Search Results (156)

Of breast cancers, the triple-negative subtype (TNBC) is characterized by aggressive behavior, poor prognosis and limited treatment options due to its high molecular heterogeneity. Since insufficient programmed cell death response is a major hallmark of cancer, here we searched for apoptosis-related biomarkers of prognostic potential in TNBC. The expression of the pro-apoptotic caspase 8, cytochrome c, caspase 3, the anti-apoptotic BCL2 and the caspase-independent mediator, apoptosis-inducing factor-1 (AIF1; gene AIFM1) was tested in TNBC both in silico at transcript and protein level using KM-Plotter, and in situ in our clinical TNBC cohort of 103 cases using immunohistochemistry. Expression data were correlated with overall survival (OS), recurrence-free survival (RFS) and distant metastasis-free survival (DMFS). We found that elevated expression of the executioner apoptotic factors AIF1 and caspase 3, and of BCL2, grants significant OS advantage within TNBC, both at the mRNA and protein level, particularly for chemotherapy-treated vs untreated patients. The dominantly cytoplasmic localization of AIF1 and cleaved-caspase 3 proteins in primary TNBC suggests that chemotherapy may recruit them from the cytoplasmic/mitochondrial stocks to contribute to improved patient survival in proportion to their expression. Our results suggest that testing for the expression of AIF1, caspase 3 and BCL2 may identify partly overlapping TNBC subgroups with favorable prognosis, warranting further research into the potential relevance of apoptosis-targeting treatment strategies. Full article

Background: Necroptosis, a regulated form of inflammatory cell death, is increasingly recognized as a key driver of sepsis and critical illness. The balance between necroptosis and apoptosis may influence immune responses and outcomes in ICU patients. Objective: To evaluate necroptosis- and apoptosis-related protein expression in critically ill pediatric and adult patients with sepsis/septic shock, trauma/SIRS, or cardiac conditions, and assess their association with clinical outcomes. Methods: In this prospective, observational study, 88 patients admitted to a tertiary ICU were categorized into four groups: sepsis/septic shock, trauma/SIRS, cardiac disease, and healthy controls. Serum levels of RIPK1, RIPK3, MLKL, A20, caspase-8, IL-1β, and IL-18 were measured within 24 h of admission using ELISA. Biomarkers were analyzed by disease group, age, and severity indices. Results: Patients with sepsis—both adults and children—exhibited significantly elevated levels of RIPK1, IL-1β, and IL-18 (p < 0.001) and reduced levels of caspase-8 (p = 0.015), indicating activation of the necroptosis pathway. A20 was significantly upregulated (p < 0.001) and independently associated with lactate levels. RIPK1, IL-1β, and IL-18 were positively correlated with ICU length of stay and illness severity, whereas caspase-8 showed an inverse correlation. ROC analysis demonstrated strong predictive performance for sepsis/septic shock using RIPK1 (AUC = 0.81), IL-18 (AUC = 0.71), and A20 (AUC = 0.71); conversely, caspase-8 was inversely associated with sepsis (AUC = 0.32). Conclusions: Necroptosis appears to play a central role in the pathophysiology of sepsis across age groups. Elevated levels of RIPK1, IL-1β, IL-18, and A20 may serve as biomarkers of disease severity, while reduced caspase-8 supports a shift away from apoptosis toward necroptotic cell death. These findings highlight the potential of necroptosis-related pathways as targets for risk stratification and therapeutic intervention in critically ill patients of all ages. Full article

