Mechanism Of Death Research Articles

Targeting Ferroptosis with Small Molecule Atranorin (ATR) as a Novel Therapeutic Strategy and Providing New Insight into the Treatment of Breast Cancer

Background/Objectives: Ferroptosis results from the accumulation of iron-dependent lipid peroxides and reactive oxygen species (ROS). Previous research has determined the effect of atranorin (ATR) on other cell death mechanisms, but its potential for a ferroptotic effect depending on ROS levels is unclear. This study details the therapeutic role of small-molecule ATR through ferroptosis by suppressing MDA-MB-231, MCF-7, BT-474, and SK-BR-3 breast cancer cells. Methods: The anti-proliferative effect of ATR on cells was evaluated by xCELLigence analysis, and ferroptotic activity was evaluated by enzymatic assay kits. The changes in gene and protein expression levels of ATR were investigated by the qRT-PCR and western blot. In addition, mitochondrial changes were examined by transmission electron microscopy. Results: ATR was found to reduce cell viability in cancer cells in a dose- and time-dependent manner without showing cytotoxic effects on normal breast cells. In BT-474 and MDA-MB-231 cells, ATR, which had a higher anti-proliferative effect, increased iron, lipid peroxidation, and ROS levels in cells and decreased the T-GSH/GSSG ratio. The results revealed for the first time that small-molecule ATR exhibited anti-cancer activity by inducing the glutathione pathway and ferroptosis. Conclusions: This study highlights the potential of ATR as a drug candidate molecule that can be used in the development of new therapeutic strategies for the treatment of triple-negative and luminal-B breast cancer subtypes.

Role of Exogenous Pyruvate in Maintaining Adenosine Triphosphate Production under High-Glucose Conditions through PARP-Dependent Glycolysis and PARP-Independent Tricarboxylic Acid Cycle.

Pyruvate serves as a key metabolite in energy production and as an anti-oxidant. In our previous study, exogenous pyruvate starvation under high-glucose conditions induced IMS32 Schwann cell death because of the reduced glycolysis-tricarboxylic acid (TCA) cycle flux and adenosine triphosphate (ATP) production. Thus, this study focused on poly-(ADP-ribose) polymerase (PARP) to investigate the detailed molecular mechanism of cell death. Rucaparib, a PARP inhibitor, protected Schwann cells against cell death and decreased glycolysis but not against an impaired TCA cycle under high-glucose conditions in the absence of pyruvate. Under such conditions, reduced pyruvate dehydrogenase (PDH) activity and glycolytic and mitochondrial ATP production were observed but not oxidative phosphorylation or the electric transfer chain. In addition, rucaparib supplementation restored glycolytic ATP production but not PDH activity and mitochondrial ATP production. No differences in the increased activity of caspase 3/7 and the localization of apoptosis-inducing factor were found among the experimental conditions. These results indicate that Schwann cells undergo necrosis rather than apoptosis or parthanatos under the aforementioned conditions. Exogenous pyruvate plays a pivotal role in maintaining the flux in PARP-dependent glycolysis and the PARP-independent TCA cycle in Schwann cells under high-glucose conditions.

Histidine phosphatase-ferroptosis crosstalk modulation for efficient hepatocellular carcinoma treatment

Altering the mechanisms of tumor cell death and overcoming the limitations of traditional chemotherapy is pivotal to contemporary tumor treatment. Inducing ferroptosis, while circumventing safety concerns associated with ferrous vectors, through nonferrous ferroptosis is a promising but underexplored frontier in cancer therapy. Histidine phosphatase (LHPP) has emerged as a novel therapeutic target in treating hepatocellular carcinoma (HCC), but the precise mechanism of LHPP against HCC remains unclear. Herein, we explore the effects of upregulating LHPP expression on ferroptosis and tumor immunogenicity induction by simply delivering a miRNA-363-5p inhibitor (miR-363-5pi) via a previously optimized gemcitabine-oleic acid (GOA) prodrug. Efficient miRNA encapsulation was achieved through hydrogen bonding at an optimized GOA/miRNA molar feed ratio of 250:1, affording spherical nanoparticles with a uniform hydrodynamic size of 147.1 nm and a negative potential of -21.5 mV. The mechanism of this LHPP-ferroptosis crosstalk is disclosed to be an inhibited phosphorylation of the PI3K/Akt pathway, leading to a remarkable tumor inhibition rate of 88.2% in nude mice bearing Bel-7402 tumor xenografts via a combination of LHPP-triggered nonferrous ferroptosis and GOA-induced chemotherapy. The biocompatibility of GOA/miR-363-5pi is strongly supported by their non-hematologic toxicity and insignificant organ damage. In addition, the tumor immunogenic activation potential of GOA/miR-363-5pi was finally explored. Overall, this study is the first work that elucidates the precise mechanism of LHPP for treating HCC via ferroptosis induction and achieves the transformation of chemotherapy and gene therapy into ferroptosis activation with tumor cell immunogenicity, which lays a new therapeutic foundation for the clinical treatment of HCC.Graphical

