in treating HCC.

Unraveling ETC complex I function in ferroptosis reveals a potential ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

The role of the mitochondrial electron transport chain (ETC) in regulating ferroptosis is not fully elucidated. Here, we reveal that pharmacological inhibition of the ETC complex I reduces ubiquinol levels while decreasing ATP levels and activating AMP-activated protein kinase (AMPK), the two effects known for their roles in promoting and suppressing ferroptosis, respectively. Consequently, the impact of complex I inhibitors on ferroptosis induced by glutathione peroxidase 4 (GPX4) inhibition is limited. The pharmacological inhibition of complex I in LKB1-AMPK-inactivated cells, or genetic ablation of complex I (which does not trigger apparent AMPK activation), abrogates the AMPK-mediated ferroptosis-suppressive effect and sensitizes cancer cells to GPX4-inactivation-induced ferroptosis. Furthermore, complex I inhibition synergizes with radiotherapy (RT) to selectively suppress the growth of LKB1-deficient tumors by inducing ferroptosis in mouse models. Our data demonstrate a multifaceted role of complex I in regulating ferroptosis and propose a ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

ARHGAP10, Transcriptionally Regulated by Sodium Butyrate, Promotes Ferroptosis of Ovarian Cancer Cells.

BACKGROUND: Ovarian cancer is a highly lethal gynecologic malignancy. ARHGAP10, a member of Rho GTPase-activating proteins, is a potential tumor suppressor in ovarian cancer. However, its role and the involved mechanism need further examination. Here, we investigated whether ARHGAP10 is also associated with ferroptosis. METHODS: Lentivirus infection was used for gene overexpression or silencing. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to assess mRNA and protein levels, respectively. Cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. Lipid reactive oxygen species level was measured by flow cytometry. A tumorigenicity assay was performed to evaluate tumor growth in vivo, and sections of mouse tumor tissues were examined by immunofluorescence microscopy. Chromatin Immunoprecipitation (ChIP) assay was used to assess the binding of H3K9ac to the promoter region of ARHGAP10. RESULTS: ARHGAP10 overexpression promoted ferroptosis in ovarian cancer cells, resulting in decreased cell viability, and increased lipid reactive oxygen species (ROS) level. Further, it decreased and increased GPX4 and PTGS2 expression, respectively, and also induced suppression of tumor growth in mice. Fer-1, a potent inhibitor of ferroptosis, suppressed the above effects of ARHGAP10. Contrarily, ARHGAP10 silencing alleviated ferroptosis in ovarian cancer cells, which was reversed by RSL3, a ferroptosis-inducing agent. Lastly, sodium butyrate (SB) was found to transcriptionally regulate ARHGAP10, thereby also contributing to the ferroptosis of ovarian cancer cells. CONCLUSIONS: Our results suggest that SB/ARHGAP10/GPX4 is a new signaling axis involved in inducing ferroptosis in ovarian cancer cells and suppressing tumor growth, which has potential clinical significance.

Asiaticoside promoted ferroptosis and suppressed immune escape in gastric cancer cells by downregulating the Wnt/ß-catenin pathway.

BACKGROUND: Our previous study has revealed that asiaticoside (AC) promotes endoplasmic reticulum stress and antagonizes proliferation and migration of gastric cancer (GC) via miR-635/HMGA1 axis. However, the effect and mechanism of AC on other progressions of GC, such as ferroptosis and immune escape, are still unknown. METHODS: AGS and HGC27 cells were incubated with 1, 2 and 4 µM of AC for 24 h. Mice xenografted with AGS cells were intragastrically injected with AC. The effect and mechanism of AC on GC were determined by the measurement of the ferrous iron level, the ROS level and the glutathione peroxidase (GSH) content, flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and western blotting assays. RESULTS: AC increased the Fe2+ level and the ROS level, but decreased the expression of GPX4 and SLC7A11 and the GSH level. Besides, AC enhanced the percent of CD8+ T cells and the IFN-γ concentration, but reduced the PD-L1 expression and the IL-10 level. Mechanically, AC downregulated the relative levels of ß-catenin, active-ß-catenin, p-GSK3ß/GSK3ß, cyclin D1 and c-Myc in GC cells, which were rescued with the application of LiCl (an activator of Wnt/ß-catenin pathway) in AGS cells. Moreover, activation of Wnt/ß-catenin pathway by LiCl or the ß-catenin overexpression inverted the effect of AC on ferroptosis and immune escape in GC cells. In vivo, AC treatment declined the tumor size and weight, the level of GPX4, SLC7A11, PD-L1 and IFN-γ, and the expression of Wnt/ß-catenin pathway. CONCLUSION: AC enhanced ferroptosis and repressed immune escape by downregulating the Wnt/ß-catenin signaling in GC.

