APAF1 Silencing Ameliorates Diabetic Retinopathy by Suppressing Inflammation, Oxidative Stress, and Caspase-3/GSDME-Dependent Pyroptosis.

Objective: Diabetic retinopathy (DR) can cause permanent blindness with unstated pathogenesis. We aim to find novel biomarkers and explore the mechanism of apoptotic protease activating factor 1 (APAF1) in DR. Methods: Differential expression genes (DEGs) were screened based on GSE60436 dataset to find hub genes involved in pyroptosis after comprehensive bioinformatics analysis. DR mice model was constructed by streptozotocin injection. The pathological structure of retina was observed using hematoxylin-eosin staining. The enzyme-linked immunosorbent assay was applied to assess inflammatory factors, vascular endothelial growth factor (VEGF), and oxidative stress. The mRNA and protein expression levels were detected using quantitative real-time polymerase-chain reaction and Western blot. Cell counting kit and flow cytometry were employed to detect proliferation and apoptosis in high glucose-induced ARPE-19 cells. Results: Total 71 pyroptosis-related DEGs were screened. BIRC2, CXCL8, APAF1, PPARG, TP53, and CYCS were identified as hub genes of DR. APAF1 was selected as a potential regulator of DR, which was up-regulated in DR mice. APAF1 silencing alleviated retinopathy and inhibited pyroptosis in DR mice with decreased levels of inflammatory factors, VEGF, and oxidative stress. Moreover, APAF1 silencing promoted proliferation while inhibiting apoptosis and caspase-3/GSDME-dependent pyroptosis with a decrease in TNF-α, IL-1ß, IL-18, and lactate dehydrogenase in high glucose-induced ARPE-19 cells. Additionally, caspase-3 activator reversed the promotion effect on proliferation and inhibitory effect on apoptosis and pyroptosis after APAF1 silencing in high glucose-induced ARPE-19 cells. Conclusion: APAF1 is a novel biomarker for DR and APAF1 silencing inhibits the development of DR by suppressing caspase-3/GSDME-dependent pyroptosis.

Altered conformational dynamics contribute to species-specific effects of cytochrome c mutations on caspase activation.

Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human cytochrome c, this phenotype is not recapitulated in mice for the sole mutant (G41S) that has been investigated. The effect of the G41S mutation on the in vitro activities of cytochrome c is also not conserved between human and mouse. Peroxidase activity is increased in both mouse and human G41S variants, whereas apoptosome activation is increased for human G41S cytochrome c but decreased for mouse G41S cytochrome c. These apoptotic activities of cytochrome c are regulated at least in part by conformational dynamics of the main chain. Here we use computational and in vitro approaches to understand why the impact of the G41S mutation differs between mouse and human cytochromes c. The G41S mutation increases the inherent entropy and main chain mobility of human but not mouse cytochrome c. Exclusively in human G41S cytochrome c this is accompanied by a decrease in occupancy of H-bonds between protein and heme during simulations. These data demonstrate that binding of cytochrome c to Apaf-1 to trigger apoptosome formation, but not the peroxidase activity of cytochrome c, is enhanced by increased mobility of the native protein conformation.

Gestational exposure to haloperidol changes Cdkn1a and Apaf1 mRNA expressions in mouse hippocampus.

BACKGROUND: The onset of schizophrenia is associated with both genetic and environmental risks during brain development. Environmental factors during pregnancy can represent risk factors for schizophrenia, and we have previously reported that several microRNA and mRNA expression changes in fetal brains exposed to haloperidol during pregnancy may be related to the onset of this disease. This study aimed to replicate that research and focused on apoptotic-related gene expression changes. METHODS: Haloperidol (1 mg/kg) or aripiprazole (1 mg/kg) was injected into pregnant mice. Using RNA sequencing for the hippocampus of each offspring born from pregnant mice exposed to haloperidol, we analyzed genes identified as changed in our previous report and validated two apoptosis-related genes (Cdkn1a and Apaf1) using quantitative polymerase chain reaction (qPCR) methods. Furthermore, we attempted to elucidate the direct effects of haloperidol and aripiprazole on those mRNA expressions in in vitro experiments. RESULTS: RNA sequencing successfully replicated 16 up-regulated and 5 down-regulated genes in this study. Of those, up-regulations of Cdkn1a and Apaf1 mRNA expression were successfully validated by direct quantification. Moreover, haloperidol and aripiprazole dose-dependent upregulation of both mRNA expressions were confirmed in a Neuro2a cell line. CONCLUSIONS: In the hippocampus of offspring, intraperitoneal injection of haloperidol to pregnant mice induced up-regulation of apoptotic genes that representing the phenotypic change without apoptosis. These findings will be useful for understanding the molecular biological mechanisms underlying the effects of antipsychotics on the fetal brain.

