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Sample records for host cell apoptosis

  1. Neisseria gonorrhoeae infection protects human endocervical epithelial cells from apoptosis via expression of host antiapoptotic proteins.

    PubMed

    Follows, S A; Murlidharan, J; Massari, P; Wetzler, L M; Genco, C A

    2009-09-01

    Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-kappaB activation and resulted in increased gene expression of the NF-kappaB-regulated antiapoptotic genes bfl-1, cIAP-2, and c-FLIP. Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells. PMID:19546192

  2. Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis.

    PubMed

    Ruhland, Aaron; Leal, Nicole; Kima, Peter E

    2007-01-01

    Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NFkappaB and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NFkappaB signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.

  3. Anaplasma phagocytophilum Ats-1 Is Imported into Host Cell Mitochondria and Interferes with Apoptosis Induction

    PubMed Central

    Niu, Hua; Kozjak-Pavlovic, Vera; Rudel, Thomas; Rikihisa, Yasuko

    2010-01-01

    Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, infects human neutrophils and inhibits mitochondria-mediated apoptosis. Bacterial factors involved in this process are unknown. In the present study, we screened a genomic DNA library of A. phagocytophilum for effectors of the type IV secretion system by a bacterial two-hybrid system, using A. phagocytophilum VirD4 as bait. A hypothetical protein was identified as a putative effector, hereby named Anaplasma translocated substrate 1 (Ats-1). Using triple immunofluorescence labeling and Western blot analysis of infected cells, including human neutrophils, we determined that Ats-1 is abundantly expressed by A. phagocytophilum, translocated across the inclusion membrane, localized in the host cell mitochondria, and cleaved. Ectopically expressed Ats-1 targeted mitochondria in an N-terminal 17 residue-dependent manner, localized in matrix or at the inner membrane, and was cleaved as native protein, which required residues 55–57. In vitro-translated Ats-1 was imported in a receptor-dependent manner into isolated mitochondria. Ats-1 inhibited etoposide-induced cytochrome c release from mitochondria, PARP cleavage, and apoptosis in mammalian cells, as well as Bax-induced yeast apoptosis. Ats-1(55–57) had significantly reduced anti-apoptotic activity. Bax redistribution was inhibited in both etoposide-induced and Bax-induced apoptosis by Ats-1. Taken together, Ats-1 is the first example of a bacterial protein that traverses five membranes and prevents apoptosis at the mitochondria. PMID:20174550

  4. Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis.

    PubMed

    Kozjak-Pavlovic, Vera; Dian-Lothrop, Elke A; Meinecke, Michael; Kepp, Oliver; Ross, Katharina; Rajalingam, Krishnaraj; Harsman, Anke; Hauf, Eva; Brinkmann, Volker; Günther, Dirk; Herrmann, Ines; Hurwitz, Robert; Rassow, Joachim; Wagner, Richard; Rudel, Thomas

    2009-10-01

    The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m)). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m). The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m) loss and apoptosis, demonstrating that dissipation of DeltaPsi(m) is a requirement for cell death caused by neisserial infection. PMID:19851451

  5. Macrophage Activation Redirects Yersinia-Infected Host Cell Death from Apoptosis to Caspase-1-Dependent Pyroptosis

    PubMed Central

    Bergsbaken, Tessa; Cookson, Brad T

    2007-01-01

    Infection of macrophages by Yersinia species results in YopJ-dependent apoptosis, and naïve macrophages are highly susceptible to this form of cell death. Previous studies have demonstrated that macrophages activated with lipopolysaccharide (LPS) prior to infection are resistant to YopJ-dependent cell death; we found this simultaneously renders macrophages susceptible to killing by YopJ− Yersinia pseudotuberculosis (Yptb). YopJ− Yptb-induced macrophage death was dependent on caspase-1 activation, resulting in rapid permeability to small molecules, followed by membrane breakdown and DNA damage, and accompanied by cleavage and release of proinflammatory interleukin-18. Induction of caspase-1-dependent death, or pyroptosis, required the bacterial type III translocon but none of its known translocated proteins. Wild-type Yptb infection also triggered pyroptosis: YopJ-dependent activation of proapoptotic caspase-3 was significantly delayed in activated macrophages and resulted in caspase-1-dependent pyroptosis. The transition to susceptibility was not limited to LPS activation; it was also seen in macrophages activated with other Toll-like receptor (TLR) ligands and intact nonviable bacteria. Yptb infection triggered macrophage activation and activation of caspase-1 in vivo. Y. pestis infection of activated macrophages also stimulated caspase-1 activation. These results indicate that host signaling triggered by TLR and other activating ligands during the course of Yersinia infection redirects both the mechanism of host cell death and the downstream consequences of death by shifting from noninflammatory apoptosis to inflammatory pyroptosis. PMID:17983266

  6. NF-κB-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection

    PubMed Central

    Clifton, Dawn R.; Goss, Rachel A.; Sahni, Sanjeev K.; van Antwerp, Daniel; Baggs, Raymond B.; Marder, Victor J.; Silverman, David J.; Sporn, Lee Ann

    1998-01-01

    The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-κB and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-κB was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit IκB that rendered NF-κB inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-κB activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-κB activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell’s apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection. PMID:9539792

  7. The cyclomodulin Cif of Photorhabdus luminescens inhibits insect cell proliferation and triggers host cell death by apoptosis.

    PubMed

    Chavez, Carolina Varela; Jubelin, Grégory; Courties, Gabriel; Gomard, Aurélie; Ginibre, Nadège; Pages, Sylvie; Taïeb, Frédéric; Girard, Pierre-Alain; Oswald, Eric; Givaudan, Alain; Zumbihl, Robert; Escoubas, Jean-Michel

    2010-12-01

    Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G(2)/M phase transition in human cell lines. We report here the first direct functional analysis of Cif(Pl), from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cif(Pl) gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cif(Pl) was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. Cif(Pl) inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G(2)/M phase transition. It also triggered host cell death by apoptosis. The integrity of the Cif(Pl) catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line. PMID:20870031

  8. B7H1/CD80 interaction augments PD-1-dependent T cell apoptosis and ameliorates graft versus host disease

    PubMed Central

    Deng, Ruishu; Cassady, Kaniel; Li, Xiaofan; Yao, Sheng; Zhang, Mingfeng; Racine, Jeremy; Lin, Jeffrey; Chen, Lieping; Zeng, Defu

    2014-01-01

    Interactions of B7H1 (PD-L1) with its two ligands, PD-1 and CD80, on T cells play a pivotal role in controlling T cell activation, proliferation, anergy, and apoptosis. However, the interactions between the two pathways remain unknown. Using an alloimmune response model of graft-versus-host disease (GVHD), we report here that: 1) Comparison of proliferation and apoptosis of wild-type (WT) and PD-1−/− CD4+ conventional T (Tcon) cells in WT and B7H1−/− recipients has revealed that B7H1/CD80 interaction per se augments T cell proliferation, and this interaction augments T cell apoptosis mediated by B7H1/PD-1 interaction. This observation was recapitulated in an in vitro mixed lymphocyte reaction assay. 2) Specific blockade of the B7H1/CD80 axis by anti-B7H1 mAb reduces WT-alloreactive Tcon cell proliferation, IL-2 production, expression of PD-1, and apoptosis, resulting in worsening GVHD. In contrast, specific blockade of B7H1/CD80 interaction reduces donor PD-1−/− Tcon cell proliferation without impact on apoptosis, resulting in ameliorating GVHD. 3) B7H1 fused to an immunoglobulin Fc domain (B7H1-Ig), when produced in vivo by hydrodynamic injection of B7H1-Ig plasmid, ameliorates GVHD by augmenting proliferation and apoptosis of WT- alloreactive Tcon cells. Conversely, B7H1-Ig treatment has no impact on apoptosis but augments PD-1−/− T cell proliferation and worsens GVHD. These results indicate that B7H1/CD80 interaction augments Tcon cell proliferation, IL-2 production, and expression of PD-1, which leads to increased apoptosis mediated by the B7H1/PD1 pathway. Additionally, by engaging both PD-1 and CD80, B7H1-Ig can be a powerful therapeutic reagent for down-regulating the T cell immune response. PMID:25488990

  9. An Alternative Kaposi's Sarcoma-Associated Herpesvirus Replication Program Triggered by Host Cell Apoptosis

    PubMed Central

    Prasad, Alka; Lu, Michael; Lukac, David M.

    2012-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several neoplastic diseases: Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). KSHV replicates actively, via a controlled gene expression program, but can also remain latent. It had been thought that the transition from latent to lytic replication was controlled exclusively by the replication and transcription activator protein RTA (open reading frame 50 [ORF50] gene product). A dominant-negative (DN) ORF50 mutant, ORF50ΔSTAD, blocks gene expression and replication. We produced a PEL cell line derivative containing both latent KSHV genomes and an inducible ORF50ΔSTAD. We unexpectedly found that induction of apoptosis triggered high-level viral replication, even when DN ORF50ΔSTAD was present, suggesting that apoptosis triggers KSHV replication through a distinct RTA-independent pathway. We verified that apoptosis triggers KSHV replication independent of RTA using ORF50 small interfering RNA (siRNA) and also showed that caspase activity is required to trigger KSHV replication. We showed that when apoptosis triggers KSHV replication, the kinetics of late gene expression is accelerated by 12 to 24 h and that virus produced following apoptosis has reduced infectivity. KSHV therefore appears to replicate via two distinct pathways, a conventional pathway requiring RTA, with slower replication kinetics, producing virus with higher infectivity, and an alternative apoptosis-triggered pathway that does not require RTA, has faster replication kinetics, and produces virus with lower infectivity. The existence of a distinct apoptosis-triggered, accelerated replication pathway may have evolutionary advantages for the virus and clinical significance for the treatment of KSHV-associated neoplasms. It also provides further evidence that KSHV can sense and react to its environment. PMID:22345480

  10. Apoptosis: A Functional Paradigm for Programmed Plant Cell Death Induced by a Host-Selective Phytotoxin and Invoked during Development.

    PubMed Central

    Wang, H; Li, J; Bostock, RM; Gilchrist, DG

    1996-01-01

    The host-selective AAL toxins secreted by Alternaria alternata f sp lycopersici are primary chemical determinants in the Alternaria stem canker disease of tomato. The AAL toxins are members of a new class of sphinganine analog mycotoxins that cause cell death in both animals and plants. Here, we report detection of stereotypic hallmarks of apoptosis during cell death induced by these toxins in tomato. DNA ladders were observed during cell death in toxin-treated tomato protoplasts and leaflets. The intensity of the DNA ladders was enhanced by Ca2+ and inhibited by Zn2+. The progressive delineation of fragmented DNA into distinct bodies, coincident with the appearance of DNA ladders, also was observed during death of toxin-treated tomato protoplasts. In situ analysis of cells dying during development in both onion root caps and tomato leaf tracheary elements revealed DNA fragmentation localized to the dying cells as well as the additional formation of apoptotic-like bodies in sloughing root cap cells. We conclude that the fundamental elements of apoptosis, as characterized in animals, are conserved in plants. The apoptotic process may be expressed during some developmental transitions and is the functional process by which symptomatic lesions are formed in the Alternaria stem canker disease of tomato. Sphinganine analog mycotoxins may be used to characterize further signaling pathways leading to apoptosis in plants. PMID:12239387

  11. Endothelial Cell Apoptosis Induces TGF-β Signaling-Dependent Host Endothelial-Mesenchymal Transition to Promote Transplant Arteriosclerosis.

    PubMed

    Li, J; Xiong, J; Yang, B; Zhou, Q; Wu, Y; Luo, H; Zhou, H; Liu, N; Li, Y; Song, Z; Zheng, Q

    2015-12-01

    Endothelial cells (ECs) apoptosis is an initial event in transplant arteriosclerosis (TA), resulting in allograft function loss. To elucidate the precise mechanisms of ECs apoptosis leading to neointimal smooth muscle cells (SMCs) accumulation during TA. We induced apoptosis in cultured ECs by overexpressing p53 through lentivirus-mediated transfection. ECs apoptosis induced the production of transforming growth factor (TGF)-β1 in both apoptotic and neighboring viable cells, leading to increased TGF-β1 in the culture media. Conditioned media from Ltv-p53-transfected ECs further promoted transition of cultured ECs to SM-like cells by activating TGF-β/Smad3, PI3K/Akt/mTOR, and MAPK/ERK signaling in a TGF-β-dependent manner. In transgenic rat aorta transplantation models, inhibition of ECs apoptosis in Bcl-xL(+/+) knock-in rat aortic allografts significantly reduced TGF-β1 production both in allograft endothelia and in blood plasma, which in turn decreased accumulation of SM22α+ cells from transgenic recipient ECs originally marked with EGFP knock-in in neointima and alleviated TA. Systemic treatment with SIS3, AP23573, or PD98059 also prevented recipient ECs-originated SM-like cells accumulation and intima hyperplasia in aortic allografts. These data suggest that allograft EC apoptosis induced recipient endothelial-mesenchymal (smooth muscle) transition via TGF-β signaling, resulting in recipient EC-derived SMC accumulation as a major mechanism of vascular remodeling during TA.

  12. Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin

    PubMed Central

    Wood, Stephen; Goldufsky, Josef; Shafikhani, Sasha H.

    2015-01-01

    Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38β and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell. PMID:26020630

  13. Infection of human urethral epithelium with Neisseria gonorrhoeae elicits an upregulation of host anti-apoptotic factors and protects cells from staurosporine-induced apoptosis.

    PubMed

    Binnicker, Matthew J; Williams, Richard D; Apicella, Michael A

    2003-08-01

    In order to better understand the host response to an infection with Neisseria gonorrhoeae, microarray technology was used to analyse the gene expression profile between uninfected and infected human urethral epithelium. The anti-apoptotic genes bfl-1, cox-2 and c-IAP-2 were identified to be upregulated approximately eight-, four- or twofold, respectively, following infection. Subsequent assays including RT-PCR, real time RT-PCR and RNase protection confirmed the increased expression of these apoptotic regulators, and identified that a fourth anti-apoptotic factor, mcl-1, is also upregulated. RT-PCR and RNase protection also showed that key pro-apoptotic factors including bax, bad and bak do not change in expression. Furthermore, our studies demonstrated that infection with the gonococcus partially protects urethral epithelium from apoptosis induced by the protein kinase inhibitor, staurosporine (STS). This work shows that following infection with Neisseria gonorrhoeae, several host anti-apoptotic factors are upregulated. In addition, a gonococcal infection protects host cells from subsequent STS-induced death. The regulation of host cell death by the gonococcus may represent a mechanism employed by this pathogen to survive and proliferate in host epithelium. PMID:12864814

  14. Premature apoptosis of Chlamydia-infected cells disrupts chlamydial development.

    PubMed

    Ying, Songmin; Pettengill, Matthew; Latham, E Ray; Walch, Axel; Ojcius, David M; Häcker, Georg

    2008-11-15

    The obligate intracellular development of Chlamydia suggests that the bacteria should be vulnerable to premature host cell apoptosis, but because Chlamydia-infected cells are apoptosis resistant, this has never been able to be tested. We have devised a system to circumvent the apoptotic block imposed by chlamydial infection. When the proapoptotic protein Bim(S) was experimentally induced, epithelial cells underwent apoptosis that was not blocked by chlamydial infection. Apoptosis during the developmental cycle prevented the generation of infectious bacteria and caused transcriptional changes of bacterial genes and loss of intracellular ATP. Intriguingly, although apoptosis resulted in destruction of host cell structures and of the Chlamydia inclusion, and prevented generation of elementary bodies, Bim(S) induction in the presence of a caspase inhibitor allowed differentiation into morphologically normal but noninfectious elementary bodies. These data show that chlamydial infection renders host cells apoptosis resistant at a premitochondrial step and demonstrate the consequences of premature apoptosis for development of the bacteria.

  15. Involvement of apoptosis in host-parasite interactions in the zebra mussel.

    PubMed

    Minguez, Laëtitia; Brulé, Nelly; Sohm, Bénédicte; Devin, Simon; Giambérini, Laure

    2013-01-01

    The question of whether cell death by apoptosis plays a biological function during infection is key to understanding host-parasite interactions. We investigated the involvement of apoptosis in several host-parasite systems, using zebra mussels Dreissena polymorpha as test organisms and their micro- and macroparasites. As a stress response associated with parasitism, heat shock proteins (Hsp) can be induced. In this protein family, Hsp70 are known to be apoptosis inhibitors. Mussels were diagnosed for their respective infections by standard histological methods; apoptosis was detected using the TUNEL methods on paraffin sections and Hsp70 by immunohistochemistry on cryosections. Circulating hemocytes were the main cells observed in apoptosis whereas infected tissues displayed no or few apoptotic cells. Parasitism by intracellular bacteria Rickettsiales-like and the trematode Bucephalus polymorphus were associated with the inhibition of apoptosis whereas ciliates Ophryoglena spp. or the trematode Phyllodistomum folium did not involve significant differences in apoptosis. Even if some parasites were able to modulate apoptosis in zebra mussels, we did not see evidence of any involvement of Hsp70 on this mechanism. PMID:23785455

  16. Involvement of Apoptosis in Host-Parasite Interactions in the Zebra Mussel

    PubMed Central

    Minguez, Laëtitia; Brulé, Nelly; Sohm, Bénédicte; Devin, Simon; Giambérini, Laure

    2013-01-01

    The question of whether cell death by apoptosis plays a biological function during infection is key to understanding host-parasite interactions. We investigated the involvement of apoptosis in several host-parasite systems, using zebra mussels Dreissena polymorpha as test organisms and their micro- and macroparasites. As a stress response associated with parasitism, heat shock proteins (Hsp) can be induced. In this protein family, Hsp70 are known to be apoptosis inhibitors. Mussels were diagnosed for their respective infections by standard histological methods; apoptosis was detected using the TUNEL methods on paraffin sections and Hsp70 by immunohistochemistry on cryosections. Circulating hemocytes were the main cells observed in apoptosis whereas infected tissues displayed no or few apoptotic cells. Parasitism by intracellular bacteria Rickettsiales-like and the trematode Bucephalus polymorphus were associated with the inhibition of apoptosis whereas ciliates Ophryoglena spp. or the trematode Phyllodistomum folium did not involve significant differences in apoptosis. Even if some parasites were able to modulate apoptosis in zebra mussels, we did not see evidence of any involvement of Hsp70 on this mechanism. PMID:23785455

  17. Apoptosis in Cryopreserved Eukaryotic Cells.

    PubMed

    Savitskaya, M A; Onishchenko, G E

    2016-05-01

    This review considers apoptosis mechanisms that have been revealed in cryopreserved cells and which can be controlled using different chemical agents, thereby improving the viability of cells after their return to normal conditions. The role of oxidative stress as of the most significant damaging factor is discussed, as well as the reasonability of including antioxidants into cryopreservation/thawing protocols as independent agents or in combination with other compounds.

  18. Apoptosis of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae), which failed to migrate within its natural host.

    PubMed

    Chen, Wei-June; Huang, Ching-Gi; Fan-Chiang, Mei-Huei; Liu, Yu-Han; Lee, Yi-Feng

    2013-01-15

    Sexual reproduction of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae), a parasite specific to the mosquito Aedes albopictus, in Malpighian tubules is initiated by the entry of the trophotozoites developed in the midgut shortly after pupation (usually <5 h). However, only a low proportion of trophozoites are able to migrate; others end up dying. In this study, we demonstrated that those trophozoites that failed to migrate eventually died of apoptosis. Morphological changes such as shrinkage, chromatin aggregations and formation of blunt ridges on the surface were seen in moribund trophozoites. In addition, DNA fragmentation of trophozoites isolated from the midgut of pupae was demonstrated by the presence of DNA ladders, Annexin V staining and TUNEL assays. Detection of caspase-like activity suggests that apoptosis of those trophozoites may have occurred through a mechanism of an intrinsic or mitochondrial-mediated pathway. Although apoptosis has been observed in various protozoan species, it is not clear how apoptosis in single-celled organisms might result from evolution by natural selection. However, we speculate that apoptosis may regulate the parasite load of A. taiwanensis within its natural mosquito host, leading to an optimized state of the survival rate for both parasite and host.

