Upregulation Of Reactive Oxygen Species Research Articles

CSIG-03. MT-125 INHIBITS NON-MUSCLE MYOSIN IIA AND IIB, SYNERGIZES WITH ONCOGENIC KINASE INHIBITORS, AND PROLONGS SURVIVAL IN GLIOBLASTOMA

Abstract We have shown that members of the non-muscle myosin II (NMII) family of molecular motors are non-redundant drivers of both invasion and proliferation in glioblastoma (GBM), consistent with the canonical roles NMII isoforms play in cell motility and cytokinesis. However, we have also found that NMII also has a non-canonical role by regulating oncogenic signaling pathways relevant to GBM pathogenesis. This unexpected finding has led us to identify and characterize MT-125, a highly specific small molecule inhibitor of the two predominant NMII isoforms in GBM (NMIIA and NMIIB), and we have studied its effects in pre-clinical GBM models. MT-125 has high brain penetrance and retention; an excellent safety profile in rodents and canines with no changes in metabolic or hematologic indices after prolonged administration; is well-tolerated with daily dosing for at least 85 consecutive days; blocks GBM invasion and cytokinesis, consistent with the canonical roles of NMII; and prolongs median survival by 35% as a single agent in murine GBM models. MT-125 increases signaling along both the PDGFR- and MAPK-driven pathways through a mechanism that involves the upregulation of reactive oxygen species, leading to oncogene addiction to these pathways. As a consequence, MT-125 is synthetically lethal when combined with FDA-approved, CNS-permeant inhibitors of PDGFR and mTOR in vitro. Combining MT-125 with sunitinib, a PDGFR inhibitor, or paxalisib, a combined PI3 Kinase/mTOR inhibitor, in vivo doubles median survival in a highly aggressive murine orthotopic GBM model. Furthermore, combining MT-125 with sunitinib produces tumor free remissions of >100 days--2.5-fold greater than median survival of either drug alone—in approximately 40% of mice. Our results provide a powerful rationale for developing NMII targeting strategies to treat cancer and demonstrate that MT-125 has strong clinical potential for the treatment of GBM.

Exploring the response of yellow lupine (Lupinus luteus L.) root to drought mediated by pathways related to phytohormones, lipid, and redox homeostasis.

Yellow lupine (Lupinus luteus L.) is a high-protein crop of considerable economic and ecological significance. It has the ability to fix atmospheric nitrogen in symbiosis with Rhizobium, enriching marginal soils with this essential nutrient and reducing the need for artificial fertilizers. Additionally, lupine produces seeds with a high protein content, making it valuable for animal feed production. However, drought negatively affects lupine development, its mutualistic relationship with bacteria, and overall yield. To understand how lupine responds to this stress, global transcriptome sequencing was conducted, along with in-depth biochemical, chromatography, and microscopy analyses of roots subjected to drought. The results presented here contribute to strategies aimed at mitigating the effects of water deficit on lupine growth and development. Based on RNA-seq, drought-specific genes were identified and annotated to biological pathways involved in phytohormone biosynthesis/signaling, lipid metabolism, and redox homeostasis. Our findings indicate that drought-induced disruption of redox balance characterized by the upregulation of reactive oxygen species (ROS) scavenging enzymes, coincided with the accumulation of lipid-metabolizing enzymes, such as phospholipase D (PLD) and lipoxygenase (LOX). This disruption also led to modifications in lipid homeostasis, including increased levels of triacylglycerols (TAG) and free fatty acids (FFA), along with a decrease in polar lipid content. Additionally, the stress response involved alterations in the transcriptional regulation of the linolenic acid metabolism network, resulting in changes in the composition of fatty acids containing 18 carbons. The first comprehensive global transcriptomic profiles of lupine roots, combined with the identification of key stress-responsive molecules, represent a significant advancement in understanding lupine's responses to abiotic stress. The increased expression of the Δ12DESATURASE gene and enhanced PLD activity lead to higher level of linoleic acid (18:2), which is subsequently oxidized by LOX, resulting in membrane damage and malondialdehyde (MDA) accumulation. Oxidative stress elevates the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), while the conversion of FFAs into TAGs provides protection against ROS. This research offers valuable molecular and biochemical candidates with significant potential to enhance drought tolerance . It enables innovative strategies in lupine breeding and crop improvement to address critical agricultural challenges.

Bilirubin-Modified Chondroitin Sulfate-Mediated Multifunctional Liposomes Ameliorate Acute Kidney Injury by Inducing Mitophagy and Regulating Macrophage Polarization.

