ABSTRACT
SUMMARY: Cadmium is a highly toxic metal and affects the respiratory mucosa. The aim of the study is to show the inflammation and degenerative effect of cadmium on the olfactory mucosa. In this study, eight-week-old Wistar rats with an average weight of 170-190 g were divided into two groups (control and experiment) with 20 animals in each group and used in the experiments. The rats in the experimental group were given 2 mg/kg/day powdered cadmium chloride dissolved in water intraperitoneally every day for two weeks. At the end of the experiment, the nasal cavity was completely removed with anesthesia. Concha nasalis superior was separated, fixed with zinc-Formalin solution and decalcified with 5 % EDTA (Ethylene-diaminetetraacetic acid). After routine histopathological procedure, APAF-1 antibody was used for expression of Hematoxylin-Eosin (HE) and immunohistochemistry. Histopathological examination revealed interruptions in the basement membrane structure due to cadmium and degenerative changes in stem cells, degeneration in sensory cells and pycnosis in nuclei, dilatation in blood vessels and increased inflammation in connective tissue. APAF-1 expression was found to increase in epithelial cells and olfactory glands (Bowman gland) cells. It has been thought that cadmium toxicity increases cell degeneration and inflammation in the olfactory mucosa and may significantly affect cell death and olfactory metabolism by inducing the pro-apoptotic process.
El cadmio es un metal altamente tóxico que afecta la mucosa respiratoria. El objetivo fue mostrar el efecto inflamatorio y degenerativo del cadmio sobre la mucosa olfativa. En este estudio, ratas Wistar de ocho semanas de edad con un peso promedio de 170-190 g se dividieron en dos grupos (control y experimental) con 20 animales en cada grupo. Las ratas del grupo experimental recibieron 2 mg/kg/día de cloruro de cadmio en polvo disuelto en agua por vía intraperitoneal todos los días durante dos semanas. En los animales se exirpó la cavidad nasal bajo anestesia. Se separó la concha nasal superior, se fijó con solución de zinc-Formalina y se descalcificó con EDTA (ácido etilendiaminotetraacético) al 5 %. Después del procedimiento histopatológico de rutina, Hematoxilina- Eosina (HE) e inmunohistoquímica, se utilizó el anticuerpo APAF-1. El examen histopatológico reveló interrupciones en la estructura de la membrana basal debido al cadmio y cambios degenerativos en las células madre, degeneración en las células sensoriales y picnosis en los núcleos, dilatación de los vasos sanguíneos y aumento de la inflamación en el tejido conjuntivo. Se encontró que la expresión de APAF-1 aumenta en las células epiteliales y en las células de las glándulas olfatorias (glándulas de Bowman). Se ha pensado que la toxicidad del cadmio aumenta la degeneración celular y la inflamación en la mucosa olfativa y puede afectar significativamente la muerte celular y el metabolismo olfativo al inducir el proceso proapoptótico.
Subject(s)
Animals , Rats , Olfactory Mucosa/drug effects , Olfactory Mucosa/pathology , Cadmium Chloride/toxicity , Administration, Intranasal , Immunohistochemistry , Rats, Wistar , Apoptotic Protease-Activating Factor 1ABSTRACT
@#[Abstract] Objective:ToinvestigatetheroleofmiR-125a-5pininducingthegefitinib(Gef)-resistance of non-small cell lung carcinoma (NSCLC) cells and its possible mechanism. Methods: Human NSCLC drug-resistant cell line A549/GR and NSCLC cell line A549 were chosen for this study. miR-125a-5p mimic, miR-125a-5p inhibitor, pcDNA3.1-APAF1 and empty vector pcDNA3.1 were transfected into A549/GR cells. The expression level of miR-125a-5p in cell lines was detected by qPCR. MTT, Transwell and Flow cytometry were used to detect the effects of Gef on proliferation, migration and apoptosis of cell lines, respectively. The targeting relationship between miR-125a-5p and APAF1 (apoptotic peptidase activating factor 1) was verified by Dual-luciferase reporter gene system. In addition, the expression of APAF1 protein in A549/GR cells was detected by Western blotting. The expression levels of caspase-3 and caspase-9 were assessed by colorimetry. Results: Expression level of miR-125a-5p was upregulated significantly in Gefresistant A549/GR cells (P<0.01). AndtheinfluencesofGefonA549/GRcellswereenhancedby knockdown of miR-125a-5p, including inhibiting cell proliferation and migration (all P<0.05) and inducing apoptosis (P<0.01). Dual luciferase reporter gene assay confirmed that miR-125a-5p targeted APAF1 and negatively regulated its expression. Furthermore, by targetedly downregulating APAF1, miR-125a-5p alleviated the inhibition of proliferation and migration (all P<0.05) and promotion of apoptosis (P<0.05) of A549/GR cells caused by Gef, and attenuated Gef-induced upregulation of apoptosis-related proteins caspase-3 and caspase-9 (all P<0.05). Conclusion: miR-125a-5p promotes Gef-resistance of A549/GR cells, and the underlying mechanisms are promotion of proliferation, migration and inhibition of apoptosis of non-small cell lung cancer cells by targetingAPAF1.