FGF10 protects against particulate matter-induced lung injury by inhibiting ferroptosis via Nrf2-dependent signaling.

Particulate matter (PM) is considered the fundamental component of atmospheric pollutants and is associated with the pathogenesis of many respiratory diseases. Fibroblast growth factor 10 (FGF10) mediates mesenchymal-epithelial signaling and has been linked with the repair process of PM-induced lung injury (PMLI). However, the pathogenic mechanism of PMLI and the specific FGF10 protective mechanism against this injury are still undetermined. PM was administered in vivo into murine airways or in vitro to human bronchial epithelial cells (HBECs), and the inflammatory response and ferroptosis-related proteins SLC7A11 and GPX4 were assessed. The present research investigates the FGF10-mediated regulation of ferroptosis in PMLI mice models in vivo and HBECs in vitro. The results showed that FGF10 pretreatment reduced PM-mediated oxidative damage and ferroptosis in vivo and in vitro. Furthermore, FGF10 pretreatment led to reduced oxidative stress, decreased secretion of inflammatory mediators, and activation of the Nrf2-dependent antioxidant signaling. Additionally, silencing of Nrf2 using siRNA in the context of FGF10 treatment attenuated the effect on ferroptosis. Altogether, both in vivo and in vitro assessments confirmed that FGF10 protects against PMLI by inhibiting ferroptosis via the Nrf2 signaling. Thus, FGF10 can be used as a novel ferroptosis suppressor and a potential treatment target in PMLI.

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Bladder cancer (BC) is one of the 3 common malignant tumors in the urinary system, with high incidence, easy metastasis, poor therapeutic efficacy, and poor prognosis, which seriously threatens the health of human. Tumor cells exhibit a strong demand for iron, and iron overload can induce ferroptosis, which is an iron dependent cell death caused by lipid peroxidation and cell membrane damage. Therefore, ferroptosis has strong anti-tumor potential. The molecular mechanisms of ferroptosis is associated with abnormalities in cellular phospholipid metabolism and iron metabolism, and dysregulation of antioxidant and non-antioxidant systems Xc-/glutathione (GSH)/glutathione peroxidase 4 (GPX4). Ferroptosis relevant molecules play important roles in the occurrence and development, metastasis, drug resistance, and immune response of BC, and are expected to become targets for the treatment of BC.

PITX2 functions as a transcription factor for GPX4 and protects pancreatic cancer cells from ferroptosis.

Ferroptosis inhibits tumor progression in pancreatic cancer cells, while PITX2 is known to function as a pro-oncogenic factor in various tumor types, protecting them from ferroptosis and thereby promoting tumor progression. In this study, we sought to investigate the regulatory role of PITX2 in tumor cell ferroptosis within the context of pancreatic cancer. We conducted PITX2 knockdown experiments using lentiviral infection in two pancreatic cancer cell lines, namely PANC-1 and BxPC-3. We assessed protein expression through immunoblotting and mRNA expression through RT-PCR. To confirm PITX2 as a transcription factor for GPX4, we employed Chromatin Immunoprecipitation (ChIP) and Dual-luciferase assays. Furthermore, we used flow cytometry to measure reactive oxygen species (ROS), lipid peroxidation, and apoptosis and employed confocal microscopy to assess mitochondrial membrane potential. Additionally, electron microscopy was used to observe mitochondrial structural changes and evaluate PITX2's regulation of ferroptosis in pancreatic cancer cells. Our findings demonstrated that PITX2, functioning as a transcription factor for GPX4, promoted GPX4 expression, thereby exerting an inhibitory effect on ferroptosis in pancreatic cancer cells and consequently promoting tumor progression. Moreover, PITX2 enhanced the invasive and migratory capabilities of pancreatic cancer cells by activating the WNT signaling pathway. Knockdown of PITX2 increased ferroptosis and inhibited the proliferation of PANC-1 and BxPC-3 cells. Notably, the inhibitory effect on ferroptosis resulting from PITX2 overexpression in these cells could be countered using RSL3, an inhibitor of GPX4. Overall, our study established PITX2 as a transcriptional regulator of GPX4 that could promote tumor progression in pancreatic cancer by reducing ferroptosis. These findings suggest that PITX2 may serve as a potential therapeutic target for combating ferroptosis in pancreatic cancer.

