Apoptin and apoptotic protease-activating factor 1 plasmid-assisted multi-functional nanoparticles in hepatocellular carcinoma therapy.

Cancer drugs usually have side effects in chemotherapy. Apoptin, a protein recognized by its good therapeutical effect on tumors and innocuous to body, is employed to treat hepatocellular carcinoma (HCC). As our previous data shown, the efficiency of apoptin protein might be limited by the protein of apaf-1. Therefore, we designed the multi-functional nanoparticles (MFNPs) encapsulating apoptin and apaf-1 plasmids by layer-by layer assembly. The NPs could release drugs into tumor site specifically and had good compatibility to normal cells and tissues. The groups of biotin, ε-polylysine, and nuclear localization signal in MFNPs conferred NPs the capabilities to enter cancer cells specifically, escape lysosome and enter the nucleus, respectively. In vitro inhibition experiment and in vivo anti-tumor therapy confirmed MFNPs as an excellent carrier to treat HCC. In addition, the dual-drug system was superior to any of the single-drug system. The mechanism analysis proved that supplement of the protein of apaf-1 might enhance apoptosome formation, causing the increase of therapeutical efficacy.

Cdyl2-60aa encoded by CircCDYL2 accelerates cardiomyocyte death by blocking APAF1 ubiquitination in rats.

The loss of cardiomyocytes (CMs) after myocardial infarction (MI) is widely acknowledged to initiate the development of heart failure (HF). Herein, we found that circCDYL2 (583 nt) derived from chromodomain Y-like 2 (Cdyl2) is significantly upregulated in vitro (oxygen-glucose deprivation (OGD)-treated CMs) and in vivo (failing heart post-MI) and can be translated into a polypeptide termed Cdyl2-60aa (~7 kDa) in the presence of internal ribosomal entry sites (IRES). Downregulation of circCDYL2 significantly decreased the loss of OGD-treated CMs or the infarcted area of the heart post-MI. Additionally, elevated circCDYL2 significantly accelerated CM apoptosis via Cdyl2-60aa. We then discovered that Cdyl2-60aa could stabilize protein apoptotic protease activating factor-1 (APAF1) and promote CM apoptosis; heat shock protein 70 (HSP70) mediated APAF1 degradation in CMs by ubiquitinating APAF1, which Cdyl2-60aa could competitively block. In conclusion, our work substantiated the claim that circCDYL2 could promote CM apoptosis via Cdyl2-60aa, which enhanced APAF1 stability by blocking its ubiquitination by HSP70, suggesting that it is a therapeutic target for HF post-MI in rats.

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.

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.

LncRNA UCA1 epigenetically suppresses APAF1 expression to mediate the protective effect of sevoflurane against myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MI/RI) is a leading cause of death globally. Whereas some long noncoding RNAs (lncRNAs) are known to participate in the progression of MI/RI, the role of urothelial carcinoma associated 1 (UCA1) in conjunction with sevoflurane treatment remains largely unknown. H9C2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to establish an in vitro MI/RI model, and sevoflurane was then added. Cell viability, apoptosis, SOD activity, and MDA levels were measured. Levels of inflammatory cytokines and methylation of apoptosis protease-activating factor 1 (APAF1) were determined. Interactions among lncRNA UCA1, enhancer of zeste homologue 2 (EZH2), DNA methyltransferase-1 (DNMT1), and APAF1 were analyzed. After H/R treatment, the viability of H9C2 cardiomyocytes decreased and apoptosis rate, oxidative stress factor levels, inflammatory cytokine levels, and apoptosis-related protein levels all increased. Sevoflurane treatment reversed these changes. LncRNA UCA1 knockdown attenuated the therapeutic effect of sevoflurane on H/R-treated cardiomyocytes, and silencing of APAF1 reversed this role of UCA1 knockdown. Moreover, lncRNA UCA1 recruited DNMT1 through EZH2, thus promoting methylation of the APAF1 promoter region. LncRNA UCA1 recruits DNMT1 to promote methylation of the APAF1 promoter through EZH2, thus strengthening the protective effect of sevoflurane on H/R-induced cardiomyocyte injury.

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.

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.

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.

Methylation of Promoters of Apoptosis-Related Genes in Blood Lymphocytes of Workers Exposed to Occupational External Irradiation.

