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Sample records for benzopyrene-diol-epoxide bpde-induced bax

  1. Inhibition of benzopyrene-diol-epoxide (BPDE)-induced bax and caspase-9 by cadmium: Role of mitogen activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Jagat J.; Gupta, Suresh K. [State University of New York College at Buffalo, Environ. Toxicol. and Chem., Great Lakes Center, 1300 Elmwood Avenue, Buffalo, NY 14222 (United States); Kumar, Subodh [State University of New York College at Buffalo, Environ. Toxicol. and Chem., Great Lakes Center, 1300 Elmwood Avenue, Buffalo, NY 14222 (United States)], E-mail: kumars@buffalostate.edu

    2009-02-10

    Cadmium, a major metal constituent of tobacco smoke, elicits synergistic enhancement of cell transformation when combined with benzo[a]pyrene (BP) or other polynuclear aromatic hydrocarbons (PAHs). The mechanism underlying this synergism is not clearly understood. Present study demonstrates that (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogen of BP, induces apoptosis in human leukemic HL-60 cells and others, and cadmium at non-cytotoxic concentration inhibits BPDE-induced apoptosis. We observed that BPDE treatment also activates all three MAP kinases e.g. ERK1/2, p38 and JNK in HL-60 cells, and inhibition of BPDE-induced apoptosis by cadmium is associated with down-regulation of pro-apoptotic bax induction/caspase-9 activation and up-regulation of ERK phosphorylation, whereas p38 MAP kinase and c-Jun phosphorylation (indicative of JNK activation) remain unaffected. Inhibition of ERKs by prior treatment of cells with 10 {mu}M U0126 relieves cadmium-mediated inhibition of apoptosis/bax induction/caspase-9 activation. Our results suggest that cadmium inhibits BPDE-induced apoptosis by modulating apoptotic signaling through up-regulation of ERK, which is known to promote cell survival.

  2. Bax

    National Research Council Canada - National Science Library

    Cui, Qinghua; Valentin, Mayda; Janumyan, Yelena; Yang, Elizabeth

    2009-01-01

    .... We also discovered that the cell cycle function of Bcl-2 and Bcl-xL was dependent on Bax and Bak, and in bax -/- bak -/- double knockout cells, features of G0 quiesecence were already present and p27...

  3. Further characterization of benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects.

    Science.gov (United States)

    Bausinger, Julia; Schütz, Petra; Piberger, Ann Liza; Speit, Günter

    2016-03-01

    The present study aims to further characterize benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects. Therefore, we measured DNA effects by the comet assay and adduct levels by high-performance liquid chromatography (HPLC) in human lymphocytes and A549 cells exposed to (±)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(±)-anti-BPDE] or (+)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(+)-anti-BPDE]. Both, the racemic form and (+)-anti-BPDE, which is the most relevant metabolite with regard to mutagenicity and carcinogenicity, induced DNA migration in cultured lymphocytes in the same range of concentrations to a similar extent in the alkaline comet assay after exposure for 2h. Nevertheless, (+)-anti-BPDE induced significantly enhanced DNA migration after 16 and 18h post-cultivation which was not seen in response to (±)-anti-BPDE. Combination of the comet assay with the Fpg (formamidopyrimidine-DNA glycosylase) protein did not enhance BPDE-induced effects and thus indicated the absence of Fpg-sensitive sites (oxidized purines, N7-guanine adducts, AP-sites). The aphidicolin (APC)-modified comet assay suggested significant excision repair activity of cultured lymphocytes during the first 18h of culture after a 2 h-exposure to BPDE. In contrast to these repair-related effects measured by the comet assay, HPLC analysis of stable adducts did not reveal any significant removal of (+)-anti-BPDE-induced adducts from lymphocytes during the first 22h of culture. On the other hand, HPLC measurements indicated that A549 cells repaired about 70% of (+)-anti-BPDE-induced DNA-adducts within 22h of release. However, various experiments with the APC-modified comet assay did not indicate significant repair activity during this period in A549 cells. The conflicting results obtained with the comet assay and the HPLC-based adduct analysis question the real cause for BPDE-induced DNA migration in the comet assay and the reliability of the APC-modified comet assay for the

  4. MUC1 contributes to BPDE-induced human bronchial epithelial cell transformation through facilitating EGFR activation.

    Directory of Open Access Journals (Sweden)

    Xiuling Xu

    Full Text Available Although it is well known that epidermal growth factor receptor (EGFR is involved in lung cancer progression, whether EGFR contributes to lung epithelial cell transformation is less clear. Mucin 1 (MUC1 in human and Muc1 in animals, a glycoprotein component of airway mucus, is overexpressed in lung tumors; however, its role and underlying mechanisms in early stage lung carcinogenesis is still elusive. This study provides strong evidence demonstrating that EGFR and MUC1 are involved in bronchial epithelial cell transformation. Knockdown of MUC1 expression significantly reduced transformation of immortalized human bronchial epithelial cells induced by benzo[a]pyrene diol epoxide (BPDE, the active form of the cigarette smoke (CS carcinogen benzo(apyrene (BaPs. BPDE exposure robustly activated a pathway consisting of EGFR, Akt and ERK, and blocking this pathway significantly increased BPDE-induced cell death and inhibited cell transformation. Suppression of MUC1 expression resulted in EGFR destabilization and inhibition of the BPDE-induced activation of Akt and ERK and increase of cytotoxicity. These results strongly suggest an important role for EGFR in BPDE-induced transformation, and substantiate that MUC1 is involved in lung cancer development, at least partly through mediating carcinogen-induced activation of the EGFR-mediated cell survival pathway that facilitates cell transformation.

  5. Benzo(a)pyrene-7,8-diol-9,10-epoxide induced p53-independent necrosis via the mitochondria-associated pathway involving Bax and Bak activation.

    Science.gov (United States)

    Zhang, W; Liu, N; Wang, X; Jin, X; Du, H; Peng, G; Xue, J

    2015-02-01

    Benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) is a highly reactive DNA damage agent and can induce cell death through both p53-independent and -dependent pathways. However, little is known about the molecular mechanisms of p53-independent pathways in BPDE-induced cell death. To understand the p53-independent mechanisms, we have now examined BPDE-induced cytotoxicity in p53-deficient baby mouse kidney (BMK) cells. The results showed that BPDE could induce Bax and Bak activation, cytochrome c release, caspases activation, and necrotic cell death in the BMK cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore, were interdependently activated by BPDE, with Bax and Bak translocation to and Bax/Bak homo-oligomerization in mitochondria, release of cytochrome c was induced. Importantly, cytochrome c release and necrotic cell death were diminished in BMK cells (Bax(-/-)), BMK cells (Bak(-/-)), and BMK cells (Bax(-/-)/Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate BPDE-induced cytochrome c release and necrosis. Together the findings suggested that BPDE-induced necrosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases. © The Author(s) 2015.

  6. Attenuation of BPDE-induced p53 accumulation by TPA is associated with a decrease in stability and phosphorylation of p53 and down-regulation of NF-κB activation: Role of p38 MAP kinase

    Science.gov (United States)

    Mukherjee, Jagat J.; Sikka, Harish C.

    2005-01-01

    DNA damage caused by benzo[a]pyrene (BP) or other PAHs induce p53 protein as a protective measure to eliminate the possibility of mutagenic fixation of the DNA damage. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits p53 response induced by BP and other DNA-damaging agents and may cause tumor promotion. The molecular mechanism of attenuation of BP-induced p53 response by TPA is not known. We investigated the effect of TPA on p53 response in BPDE-treated mouse epidermal JB6(P+) Cl 41 cells. BPDE treatment induced p53 accumulation which was attenuated significantly by TPA. Cells treated with BPDE and TPA showed increased ratio of Mdm2 to p53 proteins in p53 immunoprecipitate and decreased p53 life span compared to BPDE-treated cells indicating p53 destabilization by TPA. TPA also inhibited BPDE-induced p53 phosphorylation at serine15. Activation of both ERKs and p38 MAPK by BPDE and attenuation of BPDE-induced p53 accumulation by U0126 or SB202190, specific inhibitor of MEK1/2 or p38 MAPK, indicate the role of ERKs and p38 MAPK in p53 accumulation. Interestingly, TPA potentiated BPDE-induced activation of ERKs whereas p38 MAPK activation was significantly inhibited by TPA, suggesting that inhibition of p38 MAPK is involved in p53 attenuation by TPA. Furthermore SB202190 treatment caused decreased p53 stability and inhibition of phosphorylation of p53 at serine 15 in BPDE-treated cells. We also observed that TPA or SB202190 attenuated BPDE-induced NF-κB activation in JB6 (Cl 41) cells harboring NF-κB reporter plasmid. To our knowledge this is the first report that TPA inhibits chemical carcinogen-induced NF-κB activation. Interference of TPA with BPDE-induced NF-κB activation implicates abrogation of p53 function which has been discussed. Overall our data suggest that abrogation of BPDE-induced p53 response and of NF-κB activation by TPA is mediated by impairment of signaling pathway involving p38 MAPK. PMID:16244358

  7. Cytosolic Bax

    Science.gov (United States)

    Vogel, Sandra; Raulf, Nina; Bregenhorn, Stephanie; Biniossek, Martin L.; Maurer, Ulrich; Czabotar, Peter; Borner, Christoph

    2012-01-01

    Bax is kept inactive in the cytosol by refolding its C-terminal transmembrane domain into the hydrophobic binding pocket. Although energetic calculations predicted this conformation to be stable, numerous Bax binding proteins were reported and suggested to further stabilize inactive Bax. Unfortunately, most of them have not been validated in a physiological context on the endogenous level. Here we use gel filtration analysis of the cytosol of primary and established cells to show that endogenous, inactive Bax runs 20–30 kDa higher than recombinant Bax, suggesting Bax dimerization or the binding of a small protein. Dimerization was excluded by a lack of interaction of differentially tagged Bax proteins and by comparing the sizes of dimerized recombinant Bax with cytosolic Bax on blue native gels. Surprisingly, when analyzing cytosolic Bax complexes by high sensitivity mass spectrometry after anti-Bax immunoprecipitation or consecutive purification by gel filtration and blue native gel electrophoresis, we detected only one protein, called p23 hsp90 co-chaperone, which consistently and specifically co-purified with Bax. However, this protein could not be validated as a crucial inhibitory Bax binding partner as its over- or underexpression did not show any apoptosis defects. By contrast, cytosolic Bax exhibits a slight molecular mass shift on SDS-PAGE as compared with recombinant Bax, which suggests a posttranslational modification and/or a structural difference between the two proteins. We propose that in most healthy cells, cytosolic endogenous Bax is a monomeric protein that does not necessarily need a binding partner to keep its pro-apoptotic activity in check. PMID:22277657

  8. Bax activation by Bim?

    National Research Council Canada - National Science Library

    Czabotar, P E; Colman, P M; Huang, D C S

    2009-01-01

    .... Although some data support a role for certain BH3-only proteins, such as Bim or tBid, to directly activate Bax, others have led to the conclusion that BH3-only proteins act indirectly by antagonizing...

  9. Allostery in BAX protein activation.

    Science.gov (United States)

    Jiang, Zhenyan; Zhang, Hansi; Böckmann, Rainer A

    2016-11-01

    BAX is a member of the proapoptotic BCL-2 family of proteins, which is involved in the regulation of the intrinsic pathway of apoptosis. In the process of apoptosis, BH3-only molecules activate cytosolic BAX. Activated BAX molecules insert into the mitochondrial outer membrane with their [Formula: see text]-helix and form oligomers that lead to membrane poration, resulting in the release of apoptogenic factors including cytochrome c. Recently, a novel interaction site for the binding of the BIM SAHB ligand to BAX was reported. BIM SAHB binding was shown to invoke the exposure of the 6A7 epitope (amino acids 13-19) and of the BH3 domain of BAX, followed by mobilization of the BAX [Formula: see text]-helix. However, the intramolecular pathway for signal transmission in BAX, from BIM SAHB binding to mobilization of the [Formula: see text]-helix largely remained elusive. For a molecular understanding of the activation of BAX, and thus the first steps in apoptosis, we performed microsecond atomistic molecular dynamics simulations both of the BAX protein and of the BAX:BIM SAHB complex in aqueous solution. In agreement with experiment, the 6A7 and BH3 domains adopt a more solvent-exposed conformation within the BAX:BIM SAHB complex. BIM SAHB binding was found to stabilize the secondary structure of the [Formula: see text]9-helix. A force distribution analysis revealed a force network of residue-residue interactions responsible for signal transmission from the BIM SAHB binding site predominantly via the [Formula: see text]4- and [Formula: see text]6-helices to the [Formula: see text]9-helix on the opposite site of the protein.

  10. Allosteric sensitization of proapoptotic BAX.

    Science.gov (United States)

    Pritz, Jonathan R; Wachter, Franziska; Lee, Susan; Luccarelli, James; Wales, Thomas E; Cohen, Daniel T; Coote, Paul; Heffron, Gregory J; Engen, John R; Massefski, Walter; Walensky, Loren D

    2017-09-01

    BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.

  11. Parkin promotes proteasomal degradation of misregulated BAX.

    Science.gov (United States)

    Cakir, Zeynep; Funk, Kathrin; Lauterwasser, Joachim; Todt, Franziska; Zerbes, Ralf M; Oelgeklaus, Aline; Tanaka, Atsushi; van der Laan, Martin; Edlich, Frank

    2017-09-01

    The pro-apoptotic BCL-2 protein BAX commits human cells to apoptosis by permeabilizing the outer mitochondrial membrane. BAX activation has been suggested to require the separation of helix α5 from α6 - the 'latch' from the 'core' domain - among other conformational changes. Here, we show that conformational changes in this region impair BAX translocation to the mitochondria and retrotranslocation back into the cytosol, and therefore BAX inhibition, but not activation. Redirecting misregulated BAX to the mitochondria revealed an alternative mechanism of BAX inhibition. The E3 ligase parkin, which is known to trigger mitochondria-specific autophagy, ubiquitylates BAX K128 and targets the pro-apoptotic BCL-2 protein for proteasomal degradation. Retrotranslocation-deficient BAX is completely degraded in a parkin-dependent manner. Although only a minor pool of endogenous BAX escapes retrotranslocation into the cytosol, parkin-dependent targeting of misregulated BAX on the mitochondria provides substantial protection against BAX apoptotic activity. © 2017. Published by The Company of Biologists Ltd.

  12. Differential retrotranslocation of mitochondrial Bax and Bak

    Science.gov (United States)

    Todt, Franziska; Cakir, Zeynep; Reichenbach, Frank; Emschermann, Frederic; Lauterwasser, Joachim; Kaiser, Andrea; Ichim, Gabriel; Tait, Stephen WG; Frank, Stephan; Langer, Harald F; Edlich, Frank

    2015-01-01

    The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death. PMID:25378477

  13. Apopotic gene Bax expression in carotid plaque

    Institute of Scientific and Technical Information of China (English)

    Bao-Zhong MEN; Ding-Biao ZHOU; Huai-Yin SHI; Xiao-Ming ZHANG

    2006-01-01

    The expression of BAX in carotid atherosclerosis and its regulation is far from defined. Objectives To investigate BAX expression in stable/fibrous and instable/vulnerable carotid plaque and its clinical significance. Methods 25 cases of carotid plaque specimens obtained from endarterectomy were divided into two groups, stable/fibrous 14 cases, vulnerable/instable 11 cases; aortic artery and its branches from hepatic transplantation donors 6 case as control. The expression of proapoptotic BAX was detected by immunohistochemistry(IHC), in situ hybridization(ISH) and in situ TdT dUTP nick end labeling (TUNEL). Results 5 cases of BAX ( + ) were detected by ICH and ISH, 4 case of TUNEL ( + ) were detected by TUNEL in stable/fibrous carotid plaque , while 10 cases were BAX ( + )by IHC(P < 0.05) , 11case by ISH and 9 case by TUNEL were detected in instable/vulnerable carotid plaque ( P < 0.01 ), respectively. The intensity of BAX ( + ) cells by IHC and ISH was 8.63 ± 2.62 and 10.32 ± 3.12 in fibrous plaques, whereas 122 ± 21.64and 152 ± 23.35 in vulnerable plaques, respectively. No expression of BAX was found in controlled group. Conclusion The higher expression of Bax in vulnerable carotid plaque may be one mechanisms in molecular pathogenesis of carotid atherosclerosis which affect plaque stability and be the cause of higher incidence of stroke than fibrous carotid plaques, the regulation of BAX expression in different stage of atherosclerosis may provide targets in gene therapy for carotid atherosclerosis.

  14. An Autoinhibited Dimeric Form of BAX Regulates the BAX Activation Pathway.

    Science.gov (United States)

    Garner, Thomas P; Reyna, Denis E; Priyadarshi, Amit; Chen, Hui-Chen; Li, Sheng; Wu, Yang; Ganesan, Yogesh Tengarai; Malashkevich, Vladimir N; Almo, Steve S; Cheng, Emily H; Gavathiotis, Evripidis

    2016-08-04

    Pro-apoptotic BAX is a cell fate regulator playing an important role in cellular homeostasis and pathological cell death. BAX is predominantly localized in the cytosol, where it has a quiescent monomer conformation. Following a pro-apoptotic trigger, cytosolic BAX is activated and translocates to the mitochondria to initiate mitochondrial dysfunction and apoptosis. Here, cellular, biochemical, and structural data unexpectedly demonstrate that cytosolic BAX also has an inactive dimer conformation that regulates its activation. The full-length crystal structure of the inactive BAX dimer revealed an asymmetric interaction consistent with inhibition of the N-terminal conformational change of one protomer and the displacement of the C-terminal helix α9 of the second protomer. This autoinhibited BAX dimer dissociates to BAX monomers before BAX can be activated. Our data support a model whereby the degree of apoptosis induction is regulated by the conformation of cytosolic BAX and identify an unprecedented mechanism of cytosolic BAX inhibition. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells

    Science.gov (United States)

    Boohaker, Rebecca J.; Zhang, Ge; Carlson, Adina Loosley; Nemec, Kathleen N.

    2011-01-01

    The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration. PMID:21289292

  16. Traveling Bax and Forth from Mitochondria to Control Apoptosis

    Science.gov (United States)

    Soriano, Maria Eugenia; Scorrano, Luca

    2011-01-01

    Antiapoptotic Bcl-2 proteins on mitochondria inhibit prodeath proteins, such as Bax, which are found primarily in the cytosol. In this issue, Edlich et al., (2011) show that Bax and Bcl-xL interact on the mitochondrial surface and then retrotranslocate to the cytosol, effectively preventing Bax-induced permeabilization of mitochondria. PMID:21458662

  17. Small molecules reveal an alternative mechanism of Bax activation.

    Science.gov (United States)

    Brahmbhatt, Hetal; Uehling, David; Al-Awar, Rima; Leber, Brian; Andrews, David

    2016-04-15

    The pro-apoptotic protein Bax commits a cell to death by permeabilizing the mitochondrial outer membrane (MOM). To obtain small-molecule probes for elucidating the molecular mechanism(s) of Bax activation, we screened for compounds that induced Bax-mediated liposome permeabilization. We identified five structurally different small molecules that promoted both Bax targeting to and oligomerization at membranes. All five compounds initiated Bax oligomerization in the absence of membranes by a mechanism unlike Bax activation by Bcl-2 homology 3 domain (BH3) proteins. Some of the compounds induced Bax/Bak-dependent apoptosis in cells. Activation of Bax by the most active compound was poorly inhibited by the anti-apoptotic protein Bcl-XL and requires a cysteine residue at position 126 of Bax that is not required for activation by BH3 proteins. Our results reveal a novel pathway for Bax activation independent of pro-apoptotic BH3 proteins that may have important implications for the regulation of Bax activity in cells. © 2016 The Author(s).

  18. Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

    Science.gov (United States)

    Kushnareva, Yulia; Andreyev, Alexander Y.; Kuwana, Tomomi; Newmeyer, Donald D.

    2012-01-01

    Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event. PMID:23049480

  19. Conformational Heterogeneity in the Activation Mechanism of Bax.

    Science.gov (United States)

    Barnes, C Ashley; Mishra, Pushpa; Baber, James L; Strub, Marie-Paule; Tjandra, Nico

    2017-08-01

    Bax is known for its pro-apoptotic role within the mitochondrial pathway of apoptosis. However, the mechanism for transitioning Bax from cytosolic to membrane-bound oligomer remains elusive. Previous nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) studies defined monomeric Bax as conformationally homogeneous. Yet it has recently been proposed that monomeric Bax exists in equilibrium with a minor state that is distinctly different from its NMR structure. Here, we revisited the structural analysis of Bax using methods uniquely suited for unveiling "invisible" states of proteins, namely, NMR paramagnetic relaxation enhancements and EPR double electron-electron resonance (DEER). Additionally we examined the effect of glycerol, the co-solvent of choice in DEER studies, on the structure of Bax using NMR chemical-shift perturbations and residual dipolar couplings. Based on our combined NMR and EPR results, Bax is a conformationally homogeneous protein prior to its activation. Published by Elsevier Ltd.

  20. Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation.

    Science.gov (United States)

    Düssmann, H; Rehm, M; Concannon, C G; Anguissola, S; Würstle, M; Kacmar, S; Völler, P; Huber, H J; Prehn, J H M

    2010-02-01

    Mitochondrial outer membrane permeabilisation (MOMP) during apoptosis is triggered by the activation and oligomerisation of Bax and Bak, but a quantification of these processes in individual cells has not yet been performed. Single-cell imaging of Bax translocation and oligomerisation in Bax-deficient DU-145 cells expressing CFP-Bax and YFP-Bax revealed that both processes started only minutes before or concomitantly with MOMP, with the majority of Bax translocation and oligomerisation occurring downstream of MOMP. Quantification of YFP-Bax concentrations at mitochondria revealed an increase of only 1.8 + or - 1.5% at MOMP onset. This was increased to 11.2 + or - 3.6% in bak-silenced cells. These data suggested that Bax activation exceeded by far the quantities required for MOMP induction, and that minimal Bax or Bak activation may be sufficient to trigger rapid pore formation. In a cellular automaton modelling approach that incorporated the quantities and movement probabilities of Bax and its inhibitors, activators and enablers in the mitochondrial membrane, we could re-model rapid pore formation kinetics at submaximal Bax activation.

  1. Hyperactivity and depression-like traits in Bax KO mice.

    Science.gov (United States)

    Krahe, Thomas E; Medina, Alexandre E; Lantz, Crystal L; Filgueiras, Cláudio C

    2015-11-02

    The Bax gene is a member of the Bcl-2 gene family and its pro-apoptotic Bcl-associated X (Bax) protein is believed to be crucial in regulating apoptosis during neuronal development as well as following injury. With the advent of mouse genomics, mice lacking the pro-apoptotic Bax gene (Bax KO) have been extensively used to study how cell death helps to determine synaptic circuitry formation during neurodevelopment and disease. Surprisingly, in spite of its wide use and the association of programmed neuronal death with motor dysfunctions and depression, the effects of Bax deletion on mice spontaneous locomotor activity and depression-like traits are unknown. Here we examine the behavioral characteristics of Bax KO male mice using classical paradigms to evaluate spontaneous locomotor activity and depressive-like responses. In the open field, Bax KO animals exhibited greater locomotor activity than their control littermates. In the forced swimming test, Bax KO mice displayed greater immobility times, a behavior despair state, when compared to controls. Collectively, our findings corroborate the notion that a fine balance between cell survival and death early during development is critical for normal brain function later in life. Furthermore, it points out the importance of considering depressive-like and hyperactivity behavioral phenotypes when conducting neurodevelopmental and other studies using the Bax KO strain. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia.

    Science.gov (United States)

    Vucicevic, Ksenija; Jakovljevic, Vladimir; Colovic, Natasa; Tosic, Natasa; Kostic, Tatjana; Glumac, Irena; Pavlovic, Sonja; Karan-Djurasevic, Teodora; Colovic, Milica

    2016-04-01

    In chronic lymphocytic leukemia (CLL), in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role. The aim of this study was to investigate the association of pro-apoptotic Bax gene expression and Bcl2/Bax ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (LPL) gene expression. We analyzed the expression of Bax mRNA and Bcl2/Bax mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction. We detected significant Bax gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated Bcl2/Bax ratio (p=Bax ratio showed a negative correlation to lymphocyte doubling time (r=-0.307; p=0.0451), while high-level Bax expression was associated with LPL-positive status (p=0.035). Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance. Our results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL.

  3. Bcl-xL retrotranslocates Bax from the mitochondria into the cytosol

    Science.gov (United States)

    Edlich, Frank; Banerjee, Soojay; Suzuki, Motoshi; Cleland, Megan M.; Arnoult, Damien; Wang, Chunxin; Neutzner, Albert; Tjandra, Nico; Youle, Richard J.

    2011-01-01

    Summary The Bcl-2 family member Bax translocates from the cytosol to mitochondria where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by pro-survival Bcl-2 proteins, but how they inhibit Bax remains controversial, because they neither co-localize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-xL in detergents and cell free MOMP activity, but unexpectedly induce Bax accumulation on mitochondria. Fluorescence Loss in Photobleaching (FLIP) reveals constant retrotranslocation of wt Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on pro-survival Bcl-2 family proteins and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-xL inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax. PMID:21458670

  4. Cytosolic Ku70 regulates Bax-mediated cell death.

    Science.gov (United States)

    Hada, Manila; Subramanian, Chitra; Andrews, Phillip C; Kwok, Roland P S

    2016-10-01

    The first known function of Ku70 is as a DNA repair factor in the nucleus. Using neuronal neuroblastoma cells as a model, we have established that cytosolic Ku70 binds to the pro-apoptotic protein Bax in the cytosol and blocks Bax's cell death activity. Ku70-Bax binding is regulated by Ku70 acetylation in that when Ku70 is acetylated Bax dissociates from Ku70, triggering cell death. We propose that Ku70 may act as a survival factor in these cells such that Ku70 depletion triggers Bax-dependent cell death. Here, we addressed two fundamental questions about this model: (1) Does all Bax, which is a cytosolic protein, bind to all cytosolic Ku70? and (2) Is Ku70 a survival factor in cells types other than neuronal neuroblastoma cells? We show here that, in neuronal neuroblastoma cells, only a small fraction of Ku70 binds to a small fraction of Bax; most Bax is monomeric. Interestingly, there is no free or monomeric Ku70 in the cytosol; most cytosolic Ku70 is in complex with other factors forming several high molecular weight complexes. A fraction of cytosolic Ku70 also binds to cytosolic Ku80, Ku70's binding partner in the nucleus. Ku70 may not be a survival factor in some cell types (Ku70-depletion less sensitive) because Ku70 depletion does not affect survival of these cells. These results indicate that, in addition to Ku70 acetylation, other factors may be involved in regulating Ku70-Bax binding in the Ku70-depletion less sensitive cells because Ku70 acetylation in these cells is not sufficient to dissociate Bax from Ku70 or to activate Bax.

  5. Direct Activation of Bax Protein for Cancer Therapy

    Science.gov (United States)

    Liu, Zhiqing; Ding, Ye; Ye, Na; Wild, Christopher; Chen, Haiying; Zhou, Jia

    2015-01-01

    Bax, a central cell death regulator, is an indispensable gateway to mitochondrial dysfunction and a major pro-apoptotic member of the Bcl-2 family proteins that control apoptosis in normal and cancer cells. Dysfunction of apoptosis renders the cancer cell resistant to treatment as well as promotes tumorigenesis. Bax activation induces mitochondrial membrane permeabilization, thereby leading to the release of apoptotic factor cytochrome c and consequently cancer cell death. A number of drugs in clinical use are known to indirectly activate Bax. Intriguingly, recent efforts demonstrate that Bax can serve as a promising direct target for small-molecule drug discovery. Several direct Bax activators have been identified to hold promise for cancer therapy with the advantages of specificity and the potential of overcoming chemo- and radioresistance. Further investigation of this new class of drug candidates will be needed to advance them into the clinic as a novel means to treat cancer. PMID:26395559

  6. Bax, Bak and beyond - mitochondrial performance in apoptosis.

    Science.gov (United States)

    Peña-Blanco, Aida; García-Sáez, Ana J

    2017-07-29

    Bax and Bak are members of the Bcl-2 family and core regulators of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, they are activated and oligomerize at the mitochondrial outer membrane (MOM) to mediate its permeabilization, which is considered a key step in apoptosis. However, the molecular mechanism underlying Bax and Bak function has remained a key question in the field. Here, we review recent structural and biophysical evidence that has changed our understanding of how Bax and Bak promote MOM permeabilization. We also discuss how the spatial regulation of Bcl-2 family preference for binding partners contributes to regulate Bax and Bak activation. Finally, we consider the contribution of mitochondrial composition, dynamics and interaction with other organelles to apoptosis commitment. A new perspective is emerging, in which the control of apoptosis by Bax and Bak goes beyond them and is highly influenced by additional mitochondrial components. © 2017 Federation of European Biochemical Societies.

  7. Bax and Bak Pores: Are We Closing the Circle?

    Science.gov (United States)

    Cosentino, Katia; García-Sáez, Ana J

    2017-04-01

    Bax and its homolog Bak are key regulators of the mitochondrial pathway of apoptosis. On cell stress Bax and Bak accumulate at distinct foci on the mitochondrial surface where they undergo a conformational change, oligomerize, and mediate cytochrome c release, leading to cell death. The molecular mechanisms of Bax and Bak assembly and mitochondrial permeabilization have remained a longstanding question in the field. Recent structural and biophysical studies at several length scales have shed light on key aspects of Bax and Bak function that have shifted how we think this process occurs. These discoveries reveal an unexpected molecular mechanism in which Bax (and likely Bak) dimers assemble into oligomers with an even number of molecules that fully or partially delineate pores of different sizes to permeabilize the mitochondrial outer membrane (MOM) during apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. CONSTRUCTION OF RECOMBINANT PLASMID PIRES-BAX-HGF%pIRES-Bax-HGF重组质粒的构建

    Institute of Scientific and Technical Information of China (English)

    黄涛; 常青; 徐平

    2011-01-01

    Objective To construct the plasmid vector of pIRES-Bax-HGF. Methods The Bax gene sequence was cloned from ovarian cancer samples, and HGF gene sequence from pBluescript Ⅱ SK+HGF plasmid derived from ATCC. Bax and HGF coding sequence were inserted into pIRES plasmid by step double enzyme digestion. Results Enzyme digestion and sequencing indicated that the construction of recombinant pIRES-Bax-HGF plasmid was successful. Conclusion Obtaining the coding genes of both Bax and HGF, and successfully creating the plasmid vector of plRES-Bax-HGF establish a foundation for further study of the effects of HGF and Bax on vascular endothelial cells, smooth muscle cells, and fibrocytes, which steps out an important step to treat post-CABG restenosis at gene level.%目的 构建pIRES-Bax-HGF质粒载体.方法 采用RT-PCR方法从卵巢癌标本中克隆Bax的基因序列,从ATCC来源的pBlueseriptⅡ SK+HGF质粒中克隆HGF的基因序列.分步双酶切插入到plRES载体质粒中.结果 酶切鉴定及测序表明,重组pIRES-Bax-HGF质粒构建成功.结论 获取HGF及Bax编码基因并成功构建重组pIRES-Bax-HGF质粒载体,为进一步研究HGF及Bax对血管内皮细胞、平滑肌细胞、纤维细胞等的影响奠定了基础,也向基因水平干预冠状动脉旁路移植术术后再狭窄的形成迈出了重要一步.

  9. The dynamics of Bax channel formation: influence of ionic strength.

    Science.gov (United States)

    Ganesan, Vidyaramanan; Walsh, Timothy; Chang, Kai-Ti; Colombini, Marco

    2012-08-08

    Mitochondrial outer membrane permeabilization (MOMP) is a complex multistep process. Studies of MOMP in vivo are limited by the stochastic variability of MOMP between cells and rapid completion of IMS protein release within single cells. In vitro models have provided useful insights into MOMP. We have investigated the dynamics of Bax-mediated MOMP in isolated mitochondria using ionic strength as a tool to control the rate of MOMP. We find that Bax can induce both transient permeabilization, detected by protein release, and more substantial long-lasting permeabilization, measured by the rate of oxidation of added cytochrome c. We found that higher ionic strength causes Bax to form small channels quickly but the expansion of these early channels is impeded. This inhibitory effect of ionic strength is independent of tBid. Channels formed under low ionic strength are not destabilized by raising the ionic strength. Increase in ionic strength also increases the ability of Bcl-xL to inhibit Bax-mediated MOMP. Ionic strength does not affect Bax insertion into mitochondria. Thus, ionic strength influences the assembly of Bax molecules already in membrane into channels. Ionic strength can be used as an effective biophysical tool to study Bax-mediated channel formation.

  10. Modulation of Bax and mTOR for Cancer Therapeutics.

    Science.gov (United States)

    Li, Rui; Ding, Chunyong; Zhang, Jun; Xie, Maohua; Park, Dongkyoo; Ding, Ye; Chen, Guo; Zhang, Guojing; Gilbert-Ross, Melissa; Zhou, Wei; Marcus, Adam I; Sun, Shi-Yong; Chen, Zhuo G; Sica, Gabriel L; Ramalingam, Suresh S; Magis, Andrew T; Fu, Haian; Khuri, Fadlo R; Curran, Walter J; Owonikoko, Taofeek K; Shin, Dong M; Zhou, Jia; Deng, Xingming

    2017-06-01

    A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non-small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment. Cancer Res; 77(11); 3001-12. ©2017 AACR. ©2017 American Association for Cancer Research.

  11. Direct and selective small-molecule activation of proapoptotic BAX

    Science.gov (United States)

    Gavathiotis, Evripidis; Reyna, Denis E; Bellairs, Joseph A; Leshchiner, Elizaveta S; Walensky, Loren D

    2013-01-01

    BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX’s ‘on switch’. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis. PMID:22634637

  12. Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

    Directory of Open Access Journals (Sweden)

    Hyunmi Lee

    Full Text Available Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP, the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM, which we visualize and isolate, into which Bax integrates.MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

  13. Mitochondrial BAX Determines the Predisposition to Apoptosis in Human AML.

    Science.gov (United States)

    Reichenbach, Frank; Wiedenmann, Cornelius; Schalk, Enrico; Becker, Diana; Funk, Kathrin; Scholz-Kreisel, Peter; Todt, Franziska; Wolleschak, Denise; Döhner, Konstanze; Marquardt, Jens U; Heidel, Florian; Edlich, Frank

    2017-08-15

    Purpose: Cell-to-cell variability in apoptosis signaling contributes to heterogenic responses to cytotoxic stress in clinically heterogeneous neoplasia, such as acute myeloid leukemia (AML). The BCL-2 proteins BAX and BAK can commit mammalian cells to apoptosis and are inhibited by retrotranslocation from the mitochondria into the cytosol. The subcellular localization of BAX and BAK could determine the cellular predisposition to apoptotic death.Experimental Design: The relative localization of BAX and BAK was determined by fractionation of AML cell lines and patient samples of a test cohort and a validation cohort.Results: This study shows that relative BAX localization determines the predisposition of different AML cell lines to apoptosis. Human AML displays a surprising variety of relative BAX localizations. In a test cohort of 48 patients with AML, mitochondria-shifted BAX correlated with improved patient survival, FLT3-ITD status, and leukocytosis. Analysis of a validation cohort of 80 elderly patients treated with myelosuppressive chemotherapy confirmed that relative BAX localization correlates with probability of disease progression, FLT3-ITD status, and leukocytosis. Relative BAX localization could therefore be helpful to identify elderly or frail patients who may benefit from cytotoxic therapy.Conclusions: In this retrospective analysis of two independent AML cohorts, our data suggest that Bax localization may predict prognosis of patients with AML and cellular predisposition to apoptosis, combining the actual contribution of known and unknown factors to a final "common path." Clin Cancer Res; 23(16); 4805-16. ©2017 AACR. ©2017 American Association for Cancer Research.

  14. A nonapoptotic role for BAX and BAK in eicosanoid metabolism.

    Science.gov (United States)

    Zhang, Tejia; Walensky, Loren D; Saghatelian, Alan

    2015-06-19

    BCL-2 proteins are key regulators of programmed cell death. The interplay between pro and antiapoptotic BCL-2 members has important roles in many cancers. In addition to their apoptotic function, recent evidence supports key nonapoptotic roles for several BCL-2 proteins. We used an unbiased lipidomics strategy to reveal that the proapoptotic proteins BAX, and to a lesser extent BAK, regulate the cellular inflammatory response by mediating COX-2 expression and prostaglandin biosynthesis. COX-2 upregulation in response to the bacterial endotoxin lipopolysaccharide is blunted in the absence of BAX, and Bax(-/-) mouse embryonic fibroblasts display altered kinetics of NFκB and MAPK signaling following endotoxin treatment. Our approach uncovers a novel, nonapoptotic function for BAX in regulation of the cellular inflammatory response and suggests that inflammation and apoptosis are more tightly connected than previously anticipated.

  15. The Dynamics of Bax Channel Formation: Influence of Ionic Strength

    OpenAIRE

    Ganesan, Vidyaramanan; Walsh, Timothy; Chang, Kai-Ti; Colombini, Marco

    2012-01-01

    Mitochondrial outer membrane permeabilization (MOMP) is a complex multistep process. Studies of MOMP in vivo are limited by the stochastic variability of MOMP between cells and rapid completion of IMS protein release within single cells. In vitro models have provided useful insights into MOMP. We have investigated the dynamics of Bax-mediated MOMP in isolated mitochondria using ionic strength as a tool to control the rate of MOMP. We find that Bax can induce both transient permeabilization, d...

  16. Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release

    National Research Council Canada - National Science Library

    Mikhailov, Valery; Mikhailova, Margarita; Degenhardt, Kurt; Venkatachalam, Manjeri A; White, Eileen; Saikumar, Pothana

    2003-01-01

    ATP depletion induced by hypoxia or mitochondrial inhibitors results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured rat proximal tubule cells...

  17. BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

    Science.gov (United States)

    Bové, Jordi; Martínez-Vicente, Marta; Dehay, Benjamin; Perier, Celine; Recasens, Ariadna; Bombrun, Agnes; Antonsson, Bruno; Vila, Miquel

    2014-05-01

    Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

  18. Activation of the Proapoptotic Bcl-2 Protein Bax by a Small Molecule Induces Tumor Cell Apoptosis

    Science.gov (United States)

    Zhao, Guoping; Zhu, Yanglong; Eno, Colins O.; Liu, Yanlong; DeLeeuw, Lynn; Burlison, Joseph A.; Chaires, Jonathan B.; Trent, John O.

    2014-01-01

    The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer. PMID:24421393

  19. Pro-apoptotic Bax molecules densely populate the edges of membrane pores.

    Science.gov (United States)

    Kuwana, Tomomi; Olson, Norman H; Kiosses, William B; Peters, Bjoern; Newmeyer, Donald D

    2016-06-03

    How the pro-apoptotic Bax protein permeabilizes the mitochondrial outer membrane is not fully understood. Previously, using cryo-electron microscopy (cryo-EM), we showed that activated Bax forms large, growing pores. Whether formed in liposomes or in mitochondrial outer membranes, Bax-induced pores exhibit the same morphology, with negative curvature flanking the edges and with no visible protein structure protruding from the membranes. Here we used cryo-EM to show that gold-labeled Bax molecules, after activation by Bid, became localized strictly at pore edges. This argues that Bax acts at short range to deform the membrane. Also, Bax molecules populated the walls of both small and large pores at the same density, implying that Bax is continuously recruited to the pores as they widen. Moreover, because all Bax molecules became oligomerized after membrane insertion, we infer that Bax oligomers are present at pore edges. We suggest that oligomerization may promote pore enlargement.

  20. The C-terminal helix of Bcl-xL mediates Bax retrotranslocation from the mitochondria

    Science.gov (United States)

    Todt, F; Cakir, Z; Reichenbach, F; Youle, R J; Edlich, F

    2013-01-01

    The proapoptotic Bcl-2 protein Bax can commit a cell to apoptosis by translocation from the cytosol to the mitochondria and permeabilization of the outer mitochondrial membrane. Prosurvival Bcl-2 family members, such as Bcl-xL, control Bax activity. Bcl-xL recognizes Bax after a conformational change in the N-terminal segment of Bax on the mitochondria and retrotranslocates it back into the cytoplasm, stabilizing the inactive form of Bax. Here we show that Bax retrotranslocation depends on the C-terminal helix of Bcl-xL. Deletion or substitution of this segment reduces Bax retrotranslocation and correlates with the accumulation of GFP-tagged or endogenous Bax on the mitochondria of non-apoptotic cells. Unexpectedly, the substitution of the Bcl-xL membrane anchor by the corresponding Bax segment reverses the Bax retrotranslocation activity of Bcl-xL, but not that of Bcl-xL shuttling. Bax retrotranslocation depends on interaction to the Bcl-xL membrane anchor and interaction between the Bax BH3 domain and the Bcl-xL hydrophobic cleft. Interference with either interaction increases mitochondrial levels of endogenous Bax. In healthy cells, mitochondrial Bax does not permeabilize the outer mitochondrial membrane, but increases cell death after apoptosis induction. PMID:23079612

  1. The MUC1-C Oncoprotein Binds to the BH3 Domain of the Pro-apoptotic BAX Protein and Blocks BAX Function*

    Science.gov (United States)

    Ahmad, Rehan; Alam, Maroof; Rajabi, Hasan; Kufe, Donald

    2012-01-01

    The pro-apoptotic BAX protein contains a BH3 domain that is necessary for its dimerization and for activation of the intrinsic apoptotic pathway. The MUC1 (mucin 1) heterodimeric protein is overexpressed in diverse human carcinomas and blocks apoptosis in the response to stress. In this study, we demonstrate that the oncogenic MUC1-C subunit associates with BAX in human cancer cells. MUC1-C·BAX complexes are detectable in the cytoplasm and mitochondria and are induced by genotoxic and oxidative stress. The association between MUC1-C and BAX is supported by the demonstration that the MUC1-C cytoplasmic domain is sufficient for the interaction with BAX. The results further show that the MUC1-C cytoplasmic domain CQC motif binds directly to the BAX BH3 domain at Cys-62. Consistent with binding to the BAX BH3 domain, MUC1-C blocked BAX dimerization in response to (i) truncated BID in vitro and (ii) treatment of cancer cells with DNA-damaging agents. In concert with these results, MUC1-C attenuated localization of BAX to mitochondria and the release of cytochrome c. These findings indicate that the MUC1-C oncoprotein binds directly to the BAX BH3 domain and thereby blocks BAX function in activating the mitochondrial death pathway. PMID:22544745

  2. A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast

    DEFF Research Database (Denmark)

    Kampranis, S C; Damianova, R; Atallah, M

    2000-01-01

    for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed......The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows...

  3. Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

    Science.gov (United States)

    Ma, S B; Nguyen, T N; Tan, I; Ninnis, R; Iyer, S; Stroud, D A; Menard, M; Kluck, R M; Ryan, M T; Dewson, G

    2014-01-01

    In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-xL. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family

  4. The porin VDAC2 is the mitochondrial platform for Bax retrotranslocation.

    Science.gov (United States)

    Lauterwasser, Joachim; Todt, Franziska; Zerbes, Ralf M; Nguyen, Thanh Ngoc; Craigen, William; Lazarou, Michael; van der Laan, Martin; Edlich, Frank

    2016-09-13

    The pro-apoptotic Bcl-2 protein Bax can permeabilize the outer mitochondrial membrane and therefore commit human cells to apoptosis. Bax is regulated by constant translocation to the mitochondria and retrotranslocation back into the cytosol. Bax retrotranslocation depends on pro-survival Bcl-2 proteins and stabilizes inactive Bax. Here we show that Bax retrotranslocation shuttles membrane-associated and membrane-integral Bax from isolated mitochondria. We further discover the mitochondrial porin voltage-dependent anion channel 2 (VDAC2) as essential component and platform for Bax retrotranslocation. VDAC2 ensures mitochondria-specific membrane association of Bax and in the absence of VDAC2 Bax localizes towards other cell compartments. Bax retrotranslocation is also regulated by nucleotides and calcium ions, suggesting a potential role of the transport of these ions through VDAC2 in Bax retrotranslocation. Together, our results reveal the unanticipated bifunctional role of VDAC2 to target Bax specifically to the mitochondria and ensure Bax inhibition by retrotranslocation into the cytosol.

  5. DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX

    Science.gov (United States)

    Guan, J-J; Zhang, X-D; Sun, W; Qi, L; Wu, J-C; Qin, Z-H

    2015-01-01

    DRAM1 (DNA damage-regulated autophagy modulator 1) is a TP53 target gene that modulates autophagy and apoptosis. We previously found that DRAM1 increased autophagy flux by promoting lysosomal acidification and protease activation. However, the molecular mechanisms by which DRAM1 regulates apoptosis are not clearly defined. Here we report a novel pathway by which DRAM1 regulates apoptosis involving BAX and lysosomes. A549 or HeLa cells were treated with the mitochondrial complex II inhibitor, 3-nitropropionic acid (3NP), or an anticancer drug, doxorubicin. Changes in the protein and mRNA levels of BAX and DRAM1 and the role of DRAM1 in BAX induction were determined. The interaction between DRAM1 and BAX and its effect on BAX degradation, BAX lysosomal localization, the release of cathepsin B and cytochrome c by BAX and the role of BAX in 3NP- or doxorubicin-induced cell death were studied. The results showed that BAX, a proapoptotic protein, was induced by DRAM1 in a transcription-independent manner. BAX was degraded by autophagy under basal conditions; however, its degradation was inhibited when DRAM1 expression was induced. There was a protein interaction between DRAM1 and BAX and this interaction prolonged the half-life of BAX. Furthermore, upregulated DRAM1 recruited BAX to lysosomes, leading to the release of lysosomal cathepsin B and cleavage of BID (BH3-interacting domain death agonist). BAX mediated the release of mitochondrial cytochrome c, activation of caspase-3 and cell death partially through the lysosome-cathepsin B-tBid pathway. These results indicate that DRAM1 regulates apoptosis by inhibiting BAX degradation. In addition to mitochondria, lysosomes may also be involved in BAX-initiated apoptosis. PMID:25633293

  6. Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis.

    Science.gov (United States)

    Maes, Margaret E; Schlamp, Cassandra L; Nickells, Robert W

    2017-01-01

    The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.

  7. Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.

    Science.gov (United States)

    D'Orsi, Beatrice; Bonner, Helena; Tuffy, Liam P; Düssmann, Heiko; Woods, Ina; Courtney, Michael J; Ward, Manus W; Prehn, Jochen H M

    2012-02-01

    Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

  8. BH3-Triggered Structural Reorganization Drives the Activation of Pro-apoptotic BAX

    Science.gov (United States)

    Gavathiotis, Evripidis; Reyna, Denis E.; Davis, Marguerite L.; Bird, Gregory H.; Walensky, Loren D.

    2010-01-01

    Summary BAX is a pro-apoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. Having recently identified the elusive trigger site for direct BAX activation, we now delineate by NMR and biochemical methods the essential allosteric conformational changes that transform ligand-triggered BAX into a fully activated monomer capable of propagating its own activation. Upon BAX engagement by a triggering BH3 helix, the unstructured loop between α-helices 1 and 2 is displaced, the carboxy terminal helix 9 is mobilized for membrane translocation, and the exposed BAX BH3 domain propagates the death signal through an auto-activating interaction with the trigger site of inactive BAX monomers. Our structure-activity analysis of this seminal apoptotic process reveals new pharmacologic opportunities to modulate cell death by interceding at key steps of the BAX activation pathway. PMID:21070973

  9. Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes.

    Science.gov (United States)

    Andreu-Fernández, Vicente; Sancho, Mónica; Genovés, Ainhoa; Lucendo, Estefanía; Todt, Franziska; Lauterwasser, Joachim; Funk, Kathrin; Jahreis, Günther; Pérez-Payá, Enrique; Mingarro, Ismael; Edlich, Frank; Orzáez, Mar

    2017-01-10

    The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation.

  10. Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores.

    Science.gov (United States)

    Salvador-Gallego, Raquel; Mund, Markus; Cosentino, Katia; Schneider, Jale; Unsay, Joseph; Schraermeyer, Ulrich; Engelhardt, Johann; Ries, Jonas; García-Sáez, Ana J

    2016-02-15

    Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane. © 2016 The Authors.

  11. Clinicopathological significance of Bcl-2 and Bax protein expression in human pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Ming Dong; Jian-Ping Zhou; Hao Zhang; Ke-Jian Guo; Yu-Lin Tian; Yu-Ting Dong

    2005-01-01

    AIM: To assess the clinicopathological significance of the expression of the apoptosis-inhibitory Bcl-2 protein (pBcl-2) and the apoptosis-promoting Bax protein (pBax) in human invasive ductal carcinomas (IDCs) of the pancreas. METHODS: Fifty-nine surgical specimens of IDCs of the pancreas were stained immunohistochemically to detectpBcl-2 and pBax expressions whose correlation to tumor classification, staging, and prognosis was analyzed by univariate and multivariate analyses. RESULTS: The expression of pBcl-2 and pBax was detected in 21 of 59 (35.6%) and in 29 of 59 (49.2%) patients with IDCs of the pancreas, respectively. Neither pBcl-2 nor pBax alone was correlated to TNM staging and differentiation degree of IDCs of the pancreas according to univariate analysis. By Mantel-Cox test, the median survival time after surgery for pBcl-2(+) and pBcl-2(-) groups were 14.3 and 7.3 mo, respectively (χ2= 9.357, P = 0.002) and that for pBax(+) and pBax(-) groups were 12.9 and 10.2 mo, respectively (χ2= 0.285, P>0.05).Contingency coefficient between pBd-2 and pBax expression was 0.298, indicating that there was correlation between them (χ2= 5.74, P<0.05). The median survival time after surgery for pBd-2(+)pBax(+) and pBcl-2(+)pBax(-) groups were 14.3 and 14.1 mo, respectively, and that for pBcl-2 (-)pBax(+) and pBcl-2(-)pBax(-) groups were 5.9 and 9.9 mo, respectively. There was a significant difference between pBcl-2(+)pBax(+) and pBcl-2(-)pBax(+) (χ2 = 5.06,P<0.05), such was the case for pBcl-2(+)pBax(+) andpBcl-2(-)pBax(-) (χ2= 7.18, P<0.01). Cox proportional hazards model for multivariate analysis was applied, indicating that pBcl-2, TNM staging, age and pBax were high risk factors of post-surgical survival time. CONCLUSION: Both pBcl-2 and pBax have high expression in IDCs of the pancreas, indicating that co-expression of pBcl-2 and pBax is a good indicator of favorable prognosis in IDCs of the pancreas.

  12. Impact of the combined loss of BOK, BAX and BAK on the hematopoietic system is slightly more severe than compound loss of BAX and BAK.

    Science.gov (United States)

    Ke, F; Grabow, S; Kelly, G L; Lin, A; O'Reilly, L A; Strasser, A

    2015-10-22

    It is well established that BAX and BAK play crucial, overlapping roles in the intrinsic pathway of apoptosis. Gene targeted mice lacking both BAX and BAK have previously been generated, but the majority of these animals died perinatally. BOK is a poorly studied relative of BAX and BAK that shares extensive amino acid sequence homology to both proteins, but its function remains largely unclear to date. To determine whether BOK plays an overlapping role with BAX and BAK, we utilized a hematopoietic reconstitution model where lethally irradiated wild type mice were transplanted with Bok(-/-)Bax(-/-)Bak(-/-) triple knockout (TKO) fetal liver cells, and compared alongside mice reconstituted with a Bax(-/-)Bak(-/-) double knockout (DKO) hematopoietic compartment. We report here that mice with a TKO and DKO hematopoietic system died at a similar rate and much earlier than control animals, mostly due to severe autoimmune pathology. Both TKO and DKO reconstituted mice also had altered frequencies of various leukocyte subsets in the thymus, bone marrow and spleen, displayed leukocyte infiltrates and autoimmune pathology in multiple tissues, as well as elevated levels of anti-nuclear autoantibodies. Interestingly, the additional deletion of BOK (on top of BAX and BAK loss) led to a further increase in peripheral blood lymphocytes, as well as enhanced lymphoid infiltration in some organs. These findings suggest that BOK may have some functions that are redundant with BAX and BAK in the hematopoietic system.

  13. Bax function in the absence of mitochondria in the primitive protozoan Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Adrian B Hehl

    Full Text Available Bax-induced permeabilization of the mitochondrial outer membrane and release of cytochrome c are key events in apoptosis. Although Bax can compromise mitochondria in primitive unicellular organisms that lack a classical apoptotic machinery, it is still unclear if Bax alone is sufficient for this, or whether additional mitochondrial components are required. The protozoan parasite Giardia lamblia is one of the earliest branching eukaryotes and harbors highly degenerated mitochondrial remnant organelles (mitosomes that lack a genome. Here we tested whether human Bax expressed in Giardia can be used to ablate mitosomes. We demonstrate that these organelles are neither targeted, nor compromised, by Bax. However, specialized compartments of the regulated secretory pathway are completely ablated by Bax. As a consequence, maturing cyst wall proteins that are sorted into these organelles are released into the cytoplasm, causing a developmental arrest and cell death. Interestingly, this ectopic cargo release is dependent on the carboxy-terminal 22 amino acids of Bax, and can be prevented by the Bax-inhibiting peptide Ku70. A C-terminally truncated Bax variant still localizes to secretory organelles, but is unable to permeabilize these membranes, uncoupling membrane targeting and cargo release. Even though mitosomes are too diverged to be recognized by Bax, off-target membrane permeabilization appears to be conserved and leads to cell death completely independently of mitochondria.

  14. Connexin 43 enhances Bax activation via JNK activation in sunitinib-induced apoptosis in mesothelioma cells.

    Science.gov (United States)

    Uzu, Miaki; Sato, Hiromi; Shimizu, Ayaka; Shibata, Yukihiro; Ueno, Koichi; Hisaka, Akihiro

    2017-06-01

    The constituent protein of gap junctions, connexin (Cx), interacts with various proteins via its C-terminus region, including kinases, cell-adhesion proteins, and a pro-apoptotic protein, Bax. This molecular interaction may affect expression and functioning of the interacting proteins and modulate the cellular physiology. In our previous work, Cx43 was found to interact directly with Bax and in the presence of sunitinib, lead to the Bax-mediated apoptosis in mesothelioma cells. In this study, we investigated the mechanism of how Cx43 promotes Bax-mediated apoptosis using the same cell line. Treatment with sunitinib increased the expression of the active conformation of the Bax protein, which was predominantly localized at the mitochondria, only in Cx43-transfected cells. Bax oligomerization and decrease in the mitochondrial membrane potential were also observed. The involvement of c-Jun N-terminal kinase (JNK) in the interaction of Cx43 and Bax was further examined. Treatment with sunitinib increased the expression of phosphorylated (active) form of JNK only in the Cx43-transfected cells. Phosphorylated JNK and active Bax were co-localized, and the co-localization was suppressed by the knockdown of Cx43. Moreover, JNK inhibition clearly suppressed Bax activation. In conclusion, we identified a novel Cx43-JNK-Bax axis regulating the process of apoptosis for the first time. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  15. Expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia

    Institute of Scientific and Technical Information of China (English)

    Sheng-Mian Li; Shu-Kun Yao; Nobuyoshi Yamamura; Toshitsugu Nakamura

    2003-01-01

    AIM: To compare the difference of expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia, and to analyze the role of Bcl-2 and Bax proteins in the progression from dysplasia to carcinoma and to evaluate the correlation of Bcl-2/Bax protein expression with the biological behaviors.METHODS: Expressions of Bcl-2 and Bax were examined immunohistochemically in 27 cases of extrahepatic biliary tract carcinomas (bile duct carcinoma: n=21, carcinoma of ampulla of Vater: n=6), and 10 cases of atypical dysplasia.Five cases of normal biliary epithelial tissues were used as controls. A semiquantitative scoring system was used to assess the Bcl-2 and Bax reactivity.RESULTS: The expression of Bd-2 was observed in 10 out of 27 (37.0 %) invasive carcinomas, 1 out of 10 clysplasias, none out of 5 normal epithelial tissues. Bax expression rate was 74.1% (20/27) in invasive carcinoma, 30 % (3/10) in dysplasia,and 40 % (2/5) in normal biliary epithelium. Bcl-2 and Bax activities were more intense in carcinoma than in dysplasia,with no significant difference in Bcl-2 expression (P=0.1:10),and significant difference in Bax expression (P=0.038). Level of Bax expression was higher in invasive carcinoma than in dysplasia and normal tissue (P=0.012). Bcl-2 expression was correlated to Bax expression (P=0.0059). However, Bcl-2/Bax expression had no correlation with histological subtype,grade of differentiation, or level of invasion.CONCLUSION: Increased Bcl-2/Bax expression from dysplasia to invasive tumors supports the view that this is the usual route for the development of extrahepatic biliary tract carcinoma. Bcl-2/Bax may be involved, at least in part,in the apoptotic activity in extrahepatic biliary carcinoma.

  16. Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX

    Institute of Scientific and Technical Information of China (English)

    Bonsu Ku; Chengyu Liang; Jae U Jung; Byung-Ha Oh

    2011-01-01

    Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic Bc1-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague.Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, hut was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-XL, MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could he expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.

  17. JNK3 phosphorylates Bax protein and induces ability to form pore on bilayer lipid membrane

    Directory of Open Access Journals (Sweden)

    Rajeev Gupta

    2017-06-01

    Full Text Available Bax is a pro-apoptotic cytosolic protein. In this work native (unphosphorylated and JNK3 phosphorylated Bax proteins are studied on artificial bilayer membranes for pore formation. Phosphorylated Bax formed pore on the bilayer lipid membrane whereas native one does not. In cells undergoing apoptosis the pore formed by the phosphorylated Bax could be important in cytochrome c release from the mitochondrial intermembrane space to the cytosol. The low conductance (1.5 nS of the open state of the phosphorylated Bax pore corresponds to pore diameter of 0.9 nm which is small to release cytochrome c (∼3.4 nm. We hypothesized that JNK3 phosphorylated Bax protein can form bigger pores after forming complexes with other mitochondrial proteins like VDAC, t-Bid etc. to release cytochrome c.

  18. Bax mitochondrial relocation is linked to its phosphorylation and its interaction with Bcl-xL.

    Science.gov (United States)

    Garenne, David; Renault, Thibaud T; Manon, Stéphen

    2016-12-05

    The heterologous expression of Bax, and other Bcl-2 family members, in the yeast Saccharomyces cerevisiae, has proved to be a valuable reporter system to investigate the molecular mechanisms underlying their interaction with mitochondria. By combining the co-expression of Bax and Bcl-xL mutants with analyzes of their localization and interaction in mitochondria and post-mitochondrial supernatants, we showed that the ability of Bax and Bcl-xL to interact is dependent both on Bax phosphorylation - mimicked by a substitution S184D - and by Bax and Bcl-xL localization. This, and previous data, provide the molecular basis for a model of dynamic equilibrium for Bax localization and activation, regulated both by phosphorylation and Bcl-xL.

  19. Bax and Bif-1 proteins interact on Bilayer Lipid Membrane and form pore.

    Science.gov (United States)

    Gupta, Rajeev; Ghosh, Subhendu

    2015-08-07

    Bax and Bax interacting factor-1(Bif-1) are cytosolic proteins, which translocate towards mitochondria during mitochondria-mediated apoptosis. Bif-1 has been identified to co-immunoprecipitate with Bax in apoptotic cells. We have studied the interaction of Bax and Bif-1 on Bilayer Lipid Membrane (BLM) through electrophysiological experiments. It has been observed that Bax-Bif-1 equimolar mixture can form a pore. The pore conductance is in the range of 4.96-5.41 nS. It also displays a sub-state with a conductance of 2.6 nS. No pore activity is observed on BLM when monomeric Bax and Bif-1 proteins are tested independently. The above-mentioned pore forming activity could be relevant in mitochondria-mediated apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax.

    Science.gov (United States)

    Lidman, Martin; Pokorná, Šárka; Dingeldein, Artur P G; Sparrman, Tobias; Wallgren, Marcus; Šachl, Radek; Hof, Martin; Gröbner, Gerhard

    2016-06-01

    Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Role of Bax in death of uninfected retinal cells during murine cytomegalovirus retinitis.

    Science.gov (United States)

    Mo, Juan; Marshall, Brendan; Covar, Jason; Zhang, Nancy Y; Smith, Sylvia B; Atherton, Sally S; Zhang, Ming

    2014-10-08

    Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3-dependent and -independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells. BALB/c mice, Bax(-/-) mice, or Bax(+/+) mice were immunosuppressed with methylprednisolone and infected with 5 × 10(3) plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3. Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax(-/-) eyes than in MCMV-infected Bax(+/+) eyes. However, more caspase 3-independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax(-/-) than in Bax(+/+) eyes. During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3-dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3-independent pathway. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  2. Integration and oligomerization of Bax protein in lipid bilayers characterized by single molecule fluorescence study.

    Science.gov (United States)

    Luo, Lu; Yang, Jun; Liu, Dongxiang

    2014-11-14

    Bax is a pro-apoptotic Bcl-2 family protein. The activated Bax translocates to mitochondria, where it forms pore and permeabilizes the mitochondrial outer membrane. This process requires the BH3-only activator protein (i.e. tBid) and can be inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-xL. Here by using single molecule fluorescence techniques, we studied the integration and oligomerization of Bax in lipid bilayers. Our study revealed that Bax can bind to lipid membrane spontaneously in the absence of tBid. The Bax pore formation undergoes at least two steps: pre-pore formation and membrane insertion. The activated Bax triggered by tBid or BH3 domain peptide integrates on bilayers and tends to form tetramers, which are termed as pre-pore. Subsequent insertion of the pre-pore into membrane is highly dependent on the composition of cardiolipin in lipid bilayers. Bcl-xL can translocate Bax from membrane to solution and inhibit the pore formation. The study of Bax integration and oligomerization at the single molecule level provides new evidences that may help elucidate the pore formation of Bax and its regulatory mechanism in apoptosis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Cell death induced by 2-phenylethynesulfonamide uncovers a pro-survival function of BAX.

    Science.gov (United States)

    Mattiolo, Paolo; Barbero-Farran, Ares; Amigó, Josep; Ripamonti, Marta; Ribas, Judit; Boix, Jacint

    2014-11-01

    PES (2-phenylethynesulfonamide) was initially identified as an inhibitor of p53 translocation to mitochondria and named Pifithrin-µ. Further studies showed that PES selectively killed tumour cells and was thus a promising anticancer agent. PES-induced cell death was characterised by a non-apoptotic, autophagosome-rich phenotype. We observed this phenotype via electron microscopy in wild type (wt) and double Bax-/- Bak-/- (DKO) mouse embryonic fibroblasts (MEFs) treated with PES. We excluded the involvement of effector caspases, BAX and BAK, in causing PES-triggered cell death. Therefore, apoptosis was ruled out as the lethal mode of action of PES. Surprisingly, MEFs containing BAX were significantly protected from PES treatments. BAX overexpression in Bax-/- MEFs confirmed this pro-survival effect. Moreover, this protective effect required the ability of BAX to localise to mitochondrial membranes. Conversely, mitochondrial fusion induced by treatment with Mdivi-1 conferred increased resistance to MEFs subjected to PES treatment. The involvement of BAX in the regulation of mitochondrial dynamics has been reported. We propose the promotion of mitochondrial fusion by BAX to be the pro-survival function attributed to BAX. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis.

    Science.gov (United States)

    Große, Lena; Wurm, Christian A; Brüser, Christian; Neumann, Daniel; Jans, Daniel C; Jakobs, Stefan

    2016-02-15

    The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  5. Bak and Bax function to limit adenovirus replication through apoptosis induction.

    Science.gov (United States)

    Cuconati, Andrea; Degenhardt, Kurt; Sundararajan, Ramya; Anschel, Alan; White, Eileen

    2002-05-01

    Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.

  6. Expression of Bax in yeast affects not only the mitochondria but also vacuolar integrity and intracellular protein traffic

    DEFF Research Database (Denmark)

    Dimitrova, Irina; Toby, Garabet G; Tili, Esmerina

    2004-01-01

    Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules. Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S-transfer......Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules. Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S......-transferase (BI-GST) leads to aggregation, but not fusion of the mitochondria. In addition, Bax affects the integrity of yeast vacuoles, resulting in the disintegration and eventual loss of the organelles, and the disruption of intracellular protein traffic. While Bcl-2 coexpression only partially corrects...

  7. Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation

    Science.gov (United States)

    Peng, R; Tong, J-S; Li, H; Yue, B; Zou, F; Yu, J; Zhang, L

    2013-01-01

    Bax is a proapoptotic Bcl-2 family member that has a central role in the initiation of mitochondria-dependent apoptosis. However, the mechanism of Bax activation during apoptosis remains unsettled. It is believed that the activation of Bax is mediated by either dissociation from prosurvival Bcl-2 family members, or direct association with BH3-only members. Several interaction sites on Bax that mediate its interactions with other Bcl-2 family members, as well as its proapoptotic activity, have been identified in previous studies by other groups. To rigorously investigate the functional role of these interaction sites, we knocked in their respective mutants using HCT116 colon cancer cells, in which apoptosis induced by several stimuli is strictly Bax-dependent. Bax-mediated apoptosis was intact upon knock-in (KI) of K21E and D33A, which were shown to block the interaction of Bax with BH3-only activators. Apoptosis was partially reduced by KI of D68R, which impairs the interaction of Bax with prosurvival members, and S184V, a constitutively mitochondria-targeting mutant. In contrast, apoptosis was largely suppressed by KI of L70A/D71A, which blocks homo-oligomerization of Bax and its binding to prosurvival Bcl-2 family proteins. Collectively, our results suggest that the activation of endogenous Bax in HCT116 cells is dependent on its homo-oligomerization sites, but not those previously shown to interact with BH3-only activators or prosurvival proteins only. We therefore postulate that critical interaction sites yet to be identified, or mechanisms other than protein-protein interactions, need to be pursued to delineate the mechanism of Bax activation during apoptosis. PMID:23392123

  8. The Expression of Bcl-2 and Bax in Normal,Hyperplastic,and Malignant Endometrium

    Institute of Scientific and Technical Information of China (English)

    ZHONGGang; TANLingfang; 等

    2002-01-01

    Objective:To investigate the expression of Bcl-2 and Bax proteins in normal,hyperplastic,and malignant endometrium.Methods:Endometrial tissues were obtained from 14 proliferative endometrial samples;simple(n=30)and complex hyperplasia without atypia(n=13);complex hyperplasia with atypia(n=20)and endometrial adenocarcinoma(n=17).The expression of Bcl-2and Bax proteins was detected by using immunohistochemical staining with appropriate antibodies.Results:The intensity of Bcl-2 staining was gradually increased from proliferative to simple and complex hyperplasia,but it was gradually decreased from atypia hyperplasia to endometrial adenocarcinoma(P<0.05).The intensity of Bax staining was gradually increased from proliferative endometrium to simple and complex hyperplasia,but in atypia hyperplasia it was obviously lower than simple hyperplasia,the ratio of Bco-2;Bax staining intensity was changed with the endometrium from proliferative,hyperplastic endo-metrium to endometrial adenocarcinoma.The ratio of Bcl-2;Bax staining intensity was obviously decreased in atypia hyperplasia and endometrial adenocarcinoma.Conclusion:The survival time of the cells in hyperplasia expressing Bcl-2 might be prolonged.Neoplastic cells in atypia hyperplasia and adenocarcinoma might show a decreased expression of Bcl-2 and Bax,suggesting that Bcl-2 and Bax might be important indexes and prognosis factors and the expression of Bcl-2 and Bax might be correlated with carcinogenesis in the uterine endometrium of hu-mans.

  9. Oncogenic Mutations Differentially Affect Bax Monomer, Dimer, and Oligomeric Pore Formation in the Membrane

    Science.gov (United States)

    Zhang, Mingzhen; Zheng, Jie; Nussinov, Ruth; Ma, Buyong

    2016-09-01

    Dysfunction of Bax, a pro-apoptotic regulator of cellular metabolism is implicated in neurodegenerative diseases and cancer. We have constructed the first atomistic models of the Bax oligomeric pore consisting with experimental residue-residue distances. The models are stable, capturing well double electron-electron resonance (DEER) spectroscopy measurements and provide structural details in line with the DEER data. Comparison with the latest experimental results revealed that our models agree well with both Bax and Bak pores, pointed to a converged structural arrangement for Bax and Bak pore formation. Using multi-scale molecular dynamics simulations, we probed mutational effects on Bax transformation from monomer → dimer → membrane pore formation at atomic resolution. We observe that two cancer-related mutations, G40E and S118I, allosterically destabilize the monomer and stabilize an off-pathway swapped dimer, preventing productive pore formation. This observation suggests a mechanism whereby the mutations may work mainly by over-stabilizing the monomer → dimer transformation toward an unproductive off-pathway swapped-dimer state. Our observations point to misfolded Bax states, shedding light on the molecular mechanism of Bax mutation-elicited cancer. Most importantly, the structure of the Bax pore facilitates future study of releases cytochrome C in atomic detail.

  10. Bax-deficiency prolongs cerebellar neurogenesis, accelerates medulloblastoma formation and paradoxically increases both malignancy and differentiation

    Science.gov (United States)

    Garcia, Idoia; Crowther, Andrew J.; Gama, Vivian; Miller, C. Ryan; Deshmukh, Mohanish; Gershon, Timothy R.

    2012-01-01

    Neurogenesis requires negative regulation through differentiation of progenitors or their programmed cell death (PCD). Growth regulation is particularly important in the postnatal cerebellum, where excessive progenitor proliferation promotes medulloblastoma, the most common malignant brain tumor in children. We present evidence that PCD operates alongside differentiation to regulate cerebellar granule neuron progenitors (CGNPs) and to prevent medulloblastoma. Here we show that genetic deletion of pro-apoptotic Bax disrupts regulation of cerebellar neurogenesis and promotes medulloblastoma formation. In Bax−/− mice, the period of neurogenesis was extended into the third week of postnatal life, and ectopic neurons and progenitors collected in the molecular layer of the cerebellum and adjacent tectum. Importantly, genetic deletion of Bax in medulloblastoma-prone ND2:SmoA1 transgenic mice greatly accelerated tumorigenesis. Bax-deficient medulloblastomas exhibited strikingly distinct pathology, with reduced apoptosis, increased neural differentiation and tectal migration. Comparing Bax+/+ and Bax−/− medulloblastomas, we were able to identify up-regulation of Bcl-2 and nuclear exclusion of p27 as tumorigenic changes that are required to mitigate the tumor suppressive effect of Bax. Studies on human tumors confirmed the importance of modulating Bax in medulloblastoma pathogenesis. Our results demonstrate that Bax-dependent apoptosis regulates postnatal cerebellar neurogenesis, suppresses medulloblastoma formation, and imposes selective pressure on tumors that form. Functional resistance to Bax-mediated apoptosis, required for medulloblastoma tumorigenesis, may be a tumor-specific vulnerability to be exploited for therapeutic benefit. PMID:22710714

  11. [The Mechanisms by which Bax Induces the Apoptosis of Human Ovarian Cancer Cells].

