Apoptosis-inducing Factor Levels Research Articles

A PILOT IN VITRO STUDY ON THE EFFECT OF BLUE HONEYSUCKLE EXTRACT ON HUMAN NON-CANCEROUS AND CANCEROUS OVARIAN CELLS

Honeysuckle (Lonicera caerulea L.) is utilized in Chinese, Japanese, and Russian folk medicines for centuries. The berries referred to as blue honeysuckle are recently gaining popularity in central and southern Europe due to remarkable therapeutic potential and widespread use as an ingredient in dietary supplements. The primary objective of this investigation was to examine the impact of blue honeysuckle extract on human ovarian cells – both non-cancerous HGL5 (immortalized human ovarian granulosa cell line) and cancerous OVCAR-3 (human ovarian carcinoma cell line). Cell viability, secretion of steroid hormones (17ß-estradiol and progesterone), human transforming growth factor-β2 (TGFβ-2) and its receptor (TGFBR2), and the level of apoptosis-inducing factor (AIF) were examined in ovarian granulosa cells HGL5, and the same parameters (excepting the steroid hormones) were assessed in ovarian epithelial cancer cells OVCAR-3. Blue honeysuckle ethanolic extract was applied to cells for 24 hours. AlamarBlueTM assay was used to assess cell viability, whereas secretion of steroid hormones was evaluated by ELISA. ELISA was also used to assess the levels of TGF-β2, its receptor TGFBR2 and AIF in HGL5 and OVCAR-3 cell lysates. Based on a range of doses ranging from 5 – 100 μg/mL, the extract from blue honeysuckle had no adverse effects on the viability of human ovarian cells. Futuremore, it did not affect their secretory activity – be it the release of steroid hormones or that of growth factor TGF-β2 and its receptor TGFBR2, nor did induce apoptosis (as revealed by the AIF). The present pilot study for the first time reports the effect of blue honeysuckle extract on ovarian cell models using both non-cancerous cells HGL5 and cancerous OVCAR-3 cells. These findings provide initial insights into the safety and potential utility of blue honeysuckle extract, warranting further investigation into its therapeutic applications. In further research, it is possible to examine a longer exposure time to see if it affects the cells.

Parthanatos type programmed cell death and septic patient mortality

ObjectiveParthanatos is a form of programmed cell death mediated by apoptosis-inducing factor (AIF). However, there are not data on parthanatos in septic patients. The objective of the current study was to explore whether parthanatos is associated with mortality of septic patients. DesignObservational and prospective study. SettingThree Spanish Intensive Care Units during 2017. PatientsPatients with sepsis according to Sepsis-3 Consensus criteria. InterventionsSerum AIF concentrations were determined at moment of sepsis diagnosis. Main variable of interestMortality at 30 days. ResultsThere were included 195 septic patients, and non-surviving (n=72) had serum AIF levels (p<0.001), lactic acid (p<0.001) and APACHE-II (p<0.001) that surviving (n=123). Multiple logistic regression analysis showed that patients with serum AIF levels>55.6ng/mL had higher mortality risk (OR=3.290; 95% CI=1.551−6.979; p=0.002) controlling for age, SOFA and lactic acid. ConclusionsParthanatos is associated with mortality of septic patients.

Parthanatos type programmed cell death and septic patient mortality

ObjectiveParthanatos is a form of programmed cell death mediated by apoptosis-inducing factor (AIF). However, there are not data on parthanatos in septic patients. The objective of the current study was to explore whether parthanatos is associated with mortality of septic patients. DesignObservational and prospective study. SettingThree Spanish Intensive Care Units during 2017. PatientsPatients with sepsis according to Sepsis-3 Consensus criteria. InterventionsSerum AIF concentrations were determined at moment of sepsis diagnosis. Main variable of interestMortality at 30 days. ResultsThere were included 195 septic patients, and non-surviving (n=72) had serum AIF levels (p<0.001), lactic acid (p<0.001) and APACHE-II (p<0.001) that surviving (n=123). Multiple logistic regression analysis showed that patients with serum AIF levels>55.6ng/mL had higher mortality risk (OR=3.290; 95% CI=1.551−6.979; p=0.002) controlling for age, SOFA and lactic acid. ConclusionsParthanatos is associated with mortality of septic patients.

Permissive hypercapnia and hypercapnic hypoxia inhibit signaling pathways of neuronal apoptosis in ischemic/hypoxic rats.