Acute kidney injury (AKI) is a dynamic process associated with inflammation, oxidative stress, and lipid peroxidation, in which mitochondrial mitophagy and macrophage polarization play a critical role in the pathophysiology. Based on the expression of the CD44 receptor on renal tubular epithelial cells (RTECs) and activated M1 macrophages being abnormally increased, accompanied by up-regulation of reactive oxygen species (ROS) during AKI, the conjugates of bilirubin (BR), an endogenous antioxidant which has the property of anti-inflammation, and chondroitin sulfate (CS) with CD44-targeting property could be a promising therapeutic carrier. In this study, we develop a CD44-targeted/ROS-responsive CS-BR-mediated multifunctional liposome loading celastrol (CS-BR@CLT) for the targeted therapy of AKI. CS-BR@CLT is shown to selectively accumulate in AKI mouse kidneys via targeting of CD44 receptors. Treatment with CS-BR@CLT significantly ameliorates acute kidney injury caused by ischemia-reperfusion and protects renal function. Mechanistically, CS-BR@CLT inhibits apoptosis, protects mitochondria, promotes autophagy, regulates macrophage polarization, and alleviates interstitial inflammation. Overall, our study demonstrates that CS-BR@CLT could be a promising strategy to ameliorate acute kidney injury.

MG132-mediated Suppression of the Ubiquitin-proteasome Pathway Enhances the Sensitivity of Endometrial Cancer Cells to Cisplatin.

Tumor cell resistance to cisplatin is a common challenge in endometrial cancer chemotherapy, stemming from various mechanisms. Targeted therapies using proteasome inhibitors, such as MG132, have been investigated to enhance cisplatin sensitivity, potentially offering a novel treatment approach. The aim of this study was to investigate the effects of MG132 on cisplatin sensitivity in the human endometrial cancer (EC) cell line RL95-2, focusing on cell proliferation, apoptosis, and cell signaling. Human endometrial cancer RL95-2 cells were exposed to MG132, and cell viability was assessed in a dose-dependent manner. The study evaluated the effect of MG132 on cisplatin-induced proliferation inhibition and apoptosis, correlating with caspase-3 activation and reactive oxygen species (ROS) upregulation. Additionally, we examined the inhibition of the ubiquitin-proteasome system and the expression of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and IL-13 during MG132 and cisplatin co-administration. MG132 exposure significantly reduced cell viability in a dose-dependent manner. It augmented cisplatin- induced proliferation inhibition and enhanced apoptosis, correlating with caspase-3 activation and ROS upregulation. Molecular analysis revealed a profound inhibition of the ubiquitin-proteasome system. MG132 also significantly increased the expression of cisplatin-induced pro-inflammatory cytokines, suggesting a transition from chronic to acute inflammation. MG132 enhances the therapeutic efficacy of cisplatin in human EC cells by suppressing the ubiquitin- proteasome pathway, reducing cell viability, enhancing apoptosis, and shifting the inflammatory response. These findings highlighted the potential of MG132 as an adjuvant in endometrial cancer chemotherapy. Further research is needed to explore detailed mechanisms and clinical applications of this combination therapy.

Closed-loop theranostic microgels for immune microenvironment modulation and microbiota remodeling in ulcerative colitis

Inflammatory bowel disease (IBD) is characterized by the upregulation of reactive oxygen species (ROS) and dysfunction of gut immune system, and microbiota. The conventional treatments mainly focus on symptom control with medication by overuse of drugs. There is an urgent need to develop a closed-loop strategy that combines in situ monitoring and precise treatment. Herein, we innovatively designed the ‘cluster munition structure’ theranostic microgels to realize the monitoring and therapy for ulcerative colitis (a subtype of IBD). The superoxide anion specific probe (tetraphenylethylene-coelenterazine, TPC) and ROS-responsive nanogels consisting of postbiotics urolithin A (UA) were loaded into alginate and ion-crosslinked to obtain the theranostic microgels. The theranostic microgels could be delivered to the inflammatory site, where the environment-triggered breakup of the microgels and release of the nanogels were achieved in sequence. The TPC-UA group had optimal results in reducing inflammation, repairing colonic epithelial tissue, and remodeling microbiota, leading to inflammation amelioration and recovery of tight junction between the colonic epithelium, and maintenance of gut microbiota. During the recovery process, the local chemiluminescence intensity, which is proportional to the degree of inflammation, was gradually inhibited. The cluster munition of theranostic microgels displayed promising outcomes in monitoring inflammation and precise therapy, and demonstrated the potential for inflammatory disease management.

