Cell Death Pathways Research Articles

Oxidative Stress in Cataract Formation: Is There a Treatment Approach on the Horizon?

Cataracts, a leading cause of blindness worldwide, are closely linked to oxidative stress-induced damage to lens epithelial cells (LECs). Key factors contributing to cataract formation include aging, arterial hypertension, and diabetes mellitus. Given the high global prevalence of cataracts, the burden of cataract-related visual impairment is substantial, highlighting the need for pharmacological strategies to supplement surgical interventions. Understanding the molecular pathways involved in oxidative stress during cataract development may offer valuable insights for designing novel therapeutic approaches. This review explores the role of oxidative stress in cataract formation, focusing on critical mechanisms, such as mitochondrial dysfunction, endoplasmic reticulum stress, loss of gap junctions, and various cell death pathways in LECs. Additionally, we discuss emerging therapeutic strategies and potential targeting options, including antioxidant-based treatments.

Transcriptome profiling of Canine Parvovirus 2 Nonstructural gene 1(CPV2.NS1) transfected 4T1 mice mammary tumor cells to elucidate its oncolytic effects

Oncolytic viral gene therapy is a directed approach to target cancer cells without affecting healthy cells of the body. Canine parvovirus (CPV2) is an oncolytic virus that precisely targets and destroys neoplastic cells by causing DNA damage, mitochondrial damage, and apoptosis. Non-structural gene 1 (NS1) of CPV, concerned with viral DNA replication is a key mediator of cytotoxicity of CPV and can specifically cause tumor cell lysis. In the present study, by using the transcriptomics approach, we tried to identify molecular pathways and key genes involved in CPV2.NS1 mediated 4T1 mice mammary tumor cell death. We identified necroptosis and mitochondrial damage-mediated apoptosis as major cell death pathways leading to CPV2.NS1 transfected 4T1 cancer cell death. Various DEGs identified in our study play an important role in pathways like the PI3K/AKT pathway, diverse metabolic pathways, MAPK signaling pathway, and FGF signaling pathway, whichare mostly dysregulated in cancerous conditions. Histone variant H2A.X genes, Capn2, and Mapk10/JNK are predicted as key genes that play a role in causing endoplasmic reticulum stress and mitochondrial damage, thereby leading to necroptosis and apoptosis. This study is a preliminary work done to identify key genes and molecular pathways involved in CPV2.NS1 mediated 4T1 cancer cells death which need to be further validated to establish this viral gene as a potent oncolytic agent.

Hollow Calcium/Copper Bimetallic Amplifier for Cuproptosis/Paraptosis/Apoptosis Cancer Therapy via Cascade Reinforcement of Endoplasmic Reticulum Stress and Mitochondrial Dysfunction.

The endoplasmic reticulum (ER) and mitochondria are essential organelles that play crucial roles in maintaining cellular homeostasis. The simultaneous induction of ER stress and mitochondrial dysfunction represents a promising yet challenging strategy for cancer treatment. Herein, a hollow calcium-copper bimetallic nanoplatform is developed as a cascade amplifier to reinforce ER stress and mitochondrial dysfunction for breast cancer treatment. For this purpose, we report a facile method for preparing hollow CaCO3 (HCC) nanoparticles by regulating the dissolution-recrystallization process of amorphous CaCO3, and the amplifier D@HCC-CuTH is meticulously fabricated by sequentially coating disulfiram-loaded HCC nanoparticles with a copper coordination polymer and hyaluronan. In tumor cells, the dithiocarbamate-copper complex generated in situ by liberated disulfiram and Cu2+ inhibits the ubiquitin-proteasome system, causing irreversible ER stress and intracellular Ca2+ redistribution. Meanwhile, the amplifier induces mitochondrial dysfunction via triggering a self-amplifying loop of mitochondrial Ca2+ burst, and reactive oxygen species augment. Additionally, Cu2+ induces dihydrolipoamide S-acetyltransferase oligomerization in mitochondria, further exacerbating mitochondrial damage via cuproptosis. Collectively, ER stress amplification and mitochondrial dysfunction synergistically induce a cuproptosis-paraptosis-apoptosis trimodal cell death pathway, which demonstrates significant efficacy in suppressing tumor growth. This study presents a paradigm for synchronously inducing subcellular organelle disorders to boost cancer multimodal therapy.

