Oxidative Damage Research Articles

Abstract 7063: Monoclonal antibody evaluation of oxidative DNA base damage in normal and neoplastic breast tissues: development of antibodies specific for 8-OH-adenine and 4,6-diamino-5-formamidopyrimidine (FAPY-adenine)

Abstract Breast cancer affects 1 in 8 women and often results from mutations caused by oxidative DNA damage from reactive oxygen species. Two key lesions, 8-hydroxy-adenine (8-OH-Ade) and 4, 6-diamino-5-formamidopyrimidine (FAPY-Ade), exhibit two distinct patterns: FAPY-Ade lesions are predominant in normal tissues, while 8-OH-Ade lesions are abundant in cancerous tissues. These shifts reflect tissue redox status, which may provide a pathway to assess future breast cancer risk. Quantitation of these lesions by chemical detection methods is precise but time-intensive and impractical for high-throughput clinical applications. To address this, monoclonal antibodies have been developed and offer a rapid, scalable, and clinically practical alternative for efficiently detecting and quantifying these biomarkers. This innovation can potentially support personalized breast cancer screening strategies. BALB/c mice were immunized with 8-OH-Ade or FAPY-Ade haptens coupled to Keyhole Limpet Hemocyanin (KLH). After three monthly immunizations, the serums of IgG and IgM titers were evaluated. The mice having the highest IgG titer for each antigen were given an intravenous boost, and the spleens were removed three days later for fusions according to standard methods. Using hapten-conjugated BSA-coated 96-well Pro-Bind plates, the ELISA results demonstrated 30-40 positive wells for each antigen. Up to ten positive wells for each antigen were selected and cloned to yield monoclonal antibody-producing hybridomas. The ELISA of the anti-FAPY-Ade antibody, FA5, showed concentration-dependent reduced binding to immobilized BSA-FAPY-Ade hapten conjugate in increasing FAPY-Ade, confirming its FAPY-Ade binding specificity. Immunohistochemistry using normal and cancerous breast tissue revealed significant staining of normal breast tissue compared to cancerous breast tissue using the FA5 antibody. Conversely, the anti-8-OH-Ade antibody, 8A6, displayed weak staining of normal breast tissue with higher staining of cancerous breast tissues. The results demonstrate that monoclonal antibodies specific for 8-OH-Ade and a novel antibody specific for the ring-opening reactive oxygen product, FAPY-Ade, are practical and rapid alternatives to chemical detection methods for their analysis. Immunohistochemistry mirrored past results using chemical detection methods for these oxidized bases, with high FAPY-Ade compared to 8-OH-Ade in normal tissues and the opposite result in cancer-derived tissues. The potential for high-throughput analysis of 8-OH-Ade and FAPY-Ade levels in the DNA of breast tissues may provide information on future breast cancer risk. Further research is underway to evaluate this hypothesis. Citation Format: Zenzeale A. Hudson, Sarah McLaughlin, Alfredo A. Paredes, Gary K. Ostrander, Eric H. Holmes. Monoclonal antibody evaluation of oxidative DNA base damage in normal and neoplastic breast tissues: development of antibodies specific for 8-OH-adenine and 4, 6-diamino-5-formamidopyrimidine (FAPY-adenine) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7063.

Abstract 5402: Metabolic shift towards the de novo serine pathway in non-transformed breast cells drives epigenetic plasticity, oxidative DNA damage, and pro-tumorigenic cChanges associated with aging