Recent Advances in the Inhibition of Membrane Lipid Peroxidation by Food-Borne Plant Polyphenols via the Nrf2/GPx4 Pathway.

Lipid peroxidation (LP) leads to changes in the fluidity and permeability of cell membranes, affecting normal cellular function and potentially triggering apoptosis or necrosis. This process is closely correlated with the onset of many diseases. Evidence suggests that the phenolic hydroxyl groups in food-borne plant polyphenols (FPPs) make them effective antioxidants capable of preventing diseases triggered by cell membrane LP. Proper dietary intake of FPPs can attenuate cellular oxidative stress, especially damage to cell membrane phospholipids, by activating the Nrf2/GPx4 pathway. Nuclear factor E2-related factor 2 (Nrf2) is an oxidative stress antagonist. The signaling pathway regulated by Nrf2 is a defense transduction pathway of the organism against external stimuli such as reactive oxygen species and exogenous chemicals. Glutathione peroxidase 4 (GPx4), under the regulation of Nrf2, is the only enzyme that reduces cell membrane lipid peroxides with specificity, thus playing a pivotal role in regulating cellular ferroptosis and counteracting oxidative stress. This study explored the Nrf2/GPx4 pathway mechanism, antioxidant activity of FPPs, and mechanism of LP. It also highlighted the bioprotective properties of FPPs against LP and its associated mechanisms, including (i) activation of the Nrf2/GPx4 pathway, with GPx4 potentially serving as a central target protein, (ii) regulation of antioxidant enzyme activities, leading to a reduction in the production of ROS and other peroxides, and (iii) antioxidant effects on LP and downstream phospholipid structure. In conclusion, FPPs play a crucial role as natural antioxidants in preventing LP. However, further in-depth analysis of FPPs coregulation of multiple signaling pathways is required, and the combined effects of these mechanisms need further evaluation in experimental models. Human trials could provide valuable insights into new directions for research and application.

[Mechanism of acupotomy on chondrocyte ferroptosis in rabbits with knee osteoarthritis based on HSPA5/GPX4 signaling pathway].

