Nuclear Apoptosis-inducing Factor Research Articles

Synaptic vesicle protein 2A mitigates parthanatos via apoptosis-inducing factor in a rat model of pharmacoresistant epilepsy.

Synaptic vesicle protein 2A (SV2A) is a unique therapeutic target for pharmacoresistant epilepsy (PRE). As seizure-induced neuronal programmed death, parthanatos was rarely reported in PRE. Apoptosis-inducing factor (AIF), which has been implicated in parthanatos, shares a common cytoprotective function with SV2A. We aimed to investigate whether parthanatos participates in PRE and is mitigated by SV2A via AIF. An intraperitoneal injection of lithium chloride-pilocarpine was used to establish an epileptic rat model, and phenytoin and phenobarbital sodium were utilized to select PRE and pharmacosensitive rats. The expression of SV2A was manipulated via lentivirus delivery into the hippocampus. Video surveillance was used to assess epileptic ethology. Biochemical tests were employed to test hippocampal tissues following a successful SV2A infection. Molecular dynamic calculations were used to simulate the interaction between SV2A and AIF. Parthanatos core index, PARP1, PAR, nuclear AIF and MIF, γ-H2AX, and TUNEL staining were all increased in PRE. SV2A is bound to AIF to form a stable complex, successfully inhibiting AIF and MIF nuclear translocation and parthanatos and consequently mitigating spontaneous recurrent seizures in PRE. Moreover, parthanatos deteriorated after the SV2A reduction. SV2A protected hippocampal neurons and mitigated epileptic seizures by inhibiting parthanatos via binding to AIF in PRE.

NAIF1 EXPRESSION IN COLORECTAL CANCER PATIENTS: CLINICAL AND PATHOLOGICAL IMPLICATIONS

Background: In advanced human malignancies, the nuclear apoptosis-inducing factor 1 (NAIF1) is commonly silenced or not expressed. The purpose of this study was to look into potential relationships between clinical, pathological characteristics and NAIF1 expression. Methods: This cross-sectional study used western blot and immunohistochemistry staining to assess the level of NAIF1 expression in 100 colorectal cancer samples. The outcomes of the immunohistochemistry staining were then contrasted with those of the clinicopathological characteristics. Results: 68 out of 100 colorectal cancer samples were negative for NAIF1 expression. In contrast to other clinicopathological parameters, loss of NAIF1 expression was significantly linked with lymphovascular invasion (P=< 0.00001), angiovascular invasion (P= 0.004356) and advanced TNM tumor stage (P=0. 004077). Conclusion: According to the current study, a higher stage and a bad prognosis may be linked to decreased or negative NAIF1 expression. Keywords: Clinical, Colorectal Cancer, Expression, NAIF1, Pathological

AATF Competitively Interacts with Nuclear AIF and Inhibits Parthanatos of Neurons in dMCAO/R and OGD/R Models.

Ischemic stroke (IS) poses a heavy burden on the healthcare system, and revascularization is the most effective treatment. However, ischemia/reperfusion (I/R) injury, one main cause of revascularization complications, significantly hinders IS recovery. Unfortunately, none of the neuroprotectants tested to date has been successfully translated clinically for post-revascularization I/R injury therapy. In multiple pathophysiological processes, apoptosis antagonizing transcription factor (AATF) serves as a cell protector, but its role in neuronal I/R injury is unknown. Therefore, we firstly demonstrated the expression profiles of AATF in a distal middle cerebral artery occlusion/reperfusion (dMCAO/R) model and found that AATF expression was increased in cortical neuron after dMCAO/R. Over-expressing AATF reduced infarct volume, alleviated neuronal death, and promoted neurological functions. Next, we used an oxygen-glucose deprivation/reoxygenation (OGD/R) model to investigate the mechanism of AATF. Results indicated that AATF alleviated OGD/R-induced large-scale DNA fragmentation, which suggested that the protective effect of AATF may be attributed to parthanatos inhibition. After that, we examined the regulatory mechanism of AATF. We found that AATF did not affect poly (ADP-ribose) accumulation and apoptosis-inducing factor (AIF) nucleus translocation. AATF competitively interacted with nuclear AIF, which inhibited AIF from binding DNA. At last, we verified the effect and mechanism of AATF in dMCAO/R model. The present study, for the first time, demonstrates the expression, function, and mechanism of AATF in the context of neuronal I/R injury via dMCAO/R and OGD/R model, which provides new evidence in this area and may facilitate exploring new therapeutic targets.