Upregulation of APAF1 and CSF1R in Peripheral Blood Mononuclear Cells of Parkinson's Disease.

Increased oxidative stress and neuroinflammation play a crucial role in the pathogenesis of Parkinson's disease (PD). In this study, the expression levels of 52 genes related to oxidative stress and inflammation were measured in peripheral blood mononuclear cells of the discovery cohort including 48 PD patients and 25 healthy controls. Four genes, including ALDH1A, APAF1, CR1, and CSF1R, were found to be upregulated in PD patients. The expression patterns of these genes were validated in a second cohort of 101 PD patients and 61 healthy controls. The results confirmed the upregulation of APAF1 (PD: 0.34 ± 0.18, control: 0.26 ± 0.11, p < 0.001) and CSF1R (PD: 0.38 ± 0.12, control: 0.33 ± 0.10, p = 0.005) in PD patients. The expression level of APAF1 was correlated with the scores of the Unified Parkinson's Disease Rating Scale (UPDRS, r = 0.235, p = 0.018) and 39-item PD questionnaire (PDQ-39, r = 0.250, p = 0.012). The expression level of CSF1R was negatively correlated with the scores of the mini-mental status examination (MMSE, r = -0.200, p = 0.047) and Montréal Cognitive Assessment (MoCA, r = -0.226, p = 0.023). These results highly suggest that oxidative stress biomarkers in peripheral blood may be useful in monitoring the progression of motor disabilities and cognitive decline in PD patients.

Impairment of apoptosis pathway via Apaf1 downregulation during chlorpyrifos and/or cypermethrin induced lung damage.

Chlorpyrifos is an organophosphate and the cypermethrin is type 2 pyrethroid insecticide that are used for indoor and outdoor pest control. The present study aimed to investigate differential transcriptional profiling to identify the candidate gene associated with lung injury following exposure to chlorpyrifos and/or cypermethrin in a mouse model system. Swiss male albino mice (n = 24) were divided into three treatment groups (n = 6 each) that were given chlorpyrifos (2.76 mg kg-1 body weight), cypermethrin (2 mg kg-1 body weight) and the combination of both pesticides orally dissolved in corn oil and one control group (n = 6) that received corn oil for 90 days. The pulmonary expression of the Apaf1 was observed using RT2 Profiler PCR Array. The results showed that chronic exposure to chlorpyrifos, cypermethrin and their combination downregulated (67, 63 and 66 genes) and upregulated (4, 2 and 2 genes), respectively. The pulmonary expression of Apaf1 that plays important role in apoptosis was found to be downregulated. The immunohistochemistry depicted reduced expression of Apaf1 in both airway epithelium and alveolar septa following exposure to chlorpyrifos and/or cypermethrin. In conclusion, results demonstrated that exposure to chlorpyrifos, cypermethrin and their combination cause lung damage by the dysregulation of Apaf1 gene expression.

Effect of cadmium chloride toxicity on olfactory mucosa / Efecto de la toxicidad del cloruro de cadmio en la mucosa olfativa

SUMMARY: Cadmium is a highly toxic metal and affects the respiratory mucosa. The aim of the study is to show the inflammation and degenerative effect of cadmium on the olfactory mucosa. In this study, eight-week-old Wistar rats with an average weight of 170-190 g were divided into two groups (control and experiment) with 20 animals in each group and used in the experiments. The rats in the experimental group were given 2 mg/kg/day powdered cadmium chloride dissolved in water intraperitoneally every day for two weeks. At the end of the experiment, the nasal cavity was completely removed with anesthesia. Concha nasalis superior was separated, fixed with zinc-Formalin solution and decalcified with 5 % EDTA (Ethylene-diaminetetraacetic acid). After routine histopathological procedure, APAF-1 antibody was used for expression of Hematoxylin-Eosin (HE) and immunohistochemistry. Histopathological examination revealed interruptions in the basement membrane structure due to cadmium and degenerative changes in stem cells, degeneration in sensory cells and pycnosis in nuclei, dilatation in blood vessels and increased inflammation in connective tissue. APAF-1 expression was found to increase in epithelial cells and olfactory glands (Bowman gland) cells. It has been thought that cadmium toxicity increases cell degeneration and inflammation in the olfactory mucosa and may significantly affect cell death and olfactory metabolism by inducing the pro-apoptotic process.

