Mode Of Death Research Articles

Host‐Pathogen Dialogues in Different Cell Death Modes during Mycobacterium Tuberculosis Infection (2/2024)

Host‐Pathogen Dialogues in Different Cell Death Modes during Mycobacterium Tuberculosis Infection (2/2024)

Apoptosis and Autophagy: Therapeutic Implications in Cancer

Despite the advances in the medical field so far, cancer remains a global health priority even now. Considering the drug resistance and the failure of cancer therapies to achieve complete eradication of cancer cells in certain populations, developing molecules that induce programmed cell death or apoptosis has been the focus of cancer research for several decades. Apoptosis evasion is one of the hallmarks of cancer cells, and efforts continue to achieve complete annihilation of cancer cells through selective killing. On the other hand, autophagy, a mode of cell degradation, is considered a double-edged sword. Recent studies show that autophagy also can be manipulated to selectively target cancer cells based on the tumor microenvironment and cellular context. Studies show that autophagy is an evolutionarily conserved process initiated during stress response and has enormous importance in maintaining physiological balance. Most importantly, the dynamic equilibrium between apoptosis and autophagy is crucial in maintaining cellular homeostasis. Although a ‘cell eating’ process, the fate of autophagic cells depends entirely on the nature of stress and the extent of crosstalk between autophagy. This understanding is of immense significance when designing therapeutic interventions targeting apoptosis and autophagy. Currently, several studies are ongoing to gain insights into the role of autophagy in cancer initiation, invasion, progression, angiogenesis, and metastasis. This review focuses on the two major cell death mechanisms, apoptosis and autophagy, in the context of cancer, their crosstalk, and the therapeutic interventions targeting both modes of cell death.

NK-cell-elicited gasdermin-D-dependent hepatocyte pyroptosis induces neutrophil extracellular traps that facilitate HBV-related acute-on-chronic liver failure.

HBV infection is a major etiology of acute-on-chronic liver failure (ACLF). At present, the pattern and regulation of hepatocyte death during HBV-ACLF progression are still undefined. Evaluating the mode of cell death and its inducers will provide new insights for developing therapeutic strategies targeting cell death. In this study, we aimed to elucidate whether and how immune landscapes trigger hepatocyte death and lead to the progression of HBV-related ACLF. We identified that pyroptosis represented the main cell death pattern in the liver of patients with HBV-related ACLF. Deficiency of MHC-I in HBV-reactivated hepatocytes activated cytotoxic NK cells, which in turn operated in a perforin/granzyme-dependent manner to trigger GSDMD/caspase-8-dependent pyroptosis of hepatocytes. Neutrophils selectively accumulated in the pyroptotic liver, and HMGB1 derived from the pyroptotic liver constituted an important factor triggering the generation of pathogenic extracellular traps in neutrophils (NETs). Clinically, elevated plasma levels of myeloperoxidase-DNA complexes were a promising prognostic biomarker for HBV-related ACLF. More importantly, targeting GSDMD pyroptosis-HMGB1 release in the liver abrogates NETs that intercept the development of HBV-related ACLF. Studying the mechanisms that selectively modulate GSDMD-dependent pyroptosis, as well as its immune landscapes, will provide a novel strategy for restoring the liver function of patients with HBV-related ACLF.

Combination treatment with ferroptosis and autophagy inducers significantly inhibit the proliferation and migration of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC), a malignancy originating from mucosal epithelial cells. Currently, triggering apoptotic cell death with anticancer drugs is the main way to inhibit OSCC cells. However, the capability to trigger apoptosis in tumors is constrained by the intrinsic resistance of tumor cells to apoptosis, hampering its effectiveness. Thus, utilizing alternative modes of non-apoptotic cell death offers new therapeutic possibilities, such as using a drug combination strategy to simultaneously induce ferroptosis and autophagy has the potential to improve OSCC therapy. In this study, we found the ferroptosis inducer RSL3 has certain inhibitory effects on the proliferation and migration of OSCC cells. Interestingly, our studies showed that RSL3 is also associated with autophagy activation. Based on this finding, we tried to combine RSL3 with the autophagy inducer LYN-1604 to improve the therapeutic effect. The results demonstrated that simultaneous regulation of autophagy and ferroptosis significantly reduced the proliferation and migration of OSCC cells. Taken together, we demonstrated the therapeutic potential of RSL3 in OSCC cells and proposed that simultaneous activation of autophagy and ferroptosis have synergistic effects, which would provide valuable clues for further exploration of targeted therapy for OSCC.

