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

The purpose of this study was to investigate the effect of inhibition of calpain on retinal ganglion cell-5 (RGC-5) necroptosis following oxygen glucose deprivation (OGD). RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8-h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. The calpain expression was detected by Western blotting and immunofluorescence staining. The calpain activity was tested by activity detection kit. Flow cytometry was used to detect the effect of calpain on RGC-5 necroptosis following OGD with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN) pre-treatment. Western blot was used to detect the protein level of truncated apoptosis inducing factor (tAIF) in RGC-5 cells following OGD. The results showed that there was an up-regulation of the calpain expression and activity following OGD. Upon adding ALLN, the calpain activity was inhibited and tAIF was reduced following OGD along with the decreased number of RGC-5 necroptosis. In conclusion, calpain was involved in OGD-induced RGC-5 necroptosis with the increased expression of its downstream molecule tAIF.
Animals ; Apoptosis Inducing Factor ; biosynthesis ; genetics ; Calpain ; biosynthesis ; genetics ; Gene Expression Regulation ; drug effects ; Glucose ; metabolism ; Humans ; Leupeptins ; administration & dosage ; Mice ; Oxygen ; metabolism ; Retinal Ganglion Cells ; metabolism ; pathology ; Retinal Necrosis Syndrome, Acute ; genetics ; pathology

OBJECTIVETo explore the enhanced sensitivity of leukemia cell line KG-1a to activated immune cell-mediated cytolysis after treated with resveratrol.

METHODSThe value of 50% inhibition concentration (IC₅₀) for KG-1a by resveratrol was analyzed using trypan blue staining. Peripheral blood mononuclear cells were separated, and then activated by interleukin (IL)-2 and IL-15. The sensitivity of KG-1a treated with and without resveratrol to activated immune cell-mediated cytolysis was assayed by lactate dehydrogenase (LDH) -releasing assay. The expression of tumor necrosis factor related apoptosis inducing ligand (TRAIL) on the surface of activated immune cells and its receptors (DR4/5 and DcR1/2) on the surface of KG-1a were detected by flow cytometry.

RESULTSResveratrol could inhibit the proliferation of KG-1a and IC50 at 24 h was 25 mmol/L. At a ratio of 10:1 or 20:1 between effect and target, the cytolytic rates of treated KG-1a by activated immune cells were (55.80 ± 10.88)% and (72.31 ± 13.06)%, significantly higher than (24.96 ± 9.25)% and (37.93 ± 5.21)% of untreated KG-1a (P<0.05). The expression of DR5 on the surface of KG-1a treated with resveratrol was (9.05 ± 3.57)%, significantly higher than (3.11 ± 0.54)% of untreated KG-1a (P<0.05). Conversely, the expression of DcR1 on the surface of treated KG-1a was (13.23 ± 3.56)%, lower than (53.75 ± 10.51)% of KG-1a (P<0.05). When TRAIL pathway on the surface of activated immune cells was blocked, the cytolytic rates of treated KG-1a were (35.97 ± 6.36)% and (49.80 ± 10.68)%, significantly lower than (52.92 ± 6.98)% and (70.73 ± 9.79)% of untreated KG-1a (P<0.05) at the same ratio of effector and target.

CONCLUSIONResveratrol could enhance cytolytic sensitivity of KG-1a by activated immune cells through TRAIL pathway.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia ; metabolism ; pathology ; Leukocytes, Mononuclear ; drug effects ; immunology ; metabolism ; Male ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Receptors, Tumor Necrosis Factor, Member 10c ; metabolism ; Stilbenes ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; metabolism

OBJECTIVETo investigate the effect of doxorubicin on TRAIL resistance and TRAIL receptor expression in lymphoma cell line SNK-6 cells.

METHODSSNK-6 cells treated with doxorubicin at different concentrations alone or in combination with tumor necrosis factor related apoptosis inducing ligand (TRAIL). Cell proliferation was evaluated by MTT assay. Apoptosis and the expression of TRAIL receptors were determined by flow cytometry.