  19. Poxvirus host cell entry.

    PubMed

    Schmidt, Florian Ingo; Bleck, Christopher Karl Ernst; Mercer, Jason

    2012-02-01

    Poxviruses are characterized by their large size, complex composition, and cytoplasmic life cycle. They produce two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). Both MVs and EVs of vaccinia virus, the model poxvirus, take advantage of host cell endocytosis for internalization: they activate macropinocytosis-the most suitable form of endocytosis for large particles. Although largely dependent on the same cellular machinery, MV and EV entry differs with regard to the mechanisms used to trigger macropinocytosis and to undergo fusion. While EVs have to shed an additional membrane to expose the fusion complex, MV fusion requires the inactivation of fusion inhibitory proteins absent in EVs. This review highlights recent advances in the understanding of poxvirus MV and EV cell entry. PMID:22440962

  20. Male-killing symbiont damages host's dosage-compensated sex chromosome to induce embryonic apoptosis

    PubMed Central

    Harumoto, Toshiyuki; Anbutsu, Hisashi; Lemaitre, Bruno; Fukatsu, Takema

    2016-01-01

    Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. Here, we uncover the molecular and cellular mechanisms underlying Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos. Detailed genetic and cytological analyses demonstrate that male-killing Spiroplasma causes DNA damage on the male X chromosome interacting with the male-specific lethal (MSL) complex. The damaged male X chromosome exhibits a chromatin bridge during mitosis, and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways. Notably, the MSL complex is not only necessary but also sufficient for this cytotoxic process. These results highlight symbiont's sophisticated strategy to target host's sex chromosome and recruit host's molecular cascades toward massive apoptosis in a sex-specific manner. PMID:27650264

  1. Male-killing symbiont damages host's dosage-compensated sex chromosome to induce embryonic apoptosis.

    PubMed

    Harumoto, Toshiyuki; Anbutsu, Hisashi; Lemaitre, Bruno; Fukatsu, Takema

    2016-01-01

    Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. Here, we uncover the molecular and cellular mechanisms underlying Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos. Detailed genetic and cytological analyses demonstrate that male-killing Spiroplasma causes DNA damage on the male X chromosome interacting with the male-specific lethal (MSL) complex. The damaged male X chromosome exhibits a chromatin bridge during mitosis, and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways. Notably, the MSL complex is not only necessary but also sufficient for this cytotoxic process. These results highlight symbiont's sophisticated strategy to target host's sex chromosome and recruit host's molecular cascades toward massive apoptosis in a sex-specific manner. PMID:27650264

  2. Umbelliprenin Induces Apoptosis in CLL Cell Lines.

    PubMed

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V-FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate.

  3. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

  4. Measuring Apoptosis at the Single Cell Level

    PubMed Central

    Bouchier-Hayes, Lisa; Muñoz-Pinedo, Cristina; Connell, Samuel; Green, Douglas R.

    2008-01-01

    The use of live cell microscopy has made a number of contributions to the study of apoptosis. Many of the tools and techniques are available that allow us to image the key events that occur during cell death including mitochondrial outer membrane permeabilization, mitochondrial transmembrane potential changes, translocation of Bcl-2 family members, caspase activation, phosphatidylserine flip and plasma membrane rupture. We discuss these techniques here and highlight the advantages and drawbacks of using such approaches to study apoptosis. PMID:18314052

  5. Solamargine triggers hepatoma cell death through apoptosis

    PubMed Central

    XIE, XIAODONG; ZHU, HAITAO; YANG, HUIJIAN; HUANG, WENSI; WU, YINGYING; WANG, YING; LUO, YANLING; WANG, DONGQING; SHAO, GENBAO

    2015-01-01

    Solamargine (SM), a steroidal alkaloid glycoside extracted from the traditional Chinese herb Solanum incanum, has been evidenced to inhibit the growth and induce apoptosis in a number of human cancer cell lines. In the present study, the anticancer effect of SM and underlying molecular mechanism of SM-induced apoptosis were investigated on the human hepatocellular carcinoma cells, SMMC7721 and HepG2. The proliferation effects of SM on the SMMC7721 and HepG2 cell lines were evaluated using MTT and colony formation assays. In addition, the percentage of apoptosis was measured using an Annexin V/propidium iodide staining method and the cell cycle distribution mediated by SM was analyzed using flow cytometry. The expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, caspase-9, proliferating cell nuclear antigen (pcna) and Ki67 proteins were examined to further demonstrate the proliferate and apoptosis effects of SM on the hepatoma cells. The results indicated that SM effectively inhibited hepatoma cell proliferation and promoted apoptosis. SM resulted in cell cycle arrest at the G2/M phase in the two cell lines. In addition, SM downregulated the levels of proliferation-associated (Ki67 and pcna) and anti-apoptotic (Bcl-2) proteins, and promoted the activity of apoptosis-associated proteins (Bax, caspase-3 and caspase-9). Therefore, the activation of the Bcl-2/Bax and caspase signaling pathways may be involved in the SM-induced apoptosis of hepatoma cells. PMID:26170994

  6. Stress and death of cnidarian host cells play a role in cnidarian bleaching.

    PubMed

    Paxton, Camille W; Davy, Simon K; Weis, Virginia M

    2013-08-01

    Coral bleaching occurs when there is a breakdown of the symbiosis between cnidarian hosts and resident Symbiodinium spp. Multiple mechanisms for the bleaching process have been identified, including apoptosis and autophagy, and most previous work has focused on the Symbiodinium cell as the initiator of the bleaching cascade. In this work we show that it is possible for host cells to initiate apoptosis that can contribute to death of the Symbiodinium cell. First we found that colchicine, which results in apoptosis in other animals, causes cell death in the model anemone Aiptasia sp. but not in cultured Symbiodinium CCMP-830 cells or in cells freshly isolated from host Aiptasia (at least within the time frame of our study). In contrast, when symbiotic Aiptasia were incubated in colchicine, cell death in the resident Symbiodinium cells was observed, suggesting a host effect on symbiont mortality. Using live-cell confocal imaging of macerated symbiotic host cell isolates, we identified a pattern where the initiation of host cell death was followed by mortality of the resident Symbiodinium cells. This same pattern was observed in symbiotic host cells that were subjected to temperature stress. This research suggests that mortality of symbionts during temperature-induced bleaching can be initiated in part by host cell apoptosis. PMID:23619418

  7. Effects of conidia of various Aspergillus species on apoptosis of human pneumocytes and bronchial epithelial cells.

    PubMed

    Féménia, F; Huet, D; Lair-Fulleringer, S; Wagner, M C; Sarfati, J; Shingarova, L; Guillot, J; Boireau, P; Chermette, R; Berkova, N

    2009-05-01

    Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species. PMID:19117118

  8. Ambivalent Outcomes of Cell Apoptosis: A Barrier or Blessing in Malaria Progression

    PubMed Central

    Kakani, Parik; Suman, Sneha; Gupta, Lalita; Kumar, Sanjeev

    2016-01-01

    The life cycle of Plasmodium in two evolutionary distant hosts, mosquito, and human, is a complex process. It is regulated at various stages of developments by a number of diverged mechanisms that ultimately determine the outcome of the disease. During the development processes, Plasmodium invades a variety of cells in two hosts. The invaded cells tend to undergo apoptosis and are subsequently removed from the system. This process also eliminates numerous parasites along with these apoptotic cells as a part of innate defense against the invaders. Plasmodium should escape the invaded cell before it undergoes apoptosis or it should manipulate host cell apoptosis for its survival. Interestingly, both these phenomena are evident in Plasmodium at different stages of development. In addition, the parasite also exhibits altruistic behavior and triggers its own killing for the selection of the best ‘fit’ progeny, removal of the ‘unfit’ parasites to conserve the nutrients and to support the host survival. Thus, the outcomes of cell apoptosis are ambivalent, favorable as well as unfavorable during malaria progression. Here we discuss that the manipulation of host cell apoptosis might be helpful in the regulation of Plasmodium development and will open new frontiers in the field of malaria research. PMID:27014225

  9. Apoptosis of beta cells in diabetes mellitus.

    PubMed

    Anuradha, Rachakatla; Saraswati, Mudigonda; Kumar, Kishore G; Rani, Surekha H

    2014-11-01

    Diabetes mellitus is a multifactorial metabolic disorder characterized by hyperglycemia. Apoptosis in beta cells has been observed in response to diverse stimuli, such as glucose, cytokines, free fatty acids, leptin, and sulfonylureas, leading to the activation of polyol, hexosamine, and diacylglycerol/protein kinase-C (DAG/PKC) pathways that mediate oxidative and nitrosative stress causing the release of different cytokines. Cytokines induce the expression of Fas and tumor necrosis factor-alpha (TNF-α) by activating the transcription factor, nuclear factor-κb, and signal transducer and activator of transcription 1 (STAT-1) in the β cells in the extrinsic pathway of apoptosis. Cytokines produced in beta cells also induce proapoptotic members of the intrinsic pathway of apoptosis. The genetic alterations in apoptosis signaling machinery and the pathogenesis of diabetes include Fas, FasL, Akt, caspases, calpain-10, and phosphatase and tensin homolog (Pten). The other gene products that are involved in diabetes are nitric oxide synthase-2 (NOS2), small ubiquitin-like modifier (SUMO), apolipoprotein CIII (ApoCIII), forkhead box protein O1 (FOXO1), and Kruppel-like zinc finger protein Gli-similar 3 (GLIS3). The gene products having antiapoptotic nature are Bcl-2 and Bcl-XL. Epigenetic mechanisms play an important role in type I and type II diabetes. Further studies on the apoptotic genes and gene products in diabetics may be helpful in pharmacogenomics and individualized treatment along with antioxidants targeting apoptosis in diabetes. PMID:25093391

  10. Drosophila grim induces apoptosis in mammalian cells.

    PubMed Central

    Clavería, C; Albar, J P; Serrano, A; Buesa, J M; Barbero, J L; Martínez-A, C; Torres, M

    1998-01-01

    Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway. PMID:9857177

  11. Modified host cells with efflux pumps

    DOEpatents

    Dunlop, Mary J.; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-08-30

    The present invention provides for a modified host cell comprising a heterologous expression of an efflux pump capable of transporting an organic molecule out of the host cell wherein the organic molecule at a sufficiently high concentration reduces the growth rate of or is lethal to the host cell.

  12. CASPASE CONTROL: PROTAGONISTS OF CANCER CELL APOPTOSIS

    PubMed Central

    Fiandalo, M.V.; Kyprianou, N.

    2013-01-01

    Emergence of castration-resistant metastatic prostate cancer is due to activation of survival pathways, including apoptosis suppression and anoikis resistance, and increased neovascularization. Thus targeting of apoptotic players is of critical significance in prostate cancer therapy since loss of apoptosis and resistance to anoikis are critical in aberrant malignant growth, metastasis and conferring therapeutic failure. The majority of therapeutic agents act through intrinsic mitochondrial, extrinsic death receptor pathways or endoplasmic reticulum stress pathways to induce apoptosis. Current therapeutic strategies target restoring regulatory molecules that govern the pro-survival pathways such as PTEN which regulates AKT activity. Other strategies focus on reactivating the apoptotic pathways either by down-regulating anti-apoptotic players such as BCL-2 or by up-regulating pro-apoptotic protein families, most notably, the caspases. Caspases are a family of cystine proteases which serve critical roles in apoptotic and inflammatory signaling pathways. During tumorigenesis, significant loss or inactivation of lead members in the caspase family leads to impairing apoptosis induction, causing a dramatic imbalance in the growth dynamics, ultimately resulting in aberrant growth of human cancers. Recent exploitation of apoptosis pathways towards re-instating apoptosis induction via caspase re-activation has provided new molecular platforms for the development of therapeutic strategies effective against advanced prostate cancer as well as other solid tumors. This review will discuss the current cellular landscape featuring the caspase family in tumor cells and their activation via pharmacologic intervention towards optimized anti-cancer therapeutic modalities. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”. PMID:23070001

  13. Uropathogenic Escherichia coli Epigenetically Manipulate Host Cell Death Pathways.

    PubMed

    Zhang, Zhengguo; Wang, Ming; Eisel, Florian; Tchatalbachev, Svetlin; Chakraborty, Trinad; Meinhardt, Andreas; Bhushan, Sudhanshu

    2016-04-01

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in human. It is well established that UPEC can subvert innate immune responses, but the role of UPEC in interfering with host cell death pathways is not known. Here, we show that UPEC abrogates activation of the host cell prosurvival protein kinase B signaling pathway, which results in the activation of mammalian forkhead box O (FOXO) transcription factors. Although FOXOs were localized in the nucleus and showed increased DNA-binding activity, no change in the expression levels of FOXO target genes were observed. UPEC can suppress BIM expression induced by LY249002, which results in attenuation of caspase 3 activation and blockage of apoptosis. Mechanistically, BIM expression appears to be epigenetically silenced by a decrease in histone 4 acetylation at the BIM promoter site. Taken together, these results suggest that UPEC can epigenetically silence BIM expression, a molecular switch that prevents apoptosis.

  14. [How does the apicomplexan parasite Theileria control host cell identity?].

    PubMed

    Marsolier, Justine; Weitzman, Jonathan B

    2014-01-01

    Infectious agents, like bacteria or virus, are responsible for a large number of pathologies in mammals. Microbes have developed mechanisms for interacting with host cell pathways and hijacking cellular machinery to change the phenotypic state. In this review, we focus on an interesting apicomplexan parasite called Theileria. Infection by the tick-transmitted T. annulata parasite causes Tropical Theileriosis in North Africa and Asia, and the related T. parva parasite causes East Coast Fever in Sub-Saharan Africa. This parasite is the only eukaryote known to induce the transformation of its mammalian host cells. Indeed, T. annulata and T. parva infect bovine leukocytes leading to transforming phenotypes, which partially mirror human lymphoma pathologies. Theileria infection causes hyperproliferation, invasiveness and escape from apoptosis, presumably through the manipulation of host cellular pathways. Several host-signaling mechanisms have been implicated. Here we describe the mechanisms involved in parasite-induced transformation phenotypes.

  15. Mitochondria in human pluripotent stem cell apoptosis.

    PubMed

    TeSlaa, Tara; Setoguchi, Kiyoko; Teitell, Michael A

    2016-04-01

    Human pluripotent stem cells (hPSCs) have great potential in regenerative medicine because they can differentiate into any cell type in the body. Genome integrity is vital for human development and for high fidelity passage of genetic information across generations through the germ line. To ensure genome stability, hPSCs maintain a lower rate of mutation than somatic cells and undergo rapid apoptosis in response to DNA damage and additional cell stresses. Furthermore, cellular metabolism and the cell cycle are also differentially regulated between cells in pluripotent and differentiated states and can aid in protecting hPSCs against DNA damage and damaged cell propagation. Despite these safeguards, clinical use of hPSC derivatives could be compromised by tumorigenic potential and possible malignant transformation from failed to differentiate cells. Since hPSCs and mature cells differentially respond to cell stress, it may be possible to specifically target undifferentiated cells for rapid apoptosis in mixed cell populations to enable safer use of hPSC-differentiated cells in patients.

  16. Mitochondria in human pluripotent stem cell apoptosis.

    PubMed

    TeSlaa, Tara; Setoguchi, Kiyoko; Teitell, Michael A

    2016-04-01

    Human pluripotent stem cells (hPSCs) have great potential in regenerative medicine because they can differentiate into any cell type in the body. Genome integrity is vital for human development and for high fidelity passage of genetic information across generations through the germ line. To ensure genome stability, hPSCs maintain a lower rate of mutation than somatic cells and undergo rapid apoptosis in response to DNA damage and additional cell stresses. Furthermore, cellular metabolism and the cell cycle are also differentially regulated between cells in pluripotent and differentiated states and can aid in protecting hPSCs against DNA damage and damaged cell propagation. Despite these safeguards, clinical use of hPSC derivatives could be compromised by tumorigenic potential and possible malignant transformation from failed to differentiate cells. Since hPSCs and mature cells differentially respond to cell stress, it may be possible to specifically target undifferentiated cells for rapid apoptosis in mixed cell populations to enable safer use of hPSC-differentiated cells in patients. PMID:26828436

  17. Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection.

    PubMed Central

    Müller, A; Hacker, J; Brand, B C

    1996-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease and Pontiac fever, replicates within and eventually kills human macrophages. In this study, we show that L. pneumophila is cytotoxic to HL-60 cells, a macrophage-like cell line. We demonstrate that cell death mediated by L. pneumophila occurred at least in part through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented host cell DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether potential virulence factors like the metalloprotease and the macrophage infectivity potentiator of L. pneumophila are involved in the induction of apoptosis. None of these factors are essential for the induction of apoptosis in HL-60 cells but may be involved in other cytotoxic mechanisms that lead to accidental cell death (necrosis). The ability of L. pneumophila to promote cell death may be important for the initiation of infection, bacterial survival, and escape from the host immune response. Alternatively, the triggering of apoptosis in response to bacterial infection may have evolved as a means of the host immune system to reduce or inhibit bacterial replication. PMID:8945524

  18. Infection of human fallopian tube epithelial cells with Neisseria gonorrhoeae protects cells from tumor necrosis factor alpha-induced apoptosis.

    PubMed

    Morales, Priscilla; Reyes, Paz; Vargas, Macarena; Rios, Miguel; Imarai, Mónica; Cardenas, Hugo; Croxatto, Horacio; Orihuela, Pedro; Vargas, Renato; Fuhrer, Juan; Heckels, John E; Christodoulides, Myron; Velasquez, Luis

    2006-06-01

    Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-alpha). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-alpha was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-alpha antibodies; and (iii) the addition of exogenous TNF-alpha induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-alpha-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-alpha-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease. PMID:16714596

  19. Infection of Human Fallopian Tube Epithelial Cells with Neisseria gonorrhoeae Protects Cells from Tumor Necrosis Factor Alpha-Induced Apoptosis

    PubMed Central

    Morales, Priscilla; Reyes, Paz; Vargas, Macarena; Rios, Miguel; Imarai, Mónica; Cardenas, Hugo; Croxatto, Horacio; Orihuela, Pedro; Vargas, Renato; Fuhrer, Juan; Heckels, John E.; Christodoulides, Myron; Velasquez, Luis

    2006-01-01

    Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-α). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-α was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-α antibodies; and (iii) the addition of exogenous TNF-α induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-α-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-α-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease. PMID:16714596

  20. Comparison of apoptosis between adult worms of Schistosoma japonicum from susceptible (BALB/c mice) and less-susceptible (Wistar rats) hosts.

    PubMed

    Wang, Tao; Guo, Xiaoyong; Hong, Yang; Han, Hongxiao; Cao, Xiaodan; Han, Yanhui; Zhang, Min; Wu, Miaoli; Fu, Zhiqiang; Lu, Ke; Li, Hao; Zhao, Zhixin; Lin, Jiaojiao

    2016-10-30

    Schistosomiasis remains a serious public health concern in China. BALB/c mice are susceptible to Schistosoma japonicum infection, whereas the Wistar rats are less susceptible. Apoptosis phenomenon was observed in 42d adult worms of S. japonicum from both rats and mice at the morphologic, DNA, cellular, and gene levels by transmission electron microscopy (TEM), fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, fluorescein isothiocyanate-annexin-V/propidium iodide staining flow cytometry (FCM) analysis, and real-time PCR. The results showed that the apoptotic state in worms from two different susceptible hosts was diverse. Several classical hallmarks of apoptosis, including cell shrinkage, chromatin condensation and lunate marginalization, splitting of the nucleoli, nuclear shrinkage and apoptotic body formation were observed by TEM. TUNEL analysis showed that there were much more apoptosis spots in adult worms from rats than those from mice. Statistical analysis revealed that the degree of apoptosis and percentage of necrotic cells in adult worms from Wistar rats were significantly greater (P<0.01) than those from BALB/c mice by flow cytometry. A total of 15 apoptosis-associated genes including the major components of an intrinsic cell-death pathway were identified from S. japonicum in this study, suggested that a similar apoptosis pathway might occur in S. japonicum. Real-time PCR analyses revealed that the expression levels of most of the tested apoptosis-associated genes, except CASP7, were significantly higher or at the similar level in adult worms from Wistar rats, as compared to those from BALB/c mice. The results obtained in this study collectively demonstrated that differential development of adult S. japonicum in less-susceptible rats and susceptible mice was significantly associated with apoptosis in the worm, and provided valuable information to guide further investigations of the mechanisms governing

  1. Comparison of apoptosis between adult worms of Schistosoma japonicum from susceptible (BALB/c mice) and less-susceptible (Wistar rats) hosts.

    PubMed

    Wang, Tao; Guo, Xiaoyong; Hong, Yang; Han, Hongxiao; Cao, Xiaodan; Han, Yanhui; Zhang, Min; Wu, Miaoli; Fu, Zhiqiang; Lu, Ke; Li, Hao; Zhao, Zhixin; Lin, Jiaojiao

    2016-10-30

    Schistosomiasis remains a serious public health concern in China. BALB/c mice are susceptible to Schistosoma japonicum infection, whereas the Wistar rats are less susceptible. Apoptosis phenomenon was observed in 42d adult worms of S. japonicum from both rats and mice at the morphologic, DNA, cellular, and gene levels by transmission electron microscopy (TEM), fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, fluorescein isothiocyanate-annexin-V/propidium iodide staining flow cytometry (FCM) analysis, and real-time PCR. The results showed that the apoptotic state in worms from two different susceptible hosts was diverse. Several classical hallmarks of apoptosis, including cell shrinkage, chromatin condensation and lunate marginalization, splitting of the nucleoli, nuclear shrinkage and apoptotic body formation were observed by TEM. TUNEL analysis showed that there were much more apoptosis spots in adult worms from rats than those from mice. Statistical analysis revealed that the degree of apoptosis and percentage of necrotic cells in adult worms from Wistar rats were significantly greater (P<0.01) than those from BALB/c mice by flow cytometry. A total of 15 apoptosis-associated genes including the major components of an intrinsic cell-death pathway were identified from S. japonicum in this study, suggested that a similar apoptosis pathway might occur in S. japonicum. Real-time PCR analyses revealed that the expression levels of most of the tested apoptosis-associated genes, except CASP7, were significantly higher or at the similar level in adult worms from Wistar rats, as compared to those from BALB/c mice. The results obtained in this study collectively demonstrated that differential development of adult S. japonicum in less-susceptible rats and susceptible mice was significantly associated with apoptosis in the worm, and provided valuable information to guide further investigations of the mechanisms governing

  2. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    SciTech Connect

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-03-07

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.