Treatment of polyethylene microplastics degraded by ultraviolet light irradiation causes lysosome-deregulated cell death

BackgroundMicroplastics (MPs), plastic particles < 5 mm in size, are prevalent in the environment, and human exposure to them is inevitable. To assess the potential risk of MPs on human health, it is essential to consider the physicochemical properties of environmental MPs, including polymer types, size, shape, and surface chemical modifications. Notably, environmental MPs undergo degradation due to external factors such as ultraviolet (UV) rays and waves, leading to changes in their surface characteristics. However, limited knowledge exists regarding the health effects of MPs, with a specific focus on their surface degradation. This study concentrates on cytotoxic MPs with surface degradation through UV irradiation, aiming to identify the mechanisms underlying their cell toxicity.ResultsPolyethylene (PE) and surface-degraded PE achieved through UV light irradiation were employed as model MPs in this study. We explored the impact of PE and degraded PE on cell death in murine macrophage cell line RAW264.7 cells and human monocyte cell line THP-1 cells. Flow cytometric analysis revealed that degraded PE induced programmed cell death without activating caspase 3, while non-degraded PE did not trigger programmed cell death. These findings suggest that degraded PE might induce programmed cell death through mechanisms other than caspase-driven apoptosis. To understand the mechanisms of cell death, we investigated how cells responded to degraded PE-induced cellular stress. Immunofluorescence and western blotting analyses demonstrated that degraded PE induced autophagosome formation and increased p62 expression, indicating inhibited autophagy flux after exposure to degraded PE. Furthermore, degraded PE exposure led to a decrease in acidic lysosomes, indicating lysosomal dysregulation. These results imply that degraded PE induces lysosomal dysfunction, subsequently causing autophagy dysregulation and cell death.ConclusionsThis study unveils that UV-induced degradation of PE results in cell death attributed to lysosomal dysfunction. The findings presented herein provide novel insights into the effects of surface-degraded MPs on biological responses.

Identification of PANoptosis Subtypes to Assess the Prognosis and Immune Microenvironment of Lung Adenocarcinoma Patients: A Bioinformatics Combined Machine Learning Study.

PANoptosis, a novelty mechanism of cell death involving crosstalk between apoptosis, pyroptosis, and necroptosis, is strongly associated with tumor cell death and immunotherapy efficacy. However, its relevance in lung adenocarcinoma (LUAD) remains to be elucidated. In this study, we acquired 18 PANoptosis-related differentially expressed gene (PRDEG) of LUAD. Based on these genes, LUAD samples were identified with different sub-types by unsupervised clustering. Next, we compared the differences between the subtypes, including clinical features, immune microenvironment, and potentially sensitive drugs. Further-more, we used machine learning to identify hub prognostic PRDEGs, construct a risk score, and validate it on other external datasets. We incorporated the patient's clinical information and risk score into the proportional hazards model and lasso-cox models to find key prognostic features and constructed five prognostic models. The best model was identified via the area under the curve and validated on an external dataset. LUAD patients were divided into two clusters named C1 and C2, respectively. The C2 cluster exhibited shorter survival time, more advanced tumor stage, higher suppressive immune cell scores, such as dendritic cells, and higher expression of inhibitory immune checkpoints, such as LAG3 and CD86. TIMP1, CAV1, and CD69 were recognized as key prognostic factors, and risk scores predicted survival with significant differences in the external validation set. Risk score and N-stage were identified as critical prognostic features. The Coxph model outper-formed other machine learning clinical models. The 1-, 3-, and 5-year time-ROCs in the exter-nal validation set were 0.55, 0.59, and 0.60, respectively. We demonstrated the potential of PANoptosis-based molecular clustering and prognostic features in predicting the survival of patients with LUAD as well as the tumor mi-croenvironment.