ABSTRACT
OBJECTIVE@#To investigate apoptotic effects of berberine, a significant alkaloids component existing in Rhizoma coptidis, and its possible acting mechanism in insulinoma cells.@*METHODS@#Different concentrations of berberine were used to treat mouse insulinoma (MIN6) cells for various period of time. The viability and apoptosis of the cells were analyzed using methylthiazolyldiphenvl-tetrazolium bromide assay, flow cytometry and enzyme-linked immuno sorbent assay. Changes in the relating pro- and anti-apoptosis proteins were detected by western-blotting.@*RESULTS@#The half-maximal inhibitory concentration (IC) of berberine was 5.7 μmol/L on MIN6 cells viability for 16 h. Berberine caused a 20% reduction (P<0.05) in cell number after only 4-h incubation; which reached 50% after 24 h (P<0.01). Berberine treatment for 16 h significantly increased the level of DNA fragmentation. The flow cytometry showed the apoptotic rate increased 2.9- and 4.6-fold after treating with berberine (5 μmol/L) for 8 and 16 h, while 3- and 8.7-fold after 10 μmol/L treatment for 8 and 16 h (P<0.01). Berberine treatment dramatically elevated the expression ratio of Bax to Bcl-2. Meanwhile, berberine notably increased the apoptosis-inducing factors and cytochrome C transforming from the mitochondria to the cytoplasm. Apoptotic protease-activating factor 1 (Apaf-1) was subsequently activated after cytochrome C release. Furthermore, caspase-3 and poly adenosine diphosphate-ribose polymerase were also activated to trigger apoptosis cascade.@*CONCLUSION@#High concentration (5 and 10 μmol/L) of berberine could induce the apoptosis of MIN6 cells through cytochrome C/Apaf-1/caspase-3 and apoptosis inducing factor (AIF) pathway.
ABSTRACT
Objective To study the rule of Apaf-1 in wnt/β-catenin signaling pathway and its regulatory expression mechanism in hepatocellular carcinoma cells.Methods The regulatory mechanism of Apaf-1 gene in Wnt/β-catenin signaling pathway was verified by the TOPflash experiment;real-time PCR was used to detect the expression amounts of Apaf-1 in various hepatoma cell lines (HepG2,H HCC,HB611) and normal liver cell line(LO2);the RNAi interference plasmid of Apaf-1 was constructed and transfected into the HepG2 cell line.Then the transfection efficiency and expression of related genes and proteins were detected.Results The TOPFlash experiment found that Apaf-1 gene could inhibit the wnt/β-catenin signal pathway in a dose dependent manner;the Apaf-1 expression level in hepatoma cells was decreased compared with the normal liver cells,moreover its expression level was lowest in HepG2 cell line;RNA interfering Apaf-1 gene in HepG2 cell line,the expression level of Apaf-1 gene was significantly decreased,and the expression levels of downstream genes and protein(β-catenin,Cyclin A,CDK2,wnt5a,STAT3,EGFR,APC) in wnt signal pathway were significantly increased or decreased,the difference was statistically significant(P＜0.05).Conclusion Apaf-1 gene plays an important role in the formation process of hepatocarcinoma cells by wnt/β-catenin signaling pathway.