El cadmio es un metal altamente tóxico que afecta la mucosa respiratoria. El objetivo fue mostrar el efecto inflamatorio y degenerativo del cadmio sobre la mucosa olfativa. En este estudio, ratas Wistar de ocho semanas de edad con un peso promedio de 170-190 g se dividieron en dos grupos (control y experimental) con 20 animales en cada grupo. Las ratas del grupo experimental recibieron 2 mg/kg/día de cloruro de cadmio en polvo disuelto en agua por vía intraperitoneal todos los días durante dos semanas. En los animales se exirpó la cavidad nasal bajo anestesia. Se separó la concha nasal superior, se fijó con solución de zinc-Formalina y se descalcificó con EDTA (ácido etilendiaminotetraacético) al 5 %. Después del procedimiento histopatológico de rutina, Hematoxilina- Eosina (HE) e inmunohistoquímica, se utilizó el anticuerpo APAF-1. El examen histopatológico reveló interrupciones en la estructura de la membrana basal debido al cadmio y cambios degenerativos en las células madre, degeneración en las células sensoriales y picnosis en los núcleos, dilatación de los vasos sanguíneos y aumento de la inflamación en el tejido conjuntivo. Se encontró que la expresión de APAF-1 aumenta en las células epiteliales y en las células de las glándulas olfatorias (glándulas de Bowman). Se ha pensado que la toxicidad del cadmio aumenta la degeneración celular y la inflamación en la mucosa olfativa y puede afectar significativamente la muerte celular y el metabolismo olfativo al inducir el proceso proapoptótico.

Targeting the MITF/APAF-1 axis as salvage therapy for MAPK inhibitors in resistant melanoma.

Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in in vitro and in vivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma.

APAF1-Binding Long Noncoding RNA Promotes Tumor Growth and Multidrug Resistance in Gastric Cancer by Blocking Apoptosome Assembly.

Chemotherapeutics remain the first choice for advanced gastric cancers (GCs). However, drug resistance and unavoidable severe toxicity lead to chemotherapy failure and poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in tumor progression in many cancers, including GC. Here, through RNA screening, an apoptotic protease-activating factor 1 (APAF1)-binding lncRNA (ABL) that is significantly elevated in cancerous GC tissues and an independent prognostic factor for GC patients is identified. Moreover, ABL overexpression inhibits GC cell apoptosis and promotes GC cell survival and multidrug resistance in GC xenograft and organoid models. Mechanistically, ABL directly binds to the RNA-binding protein IGF2BP1 via its KH1/2 domain, and then IGF2BP1 further recognizes the METTL3-mediated m6A modification on ABL, which maintains ABL stability. In addition, ABL can bind to the WD1/WD2 domain of APAF1, which competitively prevent cytochrome c from interacting with APAF1, blocking apoptosome assembly and caspase-9/3 activation; these events lead to resistance to cell death in GC cells. Intriguingly, targeting ABL using encapsulated liposomal siRNA can significantly enhance the sensitivity of GC cells to chemotherapy. Collectively, the results suggest that ABL can be a potential prognostic biomarker and therapeutic target in GC.

Novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death.

Background & Aims: The stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress. Methods: A mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice. Results: The TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-ß (IFN-ß) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis. Conclusions: Macrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases. Lay summary: Liver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.

Neuroprotective effects of combined trimetazidine and progesterone on cerebral reperfusion injury.