Identification of necroptosis-related gene expression and the immune response in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common reproductive and endocrine disorder; however, the understanding of the pathogenesis of PCOS remains unclear. Necroptosis is a newly discovered mechanism of cell death, and it is closely related to reproductive endocrine-related diseases. This study aimed to investigate the hub necroptosis-related genes in PCOS patients and its correlation with immune cell infiltration by bioinformatics methods. The gene expression chip result matrix and the annotation matrix files of the GSE34526, GSE8157, and GSE5090 datasets were downloaded from the GEO database. We analyzed the expression and correlation of the necroptosis-related genes in all samples, constructed a diagnostic model based on all necroptosis-related genes and genes with significant differences, performed unsupervised clustering of samples and gene enrichment analysis, and evaluated the correlations between the hub gene and immune cell infiltration levels by the R packages GSVA and CIBERSORT. Finally, PPI networks were constructed using the Cytoscape software GeneMANIA plug-in, and the miRNA, transcription factors, RBP, and drugs were predicted. Necroptosis-related genes have important relationships in the development of PCOS and are potentially associated with immune infiltration in PCOS patients.

Mechanisms of drug resistance to neoadjuvant chemotherapy in breast cancer

Aim. Тo study the molecular genetic characteristics of the tumor microenvironment and the mechanisms of cell death in resistant locally advanced breast cancer.Materials and methods. The study included 48 patients with breast cancer T2–4N0–3M0–1 (mean age 55.6 ± 9.8 years), and 29 patients of comparable age with breast fibroadenoma. According to the design of the study, patients were divided into groups: Group 1 included women with breast cancer resistant to neoadjuvant chemotherapy (n = 23), Group 2 – with breast cancer and a complete response to neoadjuvant chemotherapy (n = 25), control Group – with fibroadenoma (n = 29). The expression of markers CD4+, CD8+, CD20+, CD68+, tumor necrosis factor α (TNF-α), vascular endothelial growth factor A (VEGF A), Ang-2, matrix metalloproteinase 12 (MMP-12), inducible nitric oxide synthase (iNOS), bcl-2, p53, CD95 was assessed using immunohistochemistry.Results. When phenotyping immune cells, the following differences were obtained: in the tumor tissue of patients in Group 1, a significant decrease in the number of cytotoxic CD8+ cells was noted compared to Group 2 (p = 0.001) and control (p = 0.032). In Group 2, a significant increase in the number of CD68+ cells was revealed in relation to Group 1 (p = 0.027). The cytokine profile of the tumor microenvironment in Group 1 is characterized by statistically significant overexpression of TNF-α compared to Group 2 (p &gt;0.001) and the control Group (p = 0.01). With regard to apoptotic factors, noteworthy is the significant decrease in the expression of bcl-2 and p53 in Group 1 compared to Group 2 (p = 0.001 and p = 0.02 accordingly).Conclusion. The presented results can serve as the basis for the creation of diagnostic algorithms that have predictive value regarding the effectiveness of NCT, and also to help identify new targets to justify the use of combined breast cancer treatments in the early stage.

Synergistic ferroptosis in triple-negative breast cancer cells: Paclitaxel in combination with Erastin induced oxidative stress and Ferroportin-1 modulation in MDA-MB-231 cells.