OBJECTIVE: To observe the effect of acupotomy on heat shock protein A family member 5 (HSPA5)/glutathione peroxidase 4 (GPX4) signaling pathway in the chondrocytes of the rabbits with knee osteoarthritis (KOA) and explore the mechanism of acupotomy on chondrocyte ferroptosis in KOA. METHODS: Twenty-seven New Zealand rabbits were randomly divided into a normal group, a model group and an acupotomy group, with 9 rabbits in each group. The left hind limb was fixed by the modified Videman method for 6 weeks to establish KOA model. After modeling, acupotomy was given in the acupotomy group, once a week and for consecutive 3 weeks. Using Lequesne MG score, the local symptoms, physical signs and functions of knee joint were evaluated. With HE staining and saffrane-solid green staining adopted, the morphology of chondrocytes and cartilage tissue was observed. Under transmission electron microscope, the mitochondrial structure of chondrocytes was observed. The iron content of cartilage tissue was detected by iron ion kit. The mitochondrial membrane potential (Δψm) and the reactive oxygen species (ROS) level in cartilage tissue were determined by flow cytometry, and the mitochondrial damage rate was calculated. The mRNA expression of HSPA5, GPX4, type Ⅱ collagen α1 chain (COL2A1), matrix metalloproteinases (MMP) 3 and MMP13 was detected by the real-time quantitative PCR; and the protein expression of HSPA5, GPX4, type Ⅱ collagen (COL-Ⅱ), MMP3 and MMP13 was detected by Western blot. The mean flourscence intensity of HSPA5 and GPX4 in cartilage tissue was determined by immunofluorescence. RESULTS: Before intervention, compared with the normal group, the Lequesne MG scores were increased in the model group and the acupotomy group (P<0.01). After intervention, the Lequesne MG score in the acupotomy group was decreased when compared with that in the model group. In comparison with that in the normal group, the number of chondrocytes was reduced and the cells were disarranged; the layers of cartilage structure were unclear, the tide lines disordered and blurred; the mitochondria were wrinkled and the mitochondrial crista decreased or even disappeared in the model group. Compared with the model group, the number of chondrocytes was increased, the layers of cartilage structure were clear, the tide lines recovered, the number of mitochondria elevated, with normal structure and more crista in the acupotomy group. The iron content of cartilage tissue was increased (P<0.01), the Δψm of chondrocytes was declined, the mitochondrial damage rate was increased (P<0.01), the average fluorescence intensity of ROS was increased (P<0.01); the mRNA and corresponding protein expression of HSPA5, GPX4 and COL2A1 was decreased (P<0.01), the mRNA and protein expression of MMP3 and MMP13 was increased (P<0.01) and the average fluorescence intensity of HSPA5, GPX4 was decreased (P<0.01) in the model group when compared with those in the normal group. Compared with the model group, the iron content in cartilage tissue was reduced (P<0.01), the Δψm of chondrocytes was increased, the mitochondrial damage rate was decreased (P<0.01), and the average fluorescence intensity of ROS was decreased (P<0.01); the mRNA and corresponding protein expression of HSPA5, GPX4 and COL2A1 was higher (P<0.01), and the mRNA and protein expression of MMP3 and MMP13 was lower, and the average fluorescence intensity of HSPA5, GPX4 was increased (P<0.01) in the acupotomy group. CONCLUSION: Acupotomy can alleviate cartilage injury of KOA rabbits, and its mechanism may be related to the regulation of HSPA5/GPX4 signaling pathway to maintain iron homeostasis in articular cartilage, thus inhibiting chondrocyte ferroptosis and relieving extracellular matrix degradation.

Expression of Glutathione Peroxidase4 in Patients with Esophageal Squamous Carcinoma and Its Impact on the Radiosensitivity of Esophageal Squamous Carcinoma.

OBJECTIVE: Glutathione peroxidase-4 (GPX4) is a member of Ferroptosis and lipid circulation. This study aims to investigate the expression of GPX4 in esophageal squamous cell carcinoma and its impact on radiosensitivity. METHOD: Immunohistochemistry staining was used to detect GPX4 expression in 180 samples of ESCC tissues and adjacent tissues. We analyzed the relationship between GPX4 expression and ESCC clinical parameters. In vitro experiments were conducted using apoptosis assays and colony formation assays to investigate the effect of GPX4 on the radiosensitivity of ESCC cells. In vivo experiments were carried out using a nude mouse xenograft model to evaluate the impact of GPX4 on the radiosensitivity of ESCC. RESULTS: GPX4 expression was lower in adjacent tissues than tumor tissues. The expression of GPX4 was significantly associated with the pathological grade of ESCC. The overall survival time (OS) of ESCC patients with low GPX4 expression was significantly longer than that of patients with high GPX4 expression. GPX4 could be used as independent prognostic factors in patients with ESCC. In vivo experiments, silencing of GPX4 or using GPX4 inhibitors significantly inhibits the viability and colony formation of ESCC cells after radiation exposure while increasing intracellular reactive oxygen species (ROS) levels, and significantly suppresses the tumorigenic ability of ESCC cells in subcutaneous xenografts after radiation exposure. CONCLUSION: GPX4 is highly expressed in ESCC, which has the potential value for prognostic assessment of ESCC. Silencing or inhibiting GPX4 can enhance the radiosensitivity of ESCC.

Construction and Validation of Novel Ferroptosis-related Risk Score Signature and Prognostic Prediction Nomogram for Patients with Colorectal Cancer.