In the present study, we aimed to test the hypothesis that hypercapnia, independently and/or in combination with hypoxia, can activate signaling pathways related to the inhibition of proapoptotic (caspase-dependent and caspase-independent) factors and the induction of antiapoptotic factors in facilitating adaptation to hypoxia/ischemia. Following exposure to permissive hypercapnia and/or normobaric hypoxia, the degree of apoptosis was evaluated in experimental ischemia models in vivoand in vitro. The percentages of caspase-3, apoptosis-inducing factor (AIF), Bax, and Bcl-2 in astrocytes and neurons derived from male Wistar rats were also calculated. In vitro, cells were subjected to various types of respiratory exposure (hypoxia and/or hypercapnia for 24 or 12h) as well as further sublethal chemical hypoxia. The percentages of these molecules in nerve cells in the ischemic penumbra of the brain after photothrombotic injury were also calculated. The degree of apoptosis was found to decrease in ischemic penumbra, mostly due to the hypercapnic component. It was also discovered that the levels of caspase-3, AIF, and Bax decreased in this region, whereas the Bcl-2 levels increased following exposure to hypercapnia and hypercapnic hypoxia. This integrative assessment of the rate of apoptosis/necrosis in astrocyte and neuron cultures shows that the combination of hypercapnia and hypoxia resulted in the maximum neuroprotective effect. The levels of apoptosis mediators in astrocyte and neuron cultures were calculated after modeling chemical hypoxia in vitro. These results show that the exposure models where permissive hypercapnia and normobaric hypoxia were combined also had the most pronounced inhibitory effects on apoptotic signaling pathways.

The Protective Effects of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells in Noise-Induced Hearing Loss of Rats.

A few prior animal studies have suggested the transplantation or protective effects of mesenchymal stem cells (MSCs) in noise-induced hearing loss. This study intended to evaluate the fates of administered MSCs in the inner ears and the otoprotective effects of MSCs in the noise-induced hearing loss of rats. Human embryonic stem cell-derived MSCs (ES-MSCs) were systematically administered via the tail vein in adult rats. Eight-week-old Sprague-Dawley rats were randomly allocated to the control (n = 8), ES-MSC (n = 4), noise (n = 8), and ES-MSC+noise (n = 10) groups. In ES-MSC and ES-MSC+noise rats, 5 × 105 ES-MSCs were injected via the tail vein. In noise and ES-MSC+noise rats, broadband noise with 115 dB SPL was exposed for 3 h daily for 5 days. The hearing levels were measured using auditory brainstem response (ABR) at 4, 8, 16, and 32 kHz. Cochlear histology was examined using H&E staining and cochlear whole mount immunofluorescence. The presence of human DNA was examined using Sry PCR, and the presence of human cytoplasmic protein was examined using STEM121 immunofluorescence staining. The protein expression levels of heat shock protein 70 (HSP70), apoptosis-inducing factor (AIF), poly (ADP-ribose) (PAR), PAR polymerase (PARP), caspase 3, and cleaved caspase 3 were estimated. The ES-MSC rats did not show changes in ABR thresholds following the administration of ES-MSCs. The ES-MSC+ noise rats demonstrated lower ABR thresholds at 4, 8, and 16 kHz than the noise rats. Cochlear spiral ganglial cells and outer hair cells were more preserved in the ES-MSC+ noise rats than in the noise rats. The Sry PCR bands were highly detected in lung tissue and less in cochlear tissue of ES-MSC+noise rats. Only a few STEM121-positivities were observed in the spiral ganglial cell area of ES-MSC and ES-MSC+noise rats. The protein levels of AIF, PAR, PARP, caspase 3, and cleaved caspase 3 were lower in the ES-MSC+noise rats than in the noise rats. The systemic injection of ES-MSCs preserved hearing levels and attenuated parthanatos and apoptosis in rats with noise-induced hearing loss. In addition, a tiny number of transplanted ES-MSCs were observed in the spiral ganglial areas.

Realgar (As4S4), a traditional Chinese medicine, induces acute promyelocytic leukemia cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway in vitro.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myelogenous leukemia (AML) characterized by the proliferation of abnormal promyelocytes. Realgar, a Chinese medicine containing arsenic, can be taken orally. Traditional Chinese medicine physicians have employed realgar to treat APL for over a thousand years. Therefore, realgar may be a promising candidate for the treatment of APL. Nevertheless, the underlying mechanism behind realgar therapy is largely unclear. The present study aimed to investigate the effect of realgar on cell death in the APL cell line (NB4) in vitro and to elucidate the underlying mechanism. In this study, after APL cells were treated with different concentrations of realgar, the cell survival rate, apoptotic assay, morphological changes, ATP levels and cell cycle arrest were assessed. The expression of Bcl-2, Bax, Cytochrome C (Cyt-C) and apoptosis-inducing factor (AIF) at the mRNA and protein levels were also measured by immunofluorescence, quantitative PCR (qPCR) and Western blotting. We found that realgar could significantly inhibit APL cell proliferation and cell death in a time- and dose-dependent manner. Realgar effectively decreased the ATP levels in APL cells. Realgar also induced APL cell cycle arrest at the S and G2/M phases. Following realgar treatment, the mRNA and protein levels of Bcl-2 were significantly downregulated, whereas the levels of Bax, Cyt-C, and AIF were significantly upregulated. In summary, realgar can induce APL cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway, suggesting that realgar may be an effective therapeutic for APL.