Ferroptosis is an important regulated cell death mechanism characterized by iron-dependent lipid peroxidation and oxidative stress. In this study, we examined the ferroptosis-inducing effect of the combined use of Paclitaxel, a microtubule-stabilizing agent, and Erastin, a ferroptosis inducer, in breast cancer cells. In this context, the combination of the compounds in question was applied to the cells and the presence of a synergistic effect was determined by calculating the combination index. Glutathione (GSH) levels and glutathione peroxidase (GPX) activity were determined by commercial assay kits, and the effect of the compounds on lipid peroxidation was determined by measurement of malondialdehyde (MDA) levels. Additionally, the effect of combination treatment on ferroptotic protein expression was determined by western blot. Our findings revealed that the combination treatment caused a significant change in mitochondrial function by causing an increase in the depolarized/viable cell population. Additionally, there was a significant increase in intracellular reactive oxygen species (ROS) levels compared to single applications of the compounds. The significant increase observed in malondialdehyde (MDA) levels revealed that the combination treatment increased lipid peroxidation. Moreover, intracellular GSH levels and glutathione peroxidase (GPX) activity significantly decreased by Paclitaxel-Erastin combination. The expression of ferroptosis-regulating proteins was significantly downregulated. The findings showed that the Paclitaxel-Erastin combination synergistically contributed to the accumulation of lipid reactive oxygen species and induced the ferroptotic cell death pathway in breast cancer cells.

Pro-aromatic Natural Terpenes as Unusual "Slingshot" Antioxidants with Promising Ferroptosis Inhibition Activity.

Ferroptosis is a cell death mechanism based on extensive cellular membrane peroxidation, implicated in neurodegenerative and other diseases. The essential oil component γ-terpinene, a natural monoterpene with a unique highly oxidizable pro-aromatic 1,4-cyclohexadiene skeleton, inhibits peroxidation of polyunsaturated lipid in model heterogeneous systems (micelles and liposomes). Upon H-atom abstraction, an unstable γ-terpinene-derived peroxyl radical is formed, that aromatizes to p-cymene generating HOO• radicals. As HOO• are small and hydrophilic radicals, they quickly diffuse outside the lipid core, blocking the radical chain propagation of polyunsaturated lipids. This unprecedented antioxidant "slingshot" mechanism explains why γ-terpinene shows a protective activity against ferroptosis, being effective at submicromolar concentrations in human neuroblastoma (SH-SY5Y) cells.

Anti-staphylococcal, antibiofilm and trypanocidal activities of CrataBL encapsulated into liposomes: Lectin with potential against infectious diseases

The present study aimed to evaluate the anti-staphylococcal, antibiofilm, cytotoxicity and trypanocidal activity, mechanisms of parasite death and immunomodulatory effect of CrataBL encapsulated into liposomes (CrataBL-Lipo). CrataBL-Lipo were prepared by the freeze-thaw technique and characterized. Anti-staphylococcal and antibiofilm activities of CrataBL and CrataBL-Lipo were evaluated against standard and clinical strains of Staphylococcus aureus susceptible and resistant. Thus, broth microdilution method was performed to determine the Minimum Inhibitory Concentration (MIC). Antibiofilm activity at subinhibitory concentrations was evaluated using the crystal violet staining method. Cytotoxicity of CrataBL-Lipo was verified in L929 fibroblasts and J774A.1 macrophages by determining the inhibitory concentration necessary to kill 50 % of cells (IC50). Trypanocidal activities of CrataBL-Lipo was evaluated in Trypanosoma cruzi and the efficacy was expressed as the concentration necessary to kill 50 % of parasites (EC50). The mechanisms of parasite death and immunomodulatory effect of CrataBL-Lipo were evaluated using flow cytometry analysis. CrataBL-Lipo presented Ø of 101.9 ± 1.3 nm (PDI = 0.245), ζ of +33.8 ± 1.3 mV and %EE = 80 ± 0.84 %. CrataBL-Lipo presented anti-staphylococcal activity (MIC = 0.56 mg/mL to 0.72 mg/mL). CrataBL-Lipo inhibited 45.4 %–75.6 % of biofilm formation. No cytotoxicity of CrataBL-Lipo was found (IC50 > 100 mg/L). CrataBL-Lipo presented EC50 of 1.1 mg/L, presenting autophagy, apoptosis and necrosis as death profile. In addition, CrataBL-Lipo reduced the production of IL-10 and TNF-α levels, causing an immunomodulatory effect. CrataBL-Lipo has a therapeutic potential for the treatment of staphylococcal infections and Chagas disease exhibiting a high degree of selectivity for the microorganism, and immunomodulatory properties.