Background: Colorectal cancer (CRC) has a high morbidity and mortality. Ferroptosis is a phenomenon in which metabolism and cell death are closely related. The role of ferroptosis-related genes in the progression of CRC is still not clear. Therefore, we screened and validated the ferroptosis-related genes which could determine the prevalence, risk and prognosis of patients with CRC. Methods: We firstly screened differentially expressed ferroptosis-related genes by The Cancer Genome Atlas (TCGA) database. Then, these genes were used to construct a risk-score model using the least absolute shrinkage and selection operator (LASSO) regression algorithm. The function and prognosis of the ferroptosis-related genes were confirmed using multi-omics analysis. The gene expression results were validated using publicly available databases and qPCR. We also used publicly available data and ferroptosis-related genes to construct a prognostic prediction nomogram. Results: A total of 24 differential expressed genes associated with ferroptosis were screened in this study. A three-gene risk score model was then established based on these 24 genes and GPX3, CDKN2A and SLC7A11 were selected. The significant prognostic value of this novel three-gene signature was also assessed. Furthermore, we conducted RT-qPCR analysis on cell lines and tissues, and validated the high expression of CDKN2A, GPX3 and low expression of SLC7A11 in CRC cells. The observed mRNA expression of GPX3, CDKN2A and SLC7A11 was consistent with the predicted outcomes. Besides, eight variables including selected ferroptosis related genes were included to establish the prognostic prediction nomogram for patients with CRC. The calibration plots showed favorable consistency between the prediction of the nomogram and actual observations. Also, the time-dependent AUC (>0.7) indicated satisfactory discriminative ability of the nomogram. Conclusions: The present study constructed and validated a novel ferroptosis-related three-gene risk score signature and a prognostic prediction nomogram for patients with CRC. Also, we screened and validated the ferroptosis-related genes GPX3, CDKN2A, and SLC7A11 which could serve as novel biomarkers for patients with CRC.

Dipeptidase 1 promotes ferroptosis in renal tubular epithelial cells in diabetic nephropathy via inhibition of the GSH/GPX4 axis.

Renal tubular injury is an important pathological change associated with diabetic nephropathy (DN), in which ferroptosis of renal tubular epithelial cells is critical to its pathogenesis. Inhibition of the glutathione/glutathione peroxidase 4 (GSH/GPX4) axis is the most important mechanism in DN tubular epithelial cell ferroptosis, but the underlying reason for this is unclear. Our biogenic analysis showed that a zinc-dependent metalloproteinase, dipeptidase 1 (DPEP1), is associated with DN ferroptosis. Here, we investigated the role and mechanism of DPEP1 in DN tubular epithelial cell ferroptosis. DPEP1 upregulation was observed in the renal tubular epithelial cells of DN patients and model mice, as well as in HK-2 cells stimulated with high glucose. Furthermore, the level of DPEP1 upregulation was associated with the degree of tubular injury in DN patients and HK-2 cell ferroptosis. Mechanistically, knocking down DPEP1 expression could alleviate the inhibition of GSH/GPX4 axis and reduce HK-2 cell ferroptosis levels in a high glucose environment. HK-2 cells with stable DPEP1 overexpression also showed GSH/GPX4 axis inhibition and ferroptosis, but blocking the GSH/GPX4 axis could mitigate these effects. Additionally, treatment with cilastatin, a DPEP1 inhibitor, could ameliorate GSH/GPX4 axis inhibition and relieve ferroptosis and DN progression in DN mice. These results revealed that DPEP1 can promote ferroptosis in DN renal tubular epithelial cells via inhibition of the GSH/GPX4 axis.

Icariin inhibits chondrocyte ferroptosis and alleviates osteoarthritis by enhancing the SLC7A11/GPX4 signaling.