The impact of media reporting of suicides on subsequent suicides in Asia: A systematic review

Introduction: This systematic review is aimed at (1) evaluating the association between media portrayals of suicides and subsequent copycat suicides or attempts among the general public in Asia, (2) understanding the factors associated with copycat suicides and (3) determining the positive impacts of the media reporting of suicides (e.g. increased help-seeking, coping). Method: A systematic review and narrative synthesis of English and Chinese articles from 8 electronic databases (i.e. PsycINFO, MEDLINE, Embase, CINAHL, Web of Science, Ariti, China National Knowledge Infrastructure and OpenGrey) from January 2000 to May 2023 was conducted. Observational studies were included, and the data were analysed through narrative synthesis. The protocol was registered with PROSPERO (CRD42021281535). Results: Among the 32 studies included (n=29 for evidence synthesis) in the review, there is good-quality evidence to show that copycat suicides and suicide attempts increase after media reports of a suicide, regardless of country, celebrity status, study design, type of media, mode of suicide or follow-up period. Females, younger age groups and those sharing similar characteristics as the deceased in publicised suicides (age, gender) were more susceptible to negative impact. Reporting of the mode of death of the deceased increased suicides by the same method among the public. Conclusion: Media portrayals of suicide appear to have a negative impact on copycat suicides at the population level in Asia. Thus, in addition to tighter media control, healthcare systems, professional medical bodies and community outreach services should work collaboratively to promote early help-seeking in those with psychological distress.

The engineered exosomes targeting ferroptosis: A novel approach to reverse immune checkpoint inhibitors resistance.

Immune checkpoint inhibitors (ICIs) have been extensively used in immunological therapy primarily due to their ability to prolong patient survival. Although ICIs have achieved success in cancer treatment, the resistance of ICIs should not be overlooked. Ferroptosis is a newly found cell death mode characterized by the accumulation of reactive oxygen species (ROS), glutathione (GSH) depletion, and glutathione peroxidase 4 (GPX4) inactivation, which has been demonstrated to be beneficial to immunotherapy and combining ferroptosis and ICIs to exploit new immunotherapies may reverse ICIs resistance. Exosomes act as mediators in cell-to-cell communication that may regulate ferroptosis to influence immunotherapy through the secretion of biological molecules. Thus, utilizing exosomes to target ferroptosis has opened up exciting possibilities for reversing ICIs resistance. In this review, we summarize the mechanisms of ferroptosis improving ICIs therapy and how exosomes regulate ferroptosis through adjusting iron metabolism, blocking the ROS accumulation, controlling ferroptosis defense systems, and influencing classic signaling pathways and how engineered exosomes target ferroptosis and improve ICIs efficiency.

Novel methyltransferase G9a inhibitor induces ferroptosis in multiple myeloma through Nrf2/HO-1 pathway.

Multiple myeloma (MM) is a common malignant hematologic neoplasm, and the involvement of epigenetic modifications in its development and drug resistance has received widespread attention. Ferroptosis, a new ferroptosis-dependent programmed death mode, is closely associated with the development of MM. The novel methyltransferase inhibitor DCG066 has higher cell activity, but its mechanism of action in MM has not been clarified. Here, we found that DCG066 (5µM) inhibited the proliferation and induced ferroptosis in MM cells; the intracellular levels of ROS, iron, and MDA were significantly elevated, and the level of GSH was reduced after the treatment of DCG066; The protein expression levels of SLC7A11, GPX4, Nrf2 and HO-1 were significantly reduced, and these phenomena could be reversed by ferroptosis inhibitor Ferrostatin-1 (Fer-1) and Nrf2 activator Tert-butyl hydroquinone (TBHQ). Meanwhile, the protein expression levels of Keap1 was increased, and heat shock proteins (HSP70, HSP90 and HSPB1) were reduced after DCG066 treatment. In conclusion, this study confirmed that DCG066 inhibits MM proliferation and induces ferroptosis via the Nrf2/HO-1 pathway.

Focus on the Role of Inflammation as a Bridge between Ferroptosis and Atrial Fibrillation: A Narrative Review and Novel Perspective.