ABSTRACT
Objective To explore the relationship between apoptosis promoting effector molecules TFAR19,Apaf-1 and lubar intervertebral disc protrusion.Methods 99 of operation patients with lubar intervertebral disc protrusion in the First Affiliated Hospital of Guangxi Medical University Department of Spine and Osteoputhy were recruited.Among them,single segment of lubar intervertebral disc protrusion were 70 (Group A),more than one segments of lubar intervertebral disc protrusion were 29 (Group B).In addition,40 unrelated healthy people from physical examination center were enrolled as controls (Group C).Enzyme-linked immunosorbent assay (ELISA) was used to examine serum TFAR19,Apaf-1 levels in lubar intervertebral disc protrusion patients.Results The level of TFAR19 in Group A,Group B and Group C respectively were 1.85±0.14,2.33±0.25 and 1.30±0.09 ng/ml.The TFAR19 activity in each group was statistically significant difference (F=7.979,P＜0.01).Compared with Group C,the TFAR19 activity in Group B (q=5.59,P＜0.01),Group A (q=3.60,P=0.012) was statistically significant difference.The TFAR19 activity in lubar intervertebral disc protrusion patients subgroups (Group A,Group B) was statistically significant difference (q =2.93,P =0.012).The level of Apaf-1 in Group A,Group B and Group C respectively were 159.22±11.87,203.20±20.21 and 107.52±11.58 pg/ml.The Apaf-1 activity in each group was statistically significant difference (F=8.828,P＜0.01).Compared with Group C,the Apaf-1 activity in Group B (q=5.86,P＜0.01),Group A (q=3.89,P=0.007) was statistically significant difference.The Apaf-1 activity in lubar intervertebral disc protrusion patients subgroups (Group A,Group B) was statistically significant difference (q =2.97,P=0.037).Male/female ratio between each groups was not statistically significant difference (x2=0.229,P =0.892).Age between each groups was not statistically significant difference (F=0.091,P =0.91).Conclusion The augment of TFAR19 and Apaf-1 promotes apoptosis of lubar intervertebral disc protrusion.It is connected with quantity of protrusive segments.The more segments of protrusion,the higher TFAR19 and Apaf-1 level of examination will be.
ABSTRACT
AIM: To investigate the synergistic effect of resveratrol and 5-fluorouracil on osteosarcoma CD133+ cell subset.METHODS: Human osteosarcoma cell line MG-63 CD133+ cell subset and the corresponding CD133-cell subset were treated with resveratrol and 5-fluorouracil.After treatment, the viability of MG-63 cells was measured by MTT assay.The apoptosis of MG-63 cells was analyzed by flow cytometry.Activation of caspase-9 and caspase-3, the expression of Apaf-1, and the release of cytochrome C were evaluated by Western blot.The interaction between Apaf-1 and pro-caspase-9 was detected by co-immunoprecipitation.RESULTS: The cell death and apoptosis of MG-63 CD133+ cell subset induced by 5-fluorouracil were significantly weaker than those in the corresponding MG-63 CD133-cell subset.However, co-treatment with resveratrol significantly enhanced the effect of 5-fluorouracil on inhibiting the viability of MG-63 CD133+ cell subset.Mechanically, treatment with resveratrol upregulated the expression of Apaf-1.Transfection with Apaf-1 siRNA abolished the synergistic effect of resveratrol and 5-fluorouracil in MG-63 CD133+ cell subset.In addition, the results of co-immunoprecipitation indicated that the combination of resveratrol and 5-fluorouracil significantly induced the formation of Apaf-1/pro-caspase-9 complex, leading to the activation of caspase-9 in MG-63 CD133+ cell subset.CONCLUSION: Resveratrol enhances 5-fluorouracil-induced apoptosis of osteosarcoma CD133+ cell subset by promoting the formation of Apaf-1/caspase-9 complex.