    Science.gov (United States)

    Zeng, Jun; Yang, Jing; Chen, Deng-bang; Cao, Kang

    2015-09-01

    The purpose of this study was to observe the apoptosis of A2780 cells transfected with the recombinant plasmid of pcDNA-Bax and to observe the release of cytochrome C from the mitochondria. The recombinant plasmid of pcDNA-Bax was constructed and transfected into A2784 cells. The Hoechst 33258 stain method was applied to evaluate the apoptosis of the transfected cells and MTT mothod was used to test the cell viability. Western blot analysis was performed to determine the overexpression of Bax and the release of cytochrome C from the mitochondria. The recombinant plasmid of pcDNA-Bax was successfully constructed by using endonuclease digestion and the sequence analysis. The apoptosis of A2780 cells was induced after transfected with pcDNA3. 1-Bax as demonstrated with Hoechst staining. The cell viability were decreased in the pcDNA3. 1-Bax transfected group by MTT assay. The release of cytochrome C from the mitochondria was observed when using Western blotting analysis. And the caspase-9 and the caspase-3 were activated. Our data suggestted that Bax exhibited potent pro-apoptotic activity against the ovarian cancer cells. This study is a foundation for the further research in the pro-apoptotic activity of Bax.

  12. Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression.

    Science.gov (United States)

    Wang, Weicheng; Guo, Wenjie; Li, Lele; Fu, Zan; Liu, Wen; Gao, Jian; Shu, Yongqian; Xu, Qiang; Sun, Yang; Gu, Yanhong

    2016-12-01

    5-FU is the first line therapy for colorectal cancer, however, treatment effect is often hampered by the development of drug resistance or toxicity at high doses. Andrographolide is a natural diterpenoid from Andrographis paniculata which has anti-bacterial, anti-antiviral and anti-inflammation activities. In the current study, we test the hypothesis that Andrographolide reverses 5-FU resistance in colorectal cancer and examine the underlying mechanism. In vitro and vivo studies indicated that Andrographolide treatment significantly re-sensitizes HCT116/5-FUR cells (HCT116 cells which are 5-FU resistant) to cytotoxicity of 5-FU. Mechanism analysis showed that Andrographolide/5-FU co-treatment elevated apoptosis level of HCT116/5-FUR cells with highly increased level of BAX. By using biotin-Andrographolide pull down and cellular thermal shift assay, we found out that Andrographolide can directly target to BAX. Andrographolide-BAX interaction prevented BAX degradation, enhancing mitochondria-mediated apoptosis thus reversed 5-FU resistance while BAX silence diminished this effect. Further, by analyzing patient samples who received 5-FU involved chemotherapy, we found that expression level of BAX is correlated with PFS. Our results here provide a novel combination treatment strategy, especially for patients with 5-FU-resistant tumors expressing low level of BAX. Meanwhile, we also proposed that BAX expression may be a predicted and prognosis marker of 5-FU involved chemotherapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.

    Science.gov (United States)

    Teijido, Oscar; Ganesan, Yogesh Tengarai; Llanos, Raul; Peton, Ashley; Urtecho, Jean-Baptiste; Soprani, Adauri; Villamayor, Aimee; Antonsson, Bruno; Manon, Stéphen; Dejean, Laurent

    2016-03-15

    Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Quantitative assessment of BAX transcript and flow cytometric expression in acute myeloid leukemia: a prospective study.

    Science.gov (United States)

    Sharawat, Surender Kumar; Raina, Vinod; Kumar, Lalit; Sharma, Atul; Bakhshi, Radhika; Vishnubhatla, Sreenivas; Gupta, Ritu; Bakhshi, Sameer

    2014-10-01

    Quantitative assessment of BAX transcripts and protein in acute myeloid leukemia (AML). We quantitatively evaluated BAX gene transcripts by real-time polymerase chain reaction (TaqMan probe chemistry) and protein expression by flow cytometry. Consecutive 112 AML patients with a median age of 16 (1-59) years were recruited in the study. By flow cytometry, the percentage expression was in linear correlation with relative median fluorescent intensity (RMFI; R = 0.4425; P BAX with its RMFI (R = -0.0559; P = 0.586). The expression of the BAX at both protein and transcript level was significantly higher in AML patients as compared with normal control. RMFI of the BAX were higher in the cohort with lower white blood cell count (P = 0.029). None of the other baseline characteristics correlated with either the BAX transcript or the RMFI. BAX expression did not correlate with complete remission rate, event free, disease free, and overall survival. BAX gene expression in AML was evaluated first time with two different methods but did not correlate with the survival outcome.

  15. Effect of Helicobacter pylori infection on Bax protein expression in patients with gastric precancerous lesions

    Institute of Scientific and Technical Information of China (English)

    Hai-Feng Liu; Wei-Wen Liu; Guo-An Wang; Xiao-Chun Teng

    2005-01-01

    AIM: To investigate the effect of Helicobacter pylori (H pylori) infection on Bax protein expression, and explore the role of H pylori in gastric carcinogenesis.METHODS: H pylori was assessed by rapid urease test and Warthin-Starry method, and expression of Bax protein was examined immunohistochemically in 72 patients with pre-malignant lesions.RESULTS: Bax protein was differently expressed in intestinal metaplasia and gastric dysplasia, and showed 63.99% positivity. The positivity of Bax protein expression in H pylori-positive gastric precancerous lesions (72.3%) was significantly higher than that in H pylori-negative gastric precancerous lesions (48.0%, χ2 = 4.191, P<0.05).H pylori infection was well correlated with the expression of Bax protein in gastric precancerous lesions (r = 0.978,P<0.01). After eradication of H pylori, the positivity of Bax protein expression significantly decreased in H pylori-positive gastric precancerous lesions (χ2= 5.506,P<0.05). In the persisting H pylori-infected patients,the positivity of Bax protein expression was not changed.CONCLUSION: H pylori infection may be involved in the upregulation of Bax gene, which might be one of the mechanisms of H pylori infection-induced gastric epithelial cell apoptosis. H pylori might act as a tumor promoter in the genesis of gastric carcinoma and eradication of H pylori could inhibit gastric carcinogenesis.

  16. Higher expression of Bax in regulatory T cells increases vascular inflammation.

    Science.gov (United States)

    Xiong, Zeyu; Song, Jian; Yan, Yan; Huang, Yajue; Cowan, Alan; Wang, Hong; Yang, Xiao-Feng

    2008-05-01

    This study is to examine our hypothesis that CD4+CD25(high)Foxp3+ regulatory T cells (Tregs) have an interleukin-2 (IL-2) withdrawal-triggered apoptosis pathway, and modulation of Treg apoptosis pathway affects development of vascular inflammation. We found that pro-apoptotic protein Bax upregulation in Tregs is induced by IL-2 withdrawal. Treg apoptosis induced by IL-2 withdrawal is inhibited by a Bax inhibitor, suggesting that highly expressed Bax is functional. To define the role of upregulated Bax in Treg apoptosis, we established a Tregs-specific Bax transgenic mouse model. Enforced expression of Bax in Tregs promotes Treg apoptosis triggered by IL-2 withdrawal and other apoptosis stimuli, suggesting pro-apoptotic role of highly expressed Bax in wild-type Tregs. Finally, higher expression of Bax in Tregs decreases the striking threshold of vascular inflammation due to the failure of suppression of inflammatory cells resulting from Treg apoptosis. These results have demonstrated the proof of principle that the modulation of Tregs apoptosis/survival could be used as a new therapeutic approach for inflammatory cardiovascular diseases.

  17. The substitution of Proline 168 favors Bax oligomerization and stimulates its interaction with LUVs and mitochondria.

    Science.gov (United States)

    Simonyan, Lilit; Légiot, Alexandre; Lascu, Ioan; Durand, Grégory; Giraud, Marie-France; Gonzalez, Cécile; Manon, Stéphen

    2017-06-01

    Bax is a major player in the apoptotic process, being at the core of the mitochondria permeabilization events. In spite of the major recent advances in the knowledge of Bax organization within the membrane, the precise behavior of the C-terminal helix α9 remains elusive, since it was absent from the resolved structure of active Bax. The Proline 168 (P168) residue, located in the short loop between α8 and α9, has been the target of site-directed mutagenesis experiments, with conflicting results. We have produced and purified a recombinant mutant Bax-P168A, and we have compared its behavior with that of wild-type Bax in a series of tests on Large Unilamellar Vesicles (LUVs) and isolated mitochondria. We conclude that Bax-P168A had a greater ability to oligomerize and bind to membranes. Bax-P168A was not more efficient than wild-type Bax to permeabilize liposomes to small molecules but was more prone to release cytochrome c from mitochondria. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Involvement of nitric oxide signaling in mammalian Bax-induced terpenoid indole alkaloid production of Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bax, a mammalian pro-apoptotic member of the Bcl-2 family, has been demonstrated to be a potential regulatory factor for plant secondary metabolite biosynthesis recently. To investigate the molecular mechanism of Bax-induced secondary metabolite biosynthesis, we determined the contents of nitric oxide (NO) of the transgenic Catharanthus roseus cells overexpressing a mouse Bax protein and checked the effects of NO specific scavenger 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide (cPITO) on Bax-induced terpenoid indole alkaloid (TIA) production of the cells. The data showed that overexpression of the mouse Bax in C. roseus cells triggered NO generation of the cells. Treatment of cPITO not only inhibited the Bax-triggered NO burst but also suppressed the Bax-induced TIA production. The results indicated that the mouse Bax might activate the NO signaling in C. roseus cells and induce TIA production through the NO-dependent signal pathway in the cells. Furthermore, the activities of nitric oxide synthase (NOS) were significantly increased in the transgenic Bax cells as compared to those in the control cells, showing that the mouse Bax may induce NOS of C. roseus cells. Treatment of the transgenic Bax cells with NOS inhibitor PBITU blocked both Bax-induced NO generation and TIA production, which suggested that the mouse Bax might trigger NO generation and TIA production through NOS. However, the NOS-like activities and NO generation in the transgenic Bax cells did not match kinetically and the Bax-induced NOS-like activity was much later and lower than NO production. Moreover, the Bax-induced NO generation and TIA production were only partially inhibited by PBITU. Thus, our results suggested that the Bax-induced NO production and secondary metabolite biosynthesis in C. roseus cells was not entirely dependent on NOS or NOS-like enzymes.

  19. S-palmitoylation represents a novel mechanism regulating the mitochondrial targeting of BAX and initiation of apoptosis

    Science.gov (United States)

    Fröhlich, M; Dejanovic, B; Kashkar, H; Schwarz, G; Nussberger, S

    2014-01-01

    The intrinsic pathway of apoptotic cell death is mainly mediated by the BCL-2-associated X (BAX) protein through permeabilization of the mitochondrial outer membrane (MOM) and the concomitant release of cytochrome c into the cytosol. In healthy, non-apoptotic cells, BAX is predominantly localized in the cytosol and exhibits a dynamic shuttle cycle between the cytosol and the mitochondria. Thus, the initial association with mitochondria represents a critical regulatory step enabling BAX to insert into MOMs, promoting the release of cytochrome c and ultimately resulting in apoptosis. However, the molecular mode of how BAX associates with MOMs and whether a cellular regulatory mechanism governs this process is poorly understood. Here we show that in both primary tissues and cultured cells, the association with MOMs and the proapoptotic action of BAX is controlled by its S-palmitoylation at Cys-126. A lack of BAX palmitoylation reduced BAX mitochondrial translocation, BAX oligomerization, caspase activity and apoptosis. Furthermore, ectopic expression of specific palmitoyl transferases in cultured healthy cells increases BAX S-palmitoylation and accelerates apoptosis, whereas malignant tumor cells show reduced BAX S-palmitoylation consistent with their reduced BAX-mediated proapoptotic activity. Our findings suggest that S-palmitoylation of BAX at Cys126 is a key regulatory process of BAX-mediated apoptosis. PMID:24525733

  20. Activation of JNK/Bim/Bax pathway in UV-induced apoptosis

    Science.gov (United States)

    Liu, Lei; Hui, Li; Zhang, Zhen-zhen

    2011-03-01

    Cell apoptosis induced by UV irradiation is a highly complex process in which different molecular signaling pathways are involved. JNK has been proposed as an important regulator in UV irradiation-induced apoptosis. However, the molecular mechanism through which JNK regulates apoptosis, especially how JNK activates Bax in response to UV irradiation is still controversial. In this study, using real-time single-cell analysis, we studied the machinery of Bax activation during UV-induced apoptosis. UV treatment resulted in a series of events: phosphorylation of JNK, mitochondrial translocation of Bim, and subsequent activation of Bax. The activation of Bim and Bax could be inhibited in the presence of SP600125 (a specific inhibitor of JNK), suggesting that UV irradiation activated the JNK/Bim/Bax pathway.

  1. Pin1-Induced Proline Isomerization in Cytosolic p53 Mediates BAX Activation and Apoptosis.

    Science.gov (United States)

    Follis, Ariele Viacava; Llambi, Fabien; Merritt, Parker; Chipuk, Jerry E; Green, Douglas R; Kriwacki, Richard W

    2015-08-20

    The cytosolic fraction of the tumor suppressor p53 activates the apoptotic effector protein BAX to trigger apoptosis. Here we report that p53 activates BAX through a mechanism different from that associated with activation by BH3 only proteins (BIM and BID). We observed that cis-trans isomerization of proline 47 (Pro47) within p53, an inherently rare molecular event, was required for BAX activation. The prolyl isomerase Pin1 enhanced p53-dependent BAX activation by catalyzing cis-trans interconversion of p53 Pro47. Our results reveal a signaling mechanism whereby proline cis-trans isomerization in one protein triggers conformational and functional changes in a downstream signaling partner. Activation of BAX through the concerted action of cytosolic p53 and Pin1 may integrate cell stress signals to induce a direct apoptotic response. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Overexpression of Bax sensitizes prostate cancer cells to TGF-β induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    Pei Hui LIN; Zui PAN; Lin ZHENG; Na LI; David DANIELPOUR; Jian Jie MA

    2005-01-01

    NRP-154 is a tumorigenic epithelial cell line derived from the preneoplastic dorsal-lateral prostate of rats. These cells are exquisitely sensitive to TGF-β induced apoptosis. In contrast, we find that NRP-154 cells can sustain overexpression of exogenous Bax protein, which is different from non-tumor cells where Bax functions as a ubiquitous stimulator of apoptosis. NRP-154 cells stably overexpressing Bax show increased sensitivity to TGF-β induced apoptosis. The degree of TGF-β induced apoptosis displays high correlation with cleavage of Bax at the amino-terminus. Our data indicate that prostate cancer cells can host high levels of latent Bax which can be activated through post-translational modification.

  3. Quercetin induces tumor-selective apoptosis through downregulation of Mcl-1 and activation of Bax.

    Science.gov (United States)

    Cheng, Senping; Gao, Ning; Zhang, Zhuo; Chen, Gang; Budhraja, Amit; Ke, Zunji; Son, Young-ok; Wang, Xin; Luo, Jia; Shi, Xianglin

    2010-12-01

    To investigate the in vivo antitumor efficacy of quercetin in U937 xenografts and the functional roles of Mcl-1 and Bax in quercetin-induced apoptosis in human leukemia. Leukemia cells were treated with quercetin, after which apoptosis, Mcl-1 expression, and Bax activation and translocation were evaluated. The efficacy of quercetin as well as Mcl-1 expression and Bax activation were investigated in xenografts of U937 cells. Administration of quercetin caused pronounced apoptosis in both transformed and primary leukemia cells but not in normal blood peripheral mononuclear cells. Quercetin-induced apoptosis was accompanied by Mcl-1 downregulation and Bax conformational change and mitochondrial translocation that triggered cytochrome c release. Knockdown of Bax by siRNA reversed quercetin-induced apoptosis and abrogated the activation of caspase and apoptosis. Ectopic expression of Mcl-1 attenuated quercetin-mediated Bax activation, translocation, and cell death. Conversely, interruption of Mcl-1 by siRNA enhanced Bax activation and translocation, as well as lethality induced by quercetin. However, the absence of Bax had no effect on quercetin-mediated Mcl-1 downregulation. Furthermore, in vivo administration of quercetin attenuated tumor growth in U937 xenografts. The TUNEL-positive apoptotic cells in tumor sections increased in quercetin-treated mice as compared with controls. Mcl-1 downregulation and Bax activation were also observed in xenografts. These data suggest that quercetin may be useful for the treatment of leukemia by preferentially inducing apoptosis in leukemia versus normal hematopoietic cells through a process involving Mcl-1 downregulation, which, in turn, potentiates Bax activation and mitochondrial translocation, culminating in apoptosis. ©2010 AACR.

  4. Bax/Bak activation in the absence of Bid, Bim, Puma, and p53.

    Science.gov (United States)

    Zhang, J; Huang, K; O'Neill, K L; Pang, X; Luo, X

    2016-06-16

    How BH3-only proteins activate Bax/Bak, the two gateway proteins of the mitochondria-dependent apoptotic pathway, remains incompletely understood. Although all pro-apoptotic BH3-only proteins are known to bind/neutralize the anti-apoptotic Bcl-2 proteins, the three most potent ones, Bid (tBid), Bim, and Puma, possess an additional activity of directly activating Bax/Bak in vitro. This latter activity has been proposed to be responsible for triggering Bax/Bak activation following apoptotic stimulation. To test this hypothesis, we generated Bid(-/)(-)Bim(-/)(-)Puma(-/)(-) (TKO), TKO/Bax(-/)(-)/Bak(-/)(-) (PentaKO), and PentaKO/Mcl-1(-/-) (HexaKO) HCT116 cells through gene editing. Surprisingly, although the TKO cells were resistant to several apoptotic stimuli, robust apoptosis was induced upon the simultaneous inactivation of Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins known to suppress Bax/Bak activation and activity. Importantly, such apoptotic activity was completely abolished in the PentaKO cells. In addition, ABT-737, a BH3 mimetic that inhibits Bcl-xL/Bcl-w/Bcl-2, induced Bax activation in HexaKO cells reconstituted with endogenous level of GFP-Bax. Further, by generating TKO/p53(-/-) (QKO) cells, we demonstrated that p53, a tumor suppressor postulated to directly activate Bax, is not required for Bid/Bim/Puma-independent Bax/Bak activation. Together, these results strongly suggest that the direct activation activities of Bid (tBid), Bim, Puma, and p53 are not essential for activating Bax/Bak once the anti-apoptotic Bcl-2 proteins are neutralized.

  5. Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species

    Science.gov (United States)

    Subburaj, Yamunadevi; Cosentino, Katia; Axmann, Markus; Pedrueza-Villalmanzo, Esteban; Hermann, Eduard; Bleicken, Stephanie; Spatz, Joachim; García-Sáez, Ana J.

    2015-08-01

    Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore.

  6. HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis.

    Science.gov (United States)

    Deng, Lin; Chen, Ming; Tanaka, Motofumi; Ku, Yonson; Itoh, Tomoo; Shoji, Ikuo; Hotta, Hak

    2015-09-01

    We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.

  7. Bax phosphorylation association with nucleus and oligomerization after neonatal hypoxia-ischemia.

    Science.gov (United States)

    Infante, Smitha Krishna; Oberhauser, Andres F; Perez-Polo, J Regino

    2013-09-01

    Neonatal hypoxia-ischemia (HI) is a common occurrence in preterm and low-birth-weight infants, and the incidence of low-birth-weight and preterm births is increasing. Characterization of brain injury after HI is of critical importance in developing new treatments that more accurately target the injury. After severe HI, neuronal cells undergo necrosis and secondary apoptosis of the surrounding cells as a result of neuroinflammation. We sought to characterize the biochemical pathways associated with cell death after HI. Bax, a cell death signaling protein, is activated after HI and translocates to the nucleus, endoplasmic reticulum, and mitochondria. The translocation patterns of Bax affect the resultant cell death phenotype (necrotic or apoptotic) observed. Although Bax is known to oligomerize once it is activated, less is known about the factors that control its translocation and oligomerization. We hypothesize that Bax kinase-specific phosphorylation determines its oligomerization and intracellular localization. Using well-established in vivo and in vitro models of neonatal HI, we characterized Bax oligomerization and multiorganelle translocation. We found that HI-dependent phosphorylation of Bax determines its oligomerization status and multiorganelle localization, and, ultimately, the cell death phenotype observed. Understanding the mechanisms of Bax translocation will aid in the rational design of therapeutic strategies that decrease the trauma resulting from HI-associated inflammation.

  8. Insights into the structural stability of Bax from molecular dynamics simulations at high temperatures

    Science.gov (United States)

    Rosas-Trigueros, Jorge Luis; Correa-Basurto, José; Guadalupe Benítez-Cardoza, Claudia; Zamorano-Carrillo, Absalom

    2011-01-01

    Bax is a member of the Bcl-2 protein family that participates in mitochondrion-mediated apoptosis. In the early stages of the apoptotic pathway, this protein migrates from the cytosol to the outer mitochondrial membrane, where it is inserted and usually oligomerizes, making cytochrome c-compatible pores. Although several cellular and structural studies have been reported, a description of the stability of Bax at the molecular level remains elusive. This article reports molecular dynamics simulations of monomeric Bax at 300, 400, and 500 K, focusing on the most relevant structural changes and relating them to biological experimental results. Bax gradually loses its α-helices when it is submitted to high temperatures, yet it maintains its globular conformation. The resistance of Bax to adopt an extended conformation could be due to several interactions that were found to be responsible for maintaining the structural stability of this protein. Among these interactions, we found salt bridges, hydrophobic interactions, and hydrogen bonds. Remarkably, salt bridges were the most relevant to prevent the elongation of the structure. In addition, the analysis of our results suggests which conformational movements are implicated in the activation/oligomerization of Bax. This atomistic description might have important implications for understanding the functionality and stability of Bax in vitro as well as within the cellular environment. PMID:21936009

  9. Bax/bcl-2: cellular modulator of apoptosis in feline skin and basal cell tumours.

    Science.gov (United States)

    Madewell, B R; Gandour-Edwards, R; Edwards, B F; Matthews, K R; Griffey, S M

    2001-01-01

    Bcl-2 and bax are two members of the BCL-2 gene family that play a prominent role in the regulation of apoptosis. Bax and bcl-2 expression were examined immunohistochemically in normal (healthy) feline skin and in 24 benign feline cutaneous basal cell tumours. The tumours were also examined for cellular proliferation by measurement of reactivity for the proliferation marker Ki-67, and for apoptosis by in-situ labelling for fragmented DNA. Bcl-2 was detected in normal basal epithelium and in 23 of 24 basal cell tumours. Bax was detected in both basal and suprabasal epithelium, but in only seven of 24 tumours. For tumours that expressed both bax and bcl-2, the bax:bcl-2 ratio was low. Neither bax nor bcl-2 expression was detected in 14 feline cutaneous squamous cell carcinomas. Basal cell tumours showed modest cellular proliferation (median, 17.5% Ki-67- reactive cells), but few (less than 1%) apoptotic cells. The slow, indolent growth of feline cutaneous basal cells in these benign skin tumours may be a response, at least in part, to opposing regulatory expressions of bcl-2 and bax.

  10. Inhibition of Pro-Apoptotic BAX by a Noncanonical Interaction Mechanism

    Science.gov (United States)

    Barclay, Lauren A.; Wales, Thomas E.; Garner, Thomas P.; Wachter, Franziska; Lee, Susan; Guerra, Rachel M.; Stewart, Michelle L.; Braun, Craig R.; Bird, Gregory H.; Gavathiotis, Evripidis; Engen, John R.; Walensky, Loren D.

    2015-01-01

    SUMMARY BCL-2 is a negative regulator of apoptosis implicated in homeostatic and pathologic cell survival. The canonical anti-apoptotic mechanism involves entrapment of activated BAX by a groove on BCL-2, preventing BAX homo-oligomerization and mitochondrial membrane poration. The BCL-2 BH4 domain also confers anti-apoptotic functionality, but the mechanism is unknown. We find that a synthetic α-helical BH4 domain binds to BAX with nanomolar affinity and independently inhibits the conformational activation of BAX. Hydrogen-deuterium exchange mass spectrometry demonstrated that the N-terminal conformational changes in BAX induced by a triggering BIM BH3 helix were suppressed by the BCL-2 BH4 helix. Structural analyses localized the BH4 interaction site to a groove formed by residues of α1, α1–α2 loop, and α2–α3 and α5–α6 hairpins on the BAX surface. These data reveal a previously unappreciated binding site for targeted inhibition of BAX and suggest that the BCL-2 BH4 domain may participate in apoptosis blockade by a noncanonical interaction mechanism. PMID:25684204

  11. Identification and expression analysis of alternatively spliced new transcript isoform of Bax gene in mouse.

    Science.gov (United States)

    Husain, Mohammed Amir; Ishqi, Hassan Mubarak; Sarwar, Tarique; Rehman, Sayeed Ur; Tabish, Mohammad

    2017-07-20

    Bax, a pro-apoptotic member of Bcl-2 family regulates apoptosis through homodimerization/heterodimerization with Bcl-2. Bax-α is the only product of the Bax gene that has been extensively studied. Bax-α exists in inactive form and several conformational changes are required during apoptosis to activate it. Here, we have identified a novel transcript variant of Bax gene in mouse which contains alternatively spliced new first exon that is different from the first exon of previously reported transcript. Conceptual translation of new transcript encodes a protein (Bax-α1), having different N-terminus. The existence of the new transcript variant was confirmed by reverse transcriptase-PCR, semi-nested PCR using primers designed for the newly identified transcript variant. The identity of PCR product obtained after semi-nested PCR was confirmed by DNA sequencing. Relative expression of new transcript variant with respect to reported transcript was also studied with the help of real time PCR. The existence of new transcript variant was further supported by the presence of clusters of overlapping ESTs from the database. Bax-α1 possibly displays heterogeneous properties as predicted by post-translational modification analysis tools. The differences in post-translational modifications might play important roles in divergent function of the new isoform. The three dimensional structure was generated by homology modelling to visualize the differences at N termini of known and newly identified variant. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Bax Exists in a Dynamic Equilibrium between the Cytosol and Mitochondria to Control Apoptotic Priming

    Science.gov (United States)

    Schellenberg, Barbara; Wang, Pengbo; Keeble, James A.; Rodriguez-Enriquez, Ricardo; Walker, Scott; Owens, Thomas W.; Foster, Fiona; Tanianis-Hughes, Jolanta; Brennan, Keith; Streuli, Charles H.; Gilmore, Andrew P.

    2013-01-01

    Summary The proapoptotic Bcl-2 protein Bax is predominantly found in the cytosol of nonapoptotic cells and is commonly thought to translocate to mitochondria following an apoptotic stimulus. The current model for Bax activation is that BH3 proteins bind to cytosolic Bax, initiating mitochondrial targeting and outer-membrane permeabilization. Here, we challenge this and show that Bax is constitutively targeted to mitochondria but in nonapoptotic cells is constantly translocated back to the cytosol. Using live-cell spinning-disk confocal imaging with a combination of FLIP, FRAP, and photoactivatable GFP-Bax, we demonstrate that disrupting adhesion-dependent survival signals slows the rate of Bax’s dissociation from mitochondria, leading to its accumulation on the outer mitochondrial membrane. The overall accumulation of mitochondrial Bax following loss of survival signaling sensitizes cells to proapoptotic BH3 proteins. Our findings show that Bax is normally in a dynamic equilibrium between cytosol and mitochondria, enabling fluctuations in survival signals to finely adjust apoptotic sensitivity. PMID:23375500

  13. Bax-PGAM5L-Drp1 complex is required for intrinsic apoptosis execution.

    Science.gov (United States)

    Xu, Wenjuan; Jing, Linlin; Wang, Quanshi; Lin, Chung-Chih; Chen, Xiaoting; Diao, Jianxin; Liu, Yuanliang; Sun, Xuegang

    2015-10-06

    Intrinsic apoptosis eliminates cells with damaged DNA and cells with dysregulated expression of oncogene. PGAM5, a member of the phosphoglycerate mutase family, has two splicing variants: PGAM5L (the long form) and PGAM5S (the short form). It has been well established that PGAM5 is at the convergent point of multiple necrosis pathways. However, the role of PGAM5 in intrinsic apoptosis is still controversial. Here we report that the PGAM5L, but not PGAM5S is a prerequisite for the activation of Bax and dephosphorylation of Drp1 in arenobufagin and staurosporine induced intrinsic apoptosis. Knockdown of PGAM5L inhibits the translocation of Bax to the mitochondria and reduces mitochondrial fission. The interaction between PGAM5L and Drp1 was observed in both arenobufagin and staurosporine treated HCT116 cells, but not in HCT116 Bax(-/-) cells. Bax transfection rescues the formation of the triplex in both arenobufagin and staurosporine stimulated HCT116 Bax(-/-) cells. Arenobufagin shows remarkable anti-cancer effects both in orthotropic and heterotropic CRC models and demonstrates less toxic effects as compared with that of cisplatin. Bax-PGAM5L-Drp1 complex is detected in arenobufagin and staurosporine treated CRC cells in vitro and in arenobufagin and cisplatin treated tumor in vivo as well. In summary, our results demonstrate that Bax-PGAM5L-Drp1 complex is required for intrinsic apoptosis execution.

  14. Pro-apoptotic cBid and Bax exhibit distinct membrane remodeling activities: An AFM study.

    Science.gov (United States)

    Unsay, Joseph D; Cosentino, Katia; Sporbeck, Katharina; García-Sáez, Ana J

    2017-01-01

    Bcl-2 proteins are key regulators of the mitochondrial outer membrane (MOM) permeabilization that mediates apoptosis. During apoptosis, Bid is cleaved (cBid) and translocates to the MOM, where it activates Bax. Bax then oligomerizes and induces MOM permeabilization. However, little is known about how these proteins affect membrane organization aside from pore formation. In previous studies, we have shown that both cBid and Bax are able to remodel membranes and stabilize curvature. Here, we dissected the independent effects of Bax and cBid on supported lipid structures mimicking the mitochondrial composition by means of atomic force spectroscopy. We show that cBid did not permeabilize the membrane but lowered the membrane breakthrough force. On the other hand, Bax effects were dependent on its oligomeric state. Monomeric Bax did not affect the membrane properties. In contrast, oligomeric Bax lowered the breakthrough force of the membrane, which in the context of pore formation, implies a lowering of the line tension at the edge of the pore. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Deacetylation of Ku70 by SIRT6 attenuates Bax-mediated apoptosis in hepatocellular carcinoma.

    Science.gov (United States)

    Tao, Na-Na; Ren, Ji-Hua; Tang, Hua; Ran, Long-Kuan; Zhou, Hong-Zhong; Liu, Bo; Huang, Ai-Long; Chen, Juan

    2017-04-15

    SIRT6 is a class III histone deacetylase that has been implicated in HCC development. We previously reported that SIRT6 potentiated apoptosis evasion in hepatocellular carcinoma by inhibiting both Bax expression and mitochondrial translocalization. However, the mechanism underlying SIRT6-mediated inhibition of Bax mitochondrial localization remains elusive. In this study, we found that although SIRT6 had no effect on the expression level of Ku70, SIRT6 could interact with Ku70 and deacetylate it. The increased acetylation of Ku70 in SIRT6-depleted cells disrupt its interaction with Bax, which finally resulted in Bax mitochondrial translocalization. Furthermore, lysine K542 on Ku70 was the target for deacetylation by SIRT6. Ku70(K542Q) mutation abolished suppression of association between Ku70 and Bax and caused redistribution of Bax to the cytosol in SIRT6-depleted cells. Finally, Ku70(K542Q) mutation could reversed the inhibition of growth and apoptosis promotion mediated by SIRT6 silencing. Together, our findings revealed SIRT6 could block the mitochondrial translocation of Bax and decrease the apoptotic ratio of HCC cells by deacetylation of Ku70. SIRT6 may serve as a promising target for developing targeted therapies for HCC in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    XU MaoJun; DONG JuFang

    2007-01-01

    Bax, a mammalian pro-apoptotic member of the Bcl-2 family, triggers hypersensitive reactions when expressed in plants. To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells, we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter. The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied. Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0- and 5.5-fold of the control cells. Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str, two key genes in terpenoid indole alkaloid biosynthetic pathway of Catharanthus roseus cells, and stimulates the accumulation of defense-related protein PR1 in the cells, showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway. Thus, our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

  17. Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bax,a mammalian pro-apoptotic member of the Bcl-2 family,triggers hypersensitive reactions when expressed in plants.To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells,we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter.The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied.Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0-and 5.5-fold of the control cells.Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str,two key genes in terpenoid indole alkaloid bio-synthetic pathway of Catharanthus roseus cells,and stimulates the accumulation of defense-related protein PR1 in the cells,showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway.Thus,our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

  18. A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Claudia Fabiola Méndez-Catalá

    2013-08-01

    Full Text Available We previously reported the association of elevated levels of the multifunctional transcription factor, CCCTC binding factor (CTCF, in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc was generally increased in non-breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.

  19. Isatin decreases Bax protein expression in the substantia nigra of a mouse model of Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    Jiguo Zhang; Fang Zhang; Yanlong Qiu; Wang Yue

    2011-01-01

    The present study observed the action of 1H-indole-2, 3-dione (isatin) on Bax protein expression in the substantia nigra of a Parkinson's disease animal model. Parkinson's disease-like behaviors were induced in C57BL/6J mice treated with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Bax protein expression was significantly reduced in isatin (100, 200 mg/kg)-pretreated mice. Results demonstrate that isatin plays a neuroprotective role in mice treated with MPTP by down-regulating Bax protein expression.

  20. Bcl-2/Bax protein and mRNA expression in yak (Bos grunniens) placentomes.

    Science.gov (United States)

    Fan, JiangFeng; Yu, SiJiu; Cui, Yan; Xu, Gengquan; Wang, Libin; Pan, Yangyang; He, Honghong

    2017-07-29

    Placental function is complex and influenced by various factors; furthermore, it depends on a delicate balance between cell proliferation, cell differentiation, and cell death. Bcl-2 and Bax proteins are key apoptosis regulators and are considered to play an important role in the maintenance of both dynamic balance and integrity of many tissues. Changes in Bcl-2 and Bax expressions have been described during different developmental stages in normal human placentas. Studies furthermore indicated several pathological placental changes to be related to abnormal Bcl-2 and Bax expressions. In the present study, we investigated both expression and distribution of Bcl-2 and Bax in yak placentas. For this, we collected placentas of 35 yaks at different stages of pregnancy as well as cotyledonary villi of four postpartum yaks. Protein and mRNA expressions of both Bcl-2 and Bax were investigated via immunohistochemistry, Western blot, and real-time PCR. Immunoreactive Bcl-2 protein was mainly localized near the fetal villous trophoblast at various gestational stages and post-partum. The Positive Index (PI) of Bcl-2 protein expression significantly decreased with increasing gestational age. Early during pregnancy (≤2 months), the Bax protein was widely distributed in the fetal villous trophoblast layer, the maternal caruncular crypt epithelium, and the stroma. Subsequently, the Bax protein distribution gradually concentrated in the fetal villous trophoblast layer. The staining intensity of Bax increased from the 3rd month to the prepartum of gestation. The PI reached a minimum of 9.4 ± 2.2 in fetal chorionic villi (FCV) and 1.3 ± 0.8 in maternal caruncular crypts (MCC) of the three months group. Both Bcl-2 and Bax had maximum immunoreactivity in the fetal villous trophoblast layer of placentas collected form postpartum yaks (with PIs of 36.6 ± 5.7 and 38.2 ± 4.8, respectively). Protein and mRNA expression of Bcl-2 and Bax investigated via Western blot and real

  1. Expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins in human retinoblastoma.

    Science.gov (United States)

    Singh, Lata; Pushker, Neelam; Saini, Neeru; Sen, Seema; Sharma, Anjana; Bakhshi, Sameer; Chawla, Bhavna; Kashyap, Seema

    2015-04-01

    Regulation of apoptosis is a complex process that involves a number of genes, including Bcl-2, Bcl-x, Bax and other Bcl-2 family members. The aim of the present study is to assess the expression of Bcl- 2 and Bax in retinoblastoma, and correlate them with clinical and histopathological parameters. The expression of Bcl-2 and Bax proteins were examined using immunohistochemistry, Western blotting and reverse transcriptase-polymerase chain reaction in a series of 60 prospective cases of primary retinoblastoma tissues. Immunohistochemistry showed expression of Bcl-2 in 40/60 (66.6%), whereas Bax expression was found only in 18/60 (30%) cases, and these correlated with mRNA expression. The Western blotting results also correlated well with the immunohistochemical expression of Bcl-2 (25 kDa) and Bax (21 kDa) proteins. Bcl-2 was expressed in 96% (24/25) of invasive tumours and in 45.7% (16/35) of non-invasive tumours. Expression of Bcl-2 significantly correlated with tumour invasiveness (P = 0.0274) and poor differentiation (P = 0.0163), whereas loss of Bax correlated with massive choroidal invasion and Pathological Tumor-Node-Metastasis (pTNM) (P = 0.0341). However, no correlation was found between Bax and Bcl-2 expression. Our findings suggest that these apoptotic regulatory proteins may serve as poor prognostic markers and can be used as a therapeutic target for the treatment of invasive retinoblastoma. Further functional studies are required to explore the role of Bax and Bcl-2 in retinoblastoma. © 2014 Royal Australian and New Zealand College of Ophthalmologists.

  2. Driving p53 Response to Bax Activation Greatly Enhances Sensitivity to Taxol by Inducing Massive Apoptosis

    Directory of Open Access Journals (Sweden)

    Paola De Feudis

    2000-05-01

    Full Text Available The proapoptotic gene bax is one of the downstream effectors of p53. The p53 binding site in the bax promoter is less responsive to p53 than the one in the growth arrest mediating gene p21. We introduced the bax gene under the control of 13 copies of a strong p53 responsive element into two ovarian cancer cell lines. The clones expressing bax under the control of p53 obtained from the wild-type (wt p53-expressing cell line A2780 were much more sensitive (500- to 1000-fold to the anticancer agent taxol than the parent cell line, with a higher percentage of cells undergoing apoptosis after drug treatment that was clearly p53-dependent and bax-mediated. Xenografts established in nude mice from one selected clone (A2780/C3 were more responsive to taxol than the parental line and the apoptotic response of A2780/C3 tumors was also increased after treatment. Introduction of the same plasmid into the p53 null SKOV3 cell line did not alter the sensitivity to taxol or the induction of apoptosis. In conclusion, driving the p53 response (after taxol treatment by activating the bax gene rather than the p21 gene results in induction of massive apoptosis, in vitro and in vivo, and greatly enhances sensitivity to the drug.

  3. Interaction of caveolin-1 with Ku70 inhibits Bax-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Huafei Zou

    Full Text Available Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101 to the caveolin-binding domain (CBD of Ku70 (amino acids 471-478. Cell culture data show that caveolin-1 binds Ku70 after treatment with chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug-induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 Φ→A, limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death.

  4. THE EXPRESSION AND CLINICAL VALUE OF APOPTOSIS CONTROL GENE Bcl-2 AND Bax IN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jun; YAO Zhen-xiang; ZHANG Jing

    1999-01-01

    Objective: To study the expression and clinical value of apoptosis control gene bcl-2 and bax in breast cancer.Methods: Protein bax and bcl-2 in 41 breast cancers obtained from operations in our hospital in 1996 were detected using ABC immunohistochemical stain assay and compared with 10 cases with normal breast tissues.Results: The positive rate of bax in normal breast tissue was 90% and in breast cancer was 59%, with a significant statistical difference between them (P<0.05), but there was no statistical difference in bcl-2 protein expression. Among the 41 breast cancer, the group with lymph node metastasis (21 cases) had obviously low bax expression (43%) and high bcl-2 expression (76%), showing significant difference to the group without lymph node metastasis (P<0.05).Conclusion: The antiapoptosis function of bcl-2 was stronger than bax in breast cancer. Protein bax and bcl-2 assay may be useful in understanding the biological behaviors of breast cancer.

  5. A New Fungal Diterpene Induces VDAC1-dependent Apoptosis in Bax/Bak-deficient Cells.

    Science.gov (United States)

    Huang, Li; Han, Junjie; Ben-Hail, Danya; He, Luwei; Li, Baowei; Chen, Ziheng; Wang, Yueying; Yang, Yanlei; Liu, Lei; Zhu, Yushan; Shoshan-Barmatz, Varda; Liu, Hongwei; Chen, Quan

    2015-09-25

    The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release. Diphenylamine-2-carboxilic acid, an inhibitor of VDAC1 conductance and oligomerization, inhibited cyathin-R-induced VDAC1 oligomerization and apoptosis. Similarly, Bcl-2 overexpression conferred resistance to cyathin-R-induced apoptosis and VDAC1 oligomerization. Silencing of VDAC1 expression prevented cyathin-R-induced apoptosis. Finally, cyathin-R effectively attenuated tumor growth and induced apoptosis in Bax/Bak-deficient cells implanted into a xenograft mouse model. Hence, this study identified a new compound promoting VDAC1-dependent apoptosis as a potential therapeutic option for cancerous cells lacking or presenting inactivated Bax/Bak.

  6. A New Fungal Diterpene Induces VDAC1-dependent Apoptosis in Bax/Bak-deficient Cells*

    Science.gov (United States)

    Huang, Li; Han, Junjie; Ben-Hail, Danya; He, Luwei; Li, Baowei; Chen, Ziheng; Wang, Yueying; Yang, Yanlei; Liu, Lei; Zhu, Yushan; Shoshan-Barmatz, Varda; Liu, Hongwei; Chen, Quan

    2015-01-01

    The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release. Diphenylamine-2-carboxilic acid, an inhibitor of VDAC1 conductance and oligomerization, inhibited cyathin-R-induced VDAC1 oligomerization and apoptosis. Similarly, Bcl-2 overexpression conferred resistance to cyathin-R-induced apoptosis and VDAC1 oligomerization. Silencing of VDAC1 expression prevented cyathin-R-induced apoptosis. Finally, cyathin-R effectively attenuated tumor growth and induced apoptosis in Bax/Bak-deficient cells implanted into a xenograft mouse model. Hence, this study identified a new compound promoting VDAC1-dependent apoptosis as a potential therapeutic option for cancerous cells lacking or presenting inactivated Bax/Bak. PMID:26253170

  7. Dynamin-related protein Drp1 is required for Bax translocation to mitochondria in response to irradiation-induced apoptosis.

    Science.gov (United States)

    Wang, Ping; Wang, Peiguo; Liu, Becky; Zhao, Jing; Pang, Qingsong; Agrawal, Samir G; Jia, Li; Liu, Feng-Ting

    2015-09-08

    Translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondria is a crucial step in DNA damage-mediated apoptosis, and is also found to be involved in mitochondrial fragmentation. Irradiation-induced cytochrome c release and apoptosis was associated with Bax activation, but not mitochondrial fragmentation. Both Bax and Drp1 translocated from the cytosol to the mitochondria in response to irradiation. However, Drp1 mitochondrial translocation and oligomerization did not require Bax, and failed to induce apoptosis in Bax deficient diffuse large B-cell lymphoma (DLBCL) cells. Using fluorescent microscopy and the intensity correlation analysis, we demonstrated that Bax and Drp1 were colocalized and the levels of colocalization were increased by UV irradiation. Using co-immuno-precipitation, we confirmed that Bax and Drp1 were binding partners. Irradiation induced a time-associated increase in the interaction between active Bax and Drp1. Knocking down Drp1 using siRNA blocked UV irradiation-mediated Bax mitochondrial translocation. In conclusion, our findings demonstrate for the first time, that Drp1 is required for Bax mitochondrial translocation, but Drp1-induced mitochondrial fragmentation alone is not sufficient to induce apoptosis in DLBCL cells.

  8. Bcl-xS and Bax induce different apoptotic pathways in PC12 cells.

    Science.gov (United States)

    Lindenboim, L; Yuan, J; Stein, R

    2000-03-30

    Apoptosis is regulated by the action of the Bcl-2 family of proteins, which includes anti- and pro-apoptotic members such as Bcl-xS and Bax. These proteins may differ from each other in structure, mechanism of action and interactions with anti-apoptotic signaling. The mechanism whereby Bax induces cell death has been studied in some cellular systems, but the mechanism of Bcl-xS-induced apoptosis is largely unknown. In this study we investigated and compared the apoptotic effects of Bcl-xS and Bax in the pheochromocytoma cell line, PC12 (a useful model system for studying neuronal apoptosis), and the extent to which they are protected by the survival factor, nerve growth factor (NGF). PC12 cells express endogenous Bcl-xS, Bax and Bcl-xL proteins. Subcellular fractionation revealed that Bax is presented mainly in the cytosolic and the heavy membrane fractions, Bcl-xS is present only in the cytosol, and the anti-apoptotic protein Bcl-xL is located mainly in the heavy membrane fraction. In contrast to the cytosolic localization of endogenous Bcl-xS, the exogenously overexpressed Bcl-xS is localized to the mitochondria. Overexpression of Bcl-xS or Bax induces cell death in the transfected cells. The cell death induced by overexpression of Bcl-xS was inhibited by coexpression of Bcl-xS with Bcl-2 or Bcl-xL, or by treatment with the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone (Z-VAD-FMK) or with NGF. The Bcl-2 mutants deltaC22, which lacks the transmembrane domain, and G145A (mI-3) were able to inhibit the death-inducing effect of Bcl-xS. These results therefore suggest that the apoptotic pathway induced by overexpression of Bcl-xS in PC12 cells can be controlled by Bcl-2 and Bcl-xL, is mediated by caspases, and can be inhibited by the NGF signaling pathway. The Bax-induced cell death was inhibited by co-expression of Bax with Bcl-2 or Bcl-xL, but was not inhibited by Z-VAD-FMK, NGF, or the Bcl-2 ml-3 or deltaC22 mutants. These

  9. BAX insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and SH-redox regulation

    Science.gov (United States)

    Brustovetsky, Tatiana; Li, Tsyregma; Yang, Youyun; Zhang, Jiang-Ting; Antonsson, Bruno; Brustovetsky, Nickolay

    2010-01-01

    BAX cooperates with truncated BID (tBID) and Ca2+ in permeabilizing the outer mitochondrial membrane (OMM) and releasing mitochondrial apoptogenic proteins. The mechanisms of this cooperation are still unclear. Here we show that in isolated brain mitochondria, recombinant BAX readily self-integrates/oligomerizes in the OMM but produces only a minuscule release of cytochrome c, indicating that BAX insertion/oligomerization in the OMM does not always lead to massive OMM permeabilization. Ca2+ in a mitochondrial permeability transition (mPT)-dependent and recombinant tBID in an mPT-independent manner promoted BAX insertion/oligomerization in the OMM and augmented cytochrome c release. Neither tBID nor Ca2+ induced BAX oligomerization in the solution without mitochondria, suggesting that BAX oligomerization required interaction with the organelles and followed rather than preceded BAX insertion in the OMM. Recombinant Bcl-xL failed to prevent BAX insertion/oligomerization in the OMM but strongly attenuated cytochrome c release. On the other hand, a reducing agent, dithiothreitol (DTT), inhibited BAX insertion/oligomerization augmented by tBID or Ca2+ and suppressed the BAX-mediated release of cytochrome c and Smac/DIABLO but failed to inhibit Ca2+-induced swelling. Altogether, these data suggest that in brain mitochondria, BAX insertion/oligomerization can be dissociated from OMM permeabilization and that tBID and Ca2+ stimulate BAX insertion/oligomerization and BAX-mediated OMM permeabilization by different mechanisms involving mPT induction and modulation of the SH-redox state. PMID:20655869

  10. Clinical significance of bax/bcl-2 ratio in chronic lymphocytic leukemia.

    Science.gov (United States)

    Del Principe, Maria Ilaria; Dal Bo, Michele; Bittolo, Tamara; Buccisano, Francesco; Rossi, Francesca Maria; Zucchetto, Antonella; Rossi, Davide; Bomben, Riccardo; Maurillo, Luca; Cefalo, Mariagiovanna; De Santis, Giovanna; Venditti, Adriano; Gaidano, Gianluca; Amadori, Sergio; de Fabritiis, Paolo; Gattei, Valter; Del Poeta, Giovanni

    2016-01-01

    In chronic lymphocytic leukemia the balance between the pro-apoptotic and anti-apoptotic members of the bcl-2 family is involved in the pathogenesis, chemorefractoriness and clinical outcome. Moreover, the recently proposed anti-bcl-2 molecules, such as ABT-199, have emphasized the potential role of of bcl-2 family proteins in the context of target therapies. We investigated bax/bcl-2 ratio by flow cytometry in 502 patients and identified a cut off of 1.50 to correlate bax/bcl-2 ratio with well-established clinical and biological prognosticators. Bax/bcl-2 was 1.50 or over in 263 patients (52%) with chronic lymphocytic leukemia. Higher bax/bcl-2 was associated with low Rai stage, lymphocyte doubling time over 12 months, beta-2 microglobulin less than 2.2 mg/dL, soluble CD23 less than 70 U/mL and a low risk cytogenetic profile (Pbax/bcl-2 was correlated with unmutated IGHV (Pbax/bcl-2 (Pbax/bcl-2 identified cases with significant longer PFS (P=0.00002 and P=0.039). In multivariate analysis of progression-free survival and overall survival, bax/bcl-2 was an independent prognostic factor (P=0.0002 and P=0.002). In conclusion, we defined the prognostic power of bax/bcl-2 ratio, as determined by a flow cytometric approach, and highlighted a correlation with chemoresistance and outcome in chronic lymphocytic leukemia. Finally, the recently proposed new therapies employing bcl-2 inhibitors prompted the potential use of bax/bcl-2 ratio to identify patients putatively resistant to these molecules. Copyright© Ferrata Storti Foundation.

  11. Doxorubicin Changes Bax /Bcl-xL Ratio, Caspase-8 and 9 in Breast Cancer Cells.

    Science.gov (United States)

    Sharifi, Simin; Barar, Jaleh; Hejazi, Mohammad Saeid; Samadi, Nasser

    2015-09-01

    Doxorubicin is administrated as a single agent in first-line therapy of breast cancer to induce apoptosis in tumor cells. Bax, Bcl-xL, Caspase-8 and 9 proteins are involved in induction of apoptosis. The present study describes Bax, Bcl-xL gene expression and Caspase-8 and 9 protein levels in MCF-7 cells incubated with doxorubicin at different doses an incubation times. The cytotoxic effects of doxorubicin were studied using MTT assay. MCF-7 cells were treated with three concentrations of doxorubicin (0.1, 0.5, 1 μM) and incubated for 24, 48 and 72 hours then expression levels of Bax and Bcl-xL genes were elucidated by Real-time RT-PCR technique and protein levels of caspase-8 and caspase-9 proteins were measured using ELISA method. Morphological modifications of the cells were also monitored via light microscopic images. Doxorubicin decreased the anti-apoptotic Bcl-xL and increased pro-apoptotic Bax mRNA levels. Doxorubicin induced a significant increase in Bax /Bcl-xL ratio in all doses and incubation times (pBax /Bcl-xL ratio was revealed after 48 h incubation of the cells with in all doses of doxorubicin. Doxorubicin also increased caspase-9 level in a time and dose-dependent manner, while caspase-8 level didn't follow time and dose dependency pattern. Our results confirm that doxorubicin induces mitochondrial-dependent apoptosis by down-regulation of Bcl-xL and up- regulation of Bax and caspase-9 expressions.

  12. Effect of β Radiation on Bcl-2 and Bax Expressions in Benign Prostate Hyperplasia Tissues

    Institute of Scientific and Technical Information of China (English)

    MA Qing-jie; GAO Shi; ZHAO Jie; GU Xin-quan; CAI Shan-yu; ZHAO Guo-qing

    2008-01-01

    The authors chose specimens from nine normal prostate tissues(NP group),15 benign prostate hyperplasia(BPH) prostates(BPH group),and 35 BPH prostates that had been treated with 90Sr/90Y Prostatic Hyperplasia Applicator(exposure group),The expressions of bcl-2 and bax in stroma and epithelia of prostate tissues were demonstrated by means of immunohistochemical staining,and the staining positive rate was semiquantatively determined,so as to observe the expression of bcl-2 and bax genes in the prostate tissues of normal individuals and BPH patients,before and after β radiation,and to evaluate the influence of β radiation on bcl-2 and bax expressions,The expressions of gene bcl-2 in the prostate epithelia of NP and BPH are significantly higher than those in the prostate stroma(P<0.01),However,the expressions of bcl-2 in the prostate epithelia and stroma of the BPH group are obviously higher than those in the NP group(P<0.01),The expression of gene bax in the prostate epithelia of the NP group is higher than that in the BPH group(P<0.05),However,bcl-2 expressions in the prostate epithelia and stroma of the BPH group are significantly higher than the bax expressions(P<0.01),Compared with those of the NP group,the expressions of bcl-2 in the prostate epithelia and stroma of the exposure group decrease remarkably,even as the expressions of the bax notably increase(P<0.01),Thus,the administration of β radiation can remarkably affect bcl-2 and bax gene expressions,to regulate cell apoptosis,in the prostate tissues of BPH.

  13. Prostaglandin E2 reduces radiation-induced epithelial apoptosis through a mechanism involving AKT activation and bax translocation.

    Science.gov (United States)

    Tessner, Teresa G; Muhale, Filipe; Riehl, Terrence E; Anant, Shrikant; Stenson, William F

    2004-12-01

    Prostaglandin E2 (PGE2) synthesis modulates the response to radiation injury in the mouse intestinal epithelium through effects on crypt survival and apoptosis; however, the downstream signaling events have not been elucidated. WT mice receiving 16,16-dimethyl PGE2 (dmPGE2) had fewer apoptotic cells per crypt than untreated mice. Apoptosis in Bax(-/-) mice receiving 12 Gy was approximately 50% less than in WT mice, and the ability of dmPGE2 to attenuate apoptosis was lost in Bax(-/-) mice. Positional analysis revealed that apoptosis in the Bax(-/-) mice was diminished only in the bax-expressing cells of the lower crypts and that in WT mice, dmPGE2 decreased apoptosis only in the bax-expressing cells. The HCT-116 intestinal cell line and Bax(-/-) HCT-116 recapitulated the apoptotic response of the mouse small intestine with regard to irradiation and dmPGE2. Irradiation of HCT-116 cells resulted in phosphorylation of AKT that was enhanced by dmPGE2 through transactivation of the EGFR. Inhibition of AKT phosphorylation prevented the reduction of apoptosis by dmPGE2 following radiation. Transfection of HCT-116 cells with a constitutively active AKT reduced apoptosis in irradiated cells to the same extent as in nontransfected cells treated with dmPGE2. Treatment with dmPGE2 did not alter bax or bcl-x expression but suppressed bax translocation to the mitochondrial membrane. Our in vivo studies indicate that there are bax-dependent and bax-independent radiation-induced apoptosis in the intestine but that only the bax-dependent apoptosis is reduced by dmPGE2. The in vitro studies indicate that dmPGE2, most likely by signaling through the E prostaglandin receptor EP2, reduces radiation-induced apoptosis through transactivation of the EGFR and enhanced activation of AKT and that this results in reduced bax translocation to the mitochondria.

  14. Identification of Bax-Interacting Proteins in Oligodendrocyte Progenitors during Glutamate Excitotoxicity and Perinatal Hypoxia–Ischemia

    Directory of Open Access Journals (Sweden)

    Sopio Simonishvili

    2013-11-01

    Full Text Available OPC (oligodendrocyte progenitor cell death contributes significantly to the pathology and functional deficits following hypoxic-ischemic injury in the immature brain and to deficits resulting from demyelinating diseases, trauma and degenerative disorders in the adult CNS. Glutamate toxicity is a major cause of oligodendroglial death in diverse CNS disorders, and previous studies have demonstrated that AMPA/kainate receptors require the pro-apoptotic protein Bax in OPCs undergoing apoptosis. The goal of the present study was to define the pro-apoptotic and anti-apoptotic effectors that regulate Bax in healthy OPCs and after exposure to excess glutamate in vitro and following H–I (hypoxia–ischemia in the immature rat brain. We show that Bax associates with a truncated form of Bid, a BH3-only domain protein, subsequent to glutamate treatment. Furthermore, glutamate exposure reduces Bax association with the anti-apoptotic Bcl family member, Bcl-xL. Cell fractionation studies demonstrated that both Bax and Bid translocate from the cytoplasm to mitochondria during the early stages of cell death consistent with a role for Bid as an activator, whereas Bcl-xL, which normally complexes with both Bax and Bid, disassociates from these complexes when OPCs are exposed to excess glutamate. Bax remained unactivated in the presence of insulin-like growth factor-1, and the Bcl-xL complexes were protected. Our data similarly demonstrate loss of Bcl-xL–Bax association in white matter following H–I and implicate active Bad in Bax-mediated OPC death. To identify other Bax-binding partners, we used proteomics and identified cofilin as a Bax-associated protein in OPCs. Cofilin and Bax associated in healthy OPCs, whereas the Bax–cofilin association was disrupted during glutamate-induced OPC apoptosis.

  15. Identification of Bax-interacting proteins in oligodendrocyte progenitors during glutamate excitotoxicity and perinatal hypoxia–ischemia

    Directory of Open Access Journals (Sweden)

    Sopio Simonishvili

    2013-12-01

    Full Text Available OPC (oligodendrocyte progenitor cell death contributes significantly to the pathology and functional deficits following hypoxic-ischemic injury in the immature brain and to deficits resulting from demyelinating diseases, trauma and degenerative disorders in the adult CNS. Glutamate toxicity is a major cause of oligodendroglial death in diverse CNS disorders, and previous studies have demonstrated that AMPA/kainate receptors require the pro-apoptotic protein Bax in OPCs undergoing apoptosis. The goal of the present study was to define the pro-apoptotic and anti-apoptotic effectors that regulate Bax in healthy OPCs and after exposure to excess glutamate in vitro and following H–I (hypoxia–ischemia in the immature rat brain. We show that Bax associates with a truncated form of Bid, a BH3-only domain protein, subsequent to glutamate treatment. Furthermore, glutamate exposure reduces Bax association with the anti-apoptotic Bcl family member, Bcl-xL. Cell fractionation studies demonstrated that both Bax and Bid translocate from the cytoplasm to mitochondria during the early stages of cell death consistent with a role for Bid as an activator, whereas Bcl-xL, which normally complexes with both Bax and Bid, disassociates from these complexes when OPCs are exposed to excess glutamate. Bax remained unactivated in the presence of insulin-like growth factor-1, and the Bcl-xL complexes were protected. Our data similarly demonstrate loss of Bcl-xL–Bax association in white matter following H–I and implicate active Bad in Bax-mediated OPC death. To identify other Bax-binding partners, we used proteomics and identified cofilin as a Bax-associated protein in OPCs. Cofilin and Bax associated in healthy OPCs, whereas the Bax–cofilin association was disrupted during glutamate-induced OPC apoptosis.

  16. BAX OVEREXPRESSION ENHANCES APOPTOSIS INDUCED BY ADRIAMYCIN IN HCC-9204 CELLS

    Institute of Scientific and Technical Information of China (English)

    郑建勇; 李江; 李开宗; 王文亮; 王为忠

    2004-01-01

    Objective: To investigate the effect of overexpression of Bax to the sensitivity of human HCC-9204 cells to adriamycin (ADR). Methods: Human cultured hepatocellular carcinoma cell line HCC-9204 was exposed in vitro to adriamycin for various time. An inducible vector containing Bax gene, with ZnSO4 as external inducer was constructed. Cell apoptosis was ascertained by morphological criteria, detection of apoptotic DNA fragmentation by TUNEL assay and flow cytometry. Tetrazolium blue (MTT) assay was used to evaluate the differences in drug sensitivity of HCC-9204 cells after Bax-transfection. Results: HCC-9204 cells treated with adriamycin at 20μmol/L showed extensive cell death. TUNEL assay showed nucleus fragmentation. And apoptotic peak was also shown by flow cytometry. FACS analyses showed a significant sub-G1 peak and apoptosis in 31% cells at 24h after treatment. Furthermore, the time-course of cell viability following exposure of HCC-9204/Bax cells to adriamycin showed that Bax was able to significantly decrease cell survival following exposure to adriamycin.

  17. Immunohistochemical localization of Bcl-2 and Bax proteins in in situ and invasive duct breast carcinomas.

    Science.gov (United States)

    Kapucuoglu, N; Losi, L; Eusebi, V

    1997-01-01

    Bcl-2 and Bax proteins are coded by a family of genes that take part in the manteinance of the balance between cell proliferation rate and programmed cell death in multicellular organisms. The Bax gene acts as promoter of cell death by opposing the death protector effect of the Bcl-2 gene. Expression of the Bcl-2 and Bax proteins has been investigated in 58 cases of duct carcinoma in situ (DCIS) and duct invasive and invasive lobular carcinomas (IC) of the breast. While both proteins were expressed at the same time in normal and benign epithelium, different staining patterns were observed according to the degree of differentiation of the neoplastic epithelium. In well-differentiated DCIS and grade I IC there was a predominance of Bcl-2 protein staining. Grade II lesions co-expressed both proteins. Poorly differentiated DCIS displayed a predominantly Bax protein staining pattern. Therefore, it appears that Bax protein expression, especially in DCIS, relates to more aggressive neoplasms while Bcl-2 protein expression is associated with less aggressive malignant lesions.

  18. Effects of cadmium on Bcl-2/ Bax expression ratio in rat cortex brain and hippocampus.

    Science.gov (United States)

    Mahdavi, S; Khodarahmi, P; Roodbari, N H

    2017-01-01

    To investigate the underlying mechanism of neurotoxicity of cadmium, we examined the effects of intraperitoneal injection of cadmium on messenger RNA (mRNA) expression of Bcl-2 (B-cell lymphoma 2) and Bax (Bcl2-associated x) genes and caspase-3/7 activation in rat hippocampus and frontal cortex. Twenty-eight male Wistar rats weighing 200-250 g were randomly divided into four groups. Control group received saline and three other groups received cadmium at doses of 1, 2 and 4 mg/kg (body weight) for 15 successive days. One day after the last injection, the hippocampus and frontal cortex were dissected and removed and then the expression of Bcl-2 and Bax genes was evaluated using real-time polymerase chain reaction and apoptotic studies was done using caspase-3/7 activation assay. Cadmium reduced the mRNA level of Bcl-2 in the control group at doses of 1 ( p Bax increased significantly compared to the control group at the doses of 1 ( p Bax was increased significantly compared to the control group at the doses of 2 and 4 mg/kg ( p Bax mRNA ratio induces cell apoptosis. Apoptotic effect of cadmium may be through the mitochondrial pathway by the activation of caspase-3/7.

  19. Bax Activation Blocks Self-Renewal and Induces Apoptosis of Human Glioblastoma Stem Cells.

    Science.gov (United States)

    Daniele, Simona; Pietrobono, Deborah; Costa, Barbara; Giustiniano, Mariateresa; La Pietra, Valeria; Giacomelli, Chiara; La Regina, Giuseppe; Silvestri, Romano; Taliani, Sabrina; Trincavelli, Maria Letizia; Da Settimo, Federico; Novellino, Ettore; Martini, Claudia; Marinelli, Luciana

    2017-04-11

    Glioblastoma (GBM) is characterized by a poor response to conventional chemotherapeutic agents, attributed to the insurgence of drug resistance mechanisms and to the presence of a subpopulation of glioma stem cells (GSCs). GBM cells and GSCs present, among others, an overexpression of antiapoptotic proteins and an inhibition of pro-apoptotic ones, which help to escape apoptosis. Among pro-apoptotic inducers, the Bcl-2 family protein Bax has recently emerged as a promising new target in cancer therapy along with first BAX activators (BAM7, Compound 106, and SMBA1). Herein, a derivative of BAM-7, named BTC-8, was employed to explore the effects of Bax activation in different human GBM cells and in their stem cell subpopulation. BTC-8 inhibited GBM cell proliferation, arrested the cell cycle, and induced apoptosis through the induction of mitochondrial membrane permeabilization. Most importantly, BTC-8 blocked proliferation and self-renewal of GSCs and induced their apoptosis. Notably, BTC-8 was demonstrated to sensitize both GBM cells and GSCs to the alkylating agent Temozolomide. Overall, our findings shed light on the effects and the relative molecular mechanisms related to Bax activation in GBM, and they suggest Bax-targeting compounds as promising therapeutic tools against the GSC reservoir.

  20. Eucommia ulmoides cortex, geniposide and aucubin regulate lipotoxicity through the inhibition of lysosomal BAX.

    Science.gov (United States)

    Lee, Geum-Hwa; Lee, Mi-Rin; Lee, Hwa-Young; Kim, Seung Hyun; Kim, Hye-Kyung; Kim, Hyung-Ryong; Chae, Han-Jung

    2014-01-01

    In this study we examined the inhibition of hepatic dyslipidemia by Eucommia ulmoides extract (EUE). Using a screening assay for BAX inhibition we determined that EUE regulates BAX-induced cell death. Among various cell death stimuli tested EUE regulated palmitate-induced cell death, which involves lysosomal BAX translocation. EUE rescued palmitate-induced inhibition of lysosomal V-ATPase, α-galactosidase, α-mannosidase, and acid phosphatase, and this effect was reversed by bafilomycin, a lysosomal V-ATPase inhibitor. The active components of EUE, aucubin and geniposide, showed similar inhibition of palmitate-induced cell death to that of EUE through enhancement of lysosome activity. Consistent with these in vitro findings, EUE inhibited the dyslipidemic condition in a high-fat diet animal model by regulating the lysosomal localization of BAX. This study demonstrates that EUE regulates lipotoxicity through a novel mechanism of enhanced lysosomal activity leading to the regulation of lysosomal BAX activation and cell death. Our findings further indicate that geniposide and aucubin, active components of EUE, may be therapeutic candidates for non-alcoholic fatty liver disease.

  1. Bax Inhibitor-1 down-regulation in the progression of chronic liver diseases

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    Burra Patrizia

    2010-04-01

    Full Text Available Abstract Background Bax inhibitor-1 (BI-1 is an evolutionary conserved endoplasmic reticulum protein that, when overexpressed in mammalian cells, suppresses the apoptosis induced by Bax, a pro-apoptotic member of the Bcl-2 family. The aims of this study were: (1 to clarify the role of intrinsic anti- and pro-apoptotic mediators, evaluating Bax and BI-1 mRNA and protein expressions in liver tissues from patients with different degrees of liver damage; (2 to determine whether HCV and HBV infections modulate said expression. Methods We examined 62 patients: 39 with chronic hepatitis (CH (31 HCV-related and 8 HBV-related; 7 with cirrhosis (6 HCV-related and 1 HBV-related; 13 with hepatocellular carcinoma (HCC [7 in viral cirrhosis (6 HCV- and 1 HBV-related, 6 in non-viral cirrhosis]; and 3 controls. Bax and BI-1 mRNAs were quantified by real-time PCR, and BI-1 protein expression by Western blot. Results CH tissues expressed significantly higher BI-1 mRNA levels than cirrhotic tissues surrounding HCC (P Conclusions BI-1 expression is down-regulated as liver damage progresses. The high BI-1 mRNAs levels observed in early liver disease may protect virus-infected cells against apoptosis, while their progressive downregulation may facilitate hepatocellular carcinogenesis. HCV genotype seems to have a relevant role in Bax transcript expression.

  2. THE EXPRESSION OF BCL-2 AND BAX PROTEINS IN GASTRIC CARCINOMA AND PRECANCEROUS LESIONS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate the variance of expression of bcl-2 and bax genes in the genesis of gastric carcinoma as well as their relationship. Methods Thirty-five cases of early-stage gastric carcinoma and Twenty-four cases of chronic atrophic gastritis were studied by immunohistochemical method. Results There were no statistical differences of bcl-2 expression levels between gastric carcinoma and atypical hyperplasia or paracancerous intestinalepithelial metaplasia(IEM)(P>0. 05). There were statistical differences of bcl-2 expression between normal epithelial tissues (or non-cancerous IEM) and the other three groups (P<0.05),but no statistical difference between the normal epithelial and the non-cancerous IEM group was observed (P>0. 05). The expressions of bax protein were found in the normal epithelial and the other groups in varying degrees,but there were no statistical differences between either two of the groups (P>0.05). The bcl-2/bax ratio was higher in early-stage gastric carcinoma,atypical hyperplasia and paracancerous intestinai-metaplasia than in the non-cancerous intestinal-metaplasia (P<0. 05) and normal epithelial tissues(P<0. 01). Conclusion The abnormal expression of bcl-2 protein and bax protein ,especially the increased bcl-2/bax ratio, probably play an important role in the course of carcinogenesis of gastric carcinoma.

  3. Is Bax/Bcl-2 ratio considered as a prognostic marker with age and tumor location in colorectal cancer?

    Science.gov (United States)

    Khodapasand, Ehsan; Jafarzadeh, Narges; Farrokhi, Farid; Kamalidehghan, Behnam; Houshmand, Massoud

    2015-01-01

    Bax and Bcl-2 are the major members of Bcl-2 family whose play a key role in tumor progression or inhibition of intrinsic apoptotic pathway triggered by mitochondrial dysfunction. Therefore, the balance between pro- and anti-apoptotic members of this family can determine the cellular fate. In this study, the relative level of mRNA expression of Bax and Bcl-2 genes was determined using RNA extraction, cDNA synthesis and RT-qPCR technique from 22 tumoral tissues and adjacent non-tumoral tissues from adenocarcinoma colorectal cancer. The potential prognostic and predictive significance of Bax and Bcl-2 gene expression and Bax/Bcl-2 ratio were demonstrated in colorectal cancer. The significant correlation between qPCR data and different clinicopathologic parameters of colorectal carcinoma, including age, gender, tumor size, tumor stage, tumor location, and tumor differentiation was also examined. Interestingly, no significant correlation was seen between Bax and Bcl-2 expressions and clinicopathological parameters of colorectal cancer. However, Bax/Bcl-2 ratio was statistically correlated with age and tumor location. Patients with age above 50 showed decreased levels of Bax/Bcl-2 ratio. Moreover, the Bax/Bcl-2 ratio was significantly lower in tumors resected from colon compared to sigmoid colon, rectosigmoid and rectum tumors. This study indicates a significant correlation between age and tumor location with Bax/Bcl-2 expression ratio, suggesting predictive value as a potential molecular marker of colorectal cancer.

  4. Bax is not involved in the resveratrol-induced apoptosis in human lung adenocarcinoma cells

    Science.gov (United States)

    Zhang, Wei-wei; Wang, Zhi-ping; Chen, Tong-sheng

    2010-02-01

    Resveratrol (RV) is a natural plant polyphenol widely present in foods such as grapes, wine, and peanuts. Previous studies indicate that RV has an ability to inhibit various stages of carcinogenesis and eliminate preneoplastic cells in vitro and in vivo. However, little is known about the molecular mechanism of RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cell. In this report, we analyzed whether Bax translocation from cytoplasm to mitochondria during RV-induced apoptosis in single living cell using onfocal microscopey. Cells were transfected with GFP-Bax plasmid. Cell counting kit (CCK-8) assay was used to assess the inhibition of RV on the cells viability. Apoptotic activity of RV was detected by Hoechst 33258 and propidium iodide (PI) staining. Our results showed that RV induced a dose-dependent apoptosis in which Bax did not translocate to mitochondrias.