Doramectin Induces Apoptosis in B16 Melanoma Cells.

Metastatic melanoma resists current pharmacological regimens that act through apoptosis. This indicates that therapies acting via non-apoptotic cell-death pathways could be pursued. Doramectin has shown promising results in another cancer of neural crest origin, neuroblastoma, through the inhibition of growth via autophagy. Our research hypothesis is that doramectin induces autophagy in B16F10 melanoma cells. Cells were treated with doramectin (15 uM) or a combination of both doramectin and a cell-death inhibitor, compared to untreated control cells (media), and then analyzed with MTT analysis. Likewise, MDC analysis was completed to detect autophagy involvement with doramectin treatment. Flow cytometry and TUNEL Assay were conducted to observe cell death-related effects. MTT analysis of doramectin-treated cells displayed a decrease in cell growth compared to control. Apoptotic morphology was prominent in melanoma cells treated with doramectin. Increased autophagy was not detected by fluorometric microscopic analysis. Flow cytometry analysis of doramectin-treated cells showed apoptosis as a major mode of cell death with some necrosis. Doramectin induces a novel cell-death mechanism in melanoma compared to other forms of cancer and should be studied as an effective anti-cancer agent for melanoma treatment.

Neonatal overfeeding attenuates microgliosis and hippocampal damage in an infant rat model of pneumococcal meningitis.

Pneumococcal meningitis (PM) triggers apoptotic neuronal and progenitor cell death in the hippocampal dentate gyrus (DG), resulting in subsequent cognitive impairment. Microglia play a crucial role in PM-induced hippocampal damage. While the lasting effects of neonatal nutrition on health are well documented, the influence of early-life overfeeding on the host response to neuroinfections remains uncertain. This study aimed to examine whether neonatal overfeeding affects the outcome of PM in the hippocampus (HC). Overfeeding was induced by adjusting litter size immediately after birth. On the eleventh day of life, rats were intracisternally injected with Streptococcus pneumoniae or saline, followed by euthanasia after 24 hours for brain dissection. Histological analysis evaluated apoptosis in the DG and the extent of inflammatory infiltrate in the hippocampal fissure, while microgliosis was assessed by immunohistochemistry. The hippocampal transcriptome was analyzed using RNAseq, and the mRNA levels of specific inflammatory biomarkers were evaluated via RT-qPCR. Overfed rats exhibited 40.5% greater body mass compared to their normal-fed counterparts. Intriguingly, PM-induced apoptosis in the DG was 50% lower in overfed rats. This effect was accompanied by significant alterations in the hippocampal transcriptional profile, particularly the lack of activation of the Programmed cell death pathway in overfed infected animals. RT-qPCR analysis of Aif1 and examination of Iba1-immunostained cells revealed mild microgliosis in the HC of infected-overfed animals. This reduced microglial reaction may be attributed to the diminished activation of interferon signaling pathways, as disclosed by the transcriptome analysis, potentially preventing microglial priming. Additionally, evidence of reduced neuroinflammation in overfed rats with PM was observed through the milder activation of pathways associated with Toll-like receptors, interleukins, and chemokine signaling. Furthermore, overfed animals exhibited increased transcription of proinflammatory Il6 and anti-inflammatory Il10 genes, with the latter showing higher expression even in the absence of PM, suggesting a priming effect of overfeeding on hippocampal immune cells. This study sheds light on the complex interplay between early-life overfeeding, immune response, and neuroprotection in infant rats with PM. The findings demonstrate the neuroprotective impact of early-life overfeeding in the context of PM, linked to the modulation of microglial function.