Background/Objectives: Death-associated protein kinase 1 (DAPK1) is a serine/threonine kinase that plays a crucial role in cancer by regulating apoptosis through interactions with TP53. Aberrant expression of DAPK1 was shown in certain types of human cancer contributing to tumor progression and chemoresistance. This study aimed to investigate the role of DAPK1 in high-grade serous ovarian cancer (HGSOC) and to evaluate the therapeutic potential of restoring its kinase activity, including the use of truncated DAPK1 variants, to overcome chemoresistance and enhance tumor suppression. Methods: Gene expression analysis was performed on ovarian cancer tissues compared to benign controls to assess DAPK1 downregulation and its epigenetic regulation. Prognostic relevance was evaluated in a cohort of 1436 HGSOC patient samples. Functional restoration of DAPK1 was conducted in HGSOC cell lines and patient-derived primary tumor cells using vector-based expression or in vitro-transcribed (IVT) DAPK1 mRNA, including the application of truncated DAPK1 (ΔDAPK1) forms. To assess apoptosis, Caspase activation assays, 2D-colony formation assays, and cell survival assays were performed. To analyze the reactivation of DAPK1 downstream signaling, phosphorylation of p53 at Ser20 and the expression of p53 target proteins were examined. Chemosensitivity to Paclitaxel and Cisplatin was quantified by changes in IC₅₀ values. Results: DAPK1 expression was significantly downregulated in ovarian cancer compared to benign tissue, correlating with epigenetic silencing, and showed prognostic value in early-stage HGSOC. Restoration of DAPK1 activity, including ΔDAPK1 variants, led to phosphorylation of p53 Ser20, increased expression of p53 target proteins, and Caspase-dependent apoptosis. Reactivation of DAPK1 sensitized both established HGSOC cell lines and patient-derived ascites cells to Paclitaxel and Cisplatin. These effects occurred through both p53-dependent and p53-independent pathways, enabling robust tumor suppression even in p53-mutant contexts. Conclusions: Reactivation of DAPK1, particularly through truncated variants, represents a promising therapeutic strategy to overcome chemoresistance in HGSOC. The dual mechanisms of tumor suppression provide a strong rationale for developing DAPK1-based therapies to enhance the efficacy of standard chemotherapy, especially in patients with chemoresistant or p53-deficient tumors. Future work should focus on optimizing delivery approaches for DAPK1 variants and assessing their synergistic potential with emerging targeted treatments in clinical settings. Full article

Cervical cancer remains a significant global health concern, causing more than 300,000 deaths annually. Erythrina velutina, a tree native to north-eastern Brazil, contains bioactive alkaloids with potential anticancer properties. This study aimed to characterize the alkaloid-enriched fraction of Erythrina velutina leaves and investigate the effects of the alkaloid erythraline on apoptosis and cell cycle in SiHa cervical cancer cells. Using Gas Chromatography–Mass Spectrometry (GC-MS), six alkaloids, including erythraline, were identified. Cytotoxicity was assessed through proliferation assays on SiHa cells and peripheral blood mononuclear cells (PBMCs). Apoptosis and cell cycle analyses were performed using flow cytometry, and in silico virtual screening identified potential protein targets of erythraline. Erythraline showed time- and concentration-dependent inhibitory effects on SiHa cell proliferation, with significant cytotoxicity observed at 50 µg/mL. Morphological changes, chromatin condensation, and increased apoptotic cell percentages confirmed the induction of caspase-independent apoptosis. Erythraline also induced G2/M cell cycle arrest, with 22% of cells in the G2/M phase compared with 7.25% in the untreated controls. In silico analysis identified polyamine oxidase, pyruvate kinase M2, and tankyrase as potential targets that contribute to the antitumor activity of erythraline. These findings suggest that erythraline is a promising candidate for anticancer therapy, warranting further investigation. Full article

Background: Most newly developed anticancer treatments trigger tumor cell death through apoptosis, for which involvement of caspases activity is essential. However, numerous mutations in apoptotic pathways that lead to cancer and favor resistance to apoptosis are known; most are related to caspase-dependent apoptosis pathways and thus have low efficacy. To overcome these limitations, we constructed a novel chimeric protein, GnRH-AIF, using a gonadotropin-releasing hormone (GnRH) analog as a targeting moiety and the apoptosis-inducing factor (AIF) in its cleaved form as a killing moiety, fused at the cDNA level. AIF has a crucial role in the caspase-independent apoptotic pathway. A wide variety of solid tumors overexpress GnRH-receptors (GnRH-R) that are targeted by the new GnRH-AIF chimeric protein. Methods and Results: In this study, we constructed, expressed, and highly purified GnRH-AIF chimeric proteins. We demonstrated the ability of the chimera to enter and specifically and very efficiently kill solid cancer cell lines overexpressing GnRH-R. Most importantly, upon its entry, GnRH-AIFs translocate to the nucleus where it causes DNA fragmentation leading to a direct caspase-independent apoptotic death. As AIFs lack nuclease activity, our findings also emphasize that cell death induced by GnRH-AIF is dependent on the presence of the ENDOG and PPIA proteins, known to participate in the formation of a DNA–degradosome complex. Finally, we demonstrated the high anti-tumor efficacy of the GnRH-AIF ex vivo, in a human, colon cancer organoid model. Conclusions: Our study shows the potential of using a GnRH-AIF chimeric protein as a novel approach to treat solid cancers that overexpress GnRH-R, via a caspase-independent apoptotic pathway. Full article