  5. A non-apoptotic role for BAX and BAK in eicosanoid metabolism

    Science.gov (United States)

    Zhang, Tejia; Walensky, Loren D.; Saghatelian, Alan

    2015-01-01

    BCL-2 proteins are key regulators of programmed cell death. The interplay between pro- and anti-apoptotic BCL-2 members has important roles in many cancers. In addition to their apoptotic function, recent evidence supports key non-apoptotic roles for several BCL-2 proteins. We used an unbiased lipidomics strategy to reveal that the pro-apoptotic proteins BAX, and to a lesser extent BAK, regulate the cellular inflammatory response by mediating COX-2 expression and prostaglandin biosynthesis. COX-2 upregulation in response to the bacterial endotoxin lipopolysaccharide is blunted in the absence of BAX, and Bax−/− mouse embryonic fibroblasts display altered kinetics of NFκB and MAPK signaling following endotoxin treatment. Our approach uncovers a novel, non-apoptotic function for BAX in regulation of the cellular inflammatory response and suggests that inflammation and apoptosis are more tightly connected than previously anticipated. PMID:25815636

  6. Influence of Apoptin on Up-regulation of the Expression of Bad and Bax

    Institute of Scientific and Technical Information of China (English)

    GUO Tai; YANG Qian

    2005-01-01

    The chicken anemia virus protein, apoptin, which manifests selectivity and specificity to tumor cells, induces a p53-independent and Bcl-2-insensitive type of apoptosis in various human tumor cells. In this study, the apoptin gene was cloned from the total DNA of chicken anemia virus, and the recombinant vector was constructed. We used oligonucleotide microarray to study the changes of four genes, including Bcl-2, Bcl-xL, Bad and Bax. The post-transfection with the recombinant was also studied. The pro-apoptotic genes(Bad and Bax) and anti-apoptosis genes(Bcl-2 and Bcl-xL) were up-regulated in contrast to the controls. According to the published data, either Bcl-2 or Bcl-xL can form non-functional heterodimers by Bad and Bax binding together, resulting in blocking partly the release of cytochrome c from mitochondria. However, apoptosis could be inhibited by neither the endogenous Bcl-xL nor Bcl-2 over-expression. The experiments show that the apoptin-induced apoptotic pathway is related to the up-regulation of Bad and Bax. Bad was up-regulated by apoptin; then this up-regulated product of Bad was in favor of displacing Bax from binding to Bcl-xL or Bcl-2. Consequently, Bax exerted a pro-apoptotic dysfunction to mitochondria, thereby inducing the release of cytochrome c. Finally, apoptin induced the apoptosis of HHCC cells. These results indicate that the oligonucleotide microarray can reveal the genes related to the apoptosis induced by apoptin in HHCC cells.

  7. Differential expression of Bcl-2 and Bax during gastric ischemia-reperfusion of rats

    Institute of Scientific and Technical Information of China (English)

    Wei-Li Qiao; Guang-Ming Wang; Yue Shi; Jin-Xia Wu; You-Jian Qi; Jian-Fu Zhang; Hong Sun; Chang-Dong Yan

    2011-01-01

    AIM: To investigate expression of Bcl-2 and Bax in gastric ischemia-reperfusion (GI-R) and involvement of extracellular signal-regulated kinase (ERK) 1/2 activation.METHODS: The GI-R model was established by ligature of the celiac artery for 30 min and reperfusion in Sprague-Dawley rats. Rats were assigned to groups in accordancewith their evaluation period: control, 0, 0.5, 1, 3, 6, 24,48, and 72 h. Expression and distribution of Bcl-2 and Bax proteins were analyzed by immunohistochemistry and western blotting in gastric tissue samples after sacrifice.RESULTS: Compared with controls, the percentage of positive cells and protein levels of Bcl-2 decreased in the early phases of reperfusion, reached its minimumat 1 h (P < 0.05); it then increased, reaching its peak at 24 h of reperfusion (P < 0.05). The pattern of Bax expression was opposite to that of Bcl-2. Bax expressionincreased after reperfusion, with its peak at 1 h of reperfusion (P < 0.05), and then it decreased gradually to a minimum at 24 h after reperfusion (P < 0.05).On the other hand, inhibition of activation of ERK1/2 induced by PD98059, a specific upstream MEK inhibitor,had significant effects on Bcl-2 and Bax in GI-R.Compared with GI-R treatment only at 3 h of reperfusion,PD98059 reduced the number of Bcl-2 positive cells (0.58% of R3h group, P < 0.05) and Bcl-2 proteinlevel (74% of R3h group, P < 0.05) but increased the number of Bax-positive cells (1.33-fold vs R3h group, P< 0.05) and Bax protein level (1.35-fold of R3h group,P < 0.05).CONCLUSION: These results indicated that the Bcl-2 and Bax played a pivotal role in the gastric mucosal I-R injury and repair by activation of ERK1/2.

  8. Quercetin induces tumor-selective apoptosis through down-regulation of Mcl-1 and activation of Bax

    Science.gov (United States)

    Cheng, Senping; Gao, Ning; Zhang, Zhuo; Chen, Gang; Budhraja, Amit; Ke, Zunji; Son, Young-ok; Wang, Xin; Luo, Jia; Shi, Xianglin

    2010-01-01

    Purpose To investigate the in vivo antitumor efficacy of querctin in U937 xenografts and the functional role of Mcl-1 and Bax in quercetin-induced apoptosis in human leukemia cells. Experimental Design Leukemia cells were treated with quercetin, after which apoptosis, Mcl-1 expression, and Bax activation and translocation were evaluated. The efficacy of quercein, as well as Mcl-1 expression and Bax activation were investigated in xenografts of leukemia cells. Results Administration of quercetin caused pronounced apoptosis in both transformed and primary leukemia cells, but not in normal blood peripheral mononuclear cells. Quercetin-induced apoptosis was accompanied by Mcl-1 down-regulation and Bax conformational change and mitochondrial translocation which triggered cytochrome c release. Knockdown of Bax by siRNA reversed querctin-induced apoptosis. Knockout of Bax abrogated the activation of caspase and apoptosis. Ectopic expression of Mcl-1 attenuated quercetin-mediated Bax activation, translocation and cell death. Conversely, interruption of Mcl-1 by siRNA enhanced Bax activation and translocation, as well as lethality induced by quercetin. However, the absence of Bax had no effect on quercetin-mediated Mcl-1 down-regulation. Furthermore, in vivo administration of quercetin attenuated tumor growth in U937 xenografts. The TUNEL positive apoptotic cells in tumor sections increased in quercetin-treated mice as compared with controls. Mcl-1 down-regulation and Bax activation were observed in xenografts. Conclusions These data suggest that quercetin may be useful for the treatment of leukemia by preferentially inducing apoptosis in leukemia versus normal hematopoietic cells, through a process involving Mcl-1 down-regulation, which in turn potentiates Bax activation and mitochondrial translocation, culminating in apoptosis. PMID:21138867

  9. 杏仁核点燃鼠海马不同亚区Bax mRNA的表达%The expression of Bax mRNA in the hippocampus-subareas in amygdala-kindled rats

    Institute of Scientific and Technical Information of China (English)

    梁代义; 伍国锋; 庞成

    2006-01-01

    目的:观察细胞凋亡调控基因Bax mRNA在癫(癎)鼠海马不同亚区的表达.方法:利用电极植入鼠脑杏仁核的方法建立点燃癫(癎)模型,采用原位杂交法检测鼠脑海马CA1、CA2、CA3及齿状回(DGL)区Bax mRNA的表达.结果:正常大鼠海马各区少见Bax mRNA阳性细胞表达,杏仁核植入电极大鼠及其点燃后海马各区Bax mRNA表达阳性细胞数增多,点燃鼠Bax mRNA阳性细胞平均光密度高于仅仅植入电极鼠,海马不同亚区Bax mRNA阳性细胞平均光密度值不同.结论:杏仁核点燃癫(癎)模型鼠的海马不同亚区均存在Bax mRNA表达增强,但不同亚区对Bax mRNA表达敏感性不同,以DGL区为最高.

  10. NDV-induced apoptosis in absence of Bax; evidence of involvement of apoptotic proteins upstream of mitochondria

    Directory of Open Access Journals (Sweden)

    Molouki Aidin

    2012-08-01

    Full Text Available Abstract Background Recently it was shown that following infection of HeLa cells with Newcastle disease virus (NDV, the matrix (M protein binds to Bax and subsequently the intrinsic pathway of apoptosis is activated. Moreover, there was very little alteration on mRNA and protein levels of Bax and Bcl-2 after infection with NDV. Finding In order to further investigate the role of members of the Bcl-2 family, Bax-knockout and wild-type HCT116 cells were infected with NDV strain AF2240. Although both cells underwent apoptosis through the activation of the intrinsic pathway and the release of cytochrome c from mitochondria, the percentage of dead Bax-knockout cells was significantly lower than wt cells (more than 10% at 48 h post-infection. In a parallel experiment, the effect of NDV on HT29 cells, that are originally Bcl-2-free, was studied. Apoptosis in HT29 cells was associated with Bax redistribution from cytoplasm to mitochondria, similar to that of HeLa and wt HCT116 cells. Conclusion Although the presence of Bax during NDV-induced apoptosis contributes to a faster cell death, it was concluded that other apoptotic protein(s upstream of mitochondria are also involved since cancer cells die whether in the presence or absence of Bax. Therefore, the classic Bax/Bcl-2 ratio may not be a major determinant in NDV-induced apoptosis.

  11. Imbalanced expression of Bcl-xL and Bax in platelets treated with plasma from immune thrombocytopenia.

    Science.gov (United States)

    Qiao, Jianlin; Liu, Yun; Li, Depeng; Wu, Yulu; Li, Xiaoqian; Yao, Yao; Niu, Mingshan; Fu, Chunling; Li, Hongchun; Ma, Ping; Li, Zhenyu; Xu, Kailin; Zeng, Lingyu

    2016-04-01

    Immune thrombocytopenia is a heterogeneous autoimmune disease, characterized by accelerated platelet destruction and impaired platelet production. Bcl-xL and Bax play an opposite role in the regulation of apoptotic process with Bcl-xL for cell survival and Bax for cell apoptosis. Given the critical roles in the regulation of platelet apoptosis, whether Bcl-xL or Bax was involved in the pathogenesis of ITP remains unknown. The aim of this study is to evaluate the expression profile of Bcl-xL and Bax in platelets treated with ITP plasma. Normal washed platelets were treated with plasma from 20 active ITP patients or 10 age and gender-matched control to mimic the ITP in vivo environment. Mitochondrial depolarization, platelet apoptosis and activation were measured by flow cytometry. Expression of Bcl-xL, Bax and caspase-3 were also measured by quantitative real-time PCR and western blot. Our results demonstrated increased mitochondrial depolarization, platelet apoptosis and activation in platelets after treated with ITP plasma in comparison to control. In addition, decreased expression of Bcl-xL, increased expression of Bax and activity of caspase-3 were also observed. Furthermore, a negative correlation of Bcl-xL with Bax was found in platelets treated with ITP plasma. In conclusion, imbalanced expression of Bcl-xL and Bax might be associated with platelet apoptosis in ITP and therapeutically targeting them might be a novel approach in the treatment of ITP.

  12. Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins.

    Science.gov (United States)

    Goodall, Katharine J; Finch-Edmondson, Megan L; van Vuuren, Joanne; Yeoh, George C; Gentle, Ian E; Vince, James E; Ekert, Paul G; Vaux, David L; Callus, Bernard A

    2016-01-01

    Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.

  13. A Small-Molecule Inhibitor of Bax and Bak Oligomerization Prevents Genotoxic Cell Death and Promotes Neuroprotection.

    Science.gov (United States)

    Niu, Xin; Brahmbhatt, Hetal; Mergenthaler, Philipp; Zhang, Zhi; Sang, Jing; Daude, Michael; Ehlert, Fabian G R; Diederich, Wibke E; Wong, Eve; Zhu, Weijia; Pogmore, Justin; Nandy, Jyoti P; Satyanarayana, Maragani; Jimmidi, Ravi K; Arya, Prabhat; Leber, Brian; Lin, Jialing; Culmsee, Carsten; Yi, Jing; Andrews, David W

    2017-04-20

    Aberrant apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeabilization (MOMP) triggered by the oligomerization of the Bcl-2 family proteins Bax/Bak is an irreversible step leading to execution of apoptosis. Here, we describe the discovery of small-molecule inhibitors of Bax/Bak oligomerization that prevent MOMP. We demonstrate that these molecules disrupt multiple, but not all, interactions between Bax dimer interfaces thereby interfering with the formation of higher-order oligomers in the MOM, but not recruitment of Bax to the MOM. Small-molecule inhibition of Bax/Bak oligomerization allowed cells to evade apoptotic stimuli and rescued neurons from death after excitotoxicity, demonstrating that oligomerization of Bax is essential for MOMP. Our discovery of small-molecule Bax/Bak inhibitors provides novel tools for the investigation of the mechanisms leading to MOMP and will ultimately facilitate development of compounds inhibiting Bax/Bak in acute and chronic degenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway.

    Directory of Open Access Journals (Sweden)

    Sebastián Zamorano

    Full Text Available Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.

  15. Label-Free Classification of Bax/Bak Expressing vs. Double-Knockout Cells.

    Science.gov (United States)

    Naser, Mohammad; Graham, Michelle T; Pierre, Kamau; Boustany, Nada N

    2016-11-01

    We combine optical scatter imaging with principal component analysis (PCA) to classify apoptosis-competent Bax/Bak-expressing, and apoptosis resistant Bax/Bak-null immortalized baby mouse kidney cells. We apply PCA to 100 stacks each containing 236 dark-field cell images filtered with an optically implemented Gabor filter with period between 0.3 and 2.9 μm. Each stack yields an "eigencell" image corresponding to the first principal component obtained at one of the 100 Gabor filter periods used. At each filter period, each cell image is multiplied by (projected onto) the eigencell image. A Feature Matrix consisting of 236 × 100 scalar values is thus constructed with significantly reduced dimension compared to the initial dataset. Utilizing this Feature Matrix, we implement a supervised linear discriminant analysis and classify successfully the Bax/Bak-expressing and Bax/Bak-null cells with 94.7% accuracy and an area under the curve (AUC) of 0.993. Applying a feature selection algorithm further reveals that the Gabor filter period ranges most significant for the classification correspond to both large (likely nuclear) features as well as small sized features (likely organelles present in the cytoplasm). Our results suggest that cells with a genetic defect in their apoptosis pathway can be differentiated from their normal counterparts by label-free multi-parametric optical scatter data.

  16. Conformational Rearrangements in the Pro-apoptotic Protein, Bax, as It Inserts into Mitochondria

    Science.gov (United States)

    Gahl, Robert F.; He, Yi; Yu, Shiqin; Tjandra, Nico

    2014-01-01

    The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the activation of apoptosis through the mitochondria pathway. Pro- and anti-apoptotic members of this family keep each other in check until the correct time to commit to apoptosis. The point of no return for this commitment is the permeabilization of the outer mitochondrial membrane. Translocation of the pro-apoptotic member, Bax, from the cytosol to the mitochondria is the molecular signature of this event. We employed a novel method to reliably detect Förster resonance energy transfer (FRET) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in Bax as this translocation occurs in live cells. In the cytosol, our FRET measurement indicated that the C-terminal helix is exposed instead of tucked away in the core of the protein. In addition fluorescence correlation spectroscopy (FCS) showed that cytosolic Bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. Cross-linking the C-terminal helix (α9) to helix α4 reduced the potential of those interactions to occur. After translocation, our FRET measurements showed that Bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the BH3 domain (helix α2) and the C-terminal helix. These findings have implications for possible contacts with other Bcl-2 proteins necessary for the regulation of apoptosis. PMID:25315775

  17. Wheat BAX inhibitor-1 contributes to wheat resistance to Puccinia striiformis

    Science.gov (United States)

    BAX inhibitor-1 (BI-1) is proposed to be a cell death suppressor conserved in both animals and plants. The ability of BI-1 genes to inhibit programmed cell death (PCD) has been well studied in animals, but the physiological importance of BI-1 in plant-microbe interactions remains unclear. This study...

  18. cBid, Bax and Bcl-xL exhibit opposite membrane remodeling activities

    Science.gov (United States)

    Bleicken, S; Hofhaus, G; Ugarte-Uribe, B; Schröder, R; García-Sáez, A J

    2016-01-01

    The proteins of the Bcl-2 family have a crucial role in mitochondrial outer membrane permeabilization during apoptosis and in the regulation of mitochondrial dynamics. Current models consider that Bax forms toroidal pores at mitochondria that are responsible for the release of cytochrome c, whereas Bcl-xL inhibits pore formation. However, how Bcl-2 proteins regulate mitochondrial fission and fusion remains poorly understood. By using a systematic analysis at the single vesicle level, we found that cBid, Bax and Bcl-xL are able to remodel membranes in different ways. cBid and Bax induced a reduction in vesicle size likely related to membrane tethering, budding and fission, besides membrane permeabilization. Moreover, they are preferentially located at highly curved membranes. In contrast, Bcl-xL not only counterbalanced pore formation but also membrane budding and fission. Our findings support a mechanism of action by which cBid and Bax induce or stabilize highly curved membranes including non-lamellar structures. This molecular activity reduces the energy for membrane remodeling, which is a necessary step in toroidal pore formation, as well as membrane fission and fusion, and provides a common mechanism that links the two main functions of Bcl-2 proteins. PMID:26913610

  19. [Influence of TIEG1 on apoptosis of HL-60 cells and expression of Bcl-2/Bax].

    Science.gov (United States)

    Yao, Kun; Yang, Ying; Hu, Rong; Miao, Miao; Liao, Ai-Jun; Yang, Wei; Liu, Zhuo-Gang

    2013-06-01

    This study was aimed to investigate the influence of TIEG1 on apoptosis of HL-60 cells and the expression of Bcl-2/Bax. Different concentration of TIEG1 were used to treat HL-60 cells, the cell growth inhibition rate was detected by MTT method. After treating HL-60 cells with 12.03 ng/ml TIEG1, cell apoptosis was detected with flow cytometry. Bcl-2 and Bax was detected with RT-PCR. The results showed that TIEG1 had inhibitory effect on HL-60 cell proliferation, and in time-and dose-dependent manners. The more obvious inhibitory effect was observed in HL-60 cells treated with TIEG1 of 12.03 ng/ml. During the course of cell apoptosis, Bax expression increased, but Bcl-2 expression decreased (P HL-60 cell proliferation and induces apoptosis in time and dose-dependent manners. During the course of HL-60 cells apoptosis induced by TIEG1, Bcl-2/Bax are associated with HL-60 cell apoptosis induced by TIEG1.

  20. A Single Nucleotide Polymorphism in the Bax Gene Promoter Affects Transcription and Influences Retinal Ganglion Cell Death

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    Sheila J Semaan

    2010-03-01

    Full Text Available Pro-apoptotic Bax is essential for RGC (retinal ganglion cell death. Gene dosage experiments in mice, yielding a single wild-type Bax allele, indicated that genetic background was able to influence the cell death phenotype. DBA/2J Bax+/− mice exhibited complete resistance to nerve damage after 2 weeks (similar to Bax −/− mice, but 129B6 Bax+/− mice exhibited significant cell loss (similar to wild-type mice. The different cell death phenotype was associated with the level of Bax expression, where 129B6 neurons had twice the level of endogenous Bax mRNA and protein as DBA/2J neurons. Sequence analysis of the Bax promoters between these strains revealed a single nucleotide polymorphism (T129B6 to CDBA/2J at position −515. A 1.5- to 2.5-fold increase in transcriptional activity was observed from the 129B6 promoter in transient transfection assays in a variety of cell types, including RGC5 cells derived from rat RGCs. Since this polymorphism occurred in a p53 half-site, we investigated the requirement of p53 for the differential transcriptional activity. Differential transcriptional activity from either 129B6 or DBA/2J Bax promoters were unaffected in p53−/− cells, and addition of exogenous p53 had no further effect on this difference, thus a role for p53 was excluded. Competitive electrophoretic mobility-shift assays identified two DNA-protein complexes that interacted with the polymorphic region. Those forming Complex 1 bound with higher affinity to the 129B6 polymorphic site, suggesting that these proteins probably comprised a transcriptional activator complex. These studies implicated quantitative expression of the Bax gene as playing a possible role in neuronal susceptibility to damaging stimuli.

  1. Opposite role of Bax and BCL-2 in the anti-tumoral responses of the immune system

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    Bougras Gwenola

    2004-08-01

    Full Text Available Abstract Background The relative role of anti apoptotic (i.e. Bcl-2 or pro-apoptotic (e.g. Bax proteins in tumor progression is still not completely understood. Methods The rat glioma cell line A15A5 was stably transfected with human Bcl-2 and Bax transgenes and the viability of theses cell lines was analyzed in vitro and in vivo. Results In vitro, the transfected cell lines (huBax A15A5 and huBcl-2 A15A5 exhibited different sensitivities toward apoptotic stimuli. huBax A15A5 cells were more sensitive and huBcl-2 A15A5 cells more resistant to apoptosis than mock-transfected A15A5 cells (pCMV A15A5. However, in vivo, in syngenic rat BDIX, these cell lines behaved differently, as no tumor growth was observed with huBax A15A5 cells while huBcl-2 A15A5 cells formed large tumors. The immune system appeared to be involved in the rejection of huBax A15A5 cells since i huBax A15A5 cells were tumorogenic in nude mice, ii an accumulation of CD8+ T-lymphocytes was observed at the site of injection of huBax A15A5 cells and iii BDIX rats, which had received huBax A15A5 cells developed an immune protection against pCMV A15A5 and huBcl-2 A15A5 cells. Conclusions We show that the expression of Bax and Bcl-2 controls the sensitivity of the cancer cells toward the immune system. This sensitization is most likely to be due to an increase in immune induced cell death and/or the amplification of an anti tumour immune response

  2. Effect of Bcl-2 and Bax on survival of side population cells from hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To understand the role and significance of side population (SP) cells from hepatocellular carcinoma (HCC) in hepatocarcinogenesis, development, relapse and metastasis, we simulated the denutrition conditions that cancer cells experience in clinical therapy, observed the different anti-apoptosis ability of SP cells and non-SP cells under such conditions, and established the possible effects of P53, Bcl-2 and Bax on survival of SP cells.METHODS: We used flow cytometry to analyze and sort the SP and non-SP cells in established HCC lines MHCC97and hHCC. We evaluated cell proliferation by methyl thiazolyl tetrazolium (MTT) assay and investigated the expression of p53, bd-2 and bax genes during denutrition,by RT-PCR and immunofluorescence staining.RESULTS: The percentage of SP cells in the two established HCC lines was 0.25% and 0.5%, respectively.SP cells had greater anti-apoptosis and proliferation ability than non-SP cells. Expression of Bcl-2 and Bax in SP and non-SP cells differed during denutrition. The former was up-regulated in SP cells, and the latter was up-regulated in non-SP cells.CONCLUSION: It may be that different upstream molecules acted and led to different expression levels of Bcl-2 and Bax in these two cell lines. There was a direct relationship between up-regulation of Bcl-2 and down-regulation of Bax and higher anti-apoptosis ability in SP cells. It may be that the existence and activity of SP cells are partly responsible for some of the clinical phenomena which are seen in HCC, such as relapse or metastasis. Further research on SP cells may have potential applications in the field of anticancer therapy.

  3. WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zeilstra, Jurrit; Joosten, Sander P.J. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Wensveen, Felix M. [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Dessing, Mark C.; Schuetze, Denise M. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Eldering, Eric [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Spaargaren, Marcel [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Pals, Steven T., E-mail: s.t.pals@amc.uva.nl [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands)

    2011-03-04

    Research highlights: {yields} Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. {yields} Expression profiling of apoptosis-related genes in Apc{sup Min/+} mice revealed the differential expression of pro-apoptotic Bok and Bax. {yields} APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. {yields} Blocking of {beta}-catenin/TCF-4-mediated signaling in colon cancer cells reduced the expression of BOK and BAX. -- Abstract: In a majority of cases, colorectal cancer is initiated by aberrant activation of the WNT signaling pathway. Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or {beta}-catenin causes constitutively active {beta}-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to cancer precursor lesions, called dysplastic aberrant crypt foci. Deregulated apoptosis is a hallmark of adenomatous colon tissue. However, the contribution of WNT signaling to this process is not fully understood. We addressed this role by analyzing the rate of epithelial apoptosis in aberrant crypts and adenomas of the Apc{sup Min/+} mouse model. In comparison with normal crypts and adenomas, aberrant crypts displayed a dramatically increased rate of apoptotic cell death. Expression profiling of apoptosis-related genes along the crypt-villus axis and in Apc mutant adenomas revealed increased expression of two pro-apoptotic Bcl-2 family members in intestinal adenomas, Bok and Bax. Analysis of the colon of familial adenomatous polyposis (FAP) patients along the crypt-to-surface axis, and of dysplastic crypts, corroborated this expression pattern. Disruption of {beta}-catenin/TCF-4-mediated signaling in the colorectal cancer cell line Ls174T significantly decreased BOK and BAX expression, confirming WNT-dependent regulation in intestinal epithelial cells. Our results suggest a feedback mechanism by which

  4. Bax and Bcl-2 are focally overexpressed in the normal epithelium of cancerous prostates.

    Science.gov (United States)

    Tolonen, Teemu T; Tommola, Satu; Jokinen, Samuli; Parviainen, Tiina; Martikainen, Paula M

    2007-01-01

    Development of prostate cancer is connected with a disturbance of apoptosis. Prostate cancer is multifocal, suggesting that the control of apoptosis is impaired at multiple foci. We wanted to know whether apoptosis is generally disturbed in cancerous prostates and if changes in apoptotic control could be detected even in the absence of any morphologically visible changes. Therefore, we compared expression of two common apoptotic markers, Bax and Bcl-2, in normal epithelium of cancerous prostates and controls. We also evaluated the expression of these proteins in hyperplasia, prostatic intraepithelial neoplasia (PIN), carcinomas of different Gleason grades and capsular perineural invasion. The tissue material was obtained from radical prostatectomies, transurethral resection chips and autopsies. Individual tissue arrays were done for each patient. The intensity of Bax and Bcl-2 immunostaining was estimated semiquantitatively. The data were analyzed using a linear mixed-models analysis as well as dichotomized staining indices. Normal epithelium of cancerous prostates contained foci with high expression of Bax and Bcl-2. The expression of Bax in Gleason grades 3-5 carcinoma was significantly higher than that in Gleason grade 2, and was highest in foci with perineural invasion. The expression of Bcl-2 was strongest in PIN foci. Expression of Bax and Bcl-2 in normal epithelium of cancerous prostates suggests that increases in these indirect markers may reflect altered apoptotic control in these foci. Further studies are needed to show whether these changes represent the earliest step of the multifocal carcinogenetic process. Control of apoptosis seems to be involved and modulated during local progression of prostate cancer.

  5. Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio

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    Azimahtol Hawariah LP

    2007-04-01

    Full Text Available Abstract Background Zerumbone is a cytotoxic component isolated from Zingiber zerumbet Smith, a herbal plant which is also known as lempoyang. This new anticancer bioactive compound from Z. zerumbet was investigated for its activity and mechanism in human liver cancer cell lines. Results Zerumbone significantly showed an antiproliferative activity upon HepG2 cells with an IC50 of 3.45 ± 0.026 μg/ml. Zerumbone was also found to inhibit the proliferation of non-malignant Chang Liver and MDBK cell lines. However the IC50 obtained was higher compared to the IC50 for HepG2 cells (> 10 μg/ml. The extent of DNA fragmentation was evaluated by the Tdt-mediated dUTP nick end labelling assay which showed that, zerumbone significantly increased apoptosis in HepG2 cells in a time-course manner. In detail, the apoptotic process triggered by zerumbone involved the up-regulation of pro-apoptotic Bax protein and the suppression of anti-apoptotic Bcl-2 protein expression. The changes that occurred in the levels of this antagonistic proteins Bax/Bcl-2, was independent of p53 since zerumbone did not affect the levels of p53 although this protein exists in a functional form. Western blotting analysis for Bax protein was further confirmed qualitatively with an immunoassay that showed the distribution of Bax protein in zerumbone-treated cells. Conclusion Therefore, zerumbone was found to induce the apoptotic process in HepG2 cells through the up and down regulation of Bax/Bcl-2 protein independently of functional p53 activity.

  6. Altered mitochondrial morphology and defective protein import reveal novel roles for Bax and/or Bak in skeletal muscle.

    Science.gov (United States)

    Zhang, Yuan; Iqbal, Sobia; O'Leary, Michael F N; Menzies, Keir J; Saleem, Ayesha; Ding, Shuzhe; Hood, David A

    2013-09-01

    The function Bax and/or Bak in constituting a gateway for mitochondrial apoptosis in response to apoptotic stimuli has been unequivocally demonstrated. However, recent work has suggested that Bax/Bak may have unrecognized nonapoptotic functions related to mitochondrial function in nonstressful environments. Wild-type (WT) and Bax/Bak double knockout (DKO) mice were used to determine alternative roles for Bax and Bak in mitochondrial morphology and protein import in skeletal muscle. The absence of Bax and/or Bak altered mitochondrial dynamics by regulating protein components of the organelle fission and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial protein import, both into the matrix and outer membrane compartments, which was consistent with our observations of impaired membrane potential and attenuated expression of protein import machinery (PIM) components in intermyofibrillar mitochondria. Furthermore, the cytosolic chaperones heat-shock protein 90 (Hsp90) and binding immunoglobulin protein (BiP) were markedly increased with the deletion of Bax/Bak, indicating that the cytosolic environment related to protein folding may be changed in DKO mice. Interestingly, endurance training fully restored the deficiency of protein import in DKO mice, likely via the upregulation of PIM components and through improved cytosolic chaperone protein expression. Thus our results emphasize novel roles for Bax and/or Bak in mitochondrial function and provide evidence, for the first time, of a curative function of exercise training in ameliorating a condition of defective mitochondrial protein import.

  7. Loss of BAX by miR-365 Promotes Cutaneous Squamous Cell Carcinoma Progression by Suppressing Apoptosis.

    Science.gov (United States)

    Zhou, Liang; Gao, Ruirui; Wang, Yinghui; Zhou, Meijuan; Ding, Zhenhua

    2017-05-30

    Pro-apoptotic BCL2 associated X (BAX) is traditionally thought to be regulated by anti-apoptotic BCL-2 family members, like BCL2-like 1 (BCL-XL), at the protein level. However, the posttranscriptional regulation of BAX is under explored. In this study, we identified BAX as the novel downstream target of miR-365, which is supported by gain- and loss-of-function studies of onco-miR-365. Loss of BAX by either RNA interference or highly-expressed miR-365 in cells of cutaneous squamous cell carcinoma (CSCC) enhanced the tumor resistance against apoptosis, while repressing cell proliferation, migration, and invasiveness. In vivo experiment confirmed that BAX knockdown promotes the growth of CSCC xenografts. Collectively, our results find a miR-365-BAX axis for alleviating the pro-apoptotic effects of BAX, which promotes CSCC development and may facilitate the generation of novel therapeutic regimens to the clinical treatment of CSCC.

  8. Ghrelin Administration Increases the Bax/Bcl-2 Gene Expression Ratio in the Heart of Chronic Hypoxic Rats.

    Science.gov (United States)

    Aliparasti, Mohammad Reza; Alipour, Mohammad Reza; Almasi, Shohreh; Feizi, Hadi

    2015-06-01

    Programmed cell death or apoptosis, is a biochemical procedure that initiates due to some conditions, including hypoxia. Bax and Bcl-2 are among the agents that regulate apoptosis. The amplification of the first one triggers the initiation of apoptosis, and the second one prevents it. Ghrelin is an endogenous peptide that antiapoptosis is its new effect. The aim of this study is to examine the effect of ghrelin on the Bax/Bcl-2 ratio. Twenty four wistar rats were divided randomly in three groups; control, hypoxic + saline and hypoxic + ghrelin. Hypoxic animals lived in O2 11% for 2 weeks and received either saline or ghrelin subcutaneously daily. The bax and Bcl-2 gene expression were measured by Real-Time RT-PCR. Chronic hypoxia increased the Bax gene expression significantly compared with normal animals (P = 0.008), but the Bcl-2 was not affected by hypoxia. The Bax/Bcl-2 ratio also amplified significantly (P=0.005). Ghrelin administration significantly increased the Bax/Bcl-2 ratio in the hypoxic animals compared to the hypoxic + saline and normal groups (p=0.042 and P= 0.001, respectively). In the present study, animals' treatment with ghrelin leads to an increment of Bax/Bcl-2 ratio, which indicates a controversy related to cardioprotection of ghrelin.

  9. Ghrelin Administration Increases the Bax/Bcl-2 Gene Expression Ratio in the Heart of Chronic Hypoxic Rats

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    Mohammad Reza Aliparasti

    2015-06-01

    Full Text Available Purpose: Programmed cell death or apoptosis, is a biochemical procedure that initiates due to some conditions, including hypoxia. Bax and Bcl-2 are among the agents that regulate apoptosis. The amplification of the first one triggers the initiation of apoptosis, and the second one prevents it. Ghrelin is an endogenous peptide that antiapoptosis is its new effect. The aim of this study is to examine the effect of ghrelin on the Bax/Bcl-2 ratio. Methods: Twenty four wistar rats were divided randomly in three groups; control, hypoxic + saline and hypoxic + ghrelin. Hypoxic animals lived in O2 11% for 2 weeks and received either saline or ghrelin subcutaneously daily. The bax and Bcl-2 gene expression were measured by Real-Time RT-PCR. Results: Chronic hypoxia increased the Bax gene expression significantly compared with normal animals (P = 0.008, but the Bcl-2 was not affected by hypoxia. The Bax/Bcl-2 ratio also amplified significantly (P=0.005. Ghrelin administration significantly increased the Bax/Bcl-2 ratio in the hypoxic animals compared to the hypoxic + saline and normal groups (p=0.042 and P= 0.001, respectively. Conclusion: In the present study, animals’ treatment with ghrelin leads to an increment of Bax/Bcl-2 ratio, which indicates a controversy related to cardioprotection of ghrelin.

  10. Ghrelin Administration Increases the Bax/Bcl-2 Gene Expression Ratio in the Heart of Chronic Hypoxic Rats

    Science.gov (United States)

    Aliparasti, Mohammad Reza; Alipour, Mohammad Reza; Almasi, Shohreh; Feizi, Hadi

    2015-01-01

    Purpose: Programmed cell death or apoptosis, is a biochemical procedure that initiates due to some conditions, including hypoxia. Bax and Bcl-2 are among the agents that regulate apoptosis. The amplification of the first one triggers the initiation of apoptosis, and the second one prevents it. Ghrelin is an endogenous peptide that antiapoptosis is its new effect. The aim of this study is to examine the effect of ghrelin on the Bax/Bcl-2 ratio. Methods: Twenty four wistar rats were divided randomly in three groups; control, hypoxic + saline and hypoxic + ghrelin. Hypoxic animals lived in O2 11% for 2 weeks and received either saline or ghrelin subcutaneously daily. The bax and Bcl-2 gene expression were measured by Real-Time RT-PCR. Results: Chronic hypoxia increased the Bax gene expression significantly compared with normal animals (P = 0.008), but the Bcl-2 was not affected by hypoxia. The Bax/Bcl-2 ratio also amplified significantly (P=0.005). Ghrelin administration significantly increased the Bax/Bcl-2 ratio in the hypoxic animals compared to the hypoxic + saline and normal groups (p=0.042 and P= 0.001, respectively). Conclusion: In the present study, animals’ treatment with ghrelin leads to an increment of Bax/Bcl-2 ratio, which indicates a controversy related to cardioprotection of ghrelin. PMID:26236657

  11. Bax-induced apoptosis in Leber's congenital amaurosis: a dual role in rod and cone degeneration.

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    Séverine Hamann

    Full Text Available Pathogenesis in the Rpe65(-/- mouse model of Leber's congenital amaurosis (LCA is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65(-/- mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65, was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65(-/- mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65(-/-/Gnat1(-/- mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65(-/-/Bax(-/- mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65(-/- mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65(-/-/Bax(-/- mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice

  12. Expression and Prognostic Significance of EP300, TP53 and BAX in Clear Cell Renal Cell Carcinoma.

    Science.gov (United States)

    Godlewski, Janusz; Krazinski, Bartlomiej E; Kowalczyk, Anna E; Kiewisz, Jolanta; Kiezun, Jacek; Kwiatkowski, Przemyslaw; Sliwińska-Jewsiewicka, Agnieszka; Wierzbicki, Piotr W; Kmieć, Zbigniew

    2017-06-01

    Histone acetyltransferase E1A-binding protein p300 (EP300), tumor protein p53 (TP53) and B-cell lymphoma-2-associated X protein (BAX) contribute to the regulation of the cell cycle and apoptosis, cellular processes that are often impaired in cancer cells. The aim of this study was to determine the expression levels of EP300, TP53 and BAX genes and their respective proteins in clear cell renal cell carcinoma (ccRCC) and evaluate the value of these factors as prognostic factors. EP300, TP53 and BAX expression at the transcript and protein levels were determined by quantitative polymerase-chain reaction (QPCR) and immunohistochemistry (IHC) in paired tumor and kidney specimens from 31 patients with ccRCC. Levels of EP300, TP53 and BAX transcripts were found increased in tumor tissues. Immunoreactivity for TP53 was elevated in cancer cells when compared to unchanged kidney, while EP300 and BAX immunoexpression in ccRCC did not differ from that of normal renal tissue. Immunoreactivity for TP53 was positively associated with larger tumor size. In contrast, stronger BAX immunoexpression correlated with smaller tumor diameters. The average immunoreactivity for BAX was higher in localized, kidney-confined tumor than in advanced/recurrent tumors. None of the analyzed transcripts or proteins correlated with the overall survival of patients. Although TP53 and BAX immunoreactivity levels were associated with some clinicopathological parameters of the patients, the expression of EP300, TP53 and BAX did not reveal any prognostic significance in ccRCC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  13. The Proapoptotic Bcl-2 Protein Bax Plays an Important Role in the Pathogenesis of Reovirus Encephalitis ▿

    Science.gov (United States)

    Berens, Heather M.; Tyler, Kenneth L.

    2011-01-01

    Encephalitis induced by reovirus serotype 3 (T3) strains results from the apoptotic death of infected neurons. Extrinsic apoptotic signaling is activated in reovirus-infected neurons in vitro and in vivo, but the role of intrinsic apoptosis signaling during encephalitis is largely unknown. Bax plays a key role in intrinsic apoptotic signaling in neurons by allowing the release of mitochondrial cytochrome c. We found Bax activation and cytochrome c release in neurons following infection of neonatal mice with T3 reoviruses. Bax−/− mice infected with T3 Abney (T3A) have reduced central nervous system (CNS) tissue injury and decreased apoptosis, despite viral replication that is similar to that in wild-type (WT) Bax+/+ mice. In contrast, in the heart, T3A-infected Bax−/− mice have viral growth, caspase activation, and injury comparable to those in WT mice, indicating that the role of Bax in pathogenesis is organ specific. Nonmyocarditic T3 Dearing (T3D)-infected Bax−/− mice had delayed disease and enhanced survival compared to WT mice. T3D-infected Bax−/− mice had significantly lower viral titers and levels of activated caspase 3 in the brain despite unaffected transneuronal spread of virus. Cytochrome c and Smac release occurred in some reovirus-infected neurons in the absence of Bax; however, this was clearly reduced compared to levels seen in Bax+/+ wild-type mice, indicating that Bax is necessary for efficient activation of proapoptotic mitochondrial signaling in infected neurons. Our studies suggest that Bax is important for reovirus growth and pathogenesis in neurons and that the intrinsic pathway of apoptosis, mediated by Bax, is important for full expression of disease, CNS tissue injury, apoptosis, and viral growth in the CNS of reovirus-infected mice. PMID:21307199

  14. Low levels of Bax inhibitor-1 gene expression increase tunicamycin-induced apoptosis in human neuroblastoma SY5Y cells

    Institute of Scientific and Technical Information of China (English)

    Dan Wu; Peirong Wang; Shiyao Wang

    2012-01-01

    A human SH-SY5Y neuroblastoma cell line with a low level of Bax inhibitor-1 expression was established by lentivirus-mediated RNA interference and fluorescence-activated cell sorting. In control SH-SY5Y cells, tunicamycin treatment induced endoplasmic reticulum stress-mediated apoptosis; however, after Bax inhibitor-1 gene knockdown, cell survival rates were significantly decreased and the degree of apoptosis was significantly increased following tunicamycin treatment. In addition, chromatin condensation and apparent apoptotic phenomena, such as marginalization and cytoplasmic vesicles, were observed. Our findings indicate that Bax inhibitor-1 can delay apoptosis induced by endoplasmic reticulum stress.

  15. Quercetin inhibits human breast cancer cell proliferation and induces apoptosis via Bcl-2 and Bax regulation.

    Science.gov (United States)

    Duo, Jian; Ying, Guo-Guang; Wang, Guo-Wen; Zhang, Li

    2012-06-01

    Breast cancer is a disease in which cancer cells form in the tissues of the breast. The present study aimed to explore the effect of the flavonoid compound quercetin on the growth and apoptosis of human breast cancer cells. Varying concentrations (12.5, 25, 50, 100, 200 µM) of quercetin were applied to cultured MCF-7 human breast cancer cells for defined lengths of time. At 50 to 200 µM doses, quercetin significantly inhibited the proliferation of MCF-7 cells assessed by MTT colorimetry, in both dose- and time-dependent manners (Papoptosis after 48 h of exposure (Pquercetin treatment Bcl-2 expression decreased significantly while Bax expression increased significantly (Pquercetin inhibits cell growth and induces apoptosis in MCF-7 human breast cancer cells. The mechanisms behind these effects may stem from the downregulation of Bcl-2 protein expression and upregulation of Bax expression.

  16. The Bax Inhibitor-1 (BI-1) family in apoptosis and tumorigenesis.

    Science.gov (United States)

    Reimers, Kerstin; Choi, Claudia Y U; Bucan, Vesna; Vogt, Peter M

    2008-03-01

    The signaling pathways that determine the fate of a cell regarding death or survival depend on a large number of regulatory proteins. The Bax Inhibitor-1 (BI-1) family is a highly preserved family of small transmembrane proteins located mostly in the endoplasmic reticulum (ER). Although most members of this family are still not characterized an antiapoptotic effect has been described for BI-1, Lifeguard (LFG), and the Golgi anti-apoptotic protein (GAAP). The cytoprotective activity has been associated to the control of ion homeostasis and ER stress but includes other cell death stimuli as well. Recent data describes multiple interactions between the proteins of the BI-1 family and the Bcl-2 family either stimulating the antiapoptotic function of Bcl-2 or inhibiting the proapoptotic effect of Bax. The potent cell death suppression makes this protein family an interesting target for the development of new drugs and gene therapeutic approaches for diseases caused by apoptotic dysregulation, such as cancer.

  17. Kazrin F is involved in apoptosis and interacts with BAX and ARC

    Institute of Scientific and Technical Information of China (English)

    Qiong Wang; Min Liu; Xin Li; Lu Chen; Hua Tang

    2009-01-01

    Kazrin has recently been identified as a functional protein that is involved in cell-cell junctions and in signal transduction. Here, we identified a new isoform, Kazrin F, which is 518 aa in length and has 97 aa unique at the N-terminus. Knockdown of Kazrin F using siRNA caused cell apoptosis and a marked decrease in cell viability measured by MTT and TUNEL assays. Co-immunoprecipitation analysis revealed that Kazrin F interacts with ARC (apoptosis repressor with caspase recruitment domain) and Bas (Bcl-2-associated X protein). Co-localization of Kazriin F with ARC and Bax in the cytoplasm was determined by immunofluorescence analysis. These results suggested that Kazrin F might play an important role in regulating cellular apoptosis by interacting with ARC and Bax.

  18. Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

    Science.gov (United States)

    Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

    2016-07-01

    Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

  19. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis

    Science.gov (United States)

    Westphal, D; Kluck, R M; Dewson, G

    2014-01-01

    The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different ‘modes' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane. PMID:24162660

  20. Influence of neurotrophin-3 on Bcl-2 and Bax expressions in spinal cord injury of rats

    Institute of Scientific and Technical Information of China (English)

    GUO Shu-zhang; JIANG Tao; REN Xian-jun

    2007-01-01

    Objective:To study the protective mechanisms of neurotrophin-3 (NT-3) on the spinal cord injury.Methods:Totally 105 SD rats were randomly divided into 3 groups:control group,experimental group and sham operation group.Rats from the former 2 groups were inflicted to animal model of acute spinal cord injury according to Allen's (WD) by situating a thin plastic tube in the subarachnoid space below the injury level for perfusion.Rats in experimental group received 20μl NT-3 (200 ng) from the tube at 0,4,8,12,24 h and 3,7 d after injury,and those in control group got an equal volume of normal saline at the same time.The animals in sham operation group only received opening vertebral plate and tube was put in subarachnoid space.The rats were sacrificed at 4,8,12,24 h and 3,7,14 d post injury (n=5).The expression levels of Bcl-2 and Bax proteins in spinal cord of rats were detected by immunohistochemistry assay.Results:The level of Bax protein in control group significantly increased as compared with those in sham operation group, and the peak reached at 8 h after spinal cord injury.The Bcl-2 proteins were always weakly positive.The Bax proteins in NT-3 group significantly decreased but the Bcl-2 proteins obviously increased as compared with those in control group.Conclusion:NT-3 can protect spinal cord from injury in vivo.One of the mechanisms is that NT-3 can inhibit abnormal expression of Bax protein,and increase the expression of Bcl-2 protein,then inhibit apoptosis after spinal cord injury.

  1. DuPont Qualicon BAX System assay for genus Listeria 24E.

    Science.gov (United States)

    Wallace, F Morgan; Fallon, Dawn; DeMarco, Daniel; Varkey, Stephen

    2011-01-01

    The new BAX System PCR Assay for Genus Listeria 24E was evaluated for detecting Listeria spp. in frankfurters, spinach, cooked shrimp, queso fresco cheese, and on stainless steel surfaces with a single-stage enrichment in BAX System 24 Listeria Enrichment Broth (24 LEB). Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent or superior to the U.S. Food and Drug Administration's Bacteriological Analytical Manual and the U.S. Department of Agriculture-Food Safety and Inspection Service culture methods, but with a significantly shorter time to result. Tests to evaluate inclusivity and exclusivity returned no false-negative and no false-positive results on a diverse panel of isolates, and tests for lot-to-lot variability and tablet stability demonstrated consistent performance. Ruggedness studies determined that none of the factors examined, within the range of deviations from specified parameters examined, affect the performance of the assay.

  2. Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion

    Energy Technology Data Exchange (ETDEWEB)

    Ueda, Masashi; Li, Shimo; Itoh, Masanori; Wang, Miao-xing; Hayakawa, Miki; Islam, Saiful; Tana; Nakagawa, Kiyomi [Department of Neurobiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan); Chen, Huayue [Department of Anatomy, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan); Nakagawa, Toshiyuki, E-mail: tnakagaw@gifu-u.ac.jp [Department of Neurobiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan)

    2016-05-27

    The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ER luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax. -- Highlights: •We characterized polyglutamine embedded in the ER membrane. •The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. •The ER membrane may be a target of polyglutamine, which triggers cell death.

  3. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation.

    Science.gov (United States)

    Chen, Fang; Zhang, Bing; Sun, Yong; Xiong, Zeng-Xing; Peng, Han; Deng, Ze-Yuan; Hu, Jiang-Ning

    2016-04-25

    Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O), has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu) and Cat D inhibitor (pepA) inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome.

  4. Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax.

    Science.gov (United States)

    Cong, Haolong; Du, Ning; Yang, Yang; Song, Lei; Zhang, Wenliang; Tien, Po

    2016-11-01

    To survive and replicate within a host, many viruses have evolved strategies that target crucial components within the apoptotic cascade, leading to either inhibition or induction of cell apoptosis. Enterovirus 71 (EV71) infections have been demonstrated to impact the mitochondrial apoptotic pathway and induce apoptosis in many cell lines. However, the detailed mechanism of EV71-induced apoptosis remains to be elucidated. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. 2B recruited Bax to the mitochondria and induced Bax conformational activation. In addition, mitochondria isolated from 2B-expressing cells that were treated with a recombinant Bax showed increased Bax interaction and cytochrome c (Cyt c) release. Importantly, apoptosis in cells with either EV71 infection or 2B expression was dramatically reduced in Bax knockdown cells but not in Bak knockdown cells, suggesting that Bax played a pivotal role in EV71- or 2B-induced apoptosis. Further studies indicate that a hydrophobic region of 18 amino acids (aa) in the C-terminal region of 2B (aa 63 to 80) was responsible for the location of 2B in the mitochondria. A hydrophilic region of 14 aa in the N-terminal region of 2B was functional in Bax interaction and its subsequent activation. Moreover, overexpression of the antiapoptotic protein Bcl-XL abrogates 2B-induced release of Cyt c and caspase activation. Therefore, this study provides direct evidence that EV71 2B induces cell apoptosis and impacts the mitochondrial apoptotic pathway by directly modulating the redistribution and activation of proapoptotic protein Bax. EV71 infections are usually accompanied by severe neurological complications. It has also been postulated that the induction of cell apoptosis resulting from tissue damage is a possible process of EV71-related pathogenesis. In this study, we report that EV71 2B

  5. Expression and Significance of Bcl-2, Bax, Fas and Caspase-3 in Different Phases of Human Hemangioma

    Institute of Scientific and Technical Information of China (English)

    YANG Hong; DENG Chenguo; SHEN Shengguo; ZHANG Duanlian; YUYing

    2006-01-01

    The relationship between Bcl-2, Bax, Fas, caspase-3 and development of hemangioma and the molecular mechanism was investigated. By using immunohistochemical S-P method, proliferating cell nuclear antigen was detected. According to the classification of Mulliken in combination with PCNA expression, 27 cases were identified as proliferating hemangioma and 22 cases as involutive hemangioma. Five normal skin tissues around the tumor tissue served as controls. By using immunohistochemical technique, the expression of Bcl-2, Bax, Fax and Caspase-3 was detected. The cells expressing Bcl-2, Bax, Fax and cappase-3 were identified as hemangioma endothelia by immunohistochemical staining of Ⅷ factor. The average absorbance (A) and average positive area rate of Bcl-2, Bax, Fas and caspase-3 expression were measured by using HPIAS-2000 imaging analysis system. The results showed that the expression of Bcl-2 in the endothelia of proliferating hemangioma was significantly higher that in involutive degenerative hemangioma endothelia and vascular endothelia of normal skin tissue (P<0.01). The expression of Bax, Fas and Caspase-3 in the endothelia of involutive hemangioma was obviously higher than in the endothelia of proliferating hemangioma and normal skin tissue (P<0.01). The expression of BAx and Fas in endothelia of proliferating hemangioma was higher than in those of normal skin tissue (P<0.05). It was suggested that Bcl-2,Bax, Fas and caspase-3 might be involved in the development and involution of hemangioma. Bcl-2 could promote the growth of hemangioma by inhibiting apoptosis of endothelia. Bax, Fas and caspase-3 promote the switch of hemangioma from proliferation to involution by inducing the apoptosis of hemangioma endothelia.

  6. Bax-induced apoptosis shortens the life span of DNA repair defect Ku70-knockout mice by inducing emphysema.

    Science.gov (United States)

    Matsuyama, Shigemi; Palmer, James; Bates, Adam; Poventud-Fuentes, Izmarie; Wong, Kelvin; Ngo, Justine; Matsuyama, Mieko

    2016-06-01

    Cells with DNA damage undergo apoptosis or cellular senescence if the damage cannot be repaired. Recent studies highlight that cellular senescence plays a major role in aging. However, age-associated diseases, including emphysema and neurodegenerative disorders, are caused by apoptosis of lung alveolar epithelial cells and neurons, respectively. Therefore, enhanced apoptosis also promotes aging and shortens the life span depending on the cell type. Recently, we reported that ku70(-) (/) (-)bax(-) (/) (-) and ku70(-) (/) (-)bax(+/) (-) mice showed significantly extended life span in comparison with ku70(-) (/) (-)bax(+/+) mice. Ku70 is essential for non-homologous end joining pathway for DNA double strand break repair, and Bax plays an important role in apoptosis. Our study suggests that Bax-induced apoptosis has a significant impact on shortening the life span of ku70(-) (/) (-) mice, which are defective in one of DNA repair pathways. The lung alveolar space gradually enlarges during aging, both in mouse and human, and this age-dependent change results in the decrease of respiration capacity during aging that can lead to emphysema in more severe cases. We found that emphysema occurred in ku70(-) (/) (-) mice at the age of three-months old, and that Bax deficiency was able to suppress it. These results suggest that Bax-mediated apoptosis induces emphysema in ku70(-) (/) (-) mice. We also found that the number of cells, including bronchiolar epithelial cells and type 2 alveolar epithelial cells, shows a higher DNA double strand break damage response in ku70 KO mouse lung than in wild type. Recent studies suggest that non-homologous end joining activity decreases with increased age in mouse and rat model. Together, we hypothesize that the decline of Ku70-dependent DNA repair activity in lung alveolar epithelial cells is one of the causes of age-dependent decline of lung function resulting from excess Bax-mediated apoptosis of lung alveolar epithelial cells (and their

  7. Expression of the EP300, TP53 and BAX genes in colorectal cancer: Correlations with clinicopathological parameters and survival.

    Science.gov (United States)

    Kowalczyk, Anna E; Krazinski, Bartlomiej E; Godlewski, Janusz; Kiewisz, Jolanta; Kwiatkowski, Przemyslaw; Sliwinska-Jewsiewicka, Agnieszka; Kiezun, Jacek; Sulik, Marian; Kmiec, Zbigniew

    2017-07-01

    E1A binding protein P300 (EP300), tumor protein P53 (TP53) and BCL2 associated X, apoptosis regulator (BAX) genes encode proteins which cooperate to regulate important cellular processes. The present study aimed to determine the expression levels of EP300, TP53 and BAX in colorectal cancer (CRC) and to investigate their prognostic value and association with the progression of CRC. Tumor and matched unchanged colorectal tissues were collected from 121 CRC patients. Quantitative polymerase chain reaction and immunohistochemistry were used to assess the mRNA and protein levels of the studied genes. Altered expression of the studied genes in CRC tissues was observed at both the mRNA and protein levels. The depth of invasion was associated with TP53 mRNA levels and was correlated negatively with BAX mRNA expression. Moreover, a relationship between tumor location and BAX mRNA content was noted. BAX immunoreactivity was correlated positively with the intensity of p300 immunostaining and was associated with lymph node involvement and tumor-node-metastasis (TNM) disease stage. Univariate regression analysis revealed that overexpression of p53 and BAX in CRC tissues was associated with poor patient outcome. In conclusion, dysregulation of the expression of the studied genes was found to contribute to CRC pathogenesis. The association between p300 and BAX levels suggests the existence of an interdependent regulatory mechanism of their expression. Moreover, BAX expression may be regulated alternatively, in a p53-independent manner, since the lack of correlations between expression of these factors was observed.

  8. EGR-1/Bax pathway plays a role in vitamin E δ-tocotrienol-induced apoptosis in pancreatic cancer cells.

    Science.gov (United States)

    Wang, Chen; Husain, Kazim; Zhang, Anying; Centeno, Barbara A; Chen, Dung-Tsa; Tong, Zhongsheng; Sebti, Säid M; Malafa, Mokenge P

    2015-08-01

    The anticancer activity of δ-tocotrienol, a bioactive vitamin E present in whole grain cereals, annatto beans and palm fruit, is strongly dependent on its effect on the induction of apoptosis. δ-Tocotrienol-induced apoptosis is associated with consistent induction in the expression of the proapoptotic protein Bcl-2-associated X protein (Bax). The molecular mechanism by which δ-tocotrienol regulates Bax expression is unknown. We carried out a DNA microarray study that identified δ-tocotrienol induction of the zinc finger transcription factor EGR-1 in pancreatic cancer cells. Here, we provide evidence linking δ-tocotrienol-induced apoptosis in pancreatic cancer cells to EGR-1 regulation of Bax expression. Forced expression of EGR-1 induces Bax expression and apoptosis in pancreatic cancer cells. In contrast, knockdown of δ-tocotrienol-induced EGR-1 by small interfering RNA attenuated δ-tocotrienol-induced Bax expression and reduced δ-tocotrienol-induced apoptosis. Further analyses showed that de novo protein synthesis was not required for δ-tocotrienol-induced EGR-1 expression, suggesting a direct effect of δ-tocotrienol on EGR-1 expression. Furthermore, a chromatin immunoprecipitation assay demonstrated that EGR-1 binds to the Bax gene promoter. Finally, δ-tocotrienol treatment induced Bax expression and activated EGR-1 in the pancreatic neoplastic cells of the PDX-Cre Kras genetically engineered model of pancreatic cancer. Our study provides the first evidence for EGR-1 as a direct target of vitamin E δ-tocotrienol, suggesting that EGR-1 may act as a proapoptotic factor in pancreatic cancer cells via induction of Bax. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. MAC inhibitors antagonize the pro-apoptotic effects of tBid and disassemble Bax / Bak oligomers.

    Science.gov (United States)

    Peixoto, Pablo M; Teijido, Oscar; Mirzalieva, Oygul; Dejean, Laurent M; Pavlov, Evgeny V; Antonsson, Bruno; Kinnally, Kathleen W

    2017-02-01

    Mitochondrial Apoptotic Channel inhibitors or iMACs are di-bromocarbazole derivatives with anti-apoptotic function which have been tested and validated in several mouse models of brain injury and neurodegeneration. Owing to the increased therapeutic potential of these compounds, we sought to expand our knowledge of their mechanism of action. We investigated the kinetics of MAC inhibition in mitochondria from wild type, Bak, and Bax knockout cell lines using patch clamp electrophysiology, fluorescence microscopy, ELISA, and semiquantitative western blot analyses. Our results show that iMACs work through at least two mechanisms: 1) by blocking relocation of the cytoplasmic Bax protein to mitochondria and 2) by disassembling Bax and Bak oligomers in the mitochondrial outer membrane. iMACs exert comparable effects on channel conductance of Bax or Bak and similarly affect cytochrome c release from Bax or Bak-containing mitochondria. Interestingly, wild type mitochondria were more susceptible to inhibition than the Bak or Bax knockouts. Western blot analysis showed that wild type mitochondria had lower steady state levels of Bak in the absence of apoptotic stimulation.

  10. Resveratrol ameliorates hypoxia/ischemia-induced brain injury in the neonatal rat via the miR-96/Bax axis.

    Science.gov (United States)

    Bian, Hongen; Shan, Haijun; Chen, Tuanying

    2017-07-18

    This study was aimed to investigate the mechanism of resveratrol on amelioration of hypoxia/ischemia (H/I)-induced brain injury. The RT-PCR and western blot were used to detect the mRNA and protein expressions, respectively. The PC12 cell induced by OGD/R was as in vitro H/I brain injury model. The luciferase reporter assay was used to prove the relationship between Bax and miR-96, and the cell apoptosis was detected by MTT assay. The loss of MBP+ area in neonatal rats analyzed by immunohistochemistry was to evaluate the extent of brain injury. The miR-96 expression was decreased in the hippocampus and cerebral cortex of neonatal rats with H/I brain injury and the oxygenglucose deprivation/re-oxygenation (OGD/R)-induced PC12 cell, while Bax expression was opposite. And then the H/I rats and OGD/R-induced PC12 cell were treated with resveratrol (RSV); the results showed that the RSV could reverse the miR-96 and Bax expressions. Next, the luciferase reporter assay proved that Bax was a target of miR-96. We used the miR-96 inhibitor to suppress miR-96 expression in OGD/R-induced PC12 cell, and found that RSV regulated Bax expression and prevented OGD/R-induced PC12 cell apoptosis via miR-96. In addition, the immunohistochemistry was used to analyze the loss of MBP+ area in neonatal rats, and the result showed that the RSV significantly reduced the brain damage, increased miR-96 expression, and decreased Bax expression, while inhibition of miR-96 aggravated the brain damage and reversed the effect of RSV. Resveratrol ameliorates hypoxia/ischemia-induced brain injury in neonatal rat via the miR-96/ Bax axis.

  11. Germ cell apoptosis and expression of Bcl-2 and Bax in porcine testis under normal and heat stress conditions.

    Science.gov (United States)

    Fan, Xiaorui; Xi, Huaming; Zhang, Zhen; Liang, Yajun; Li, Qinghong; He, Junping

    2017-04-01

    The aim of this study was to examine whether an elevated ambient temperature (37-40°C) had an effect on the apoptosis of germ cells and the expression of Bcl-2 and Bax in porcine testis. Six boars were used. Three boars were subjected to an elevated ambient temperature (37-40°C, 7days, 3h per day) as a heat stress (HS) group. The other 3 boars were kept in a room temperature house (20-27°C) as a control group. All boars were castrated and the testes were harvested. TUNEL assay was used for the detection of apoptotic cells. Immunohistochemistry, Western blotting and quantitative real-time PCR were used to analyze protein and mRNA levels of Bcl-2 and Bax in response to heat treatment. The results showed that apoptotic signals increased under heat stress conditions compared with the control (PBax protein and mRNA did not show significant changes between the control and experimental group. Low to moderate Bax immunoreactivity staining was observed in all kinds of germ cells in the control group. Strong staining was observed in spermatogonia, and low to moderate Bax staining was observed in spermatocytes and spermatids. A redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed in the spermatogonia, spermatocytes and spermatids obtained in the heat treated group. These results showed that elevated ambient temperatures induced germ cell apoptosis. In response to heat stress, the expression of Bcl-2 increased and a redistribution of Bax from a cytoplasmic to a perinuclear or nuclear localization. This indicates that Bcl-2 and Bax may be involved in regulation of germ cell apoptosis induced by heat stress in boars. Copyright © 2016. Published by Elsevier GmbH.

  12. Expressão da proteína Bax no tecido mamário normal de mulheres no menacme tratadas com raloxifeno Expression of Bax protein in normal tissue of premenopausal women treated with raloxifene

    Directory of Open Access Journals (Sweden)

    Ana Maria Furtado-Veloso

    2008-04-01

    Full Text Available OBJETIVO: avaliar a expressão do antígeno Bax no epitélio mamário normal de mulheres na pré-menopausa tratadas com raloxifeno. MÉTODOS: estudo randomizado duplo-cego, envolvendo 33 mulheres pré-menopáusicas com fibroadenoma. As pacientes foram divididas em dois grupos: Placebo, (n=18 e Raloxifeno 60 mg, (n=15. A medicação foi usada durante 22 dias, começando no primeiro dia do ciclo menstrual. Uma biópsia foi realizada no 23º dia do ciclo menstrual, durante a qual uma amostra do tecido mamário normal adjacente ao fibroadenoma foi coletada e submetida a estudo imuno-histoquímico utilizando o anticorpo policlonal anti-Bax para avaliar a expressão da proteína Bax. A imunorreação para a proteína Bax foi avaliada, levando-se em consideração a intensidade e a fração de células coradas, cuja combinação resultou em um escore final de 0 a 6. Os casos com escore final >3 foram classificados como positivos para proteína Bax. O teste do c2 foi usado para análise estatística dos dados (pPURPOSE: to evaluate the expression of Bax antigen in the normal mammary epithelium of premenopausal women treated with raloxifene. METHODS: a randomized double-blind study was conducted in 33 ovulatory premenopausal women with fibroadenoma. Patients were divided into two groups: Placebo, (n=18 and Raloxifene 60 mg, (n=15. The medication was used for 22 days, beginning on the first day of the menstrual cycle. An excisional biopsy was carried out on the 23rd day of the menstrual cycle and a sample of normal breast tissue adjacent to the fibroadenoma was collected and submitted to immunohistochemical study using anti-Bax polyclonal antibody to evaluate the expression of Bax protein. Immunoreaction for Bax was evaluated taking into consideration intensity and fraction of stained cells, whose combination resulted in a final score ranging from 0 to 6. Cases with a final score >3 were classified as positive for Bax. The c2 test was used for statistical

  13. Grape Seed Proanthocyanidins Induce Apoptosis through p53, Bax, and Caspase 3 Pathways

    Directory of Open Access Journals (Sweden)

    Anshu M. Roy

    2005-01-01

    Full Text Available Grape seed proanthocyanidins (GSP have been shown to inhibit skin chemical carcinogenesis and photocarcinogenesis in mice. The mechanisms responsible for the anticarcinogenic effects of GSP are not clearly understood. Here, we report that treatment of JB6 C141 cells (a well-developed cell culture model for studying tumor promotion in keratinocytes and p53+/+ fibroblasts with GSP resulted in a dose-dependent induction of apoptosis. GSP-induced (20–80 g/ml apoptosis was observed by using immunofluorescence (27–90% apoptosis and flow cytometry (18–87% apoptosis. The induction of apoptosis by GSP was p53-dependent because it occurred mainly in cells expressing wild-type p53 (p53+/+; 15–80% to a much greater extent than in p53-deficient cells (p53-/-; 6–20%. GSP-induced apoptosis in JB6 C141 cells was associated with increased expression of the tumorsuppressor protein, p53, and its phosphorylation at Ser15. The antiapoptotic proteins, Bcl-2 and Bcl-xl, were downregulated by GSP, whereas the expression of the pro-apoptotic protein, Bax, and the levels of cytochrome c release, Apaf-1, caspase-9, and cleaved caspase 3 (p19 and p17 were markedly increased in JB6 C141 cells. The downregulation of Bcl-2 and upregulation of Bax were also observed in wild-type p53 (p53+/+ fibroblasts but was not observed in their p53-deficient counterparts. These data clearly demonstrate that GSP-induced apoptosis is p53-dependent and mediated through the Bcl-2, Bax, and caspase 3 pathways.

  14. Grape Seed Proanthocyanidins Induce Apoptosis through p53, Bax, and Caspase 3 Pathways1

    Science.gov (United States)

    Roy, Anshu M; Baliga, Manjeshwar S; Elmets, Craig A; Katiyar, Santosh K

    2005-01-01

    Abstract Grape seed proanthocyanidins (GSP) have been shown to inhibit skin chemical carcinogenesis and photocarcinogenesis in mice. The mechanisms responsible for the anticarcinogenic effects of GSP are not clearly understood. Here, we report that treatment of JB6 C141 cells (a well-developed cell culture model for studying tumor promotion in keratinocytes) and p53+/+ fibroblasts with GSP resulted in a dose-dependent induction of apoptosis. GSP-induced (20–80 g/ml) apoptosis was observed by using immunofluorescence (27–90% apoptosis) and flow cytometry (18–87% apoptosis). The induction of apoptosis by GSP was p53-dependent because it occurred mainly in cells expressing wild-type p53 (p53+/+; 15–80%) to a much greater extent than in p53-deficient cells (p53-/-; 6–20%). GSP-induced apoptosis in JB6 C141 cells was associated with increased expression of the tumor-suppressor protein, p53, and its phosphorylation at Ser15. The antiapoptotic proteins, Bcl-2 and Bcl-xl, were downregulated by GSP, whereas the expression of the pro-apoptotic protein, Bax, and the levels of cytochrome c release, Apaf-1, caspase-9, and cleaved caspase 3 (p19 and p17) were markedly increased in JB6 C141 cells. The downregulation of Bcl-2 and upregulation of Bax were also observed in wild-type p53 (p53+/+) fibroblasts but was not observed in their p53-deficient counterparts. These data clearly demonstrate that GSP-induced apoptosis is p53-dependent and mediated through the Bcl-2, Bax, and caspase 3 pathways. PMID:15720815

  15. Enhancing survival of mouse oocytes following chemotherapy or aging by targeting Bax and Rad51.

    Directory of Open Access Journals (Sweden)

    Loro L Kujjo

    Full Text Available BACKGROUND: Therapeutic approaches to preserve fertility in females undergoing cancer treatments are currently ineffective. This is partly due to limited knowledge of the molecular mechanisms that injured germ cells elicit to repair damage and survive or to abort repair and activate biochemical pathways leading to death. So far, we know that following spontaneously occurring or drug-induced DNA damage, the efficiency of DNA repair is a critical determinant of the cell's fate. The protein encoded by the Rad51 gene is one of several components recruited for homologous recombination-dependent DNA double-strand break repair in both somatic cells and germ cells. Recently, we showed that microinjection of recombinant Rad51 into AKR/J mouse oocytes decreased the extent of spontaneous DNA double-strand breaks, suppressed apoptosis, and restored the developmental competence in AKR/J embryos. Herein we characterized the nature of chemotherapy-induced lesions in oocytes, and the associated individual components of the DNA damage sensor and repair apparatus. For comparison, we also assessed parallel spontaneous changes in aging oocytes. METHODS: Data collected were derived from: analysis of apoptosis; immunodepletion; oocyte microinjections; immunocytochemistry; immunofluorescence; and CHIP-like assays. RESULTS: Our data show that: (i DNA damage in oocytes can be induced by both chemotherapy and spontaneously by the aging process; (ii oocytes possess the machinery and capability for repairing such DNA damage; (iii Rad51 is a critical player in the repair of both chemotherapy-induced and spontaneously-sustained DNA damage; and (iv in response to damage, oocytes exhibit an inverse functional relationship between presence of Bax and activity of Rad51. CONCLUSION/SIGNIFICANCE: Our results establish Rad51 and/or Bax as potential candidates that can be targeted for development of individualized chemotherapeutic interventions that are effective, but minimal in

  16. BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

    Science.gov (United States)

    Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

    2010-12-01

    During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

  17. Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax.

    Science.gov (United States)

    Starkov, Anatoly A; Polster, Brian M; Fiskum, Gary

    2002-10-01

    Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.

  18. Influence of Stress on the Expression of bcl-2/bax Protein in Spontaneously Hypertensive Rats

    Institute of Scientific and Technical Information of China (English)

    刘巍; 李为民; 孙宁玲; 陈源源; 任哲; 虞有智

    2002-01-01

    Objective To explore the influence of stress-induced increased sympathetic nerve activity on cardiomyocyte apoptosis and on the development of congestive heart failure. Methods 45male, 16-week-old spontaneously hypertensive rats (SHRs) were studied, in which 6 as controls. After the 6 controlled SHRs were examined by echocardiography, they were anesthetized and killed by decapitation.The other 39 were divided into the stress group ( n =20) and the control group ( n = 19), and both groups were observed from 16-week-old to 36-week-old. In the stress group, binding- stress model was used. Till 36week, all animals were echocardiographied, weighed and killed as described above. Cardiac bcl-2 and bax protein were quantified by western blot. Circulating catecholamine and angiotensin II (Ang II) were detected by radioimmunoasssy. Results Left ventricular volume ( P < 0.05), left ventricular mass ( P<0.05) and the ratio of ventricular mass to body weight were higher in 36 week than those of the 16 week SHRs, whereas the volumes of eject fraction (EF)manifested the trend of decline, P< 0.05, bindingstress for 20 weeks made this trend significantly, P<0.05. With the increase of age, the serum nore pinephrine (NE), epinephrine (E) and Ang Ⅱ in creased, suggesting that the binding- stress triggered the activity of central sympathetic nerve system. The cardiac bcl-2 protein was higher in 36 week than 16week, P >0.05, whereas the bax protein increased significantly with the increase of age, P < 0.05, and so was the ratio of bax to bcl-2, P < 0.05. Conclusions The model of binding-stress can effectivelyactivate central sympathetic system, thus and mimic the neuroendocrine states. From 16 to 36 week, the process of cardiac apoptosis aggravated and the increased sympathetic activity would exacerbate rather than relieve this trend.