BACKGROUND: Chondrocyte ferroptosis plays a critical role in the pathogenesis of osteoarthritis (OA), regulated by the SLC7A11/GPX4 signaling pathway. Icariin (ICA), a flavonoid glycoside, exhibits strong anti-inflammatory and antioxidant activities. This study investigated whether ICA could modulate the SLC7A11/GPX4 signaling to inhibit chondrocyte ferroptosis and alleviate OA. PURPOSE: The objective was to explore the impact of ICA on chondrocyte ferroptosis in OA and its modulation of the SLC7A11/GPX4 signaling pathway. METHODS: The anti-ferroptosis effects of ICA were evaluated in an interleukin-1ß (IL-1ß)-treated SW1353 cell model, using Ferrostatin-1 (Fer-1) and Erastin (Era) as ferroptosis inhibitor and inducer, respectively, along with GPX4 knockdown via lentivirus-based shRNA. Additionally, the therapeutic efficacy of ICA on OA-related articular cartilage damage was assessed in rats through histopathology and immunohistochemistry (IHC). RESULTS: IL-1ß treatment upregulated the expression of OA-associated matrix metalloproteinases (MMP3 and MMP1), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5), and increased intracellular ROS, lipid ROS, and MDA levels while downregulating collagen II and SOX9 expression in SW1353 cells. ICA treatment countered the IL-1ß-induced upregulation of MMPs and ADAMTS-5, restored collagen II and SOX9 expression, and reduced intracellular ROS, lipid ROS, and MDA levels. Furthermore, IL-1ß upregulated P53 but downregulated SLC7A11 and GPX4 expression in SW1353 cells, effects that were mitigated by ICA or Fer-1 treatment. Significantly, ICA also alleviated Era-induced ferroptosis, whereas it had no effect on GPX4-silenced SW1353 cells. In vivo, ICA treatment reduced articular cartilage damage in OA rats by partially restoring collagen II and GPX4 expression, inhibiting cartilage extracellular matrix (ECM) degradation and chondrocyte ferroptosis. CONCLUSION: ICA treatment mitigated chondrocyte ferroptosis and articular cartilage damage by enhancing the SLC7A11/GPX4 signaling, suggesting its potential as a therapeutic agent for OA interventions.

Glutathione peroxidase 4 restrains temporomandibular joint osteoarthritis progression by inhibiting ferroptosis.

There are few effective therapeutic strategies for temporomandibular joint osteoarthritis (TMJOA) due to the unclear pathology and mechanisms. We aimed to confirm the roles of GPX4 and ferroptosis in TMJOA progression. ELISA assay was hired to evaluate concentrations of ferroptosis-related markers. The qRT-PCR assay was hired to assess gene mRNA level. Western blot assay and immunohistochemistry were hired to verify the protein level. CCK-8 assay was hired to detect cell viability. Human fibroblast-like synoviocytes (FLSs) were cultured to confirm the effects of GPX4 and indicated inhibitors, and further verified the effects of GPX4 and ferroptosis inhibitors in TMJOA model rats. Markers of ferroptosis including 8-hidroxy-2-deoxyguanosine (8-OHdG) and iron were notably increased in TMJOA tissues and primary OA-FLSs. However, the activity of the antioxidant system including the glutathione peroxidase activity, glutathione (GSH) contents, and glutathione/oxidized glutathione (GSH/GSSG) ratio was notably inhibited in TMJOA tissues, and the primary OA-FLSs. Furthermore, the glutathione peroxidase 4 (GPX4) expression was down-regulated in TMJOA tissues and primary OA-FLSs. Animal and cell experiments have shown that ferroptosis inhibitors notably inhibited ferroptosis and promoted HLS survival as well as up-regulated GPX4 expression. Also, GPX4 knockdown promoted ferroptosis and GPX4 overexpression inhibited ferroptosis. GPX4 also positively regulated cell survival which was the opposite with ferroptosis. In conclusion, GPX4 and ferroptosis regulated the progression of TMJOA. Targeting ferroptosis might be an effective therapeutic strategy for TMJOA patients in the clinic.

Downregulated GPX4 in salivary gland epithelial cells contributes to salivary secretion dysfunction in Sjogren's syndrome via lipid ROS/pSTAT4/AQP5 axis.

Sjogren's syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.

Orexin-A mediates glioblastoma proliferation inhibition by increasing ferroptosis triggered by unstable iron pools and GPX4 depletion.

Glioblastoma (GBM) represents a prevalent form of primary malignant tumours in the central nervous system, but the options for effective treatment are extremely limited. Ferroptosis, as the most enriched programmed cell death process in glioma, makes a critical difference in glioma progression. Consequently, inducing ferroptosis has become an appealing strategy for tackling gliomas. Through the utilization of multi-omics sequencing data analysis, flow cytometry, MDA detection and transmission electron microscopy, the impact of orexin-A on ferroptosis in GBM was assessed. In this report, we provide the first evidence that orexin-A exerts inhibitory effects on GBM proliferation via the induction of ferroptosis. This induction is achieved by instigating an unsustainable increase in iron levels and depletion of GPX4. Moreover, the regulation of TFRC, FTH1 and GPX4 expression through the targeting of NFE2L2 appears to be one of the potential mechanisms underlying orexin-A-induced ferroptosis.