Abstract Introduction: A lipid metabolism gene signature is enriched in breast tissue at risk for estrogen receptor negative (ERneg) breast cancer (BC). Fatty acid (FA) exposure alters histone methylation, gene expression and increases metabolic flux through serine, one-carbon, glycine (SOG) and methionine pathways. We hypothesize that FA exposure induces a metabolic shift towards the SOG, increasing S-adenosylmethionine (SAM), altering histone methylation, gene expression, and promoting ERneg BC. Methods: Proteomics, metabolomics, Reactive Oxygen Species (ROS) measurement, comet assay and H3K4me3 CUT&RUN were performed in MCF-10A cells exposed to octanoic acid (OA). Single-cell RNA-seq (scRNAseq) was performed in breast tissue derived microstructures exposed to OA. Intracellular communication was analyzed using CellChat, and metabolic flux with Compass. Results: OA increased SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG); blocking the serine pathway (SSP) prevented these increases. ScRNAseq revealed that OA increased expression of the SSP transcription factor ATF3 and genes PHGDH and PSAT1 in epithelial and stromal compartments. Metabolic flux analysis revealed a significant increase in flux through SSP in Basal BSL1, Luminal Progenitor LP3, and Hormone Sensing HS1 cells after OA exposure. Differential proteomics reveals PHGDH overexpression and downregulation of proteins involved in extracellular matrix (ECM)-receptor interaction and focal adhesion post-OA exposure, along with significant increase in mitochondrial and nuclear ROS (p < 0.01). OA exposure also induced DNA damage, likely due to elevated nuclear ROS. OA increased GSH metabolism and ROS detoxification in BSL1. CUT&RUN identified 661 peaks significantly enriched upon OA (FDR < 0.01) in regulatory regions of OA-induced genes involved in neural pathways and BC, including MDK, NGF, and NGFR. CellChat predicted a decrease in ECM-cell interactions, a reduction in cell-cell adhesions, and an increase of secreted signaling upon OA exposure. The strongest secreted signals in OA were AREG (linked to proliferation, growth, and invasiveness), GDF15 (involved in EMT, invasion, and aging), and MDK (linked to neurogenesis, and aging). Conclusions: We demonstrate an FA-induced shift towards the SOG and methionine pathways that promotes epigenetic plasticity, regulates ROS, and supports the survival of cells with 'inappropriate' phenotypes. These accumulate DNA damage, leading to age-related changes in the mammary gland (elevated ROS, disrupted junctions, altered ECM interactions, and increased MDK/GDF15 expression), all supporting carcinogenesis. Our findings also provide a metabolic explanation for the elevation of PHGDH in 70% of ERneg BCs, despite gene amplification in only 6%, and point to preventive strategies targeting the SSP. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Curtis McCloskey, Flavio Palma, Shiyu Liu, Maria Paula Zappia, Abul B.M.M.K. Islam, Elizaveta Benevolenskaya, Maxim Frolov, Jason Locasale, Marcelo Bonini, Rama Khokha, Navdeep Chandel, Seema A. Khan, Susan E. Clare. Metabolic shift towards the de novo serine pathway in non-transformed breast cells drives epigenetic plasticity, oxidative DNA damage, and pro-tumorigenic cChanges associated with aging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5402.

Abstract 1509: Discovery of choroid plexus targeted gene therapy to protect cancer chemotherapy-related cognitive impairment

Abstract Chemotherapy-induced neurotoxicity in non-cancerous cells can cause lifelong complications including cognitive impairment, also known as chemobrain, which is a devastating side effect for cancer patients and survivors. One mechanism by which chemotherapy damages healthy cells is through oxidative stress. The choroid plexus (ChP)-cerebrospinal fluid (CSF) system delivers important health-promoting factors, such as the antioxidant superoxide dismutase 3 (SOD3), to increase survival in response to oxidative stress, while cell-intrinsic pathways protect neurons from oxidative damage. However, the consequences of chemotherapy on ChP and CSF composition are unknown. Here, we show that the chemotherapeutic drug methotrexate (MTX) induces a global metabolic change in the brain, which is reflected in the CSF. Leveraging metabolomics, we found that MTX caused oxidative stress in the CSF of MTX-treated mice, including a reduction in important antioxidant metabolites. The ChP is the main source of secreted SOD3 in the brain. Following MTX treatment in mice, ChP-SOD3 expression was diminished as well as CSF-SOD3 was decreased. Moreover, overall CSF-antioxidant activity was compromised, indicating that cancer chemotherapy damaged the ChP, resulting in an impoverished CSF with reduced antioxidant capability. MTX-induced oxidative stress may also be tracked to tissues in contact with CSF that are functionally vulnerable to the toxicity of cancer chemotherapy. For example, glutathione peroxidase (GPx) enzyme activity was decreased in the hippocampus, where lipid peroxidation was elevated following MTX treatment. Analyses of CSF samples from primary and secondary central nervous system (CNS) lymphoma patients supported our hypothesis that MTX treatment is associated with diminished antioxidant levels in patient CSF. We employed an intraventricular adeno-associated viral (AAV) gene delivery technique to augment SOD3 production by the ChP. ChP-SOD3 gene therapy inhibited oxidative stress indicators in CSF and mitigated MTX-induced toxicity in the adjacent hippocampus. Furthermore, this gene therapy prevented MTX-induced anxiety as well as short-term learning and memory deficits. Collectively, our findings on AAV-based gene therapy via the ChP-CSF system may represent an underutilized approach for widespread and long-term delivery of health-promoting factors to the brain. Citation Format: Ahram Jang, Boryana Petrova, Taek-Chin Cheong, Miriam E. Zawadzki, Jill K. Jones, Andrew J. Culhane, Frederick B. Shipley, Roberto Chiarle, Eric T. Wong, Naama Kanarek, Maria K. Lehtinen. Discovery of choroid plexus targeted gene therapy to protect cancer chemotherapy-related cognitive impairment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1509.