Effects of moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) combined with benazepril on myocardial cells apoptosis index and apoptosis-related proteins cytochrome c and apoptosis-inducing factor in rats with chronic heart failure.

To observe the effects of moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) combined with benazepril on myocardial cells apoptosis index, the expression levels of apoptosis-related proteins cytochrome c (Cyt-C) and apoptosis-inducing factor (AIF) in chronic heart failure (CHF) rats. Sixty-five rats were randomly divided into normal group () and model-I group (). After modeling, CHF rats in model-I group were divided into model group, moxibustion group, benazepril group, moxibustion plus benazepril group (abbreviated as aibei group, the same below), 10 rats in each group. Echocardiogram index was examined by echocardiography. Hemodynamic indices were measured by rat cardiac function meter. Serum B-type brain natriuretic peptide (BNP) was detected by enzyme-linked immunosorbent assay. Myocardial cells apoptosis index was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining. Pathological changes of myocardial tissues were observed by hematoxylin and eosin staining. The expression levels of Cyt-C and AIF in myocardial tissues were detected by Western blot. Compared with normal group, ejection fraction and left ventricular diameter shortening rate in model-Ⅰ group were significantly reduced, myocardial cells of rats in model group exhibited unclear transverse striations, cells swellings and vacuoles, cardiac functions were deteriorated, serum BNP level, myocardial cells apoptosis index, and the expression levels of Cyt-C and AIF were significantly increased. Compared with model group, myocardial cells of rats in moxibustion group, benazepril group, and aibei group were dyed more evenly, muscle fibers were arranged relatively neatly, cardiac functions were improved, serum BNP level, myocardial cells apoptosis index, and the expression levels of Cyt-C and AIF were significantly decreased. Compared with aibei group, cardiac functions were worsened, myocardial cells apoptosis index, and the expression levels of Cyt-C and AIF were increased. Moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) combined with benazepril could improve CHF better than moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) or benazepril alone. The mechanisms might be that they can inhibit the expressions of Cyt-C and AIF, and inhibit the apoptosis of cardiomyocytes.

Venetoclax Reverses Metabolic Reprogramming Induced By S1P Modulator FTY720, Suppresses Oxidative Phosphorylation and Synergistically Targets Multiple Myeloma

Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic modalities that bypass these resistance mechanisms. FTY720, also known as fingolimod, is an S1P modulator approved by the FDA to treat the relapsing form of multiple sclerosis. Previously we reported that FTY720 exhibits potent anti-myeloma effect in vitro and in vivo in disseminated xenograft model of MM (Beider et al., Clin Cancer Res 2017). Cytotoxic activity of FTY720 was associated with down-regulation of anti-apoptotic protein MCL-1 while not affecting BCL-2 levels. It is therefore conceivable that BCL-2 inhibition using BH3-mimetic venetoclax may improve responses to FTY720.Incubation of human MM cell lines (n=8) and primary MM cells (n=3) with venetoclax and FTY720 combination synergistically potentiated cell death (CI<0.02), regardless of the MM cells t (11; 14) status. The robust apoptosis induced by venetoclax /FTY720 treatment was accompanied by cytochrome C release, activation of caspase-3 and extensive DNA damage, demonstrated by increased TUNEL staining and elevated levels of phosphorylated histone H2AX, respectively . These effects were associated with down-regulation of BCL-2 protein, stabilization of pro-apoptotic Bak protein, loss of mitochondrial membrane potential, ER stress induction, and inhibition of the AKT/mTOR signaling pathway. Furthermore, the venetoclax /FTY720 combination markedly induced mitochondrial calcium flux and mitochondrial ROS generation.Corresponding with mitochondrial destabilization, venetoclax/FTY720 combination promoted the release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol and subsequently increased AIF nuclear localization, suggesting its functional role in the execution phase of the apoptosis in response to the dual treatment. AIF is a mitochondrial oxidoreductase that contributes to cell death programs and participates in the assembly of the respiratory chain. Of note, single-agent treatment with FTY720 profoundly up-regulated mitochondrial AIF levels. Given the regulative role of AIF in mitochondrial bioenergetics, we could suggest that increased mitochondrial levels of AIF upon FTY720 exposure may support adaptive responses and promote MM survival upon mitochondrial stress. We thus investigated a possible effect of venetoclax and FTY720 separately or in combination on the metabolic activity of MM cells, observing distinct metabolic profiles of single versus combined exposures. FTY720 significantly suppressed glycolysis, down-regulating the transcript levels of the glycolytic enzymes HK2, PDK1, and LDHA. Glycolytic suppression may result in upregulation of mitochondrial content, which maintains cell survival. In accordance, increased mitochondrial activity was detected in FTY720-treated MM cells, detected by high uptake of MitoSpy Red, a dye that stains mitochondria in a membrane potential-dependent manner. To determine if the changes in the mitochondrial content also altered mitochondrial function, bioenergetic analysis was undertaken. FTY720-treated MM cells demonstrated increased levels of NDUFB8 and UQCRC2 (subunits of mitochondrial respiratory complexes I and III, respectively). Furthermore, FTY720 exposure up-regulated ATP production, suggesting an increase in tumor-protective oxidative phosphorylation (OXPHOS). In agreement, inhibition of mitochondrial electron transport chain using rotenone sensitized MM cells to FTY720, synergistically promoting cell death.Notably, co-treatment with venetoclax effectively reversed the metabolic changes mediated by FTY720, reducing mitochondrial mass, suppressing mitochondrial activity and strongly down-regulating the pathways related to OXPHOS. Furthermore, venetoclax/FTY720 combination significantly reduced glutathione (GSH) levels, therefore suppressing antioxidative cell responses.To conclude, we unveil venetoclax role in the metabolic regulation in MM cells. Venetoclax reverses metabolic reprogramming induced by FTY720, suppresses mitochondrial respiration, induces vigorous mitochondrial damage and preferentially targets MM cells in combination with FTY720. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. [Display omitted] DisclosuresPeled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding.