Regulatory Peptide Pro-Gly-Pro Accelerates Neuroregeneration of Primary Neuroglial Culture after Mechanical Injury in Scratch Test

The scratch test is used as an experimental in vitro model of mechanical damage to primary neuronal cultures to study the mechanisms of cell death in damaged areas. The involvement of NMDA receptors in processes leading to delayed neuronal death, due to calcium dysregulation and synchronous mitochondrial depolarization, has been previously demonstrated. In this study, we explored the neuroregenerative potential of Pro-Gly-Pro (PGP)—an endogenous regulatory peptide with neuroprotective and anti-inflammatory properties and a mild chemoattractant effect. Mechanical injury to the primary neuroglial culture in the form of a scratch caused acute disruption of calcium homeostasis and mitochondrial functions. This was accompanied by neuronal death alongside changes in the profile of neuronal markers (BDNF, NSE and GFAP). In another series of experiments, under subtoxic doses of glutamate (Glu, 33 μM), delayed changes in [Ca2+]i and ΔΨm, i.e., several days after scratch application, were more pronounced in cells in damaged neuroglial cultures. The percentage of cells that restored the initial level of [Ca2+]i (p &lt; 0.05) and the rate of recovery of ΔΨm (p &lt; 0.01) were decreased compared with undamaged cells. Prophylactic application of PGP (100 μM, once) prevented the increase in [Ca2+]i and the sharp drop in mitochondrial potential [ΔΨm] at the time of scratching. Treatment with PGP (30 μM, three or six days) reduced the delayed Glu-induced disturbances in calcium homeostasis and cell death. In the post-glutamate period, the surviving neurons more effectively restored the initial levels of [Ca2+]i (p &lt; 0.001) and Ψm (p &lt; 0.0001). PGP also increased intracellular levels of BDNF and reduced extracellular NSE. In the context of the peptide’s therapeutic effect, the recovery of the damaged neuronal network occurred faster due to reduced astrogliosis and increased migration of neurons to the scratch area. Thus, the peptide PGP has a neuroprotective effect, increasing the survival of neuroglial cells after mechanical trauma in vitro by reducing cellular calcium overload and preventing mitochondrial dysfunction. Additionally, the tripeptide limits the post-traumatic consequences of mechanical damage: it reduces astrogliosis and promotes neuronal regeneration.

SLIM-assisted automatic cartography of cell death types and rates resulting from localized photodynamic treatment

We report a spatial light interference microscopy (SLIM)-based methodology aimed at automatic monitoring and analysis of changes in cellular morphology within extended fields of view in cytological samples. The experimental validation was performed on HeLa cells in vitro subjected to localized photodynamic treatment. The performed long-term noninvasive monitoring using the SLIM technique allowed us to estimate quantitative parameters characterizing the dynamics of average phase shift in individual cells and to reveal changes in their morphology specific for different mechanisms of cell death. The results obtained evidenced that the proposed SLIM-based methodology provides an opportunity for identification of cell death type and quantification of cell death rate in an automatic mode. The major sources of potential errors that can affect the results obtained are discussed. The developed methodology is promising for automatic monitoring of large ensembles of individual cells and for quantitative characterization of their response to various treatment modalities.

Tumor integrin-targeted glucose oxidase enzyme promotes ROS-mediated cell death that combines with interferon alpha therapy for tumor control.

While heightened intratumoral levels of reactive oxygen species (ROS) are typically associated with a suppressive tumor microenvironment, under certain conditions ROS contribute to tumor elimination. Treatment approaches, including some chemotherapy and radiation protocols, increase cancer cell ROS levels that influence their mechanism of cell death and subsequent recognition by the immune system. Furthermore, activated myeloid cells rapidly generate ROS upon encounter with pathogens or infected cells to eliminate disease, and recently, this effector function has been noted in cancer contexts as well. Collectively, ROS-induced cancer cell death may help initiate adaptive anti-tumor immune responses that could synergize with current approved immunotherapies, for improved control of solid tumors. In this work, we explore the use of glucose oxidase, an enzyme which produces hydrogen peroxide, a type of ROS, to therapeutically mimic the endogenous oxidative burst from myeloid cells to promote antigen generation within the tumor microenvironment. We engineer the enzyme to target pan-tumor expressed integrins both as a tumor-agnostic therapeutic approach, but also as a strategy to prolong local enzyme activity following intratumoral administration. We found the targeted enzyme potently induced cancer cell death and enhanced cross-presentation by dendritic cells in vitro, and further combined with interferon alpha for long-term tumor control in murine MC38 tumors in vivo. Optimizing the single-dose administration of this enzyme overcomes limitations with immunogenicity noted for other pro-oxidant enzyme approaches. Overall, our results suggest ROS-induced cell death can be harnessed for tumor control, and highlight the potential use of designed enzyme therapies alongside immunotherapy against cancer.