  3. Entamoeba histolytica-induced dephosphorylation in host cells.

    PubMed

    Teixeira, José E; Mann, Barbara J

    2002-04-01

    Activation of host cell protein tyrosine phosphatases (PTPases) and protein dephosphorylation is an important mechanism used by various microorganisms to deactivate or kill host defense cells. To determine whether protein tyrosine dephosphorylation played a role in signaling pathways affecting Entamoeba histolytica-mediated host cell killing, we investigated the involvement of PTPases during the attachment of E. histolytica to target cells. We observed a rapid decrease in cellular protein tyrosine levels in Jurkat cells, as measured with an antiphosphotyrosine monoclonal antibody, following adherence to E. histolytica. Ameba-induced protein dephosphorylation was contact dependent and required intact parasite, since blocking amebic adherence with galactose inhibited tyrosine dephosphorylation and amebic lysates had no effect on phosphotyrosine levels. Moreover, disruption of amebic adherence with galactose promoted recovery of phosphorylation in Jurkat cells, indicating that dephosphorylation precedes target cell death. The evidence suggests that ameba-induced dephosphorylation is mediated by host cell phosphatases. Prior treatment of Jurkat cells with phenylarsine oxide, a PTPase inhibitor, inhibited ameba-induced dephosphorylation. We also found proteolytic cleavage of the PTPase 1B (PTP1B) in Jurkat cells after contact with amebae. The calcium-dependent protease calpain is responsible for PTP1B cleavage and enzymatic activation. Pretreatment of Jurkat cells with calpeptin, a calpain inhibitor, blocked PTP1B cleavage and inhibited ameba-induced dephosphorylation. In addition, inhibition of Jurkat cell PTPases with phenylarsine oxide blocked Jurkat cell apoptosis induced by E. histolytica. These results suggest that E. histolytica-mediated host cell death occurs by a mechanism that involves PTPase activation. PMID:11895943

  4. Characterization of Oyster Voltage-Dependent Anion Channel 2 (VDAC2) Suggests Its Involvement in Apoptosis and Host Defense.

    PubMed

    Li, Yingxiang; Zhang, Linlin; Qu, Tao; Li, Li; Zhang, Guofan

    2016-01-01

    Genomic and transcriptomic studies have revealed a sophisticated and powerful apoptosis regulation network in oyster, highlighting its adaptation to sessile life in a highly stressful intertidal environment. However, the functional molecular basis of apoptosis remains largely unexplored in oysters. In this study, we focused on a representative apoptotic gene encoding voltage-dependent anion channel 2 (VDAC2), a porin that abounds at the mitochondrial outer membrane. This is the first report on the identification and characterization of a VDAC gene in the Pacific oyster, Crassostrea gigas (CgVDAC2). The full length of CgVDAC2 was 1,738 bp with an open reading frame of 843 bp that encoded a protein of 281 amino acids. A four-element eukaryotic porin signature motif, a conserved ATP binding motif, and a VKAKV-like sequence were identified in the predicted CgVDAC2. Expression pattern analysis in different tissues and developmental stages as well as upon infection by ostreid herpesvirus 1 revealed the energy supply-related and immunity-related expression of CgVDAC2. CgVDAC2 was co-localized with mitochondria when it was transiently transfected into HeLa cells. Overexpression of CgVDAC2 in HEK293T cells suppressed the UV irradiation-induced apoptosis by inhibiting the pro-apoptotic function of CgBak. RNA interference induced reduction in CgVDAC2 expression showed a promoted apoptosis level upon UV light irradiation in hemocytes. The yeast two-hybrid system and co-immunoprecipitation assay indicated a direct interaction between CgVDAC2 and the pro-apoptotic protein CgBak. This study revealed the function of VDAC2 in oyster and provided new insights into its involvement in apoptosis modulation and host defense in mollusks.

  5. Characterization of Oyster Voltage-Dependent Anion Channel 2 (VDAC2) Suggests Its Involvement in Apoptosis and Host Defense.

    PubMed

    Li, Yingxiang; Zhang, Linlin; Qu, Tao; Li, Li; Zhang, Guofan

    2016-01-01

    Genomic and transcriptomic studies have revealed a sophisticated and powerful apoptosis regulation network in oyster, highlighting its adaptation to sessile life in a highly stressful intertidal environment. However, the functional molecular basis of apoptosis remains largely unexplored in oysters. In this study, we focused on a representative apoptotic gene encoding voltage-dependent anion channel 2 (VDAC2), a porin that abounds at the mitochondrial outer membrane. This is the first report on the identification and characterization of a VDAC gene in the Pacific oyster, Crassostrea gigas (CgVDAC2). The full length of CgVDAC2 was 1,738 bp with an open reading frame of 843 bp that encoded a protein of 281 amino acids. A four-element eukaryotic porin signature motif, a conserved ATP binding motif, and a VKAKV-like sequence were identified in the predicted CgVDAC2. Expression pattern analysis in different tissues and developmental stages as well as upon infection by ostreid herpesvirus 1 revealed the energy supply-related and immunity-related expression of CgVDAC2. CgVDAC2 was co-localized with mitochondria when it was transiently transfected into HeLa cells. Overexpression of CgVDAC2 in HEK293T cells suppressed the UV irradiation-induced apoptosis by inhibiting the pro-apoptotic function of CgBak. RNA interference induced reduction in CgVDAC2 expression showed a promoted apoptosis level upon UV light irradiation in hemocytes. The yeast two-hybrid system and co-immunoprecipitation assay indicated a direct interaction between CgVDAC2 and the pro-apoptotic protein CgBak. This study revealed the function of VDAC2 in oyster and provided new insights into its involvement in apoptosis modulation and host defense in mollusks. PMID:26727366

  6. Characterization of Oyster Voltage-Dependent Anion Channel 2 (VDAC2) Suggests Its Involvement in Apoptosis and Host Defense

    PubMed Central

    Li, Yingxiang; Zhang, Linlin; Qu, Tao; Li, Li; Zhang, Guofan

    2016-01-01

    Genomic and transcriptomic studies have revealed a sophisticated and powerful apoptosis regulation network in oyster, highlighting its adaptation to sessile life in a highly stressful intertidal environment. However, the functional molecular basis of apoptosis remains largely unexplored in oysters. In this study, we focused on a representative apoptotic gene encoding voltage-dependent anion channel 2 (VDAC2), a porin that abounds at the mitochondrial outer membrane. This is the first report on the identification and characterization of a VDAC gene in the Pacific oyster, Crassostrea gigas (CgVDAC2). The full length of CgVDAC2 was 1,738 bp with an open reading frame of 843 bp that encoded a protein of 281 amino acids. A four-element eukaryotic porin signature motif, a conserved ATP binding motif, and a VKAKV-like sequence were identified in the predicted CgVDAC2. Expression pattern analysis in different tissues and developmental stages as well as upon infection by ostreid herpesvirus 1 revealed the energy supply-related and immunity-related expression of CgVDAC2. CgVDAC2 was co-localized with mitochondria when it was transiently transfected into HeLa cells. Overexpression of CgVDAC2 in HEK293T cells suppressed the UV irradiation-induced apoptosis by inhibiting the pro-apoptotic function of CgBak. RNA interference induced reduction in CgVDAC2 expression showed a promoted apoptosis level upon UV light irradiation in hemocytes. The yeast two-hybrid system and co-immunoprecipitation assay indicated a direct interaction between CgVDAC2 and the pro-apoptotic protein CgBak. This study revealed the function of VDAC2 in oyster and provided new insights into its involvement in apoptosis modulation and host defense in mollusks. PMID:26727366

  7. The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43

    PubMed Central

    Nailwal, H; Sharma, S; Mayank, A K; Lal, S K

    2015-01-01

    The interplay between influenza virus and host factors to support the viral life cycle is well documented. Influenza A virus (IAV) proteins interact with an array of cellular proteins and hijack host pathways which are at the helm of cellular responses to facilitate virus invasion. The multifaceted nature of the ubiquitination pathway for protein regulation makes it a vulnerable target of many viruses including IAV. To this end we conducted a yeast two-hybrid screen to search for cellular ubiquitin ligases important for influenza virus replication. We identified host protein, RING finger protein 43 (RNF43), a RING-type E3 ubiquitin ligase, as a novel interactor of nucleoprotein (NP) of IAV and an essential partner to induce NP-driven p53-mediated apoptosis in IAV-infected cells. In this study, we demonstrate that IAV leads to attenuation of RNF43 transcripts and hence its respective protein levels in the cellular milieu whereas in RNF43 depleted cells, viral replication was escalated several folds. Moreover, RNF43 polyubiquitinates p53 which further leads to its destabilization resulting in a decrease in induction of the p53 apoptotic pathway, a hitherto unknown process targeted by NP for p53 stabilization and accumulation. Collectively, these results conclude that NP targets RNF43 to modulate p53 ubiquitination levels and hence causes p53 stabilization which is conducive to an enhanced apoptosis level in the host cells. In conclusion, our study unravels a novel strategy adopted by IAV for utilizing the much conserved ubiquitin proteasomal pathway. PMID:25996295

  8. Signal transduction and metabolic changes during tumor cell apoptosis following phthalocyanine-sensitized photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Oleinick, Nancy L.; Agarwal, Munna L.; Berger, Nathan A.; Cheng, Ming-Feng; Chatterjee, Satadel; He, Jin; Kenney, Malcolm E.; Larkin, Hedy E.; Mukhter, Hasan; Rihter, Boris D.; Zaidi, Syed I. A.

    1993-06-01

    Mechanisms of cell death have been explored in cells and tumors treated with photodynamic therapy (PDT). Photosensitizers used for these studies were Photofrin, tetrasulfonated and nonsulfonated aluminum phthalocyanine, and a new silicon phthalocyanine [SiPc(OH)OSi(CH3)2(CH2)3N(CH3)2], referred to as PcIV. In mouse lymphoma L5178Y cells, a dose of PDT sensitized by PcIV which causes a 90% loss of cell survival induces apoptosis (programmed cell death) over a several-hour time course, beginning within 10 minutes of irradiation. Apoptosis is a metabolic process initiated by PDT-induced damage to membranes and triggered by the activation of phospholipases A2 and C and the release of Ca++ from intracellular stores. An endogenous endonuclease is activated and cleaves nuclear DNA in the internucleosomal region of chromatin. Subsequent metabolic events now appear to cause the loss of cellular NAD and ATP, the former a result of the activation of a second nuclear enzyme, poly(ADP-ribose) polymerase, by the endonucleolytically generated DNA strand breaks. Loss of ATP follows upon the loss of NAD needed for energy metabolism. Although the induction of apoptosis is efficiently produced by direct PDT damage to L5178Y cells, we now find that apoptosis is also produced by treatment of certain other lymphoid-derived cells and cells of epithelial origin. Under the limited set of conditions tested, there was no evidence for PDT-induced apoptosis in a fibroblast cell line, in mouse fibrosarcoma RIF-1 and L929 cells, in human adenocarcinoma A549 cells, or in human squamous cell carcinoma cells in culture. The evidence suggests that apoptosis, a form of metabolic cell death, is an important mechanism of tumor ablation in PDT-treated tumors, and that the induction of apoptosis may involve the interaction of direct PDT damage to malignant cells with factors produced by PDT action on vascular and other host cells.

  9. Review on Trypanosoma cruzi: Host Cell Interaction

    PubMed Central

    de Souza, Wanderley; de Carvalho, Tecia Maria Ulisses; Barrias, Emile Santos

    2010-01-01

    Trypanosoma cruzi, the causative agent of Chagas' disease, which affects a large number of individuals in Central and South America, is transmitted to vertebrate hosts by blood-sucking insects. This protozoan is an obligate intracellular parasite. The infective forms of the parasite are metacyclic and bloodstream trypomastigote and amastigote. Metacyclic trypomastigotes are released with the feces of the insect while amastigotes and bloodstream trypomastigotes are released from the infected host cells of the vertebrate host after a complex intracellular life cycle. The recognition between parasite and mammalian host cell involves numerous molecules present in both cell types. Here, we present a brief review of the interaction between Trypanosoma cruzi and its host cells, mainly emphasizing the mechanisms and molecules that participate in the T. cruzi invasion process of the mammalian cells. PMID:20811486

  10. Artesunate induces AIF-dependent apoptosis in A549 cells

    NASA Astrophysics Data System (ADS)

    Zhou, Chen-juan; Chen, Tong-Sheng

    2012-03-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

  11. Detection of apoptosis of bone cells in vitro.

    PubMed

    Bellido, Teresita; Plotkin, Lilian I

    2008-01-01

    Studies during the last decade demonstrated that apoptosis is as important as mitosis for the growth and maintenance of the skeleton and provided information on the significance and molecular regulation of apoptosis of bone cells. It is now known that: (1) all osteoclasts die by apoptosis after completing a bone resorption cycle; (2) the majority of osteoblasts also die, whereas the remainder become lining cells or osteocytes; and (3) osteocytes, although long-living cells, also can die prematurely. Furthermore, mounting evidence indicates that systemic hormones, local growth factors, cytokines, and pharmacological agents, as well as mechanical forces regulate the rate of bone cell apoptosis. This chapter summarizes the methods developed in the last few years to examine apoptosis of cultured bone cells and identify the signaling pathways and molecules involved in apoptosis regulation by diverse skeletal stimuli.

  12. Block of Death-Receptor Apoptosis Protects Mouse Cytomegalovirus from Macrophages and Is a Determinant of Virulence in Immunodeficient Hosts

    PubMed Central

    Ebermann, Linda; Ruzsics, Zsolt; Guzmán, Carlos A.; van Rooijen, Nico; Casalegno-Garduño, Rosaely; Koszinowski, Ulrich; Čičin-Šain, Luka

    2012-01-01

    The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC−/−), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system. PMID:23271968

  13. The New World arenavirus Tacaribe virus induces caspase-dependent apoptosis in infected cells.

    PubMed

    Wolff, Svenja; Groseth, Allison; Meyer, Bjoern; Jackson, David; Strecker, Thomas; Kaufmann, Andreas; Becker, Stephan

    2016-04-01

    The Arenaviridae is a diverse and growing family of viruses that already includes more than 25 distinct species. While some of these viruses have a significant impact on public health, others appear to be non-pathogenic. At present little is known about the host cell responses to infection with different arenaviruses, particularly those found in the New World; however, apoptosis is known to play an important role in controlling infection of many viruses. Here we show that infection with Tacaribe virus (TCRV), which is widely considered the prototype for non-pathogenic arenaviruses, leads to stronger induction of apoptosis than does infection with its human-pathogenic relative Junín virus. TCRV-induced apoptosis occurred in several cell types during late stages of infection and was shown to be caspase-dependent, involving the activation of caspases 3, 7, 8 and 9. Further, UV-inactivated TCRV did not induce apoptosis, indicating that the activation of this process is dependent on active viral replication/transcription. Interestingly, when apoptosis was inhibited, growth of TCRV was not enhanced, indicating that apoptosis does not have a direct negative effect on TCRV infection in vitro. Taken together, our data identify and characterize an important virus-host cell interaction of the prototypic, non-pathogenic arenavirus TCRV, which provides important insight into the growing field of arenavirus research aimed at better understanding the diversity in responses to different arenavirus infections and their functional consequences.

  14. The New World arenavirus Tacaribe virus induces caspase-dependent apoptosis in infected cells.

    PubMed

    Wolff, Svenja; Groseth, Allison; Meyer, Bjoern; Jackson, David; Strecker, Thomas; Kaufmann, Andreas; Becker, Stephan

    2016-04-01

    The Arenaviridae is a diverse and growing family of viruses that already includes more than 25 distinct species. While some of these viruses have a significant impact on public health, others appear to be non-pathogenic. At present little is known about the host cell responses to infection with different arenaviruses, particularly those found in the New World; however, apoptosis is known to play an important role in controlling infection of many viruses. Here we show that infection with Tacaribe virus (TCRV), which is widely considered the prototype for non-pathogenic arenaviruses, leads to stronger induction of apoptosis than does infection with its human-pathogenic relative Junín virus. TCRV-induced apoptosis occurred in several cell types during late stages of infection and was shown to be caspase-dependent, involving the activation of caspases 3, 7, 8 and 9. Further, UV-inactivated TCRV did not induce apoptosis, indicating that the activation of this process is dependent on active viral replication/transcription. Interestingly, when apoptosis was inhibited, growth of TCRV was not enhanced, indicating that apoptosis does not have a direct negative effect on TCRV infection in vitro. Taken together, our data identify and characterize an important virus-host cell interaction of the prototypic, non-pathogenic arenavirus TCRV, which provides important insight into the growing field of arenavirus research aimed at better understanding the diversity in responses to different arenavirus infections and their functional consequences. PMID:26769540

  15. Activation-induced apoptosis in peripheral blood mononuclear cells during hepatosplenic Schistosoma mansoni infections.

    PubMed

    Ghoneim, H M; Demian, S R; Heshmat, M G; Ismail, N S; El-Sayed, Laila H

    2008-01-01

    It is well established that programmed cell death (apoptosis) is an important regulator of host responses during infection with a variety of intra- and extra-cellular pathogens. The present work aimed at assessment of in vitro spontaneous and phytohemagglutinin (PHA)-induced apoptosis in mononuclear cells isolated from patients with hepatosplenic form of S. mansoni infections. Cell death data were correlated to the degree of lymphoproliferative responses to PHA as well as to the serum anti-schistosomal antibody titers. A markedly significant increase in PHA-induced apoptosis in lymphocytes isolated from S. mansoni-infected patients was seen when compared to the corresponding healthy controls. However, a slight difference was recorded between the two studied groups regarding the spontaneous apoptosis. This was accompanied with a significant impairment of in vitro PHA-induced lymphoproliferation of T cells from S. mansoni patients. Data of the present study supports the hypothesis that activation-induced cell death (AICD) is a potentially contributing factor in T helper (Th) cell regulation during chronic stages of schistosomiasis, which represents a critically determinant factor in the host-parasite interaction and might influence the destiny of parasitic infections either towards establishment of chronic infection or towards host death.

  16. Activation-induced apoptosis in peripheral blood mononuclear cells during hepatosplenic Schistosoma mansoni infections.

    PubMed

    Ghoneim, H M; Demian, S R; Heshmat, M G; Ismail, N S; El-Sayed, Laila H

    2008-01-01

    It is well established that programmed cell death (apoptosis) is an important regulator of host responses during infection with a variety of intra- and extra-cellular pathogens. The present work aimed at assessment of in vitro spontaneous and phytohemagglutinin (PHA)-induced apoptosis in mononuclear cells isolated from patients with hepatosplenic form of S. mansoni infections. Cell death data were correlated to the degree of lymphoproliferative responses to PHA as well as to the serum anti-schistosomal antibody titers. A markedly significant increase in PHA-induced apoptosis in lymphocytes isolated from S. mansoni-infected patients was seen when compared to the corresponding healthy controls. However, a slight difference was recorded between the two studied groups regarding the spontaneous apoptosis. This was accompanied with a significant impairment of in vitro PHA-induced lymphoproliferation of T cells from S. mansoni patients. Data of the present study supports the hypothesis that activation-induced cell death (AICD) is a potentially contributing factor in T helper (Th) cell regulation during chronic stages of schistosomiasis, which represents a critically determinant factor in the host-parasite interaction and might influence the destiny of parasitic infections either towards establishment of chronic infection or towards host death. PMID:20306689

  17. Penetration of Bdellovibrio bacteriovorus into Host Cells

    PubMed Central

    Abram, Dinah; e Melo, J. Castro; Chou, D.

    1974-01-01

    Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite. Images PMID:4208138

  18. Aspartame-induced apoptosis in PC12 cells.

    PubMed

    Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR. Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity.

  19. Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

    SciTech Connect

    Shu, Guangwen; Yang, Jing; Zhao, Wenhao; Xu, Chan; Hong, Zongguo; Mei, Zhinan; Yang, Xinzhou

    2014-12-01

    Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals.

  20. Differential effects of alloherpesvirus CyHV-3 and rhabdovirus SVCV on apoptosis in fish cells.

    PubMed

    Miest, Joanna J; Adamek, Mikolaj; Pionnier, Nicolas; Harris, Sarah; Matras, Marek; Rakus, Krzysztof Ł; Irnazarow, Ilgiz; Steinhagen, Dieter; Hoole, Dave

    2015-03-23

    Whilst Herpesviridae, which infect higher vertebrates, actively influence host immune responses to ensure viral replication, it is mostly unknown if Alloherpesviridae, which infect lower vertebrates, possess similar abilities. An important antiviral response is clearance of infected cells via apoptosis, which in mammals influences the outcome of infection. Here, we utilise common carp infected with CyHV-3 to determine the effect on the expression of genes encoding apoptosis-related proteins (p53, Caspase 9, Apaf-1, IAP, iNOS) in the pronephros, spleen and gills. The influence of CyHV-3 on CCB cells was also studied and compared to SVCV (a rhabdovirus) which induces apoptosis in carp cell lines. Although CyHV-3 induced iNOS expression in vivo, significant induction of the genetic apoptosis pathway was only seen in the pronephros. In vitro CyHV-3 did not induce apoptosis or apoptosis-related expression whilst SVCV did stimulate apoptosis. This suggests that CyHV-3 possesses mechanisms similar to herpesviruses of higher vertebrates to inhibit the antiviral apoptotic process.

