Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.‍T260A, p.‍R422W, and p.‍R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%‍‒‍49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%‍‒‍17.9%, which was significantly higher than that (6.9%‍‒‍7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.
Humans ; Apoptosis Inducing Factor/metabolism* ; NAD/metabolism* ; Dimerization ; Apoptosis

Cardiomyocyte apoptosis leads to the functional incapacitation of myocardial plasmodium and plays an important role in the pathogenesis of heart failure transformed from compensable cardiac hypertrophy. Mitochondria are the main source of apoptosis-inducing molecule of various cells, and the role of caspartate-specific cysteinyl proteinase (caspase)-dependent mechanism has generally been accepted in the cardiomyocyte apoptosis. However, the significance of caspase-independent apoptosis-inducing factor (AIF) mechanism is not yet understood. The purpose of this study was to evaluate hypoxia-reperfusion-induced alterations of AIF mRNA and protein expressions in hypertrophic cardiomyocytes. Cardiomyocyte hypertrophy was produced by angiotensin II (0.1 mumol/L). The cells were cultured under the condition of hypoxia (95% N2 and 5% CO2; the O2 partial pressure was lower than 5 mmHg) for 8 h or 12 h (named as H8h and H12h groups, respectively), and then exposed to normal culture environment (named as H8h/R and H12h/R groups, respectively). Apoptosis was detected with Hoechst 33258 staining. The AIF mRNA and protein expressions were detected by RT-PCR and Western blot and quantified by gel scanning. The results were as follows: (1) The level of AIF mRNA expression was 0.29+/-0.08 (optical density, relative value) in the control group (hypertrophic cardiomyocytes cultured in normal environment). Compared with that in the control group, the levels of AIF mRNA expression were significantly higher in the groups of H8h and H12h (0.52+/-0.04 and 0.85+/-0.10), indicating that this effect was time-dependent. A further increase of AIF mRNA expression was observed in the groups of H8h/R (1.09+/-0.12) and H12h/R (1.41+/-0.23). (2) The level of AIF protein expression was 0.29+/-0.04 in the control group. Compared with that in the control group, the levels of AIF protein expression were significantly higher in the groups of H8h and H12h (2.07+/-0.15 and 3.12+/-0.19). The AIF protein expression was increased further in the groups of H8h/R (4.57+/-0.25) and H12h/R (5.71+/-0.27). The nuclear translocation of AIF protein was obvious only in the groups of H8h/R and H12h/R. (3) The expressions of AIF mRNA and protein were almost completely inhibited by AIF siRNA transfection. The siRNA transfection also reduced the apoptosis of hypertrophic cardiomyocytes in the groups of H8h/R and H12h/R but not in the groups of H8h and H12h. The apoptosis rate was significantly reduced by both AIF siRNA transfection and Ac-DEVD-cmk, an inhibitor of caspase-3. This reduction induced by two factors was more evident than that by one factor. (4) AIF nuclear translocation induced by hypoxia-reperfusion was not affected by inhibition of the activity of caspase-3. These data suggest that AIF plays a pivotal role in the apoptosis of hypertrophic cardiomyocytes induced by hypoxia-reperfusion.
Apoptosis ; Apoptosis Inducing Factor ; metabolism ; Cardiomegaly ; Cell Hypoxia ; Myocytes, Cardiac ; cytology ; Reperfusion Injury

OBJECTIVE: To investigate the effect of lycium barbarum polysaccharide (LBP) alone or combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the apoptosis of leukemia cell lines with MLL gene-rearrangement, and to explore the cell apoptotic pathway after the combined action. METHODS: MLL-ALL cell line KOCL44 and KOCL45 were selected as the research object, then the control and experimental groups were set up. The cell survival rate was measured by the trypan blue dye exclusion method, the cell early apoptosis and expression of death receptors on the cell surface were detected by flow cytometry with Annexin-V/PI double staining. The protein level of caspase-8, BID, caspase-3, caspase-9, BAD, BCL-2, as well as mitochondrial and cytosol Cyto-C were detected by Western blot. RESULTS: LBF combined with TRAIL inhibited the growth of KOCL44 and KOCL-45 cells and showed the synergistic effect, the results of flow cytometry with Amnexiu V/PI double staining were consistent with above-mentioned results. After treatment of KOCL44 and KOCL45 cells with LBF plus TRAIL, the significant expression of DR4 on cell surface was not found, while the expression of DR4 receptor was enhanced significantly, the pro-apoptotic proteins including caspase-8, BID, caspase-3, caspase-9 and BAD were activated significantly and BCL-2 was suppressed significantly with time-dependent manner. The expression of mitochondria cyto-C in KOCL44 and KOCL45 decreased along with prolonging of treatment time (r=-0.95, r=-0.866), while the expression of cytosol cyto-C in KOCL44 and KOCL45 increased along with prolonging of treatment time (r=0.883, r=0.903). CONCLUSION The combination of LBP and TRAIL significantly increases the apoptosis of KOCL44 and KOCL45, and the LBP and TRAIL can up-regulate the expression of TRAIL death receptor-DR5 on the cell surface, activate the pathway of caspase and mito-chrondia mitachondria, thus enhance the sensitivity of KOCL44 and KOCL45 to TRAIL induced apoptosis through both mitochondrial and apoptotic pathway.
Apoptosis ; Caspase 8 ; Drugs, Chinese Herbal ; Leukemia, Myeloid, Acute ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha

The objective of this study was to investigate the synergistic effect of soluble human recombinant tumor necrosis factor related apoptosis inducing ligand (TRAIL) protein combined with anti-vascular endothelial growth factor (anti-VEGF) antibody on inducing apoptosis of leukemia K562 cells. The inhibitory rates and apoptotic rates of K562 cells treated with TRAIL and anti-VEGF antibody alone and their combination for 48 hours were examined by CCK-8 assay and flow cytometry respectively. The results indicated that the apoptotic rates of K562 cells induced with 75, 100 and 150 ng/ml TRAIL after culture for 48 hours were (4.26±0.67)%, (8.91±0.55)% and (11.71±0.78)% respectively. The apoptotic rates of K562 cells induced with 2.5, 5 and 7.5 µg/ml anti-VEGF antibody after culture for 48 hours were (3.95±0.69)%, (7.98±0.74)% and (10.26±0.83)% respectively. The apoptotic rates of K562 cells treated with combination use of 2.5 µg/ml anti-VEGF antibody and 75 ng/ml TRAIL, 5 µg/ml anti-VEGF antibody and 100 ng/ml TRAIL, and 7.5 µg/ml anti-VEGF antibody and 150 ng/ml TRAIL for 48 hours were (22.16±0.93)%, (36.32±1.31)% and (49.19±0.71)% respectively. The combined use of above mentioned agents induced significantly higher apoptosis and cytotoxicity than that of TRAIL or anti-VEGF antibody alone (p<0.05). It is concluded that the combination use of TRAIL and anti-VEGF antibody can significantly increase the sensitivity of K562 cells to apoptosis.
Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Humans ; K562 Cells ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Vascular Endothelial Growth Factor A ; immunology

Apoptosis is a tightly regulated, cell deletion process that plays an important role in various cardiovascular diseases, such as myocardial infarction, reperfusion injury, and heart failure. Since cardiomyocyte loss is the most important determinant of patient morbidity and mortality, fully understanding the regulatory mechanisms of apoptotic signaling is crucial. In fact, the inhibition of cardiac apoptosis holds promise as an effective therapeutic strategy for cardiovascular diseases. Caspase, a critical enzyme in the induction and execution of apoptosis, has been the main potential target for achieving anti-apoptotic therapy. Studies suggest, however, that a caspase-independent pathway may also play an important role in cardiac apoptosis, although the mechanism and potential significance of caspase-independent apoptosis in the heart remain poorly understood. Herein we discuss the role of apoptosis in various cardiovascular diseases, provide an update on current knowledge about the molecular mechanisms that govern apoptosis, and discuss the clinical implications of anti-apoptotic therapies.
Apoptosis ; Apoptosis Inducing Factor ; Cardiovascular Diseases ; Cell Death ; Heart ; Heart Failure ; Humans ; Myocardial Infarction ; Myocytes, Cardiac ; Necrosis ; Reperfusion Injury

OBJECTIVETo investigate the pathway and mechanism of noise exposure induced out hair cells (OHC) apoptosis.

METHODSThe cochleae of control and noise exposure group were dissected. The activity of caspase 3, an important mediator of apoptosis, in OHC, was examined with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The apoptosis inducing factor (AIF) translocation from mitochondria in OHC were further examined by immunohistology method. The nuclei were labeled with PI and the mitochondrion was labeled with Mito-tracker. Whole mount organ of Corti was prepared. Morphological and fluorescent change was observed use confocal microscope.

RESULTSIn the normal OHC, AIF is distributed where the mitochondria were located and no activated caspase 3 was observed. After the animals exposed to broadband noise at 122 dB in 4 h/day for 2 days, both apoptosis and necrosis were appeared in OHC. AIF translocated from mitochondrion to nuclei in apoptotic and necrotic OHC following noise exposure. The noise exposure triggered activation of caspase 3 in apoptic hair cells. But no caspase 3 activation appeared in necrotic OHC.

CONCLUSIONSThese findings indicated that the caspase-dependent pathway is an important pathway in noise exposure induced apoptosis. And AIF also involves OHC death pathway following noise exposure.