ABSTRACT
Objective To investigate the expressions of Apaf-1 and Caspase-9 protein in ovarian serous carcinoma(OSC) and their clinicopathological significance.Methods The expressions of Apaf-1 and Caspase-9 protein in 45 cases of OSC,60 cases of ovarian serous cystadenomas and 32 cases of ovarian borderline serous cystadenomas were detected by immunohistochemical SP method.The relationship between the expressions of these two proteins and the clinicopathological features of OSC and the correlation between Apaf-1 and Caspase-9 expressions in OSC were analyzed.Results The positive rates of Apaf-1 in OSC,ovarian serous cystadenoma and ovarian borderline serous cystadenoma were 24.4%(11/45),75.0%(45/60) and 46.9%(15/32),the difference was statistically significant (χ2=26.734,P<0.01).Apaf-1 expression was correlated with pathological grade,clinical stage and lymph nodes metastasis (χ2=6.318,7.565,5.554,P<0.05).The expression rates of Caspase-9 in OSC,ovarian serous cystadenoma and ovarian borderline serous cystadenoma were 28.9%(13/45),83.3%(50/60) and 56.3%(18/32),the difference was statistically significant (χ2=31.682,P<0.01).The expression of Caspase-9 was correlated with pathological grade,clinical stage and lymph nodes metastasis (χ2=5.750,4.391,5.466,P<0.05).In OSC,the expressions of Apaf-1 and Capase-9 were positively correlated (k=0.433,P=0.003).Conclusion The low expressions of Apaf-1 and Caspase-9 in OSC may be related to the occurrence and development of OSC.
ABSTRACT
Purpose To investigate the expression of Apaf-1 and Caspase-9 in prostate cancer ( PCa) and benign prostatic hyperplasia ( BPH) , and to analyze their correlation with clinicopathological parameters of PCa. Methods Immunohistochemistry was used to de-tect Apaf-1 and Caspase-9 protein in 45 cases of PCa and 60 cases of BPH. Results The positive rate of Apaf-1 and Caspase-9 in PCa tissues was significantly lower than that in BPH (P0. 05), but it was correlated with the pathological grade and clinical stage of PCa (P<0. 05). Conclusion Apaf-1 and Caspase-9 are lowly expressed in PCa. There is positive correlation between the expression of Apaf-1 and Caspase-9 (rs =0. 645, P<0. 01). Apaf-1 and Caspase-9 might be correlated with the carcinogenesis and development of PCa.
ABSTRACT
The protein apoptotic protease activating factor 1 (Apaf1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. We have developed a vital method that allows fluorescence-activated cell sorting of cells at different stages of the apoptotic pathway and demonstrated that upon pharmacological inhibition of Apaf1, cells recover from doxorubicin- or hypoxia-induced early apoptosis to normal healthy cell. Inhibiting Apaf1 not only prevents procaspase-9 activation but delays massive mitochondrial damage allowing cell recovery.
Subject(s)
Humans , Adenosine Triphosphate , Metabolism , Apoptosis , Apoptotic Protease-Activating Factor 1 , Genetics , Metabolism , Cell Hypoxia , Cell Line, Tumor , Doxorubicin , Pharmacology , HeLa Cells , Microscopy, Electron, TransmissionABSTRACT
Objective To investigate the effects of astragalus injection on the morphology and expression of Apaf-1 in hippocampal neurons after cerebral ischemia reperfusion in rats. Methods The male SD rats were randomly divided into 3 groups, sham-operated group, cerebral ischemia-reperfusion group (reperfusion group) and astragalus injection interven-tion group (experiment group). The global cerebral ischemia-reperfusion rat model was established by Pulsinelli four-vessel occlusion method. The astragalus injection group was intraperitoneally injected with astragalus 6 mL/kg, 30 mins before sur-gery and repeated every 24 h. Rat brains were removed 24 h after reperfusion in each group. HE staining was used to observe the pathological changes of the hippocampal neurons under the light microscope. The ultrastructural changes of hippocam-pal neurons were observed by transmission electron microscopy. Immunohistochemistry and Western blot methods were used to measure the expression of apoptotic protease activating factor-1(Apaf-1) protein. Results Compared with sham-operat-ed group, nuclear and mitochondrial damage was found in reperfusion group, and the expression of Apaf-1 protein increased obviously in hippocampus(Immunohistochemistry result:0.024 ± 0.001 vs 0.109 ± 0.011;Western blot result:0.270 ± 0.018 vs 0.894±0.072, P<0.01). Compared with reperfusion group, the damage in nuclear and mitochondria was relieved obviously in experiment group, and the expression of Apaf-1 protein in hippocampus was significantly decreased (Immunohistochemistry result:0.048±0.005;Western blot result:0.392±0.046, P<0.01). Conclusion Astragalus injection can reduce pathological damage of hippocampal neurons after cerebral ischemia and reperfusion in rats, and the mechanism is related with inhibiting of Apaf-1 protein.
