Apoptosis-inducing Factor Expression Research Articles

Drp1 mediates compression-induced programmed necrosis of rat nucleus pulposus cells by promoting mitochondrial translocation of p53 and nuclear translocation of AIF

Compression-induced programmed cell death of nucleus pulposus (NP) cells is an important contributor to intervertebral disc degeneration (IDD). Dynamin-related protein 1 (Drp1), a crucial mitochondrial fission protein, triggers programmed necrosis upon cellular injury. However, limited information is available about the role of Drp1 in compression-induced programmed necrosis of NP cells. In the present study, we found that compression resulted in upregulation and mitochondrial translocation of Drp1. Inhibition of Drp1 by siRNA or mitochondrial division inhibitor 1 (mdivi-1) effectively prevented the programmed necrosis of NP cells treated with compression. Furthermore, Drp1 promoted mitochondrial translocation of p53 and nuclear translocation of apoptosis-inducing factor (AIF) in compression-treated NP cells. Inhibition of p53 mitochondrial translocation by pifithrin-μ (PFT-μ) and silencing of AIF expression by siRNA significantly alleviated compression-induced NP cell programmed necrosis. These data indicates that Drp1 mediates compression-induced programmed necrosis of NP cells by promoting mitochondrial translocation of p53 and nuclear translocation of AIF.

Death associated protein 1 (DAP 1) positively regulates virus replication and apoptosis of hemocytes in shrimp Marsupenaeus japonicus

Death-associated protein 1 (DAP1) is a small proline-rich cytoplasmic protein that functions both in the apoptosis and autophage process of mammalian and in the clinical cancer of human. However, little knowledge is known about the homologue gene of DAP1 and its roles in the physiological process of invertebrates. In this paper, we report a novel function of DAP1 in the antivirus immunity of shrimp. A homologue gene of DAP1 was cloned from Marsupenaeus japonicus and named as Mjdap-1. The full-length of Mjdap-1 was 1761 bp with a 309 bp open reading frame that encoded 102 amino acids. Reverse transcription-PCR results showed that Mjdap-1 was expressed in all tested tissues, including hemocytes, gills, intestines, stomach, heart, hepatopancreas, testes, and ovaries. In shrimp, Mjdap-1 transcripts were up-regulated by white spot syndrome virus (WSSV) infection; Mjdap-1 knockdown decreased the virus copy in vivo and the mortality of M. japonicus to WSSV challenge. Conversely, injecting the purified recombinant MjDAP1 protein promoted the amplification of virus in shrimp. Flow cytometric assay showed, the virus infection-induced apoptosis of hemocytes was enhanced by MjDAP1 protein injection and inhibited in MjDAP1 knockdown shrimp. Furthermore, the expression of apoptosis-inducing factor (AIF) was regulated by Mjdap-1, but the caspase transcripts were not affected. Our results suggested that MjDAP1 facilitated the amplification of virus in shrimp, which may be attributed to the promotion of hemocyte apoptosis in an AIF-dependent manner. These results provided a new insight into the function of this protein that may be used for virus disease control.

Downregulation of Iduna is associated with AIF nuclear translocation in neonatal brain after hypoxia–ischemia

In adult stroke models, the neuroprotective protein, Iduna, inhibits poly (ADP-ribose) polymerase-1 (PARP-1)-dependent cell death by decreasing apoptosis-inducing factor (AIF) nuclear translocation. Because the PARP1-dependent pathway and Iduna, which promotes AIF degradation, contribute to hypoxic–ischemic (HI) brain damage in the immature brain, we examined the relationship between Iduna expression and AIF nuclear translocation in the cerebral cortex of postnatal day 7 rats after HI. Ninety rats were divided into three groups: sham, 1-h hypoxia and 2-h hypoxia. The HI insult was induced by permanent ligation of the left common carotid artery plus 1 or 2h of hypoxia. Brain damage pathological features were evaluated by hematoxylin and eosin staining, Nissl staining, transmission electron microscopy, TUNEL staining and immunofluorescence. Immunohistochemistry and western blot analysis were used to assess protein expression and ubiquitination status of AIF. The interaction between Iduna and AIF was tested by co-immunoprecipitation. Learning and memory were analyzed by the Morris water maze test. Compared with sham animals, the number of surviving neurons in the cerebral cortex decreased, and cell damage and DNA breakage were severe in rats with HI, with worse damage in the 2-h group. Iduna expression significantly decreased, whereas nuclear AIF expression increased. Furthermore, Iduna downregulation negatively correlated with nuclear AIF abundance in the 2-h HI group (r=−0.950; P<0.0001). Additionally, learning and memory ability decreased with hypoxic time. These results suggest that AIF nuclear translocation and neuronal cell death are associated with Iduna loss after severe HI in the immature brain.