In this comprehensive review, we examine the intricate interplay between inflammation, ferroptosis, and atrial fibrillation (AF), highlighting their significant roles in AF pathophysiology and pathogenesis. Augmented inflammatory responses are pivotal to AF, potentially leading to atrial remodeling and reentry phenomena by impacting calcium channels and atrial tissue fibrosis. A strong correlation exists between inflammatory cytokines and AF, underscoring the importance of inflammatory signaling pathways, such as NOD-like receptor thermal protien domain associated protein 3 (NLRP3) inflammasome, Nuclear Factor kappa B (NF- B) signaling, and Tumor necrosis factor- (TNF- ) signaling in AF development. Ferroptosis, a non-apoptotic regulated mode of cell death, has been widely studied in relation to cardiovascular diseases including heart failure, myocardial infarction, cardiomyopathy, and reperfusion injury. The interaction between ferroptosis and inflammation is complex and mutually influential. While significant progress has been made in understanding the inflammation-AF relationship, the role of inflammation as a conduit linking ferroptosis and AF remains underexplored. The specific pathogenesis and key molecules of atrial fibrosis caused by ferroptosis are still not fully understood. Here we review the role of inflammatory signaling in ferroptosis and AF. We elucidated the association between ferroptosis and AF, aiming to unveil mechanisms for targeted inhibition of atrial cell fibrosis and to propose novel therapeutic strategies for AF. This exploration is vital for advancing our knowledge and developing more effective interventions for AF, a condition deeply intertwined with inflammatory processes and ferroptotic pathways.

Abstract 7156: Targeting the β-catenin signaling axis in osteosarcoma

Abstract Osteosarcoma, an aggressive bone malignancy predominantly affecting children, poses a formidable challenge for therapeutic interventions. Here we evaluated a recently approved FDA drug, PCT, for potential anti-cancer effect in osteosarcoma model. Initial assessments of cytotoxicity encompassing various human and murine osteosarcoma cell lines—OS-17, OS-25, U2OS, SAOS2, MG63, and K7M3—revealed an IC50 ranging 1-3 µM. Subsequent investigations demonstrated significant anti-clonogenic activity and G2M phase cell cycle arrest. Annexin-PI assay showed a notable apoptotic effect of PCT, shedding light on the mode of cell death. Given the pivotal role of β-catenin signaling in human osteosarcoma pathogenesis and metastasis, targeting β-catenin components holds promise for therapeutic intervention. In silico docking studies suggested that PCT may have similar anti-cancer effect compared to β-catenin inhibitors (e.g., MSAB, ICG001). Our western blot analyses in multiple osteosarcoma cell lines demonstrated that PCT inhibits β-catenin pathway. Fluorescence microscopy and PCR analyses substantiated PCT's ability to hinder the nuclear translocation of β-catenin, providing mechanistic insights into its anti-tumor properties. Our preliminary data also suggests that AXL, an upstream regulator of β-catenin pathway, was modulated by PCT. In summary, our findings indicate that PCT exhibits therapeutic potential against osteosarcoma by targeting the β-catenin pathway. Further mechanistic studies are currently underway in our lab. Citation Format: Marina Curcic, Shreyas Gaikwad, Sanjay K Srivastava. Targeting the β-catenin signaling axis in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7156.

Abstract 5849: Integrated multi-omics of hydroxychloroquine evolved cancer cells reveal survival requires transcriptional upregulation of glycolysis, exocytosis, and chromosome fidelity, but not autophagy