We studied the effect of technogenic radiation on the degree of promoter methylation in genes involved in apoptosis in blood lymphocytes of workers exposed to long-term γ-radiation during their professional activities. Blood samples for the analysis were obtained from 11 conventionally healthy men aged from 54 to 71 years (mean 66 years), workers of the Siberian Group of Chemical Enterprises working experience from 27 to 40 years (mean 30 years); the external exposure dose was 175.88 mSv (158.20-207.81 mSv). In all examined subjects, the degree of methylation of the promoters of apoptosis-related genes ranged from 0.22 to 50.00%. A correlation was found between the degree of methylation of BCLAF1 promoters (p=0.035) with the age of workers, BAX promoters (p=0.0289) with high content of aberrant cells, and APAF1 promoters (p=0.0152) with increased number of dicentric chromosomes. A relationship was found between the dose of external irradiation and the degree of methylation of gene promoters of BAD (p=0.0388), BID (р=0.0426), and HRK (р=0.0101) genes.

Contribution of Apaf-1 to the pathogenesis of cancer and neurodegenerative diseases.

Deregulation of apoptosis is associated with various pathologies, such as neurodegenerative disorders at one end of the spectrum and cancer at the other end. Generally speaking, differentiated cells like cardiomyocytes, skeletal myocytes and neurons exhibit low levels of Apaf-1 (Apoptotic protease activating factor 1) protein suggesting that down-regulation of Apaf-1 is an important event contributing to the resistance of these cells to apoptosis. Nonetheless, upregulation of Apaf-1 has not emerged as a common phenomenon in pathologies associated with enhanced neuronal cell death, i.e., neurodegenerative diseases. In cancer, on the other hand, Apaf-1 downregulation is a common phenomenon, which occurs through various mechanisms including mRNA hyper-methylation, gene methylation, Apaf-1 localization in lipid rafts, inhibition by microRNAs, phosphorylation, and interaction with specific inhibitors. Due to the diversity of these mechanisms and involvement of other factors, defining the exact contribution of Apaf-1 to the development of cancer in general and neurodegenerative disorders, in particular, is complicated. The current review is an attempt to provide a comprehensive image of Apaf-1's contribution to the pathologies observed in cancer and neurodegenerative diseases with the emphasis on the therapeutic aspects of Apaf-1 as an important target in these pathologies.

PTB domain and leucine zipper motif 1 (APPL1) inhibits myocardial ischemia/hypoxia-reperfusion injury via inactivation of apoptotic protease activating factor-1 (APAF-1)/Caspase9 signaling pathway.

Myocardial ischemia/hypoxia-reperfusion injury mediates the progression of multiple cardiovascular diseases. It has been reported that knockdown of adaptor protein containing a PH domain, PTB domain and leucine zipper motif 1 (APPL1) is a significant factor for the progression of myocardial injury. However, the role of APPL1 in myocardial ischemia remains unclear. Hence, the aim of the present study was to investigate the specific mechanism underlying the role of APPL1 in myocardial ischemia.In our study, the mRNA level of APPL1 was detected by quantitative real-time PCR (RT-qPCR). The expressions of APPL1, Apoptotic protease activating factor-1 (APAF-1), cleaved caspase9 and other inflammation- and apoptosis-related proteins were determined by western blotting. The secretion of inflammatory cytokines and lactate dehydrogenase (LDH) levels were measured by commercial assay kits. The H9C2 cell viability was analyzed by cell counting kit-8 (CCK-8) assay. The apoptosis rate of H9C2 cells was analyzed by TUNEL assay. The interaction between APPL1 and APAF-1/caspase9 was determined by Immunoprecipitation (IP).Our findings demonstrated that APPL1 was low expressed in myocardial ischemia tissues and cells. APPL1 knockdown suppressed the viability of myocardial ischemia cells and aggravated hypoxia/reperfusion-induced LDH hypersecretion, inflammation and apoptosis. In addition, the overexpression of APPL1 induced inactivation of APAF-1/Caspase9 signaling pathway. Significantly, APAF1 inhibitor reversed the effect of APPL1 knockdown on viability, LDH secretion, inflammation and apoptosis.We conclude that APPL1 inhibits myocardial ischemia/hypoxia-reperfusion injury via inactivation of APAF-1/Caspase9 signaling pathway. Hence, APPL1 may be a novel and effective target for the treatment of myocardial ischemia.