Effect of electroacupuncture preconditioning on myocyte apoptosis in myocardial ischemia-reperfusion injury rats based on FXR/SHP pathway

To observe the effect of electroacupuncture (EA) preconditioning on left-cardiac function, contents of serum TNF-α and IL-6 and expression of myocardial farnesoid X receptor(FXR), small heterodimer partner (SHP), apoptosis inducing factor (AIF) and heat shock proteins 70 (HSP70) genes in myocardial ischemia-reperfusion injury (MIRI) rats, so as to explore its mechanisms underlying improvement of ischemic myocardial injury. Forty male Wistar rats were randomly divided into normal control, sham operation, MIRI model and EA pretreatment groups, with 10 rats in each group. Rats of the sham operation group received exposure of the thorax and heart. The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery (LAD). EA (2 Hz/100 Hz and 1 mA) was applied to bilateral "Neiguan" (PC6), "Zusanli" (ST36) and "Guanyuan" (CV4) for 20 min, once a day for 7 days. The left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP)and maximal rates of rise and fall of left ventricular pressure (±dp/dtmax) were detected, the contents of serum TNF-α and IL-6 were detected by using enzyme-linked immunosorbent assay (ELISA), and the expression of FXR, SHP, AIF and HSP70 apoptotic genes in the myocardial tissue were measured by fluorescent quantitative RT-PCR. Compared with the normal control group, the LVEDP, contents of serum TNF-α and IL-6, and the expression levels of myocardial FXR, SHP, AIF and HSP70 mRNAs were significantly increased (P<0.05), while LVSP and ±dp/dtmax levels were obviously decreased in the model group (P<0.05). In comparison with the model group, MIRI-induced increases of LVEDP, TNF-α and IL-6 contents, and FXR, SHP and AIF mRNA expression and decreases of ±dp/dtmax and LVSP levels were reversed(P<0.05), except HSP70 mRNA expression with significantly increased (P<0.05) in the EA pretreatment group. EA pretreatment can protect the left ventricular function of the ischemic heart in MIRI rats, which may be related to its effects in reliving peripheral inflammation and regulating the expression levels of apoptosis-related factors FXR, SHP, AIF and HSP70 in the myocardium.

Impact of Parthanatos on the Increased Risk of Onset and Mortality in Male Patients With Pulmonary Hypertension.