NADH:ubiquinone oxidoreductase core subunit S8 expression and functional significance in non-small cell lung cancer

Abstract Hyperfunctional mitochondria provide a growth advantage by supporting the energy-intensive processes essential for non-small cell lung cancer (NSCLC). NADH:ubiquinone oxidoreductase core subunit S8 (NDUFS8) is a key subunit of mitochondrial complex I involved in oxidative phosphorylation (OXPHOS) and cellular energy production. Bioinformatics and local tissue examinations show that NDUFS8 expression is elevated in NSCLC compared to normal lung tissue. Both immortalized and primary human NSCLC cells exhibit higher NDUFS8 levels. Single-cell RNA sequencing confirmed NDUFS8 upregulation in cancerous cells of NSCLC tumor. Silencing NDUFS8 via shRNA or Cas9/sgRNA-mediated knockout (KO) disrupted mitochondrial functions, leading to decreased complex I activity, ATP depletion, mitochondrial depolarization, increased reactive oxygen species (ROS) production, and heightened lipid peroxidation. Furthermore, NDUFS8 silencing/KO triggered apoptosis and significantly reduced Akt-mTOR activation, cell viability, proliferation, and motility in various NSCLC cells. In contrast, ectopic overexpression of NDUFS8 boosted mitochondrial complex I activity and ATP levels, promoting Akt-mTOR activation, and enhancing NSCLC cell proliferation and motility. NDUFS8 also contributes to radioresistance in NSCLC; silencing or KO enhanced ionizing radiation (IR)-induced cytotoxicity, while overexpression mitigated it. Intratumoral injection of NDUFS8 shRNA-expressing adeno-associated virus significantly inhibited growth of primary NSCLC xenografts in nude mice, with observed NDUFS8 silencing, ATP reduction, oxidative damage, proliferation inhibition, Akt-mTOR inactivation and apoptosis in treated tissues. These findings highlight the pivotal pro-tumorigenic role of NDUFS8 in NSCLC.

Enhanced Renal Protection in Acute Kidney Injury with ROS-Activated Nanoparticles Targeting Oxidative Stress and Inflammation.

Acute kidney injury (AKI) is often associated with oxidative stress, which leads to a range of pathological changes, including inflammation and cell apoptosis. These mechanisms highlight the crucial role of eliminating ROS in the pathogenesis of AKI. This study presented a ROS-activated drug delivery system, NPSPBA@Hib, designed for the targeted delivery of the anti-inflammatory and antioxidant drug hibifolin (Hib) to the kidneys, marking its inaugural application in AKI therapy. The drug loading of Hib was up to be 15% by conversely binding with the phenylboronic acid parts in the nanoparticles. NPSPBA@Hib increased cellular uptake of drugs in HK-2 cells and reduced oxidative stress-induced damage by scavenging ROS. The nanoparticles notably extended the retention of Hib in AKI kidneys when compared to healthy kidneys, leading to heightened accumulation in the renal tubules. NPSPBA@Hib demonstrated Hib's reno-protective effects by reducing oxidative stress and inflammation. In essence, this research serves as the primary confirmation of Hib's efficacy in inhibiting NLRP3 signaling pathway for the AKI treatment. The findings suggest that NPSPBA@Hib nanoparticles are effective in treating AKI, highlighting the promising potential of utilizing Hib as a natural antioxidant nanoplatform for AKI, as well as other ROS-related diseases.

Research progress on cuproptosis and copper related anti-tumor therapy.

Copper is a trace element which is essential for biological organisms, and its homeostatic balance is important for living organisms to maintain the normal function. When the copper homeostasis is disordered, the cellular function and structure will be disrupted. Excess copper cause oxidative stress and DNA damage in cells, thereby inducing regulated cell death such as apoptosis and necroptosis. Excess copper in mitochondria can bind to lipoylated proteins in the tricarboxylic acid (TCA) cycle and cause them to aggregate, resulting in proteotoxic stress and eliciting a novel cell death modality: cuproptosis. Cancer cells have a greater demand for copper compared to normal tissue, and high levels of copper ions are closely associated with tumour proliferation and metastasis. The anti-tumor mechanisms of copper include the production of oxidative stress, inhibition of the ubiquitin-proteasome system, suppression of angiogenesis, and induction of copper-dependent cell death. Targeting copper is one of the current directions in oncology research, including the use of copper ion carriers to increase intracellular copper levels to induce oxidative stress and cuproptosis, as well as the use of copper ion chelators to reduce copper bioavailability. However, copper complexes have certain toxicity, so their biosafety needs to be improved. Emerging nanotechnology is expected to solve this problem by utilizing copper-based nanomaterials (Cu-based NMs) to deliver copper ions and a variety of drugs with different functions, thereby improving the anti-tumor efficacy and reducing the side effects. Therefore, a thorough understanding of copper metabolic processes and the mechanism of cuproptosis will greatly benefit anti-tumor therapy. This review summarizes the processes of copper metabolism and the mechanism of cuproptosis. In addition, we discuss the current anti-tumor paradigms related to copper, we also discuss current nanotherapeutic approaches to copper mortality and provide prospective insights into the future copper-mediated cancer therapy.