Cerebral ischemia-reperfusion injury induces multi-dimensional damage to neuronal cells through exacerbation of critical protective mechanisms. Targeting more than one mechanism simultaneously namely, inflammatory responses and metabolic energy homeostasis could provide additional benefits to restrict or manage cerebral injury. Being proven neuroprotective agents both, progesterone (PG) and trimetazidine (TMZ) has the potential to add on the individual therapeutic outcomes. We hypothesized the simultaneous administration of PG and TMZ could complement each other to synergize, or at least enhance neuroprotection in reperfusion injury. We investigated the combination of PG and TMZ on middle cerebral artery occlusion (MCAO) induced cerebral reperfusion injury in rats. Molecular docking on targets of energy homeostasis and apoptosis assessed the initial viability of PG and TMZ for neuroprotection. Animal experimentation with MCA induced ischemia-reperfusion (I/R) injury in rats was performed on five randomized groups. Sham operated control group received vehicle (saline) while the other four I-R groups were pre-treated with vehicle (saline), PG (8 â€‹mg/kg), TMZ treated (25 â€‹mg/kg), and PG â€‹+ â€‹TMZ (8 and 25 â€‹mg/kg) for 7 days by intraperitoneal route. Neurological deficit, infarct volume, and oxidative stress were evaluated to assess the extent of injury in rats. Inflammatory reactivity and apoptotic activity were determined with alterations in myeloperoxidase (MPO) activity, blood-brain barrier (BBB) permeability, and DNA fragments. Reperfusion injury inflicted cerebral infarct, neurological deficit, and shattered BBB integrity. The combination treatment of PG and TMZ restricted cellular damage indicated by significant (p â€‹< â€‹0.05) decrease in infarct volume and improvement in free radical scavenging ability (SOD activity and GSH level). MPO activity and LPO decreased which contributed in improved BBB integrity in treated rats. We speculate that inhibition of inflammatory and optimum energy utilization would critically contribute to observed neuroprotection with combined PG and TMZ treatment. Further exploration of this neuroprotective approach for post-recovery cognitive improvement is worth investigating.

Allele Frequency of APAF1 Mutation in Holstein Cattle in Brazil.

APAF1 is an autosomal recessive inherited mutation, associated with Holstein haplotype 1 (HH1) and characterized by a substitution of cytosine for a thymine (c.1741C>T) in chromosome 5. The mutation causes fetal and embryonic loss, between 60 and 200 days of gestation, and reduced conception rate. The ARMS-PCR is considered a simple and low-cost method to determine single nucleotide polymorphism (SNP) with no need for genetic sequencing of the animal genome. This study aimed to verify the allelic frequency of APAF1 mutation in Brazilian Holstein cattle. A total of 248 Holstein DNA samples (210 cows and 38 bulls) were analyzed, and synthetic genes were manufactured to validate the primers developed by the authors. All animals assessed in this study were classified as wild-type for APAF1 mutation. The primers and protocol developed for the ARMS-PCR technique work with 100% specificity and efficiency since the amplicon formations are as expected according to the genotypes. In conclusion, the mutation responsible for APAF1 was not detected in the Brazilian Holstein cattle population assessed in this prevalence study, although it is not possible to affirm that APAF1 does not occur in Brazilian Holstein animals. The tetra-primer ARMS-PCR protocol for APAF1 mutation that has been validated here may be a relatively simple and economical method to determine the animals' genotype.

Non-Canonical Roles of Apoptotic Caspases in the Nervous System.

Caspases are a family of cysteine proteases that predominantly cleave their substrates after aspartic acid residues. Much of what we know of caspases emerged from investigation a highly conserved form of programmed cell death called apoptosis. This form of cell death is regulated by several caspases, including caspase-2, caspase-3, caspase-7, caspase-8 and caspase-9. However, these "killer" apoptotic caspases have emerged as versatile enzymes that play key roles in a wide range of non-apoptotic processes. Much of what we understand about these non-apoptotic roles is built on work investigating how "killer" caspases control a range of neuronal cell behaviors. This review will attempt to provide an up to date synopsis of these roles.

Apoptotic protease activating factor-1 gene and MicroRNA-484: A possible interplay in relapsing remitting multiple sclerosis.