Comprehensive analysis reveals the differentiated influential mechanism of biodegradable and non-biodegradable microplastics on core microbial metabolisms and functional genes in an adsorption-biological coupling wastewater treatment system

The effects of different types of microplastics (MPs) on the wastewater treatment efficiency of an adsorption-biological coupling system were investigated. The distribution of the microbial community and metabolic pathways in the coupled system were also analyzed. Biodegradable polybutylene succinate microplastics (PBS MPs) were added to reactor A, while non-biodegradable polystyrene microplastics (PS MPs) was added to reactor B. The average chemical oxygen demand (COD) removal rates in reactor A with 0, 50, and 100 mg/L of MPs were 95.34 %, 89.13 %, and 86.08 %, respectively; the average COD removal rates in reactor B were 96.84 %, 88.43 %, and 87.02 %, respectively. The average total phosphorus (TP) removal rates in reactor A were 74.14 %, 38.94 %, and 36.45 %, respectively, while those in reactor B were 71.73 %, 39.50 %, and 33.09 %, respectively. In the two reactors, Proteobacteria had the highest relative abundance (53 %–62 %), followed by Actinobacteria. PS MPs inhibited the expression of quorum sensing genes by influencing SecA and SecY. Horizontal transfer genes illustrated that both MPs led to up-regulation of reactive oxygen species (ROS)-related functional genes. During glycolysis, the abundances of glucokinase (GLK), pyruvate kinase (PK), and phosphofructokinase (PFK) in reactor A were 0.0179 %, 0.0133 %, and 0.0387 % higher than that in reactor B, respectively. Meanwhile, in nitrogen metabolism, the abundance of key genes such as nasA (A: 40.8 % B: 37.9 %) was greater in reactor A than in reactor B.

Matrine Suppresses Arsenic-Induced Malignant Transformation of SV-HUC-1 Cells via NOX2.

Arsenic (As) has been classified as a carcinogen for humans. There is abundant evidence indicating that arsenic increases the risk of bladder cancer among human populations. However, the underlying mechanisms have yet to be fully understood and elucidated. NADPH oxidases (NOXs) are the main enzymes for ROS production in the body. NADPH Oxidase 2 (NOX2), which is the most distinctive and ubiquitously expressed subunit of NOXs, can promote the formation and development of tumors. The utilization of NOX2 as a therapeutic target has been proposed to modulate diseases resulting from the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3). Matrine has been reported to exhibit various pharmacological effects, including anti-inflammatory, antifibrotic, antitumor, and analgesic properties. However, it has not been reported whether matrine can inhibit malignant transformation induced by arsenic in uroepithelial cells through NOX2. We have conducted a series of experiments using both a sub-chronic NaAsO2 exposure rat model and a long-term NaAsO2 exposure cell model. Our findings indicate that arsenic significantly increases cell proliferation, migration, and angiogenesis in vivo and in vitro. Arsenic exposure resulted in an upregulation of reactive oxygen species (ROS), NOX2, and NLRP3 inflammasome expression. Remarkably, both in vivo and in vitro, the administration of matrine demonstrated a significant improvement in the detrimental impact of arsenic on bladder epithelial cells. This was evidenced by the downregulation of proliferation, migration, and angiogenesis, as well as the expression of the NOX2 and NLRP3 inflammasomes. Collectively, these findings indicate that matrine possesses the ability to reduce NOX2 levels and inhibit the transformation of bladder epithelial cells.

NOX2-TRPM2 coupling promotes Zn2+ inhibition of complex III to exacerbate ROS production in a cellular model of Parkinson’s disease

