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Sample records for pro-apoptotic protein bax

  1. 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.

  2. 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.

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

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

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    Zeilstra, Jurrit; Joosten, Sander P.J.; Wensveen, Felix M.; Dessing, Mark C.; Schuetze, Denise M.; Eldering, Eric; Spaargaren, Marcel; Pals, Steven T.

    2011-01-01

    Research highlights: → Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. → Expression profiling of apoptosis-related genes in Apc Min/+ mice revealed the differential expression of pro-apoptotic Bok and Bax. → APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. → Blocking of β-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 β-catenin causes constitutively active β-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 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 β-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 uncontrolled epithelial cell proliferation in the

  5. RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

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    Subauste, M Cecilia; Ventura-Holman, Tereza; Du, Liqin; Subauste, Jose S; Chan, Shing-Leng; Yu, Victor C; Maher, Joseph F

    2009-12-01

    Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is upregulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1 and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be upregulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 overexpression in SW620 cells led to downregulation of Fem1b protein levels. Conversely, downregulation of RACK1 led to upregulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein upregulation. In conclusion, RACK1 downregulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

  6. BAX protein expression and clinical outcome in epithelial ovarian cancer.

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    Tai, Y T; Lee, S; Niloff, E; Weisman, C; Strobel, T; Cannistra, S A

    1998-08-01

    Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

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

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

    2013-11-01

    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.

  8. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

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    Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

    2015-01-01

    The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  9. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM.

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    Daniel O Frank

    Full Text Available The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM. In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20 by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  10. Endoplasmic Reticulum Stress Induces the Early Appearance of Pro-apoptotic and Anti-apoptotic Proteins in Neurons of Five Familial Alzheimer′s Disease Mice

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    Hui Shen

    2016-01-01

    Conclusions: These findings suggest that compared with those of age-matched WT mice, ERS-associated pro-apoptotic and anti-apoptotic proteins are upregulated in 2-month-old 5×FAD mice, consistent with intracellular Aβ aggregation in neurons.

  11. Human Thyroid Cancer-1 (TC-1 is a vertebrate specific oncogenic protein that protects against copper and pro-apoptotic genes in yeast

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    Natalie K. Jones

    2015-07-01

    Full Text Available The human Thyroid Cancer-1 (hTC-1 protein, also known as C8orf4 was initially identified as a gene that was up-regulated in human thyroid cancer. Here we show that hTC-1 is a peptide that prevents the effects of over-expressing Bax in yeast. Analysis of the 106 residues of hTC-1 in available protein databases revealed direct orthologues in jawed-vertebrates, including mammals, frogs, fish and sharks. No TC-1 orthologue was detected in lower organisms, including yeast. Here we show that TC-1 is a general pro-survival peptide since it prevents the growth- and cell death-inducing effects of copper in yeast. Human TC-1 also prevented the deleterious effects that occur due to the over-expression of a number of key pro-apoptotic peptides, including YCA1, YBH3, NUC1, and AIF1. Even though the protective effects were more pronounced with the over-expression of YBH3 and YCA1, hTC-1 could still protect yeast mutants lacking YBH3 and YCA1 from the effects of copper sulfate. This suggests that the protective effects of TC-1 are not limited to specific pathways or processes. Taken together, our results indicate that hTC-1 is a pro-survival protein that retains its function when heterologously expressed in yeast. Thus yeast is a useful model to characterize the potential roles in cell death and survival of cancer related genes.

  12. EFP1 is an ER stress-induced glycoprotein which interacts with the pro-apoptotic protein Par-4

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    Sarah Appel

    2009-05-01

    Full Text Available Sarah Appel1,2,6, Susanne Vetterkind1,2,6, Ansgar Koplin1,3, Barbara Maertens1,4, Meike Boosen1,5, Ute Preuss11The Institute of Genetics, University of Bonn, Bonn, Germany; 2Department of Health Sciences, Sargent College of Health and Rehabilitation Sciences, Boston University, Boston, MA, USA; 3Center for Molecular Biology Heidelberg (ZMBH, Heidelberg, Germany; 4Institute of Biochemistry II, University of Cologne, Cologne, Germany; 5Institute of Pharmacology and Toxicology, University Hospital of Johann Wolfgang Goethe-University Frankfurt am Main, Frankfurt am Main, Germany; 6These authors contributed equally to this work.Abstract: We have isolated the rat ortholog of EFP1 (EF-hand binding protein 1 as a novel interaction partner of the pro-apoptotic protein Par-4 (prostate apoptosis response-4. Rat EFP1 contains two thioredoxin domains, the COOH-terminal one harboring a CGFC motif, and has a similar protein domain structure as members of the protein disulfide isomerase (PDI family. In REF52.2 and CHO cells, EFP1 colocalized with the endoplasmic reticulum (ER marker PDI. Furthermore, EFP1 possesses catalytic activity as demonstrated by an insulin disulfide reduction assay. Western blot analysis revealed two EFP1 protein bands of approximately 136 and 155 kDa, representing different glycosylation states of the protein. Complex formation between EFP1 and Par-4 was confirmed in vitro and in vivo by co-immunoprecipitation, dot blot overlay and pull-down experiments. In CHO cells, coexpression of EFP1 and Par-4 resulted in enhanced Par-4-mediated apoptosis, which required the catalytic activity of EFP1. Interestingly, EFP1 was specifically upregulated in NIH3T3 cells after induction of ER stress by thapsigargin, tunicamycin, and brefeldin A, but not by agents that induce oxidative stress or ER-independent apoptosis. Furthermore, we could show that the induction of apoptosis by Ca2+ stress-inducing agents was significantly decreased after si

  13. Ethyl acetate extract of Chinese medicinal herb Sarcandra glabra induces growth inhibition on human leukemic HL-60 cells, associated with cell cycle arrest and up-regulation of pro-apoptotic Bax/Bcl-2 ratio.

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    Li, W Y; Chiu, Lawrence C M; Lam, W S; Wong, W Y; Chan, Y T; Ho, Y P; Wong, Elaine Y L; Wong, Y S; Ooi, Vincent E C

    2007-02-01

    Sarcandra glabra (Thunb.) Nakai, colloquially known as Caoshanhu, is a Chinese medicinal herb with reported anti-tumor, anti-inflammatory, anti-viral and non-specific immunoenhancing properties. Although the plant has been clinically used for treating a variety of diseases, its bioactive ingredients are largely unknown and its mode of action has never been investigated. In this study, the anti-tumor property of ethyl acetate (EA) extract of S. glabra was investigated by determining its in vitro growth-inhibitory effects on a panel of human cancer cell lines of different histotypes. Growth inhibition of the EA extract on the cancer cells seemed to be selective, and the leukemic HL-60 was found to be the most responsive after 48 h of treatment (IC50=58 microg/ml). Flow cytometric studies further illustrated that the extract might interfere with DNA replication and thus arrested the cell cycle at S phase in the leukemic cells, followed by DNA fragmentation and loss of phospholipid asymmetry in the plasma membrane after 72 h of treatment. Concurrently, the pro-apoptotic Bax/Bcl-2 ratio was also up-regulated by more than 178% of the control level. All these findings suggested that the extract had initiated apoptosis to kill the leukemic cells. Results from this pioneer study help to establish a scientific foundation for future research and development of the bioactive ingredients in EA extract of S. glabra as efficacious anti-cancer agents.

  14. β3 integrin promotes chemoresistance to epirubicin in MDA-MB-231 through repression of the pro-apoptotic protein, BAD

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    Nair, Madhumathy G.; Desai, Krisha; Prabhu, Jyothi S.; Hari, P.S.; Remacle, Jose; Sridhar, T.S., E-mail: tssridhar@sjri.res.in

    2016-08-01

    Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin β3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin β3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin β3 helps inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin β3 brought about a reversal of this effect and chemosensitized the cells. These results identify β3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress. - Highlights: • Integrin β3 signaling promotes chemoresistance to epirubicin in breast cancer cells. • Integrin β3 promotes cell survival and proliferation in drug treated cells through the PI3K and MAPK pathways. • Integrin signaling helps evade drug induced cytotoxicity by repression of pro-apoptotic molecule; BAD.

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

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    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.

  16. Ethanol Influences on Bax Associations with Mitochondrial Membrane Proteins in Neonatal Rat Cerebellum

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    Heaton, Marieta Barrow; Siler-Marsiglio, Kendra; Paiva, Michael; Kotler, Alexandra; Rogozinski, Jonathan; Kubovec, Stacey; Coursen, Mary; Madorsky, Vladimir

    2012-01-01

    These studies investigated interactions taking place at the mitochondrial membrane in neonatal rat cerebellum following ethanol exposure, and focused on interactions between pro-apoptotic Bax and proteins of the permeability transition pore (PTP), voltage-dependent anion channel (VDAC), and adenine nucleotide translocator (ANT), of the outer and inner mitochondrial membranes, respectively. Cultured cerebellar granule cells were used to assess the role of these interactions in ethanol neurotoxicity. Analyses were made at the age of maximal cerebellar ethanol vulnerability (P4), compared to the later age of relative resistance (P7), to determine whether differential ethanol sensitivity was mirrored by differences in these molecular interactions. We found that following ethanol exposure, Bax pro-apoptotic associations with both VDAC and ANT were increased, particularly at the age of greater ethanol sensitivity, and these interactions were sustained at this age for at least two hours post-exposure. Since Bax:VDAC interactions disrupt protective VDAC interactions with mitochondrial hexokinase (HXK), we also assessed VDAC:HXK associations following ethanol treatment, and found such interactions were altered by ethanol treatment, but only at two-hours post-exposure, and only in the P4, ethanol-sensitive cerebellum. Ethanol neurotoxicity in cultured neuronal preparations was abolished by pharmacological inhibition of both VDAC and ANT interactions with Bax, but not by a Bax channel blocker. Therefore, we conclude that at this age, within the constraints of our experimental model, a primary mode of Bax-induced initiation of the apoptosis cascade following ethanol insult involves interactions with proteins of the PTP complex, and not channel formation independent of PTP constituents. PMID:22767450

  17. The effect of N-nitrosodimethylamine (NDMA) on Bax and Mcl-1 expression in human neutrophils.

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    Jablonski, Jakub; Jablonska, Ewa; Leonik, Agnieszka

    2011-12-01

    In the present study we examined a role of pro-apoptotic Bax and anti-apoptotic Mcl-1 proteins, participating in the regulation of intrinsic apoptosis pathway in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA), the environmental xenobiotic. For the purpose comparison, the same studies were conducted in autologous peripheral blood mononuclear cells (PBMCs). The production of cytochrome c by PMNs was also determined. A deficit of anti-apoptotic Mcl-1 and overexpression of the pro-apoptotic protein Bax suggest that the apoptosis process in human neutrophils exposed to NDMA is dependent on changes in the expression of these proteins. PMNs were more sensitive to NDMA than PBMCs.

  18. Identification of residues within the African swine fever virus DP71L protein required for dephosphorylation of translation initiation factor eIF2α and inhibiting activation of pro-apoptotic CHOP

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    Barber, Claire; Netherton, Chris; Goatley, Lynnette [The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF (United Kingdom); Moon, Alice; Goodbourn, Steve [Institute for Infection and Immunity, St. George' s, University of London, London SW17 0RE (United Kingdom); Dixon, Linda, E-mail: linda.dixon@pirbright.ac.uk [The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF (United Kingdom)

    2017-04-15

    The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate the translation initiation factor 2α (eIF2α) and avoid shut-off of global protein synthesis and downstream activation of the pro-apoptotic factor CHOP. Residues V16 and F18A were critical for binding of DP71L to PP1. Mutation of this PP1 binding motif or deletion of residues between 52 and 66 reduced the ability of DP71L to cause dephosphorylation of eIF2α and inhibit CHOP induction. The residues LSAVL, between 57 and 61, were also required. PP1 was co-precipitated with wild type DP71L and the mutant lacking residues 52- 66 or the LSAVL motif, but not with the PP1 binding motif mutant. The residues in the LSAVL motif play a critical role in DP71L function but do not interfere with binding to PP1. Instead we propose these residues are important for DP71L binding to eIF2α. - Highlights: •The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate translation initiation factor eIF2α (eIF2α). •The residues V{sup 16}, F{sup 18} of DP71L are required for binding to the α, β and γ isoforms of PP1 and for DP71L function. •The sequence LSAVL downstream from the PP1 binding site (residues 57–61) are also important for DP71L function. •DP71L mutants of the LSAVL sequence retain ability to co-precipitate with PP1 showing these sequences have a different role to PP1 binding.

  19. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

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    Mas-Oliva, Jaime; Navarro-Vidal, Enrique; Tapia-Vieyra, Juana Virginia

    2014-01-01

    Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+)-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2) associated to a lethal influx of Ca(2+) in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line) transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  20. Irigenin sensitizes TRAIL-induced apoptosis via enhancing pro-apoptotic molecules in gastric cancer cells.

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    Xu, Ying; Gao, Cheng-Cheng; Pan, Zhen-Guo; Zhou, Chuan-Wen

    2018-02-12

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds promising value for cancer therapy due to its capacity to induce apoptosis in cancer cells. Nevertheless, TRAIL therapy is greatly hampered by its resistance. Irigenin (Iri), isoflavonoids, can be isolated from the rhizome of Belamcanda chinensis, and has been shown anti-cancer properties. In this study, we explored if Iri could enhance TRAIL-regulated apoptosis in TRAIL resistant gastric cancer cells. Iri significantly potentiated TRAIL-triggered cytotoxicity. Iri alone and TRAIL alone showed no effective role in apoptosis induction, whereas combined treatment with Iri and TRAIL markedly induced apoptosis in cancer cells, as evidenced by the up-regulation of cleaved Caspase-8/-9/-3 and PARP. Additionally, the sensitization to TRAIL was along with the enhancement of pro-apoptotic proteins, including FAS-associated protein with death domain (FADD), death receptor 5 (DR5) and Bax. And suppressing FADD, DR5 and Bax by si RNA significantly reduced the apoptosis and enhanced the cell viability induced by the co-application of Iri and TRAIL. Moreover, the sensitization to TRAIL was accompanied by the decrease of Cellular-FLICE inhibitory protein (c-FLIP), Bcl-2 and Survivin. Additionally, Iri could sensitize TRAIL to produce reactive oxygen species (ROS). Pre-treatment of N-acetyl-cysteine (NAC), ROS scavenger, attenuated Iri plus TRAIL-induced apoptosis and improved cell viability. Finally, combination of Iri and TRAIL inhibited tumor growth in the xenograft model. Collectively, our present study gave new insights into the effects of Iri on potentiating TRAIL-sensitivity, and suggested that Iri could be a potential candidate for sensitizer of TRAIL-resistant cancer cell treatment. Copyright © 2018. Published by Elsevier Inc.

  1. 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

  2. The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), 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

    2011-03-18

    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-X(L), 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-X(L) 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-X(L), 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-X(L)/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-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.

  3. Exon-skipping strategy by ratio modulation between cytoprotective versus pro-apoptotic clusterin forms increased sensitivity of LNCaP to cell death.

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    Abdellatif Essabbani

    Full Text Available BACKGROUND: In prostate cancer the secreted form of clusterin (sCLU has been described as an anti-apoptotic protein whose expression is increased after therapeutic intervention, whereas, the nuclear protein form nCLU was reported to have pro-apoptotic properties. METHODOLOGY: In order to provide new therapeutic approaches targeting CLU, we developed a strategy based on exon skipping by using a lentiviral construct to preferentially induce the nuclear spliced form of the protein. The molecular construct was transduced in LNCaP cells for testing the modulation of sensitivity of the transduced cells to pro-apoptotic stress. RESULTS AND CONCLUSIONS: We showed an increase of nCLU/sCLU expression ratio in the prostate cancer cell line "LNCaP" after lentiviral vector-U7 nCLU transduction. Moreover, we showed a significant inhibition of cell proliferation in nCLU-U7 LNCaP cells after treatment with cisplatin and after exposure to ionizing radiation compared to control cells. Finally, we showed that nCLU-U7 LNCaP cells exposure to UV-C significantly reduced an increase of cell death compared to control. Finally, we showed that modulating nCLU expression had profound impact on Ku70/Bax interaction as well as Rad17 expression which could be a key mechanism in sensitizing cells to cell death. In conclusion, this is the first report showing that increasing of nCLU/sCLU expression ratio by using an "on demand alternative splicing" strategy successfully increased sensitivity to radiotherapy and chemotherapy of prostate cancer cells.

  4. Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells

    Science.gov (United States)

    Rivera, Mariela; Ramos, Yanilda; Rodríguez-Valentín, Madeline; López-Acevedo, Sheila; Cubano, Luis A.; Zou, Jin; Zhang, Qiang; Wang, Guangdi

    2017-01-01

    Curcumin, an extract from the turmeric rhizome (Curcuma longa), is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12) and Poly (ADP-ribose) polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses. PMID:28628644

  5. Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Mariela Rivera

    Full Text Available Curcumin, an extract from the turmeric rhizome (Curcuma longa, is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12 and Poly (ADP-ribose polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses.

  6. Arsenic trioxide (AT) is a novel human neutrophil pro-apoptotic agent: effects of catalase on AT-induced apoptosis, degradation of cytoskeletal proteins and de novo protein synthesis.

    Science.gov (United States)

    Binet, François; Cavalli, Hélène; Moisan, Eliane; Girard, Denis

    2006-02-01

    The anti-cancer drug arsenic trioxide (AT) induces apoptosis in a variety of transformed or proliferating cells. However, little is known regarding its ability to induce apoptosis in terminally differentiated cells, such as neutrophils. Because neutropenia has been reported in some cancer patients after AT treatment, we hypothesised that AT could induce neutrophil apoptosis, an issue that has never been investigated. Herein, we found that AT-induced neutrophil apoptosis and gelsolin degradation via caspases. AT did not increase neutrophil superoxide production and did not induce mitochondrial generation of reactive oxygen species. AT-induced apoptosis in PLB-985 and X-linked chronic granulomatous disease (CGD) cells (PLB-985 cells deficient in gp91(phox) mimicking CGD) at the same potency. Addition of catalase, an inhibitor of H2O2, reversed AT-induced apoptosis and degradation of the cytoskeletal proteins gelsolin, alpha-tubulin and lamin B1. Unexpectedly, AT-induced de novo protein synthesis, which was reversed by catalase. Cycloheximide partially reversed AT-induced apoptosis. We conclude that AT induces neutrophil apoptosis by a caspase-dependent mechanism and via de novo protein synthesis. H2O2 is of major importance in AT-induced neutrophil apoptosis but its production does not originate from nicotinamide adenine dinucleotide phosphate dehydrogenase activation and mitochondria. Cytoskeletal structures other than microtubules can now be considered as novel targets of AT.

  7. The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

    International Nuclear Information System (INIS)

    Semaan, Sheila J; Nickells, Robert W

    2010-01-01

    Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116 BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. HCT116 BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of BAX aggregation at sub-saturation levels suggests that the

  8. The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

    Directory of Open Access Journals (Sweden)

    Semaan Sheila J

    2010-10-01

    Full Text Available Abstract Background Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Methods Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. Results HCT116BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Conclusion Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of

  9. Differential Effects of Ethanol on c-Jun N-Terminal Kinase, 14-3-3 Proteins, and Bax in Postnatal Day 4 and Postnatal Day 7 Rat Cerebellum

    Science.gov (United States)

    Heaton, Marieta Barrow; Paiva, Michael; Kubovic, Stacey; Kotler, Alexandra; Rogozinski, Jonathan; Swanson, Eric; Madorsky, Vladimir; Posados, Michelle

    2011-01-01

    These studies investigated ethanol effects on upstream cellular elements and interactions which contribute to Bax-related apoptosis in neonatal rat cerebellum at ages of peak ethanol sensitivity (postnatal day 4 [P4]), compared to later ages of relative resistance (P7). Analyses were made of basal levels of the pro-apoptotic c-jun N-termimal kinase (JNK), Bax, and the 14-3-3 anchoring proteins, as well as the responsiveness of these substances to ethanol at P4 versus P7. Dimerization of Bax with 14-3-3 was also investigated at the two ages following ethanol treatment, a process which sequesters Bax in the cytosol, thus inhibiting its mitochondrial translocation and disruption of the mitochondrial membrane potential. Cultured cerebellar granule cells were used to examine the protective potential of JNK inhibition on ethanol-mediated cell death. Basal levels of JNK were significantly higher at P4 than P7, but no differences in the other proteins were found. Activated JNK, and cytosolic and mitochondrially-translocated Bax were increased in P4 but not P7 animals following ethanol exposure, while protective 14-3-3 proteins were increased only at P7. Ethanol treatment resulted in decreases in Bax:14-3-3 heterodimers at P4, but not at P7. Inhibition of JNK activity in vitro provided partial protection against ethanol neurotoxicity. Thus, differential temporal vulnerability to ethanol in this CNS region correlates with differences in both levels of apoptosis-related substances (e.g., JNK), and differential cellular responsiveness, favoring apoptosis at the most sensitive age and survival at the resistant age. The upstream elements contributing to this vulnerability can be targets for future therapeutic strategies. PMID:22169498

  10. Intrinsic, pro-apoptotic effects of IGFBP-3 on breast cancer cells are reversible: Involvement of PKA, Rho and ceramide.

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    Claire M Perks

    2011-05-01

    Full Text Available We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signalling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signalling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A(PKA, Rho associated kinase (ROCK and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3.

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

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    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.

  12. Baculovirus PTP2 functions as a pro-apoptotic protein

    NARCIS (Netherlands)

    Han, Yue; Houte, van Stineke; Oers, van Monique M.; Ros, Vera I.D.

    2018-01-01

    The family Baculoviridae encompasses a large number of invertebrate viruses, mainly infecting caterpillars of the order Lepidoptera. The baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces physiological and behavioral changes in its host Spodoptera exigua, as well as

  13. Role of Bax in resveratrol-induced apoptosis of colorectal carcinoma cells

    International Nuclear Information System (INIS)

    Mahyar-Roemer, Mojgan; Köhler, Hans; Roemer, Klaus

    2002-01-01

    The natural plant polyphenol resveratrol present in some foods including grapes, wine, and peanuts, has been implicated in the inhibition, delay, and reversion of cellular events associated with heart diseases and tumorigenesis. Recent work has suggested that the cancer chemoprotective effect of the compound is primarily linked to its ability to induce cell division cycle arrest and apoptosis, the latter possibly through the activation of pro-apoptotic proteins such as Bax. The expression, subcellular localization, and importance of Bax for resveratrol-provoked apoptosis were assessed in human HCT116 colon carcinoma cells and derivatives with both bax alleles inactivated. Low to moderate concentrations of resveratrol induced co-localization of cellular Bax protein with mitochondria, collapse of the mitochondrial membrane potential, activation of caspases 3 and 9, and finally, apoptosis. In the absence of Bax, membrane potential collapse was delayed, and apoptosis was reduced but not absent. Resveratrol inhibited the formation of colonies by both HCT116 and HCT116 bax -/- cells. Resveratrol at physiological doses can induce a Bax-mediated and a Bax-independent mitochondrial apoptosis. Both can limit the ability of the cells to form colonies

  14. Expression and significance of Bax protein in model of radiation injury in mouse skin

    International Nuclear Information System (INIS)

    Feng Yizhong; Mo Yahong

    2002-01-01

    Objective: The study is to find some valuable criteria for diagnosis and treatment of radiation injury in skin. Methods: The expression of Bax protein was studied by SP immunohistochemistry in 40 cases of model of radiation injury in mouse skin. Their relationship relating to radiation dose was also investigated. Results: The expression rates of Bax were 30%, 30%, 70%, 70% in 5 Gy group, 15 Gy group, 30 Gy group, 45 Gy group respectively. There was no significant correlation between the expression of Bax and radiation groups. Conclusions: The experiment shows that radiation can increase the expression of Bax protein which might be related to poor healing in radiation skin injury

  15. Thiamine deficiency activates hypoxia inducible factor-1α to facilitate pro-apoptotic responses in mouse primary astrocytes.

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    Kristy Zera

    Full Text Available Thiamine is an essential enzyme cofactor required for proper metabolic function and maintenance of metabolism and energy production in the brain. In developed countries, thiamine deficiency (TD is most often manifested following chronic alcohol consumption leading to impaired mitochondrial function, oxidative stress, inflammation and excitotoxicity. These biochemical lesions result in apoptotic cell death in both neurons and astrocytes. Comparable histological injuries in patients with hypoxia/ischemia and TD have been described in the thalamus and mammillary bodies, suggesting a congruency between the cellular responses to these stresses. Consistent with hypoxia/ischemia, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α under physiological oxygen levels. However, the role of TD-induced HIF-1α in neurological injury is currently unknown. Using Western blot analysis and RT-PCR, we have demonstrated that TD induces HIF-1α expression and activity in primary mouse astrocytes. We observed a time-dependent increase in mRNA and protein expression of the pro-apoptotic and pro-inflammatory HIF-1α target genes MCP1, BNIP3, Nix and Noxa during TD. We also observed apoptotic cell death in TD as demonstrated by PI/Annexin V staining, TUNEL assay, and Cell Death ELISA. Pharmacological inhibition of HIF-1α activity using YC1 and thiamine repletion both reduced expression of pro-apoptotic HIF-1α target genes and apoptotic cell death in TD. These results demonstrate that induction of HIF-1α mediated transcriptional up-regulation of pro-apoptotic/inflammatory signaling contributes to astrocyte cell death during thiamine deficiency.

  16. 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.

  17. Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p53 and p21/WAF1 proteins in skin of mice

    International Nuclear Information System (INIS)

    Saxena, Neha; Ansari, Kausar M.; Kumar, Rahul; Dhawan, Alok; Dwivedi, Premendra D.; Das, Mukul

    2009-01-01

    Patulin (PAT), a mycotoxin found in apples, grapes, oranges, pear and peaches, is a potent genotoxic compound. WHO has highlighted the need for the study of cutaneous toxicity of PAT as manual labour is employed during pre and post harvest stages, thereby causing direct exposure to skin. In the present study cutaneous toxicity of PAT was evaluated following topical application to Swiss Albino mice. Dermal exposure of PAT, to mice for 4 h resulted in a dose (40-160 μg/animal) and time (up to 6 h) dependent enhancement of ornithine decarboxylase (ODC), a marker enzyme of cell proliferation. The ODC activity was found to be normal after 12 and 24 h treatment of patulin. Topical application of PAT (160 μg/100 μl acetone) for 24-72 h caused (a) DNA damage in skin cells showing significant increase (34-63%) in olive tail moment, a parameter of Comet assay (b) significant G 1 and S-phase arrest along with induction of apoptosis (2.8-10 folds) as shown by annexin V and PI staining assay through flow cytometer. Moreover PAT leads to over expression of p 21/WAF1 (3.6-3.9 fold), pro apoptotic protein Bax (1.3-2.6) and tumor suppressor wild type p 53 (2.8-3.9 fold) protein. It was also shown that PAT induced apoptosis was mediated through mitochondrial intrinsic pathway as revealed through the release of cytochrome C protein in cytosol leading to enhancement of caspase-3 activity in skin cells of mice. These results suggest that PAT has a potential to induce DNA damage leading to p 53 mediated cell cycle arrest along with intrinsic pathway mediated apoptosis that may also be correlated with enhanced polyamine production as evident by induction of ODC activity, which may have dermal toxicological implications

  18. Inhibition of Hepatocarcinogenesis by ArtinM via Anti-proliferative and Pro-apoptotic Mechanisms.

    Science.gov (United States)

    Braz, Mariana M; Roque-Barreira, Maria Cristina; Ramalho, Fernando S; Oliveira, Carlos A; Augusto, Marlei J; Ramalho, Leandra N Z

    ArtinM is a d-mannose-binding lectin found in the seeds of Artocarpus heterophyllus (jackfruit) that interacts with N-glycans, that is associated with receptors on the surface of phagocytic cells and induces the production of inflammatory mediators. Some of them are especially important because they may be required for antitumor immune response. This study aimed to evaluate the effect of ArtinM on hepatocellular preneoplastic foci. Wistar rats received 50 mg/kg of diethyl-nitrosamine (DEN) intraperitoneal weekly for 12 weeks. From the 14th week, the treated animals received 50 μg/kg of ArtinM subcutaneous every 2 weeks until the 18th week, whereas control animals were injected with vehicle alone. Preneoplastic-related factors were estimated using histological, western blotting and RT-PCR analysis. In comparison to the groups exposed to DEN, the ArtinM-treated rats showed diminution of preneoplastic foci, decreased expression of proliferating cell nuclear antigen (PCNA), increased number of nuclear p21 and p27 stained cells, augmented number of apoptotic cells, increased expression of p53, p42/44 MAPK and p21 proteins, reduced cyclin D1 (CCND1) protein levels and increased expression of TNFα and IFNγ genes. No difference was observed in interleukin 12 (IL12) protein levels. These findings indicate that ArtinM may provide protection against hepatocarcinogenesis as a result of the induction of cell-cycle blockage and pro-apoptotic mechanisms. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  19. Effects of Helicobacter pylori infection on the expressions of Bax and Bcl-2 in patients with chronic gastritis and gastric cancer.

    Science.gov (United States)

    Bartchewsky, Waldemar; Martini, Mariana R; Squassoni, Aline C; Alvarez, Marisa C; Ladeira, Marcelo S P; Salvatore, Daisy M F; Trevisan, Miriam A; Pedrazzoli, José; Ribeiro, Marcelo L

    2010-01-01

    The aim of the present study is to evaluate the influence of Helicobacter pylori on Bax and Bcl-2 mRNA and protein levels in patients with chronic gastritis and gastric cancer. The study included 217 patients, of which 26 were uninfected; 127 had chronic gastritis and were H. pylori-positive, and 64 had gastric cancer. Bacterial genotypes were evaluated by PCR, and the expression values were determined by quantitative real-time PCR and immunohistochemistry. Our data showed that the up-regulationary effects of H. pylori infection on the pro-apoptotic gene, Bax, were stronger than its induction of Bcl-2; this effect may increase apoptosis in patients with chronic gastritis. In patients with gastric cancer, the up-regulation of the anti-apoptotic gene, Bcl-2, counteracted the pro-apoptotic effects of Bax, leading to a deregulation of apoptosis-associated gene expression, favoring cell proliferation. Thus, the disturbance in Bax and Bcl-2 balance, induced by H. pylori, might be important in gastric cancer development.

  20. Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.

    Science.gov (United States)

    Rexford, Alix; Zorio, Diego A R; Miller, Brian G

    2017-01-01

    The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic β-cells. Photochemical crosslinking studies using a synthetic analog of BAD's BH3 domain and in vitro transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method for the production of recombinant human BAD. Consistent with published reports, recombinant BAD displays high affinity for Bcl-xL (KD = 7 nM), and phosphorylation of BAD at S118, within the BH3 domain, abolishes this interaction. Unexpectedly, we do not detect association of recombinant, full-length BAD with recombinant human pancreatic GCK over a range of protein concentrations using various biochemical methods including size-exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and isothermal titration calorimetry. Furthermore, fluorescence polarization assays and isothermal titration calorimetry detect no direct interaction between GCK and BAD BH3 peptides. Kinetic characterization of GCK in the presence of high concentrations of recombinant BAD show modest (BAD BH3 peptides. These results raise questions as to the mechanism of action of stapled peptide analogs modeled after the BAD BH3 domain, which reportedly enhance the Vmax value of GCK and stimulate insulin release in BAD-deficient islets. Based on our results, we postulate that the BAD:GCK interaction, and any resultant regulatory effect(s) upon GCK activity, requires the participation of additional members of the mitochondrial complex.

  1. Bioactives in cactus (Opuntia ficus-indica) stems possess potent antioxidant and pro-apoptotic activities through COX-2 involvement.

    Science.gov (United States)

    Kim, Jinhee; Soh, Soon Yil; Shin, Juha; Cho, Chi-Woung; Choi, Young Hee; Nam, Sang-Yong

    2015-10-01

    Bioactives extracted from cactus (Opuntia ficus-indica) stems were investigated for their chemopreventive activities using human cancer cells in vitro. The bioactives present in crude extracts were detected and quantified using high-performance liquid chromatography. Among all the extracts, such as hexane, ethyl acetate (EtOAc), acetone, methanol (MeOH), and MeOH:water (80:20), the MeOH extract had the highest amount of polyphenolic compounds and the acetone extract exhibited the most potent effect at scavenging the 2,2,-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS(•+) ) radical. In addition, most of the extracts, with the exception of hexane, exhibited significant cytotoxicity in human SW480 colon and MCF7 breast cancer cells. Overall, the SW480 cells were more sensitive than the MCF7 cells to the cytotoxic effect of the O. ficus-indica extracts (OFEs). Cell death by OFE treatment caused significant inhibition of cyclooxygenase-2 and increased the Bax/Bcl2 ratio in both SW480 and MCF7 cell lines. However, degradation of poly (ADP-ribose) polymerase was significantly increased by OFE only in the MCF7 cells, thereby inducing apoptosis. These findings demonstrate the health-benefit roles, including anti-oxidative and anti-proliferative activities as well as pro-apoptotic effects, of bioactive compounds in OFEs, suggesting a chemopreventive role in human cancer cells. © 2014 Society of Chemical Industry.

  2. The associated expression of Maspin and Bax proteins as a potential prognostic factor in intrahepatic cholangiocarcinoma

    International Nuclear Information System (INIS)

    Romani, Antonello A; Soliani, Paolo; Desenzani, Silvia; Borghetti, Angelo F; Crafa, Pellegrino

    2006-01-01

    Maspin, a member of the serpin family, is a suppressor of tumor growth, an inhibitor of angiogenesis and an inducer of apoptosis. Maspin induces apoptosis by increasing Bax, a member of the Bcl-2 family of apoptosis-regulating proteins. In this exploratory study, we investigated the associated expression of Maspin and Bax proteins as a potential prognostic factor in intrahepatic cholangiocarcinoma (IHCCA). Twenty-two paraffin-embedded samples were analyzed by immunohistochemical methods using Maspin, Bax and CD34 antibodies. Maspin was scored semiquantitatively (HSCORE). Apoptosis was assessed using an antibody against cleaved caspase-3. The strong relationship observed between the expression of Maspin and Bax, indicates that Bax is likely to be the key effector of Maspin-mediated induction of apoptosis as indicated by the activation of cleaved caspase-3. We categorized Maspin HSCORE by calculating the optimal cutpoint. A Maspin HSCORE above the cutpoint was inversely related with tumor dimension, depth of tumor and vascular invasion. Uni/multivariate analysis suggests that a Maspin HSCORE below the cutpoint significantly worsens the patients' prognosis. Tumors with Maspin HSCORE below the cutpoint had a shorter survival (11+/-5 months) than did patients with Maspin HSCORE above the cutpoint (27+/-4 months), whereas Kaplan-Meier analysis and logrank test showed no significant difference in overall survival between the patients. The associated expression of Maspin and Bax might delay tumor progression in IHCCA. Maspin above the cutpoint might counteract tumor development by increasing cell apoptosis, and by decreasing tumor mass and cell invasion. The combined expression of Maspin and Bax appears to influence the susceptibility of tumor cholangiocytes to apoptosis and thus may be involved in delaying IHCCA progression

  3. Pro-apoptotic effect of a Mycoplasma hyopneumoniae putative type I signal peptidase on PK(15) swine cells.

    Science.gov (United States)

    Paes, Jéssica A; Virginio, Veridiana G; Cancela, Martín; Leal, Fernanda M A; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Schrank, Irene S; Ferreira, Henrique B

    2017-03-01

    Mycoplasma hyopneumoniae is an economically significant swine pathogen that causes porcine enzootic pneumonia (PEP). Important processes for swine infection by M. hyopneumoniae depend on cell surface proteins, many of which are secreted by secretion pathways not completely elucidated so far. A putative type I signal peptidase (SPase I), a possible component of a putative Sec-dependent pathway, was annotated as a product of the sipS gene in the pathogenic M. hyopneumoniae 7448 genome. This M. hyopneumoniae putative SPase I (MhSPase I) displays only 14% and 23% of sequence identity/similarity to Escherichia coli bona fide SPase I, and, in complementation assays performed with a conditional E. coli SPase I mutant, only a partial restoration of growth was achieved with the heterologous expression of a recombinant MhSPase I (rMhSPase I). Considering the putative surface location of MhSPase I and its previously demonstrated capacity to induce a strong humoral response, we then assessed its potential to elicit a cellular and possible immunomodulatory response. In assays for immunogenicity assessment, rMhSPase I unexpectedly showed a cytotoxic effect on murine splenocytes. This cytotoxic effect was further confirmed using the swine epithelial PK(15) cell line in MTT and annexin V-flow cytometry assays, which showed that rMhSPase I induces apoptosis in a dose dependent-way. It was also demonstrated that this pro-apoptotic effect of rMhSPase I involves activation of a caspase-3 cascade. The potential relevance of the rMhSPase I pro-apoptotic effect for M. hyopneumoniae-host interactions in the context of PEP is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. 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.

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

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    Katharine J Goodall

    Full Text Available 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.

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

    International Nuclear Information System (INIS)

    Bougras, Gwenola; Cartron, Pierre-François; Gautier, Fabien; Martin, Stéphane; LeCabellec, Marité; Meflah, Khaled; Gregoire, Marc; Vallette, François M

    2004-01-01

    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. 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. 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. 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

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

    Science.gov (United States)

    Dyugovskaya, Larissa; Polyakov, Andrey; Cohen-Kaplan, Victoria; Lavie, Peretz; Lavie, Lena

    2012-10-22

    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. 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. 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 the IH-induced Mcl-1 increase. In SH, only p38MAPK

  8. Effect of polysaccharides from Angelica sinensis on Bcl-2 and Bax protein expression of irradiated liver cells

    International Nuclear Information System (INIS)

    Sun Yuanlin; Tang Jian; Gu Xiaohong; Li Deyuan

    2009-01-01

    Objective: To investigate the effect of polysaccharides from Angelica sinensis (ASP3) on Bcl-2 and Bax protein expression of irradiated liver cells from mice. Methods: Bcl-2 and Bax protein expression of liver cells in vitro exposed to 2.0 Gy rays were examined by using immunohistochemistry method. Results: The expression of apoptosis-accelerating protein Bax in the irradiation group was enhanced obviously (70.83%), while apoptosis inhibiting protein Bcl-2 tended to decline (55.60%), with the statistically significant difference (P <0.01) compared with that of the control. ASP3 pretreatment could regulate Bcl-2 and Bax protein expression of liver cells, inhibiting Bax protein expression(64.14/58.37%) and increasing Bcl-2 protein expression(59.21%/ 67.45%). The differences between the high dosage (100 mg/L of ASP3) and the irradiation group were statistically significant (P<0.05). Conclusions: ASP3 pretreatment could prohibit the apoptosis of radiation- damaged liver cells due to abnormal expression of Bcl-2 and Bax, and reduce the cell apoptosis by increasing Bcl-2/Bax protein expression so as to enhance the radiation endurance of liver cells. (authors)

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

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    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.

  10. Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling

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    Takehara Tadamichi

    2006-03-01

    Full Text Available Abstract Background Porphyromonas gingivalis is the foremost oral pathogen of adult periodontitis in humans. However, the mechanisms of bacterial invasion and the resultant destruction of the gingival tissue remain largely undefined. Results We report host-P. gingivalis interactions in primary human gingival fibroblast (HGF cells. Quantitative immunostaining revealed the need for a high multiplicity of infection for optimal infection. Early in infection (2–12 h, P. gingivalis activated the proinflammatory transcription factor NF-kappa B, partly via the PI3 kinase/AKT pathway. This was accompanied by the induction of cellular anti-apoptotic genes, including Bfl-1, Boo, Bcl-XL, Bcl2, Mcl-1, Bcl-w and Survivin. Late in infection (24–36 h the anti-apoptotic genes largely shut down and the pro-apoptotic genes, including Nip3, Hrk, Bak, Bik, Bok, Bax, Bad, Bim and Moap-1, were activated. Apoptosis was characterized by nuclear DNA degradation and activation of caspases-3, -6, -7 and -9 via the intrinsic mitochondrial pathway. Use of inhibitors revealed an anti-apoptotic function of NF-kappa B and PI3 kinase in P. gingivalis-infected HGF cells. Use of a triple protease mutant P. gingivalis lacking three major gingipains (rgpA rgpB kgp suggested a role of some or all these proteases in myriad aspects of bacteria-gingival interaction. Conclusion The pathology of the gingival fibroblast in P. gingivalis infection is affected by a temporal shift from cellular survival response to apoptosis, regulated by a number of anti- and pro-apoptotic molecules. The gingipain group of proteases affects bacteria-host interactions and may directly promote apoptosis by intracellular proteolytic activation of caspase-3.

  11. Induction of Pro-Apoptotic Endoplasmic Reticulum Stress in Multiple Myeloma Cells by NEO214, Perillyl Alcohol Conjugated to Rolipram.

    Science.gov (United States)

    Chen, Thomas C; Chan, Nymph; Labib, Shirin; Yu, Jiali; Cho, Hee-Yeon; Hofman, Florence M; Schönthal, Axel H

    2018-01-17

    Despite the introduction of new therapies for multiple myeloma (MM), many patients are still dying from this disease and novel treatments are urgently needed. We have designed a novel hybrid molecule, called NEO214, that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH), an inducer of endoplasmic reticulum (ER) stress, to rolipram (Rp), an inhibitor of phosphodiesterase-4 (PDE4). Its potential anticancer effects were investigated in a panel of MM cell lines. We found that NEO214 effectively killed MM cells in vitro with a potency that was over an order of magnitude stronger than that of its individual components, either alone or in combination. The cytotoxic mechanism of NEO214 involved severe ER stress and prolonged induction of CCAAT/enhancer-binding protein homologous protein (CHOP), a key pro-apoptotic component of the ER stress response. These effects were prevented by salubrinal, a pharmacologic inhibitor of ER stress, and by CHOP gene knockout. Conversely, combination of NEO214 with bortezomib, a drug in clinical use for patients with MM, resulted in synergistic enhancement of MM cell death. Combination with the adenylate cyclase stimulant forskolin did not enhance NEO214 impact, indicating that cyclic adenosine 3',5'-monophosphate (AMP) pathways might play a lesser role. Our study introduces the novel agent NEO214 as a potent inducer of ER stress with significant anti-MM activity in vitro. It should be further investigated as a potential MM therapy aimed at exploiting this tumor's distinct sensitivity to ER stress.

  12. Antiproliferative and Pro-Apoptotic Effect of Novel Nitro-Substituted Hydroxynaphthanilides on Human Cancer Cell Lines

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    Tereza Kauerova

    2016-07-01

    Full Text Available Ring-substituted hydroxynaphthanilides are considered as cyclic analogues of salicylanilides, compounds possessing a wide range of pharmacological activities, including promising anticancer properties. The aim of this study was to evaluate the potential anticancer effect of novel nitro-substituted hydroxynaphthanilides with a special focus on structure-activity relationships. The antiproliferative effect was assessed by Water Soluble Tetrazolium Salts-1 (WST-1 assay, and cytotoxicity was evaluated via dye exclusion test. Flow cytometry was used for cell cycle analysis and detection of apoptosis using Annexin V-FITC/PI assay. Protein expression was estimated by Western blotting. Our data indicate that the potential to cause the antiproliferative effect increases with the shift of the nitro substituent from the ortho- to the para-position. The most potent compounds, 3-hydroxy-N-(3-nitrophenylnaphthalene-2-carboxamide (2, and 2-hydroxy-N-(4-nitrophenyl-naphthalene-1-carboxamide (6 showed antiproliferative activity against THP-1 and MCF-7 cancer cells without affecting the proliferation of 3T3-L1 non-tumour cells. Compounds 2 and 6 induced the accumulation of THP-1 and MCF-7 cells in G1 phase associated with the downregulation of cyclin E1 protein levels, while the levels of cyclin B1 were not affected. Moreover, compound 2 was found to exert the pro-apoptotic effect on the THP-1 cells. These results suggest that hydroxynaphthanilides might represent a potential model structure for the development of novel anticancer agents.

  13. Ebi/AP-1 suppresses pro-apoptotic genes expression and permits long-term survival of Drosophila sensory neurons.

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    Young-Mi Lim

    Full Text Available Sensory organs are constantly exposed to physical and chemical stresses that collectively threaten the survival of sensory neurons. Failure to protect stressed neurons leads to age-related loss of neurons and sensory dysfunction in organs in which the supply of new sensory neurons is limited, such as the human auditory system. Transducin β-like protein 1 (TBL1 is a candidate gene for ocular albinism with late-onset sensorineural deafness, a form of X-linked age-related hearing loss. TBL1 encodes an evolutionarily conserved F-box-like and WD40 repeats-containing subunit of the nuclear receptor co-repressor/silencing mediator for retinoid and thyroid hormone receptor and other transcriptional co-repressor complexes. Here we report that a Drosophila homologue of TBL1, Ebi, is required for maintenance of photoreceptor neurons. Loss of ebi function caused late-onset neuronal apoptosis in the retina and increased sensitivity to oxidative stress. Ebi formed a complex with activator protein 1 (AP-1 and was required for repression of Drosophila pro-apoptotic and anti-apoptotic genes expression. These results suggest that Ebi/AP-1 suppresses basal transcription levels of apoptotic genes and thereby protects sensory neurons from degeneration.

  14. Quantitative analysis of receptor-mediated uptake and pro-apoptotic activity of mistletoe lectin-1 by high content imaging.

    Science.gov (United States)

    Beztsinna, N; de Matos, M B C; Walther, J; Heyder, C; Hildebrandt, E; Leneweit, G; Mastrobattista, E; Kok, R J

    2018-02-09

    Ribosome inactivating proteins (RIPs) are highly potent cytotoxins that have potential as anticancer therapeutics. Mistletoe lectin 1 (ML1) is a heterodimeric cytotoxic protein isolated from European Mistletoe and belongs to RIP class II. The aim of this project was to systematically study ML1 cell binding, endocytosis pathway(s), subcellular processing and apoptosis activation. For this purpose, state of the art cell imaging equipment and automated image analysis algorithms were used. ML1 displayed very fast binding to sugar residues on the membrane and energy-dependent uptake in CT26 cells. The co-staining with specific antibodies and uptake blocking experiments revealed involvement of both clathrin-dependent and -independent pathways in ML1 endocytosis. Co-localization studies demonstrated the toxin transport from early endocytic vesicles to Golgi network; a retrograde road to the endoplasmic reticulum. The pro-apoptotic and antiproliferative activity of ML1 were shown in time lapse movies and subsequently quantified. ML1 cytotoxicity was less affected in multidrug resistant tumor cell line 4T1 in contrast to commonly used chemotherapeutic drug (ML1 resistance index 6.9 vs 13.4 for doxorubicin; IC 50 : ML1 1.4 ng/ml vs doxorubicin 24000 ng/ml). This opens new opportunities for the use of ML1 as an alternative treatment in multidrug resistant cancers.

  15. 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.

  16. Reduced expression of bax in small cell lung cancer cells is not sufficient to induce cisplatin-resistance

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    Biagosch J

    2010-10-01

    Full Text Available Abstract Resistance to cisplatin in the course of chemotherapy contributes to the poor prognosis of small cell lung cancer (SCLC. B cell lymphoma-2 is the founding member of a large family of proteins that either promote or inhibit apoptosis. We aimed at investigating if the pro-apoptotic members Bad, Bax, Bim and Bid are involved in cisplatin-resistance. Cisplatin-resistance in the SCLC cell line H1339 was induced by repetitive exposure to cisplatin. Protein expression was quantified by Western Blot and immuno-fluorescence analysis. Protein expression was altered using siRNA interference. Four "cycles" of 0.5 μg/ml cisplatin led to partial cisplatin-resistance in H1339 cells. The expression of Bad, Bim and Bid was comparable in naïve and resistant cells while the expression of Bax was reduced in the resistant clone. But, reducing Bax expression in naïve cells did not lead to altered cisplatin sensitivity neither in H1339 nor in H187 SCLC cells. We conclude that the reduced Bax expression after exposure to cisplatin is not sufficient to induce cis-platin-resistance in SCLC cells.

  17. Loss of anti-Bax function in Gerstmann-Sträussler-Scheinker syndrome-associated prion protein mutants.

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    Julie Jodoin

    2009-08-01

    Full Text Available Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD-associated prion protein (PrP mutants that are unable to generate cytosolic PrP (CyPrP. To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS-associated PrP mutants. These PrP mutants contained their respective methionine ((M or valine ((V at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S(V, D202N(V, P102L(V and Q217R(V retained, whereas the P102L(M, P105L(V, Y145stop(M and Q212P(M PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L(V, none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.

  18. Correlation of mammographical imaging signs with the expression of bcl-2 and bax proteins in breast cancer

    International Nuclear Information System (INIS)

    Zhang Yili; Du Hongwen; Zhang Yun; Zhang Yuelang; Kuang Fangjun; Guo Zuomin

    2004-01-01

    Objective: To discuss the correlation of mammographical imaging signs with the expression of bcl-2 and bax proteins in breast cancer for early diagnosis and forecast of its prognoses. Methods: Fifty-four breast cancers and 26 benign diseases were proved by pathologic methods and all cases underwent mammography. Immunohistochemical technique was used to measure the expression of bcl-2 and bax proteins in these tissues. The correlation of imaging signs with the expression of bcl-2 and bax proteins in breast cancer and benign lesion was analyzed. Results: The expression of bcl-2 or bax protein in the breast cancer was higher than that in breast benign diseases (χ 2 =15.116, 11.361, P 2 =10.358, 12.818, P 2 =10.996, 10.667, P 2 =10.405, P 2 =6.841, P<0.05). Conclusion: Some imaging signs of breast cancer were closely related to the expression of bcl-2 and bax proteins and these signs could reflect the biological behavior of tumor cells and prognoses. Therefore it could be helpful to the early diagnosis and treatment of breast cancer. (authors)

  19. Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

    Science.gov (United States)

    Zareba, Ilona; Surazynski, Arkadiusz; Chrusciel, Marcin; Miltyk, Wojciech; Doroszko, Milena; Rahman, Nafis; Palka, Jerzy

    2017-01-01

    The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

  20. The pro-apoptotic action of the peptide hormone Neb-colloostatin on insect haemocytes.

    Science.gov (United States)

    Czarniewska, E; Mrówczynska, L; Kuczer, M; Rosinski, G

    2012-12-15

    The gonadoinhibitory peptide hormone Neb-colloostatin was first isolated from ovaries of the flesh fly Neobellieria bullata. This 19-mer peptide is thought to be a cleaved product of a collagen-like precursor molecule that is formed during remodelling of the extracellular matrix. In this study, we report that upon injection of picomolar and nanomolar doses, this peptide exerts a pro-apoptotic action on haemocytes of Tenebrio molitor adults, as visualized by changes in morphology and viability. The F-actin cytoskeleton was found to aggregate into distinctive patches. This may be responsible for the observed inhibition of adhesion of haemocytes and for the stimulation of filopodia formation. However, Neb-colloostatin injection did not induce the formation of autophagic vacuoles. Our results suggest that physiological concentrations of Neb-colloostatin play an important role in controlling the quantity and activity of haemocytes in insect haemolymph. They also suggest that during periods in which Neb-colloostatin is released, this peptide may cause a weakening of the insects' immune system. This is the first report that exposure to a peptide hormone causes apoptosis in insect haemocytes.

  1. 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

  2. Involvement of Bax and Bcl2 in Neuroprotective Effect of Curcumin in Kainic Acid-Induced Model of Temporal Lobe Epilepsy in Male Rat

    Directory of Open Access Journals (Sweden)

    zahra Kiasalari

    2016-04-01

    Full Text Available Background & objectives: Temporal lobe epilepsy is associated with neuronal apoptosis. Curcumin has antioxidant and anticonvulsant activities, therefore this study was conducted to assess involvement of Bax and Bcl2 in protective effect of curcumin in epileptic rats. Methods: 28 rats were divided into sham, curcumin-pretreated sham, epileptic (kainate, and curcumin-pretreated epileptic groups. Experimental model of epilepsy was induced by intrahippocampal administration of kainic acid. Rats received curcumin at a dose of 100 mg/kg. Finally, Nissl staining and Bax and Bcl2 immunohistochemistry were conducted on hippocampal sections and data were analyzed using one-way ANOVA and unpaired t-test. The p-value less than 0.05was considered statistically significant. Results: Induction of epilepsy was followed by a significant seizure and curcumin pretreatment significantly reduced seizure intensity (p<0.01. In addition, there were no significant differences between the groups in Nissl staining of CA3 area neurons. In addition, Bax positive neurons were observed in CA3 area in kainate group and significantly decreased in curcumin pretreated rats (p<0.05. Meanwhile, Bcl2 positive neurons were also moderately observed in kainate group and curcumin pretreatment significantly increased it (p<0.05. Conclusion: Curcumin pretreatment exhibits anticonvulsant activity in epileptic rats. It also decreases the expression of pro-apoptotic protein Bax and significantly enhances the expression of anti-apoptotic protein Bcl2 and hence could reduce neuronal apoptosis.

  3. 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

    -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...

  4. 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.

  5. Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress.

    Science.gov (United States)

    Ishikawa, Toshiki; Aki, Toshihiko; Yanagisawa, Shuichi; Uchimiya, Hirofumi; Kawai-Yamada, Maki

    2015-10-01

    BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death. © 2015 American Society of Plant Biologists. All Rights Reserved.

  6. Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Krüger, Katharina; Ziegler, Verena; Hartmann, Christina; Henninger, Christian [Institute of Toxicology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf (Germany); Thomale, Jürgen [Institute of Cell Biology, University Duisburg-Essen, 45122 Essen (Germany); Schupp, Nicole [Institute of Toxicology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf (Germany); Fritz, Gerhard, E-mail: fritz@uni-duesseldorf.de [Institute of Toxicology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf (Germany)

    2016-02-01

    The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage of Cis

  7. Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

    International Nuclear Information System (INIS)

    Krüger, Katharina; Ziegler, Verena; Hartmann, Christina; Henninger, Christian; Thomale, Jürgen; Schupp, Nicole; Fritz, Gerhard

    2016-01-01

    The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage of Cis

  8. 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.

  9. Design and synthesis of thienopyrimidine urea derivatives with potential cytotoxic and pro-apoptotic activity against breast cancer cell line MCF-7.