BACKGROUND: Emerging evidence suggests that dysregulated apoptosis might be implicated in the pathogenesis of multiple sclerosis (MS). The aim of the current study was to evaluate the expression of Apoptotic protease activating factor-1 (APAF1) mRNA and its potential regulator miR-484 in relapsing remitting MS patients (RRMS) and to investigate their role as potential disease biomarkers. METHODS: After Bioinformatic analysis was conducted and revealed miR-484 involvement in the regulation of APAF-1 gene expression. Reverse Transcription-quantitative Real-Time PCR (RT-qPCR) was performed to detect the expression levels of APAF-1 and miR-484 in the peripheral blood mononuclear cells (PBMCs) of 34 RRMS patients recruited from the MS clinic of kasr al ainy hospital- faculty of medicine-Egypt and 34 healthy controls. RESULTS: APAF-1 mRNA was significantly downregulated in patients whereas miR-484 expression was upregulated compared to controls (p < 0.01). Sensitivity and specificity of APAF-1 and miR-484 to diagnose MS was (85.3%, 76.5%) and (88.2% and 86.7%) respectively. CONCLUSION: APAF-1 and miR-484 could play a role as potential MS diagnostic biomarkers. However, absence of a control group of patients with other inflammatory diseases in our study warrants further research to corroborate our findings.

Exosomal microRNA⁃93⁃3p secreted by bone marrow mesenchymal stem cells downregulates apoptotic peptidase activating factor 1 to promote wound healing.

Wounds are soft tissue injuries, which are difficult to heal and can easily lead to other skin diseases. Bone marrow mesenchymal stem cells (BMSCs) and the secreted exosomes play a key role in skin wound healing. This study aims to clarify the effects and mechanisms of exosomes derived from BMSCs in wound healing. Exosomes were extracted from the supernatant of the BMSCs. The expression of the micro-RNA miR-93-3p was determined by qRT-PCR analysis. HaCaT cells were exposed to hydrogen peroxide (H2O2) to establish a skin lesion model. MTT, flow cytometry, and transwell assays were conducted to determine cellular functions. The binding relationship between miR-93-3p and apoptotic peptidase activating factor 1 (APAF1) was measured using a dual luciferase reporter gene assay. The results showed that BMSC-derived exosomes or BMSC-exos promoted proliferation and migration and suppressed apoptosis in HaCaT cells damaged by H2O2. However, the depletion of miR-93-3p in BMSC-exos antagonized the effects of BMSC-exos on HaCaT cells. In addition, APAF1 was identified as a target of miR-93-3p. Overexpression of APAF1 induced the dysfunction of HaCaT cells. Collectively, the results indicate that BMSC-derived exosomes promote skin wound healing via the miR-93-3p/APAF1 axis. This finding may help establish a new therapeutic strategy for skin wound healing.

Global Reprogramming of Apoptosis-Related Genes during Brain Development.

To enable long-term survival, mammalian adult neurons exhibit unique apoptosis competence. Questions remain as to whether and how neurons globally reprogram the expression of apoptotic genes during development. We systematically examined the in vivo expression of 1923 apoptosis-related genes and associated histone modifications at eight developmental ages of mouse brains. Most apoptotic genes displayed consistent temporal patterns across the forebrain, midbrain, and hindbrain, suggesting ubiquitous robust developmental reprogramming. Although both anti- and pro-apoptotic genes can be up- or downregulated, half the regulatory events in the classical apoptosis pathway are downregulation of pro-apoptotic genes. Reduced expression in initiator caspases, apoptosome, and pro-apoptotic Bcl-2 family members restrains effector caspase activation and attenuates neuronal apoptosis. The developmental downregulation of apoptotic genes is attributed to decreasing histone-3-lysine-4-trimethylation (H3K4me3) signals at promoters, where histone-3-lysine-27-trimethylation (H3K27me3) rarely changes. By contrast, repressive H3K27me3 marks are lost in the upregulated gene groups, for which developmental H3K4me3 changes are not predictive. Hence, developing brains remove epigenetic H3K4me3 and H3K27me3 marks on different apoptotic gene groups, contributing to their downregulation and upregulation, respectively. As such, neurons drastically alter global apoptotic gene expression during development to transform apoptosis controls. Research into neuronal cell death should consider maturation stages as a biological variable.

Over Fifty Years of Life, Death, and Cannibalism: A Historical Recollection of Apoptosis and Autophagy.