Reactive oxygen species (ROS) serve vital physiological functions, but aberrant ROS production contributes to numerous diseases. Unfortunately, therapeutic progress targeting pathogenic ROS has been hindered by the limited understanding of whether the mechanisms driving pathogenic ROS differ from those governing physiological ROS generation. To address this knowledge gap, we utilised a cellular model of Parkinson’s disease (PD), as an exemplar of ROS-associated diseases. We exposed SH-SY5Y neuroblastoma cells to the PD-toxin, MPP+ (1-methyl-4-phenylpyridinium) and studied ROS upregulation leading to cell death, the primary cause of PD. We demonstrate: (1) MPP+ stimulates ROS production by raising cytoplasmic Ca2+ levels, rather than acting directly on mitochondria. (2) To raise the Ca2+, MPP+ co-stimulates NADPH oxidase-2 (NOX2) and the Transient Receptor Potential Melastatin2 (TRPM2) channel that form a positive feedback loop to support each other’s function. (3) Ca2+ exacerbates mitochondrial ROS (mtROS) production not directly, but via Zn2+. (4) Zn2+ promotes electron escape from respiratory complexes, predominantly from complex III, to generate mtROS. These conclusions are drawn from data, wherein inhibition of TRPM2 and NOX2, chelation of Ca2+ and Zn2+, and prevention of electron escape from complexes -all abolished the ability of MPP+ to induce mtROS production and the associated cell death. Furthermore, calcium ionophore mimicked the effects of MPP+, while Zn2+ ionophore replicated the effects of both MPP+ and Ca2+. Thus, we unveil a previously unrecognized signalling circuit involving NOX2, TRPM2, Ca2+, Zn2+, and complex III that drives cytotoxic ROS production. This circuit lies dormant in healthy cells but is triggered by pathogenic insults and could therefore represent a safe therapeutic target for PD and other ROS-linked diseases.

A preoperative dose of the pyridoindole AC102 improves the recovery of residual hearing in a gerbil animal model of cochlear implantation

Sensorineural hearing loss (SNHL) is the most common sensory deficit worldwide. Due to the heterogeneity of causes for SNHL, effective treatment options remain scarce, creating an unmet need for novel drugs in the field of otology. Cochlear implantation (CI) currently is the only established method to restore hearing function in profound SNHL and deaf patients. The cochlear implant bypasses the non-functioning sensory hair cells (HCs) and electrically stimulates the neurons of the cochlear nerve. CI also benefits patients with residual hearing by combined electrical and auditory stimulation. However, the insertion of an electrode array into the cochlea induces an inflammatory response, characterized by the expression of pro-inflammatory cytokines, upregulation of reactive oxygen species, and apoptosis and necrosis of HCs, putting residual hearing at risk. Here, we characterize the small molecule AC102, a pyridoindole, for its protective effects on residual hearing in CI. In a gerbil animal model of CI, AC102 significantly improves the recovery of hearing thresholds across multiple frequencies and confines the cochlear trauma to the directly mechanically injured area. In addition, AC102 significantly preserves auditory nerve fibers and inner HC synapses throughout the whole cochlea. In vitro experiments in an ethanol challenged HT22 cell-line revealed significant and dose-responsive anti-apoptotic effects following the treatment of with AC102. Further, AC102 treatment resulted in significant downregulation of the expression of pro-inflammatory cytokines in an organotypic ex vivo model of electrode insertion trauma (EIT). These results suggest that AC102’s effects are likely elicited during the inflammatory phase of EIT and mediated by anti-apoptotic and anti-inflammatory properties, highlighting AC102 as a promising compound for hearing preservation during CI. Moreover, since the inflammatory response in CI shares similarities to that in other etiologies of SNHL, AC102 may be inferred as a potential general treatment option for various inner ear conditions.

MitoNEET preserves muscle insulin sensitivity during iron overload by regulating mitochondrial iron, reactive oxygen species and fission.

Iron overload (IO) is known to contribute to metabolic dysfunctions such as type 2 diabetes and insulin resistance. Using L6 skeletal muscle cells overexpressing the CDGSH iron-sulfur domain-containing protein 1 (CISD1, also known as mitoNEET) (mitoN) protein, we examined the potential role of MitoN in preventing IO-induced insulin resistance. In L6 control cells, IO resulted in insulin resistance which could be prevented by MitoN as demonstrated by western blot of p-Akt and Akt biosensor cells. Mechanistically, IO increased; mitochondrial iron accumulation, mitochondrial reactive oxygen species (ROS), Fis1-dependent mitochondrial fission, mitophagy, FUN14 domain-containing protein 1 (FUNDC1) expression, and decreased Parkin. MitoN overexpression was able to reduce increases in mitochondrial iron accumulation, mitochondrial ROS, mitochondrial fission, mitophagy and FUNDC1 upregulation due to IO. MitoN did not have any effect on the IO-induced downregulation of Parkin. MitoN alone also upregulated peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) protein levels, a master regulator of mitochondrial biogenesis. The use of mitochondrial antioxidant, Skq1, or fission inhibitor, Mdivi-1, prevented IO-induced insulin resistance implying both mitochondrial ROS and fission play a causal role in the development of insulin resistance. Taken together, MitoN is able to confer protection against IO-induced insulin resistance in L6 skeletal muscle cells through regulation of mitochondrial iron content, mitochondrial ROS, and mitochondrial fission.