Prognostic value of apoptosis inducing factor in uveal melanoma.

In malignant tumors including uveal melanoma there is acontinuous effort in search for additional and relevant factors with predictive value and possible therapeutic indications. In the present work we evaluated the 5-year mortality in agroup of patients with surgically treated uveal melanoma and its relation to selected demographic, clinical and histopathological parameters, including the expression of apoptosis inducing factor (AIF) in the neoplastic tissue.We analyzed retrospectively the clinical data of patients with uveal melanoma treated surgically (enucleation, endoresection, exenteration) in the period from 2001 to 2007 (n=54). Immunohistochemical detection of AIF expression in formalin fixed and in paraffin embedded tissue samples was evaluated semiquantitatively, intensity and percentage multiplicative Quick Score (QS) was calculated and compared between patients with over 5 year (n=32) and less than 5 year (n=22) survival. In the analyzed group of 54 patients the 5 year mortality was 41%. We confirmed the negative prognostic significance of some of the known prognostic factors as the tumor size and volume, T3 and T4 stage in the TNM classification and the mixed histological type of the tumor. Immunohistochemistry performed on 49 melanoma specimens showed AIF cytoplasmic positivity, no nuclear translocation was detected. The cut-off value of AIF expression QS ≥ 4 (18) in tumor cells separated the 5 year survival of patients (P = 0.018), odds ratio 5.2 (1.24 - 21.73). Moderate and strong expression of AIF in tumor cells also correlated with less favorable prognosis. Confocal microscopy proved colocalization of AIF with mitochondrial marker in neoplastic cells.The prognosis of patients with uveal melanoma can be more accurate with inclusion of immunohistochemical detection of AIF expression. Increased expression of the AIF protein appears as anew negative prognostic factor predicting the 5 year survival.

Apoptosis inducing factor gene depletion inhibits zearalenone-induced cell death in a goat Leydig cell line

Zearalenone (ZEA) is a contaminant of human food and animal feedstuffs that causes health hazards. However, the signal pathways underlying ZEA toxicity remain elusive. The aims of this study were to determine which pathways are involved in ZEA-induced cell death and investigate the effect of apoptosis inducing factor (AIF) on cell death during ZEA treatment in the immortalized goat Leydig cell line hTERT-GLC. This study showed that ZEA-induced cell death in hTERT-GLCs works via endoplasmic reticulum (ER) stress, the caspase-dependent pathway, the caspase-independent pathway and autophagy. Recombinant lentiviral vectors were constructed to silence AIF expression in hTERT-GLCs. Flow cytometry results showed that knockdown of AIF diminished ZEA-induced cell apoptosis in hTERT-GLCs. Furthermore, we found AIF depletion down-regulated phosphoIRE1α, GRP78, CHOP and promoted the switch of LC3-I to LC3-II. Therefore, ZEA induces cytotoxicity in hTERT-GLCs via different pathways, while AIF-mediated signaling plays a critical role in ZEA-induced cell death in hTERT-GLCs.