Research in biomedical sciences has changed dramatically over the past fifty years. There is no doubt that the discovery of apoptosis and autophagy as two highly synchronized and regulated mechanisms in cellular homeostasis are among the most important discoveries in these decades. Along with the advancement in molecular biology, identifying the genetic players in apoptosis and autophagy has shed light on our understanding of their function in physiological and pathological conditions. In this review, we first describe the history of key discoveries in apoptosis with a molecular insight and continue with apoptosis pathways and their regulation. We touch upon the role of apoptosis in human health and its malfunction in several diseases. We discuss the path to the morphological and molecular discovery of autophagy. Moreover, we dive deep into the precise regulation of autophagy and recent findings from basic research to clinical applications of autophagy modulation in human health and illnesses and the available therapies for many diseases caused by impaired autophagy. We conclude with the exciting crosstalk between apoptosis and autophagy, from the early discoveries to recent findings.

Immunohistochemical Markers of Apoptotic and Hypoxic Damage Facilitate Evidence-Based Assessment in Pups with Neurological Disorders.

Seizures in puppies often present a diagnostic challenge in terms of identifying and treating the underlying cause. Dog breeds with mutations of the MDR1-gene are known to show adverse reactions to certain drugs, yet metabolic imbalance exacerbated by physiologically immature organs and other contributing pathologies require consideration before arriving at a diagnosis. This study analysed the brains of two male, 5-week-old Australian Shepherd siblings that died after displaying severe neurological symptoms upon administration of MilproVet® to treat severe intestinal helminth infection. Despite the initial symptoms being similar, their case histories varied in terms of the symptom duration, access to supportive therapy and post-mortem interval. Histopathology and immunohistochemistry were used to obtain more information about the phase of the pathological processes in the brain, employing protein markers associated with acute hypoxic damage (ß-amyloid precursor protein/APP) and apoptosis (diacylglycerolkinase-ζ/DGK-ζ, apoptotic protease activating factor 1/Apaf1, and B-cell lymphoma related protein 2/Bcl-2). The results seem to reflect the course of the animals' clinical deterioration, implicating that the hypoxic damage to the brains was incompatible with life, and suggesting the usefulness of the mentioned immunohistochemical markers in clarifying the cause of death in animals with acute neurological deficits.

Solitary Fibrous Tumor: Integration of Clinical, Morphologic, Immunohistochemical and Molecular Findings in Risk Stratification and Classification May Better Predict Patient outcome.

Although solitary fibrous tumors (SFTs) have an unpredictable evolution, some specific clinicopathologic factors have been associated with the final outcome. We retrieved clinical, pathological and molecular data of 97 patients with a histological diagnosis of SFT and Signal transducer and activator of transcription 6 (STAT6) positivity. We retrospectively studied the pathological factors predictive of recurrence/metastasis and compared them with the clinical outcome. A wide immunohistochemical study and molecular analysis to detect NAB2/STAT6 gene fusion, tumor protein-53 (TP53) and/or (telomerase reverse transcriptase) TERT promotor mutation were performed. The risk of metastasis was calculated using the Demicco risk stratification system (RSS). The results were combined and examined to assess the accuracy of risk stratification and classification. The most common location was in non-extremities; 66% were located in soft tissue or subcutaneous areas and 92.8% in deep locations. On microscopic analysis, 38.1% of tumors revealed hypercellularity with a predominant patternless and/or hemangiopericytic growth pattern; 13.4% had ≥4 mitoses/10HPF; 16.5% showed necrosis, and almost half the tumors showed at least focal myxoid areas. Dedifferentiation was observed in three tumors. Immunomarker expression in SFTs was as follows: CD34 92.9%, CD99 57.1%, Bcl2 67.9%, neuroendocrine markers (at least 1) 25.7%, Desmin 14.3%, CK(AE1/AE3) 3%, Apoptotic Protease Activating Factor (APAF-1) 87% and finally Ki-67 ≥ 10% in 14.4%. The NAB2/STAT6 gene fusion was detected in 50 tumors. After a median follow-up of 90 months, 9.3% recurred, 11.3% metastasized, 10.3% died of disease and 76.2% were free of disease. TERT mutations were detected in 40.6% of the SFTs; the TP53 mutation was detected in 17%, and only 9.3% showed both mutations. According to the Demicco RSS, 6.1%, 11.3% and 82.4% of the tumors were classified as high, intermediate or low-risk of metastasis, respectively. All high-risk tumors had ≥4 mitoses/10HPF, necrosis, Ki-67 ≥ 10, HTER and/or TP53 mutation and poor evolution. The intermediate risk SFTs with worse evolution displayed the HTER mutation. Almost all low-risk tumors had a favorable evolution, although four showed at least one adverse factor (Ki-67 ≥ 10, ≥4 mitoses/10HPF or high tumor size) and had a worse evolution. An integration of clinical, morphologic, immunohistochemical and molecular findings may improve risk stratification and classification and better predict patient outcome. The unfavorable course seems to be more frequent in high-risk SFTs, although it is not exceptional in low-risk SFTs either; hence, a long-term follow-up is required independently of the assigned risk stratification score. The inclusion of molecular findings in risk stratification systems could improve the precision in the classification of SFTs, especially those of intermediate risk. Future studies will be required to determine the most effective way to incorporate molecular analyses into RSS on SFTs. The coexistence of several adverse factors such as ≥4 mitoses/10HPF, necrosis, Ki-67 ≥ 10%, mutations in HTER and/or p53 may suggest a closer clinical follow-up regardless of the histological appearance of the tumor.