Design, Synthesis, and Biological Evaluation of Novel Heterocyclic Derivatives of 2,4‐Thiazolidine Dione as Anti‐Cancer Agents

ABSTRACTThis study explores the development of novel 2,4‐thiazolidinedione derivatives as potential anticancer agents. We report the synthesis of 15 novel analogs utilizing a strategic approach focused on intermediate diversification. Key intermediates, (Z)‐5‐([1,1′‐biphenyl]‐4‐ylmethylene)thiazolidine‐2,4‐dione 5a and (Z)‐5‐(benzo[1,3]dioxol‐5‐ylmethylene)thiazolidine‐2,4‐dione 5b, were synthesized via Knoevenagel condensation and acetal formation reactions, respectively. Subsequent N‐alkylation reactions provided a platform for introducing diverse functionalities and exploring structure–activity relationships to optimize the medicinal chemistry profile of these novel compounds. The synthesized compounds were tested against various human cancer cell lines, including for breast (MCF‐7, MDA‐MB‐453), lung (A549), and prostate (PC‐3) cancers, using an MTT assay. Compounds 8c and 10g emerged as particularly promising, exhibiting activity against all tested cell lines with IC50 values between 3.41 ± 0.51 and 40 μM. These compounds also induced apoptosis, suggesting that they inhibit cell proliferation through this cell death pathway. Moreover, relative molecular docking studies provided evidence that compound 8c likely functions by intercalating with DNA.

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.

Mechanisms regulating host cell death during Leishmania infection.

Parasites from the Leishmania genus are the causative agents of leishmaniasis and primarily reside within macrophages during mammalian infection. Their ability to establish intracellular infection provides a secure niche for proliferation while evading detection. However, successful multiplication within mammalian cells requires the orchestration of multiple mechanisms that control host cell viability. In contrast, innate immune cells, such as macrophages, can undergo different forms of cell death in response to pathogenic intracellular microbes. Thus, modulation of these different forms of host cell death is crucial for Leishmaniasis development. The regulation of host cell apoptosis, a form of programmed cell death, is crucial for sustaining parasites within viable host cells. Accordingly, several studies have demonstrated evasion of apoptosis induced by dermotropic and viscerotropic Leishmania species. Conversely, the prevention of pyroptosis, an inflammatory form of cell death, ensures the establishment of infection by silencing the release of mediators that could trigger massive proinflammatory responses. This manuscript explores how Leishmania regulates various host cell death pathways and overviews seminal studies on regulating host cell apoptosis by different Leishmania species.

Calcium signaling in mitochondrial intermembrane space.

The mitochondrial intermembrane space (IMS) is a highly protected compartment, second only to the matrix. It is a crucial bridge, coordinating mitochondrial activities with cellular processes such as metabolites, protein, lipid, and ion exchange. This regulation influences signaling pathways for metabolic activities and cellular homeostasis. The IMS harbors various proteins critical for initiating apoptotic cascades and regulating reactive oxygen species production by controlling the respiratory chain. Calcium (Ca2+), a key intracellular secondary messenger, enter the mitochondrial matrix via the IMS, regulating mitochondrial bioenergetics, ATP production, modulating cell death pathways. IMS acts as a regulatory site for Ca2+ entry due to the presence of different Ca2+ sensors such as MICUs, solute carriers (SLCs); ion exchangers (LETM1/SCaMCs); S100A1, mitochondrial glycerol-3-phosphate dehydrogenase, and EFHD1, each with unique Ca2+ binding motifs and spatial localizations. This review primarily emphasizes the role of these IMS-localized Ca2+ sensors concerning their spatial localization, mechanism, and molecular functions. Additionally, we discuss how these sensors contribute to the progression and pathogenesis of various human health conditions and diseases.

Protein profile of extracellular vesicles derived from adult Parascaris spp.