Apoptosis-inducing factor (AIF) nuclear translocation mediated caspase-independent mechanism involves in X-ray-induced MCF-7 cell death

Purpose: Breast cancer is the most common cancer among women and radiotherapy is a conventional therapy following surgery. Previous studies have demonstrated that except the caspase-dependent pathway, caspase-independent pathway is also involved in the cell death responding to irradiation, despite the unclear mechanism. The purpose of the present study was to observe the role of apoptosis-inducing factor (AIF), the first identified caspase-independent molecule, in X-ray-induced breast cancer cell (MCF-7) cell death.Materials and methods: In this study, WST-1 assay, DAPI nuclear staining and clonogenic survival assay were used to test the cell response to different treatments; Western blot was used to detect the protein expression; RT-PCR and plasmid transfection were used to observe the role of AIF.Results: X-ray-induced AIF transferred from the mitochondrion to the nucleus. Inhibition of AIF expression reduced X-ray-induced MCF-7 cell death. Further, AIF nuclear translocation is in a caspase-independent manner in this process, but not caspase-dependent manner.Conclusions: The present study revealed that AIF nuclear translocation proceeded in X-ray-induced MCF-7 cell death in a caspase-independent manner.

Influenza virus infection induces translocation of apoptosis-inducing factor (AIF) in A549 cells: role of AIF in apoptosis and viral propagation.

It is recognized that influenza virus induces caspase-dependent apoptosis by activating caspase-3. Apoptosis-inducing factor (AIF) is a caspase-independent cell death effector, and its mitochondrial-nuclear translocation plays an important role in apoptosis. It is demonstrated in this study how influenza virus infection can induce caspase-independent apoptosis in the human alveolar epithelial cell line A549. AIF is translocated from the mitochondria to the nucleus in a caspase-independent manner in response to infection with influenza virus. Knockdown of AIF expression by small interfering RNA (siRNA) led to a reduction in virus-infection-induced apoptosis and virus yield. These results indicate that AIF translocation has a role in influenza-virus-induced apoptosis.

Characteristic of apoptosis of coronary artery cells with atherosclerotic lesions

To study apoptosis of endothelial cells (EC), macrophages (MF) and smooth muscle cells (SMC) in the early atherosclerotic process (prior to plaque formation), elucidate mechanisms of its realization and evaluate effect on progression of atherosclerosis. Histopathological studies were performed on coronary arteries affected by atherosclerosis taken at autopsy of patients with coronary heart disease (n = 63). To detect apoptosis, the TUNEL method was used for calculating the apoptotic index (AI) in paraffin sections. Phenotyping the cells and test for expression of active caspase 3 (AC-3) and apoptosis-inducing factor (AIF) were performed using sections of coronary arteries stained by immunohistochemistry. The study of apoptosis showed a significant (p <0.0001) increase in AI of SMC, EC, MF coronary arteries affected by atherosclerosis compared with unaffected vessels. Progression of the atherosclerotic process led to a decrease of AI of SMC and EC. Statistical analysis revealed moderate feedback between AI of SMC and neointimal thickness in the development of atherosclerotic lesions (r = -0,44, p <0.0001). The amount of the AC-positive SMC and EC at the stage of lipoidosis significantly (p <0.0001) exceeded the number of AC -positive SMC at the liposclerosis stage. Expression of AIF was observed in the nuclei of EC in the newly formed vessels of hyperplastic intima. Early atherosclerotic lesions of the coronary arteries are accompanied by intense apoptosis of SMC, EC and MF. The intensity of apoptosis of SMC and EC decreases with the development of atherosclerosis. Progression of intimal hyperplasia at the early stages of atherosclerosis is associated with reduced apoptosis of smooth muscle cells and enhanced macrophage apoptosis. Apoptosis of SMC and MF is caspase-dependent. Apoptosis of endothelial cells can occur both with the participation of caspases and independently of them (with AIF).

Apoptosis Inducing Factor Is Involved in Stretch-Induced Apoptosis of Myoblast via a Caspase-9 Independent Pathway.