RESULTSMTT assay showed that treatment with 100 and 1000 ng/ml doxorubicin for 24 h, the survival rates of SNK-6 cells were (80.9 ± 7.2)% and (53.7 ± 2.8)%, significantly higher than that by treatment combined with 500 ng/ml TRAIL (64.9 ± 1.1)% and (34.0 ± 3.9)%, respectively (P < 0.05). Flow cytometry showed that after treatment with 100 and 1000 ng/ml doxorubicin for 48 h, the survival rates of SNK-6 cells were (69.9 ± 6.1)% and (31.1 ± 1.9)%, while treated in combination with 500 ng/ml TRAIL, the cell survival rates were (37.5 ± 6.4)% and (15.0 ± 1.8)%, respectively. The early apoptosis rate was (14.8 ± 0.6)% and (30.8 ± 1.5)%, significantly lower than that [(28.7 ± 0.6)% and (46.6 ± 2.8)%] after treatment in combination with TRAIL (P < 0.05). The expressions of TRAIL receptors and decoy receptors were increased when SNK-6 cells were treated with 100 ng/ml doxorubicin for 24 hours.

CONCLUSIONSDoxorubicin can overcome to a certain extent the TRAIL resistance of SNK-6 cells and induce upregulation of TRAIL death receptors and decoy receptors on the surface of SNK-6 cells. However, a higher dose is needed.

Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Doxorubicin ; administration & dosage ; pharmacology ; Drug Resistance, Neoplasm ; Drug Synergism ; Humans ; Lymphoma, Extranodal NK-T-Cell ; metabolism ; pathology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Tumor Necrosis Factor Decoy Receptors ; metabolism

OBJECTIVETo observe the expression of the C-terminal truncated human apoptosis-inducing factor (AIF) and its biological effect on MCF-7 cells.

METHODSPcDNA3.0-FDT-AIFδ1-480 was transfected into human breast carcinoma MCF-7 cells with lipofectamine. The expression of the truncated AIF gene was detected by Western blotting, and its effects on the biological behaviors of MCF-7 cells and on the expression of cytochrome c (cytC) were evaluated using flow cytometry, MTT assay, colony-forming assay, and mitochondrial membrane potential measurement.

RESULTSPcDNA3.0-FDT-AIFδ1-480 enhanced AIF expression in MCF-7 cells, obviously inhibited the cell proliferation, and significantly reduced the mitochondrial membrane potentials (P<0.05). Transfection of the cells with PcDNA3.0-FDT-AIFδ1-480 promoted the expression of cytC and resulted in significantly increased apoptosis of MCF-7 cells (P<0.05).

CONCLUSIONThe expression of C-terminal truncated human AIF gene can induce apoptosis of human MCF-7 cells by promoting cytC release from mitochondria.

Apoptosis ; Apoptosis Inducing Factor ; genetics ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; Cell Proliferation ; Cytochromes c ; genetics ; metabolism ; Female ; Humans ; MCF-7 Cells ; Membrane Potential, Mitochondrial ; Mitochondria ; metabolism

OBJECTIVETo in vitro compare the induction of extracts of Stellera chamaejasme ESC, ESC-1 and ESC-2 on NCI-H157 cell apoptotic.

METHODThe apoptosis rate was inspected by flow cytometry; caspase-3, 8, 9 activities was measured by spectrophotometry. Fas, Fas-L, TNF-alpha, Trail-R, Cyto-C, Smac/diablo protein expressions of apoptosis pathway was observed by Elisa method.

RESULTCompared with the control group, ESC, ESC-1, ESC-2 can significantly improve the apoptosis rate of NCI-H157 cell. ESC significantly improved cells caspase-3, 8 activity, ESC-2 can significantly improve the activity of caspase-3, 8, 9. ESC, ESC-1, ESC-2 significantly increased Fas expression and ESC significantly increased Fas/Fas-L ratio. ESC, ESC-1, ESC-2 significantly increased TNF-alpha protein expression. ESC-1 significantly lowered TRAIL-R expression. ESC, ESC-1, ESC-2 had no significant effect on Cyto-C. ESC-1, ESC-2 significantly reduced Smac protein expression.