Apaf1 nanoLuc biosensors identified lentinan as a potent synergizer of cisplatin in targeting hepatocellular carcinoma cells.

Liver cancer is one of the most common malignancies that is difficult to treat due to late diagnosis and chemo-resistance. In the present study, we developed and validated a cell based split nanoLuc biosensor to monitor the Apaf1-Apaf1 interactions in response to apoptosis-inducing drugs such as cisplatin. We showed that the activity of split nanoLuc is reconstituted only in response to apoptotic inducer, cisplatin and in a dose-dependent manner. Apaf1 mutants which were unable to oligomerize failed to recover nanoLuc activity while constitutively active variant increased the nanoLuc activity. Generation of Apaf1 knockout HepG2 and treatment with cisplatin showed dramatic reduction in cell death suggesting that cisplatin mainly targets liver cancer cells through apoptosis. As the natural products are potent sources of compounds for adjuvant therapy, we screened a collection of natural products and identified lentinan as an inducer of apoptosome formation, a key step for induction of apoptosis. Lentinan is a polysaccharide with antitumor, pro-apoptotic properties that functions with poorly understood mechanisms. Lentinan was shown to have cytotoxic effects with the IC50 of 650 µM. Sub-lethal lentinan concentration doubled the nanoLuc activity when co-treated with cisplatin. We also showed that lentinan hugely reduced the dose of cisplatin to induce certain amount of death and that lentinan co-treatment with cisplatin enhanced the Apaf1 transcription in HepG2 cells while lentinan or cisplatin alone failed to alter the transcription. In addition, lentinan and cisplatin co-treatment induced mitochondrial depolarization. This suggested that lentinan combinatorial therapy with cisplatin engaged a different signalling pathway to kill the liver cancer cells and that adjuvant therapy with lentinan can reduce the dose of cisplatin and thus reduce the possibility of chemo-resistance.

Comprehensive Profiling of Hypoxia-Related miRNAs Identifies miR-23a-3p Overexpression as a Marker of Platinum Resistance and Poor Prognosis in High-Grade Serous Ovarian Cancer.

The onset of chemo-resistant recurrence represents the principal cause of high-grade serous ovarian carcinoma (HGSOC) death. HGSOC masses are characterized by a hypoxic microenvironment, which contributes to the development of this chemo-resistant phenotype. Hypoxia regulated-miRNAs (HRMs) represent a molecular response of cancer cells to hypoxia and are involved in tumor progression. We investigated the expression of HRMs using miRNA expression data from a total of 273 advanced-stage HGSOC samples. The miRNAs associated with chemoresistance and survival were validated by RT-qPCR and target prediction, and comparative pathway analysis was conducted for target gene identification. Analysis of miRNA expression profiles indicated miR-23a-3p and miR-181c-5p over-expression as associated with chemoresistance and poor PFS. RT-qPCR data confirmed upregulation of miR-23a-3p in tumors from chemoresistant HGSOC patients and its significant association with shorter PFS. In silico miR-23a-3p target prediction and comparative pathway analysis identified platinum drug resistance as the pathway with the highest number of miR-23a-3p target genes. Among them, APAF-1 emerged as the most promising, being downregulated in platinum-resistant patients and in HGSOC chemo-resistant cells. These results highlight miR-23a-3p as a potential biomarker for HGSOC platinum response and prognosis and the miR23a-3p/APAF1 axis as a possible target to overcome platinum-resistance.