The apoptosis of myoblast in response to excessive cyclic stretch is crucial in adaptive construction of skeletal muscles in orthopedic functional therapy. Mitochondria signaling pathway is the central links in the execution of the intrinsic apoptotic cascade, but its molecular mechanism in stretch-induced apoptosis in myoblasts remains incompletely understood. The aim of this study was to investigate the mechanobiological roles of caspase-9 and Apoptosis Inducing Factor (AIF), two important components in mitochondrial pathway, in stretch-induced apoptosis of myoblast. Hoechst 33258 was used to observe apoptotic cells morphologically and flow cytometry to analyze apoptosis rate of myoblasts. Protein and mRNA of caspase-9 and AIF were detected by Western blotting and RT-PCR. Furthermore, caspase-9 specific inhibitor z-LEHD-fmk was applied to clear the mechanism of caspase-9 pathway in stretch-induced apoptosis. We found that apoptotic rate induced by cyclic stretch increased in a time-dependent manner, and the same tendency of mRNA and protein of caspase-9 and AIF. Caspase-9 inhibition reduced stretch-induced apoptosis, but had no effect on expression of AIF. We concluded that caspase-9 and AIF played an important role in stretch-induced apoptosis in myoblast, and AIF was involved in the process in a caspase-9 independent way. J. Cell. Biochem. 118: 829-838, 2017. © 2016 Wiley Periodicals, Inc.

Melatonin ameliorates the adverse effects of leptin on sperm.

This study examined the effects of melatonin on leptin-induced changes in sperm parameters in adult rats. Five groups of Sprague-Dawley rats were treated with either leptin or leptin and melatonin or melatonin for 6 weeks. Leptin was given daily via the intraperitoneal route (60 μg kg−1 body weight) and melatonin was given in drinking water (10 mg kg−1 or 20 mg kg−1 body weight per day). Upon completion, sperm count, sperm morphology, 8-hydroxy-2-deoxyguanosine, Comet assay, TUNEL assay, gene expression profiles of antioxidant enzymes, respiratory chain reaction enzymes, DNA damage, and apoptosis genes were estimated. Data were analyzed using ANOVA. Sperm count was significantly lower whereas the fraction of sperm with abnormal morphology, the level of 8-hydroxy-2-deoxyguanosine, and sperm DNA fragmentation were significantly higher in rats treated with leptin only. Microarray analysis revealed significant upregulation of apoptosis-inducing factor, histone acetyl transferase, respiratory chain reaction enzyme, cell necrosis and DNA repair genes, and downregulation of antioxidant enzyme genes in leptin-treated rats. Real-time polymerase chain reaction showed significant decreases in glutathione peroxidase 1 expression with increases in the expression of apoptosis-inducing factor and histone acetyl transferase in leptin-treated rats. There was no change in the gene expression of caspase-3 (CASP-3). In conclusion, the adverse effects of leptin on sperm can be prevented by concurrent melatonin administration.

Knock-down of apoptosis inducing factor gene protects endoplasmic reticulum stress-mediated goat granulosa cell apoptosis

The apoptosis of granulosa cells is the main cause of follicular atresia, and endoplasmic reticulum (ER) stress is involved in the apoptosis of granulosa cells. Apoptosis inducing factor (AIF) mediates caspase-independent apoptosis and causes chromatin condensation and DNA fragmentation, but its role in ER stress–mediated granulosa cell apoptosis during goat follicular atresia remains largely unknown. The aim of this study was to investigate the function of AIF in the apoptosis of goat granulosa cells mediated by ER stress. The results of immunohistochemical and Western blot analyses demonstrated that AIF was mainly located in granulosa cells, and the expression of AIF significantly increased during follicular atresia. Then, AIF-short hairpin RNA recombinant lentiviral vectors were constructed successfully and transfected into human telomerase reverse transcriptase-goat granulosa cells (hTERT-GGCs). Real-time quantitative polymerase chain reaction and Western blot analysis confirmed that AIF was effectively knocked down in hTERT-GGCs. Flow cytometry results showed that the knockdown of AIF in hTERT-GGCs reduced apoptosis due to serum starvation or thapsigargin (Tg) treatment. In addition, AIF depletion changed the expression of related molecular marker molecules of ER stress under Tg treatment. In conclusion, AIF may serve as a key factor during follicular atresia, and AIF depletion protects ER stress–mediated goat granulosa cell apoptosis.

Downregulation of AIF by HIF-1 contributes to hypoxia-induced epithelial-mesenchymal transition of colon cancer.