  19. Expression of Ki-67, Bcl-2 and Bax in the First Trimester Abortion Materials

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    Ender DÜZCAN

    2010-01-01

    Full Text Available Objective: The aim of this study was to investigate possible similar or different mechanisms in recurrent and spontaneous abortion by evaluating immunohistochemical correlation between proliferation marker Ki-67, and apoptosis markers Bcl-2 and Bax in the fetal trophoblasts and maternal deciduas from abortion material.Material and Method: Eighty samples of curettage materials from 65 abortion patients histopathologically diagnosed “decidua showing Arias-Stella reaction and chorionic villi” or only “decidua showing Arias-Stella reaction” were included in the study. Hematoxylin&Eosin stained sections from all cases were re-evaluated and further stained immunohistochemically using antibodies against Ki-67, Bcl-2 and Bax.Results: Proliferation rate evaluated by Ki-67 expression both in the cytotrophoblastic cells and decidua was found to be significantly lower in spontaneous and recurrent abortions compared to evacuation abortion. The extent of Bcl-2 expression in syncytiotrophoblastic cells covering villous stroma was also decreased in spontaneous abortion. There were no significant differences between spontaneous and recurrent abortions in terms of Bcl-2 expression in syncytiotrophoblasts and Ki-67 proliferation index in cytotrophoblastic cells or decidua. Bax staining showed minimal decidual expression in a few spontaneous and recurrent abortions.Conclusion: We concluded that proliferation rate was decreased in fetal villous cytotrophoblasts and maternal deciduas in spontaneous and recurrent abortions. We also proposed that loss of Bcl-2 expression in syncytiotrophoblasts may cause abortion in a subset of cases. However, the data from spontaneous and recurrent abortions did not not support the presence of different mechanisms in both groups.

  20. Effect of Flunarizine on the Expression of Bax mRNA in Hippocampus of Amygdala Kindling Seizures Rat%氟桂利嗪对杏仁核点燃鼠海马Bax mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    梁代义; 伍国锋; 董佑忠; 庞成

    2005-01-01

    目的:观察氟桂利嗪对杏仁核点燃鼠癎性发作及海马促凋亡基因Bax mRNA表达的影响.方法:建立杏仁核点燃模型,予不同剂量氟桂利嗪灌喂点燃鼠.原位杂交法检测鼠脑海马Bax mRNA表达,图像分析软件测量阳性细胞平均吸光度.结果:正常大鼠海马存在少量Bax mRNA表达阳性细胞,点燃鼠海马各区Bax mRNA表达阳性细胞数及平均吸光度增加,氟桂利嗪处理后平均吸光度下降与剂量有关.结论:氟桂利嗪具有抗癫癎效应和拮抗点燃鼠海马Bax mRNA表达的作用.

  1. Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status

    OpenAIRE

    Mirjolet, J-F; Barberi-Heyob, M; Didelot, C; Peyrat, J-P; Abecassis, J; Millon, R.; Merlin, J-L

    2000-01-01

    p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53...

  2. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ruizhao, E-mail: liruizhao1979@126.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Zhang, Li, E-mail: Zhanglichangde@163.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Southern Medical University, Guangzhou, Guangdong (China); Shi, Wei, E-mail: shiwei.gd@139.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Zhang, Bin, E-mail: zhangbinyes@yahoo.com.cn [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Liang, Xinling, E-mail: xinlingliang@yahoo.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Liu, Shuangxin, E-mail: mplsxi@yahoo.com.cn [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Wang, Wenjian, E-mail: wwjph@yahoo.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China)

    2013-04-15

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca{sup 2+} was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca{sup 2+}]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway

  3. Robotic assistance for performing vocational rehabilitation activities using BaxBot.

    Science.gov (United States)

    Ashley, Kyle; Alqasemi, Redwan; Dubey, Rajiv

    2017-07-01

    Activities of Daily Living (ADL's) refer to tasks that people do on a daily basis, such as self-feeding, cleaning the house, or bathing. These activities often require a degree of functional mobility that may be outside the ability of a person suffering from cognitive or physical impairment. This work describes methods of performing ADL's with a mobile robotic system. We examined the needs of potential users and caregivers through surveys to determine the most needed applications for robotic assistance. Using this information, we extended the functionality of our BaxBot mobile robotic system to provide meaningful, autonomous assistance in performing three specific ADL's with minimal user interaction.

  4. NOXA-induced alterations in the Bax/Smac axis enhance sensitivity of ovarian cancer cells to cisplatin.

    Directory of Open Access Journals (Sweden)

    Chao Lin

    Full Text Available Ovarian cancer is the most common cause of death from gynecologic malignancy. Deregulation of p53 and/or p73-associated apoptotic pathways contribute to the platinum-based resistance in ovarian cancer. NOXA, a pro-apoptotic BH3-only protein, is identified as a transcription target of p53 and/or p73. In this study, we found that genetic variants of Bcl-2 proteins exist among cisplatin-sensitive and -resistant ovarian cancer cells, and the responses of NOXA and Bax to cisplatin are regulated mainly by p53. We further evaluated the effect of NOXA on cisplatin. NOXA induced apoptosis and sensitized A2780s and SKOV3 cells to cisplatin in vitro and in vivo. The effects were mediated by elevated Bax expression, enhanced caspase activation, release of Cyt C and Smac into the cytosol. Furthermore, gene silencing of Bax or Smac significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells, whereas overexpression of Bax or addition of Smac-N7 peptide significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells. To our knowledge, these data suggest a new mechanism by which NOXA chemosensitized ovarian cancer cells to cisplatin by inducing alterations in the Bax/Smac axis. Taken together, our findings show that NOXA is potentially useful as a chemosensitizer in ovarian cancer therapy.

  5. NOXA-induced alterations in the Bax/Smac axis enhance sensitivity of ovarian cancer cells to cisplatin.

    Science.gov (United States)

    Lin, Chao; Zhao, Xin-yu; Li, Lei; Liu, Huan-yi; Cao, Kang; Wan, Yang; Liu, Xin-yu; Nie, Chun-lai; Liu, Lei; Tong, Ai-ping; Deng, Hong-xin; Li, Jiong; Yuan, Zhu; Wei, Yu-quan

    2012-01-01

    Ovarian cancer is the most common cause of death from gynecologic malignancy. Deregulation of p53 and/or p73-associated apoptotic pathways contribute to the platinum-based resistance in ovarian cancer. NOXA, a pro-apoptotic BH3-only protein, is identified as a transcription target of p53 and/or p73. In this study, we found that genetic variants of Bcl-2 proteins exist among cisplatin-sensitive and -resistant ovarian cancer cells, and the responses of NOXA and Bax to cisplatin are regulated mainly by p53. We further evaluated the effect of NOXA on cisplatin. NOXA induced apoptosis and sensitized A2780s and SKOV3 cells to cisplatin in vitro and in vivo. The effects were mediated by elevated Bax expression, enhanced caspase activation, release of Cyt C and Smac into the cytosol. Furthermore, gene silencing of Bax or Smac significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells, whereas overexpression of Bax or addition of Smac-N7 peptide significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells. To our knowledge, these data suggest a new mechanism by which NOXA chemosensitized ovarian cancer cells to cisplatin by inducing alterations in the Bax/Smac axis. Taken together, our findings show that NOXA is potentially useful as a chemosensitizer in ovarian cancer therapy.

  6. Cytosolic BNIP3 Dimer Interacts with Mitochondrial BAX Forming Heterodimers in the Mitochondrial Outer Membrane under Basal Conditions.

    Science.gov (United States)

    Hendgen-Cotta, Ulrike B; Esfeld, Sonja; Rudi, Katharina; Miinalainen, Ilkka; Klare, Johann P; Rassaf, Tienush

    2017-03-23

    The primary function of mitochondria is energy production, a task of particular importance especially for cells with a high energy demand like cardiomyocytes. The B-cell lymphoma (BCL-2) family member BCL-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is linked to mitochondrial targeting after homodimerization, where it functions in inner membrane depolarization and permeabilization of the mitochondrial outer membrane (MOM) mediating cell death. We investigated the basal distribution of cardiac BNIP3 in vivo and its physical interaction with the pro-death protein BCL2 associated X, apoptosis regulator (BAX) and with mitochondria using immunoblot analysis, co-immunoprecipitation, and continuous wave and pulsed electron paramagnetic resonance spectroscopy techniques. We found that BNIP3 is present as a dimer in the cytosol and in the outer membrane of cardiac mitochondria under basal conditions. It forms disulfide-bridged, but mainly non-covalent dimers in the cytosol. Heterodimers with BAX are formed exclusively in the MOM. Furthermore, our results suggest that BNIP3 interacts with the MOM directly via mitochondrial BAX. However, the physical interactions with BAX and the MOM did not affect the membrane potential and cell viability. These findings suggest that another stimulus other than the mere existence of the BNIP3/BAX dimer in the MOM is required to promote BNIP3 cell-death activity; this could be a potential disturbance of the BNIP3 distribution homeostasis, namely in the direction of the mitochondria.

  7. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly.

    Science.gov (United States)

    Kuwana, Tomomi; Bouchier-Hayes, Lisa; Chipuk, Jerry E; Bonzon, Christine; Sullivan, Barbara A; Green, Douglas R; Newmeyer, Donald D

    2005-02-18

    Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.

  8. PUMA promotes Bax translocation in FOXO3a-dependent pathway during STS-induced apoptosis

    Science.gov (United States)

    Zhang, Yingjie; Chen, Qun

    2009-08-01

    PUMA (p53 up-regulated modulator of apoptosis, also called Bbc3) was first identified as a BH3-only Bcl-2 family protein that is transcriptionally up-regulated by p53 and activated upon p53-dependent apoptotic stimuli, such as treatment with DNA-damaging drugs or UV irradiation. Recently studies have been shown that Puma is also up-regulated in response to certain p53-independent apoptotic stimuli, such as growth factor deprivation or treatment with glucocorticoids or STS (staurosporine). However, the molecular mechanisms of PUMA up-regulation and how PUMA functions in response to p53-independent apoptotic stimuli remain poorly understood. In this study, based on real-time single cell analysis, flow cytometry and western blotting technique, we investigated the function of PUMA in living human lung adenocarcinoma cells (ASTC-a-1) after STS treatment. Our results show that FOXO3a was activated by STS stimulation and then translocated from cytosol to nucleus. The expression of PUMA was up-regulated via a FOXO3a-dependent manner after STS treatment, while p53 had little function in this process. Moreover, cell apoptosis and Bax translocation induced by STS were not blocked by Pifithrin-α (p53 inhibitor), which suggested that p53 was not involved in this signaling pathway. Taken together, these results indicate that PUMA promoted Bax translocation in a FOXO3a-dependment pathway during STS-induced apoptosis, while p53 was dispensable in this process.

  9. Killing of Brain Tumor Cells by Hypoxia-Responsive Element Mediated Expression of BAX

    Directory of Open Access Journals (Sweden)

    Hangjun Ruan

    1999-11-01

    Full Text Available The presence of radioresistant hypoxic cells in human brain tumors limits the overall effectiveness of conventional fractionated radiation therapy. Tumor-specific therapies that target hypoxic cells are clearly needed. We have investigated the expression of suicide genes under hypoxia by a hypoxia-responsive element (HRE, which can be activated through hypoxia-inducible factor-1 (HIF-1. We transfected plasmids containing multiple copies of HIRE into U-87 MG and U-251 MG-NCI human brain tumor cells and tested their ability to induce LacZ gene expression under anoxia. Gene expression under anoxia versus oxia was increased about 12-fold for U-87 MG cells and about fourfold for U-251 MG-NCI cells. At intermediate hypoxic conditions, increased LacZ gene expression in U-87 MG cells was induced by the plasmid that contained three HREs, but not by the plasmid with two HREs. Lastly, when we placed a suicide gene BAX under the control of HREs, cells transfected with the BAX plasmids were preferentially killed through apoptosis under anoxia. Our studies demonstrate that HRE-regulated gene expression is active in brain tumor cells, and that the amount of increased gene expression obtained is dependent on the cell line, the HIRE copy number, and the degree of hypoxia.

  10. Bz-423 superoxide signals B cell apoptosis via Mcl-1, Bak, and Bax.

    Science.gov (United States)

    Blatt, Neal B; Boitano, Anthony E; Lyssiotis, Costas A; Opipari, Anthony W; Glick, Gary D

    2009-10-15

    Bz-423 is a pro-apoptotic 1,4-benzodiazepine with therapeutic properties in murine models of lupus demonstrating selectivity for autoreactive lymphocytes. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. In order to understand some of the features that contribute to the increased sensitivity of lymphocytes, we report the signaling pathway engaged by Bz-423 in a Burkitt lymphoma cell line (Ramos). Following the generation of superoxide, Bz-423-induced apoptosis requires the activation of Bax and Bak to induce mitochondrial outer membrane permeabilization and cytochrome c release. Knockdown of the BH3-only proteins Bad, Bim, Bik, and Puma inhibits Bz-423 apoptosis, suggesting that these proteins serve as upstream sensors of the oxidant stress induced by Bz-423. Treatment with Bz-423 results in superoxide-dependent Mcl-1 degradation, implicating this protein as the link between Bz-423-induced superoxide and Bax and Bak activation. In contrast to fibroblasts, B cell death induced by Bz-423 is independent of c-Jun N-terminal kinase. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a specific apoptotic response that differs across cell types.

  11. Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax.

    Science.gov (United States)

    Blatt, Neal B; Boitano, Anthony E; Lyssiotis, Costas A; Opipari, Anthony W; Glick, Gary D

    2008-11-01

    Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.

  12. CO-EXPRESSIONS OF SURVIVIN GENE,BCL-2 AND BAX PROTEINS IN OVARIAN CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    林蓓; 张淑兰; 赵长清

    2004-01-01

    Objective To characterize the cellular properties of ovarian cancer, we examined the correlation between the expression of apoptosis-related gene survivin and those of Bcl-2 and Bar proteins. Methods Expressions of survivin mRNA, and Bcl-2 and Bax proteins in 35 cases of ovarian carcinoma, 10 cases of borderline carcinoma, 10 cases of benign tumors and 10 cases of normal tissue were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry SABC method, respectively. Results Expression of survivin gene was detected in a significantly greater proportion in ovarian carcinoma and borderline carcinoma than those in benign tumors and normal tissues. Although there was no relationship between expression of survivin gene and FIGO stage, histologic grade, pathological type and lymphatic metastasis, expressions of Bcl-2 and Bar proteins were positively and negatively correlated with that of survivin gene, respectively. Conclusion Survivin may play an important role in pathogenesis of ovarian carcinoma, with a synergistic role of apoptosis-related gene Bcl-2protein and an antagonistic role of Bax protein in formation and progression of ovarian carcinoma.

  13. Ionic conduction in BaxCe0.8Pr0.2O3-

    Institute of Scientific and Technical Information of China (English)

    WANG; Maoyuan

    2009-01-01

    [1]Iwahara H,Yajima T,Ushida H.Effect of ionic radii of dopants on mixed ionic conductions (H++O2) in BaCeO3-based electrolytes.SolidState Ionics,1994,70/71:267.[2]Taniguchi N,Yasumoto E,Nakagiri Y,Gamo T.Sensing properties of an oxygen sensor using BaCe0.8Gd0.2O3-a ceram-ics as electrolytes.J.Electrochem.Soc.,1998,145(5):1744.[3]de Arcllano-Lopez A R,Goretta K C,Park E T,Dorris S E,Balchandran U,Routbort J L.High-temperature deformation of a BaCe0.8Y0.2O3-y+Ni composite.J.Fur.Ceram.Soc.,2002,22:2555.[4]Ma G L,Shimura T,Iwahara H.Ionic conduction and non-stoichiometry in BaxCe0.90Y0.10O3-a.Solid State lonics,1998,110:103.[5]Ma G L,Matsumoto H,Iwahara H.Ionic conduction and non-stoichiometry in non-doped BaxCeO3-a.Solid State Ionics,1999,122:237.[6]Ma Guilin,Qiu Ligan,Chen Rong.Performance of the solid oxide fuel cell based on BaxCe0.8Sm0.2O3-a.Acta Chimica Sinica (in Chin.),2002,60(12):2135.[7]Ma G L,Qiu L G,Tao W H,Zhou L,Chen R.Ionic conduc-tion in BaxCe0.8Sm0.2O3-a solid electrolyte.J.Chin.Rare Earths Soc.(in Chin.),2003,21(2):236.[8]Qiu L G,Ma G L,Wen D J.Study on preparation and electri-cal properties of Ba1.03Ce0.8Eu0.2O3-a solid electrolyte.J.Rare Earths,2004,22(5):678.[9]Qiu L G,Ma G L,Wen D J.Ionic conduction in BaxCe0.8Er0.2O3-a.Solid State lonics,2004,166:69.[10]Wang M Y,Qin L G.Mixed conduction in BaCe0.8Pr0.2O3-a ceramic.Chin.J.Chem.Phys.,2008,21:286.[11]Bonanos N.Transport properties and conduction mechanism in high-temperature protonic conductots.Solid State Ionics,1992,53-56:967.[12]Ma G L,Shimura T,Iwahara H.Simultaneous doping with La3+ and y3+ for Ba2+-and Ce4+-sites in BaCeO3 and the ionic conduction.Solid State Ionics,1999,120:51.[13]Shima D,Halle S M.The influence of cation non-stoichiome-try on the properties of undoped and gadolinia-doped barium ceres.Solid State Ionics,1997,97:443.

  14. BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy.

    Science.gov (United States)

    Pedro, Jose Manuel Bravo-San; Wei, Yongjie; Sica, Valentina; Maiuri, Maria Chiara; Zou, Zhongju; Kroemer, Guido; Levine, Beth

    2015-01-01

    Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-XL is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-XL may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.

  15. Exhaustive Training Increases Uncoupling Protein 2 Expression and Decreases Bcl-2/Bax Ratio in Rat Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    W. Y. Liu

    2013-01-01

    Full Text Available This work investigates the effects of oxidative stress due to exhaustive training on uncoupling protein 2 (UCP2 and Bcl-2/Bax in rat skeletal muscles. A total of 18 Sprague-Dawley female rats were randomly divided into three groups: the control group (CON, the trained control group (TC, and the exhaustive trained group (ET. Malondialdehyde (MDA, superoxide dismutase (SOD, xanthine oxidase (XOD, ATPase, UCP2, and Bcl-2/Bax ratio in red gastrocnemius muscles were measured. Exhaustive training induced ROS increase in red gastrocnemius muscles, which led to a decrease in the cell antiapoptotic ability (Bcl-2/Bax ratio. An increase in UCP2 expression can reduce ROS production and affect mitochondrial energy production. Thus, oxidative stress plays a significant role in overtraining.

  16. Effects of curcumin on hippocampal Bax and Bcl-2 expression and cognitive function of a rat model of Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    Yunliang Wang; Honglei Yin; Jiyu Lou; Bing Han; Xinyue Qin; Fanchao Meng; Shuang Geng; Yajun Liu

    2011-01-01

    We tested the effects of curcumin treatment on a rat model of Alzheimer's disease induced by beta-amlyoid (Aβ1-40) expression. We investigated alterations in the expression of the apoptosis-related genes Bax and Bcl-2 in the hippocampus, as well as changes in the spatial memory and cognitive function of the rats. Reverse transcription-polymerase chain reaction and immunohistochemistry results showed that Bax expression was remarkably decreased and Bcl-2 expression was increased in the rat Alzheimer's disease model after curcumin treatment. Morris water maze results showed that the average time of escape latency was shortened in the curcumin treated model rats. Our study shows that curcumin can significantly improve spatial learning and memory functions in rats with Aβ1-40-induced Alzheimer's disease by modulating Bax and Bcl-2 expression.

  17. Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 colon cancer cells.

    Science.gov (United States)

    Coker-Gurkan, Ajda; Arisan, Elif Damla; Obakan, Pinar; Guvenir, Esin; Unsal, Narcin Palavan

    2014-10-15

    The purine-derived analogs, roscovitine and purvalanol are selective synthetic inhibitors of cyclin-dependent kinases (CDKs) induced cell cycle arrest and lead to apoptotic cell death in various cancer cells. Although a number of studies investigated the molecular mechanism of each CDK inhibitor on apoptotic cell death mechanism with their therapeutic potential, their regulatory role on autophagy is not clarified yet. In this paper, our aim was to investigate molecular mechanism of CDK inhibitors on autophagy and apoptosis in wild type (wt) and Bax deficient HCT 116 cells. Exposure of HCT 116 wt and Bax(-/-) cells to roscovitine or purvalanol for 24h decreased cell viability in dose-dependent manner. However, Bax deficient HCT 116 cells were found more resistant against purvalanol treatment compared to wt cells. We also established that both CDK inhibitors induced apoptosis through activating mitochondria-mediated pathway in caspase-dependent manner regardless of Bax expression in HCT 116 colon cancer cells. Concomitantly, we determined that purvalanol was also effective on autophagy in HCT 116 colon cancer cells. Inhibition of autophagy by 3-MA treatment enhanced the purvalanol induced apoptotic cell death in HCT 116 Bax(-/-) cells. Our results revealed that mechanistic action of each CDK inhibitor on cell death mechanism differs. While purvalanol treatment activated apoptosis and autophagy in HCT 116 cells, roscovitine was only effective on caspase-dependent apoptotic pathway. Another important difference between two CDK inhibitors, although roscovitine treatment overcame Bax-mediated drug resistance in HCT 116 cells, purvalanol did not exert same effect. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70-Bax interaction in prostate cancer.

    Science.gov (United States)

    Shukla, Sanjeev; Fu, Pingfu; Gupta, Sanjay

    2014-05-01

    Dysfunction of the apoptotic pathway in prostate cancer cells confers apoptosis resistance towards various therapies. A novel strategy to overcome resistance is to directly target the apoptotic pathway in cancer cells. Apigenin, an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms which are not fully explored. In the present study we provide novel insight into the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment resulted in significant decrease in cell viability and apoptosis induction with the increase of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. These results suggest that apigenin targets inhibitor of apoptosis proteins and Ku70-Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy.

  19. Safe switching from a pdFIX (Immunine®) to a rFIX (Bax326).

    Science.gov (United States)

    Solano Trujillo, M H; Stasyshyn, O; Rusen, L; Serban, M; Lamas, J L; Perina, F G; Urasinski, T; Oh, M; Knowlton, W B; Valenta-Singer, B; Pavlova, B G; Abbuehl, B

    2014-09-01

    The ability to switch between coagulation factors safely is of common interest to haemophilia patients and treating physicians. This is the first formal prospective comparative evaluation of safety, efficacy and incremental recovery of a plasma-derived FIX (pdFIX) and a recombinant FIX (rFIX) in the same haemophilia B patients following a switch from pdFIX Immunine® to a recently developed rFIX Bax326 product. Patients (aged <65 years) who completed a pretreatment study which prospectively documented the exposure to Immunine® and monitored FIX inhibitors while receiving prophylactic treatment were transitioned into pivotal (patients aged 12-65 years) and paediatric (patients aged <12 years) clinical studies investigating prophylaxis and treatment of bleeding episodes with Bax326. None of the 44 patients developed inhibitory or specific binding anti-FIX antibodies during the course of the studies. A total of 38 unrelated adverse events (AEs) were occurred in 20/44 (45.5%) subjects during the Immunine® study. Following a switch to Bax326, 51 AEs were reported in 25/44 (56.8%) subjects. The incidence of AEs related to Bax326 treatment (two episodes of dysgeusia in one patient) was low (2.3%); there were no serious adverse reactions. The comparison between Immunine® and Bax326 demonstrated analogous haemostatic characteristics and annualized bleeding rates. Overall, there is direct evidence indicating a safe and clinically effective transition from a pdFIX (Immunine®) to a newly developed rFIX (Bax326(1) ) for prophylaxis and treatment of bleeding in previously treated patients of all age cohorts with severe or moderately severe haemophilia B.

  20. Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70–Bax interaction in prostate cancer

    Science.gov (United States)

    Shukla, Sanjeev; Fu, Pingfu; Gupta, Sanjay

    2014-01-01

    Dysfunction of the apoptotic pathway in prostate cancer cells confers apoptosis resistance towards various therapies. A novel strategy to overcome resistance is to directly target the apoptotic pathway in cancer cells. Apigenin, an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms which are not fully explored. In the present study we provide novel insight into the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment resulted in significant decrease in cell viability and apoptosis induction with the increase of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. These results suggest that apigenin targets inhibitor of apoptosis proteins and Ku70–Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy. PMID:24563225

  1. Recombinant factor IX (BAX326) in previously treated paediatric patients with haemophilia B: a prospective clinical trial.

    Science.gov (United States)

    Urasinski, T; Stasyshyn, O; Andreeva, T; Rusen, L; Perina, F G; Oh, M S; Chapman, M; Pavlova, B G; Valenta-Singer, B; Abbuehl, B E

    2015-03-01

    A newly developed recombinant factor IX (BAX326(1) ) was investigated for prophylactic use in paediatric patients aged 96% of bleeds (100% of minor, 88.9% of moderate and 100% of major bleeds); the majority (88.5%) resolved after 1-2 infusions. Longer T1/2 and lower IR were observed in younger children (<6 years) compared to those aged 6 to 12 years. BAX326 administered as prophylactic treatment as well as for controlling bleeds is efficacious and safe in paediatric patients aged <12 years with haemophilia B.

  2. Stripe order in La2-xBaxCuO4 in high magnetic fields

    Science.gov (United States)

    Huecker, M.; von Zimmermann, M.; Xu, Z. J.; Wen, J. S.; Gu, G. D.; Zaliznyak, I. A.; Chung, J.-H.; Choi, E. S.; Tranquada, J. M.

    2012-02-01

    The observation of enhanced spin stripe order in the vortex cores of La2-xSrxCuO4 has been a landmark experiment that revealed the intimate connection between superconductivity and incommensurate antiferromagnetism. Only recently we have observed a corresponding field dependence of the spin and, more importantly, of the charge stripe order in La2-xBaxCuO4. Here we present our recent results from neutron diffraction, x-ray diffraction, and torque measurements in high magnetic fields. These helped us to establish the field versus temperature and doping phase diagrams for spin and charge order, and to further corroborate the stripe model as the more appropriate description than for example spiral and vortex states.

  3. Immortalization of mouse myogenic cells can occur without loss of p16INK4a, p19ARF, or p53 and is accelerated by inactivation of Bax

    Directory of Open Access Journals (Sweden)

    Kravetz Amanda J

    2004-01-01

    Full Text Available Abstract Background Upon serial passaging of mouse skeletal muscle cells, a small number of cells will spontaneously develop the ability to proliferate indefinitely while retaining the ability to differentiate into multinucleate myotubes. Possible gene changes that could underlie myogenic cell immortalization and their possible effects on myogenesis had not been examined. Results We found that immortalization occurred earlier and more frequently when the myogenic cells lacked the pro-apoptotic protein Bax. Furthermore, myogenesis was altered by Bax inactivation as Bax-null cells produced muscle colonies with more nuclei than wild-type cells, though a lower percentage of the Bax-null nuclei were incorporated into multinucleate myotubes. In vivo, both the fast and slow myofibers in Bax-null muscles had smaller cross-sectional areas than those in wild-type muscles. After immortalization, both Bax-null and Bax-positive myogenic cells expressed desmin, retained the capacity to form multinucleate myotubes, expressed p19ARF protein, and retained p53 functions. Expression of p16INK4a, however, was found in only about half of the immortalized myogenic cell lines. Conclusions Mouse myogenic cells can undergo spontaneous immortalization via a mechanism that can include, but does not require, loss of p16INK4a, and also does not require inactivation of p19ARF or p53. Furthermore, loss of Bax, which appears to be a downstream effector of p53, accelerates immortalization of myogenic cells and alters myogenesis.

  4. Comparison of automated BAX polymerase chain reaction and standard culture methods for detection of Listeria monocyogenes in blue crab meat (Callinectus sapidus) and blue crab processing plants

    Science.gov (United States)

    This study compared the BAX Polymerase Chain Reaction method (BAX PCR) with the Standard Culture Method (SCM) for detection of L. monocytogenes in blue crab meat and crab processing plants. The aim of this study was to address this data gap. Raw crabs, finished products and environmental sponge samp...

  5. Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells

    DEFF Research Database (Denmark)

    Nielsen, M; Kaestel, C G; Eriksen, K W

    1999-01-01

    promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic...

  6. Expressão das proteínas BCL-2 e BAX em tumores astrocíticos humanos Expression of BCL-2 and BAX proteins in human astrocytic tumors

    Directory of Open Access Journals (Sweden)

    Mário Henrique Girão Faria

    2006-08-01

    Full Text Available INTRODUÇÃO: Os astrocitomas constituem os mais freqüentes tumores primários do sistema nervoso central (SNC. Admite-se que parte do crescimento tumoral seja resultante da inibição da morte celular programada: a apoptose. Tal fenômeno é basicamente regulado pelo equilíbrio entre moléculas antiapoptóticas (ex.: B-cell lymphoma protein 2 [BCL-2] e pró-apoptóticas (ex.: BCL-2 associated protein X [BAX]. OBJETIVO: O presente estudo objetivou avaliar a expressão de BCL-2 e BAX em tumores astrocíticos humanos. MATERIAL E MÉTODOS: Procedeu-se ao estudo imuno-histoquímico dessas proteínas utilizando-se o método da avidina-biotina-peroxidase em 55 astrocitomas (13 do grau I, 14 do II, sete do III e 21 do grau IV e cinco amostras de tecido cerebral não-tumoral (grupo controle. RESULTADOS: Os índices de positividade para BCL-2 e BAX demonstraram propensão ao acréscimo, de acordo com a gradação tumoral, com positividade geral de 43,26% e 24,67%, respectivamente. Essas proteínas não foram detectadas entre os espécimes não-tumorais. Os escores de marcação para BCL-2 apresentaram tendência ao aumento conforme a progressão histológica, enquanto os para BAX mostraram-se semelhantes nas diversas graduações. A análise conjunta dessas proteínas demonstrou significativa correlação com a gradação tumoral (p BACKGROUND: Astrocytomas represent the most frequent primary tumors of the central nervous system. Admittedly, part of tumor growth is due to inhibition of programmed cell death: the apoptosis. This phenomenon is basically regulated by the balance between anti-apoptotic (e.g.: B-cell lymphoma protein 2 [BCL-2] and pro-apoptotic (e.g.: BCL-2 associated protein X [BAX] molecules. OBJECTIVE: The present study aimed to evaluate the expression of BCL-2 and BAX in human astrocytic tumors of different histopathological grades. MATERIAL AND METHOD: An immunohistochemical study of those proteins using the avidin

  7. DuPont Qualicon BAX System polymerase chain reaction assay. Performance Tested Method 100201.

    Science.gov (United States)

    Tice, George; Andaloro, Bridget; Fallon, Dawn; Wallace, F Morgan

    2009-01-01

    A recent outbreak of Salmonella in peanut butter has highlighted the need for validation of rapid detection methods. A multilaboratory study for detecting Salmonella in peanut butter was conducted as part of the AOAC Research Institute Emergency Response Validation program for methods that detect outbreak threats to food safety. Three sites tested spiked samples from the same master mix according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) method and the BAX System method. Salmonella Typhimurium (ATCC 14028) was grown in brain heart infusion for 24 h at 37 degrees C, then diluted to appropriate levels for sample inoculation. Master samples of peanut butter were spiked at high and low target levels, mixed, and allowed to equilibrate at room temperature for 2 weeks. Spike levels were low [1.08 most probable number (MPN)/25 g]; high (11.5 MPN/25 g) and unspiked to serve as negative controls. Each master sample was divided into 25 g portions and coded to blind the samples. Twenty portions of each spiked master sample and five portions of the unspiked sample were tested at each site. At each testing site, samples were blended in 25 g portions with 225 mL prewarmed lactose broth until thoroughly homogenized, then allowed to remain at room temperature for 55-65 min. Samples were adjusted to a pH of 6.8 +/- 0.2, if necessary, and incubated for 22-26 h at 35 degrees C. Across the three reporting laboratories, the BAX System detected Salmonella in 10/60 low-spike samples and 58/60 high-spike samples. The reference FDA-BAM method yielded positive results for 11/60 low-spike and 58/60 high-spike samples. Neither method demonstrated positive results for any of the 15 unspiked samples.

  8. ISCHEMIC PRECONDITIONING RELIEVES ISCHEMIA/REPERFUSION INJURY OF HIPPOCAMPUS NEURONS IN RAT BY INHIBITING p53 AND BAX EXPRESSIONSA

    Institute of Scientific and Technical Information of China (English)

    Hui-min Liu; Jing-xin Li; Lian-bi Chen

    2007-01-01

    Objective To examine whether ischemic preconditioning (IPC) can protect neuron against delayed death in CA1 subfield of hippocampus following reperfusion of a lethal ischemia in rats, and explore the role of p53 and bax in this process.Methods We examined the effect of IPC on delayed neuron death, neuron apoptosis, expressions of p53 and bax gene in the CA1 area of hippocampus in the rats using HE staining, flow cytometry, RT-PCR, and immunohistochemis-try technique.Results IPC enhanced the quantity of survival cells in the CA1 region of hippocampus (216 ±9 cells/0. 72 mm2 vs. 30 ±5 cells/0. 72 mm2, P<0. 01), decreased the percentages of apoptotic neurons of hippocampus caused by is-chemia/reperfusion (2. 06% ±0.21% vs. 4.27% ±0. 08% , P<0. 01), and weakened the expressions of p53 and bax gene of hippocampus compared with ischemia/reperrusion without IPC.Conclusion IPC can protect the neurons in the CA1 region of hippocampus against apoptosis caused by ischemia/reperfusion, and this process may be related to the reduced expressions of p53 and bax.

  9. Apoptosis and Bax expression are increased by coal dust in the polycyclic aromatic hydrocarbon-exposed lung

    Energy Technology Data Exchange (ETDEWEB)

    Ghanem, M.M.; Battelli, L.A.; Mercer, R.R.; Scabilloni, J.F.; Kashon, M.L.; Ma, J.Y.C.; Nath, J.; Hubbs, A.F.

    2006-09-15

    Miners inhaling respirable coal dust (CD) frequently develop coal workers' pneumoconiosis. Many coal miners are also exposed to polycyclic aromatic hydrocarbon (PAH) components of diesel engine exhaust and cigarette smoke, which may contribute to lung disease in these workers. Recently, apoptosis was reported to play a critical role in the development of another pneumoconiosis of miners, silicosis. In addition, CID was reported to suppress cytochrome P450 1A1 (CYP1A1) induction by PAHs. We exposed rats intratracheally to 0.0, 2.5, 10.0, 20.0, or 40.0 mg/rat CD and, 11 days later, to intraperitoneal P-naphthoflavone (BNF), a PAH. In another group of rats exposed to CD and BNF, caspase activity was inhibited by injection of the pan-caspase inhibitor Q-VD-OPH (quinoline-Val-Asp (OMe)-CH{sub 2}-OPH). In rats exposed to BNF, CD exposure increased alveolar expression of the proapoptotic mediator Bax but decreased CYP1A1 induction relative to BNF exposure alone. Pan-caspase inhibition decreased CD-associated Bax expression and apoptosis but did not restore CYP1A1 activity. Further, CD-induced lung inflammation and alveolar epithelial cell hypertrophy and hyperplasia were not suppressed by caspase inhibition. It is concluded that combined BNF and CD exposure increased Bax expression and apoptosis in the lung, but Bax and apoptosis were not the major determinants of early lung injury in this model.

  10. Immunohistochemical study of epidermal and dermal expression of Bcl-X, Bcl-2 and bax in psoriasis.

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the regulation of cell proliferation and apoptosis in psoriasis. Methods: The expressions of Bcl-X, Bcl-2 and Bax were studied with immunohistochemical technique (SP) in the lesional and non-lesional psoriatic skin. Results: There were significant overexpressions of Bcl-X in all layers of epidermis, inflammatory cells and vascular endothelia in dermis;

  11. Relationship between expression of Bax and Bcl-2 proteins and apoptosis in radiation compound wound healing of rats

    Institute of Scientific and Technical Information of China (English)

    崔玉芳; 夏国伟; 付小兵; 杨红; 彭瑞云; 张莹; 谷庆阳; 高亚兵; 崔雪梅; 胡文华

    2003-01-01

    Objective: To study the relationship between the expression of Bax, Bcl-2 proteins, and apoptosis in radiation compound wound healing of rats.Methods: Apoptosis, Bax and Bcl-2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical methods. Results: (1) Changes of the apoptosis in wound healing showed three typical characteristics: early occurrence, high frequency and delayed disappearance after radiation to rats when compared with those of simple wound group, which might be an important reason for radiation-induced delayed wound healing. (2) The expression of Bax protein increased evidently with the increment of apoptosis and showed a good corresponding relationship with the apoptotic frequency in the process of wound healing. While the expression of Bcl-2 protein decreased obviously as the apoptosis reached a maximum and showed increasing tendency up to normal level when the apoptosis decreased distinctively. Conclusions: Bax and Bcl-2 proteins play an important role in the apoptotic regulation of radiation compound wound healing in rats.

  12. EXPRESSION OF BAX AND BCL-2 IN MOUSE OFFSPRING BRAIN AFTER MATERNAL ORAL ADMINIS TRATION OF MONOSODIUM GLUTAMATE

    Institute of Scientific and Technical Information of China (English)

    徐磊; 赵晏; 展淑琴; 王会生; 史文春

    2002-01-01

    Objective To analyze the excitotoxicity of monoso dium glutamate (MSG) in the offspring cerebral cortex and hippocampal subregions after maternal oral administration of MSG. Methods Kunming mi ce were given per os MSG ( 4.0 g/kg ) at 17~21 days of pregnancy and their offs pring behaviors were studied at 10, 20 , 30 days postnatally. By using immunohis tochemical means, the involvement of Bcl-2 and Bax in the glutamate-induced c ell death in cortical and hippocampal neur ons were examined. Cell damage was assessed by direct cell counting. Res ults Administration of monosodium glutamate during the fetal period in mice resulted in a moderate increase in the expression of Bax in principal neuro ns in CA1, CA2, CA3, CA4 and in the cerebral cortex at postpartum 10, 20, 30 day s in the offspring mice, whereas Bcl-2 protein expressions were reduced signif icantly in the same regions as compared with those of controls. Conclusi on These findings suggest that glutamate toxicity results in cellular d eath via an apoptotic mechanism in which the Bcl-2/Bax-alpha molecular comple x may be involved. The glutamate-induced apoptosis appears to be related to the modulation of Bcl-2 family gene products such as Bcl-2 and Bax.

  13. EXPRESSION OF BAX AND BCL-2 IN MOUSE OFFSPRING BRAIN AFIER MATERNAL ORAL ADMINISTRATION OF MONOSODIUM GLUTAMATE

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To analyze the excitotoxicity of monosodium glutamate(MSG)in the offspring crebral cortex and hippocampal subresions after maternal oral administration of MSG.Methods:Kunming mice were given per os MSG(4.0g/kg)at 17-21 days of pregnancy and their offspring behaviors were studied at 10,20,30days postnatally.By using inmunohistochemical means,the involvment of Bcl-2 and bax in the glutamate-induced cell death in cortical and hippocampal neurons were examined.Cell damage was assessed by direct cell counting.Results:administration of monosodium glutamate during the fetal period in mice resulted in a moderate increase in the expression of Bax in principal neurons in CA1,CA2,CA3,CA4 and in the cerebral cortex at postpartum 10,20,30 days in the offspring mice,whereas Bcl-2 protein expressions were reduced significantly in the same regions as compared with those of controls.Conclusion:These findings suggest that glutamate toxicity results in cellular death via an apoptotic mechanism in which the Bcl-2/Bax-alpha molecular complex may be involved.The glutamate-induced apoptosis appears to be related to the modulation of Bcl-2 family gene products such as Bcl-2 and Bax.

  14. ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells.

    Science.gov (United States)

    Song, Shanshan; Jacobson, Krista N; McDermott, Kimberly M; Reddy, Sekhar P; Cress, Anne E; Tang, Haiyang; Dudek, Steven M; Black, Stephen M; Garcia, Joe G N; Makino, Ayako; Yuan, Jason X-J

    2016-01-15

    Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. Copyright

  15. p38(MAPK)/p53-Mediated Bax induction contributes to neurons degeneration in rotenone-induced cellular and rat models of Parkinson's disease.

    Science.gov (United States)

    Wu, Feng; Wang, Zhong; Gu, Jin-Hua; Ge, Jian-Bin; Liang, Zhong-Qin; Qin, Zheng-Hong

    2013-09-01

    Rotenone is an environmental neurotoxin that induces degeneration of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc), which ultimately results in parkinsonism, but the molecular mechanisms of selective degeneration of nigral DA neurons are not fully understood. In the present study, we investigated the induction of p38(MAPK)/p53 and Bax in SNpc of Lewis rats after chronic treatment with rotenone and the contribution of Bax to rotenone-induced apoptotic commitment of differentiated PC12 cells. Lewis rats were subcutaneously treated with rotenone (1.5mg/kg) twice a day for 50days and the loss of tyrosine hydroxylase (THase), motor function impairment, and expression of p38(MAPK), P-p38(MAPK), p53, and Bax were assessed. After differentiated PC cells were treated with rotenone (500nM) for 6-36h, protein levels of p38(MAPK) and P-p38(MAPK), p53 nuclear translocation, Bax induction and cell death were measured. The results showed that rotenone administration significantly reduced motor activity and caused a loss of THase immunoreactivity in SNpc of Lewis rats. The degeneration of nigral DA neurons was accompanied by the increases in p38(MAPK), P-p38(MAPK), p53, and Bax protein levels. In cultured PC12 cells, rotenone also induced an upregulation of p38(MAPK), P-p38(MAPK), p53 and Bax. Pharmacological inhibition of p38(MAPK) with SB203580 (25μM) blunted rotenone-induced cell apoptosis. Treatment with SB203580 prevented the p53 nuclear translocation and upregulation of Bax. Inhibition of p53 with pifthrin-alpha or Bax with siRNAs significantly reduced rotenone-induced Bax induction and apoptotic cell death. These results suggest that the p38(MAPK)/p53-dependent induction of Bax contributes to rotenone's neurotoxicity in PD models.

  16. After Embedding in Membranes Antiapoptotic Bcl-XL Protein Binds Both Bcl-2 Homology Region 3 and Helix 1 of Proapoptotic Bax Protein to Inhibit Apoptotic Mitochondrial Permeabilization*

    Science.gov (United States)

    Ding, Jingzhen; Mooers, Blaine H. M.; Zhang, Zhi; Kale, Justin; Falcone, Domina; McNichol, Jamie; Huang, Bo; Zhang, Xuejun C.; Xing, Chengguo; Andrews, David W.; Lin, Jialing

    2014-01-01

    Bcl-XL binds to Bax, inhibiting Bax oligomerization required for mitochondrial outer membrane permeabilization (MOMP) during apoptosis. How Bcl-XL binds to Bax in the membrane is not known. Here, we investigated the structural organization of Bcl-XL·Bax complexes formed in the MOM, including the binding interface and membrane topology, using site-specific cross-linking, compartment-specific labeling, and computational modeling. We found that one heterodimer interface is formed by a specific interaction between the Bcl-2 homology 1–3 (BH1–3) groove of Bcl-XL and the BH3 helix of Bax, as defined previously by the crystal structure of a truncated Bcl-XL protein and a Bax BH3 peptide (Protein Data Bank entry 3PL7). We also discovered a novel interface in the heterodimer formed by equivalent interactions between the helix 1 regions of Bcl-XL and Bax when their helical axes are oriented either in parallel or antiparallel. The two interfaces are located on the cytosolic side of the MOM, whereas helix 9 of Bcl-XL is embedded in the membrane together with helices 5, 6, and 9 of Bax. Formation of the helix 1·helix 1 interface partially depends on the formation of the groove·BH3 interface because point mutations in the latter interface and the addition of ABT-737, a groove-binding BH3 mimetic, blocked the formation of both interfaces. The mutations and ABT-737 also prevented Bcl-XL from inhibiting Bax oligomerization and subsequent MOMP, suggesting that the structural organization in which interactions at both interfaces contribute to the overall stability and functionality of the complex represents antiapoptotic Bcl-XL·Bax complexes in the MOM. PMID:24616095

  17. BAX, a novel cell pro-apoptotic protein, involved in hemocytes early antiviral immune response in fresh water crayfish, Procambarus clarkii.

    Science.gov (United States)

    du, Zhi-Qiang

    2016-08-01

    Apoptosis plays an important role in various biological processes and acts as a host defending mechanism by which infected cells are eliminated to restrict the virus propagation scale. Bax is a crucial pro-apoptotic protein, which mediates the release of cytochrome c from mitochondrion to cytosol in mammalian. However, its role in invertebrate is still obscure. Here, a novel pro-apoptotic protein gene was identified from hemocytes of red swamp crayfish. There was a Bcl-2 domain in the C-terminus of Pc-Bax, which possessed 497 amino acids residues. And an important transmembrane region existed in the C-terminus of Pc-Bax, which implied that Pc-Bax located in mitochondrial membrane. Besides, Pc-Bax was expressed at a relative high level in hemocytes, and a relative low expression levels in hepatopancreas, gills, and intestine. In hemocytes, Pc-Bax transcript was rapidly up-regulated from 12 h to 36 h after WSSV infection. And there was the same trend for Pc-Bax protein expression level in hemocytes after WSSV infection. Results of qRT-PCR testing for VP28 gene showed WSSV replication was obviously enhanced after Pc-Bax knockdown. Meantime, hemocytes apoptosis was suppressed in Pc-Bax knockdown crayfish after WSSV injection, compared with the dsGFP injection group and normal group. Taken together, these results revealed that crayfish hemocytes apoptosis scale was enhanced to suppress WSSV replication by up-regulating Bax protein expression level after WSSV infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax.

    Science.gov (United States)

    Wilfling, F; Weber, A; Potthoff, S; Vögtle, F-N; Meisinger, C; Paschen, S A; Häcker, G

    2012-08-01

    During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.

  19. EGR-1 forms a complex with YAP-1 and upregulates Bax expression in irradiated prostate carcinoma cells.

    Science.gov (United States)

    Zagurovskaya, M; Shareef, M M; Das, A; Reeves, A; Gupta, S; Sudol, M; Bedford, M T; Prichard, J; Mohiuddin, M; Ahmed, M M

    2009-02-26

    In this study, we investigated the functional role of early growth response-1 (Egr1 gene) in the regulation of radiation-induced clonogenic inhibition and apoptosis in p53 wild-type and mutant prostate cancer cells 22Rv1 and DU145, respectively. 22Rv1 cells were more sensitive to irradiation compared with DU145 cells, and the sensitivity was enhanced by overexpression of EGR-1 in both cells. Dominant-negative EGR-1 mutant (dnEGR-1) or repressor of EGR-1, NGFIA binding protein 1 (NAB1), increased radioresistance of these cells. Significant activation of caspases 3 and 9 and Bcl2-associated X (Bax) with increased poly(ADP-ribose) polymerase (PARP) cleavage and cytochrome c release was observed in radiation-exposed EGR-1 overexpressing cells. Gel shift analysis and chloramphenicol acetyl transferase (CAT) reporter assays indicate that EGR-1 transactivates the promoter of the Bax gene. Interaction of EGR-1 and Yes kinase-associated protein 1 (YAP-1) through the WW domain of YAP-1 enhances the transcriptional activity of EGR-1 on the Bax promoter as shown by chromatin immunoprecipitation and reporter assays. Irradiation of PC3 cell xenografts that were treated with adenoviral EGR-1 showed significant regression in tumor volume. These findings establish the radiation-induced pro-apoptotic action of EGR-1, in a p53-independent manner, by directly transactivating Bax, and prove that alters the B-cell CLL/lymphoma 2 (Bcl-2)/Bax ratio as one of the mechanisms resulting in significant activation of caspases, leading to cell death through the novel interaction of EGR-1 with YAP-1.

  20. Calpain and reactive oxygen species targets Bax for mitochondrial permeabilisation and caspase activation in zerumbone induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Praveen K Sobhan

    Full Text Available Fluorescent protein based signaling probes are emerging as valuable tools to study cell signaling because of their ability to provide spatio- temporal information in non invasive live cell mode. Previously, multiple fluorescent protein probes were employed to characterize key events of apoptosis in diverse experimental systems. We have employed a live cell image based approach to visualize the key events of apoptosis signaling induced by zerumbone, the active principle from ginger Zingiber zerumbet, in cancer cells that enabled us to analyze prominent apoptotic changes in a hierarchical manner with temporal resolution. Our studies substantiate that mitochondrial permeabilisation and cytochrome c dependent caspase activation dominate in zerumbone induced cell death. Bax activation, the essential and early event of cell death, is independently activated by reactive oxygen species as well as calpains. Zerumbone failed to induce apoptosis or mitochondrial permeabilisation in Bax knockout cells and over-expression of Bax enhanced cell death induced by zerumbone confirming the essential role of Bax for mitochondrial permeabilsation. Simultaneous inhibition of reactive oxygen species and calpain is required for preventing Bax activation and cell death. However, apoptosis induced by zerumbone was prevented in Bcl 2 and Bcl-XL over-expressing cells, whereas more protection was afforded by Bcl 2 specifically targeted to endoplasmic reticulum. Even though zerumbone treatment down-regulated survival proteins such as XIAP, Survivin and Akt, it failed to affect the pro-apoptotic proteins such as PUMA and BIM. Multiple normal diploid cell lines were employed to address cytotoxic activity of zerumbone and, in general, mammary epithelial cells, endothelial progenitor cells and smooth muscle cells were relatively resistant to zerumbone induced cell death with lesser ROS accumulation than cancer cells.

  1. The human septin7 and the yeast CDC10 septin prevent Bax and copper mediated cell death in yeast.

    Science.gov (United States)

    Horowitz, Avital; Lapointe, Jason F; Eid, Rawan; Sheibani, Sara; Gharib, Nada; Jones, Natalie K; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2013-12-01

    The mechanisms of programmed cell death activate genetically encoded intracellular programs in a controlled manner, the most common form being apoptosis. Apoptosis is carried out through a cascade of caspase mediated proteolytic cleavages initiated by the oligomerization of Bax, a cardinal regulator of mitochondrial-mediated apoptosis. Heterologous expression of Bax in yeast causes cell death that shares a number of similarities to processes that occur in mammalian apoptosis. A screen of a cardiac cDNA library for suppressors of Bax-mediated apoptosis identified human septin7, a protein that belongs to the septin superfamily of conserved GTP-binding proteins that share a conserved cdc/septin domain. Analysis of the amino acid sequence deduced from the septin7 clone as well as the corresponding human septin7 gene revealed that a novel alternatively spliced transcript called septin7 variant4 (v4) was uncovered. Yeast cells overexpressing the human septin7 v4 cDNA were also capable of resisting copper-mediated cell death suggesting that it is not only a Bax suppressor but also an anti-apoptotic sequence. Analysis of septin7 function in a MCA1Δ yeast strain suggests that septin7 inhibits apoptosis in a caspase independent pathway. Overexpression of the yeast septin7 ortholog CDC10 also conferred resistance to the negative effects of copper as well as protecting cells from the overexpression of Bax. In contrast, septin7 was unable to prevent the increase in cell size associated with mutants lacking the endogenous yeast CDC10 gene. Taken together, our analysis suggests that anti-apoptosis is a novel yet evolutionarily conserved property of the septin7 sub-family of septins.

  2. Erythropoietin can promote survival of cerebral cells by downregulating Bax gene after traumatic brain injury in rats

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    Liao Z

    2009-01-01

    Full Text Available Background : Traumatic brain injury (TBI is an important cause of adult mortality and morbidity. Erythropoietin (Epo has been shown to promote the viability of cerebral cells by upregulating Bcl-2 gene; however, Epo may exert its antiapoptotic effect via the differential regulation of the expression of genes involved in the apoptotic process. Aim : The present study examined the neuroprotective effect of Epo as a survival factor through the regulation of the Bax. Materials and Methods : Wistar rats were randomly divided into three groups: Recombinant human EPO treated (rhEPO TBI, vehicle-treated TBI, and sham-operated. Traumatic brain injury was induced by the Feeney free-falling model. Rats were killed 5, 12, 24, 72, 120, or 168 h after TBI. Regulation of Bcl-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR, western blotting and immunofluorescence. Results : Bax mRNA and protein levels were lower in the rhEPO-treated rat brains than in the vehicle-treated rat brains. Induction of Bax expression peaked at 24 h and remained stable for 72-120 h in vehicle-treated rat brains, whereas induction of Bax expression was only slightly elevated in rhEPO-treated rat brains. The number of TdT-mediated dUTP Nick-End Labeling(TUNEL-positive cells in the rhEPO-treated rat brains was far fewer than in the vehicle-treated rat brains. Conclusions : Epo exerts neuroprotective effect against traumatic brain injury via reducing Bax gene expression involved in inhibiting TBI-induced neuronal cell death.

  3. BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) not always convinces BAX (BCL-2-associated X protein) for apoptosis.

    Science.gov (United States)

    Verma, Sharad; Goyal, Sukriti; Tyagi, Chetna; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-06-01

    The interaction of BAX (BCL-2-associated X protein) with BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) directly initiates BAX-mediated mitochondrial apoptosis. This molecular dynamics study reveals that BIM SAHB forms a stable complex with BAX but it remains in a non-functional conformation. N terminal of BAX folds towards the core which has been reported exposed in the functional monomer. The α1-α2 loop, which has been reported in open conformation in functional BAX, acquires a closed conformation during the simulation. BH3/α2 remains less exposed as compared to initial structure. The hydrophobic residues of BIM accommodates in the rear pocket of BAX during the simulation. A steep decrease in radius of gyration and solvent accessible surface area (SASA) indicates the complex folding to acquire a more stable but inactive conformation. Further the covariance matrix reveals that the backbone atoms' motions favour the inactive conformation of the complex. This is the first report on the non-functional BAX-BIM SAHB complex by molecular dynamics simulation in the best of our knowledge.

  4. Modification of the BAX Salmonella test kit to include a hot start functionality (modification of AOAC Official Method 2003.09).

    Science.gov (United States)

    Wallace, F Morgan; DiCosimo, Deana; Farnum, Andrew; Tice, George; Andaloro, Bridget; Davis, Eugene; Burns, Frank R

    2011-01-01

    In 2010, the BAX System PCR assay for Salmonella was modified to include a hot start functionality designed to keep the reaction enzyme inactive until PCR begins. To validate the assay's Official Methods of Analysis status to include this procedure modification, an evaluation was conducted on four food types that were simultaneously analyzed with the BAX System and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference method for detecting Salmonella. Identical performance between the BAX System method and the reference methods was observed. Additionally, lysates were analyzed using both the BAX System Classic and BAX System Q7 instruments with identical results using both platforms for all samples tested. Of the 100 samples analyzed, 34 samples were positive for both the BAX System and reference methods, and 66 samples were negative by both the BAX System and reference methods, demonstrating 100% correlation. No instrument platform variation was observed. Additional inclusivity and exclusivity testing using the modified test kit demonstrated the test kit to be 100% accurate in evaluation of test panels of 352 Salmonella strains and 46 non-Salmonella strains.

  5. Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201.

    Science.gov (United States)

    Peng, Linda X; Wallace, Morgan; Andaloro, Bridget; Fallon, Dawn; Fleck, Lois; Delduco, Dan; Tice, George

    2011-01-01

    The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

  6. Boron neutron capture therapy induces apoptosis of glioma cells through Bcl-2/Bax

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    Mao Xinggang

    2010-12-01

    Full Text Available Abstract Background Boron neutron capture therapy (BNCT is an alternative treatment modality for patients with glioma. The aim of this study was to determine whether induction of apoptosis contributes to the main therapeutic efficacy of BNCT and to compare the relative biological effect (RBE of BNCT, γ-ray and reactor neutron irradiation. Methods The neutron beam was obtained from the Xi'an Pulsed Reactor (XAPR and γ-rays were obtained from [60Co] γ source of the Fourth Military Medical University (FMMU in China. Human glioma cells (the U87, U251, and SHG44 cell lines were irradiated by neutron beams at the XAPR or [60Co] γ-rays at the FMMU with different protocols: Group A included control nonirradiated cells; Group B included cells treated with 4 Gy of [60Co] γ-rays; Group C included cells treated with 8 Gy of [60Co] γ-rays; Group D included cells treated with 4 Gy BPA (p-borono-phenylalanine-BNCT; Group E included cells treated with 8 Gy BPA-BNCT; Group F included cells irradiated in the reactor for the same treatment period as used for Group D; Group G included cells irradiated in the reactor for the same treatment period as used for Group E; Group H included cells irradiated with 4 Gy in the reactor; and Group I included cells irradiated with 8 Gy in the reactor. Cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT cytotoxicity assay. The morphology of cells was detected by Hoechst33342 staining and transmission electron microscope (TEM. The apoptosis rate was detected by flow cytometer (FCM. The level of Bcl-2 and Bax protein was measured by western blot analysis. Results Proliferation of U87, U251, and SHG44 cells was much more strongly inhibited by BPA-BNCT than by irradiation with [60Co] γ-rays (P 60Co] γ-rays (P P Conclusions Compared with ��-ray and reactor neutron irradiation, a higher RBE can be achieved upon treatment of glioma cells with BNCT. Glioma cell apoptosis induced by

  7. Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

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    Yoon TH

    2012-03-01

    Full Text Available Ki-Chun Yoo1, Chang-Hwan Yoon1, Dongwook Kwon2, Kyung-Hwan Hyun1, Soo Jung Woo1, Rae-Kwon Kim1, Eun-Jung Lim1, Yongjoon Suh1, Min-Jung Kim1, Tae Hyun Yoon2, Su-Jae Lee11Laboratory of Molecular Biochemistry, 2Laboratory of Nanoscale Characterization and Environmental Chemistry, Department of Chemistry, Hanyang University, Seoul, Republic of KoreaBackground: Titanium dioxide (TiO2 has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO2 particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO2 has yet to be defined.Methods and results: In this study, we demonstrated that combined treatment with TiO2 nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO2 nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO2P25–70 together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO2-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO2P25–70 and ultraviolet A irradiation.Conclusion: These results indicate that sub-100 nm sized TiO2 treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein

  8. Effects of erythropoietin on the expression of tumor necrosis factor-alpha and Bax after facial nerve axotomy in rats

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Shengyu Lü; Ziying Yu; Ming Bi; Bin Sun

    2011-01-01

    This study sought to evaluate the effect of high-dose erythropoietin (EPO; 5 000 IU/kg) on the expression of tumor necrosis factor-alpha (TNF-α) and Bax in the facial nucleus after facial nerve transection in rats. A total of 42 Wistar rats of both genders were used in this study, and 40 rats were randomly divided into 2 groups: EPO group and model group. The EPO group was treated with EPO once a day for 5 days at a dose of 5 000 IU/kg body weight. The model group was treated with saline of the same amount. At day 3 after EPO (or saline) treatment, the right facial nerves of the 40 rats were transected at the level of the stylomastoid foramen, with the left sides untreated. The remaining 2 rats that did not undergo axotomy served as the control group. The surviving motor neurons in operated rats were counted in coronal paraffin sections of the facial nucleus. The expression of TNF-α and Bax in the facial nucleus was detected by immunohistochemical staining at days 3, 7, 14, 21, and 28 after axotomy. At days 14, 21, and 28 after facial nerve axotomy, a significantly greater proportion of facial motor neurons survived in the EPO group than in the model group. After axotomy, the expression of TNF-α and Bax increased in motor neurons in both the EPO and the model groups. TNF-α expression reached its peak level at day 14 after axotomy, while Bax expression reached its peak level at day 21. TNF-α expression was much lower in the EPO group than in the model group at all time points. No significant difference in Bax expression was found between the EPO and the model groups. These results indicate that high-dose EPO treatment attenuates the increase in TNF-α expression in the facial nucleus and reduces the loss of motor neurons after facial nerve transection in rats. However, high-dose EPO treatment has little effect on Bax expression.

  9. Potential impact of (rs 4645878) BAX promoter -248G>A and (rs 1042522) TP53 72Arg>pro polymorphisms on epithelial ovarian cancer patients.

    Science.gov (United States)

    Dholariya, S; Mir, R; Zuberi, M; Yadav, P; Gandhi, G; Khurana, N; Saxena, A; Ray, P C

    2016-01-01

    In India, Epithelial ovarian cancer has emerged as one of the most common malignancies affecting women. Tumor protein 53 (TP53) induces expression of the B cell lymphoma 2-associated X protein (BAX) gene by directly binding to the TP53-binding element in the BAX promoter. Therefore, we hypothesized that single-nucleotide polymorphism of BAX promoter -248G>A and TP53 72Arg>Pro gene may jointly contribute to ovarian cancer risk. This study aimed at exploring the association of BAX promoter -248G>A and TP53 72Arg>Pro gene polymorphism with risk of developing EOC and its clinicopathological features and to evaluate gene-gene interaction of these two polymorphisms with risk of developing EOC. The study was conducted on 70 Epithelial ovarian cancer patients and 70 healthy controls. Genotyping of p53 codon 72 and BAX promoter gene was examined by ASO-PCR and PICA-PCR, respectively. Odds ratios and 95 % confidence intervals were calculated. We found an increased cancer risk associated with the BAX AA (ORs = 4.1, 95 %, CI = 1.23-13.97) genotype. An increased risk was also associated with the TP53 Pro/Pro (OR = 4.4, 95 % CI = 1.40-13.99) and Arg/Pro genotype (OR = 2.3, 95 % CI = 1.13-4.86). The gene-gene interaction of these polymorphisms increased EOC risk in a more than additive manner (ORs for the presence of both BAX AA and TP53 Arg/Pro genotypes = 8.7, 95 % CI = 1.66-45.48). BAX GG genotype was associated with adverse staging of cancer (P = 0.01). The findings suggest that polymorphism of BAX and TP53 genes may be potential genetic modifiers for developing ovarian cancer.

  10. Methylglyoxal reduces mitochondrial potential and activates Bax and caspase-3 in neurons: Implications for Alzheimer's disease.

    Science.gov (United States)

    Tajes, Marta; Eraso-Pichot, Abel; Rubio-Moscardó, Fanny; Guivernau, Biuse; Bosch-Morató, Mònica; Valls-Comamala, Victòria; Muñoz, Francisco J

    2014-09-19

    Alzheimer's disease (AD) is characterized by the oxidative stress generated from amyloid β-peptide (Aβ) aggregates. It produces protein nitrotyrosination, after the reaction with nitric oxide to form peroxynitrite, being triosephosphate isomerase (TPI) one of the most affected proteins. TPI is a glycolytic enzyme that catalyzes the interconversion between glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Methylglyoxal (MG) is a by-product of TPI activity whose production is triggered when TPI is nitrotyrosinated. MG is harmful to cells because it glycates proteins. Here we found protein glycation when human neuroblastoma cells were treated with Aβ. Moreover glycation was also observed when neuroblastoma cells overexpressed mutated TPI where Tyr165 or Tyr209, the two tyrosines close to the catalytic center, were changed by Phe in order to mimic the effect of nitrotyrosination. The pathological relevance of these findings was studied by challenging cells with Aβ oligomers and MG. A significant decrease in mitochondrial transmembrane potential, one of the first apoptotic events, was obtained. Therefore, increasing concentrations of MG were assayed searching for MG effect in neuronal apoptosis. We found a decrease of the protective Bcl2 and an increase of the proapoptotic caspase-3 and Bax levels. Our results suggest that MG is triggering apoptosis in neurons and it would play a key role in AD neurodegeneration.

  11. Direct proof of static charge stripe correlations in La2-xBaxCuO4

    Science.gov (United States)

    Chen, X. M.; Thampy, V.; Mazzoli, C.; Barbour, A.; Gu, G.; Hill, J. P.; Tranquada, J. M.; Dean, M. P. M.; Wilkins, S. B.

    The nature of charge stripe order in the cuprates, and in particular whether the stripes are static or dynamic, is a key issue in understanding the relationship between stripes and superconductivity. In La2-xBaxCuO4 (LBCO) a low temperature structural distortion is widely believed to pin stripes into fixed, static domains, but such an assertion has never been directly verified. We performed resonant soft x-ray photon correlation spectroscopy (XPCS) to probe the charge order Bragg peak of 1/8 doped LBCO. At low temperatures, we observe time-independent x-ray speckle patterns persisting for more than three hours, proving the static nature of the stripes and we go on to discuss how stripe order melts with increasing temperature. Our results demonstrate that the combination of XPCS with diffraction limited light sources such as the National Synchrotron Light Source II can probe the dynamics of even subtle order parameters such as stripes in the cuprates. Work performed at Brookhaven National Laboratory was supported by the US Department of Energy, Division of Materials Science, under Contract No. DE-AC02-98CH10886. Use of the National Synchrotron Light Source II was supported under Contract No. DE-SC0012704.

  12. Stripe order in La2-xBaxCuO4 at ambient and high pressure.

    Science.gov (United States)

    Huecker, M.; Wen, J. S.; Xu, Z. J.; Gu, G. D.; Tranquada, J. M.; Zimmermann, M. V.

    2009-03-01

    The pronounced stability of the charge and spin stripe order in La2-xBaxCuO4 at x=1/8 doping still is a poorly understood peculiarity. A combination of electronic and structural interactions is likely, however it has been difficult to clearly separate the involved mechanisms. One approach is to explore how stripe order fades away for dopings x !=1/8. We have performed high energy (100 keV) x-ray diffraction and static magnetization experiments on single crystals between x=0.095 and 0.155. To our surprise, at ambient pressure stripes exist in a much broader range of doping around x=1/8 than expected. In the underdoped region charge stripe order always coincides with a structural transition associated with a rotation of the octahedral tilt axis. However, for x=1/8 and high pressure we have been able to show that stripe order also occurs in the absence of this structural phase, which motivates us to discuss stripes in terms of an electronic liquid crystal phase.

  13. Infrared Spectra of La0.65BaxMnO3-δ Oxides

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The infrared spectra of La0.65BaxMnO3-d (x = 0.35, 0.33 and 0.30) were investigated experimentally. The result shows that the sample La0.65Ba0.33MnO3-d has the largest Curie temperature and the smallest resistivity and wave number of the stretching vibration mode of MnO6 octahedron at 300 K among the investigated samples. However, the absorption strength for the stretching vibration mode of MnO6 octahedron in La0.65Ba0.35MnO3-d is stronger for paramagnetic phase than that for ferromagnetic phase, which may be connected with the reducing of the dynamic incoherent Jahn-Teller distortion below Curie temperature. In addition, the large shift of wave number for the stretching mode at the temperatures from 293 to 423 K has been observed in La0.65Ba0.35MnO3-d, which may be due to the increase of the Mn-O bond length with temperature increasing.

  14. Integration of BaxSr1-xTiO3 thin film for DRAM application

    Science.gov (United States)

    Kashihara, Keiichiro; Okudaira, Tomonori; Tsunemine, Yoshikazu; Ohno, Yoshikazu; Itoh, Hiromi; Nishimura, Tadashi; Hirayama, Makoto; Horikawa, Tsuyoshi; Shibano, Teruo; Horie, Kazuo

    1995-09-01

    This paper presents a newly developed process technology to integrate the BaxSr1-xTiO3(BST) thin film prepared by an rf magnetron sputtering. Evaluations of the integrated BST capacitors on a test element group (TEG) structure revealed some of key issues for a successful integration. A two-step sputtering method comprising the first step to form a nucleation layer and the second step to form the main part of the BST film was found to be useful for preventing the dielectric properties of the integrated BST thin film from the degradation. A careful control of the shape of the lower structures such as the edge of the bottom electrode or the poly Si plug of the storage node turned to be indispensable to obtain the reliable capacitor and this recommends the extensive use of the process that can provide a flush surface such as the Chemical Mechanical Polishing (CMP) in the future integration. The interlevel dielectrics over the BST capacitor was shown to seriously affect the leakage characteristics and an undoped SiO2 film was most suitable for the integrity, implying the needs for another planarization technique instead of the glass reflow. Finally, a preliminary evaluation of the reliability and the normal bit function of a 4 Mbits DRAM, made of fully flat BST stacked capacitors, demonstrated the utility of the developed integration technology.

  15. Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

    Science.gov (United States)

    Eid, Rawan; Sheibani, Sara; Gharib, Nada; Lapointe, Jason F; Horowitz, Avital; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2014-05-01

    The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival.

  16. Two-Dimensional Superconducting Fluctuations in Stripe-Ordered La2-xBaxCuO4

    Science.gov (United States)

    Li, Qiang; Hucker, Markus; Gu, Genda; Tsvelik, Alexei; Tranquada, John

    2008-03-01

    Recent spectroscopic observations of a d-wave-like gap in stripe-ordered La2-xBaxCuO4 with x=1/8 have led us to critically analyze the anisotropic transport and magnetization properties of this material. The data suggest that concomitant with the spin ordering is an electronic decoupling of the CuO2 planes. We observe a transition (or crossover) to a state of two-dimensional (2D) fluctuating superconductivity. Thus, it appears that the stripe order in La2-xBaxCuO4 frustrates three-dimensional superconducting phase order, but is fully compatible with 2D superconductivity and an enhanced Tc --[Ref. Q. Li, et al., PRL 99, 067001 (2007)

  17. Double knockout of Bax and Bak from kidney proximal tubules reduces unilateral urethral obstruction associated apoptosis and renal interstitial fibrosis

    Science.gov (United States)

    Mei, Shuqin; Li, Lin; Wei, Qingqing; Hao, Jielu; Su, Yunchao; Mei, Changlin; Dong, Zheng

    2017-01-01

    Interstitial fibrosis, a common pathological feature of chronic kidney diseases, is often associated with apoptosis in renal tissues. To determine the associated apoptotic pathway and its role in renal interstitial fibrosis, we established a mouse model in which Bax and Bak, two critical genes in the intrinsic pathway of apoptosis, were deleted specifically from kidney proximal tubules and used this model to examine renal apoptosis and interstitial fibrosis following unilateral urethral obstruction (UUO). It was shown that double knockout of Bax and Bak from proximal tubules attenuated renal tubular cell apoptosis and suppressed renal interstitial fibrosis in UUO. The results indicate that the intrinsic pathway of apoptosis contributes significantly to the tubular apoptosis and renal interstitial fibrosis in kidney diseases. PMID:28317867

  18. A brewing understanding of the regulation of Bax function by Bcl-xL and Bcl-2.

    Science.gov (United States)

    Renault, Thibaud T; Dejean, Laurent M; Manon, Stéphen

    2017-01-01

    Bcl-2 family members form a network of protein-protein interactions that regulate apoptosis through permeabilization of the mitochondrial outer membrane. Deciphering this intricate network requires streamlined experimental models, including the heterologous expression in yeast. This approach had previously enabled researchers to identify domains and residues that underlie the conformational changes driving the translocation, the insertion and the oligomerization of the pro-apoptotic protein Bax at the level of the mitochondrial outer membrane. Recent studies that combine experiments in yeast and in mammalian cells have shown the unexpected effect of the anti-apoptotic protein Bcl-xL on the priming of Bax. As demonstrated with the BH3-mimetic molecule ABT-737, this property of Bcl-xL, and of Bcl-2, is crucial to elaborate about how apoptosis could be reactivated in tumoral cells.

  19. Interaction between Hsp60 and Bax in normal human myocardium and in myocardium affected by dilated cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Tykhonkova I. O.