Boswellia carterii n-hexane extract suppresses breast cancer growth via induction of ferroptosis by downregulated GPX4 and upregulated transferrin

Breast cancer (BC) remains a significant health concern for women globally, prompting the relentless pursuit of novel therapeutic modalities. As a traditional Chinese medicine, Boswellia carterii has been extensively used to treat various cancers, such as BC. However, the anti-BC effect and underlying mechanism of Boswellia carterii remain largely unclear. The aim of this study is to explore the therapeutic effect of Boswellia carterii n-hexane extract (BCHE) against BC as well as its underlying mechanism. The present study showed that BCHE significantly suppressed the viability of human BC cells. Moreover, BCHE exhibited potent anti-BC activity in vivo with no significant toxic effects. Additionally, BCHE induced ferroptosis via increased Transferrin expression and the intracellular accumulation of Fe2+, as well as decreased glutathione peroxidase 4 (GPX4) expression and the upregulation of reactive oxygen species (ROS)-induced lipid peroxidation in BC cells. In vivo experimental results also demonstrated that BCHE effectively induced ferroptosis through GPX4 downregulation and Transferrin upregulation in tumor-bearing mice. Overall, BCHE inhibited the growth of BC cells by inducing ferroptosis mediated by modulating the iron accumulation pathway and the lipid peroxidation pathway. Therefore, BCHE could serve as a potential ferroptosis-targeting drug for treating BC.

Yttrium oxide nanoparticles ameliorates calcium hydroxide and calcium titanate nanoparticles induced genomic DNA and mitochondrial damage, ROS generation and inflammation

Calcium hydroxide (Ca(OH)2NPs), calcium titanate (CaTiO3NPs) and yttrium oxide (Y2O3NPs) nanoparticles are prevalent in many industries, including food and medicine, but their small size raises concerns about potential cellular damage and genotoxic effects. However, there are very limited studies available on their genotoxic effects. Hence, this was done to investigate the effects of multiple administration of Ca(OH)2NPs, CaTiO3NPs or/and Y2O3NPs on genomic DNA stability, mitochondrial membrane potential integrity and inflammation induction in mouse brain tissues. Mice were orally administered Ca(OH)2NPs, CaTiO3NPs or/and Y2O3NPs at a dose level of 50 mg/kg b.w three times a week for 2 weeks. Genomic DNA integrity was studied using Comet assay and the level of reactive oxygen species (ROS) within brain cells was analyzed using 2,7 dichlorofluorescein diacetate dye. The expression level of Presenilin-1, tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) genes and the integrity of the mitochondrial membrane potential were also detected. Oral administration of Ca(OH)2NPs caused the highest damage to genomic DNA and mitochondrial membrane potential, less genomic DNA and mitochondrial damage was induced by CaTiO3NPs administration while administration of Y2O3NPs did not cause any remarkable change in the integrity of genomic DNA and mitochondrial membrane potential. Highest ROS generation and upregulation of presenilin-1, TNF-α and IL-6 genes were also observed within the brain cells of mice administrated Ca(OH)2NPs but Y2O3NPs administration almost caused no changes in ROS generation and genes expression compared to the negative control. Administration of CaTiO3NPs alone slightly increased ROS generation and the expression level of TNF-α and IL-6 genes. Moreover, no remarkable changes in the integrity of genomic DNA and mitochondrial DNA potential, ROS level and the expression level of presenilin-1, TNF-α and IL-6 genes were noticed after simultaneous coadministration of Y2O3NPs with Ca(OH)2NPs and CaTiO3NPs. Coadministration of Y2O3NPs with Ca(OH)2NPs and CaTiO3NPs mitigated Ca(OH)2NPs and CaTiO3NPs induced ROS generation, genomic DNA damage and inflammation along with restoring the integrity of mitochondrial membrane potential through Y2O3NPs scavenging free radicals ability. Therefore, further studies are recommended to study the possibility of using Y2O3NPs to alleviate Ca(OH)2NPs and CaTiO3NPs induced genotoxic effects.

Mechanism of Purinergic Regulation of Neurotransmission in Mouse Neuromuscular Junction: The Role of Redox Signaling and Lipid Rafts.