Recently, we have reported that apoptosis-inducing factor (AIF) regulates the epithelial-mesenchymal transition (EMT) process of cancers, but the mechanisms underlying the regulation of AIF expression in cancers remain greatly unknown. Here, we report that hypoxia inversely correlates with the expression of AIF in tumor tissues from a cohort of colon cancer patients and inhibits AIF expression in multiple colon cancer cell lines. This inhibition is mediated by hypoxia-inducible factor-1 (HIF-1), which transcriptionally represses AIF through direct binding to the hypoxia-response element in AIF promoter as revealed by luciferase reporter and chromatin immunoprecipitation assays. We also show that downregulation of AIF contributes to hypoxia-induced EMT as overexpression or silencing of AIF partially reverses or potentiates the EMT program initiated by hypoxic treatment. Mechanistic study reveals that downregulation of AIF by hypoxia causes oxidative inactivation of the lipid phosphatase activity of phosphatase and tensin homolog on chromosome 10 (PTEN), with ensuing activation of Akt kinase, phosphorylation of the Akt substrate GSK-3β and activation of WNT/β-catenin signaling in colon cancer cells. These results identify AIF as a novel target gene of HIF-1 and reveal the role of AIF downregulation in hypoxia-induced EMT.

Role of hydrogen gas in regulating of poly (ADP-ribose) polymerase-1 dependent cell death in rat Schwann cells

To investigate the protective effects and underlying molecular mechanisms of hydrogen (H2) on high glucose-induced poly (ADP-ribose) polymerase-1 (PARP-1) dependent cell death (PARthanatos) in primary rat Schwann cells. Cultured primary rat Schwann cells were randomly divided into five groups: blank control group (C group), H2 control group (H2 group), high osmotic control group (M group), high glucose treatment group (HG group), and H2 treatment group (HG+H2 group). The cells in H2 group and HG+H2 group were cultured with saturated hydrogen-rich medium containing 0.6 mmol/L of H2, and those in three control groups were cultured with low sugar DMEM medium containing 5.6 mmol/L of sugar, and the cells in HG and HG+H2 groups were given 44.4 mmol/L of glucose in addition (the medium containing 50 mmol/L of glucose), the cells in C group and H2 group were given the same volume of normal saline, and the cells in M group were given the same volume of mannitol. Cytotoxicity was evaluated using lactate dehydrogenase (LDH) release rate assays after treatment for 48 hours in each group. The contents of peroxynitrite (ONOO(-)) and 8-hydroxy-2-deoxyguanosine (8-OHdG) reflecting oxidative stress injury and DNA damage were detected by enzyme linked immunosorbent assay (ELISA). Poly (ADP-ribose) (PAR) protein expression was analyzed by Western Blot, and immunofluorescence staining was used to determine the nuclear translocation of the apoptosis-inducing factor (AIF). The cytotoxicity in HG and HG+H2 groups was significantly increased as compared with that of C group [LDH release rate: (61.40±2.89)%, (42.80±2.32)% vs. (9.92±0.38)%, both P < 0.01], the levels of ONOO(-) and 8-OHdG were markedly elevated [ONOO(-) (ng/L): 853.58±51.00, 553.11±38.66 vs. 113.56±14.22; 8-OHdG (ng/L): 1?177.37±60.97, 732.06±54.29 vs. 419.67±28.77, all P < 0.01], and the PAR protein expression was up-regulated (A value: 0.603±0.028, 0.441±0.010 vs. 0.324±0.021, both P < 0.01). The cytotoxicity, the levels of ONOO(-) and 8-OHdG, and PAR expression in HG+H2 group were significantly lower than those of the HG group (all P < 0.01). There were no significant differences in above parameters between H2 group as well as M group and C group. It was shown by immunofluorescence that AIF was expressed in the cytoplasm in C group, H2 group and M group, AIF was expressed in the whole cell in HG group, and the expression in the nucleus was particularly increased. A small amount of AIF expression was found in the nucleus of HG+H2 group, which indicated that high glucose could promote the AIF nuclear translocation, and that hydrogen-rich medium could prevent the process of translocation. High glucose levels could enhance DNA damage that enhance PARthanatos in primary rat Schwann cells. However, H2 can not only reduce DNA damage of injured cells, but also inhibit the special death process, reduce the cell toxicity, all of which have protective effects.