Revealing the mechanism of natural product-induced immunogenic cell death: opening a new chapter in tumor immunotherapy.

This review underscores the role of natural products in inducing immunogenic cell death (ICD) as a key strategy in tumor immunotherapy. It reveals that natural products can activate ICD through multiple pathways-apoptosis, autophagy, pyroptosis, and necroptosis-leading to the release of danger-associated molecular patterns (DAMPs), dendritic cell activation, and improved antigen presentation, which together stimulate a potent anti-tumor immune response. The study also demonstrates the enhanced therapeutic potential of combining natural products with immune checkpoint inhibitors. With a focus on translating preclinical findings into clinical practice, this review consolidates recent discoveries and suggests future research paths, offering both theoretical insights and practical guidance for advancing cancer immunotherapy.

Mitophagy in Cell Death Regulation: Insights into Mechanisms and Disease Implications.

Mitophagy, a selective form of autophagy, plays a crucial role in maintaining optimal mitochondrial populations, normal function, and intracellular homeostasis by monitoring and removing damaged or excess mitochondria. Furthermore, mitophagy promotes mitochondrial degradation via the lysosomal pathway, and not only eliminates damaged mitochondria but also regulates programmed cell death-associated genes, thus preventing cell death. The interaction between mitophagy and various forms of cell death has recently gained increasing attention in relation to the pathogenesis of clinical diseases, such as cancers and osteoarthritis, neurodegenerative, cardiovascular, and renal diseases. However, despite the abundant literature on this subject, there is a lack of understanding regarding the interaction between mitophagy and cell death. In this review, we discuss the main pathways of mitophagy, those related to cell death mechanisms (including apoptosis, ferroptosis, and pyroptosis), and the relationship between mitophagy and cell death uncovered in recent years. Our study offers potential directions for therapeutic intervention and disease diagnosis, and contributes to understanding the molecular mechanism of mitophagy.

Exploring the diagnostic and immune infiltration roles of disulfidptosis related genes in pulmonary hypertension

BackgroundPulmonary hypertension (PH) is marked by elevated pulmonary artery pressures due to various causes, impacting right heart function and survival. Disulfidptosis, a newly recognized cell death mechanism, may play a role in PH, but its associated genes (DiGs) are not well understood in this context. This study aims to define the diagnostic relevance of DiGs in PH.MethodsUsing GSE11726 data, we analyzed DiGs and their immune characteristics to identify core genes influencing PH progression. Various machine learning models, including RF, SVM, GLM, and XGB, were compared to determine the most effective diagnostic model. Validation used datasets GSE57345 and GSE48166. Additionally, a CeRNA network was established, and a hypoxia-induced PH rat model was used for experimental validation with Western blot analysis.Results12 DiGs significantly associated with PH were identified. The XGB model excelled in diagnostic accuracy (AUC = 0.958), identifying core genes DSTN, NDUFS1, RPN1, TLN1, and MYH10. Validation datasets confirmed the model’s effectiveness. A CeRNA network involving these genes, 40 miRNAs, and 115 lncRNAs was constructed. Drug prediction suggested therapeutic potential for folic acid, supported by strong molecular docking results. Experimental validation in a rat model aligned with these findings.ConclusionWe uncovered the distinct expression patterns of DiGs in PH, identified core genes utilizing an XGB machine-learning model, and established a CeRNA network. Drugs targeting the core genes were predicted and subjected to molecular docking. Experimental validation was also conducted for these core genes.

Ferroptosis and pathogenesis of neuritic plaques in Alzheimer's Disease.