Abstract 2824: Physiologic regulation of lipid oxidation and ferroptosis by vitamin E, high-density lipoprotein and scavenger receptor class B type 1

Abstract Introduction: Ferroptosis is a form of cell death caused by oxidative (-OOH) damage to phospholipids (PL) in the cell membrane. Understanding mechanisms of ferroptosis is important because of the role it plays in cancer. Previous work has focused on the intracellular antioxidant enzyme glutathione peroxidase 4 (GPx4), which detoxifies PL-peroxides (PL-OOH) and prevents ferroptosis. Studies show that alpha-tocopherol (α-toc), the active form of Vitamin E, is an exogenous lipophilic antioxidant that inhibits ferroptosis in cancer cells in vitro. The mechanisms by which α-toc engages cells to manipulate PL redox balance and ferroptosis are unknown. Lipoproteins, like high- and low-density lipoproteins (HDL and LDL), carry α-toc in the circulation. Using diffuse large B cell lymphoma (DLBCL) and clear cell renal cell carcinoma (ccRCC) models, two malignancies sensitive to ferroptosis, we show that lipoproteins, particularly HDL, deliver α-toc by binding the cell membrane receptor scavenger receptor class B type 1 (SR-B1) to prevent ferroptosis. Methods: Ramos (lymphoma), SUDHL4 (lymphoma) and 786-O (ccRCC) cell lines were used in these experiments. Ferroptosis was induced using ML210 and erastin. α-toc loading of native HDL and LDL was increased by incubation with free α-toc and spin filter purification. To isolate α-toc as the component responsible for modulating ferroptosis, we developed synthetic α-toc-loaded HDLs (sHDL). siRNAs were used to reduce SR-B1 and LDL Receptor (LDLR) expression. Blocker of lipid transport-1 (BLT-1) was used to inhibit uptake of lipoprotein cargo via SR-B1. Cell viability was measured by MTS assay and lipid peroxidation was measured by C11-BODIPY. Results: The α-toc level was higher in LDLs compared to HDLs (6.0 ± 0.1 α-toc/ apoB-100 vs. 1.0 ± 0.1 α-toc/ apoA-I) and increased in both HDL and LDL following incubation with free α-toc (α-toc LDL, 10.1 ± 0.1; α-toc HDL, 1.6 ± 0.3). HDL, α-toc HDL and α-toc LDL, but not LDL, rescued DLBCL and ccRCC cells from ferroptosis induced by the addition of ML210 and erastin, with a corresponding decrease in PL-OOH accumulation. Similarly, α-toc sHDLs, but not empty sHDLs, prevented ferroptosis in DLBCL and ccRCC. Knockdown of SR-B1 resulted in down-regulation of GPx4 expression and ferroptosis in ccRCC cells, requiring the use of BLT-1 to investigate SR-B1’s role in α-toc delivery. Addition of BLT-1 prevented α-toc lipoprotein rescue from ferroptosis. Knockdown of LDLR expression did not alter the ability of HDL and α-toc HDL to prevent ferroptosis and enhanced the ability of α-toc LDL to prevent ferroptosis. Conclusion: We reveal a tunable cellular redox axis whereby HDLs containing α-toc target SR-B1 to reduce PL-OOH and prevent ferroptosis. Accordingly, this work reveals that the α-toc / HDL/ SR-B1 axis of ferroptosis prevention must be accounted for when targeting ferroptosis in vivo. Citation Format: Jonathan S. Rink, Andrea E. Calvert, Adam Y. Lin, SonBihn T. Nguyen, Leo I. Gordon, C Shad Thaxton. Physiologic regulation of lipid oxidation and ferroptosis by vitamin E, high-density lipoprotein and scavenger receptor class B type 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2824.