    2009-04-01

    Full Text Available The main functional compartments of molecular chaperone Hsp60 are mitochondria and cytoplasm. Up to 30 % of Hsp60 are located in cytoplasm of cardiomyocytes. The interaction between molecular chaperone Hsp60 and proapoptotic Bax protein in the cytoplasmic fraction from normal human heart tissue has been revealed by co-immunoprecipitation in contrast to myocardium affected by dilated cardiomyopathy, where this interaction has not been observed

  20. The effect of octreotide and bromocriptine on expression of a pro-apoptotic Bax protein in rat prolactinoma.

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    Jolanta Kunert-Radek

    2004-03-01

    Full Text Available It is well established that disruption of apoptosis may lead to tumor initiation, progression or metastasis. It is also well documented that many anticancer drugs induce apoptosis. In the earlier studies, the dopamine D2 receptor agonist bromocriptine (BC and somatostatin analog octreotide (OCT were found to inhibit the growth of the estrogen-induced rat prolactinoma. Our previous investigations, applying the TUNEL method showed the involvement of the pro-apoptotic effect in the action of BC, and to a lesser degree, in the action of OCT. The aim of the present study was to investigate whether the pro-apoptotic action of these drugs involves the increased expression of Bax--a member of Bcl-2 protein family which is known to play an important role in the regulation of apoptosis. Male four-week Fisher 344 rats were used in the experiment. Capsules containing diethylstilboestrol (DES were implanted subcutaneously. Six weeks after the implantation the rats were given OCT (2 x 25 microg/animal/24, BC (3 mg/kg b.w./24 h or OCT and BC at the above doses for 10 days. Bax expression was detected by immunohistochemistry. Prolactin (PRL in blood serum was measured by radioimmunoassay (RIA. It has been found that both OCT and BC, alone or in combination, significantly reduce the tumor weight. Both OCT and BC suppressed PRL levels, but the inhibitory effect of BC was stronger than that of OCT. It has been found that the treatment with OCT and BC, alone or in combination, causes a significant increase in Bax expression in the rat prolactinoma cells. Our findings indicate that anti-tumoral action of bromocriptine and to some extent the action of octreotide in the experimental rat prolactinoma is connected with the induction of apoptosis and is associated with increased Bax expression.

  1. Enhanced Superconductivity in Close Proximity to the Structural Phase Transition of Sr1-xBaxNi2P2

    Science.gov (United States)

    Kudo, Kazutaka; Kitahama, Yutaka; Iba, Keita; Takasuga, Masaya; Nohara, Minoru

    2017-03-01

    The structural evolution and superconductivity of a 122-type solid solution Sr1-xBaxNi2P2 were studied. We found that an orthorhombic-tetragonal structural phase transition takes place at x = 0.5, and is characterized by the P-P dimers breaking. The superconducting transition temperature exhibited its highest value of 2.85 K at x = 0.4.

  2. Preparation and Photocatalytic Properties of Sr2−xBaxTa3O10−yNz Nanosheets

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    Tatsumi Ishihara

    2013-01-01

    Full Text Available Sr2−xBaxTa3O10−yNz (x = 0.0, 0.5, 1.0 nanosheets were prepared by exfoliating layered perovskite compounds (CsSr2−xBaxTa3O10−yNz. The Sr1.5Ba0.5Ta3O9.7N0.2 nanosheet showed the highest photocatalytic activity for H2 production from the water/methanol system among the Sr2−xBaxTa3O9.7N0.2 nanosheets prepared. In addition, Rh-loaded Sr1.5Ba0.5Ta3O9.6N0.3 nanosheet showed the photocatalytic activity for oxygen and hydrogen production from water. The ratio of hydrogen to oxygen evolved was around two. These results indicate that the Rh-loaded Sr1.5Ba0.5Ta3O9.6N0.3 nanosheet is a potential catalyst for photocatalytic water splitting.

  3. Paclitaxel-induced apoptosis is BAK-dependent, but BAX and BIM-independent in breast tumor.

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    Anna V Miller

    Full Text Available Paclitaxel (Taxol-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim(-/- MEFs (mouse embryonic fibroblasts, the bim(-/- mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak (-/- MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax(-/- MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance.

  4. Emodin inhibits LOVO colorectal cancer cell proliferation via the regulation of the Bcl-2/Bax ratio and cytochrome c.

    Science.gov (United States)

    Ma, Liang; Li, Wusheng

    2014-10-01

    In this study, the effect of emodin and its mechanism of action were investigated in LOVO colorectal cancer cells. Cell growth was determined using a Cell Counting kit-8 assay, and the results demonstrated that emodin significantly inhibited the growth of LOVO cells in a concentration-dependent manner. In order to investigate the anticancer mechanism of emodin, reverse transcription polymerase chain reaction assays were performed to determine the B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X protein (Bax) expression ratio in LOVO colorectal cancer cells following treatment with emodin. The results showed that emodin induced a significant increase in the Bax expression level and a marked reduction of the Bcl-2 expression level in LOVO cells. In addition, emodin was found to have an inhibitory effect on the mitochondrial membrane potential and the results from the western blot analysis revealed that cytochrome c was released from the mitochondria to the cytoplasm. In combination, these results suggest that emodin inhibits cancer cell growth via the regulation of the Bcl-2/Bax ratio and by its effect on the mitochondrial apoptosis pathway.

  5. Associations of NF-kappaB and Bax with Apoptosis in Varicose Veins of Women of Different Age Groups

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    Helle Evi Simovart

    2011-01-01

    Full Text Available The study aimed at detecting apoptotic endothelial cells (ECs and smooth muscle cells (SMCs together with determining expression of NF-kappaB (p105/p50 and Bax in varicose vein walls. Women (n=35 undergoing the excision of varicose veins were divided into 3 groups: younger than 35 years (I, 36–50 years (II, and older than 50 years (III. Apoptosis was determined by the TUNEL method, NF-kappaB and Bax expression by immunohistochemistry. The percentage of apoptotic ECs and SMCs in the layers of varicose vein wall increased in groups II and III. NF-kappaB expression had the lowest level in Group II with particularly low level in the media. Contrariwise, Bax expression levels in Group II were increased. The study revealed that in varicose veins ECs and SMCs apoptosis increased with advancing age. If increase in apoptosis during earlier stages of varicosities is probably regulated by intrinsic pathway, then in older patients other signaling pathways may be involved.

  6. Expression of apoptosis-Related genes bcl-2 and bax in rat brain hippocampus, followed by intraperitoneal injection of nanosilver

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    Maryam Ghoshcian

    2016-05-01

    Full Text Available Background: Silver nanoparticles are small scale substance (<100 nm used in food technology and medical industry. The data suggest that nanosilver may produce neurotoxicity by generating free radical-induced oxidative stress and by altering gene expression producing apoptosis and neurotoxicity. In this study, the apoptotic effects of Nano silver on apoptosis- related genes expression bcl-2 and bax on rat hippocampus, which is involved in memory and learning, was investigated. Materials & Methods: 28 male Wistar rats were divided into four groups of control and three groups of the treatment. The control group received saline and the treatment groups received intraperitoneal injections of silver nanoparticles at doses of 100, 200 and 400ppm. Ten days after the last injection, the hippocampal region was dissected and removed and then the expression of bcl-2 and bax genes was evaluated using semi-qualitative RT-PCR and Densitometry assay. Results: The expression of anti- apoptotic b-cl2 gene was reduced in the treatment groups compared to the control group. In comparison, the expression of pro- apoptotic bax gene was increased in the treatment groups compared to the control group. This apoptotic affects was increased at higher doses. Conclusion: The data suggest that silver nanoparticles may produce apoptosis by altering apoptosis- related genes expression, in rat brain hippocampus cells.

  7. Schiff Base Metal Derivatives Enhance the Expression of HSP70 and Suppress BAX Proteins in Prevention of Acute Gastric Lesion

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    Shahram Golbabapour

    2013-01-01

    Full Text Available Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg, the positive control (Tween 20 5% v/v, 5 mL/kg, and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg. After 1 h, all of the groups received ethanol 95% (5 mL/kg but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg. The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E, immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

  8. Virosecurinine induces apoptosis by affecting Bcl-2 and Bax expression in human colon cancer SW480 cells.

    Science.gov (United States)

    Chen, Chuan-Rong; Xia, Yong-Hui; Yao, Shu-Yan; Zhang, Qing; Wang, Ying; Ji, Zhao-Ning

    2012-04-01

    Virosecurinine, the major alkaloid isolated from Securinega suffruticosa Pall Rehd was found to exhibit growth inhibition and cytotoxicity against huaman colon cancer SW480 cells via the microculture tetrazolium (MTT) assay. Due to its greater cytotoxic potency and selectivity towards SW480 cells, flow cytometry was used to analyze the cell cycle distribution of control and treated SW480 cells whereas Annexin V-FITC/PI flow cytometry analysis was carried out to confirm apoptosis induced by virosecurinine in SW480 cells. Apoptotic regulatory genes were determined by RT-PCR analysis. Virosecurinine was found to induce G1/S cell cycle arrest which led to predominantly apoptotic mode of cell death. Mechanistically, virosecurinine was found to up-regulated the Bax gene expression and down-regulated the Bcl-2 expression in SW480, The ratio of Bcl-2 to Bax was significantly decreased. Hence, we suggest that virosecurinine induced apoptosis in SW480 cells by affecting the expression of bcl-2 and bax.

  9. p53's mitochondrial translocation and MOMP action is independent of Puma and Bax and severely disrupts mitochondrial membrane integrity

    Institute of Scientific and Technical Information of China (English)

    Sonja Wolff; Susan Erster; Gustavo Palacios; Ute M Moll

    2008-01-01

    p53's apoptotic program consists of transcription-dependent and transcription-independent pathways. In the latter, physical interactions between mitochondrial p53 and anti-and pro-apoptotic members of the Bcl2 family of mitochondrial permeability regulators are central. Using isogenic cell systems with defined deficiencies, we characterize in detail how mitochondrial p53 contributes to mitochondrial permeabilization, to what extent its action depends on other key Bcl2 family members and define its release activity. We show that mitochondrial p53 is highly efficient in inducing the release of soluble and insoluble apoptogenic factors by severely disrupting outer and inner mitochondrial membrane integrity. This action is associated with wild-type p53-induced oligomerization of Bax, Bak and VDAC and the formation of a stress-induced endogenous complex between p53 and cyclophilin D, normally located at the inner membrane. Tumor-derived p53 mutants are deficient in activating the Bax/Bak lipid pore. These actions are independent of Puma and Bax. Importantly, the latter distinguishes the mitochondrial from the cytosolic p53 death pathway.

  10. Melatonin restores normal Bax and Bcl-2 protein expression in the subgranular zone of the dentate gyrus in pinealectomized rats

    Institute of Scientific and Technical Information of China (English)

    Shengchang Zhang; Shuang Zhao; Lu Bai; Mingming Guan; Jielin Mo; Ling Lan

    2011-01-01

    In this study, we sought to elucidate the effects of melatonin on learning and memory as well as apoptosis and expression of the Bax or Bcl-2 proteins in the subgranular zone of the dentate gyrus in pinealectomized rats. Using the Morris water maze and the olfactory memory tests, we found that the average escape latency in pinealectomized rats was clearly increased compared with sham-operated rats. Moreover, the average escape latency in the melatonin-treated and pinealectomized rats was longer than that in the sham-operated rats and shorter than that in the pinealectomized and untreated rats. Immunohistochemistry and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) showed that there were fewer Bax immunoreactive cells and TUNEL-positive (apoptotic) cells but more Bcl-2 immunoreactive cells in the melatonin-treated rats compared with the pinealectomized rats. The sham-operated rats showed numbers of these cells similar to the melatonin-treated rats. These experimental findings demonstrate that melatonin treatment may reduce abnormal apoptosis by promoting gene expression of Bax and suppressing gene expression of Bcl-2 in the subgranular zone of the dentate gyrus in pinealectomized rats. These effects appear to result in the inhibition of cellular apoptosis and the improvement of spatial learning and memory in pinealectomized rats.

  11. Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

    KAUST Repository

    Radogna, Flavia

    2015-03-01

    Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10-9 vs. 10-5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10-5M, but drops to 10-9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

  12. Effect of hyperbaric oxygen on cytochrome C, Bcl-2 and bax expression after experimental traumatic brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    LIU Zhan; JIAO Qing-fang; YOU Chao; CHE Yan-jun; SU Fang-zhong

    2006-01-01

    Objective: To explore the effects of hyperbaric oxygen (HBO) treatment on the neuronal apoptosis at an earlier stage and the expressions of Cytochrome C (Cyt C), Bcl-2 (B-cell lymphoma-2 family) and Bax (Bcl-2associated X protein) in rat brain tissues after traumatic brain injury (TBI).Methods: Forty adult rats were divided into two groups, i.e., Group A ( the rats with untreated TBI) and Group B ( rats with HBO treatment after TBI). Sections of brain tissues of these two groups were then detected at 3,6,12,24,72 hours after TBI by immunohistochemistry and electronmicroscope, respectively.Results: HBO treatment could up-regulate the expression of Bcl-2 within 72 hours, reduce the release of Cyt C from mitochondria, attenuate the formation of dimeric Bax and alleviate the mitochondrial edema within 24 hours after TBI.Conclusions: HBO treatment can alleviate neuronal apoptosis after TBI by reducing the release of Cyt C and the dimers of Bax and up-regulating the expression of Bcl-2.

  13. Expression of Bax/Bcl-2 in renal tissue of rats with lymphatic flow barrier%Bax/Bcl-2在淋巴回流障碍大鼠肾组织中的表达

    Institute of Scientific and Technical Information of China (English)

    张桃艳; 李德祥; 柳刚; 关广聚

    2014-01-01

    目的:探讨阻断肾淋巴循环对大鼠肾脏细胞Bax、Bcl-2表达的影响及与大鼠肾脏功能的关系。方法选取雄性Wistar大鼠48只,将其随机分为模型组和对照组,各24只。各组大鼠分别于术后第1、7、14、28天各处死6只,留取肾组织标本提取组织蛋白、mRNA和制作石蜡切片。运用Real-time PCR、Western blot和免疫组织化学检测Bax、Bcl-2在肾组织中的表达,并测定24 h尿蛋白和血肌酐水平。结果模型组大鼠的肾功能逐渐减退,随着术后时间的延长,肾功能损害逐渐加重。模型组大鼠的Bax表达明显强于对照组,免疫组织化学显示,Bax的表达主要在肾小管及肾间质,远端小管的表达尤其明显,相反,模型组大鼠的Bcl-2的表达明显减弱。结论阻断肾淋巴循环可导致大鼠肾功能及肾小管间质的损害,并随时间延长而加重,肾细胞凋亡与此密切相关,其中Bax/Bcl-2途径发挥了积极作用。%Objective To investigate the influence of blocking renal lymph circulation on the expression of Bax/Bcl-2 in kidney cells of rats and the relationship of between the expression of Bax/Bcl-2 and kidney function of rats. Meth-ods 48 male Wistar rats were randomly divided into the model group (n=24) and the Control group (n=24).6 rats in each group were put to death after 1,7.14,28 days reapectively,and nephridial tissue sample were obtained for extracting protein,mRNA and making paraffin section.The expression of Bax/Bcl-2 in renal tissue was tested using Real-time PCR,Western blot and immunohistochemistry.24 hours urine protein and serum creatinine level were determined. Re-sults The renal function of rats in the model group decreased gradually,and with prolonging of postoperative time,renal function injury aggravated gradually.The expression of Bax of rats in the model group was stronger than that of the con-trol group,immunohistochemistry showed that Bax mainly expressed in renal tubule and interstitium

  14. Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during Withanolide D-induced apoptosis.

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    Susmita Mondal

    Full Text Available The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD, a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53-/- cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53-/- over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53-/- cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax-Bak- reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax-Bak+/HCT116Bax+Bak- was only marginally effective after WithaD treatment. In HCT116p53-/- cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells.

  15. Reconstitution of the anti-apoptotic Bcl-2 protein into lipid membranes and biophysical evidence for its detergent-driven association with the pro-apoptotic Bax protein.

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    Marcus Wallgren

    Full Text Available The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2 protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax, are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23-lauryl-ether (Brij-35 detergent at a level below its critical micelle concentration (CMC. Additional surface plasmon resonance (SPR measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A2 (a known inhibitory ligand of Bcl-2 to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC. Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.

  16. The prognostic significance of p53, Bax, Bcl-2 and cyclin E protein overexpression in colon cancer - an immunohistochemical study using the tissue microarray technique.

    Science.gov (United States)

    Melincovici, Carmen Stanca; Mihu, Carmen Mihaela; Mărginean, Mariana; Boşca, Adina Bianca; Coneac, Andrei; Moldovan, Ioana; Crişan, Maria

    2016-01-01

    In colon cancer, biological markers continue to have a limited prognostic value, the results being controversial. Studies of cell-cycle regulatory proteins and anti-apoptotic proteins aim to identify groups of patients that develop more aggressive tumors and might benefit from an individualized therapy management. The present study evaluates the prognostic role of the p53, Bax, Bcl-2 and cyclin E immunoexpression in colon cancer, using the tissue microarray (TMA) method. Tissue samples were obtained from 31 patients operated for colon cancer, embedded in TMA paraffin blocks and immunohistochemically stained for p53, Bax, Bcl-2 and cyclin E. We evaluated the relationship between the overexpression of these proteins and the clinico-pathological parameters, as well as the effect of these molecular markers on the survival rate. 65.22% of the patients were p53 positive, 39.13% Bcl-2 positive, 78.26% Bax positive and 34.78% cyclin E positive. Bcl-2(+) patients had significantly better differentiated tumors (p=0.043). Significantly poorly differentiated tumors were: Bax(+) patients (p=0.031), Bcl-2(-)÷p53(-) patients (p=0.042), Bcl-2(-)÷Bax(+) patients (p=0.029), and Bcl-2(-)÷p53(-)÷Bax(+) patients (p=0.016). The individual expression of the studied proteins did not influence the survival rate. A significantly lower survival rate was found in the following groups of patients: Bcl-2(-)÷p53(-) (40% vs. 83.3%, p=0.027), p53(-)÷Bax(+) (40% vs. 83.3%, p=0.027), Bcl-2(-)÷p53(-)÷Bax(+) (25% vs. 84.2%, p=0.003). The current study identified groups of patients with a significantly lower survival rate, which consequently are at an increased risk to develop tumors with a more aggressive biological behavior.

  17. Bax and Bak expression in cervical smears of women with low-and high-risk HPV types: A study of 120 cases.

    Science.gov (United States)

    Klapsinou, Eirini; Argyri, Elena; Panotopoulou, Efstathia; Daskalopoulou, Dimitra; Patsouris, Efstratios; Nonni, Afroditi; Lazaris, Andreas C; Thomopoulou, Georgia-Heleni

    2015-01-01

    Human papillomavirus (HPV) is known to be involved in the carcinogenesis of squamous cells in uterine cervix cancer, mostly by binding and inactivating the p53 and pRb tumor suppressor genes. Lately, evidence has emerged suggesting that HPV oncoproteins may interact with proteins involved in cellular apoptosis as well. This study aimed to investigate the expression of proapoptotic proteins Bax and Bak in women with low-risk and high-risk HPV types as opposed to HPV-negative women, and in women with normal pap smear compared to women with abnormal Papanicolau test (Pap) smear. A total of 120 liquid-based cervical samples were subtyped for HPV types with microarray hybridization and then stained and evaluated immunocytochemically for Bax and Bak expression. Statistical analysis was performed on the Bax and Bak scores (percentage of positive cells × staining intensity), the overall percentage of positive cells, and the most prevalent staining intensity group found in each sample. A weak association between negative Bax staining and cytologically normal Pap smears was discovered, whereas cytologically abnormal samples tended to stain weakly or moderately positive. No other statistically significant difference was found in the other analyzed parameters. Cytologically normal pap smears seem to have a slight tendency to stain negative for Bax as opposed to cytologically abnormal pap smears. Although the association is weak, it is an indication that there might be a connection between the expression of Bax and the development of cervical intraepithelial dysplasia, which warrants further investigation in larger-scale studies.

  18. Structural studies of the protein endostatin in fusion with BAX BH3 death domain, a hybrid that presents enhanced antitumoral activity.

    Science.gov (United States)

    Chura-Chambi, Rosa Maria; Arcuri, Helen Andrade; Lino, Felipe; Versati, Natan; Palma, Mario Sergio; Favaro, Denize C; Morganti, Ligia

    2017-05-01

    Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. (15) N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  19. Spatiotemporal Expression of Bcl-2/Bax and Neural Cell Apoptosis in the Developing Lumbosacral Spinal Cord of Rat Fetuses with Anorectal Malformations.

    Science.gov (United States)

    Yang, Zhonghua; Geng, Yuanyuan; Yao, Zhiya; Jia, Huimin; Bai, Yuzuo; Wang, Weilin

    2017-07-15

    Fecal incontinence and constipation still remain the major complications after procedures for anorectal malformations (ARMs). Previous studies have demonstrated a decrease of neural cell in lumbosacral spinal cord of ARMs patients and rat models. However, the underlying mechanism remains elusive. In this study, the neural cell apoptosis and Bcl-2/Bax expression were explored during lumbosacral spinal cord development in normal and ARMs fetuses. ARMs rat fetuses were induced by treating pregnant rats with ethylenethiourea on embryonic day 10. TUNEL staining was performed to identify apoptosis, and the expression of Bcl-2/Bax was confirmed with immunohistochemical staining, RT-qPCR and Western blot analysis on E16, E17, E19 and E21. Apoptosis index (AI) in the ARMs group was significantly higher compared to normal group. Our results showed that TUNEL-positive cells were mainly localized in the ventral horn, which is the location of neural cells controlling defecation. And the expression of Bcl-2 decreased, whereas the level of Bax increased in the ARMs fetuses. In addition, there was a significantly negative correlation between protein expression of Bcl-2/Bax ratio and AI in the ARMs group. Abnormal apoptosis might be a fundamental pathogenesis for the number decrease of neural cells in lumbosacral spinal cord, which leads to complications after procedures for ARMs. The negative correlation between the ratio of Bcl-2/Bax and AI manifested that Bcl-2/Bax pathway might be the mechanism for neural cell apoptosis in ARMs.

  20. Early apoptosis and cell death induced by ATX-S10Na ( Ⅱ)-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Makoto Mitsunaga; Akihito Tsubota; Kohichi Nariai; Yoshihisa Namiki; Makoto Sumi; Tetsuya Yoshikawa; Kiyotaka Fujise

    2007-01-01

    AIM: To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na (Ⅱ).METHODS: HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin V assays, transmission electron microscopy assays, caspase assays and western blotting.RESULTS: Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-XL,were decreased in Bax- and p53-independent manner.CONCLUSION: Our results indicate that early apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizingphotosensitizer ATX-S10Na (Ⅱ) are mediated by p53-Bax network and Iow levels of Bcl-2 and Bcl-XL proteins.Our results might help in formulating new therapeutic approaches in photedynamic therapy.

  1. In-house validation study of the DuPont Qualicon BAX system Q7 instrument with the BAX system PCR Assay for Salmonella (modification of AOAC Official Method 2003.09 and AOAC Research Institute Performance-Tested Method 100201).

    Science.gov (United States)

    Tice, George; Andaloro, Bridget; White, H Kirk; Bolton, Lance; Wang, Siqun; Davis, Eugene; Wallace, Morgan

    2009-01-01

    In 2006, DuPont Qualicon introduced the BAX system Q7 instrument for use with its assays. To demonstrate the equivalence of the new and old instruments, a validation study was conducted using the BAX system PCR Assay for Salmonella, AOAC Official Method 2003.09, on three food types. The foods were simultaneously analyzed with the BAX system Q7 instrument and either the U.S. Food and Drug Administration Bacteriological Analytical Manual or the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference method for detecting Salmonella. Comparable performance between the BAX system and the reference methods was observed. Of the 75 paired samples analyzed, 39 samples were positive by both the BAX system and reference methods, and 36 samples were negative by both the BAX system and reference methods, demonstrating 100% correlation. Inclusivity and exclusivity for the BAX system Q7 instrument were also established by testing 50 Salmonella strains and 20 non-Salmonella isolates. All Salmonella strains returned positive results, and all non-Salmonella isolates returned a negative response.

  2. Alterations in the characteristic size distributions of subcellular scatterers at the onset of apoptosis: effect of Bcl-xL and Bax/Bak

    Science.gov (United States)

    Zheng, Jing-Yi; Boustany, Nada N.

    2010-07-01

    Optical scatter imaging is used to estimate organelle size distributions in immortalized baby mouse kidney cells treated with 0.4 μM staurosporine to induce apoptosis. The study comprises apoptosis competent iBMK cells (W2) expressing the proapoptotic proteins Bax/Bak, apoptosis resistant Bax/Bak null cells (D3), and W2 and D3 cells expressing yellow fluorescent protein (YFP) or YFP fused to the antiapoptotic protein Bcl-xL (YFP-Bcl-xL). YFP expression is diffuse within the transfected cells, while YFP-Bcl-xL is localized to the mitochondria. Our results show a significant increase in the mean subcellular particle size from approximately 1.1 to 1.4 μm in both Bax/Bak expressing and Bax/Bak null cells after 60 min of STS treatment compared to DMSO-treated control cells. This dynamic is blocked by overexpression of YFP-Bcl-xL in Bax/Bak expressing cells, but is less significantly inhibited by YFP-Bcl-xL in Bax/Bak null cells. Our data suggest that the increase in subcellular particle size at the onset of apoptosis is modulated by Bcl-xL in the presence of Bax/Bak, but it occurs upstream of the final commitment to programmed cell death. Mitochondrial localization of YFP-Bcl-xL and the finding that micron-sized particles give rise to the scattering signal further suggest that alterations in mitochondrial morphology may underlie the observed changes in light scattering.

  3. Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner.

    Science.gov (United States)

    Guo, W; Zhang, Y; Ling, Z; Liu, X; Zhao, X; Yuan, Z; Nie, C; Wei, Y

    2015-10-15

    Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.

  4. Curcuma purpurascens BI. rhizome accelerates rat excisional wound healing: involvement of Hsp70/Bax proteins, antioxidant defense, and angiogenesis activity

    Science.gov (United States)

    Rouhollahi, Elham; Moghadamtousi, Soheil Zorofchian; Hajiaghaalipour, Fatemeh; Zahedifard, Maryam; Tayeby, Faezeh; Awang, Khalijah; Abdulla, Mahmood Ameen; Mohamed, Zahurin

    2015-01-01

    Purpose Curcuma purpurascens BI. is a member of Zingiberaceae family. The purpose of this study is to investigate the wound healing properties of hexane extract of C. purpurascens rhizome (HECP) against excisional wound healing in rats. Materials and methods Twenty four rats were randomly divided into 4 groups: A) negative control (blank placebo, acacia gum), B) low dose of HECP, C) high dose of HECP, and D) positive control, with 6 rats in each group. Full-thickness incisions (approximately 2.00 cm) were made on the neck area of each rat. Groups 1–4 were treated two-times a day for 20 days with blank placebo, HECP (100 mg/kg), HECP (200 mg/kg), and intrasite gel as a positive control, respectively. After 20 days, hematoxylin and eosin and Masson’s trichrome stainings were employed to investigate the histopathological alterations. Protein expressions of Bax and Hsp70 were examined in the wound tissues using immunohistochemistry analysis. In addition, levels of enzymatic antioxidants and malondialdehyde representing lipid peroxidation were measured in wound tissue homogenates. Results Macroscopic evaluation of wounds showed conspicuous elevation in wound contraction after topical administration of HECP at both doses. Moreover, histopathological analysis revealed noteworthy reduction in the scar width correlated with the enhanced collagen content and fibroblast cells, accompanied by a reduction of inflammatory cells in the granulation tissues. At the molecular level, HECP facilitates wound-healing process by downregulating Bax and upregulating Hsp70 protein at the wound site. The formation of new blood vessel was observed in Masson’s trichrome staining of wounds treated with HECP (100 and 200 mg/kg). In addition, HECP administration caused a significant surge in enzymatic antioxidant activities and a decline in lipid peroxidation. Conclusion These findings suggested that HECP accelerated wound-healing process in rats via antioxidant activity, angiogenesis

  5. Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Li-Hsuen Chen; Chia-Yu Hsu; Ching-Feng Weng

    2006-01-01

    AIM: Thioacetamide (TAA) has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death. METHODS: The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAAinduced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAAinduced clone 9 cells were measured by Western blot. RESULTS: The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died (MTT assay) within 24 h after 100 mg/L TAA was applied. Apoptotic cell percentage (TUNEL assay) and caspase 3 activities were highest after 100 mg/L TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspasedependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment.CONCLUSION: These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents,perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.

  6. First Principles Calculations of Structural, Electronic, Thermodynamic and Thermal Properties of BaxSr1-xTe Ternary Alloys

    Science.gov (United States)

    Chelli, S.; Meradji, H.; Amara Korba, S.; Ghemid, S.; El Haj Hassan, F.

    2014-12-01

    The structural, electronic thermodynamic and thermal properties of BaxSr1-xTe ternary mixed crystals have been studied using the ab initio full-potential linearized augmented plane wave (FP-LAPW) method within density functional theory (DFT). In this approach, the Perdew-Burke-Ernzerhof-generalized gradient approximation (PBE-GGA) was used for the exchange-correlation potential. Moreover, the recently proposed modified Becke Johnson (mBJ) potential approximation, which successfully corrects the band-gap problem was also used for band structure calculations. The ground-state properties are determined for the cubic bulk materials BaTe, SrTe and their mixed crystals at various concentrations (x = 0.25, 0.5 and 0.75). The effect of composition on lattice constant, bulk modulus and band gap was analyzed. Deviation of the lattice constant from Vegard's law and the bulk modulus from linear concentration dependence (LCD) were observed for the ternary BaxSr1-xTe alloys. The microscopic origins of the gap bowing were explained by using the approach of Zunger and co-workers. On the other hand, the thermodynamic stability of these alloys was investigated by calculating the excess enthalpy of mixing, ΔHm as well as the phase diagram. It was shown that these alloys are stable at high temperature. Thermal effects on some macroscopic properties of BaxSr1-xTe alloys were investigated using the quasi-harmonic Debye model, in which the phononic effects are considered.

  7. Engineering of the Curie temperature of epitaxial Sr1-xBaxTiO3 films via strain

    Science.gov (United States)

    Dai, Y.; Schubert, J.; Hollmann, E.; Mussler, G.; Wördenweber, R.

    2016-09-01

    The impact of strain on the structural and electrical properties of epitaxial Sr1-xBaxTiO3 films grown on single crystalline DyScO3 (110), TbScO3 (110), and GdScO3 (110) substrates is presented. X-ray diffraction measurements demonstrate that all films are grown epitaxially. The tensile in-plane strain is only partially compensated by a contraction of the out-of-plane lattice parameter. As a result, the volume of the unit cell of the Sr1-xBaxTiO3 film increases due to the tensile strain, and the resulting Poisson ratio of the film is ν ≈ 0.33, which is larger than but still close to the literature values of ν ≈ 0.23 for unstrained defect-free SrTiO3. The Curie temperature derived from the temperature dependence of the in-plane dielectric response leads to a strain-temperature phase diagram for the epitaxial Sr1-xBaxTiO3 films. The experimental data show a deviation from the linear dependence predicted by the Landau thermodynamic theory for large strain (>1.2%). However, using the equilibrium thermodynamic analysis, we can demonstrate that this deviation arises from the relaxation of the strain due to defect formation in the film. The result reveals that in addition to the nominal misfit strain, the defect formation strongly affects the effective strain and, thus, the dielectric response of epitaxially grown ferroelectric films.

  8. Astilbic Acid Induced COLO 205 cell Apoptosis by Regulating Bcl-2 and Bax Expression and Activating Caspase-3

    Institute of Scientific and Technical Information of China (English)

    ZhengXiao-liang; SunHong-xiang; LiuXue-li; ChenYun-xiang; QianBo-chu

    2005-01-01

    To investigate the effect of astilbic acid (3β,6β-dihydroxyolean-12-en-27-oic acid, AA) on human colorectal carcinoma COLO 205 cell proliferation and apoptosis.Methods Proliferation of COLO 205 cells was measued by MTT assay. Content of DNA in COLO 205 cell was measued by modified diphenylamine assay. AA-induced morphological changes was observed with fluorescence microscope and transmission electron microscope.DNA fragmentation was visualized by agarose gel electrophoresis.Apoptosis rate and cell cycle distribution were deter-mined by flow cytometric analysis.Expressions of Bcl-2 and Bax proteins were visioned by immunohistochemical analysis.The change of relative mitochondral transmembrane potential (MTP) in COLO 205 cell was analyzed with FCM after rhodamine 123 staining. Results The IC50 (96h) of AA for inhibiting COLO 205 cell proliferation was 61.56±0.34 μmol/L.AA induced a marked concentration- and time-dependent inhibition of COLO 205 cell proliferation and reduced the DNA content in COLO 205 cell. Cells treated with AA 64 μmol/L showed typical morphological changes of apoptosis and DNA “ladder” pattern. The cell cycle was arrested in G0/G1 phase, and the apoptosis rate was 28.25% for COLO 205 cells treated with AA 64 μmol/L for 48h. Meanwhile the expression of Bcl-2 protein was decreased while that of Bax was increased and relative MTP was decreased as well. DEVD-CHO 1μmol/L could increase the viability of COLO 205 cells treated with AA for 48h.Conclusion AA showed potent inhibitory activity on COLO 205 cells proliferation,and could induce COLO 205 cells apoptosis through disturbing DNA replication, down-regulating Bcl-2 expression, and up-regulating Bax expression, lowering relative MTP, and activating caspase-3 pathway.

  9. Astilbic acid induced COLO 205 cell apoptosis by regulating Bcl-2 and Bax expression and activating caspase-3

    Institute of Scientific and Technical Information of China (English)

    Xiao-liang ZHENG; Hong-xiang SUN; Xue-li LIU; Yun-xiang CHEN; Bo-chu QIAN

    2004-01-01

    AIM: To investigate the effect of astilbic acid (3β, 6β-dihydroxyolean-12-en-27-oic acid, AA) on human colorectal carcinoma COLO 205 cell proliferation and apoptosis. METHODS: Proliferation of COLO 205 cells was measued by MTT assay. Content of DNA in COLO 205 cell was measued by modified diphenylamine assay. AA-induced morphological changes was observed with fluorescence microscope and transmission electron microscope. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptosis rate and cell cycle distribution were determined by flow cytometric analysis. Expressions of Bcl-2 and Bax proteins were visioned by immunohistochemical analysis. The change of relative mitochondral transmembrane potential (MTP) in COLO 205 cell was analyzed with FCM after rhodamine 123 staining. RESULTS: The ICs0 (96 h) of AA for inhibiting COLO 205 cell proliferation was 61.56±0.34 μmol/L. AA induced a marked concentration- and time-dependent inhibition of COLO 205 cell proliferation and reduced the DNA content in COLO 205 cell. Cells treated with AA 64 μmol/L showed typical morphological changes of apoptosis and DNA "ladder" pattern. The cell cycle was arrested in G0/G1 phase, and the apoptosis rate was 28.25 % for COLO 205 cells treated with AA 64 μmol/L for 48 h. Meanwhile the expression of Bcl-2 protein was decreased while that of Bax was increased and relative MTP was decreased as well. DEVD-CHO 1 μmol/L could increase the viability of COLO 205 cells treated with AA for 48 h. CONCLUSION: AA showed potent inhibitory activity on COLO 205 cells proliferation, and could induce COLO 205 cells apoptosis through disturbing DNA replication, down-regulatin Bcl-2 expression,and up-regulating Bax expression,lowering relative MTP, and activating caspase-3 pathway.

  10. Changes of bcl-xL and bax mRNA expression following traumatic brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    骆纯; 卢亦成; 江基尧; 朱诚

    2002-01-01

    Objective: To investigate the changes of bcl-2 gene family and the molecular mechanism of neuronal apoptosis following traumatic brain injury (TBI) in rats.Methods: Male Sprague-Dawley (SD) rats were subjected to lateral fluid percussion brain injury (FPBI) of moderate severity. The bcl-xL and bax mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). In addition to morphological evidence of apoptosis, terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling (TUNEL) histochemistry was used to identify the DNA fragmentation in situ at both light and electron microscope levels, whereas characteristic internucleosomal DNA fragmentation of apoptosis was demonstrated by DNA gel electrophoresis.Results: The apoptotic response to trauma was regionally distinct and may be involved in both acute and delayed cell death. The bcl-xL mRNA expression of the impact site was significantly lower (67.42%±7.54%) than that of the ipsilateral hemisphere at 6 hours after injury (P<0.01). The decrease of bcl-xL mRNA expression preceded apoptosis at 24 hours after injury. The bax mRNA expression rose slowly, doubled at 3 days after injury and returned to the sham level slowly.Conclusions: Decreased expression of bcl-xL mRNA and increased expression of bax mRNA coincides with apoptosis following brain injury. The bcl-2 gene family is involved in neuronal apoptosis after TBI, and the changes of mRNA expression of the family members lead the neuronal cells to apoptosis.

  11. Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts compared with normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reduce myocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl

  12. Hydro(Solvo) Thermal Synthesis and Characterization of Bax Sr1-x TiO3 Nanopowders

    Institute of Scientific and Technical Information of China (English)

    XING Guang-jian; YANG Zhi-min; YANG Jian; MAO Chang-hui; DU Jun

    2004-01-01

    Nano-sized barium strontium titanate (BaxSr1-x TiO3, BST) powders were synthesized with hydrothermal and solvothermal methods at 120 ℃ for 4 h based on the reaction precursor of barium acetate, strontium acetate, tetrabutyl titanium and (CH3 )4NOH. The crystalline phase, particle size and morphology of the BST powders were characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM). Influences of reaction temperature and reaction time on the morphologies of powders were also discussed.

  13. Improvement of cytomegalovirus pp65 DNA vaccine efficacy by co-administration of siRNAs targeting BAK and BAX.

    Science.gov (United States)

    Liu, Jixiao; Feng, Keke; Zhao, Lu; Luo, Haining; Zhu, Yingjun

    2017-06-01

    The efficacy of DNA vaccines may be improved by small interfering (si)RNA adjuvants targeting pro-apoptotic genes. The aim of the present study was to investigate the capacity of siRNAs targeting B-cell lymphoma 2 homologous antagonist killer (BAK) and B-cell lymphoma 2-associated X protein (BAX) to improve the efficacy of a cytomegalovirus (CMV) vaccine. BALB/c mice were divided into four groups (n=18 in each): unimmunized and immunized with pcDNA 3.1-pp65 expressing CMV 65 kDa matrix phosphoprotein and BAK + BAX siRNAs, pcDNA 3.1-pp65 and control siRNA, or control pcDNA 3.1 and BAK + BAX siRNAs. Immunizations were performed twice with an interval of 3 weeks. CMV-specific mouse splenocyte interferon (IFN)-γ secretion was assessed by ELISPOT; furthermore, an in vivo cytotoxic T lymphocyte assay was performed 2 weeks after the last immunization. After lethal CMV challenge of the mice, body weight, virus titers in the spleens and salivary glands as well as survival were recorded. The amount of splenocytes secreting IFN-γ in response to CMV pp65 peptides and specific lysis of peptide-pulsed target cells were significantly higher in mice administered pcDNA3.1-pp65 and BAK + BAX siRNAs than those in mice administered pcDNA3.1-pp65 and control siRNA (PBAX siRNAs were significantly lower than those in mice immunized with pcDNA3.1-pp65 and control siRNA (PBAX siRNAs survived for longer, and at 21 days after lethal CMV challenge, 66 and 100% of these mice survived, respectively. These mice also experienced less weight loss compared with mice immunized with pcDNA3.1-pp65 and control siRNA (PBAX improved the efficacy of CMV pp65 DNA vaccine.

  14. Glycyrrhizin protects rat heart against ischemia-reperfusion injury through blockade of HMGB1-dependent phospho-JNK/Bax pathway

    Institute of Scientific and Technical Information of China (English)

    Chang-lin ZHAI; Mei-qi ZHANG; Yun ZHANG; Hong-xia XU; Jing-min WANG; Gui-peng AN; Yuan-yuan WANG; Li LI

    2012-01-01

    Aim: Glycyrrhizin (GL) has been found to inhibit extracellular HMGB1 cytokine's activity,and protect spinal cord,liver and brain against I/R-induced injury in experimental animals.The purpose of this study was to investigate the protective effect of GL in rat myocardial I/R-induced injury and to elucidate the underlying mechanisms.Methods: Male adult Sprague-Dawley rats underwent a 30-min left coronary artery occlusion followed by a 24-h reperfusion.The rats were treated with glycyrrhizin or glycyrrhizin plus recombinant HMGB1 after 30 min of ischemia and before reperfusion.Serum HMGB1,TNF-α and IL-6 levels,and hemodynamic parameters were measured at the onset and different time points of reperfusion.At the end of the experiment,the heart was excised,and the infarct size and histological changes were examined.The levels of Bcl2,Bax and cytochrome c,as well as phospho-ERK1/2,phospho-JNK and phospho-P38 in the heart tissue were evaluated using Western blot analysis,and myocardial caspase-3 activity was measured colorimetrically using BD pharmingen caspase 3 assay kit.Results: Intravenous administration of GL (10 mg/kg) significantly reduced the infarct size,but did not change the hemodynamic parameters at different time points during reperfusion.GL significantly decreased the levels of serum HMGB1,TNF-α and IL-6.GL changed the distribution of Bax and cytochrome c expression between the mitochondrial and cytosolic fractions in the heart tissue,resulting in inhibition of myocardial apoptosis.Moreover,expression of phospho-JNK,but not ERK1/2 and P38 was decreased by GL in the heart tissue.All of the effects produced by GL treatment were reversed by co-administration with the recombinant HMGB1 (100 μg).Intravenous administration of SP600125,a selective phospho-JNK inhibitor (0.5 mg/kg),attenuated HMGB1-dependent Bax translocation and the subsequent apoptosis.Conclusion: These results demonstrate that GL alleviates rat myocardial I/R-induced injury via directly

  15. Apoptosis Induction by Targeting Interferon Gamma Receptor 2 (IFNgammaR2) in Prostate Cancer: Ligand (IFNgamma)-Independent Novel Function of IFNgammaR2 as a Bax Inhibitor

    Science.gov (United States)

    2015-08-01

    in Mitochondria and ER membranes! Cell Death ER# Bax! Bax! Bax! Mitochondria ! IFNγR 2! IFNγR1! IFNγ" Jak! STA T! Gene Expression! (IFNγ Response...planned to determine what kind of cell type(s) in prostate cancer tissue expresses IFNγR2 by performing immunohistochemistry. Another important...proposed experiment is to determine whether IFNγR2 expression profile (expression levels and expression type (cytosol or membrane expression, or cell type

  16. Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2 associated X protein) Bax in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge.

    Science.gov (United States)

    Luo, Sheng-Wei; Wang, Wei-Na; Sun, Zuo-Ming; Xie, Fu-Xing; Kong, Jing-Rong; Liu, Yuan; Cheng, Chang-Hong

    2016-07-01

    Bax is a pro-apoptotic member of Bcl-2 like superfamily, playing an important role in regulating the apoptosis. In this study, the full-length Bax (EcBax) was obtained, containing a 5'UTR of 64 bp, an ORF of 579 bp and a 3'UTR of 1021 bp. The EcBax gene encoded a polypeptide of 192 amino acids with an estimated molecular mass of 21.55 KDa and a predicted isoelectric point (pI) of 6.75. The deduced amino acid sequence analysis showed that EcBax comprised the conserved residues and the characteristic domains known to the critical function of Bax. qRT-PCR analysis revealed that EcBax mRNA was broadly expressed in all of the examined tissues, while the highest expression level was observed in blood, followed by the expression in liver, gill, spleen, kidney, heart, muscle and intestine. A sharp increase of EcBax expression was observed in the vibrio challenge group by comparing with those in the control. Subcellular localization analysis revealed that EcBax was predominantly localized in the cytoplasm. EcBax exerted a regulatory role in modulating the mitochondrial membrane potential, promoting the cytochrome c release, and then activating the downstream caspase signaling. Moreover, the overexpression of EcBax can decrease the cell viability and antagonize NF-kB, AP-1, Stat3 promoter activity in Hela cells. These results indicate that EcBax containing the conserved domain of pro-apoptotic member of Bcl-2 family may disrupt the mammalian signaling and play a regulative role in the apoptotic process.

  17. The Impact of Adenosine Fast Induction of Myocardial Arrest during CABG on Myocardial Expression of Apoptosis-Regulating Genes Bax and Bcl-2

    Directory of Open Access Journals (Sweden)

    Ahmed Shalaby

    2009-01-01

    Full Text Available Background. We studied the effect of fast induction of cardiac arrest with denosine on myocardial bax and bcl-2 expression. Methods and Results. 40 elective CABG patients were allocated into two groups. The adenosine group (n=20 received 250 μg/kg adenosine into the aortic root followed by blood potassium cardioplegia. The control group received potassium cardioplegia in blood. Bcl-2 and bax were measured. Bax was reduced in the postoperative biopsies (1.38 versus 0.47, P=.002 in the control group. Bcl-2 showed a reducing tendency (0.14 versus 0.085, P=.07. After the adenosine treatment, the expression of both bax (0.52 versus 0.59, P=.4 and bcl-2 (0.104 versus 0.107, P=.4 remained unaltered after the operation. Conclusion. Open heart surgery is associated with rapid reduction in the expression of apoptosis regulating genes bax and bcl-2. Fast Adenosine induction abolished changes in their expression.

  18. Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells.

    Science.gov (United States)

    Papaianni, Emanuela; El Maadidi, Souhayla; Schejtman, Andrea; Neumann, Simon; Maurer, Ulrich; Marino-Merlo, Francesca; Mastino, Antonio; Borner, Christoph

    2015-01-01

    Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells.

  19. Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells.

    Directory of Open Access Journals (Sweden)

    Emanuela Papaianni

    Full Text Available Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP. The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1 and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-. Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells.

  20. Molecular and immunohistochemical expression of apoptotic proteins Bax, Bcl-2 and Caspase 3 in infantile hemangioma tissues as an effect of propranolol treatment.

    Science.gov (United States)

    Wnęk, Aneta; Andrzejewska, Ewa; Kobos, Józef; Taran, Katarzyna; Przewratil, Przemysław

    2017-05-01

    Infantile hemangiomas (IHs) are the most common benign tumors of childhood. They are characterized by a unique clinical course with two phases, proliferation and involution, which are followed by regression. The therapy of infantile hemangiomas was revolutionized in 2008 by the introduction of propranolol, however, the mechanism of its influence on hemangiomas remains unclear. The study included 71 patients with IHs, 27 of whom were treated with propranolol while the remaining 44 were used as a comparative group. The expression of Bcl-2, Bax and Caspase3 was determined with immunohistochemistry and mRNA of Bax, Bcl-2 and Caspase3 were assessed with the use of RT-PCR. Both methods revealed a statistically significant decrease in Bcl-2 expression and an increase in Bax in IHs tissues after propranolol treatment. The results obtained for Bax and Bcl-2 proteins may indicate a link between the effect of propranolol and apoptosis. Higher Bax and lower Bcl-2 expression in the propranolol treated group indicates a strong pro- apoptotic action countering any anti-apoptotic activity; apoptosis was indicted in IH tissue as a potential result of propranolol treatment, with potential clinical impact in other tumors. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  1. Glycogen synthase kinase-3β facilitates cell apoptosis induced by high fluence low-power laser irradiation through acceleration of Bax translocation

    Science.gov (United States)

    Huang, Lei; Wu, Shengnan; Xing, Da

    2011-03-01

    Glycogen synthase kinase-3β (GSK-3β) is a critical activator of cell apoptosis induced by a diverse array of insults. However, the effects of GSK-3β on the human lung adenocarcinoma cell (ASTC-a-1) apoptosis induced by high fluence low-power laser irradiation (HF-LPLI) are not clear. Here, we showed that GSK-3β was constantly translocated from cytoplasm to nucleus and activated during HF-LPLI-induced cell apoptosis. In addition, we found that co-overexpression of YFP-GSK-3β and CFP-Bax in ASTC-a-1 cells accelerated both Bax translocations to mitochondria and cell apoptosis, compared to the cells expressed CFP-Bax only under HF-LPLI treatment, indicating that GSK-3β facilitated ASTC-a-1 cells apoptosis through acceleration mitochondrial translocation of Bax. Our results demonstrate that GSK-3β exerts some of its pro-apoptotic effects in ASTC-a-1 cells by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.

  2. Mode of cell death induced by the HSP90 inhibitor 17-AAG (tanespimycin) is dependent on the expression of pro-apoptotic BAX

    Science.gov (United States)

    Powers, Marissa V; Valenti, Melanie; Miranda, Susana; Maloney, Alison; Eccles, Suzanne A.; Thomas, George; Clarke, Paul A; Workman, Paul

    2013-01-01

    Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) are of considerable current interest as targeted cancer therapeutic agents because of the ability to destabilize multiple oncogenic client proteins. Despite their resulting pleiotropic effects on multiple oncogenic pathways and hallmark traits of cancer, resistance to HSP90 inhibitors is possible and their ability to induce apoptosis is less than might be expected. Using an isogenic model for BAX knockout in HCT116 human colon carcinoma cells, we demonstrate the induction of BAX-dependent apoptosis at pharmacologically relevant concentrations of the HSP90 inhibitor 17-AAG both in vitro and in tumor xenografts in vivo. Removal of BAX expression by homologous recombination reduces apoptosis in vitro and in vivo but allows a lower level of cell death via a predominantly necrotic mechanism. Despite reducing apoptosis, the loss of BAX does not alter the overall sensitivity to 17-AAG in vitro or in vivo. The results indicate that 17-AAG acts predominantly to cause a cytostatic antiproliferative effect rather than cell death and further suggest that BAX status may not alter the overall clinical response to HSP90 inhibitors. Other agents may be required in combination to enhance tumor-selective killing by these promising drugs. In addition, there are implications for the use of apoptotic endpoints in the assessment of the activity of molecularly targeted agents. PMID:24185264

  3. Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells

    Science.gov (United States)

    Papaianni, Emanuela; El Maadidi, Souhayla; Schejtman, Andrea; Neumann, Simon; Maurer, Ulrich; Marino-Merlo, Francesca; Mastino, Antonio; Borner, Christoph

    2015-01-01

    Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells. PMID:26030884

  4. Effects of Ethyl Pyruvate on Myocardial Apoptosis and Expression of Bcl-2 and Bax Proteins after Ischemia-reperfusion in Rats

    Institute of Scientific and Technical Information of China (English)

    Jialong GUO; Kailun ZHANG; Yanmei JI; Xionggang JIANG; Shunqing ZUO

    2008-01-01

    In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion (I/R) in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups (n=8 in each group): control group was perfused for 120min. In the I/R group, after 30min stabilization the injury was induced by 30min global ischemia followed by 60min reperfusion. Ethyl pyruvate (EP) group was set up with the same protocol as I/R group except that it was supplied with 2mmol/L EP 15min before ischemia and throughout reperfusion. Myocardial malonaldehyde (MDA) content Was measured. Myocardial apoptotic index (AI) was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group (P<0.05). These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.

  5. Prognostic value of Bcl-2 and Bax tumor cell expression in patients with non muscle-invasive bladder cancer receiving bacillus Calmette-Guerin immunotherapy.

    Science.gov (United States)

    Ajili, Faouzia; Kaabi, Belhassen; Darouiche, Amine; Tounsi, Haifa; Kourda, Nadia; Chebil, Mohamed; Manai, Mohamed; Boubaker, Samir

    2012-02-01

    Apoptosis is the distinctive form of programmed cell death that complements cell proliferation in maintaining normal tissue homeostasis. The significance of constitutive apoptosis in the recurrence of Non Muscle Invasive Bladder Cancer has yet to be investigated. The aim of this study is to investigate the prognostic significance of Bax and Bcl-2 in terms of recurrence after BCG immunotherapy. Immunohistochemical analysis was performed on frozen biopsies to evaluate bcl-2 and Bax proteins expression in 28 cases of NMIBC. All patients with confirmed NMIBC were treated with intravesical BCG-immunotherapy. The follow up was performed for 26 months. The correlation between clinicopathological, immunohistochemical data and the response to BCG therapy was performed. Univariate analysis showed that, PT1 stage, High grade and Bax expression increased significantly the risk of recurrence (P = 0.015, P = 0.015 and P= 0.034 respectively). In addition, multivariate analysis selected the model involving stage, age, Bax and Bcl-2 expression as the best independent variables of recurrence. In conclusion, the expression of Bcl-2 and Bax in NMIBC could have a prognostic value in assessing the risk of recurrence after BCG immunotherapy. These findings require further investigations on larger cohort in order to ascertain new molecular markers of the response to BCG immunotherapy.

  6. Structure evolution upon chemical and physical pressure in (Sr1-xBax)2FeSbO6

    Science.gov (United States)

    Tiittanen, T.; Karppinen, M.

    2017-02-01

    Here we demonstrate the gradual structural transformation from the monoclinic I2/m to tetragonal I4/m, cubic Fm-3m and hexagonal P63/mmc structure upon the isovalent larger-for-smaller A-site cation substitution in the B-site ordered double-perovskite system (Sr1-xBax)2FeSbO6. This is the same transformation sequence previously observed up to Fm-3m upon heating the parent Sr2FeSbO6 phase to high temperatures. High-pressure treatment, on the other hand, transforms the hexagonal P63/mmc structure of the other end member Ba2FeSbO6 back to the cubic Fm-3m structure. Hence we may conclude that chemical pressure, physical pressure and decreasing temperature all work towards the same direction in the (Sr1-xBax)2FeSbO6 system. Also shown is that with increasing Ba-for-Sr substitution level, i.e. with decreasing chemical pressure effect, the degree-of-order among the B-site cations, Fe and Sb, decreases.

  7. Enhanced charge stripe order of superconducting La2-xBaxCuO4 in a magnetic field

    Science.gov (United States)

    Hücker, M.; v. Zimmermann, M.; Xu, Z. J.; Wen, J. S.; Gu, G. D.; Tranquada, J. M.

    2013-01-01

    The effect of a magnetic field on the charge stripe order in La2-xBaxCuO4 has been studied by means of high-energy (100 keV) x-ray diffraction for charge carrier concentrations ranging from strongly underdoped to optimally doped. We find that charge stripe order can be significantly enhanced by a magnetic field applied along the c axis, but only at temperatures and dopings where it coexists with bulk superconductivity at zero field. The field also increases stripe correlations between the planes, which can result in an enhanced frustration of the interlayer Josephson coupling. Close to the famous x=(1)/(8) compound, where zero field stripe order is pronounced and bulk superconductivity is suppressed, charge stripe order is independent of a magnetic field. The results for La2-xBaxCuO4 resemble recent observations in YBa2Cu3O6+δ and, independent of potential differences in the microscopic origin of charge order in these two compounds, imply a very similar competition with three-dimensionally coherent superconductivity.

  8. Gynostemma pentaphyllum protects mouse male germ cells against apoptosis caused by zearalenone via Bax and Bcl-2 regulation.

    Science.gov (United States)

    Yuan, Hui; Deng, Youtian; Yuan, Liyun; Wu, Jing; Yuan, Zhihang; Yi, Jine; Zhang, Ming; Guo, Chengzhi; Wen, Lixin; Li, Rongfang; Zhu, Li; He, Zuping

    2010-03-01

    The objective of this study was to explore the effects of Gynostemma pentaphyllum on Zearalenone-induced apoptosis in mouse male germ cells. Fifty Kunming male mice at 25-days-old were classified into five groups: group A was the control (10% ethanol, 0.5 ml/day); group B with 10 microg Zearalenone/day; group C with 10 microg Zearalenone and 50 mg/kg/day Gynostemma pentaphyllum; group D with 10 microg Zearalenone and 100 mg/kg/day Gynostemma pentaphyllum; and group E with 10 microg Zearalenone and 200 mg/kg/day Gynostemma pentaphyllum. It was found that Gynostemma pentaphyllum has a marked effect on protecting male germ cells against Zearalenone-induced apoptosis, as evidenced by a reduced apoptosis rate of male germ cells and Bax expression as well as an enhancement of Bcl-2 expression in Gynostemma pentaphyllum-treated groups compared to the control. In addition, Gynostemma pentaphyllum remarkably improved pathologic changes of testicular tissue, reduced the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD) caused by Zearalenone. Taken together, these results suggest that Gynostemma pentaphyllum protects against toxicity caused by Zearalenone through anti-oxidation and anti-apoptosis via the regulation of Bax and Bcl-2 expression.

  9. BaP-induced DNA damage initiated p53-independent necroptosis via the mitochondrial pathway involving Bax and Bcl-2.

    Science.gov (United States)

    Jiang, Y; Chen, X; Yang, G; Wang, Q; Wang, J; Xiong, W; Yuan, J

    2013-12-01

    Benzo(a)pyrene (BaP), a typical environmental carcinogen, can induce cell death both by protein 53 or tumor protein 53 (p53)-independent and -dependent pathways. However, little is known about the molecular mechanisms of p53-independent pathways in BaP-induced cell death. In this study, cells with different genetic background (including p53-proficient human fetal lung fibroblast cell lines (MRC-5), p53-deficient human non-small-cell lung carcinoma cell lines (H1299), and p53-knockdown cell lines (MRC-5(p53-/-))) were used to establish models of BaP-induced cell death. The results showed that BaP (8, 16, 32, and 64 μM) induced necroptotic cell death in the cell lines. The necroptotic cell death and DNA damage were concurrently observed. In the three cell lines, at 24 h after treatment, BaP (8-64 μM) upregulated expressions of BAX, BCL-2, and cleaved caspase-3 proteins, but not their messenger RNA levels. The findings suggested that BaP-induced necroptosis was modulated by the p53-independent pathway, which was related to the induction of BAX, decreased expression of BCL-2, and activation of caspase-3.

  10. Expression of p53, Bax and Bcl-2 proteins in hepatocytes in non-alcoholic fatty liver disease

    Institute of Scientific and Technical Information of China (English)

    Anatol Panasiuk; Janusz Dzieciol; Bozena Panasiuk; Danuta Prokopowicz

    2006-01-01

    AIM: To analyze the protein expression essential for apoptosis in liver steatosis.METHODS: The expression of proapoptotic proteinsp53, Bax, and antiapoptotic Bcl-2 in hepatocytes with steatosis (SH) and without steatosis (NSH) was evaluated in 84 patients at various stages of non-alcoholic fatty liver disease (NAFLD).RESULTS: Immunohistochemical staining of liver tissue showed the activation of p53 protein in SH and NSH with increased liver steatosis, diminished Bcl-2 and slightly decreased Bax protein. Positive correlation was found between the stage of liver steatosis with p53 expression in SH (r = 0.54, P < 0.01) and NSH (r = 0.49,P < 0.01).The antiapoptotic protein Bcl-2 was diminished together with the advancement of liver steatosis, especially in non-steatosed hepatocytes (r =0.43, P < 001).CONCLUSION: Apoptosis is one of the most important mechanisms leading to hepatocyte elimination in NAFLD. The intensification of inflammation in NAFLD induces proapoptotic protein p53 with the inhibition of antiapoptotic Bcl-2.

  11. Effect of bax, bcl-2 and bcl-xL on regulating apoptosis in tissues of normal liver and hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Xiao-Zhong Guo; Xiao-Dong Shao; Min-Pei Liu; Jian-Hua Xu; Li-Nan Ren; Jia-Jun Zhao; Hong-Yu Li; Di Wang

    2002-01-01

    AIM: To investigate the expression of bax, bcl-2 and bcl-xL mRNA in the tissues of normal liver and hepatocellular carcinoma (HCC), and analyze the relationship between the expression of bax, bcl-2 and bcl-xL mRNA and clinical parameters of HCC patients.METHODS: The expression of bax, bcl-2 and bcl-xL mRNA of normal liver and HCC was measured by Northern blot. Statistical analyses were made by t test and correlation analysis.RESULTS: A very low mRNA level was indicated at bax,bcl-2 and bcl-xL in the HCC tissues in contrast to the tissues of normal liver by Northern blot analysis. The analyses of mRNA level revealed that HCC tissues exhibited a mean 7.6-fold decrease in bax, 4.2-fold in bcl-2 and 3.5-fold in bcl-xL in comparison with normal control tissues, respectively. Positive correlation was found between bax and bcl-xL (r=0.7061,P<0.01). There was no significance between the mRNA expression of these three genes and age, gender, tumor differentiation and tumor stage of HCC patients. CONCLUSION: The results are consistent with the fact that apoptosis rarely occurs in normal livers but increases in HCC, indicating that bcl-2 and bcl-xL may play a very important role in regulating the apoptosis of normal liver and HCC.

  12. JNK-Bcl-2/Bcl-xL-Bax/Bak Pathway Mediates the Crosstalk between Matrine-Induced Autophagy and Apoptosis via Interplay with Beclin 1.

    Science.gov (United States)

    Yang, Jiong; Yao, Shukun

    2015-10-27

    Autophagy is associated with drug resistance which has been a threat in chemotherapy of hepatocellular carcinoma (HCC). The interconnected molecular regulators between autophagy and apoptosis serve as switching points critical to the ultimate outcome of the cell. Our study was performed to investigate the crosstalk between autophagy and apoptosis in HCC after the treatment of matrine. Flow cytometry and TUNEL (terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling) assay were used to detect apoptosis in vitro and in vivo, respectively. Bax oligomerization and Cytochrome c release assay were performed. Immunoprecipitation and siRNA transfection were used to detect the interplay between Bcl-2/Bcl-xL,Bax, and Beclin 1. Our results showed that: (1) matrine not only activated caspase and PARP (poly ADP-ribose polymerase) cleavage, but also triggered autophagy as shown by the increased levels of LC3II, Beclin 1, and PI3KC3, and the decreased level of p62; (2) matrine treatment promoted the JNK-Bcl-2/ Bcl-xL-Bax/Bak pathway; (3) Bax was oligomerized, the mitochondrial membrane potential altered, and Cytochrome c was released subsequently; (4) Bax interacts with Beclin 1 and inhibits autophagy, which may be a new crosstalk point; and (5) finally, we showed that matrine suppressed the growth of a MHCC97L xenograft in vivo for the first time. In conclusion, the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates the crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin 1.

  13. Bax and Bak expression in cervical smears of women with low-and high-risk HPV types: A study of 120 cases

    Directory of Open Access Journals (Sweden)

    Eirini Klapsinou

    2015-01-01

    Full Text Available Background: Human papillomavirus (HPV is known to be involved in the carcinogenesis of squamous cells in uterine cervix cancer, mostly by binding and inactivating the p53 and pRb tumor suppressor genes. Lately, evidence has emerged suggesting that HPV oncoproteins may interact with proteins involved in cellular apoptosis as well. Aim: This study aimed to investigate the expression of proapoptotic proteins Bax and Bak in women with low-risk and high-risk HPV types as opposed to HPV-negative women, and in women with normal pap smear compared to women with abnormal Papanicolau test (Pap smear. Materials and Methods: A total of 120 liquid-based cervical samples were subtyped for HPV types with microarray hybridization and then stained and evaluated immunocytochemically for Bax and Bak expression. Statistical analysis was performed on the Bax and Bak scores (percentage of positive cells × staining intensity, the overall percentage of positive cells, and the most prevalent staining intensity group found in each sample. Results: A weak association between negative Bax staining and cytologically normal Pap smears was discovered, whereas cytologically abnormal samples tended to stain weakly or moderately positive. No other statistically significant difference was found in the other analyzed parameters. Conclusion: Cytologically normal pap smears seem to have a slight tendency to stain negative for Bax as opposed to cytologically abnormal pap smears. Although the association is weak, it is an indication that there might be a connection between the expression of Bax and the development of cervical intraepithelial dysplasia, which warrants further investigation in larger-scale studies.

  14. JNK-Bcl-2/Bcl-xL-Bax/Bak Pathway Mediates the Crosstalk between Matrine-Induced Autophagy and Apoptosis via Interplay with Beclin 1

    Directory of Open Access Journals (Sweden)

    Jiong Yang

    2015-10-01

    Full Text Available Autophagy is associated with drug resistance which has been a threat in chemotherapy of hepatocellular carcinoma (HCC. The interconnected molecular regulators between autophagy and apoptosis serve as switching points critical to the ultimate outcome of the cell. Our study was performed to investigate the crosstalk between autophagy and apoptosis in HCC after the treatment of matrine. Flow cytometry and TUNEL (terminal dexynucleotidyl transferase (TdT-mediated dUTP nick end labeling assay were used to detect apoptosis in vitro and in vivo, respectively. Bax oligomerization and Cytochrome c release assay were performed. Immunoprecipitation and siRNA transfection were used to detect the interplay between Bcl-2/Bcl-xL,Bax, and Beclin 1. Our results showed that: (1 matrine not only activated caspase and PARP (poly ADP-ribose polymerase cleavage, but also triggered autophagy as shown by the increased levels of LC3II, Beclin 1, and PI3KC3, and the decreased level of p62; (2 matrine treatment promoted the JNK-Bcl-2/ Bcl-xL-Bax/Bak pathway; (3 Bax was oligomerized, the mitochondrial membrane potential altered, and Cytochrome c was released subsequently; (4 Bax interacts with Beclin 1 and inhibits autophagy, which may be a new crosstalk point; and (5 finally, we showed that matrine suppressed the growth of a MHCC97L xenograft in vivo for the first time. In conclusion, the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates the crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin 1.

  15. Bax and Bak expression in cervical smears of women with low-and high-risk HPV types: A study of 120 cases

    Science.gov (United States)

    Klapsinou, Eirini; Argyri, Elena; Panotopoulou, Efstathia; Daskalopoulou, Dimitra; Patsouris, Efstratios; Nonni, Afroditi; Lazaris, Andreas C; Thomopoulou, Georgia-Heleni

    2015-01-01

    Background: Human papillomavirus (HPV) is known to be involved in the carcinogenesis of squamous cells in uterine cervix cancer, mostly by binding and inactivating the p53 and pRb tumor suppressor genes. Lately, evidence has emerged suggesting that HPV oncoproteins may interact with proteins involved in cellular apoptosis as well. Aim: This study aimed to investigate the expression of proapoptotic proteins Bax and Bak in women with low-risk and high-risk HPV types as opposed to HPV-negative women, and in women with normal pap smear compared to women with abnormal Papanicolau test (Pap) smear. Materials and Methods: A total of 120 liquid-based cervical samples were subtyped for HPV types with microarray hybridization and then stained and evaluated immunocytochemically for Bax and Bak expression. Statistical analysis was performed on the Bax and Bak scores (percentage of positive cells × staining intensity), the overall percentage of positive cells, and the most prevalent staining intensity group found in each sample. Results: A weak association between negative Bax staining and cytologically normal Pap smears was discovered, whereas cytologically abnormal samples tended to stain weakly or moderately positive. No other statistically significant difference was found in the other analyzed parameters. Conclusion: Cytologically normal pap smears seem to have a slight tendency to stain negative for Bax as opposed to cytologically abnormal pap smears. Although the association is weak, it is an indication that there might be a connection between the expression of Bax and the development of cervical intraepithelial dysplasia, which warrants further investigation in larger-scale studies. PMID:26811568

  16. Apoptosis and the activity of ceramide, Bax and Bcl-2 in the lungs of neonatal rats exposed to limited and prolonged hyperoxia

    Directory of Open Access Journals (Sweden)

    Bitar Fadi F

    2006-07-01

    Full Text Available Abstract Background The aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1β and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2. Methods Newborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1β expression. Results TUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1β expression was significantly increased at 14 days. Conclusion Exposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.

  17. On the structural properties and superconductivity of room-temperature chemically oxidized La2-xBaxCuO4+y (0<=x<=0.15)

    DEFF Research Database (Denmark)

    Rial, C.; Moran, E.; Alario-Franco, M.A.;

    1996-01-01

    The insertion of oxygen within the structure of La2-xBaxCuO4+y (x less than or equal to 0.15), by means of room-temperature chemical oxidation, modifies both the physical and the structural features of these materials, Concerning the superconducting properties, the extra oxygen gives rise...... and differences concerning the modifications induced by this oxidation process in the present series of La2-xBaxCuO4+y compounds and in equivalent La2-xSrxCuO4+y materials are discussed....

  18. Determination of stoichiometry and concentration of trace elements in thin BaxSr1-xTiO3 perovskite layers.

    Science.gov (United States)

    Becker, J S; Boulyga, S F

    2001-07-01

    This paper describes an analytical procedure for determining the stoichiometry of BaxSr1-xTiO3 perovskite layers using inductively coupled plasma mass spectrometry (ICP-MS). The analytical results of mass spectrometry measurements are compared to those of X-ray fluorescence analysis (XRF). The performance and the limits of solid-state mass spectrometry analytical methods for the surface analysis of thin BaxSr1-xTiO3 perovskite layers sputtered neutral mass spectrometry (SNMS)--are investigated and discussed.

  19. The IRE1/bZIP60 pathway and bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana Plants

    DEFF Research Database (Denmark)

    Gaguancela, Omar Arias; Zúñiga, Lizbeth Peña; Arias, Alexis Vela

    2016-01-01

    The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor that reg......The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor...

  20. Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance

    DEFF Research Database (Denmark)

    Erler, Janine Terra; Cawthorne, Christopher J; Williams, Kaye J;

    2004-01-01

    of the Bcl-2 protein family. Oxygen deprivation of human colon cancer cells in vitro provoked decreased mRNA and protein levels of proapoptotic Bid and Bad. Hypoxia-inducible factor 1 (HIF-1) was dispensable for the down-regulation of Bad but required for that of Bid, consistent with the binding of HIF-1......alpha to a hypoxia-responsive element (positions -8484 to -8475) in the bid promoter. Oxygen deprivation resulted in proteosome-independent decreased expression of Bax in vitro, consistent with a reduction in global translation efficiency. The physiological relevance of Bid and Bax down...