Abstract Repurposed drugs are undergoing a renaissance in the era of genomics, particularly in oncology. Over 2,300 FDA-approved and natural product small molecules now exist, but challenges remain in identifying which cancer patients may best benefit from such drugs. To best position repurposed drugs, mechanisms of action and resistance to the drug in a cancer cell setting must be determined. Hydroxychloroquine (HCQ) and chloroquine are repurposed drugs reported to disrupt autophagy, a molecular recycling pathway which is essential for tumor cell survival, chemotherapeutic resistance, and stem cell phenotypes. However, oncology trials with chloroquine have no biomarker or other genetic test to categorize patients who may best respond to HCQ therapy. It is unclear how tumors mechanistically evolve resistance to HCQ. We pursued a multi-omic strategy in OVCAR3 ovarian cancer and CCL218 colorectal cancer cells. Two genome-scale screens were performed. In the forward genetic screen, cell populations were passaged in 93-independent cultures for 15 drug pulse-chases with HCQ or vehicle control. Evolved cells were collected and processed for bulk RNA-seq, exome-seq, and single cell RNA-seq (scRNA-seq). In the reverse genetic screen, a pooled Brunello CRISPR-Cas9 library of 19,114 genes targeted by multiple sgRNAs was used in cells over three pulse-chases of HCQ or vehicle control treatments. HCQ evolved cells displayed remarkably few mutational differences, but substantial transcriptional differences. Bulk transcriptomes revealed multiple broad-spectrum pathways associated with resistance to HCQ, including upregulation of glycolysis, exocytosis, and chromosome condensation/segregation, as well as downregulation of translation and apoptosis. Analysis of scRNA-seq data indicated cell clusters were differentially reliant on cytoskeletal regulation, DNA repair, and metabolism. The genome-scale Cas9 screen identified remarkably few autophagy genes. However, an integrated analysis of Cas9 hits with RNA-seq upregulated genes confirmed glycolytic genes KDM3A, ENO2, and PDK3 were essential for HCQ resistance. Additional integrated hits were NEK2, involved in exocytosis, and chromosome condensation/segregation genes CDK1 and CENPA. Transcriptional plasticity was the primary mechanism by which cells evolved resistance to HCQ. Contrary to the widely presumed autophagy-related mode of cell death, the multi-omic analysis instead prioritized glycolytic metabolism, exocytosis, and chromosome condensation/segregation as most involved in cancer cell survival with HCQ. Integrated multi-omic analysis defined high-value biomarkers of HCQ sensitivity and resistance. Our analysis may serve as a model for how to better define the effects and positioning of repurposed drugs in oncology. Citation Format: Madison Clark, Silvia G. Vaena, Joe R. Delaney. Integrated multi-omics of hydroxychloroquine evolved cancer cells reveal survival requires transcriptional upregulation of glycolysis, exocytosis, and chromosome fidelity, but not autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5849.

Ferritinophagy-Mediated Hippocampus Ferroptosis is Involved in Cognitive Impairment in Immature Rats Induced by Hypoxia Combined with Propofol.

Propofol is a clinically common intravenous general anesthetic and is widely used for anesthesia induction, maintenance and intensive care unit (ICU) sedation in children. Hypoxemia is a common perioperative complication. In clinical work, we found that children with hypoxemia who received propofol anesthesia experienced significant postoperative cognitive changes. To explore the causes of this phenomenon, we conducted the study. In this study, our in vivo experiments found that immature rats exposed to hypoxia combined with propofol (HCWP) could develop cognitive impairment. We performed the RNA-seq analysis of its hippocampal tissues and found that autophagy and ferroptosis may play a role in our model. Next, we verified the participation of the two modes of death by detecting the expression of autophagy-related indexes Sequestosome 1 (SQSTM1) and Beclin1, and ferroptosis-related indicators Fe2+, reactive oxygen species (ROS) and glutathione peroxidase 4 (GPX4). Meanwhile, we found that ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, could improve cognitive impairment in immature rats caused by HCWP. In addition, we found that nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, which acted as a key junction between autophagy and ferroptosis, was also involved. Finally, our in vitro experiments concluded that autophagy activation was an upstream factor in HCWP-induced hippocampus ferroptosis through the intervention of autophagy inhibitor 3-methyladenine (3-MA). Our study was expected to provide an attractive therapeutic target for cognitive impairment that occurred after HCWP exposures.