BackgroundParascaris spp. represent a significant threat to equine health worldwide, particularly in foals. The long-term survival of parasites in the host necessitates persistent modulation of the host immune response. Intercellular communication achieved through the exchange of molecules via extracellular vesicles (EVs) released from the parasite could be a crucial factor in this regard. This study aimed to isolate and characterize EVs released by adult male and female Parascaris worms and conduct a proteomic analysis to identify sex-specific proteins and potential immunomodulatory factors.MethodsLive adult Parascaris worms were collected, and EVs were isolated from spent culture media using differential ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy confirmed the size, concentration, and morphology of the isolated EVs. Proteins within the isolated EVs were analyzed using mass spectrometry-based proteomics (LC–MS/MS).ResultsProteomic analysis revealed a total of 113 proteins in Parascaris EVs, with several proteins showing homology to known helminth exosome proteins and exhibiting immunomodulatory functions. Sex-specific differences in EV protein composition were observed, with a distinct abundance of C-type lectins in female EVs, suggesting potential sex-specific roles or regulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed metabolic pathways shared between male and female Parascaris EVs, as well as differences in signal transduction, and cell growth and death pathways, indicating sex-specific variations.ConclusionsThese findings imply that Parascaris EVs and their protein cargo are complex. This data potentially opens avenues for discovering innovative approaches to managing and understanding helminth infection.Graphical

Integrated analysis of gene expressions and targeted mirnas for explaining crosstalk between oral and esophageal squamous cell carcinomas through an interpretable machine learning approach.

This study explores the bidirectional relation of esophageal squamous cell carcinoma (ESCC) and oral squamous cell carcinoma (OSCC), examining shared risk factors and underlying molecular mechanisms. By employing random forest (RF) classifier, enhanced with interpretable machine learning (IML) through SHapley Additive exPlanations (SHAP), we analyzed gene expression from two GEO datasets (GSE30784 and GSE44021). The GSE30784 dataset comprises 167 OSCC samples and 45 control group, whereas the GSE44021 dataset encompasses 113 ESCC samples and 113 control group. Our analysis led to identification of 20 key genes, such as XBP1, VGLL1, and RAD1, which are significantly associated with development of ESCC and OSCC. Further investigations were conducted using tools like NetworkAnalyst 3.0, Single Cell Portal, and miRNET 2.0, which highlighted complex interactions between these genes and specific miRNA targets including hsa-mir-124-3p and hsa-mir-1-3p. Our model achieved high precision in identifying genes linked to crucial processes like programmed cell death and cancer pathways, suggesting new avenues for diagnosis and treatment. This study confirms the bidirectional relationship between OSCC and ESCC, laying groundwork for targeted therapeutic approaches. This study helps to identify shared biological pathways and genetic factors of these conditions for designing personalized medicine strategies and to improve disease management.

Exploring MPC1 as a potential ferroptosis-linked biomarker in the cervical cancer tumor microenvironment: a comprehensive analysis

BackgroundThe increasing problems of drug and radiotherapy resistance in cervical cancer underscores the need for novel methods for its management. Reports indicate that the expression of MPC1 may be associated with the tumor microenvironment and the occurrence of ferroptosis in cervical cancer. The objective of this study was to visually illustrate the prognostic significance and immunological characterization of MPC1 in cervical cancer.MethodsThe expression profile and prognostic significance of MPC1 were analyzed using various databases, including UALCAN, TIMER2, GEPIA2, and Kaplan–Meier Plotter. TISIDB, TIMER2, and immunohistochemical analysis were used to investigate the correlation between MPC1 expression and immune infiltration. GO enrichment analysis, KEGG analysis, Reactome analysis, ConsensusPathDB, and GeneMANIA were used to visualize the functional enrichment of MPC1 and signaling pathways related to MPC1. The correlation analysis was carried out to examine the relationship between MPC1 and Ferroptosis gene in TIMER 2.0, ncFO, GEPIA Database and Kaplan–Meier Plotter.ResultsWe demonstrated that the expression levels of MPC1 in cervical cancer tissues were lower than those in normal cervical tissues. Kaplan–Meier survival curves showed shorter overall survival in cervical cancer patients with low levels of MPC1 expression. The expression of MPC1 was related to the infiltrating levels of tumor-infiltrating immune cells in cervical cancer. Moreover, MPC1 expression was associated with the iron-mediated cell death pathway, and several important ferroptosis genes were upregulated in cervical cancer cells. Furthermore, after knocking down MPC1 in HeLa cells, the expression of these genes decreased.ConclusionThese findings indicate that MPC1 functions as a prognostic indicator and plays a role in the regulation of the ferroptosis pathway in cervical cancer.