Abstract 4198: Unraveling the molecular interactions between DDR and autophagy in response to oxidative stress

Abstract The DNA damage response (DDR) is essential for maintaining genomic integrity by detecting and repairing DNA damage. In conjunction with autophagy, DDR functions as a critical protective mechanism under oxidative stress, mitigating the risk of cancer development. Our study investigates the link between oxidative stress and DDR, particularly focusing on ATM kinase's role in reactive oxygen species (ROS)-mediated genotoxic stress for double-strand break (DSB) repair. Cancer cells' heightened reliance on DDR, due to high proliferation and genomic instability, underscores their susceptibility to DDR inhibitors. However, the effectiveness of DDR inhibitors is often limited by the evolution of resistance in tumors. Resistance mechanisms can include the restoration of DNA repair pathways or activation of compensatory pathways. This adaptability limits the long-term efficacy of DDR-targeted therapies, emphasizing the need to understand the molecular drivers underlying resistance to enable the development of more effective strategies. Our study focuses on the potential interaction between Mediator of DNA damage checkpoint 1 (MDC1), a key DDR protein, and Beclin-1, a vital autophagy inducer. We have employed a comprehensive approach using the HeLa cell line, including wild type, MDC1 knockdown (KD), and MDC1 overexpressing (MDC1-Ov) variants. Oxidative stress was induced using hydrogen peroxide (H2O2), and we conducted co-immunoprecipitation, immunofluorescence, and immunoblotting to investigate the interactions between MDC1 and Beclin-1 under these conditions. Our results demonstrate that MDC1 interacts with Beclin-1, with the interaction enhanced over time, indicating its essential role in cellular responses to oxidative stress. We also show that oxidative stress prompts the relocation of Beclin-1 from the cytoplasm to the nucleus, suggesting that Beclin-1 may be involved in regulating DDR in response to oxidative stress-induced DNA damage. Given the central role of MDC1 in recruiting various effector proteins at the site of DNA repair, we hypothesized that MDC1 may facilitate the nuclear translocation of Beclin-1. Consistent with this hypothesis, we found that nuclear localization of Beclin-1 was significantly reduced in MDC1 knockdown cells compared to wild type when treated with H2O2. These findings underscore the critical role of MDC1 in modulating the nuclear localization of Beclin-1 in response to oxidative stress. The significance of our study lies in its potential implications for chemotherapy. Given the prevalence of oxidative stress in various cancers and the therapeutic reliance on chemotherapy-induced oxidative stress, understanding this novel interplay between DDR and autophagy pathways may provide insights for developing constructive therapeutic strategies. Citation Format: Kavya Ajit Pandya, Neeru Singh. Unraveling the molecular interactions between DDR and autophagy in response to oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4198.

Abstract 2558: Ferroptosis modulates melanoma progression in an age-specific manner through changes in the tumor microenvironment

Abstract Aging is an independent prognostic factor for melanoma, the most aggressive form of skin cancer. Although immune therapy has shown a dramatic improvement in melanoma treatment, resistance to it still occurs in some patients. Higher oxidative damage in tumor cells and lipid and iron accumulation are singular characteristics of the aged tumor microenvironment (TME). Here, we investigate the impact of these characteristics of the aged TME in promoting ferroptosis, a caspase-independent iron-mediated type of cell death, and as a possible therapeutic opportunity for older patients. Lipid peroxidation levels, resistance to ferroptosis inducers, and the impact of the aged TME secretome were assessed through in vitro and in vivo modeling. Initially, correlation analysis between transcriptomic data (TCGA-SKCM) and ferroptosis sensitivity (CTD^2) was performed to identify common denominators among transcripts associated with the aged TME and ferroptosis pathway. Phenotype-switching markers, such as AXL and Wnt5a, showed a strong correlation with several ferroptosis-related transcripts and an inverse correlation with ferroptosis resistance. Using a panel of melanoma cell lines with distinct Wnt5a levels, we confirmed this association and observed that three critical regulators of lipid peroxidation are downregulated in Wnt5a high lines: GPX4, AIFM2, and GCH1. The comparison through IHC of young and aged mouse skin samples highlighted a pro-ferroptosis signature increasing with age. Senescence markers such as p16 and p21 could be modulated in dermal fibroblasts through induction or inhibition of lipid peroxidation. When associating conditioned media (CM) derived from these dermal fibroblasts (capable of inducing phenotype switching), RSL-3, a GPX4 inhibitor, induced a higher lipid peroxidation and cell death in melanoma cells exposed to aged CM. To understand how ferroptosis impacts tumor growth in young and aged mice, we used a syngeneic tumor model injecting Yumm1.7 cells intra-dermally and the treatment with a lipid peroxidation inducer (imidazole ketone erastin, IKE) or an inhibitor (Liproxstatin-1). Tumors from aged mice showed stronger staining with 4-HNE antibody when compared to young, correlating with AXL and Wnt5a staining. Interestingly, both IKE and Liproxstatin-1 lead to age-specific reduced tumor growth, followed by changes in immune cell populations such as monocytes, macrophages and NK cells. We are currently exploring how these treatments impacted metastatic dissemination, stromal architecture and sensitivity in BRAF/MEK-resistant tumors. These results indicate that ferroptosis modulation is a promising avenue for combined treatment with existing clinical strategies. Citation Format: Murilo Ramos Rocha, Yash Chhabra, Alexis E. Carey, Cheyenne Palm, Kevin Zhang, Edwin Kumah, Vania Wang, Laura Huser, Elizabeth I. Harper, Fan Huang, Ashani Weeraratna. Ferroptosis modulates melanoma progression in an age-specific manner through changes in the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2558.