    Science.gov (United States)

    Abdelhaleem, Eman F; Abdelhameid, Mohammed K; Kassab, Asmaa E; Kandeel, Manal M

    2018-01-01

    A series of novel tetrahydrobenzothieno[2,3-d]pyrimidine urea derivatives was synthesized according to fragment-based design strategy. They were evaluated for their anticancer activity against MCF-7 cell line. Three compounds 9c, 9d and 11b showed 1.5-1.03 folds more potent anticancer activity than doxorubicin. In this study, a promising multi-sited enzyme small molecule inhibitor 9c, which showed the most potent anti-proliferative activity, was identified. The anti-proliferative activity of this compound appears to correlate well with its ability to inhibit topoisomerase II (IC 50  = 9.29 μM). Moreover, compound 9c showed excellent VEGFR-2 inhibitory activity, at the sub-micromolar level with IC 50 value 0.2 μM, which is 2.1 folds more potent than sorafenib. Moreover, activation of damage response pathway of the DNA leads to cell cycle arrest at G2/M phase, accumulation of cells in pre-G1 phase and annexin-V and propidium iodide staining, indicating that cell death proceeds through an apoptotic mechanism. Compound 9c showed potent pro-apoptotic effect through induction of the intrinsic mitochondrial pathway of apoptosis. This mechanistic pathway was confirmed by a significant increase in the expression of the tumor suppressor gene p53, elevation in Bax/BCL-2 ratio and a significant increase in the level of active caspase-3. Quantitative structure-activity relationship (QSAR) studies delivered equations of five 3D descriptors with R 2  = 0.814. This QSAR model provides an effective technique for understanding the observed antitumor properties and thus could be adopted for developing effective lead structures. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  10. Pro-apoptotic protein Noxa regulates memory T cell population size and protects against lethal immunopathology

    NARCIS (Netherlands)

    Wensveen, Felix M.; Klarenbeek, Paul L.; van Gisbergen, Klaas P. J. M.; Pascutti, Maria F.; Derks, Ingrid A. M.; van Schaik, Barbera D. C.; ten Brinke, Anja; de Vries, Niek; Cekinovic, Durdica; Jonjic, Stipan; van Lier, René A. W.; Eldering, Eric

    2013-01-01

    Memory T cells form a highly specific defense layer against reinfection with previously encountered pathogens. In addition, memory T cells provide protection against pathogens that are similar, but not identical to the original infectious agent. This is because each T cell response harbors multiple

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Schiff base metal derivatives enhance the expression of HSP70 and suppress BAX proteins in prevention of acute gastric lesion.

    Science.gov (United States)

    Golbabapour, Shahram; Gwaram, Nura Suleiman; Al-Obaidi, Mazen M Jamil; Soleimani, A F; Ali, Hapipah Mohd; Abdul Majid, Nazia

    2013-01-01

    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.

  13. 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

  14. BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori.

    Science.gov (United States)

    Yi, Hua-Shan; Pan, Cai-Xia; Pan, Chun; Song, Juan; Hu, Yan-Fen; Wang, La; Pan, Min-Hui; Lu, Cheng

    2014-02-28

    In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a (169)QACRG(173) sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Association of Pro-apoptotic Bad Gene Expression Changes with Benign Thyroid Nodules.

    Science.gov (United States)

    Gül, Nurdan; Temel, Berna; Ustek, Duran; Sirma-Ekmekçi, Sema; Kapran, Yersu; Tunca, Fatih; Giles-Şenyürek, Yasemin; Özbek, Uğur; Alagöl, Faruk

    2018-01-01

    This study aimed to investigate the role of the mitochondrial apoptotic pathway in benign thyroid nodules. Paired samples of nodular and normal tissues were collected from 26 patients with nodular goiters undergoing thyroidectomy. Variable expression of Bcl-2, Bax and Bad genes were evaluated by quantitative PCR. Expression level of Bad gene in nodules was found to be significantly decreased compared to normal tissues (p=0.049). A positive correlation was observed between nodule size and Bad expression levels (correlation coefficient=0.563, p=0.004); and this correlation was stronger in hot nodules (n=18, correlation coefficient=0.689, p=0.003). No significant difference was observed between nodular and normal tissue expressions of Bax and Bcl-2. These results suggest that Bad expression correlates with the size of benign thyroid nodules and also its relatively lower expression in nodules, warrant further investigation. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  16. Inhibition of Xenograft tumor growth by gold nanoparticle-DNA oligonucleotide conjugates-assisted delivery of BAX mRNA.

    Directory of Open Access Journals (Sweden)

    Ji-Hyun Yeom

    Full Text Available Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

  17. 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.

  18. 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

  19. 1800 MHz mobile phone irradiation induced oxidative and nitrosative stress leads to p53 dependent Bax mediated testicular apoptosis in mice, Mus musculus.

    Science.gov (United States)

    Shahin, Saba; Singh, Surya P; Chaturvedi, Chandra M

    2018-09-01

    Present study was carried out to investigate the effect of long-term mobile phone radiation exposure in different operative modes (Dialing, Receiving, and Stand-by) on immature male mice. Three-week old male mice were exposed to mobile phone (1800 MHz) radiation for 3 hr/day for 120 days in different operative modes. To check the changes/alteration in testicular histoarchitecture and serum testosterone level, HE staining and ELISA was performed respectively. Further, we have checked the redox status (ROS, NO, MDA level, and antioxidant enzymes: SOD, CAT, and GPx) by biochemical estimation, alteration in the expression of pro-apoptotic proteins (p53 and Bax), active executioner caspase-3, full length/uncleaved PARP-1 (DNA repair enzyme), anti-apoptotic proteins (Bcl-2 and Bcl-x L ) in testes by immunofluorescence and cytosolic cytochrome-c by Western blot. Decreased seminiferous tubule diameter, sperm count, and viability along with increased germ cells apoptosis and decreased serum testosterone level, was observed in the testes of all the mobile phone exposed mice compared with control. We also observed that, mobile phone radiation exposure in all the three different operative modes alters the testicular redox status via increasing ROS, NO, and MDA level, and decreasing antioxidant enzymes levels leading to enhanced apoptosis of testicular cells by increasing the expression of pro-apoptotic and apoptotic proteins along with decreasing the expression of anti-apoptotic protein. On the basis of results, it is conclude that long-term mobile phone radiation exposure induced oxidative stress leads to apoptosis of testicular cells and thus impairs testicular function. © 2018 Wiley Periodicals, Inc.

  20. 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

  1. Pro-Apoptotic Role of the Human YPEL5 Gene Identified by Functional Complementation of a Yeast moh1Δ Mutation.

    Science.gov (United States)

    Lee, Ji Young; Jun, Do Youn; Park, Ju Eun; Kwon, Gi Hyun; Kim, Jong-Sik; Kim, Young Ho

    2017-03-28

    To examine the pro-apoptotic role of the human ortholog (YPEL5) of the Drosophila Yippee protein, the cell viability of Saccharomyces cerevisiae mutant strain with deleted MOH1 , the yeast ortholog, was compared with that of the wild-type (WT)- MOH1 strain after exposure to different apoptogenic stimulants, including UV irradiation, methyl methanesulfonate (MMS), camptothecin (CPT), heat shock, and hyperosmotic shock. The moh1 Δ mutant exhibited enhanced cell viability compared with the WT- MOH1 strain when treated with lethal UV irradiation, 1.8 mM MMS, 100 µ CPT, heat shock at 50°C, or 1.2 M KCl. At the same time, the level of Moh1 protein was commonly up-regulated in the WT- MOH1 strain as was that of Ynk1 protein, which is known as a marker for DNA damage. Although the enhanced UV resistance of the moh1 Δ mutant largely disappeared following transformation with the yeast MOH1 gene or one of the human YPEL1-YPEL5 genes, the transformant bearing pYES2- YPEL5 was more sensitive to lethal UV irradiation and its UV sensitivity was similar to that of the WT- MOH1 strain. Under these conditions, the UV irradiation-induced apoptotic events, such as FITC-Annexin V stainability, mitochondrial membrane potential (ΔΨm) loss, and metacaspase activation, occurred to a much lesser extent in the moh1 Δ mutant compared with the WT- MOH1 strain and the mutant strain bearing pYES2- MOH1 or pYES2- YPEL5 . These results demonstrate the functional conservation between yeast Moh1 and human YPEL5, and their involvement in mitochondria-dependent apoptosis induced by DNA damage.

  2. cAMP signalling of Bordetella adenylate cyclase toxin through the SHP-1 phosphatase activates the BimEL-Bax pro-apoptotic cascade in phagocytes

    Czech Academy of Sciences Publication Activity Database

    Ahmad, Jawid Nazir; Černý, Ondřej; Linhartová, Irena; Mašín, Jiří; Osička, Radim; Šebo, Peter

    2016-01-01

    Roč. 18, č. 3 (2016), s. 384-398 ISSN 1462-5814 R&D Projects: GA ČR GA13-14547S; GA ČR(CZ) GAP302/11/0580; GA ČR GAP302/12/0460 Institutional support: RVO:61388971 Keywords : FORKHEAD TRANSCRIPTION FACTOR * HUMAN T- CELL S * DENDRITIC CELL S Subject RIV: EE - Microbiology, Virology Impact factor: 4.554, year: 2016

  3. Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2014-01-15

    7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4 days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and cultured for 4 or 8 days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P < 0.05) at 4 days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P < 0.05) but CX43 protein increased (P < 0.05) at 4 days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P < 0.05) but no changes in Bax expression were observed after 4 days of DMBA exposures. In contrast, after 8 days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia. - Highlights: • DMBA increases Cx37 and Cx43 expression prior to follicle loss. • During follicle loss both Cx37 and Cx43 expressions are reduced. • CX43 protein is absent in follicle remnants lacking an oocyte.

  4. A novel assay for discovery and characterization of pro-apoptotic drugs and for monitoring apoptosis in patient sera.

    Science.gov (United States)

    Bivén, K; Erdal, H; Hägg, M; Ueno, T; Zhou, R; Lynch, M; Rowley, B; Wood, J; Zhang, C; Toi, M; Shoshan, M C; Linder, S

    2003-06-01

    We have developed an apoptosis assay based on measurement of a neoepitope of cytokeratin-18 (CK18-Asp396) exposed after caspase-cleavage and detected by the monoclonal antibody M30. The total amount of caspase-cleaved CK18 which has accumulated in cells and tissue culture media during apoptosis is measured by ELISA. The sensitivity is sufficient for use in the 96-well format to allow high-through-put screening of drug libraries. We here describe strategies allowing classification of pro-apoptotic compounds according to their profiles of induction of apoptosis in the presence of pharmacological inhibitors. The time course of induction of CK18 cleavage can furthermore be used to distinguish structurally similar compounds. We propose that compounds that induce rapid CK18 cleavage have mechanisms of actions distinct from conventional genotoxic and microtubuli-targeting agents, and we present one example of an agent that induces almost immediate mitochondrial depolarization and cytochrome c release. Finally, CK18-Asp396 cleavage products are released from cells in tissue culture, and presumably from tumor cells in vivo. These products can be measured in sera from cancer patients. We present evidence suggesting that it will be possible to use the M30-ELISA assay for measuring chemotherapy-induced apoptosis in patient sera, opening possibilities for monitoring therapy.

  5. Long noncoding RNA TUG1 is a diagnostic factor in lung adenocarcinoma and suppresses apoptosis via epigenetic silencing of BAX.

    Science.gov (United States)

    Liu, Huan; Zhou, Guizhi; Fu, Xin; Cui, Haiyan; Pu, Guangrui; Xiao, Yao; Sun, Wei; Dong, Xinhua; Zhang, Libin; Cao, Sijia; Li, Guiqin; Wu, Xiaowei; Yang, Xu

    2017-11-24

    Lung cancer is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel diagnostic markers and therapeutic targets. Increasing evidences have indicated that long non-coding RNAs (lncRNAs) play an important role in initiation and progression of lung cancer. However, the role of lncRNA Taurine upregulated 1 (TUG1) in lung adenocarcinoma (LAD) progression is not well known. In this study, we determined the diagnostic value of TUG1 in LAD patients, and further uncovered the underlying functional mechanism. Our results showed that TUG1 was significantly upregulated in LAD cells and serum samples. Receiver operator characteristic (ROC) analysis suggested a relatively higher area under the curve (AUC) of TUG1 (0.756) contrast to cyfra21-1 (0.619). In addition, high TUG1 level was associated with enhanced tumor size, degree of differentiation, lymph node metastases, distant metastasis and TNM stage. Cell functional assays showed that knockdown of TUG1 suppressed LAD cell viability and promoted cell apoptosis. We then sought to reveal the underlying regulatory mechanism, and the pro-apoptotic protein BAX was then identified as the downstream target of TUG1. Gain and loss functional assays showed that inhibition of BAX reversed the induced apoptosis by TUG1 knockdown. Finally, RNA immunoprecipitation and Chromatin immunoprecipitation revealed that TUG1 suppressed BAX expression through physically interacting with EZH2. In conclusion, lncRNA TUG1 is a promising diagnostic marker for LAD patients and suppression of TUG1 levels could be a future direction to promote the prognosis of LAD patients.

  6. Development of pro-apoptotic peptides as potential therapy for peritoneal endometriosis.

    Science.gov (United States)

    Sugihara, K; Kobayashi, Y; Suzuki, A; Tamura, N; Motamedchaboki, K; Huang, C-T; Akama, T O; Pecotte, J; Frost, P; Bauer, C; Jimenez, J B; Nakayama, J; Aoki, D; Fukuda, M N

    2014-07-22

    Endometriosis is a common gynaecological disease associated with pelvic pain and infertility. Current treatments include oral contraceptives combined with nonsteroidal anti-inflammatory drugs or surgery to remove lesions, all of which provide a temporary but not complete cure. Here we identify an endometriosis-targeting peptide that is internalized by cells, designated z13, using phage display. As most endometriosis occurs on organ surfaces facing the peritoneum, we subtracted a phage display library with female mouse peritoneum tissue and selected phage clones by binding to human endometrial epithelial cells. Proteomics analysis revealed the z13 receptor as the cyclic nucleotide-gated channel β3, a sorting pathway protein. We then linked z13 with an apoptosis-inducing peptide and with an endosome-escaping peptide. When these peptides were co-administered into the peritoneum of baboons with endometriosis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs. Thus, this study presents a strategy that could be useful to treat peritoneal endometriosis in humans.

  7. Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

    Science.gov (United States)

    Trotta, Andrew P; Gelles, Jesse D; Serasinghe, Madhavika N; Loi, Patrick; Arbiser, Jack L; Chipuk, Jerry E

    2017-07-14

    The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAF V600E ), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAF V600E is pharmacologically inhibited by targeted therapies ( e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAF V600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G 1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

  8. Cytotoxic, Antiproliferative and Pro-Apoptotic Effects of 5-Hydroxyl-6,7,3′,4′,5′-Pentamethoxyflavone Isolated from Lantana ukambensis

    Directory of Open Access Journals (Sweden)

    Wamtinga Richard Sawadogo

    2015-12-01

    Full Text Available Lantana ukambensis (Vatke Verdc. is an African food and medicinal plant. Its red fruits are eaten and highly appreciated by the rural population. This plant was extensively used in African folk medicinal traditions to treat chronic wounds but also as anti-leishmanial or cytotoxic remedies, especially in Burkina Faso, Tanzania, Kenya, or Ethiopia. This study investigates the in vitro bioactivity of polymethoxyflavones extracted from a L. ukambensis as anti-proliferative and pro-apoptotic agents. We isolated two known polymethoxyflavones, 5,6,7,3′,4′,5′-hexamethoxyflavone (1 and 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone (2 from the whole plant of L. ukambensis. Their chemical structures were determined by spectroscopic analysis and comparison with published data. These molecules were tested for the anti-proliferative, cytotoxic and pro-apoptotic effects on human cancer cells. Among them, 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone (2 was selectively cytotoxic against monocytic lymphoma (U937, acute T cell leukemia (Jurkat, and chronic myelogenous leukemia (K562 cell lines, but not against peripheral blood mononuclear cells (PBMCs from healthy donors, at all tested concentrations. Moreover, this compound exhibited significant anti-proliferative and pro-apoptotic effects against U937 acute myelogenous leukemia cells. This study highlights the anti-proliferative and pro-apoptotic effects of 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone (2 and provides a scientific basis of traditional use of L. ukambensis.

  9. Cytotoxic and Pro-Apoptotic Effects of Honey Bee Venom and Chrysin on Human Ovarian Cancer Cells

    Directory of Open Access Journals (Sweden)

    Elaheh Amini

    2015-06-01

    Full Text Available Background: The anti-cancer effects of honey bee venom (BV and chrysin might open a new window for treatment of chemo-resistant cancers. This study was designed to evaluate cytotoxic and pro-apoptotic effects of BV and chrysin on A2780cp cistplatin- resistant human ovarian cancer cells. Methods: As per the study objectives, A2780cp cells were categorized to 4 groups: 3 experiment groups (treated either with BV or chrysin or BV + chrysin and 1 control group (untreated cells.  Experiment group cells were cultured and treated by different concentrations of BV and chrysin for 24 hours. Then, experiment and control cells were studied with MTT assay, Annexin V-FITC, DAPI and Acridine Orange / Propidium Iodide statining, flow cytometry, caspase-3 and -9 assay, measurement of intracellular level of reactive oxygen species (ROS and RT-PCR. Results: MTT assay showed that 8 μg/mL BV, 40 µg/ml chrysin and 6 + 15 μg/mL BV + chrysin co-treatment induced 50% cell death on A2780cp cells compared with controls (P < 0.001. Morphological observations by inverted and fluorescent microscopy revealed ROS generation and apoptotic cell death under exposure to BV or chrysin or BV + chrysin co-treatment. Caspase-3 and -9 assay demonstrated that BV and chrysin triggered apoptosis through intrinsic pathway and RT-PCR demonstrated down-regulation of Bcl-2. Conclusion: Honey bee venom and chrysin are effective for destroying chemoresistant ovarian cancer cells through activation of intrinsic apoptosis, which propose them as potential candidates to be used in development of improved chemotherapeutic agents in the future.

  10. Non-THC cannabinoids inhibit prostate carcinoma growth in vitro and in vivo: pro-apoptotic effects and underlying mechanisms.

    Science.gov (United States)

    De Petrocellis, Luciano; Ligresti, Alessia; Schiano Moriello, Aniello; Iappelli, Mariagrazia; Verde, Roberta; Stott, Colin G; Cristino, Luigia; Orlando, Pierangelo; Di Marzo, Vincenzo

    2013-01-01

    Cannabinoid receptor activation induces prostate carcinoma cell (PCC) apoptosis, but cannabinoids other than Δ(9) -tetrahydrocannabinol (THC), which lack potency at cannabinoid receptors, have not been investigated. Some of these compounds antagonize transient receptor potential melastatin type-8 (TRPM8) channels, the expression of which is necessary for androgen receptor (AR)-dependent PCC survival. We tested pure cannabinoids and extracts from Cannabis strains enriched in particular cannabinoids (BDS), on AR-positive (LNCaP and 22RV1) and -negative (DU-145 and PC-3) cells, by evaluating cell viability (MTT test), cell cycle arrest and apoptosis induction, by FACS scans, caspase 3/7 assays, DNA fragmentation and TUNEL, and size of xenograft tumours induced by LNCaP and DU-145 cells. Cannabidiol (CBD) significantly inhibited cell viability. Other compounds became effective in cells deprived of serum for 24 h. Several BDS were more potent than the pure compounds in the presence of serum. CBD-BDS (i.p.) potentiated the effects of bicalutamide and docetaxel against LNCaP and DU-145 xenograft tumours and, given alone, reduced LNCaP xenograft size. CBD (1-10 µM) induced apoptosis and induced markers of intrinsic apoptotic pathways (PUMA and CHOP expression and intracellular Ca(2+)). In LNCaP cells, the pro-apoptotic effect of CBD was only partly due to TRPM8 antagonism and was accompanied by down-regulation of AR, p53 activation and elevation of reactive oxygen species. LNCaP cells differentiated to androgen-insensitive neuroendocrine-like cells were more sensitive to CBD-induced apoptosis. These data support the clinical testing of CBD against prostate carcinoma. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  11. 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.

  12. BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Sanja Aveic

    Full Text Available BCL2 associated Athano-Gene 1 (BAG1 is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.

  13. Relationship between bcl-2, bax, beclin-1, and cathepsin-D proteins during postovulatory follicular regression in fish ovary.

    Science.gov (United States)

    Morais, Roberto D V S; Thomé, Ralph G; Santos, Hélio B; Bazzoli, Nilo; Rizzo, Elizete

    2016-04-01

    In fish ovaries, postovulatory follicles (POFs) are key biomarkers of breeding and provide an interesting model for studying the relationship between autophagy and apoptosis. In this study, we investigated the immunohistochemical expression of autophagic and apoptotic proteins to improve the knowledge on the mechanisms regulating ovarian remodeling after spawning. Females from three neotropical fish species kept in captivity were submitted to hormonal induction. After ova stripping, ovarian sections were sampled daily until 5 days postspawning (dps). Similar events of POF regression were detected by histology, terminal transferase-mediated dUTP nick-end labeling (TUNEL), and electron microscopy in the three species: follicular cells hypertrophy, progressive disintegration of the basement membrane, gradual closing of the follicular lumen, theca thickening, and formation of large autophagic vacuoles preceding apoptosis of the follicular cells. Autophagic and apoptotic proteins were assessed by immunohistochemistry. Morphometric analysis of the immunolabeling revealed a more intense reaction for bcl-2 and beclin-1 (BECN1) in POFs at 0 to 1 dps and for bax at 2 to 3 dps (P family, BECN1, and cathepsin-D can be involved in the regulation of ovarian remodeling in teleost fish. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation

    International Nuclear Information System (INIS)

    Hao, Hongying; Dong, Yanbin; Bowling, Maria T; Gomez-Gutierrez, Jorge G; Zhou, H Sam; McMasters, Kelly M

    2007-01-01

    PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling

  15. 14-3-3theta protects against neurotoxicity in a cellular Parkinson's disease model through inhibition of the apoptotic factor Bax.

    Directory of Open Access Journals (Sweden)

    Sunny R Slone

    Full Text Available Disruption of 14-3-3 function by alpha-synuclein has been implicated in Parkinson's disease. As 14-3-3s are important regulators of cell death pathways, disruption of 14-3-3s could result in the release of pro-apoptotic factors, such as Bax. We have previously shown that overexpression of 14-3-3θ reduces cell loss in response to rotenone and MPP(+ in dopaminergic cell culture and reduces cell loss in transgenic C. elegans that overexpress alpha-synuclein. In this study, we investigate the mechanism for 14-3-3θ's neuroprotection against rotenone toxicity. While 14-3-3s can inhibit many pro-apoptotic factors, we demonstrate that inhibition of one factor in particular, Bax, is important to 14-3-3s' protection against rotenone toxicity in dopaminergic cells. We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation. Pharmacological inhibition or shRNA knockdown of Bax provided protection against rotenone, comparable to 14-3-3θ's neuroprotective effects. A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone. These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease.

  16. 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

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    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

  17. Identification of human ferritin, heavy polypeptide 1 (FTH1) and yeast RGI1 (YER067W) as pro-survival sequences that counteract the effects of Bax and copper in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Eid, Rawan [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada); Department of Biology, Queen' s University, Kingston, Ontario (Canada); Boucher, Eric [Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec (Canada); Gharib, Nada [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada); Khoury, Chamel [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada); Department of Biology, Queen' s University, Kingston, Ontario (Canada); Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec (Canada); Arab, Nagla T.T. [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada); Department of Biology, Queen' s University, Kingston, Ontario (Canada); Murray, Alistair [Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec (Canada); Young, Paul G. [Department of Biology, Queen' s University, Kingston, Ontario (Canada); Mandato, Craig A. [Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec (Canada); Greenwood, Michael T., E-mail: michael.greenwood@rmc.ca [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada)

    2016-03-01

    Ferritin is a sub-family of iron binding proteins that form multi-subunit nanotype iron storage structures and prevent oxidative stress induced apoptosis. Here we describe the identification and characterization of human ferritin, heavy polypeptide 1 (FTH1) as a suppressor of the pro-apoptotic murine Bax sequence in yeast. In addition we demonstrate that FTH1 is a general pro-survival sequence since it also prevents the cell death inducing effects of copper when heterologously expressed in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Bacteria, Archaea and Eukarya, ferritin is conspicuously absent in most fungal species including Saccharomyces cerevisiae. An in silico analysis of the yeast proteome lead to the identification of the 161 residue RGI1 (YER067W) encoded protein as a candidate for being a yeast ferritin. In addition to sharing 20% sequence identity with the 183 residue FTH1, RGI1 also has similar pro-survival properties as ferritin when overexpressed in yeast. Analysis of recombinant protein by SDS-PAGE and by electron microscopy revealed the expected formation of higher-order structures for FTH1 that was not observed with Rgi1p. Further analysis revealed that cells overexpressing RGI1 do not show increased resistance to iron toxicity and do not have enhanced capacity to store iron. In contrast, cells lacking RGI1 were found to be hypersensitive to the toxic effects of iron. Overall, our results suggest that Rgi1p is a novel pro-survival protein whose function is not related to ferritin but nevertheless it may have a role in regulating yeast sensitivity to iron stress. - Highlights: • Human ferritin, heavy polypeptide 1 (FTH1) was identified as a suppressor of the pro-apoptotic Bax in yeast. • Based on its similarity to ferritin we examined Rgi1p/YER067W for potential ferritin like functions. • Like human H-ferritin, RGI1 confers increased resistance to apoptotic inducing stresses in yeast.

  18. Identification of human ferritin, heavy polypeptide 1 (FTH1) and yeast RGI1 (YER067W) as pro-survival sequences that counteract the effects of Bax and copper in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Eid, Rawan; Boucher, Eric; Gharib, Nada; Khoury, Chamel; Arab, Nagla T.T.; Murray, Alistair; Young, Paul G.; Mandato, Craig A.; Greenwood, Michael T.

    2016-01-01

    Ferritin is a sub-family of iron binding proteins that form multi-subunit nanotype iron storage structures and prevent oxidative stress induced apoptosis. Here we describe the identification and characterization of human ferritin, heavy polypeptide 1 (FTH1) as a suppressor of the pro-apoptotic murine Bax sequence in yeast. In addition we demonstrate that FTH1 is a general pro-survival sequence since it also prevents the cell death inducing effects of copper when heterologously expressed in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Bacteria, Archaea and Eukarya, ferritin is conspicuously absent in most fungal species including Saccharomyces cerevisiae. An in silico analysis of the yeast proteome lead to the identification of the 161 residue RGI1 (YER067W) encoded protein as a candidate for being a yeast ferritin. In addition to sharing 20% sequence identity with the 183 residue FTH1, RGI1 also has similar pro-survival properties as ferritin when overexpressed in yeast. Analysis of recombinant protein by SDS-PAGE and by electron microscopy revealed the expected formation of higher-order structures for FTH1 that was not observed with Rgi1p. Further analysis revealed that cells overexpressing RGI1 do not show increased resistance to iron toxicity and do not have enhanced capacity to store iron. In contrast, cells lacking RGI1 were found to be hypersensitive to the toxic effects of iron. Overall, our results suggest that Rgi1p is a novel pro-survival protein whose function is not related to ferritin but nevertheless it may have a role in regulating yeast sensitivity to iron stress. - Highlights: • Human ferritin, heavy polypeptide 1 (FTH1) was identified as a suppressor of the pro-apoptotic Bax in yeast. • Based on its similarity to ferritin we examined Rgi1p/YER067W for potential ferritin like functions. • Like human H-ferritin, RGI1 confers increased resistance to apoptotic inducing stresses in yeast.

  19. The co-repressor SMRT delays DNA damage-induced caspase activation by repressing pro-apoptotic genes and modulating the dynamics of checkpoint kinase 2 activation.

    Directory of Open Access Journals (Sweden)

    Claudio Scafoglio

    Full Text Available Checkpoint kinase 2 (Chk2 is a major regulator of DNA damage response and can induce alternative cellular responses: cell cycle arrest and DNA repair or programmed cell death. Here, we report the identification of a new role of Chk2 in transcriptional regulation that also contributes to modulating the balance between survival and apoptosis following DNA damage. We found that Chk2 interacts with members of the NCoR/SMRT transcriptional co-regulator complexes and serves as a functional component of the repressor complex, being required for recruitment of SMRT on the promoter of pro-apoptotic genes upon DNA damage. Thus, the co-repressor SMRT exerts a critical protective action against genotoxic stress-induced caspase activation, repressing a functionally important cohort of pro-apoptotic genes. Amongst them, SMRT is responsible for basal repression of Wip1, a phosphatase that de-phosphorylates and inactivates Chk2, thus affecting a feedback loop responsible for licensing the correct timing of Chk2 activation and the proper execution of the DNA repair process.

  20. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized

    OpenAIRE

    van Delft, Mark F.; Wei, Andrew H.; Mason, Kylie D.; Vandenberg, Cassandra J.; Chen, Lin; Czabotar, Peter E.; Willis, Simon N.; Scott, Clare L.; Day, Catherine L.; Cory, Suzanne; Adams, Jerry M.; Roberts, Andrew W.; Huang, David C.S.

    2006-01-01

    Since apoptosis is impaired in malignant cells overexpressing pro-survival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-xL and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects its inability to target another pro-survival relative, Mcl-1. Down-regulation of Mc...

  1. Pro-apoptotic and anti-proliferative effects of corn silk extract on human colon cancer cell lines.

    Science.gov (United States)

    Guo, Hao; Guan, Hong; Yang, Wenqin; Liu, Han; Hou, Huiling; Chen, Xue; Liu, Zhenyan; Zang, Chuangang; Liu, Yuchao; Liu, Jicheng

    2017-02-01

    Corn silk is an economically and nutritionally significant natural product as it represents a staple food for a large proportion of the world population. This study investigated the anticancer activity of corn silk extract in human colon cancer cells and human gastric cancer cells. Following treatment with corn silk extract, certain apoptosis-related events were observed, including inhibition of cell proliferation, loss of mitochondrial membrane potential (ΔΨm), release of Ca2+ and release of cytochrome c from the mitochondria into the cytosol. Our results revealed that corn silk extract inhibited the proliferation of cancer cells and increased the level of apoptosis in a concentration-dependent manner. Western blot analysis revealed that corn silk extract upregulated the levels of Bax, cytochrome c , caspase-3 and caspase-9, but downregulated the levels of B-cell lymphoma 2. These results suggest that corn silk extract may induce apoptosis through the mitochondria-mediated pathway.

  2. Pro-apoptotic signaling induced by Retinoic acid and dsRNA is under the control of Interferon Regulatory Factor-3 in breast cancer cells.

    Science.gov (United States)

    Bernardo, Ana R; Cosgaya, José M; Aranda, Ana; Jiménez-Lara, Ana M

    2017-07-01

    Breast cancer is one of the most lethal malignancies for women. Retinoic acid (RA) and double-stranded RNA (dsRNA) are considered signaling molecules with potential anticancer activity. RA, co-administered with the dsRNA mimic polyinosinic-polycytidylic acid (poly(I:C)), synergizes to induce a TRAIL (Tumor-Necrosis-Factor Related Apoptosis-Inducing Ligand)- dependent apoptotic program in breast cancer cells. Here, we report that RA/poly(I:C) co-treatment, synergically, induce the activation of Interferon Regulatory Factor-3 (IRF3) in breast cancer cells. IRF3 activation is mediated by a member of the pathogen recognition receptors, Toll-like receptor-3 (TLR3), since its depletion abrogates IRF3 activation by RA/poly(I:C) co-treatment. Besides induction of TRAIL, apoptosis induced by RA/poly(I:C) correlates with the increased expression of pro-apoptotic TRAIL receptors, TRAIL-R1/2, and the inhibition of the antagonistic receptors TRAIL-R3/4. IRF3 plays an important role in RA/poly(I:C)-induced apoptosis since IRF3 depletion suppresses caspase-8 and caspase-3 activation, TRAIL expression upregulation and apoptosis. Interestingly, RA/poly(I:C) combination synergizes to induce a bioactive autocrine/paracrine loop of type-I Interferons (IFNs) which is ultimately responsible for TRAIL and TRAIL-R1/2 expression upregulation, while inhibition of TRAIL-R3/4 expression is type-I IFN-independent. Our results highlight the importance of IRF3 and type-I IFNs signaling for the pro-apoptotic effects induced by RA and synthetic dsRNA in breast cancer cells.

  3. p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD

    DEFF Research Database (Denmark)

    Harada, H; Andersen, Jens S.; Mann, M

    2001-01-01

    Cytokines often deliver simultaneous, yet distinct, cell growth and cell survival signals. The 70-kDa ribosomal protein S6 kinase (p70S6K) is known to regulate cell growth by inducing protein synthesis components. We purified membrane-based p70S6K as a kinase responsible for site-specific phospho...

  4. Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

    International Nuclear Information System (INIS)

    Yamagishi, Nobuyuki; Ishihara, Keiichi; Saito, Youhei; Hatayama, Takumi

    2006-01-01

    Hsp105 (Hsp105α and Hsp105β), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105α has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105α regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105α or Hsp105β by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105α or Hsp105β. In addition, we found that overexpression of Hsp105α or Hsp105β suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105α or Hsp105β. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells

  5. PKCη confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

    International Nuclear Information System (INIS)

    Rotem-Dai, Noa; Oberkovitz, Galia; Abu-Ghanem, Sara; Livneh, Etta

    2009-01-01

    Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKCη, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug - Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKCη in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKCη. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKCη expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKCη is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKCη could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.

  6. Effect of hrHPV infection on anti-apoptotic gene and pro-apoptotic gene expression in cervical cancer tissue

    Directory of Open Access Journals (Sweden)

    Min-Er Tang

    2016-09-01

    Full Text Available Objective: To study the effect of hrHPV infection on anti-apoptotic gene and pro-apoptotic gene expression in cervical cancer tissue. Methods: A total of 56 patients with cervical cancer, 94 cases of patients with cervical intraepithelial neoplasia and 48 cases of patients with chronic cervicitis who were treated in our hospital from May 2013 to December 2015 were selected for study and included in malignant group, precancerous lesion group and benign group respectively. hrHPV infection as well as the expression of anti-apoptotic genes and proapoptotic genes in cervical tissue were detected. Results: hrHPV infection rate and viral load in cervical tissue of malignant group were significantly higher than those of precancerous lesion group and benign group; P27 and p16 levels in cervical tissue of malignant group were significantly lower than those of precancerous lesion group and benign group, and K-ras, c-myc, Prdx4 and TNFAIP8 levels were significantly higher than those of precancerous lesion group and benign group; the greater the HPV virus load, the lower the p27 and p16 levels and the higher the K-ras, c-myc, Prdx4 and TNFAIP8 levels in cervical tissue. Conclusions: hrHPV infection can result in tumor suppressor genes p27 and p16 expression deletion and increase the expression of proto-oncogene and apoptosis-inhibiting genes, and it is associated with the occurrence and development of cervical cancer.

  7. Human colon cancer targeted pro-apoptotic, anti-metastatic and cytostatic effects of binuclear Silver(I)-N-Heterocyclic carbene (NHC) complexes.

    Science.gov (United States)

    Asif, Muhammad; Iqbal, Muhammad Adnan; Hussein, Mouayed A; Oon, Chern Ein; Haque, Rosenani A; Khadeer Ahamed, Mohamed B; Abdul Majid, Aman Shah; Abdul Majid, Amin Malik Shah

    2016-01-27

    The current mechanistic study was conducted to explore the effects of increased lipophilicity of binuclear silver(I)-NHC complexes on cytotoxicity. Two new silver(I)-N-Heterocyclic Carbene (NHC) complexes (3 and 4), having lypophilic terminal alkyl chains (Octyl and Decyl), were derived from meta-xylyl linked bis-benzimidazolium salts (1 and 2). Each of the synthesized compounds was characterized by microanalysis and spectroscopic techniques. The complexes were tested for their cytotoxicity against a panel of human cancer c as well normal cell lines using MTT assay. Based on MTT assay results, complex 4 was found to be selectively toxic towards human colorectal carcinoma cell line (HCT 116). Complex 4 was further studied in detail to explore the mechanism of cell death and findings of the study revealed that complex 4 has promising pro-apoptotic and anti-metastatic activities against HCT 116 cells. Furthermore, it showed pronounced cytostatic effects in HCT 116 multicellular spheroid model. Hence, binuclear silver(I)-NHC complexes with longer terminal aliphatic chains have worth to be further studied against human colon cancer for the purpose of drug development. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  8. Polysaccharide peptide isolated from grass-cultured Ganoderma lucidum induces anti-proliferative and pro-apoptotic effects in the human U251 glioma cell line.

    Science.gov (United States)

    Wang, Chunhua; Lin, Dongmei; Chen, Quan; Lin, Shuqian; Shi, Songsheng; Chen, Chunmei

    2018-04-01

    The Ganoderma lucidum ( G. lucidum ) mushroom is one of the most extensively studied functional foods, known for its numerous health benefits, including the inhibition of tumor cell growth. The present study assessed the anti-proliferative and pro-apoptotic activity of a novel G. lucidum polysaccharide peptide (GL-PP) in human glioma U251 cells, which was purified from grass-cultured G. lucidum . GL-PP is a glycopeptide with an average molecular weight of 42,635 Da and a polysaccharide-to-peptide ratio of 88.70:11.30. The polysaccharides were composed of l-arabinose, d-mannose and d-glucose at a molar ratio of 1.329:0.372:2.953 and a total of 17 amino acids were detected. The results of the current study demonstrated that GL-PP significantly inhibited U251 cellular proliferation. The proportion of G 0 /G 1 phase cells and sub-G 1 phase cells significantly increased as the concentration of GL-PP increased, as did the activity of caspase-3. These results indicate that GL-PP directly inhibited human glioma U251 proliferation by inducing cell cycle arrest and promoting apoptosis.

  9. The Wheat Bax Inhibitor-1 Protein Interacts with an Aquaporin TaPIP1 and Enhances Disease Resistance in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Pan-Pan Lu

    2018-01-01

    Full Text Available Bax inhibitor-1 (BI-1 is an endoplasmic reticulum (ER-resident cell death suppressor evolutionarily conserved in eukaryotes. The ability of BI-1 to inhibit the biotic and abiotic stresses have been well-studied in Arabidopsis, while the functions of wheat BI-1 are largely unknown. In this study, the wheat BI-1 gene TaBI-1.1 was isolated by an RNA-seq analysis of Fusarium graminearum (Fg-treated wheat. TaBI-1.1 expression was induced by a salicylic acid (SA treatment and down-regulated by an abscisic acid (ABA treatment. Based on β-glucuronidase (GUS staining, TaBI-1.1 was expressed in mature leaves and roots but not in the hypocotyl or young leaves. Constitutive expression of TaBI-1.1 in Arabidopsis enhanced its resistance to Pseudomonas syringae pv. Tomato (Pst DC3000 infection and induced SA-related gene expression. Additionally, TaBI-1.1 transgenic Arabidopsis exhibited an alleviation of damage caused by high concentrations of SA and decreased the sensitivity to ABA. Consistent with the phenotype, the RNA-seq analysis of 35S::TaBI-1.1 and Col-0 plants showed that TaBI-1.1 was involved in biotic stresses. These results suggested that TaBI-1.1 positively regulates SA signals and plays important roles in the response to biotic stresses. In addition, TaBI-1.1 interacted with the aquaporin TaPIP1, and both them were localized to ER membrane. Furthermore, we demonstrated that TaPIP1 was up-regulated by SA treatment and TaPIP1 transgenic Arabidopsis enhanced the resistance to Pst DC3000 infection. Thus, the interaction between TaBI-1.1 and TaPIP1 on the ER membrane probably occurs in response to SA signals and defense response.

  10. [Distribution of Pathogenic Bacteria and Its Influence on Expression of BCL-2 and BAX Protein after HSCT in the Patients with Hematological Malignancies].

    Science.gov (United States)

    Su, Gui-Ping; Dai, Yan; Huang, Lai-Quan; Jiang, Yi-Zhi; Geng, Liang-Quan; Ding, Kai-Yang; Huang, Dong-Ping

    2016-06-01

    To investigate the distribution of pathogenic bacteria in the patients with hematologic malignancies received hematopoietic stem cell transplantation (HSCT) and its influence on the expression of BCL-2 and BAX proteins. The clinical data of 64 patients with malignant lymphoma (ML) received auto-HSCT from January 2011 to December 2015 in our hospital were analyzed. On basis of post-treansplant infection, the patients were divided into infection group (36 cases) and non-infection group (28 cases). The distribution of pathogenic bacteria in 2 groups was identified, the T lymphocyte subsets of peripheral blood, expression level of apoptotic proteins and C-reaction protein (CRP) in 2 group were detected. Thirty-six strains of pathogenic bacteria were isolated from 36 case of hematological malignancy after HSCT, including 24 strains of Gram-negative bacteria (66.67%) with predominamce of klebsiella pneumoniae (19.44%). The periperal blood CD4+ (t=2.637, Ppathogenic bacteria infecting ML patients after HSCT were mainly Gram-negative bacteria. The post-transplant infection can promote the expression up-regulation of related inflammatory factors and apoptotic proteins. The pathogens may be involved in cell apoptisis that provides a new strategy to treat the hematologic malignancies.

  11. Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1

    Science.gov (United States)

    Bauer, William J.; Heath, Jason; Jenkins, Jermaine L.; Kielkopf, Clara L.

    2012-01-01

    T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering (SAXS) to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. SAXS analyses demonstrated a `V' shape for a TIA-1 construct comprising the three RRMs, and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed. PMID:22154808

  12. Novel Acylguanidine Derivatives Targeting Smoothened Induce Antiproliferative and Pro-Apoptotic Effects in Chronic Myeloid Leukemia Cells.

    Directory of Open Access Journals (Sweden)

    Alessandra Chiarenza

    Full Text Available The most relevant therapeutic approaches to treat CML rely on the administration of tyrosine kinase inhibitors (TKIs like Imatinib, which are able to counteract the activity of Bcr-Abl protein increasing patient's life expectancy and survival. Unfortunately, there are some issues TKIs are not able to address; first of all TKIs are not so effective in increasing survival of patients in blast crisis, second they are not able to eradicate leukemic stem cells (LSC which represent the major cause of disease relapse, and third patients often develop resistance to TKIs due to mutations in the drug binding site. For all these reasons it's of primary interest to find alternative strategies to treat CML. Literature shows that Hedgehog signaling pathway is involved in LSC maintenance, and pharmacological inhibition of Smoothened (SMO, one of the key molecules of the pathway, has been demonstrated to reduce Bcr-Abl positive bone marrow cells and LSC. Consequently, targeting SMO could be a promising way to develop a new treatment strategy for CML overcoming the limitations of current therapies. In our work we have tested some compounds able to inhibit SMO, and among them MRT92 appears to be a very potent SMO antagonist. We found that almost all our compounds were able to reduce Gli1 protein levels in K-562 and in KU-812 CML cell lines. Furthermore, they were also able to increase Gli1 and SMO RNA levels, and to reduce cell proliferation and induce apoptosis/autophagy in both the tested cell lines. Finally, we demonstrated that our compounds were able to modulate the expression of some miRNAs related to Hedgehog pathway such as miR-324-5p and miR-326. Being Hedgehog pathway deeply implicated in the mechanisms of CML we may conclude that it could be a good therapeutic target for CML and our compounds seem to be promising antagonists of such pathway.

  13. JNK Promotes Epithelial Cell Anoikis by Transcriptional and Post-translational Regulation of BH3-Only Proteins

    Directory of Open Access Journals (Sweden)

    Nomeda Girnius

    2017-11-01

    Full Text Available Summary: Developmental morphogenesis, tissue injury, and oncogenic transformation can cause the detachment of epithelial cells. These cells are eliminated by a specialized form of apoptosis (anoikis. While the processes that contribute to this form of cell death have been studied, the underlying mechanisms remain unclear. Here, we tested the role of the cJUN NH2-terminal kinase (JNK signaling pathway using murine models with compound JNK deficiency in mammary and kidney epithelial cells. These studies demonstrated that JNK is required for efficient anoikis in vitro and in vivo. Moreover, JNK-promoted anoikis required pro-apoptotic members of the BCL2 family of proteins. We show that JNK acts through a BAK/BAX-dependent apoptotic pathway by increasing BIM expression and phosphorylating BMF, leading to death of detached epithelial cells. : Developmental morphogenesis, tissue injury, and oncogenic transformation can cause epithelial cell detachment. These cells are eliminated by a specialized form of apoptosis termed anoikis. Girnius and Davis show that anoikis is mediated by the cJUN NH2-terminal kinase (JNK, which increases BIM expression and phosphorylates BMF to engage BAK/BAX-dependent apoptosis. Keywords: apoptosis, anoikis, epithelial cell, mammary gland, JNK, BAX, BAK, BH3-only protein, BIM, BMF

  14. Clusterin Reduces Cold Ischemia-Reperfusion Injury in Heart Transplantation Through Regulation of NF-kB Signaling and Bax/Bcl-xL Expression

    Directory of Open Access Journals (Sweden)

    Guodong Liu

    2018-02-01

    the pro-apoptotic gene Bax, while it enhanced the expression of the anti-apoptotic gene Bcl-xL in vitro and in vivo. Clusterin inhibited cell apoptosis/death and reduced pre-inflammatory. Conclusion: Clusterin is a promising target for preventing cold I/R injury in heart transplantation. This study also shows that the resultant protective effects of clusterin are mediated by NF-κB signaling and Bax/Bcl-xL expression.

  15. Clusterin Reduces Cold Ischemia-Reperfusion Injury in Heart Transplantation Through Regulation of NF-kB Signaling and Bax/Bcl-xL Expression.

    Science.gov (United States)

    Liu, Guodong; Zhang, Hongmei; Hao, Fengyun; Hao, Jing; Pan, Lixiao; Zhao, Qing; Wo, Jinshan

    2018-01-01

    Ischemia-reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation and is a key factor in graft failure and the long-term survival rate of recipients. Therefore, there is an urgent need for the development of new therapies to prevent I/R injury. Clusterin is a hetero-dimeric glycoprotein with an antiapoptotic function. In this study, we investigated whether clusterin was cardioprotective in heart transplantation against I/R injury using an in vivo rat model and an in vitro cell culture system, and examined the underlying mechanisms of I/R injury. Heart grafts from wild-type C57BL/6 mice were preserved in UW solution (control) or UW solution containing recombinant human apolipoprotein-J (hr clusterin) for 24 h. The preserved hearts were implanted into recipient mice of the same strain as the donors for 72 h, and the heart grafts were then taken for histopathological and gene expression analyses. An in vitro ischemia reperfusion model using H9C2 cells or H9C2/clusterin cDNA cells was constructed. The expression of clusterin, p65, Bax, Bcl-xL, IL-1β, and TNF-α protein and mRNA in heart tissue and H9C2 cells was detected by western blot, reverse transcription-polymerase chain reaction (RT-PCR), and quantitative RT-PCR assays; IL-1β and TNF-α protein was detected by enzyme-linked immunosorbent assays; NF-kB activity was detected by an electrophoretic mobility shift assay; cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometric analyses. Cold I/R caused severe morphologic myocardial injury to heart grafts from wild-type C57BL/6 mice, whereas grafts from hr clusterin preservation showed less damage, as demonstrated by decreased cell apoptosis/death, decreased neutrophil infiltration, and the preservation of the normal structure of the heart. Clusterin reduced the expression of p65, pre-inflammatory IL-1β, and TNF-α, and the pro-apoptotic gene Bax, while it enhanced the

  16. Dapoxetine attenuates testosterone-induced prostatic hyperplasia in rats by the regulation of inflammatory and apoptotic proteins

    International Nuclear Information System (INIS)

    Sayed, Rabab H.; Saad, Muhammed A.; El-Sahar, Ayman E.

    2016-01-01

    Serotonin level plays a role in suppressing the pathological findings of benign prostatic hyperplasia (BPH). Thus a new selective serotonin reuptake inhibitor, dapoxetine was used to test its ability to ameliorate the pathological changes in the rat prostate. A dose response curve was constructed between the dose of dapoxetine and prostate weight as well as relative prostate weight, then a 5 mg/kg dose was used as a representative dose for dapoxetine administration. Rats were divided into four groups; the control group that received the vehicle; the BPH-induced group received daily s.c injection of 3 mg/kg testosterone propionate dissolved in olive oil for four weeks; BPH-induced group treated with finasteride 5 mg/kg/day p.o and BPH-induced group treated with dapoxetine 5 mg/kg/day p.o. Injection of testosterone increased prostate weight and relative prostate weight which were both returned back to the normal value after treatment with dapoxetine as well as finasteride. Testosterone also upregulated androgen receptor (AR) and proliferating cell nuclear antigen gene expression. Furthermore, testosterone injection elevated cyclooxygenase-II (COX II), inducible nitric oxide synthase (iNOS), B-cell lymphoma-2 (Bcl2) expression and tumor necrosis factor alpha content and reduced caspase-3 activity, Bcl-2-associated X protein (Bax) expression and Bax/Bcl2 ratio. Dapoxetine and finasteride administration reverted most of the changes made by testosterone injection. In conclusion, the current study provides an evidence for the protective effects of dapoxetine against testosterone-induced BPH in rats. This can be attributed, at least in part, to decreasing AR expression, and the anti-proliferative, anti-inflammatory and pro-apoptotic activities of dapoxetine in BPH. - Highlights: • Dapoxetine attenuates testosterone-induced prostatic hyperplasia in rats. • Dapoxetine decreased androgen receptor gene expression in rat prostate. • Dapoxetine possess anti

  17. Dapoxetine attenuates testosterone-induced prostatic hyperplasia in rats by the regulation of inflammatory and apoptotic proteins

    Energy Technology Data Exchange (ETDEWEB)

    Sayed, Rabab H., E-mail: rabab.sayed@pharma.cu.edu.eg; Saad, Muhammed A.; El-Sahar, Ayman E.

    2016-11-15

    Serotonin level plays a role in suppressing the pathological findings of benign prostatic hyperplasia (BPH). Thus a new selective serotonin reuptake inhibitor, dapoxetine was used to test its ability to ameliorate the pathological changes in the rat prostate. A dose response curve was constructed between the dose of dapoxetine and prostate weight as well as relative prostate weight, then a 5 mg/kg dose was used as a representative dose for dapoxetine administration. Rats were divided into four groups; the control group that received the vehicle; the BPH-induced group received daily s.c injection of 3 mg/kg testosterone propionate dissolved in olive oil for four weeks; BPH-induced group treated with finasteride 5 mg/kg/day p.o and BPH-induced group treated with dapoxetine 5 mg/kg/day p.o. Injection of testosterone increased prostate weight and relative prostate weight which were both returned back to the normal value after treatment with dapoxetine as well as finasteride. Testosterone also upregulated androgen receptor (AR) and proliferating cell nuclear antigen gene expression. Furthermore, testosterone injection elevated cyclooxygenase-II (COX II), inducible nitric oxide synthase (iNOS), B-cell lymphoma-2 (Bcl2) expression and tumor necrosis factor alpha content and reduced caspase-3 activity, Bcl-2-associated X protein (Bax) expression and Bax/Bcl2 ratio. Dapoxetine and finasteride administration reverted most of the changes made by testosterone injection. In conclusion, the current study provides an evidence for the protective effects of dapoxetine against testosterone-induced BPH in rats. This can be attributed, at least in part, to decreasing AR expression, and the anti-proliferative, anti-inflammatory and pro-apoptotic activities of dapoxetine in BPH. - Highlights: • Dapoxetine attenuates testosterone-induced prostatic hyperplasia in rats. • Dapoxetine decreased androgen receptor gene expression in rat prostate. • Dapoxetine possess anti

  18. Cambogin exerts anti-proliferative and pro-apoptotic effects on breast adenocarcinoma through the induction of NADPH oxidase 1 and the alteration of mitochondrial morphology and dynamics.

    Science.gov (United States)

    Shen, Kaikai; Lu, Fangfang; Xie, Jianling; Wu, Minfeng; Cai, Bo; Liu, Yurong; Zhang, Hong; Tan, Hongsheng; Pan, Yingyi; Xu, Hongxi

    2016-08-02

    Cambogin, a bioactive polycyclic polyprenylated acylphoroglucinol (PPAP) derived from the Garcinia genus, possesses proapoptotic effect in medulloblastoma and breast cancer cells. We have previously demonstrated that the proapoptotic effect of cambogin is driven by the production of reactive oxygen species (ROS). Here we have shown that the inhibitory effect of cambogin on cell proliferation is associated with the loss of mitochondrial transmembrane potential (ΔΨm) and mitochondrial fragmentation. Cambogin also promotes the mutual complex formation of the membrane-bound subunit p22phox of NADPH oxidase 1 (NOX1), as well as the phosphorylation of the cytosolic subunit p47phox, subsequently enhancing membrane-bound NOX1 activity, which leads to increases in intracellular and mitochondrial levels of O2.- and H2O2. Pharmacological inhibition of NOX1 using apocynin (pan-NOX inhibitor), ML171 (NOX1 inhibitor) or siRNA against NOX1 prevents the increases in O2.- and H2O2 levels and the anti-proliferative effect of cambogin. Antioxidants, including SOD (superoxide dismutase), CAT (catalase) and EUK-8, are also able to restore cell viability in the presence of cambogin. Besides, cambogin increases the dissociation of thioredoxin-1 (Trx1) from ASK1, switching the inactive form of ASK1 to the active kinase, subsequently leads to the phosphorylation of JNK/SAPK, which is abolished upon ML171 treatment. The proapoptotic effect of cambogin in breast cancer cells is also aggravated upon knocking down Trx1 in MCF-7 cells. Taken in conjunction, these data indicate that the anti-proliferative and pro-apoptotic effect of cambogin is mediated via inducing NOX1-dependent ROS production and the dissociation of ASK1 and Trx1.

  19. Vicrostatin - an anti-invasive multi-integrin targeting chimeric disintegrin with tumor anti-angiogenic and pro-apoptotic activities.

    Directory of Open Access Journals (Sweden)

    Radu O Minea

    2010-06-01

    Full Text Available Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., alphavbeta3, alphavbeta5, and alpha5beta1, VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis. Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.

  20. Neurobehavioral impairments, generation of oxidative stress and release of pro-apoptotic factors after chronic exposure to sulphur mustard in mouse brain

    International Nuclear Information System (INIS)

    Sharma, Deep Raj; Sunkaria, Aditya; Bal, Amanjit; Bhutia, Yangchen D.; Vijayaraghavan, R.; Flora, S.J.S.; Gill, Kiran Dip

    2009-01-01

    Recent global events have focused attention on the potential threat of international and domestic chemical terrorism, as well as the possibility of chemical warfare proliferation. Sulphur mustard (SM) is one of the potent chemical warfare agents (CWA), which initiates a cascade of events that converge on the redox mechanisms common to brain injury. The present study was designed to examine the effects of chronic SM exposure on neurobehavioral impairments, mitochondrial oxidative stress in male Swiss Albino mice and its role in inducing apoptotic neuronal cell death. The animals were divided into four groups (control, low, medium and high dose) of 5 animals each. Exposure to SM was given percutaneously daily for 12 weeks. The results demonstrated impairment in neurobehavioral indices viz. rota rod, passive avoidance and water maze tests in a dose dependent manner. There was a significant increase in lipid peroxidation and protein carbonyl content whereas, decrease in the activity of manganese superoxide dismutase (MnSOD), glutathione reductase and glutathione peroxidase suggesting impaired antioxidant defense system. Immunoblotting of cytochrome c, Bcl-2, Bax and activation of caspase-3 suggest induction of apoptosis in a dose dependent manner. Finally, increased p53 expression suggests that it may target the mitochondrial pathway for inducing apoptosis in response to DNA damage signals. In conclusion, chronic SM exposure may have the potential to generate oxidative stress which may trigger the release of cytochrome c as well as caspase-3 activation in neurons leading to cell death by apoptosis in a dose dependent manner which may in the end be responsible for the disruption of cognitive functions in mice.