  1. Ethanol promotes T cell apoptosis through the mitochondrial pathway

    PubMed Central

    Kapasi, Aditi A; Patel, Geeta; Goenka, Anuj; Nahar, Nilay; Modi, Neeraj; Bhaskaran, Madhu; Reddy, Krishna; Franki, Nicholas; Patel, Jaimita; Singhal, Pravin C

    2003-01-01

    Clinical reports suggest that acute ethanol intoxication is often associated with lymphopenia. Previously, ethanol was reported to invoke thymocyte apoptosis. We studied the effect of ethanol on T cell apoptosis. In addition, we evaluated the molecular mechanism of ethanol-induced T cell apoptosis. Human T cells harvested from healthy subjects after an alcohol drinking binge showed enhanced T cell apoptosis (before, 0·4 ± 0·2% versus after, 19·6 ± 2·5% apoptotic lymphocytes/field; P < 0·001). In in vitro studies, ethanol in a concentration of 50 mm and higher enhanced the apoptosis of Jurkat cells. DNA isolated from ethanol-treated Jurkat cells displayed integer multiples of 180 base pairs. Ethanol decreased Jurkat cell expression of Bcl-2, whereas ethanol increased Jurkat cell expression of Bax. Jurkat cells treated with ethanol also showed translocation of cytochrome C into cytosol. Moreover, a caspase-9 inhibitor partially inhibited ethanol-induced Jurkat cell apoptosis. In in vivo studies, after binge drinking, T cell expression of Bcl-2 also decreased. In addition, binge drinking induced the cleavage of caspase-3, suggesting activation of caspase-3 in T cells. These results suggest that ethanol promotes T cell apoptosis through the activation of intrinsic or mitochondrial pathway. PMID:12603597

  2. Proteinase-activated receptor-1 mediates allogeneic CD8(+) T cell-induced apoptosis of vascular endothelial cells.

    PubMed

    Quan, Li; Jian, Zhang; Ping, Zou; Weiming, Li

    2009-12-01

    Vascular endothelial-cells injury plays a pivotal role in the pathogenesis of graft-versus-host disease (GVHD) and transplant-associated endothelial injury syndrome. Vascular endothelial cells are an exposed target tissue for immune-mediated injury during GVHD. Early endothelial injury syndromes share common features with acute GVHD. Chronic GVHD leads to a rarefaction of microvessels caused by the infiltration of alloreactive cytotoxic T lymphocytes. In this context, allogeneic reactive cytotoxic T cell may contribute to apoptosis of vascular endothelial cells. The involvement of proteinase-activated receptor (PAR-1) in regulation of apoptosis has been recently recognized in many cell types. We hypothesized that apoptosis of vascular endothelial cells induced by allogeneic cytotoxic T cell are mediated via the PAR-1. Allogeneic CD8(+) T cell, PAR-1 agonist peptide (SFLLRN) induced apoptosis of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs) as assessed by AnnexinV-FITC labeling. To ascertain the mechanism of endothelial apoptosis, we determined that allogeneic CD8(+) T cell, SFLLRN enhanced cleavage of caspase-3 and led to p38MAPK activation as assessed by Western blot. The effects of allogeneic CD8(+) T cell and SFLLRN on apoptosis of vascular endothelial cells were largely prevented by a cleavage-blocking anti-human PAR-1-antibody (ATAP2) and a specific inhibitor of p38MAPK. In concert, these observations provide strong evidence that allogeneic CD8(+) T cell induces apoptosis of human vascular endothelial cells through PAR-1-dependent modulation of intrinsic apoptotic pathway via alterations of p38MAPK and caspase-3. PMID:19082770

  3. Serratia marcescens induces apoptotic cell death in host immune cells via a lipopolysaccharide- and flagella-dependent mechanism.

    PubMed

    Ishii, Kenichi; Adachi, Tatsuo; Imamura, Katsutoshi; Takano, Shinya; Usui, Kimihito; Suzuki, Kazushi; Hamamoto, Hiroshi; Watanabe, Takeshi; Sekimizu, Kazuhisa

    2012-10-19

    Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH(2)-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity.

  4. Apoptosis and T-cell depletion during feline infectious peritonitis.

    PubMed

    Haagmans, B L; Egberink, H F; Horzinek, M C

    1996-12-01

    Cats that have succumbed to feline infectious peritonitis, an immune-mediated disease caused by variants of feline coronaviruses, show apoptosis and T-cell depletion in their lymphoid organs. The ascitic fluid that develops in the course of the condition causes apoptosis in vitro but only in activated T cells. Since feline infectious peritonitis virus does not infect T cells, and viral proteins did not inhibit T-cell proliferation, we postulate that soluble mediators released during the infection cause apoptosis and T-cell depletion.

  5. Probing of cancer cell apoptosis by SERS and LSCM

    NASA Astrophysics Data System (ADS)

    Kang, Jian; Gu, Huaimin

    2009-07-01

    Surface enhanced Raman spectroscopy (SERS) can provide information of internal structures and chemical components from different kinds of samples. Laser scanning confocal microscopy (LSCM) can show morphologic information of samples by high-resolution optical images with different focal planes. In this paper, the dynamic variation of cancer cells (HELA cells) in the apoptosis was first studied by combining SERS and LSCM. After gold nanoparticles (GNPS) uptake, HELA cells were divided into two groups, and were respectively studied at six different time points of cell apoptosis period by SERS and LSCM. The LSCM images of HELA cells obtained at different time points were analyzed, and the morphology varieties of HELA cells apoptosis were obtained. It suggests that HELA cells apoptosis gradually in the apoptosis period until they died. In addition, Raman spectra of HELA cells measured at different time points were also compared. It shows that some Raman signal peaks shift, and FWHM of Raman peaks change too. The variation of internal structures and chemical constituents were analyzed according to the shifts and FWHM of the Raman peaks. The internal dynamic information and morphologic varieties from HELA cells apoptosis gained by combining SERS and LSCM will make us to understand cancer cell apoptosis throughly.

  6. Apoptosis in capillary endothelial cells in ageing skeletal muscle

    PubMed Central

    Wang, Huijuan; Listrat, Anne; Meunier, Bruno; Gueugneau, Marine; Coudy-Gandilhon, Cécile; Combaret, Lydie; Taillandier, Daniel; Polge, Cécile; Attaix, Didier; Lethias, Claire; Lee, Kijoon; Goh, Kheng Lim; Béchet, Daniel

    2014-01-01

    The age-related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein-7 (Pax7) or laminin-2α, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age-related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre-associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7+ satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age-dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing. PMID:24245531

  7. Apoptosis and the selective survival of host animals following thermal bleaching in zooxanthellate corals

    PubMed Central

    Tchernov, Dan; Kvitt, Hagit; Haramaty, Liti; Bibby, Thomas S.; Gorbunov, Maxim Y.; Rosenfeld, Hanna; Falkowski, Paul G.

    2011-01-01

    During the past several decades, numerous reports from disparate geographical areas have documented an increased frequency of “bleaching” in reef-forming corals. The phenomenon, triggered by increased sea surface temperatures, occurs when the cnidarian hosts digest and/or expel their intracellular, photosynthetic dinoflagellate symbionts (“zooxanthellae” in the genus Symbiodinium). Although coral bleaching is often followed by the death of the animal hosts, in some cases, the animal survives and can be repopulated with viable zooxanthellae. The physiological factors determining the ability of the coral to survive bleaching events are poorly understood. In this study, we experimentally established that bleaching and death of the host animal involve a caspase-mediated apoptotic cascade induced by reactive oxygen species produced primarily by the algal symbionts. In addition, we demonstrate that, although some corals naturally suppress caspase activity and significantly reduce caspase concentration under high temperatures as a mechanism to prevent colony death from apoptosis, even sensitive corals can be prevented from dying by application of exogenous inhibitors of caspases. Our results indicate that variability in response to thermal stress in corals is determined by a four-element, combinatorial genetic matrix intrinsic to the specific symbiotic association. Based on our experimental data, we present a working model in which the phenotypic expression of this symbiont/host relationship places a selective pressure on the symbiotic association. The model predicts the survival of the host animals in which the caspase-mediated apoptotic cascade is down-regulated. PMID:21636790

  8. Contrasting Lifestyles Within the Host Cell.

    PubMed

    Di Russo Case, Elizabeth; Samuel, James E

    2016-02-01

    Intracellular bacterial pathogens have evolved to exploit the protected niche provided within the boundaries of a eukaryotic host cell. Upon entering a host cell, some bacteria can evade the adaptive immune response of its host and replicate in a relatively nutrient-rich environment devoid of competition from other host flora. Growth within a host cell is not without their hazards, however. Many pathogens enter their hosts through receptor-mediated endocytosis or phagocytosis, two intracellular trafficking pathways that terminate in a highly degradative organelle, the phagolysosome. This usually deadly compartment is maintained at a low pH and contains degradative enzymes and reactive oxygen species, resulting in an environment to which few bacterial species are adapted. Some intracellular pathogens, such as Shigella, Listeria, Francisella, and Rickettsia, escape the phagosome to replicate within the cytosol of the host cell. Bacteria that remain within a vacuole either alter the trafficking of their initial phagosomal compartment or adapt to survive within the harsh environment it will soon become. In this chapter, we focus on the mechanisms by which different vacuolar pathogens either evade lysosomal fusion, as in the case of Mycobacterium and Chlamydia, or allow interaction with lysosomes to varying degrees, such as Brucella and Coxiella, and their specific adaptations to inhabit a replicative niche. PMID:26999394

  9. Contrasting Lifestyles Within the Host Cell

    PubMed Central

    Case, Elizabeth Di Russo; Samuel, James E.

    2015-01-01

    CHAPTER SUMMARY Intracellular bacterial pathogens have evolved to exploit the protected niche provided within the boundaries of a eukaryotic host cell. Upon entering a host cell, some bacteria can evade the adaptive immune response of its host, and replicate in a relatively nutrient-rich environment devoid of competition from other host flora. Growth within a host cell is not without its hazards, however. Many pathogens enter their hosts through receptor-mediated endocytosis or phagocytosis, two intracellular trafficking pathways that terminate in a highly degradative organelle, the phagolysosome. This usually deadly compartment is maintained at a low pH, and contains degradative enzymes, and reactive oxygen species resulting in an environment to which few bacterial species are adapted. Some intracellular pathogens, like Shigella, Listeria, Francisella, and Rickettsia escape the phagosome to replicate within the cytosol of the host cell. Bacteria that remain within a vacuole either alter the trafficking of their initial phagosomal compartment or adapt to survive within the harsh environment it will soon become. In this chapter, we focus on the mechanisms by which different vacuolar pathogens either evade lysosomal fusion, as in the case of Mycobacterium and Chlamydia, or allow interaction with lysosomes to varying degrees, such as Brucella and Coxiella, and their specific adaptations to inhabit a replicative niche. PMID:26999394

  10. Rac1 mediates intestinal epithelial cell apoptosis via JNK.

    PubMed

    Jin, Shi; Ray, Ramesh M; Johnson, Leonard R

    2006-12-01

    Apoptosis plays a key role in the maintenance of a constant cell number and a low incidence of cancer in the mucosa of the intestine. Although the small GTPase Rac1 has been established as an important regulator of migration of intestinal epithelial cells, whether Rac1 is also involved in apoptosis is unclear. The present study tested the hypothesis that Rac1 mediates TNF-alpha-induced apoptosis in IEC-6 cells. Rac1 is activated during TNF-alpha-induced apoptosis as judged by the level of GTP-Rac1, the level of microsomal membrane-associated Rac1, and lamellipodia formation. Although expression of constitutively active Rac1 does not increase apoptosis in the basal condition, inhibition of Rac1 either by NSC-23766 (Rac1 inhibitor) or expression of dominant negative Rac1 protects cells from TNF-alpha-induced apoptosis by inhibiting caspase-3, -8, and -9 activities. Inhibition of Rac1 before the administration of apoptotic stimuli significantly prevents TNF-alpha-induced activation of JNK1/2, the key proapoptotic regulator in IEC-6 cells. Inhibition of Rac1 does not modulate TNF-alpha-induced ERK1/2 and Akt activation. Inhibition of ERK1/2 and Akt activity by U-0126 and LY-294002, respectively, increased TNF-alpha-induced apoptosis. However, inhibition of Rac1 significantly decreased apoptosis in the presence of ERK1/2 and Akt inhibitors, similar to the effect observed with NSC-23766 alone in response to TNF-alpha. Thus, Rac1 inhibition protects cells independently of ERK1/2 and Akt activation during TNF-alpha-induced apoptosis. Although p38 MAPK is activated in response to TNF-alpha, inhibition of p38 MAPK did not decrease apoptosis. Rac1 inhibition did not alter p38 MAPK activity. Thus, these results indicate that Rac1 mediates apoptosis via JNK and plays a key role in proapoptotic pathways in intestinal epithelial cells.

  11. Apoptosis, oncosis, and necrosis. An overview of cell death.

    PubMed Central

    Majno, G.; Joris, I.

    1995-01-01

    The historical development of the cell death concept is reviewed, with special attention to the origin of the terms necrosis, coagulation necrosis, autolysis, physiological cell death, programmed cell death, chromatolysis (the first name of apoptosis in 1914), karyorhexis, karyolysis, and cell suicide, of which there are three forms: by lysosomes, by free radicals, and by a genetic mechanism (apoptosis). Some of the typical features of apoptosis are discussed, such as budding (as opposed to blebbing and zeiosis) and the inflammatory response. For cell death not by apoptosis the most satisfactory term is accidental cell death. Necrosis is commonly used but it is not appropriate, because it does not indicate a form of cell death but refers to changes secondary to cell death by any mechanism, including apoptosis. Abundant data are available on one form of accidental cell death, namely ischemic cell death, which can be considered an entity of its own, caused by failure of the ionic pumps of the plasma membrane. Because ischemic cell death (in known models) is accompanied by swelling, the name oncosis is proposed for this condition. The term oncosis (derived from ónkos, meaning swelling) was proposed in 1910 by von Reckling-hausen precisely to mean cell death with swelling. Oncosis leads to necrosis with karyolysis and stands in contrast to apoptosis, which leads to necrosis with karyorhexis and cell shrinkage. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7856735

  12. Single-Cell-Precision Microplasma-Induced Cancer Cell Apoptosis

    PubMed Central

    Lu, Xinpei; He, Guangyuan; Ostrikov, Kostya

    2014-01-01

    The issue of single-cell control has recently attracted enormous interest. However, in spite of the presently achievable intracellular-level physiological probing through bio-photonics, nano-probe-based, and some other techniques, the issue of inducing selective, single-cell-precision apoptosis, without affecting neighbouring cells remains essentially open. Here we resolve this issue and report on the effective single-cell-precision cancer cell treatment using the reactive chemistry of the localized corona-type plasma discharge around a needle-like electrode with the spot size ∼1 µm. When the electrode is positioned with the micrometer precision against a selected cell, a focused and highly-localized micro-plasma discharge induces apoptosis in the selected individual HepG2 and HeLa cancer cells only, without affecting any surrounding cells, even in small cell clusters. This is confirmed by the real-time monitoring of the morphological and structural changes at the cellular and cell nucleus levels after the plasma exposure. PMID:24971517

  13. Role of Fas/Fas-L in vascular cell apoptosis.

    PubMed

    Stoneman, Victoria E A; Bennett, Martin R

    2009-02-01

    Apoptosis of vascular cells is observed in vivo in normal vessel development and a variety of vascular pathologies. Apoptosis occurs in all cell types within the vessel wall, the consequences of which depend on both cell type and the pathology under study. The death receptor Fas is expressed throughout the vessel wall, and increasingly Fas-Fas-L-induced killing has been recognized in the vasculature. This review outlines the current developments in understanding the role, regulation, and consequences of Fas-Fas-L-induced apoptosis in vascular cells.

  14. Morphological and cytochemical determination of cell death by apoptosis

    PubMed Central

    Sobel, Burton E.; Budd, Ralph C.

    2007-01-01

    Several modes of cell death are now recognized, including necrosis, apoptosis, and autophagy. Oftentimes the distinctions between these various modes may not be apparent, although the precise mode may be physiologically important. Accordingly, it is often desirable to be able to classify the mode of cell death. Apoptosis was originally defined by structural alterations in cells observable by transmitted light and electron microscopy. Today, a wide variety of imaging and cytochemical techniques are available for the investigation of apoptosis. This review will highlight many of these methods, and provide a critique on the advantages and disadvantages associated with them for the specific identification of apoptotic cells in culture and tissues. PMID:18000678

  15. Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection.

    PubMed

    Silva, Elisabeth M; Guillermo, Landi V C; Ribeiro-Gomes, Flávia L; De Meis, Juliana; Nunes, Marise P; Senra, Juliana F V; Soares, Milena B P; DosReis, George A; Lopes, Marcela F

    2007-03-01

    In experimental Chagas' disease, lymphocytes from mice infected with Trypanosoma cruzi show increased apoptosis in vivo and in vitro. Treatment with a pan-caspase blocker peptide inhibited expression of the active form of effector caspase-3 in vitro and rescued both B and T cells from cell death. Injection of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone, but not a control peptide, reduced parasitemia and lymphocyte apoptosis in T. cruzi-infected mice. Moreover, treatment with caspase inhibitor throughout acute infection increased the absolute numbers of B and T cells in the spleen and lymph nodes, without affecting cell infiltrates in the heart. Following treatment, we found increased accumulation of memory/activated CD4 and CD8 T cells, and secretion of IFN-gamma by splenocytes stimulated with T. cruzi antigens. Caspase inhibition in the course of infection reduced the intracellular load of parasites in peritoneal macrophages, and increased the production of TNF-alpha and nitric oxide upon activation in vitro. Our results indicate that inhibition of caspases with a pan-caspase blocker peptide improves protective type-1 immune responses to T. cruzi infection. We suggest that mechanisms of apoptosis are potential therapeutic targets in Chagas' disease.

  16. Epstein-Barr virus interactions with the Bcl-2 protein family and apoptosis in human tumor cells*

    PubMed Central

    Fu, Qin; He, Chen; Mao, Zheng-rong

    2013-01-01

    Epstein-Barr virus (EBV), a human gammaherpesvirus carried by more than 90% of the world’s population, is associated with malignant tumors such as Burkitt’s lymphoma (BL), Hodgkin lymphoma, post-transplant lymphoma, extra-nodal natural killer/T cell lymphoma, and nasopharyngeal and gastric carcinomas in immune-compromised patients. In the process of infection, EBV faces challenges: the host cell environment is harsh, and the survival and apoptosis of host cells are precisely regulated. Only when host cells receive sufficient survival signals may they immortalize. To establish efficiently a lytic or long-term latent infection, EBV must escape the host cell immunologic mechanism and resist host cell apoptosis by interfering with multiple signaling pathways. This review details the apoptotic pathway disrupted by EBV in EBV-infected cells and describes the interactions of EBV gene products with host cellular factors as well as the function of these factors, which decide the fate of the host cell. The relationships between other EBV-encoded genes and proteins of the B-cell leukemia/lymphoma (Bcl) family are unknown. Still, EBV seems to contribute to establishing its own latency and the formation of tumors by modifying events that impact cell survival and proliferation as well as the immune response of the infected host. We discuss potential therapeutic drugs to provide a foundation for further studies of tumor pathogenesis aimed at exploiting novel therapeutic strategies for EBV-associated diseases. PMID:23303627

  17. Apoptosis in vascular cells induced by cold atmospheric plasma treatment

    NASA Astrophysics Data System (ADS)

    Sladek, Raymond; Stoffels, Eva

    2006-10-01

    Apoptosis is a natural mechanism of cellular self-destruction. It can be triggered by moderate, yet irreversible damage. Apoptosis plays a major role in tissue renewal. Artificial apoptosis induction will become a novel therapy that meets all requirements for tissue-saving surgery. Diseased tissues can disappear without inflammation and scarring. This is particularly important in treatment of blockages in body tracts (e.g. cardiovascular diseases). Artificial induction of apoptosis can be achieved by means of cold plasma treatment. In this work an atmospheric micro-plasma operated in helium/air has been used to induce apoptosis in vascular cells. Parametric studies of apoptosis induction have been conducted; the efficiency is almost 100%. The apoptotic factors are ROS/RNS (reactive oxygen and nitrogen species). Their densities in the plasma have been measured by mass spectrometry. For apoptosis induction, RNS seem to be more important than ROS, because of their relative abundance. Moreover, addition of a ROS scavenger (ascorbic acid) to the cell culture medium does not reduce the occurrence of apoptosis. Cold plasma is a very efficient tool for fundamental studies of apoptosis, and later, for controlled tissue removal in vivo.