Animals ; Apoptosis ; Apoptosis Inducing Factor ; metabolism ; Caspase 3 ; metabolism ; Female ; Guinea Pigs ; Hair Cells, Auditory, Outer ; metabolism ; pathology ; Male ; Noise

TNF-related apoptosis-inducing ligand (TRAIL) is a newly identified member of the tumor necrosis factor (TNF) family. TRAIL induces apoptosis by activating caspase cascades, stimulating a loss of mitochondrial membrane potential (Delta Psim) and cytochrome C release in the FADD/caspase-8 dependent pathway. However, TRAIL can also trigger transcriptional activations of the pro-oncogene of c-fos, JNK, and NF-kappaB by other signaling pathways downstream of FADD/caspase-8. MAPK/ERK activation has a dominant protecting effect over apoptotic signaling from the death receptors. The functional expression of TRAIL by leukemic cells may be involved in tumor cells evasion of immunosurveillance. Somatic mutations of TRAIL-R1 and TRAIL-R2 genes may play a role in the pathogenesis of some tumors. TRAIL can induce apoptosis on various continuous transformed cell lines and primary tumor cells, including several of hematopoietic origin, displaying minimal toxic effects on normal tissues. Because of the abilities of induction of both cytotoxic (apoptosis) and cytostatic (cell cycle perturbation) effects on the leukemic cells, TRAIL is currently considered as a potential(co) therapeutic drug against tumors.
Animals ; Apoptosis Regulatory Proteins ; Hematologic Neoplasms ; etiology ; therapy ; Humans ; Membrane Glycoproteins ; physiology ; Mutation ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; Receptors, Tumor Necrosis Factor ; genetics ; physiology ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha ; physiology

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF super family found in recent years, which widely exists in the body tissues and participates in the immune regulation, immune stability, and immune surveillance of the human body. The TRAIL receptor is expressed in the surface of a variety of cells. Recent studies show that TRAIL induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. Its anti-tumor activity and safety have been widely recognized. The development of prostate cancer is regulated by the mechanisms of cell apoptosis. TRAIL can induce the apoptosis of prostate cancer cells, and therefore has a great application value in the treatment of prostate cancer.
Antineoplastic Agents ; therapeutic use ; Apoptosis ; Apoptosis Regulatory Proteins ; Humans ; Male ; Membrane Glycoproteins ; Prostatic Neoplasms ; drug therapy ; pathology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; physiology ; therapeutic use ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha

OBJECTIVE: Patients with bipolar disorder (BD) exhibit peripheral low-grade inflammation. The aim of the current study was to investigate the involvement of hitherto unexplored components of the tumor necrosis factor (TNF) superfamily in BD. METHODS: Eighty patients with type I BD and 50 healthy controls matched for age and gender were enrolled in this study. All subjects were assessed with the Mini-Plus to evaluate psychiatric comorbidities; the Young Mania Rating Scale and the Hamilton Depression Rating Scale to evaluate manic and depressive symptoms severity, respectively. TNF superfamily molecules (TNF, TNF-related weak inducer of apoptosis [TWEAK], TNF-related apoptosis-inducing ligand [TRAIL], soluble TNF receptor type 1 [sTNFR1], and soluble TNF receptor type 2 [sTNFR2]) levels were measured by ELISA. RESULTS: Patients with BD, regardless of mood state, presented increased plasma levels of sTNFR1 and TWEAK in comparison with controls. CONCLUSION: These findings corroborate the view that TNF superfamily may play a role in BD pathophysiology.
Apoptosis ; Bipolar Disorder* ; Comorbidity ; Depression ; Enzyme-Linked Immunosorbent Assay ; Humans ; Inflammation ; Plasma* ; Receptors, Tumor Necrosis Factor ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha*

OBJECTIVETo investigate the molecular mechanism of doxorubicin enhancement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) inducing apoptotic effect on multiple myeloma cell line KM3.

METHODSApoptosis was studied independently through flow cytometry analysis and TUNEL staining. The expression of death receptor 5 (DR5) and nuclear factor P65 in nuclear was examined by Western blot.

RESULTSThe apoptosis ratio of KM3 cells was 20.88%, 40.03%, 57.87%, 60.82% respectively when treated with different concentration of TRAIL (10, 20, 50, 100 ng/ml) combining with doxorubicin. It is markedly higher than the group treated with TRAIL or doxorubicin alone. DR5 expression increased while P65 decreased as the doses of doxorubicin increased when KM3 cells treated with doxorubicin (0.5, 1.0, 2.0 and 4.0 microg/ml) plus 20 ng/ml TRAIL.

CONCLUSIONIncreasing the expression of DR5 and nuclear transferring of P65 are the important molecular mechanism by which doxorubicin enhances TRAIL-inducing apoptosis of KM3 cells.

Apoptosis ; drug effects ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Interactions ; Humans ; Multiple Myeloma ; metabolism ; pathology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Transcription Factor RelA ; genetics ; metabolism