  1. 15, 16-Dihydrotanshinone I Inhibits Hemangiomas through Inducing Pro-apoptotic and Anti-angiogenic Mechanisms in Vitro and in Vivo

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    Yihong Cai

    2018-01-01

    Full Text Available Infantile hemangioma (IH is a common and benign vascular neoplasms, which has a high incidence in children. Although IH is benign, some patients experience complications such as pain, functional impairment, and permanent disfigurement. Treatment options for IH include corticosteroids, surgery, vincristine, interferon or cyclophosphamide. However, none of these modalities are ideal due to restrictions or potential serious side effects. There is thus a great need to explore novel treatments for IH with less side effects. Angiogenesis, vasculogenesis and tumorigenesis are the main features of IH. Tanshen is mostly used in Chinese traditional medicine to treat hematological abnormalities. Therefore, the aim of our study was to evaluate anti-proliferation and anti-angiogenesis effects on hemangiomas cells by extracted Tanshen compounds compared with propranolol, the first-line treatment for IH currently, both in vitro and in vivo. Cell viability, apoptosis, protein expression and anti-angiogenesis were analyzed by CCK8, Annexin V staining, Western blot and tube formation, respectively. The anti-tumor activity in vivo was evaluated using a mouse xenograft model. Fourteen major compounds extracting from Tanshen were screened for their ability to inhibit hemangiomas cells. Of the 14 compounds investigated, 15,16-Dihydrotanshinone I (DHTS was the most potent modulator of EOMA cell biology. DHTS could significantly decrease EOMA cells proliferation by inducing cell apoptosis, which is much more efficient than propranolol in vitro. DHTS increased the expression of several apoptosis-related proteins, including caspase9, caspase3, PARP, AIF, BAX, cytochrome c, caspase8 and FADD and significantly inhibited angiogenesis, as indicated by reduced tube formation and diminished expression of vascular endothelial cell growth factor receptor 2 and matrix metalloproteinase 9. In nude mice xenograft experiment, DHTS (10 mg/kg could significantly inhibit the tumor

  2. 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

    Directory of Open Access Journals (Sweden)

    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.

  3. Deregulated expression of A1, Bcl-2, Bcl-xL, and Mcl-1 antiapoptotic proteins and Bid, Bad, and Bax proapoptotic genes in polycythemia vera patients

    Directory of Open Access Journals (Sweden)

    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

  4. 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.

  5. SPATA4 Counteracts Etoposide-Induced Apoptosis via Modulating Bcl-2 Family Proteins in HeLa Cells.

    Science.gov (United States)

    Jiang, Junjun; Li, Liyuan; Xie, Mingchao; Fuji, Ryosuke; Liu, Shangfeng; Yin, Xiaobei; Li, Genlin; Wang, Zhao

    2015-01-01

    Spermatogenesis associated 4 (SPATA4) is a testis-specific gene first cloned by our laboratory, and plays an important role in maintaining the physiological function of germ cells. Accumulated evidence suggests that SPATA4 might be associated with apoptosis. Here we established HeLa cells that stably expressed SPATA4 to investigate the function of SPATA4 in apoptosis. SPATA4 protected HeLa cells from etoposide-induced apoptosis through the mitochondrial apoptotic pathway, in the way that SPATA4 suppressed decrease of the mitochondrial membrane potential, the release of cytochrome c, and subsequent activation of caspase-9 and -3. We further demonstrated that SPATA4 upregulated anti-apoptotic members of Bcl-2 family proteins, Bcl-2, and downregulated the pro-apoptotic member of Bcl-2 family proteins, Bax. Knockdown of SPATA4 in HeLa/SPATA4 cells could partially rescue expression levels of bcl-2 and bax. In conclusion, SPATA4 protects HeLa cells against etoposide-induced apoptosis through the mitochondrial apoptotic pathway. Our findings provide further evidence that SPATA4 plays a role in regulating apoptosis.

  6. The dimethylthiourea-induced attenuation of cisplatin nephrotoxicity is associated with the augmented induction of heat shock proteins

    International Nuclear Information System (INIS)

    Tsuji, Takayuki; Kato, Akihiko; Yasuda, Hideo; Miyaji, Takehiko; Luo, Jinghui; Sakao, Yukitoshi; Ito, Hideaki; Fujigaki, Yoshihide; Hishida, Akira

    2009-01-01

    Dimethylthiourea (DMTU), a potent hydroxyl radical scavenger, affords protection against cisplatin (CDDP)-induced acute renal failure (ARF). Since the suppression of oxidative stress and the enhancement of heat shock proteins (HSPs) are both reported to protect against CDDP-induced renal damage, we tested whether increased HSP expression is involved in the underlying mechanisms of the DMTU-induced renal protection. We examined the effect of DMTU treatment on the expression of HSPs in the kidney until day 5 following a single injection of CDDP (5 mg/kg BW). DMTU significantly inhibited the CDDP-induced increments of serum creatinine, the number of 8-hydroxyl-2'-deoxyguanosine (8-OHdG)- and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive tubular cells, and tubular damage score (p < 0.05). CDDP significantly increased renal abundances of HO-1, HSP60, HSP72 and HSP90 at days 1, 3, and 5. DMTU significantly augmented only the expression of HSP60 expression mainly in the cytoplasm of the proximal tubular cells at days 1 and 3 in CDDP-induced ARF. DMTU also inhibited the CDDP-induced increment of Bax, a pro-apoptotic protein, in the fraction of organelles/membranes at day 3. The findings suggest that DMTU may afford protection against CDDP-induced ARF, partially through the early induction of cytoplasmic HSP60, thereby preventing the Bax-mediated apoptosis in renal tubular cells

  7. A comparison of the effects of tributyltin chloride and triphenyltin chloride on cell proliferation, proapoptotic p53, Bax, and antiapoptotic Bcl-2 protein levels in human breast cancer MCF-7 cell line.

    Science.gov (United States)

    Fickova, Maria; Macho, Ladislav; Brtko, Julius

    2015-06-01

    In recent years it was disclosed, that numerous organotin(IV) derivatives have remarkable cytotoxicity against several types of cancer cells. The property to inhibit cell growth makes these compounds promising for antitumor therapy, as the clinical effectiveness of cisplatin is limited by drug resistance and significant side effects. Tributyltin and triphenyltin are known as endocrine disruptors. Moreover, the compounds exert their toxicity in mammals predominantly through nuclear receptor signaling. Here we present the effects of tributyltin chloride (TBT-Cl) and triphenyltin chloride (TPT-Cl) on cell proliferation, expression of proapoptotic p53, Bax, and antiapoptotic Bcl-2 proteins in human breast cancer MCF-7 cell line. Dose and time dependent (24, 48 and 72 h) cell expositions have demonstrated TBT-Cl as more effective in inhibiting MCF-7 cell proliferation than TPT-Cl. Short time treatment with TBT-Cl displayed marked stimulation of p53 protein expression when compared to TPT-Cl. Both organotin compounds displayed similar mild enhancement of Bax protein expression. The 24h exposition of TPT-Cl induced substantial diminution of Bcl-2 protein expression in comparison with both, untreated cells and TBT-Cl treated cells. Our observations indicate that TBT-Cl and TPT-Cl have different antiproliferative potency and distinct impact on expression of apoptosis marker proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Alpha lipoic acid (ALA modulates expression of apoptosis associated proteins in hippocampus of rats exposed during postnatal period to sodium arsenite (NaAsO2

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    Shilpi Dixit

    2015-01-01

    Full Text Available The present study focused on the role of exogenous alpha lipoic acid (ALA in amelioration of inorganic arsenic (iAs induced effects on apoptosis and apoptosis associated proteins in developing rat hippocampus. NaAsO2 (1.5/2.0 mg/kg bw alone or along with ALA (70 mg/kg bw was administered to rat pups (experimental groups by intraperitoneal (i.p. route from postnatal day (PND 4–15. Controls received no treatment/distilled water/ALA. On PND 16, the animals were perfusion fixed and the brains were processed for paraffin embedding (CV and TUNEL staining and cryopreservation (immunohistochemistry. The fresh brain tissue was used for Western blotting. Significant increase was observed in TUNEL positive cells and Bax (pro-apoptotic protein expression in hippocampal sub-regions of iAs alone treated groups, whereas Bcl-2 expression was intensified in animals receiving ALA with iAs. Densitometric analysis (Western blots revealed optimal restoration of Bax and Bcl-2 ratio in animals receiving ALA with iAs, thereby suggesting the protective role of ALA in iAs induced developmental neurotoxicity.

  9. Alpha lipoic acid (ALA) modulates expression of apoptosis associated proteins in hippocampus of rats exposed during postnatal period to sodium arsenite (NaAsO2).

    Science.gov (United States)

    Dixit, Shilpi; Dhar, Pushpa; Mehra, Raj D

    2015-01-01

    The present study focused on the role of exogenous alpha lipoic acid (ALA) in amelioration of inorganic arsenic ( iAs ) induced effects on apoptosis and apoptosis associated proteins in developing rat hippocampus. NaAsO 2 (1.5/2.0 mg/kg bw) alone or along with ALA (70 mg/kg bw) was administered to rat pups (experimental groups) by intraperitoneal (i.p.) route from postnatal day (PND) 4-15. Controls received no treatment/distilled water/ALA. On PND 16, the animals were perfusion fixed and the brains were processed for paraffin embedding (CV and TUNEL staining) and cryopreservation (immunohistochemistry). The fresh brain tissue was used for Western blotting. Significant increase was observed in TUNEL positive cells and Bax (pro-apoptotic protein) expression in hippocampal sub-regions of iAs alone treated groups, whereas Bcl-2 expression was intensified in animals receiving ALA with iAs . Densitometric analysis (Western blots) revealed optimal restoration of Bax and Bcl-2 ratio in animals receiving ALA with iAs , thereby suggesting the protective role of ALA in iAs induced developmental neurotoxicity.

  10. Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro.

    Science.gov (United States)

    Bai, Fangfang; Ni, Bo; Liu, Maojun; Feng, Zhixin; Xiong, Qiyan; Shao, Guoqing

    2015-01-30

    Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Effects of voluntary and treadmill exercise on spontaneous withdrawal signs, cognitive deficits and alterations in apoptosis-associated proteins in morphine-dependent rats.

    Science.gov (United States)

    Mokhtari-Zaer, Amin; Ghodrati-Jaldbakhan, Shahrbanoo; Vafaei, Abbas Ali; Miladi-Gorji, Hossein; Akhavan, Maziar M; Bandegi, Ahmad Reza; Rashidy-Pour, Ali

    2014-09-01

    Chronic exposure to morphine results in cognitive deficits and alterations of apoptotic proteins in favor of cell death in the hippocampus, a brain region critically involved in learning and memory. Physical activity has been shown to have beneficial effects on brain health. In the current work, we examined the effects of voluntary and treadmill exercise on spontaneous withdrawal signs, the associated cognitive defects, and changes of apoptotic proteins in morphine-dependent rats. Morphine dependence was induced through bi-daily administrations of morphine (10mg/kg) for 10 days. Then, the rats were trained under two different exercise protocols: mild treadmill exercise or voluntary wheel exercise for 10 days. After exercise training, their spatial learning and memory and aversive memory were examined by a water maze and by an inhibitory avoidance task, respectively. The expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 in the hippocampus were determined by immunoblotting. We found that chronic exposure to morphine impaired spatial and aversive memory and remarkably suppressed the expression of Bcl-2, but Bax expression remained constant. Both voluntary and treadmill exercise alleviated memory impairment, increased the expression of Bcl-2 protein, and only the later suppressed the expression of Bax protein in morphine-dependent animals. Moreover, both exercise protocols diminished the occurrence of spontaneous morphine withdrawal signs. Our findings showed that exercise reduces the spontaneous morphine-withdrawal signs, blocks the associated impairment of cognitive performance, and overcomes morphine-induced alterations in apoptotic proteins in favor of cell death. Thus, exercise may be a useful therapeutic strategy for cognitive and behavioral deficits in addict individuals. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Suppression of chondrosarcoma cells by 15-deoxy-Δ12,14-prostaglandin J2 is associated with altered expression of Bax/Bcl-xL and p21

    International Nuclear Information System (INIS)

    Shen, Zheng-Nan; Nishida, Keiichiro; Doi, Hideyuki; Oohashi, Toshitaka; Hirohata, Satoshi; Ozaki, Toshifumi; Yoshida, Aki; Ninomiya, Yoshifumi; Inoue, Hajime

    2005-01-01

    We previously reported that 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), the most potent agonist for peroxisome proliferator-activated receptor γ (PPARγ), induces apoptosis of human chondrosarcoma cell line OUMS-27. The current study aimed to explore the mechanism of 15d-PGJ 2 -induced apoptosis and inhibition of cell proliferation in OUMS-27 cells. The preliminary results of cDNA microarray analysis showed the down-regulation of anti-apoptotic Bcl-xL and up-regulation of pro-apoptotic Bax in the process of 15d-PGJ 2 -induced apoptosis. These changes were further confirmed at mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Among cyclin-dependent kinase inhibitors, p21 was induced and up-regulated by 15d-PGJ 2 , but p16 and p27 were not changed, suggesting that the involvement of p21 in inhibition of cell proliferation. Activation of caspase-3 by 15d-PGJ 2 was partly, but not completely, blocked by PPARγ antagonist (GW9662) suggesting the 15d-PGJ 2 exerted its effect by PPARγ-dependent and -independent pathways. Interestingly, immunohistochemical study on human chondrosarcoma samples revealed that Bcl-xL is frequently expressed by tumor cells. The results of the current study suggest that the potential ability of 15d-PGJ 2 in regulation of cell cycle and inhibition of Bcl-xL expression might be beneficial in the development of novel pharmacological agents for chondrosarcoma

  13. 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

    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...... 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...... in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane...

  14. In Vitro Pro-apoptotic and Anti-migratory Effects of Ficus deltoidea L. Plant Extracts on the Human Prostate Cancer Cell Lines PC3

    Science.gov (United States)

    Hanafi, Mohd M. M.; Afzan, Adlin; Yaakob, Harisun; Aziz, Ramlan; Sarmidi, Mohamad R.; Wolfender, Jean-Luc; Prieto, Jose M.

    2017-01-01

    This study aims to evaluate the in vitro cytotoxic and anti-migratory effects of Ficus deltoidea L. on prostate cancer cells, identify the active compound/s and characterize their mechanism of actions. Two farmed varieties were studied, var. angustifolia (FD1) and var. deltoidea (FD2). Their crude methanolic extracts were partitioned into n-hexane (FD1h, FD2h) chloroform (FD1c, FD2c) and aqueous extracts (FD1a, FD2a). Antiproliferative fractions (IC50 < 30 μg/mL, SRB staining of PC3 cells) were further fractionated. Active compound/s were dereplicated using spectroscopic methods. In vitro mechanistic studies on PC3 and/or LNCaP cells included: annexin V-FITC staining, MMP depolarization measurements, activity of caspases 3 and 7, nuclear DNA fragmentation and cell cycle analysis, modulation of Bax, Bcl-2, Smac/Diablo, and Alox-5 mRNA gene expression by RT-PCR. Effects of cytotoxic fractions on 2D migration and 3D invasion were tested by exclusion assays and modified Boyden chamber, respectively. Their mechanisms of action on these tests were further studied by measuring the expression VEGF-A, CXCR4, and CXCL12 in PC3 cells by RT-PCR. FD1c and FD2c extracts induced cell death (P < 0.05) via apoptosis as evidenced by nuclear DNA fragmentation. This was accompanied by an increase in MMP depolarization (P < 0.05), activation of caspases 3 and 7 (P < 0.05) in both PC3 and LNCaP cell lines. All active plant extracts up-regulated Bax and Smac/DIABLO, down-regulated Bcl-2 (P < 0.05). Both FD1c and FD2c were not cytotoxic against normal human fibroblast cells (HDFa) at the tested concentrations. Both plant extracts inhibited both migration and invasion of PC3 cells (P < 0.05). These effects were accompanied by down-regulation of both VEGF-A and CXCL-12 gene expressions (P < 0.001). LC–MS dereplication using taxonomy filters and molecular networking databases identified isovitexin in FD1c; and oleanolic acid, moretenol, betulin, lupenone, and lupeol in FD2c. In conclusion

  15. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article...

  16. Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.

    Science.gov (United States)

    Renault, Thibaud T; Floros, Konstantinos V; Elkholi, Rana; Corrigan, Kelly-Ann; Kushnareva, Yulia; Wieder, Shira Y; Lindtner, Claudia; Serasinghe, Madhavika N; Asciolla, James J; Buettner, Christoph; Newmeyer, Donald D; Chipuk, Jerry E

    2015-01-08

    Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Bax/Bcl-2 protein expression ratio and leukocyte function are related to reduction of Walker-256 tumor growth after β-hydroxy-β-methylbutyrate (HMB) administration in Wistar rats.

    Science.gov (United States)

    Kuczera, Diogo; Paro de Oliveira, Heloísa Helena; Fonseca Guimarães, Fernando de Souza; de Lima, Carina; Alves, Luciana; Machado, Andressa Franzói; Coelho, Isabela; Yamaguchi, Adriana; Donatti, Lucélia; Naliwaiko, Katya; Fernandes, Luiz Claudio; Nunes, Everson Araújo

    2012-01-01

    This study investigated the mechanisms by which β-hydroxy-β-methylbutyrate (HMB) administration in rats reduces Walker-256 tumor growth. Male Wistar rats were supplemented with HMB (76 mg/kg/day) (HW), or a placebo (W), during 8 wk by gavage. At the 6th wk, rats were inoculated with a suspension of Walker 256 tumor cells (3 × 10(7)/mL). Fifteen days after inoculation, the HW group showed higher glycemia (109.4 ± 5.53 vs. 89.87 ± 7.02 mg/dL, P HMB-treated rats displayed a 36.9% decrement in rates of proliferation ex vivo and a significant increase in the Bax/Bcl-2 protein expression ratio in comparison to those extracted from the placebo-treated rats (P HMB supplementation decreases tumor burden by modifying the inner environment of tumor cells and by interfering with blood leukocyte function.

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

    Science.gov (United States)

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

    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 Bcl-2 was observed in 10 out of 27 (37.0%) invasive carcinomas, 1 out of 10 dysplasias, 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 = 0.110), 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. PMID:14606101

  19. The catechin flavonoid reduces proliferation and induces apoptosis of murine lymphoma cells LB02 through modulation of antiapoptotic proteins

    Directory of Open Access Journals (Sweden)

    Daniela Laura Papademetrio

    2013-06-01

    Full Text Available Flavonoids are products of secondary metabolism of plants. They are present in herbs and trees and also act as natural chemopreventives and anticancer agents. Ligaria cuneifolia (Ruiz & Pav. Tiegh., Loranthaceae, is a hemiparasite species that belongs to Argentine flora. Phytochemical studies have disclosed the presence of quercetin, catechin-4β-ol and pro-anthocyanidine as polyphenolic compounds in the active extracts. We previously demonstrated that ethyl acetate extract was capable of reducing cell proliferation and inducing apoptotic death of lymphoid tumor cells. The aim of the current study is to determine whether or not catechin, isolated from L. cuneifolia extracts can induce leukemia cell death and to determine its effect on the cytoplasmatic proteins that modulate cell survival. Our results show that catechin can reduce proliferation of murine lymphoma cell line LB02. The effect is mediated by apoptosis at concentrations upper to 100 µg/mL. Cell death is related to the loss of mitochondrial membrane potential (ΔΨm and a down regulation of survivin and Bcl-2 together with the increase of pro-apoptotic protein Bax. In summary, the current study indicates that catechin present in the extract of L. cuneifolia is in part, responsible for the anti-proliferative activity of whole extracts by induction of ΔΨm disruption and modulation of the anti-apoptotic proteins over expressed in tumor cells. These results give new findings into the potential anticancer and chemopreventive activities of L. cuneifolia.

  20. The catechin flavonoid reduces proliferation and induces apoptosis of murine lymphoma cells LB02 through modulation of antiapoptotic proteins

    Directory of Open Access Journals (Sweden)

    Daniela Laura Papademetrio

    2013-03-01

    Full Text Available Flavonoids are products of secondary metabolism of plants. They are present in herbs and trees and also act as natural chemopreventives and anticancer agents. Ligaria cuneifolia (Ruiz & Pav. Tiegh., Loranthaceae, is a hemiparasite species that belongs to Argentine flora. Phytochemical studies have disclosed the presence of quercetin, catechin-4β-ol and pro-anthocyanidine as polyphenolic compounds in the active extracts. We previously demonstrated that ethyl acetate extract was capable of reducing cell proliferation and inducing apoptotic death of lymphoid tumor cells. The aim of the current study is to determine whether or not catechin, isolated from L. cuneifolia extracts can induce leukemia cell death and to determine its effect on the cytoplasmatic proteins that modulate cell survival. Our results show that catechin can reduce proliferation of murine lymphoma cell line LB02. The effect is mediated by apoptosis at concentrations upper to 100 µg/mL. Cell death is related to the loss of mitochondrial membrane potential (ΔΨm and a down regulation of survivin and Bcl-2 together with the increase of pro-apoptotic protein Bax. In summary, the current study indicates that catechin present in the extract of L. cuneifolia is in part, responsible for the anti-proliferative activity of whole extracts by induction of ΔΨm disruption and modulation of the anti-apoptotic proteins over expressed in tumor cells. These results give new findings into the potential anticancer and chemopreventive activities of L. cuneifolia.

  1. 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.

  2. Anti-inflammatory drugs suppress proliferation and induce apoptosis through altering expressions of cell cycle regulators and pro-apoptotic factors in cultured human osteoblasts

    International Nuclear Information System (INIS)

    Chang, J.-K.; Li, C.-J.; Liao, H.-J.; Wang, C.-K.; Wang, G.-J.; Ho, M.-L.

    2009-01-01

    It has been reported that anti-inflammatory drugs (AIDs) inhibited bone repair in animal studies, and suppressed proliferation and induced cell death in rat osteoblast cultures. In this study, we further investigated the molecular mechanisms of AID effects on proliferation and cell death in human osteoblasts (hOBs). We examined the effects of dexamethasone (10 -7 and 10 -6 M), non-selective non-steroidal anti-inflammatory drugs (NSAIDs): indomethacin, ketorolac, piroxicam and diclofenac (10 -5 and 10 -4 M), and COX-2 inhibitor: celecoxib (10 -6 and 10 -5 M) on proliferation, cytotoxicity, cell death, and mRNA and protein levels of cell cycle and apoptosis-related regulators in hOBs. All the tested AIDs significantly inhibited proliferation and arrested cell cycle at G0/G1 phase in hOBs. Celecoxib and dexamethasone, but not non-selective NSAIDs, were found to have cytotoxic effects on hOB, and further demonstrated to induce apoptosis and necrosis (at higher concentration) in hOBs. We further found that indomethacin, celecoxib and dexamethasone increased the mRNA and protein expressions of p27 kip1 and decreased those of cyclin D2 and p-cdk2 in hOBs. Bak expression was increased by celecoxib and dexamethasone, while Bcl-XL level was declined only by dexamethasone. Furthermore, the replenishment of PGE1, PGE2 or PGF2α did not reverse the effects of AIDs on proliferation and expressions of p27 kip1 and cyclin D2 in hOBs. We conclude that the changes in expressions of regulators of cell cycle (p27 kip1 and cyclin D2) and/or apoptosis (Bak and Bcl-XL) by AIDs may contribute to AIDs caused proliferation suppression and apoptosis in hOBs. This effect might not relate to the blockage of prostaglandin synthesis by AIDs

  3. Cytotoxicity of CdTe quantum dots in human umbilical vein endothelial cells: the involvement of cellular uptake and induction of pro-apoptotic endoplasmic reticulum stress

    Directory of Open Access Journals (Sweden)

    Yan M

    2016-02-01

    activation of protein kinase RNA-like ER kinase-eIF2α-activating transcription factor 4 pathway (including phosphorylation of both protein kinase RNA-like ER kinase and eIF2α and elevated level of activating transcription factor 4. CdTe QDs further promoted an increased C/EBP homologous protein expression, phosphorylation of c-JUN NH2-terminal kinase, and cleavage of ER-resident caspase-4, while the specific inhibitor (SP600125, Z-LEVD-fmk, or salubrinal significantly attenuated QDs-triggered apoptosis, indicating that all three ER stress-mediated apoptosis pathways were activated and the direct participation of ER in the CdTe QDs-caused apoptotic cell death in HUVECs. Our findings provide important new insights into QDs toxicity and reveal potential cardiovascular risks for the future applications of QDs. Keywords: quantum dots, human umbilical vein endothelial cells, endocytosis, ER stress, apoptosis

  4. A new l-amino acid oxidase from Bothrops jararacussu snake venom: Isolation, partial characterization, and assessment of pro-apoptotic and antiprotozoal activities.

    Science.gov (United States)

    Carone, Sante E I; Costa, Tássia R; Burin, Sandra M; Cintra, Adélia C O; Zoccal, Karina F; Bianchini, Francine J; Tucci, Luiz F F; Franco, João J; Torqueti, Maria R; Faccioli, Lúcia H; Albuquerque, Sérgio de; Castro, Fabíola A de; Sampaio, Suely V

    2017-10-01

    A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95-97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC 50 =4.56μg/mL) and Trypanosoma cruzi (IC 50 =4.85μg/mL). This toxin also induced cytotoxicity (IC 50 =1.80μg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. The innate immunity adaptor SARM translocates to the nucleus to stabilize lamins and prevent DNA fragmentation in response to pro-apoptotic signaling.

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    Chad R Sethman

    Full Text Available Sterile alpha and armadillo-motif containing protein (SARM, a highly conserved and structurally unique member of the MyD88 family of Toll-like receptor adaptors, plays an important role in innate immunity signaling and apoptosis. Its exact mechanism of intracellular action remains unclear. Apoptosis is an ancient and ubiquitous process of programmed cell death that results in disruption of the nuclear lamina and, ultimately, dismantling of the nucleus. In addition to supporting the nuclear membrane, lamins serve important roles in chromatin organization, epigenetic regulation, transcription, nuclear transport, and mitosis. Mutations and other damage that destabilize nuclear lamins (laminopathies underlie a number of intractable human diseases. Here, we report that SARM translocates to the nucleus of human embryonic kidney cells by using its amino-terminal Armadillo repeat region. Within the nucleus, SARM forms a previously unreported lattice akin to the nuclear lamina scaffold. Moreover, we show that SARM protects lamins from apoptotic degradation and reduces internucleosomal DNA fragmentation in response to signaling induced by the proinflammatory cytokine Tumor Necrosis Factor alpha. These findings indicate an important link between the innate immunity adaptor SARM and stabilization of nuclear lamins during inflammation-driven apoptosis in human cells.

  6. Mutational analysis of Bax and Bcl-2 in childhood acute lymphoblastic leukaemia

    NARCIS (Netherlands)

    Salomons, G. S.; Buitenhuis, C. K.; Martínez Muñoz, C.; Verwijs-Jassen, M.; Behrendt, H.; Zsiros, J.; Smets, L. A.

    1998-01-01

    In childhood acute lymphoblastic leukaemia there are large interpatient variations in levels of the apoptosis-regulating proteins Bax and Bcl-2, but the molecular basis for this variation is unknown. Point-mutations in bax have been reported in cell lines derived from haematological malignancies.

  7. All-trans retinoic acid inhibits KIT activity and induces apoptosis in gastrointestinal stromal tumor GIST-T1 cell line by affecting on the expression of survivin and Bax protein

    Directory of Open Access Journals (Sweden)

    Taguchi Takahiro

    2010-12-01

    Full Text Available Abstract Background Imatinib, a selective tyrosine kinase inhibitor, has been used as a standard first-line therapy for irresectable and metastasized gastrointestinal stromal tumor (GIST patients. Unfortunately, most patients responding to imatinib will eventually exhibit imatinib-resistance, the cause of which is not fully understood. The serious clinical problem of imatinib-resistance demands alternative therapeutic strategy. This study was conducted to investigate the effect of all-trans retinoic acid (ATRA on GIST cell lines. Methods Cell proliferation was determined by trypan blue dye exclusion test. Western blot analysis was performed to test the expression of activated KIT, its downstream proteins, and apoptosis associated proteins. The cytotoxic interactions of imatinib with ATRA were evaluated using the isobologram of Steel and Peckham. Results and conclusion In this work, for the first time we have demonstrated that ATRA affected on cell proliferation of GIST-T1 and GIST-882 cell line through inhibition of cell growth in a dose dependent manner and induced apoptosis. High dose of ATRA induced morphologic change in GIST-T1 cells, rounded-up cells, and activated the caspase-3 protein. In further examination, we found that the ATRA-induced apoptosis in GIST-T1 cells was accompanied by the down-regulated expression of survivin and up-regulated expression of Bax protein. Moreover, ATRA suppressed the activity of KIT protein in GIST-T1 cells and its downstream signal, AKT activity, but not MAPK activity. We also have demonstrated that combination of ATRA with imatinib showed additive effect by isobologram, suggesting that the combination of ATRA and imatinib may be a novel potential therapeutic option for GIST treatment. Furthermore, the scracht assay result suggested that ATRA was a potential reagent to prevent the invasion or metastasis of GIST cells.

  8. Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737.

    Science.gov (United States)

    Renault, Thibaud T; Teijido, Oscar; Missire, Florent; Ganesan, Yogesh Tengarai; Velours, Gisèle; Arokium, Hubert; Beaumatin, Florian; Llanos, Raul; Athané, Axel; Camougrand, Nadine; Priault, Muriel; Antonsson, Bruno; Dejean, Laurent M; Manon, Stéphen

    2015-07-01

    Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. An Involvement of PI3-K/Akt Activation and Inhibition of AIF Translocation in Neuroprotective Effects of Undecylenic Acid (UDA) Against Pro-Apoptotic Factors-Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells.

    Science.gov (United States)

    Jantas, Danuta; Piotrowski, Marek; Lason, Wladyslaw

    2015-12-01

    Undecylenic acid (UDA), a naturally occurring 11-carbon unsaturated fatty acid, has been used for several years as an economical antifungal agent and a nutritional supplement. Recently, the potential usefulness of UDA as a neuroprotective drug has been suggested based on the ability of this agent to inhibit μ-calpain activity. In order to verify neuroprotective potential of UDA, we tested protective efficacy of this compound against cell damage evoked by pro-apoptotic factors (staurosporine and doxorubicin) and oxidative stress (hydrogen peroxide) in human neuroblastoma SH-SY5Y cells. We showed that UDA partially protected SH-SY5Y cells against the staurosporine- and doxorubicin-evoked cell death; however, this effect was not connected with its influence on caspase-3 activity. UDA decreased the St-induced changes in mitochondrial and cytosolic AIF level, whereas in Dox-model it affected only the cytosolic AIF content. Moreover, UDA (1-40 μM) decreased the hydrogen peroxide-induced cell damage which was connected with attenuation of hydrogen peroxide-mediated necrotic (PI staining, ADP/ATP ratio) and apoptotic (mitochondrial membrane potential, caspase-3 activation, AIF translocation) changes. Finally, we demonstrated that an inhibitor of PI3-K/Akt (LY294002) but not MAPK/ERK1/2 (U0126) pathway blocked the protection mediated by UDA in all tested models of SH-SY5Y cell injury. These in vitro data point to UDA as potentially effective neuroprotectant the utility of which should be further validated in animal studies. © 2015 Wiley Periodicals, Inc.

  10. Pro-Apoptotic Breast Cancer Nanotherapeutics

    Science.gov (United States)

    2013-07-01

    R.; Hagooly, A.; Chen, Z.; Welch, M. J.; Wooley, K. L. Folate -Mediated Cell Uptake of Shell-Cross- linked Spheres and Cylinders. J. Polym. Sci. Part...Cancer Res. 1995, 55, 3825–3834. 44. Masson, C.; Scherman, D.; Bessodes, M. 2,2,6,6-Tetra- methyl -1-Piperidinyl-Oxyl/[Bis(Acetoxy)-Iodo]Benzene

  11. BH3-only protein Bim is associated with the degree of Helicobacter pylori-induced gastritis and is localized to the mitochondria of inflammatory cells in the gastric mucosa.

    Science.gov (United States)

    Akazawa, Yuko; Matsuda, Katsuya; Isomoto, Hajime; Matsushima, Kayoko; Kido, Yoko; Urabe, Shigetoshi; Yamaghchi, Naoyuki; Ohnita, Ken; Takeshima, Fuminao; Kondo, Hisayoshi; Tsugawa, Hitoshi; Suzuki, Hidekazu; Moss, Joel; Nakao, Kazuhiko; Nakashima, Masahiro

    2015-09-01

    BH3-only protein, Bim, is a pro-apoptotic protein that mediates mitochondria-dependent cell death. However, the role of Bim in Helicobacter pylori-associated gastritis remains unclear. This study aimed to assess the cellular localization of Bim and its possible role in H. pylori-induced gastritis. The study was conducted on biopsy specimens obtained from 80 patients who underwent upper gastrointestinal endoscopy (H. pylori-negative: n=30, positive: n=50). Association between Bim mRNA expression and severity of gastritis was evaluated and the localization of Bim was examined by immunofluorescence. Bim mRNA expression was positively correlated with the degree of gastritis, as defined by the Sydney system. Immunohistochemical analysis confirmed increased Bim expression in H. pylori-infected gastric mucosa compared with uninfected mucosa in both humans and mice. Bim localized in myeloperoxidase- and CD138-positive cells of H. pylori-infected lamina propria and submucosa of the gastric tract, indicating that this protein is predominantly expressed in neutrophils and plasma cells. In contrast, Bim did not localize in CD20-, CD3-, or CD68-positive cells. Bim was expressed in the mitochondria, where it was partially co-localized with activated Bax and cleaved-PARP. In conclusion, Bim is expressed in neutrophils and plasma cells in H. pylori-associated gastritis, where it may participate in the termination of inflammatory response by causing mitochondria-mediated apoptosis in specific leucocytes. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. Effect of acetone extract of Rumex japonicas Houtt on hydrogen ...

    African Journals Online (AJOL)

    is the most important executor in the apoptotic process [25]. Furthermore, Bax is a pro-apoptotic protein in the mitochondria-mediated apoptosis, whereas Bcl-2 is an anti-apoptotic protein [24]. In the present study, AER down-regulated the caspase-3 and Bax, but up-regulated Bcl-2, indicating that AER suppresses apoptosis ...

  13. Pro-apoptotic and pro-autophagic effects of the Aurora kinase A inhibitor alisertib (MLN8237 on human osteosarcoma U-2 OS and MG-63 cells through the activation of mitochondria-mediated pathway and inhibition of p38 MAPK/PI3K/Akt/mTOR signaling pathway

    Directory of Open Access Journals (Sweden)

    Niu NK

    2015-03-01

    mesenchymal transition (EMT and the underlying mechanisms in two human OS cell lines U-2 OS and MG-63. The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells. ALS remarkably induced G2/M arrest and down-regulated the expression levels of cyclin-dependent kinases 1 and 2 and cyclin B1 in both U-2 OS and MG-63 cells. ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins. Furthermore, ALS promoted autophagic cell death via the inhibition of phosphatidylinositol 3-kinase (PI3K/protein kinase B (Akt/mammalian target of rapamycin (mTOR and p38 mitogen-activated protein kinase (p38 MAPK signaling pathways, and activation of 5'-AMP-dependent kinase (AMPK signaling pathway. Inducers or inhibitors of apoptosis or autophagy simultaneously altered ALS-induced apoptotic and autophagic death in both U-2 OS and MG-63 cells, suggesting a crosstalk between these two primary modes of programmed cell death. Moreover, ALS suppressed EMT-like phenotypes with a marked increase in the expression of E-cadherin but a decrease in N-cadherin in U-2 OS and MG-63 cells. ALS treatment also induced reactive oxygen species (ROS generation but inhibited the expression levels of sirtuin 1 and nuclear factor-erythroid-2-related factor 2 (Nrf2 in both cell lines. Taken together, these findings show that ALS promotes apoptosis and autophagy but inhibits EMT via PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways with involvement of ROS- and sirtuin 1-associated pathways in U-2 OS and MG-63 cells. ALS is a promising anticancer agent in OS treatment and further studies are needed to confirm its efficacy and safety in OS chemotherapy. Keywords: ALS, autophagy, apoptosis, osteosarcoma, PI3K/Akt/mTOR pathway, EMT

  14. 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.

  15. Bax regulates neuronal Ca2+ homeostasis.

    Science.gov (United States)

    D'Orsi, Beatrice; Kilbride, Seán M; Chen, Gang; Perez Alvarez, Sergio; Bonner, Helena P; Pfeiffer, Shona; Plesnila, Nikolaus; Engel, Tobias; Henshall, David C; Düssmann, Heiko; Prehn, Jochen H M

    2015-01-28

    Excessive Ca(2+) entry during glutamate receptor overactivation ("excitotoxicity") induces acute or delayed neuronal death. We report here that deficiency in bax exerted broad neuroprotection against excitotoxic injury and oxygen/glucose deprivation in mouse neocortical neuron cultures and reduced infarct size, necrotic injury, and cerebral edema formation after middle cerebral artery occlusion in mice. Neuronal Ca(2+) and mitochondrial membrane potential (Δψm) analysis during excitotoxic injury revealed that bax-deficient neurons showed significantly reduced Ca(2+) transients during the NMDA excitation period and did not exhibit the deregulation of Δψm that was observed in their wild-type (WT) counterparts. Reintroduction of bax or a bax mutant incapable of proapoptotic oligomerization equally restored neuronal Ca(2+) dynamics during NMDA excitation, suggesting that Bax controlled Ca(2+) signaling independently of its role in apoptosis execution. Quantitative confocal imaging of intracellular ATP or mitochondrial Ca(2+) levels using FRET-based sensors indicated that the effects of bax deficiency on Ca(2+) handling were not due to enhanced cellular bioenergetics or increased Ca(2+) uptake into mitochondria. We also observed that mitochondria isolated from WT or bax-deficient cells similarly underwent Ca(2+)-induced permeability transition. However, when Ca(2+) uptake into the sarco/endoplasmic reticulum was blocked with the Ca(2+)-ATPase inhibitor thapsigargin, bax-deficient neurons showed strongly elevated cytosolic Ca(2+) levels during NMDA excitation, suggesting that the ability of Bax to support dynamic ER Ca(2+) handling is critical for cell death signaling during periods of neuronal overexcitation. Copyright © 2015 the authors 0270-6474/15/351706-17$15.00/0.

  16. An ANNEXIN-like protein from the cereal cyst nematode Heterodera avenae suppresses plant defense.

    Directory of Open Access Journals (Sweden)

    Changlong Chen

    Full Text Available Parasitism genes encoding secreted effector proteins of plant-parasitic nematodes play important roles in facilitating parasitism. An annexin-like gene was isolated from the cereal cyst nematode Heterodera avenae (termed Ha-annexin and had high similarity to annexin 2, which encodes a secreted protein of Globodera pallida. Ha-annexin encodes a predicted 326 amino acid protein containing four conserved annexin domains. Southern blotting revealed that there are at least two homologies in the H. avenae genome. Ha-annexin transcripts were expressed within the subventral gland cells of the pre-parasitic second-stage juveniles by in situ hybridization. Additionally, expression of these transcripts were relatively higher in the parasitic second-stage juveniles by quantitative real-time RT-PCR analysis, coinciding with the time when feeding cell formation is initiated. Knockdown of Ha-annexin by method of barley stripe mosaic virus-based host-induced gene silencing (BSMV-HIGS caused impaired nematode infections at 7 dpi and reduced females at 40 dpi, indicating important roles of the gene in parasitism at least in early stage in vivo. Transiently expression of Ha-ANNEXIN in onion epidermal cells and Nicotiana benthamiana leaf cells showed the whole cell-localization. Using transient expression assays in N. benthamiana, we found that Ha-ANNEXIN could suppress programmed cell death triggered by the pro-apoptotic mouse protein BAX and the induction of marker genes of PAMP-triggered immunity (PTI in N. benthamiana. In addition, Ha-ANNEXIN targeted a point in the mitogen-activated protein kinase (MAPK signaling pathway downstream of two kinases MKK1 and NPK1 in N. benthamiana.

  17. 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.

  18. Novel Functional Role of Heat Shock Protein 90 in Mitochondrial Connexin 43-Mediated Hypoxic Postconditioning

    Directory of Open Access Journals (Sweden)

    Rong-Hui Tu

    2017-11-01

    Full Text Available Background/Aims: Previous studies have shown that heat shock protein 90 (HSP90-mediated mitochondrial import of connexin 43 (Cx43 is critical in preconditioning cardioprotection. The present study was designed to test whether postconditioning has the same effect as preconditioning in promoting Cx43 translocation to mitochondria and whether mitochondrial HSP90 modulates this effect. Methods: Cellular models of hypoxic postconditioning (HPC from rat heart-derived H9c2 cells and neonatal rat cardiomyocytes were employed. The effects of HPC on cardiomyocytes apoptosis were examined by flow cytometry and Hoechst 33342 fluorescent staining. Reactive oxidative species (ROS production was assessed with the peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescin in diacetate (DCFH-DA. The anti- and pro-apoptotic markers Bcl-2 and Bax, HSP90 and Cx43 protein levels were studied by Western blot analysis in total cell homogenate and sarcolemmal and mitochondrial fractions. The effects on HPC of the HSP90 inhibitor geldanamycin (GA, ROS scavengers superoxide dismutase (SOD and catalase (CAT, and small interfering RNA (siRNA targeting Cx43 and HSP90 were also investigated. Results: HPC significantly reduced hypoxia/reoxygenation (H/R-induced cardiomyocyte apoptosis. These beneficial effects were accompanied by an increase in Bcl-2 levels and a decrease in Bax levels in both sarcolemmal and mitochondrial fractions. HPC with siRNA targeting Cx43 or the ROS scavengers SOD plus CAT significantly prevented ROS generation and HPC cardioprotection, but HPC with either SOD or CAT did not. These data strongly supported the involvement of Cx43 in HPC cardioprotection, likely via modulation of the ROS balance which plays a central role in HPC protection. Furthermore, HPC increased total and mitochondrial levels of HSP90 and the mitochondria-to-sarcolemma ratio of Cx43; blocking the function of HSP90 with the HSP90 inhibitor geldanamycin (GA or siRNA targeting

  19. 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.

  20. Ginsenoside Rh2 Induces Human Hepatoma Cell Apoptosisvia Bax/Bak Triggered Cytochrome C Release and Caspase-9/Caspase-8 Activation

    Directory of Open Access Journals (Sweden)

    Xiao-Xi Guo

    2012-11-01

    Full Text Available Ginsenoside Rh2 (G-Rh2 has been shown to induce apoptotic cell death in a variety of cancer cells. However, the details of the signal transduction cascade involved in G-Rh2-induced cell death is unclear. In this manuscript we elucidate the molecular mechanism of G-Rh2-induced apoptosis in human hepatoma SK-HEP-1 cells by demonstrating that G-Rh2 causes rapid and dramatic translocation of both Bak and Bax, which subsequently triggers mitochondrial cytochrome c release and consequent caspase activation. Interestingly, siRNA-based gene inactivation of caspase-8 effectively delays caspase-9 activation and apoptosis induced by G-Rh2, indicating that caspase-8 also plays an important role in the G-Rh2-induced apoptosis program. Taken together, our results indicate that G-Rh2 employs a multi pro-apoptotic pathway to execute cancer cell death, suggesting a potential role for G-Rh2 as a powerful chemotherapeutic agent.

  1. 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.

  2. Modulation of Signal Proteins: A Plausible Mechanism to Explain How a Potentized Drug Secale Cor 30C Diluted beyond Avogadro's Limit Combats Skin Papilloma in Mice.

    Science.gov (United States)

    Khuda-Bukhsh, Anisur Rahman; Bhattacharyya, Soumya Sundar; Paul, Saili; Dutta, Suman; Boujedaini, Naoual; Belon, Philippe

    2011-01-01

    In homeopathy, ability of ultra-high diluted drugs at or above potency 12C (diluted beyond Avogadro's limit) in ameliorating/curing various diseases is often questioned, particularly because the mechanism of action is not precisely known. We tested the hypothesis if suitable modulations of signal proteins could be one of the possible pathways of action of a highly diluted homeopathic drug, Secale cornutum 30C (diluted 10(60) times; Sec cor 30). It could successfully combat DMBA + croton oil-induced skin papilloma in mice as evidenced by histological, cytogenetical, immunofluorescence, ELISA and immunoblot findings. Critical analysis of several signal proteins like AhR, PCNA, Akt, Bcl-2, Bcl-xL, NF-κB and IL-6 and of pro-apoptotic proteins like cytochrome c, Bax, Bad, Apaf, caspase-3 and -9 revealed that Sec cor 30 suitably modulated their expression levels along with amelioration of skin papilloma. FACS data also suggested an increase of cell population at S and G2 phases and decrease in sub-G1 and G1 phages in carcinogen-treated drug-unfed mice, but these were found to be near normal in the Sec cor 30-fed mice. There was reduction in genotoxic and DNA damages in bone marrow cells of Sec Cor 30-fed mice, as revealed from cytogenetic and Comet assays. Changes in histological features of skin papilloma were noted. Immunofluorescence studies of AhR and PCNA also suggested reduced expression of these proteins in Sec cor 30-fed mice, thereby showing its anti-cancer potentials against skin papilloma. Furthermore, this study also supports the hypothesis that potentized homeopathic drugs act at gene regulatory level.

  3. Modulation of Signal Proteins: A Plausible Mechanism to Explain How a Potentized Drug Secale Cor 30C Diluted beyond Avogadro's Limit Combats Skin Papilloma in Mice

    Directory of Open Access Journals (Sweden)

    Anisur Rahman Khuda-Bukhsh

    2011-01-01

    Full Text Available In homeopathy, ability of ultra-high diluted drugs at or above potency 12C (diluted beyond Avogadro's limit in ameliorating/curing various diseases is often questioned, particularly because the mechanism of action is not precisely known. We tested the hypothesis if suitable modulations of signal proteins could be one of the possible pathways of action of a highly diluted homeopathic drug, Secale cornutum 30C (diluted 1060 times; Sec cor 30. It could successfully combat DMBA + croton oil-induced skin papilloma in mice as evidenced by histological, cytogenetical, immunofluorescence, ELISA and immunoblot findings. Critical analysis of several signal proteins like AhR, PCNA, Akt, Bcl-2, Bcl-xL, NF-κB and IL-6 and of pro-apoptotic proteins like cytochrome c, Bax, Bad, Apaf, caspase-3 and -9 revealed that Sec cor 30 suitably modulated their expression levels along with amelioration of skin papilloma. FACS data also suggested an increase of cell population at S and G2 phases and decrease in sub-G1 and G1 phages in carcinogen-treated drug-unfed mice, but these were found to be near normal in the Sec cor 30-fed mice. There was reduction in genotoxic and DNA damages in bone marrow cells of Sec Cor 30-fed mice, as revealed from cytogenetic and Comet assays. Changes in histological features of skin papilloma were noted. Immunofluorescence studies of AhR and PCNA also suggested reduced expression of these proteins in Sec cor 30-fed mice, thereby showing its anti-cancer potentials against skin papilloma. Furthermore, this study also supports the hypothesis that potentized homeopathic drugs act at gene regulatory level.

  4. Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation.

    Science.gov (United States)

    Romero-Fernandez, Wilber; Borroto-Escuela, Dasiel O; Alea, Mileidys Perez; Garcia-Mesa, Yoelvis; Garriga, Pere

    2011-12-01

    The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity. © The Author 2011. Published by Oxford University Press. All rights reserved.

  5. The HIV-1 Vpu protein induces apoptosis in Drosophila via activation of JNK signaling.

    Directory of Open Access Journals (Sweden)

    Christelle Marchal

    Full Text Available The genome of the human immunodeficiency virus type 1 (HIV-1 encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu, in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1. Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.

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

    Czech Academy of Sciences Publication Activity Database

    Lidman, M.; Pokorná, Šárka; Dingeldein, A. P. G.; Sparrman, T.; Wallgren, M.; Šachl, Radek; Hof, Martin; Gröbner, G.

    2016-01-01

    Roč. 1858, č. 6 (2016), s. 1288-1297 ISSN 0005-2736 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : Apoptosis * Bax-protein * Calorimetry Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.498, year: 2016

  7. Estradiol increases the Bax/Bcl-2 ratio and induces apoptosis in the anterior pituitary gland.

    Science.gov (United States)

    Zaldivar, Verónica; Magri, María Laura; Zárate, Sandra; Jaita, Gabriela; Eijo, Guadalupe; Radl, Daniela; Ferraris, Jimena; Pisera, Daniel; Seilicovich, Adriana

    2009-01-01

    Estrogens are recognized as acting as modulators of pituitary cell renewal, sensitizing cells to mitogenic and apoptotic signals, thus participating in anterior pituitary homeostasis during the estrous cycle. The balance of pro- and antiapoptotic proteins of the Bcl-2 family is known to regulate cell survival and apoptosis. In order to understand the mechanisms underlying apoptosis during the estrous cycle, we evaluated the expression of the proapoptotic protein Bax and the antiapoptotic proteins Bcl-2 and Bcl-xL in the anterior pituitary gland in cycling female rats as well as the influence of estradiol on the expression of these proteins in anterior pituitary cells of ovariectomized rats. As determined by Western blot, the expression of Bax was higher in anterior pituitary glands from rats at proestrus than at diestrus I, Bcl-2 protein levels showed no difference and Bcl-xL expression was lower, thus increasing the Bax/Bcl-2 ratio at proestrus. Assessed by annexin V binding and flow cytometry, the percentage of apoptotic anterior pituitary cells was higher in rats at proestrus than at diestrus I. Chronic estrogen treatment in ovariectomized rats enhanced the Bax/Bcl-2 ratio and induced apoptosis. Moreover, incubation of cultured anterior pituitary cells from ovariectomized rats with 17beta-estradiol for 24 h increased the Bax/Bcl-2 ratio, decreased Bcl-xL expression and induced apoptosis. Our results demonstrate that estradiol increases the ratio between proapoptotic and antiapoptotic proteins of the Bcl-2 family. This effect could participate in the sensitizing action of estrogens to proapoptotic stimuli and therefore be involved in the high apoptotic rate observed at proestrus in the anterior pituitary gland.

  8. Inhibition of early 99mTc-MIBI uptake by Bcl-2 anti-apoptotic protein overexpression in untreated breast carcinoma

    International Nuclear Information System (INIS)

    Del Vecchio, Silvana; Zannetti, Antonella; Aloj, Luigi; Caraco, Corradina; Ciarmiello, Andrea; Salvatore, Marco

    2003-01-01

    Lack of technetium-99m methoxyisobutylisonitrile ( 99m Tc-MIBI) uptake is consistently reported to predict poor response to subsequent chemotherapy in a variety of human malignant tumours. Since 99m Tc-MIBI accumulates within mitochondria, which also play a central role in apoptosis through the integration of death signals by Bcl-2 family members, we tested whether early 99m Tc-MIBI uptake is affected by alterations of the apoptotic pathway. Forty-two breast cancer patients were intravenously injected with 740 MBq of 99m Tc-MIBI and planar images were obtained 10 min post injection with the patients in the prone lateral position. Ten carcinomas failed to accumulate 99m Tc-MIBI and could not be visualised on scintigraphic images despite being larger than 1.8 cm (MIBI negative). Thirty-two of the 42 breast carcinomas showed focal uptake of 99m Tc-MIBI (MIBI positive), and 10 min tumour-to-background ratios (T/B) varied between 1.14 and 6.93. The apoptotic index, the rate of proliferation, and the expression of the anti-apoptotic Bcl-2 protein and pro-apoptotic Bax protein were assessed in surgically excised tumours. All MIBI-negative carcinomas showed a dramatic and statistically significant reduction in the apoptotic index as compared with MIBI-positive lesions (mean±SD, 0.14±0.15 vs 1.28±0.83, P 99m Tc-MIBI in breast carcinomas is affected by alterations of apoptotic pathway. High levels of Bcl-2, despite the stabilisation of mitochondrial membrane potentials, prevent accumulation of 99m Tc-MIBI in tumour cells. In conclusion, absent or reduced early 99m Tc-MIBI uptake in large tumours may indicate a Bcl-2-mediated resistance to chemo- and radiotherapy. (orig.)

  9. Bcl-2 family of proteins as drug targets for cancer chemotherapy: the long way of BH3 mimetics from bench to bedside.

    Science.gov (United States)

    Vela, Laura; Marzo, Isabel

    2015-08-01

    Bcl-2 proteins are key determinants in the life-death balance. In recent years, proteins in this family have been identified as drug targets in the design of new anti-tumor therapies. Advances in the knowledge of the mechanism of action of anti-apoptotic and pro-apoptotic members of the Bcl-2 family have enabled the development of the so-called 'BH3 mimetics'. These compounds act by inhibiting anti-apoptotic proteins of the family, imitating the function of the BH3-only subset of pro-apoptotic members. Combinations of BH3-mimetics with anti-tumor drugs are being evaluated in both preclinical models and clinical trials. Recent advances in these approaches will be reviewed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. BAX/BAK–Independent Mitoptosis during Cell Death Induced by Proteasome Inhibition?

    OpenAIRE

    Lomonosova, Elena; Ryerse, Jan; Chinnadurai, G.

    2009-01-01

    Proteasome inhibitors induce rapid death of cancer cells. We show that in epithelial cancer cells, such death is associated with dramatic and simultaneous up-regulation of several BH3-only proteins, including BIK, BIM, MCL-1S, NOXA, and PUMA, as well as p53. Elevated levels of these proteins seem to be the result of direct inhibition of their proteasomal degradation, induction of transcription, and active translation. Subsequent cell death is independent of BAX, and probably BAK, and proceeds...

  11. Proapoptotic Bak and Bax guard against fatal systemic and organ-specific autoimmune disease

    Science.gov (United States)

    Mason, Kylie D.; Lin, Ann; Robb, Lorraine; Josefsson, Emma C.; Henley, Katya J.; Gray, Daniel H. D.; Kile, Benjamin T.; Roberts, Andrew W.; Strasser, Andreas; Huang, David C. S.; Waring, Paul; O’Reilly, Lorraine A.