There have been no studies as to whether parthanatos, a poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1)-dependent and apoptosis-inducing factor (AIF)-mediated caspase-independent programmed cell death, is present in pulmonary hypertension (PH). Basic studies have, however, been conducted on several of the key molecules in parthanatos, such as PARP-1, AIF, and macrophage migration inhibitory factor (MIF). For this study, we collected blood samples from 88 incident male patients with PH and 50 healthy controls at the Shanghai Pulmonary Hospital. We measured the key factors of parthanatos (PARP-1, PAR, AIF, and MIF) by enzyme-linked immunosorbent assay and performed a logistic regression, Cox proportional hazards analysis, and Kaplan–Meier test to assess the prognostic value of the key molecules in diagnosing and predicting survival. The patients who ultimately died had a significantly poorer clinical status during the study than those who survived. The PARP-1, PAR, AIF, and MIF levels were significantly higher in the patients than in the controls (all p < .0001), and the PARP-1, PAR, and AIF levels were higher in the nonsurvivors than in the survivors (all p < .0001). PARP-1 and AIF levels served as independent predictors of disease onset and mortality in these patients (all p < .005). Patients with PARP-1 levels <11.24 ng/mL or AIF levels <1.459 pg/mL had significantly better survival than those with higher PARP-1 or AIF levels (p < .0001). Circulating levels of PARP-1 and AIF were independent predictors for PH onset and mortality, which indicated that parthanatos might be associated with the pathogenesis of PH.

Activation of Cell Death Mediated by the Crosstalk between Caspase‐3 and Apoptosis Inducing Factor in the Brainstem upon Exposure to Monosodium Glutamate and Corticosterone

Cellular stress induced by excessive glutamate or corticosterone leads to apoptosis. Our previous studies showed that Monosodium Glutamate (MSG) and Corticosterone (CORT) potentially activated the neuroendocrine stress axis and induced microvascular disruption, neuronal and glial cell apoptosis. The present study tested the hypothesis that whether specific death programs were activated according to cell types and further delineated the intracellular mechanisms involved in the apoptotic signaling cascade in the brain stem. We used glutamate and CORT paradigms to test this hypothesis. Young adult (2 months old) male Wistar rats were injected subcutaneously with MSG or CORT for 7 days and sacrificed on 8th, 15th and 30th days of the treatment to monitor recovery responses after 1, 8 and 23 days. Brain stem (BS) was used for expression of Glucocorticoid receptor (Nr3c1) and Caspase-3 by qPCR protocols. MSG upregulated Nr3c1 expression on day 8 and 30, and downregulated it on day 15, though not significant. The CORT treatment showed downregulation of Nr3c1 expression across the 3 time points, significant only on day 8. In the MSG treated group, Caspase-3 expression displayed an insignificant upregulation on day 8 and 30, and an insignificant downregulation on day 15. The CORT treatment displayed downregulated Caspase-3 expression on day 8 and 30 and an upregulation on day 15 though not significant. Immunohistochemical staining of the brain stem showed an increase in Caspase-3 immunoreactivity in microvasculature, and neuron and glia-like cells in both MSG and CORT groups. Cells from control rats were with either no signal or less immunoreactivity. Since the Caspase-3 apoptotic marker gene was not activated in the BS, there is a possibility that cell death in the BS might be mediated by a caspase-independent apoptotic pathway involving apoptosis inducing factor (AIF) protein. The mitochondrial AIF protein is an important cell death effector upon cellular stress and is redistributed from the mitochondria to the nucleus to function as a proapoptotic factor mostly independent of caspase activity. Immunolabeling with anti-Caspase-3 and anti-AIF showed that AIF was colocalized with Caspase-3 in neuron and glia-like cells that underwent apoptosis, whereas the AIF labeling was not detected in microvasculature of the BS upon exposure to MSG and CORT. No signal was detected in control groups. The results suggest that microvessels may undergo disruption through a spontaneous active caspase-3 dependent mechanism whereas neurons or glial cells undergo AIF mediated caspase-3 apoptosis execution pathway. The protein levels of AIF and Caspase-3 in cytosol, mitochondria, nucleus and whole cell lysates were detected by Western blot analysis. Our study is the first to report that multiple and distinctive death programs involving only Caspase-3 dependent microvascular disruption and AIF - Caspase-3 mediated neuronal and glial cell death occur in the BS. Furthermore potential disruption in blood-brain barrier integrity and neural connectivity occurs upon exposure to MSG and CORT.

Apoptosis-inducing factor plays a role in the pathogenesis of hepatic and renal injury during cholestasis.