High expression of apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) in human cholangiocarcinoma.

Prognosis of cholangiocarcinoma (CCA) patients is absolutely poor in spite of extensive efforts for the development of chemotherapy. Mitochondrial proteins play key roles in carcinogenesis of various cancers. Therefore, mitochondria are considered as the target organelles for chemotherapy of several cancers including CCA. The purpose of this study is to identify potential candidate proteins for chemotherapy using mitochondrial proteome analysis for CCA tissues. A shotgun proteomic approach using SDS-PAGE coupled with LC-MS/MS was applied to compare the expression of mitochondrial proteins in CCA and the adjacent non-cancerous tissues. Using the proteomic analysis for the pooled mitochondrial proteins purified from three each of papillary and non-papillary types of CCA and their adjacent tissues, 281 proteins were identified as mitochondrial proteins, and 105 of them have significantly different expression levels compared with the corresponding counterparts. Among the 105 proteins, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) was a unique protein commonly over-expressed in both papillary and non-papillary types of CCA tissues but not in the adjacent non-cancerous tissues. In conclusion, AIFM3 was aberrantly expressed only in the mitochondria of CCA tissues. This finding suggests that AIFM3 could be a potential target molecule for CCA chemotherapy.

Effect of propofol on mitochondrial fission in a rat hippocampal neuron model of hypoxia/reoxygenation injury

Objective To evaluate the effect of propofol on mitochondrial fission in a rat hippocampal neuron model of hypoxia/reoxygenation (H/R) injury. Methods Primarily cultured hippocampal neurons of neonatal Sprague-Dawley rats were randomly divided into 4 groups (n= 42 each) using a random number table: control group (group C); vehicle group (group V); H/R group; H/R+ propofol group (group H/R+ P). In group V, H/R was not produced, the vehicle dimethyl sulfoxide with the final concentration of 0.01% was added, and the cells were then incubated for 6 h. In group H/R, the hippocampal neurons were subjected to oxygen-glucose deprivation for 6 h followed by 20 h reoxygenation.In group H/R+ P, propofol with the final concentration of 1 μmol/L was added at 6 h of hypoxia.At 20 h of reoxygenation, the cell apoptosis (using flow cytometry), Ca2+ concentrations in cytoplasm (with the laser scanning confocal microscope), calcineurin (CaN) activities (by enzyme-linked immunosorbent assay), and expression of mitochondrial fission proteins dynamin-related protein 1 (Drp1) and fission 1 (Fis1), and apoptosis-related proteins cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) (by Western blot) were measured.The apoptotic rate was calculated. Results Compared with group C, the apoptotic rate, Ca2+ concentrations, CaN activities, and expression of Drp1, Fis1, Cyt c and AIF were significantly increased in H/R and H/R+ P groups (P 0.05). Compared with group H/R, the apoptotic rate, Ca2+ concentrations, CaN activities, and expression of Drp1, Fis1, Cyt c and AIF were significantly decreased in group H/R+ P (P<0.05=. Conclusion Propofol can reduce the H/R injury to rat hippocampal neurons through inhibiting mitochondrial fission. Key words: Propofol; Anoxia; Mitochondria; Hippocampus; Neurons

Mda-7/IL-24 Induces Cell Death in Neuroblastoma through a Novel Mechanism Involving AIF and ATM.