    2013-01-01

    Dysregulation of the “intrinsic” apoptotic pathway is associated with the development of cancer and autoimmune disease. Bak and Bax are two proapoptotic members of the Bcl-2 protein family with overlapping, essential roles in the intrinsic apoptotic pathway. Their activity is critical for the control of cell survival during lymphocyte development and homeostasis, best demonstrated by defects in thymic T-cell differentiation and peripheral lymphoid homeostasis caused by their combined loss. Because most bak−/−bax−/− mice die perinatally, the roles of Bax and Bak in immunological tolerance and prevention of autoimmune disease remain unclear. We show that mice reconstituted with a Bak/Bax doubly deficient hematopoietic compartment develop a fatal systemic lupus erythematosus-like autoimmune disease characterized by hypergammaglobulinemia, autoantibodies, lymphadenopathy, glomerulonephritis, and vasculitis. Importantly, these mice also develop a multiorgan autoimmune disease with autoantibodies against most solid glandular structures and evidence of glandular atrophy and necrotizing vasculitis. Interestingly, similar albeit less severe pathology was observed in mice containing a hematopoietic compartment deficient for only Bak, a phenotype reminiscent of the disease seen in patients with point mutations in BAK. These studies demonstrate a critical role for Bak and an ancillary role for Bax in safeguarding immunological tolerance and prevention of autoimmune disease. This suggests that direct activators of the intrinsic apoptotic pathway, such as BH3 mimetics, may be useful for treatment of diverse autoimmune diseases. PMID:23349374

  12. An RNAi-Enhanced Logic Circuit for Cancer Specific Detection and Destruction

    Science.gov (United States)

    2013-02-01

    monomeric protein secreted by Corynebacterium diphtheriae, and pro-apoptotic members of Bcl-2 family: mBax (Mus musculus), hBax ( Homo sapiens ), and its...Gata3 mStaple. Intron- feature sequences – donor site, branch point, poly- pyrimidine tract, and acceptor site – were selected based on previously...sequences found in literature our intron features were chosen according SplicePort [4], an online analyzer that detects the likelihood of splicing to

  13. The pro-apoptotic effects of TIPE2 on AA rat fibroblast-like synoviocytes via regulation of the DR5–caspase–NF-κB pathway in vitro

    Directory of Open Access Journals (Sweden)

    Shi C

    2016-02-01

    Full Text Available Chunyan Shi,1,2,* Shifeng Zhang,1,3,* Shifu Hong,1 Jinglong Pang,1 Yeletai Yesibulati,1 Ping Yin,4 Guohong Zhuang1 1Organ Transplantation Institute, Anti-Cancer Research Center, Medical College, Xiamen University, Xiamen, Fujian, 2The Department of Oncology, Jiujiang No.1 People’s Hospital, Jiujiang, Jiangxi City, 3Division of Gastroenterology Surgery, Zhongshan Hospital, Gastroenterology Institute of Xiamen University, Gastroenterology Center of Xiamen, 4The Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen, Fujian, People’s Republic of China *These authors contributed equally to this work Abstract: TIPE2, also known as TNFAIP8L2, a member of the tumor necrosis factor- alpha-induced protein-8 (TNFAIP8 family, is known as an inhibitor in inflammation and cancer, and its overexpression induces cell death. We examined the role of TIPE2 with respect to adjuvant arthritis (AA-associated pathogenesis by analyzing the TIPE2 regulation of death receptor (DR5-mediated apoptosis in vitro. The results showed that TIPE2 was detected in normal fibroblast-like synoviocytes (FLSs, but scarcely observed in AA-FLSs. Therefore, recombinant MIGR1/TIPE2+/+ and control MIGR1 lentivirus vectors were transfected to AA-FLSs, which were denoted as TIPE2+/+-FLSs and MIGR1-FLSs, respectively. Our results showed that TIPE2+/+-FLSs were highly susceptible to ZF1-mediated apoptosis, and ZF1 was our own purification of an anti-DR5 single chain variable fragment antibody. Under the presence of TIPE2, the expression of DR5 was significantly increased compared with that of the MIGR1-FLS group. In contrast, the level of phosphorylated nuclear factor-kappa B (pNF-κB was lower in the TIPE2+/+-FLS group treated with ZF1, whereas the activity of caspase was higher. Moreover, the rate of apoptosis in the TIPE2+/+-FLS group, which was pretreated with caspase inhibitor Z-VAD-FMK, was significantly decreased. In contrast, the apoptosis occurrence in the MIGR1

  14. 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.

  15. Effect of Bcl-2/Bax gene expression on apoptosis of spermatogenic cells of mouse testes induced by low dose radiation

    International Nuclear Information System (INIS)

    Liu Guangwei; Wang Chunyan; Lu Zhe; Liu Shunchun; Gong Shouliang

    2003-01-01

    The different kinds of spermatogenic cells were separated using density gradient centrifugation and their apoptosis and Bcl-2 and Bax protein expression were measured with flow cytometry and immunohistochemical method, respectively. The results showed the apoptosis in all kinds of spermatogenic cells induced by low dose radiation (LDR) had a obvious regularity. When the doses were 0.025 and 0.05 Gy, spermatogonia apoptosis was dominant. With the increase of irradiation dose (0.075-0.2 Gy), spermatocytes also showed an apoptotic change, but the apoptotic percentage of spermatogonia was significantly higher than that of spermatocytes. Moreover, the apoptosis of spermatids and spermatozoa scarcely occurred after LDR. Bax protein was primarily expressed in spermatogonia and spermatocytes, and the former was significantly higher than that of the latter after LDR. With the increase of irradiation dose, Bax protein expression showed a upgrading tendency, but that of spermatids and spermatozoa scarcely occurred. Bcl-2 protein was primarily expressed in spermatids and spermatozoa, but the Bcl-2 protein expressions of spermatogonia and spermatocytes scarcely occurred after LDR. These results imply that the interacting regulation of Bcl-2 and Bax gene expression might be involved in selective apoptosis of spermatogenic cells induced by LDR, which provided an experimental evidence for further exploring the apoptotic mechanism of adaptive response of spermatogenic cells by LDR

  16. faloabi@uniben.edu Antiproliferative and Pro-apoptotic activities

    African Journals Online (AJOL)

    MICHAEL HORSFALL

    Keyword: Persea americana, antiproliferative activity, apoptotic effect, flow ... of the stem bark of Persea americana in MCF-7 cell line by flow cytometer. .... of an electric milling machine. ... Flow Cytometric Measurement Of Cell Proliferation:.

  17. Bcl-2 and bax expression and prostate cancer outcome in men treated with radiotherapy in Radiation Therapy Oncology Group protocol 86-10

    International Nuclear Information System (INIS)

    Khor, L.-Y.; De Silvio, Michelle; Li, Rile; McDonnell, Timothy J.; Hammond, M. Elizabeth H.; Sause, William T.; Pilepich, Miljenko V.; Okunieff, Paul; Sandler, Howard M.; Pollack, Alan

    2006-01-01

    Purpose: Bcl-2 and bax are proteins with opposing roles in apoptosis regulation; yet abnormal expression of either has been associated with failure after radiotherapy (RT). In this study we examined bcl-2 and bax expression as predictive markers in men treated with radiotherapy ± androgen deprivation on Radiation Therapy Oncology Group (RTOG) protocol 86-10. Experimental Design: Suitable archival diagnostic tissue was obtained from 119 (26%) patients for bcl-2 analysis and 104 (23%) patients for bax analysis. Cox proportional hazards multivariate analysis was used to determine the relationship of abnormal bcl-2 and bax expression to the end points of local failure, distant metastasis, cause-specific mortality, and overall mortality. Bcl-2 overexpression was classified as any tumor cell cytoplasmic staining and altered bax expression was classified as greater or lesser cytoplasmic staining intensity of tumor cells as compared with adjacent normal prostate epithelium. Results: The study cohort exhibited bcl-2 overexpression in 26% (n = 30) of cases and abnormal bax expression in 47% (n = 49) of cases. A borderline significant relationship was observed between abnormal bax expression and higher Gleason score (p = 0.08). In univariate and multivariate analyses, there was no statistically significant relationship seen between abnormal bcl-2 or bax expression and outcome. Conclusions: Abnormal bcl-2 and bax expression were not related to any of the end points tested. The cohort examined was comprised of patients with locally advanced disease and it is possible that these markers may be of greater value in men with earlier-stage prostate cancer

  18. Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dong-Hwa; Ha, Ji-Hyang [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Kim, Yul [Department of Bio and Brain Engineering, KAIST, Daejeon 305-701 (Korea, Republic of); Bae, Kwang-Hee [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Park, Jae-Yong [Department of Physiology, Institute of Health Science, School of Medicine, Gyeongsang National University, Jinju, Gyeongnam 660-751 (Korea, Republic of); Choi, Wan Sung [Department of Anatomy and Neurobiology, Institute of Health Science, School of Medicine, Gyeongsang National University, Jinju, Gyeongnam 660-751 (Korea, Republic of); Yoon, Ho Sup [Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637511 (Singapore); Park, Sung Goo; Park, Byoung Chul [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Yi, Gwan-Su, E-mail: gsyi@kaist.ac.kr [Department of Bio and Brain Engineering, KAIST, Daejeon 305-701 (Korea, Republic of); Chi, Seung-Wook, E-mail: swchi@kribb.re.kr [Medical Proteomics Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of)

    2011-05-20

    Highlights: {yields} Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. {yields} The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. {yields} A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-X{sub L}. {yields} The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-X{sub L.} {yields} Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. A structural model of the Bcl-X{sub L}/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X{sub L}/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X{sub L}. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.

  19. p53-Dependent radiation-induced apoptosis in vivo: relationship to Bcl-2 and Bax expression

    International Nuclear Information System (INIS)

    Hasegawa, Masatoshi; Suzuki, Yoshiyuki; Furuta, Masaya; Yamakawa, Michitaka; Maebayashi, Katsuya; Hayakawa, Kayoko; Saito, Yoshihiro; Mitsuhashi, Norio; Niibe, Hideo

    1997-01-01

    Purpose: A close correlation between p53 protein expression and radiation-induced apoptosis has already been reported, however, Bcl-2 and Bax expression and the ratio of Bcl-2 to Bax have been also suggested to play an important role in the regulation of apoptotic cell death. In this study, we investigated the relationship between p53-dependent radiation-induced apoptosis and expression of Bcl-2 and Bax by using human tumors transplanted into nude mice. Materials and Methods: Three human tumors (an ependymoblastoma, a glioblastoma, and a small cell lung cancer) were subcutaneously transplanted into nude mice and irradiated with single doses of 1, 2, 5, or 10 Gy. The tumors were excised 1, 3, 6, 12, 24, and 48 hours after irradiation, fixed in 10% formalin for 24 hours, and embedded in paraffin. Slides were stained with hematoxylin and eosin for morphologic examination. Immunohistochemical studies were performed with mouse monoclonal antibodies to demonstrate p53, p21 (WAF-1), Bcl-2, and Bax expression. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) and electron microscopic studies were performed to identify apoptosis, and PCR-SSCP analysis was used to evaluate p53 gene mutation. Results: All of the tumors showed only a few cells undergoing apoptosis before irradiation. Beginning several hours after irradiation, only the ependymoblastoma showed a large increase in the number of cells undergoing apoptosis, peaking at 6 hours after irradiation, and there was a clear dose-effect relationship. In contrast, the other tumors showed much less change following irradiation, and the dose-effect relationship was not as clear as in the ependymoblastoma. Immunohistochemically, the non-irradiated ependymoblastoma was negative for p53, p21, Bcl-2, and Bax. Following irradiation, however, many of the tumor cells became positive for p53 and p21, and a few cells became positive for bcl-2. In contrast, the glioblastoma and the small cell lung cancer were positive for p53 and Bcl-2

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    Fang Chen

    2016-04-01

    Full Text Available 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 < 0.05 in 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.

  2. Apoptosis, proliferation, Bax, Bcl-2 and p53 status prior to and after preoperative radiochemotherapy for locally advanced rectal cancer

    International Nuclear Information System (INIS)

    Tannapfel, Andrea; Nuesslein, Siegfried; Fietkau, Rainer; Katalinic, Alexander; Koeckerling, Ferdinand; Wittekind, Christian

    1998-01-01

    Purpose: To investigate the relationship between apoptotic cell death, proliferative activity, and the expression of apoptosis regulating proteins in rectal cancer prior to and after radiochemotherapy. Materials and Methods: In 32 patients dispositioned to receive preoperative radiochemotherapy for locally advanced rectal carcinoma, pretherapy biopsies and the final resected specimen after radiochemotherapy were available for analyses. Apoptotic cells were identified and quantified using in situ end labeling (ISEL) technique. The expression of the bax protein was assessed immunohistochemically. Additionally, double immunostaining was performed for apoptotic cells and bax expression. The proliferative activity was determined by immunohistochemical assessment of the Ki67 (MIB-1) and the proliferating cell nuclear antigen (PCNA). p53- and bcl-2 expression was analyzed immunohistochemically. A clinical-to-pathologic downstaging after radiochemotherapy was achieved in 25 of 32 patients (78%). During follow-up, tumor recurrence was observed in six cases. In one case, no residual tumor was detected after radiochemotherapy. Results: After radiochemotherapy, the apoptotic index increased significantly in almost every case examined. In contrast, the proliferative activity was significantly decreased in resected specimens as compared to biopsies. Bax immunostaining was detected in 12/31 (39%) biopsies and in 26/31 (84%) resected specimens. In the resected specimen, significantly more apoptotic cells that were bax-positive were found than in biopsies. Bcl-2 immunostaining occurred in 15/31 biopsies and 12/31 resected specimens, respectively. Tumors that were immunohistochemically negative for p53 (20/31 [65%]) generally exhibited a higher apoptotic index and a high expression level of bax than p53-positive tumors (11/31 [35%]). However, we did not find any correlation between the (pre- and post-therapeutic) rate of apoptosis or the level of bax expression and the degree of

  3. Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer.

    Science.gov (United States)

    Michie, Alison M; McCaig, Alison M; Nakagawa, Rinako; Vukovic, Milica

    2010-01-01

    Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post-translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK.

  4. Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ginkel, Paul R. van; Yan, Michael B. [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Bhattacharya, Saswati [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Department of Pediatrics, University of Wisconsin, Madison, WI 53792 (United States); Polans, Arthur S., E-mail: aspolans@wisc.edu [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Kenealey, Jason D. [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Department of Nutrition, Dietetics and Food Science, Brigham Young University, Provo, UT 84602 (United States)

    2015-11-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. - Highlights: • Natural products having low toxicity increase cytoplasmic calcium in cancer cells. • A G-protein/IP{sub 3} pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca{sup 2+}-dependent pro-apoptotic pathways inhibit cancer cell growth.

  5. Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells

    International Nuclear Information System (INIS)

    Ginkel, Paul R. van; Yan, Michael B.; Bhattacharya, Saswati; Polans, Arthur S.; Kenealey, Jason D.

    2015-01-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. - Highlights: • Natural products having low toxicity increase cytoplasmic calcium in cancer cells. • A G-protein/IP 3 pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca 2+ -dependent pro-apoptotic pathways inhibit cancer cell growth.

  6. Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary

    NARCIS (Netherlands)

    Slot, K.A.; Voorendt, M.; Boer-Brouwer, de M.; Vugt, van H.H.; Teerds, K.J.

    2006-01-01

    In the present investigation, the localization of proteins involved in ovarian apoptosis were studied throughout the estrous cycle in the presence of fluctuating hormone levels. Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA expression and proteins were detected in all ovarian tissue extracts,

  7. Coenzyme Q10 Protects Hippocampal Neurons against Ischemia/ Reperfusion Injury via Modulation of BAX/Bcl-2 Expression

    Directory of Open Access Journals (Sweden)

    Mohammad Zamani

    2012-09-01

    Full Text Available Introduction: Preliminary studies have con.rmed reduction in cell death following treatment with antioxidants. According to this .nding we study the relationship between consumption of CoQ10 and expression of Bax and Bcl2 in hippocampus following ischemia/reperfusion as proteins involved in cell programmed death or apoptosis. Methods: We studied the protective role of CoQ10 against ischemia-reperfusion. Experimental design includes four groups:  intact, ischemic control, sham control and treatment group with CoQ10. The mice were pre-treated with CoQ10 for a week, then ischemia was induced by common carotid artery ligation and following the reduction in in.ammation (a week the mice was treated with CoQ10.  Nissl staining was applied for counting the necrotic cells of hippocampus and the western blot was performed to measure the Bax and Bcl2 expression.Results: Cell death was signi.cantly lower when mice were treated with CoQ10. Bax expression was signi.cantly high in the ischemic group but low in the treatment group, and the bcl2 expression was lower in the ischemic group than the treatment and the vehicle groups.Discussion: Ischemia for 15 minutes induced cell death in hippocampus with more potent effect on CA1. CoQ10 intake signi.cantly reduced cell death and prevented the expression of Bax while inducing an increase in expression of bcl2.

  8. Coenzyme Q10 Protects Hippocampal Neurons Against Ischemia/Reperfusion Injury via Modulation of BAX/Bcl-2 Expression

    Directory of Open Access Journals (Sweden)

    M Zamani

    2012-12-01

    Full Text Available Introduction : Preliminary studies confirmed reduction in cell death following treatment with antioxidants. According to this finding we study the relationship between consumption of CoQ10 and expression of bax and bcl2 in hippocampus following ischemia/reperfusion as proteins involved in cell programmed death or apoptosis.Material & methods : We studied the protective role of CoQ10 against Ischemia-Reperfusion. Experimental design includes four groups: intact, ischemic control, sham control and treatment groups with CoQ10. The mice 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 mice post-treated with CoQ10.Nissl staining applied to counting necrotic cells of hippocampus and the western blotting performed to measurement the bax and bcl2 expression.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.Conclusion : Ischemia for 15 minutes induced cell death in hippocampus with more potent effect on CA1. CoQ10 intake significantly reduced cell death and prevented the expression of bax while inducing an increase in expression of bcl2.

  9. CoQ10 Augments Rosuvastatin Neuroprotective Effect in a Model of Global Ischemia via Inhibition of NF-κB/JNK3/Bax and Activation of Akt/FOXO3A/Bim Cues

    Directory of Open Access Journals (Sweden)

    Sarah A. Abd El-Aal

    2017-10-01

    Full Text Available Statins were reported to lower the Coenzyme Q10 (CoQ10 content upon their inhibition of HMG-CoA reductase enzyme and both are known to possess neuroprotective potentials; therefore, the aim is to assess the possible use of CoQ10 as an adds-on therapy to rosuvastatin to improve its effect using global I/R model. Rats were allocated into sham, I/R, rosuvastatin (10 mg/kg, CoQ10 (10 mg/kg and their combination. Drugs were administered orally for 7 days before I/R. Pretreatment with rosuvastatin and/or CoQ10 inhibited the hippocampal content of malondialdehyde, nitric oxide, and boosted glutathione and superoxide dismutase. They also opposed the upregulation of gp91phox, and p47phox subunits of NADPH oxidase. Meanwhile, both agents reduced content/expression of TNF-α, iNOS, NF-κBp65, ICAM-1, and MPO. Besides, all regimens abated cytochrome c, caspase-3 and Bax, but increased Bcl-2 in favor of cell survival. On the molecular level, they increased p-Akt and its downstream target p-FOXO3A, with the inhibition of the nuclear content of FOXO3A to downregulate the expression of Bim, a pro-apoptotic gene. Additionally, both treatments downregulate the JNK3/c-Jun signaling pathway. The effect of the combination regimen overrides that of either treatment alone. These effects were reflected on the alleviation of the hippocampal damage in CA1 region inflicted by I/R. Together, these findings accentuate the neuroprotective potentials of both treatments against global I/R by virtue of their rigorous multi-pronged actions, including suppression of hippocampal oxidative stress, inflammation, and apoptosis with the involvement of the Akt/FOXO3A/Bim and JNK3/c-Jun/Bax signaling pathways. The study also nominates CoQ10 as an adds-on therapy with statins.

  10. Erdosteine protects HEI-OC1 auditory cells from cisplatin toxicity through suppression of inflammatory cytokines and induction of Nrf2 target proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Se-Jin [Department of Microbiology, Center for Metabolic Function Regulation (CMFR), Wonkwang University, College of Medicine, 460 Iksandae-ro, Iksan, Jeonbuk 570-749 (Korea, Republic of); Park, Channy [Department of Head and Neck Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA (United States); Lee, Joon No [Department of Microbiology, Center for Metabolic Function Regulation (CMFR), Wonkwang University, College of Medicine, 460 Iksandae-ro, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lim, Hyewon; Hong, Gi-yeon [Department of Obstetrics and Gynecology, Wonkwang University, College of Medicine, 460 Iksandae-ro, Iksan, Jeonbuk 570-749 (Korea, Republic of); Moon, Sung K.; Lim, David J. [Department of Head and Neck Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA (United States); Choe, Seong-Kyu, E-mail: seongkyu642@wku.ac.kr [Department of Microbiology, Center for Metabolic Function Regulation (CMFR), Wonkwang University, College of Medicine, 460 Iksandae-ro, Iksan, Jeonbuk 570-749 (Korea, Republic of); Park, Raekil, E-mail: rkpark@wku.ac.kr [Department of Microbiology, Center for Metabolic Function Regulation (CMFR), Wonkwang University, College of Medicine, 460 Iksandae-ro, Iksan, Jeonbuk 570-749 (Korea, Republic of)

    2015-10-15

    Cisplatin has many adverse effects, which are a major limitation to its use, including ototoxicity, neurotoxicity, and nephrotoxicity. This study aims to elucidate the protective mechanisms of erdosteine against cisplatin in HEI-OC1 cells. Pretreatment with erdosteine protects HEI-OC1 cells from cisplatin-medicated apoptosis, which is characterized by increase in nuclear fragmentation, DNA laddering, sub-G{sub 0}/G{sub 1} phase, H2AX phosphorylation, PARP cleavage, and caspase-3 activity. Erdosteine significantly suppressed the production of reactive nitrogen/oxygen species and pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in cisplatin-treated cells. Studies using pharmacologic inhibitors demonstrated that phosphatidylinositol-3-kinases (PI3K) and protein kinase B (Akt) have protective roles in the action of erdosteine against cisplatin in HEI-OC1 cells. In addition, pretreatment with erdosteine clearly suppressed the phosphorylation of p53 (Ser15) and expression of p53-upregulated modulator of apoptosis. Erdosteine markedly induces expression of NF-E2-related factor 2 (Nrf2), which may contribute to the increase in expression of glutathione redox genes γ-L-glutamate-L-cysteine-ligase catalytic and γ-L-glutamate-L-cysteine-ligase modifier subunits, as well as in the antioxidant genes HO-1 and SOD2 in cisplatin-treated HEI-OC1 cells. Furthermore, the increase in expression of phosphorylated p53 induced by cisplatin is markedly attenuated by pretreatment with erdosteine in the mitochondrial fraction. This increased expression may inhibit the cytosolic expression of the apoptosis-inducing factor, cytochrome c, and Bax/Bcl-xL ratio. Thus, our results suggest that treatment with erdosteine is significantly attenuated cisplatin-induced damage through the activation of Nrf2-dependent antioxidant genes, inhibition of pro-inflammatory cytokines, activation of the PI3K/Akt signaling, and mitochondrial-related inhibition of pro-apoptotic

  11. Erdosteine protects HEI-OC1 auditory cells from cisplatin toxicity through suppression of inflammatory cytokines and induction of Nrf2 target proteins

    International Nuclear Information System (INIS)

    Kim, Se-Jin; Park, Channy; Lee, Joon No; Lim, Hyewon; Hong, Gi-yeon; Moon, Sung K.; Lim, David J.; Choe, Seong-Kyu; Park, Raekil

    2015-01-01

    Cisplatin has many adverse effects, which are a major limitation to its use, including ototoxicity, neurotoxicity, and nephrotoxicity. This study aims to elucidate the protective mechanisms of erdosteine against cisplatin in HEI-OC1 cells. Pretreatment with erdosteine protects HEI-OC1 cells from cisplatin-medicated apoptosis, which is characterized by increase in nuclear fragmentation, DNA laddering, sub-G 0 /G 1 phase, H2AX phosphorylation, PARP cleavage, and caspase-3 activity. Erdosteine significantly suppressed the production of reactive nitrogen/oxygen species and pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in cisplatin-treated cells. Studies using pharmacologic inhibitors demonstrated that phosphatidylinositol-3-kinases (PI3K) and protein kinase B (Akt) have protective roles in the action of erdosteine against cisplatin in HEI-OC1 cells. In addition, pretreatment with erdosteine clearly suppressed the phosphorylation of p53 (Ser15) and expression of p53-upregulated modulator of apoptosis. Erdosteine markedly induces expression of NF-E2-related factor 2 (Nrf2), which may contribute to the increase in expression of glutathione redox genes γ-L-glutamate-L-cysteine-ligase catalytic and γ-L-glutamate-L-cysteine-ligase modifier subunits, as well as in the antioxidant genes HO-1 and SOD2 in cisplatin-treated HEI-OC1 cells. Furthermore, the increase in expression of phosphorylated p53 induced by cisplatin is markedly attenuated by pretreatment with erdosteine in the mitochondrial fraction. This increased expression may inhibit the cytosolic expression of the apoptosis-inducing factor, cytochrome c, and Bax/Bcl-xL ratio. Thus, our results suggest that treatment with erdosteine is significantly attenuated cisplatin-induced damage through the activation of Nrf2-dependent antioxidant genes, inhibition of pro-inflammatory cytokines, activation of the PI3K/Akt signaling, and mitochondrial-related inhibition of pro-apoptotic

  12. Safety of PEGylated recombinant human full-length coagulation factor VIII (BAX 855) in the overall context of PEG and PEG conjugates.

    Science.gov (United States)

    Stidl, R; Fuchs, S; Bossard, M; Siekmann, J; Turecek, P L; Putz, M

    2016-01-01

    BAX 855 is a PEGylated human full-length recombinant factor VIII (rFVIII) based on licensed rFVIII (ADVATE). The applied PEGylation technology has been optimized to retain functionality of the FVIII molecule, improve its pharmacokinetic properties and allow less frequent injections while maintaining efficacy. The aim of this study was to confirm that the excellent safety profile of ADVATE remains unchanged after PEGylation. Non-clinical safety studies with BAX 855 and its respective unbound polyethylene glycol (PEG) were conducted in several species. The distribution of a single dose of radiolabelled BAX 855 was further investigated in rats. Publically available safety data on PEG alone and PEGylated biomolecules were summarized and reviewed for specific safety findings attributable to PEG or PEGylated biopharmaceuticals. Safety pharmacology studies in rabbits and macaques and repeated dose toxicity studies in rats and macaques identified no safety issues. Results of a distribution study in rats administered radiolabelled BAX 855 showed that radioactivity was completely excreted; urine was the major elimination route. A 28-day study in rats dosed with the unbound PEG constituent (PEG2ru20KCOOH) of BAX 855 showed no adverse or non-adverse effects. Safety data for PEG and PEG-protein conjugates indicate no safety concerns associated with PEG at clinically relevant dose levels. Although vacuolation of certain cell types has been reported in mammals, no such vacuolation was observed with BAX 855 or with the unbound PEG constituent. Non-clinical safety evaluation of PEG and BAX 855 identified no safety signals; the compound is now in clinical development for the treatment of patients with haemophilia A. © 2015 Baxalta Innovations GmbH. Haemophilia Published by John Wiley & Sons Ltd.

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

    International Nuclear Information System (INIS)

    Li, Ruizhao; Zhang, Li; Shi, Wei; Zhang, Bin; Liang, Xinling; Liu, Shuangxin; Wang, Wenjian

    2013-01-01

    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 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 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, which may

  14. 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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Borax-induced apoptosis in HepG2 cells involves p53, Bcl-2, and Bax.

    Science.gov (United States)

    Wei, Y; Yuan, F J; Zhou, W B; Wu, L; Chen, L; Wang, J J; Zhang, Y S

    2016-06-21

    Borax, a boron compound and a salt of boric acid, is known to inhibit the growth of tumor cells. HepG2 cells have been shown to be clearly susceptible to the anti-proliferative effects of borax. However, the specific mechanisms regulating this effect are poorly understood. This study aimed to investigate the pathways underlying the growth inhibition induced by borax in HepG2 cells. The effects of borax on HepG2 cell viability were characterized using MTT. Apoptosis was also verified by annexin V/propidium iodide staining. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and p53, Bax, and Bcl-2 protein expression, respectively. Relevant mRNA levels were measured by qRT-PCR. Borax inhibited the proliferation of HepG2 cells in a time- and dose-dependent manner in vitro. The apoptotic process triggered by borax involved the upregulation of p53 and Bax and the downregulation of Bcl-2, which was confirmed by a change in the mitochondrial membrane potential. These results elucidate a borax-induced apoptotic pathway in HepG2 cells that involves the upregulation of p53 and Bax and the downregulation of Bcl-2.

  18. [Effects of blueberry on apoptosis and expression of Bcl-2 and Bax in HSC-T6].

    Science.gov (United States)

    Lu, Shuang; Cheng, Mingliang; Yang, Demeng; Liu, Yang; Guan, Li; Wu, Jun

    2015-08-18

    To investigate the effects of blueberry on the apoptosis, expression of Bcl-2 and Bax in rat hepatic stellate cell (HSC-T6). 10% blueberry serum at low, middle and high dose, 10% Fu-Fang-Bie-Jia-Ruan-Gan tablet serum and 10% saline serum were prepared by method of serum pharmacology. Subcultured HSC-T6 was divided into saline serum control group, blueberry serum at low, middle, high dose and Fu-Fang-Bie-Jia-Ruan-Gan tablet serum group, and then was respectively incubated at different dose of 10% blueberry serum, 10% Fu-Fang-Bie-Jia-Ruan-Gan tablet serum and 10% saline serum for 72 hours.Apoptosis of HSC-T6 was detected using flow cytometry with annexin V FITC/PI double staining. The expression of Bcl-2 and Bax in HSC-T6 were examined using immunocytochemistry and Western blotting, respectively. There was no significant difference for HSC-T6 Bax protein expression in the low, middle and high dose blueberry serum groups, compared with saline serum control group, respectively.In the high-dose blueberry serum group HSC-T6 early and total apoptosis rate increased significantly compared with the saline serum control group (5.55% ± 0.98% vs 2.53% ± 0.46%, 7.01% ± 1.05% vs 2.96% ± 0.81%, both Pblueberry serum group showed no significant difference with the saline serum control group. Blueberry can induce HSC-T6 apoptosis by down-regulating Bcl-2 expression and decreasing the ratio of Bcl-2/Bax in HSC-T6 cells, so it may have potential interference effects on hepatic fibrosis.

  19. Pushing the size limit of de novo structure ensemble prediction guided by sparse SDSL-EPR restraints to 200 residues: The monomeric and homodimeric forms of BAX

    Science.gov (United States)

    Fischer, Axel W.; Bordignon, Enrica; Bleicken, Stephanie; García-Sáez, Ana J.; Jeschke, Gunnar; Meiler, Jens

    2016-01-01

    Structure determination remains a challenge for many biologically important proteins. In particular, proteins that adopt multiple conformations often evade crystallization in all biologically relevant states. Although computational de novo protein folding approaches often sample biologically relevant conformations, the selection of the most accurate model for different functional states remains a formidable challenge, in particular, for proteins with more than about 150 residues. Electron paramagnetic resonance (EPR) spectroscopy can obtain limited structural information for proteins in well-defined biological states and thereby assist in selecting biologically relevant conformations. The present study demonstrates that de novo folding methods are able to accurately sample the folds of 192-residue long soluble monomeric Bcl-2-associated X protein (BAX). The tertiary structures of the monomeric and homodimeric forms of BAX were predicted using the primary structure as well as 25 and 11 EPR distance restraints, respectively. The predicted models were subsequently compared to respective NMR/X-ray structures of BAX. EPR restraints improve the protein-size normalized root-mean-square-deviation (RMSD100) of the most accurate models with respect to the NMR/crystal structure from 5.9 Å to 3.9 Å and from 5.7 Å to 3.3 Å, respectively. Additionally, the model discrimination is improved, which is demonstrated by an improvement of the enrichment from 5% to 15% and from 13% to 21%, respectively. PMID:27129417

  20. Pattern of MAP kinases p44/42 and JNK activation by non-lethal doses of tributyltin in human natural killer cells

    Energy Technology Data Exchange (ETDEWEB)

    Aluoch, Aloice O. [Tennessee State University, Department of Biological Sciences, Nashville, TN (United States); Odman-Ghazi, Sabah O.; Whalen, Margaret M. [Tennessee State University, Department of Chemistry, Nashville, TN (United States)

    2007-04-15

    Tributyltin (TBT) has been shown to disrupt the ability of natural killer (NK) cells to destroy tumor targets in vitro even at exposures of 25 nM for 24 h, but cell viability was not significantly impacted. Thus, evaluation of intracellular molecular events that regulate cell viability in TBT exposed NK cells are of interest. It has been suggested that activation of the mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), may promote apoptosis while activation of the MAPK p44/42 may be crucial in mediating anti-apoptotic stimuli. However, it is well established that increases in pro-apoptotic BCL-2 family members, such as Bax, results in cell death. We have set out to study the effects of a range of TBT concentrations on the MAPKs, JNK and p44/42. Additionally, we examined the effects of TBT on the levels of pro-apoptotic proteins Bax and p53 as well as anti-apoptotic protein Bcl-2. The results show that 300-25 nM TBT activated JNK within 10 min. MAPK p44/42 was also activated by 300-50 nM TBT within 10 min. These data show that while 300-200 nM TBT activates p44/42 significantly more than JNK, the pattern of 100-25 nM TBT activation of these MAPKs may be similar. TBT exposure alters neither pro-apoptotic proteins Bax and p53 nor anti-apoptotic protein Bcl-2 levels at any exposure studied. The results suggest that exposure to TBT activated the anti-apoptotic regulatory p44/42 pathway to a greater extent than the pro-apoptotic JNK pathway, which may explain to some extent how NK cell viability is maintained. (orig.)

  1. Differential induction of p53-mediated apoptosis in medulloblastomas and gliomas correlates with their ability to induce bax

    International Nuclear Information System (INIS)

    Shu, H.-K.G.; Furman, Felix; Dee, Suzanne; Israel, Mark A.

    1997-01-01

    Purpose/Objective: Medulloblastoma cell lines readily undergo a p53-mediated apoptosis following exposure to ionizing radiation, while glioma cell lines do not undergo significant levels of apoptosis following irradiation. This study attempts to define some of the molecular events that characterize the differential ability medulloblastomas and gliomas to undergo radiation-induced apoptosis. Materials and Methods: The medulloblastoma cell lines D283 and D341 and the glioma cell lines U87, U343 and U563 were used in this study. All five cell lines were confirmed to have a wild type p53 by their ability to induce p21 protein levels and to undergo a cell-cycle arrest in G1 following treatment with ionizing radiation. Also, 3 clonal derivatives of D283 were used. Two of the clones were derived following transfection with an expression plasmid containing a dominant negative mutant p53 (Arg175 --> His) expressed from the CMV promoter (D283/53.6 and D283/53.7), while the remaining clone was derived following transfection with that same expression plasmid without mutant p53 (D283/vec). All irradiation experiments were performed on Phillips RT-250 X-ray unit using 250 Kvp X-rays. In each case, 5 Gy of ionizing radiation was given at a dose rate of 250 cGy/minute. Apoptosis was quantitated by staining fixed cells with propidium iodide and determining the percentage of cells with subdiploid DNA content by flow cytometry. Northern blot analysis was performed using standard methods. Results: The D283 and D341 cell lines exhibited a significant induction of apoptosis when assayed 2 days following treatment with radiation while the U87, U343 and U563 cell lines displayed only minimal induction of apoptosis when assayed following treatment at that time. RNA was prepared from the different cell lines that were unirradiated, 6 hours or 24 hours post-irradiation. Northern blots were made of the total RNAs and probed for bax, bcl-2 and bcl-x mRNA. This analysis detected no significant

  2. 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.

  3. ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death.

    Science.gov (United States)

    Darling, Nicola J; Balmanno, Kathryn; Cook, Simon J

    2017-01-01

    Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway.

  4. ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death.

    Directory of Open Access Journals (Sweden)

    Nicola J Darling

    Full Text Available Disruption of protein folding in the endoplasmic reticulum (ER causes ER stress. Activation of the unfolded protein response (UPR acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2 signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway.

  5. 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.

  6. Electromagnetic radiation at 900 MHz induces sperm apoptosis through bcl-2, bax and caspase-3 signaling pathways in rats.

    Science.gov (United States)

    Liu, Qi; Si, Tianlei; Xu, Xiaoyun; Liang, Fuqiang; Wang, Lufeng; Pan, Siyi

    2015-08-04

    The decreased reproductive capacity of men is an important factor contributing to infertility. Accumulating evidence has shown that Electromagnetic radiation potentially has negative effects on human health. However, whether radio frequency electromagnetic radiation (RF-EMR) affects the human reproductive system still requires further investigation. Therefore, The present study investigates whether RF-EMR at a frequency of 900 MHz can trigger sperm cell apoptosis and affect semen morphology, concentration, and microstructure. Twenty four rats were exposed to 900 MHz electromagnetic radiation with a special absorption rate of 0.66 ± 0.01 W/kg for 2 h/d. After 50d, the sperm count, morphology, apoptosis, reactive oxygen species (ROS), and total antioxidant capacity (TAC), representing the sum of enzymatic and nonenzymatic antioxidants, were investigated. Western blotting and reverse transcriptase PCR were used to determine the expression levels of apoptosis-related proteins and genes, including bcl-2, bax, cytochrome c, and capase-3. In the present study, the percentage of apoptotic sperm cells in the exposure group was significantly increased by 91.42% compared with the control group. Moreover, the ROS concentration in exposure group was increased by 46.21%, while the TAC was decreased by 28.01%. Radiation also dramatically decreased the protein and mRNA expression of bcl-2 and increased that of bax, cytochrome c, and capase-3. RF-EMR increases the ROS level and decreases TAC in rat sperm. Excessive oxidative stress alters the expression levels of apoptosis-related genes and triggers sperm apoptosis through bcl-2, bax, cytochrome c and caspase-3 signaling pathways.

  7. The effect of radiation on bcl-2 and bax in hyperplastic prostatic tissues

    International Nuclear Information System (INIS)

    Ma Qingjie; Li Yuxin; Gu Xinquan; Cao Xia; Zhao Jie; Kong Xiangbo; Cai Shanyu

    2004-01-01

    Aim: To investigate the expressions of bcl-2 and bax in benign prostatic hyperplasia (BPH) and the effect of β-rays on bcl-2 and bax. Methods: The expressions of bcl-2 and bax are studied by means of immunohistochemical method in 9 normal prostate (NP) and 15 BPH and 35 patients treated with 90Sr/90Y Prostatic Hyperplasia Applicator. Results: The expressions of bcl-2 in epithelia of NP and BPH are higher than that in stroma P<0.01=. The expressions of bcl-2 in epithelia and stroma of BPH are higher than that in NP P<0.01=. The expressions of bax in epithelia of NP are higher than that in BPH P<0.05=. However ,the expressions of bcl-2 in epithelia and stroma of BPH are higher than bax P<0.01 =. Compared with the control group, the expressions of bcl-2 in epithelia and stroma of BPH treated with 90Sr/90Y Prostatic Hyperplasia Applicator decreased and the expressions of bax increased P<0.01=. Conclusion: bcl-2 gene and bax gene play an important role in the regulation of prostatic apoptosis and the treatment of β-rays can accelerate the apoptosis of prostatic tissues. (authors)

  8. 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.

  9. Apoptosis and the FLIP and NF-kappa B proteins as pharmacodynamic criteria for biosimilar TNF-alpha antagonists

    Directory of Open Access Journals (Sweden)

    Urbano PCM

    2014-07-01

    Full Text Available Paulo César Martins Urbano,1 Vanete Thomaz Soccol,1 Valderilio Feijó Azevedo2 1Biotechnology and Bioprocess Engineering Program, Federal University of Parana, Curitiba, Parana, Brazil; 2Hospital de Clínicas, Federal University of Parana, Curitiba, Parana, Brazil Abstract: Various criteria are necessary to assess the efficacy and safety of biological medications in order to grant companies the right to register these medications with the appropriate bodies that regulate their sale. The imminent expiration of the patents on reference biological products which block the cytokine TNF-α (tumor necrosis factor-α raises the possibility of bringing so-called biosimilars to the market (similar to the biologicals of reference products. This occurrence is inevitable, but criteria to adequately evaluate these medications are now needed. Even among controversy, there is a demand from publications correlating the pro-apoptotic mechanism of the original TNF-α antagonists (etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol in the treatment of rheumatoid arthritis and other diseases. In this article, the authors discuss the possibility of utilizing the pro-apoptotic effect correlated with the regulation of the anti-apoptotic proteins FLIP and NF-κB as new criteria for analyzing the pharmacodynamics of possible biosimilar TNF-α antagonists which should be submitted to regulatory agencies for evaluation. Keywords: anti-TNF drugs, rheumatoid arthritis, apoptosis, NF-κB, FLIP

  10. Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A.

    Science.gov (United States)

    Siu, Woen Ping; Pun, Pamela Boon Li; Latchoumycandane, Calivarathan; Boelsterli, Urs A

    2008-03-15

    Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (>500 microM) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 microM) fully inhibited diclofenac-induced cell injury, suggesting that mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca2+ chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca2+-Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.

  11. Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A

    International Nuclear Information System (INIS)

    Siu, W.P.; Pun, Pamela Boon Li; Latchoumycandane, Calivarathan; Boelsterli, Urs A.

    2008-01-01

    Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (> 500 μM) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 μM) fully inhibited diclofenac-induced cell injury, suggesting that mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca 2+ chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca 2+ -Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury

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

    Directory of Open Access Journals (Sweden)

    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

  13. Quantifying export production in the Southern Ocean: Implications for the Baxs proxy

    Science.gov (United States)

    Hernandez-Sanchez, Maria T.; Mills, Rachel A.; Planquette, HéLèNe; Pancost, Richard D.; Hepburn, Laura; Salter, Ian; Fitzgeorge-Balfour, Tania

    2011-12-01

    The water column and sedimentary Baxs distribution around the Crozet Plateau is used to decipher the controls and timing of barite formation and to evaluate how export production signals are recorded in sediments underlying a region of natural Fe fertilization within the Fe limited Southern Ocean. Export production estimated from preserved, vertical sedimentary Baxs accumulation rates are compared with published export fluxes assessed from an integrated study of the biological carbon pump to determine the validity of Baxs as a quantitative proxy under different Fe supply conditions typical of the Southern Ocean. Detailed assessment of the geochemical partitioning of Ba in sediments and the lithogenic end-member allows appropriate correction of the bulk Ba content and determination of the Baxs content of sediments and suspended particles. The upper water column distribution of Baxs is extremely heterogeneous spatially and temporally. Organic carbon/Baxs ratios in deep traps from the Fe fertilized region are similar to other oceanic settings allowing quantification of the inferred carbon export based on established algorithms. There appears to be some decoupling of POC and Ba export in the Fe limited region south of the Plateau. The export production across the Crozet Plateau inferred from the Baxs sedimentary proxy indicates that the Fe fertilized area to the north of the Plateau experiences enhanced export relative to equivalent Southern Ocean settings throughout the Holocene and that this influence may also have impacted the site to the south for significant periods. This interpretation is corroborated by alternative productivity proxies (opal accumulation, 231Paxs/230Thxs). Baxs can be used to quantify export production in complex settings such as naturally Fe-fertilized (volcanoclastic) areas, providing appropriate lithogenic correction is undertaken, and sediment focusing is corrected for along with evaluation of barite preservation.

  14. Increase in intracellular PGE2 induces apoptosis in Bax-expressing colon cancer cell

    International Nuclear Information System (INIS)

    Lalier, Lisenn; Pedelaborde, François; Braud, Christophe; Menanteau, Jean; M Vallette, François; Olivier, Christophe

    2011-01-01

    NSAIDs exhibit protective properties towards some cancers, especially colon cancer. Yet, it is not clear how they play their protective role. PGE 2 is generally shown as the only target of the NSAIDs anticancerous activity. However, PGE 2 known targets become more and more manifold, considering both the molecular pathways involved and the target cells in the tumour. The role of PGE 2 in tumour progression thus appears complex and multipurpose. To gain understanding into the role of PGE 2 in colon cancer, we focused on the activity of PGE 2 in apoptosis in colon cancer cell lines. We observed that an increase in intracellular PGE 2 induced an apoptotic cell death, which was dependent on the expression of the proapoptotic protein Bax. This increase was induced by increasing PGE 2 intracellular concentration, either by PGE 2 microinjection or by the pharmacological inhibition of PGE 2 exportation and enzymatic degradation. We present here a new sight onto PGE 2 in colon cancer cells opening the way to a new prospective therapeutic strategy in cancer, alternative to NSAIDs

  15. BAX INHIBITOR-1 is required for full susceptibility of barley to powdery mildew.

    Science.gov (United States)

    Eichmann, Ruth; Bischof, Melanie; Weis, Corina; Shaw, Jane; Lacomme, Christophe; Schweizer, Patrick; Duchkov, Dimitar; Hensel, Götz; Kumlehn, Jochen; Hückelhoven, Ralph

    2010-09-01

    BAX INHIBITOR-1 (BI-1) is one of the few proteins known to have cross-kingdom conserved functions in negative control of programmed cell death. Additionally, barley BI-1 (HvBI-1) suppresses defense responses and basal resistance to the powdery mildew fungus Blumeria graminis f. sp. hordei and enhances resistance to cell death-provoking fungi when overexpressed in barley. Downregulation of HvBI-1 by transient-induced gene silencing or virus-induced gene silencing limited susceptibility to B. graminis f. sp. hordei, suggesting that HvBI-1 is a susceptibility factor toward powdery mildew. Transient silencing of BI-1 did not limit supersusceptibility induced by overexpression of MLO. Transgenic barley plants harboring an HvBI-1 RNA interference (RNAi) construct displayed lower levels of HvBI-1 transcripts and were less susceptible to powdery mildew than wild-type plants. At the cellular level, HvBI-1 RNAi plants had enhanced resistance to penetration by B. graminis f. sp. hordei. These data support a function of BI-1 in modulating cell-wall-associated defense and in establishing full compatibility of B. graminis f. sp. hordei with barley.

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

    International Nuclear Information System (INIS)

    Wang, Peng; Zhen, Haining; Jiang, Xinbiao; Zhang, Wei; Cheng, Xin; Guo, Geng; Mao, Xinggang; Zhang, Xiang

    2010-01-01

    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. The neutron beam was obtained from the Xi'an Pulsed Reactor (XAPR) and γ-rays were obtained from [ 60 Co] 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 [ 60 Co] γ-rays at the FMMU with different protocols: Group A included control nonirradiated cells; Group B included cells treated with 4 Gy of [ 60 Co] γ-rays; Group C included cells treated with 8 Gy of [ 60 Co] 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. Proliferation of U87, U251, and SHG44 cells was much more strongly inhibited by BPA-BNCT than by irradiation with [ 60 Co] γ-rays (P < 0.01). Nuclear condensation was determined using both a fluorescence technique and electron microscopy in all cell lines treated with BPA-BNCT. Furthermore, the cellular apoptotic rates in Group D and Group E treated with

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

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    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.

  18. Cycloheximide and actinomycin D delay death and affect bcl-2, bax, and Ice gene expression in astrocytes under in vitro ischemia.

    Science.gov (United States)

    Yu, Albert Cheung Hoi; Yung, Hon Wa; Hui, Michael Hung Kit; Lau, Lok Ting; Chen, Xiao Qian; Collins, Richard A

    2003-10-15

    An in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT-mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL-positive cells was reduced by 38%. The nuclear shrinkage in TUNEL-positive astrocytes in control cultures did not occur in ActD-treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl-2 (alpha and beta), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription-polymerase chain reaction, indicated that ischemia down-regulated bcl-2 (alpha and beta) and bax. Ice was initially down-regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl-2 (alpha and beta) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl-2, Bcl-Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl-2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl-2 family members that plays a critical role in determining ischemia-induced apoptosis. It is also important to note that ischemia-induced apoptosis involves the regulation of RNA and protein synthesis. Copyright 2003 Wiley-Liss, Inc.

  19. 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. Copyright © 2015 Elsevier GmbH. All rights reserved.

  20. Bax/Bcl-2 expression ratio in prediction of response to breast cancer radiotherapy

    Directory of Open Access Journals (Sweden)

    Hosein Azimian

    2018-03-01

    Full Text Available Objective(s: Radiotherapy is one of the most effective modalities of cancer therapy, but clinical responses of individual patients varies considerably. To enhance treatment efficiency it is essential to implement an individual-based treatment. The aim of present study was to identify the mechanism of intrinsic apoptosis pathway on radiosensitivity and normal tissue complications caused by the radiotherapy. Materials and Methods: Peripheral blood mononuclear cells from ten breast cancer patients were exposed to 6MV X-rays to deliver 1 and 2 Gy. Expression levels of Bax, Bcl-2, and Bax/Bcl-2 ratio were examined by relative quantitative RT-PCR. All the patients received similar tangential irradiation of the whole breast and conventional fractionation. Skin dosimetry was done by GAFChromic EBT-3 film and clinical radiosensitivity was determined using the acute reactions to radiotherapy of the skin according to Radiation Therapy Oncology Group score. All statistical analyses were performed using GraphPad Prism, version 7.01. Results: In the in-vitro experiment, Bax and Bax/Bcl-2 ratios were significantly increased with 1 and 2 Gy doses (PP0.05 for all patients. Conclusion: Significant correlation between Bax/Bcl-2 ratio determined before radiation therapy and clinical response in the patients, can be used as a biomarker to identify radiosensitive individuals. However, further studies are required to validate radiation-induced apoptotic biomarkers.

  1. The studies on thyrocyte apoptosis and expression of Bcl-2 and Bax in Hashimoto's thyroiditis

    International Nuclear Information System (INIS)

    Zhao Yaping; Wang Jialing; Fan Zhiyong; Liu Zehong; Wu HeJun; Zhou Wei; Jia Meizhai

    2003-01-01

    To investigate the thyrocyte apoptosis, the expression of Bcl-2, Bax and the relationship between apoptosis and the pathogenesis in Hashimoto's thyroiditis (HT), 41 HT thyroid and 10 normal thyroid specimens were selected. The level of apoptosis was detected by TUNEL methods. The expression and distribution of Bcl-2 and Bax were detected using immunohistochemical methods and analyzed by Mias99 pathological image system. Immunohistochemical staining was carried out using S-P kit. The Result showed that an increased level of apoptosis was observed in Hashimoto's glands. The apoptosis mainly distributed in thyroid follicles destruction area. This was associated with increased Bax expression. The strongly positive Bcl-2 staining was observed in the thyrocyte of intact thyroid follicles. The ratios of positive granule area and total light density of Bcl-2 to those of Bax in HT thyroid follicle area were lower than those in normal thyroid. The apoptosis of thyrocyte induced by dysregulation of Bcl-2 and Bax may be involved in the pathogeneses of HT

  2. Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway.

    Science.gov (United States)

    Deng, Lin; Adachi, Tetsuya; Kitayama, Kikumi; Bungyoku, Yasuaki; Kitazawa, Sohei; Ishido, Satoshi; Shoji, Ikuo; Hotta, Hak

    2008-11-01

    We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).

  3. 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. PMID:27845306

  4. The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor.

    Directory of Open Access Journals (Sweden)

    Rinki Minakshi

    2009-12-01

    Full Text Available The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR, which includes the inositol-requiring enzyme 1 (IRE-1, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1 increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha and inhibitory effects of a dominant-negative form of eIF2alpha on GRP78 promoter activity, (2 increased translation of activating transcription factor 4 (ATF4 mRNA, and (3 ATF4-dependent activation of the C/EBP homologous protein (CHOP gene promoter. Activation of PERK affects innate immunity by suppression of type 1 interferon (IFN signaling. The 3a protein was found to induce serine phosphorylation within the IFN alpha-receptor subunit 1 (IFNAR1 degradation motif and to increase IFNAR1 ubiquitination. Confocal microscopic analysis showed increased translocation of IFNAR1 into the lysosomal compartment and flow cytometry showed reduced levels of IFNAR1 in 3a-expressing cells. These results provide further mechanistic details of the pro-apoptotic effects of the SARS-CoV 3a protein, and suggest a potential role for it in attenuating interferon responses and innate immunity.

  5. TGFBR2 and BAX mononucleotide tract mutations, microsatellite instability, and prognosis in 1072 colorectal cancers.

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    Kaori Shima

    Full Text Available Mononucleotide tracts in the coding regions of the TGFBR2 and BAX genes are commonly mutated in microsatellite instability-high (MSI-high colon cancers. The receptor TGFBR2 plays an important role in the TGFB1 (transforming growth factor-β, TGF-β signaling pathway, and BAX plays a key role in apoptosis. However, a role of TGFBR2 or BAX mononucleotide mutation in colorectal cancer as a prognostic biomarker remains uncertain.We utilized a database of 1072 rectal and colon cancers in two prospective cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study. Cox proportional hazards model was used to compute mortality hazard ratio (HR, adjusted for clinical, pathological and molecular features including the CpG island methylator phenotype (CIMP, LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. MSI-high was observed in 15% (162/1072 of all colorectal cancers. TGFBR2 and BAX mononucleotide mutations were detected in 74% (117/159 and 30% (48/158 of MSI-high tumors, respectively. In Kaplan-Meier analysis as well as univariate and multivariate Cox regression analyses, compared to microsatellite stable (MSS/MSI-low cases, MSI-high cases were associated with superior colorectal cancer-specific survival [adjusted HR, 0.34; 95% confidence interval (CI, 0.20-0.57] regardless of TGFBR2 or BAX mutation status. Among MSI-high tumors, TGFBR2 mononucleotide mutation was associated with CIMP-high independent of other variables [multivariate odds ratio, 3.57; 95% CI, 1.66-7.66; p = 0.0011].TGFBR2 or BAX mononucleotide mutations are not associated with the patient survival outcome in MSI-high colorectal cancer. Our data do not support those mutations as prognostic biomarkers (beyond MSI in colorectal carcinoma.

  6. 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.

  7. Anti-mutagenic and Pro-apoptotic Effects of Apigenin on Human Chronic Lymphocytic Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Mehrdad Hashemi

    2010-09-01

    Full Text Available "nDiet can play a vital role in cancer prevention. Nowadays the scientists are looking for food materials which can potentially prevent the cancer occurrence. The purpose of this research is to examine anti-mutagenic and apoptotic effects of apigenin in human lymphoma cells. In present study human chronic lymphocytic leukemia (Eheb cell line were cultured in RPMI 1640 (Sigma, supplemented with 10% fetal calf serum, penicillin-streptomycin, L-glutamine and incubated at 37 ºC for 2 days. In addition cancer cell line was treated by and apigenin and cellular vital capacity was determined by MTT assay. Then effect of apigenin in human lymphoma B cells was examined by flow cytometry techniques. The apigenin was subsequently evaluated in terms of anti-mutagenic properties by a standard reverse mutation assay (Ames test. This was performed with histidine auxotroph strain of Salmonella typhimurium (TA100. Thus, it requires histidine from a foreign supply to ensure its growth. The aforementioned strain gives rise to reverted colonies when expose to sodium azide as a carcinogen substance. During MTT assay, human chronic lymphocytic leukemia revealed to have a meaningful cell death when compared with controls (P<0.01 Apoptosis was induced suitably after 48 hours by flow cytometry assay. In Ames test apigenin prevented the reverted mutations and the hindrance percent of apigenin was 98.17%.These results have revealed apigenin induced apoptosis in human lymphoma B cells in vitro.

  8. Anti-mutagenic and Pro-apoptotic Effects of Apigenin on Human Chronic Lymphocytic Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Mehrdad Hashemi

    2010-10-01

    Full Text Available Diet can play a vital role in cancer prevention. Nowadays the scientists are looking for food materials which can potentially prevent the cancer occurrence. The purpose of this research is to examine anti-mutagenic and apoptotic effects of apigenin in human lymphoma cells. In present study human chronic lymphocytic leukemia (Eheb cell line were cultured in RPMI 1640 (Sigma, supplemented with 10% fetal calf serum, penicillin-streptomycin, L-glutamine and incubated at 37 ºC for 2 days. In addition cancer cell line was treated by and apigenin and cellular vital capacity was determined by MTT assay. Then effect of apigenin in human lymphoma B cells was examined by flow cytometry techniques. The apigenin was subsequently evaluated in terms of anti-mutagenic properties by a standard reverse mutation assay (Ames test. This was performed with histidine auxotroph strain of Salmonella typhimurium (TA100. Thus, it requires histidine from a foreign supply to ensure its growth. The aforementioned strain gives rise to reverted colonies when expose to sodium azide as a carcinogen substance. During MTT assay, human chronic lymphocytic leukemia revealed to have a meaningful cell death when compared with controls (P

  9. Antiproliferative and Pro-apoptotic activities of the stem bark of ...

    African Journals Online (AJOL)

    Persea americana (Lauraceae) have been used in traditional medicine for a wide range of illness and some of these uses have been proven scientifically. The aim of this present study is to screen for the phytochemical content, determine the proximate parameter and determine the antiproliferative and apoptotic effects of ...