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1.

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.

Fusobacterium nucleatum induces oxaliplatin resistance by inhibiting ferroptosis through E-cadherin/ß-catenin/GPX4 axis in colorectal cancer.

Fusobacterium (F.) nucleatum is a carcinogenesis microbiota in colorectal cancer (CRC). Growing evidence shows that F. nucleatum contributes to chemoresistance. Ferroptosis is reported to restore the susceptibility of resistant cells to chemotherapy. However, the role of gut microbiota affecting ferroptosis in chemoresistance remains unclear. Here, we examined the CRC tissues of patients using 16S rRNA sequencing to investigate the possible connection between gut microbiota dysbiosis and the relapse of CRC. We found that a high abundance of F. nucleatum in CRC tissue is associated with relapse. We further demonstrated that F. nucleatum induced oxaliplatin resistance in vitro and in vivo. The transcriptome of an F. nucleatum-infected cell revealed ferroptosis was associated with F. nucleatum infection. We perform malondialdehyde, ferrous iron, and glutathione assays to verify the effect of F. nucleatum on ferroptosis under oxaliplatin treatment in vivo and in vitro. Mechanistically, F. nucleatum promoted oxaliplatin resistance by overexpressing GPX4 and then inhibiting ferroptosis. E-cadherin/ß-catenin/TCF4 pathway conducted the GPX4 overexpression effect of F. nucleatum. The chromatin immuno-precipitation quantitative PCR (CHIP-qPCR) and dual-luciferase reporter assay showed that F. nucleatum promoted TCF4 binding with GPX4. We also determined the E-cadherin/ß-catenin/TCF4/GPX4 axis related to tumor tissue F. nucleatum status and CRC relapse clinically. Here, we revealed the contribution of F. nucleatum to oxaliplatin resistance by inhibiting ferroptosis in CRC. Targeting F. nucleatum and ferroptosis will provide valuable insight into chemoresistance management and may improve outcomes for patients with CRC.

Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells.

OBJECTIVE: Research has shown that celastrol can effectively treat a variety of diseases, yet when passing a certain dosage threshold, celastrol becomes toxic, causing complications such as liver and kidney damage and erythrocytopenia, among others. With this dichotomy in mind, it is extremely important to find ways to preserve celastrol's efficacy while reducing or preventing its toxicity. METHODS: In this study, insulin-resistant HepG2 (IR-HepG2) cells were prepared using palmitic acid and used for in vitro experiments. IR-HepG2 cells were treated with celastrol alone or in combination with N-acetylcysteine (NAC) or ferrostatin-1 (Fer-1) for 12, 24 or 48 h, at a range of doses. Cell counting kit-8 assay, Western blotting, quantitative reverse transcription-polymerase chain reaction, glucose consumption assessment, and flow cytometry were performed to measure celastrol's cytotoxicity and whether the cell death was linked to ferroptosis. RESULTS: Celastrol treatment increased lipid oxidation and decreased expression of anti-ferroptosis proteins in IR-HepG2 cells. Celastrol downregulated glutathione peroxidase 4 (GPX4) mRNA. Molecular docking models predicted that solute carrier family 7 member 11 (SLC7A11) and GPX4 were covalently bound by celastrol. Importantly, we found for the first time that the application of ferroptosis inhibitors (especially NAC) was able to reduce celastrol's toxicity while preserving its ability to improve insulin sensitivity in IR-HepG2 cells. CONCLUSION: One potential mechanism of celastrol's cytotoxicity is the induction of ferroptosis, which can be alleviated by treatment with ferroptosis inhibitors. These findings provide a new strategy to block celastrol's toxicity while preserving its therapeutic effects. Please cite this article as: Liu JJ, Zhang X, Qi MM, Chi YB, Cai BL, Peng B, Zhang DH. Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells. J Integr Med. 2024; 22(3): 286-294.