  1. Immunohistochemical expression of p53, BCL-2, BAX and VEGFR1 proteins in nephroblastomas A expressão imuno-histoquímica das proteínas p53, BCL-2, BAX e VEGFR1 em nefroblastomas

    Directory of Open Access Journals (Sweden)

    Ana Paula Percicote

    2013-02-01

    Full Text Available INTRODUCTION: Nephroblastoma or Wilms' tumor is the most frequent renal cancer in children. Although its prognosis is favorable for most patients, it may relapse or have a fatal outcome. The characterization of risk groups by applying immunohistochemical biomarkers aims to adapt the treatment to its corresponding group as well as to reduce relapses and fatal outcome. p53, B-cell lymphoma 2 (BCL-2, BCL-2 associated protein X (BAX and vascular endothelial growth factor receptor 1 (VEGFR1 are among the most widely studied biomarkers, which are related to the apoptotic pathway, DNA repair and neovascularization. OBJECTIVE: The objective of this study is to assess the immunohistochemical expression of p53, BCL-2, BAX and VEGFR1 in samples of human nephroblastoma and to correlate them with clinicopathological prognostic factors. MATERIAL AND METHODS: Twenty-nine surgical specimens of nephroblastoma diagnosed from 1994 to 2007 were selected from the Anatomopathological Service of two hospitals in Curitiba. The immunohistochemical analysis of tissue microarrays was performed through immunoperoxidase staining and the yielded results were compared with clinicopathological prognostic factors. RESULTS: The major immunohistochemical expression of VEGFR1 in blastema and epithelium presented positive association with the risk group. Hence this may be related to higher vascular neoplastic invasion apparently caused by the endothelial growth factor, which maximizes the chances of metastasis and ultimately changes tumor staging, risk group and clinical evolution. CONCLUSIONS: The immunohistochemical expression of VEGFR1 substantiated a directly proportional association with the nephroblastoma risk group.INTRODUÇÃO: O nefroblastoma, ou tumor de Wilms, é a neoplasia renal mais frequente na infância. Embora o prognóstico seja favorável para a maioria dos pacientes, muitos evoluem para recidiva ou óbito. A caracterização de grupos de risco por meio de

  2. Bax/Mcl-1 balance affects neutrophil survival in intermittent hypoxia and obstructive sleep apnea: effects of p38MAPK and ERK1/2 signaling

    Directory of Open Access Journals (Sweden)

    Dyugovskaya Larissa

    2012-10-01

    Full Text Available Abstract Background Prolonged neutrophil survival is evident in various cardiovascular and respiratory morbidities, in hypoxic conditions in-vitro and in patients with obstructive sleep apnea (OSA characterized by nightly intermittent hypoxia (IH. This may lead to persistent inflammation, tissue injury and dysfunction. We therefore investigated by a translational approach the potential contribution of the intrinsic stress-induced mitochondrial pathway in extending neutrophil survival under IH conditions. Thus, neutrophils of healthy individuals treated with IH in-vitro and neutrophils of OSA patients undergoing nightly IH episodes in-vivo were investigated. Specifically, the balance between pro-apoptotic Bax and anti-apoptotic Mcl-1 protein expression, and the potential involvement of p38MAPK and ERK1/2 signaling pathways in the control of Mcl-1 expression were investigated. Methods Purified neutrophils were exposed to IH and compared to normoxia and to sustained hypoxia (SH using a BioSpherix-OxyCycler C42 system. Bax and Mcl-1 levels, and p38MAPK and ERK1/2 phosphorylation were determined by western blotting. Also, Bax/Mcl-1 expression and Bax translocation to the mitochondria were assessed by confocal microscopy in pre-apoptotic neutrophils, before the appearance of apoptotic morphology. Co-localization of Bax and mitochondria was quantified by LSM 510 CarlZeiss MicroImaging using Manders Overlap Coefficient. A paired two-tailed t test, with Bonferroni correction for multiple comparisons, was used for statistical analysis. Results Compared to normoxia, IH and SH up-regulated the anti-apoptotic Mcl-1 by about 2-fold, down-regulated the pro-apoptotic Bax by 41% and 27%, respectively, and inhibited Bax co-localization with mitochondria before visible morphological signs of apoptosis were noted. IH induced ERK1/2 and p38MAPKs phosphorylation, whereas SH induced only p38MAPK phosphorylation. Accordingly, both ERK and p38MAPK inhibitors attenuated

  3. Charge order, metallic behavior, and superconductivity in La2-xBaxCuO4 with x=1/8.

    Science.gov (United States)

    Homes, C C; Dordevic, S V; Gu, G D; Li, Q; Valla, T; Tranquada, J M

    2006-06-30

    The ab-plane optical properties of a cleaved single crystal of La2-xBaxCuO4 for x=1/8 (Tc approximately =2.4 K) have been measured over a wide frequency and temperature range. The low-frequency conductivity is Drude-like and shows a metallic response with decreasing temperature. However, below approximately =60 K, corresponding to the onset of charge-stripe order, there is a rapid loss of spectral weight below about 40 meV. The behavior is quite different from that typically associated with the pseudogap in the normal state of the cuprates. Instead, the gapping of the normal-state single-particle excitations looks surprisingly similar to that observed in superconducting La2-xSrxCuO4, including the presence of a residual Drude peak with reduced weight.

  4. Large-scale synthesis of BaxSr1-xTiO3 nanowires with controlled stoichiometry

    Science.gov (United States)

    Tang, Haixiong; Zhou, Zhi; Sodano, Henry A.

    2014-04-01

    This study demonstrates a highly efficient method for large-scale synthesis BaTiO3 nanowires (NWs) using a two-step hydrothermal reaction. This synthesis process provides a facile approach to the growth of BaTiO3 NWs with high yield and control over the stoichiometry of the BaxSr1-xTiO3 solid solution. The ferroelectricity of the BaTiO3 NWs is directly characterized using atomic force microscopy with the piezoelectric strain coupling coefficient (d33) reaching 31.1 pm/V. This work provide an avenue for high volume manufacturing of ferroelectric NWs, allowing both fundamental investigation of nanoscale ferroelectricity as well as their future application in the electrical devices.

  5. Study of BaxSr1-xTiO3 thin films using transverse-field Ising model

    Institute of Scientific and Technical Information of China (English)

    Tao Yong-Mei; Jiang Qing

    2004-01-01

    In this paper, the effects of doping on the thermodynamic properties of BaxSr1-xTiO3 (BST) thin film are investigated, based on the transverse-field Ising model (TIM) within the framework of mean field theory. We apply the double-peak distribution model of related parameters to mimic doping. The lattice expansion arising from doping with large Ba2+ was also taken into account. We concentrate on the doping concentration dependence of peak temperature (Tm), spontaneous polarization and dielectric susceptibility. It is found that the doping concentration has great influence on the dielectric properties and phase transition properties of BST thin films. We also discuss the quantum effect arising from doping.

  6. Effects of aspartame on hsp70, bcl-2 and bax expression in immune organs of Wistar albino rats

    Science.gov (United States)

    Choudhary, Arbind Kumar; Devi, Rathinasamy Sheela

    2016-01-01

    Abstract Aspartame, a “first generation sweetener”, is widely used in a variety of foods, beverages, and medicine. The FDA has determined the acceptable daily intake (ADI) value of aspartame to be 50 mg/kg·day, while the JECFA (Joint FAO/WHO Expert Committee on Food Additives) has set this value at 40 mg/kg of body weight/day. Safety issues have been raised about aspartame due to its metabolites, specifically toxicity from methanol and/or its systemic metabolites formaldehyde and formic acid. The immune system is now recognized as a target organ for many xenobiotics, such as drugs and chemicals, which are able to trigger unwanted apoptosis or to alter the regulation of apoptosis. Our previous studies has shown that oral administration of aspartame [40 mg/(kg·day)] or its metabolites for 90 days increased oxidative stress in immune organs of Wistar albino rats. In this present study, we aimed to clarify whether aspartame consumption over a longer period (90-days) has any effect on the expression of hsp70, bcl-2 and bax at both mRNA transcript and protein expression levels in immune organs. We observed that oral administration of aspartame for 90 days did not cause any apparent DNA fragmentation in immune organs of aspartame treated animals; however, there was a significant increase in hsp70 expression, apart from significant alteration in bcl-2 and bax at both mRNA transcript and protein expression level in the immune organs of aspartame treated animals compared to controls. Hence, the results indicated that hsp70 levels increased in response to oxidative injury induced by aspartame metabolites; however, these metabolites did not induce apoptosis in the immune organs. Furthermore, detailed analyses are needed to elucidate the precise molecular mechanisms involved in these changes.

  7. Effects of aspartame on hsp70, bcl-2 and bax expression in immune organs of Wistar albino rats.

    Science.gov (United States)

    Choudhary, Arbind Kumar; Devi, Rathinasamy Sheela

    2016-09-01

    Aspartame, a "first generation sweetener", is widely used in a variety of foods, beverages, and medicine. The FDA has determined the acceptable daily intake (ADI) value of aspartame to be 50 mg/kg·day, while the JECFA (Joint FAO/WHO Expert Committee on Food Additives) has set this value at 40 mg/kg of body weight/day. Safety issues have been raised about aspartame due to its metabolites, specifically toxicity from methanol and/or its systemic metabolites formaldehyde and formic acid. The immune system is now recognized as a target organ for many xenobiotics, such as drugs and chemicals, which are able to trigger unwanted apoptosis or to alter the regulation of apoptosis. Our previous studies has shown that oral administration of aspartame [40 mg/(kg·day)] or its metabolites for 90 days increased oxidative stress in immune organs of Wistar albino rats. In this present study, we aimed to clarify whether aspartame consumption over a longer period (90-days) has any effect on the expression of hsp70, bcl-2 and bax at both mRNA transcript and protein expression levels in immune organs. We observed that oral administration of aspartame for 90 days did not cause any apparent DNA fragmentation in immune organs of aspartame treated animals; however, there was a significant increase in hsp70 expression, apart from significant alteration in bcl-2 and bax at both mRNA transcript and protein expression level in the immune organs of aspartame treated animals compared to controls. Hence, the results indicated that hsp70 levels increased in response to oxidative injury induced by aspartame metabolites; however, these metabolites did not induce apoptosis in the immune organs. Furthermore, detailed analyses are needed to elucidate the precise molecular mechanisms involved in these changes.

  8. Cancer-selective death of human breast cancer cells by leelamine is mediated by bax and bak activation.

    Science.gov (United States)

    Sehrawat, Anuradha; Kim, Su-Hyeong; Hahm, Eun-Ryeong; Arlotti, Julie A; Eiseman, Julie; Shiva, Sruti S; Rigatti, Lora H; Singh, Shivendra V

    2017-02-01

    The present study is the first to report inhibition of breast cancer cell growth in vitro and in vivo and suppression of self-renewal of breast cancer stem cells (bCSC) by a pine bark component (leelamine). Except for a few recent publications in melanoma, anticancer pharmacology of this interesting phytochemical is largely elusive. Leelamine (LLM) dose-dependently inhibited viability of MDA-MB-231 (triple-negative), MCF-7 (estrogen receptor-positive), and SUM159 (triple-negative) human breast cancer cells in association with apoptotic cell death induction. To the contrary, a normal mammary epithelial cell line derived from fibrocystic breast disease and spontaneously immortalized (MCF-10A) was fully resistant to LLM-mediated cell growth inhibition and apoptosis induction. LLM also inhibited self-renewal of breast cancer stem cells. Apoptosis induction by LLM in breast cancer cells was accompanied by a modest increase in reactive oxygen species production, which was not due to inhibition of mitochondrial electron transport chain complexes. Nevertheless, ectopic expression of manganese superoxide dismutase conferred partial protection against LLM-induced cell death but only at a lower yet pharmacologically relevant concentration. Exposure of breast cancer cells to LLM resulted in (a) induction and/or activation of multidomain proapoptotic proteins Bax and Bak, (b) caspase-9 activation, and (c) cytosolic release of cytochrome c. Bax and Bak deficiency in immortalized fibroblasts conferred significant protection against cell death by LLM. Intraperitoneal administration of LLM (7.5 mg/kg; 5 times/wk) suppressed the growth of orthotopic SUM159 xenografts in mice without any toxicity. In conclusion, the present study provides critical preclinical data to warrant further investigation of LLM. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Changes of bcl—XL and bax mRNA expression following traumatic brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    骆纯; 卢亦成; 等

    2002-01-01

    Objective:To investigate the changes of bcl-2 gene family and the molecular mechanism of neuromal apoptosis following traumatic brain injury(TBI)in rats.Methods:Male Sprague-Dawley(SD)rats were subjected to lateral fluid percussion brain injury(FPBI)of moderate severity.Thebcl-XLand baxmRNA expression was detected by reverse transcription polymerase chain reaction(RT-PCR)bax mRNA expression rose slowly,doubled at 3days after injury and returned to the sham level slowly.Conclusions:Decreased expression of bcl-XLmRNA and increased expression of bax mRNA coincides tith apoptosis followwin brain injury.The bcl-2gene family is involved in neuronal apoptosis after TBI,and the changes of mRNA expression of the family members lead the neuronal cells to apoptosis.

  10. Ganoderma lucidum spore powder modulates Bcl-2 and Bax expression in the hippocampus and cerebral cortex, and improves learning and memory in pentylenetetrazole-kindled rats

    Institute of Scientific and Technical Information of China (English)

    Shuang Zhao; Shengchang Zhang; Shuqiu Wang

    2011-01-01

    We studied the effects of Ganoderma lucidum spore powder on Bax and Bcl-2 expression and neuronal apoptosis in pentylenetetrazole-kindled epileptic rats. Sixty adult rats were randomly divided into a control group, an epileptic group (kindled) and three medication groups ( 150, 300,450 mg/kg given to kindled rats). Bax and Bcl-2 immunohistochemistry and TUNEL labeling show ed that the number of Bax- and TUNEL-positive cells in the hippocampus and cerebral cortex decreased significantly in the high-dose medication group, while the number of Bcl-2immunoreactive cells increased. The Morris water maze test showed that high-dose treatment significantly shortened escape latency and increased spatial probe trial performance. Our findings indicate that a high dose of Ganoderma lucidum spore powder upregulates the expressionof antiapoptotic Bcl-2 protein in the hippocampus and cerebral cortex, inhibits proapoptotic Bax expression, and decreases seizure-induced neuronal apoptosis. Further,Ganoderma lucidum appears to protect against epilepsy-related learning and memory impairments.

  11. Hypoxia-Mediated Down-Regulation of Bid and Bax in Tumors Occurs via Hypoxia-Inducible Factor 1-Dependent and -Independent Mechanisms and Contributes to Drug Resistance

    Science.gov (United States)

    Erler, Janine T.; Cawthorne, Christopher J.; Williams, Kaye J.; Koritzinsky, Marianne; Wouters, Bradley G.; Wilson, Clare; Miller, Crispin; Demonacos, Costas; Stratford, Ian J.; Dive, Caroline

    2004-01-01

    Solid tumors with disorganized, insufficient blood supply contain hypoxic cells that are resistant to radiotherapy and chemotherapy. Drug resistance, an obstacle to curative treatment of solid tumors, can occur via suppression of apoptosis, a process controlled by pro- and antiapoptotic members of the Bcl-2 protein family. Oxygen deprivation of human colon cancer cells in vitro provoked decreased mRNA and protein levels of proapoptotic Bid and Bad. Hypoxia-inducible factor 1 (HIF-1) was dispensable for the down-regulation of Bad but required for that of Bid, consistent with the binding of HIF-1α to a hypoxia-responsive element (positions −8484 to −8475) in the bid promoter. Oxygen deprivation resulted in proteosome-independent decreased expression of Bax in vitro, consistent with a reduction in global translation efficiency. The physiological relevance of Bid and Bax down-regulation was confirmed in tumors in vivo. Oxygen deprivation resulted in decreased drug-induced apoptosis and clonogenic resistance to agents with different mechanisms of action. The contribution of Bid and/or Bax down-regulation to drug responsiveness was demonstrated by the relative resistance of normoxic cells that had no or reduced expression of Bid and/or Bax and by the finding that forced expression of Bid in hypoxic cells resulted in increased sensitivity to the topoisomerase II inhibitor etoposide. PMID:15024076

  12. Bcl2 at the endoplasmic reticulum protects against a Bax/Bak-independent paraptosis-like cell death pathway initiated via p20Bap31.

    Science.gov (United States)

    Heath-Engel, Hannah M; Wang, Bing; Shore, Gordon C

    2012-02-01

    Bap31 is an integral ER membrane protein which functions as an escort factor in the sorting of newly synthesized membrane proteins within the endoplasmic reticulum (ER). During apoptosis signaling, Bap31 is subject to early cleavage by initiator caspase-8. The resulting p20Bap31 (p20) fragment has been shown to initiate proapoptotic ER-mitochondria Ca2+ transmission, and to exert dominant negative (DN) effects on ER protein trafficking. We now report that ectopic expression of p20 in E1A/DNp53-transformed baby mouse kidney epithelial cells initiates a non-apoptotic form of cell death with paraptosis-like morphology. This pathway was characterized by an early rise in ER Ca2+ stores and massive dilation of the ER/nuclear envelope, dependent on intact ER Ca2+ stores. Ablation of the Bax/Bak genes had no effect on these ER/nuclear envelope transformations, and delayed but did not prevent cell death. ER-restricted expression of Bcl2 in the absence of Bax/Bak, however, delayed both ER/nuclear envelope dilation and cell death. This prosurvival role of Bcl2 at the ER thus extended beyond inhibition of Bax/Bak, and correlated with its ability to lower ER Ca2+ stores. Furthermore, these results indicate that ER restricted Bcl2 is capable of antagonizing not only apoptosis, but also a non-apoptotic, Bax/Bak independent, paraptosis-like form of cell death.

  13. Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice

    Science.gov (United States)

    Karch, Jason; Kwong, Jennifer Q; Burr, Adam R; Sargent, Michelle A; Elrod, John W; Peixoto, Pablo M; Martinez-Caballero, Sonia; Osinska, Hanna; Cheng, Emily H-Y; Robbins, Jeffrey; Kinnally, Kathleen W; Molkentin, Jeffery D

    2013-01-01

    A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death. DOI: http://dx.doi.org/10.7554/eLife.00772.001 PMID:23991283

  14. The IRE1/bZIP60 pathway and Bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana plants

    Science.gov (United States)

    The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor and when activated it splices the bZIP60 mRNA producing a truncated transcription factor that upregulates expression of genes involved in the unfolded protein response (UPR). Bax inhibitor 1 (BI-1) is another ER stre...

  15. Expression of apoptosis related genes bax, bcl-2 and bcl-X in human gastric cancer: early results of an investigation

    Directory of Open Access Journals (Sweden)

    Domenico D'Ugo

    2005-03-01

    Full Text Available

    Background. Evidences indicate an involvement of apoptosis related genes in gastric carcinogenesis. We studied the gene and protein expression patterns of bcl-2, bax and bcl-X in samples of gastric adenocarcinoma. The apoptotic index values, histological type, differentiation grade, cancer stage and lymph node status were statistically analysed for possible correlations with expressional data.

    Methods. Thirty specimens of gastric cancer and respective normal control gastric mucosa were collected from patients with the diagnosis of gastric adenocarcinoma who underwent a curative gastrectomy. bcl-2, bax and bcl-XL mRNA and protein levels were respectively determined by reverse transcription PCR (RT-PCR and western blot using monoclonal antibodies for immunodetection.

    Results and conclusions.We observed a significant suppression of bax with an increase of bcl-XL at protein and mRNA levels. The presence of lymph node metastases was statistically related to the loss of bax overexpression. Bcl-XL was mostly up-regulated in intestinal/mixed types of gastric carcinoma. The expression patterns described confirm the role for these apoptosis genes in gastric adenocarcinoma.

  16. The correlation between telomerase activity and Bax/Bcl-2 ratio in valproic acid-treated MCF-7 breast cancer cell line

    Directory of Open Access Journals (Sweden)

    Zahra Vafaiyan

    2015-07-01

    Conclusion: Our study demonstrated that cell viability of MCF-7 cells was decreased after treatment with VPA, probably through a reduction of telomerase activity and an increase in Bax/bcl-2 ratio. Therefore, it could be concluded that VPA is a potent anti-cancer agent for breast cancer cells through inhibition of telomerase activity and induction of apoptosis.

  17. Neohesperidin induces cellular apoptosis in human breast adenocarcinoma MDA-MB-231 cells via activating the Bcl-2/Bax-mediated signaling pathway.

    Science.gov (United States)

    Xu, Fei; Zang, Jia; Chen, Daozhen; Zhang, Ting; Zhan, Huiying; Lu, Mudan; Zhuge, Hongxiang

    2012-11-01

    Neohesperidin, a flavonoid compound found in high amounts in Poncirus trifoliata, has free radical scavenging activity. For the first time, our study indicated that neohesperidin also induces cell apoptosis in human breast adenocarcinoma MDA-MB-231 cells, which was possibly mediated by regulating the P53/Bcl-2/Bax pathway. MDA-MB-231 cells were subjected to treatment with neohesperidin. MTT and Trypan blue exclusion assays were applied to assess the cell viability. The morphological changes of cells were observed using an inverted microscope, and cell apoptosis was detected by flow cytometric analysis. Immunoblot analysis was conducted to evaluate the protein expressions of apoptosis-related genes, including P53, Bcl-2 and Bax. Our results indicated that the proliferation of MDA-MB-231 cells was inhibited by the treatment with neohesperidin in a time- and dose-dependent manner. The IC50 values of neohesperidin at 24 and 48 h were 47.4 +/- 2.6 microM and 32.5 +/- 1.8 microM, respectively. The expressions of P53 and Bax in the neohesperidin-treated cells were significantly up-regulated, while that of Bcl-2 was down-regulated. Our study suggested that neohesperidin could induce apoptosis of MDA-MB-231 cells, a process which was associated with the activation of the Bcl-2/Bax-mediated signaling pathway.

  18. Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice.

    Science.gov (United States)

    Karch, Jason; Kwong, Jennifer Q; Burr, Adam R; Sargent, Michelle A; Elrod, John W; Peixoto, Pablo M; Martinez-Caballero, Sonia; Osinska, Hanna; Cheng, Emily H-Y; Robbins, Jeffrey; Kinnally, Kathleen W; Molkentin, Jeffery D

    2013-08-27

    A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death. DOI:http://dx.doi.org/10.7554/eLife.00772.001.

  19. Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

    Science.gov (United States)

    Karaliotas, Georgios I; Mavridis, Konstantinos; Scorilas, Andreas; Babis, George C

    2015-09-01

    Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the

  20. Study on Apoptosis and Expression of P53, Bcl-2, Bax in Cardiac Myocytys of Congestive Heart Failure Induced by Ventricular Pacing

    Institute of Scientific and Technical Information of China (English)

    QI; Benling; CAO; Linsheng; WANG; Lin; ZHOU; Jingqun

    2001-01-01

    The apoptosis and the expression of p53, bcl-2 and Bax in myocytes of chronic rapid ventricular pacing-induced congestive heart failure (CHF) in rabbits were investigated. The CHF rabbit model (P, n= 7) was established by chronic rapid ventricular pacing for 3 weeks. By using TUNEL technique the apoptosis in the myocytes in the rabbit model was studied and the expression of p53,bcl-2 and Bax in myocytes was detected by using immunohistochemical method. Sham-operated (C,n = 9) group served as control group. The results showed that there were about 4033± 884.56 apoptotic cells/106 myocytes in P group, but no apoptotic cells were found in C group. Myocytes positive for p53 immunoreactivity (18. 86±8. 48 vs 5. 06±0. 87, P<0.01) and positive for Bax immunoreactivity (7. 15±1.91 vs 0. 43±0. 09, P<0.01) were increased in P group as compared with those in C group, while the myocytes positive for bcl-2 immunoreactivity (7. 08±1.05 vs 14. 97±4.47,P<0. 01) and the ratio of bcl-2/Bax were decreased in P group as compared with those in C group.Apoptosis was involved in the development of CHF induced by continuously rapid ventricular pacing in rabbit. The expression of p53 and Bax was increased, while the expression of bcl-2 was inhibited.These might play an important role in the acceleration of the apoptosis.

  1. Photobiomodulation on Bax and Bcl-2 Proteins and SIRT1/PGC-1α Axis mRNA Expression Levels of Aging Rat Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Fang-Hui Li

    2014-01-01

    Full Text Available Objective. This study aimed to analyze the effects of low level laser irradiation (LLLI on Bax and IGF-1 and Bcl-2 protein contents and SIRT1/PGC-1α axis mRNA expression levels to prevent sarcopenia in aged rats. Material and Methods. Twenty female Sprague Dawley rats (18 months old were randomly divided into two groups (n=10 per group: control (CON and LLLI groups. The gallium-aluminum-arsenium (GaAlAs laser irradiation at 810 nm was used in the single point contact mode (3.75 J/cm2; 0.4 cm2; 125 mW/cm2; 30 s. Bax, Bcl-2, and IGF-1 proteins and SIRT1/PGC-1α axis mRNA expression were assessed 24 h after LLLI on gastrocnemius in aged rat. Results. Gastrocnemius muscle weights, gastrocnemius mass/body mass, Bcl-2/BAX ratio, Bcl-2 protein, IGF-1 protein, and the mRNA contents in SIRT1, PGC-1α, NRF1, TMF, and SOD2 were significantly (P<0.05 increased by LLLI compared to CON group without LLLI. However, levels of BAX protein and caspase 3 mRNA were significantly attenuated by LLLI compared to CON group (P<0.05. Conclusion. LLLI at 810 nm inhibits sarcopenia associated with upregulation of Bcl-2/BAX ratio and IGF-1 and SIRT1/PGC-1α axis mRNA expression in aged rats. This indicates that LLLI has potential to decrease progression of myocyte apoptosis in sarcopenic muscles.

  2. Melatonin may play a role in modulation of bax and bcl-2 expression levels to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Mohseni, Mehran [Department of Radiology and Medical Physics, Faculty of Paramedicine, Kashan University of Medical Sciences, Kashan (Iran, Islamic Republic of); Mihandoost, Ehsan, E-mail: mihandoost.e@gmail.com [Department of Medical Radiation Engineering, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of); Shirazi, Alireza [Department of Medical Physics and Biomedical Engineering, Faculty of Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Sepehrizadeh, Zargham [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Bazzaz, Javad Tavakkoly [Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ghazi-khansari, Mahmoud [Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2012-10-15

    The close relationship between free radicals effects and apoptosis process has been proved. Melatonin has been reported as a direct free radical scavenger. We investigated the capability of melatonin in the modification of radiation-induced apoptosis and apoptosis-associated upstream regulators expression in rat peripheral blood lymphocytes. Rats were irradiated with a single whole body Cobalt 60-gamma radiation dose of 8 Gy at a dose rate of 101 cGy/min with or without melatonin pretreatments at different concentrations of 10 and 100 mg/kg body weight. The rats were divided into eight groups of control, irradiation-only, vehicle-only, vehicle plus irradiation, 10 mg/kg melatonin alone, 10 mg/kg melatonin plus irradiation, 100 mg/kg melatonin alone and 100 mg/kg melatonin plus irradiation. Rats were given an intraperitoneal (IP) injection of melatonin or the same volume of vehicle alone 1 h prior to irradiation. Blood samples were taken 4, 24, 48 and 72 h after irradiation for evaluation of flow cytometric analysis of apoptotic lymphocytes using Annexin V/PI assay and measurement of bax and bcl-2 expression using quantitative real-time PCR (RT{sup 2}qPCR). Irradiation-only and vehicle plus irradiation showed an increase in the percentage of apoptotic lymphocytes significantly different from control group (P < 0.01), while melatonin pretreatments in a dose-dependent manner reduced it as compared with the irradiation-only and vehicle plus irradiation groups (P < 0.01) in all time points. This reduced apoptosis by melatonin was related to the downregulation of bax, upregulation of bcl-2, and therefore reduction of bax/bcl-2 ratio. Our results suggest that melatonin in these doses may provide modulation of bax and bcl-2 expression as well as bax/bcl-2 ratio to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis.

  3. MiR-467a is upregulated in radiation-induced mouse thymic lymphomas and regulates apoptosis by targeting Fas and Bax.

    Science.gov (United States)

    Gao, Fu; Chen, Song; Sun, Mingjuan; Mitchel, Ronald E J; Li, Bailong; Chu, Zhiyong; Cai, Jianming; Liu, Cong

    2015-01-01

    It has been reported dysregulation of certain microRNAs (miRNAs / miRs) is involved in tumorigenesis. However, the miRNAs associated with radiocarcinogenesis remain undefined. In this study, we validated the upregulation of miR-467a in radiation-induced mouse thymic lymphoma tissues. Then, we investigated whether miR-467a functions as an oncogenic miRNA in thymic lymphoma cells. For this purpose, we assessed the biological effect of miR-467a on thymic lymphoma cells. Using miRNA microarray, we found four miRNAs (miR-467a, miR-762, miR-455 and miR-714) were among the most upregulated (>4-fold) miRNAs in tumor tissues. Bioinformatics prediction suggests miR-467a may potentially regulate apoptosis pathway via targeting Fas and Bax. Consistently, in miR-467a-transfected cells, both proliferation and colony formation ability were significantly increased with decrease of apoptosis rate, while, in miR-467a-knockdown cells, proliferation was suppressed with increase of apoptosis rate, indicating that miR-467a may be involved in the regulation of apoptosis. Furthermore, miR-467a-knockdown resulted in smaller tumors and better prognosis in an in vivo tumor-transplanted model. To explain the mechanism of apoptosis suppression by miR-467a, we explore the expression of candidate target genes (Fas and Bax) in miR-467a-transfected relative to negative control transfected cells using flow cytometry and immunoblotting. Fas and Bax were commonly downregulated in miR-467a-transfected EL4 and NIH3T3 cells, and all of the genes harbored miR-467a target sequences in the 3'UTR of their mRNA. Fas and Bax were actually downregulated in radiation-induced thymic lymphoma tissues, and therefore both were identified as possible targets of miR-467a in thymic lymphoma. To ascertain whether downregulation of Fas and / or Bax is involved in apoptosis suppression by miR-467a, we transfected vectors expressing Fas and Bax into miR-467a-upregulated EL4 cells. Then we found that both Fas- and Bax

  4. Lack of association between Bax promoter (-248G>A single nucleotide polymorphism and susceptibility towards cancer: evidence from a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Sushil Kumar Sahu

    Full Text Available BACKGROUND: The Bcl-2-associated X protein (Bax is a proapoptotic member of the Bcl-2 family known to be activated and upregulated during apoptosis. Single nucleotide polymorphisms (SNPs in Bax promoter may participate in the process of carcinogenesis by altering its own expression and the cancer related genes. Bax-248G>A polymorphism has been implicated to alter the risk of cancer, but the listed results are inconsistent and inconclusive. In the present study, we performed a meta-analysis to systematically summarize the possible association of this polymorphism with the risk of cancer. METHODOLOGY: We conducted a search of case-control studies on the associations of Bax-248G>A polymorphism with susceptibility to cancer in Pub Med, Science Direct, Wiley Online Library and hand search. Data from all eligible studies based on some key search terms, inclusion and exclusion criteria were extracted for this meta-analysis. Hardy-Weinberg equilibrium (HWE in controls, power calculation, heterogeneity analysis, Begg's funnel plot, Egger's linear regression test, forest plot and sensitivity analysis were performed in the present study. RESULTS: Cancer risk associated with Bax-248G>A polymorphism was estimated by pooled odds ratios (ORs and 95% confidence intervals (95% CIs. The pooled ORs were calculated in allele contrast, homozygous comparison, heterozygous comparison, dominant and recessive model. Statistical significance was checked through Z and p-value in forest plot. A total of seven independent studies including 1772 cases and 1708 controls were included in our meta-analysis. Our results showed that neither allele frequency nor genotype distributions of this polymorphism were associated with risk for cancer in any of the genetic model. Furthermore, Egger's test did not show any substantial evidence of publication bias. CONCLUSIONS/SIGNIFICANCE: This meta-analysis suggests that the Bax-248G>A polymorphism is not an important cancer risk factor

  5. GSIV serine/threonine kinase can induce apoptotic cell death via p53 and pro-apoptotic gene Bax upregulation in fish cells.

    Science.gov (United States)

    Reshi, Latif; Wu, Horng-Cherng; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-04-01

    Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these

  6. Cyclophilin A affects Bcl-2 and Bax expression following beta-amyloid fragment 25-35-induced injury to PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Li Cheng; Chaodong Zhang

    2008-01-01

    BACKGROUND: Cyclophilin A can protect neurons against oxidative stress.OBJECTIVE: To investigate the effect of cyclophilin A on Bcl-2 and Bax protein expression in pheochromocytoma (PCI2) cells treated with beta-amyloid fragment 25-35 (A β25-35), and to verify the protection pathway ofcyclophilin A.DESIGN, TIME AND SETTING: The initial experiment was performed at the Laboratory of Department of Neurology, First Clinical College, China Medical University from November 2006 to July 2007.MATERIALS: PCI2 cells were cultured at the Cell Center of Peking Union Medical College. A β25-35 (Sigma, USA), antibodies of Bcl-2 and Bax (Wuhan Boster, China), and recombinant human cyclophilin A (Biomol, USA) were used in this study.METHODS: PC12 cells were divided into three groups. Cells in the control group were incubated in culture medium. Cells in the Aβ25-35 injury group were incubated in medium containing a final concentration of 10 μ mol/L of Aβ25-35. Cells in the cyclophilin A group were incubated in medium containing a final concentration of 10 nmol/L of cyclophilin A for 30 minutes, and then treated with 10 μmol/L Aβ25-35. MAIN OUTCOME MEASURES: After 24 hours of culture, immunohistochemistry was used to detect Bcl-2 and Bax expression in PC12 cells. Annexin-V flow cytometry was employed to measure the apoptosis rate of PC12 cells. The MTT method was applied to examine the survival rate of PC12 cells.RESULTS: Bcl-2 expression decreased, whereas Bax expression increased in PCI2 cells treated with Aβ25-35 (t = 2.277, 5.957, P<0.05). However, in PC12 cells treated with Aβ25-35 and cyclophilin A, Bcl-2 expression increased and Bax expression decreased (t = 4.497, 2.531, P < 0.05). The survival rate of PC12 cells significantly decreased and the apoptosis rate increased (t=8.509, 22.886, P < 0.05) following Aβ25-35 treatment. Cyclophilin A enhanced the survival rate of PC12 cells to Aβ25-35-induced apoptosis (t = 4.895, 10.042, P< 0.05).CONCLUSION: Cyclophilin A can

  7. Apoptosis Induction by Targeting Interferon Gamma Receptor 2 (IFNgammaR2) in Prostate Cancer: Ligand (IFNgamma)-Independent Novel Function of IFNgammaR2 as a Bax Inhibitor

    Science.gov (United States)

    2015-08-01

    inhibitor of Bax. Bax is a key mediator of apoptosis. We found that IFNγR2 is overexpressed in prostate cancer, and we hypothesize that abnormally high...We found that IFNγR2 levels are abnormally elevated in prostate cancer cell lines. Short hairpin (sh) RNA- mediated knockdown of IFNγR2 was able to... enhances Bax activation. (Months 1-24) Task 2: To identify the subtype of prostate cancer that can be effectively treated by IFNγR2-targeting

  8. Grape Seed Procyanidin Extract Improves Insulin Production but Enhances Bax Protein Expression in Cafeteria-Treated Male Rats.

    Science.gov (United States)

    Cedó, Lídia; Castell-Auví, Anna; Pallarès, Victor; Blay, Mayte; Ardévol, Anna; Pinent, Montserrat

    2013-01-01

    In a previous study, the administration of a grape seed procyanidin extract (GSPE) in female Wistar rats improved insulin resistance, reduced insulin production, and modulated apoptosis biomarkers in the pancreas. Considering that pharmacokinetic and pharmacodynamic parameters in females are different from these parameters in males, the aim of the present study was to evaluate the effects of GSPE on male Wistar cafeteria-induced obese rats. The results have confirmed that the cafeteria model is a robust model mimicking a prediabetic state, as these rats display insulin resistance, increased insulin synthesis and secretion, and increased apoptosis in the pancreas. In addition, GSPE treatment (25 mg/kg of GSPE for 21 days) in male rats improves insulin resistance and counteracts the cafeteria-induced effects on insulin synthesis. However, the administration of the extract enhances the cafeteria-induced increase in Bax protein levels, suggesting increased apoptosis. This result contradicts previous results from cafeteria-fed female rats, in which GSPE seemed to counteract the increased apoptosis induced by the cafeteria diet.

  9. Two Dimensional Incommensurate Spin Excitations and Lattice Fluctuations in La2 - x Bax CuO4

    Science.gov (United States)

    Wagman, J. J.; Carlo, J. P.; van Gastel, G.; Zhao, Y.; Kallin, A. B.; Mazurek, E.; Dabkowska, H. A.; Savicii, A.; Granroth, G. E.; Yamani, Z.; Tun, Z.; Gaulin, B. D.

    2013-03-01

    'Hour-glass' shaped dispersions of antiferromagnetic (AF) spin fluctuations are a robust feature common to many high temperature superconductors. In 214 cuprates, these phenomena are well known to display a strong dependence on the concentration of holes that are introduced into the copper oxide planes by doping. The incommensurability (IC) of the two dimensional magnetic order in this system is sensitive to hole concentration. Here, we present a series of neutron scattering measurements on single crystals of La2 - x Bax CuO4 (LBCO), with 0 . 035 <= x <= 0 . 095 , a doping range that spans the transition from diagonal to parallel IC ordering wavevectors, and from non-superconducting to superconducting ground states. Our measurements map out the evolution of the spin excitations for energies below ~ 50 meV, and focus on an enhancement in the scattered intensity centered in the 17-20 meV at the AF IC positions. This regime corresponds to the approximate crossing of very dispersive spin excitations and weakly dispersive low lying optic phonons in LBCO. NSERC, Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy

  10. Enhanced charge stripe order in superconducting La2-xBaxCuO4 in high magnetic fields

    Science.gov (United States)

    Huecker, M.; Zimmermann, M. V.; Xu, Z. J.; Wen, J. S.; Gu, G. D.; Tranquada, J. M.

    2013-03-01

    There is mounting evidence for a proximity of the superconducting ground state in the cuprates to competing states with static spin and/or charge density modulations. One such competing state is the spin and charge stripe phase in La2-xBaxCuO4. By means of high energy (100 keV) x-ray diffraction we have studied the effect of a high magnetic field (H||c) on the charge stripe order in a broad range of doping (0.095 <= x <= 0.155). We find that the field can significantly enhance the charge stripe order, but only at temperatures and dopings where it coexists with bulk superconductivity at zero field. The field also increases stripe correlations between the planes, which can result in an enhanced frustration of the interlayer Josephson coupling. Close to the famous x =1/8 compound, where zero field stripe order is pronounced and bulk superconductivity is suppressed, charge stripe order is independent of the field. The results imply that static stripe order and three-dimensionally coherent superconductivity are competing ground states. The work at Brookhaven was supported by the Office of Basic Energy Sciences, Division of Materials Science and Engineering, U.S. Department of Energy (DOE), under Contract No. DE-AC02-98CH10886.

  11. Role of the lattice dynamics in La2-xBaxCuO4 superconductor based on DFT method

    Directory of Open Access Journals (Sweden)

    A Tavana

    2010-09-01

    Full Text Available Electron-phonon coupling parameters are calculated for La2-x BaxCuO4 cuprate superconductor in a wide range of dopings, from undoped to overdoped compounds. In this study we aim to study the quality of such calculations based on DFT method so, the results of σ GGA+U electronic structure calculations are also investigated. The obtained value for electron-phonon coupling is in the same order of previous calculations but, the value obtained for the Hubbard U parameter shows that, such methods are poor in the estimation of electronic correlations to decide about the role of phonons in these compounds based on their results. Moreover, existence of several structural phase transitions with temperature and doping, lead to larger error in these calculations. Based on the calculated phonon dispersions, structural phase transitions can be resulted which shows the ability of DFT in the study of structural properties and the weakness of the strongly correlations in this properties.

  12. Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

    Science.gov (United States)

    Faião-Flores, Fernanda; Coelho, Paulo Rogério Pinto; Toledo Arruda-Neto, João Dias; Maria-Engler, Silvya Stuchi; Tiago, Manoela; Capelozzi, Vera Luiza; Giorgi, Ricardo Rodrigues; Maria, Durvanei Augusto

    2013-01-01

    Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and 7Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT. PMID:23527236

  13. Apoptosis through Bcl-2/Bax and cleaved caspase up-regulation in melanoma treated by boron neutron capture therapy.

    Directory of Open Access Journals (Sweden)

    Fernanda Faião-Flores

    Full Text Available Boron neutron capture therapy (BNCT is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET particles, alpha and (7Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.

  14. Evidence for a second messenger function of dUTP during Bax mediated apoptosis of yeast and mammalian cells.

    Science.gov (United States)

    Williams, Drew; Norman, Grant; Khoury, Chamel; Metcalfe, Naomi; Briard, Jennie; Laporte, Aimee; Sheibani, Sara; Portt, Liam; Mandato, Craig A; Greenwood, Michael T

    2011-02-01

    The identification of novel anti-apoptotic sequences has lead to new insights into the mechanisms involved in regulating different forms of programmed cell death. For example, the anti-apoptotic function of free radical scavenging proteins supports the pro-apoptotic function of Reactive Oxygen Species (ROS). Using yeast as a model of eukaryotic mitochondrial apoptosis, we show that a cDNA corresponding to the mitochondrial variant of the human DUT gene (DUT-M) encoding the deoxyuridine triphosphatase (dUTPase) enzyme can prevent apoptosis in yeast in response to internal (Bax expression) and to exogenous (H(2)O(2) and cadmium) stresses. Of interest, cell death was not prevented under culture conditions modeling chronological aging, suggesting that DUT-M only protects dividing cells. The anti-apoptotic function of DUT-M was confirmed by demonstrating that an increase in dUTPase protein levels is sufficient to confer increased resistance to H(2)O(2) in cultured C2C12 mouse skeletal myoblasts. Given that the function of dUTPase is to decrease the levels of dUTP, our results strongly support an emerging role for dUTP as a pro-apoptotic second messenger in the same vein as ROS and ceramide.

  15. Electromagnon in the Z -type hexaferrite (BaxSr1-x) 3Co2Fe24O41

    Science.gov (United States)

    Kadlec, Filip; Kadlec, Christelle; Vít, Jakub; Borodavka, Fedir; Kempa, Martin; Prokleška, Jan; Buršík, Josef; Uhrecký, Róbert; Rols, Stéphane; Chai, Yi Sheng; Zhai, Kun; Sun, Young; Drahokoupil, Jan; Goian, Veronica; Kamba, Stanislav

    2016-07-01

    We studied experimentally the high-temperature magnetoelectric (BaxSr1-x) 3Co2Fe24O41 prepared as ceramics (x =0 , 0.2) and a single crystal (x =0.5 ) using inelastic neutron scattering, THz time-domain, Raman, and far-infrared spectroscopies. The spectra, measured with varying temperature and magnetic field, reveal rich information about the collective spin and lattice excitations. In the ceramics, we observed an infrared-active magnon which is absent in Eω⊥z polarized THz spectra of the crystal, and we assume that it is an electromagnon active in Eω∥z polarized spectra. On heating from 7 to 250 K, the frequency of this electromagnon drops from 36 to 25 cm-1 and its damping gradually increases, so it becomes overdamped at room temperature. Applying external magnetic field has a similar effect on the damping and frequency of the electromagnon, and the mode is no longer observable in the THz spectra above 2 T, as the transverse-conical magnetic structure transforms into a collinear one. Raman spectra reveal another spin excitation with a slightly different frequency and much higher damping. Upon applying magnetic field higher than 3 T, in the low-frequency part of the THz spectra, a narrow excitation appears whose frequency linearly increases with magnetic field. We interpret this feature as the ferromagnetic resonance.

  16. Ab-initio study of structural, elastic, electronic and thermodynamic properties of BaxSr1−xS ternary alloys

    Directory of Open Access Journals (Sweden)

    Chelli S.

    2015-12-01

    Full Text Available The structural, elastic, electronic and thermodynamic properties of BaxSr1−xS ternary alloys have been investigated using the full-potential (linearized augmented plane wave method. The ground state properties, such as lattice constant, bulk modulus and elastic constants, are in good agreement with numerous experimental and theoretical data. The dependence of the lattice parameters, bulk modulus and band gap on the composition x was analyzed. Deviation of the lattice constant from Vegard’s law and the bulk modulus from linear concentration dependence (LCD was observed. The microscopic origins of the gap bowing were explained by using the approach of Zunger et al. The thermodynamic stability of BaxSr1−xS alloy was investigated by calculating the excess enthalpy of mixing, ΔHm and the calculated phase diagram showed a broad miscibility gap with a critical temperature.

  17. Injury of mitochondria and the expressions of fas and bax mRNA in the hip-pus of epileptic rats of different latency

    Institute of Scientific and Technical Information of China (English)

    Shuhai Tang; Jianying Sun; Xiaojun Pan; Li Zhang

    2006-01-01

    BACKGROUND: It has been confirmed that Fas and Bax respectively mediate the exogenous and endogenous pathways of neuronal apoptosis, and then mediate the neuronal injury after status epilepticus.OBJECTIVE: To comparatively observe the injury of mitochondrial ultrastructure and the expressions of fas and bax in hippocampal tissue of rats with status epilepticus of different latency.DESIGN: A randomized control study.SETTING: Department of Anesthesiology and Department of Neurology, Qilu Hospital of Shandong University.MATERIALS: Totally 110 male adult SD rats of 260-300 g were used. Kainic acid was purchased from American Sigma Company.METHODS: The experiment was carried out in the Pathological Laboratory of Shandong Academy of Medical Sciences between March and July 2005.① Totally 100 SD rats were divided into two groups according to the random number table method:intraperitoneal injection group and caudal venous injection group.The rats were given kainic acid injected intraperitoneally(12 mg/kg)and through caudal vein (10 mg/kg) respectively. Each group was observed at 3, 6, 24, 48 and 72 hours after status epilepticus respectively.Ten rats were selected for each time point, including 2 for examination of electron microscope and 8 for the diction of the fas and bax mRNA expressions. The time and manifestations of seizure were observed, and the seizure was lasted for 2 hours, and then it was terminated by intraperitoneal injection of diazepam (10 mg/kg). Another 10 rats were used as the normal control group, and the materials were taken at 24 hours after status epilepticus, 2 of rats for the examination of electron microscope and 8 of them for the reverse transcription-polymerase chain reaction (RT-PCR). ② The ultrastructure of hippocampal neurons and its mitochondria were observed with transmission electron microscope. ③ The fas and bax mRNA expressions were detected with reverse transcription-polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: The

  18. Mangiferin attenuates contusive spinal cord injury in rats through the regulation of oxidative stress, inflammation and the Bcl‑2 and Bax pathway.

    Science.gov (United States)

    Luo, Yang; Fu, Changfeng; Wang, Zhenyu; Zhang, Zhuo; Wang, Hongxia; Liu, Yi

    2015-11-01

    Mangiferin has antioxidant, antiviral, apoptosis regulating, anti‑inflammatory, antitumor and antidiabetic effects, which can also inhibit osteoclast formation and bone resorption. However, whether mangiferin ameliorates the neurological pain of spinal cord injury (SCI) in ratS remains to be elucidated. The present study investigated the therapeutic effects of mangiferin on neurological function, the water content of spinal cord, oxidative stress, the expression of inflammatory cytokines and the protein expression of Bcl‑2/Bax in a SCI rat model. In the present study, the Basso, Beattie and Bresnahan scores, and the water content of the spinal cord were used to analyze the therapeutic effects of mangiferin on neurological pain in the SCI rat. The concentrations of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and the serum levels of glutathione peroxidase (GSH‑PX), nuclear factor‑κB p65 unit, tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6 and caspase‑3/9 were detected using commercial kits. The expression levels of Bcl‑2 and Bax were measured using western blot analysis. The results demonstrated that administrating mangiferin began to ameliorate neurological function and the water content of the spinal cord in the SCI rat. The mangiferin‑treated group were found to have lower oxidative stress activity and lower expression levels of inflammatory cytokines, compared with the SCI rat. In addition, mangiferin significantly reduced the protein expression of Bax and promoted the protein expression of Bcl-2 in the SCI rat model. Finally, mangiferin markedly suppressed the expression of caspase‑3/9, indicating that the protective action of mangiferin may be associated with anti‑apoptosis activation. In conclusion, mangiferin attenuated contusive SCI in the rats through regulating oxidative stress, inflammation and the Bcl‑2 and Bax pathway.

  19. Unusual Nernst Effect Suggesting Time-Reversal Violation in the Striped Cuprate Superconductor La2-xBaxCuO4

    Science.gov (United States)

    Li, Lu; Alidoust, N.; Tranquada, J. M.; Gu, G. D.; Ong, N. P.

    2011-12-01

    The striped cuprate La2-xBaxCuO4 (x=(1)/(8)) undergoes several transitions below the charge-ordering temperature Tco=54K. From Nernst experiments, we find that, below Tco, there exists a large, anomalous Nernst signal eN,even(H,T) that is symmetric in field H, and remains finite as H→0. The time-reversal violating signal suggests that, below Tco, vortices of one sign are spontaneously created to relieve interlayer phase frustration.

  20. An Assay of Bax and Bcl2 Expression in Mice Hippocampus Following Ischemia-Reperfusion Treatment with CoQ10

    Directory of Open Access Journals (Sweden)

    Jalal Hassanshahi

    2013-10-01

    Full Text Available Introduction: Preliminary studies confirmed reduction of cell death following treatment with antioxidants. According to this finding we investigated the relationship between consumption of CoQ10 and expression of bax and bcl2 in hippocampus ischemia that this expression related to cell programmed death.Material and Methods: We studied the protective role of CoQ10 against ischemia-reperfusion. Experimental design includes four groups: intact (N=7, ischemic control (N=7, sham control (N=7 and treatment groups with CoQ10 (N=7. The mice (treatment group treated with CoQ10 as Pre-Treatment for a week. Then, ischemia induced by common carotid artery ligation and following the reduction in inflammation (a week the treatment group post-treated with CoQ10 for a week. Nissl staining applied to counting necrotic cells of hippocampus and the western blotting performed to measurement the bax and bcl2 expression. Tunnel kit was used to quantify apoptotic cell death while to short term memory scale, we apply Y-maze.Results: Cell death was significantly lower when mice treated with CoQ10. Bax expression was significantly high in ischemic group but in treatment group was less and reversely the bcl2 expression in ischemic group was lower than treatment and vehicle groups. The memory test results were consistent with cell death results. Conclusion: Ischemia for 15 minutes induced cell death in hippocampus with more potent effect on CA1. CoQ10 intake significantly reduced cell death and decreased memory loss. with prevent of expression of bax and increase in expression of bcl2.

  1. Doxycycline Protects Thymic Epithelial Cells from Mitomycin C-Mediated Apoptosis In Vitro via Trx2-NF-κB-Bcl-2/Bax Axis

    Directory of Open Access Journals (Sweden)

    Jun Wang

    2016-02-01

    Full Text Available Background/Aims: Age-associated and stress-induced involution of the thymus is accompanied by reduced numbers of thymic epithelial cells (TECs and severe reduction in peripheral T cell repertoire specificities. These events seriously affect immune function, but the mechanisms involved are unclear. Our preliminary findings showed that doxycycline (Dox could drive the proliferation of a TEC line (MTEC1 cells partially via the MAPK signaling pathway. Dox can also up-regulate IL-6 and GM-CSF expression via the NF-κB and MAPK/ERK pathways. Herein, we investigate the effects and mechanisms used by Dox that protect against mitomycin C (MMC-induced MTEC1 cell apoptosis. Methods: MTEC1 cells were treated with Dox, MMC, and Dox plus MMC for different amounts of time. The expression of Trx2, NF-κB, Bcl-2, and Bax proteins were then detected by western blotting. Results: Our findings show that Dox protects MTEC1 cells from MMC-induced apoptosis. Dox up-regulated the expression of Trx2 and promoted NF-κB phosphorylation. Meanwhile, Dox also increased the expression of Bcl-2, partially reduced the expression of Bax, and normalized the ratio of Bcl-2 to Bax. Conclusion: Dox exerts an anti-apoptosis function via the NF-κB-Bcl-2/Bax and Trx2-ASK1/JNK pathways in vitro. Therefore, Dox may represent a drug that could be used to attenuate thymic senescence, rescue thymic function, and promote T cell reconstitution.

  2. Disturbance of Bcl-2, Bax, Caspase-3, Ki-67 and C-myc expression in acute and subchronic exposure to benzo(a)pyrene in cervix.

    Science.gov (United States)

    Gao, Meili; Li, Yongfei; Ji, Xiaoying; Xue, Xiaochang; Chen, Lan; Feng, Guodong; Zhang, Huqin; Wang, Huichun; Shah, Walayat; Hou, Zhanwu; Kong, Yu

    2016-03-01

    Epidemiological studies have demonstrated that cigarette smoking is an important cofactor or an independent risk factor for the development of cervical cancer. Benzo(a)pyrene (BaP) is one of the most potent tobacco smoke carcinogens in tobacco smoke. BaP induced DNA damage and over expression in p53 cervical tissue of mice as demonstrated in our previous study. Here we present the findings of exposure to BaP on the expression of Bcl-2, C-myc, Ki-67, Caspase-3 and Bax genes in mouse cervix. Acute intraperitoneal administration of BaP (12.5, 25, 50, 100mg/kg body weight) to ICR female mice induced a significant increase in Bcl-2, C-myc, Ki-67 mRNA and protein level till 72h except in Bcl-2 at 24h with 12.5, 25, 50mg/kg as well as at 48h with 12.5mg/kg body weight post treatment. A significant increase was also seen in Caspase-3 and Bax mRNA and protein level with peak level at 24h and gradual decrease till 72h, however, the expression of caspase-3 increased while that of Bax decreased with increasing dose of Bap after 24h. In sub chronic intraperitoneal and oral gavage administration of BaP (2.5, 5, 10mg/kg body weight), similar significant increase was observed for all the examined genes as compared to the control and vehicle groups, however the expression of Bax decreased in a dose dependent manner. The findings of this study will help in further understanding the molecular mechanism of BaP induced carcinogenesis of cervical cancer.

  3. Long-term exposure of human gingival fibroblasts to cigarette smoke condensate reduces cell growth by modulating Bax, caspase-3 and p53 expression.

    Science.gov (United States)

    Alamri, A; Semlali, A; Jacques, É; Alanazi, M; Zakrzewski, A; Chmielewski, W; Rouabhia, M

    2015-08-01

    Smoking cigarettes increases the risk of oral tissue damage leading to periodontal disease. Gingival fibroblasts, the predominant cell type inhabiting gingival connective tissue, play a critical role in remodeling and maintaining gingival structure. The objective of this study was to investigate the effect of long-term exposure to cigarette smoke on human gingival fibroblast survival/apoptosis and the molecular pathways involved in these cell responses. Human gingival fibroblasts were extracted from healthy non-smokers and cultured in the presence of cigarette smoke condensate (CSC). At the end of each time point, cell growth was evaluated by means of MTT assay. Apoptotic and necrotic gene's expression was investigated by polymerase chain reaction array and by annexin V/propidium iodide staining and cell cycle assays. Western blot was used to investigate Bax and p53 proteins. These tests were supported by caspase 3 activity analyses. High levels of CSC decreased cell growth and deregulated cell cycle progression by increasing the G(0)/G(1) and reducing the S and G(2)/M phases of the gingival fibroblasts. Polymerase chain reaction arrays revealed the activation of several apoptotic genes by CSC, including TNF receptors, caspases, Bax and p53. This was supported by increases in the Bax and p53 protein levels as well as by an elevated activity of caspase-3 in the CSC-exposed cells. Immunofluorescence staining demonstrated that both Bax and caspase-3 displayed a cytosolic and mitochondrial distribution in the CSC-exposed gingival fibroblasts, compared to controls. The damaging effect of CSC on gingival fibroblast growth was also supported by the decrease in interleukin 6 and 8 secretion by the gingival fibroblasts. These results suggest that CSC may contribute to deregulating fibroblast functions. This can compromise fibroblast-epithelial cell interactions, which ultimately increases the risk of gingival tissue damage and the onset of periodontitis. © 2014 John Wiley

  4. Morphological Aspect and iNOS and Bax Expression Modification in Bone Tissue Around Dental Implants Positioned Using Piezoelectric Bone Surgery Versus Conventional Drill Technique.

    Science.gov (United States)

    Zizzari, Vincenzo Luca; Berardi, Davide; Congedi, Francesca; Tumedei, Margherita; Cataldi, Amelia; Perfetti, Giorgio

    2015-05-01

    The aim of this work is to evaluate differences occurring in bone tissue around dental implants positioned using piezoelectric or conventional drill technique. Twenty-four implants were inserted bilaterally in the iliac crest of 6 sheep after site preparation through a piezoelectric instrument (Test) or after site preparation through conventional drill technique with rotary instruments (Control). Animals were randomly divided to be euthanized at 15 and 30 days post-intervention (p.i.); peri-implant bone samples were withdrawn and processed for histological analysis and immunohistochemical evaluation of iNOS and Bax expression. Active remodeling phenomena in both Test and Control samples are showed at 15 days p.i., while at 30 days p.i., the overall organization of the peri-implant bone resembles native bone tissue. Immunohistochemical evaluation reveals a statistically significant increase of both iNOS and Bax expression at 15 days p.i. compared to samples obtained 30 days p.i. and to native bone. At both healing times, a higher but not statistically significant iNOS and Bax expression is recorded in samples from Control compared to Test Group. Even if the insertion protocol does not seem to significantly interfere with the long-term healing process, implant site preparation through the piezoelectric bone surgery technique may allow a reduction of peri-implant bone tissue inflammation and support a more rapid bone tissue healing phase.

  5. Aflatoxin B1 affects apoptosis and expression of Bax, Bcl-2, and Caspase-3 in thymus and bursa of fabricius in broiler chickens.

    Science.gov (United States)

    Peng, Xi; Chen, Kejie; Chen, Jin; Fang, Jing; Cui, Hengmin; Zuo, Zhicai; Deng, Junliang; Chen, Zhengli; Geng, Yi; Lai, Weimin

    2016-09-01

    Aflatoxin B1 is known as a mycotoxin that develops various health problems of animals, the effects of AFB1 on thymus and bursa of Fabricius in chickens are not clear. The objective of this study was to investigate the apoptosis of thymus and bursa of Fabricius in broilers fed with AFB1 . Two hundred Avian broilers were randomly divided into four groups of 50 each, namely control group and three AFB1 groups fed with 0.15 mg, 0.3 mg, and 0.6 mg AFB1 /kg diet, respectively. In this study, flow cytometer and immunohistochemical approaches were used to determine the percentage of apoptotic cells and the expression of Bax, Bcl-2, and Caspase-3. The results showed that consumption of AFB1 diets results in increased percentage of apoptotic cells and increased expression of Caspase-3 in both thymus and bursa of Fabricius. The expression of Bax was increased and the expression of Bcl-2 was decreased in the thymus, but no significant changes in Bax and Bcl-2 expression were observed in the bursa of Fabricius when broilers fed with AFB1 . These findings suggest that adverse effects of AFB1 on thymus and bursa of Fabricius in broilers were confirmed by increased apoptotic cells and abnormal expression of Caspase-3. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1113-1120, 2016.

  6. Targeted therapy of the XIAP/proteasome pathway overcomes TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent 'type I' mode.

    Science.gov (United States)

    Gillissen, B; Richter, A; Richter, A; Overkamp, T; Essmann, F; Hemmati, P G; Preissner, R; Belka, C; Daniel, P T

    2013-05-23

    TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting 'type II' to 'type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

  7. An investigation of the optical properties and water splitting potential of the coloured metallic perovskites Sr1-xBaxMoO3

    Science.gov (United States)

    Hopper, H. A.; Le, J.; Cheng, J.; Weller, T.; Marschall, R.; Bloh, J. Z.; Macphee, D. E.; Folli, A.; Mclaughlin, A. C.

    2016-02-01

    The solid solution Sr1-xBaxMoO3 (x=0.00, 0.025, 0.050, 0.075, 0.100 and 1.00) has been synthesised. Rietveld refinement of X-ray diffraction data shows that all materials crystallise with cubic (Pm-3m) symmetry and that a miscibility gap exists from x=0.1-1.0. The optical properties of the metallic perovskites Sr1-xBaxMoO3 have been investigated by a combination of UV-vis spectroscopy and density functional theory (DFT). Upon increasing x from 0 to 1 in Sr1-xBaxMoO3 there is a reduction in the measured band gap from 2.20 eV to 2.07 eV. The measured band gap is attributed to the electronic transition from the Mo 4d t2g band to the eg band. The potential of SrMoO3 and BaMoO3 as water-splitting photocatalysts was explored but there was no evidence of hydrogen or oxygen evolution, even with the presence of a Pt co-catalyst.

  8. Structure of p53 binding to the BAX response element reveals DNA unwinding and compression to accommodate base-pair insertion

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Y.; Zhang, X.; Dantas Machado, A. C.; Ding, Y.; Chen, Z.; Qin, P. Z.; Rohs, R.; Chen, L.

    2013-07-08

    The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein–DNA interactions as previously observed, and the two dimers retain all the protein–protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.

  9. Nano-SiO2 induces apoptosis via activation of p53 and Bax mediated by oxidative stress in human hepatic cell line.

    Science.gov (United States)

    Ye, Yiyi; Liu, Jianwen; Xu, Jianhe; Sun, Lijuan; Chen, Mingcang; Lan, Minbo

    2010-04-01

    Nanoparticles such as nano-SiO(2) are increasingly used in food, cosmetics, diagnosis, imaging and drug delivery. However, toxicological data of nano-SiO(2) on hepatic cells in vitro and their detailed molecular mechanisms still remain unclear. In order to assess toxicity of nano-SiO(2), L-02 cells were exposed to 0.2, 0.4 and 0.6 mg/ml of SiO(2) colloids (21, 48 and 86 nm) for 12, 24, 36 and 48h. Lactate dehydrogenase released from damaged cells were quantified, cellular ultrastructural organization was observed, and the levels of reactive oxygen species (ROS), lipid peroxidation and glutathione were measured. Apoptosis induced by 21 nm SiO(2) was characterized by annexin V-FITC/PI staining and DNA ladder assay. Furthermore, apoptosis related proteins such as p53, Bax and Bcl-2 were analyzed by using western blot analysis. Our data indicated that nano-SiO(2) caused cytotoxicity in size, dose and time dependent manners. Oxidative stress and apoptosis were induced by exposure to 21 nm SiO(2). Moreover, the expression of p53 and Bax was increased in time and dose dependent patterns, whereas the expression of Bcl-2 was not significantly changed. In conclusion, ROS-mediated oxidative stress, the activation of p53 and up-regulation of Bax/Bcl-2 ratio are involved in mechanistic pathways of 21 nm SiO(2) induced apoptosis in L-02 cells.

  10. [Salidroside protects cultured rat subventricular zone neural stem cells against hypoxia injury by inhibiting Bax, Bcl-2 and caspase-3 expressions].

    Science.gov (United States)

    Qi, Cunfang; Zhang, Junfeng; Chen, Xinlin; Zhang, JiansHui; Yang, Pengbo; Jiao, Qian; Zhang, Pengbo; Lu, Hai-Xia; Liu, Yong

    2013-07-01

    To explore the effects of salidroside (sal) on the expressions of Bcl-2, Bax and caspases-3 proteins in cultured rat subventricular zone (SVZ) neural stem cells (NSCs) exposed to hypoxia injury. Primarily cultured SVZ NSCs from adult SD rats were incubated with salidroside (120 and 240 µmol/L) for 24 h prior to exposure to hypoxia. The cell viability was assessed with MTT assay, and the cell apoptosis was analyzed using TUNEL staining and flow cytometry. Western blotting was performed to detect the expressions of Bcl-2, Bax and caspase-3 in the cells. Salidroside pretreatment of the cells for 24 h resulted in an obvious resistance to hypoxia-induced cell apoptosis and decrement of cell viability (PSalidroside also antagonized the effect of hypoxia exposure in lowering Bcl-2/Bax ratio apoptosis of rat neural stem cells and decreased the expression of caspases-3 protein (PSalidroside can significantly resist hypoxia-induced. The neuroprotective effect of salidroside may be related to the modulation of expressions of apoptosis-related proteins.

  11. Piracetam ameliorated oxygen and glucose deprivation-induced injury in rat cortical neurons via inhibition of oxidative stress, excitatory amino acids release and P53/Bax.

    Science.gov (United States)

    He, Zhi; Hu, Min; Zha, Yun-hong; Li, Zi-cheng; Zhao, Bo; Yu, Ling-ling; Yu, Min; Qian, Ying

    2014-05-01

    Our previous work has demonstrated that piracetam inhibited the decrease in amino acid content induced by chronic hypoperfusion, ameliorated the dysfunction of learning and memory in a hypoperfusion rat model, down-regulated P53, and BAX protein, facilitated the synaptic plasticity, and may be helpful in the treatment of vascular dementia. To explore the precise mechanism, the present study further evaluated effects of piracetam on Oxygen and glucose deprivation (OGD)-induced neuronal damage in rat primary cortical cells. The addition of piracetam to the cultured cells 12 h before OGD for 4 h significantly reduced neuronal damage as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release experiments. Piracetam also lowered the levels of malondialdehyde, nitrogen monoxidum, and xanthine oxidase which was increased in the OGD cells, and enhanced the activities of superoxide dismutase and glutathione peroxidase, which were decreased in the OGD cells. We also demonstrated that piracetam could decrease glutamate and aspartate release when cortical cells were subjected to OGD. Furthermore, Western blot study demonstrated that piracetam attenuated the increased expression of P53 and BAX protein in OGD cells. These observations demonstrated that piracetam reduced OGD-induced neuronal damage by inhibiting the oxidative stress and decreasing excitatory amino acids release and lowering P53/Bax protein expression in OGD cells.

  12. Oleanolic acid from Prunella Vulgaris L. induces SPC-A-1 cell line apoptosis via regulation of Bax, Bad and Bcl-2 expression.

    Science.gov (United States)

    Feng, Liang; Au-Yeung, Wai; Xu, You-Hua; Wang, Shan-Shan; Zhu, Quan; Xiang, Ping

    2011-01-01

    Prunella vulgaris L. (PV) has been used as a herb for chemoprevention of lung cancer. In this study, the main active compound, oleanolic acid (OA) was isolated from an ethanol extract and its chemical structure was identified according to the results of high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC) and liquid chromatography-mass spectrography (LC-MS). Results for cell viability indictated no notable differences between OA and ethanol extract of PV in lung adenocarcinoma SPC-A-1 cells measured by MTT assay. Consistent concentration-response curves. Fluorescence detection with acridine orange-ethidium bromide was used to evaluate apoptosis of SPC-A-1 cells. OA at 16 and 8 microM group increased significantly the apoptosis rate compared with normal and 1% DMSO groups (p<0.05). In addition, immunocytochemistry assays showed increase in Bax and Bad protein expression while Bcl-2 decreased. Moreover, the ratio of Bax/Bcl-2 was heightened by OA treatment. The results suggest OA induced apoptosis of lung adenocarcinoma cells through down-regulating Bcl-2 expression, and up-regulating Bax and Bad expression.

  13. Oridonin induces apoptosis of HeLa cells via altering expres sion of Bcl-2/Bax and activating caspase-3/ICAD pathway

    Institute of Scientific and Technical Information of China (English)

    Chun-ling ZHANG; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2004-01-01

    AIM: To study the mechanisms by which oridonin inhibited HeLa cell growth in vitro. METHODS: Viability of oridonin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis.Caspase activity was assayed using fiuorometric protease assay. ICAD, Bcl-2, and Bax proteins expression were detected by Western blot analysis. RESULTS: Oridonin induced oligonucleosomal fragmentation of DNA and increased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-activated DNase (ICAD), a caspase-3 substrate, at 12 h in HeLa cells. Oridonin-induced DNA fragmentation, caspase-3 activation and down-regulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-AspGlu-Val-Asp-fmk). However, pretreatment with an inhibitor of poly (ADP-ribose) polymerase (PARP), 3, 4-dihydro5-[4-(1-piperidinyl)butoxy]-1 (2H)-isoquinolinone (DPQ), did not suppress oridonin-induced HeLa cell death. In addition, oridonin-induced apoptosis was associated with an increase in the expression of the apoptosis inducer Bax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria. CONCLUSION:Oridonin induces HeLa cells apoptosis by altering balance of Bcl-2 and Bax protein expression and activation of caspase-3/ICAD pathway.

  14. Study on the Expression of Apoptosis - Controlled Gene Bcl - 2/Bax mRNA in the Uterine Leiomyoma%凋亡调控基因Bcl2/Bax mRNA在子宫平滑肌瘤中的表达

    Institute of Scientific and Technical Information of China (English)

    李卫平; 严隽鸿; 林其德; 裴军; 丁传伟

    2000-01-01

    Objective To investigate the expression of apoptosis - controlled gene Bcl - 2/BaxmRNA in leiomyoma and it's relationship with ER、PR. Methods To measure ER, PR volume (28cases) in leiomyoma and the ratio of Bcl - 2/Bax mRNA (21 cases) by immunochemistry and RT -PCR, respectively, and use the myometrium as control. Results The content of ER, PR and the ra-tio of Bcl- 2/Bax mRNA in leiomyoma were significantly higher than those in the myometrium (P< 0.05 and 0.01); there was positive correlation for ER, PR versus ratio of Bcl- 2/Bax mRNA in leiomy-oma Conclusion Apoptosic-controlled gene Bcl - 2/Bax may play a role in the pathogenesis ofleiomyoma; the stimalating effect of estrogen and progesterone combining with ER, PR on leiomyomamay be related with their regulation of the expression of Bcl - 2/Bax mRNA.%目的 探讨细胞凋亡调控基因Bcl-2/Bax mRNA在子宫肌瘤中的表达及与ER、PR的关系。方法应用免疫组化法测定子宫肌瘤内ER、PR的含量(28例)。用RT-PCR法测定肌瘤内Bcl-2/Bax mRNA(21例)。以肌瘤邻近的正常子宫平滑肌作对照。结果子宫肌瘤内ER、PR,Bcl-2/Bax比值显著高于正常子宫平滑肌(P<0.05和P<0.01);肌瘤ER、PR与Bcl-2/BaxmRNA比值呈正相关。结论细胞凋亡调控基因Bcl-2/Bax可能参与子宫肌瘤的发生;而雌、孕激素与ER、PR结合刺激肌瘤生长则可能与影响这一基因的表达有关。

  15. Apoptosis Induction by Targeting Interferon Gamma Receptor 2 (IFNgammaR2) in Prostate Cancer: Ligand (IFNgamma)-Independent Novel Function of IFNgammaR2 as a Bax Inhibitor

    Science.gov (United States)

    2015-08-01

    AWARD NUMBER: W81XWH-12-1-0331 TITLE: Apoptosis Induction by Targeting Interferon Gamma Receptor 2 (IFNgammaR2) in Prostate Cancer: Ligand...DATE August 2015 2. REPORT TYPE Annual 3. DATES COVERED 1Aug2014 - 31Jul2015 4. TITLE AND SUBTITLE Apoptosis Induction by Targeting Interferon ...Introduction In our preliminary study, we identified interferon γ receptor 2 (IFNγR2) as a Bax suppressor using yeast-based functional screening of Bax

  16. Bax-inhibitor-1 knockdown phenotypes are suppressed by Buffy and exacerbate degeneration in a Drosophila model of Parkinson disease

    Science.gov (United States)

    2017-01-01

    Background Bax inhibitor-1 (BI-1) is an evolutionarily conserved cytoprotective transmembrane protein that acts as a suppressor of Bax-induced apoptosis by regulation of endoplasmic reticulum stress-induced cell death. We knocked down BI-1 in the sensitive dopa decarboxylase (Ddc) expressing neurons of Drosophila melanogaster to investigate its neuroprotective functions. We additionally sought to rescue the BI-1-induced phenotypes by co-expression with the pro-survival Buffy and determined the effect of BI-1 knockdown on the neurodegenerative α-synuclein-induced Parkinson disease (PD) model. Methods We used organismal assays to assess longevity of the flies to determine the effect of the altered expression of BI-1 in the Ddc-Gal4-expressing neurons by employing two RNAi transgenic fly lines. We measured the locomotor ability of these RNAi lines by computing the climbing indices of the climbing ability and compared them to a control line that expresses the lacZ transgene. Finally, we performed biometric analysis of the developing eye, where we counted the number of ommatidia and calculated the area of ommatidial disruption. Results The knockdown of BI-1 in these neurons was achieved under the direction of the Ddc-Gal4 transgene and resulted in shortened lifespan and precocious loss of locomotor ability. The co-expression of Buffy, the Drosophila anti-apoptotic Bcl-2 homologue, with BI-1-RNAi resulted in suppression of the reduced lifespan and impaired climbing ability. Expression of human α-synuclein in Drosophila dopaminergic neurons results in neuronal degeneration, accompanied by the age-dependent loss in climbing ability. We exploited this neurotoxic system to investigate possible BI-1 neuroprotective function. The co-expression of α-synuclein with BI-1-RNAi results in a slight decrease in lifespan coupled with an impairment in climbing ability. In supportive experiments, we employed the neuron-rich Drosophila compound eye to investigate subtle phenotypes

  17. Gastroprotective activity of Annona muricata leaves against ethanol-induced gastric injury in rats via Hsp70/Bax involvement.