Advanced stages of neuroblastoma, the most common extracranial malignant solid tumor of the central nervous system in infants and children, are refractive to therapy. Ectopic expression of melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) promotes broad-spectrum antitumor activity in vitro, in vivo in preclinical animal models, and in a phase I clinical trial in patients with advanced cancers without harming normal cells. mda-7/IL-24 exerts cancer-specific toxicity (apoptosis or toxic autophagy) by promoting endoplasmic reticulum stress and modulating multiple signal transduction pathways regulating cancer cell growth, invasion, metastasis, survival, and angiogenesis. To enhance cancer-selective expression and targeted anticancer activity of mda-7/IL-24, we created a tropism-modified cancer terminator virus (Ad.5/3-CTV), which selectively replicates in cancer cells producing robust expression of mda-7/IL-24 We now show that Ad.5/3-CTV induces profound neuroblastoma antiproliferative activity and apoptosis in a caspase-3/9-independent manner, both in vitro and in vivo in a tumor xenograft model. Ad.5/3-CTV promotes these effects through a unique pathway involving apoptosis-inducing factor (AIF) translocation into the nucleus. Inhibiting AIF rescued neuroblastoma cells from Ad.5/3-CTV-induced cell death, whereas pan-caspase inhibition failed to promote survival. Ad.5/3-CTV infection of neuroblastoma cells increased ATM phosphorylation instigating nuclear translocation and increased γ-H2AX, triggering nuclear translocation and intensified expression of AIF. These results were validated further using two ATM small-molecule inhibitors that attenuated PARP cleavage by inhibiting γ-H2AX, which in turn inhibited AIF changes in Ad.5/3-CTV-infected neuroblastoma cells. Taken together, we elucidate a novel pathway for mda-7/IL-24-induced caspase-independent apoptosis in neuroblastoma cells mediated through modulation of AIF, ATM, and γ-H2AX. Cancer Res; 76(12); 3572-82. ©2016 AACR.

Apoptosis Inducing Factor Binding Protein PGAM5 Triggers Mitophagic Cell Death That Is Inhibited by the Ubiquitin Ligase Activity of X-Linked Inhibitor of Apoptosis.

Apoptosis inducing factor (AIF) plays a well-defined role in controlling cell death but is also a critical factor for maintaining mitochondrial energy homeostasis; how these dueling activities are balanced has remained largely elusive. To identify new AIF binding partners that may define the continuum of AIF cellular regulation, a biochemical screen was performed that identified the mitochondrial phosphoglycerate mutase 5 (PGAM5) as an AIF associated factor. AIF binds both the short and long isoforms of PGAM5 and can reduce the ability of PGAM5 to control antioxidant responses. Transient overexpression of either PGAM5 isoform triggers caspase activation and cell death, and while AIF could reduce this caspase activation neither AIF expression nor caspase activity is required for PGAM5-mediated death. PGAM5 toxicity morphologically and biochemically resembles mitophagic cell death and is inhibited by the AIF binding protein X-linked inhibitor of apoptosis (XIAP) in a manner that depends on the ubiquitin ligase activity of XIAP. The phosphatase activity of PGAM5 was not required for cell death, and comparison of phosphatase activity between short and long PGAM5 isoforms suggested that only the long isoform is catalytically competent. This property correlated with an increased ability of PGAM5L to form dimers and/or higher order oligomers in intact cells compared to PGAM5S. Overall this study identifies an AIF/PGAM5/XIAP axis that can regulate PGAM5 activities related to the antioxidant response and mitophagy.

Toxicity of Pekinenin C from Euphorbia Pekinensis Radix on Rat Small Intestinal Crypt Epithelial Cell and Its Apoptotic Mechanism.

Pekinenin C is a casbane diterpenoid separated from the root of the traditional Chinese medicine, Euphorbia pekinensis Rupr., which is used as drug for the treatment of edema, ascites, and hydrothorax. Whereas pekinenin C exhibits severe cytotoxicity, the exact toxicity mechanism is unclear. In this study, the effects of pekinenin C on cell inhibition, cell cycle, and cell apoptosis were examined to explain its toxic mechanism. The proliferation of IEC-6 cells was accessed via MTT colorimetric assay after incubated with different concentrations of pekinenin C. Pekinenin C-treated IEC-6 cells labeled with RNase/PI and Annexin V/PI were analyzed by flow cytometric analyses for evaluation of cell cycle distribution and cell apoptosis, respectively. The apoptosis mechanism of pekinenin C on IEC-6 was investigated through assaying the activities of caspase-3, 8, 9 by enzyme-linked immunosorbent assay (ELISA), protein expression of Bax, Bcl-2, apoptosis-inducing factor (AIF), Apaf-1, Fas-associated death domain (FADD) and type 1-associated death domain (TRADD) by Western-blot, mRNA expression of Fas receptor (FasR), Fas ligand (FasL), tumor necrosis factor receptor (TNFR1) and NF-κB by RT-PCR. The results showed that pekinenin C has exhibited obvious IEC-6 cells toxicity and the IC50 value was 2.1 μg·mL−1. Typical apoptosis characteristics were observed under a transmission electron microscopy, and it was found that pekinenin C could cause G0/G1 phase arrest in IEC-6 cells in a dose-dependent manner and induce apoptosis of IEC-6 cells. Additionally, pekinenin C could increase the expressions of Bax, AIF, Apaf-1, FasR, FasL, TNFR1 and NF-κB, suppress the expression of Bcl-2, FADD and TRADD, then activate caspase-3, 8, 9 cascades, and at last result in apoptosis. These results demonstrated that pekinenin C effectively promoted cell apoptosis, and induced IEC-6 cells apoptosis through both the mitochondrial and death receptor pathways.