Neuritic plaques are pathognomonic and terminal lesions of Alzheimer's Disease (AD). They embody AD pathogenesis because they harbor in one space critical pathologic features of the disease: amyloid deposits, neurofibrillary degeneration (NFD), neuroinflammation, iron accumulation. Neuritic plaques are thought to arise from the conversion of diffuse extracellular deposits of amyloid beta protein (Aβ), and it is believed that during conversion amyloid toxicity creates the dystrophic neurites of neuritic plaques, as well as neurofibrillary tangles (NFTs). However, recent evidence from human post-mortem studies suggests a much different mechanism of neuritic plaque formation where the first step in their creation is neuronal degeneration driven by iron overload and ferroptosis. Similarly, NFTs represent corpses of iron-laden neurons that develop independent of Aβ deposits. In this review, we will focus on the role of free redox-active iron in the development of typical AD pathology, as determined largely by evidence obtained in human temporal lobe during early, preclinical stages of AD. The findings have allowed construction of a scheme of AD pathogenesis where brain iron is center stage and is involved in every step of the sequence of events that produce characteristic AD pathology. We will discuss how the study of preclinical AD has produced a fresh and revised assessment of AD pathogenesis that may be important for reconsidering current therapeutic efforts and guiding future ones. Significance Statement This review offers a novel perspective on AD pathogenesis where elevated brain iron plays a central role and is involved throughout the development of lesions. We review arguments against the amyloid cascade theory and explain how recent findings in humans during early preclinical disease support iron-mediated cell death and endogenous iron containment mechanisms as critical components of neuritic plaque formation and the ensuing dementia.

7888 Mitotane Induces Different Modes Of Cell Death In Treatment Resistant And Sensitive Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. C. Hong: None. C. Hantel: None. W.E. Rainey: None. M.C. Dennedy: None. Adrenocortical carcinoma (ACC) is a rare aggressive cancer with poor survival. Adjuvant mitotane is the only approved drug for treatment of ACC. It improves survival but its use is limited by poor tolerability and drug resistance. For metastatic ACC, combination chemotherapy, using mitotane and etoposide, doxorubicin, and cisplatin improves survival, but efficacy is limited. Better understating the cytotoxic mechanisms of mitotane offers potential to develop improved therapeutic options for ACC. To investigate the underlying mechanisms in vitro, human ACC cells (HAC15 and H295R), in monolayer cell culture were treated with increasing concentrations of mitotane. Cell death was evaluated at 6 and 24 hours using flow cytometry analysis of Annexin V and Sytox Blue. This was complemented by Incucyte ® Live-Cell Analysis System imaging of annexin V and Sytox Blue staining. Expression of the markers of apoptosis, cleaved caspase 3, and necroptosis, phosphorylated MLKL and RIPK1 was measured at 6 and 24 hours. Mitotane significantly reduced the viability of H295R cells at 6 and 24 hours at the therapeutic concentration of 50uM (p&amp;gt;0.0001), thereby demonstrating treatment-sensitivity. In HAC15 cells, a supratherapeutic dose of 200uM was needed to significantly reduce viability (p&amp;gt;0.05) at 24 hours, indicating resistance to mitotane treatment at therapeutic concentrations. There was increasing Annexin V fluorescence with increasing mitotane concentration in H295R and HAC15 cells ar 6 and 24 hours without higher coinciding staining with Sytox Blue. Video analysis demonstrated these findings. Cleaved caspase 3 expression was associated with mitotane-induced cell death in H295R cells at 6 and 24 hours, indicating apoptosis. HAC15 cells demonstrated cleaved caspase 3 expression only at the highest concentration of mitotane exposure (600uM) at 6 hours, without appreciable expression at 24 hours. Phosphorylated-MLKL and RIPK1 expression was seen in controls and all mitotane doses in both cell lines at both timepoints, indicating necroptosis. In this study, H295R cells, representing a treatment-sensitive model of Mitotane undergo both apoptotic and necroptotic cell death at 6 and 24 hours of mitotane exposure. In contrast, HAC15 cells, demonstrated relative treatment resistance to mitotane, and undergo necroptotic cell death only. These results highlight the importance of understanding and targeting non-apoptotic and necroptotic cell death pathways, particularly when evaluating mechanisms of treatment resistance in ACC. Given the difference in the mechanism of cell death in treatment-sensitive and treatment-resistant cells, further exploration of necroptosis is necessary to better understand how ACC cells may manifest treatment resistance. Presentation: 6/1/2024