  10. Activation of Pro-apoptotic Caspases in Non-apoptotic Cells During Odontogenesis and Related Osteogenesis

    Czech Academy of Sciences Publication Activity Database

    Švandová, Eva; Veselá, Barbora; Tucker, A. S.; Matalová, Eva

    2018-01-01

    Roč. 9, č. 1 (2018), č. článku 174. ISSN 1664-042X Institutional support: RVO:67985904 Keywords : caspase * differentiation * apoptosis Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Developmental biology Impact factor: 4.134, year: 2016

  11. Pro-apoptotic Effect of Pifithrin-α on Preimplantation Porcine Fertilized Embryo Development

    Directory of Open Access Journals (Sweden)

    Brendan Mulligan

    2012-12-01

    Full Text Available The aim of this study was to investigate the impact of a reported p53 inhibitor, pifithrin-α (PFT-α, on preimplantation porcine in vitro fertilized (IVF embryo development in culture. Treatment of PFT-α was administered at both early (0 to 48 hpi, and later stages (48 to 168 hpi of preimplantation development, and its impact upon the expression of five genes related to apoptosis (p53, bak, bcl-xL, p66Shc and caspase3, was assessed in resulting d 7 blastocysts, using real-time quantitative PCR. Total cell numbers, along with the number of apoptotic nuclei, as detected by the in situ cell death detection assay, were also calculated on d 7 in treated and non-treated control embryos. The results indicate that PFT-α, when administered at both early and later stages of porcine IVF embryo development, increases the incidence of apoptosis in resulting blastocysts. When administered at early cleavage stages, PFT-α treatment was shown to reduce the developmental competence of porcine IVF embryos, as well as reducing the quality of resulting blastocysts in terms of overall cell numbers. In contrast, at later stages, PFT-α administration resulted in marginally increased blastocyst development rates amongst treated embryos, but did not affect cell numbers. However, PFT-α treatment induced apoptosis and apoptotic related gene expression, in all treated embryos, irrespective of the timing of treatment. Our results indicate that PFT-α may severely compromise the developmental potential of porcine IVF embryos, and is a potent apoptotic agent when placed into porcine embryo culture media. Thus, caution should be exercised when using PFT-α as a specific inhibitor of p53 mediated apoptosis, in the context of porcine IVF embryo culture systems.

  12. P53-dependent antiproliferative and pro-apoptotic effects of trichostatin A (TSA) in glioblastoma cells.

    Science.gov (United States)

    Bajbouj, K; Mawrin, C; Hartig, R; Schulze-Luehrmann, J; Wilisch-Neumann, A; Roessner, A; Schneider-Stock, R

    2012-05-01

    Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.

  13. Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells

    International Nuclear Information System (INIS)

    Michels, G.; Waetjen, W.; Niering, P.; Steffan, B.; Thi, Q.-H. Tran; Chovolou, Y.; Kampkoetter, A.; Bast, A.; Proksch, P.; Kahl, R.

    2005-01-01

    Polyphenols are ubiquitous substances in the diet. Their anti-oxidative, anti-inflammatory and anti-viral effects are of interest for human health, and polyphenols such as luteolin are used at high concentrations in food supplements. The aim of this project was to determine the intrinsic effects of luteolin in H4IIE rat hepatoma cells. Luteolin is relatively toxic, cell death was caused via induction of apoptosis as detected by DNA-ladder formation, by nuclear fragmentation and activation of apoptotic enzymes (caspase-2, -3/7, -9 and -8/10). Luteolin (250 μM, 24 h) increased the caspase-3/7 activity four-fold and the caspase-9 activity six-fold. In a time course experiment caspase-9 is activated after 6 h, while caspase-2 and -3/7 are activated after 12 h. After 24 h, caspase-8/10 also displays activation. We found a concentration-dependent increase in malondialdehyde release suggesting a prooxidative effect of luteolin. Furthermore, we analysed DNA strand break formation by luteolin and found a distinct increase of DNA strand breaks after incubation for 3 h with 100 μM luteolin, a concentration which induces oligonucleosomal DNA cleavage at 24 h. In conclusion, the sequence of events is compatible with the assumption that luteolin triggers the mitochondrial pathway of apoptosis, probably by inducing DNA damage

  14. Biological Evaluation of Dipyrromethanes in Cancer Cell Lines: Antiproliferative and Pro-apoptotic Properties

    Czech Academy of Sciences Publication Activity Database

    Jorda, Radek; Lopes, S. M.M.; Řezníčková, Eva; Kryštof, Vladimír; Pinho e Melo, T. M.V.D.

    2017-01-01

    Roč. 12, č. 9 (2017), s. 701-711 ISSN 1860-7179 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : anticancer agents * apoptosis * cell cycle * cytotoxicity * dipyrromethanes Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Oncology Impact factor: 3.225, year: 2016

  15. Pro-apoptotic effect of Persea americana var. Hass (avocado) on Jurkat lymphoblastic leukemia cells.

    Science.gov (United States)

    Bonilla-Porras, Angelica R; Salazar-Ospina, Andrea; Jimenez-Del-Rio, Marlene; Pereañez-Jimenez, Andres; Velez-Pardo, Carlos

    2013-11-05

    Abstract Context: Therapy for leukemia has a limited efficacy. There is a need to search for alternative anti-leukemia therapies. Persea americana Mill var. Hass (Lauraceae) is a tropical fruit (avocado) that might be used against cancer. Objective: To investigate whether P. americana induces death in Jurkat lymphoblastic leukemia cells. Materials and methods: Four ethanol extracts (0.1, 0.5, 1, 2 and 5 mg/mL) from avocado fruit (endocarp, whole seed, seed and leaves) were analyzed against Jurkat cells. Hydrogen peroxide generation by oxidation of 2',7'-dichlorodihydrofluorescein diacetate to the fluorescent compound 2',7'-dichlorfluorescein assay, acridine orange/ethidium bromide staining, flow cytometry analysis of annexin-V/7-amino-actinomycin, mitochondrial membrane potential and immunocytochemistry detection of transcription factor p53, caspase-3 and apoptosis-inducing factor (AIF) were evaluated. Results: Endocarp, seed, whole seed, and leaf (0.1 mg/mL) extracts induced significant apoptosis in Jurkat cells (p avocado and its therapeutic action on leukemia.

  16. The unfolded protein response and the role of protein disulphide isomerase in neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Emma ePerri

    2016-01-01

    Full Text Available The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and dysregulation of proteostasis is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR, distinct signalling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulphide Isomerase (PDI is an ER chaperone induced during ER stress that is responsible for the formation of disulphide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions.

  17. Tempo enhances heat-induced apoptosis by mitochondrial targeting of Bax

    International Nuclear Information System (INIS)

    Zhao, Q.-L.; Fujiwara, Y.; Kondo, T.

    2003-01-01

    A stable membrane-permeable nitroxide, Tempo, exerts an SOD-like antioxidant activity against ROS. Reportedly, Tempo inhibits ROS-induced thymocyte apoptosis, while 10 mM Tempo activates JNK1 to induce apoptosis in breast cancer cells. We have observed that nontoxic 5 mM Tempo enhances suboptimal hyperthermia (44 deg C/10 min)-induced apoptosis in U937 cells. Here we report the enhancing mechanism, focusing on activation and targeting of Bax to mitochondria and cytochrome c release. Methods: U937 cells were treated with either Tempo (5 mM, 37 deg C/10 min), heating (44 deg C/10 min), or Tempo-plus-heating, washed and incubated for various times up to 6 h, until assessing apoptosis, mitochondrial potential (ΔΨ>), and amount of superoxide by flow cytometry using Annexin V-FITC/PI, TMRM, and dihydroethidium, respectively. Bax, Bcl-2 and Bcl-XL, and cytochrome c were detected by western blotting, activated Bax was by immunoprecipitation, and ATP was by a luciferase assay. Bax targeting to and cytochrome c release from mitochorndria were also detected immunocytochemically under fluorescent microscopy. Results and Discussion: Treatment of U937 cells with 5 mM Tempo for 10 min at 37 deg C or suboptimal heating (44 deg C/ 10 min) alone did not induce apoptosis. The combined treatment with 5 mM Tempo and 44 deg C for 10 min dramatically induced ∼90% apoptosis in 6 h, as did the 44 deg C/30 min heating. During the enhanced apoptosis, cytochrome c release progressed. Although signals of Bcl-2, Bcl-XL and Bax in cell lysates were not altered, Bax was specifically activated and translocated to mitochondria after the combined treatment. Further, loss of ΔΨ>and decreases in superoxide and ATP progressed after the combined treatment, suggesting that the treatment may disturb mitochondrial electron transport. Thus, Tempo sensitizes the heat-induced apoptosis through (1) targeting of Bax to mitochondria and releasing cytochrome c, and (2) mitochondrial dysfunction

  18. Lithium chloride attenuates mitomycin C induced necrotic cell death in MDA-MB-231 breast cancer cells via HMGB1 and Bax signaling.

    Science.gov (United States)

    Razmi, Mahdieh; Rabbani-Chadegani, Azra; Hashemi-Niasari, Fatemeh; Ghadam, Parinaz

    2018-07-01

    The clinical use of potent anticancer drug mitomycin C (MMC) has limited due to side effects and resistance of cancer cells. The aim of this study was to investigate whether lithium chloride (LiCl), as a mood stabilizer, can affect the sensitivity of MDA-MB-231 breast cancer cells to mitomycin C. The cells were exposed to various concentrations of mitomycin C alone and combined with LiCl and the viability determined by trypan blue and MTT assays. Proteins were analyzed by western blot and mRNA expression of HMGB1 MMP9 and Bcl-2 were analyzed by RT-PCR. Flow cytometry was used to determine the cell cycle arrest and percent of apoptotic and necrotic cells. Concentration of Bax assessed by ELISA. Exposure of the cells to mitomycin C revealed IC 50 value of 20 μM, whereas pretreatment of the cells with LiCl induced synergistic cytotoxicity and IC 50 value declined to 5 μM. LiCl combined with mitomycin C significantly down-regulated HMGB1, MMP9 and Bcl-2 gene expression but significantly increased the level of Bax protein. In addition, the content of HMGB1 in the nuclei decreased and pretreatment with LiCl reduced the content of HMGB1 release induced by MMC. LiCl increased mitomycin C-induced cell shrinkage and PARP fragmentation suggesting induction of apoptosis in these cells. LiCl prevented mitomycin C-induced necrosis and changed the cell death arrest at G2/M-phase. Taking all together, it is suggested that LiCl efficiently enhances mitomycin C-induced apoptosis and HMGB1, Bax and Bcl-2 expression may play a major role in this process, the findings that provide a new therapeutic strategy for LiCl in combination with mitomycin C. Copyright © 2018 Elsevier GmbH. All rights reserved.

  19. The radiosensitivity of glioblastoma cell lines after hypoxia-induced Bax expression

    International Nuclear Information System (INIS)

    Chen, J.K.; Hu, L.J.; Kong, E.L.; Lamborn, K.R.; Deen, D.F.

    2003-01-01

    Full text: Radiation therapy is the most effective treatment after surgery for patients with malignant gliomas. However, the hypoxic cells exclusive to tumor tissue have proven resistant to both radiotherapy and many forms of chemotherapy. In order to specifically target these hypoxic cells, U-251 MG and U-87 MG human glioblastoma cells were stably transfected with constructs containing the suicide gene Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible-factor 1 (HIF-1) binds to HRE and facilitates the transcription of downstream genes. Previously, hypoxia-induced Bax expression in transfected U-251 and U-87 clone cells has been shown to increase cell killing. The benefits of the gene therapy could be further expanded if Bax also acted to increase the sensitivity of these clone cells to radiation. To determine whether this was the case, parent and clone cells were irradiated with graded doses of X-rays under hypoxic conditions. These cells were then left hypoxic for varying durations of time, after which they were incubated for two weeks under aerated conditions to assay for clonogenic cell survival. After less than an hour under hypoxia, both U-251 and U-87 clone cells appeared significantly more sensitive to radiation than their respective parent cells. However, after longer amounts of time under anoxia, higher surviving fractions were found in each clone that were consistent with those of their respective parent cell line, showing that potentially lethal damage repair (PLDR) had occurred in the clone cells. Parent cells did not exhibit PLDR. Results are inconclusive at this point in time. Western blot analyses detailing the amount of Bax expression at each time point as well as further research exploring different durations of hypoxia will be necessary to reveal the nature of the correlation between Bax expression and radiosensitivity. Supported by NS-42927 and CA-85356

  20. XIAP impairs mitochondrial function during apoptosis by regulating the Bcl-2 family in renal cell carcinoma.

    Science.gov (United States)

    Chen, Chao; Liu, Tian Shu; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

    2018-05-01

    Efficient apoptosis requires Bcl-2 family-mediated mitochondrial outer membrane permeabilization (MOMP), which releases pro-apoptotic proteins to the cytosol, activating apoptosis and inhibiting X-linked inhibitor of apoptosis protein (XIAP). XIAP is a member of the inhibitors of apoptosis protein family whose expression is elevated in many cancer types and participates in the release of pro-apoptotic proteins. To explore the association between XIAP and the Bcl-2 family, and the influence of XIAP on mitochondria, RNA interference of XIAP was performed in Caki-1 cells and the dynamic change in the levels of related proteins was compared with the original Caki-1 cells upon induction of apoptosis. Upon knockdown of XIAP, the release of cytochrome c (Cyt-c), second mitochondria-derived activator of caspase (Smac) and apoptotic protease activating factor 1 (Apaf-1) from mitochondria proceeded normally, whereas in Caki-1 cells, the release of these pro-apoptotic proteins was significantly prolonged, and incomplete. Downregulation of XIAP through small interfering RNA resulted in an increase of apoptosis and a marked decrease in Bcl-2 and Bcl-xl levels at 3 h. Additionally, the regulation of the level of XIAP protein affected the specific ratios of Bcl-2/Bax and Bcl-xl/Bax, which play decisive roles in cell death. In the present study, it was revealed that XIAP can feed back to mitochondria, delaying Cyt-c and Apaf-1 release. Furthermore, XIAP can limit the release of its inhibitor Smac with the involvement of Bcl-2 family proteins.

  1. The sensitivity of diffuse large B-cell lymphoma cell lines to histone deacetylase inhibitor-induced apoptosis is modulated by BCL-2 family protein activity.

    Directory of Open Access Journals (Sweden)

    Ryan C Thompson

    Full Text Available BACKGROUND: Diffuse large B-cell lymphoma (DLBCL is a genetically heterogeneous disease and this variation can often be used to explain the response of individual patients to chemotherapy. One cancer therapeutic approach currently in clinical trials uses histone deacetylase inhibitors (HDACi's as monotherapy or in combination with other agents. METHODOLOGY/PRINCIPAL FINDINGS: We have used a variety of cell-based and molecular/biochemical assays to show that two pan-HDAC inhibitors, trichostatin A and vorinostat, induce apoptosis in seven of eight human DLBCL cell lines. Consistent with previous reports implicating the BCL-2 family in regulating HDACi-induced apoptosis, ectopic over-expression of anti-apoptotic proteins BCL-2 and BCL-XL or pro-apoptotic protein BIM in these cell lines conferred further resistance or sensitivity, respectively, to HDACi treatment. Additionally, BCL-2 family antgonist ABT-737 increased the sensitivity of several DLBCL cell lines to vorinostat-induced apoptosis, including one cell line (SUDHL6 that is resistant to vorinostat alone. Moreover, two variants of the HDACi-sensitive SUDHL4 cell line that have decreased sensitivity to vorinostat showed up-regulation of BCL-2 family anti-apoptotic proteins such as BCL-XL and MCL-1, as well as decreased sensitivity to ABT-737. These results suggest that the regulation and overall balance of anti- to pro-apoptotic BCL-2 family protein expression is important in defining the sensitivity of DLBCL to HDACi-induced apoptosis. However, the sensitivity of DLBCL cell lines to HDACi treatment does not correlate with expression of any individual BCL-2 family member. CONCLUSIONS/SIGNIFICANCE: These studies indicate that the sensitivity of DLBCL to treatment with HDACi's is dependent on the complex regulation of BCL-2 family members and that BCL-2 antagonists may enhance the response of a subset of DLBCL patients to HDACi treatment.

  2. Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria

    International Nuclear Information System (INIS)

    Grondin, Melanie; Marion, Michel; Denizeau, Francine; Averill-Bates, Diana A.

    2007-01-01

    Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad from the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl - /HCO 3 - exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-n-butyltin-induced apoptotic signaling in rat hepatocytes

  3. American Ginseng Stimulates Insulin Production and Prevents Apoptosis through Regulation of Uncoupling Protein-2 in Cultured β Cells

    Directory of Open Access Journals (Sweden)

    John Zeqi Luo

    2006-01-01

    Full Text Available American ginseng root displays the ability to achieve glucose homeostasis both experimentally and clinically but the unknown mechanism used by ginseng to achieve its therapeutic effects on diabetes limits its application. Disruption in the insulin secretion of pancreatic β cells is considered the major cause of diabetes. A mitochondrial protein, uncoupling protein-2 (UCP-2 has been found to play a critical role in insulin synthesis and β cell survival. Our preliminary studies found that the extracts of American ginseng inhibit UCP-2 expression which may contribute to the ability of ginseng protecting β cell death and improving insulin synthesis. Therefore, we hypothesized that ginseng extracts suppress UCP-2 in the mitochondria of pancreatic β cells, promoting insulin synthesis and anti-apoptosis (a programmed cell-death mechanism. To test the hypothesis, the serum-deprived quiescent β cells were cultured with or without interleukin-1β (IL-1β, (200 pg ml−1, a cytokine to induce β cell apoptosis and water extracts of American ginseng (25 μg per 5 μl administered to wells of 0.5 ml culture for 24 h. We evaluated effects of ginseng on UCP-2 expression, insulin production, anti-/pro-apoptotic factors Bcl-2/caspase-9 expression and cellular ATP levels. We found that ginseng suppresses UCP-2, down-regulates caspase-9 while increasing ATP and insulin production/secretion and up-regulates Bcl-2, reducing apoptosis. These findings suggest that stimulation of insulin production and prevention of β cell loss by American ginseng extracts can occur via the inhibition of mitochondrial UCP-2, resulting in increase in the ATP level and the anti-apoptotic factor Bcl-2, while down-regulation of pro-apoptotic factor caspase-9 occurs, lowering the occurrence of apoptosis, which support the hypothesis.

  4. Tumor protein 53-induced nuclear protein 1 (TP53INP1 enhances p53 function and represses tumorigenesis

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    Jeyran eShahbazi

    2013-05-01

    Full Text Available Tumor protein 53-induced nuclear protein 1 (TP53INP1 is a stress-induced p53 target gene whose expression is modulated by transcription factors such as p53, p73 and E2F1. TP53INP1 gene encodes two isoforms of TP53INP1 proteins, TP53INP1α and TP53INP1β, both of which appear to be key elements in p53 function. When associated with homeodomain-interacting protein kinase-2 (HIPK2, TP53INP1 phosphorylates p53 protein at Serine 46, enhances p53 protein stability and its transcriptional activity, leading to transcriptional activation of p53 target genes such as p21, PIG-3 and MDM2, cell growth arrest and apoptosis upon DNA damage stress. The anti-proliferative and pro-apoptotic activities of TP53INP1 indicate that TP53INP1 has an important role in cellular homeostasis and DNA damage response. Deficiency in TP53INP1 expression results in increased tumorigenesis; while TP53INP1 expression is repressed during early stages of cancer by factors such as miR-155. This review aims to summarize the roles of TP53INP1 in blocking tumor progression through p53-dependant and p53-independent pathways, as well as the elements which repress TP53INP1 expression, hence highlighting its potential as a therapeutic target in cancer treatment.

  5. Lithium-Induced Neuroprotection is Associated with Epigenetic Modification of Specific BDNF Gene Promoter and Altered Expression of Apoptotic-Regulatory Proteins

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    Tushar eDwivedi

    2015-01-01

    Full Text Available Bipolar disorder (BD, one of the most debilitating mental disorders, is associated with increased morbidity and mortality. Lithium is the first line of treatment option for BD and is often used for maintenance therapy. Recently, the neuroprotective action of lithium has gained tremendous attention, given that BD is associated with structural and functional abnormalities of the brain. However, the precise molecular mechanism by which lithium exerts its neuroprotective action is not clearly understood. In hippocampal neurons, the effects of lithium on neuronal viability against glutamate-induced cytotoxicity, dendritic length and number, and expression and methylation of BDNF promoter exons and expression of apoptotic regulatory genes were studied. In rat hippocampal neurons, lithium not only increased dendritic length and number, but also neuronal viability against glutamate-induced cytotoxicity. While lithium increased the expression of BDNF as well as genes associated with neuroprotection such as Bcl2 and Bcl-XL, it decreased the expression of pro-apoptotic genes Bax, Bad, and caspases 3. Interestingly, lithium activated transcription of specific exon IV to induce BDNF gene expression. This was accompanied by hypomethylation of BDNF exon IV promoter. This study delineates mechanisms by which lithium mediates its effects in protecting neurons.

  6. BH3-only protein Bim inhibits activity of antiapoptotic members of Bcl-2 family when expressed in yeast.

    Science.gov (United States)

    Juhásová, Barbora; Mentel, Marek; Bhatia-Kiššová, Ingrid; Zeman, Igor; Kolarov, Jordan; Forte, Michael; Polčic, Peter

    2011-09-02

    Proteins of the Bcl-2 family regulate programmed cell death in mammals by promoting the release of cytochrome c from mitochondria in response to various proapoptotic stimuli. The mechanism by which BH3-only members of the family activate multidomain proapoptotic proteins Bax and Bak to form a pore in mitochondrial membranes remains under dispute. We report that cell death promoting activity of BH3-only protein Bim can be reconstituted in yeast when both Bax and antiapoptotic protein Bcl-X(L) are present, suggesting that Bim likely activates Bax indirectly by inhibiting antiapoptotic proteins. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

    Science.gov (United States)

    Godoi, Paulo H C; Wilkie-Grantham, Rachel P; Hishiki, Asami; Sano, Renata; Matsuzawa, Yasuko; Yanagi, Hiroko; Munte, Claudia E; Chen, Ya; Yao, Yong; Marassi, Francesca M; Kalbitzer, Hans R; Matsuzawa, Shu-Ichi; Reed, John C

    2016-07-01

    B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway.

    Science.gov (United States)

    Kaur, Anuvinder; Riaz, Muhammad Suleman; Murugaiah, Valarmathy; Varghese, Praveen Mathews; Singh, Shiv K; Kishore, Uday

    2018-01-01

    Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53 mt ), MiaPaCa-2 (p53 mt ), and Capan-2 (p53 wt ) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

  9. Inhibitor of apoptosis-stimulating protein of p53 (iASPP is required for neuronal survival after axonal injury.

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    Ariel M Wilson

    Full Text Available The transcription factor p53 mediates the apoptosis of post-mitotic neurons exposed to a wide range of stress stimuli. The apoptotic activity of p53 is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP family members: ASPP1, ASPP2 and iASPP. We previously showed that the pro-apoptotic members ASPP1 and ASPP2 contribute to p53-dependent death of retinal ganglion cells (RGCs. However, the role of the p53 inhibitor iASPP in the central nervous system (CNS remains to be elucidated. To address this, we asked whether iASPP contributes to the survival of RGCs in an in vivo model of acute optic nerve damage. We demonstrate that iASPP is expressed by injured RGCs and that iASPP phosphorylation at serine residues, which increase iASPP affinity towards p53, is significantly reduced following axotomy. We show that short interference RNA (siRNA-induced iASPP knockdown exacerbates RGC death, whereas adeno-associated virus (AAV-mediated iASPP expression promotes RGC survival. Importantly, our data also demonstrate that increasing iASPP expression in RGCs downregulates p53 activity and blocks the expression of pro-apoptotic targets PUMA and Fas/CD95. This study demonstrates a novel role for iASPP in the survival of RGCs, and provides further evidence of the importance of the ASPP family in the regulation of neuronal loss after axonal injury.

  10. The ethanol extract of Scutellaria baicalensis and the active compounds induce cell cycle arrest and apoptosis including upregulation of p53 and Bax in human lung cancer cells

    International Nuclear Information System (INIS)

    Gao Jiayu; Morgan, Winston A.; Sanchez-Medina, Alberto; Corcoran, Olivia

    2011-01-01

    Despite a lack of scientific authentication, Scutellaria baicalensis is clinically used in Chinese medicine as a traditional adjuvant to chemotherapy of lung cancer. In this study, cytotoxicity assays demonstrated that crude ethanolic extracts of S. baicalensis were selectively toxic to human lung cancer cell lines A549, SK-LU-1 and SK-MES-1 compared with normal human lung fibroblasts. The active compounds baicalin, baicalein and wogonin did not exhibit such selectivity. Following exposure to the crude extracts, cellular protein expression in the cancer cell lines was assessed using 2D gel electrophoresis coupled with MALDI-TOF-MS/Protein Fingerprinting. The altered protein expression indicated that cell growth arrest and apoptosis were potential mechanisms of cytotoxicity. These observations were supported by PI staining cell cycle analysis using flow cytometry and Annexin-V apoptotic analysis by fluorescence microscopy of cancer cells treated with the crude extract and pure active compounds. Moreover, specific immunoblotting identification showed the decreased expression of cyclin A results in the S phase arrest of A549 whereas the G 0 /G 1 phase arrest in SK-MES-1 cells results from the decreased expression of cyclin D1. Following treatment, increased expression in the cancer cells of key proteins related to the enhancement of apoptosis was observed for p53 and Bax. These results provide further insight into the molecular mechanisms underlying the clinical use of this herb as an adjuvant to lung cancer therapy. - Research highlights: → Scutellaria baicalensis is a clinical adjuvant to lung cancer chemotherapy in China. → Scutellaria ethanol extracts selectively toxic to A549, SK-LU-1 and SK-MES-1. → Baicalin, baicalein and wogonin were toxic to all lung cancer cell lines. → Proteomics identified increased p53 and BAX in response to Scutellaria extracts.

  11. Prognostic value of TP53 transcriptional activity on p21 and bax in patients with esophageal squamous cell carcinomas treated by definitive chemoradiotherapy

    International Nuclear Information System (INIS)

    Michel, Pierre; Magois, Karine; Robert, Valerie; Chiron, Anne; Lepessot, Florence; Bodenant, Corinne; Roque, Isabelle; Seng, Sok H.; Frebourg, Thierry; Paillot, Bernard

    2002-01-01

    Purpose: The aim of this study was to evaluate biologic factors on survival and clinical response after definitive concomitant chemoradiotherapy (CRT) in patients with esophageal squamous cell carcinoma (ESCC). Methods and Materials: TP53 protein hyperexpression (immunochemistry [IHC]) and functional assay (FA) of TP53, measuring the ability of TP53 to transactivate p21 and bax reporter systems, were performed in patients with ESCC treated by CRT. The impact of parameters studied on survival and clinical response to CRT was assessed. Results: Thirty-eight patients with ESCC were included. TP53 alterations were detected in 84.2% of cases with FA. All TP53 mutations abolished the transactivation of p21 and bax reporter systems. After CRT, complete response rate was 55.3%. The median survival of the population was 17.5 months. Serum albumin (p=0.002), weight loss <10% (p=0.005), and response to treatment (p<0.001) were significantly linked with survival. TP53 alteration in FA was not significantly predictive of response to CRT (p=0.132) nor survival (p=0.154). Conclusions: Our results suggest that wild-type TP53 in ESCC could be associated with good response to definitive CRT. However, the small rate of ESCC with wild-type TP53 suggests that systematic determination of TP53 status is not appropriate for the management of the ESCC population

  12. USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

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    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.

  13. Bax and Bak Do Not Exhibit Functional Redundancy in Mediating Radiation-Induced Endothelial Apoptosis in the Intestinal Mucosa

    International Nuclear Information System (INIS)

    Rotolo, Jimmy A.; Maj, Jerzy G.; Feldman, Regina; Ren, Decheng; Haimovitz-Friedman, Adriana; Cordon-Cardo, Carlos; Cheng, Emily H.-Y.; Kolesnick, Richard; Fuks, Zvi

    2008-01-01

    Purpose: To address in vivo the issue of whether Bax and Bak are functionally redundant in signaling apoptosis, capable of substituting for each other. Methods and Materials: Mice were exposed to whole-body radiation, and endothelial cell apoptosis was quantified using double immunostaining with TUNEL and anti-CD31 antibody. Crypt survival was determined at 3.5 days after whole-body radiation by the microcolony survival assay. Actuarial animal survival was calculated by the product-limit Kaplan-Meier method, and autopsies were performed to establish cause of death. Results: Radiation exposure of Bax- and Bak-deficient mice, both expressing a wild-type acid sphingomyelinase (ASMase) phenotype, indicated that Bax and Bak are both mandatory, though mutually independent, for the intestinal endothelial apoptotic response. However, neither affected epithelial apoptosis at crypt positions 4-5, indicating specificity toward endothelium. Furthermore, Bax deficiency and Bak deficiency each individually mimicked ASMase deficiency in inhibiting crypt lethality in the microcolony assay and in rescuing mice from the lethal gastrointestinal syndrome. Conclusions: The data indicate that Bax and Bak have nonredundant functional roles in the apoptotic response of the irradiated intestinal endothelium. The observation that Bax deficiency and Bak deficiency also protect crypts in the microcolony assay provides strong evidence that the microvascular apoptotic component is germane to the mechanism of radiation-induced damage to mouse intestines, regulating reproductive cell death of crypt stem cell clonogens

  14. An Integrated Bioinformatics and Computational Biology Approach Identifies New BH3-Only Protein Candidates.

    Science.gov (United States)

    Hawley, Robert G; Chen, Yuzhong; Riz, Irene; Zeng, Chen

    2012-05-04

    In this study, we utilized an integrated bioinformatics and computational biology approach in search of new BH3-only proteins belonging to the BCL2 family of apoptotic regulators. The BH3 (BCL2 homology 3) domain mediates specific binding interactions among various BCL2 family members. It is composed of an amphipathic α-helical region of approximately 13 residues that has only a few amino acids that are highly conserved across all members. Using a generalized motif, we performed a genome-wide search for novel BH3-containing proteins in the NCBI Consensus Coding Sequence (CCDS) database. In addition to known pro-apoptotic BH3-only proteins, 197 proteins were recovered that satisfied the search criteria. These were categorized according to α-helical content and predictive binding to BCL-xL (encoded by BCL2L1) and MCL-1, two representative anti-apoptotic BCL2 family members, using position-specific scoring matrix models. Notably, the list is enriched for proteins associated with autophagy as well as a broad spectrum of cellular stress responses such as endoplasmic reticulum stress, oxidative stress, antiviral defense, and the DNA damage response. Several potential novel BH3-containing proteins are highlighted. In particular, the analysis strongly suggests that the apoptosis inhibitor and DNA damage response regulator, AVEN, which was originally isolated as a BCL-xL-interacting protein, is a functional BH3-only protein representing a distinct subclass of BCL2 family members.

  15. Preclinical assessment of a new recombinant ADAMTS-13 drug product (BAX930) for the treatment of thrombotic thrombocytopenic purpura.

    Science.gov (United States)

    Kopić, A; Benamara, K; Piskernik, C; Plaimauer, B; Horling, F; Höbarth, G; Ruthsatz, T; Dietrich, B; Muchitsch, E-M; Scheiflinger, F; Turecek, M; Höllriegl, W

    2016-07-01

    Essentials ADAMTS-13-deficiency is a cause of thrombotic thrombocytopenic purpura (TTP). Preclinical safety of recombinant human ADAMTS-13 (BAX930) was shown in animal models. Preclinical efficacy of BAX930 was shown in a mouse model of TTP. BAX930 showed advantageous efficacy over fresh frozen plasma, the current standard of care. Click to hear Dr Cataland and Prof. Lämmle present a seminar on Thrombotic Thrombocytopenic Purpura (TTP): new Insights in Pathogenesis and Treatment Modalities. Background Thrombotic thrombocytopenic purpura (TTP) is a rare blood disorder characterized by microthrombosis in small blood vessels of the body, resulting in a low platelet count. Baxalta has developed a new recombinant ADAMTS-13 (rADAMTS-13) product (BAX930) for on-demand and prophylactic treatment of patients with hereditary TTP (hTTP). Objectives To evaluate the pharmacokinetics, efficacy and safety of BAX930 in different species, by use of an extensive preclinical program. Methods The prophylactic and therapeutic efficacies of BAX930 were tested in a previously established TTP mouse model. Pharmacokinetics were evaluated after single intravenous bolus injection in mice and rats, and after repeated dosing in cynomolgus monkeys. Toxicity was assessed in rats and monkeys, safety pharmacology in monkeys, and local tolerance in rabbits. Results BAX930 was shown to be efficacious, as demonstrated by a stabilized platelet count in ADAMTS-13 knockout mice that were thrombocytopenic when treated. Prophylactic efficacy was dose-dependent and comparable with that achieved by treatment with fresh frozen plasma, the mainstay of hTTP treatment. Therapeutic efficacy was treatment interval-dependent. Safety pharmacology evaluation did not show any deleterious effects of BAX930 on cardiovascular and respiratory functions in monkeys. The compound's pharmacokinetics were similar and dose-proportional in mice, rats, and monkeys. BAX930 was well tolerated in rats, monkeys, and rabbits, even

  16. Atractylenolide-I Protects Human SH-SY5Y Cells from 1-Methyl-4-Phenylpyridinium-Induced Apoptotic Cell Death

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    Sandeep Vasant More

    2017-05-01

    Full Text Available Oxidative stress and apoptosis are the major mechanisms that induce dopaminergic cell death. Our study investigates the protective effects of atractylenolide-I (ATR-I on 1-methyl-4-phenylpyridinium (MPP+-induced cytotoxicity in human dopaminergic SH-SY5Y cells, as well as its underlying mechanism. Our experimental data indicates that ATR-I significantly inhibits the loss of cell viability induced by MPP+ in SH-SY5Y cells. To further unravel the mechanism, we examined the effect of ATR-I on MPP+-induced apoptotic cell death characterized by an increase in the Bax/Bcl-2 mRNA ratio, the release of cytochrome-c, and the activation of caspase-3 leading to elevated levels of cleaved poly(ADP-ribose polymerase (PARP resulting in SH-SY5Y cell death. Our results demonstrated that ATR-I decreases the level of pro-apoptotic proteins induced by MPP+ and also restored Bax/Bcl-2 mRNA levels, which are critical for inducing apoptosis. In addition, ATR-I demonstrated a significant increase in the protein expression of heme-oxygenase in MPP+-treated SH-SY5Y cells. These results suggest that the pharmacological effect of ATR-I may be, at least in part, caused by the reduction in pro-apoptotic signals and also by induction of anti-oxidant protein.

  17. 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.

  18. The Unfolded Protein Response in Amelogenesis and Enamel Pathologies

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    Steven J. Brookes

    2017-09-01

    Full Text Available During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other “professional” secretory cells, ameloblasts employ the unfolded protein response (UPR to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed.

  19. The Unfolded Protein Response in Amelogenesis and Enamel Pathologies.

    Science.gov (United States)

    Brookes, Steven J; Barron, Martin J; Dixon, Michael J; Kirkham, Jennifer

    2017-01-01

    During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other "professional" secretory cells, ameloblasts employ the unfolded protein response (UPR) to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum)/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI) and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed.

  20. Detection of cow's milk proteins and minor components in human milk using proteomics techniques.

    Science.gov (United States)

    Coscia, A; Orrù, S; Di Nicola, P; Giuliani, F; Varalda, A; Peila, C; Fabris, C; Conti, A; Bertino, E

    2012-10-01

    Cow's milk proteins (CMPs) are the best characterized food allergens. The aim of this study was to investigate cow's milk allergens in human colostrum of term and preterm newborns' mothers, and other minor protein components by proteomics techniques, more sensitive than other techniques used in the past. Sixty-two term and 11 preterm colostrum samples were collected, subjected to a treatment able to increase the concentration of the most diluted proteins and simultaneously to reduce the concentration of the proteins present at high concentration (Proteominer Treatment), and subsequently subjected to the steps of proteomic techniques. The most relevant finding in this study was the detection of the intact bovine alpha-S1-casein in human colostrum, then bovine alpha-1-casein could be considered the cow's milk allergen that is readily secreted in human milk and could be a cause of sensitization to cow's milk in exclusively breastfed predisposed infants. Another interesting result was the detection, at very low concentrations, of proteins previously not described in human milk (galectin-7, the different isoforms of the 14-3-3 protein and the serum amyloid P-component), probably involved in the regulation of the normal cell growth, in the pro-apoptotic function and in the regulation of tissue homeostasis. Further investigations are needed to understand if these families of proteins have specific biological activity in human milk.

  1. Modafinil abrogates methamphetamine-induced neuroinflammation and apoptotic effects in the mouse striatum.

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    Mariana Raineri

    Full Text Available Methamphetamine is a drug of abuse that can cause neurotoxic damage in humans and animals. Modafinil, a wake-promoting compound approved for the treatment of sleeping disorders, is being prescribed off label for the treatment of methamphetamine dependence. The aim of the present study was to investigate if modafinil could counteract methamphetamine-induced neuroinflammatory processes, which occur in conjunction with degeneration of dopaminergic terminals in the mouse striatum. We evaluated the effect of a toxic methamphetamine binge in female C57BL/6 mice (4 × 5 mg/kg, i.p., 2 h apart and modafinil co-administration (2 × 90 mg/kg, i.p., 1 h before the first and fourth methamphetamine injections on glial cells (microglia and astroglia. We also evaluated the striatal expression of the pro-apoptotic BAX and anti-apoptotic Bcl-2 proteins, which are known to mediate methamphetamine-induced apoptotic effects. Modafinil by itself did not cause reactive gliosis and counteracted methamphetamine-induced microglial and astroglial activation. Modafinil also counteracted the decrease in tyrosine hydroxylase and dopamine transporter levels and prevented methamphetamine-induced increases in the pro-apoptotic BAX and decreases in the anti-apoptotic Bcl-2 protein expression. Our results indicate that modafinil can interfere with methamphetamine actions and provide protection against dopamine toxicity, cell death, and neuroinflammation in the mouse striatum.

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

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    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.

  3. MAGE-A inhibits apoptosis in proliferating myeloma cells through repression of Bax and maintenance of survivin.

    Science.gov (United States)

    Nardiello, Tricia; Jungbluth, Achim A; Mei, Anna; Diliberto, Maurizio; Huang, Xiangao; Dabrowski, Ania; Andrade, Valéria C C; Wasserstrum, Rebecca; Ely, Scott; Niesvizky, Ruben; Pearse, Roger; Coleman, Morton; Jayabalan, David S; Bhardwaj, Nina; Old, Lloyd J; Chen-Kiang, Selina; Cho, Hearn Jay

    2011-07-01

    The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67(+) malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells. The impact of MAGE-A3 expression on survival and proliferation in vivo was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups: newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated in vitro by silencing its expression by short hairpin RNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis. MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared with newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A showed that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of proapoptotic Bax expression and by reduction of survivin expression through both p53-dependent and -independent mechanisms. These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms.

  4. Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin

    OpenAIRE

    Matsuura, K; Huang, N-J; Cocce, K; Zhang, L; Kornbluth, S

    2016-01-01

    Evasion of apoptosis allows many cancers to resist chemotherapy. Apoptosis is mediated by the serial activation of caspase family proteins. These proteases are often activated upon the release of cytochrome c from the mitochondria, which is promoted by the proapoptotic Bcl-2 family protein, Bax. This function of Bax is enhanced by the MOAP-1 (modulator of apoptosis protein 1) protein in response to DNA damage. Previously, we reported that MOAP-1 is targeted for ubiquitylation and degradation ...

  5. Expression of measles virus nucleoprotein induces apoptosis and modulates diverse functional proteins in cultured mammalian cells.

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    Ashima Bhaskar

    Full Text Available BACKGROUND: Measles virus nucleoprotein (N encapsidates the viral RNA, protects it from endonucleases and forms a virus specific template for transcription and replication. It is the most abundant protein during viral infection. Its C-terminal domain is intrinsically disordered imparting it the flexibility to interact with several cellular and viral partners. PRINCIPAL FINDINGS: In this study, we demonstrate that expression of N within mammalian cells resulted in morphological transitions, nuclear condensation, DNA fragmentation and activation of Caspase 3 eventuating into apoptosis. The rapid generation of intracellular reactive oxygen species (ROS was involved in the mechanism of cell death. Addition of ascorbic acid (AA or inhibitor of caspase-3 in the extracellular medium partially reversed N induced apoptosis. We also studied the protein profile of cells expressing N protein. MS analysis revealed the differential expression of 25 proteins out of which 11 proteins were up regulated while 14 show signs of down regulation upon N expression. 2DE results were validated by real time and semi quantitative RT-PCR analysis. CONCLUSION: These results show the pro-apoptotic effects of N indicating its possible development as an apoptogenic tool. Our 2DE results present prima facie evidence that the MV nucleoprotein interacts with or causes differential expression of a wide range of cellular factors. At this stage it is not clear as to what the adaptive response of the host cell is and what reflects a strategic modulation exerted by the virus.

  6. Air pollution induces enhanced mitochondrial oxidative stress in cystic fibrosis airway epithelium.

    Science.gov (United States)

    Kamdar, O; Le, Wei; Zhang, J; Ghio, A J; Rosen, G D; Upadhyay, D

    2008-10-29

    We studied the effects of airborne particulate matters (PM) on cystic fibrosis (CF) epithelium. We noted that PM enhanced human CF bronchial epithelial apoptosis, activated caspase-9 and PARP-1; and reduced mitochondrial membrane potential. Mitochondrial inhibitors (4,4-diisothiocyanatostilbene-2,2'disulfonic acid, rotenone and thenoyltrifluoroacetone) blocked PM-induced generation of reactive oxygen species and apoptosis. PM upregulated pro-apoptotic Bad, Bax, p53 and p21; and enhanced mitochondrial localization of Bax. The anti-apoptotic Bcl-2, Bcl-xl, Mcl-1 and Xiap remained unchanged; however, overexpression of Bcl-xl blocked PM-induced apoptosis. Accordingly, we provide the evidence that PM enhances oxidative stress and mitochondrial signaling mediated apoptosis via the modulation of Bcl family proteins in CF.

  7. St. John’s Wort Regulates Proliferation and Apoptosis in MCF-7 Human Breast Cancer Cells by Inhibiting AMPK/mTOR and Activating the Mitochondrial Pathway

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    Mi-Kyoung You

    2018-03-01

    Full Text Available St. John’s Wort (SJW has been used as an estrogen agonist in the systems affected by menopause. Also, hypericin, a bioactive compound of SJW, has been used as a photosensitizer in photodynamic therapy. In the present study, we investigate the anti-proliferative and pro-apoptotic effects of SJW to demonstrate the chemo-preventive effect in human breast cancer cells. MCF-7 cells were cultured with DMSO or various concentrations of SJW ethanol extract (SJWE. Cell viability, proliferation, apoptosis, the expression of proteins involved in cell growth and apoptosis, and caspase-3/7 activity were examined. SJWE dose-dependently suppressed cell growth and induced apoptosis of MCF-7 cells. Mechanistically, SJWE enhanced the phosphorylation of AMP-activated protein kinase (AMPK and decreased the expression of p-mammalian target of rapamycin (p-mTOR and p-eukaryotic translation initiation factor 4E (eIF4E-binding protein 1 (4E-BP1. Also, SJWE inhibited the phosphorylation of protein kinase B (Akt and showed increases in the expression of pro-apoptotic proteins Bax and Bad with decreases in the expression of anti-apoptotic proteins including B-cell lymphoma 2 (Bcl-2, B-cell lymphoma-extra large (Bcl-xL, and p-Bcl-2-associated death promoter (p-Bad. SJWE at 50 μg/mL showed markedly enhanced caspase-7 activation. Taken together, our results provide evidence that SJWE shows anti-proliferative and pro-apoptotic effects via inhibition of AMPK/mTOR and activation of a mitochondrial pathway. Therefore, SJWE can be used as a chemo-preventive agent without photo-activation.

  8. Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs.

    Science.gov (United States)

    Chen, Mo; Qiu, Tao; Wu, Jiajie; Yang, Yang; Wright, Graham D; Wu, Min; Ge, Ruowen

    2018-03-09

    Classic endocytosis destinations include the recycling endosome returning to the plasma membrane or the late endosome (LE) merging with lysosomes for cargo degradation. However, the anti-angiogenic proteins angiostatin and isthmin, are endocytosed and trafficked to mitochondria (Mito) to execute apoptosis of endothelial cells. How these extracellular proteins reach mitochondria remains a mystery. Through confocal and super-resolution fluorescent microscopy, we demonstrate that angiostatin and isthmin are trafficked to mitochondria through the interaction between LE and Mito. Using purified organelles, the LE-Mito interaction is confirmed through in vitro lipid-fusion assay, as well as single vesicle total internal reflection fluorescent microscopy. LE-Mito interaction enables the transfer of not only lipids but also proteins from LE to Mito. Angiostatin and isthmin augment this endosomal protein trafficking pathway and make use of it to reach mitochondria to execute apoptosis. Cell fractionation and biochemical analysis identified that the cytosolic scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) associated with LE and the t-SNARE protein synaptosome-associated protein 25 kDa (SNAP25) associated with Mito form an interaction complex to facilitate LE-Mito interaction. Proximity ligation assay coupled with fluorescent microscopy showed that both NHERF1 and SNAP25 are located at the contacting face between LE and Mito. RNAi knockdown of either NHERF1 or SNAP25 suppressed not only the mitochondrial trafficking of angiostatin and isthmin but also their anti-angiogenic and pro-apoptotic functions. Hence, this study reveals a previously unrealized endosomal protein trafficking pathway from LE to Mito that allows extracellular proteins to reach mitochondria and execute apoptosis.

  9. Anticancer copper(II) phosphorus dendrimers are potent proapoptotic Bax activators.

    Science.gov (United States)

    Mignani, Serge; El Brahmi, Nabil; Eloy, Laure; Poupon, Joel; Nicolas, Valérie; Steinmetz, Anke; El Kazzouli, Said; Bousmina, Mosto M; Blanchard-Desce, Mireille; Caminade, Anne-Marie; Majoral, Jean-Pierre; Cresteil, Thierry

    2017-05-26

    A multivalent phosphorus dendrimer 1G 3 and its corresponding Cu-complex, 1G 3 -Cu have been recently identified as agents retaining high antiproliferative potency. This antiproliferative capacity was preserved in cell lines overexpressing the efflux pump ABC B1, whereas cross-resistance was observed in ovarian cancer cell lines resistant to cisplatin. Theoretical 3D models were constructed: the dendrimers appear as irregularly shaped disk-like nano-objects of about 22 Å thickness and 49 Å diameter, which accumulated in cells after penetration by endocytosis. To get insight in their mode of action, cell death pathways have been examined in human cancer cell lines: early apoptosis was followed by secondary necrosis after multivalent phosphorus dendrimers exposure. The multivalent plain phosphorus dendrimer 1G 3 moderately activated caspase-3 activity, in contrast with the multivalent Cu-conjugated phosphorus dendrimer 1G 3 -Cu which strikingly reduced the caspase-3 content and activity. This decrease of caspase activity is not related to the presence of copper, since inorganic copper has no or little effect on caspase-3. Conversely the potent apoptosis activation could be related to a noticeable translocation of Bax to the mitochondria, resulting in the release of AIF into the cytosol, its translocation to the nucleus and a severe DNA fragmentation, without alteration of the cell cycle. The multivalent Cu-conjugated phosphorus dendrimer is more efficient than its non-complexed analog to activate this pathway in close relationship with the higher antiproliferative potency. Therefore, this multivalent Cu-conjugated phosphorus dendrimer 1G 3 -Cu can be considered as a new and promising first-in-class antiproliferative agent with a distinctive mode of action, inducing apoptosis tumor cell death through Bax activation pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  10. Interacciones hiperfinas en BaxSr1-xHfO3

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    López García, A.

    1999-10-01

    Full Text Available In order to determine the microscopic role of cation A in ABO3 perovskites the hyperfine interaction in the Sr1-xBaxHfO3 system using Perturbed Angular Correlation spectroscopy (PAC is studied. As a previous result the compositions x = 0.50 and 0.25 are shown. The PAC spectra were obtained in the temperature range from 20 to 1000ºC. In both compounds a phase transition to higher symmetry structure was observed at T≈400 and 200ºC, respectively. This structure is proposed to be cubic in both compositions. The results are compared with those obtained in SrHfO3 and BaHfO3.Con el fin de determinar el papel microscópico que juega el catión A en las perovskitas ABO3 se estudia la interacción hiperfina el sistema Sr1-xBaxHfO3 usando la espectroscopia Correlaciones Angulares Perturbadas (PAC en función de la temperatura y la composición. Como resultado previo se muestra en este trabajo, el estudio de las composiciones x = 0.5 y 0.75. Las medidas PAC se realizaron en el intervalo de temperaturas que va desde 20 a 1000ºC. Para x=0.50 y 0.75 se determinó una transición a una fase de mayor simetría a T≈400 y 200ºC, respectivamente. Se propone que esta fase de mayor simetría es cúbica en ambas composiciones. Los resultados obtenidos son comparados con los compuestos puros SrHfO3 y BaHfO3.

  11. Piperine attenuates UV-R induced cell damage in human keratinocytes via NF-kB, Bax/Bcl-2 pathway: An application for photoprotection.

    Science.gov (United States)

    Verma, Ankit; Kushwaha, Hari N; Srivastava, Ajeet K; Srivastava, Saumya; Jamal, Naseem; Srivastava, Kriti; Ray, Ratan Singh

    2017-07-01

    Chronic ultraviolet radiation (UV-R) exposure causes skin disorders like erythema, edema, hyperpigmentation, photoaging and photocarcinogenesis. Recent research trends of researchers have focused more attention on the identification and use of photo stable natural agents with photoprotective properties. Piperine (PIP), as a plant alkaloid, is an important constituent present in black pepper (Piper nigrum), used widely in ayurvedic and other traditional medicines and has broad pharmacological properties. The study was planned to photoprotective efficacy of PIP in human keratinocyte (HaCaT) cell line. We have assessed the UV-R induced activation of transcription factor NF-κB in coordination with cell death modulators (Bax/Bcl-2 and p21). The LC-MS/MS analysis revealed that PIP was photostable under UV-A/UV-B exposure. PIP (10μg/ml) attenuates the UV-R (A and B) induced phototoxicity of keratinocyte cell line through the restoration of cell viability, inhibition of ROS, and malondialdehyde generation. Further, PIP inhibited UV-R mediated DNA damage, prevented micronuclei formation, and reduced sub-G1 phase in cell cycle, which supported against photogenotoxicity. This study revealed that PIP pretreatment strongly suppressed UV-R induced photodamages. Molecular docking studies suggest that PIP binds at the active site of NF-κB, and thus, preventing its translocation to nucleus. In addition, transcriptional and translational analysis advocate the increased expression of NF-κB and concomitant decrease in IkB-α expression under UV-R exposed cells, favouring the apoptosis via Bax/Bcl-2 and p21 pathways. However, PIP induced expression of IkB-α suppress the NF-κB activity which resulted in suppression of apoptotic marker genes and proteins that involved in photoprotection. Therefore, we suggest the applicability of photostable PIP as photoprotective agent for human use. Copyright © 2017. Published by Elsevier B.V.

  12. Association of anti-apoptotic Mcl-1L isoform expression with radioresistance of oral squamous carcinoma cells

    International Nuclear Information System (INIS)

    Palve, Vinayak C; Teni, Tanuja R

    2012-01-01

    Oral cancer is a common cancer and a major health problem in the Indian subcontinent. At our laboratory Mcl-1, an anti-apoptotic member of the Bcl-2 family has been demonstrated to be overexpressed in oral cancers and to predict outcome in oral cancer patients treated with definitive radiotherapy. To study the role of Mcl-1 isoforms in radiation response of oral squamous carcinoma cells (OSCC), we investigated in the present study, the association of Mcl-1 isoform expression with radiosensitivity of OSCC, using siRNA strategy. The time course expression of Mcl-1 splice variants (Mcl-1L, Mcl-1S & Mcl-1ES) was studied by RT-PCR, western blotting & immunofluorescence, post-irradiation in oral cell lines [immortalized FBM (radiosensitive) and tongue cancer AW8507 & AW13516 (radioresistant)]of relatively differing radiosensitivities. The effect of Mcl-1L knockdown alone or in combination with ionizing radiation (IR) on cell proliferation, apoptosis & clonogenic survival, was investigated in AW8507 & AW13516 cells. Further the expression of Mcl-1L protein was assessed in radioresistant sublines generated by fractionated ionizing radiation (FIR). Three to six fold higher expression of anti-apoptotic Mcl-1L versus pro-apoptotic Mcl-1S was observed at mRNA & protein levels in all cell lines, post-irradiation. Sustained high levels of Mcl-1L, downregulation of pro-apoptotic Bax & Bak and a significant (P < 0.05) reduction in apoptosis was observed in the more radioresistant AW8507, AW13516 versus FBM cells, post-IR. The ratios of anti to pro-apoptotic proteins were high in AW8507 as compared to FBM. Treatment with Mcl-1L siRNA alone or in combination with IR significantly (P < 0.01) increased apoptosis viz. 17.3% (IR), 25.3% (siRNA) and 46.3% (IR plus siRNA) and upregulated pro-apoptotic Bax levels in AW8507 cells. Combination of siRNA & IR treatment significantly (P < 0.05) reduced cell proliferation and clonogenic survival of radioresistant AW8507 & AW13516 cells

  13. 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.

  14. NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

    Science.gov (United States)

    de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

    2013-01-01

    NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1–33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species. PMID:23516598

  15. Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

    International Nuclear Information System (INIS)

    Sun, Lihua; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Yang, Yang; Yang, Hua

    2012-01-01

    Highlights: ► Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. ► The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. ► GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. ► GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.

  16. Regulation of radiation-induced apoptosis by early growth response-1 gene in solid tumors

    International Nuclear Information System (INIS)

    Ahmed, M.