The Proteomic Expression of Nuclear Apoptosis-Inducing Factor1 (NAIF1) in Colorectal Tissues

The neogenic recombinases are potentially a source of genetic variability possibly implicated in themechanisms of genetic instability involved in the processes of carcinogenesis. One of these neogenicrecombinases is the nuclear apoptosis-inducing factor 1(NAIF1), which codes the protein NAIF1 thatinduces apoptosis in various human cancers.The aim of this work is to study the expression of NAIF1 neogene in cancerous and non-cancerous colorectaltissues.The protein expression of NAIF1gene has been studied by western blot method in the samples of proteinextracted from 29 patients with colorectal cancer.The result of this study showed that the protein expression of NAIF1 was found in both cancerous andnon-cancerous colorectal tissues, but it is highly expressed in non-cancerous tissues adjacent to cancerousone. Its expression is higher in non-metastatic cancerous tissues compared with metastatic one. Also thisexpression is higher in microsatellite instable (MSI) group of cancer compared with microsatellite stable(MSS) group.It can be concluded that NAIF1 protein expression inversely related with more advanced stage or grade ofcolorectal cancer and it may have a role in inhibition of proliferation, migration and invasion of colorectalcancer by inducing apoptosis.

Anti-Interleukin-16 Neutralizing Antibody Treatment Alleviates Sepsis-Induced Cardiac Injury and Dysfunction via the Nuclear Factor Erythroid-2 Related Factor 2 Pathway in Mice

Several interleukin (IL) members have been reported to participate in sepsis. In this study, the effects of IL-16 on sepsis-induced cardiac injury and dysfunction were examined, and the related mechanisms were detected. IL-16 expression in septic mice was first measured, and the results showed that both cardiac and serum IL-16 expression levels were increased in mice with sepsis induced by LPS or cecal ligation and puncture (CLP) compared with control mice. Then, IL-16 was neutralized, and the effects on lipopolysaccharide- (LPS-) induced cardiac injury were detected. The results showed that an anti-IL-16 neutralizing antibody (nAb) significantly reduced mortality and increased serum lactate dehydrogenase (LDH), creatine kinase myocardial bound (CK-MB), and cardiac troponin T (cTnT) levels while improving cardiac function in mice with LPS-induced sepsis. Neutralization of IL-16 also increased the activation of antioxidant pathways and the expression of antioxidant factors in septic mice while decreasing the activation of prooxidant pathways and the expression of prooxidants. Treatment with the anti-IL-16 nAb increased mitochondrial apoptosis-inducing factor (AIF) expression, decreased nuclear AIF and cleaved poly-ADP-ribose polymerase (PARP) expression, and decreased TUNEL-positive cell percentages in LPS-treated mice. Additionally, treatment with CPUY192018, the nuclear factor erythroid-2 related factor 2 (Nrf2) pathway, significantly increased mortality and reversed the above effects in mice treated with LPS and the anti-IL-16 nAb. Our results showed that the anti-IL-16 nAb regulates oxidative stress through the Nrf2 pathway and participates in the regulation of cardiac injury in septic mice. Neutralization of IL-16 may be a beneficial strategy for the prevention of cardiac injury and dysfunction in sepsis patients.

Long noncoding RNA SRY-box transcription factor 2 overlapping transcript participates in Parkinson’s disease by regulating the microRNA-942-5p/nuclear apoptosis-inducing factor 1 axis