An Integrated Machine Learning Framework Identifies Prognostic Gene Pair Biomarkers Associated with Programmed Cell Death Modalities in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a common and lethal urological malignancy for which there are no effective personalized therapeutic strategies. Programmed cell death (PCD) patterns have emerged as critical determinants of clinical prognosis and immunotherapy responses. However, the actual clinical relevance of PCD processes in ccRCC is still poorly understood. We screened for PCD-related gene pairs through single-sample gene set enrichment analysis (ssGSEA), consensus cluster analysis, and univariate Cox regression analysis. A novel machine learning framework incorporating 12 algorithms and 113 unique combinations were used to develop the cell death-related gene pair score (CDRGPS). Additionally, a radiomic score (Rad_Score) derived from computed tomography (CT) image features was used to classify the CDRGPS status as high or low. Finally, we conclusively verified the function of PRSS23 in ccRCC. The CDRGPS was developed through an integrated machine learning approach that leveraged 113 algorithm combinations. CDRGPS represents an independent prognostic biomarker for overall survival and demonstrated consistent performance between training and external validation cohorts. Moreover, CDRGPS showed better prognostic accuracy compared to seven previously published cell death-related signatures. In addition, patients classified as high-risk by CDRGPS exhibited increased responsiveness to tyrosine kinase inhibitors (TKIs), mammalian Target of Rapamycin (mTOR) inhibitors, and immunotherapy. The Rad_Score demonstrated excellent discrimination for predicting high versus low CDRGPS status, with an area under the curve (AUC) value of 0.813 in the Cancer Imaging Archive (TCIA) database. PRSS23 was identified as a significant factor in the metastasis and immune response of ccRCC, thereby validating experimental in vitro results. CDRGPS is a robust and non-invasive tool that has the potential to improve clinical outcomes and enable personalized medicine in ccRCC patients.

Comprehensive analysis of scRNA-seq and bulk RNA-seq reveal the characteristics of disulfidptosis and a prognostic signature in BLCA.

Disulfidptosis is a newly discovered mode of cell death. However, its biological mechanism in bladder cancer (BLCA) is still uncharacterized. In this investigation, we firstly examined the expression and mutation of disulfidptosis-related genes (DRGs) in BLCA. Two disulfidptosis phenotypes associated with DRGs expression patterns and immune cell infiltration were built. A disulfidptosis risk score signature was constructed based on ten differentially expressed genes (DEGs) between the disulfidptosis subtypes, which allowed patients to be stratified into high- and low-risk groups. We further confirmed that the disulfidptosis risk score signature has great power to predict prognosis, immune cell infiltration, and immunotherapy efficacy in BLCA. Additionally, we analyzed the differences in therapeutic sensitivities between high- and low-risk groups concerning targeted inhibitor therapy and immunotherapy. Analysis of single-cell RNA sequencing was conducted of the ten hub DRGs. Of the ten genes, we found that DUSP2 and SLCO1B3 were differentially expressed in BLCA tissues and adjacent normal tissues, and were markedly associated with patients' prognosis. Functional experiments revealed that overexpression of DUSP2 or knockdown of SLCO1B3 significantly inhibited cell proliferation, migration, and invasion in BLCA cells. In all, we present a fresh disulfidptosis-related prognostic signature, which has a remarkable capacity to characterize the immunological landscape and prognosis of BLCA patients.

Research progress on pyroptosis in liver diseases

Pyroptosis is a newly discovered kind of cell death modality that, due to its association with innate immunity, plays a crucial role in cytolysis and inflammatory cytokine release during host defense against infection. In recent years, studies have shown that pyroptosis plays an important role in the occurrence and development of liver diseases. This article introduces and elaborates on the most recent research progress on pyroptosis in liver diseases based on the morphological features, molecular and pathophysiological mechanisms.

Mitochondrial involvement in ferroptotic cell death

Ferroptosis is a regulated cell death pathophysiologically associated with the depletion of the antioxidant system due to iron overload, which results in excess lipid peroxidation. Mitochondria are crucial organelles known for their prominent involvement in various cellular metabolic activities and cell death processes. While our understanding of ferroptotic signaling pathways is advancing, further investigation into the intricate relationship between mitochondrial bio-process and this mode of cell death is necessary to identify effective biomedical therapeutic options targeting this organelle. However, the direct involvement of mitochondria in ferroptosis has remained a topic of debate due to the limited availability of concrete information to date. This review aims to elucidate the pathophysiological perspectives of mitochondria during ferroptotic cell death.

A deep insight into ferroptosis in lung disease: facts and perspectives.