Treating MCF7 breast cancer cell with proteasome inhibitor Bortezomib restores apoptotic factors and sensitizes cell to Docetaxel.

Chemoresistance is the leading cause of limiting long-term treatment success in cancer cells. Anticancer drugs usually kill cells through apoptosis induction and defects in this signaling pathway lead to chemoresistance. Apoptotic protease activating factor 1 regulates cellular stress evoked by chemotherapeutic agents through facilitating apoptosome assembling but can be degraded by proteasome. This study examined the role of proteasome inhibitor Bortezomib in the cytotoxic effects of Docetaxel on MCF7 cells response and its correlation with Apaf-1 expression level. MTT assay, caspase 3/7 activity assay, propidium iodide staining, adenosine triphosphate and reactive oxygen species amount measurements were utilized to demonstrate the role of Bortezomib in Docetaxel efficacy with and without Apaf-1 overexpressing. Meanwhile, two-dimensional cell migration assay was performed by scratch wound assay. The combination of Docetaxel with Bortezomib was significantly more cytotoxic compared single drug, more effectively delayed cell growth, reduced ATP level and increased ROS production. In Apaf-1 overexpressing, Docetaxel was more efficient in preventing cell migration, however, Docetaxel plus Bortezomib were not significantly effective; and fluorescence images supported the interpretation. Our findings demonstrated MCF7 resistance to Docetaxel is due in part to low Apaf-1 level and Apaf-1 overexpression resulted in the increase of cell susceptibility to Docetaxel stimulus. We assume that proteasome inhibitor may restore apoptotic proteins like Apaf-1 and prevent the degradation of cytosolic cytochrome c released by Docetaxel, consequently triggering intrinsic apoptosis and promoting cancer cell death. Collectively, treating MCF7 breast cells with proteasome inhibitor sensitizes cells to Docetaxel-induced apoptosis and possibly overcomes chemoresistance.

Intracellular leucine-rich alpha-2-glycoprotein-1 competes with Apaf-1 for binding cytochrome c in protecting MCF-7 breast cancer cells from apoptosis.

Leucine-rich alpha-2-glycoprotein-1 (LRG1) has been shown to compete with apoptosis activating factor-1 (Apaf-1) for binding cytochrome c (Cyt c) and could play a role in inhibition of apoptosis. Employing MCF-7 breast cancer cells, we report that intracellular LRG1 does protect against apoptosis. Thus, cells transfected with the lrg1 gene and expressing higher levels of LRG1 were more resistant to hydrogen peroxide-induced apoptosis than parental cells, while cells in which LRG mRNA was knocked down by short hairpin (sh) RNA-induced degradation were more sensitive. The amount of Cyt c co-immunoprecipitated with Apaf-1 from the cytosol of apoptotic cells was inversely related to the level of LRG1 expression. In lrg1-transfected cells partially-glycosylated LRG1 was found in the cytosol and there was an increase in cytosolic Cyt c in live lrg1-transfected cells relative to parental cells. However, apoptosis was not spontaneously induced because Cyt c was bound to LRG1 and not to Apaf-1. Cyt c was the only detectable protein co-immunoprecipitated with LRG1. Following hydrogen peroxide treatment degradation of LRG1 allowed for induction of apoptosis. We propose that intracellular LRG1 raises the threshold of cytoplasmic Cyt c required to induce apoptosis and, thus, prevents onset of the intrinsic pathway in cells where Cyt c release from mitochondria does not result from committed apoptotic signaling. This mechanism of survival afforded by LRG1 is likely to be distinct from its extracellular survival function that has been reported by several research groups.

Hepatitis B virus surface protein induces sperm dysfunction through the activation of a Bcl2/Bax signaling cascade triggering AIF/Endo G-mediated apoptosis.