    Science.gov (United States)

    Moghadamtousi, Soheil Zorofchian; Rouhollahi, Elham; Karimian, Hamed; Fadaeinasab, Mehran; Abdulla, Mahmood Ameen; Kadir, Habsah Abdul

    2014-01-01

    The popular fruit tree of Annona muricata L. (Annonaceae), known as soursop and graviola, is a widely distributed plant in Central and South America and tropical countries. Leaves of A. muricata have been reported to possess antioxidant and anti-inflammatory activities. In this study, the gastroprotective effects of ethyl acetate extract of A. muricata leaves (EEAM) were investigated against ethanol-induced gastric injury models in rats. The acute toxicity test of EEAM in rats, carried out in two doses of 1 g/kg and 2 g/kg, showed the safety of this plant, even at the highest dose of 2 g/kg. The antiulcer study in rats (five groups, n=6) was performed with two doses of EEAM (200 mg/kg and 400 mg/kg) and with omeprazole (20 mg/kg), as a standard antiulcer drug. Gross and histological features showed the antiulcerogenic characterizations of EEAM. There was significant suppression on the ulcer lesion index of rats pretreated with EEAM, which was comparable to the omeprazole effect in the omeprazole control group. Oral administration of EEAM to rats caused a significant increase in the level of nitric oxide and antioxidant activities, including catalase, glutathione, and superoxide dismutase associated with attenuation in gastric acidity, and compensatory effect on the loss of gastric wall mucus. In addition, pretreatment of rats with EEAM caused significant reduction in the level of malondialdehyde, as a marker for oxidative stress, associated with an increase in prostaglandin E2 activity. Immunohistochemical staining also demonstrated that EEAM induced the downregulation of Bax and upregulation of Hsp70 proteins after pretreatment. Collectively, the present results suggest that EEAM has a promising antiulcer potential, which could be attributed to its suppressive effect against oxidative damage and preservative effect toward gastric wall mucus.

  18. Transient activation of microglia following acute alcohol exposure in developing mouse neocortex is primarily driven by BAX-dependent neurodegeneration.

    Science.gov (United States)

    Ahlers, Katelin E; Karaçay, Bahri; Fuller, Leah; Bonthius, Daniel J; Dailey, Michael E

    2015-10-01

    Fetal alcohol exposure is the most common known cause of preventable mental retardation, yet we know little about how microglia respond to, or are affected by, alcohol in the developing brain in vivo. Using an acute (single day) model of moderate (3 g/kg) to severe (5 g/kg) alcohol exposure in postnatal day (P) 7 or P8 mice, we found that alcohol-induced neuroapoptosis in the neocortex is closely correlated in space and time with the appearance of activated microglia near dead cells. The timing and molecular pattern of microglial activation varied with the level of cell death. Although microglia rapidly mobilized to contact and engulf late-stage apoptotic neurons, apoptotic bodies temporarily accumulated in neocortex, suggesting that in severe cases of alcohol toxicity the neurodegeneration rate exceeds the clearance capacity of endogenous microglia. Nevertheless, most dead cells were cleared and microglia began to deactivate within 1-2 days of the initial insult. Coincident with microglial activation and deactivation, there was a transient increase in expression of pro-inflammatory factors, TNFα and IL-1β, after severe (5 g/kg) but not moderate (3 g/kg) EtOH levels. Alcohol-induced microglial activation and pro-inflammatory factor expression were largely abolished in BAX null mice lacking neuroapoptosis, indicating that microglial activation is primarily triggered by apoptosis rather than the alcohol. Therefore, acute alcohol exposure in the developing neocortex causes transient microglial activation and mobilization, promoting clearance of dead cells and tissue recovery. Moreover, cortical microglia show a remarkable capacity to rapidly deactivate following even severe neurodegenerative insults in the developing brain.

  19. 复方参芩对犬细小病毒致心肌组织Bcl-2和Bax mRNA的影响%Effect of Shenqin Compound on mRNA Expression of Bcl-2 and Bax Genes in Canine Myocardium Infected by Canine Parvovirus

    Institute of Scientific and Technical Information of China (English)

    刘俊玮; 刘娟; 杜林林; 郭志兴; 凌榕镔

    2013-01-01

    为了探讨复方参芩对犬细小病毒致犬心肌组织Bcl-2和Bax基因表达的影响,将丹参、黄芩、甘草等中药配伍并制备成为复方参芩针剂,人工感染犬细小病毒建立模型;将犬分为空白对照组、模型组、黄芪多糖组、复方参芩组;给药7d后,接种病毒,观察各组犬临床症状,取心肌组织,电镜观察心脏组织超微结构变化,采用荧光实时定量PCR法检测Bcl-2和Bax mRNA表达.结果表明,模型组犬死亡率高,心肌组织结构损伤严重,与空白对照组比较,心肌组织细胞Bcl-2 mRNA表达下调(P<0.05),Bax mRNA表达增加(P<0.01).复方参芩组犬存活率较高,心肌组织损伤轻,与模型组相比,心肌组织细胞Bax mRNA表达下调(P<0.01),Bcl-2 mRNA表达上调(P<0.01).通过本试验证明复方参芩可通过上调犬心肌组织细胞Bcl-2 mRNA表达,下调Bax mRNA的表达,抑制细小病毒引起的心肌细胞凋亡,保护犬心肌组织免受细小病毒损害.%To investigate the effect of Shenqin compound to Bcl-2 and Bax genes in canine myocardium infected by canine parvovirus. Compatibility of salvia, scutellaria, glycyrrhiza and other Chinese herbal to prepare Shenqin compound injection, and to build model by artificial infection of canine parvovirus, the canines were divided into blank control group, model group, astragalus polysaccharide group and Shenqin compound group, after injecting for 7 days, inoculated canine parvovirus, observed clinical symptom in each group, the change of ultrastructure in myocardium was observed through electron microscope. The RT-PCR method were used to test the expression of Bcl-2 and Bax mRNA. The results showed: the model group had high mortality and the myocardium was seriously damaged, compared with blank control group, the expression of Bcl-2 mRNA was down-regulated at P<0. 05 level, and the expression of Bax mRNA was up-regulated at P<0. 01 level. In Shenqin compound group, the protective rates were high

  20. Influence of BCL2-938C>A and BAX-248G>A promoter polymorphisms in the development of AML: case-control study from South India.

    Science.gov (United States)

    Cingeetham, Anuradha; Vuree, Sugunakar; Dunna, Nageswara Rao; Gorre, Manjula; Nanchari, Santhoshi Rani; Edathara, Prajitha Mohandas; Meka, Phannibhushann; Annamaneni, Sandhya; Digumarthi, Raghunadharao; Sinha, Sudha; Satti, Vishnupriya

    2015-09-01

    B-cell lymphoma 2 (BCL2) and BCL2-associated X protein (BAX) proteins are anti-apoptotic and pro-apoptotic determinants of mitochondrial-mediated apoptosis, and their relative expression determines the cell fate. The promoter polymorphisms in these genes were shown to alter the protein function or expression and exert an impact on apoptosis regulation. Deregulation in the expression of any of these genes leads to disruption of cellular homeostasis and malignant transformation. The present study was aimed to determine the association of BCL2-938C>A and BAX-248G>A promoter polymorphisms with origin and progression of acute myeloid leukemia (AML). We also have performed combined genotype analysis to evaluate the cumulative effect of risk genotypes in the AML development. These polymorphisms were genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) in 221 AML patients and 305 age- and sex-matched healthy controls. Our study revealed that BCL2-938CA (p = 0.018) and BAX-248GG (0.043) genotypes were significantly associated with increased risk for AML occurrence. BAX-248A allele had shown decreased risk for AML. The combined analysis had shown that BCL2-938CA+AA-BAX-248GG group had a 1.63-fold (95 % CI: 1.08-2.45, p = 0.02) increased risk for AML. None of the clinical variables had shown any significant association with both polymorphisms. With respect to complete remission (CR) rate, BAX-248GG genotype (p = 0.002) and G allele (p = 0.009) had conferred significant risk for complete remission failure. Although the log rank test was not significant, survival analysis had shown a trend where BCL2-938CA genotype, and BAX-248GG had reduced median disease-free survival (DFS) of 9 and 10 months, respectively. In conclusion, BCL2-938C>A and BAX-248G>A gene polymorphisms might contribute to the origin of AML. Moreover, influence of BAX-248GG genotype on CR and DFS rate suggests that the BAX-248G>A polymorphism can serve as

  1. Preparation and Humidity-sensitive Properties ofNanometer-sized Semiconductor Ceramics La1-xBaxCoO3%纳米级La1-xBaxCoO3半导体陶瓷的制备与湿敏性能

    Institute of Scientific and Technical Information of China (English)

    陈秀芳; 莫茂松; 王银海; 刘艳美

    2001-01-01

    Nanometer-sized composite oxided semiconductor ceramics La1-xBaxCoO3(X=0,0.1≤X≤0.2)were successfully prepared by sol-gel technology. Phase composition and microstructure of the prepared nanoceramics were characterized by means of XRD, specific surface area absorption(BET)and Archimede replacement water method. The results of humidity-sensitive characteristic investigation showed that LaCoO3 nanoceramic sensor exhibits a negative humidity-resistance property and La1-xBaxCoO3 nanoceramic sensor exhibits a positive humidity-resistance property. It is proposed that negative humidity-resistance property of LaCoO3 nanoceramic sensor may be explained with proton(H+)theory, and positive humidity-resistance property of La1-xBaxCoO3(0.1≤X≤0.2)nanoceramic sensor may be explained with P-type carrier(⊕)quantity in grain boundary.%采用Sol-gel法化学工艺在较低温度下合成纳米级La1-xBaxCoO3(X=0,0.1≤X≤0.2)湿敏半导体陶瓷.用XRD、BET比表面吸附、Archimede排水法等技术对所合成陶瓷物相和结构进行了表征和分析.湿敏特性测试结果表明,La1-xBaxCoO3半导体纳米陶瓷有一定的湿敏性能,当X=0,材料呈负湿-阻特性,当0.1≤X≤0.2,材料呈正湿-阻特性.从晶界层空穴载流子数角度合理解释了La1-xBaxCoO3纳米陶瓷的正湿-阻特性.

  2. Ginkgo biloba leaf extract effects on inducible nitric oxide synthase, Bcl-2, and Bax expression in rat models of spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Jiejun Jiao; Bin Du

    2008-01-01

    BACKGROUND: Ginkgo biloba leaf extract exhibits neuroprotective effects in spinal cord injury. However,the mechanisms of action remain unclear.OBJECTIVE: To investigate inducible nitric oxide synthase (iNOS) and Bcl-2/Bax expression in the injured spinal cord, and to explore the neuroprotective mechanisms of ginkgo biloba leaf extract in rats with spinal cord injury.DESIGN, TIME AND SETTING: The randomized, controlled, cell molecular biology experiment was performed at Soochow University, China from March 2007 to March 2008.MATERIALS: A total of 120 healthy, adult Sprague Dawley rats were selected for this study. Rat models of moderate acute thoracic (T9) spinal cord injury were established using the modified Allen method.Shuxuening injection was obtained from Zhenbaodao Pharmaceutical Co., Ltd., China. Methylprednisolone was purchased from North China Pharmaceutical Co., Ltd.METHODS: All rats were equally and randomly divided into four groups. Only the spinal cord was exposed in the sham operation group rats. In the trauma group, rats were not treated with drugs following spinal cord injury. Rats in the hormone group were intraperitoneally injected with 30 mg/kg methylprcdnisolone following spinal cord injury. Rats in the ginkgo biloba leaf extract group were intraperitoneally infused with a 1.0 mL/kg Shuxuening injection per day.MAIN OUTCOME MEASURES: At l hour, as well as 1, 3, 5, 7, and 14 days after spinal cord injury,iNOS- and Bcl-2/Bax-positive cells were quantified with immunohistochemistry. Pathological changes were detected using hematoxylin-eosin staining under an optical microscope.RESULTS: Spinal cord injury in the ginkgo biloba leaf extract and hormone groups was milder compared with the trauma group. Demyelination was significantly ameliorated and the necrotic cavity was obviously reduced in the injured spinal cord of rats in the ginkgo biloba leaf extract and hormone groups at each time point, iNOS expression was increased in the injured spinal cord

  3. Ginkgo biloba leaf extract and alpha-tocopherol attenuate haloperidol-induced orofacial dyskinesia in rats: Possible implication of antiapoptotic mechanisms by preventing Bcl-2 decrease and Bax elevation.

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    An, Hui Mei; Tan, Yun Long; Shi, Jing; Wang, Zhiren; Lv, Meng Han; Soares, Jair C; Zhou, Dongfeng; Yang, Fude; Zhang, Xiang Yang

    2016-12-01

    Tardive dyskinesia (TD) is a serious side effect of long-term administration of typical neuroleptics, such as haloperidol. The pathophysiology of TD remains unclear, but the experimental evidence suggests that free radical-induced neuronal apoptosis in the basal ganglia may play an important role. This study was to investigate changes in Bax and Bcl-2 expression levels in TD-associated brain regions and the effects of the antioxidant EGb761 on Bax and Bcl-2 levels in an animal model of TD. Thirty-two rats were randomly divided into four study groups: saline control (saline), haloperidol-alone (haloperidol), EGb761-haloperidol (EGb), and alpha-tocopherol-haloperidol (vitamin E). Rats were treated with daily intraperitoneal haloperidol injections (2 mg/kg/day) for 5 weeks. EGb761 (50 mg/kg/day) and alpha-tocopherol (20 mg/kg/day) were then administered for another 5 weeks during the withdrawal period. Behavioral assessments were performed, and Bax and Bcl-2 protein expression levels were immunohistochemically analyzed in four brain regions, including the prefrontal cortex, striatum, substantia nigra, and globus pallidum. We found that increased vacuous chewing movements (VCMs) were associated with increased proapoptotic Bax protein expression, decreased antiapoptotic Bcl-2 protein expression, and an increased Bax/Bcl-2 ratio. EGb761 and alpha-tocopherol treatment reversed the increase in VCMs, decreased Bax expression, increased Bcl-2 expression, and decreased the Bax/Bcl-2 ratio. These results demonstrate that long-term haloperidol administration may affect Bcl-2 protein family expression and promote neuronal apoptosis in the basal ganglia. In combination with their antioxidant capacity, EGb761 and alpha-tocopherol's antiapoptotic effects through Bcl-2 might account for the symptom improvement observed in haloperidol-induced TD rats. Copyright © 2016 Elsevier GmbH. All rights reserved.

  4. High BAX/BCL2 mRNA ratio predicts favorable prognosis in laryngeal squamous cell carcinoma, particularly in patients with negative lymph nodes at the time of diagnosis.

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    Giotakis, Aris I; Kontos, Christos K; Manolopoulos, Leonidas D; Sismanis, Aristides; Konstadoulakis, Manousos M; Scorilas, Andreas

    2016-08-01

    Laryngeal squamous cell carcinoma (LSCC), a common type of head and neck cancer, is associated with high rates of metastasis and recurrence. Therefore, accurate prognostic stratification of LSCC patients based on molecular prognostic tumor biomarkers would definitely lead to a better clinical management of this malignancy. The aim of this study was the investigation of the potential combinatorial prognostic value of BCL2 and BAX mRNA expression in LSCC. Total RNA was isolated from 105 cancerous laryngeal tissue specimens obtained from patients having undergone surgical treatment for primary LSCC. After cDNA preparation, a low-cost, in-house developed, sensitive and accurate real-time quantitative PCR (qPCR) methodology was applied for the quantification of BCL2 and BAX mRNA levels. Then, we carried out a biostatistical analysis to assess the prognostic value of the BAX/BCL2 mRNA expression ratio. High BAX/BCL2 mRNA expression constitutes a favorable prognosticator in LSCC, predicting significantly longer disease-free survival (P=0.011) and overall survival (P=0.014) of patients. More importantly, the significant prognostic value of the BAX/BCL2 mRNA expression appeared to be independent of the histological grade and size of the malignant laryngeal tumor as well as TNM stage, as revealed by the multivariate bootstrap Cox regression analysis. Kaplan-Meier survival analysis demonstrated also that the BAX/BCL2 ratio can stratify node-negative (N0) LSCC patients into two subgroups with significantly different DFS and OS (P=0.021 and P=0.009, respectively). The BAX/BCL2 mRNA ratio is a putative molecular tissue biomarker in CLL and hence deserves further validation in larger cohorts of LSCC patients. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  5. Dexamethasone protected human glioblastoma U87MG cells from temozolomide induced apoptosis by maintaining Bax:Bcl-2 ratio and preventing proteolytic activities

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    Patel Sunil J

    2004-12-01

    Full Text Available Abstract Background Glioblastoma is the deadliest and most prevalent brain tumor. Dexamethasone (DXM is a commonly used steroid for treating glioblastoma patients for alleviation of vasogenic edema and pain prior to treatment with chemotherapeutic drugs. Temozolomide (TMZ, an alkylating agent, has recently been introduced in clinical trials for treating glioblastoma. Here, we evaluated the modulatory effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells. Results Freshly grown cells were treated with different doses of DXM or TMZ for 6 h followed by incubation in a drug-free medium for 48 h. Wright staining and ApopTag assay showed no apoptosis in cells treated with 40 μM DXM but considerable amounts of apoptosis in cells treated with 100 μM TMZ. Apoptosis in TMZ treated cells was associated with an increase in intracellular free [Ca2+], as determined by fura-2 assay. Western blot analyses showed alternations in the levels of Bax (pro-apoptotic and Bcl-2 (anti-apoptotic proteins resulting in increased Bax:Bcl-2 ratio in TMZ treated cells. Western blot analyses also detected overexpression of calpain and caspase-3, which cleaved 270 kD α-spectrin at specific sites for generation of 145 and 120 kD spectrin break down products (SBDPs, respectively. However, 1-h pretreatment of cells with 40 μM DXM dramatically decreased TMZ induced apoptosis, decreasing Bax:Bcl-2 ratio and SBDPs. Conclusion Our results revealed an antagonistic effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells, implying that treatment of glioblastoma patients with DXM prior to chemotherapy with TMZ might result in an undesirable clinical outcome.

  6. Hyperhomocysteinemia induces cardiac injury by up-regulation of p53-dependent Noxa and Bax expression through the p53 DNA methylation in ApoE-/-mice

    Institute of Scientific and Technical Information of China (English)

    Shengchao Ma; Huiping Zhang; Weiwei Sun; HuiHui Gong; Yanhua Wang; Changjian Ma; Ju Wang

    2013-01-01

    Hyperhomocysteinemia (HHcy) is a risk factor for cardiovascular disease and has a strong correlation with heart failure.However,the effects of HHcy on cardiac tissue remain less well understood.To elucidate the role of p53-dependent apoptosis in HHcy-induced cardiac injury,we fed ApoE-/-mice with high methionine diet to establish HHcy model.Serum Hcy,cardiac enzymes,and lipids were measured.The protein levels of Noxa,DNMT1,caspases-3/9,and p53 were determined by enzyme-linked immunosorbent assay.Bcl-2 and Bax proteins were detected by immunohistochemistry staining.S-adenosyl methionine and S-adenosyl homocysteine concentrations were determined by high-performance liquid chromatography.The mRNA levels of p53 and DNMT1 were analyzed by real-time polymerase chain reaction (PCR) and the methylation levels of p53 were analyzed by nested methylation-specific-PCR.Our data showed that the concentrations of serum Hey and lipids were increased in Meth group compared with the N-control group,which indicated that the model was established successfully.The expression levels of p53 and Noxa were increased in Meth group,while the methylation status of p53 was hypomethylation.The activities of caspase-3/9 were increased in Meth group compared with the N-control group.In addition,immunohistochemistry staining showed that the expression of Bax was significantly increased in Meth and Meth-F group compared with the N-control group.In summary,HHcy induces cardiac injury by up-regulation of p53-dependent pro-apoptotic related genes Noxa and Bax,while p53 DNA hypomethylation is a key molecular mechanism in pathological process induced by HHcy.

  7. Suppression of the Arboviruses Dengue and Chikungunya Using a Dual-Acting Group-I Intron Coupled with Conditional Expression of the Bax C-Terminal Domain.

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    James R Carter

    Full Text Available In portions of South Asia, vectors and patients co-infected with dengue (DENV and chikungunya (CHIKV are on the rise, with the potential for this occurrence in other regions of the world, for example the United States. Therefore, we engineered an antiviral approach that suppresses the replication of both arboviruses in mosquito cells using a single antiviral group I intron. We devised unique configurations of internal, external, and guide sequences that permit homologous recognition and splicing with conserved target sequences in the genomes of both viruses using a single trans-splicing Group I intron, and examined their effectiveness to suppress infections of DENV and CHIKV in mosquito cells when coupled with a proapoptotic 3' exon, ΔN Bax. RT-PCR demonstrated the utility of these introns in trans-splicing the ΔN Bax sequence downstream of either the DENV or CHIKV target site in transformed Aedes albopictus C6/36 cells, independent of the order in which the virus specific targeting sequences were inserted into the construct. This trans-splicing reaction forms DENV or CHIKV ΔN Bax RNA fusions that led to apoptotic cell death as evidenced by annexin V staining, caspase, and DNA fragmentation assays. TCID50-IFA analyses demonstrate effective suppression of DENV and CHIKV infections by our anti-arbovirus group I intron approach. This represents the first report of a dual-acting Group I intron, and demonstrates that we can target DENV and CHIKV RNAs in a sequence specific manner with a single, uniquely configured CHIKV/DENV dual targeting group I intron, leading to replication suppression of both arboviruses, and thus providing a promising single antiviral for the transgenic suppression of multiple arboviruses.

  8. Antrodia camphorata Potentiates Neuroprotection against Cerebral Ischemia in Rats via Downregulation of iNOS/HO-1/Bax and Activated Caspase-3 and Inhibition of Hydroxyl Radical Formation

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    Po-Sheng Yang

    2015-01-01

    Full Text Available Antrodia camphorata (A. camphorata is a fungus generally used in Chinese folk medicine for treatment of viral hepatitis and cancer. Our previous study found A. camphorata has neuroprotective properties and could reduce stroke injury in cerebral ischemia animal models. In this study, we sought to investigate the molecular mechanisms of neuroprotective effects of A. camphorata in middle cerebral artery occlusion (MCAO rats. A selective occlusion of the middle cerebral artery (MCA with whole blood clots was used to induce ischemic stroke in rats and they were orally treated with A. camphorata (0.25 and 0.75 g/kg/day alone or combined with aspirin (5 mg/kg/day. To provide insight into the functions of A. camphorata mediated neuroprotection, the expression of Bax, inducible nitric oxide synthase (iNOS, haem oxygenase-1 (HO-1, and activated caspase-3 was determined by Western blot assay. Treatment of aspirin alone significantly reduced the expressions of HO-1 (P<0.001, iNOS (P<0.001, and Bax (P<0.01 in ischemic regions. The reduction of these expressions was more potentiated when rats treated by aspirin combined with A. camphorata (0.75 g/kg/day. Combination treatment also reduced apoptosis as measured by a significant reduction in active caspase-3 expression in the ischemic brain compared to MCAO group (P<0.01. Moreover, treatment of A. camphorata significantly (P<0.05 reduced fenton reaction-induced hydroxyl radical (OH• formation at a dose of 40 mg/mL. Taken together, A. camphorata has shown neuroprotective effects in embolic rats, and the molecular mechanisms may correlate with the downregulation of Bax, iNOS, HO-1, and activated caspase-3 and the inhibition of OH• signals.

  9. Garlic (Allium sativum Fresh Juice Induces Apoptosis in Human Oral Squamous Cell Carcinoma: The Involvement of Caspase-3, Bax and Bcl-2

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    Farrokh Farhadi

    2015-12-01

    Full Text Available Background and aims. There is no report on the apoptotic impact of Allium sativum L.(Garlic on the oral squamous cell carcinoma (KB; hence, this study was designed to survey the apoptotic effects of garlic fresh juice (GFJ on the KB cells. Materials and methods. MTTassay (MicrocultureTetrazolium Assay was carried out to evaluate the cytotoxicity of GFJ on KB cells. Furthermore, TUNEL(Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelingand DNA fragmentation tests were performed to determine if GFJ is able to induce apoptosis in KB cells. Also a standard kit was used to assess caspase-3 activity in KB cells. Also western blotting was employed to evaluate the effect of GFJ on Bax:Bcl-2 ratio. Results. Significant cytotoxic effects were observed for the minimum used concentration (1g/mL as calculated to be 77.97±2.3% for 24 h and 818±3.1% for 36h of incubation (P < 0.001. Furthermore, TUNEL and DNA fragmentation tests corroborated the apoptosis inducing activity of GFJ. Consistently, after treating KB cells with GFJ(1μg/mL, caspase-3 activity and Bax:Bcl-2 ratio were raised by 7.3±0.6 and (P <0.001 folds, respectively. Conclusion. The results of this study advanced that GFJ induces apoptosis in the KB cells through increasing caspase-3 activity and Bax:Bcl2 ratio which could be attributed to its organo-sulfurcomponents.

  10. PATZ1 expression correlates positively with BAX and negatively with BCL6 and survival in human diffuse large B cell lymphomas

    Science.gov (United States)

    Valentino, Elena; Vitiello, Michela; Luciano, Antonio; Palma, Giuseppe; Arra, Claudio; Mantia, Elvira La; Panico, Luigi; Tenneriello, Valentina; Pinto, Antonello; Frigeri, Ferdinando; Capobianco, Gaetana; Botti, Gerardo; Cerchia, Laura; De Chiara, Annarosaria; Fedele, Monica

    2016-01-01

    Non-Hodgkin lymphomas (NHLs) include a heterogeneous group of diseases, which differ in both cellular origin and clinical behavior. Among the aggressive malignancies of this group, the diffuse large B-cell lymphomas (DLBCLs) are the most frequently observed. They are themselves clinically and molecularly heterogeneous and have been further sub-divided in three sub-types according to different cell of origin, mechanisms of oncogenesis and clinical outcome. Among them, the germinal center B-cell-like (GCB) derives from the germinal center and expresses the BCL6 oncogene. We have previously shown that Patz1-knockout mice develop B-cell neoplasias, suggesting a tumor suppressor role for PATZ1 in human NHLs. Here, by immunohistochemical analysis of a tissue-microarray including 170 NHLs, we found that PATZ1 nuclear expression is down-regulated in follicular lymphomas and DLBCLs. Moreover, consistent with our previous results showing a PATZ1-dependent regulation of BCL6 and BAX transcription, we show that low PATZ1 nuclear expression significantly correlates with high BCL6 expression, mainly in DLBCLs, and with low BAX expression, also considering separately follicular lymphomas and DLBCLs. Finally, by analyzing overall and progression-free survival in DLBCL patients that underwent rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy, low levels of PATZ1 were significantly associated to a worst outcome and demonstrated an independent prognostic factor in multivariate analysis, including known prognostic factors of DLBCL, IPI score and cell of origin (GCB/non-GCB). Therefore, we propose PATZ1 as a new prognostic marker of DLBCLs, which may act as a tumor suppressor by enhancing apoptosis through inhibiting and enhancing transcription of BCL6 and BAX, respectively. PMID:27494852

  11. Bax, Bcl2, and p53 differentially regulate neomycin- and gentamicin-induced hair cell death in the zebrafish lateral line.

    Science.gov (United States)

    Coffin, Allison B; Rubel, Edwin W; Raible, David W

    2013-10-01

    Sensorineural hearing loss is a normal consequence of aging and results from a variety of extrinsic challenges such as excessive noise exposure and certain therapeutic drugs, including the aminoglycoside antibiotics. The proximal cause of hearing loss is often death of inner ear hair cells. The signaling pathways necessary for hair cell death are not fully understood and may be specific for each type of insult. In the lateral line, the closely related aminoglycoside antibiotics neomycin and gentamicin appear to kill hair cells by activating a partially overlapping suite of cell death pathways. The lateral line is a system of hair cell-containing sense organs found on the head and body of aquatic vertebrates. In the present study, we use a combination of pharmacologic and genetic manipulations to assess the contributions of p53, Bax, and Bcl2 in the death of zebrafish lateral line hair cells. Bax inhibition significantly protects hair cells from neomycin but not from gentamicin toxicity. Conversely, transgenic overexpression of Bcl2 attenuates hair cell death due to gentamicin but not neomycin, suggesting a complex interplay of pro-death and pro-survival proteins in drug-treated hair cells. p53 inhibition protects hair cells from damage due to either aminoglycoside, with more robust protection seen against gentamicin. Further experiments evaluating p53 suggest that inhibition of mitochondrial-specific p53 activity confers significant hair cell protection from either aminoglycoside. These results suggest a role for mitochondrial p53 activity in promoting hair cell death due to aminoglycosides, likely upstream of Bax and Bcl2.

  12. Bone Marrow Stromal Cells Promote Neuronal Restoration in Rats with Traumatic Brain Injury: Involvement of GDNF Regulating BAD and BAX Signaling

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    Qin Shen

    2016-02-01

    Full Text Available Background/Aims: To investigate the effects of bone marrow stromal cells (BMSCs and underlying mechanisms in traumatic brain injury (TBI. Methods: Cultured BMSCs from green fluorescent protein-transgenic mice were isolated and confirmed. Cultured BMSCs were immediately transplanted into the regions surrounding the injured-brain site to test their function in rat models of TBI. Neurological function was evaluated by a modified neurological severity score on the day before, and on days 7 and 14 after transplantation. After 2 weeks of BMSC transplantation, the brain tissue was harvested and analyzed by microarray assay. And the coronal brain sections were determined by immunohistochemistry with mouse anti-growth-associated protein-43 kDa (anti-GAP-43 and anti-synaptophysin to test the effects of transplanted cells on the axonal regeneration in the host brain. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay and Western blot were used to detect the apoptosis and expression of BAX and BAD. Results: Microarray analysis showed that BMSCs expressed growth factors such as glial cell-line derived neurotrophic factor (GDNF. The cells migrated around the injury sites in rats with TBI. BMSC grafts resulted in an increased number of GAP-43-immunopositive fibers and synaptophysin-positive varicosity, with suppressed apoptosis. Furthermore, BMSC transplantation significantly downregulated the expression of BAX and BAD signaling. Moreover, cultured BMSC transplantation significantly improved rat neurological function and survival. Conclusion: Transplanted BMSCs could survive and improve neuronal behavior in rats with TBI. Mechanisms of neuroprotection and regeneration were involved, which could be associated with the GDNF regulating the apoptosis signals through BAX and BAD.

  13. Multiple doses of erythropoietin impair liver regeneration by increasing TNF-alpha, the Bax to Bcl-xL ratio and apoptotic cell death.

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    Katja Klemm

    Full Text Available BACKGROUND: Liver resection and the use of small-for-size grafts are restricted by the necessity to provide a sufficient amount of functional liver mass. Only few promising strategies to maximize liver regeneration are available. Apart from its erythropoiesis-stimulating effect, erythropoietin (EPO has meanwhile been recognized as mitogenic, tissue-protective, and anti-apoptotic pleiotropic cytokine. Thus, EPO may support regeneration of hepatic tissue. METHODOLOGY: Rats undergoing 68% hepatectomy received daily either high dose (5000 IU/kg bw i.v. or low dose (500 IU/kg bw i.v. recombinant human EPO or equal amounts of physiologic saline. Parameters of liver regeneration and hepatocellular apoptosis were assessed at 24 h, 48 h and 5 d after resection. In addition, red blood cell count, hematocrit and serum EPO levels as well as plasma concentrations of TNF-alpha and IL-6 were evaluated. Further, hepatic Bcl-x(L and Bax protein expression were analyzed by Western blot. PRINCIPAL FINDINGS: Administration of EPO significantly reduced the expression of PCNA at 24 h followed by a significant decrease in restitution of liver mass at day 5 after partial hepatectomy. EPO increased TNF-alpha levels and shifted the Bcl-x(L to Bax ratio towards the pro-apoptotic Bax resulting in significantly increased hepatocellular apoptosis. CONCLUSIONS: Multiple doses of EPO after partial hepatectomy increase hepatocellular apoptosis and impair liver regeneration in rats. Thus, careful consideration should be made in pre- and post-operative recombinant human EPO administration in the setting of liver resection and transplantation.

  14. Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis.

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    Srivastava, M; Ahmad, N; Gupta, S; Mukhtar, H

    2001-05-04

    Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.

  15. PCSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax-caspase9-caspase3 pathway.

    Science.gov (United States)

    Wu, Chun-Yan; Tang, Zhi-Han; Jiang, Lu; Li, Xue-Fei; Jiang, Zhi-Sheng; Liu, Lu-Shan

    2012-01-01

    This paper investigated the effects of ox-LDL on PCSK9, and the molecular mechanisms of PCSK9 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cells (HUVECs), to clarify the role of PCSK9 in atherosclerogenesis. HUVECs were incubated with ox-LDL for 24 h. The apoptosis was observed by Hoechst 33258 staining. The expression of PCSK9, LOX-1 mRNAs and proteins was detected by RT-PCR, western blot, respectively. The PCSK9 siRNAs labeled with fluorescence were transfected into HUVECs by Lipofectamine 2000. After transfection for 24 h, cells were treated with ox-LDL for 24 h, HUVECs apoptosis transfected siRNA was detected by Hoechst 33258 staining and flow cytometer. The expression of Bcl-2, Bax, caspase3, 8, 9 was detected by western blot. The activity of caspase3, 9 was detected by kits. Our results showed that apoptosis of HUVECs and the expressions of PCSK9 and LOX-1 were upregulated secondary to induction by ox-LDL in a concentration-dependent manner. However, ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression, were significantly reduced by PCSK9 siRNA. These results demonstrate a linkage between HUVEC apoptosis and PCSK9 expression. Furthermore, we detected the possible pathway involved in apoptotic regulation by PCSK9 siRNA; our results showed that the expression of Bcl-2 decreased, whereas that of Bax increased. In addition, ox-LDL enhanced the activity of caspase9 and then caspase3. Pretreatment of HUVECs with PCSK9 siRNA blocked these effects of ox-LDL. These findings suggest that ox-LDL-induced HUVECs apoptosis could be inhibited by PCSK9 siRNA, in which Bcl/Bax-caspase9-caspase3 pathway maybe was involved through reducing the Bcl-2/Bax ratio and inhibited the activation of both caspase9 and 3.

  16. Electric field-tunable BaxSr1-xTiO3 films with high figures of merit grown by molecular beam epitaxy

    Science.gov (United States)

    Mikheev, Evgeny; Kajdos, Adam P.; Hauser, Adam J.; Stemmer, Susanne

    2012-12-01

    We report on the dielectric properties of BaxSr1-xTiO3 (BST) films grown by molecular beam epitaxy on epitaxial Pt bottom electrodes. Paraelectric films (x ≲ 0.5) exhibit dielectric losses that are similar to those of BST single crystals and ceramics. Films with device quality factors greater than 1000 and electric field tunabilities exceeding 1:5 are demonstrated. The results provide evidence for the importance of stoichiometry control and the use of a non-energetic deposition technique for achieving high figures of merit of tunable devices with BST thin films.

  17. Nerve growth factor affects focal cerebral cortical neuronal Bcl-2 and Bax expression in a mouse model of oxyhemoglobin-induced subarachnoid hemorrhage

    Institute of Scientific and Technical Information of China (English)

    Xianfeng Jiang; Wei Shi; Jin Liang

    2008-01-01

    BACKGROUND: Studies have demonstrated that oxyhemoglobin (OxyHb) can induce brain cell apoptosis in vivo.OBJECTIVE: To observe the effects of exogenous nerve growth factor (NGF) on cerebral cortical neuronal Bcl-2 and Bax expression in mice with OxyHb-induced subarachnoid hemorrhage.DESIGN, TIME AND SETTING: A completely randomized grouping, controlled animal experiment was performed at the Experimental Center for Biomedicine, College of Medicine, Xi'an Jiaotong University between February and April 2005.MATERIALS: Fifty-four healthy, male, adult, ICR mice were included in this study. Subarachnoid hemorrhage was induced by a subarachnoid injection of OxyHb in 48 mice. Mouse NGF was obtained from Xiamcn Beidazhilu Bioengineering Co., Ltd., China.METHODS: All 54 mice were randomly divided into three groups: control (n = 6), injury (n = 24), and NGF (n = 24). The NGF group received a subarachnoidal administration of OxyHb, immediately followed by a caudal vein injection of NGF (1 μg). The injury group was injected with OxyHb, and subsequently with physiological saline. Thc control group only received intravenous physiological saline.MAIN OUTCOME MEASURES: At 1, 6, 24, and 48 hours following subarachnoid hemorrhage induction,expression levels of Bcl-2 and Bax were detected by immunohistochemistry in the cerebral cortex 3 mm anterior and posterior to the injection site.RESULTS: At all time points following OxyHb injection, cerebral cortical Bax levels were significantly higher in the injured group than in the control and NGF groups (P < 0.01). During the first 24 hours following OxyHb injection, cerebral cortical Bcl-2 levels were significantly lower in the injury group compared to the control group (P < 0.05 0.01). Between 1 and 48 hours, Bcl-2 levels were significantly higher in the NGF group than in the injury group (P < 0.01).CONCLUSION: Exogenous NGF can inhibit increased neuronal Bax expression and decreased Bcl-2expression in the cerebral cortex of mice with Oxy

  18. 前胡甲素对缺血再灌注心肌IL-6水平及Fas,bax,bcl-2蛋白表达的影响%Effects of dl-praeruptorin A on interleukin-6 level and Fas,bax, bcl-2 protein expression in ischemia-reperfusion myocardium

    Institute of Scientific and Technical Information of China (English)

    常天辉; 刘晓阳; 章新华; 王怀良

    2002-01-01

    AIM: To investigate the effects of dl-praeruptorin (Pd-Ia) on interleukin-6 (IL-6) level and apoptosis-relatedprotein expression in ischemia-reperfusion myocardium. METHODS: Left anterior descending coronary arterywas subjected to 30 min ischemia followed by 120 min reperfusion in open-chest anesthetized rats. Serum IL-6level was measured by radioimmunoassay. Apoptosis-related protein Fas, bax, and bcl-2 expression was detectedby immunohistochemistry and computer image analysis system. Infiltration of neutrophils was observed usingHematoxylin-Eosin staining under optical microscope. RESULTS: Pd-Ia 2.0 mg.kg -1 iv lowered serum 1L-6 level andFas, bax, bcl-2 expression under conditions with hypotension and without changes on heart rate, but increased theratio of bcl-2/bax. There existed a close linearity and positive correlation between IL-6 level and Fas, bax, bcl-2expression. Whereas, the infiltration of neutrophils was mild. CONCLUSION: Pd-Ia elicits a novel target in thetherapeutic prevention of postischemic cardiomyocyte death. The reason might be associated with modulating theexpression of some immediate-early genes including IL-6, Fas, bax, and bcl-2 in ischemia-reperfusion myocardium.%目的:研究前胡甲素对缺血再灌注心肌IL-6水平及凋亡相关蛋白表达的影响.方法:麻醉开胸大鼠左前降枝冠状动脉蒙受30分钟缺血及120分钟再灌注.放射免疫法测定血清IL-6水平;免疫组化法和计算机图像分析系统检测凋亡相关蛋白Fas,bax及bcl-2的表达:苏木精一依红染色法染色并于光镜下观测嗜中性白细胞的浸润.结果:前胡甲素2.0 mg.kg-1 iv,在降压和不影响心率的情况下,减少IL-6水平及Fas,bax,bcl-2蛋白的表达,但增加bcl-2/bax的比率.IL-6水平及Fas,bax,bcl-2蛋白表达之间有密切的线性正相关,而嗜中性白细胞只有轻微浸润.结论:前胡甲素防治缺血后心肌细胞死亡出现新靶位,可能与机体在心肌缺血再灌

  19. The BH3 α-Helical Mimic BH3-M6 Disrupts Bcl-XL, Bcl-2, and MCL-1 Protein-Protein Interactions with Bax, Bak, Bad, or Bim and Induces Apoptosis in a Bax- and Bim-dependent Manner*

    Science.gov (United States)

    Kazi, Aslamuzzaman; Sun, Jiazhi; Doi, Kenichiro; Sung, Shen-Shu; Takahashi, Yoshinori; Yin, Hang; Rodriguez, Johanna M.; Becerril, Jorge; Berndt, Norbert; Hamilton, Andrew D.; Wang, Hong-Gang; Sebti, Saïd M.

    2011-01-01

    A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-XL and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-XL, Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-XL/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-XL, Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612. PMID:21148306

  20. Structural and energetical studies of the adsorption of para and meta-isomers of xylene on pre-hydrated zeolite BaX. Characterization by neutron diffraction and temperature programmed desorption; Etude structurale et energetique de l'adsorption des isomeres para- et meta- du xylene dans la zeolithe BaX prehydratee. Caracterisation par diffraction des neutrons et thermodesorption programmee

    Energy Technology Data Exchange (ETDEWEB)

    Pichon, Ch.

    1999-10-19

    The separation of p-xylene from C{sub 8} aromatics is performed industrially by selective adsorption on zeolitic materials. FAU-type zeolites are currently used for this separation and especially the partially hydrated BaX. The aim of this work is to characterize from a structural (by low temperature neutron powder diffraction) and an energetical (by temperature programmed desorption) point of view, the adsorption of para- and meta- isomers of xylene, for different fillings, as pure substances as well as mixtures, on pre-hydrated zeolite BaX. The influence of the water pre-adsorption on xylene adsorption selectivity is carefully discussed. The crystalline structure of the zeolite BaX (framework and compensation of charge cations) and of the adsorbed phase (water, p- and m-xylene molecules) are completely characterized by neutron diffraction. The location and the distribution of water and xylene molecules on their adsorption sites is especially followed as a function of the filling of the zeolite and of the composition of the adsorbed phase. Microscopic measurements were correlated to the energetical analysis (at a macroscopic level) in order to obtain a consistent description of adsorption phenomenon and to propose a possible origin for adsorption selectivity.

  1. Knockdown of miR-221 promotes the cisplatin-inducing apoptosis by targeting the BIM-Bax/Bak axis in breast cancer.

    Science.gov (United States)

    Ye, Zhiqiang; Hao, Rutian; Cai, Yefeng; Wang, Xiaobo; Huang, Guanli

    2016-04-01

    Accumulating evidence shows that microRNAs (miRNAs) have a critical role in the initiation and progression of types of human cancer, including breast cancer. Recent research indicated that miRNAs are also related with the chemotherapy on cancers. In this study, the expression of miR-221 in breast cancer (BC) patients' serum and cancer tissues was found to be significantly up-regulated. The results of in vitro MTT assay indicated that although the anti-miR-221 oligonucleotide alone did not influence the viability of BC cell lines markedly, it significantly promoted the cytotoxicity of cisplatin (DDP) to BC cells. Mechanistic studies demonstrated that the gene of BIM (Bcl-2 interacting mediator of cell death), a pro-apoptotic Bcl-2 family protein, was up-regulated by the knockdown of miR-221. We found that the synergetic effect of anti-miR-221 on increasing the sensitivity of MDA-MB-231 was BIM dependant. Furthermore, results of immunoprecipitation showed the up-regulated BIM directly combined with the Bax and Bak, leading to mitochondrial dysfunction. Our results suggest the anti-miR-221 could promote the cisplatin-inducing apoptosis by targeting the Bim-Bax/Bak axis in breast cancer.

  2. Apigenin induces caspase-dependent apoptosis in human lung cancer A549 cells through Bax- and Bcl-2-triggered mitochondrial pathway.

    Science.gov (United States)

    Lu, Hsu-Feng; Chie, Yu-Jie; Yang, Ming-Sung; Lee, Ching-Sung; Fu, Jene-John; Yang, Jai-Sing; Tan, Tzu-Wei; Wu, Shin-Hwar; Ma, Yi-Shih; Ip, Siu-Wan; Chung, Jing-Gung

    2010-06-01

    The molecular mechanism and possible signaling pathway of apigenin-induced cytotoxicity and apoptosis in human lung cancer cells has not been reported. We investigated the role of ROS, Ca2+, caspases and Bax proteins and mitochondria membrane potential in apigenin-induced apoptosis in A549 cells. Cells were incubated with different concentrations of apigenin then cell morphological changes, DNA damage, cell viability and apoptosis were determined by Comet assay, and flow cytometric analysis. Sub-G1 phase was also examined. Western blot analysis was used to determined the levels of Bax and Bcl-2 and apoptosis associated proteins, and confocal laser microscope for examining the translocation of associated protein after exposed to apigenin. The results indicated that apigenin induced morphological changes, decreased percentage of viable cells and induced apoptosis dose- and time-dependently. DAPI staining and Comet assay also confirmed that apigenin-induced DNA condensation and damage. The levels of caspase-3, -8 and -9 involved in apigenin-induced apoptosis indicating caspase-dependent pathway was induced by apigenin. Western blotting showed that apigenin promoted cytochrome c levels and also induced dysfunction of mitochondria leading to the release of cytochrome c, AIF and Endo G, causing the activation of caspase-9 and -3, then apoptosis in A549 cells.

  3. PCR detection of Salmonella typhimurium in pharmaceutical raw materials and products contaminated with a mixed bacterial culture using the BAX system.

    Science.gov (United States)

    Jimenez, L; Scalici, C; Smalls, S; Bosko, Y; Ignar, R

    2001-01-01

    The BAX system, a PCR-based assay, was evaluated for detecting Salmonella typhimurium in pharmaceutical raw materials and products contaminated with mixed bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhimurium. Artificially contaminated samples were preenriched in lactose broth with and without Tween 20. After preenrichment, samples were analyzed by PCR and standard methods. Ten of 25 samples did not show presence of the specific Salmonella spp. 740-base pair DNA fragment. However, S. typhimurium was isolated and identified by standard methods from all 25 samples. To optimize S. typhimurium detection in PCR negative samples, lactose broth was replaced by buffered peptone water (BPW) as the preenrichment broth. When BPW was used, all 10 samples were PCR positive. BPW enrichments increased S. typhimurium growth resulting in rapid PCR detection. The presence of non-Salmonella bacteria influenced the performance of the PCR-based assay. Optimization of S. typhimurium PCR detection in mixed culture required the use of different preenrichment broths. However, the BAX system detected S. typhimurium within 27 hours while standard methods required 5-7 days.

  4. Asiaticoside: attenuation of neurotoxicity induced by MPTP in a rat model of Parkinsonism via maintaining redox balance and up-regulating the ratio of Bcl-2/Bax.

    Science.gov (United States)

    Xu, Chang-Liang; Wang, Qi-Zhi; Sun, Ling-Mei; Li, Xiu-Min; Deng, Ji-Min; Li, Lu-Fan; Zhang, Jin; Xu, Rong; Ma, Shi-Ping

    2012-01-01

    In this study, we investigated the neuroprotective effects of asiaticoside, a triterpenoid saponin isolated from the Chinese medicinal herb Centella asiatica, in the rats model of Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Rats were first injected with MPTP. One day after surgery, asiaticoside was administered and the behavioral tests were assessed. On 14th day, the rats were sacrificed, substantia nigra (SN) and striatum were dissected, and then dopamine (DA) and its metabolites in striatum and malonyldialdehyde (MDA) contents, reduced glutathione (GSH) level and gene expression level in SN were estimated. Treatment with asiaticoside was found to protect dopaminergic neuron by antagonizing MPTP induced neurotoxicity and to improve locomotor dysfunction. Asiaticoside significantly attenuated the MPTP-induced reduction of dopamine in the striatum. The content of MDA was significantly decreased while the GSH level was significantly increased in asiaticoside-treated groups. In addition, asiaticoside increased the Bcl-2/Bax ratio. These results indicated that asiaticoside was effective in reversing MPTP induced Parkinsonism via its neuroprotective effects including antioxidant activity, maintaining the metabolic balance of DA, and increasing ratio of Bcl-2/Bax.

  5. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression

    Science.gov (United States)

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC. PMID:27642320

  6. Lack of ligand-selective binding of the aryl hydrocarbon receptor to putative DNA binding sites regulating expression of Bax and paraoxonase 1 genes.

    Science.gov (United States)

    DeGroot, Danica E; Hayashi, Ai; Denison, Michael S

    2014-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals through its ability to bind specific DNA recognition sites (dioxin responsive elements (DREs)), and activate transcription of adjacent genes. While the DRE has a highly conserved consensus sequence, it has been suggested that the nucleotide specificity of AhR DNA binding may be ligand-dependent. The upstream regulatory regions of the murine Bax and human paraoxonase 1 (PON1) genes reportedly contain unique DRE-like sequences that respond to AhRs activated by some ligands but not others. Given the significant implications of this observation to understanding the diversity in AhR responses and that of other ligand-dependent nuclear receptors, a combination of DNA binding, nuclear translocation and gene expression analysis was used to investigate the molecular mechanisms underlying these ligand-selective responses. Although known AhR agonists stimulated AhR nuclear translocation, DRE binding and gene expression, the ligand-selective DRE-like DNA elements identified in the Bax and PON1 upstream regulatory regions failed to bind ligand-activated AhR or confer AhR-responsiveness upon a reporter gene. These results argue against the reported ligand-selectivity of AhR DNA binding and suggest DNA binding by ligand activated AhR involves DRE-containing DNA.

  7. Inotodiol inhabits proliferation and induces apoptosis through modulating expression of cyclinE, p27, bcl-2, and bax in human cervical cancer HeLa cells.

    Science.gov (United States)

    Zhao, Li-Wei; Zhong, Xiu-Hong; Yang, Shu-Yan; Zhang, Yi-Zhong; Yang, Ning-Jiang

    2014-01-01

    Inonotus obliquus is a medicinal mushroom that has been used as an effective agent to treat various diseases such as diabetes, tuberculosis and cancer. Inotodiol, an included triterpenoid shows significant anti-tumor effect. However, the mechanisms have not been well documented. In this study, we aimed to explore the effect of inotodiol on proliferation and apoptosis in human cervical cancer HeLa cells and investigated the underlying molecular mechanisms. HeLa cells were treated with different concentrations of inotodiol. The MTT assay was used to evaluate cell proliferating ability, flow cytometry (FCM) was employed for cell cycle analysis and cell apoptosis, while expression of cyclinE, p27, bcl-2 and bax was detected by immunocytochemistry. Proliferation of HeLa cells was inhibited by inotodiolin a dose-dependent manner at 24h (r=0.9999, pHeLa cells was detected after treatment and the apoptosis rate with the concentration and longer incubation time (r=1.0, pHeLa cells and induced apoptosis in vitro. The mechanisms may be related to promoting apoptosis through increasing the expression of bax and cutting bcl-2 and affecting the cell cycle by down-regulation the expression of cyclin E and up-regulation of p27. The results further indicate the potential value of inotodiol for treatment of human cervical cancer.

  8. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression.

    Science.gov (United States)

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC.

  9. USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

    Directory of Open Access Journals (Sweden)

    Aman Wang

    2017-05-01

    Full Text Available Resistance to platinum-based chemotherapy is one of the most important reasons for treatment failure in advanced non-small cell lung cancer, but the underlying mechanism is extremely complex and unclear. The present study aimed to investigate the correlation of ubiquitin-specific peptidase 22 (USP22 with acquired resistance to cisplatin in lung adenocarcinoma. In this study, we found that overexpression of USP22 could lead to cisplatin resistance in A549 cells. USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin- resistant A549/CDDP cell line. USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. In addition, USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition in vivo and vitro. In summary, our findings reveal the dual mechanism of USP22 involvement in cisplatin resistance that USP22 can regulate γH2AX-mediated DNA damage repair and Ku70/Bax-mediated apoptosis. USP22 is a potential target in cisplatin-resistant lung adenocarcinoma and should be considered in future therapeutic practice.

  10. Artemisinin induces caspase-8/9-mediated and Bax/Bak-independent apoptosis in human lung adenocarcinoma (ASTC-a-1) cells.

    Science.gov (United States)

    Xiao, Feng-Lian; Gao, Wei-Jie; Liu, Cheng-Yi; Wang, Xiao-Ping; Chen, Tong-Sheng

    2011-01-01

    Artemisinin (ARTE), an antimalarial phytochemical component from the sweet wormwood plant, has been shown a potential anticancer activity by inducing cell apoptosis. The aim of this report is to explore the mechanism of ARTE-induced human lung adenocarcinoma (ASTC-a-1) cell apoptosis. Cell counting kit (CCK-8) assay showed that ARTE induced cytotoxcity in a dose- and time-dependent manner. Confocal microscopy fluorescence imaging of cells stained with Hoechst 33258 and flow cytometry (FCM) analysis of cells stained with Annexin V-FITC/propidium iodide (PI) showed that ARTE induced reactive oxygen species (ROS)-dependent apoptosis. Confocal fluorescence resonance energy transfer (FRET) imaging of single living cells expressing SCAT3, SCAT9 or CFP-Bid-YFP and fluorometic substrate assay showed that ARTE induced the activation of caspase-3, -8 and -9. Moreover, inhibition of caspase-8 or -9 completely blocked ARTE-induced apoptosis which was only partially attenuated by caspase-3 inhibitor. Interestingly, silencing Bax and Bak by RNA interference (RNAi) did not attenuate ARTE-induced apoptosis. Collectively, ARTE induces caspase-dependent but Bax/Bak-independent apoptosis in ASTC-a-1 cells.

  11. Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

    Science.gov (United States)

    Chen, Ying-Jung; Liu, Wen-Hsin; Kao, Pei-Hsiu; Wang, Jeh-Jeng; Chang, Long-Sen

    2010-06-15

    CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation.

  12. D-pinitol promotes apoptosis in MCF-7 cells via induction of p53 and Bax and inhibition of Bcl-2 and NF-κB.

    Science.gov (United States)

    Rengarajan, Thamaraiselvan; Nandakumar, Natarajan; Rajendran, Peramaiyan; Haribabu, Lingaiah; Nishigaki, Ikuo; Balasubramanian, Maruthaiveeran Periyasamy

    2014-01-01

    Development of drugs from natural products has been undergoing a gradual evoluation. Many plant derived compounds have excellent therapeutic potential against various human ailments. They are important sources especially for anticancer agents. A number of promising new agents are in clinical development based on their selective molecular targets in the field of oncology. D-pinitol is a naturally occurring compound derived from soy which has significant pharmacological activitites. Therefore we selected D-pinitol in order to evaluate apoptotic potential in the MCF-7 cell line. Human breast cancer cells were treated with different concentrations of D-pinitol and cytotoxicity was measured by MTT and LDH assays. The mechanism of apoptosis was studied with reference to expression of p53, Bcl-2, Bax and NF-kB proteins. The results revealed that D-pinitol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner, while upregulating the expression of p53, Bax and down regulating Bcl-2 and NF-kB. Thus the results obtained in this study clearly vindicated that D-pinitol induces apotosis in MCF-7 cells through regulation of proteins of pro- and anti-apoptotic cascades.

  13. Amygdalin induces apoptosis through regulation of Bax and Bcl-2 expressions in human DU145 and LNCaP prostate cancer cells.

    Science.gov (United States)

    Chang, Hyun-Kyung; Shin, Mal-Soon; Yang, Hye-Young; Lee, Jin-Woo; Kim, Young-Sick; Lee, Myoung-Hwa; Kim, Jullia; Kim, Khae-Hawn; Kim, Chang-Ju

    2006-08-01

    Prostate cancer is one of the most common non-skin cancers in men. Amygdalin is one of the nitrilosides, natural cyanide-containing substances abundant in the seeds of plants of the prunasin family that have been used to treat cancers and relieve pain. In particular, D-amygdalin (D-mandelonitrile-beta-D-gentiobioside) is known to exhibit selective killing effect on cancer cells. Apoptosis, programmed cell death, is an important mechanism in cancer treatment. In the present study, we prepared the aqueous extract of the amygdalin from Armeniacae semen and investigated whether this extract induces apoptotic cell death in human DU145 and LNCaP prostate cancer cells. In the present results, DU145 and LNCaP cells treated with amygdalin exhibited several morphological characteristics of apoptosis. Treatment with amygdalin increased expression of Bax, a pro-apoptotic protein, decreased expression of Bcl-2, an anti-apoptotic protein, and increased caspase-3 enzyme activity in DU145 and LNCaP prostate cancer cells. Here, we have shown that amygdalin induces apoptotic cell death in human DU145 and LNCaP prostate cancer cells by caspase-3 activation through down-regulation of Bcl-2 and up-regulation of Bax. The present study reveals that amygdalin may offer a valuable option for the treatment of prostate cancers.

  14. The correlation between mRNA and protein expression of bax and bcl-xL follo wing fluid percussion brain injury in rats%大鼠液压脑损伤后bax/bcl-xL 的表达在mRNA和蛋白质水平的相关性

    Institute of Scientific and Technical Information of China (English)

    骆纯; 卢亦成; 朱诚; 江基尧; 张光霁

    2001-01-01

    目的:探讨脑创伤后bax/bcl-xL在mRNA和蛋白水平的变化规律及其与神经细胞凋亡发生、发展的关系。方法:在液压脑损伤模型中,应用逆转录聚合酶链反应、免疫组化分别检测大鼠脑创伤后不同时程bax和bcl-xL表达;采用凋亡原位末端标记、电镜超微结构、DNA凝胶电泳观察脑创伤后细胞凋亡的形态和生化特征。结果:伤后6 h,bcl-xL mRNA表达下调[伤侧半球为对侧的(67.42±7.5 4)%],bcl-xL蛋白水平下降[伤侧为对侧的(85.85±5.72)%]。伤后3 d,bcl-xL mRNA和 bcl-xL蛋白表达分别为对侧的(39.97±3.61)%和(57.50±6.21)%;bax mRNA和bax蛋白分别为对侧半球的(203.95±17.53)%和(189.02±7.23)%。伤后bax/bcl-xL比率升高比细胞凋亡提前出现,早期由于bcl-xL的表达下降,后期主要是由于bax的升高所致。结论:细胞凋亡及其调节基因的表达间具有一致性;脑创伤对bax和 bcl-xL 的调节发生在转录水平以前的某一环节。bax/bcl-xL平衡体系的维持或紊乱影响脑创伤后神经细胞生存或死亡。%Objective: To investigate the alterations of bcl-2 gene family in the rat brain and the molecular mechanism of neuronal apoptosis follow ing traumatic brain injury (TBI). Methods: Male Sprague-Dawley rats were subjected to lateral fluid percussion brain injury(FPBI) of moderate severity. bax and bcl-xL mRNA and protein expression was detected by RT-PCR an d immunohistochemistry. In addition to morphological evidence of apoptosis, TUNE L histochemistry was used to identify DNA fragmentation in situ under both l ight and electron microscope, whereas characteristic internucleosomal DN A fragm entation of apoptosis was demonstrated by DNA gel electrophoresis. Resul ts: bcl-xL mRNA and protein decreased in the ipsilateral hemisphere t o the impact site as early as 6 h post-injury[(67.42±7.54)% and (85.85±5.72)% r espectively]. The decrease in bcl-xL mRNA and protein

  15. 苦参碱对 HaCaT 细胞 Bcl -2/Bax 和Fas/FasL 的调控%Regulation of Bcl-2/Bax and Fas/FasL by matrine in HaCaT cells

    Institute of Scientific and Technical Information of China (English)

    牟宽厚; 周艳; 韩丹; 穆欣

    2014-01-01

    目的:明确苦参碱对 HaCaT 细胞 Bcl-2/ Bax 和 Fas/ FasL 表达的影响。方法:体外培养HaCaT 细胞,选择第二代细胞对数生长期 HaCaT 细胞作为研究对象,将细胞随机分为4组:苦参碱2 mg/ mL、10 mg/ mL 和50 mg/ mL 3组及对照组(加入相同体积的0.9%盐水),孵育48 h 后,MTT 法测定各浓度下细胞增殖,RT-PCR 检测 Bcl-2/ Bax 和 Fas/ FasL 的表达。结果:与对照组相比,当苦参碱浓度为2 mg/ mL 时,HaCaT 细胞增殖活性无明显变化;Bcl -2、Bax、Fas、FasL 表达也无明显变化( P>0.05)。当苦参碱浓度为10 mg/ mL 时 HaCaT 细胞增殖活性较对照组明显下降(P0.05)。结论:苦参碱能够调控上皮细胞致炎因子的表达,抑制细胞的增殖。%To determine the effect of matrine on Bcl-2/ Bax and Fas/ FasL in keratinocytes in vitro. Methods: Second generation cultured HaCaT cells (logarithmic phase cells) were selected and divided into 4 groups:3 matrine groups (2 mg/ mL, 10 mg/ mL and 50 mg/ mL were used in each group) and the control group (0.9% Natrii Chloride). After 48-hour culture, the proliferation of HaCaT were detected by MTT and the levels of Bcl-2/ Bax and Fas/ FasL were measured by RT-PCR. Results: The viability of HaCaT cells was similar in 2 mg/ mL matrine group and control group (P>0.05). In 10 mg/ mL matrine group the proliferation of the cells was significantly decreased (P<0.001) and the Bcl-2 expression was remarkably reduced (P<0.001), while the expression of Bax, Fas and FasL was significantly increased (P<0.01 and P<0.05, respectively). When the concentration of matrine was increased to 50 mL, the viability and the expression of Bcl-2, Bax, Fas and FasL was similar to the results when 10 mL matrine was used. Conclusion: Matrine can inhibit HaCaT cells proliferation (at 10 mg/ mL or more) and may adjust expression of Bcl-2/ Bax and Fas/FasL in HaCaT cells.

  16. DHT inhibits the Aβ25-35-induced apoptosis by regulation of seladin-1, survivin, XIAP, bax, and bcl-xl expression through a rapid PI3-K/Akt signaling in C6 glial cell lines.

    Science.gov (United States)

    Bing, Lelin; Wu, Junfeng; Zhang, Jianfeng; Chen, Yinghui; Hong, Zhen; Zu, Hengbing

    2015-01-01

    Previous evidences indicate that androgen is neuroprotective in the brain. However, the underling mechanisms remain to be fully elucidated. Moreover, it is controversial whether dihydrotestosterone (DHT) modulates the expression of apoptosis-related effectors, such as survivin, XIAP, bax, and bcl-xl proteins mediated by the PI3-K/Akt pathway, which contributes to androgen neuroprotection. In this study using a C6 glial cell model, apoptotic cells were detected by flow cytometry. Akt, seladin-1, survivin, XIAP, bcl-xl, and bax protein expression is investigated by Western blot. After amyloid β-protein fragment (Aβ25-35) treatment, apoptotic cells at early (annexin V+, PI-) and late (annexin V+, PI+) stages were significantly increased. Apoptosis at early and late was obviously inhibited in the presence of DHT. The effect of DHT was markedly blocked by PI3-K inhibitor LY294002.To elicit the mechanism of DHT protection, the expression of seladin-1, survivin, XIAP, bax, and bcl-xl protein was determined in C6 cells treated with Aβ25-35, DHT, or LY294002. Aβ25-35 significantly downregulated the expression of seladin-1, survivin, XIAP, bcl-xl protein and upregulated the expression of bax protein. DHT significantly inhibited the expression of bax, seladin-1, survivin, XIAP, and bcl-xl protein induced by Aβ25-35. Further, we found the effect of DHT was significantly inhibited by LY294002. Collectively, in a C6 glial cell model, we firstly found that DHT inhibits Aβ25-35-induced apoptosis by a rapid nongenic PI-3K/Akt activation as well as regulation of seladin-1, survivin, XIAP, bcl-xl, and bax proteins.

  17. Pharmacokinetics, efficacy and safety of BAX326, a novel recombinant factor IX: a prospective, controlled, multicentre phase I/III study in previously treated patients with severe (FIX level <1%) or moderately severe (FIX level ≤2%) haemophilia B.

    Science.gov (United States)

    Windyga, J; Lissitchkov, T; Stasyshyn, O; Mamonov, V; Rusen, L; Lamas, J L; Oh, M-S; Chapman, M; Fritsch, S; Pavlova, B G; Wong, W-Y; Abbuehl, B E

    2014-01-01

    BAX326 is a recombinant factor IX (rFIX; nonacog gamma) manufactured without the addition of any materials of human or animal origin, and with two viral inactivation steps (solvent/detergent treatment and 15 nm nanofiltration). The aim of this prospective trial was to investigate the pharmacokinetics, haemostatic efficacy and safety of BAX326 in previously treated patients aged 12-65 years with severe or moderately severe haemophilia B. BAX326 was safe and well tolerated in all 73 treated subjects; adverse events considered related to treatment (2.7% incidence, all non-serious) were transient and mild, and no hypersensitivity reactions, inhibitor formation or thrombotic events were observed. Pharmacokinetic (PK) equivalence (n = 28) between BAX326 and a licensed rFIX was confirmed in terms of the ratio of geometric mean AUC(0-72) h per dose. Twice-weekly prophylaxis [mean duration 6.2 (±0.7) months; 1.8 (±0.1) infusions per week, 49.5 (±4.8) IU kg(-1) per infusion] was effective in preventing bleeding episodes, with a significantly lower (79%, P < 0.001) annualized bleed rate (4.2) compared to an on-demand treatment in a historical control group (20.0); 24 of 56 subjects on prophylaxis (43%) did not bleed throughout the study observation period. Of 249 total acute bleeds, 211 (84.7%) were controlled with one to two infusions of BAX326. Haemostatic efficacy at resolution of bleed was rated excellent or good in 96.0% of all treated bleeding episodes. The results of this study indicate that BAX326 is safe and efficacious in treating bleeds and routine prophylaxis in patients aged 12 years and older with haemophilia B.

  18. Effects of acupoint versus non-acupoint electroacupuncture on cerebral cortical neuronal Bcl-2,Bax and caspase-3 expression in a rat model of focal cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Jun Wang; Junming Fan; Yongshu Dong; Xia Huang; Hongxia Zhang

    2008-01-01

    BACKGROUND: Several studies have demonstrated that electroacupuncture by acupoint selection can inhibit cerebral cortical neuronal apoptosis following cerebral ischemia/reperfusion.OBJECTIVE: To validate the effects of electroacupuncture by acupoint selection on the expression level of cortical neuronal anti-apoptotic Bcl-2 protein and the apoptotic executive protein, caspase-3, in rat models of focal cerebral ischemia/reperfusion.DESIGN, TIME AND SETTING: This randomized grouping, neural cell and molecular biology animal experiment was performed at the Laboratory of Pharmacology of Traditional Chinese Medicine and the Laboratory Animal Center of Henan Institute of Traditional Chinese Medicine between November 2006 and May 2007.MATERIALS: Atotal of 40 healthy male adult Sprague-Dawley rats were randomly and evenly divided into four groups: sham-operated, model, electroacupuncture and non-acupoint control. G6895 electro-acupuncture instruments were purchased from Shanghai Huayi Instrument Factory, China. Caspase-3, Bcl-2 and Bax kits were provided by Wuhan Boster Bioengineering Co., Ltd., China.METHODS: Middle cerebral artery occlusion was induced in the model, electroacupuncture and non-acupoint groups. In the electroacupuncture group, the acupoints Jianyu (LI15), Waiguan (SJ5), Biguan (ST31), and Zusanli (ST36) were given electroacupuncture. In the non-acupoint control group, at each time point (immediately after ischemia and after reperfusion, or 2 hours after reperfusion), electroacupuncture was performed at the midpoints of Tianquan (PC2)-Quze (PC 3) line, Quze (PC 3)-Ximen (PC4) line, Zuwuli (LRlO)-Yinbao (LRg) line, and Xiguan (LR7)-Zhongdu (LR6) line. Electroacupuncture parameters were set with a continuous wave with a frequency of 10 Hz, wave width 0.6 ms, voltage 1.5-3.0 V, and a duration of 10 minutes. The sham-operated and model groups received only animal fixation without electroacupuncture procedure.MAIN OUTCOME MEASURES: Five rats were selected from

  19. Identification of apoptosis-related genes Bcl2 and Bax from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to waterborne and dietborne zinc exposure.

    Science.gov (United States)

    Li, Dan-Dan; Luo, Zhi; Chen, Guang-Hui; Song, Yu-Feng; Wei, Chuan-Chuan; Pan, Ya-Xiong

    2017-10-30

    Apoptosis plays a key role in the physiology of multicellular organisms, and has been well studied in mammals, but not in teleosts. Zinc (Zn) has been shown to be an important regulator of apoptosis and apoptosis involves in the regulation of lipid metabolism. Moreover, our recent study indicated that waterborne and dietborne Zn exposure differently influenced lipid metabolism in Pelteobagrus fulvidraco, but further mechanism remained unknown. The hypothesis of the present study is that apoptosis mediated the Zn-induced changes of lipid metabolism of P. fulvidraco subjected to different exposure pathways. To this end, we cloned full-length cDNA sequences of Bcl2 and three Bax subtypes involved in apoptosis in P. fulvidraco, explored their mRNA expressions in responses to different Zn exposure pathways. Bcl2 and three Bax subtypes shared similar domain structure as typical pro- and anti-apoptotic Bcl2 family members. Their mRNAs were widely expressed among various tissues, but at variable levels. Waterborne Zn exposure down-regulated mRNA levels of Baxg and ratios of Baxa/Bcl2, and Baxg/Bcl2, but showed no significant effects on mRNA abundances of Bcl2, Baxa and Baxb, and the ratio of Baxb/Bcl2. In contrast, dietborne Zn exposure up-regulated mRNA levels of Bcl2, Baxa, Baxb and Baxg, but reduced the ratios of Baxa/Bcl2, Baxb/Bcl2, and Baxg/Bcl2. Considering their important roles of these genes in apoptosis induced by Zn, apoptosis may mediate the Zn-induced changes of hepatic lipid metabolism of Pelteobagrus fulvidraco under different Zn exposure pathways. For the first time, we characterized the full-length cDNA sequences of Bcl2 and three Bax subtypes, determined their expression profiles and transcriptional responses to different Zn exposure pathways, which would contribute to our understanding of the molecular basis of apoptosis, and also provide new insights into physiological responses to different Zn exposure pathways. Copyright © 2017 Elsevier B.V. All

  20. Inhibition of nuclear factor-kappaB or Bax prevents endoplasmic reticulum stress- but not nitric oxide-mediated apoptosis in INS-1E cells

    DEFF Research Database (Denmark)

    Tonnesen, Morten F; Grunnet, Lars G; Friberg, Josefine

    2009-01-01

    and alternative splicing of the transcription factor Xbp-1 were exclusively activated by TG. TG exposure caused NFkappaB activation, as assessed by IkappaB degradation and NFkappaB DNA binding. Inhibition of NFkappaB or the Bcl-2 family member Bax pathways protected beta-cells against TG- but not SNAP....... Exposure of INS-1E cells to TG or SNAP caused caspase-3 cleavage and apoptosis. Both TG and SNAP induced activation of the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP). However, other classical ER stress-induced markers such as up-regulation of ER chaperone Bip......-induced beta-cell death. These data suggest that NO generation and direct SERCA2 inhibition cause two quantitative and qualitative different forms of ER stress. In contrast to NO, direct ER stress induced by SERCA inhibition causes activation of ER stress signaling pathways and elicit proapoptotic signaling...