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic Gi protein-coupled P2Y13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P2Y13 receptors (MRS 2211), Ca2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca2+ with subsequent elevation of ROS levels. The elevation of [Ca2+]in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of Gi protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P2Y13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P2Y13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P2Y13 receptor/Gi protein/Ca2+/protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner.

Circ_WASF2 regulates ferroptosis by miR-634/ GPX4 signaling in pancreatic cancer

PurposePancreatic cancer (PC) is one of the most lethal malignant gastrointestinal tumors (GI) characterized by a poor prognosis. Ferroptosis is an emerging programmed cell death that plays an essential role in the progression of various cancers. Ferroptosis is driven by iron-dependent phospholipid peroxidation and is regulated by mitochondrial activity, lipid peroxidation, and reactive oxygen species (ROS). The function and mechanism of ferroptosis in PC need more research.MethodsThe levels of circRNAs, miRNAs, and mRNAs were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was used for protein detection. CCK8 assays were used to detect cell proliferation. Cell death, lipid peroxidation, ROS, and Fe2+ were detected by indicted kits. Dual-luciferase reporter and RNA pull-down assays were conducted to confirm the interaction between circRNAs, miRNAs, and mRNAs.ResultsIn this research, we found that circular RNA hsa_circ_0000003(circ_WASF2) was upregulated in pancreatic cancer cells. The silence of circ_WASF2 inhibited cancer proliferation and increased cell death by increasing ferroptosis accompanied by up-regulation of lipid peroxidation, ROS, and Fe2+. Further studies showed that circ_WASF2 could attenuate ferroptosis by targeting miR-634 and the downstream glutathione peroxidase 4 (GPX4). GPX4 has been well-reported as a central factor in ferroptosis. Our research revealed a new pathway for regulating ferroptosis in PC.ConclusionIn summary, we have determined that circ_WASF2/miR-634/GPX4 contributed to ferroptosis-induced cell death, and provided a possible therapeutic target in PC.

Forced swim stress exacerbates inflammation-induced hyperalgesia and oxidative stress in the rat trigeminal ganglia.

This study investigates the impact of combining psychophysical stress, induced by forced swim (FSS), with masseter inflammation on reactive oxygen species (ROS) production in trigeminal ganglia (TG), TRPA1 upregulation in TG, and mechanical hyperalgesia. In a rat model, we demonstrate that FSS potentiates and prolongs CFA-induced ROS upregulation within TG. The ROS levels in CFA combined with FSS group surpass those in the CFA-only group on days 4 and 28 post-treatment. FSS also enhances TRPA1 upregulation in TG, with prolonged expression compared to CFA alone. Furthermore, CFA-induced mechanical hyperalgesia is significantly prolonged by FSS, persisting up to day 28. PCR array analyses reveal distinct alterations in oxidative stress genes under CFA and CFA combined with FSS conditions, suggesting an intricate regulation of ROS within TG. Notably, genes like Nox4, Hba1, Gpx3, and Duox1 exhibit significant changes, providing potential targets for managing oxidative stress and inflammatory pain. Western blot and immunohistochemistry confirm DUOX1 protein upregulation and localization in TG neurons, indicating a role in ROS generation under inflammatory and stress conditions. This study underscores the complex interplay between psychophysical stress, inflammation, and oxidative stress in the trigeminal system, offering insights into novel therapeutic targets for pain management.

Abstract 4715: Azathioprine and Z36 induced cell death in human colorectal cancer HCT-116 and HCT-15 cells