Abstract 4694: (R)evolutionary approach to predicting individual response to radiation therapy in breast cancer

Abstract Despite increasing incorporation of gene panel testing into radiotherapeutic decision-making, there is no established method to tailor prescribed radiation regimens to individual tumor radiosensitivity. We propose a novel approach to assessing evolutionary determinants of radioresistance from patient-derived tumor samples. We hypothesized that pre-existing resistance to oxidative damage in tumor microenvironment (TME) can define a cell's evolutionary trajectory and its radiosensitivity. In contrast with the current genetic panels, we use a panel catered toward heterogeneity within the tumor and the TME niches, which may harbor radioresistant populations from collateral TME mechanisms of resistance. The aim is to develop an AI-based algorithm using these eco-evolutionary results to individualize radiation dose in patients, thus minimizing the percentage of non-responders. 3D spheroids of several breast cancer cell lines were grown and collected at multiple time points after 10 Gy of radiation. MALDI data was acquired in negative and positive ionization mode for spatial lipidomics and metabolomics analysis. Additionally, spatial single cell multiplexed immunofluorescence was performed on the sequential cut to MALDI using markers associated with hypoxia, acidosis, ROS, and epithelial-to-mesenchymal transition. Then we grew patient derived organoids (PDO) of breast cancer and irradiated them and analyzed them as described above. Based on a previously validated oxygen diffusion model, spheroids were categorized according to size, with those <240 micrometers in diameter designated as O-spheroids (oxygenated) and those ≥ 240 microns designated as H-spheroids (hypoxic core). While the irradiated O-spheroid growth curves plateaued upon irradiation, H-spheroids grew at the same rate as the non-irradiated controls. To investigate this phenomenon, habitats and cell phenotypes were defined using spatial multi-omics data from MALDI and multiplexed-IF, respectively. We found differential distributions of habitats in radiated spheroids as determined by the cluster size and abundance spatio-temporally. We discovered a switch in the sphingolipid metabolism pathway. Using principles of ecology and evolution, we have developed a novel approach to identifying radiosensitivity markers. Detecting these cell states and ecological niches in patients’ pre-treatment biopsies can inform a more individualized approach to radiotherapy. Citation Format: Naheel Khatri, Raafat Chalar, Saiful Samad, Alexander Stessin, Mehdi Damaghi. (R)evolutionary approach to predicting individual response to radiation therapy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4694.

Unveiling the impact of ferroptosis on diabetes-associated cognitive decline through comprehensive single-cell RNA sequencing and experimental studies.

Diabetes-associated cognitive decline (DACD) is defined as an impairment of cognitive functions, including memory, attention and executive functions, attributed to chronic hyperglycemia and metabolic dysregulation associated with type 2 diabetes mellitus (T2DM). Ferroptosis is a regulated form of cell death that is dependent on iron and is primarily characterized by the excessive accumulation of lipid peroxides within cellular membranes, and also plays a critical role by exacerbating neuronal loss and synaptic dysfunction. The present study aims to use single-cell RNA sequencing (scRNA-seq) technology to investigate the role of ferroptosis in microglia and oligodendrocytes in DACD, thereby elucidating the pathogenesis of DACD. scRNA-seq and bulk RNA-seq datasets were analyzed for differential gene expression in hippocampus samples of T2DM and control mice, with an emphasis on oligodendrocytes and microglia cell types. We further constructed a T2DM model in mice and conducted behavioral analyses to evaluate cognitive functions. Additionally, we explored the role of ferroptosis in the progression of DACD disease by knocking down transferrin receptor 1 (Tfr1) using small interfering RNA and utilizing the ferroptosis inhibitor ferrostatin-1. The study identified significant alterations in the expression of ferroptosis-related genes Fth1, Slc40a1, Slc3a2, Trf, Tfrc and Sat1 in T2DM mice, suggesting the possible involvement of ferroptosis in DACD. Knocking down Tfr1 and inhibiting ferroptosis could significantly alleviate inflammation and oxidative stress damage in oligodendrocytes. This research provides new perspectives into the pathophysiology of DACD, emphasizing the critical role of ferroptosis and offering a potential therapeutic target to mitigate neurological damage and cognitive impairment associated with T2DM.