Cholestasis is a clinical complication with different etiologies. The liver is the primary organ influenced in cholestasis. Renal injury is also a severe clinical complication in cholestatic/cirrhotic patients. Several studies mentioned the importance of oxidative stress and mitochondrial impairment as two mechanistically interrelated events in cholestasis-induced organ injury. Apoptosis-inducing factor (AIF) is a flavoprotein located in the inner mitochondrial membrane. This molecule is involved in a distinct pathway of cell death. The current study aimed to evaluate the role of AIF in the pathophysiology of cholestasis-associated hepatic and renal injury. Bile duct ligation (BDL) was used as an animal model of cholestasis. Serum, urine, and tissue samples were collected at scheduled time intervals (3, 7, 14, and 28 days after BDL surgery). Tissues' AIF mRNA levels, as well as serum, urine, and tissue activity of AIF, were measured. Moreover, markers of DNA fragmentation and apoptosis were assessed in the liver and kidney of cholestatic animals. A significant increase in liver and kidney AIF mRNA levels, in addition to increased AIF activity in the liver, kidney, serum, and urine, was detected in BDL rats. DNA fragmentation and apoptosis were raised in the liver and kidney of cholestatic animals, especially at the early stage of the disease. The apoptotic mode of cell death in the liver and kidney was connected to a higher AIF level. These data mention the importance of AIF in the pathogenesis of cholestasis-induced organ injury, especially at the early stage of this disease. Mitochondrial release of apoptosis-inducing factor (AIF) seems to play a pathogenic role in cholestasis-associated hepatic and renal injury. AIF release is directly connected to oxidative stress and mitochondrial impairment in cholestatic animals.

Alopecia in Harlequin mutant mice is associated with reduced AIF protein levels and expression of retroviral elements

We investigated the contribution of apoptosis-inducing factor (AIF), a key regulator of mitochondrial biogenesis, in supporting hair growth. We report that pelage abnormalities developed during hair follicle (HF) morphogenesis in Harlequin (Hq) mutant mice. Fragility of the hair cortex was associated with decreased expression of genes encoding structural hair proteins, though key transcriptional regulators of HF development were expressed at normal levels. Notably, Aifm1 (R200 del) knockin males and Aifm1(R200 del)/Hq females showed minor hair defects, despite substantially reduced AIF levels. Furthermore, we cloned the integrated ecotropic provirus of the Aifm1Hq allele. We found that its overexpression in wild-type keratinocyte cell lines led to down-regulation of HF-specific Krt84 and Krtap3-3 genes without altering Aifm1 or epidermal Krt5 expression. Together, our findings imply that pelage paucity in Hq mutant mice is mechanistically linked to severe AIF deficiency and is associated with the expression of retroviral elements that might potentially influence the transcriptional regulation of structural hair proteins.

MiR-155-5p promotes metastasis and epithelial-mesenchymal transition of renal cell carcinoma by targeting apoptosis-inducing factor.

Although renal cell carcinoma remains one of the most malignant cancers, our understanding of progression and recurrence of this disease is limited. The present study explored the precise role of miR-155-5p in renal cancer metastasis. The expression of miR-155-5p in renal carcinoma clinical tissues and cells was determined using quantitative real time-polymerase chain reaction. The role of miR-155-5p on tumor cell growth were examined using CCK-8 and colony formation assays. Transwell assay was utilized to identify the role of miR-155-5p on the invasion and migration of renal cancer cells. Markers of epithelial-mesenchymal transition were determined using western blot. The in vivo effects of miR-155-5p on renal cancer cell growth, apoptosis, and metastasis were explored using xenograft mice. Luciferase reporter assay was performed to identify the potential target of miR-155-5p. Levels of miR-155-5p were significantly elevated in renal cancer tissues and cell lines. Suppression of miR-155-5p decreased the growth, colony formation, migration, and invasiveness of renal cancer cells. In contrast, overexpression of miR-155-5p led to opposite effects on renal cancer cells. Mechanically, the apoptosis-inducing factor was identified as the target of miR-155-5p. Interference of miR-155-5p significantly increased mRNA and protein expression of the apoptosis-inducing factor, whereas overexpression of miR-155-5p remarkably suppressed the apoptosis-inducing factor levels in renal cancer cells. The xenograft model identified that suppression of miR-155-5p restrained tumor growth and promoted apoptosis, whereas overexpression of miR-155-5p decreased apoptosis and accelerated tumor growth. Moreover, the number of lung metastasis nodules were decreased following injection with anti-miR-155-5p transfected cells, whereas the nodules were remarkably increased after overexpression of miR-155-5p. In addition, in vitro and in vivo assays both confirmed that suppression of miR-155-5p increased the expression of E-cadherin and decreased levels of N-cadherin and Snail, whereas overexpression of miR-155-5p accelerated epithelial-mesenchymal transition progression in renal cancer cells. These findings demonstrate that miR-155-5p enhances metastasis and epithelial-mesenchymal transition by targeting the apoptosis-inducing factor, suggesting that miR-155-5p represents a novel therapeutic target for renal cancer.

Inhaled H2 or CO2 Do Not Augment the Neuroprotective Effect of Therapeutic Hypothermia in a Severe Neonatal Hypoxic-Ischemic Encephalopathy Piglet Model.