A Specific Targeted Enhanced Nanotherapy Strategy for Inducing Ferroptosis by Regulating the Iron Pool Levels in Tumor Cells.

Ferroptosis is an iron-dependent cell death pathway triggered by the toxic accumulation of lipid peroxides on cellular membranes. However, an imbalance in iron metabolism in tumor cells leads to the insufficient accumulation of iron ions in the iron pool, which inhibits ferroptosis. Nonspecific delivery of iron species may increase the risk of promoting tumor proliferation as well as trigger undesirable detrimental effects such as anaphylactic reactions in normal tissues. This study found that a constructed self-enhancing targeted nanocarrier can accurately regulate the iron pool levels in tumor cells. We constructed a programmatic targeted enhanced nanotherapy strategy by loading the ferroptosis inducer erastin into a self-enhancing targeted nanocarrier. This nanosystem was more effective at inducing tumor ferroptosis after upregulating the iron pool levels in tumor cells with self-enhancing targeted nanocarriers. Both in vitro and in vivo outcomes demonstrated notable anticancer ferroptosis efficacy, indicating that self-enhancing targeted nanocarriers can effectively regulate the level of the iron pool in tumor cells and promote ferroptosis. The combination of this nanotherapy strategy with self-enhancing targeted chemotherapy nanomedicines can achieve complete tumor clearance. Moreover, this self-enhancing targeted ferroptosis nanotherapy strategy is expected to be beneficial for future progress in the field of cancer self-enhancing targeted therapy.

AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence1. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy; however, responses are typically short lived1,2. Thus, there is an urgent need to develop more effective treatments. Components of the PI3K pathway represent plausible therapeutic targets; more than 70% of TNBCs have alterations in PIK3CA, AKT1 or PTEN3-6. However, in contrast to hormone-receptor-positive tumours, it is still unclear whether or how triple-negative disease will respond to PI3K pathway inhibitors7. Here we describe a promising AKT-inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors synergize with agents that suppress the histone methyltransferase EZH2 and promote robust tumour regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. OnceTNBCs are differentiated, these agents kill them by hijacking signals that normally drive mammary gland involution. Using a machine learning approach, we developed a classifier that can be used to predict sensitivity. Together, these findings identify a promising therapeutic strategy for this highly aggressive tumour type and illustrate how deregulated epigenetic enzymes can insulate tumours from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit.

Integrative analysis of multiple cell death model for precise prognosis and drug response prediction in gastric cancer.