Myricetin ameliorates the effects of hydrogen peroxide-induced oxidative stress in human mesenchymal stem cells: an ultrastructural and immunocytochemical study

ABSTRACT The development of new strategies to raise the survival and viability of transplanted mesenchymal stem cells (MSCs) is very important for the therapeutic potential of stem cells. The natural flavonoid myricetin has anticancer, antioxidant, anti-inflammatory and antiapoptotic effects. The effects of myricetin on human umbilical cord-derived MSCs (HUC-MSCs) induced oxidative stress with hydrogen peroxide (H2O2) were evaluated by transmission electron microscopy (TEM) and immunocytochemistry (ICC) staining. Myricetin showed an increase in the number of live cells, a decrease in caspase-3 and tumor necrosis factor-α (TNF-α) ICC staining intensity, an increase in the translocase of the mitochondrial inner membrane 17 (TIM17) ICC staining intensity, and a decrease in degeneration of cell ultrastructure in TEM against oxidative stress damage in HUC-MSCs. The results suggest that myricetin prevents oxidative stress-induced apoptosis and inflammation in the HUC-MSCs. Myricetin can be combined with HUC-MSCs in cell culture and considered as a supportive alternative treatment option.

Metabolomics-Based Analysis of Adaptive Mechanism of Eleutheronema tetradactylum to Low-Temperature Stress

Temperature is a critical environmental factor that influences the growth, development, metabolism, and overall physiological performance of fish. Eleutheronema tetradactylum is an economically significant fish species; however, its molecular mechanism’s response to long-term cold stress is still unclear. In this study, we investigated the physiological responses of the liver in E. tetradactylum exposed to a constant temperature of 18 °C for durations of both 7 and 14 days, utilizing liquid chromatography–mass spectrometry (LC-MS), metabolomics, and conventional biochemical assays. The antioxidant status, liver histology, and metabolite profiles were examined at different time points. Our results revealed that, following sustained cold exposure, the activities of key antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)—initially increased and then decreased. Additionally, levels of malondialdehyde (MDA), a marker of oxidative damage, significantly elevated after 7 and 14 days of cold stress. Histopathological examination of liver tissues showed varying degrees of vacuolation and nuclear atrophy in hepatocytes, indicating oxidative damage. Metabolomic profiling identified 87 and 116 differentially expressed metabolites in the liver on days 7 and 14, respectively. Pathway enrichment analysis revealed significant alterations in pathways related to carbohydrate digestion and absorption, glutathione metabolism, and glycerolipid metabolism. These findings suggest that mechanisms regulating cell membrane fluidity, energy metabolism, autophagy, and antioxidant defense are crucial for the adaptation of E. tetradactylum to cold stress. Overall, this study provides valuable insights into the molecular and physiological adaptations of E. tetradactylum to low temperature, highlighting the activation of protective antioxidant responses and modifications of metabolic pathways in the liver.

Impact of climate change on the toxicity of bisphenol A in Mytilus galloprovincialis and assessment of phycoremediation using Nannochloropsis salina via a multi-biomarker strategy and modeling.

Impact of climate change on the toxicity of bisphenol A in Mytilus galloprovincialis and assessment of phycoremediation using Nannochloropsis salina via a multi-biomarker strategy and modeling.

Structural characterization and protective activity of α-glucans derived from Cynomorium songaricum Rupr. against homocysteine-induced oxidative stress in MC3T3-E1 cells.

Structural characterization and protective activity of α-glucans derived from Cynomorium songaricum Rupr. against homocysteine-induced oxidative stress in MC3T3-E1 cells.

Antioxidant and anti-apoptotic properties of heme oxygenase-1 in red swamp crayfish Procambarus clarkii.

Antioxidant and anti-apoptotic properties of heme oxygenase-1 in red swamp crayfish Procambarus clarkii.

Neuroprotective Effect of a Novel Soluble Guanylate Cyclase Activator (sGCa) Runcaciguat in Diabetes and Ischemic Retinopathy.