9316 Modulation Of Intracellular Lipid Access By ATGL Determines Ferroptosis Sensitivity In Castration-resistant Prostate Cancer

Abstract Disclosure: P. Cao: None. D. Awad: None. J. Han: None. D.E. Frigo: Grant Recipient; Self; GTx, Inc.. Other; Self; Familial relationship with Biocity Biopharmaceuticals, Hummingbird Bioscience, Bellicum Pharmaceuticals, Maia Biotechnology, Alms Therapeutics, Hinova Pharmaceuticals and Barricade Therapeutics. Ferroptosis induction has recently been recognized as a novel therapeutic approach to alter the outcomes of drug-resistant cancer. Ferroptosis is cell death that is caused by the intracellular accumulation of toxic lipid hydroperoxides. Due to their elevated metabolism, castration-resistant prostate cancer (CRPCs) are highly prone to ferroptosis; yet, many of them manage to avert this cellular fate. We have identified a key enzyme involved in lipid metabolism, adipose triglyceride lipase (ATGL), that engenders CRPCs with the ability to resist ferroptosis. Our data has shown that ATGL deletion by CRISPR-Cas9 knockout method sensitized human CRPC cell lines such as C4-2, C4-2BLT, and PC3 cells to the ferroptosis inducer RSL3. In all cases, RSL3-mediated cell death could be blocked by the antioxidant Ferrostatin-1, confirming ferroptosis as the mechanism of cell death. Knocking out ATGL also augmented the ferroptosis inducer-mediated increase in lipid peroxides as indicated by the increased ratio of oxidized to non-oxidized C11-BODIPY 581/591. With regards to the mechanism for the sensitivity of ATGL KO to ferroptosis induction, previous research has established that the enzyme long-chain acyl-CoA synthetase 4 (ACSL4) is universally essential to facilitate ferroptosis. More specifically, ATGL(PNPLA2) was found to inversely correlate with ACSL4 in the Taylor et al., the TCGA PanCancer Atlas, and Metastatic Prostate Adenocarcinoma (SU2C/PCF Dream Team) datasets in both primary and metastatic tumors. qPCR and western blot analysis of ACSL4 levels further showed that ACSL4 expression is closely tied to the lipase activity of ATGL. Addition of arachidonic acid significantly induced ferroptosis when combined with RSL3 in a dose-dependent manner only in ATGL KO cells. This indicates that PUFAs can render ATGL KO cells even more sensitive to RSL3-induced ferroptosis while cells with functional ATGL remain highly resistant, further reinforcing that ATGL is an essential safeguard of CRPC against ferroptosis. ATGL KO cells also demonstrated sensitivity to the antiandrogen Enzalutamide. The combination of Enzalutamide and RSL3 substantially decreased cell viability in ATGL KO cells as compared to control cells, indicating a strong synergy effect particularly seen in ATGL KO cells. In summary, this work demonstrates that ATGL is an important and novel regulator of ferroptosis in CRPC and supports the use of ferroptosis inducer as a new therapeutic approach in CRPC. Presentation: 6/2/2024

Ruthenium complexes with abiraterone acetate as antiproliferative agents

This study is dedicated to the development of multimodal anticancer agents. We have obtained ruthenium complexes conjugated with the steroid-type antitumor drug abiraterone acetate in order to take advantage of the dual antitumor properties of both ruthenium and abiraterone. The compounds exhibit good antiproliferative activity against cancer cells, with selectivity over primary fibroblasts. Real-time cell analysis revealed that compound dichlorido(η66-p-cymene)(abiraterone acetate)ruthenium(II) had pronounced antiproliferation activity compared to abiraterone acetate. Flow cytometric studies on the mechanism of cell death have revealed that the most active compound induces apoptosis more effectively than abiraterone acetate. Our findings demonstrate the potential of this novel dual-action compound as promising candidates for further development as anticancer agents.