ABSTRACT Parkinson’s disease (PD) is a neurodegenerative disorder. Studies have shown that long noncoding RNA SRY-box transcription factor 2 overlapping transcript (lncRNA SOX2-OT) is highly expressed in PD patients, but its specific functions and mechanisms require further research. To address this gap, this study utilized an in vitro PD cell model induced by 1-methyl-4-phenylpyridinium (MPP+). Cell viability, apoptosis, lactate dehydrogenase (LDH) activity, inflammatory factor secretion, and oxidative stress indicators were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide assay, LDH assay, flow cytometry, enzyme linked immunosorbent assay (ELISA), and corresponding kits, respectively. Gene and protein expression were measured using quantitative real-time-PCR and western blotting, respectively. The results indicated that microRNA-942-5p (miR-942-5p) was a direct target of lncRNA SOX2-OT and nuclear apoptosis-inducing factor 1 (NAIF1) was a direct target of miR-942-5p. The expression levels of lncRNA SOX2-OT and NAIF1 were increased, and miR-942-5p expression was decreased in SH-SY5Y cells following MPP+ treatment. In addition, MPP+ treatment reduced SH-SY5Y cell viability, increased apoptosis; increased cleaved caspase-3 protein expression and cleaved caspase-3/caspase-3 ratio; enhanced lactate dehydrogenase viability; increased tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and reactive oxygen species, and decreased superoxide dismutase activity in SH-SY5Y cells were inhibited by SOX2-OT-siRNA, and these inhibitions were reversed by miR-942-5p inhibitor. Moreover, the protective role of miR-942-5p mimic in MPP+-induced SH-SY5Y cells was eliminated by the NAIF1 plasmid. Overall, lncRNA SOX2-OT-mediated regulation of oxidative stress, inflammation, and neuronal apoptosis were directly controlled by the miR-942-5p/NAIF1 signal axis in MPP+-induced SH-SY5Y cells.

Estrogen receptors participate in silibinin-caused nuclear translocation of apoptosis-inducing factor in human breast cancer MCF-7 cells

Our previous studies showed that silibinin promoted activation of caspases to induce apoptosis in human breast cancer MCF-7 cells by down-regulating the protein expression level of estrogen receptor (ER) α and up-regulating ERβ. Recently, it has been reported that silibinin-induced apoptosis also involved nuclear translocation of apoptosis-inducing factor (AIF). Here we report that silibinin induces nuclear translocation of AIF through the down-regulation of ERα and up-regulation of ERβ in a concentration dependent manner in MCF-7 cells. AIF knockdown with siRNA significantly reverses silibinin-induced apoptosis. The nuclear translocation of AIF is enhanced by treatment with MPP, an ERα antagonist, and blocked with PPT, an ERα agonist. In contrast to ERα activity, the nuclear AIF is increased with an ERβ agonist, DPN and blocked with an ERβ antagonist, PHTPP. Autophagy, negatively regulated by ERα, positively controls AIF-mediated apoptosis, as evidenced by the preventive effect of autophagy inhibitor 3-MA and siRNA targeting LC3, on the nuclear translocation of AIF and cell death induced by silibinin co-treatment with or without MPP. In sum we conclude that AIF in nuclei is involved in silibinin-induced apoptosis, and the nuclear translocation of AIF is increased by down-regulated ERα pathway and/or up-regulated ERβ pathway in MCF-7 cells, accompanying up-regulation of autophagy.

MicroRNA-351 Promotes the Proliferation and Invasion of Glioma Cells through Downregulation of NAIF1.

Despite the well-characterized expression profile of miR-351 in the neural system, its molecular mechanisms in glioma still remain elusive. Here we intended to assess the regulatory function of miR-351 on nuclear apoptosis-inducing factor 1 (NAIF1) and, thereby, modulation of cancerous behaviors of human glioma cell lines. Two human glioma cell lines (U87 and U251) and normal human astroglia (NHA) cell line were cultured. The cell lines were prepared and transfected with mimic, inhibitor, and negative controls (NCs) of miR-351, then MTT and wound healing assays were performed. We extracted the total protein for western blotting assay and isolated the total RNA for real-time PCR. The miR-351 expression was significantly decreased in U87 and U251 cell lines compared with the NHA cell line (P < 0.05). NAIF1 expression was significantly higher in glioma cell lines compared with the NHA cell line (P < 0.05). Moreover, the NAIF1 expression showed a negative correlation with miR-351 (P = 0.005, r = -0.522). Apoptosis was significantly decreased in both cell lines transfected with miR-351 mimics compared with the NC group at 72 and 96h after transfection (P < 0.05) and significantly increased in the transfected group with miR-351 inhibitors compared with the NC group at 72 and 96h after transfection (P < 0.05). According to our results, after 24 and 48h, migration was increased in the mimic group compared with the miR-351 NC group and decreased in the inhibitory group compared with the miR-351 NC group in the U251 cell line. Our findings provide theoretical evidence that miR-351, which targets NAIF1, could be considered an important marker in the pathogenesis of glioma. Furthermore, miR-351 has valuable potential to serve as a new prognostic and diagnostic biomarker and could be considered a potential target for the treatment of this cancer in the near future.