CONCLUSIONThe apoptotic effect induced by ESCs may be related to the regulation of death receptor pathway proteins. Induction mechanisms of ESCs were so complicated that it may have a two-way regulatory effect. Its induction in apoptosis is a result from comprehensive regulation and control.

Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Line, Tumor ; Humans ; Neoplasms ; chemistry ; drug therapy ; pathology ; Plant Extracts ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; analysis ; Thymelaeaceae ; chemistry ; Tumor Necrosis Factor-alpha ; analysis ; fas Receptor ; analysis

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. Here, we report that paxilline, an indole alkaloid from Penicillium paxilli, can sensitize various glioma cells to TRAIL-mediated apoptosis. While treatment with TRAIL alone caused partial processing of caspase-3 to its p20 intermediate in TRAIL-resistant glioma cell lines, co-treatment with TRAIL and subtoxic doses of paxilline caused complete processing of caspase-3 into its active subunits. Paxilline treatment markedly upregulated DR5, a receptor of TRAIL, through a CHOP/GADD153-mediated process. In addition, paxilline treatment markedly downregulated the protein levels of the short form of the cellular FLICE-inhibitory protein (c-FLIPS) and the caspase inhibitor, survivin, through proteasome-mediated degradation. Taken together, these results show that paxilline effectively sensitizes glioma cells to TRAIL-mediated apoptosis by modulating multiple components of the death receptor-mediated apoptotic pathway. Interestingly, paxilline/TRAIL co-treatment did not induce apoptosis in normal astrocytes, nor did it affect the protein levels of CHOP, DR5 or survivin in these cells. Thus, combined treatment regimens involving paxilline and TRAIL may offer an attractive strategy for safely treating resistant gliomas.
Antineoplastic Agents/*pharmacology ; Apoptosis/*drug effects ; Astrocytes/metabolism ; CASP8 and FADD-Like Apoptosis Regulating Protein/genetics/*metabolism ; Caspase 3/metabolism ; Cell Line, Tumor ; Drug Discovery ; Flow Cytometry ; Glioma/*metabolism/pathology ; Humans ; Indoles/*pharmacology ; Inhibitor of Apoptosis Proteins/metabolism ; RNA, Small Interfering ; Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; TNF-Related Apoptosis-Inducing Ligand/metabolism/*pharmacology ; Transcription Factor CHOP/analysis

Apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be regulated by the epidermal growth factor (EGF) signaling pathway. In this study, recombinant adenoviral vectors that encode TRAIL gene from the hTERT/RGD promoter (AdTRAIL) was combined with drugs including gefitinib, elotinib, and cetuximab that inhibit EGFR and the EGF signaling pathway in non-small cell lung cancer (NSCLC) cell lines to investigate their antitumor activity. In vitro, compared to single reagent, AdTRAIL combined with EGFR inhibitors reduced proliferation and enhanced apoptosis in H460, A549, and SW1573 cell lines. Western blot results suggested that these effects were relative to up-regulation of pro-apoptosis protein BAX and down-regulation of p-AKT. In vivo, AdTRAIL combined with cetuximab resulted in a significant growth reduction in H460 xenografts without damage to the main organs of nude mice. Histological examination and TUNEL analyses of xenografts showed that cetuximab enhanced cell apoptosis induced by AdTRAIL. These results indicate that EGFR inhibitors enhanced AdTRAIL anti-tumor activity in NSCLC cell lines and that inhibiting the AKT pathway played an important role in this enhancement.
Adenoviridae ; genetics ; Animals ; Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cetuximab ; Drug Synergism ; Erlotinib Hydrochloride ; Female ; Genetic Therapy ; Genetic Vectors ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Mice ; Mice, Nude ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; physiology ; Transfection ; Tumor Burden ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the involvement of apoptosis inducing factor (AIF) in caspase-independent pathway mediating apoptosis of cultured renal tubular epithelial cells induced by cisplatin (CP).