  18. HIV: master of the host cell

    PubMed Central

    Arendt, Christopher W; Littman, Dan R

    2001-01-01

    The human immunodeficiency virus has evolved various mechanisms to exploit its host cells, including the interruption and augmentation of signal transduction pathways. Recently, two DNA microarray studies have illustrated a remarkably broad-based perturbation in host transcriptional responses, which is in part mediated by the HIV-encoded Nef protein. HIV therefore seems to function as a 'master regulator' of cellular gene expression. PMID:11737949

  19. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    PubMed Central

    Kuo, Chen-Tzu; Chen, Bing-Chang; Yu, Chung-Chi; Weng, Chih-Ming; Hsu, Ming-Jen; Chen, Chien-Chih; Chen, Mei-Chieh; Teng, Che-Ming; Pan, Shiow-Lin; Bien, Mauo-Ying; Shih, Chung-Hung; Lin, Chien-Huang

    2009-01-01

    In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis. PMID:19405983

  20. Protein tyrosine phosphatase regulation of endothelial cell apoptosis and differentiation.

    PubMed

    Yang, C; Chang, J; Gorospe, M; Passaniti, A

    1996-02-01

    Apoptosis, or programmed cell death, occurs during development and may also be an important factor in many diseases. However, little is known about the signal transduction pathways regulating apoptosis. In these studies, loss of endothelial cell-substrate attachment and apoptosis after removal of growth factors was associated with dephosphorylation of tyrosine residues at the cell periphery. Dephosphorylation of total cellular proteins accompanied apoptosis and was reduced by orthovanadate, an inhibitor of protein tyrosine phosphatases. Orthovanadate blocked the fragmentation of nuclear DNA, inhibited DNA laddering, and suppressed the expression of TRPM-2, an apoptosis-associated gene. The tyrosine phosphorylation levels of FAK125, erk1 (mitogen-activated kinase kinase), and cdc-2 were reduced during apoptosis. FAK125 dephosphorylation was inhibited by orthovanadate, but premature activation (tyrosine dephosphorylation) of cdc-2 was not. Orthovanadate was as effective as basic fibroblast growth factor in activating erk1 without increasing cell proliferation and in preventing the apoptosis of endothelial cells after treatment with tumor necrosis factor alpha. Endothelial cell differentiation on extracellular matrix (Matrigel) was also stimulated by orthovanadate in the absence of basic fibroblast growth factor without affecting growth arrest and inhibition of DNA synthesis. Expression of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1) was down-regulated during the early stages of differentiation, remained low for at least 6 hours as differentiation proceeded, and increased upon completion of differentiation. Cells that failed to down-regulate p21 mRNA on Matrigel in the absence of angiogenic factors underwent apoptosis. These results suggest that protein tyrosine phosphatases are actively involved in signal transduction during apoptosis and may regulate p21 expression to inhibit endothelial cell differentiation.

  1. Necrosis, and then stress induced necrosis-like cell death, but not apoptosis, should be the preferred cell death mode for chemotherapy: clearance of a few misconceptions

    PubMed Central

    Zhang, Ju; Lou, Xiaomin; Jin, Longyu; Zhou, Rongjia; Liu, Siqi; Xu, Ningzhi; Liao, D. Joshua

    2014-01-01

    Cell death overarches carcinogenesis and is a center of cancer researches, especially therapy studies. There have been many nomenclatures on cell death, but only three cell death modes are genuine, i.e. apoptosis, necrosis and stress-induced cell death (SICD). Like apoptosis, SICD is programmed. Like necrosis, SICD is a pathological event and may trigger regeneration and scar formation. Therefore, SICD has subtypes of stress-induced apoptosis-like cell death (SIaLCD) and stress-induced necrosis-like cell death (SInLCD). Whereas apoptosis removes redundant but healthy cells, SICD removes useful but ill or damaged cells. Many studies on cell death involve cancer tissues that resemble parasites in the host patients, which is a complicated system as it involves immune clearance of the alien cancer cells by the host. Cancer resembles an evolutionarily lower-level organism having a weaker apoptosis potential and poorer DNA repair mechanisms. Hence, targeting apoptosis for cancer therapy, i.e. killing via SIaLCD, will be less efficacious and more toxic. On the other hand, necrosis of cancer cells releases cellular debris and components to stimulate immune function, thus counteracting therapy-caused immune suppression and making necrosis better than SIaLCD for chemo drug development. PMID:25594039

  2. Necrosis, and then stress induced necrosis-like cell death, but not apoptosis, should be the preferred cell death mode for chemotherapy: clearance of a few misconceptions.

    PubMed

    Zhang, Ju; Lou, Xiaomin; Jin, Longyu; Zhou, Rongjia; Liu, Siqi; Xu, Ningzhi; Liao, D Joshua

    2014-01-01

    Cell death overarches carcinogenesis and is a center of cancer researches, especially therapy studies. There have been many nomenclatures on cell death, but only three cell death modes are genuine, i.e. apoptosis, necrosis and stress-induced cell death (SICD). Like apoptosis, SICD is programmed. Like necrosis, SICD is a pathological event and may trigger regeneration and scar formation. Therefore, SICD has subtypes of stress-induced apoptosis-like cell death (SIaLCD) and stress-induced necrosis-like cell death (SInLCD). Whereas apoptosis removes redundant but healthy cells, SICD removes useful but ill or damaged cells. Many studies on cell death involve cancer tissues that resemble parasites in the host patients, which is a complicated system as it involves immune clearance of the alien cancer cells by the host. Cancer resembles an evolutionarily lower-level organism having a weaker apoptosis potential and poorer DNA repair mechanisms. Hence, targeting apoptosis for cancer therapy, i.e. killing via SIaLCD, will be less efficacious and more toxic. On the other hand, necrosis of cancer cells releases cellular debris and components to stimulate immune function, thus counteracting therapy-caused immune suppression and making necrosis better than SIaLCD for chemo drug development. PMID:25594039

  3. Piscirickettsia salmonis induces apoptosis in macrophages and monocyte-like cells from rainbow trout.

    PubMed

    Rojas, Verónica; Galanti, Norbel; Bols, Niels C; Jiménez, Verónica; Paredes, Rodolfo; Marshall, Sergio H

    2010-05-15

    Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS) which causes significant losses in salmon production in Chile and other and in other regions in the southern hemisphere. As the killing of phagocytes is an important pathogenic mechanism for other bacteria to establish infections in vertebrates, we investigated whether P. salmonis kills trout macrophages by apoptosis. Apoptosis in infected macrophages was demonstrated by techniques based on morphological changes and host cell DNA fragmentation. Transmission electron microcopy showed classic apoptotic characteristics and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling showed fragmented DNA. Programmed cell death type I was further confirmed by increased binding of annexin V to externalized phosphatidylserine in infected macrophages. Moreover, significant increases of caspase 3 activation were detected in infected cells and treatment with caspase inhibitor caused a decrease in levels of apoptosis. This is the first evidence that P. salmonis induces cell death in trout macrophages. This could lead to bacterial survival and evasion of the host immune response and play an important role in the establishment of infection in the host.

  4. Triggering Apoptosis in Hematopoietic Cells with Cytotoxic Drugs.

    PubMed

    Crowley, Lisa C; Marfell, Brooke J; Scott, Adrian P; Waterhouse, Nigel J

    2016-01-01

    Cytotoxic agents are commonly added to cultured cells in the laboratory to investigate their efficacy, mechanism of action, and therapeutic potential. Most of these agents trigger cell death by apoptosis, which is also the most common form of cell death during development, aging, homeostasis, and eradication of disease. Treatment of cells with cytotoxic agents is therefore useful for investigating basic mechanisms of cell death in the human body. Actinomycin D, a cytotoxic agent isolated from Streptomyces, induces apoptosis in a variety of cell lines including the histiocytic lymphoma cell line U937. Treatment of U937 cells with actinomycin D provides an ideal model of drug-induced apoptosis that can also be used as a positive control for comparison with other treatments. PMID:27371592

  5. Mutations in ribosomal proteins: Apoptosis, cell competition, and cancer.

    PubMed

    Baker, Nicholas E; Kale, Abhijit

    2016-01-01

    Mutations affecting multiple ribosomal proteins are implicated in cancer. Using genetic mosaics in the fruit fly Drosophila, we describe 3 apoptotic mechanisms that affect Rp/Rp homozygous mutant cells, Rp/+ heterozygous cells, or Rp/+ heterozygous cells in competition with nearby wild type cells, and discuss how apoptosis might be related to cancer predisposition. PMID:27308545

  6. TOSO promotes β-cell proliferation and protects from apoptosis.

    PubMed

    Dharmadhikari, G; Mühle, M; Schulthess, F T; Laue, S; Oberholzer, J; Pattou, F; Kerr-Conte, J; Maedler, K

    2012-01-01

    Decreased β-cell mass reflects a shift from quiescence/proliferation into apoptosis, it plays a crucial role in the pathophysiology of diabetes. A major attempt to restore β-cell mass and normoglycemia is to improve β-cell survival. Here we show that switching off the Fas pathway using Fas apoptotic inhibitory protein (Faim/TOSO), which regulates apoptosis upstream of caspase 8, blocked β-cell apoptosis and increased proliferation in human islets. TOSO was clearly expressed in pancreatic β-cells and down-regulated in T2DM. TOSO expression correlated with β-cell turnover; at conditions of improved survival, TOSO was induced. In contrast, TOSO downregulation induced β-cell apoptosis. Although TOSO overexpression resulted in a 3-fold induction of proliferation, proliferating β-cells showed a very limited capacity to undergo multiple rounds of replication. Our data suggest that TOSO is an important regulator of β-cell turnover and switches β-cell apoptosis into proliferation.

  7. Exploitation of host cells by Burkholderia pseudomallei.

    PubMed

    Stevens, Mark P; Galyov, Edouard E

    2004-04-01

    Intracellular bacterial pathogens have evolved mechanisms to enter and exit eukaryotic cells using the power of actin polymerisation and to subvert the activity of cellular enzymes and signal transduction pathways. The proteins deployed by bacteria to subvert cellular processes often mimic eukaryotic proteins in their structure or function. Studies on the exploitation of host cells by the facultative intracellular pathogen Burkholderia pseudomallei are providing novel insights into the pathogenesis of melioidosis, a serious invasive disease of animals and humans that is endemic in tropical and subtropical areas. B. pseudomallei can invade epithelial cells, survive and proliferate inside phagocytes, escape from endocytic vesicles, form actin-based membrane protrusions and induce host cell fusion. Here we review current understanding of the molecular mechanisms underlying these processes.

  8. Staphylococcal Esx Proteins Modulate Apoptosis and Release of Intracellular Staphylococcus aureus during Infection in Epithelial Cells

    PubMed Central

    Korea, Charalampia G.; Balsamo, Giuliana; Pezzicoli, Alfredo; Merakou, Christina; Tavarini, Simona; Bagnoli, Fabio; Serruto, Davide

    2014-01-01

    The opportunistic pathogen Staphylococcus aureus is one of the major causes of health care-associated infections. S. aureus is primarily an extracellular pathogen, but it was recently reported to invade and replicate in several host cell types. The ability of S. aureus to persist within cells has been implicated in resistance to antimicrobials and recurrent infections. However, few staphylococcal proteins that mediate intracellular survival have been identified. Here we examine if EsxA and EsxB, substrates of the ESAT-6-like secretion system (Ess), are important during intracellular S. aureus infection. The Esx proteins are required for staphylococcal virulence, but their functions during infection are unclear. While isogenic S. aureus esxA and esxB mutants were not defective for epithelial cell invasion in vitro, a significant increase in early/late apoptosis was observed in esxA mutant-infected cells compared to wild-type-infected cells. Impeding secretion of EsxA by deleting C-terminal residues of the protein also resulted in a significant increase of epithelial cell apoptosis. Furthermore, cells transfected with esxA showed an increased protection from apoptotic cell death. A double mutant lacking both EsxA and EsxB also induced increased apoptosis but, remarkably, was unable to escape from cells as efficiently as the single mutants or the wild type. Thus, using in vitro models of intracellular staphylococcal infection, we demonstrate that EsxA interferes with host cell apoptotic pathways and, together with EsxB, mediates the release of S. aureus from the host cell. PMID:25047846

  9. Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells

    PubMed Central

    Bayles, Kenneth W.; Wesson, Carla A.; Liou, Linda E.; Fox, Lawrence K.; Bohach, Gregory A.; Trumble, W. R.

    1998-01-01

    We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes. PMID:9423876

  10. Autophagy regulates colistin-induced apoptosis in PC-12 cells.

    PubMed

    Zhang, Ling; Zhao, Yonghao; Ding, Wenjian; Jiang, Guozheng; Lu, Ziyin; Li, Li; Wang, Jinli; Li, Jian; Li, Jichang

    2015-04-01

    Colistin is a cyclic cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. Our recent study demonstrated that colistin induces apoptosis in primary chick cortex neurons and PC-12 cells. Although apoptosis and autophagy have different impacts on cell fate, there is a complex interaction between them. Autophagy plays an important role as a homeostasis regulator by removing excessive or unnecessary proteins and damaged organelles. The aim of the present study was to investigate the modulation of autophagy and apoptosis regulation in PC-12 cells in response to colistin treatment. PC-12 cells were exposed to colistin (125 to 250 μg/ml), and autophagy was detected by visualization of monodansylcadaverine (MDC)-labeled vacuoles, LC3 (microtubule-associated protein 1 light chain 3) immunofluorescence microscopic examination, and Western blotting. Apoptosis was measured by flow cytometry, Hoechst 33258 staining, and Western blotting. Autophagosomes were observed after treatment with colistin for 12 h, and the levels of LC3-II gene expression were determined; observation and protein levels both indicated that colistin induced a high level of autophagy. Colistin treatment also led to apoptosis in PC-12 cells, and the level of caspase-3 expression increased over the 24-h period. Pretreatment of cells with 3-methyladenine (3-MA) increased colistin toxicity in PC-12 cells remarkably. However, rapamycin treatment significantly increased the expression levels of LC3-II and beclin 1 and decreased the rate of apoptosis of PC-12 cells. Our results demonstrate that colistin induced autophagy and apoptosis in PC-12 cells and that the latter was affected by the regulation of autophagy. It is very likely that autophagy plays a protective role in the reduction of colistin-induced cytotoxicity in neurons.

  11. Plasma-activated medium induced apoptosis on tumor cells

    NASA Astrophysics Data System (ADS)

    Hori, Masaru; Tanaka, Hiromasa; Mizuno, Masaaki; Nakamura, Kae; Kajiyama, Hiroaki; Takeda, Keigo; Ishikawa, Kenji; Kano, Hiroyuki; Kikkawa, Fumitaka

    2013-09-01

    The non-equilibrium atmospheric pressure plasma (NEAPP) has attracted attention in cancer therapy. In this study, the fresh medium was treated with our developed NEAPP, ultra-high electron density (approximately 2 × 1016 cm-3). The medium called the plasma-activated medium (PAM) killed not normal cells but tumor cells through induction of apoptosis. Cell proliferation assays showed that the tumor cells were selectively killed by the PAM. Those cells induced apoptosis using an apoptotic molecular marker, cleaved Caspase3/7. The molecular mechanisms of PAM-mediated apoptosis in the tumor cells were also found that the PAM downregulated the expression of AKT kinase, a marker molecule in a survival signal transduction pathway. These results suggest that PAM may be a promising tool for tumor therapy by downregulating the survival signals in cancers.

  12. Taurine induces the apoptosis of breast cancer cells by regulating apoptosis-related proteins of mitochondria.

    PubMed

    Zhang, Xiali; Lu, Hongfei; Wang, Yibing; Liu, Chunju; Zhu, Weifeng; Zheng, Shuangyan; Wan, Fusheng

    2015-01-01

    Taurine (Tau), the most abundant free amino acid in humans has numerous potential health benefits through its antioxidant and anti-inflammatory properties. However, limited studies have assessed its effect on tumors and the antitumor mechanism remains unknown. The present study investigated the cellular and molecular changes induced by Tau, leading to the induction of apoptosis in human breast cancer cell lines MCF-7 and MDA-MB-231. MCF-7 is p53 proficient (p53+/+) and MDA-MB-231 is a p53 null mutant (p53-/-). Cell proliferation and viability were assessed by MTT. Flow cytometry and hoechst33342 fluorescent staining were employed to detect apoptosis. Spectrophotometry was used to detect caspase-3 activity. Reverse transcription-polymerase chain reaction and western blot analysis were used to detect the levels of mRNA and proteins of p53-upregulated modulator of apoptosis (PUMA), Bax and Bcl-2. Finally, the affect of Tau on the growth of MDA-MB-231-cell-nude mice xenografts was examined. In the study, Tau inhibited growth and induced apoptosis of the two cell lines in a concentration- and time-dependent manner. Notably, the inhibitory effect of Tau on p53-/- cancer cells was clearly significant compared to the p53+/+ cancer cells. Further studies showed that Tau promoted apoptosis in human breast cancer cells and inhibited the growth of tumor in nude mice by inducing the expression of PUMA, which further up- and downregulated the expression of Bax and Bcl-2 protein, giving rise to increased activation of caspase-3. Collectively, these results indicate that Tau is a potent candidate for the chemotherapy of breast cancer through increasing the PUMA expression independent of p53 status. PMID:25395275

  13. The trans-sialidase from Trypanosoma cruzi triggers apoptosis by target cell sialylation.

    PubMed

    Mucci, Juan; Risso, Marikena G; Leguizamón, M Susana; Frasch, Alberto C C; Campetella, Oscar

    2006-07-01

    The trans-sialidase, a modified sialidase that transfers sialyl residues among macromolecules, is a unique enzymatic activity expressed by some parasitic trypanosomes being essential for their survival in the mammalian host and/or in the insect vector. The enzyme from Trypanosoma cruzi, the agent of Chagas disease, is found in blood and able to act far from the infection site by inducing apoptosis in cells from the immune system. A central and still unsolved question is whether trans-sialidase-mediated addition or removal of sialic acid to/from host acceptor molecules is the event associated with the apoptosis induced by the enzyme. Here we show that lactitol, a competitive inhibitor that precluded the transference of the sialyl residue to endogenous acceptors but not the hydrolase activity of the enzyme, prevented ex vivo and in vivo the apoptosis caused by the trans-sialidase. By lectin histochemistry, the transference of sialyl residue to the cell surface was demonstrated in vivo and found associated with the apoptosis induction. The sialylation of the CD43 mucin, a key molecule involved in trans-sialidase-apoptotic process, was readily detected and also prevented by lactitol on thymocytes. Therefore, lesions induced by trans-sialidase on the immune system are due to the sialylation of endogenous acceptor molecules. PMID:16819962

  14. Protochlamydia Induces Apoptosis of Human HEp-2 Cells through Mitochondrial Dysfunction Mediated by Chlamydial Protease-Like Activity Factor

    PubMed Central

    Matsuo, Junji; Nakamura, Shinji; Ito, Atsushi; Yamazaki, Tomohiro; Ishida, Kasumi; Hayashi, Yasuhiro; Yoshida, Mitsutaka; Takahashi, Kaori; Sekizuka, Tsuyoshi; Takeuchi, Fumihiko; Kuroda, Makoto; Nagai, Hiroki; Hayashida, Kyoko; Sugimoto, Chihiro; Yamaguchi, Hiroyuki

    2013-01-01

    Obligate amoebal endosymbiotic bacterium Protochlamydia with ancestral pathogenic chlamydial features evolved to survive within protist hosts, such as Acanthamoba, 0.7–1.4 billion years ago, but not within vertebrates including humans. This observation raises the possibility that interactions between Protochlamydia and human cells may result in a novel cytopathic effect, leading to new insights into host-parasite relationships. Previously, we reported that Protochlamydia induces apoptosis of the immortalized human cell line, HEp-2. In this study, we attempted to elucidate the molecular mechanism underlying this apoptosis. We first confirmed that, upon stimulation with the bacteria, poly (ADP-ribose) polymerase (PARP) was cleaved at an early stage in HEp-2 cells, which was dependent on the amount of bacteria. A pan-caspase inhibitor and both caspase-3 and -9 inhibitors similarly inhibited the apoptosis of HEp-2 cells. A decrease of the mitochondrial membrane potential was also confirmed. Furthermore, lactacystin, an inhibitor of chlamydial protease-like activity factor (CPAF), blocked the apoptosis. Cytochalasin D also inhibited the apoptosis, which was dependent on the drug concentration, indicating that bacterial entry into cells was required to induce apoptosis. Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis. We also confirmed that the Protochlamydia used in this study possessed a homologue of the cpaf gene and that two critical residues, histidine-101 and serine-499 of C. trachomatis CPAF in the active center, were conserved. Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis. More interestingly, because C. trachomatis infection can block the apoptosis, our finding implies unique features of CPAF between pathogenic and primitive

  15. Protochlamydia induces apoptosis of human HEp-2 cells through mitochondrial dysfunction mediated by chlamydial protease-like activity factor.