Apoptotic Markers in the Midbrain of the Human Neonate After Perinatal Hypoxic/Ischemic Injury.

Our previous postmortem studies on neonates with neuropathological injury of perinatal hypoxia/ischemia (PHI) showed a dramatic reduction of tyrosine hydroxylase expression (dopamine synthesis enzyme) in substantia nigra (SN) neurons, with reduction of their cellular size. In order to investigate if the above observations represent an early stage of SN degeneration, we immunohistochemically studied the expression of cleaved caspase-3 (CCP3), apoptosis inducing factor (AIF), and DNA fragmentation by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin 3'-end-labeling (TUNEL) technique in the SN of 22 autopsied neonates (corrected age ranging from 34 to 46.5 gestational weeks), in relation to the severity/duration of PHI injury, as estimated by neuropathological criteria. No CCP3-immunoreactive neurons and a limited number of apoptotic TUNEL-positive neurons with pyknotic characteristics were found in the SN. Nuclear AIF staining was revealed only in few SN neurons, indicating the presence of early signs of AIF-mediated degeneration. By contrast, motor neurons of the oculomotor nucleus showed higher cytoplasmic AIF expression and nuclear translocation, possibly attributed to the combined effect of developmental processes and increased oxidative stress induced by antemortem and postmortem factors. Our study indicates the activation of AIF, but not CCP3, in the SN and oculomotor nucleus of the human neonate in the developmentally critical perinatal period.

The effect of hyperbaric oxygenation on apoptosis in rats after middle cerebral artery occlusion

Objective To observe the effect of hyperbaric oxygenation (HBO) on apoptosis-inducing factor (AIF) and Caspase-3 levels in rats with permanent middle cerebral artery occlusion (MCAO), and to elucidate the apoptosis pathways. Methods Sixty Sprague-Dawley rats were subjected to permanent MCAO and then randomly divided into a control group and an HBO group, each of 30. Three hours later the rats of the HBO group were put into a hyperbaric cabin held at a pressure of 0.2 MPa for 9 hours. They inhaled supplementary oxygen at the 1st, 3rd, 5th, 7th and 9th hour while the rats in the control group inhaled air at normal pressure. The neurological outcome was measured at the 3rd, 13th and 72nd hour after the MCAO using Garcia scores. Apoptosis in the tissue of the ischemic penumbra, nuclear and mitochondrial AIF and Caspase-3 levels were measured at the 13th and 72nd hours after the modeling. Results The scores were significantly higher at the 13th hour than after the 3rd hour in both groups, and then even higher at the 72nd hour. Apoptosis was evident in the ischemic penumbra at the 13th and 72nd hours in both groups, but the number of cells was less at the 72nd hour than at the 13th hour in the control group. There was significantly less apoptosis in the HBO group than in the control group at the 13th hour. The average AIF level had significantly decreased in the nuclei and increased in the mitochondria by the 72nd hour compared with the 13th hour in both groups. The average levels of nuclear AIF at the 13th hour and the 72nd hour were lower than those in the mitochondria. But they were significantly higher in the HBO group than in the control group at the same time points. The levels of Caspase-3, normally zero, had increased by the 13th hour in both groups. The average level of Caspase-3 was significantly lower in both groups at the 72nd hour than at the 13th hour. Conclusions HBO can improve neurological function, inhibit the transfer of AIF from the mitochondria to the nucleus and reduce Caspase-3 levels. The mechanism may involve reducing apoptosis through caspase-dependent and caspase-independent pathways in the mitochondria. Key words: Hyperbaric oxygen; Middle cerebral artery occlusion; Apoptosis; Apoptosis-inducing factors; Caspase-3

Parkin Interacts with Apoptosis-Inducing Factor and Interferes with Its Translocation to the Nucleus in Neuronal Cells.