In the last decade, ferroptosis has received much attention from the scientific research community. It differs from other modes of cell death at the morphological, biochemical, and genetic levels. Ferroptosis is mainly characterized by non-apoptotic iron-dependent cell death caused by iron-dependent lipid peroxide excess and is accompanied by abnormal iron metabolism and oxidative stress. In recent years, more and more studies have shown that ferroptosis is closely related to the occurrence and development of lung diseases. COPD, asthma, lung injury, lung fibrosis, lung cancer, lung infection and other respiratory diseases have become the third most common chronic diseases worldwide, bringing serious economic and psychological burden to people around the world. However, the exact mechanism by which ferroptosis is involved in the development and progression of lung diseases has not been fully revealed. In this manuscript, we describe the mechanism of ferroptosis, targeting of ferroptosis related signaling pathways and proteins, summarize the relationship between ferroptosis and respiratory diseases, and explore the intervention and targeted therapy of ferroptosis for respiratory diseases.

Late transition metal complexes of ferrocene‐containing nitrogen ligands: Coordination chemistry, electron transfer properties, and tumor cell viability

Bis(2‐picolyl)amine (bpa), iminodiacetamide (imda), and bis‐1,2,3‐triazole (bta) ferrocene ligands (L) with and without an aliphatic linker were prepared by multi‐step synthesis. The cis‐fac, trans‐fac, or mer stereochemistry of their ML2 complexes with Ni(II), Cu(II), Cd(II), and Zn(II) was studied in the solid state (infrared [IR] and single‐crystal X‐ray diffraction [SC‐XRD]), in solution (nuclear magnetic resonance [NMR] and cyclic voltammetry [CV]) and by density functional theory (DFT) calculations. Crystal structures were determined for bpa ligand 7, and complexes [Ni(1)2](NO3)2 (1Ni), [Cu(8)2]OTf2 (8Cu), [Ni(10b)2](NO3)2 (10bNi), and [Cu(10b)2]OTf2 (10bCu). The bond strength of the central metal ion to the ligand amine nitrogen atom was studied by NMR, electrochemistry, and DFT. The information on redox‐active centers, electron transfer properties of ferrocene ligands (L), and their in situ complexation with zinc(II) and nickel(II) ions were obtained by voltammetric analysis. In addition, DFT calculations showed that the electron ionization in ML2 complexes occurs from one of the ferrocene units, leaving the electronic structure of the other ligand intact, while some of the expelled electron density is recovered by the adjacent amine through resonance. This effect is more pronounced in the free ligands, because the eventual amine resonance in ML2 needs to balance its Zn(II) coordination participation, which justifies why they show higher ionization energies over free ligands. Moreover, due to lower steric hindrance, the N(amino)–Zn(II) coordination is additionally stronger in 1:1 ML complexes, which makes their electron depletion further more demanding. If compared with the clinical drug cisplatin, complexes of bpa 1Ni and imda 2Ni showed a better effect on the viability of different tumor cell lines and better selectivity towards normal cells. Treatment with 1Ni and 2Ni causes an increase of cells in the S phase of the cell cycle and leads to the accumulation of cells in G0/G1. A decrease in the expression level of anti‐apoptotic marker Bcl‐2 upon treatment with both compounds together with increased amount of Annexin V‐FITC positive cells implied apoptosis as the mode of cell death.

Experimental evidence for Parthanatos-like mode of cell death of heat-damaged human skin fibroblasts in a cell culture-based in vitro burn model

The cellular mechanisms of burn conversion of heat damaged tissue are center of many studies. Even if the molecular mechanisms of heat-induced cell death are controversially discussed in the current literature, it is widely accepted that caspase-mediated apoptosis plays a central role. In the current study we wanted to develop further information on the nature of the mechanism of heat-induced cell death of fibroblasts in vitro. We found that heating of human fibroblast cultures (a 10 s rise from 37 °C to 67 °C followed by a 13 s cool down to 37 °C) resulted in the death of about 50% of the cells. However, the increase in cell death started with a delay, about one hour after exposure to heat, and reached the maximum after about five hours. The lack of clear evidence for an active involvement of effector caspase in the observed cell death mechanism and the lack of observation of the occurrence of hypodiploid nuclei contradict heat-induced cell death by caspase-mediated apoptosis. Moreover, a dominant heat-induced increase in PARP1 protein expression, which correlated with a time-delayed ATP synthesis inhibition, appearance of double-strand breaks and secondary necrosis, indicate a different type of cell death than apoptosis. Indeed, increased translocation of Apoptosis Inducing Factor (AIF) and Macrophage Migration Inhibitory Factor (MIF) into cell nuclei, which correlates with the mentioned enhanced PARP1 protein expression, indicate PARP1-induced, AIF-mediated and MIF-activated cell death. With regard to the molecular actors involved, the cellular processes and temporal sequences, the mode of cell death observed in our model is very similar to the cell death mechanism via Parthanatos described in the literature.