    PubMed

    Matsuo, Junji; Nakamura, Shinji; Ito, Atsushi; Yamazaki, Tomohiro; Ishida, Kasumi; Hayashi, Yasuhiro; Yoshida, Mitsutaka; Takahashi, Kaori; Sekizuka, Tsuyoshi; Takeuchi, Fumihiko; Kuroda, Makoto; Nagai, Hiroki; Hayashida, Kyoko; Sugimoto, Chihiro; Yamaguchi, Hiroyuki

    2013-01-01

    Obligate amoebal endosymbiotic bacterium Protochlamydia with ancestral pathogenic chlamydial features evolved to survive within protist hosts, such as Acanthamoba, 0.7-1.4 billion years ago, but not within vertebrates including humans. This observation raises the possibility that interactions between Protochlamydia and human cells may result in a novel cytopathic effect, leading to new insights into host-parasite relationships. Previously, we reported that Protochlamydia induces apoptosis of the immortalized human cell line, HEp-2. In this study, we attempted to elucidate the molecular mechanism underlying this apoptosis. We first confirmed that, upon stimulation with the bacteria, poly (ADP-ribose) polymerase (PARP) was cleaved at an early stage in HEp-2 cells, which was dependent on the amount of bacteria. A pan-caspase inhibitor and both caspase-3 and -9 inhibitors similarly inhibited the apoptosis of HEp-2 cells. A decrease of the mitochondrial membrane potential was also confirmed. Furthermore, lactacystin, an inhibitor of chlamydial protease-like activity factor (CPAF), blocked the apoptosis. Cytochalasin D also inhibited the apoptosis, which was dependent on the drug concentration, indicating that bacterial entry into cells was required to induce apoptosis. Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis. We also confirmed that the Protochlamydia used in this study possessed a homologue of the cpaf gene and that two critical residues, histidine-101 and serine-499 of C. trachomatis CPAF in the active center, were conserved. Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis. More interestingly, because C. trachomatis infection can block the apoptosis, our finding implies unique features of CPAF between pathogenic and primitive

  16. Differential apoptosis-like cell death in amastigote and trypomastigote forms from Trypanosoma cruzi-infected heart cells in vitro.

    PubMed

    De Souza, E M; Nefertiti, A S G; Bailly, C; Lansiaux, A; Soeiro, M N C

    2010-07-01

    Apoptosis, type-I of programmed cell death (PCD-I), is not restricted to multicellular organisms since many apoptotic features have been described in different trypanosomatids, including Trypanosoma cruzi. Our present aim was to monitor, by different morphological markers, the occurrence of apoptosis-like death in amastigotes and trypomastigotes of T.cruzi (Y strain) during the infection of heart culture cells. We documented the differential occurrence of PCD-I in amastigotes and trypomastigotes, with distinct death rates noticed between these two parasite-distinct forms. Fluorescence microscopy and flow cytometry analysis using different hall markers of apoptosis (phosphatidylserine exposure, collapse of mitochondrial membrane potential and DNA fragmentation) showed that amastigotes present higher levels of apoptosis-like cell death as compared to trypomastigotes. It is possible that the higher levels of PCD-I in these highly multiplicative forms may contribute to the control of the parasite burden within the host cells. On the other hand, the apoptosis-like occurrence in the infective but non-proliferative stage of the parasite (trypomastigotes) may play a role in parasite evasion mechanisms as suggested for other parasites.

  17. Comparison of Types of Cell Death: Apoptosis and Necrosis.

    ERIC Educational Resources Information Center

    Manning, Francis; Zuzel, Katherine

    2003-01-01

    Cell death is an essential factor in many biological processes including development. Discusses two types of cell death: (1) necrosis (induced by sodium azide); and (2) apoptosis (induced by sodium chromate). Illustrates key features that differ between these two types of cells death including loss of membrane integrity and internucleosomal DNA…

  18. Dendritic Cell Apoptosis and the Pathogenesis of Dengue

    PubMed Central

    Martins, Sharon de T.; Silveira, Guilherme F.; Alves, Lysangela R.; dos Santos, Claudia Nunes Duarte; Bordignon, Juliano

    2012-01-01

    Dengue viruses and other members of the Flaviviridae family are emerging human pathogens. Dengue is transmitted to humans by Aedes aegypti female mosquitoes. Following infection through the bite, cells of the hematopoietic lineage, like dendritic cells, are the first targets of dengue virus infection. Dendritic cells (DCs) are key antigen presenting cells, sensing pathogens, processing and presenting the antigens to T lymphocytes, and triggering an adaptive immune response. Infection of DCs by dengue virus may induce apoptosis, impairing their ability to present antigens to T cells, and thereby contributing to dengue pathogenesis. This review focuses on general mechanisms by which dengue virus triggers apoptosis, and possible influence of DC-apoptosis on dengue disease severity. PMID:23202502

  19. Apoptosis of human seminoma cells upon disruption of their microenvironment.

    PubMed Central

    Olie, R. A.; Boersma, A. W.; Dekker, M. C.; Nooter, K.; Looijenga, L. H.; Oosterhuis, J. W.

    1996-01-01

    One of the main obstacles encountered when trying to culture human seminoma (SE) cells in vitro is massive degeneration of the tumour cells. We investigated whether dissociation of tumour tissue, to obtain single-cell suspensions for in vitro culture, results in the onset of apoptosis. Using morphological analysis and in situ end labelling, less than 4% of apoptotic tumour cells were detected in intact tissue from 11 out of 14 SEs. In these 11 tumours, apoptosis-specific DNA ladders, indicative of internucleosomal double-strand DNA cleavage, were not detected on electrophoresis gels. In contrast, three SEs with over 12% of apoptotic tumour cells in the intact tissue and all analysed (pure) SE cell suspensions, obtained after mechanical dissociation of intact tumour tissue, showed DNA ladders. Flow cytometric analysis of end labelled SE suspensions showed DNA breaks in up to 85% of the tumour cells. As indicated by cell morphology and DNA degradation, SE cells appear to rapidly enter the apoptotic pathway upon mechanical disruption of their microenvironment. No expression of p53 and of the apoptosis-inhibitor bcl-2 was detectable in intact SE tissue or cell suspensions. Our data suggest that abrogation of apoptosis might be crucial to succeed in culturing human SE cells in vitro. Images Figure 1 Figure 2 Figure 4 PMID:8624259

  20. Cell shrinkage and monovalent cation fluxes: role in apoptosis.

    PubMed

    Bortner, Carl D; Cidlowski, John A

    2007-06-15

    The loss of cell volume or cell shrinkage has been a morphological hallmark of the programmed cell death process known as apoptosis. This isotonic loss of cell volume has recently been term apoptotic volume decrease or AVD to distinguish it from inherent volume regulatory responses that occurs in cells under anisotonic conditions. Recent studies examining the intracellular signaling pathways that result in this unique cellular characteristic have determined that a fundamental movement of ions, particularly monovalent ions, underlie the AVD process and plays an important role on controlling the cell death process. An efflux of intracellular potassium was shown to be a critical aspect of the AVD process, as preventing this ion loss could protect cells from apoptosis. However, potassium plays a complex role as a loss of intracellular potassium has also been shown to be beneficial to the health of the cell. Additionally, the mechanisms that a cell employs to achieve this loss of intracellular potassium vary depending on the cell type and stimulus used to induce apoptosis, suggesting multiple ways exist to accomplish the same goal of AVD. Additionally, sodium and chloride have been shown to play a vital role during cell death in both the signaling and control of AVD in various apoptotic model systems. This review examines the relationship between this morphological change and intracellular monovalent ions during apoptosis. PMID:17321483

  1. Parasite calcineurin regulates host cell recognition and attachment by apicomplexans

    PubMed Central

    Paul, Aditya S.; Saha, Sudeshna; Engelberg, Klemens; Jiang, Rays H.Y.; Coleman, Bradley I.; Kosber, Aziz L.; Chen, Chun-Ti; Ganter, Markus; Espy, Nicole; Gilberger, Tim W.; Gubbels, Marc-Jan; Duraisingh, Manoj T.

    2015-01-01

    SUMMARY Apicomplexans invade a variety of metazoan host cells through mechanisms involving host cell receptor engagement and secretion of parasite factors to facilitate cellular attachment. We find that the parasite homolog of calcineurin, a calcium-regulated phosphatase complex central to signal transduction in eukaryotes, also contributes to host cell invasion by the malaria parasite Plasmodium falciparum and related Toxoplasma gondii. Using reverse genetic and chemical-genetic approaches, we determine that calcineurin critically regulates and stabilizes attachment of extracellular P. falciparum to host erythrocytes before intracellular entry and has similar functions in host cell engagement by T. gondii. Calcineurin-mediated Plasmodium invasion is strongly associated with host receptors required for host cell recognition and calcineurin function distinguishes this form of receptor-mediated attachment from a second mode of host-parasite adhesion independent of host receptors. This specific role of calcineurin in coordinating physical interactions with host cells highlights an ancestral mechanism for parasitism used by apicomplexans. PMID:26118996

  2. Apoptosis of epithelial cells and macrophages due to nonpigmented Serratia marcescens strains.

    PubMed

    Krzymińska, Sylwia; Ochocka, Katarzyna; Kaznowski, Adam

    2012-01-01

    Serratia marcescens strains are opportunistic pathogens that are increasingly recognized as a cause of severe nosocomial infections. In this study we observed interactions between nonpigmented strains with human epithelial and macrophage-like cells. The strains revealed hemolytic activity only after the contact of the cells with erythrocytes. The contact of the bacteria with the host cells was also essential to their cytotoxicity. Moreover, all strains revealed adherence ability and were invasive to epithelial cells. Analyses of cellular morphology and DNA fragmentation of the HEp-2 and J774 cells exhibited typical features of cells undergoing apoptosis. We observed morphological changes, including condensation of nuclear chromatin and formation of membrane-bound apoptotic bodies. The lowest apoptotic index in HEp-2 cells did not exceed 25%, whereas the highest reached 59% at 24 h and 72% at 48 h after infection. Most of the strains (60%) induced fragmentation of nuclear DNA. The process depended on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. This study provided new insights into the mechanisms of nonpigmented S. marcescens pathogenesis. The results revealed that the strains produce cell-contact toxins that facilitate bacterial invasion, induce hemolysis, cytotoxicity, and apoptosis of host cells.

  3. Induction of apoptosis in human endothelial cells by nanodiamond particles.

    PubMed

    Solarska, K; Gajewska, A; Bartosz, G; Mitura, K

    2012-06-01

    Carbon nanoparticles are a promising material which finds application in different fields in industry and medicine. For medical applications, biocompatibility of nanoparticles is of critical importance because a lot of medical implants are coated by carbon coating. Our previous results showed that nanoparticles may induce increased production of ROS by the cells so we decided to checked if nanopowders can induce apoptosis. Apoptosis was quantified by double-staining with acridine orange and ethidium bromide. For comparison, we identified apoptotic cells with annexin V-FITC/propidium iodide. Our data demonstrate that treatment of the cells with diamond nanopowders may induce apoptosis and necrosis and this effect is dependent on the time of treatment and concentration of the nanopowders. The highest level of apoptotic cells was observed after incubation with Ultrananocrystalline Detonation Diamond (UDD) suggesting that the size is the main determinant of nanoparticle cytotoxicity. PMID:22905588

  4. Diabetes and apoptosis: neural crest cells and neural tube

    PubMed Central

    Chappell, James H.; Dan Wang, Xiao

    2016-01-01

    Birth defects resulting from diabetic pregnancy are associated with apoptosis of a critical mass of progenitor cells early during the formation of the affected organ(s). Insufficient expression of genes that regulate viability of the progenitor cells is responsible for the apoptosis. In particular, maternal diabetes inhibits expression of a gene, Pax3, that encodes a transcription factor which is expressed in neural crest and neuroepithelial cells. As a result of insufficient Pax3, cardiac neural crest and neuroepithelial cells undergo apoptosis by a process dependent on the p53 tumor suppressor protein. This, then provides a cellular explanation for the cardiac outflow tract and neural tube and defects induced by diabetic pregnancy. PMID:19333760

  5. Apoptosis and necrosis in vaccinia virus-infected HeLa G and BSC-40 cells.

    PubMed

    Liskova, Jana; Knitlova, Jarmila; Honner, Richard; Melkova, Zora

    2011-09-01

    In most cells, vaccinia virus (VACV) infection is considered to cause a lytic cell death, an equivalent of necrosis. However, upon infection of the epithelial cell lines HeLa G and BSC-40 with VACV strain Western Reserve (WR), we have previously observed an increased activation of and activity attributable to caspases, a typical sign of apoptosis. In this paper, we have further analyzed the type of cell death in VACV-infected cells HeLa G and BSC-40. In a cell-based flow cytometric assay, we showed a specific activation of caspase-2 and 4 in HeLa G and BSC-40 cells infected with VACV, strain WR, while we did not find any effects of inhibitors of calpain and cathepsin D and E. The actual activity of the two caspases, but also of caspase-3, was then confirmed in lysates of infected HeLa G, but not in BSC-40 cells. Accordingly, poly(ADP)-ribose polymerase (PARP) cleavage was found increased only in infected HeLa G cells. Consequently, we have determined morphological features of apoptosis and/or activity of the executioner caspase-3 in infected HeLa G cells in situ, while only a background apoptosis was observed in infected BSC-40 cells. Finally, vaccination strains Dryvax and Praha were found to induce apoptosis in both HeLa G and BSC-40 cells, as characterized morphologically and by PARP cleavage. These findings may be important for understanding the differences in VACV-host interactions and post-vaccination complications in different individuals.

  6. Signaling pathways that regulate life and cell death: evolution of apoptosis in the context of self-defense.

    PubMed

    Muñoz-Pinedo, Cristina

    2012-01-01

    Programmed Cell Death is essential for the life cycle of many organisms. Cell death in multicellular organisms can occur as a consequence of massive damage (necrosis) or in a controlled form, through engagement of diverse biochemical programs. The best well known form of programmed cell death is apoptosis. Apoptosis occurs in animals as a consequence of a variety of stimuli including stress and social signals and it plays essential roles in morphogenesis and immune defense. The machinery of apoptosis is well conserved among animals and it is composed of caspases (the proteases which execute cell death), adapter proteins (caspase activators), Bcl-2 family proteins and Inhibitor of Apoptosis Proteins (IAPs). We will describe in this chapter the main apoptotic pathways in animals: the extrinsic (death receptor-mediated), the intrinsic/mitochondrial and the Granzyme B pathway. Other forms of non-apoptotic Programmed Cell Death which occur in animals will also be discussed. We will summarize the current knowledge about apoptotic-like and other forms of cell death in other organisms such as plants and protists.Additionally, we will discuss the hypothesis that apoptosis originated as part of a host defense mechanism. We will explore the similarities between the protein complexes which mediate apoptosis (apoptosomes) and complexes involved in immunity: inflammasomes. Additional functions of apoptotic proteins related to immune function will be summarized, in an effort to explore the evolutionary origins of cell death.

  7. Silencing CDK4 radiosensitizes breast cancer cells by promoting apoptosis

    PubMed Central

    2013-01-01

    Background The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery. Methods To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks. Results Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis. Conclusion Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways. PMID:23886499

  8. Analysis of Host Gene Expression Profile in HIV-1 and HIV-2 Infected T-Cells.

    PubMed

    Devadas, Krishnakumar; Biswas, Santanu; Haleyurgirisetty, Mohan; Wood, Owen; Ragupathy, Viswanath; Lee, Sherwin; Hewlett, Indira

    2016-01-01

    HIV replication is closely regulated by a complex pathway of host factors, many of them being determinants of cell tropism and host susceptibility to HIV infection. These host factors are known to exert a positive or negative influence on the replication of the two major types of HIV, HIV-1 and HIV-2, thereby modulating virus infectivity, host response to infection and ultimately disease progression profiles characteristic of these two types. Understanding the differential regulation of host cellular factors in response to HIV-1 and HIV-2 infections will help us to understand the apparent differences in rates of disease progression and pathogenesis. This knowledge would aid in the discovery of new biomarkers that may serve as novel targets for therapy and diagnosis. The objective of this study was to determine the differential expression of host genes in response to HIV-1/HIV-2 infection. To achieve this, we analyzed the effects of HIV-1 (MN) and HIV-2 (ROD) infection on the expression of host factors in PBMC at the RNA level using the Agilent Whole Human Genome Oligo Microarray. Differentially expressed genes were identified and their biological functions determined. Host gene expression profiles were significantly changed. Gene expression profiling analysis identified a subset of differentially expressed genes in HIV-1 and HIV-2 infected cells. Genes involved in cellular metabolism, apoptosis, immune cell proliferation and activation, cytokines, chemokines, and transcription factors were differentially expressed in HIV-1 infected cells. Relatively few genes were differentially expressed in cells infected with HIV-2.

  9. Analysis of Host Gene Expression Profile in HIV-1 and HIV-2 Infected T-Cells

    PubMed Central

    Devadas, Krishnakumar; Biswas, Santanu; Haleyurgirisetty, Mohan; Wood, Owen; Ragupathy, Viswanath; Lee, Sherwin; Hewlett, Indira

    2016-01-01

    HIV replication is closely regulated by a complex pathway of host factors, many of them being determinants of cell tropism and host susceptibility to HIV infection. These host factors are known to exert a positive or negative influence on the replication of the two major types of HIV, HIV-1 and HIV-2, thereby modulating virus infectivity, host response to infection and ultimately disease progression profiles characteristic of these two types. Understanding the differential regulation of host cellular factors in response to HIV-1 and HIV-2 infections will help us to understand the apparent differences in rates of disease progression and pathogenesis. This knowledge would aid in the discovery of new biomarkers that may serve as novel targets for therapy and diagnosis. The objective of this study was to determine the differential expression of host genes in response to HIV-1/HIV-2 infection. To achieve this, we analyzed the effects of HIV-1 (MN) and HIV-2 (ROD) infection on the expression of host factors in PBMC at the RNA level using the Agilent Whole Human Genome Oligo Microarray. Differentially expressed genes were identified and their biological functions determined. Host gene expression profiles were significantly changed. Gene expression profiling analysis identified a subset of differentially expressed genes in HIV-1 and HIV-2 infected cells. Genes involved in cellular metabolism, apoptosis, immune cell proliferation and activation, cytokines, chemokines, and transcription factors were differentially expressed in HIV-1 infected cells. Relatively few genes were differentially expressed in cells infected with HIV-2. PMID:26821323

  10. Cilostazol suppresses angiotensin II-induced apoptosis in endothelial cells.

    PubMed

    Shi, Miao-Qian; Su, Fei-Fei; Xu, Xuan; Liu, Xiong-Tao; Wang, Hong-Tao; Zhang, Wei; Li, Xue; Lian, Cheng; Zheng, Qiang-Sun; Feng, Zhi-Chun

    2016-03-01

    Patients with essential hypertension undergo endothelial dysfunction, particularly in the conduit arteries. Cilostazol, a type III phosphodiesterase inhibitor, serves a role in the inhibition of platelet aggregation and it is widely used in the treatment of peripheral vascular diseases. Previous studies have suggested that cilostazol suppresses endothelial dysfunction; however, it remains unknown whether cilostazol protects the endothelial function in essential hypertension. The aim of the present study was to investigate whether, and how, cilostazol suppresses angiotensin II (angII)‑induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) and Sprague Dawley rats were exposed to angII and treated with cilostazol. Endothelial cell apoptosis and function, nitric oxide and superoxide production, phosphorylation (p) of Akt, and caspase‑3 protein expression levels were investigated. AngII exposure resulted in the apoptosis of endothelial cells in vitro and in vivo. In vitro, cilostazol significantly suppressed the angII‑induced apoptosis of HUVECs; however, this effect was reduced in the presence of LY294002, a phosphoinositide 3 kinase (PI3K) inhibitor. Furthermore, cilostazol suppressed the angII‑induced p‑Akt downregulation and cleaved caspase‑3 upregulation. These effects were also alleviated by LY294002. In vivo, cilostazol suppressed the angII‑induced endothelial cell apoptosis and dysfunction. Cilostazol was also demonstrated to partially reduced the angII‑induced increase in superoxide production. The results of the present study suggested that cilostazol suppresses endothelial apoptosis and dysfunction by modulating the PI3K/Akt pathway.