Mutations in the PRKN gene (encoding parkin) have been linked to the most frequent known cause of recessive Parkinson’s disease (PD), and parkin dysfunction represents a risk factor for sporadic PD. Parkin is widely neuroprotective through different cellular pathways, as it protects dopaminergic neurons from apoptosis in a series of cellular and animal models of PD. The mitochondrial protein apoptosis-inducing factor (AIF) is an important cell death effector, which, upon cellular stress in many paradigms, is redistributed from the mitochondria to the nucleus to function as a proapoptotic factor, mostly independent of caspase activity, while in normal mitochondria it functions as an antiapoptotic factor. AIF is known to participate in dopaminergic neuron loss in experimental PD models and in patients with PD. We, therefore, investigated possible crosstalk between parkin and AIF. By using immunoprecipitation and proximity ligation assays, we demonstrated a physical interaction between the two proteins. Nuclear AIF translocation was significantly reduced by parkin expression in neuroblastoma SH-SY5Y cells after exposure to an apoptogenic stimulus. These results were confirmed in primary murine cortical neurons, which showed a higher nuclear translocation of AIF in parkin-deficient neurons upon an excitotoxic stimulus. Our results indicate that the interaction of parkin with AIF interferes with the nuclear translocation of AIF, which might contribute to the neuroprotective activity of parkin.

Ginsenoside Rb2 suppresses the glutamate-mediated oxidative stress and neuronal cell death in HT22 cells

BackgroundThe objective of our study was to analyze the neuroprotective effects of ginsenoside derivatives Rb1, Rb2, Rc, Rd, Rg1, and Rg3 against glutamate-mediated neurotoxicity in HT22 hippocampal mouse neuron cells. MethodsThe neuroprotective effect of ginsenosides were evaluated by measuring cell viability. Protein expressions of mitogen-activated protein kinase (MAPK), Bcl2, Bax, and apoptosis-inducing factor (AIF) were determined by Western blot analysis. The occurrence of apoptotic and death cells was determined by flow cytometry. Cellular level of Ca2+ and reactive oxygen species (ROS) levels were evaluated by image analysis using the fluorescent probes Fluor-3 and 2′,7′-dichlorodihydrofluorescein diacetate, respectively. In vivo efficacy of neuroprotection was evaluated using the Mongolian gerbil of ischemic brain injury model. ResultReduction of cell viability by glutamate (5 mM) was significantly suppressed by treatment with ginsenoside Rb2. Phosphorylation of MAPKs, Bax, and nuclear AIF was gradually increased by treatment with 5 mM of glutamate and decreased by co-treatment with Rb2. The occurrence of apoptotic cells was decreased by treatment with Rb2 (25.7 μM). Cellular Ca2+ and ROS levels were decreased in the presence of Rb2, and in vivo data indicated that Rb2 treatment (10 mg/kg) significantly diminished the number of degenerated neurons. ConclusionOur results suggest that Rb2 possesses neuroprotective properties that suppress glutamate-induced neurotoxicity. The molecular mechanism of Rb2 is by suppressing the MAPKs activity and AIF translocation.

The proteomic expression of Neogene nuclear apoptosis-inducing factor 1(NAIF1) in Leukaemia cell lines

The process of molecular domestication which had been occured in some DNA transposons (2nd class of transposable elements) led to the formation of novel genes, which are called “Neogenes”. These neogenes may play an important role in the human genetic instability, human diseases and cancer. One of these Neogene is nuclear apoptosis-inducing factor 1(NAIF1) which inducing apoptosis in various human cancers.The aim of this work is to study the expression of NAIF1 Neogene in leukaemia cell lines.The protein expression of NAIF1gene has been studied by western blot method in seven leu-kaemia cell lines (HL60, NB4, KG1, KG1a, ML2, THP1 and U937) and in a healthy tissue of blood as a control.The result of this study showed that the protein expression of NAIF1 in leukaemia cell lines were found in variable degree of expression, which was highly expressed in healthy blood tis-sue.From the results of this work, it can be concluded that NAIF1 protein may have a role in inhi-bition of proliferation, migration and invasion of leukaemia by inducing apoptosis, which need further research and confirmation.

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.