ABSTRACT
BACKGROUND: Apoptosis protease activating factor-1 (Apaf-1), caspase-9, Bcl-2, p53, and survivin are important factors in the pathway of apoptosis, but their clinicopathologic significance remains unclear in human cutaneous melanoma. We investigated the expression of these proteins and their clinical value in human cutaneous melanocytic lesions. METHODS: We performed an immunohistochemical analysis to examine the expression and distribution of Apaf-1, caspase-9, Bcl-2, p53, and survivin in 36 cases of malignant melanoma (22 cases of primary melanoma and 14 cases of metastatic melanoma) and 41 cases of melanocytic nevus. RESULTS: The expression of p53 was significantly higher in malignant melanoma than in melanocytic nevus (p<0.01), however the expressions of Apaf-1 and caspase-9 were significantly lower in malignant melanoma compared with melanocytic nevus (p<0.01 and p=0.027, respectively). Also, there was a significant difference for Bcl-2 staining between primary melanomas and metastatic lesions (p=0.004). Nuclear staining for survivin were absent in nevus, but were positive in 14 of 36 melanomas (p<0.01). CONCLUSIONS: The altered expression of Apaf-1, caspase-9, p53, and survivin are considered to be related to malignant progression in human cutaneous melanocytic lesions. Loss of Bcl-2 can be considered as a prognostic marker of malignant melanomas.
Subject(s)
Humans , Apoptosis , Caspase 9 , Melanoma , Nevus , Nevus, Pigmented , ProteinsABSTRACT
methyltransferase 3b and re-activating the Apaf-1 gene expression.
ABSTRACT
Apoptotic protease activating factor 1 (APAF-1) has a critical role in the regulation of apoptosis. In the present study, the mRNA expression analysis of different APAF-1 transcripts (APAF-1S, APAF-1LC, APAF-1LN, and APAF-1XL) was analyzed in bone marrow samples from 37 patients with acute myeloid leukemia (newly diagnosed, with no previous treatment). APAF-1XL and APAF-1LN transcripts (with and without an extra WD-40 repeat region, respectively) were detected in all samples, although the major form expressed was APAF-1XL in 65 percent of the samples (group 1), while 35 percent of the samples expressed primarily APAF-1LN (group 2). Only 46 percent of the patients presented complete remission in response to remission induction therapy (represented by less than 5 percent marrow blasts and hematological recovery), all but 2 cases being from group 1, 21.6 percent did not attain complete remission (only 1 case from group 1), and 32.4 percent of the patients died early. Lower expression of APAF-1XL (APAF-1XL/APAF-1LN ratio <1.2) was associated with a poor response to therapy (P = 0.0005, Fisher exact test). Both groups showed similar characteristics regarding white blood cell counts, cytogenetic data or presence of gene rearrangements associated with good prognosis as AML1-ETO, CBFB-MYH11 and PML/RARA. Since it has been shown that only the isoforms with the extra WD-40 repeat region activate procaspase-9, we suggest that low procaspase-9 activation may also be involved in the deregulation of apoptosis and chemotherapy resistance in acute myeloid leukemia.