Infectious bronchitis virus (IBV) poses a major challenge to poultry health and productivity. This study examined how inflammatory cell death pathways influence the replication and pathogenesis of two IBV strains—respiratory Connecticut (Conn) A5968 and nephropathogenic Delmarva (DMV)/1639—in chicken macrophages. Low serum conditions enhanced viral replication, reduced cell viability, and promoted apoptosis and necroptosis, with DMV/1639 showing more pronounced effects. Modulation of the cyclooxygenase-2/prostaglandin E2 (COX-2/PGE2) pathway displayed strain-specific effects, mitigating necroptosis in DMV/1639-infected cells but exacerbating apoptosis and necroptosis in Conn A5968-infected cells. Broad caspase inhibition (z-VAD-FMK) reduced necroptosis, while selective caspase-1/4 inhibition heightened apoptotic responses. Caspase-8 inhibition selectively reduced necroptosis in DMV/1639 infections but increased apoptosis and necroptosis in Conn A5968 infections. NLRP3 inflammasome and RIPK1 inhibition decreased cell viability and increased apoptosis in both strains but had distinct effects on necroptosis. These findings reveal the strain-specific regulation of viral replication, apoptosis, and necroptosis, underscoring the intricate interplay between IBV and host inflammatory pathways. Understanding these mechanisms provides novel insights into IBV pathogenesis and highlights potential therapeutic strategies to mitigate its impact on poultry health. Full article

Obesity has been identified as an independent risk factor for severe COVID-19 unfavorable outcomes. Several factors, such as increased ACE2 receptor expression and chronic inflammation, can contribute to this relationship, yet the activation of the NLRP3 inflammasome pathway is also a key element. Our primary goal was to determine whether chronic NLRP3 inflammasome activation in people with obesity is different in critical COVID-19 and in critical chronic conditions. A retrospective analysis was conducted using clinical data and post-mortem lung tissue samples from 14 COVID-19 patients with obesity (group A) and 9 patients with obesity who died from non-COVID-19 causes (group B). Immunohistochemical analysis assessed twelve markers related to the NLRP3 inflammasome pathway. Group A showed a significantly higher expression of ASC (p = 0.0387) and CASP-1 (p = 0.0142). No significant differences were found for IL-8, TNF-α, NF-kB, NLRP3, IL-1β, and gasdermin-D. Group B had higher levels of IL-6 (p < 0.0001), IL-18 (p = 0.002), CASP-9 (p < 0.0001), and HIF (p = 0.0327). We concluded that COVID-19 activates the NLRP3 inflammasome pathway, possibly leading to pyroptotic cell death mediated by caspase-1. In contrast, people with obesity without COVID-19, despite exhibiting some markers of the NLRP3 inflammasome, are more likely to experience necroptosis mediated by caspase-9. Full article

Lactoferrin-derived peptide chimera is a synthetic peptide that mimics the functional unit of lactoferrin with antibacterial activity. Although LF has anticancer effects, to the best of our knowledge, its effects on multiple myeloma have not yet been studied. We explored the potential of a lactoferrin-derived chimera for multiple myeloma treatment, a malignant clonal plasma cell bone marrow disease. The lactoferrin-derived chimera effectively inhibited MM1S, MM1R, and RPMI8226 multiple myeloma cell growth, and induced the early and late phases of apoptosis, but not in normal peripheral blood mononuclear cells. Furthermore, the lactoferrin-derived chimera modulates the relative expression of genes involved in survival, apoptosis, and mitochondrial dysfunction at the transcriptional level. Mitochondrial analysis revealed that lactoferrin-derived chimera triggered oxidative stress in multiple myeloma cells, leading to reactive oxygen species generation and a decline in mitochondrial membrane potential, resulting in mitochondrial dysfunction. Although lactoferrin-derived chimera did not cause caspase-dependent cell death, it induced nuclear translocation of apoptosis-inducing factor and endonuclease G, indicating the initiation of caspase-independent apoptosis. Overall, the lactoferrin-derived chimera induces caspase-independent programmed cell death in multiple myeloma cell lines by increasing the nuclear translocation of apoptosis-inducing factor/endonuclease G. Therefore, it has potential for multiple myeloma cancer therapies. Full article