Hypoxic-ischemic encephalopathy (HIE) is still a major cause of neonatal death and disability as therapeutic hypothermia (TH) alone cannot afford sufficient neuroprotection. The present study investigated whether ventilation with molecular hydrogen (2.1% H2) or graded restoration of normocapnia with CO2 for 4 h after asphyxia would augment the neuroprotective effect of TH in a subacute (48 h) HIE piglet model. Piglets were randomized to untreated naïve, control-normothermia, asphyxia-normothermia (20-min 4%O2–20%CO2 ventilation; Tcore = 38.5 °C), asphyxia-hypothermia (A-HT, Tcore = 33.5 °C, 2–36 h post-asphyxia), A-HT + H2, or A-HT + CO2 treatment groups. Asphyxia elicited severe hypoxia (pO2 = 19 ± 5 mmHg) and mixed acidosis (pH = 6.79 ± 0.10). HIE development was confirmed by altered cerebral electrical activity and neuropathology. TH was significantly neuroprotective in the caudate nucleus but demonstrated virtually no such effect in the hippocampus. The mRNA levels of apoptosis-inducing factor and caspase-3 showed a ~10-fold increase in the A-HT group compared to naïve animals in the hippocampus but not in the caudate nucleus coinciding with the region-specific neuroprotective effect of TH. H2 or CO2 did not augment TH-induced neuroprotection in any brain areas; rather, CO2 even abolished the neuroprotective effect of TH in the caudate nucleus. In conclusion, the present findings do not support the use of these medical gases to supplement TH in HIE management.

The human papillomavirus E6 protein targets apoptosis-inducing factor (AIF) for degradation

Oncoprotein E6 of high-risk human papillomavirus (HPV) plays a critical role in inducing cell immortalization and malignancy. E6 downregulates caspase-dependent pathway through the degradation of p53. However, the effect of HPV E6 on other pathways is still under investigation. In the present study, we found that HPV E6 directly binds to all three forms (precursor, mature, and apoptotic) of apoptosis-inducing factor (AIF) and co-localizes with apoptotic AIF. This binding induced MG132-sensitive reduction of AIF expression in the presence of E6 derived from HPV16 (16E6), a cancer-causing type of HPV. Conversely, E6 derived from a non-cancer-causing type of HPV, HPV6 (6E6), did not reduce the levels of AIF despite its interaction with AIF. Flow cytometric analysis revealed that 16E6, but not 6E6, suppressed apoptotic AIF-induced chromatin degradation (an indicator of caspase-independent apoptosis) and staurosporine (STS, a protein kinase inhibitor)-induced apoptosis. AIF knockdown reduced STS-induced apoptosis in both of 16E6-expressing and 6E6-expressing cells; however, the reduction in 16E6-expressing cells was lower than that in 6E6-expressing cells. These findings indicate that 16E6, but not 6E6, blocks AIF-mediated apoptosis, and that AIF may represent a novel therapeutic target for HPV-induced cervical cancer.

Mesenchymal stem cell‑derived extracellular vesicles prevent neural stem cell hypoxia injury via promoting miR‑210‑3p expression.

Neural stem cells (NSCs) have the potential to give rise to offspring cells and hypoxic injury can impair the function of NSCs. The present study investigated the effects of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) on NSC injury, as well as the underlying mechanisms. MSC-EVs were isolated and identified via morphological and particle size analysis. Cobalt chloride was used to establish a hypoxic injury model in NSCs. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was conducted to detect apoptosis. Reverse transcription-quantitative PCR was performed to detect the expression levels of miR-210-3p, and western blotting was used to detect the expression levels of apoptosis-inducing factor (AIF) and Bcl-2 19 kDa interacting protein (BNIP3). Compared with the control group, NSC apoptosis, and the expression of miR-210-3p, AIF and BNIP3 were significantly higher in the cobalt chloride-induced hypoxia group. By contrast, treatment with MSC-EVs further increased miR-210-3p expression levels, but reduced NSC apoptosis and the expression levels of AIF and BNIP3 compared with the model group (P<0.05). In addition, miR-210-3p inhibitor reduced miR-210-3p expression, but promoted hypoxia-induced apoptosis and the expression levels of AIF and BNIP3 compared with the model group (P<0.05). Collectively, the results suggested that MSC-EVs prevented NSC hypoxia injury by promoting miR-210-3p expression, which might reduce AIF and BNIP3 expression levels and NSC apoptosis.