BACKGROUND: Hepatitis B virus (HBV) was found to exist in semen and male germ cells of patients with chronic HBV infection. Our previous studies demonstrated that HBV surface protein (HBs) could induce sperm dysfunction by activating a calcium signaling cascade and triggering caspase-dependent apoptosis. However, the relationship between sperm dysfunction caused by HBs and caspase-independent apoptosis has not been investigated. OBJECTIVES: To evaluate the effects of HBs exposure on sperm dysfunction by activating caspase-independent apoptosis. MATERIALS AND METHODS: Spermatozoa were exposed to HBs at concentrations of 0, 25, 50, and 100 µg/mL for 3 h. Flow cytometry, qRT-PCR, immunofluorescence assay, ELISA, and zona-free hamster oocyte penetration assays were performed. RESULTS: With increasing concentrations of HBs, various parameters of the spermatozoa changed. The number of Bcl2-positive cells declined and that of both Bax-positive cells and Apaf-1-positive cells increased. The transcription level of Bcl2 increased and that of both Bax and Apaf-1 declined. The average levels of AIF and Endo G declined in mitochondria and increased in the cytoplasm and nucleus. The sperm DNA fragmentation index increased. The mean percentages of live spermatozoa declined and that of both injured and dead spermatozoa increased; and the sperm penetration rate declined. For the aforementioned parameters, the differences between the test and the control groups were statistically significant. CONCLUSION: HBs exposure can activate the Bax/Bcl2 signaling cascade that triggers AIF/Endo G-mediated apoptosis, resulting in sperm DNA fragmentation, sperm injury, and death, and a decrease in the sperm fertilizing capacity. This new knowledge will help to evaluate the negative impact of HBV on male fertility in HBV-infected patients.

Loss of WD2 subdomain of Apaf-1 forms an apoptosome structure which blocks activation of caspase-3 and caspase-9.

Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation of apoptosome that activates caspase-9, an activation that is a key event in the mitochondrial cell death pathway. Structural studies suggest that normally Apaf-1 is held in an inactive conformation by intramolecular interactions between Apaf-1's nucleotide binding domain and one of its WD40 domains (WD1). In the prevailing model of Apaf-1 activation, cytochrome c binds to sites in WD1 and in Apaf-1's second WD40 domain (WD2), moving WD1 and WD2 closer together and rotating WD1 away from the nucleotide binding domain. This allows Apaf-1 to bind dATP or ATP and to form the apoptosome, which activates caspase-9. This model predicts that cytochrome c binding to both WD domains is necessary for apoptosome formation and that an Apaf-1 with only WD1 will be locked in an inactive conformation that cannot be activated by cytochrome c. Here we investigated the effect of removing one WD domain (Apaf-1 1-921) on Apaf-1 interactions and caspase activation. Apaf-1 1-921 could not activate caspase-9, even in the presence of cytochrome c. These data show that a single WD domain is sufficient to lock Apaf-1 in an inactive state and this state cannot be altered by cytochrome c.

Apoptosis is involved in maintaining the character of the midbrain and the diencephalon roof plate after neural tube closure.

Apoptosis, a major form of programmed cell death, is massively observed in neural plate border and subsequently in the roof plate (RP). While deficiency of apoptosis often results in brain malformations including exencephaly and hydrocephalus, the impact of apoptosis on RP formation and maintenance remains unclear. Here we described that mouse embryos deficient in Apaf1, a gene crucial for the intrinsic apoptotic pathway, in C57BL/6 genetic background exhibited narrow and discontinuous expression of RP marker genes in the midline of the midbrain and the diencephalon. Instead, cells positive for the neuroectodermal gene SOX1 ectopically accumulated in the midline. A lineage-tracing experiment suggests that these ectopic SOX1-positive cells began to accumulate in the midline of apoptosis-deficient embryos after E9.5. These embryos further displayed malformation of the subcommissural organ, which has been discussed in the etiology of hydrocephalus. Thus, the apoptosis machinery prevents ectopic emergence of SOX1-positive cells in the midbrain and the diencephalon RP, and helps in maintaining the character of the RP in the diencephalon and midbrain, thereby ensuring proper brain development.

Long Noncoding RNA Hypoxia-Inducible Factor-1 Alpha-Antisense RNA 1 Regulates Vascular Smooth Muscle Cells to Promote the Development of Thoracic Aortic Aneurysm by Modulating Apoptotic Protease-Activating Factor 1 and Targeting let-7g.