Gastric cancer (GC) is a common upper gastrointestinal tumor. However, the evaluation of prognosis and treatment response in patients with gastric cancer remains a challenge. Programmed cell death (PCD) is one of the important terminal paths for the cells of metazoans, and is involved in a variety of biological events that include morphogenesis, maintenance of tissue homeostasis, and elimination of harmful cells. The objective of this project is to investigate the predictive significance of cell death pathways and create prognostic signatures associated to cell death, with the purpose of forecasting prognosis and providing guidance for the treatment of gastric cancer. Gene transcription profiles and corresponding clinical data of gastric cancer patients were collected from The Cancer Genome Atlas (TCGA-STAD, n = 448) and the Gene Expression Comprehensive Database (GSE84437, n = 483). Thirteen types of cell death-related genes, including apoptosis, necroptosis, pyroptosis, ferroptosis, autophagy, cuprotosis, parthanatos, entotic cell death, netotic cell death, lysosome-dependent cell death, alkaliptosis, oxeiptosis, and disulfidptosis, were analysed. Cell death-related genes associated with prognosis were identified in the TCGA-STAD training cohort using Lasso-Cox regression to generate a risk score. Patients were categorized into high and low-risk groups based on the median risk score for survival difference analysis. Cell death-related genes associated with prognosis were identified in the TCGA-STAD training cohort using Lasso-Cox regression to generate a risk score. Additionally, the response to immunotherapy in the high-risk and low-risk groups was calculated using the oncoPredict algorithm. Futhermore, the model genes were validated in the GEO validation set. A total of 324 differential programmed cell death (PCD)-related genes were identified, and 65 were selected through single-factor Cox analysis. Six PCD-related genes were ultimately identified by Lasso regression to construct a prognostic risk score model. The log-rank test revealed that patients in the high-risk group had inferior survival time compared with those in the low-risk group. The area under the ROC curve (AUC) for the training group at years 1, 3, and 5 were 0.684, 0.713, 0.743, respectively, while the AUC for the validation cohort at years 1, 3, and 5 were 0.695, 0.704, and 0.707, respectively. Unsupervised clustering identified potential subtypes included in the model, and a survival difference was also observed between the two subgroups. Multifactor Cox results, combined with clinical information, demonstrated that the prognostic risk score can serve as an independent prognostic factor, irrespective of other clinical features. By comprehensively analyzing multiple cell death patterns, we have established a novel model that accurately forecasts the clinical prognosis and drug sensitivity of gastric cancer. It was found that all 12 representative drugs may not be suitable for patients in high-risk groups.

Deletion of Transmembrane protein 184b leads to retina degeneration in mice.

Transmembrane protein 184b (Tmem184b) has been implicated in axon degeneration and neuromuscular junction dysfunction. Notably, Tmem184b exhibits high expression levels in the retina; however, its specific function within this tissue remains poorly understood. To elucidate the role of Tmem184b in the mammalian visual system, we developed a Tmem184b knockout (KO) model for further investigation. Loss of Tmem184b led to significant decreases in both a and b wave amplitudes of scotopic electroretinogram (ERG) and reduced b wave amplitudes of photopic ERG, respectively, reflecting damage to both the photoreceptors and secondary neuronal cells of the retina. Histologic analyses showed a progressive retinal thinning accompanied by the significantly loss of retinal cells including cone, rod, bipolar, horizontal and retinal ganglion cells. The expression levels of photo-transduction-related proteins were down-regulated in KO retina. TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated Uridine-5'-triphosphate [UTP]nick end labelling) and glial fibrillary acidic protein (GFAP)-labelling results suggested the increased cell death and inflammation in the KO mice. RNA-sequencing analysis and GO enrichment analysis revealed that Tmem184b deletion resulted in down-regulated genes involved in various biological processes such as visual perception, response to hypoxia, regulation of transmembrane transporter activity. Taken together, our study revealed essential roles of Tmem184b in the mammalian retina and confirmed the underlying mechanisms including cell death, inflammation and hypoxia pathway in the absence of Tmem184b, providing a potential target for therapeutic and diagnostic development.

A disulfidptosis-related lncRNA signature for analyzing tumor microenvironment and clinical prognosis in hepatocellular carcinoma.