METHODSWestern Blot analysis and real-time PCR were performed to detect cytosol AIF (cAIF), nuclear AIF (nAIF) and AIF mRNA expression in cultured renal epithelial cells (HK-2) treated with cisplatin (CP) at various concentrations (0 - 200 micromol/L) and time courses (0 - 12 h). Immunofluorescence analysis was used to detect the AIF protein distribution in HK-2 cells. Pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA treatment, TUNEL and flow cytometer were used to measure the suppression of apoptosis induced by CP in HK-2 cells.

RESULTSThe expressions of cAIF, nAIF protein and AIF mRNA were all increased to some extent in HK-2 cells treated with CP at various concentrations and time points. cAIF expression was 2.3-fold (P < 0.05) increased after 25 micromol/L CP treatment for 12 h and 1.7-fold (P < 0.01) increased after 50 micromol/L CP treatment for 3 h, compared with that of control groups, and showed a concentration- and time-dependent increment. The nAIF expression reached a peak (4.3-fold increase) (P < 0.005) after 150 micromol/L CP treatment for 12 h and 3.7-fold incease (P < 0.05) after 50 micromol/L CP treatment for 9 h, compared with that of the 25 micromol/L group and 3 h group, respectively. The expression of nAIF was approximately consistent with cleaved-PARP expressive pattern. Real-time PCR showed that AIF mRNA increased gradually with prolonged treatment with 50 micromol/L CP and reached a peak at 9 h. Immunofluorescence assay showed AIF translocation from cytosol to nuclei in some cultured HK-2 cells treated with CP. Applying pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA to CP-treated HK-2 cells, the apoptotic rates were decreased by 60.1% and 39.2%, respectively. The inhibitory effect on HK-2 cell apoptosis was even more significant with combination of both Z-VAD-FMK and AIF-siRNA.

CONCLUSIONThe AIF activation and translocation to nuclei with the increment of its mRNA expression mediates CP-induced apoptosis of renal tubular epithelial cells in vitro. It may provide a new therapeutic target for protecting from nephrotoxciity of cisplatin.

Amino Acid Chloromethyl Ketones ; pharmacology ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Inducing Factor ; genetics ; metabolism ; Caspase Inhibitors ; Cell Nucleus ; metabolism ; Cells, Cultured ; Cisplatin ; administration & dosage ; pharmacology ; Cytosol ; metabolism ; Dose-Response Relationship, Drug ; Drug Synergism ; Epithelial Cells ; cytology ; metabolism ; Humans ; Kidney Tubules ; cytology ; Protein Transport ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics

OBJECTIVETo investigate the mechanism of norcantharidin (NCTD)-induced SMMC-7721 hepatoma cell apoptosis.

METHODSSMMC-7721 cell growth inhibition was measured by the MTT method. Apoptosis was detected by Annexin V/propidium iodide staining. The mitochondrial membrane potential was measured by flow cytometry. Western blot analysis was used to evaluate the level of cytochrome c, caspase-3, AIF, Bcl-2 and Bax expression.

RESULTSNCTD inhibited SMMC-7721 cell growth in a time- and dose-dependent manner. The cells treated with NCTD showed the loss of mitochondrial membrane potential. The activities of caspase-3, cytochrome c, AIF, and Bax were up-regulated after NCTD treatment at different doses. The expression of Bcl-2 was decreased after treatment with NCTD.

CONCLUSIONSNCTD could induce SMMC-7721 cell apoptosis. The activation of the mitochondrial pathway was involved in the process of NCTD-induced SMMC-7721 cell apoptosis.

Apoptosis ; drug effects ; Apoptosis Inducing Factor ; metabolism ; Blotting, Western ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Flow Cytometry ; Humans ; Liver Neoplasms ; enzymology ; metabolism ; pathology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; metabolism ; bcl-2-Associated X Protein ; metabolism