Basal metabolic state governs AIF-dependent growth support in pancreatic cancer cells.

BackgroundApoptosis-inducing factor (AIF), named for its involvement in cell death pathways, is a mitochondrial protein that regulates metabolic homeostasis. In addition to supporting the survival of healthy cells, AIF also plays a contributory role to the development of cancer through its enzymatic activity, and we have previously shown that AIF preferentially supports advanced-stage prostate cancer cells. Here we further evaluated the role of AIF in tumorigenesis by exploring its function in pancreatic cancer, a disease setting that most often presents at an advanced stage by the time of diagnosis.MethodsA bioinformatics approach was first employed to investigate AIF mRNA transcript levels in pancreatic tumor specimens vs. normal tissues. AIF-deficient pancreatic cancer cell lines were then established via lentiviral infection. Immunoblot analysis was used to determine relative protein quantities within cells. Cell viability was measured by flow cytometry; in vitro and Matrigel™ growth/survival using Coulter™ counting and phase contrast microscopy; and glucose consumption in the absence and presence of Matrigel™ using spectrophotometric methods.ResultsArchival gene expression data revealed a modest elevation of AIF transcript levels in subsets of pancreatic tumor specimens, suggesting a possible role in disease progression. AIF expression was then suppressed in a panel of five pancreatic cancer cell lines that display diverse metabolic phenotypes. AIF ablation selectively crippled the growth of cells in vitro in a manner that directly correlated with the loss of mitochondrial respiratory chain subunits and altered glucose metabolism, and these effects were exacerbated in the presence of Matrigel™ substrate. This suggests a critical metabolic role for AIF to pancreatic tumorigenesis, while the spectrum of sensitivities to AIF ablation depends on basal cellular metabolic phenotypes.ConclusionsAltogether these data indicate that AIF supports the growth and survival of metabolically defined pancreatic cancer cells and that this metabolic function may derive from a novel mechanism so far undocumented in other cancer types.

PARP-1-modulated AIF translocation is involved in streptomycin-induced cochlear hair cell death

Conclusion SM-induced dose- and location-dependent cochlear hair cell death in vitro. AIF might be translocated from mitochondria to nucleus and cytoplasm within SM-treated hair cells. The translocation of AIF might be modulated by PARP-1.Objective Streptomycin (SM), one of the widely used aminoglycoside nowadays, is still causing significant permanent sensorineural hearing loss owing to sensory hair cell death. This study was designed to investigate the role of apoptosis-inducing factor (AIF), an important mitochondrial cell death regulator, in SM ototoxicity within neonatal rat cochleae and HEI-OC1 cells.Methods The viability of HEI-OC1 cells was quantified by MTT assay. AIF, PARP-1, and myosin VIIa distributions were achieved by immunofluorescence. mRNA and protein expression of AIF and PARP-1 were examined by q-PCR and Western-blot.Results The hair cell loss was concomitant with the SM concentration variation, and aggravated from apical to basal turn. AIF was detected in nuclear region and AIF mRNA was up-regulated after SM incubation. Besides, AIF protein expression in mitochondria was decreased, whereas in cytosol it was increased. PARP-1 mRNA and protein were also up-regulated. 3-AB could attenuate the cell death and reverse the changes of AIF distribution by blocking PARP-1.