  11. Cilostazol suppresses angiotensin II-induced apoptosis in endothelial cells

    PubMed Central

    SHI, MIAO-QIAN; SU, FEI-FEI; XU, XUAN; LIU, XIONG-TAO; WANG, HONG-TAO; ZHANG, WEI; LI, XUE; LIAN, CHENG; ZHENG, QIANG-SUN; FENG, ZHI-CHUN

    2016-01-01

    Patients with essential hypertension undergo endothelial dysfunction, particularly in the conduit arteries. Cilostazol, a type III phosphodiesterase inhibitor, serves a role in the inhibition of platelet aggregation and it is widely used in the treatment of peripheral vascular diseases. Previous studies have suggested that cilostazol suppresses endothelial dysfunction; however, it remains unknown whether cilostazol protects the endothelial function in essential hypertension. The aim of the present study was to investigate whether, and how, cilostazol suppresses angiotensin II (angII)-induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) and Sprague Dawley rats were exposed to angII and treated with cilostazol. Endothelial cell apoptosis and function, nitric oxide and superoxide production, phosphorylation (p) of Akt, and caspase-3 protein expression levels were investigated. AngII exposure resulted in the apoptosis of endothelial cells in vitro and in vivo. In vitro, cilostazol significantly suppressed the angII-induced apoptosis of HUVECs; however, this effect was reduced in the presence of LY294002, a phosphoinositide 3 kinase (PI3K) inhibitor. Furthermore, cilostazol suppressed the angII-induced p-Akt downregulation and cleaved caspase-3 upregulation. These effects were also alleviated by LY294002. In vivo, cilostazol suppressed the angII-induced endothelial cell apoptosis and dysfunction. Cilostazol was also demonstrated to partially reduced the angII-induced increase in superoxide production. The results of the present study suggested that cilostazol suppresses endothelial apoptosis and dysfunction by modulating the PI3K/Akt pathway. PMID:26862035

  12. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    PubMed

    Hirsch, Matthew L; Fagan, B Matthew; Dumitru, Raluca; Bower, Jacquelyn J; Yadav, Swati; Porteus, Matthew H; Pevny, Larysa H; Samulski, R Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

  13. Viral Single-Strand DNA Induces p53-Dependent Apoptosis in Human Embryonic Stem Cells

    PubMed Central

    Hirsch, Matthew L.; Fagan, B. Matthew; Dumitru, Raluca; Bower, Jacquelyn J.; Yadav, Swati; Porteus, Matthew H.; Pevny, Larysa H.; Samulski, R. Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication. PMID:22114676

  14. Zoledronic acid induces apoptosis and autophagy in cervical cancer cells.

    PubMed

    Wang, I-Te; Chou, Shou-Chu; Lin, Ying-Chin

    2014-12-01

    Cervical cancer is one of the most common gynecological cancers in association with high mortality and morbidity. The present study was aimed to investigate the in vitro effects of zoledronic acid (ZA) on viability and induction of apoptosis and autophagy as well as inflammatory effects in three human cervical cancer cell lines (HeLa, SiHa, and CaSki). Cell viability was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Induction of apoptosis was determined by quantitation of expression level of B cell lymphoma 2 (Bcl-2) and Bax messenger RNA (mRNA) and identification of the proteolytic cleavage of poly (ADP)-ribose polymerase (PARP) and caspase-3. Autophagic effects were examined by quantitation of mRNA expression of autophagy protein 5 (ATG5) and beclin1 and identifying accumulation of microtubule-associated protein 1 light chain 3 (LC3)-II. Inflammatory effect was determined by measuring expression and production of IL-6 and cyclooxygenase-2 (Cox-2). The results showed ZA significantly inhibited cell viability of cervical cancer cells. ZA-induced cell death displayed features characteristic to both apoptosis and autophagy and was associated with different changes in the levels of Bcl-2 and Bax in the various cervical cancer lines. Expression of metastatic cytokines, IL-6 and Cox-2, was upregulated in the presence of ZA at low concentration. Our data revealed that ZA inhibits cervical cancer cells through the synergistic effect of apoptosis induction and autophagy activation.

  15. Induction of T-cell apoptosis by human herpesvirus 6.

    PubMed Central

    Inoue, Y; Yasukawa, M; Fujita, S

    1997-01-01

    The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection. PMID:9094650

  16. Dracorhodin perchlorate induces the apoptosis of glioma cells.

    PubMed

    Chen, Xin; Luo, Junjie; Meng, Linghu; Pan, Taifeng; Zhao, Binjie; Tang, Zhen-Gang; Dai, Yongjian

    2016-04-01

    Dracorhodin perchlorate (Dp), a synthetic analogue of the antimicrobial anthocyanin red pigment, has recently been shown to induce apoptotic cell death in various types of cancer cells. Yet, the inhibitory effect of Dp on human glioma cells remains uninvestigated. Therefore, in the present study, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to detect cell viability and cell cycle progression in glioma U87MG and T98G cells, respectively. Annexin V-FITC/propidium iodide double staining and JC-1 staining were separately applied to determine cellular apoptosis and mitochondrial membrane potential damage in the cells. The expression levels of associated proteins involved in cell cycle progression and apoptosis were measured by western blotting. The activities of caspase‑9/-3 were determined by Caspase-Glo-9/3 assay. The results indicated that Dp treatment significantly inhibited cell proliferation in a dose- and time-dependent manner, and blocked cell cycle progression at the G1/S phase in the U87MG and T98G cells via the upregulation of p53 and p21 protein expression, and simultaneous downregulation of Cdc25A, Cdc2 and P-Cdc2 protein expression. Additionally, Dp treatment led to the loss of cellular mitochondrial membrane potential, and the release of cytochrome c, and strongly induced the occurence of apoptosis. Increased expression levels of Bim and Bax protein and the downregulated expression of Bcl-2 protein were observed. Caspase-9/-3 were activated and their activities were elevated after Dp treatment. These findings indicate that Dp inhibits cell proliferation, induces cell cycle arrest and apoptosis in glioma cells, and is a possible candidate for glioma treatment. PMID:26846469

  17. Increased Oxidative Stress Induces Apoptosis in Human Cystic Fibrosis Cells

    PubMed Central

    Rottner, Mathilde; Tual-Chalot, Simon; Mostefai, H. Ahmed; Andriantsitohaina, Ramaroson; Freyssinet, Jean-Marie; Martínez, María Carmen

    2011-01-01

    Oxidative stress results in deleterious cell function in pathologies associated with inflammation. Here, we investigated the generation of superoxide anion as well as the anti-oxidant defense systems related to the isoforms of superoxide dismutases (SOD) in cystic fibrosis (CF) cells. Pro-apoptotic agents induced apoptosis in CF but not in control cells that was reduced by treatment with SOD mimetic. These effects were associated with increased superoxide anion production, sensitive to the inhibition of IκB-α phosphorylation, in pancreatic but not tracheal CF cells, and reduced upon inhibition of either mitochondrial complex I or NADPH oxidase. CF cells exhibited reduced expression, but not activity, of both Mn-SOD and Cu/Zn-SOD when compared to control cells. Although, expression of EC-SOD was similar in normal and CF cells, its activity was reduced in CF cells. We provide evidence that high levels of oxidative stress are associated with increased apoptosis in CFTR-mutated cells, the sources being different depending on the cell type. These observations underscore a reduced anti-oxidant defense mechanism, at least in part, via diminished EC-SOD activity and regulation of Cu/Zn-SOD and Mn-SOD expressions. These data point to new therapeutic possibilities in targeting anti-oxidant pathways to reduce oxidative stress and apoptosis in CF cells. PMID:21931865

  18. Regulation of apoptosis of rbf mutant cells during Drosophila development

    PubMed Central

    Tanaka-Matakatsu, Miho; Xu, Jinhua; Cheng, Leping; Du, Wei

    2008-01-01

    Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells. PMID:19100727

  19. Streptococcus pneumoniae secretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

    PubMed Central

    Rai, Prashant; Parrish, Marcus; Tay, Ian Jun Jie; Li, Na; Ackerman, Shelley; He, Fang; Kwang, Jimmy; Chow, Vincent T.; Engelward, Bevin P.

    2015-01-01

    Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H2O2, plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H2O2, greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H2O2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniae-induced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia. PMID:26080406

  20. Does atorvastatin induce aortic smooth muscle cell apoptosis in vivo?

    PubMed

    Doyon, Marielle; Hale, Taben Mary; Huot-Marchand, Julie-Emilie; Wu, Rong; de Champlain, Jacques; DeBlois, Denis

    2011-01-01

    It has been reported that HMG-CoA reductase inhibitors such as atorvastatin induce vascular smooth muscle cell (SMC) apoptosis in vitro. However, this effect remains to be demonstrated in vivo. The present studies were designed to test the ability of atorvastatin to induce SMC apoptosis in vivo, using the spontaneously hypertensive rat (SHR) as a well-known reference model of SMC apoptosis induction in vivo by cardiovascular drugs including the calcium channel blocker amlodipine. Atorvastatin was administered to SHR for 3 or 6 weeks either alone or together with amlodipine, a drug combination clinically available to patients. Primary endpoints included aortic medial hypertrophy and aortic SMC hyperplasia, internucleosomal DNA fragmentation and expression of the apoptosis regulatory proteins Bax and Bcl-2. The SHR aorta showed no evidence of SMC apoptosis induction by atorvastatin, even at the high dose of 50 mg kg(-1) day(-1), although the statin significantly reduced oxidative stress after 3 weeks and blood pressure after 6 weeks of administration. Amlodipine-induced regression of aortic hypertophy and aortic SMC hyperplasia were dose- and time-dependent, but there was no interaction between atorvastatin and amlodipine in modulating the primary endpoints. These results do not support the notion that atorvastatin induces SMC apoptosis in the aortic media in vivo.

  1. A mechanism of cell apoptosis by light irradiation

    NASA Astrophysics Data System (ADS)

    Xing, Da; Gao, Xuejuan; Wang, Fang

    2006-02-01

    Light irradiation can modulate various biological processes. For instance, low-power laser irradiation (LPLI) can induce cell proliferation and differentiation. It has been used to treat diseases of regeneration limitation and to promote wound healing. The biological mechanism of light irradiation remains unclear. Our previous studies have shown that low fluence LPLI induced the proliferation of human lung adenocarcinoma cells (ASTC-a-1) through PKC channel, while high fluence LPLI induced caspase-3 activation and cell apoptosis. The mechanisms of the initiation and regulation of apoptosis are complex and diverse. There are two main pathways to initiate and regulate cell apoptosis, one is the death receptor pathway (receptor/caspase-8/caspase-3), and the other is the mitochondria pathway (mitochondria/ caspase-9/caspase-3). Using fluorescent imaging techniques, we observed a temporal sequence of events during apoptosis induced by high fluence LPLI and PDT. Both the high fluence LPLI and PDT triggers mitochondrial ROS production resulting in dissipation of ΔΨ m and activation of caspase-3. Our results also show the two treatments do not activate caspase-8. These results suggest that caspase-3 activation induced by high fluence LPLI or PDT is initiated directly from mitochondria ROS generation and dissipation of ΔΨ m, and independent of the cell death pathway involving caspase-8 activation. Because the progression of the apoptosis induced by high fluence LPLI is the same as that of PDT, we concluded that light is absorbed directly either by endogenous porphyrins or by the cytochromes in mitochondrion, resulting in initial ROS generation. During light irradiation induced apoptosis, apoptotic signals are initiated from mitochondrial ROS production due to photosensitization.

  2. Apoptosis by Direct Current Treatment in Tumor Cells and Tissues

    NASA Astrophysics Data System (ADS)

    Kim, Hongbae; Sim, Sungbo; Ahn, Saeyoung

    2003-10-01

    Electric field induces cell fusion, electroporation on biological cells, including apoptosis. Apoptosis is expressed in a series of natural enzymatic reactions for the natural elimination of unhealthy, genetically damaged, or otherwise aberrant cells that are not needed or not advantageous to the well-being of the organism. Its markers involve cell shrinkage, activation of intracellular caspase proteases, externalization of phosphatidylserine at the plasma membrane, and fragmentation of DNA. Direct electric fields using direct current have been exploited recently to investigate its effects on tumor cells and tissues, but the mechanism of direct electric fields has not been exhibited clearly other than by electroosmosis or pH changes. Direct electric field induces apoptosis in tumor cells cultured and tumor tissues as indicated by cell shrinkage, DNA fragmentation and tumor suppression. In our experiment that direct electric field was applied to tumor tissues via two needle electrodes inserted into tumor tissue 5mm at distance in parallel, pH changes resulted from electrochemical reaction, exhibiting about pH 9.0, 1.83, 2.0 in the vicinity of cathodic and anodic electrode, and at their mid-point, respectively. DNA fragmentation of tumor tissues destructed by direct electric field was analyzed by Tunel assay by ApopTag technology. As a result of this analysis, it showed that apoptosis in tumor tissue destructed was increased up to 59.1normal(control) tissues, showing 41.1, 31.1cathodic tissues. In vitro cell survival was exhibited that it was decreased with enhancing electric current intensity in the same condition of electrical charge 5C having different time applied. We will show results of apoptosis analyzed by flow cytometry in vitro.

  3. Apoptosis of postovulatory cumulus granulosa cells of the rat.

    PubMed

    Szołtys, M; Tabarowski, Z; Pawlik, A

    2000-12-01

    The process of apoptosis in the postovulatory cumulus granulosa cells was investigated in pregnant rats. Mature female Wistar rats, exhibiting a regular 4-day oestrous cycle, were placed with males on the day of pro-oestrus. The following day, on which spermatozoa were found in vaginal smears, was designated day 1 of pregnancy. The animals were killed just before ovulation (24.00 hours), on days 1 (5.00, 11.00, and 18.00 hours), and 2 ( 11.00 hours) of pregnancy. Excised ovaries and oviducts were submitted to a routine histological procedure and paraplast sections were subjected to detection of apoptotic cells using the TUNEL method. The cumulus granulosa cells of preovulatory follicles (24.00 hours) were negative for apoptotic staining. However, 5 h later a positive staining was observed in the oviduct ampulla and included the cumulus granulosa cells lying in the peripheral parts of postovulatory cumuli oophori, and the oviductal epithelial cells of this region. On the evening of day 1 almost all cumulus granulosa cells showed strong immunostaining while on day 2 at 11.00 hours only immunonegative clusters of remnants of cumulus granulosa cells were present in the distended ampulla region, while naked, two or more cell embryos were present in the further parts of oviduct. These results indicate that in the rat apoptosis of cumulus granulosa cells starts shortly after ovulation in the peripheral region. Epithelial ampullary cells surrounding ovulated cumuli show a massive apoptosis. PMID:11131018

  4. Counting Legionella cells within single amoeba host cells

    EPA Science Inventory

    Here we present the first attempt to quantify L. pneumophila cell numbers within individual amoebae hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 134...

  5. Counting Legionella cells within single amoeba host cells.

    PubMed

    Buse, Helen Y; Ashbolt, Nicholas J

    2012-03-01

    Here we present the first attempt to quantify Legionella pneumophila cell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite(-1), respectively.

  6. Retinal Endothelial Cell Apoptosis Stimulates Recruitment of Endothelial Progenitor Cells

    PubMed Central

    Bhatwadekar, Ashay D.; Glenn, Josephine V.; Curtis, Tim M.; Grant, Maria B.; Stitt, Alan W.; Gardiner, Tom A.

    2013-01-01

    Purpose Bone marrow–derived endothelial progenitor cells (EPCs) contribute to vascular repair although it is uncertain how local endothelial cell apoptosis influences their reparative function. This study was conducted to determine how the presence of apoptotic bodies at sites of endothelial damage may influence participation of EPCs in retinal microvascular repair. Methods Microlesions of apoptotic cell death were created in monolayers of retinal microvascular endothelial cells (RMECs) by using the photodynamic drug verteporfin. The adhesion of early-EPCs to these lesions was studied before detachment of the apoptotic cells or after their removal from the wound site. Apoptotic bodies were fed to normal RMECs and mRNA levels for adhesion molecules were analyzed. Results Endothelial lesions where apoptotic bodies were left attached at the wound site showed a fivefold enhancement in EPC recruitment (P < 0.05) compared with lesions where the apoptotic cells had been removed. In intact RMEC monolayers exposed to apoptotic bodies, expression of ICAM, VCAM, and E-selectin was upregulated by 5- to 15-fold (P < 0.05– 0.001). EPCs showed a characteristic chemotactic response (P < 0.05) to conditioned medium obtained from apoptotic bodies, whereas analysis of the medium showed significantly increased levels of VEGF, IL-8, IL-6, and TNF-α when compared to control medium; SDF-1 remained unchanged. Conclusions The data indicate that apoptotic bodies derived from retinal capillary endothelium mediate release of proangiogenic cytokines and chemokines and induce adhesion molecule expression in a manner that facilitates EPC recruitment. PMID:19474402

  7. Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis.

    PubMed

    Rachlin, Kenneth; Moore, Dan H; Yount, Garret

    2013-11-01

    The development of nontoxic agents that can selectively enhance the cytotoxicity of chemotherapy is an important aim in oncology. This study evaluates the ability of infrasound exposure to sensitize glioblastoma cells to cisplatin-induced apoptosis. The infrasound was delivered using a device designed to replicate the unique infrasound emissions measured during external Qigong treatments. Human glioblastoma cell lines harboring wild-type p53 (U87) or mutant p53 (U251, SF210, and SF188) were treated in culture with cisplatin, infrasound emissions, or the combination of the 2 agents. Induction of apoptosis was quantified after 24 hours by flow cytometry following annexin V/propidium iodide staining. Infrasound emissions alone, delivered at moderate levels (~10 mPa) with dynamic frequency content (7-13 Hz), did not induce apoptosis, yet combining infrasound with cisplatin augmented the induction of apoptosis by cisplatin in all the 4 cell lines (P < .05). Increased cellular uptake of the fluorophore calcein associated with infrasound exposure was quantified by fluorescence microscopy as well as flow cytometry, demonstrating increased cell membrane permeability. The 4 cell lines differed in the degree to which infrasound exposure increased calcein uptake, and these differences were predictive of the extent to which infrasound enhanced cisplatin-induced apoptosis. When exposed to specific frequencies, membrane permeabilization also appeared to be differentially responsive for each cell line, suggesting the potential for selective targeting of tissue types using isolated infrasonic frequencies. Additionally, the pressure amplitudes used in this study were several orders of magnitude less than those used in similar studies involving ultrasound and shock waves. The results of this study provide support for using infrasound to enhance the chemotherapeutic effects of cisplatin in a clinical setting. PMID:23165942

  8. Capsaicin induces apoptosis in PC12 cells through ER stress.

    PubMed

    Krizanova, Olga; Steliarova, Iveta; Csaderova, Lucia; Pastorek, Michal; Hudecova, Sona

    2014-02-01

    Capsaicin, the pungent agent in chili peppers, has been shown to act as a tumor-suppressor in cancer. In our previous study, capsaicin was shown to induce apoptosis in the rat pheochromocytoma cell line (PC12 cells). Thus, the aim of the present study was to determine the potential mechanism by which capsaicin induces apoptosis. We treated PC12 cells with 50, 100 and 500 µM capsaicin and measured the reticular calcium content and expression of the reticular calcium transport systems. These results were correlated with endoplasmic reticulum (ER) stress markers CHOP, ATF4 and X-box binding protein 1 (XBP1), as well as with apoptosis induction. We observed that capsaicin decreased reticular calcium in a concentration-dependent manner. Simultaneously, expression levels of the sarco/endoplasmic reticulum pump and ryanodin receptor of type 2 were modified. These changes were accompanied by increased ER stress, as documented by increased stress markers. Thus, from these results we propose that in PC12 cells capsaicin induces apoptosis through increased ER stress. PMID:24337105

  9. Multifaceted role of prohibitin in cell survival and apoptosis.

    PubMed

    Peng, Ya-Ting; Chen, Ping; Ouyang, Ruo-Yun; Song, Lei

    2015-09-01

    Human eukaryotic prohibitin (prohibitin-1 and prohibitin-2) is a membrane protein with different cellular localizations. It is involved in multiple cellular functions, including energy metabolism, proliferation, apoptosis, and senescence. The subcellular localization of prohibitin may determine its functions. Membrane prohibitin regulate the cellular signaling of membrane transport, nuclear prohibitin control transcription activation and the cell cycle, and mitochondrial prohibitin complex stabilize the mitochondrial genome and modulate mitochondrial dynamics, mitochondrial morphology, mitochondrial biogenesis, and the mitochondrial intrinsic apoptotic pathway. Moreover, prohibitin can translocates into the nucleus or the mitochondria under apoptotic signals and the subcellular shuttling of prohibitin is necessary for apoptosis process. Apoptosis is the process of programmed cell death that is important for the maintenance of normal physiological functions. Consequently, any alteration in the content, post-transcriptional modification (i.e. phosphorylation) or the nuclear or mitochondrial translocation of prohibitin may influence cell fate. Understanding the mechanisms of the expression and regulation of prohibitin may be useful for future research. This review provides an overview of the multifaceted and essential roles played by prohibitin in the regulation of cell survival and apoptosis.

  10. Crocetin prevents AGEs-induced vascular endothelial cell apoptosis.

    PubMed

    Xiang, Min; Yang, Min; Zhou, Chenghua; Liu, Juan; Li, Wenna; Qian, Zhiyu

    2006-10-01

    Advanced glycation end products (AGEs) are causally correlated with diabetic vascular complications. AGEs triggered oxidative reaction then accelerated endothelial cell apoptosis is a critical event in the process of vascular complications. Crocetin, a carotenoid has been previously shown to have strong antioxidant activates. Therefore, this study was designed to investigate the role of crocetin on the prevention of AGEs-mediated cell apoptosis in bovine aortic endothelial cells (BEC) and the mechanisms involved. Exposure of BEC to 200 microg/ml AGEs for 48 h results in a significant increase in apoptotic rate, compared with control. AGEs-induced DNA fragmentation preferentially occurred in the S phase cells. Crocetin prevented AGEs-induced BEC apoptosis, which correlates with crocetin attenuation of AGEs mediated increase of intracellular reactive oxygen species (ROS) formation and elevation of intracellular Ca2+ concentration ([Ca2+]i) level (P<0.01 versus AGEs group). These results demonstrate that crocetin prevents AGEs-induced BEC apoptosis through ROS inhibition and [Ca2+]i stabilization and suggest that crocetin may exert a beneficial effect in preventing diabetes-associated vascular complications. PMID:16899372

  11. Tilmicosin-induced bovine neutrophil apoptosis is cell-specific and downregulates spontaneous LTB4 synthesis without increasing Fas expression.