Derivation of a novel antimicrobial peptide from the Red Sea Brine Pools modified to enhance its anticancer activity against U2OS cells

Cancer associated drug resistance is a major cause for cancer aggravation, particularly as conventional therapies have presented limited efficiency, low specificity, resulting in long term deleterious side effects. Peptide based drugs have emerged as potential alternative cancer treatment tools due to their selectivity, ease of design and synthesis, safety profile, and low cost of manufacturing. In this study, we utilized the Red Sea metagenomics database, generated during AUC/KAUST Red Sea microbiome project, to derive a viable anticancer peptide (ACP). We generated a set of peptide hits from our library that shared similar composition to ACPs. A peptide with a homeodomain was selected, modified to improve its anticancer properties, verified to maintain high anticancer properties, and processed for further in-silico prediction of structure and function. The peptide’s anticancer properties were then assessed in vitro on osteosarcoma U2OS cells, through cytotoxicity assay (MTT assay), scratch-wound healing assay, apoptosis/necrosis detection assay (Annexin/PI assay), RNA expression analysis of Caspase 3, KI67 and Survivin, and protein expression of PARP1. L929 mouse fibroblasts were also assessed for cytotoxicity treatment. In addition, the antimicrobial activity of the peptide was also examined on E coli and S. aureus, as sample representative species of the human bacterial microbiome, by examining viability, disk diffusion, morphological assessment, and hemolytic analysis. We observed a dose dependent cytotoxic response from peptide treatment of U2OS, with a higher tolerance in L929s. Wound closure was debilitated in cells exposed to the peptide, while annexin fluorescent imaging suggested peptide treatment caused apoptosis as a major mode of cell death. Caspase 3 gene expression was not altered, while KI67 and Survivin were both downregulated in peptide treated cells. Additionally, PARP-1 protein analysis showed a decrease in expression with peptide exposure. The peptide exhibited minimal antimicrobial activity on critical human microbiome species E. coli and S. aureus, with a low inhibition rate, maintenance of structural morphology and minimal hemolytic impact. These findings suggest our novel peptide displayed preliminary ACP properties against U2OS cells, through limited specificity, while triggering apoptosis as a primary mode of cell death and while having minimal impact on the microbiological species E. coli and S. aureus.

Inhibition of lysyl oxidase by pharmacological intervention and genetic manipulation alleviates epilepsy-associated cognitive disorder

Epilepsy-associated cognitive disorder (ECD), a prevalent comorbidity in epilepsy patients, has so far uncharacterized etiological origins. Our prior work revealed that lysyl oxidase (Lox) acted as a novel contributor of ferroptosis, a recently discovered cell death mode in the regulation of brain function. However, the role of Lox-mediated ferroptosis in ECD remains unknown. ECD mouse model was established 2 months later following a single injection of kainic acid (KA) for. After chronic treatment with KA, mice were treated with different doses (30 mg/kg, 100 mg/kg and 300 mg/kg) of Lox inhibitor BAPN. Additionally, hippocampal-specific Lox knockout mice was also constructed and employed to validate the role of Lox in ECD. Cognitive functions were assessed using novel object recognition test (NOR) and Morris water maze test (MWM). Protein expression of phosphorylated cAMP-response element binding (CREB), a well-known molecular marker for evaluation of cognitive performance, was also detected by Western blot. The protein distribution of Lox was analyzed by immunofluorescence. In KA-induced ECD mouse model, ferroptosis process was activated according to upregulation of 4-HNE protein and a previously discovered ferroptosis in our group, namely, Lox was remarkably increased. Pharmacological inhibition of Lox by BAPN at the dose of 100 mg/kg significantly increased the discrimination index following NOR test and decreased escape latency as well as augmented passing times within 60 s following MWM test in ECD mouse model. Additionally, deficiency of Lox in hippocampus also led to pronounced improvement of deficits in ECD model. These findings indicate that the ferroptosis regulatory factor, Lox, is activated in ECD. Ablation of Lox by either pharmacological intervention or genetic manipulation ameliorates the impairment in ECD mouse model, which suggest that Lox serves as a promising therapeutic target for treating ECD in clinic.