Triple-negative breast cancer (TNBC) represents the most aggressive breast carcinoma subtype lacking efficient therapeutic options. A promising approach in cancer treatment is the pharmacological inhibition of murine double minute 2 (MDM2)-p53 interaction inducing apoptosis in p53 wild-type tumors. However, the role of MDM2 in TNBC with primarily mutant p53 is not well understood. We here selected the clinical-stage MDM2 inhibitors Idasanutlin and Milademetan and investigated their anti-tumoral effects in TNBC. When we analyzed anti-tumor activity in the TNBC cell lines MDA-MB-231, MDA-MB-436, and MDA-MB-468, cellular viability was efficiently reduced, with half maximal inhibitory concentration (IC₅₀) values ranging between 2.00 and 7.62 µM being up to 11-fold lower compared to the well-characterized non-clinical-stage MDM2 inhibitor Nutlin-3a. Furthermore, caspase-3/7 activity was efficiently induced. Importantly, the IC₅₀ values for MDM2 inhibition were equally observed in HCT116 p53^+/+ or HCT116 p53^−/− cells. Finally, the IC₅₀ was significantly higher in non-malignant MCF-10A cells than in TNBC cells. Taken together, Idasanutlin and Milademetan show a potent anti-tumor activity in TNBC cell culture models by efficiently inducing tumor cell death via apoptosis. This effect was observed despite an inactivating p53 mutation and was apparently independent of p53 expression. Our data suggest that MDM2 is a promising target in TNBC and clinical-stage MDM2 inhibitors should be further evaluated for their potential therapeutic application. Full article

Background/Objectives: Triple-negative breast cancer (TNBC) is the most challenging molecular subtype of breast cancer (BC) in clinical practice, associated with a worse prognosis due to limited treatment strategies and its insensitivity to conventional drugs. Zinc is an important trace element for homeostasis, and its Schiff base metal complexes have shown promise in treating advanced tumors. In this study, four new heteroleptic Zn(II) complexes (1–4) with Schiff bases were synthesized, characterized, and evaluated for their activity in BC cells. Methods: Compounds were synthesized, characterized, and their crystal structures were determined. Biological activity was assessed using MTT, clonogenic, scratch wound healing, caspase 3 and 8 activity, qPCR, and chemosensitization assays. Results: The complexes exhibited cytotoxicity against MCF-7 (luminal BC), MDA-MB-453 (HER2-positive BC), and MDA-MB-231 (TNBC) cell lines, with IC₅₀ values ranging from 0.01 to 20 µM. Complex 4 showed reduced cytotoxicity toward non-tumor cell lines. This, complexation with Zn(II) increased the cytotoxicity of the ligands, a trend not observed for complexes 1–3. Due to its favorable profile, complex 4 was selected for further assays, in which it inhibited colony formation and the cell migration of TNBC cells in a dose-dependent manner. Furthermore, this compound induced cell death independently of caspases, decreasing the activity of caspase 8. Interestingly, complex 4 sensitized TBNC cells to doxorubicin and paclitaxel, possibly modulating the epithelial–mesenchymal transition mechanism, as evidenced by increased CDH1 expression. Conclusions: Results suggest the potential of complex 4 in sensitizing aggressive BC cells to chemotherapy, proving to be a promising alternative in cases of therapeutic failure. Full article

Extremely premature infants are at significant risk for developing bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment (NDI). Although BPD is a predictor of poor neurodevelopmental outcomes, it is currently unknown how BPD contributes to brain injury and long-term NDI in pre-term infants. Extracellular vesicles (EVs) are small, membrane-bound structures released from cells into the surrounding environment. EVs are involved in inter-organ communication in diverse pathological processes. Inflammasomes are large, multiprotein complexes that are part of the innate immune system and are responsible for triggering inflammatory responses and cell death. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly and activating inflammatory caspase-1. Activated caspase-1 cleaves gasdermin D (GSDMD) to release a 30 kD N-terminal domain that can form membrane pores, leading to lytic cell death, also known as pyroptosis. Activated caspase-1 can also cleave pro-IL-1β and pro-IL-18 to their active forms, which can be rapidly released through the GSDMD pores to induce inflammation. Recent evidence has emerged that activation of inflammasomes is associated with neonatal lung and brain injury, and inhibition of inflammasomes reduces hyperoxia-induced neonatal lung and brain injury. Additionally, multiple studies have demonstrated that hyperoxia stimulates the release of lung-derived EVs that contain inflammasome cargos. Adoptive transfer of these EVs into the circulation of normal neonatal mice and rats induces brain inflammatory injury. This review focuses on EV–inflammasomes’ roles in mediating lung-to-brain crosstalk via EV-dependent and EV-independent mechanisms critical in BPD, brain injury, and NDI pathogenesis. EV–inflammasomes will be discussed as potential therapeutic targets for neonatal lung and brain injury. Full article