  1. AC Conductivity and Dielectric Relaxation Behavior of Sol-gel BaxSr1-xTiO3 Thin Films

    Institute of Scientific and Technical Information of China (English)

    Ala'eddin A. Saif; P. Poopalan

    2011-01-01

    BaxSr1-xTiO3 sol-gel thin films with x--0.5, 0.7 and 0.8 have been fabricated as AI/BST/Pt capacitor. The AC conductivity and dielectric properties over a frequency rang of 10 Hz and I MHz have been studied in order to explore the ion dynamics and relaxation mechanisms in the films. The frequency dependent conductivity plots show three regions of conduction processes. Dielectric results show that ε' at low frequencies increases as Sr content decreases, whereas at high frequencies, it shows opposite variation, which is attributed to the dipole dynamics. The electric modulus plots reveal the relaxation peaks which are not observed in the ε" plots and the contribution of the grains, grain boundaries and electrode to the relaxation mechanisms.

  2. Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Zhendong, E-mail: zdyu@hotmail.com [Department of Clinical laboratory, Peking University Shenzhen Hospital, Guangdong (China); Wang, Hao [Department of pathology, The Chinese University of Hong Kong, Hong Kong (China); Zhang, Libin; Tang, Aifa; Zhai, Qinna; Wen, Jianxiang; Yao, Li [Department of Clinical laboratory, Peking University Shenzhen Hospital, Guangdong (China); Li, Pengfei, E-mail: lipengfei@cuhk.edu.hk [Department of pathology, The Chinese University of Hong Kong, Hong Kong (China)

    2009-09-04

    CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.

  3. Dielectric dispersion of BaxSr1-xTiO3 thin film with parallel-plate and coplanar interdigital electrodes

    Science.gov (United States)

    Zhang, Xiao-Yu; Song, Qing; Xu, Feng; Sheng, Su; Wang, Peng; Ong, C. K.

    2009-03-01

    Ferroelectric BaxSr1-xTiO3 (BST) thin films with x = 0.25 and 0.5 were grown by pulsed laser deposition on single crystal LaAlO3 and Pt/Ti/SiO2/Si substrates, respectively. Capacitors were then fabricated from the BST thin films based on coplanar interdigital electrodes (CIEs) and parallel-plate electrodes (PPEs). The dielectric properties of the BST film with CIE and PPE were investigated and compared over a wide frequency range from 100 Hz to 10 GHz. The dielectric dispersion in PPE configuration, caused by the interfacial polarization in film/electrode interfaces, exhibited a strong dependence on frequency. However, the permittivity ɛCIE in CIE configuration shows a gentle variation with the frequency indicating interfacial polarization substantially suppressed. The influence upon the dielectric properties of the columnar BST grains due to the use of different forms of electrodes was discussed.

  4. Evidence of an anisotropic charge-excitation gap in stripe-ordered La2-xBaxCuO4 with x=1/8

    Science.gov (United States)

    Homes, C. C.; Dordevic, S. V.; Gu, G. D.; Li, Q.; Valla, T.; Tranquada, J. M.

    2006-03-01

    The ab-plane optical properties of a cleaved single crystal of La2-xBaxCuO4 for x=1/8 (Tc˜2.4 K) have been measured over a wide frequency and temperature range. The low-frequency conductivity is Drude-like and shows a metallic response with decreasing temperature. However, below ˜60 K, corresponding to the onset of charge-stripe order, there is a rapid loss of spectral weight below about 40 meV, resulting in a major reduction in the number of free carriers. This suggests a partial gapping of the Fermi surface. Surprisingly, the sample is still metallic and becomes a superconductor at low temperature. This material is a striking example of how charge and spin stripe order, metallic behavior and superconductivity can coexist.

  5. Diffuse phase transition and high-temperature dielectric relaxation study on (Bi0.5Na0.5)1-xBaxTiO3 ceramics

    Science.gov (United States)

    Chen, Feng; Liu, Qiu-Xiang; Tang, Xin-Gui; Jiang, Yan-Ping; Yue, Jing-Long; Li, Jin-Kai

    2016-09-01

    Lead free (Bi0.5Na0.5)1-xBaxTiO3 (x=0.6, 0.7, 0.8, 0.9) ferroelectric ceramics were synthesized by the traditional solid state reaction method. Sintering was carried out at 1200 °C for 2 h in air atmosphere. The structural, microstructure and Ferroelectric of ceramics were investigated. In dielectric studies, a diffuse phase transition was exhibited and a dielectric relaxation behavious was observed at high temperature range. Impedance analysis characterized grain and grain boundaries resistivities of the ceramics and calculated activation energy and the activation energy for conduction. Polaron theory indicates that the relaxation of the samples at high temperatures was associated with the hopping ions caused by oxygen vacancies.

  6. Ziyuglycoside II-induced apoptosis in human gastric carcinoma BGC-823 cells by regulating Bax/Bcl-2 expression and activating caspase-3 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, A.K. [Department of General Surgery, Nanjing Medical University, Affiliated Hangzhou Hospital, Hangzhou (China); Zhou, H.; Xia, J.Z. [Department of General Surgery, Nanjing Medical University, Affiliated Wuxi Second Hospital, Wuxi (China); Jin, H.C. [Department of General Surgery, Nanjing Medical University, Affiliated Hangzhou Hospital, Hangzhou (China); Wang, K. [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu Province (China); Yan, J.; Zuo, J.B. [Department of General Surgery, Nanjing Medical University, Affiliated Wuxi Second Hospital, Wuxi (China); Zhu, X. [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu Province (China); Shan, T. [Department of General Surgery, Nanjing Medical University, Affiliated Wuxi Second Hospital, Wuxi (China)

    2013-08-13

    Ziyuglycoside II is an active compound of Sanguisorba officinalis L. that has anti-inflammation, antioxidation, antibiosis, and homeostasis properties. We report here on the anticancer effect of ziyuglycoside II on human gastric carcinoma BGC-823 cells. We investigated the effects of ziyuglycoside II on cell growth, cell cycle, and cell apoptosis of this cell line. Our results revealed that ziyuglycoside II could inhibit the proliferation of BGC-823 cells by inducing apoptosis but not cell cycle arrest, which was associated with regulation of Bax/Bcl-2 expression, and activation of the caspase-3 pathway. Our study is the first to report the antitumor potential of ziyuglycoside II in BGC-823 gastric cancer cells. Ziyuglycoside II may become a potential therapeutic agent against gastric cancer in the future.

  7. On the Sr1−xBaxFeO2F Oxyfluoride Perovskites: Structure and Magnetism from Neutron Diffraction and Mössbauer Spectroscopy

    Directory of Open Access Journals (Sweden)

    Crisanto A. García-Ramos

    2016-11-01

    Full Text Available Four oxyfluorides of the title series (x = 0.00, 0.25, 0.50, 0.75 have been stabilized by topotactic treatment of perovskite precursors Sr1−xBaxFeO3−δ prepared by soft-chemistry procedures, yielding reactive materials that can easily incorporate a substantial amount of F atoms at moderate temperatures, thus avoiding the stabilization of competitive SrF2 and BaF2 parasitic phases. XRD and Neutron Powder Diffraction (NPD measurements assess the phase purity and yield distinct features concerning the unit cell parameters’ variation, the Sr and Ba distribution, the stoichiometry of the anionic sublattice and the anisotropic displacement factors for O and F atoms. The four oxyfluorides are confirmed to be cubic in all of the compositional range, the unit cell parameters displaying Vergard’s law. All of the samples are magnetically ordered above room temperature; the magnetic structure is always G-type antiferromagnetic, as shown from NPD data. The ordered magnetic moments are substantially high, around 3.5 μB, even at room temperature (RT. Temperature-dependent Mössbauer data allow identifying Fe3+ in all of the samples, thus confirming the Sr1−xBaxFeO2F stoichiometry. The fit of the magnetic hyperfine field vs. temperature curve yields magnetic ordering TN temperatures between 740 K (x = 0.00 and 683 K (x = 0.75. These temperatures are substantially higher than those reported before for some of the samples, assessing for stronger Fe-Fe superexchange interactions for these specimens prepared by fluorination of citrate precursors in mild conditions.

  8. Magnetic and thermoelectric properties of the ternary pseudo-hollandite BaxCr5Se8 (0.5 solid solution.

    Science.gov (United States)

    Lefèvre, Robin; Berthebaud, David; Bux, Sabah; Hébert, Sylvie; Gascoin, Franck

    2016-07-26

    The structure of Ba0.5Cr5Se8 has been recently resolved, and its thermoelectric and magnetic properties have been studied. A ZT of 0.12 was found at around 800 K. Here, we report a study on the pseudo-hollandite BaxCr5Se8 solid-solution with 0.5 ≤ x ≤ 0.55 and its thermoelectric and magnetic properties. There is no significant impact either on the cell parameters depending on the cation content or on the magnetic properties. However, thermoelectric properties are radically changed depending on x content. While the low thermal conductivity, around 0.8 W m(-1) K(-1), remains similar for all samples, a respective increase and decrease of the resistivity and the Seebeck coefficient are observed with increasing Ba content. The maximum Seebeck coefficient is found with Ba0.5Cr5Se8 at around 635 K with 315 μV K(-1), and the Seebeck coefficient then decreases and is correlated with an activation of minority charge carriers confirmed by Hall measurements. A similar but steeper behavior is observed for the Ba0.55Cr5Se8 temperature dependence plot at around 573 K. Finally, the best thermoelectric performances are found using the lowest content of Ba, unlike when x tends to 0.55, ZT approaches a tenth of the initial best value. BaxCr5Se8 compounds are antiferromagnetic with TN = 58 K. A large peak in thermal conductivity is observed around the antiferromagnetic transition for all stoichiometry.

  9. Adsorption of xylene para- and meta- isomers in NaX and BaX zeolites. Study of properties-structure relations; Adsorption des isomeres para- et meta- du xylene dans les zeolithes NaX et BaX. Etude des relations proprietes-structure

    Energy Technology Data Exchange (ETDEWEB)

    Descours, A.

    1997-02-14

    The separation of para-xylene from C8 aromatics is performed industrially bu adsorption process on zeolitic molecular sieves. The sorption properties of these zeolites are strongly linked to their structure, and their comprehension require an accurate knowledge of the interactions between sorbate molecules and zeolitic structure. The aim of this work is to characterise from a structural point of view the adsorption of para- and meta-xylenes in BaX and NaX zeolites. The former is selective for para-xylene, and the latter has not selective properties for para- and meta-isomers of xylene. For each zeolite, the adsorption of pure para-xylene and meta-xylene or a mixture of the two isomers, is investigated as a function of coverage. Powder neutron diffraction is used to determine the crystalline structure of these zeolites and the different crystallographic adsorption sites of the molecules. The influence of coverage on sorbate-sorbent and sorbate-sorbate interactions is investigated. Infrared spectroscopy allows to determine the chemical environment of the sorbate molecules at low coverage or when the coverage increases, and is particularly effective for the study of the binary mixture of xylenes. This study is performed by sorbing a mixture of xylene isomers, or by sorbing these isomers successively. Infrared studies and crystallographic analysis are compared in order to get a consistent description of adsorption mechanism of xylene isomers for both zeolites as a function of coverage. The role of coverage, of cation type, an the presence of the two xylene isomers is the super-cages is essential. For both zeolites, the increase of coverage actually leads to steric hindrances between sorbed molecules and molecular rearrangements. These reorganizations are connected to the cationic distribution of NaX and BaX zeolites. The sorbed molecules are connected to the cationic distribution of NaX and BaX zeolites. The sorbed molecules are particularly confined in BaX zeolite

  10. 细胞凋亡相关基因Bcl-2及Bax在骨肉瘤中的表达与自下而上质量的关系%Expression of apoptosis related gene Bcl 2 and Bax in osteosarcoma and their relationship with the prognosis

    Institute of Scientific and Technical Information of China (English)

    黄鲁豫; 刘建; 王臻; 吕荣

    2002-01-01

    Objective Apoptosis related gene Bcl 2 and Bax in osteosarcoma patients with different clinical appearance were being studied to analyze the prognosis of the patients. Method The cases were divided into two different groups according to the results of the follow up.33 cases in high risk group and 18 cases in low risk group. Expression of Bcl 2 and Bax were immunohistochemically stained by ABC method. Result Positive expression rate of Bcl 2 was 61% in high risk group (20/23) and 33% in low risk group (1/8). Positive expression of Bax was 22% in high risk group (6/27) and 67% in low risk group(12/18).Conclusion Expression of Bcl 2 and Bax was related to the prognosis of osteosarcoma. Positively expressed Bcl 2 in osteosarcoma cells may indicate bad prognosis. If Bax is highly expressed in osteosarcoma cells, this may indicated a good prognosis.

  11. Sevoflurane post-conditioning protects primary rat cortical neurons against oxygen-glucose deprivation/resuscitation via down-regulation in mitochondrial apoptosis axis of Bid, Bim, Puma-Bax and Bak mediated by Erk1/2.

    Science.gov (United States)

    Zhang, Li-Min; Zhao, Xiao-Chun; Sun, Wen-Bo; Li, Rui; Jiang, Xiao-Jing

    2015-10-15

    Temporal post-conditioning helps provide neuroprotection against brain injury secondary to ischemia-reperfusion and is considered an effective intervention, but the exact mechanism of sevoflurane post-conditioning is unclear. The essential axis involves activator Bid, Bim, Puma (BH3s), Bax, and Bak; activates the mitochondrial death program; and might be involved in a cell death signal. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in cell growth and proliferation. We hypothesized that sevoflurane post-conditioning might inhibit Bid, Bim, Puma, Bax, and Bak expression and is activated by phosphor-Erk1/2 to decrease neuronal death. To test this hypothesis, we exposed primary cortical neuron cultures to oxygen-glucose deprivation for 1h, along with resuscitation for 24h (OGD/R). MTT assays, propidium iodide uptake (PI), JC-1 fluorescence, and Western blot indicated the following: decreased cell viability (PPuma, Bax, and Bak expression with OGD/R exposure. Inhibition of Erk1/2 phosphorylation could attenuate sevoflurane post-conditioning that mediated an increase in neuronal viability and mitochondrial membrane potential, as well as a decrease in cell death and Bid, Bim, Puma, Bax, and Bak expression after OGD/R treatment. The results demonstrated that sevoflurane post-conditioning caused a marked decrease in cortical neuronal death secondary to OGD/R exposure through the downregulation of the mitochondrial apoptosis axis involving Bid, Bim, Puma, Bax, and Bak that was mediated by the phosphorylation/activation of Erk1/2.

  12. The Effect of Atorvastatin and Nicorandil on Bcl-2 and Bax in Isolated and Reperfusion Rabbit Hearts%阿托伐他汀和尼可地尔后处理对兔心肌缺血再灌注心功能及 Bcl-2、Bax 的影响

    Institute of Scientific and Technical Information of China (English)

    李艳芳; 费丽萍; 常志红

    2016-01-01

    Objective:To observe the heart function and Bcl-2,Bax of Atorvastat in and Nicorandil on ischemia and reperfusion induced injury in rabbit heart.Methods:50 rabbits were randomly divided into 5 groups,and the myocardial ischemia-reperfusion were generated by two sutures and two knots,then administrated in accordance with the experimental design,monitoring ECG and left ventricular pressure,testing CK,cTnI in serum,observing the changes of myocardial structure,testing Bcl-2 and Bax by RT-PCR.Results:The LVSP and ± dp/dt max levels of group C-E were significantly higher than that of group B(P <0.05).On the contrary,the LVEDP was lower than that of group B(P <0.01).The CK and cTnI levels of group C-E were lower than B(P <0.01).The injury of group B was worse than C-E of the cellular structure under EM.The BCL-2 and Bax mRNA levels of group B-E were significantly higher than that of group A(P <0.05 ),and BCL-2 C-E higher than B,The contrary,however,the bax was lower than that of group B.Conclusion:Myocardial isehemic postconditioning of Atorvastatin and Nicorandil induced cardio protection on ischemia reperfusion injury in rabbits.They can improve myocardial systolic and diastolic function,maybe through reducing cell apoptosis.%目的::建立缺血再灌注模型,观察阿托伐他汀和尼可地尔后处理对兔心肌缺血再灌注心功能及 Bcl-2、Bax 的影响。方法:50只大白兔随机分成5组[假手术组(A)、对照组(B)、阿托伐他汀组(C)、尼可地尔组(D)、联合后处理组(E)],采用“二线二结法”建立缺血再灌注模型。各组按照实验设计方案给药,记录心电图、血流动力学指标。采集血液标本,试剂盒检测 CK、cTnI。电镜下观察各组心肌细胞超微结构变化。双色实时荧光定量 PCR 检测 Bcl-2、Bax。结果:(1)再灌注2 h,C~E 组 LVSP 高于 B 组(P <0.05),±dp/dt max 高于 B 组(P <0.01),而 LVEDP 低于 B 组(P <0.01)。其中,C、D 组无显著性差异, E 组 LVSP

  13. 雌激素对大鼠胸腺细胞凋亡及Bcl-2、Bax表达的影响%Effects of estrogen on apoptosis and expression of Bcl-2 and Bax in rat thymus

    Institute of Scientific and Technical Information of China (English)

    李雅娜; 孙研; 崔春红; 殷彦君

    2011-01-01

    目的:探讨苯甲酸雌二醇对大鼠胸腺Bcl-2和Bax表达及细胞凋亡的影响及其机制.方法:雌性大鼠行卵巢切除术,给予苯甲酸雌二醇后,观察胸腺指数的变化,Hochest33342荧光染色及透射电镜标本观察胸腺细胞凋亡情况,免疫组织化学检测胸腺组织中Bcl-2和Bax的表达情况,原位杂交技术检测Bcl-2、Bax m RNA的表达情况.结果:双侧卵巢切除组大鼠胸腺指数较假手术组增加,双侧卵巢切除+雌激素组大鼠胸腺指数较双侧卵巢切除组减小;假手术组和双侧卵巢切除组大鼠胸腺组织中以正常胸腺细胞为主,偶见凋亡细胞或凋亡小体,双侧卵巢切除+雌激素组可见较多凋亡细胞和凋亡小体;双侧卵巢切除+雌激素组大鼠胸腺组织中Bcl-2表达较双侧卵巢切除组和假手术组增高明显降低,而Bax表达呈现相反趋势;Bcl-2 mRNA、Bax mRNA的表达与Bcl-2、Bax的表达呈一致性.结论:雌激素可以降低大鼠胸腺指数,抑制胸腺组织中Bcl-2的表达,促进Bax的表达,从而诱导大鼠胸腺细胞凋亡,促进雌性大鼠胸腺退化.%Objective-. To explore the effects of estrogen on the apoptosis and the expression of Bcl-2 and Bax in rat thymus. Methods-. The rats performed with ovariectomy were injected with estradiol benzoate. Thymus was obtained 7 days after the injection. Thymic indexes were measured. Apoptosis of the thymus was detected after Hochest 33342 staining and examined under an electron microscope. The expression of Bcl-2 and Bax in the thymus was detected with a immunohistochemical method. The expression of Bcl-2 mRNA and Bax mRNA in the thymus was detected by in situ hybridization. The test was taken in statistical treatment. Results: The thymus quality index in ovariectomy group was higher than that in the model control group. The thymus quality index of rats injected with estradiol benzoate was reduced respectively. Apoptotic cells and apoptotic bodies were found in the

  14. Study of Ganoderma lucidum spores on pentylenetetrazol activation of hippocampal neurons bax expression%灵芝孢子粉对戊四氮活化海马神经细胞bax表达的研究

    Institute of Scientific and Technical Information of China (English)

    张金波; 王淑秋; 张淑红; 金岳雷; 朱金玲; 刘爽

    2012-01-01

    目的 本实验研究灵芝孢子粉对戊四氮活化大鼠海马神经细胞bax表达变化的影响,进一步探讨灵芝孢子粉的作用机制和癫痫与海马神经细胞凋亡调控基因之间的关系.方法 通过制备癫痫模型和RT-PCR检测正常对照组、癫痫模型组和灵芝孢子粉用药组bax的表达.结果 癫痫模型组和灵芝孢子粉用药组bax的表达较正常对照组(0.17±0.04)均升高;其中癫痫模型组(0.66±0.09)bax的表达水平与对照组(0.17±0.04)比较明显升高(P<0.01),灵芝孢子粉用药组(0.47±0.1)bax的表达水平与癫痫模型组比较明显降低(P<0.01),差异有统计学意义.结论 本研究结果证实,在PTZ点燃癫痫后,模型组和治疗组促凋亡基因bax表达较正常对照组显著升高,表明bax基因在细胞凋亡的调控过程中起到促进作用,而给予灵芝孢子粉治疗后,bax的表达与模型组比较显著降低,提示灵芝孢子粉有效成份能充分作用于脑组织,可以调控bax的表达,借以发挥抗凋亡的神经保护作用.%Objective; This study is Ganoderma lucidum spores on pentylenetetrazol activation of rat hippocampal neurons bax expression changes to further explore the relationship between the action mechanism of the Ganoderma lucidum spores and epilepsy with apoptosis regulatory genes of hippocampal neural. Methods; Through the preparation of the epileptic model and RT — PCR to detect of bax expression in the normal control group, epilepsy model group and Ganoderma spore powder medication group. Results;/ Bax expression in epilepsy model group and Ganoderma lucidum spores medication group were higher than in the normal control group (0. 17 ± 0. 04) ; including bax expression level in epilepsy model group (0. 66 ± 0. 09) was significantly higher than in the control group (0. 17 ± 0.04) (P<0. 01) , bax expression level in Ganoderma spore powder medication group (0.47 ± 0. 1 ) were significantly lower than in epilepsy model group

  15. Ginkgo biloba extract mitigates liver fibrosis and apoptosis by regulating p38 MAPK, NF-κB/IκBα, and Bcl-2/Bax signaling

    Directory of Open Access Journals (Sweden)

    Wang YY

    2015-12-01

    Full Text Available Yuanyuan Wang, Rong Wang, Yujie Wang, Ruqin Peng, Yan Wu, Yongfang Yuan Department of Pharmacy, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China Background: Liver fibrosis is the consequence of diverse liver injuries and can eventually develop into liver cirrhosis. Ginkgo biloba extract (GBE is an extract from dried ginkgo leaves that has many pharmacological effects because of its various ingredients and has been shown to be hepatoprotective. Purpose and methods: Aimed to investigate the underlying protective mechanisms of GBE on carbon tetrachloride (CCl4-induced liver fibrosis in rats. Male Sprague Dawley rats were randomly divided into four groups: control group (C, model group (M, low-dose group (L, and high-dose group (H. Liver fibrosis was induced by CCl4 groups M, L, and H: group C was administered saline. In addition, GBE at different doses was used to treat groups L and H. Results: The results of hematoxylin and eosin staining, Masson’s trichrome staining, a liver function index, and a liver fibrosis index showed that GBE application noticeably mitigated fibrosis and improved the function of the liver. The western blotting and immunohistochemistry analyses indicated that GBE reduced liver fibrosis not only by inhibiting p38 MAPK and NF-κBp65 via inhibition of IκBα degradation but also by inhibiting hepatocyte apoptosis via downregulation of Bax, upregulation of Bcl-2, and subsequent inhibition of caspase-3 activation. Inflammation-associated factors and hepatic stellate cell (HSC-activation markers further demonstrated that GBE could effectively inhibit HSC activation and inflammation as a result of its regulation of p38 MAPK and nuclear factor-kappa B/IκBα signaling. Conclusion: Our findings indicated a novel role for GBE in the treatment of liver fibrosis. The potential mechanisms may be associated with the following signaling pathways: 1 the p38 MAPK

  16. Gastroprotective activity of Annona muricata leaves against ethanol-induced gastric injury in rats via Hsp70/Bax involvement

    Directory of Open Access Journals (Sweden)

    Moghadamtousi SZ

    2014-10-01

    downregulation of Bax and upregulation of Hsp70 proteins after pretreatment. Collectively, the present results suggest that EEAM has a promising antiulcer potential, which could be attributed to its suppressive effect against oxidative damage and preservative effect toward gastric wall mucus. Keywords: Annona muricata, annonaceae, gastric injury, antioxidants, Hsp70/Bax

  17. Effects of the Bax channel on cantharidin induced apoptosis of Spodoptera frugiperda Sf9 cells%Bax通道在斑蝥素诱导草地贪夜蛾Sf9细胞凋亡中的作用

    Institute of Scientific and Technical Information of China (English)

    崔高峰; 张鹏飞; 郝少东; 丁凯; 王进忠; 张志勇

    2015-01-01

    Objectives] To investigate the role of the Bax channel on cantharidin-induced cell apoptosis in lepidopteran insects [Methods] Bax channel blocker was used to determine the roles of mitochondrial membrane potential, succinate dehydrogenase activity in mitochondria, and cell morphology, on apoptosis of Sf9 cells derived from fall the armyworm, Spodoptera frugiperda (J.E. Smith) exposed to cantharidin stress. [Results] The results show that Bax channel blocker can delay the decrease of mitochondrial membrane potential and reduce changes in cell morphology, but fail to stop the decline of succinate dehydrogenase activity, in Sf9 cells after treatment with cantharidin. [Conclusion] Bax related channels play a role in cantharidin-induced changes in mitochondrial membrane potential and destruction of cell morphology. However, the Bax channel does not appear to play a role in the suppression of enzyme activity in mitochondria after cantharidin treatment.%【目的】为明确Bax通道在斑蝥素诱导鳞翅目昆虫细胞凋亡过程中的作用。【方法】本文利用Bax通道抑制法测定了斑蝥素诱导鳞翅目昆虫草地贪夜蛾Spodoptera frugiperda(J. E. Smith)细胞系Sf9细胞凋亡过程中线粒体膜电位、线粒体琥珀酸脱氢酶活性及细胞形态等方面的影响。【结果】 Bax 通道被抑制后,斑蝥素诱导造成的Sf9细胞线粒体膜电位的降低时间延迟,细胞形变率下降,但琥珀酸脱氢酶活性的下降未受影响。【结论】 Bax通道参与了斑蝥素引起的Sf9细胞线粒体膜电位改变和细胞形态变化,而与抑制线粒体有关能量代谢的酶无直接关系。

  18. Study on the in-plane electrical resistivity and thermoelectric power in single crystals of La2-xBaxCuO4

    Institute of Scientific and Technical Information of China (English)

    李鹏程; 杨宏顺; 李志权; 柴一晟; 曹烈兆

    2002-01-01

    The in-plane electrical resistivity and thermoelectric power have been measured on single crystals ofLa2-xBaxCuO4 at around x=0.125. The room temperature resistivity and thermopower have their maximum val-ues at x=0.125, indicating that the carrier concentration is the minimum and the carriers are most strongly localized atx=0.125. The observed semiconductor-like behaviour can be well described by the weak-localized quasi-two-dimensionalstate. The steep rise in electric resistivity of the sample at x=0.125 below 70K is attributed to the formation of staticstripe-order of holes and spins, which are pinned by the low-temperature tetragonal (LTT) structure, as discovered inLa1.4sNd0.4Sr0.12CuO4.The temperature dependence of electric resistivity below 70K is still well described by theformula p ∝ lnT. A definite change in the slope of thermopower is observed at the low-temperature orthorhombic-LTTstructural phase transition temperature. The origin of the 1/8 anomaly is discussed in the text.

  19. Anti-apoptotic mechanism of Bacoside rich extract against reactive nitrogen species induced activation of iNOS/Bax/caspase 3 mediated apoptosis in L132 cell line.

    Science.gov (United States)

    Anand, T; Pandareesh, M D; Bhat, Pratiksha V; Venkataramana, M

    2014-10-01

    Nitric oxide is a highly reactive free radical gas that reacts with a wide range of bio-molecules to produce reactive nitrogen species and exerts nitrative stress. Bacopa monniera is a traditional folk and ayurvedic medicine known to alleviate a variety of disorders. Aim of the present study is to evaluate the protective propensity of Bacopa monniera extract (BME) through its oxido-nitrosative and anti-apoptotic mechanism to attenuate sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132). Our results elucidate that pre-treatment of L132 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP as evidenced by MTT and LDH leakage assays. BME pre-treatment inhibited NO generation by down-regulating inducible nitric oxide synthase expression. BME exhibited potent antioxidant activity by up-regulating the antioxidant enzymes. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic biomarkers such as Bax, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. By considering all these findings, we report that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.

  20. Lycopene modulates cholinergic dysfunction, Bcl-2/Bax balance, and antioxidant enzymes gene transcripts in monosodium glutamate (E621) induced neurotoxicity in a rat model.

    Science.gov (United States)

    Sadek, Kadry; Abouzed, Tarek; Nasr, Sherif

    2016-04-01

    The effect of monosodium glutamate (MSG) on brain tissue and the relative ability of lycopene to avert these neurotoxic effects were investigated. Thirty-two male Wistar rats were distributed into 4 groups: group I, untreated (placebo); group II, injected with MSG (5 mg·kg(-1)) s.c.; group III, gastrogavaged with lycopene (10 mg·kg(-1)) p.o.; and group IV received MSG with lycopene with the same mentioned doses for 30 days. The results showed that MSG induced elevation in lipid peroxidation marker and perturbation in the antioxidant homeostasis and increased the levels of brain and serum cholinesterase (ChE), total creatine phosphokinase (CPK), creatine phosphokinase isoenzymes BB (CPK-BB), and lactate dehydrogenase (LDH). Glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT) activities and gene expression were increased and glutathione content was reduced in the MSG-challenged rats, and these effects were ameliorated by lycopene. Furthermore, MSG induced apoptosis in brain tissues reflected in upregulation of pro-apoptotic Bax while lycopene upregulated the anti-apoptotic Bcl-2. Our results indicate that lycopene appears to be highly effective in relieving the toxic effects of MSG by inhibiting lipid peroxidation and inducing modifications in the activity of cholinesterase and antioxidant pathways. Interestingly, lycopene protects brain tissue by inhibiting apoptosis signaling induced by MSG.

  1. Phenomenological theory of phase transitions in epitaxial BaxSr1-xTiO3 thin films on (111)-oriented cubic substrates

    Science.gov (United States)

    Shirokov, V. B.; Shakhovoy, R. A.; Razumnaya, A. G.; Yuzyuk, Yu. I.

    2015-07-01

    A phenomenological thermodynamic theory of BaxSr1-xTiO3 (BST-x) thin films epitaxially grown on (111)-oriented cubic substrates is developed using the Landau-Devonshire approach. The group-theoretical analysis of the low-symmetry phases was performed taking into account two order parameters: the polarization related to ionic shifts in polar zone-center F1u mode and the out-of-phase rotation of TiO6 octahedra corresponding to the R25 zone-boundary mode in the parent cubic phase P m 3 ¯ m . The eight-order thermodynamic potential for BST-x solid solutions was developed and analyzed. We constructed the "concentration-misfit strain" phase diagram for BST-x thin films at room temperature and found that polar rhombohedral R3m phase with the polarization normal to the substrate is stable for x > 0.72 and negative misfit strains, while ferroelectric monoclinic C2 and Cm phases with in-plane polarization are stable for much smaller x and positive or slightly negative misfit strains. We constructed the "temperature-misfit strain" phase diagrams for several concentrations (x = 1, 0.8, 0.6, 0.4, and 0.2). Systematic changes of the phase transition lines between the paraelectric and ferroelectric phases are discussed. The phase diagrams are useful for practical applications in thin-film engineering.

  2. Evolution of superconducting correlations within magnetic-field-decoupled La2-xBaxCuO4 (x=0.095)

    Science.gov (United States)

    Stegen, Z.; Han, Su Jung; Wu, Jie; Pramanik, A. K.; Hücker, M.; Gu, Genda; Li, Qiang; Park, J. H.; Boebinger, G. S.; Tranquada, J. M.

    2013-02-01

    We explore the evolution of superconductivity in La2-xBaxCuO4 with x=0.095 in magnetic fields of up to 35 T applied perpendicular to the CuO2 planes. Previous work on this material has shown that perpendicular fields enhance both charge- and spin-stripe order within the planes. We present measurements of the resistivity parallel and perpendicular to the planes, as well as the Hall effect. Measurements of magnetic susceptibility for fields of up to 15 T applied both parallel and perpendicular to the planes provide complementary measures of the superconductivity. We show that fields sufficient to destroy pair tunneling between the planes do not disrupt the superconducting correlations within the planes. In fact, we observe an onset of large-amplitude but phase-disordered superconductivity within the planes at approximately 30 K that is remarkably insensitive to field. With further cooling, we observe a phase-transition-like drop in the in-plane resistivity to an apparent state of superconductivity despite the lack of phase coherence between the layers. These observations raise interesting questions concerning the identification of the upper critical field, where pairing is destroyed, in underdoped cuprates.

  3. No dramatic age-related loss of hair cells and spiral ganglion neurons in Bcl-2 over-expression mice or Bax null mice

    Directory of Open Access Journals (Sweden)

    Ohlemiller Kevin K

    2010-07-01

    Full Text Available Abstract Age-related decline of neuronal function is associated with age-related structural changes. In the central nervous system, age-related decline of cognitive performance is thought to be caused by synaptic loss instead of neuronal loss. However, in the cochlea, age-related loss of hair cells and spiral ganglion neurons (SGNs is consistently observed in a variety of species, including humans. Since age-related loss of these cells is a major contributing factor to presbycusis, it is important to study possible molecular mechanisms underlying this age-related cell death. Previous studies suggested that apoptotic pathways were involved in age-related loss of hair cells and SGNs. In the present study, we examined the role of Bcl-2 gene in age-related hearing loss. In one transgenic mouse line over-expressing human Bcl-2, there were no significant differences between transgenic mice and wild type littermate controls in their hearing thresholds during aging. Histological analysis of the hair cells and SGNs showed no significant conservation of these cells in transgenic animals compared to the wild type controls during aging. These data suggest that Bcl-2 overexpression has no significant effect on age-related loss of hair cells and SGNs. We also found no delay of age-related hearing loss in mice lacking Bax gene. These findings suggest that age-related hearing loss is not through an apoptotic pathway involving key members of Bcl-2 family.

  4. Effects of Active Components of Fuzi and Gancao Compatibility on Bax, Bcl-2, and Caspase-3 in Chronic Heart Failure Rats

    Directory of Open Access Journals (Sweden)

    Liqin Wang

    2016-01-01

    Full Text Available Hypaconitine (HA and glycyrrhetinic acid (GA are active components of Fuzi (Aconitum carmichaelii and Gancao (Glycyrrhiza uralensis Fisch; they have been used in compatibility for chronic heart failure (CHF from ancient times. The purpose of the present research was to explore whether apoptosis pathways were related with the protective effects of HA + GA against CHF rats or not. The rats were progressed with transverse-aortic constriction (TAC operation for 4 weeks to build the CHF state, and then the Digoxin (1 mg/kg, HA (2.07 mg/kg, GA (25 mg/kg, and HA (2.07 mg/kg + GA (25 mg/kg were orally administrated to rats for 1 week. The levels of BNP and cTnI in the plasma were decreased in the HA + GA group, and the heart/body weight ratio (H/B and left ventricular (LV parameters of transthoracic echocardiography were also declined; moreover, the expressions of Bax, Bcl-2, and caspase-3 were all improved in the HA + GA group than other groups in the immunohistochemistry and western blot methods. In general, the data suggested that Fuzi and Gancao compatibility could protect the CHF rats from apoptosis, which provided a strong evidence for further searching for mechanisms of them.

  5. Anti-tumour activity of a novel coumarin-chalcone hybrid is mediated through intrinsic apoptotic pathway by inducing PUMA and altering Bax/Bcl-2 ratio.

    Science.gov (United States)

    Singh, Neetu; Sarkar, Jayanta; Sashidhara, Koneni V; Ali, Shakir; Sinha, Sudhir

    2014-06-01

    Coumarins and chalcones are secondary plant metabolites which have shown an array of pharmacological properties including anti-tumour activity. We have previously reported on the synthesis and anti-proliferative activity of a series of novel coumarin-chalcone hybrids. Now we report on the in vivo efficacy as well as mechanism of action of the most potent molecule of the series, S009-131. Oral administration of this molecule resulted in regression of tumours induced by HeLa cell xenografts in nod SCID mice. The molecule inhibited proliferation of cervical cancer cells (HeLa and C33A) by inducing apoptosis and arresting cell cycle at G2/M phase. Apoptosis was induced through induction of caspase-dependent intrinsic pathway and alterations in the cellular levels of Bcl-2 family proteins. The mitochondrial transmembrane potential got highly depleted in S009-131 treated cells due to an increase in Bax/Bcl-2 ratio and intracellular ROS. The molecule induced release of cytochrome c into the cytosol and activation of initiator caspase-9 and executioner caspases-3/7. Tumour suppressor protein p53 and its transcriptional target PUMA were up regulated, suggesting their role in mediating the cell death. These results suggest that S009-131 is a potent candidate for the chemotherapy of cervical carcinoma.

  6. Resistance Fluctuation Spectroscopy of Charge Stripes and Intertwined Orders in the Phase Diagram of La2-xBaxCuO4

    Science.gov (United States)

    Weis, Adam; Fizari, Mounir; Hamilton, David; Wells, Azton; Lane, Justin; Chung, So Ra; Sellappan, Pathikumar; Kriven, Waltraud; van Harlingen, Dale

    The unusual phase diagram of La2-xBaxCuO4 (LBCO) near x=1/8 doping suggests a complex intertwined relationship between high-temperature superconductivity, charge stripes, spin order, and phase coherence. The charge stripe state's short-range conductance anisotropy may be observable as fluctuations in resistance. In thin film LBCO devices grown by pulsed laser deposition, our time-resolved resistance measurements have revealed an onset of resistance noise at dopings and critical temperatures consistent with charge stripes. The phase diagram of LBCO is explored by comparing the noise onset signature of charge order to measurements of superconductivity, the Hall effect, and other phenomena. I will briefly discuss the relevance of our results in LBCO thin films and crystals to a proposed ''pair-density-wave'' state near x=1/8. This research was supported by the DOE-BES under Grant DE-SC0012368, through the Frederick Seitz Materials Research Laboratory at the University of Illinois at Urbana-Champaign. SRC was sponsored by NSF-REU 13-59126.

  7. E2F is involved in radioresistance of carbon ion induced apoptosis via Bax/caspase 3 signal pathway in human hepatoma cell.

    Science.gov (United States)

    Xie, Yi; Si, Jing; Wang, Yu-Pei; Li, Hong-Yan; Di, Cui-Xia; Yan, Jun-Fang; Ye, Yan-Cheng; Zhang, Yan-Shan; Zhang, Hong

    2017-05-13

    Deletion of p53, most common genetic alteration, is observed in human tumors and reported to lead to improve in cell radioresistance. Heavy-ion irradiation (IR) could induce p53(-/-) cancer cells apoptosis. However, little is known regarding the molecular mechanism in this type of cell apoptosis. The present studies have focused on mechanisms state of signaling pathways as an activator of the cell fate decisions induced by heavy ion IR without p53. Carbon ion IR could induce up-regulation of E2F1 expression in cancer cells. This phenomenon was not observed in X-ray IR group. Up-regulation of E2F1 could cause a higher reduction in clonogenic survival, low level of cellular activity, G2 /M phase arrest, promotion of apoptosis rate, up-regulation of phosphor-Rb, Bax, and cleaved-caspase 3 proteins expressions without p53. Changes of E2F1 expressions could partly alter radioresistance in cancer cells. The results were suggested that heavy ion IR could induce p53(-/-) cancer cells apoptosis via E2F1 signal pathway. Our study provides a scientific rationale for the clinical use of heavy ion as radiotherapy in patients with p53-deficient tumors, which are often resistant to radiotherapy. © 2017 Wiley Periodicals, Inc.

  8. Sr1-x BaxTiO3 and Sr1-x Cax TiO3 crystal growth by floating zone method

    Science.gov (United States)

    Oliveira, F. S.; Dos Santos, C. A. M.; Machado, A. J. S.

    2014-03-01

    Strontium titanate has both technological and scientific interest due to high dielectric constant, semiconducting n-type behavior after oxygen reduction, and unconventional superconductivity. Titanate single crystals are commonly grown by many routes as Verneuil commercial, self-flux, and floating zone method. Nabokin et al. were able to grow high quality SrTiO3 samples in an optical furnace. In order to enhance the dielectric constant, Sr 2+has been replaced by Ba2+ or Ca2+ atoms, but solid solutions implies lattice distortions which can affect the quality of these single crystals. This work revisits the preparation of the Sr1-xBaxTiO3 and Sr1-xCaxTiO3 single crystals using floating zone method. The crystals were grown with a Quantum Design infra-red image furnace starting with SrCO3, BaCO3, CaCO3, and TiO2 high purity powders. The quality of single crystals were analyzed by x-ray difractometry. The influence of the growth conditions on the single crystal are reported. The electrical resistivity as a function of the temperature and the influence of the oxygen contents are reported. This material based upon work supported by FAPESP, CNPq, and NAP-USP. K. Behnia is acknowledged for some suggestions.

  9. (BaxSr1-x)TiO3/Mg2TiO4组分梯度陶瓷的制备及介电性能%Preparation and Dielectric Properties of (BaxSr1-x)TiO3/Mg2TiO4 Composite Ceramics with A Functionally Graded Structure

    Institute of Scientific and Technical Information of China (English)

    李俊; 王旭升; 柴晓娜; 刘鹏

    2014-01-01

    采用叠层法制备了(BaxSr1-x)TiO3/Mg2TiO4组分梯度陶瓷.采用XRD、SEM、EDS及介电性能测试等手段分析了材料的微观结构和介电性能.结果表明:1375℃烧结3h的样品较致密、结晶良好,由钙钛矿和尖晶石两相复合而成,且材料内存在Ba/Sr比组分梯度;与不含梯度的复合陶瓷相比,梯度陶瓷不但保持了较大的介电可调度,而且具有较好的介电性能温度稳定性;在室湿(20℃)和2 kV/mm电场下,典型样品的介电可调度可达21.9%,当温度升高到60℃时,其可调度仍然保持在较大的值(9.3%).温度稳定性的改善可归因于不同组分的(BaxSr1-x)TiO3具有不同的铁电-顺电相转变温度,组分梯度的设计有利于该类材料应用温度范围的拓宽.

  10. DMFC (3,5-dimethyl-(7)H-furo[3,2-g]chromen-7-one) regulates Bim to trigger Bax and Bak activation to suppress drug-resistant human hepatoma.

    Science.gov (United States)

    Xiang, Jun; Wang, Zhe; Liu, Qianqian; Li, Xia; Sun, Jianguo; Fung, Kwok-Pui; Liu, Feiyan

    2017-03-01

    3,5-Dimethyl-(7)H-furo[3,2-g]chromen-7-one (DMFC) is a coumarin derivative with anti-cancer activity against human hepatoma cells, but the mechanisms underlying DMFC function in cancer suppression is unknown. In this study, we aimed at elucidating the molecular mechanisms underlying DMFC anti-cancer activity and determining whether DMFC is effective in suppression of drug-resistant human hepatocellular carcinoma. We show here that DMFC effectively suppresses both the parent and the multidrug-resistant hepatoma cell growth in vitro and DMFC suppresses hepatoma cell growth at least in part through inducing tumor cell apoptosis. In the molecular level, we observed that DMFC treatment decreases Bcl-2 level by a post-transcriptional mechanism and activates Bim transcription to increase Bim mRNA and protein level in hepatoma cells. Furthermore, co-immunoprecipitation studies revealed that DMFC-induced Bim interrupts interactions between Bcl-2 and Bax and between Mcl-1 and Bak, resulting in dissociation of Bax from Bcl-2 and Bak from Mcl-1 and subsequent activation of both Bax and Bak. Activation of Bax and Bak leads to mitochondrial outer membrane permeabilization and cytochrome c release. Consistent with its potent apoptosis-inducing activity, DMFC exhibited potent activity against the multidrug-resistant hepatoma xenograft growth in vivo. Therefore, we determine that DMFC suppresses hepatoma growth through decreasing Bcl-2 and increasing Bim to induce tumor cell apoptosis and hold great promise for further development as a therapeutic agent to treat chemoresistant hepatoma.

  11. Effect of morin-5'-sulfonic acid sodium salt on the expression of apoptosis related proteins caspase 3, Bax and Bcl 2 due to the mercury induced oxidative stress in albino rats.

    Science.gov (United States)

    Venkatesan, Rantham Subramaniam; Sadiq, Abdul Majeeth Mohamed

    2017-01-01

    Many environmental contaminants have been reported to disturb the pro-oxidant or antioxidant balance of the cells by inducing oxidative stress. Oxidative stress mediated by the HgCl2 induces DNA, protein and lipid oxidation resulted in necrosis or apoptosis, or both. Currently flavonoids are being emerging topic and reported to have antiviral, anti-inflammatory, anti- tumor and antioxidant activities. Morin is one of the flavonoid protects the cells from oxygen free radical damage and scavenges the free radicals and metals and also heals the injured cells commercially. Morin hydrate is sparingly soluble in water. Hence, the water soluble morin -5'- sulfonic acid sodium salt (NaMSA) was selected and synthesized. Aim of the present study was to analyze the effect of morin-5'-sulfonic acid sodium salt on the expression of apoptosis related proteins caspase 3, Bax and Bcl 2 due to the mercury induced oxidative stress in albino rats.. The experimental rats were exposed to sub lethal concentration of mercuric chloride (1.25mg/kg) and the ameliorating effect of NaMSA was studied by using apoptotic protein markers Bax and caspase-3 and Bcl-2. The obtained results were analyzed using one way analysis of variance by the Duncan's Multiple comparison test to determine the level of significance (p) and pBax and caspase-3 and a decreased expression was noted in the Bcl-2 level compared with control bands significantly (pBax, Caspase-3 and Bcl-2 levels compared with control rats. Hence, the membrane damage was protected, stopped the cell death and apoptosis. This could be due to the morin-5'-sulfonic acid sodium salt effective chelation action on the HgCl2 generated free radicals. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  12. P53,Bax,Bcl-2蛋白表达及细胞凋亡在急性放射性皮肤溃疡发生发展过程中的作用探讨%The role of P53, Bax, Bcl-2 expression and cell apoptosis in the formation and development of acute radiation-induced skin ulcers

    Institute of Scientific and Technical Information of China (English)

    谷庆阳; 曹卫红; 王德文; 高亚兵; 杨志祥; 赵坡

    2001-01-01

    目的:研究细胞凋亡及一些凋亡相关基因(p53,bcl-2,bax)的表达在急性放射性皮肤溃疡发生发展过程中的作用.方法:采用Wistar大鼠以60Co γ射线进行局部照射,建立急性放射性皮肤溃疡动物模型,观察病变40 d,然后采用免疫组化方法检测皮肤溃疡组织中P53,Bcl-2,Bax蛋白表达,并采用原位末端标记法(TUNEL)检测细胞凋亡.结果:照后14 d照射野内开始出现皮肤溃疡,之后逐渐扩大、融合、加深;照后11~40 d,P53蛋白表达明显增强,主要定位于血管内皮细胞和小血管平滑肌中;照后14~21 d为Bax蛋白表达高峰,之后逐渐减弱,主要定位于血管内皮细胞、部分成纤维细胞及新生表皮细胞中;Bcl-2则在照后1~11 d呈弱或中度阳性,定位于表皮、毛囊上皮及血管内皮中,之后为阴性或可疑阳性;照后11~35 d,上述细胞特别是血管内皮细胞凋亡率较正常伤口愈合早期增高.结论:辐射诱导的P53,Bax,Bcl-2表达的变化及细胞凋亡率特别是血管内皮细胞凋亡率的增高与放射性皮肤溃疡发生、发展及难愈合(不能形成有效肉芽组织)的分子机制相关.%Objective:To study the expression of P53, Bax, Bcl-2 proteins and the role of cell apoptosis in the formation and development of acute radiation-induced skin ulcers.Methods:A rat model which was locally irradiated with 60 Co γ-rays was used, and the pathological changes were observed for 40 days. Immunohistochemistry and TUNEL assay were performed which enabled the detection of P53, Bax, Bcl-2 and cell apoptosis during the formation and development of radiation skin ulcers.Results: Skin ulcers were found on day 14 after irradiation, and enlarged and deepened gradually during the observation period. P53 was over expressed during days 11 to 40 after irradiation and was localized in vascular endotheliocytes and smooth muscle cells. Bax was moderately positive during days 14 to 21 and weakly positive during days

  13. Cisplatin-induced apoptosis in non-small-cell lung cancer cells is dependent on Bax- and Bak-induction pathway and synergistically activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells.

    Science.gov (United States)

    Matsumoto, Masaru; Nakajima, Wataru; Seike, Masahiro; Gemma, Akihiko; Tanaka, Nobuyuki

    2016-04-29

    Cisplatin is a highly effective anticancer drug for treatment of various tumors including non-small-cell lung cancer (NSCLC), and is especially useful in cases nonresponsive to molecular-targeted drugs. Accumulating evidence has shown that cisplatin activates the p53-dependent apoptotic pathway, but it also induces apoptosis in p53-mutated cancer cells. Here we demonstrated that DNA-damage inducible proapoptotic BH3 (Bcl-2 homology region 3)-only Bcl-2 family members, Noxa, Puma, Bim and Bid, are not involved in cisplatin-induced apoptosis in human NSCLC cell lines. In contrast, the expression of proapoptotic multidomain Bcl-2-family members, Bak and Bax, was induced by cisplatin in p53-dependent and -independent manners, respectively. Moreover, in wild-type p53-expressing cells, cisplatin mainly used the Bak-dependent apoptotic pathway, but this apoptotic pathway shifted to the Bax-dependent pathway by loss-of-function of p53. Furthermore, both Bak- and Bax-induced apoptosis was enhanced by the antiapoptotic Bcl-2 family member, Bcl-XL knockdown, but not by Mcl-1 knockdown. From this result, we tested the effect of ABT-263 (Navitoclax), the specific inhibitor of Bcl-2 and Bcl-XL, but not Mcl-1, and found that ABT-263 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells in the presence or absence of p53. These results indicate a novel regulatory system in cisplatin-induced NSCLC cell apoptosis, and a candidate efficient combination chemotherapy method against lung cancers.

  14. Formononetin Induces Apoptosis of Human Osteosarcoma Cell Line U2OS by Regulating the Expression of Bcl-2, Bax and MiR-375 In Vitro and In Vivo

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    Wei Hu

    2015-09-01

    Full Text Available Background/Aims: Phytoestrogens are known to prevent tumor progression by inhibiting proliferation and inducing apoptosis in cancer cells. Formononetin is one of the main components of red clover plants, and is considered as a typical phytoestrogen. This study investigates formononetin induction of apoptosis of human osteosarcoma cell line U2OS by regulating Bcl-2 and Bax expression in vitro and in vivo. Methods: U2OS cells were treated with different concentrations of formononetin and the proliferation of the cells was measured using an MTT assay. Cell apoptosis was examined by flow cytometry. The levels of miR-375, Bax and Bcl-2 protein expression in treated cells were determined by Western blot and RT-PCR. The antitumor activity of formononetin was also evaluated in vivo in nude mice bearing orthotopic tumor implants. Results: High concentrations of formononetin significantly suppress the proliferation of U2OS cells and induce cell apoptosis. Moreover, compared to control group the expression of Bcl-2 and miR-375 decreases with formononetin in the U2OS cells, while Bax increases. Conclusion: Formononetin has inhibitory effects on the proliferation of U2SO cells, both in vitro and in vivo. This antitumor effect is directly correlated with formononetin concentration.

  15. Impact of resveratrol on the expression of apoptosis related gene survivin and bax in human cancer cells%白藜芦醇对食管癌细胞凋亡相关基因survivin和bax表达的影响

    Institute of Scientific and Technical Information of China (English)

    Yongjun Li; Xiaohui Sun; Rui Zhang

    2009-01-01

    Objective: We explored the mechanism of apoptosis in human esophageal cancer Eca109 cells by resveratrol.Methods: The suppressive ratio of resveratrol on Ecal09 cells proliferation was evaluated by MTT colorimetric assay and morphology was observed by transmission electron microscope. The expression of survivin and bax was analyzed by RT-PCR and Flow Cytometry (FCM). Results: Resveratrel inhibited the growth of Ecal09 cells in a dose-and time-dependent man-ner, and the suppressive ratio arrived at 76.42%. Morphological apoptosis could be observed after treated with resveratrol.The bulk of some drug-treated cells turned small and the nuclear chromatin became condensed and marginated. The results determined by RT-PCR and FCM showed that resveratrol could down-regulate surviving, while up-regulate bax. Conclusion:Resveratrol could induce the apoptosis of human esophageal cancer Eca109 cells, and its possible molecular mechanisms might be related to modulation the expression of survivin and bax.

  16. Anti-apoptotic genes Bcl-2 and Bcl-xL overexpression can block iridovirus serine/threonine kinase-induced Bax/mitochondria-mediated cell death in GF-1 cells.

    Science.gov (United States)

    Reshi, Latif; Wang, Hua-Ven; Hui, Cho-Fat; Su, Yu-Chin; Hong, Jiann-Ruey

    2017-02-01

    Although serine/threonine (ST) kinase is known to induce host cell death in GF-1 cells, it remains unclear how ST kinase induces mitochondrial function loss. In the present study, we addressed the issue of mitochondrial function loss by determining whether the Bcl-2 family members Bcl-2 and Bcl-xL can prevent ST kinase-induced cell death activity via interacting with the pro-apoptotic gene Bax. Grouper fin cells (GF-1) carrying EGFP-Bal-xL and EGFP-Bcl-2 fused genes were selected, established in cell culture, and used to examine the involvement of Bcl-2 and Bcl-xL overexpression in protection of GF-1 cells from the effects of the giant sea perch iridovirus (GSIV) ST kinase gene. Using the TUNEL assay, we found that EGFP-Bcl-2 and EGFP-Bcl-xL reduced GSIV ST kinase-induced apoptosis to 20% all at 24 h and 48 h post-transfection (pt). Also, Bcl-2 and Bcl-xL substantially reduced the percentage of cells with GSIV ST kinase-induced loss of mitochondrial membrane potential (Δψps) at 24 and 48 hpt, respectively, and this reduction correlated with a 30% and 50% enhancement of host cell viability at 24 and 48 hpt as compared with vector control. Moreover, analysis of the effect of Bcl-2 and Bcl-xL interaction with Bax targeted to mitochondria during ST kinase expression at 48 hpt found that Bcl-2 and Bcl-xL also interacted with Bax to block cytochrome c release. Finally, Bcl-2 and Bcl-xL overexpression caused blockage of ST kinase function at 48 hpt, which was correlated with preventing caspase-9 and -3 cleavage and activation, thereby blocking downstream death signaling events. Taken together, our results suggest that the ST kinase-induced Bax/mitochondria-mediated cell death pathway can be blocked by the interaction of Bcl-2 and Bcl-xL with Bax to inhibit cytochrome c release during MMP loss. This rescue activity also correlated with inhibition of caspase-9 and -3 activation, thereby enhancing cell viability. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Deregulated expression of A1, Bcl-2, Bcl-xL, and Mcl-1 antiapoptotic proteins and Bid, Bad, and Bax proapoptotic genes in polycythemia vera patients

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    Elainy Patricia Lino Gasparotto

    2011-12-01

    Full Text Available Apoptosis deregulation might have a role in the pathophysiology of polycythemia vera (PV. This study evaluated Bcl-2 molecule expression in CD34+ cells and leukocytes in 12 PV patients. Gene expression was investigated by real time PCR using SybrGreen Quantitect kit and protein expression was evaluated by western-blotting. JAK2 V617F mutation was detected according to Baxter et al (2005. CD34+ cells from PV patients presented higher levels of A1 and Mcl-1 expression (median: 22.6 and 5.2, respectively in comparison with controls (0.9 and 0.5, p=0.004 and p=0.020; while Bcl-2 and Bcl-xL expression decreased in PV patients (0.18 and 1.19 compared with controls (1.39 and 2.01, p=0.006 and p=0.020. CD34+ cells in PV patients showed an elevated Bid expression (14.4 in comparison with healthy subjects (1.0; p=0.002. Patients' leukocytes showed an A1 augmentation (7.41, p=0.001 and a reduced expression of Bax (0.19; p=0.040 and Bad (0.2; p=0.030. There was no correlation between JAK2 V617F allele burden and molecular expression. PV patients showed alterations in Bcl-2 members' expression, which may interfere with control of apoptotic machinery and contribute to disease pathogenesis.A desregulação da apoptose parece participar da fisiopatologia da policitemia vera (PV. Este estudo avaliou a expressão das moléculas da família Bcl-2 em células hematopoéticas CD34 + e leucócitos de 12 pacientes com PV. Foram realizados: a quantificação da expressão gênica por PCR em tempo real utilizando kit Sybrgreen Quantitect, avaliação da expressão de proteínas por western-blot e detecção da mutação JAK2 V617F segundo Baxter et al. (2005. Células CD34 + dos pacientes com PV apresentaram maior expressão de A1 e Mcl-1 (mediana: 22,6 e 5,2, respectivamente em comparação com controles (0,9 e 0,5, p = 0,004 e p = 0,020 e expressão de Bcl-2 e Bcl-xL diminuída nestes pacientes (0,18 e 1,19 em relação aos controles (1,39 e 2,01, p = 0,006 e p = 0

  18. Yeast Bax inhibitor, Bxi1p, is an ER-localized protein that links the unfolded protein response and programmed cell death in Saccharomyces cerevisiae.

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    James Cebulski

    Full Text Available Bax inhibitor-1 (BI-1 is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. Studies in both mammalian and plant cells suggest that the BI-1 protein resides in the endoplasmic reticulum and is involved in the unfolded protein response (UPR that is triggered by ER stress. It is thought to act via a mechanism involving altered calcium dynamics. In this paper, we provide evidence that the Saccharomyces cerevisiae protein encoded by the open reading frame, YNL305C, is a bona fide homolog for BI-1. First, we confirm that yeast cells from two different strain backgrounds lacking YNL305C, which we have renamed BXI1, are more sensitive to heat-shock induced cell death than wildtype controls even though they have indistinguishable growth rates at 30°C. They are also more susceptible both to ethanol-induced and to glucose-induced programmed cell death. Significantly, we show that Bxi1p-GFP colocalizes with the ER localized protein Sec63p-RFP. We have also discovered that Δbxi1 cells are not only more sensitive to drugs that induce ER stress, but also have a decreased unfolded protein response as measured with a UPRE-lacZ reporter. Finally, we have discovered that deleting BXI1 diminishes the calcium signaling response in response to the accumulation of unfolded proteins in the ER as measured by a calcineurin-dependent CDRE-lacZ reporter. In toto, our data suggests that the Bxi1p, like its metazoan homologs, is an ER-localized protein that links the unfolded protein response and programmed cell death.

  19. MicroRNA-650 was a prognostic factor in human lung adenocarcinoma and confers the docetaxel chemoresistance of lung adenocarcinoma cells via regulating Bcl-2/Bax expression.

    Directory of Open Access Journals (Sweden)

    Jia-Yuan Huang

    Full Text Available Increasing evidence shows that dysregulation of microRNAs (miRNAs is involved in malignant transformation. We investigated the clinical significance of miR-650 and its involvement in chemoresistance to docetaxel. Our results showed that the relative expression level of miR-650 was significantly higher in LAD tissues than in corresponding nontumor tissues and high level of miR-650 expression was found to be significantly associated with high incidence of lymph node metastasis, advanced clinical stage and poor prognosis of LAD patients. Univariate and multivariate analyses indicated that high miR-650 expression was an independent prognostic factor for survival. Also, we found that the level of miR-650 in LAD tissues was correlated with the response of patients to docetaxel-based chemotherapy. Silencing of miR-650 could increase the in vitro sensitivity of docetaxel-resistant LAD cells to docetaxel, while upregulation of miR-650 decreased the sensitivity of parental LAD cells to docetaxel both in vitro and in vivo. Additionally, silencing of miR-650 could enhance the caspase-3-dependent apoptosis, which might be correlated with the decreased ratio of Bcl-2/Bax. Further researches suggested that inhibitor of growth 4 (ING4 was a direct target of miR-650. Downregulated or upregulated ING4 expression could partially rescue the effects of miR-650 inhibitor or mimics in docetaxel-resistant or parental LAD cells. Furthermore, we found that ING4 was upregulated in docetaxel-responding LAD tissues, and its expression was inversely correlated with miR-650. Thus, miR-650 is a novel prognostic marker in LAD and its expression is a potential indicator of chemosensitivity to docetaxel-based chemotherapy regimen.

  20. Celecoxib aggravates cardiac apoptosis in L-NAME-induced pressure overload model in rats: Immunohistochemical determination of cardiac caspase-3, Mcl-1, Bax and Bcl-2.

    Science.gov (United States)

    Mosaad, Sarah M; Zaitone, Sawsan A; Ibrahim, Abdelazim; El-Baz, Amani A; Abo-Elmatty, Dina M; Moustafa, Yasser M

    2017-06-25

    The mechanism of celecoxib cardiovascular adverse events was earlier investigated; yet in-depth investigations are needed to assess the involvement of its pro-apoptotic effect throughout this process. An in-vivo chronic rat model of pressure overload employing Nʷ-nitro-l-arginine methyl ester (L-NAME) was tested at different time intervals to ensure the occurrence of persistent myocardial apoptosis along with pressure overload. Seven groups of male Wistar rats were assigned as (i) distilled water; (ii-iv) L-NAME (60 mg/kg) for 6, 12 or 16 weeks; (v-vii) L-NAME [16 weeks] + celecoxib (25, 50 or 100 mg/kg), from week 13 to week 16. Treatment with L-NAME for 6, 12 or 16 weeks increased systolic blood pressure, serum level of creatine kinase-MB and lactate dehydrogenase. Further, it induced cardiac hypertrophy, detected in terms of greater heart weight index and cardiomyocyte cross-sectional area and produced interstitial and perivascular fibrosis. Moreover, administration of L-NAME increased cardiac immunostaining for activated caspase-3 and Bax/Bcl-2 ratio whereas; immunostaining for Mcl-1 was decreased. Administration of celecoxib (25, 50 or 100 mg/kg) aggravated the L-NAME-induced toxicity. The work results shed the light on the putative pro-apoptotic effect of celecoxib at a risk state of pressure overload comparable to the clinical condition of essential hypertension. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts.

    Science.gov (United States)

    González-Gironès, Diana M; Moncunill-Massaguer, Cristina; Iglesias-Serret, Daniel; Cosialls, Ana M; Pérez-Perarnau, Alba; Palmeri, Claudia M; Rubio-Patiño, Camila; Villunger, Andreas; Pons, Gabriel; Gil, Joan

    2013-08-01

    5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1 (-/-) /α2 (-/-) MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.

  2. Stripe order in superconducting La2-xBaxCuO4 (0.095⩽x⩽0.155)

    Science.gov (United States)

    Hücker, M.; v. Zimmermann, M.; Gu, G. D.; Xu, Z. J.; Wen, J. S.; Xu, Guangyong; Kang, H. J.; Zheludev, A.; Tranquada, J. M.

    2011-03-01

    The correlations between stripe order, superconductivity, and crystal structure in La2-xBaxCuO4 single crystals have been studied by means of x-ray and neutron diffraction as well as static magnetization measurements. The derived phase diagram shows that charge stripe order (CO) coexists with bulk superconductivity in a broad range of doping around x=1/8, although the CO order parameter and correlation length fall off quickly for x≠1/8. Except for x=0.155, the onset of CO always coincides with the transition between the orthorhombic and the tetragonal or less orthorhombic low-temperature structures. The CO transition evolves from a sharp drop at low x to a more gradual transition at higher x, eventually falling below the structural phase boundary for optimum doping. With respect to the interlayer CO correlations, we find no qualitative change of the stripe stacking order as a function of doping, and in-plane and out-of-plane correlations disappear simultaneously at the transition. Similarly to the CO, the spin stripe order (SO) is also most pronounced at x=1/8. Truly static SO sets in below the CO and coincides with the first appearance of in-plane superconducting correlations at temperatures significantly above the bulk transition to superconductivity (SC). Indications that bulk SC causes a reduction of the spin or charge stripe order could not be identified. We argue that CO is the dominant order that is compatible with SC pairing but competes with SC phase coherence. Comparing our results with data from the literature, we find good agreement if all results are plotted as a function of x' instead of the nominal x, where x' represents an estimate of the actual Ba content, extracted from the doping dependence of the structural transition between the orthorhombic phase and the tetragonal high-temperature phase.

  3. Reconciliation of local and long-range tilt correlations in underdoped La2-xBaxCuO4 (0 ≤x ≤0.155 )

    Science.gov (United States)

    Bozin, Emil S.; Zhong, Ruidan; Knox, Kevin R.; Gu, Genda; Hill, John P.; Tranquada, John M.; Billinge, Simon J. L.

    2015-02-01

    A long-standing puzzle regarding the disparity of local and long-range CuO6 octahedral tilt correlations in the underdoped regime of La2-xBaxCuO4 is addressed by utilizing complementary neutron powder diffraction and inelastic neutron scattering (INS) approaches. This system is of interest because of the strong depression of the bulk superconducting transition at x =1 /8 in association with charge and spin stripe order. The latter unidirectional order is tied to Cu-O bond-length anisotropy present in the so-called low-temperature tetragonal (LTT) phase. On warming, the lattice exhibits two sequential structural transitions, involving changes in the CuO6 tilt pattern, first to the low-temperature orthorhombic (LTO) and then the high-temperature tetragonal (HTT) phase. Despite the changes in static order, inspection of the instantaneous local atomic structure suggests that the LTT-type tilts persist through the transitions. Analysis of the INS spectra for the x =1 /8 composition reveals the dynamic nature of the LTT-like tilt fluctuations within the LTO and HTT phases. Within the low-temperature phase, the Cu-O bond-length splitting inferred from lattice symmetry and fitted atomic position parameters reaches a maximum of 0.3 % at x =1 /8 , suggesting that electron-phonon coupling may contribute to optimizing the structure to stabilize stripe order. This splitting is much too small to be resolved in the pair distribution function, and in fact we do not resolve any enhancement of the instantaneous bond-length distribution in association with stripe order. This study exemplifies the importance of a systematic approach using complementary techniques when investigating systems exhibiting a large degree of complexity and subtle structural responses.

  4. Determination of the optical properties of La2-xBaxCuO4 for several dopings, including the anomalous x=(1)/(8) phase

    Science.gov (United States)

    Homes, C. C.; Hücker, M.; Li, Q.; Xu, Z. J.; Wen, J. S.; Gu, G. D.; Tranquada, J. M.

    2012-04-01

    The optical properties of single crystals of the high-temperature superconductor La2-xBaxCuO4 have been measured over a wide frequency and temperature range for light polarized in the a-b planes and along the c axis. Three different Ba concentrations have been examined, x=0.095 with a critical temperature Tc=32 K, x=0.125 where the superconductivity is dramatically weakened with Tc≃2.4 K, and x=0.145 with Tc≃24 K. The in-plane behavior of the optical conductivity for these materials at high temperature is described by a Drude-like response with a scattering rate that decreases with temperature. Below Tc in the x=0.095 and 0.145 materials there is a clear signature of the formation of a superconducting state in the optical properties allowing the superfluid density (ρs0) and the penetration depth to be determined. In the anomalous 1/8 phase, some spectral weight shifts from lower to higher frequency (≳300 cm-1) on cooling below the spin-ordering temperature Tso≃42 K, associated with the onset of spin-stripe order; we discuss alternative interpretations in terms of a conventional density-wave gap versus the response to pair-density-wave superconductivity. The two dopings for which a superconducting response is observed both fall on the universal scaling line ρs0/8≃4.4σdcTc, which is consistent with the observation of strong dissipation within the a-b planes. The optical properties for light polarized along the c axis reveal an insulating character dominated by lattice vibrations, superimposed on a weak electronic background. No Josephson plasma edge is observed in the low-frequency reflectance along the c axis for x=1/8; however, sharp plasma edges are observed for x=0.095 and 0.145 below Tc.

  5. The influence of sleep deprivation on expression of apoptosis regulatory proteins p53, bcl-2 and bax following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

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    Juliana Noguti

    2013-01-01

    Full Text Available Background: The aim of this study was to evaluate whether paradoxical sleep deprivation could affects the mechanisms and pathways essentials for cancer cells in tongue cancer induced by 4-nitroquinole 1-oxide in Wistar rats. Materials and Methods: For this purpose, the animals were distributed into 4 groups of 5 animals each treated with 50 ppm 4 nitroquinoline 1 oxide (4 NQO solution through their drinking water for 4 and 12 weeks. The animals were submitted to paradoxical sleep deprivation (PSD for 72 h using the modified multiple platform method, which consisted of placing 5 mice in a cage (41 × 34 × 16 cm containing 10 circular platforms (3.5 cm in diameter with water 1 cm below the upper surface. The investigations were conducted using immunohistochemistry of p53, Bax and Bcl-2 proteins related to apoptosis and its pathways. Statistical analysis was performed by Kruskal-Wallis non-parametric test followed by the Dunn′s test using SPSS software pack (version 1.0. P value < 0.05 was considered for statistic significance. Results: Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure in all groups, in 12 weeks were observed pre-neoplasic lesions. Data analysis revealed statistically significant differences ( P < 0.05 in 4 weeks group for p53 and for bcl-2 and for all immunomarkers after 12 weeks of 4NQO administration. Conclusion: Our results reveal that sleep deprivation exerted alterations in proteins associated with proliferation and apoptosis in carcinogenesis.

  6. bax和p53基因共转染对人舌癌细胞增 殖和凋亡的影晌%Effect on Proliferation and Apoptosis of Human Lingual Carc inoma Cells Cotransfected by bax and p53 Genes

    Institute of Scientific and Technical Information of China (English)

    马英红; 杨绍华; 陈俊杰; 高家让; 彭文珍; 王若菡

    2001-01-01

    Objective To investigate th e effects on prolife ration andapoptosis of human cancerous cells cotransfected by bax apoptosis-in ducing gene and p53 tumor suppressor gene. Methods The chi meric gene pSV-CIP-bax -CAT was constructed in which bax gene was flanked upstream by a 217bp fragment (+822~+1093) of the first intron and a 317bp promoter fragment of human a 1(Ⅰ) colla gen gene . Human lingual carcinoma cell line Tca 8113 (LCC) in culture was respe ctively transfected with pSV-CIP-bax-CAT, and cotransfected with pSV-p53-CA T and pSV-CIP-bax-CAT by using the transfection reagent DOSPER. Res ults Immuno- slot blot and ELISA demonstrated that the expression of bax and p53 genes incr eased remarkably in the transfected and cotransfected LCC, compared with the con trols(LCC transfected with pSV-CIP-CAT and LCC).MTT colorimetric assay,TUNE L fluorecence microscopy and flow cytometry showed that foreign bax or p53 gene inhibited the LCC growth and induced apoptosis(23.9% and 26.1% of inhibitory ra te by bax gene and p53 plus bax genes respectively). Conclusion The ectopic expression of the bax gene in the LCC promoted by the cis-act ing elements of human a1(Ⅰ)collagen gene has obviously synergistic effect on th e proliferation and apoptosis of the LCC cotransfected with p53 gene plus bax gene for 48 hours.%目的 探讨诱导凋亡的bax基因和p53抑癌基因共转染对人癌细胞增殖和凋亡的作用。方法 构建pSV-CIP-bax-CAT嵌合基因。在其bax基因上游含人a1(Ⅰ)型胶原基因的第一内含子217bp片段(+822~+1093)和317bp启动子片段。经阳离子转染试剂DOSPER介导,分别用pSV-CIP-bax-CAT转染、pSV-CIP-bax-CAT和pSV-p53-CAT共转染培养的人舌癌Tca8113细胞系(LCC)。