Abstract Rationale and Objectives: Cancer accounts for a huge global health burden and is the third highest cause of death across the globe. Cancer cells are known to have higher basal reactive oxygen species (ROS) levels, owing to their higher metabolism and proliferation. Higher basal ROS in cancer cells made them susceptible to exogenous ROS stimuli that lead to cell death. Therefore, in the present study, we have analyzed the effects of Azathioprine, a glutathione (GSH) inhibitor and an intracellular ROS producer. The effect of Azathioprine was analyzed alone or in combination with a BCL-2 family protein inhibitor Z36, on human colorectal cancer cell lines HCT-116 and HCT-15. Methods: The effects of the drugs have been investigated using various methods including cell viability assay, DNA fragmentation assay, and Annexin V-FITC/PI apoptosis assay. In addition, morphological changes evident in cell death were detected by Giemsa staining. Combination dosage for drug synergistic effects was computed through the use of CompuSyn software. Western blot was performed to detect changes in the expression of cell death-related proteins following drug treatments. Results and conclusion: Azathioprine at a dose of 5 µM has been demonstrated to induce cell death in both HCT116 and HCT15 cells in a concentration and time-dependent manner. On the contrary, Z36, a novel BCLXL inhibitor, has demonstrated similar cell viability results at an even lower dose of 2.5 µM. When both drugs were combined at lower doses, no significant effects were observed on the cell viability of either cell line, demonstrating that the combined treatment has no synergistic or additive effects. This suggests that both drugs do not augment each other and might induce cell death via upregulation of ROS using a similar mechanism. Citation Format: Yousuf Tahir Ali, Mahwish Fatima, Muhammad Areeb Ahmed, Hasan Salman Siddiqi, Azhar Hussain, Mati Ur Rehman. Azathioprine and Z36 induced cell death in human colorectal cancer HCT-116 and HCT-15 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4715.

Abstract 1107: Prospective observational study to evaluate the efficacy of oxygen saturation endoscopic imaging for radiotherapeutic response in head and neck squamous cell carcinoma

Abstract Background: Radiotherapy (RT) has a pivotal role in the management of head and neck squamous cell carcinoma (HNSCC), providing effective tumor control and functional preservation. Hypoxia is a cause of radioresistance in HNSCC. We have developed an oxygen saturation endoscopic imaging (OXEI) method to assess real-time tissue oxygenation levels. However, the clinical significance of OXEI in patients with HNSCC receiving RT remains unclear. We evaluated the association between pretreatment tumor oxygen saturation (StO2) quantified by OXEI and clinical outcomes of patients with HNSCC receiving RT. Methods: We prospectively recruited patients with HNSCC scheduled to receive RT at 2 institutions. The primary endpoint was the association between pretreatment tumor StO2 and complete response (CR) rate 8 weeks after completion of RT. Secondary endpoints evaluated the impact of StO2 on 1-year overall survival (OS) and progression-free survival (PFS) rates after RT initiation. We also evaluated the cumulative incidence of local failure (LF). To explore the relationship between tumor hypoxia assessed by OXEI and gene expression profile, we performed RNA sequencing of 4 tumor samples collected from 2 patients (2 samples/patient) before RT. The hypoxia score for each sample was calculated by gene set variation analysis based on HNSCC mRNA-based hypoxia signatures. We performed gene set enrichment analysis (GSEA) to evaluate whether the biological pathways (Hallmark, Reactome, and KEGG gene sets) showed significant associations with tumor hypoxia. Results: Of the 50 patients enrolled from March 2019 to February 2022, we analyzed 37 who underwent RT and OXEI. Thirty-six (97%) patients received current platinum-based chemotherapy. The median (range) pretreatment tumor StO2 of 60 (45-84)% was lower than that of normal mucosa, 66 (56-84)% (P < 0.01). Patients with tumor StO2 < 60% were classified as the hypoxia group (n =19), and those with tumor StO2 ≥ 60% as the non-hypoxia group (n =18). The CR rate 8 weeks after RT was not significantly different between the hypoxia and non-hypoxia groups (84% vs. 100%, respectively; P = 0.23). After a median (range) follow-up of 33 (9-45) months, the hypoxia group had worse 1-year PFS (74% vs. 94%, P = 0.017) and OS (89% vs. 100%, P = 0.015) and higher LF rates (21% vs. 6%, P = 0.036) compared with the non-hypoxia group. RNA sequencing confirmed that tumor StO2 reflected expression-based hypoxia scores. GSEA showed upregulation of reactive oxygen species (ROS) and KEAP1-NFE2L2 pathways in hypoxic samples (StO2 of 59%) compared with non-hypoxic samples (StO2 of 70%; adjusted P < 0.01), consistent with the fact that hypoxic stress increases ROS production. Conclusion: Our findings suggest that pretreatment tumor StO2 quantified by OXEI correlates with 1-year PFS, OS, and LF rates of patients with HNSCC receiving RT. Citation Format: Kento Tomizawa, Atsushi Motegi, Hidenari Hirata, Hiroki Yamashita, Hironori Sunakawa, Wataru Okano, Tomohiro Enokida, Takeshi Fujisawa, Sadamoto Zenda, Masaki Nakamura, Hidehiro Hojo, Seiichiro Abe, Madoka Sakuramachi, Hiroshi Igaki, Yutaka Saito, Kazuto Matsuura, Makoto Tahara, Tomonori Yano, Tetsuo Akimoto. Prospective observational study to evaluate the efficacy of oxygen saturation endoscopic imaging for radiotherapeutic response in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1107.