Oxidative stress has a major pathogenic role in diabetic retinopathy, and neuroretina dysfunction is recognized as an early and important problem. Soluble guanylate cyclase (sGC) has been implicated for its neuroprotective effects in the central nervous system, but its role in the retina remains unclear. Here we demonstrated expression of sGC subunits GUCY1A1 and GUCY1B1 in healthy human and rodent retina in vascular cells and neuronal elements including retinal ganglion cells, bipolar, and amacrine cells. We provided evidence using in vitro and in vivo studies that sGC function is impaired by oxidative stress-induced damage in retina. The sGC activator runcaciguat activated sGC in multiple retinal cell types and counteracted the inhibitory effect of damage induced by oxidative stress on the retina and retinal cells. In the rat retinal ischemia-reperfusion model, runcaciguat treatment improved neuroretinal and visual function as measured by electroretinography and optokinetic tracking and resulted in retinal morphologic improvement. In the STZ-induced diabetic rat model, runcaciguat significantly improved neuroretinal function and improved inner plexiform layer thickness. These studies suggest that sGC signaling is involved in neuroretinal function and vision and that diabetes negatively affects this pathway, supporting restoring sGC activation as a novel therapy for early DR.

Paeoniflorin Protects Retinal Pigment Epithelial Cells from High Glucose-Induced Oxidative Damage by Activating Nrf2-Mediated HO-1 Signaling.

Oxidative stress due to hyperglycemia damages the functions of retinal pigment epithelial (RPE) cells and is a major risk factor for diabetic retinopathy (DR). Paeoniflorin is a monoterpenoid glycoside found in the roots of Paeonia lactiflora Pall and has been reported to have a variety of health benefits. However, the mechanisms underlying its therapeutic effects on high glucose (HG)-induced oxidative damage in RPE cells are not fully understood. In this study, we investigated the protective effect of paeoniflorin against HG-induced oxidative damage in cultured human RPE ARPE-19 cells, an in vitro model of hyperglycemia. Pretreatment with paeoniflorin markedly reduced HG-induced cytotoxicity and DNA damage. Paeoniflorin inhibited HG-induced apoptosis by suppressing activation of the caspase cascade, and this suppression was associated with the blockade of cytochrome c release to cytoplasm by maintaining mitochondrial membrane stability. In addition, paeoniflorin suppressed the HG-induced production of reactive oxygen species (ROS), increased the phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key redox regulator, and the expression of its downstream factor heme oxygenase-1 (HO-1). On the other hand, zinc protoporphyrin (ZnPP), an inhibitor of HO-1, abolished the protective effect of paeoniflorin against ROS production in HG-treated cells. Furthermore, ZnPP reversed the protective effects of paeoniflorin against HG-induced cellular damage and induced mitochondrial damage, DNA injury, and apoptosis in paeoniflorin-treated cells. These results suggest that paeoniflorin protects RPE cells from HG-mediated oxidative stress-induced cytotoxicity by activating Nrf2/HO-1 signaling and highlight the potential therapeutic use of paeoniflorin to improve the symptoms of DR.

Astragalus polysaccharide alleviates oxidative stress and senescence in chondrocytes in osteoarthritis via GCN2/ATF4/TXN axis.

Astragalus polysaccharide alleviates oxidative stress and senescence in chondrocytes in osteoarthritis via GCN2/ATF4/TXN axis.

Global DNA methylation and its association with genetic instability and exposure to inorganic elements and polycyclic aromatic hydrocarbons in coal mining dust.

Coal mining has significant economic and environmental implications. The extraction and combustion of coal release harmful chemicals and dust, impacting air, soil, and water quality, as well as natural habitats and human health. This study aimed to investigate the association between global DNA methylation, DNA damage biomarkers (including telomere length), and inorganic element concentrations in the blood of individuals exposed to coal mining dust. Additionally, polycyclic aromatic hydrocarbons (PAHs) were analyzed. The study included 150 individuals exposed to coal mining and 120 unexposed controls. Results showed significantly higher global DNA hypermethylation in the exposed group compared to controls. Moreover, in the exposed group, micronucleus frequency and age showed a significant correlation with global DNA hypermethylation. Blood levels of inorganic elements, including titanium, phosphorus, sodium, aluminum, iron, sulfur, copper, chromium, zinc, chlorine, calcium, and potassium, were potentially associated with DNA methylation and oxidative damage, as indicated by comet assay results. Furthermore, exposure to PAHs such as fluoranthene, naphthalene, and anthracene, emitted in mining particulate matter, may contribute to these effects. These findings highlight the complex interplay between genetic instability, global DNA hypermethylation, and environmental exposure in coal mining areas, emphasizing the urgent need for effective mitigation strategies.