    2003-01-01

    Ionizing radiation exposure is associated with activation of certain immediate-early genes that function as transcription factors. These include members of jun or fos and early growth response (EGR) gene families. In particular, the functional role of EGR-1 in radiation-induced signaling is pivotal since the promoter of EGR-1 contains radiation-inducible CArG DNA sequences. The Egr-1 gene belongs to a family of Egr genes that includes EGR-2, EGR-3, EGR-4, EGR-α and the tumor suppressor, Wilms' tumor gene product, WT1. The Egr-1 gene product, EGR-1, is a nuclear protein that contains three zinc fingers of the C 2 H 2 subtype. The EGR-1 GC-rich consensus target sequence, 5'-GCGT/GGGGCG-3' or 5'-TCCT/ACCTCCTCC-3', has been identified in the promoter regions of transcription factors, growth factors, receptors, cell cycle regulators and pro-apoptotic genes. The gene targets mediated by Egr-1 in response to ionizing radiation include TNF-α , p53, Rb and Bax, all these are effectors of apoptosis. Based on these targets, Egr-1 is a pivotal gene that initiates early signal transduction events in response to ionizing radiation leading to either growth arrest or cell death in tumor cells. There are two potential application of Egr-1 gene in therapy of cancer. First, the Egr-1 promoter contains information for appropriate spatial and temporal expression in-vivo that can be regulated by ionizing radiation to control transcription of genes that have pro-apoptotic and suicidal function. Secondly, EGR-1 protein can eliminate 'induced-radiation resistance' by inhibiting the functions of radiation-induced pro-survival genes (NFκB activity and bcl-2 expression) and activate pro-apoptotic genes (such as bax) to confer a significant radio-sensitizing effect. Together, the reported findings from my laboratory demonstrate clearly that EGR-1 is an early central gene that confers radiation sensitivity and its pro-apoptotic functions are synergized by abrogation of induced radiation

  17. The Fanconi anemia group C protein interacts with uncoordinated 5A and delays apoptosis.

    Directory of Open Access Journals (Sweden)

    FengFei Huang

    Full Text Available The Fanconi anemia group C protein (FANCC is one of the several proteins that comprise the Fanconi anemia (FA network involved in genomic surveillance. FANCC is mainly cytoplasmic and has many functions, including apoptosis suppression through caspase-mediated proteolytic processing. Here, we examined the role of FANCC proteolytic fragments by identifying their binding partners. We performed a yeast two-hybrid screen with caspase-mediated FANCC cleavage products and identified the dependence receptor uncoordinated-5A (UNC5A protein. Here, we show that FANCC physically interacts with UNC5A, a pro-apoptotic dependence receptor. FANCC interaction occurs through the UNC5A intracellular domain, specifically via its death domain. FANCC modulates cell sensitivity to UNC5A-mediated apoptosis; we observed reduced UNC5A-mediated apoptosis in the presence of FANCC and increased apoptosis in FANCC-depleted cells. Our results show that FANCC interferes with UNC5A's functions in apoptosis and suggest that FANCC may participate in developmental processes through association with the dependence receptor UNC5A.

  18. BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

    Science.gov (United States)

    Mondragon, Albert A; Betts-Obregon, Brandi S; Moritz, Robert J; Parvathaneni, Kalpana; Navarro, Mary M; Kim, Hong Seok; Lee, Chi Fung; LeBaron, Richard G; Asmis, Reto; Tsin, Andrew T

    2015-01-01

    Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

  19. Synergistic Effect of Subtoxic-dose Cisplatin and TRAIL to Mediate Apoptosis by Down-regulating Decoy Receptor 2 and Up-regulating Caspase-8, Caspase-9 and Bax Expression on NCI-H460 and A549 Cells

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhang

    2013-05-01

    Full Text Available Objective(s: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL can selectively induce apoptosis in tumor cells, more than half of tumors including non-small cell lung cancer (NSCLC exhibit TRAIL-resistance. The purpose of this study was to determine whether subtoxic-dose cisplatin and TRAIL could synergistically enhance apoptosis on NSCLC cells and investigate its underlying mechanisms. Materials and Methods:NCI-H460 and A549 cells were treated with TRAIL alone, cisplatin alone or combination treatment in this study. The cytotoxicity was evaluated according to Sulforhodamine B assay, and apoptosis was examined using Hoechst 33342 staining and flow cytometry. The mRNA and protein levels of TRAIL receptors and apoptotic proteins including caspase-8, caspase-9, Bcl-2 and Bax were determined by RT-PCR and Western blotting, respectively. Results:Our results showed that NCI-H460 cells were sensitive to TRAIL, whereas A549 cells were resistant. However, subtoxic-dose cisplatin could enhance the both cells to TRAIL-mediated cell proliferation inhibition and apoptosis. The underlying mechanisms might be associated with the down-regulation of DcR2 and up-regulation of Caspase-8, Caspase-9 and Bax. Conclusion:Subtoxic-dose cisplatin could enhance both TRAIL- sensitive and TRAIL- resistant NSCLC cells to TRAIL-mediated apoptosis. These findings motivated further studies to evaluate such a combinatory therapeutic strategy against NSCLC in the animal models.

  20. Low-frequency Electronic Transport Noise in La2-xBaxCuO4 Nanowires

    Science.gov (United States)

    Weis, Adam; Xin, Yizhou; van Harlingen, Dale

    2013-03-01

    In the pseudogap regime, high temperature superconductors often exhibit electronic structure, such as charge stripes. Charge stripes pinned to disorder have been predicted to contribute to low-frequency resistance fluctuations when sample dimensions are comparable to the size of stripe domains (Carlson, 2006). We are extending our previous studies of resistance fluctuations in YBa2Cu3O7-δ (Bonetti, 2004; Caplan, 2010) to thin films of La-based cuprates expected to have a more stable stripe phase, particularly in the regime near 1/8-filling. We present measurements of the low-frequency electronic transport in La2-xBaxCuO4 nanowires fabricated by pulsed laser deposition and lithographic techniques. We discuss temperature dependence of the power spectral density and its relevance to correlated electron phases above Tc. This research was supported by the DOE-DMS under grant DE-FG02-07ER46453, through the Frederick Seitz Materials Research Laboratory at the University of Illinois at Urbana-Champaign.

  1. Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

    International Nuclear Information System (INIS)

    Ghezali, Lamia; Leger, David Yannick; Limami, Youness; Cook-Moreau, Jeanne; Beneytout, Jean-Louis; Liagre, Bertrand

    2013-01-01

    Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression

  2. Mechanistic Studies of Oligonucleotide Aptamers With Potent Antiproliferative and Pro-Apoptotic Activity Against Prostate Cancer Cells

    Science.gov (United States)

    2007-05-01

    Quadruplex structures in nucleic acids. Biopolymers , 2000. 56(3): p. 123-46. 16. Mills, M., et al., Unusual DNA conformations: implications for telomeres...scanning autoradiographic films and using UN-SCAN-IT gel software ( Silk Scientific Corporation, UT, USA). Band intensities were normalized as indicated in

  3. Pro-apoptotic and anti-proliferative effects of corn silk extract on human colon cancer cell lines

    OpenAIRE

    Guo, Hao; Guan, Hong; Yang, Wenqin; Liu, Han; Hou, Huiling; Chen, Xue; Liu, Zhenyan; Zang, Chuangang; Liu, Yuchao; Liu, Jicheng

    2016-01-01

    Corn silk is an economically and nutritionally significant natural product as it represents a staple food for a large proportion of the world population. This study investigated the anticancer activity of corn silk extract in human colon cancer cells and human gastric cancer cells. Following treatment with corn silk extract, certain apoptosis-related events were observed, including inhibition of cell proliferation, loss of mitochondrial membrane potential (??m), release of Ca2+ and release of...

  4. Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

    Energy Technology Data Exchange (ETDEWEB)

    Ghezali, Lamia; Leger, David Yannick; Limami, Youness [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Cook-Moreau, Jeanne [Université de Limoges, FR 3503 GEIST, UMR CNRS 7276 “Contrôle de la réponse immune B et lymphoproliférations”, Faculté de Médecine, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Beneytout, Jean-Louis [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Liagre, Bertrand, E-mail: bertrand.liagre@unilim.fr [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France)

    2013-04-15

    Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression.

  5. Mitochondrial targeting of [alpha]-tocopheryl succinate enhances its pro-apoptotic efficacy: A new paradigm for effective cancer therapy

    Czech Academy of Sciences Publication Activity Database

    Dong, L.-F.; Jameson, V.J.A.; Tilly, D.; Procházka, L.; Rohlena, Jakub; Vališ, Karel; Truksa, Jaroslav; Zobalová, Renata; Mandavian, E.; Klučková, Katarína; Stantic, M.; Štursa, Jan; Freeman, R.; Witting, P.K.; Norberg, E.; Goodwin, J.; Salvatore, B.A.; Novotná, Jana; Turánek, J.; Ledvina, Miroslav; Hozák, Pavel; Zhivotovsky, B.; Coster, M.; Ralph, S.J.; Smith, R.A.J.; Neužil, Jiří

    2011-01-01

    Roč. 50, č. 11 (2011), s. 1546-1555 ISSN 0891-5849 R&D Projects: GA ČR GA204/08/0811; GA ČR GAP301/10/1937; GA AV ČR IAA500520702; GA AV ČR KAN200520703; GA AV ČR KJB500970904; GA AV ČR KAN200100801 Institutional research plan: CEZ:AV0Z50520701; CEZ:AV0Z50520514; CEZ:AV0Z4055905 Keywords : Triphenyl phosphonium * reactive oxygen species * apoptosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.423, year: 2011

  6. Human decidual stromal cells secrete soluble pro-apoptotic factors during decidualization in a cAMP-dependent manner.

    Science.gov (United States)

    Leno-Durán, E; Ruiz-Magaña, M J; Muñoz-Fernández, R; Requena, F; Olivares, E G; Ruiz-Ruiz, C

    2014-10-10

    Is there a relationship between decidualization and apoptosis of decidual stromal cells (DSC)? Decidualization triggers the secretion of soluble factors that induce apoptosis in DSC. The differentiation and apoptosis of DSC during decidualization of the receptive decidua are crucial processes for the controlled invasion of trophoblasts in normal pregnancy. Most DSC regress in a time-dependent manner, and their removal is important to provide space for the embryo to grow. However, the mechanism that controls DSC death is poorly understood. The apoptotic response of DSC was analyzed after exposure to different exogenous agents and during decidualization. The apoptotic potential of decidualized DSC supernatants and prolactin (PRL) was also evaluated. DSC lines were established from samples of decidua from first trimester pregnancies. Apoptosis was assayed by flow cytometry. PRL production, as a marker of decidualization, was determined by enzyme-linked immunosorbent assay. DSCs were resistant to a variety of apoptosis-inducing substances. Nevertheless, DSC underwent apoptosis during decidualization in culture, with cAMP being essential for both apoptosis and differentiation. In addition, culture supernatants from decidualized DSC induced apoptosis in undifferentiated DSC, although paradoxically these supernatants decreased the spontaneous apoptosis of decidual lymphocytes. Exogenously added PRL did not induce apoptosis in DSC and an antibody that neutralized the PRL receptor did not decrease the apoptosis induced by supernatants. Further studies are needed to examine the involvement of other soluble factors secreted by decidualized DSC in the induction of apoptosis. The present results indicate that apoptosis of DSC occurs in parallel to differentiation, in response to decidualization signals, with soluble factors secreted by decidualized DSC being responsible for triggering cell death. These studies are relevant in the understanding of how the regression of decidua, a crucial process for successful pregnancy, takes place. This work was supported by the Consejería de Economía, Innovación y Ciencia, Junta de Andalucía (Grant CTS-6183, Proyectos de Investigación de Excelencia 2010 to C.R.-R.) and the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain (Grants PS09/00339 and PI12/01085 to E.G.O.). E.L.-D. was supported by fellowships from the Ministerio de Educación y Ciencia, Spain and the University of Granada. The authors have no conflict of interest. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Mitochondrial targeting of vitamin E succinate enhances its pro-apoptotic and anti-cancer activity via mitochondrial complex

    Czech Academy of Sciences Publication Activity Database

    Dong, L.F.; Jameson, V.J.A.; Tilly, D.; Černý, Jiří; Mahdavian, E.; Marin-Hernandez, A.; Hernandez-Esquivel, L.; Rodriguez-Enriquez, S.; Štursa, Jan; Witting, P.K.; Stantic, B.; Rohlena, Jakub; Truksa, Jaroslav; Klučková, Katarína; Dyason, J.C.; Ledvina, Miroslav; Salvatore, B.A.; Moreno-Sanchez, R.; Coster, M.; Ralph, S.J.; Smith, A.J.; Neužil, Jiří

    2011-01-01

    Roč. 286, č. 5 (2011), s. 3717-3728 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA204/08/0811; GA ČR(CZ) GAP301/10/1937; GA AV ČR(CZ) IAA500520702; GA AV ČR(CZ) KAN200520703; GA AV ČR(CZ) KJB500970904 Institutional research plan: CEZ:AV0Z50520701; CEZ:AV0Z4055905 Keywords : Apoptosis induction * proximal ubiquinone-binding site of mitochondrial complex II * reactive oxygen species Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.773, year: 2011

  8. A Novel Membrane-Permeable Breast-Targeting Pro-Apoptotic Peptide for Treatment of Breast Cancer

    National Research Council Canada - National Science Library

    Guo, Bin

    2005-01-01

    .... The BH3 peptide of Bid (EDIIRNIARHLAQVGDSMDR) was synthesized with eight d-arginine residues at the N-terminus with a glycine linker residue, followed by a breast-homing sequence (CPGPEGAGC) at the C-terminal...

  9. Metallic gold reduces TNFalpha expression, oxidative DNA damage and pro-apoptotic signals after experimental brain injury

    DEFF Research Database (Denmark)

    Pedersen, Mie Ostergaard; Larsen, Agnete; Pedersen, Dan Sonne

    2009-01-01

    Brain injury represents a major health problem and may result in chronic inflammation and neurodegeneration. Due to antiinflammatory effects of gold, we have investigated the cerebral effects of metallic gold particles following a focal brain injury (freeze-lesion) in mice. Gold particles 20......-45 microm in size or the vehicle (placebo) were implanted in the cortical tissue followed by a cortical freeze-lesioning. At 1-2 weeks post-injury, brains were analyzed by using immunohistochemistry and markers of inflammation, oxidative stress and apoptosis. This study shows that gold treatment...

  10. Clearance of a persistent Picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses

    Science.gov (United States)

    Immunomodulatory mechanisms associated with clearance versus persistence of foot-and-mouth disease virus (FMDV) in distinct microanatomic compartments of the bovine nasopharynx were investigated using quantitative RT-PCR and whole transcriptome microarray. Analysis of tissue samples obtained during ...

  11. Pro-apoptotic activity of new analog of anthracyclines--WP 631 in advanced ovarian cancer cell line.

    Science.gov (United States)

    Gajek, Arkadiusz; Denel, Marta; Bukowska, Barbara; Rogalska, Aneta; Marczak, Agnieszka

    2014-03-01

    In this work we investigated the mode of cell death induced by WP 631, a novel anthracycline antibiotic, in the ovarian cancer cell line (OV-90) derived from the malignant ascites of a patient diagnosed with advanced disease. The effects were compared with those of doxorubicin (DOX), a first generation anthracycline. The ability of WP 631 to induce apoptosis and necrosis was examined by double staining with Annexin V and propidium iodide, measurements of the level of intracellular calcium ions and cytochrome c, PARP cleavage. We also investigated the possible involvement of the caspases activation, DNA degradation (comet assay) and intracellular reactive oxygen species (ROS) production in the development of the apoptotic events and their significance for drug efficiency. The results obtained clearly demonstrate that antiproliferative capacity of WP 631 in tested cell line was a few times greater than that of DOX. Furthermore, ovarian cancer cells treated with WP 631 showed a higher mean level of basal DNA damage in comparison to DOX. In conclusion, WP 631 is able to induce caspase - dependent apoptosis in human ovarian cancer cells. Obtained results suggested that WP 631 may be a candidate for further evaluation as chemotherapeutic agents for human cancers. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Targeting pro-apoptotic trail receptors sensitizes HeLa cervical cancer cells to irradiation-induced apoptosis

    NARCIS (Netherlands)

    Maduro, John H.; de Vries, Elisabeth G. E.; Meersma, Gert-Jan; Hougardy, Brigitte M. T.; van der Zee, Ate G. J.; De Jong, Steven

    2008-01-01

    Purpose: To investigate the potential of irradiation in combination with drugs targeting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor (DR)4 and DR5 and their mechanism of action in a cervical cancer cell line. Methods and Materials: Recombinant human TRAIL

  13. Pro-oxidative and pro-apoptotic action of defatted seeds of Oenothera paradoxa on human skin melanoma cells.

    Science.gov (United States)

    Jaszewska, Edyta; Kośmider, Anita; Kiss, Anna K; Naruszewicz, Marek

    2009-09-23

    Three extracts of defatted seeds of Oenothera paradoxa Hudziok, aqueous extract, 60% ethanolic extract, and 30% isopropanolic extract, differing by their total content of phenolic compounds and by their contents of individual polyphenols, were investigated in this study. The extracts exerted cytotoxic action on HTB-140 human skin melanoma cells. After 24 h of incubation, IC(50) values of 169.7 +/- 5.9 micog/mL, 72.4 +/- 3.8 microg/mL, and 155.3 +/- 6.3 microg/mL were obtained for HTB-140 cells with the aqueous extract, 60% ethanolic extract, and 30% isopropanolic extract at the tested concentrations (5-200 microg/mL), respectively, while IC(50) for normal fibroblast cells NHDFs was not attained. Moreover, for HTB-140 cells, LD(50) (concentration at which 50% of cells were dead) of 89.2 +/- 4.3 microg/mL and 181.4 +/- 6.5 microg/mL were obtained with 60% ethanolic extract and 30% isopropanolic extract, respectively. In melanoma cells, all three extracts caused a concentration-dependent increase of ROS production, GSH, and ATP lowering, and appearance of phosphatidylserine on the external surface of cellular membranes where it was bound to annexin V-FITC; furthermore, apoptosis without activation of caspase-3 took place. The most effective was 60% ethanolic extract, which had the greatest total content of phenolic compounds and the greatest content of pentagalloyloglucose (PGG).

  14. Acetylation of the pro-apoptotic factor, p53 in the hippocampus following cerebral ischemia and modulation by estrogen.

    Directory of Open Access Journals (Sweden)

    Limor Raz

    Full Text Available Recent studies demonstrate that acetylation of the transcription factor, p53 on lysine(373 leads to its enhanced stabilization/activity and increased susceptibility of cells to stress. However, it is not known whether acetylation of p53 is altered in the hippocampus following global cerebral ischemia (GCI or is regulated by the hormone, 17β-estradiol (17β-E(2, and thus, this study examined these issues.The study revealed that Acetyl p53-Lysine(373 levels were markedly increased in the hippocampal CA1 region after GCI at 3 h, 6 h and 24 h after reperfusion, an effect strongly attenuated by 17β-E(2. 17β-E(2 also enhanced interaction of p53 with the ubiquitin ligase, Mdm2, increased ubiquitination of p53, and induced its down-regulation, as well as attenuated elevation of the p53 transcriptional target, Puma. We also observed enhanced acetylation of p53 at a different lysine (Lys(382 at 3 h after reperfusion, and 17β-E(2 also markedly attenuated this effect. Furthermore, administration of an inhibitor of CBP/p300 acetyltransferase, which acetylates p53, was strongly neuroprotective of the CA1 region following GCI. In long-term estrogen deprived (LTED animals, the ability of 17β-E(2 to attenuate p53 acetylation was lost, and intriguingly, Acetyl p53-Lysine(373 levels were markedly elevated in sham (non-ischemic LTED animals. Finally, intracerebroventricular injections of Gp91ds-Tat, a specific NADPH oxidase (NOX2 inhibitor, but not the scrambled tat peptide control (Sc-Tat, attenuated acetylation of p53 and reduced levels of Puma following GCI.The studies demonstrate that p53 undergoes enhanced acetylation in the hippocampal CA1 region following global cerebral ischemia, and that the neuroprotective agent, 17β-E(2, markedly attenuates the ischemia-induced p53 acetylation. Furthermore, following LTED, the suppressive effect of 17β-E(2 on p53 acetylation is lost, and p53 acetylation increases in the hippocampus, which may explain previous reports of increased sensitivity of the hippocampus to ischemic stress following LTED.

  15. Mutations in TGFbeta-RII and BAX mediate tumor progression in the later stages of colorectal cancer with microsatellite instability

    International Nuclear Information System (INIS)

    Yashiro, Masakazu; Hirakawa, Kosei; Boland, C Richard

    2010-01-01

    Microsatellite instability (MSI) occurs in 15% of colorectal cancers (CRC). The genetic targets for mutation in the MSI phenotype include somatic mutations in the transforming growth factor beta receptor typeII (TGFbetaRII), BAX, hMSH3 and hMSH6. It is not clear how mutations of these genes mediate tumor progression in the MSI pathway, and the temporal sequence of these mutations remains uncertain. In this study, early stage CRCs were examined for frameshift mutations in these target genes, and compared with late stage tumors and CRC cell lines. We investigated 6 CRC cell lines and 71 sporadic CRCs, including 61 early stage cancers and 10 late stage cancers. Mutations of repetitive mononucleotide tracts in the coding regions of TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR and Fas antigen were identified by direct sequencing. Thirteen (18.3%) of 71 CRC, including 9/61 (14.7%) early stage cancers and 4/10 (40%) late stage cancers, were identified as MSI and analyzed for frameshift mutations. No mutation in the target genes was observed in any of the 9 early stage MSI CRCs. In contrast, frameshift mutations of TGFbetaRII, BAX, hMSH3 and hMSH6 were present in 3/4 late stage MSI tumors. There is a statistical association (p = 0.014) between mutation in any one gene and tumor stage. TGFbetaRII, BAX, hMSH3 and hMSH6 mutations are relatively late events in the genesis of MSI CRCs. The frameshift mutations in these target genes might mediate progression from early to late stage cancer, rather than mediating the adenoma to carcinoma transition

  16. The Neuron-Specific Protein TMEM59L Mediates Oxidative Stress-Induced Cell Death.

    Science.gov (United States)

    Zheng, Qiuyang; Zheng, Xiaoyuan; Zhang, Lishan; Luo, Hong; Qian, Lingzhi; Fu, Xing; Liu, Yiqian; Gao, Yuehong; Niu, Mengxi; Meng, Jian; Zhang, Muxian; Bu, Guojun; Xu, Huaxi; Zhang, Yun-Wu

    2017-08-01

    TMEM59L is a newly identified brain-specific membrane-anchored protein with unknown functions. Herein we found that both TMEM59L and its homolog, TMEM59, are localized in Golgi and endosomes. However, in contrast to a ubiquitous and relatively stable temporal expression of TMEM59, TMEM59L expression was limited in neurons and increased during development. We also found that both TMEM59L and TMEM59 interacted with ATG5 and ATG16L1, and that overexpression of them triggered cell autophagy. However, overexpression of TMEM59L induced intrinsic caspase-dependent apoptosis more dramatically than TMEM59. In addition, downregulation of TMEM59L prevented neuronal cell death and caspase-3 activation caused by hydrogen peroxide insults and reduced the lipidation of LC3B. Finally, we found that AAV-mediated knockdown of TMEM59L in mice significantly ameliorated caspase-3 activation, increased mouse duration in the open arm during elevated plus maze test, reduced mouse immobility time during forced swim test, and enhanced mouse memory during Y-maze and Morris water maze tests. Together, our study indicates that TMEM59L is a pro-apoptotic neuronal protein involved in animal behaviors such as anxiety, depression, and memory, and that TMEM59L downregulation protects neurons against oxidative stress.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Ibuprofen abates cypermethrin-induced expression of pro-inflammatory mediators and mitogen-activated protein kinases and averts the nigrostriatal dopaminergic neurodegeneration.

    Science.gov (United States)

    Singh, Ashish; Tripathi, Pratibha; Prakash, Om; Singh, Mahendra Pratap

    2016-12-01

    Cypermethrin induces oxidative stress, microglial activation, inflammation and apoptosis leading to Parkinsonism in rats. While ibuprofen, a non-steroidal anti-inflammatory drug, relieves from inflammation, its efficacy against cypermethrin-induced Parkinsonism has not yet been investigated. The study aimed to explore the protective role of ibuprofen in cypermethrin-induced Parkinsonism, an environmentally relevant model of Parkinson's disease (PD), along with its underlying mechanism. Animals were treated with/without cypermethrin in the presence/absence of ibuprofen. Behavioural, immunohistochemical and biochemical parameters of Parkinsonism and expression of pro-inflammatory and pro-apoptotic proteins along with mitogen-activated protein kinases (MAPKs) were determined. Ibuprofen resisted cypermethrin-induced behavioural impairments, striatal dopamine depletion, oxidative stress in the nigrostriatal tissues and loss of the nigral dopamine producing cells and increase in microglial activation along with atypical expression of pro-inflammatory and apoptotic proteins that include cyclooxygenase-2, tumour necrosis factor-α, MAPKs (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase), B cell lymphoma 2-associated protein X, tumour suppressor protein p53, cytochrome c and caspase-3 in the nigrostriatal tissue. The results obtained thus demonstrate that ibuprofen lessens inflammation and regulates MAPKs expression thereby averts cypermethrin-induced Parkinsonism.

  19. Distribution of 99Tcm-rh-Annexin vin tumor and expression relationship of bcl-2, bax after a single dose of chemotherapy

    International Nuclear Information System (INIS)

    Zhang Xin; Li Yaming; Zhang Yanjun; Tao Li; Zhu Yi; Yang Chun; Ji Xiaopeng; Zhao Ming; Tian Aijuan; Zhang Jianying; Zhao Zhenzhen

    2007-01-01

    The expression of bcl-2 and bax after the single dose of chemotherapy with 99 Tc m -rh-Annexin V as the tracer of tumor apoptosis imaging is studied. tumor cell apoptosis is examined by TUNEL methods, and the expression of bcl-2 and bax in tumor are determined by immunohistochemical methods. Single dose of chemotherapy significantly increased the tumor uptake of 99 Tc m -rh-annexin V and the positive number of TUNEL, as well as the expression of bax (P 99 Tc m -rh-annexin V in tumor reflectes not only the degree of apoptosis of tumor cells, but also the change of bax expression after the single dose of chemotherapy. (authors)

  20. Intrinsic and extrinsic apoptotic pathways are involved in rat testis by cold water immersion-induced acute and chronic stress.

    Science.gov (United States)

    Juárez-Rojas, Adriana Lizbeth; García-Lorenzana, Mario; Aragón-Martínez, Andrés; Gómez-Quiroz, Luis Enrique; Retana-Márquez, María del Socorro

    2015-01-01

    Testicular apoptosis is activated by stress, but it is not clear which signaling pathway is activated in response to stress. The aim of this study was to investigate whether intrinsic, extrinsic, or both apoptotic signaling pathways are activated by acute and chronic stress. Adult male rats were subjected to cold water immersion-induced stress for 1, 20, 40, and 50 consecutive days. The seminiferous tubules:apoptotic cell ratio was assayed on acute (1 day) and chronic (20, 40, 50 days) stress. Apoptotic markers, including cleaved-caspase 3 and 8, the pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were also determined after acute and chronic stress induction. Additionally, epididymal sperm quality was evaluated, as well as corticosterone and testosterone levels. An increase in tubule apoptotic cell count percentage after an hour of acute stress and during chronic stress induction was observed. The apoptotic cells rate per tubule increment was only detected one hour after acute stress, but not with chronic stress. Accordingly, there was an increase in Bax, cleaved caspase-8 and caspase-3 pro-apoptotic proteins with a decrease of anti-apoptotic Bcl-2 in both acutely and chronically stressed male testes. In addition, sperm count, viability, as well as total and progressive motility were low in chronically stressed males. Finally, the levels of corticosterone increased whereas testosterone levels decreased in chronically stressed males. Activation of the extrinsic apoptotic pathway was shown by cleaved caspase-8 increase whereas the intrinsic apoptotic pathway activation was determined by the increase of Bax, along with Bcl-2 decrease, making evident a cross-talk between these two pathways with the activation of caspase-3. These results suggest that both acute and chronic stress can potentially activate the intrinsic/extrinsic apoptosis pathways in testes. Chronic stress also reduces the quality of epididymal spermatozoa, possibly due to a decrease in testosterone.

  1. Kaempferol increases apoptosis in human acute promyelocytic leukemia cells and inhibits multidrug resistance genes.

    Science.gov (United States)

    Moradzadeh, Maliheh; Tabarraei, Alijan; Sadeghnia, Hamid Reza; Ghorbani, Ahmad; Mohamadkhani, Ashraf; Erfanian, Saiedeh; Sahebkar, Amirhossein

    2018-02-01

    Acute promyelocytic leukemia (APL) is one of the most life-threatening hematological malignancies. Defects in the cell growth and apoptotic pathways are responsible for both disease pathogenesis and treatment resistance. Therefore, pro-apoptotic agents are potential candidates for APL treatment. Kaempferol is a flavonoid with antioxidant and anti-tumor properties. This study was designed to investigate the cytotoxic, pro-apoptotic, and differentiation-inducing effects of kaempferol on HL-60 and NB4 leukemia cells. Resazurin assay was used to determine cell viability following treatment with kaempferol (12.5-100 μM) and all-trans retinoic acid (ATRA; 10 μM; used as a positive control). Apoptosis and differentiation were also detected using propidium iodide and NBT staining techniques, respectively. Furthermore, the expression levels of genes involved in apoptosis (PI3 K, AKT, BCL2, BAX, p53, p21, PTEN, CASP3, CASP8, and CASP9), differentiation (PML-RAR and HDAC1), and multi-drug resistance (ABCB1 and ABCC1) were determined using quantitative real-time PCR. The protein expressions of Bax/Bcl2 and casp3 were confirmed using Western blot. The results showed that kaempferol decreased cell viability and increased subG1 population in the tested leukemic cells. This effect was associated with decreased expression of Akt, BCL2, ABCB1, and ABCC1 genes, while the expression of CASP3 and BAX/BCL-2 ratio were significantly increased at both gene and protein levels. Kaempferol promoted apoptosis and inhibited multidrug resistance in a concentration-dependent manner, without any differential effect on leukemic cells. In conclusion, this study suggested that kaempferol may be utilized as an appropriate alternative for ATRA in APL patients. © 2017 Wiley Periodicals, Inc.

  2. Maternal exposure to titanium dioxide nanoparticles during pregnancy and lactation alters offspring hippocampal mRNA BAX and Bcl-2 levels, induces apoptosis and decreases neurogenesis.

    Science.gov (United States)

    Ebrahimzadeh Bideskan, Alireza; Mohammadipour, Abbas; Fazel, Alireza; Haghir, Hossein; Rafatpanah, Houshang; Hosseini, Mahmoud; Rajabzadeh, Aliakbar

    2017-07-05

    The usage of Titanium dioxide nanoparticles (TiO 2 -NPs) covers a vast area in different fields ranging from cosmetics and food to the production of drugs. Maternal exposure to TiO 2 -NPs during developmental period has been associated with hippocampal injury and with a decrease in learning and memory status of the offspring. However, little is known about its injury mechanism. This paper describes the in vivo neurotoxic effects of TiO 2 -NPs on rat offspring hippocampus during developmental period. Pregnant and lactating Wistar rats received intragastric TiO 2 -NPs (100mg/kg body weight) daily from gestational day (GD) 2 to (GD) 21 and postnatal day (PD) 2 to (PD) 21 respectively. Animals in the control groups received an equal volume of distilled water via gavage. At the end of the treatment process, offspring were deeply anesthetized and sacrificed. Then brains of each group were collected and sections of the rat offspring's brains were stained using TUNEL staining (for detection of apoptotic cells) and immunostaining (for neurogenesis). Moreover, the right hippocampus (n=6 per each group) were removed from the right hemisphere for evaluating the expression of Bax and Bcl-2 level. Results of histopatological examination by TUNEL staining showed that maternal exposure to TiO 2 -NPs during pregnancy and lactation periods increased apoptotic cells significantly (P<0.01) in the offspring hippocampus. The immunolabeling of double cortin (DCX) protein as neurogenesis marker also showed that TiO 2 -NPs reduced neurogenesis in the hippocampus of the offspring (P<0.05). Moreover, in comparison with the control group, the mRNA levels of Bax and Bcl-2 in the TiO 2 -NPs group significantly increased and decreased, respectively (P<0.01). These findings provide strong evidence that maternal exposure to TiO 2 -NPs significantly impact hippocampal neurogenesis and apoptosis in the offspring. The potential impact of nanoparticle exposure for millions of pregnant mothers and

  3. Involvement of Bax and Bcl-2 in Induction of Apoptosis by Essential Oils of Three Lebanese Salvia Species in Human Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Alessandra Russo

    2018-01-01

    Full Text Available Prostate cancer is one of the most common forms of cancer in men, and research to find more effective and less toxic drugs has become necessary. In the frame of our ongoing program on traditionally used Salvia species from the Mediterranean Area, here we report the biological activities of Salvia aurea, S. judaica and S. viscosa essential oils against human prostate cancer cells (DU-145. The cell viability was measured by 3(4,5-dimethyl-thiazol-2-yl2,5-diphenyl-tetrazolium bromide (MTT test and lactate dehydrogenase (LDH release was used to quantify necrosis cell death. Genomic DNA, caspase-3 activity, expression of cleaved caspase-9, B-cell lymphoma 2 (Bcl-2 and Bcl-2 associated X (Bax proteins were analyzed in order to study the apoptotic process. The role of reactive oxygen species in cell death was also investigated. We found that the three essential oils, containing caryophyllene oxide as a main constituent, are capable of reducing the growth of human prostate cancer cells, activating an apoptotic process and increasing reactive oxygen species generation. These results suggest it could be profitable to further investigate the effects of these essential oils for their possible use as anticancer agents in prostate cancer, alone or in combination with chemotherapy agents.

  4. Axin1 up-regulated 1 accelerates stress-induced cardiomyocytes apoptosis through activating Wnt/β-catenin signaling.

    Science.gov (United States)

    Ye, Xing; Lin, Junyi; Lin, Zebin; Xue, Aimin; Li, Liliang; Zhao, Ziqin; Liu, Li; Shen, Yiwen; Cong, Bin

    2017-10-15

    Stress-induced cardiomyocyte apoptosis contributes to the pathogenesis of a variety of cardiovascular diseases, but how stress induces cardiomyocyte apoptosis remains largely unclear. The present study aims to investigate the effects of Axin1 up-regulated 1 (Axud1), a novel pro-apoptotic protein, on the cardiomyocyte survival and the underlying mechanisms. To this end, a rat model under restraint stress (RS) was established and in vitro stress-induced cardiomyocytes culture was achieved. Our data showed that Axud1 was upregulated in the rat myocardia after exposure to RS. Anti-apoptotic Bcl-2 was decreased, whereas pro-apoptotic Bax and Cleaved caspase-3 (Cc3) were increased in a time-dependent manner. The Wnt/β-catenin signaling was observed to be interestingly activated in heart undergoing RS. In addition, the treatment of norepinephrine (NE) to in vitro cardiomyocytes increased Axud1 level and induced cell apoptosis. Wnt/β-catenin signaling was consistently activated. Knockdown of Axud1 using specific siRNA blunted NE-induced cardiomyocytes apoptosis and also inactivated the Wnt/β-catenin signaling. XAV-939, an inhibitor of Wnt/β-catenin signaling, partially reversed the pro-apoptotic effect of NE. In conclusion, Axud1 accelerated stress-induced cardiomyocytes apoptosis through activation of Wnt/β-catenin signaling pathway. Our data provided novel evidence that therapeutic strategies against Axud1 or Wnt/β-catenin signaling might be promising in relation to RS-induced myocardial injury. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Bax/Tubulin/Epithelial-Mesenchymal Pathways Determine the Efficacy of Silybin Analog HM015k in Colorectal Cancer Cell Growth and Metastasis

    Directory of Open Access Journals (Sweden)

    Haneen Amawi

    2018-05-01

    Full Text Available The inhibition of apoptosis, disruption of cellular microtubule dynamics, and over-activation of the epithelial mesenchymal transition (EMT, are involved in the progression, metastasis, and resistance of colorectal cancer (CRC to chemotherapy. Therefore, the design of a molecule that can target these pathways could be an effective strategy to reverse CRC progression and metastasis. In this study, twelve novel silybin derivatives, HM015a-HM015k (15a−15k and compound 17, were screened for cytotoxicity in CRC cell lines. Compounds HM015j and HM015k (15k and 15j significantly decreased cell proliferation, inhibited colony formation, and produced cell cycle arrest in CRC cells. Furthermore, 15k significantly induced the formation of reactive oxygen species and apoptosis. It induced the cleavage of the intrinsic apoptotic protein (Bax p21 to its more efficacious fragment, p18. Compound 15k also inhibited tubulin expression and disrupted its structure. Compound 15k significantly decreased metastatic LOVO cell migration and invasion. Furthermore, 15k reversed mesenchymal morphology in HCT116 and LOVO cells. Additionally, 15k significantly inhibited the expression of the mesenchymal marker N-cadherin and upregulated the expression of the epithelial marker, E-cadherin. Compound 15k inhibited the expression of key proteins known to induce EMT (i.e., DVL3, β-catenin, c-Myc and upregulated the anti-metastatic protein, cyclin B1. Overall, in vitro, 15k significantly inhibited CRC progression and metastasis by inhibiting apoptosis, tubulin activity and the EMT pathways. Overall, these data suggest that compound 15k should be tested in vivo in a CRC animal model for further development.

  6. Small kinetochore associated protein (SKAP promotes UV-induced cell apoptosis through negatively regulating pre-mRNA processing factor 19 (Prp19.

    Directory of Open Access Journals (Sweden)

    Shan Lu

    Full Text Available Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19. We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.

  7. 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.

  8. Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes.

    Science.gov (United States)

    Cheng, Tsing; Orlow, Seth J; Manga, Prashiela

    2013-11-01

    Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Does melatonin influence the apoptosis in rat uterus of animals exposed to continuous light?

    Science.gov (United States)

    Ferreira, Cecília S; Carvalho, Kátia C; Maganhin, Carla C; Paiotti, Ana P R; Oshima, Celina T F; Simões, Manuel J; Baracat, Edmund C; Soares, José M

    2016-02-01

    Melatonin has been described as a protective agent against cell death and oxidative stress in different tissues, including in the reproductive system. However, the information on the action of this hormone in rat uterine apoptosis is low. Our objective was to evaluate the effects of melatonin on mechanisms of cell death in uterus of rats exposed to continuous light stress. Twenty adult Wistar rats were divided into two groups: GContr (vehicle control) and GExp which were treated with melatonin (0.4 mg/mL), both were exposed to continuous light for 90 days. The uterus was removed and processed for quantitative real time PCR (qRT-PCR), using PCR-array plates of the apoptosis pathway; for immunohistochemistry and TUNEL. The results of qRT-PCR of GEXP group showed up-regulation of 13 and 7, pro-apoptotic and anti-apoptotic genes, respectively, compared to GContr group. No difference in pro-apoptotic proteins (Bax, Fas and Faslg) expression was observed by immunohistochemistry, although the number of TUNEL-positive cells was lower in the group treated with melatonin compared to the group not treated with this hormone. Our data suggest that melatonin influences the mechanism and decreases the apoptosis in uterus of rats exposed to continuous light.

  10. Biochemical responses and mitochondrial mediated activation of apoptosis on long-term effect of aspartame in rat brain

    Directory of Open Access Journals (Sweden)

    Iyaswamy Ashok

    2014-01-01

    Full Text Available Aspartame, an artificial sweetener, is very widely used in many foods and beverages. But there are controversies about its metabolite which is marked for its toxicity. Hence it is believed to be unsafe for human use. Previous studies have reported on methanol exposure with involvements of free radicals on excitotoxicity of neuronal apoptosis. Hence, this present study is proposed to investigate whether or not chronic aspartame (FDA approved Daily Acceptable Intake (ADI,40 mg/kg bwt administration could release methanol, and whether or not it can induce changes in brain oxidative stress status and gene and protein expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax and caspase-3 in the rat brain region. To mimic the human methanol metabolism, Methotrexate (MTX-treated Wistar strain male albino rats were used and after the oral administration of aspartame, the effects were studied along with controls and MTX-treated controls. Aspartame exposure resulted with a significant increase in the enzymatic activity in protein carbonyl, lipid peroxidation levels, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase and catalase activity in (aspartame MTX-treated animals and with a significant decrease in reduced glutathione, glutathione reductase and protein thiol, pointing out the generation of free radicals. The gene and protein expression of pro apoptotic marker Bax showed a marked increase whereas the anti-apoptotic marker Bcl-2 decreased markedly indicating the aspartame is harmful at cellular level. It is clear that long term aspartame exposure could alter the brain antioxidant status, and can induce apoptotic changes in brain.

  11. Biochemical responses and mitochondrial mediated activation of apoptosis on long-term effect of aspartame in rat brain.

    Science.gov (United States)

    Ashok, Iyaswamy; Sheeladevi, Rathinasamy

    2014-01-01

    Aspartame, an artificial sweetener, is very widely used in many foods and beverages. But there are controversies about its metabolite which is marked for its toxicity. Hence it is believed to be unsafe for human use. Previous studies have reported on methanol exposure with involvements of free radicals on excitotoxicity of neuronal apoptosis. Hence, this present study is proposed to investigate whether or not chronic aspartame (FDA approved Daily Acceptable Intake (ADI),40 mg/kg bwt) administration could release methanol, and whether or not it can induce changes in brain oxidative stress status and gene and protein expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax and caspase-3 in the rat brain region. To mimic the human methanol metabolism, Methotrexate (MTX)-treated Wistar strain male albino rats were used and after the oral administration of aspartame, the effects were studied along with controls and MTX-treated controls. Aspartame exposure resulted with a significant increase in the enzymatic activity in protein carbonyl, lipid peroxidation levels, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase and catalase activity in (aspartame MTX)-treated animals and with a significant decrease in reduced glutathione, glutathione reductase and protein thiol, pointing out the generation of free radicals. The gene and protein expression of pro apoptotic marker Bax showed a marked increase whereas the anti-apoptotic marker Bcl-2 decreased markedly indicating the aspartame is harmful at cellular level. It is clear that long term aspartame exposure could alter the brain antioxidant status, and can induce apoptotic changes in brain.

  12. IGF-1 protects against Aβ25-35-induced neuronal cell death via inhibition of PUMA expression and Bax activation.

    Science.gov (United States)

    Hou, Xunyao; Jin, Yan; Chen, Jian; Hong, Yan; Luo, Dingzhen; Yin, Qingqing; Liu, Xueping

    2017-01-10

    Amyloid-β-peptide (Aβ) is considered to be the toxic species in AD and causes cell death in the affected areas of patient's brain. Insulin-like growth factor 1 (IGF-1) has been reported to attenuate Aβ toxicity in neuronal cells. However, the molecular mechanisms involved in the neuroprotective function of IGF-1 remain largely unknown. In the present study, we for the first time demonstrated that IGF-1 protects against Aβ-induced neurotoxicity via inhibition of PUMA expression and Bax activation. We found that IGF-1 could activate Akt, which in turn inhibited Aβ-induced FOXO3a nuclear translocation and thus decreased the binding ability of FOXO3a to PUMA promoter, leading to decreased PUMA expression. In addition, IGF-1 inhibited the translocation of Bax to the mitochondria induced by Aβ. Notably, addition of wortmannin, a specific inhibitor of PI3K, significantly abolished the neuroprotective effect of IGF-1, suggesting that IGF-1 exerts its anti-apoptotic effect depend on PI3K activity. Our findings may provide new insights into molecular mechanisms mediated by IGF-1 in cell survival against Aβ-induced apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin

    DEFF Research Database (Denmark)

    Liang, Y G; Jorgensen, A G; Kaestel, C G

    2000-01-01

    PURPOSE. The aim of this study was to determine the role of Bcl-2, Bcl-X L, Bax, and c-Fos in regulation of apoptosis, induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells grown on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes....... METHODS. Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl......-X L, Bax, and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. RESULTS. Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Human fetal RPE cells grown on ECM-coated dishes were significantly more resistant to UV...

  14. Ellagic acid protects against neuron damage in ischemic stroke through regulating the ratio of Bcl-2/Bax expression.

    Science.gov (United States)

    Liu, Qing-Shan; Deng, Ran; Li, Shuran; Li, Xu; Li, Keqin; Kebaituli, Gulibanumu; Li, Xueli; Liu, Rui

    2017-08-01

    An oxygen-glucose deprivation and reoxygenation model in primary cultured rat cortical neurons was developed for this study to investigate the effects of ellagic acid (EA), a low-molecular-weight polyphenol, on neuron cells and their function, and to evaluate whether EA can be safely utilized by humans as a functional food or therapeutic agent. Administration of EA significantly decreased the volume of cerebrum infarction and the neurological deficit scores of the rats; EA treatment also increased the number of Bcl-2-positive cells and the ratio of Bcl-2-positive to Bax-positive neurons in the semidarkness zone near the brain ischemic focus in the photothrombotic cerebral ischemia model. Treatment of EA resulted in increased neuron viability, cell nuclear integrity, and the ratio of Bcl-2/Bax expression in the primary cultured neuron model; EA treatment also lead to a decrease in the number of apoptotic cells. Our results therefore suggest a specific mechanism for the beneficial effects of EA, providing new insights into how it provides neuroprotection. To the best of our knowledge, these results represent new insights on the mechanisms of the brain cell protective activity of EA. Thus, EA may be used in functional foods or medicines to help treat nerve dysfunction, neurodegenerative disease, and aging.

  15. 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.

  16. SiteBinder: an improved approach for comparing multiple protein structural motifs.

    Science.gov (United States)

    Sehnal, David; Vařeková, Radka Svobodová; Huber, Heinrich J; Geidl, Stanislav; Ionescu, Crina-Maria; Wimmerová, Michaela; Koča, Jaroslav

    2012-02-27

    There is a paramount need to develop new techniques and tools that will extract as much information as possible from the ever growing repository of protein 3D structures. We report here on the development of a software tool for the multiple superimposition of large sets of protein structural motifs. Our superimposition methodology performs a systematic search for the atom pairing that provides the best fit. During this search, the RMSD values for all chemically relevant pairings are calculated by quaternion algebra. The number of evaluated pairings is markedly decreased by using PDB annotations for atoms. This approach guarantees that the best fit will be found and can be applied even when sequence similarity is low or does not exist at all. We have implemented this methodology in the Web application SiteBinder, which is able to process up to thousands of protein structural motifs in a very short time, and which provides an intuitive and user-friendly interface. Our benchmarking analysis has shown the robustness, efficiency, and versatility of our methodology and its implementation by the successful superimposition of 1000 experimentally determined structures for each of 32 eukaryotic linear motifs. We also demonstrate the applicability of SiteBinder using three case studies. We first compared the structures of 61 PA-IIL sugar binding sites containing nine different sugars, and we found that the sugar binding sites of PA-IIL and its mutants have a conserved structure despite their binding different sugars. We then superimposed over 300 zinc finger central motifs and revealed that the molecular structure in the vicinity of the Zn atom is highly conserved. Finally, we superimposed 12 BH3 domains from pro-apoptotic proteins. Our findings come to support the hypothesis that there is a structural basis for the functional segregation of BH3-only proteins into activators and enablers.

  17. Tubeimoside-1 induces glioma apoptosis through regulation of Bax/Bcl-2 and the ROS/Cytochrome C/Caspase-3 pathway

    Directory of Open Access Journals (Sweden)

    Jia G

    2015-01-01

    Full Text Available Geng Jia,1,* Qiang Wang,2,* Rong Wang,2,* Danni Deng,2 Lian Xue,2 Naiyuan Shao,1 Yi Zhang,1 Xiwei Xia,1 Feng Zhi,2 Yilin Yang1,2 1Department of Neurosurgery, Third Affiliated Hospital of Soochow University, Jiangsu, People’s Republic of China; 2Modern Medical Research Center, Third Affiliated Hospital of Soochow University, Jiangsu, People’s Republic of China * These authors contributed equally to this workBackground: Tubeimoside-1 (TBMS1 is a natural compound isolated from tubeimoside, which has been widely used as a traditional Chinese herbal medicine. The purpose of the present study is to investigate the anti-tumor effect and the underling mechanism of TBMS1 on glioma cancer cells.Methods: The MTT assay was performed to evaluate the effect of TBMS1 on glioma cell proliferation. The fluorescent microscopy and flow cytometry analysis were performed to evaluate the effect of TBMS1 on glioma cell apoptosis. The Western blot analysis was used to evaluate the protein change.Results: TBMS1 inhibited glioma cancer cell proliferation in a dose- and time-dependent manner. Fluorescent microscopy and flow cytometry analysis demonstrated that TBMS1 induced glioma cell apoptosis in a concentration-dependent manner. Western blotting showed that TBMS1 induced apoptosis by increasing the expression of Bax and downregulating the level of Bcl-2. Furthermore, we found that TBMS1 induced apoptosis by increasing the concentration of reactive oxygen species through the release of Cytochrome C and activation of Caspase-3.Conclusion: These findings indicate that TBMS1 may be developed as a possible therapeutic agent for the management of glioma. Keywords: Tubeimoside-1, glioma, proliferation, apoptosis

  18. The pathways by which mild hypothermia inhibits neuronal apoptosis following ischemia/reperfusion injury

    Directory of Open Access Journals (Sweden)

    Chun Luo

    2015-01-01

    Full Text Available Several studies have demonstrated that mild hypothermia exhibits a neuroprotective role and it can inhibit endothelial cell apoptosis following ischemia/reperfusion injury by decreasing casp-ase-3 expression. It is hypothesized that mild hypothermia exhibits neuroprotective effects on neurons exposed to ischemia/reperfusion condition produced by oxygen-glucose deprivation. Mild hypothermia significantly reduced the number of apoptotic neurons, decreased the expression of pro-apoptotic protein Bax and increased mitochondrial membrane potential, with the peak of anti-apoptotic effect appearing between 6 and 12 hours after the injury. These findings indicate that mild hypothermia inhibits neuronal apoptosis following ischemia/reperfusion injury by protecting the mitochondria and that the effective time window is 6-12 hours after ischemia/reperfusion injury

  19. Essential roles of caspases and their upstream regulators in rotenone-induced apoptosis

    International Nuclear Information System (INIS)

    Lee Jihjong; Huang, M.-S.; Yang, I-C.; Lai, T.-C.; Wang, J.-L.; Pang, V.F.; Hsiao, M.; Kuo, M.Y.P.

    2008-01-01

    In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, and tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity

  20. Species-Dependent Functionality of the Human Cytolytic Fusion Proteins Granzyme B-H22(scFv and H22(scFv-Angiogenin in Macrophages

    Directory of Open Access Journals (Sweden)

    Theo Thepen

    2013-01-01

    Full Text Available Human cytolytic fusion proteins (hCFPs are comprised of a specific cell-surface-binding moiety and an effector molecule of human origin. In contrast to common immunotoxins, including bacterial or plant toxins, they are considered not to be immunogenic. Two examples for human pro-apoptotic effector proteins are the serine protease Granzyme B and the RNase Angiogenin. Pre-clinical testing of functionality in in vitro and in vivo studies is essential for therapeutics. Establishing relevant animal models that have predictive value for therapeutic success is a great challenge in biomedical research. In this study, we investigated the species-dependent cytotoxic activity of two hCFPs prior to their application in a murine inflammation model. We found that in vitro and ex vivo either hCFP was able to kill human cells only, leaving murine cells unaffected. In contrast, no species-dependency was found for the bacterial Pseudomonas exotoxin A based immunotoxin H22(scFv-ETA’. This species-dependent functioning has to be carefully considered when performing pre-clinical studies in animal models.

  1. Anomalous metallic state with strong charge fluctuations in BaxTi8O16 +δ revealed by hard x-ray photoemission spectroscopy

    Science.gov (United States)

    Dash, S.; Kajita, T.; Okawa, M.; Saitoh, T.; Ikenaga, E.; Saini, N. L.; Katsufuji, T.; Mizokawa, T.

    2018-04-01

    We have studied a charge-orbital driven metal-insulator transition (MIT) in hollandite-type BaxTi8O16 +δ by means of hard x-ray photoemission spectroscopy (HAXPES). The Ti 2 p HAXPES indicates strong Ti3 +/Ti4 + charge fluctuation in the metallic phase above the MIT temperature. The metallic phase is characterized by a power-law spectral function near the Fermi level which would be a signature of bad metal with non-Drude polaronic behavior. The power-law spectral shape is associated with the large Seebeck coefficient of the metallic phase in BaxTi8O16 +δ .

  2. 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.

  3. Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

    Science.gov (United States)

    Schoneich, Christian; Dremina, Elena; Galeva, Nadezhda; Sharov, Victor

    2014-01-01

    Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cell but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad. PMID:24129924

  4. BaxSr1-xTi1.02O3 metal-insulator-metal capacitors on planarized alumina substrates

    NARCIS (Netherlands)

    Tiggelman, M.P.J.; Reimann, K.; Klee, M.; Mauczok, R.; Keur, W.; Hueting, Raymond Josephus Engelbart

    2010-01-01

    Nanocrystalline barium strontium titanate (BaxSr1−xTi1.02O3) thin films with a barium content of x=0.8, 0.9 and 1 have been fabricated in a metal–insulator–metal configuration on glass-planarized alumina substrates. Cost-effective processing measures have been utilized by using poly-crystalline

  5. Role of Apoptosis in the Development of Uterine Leiomyoma: Analysis of Expression Patterns of Bcl-2 and Bax in Human Leiomyoma Tissue With Clinical Correlations.

    Science.gov (United States)

    Csatlós, Éva; Máté, Szabolcs; Laky, Marcella; Rigó, János; Joó, József Gábor

    2015-07-01

    To describe gene expression patterns of the apoptotic regulatory genes Bcl and Bax in human uterine leiomyoma tissue. To investigate the relationship between alterations of gene expression patterns and several relevant clinical parameters. We obtained samples from 101 cases undergoing surgery for uterine leiomyoma for gene expression analysis of the Bcl-2 and Bax genes. Gene expression was quantified using RT-PCR technique. In the leiomyoma group, the Bcl-2 gene was significantly overexpressed compared with the control group although there was no such difference in the gene expression of Bax. Gene activity of Bcl-2 positively correlated with the tumor number in individual uterine leiomyoma cases. Although there was no significant correlation between the length of the cumulative lactation period before the development of uterine leiomyoma and Bcl-2 gene expression in the leiomyoma tissue, we observed a trend for a shorter cumulative lactation period to be associated with overexpression of the Bcl-2 gene. Overexpression of the antiapoptotic Bcl-2 gene appeared to be a factor in the development of uterine leiomyoma, whereas gene activity of the proapoptotic Bax gene did not seem to play a role in the process.

  6. Characterization of Bc1-2, Bc1-xL, and Bax Pore Formation and Their Role in Apoptosis Regulation

    Science.gov (United States)

    2002-01-01

    Bcl-2, Bcl-xL, and Bax Pore Formation and Their Role in Apoptosis Regulation PRINCIPAL INVESTIGATOR: Frank Stenner -Liewen, Ph.D. Sharon Schendel, Ph.D...AUTHOR(S) Frank Stenner -Liewen, Ph.D. Sharon Schendel, Ph.D. John C. Reed, M.D., Ph.D. 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING

  7. Synthesis and characterization of BaxMgyAl2O4: Eu,Dy nanophosphors prepared using solution-combustion method

    CSIR Research Space (South Africa)

    Kebede, MA

    2011-07-01

    Full Text Available Europium-doped barium magnesium aluminate (BaxMgyAl2O4:Eu) phosphors were obtained at low temperature using the solution-combustion of corresponding metal nitrate-urea solution mixtures. The particle sizes, morphology, structural and luminescent...