Disulfidptosis is a recently identified form of non-apoptotic programmed cell death which distinguishes itself from classical cell death pathways. However, the prognostic implications of disulfidptosis-related long non-coding RNAs (DRLs) and their underlying mechanisms in hepatocellular carcinoma (HCC) remain largely unexplored. In this study, we leveraged RNA-sequencing data and clinical information of HCC patients from the TCGA database. Through expression correlation and prognostic correlation analyses, we identified a set of top-performing long non-coding RNAs. Subsequently, a 5-DRLs predictive signature was established by conducting a Lasso regression analysis. This signature effectively stratified patients into high- and low-risk groups, revealing notable differences in survival outcomes. Further validation through univariate and multivariate Cox regression analyses confirmed that the risk score derived from our signature independently predicted the prognosis of HCC patients. Moreover, we observed significant disparities in immune cell infiltration and tumor mutation burden (TMB) between the two risk groups, shedding light on the potential connection between immune-related mechanisms and disulfidptosis. Notably, the signature also exhibited predictive value in the context of chemotherapeutic drug sensitivity and immunotherapy efficacy for HCC patients. Finally, we performed experimental validation at both cellular and patient levels and successfully induced a disulfidptosis phenotype in HCC cells. In general, this multifaceted approach provides a comprehensive overview of DRLs profiles in HCC, culminating in the establishment of a novel risk signature that holds promise for predicting prognosis and therapy outcomes of HCC patients.

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>0.0001), thereby demonstrating treatment-sensitivity. In HAC15 cells, a supratherapeutic dose of 200uM was needed to significantly reduce viability (p>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

Towards New Anti-inflammatory Agents: Design, Synthesis and Evaluation of Molecules Targeting XIAP-BIR2.

The X-chromosome-linked inhibitor of apoptosis protein (XIAP) plays a crucial role in controlling cell survival across multiple regulated cell death pathways and coordinating a range of inflammatory signalling events. The discovery of selective inhibitors for XIAP-BIR2, able to disrupt the direct physical interaction between XIAP and RIPK2, offer promising therapeutic options for NOD2-mediated diseases like Crohn's disease, sarcoidosis, and Blau syndrome. The objective of this study was to design, synthesize, and evaluate small synthetic molecules with binding selectivity to XIAP-BIR2 domain. To achieve this, we applied an interdisciplinary drug design approach and firstly we have synthesized an initial fragment library to achieve a first XIAP inhibition activity. Then using a growing strategy, larger compounds were synthesized and one of them presents a good selectivity for XIAP-BIR2 versus XIAP-BIR3 domain, compound 20c. The ability of compound 20c to block the NOD1/2 pathway was confirmed in cell models. These data show that we have synthesized molecules capable of blocking NOD1/2 signalling pathways in cellulo, and ultimately leading to new anti-inflammatory compounds.

Exploring the effects of pemetrexed on drug resistance mechanisms in human lung adenocarcinoma and its association with PGRMC1

According to the 2022 cancer statistics of the World Health Organization, lung cancer ranks among the top ten causes of death, with lung adenocarcinoma being the most prevalent type. Despite significant advancements in lung cancer therapeutics, many clinical limitations remain, primarily due to the development of drug resistance. The present study investigated the effects of pemetrexed on the drug resistance mechanisms in human lung adenocarcinoma and its association with progesterone receptor membrane component 1 (PGRMC1) expression. Given that KRAS-mutant lung adenocarcinoma cell lines (e.g., A549) exhibit a high folate synthesis activity, pemetrexed, which is structurally similar to folate, was selected as the therapeutic drug. The present study used a lung adenocarcinoma cell line (A549) and established a drug-resistant lung adenocarcinoma cell line (A549/PEM). The findings demonstrated that PGRMC1 expression was elevated in the A549/PEM cells. It has been hypothesized that PGRMC1 regulates iron absorption through heme binding, resulting in a preference for iron-related cell death pathways (ferroptosis). Our findings indicate that drug-resistant lung adenocarcinoma cells with high PGRMC1 levels exhibit elevated antioxidant activity on the cell membrane and increased reliance on iron-dependent cell death pathways. This suggests a correlation between PGRMC1 and pemetrexed-induced iron-dependent cell death. Our study contributes to the development of more effective therapeutic strategies to improve the prognosis of patients with lung adenocarcinoma, particularly those facing drug resistance challenges.