BACKGROUND: Thoracic aortic aneurysm (TAA) is a severe threat that is characterized by the increased aortic diameter. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of TAA. Previous research indicated that long noncoding RNAs (lncRNAs) were involved in the development of TAA. This article aimed to explore the role of lncRNA hypoxia-inducible factor-1 alpha-antisense RNA 1 (HIF1A-AS1) and potential action mechanisms in VSMCs. METHODS: The expression of HIF1A-AS1, collagen I, collagen III, microRNA let-7g (let-7g) and apoptotic protease-activating factor 1 (APAF1) was detected by quantitative real-time polymerase chain reaction. Cell proliferation and cell apoptosis were assessed by Cell Counting Kit-8 and flow cytometry assays, respectively. The protein levels of proliferating cell nuclear antigen, Cleaved caspase-3 (Cleaved-cas3), B cell lymphoma/leukemia-2 (Bcl-2), Collagen I, Collagen III, and APAF1 were quantified by Western blot. The relationship between let-7g and HIF1A-AS1 or APAF1 was predicted by the online bioinformatics tool and verified by dual-luciferase reporter assay and RNA pull-down assay. RESULTS: HIF1A-AS1 was upregulated in TAA tissues and was a valuable diagnostic marker of TAA. HIF1A-AS1 overexpression suppressed proliferation, induced apoptosis, and reduced the expression of extracellular matrix proteins in VSMCs. let-7 g was a target of HIF1A-AS1, and its inhibition functioned the same role as HIF1A-AS1 overexpression. APAF1 was a target of let-7g, and its knockdown played the opposite role with HIF1A-AS1 overexpression. The reintroduction of let-7g or APAF1 knockdown reversed the effects of HIF1A-AS1 overexpression in VSMCs. CONCLUSIONS: HIF1A-AS1 regulated the proliferation, apoptosis ,and the activity of extracellular matrix proteins in VSMCs through modulating APAF1 by targeting let-7g, leading to the development of TAA.

Epigenetic Regulation of APAF-1 Through DNA Methylation in Pancreatic Cancer.

BACKGROUND/AIM: Apoptotic peptidase activating factor 1 (APAF-1) is essential regulator of apoptosis and inactivation by DNA methylation is common event in numerous cancer types. We investigated the regulation of APAF-1 through DNA methylation in pancreatic cancer. MATERIALS AND METHODS: Datasets from 44 patients after pancreatoduodenectomy and the pancreatic adenocarcinoma (PDAC) cell lines Capan-2 and MIA PaCa-2 treated with decitabine were analyzed by RT-PCR, immunoblotting, methylation-specific PCR analysis, apoptosis and viability assays to identify effects of APAF-1 regulation. RESULTS: APAF-1 mRNA and protein levels were significantly down-regulated, and APAF-1 methylation status was associated with perineural invasion in PDAC. Decitabine inhibited cell viability and increased apoptosis rates, however failed to restore APAF-1 mRNA and protein levels in cells. CONCLUSION: APAF-1 gene hypermethylation may contribute to the progression of PDAC through perineural invasion. Decitabine could sensitize pancreatic cancer cells to apoptosis and growth retardation, however, not directly through the APAF-1 demethylation process.

The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway.

Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the apoptosome that we named 'Lumiptosome'. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.

Gonadotrophins modulate cell death-related genes expression in human endometrium.

Background Gonadotrophins exert their functions by binding follicle-stimulating hormone receptor (FSHR) or luteinizing hormone and human chorionic gonadotropin receptor (LHCGR) present on endometrium. Within ovaries, FSH induces autophagy and apoptosis of granulosa cells leading to atresia of non-growing follicles, whereas hCG and LH have anti-apoptotic functions. Endometrial cells express functioning gonadotrophin receptors. The objective of this study was to analyze the effect of gonadotrophins on physiology and endometrial cells survival. Materials and methods Collected endometria were incubated for 48 or 72 h with 100 ng/mL of recombinant human FSH (rhFSH), recombinant human LH (rhLH) or highly purified hCG (HPhCG) alone or combined. Controls omitted gonadotrophins. The effect of gonadotrophins on cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1), hypoxia inducible factor 1α (HIF1A), and cell-death-related genes expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Immunohistochemistry for microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and apoptotic protease activating factor 1 (APAF-1) was performed. Results Gonadotrophins are able to modulate the endometrial cells survival. FSH induced autophagy and apoptosis by increasing the relative expression of MAP1LC3B and FAS receptor. In FSH-treated samples, expression of apoptosis marker APAF-1 was detected and co-localized on autophagic cells. hCG and LH does not modulate the expression of cell-death-related genes while the up-regulation of pro-proliferative epiregulin gene was observed. When combined with FSH, hCG and LH prevent autophagy and apoptosis FSH-induced. Conclusions Different gonadotrophins specifically affect endometrial cells viability differently: FSH promotes autophagy and apoptosis while LH and hCG alone or combined with rhFSH does not.