A pH-sensitive imidazole grafted polymeric micelles nanoplatform based on ROS amplification for ferroptosis-enhanced chemodynamic therapy

Highly toxic reactive oxygen species (ROS), crucial in inducing apoptosis and ferroptosis, are pivotal for cell death pathways in cancer therapy. However, the effectiveness of ROS-related tumor therapy is impeded by the limited intracellular ROS and substrates, coupled with the presence of abundant ROS scavengers like glutathione (GSH). In this research, we developed acid-responsive, iron-coordinated polymer nanoparticles (PPA/TF) encapsulating a mitochondrial-targeting drug alpha-tocopheryl succinate (α-TOS) for enhanced synergistic tumor treatment. The imidazole grafted micelles exhibit prolonged blood circulation and improve the delivery efficiency of the hydrophobic drug α-TOS. Additionally, PPA's design aids in delivering Fe3+, supplying ample iron ions for chemodynamic therapy (CDT) and ferroptosis through the attachment of imidazole groups to Fe3+. In the tumor's weakly acidic intracellular environment, PPA/TF facilitates pH-responsive drug release. α-TOS specifically targets mitochondria, generating ROS and replenishing those depleted by the Fenton reaction. Moreover, the presence of Fe3+ in PPA/TF amplifies ROS upregulation, promotes GSH depletion, and induces oxidative damage and ferroptosis, effectively inhibiting tumor growth. This research presents an innovative ROS-triggered amplification platform that optimizes CDT and ferroptosis for effective cancer treatment.

Aberrantly downregulated FENDRR by arecoline elevates ROS and myofibroblast activation via mitigating the miR-214/MFN2 axis

Long non-coding RNA FENDRR possesses both anti-fibrotic and anti-cancer properties, but its significance in the development of premalignant oral submucous fibrosis (OSF) remains unclear. Here, we showed that FENDRR was downregulated in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs), and overexpression of FENDRR mitigated various myofibroblasts hallmarks, and vice versa. In the course of investigating the mechanism underlying the implication of FENDRR in myofibroblast transdifferentiation, we found that FENDRR can directly bind to miR-214 and exhibit its suppressive effect on myofibroblast activation via titrating miR-214. Moreover, we showed that mitofusin 2 (MFN2), a protein that is crucial to the fusion of mitochondria, was a direct target of miR-214. Our data suggested that FENDRR was positively correlated with MFN2 and MFN2 was required for the inhibitory property of FENDRR pertaining to myofibroblast phenotypes. Additionally, our results showed that the FENDRR/miR-214 axis participated in the arecoline-induced reactive oxygen species (ROS) accumulation and myofibroblast transdifferentiation. Building on these results, we concluded that the aberrant downregulation of FENDRR in OSF may be associated with chronic exposure to arecoline, leading to upregulation of ROS and myofibroblast activation via the miR-214-mediated suppression of MFN2.

Self-Assembly of 2D Polyphthalocyanine in Lysosome Enables Multienzyme Activity Enhancement to Induce Tumor Ferroptosis.

Nanozymes show great potential in facilitating tumor ferroptosis by upregulation of reactive oxygen species (ROS) and downregulation of glutathione (GSH). However, mild acidity (pH 6.5-6.9) of tumor microenvironment severely restricts the activity of nanozymes. Although lysosomes as acidic organelles (pH = 3.5-5.5) are hopeful for improving enzyme-like activity, most reported nanozymes are not capable of effectively accumulating in the lysosomes. Herein, an acid-responsive self-assembly strategy based on iron phthalocyanine-rich covalent organic framework nanosheets (COFFePc NSs) is developed, which enables lysosomal targeting aggregation of COFFePc NSs due to the existence of abundant negative hydroxyl groups and rigid structure. Meanwhile, COFFePc NSs display exceptional multienzyme-mimic performance at lower pH to efficiently generate ROS to cause lysosome damage and apoptosis by synergistic photothermal effect. Subsequently, the released COFFePc with GSH oxidase-mimicking activity can consume GSH to promote ferroptosis. This is the first report of a 2D COF using its own properties to achieve lysosomal self-assembly. Overall, the work provides a new paradigm for the development of lysosome-targeted nanosystems.