Redox-active DJ-1 sustains brainstem cardiovascular regulation via maintenance of mitochondrial function during mevinphos intoxication

DJ-1 (also known as PARK7) is a redox-active protein that protects against oxidative stress. This study evaluated the hypothesis that DJ-1 sustains brainstem cardiovascular regulation via maintaining mitochondrial function in the rostral ventrolateral medulla (RVLM), a brainstem site known to maintain blood pressure and sympathetic vasomotor tone, during cardiovascular depression elicited by the organophosphate insecticide mevinphos. In Sprague–Dawley rats, intravenous administration of mevinphos (640 μg kg−1) resulted in progressive hypotension, accompanied by an increase (Phase I) followed by a decrease (Phase II) of an experimental index for spontaneous baroreflex-mediated sympathetic vasomotor tone, alongside elevation in mitochondrial superoxide levels in the RVLM. There was concurrent activation of DJ-1 induced by oxidative stress in the RVLM, which was causally and temporally related to translocation of DJ-1 to mitochondria, reduction in mitochondrial membrane potential, increase in cytosolic apoptosis-inducing factor level, and apoptotic cell death in this brainstem site. Loss-of-function by immunoneutralization of DJ-1 in the RVLM significantly exacerbated those biochemical and cellular events, enhanced the progressive hypotension, diminished the increased and augmented the decreased spontaneous baroreflex-mediated sympathetic vasomotor tone respectively during Phases I and II, and heightened lethality during mevinphos intoxication. We conclude that DJ-1 in the RVLM sustains brainstem cardiovascular regulation induced by mevinphos via maintaining mitochondrial function.

Plasma rich in growth factors reduces blue light-induced oxidative damage on retinal pigment epithelial cells and restores their homeostasis by modulating vascular endothelial growth factor and pigment epithelium-derived factor expression.

This study analysed the effectiveness of plasma rich in growth factors (PRGF) in reducing the oxidative stress induced by blue light exposition on retinal pigment epithelial (RPE) cells. Blood from six healthy donors was collected to obtain the PRGF. Retinal pigment epithelium (ARPE-19) cells were exposed to blue light. Then, cells were incubated with PRGF or with control for 24 and 48 hours maintaining exposure to blue light. The cytoprotective effect of PRGF on ARPE cells was evaluated by measuring the cell viability, the reactive oxygen species (ROS) production and the expression of different proteins such as heme oxygenase 1 (HO-1), catalase (CAT), superoxide dismutase (SOD-1), apoptosis-inducing factor (AIF), pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF). The cell viability increased significantly at 24 and 48 hours after PRGF treatment compared to the control group. ROS synthesis was significantly reduced in PRGF-treated cells with respect to control. Furthermore, the levels of HO-1, SOD-1 and AIF were significantly reduced after PRGF treatment at both times of treatment. However, CAT levels were only significantly reduced after PRGF treatment at 48 hours. The high expression of VEGF by RPE cells exposed to blue light was only counterbalanced in the PRGF group by increasing the expression of PEDF in comparison to the control group. The present results show that PRGF treatment reduces the cytotoxic effects induced in RPE cells exposed to an oxidative stress environment. Furthermore, PRGF treatment preserves the mitochondrial activity and cell viability of RPE cells subjected to an oxidative stress.

PARP1-DNMT1-CTCF complex and the apoptotic-induced factor mRNA expressions in workers occupationally exposed to benzene.

Exposure to benzene is a common occupational hazard as well as a hematopoietic system intoxicant, but the entire picture of its molecular pathogenesis is still hazy. Its leukemogenic effect could be attributed to DNA damage, decreased repair capacity, altered methylation patterns, and defective apoptosis. Poly ADP-ribose polymerase1, DNA methyltransferase1, and CCCTC-binding factor (PARP1-DNMT1-CTCF) complex play an essential role in methylation maintenance and DNA damage repair response. This study aimed to assess the expression of PARP1, PAR glycohydrolases (PARG), DNMT1, CTCF, and apoptosis-inducing factor (AIF) in subjects occupationally exposed to benzene. A total of 200 subjects were enrolled in this study: 100 workers occupationally exposed to benzene (painters and decorators) and 100 unexposed office workers. Occupational exposure data were obtained. The biochemical and hematological evaluations were done. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to assess mRNA expression of PARP1, PARG, DNMT1, CTCF, and AIF. Both biochemical and hematological parameters were within normal limits; workplace benzene air concentration was significantly higher in exposed workers than the levels among controls (P < 0.001). Significant decrease in mRNA levels of PARP1, DNMT1, CTCF, and AIF was noticed among the exposed group (P = 0.01, P < 0.001, P = 0.004, P < 0.001, respectively) in comparison with the control group, while PARG showed non-significant difference (P = 0.16). There was a significant negative correlation between workplace benzene air concentration and expression levels of PARP1, DNMT1, and AIF. The reduced expression of PARP1, DNMT1, CTCF, and AIF observed in exposed workers may represent one of the first benzene-induced changes that might threaten erythropoiesis.