  8. Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function

    Directory of Open Access Journals (Sweden)

    Hess Otto M

    2004-04-01

    Full Text Available Abstract Background Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria. Its accumulation can be observed in aging-related diseases, e.g. senile cataract and infectious disease. We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid. Results Xanthurenic acid at 10 or 20 μM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclxs, and Bad into mitochondria. In 20 μM xanthurenic acid, Bax is also translocated to the nucleus. In isolated mitochondria xanthurenic acid leads to Bax and Bclxs oligomerization, accumulation of Ca2+, and increased oxygen consumption. Conclusion Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions.

  9. 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

    International Nuclear Information System (INIS)

    Mohseni, Mehran; Mihandoost, Ehsan; Shirazi, Alireza; Sepehrizadeh, Zargham; Bazzaz, Javad Tavakkoly; Ghazi-khansari, Mahmoud

    2012-01-01

    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 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.

  10. 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.

  11. TRB3 is involved in free fatty acid-induced INS-1-derived cell apoptosis via the protein kinase C δ pathway.

    Directory of Open Access Journals (Sweden)

    Jun Qin

    Full Text Available Chronic exposure to free fatty acids (FFAs may induce β cell apoptosis in type 2 diabetes. However, the precise mechanism by which FFAs trigger β cell apoptosis is still unclear. Tribbles homolog 3 (TRB3 is a pseudokinase inhibiting Akt, a key mediator of insulin signaling, and contributes to insulin resistance in insulin target tissues. This paper outlined the role of TRB3 in FFAs-induced INS-1 β cell apoptosis. TRB3 was promptly induced in INS-1 cells after stimulation by FFAs, and this was accompanied by enhanced INS-1 cell apoptosis. The overexpression of TRB3 led to exacerbated apoptosis triggered by FFAs in INS-1-derived cell line and the subrenal capsular transplantation animal model. In contrast, cell apoptosis induced by FFAs was attenuated when TRB3 was knocked down. Moreover, we observed that activation and nuclear accumulation of protein kinase C (PKC δ was enhanced by upregulation of TRB3. Preventing PKCδ nuclear translocation and PKCδ selective antagonist both significantly lessened the pro-apoptotic effect. These findings suggest that TRB3 was involved in lipoapoptosis of INS-1 β cell, and thus could be an attractive pharmacological target in the prevention and treatment of T2DM.

  12. Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells.

    Science.gov (United States)

    Guon, Tae Eun; Chung, Ha Sook

    2017-08-01

    The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50-100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells.

  13. Probucol Attenuates Cyclophosphamide-induced Oxidative Apoptosis, p53 and Bax Signal Expression in Rat Cardiac Tissues

    Directory of Open Access Journals (Sweden)

    Yousif A. Asiri

    2010-01-01

    Full Text Available Cyclophosphamide (CP is a widely used drug in cancer chemotherapy and immunosuppression, which could cause toxicity of the normal cells due to its toxic metabolites. Probucol, a cholesterol-lowering drug, acts as potential inhibitor of DNA damage and shows to protect against doxorubicin-induced cardiomyopathy by enhancing the endogenous antioxidant system including glutathione peroxidase, catalase and superoxide dismutase. This study examined the possible protective effects of probucol, a lipid-lowering compound with strong antioxidant properties, against CPinduced cardiotoxicity. This objective could be achieved through studying the gene expression-based on the possible protective effects of probucol against CP-induced cardiac failure in rats. Adult male Wistar albino rats were assigned into four treatment groups: Animals in the first (control and second (probucol groups were injected intraperitoneally with corn oil and probucol (61 mg/kg/day, respectively, for two weeks. Animals in the third (CP and fourth (probucol plus CP groups were injected with the same doses of corn oil and probucol (61 mg/kg/day, respectively, for one week before and one week after a single dose of CP (200 mg/kg, I.P.. The p53, Bax, Bcl2 and oxidative genes signal expression were measured by real time PCR. CP-induced cardiotoxicity was clearly observed by a significant increase in serum creatine phosphokinase isoenzyme (CK-MB (117%, lactate dehydrogenase (LDH (64%, free (69% and esterified cholesterol (42% and triglyceride (69% compared to control group. In cardiac tissues, CP significantly increases the mRNA expression levels of apoptotic genes, p53 with two-fold and Bax with 1.6-fold, and decreases the anti-apoptotic gene Bcl2 with 0.5-fold. Moreover, CP caused downregulation of antioxidant genes, glutathione peroxidase, catalase, and superoxide dismutase and increased the lipid peroxidation and decreased adenosine triphosphate (ATP (40% and ATP/ADP (44% in cardiac

  14. The RNA-binding protein PCBP2 facilitates gastric carcinoma growth by targeting miR-34a

    International Nuclear Information System (INIS)

    Hu, Cheng-En; Liu, Yong-Chao; Zhang, Hui-Dong; Huang, Guang-Jian

    2014-01-01

    Highlights: • PCBP2 is overexpressed in human gastric cancer. • PCBP2 high expression predicts poor survival. • PCBP2 regulates gastric cancer growth in vitro and in vivo. • PCBP2 regulates gastric cancer apoptosis by targeting miR-34a. - Abstract: Gastric carcinoma is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. However, the mechanism underling gastric cancer is still not fully understood. Here in the present study, we identify the RNA-binding protein PCBP2 as an oncogenic protein in human gastric carcinoma. Our results show that PCBP2 is up-regulated in human gastric cancer tissues compared to adjacent normal tissues, and that high level of PCBP2 predicts poor overall and disease-free survival. Knockdown of PCBP2 in gastric cancer cells inhibits cell proliferation and colony formation in vitro, whereas opposing results are obtained when PCBP2 is overexpressed. Our in vivo subcutaneous xenograft results also show that PCBP2 can critically regulate gastric cancer cell growth. In addition, we find that PCBP2-depletion induces apoptosis in gastric cancer cells via up-regulating expression of pro-apoptotic proteins and down-regulating anti-apoptotic proteins. Mechanically, we identify that miR-34a as a target of PCBP2, and that miR-34a is critically essential for the function of PCBP2. In summary, PCBP2 promotes gastric carcinoma development by regulating the level of miR-34a

  15. Avicin D: a protein reactive plant isoprenoid dephosphorylates Stat 3 by regulating both kinase and phosphatase activities.

    Directory of Open Access Journals (Sweden)

    Valsala Haridas

    Full Text Available Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a the cross-talk that occurs between redox and phosphorylation processes, and (b the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3. The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors. Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways.

  16. G Protein-Coupled Receptor 87 (GPR87 Promotes Cell Proliferation in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xia Zhang

    2015-10-01

    Full Text Available G protein-coupled receptor 87 (GPR87 is a newly deorphanized member of the cell surface molecule G protein-coupled receptor family. GPR signaling was shown to play a role in promotion of cell growth and survival, metastasis, and drug resistance. The overexpression of GPR87 has also been reported in many malignant tumors including bladder cancer. The aim of the present study is to examine the effect of silencing GPR87 expression with a replication-deficient recombinant adenoviral vector expressing short hairpin RNA targeting GPR87 (Ad-shGPR87 and to explore the underlying molecular mechanisms in bladder cancer cells. Six GPR87-expressing human bladder cancer cells, HT1197, HT1376, J82, RT112, TCCSUP and UMUC3, were used. Infection with Ad-shGPR87 effectively downregulated the GPR87 expression, and significantly reduced the percentage of viable cells in 4 of 6 cell lines as detected by an MTT assay. Significant inhibition on cell proliferation with Ad-shGPR87 was observed in the wild-type p53 bladder cancer cell lines (HT1197, RT112, TCCSUP and UMUC3, but not in the mutant p53 cells (HT1376 and J82. As represented by a wild-type p53 RT112 cell, Ad-shGPR87 infection significantly enhanced p53 and p21 expression and caused caspase-dependent apoptosis. Furthermore, the treatment with Ad-shGPR87 exerted a significant antitumor effect against the GPR87-expressing RT112 xenografts. GPR87 appeared to be a promising target for gene therapy, and Ad-shGPR87 had strong antitumor effects, specifically anti-proliferative and pro-apoptotic effects, against GPR87-expressing human bladder cancer cells.

  17. Free fatty acid palmitate activates unfolded protein response pathway and promotes apoptosis in meniscus cells.

    Science.gov (United States)

    Haywood, J; Yammani, R R

    2016-05-01

    Obesity is the major risk factor for the development of osteoarthritis (OA); however, the mechanisms involved are not clearly understood. Obesity is associated with increased production of adipokine and elevated levels of circulating free fatty acids (FFA). A recent study has shown that saturated fatty acid palmitate induced pro-inflammatory and pro-apoptotic pathways in chondrocytes. Meniscus has been shown to be more susceptible than articular cartilage to catabolic stimuli. Thus, the aim of this study was to determine the effect of FFA (specifically, palmitate) on meniscus cells. Cultured primary porcine meniscus cells were stimulated with 500 μM FFA (palmitate and oleate) for 24 h to induce endoplasmic reticulum (ER) stress. After treatment, cell lysates were prepared and immunoblotted for C/EBP homologous protein (CHOP). To determine the activation of unfolded protein response (UPR) signaling, cell lysates were probed for cJun n-terminal kinase (JNK), cleaved caspase -3 and Xbp-1s, an alternative mRNA splicing product generated due to Ire1α activation. Treatment of isolated primary meniscus cells with palmitate but not oleate induced expression of CHOP and Xbp-1s. Palmitate treatment of meniscus cells also activated JNK and increased expression of caspase-3, thus promoting apoptosis in meniscus cells. Palmitate induces ER stress and promotes apoptotic pathways in meniscus cells. This is the first study to establish ER stress as a key metabolic mechanistic link between obesity and OA, in addition to (or operating with) biomechanical factors. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  18. 7α-Hydroxy-β-Sitosterol from Chisocheton tomentosus Induces Apoptosis via Dysregulation of Cellular Bax/Bcl-2 Ratio and Cell Cycle Arrest by Downregulating ERK1/2 Activation

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    Mohammad Tasyriq

    2012-01-01

    Full Text Available In continuation of our interest towards the elucidation of apoptotic pathways of cytotoxic phytocompounds, we have embarked upon a study on the anticancer effects of 7α-hydroxy-β-sitosterol (CT1, a rare natural phytosterol oxide isolated from Chisocheton tomentosus. CT1 was found to be cytotoxic on three different human tumor cell lines with minimal effects on normal cell controls, where cell viability levels were maintained ≥80% upon treatment. Our results showed that cell death in MCF-7 breast tumor cells was achieved through the induction of apoptosis via downregulation of the ERK1/2 signaling pathway. CT1 was also found to increase proapoptotic Bax protein levels, while decreasing anti-apoptotic Bcl-2 protein levels, suggesting the involvement of the intrinsic pathway. Reduced levels of initiator procaspase-9 and executioner procaspase-3 were also observed following CT1 exposure, confirming the involvement of cytochrome c-mediated apoptosis via the mitochondrial pathway. These results demonstrated the cytotoxic and apoptotic ability of 7α-hydroxy-β-sitosterol and suggest its potential anti-cancer use particularly on breast adenocarcinoma cells.

  19. Effect of nerve injury on the number of dorsal root ganglion neurons and autotomy behavior in adult Bax-deficient mice

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    Lyu C

    2017-08-01

    Full Text Available Chuang Lyu,1,2 Gong-Wei Lyu,3 Aurora Martinez,4 Tie-Jun Sten Shi4 1State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People’s Republic of China; 2Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; 3Department of Neurology, 1st Hospital of Harbin Medical University, Harbin, People’s Republic of China; 4Department of Biomedicine, University of Bergen, Bergen, Norway Background: The proapoptotic molecule BAX, plays an important role in mitochondrial apoptotic pathway. Dorsal root ganglion (DRG neurons depend on neurotrophic factors for survival at early developmental stages. Withdrawal of neurotrophic factors will induce apoptosis in DRG neurons, but this type of cell death can be delayed or prevented in neonatal Bax knockout (KO mice. In adult animals, evidence also shows that DRG neurons are less dependent upon neurotrophic factors for survival. However, little is known about the effect of Bax deletion on the survival of normal and denervated DRG neurons in adult mice. Methods: A unilateral sciatic nerve transection was performed in adult Bax KO mice and wild-type (WT littermates. Stereological method was employed to quantify the number of lumbar-5 DRG neurons 1 month post-surgery. Nerve injury-induced autotomy behavior was also examined on days 1, 3, and 7 post-surgery. Results: There were significantly more neurons in contralateral DRGs of KO mice as compared with WT mice. The number of neurons was reduced in ipsilateral DRGs in both KO and WT mice. No changes in size distributions of DRG neuron profiles were detected before or after nerve injury. Injury-induced autotomy behavior developed much earlier and was more serious in KO mice. Conclusion: Although postnatal death or loss of DRG neurons is partially prevented by Bax deletion, this effect cannot interfere with long-term nerve injury-induced neuronal loss. The exaggerated self

  20. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L).

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    Maiuri, Maria Chiara; Criollo, Alfredo; Tasdemir, Ezgi; Vicencio, José Miguel; Tajeddine, Nicolas; Hickman, John A; Geneste, Olivier; Kroemer, Guido

    2007-01-01

    Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X(L)) by virtue of its BH3 domain, an amphipathic alpha-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X(L). The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X(L). This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (reticulophagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X(L) can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X(L) complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X(L) complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X(L) at the level of the endoplasmic reticulum.

  1. Chronic copper exposure causes spatial memory impairment, selective loss of hippocampal synaptic proteins, and activation of PKR/eIF2α pathway in mice.

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    Ma, Quan; Ying, Ming; Sui, Xiaojing; Zhang, Huimin; Huang, Haiyan; Yang, Linqing; Huang, Xinfeng; Zhuang, Zhixiong; Liu, Jianjun; Yang, Xifei

    2015-01-01

    Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was upregulated and hippocampal neuronal apoptosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway.

  2. Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

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    Hovland Randi

    2007-05-01

    Full Text Available Abstract Background Acute myeloid leukemia (AML cells are characterized by non-mutated TP53, high levels of Hdm2, and frequent mutation of the Flt3 receptor tyrosine kinase. The juxtamembrane mutation of FLT3 is the strongest independent marker for disease relapse and is associated with elevated Bcl-2 protein and p53 hyper-phosphorylation in AML. DNA damage forms the basic mechanism of cancer cell eradication in current therapy of AML. Hdm2 and pro-apoptotic Bcl-2 members are among the most intensely induced genes immediately after chemotherapy and Hdm2 is proposed a role in receptor tyrosine kinase regulation. Thus we examined the DNA damage related modulation of these proteins in relation to FLT3 mutational status and induction of apoptosis. Results Within one hour after exposure to ionizing radiation (IR, the AML cells (NB4, MV4-11, HL-60, primary AML cells showed an increase in Flt3 protein independent of mRNA levels, while the Hdm2 protein decreased. The FLT3 mutant MV4-11 cells were resistant to IR accompanied by presence of both Mcl-1 and Hdm2 protein three hours after IR. In contrast, the FLT3 wild type NB4 cells responded to IR with apoptosis and pre-apoptotic Mcl-1 down regulation. Daunorubicin (DNR induced continuing down regulation of Hdm2 and Mcl-1 in both cell lines followed by apoptosis. Conclusion Both IR and DNR treatment resulted in concerted protein modulations of Mcl-1, Hdm2 and Flt3. Cell death induction was associated with persistent attenuation of Mcl-1 and Hdm2. These observations suggest that defining the pathway(s modulating Flt3, Hdm2 and Mcl-1 may propose new strategies to optimize therapy for the relapse prone FLT3 mutated AML patients.

  3. Role of the lattice dynamics in La2-xBaxCuO4 superconductor based on DFT method

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    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.

  4. Displacement-type ferroelectric transition with magnetic Mn ions in perovskite Sr1-xBaxMnO3

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    Sakai, Hideaki; Fujioka, Jun; Fukuda, Tatsuo; Okuyama, Daisuke; Hashizume, Daisuke; Kagawa, Fumitaka; Nakao, Hironori; Murakami, Youich; Arima, Takahisa; Baron, Alfred Q. R.; Taguchi, Yasujiro; Tokura, Yoshinori

    2012-02-01

    Almost all the proper ferroelectrics with a perovskite structure discovered so far have no d-electrons in the off-center transition metal site, as exemplified by BaTiO3 and Pb(Zr,Ti)O3. This empirical d^0 rule is incompatible with the emergence of magnetism and has significantly restricted the variety of multiferroic materials. In this work, we have discovered a displacement-type ferroelectric transition originating from off-center Mn^4+ ions in antiferromagnetic Mott insulators Sr1-xBaxMnO3. As Ba concentration increases, the perovskite lattice shows the typical soft mode dynamics, and the ferroelectricity shows up for x .45. In addition to the large polarization and high transition temperature comparable to BaTiO3, we demonstrate that the magnetic order suppresses the ferroelectric lattice dilation by ˜70% and increases the soft-phonon energy by ˜50%, indicating gigantic magnetoelectric effects [1]. This work was supported by the FIRST program on ``Quantum Science on Strong Correlation''. [4pt] [1] H. Sakai et al., Phys. Rev. Lett. 107, 137601 (2011).

  5. Ab-initio study of structural, elastic, electronic and thermodynamic properties of BaxSr1−xS ternary alloys

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    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.

  6. Next-generation proteasome inhibitor oprozomib synergizes with modulators of the unfolded protein response to suppress hepatocellular carcinoma.

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    Vandewynckel, Yves-Paul; Coucke, Céline; Laukens, Debby; Devisscher, Lindsey; Paridaens, Annelies; Bogaerts, Eliene; Vandierendonck, Astrid; Raevens, Sarah; Verhelst, Xavier; Van Steenkiste, Christophe; Libbrecht, Louis; Geerts, Anja; Van Vlierberghe, Hans

    2016-06-07

    Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators.

  7. An Assay of Bax and Bcl2 Expression in Mice Hippocampus Following Ischemia-Reperfusion Treatment with CoQ10

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    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.

  8. AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity

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    Kim, Jae-Hwan; Choi, Soo-Youn; Kang, Byung-Hee; Lee, Soon-Min [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Park, Hyung Soon; Kang, Gum-Yong; Bang, Joo Young [Center for Biomedical Mass Spectrometry, Diatech Korea Co., Ltd., Seoul (Korea, Republic of); Cho, Eun-Jung [National Research Laboratory for Chromatin Dynamics, College of Pharmacy, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Youn, Hong-Duk, E-mail: hdyoun@snu.ac.kr [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence and Technology, Seoul National University, Seoul (Korea, Republic of)

    2013-02-01

    Highlights: ► AMPK phosphorylates CtBP1 on serine 158. ► AMPK-mediated phosphorylation of CtBP1 causes the ubiquitination and nuclear export of CtBP1. ► AMPK downregulates the CtBP1-mediated repression of Bax transcription. -- Abstract: CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

  9. The protective effects of the proteasome inhibitor bortezomib (velcade on ischemia-reperfusion injury in the rat retina.

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    Fang-Ting Chen

    Full Text Available PURPOSE: To evaluate the protective effects of bortezomib (Velcade on ischemia-reperfusion (IR injury in the rat retina. METHODS: The rats were randomized to receive treatment with saline, low-dose bortezomib (0.05 mg/kg, or high-dose bortezomib (0.2 mg/kg before the induction of IR injury. Electroretinography (ERG was used to assess functional changes in the retina. The expression of inflammatory mediators (iNOS, ICAM-1, MCP-1, TNF-α, anti-oxidant proteins (heme oxygenase, thioredoxin, peroxiredoxin, and pro-apoptotic proteins (p53, bax were quantified by PCR and western blot analysis. An immunofluorescence study was performed to detect the expression of iNOS, oxidative markers (nitrotyrosine, 8-OHdG, acrolein, NF-κB p65, and CD 68. Apoptosis of retinal cells was labeled with in situ TUNEL staining. Neu-N staining was performed in the flat-mounted retina to evaluate the density of retinal ganglion cells. RESULTS: ERG showed a decreased b-wave after IR injury, and pretreatment with bortezomib, especially the high dosage, reduced the functional impairment. Bortezomib successfully reduced the elevation of inflammatory mediators, anti-oxidant proteins, pro-apoptotic proteins and oxidative markers after IR insult in a dose-dependent manner. In a similar fashion, NF-κB p65- and CD 68-positive cells were decreased by bortezomib treatment. Retinal cell apoptosis in each layer was attenuated by bortezomib. The retinal ganglion cell density was markedly decreased in the saline and low-dose bortezomib groups but was not significantly changed in the high-dose bortezomib group. CONCLUSIONS: Bortezomib had a neuro-protective effect in retinal IR injury, possibly by inhibiting the activation of NF-κB related to IR insult and reducing the inflammatory signals and oxidative stress in the retina.

  10. Andrographolide induces vascular smooth muscle cell apoptosis through a SHP-1-PP2A-p38MAPK-p53 cascade.

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    Chen, Yu-Ying; Hsieh, Cheng-Ying; Jayakumar, Thanasekaran; Lin, Kuan-Hung; Chou, Duen-Suey; Lu, Wan-Jung; Hsu, Ming-Jen; Sheu, Joen-Rong

    2014-07-10

    The abnormal growth of vascular smooth muscle cells (VSMCs) is considered a critical pathogenic process in inflammatory vascular diseases. We have previously demonstrated that protein phosphatase 2 A (PP2A)-mediated NF-κB dephosphorylation contributes to the anti-inflammatory properties of andrographolide, a novel NF-κB inhibitor. In this study, we investigated whether andrographolide causes apoptosis, and characterized its apoptotic mechanisms in rat VSMCs. Andrographolide activated the p38 mitogen-activated protein kinase (p38MAPK), leading to p53 phosphorylation. Phosphorylated p53 subsequently transactivated the expression of Bax, a pro-apoptotic protein. Transfection with pp2a small interfering RNA (siRNA) suppressed andrographolide-induced p38MAPK activation, p53 phosphorylation, and caspase 3 activation. Andrographolide also activated the Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1), and induced PP2A dephosphorylation, both of which were inhibited by the SHP-1 inhibitor sodium stibogluconate (SSG) or shp-1 siRNA. SSG or shp-1 siRNA prevented andrographolide-induced apoptosis. These results suggest that andrographolide activates the PP2A-p38MAPK-p53-Bax cascade, causing mitochondrial dysfunction and VSMC death through an SHP-1-dependent mechanism.

  11. Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in mice.

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    Monteiro, Gaby E R; Jansen van Vuren, Petrus; Wichgers Schreur, Paul J; Odendaal, Lieza; Clift, Sarah J; Kortekaas, Jeroen; Paweska, Janusz T

    2018-04-02

    The NSs protein encoded by the S segment of Rift Valley fever virus (RVFV) is the major virulence factor, counteracting the host innate antiviral defence. It contains five highly conserved cysteine residues at positions 39, 40, 149, 178 and 194, which are thought to stabilize the tertiary and quaternary structure of the protein. Here, we report significant differences between clinical, virological, histopathological and host gene responses in BALB/c mice infected with wild-type RVFV (wtRVFV) or a genetic mutant having a double cysteine-to-serine substitution at residues 39 and 40 of the NSs protein (RVFV-C39S/C40S). Mice infected with the wtRVFV developed a fatal acute disease; characterized by high levels of viral replication, severe hepatocellular necrosis, and massive up-regulation of transcription of genes encoding type I and -II interferons (IFN) as well as pro-apoptotic and pro-inflammatory cytokines. The RVFV-C39S/C40S mutant did not cause clinical disease and its attenuated virulence was consistent with virological, histopathological and host gene expression findings in BALB/c mice. Clinical signs in mice infected with viruses containing cysteine-to-serine substitutions at positions 178 or 194 were similar to those occurring in mice infected with the wtRVFV, while a mutant containing a substitution at position 149 caused mild, non-fatal disease in mice. As mutant RVFV-C39S/C40S showed an attenuated phenotype in mice, the molecular mechanisms behind this attenuation were further investigated. The results show that two mechanisms are responsible for the attenuation; (1) loss of the IFN antagonistic propriety characteristic of the wtRVFV NSs and (2) the inability of the attenuated mutant to degrade Proteine Kinase R (PKR). Copyright © 2018. Published by Elsevier B.V.

  12. Persea declinata (Bl. Kosterm Bark Crude Extract Induces Apoptosis in MCF-7 Cells via G0/G1 Cell Cycle Arrest, Bcl-2/Bax/Bcl-xl Signaling Pathways, and ROS Generation

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    Putri Narrima

    2014-01-01

    Full Text Available Persea declinata (Bl. Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill, which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl. Kosterm bark methanolic crude extract (PDM. PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48 h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development.

  13. 1-(2,6-Dihydroxy-4-methoxyphenyl-2-(4-hydroxyphenyl Ethanone-Induced Cell Cycle Arrest in G1/G0 in HT-29 Cells Human Colon Adenocarcinoma Cells

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    Ma Ma Lay

    2014-01-01

    Full Text Available 1-(2,6-Dihydroxy-4-methoxyphenyl-2-(4-hydroxyphenyl ethanone (DMHE was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff. Boerl fruits and the structure confirmed by GC-MS (gas chromatography-mass spectrometry and NMR (nuclear magnetic resonance analysis. This compound was tested on the HT-29 human colon adenocarcinoma cell line using MTT (method of transcriptional and translational cell proliferation assay. The results of MTT assay showed that DMHE exhibited good cytotoxic effect on HT-29 cells in a dose- and time-dependent manner but no cytotoxic effect on the MRC-5 cell line after 72 h incubation. Morphological features of apoptotic cells upon treatment by DMHE, e.g., cell shrinkage and membrane blebbing, were examined by an inverted and phase microscope. Other features, such as chromatin condension and nuclear fragmentation were studied using acridine orange and propidium iodide staining under the fluorescence microscope. Future evidence of apoptosis/necrosis was provided by result fromannexin V-FITC/PI (fluorescein-isothiocyanate/propidium iodide staining revealed the percentage of early apoptotic, late apoptotic, necrotic and live cells in a dose- and time-dependent manner using flow cytometry. Cell cycle analysis showed G0/G1 arrest in a time-dependent manner. A western blot analysis indicated that cell death might be associated with the up-regulation of the pro-apoptotic proteins Bax PUMA. However, the anit-apotptic proteins Bcl-2, Bcl-xL, and Mcl-1 were also found to increase in a time-dependent manner. The expression of the pro-apoptotic protein Bak was not observed.

  14. Arrhythmia causes lipid accumulation and reduced glucose uptake.

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    Lenski, Matthias; Schleider, Gregor; Kohlhaas, Michael; Adrian, Lucas; Adam, Oliver; Tian, Qinghai; Kaestner, Lars; Lipp, Peter; Lehrke, Michael; Maack, Christoph; Böhm, Michael; Laufs, Ulrich

    2015-01-01

    Atrial fibrillation (AF) is characterized by irregular contractions of atrial cardiomyocytes and increased energy demand. The aim of this study was to characterize the influence of arrhythmia on glucose and fatty acid (FA) metabolism in cardiomyocytes, mice and human left atrial myocardium. Compared to regular pacing, irregular (pseudo-random variation at the same number of contractions/min) pacing of neonatal rat cardiomyocytes induced shorter action potential durations and effective refractory periods and increased diastolic [Ca(2+)]c. This was associated with the activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK). Membrane expression of fatty acid translocase (FAT/CD36) and (14)C-palmitic acid uptake were augmented while membrane expression of glucose transporter subtype 4 (GLUT-4) as well as (3)H-glucose uptake were reduced. Inhibition of AMPK and CaMKII prevented these arrhythmia-induced metabolic changes. Similar alterations of FA metabolism were observed in a transgenic mouse model (RacET) for spontaneous AF. Consistent with these findings samples of left atrial myocardium of patients with AF compared to matched samples of patients with sinus rhythm showed up-regulation of CaMKII and AMPK and increased membrane expression of FAT/CD36, resulting in lipid accumulation. These changes of FA metabolism were accompanied by decreased membrane expression of GLUT-4, increased glycogen content and increased expression of the pro-apoptotic protein bax. Irregular pacing of cardiomyocytes increases diastolic [Ca(2+)]c and activation of CaMKII and AMPK resulting in lipid accumulation, reduced glucose uptake and increased glycogen synthesis. These metabolic changes are accompanied by an activation of pro-apoptotic signalling pathways.

  15. Polyalthia longifolia Methanolic Leaf Extracts (PLME) induce apoptosis, cell cycle arrest and mitochondrial potential depolarization by possibly modulating the redox status in hela cells.

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    Vijayarathna, Soundararajan; Oon, Chern Ein; Chen, Yeng; Kanwar, Jagat R; Sasidharan, Sreenivasan

    2017-05-01

    Medicinal plants have been accepted as a gold mine, with respect to the diversity of their phytochemicals. Many medicinal plants extracts are potential anticancer agents. Polyalthia longifolia var. angustifolia Thw. (Annonaceae) is one of the most significant native medicinal plants and is found throughout Malaysia. Hence, the present study was intended to assess the anticancer properties of P. longifolia leaf methanolic extract (PLME) and its underlying mechanisms. The Annexin V/PI flow cytometry analysis showed that PLME induces apoptosis in HeLa cells in dose-dependent manner whereas the PI flow cytometric analysis for cell cycle demonstrated the accumulation of cells at sub G0/G1, G0/G1 and G2/M phases. Investigation with JC-1 flow cytometry analysis indicated increase in mitochondria membrane potential depolarisation corresponding to increase in PLME concentrations. PLME was also shown to influence intracellular reactive oxygen species (ROS) by exerting anti-oxidant (half IC 50 ) and pro-oxidant (IC 50 and double IC 50 ) affect against HeLa cells. PLME treatment also displayed DNA damage in HeLa cells in concentration depended fashion. The proteomic profiling array exposed the expression of pro-apoptotic and anti-apoptotic proteins upon PLME treatment at IC 50 concentration in HeLa cells. Pro-apoptotic proteins; BAX, BAD, cytochrome c, caspase-3, p21, p27 and p53 were found to be significantly up-regulated while anti-apoptotic proteins; BCL-2 and BCL-w were found to be significantly down-regulated. This investigation postulated the role of p53 into mediating apoptosis, cell cycle arrest and mitochondrial potential depolarisation by modulating the redox status of HeLa cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. Interference in plant defense and development by non-structural protein NSs of Groundnut bud necrosis virus.

    Science.gov (United States)

    Goswami, Suneha; Sahana, Nandita; Pandey, Vanita; Doblas, Paula; Jain, R K; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

    2012-01-01

    Groundnut bud necrosis virus (GBNV) infects a large number of leguminous and solanaceous plants. To elucidate the biological function of the non-structural protein encoded by the S RNA of GBNV (NSs), we studied its role in RNA silencing suppression and in viral pathogenesis. Our results demonstrated that GBNV NSs functions as a suppressor of RNA silencing using the agroinfiltration patch assay. An in silico analysis suggested the presence of pro-apoptotic protein Reaper-like sequences in the GBNV NSs, which were known to be present in animal infecting bunyaviruses. Utilizing NSs mutants, we demonstrated that a Leu-rich domain was required for RNA silencing suppression activity, but not the non-overlapping Trp/GH3 motif of the Reaper-like sequence. To investigate the role of NSs in symptom development we generated transgenic tomato expressing the GBNV NSs and showed that the expression of NSs in tomato mimics symptoms induced by infection with GBNV, such as leaf senescence and necrosis. As leaf senescence is controlled by miR319 regulation of the transcription factor TCP1, we assessed the accumulation of both RNAs in transgenic NSs-expressing and GBNV-infected tomato plants. In both types of plants the levels of miR319 decreased, while the levels of TCP1 transcripts increased. We propose that GBNV-NSs affects miRNA biogenesis through its RNA silencing suppressor activity and interferes with TCP1-regulated leaf developmental pathways. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. [Apoptosis-modulating effects of heat shock proteins: the influence of Hsp27 chaperone on TBA Bcl-2 family proteins in Jurkat cell line].

    Science.gov (United States)

    Riazantseva, N V; Kaĭgorodova, E V; Maroshkina, A N; Belkina, M V; Novitskiĭ, V V

    2012-01-01

    The in vitro phosphorylated and non-phosphorylated Hsp27 forms concentrations and Bcl-2 proteins affected by Hsp27 inhibition were studied in Jurkat-line tumor cells and healthy donor mononuclear lymphocytes by Western blotting technique. The Hsp27 inhibition causes the increase of intracellular Bax protein concentration and the decrease of Bcl-2 level leading to an increase of apoptotic changes in Jurkat line cells.

  18. PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis.

    Directory of Open Access Journals (Sweden)

    Kathrin Thedieck

    Full Text Available TOR (Target of Rapamycin is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1 and TOR complex 2 (TORC2. In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS based proteomic strategy to identify new mammalian TOR (mTOR binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40 and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5 and was therefore named PRR5-Like (PRR5L. PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1 and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

  19. Pro-apoptotic effect of the landrace Bangla Mahoba of Piper betle on Leishmania donovani may be due to the high content of eugenol.

    Science.gov (United States)

    Misra, Pragya; Kumar, Awanish; Khare, Prashant; Gupta, Swati; Kumar, Nikhil; Dube, Anuradha

    2009-08-01

    In the absence of effective and safe treatment for visceral leishmaniasis or Kala-azar - a devastating parasitic disease caused by Leishmania donovani - the search for anti-leishmanial agents from natural resources in common use is imperative. Recently, the comparative in vitro anti-leishmanial activity of methanolic extracts from two landraces of Piper betle - P. betle landrace Bangla Mahoba (PB-BM) and P. betle landrace Kapoori Vellaikodi (PB-KV) - has been reported. Here, the putative pathway responsible for death induced by the effective extract of PB-BM methanolic extract in promastigotes, as well as the intracellular amastigote form of L. donovani, was assessed using various biochemical approaches. It was found that PB-BM was capable of selectively inhibiting both stages of Leishmania parasites by accelerating apoptotic events by generation of reactive oxygen species targeting the mitochondria without any cytotoxicity towards macrophages. The study was extended to determine the presence or absence of activity of the methanolic extract of PB-BM and PB-KV on the basis of differences in essential oil composition present in the extract assessed by GC and MS. The essential oil from PB-BM was found to be rich in eugenol compared with that from PB-KV. The anti-leishmanial efficacy of PB-BM methanolic extract mediated through apoptosis is probably due to the higher content of eugenol in the active landrace. This observation emphasizes the need to extend studies related to traditional medicines from bioactive plants below the species level to the gender/landrace level for better efficacy and reproducibility.

  20. Synthesis, structural characterization, and pro-apoptotic activity of 1-indanone thiosemicarbazone platinum(II) and palladium(II) complexes: potential as antileukemic agents.

    Science.gov (United States)

    Gómez, Natalia; Santos, Diego; Vázquez, Ramiro; Suescun, Leopoldo; Mombrú, Alvaro; Vermeulen, Monica; Finkielsztein, Liliana; Shayo, Carina; Moglioni, Albertina; Gambino, Dinorah; Davio, Carlos

    2011-08-01

    In the search for alternative chemotherapeutic strategies against leukemia, various 1-indanone thiosemicarbazones, as well as eight novel platinum(II) and palladium(II) complexes, with the formula [MCl₂(HL)] and [M(HL)(L)]Cl, derived from two 1-indanone thiosemicarbazones were synthesized and tested for antiproliferative activity against the human leukemia U937 cell line. The crystal structure of [Pt(HL1)(L1)]Cl·2MeOH, where L1=1-indanone thiosemicarbazone, was solved by X-ray diffraction. Free thiosemicarbazone ligands showed no antiproliferative effect, but the corresponding platinum(II) and palladium(II) complexes inhibited cell proliferation and induced apoptosis. Platinum(II) complexes also displayed selective apoptotic activity in U937 cells but not in peripheral blood monocytes or the human hepatocellular carcinoma HepG2 cell line used to screen for potential hepatotoxicity. Present findings show that, in U937 cells, 1-indanone thiosemicarbazones coordinated to palladium(II) were more cytotoxic than those complexed with platinum(II), although the latter were found to be more selective for leukemic cells suggesting that they are promising compounds with potential therapeutic application against hematological malignancies. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Study of morphological changes in breast cancer cells MCF-7 under the action of pro-apoptotic agents with laser modulation interference microscope MIM-340

    Science.gov (United States)

    Nebogatikov, V.; Nikitiuk, A.; Konysheva, A.; Ignatyev, P.; Grishko, V.; Naimark, O.

    2017-09-01

    Quantitative phase microscopy is a new method to measure and evaluate the microlevel processes characterized by the high resolution and providing ample opportunities to quantitatively analyze various parameters, including specimens from biological matter. In this study, a laser interference microscope was used to evaluate the state of cancer cells (living and apoptotic). Apoptotic cancer cells were obtained by treatment of MCF-7 cells with the use of betulin-based α-bromomethyl ketone (BMK) derivative. When using the microscope, the main differences in the morphometric parameters of living and apoptotic cells such as height, diameter, perimeter, area and volume were appraised. The criteria that can be used as markers of apoptosis activation were identified.

  2. Mitochondria play a central role in apoptosis induced by alpha-tocopheryl succinate, an agent with antineoplastic activity: comparison with receptor-mediated pro-apoptotic signaling

    Czech Academy of Sciences Publication Activity Database

    Weber, T. D.; Dalen, H.; Anděra, Ladislav; Negre-Salvayre, A.; Auge, N.; Sticha, M.; Lloret, A.; Terman, A.; Witting, P. K.; Higuchi, M.; Plasilova, M.; Zivny, J.; Gellert, N.; Weber, C.; Neuzil, J.

    2003-01-01

    Roč. 42, č. 14 (2003), s. 4277-91 ISSN 0006-2960 R&D Projects: GA MŠk LN00A026 Institutional research plan: CEZ:AV0Z5052915 Keywords : apoptosis, TRAIL, TOS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.922, year: 2003

  3. SLC5A8-Mediated Switching of STAT3 from a Pro-Oncogenic Signal into a Pro-Apoptotic Signal in Breast Cancer

    Science.gov (United States)

    2011-06-01

    provokes lung metastasis. (5) Both STAT3 and SLC5A8 knockout showed the similar phenotype, like mammary gland involution delay, mastitis and...100 ng/ml cholera toxin, 0.01mg/ml bovine insulin and 500 ng/ml hydrocortisone. HBL100 cells was grown in McCoy 5A with 10% FBS. MCF7 and BT20

  4. Evidence of a pro-apoptotic effect of specific antibodies in a bovine macrophage model of infection with Mycobacterium avium subsp. paratuberculosis.

    Science.gov (United States)

    Jolly, Ana; Lompardía, Silvina; Hajos, Silvia E; Mundo, Silvia L

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis in ruminants. Understanding the protective immune response following infection is crucial to improve the diagnosis and the development of vaccines against this disease. The goal of this work was to assess whether specific antibodies were able to modulate the macrophage response to MAP infection by evaluating apoptosis and TNF-α secretion in an in vitro model. Sera from healthy (n=2), MAP-infected (n=3) and lipoarabinomannan (LAM)-immunized (n=3) bovines were evaluated. LAM was chosen as immunogen due to its relevant role in mycobacterial pathogenesis. We demonstrated by two different techniques (Acridine Orange/Ethidium Bromide microscopy and Annexin V/7-Amino-Actinomycin D flow cytometry) that the immune sera from both, MAP-infected and LAM-immunized bovines, significantly increased macrophage apoptosis in infected cultures. Comparable levels of apoptosis were detected when MAP was pre-incubated with purified specific antibodies instead of whole serum. Furthermore, this effect was accompanied by a significantly higher secretion of TNF-α. These results strongly suggest that specific antibodies could limit the impact of MAP on the apoptosis of bovine cells. This work would contribute to elucidate the role of the specific antibody response in bovine JD and its prevention. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Anti-adhesive and pro-apoptotic effects of 2-hydroxyethyl methacrylate on human gingival fibroblasts co-cultured with Streptococcus mitis strains

    Science.gov (United States)

    Zara, S; Di Giulio, M; D’Ercole, S; Cellini, L; Cataldi, A

    2011-01-01

    Aim To evaluate and observe the cellular reactions that occur during the interaction/integration between 2-hydroxyethyl methacrylate/host tissue/microbial environment, in a co-culture of human gingival fibroblasts (HGF) and Streptococcus mitis strains. Methodology Streptococcus mitis were cultured with strains in the presence of 3 mmol L−1 HEMA for 48 h and 72 h. Cytotoxicity was evaluated by the trypan blue dye exclusion test. Apoptosis was evaluated by TUNEL analysis. Adhesion was evaluated by immunofluorescence and western blot analyses. Quantitative analyses of the results were acquired by Qwin Plus 3.5 and QuantityOne I-D analysis software, respectively. The statistical significance of the results was evaluated using t-tests and linear regression tests. Results The trypan blue dye test revealed 47.3% and 46.5% of dead fibroblasts after 48 and 72 h HEMA treatment, respectively, while bacterial viability was not influenced by the presence of HEMA and fibroblasts. The expression of pro-collagen I, involved in fibroblast adhesion, in untreated samples ranged from 12.49% to 6.91% of the positive area after 48 and 72 h, respectively, dropping to below 2% of the positive area in the other experimental conditions. Unlike the trypan blue test, co-cultured samples treated with HEMA showed 20% and 25% versus 17% and 21% (after 48 and 72 h, respectively) of apoptotic cells. Conclusions The evidence for HEMA toxicity and anti-adhesive effects against eukaryotic cells was reduced in the presence of bacteria, suggesting that dental resins should be well polymerized to avoid the spread of toxic monomers within the mouth. PMID:21902700

  6. Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2

    Science.gov (United States)

    Yue, Wen; Zheng, Xi; Lin, Yong; Yang, Chung S.; Xu, Qing; Carpizo, Darren; Huang, Huarong; DiPaola, Robert S.; Tan, Xiang-Lin

    2015-01-01

    Metformin and aspirin have been studied extensively as cancer preventive or therapeutic agents. However, the effects of their combination on pancreatic cancer cells have not been investigated. Herein, we evaluated the effects of metformin and aspirin, alone or in combination, on cell viability, migration, and apoptosis as well as the molecular changes in mTOR, STAT3 and apoptotic signaling pathways in PANC-1 and BxPC3 cells. Metformin and aspirin, at relatively low concentrations, demonstrated synergistically inhibitory effects on cell viability. Compared to the untreated control or individual drug, the combination of metformin and aspirin significantly inhibited cell migration and colony formation of both PANC-1 and BxPC-3 cells. Metformin combined with aspirin significantly inhibited the phosphorylation of mTOR and STAT3, and induced apoptosis as measured by caspase-3 and PARP cleavage. Remarkably, metformin combined with aspirin significantly downregulated the anti-apoptotic proteins Mcl-1 and Bcl-2, and upregulated the pro-apoptotic proteins Bim and Puma, as well as interrupted their interactions. The downregulation of Mcl-1 and Bcl-2 was independent of AMPK or STAT3 pathway but partially through mTOR signaling and proteasome degradation. In a PANC-1 xenograft mouse model, we demonstrated that the combination of metformin and aspirin significantly inhibited tumor growth and downregulated the protein expression of Mcl-1 and Bcl-2 in tumors. Taken together, the combination of metformin and aspirin significantly inhibited pancreatic cancer cell growth in vitro and in vivo by regulating the pro- and anti-apoptotic Bcl-2 family members, supporting the continued investigation of this two drug combination as chemopreventive or chemotherapeutic agents for pancreatic cancer. PMID:26056043

  7. Human-gyrovirus-Apoptin triggers mitochondrial death pathway--Nur77 is required for apoptosis triggering.

    Science.gov (United States)

    Chaabane, Wiem; Cieślar-Pobuda, Artur; El-Gazzah, Mohamed; Jain, Mayur V; Rzeszowska-Wolny, Joanna; Rafat, Mehrdad; Stetefeld, Joerg; Ghavami, Saeid; Los, Marek J

    2014-09-01

    The human gyrovirus derived protein Apoptin (HGV-Apoptin) a homologue of the chicken anemia virus Apoptin (CAV-Apoptin), a protein with high cancer cells selective toxicity, triggers apoptosis selectively in cancer cells. In this paper, we show that HGV-Apoptin acts independently from the death receptor pathway as it induces apoptosis in similar rates in Jurkat cells deficient in either FADD (fas-associated death domain) function or caspase-8 (key players of the extrinsic pathway) and their parental clones. HGV-Apoptin induces apoptosis via the activation of the mitochondrial intrinsic pathway. It induces both mitochondrial inner and outer membrane permebilization, characterized by the loss of the mitochondrial potential and the release into cytoplasm of the pro-apoptotic molecules including apoptosis inducing factor and cytochrome c. HGV-Apoptin acts via the apoptosome, as lack of expression of apoptotic protease-activating factor 1 in murine embryonic fibroblast strongly protected the cells from HGV-Apoptin-induced apoptosis. Moreover, QVD-oph a broad-spectrum caspase inhibitor delayed HGV-Apoptin-induced death. On the other hand, overexpression of the anti-apoptotic BCL-XL confers resistance to HGV-Apoptin-induced cell death. In contrast, cells that lack the expression of the pro-apoptotic BAX and BAK are protected from HGV-Apoptin induced apoptosis. Furthermore, HGV-Apoptin acts independently from p53 signal but triggers the cytoplasmic translocation of Nur77. Taking together these data indicate that HGV-Apoptin acts through the mitochondrial pathway, in a caspase-dependent manner but independently from the death receptor pathway. Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

  8. Caspase-3/-8/-9, Bax and Bcl-2 expression in the cerebellum, lymph nodes and leukocytes of dogs naturally infected with canine distemper virus.

    Science.gov (United States)

    Del Puerto, H L; Martins, A S; Moro, L; Milsted, A; Alves, F; Braz, G F; Vasconcelos, A C

    2010-01-26

    Canine distemper is an immunosuppressive disease caused by the canine distemper virus (CDV). Pathogenesis mainly involves the central nervous system and immunosuppression. Dogs naturally infected with CDV develop apoptotic cells in lymphoid tissues and the cerebellum, but this apoptotic mechanism is not well characterized. To better understand this process, we evaluated the expression of Bax, Bcl-2, and caspase-3, -8 and -9, by evaluating mRNA levels in the peripheral blood, lymph nodes and cerebellum of CDV-infected (CDV+) and uninfected (CDV-) dogs by real-time polymerase chain reaction (PCR). Blood samples from 12 CDV+ and 8 CDV- dogs, diagnosed by reverse transcription-PCR, were subjected to hematological analysis and apoptotic gene expression was evaluated using real-time-PCR. Tissues from the cerebellum and lymph nodes of four CDV+ and three CDV-dogs were also subjected to real time-PCR. No significant differences were found between CDV+ and CDV- dogs in the hemotological results or in the expression of caspase-3, -8, -9, Bax, and Bcl-2 in the peripheral blood. However, expression of Bax, caspase-3, -8 and -9 was significantly higher in the cerebellum of CDV+ compared to CDV- dogs. Expression of caspase-3 and -8 was significantly higher in the lymph nodes of CDV+ compared to CDV- dogs. We concluded that infection with CDV induces apoptosis in the cerebellum and lymph nodes in different ways. Lymph node apoptosis apparently occurs via caspase-3 activation, through the caspase-8 pathway, and cerebellum apoptosis apparently occurs via caspase-3 activation, through the caspase-8 and mitochondrial pathways.

  9. 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

    Directory of Open Access Journals (Sweden)

    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.

  10. Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

    Directory of Open Access Journals (Sweden)

    Tognon Raquel

    2012-02-01

    Full Text Available Abstract Background Essential Thrombocythemia (ET and Primary Myelofibrosis (PMF are Chronic Myeloproliferative Neoplasms (MPN characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34+ hematopoietic stem cells (HSC and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. Results By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34+ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34+ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. Conclusions Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation

  11. Roles of some antioxidants in modulation of cardiac myopathy induced by sodium nitrite via down-regulation of mRNA expression of NF-κB, Bax, and flt-1 and suppressing DNA damage

    Directory of Open Access Journals (Sweden)

    Laila Mohamed Fadda

    2018-02-01

    Full Text Available The underlying pathology of cardiac damage involves various molecular and signaling pathways. Therefore, this study aimed to explore the role of Quercetin (Querc, alone or in combination with Melatonin (Melat against cardiac damage induced by sodium nitrite (Sod nit, as well as to elucidate different signaling pathways. Querc and Melat were injected intraperitoneally (i.p., followed by induction of hypoxia in rats by using a single dose of Sod nit (60 mg/kg, s.c.. Treatment with Sod nit significantly decreased hemoglobin (Hb levels in blood. Pretreatment of hypoxic rats with Querc and/or Melat elevated the declined Hb concentration. The forementioned antioxidants also successfully ameliorated the alteration of heat shock protein 70 (HSP-70 and markers of cardiac injury, including troponin T (Trop. T, creatine kinase-MB (CK-MB, tumor necrosis factor-α (TNF α, and C-reactive protein (CRP in the rats serum. Furthermore, RT-PCR revealed that these antioxidants successfully modulated mRNA expression of NF-κB, Bax, Bcl-2, and flt-1. They also regulated vascular endothelial growth factor (VEGF, the apoptosis marker caspase 3, and oxidative DNA damage in cardiac tissue, compared to Sod nit-intoxicated rats. The present biochemical results are reinforced by histopathological examination. In Conclusion: The results reflected that treatment with Querc in combination with Melat was most effective in improving Sod nit-toxicity induced cardiac damage, thus confirming the promising role of this combination as an effective treatment for cardiac damage induced by other cardio-toxic agents.

  12. Increases in apoptosis, caspase activity and expression of p53 and bax, and the transition between two types of mitochondrion-rich cells, in the gills of the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater

    Science.gov (United States)

    Ching, Biyun; Chen, Xiu L.; Yong, Jing H. A.; Wilson, Jonathan M.; Hiong, Kum C.; Sim, Eugene W. L.; Wong, Wai P.; Lam, Siew H.; Chew, Shit F.; Ip, Yuen K.

    2013-01-01

    This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na+/K+-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na+:K+:2Cl− cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl− channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater

  13. Subclinical hypothyroidism in pregnant rats impaired learning and memory of their offspring by promoting the p75NTR signal pathway

    Directory of Open Access Journals (Sweden)

    Fan Zhang

    2018-05-01

    Full Text Available Objective: Maternal hypothyroidism during pregnancy can affect the neurodevelopment of their offspring. This study aimed to investigate the effects of maternal subclinical hypothyroidism (SCH on spatial learning and memory, and its relationship with the apoptotic factors in cerebral cortex of the offspring. Methods: Female adult Wistar rats were randomly divided into three groups (n = 15 per group: control (CON group, SCH group and overt hypothyroidism (OH group. Spatial learning and memory in the offspring were evaluated by long-term potentiation (LTP and Morris water-maze (MWM test. The protein expression of the p75 neurotrophin receptor (p75NTR, phospho-c-Jun N-terminal kinase (p-JNK, the pro-apoptotic protein p53 and Bax were detected by Western blotting. Results: The Pups in the SCH and OH groups showed longer escape latencies in the MWM and decreased field-excitatory post synaptic potentials in LTP tests compared with those in the CON group. p75NTR, p-JNK, p53 and Bax expression levels in the cerebral cortex increased in pups in the SCH and OH groups compared with those in the CON group. Conclusions: Maternal SCH during pregnancy may impair spatial learning and memory in the offspring and may be associated with the increased apoptosis in the cerebral cortex.

  14. Synthesis of new heterocyclic compounds based on pyrazolopyridine scaffold and evaluation of their neuroprotective potential in MPP+-induced neurodegeneration.

    Science.gov (United States)

    Jouha, Jabrane; Loubidi, Mohammed; Bouali, Jamila; Hamri, Salha; Hafid, Abderrafia; Suzenet, Franck; Guillaumet, Gérald; Dagcı, Taner; Khouili, Mostafa; Aydın, Fadime; Saso, Luciano; Armagan, Güliz

    2017-03-31

    Neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and Huntington's disease affect millions of people in the world. Thus several new approaches to treat brain disorders are under development. The aim of the present study is to synthesize potential neuroprotective heterocyclic compounds based on pyrazolopyridine derivatives and then to evaluate their effects in MPP + -induced neurodegeneration in human neuroblastoma cell line (SH-SY5Y cells). The effects of the compounds on cell viability were measured by MTT assay and the changes in apoptosis-related proteins including bax, Bcl-2, Bcl-xl and caspase-3 were investigated by western blot technique. Based on the cell viability results obtained by MTT assay, the percentage of neuroprotection-induced by compounds against MPP + -induced neurotoxicity in SH-SY5Y cells was between 20% and 30% at 5 μM concentrations of all synthesized compounds. Moreover, the downregulation in pro-apoptotic proteins including bax and caspase-3 were found following the novel synthesized compounds treatments and these effects were observed in a dose-dependent manner. Our results provide an evidence that these heterocyclic compounds based on pyrazolopyridine derivatives may have a role on dopaminergic neuroprotection via antiapoptotic pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  15. Endogenous IL-6 of mesenchymal stem cell improves behavioral outcome of hypoxic-ischemic brain damage neonatal rats by supressing apoptosis in astrocyte.

    Science.gov (United States)

    Gu, Yan; He, Mulan; Zhou, Xiaoqin; Liu, Jinngjing; Hou, Nali; Bin, Tan; Zhang, Yun; Li, Tingyu; Chen, Jie

    2016-01-14

    Mesenchymal stem cell (MSC) transplantation reduces the neurological impairment caused by hypoxic-ischemic brain damage (HIBD) via immunomodulation. In the current study, we found that MSC transplantation improved learning and memory function and enhanced long-term potentiation in neonatal rats subjected to HIBD and the amount of IL-6 released from MSCs was far greater than that of other cytokines. However, the neuroprotective effect of MSCs infected with siIL-6-transduced recombinant lentivirus (siIL-6 MSCs) was significantly weakened in the behavioural tests and electrophysiological analysis. Meanwhile, the hippocampal IL-6 levels were decreased following siIL-6 MSC transplantation. In vitro, the levels of IL-6 release and the levels of IL-6R and STAT3 expression were increased in both primary neurons and astrocytes subjected to oxygen and glucose deprivation (OGD) following MSCs co-culture. The anti-apoptotic protein Bcl-2 was upregulated and the pro-apoptotic protein Bax was downregulated in OGD-injured astrocytes co-cultured with MSCs. However, the siIL-6 MSCs suppressed ratio of Bcl-2/Bax in the injured astrocytes and induced apoptosis number of the injured astrocytes. Taken together, these data suggest that the neuroprotective effect of MSC transplantation in neonatal HIBD rats is partly mediated by IL-6 to enhance anti-apoptosis of injured astrocytes via the IL-6/STAT3 signaling pathway.