Augmentation of poly(ADP-ribose) polymerase-dependent neuronal cell death by acidosis

Tissue acidosis is a key component of cerebral ischemic injury, but its influence on cell death signaling pathways is not well defined. One such pathway is parthanatos, in which oxidative damage to DNA results in activation of poly(ADP-ribose) polymerase and generation of poly(ADP-ribose) polymers that trigger release of mitochondrial apoptosis-inducing factor. In primary neuronal cultures, we first investigated whether acidosis per sé is capable of augmenting parthanatos signaling initiated pharmacologically with the DNA alkylating agent, N-methyl-N′-nitro-N-nitrosoguanidine. Exposure of neurons to medium at pH 6.2 for 4 h after N-methyl-N′-nitro-N-nitrosoguanidine washout increased intracellular calcium and augmented the N-methyl-N′-nitro-N-nitrosoguanidine-evoked increase in poly(ADP-ribose) polymers, nuclear apoptosis-inducing factor , and cell death. The augmented nuclear apoptosis-inducing factor and cell death were blocked by the acid-sensitive ion channel-1a inhibitor, psalmotoxin. In vivo, acute hyperglycemia during transient focal cerebral ischemia augmented tissue acidosis, poly(ADP-ribose) polymers formation, and nuclear apoptosis-inducing factor , which was attenuated by a poly(ADP-ribose) polymerase inhibitor. Infarct volume from hyperglycemic ischemia was decreased in poly(ADP-ribose) polymerase 1-null mice. Collectively, these results demonstrate that acidosis can directly amplify neuronal parthanatos in the absence of ischemia through acid-sensitive ion channel-1a . The results further support parthanatos as one of the mechanisms by which ischemia-associated tissue acidosis augments cell death.

Rationally Repurposing Ruxolitinib (Jakafi (®)) as a Solid Tumor Therapeutic.

We determined whether the approved myelofibrosis drug ruxolitinib (Jakafi®), an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could be repurposed as an anti-cancer agent for solid tumors. Ruxolitinib synergistically interacted with dual ERBB1/2/4 inhibitors to kill breast as well as lung, ovarian and brain cancer cells. Knock down of JAK1/2 or of ERBB1/2/3/4 recapitulated on-target drug effects. The combination of (ruxolitinib + ERBB1/2/4 inhibitor) rapidly inactivated AKT, mTORC1, mTORC2, STAT3, and STAT5, and activated eIF2α. In parallel, the drug combination reduced expression of MCL-1, BCL-XL, HSP90, HSP70, and GRP78, and increased expression of Beclin1. Activated forms of STAT3, AKT, or mTOR prevented the drug-induced decline in BCL-XL, MCL-1, HSP90, and HSP70 levels. Over-expression of chaperones maintained AKT/mTOR activity in the presence of drugs and protected tumor cells from the drug combination. Expression of dominant negative eIF2α S51A prevented the increase in Beclin1 expression and protected tumor cells from the drug combination. Loss of mTOR activity was associated with increased ATG13 S318 phosphorylation and with autophagosome formation. Autophagosomes initially co-localized with mitochondria and subsequently with lysosomes. Knock down of Beclin1 suppressed: drug-induced mitophagy; the activation of the toxic BH3 domain proteins BAX and BAK; and tumor cell killing. Knock down of apoptosis-inducing factor (AIF) protected tumor cells from the drug combination, whereas blockade of caspase 9 signaling did not. The drug combination released AIF into the cytosol and increased nuclear AIF: eIF3A co-localization. A 4-day transient exposure of orthotopic tumors to (ruxolitinib + afatinib) profoundly reduced mammary tumor growth over the following 35 days. Re-grown tumors exhibited high levels of BAD S112 phosphorylation and activation of ERK1/2 and NFκB. Our data demonstrate that mitophagy is an essential component of (ruxolitinib + ERBB inhibitor) lethality and that this drug combination should be explored in a phase I trial in solid tumor patients.

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.

Role of HSP70 in motoneuron survival after excitotoxic stress in a rat spinal cord injury model in vitro.