During virus infection, the activation of the antiviral endoribonuclease, ribonuclease L (RNase L), by a unique ligand 2′-5′-oilgoadenylate (2-5A) causes the cleavage of single-stranded viral and cellular RNA targets, restricting protein synthesis, activating stress response pathways, and promoting cell death to establish broad antiviral effects. The immunostimulatory dsRNA cleavage products of RNase L activity (RL RNAs) recruit diverse dsRNA sensors to activate signaling pathways to amplify interferon (IFN) production and activate inflammasome, but the sensors that promote cell death are not known. In this study, we found that DEAH-box polypeptide 15 (DHX15) and retinoic acid-inducible gene I (Rig-I) are essential for apoptosis induced by RL RNAs and require mitochondrial antiviral signaling (MAVS), c-Jun amino terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) for caspase-3-mediated intrinsic apoptosis. In RNase L-activated cells, DHX15 interacts with Rig-I and MAVS, and cells lacking MAVS expression were resistant to apoptosis. RL RNAs induced the transcription of genes for IFN and proinflammatory cytokines by interferon regulatory factor 3 (IRF-3) and nuclear factor kB (NF-kB), while cells lacking both DHX15 and Rig-I showed a reduced induction of cytokines. However, apoptotic cell death is independent of both IRF-3 and NF-kB, suggesting that cytokine and cell death induction by RL RNAs are uncoupled. The RNA binding of both DHX15 and Rig-I is required for apoptosis induction, and the expression of both single proteins in cells lacking both DHX15 and Rig-I is insufficient to promote cell death by RL RNAs. Cell death induced by RL RNAs suppressed Coxsackievirus B3 (CVB3) replication, and inhibiting caspase-3 activity or cells lacking IRF-3 showed that the induction of apoptosis directly resulted in the CVB3 antiviral effect, and the effects were independent of the role of IRF-3. Full article

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

Several studies have demonstrated, both in vitro and in animal models, the anti-tumor efficacy of high-dose ascorbate treatment against a variety of tumor entities, including glioblastoma, the most common and aggressive primary malignant brain tumor. The aim of this study was to investigate the effects of high-dose ascorbate as well as dehydroascorbic acid on human glioblastoma cell lines and to evaluate different treatment conditions for the combined administration of ascorbate with magnesium (Mg²⁺) and iron (Fe³⁺). Intracellular levels of reactive oxygen species and the induction of cell death following ascorbate treatment were also investigated. We demonstrated high cytotoxicity and antiproliferative efficacy of high-dose ascorbate in human glioblastoma cells, whereas much weaker effects were observed for dehydroascorbic acid. Ascorbate-induced cell death was independent of apoptosis. Both the reduction in cell viability and the ascorbate-induced generation of intracellular reactive oxygen species could be significantly increased by incubating the cells with Fe³⁺ before ascorbate treatment. This work demonstrates, for the first time, an increase in ascorbate-induced intracellular ROS formation and cytotoxicity in human glioblastoma cells by pre-treatment of the tumor cells with ferric iron, as well as caspase-3 independence of cell death induced by high-dose ascorbate. Instead, the cell death mechanism caused by high-dose ascorbate in glioblastoma cells shows evidence of ferroptosis. The results of the present work provide insights into the efficacy and mode of action of pharmacological ascorbate for the therapy of glioblastoma, as well as indications for possible approaches to increase the effectiveness of ascorbate treatment. Full article

Chronic pancreatitis (CP) in young individuals may lead to disease-related secondary sarcopenia (SSARC), characterized by muscle loss and systemic inflammation. In this study, CP was induced in young pigs, and serum levels of key hormones, muscle fiber diameters in various muscles, and the mRNA expression of genes related to oxidative stress and programmed cell death were assessed. A decrease in muscle fiber diameters was observed in SSARC pigs, particularly in the longissimus and diaphragm muscles. Hormonal analysis revealed alterations in dehydroepiandrosterone, testosterone, oxytocin, myostatin, and cortisol levels, indicating a distinct hormonal response in SSARC pigs compared to controls. Oxytocin levels in SSARC pigs were significantly lower and myostatin levels higher. Additionally, changes in the expression of catalase (CAT), caspase 8 (CASP8), B-cell lymphoma 2 (BCL2), and BCL2-associated X protein (BAX) mRNA suggested a downregulation of oxidative stress response and apoptosis regulation. A reduced BAX/BCL2 ratio in SSARC pigs implied potential caspase-independent cell death pathways. The findings highlight the complex interplay between hormonal changes and muscle degradation in SSARC, underscoring the need for further research into the apoptotic and inflammatory pathways involved in muscle changes due to chronic organ inflammation in young individuals. Full article