  16. Effect of β,β-Dimethylacrylshikonin on Inhibition of Human Colorectal Cancer Cell Growth in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Ting Feng

    2012-07-01

    Full Text Available In traditional Chinese medicine, shikonin and its derivatives, has been used in East Asia for several years for the prevention and treatment of several diseases, including cancer. We previously identified that β,β-dimethylacrylshikonin (DA could inhibit hepatocellular carcinoma growth. In the present study, we investigated the inhibitory effects of DA on human colorectal cancer (CRC cell line HCT-116 in vitro and in vivo. A viability assay showed that DA could inhibit tumor cell growth in a time- and dose-dependent manner. Flow cytometry showed that DA blocks the cell cycle at G0/G1 phase. Western blotting results demonstrated that the induction of apoptosis by DA correlated with the induction of pro-apoptotic proteins Bax, and Bid, and a decrease in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. Furthermore, treatment of HCT-116 bearing nude mice with DA significantly retarded the growth of xenografts. Consistent with the results in vitro, the DA-mediated suppression of HCT-116 xenografts correlated with Bax and Bcl-2. Taken together, these results suggest that DA could be a novel and promising approach to the treatment of CRC.

  17. Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay.

    Science.gov (United States)

    Weber, Alfred; Engelmaier, Andrea; Mohr, Gabriele; Haindl, Sonja; Schwarz, Hans Peter; Turecek, Peter L

    2017-01-05

    BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. 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.

  19. 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. 2010 Elsevier Ltd. All rights reserved.

  20. Oxcarbazepine causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

    Science.gov (United States)

    Song, Y; Zhong, M; Cai, F-C

    2018-01-01

    Anti-epileptic drugs (AEDs) are the main methods for treatment of neonatal seizures; however, a few AEDs may cause developing brain damage of neonate. This study aims to investigate effects of oxcarbazepine (OXC) on developing brain damage of neonatal rats. Both of neonatal and adult rats were divided into 6 groups, including Control, OXC 187.5 mg/kg, OXC 281.25 mg/kg, OXC 375 mg/kg group, LEV and PHT group. Body weight and brain weight were evaluated. Hematoxylin and eosin (HE) and Nissl staining were used to observe neurocyte morphology and Nissl bodies, respectively. Apoptosis was examined using TUNEL assay, and caspase 8 activity was evaluated using spectrophotometer method. Cytochrome C-release was evaluated using flow cytometry. Western blot was used to examine Bax and Bcl-2 expression. OXC 375 mg/kg treatment significantly decreased brain weight compared to Control group in neonatal rats (P5 rats) (pOxcarbazepine at a concentration of 281.25 mg/kg or more causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

  1. BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1(Ter/Ter) mice.

    Science.gov (United States)

    Cook, Matthew S; Coveney, Douglas; Batchvarov, Iordan; Nadeau, Joseph H; Capel, Blanche

    2009-04-15

    A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1(Ter/Ter)) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1(Ter/Ter) embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1(Ter/Ter) XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.

  2. Enhanced thermoelectric figure-of-merit in environmentally benign BaxSr2-xTiCoO6 double perovskites

    Science.gov (United States)

    Saxena, Mandvi; Roy, Pinku; Acharya, Megha; Bose, Imon; Tanwar, Khagesh; Maiti, Tanmoy

    2016-12-01

    Environmental friendly, non-toxic double perovskite BaxSr2-xTiCoO6 compositions with 0 ≤ x ≤ 0.2 were synthesized using solid-state reaction route for high temperature thermoelectric (TE) applications. XRD and SEM studies confirmed the presence of single-phase solid solution with highly dense microstructure for all the oxide compositions. Temperature dependent electrical conductivity measurement showed semiconductor to metal (M-S) transition in these double perovskites. Incorporation of barium in Sr2TiCoO6 pushed M-S transition to higher temperature making it a potential candidate for high temperature TE applications. Conductivity behaviors of these oxides were explained by small polaron model. Furthermore, these oxides exhibit a glass like behavior resulting in low thermal conductivity. Low temperature dielectric measurement revealed relaxor ferroelectric behavior in these oxides below room temperature. Transition of these relaxors into a glassy state beyond Burns temperature (TD) was found responsible for having low thermal conductivity in these oxides. Maximum dimensionless TE figure-of-merit ZT = 0.29 at 1223 K was achieved for BaxSr2-xTiCoO6 composition with x = 0.2.

  3. Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

    Energy Technology Data Exchange (ETDEWEB)

    Inadomi, Chiaki, E-mail: inadomic@nagasaki-u.ac.jp [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Murata, Hiroaki [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Ihara, Yoshito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Biochemistry, Wakayama Medical University, Wakayama 641-8509 (Japan); Goto, Shinji; Urata, Yoshishige [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Yodoi, Junji [Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto 606-8507 (Japan); Kondo, Takahito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Sumikawa, Koji [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan)

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. Black-Right-Pointing-Pointer NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. Black-Right-Pointing-Pointer Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

  4. Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

    International Nuclear Information System (INIS)

    Inadomi, Chiaki; Murata, Hiroaki; Ihara, Yoshito; Goto, Shinji; Urata, Yoshishige; Yodoi, Junji; Kondo, Takahito; Sumikawa, Koji

    2012-01-01

    Highlights: ► GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. ► NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. ► Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

  5. Oxidative stress and hepatic stellate cell activation are key events in arsenic induced liver fibrosis in mice

    International Nuclear Information System (INIS)

    Ghatak, Subhadip; Biswas, Ayan; Dhali, Gopal Krishna; Chowdhury, Abhijit; Boyer, James L.; Santra, Amal

    2011-01-01

    Arsenic is an environmental toxicant and carcinogen. Exposure to arsenic is associated with development of liver fibrosis and portal hypertension through ill defined mechanisms. We evaluated hepatic fibrogenesis after long term arsenic exposure in a murine model. BALB/c mice were exposed to arsenic by daily gavages of 6 μg/gm body weight for 1 year and were evaluated for markers of hepatic oxidative stress and fibrosis, as well as pro-inflammatory, pro-apoptotic and pro-fibrogenic factors at 9 and 12 months. Hepatic NADPH oxidase activity progressively increased in arsenic exposure with concomitant development of hepatic oxidative stress. Hepatic steatosis with occasional collection of mononuclear inflammatory cells and mild portal fibrosis were the predominant liver lesion observed after 9 months of arsenic exposure, while at 12 months, the changes included mild hepatic steatosis, inflammation, necrosis and significant fibrosis in periportal areas. The pathologic changes in the liver were associated with markers of hepatic stellate cells (HSCs) activation, matrix reorganization and fibrosis including α-smooth muscle actin, transforming growth factor-β1, PDGF-Rβ, pro-inflammatory cytokines and enhanced expression of tissue inhibitor of metalloproteinase-1 and pro(α) collagen type I. Moreover, pro-apoptotic protein Bax was dominantly expressed and Bcl-2 was down-regulated along with increased number of TUNEL positive hepatocytes in liver of arsenic exposed mice. Furthermore, HSCs activation due to increased hepatic oxidative stress observed after in vivo arsenic exposure was recapitulated in co-culture model of isolated HSCs and hepatocytes exposed to arsenic. These findings have implications not only for the understanding of the pathology of arsenic related liver fibrosis but also for the design of preventive strategies in chronic arsenicosis.

  6. Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation

    International Nuclear Information System (INIS)

    Cho, Seung-Woo; Hartle, Lauren; Son, Sun Mi; Yang, Fan; Goldberg, Michael; Xu, Qiaobing; Langer, Robert; Anderson, Daniel G.

    2008-01-01

    RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-α (TNF-α). siRNA was designed and synthesized targeting tumor necrosis factor-α receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-α expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-α expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia

  7. Nutrient-intake-level-dependent regulation of intestinal development in newborn intrauterine growth-restricted piglets via glucagon-like peptide-2.

    Science.gov (United States)

    Liu, J; Liu, Z; Gao, L; Chen, L; Zhang, H

    2016-10-01

    The objective of the present study was to investigate the intestinal development of newborn intrauterine growth-restricted (IUGR) piglets subjected to normal nutrient intake (NNI) or restricted nutrient intake (RNI). Newborn normal birth weight (NBW) and IUGR piglets were allotted to NNI or RNI levels for 4 weeks from day 8 postnatal. IUGR piglets receiving NNI had similar growth performance compared with that of NBW piglets. Small intestine length and villous height were greater in IUGR piglets fed the NNI than that of piglets fed the RNI. Lactase activity was increased in piglets fed the NNI compared with piglets fed the RNI. Absorptive function, represented by active glucose transport by the Ussing chamber method and messenger RNA (mRNA) expressions of two main intestinal glucose transporters, Na+-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2), were greater in IUGR piglets fed the NNI compared with piglets fed the RNI regimen. The apoptotic process, characterized by caspase-3 activity (a sign of activated apoptotic cells) and mRNA expressions of p53 (pro-apoptotic), bcl-2-like protein 4 (Bax) (pro-apoptotic) and B-cell lymphoma-2 (Bcl-2) (anti-apoptotic), were improved in IUGR piglets fed the NNI regimen. To test the hypothesis that improvements in intestinal development of IUGR piglets fed NNI might be mediated through circulating glucagon-like peptide-2 (GLP-2), GLP-2 was injected subcutaneously to IUGR piglets fed the RNI from day 8 to day 15 postnatal. Although the intestinal development of IUGR piglets fed the RNI regimen was suppressed compared with those fed the NNI regimen, an exogenous injection of GLP-2 was able to bring intestinal development to similar levels as NNI-fed IUGR piglets. Collectively, our results demonstrate that IUGR neonates that have NNI levels could improve intestinal function via the regulation of GLP-2.

  8. Deletion of the N-terminus of IKKγ induces apoptosis in keratinocytes and impairs the AKT/PTEN signaling pathway

    International Nuclear Information System (INIS)

    Leis, Hugo; Sanchis, Ana; Perez, Paloma

    2007-01-01

    The regulatory subunit IKKγ/NEMO is crucial for skin development and function and although devoid of kinase activity, loss of IKKγ function completely abolishes the activation of NF-κB by all pro-inflammatory cytokines. To inhibit the IκB kinase (IKK) complex in keratinocytes, we have used a dominant negative approach by generating stable transfectants of an N-terminal deletion of IKKγ (IKKγ-DN97) that uncouples formation of the IKK complex. Expression of this mutant in PB keratinocytes (PB-IKKγ-DN97) delayed growth kinetics, caused morphological changes and dramatically augmented apoptosis even in the absence of pro-apoptotic stimuli, as determined by cell morphology, TUNEL and caspase-3 cleavage. Moreover, in PB-IKKγ-DN97 cells, TNF-α and IL-1 treatment failed to induce degradation of IκBα, phosphorylation of p65 on Ser 536 and nuclear translocation which, consequently, reduced κB-binding activity. In PB-IKKγ-DN97 cells, accumulation of IκBα correlated with a downregulation of AKT activity and an increase of PTEN protein levels whereas pro-apoptotic p53 target genes Bax and Puma were upregulated. These effects were most likely mediated through IKK since coexpression of the wild-type form of IKKγ in keratinocytes partially reversed apoptosis and reduced PTEN expression. Thus, our data suggest a negative cross-talk mechanism involving PTEN and NF-κB, critical for the anti-apoptotic role of NF-κB in keratinocytes

  9. Endoplasmic reticulum stress and IRE-1 signaling cause apoptosis in colon cancer cells in response to andrographolide treatment.

    Science.gov (United States)

    Banerjee, Aditi; Ahmed, Hafiz; Yang, Peixin; Czinn, Steven J; Blanchard, Thomas G

    2016-07-05

    The plant metabolite andrographolide induces cell cycle arrest and apoptosis in cancer cells. The mechanism(s) by which andrographolide induces apoptosis however, have not been elucidated. The present study was performed to determine the molecular events that promote apoptosis in andrographolide treated cells using T84, HCT116 and COLO 205 colon cancer cell lines. Andrographolide was determined to limit colony formation and Ki67 expression, alter nuclear morphology, increase cytoplasmic histone-associated-DNA-fragments, and increase cleaved caspase-3 levels. Andrographolide also induced significantly higher expression of endoplasmic reticulum (ER) stress proteins GRP-78 and IRE-1 by 48 h but not PERK or ATF6. Apoptosis signaling molecules BAX, spliced XBP-1 and CHOP were also significantly increased. Moreover, chemical inhibition of ER stress or IRE-1 depletion with siRNA in andrographolide treated cells significantly limited expression of IRE-1 and CHOP as determined by immunofluorescence staining, real time PCR, or immunobloting. This was accompanied by a decreased BAX/Bcl-2 ratio. Andrographolide significantly promotes cancer cell death compared to normal cells. These data demonstrate that andrographolide associated ER stress contributes to apoptosis through the activation of a pro-apoptotic GRP-78/IRE-1/XBP-1/CHOP signaling pathway.

  10. Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoposis

    International Nuclear Information System (INIS)

    Zhou Yijun; Wang Jiahe; Zhang Jin

    2006-01-01

    Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-κB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-κB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease

  11. Altholactone Inhibits NF-κB and STAT3 Activation and Induces Reactive Oxygen Species-Mediated Apoptosis in Prostate Cancer DU145 Cells

    Directory of Open Access Journals (Sweden)

    Chunwa Jiang

    2017-02-01

    Full Text Available Altholactone, a natural compound isolated from Goniothalamus spp., has demonstrated anti-inflammatory and anticancer activities, but its molecular mechanisms are still not fully defined. Nuclear factor kappa B (NF-κB and signal transducer and activator of transcription 3 (STAT3 play pivotal roles in the cell survival of many human tumors. The objective of this study was to elucidate the mechanism of action of altholactone against prostate cancer DU145 cells and to evaluate whether its effects are mediated by inhibition of NF-κB and STAT3 activity. Altholactone inhibited proliferation of DU145 cells and induced cell cycle arrest in S phase and triggered apoptosis. Reporter assays revealed that altholactone repressed p65- and TNF-α-enhanced NF-κB transcriptional activity and also inhibited both constitutive and IL-6-induced transcriptional activity of STAT3. Consistent with this, altholactone down-regulated phosphorylation of STAT3 and moreover, decreased constitutively active mutant of STAT3 (STAT3C-induced transcriptional activity. Altholactone treatment also results in down-regulation of STAT3 target genes such as survivin, and Bcl-2 followed by up regulation of pro-apoptotic Bax protein. However, pre-treatment with the antioxidant N-acetylcysteine (NAC significantly inhibited the activation of Bax and prevented down-regulation of STAT3 target genes. Collectively, our findings suggest that altholactone induces DU145 cells death through inhibition of NF-κB and STAT3 activity.

  12. Baicalein antagonizes rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to Parkinsonism.

    Science.gov (United States)

    Song, Ju-Xian; Choi, Mandy Yuen-Man; Wong, Kavin Chun-Kit; Chung, Winkie Wing-Yan; Sze, Stephen Cho-Wing; Ng, Tzi-Bun; Zhang, Kalin Yan-Bo

    2012-01-21

    Two active compounds, baicalein and its glycoside baicalin were found in the dried root of Scutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluate the protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to parkinsonism. Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis was evaluated with a fluorescent DNA-binding probe Hoechst 33258. The production of reactive oxidative species (ROS) and loss of mitochondrial membrane potential (ΔΨm) were determined by fluorescent staining with DCFH-DA and Rhodanmine 123, respectively. The expression of Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 was determined by the Western blots. Baicalein significantly increased viability and decreased rotenone-induced death of SH-SY5Y cells in a dose-dependent manner. Pre- and subsequent co-treatment with baicalein preserved the cell morphology and attenuated the nuclear apoptotic characteristics triggered by rotenone. Baicalein antagonized rotenone-induced overproduction of ROS, loss of ΔΨm, the increased expression of Bax, cleaved caspase-3 and phosphorylated ERK1/2 and the decreased expression of Bcl-2. The antioxidative effect, mitochondrial protection and modulation of anti-and pro-apoptotic proteins are related to the neuroprotective effects of baicalein against rotenone induced cell death in SH-SY5Y cells.

  13. Baicalein antagonizes rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to Parkinsonism

    Directory of Open Access Journals (Sweden)

    Song Ju-Xian

    2012-01-01

    Full Text Available Abstract Background Two active compounds, baicalein and its glycoside baicalin were found in the dried root of Scutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluate the protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to parkinsonism. Methods Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis was evaluated with a fluorescent DNA-binding probe Hoechst 33258. The production of reactive oxidative species (ROS and loss of mitochondrial membrane potential (ΔΨm were determined by fluorescent staining with DCFH-DA and Rhodanmine 123, respectively. The expression of Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 was determined by the Western blots. Results Baicalein significantly increased viability and decreased rotenone-induced death of SH-SY5Y cells in a dose-dependent manner. Pre- and subsequent co-treatment with baicalein preserved the cell morphology and attenuated the nuclear apoptotic characteristics triggered by rotenone. Baicalein antagonized rotenone-induced overproduction of ROS, loss of ΔΨm, the increased expression of Bax, cleaved caspase-3 and phosphorylated ERK1/2 and the decreased expression of Bcl-2. Conclusion The antioxidative effect, mitochondrial protection and modulation of anti-and pro-apoptotic proteins are related to the neuroprotective effects of baicalein against rotenone induced cell death in SH-SY5Y cells.

  14. Effects of amifostine on radiation-induced apoptosis in mouse ovary

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of); Lee, Kyoung Hee; Yoon Yong Dal [Hanyang Univ., Seoul (Korea, Republic of)

    2002-07-01

    The present study was designed to assess the radioprotective effects of amifostine on ovarian follicles. Three week-old female mice with or without pretreatment of amifostine were irradiated with 6.42 Gy of {gamma} -ray. Ovaries were collected 0 and 6h after irradiation. DNA fragmentation pattern and expression of genes and activity of proteins related with apoptosis were investigated by means of RT-PCR and Western blot. Proliferation of granulosa cells was reduced and incidence rate of follicular atresia was increased in ovarian follicles in {gamma} -ray irradiated mice compared to those in control or amifostine-treated group. DNA fragmentation was increased in time-dependent manner in granulosa cells of all irradiated groups. However, no difference between amifostine pre-treated group and irradiated groups was found and the expression of p53 as tumor suppressor gene and Bax as one of pro-apoptotic Bcl-2 family was increased in irradiated mice ovaries. PARP as DNA damage sensor was cleaved and its upstream regulator, Caspase-3 was activated. Fas (CD95), one of membrane bound receptor which initiated apoptosis pathway was expressed by {gamma} -ray. Pretreatment of amifostine before irradiation, however, inhibited the expression of p53 and Bax, the cleavage of PARP and the activation of Caspase-3. In conclusion, amifostine has an inhibitory effect on gonadal cell apoptosis induced by {gamma} -ray irradiation, through lowering the expression of apoptotic signaling peptide and the activation of DNA repair enzymes.

  15. Suppression of STAT3 NH2 -terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

    Science.gov (United States)

    Ray, Sutapa; Coulter, Don W; Gray, Shawn D; Sughroue, Jason A; Roychoudhury, Shrabasti; McIntyre, Erin M; Chaturvedi, Nagendra K; Bhakat, Kishor K; Joshi, Shantaram S; McGuire, Timothy R; Sharp, John G

    2018-04-01

    Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH 2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients. © 2017 Wiley Periodicals, Inc.

  16. Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins.

    Science.gov (United States)

    Chowdhury, Indrajit; Thompson, Winston E; Welch, Crystal; Thomas, Kelwyn; Matthews, Roland

    2013-12-01

    Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB → Mek-Erk1

  17. Musa paradisiaca inflorescence induces human colon cancer cell death by modulating cascades of transcriptional events.

    Science.gov (United States)

    K B, Arun; Madhavan, Aravind; T R, Reshmitha; Thomas, Sithara; Nisha, P

    2018-01-24

    Colorectal cancer (CRC) is one of the leading causes of cancer death, and diet plays an important role in the etiology of CRC. Traditional medical practitioners in many South Asian countries use plantain inflorescence to treat various gastro-intestinal ailments. The aim of the present study was to investigate the anticancer effects of extracts of inflorescence of Musa paradisiaca against HT29 human colon cancer cells and elucidate the mechanism of these effects by studying the modulation of cascades of transcriptional events. In vitro assays depicted that methanol extract of Musa paradisiaca inflorescence (PIMET) was cytotoxic to HT29 cells. PIMET induced DNA damage and arrested the cell cycle at the G2/M phase. Expression studies showed that PIMET pretreatment upregulates pro-apoptotic Bcl2 and downregulates anti-apoptotic Bax proteins. Different assays showed that the deregulation of pro/antiapoptotic proteins reduces the mitochondrial membrane potential and ATP production; moreover, it enhances cytochrome c release, which triggers the apoptotic pathway, and further cleaves caspase 3 and PARP proteins, resulting in apoptosis. Changes in the protein expression profile of HT29 cells after PIMET treatment were analyzed using mass-spectrometry-based proteomics. PIMET treatment significantly altered the expression of HT29 protein; interestingly, X-linked inhibitor of apoptosis protein was also downregulated. Alteration in the expression of this protein has significant effects, leading to HT29 cell death.

  18. Hilar granule cells of the mouse dentate gyrus: effects of age, septotemporal location, strain, and selective deletion of the proapoptotic gene BAX.

    Science.gov (United States)

    Bermudez-Hernandez, Keria; Lu, Yi-Ling; Moretto, Jillian; Jain, Swati; LaFrancois, John J; Duffy, Aine M; Scharfman, Helen E

    2017-09-01

    The dentate gyrus (DG) principal cells are glutamatergic granule cells (GCs), and they are located in a compact cell layer. However, GCs are also present in the adjacent hilar region, but have been described in only a few studies. Therefore, we used the transcription factor prospero homeobox 1 (Prox1) to quantify GCs at postnatal day (PND) 16, 30, and 60 in a common mouse strain, C57BL/6J mice. At PND16, there was a large population of Prox1-immunoreactive (ir) hilar cells, with more in the septal than temporal hippocampus. At PND30 and 60, the size of the hilar Prox1-ir cell population was reduced. Similar numbers of hilar Prox1-expressing cells were observed in PND30 and 60 Swiss Webster mice. Prox1 is usually considered to be a marker of postmitotic GCs. However, many Prox1-ir hilar cells, especially at PND16, were not double-labeled with NeuN, a marker typically found in mature neurons. Most hilar Prox1-positive cells at PND16 co-expressed doublecortin (DCX) and calretinin, markers of immature GCs. Double-labeling with a marker of actively dividing cells, Ki67, was not detected. These results suggest that, surprisingly, a large population of cells in the hilus at PND16 are immature GCs (Type 2b and Type 3 cells). We also asked whether hilar Prox1-ir cell numbers are modifiable. To examine this issue, we conditionally deleted the proapoptotic gene BAX in Nestin-expressing cells at a time when there are numerous immature GCs in the hilus, PND2-8. When these mice were examined at PND60, the numbers of Prox1-ir hilar cells were significantly increased compared to control mice. However, deletion of BAX did not appear to change the proportion that co-expressed NeuN, suggesting that the size of the hilar Prox1-expressing population is modifiable. However, deleting BAX, a major developmental disruption, does not appear to change the proportion that ultimately becomes neurons.

  19. Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

    International Nuclear Information System (INIS)

    Yu, Zhendong; Wang, Hao; Zhang, Libin; Tang, Aifa; Zhai, Qinna; Wen, Jianxiang; Yao, Li; Li, Pengfei

    2009-01-01

    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.

  20. 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.

  1. High levels of X-linked Inhibitor-of-Apoptosis Protein (XIAP) are indicative of radio chemotherapy resistance in rectal cancer

    International Nuclear Information System (INIS)

    Flanagan, L.; Kehoe, J.; Fay, J.; Bacon, O.; Lindner, A.U.; Kay, E.W.; Deasy, J.; McNamara, D.A.; Prehn, J.H.M.

    2015-01-01

    The mainstay of treatment in rectal cancer is neoadjuvant radio chemotherapy prior to surgery, in an attempt to downstage the tumour, allowing for more complete removal during surgery. In 40 % of cases however, this neoadjuvant radio chemotherapy fails to achieve tumour regression, partly due insufficient apoptosis signaling. X-linked Inhibitor of Apoptosis Protein (XIAP) is an anti-apoptotic protein that has been reported to contribute to disease progression and chemotherapy resistance. We obtained rectal biopsy normal and matched tumour tissue from 29 rectal cancer patients with varying degrees of tumour regression, and using Western blot, examined anti-apoptotic XIAP and pro-apoptotic Smac protein levels in these tissues, with the aim to examine whether disturbed XIAP/Smac levels may be an indicator of neoadjuvant radio chemotherapy resistance. Expression of inhibitor of apoptosis proteins cIAP-1 and cIAP-2 was also examined. We found that levels of XIAP increased in accordance with the degree of radio chemotherapy resistance of the tissue. Levels of this protein were also significantly higher in tumour tissue, compared to matched normal tissue in highly resistant tissue. In contrast, Smac protein levels did not increase with radio chemotherapy resistance, and the protein was similarly expressed in normal and tumour tissue, indicating a shift in the balance of these proteins. Post treatment surgical resection tissue was available for 8 patients. When we compared matched tissue pre- and post- radio chemotherapy we found that XIAP levels increased significantly during treatment in both normal and tumour tissue, while Smac levels did not change. cIAP-1 and cIAP-2 levels were not differentially expressed in varying degrees of radio chemotherapy resistance, and neoadjuvant therapy did not alter expression of these proteins. These data indicate that disturbance of the XIAP/Smac balance may be a driver of radio chemotherapy resistance, and hence high levels of XIAP may

  2. On the Sr1−xBaxFeO2F Oxyfluoride Perovskites: Structure and Magnetism from Neutron Diffraction and Mössbauer Spectroscopy

    Science.gov (United States)

    García-Ramos, Crisanto A.; Retuerto, María; Alonso, José Antonio

    2016-01-01

    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. PMID:28774089

  3. The effects of escitalopram on myocardial apoptosis and the expression of Bax and Bcl-2 during myocardial ischemia/reperfusion in a model of rats with depression.

    Science.gov (United States)

    Wang, Yiming; Zhang, Hongming; Chai, Fangxian; Liu, Xingde; Berk, Michael

    2014-12-04

    Major depressive disorder (MDD) is an independent risk factor for coronary heart disease (CHD), and influences the occurrence and prognosis of cardiovascular events. Although there is evidence that antidepressants may be cardioprotective after acute myocardial infarction (AMI) comorbid with MDD, the operative pathophysiological mechanisms remain unclear. Our aim was therefore to explore the molecular mechanisms of escitalopram on myocardial apoptosis and the expression of Bax and Bcl-2 in a rat model of depression during myocardial ischemia/reperfusion (I/R). Rats were divided randomly into 3 groups (n = 8): D group (depression), DI/R group (depression with myocardial I/R) and escitalopram + DI/R group. The rats in all three groups underwent the same chronic mild stress and separation for 21 days, at the same time, in the escitalopram + DI/R group, rats were administered escitalopram by gavage (10 mg/kg/day). Ligation of the rat's left anterior descending branch was done in the myocardial I/R model. Following which behavioral tests were done. The size of the myocardial infarction was detected using 1.5% TTC dye. The Tunel method was used to detect apoptotic myocardial cells, and both the Rt-PCR method and immunohistochemical techniques were used to detect the expression of Bcl-2 and Bax. Compared with the D and DI/R groups, rats in Escitalopram + DI/R group showed significantly increased movements and sucrose consumption (P escitalopram + DI/R group was significantly decreased (P escitalopram + DI/R groups (P escitalopram + DI/R group were significantly decreased (P escitalopram + DI/R group (P escitalopram. This suggests that clinically escitalopram may have a direct cardioprotective after acute myocardial infarction.

  4. 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.

  5. Benfotiamine accelerates the healing of ischaemic diabetic limbs in mice through protein kinase B/Akt-mediated potentiation of angiogenesis and inhibition of apoptosis.

    Science.gov (United States)

    Gadau, S; Emanueli, C; Van Linthout, S; Graiani, G; Todaro, M; Meloni, M; Campesi, I; Invernici, G; Spillmann, F; Ward, K; Madeddu, P

    2006-02-01

    Benfotiamine, a vitamin B1 analogue, reportedly prevents diabetic microangiopathy. The aim of this study was to evaluate whether benfotiamine is of benefit in reparative neovascularisation using a type I diabetes model of hindlimb ischaemia. We also investigated the involvement of protein kinase B (PKB)/Akt in the therapeutic effects of benfotiamine. Streptozotocin-induced diabetic mice, given oral benfotiamine or vehicle, were subjected to unilateral limb ischaemia. Reparative neovascularisation was analysed by histology. The expression of Nos3 and Casp3 was evaluated by real-time PCR, and the activation state of PKB/Akt was assessed by western blot analysis and immunohistochemistry. The functional importance of PKB/Akt in benfotiamine-induced effects was investigated using a dominant-negative construct. Diabetic muscles showed reduced transketolase activity, which was corrected by benfotiamine. Importantly, benfotiamine prevented ischaemia-induced toe necrosis, improved hindlimb perfusion and oxygenation, and restored endothelium-dependent vasodilation. Histological studies revealed the improvement of reparative neovascularisation and the inhibition of endothelial and skeletal muscle cell apoptosis. In addition, benfotiamine prevented the vascular accumulation of advanced glycation end products and the induction of pro-apoptotic caspase-3, while restoring proper expression of Nos3 and Akt in ischaemic muscles. The benefits of benfotiamine were nullified by dominant-negative PKB/Akt. In vitro, benfotiamine stimulated the proliferation of human EPCs, while inhibiting apoptosis induced by high glucose. In diabetic mice, the number of circulating EPCs was reduced, with the deficit being corrected by benfotiamine. We have demonstrated, for the first time, that benfotiamine aids the post-ischaemic healing of diabetic animals via PKB/Akt-mediated potentiation of angiogenesis and inhibition of apoptosis. In addition, benfotiamine combats the diabetes-induced deficit in

  6. Genome wide gene expression regulation by HIP1 Protein Interactor, HIPPI: Prediction and validation

    Directory of Open Access Journals (Sweden)

    Lahiri Ansuman

    2011-09-01

    Full Text Available Abstract Background HIP1 Protein Interactor (HIPPI is a pro-apoptotic protein that induces Caspase8 mediated apoptosis in cell. We have shown earlier that HIPPI could interact with a specific 9 bp sequence motif, defined as the HIPPI binding site (HBS, present in the upstream promoter of Caspase1 gene and regulate its expression. We also have shown that HIPPI, without any known nuclear localization signal, could be transported to the nucleus by HIP1, a NLS containing nucleo-cytoplasmic shuttling protein. Thus our present work aims at the investigation of the role of HIPPI as a global transcription regulator. Results We carried out genome wide search for the presence of HBS in the upstream sequences of genes. Our result suggests that HBS was predominantly located within 2 Kb upstream from transcription start site. Transcription factors like CREBP1, TBP, OCT1, EVI1 and P53 half site were significantly enriched in the 100 bp vicinity of HBS indicating that they might co-operate with HIPPI for transcription regulation. To illustrate the role of HIPPI on transcriptome, we performed gene expression profiling by microarray. Exogenous expression of HIPPI in HeLa cells resulted in up-regulation of 580 genes (p HIP1 was knocked down. HIPPI-P53 interaction was necessary for HIPPI mediated up-regulation of Caspase1 gene. Finally, we analyzed published microarray data obtained with post mortem brains of Huntington's disease (HD patients to investigate the possible involvement of HIPPI in HD pathogenesis. We observed that along with the transcription factors like CREB, P300, SREBP1, Sp1 etc. which are already known to be involved in HD, HIPPI binding site was also significantly over-represented in the upstream sequences of genes altered in HD. Conclusions Taken together, the results suggest that HIPPI could act as an important transcription regulator in cell regulating a vast array of genes, particularly transcription factors and at least, in part, play a

  7. 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

  8. TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells.

    Directory of Open Access Journals (Sweden)

    Hyou-Ju Jin

    Full Text Available Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA and cryptotanshinone (CT on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak. TIIA and CT (0.3 and 3 μΜ, in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.

  9. Bad and Bid - potential background players in preneoplastic to neoplastic shift in human endometrium.

    Science.gov (United States)

    Driak, D; Dvorska, M; Bolehovska, P; Svandova, I; Novotny, J; Halaska, M

    2014-01-01

    The most common malignancies of the female genital tract are endometrial carcinomas, whose are generally proceeded by hyperplasia. The maintenance of tissue homeostasis is to great extent governed by apoptosis, whose defects can lead to the preneoplastic and/or cancerous changes. Endometrial apoptosis involves among others three groups of proteins of the Bcl-2 family. First group contains anti-apoptotic proteins (e. g. Bcl-2, Bcl-xL). The other two groups belong to the pro-apoptotic proteins with three (e. g. Bax, Bak) or one (e. g. Bad, Bid) so-called BH domains. Bad and Bid trigger the oligomerization of Bak and Bax protein, which permeabilize the outer mitochondrial wall. Unlike Bid, Bad cannot directly trigger apoptosis. Instead, Bad lowers the threshold at which apoptosis is induced, by binding anti-apoptotic Bcl-2 proteins. However, their mutual counterbalance or synergism in the human endometrium has not been reported yet.In this study, the levels of Bid and Bad were measured using SDS-PAGE and Western blotting with specific antibodies, with the aim to analyse expression of Bid and Bad proteins in normal (NE), hyperplastic (HE) and cancerous (CE) endometrium. We demonstrated that Bid expression in CE reached only 47% and 50% of this observed in NE and HE. Conversely, Bad expression in HE reached only 40% and 36% of this observed in NE and CE, respectively. We detected no significant changes of Bid expression between HE and NE, and levels of Bad protein were not different between CE and NE.Trend of Bid and Bad protein expression is clearly opposite in HE and CE. We hypothesise that disrupted apoptotic program in CE seems to be reduced further by lowering levels of direct apoptotic trigger protein Bid. We suggest that the adenocarcinoma tissue of human endometrium thus tries to strengthen its apoptotic effort by lowering the apoptotic threshold via higher Bad levels.

  10. Self-assembled silk sericin/poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery

    International Nuclear Information System (INIS)

    Mandal, Biman B; Kundu, S C

    2009-01-01

    In recent times self-assembled micellar nanoparticles have been successfully employed in tissue engineering for targeted drug delivery applications. In this review, silk sericin protein from non-mulberry Antheraea mylitta tropical tasar silk cocoons was blended with pluronic F-127 and F-87 in the presence of solvents to achieve self-assembled micellar nanostructures capable of carrying both hydrophilic (FITC-inulin) and hydrophobic (anticancer drug paclitaxel) drugs. The fabricated nanoparticles were subsequently characterized for their size distribution, drug loading capability, cellular uptake and cytotoxicity. Nanoparticle sizes ranged between 100 and 110 nm in diameter as confirmed by dynamic light scattering. Rapid uptake of these particles into cells was observed in in vitro cellular uptake studies using breast cancer MCF-7 cells. In vitro cytotoxicity assay using paclitaxel-loaded nanoparticles against breast cancer cells showed promising results comparable to free paclitaxel drugs. Drug-encapsulated nanoparticle-induced apoptosis in MCF-7 cells was confirmed by FACS and confocal microscopic studies using Annexin V staining. Up-regulation of pro-apoptotic protein Bax, down-regulation of anti-apoptotic protein Bcl-2 and cleavage of regulatory protein PARP through Western blot analysis suggested further drug-induced apoptosis in cells. This study projects silk sericin protein as an alternative natural biomaterial for fabrication of self-assembled nanoparticles in the presence of poloxamer for successful delivery of both hydrophobic and hydrophilic drugs to target sites.

  11. ROS-mediated apoptotic cell death in prostate cancer LNCaP cells induced by biosurfactant stabilized CdS quantum dots.

    Science.gov (United States)

    Singh, Braj R; Singh, Brahma N; Khan, W; Singh, H B; Naqvi, A H

    2012-08-01

    Cadmium sulfide (CdS) quantum dots (QDs) have raised great attention because of their superior optical properties and wide utilization in biological and biomedical studies. However, little is known about the cell death mechanisms of CdS QDs in human cancer cells. This study was designed to investigate the possible mechanisms of apoptosis induced by biosurfactant stabilized CdS QDs (denoted as "bsCdS QDs") in human prostate cancer LNCaP cells. It was also noteworthy that apoptosis correlated with reactive oxygen species (ROS) production, mitochondrial damage, oxidative stress and chromatin condensation in a dose- and time-dependent manner. Results also showed involvement of caspases, Bcl-2 family proteins, heat shock protein 70, and a cell-cycle checkpoint protein p53 in apoptosis induction by bsCdS QDs in LNCaP cells. Moreover, pro-apoptotic protein Bax was upregulated and the anti-apoptotic proteins, survivin and NF-κB were downregulated in bsCdS QDs exposed cells. Protection of N-acetyl cysteine (NAC) against ROS clearly suggested the implication of ROS in hyper-activation of apoptosis and cell death. It is encouraging to conclude that biologically stabilized CdS QDs bear the potential of its applications in biomedicine, such as tumor therapy specifically by inducing caspase-dependent apoptotic cell death of human prostate cancer LNCaP cells. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  12. Self-assembled silk sericin/poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery

    Energy Technology Data Exchange (ETDEWEB)

    Mandal, Biman B; Kundu, S C, E-mail: kundu@hijli.iitkgp.ernet.i [Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302 (India)

    2009-09-02

    In recent times self-assembled micellar nanoparticles have been successfully employed in tissue engineering for targeted drug delivery applications. In this review, silk sericin protein from non-mulberry Antheraea mylitta tropical tasar silk cocoons was blended with pluronic F-127 and F-87 in the presence of solvents to achieve self-assembled micellar nanostructures capable of carrying both hydrophilic (FITC-inulin) and hydrophobic (anticancer drug paclitaxel) drugs. The fabricated nanoparticles were subsequently characterized for their size distribution, drug loading capability, cellular uptake and cytotoxicity. Nanoparticle sizes ranged between 100 and 110 nm in diameter as confirmed by dynamic light scattering. Rapid uptake of these particles into cells was observed in in vitro cellular uptake studies using breast cancer MCF-7 cells. In vitro cytotoxicity assay using paclitaxel-loaded nanoparticles against breast cancer cells showed promising results comparable to free paclitaxel drugs. Drug-encapsulated nanoparticle-induced apoptosis in MCF-7 cells was confirmed by FACS and confocal microscopic studies using Annexin V staining. Up-regulation of pro-apoptotic protein Bax, down-regulation of anti-apoptotic protein Bcl-2 and cleavage of regulatory protein PARP through Western blot analysis suggested further drug-induced apoptosis in cells. This study projects silk sericin protein as an alternative natural biomaterial for fabrication of self-assembled nanoparticles in the presence of poloxamer for successful delivery of both hydrophobic and hydrophilic drugs to target sites.

  13. Involvement of p63 in the herpes simplex virus-1-induced demise of corneal cells.

    Science.gov (United States)

    Orosz, László; Gallyas, Eva; Kemény, Lajos; Mándi, Yvette; Facskó, Andrea; Megyeri, Klára

    2010-06-07

    The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues, including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1, we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line (SIRC). SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of p63 and Bax proteins. Indirect immunofluorescence assays and Western blot analyses demonstrated the presence of HSV-1 glycoprotein D (gD) in the infected SIRC cell line, and the pattern of gD expression was consistent with efficient viral replication. The results of MTT and ELISA assays showed that HSV-1 elicited a strong cytopathic effect, and apoptosis played an important role in the demise of the infected cells. Mock-infected SIRC cells displayed the constitutive expression of DeltaNp63alpha. The expressions of the Bax-beta and TAp63gamma isoforms were considerably increased, whereas the level of DeltaNp63alpha was decreased in the HSV-1-infected SIRC cells. Experiments involving the use of acyclovir showed that viral DNA replication was necessary for the accumulation of TAp63gamma. These data suggest that a direct, virus-mediated cytopathic effect may play an important role in the pathogenic mechanism of herpetic keratitis. By disturbing the delicate balance between the pro-survival DeltaN and the pro-apoptotic TA isoforms, HSV-1 may cause profound alterations in the viability of the ocular cells and in the tissue homeostasis of the ocular surface.

  14. Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection.

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    Oliver Kepp

    2009-03-01

    Full Text Available Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo. While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1, which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L, respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

  15. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

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    Lee, Dae-Hee, E-mail: leedneo@gmail.com [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Kim, Dong-Wook [Department of Microbiology, Immunology, and Cancer Biology, University of VA (United States); Jung, Chang-Hwa [Division of Metabolism and Functionality Research, Korea Food Research Institute (Korea, Republic of); Lee, Yong J. [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Park, Daeho, E-mail: daehopark@gist.ac.kr [School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of)

    2014-09-15

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.

  16. Pectenotoxin-2 from Marine Sponges: A Potential Anti-Cancer Agent—A Review

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    Wun-Jae Kim

    2011-11-01

    Full Text Available Pectenotoxin-2 (PTX-2, which was first identified as a cytotoxic entity in marine sponges, has been reported to display significant cytotoxicity to human cancer cells where it inhibits mitotic separation and cytokinesis through the depolymerization of actin filaments. In the late stage of endoreduplication, the effects of PTX-2 on different cancer cells involves: (i down-regulation of anti-apoptotic Bcl-2 members and IAP family proteins; (ii up-regulation of pro-apoptotic Bax protein and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL-receptor 1/receptor 2 (DR4/DR5; and (iii mitochondrial dysfunction. In addition, PTX-2 induces apoptotic effects through suppression of the nuclear factor κB (NF-κB signaling pathway in several cancer cells. Analysis of cell cycle regulatory proteins showed that PTX-2 increases phosphorylation of Cdc25c and decreases protein levels of Cdc2 and cyclin B1. Cyclin-dependent kinase (Cdk inhibitor p21 and Cdk2, which are associated with the induction of endoreduplication, were upregulated. Furthermore, it was found that PTX-2 suppressed telomerase activity through the transcriptional and post-translational suppression of hTERT. The purpose of this review was to provide an update regarding the anti-cancer mechanism of PTX-2, with a special focus on its effects on different cellular signaling cascades.

  17. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    International Nuclear Information System (INIS)

    Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J.; Park, Daeho

    2014-01-01

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway

  18. Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

    International Nuclear Information System (INIS)

    Clapp, Caitlin; Portt, Liam; Khoury, Chamel; Sheibani, Sara; Eid, Rawan; Greenwood, Matthew; Vali, Hojatollah; Mandato, Craig A.; Greenwood, Michael T.

    2012-01-01

    Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti

  19. Interdependence of Bad and Puma during ionizing-radiation-induced apoptosis.

    Science.gov (United States)

    Toruno, Cristhian; Carbonneau, Seth; Stewart, Rodney A; Jette, Cicely

    2014-01-01

    Ionizing radiation (IR)-induced DNA double-strand breaks trigger an extensive cellular signaling response that involves the coordination of hundreds of proteins to regulate DNA repair, cell cycle arrest and apoptotic pathways. The cellular outcome often depends on the level of DNA damage as well as the particular cell type. Proliferating zebrafish embryonic neurons are highly sensitive to IR-induced apoptosis, and both p53 and its transcriptional target puma are essential mediators of the response. The BH3-only protein Puma has previously been reported to activate mitochondrial apoptosis through direct interaction with the pro-apoptotic Bcl-2 family proteins Bax and Bak, thus constituting the role of an "activator" BH3-only protein. This distinguishes it from BH3-only proteins like Bad that are thought to indirectly promote apoptosis through binding to anti-apoptotic Bcl-2 family members, thereby preventing the sequestration of activator BH3-only proteins and allowing them to directly interact with and activate Bax and Bak. We have shown previously that overexpression of the BH3-only protein Bad in zebrafish embryos supports normal embryonic development but greatly sensitizes developing neurons to IR-induced apoptosis. While Bad has previously been shown to play only a minor role in promoting IR-induced apoptosis of T cells in mice, we demonstrate that Bad is essential for robust IR-induced apoptosis in zebrafish embryonic neural tissue. Moreover, we found that both p53 and Puma are required for Bad-mediated radiosensitization in vivo. Our findings show the existence of a hierarchical interdependence between Bad and Puma whereby Bad functions as an essential sensitizer and Puma as an essential activator of IR-induced mitochondrial apoptosis specifically in embryonic neural tissue.

  20. Chalcone-imidazolone conjugates induce apoptosis through DNA damage pathway by affecting telomeres

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    Kamal Ahmed

    2011-04-01

    Full Text Available Abstract Background Breast cancer is one of the most prevalent cancers in the world and more than one million women are diagnosed leading to 410,000 deaths every year. In our previous studies new chalcone-imidazolone conjugates were prepared and evaluated for their anticancer activity in a panel of 53 human tumor cell lines and the lead compounds identified were 6 and 8. This prompted us to investigate the mechanism of apoptotic event. Results Involvement of pro-apoptotic protein (Bax, active caspase-9 and cleavage of retinoblastoma protein was studied. Interestingly, the compounds caused upregulation of p21, check point proteins (Chk1, Chk2 and as well as their phosphorylated forms which are known to regulate the DNA damage pathway. Increased p53BP1 foci by immunolocalisation studies and TRF1 suggested the possible involvement of telomere and associated proteins in the apoptotic event. The telomeric protein such as TRF2 which is an important target for anticancer therapy against human breast cancer was extensively studied along with proteins involved in proper functioning of telomeres. Conclusions The apoptotic proteins such as Bax, active caspase-9 and cleaved RB are up-regulated in the compound treated cells revealing the apoptotic nature of the compounds. Down regulation of TRF2 and upregulation of the TRF1 as well as telomerase assay indicated the decrease in telomeric length revealing telomeric dysfunction and thereby controlling the rapid rate of cell proliferation. In summary, chalcone-imidazolone conjugates displayed significant DNA damage activity particularly at telomeres and caused both apoptosis and senescence-like growth arrest which suggested that these compounds have potential activity against breast carcinoma.

  1. Ginkgo biloba extract mitigates liver fibrosis and apoptosis by regulating p38 MAPK, NF-κB/IκBα, and Bcl-2/Bax signaling

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    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

  2. AMP-activated protein kinase α2 and E2F1 transcription factor mediate doxorubicin-induced cytotoxicity by forming a positive signal loop in mouse embryonic fibroblasts and non-carcinoma cells.

    Science.gov (United States)

    Yang, Wookyeom; Park, In-Ja; Yun, Hee; Im, Dong-Uk; Ock, Sangmi; Kim, Jaetaek; Seo, Seon-Mi; Shin, Ha-Yeon; Viollet, Benoit; Kang, Insug; Choe, Wonchae; Kim, Sung-Soo; Ha, Joohun

    2014-02-21

    Doxorubicin is one of the most widely used anti-cancer drugs, but its clinical application is compromised by severe adverse effects in different organs including cardiotoxicity. In the present study we explored mechanisms of doxorubicin-induced cytotoxicity by revealing a novel role for the AMP-activated protein kinase α2 (AMPKα2) in mouse embryonic fibroblasts (MEFs). Doxorubicin robustly induced the expression of AMPKα2 in MEFs but slightly reduced AMPKα1 expression. Our data support the previous notion that AMPKα1 harbors survival properties under doxorubicin treatment. In contrast, analyses of Ampkα2(-/-) MEFs, gene knockdown of AMPKα2 by shRNA, and inhibition of AMPKα2 activity with an AMPK inhibitor indicated that AMPKα2 functions as a pro-apoptotic molecule under doxorubicin treatment. Doxorubicin induced AMPKα2 at the transcription level via E2F1, a transcription factor that regulates apoptosis in response to DNA damage. E2F1 directly transactivated the Ampkα2 gene promoter. In turn, AMPKα2 significantly contributed to stabilization and activation of E2F1 by doxorubicin, forming a positive signal amplification loop. AMPKα2 directly interacted with and phosphorylated E2F1. This signal loop was also detected in H9c2, C2C12, and ECV (human epithelial cells) cells as well as mouse liver under doxorubicin treatment. Resveratrol, which has been suggested to attenuate doxorubicin-induced cytotoxicity, significantly blocked induction of AMPKα2 and E2F1 by doxorubicin, leading to protection of these cells. This signal loop appears to be non-carcinoma-specific because AMPKα2 was not induced by doxorubicin in five different tested cancer cell lines. These results suggest that AMPKα2 may serve as a novel target for alleviating the cytotoxicity of doxorubicin.

  3. Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB.

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    Yao Dai

    Full Text Available Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1, with IC₅₀ in the range of 1-2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be

  4. Composition-control of magnetron-sputter-deposited (BaxSr1-x)Ti1+yO3+z thin films for voltage tunable devices

    Science.gov (United States)

    Im, Jaemo; Auciello, O.; Baumann, P. K.; Streiffer, S. K.; Kaufman, D. Y.; Krauss, A. R.

    2000-01-01

    Precise control of composition and microstructure is critical for the production of (BaxSr1-x)Ti1+yO3+z (BST) dielectric thin films with the large dependence of permittivity on electric field, low losses, and high electrical breakdown fields that are required for successful integration of BST into tunable high-frequency devices. Here, we present results on composition-microstructure-electrical property relationships for polycrystalline BST films produced by magnetron-sputter deposition, that are appropriate for microwave and millimeter-wave applications such as varactors and frequency triplers. Films with controlled compositions were grown from a stoichiometric Ba0.5Sr0.5TiO3 target by control of the background processing gas pressure. It was determined that the (Ba+Sr)/Ti ratios of these BST films could be adjusted from 0.73 to 0.98 by changing the total (Ar+O2) process pressure, while the O2/Ar ratio did not strongly affect the metal ion composition. Film crystalline structure and dielectric properties as a function of the (Ba+Sr)/Ti ratio are discussed. Optimized BST films yielded capacitors with low dielectric losses (0.0047), among the best reported for sputtered BST, while still maintaining tunabilities suitable for device applications.

  5. 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.

  6. Flavanols from evening primrose (Oenothera paradoxa) defatted seeds inhibit prostate cells invasiveness and cause changes in Bcl-2/Bax mRNA ratio.

    Science.gov (United States)

    Lewandowska, Urszula; Szewczyk, Karolina; Owczarek, Katarzyna; Hrabec, Zbigniew; Podsędek, Anna; Koziołkiewicz, Maria; Hrabec, Elżbieta

    2013-03-27

    In this study, we assessed the influence of an evening primrose flavanol preparation (EPFP) on proliferation and invasiveness of human prostate cancer cells (DU 145) and immortalized prostate epithelial cells (PNT1A). We report for the first time that EPFP reduces DU 145 cell proliferation (IC50 = 97 μM GAE for 72 h incubation) and invasiveness (by 24% versus control at 75 μM GAE). EPFP strongly inhibited PNT1A invasiveness in a concentration-dependent manner (by 67% versus control at 75 μM GAE) and did not cause a reduction in their proliferation. Furthermore, EPFP inhibited the activities of MMP-2 and MMP-9 secreted to culture medium by PNT1A cells by 84% and 34% versus control at 100 μM GAE, respectively. In the case of DU 145, MMP-9 activity at 100 μM GAE was reduced by 37% versus control. Moreover, the evening primrose seed flavanols suppressed the expression of selected genes (MMP-1, MMP-9, MMP-14, c-Fos, c-Jun, and VEGF) and also caused favorable changes in Bcl-2/Bax mRNA ratio which render DU 145 cells more sensitive to apoptosis-triggering agents. An additional confirmation of the proapoptotic activity of EPFP toward DU 145 was visualization of characteristic apoptotic bodies by DAPI staining. In conclusion, this study suggests that EPFP may increase apoptosis and reduce angiogenesis of prostate cancer cells.

  7. Structural and ferroelectric properties of Sr1−xBaxBi2Nb2O9 thin films obtained by dip-coating

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    Y. González-Abreu

    2017-10-01

    Full Text Available The paper presents the structural and ferroelectric results for Sr1−xBaxBi2Nb2O9(x=0.30; 0.85 thin films, which were obtained by using dip-coating. The solutions containing the desirable ions were prepared from the powders of the previous studied ceramic samples. The films were deposited at room temperature on Fluorine-doped Tin Oxide (FTO substrates and submitted to a heat treatment for crystallization. The films were characterized by using scanning microscopy electronic, energy dispersive spectroscopy and ellipsometry. Hysteresis ferroelectric loops were obtained, at room temperature, by using a Sawyer-Tower circuit at several frequencies. A well-defined grain structure was observed for both compositions. The energy dispersive spectroscopy (EDS measurements revealed the presence of the corresponding elements from the chemical composition of the ceramic systems. The band-gap energy was around 3.3eV for both samples. Typical hysteresis loops for normal and relaxor ferroelectrics were obtained for x=0.30 and 0.85, respectively.

  8. Born to be Alive: A Role for the BCL-2 Family in Melanoma Tumor Cell Survival, Apoptosis, and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Anvekar, Rina A.; Asciolla, James J.; Missert, Derek J.; Chipuk, Jerry E., E-mail: jerry.chipuk@mssm.edu [Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY (United States); Department of Dermatology, Mount Sinai School of Medicine, New York, NY (United States); The Tisch Cancer Institute, Mount Sinai Medical Center, New York, NY (United States)

    2011-10-13

    The global incidence of melanoma has dramatically increased during the recent decades, yet the advancement of primary and adjuvant therapies has not kept a similar pace. The development of melanoma is often centered on cellular signaling that hyper-activates survival pathways, while inducing a concomitant blockade to cell death. Aberrations in cell death signaling not only promote tumor survival and enhanced metastatic potential, but also create resistance to anti-tumor strategies. Chemotherapeutic agents target melanoma tumor cells by inducing a form of cell death called apoptosis, which is governed by the BCL-2 family of proteins. The BCL-2 family is comprised of anti-apoptotic proteins (e.g., BCL-2, BCL-xL, and MCL-1) and pro-apoptotic proteins (e.g., BAK, BAX, and BIM), and their coordinated regulation and function are essential for optimal responses to chemotherapeutics. Here we will discuss what is currently known about the mechanisms of BCL-2 family function with a focus on the signaling pathways that maintain melanoma tumor cell survival. Importantly, we will critically evaluate the literature regarding how chemotherapeutic strategies directly impact on BCL-2 family function and offer several suggestions for future regimens to target melanoma and enhance patient survival.

  9. Virus-directed enzyme prodrug therapy and the assessment of the cytotoxic impact of some benzimidazole derivatives.

    Science.gov (United States)

    Szewczuk, Michał; Boguszewska, Karolina; Żebrowska, Marta; Balcerczak, Ewa; Stasiak, Marta; Świątkowska, Maria; Błaszczak-Świątkiewicz, Katarzyna

    2017-07-01

    Virus-directed enzyme prodrug therapy is one of the major strategy of increasing cytotoxicity of bioreductive agents. This research intended to examine new selected benzimidazole derivatives as a substrate for nitroreductase, the enzyme involved in nitroreduction which is responsible to the production of cytotoxic metabolites. In this way, the selectivity and strength of cytotoxicity can be raised. The effect of benzimidazoles on virus transfected cells and non-virus transfected cells A549 cell line was established by Annexin V + propidium iodide test, western blot, and polymerase chain reaction analysis of specific pro- and anti-apoptotic proteins in the corresponding gene expression and additionally nitroreductase gene expression. Our results proved the pro-apoptotic properties of all tested compounds in normoxia and hypoxia, especially according to virused A549 cells where the time of exposition was reduced from 48 to 4 h. In this shorten period of time, the strongest activity was shown by N-oxide compounds with nitro-groups. The apoptosis was confirmed by generation of BAX gene and protein and reduction of BCL2 gene and protein.

  10. Blocking RhoA/ROCK inhibits the pathogenesis of pemphigus vulgaris by suppressing oxidative stress and apoptosis through TAK1/NOD2-mediated NF-κB pathway.

    Science.gov (United States)