The outcome for gait recovery from paralysis due to spinal lesion remains uncertain even when damage is limited. One critical factor is the survival of motoneurons, which are very vulnerable cells. To clarify the early pathophysiological mechanisms of spinal damage, an in vitro injury model of the rat spinal cord caused by moderate excitotoxicity was used. With this preparation we investigated whether motoneuron survival was dependent on the expression of the neuroprotective protein HSP70. In the present study excitotoxicity evoked by kainate induced delayed (24 h) loss (35%) of motoneurons, which became pyknotic with translocation of the cell death biomarker apoptosis-inducing factor (AIF) to the nucleus. This process was concomitant with suppression of locomotor network electrical activity. Surviving cells showed strong expression of HSP70 without nuclear AIF. The HSP70 inhibitor VER155008 per se induced neurotoxicity similar to that of kainate, while the HSP90 inhibitor geldanamycin did not damage spinal tissue. Electrophysiological recording following kainate or VER155008 indicated depression of motoneuron field potentials, with decreased excitability and impaired synaptic transmission. When these two drugs were applied together, more intense neurotoxicity emerged. Our data indicate that HSP70 was one important contributor to motoneuron survival and suggest that enhancing HSP70 activity is a potential future strategy for neuroprotecting these cells.

Heat Shock Protein 70 Prevents Hyperoxia-Induced Disruption of Lung Endothelial Barrier via Caspase-Dependent and AIF-Dependent Pathways.

Exposure of pulmonary artery endothelial cells (PAECs) to hyperoxia results in a compromise in endothelial monolayer integrity, an increase in caspase-3 activity, and nuclear translocation of apoptosis-inducing factor (AIF), a marker of caspase-independent apoptosis. In an endeavor to identify proteins involved in hyperoxic endothelial injury, we found that the protein expression of heat-shock protein 70 (Hsp70) was increased in hyperoxic PAECs. The hyperoxia-induced Hsp70 protein expression is from hspA1B gene. Neither inhibition nor overexpression of Hsp70 affected the first phase barrier disruption of endothelial monolayer. Nevertheless, inhibition of Hsp70 by using the Hsp70 inhibitor KNK437 or knock down Hsp70 using siRNA exaggerated and overexpression of Hsp70 prevented the second phase disruption of lung endothelial integrity. Moreover, inhibition of Hsp70 exacerbated and overexpression of Hsp70 prevented hyperoxia-induced apoptosis, caspase-3 activation, and increase in nuclear AIF protein level in PAECs. Furthermore, we found that Hsp70 interacted with AIF in the cytosol in hyperoxic PAECs. Inhibition of Hsp70/AIF association by KNK437 correlated with increased nuclear AIF level and apoptosis in KNK437-treated PAECs. Finally, the ROS scavenger NAC prevented the hyperoxia-induced increase in Hsp70 expression and reduced the interaction of Hsp70 with AIF in hyperoxic PAECs. Together, these data indicate that increased expression of Hsp70 plays a protective role against hyperoxia-induced lung endothelial barrier disruption through caspase-dependent and AIF-dependent apoptotic pathways. Association of Hsp70 with AIF prevents AIF nuclear translocation, contributing to the protective effect of Hsp70 on hyperoxia-induced endothelial apoptosis. The hyperoxia-induced increase in Hsp70 expression and Hsp70/AIF interaction is contributed to ROS formation.

Upregulation of miR-24 promotes cell proliferation by targeting NAIF1 in non-small cell lung cancer.

Recent studies have implied that aberration of miR-24 is linked to various human cancers. However, its role in non-small cell lung cancer (NSCLC) remains obscure. Here, we found that miR-24 was significantly upregulated in NSCLC tissues and patients' serum. High expression of miR-24 in patients' serum was independently correlated with a shorter overall survival of NSCLC patients. Depletion of miR-24 inhibited cell proliferation and anchorage-independent survival ability in lung cancer cell lines and reduced tumor formation ability in nude mice. Nuclear apoptosis-inducing factor 1 (NAIF1) was identified to be a functional target of miR-24 in the human lung. Next, we observed that the NAIF1 mRNA expression level in NSCLC tissues was suppressed in comparison to that in adjacent normal tissues. Restoration of NAIF1 in lung cancer cell inhibited cell proliferation and anchorage-independent survival ability, which were found to be similar with those from transfecting a miR-24 inhibitor into lung cancer cells. In conclusion, our study demonstrated that miR-24 was upregulated in NSCLC, and suppressing the expression of miR-24 inhibited tumor characteristics. MiR-24 acted as an oncomir, at least partially through regulation of its functional target NAIF1 in NSCLC. MiR-24 may serve as a novel potential biomarker for NSCLC diagnosis and prognosis.