    PubMed

    Lee, Wilson D; Flynn, Andrew N; LeBlanc, Justin M; Merrill, John K; Dick, Paul; Morck, Douglas W; Buret, Andre G

    2004-01-01

    The pathology of bacterial pneumonia, such as seen in the bovine lung infected with Mannheimia haemolytica, is due to pathogen virulence factors and to inflammation initiated by the host. Tilmicosin is a macrolide effective in treating bacterial pneumonia and recent findings suggest that this antibiotic may provide anti-inflammatory benefits by inducing polymorphonuclear neutrophilic leukocyte (PMN) apoptosis. Using an in vitro bovine system, we examined the cell-specificity of tilmicosin, characterized the changes in spontaneous leukotriene B4 (LTB4) synthesis by PMN exposed to the macrolide, and assessed its effects on PMN Fas expression. Previous findings demonstrated that tilmicosin is able to induce PMN apoptosis. These results were confirmed in this study by the Annexin-V staining of externalized phosphatidylserine and the analysis with flow cytometry. The cell-specificity of tilmicosin was assessed by quantification of apoptosis in bovine PMN, mononuclear leukocytes, monocyte-derived macrophages, endothelial cells, epithelial cells, and fibroblasts cultured with the macrolide. The effect of tilmicosin on spontaneous LTB4 production by PMN was evaluated via an enzyme-linked immunosorbent assay. Finally, the mechanisms of tilmicosin-induced PMN apoptosis were examined by assessing the effects of tilmicosin on surface Fas expression on PMN. Tilmicosin-induced apoptosis was found to be at least partially cell-specific, as PMN were the only cell type tested to die via apoptosis in response to incubation with tilmicosin. PMN incubated with tilmicosin under conditions that induce apoptosis spontaneously produced less LTB4, but did not exhibit altered Fas expression. In conclusion, tilmicosin-induced apoptosis is specific to PMN, inhibits spontaneous LTB4 production, and occurs through a pathway independent of Fas upregulation.

  12. Ouabain Enhances ADPKD Cell Apoptosis via the Intrinsic Pathway.

    PubMed

    Venugopal, Jessica; Blanco, Gustavo

    2016-01-01

    Progression of autosomal dominant polycystic kidney disease (ADPKD) is highly influenced by factors circulating in blood. We have shown that the hormone ouabain enhances several characteristics of the ADPKD cystic phenotype, including the rate of cell proliferation, fluid secretion and the capacity of the cells to form cysts. In this work, we found that physiological levels of ouabain (3 nM) also promote programmed cell death of renal epithelial cells obtained from kidney cysts of patients with ADPKD (ADPKD cells). This was determined by Alexa Fluor 488 labeled-Annexin-V staining and TUNEL assay, both biochemical markers of apoptosis. Ouabain-induced apoptosis also takes place when ADPKD cell growth is blocked; suggesting that the effect is not secondary to the stimulatory actions of ouabain on cell proliferation. Ouabain alters the expression of BCL family of proteins, reducing BCL-2 and increasing BAX expression levels, anti- and pro-apoptotic mediators respectively. In addition, ouabain caused the release of cytochrome c from mitochondria. Moreover, ouabain activates caspase-3, a key "executioner" caspase in the cell apoptotic pathway, but did not affect caspase-8. This suggests that ouabain triggers ADPKD cell apoptosis by stimulating the intrinsic, but not the extrinsic pathway of programmed cell death. The apoptotic effects of ouabain are specific for ADPKD cells and do not occur in normal human kidney cells (NHK cells). Taken together with our previous observations, these results show that ouabain causes an imbalance in cell growth/death, to favor growth of the cystic cells. This event, characteristic of ADPKD, further suggests the importance of ouabain as a circulating factor that promotes ADPKD progression. PMID:27047392

  13. Ouabain Enhances ADPKD Cell Apoptosis via the Intrinsic Pathway

    PubMed Central

    Venugopal, Jessica; Blanco, Gustavo

    2016-01-01

    Progression of autosomal dominant polycystic kidney disease (ADPKD) is highly influenced by factors circulating in blood. We have shown that the hormone ouabain enhances several characteristics of the ADPKD cystic phenotype, including the rate of cell proliferation, fluid secretion and the capacity of the cells to form cysts. In this work, we found that physiological levels of ouabain (3 nM) also promote programmed cell death of renal epithelial cells obtained from kidney cysts of patients with ADPKD (ADPKD cells). This was determined by Alexa Fluor 488 labeled-Annexin-V staining and TUNEL assay, both biochemical markers of apoptosis. Ouabain-induced apoptosis also takes place when ADPKD cell growth is blocked; suggesting that the effect is not secondary to the stimulatory actions of ouabain on cell proliferation. Ouabain alters the expression of BCL family of proteins, reducing BCL-2 and increasing BAX expression levels, anti- and pro-apoptotic mediators respectively. In addition, ouabain caused the release of cytochrome c from mitochondria. Moreover, ouabain activates caspase-3, a key “executioner” caspase in the cell apoptotic pathway, but did not affect caspase-8. This suggests that ouabain triggers ADPKD cell apoptosis by stimulating the intrinsic, but not the extrinsic pathway of programmed cell death. The apoptotic effects of ouabain are specific for ADPKD cells and do not occur in normal human kidney cells (NHK cells). Taken together with our previous observations, these results show that ouabain causes an imbalance in cell growth/death, to favor growth of the cystic cells. This event, characteristic of ADPKD, further suggests the importance of ouabain as a circulating factor that promotes ADPKD progression. PMID:27047392

  14. Bisphosphonates induce apoptosis in human breast cancer cell lines

    PubMed Central

    Senaratne, S G; Pirianov, G; Mansi, J L; Arnett, T R; Colston, K W

    2000-01-01

    Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50values of 15, 20 and 3 μM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 μM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. © 2000 Cancer Research Campaign PMID:10780527

  15. Doxorubicin-mediated Apoptosis in Glioma Cells Requires NFAT3

    PubMed Central

    Gopinath, Sreelatha; Vanamala, Sravan K.; Gujrati, Meena; Klopfenstein, Jeffrey D.; Dinh, Dzung H.; Rao, Jasti S.

    2009-01-01

    Nuclear Factor of Activated T cells (NFAT), a family of transcription factors, has been implicated in many cellular processes, including some cancers. For the first time, the present study characterizes the role of NFAT3 in doxorubicin (DOX) mediated apoptosis, migration, and invasion in SNB19 and U87 glioma cells. This study demonstrates specific knockdown of NFAT3 results in a dramatic inhibition of the apoptotic effect, induced by DOX, and favors cell survival. Inhibition of NFAT3 activation by shNFAT3 (shNF3) significantly downregulated TNF-α induction, its receptor TNFR1, caspase 10, caspase 3 and PARP, abrogating DOX-mediated apoptosis in glioma cells. DOX treatment resulted in NFAT3 translocation to the nucleus. Similarly, shNF3 treatment in SNB19 and U87 cells reversed DOX-induced inhibition of cell migration and invasion as determined by wound healing and matrigel invasion assays. Taken together, these results indicate that NFAT3 is a prerequisite for the induction of DOX-mediated apoptosis in glioma cells. PMID:19784808

  16. Prodigiosin-induced apoptosis in human colon cancer cells.

    PubMed

    Montaner, B; Pérez-Tomás, R

    2001-03-16

    Prodigiosin is a red pigment produced by various bacteria including Serratia marcescens. Colorectal cancer is one of the most frequent malignancies and one of the most frequent causes of cancer death in the Western world. Its treatment is far from satisfactory and the challenge to oncologists is to find novel chemical entities with less toxicity and greater effectiveness than those used in current chemotherapy. Here we characterize the apoptotic action of prodigiosin in colon cancer cells. DLD-1 and SW-620 human colon adenocarcinoma cells, NRK and Swiss-3T3 nonmalignant cells were assayed by the MTT assay, fragmentation pattern of DNA, Hoechst 33342 staining and study of PARP cleavage by Western blot, in order to characterize the prodigiosin-induced apoptosis. Prodigiosin was purified and its structure was confirmed. Metastatic SW-620 cells were more sensitive to prodigiosin (IC50: 275 nM) than DLD-1. We did not observe a significant decrease in the viability of NRK cells. We confirmed that prodigiosin induces apoptosis in both cancer cell lines by the characteristic DNA laddering pattern and condensed nuclei or apoptotic bodies identified by fluorescence microscopy. These results indicate that prodigiosin induces apoptosis in colon cancer cells.

  17. Targeting SLUG sensitizes leukemia cells to ADR-induced apoptosis

    PubMed Central

    Wei, Chang-Rong; Liu, Jun; Yu, Xiao-Jun

    2015-01-01

    Background and Aims: Slug is an E-cadherin repressor and a suppressor of PUMA (p53 upregulated modulator of apoptosis) and it has recently been demonstrated that Slug plays an important role in controlling apoptosis. In this study, we examined whether Slug’s ability to silence expression suppresses the growth of leukemia HL-60 cells and/or sensitizes leukemia HL-60 cells to adriamycin (ADR) through induction of apoptosis. Methods: SLUG siRNA was transfected into the HL-60 and HL-60ADR cell lines (an adriamycin resistant cell line). The stably SLUG siRNA transfected HL-60 and HL-60ADR cells was transiently transfected with PUMA siRNA. The mRNA and protein expression of SLUG and PUMA were determined by Quantitative real-time RT-PCR and Western blot assay. The effects of SLUG siRNA alone or combined with ADR or PUMA siRNA on growth and apoptosis in HL-60 and HL-60ADR cells was detected by MTT, ELISA and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Results: The results showed that SLUG was less expressed in the HL-60 cells, and high expressed in the HL-60ADR cells. Obvious down-regulation of SLUG mRNA and protein levels and up-regulation of PUMA mRNA and protein levels after SLUG siRNA transfection was showed in the HL-60ADR cells. Treatment with ADR induced SLUG mRNA and protein in the HL-60 cells. Significant positive correlation was observed between basal SLUG mRNA and protein and ADR sensitivity. SLUG gene silencing by SLUG siRNA transfection inhibited growth and induced apoptosis, and increased ADR killing of the HL-60 and HL-60ADR cell lines. After the SLUG siRNA transfected HL-60 and HL-60ADR cells was transiently transfected with PUMA siRNA, did not increase ADR killing of the HL-60 and HL-60ADR cell lines. Conclusion: SLUG level positively correlated with sensitivity to ADR. SLUG siRNA could effectively reduce SLUG expression and induce PUMA expression and restore the drug sensitivity of resistant leukemic cells to

  18. Prepubertal male rats with high rates of germ-cell apoptosis present exacerbated rates of germ-cell apoptosis after serotonin depletion.

    PubMed

    Méndez Palacios, Néstor; Escobar, María Elena Ayala; Mendoza, Maximino Méndez; Crispín, Rubén Huerta; Andrade, Octavio Guerrero; Melández, Javier Hernández; Martínez, Andrés Aragón

    2016-04-01

    Male germ-cell apoptosis occurs naturally and can be increased by exposure to drugs and toxic chemicals. Individuals may have different rates of apoptosis and are likely to also exhibit differential sensitivity to outside influences. Previously, we reported that p-chloroamphetamine (pCA), a substance that inhibits serotonin synthesis, induced germ-cell apoptosis in prepubertal male rats. Here, we identified prepubertal rats with naturally high or low rates of germ-cell apoptosis and evaluated gene expression in both groups. Bax and Shbg mRNA levels were higher in rats with high rates of germ-cell apoptosis. Rats were then treated with pCA and the neuro-hormonal response and gene expression were evaluated. Treatment with pCA induced a reduction in serotonin concentrations but levels of sex hormones and gonadotrophins were not changed. Rats with initially high rates of germ-cell apoptosis had even higher rates of germ-cell apoptosis after treatment with pCA. In rats with high rates of germ-cell apoptosis Bax mRNA expression remained high after treatment with pCA. On the basis of category, an inverse relationship between mRNA expression of Bax and Bcl2, Bax and AR and Bax and Hsd3b2 was found. Here we provide evidence that innate levels of germ-cell apoptosis could be explained by the level of mRNA expression of genes involved with apoptosis and spermatogenesis.

  19. Ribavirin and alpha interferon enhance death receptor-mediated apoptosis and caspase activation in human hepatoma cells.

    PubMed

    Schlosser, Stephan F; Schuler, Markus; Berg, Christoph P; Lauber, Kirsten; Schulze-Osthoff, Klaus; Schmahl, Friedrich Wilhelm; Wesselborg, Sebastian

    2003-06-01

    The molecular mechanisms underlying the clinical effects of alpha interferon (IFN) and ribavirin are not understood. Elimination of infected cells occurs in part by cytotoxic T lymphocytes (CTLs) expressing CD95 ligand and thereby attacking target cells which are positive for the death receptor CD95. Since many viruses have evolved mechanisms to inhibit apoptosis, the opposite, namely, promotion of apoptosis, could be a strategy to strengthen the host antiviral response. In the present study, we have asked whether the antiviral substances IFN and ribavirin could support CD95-mediated apoptosis by interfering with the activation of caspases, a family of proteases known for their essential role in apoptosis. HepG2 cells, stimulated with the agonistic anti-CD95 antibody, served as a minimal model to mimic the CD95 stimulation occurring during a CTL attack of target cells in vivo. Apoptosis was quantitated by flow cytometric detection of hypodiploid nuclei. Caspase activity was measured by cytofluorometry, immunocytochemistry, and immunoblot analysis. IFN and ribavirin sensitized HepG2 cells for CD95-mediated apoptosis. This effect was correlated with an increase in CD95-mediated caspase activation and enhanced cleavage of the caspase substrate poly(ADP-ribose) polymerase. Furthermore, the positive effect on CD95-mediated caspase activation by IFN and ribavirin was confirmed by immunocytochemistry for activated caspase-3 and by immunoblot detection of activated caspase-3, caspase-7, and caspase-8. Our data demonstrate that the antiviral substances IFN and ribavirin are able to sensitize for CD95-mediated apoptosis. IFN and ribavirin also enhance CD95-mediated caspase activation, which might in part be responsible for the apoptosis-promoting effect of these antiviral compounds. PMID:12760867

  20. Cell fusion to study nuclear-cytoplasmic interactions in endothelial cell apoptosis.

    PubMed Central

    Polunovsky, V. A.; Ingbar, D. H.; Peterson, M.; Bitterman, P. B.

    1996-01-01

    Studies examining the regulation of nuclear rearrangements during apoptosis have led to conflicting results. Cytoplasmic control of nuclear events has been strongly suggested by cell-free experimental systems. In contrast, strict cytoplasmic control cannot account for the results of fibroblast-thymocyte fusion experiments in which dexamethasone induction of polykaryons led only to thymocyte nuclear apoptosis. Unresolved by these fusion studies was whether fibroblast nuclei were indifferent to heterologous cytoplasmic signals. Our objective was to resolve this discrepancy using cell fusion in a homologous system. Our strategy was to fuse endothelial cells with high levels of susceptibility to the induction of apoptosis (log phase cells arrested in G1 for 48 hours by isoleucine deprivation) with those manifesting low levels of susceptibility (serum-deprived, G0). Resultant fused and unfused cells were induced to undergo apoptosis by incubation with tumor necrosis factor-alpha and cycloheximide. Depending on the parental cell of origin, between 14 and 30% of dikaryons contained one apoptotic and one intact nucleus, indicating that strict cytoplasmic control was not occurring. In accord with this, the total frequency of nuclear apoptosis was unchanged after fusion. However, the distribution of apoptotic nuclei revealed a pronounced cytoplasmic influence, with a two- to fivefold increase in coordinate nuclear behavior. This pattern of nuclear apoptosis was consistent with a model of control in which both the state of nuclear susceptibility to apoptosis and expression of cytoplasmic pro-apoptotic regulators determined whether nuclear apoptosis would eventuate. Images Figure 3 Figure 4 PMID:8686735

  1. Apoptotic-like Leishmania exploit the host's autophagy machinery to reduce T-cell-mediated parasite elimination.

    PubMed

    Crauwels, Peter; Bohn, Rebecca; Thomas, Meike; Gottwalt, Stefan; Jäckel, Florian; Krämer, Susi; Bank, Elena; Tenzer, Stefan; Walther, Paul; Bastian, Max; van Zandbergen, Ger

    2015-01-01

    Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed--in contrast to viable parasites--that apoptotic-like parasites enter an LC3(+), autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4(+) T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells' autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.

  2. Apoptotic-like Leishmania exploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

    PubMed Central

    Crauwels, Peter; Bohn, Rebecca; Thomas, Meike; Gottwalt, Stefan; Jäckel, Florian; Krämer, Susi; Bank, Elena; Tenzer, Stefan; Walther, Paul; Bastian, Max; van Zandbergen, Ger

    2015-01-01

    Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed—in contrast to viable parasites—that apoptotic-like parasites enter an LC3+, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4+ T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´ autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis. PMID:25801301

  3. The type III secretion system of Vibrio alginolyticus induces rapid apoptosis, cell rounding and osmotic lysis of fish cells.

    PubMed

    Zhao, Zhe; Chen, Chang; Hu, Chao-Qun; Ren, Chun-Hua; Zhao, Jing-Jing; Zhang, Lv-Ping; Jiang, Xiao; Luo, Peng; Wang, Qing-Bai

    2010-09-01

    Vibrio alginolyticus is a Gram-negative bacterium and has been recognized as an opportunistic pathogen in humans as well as marine animals. However, the virulence mechanisms for this species of Vibrio have not been elucidated. This study characterized multiple mechanisms that induce cell death in fish cells upon infection with a V. alginolyticus strain, ZJO. The bacterium required its type III secretion system (T3SS) to cause rapid death of infected fish cells. Dying cells exhibited some features of apoptotic cells, such as membrane blebbing, nuclear condensation and DNA fragmentation. Further studies showed that caspase-3 was activated by the T3SS of the ZJO strain, confirming that infection with V. alginolyticus rapidly induces T3SS-dependent apoptosis in fish cells. Infection with the ZJO strain also led to membrane pore formation and release of cellular contents from infected fish cells, as evidenced by lactate dehydrogenase release and the uptake of a membrane-impermeable dye. Importantly, inhibition of apoptosis did not prevent ZJO-infected cells from releasing cellular contents and did not block cell rounding. Taken together, these data demonstrate that infection with V. alginolyticus may promote at least three different T3SS-dependent events, which lead to the death of fish cells. This study provides an important insight into the mechanism used by Vibrio species to cause host-cell death.

  4. Implications of Sertoli cell induced germ cell apoptosis to testicular pathology

    PubMed Central

    Murphy, Caitlin J; Richburg, John H

    2014-01-01

    After exposure to toxicants, degenerating germ cells represents the most common testicular histopathological alteration, regardless of the mechanism of toxicity. Therefore, deciphering the primary toxicant cellular target and mechanism of action can be extremely difficult. However, most testicular toxicants display a cell-specific and a stage-specific pattern of damage, which is the best evidence for identifying the primary cellular target (i.e. germ cell, Sertoli cell, peritubular myoid cell, or Leydig cell). Some toxicant-induced Sertoli cell injury presents with germ cell apoptosis occurring primarily in spermatocytes in rats in stages XI-XIV, I and II. Although some toxicants result in spermatid degeneration and apoptosis, it is still unclear if spermatid apoptosis is a result of Sertoli cell-selective apoptosis or a direct effect of toxicants on spermatids, therefore if this is seen as the earliest change, one cannot infer the mechanism of apoptosis. This review summarizes some of the distinguishing features of Sertoli cell-induced germ cell apoptosis and the associated mechanisms of cell death to provide the toxicologist observing similar cell death, with evidence about a potential mode of action. PMID:26413394

  5. Apoptosis in response to microbial infection induces autoreactive TH17 cells.

    PubMed

    Campisi, Laura; Barbet, Gaetan; Ding, Yi; Esplugues, Enric; Flavell, Richard A; Blander, J Magarian

    2016-09-01

    Microbial infections often precede the onset of autoimmunity. How infections trigger autoimmunity remains poorly understood. We investigated the possibility that infection might create conditions that allow the stimulatory presentation of self peptides themselves and that this might suffice to elicit autoreactive T cell responses that lead to autoimmunity. Self-reactive CD4(+) T cells are major drivers of autoimmune disease, but their activation is normally prevented through regulatory mechanisms that limit the immunostimulatory presentation of self antigens. Here we found that the apoptosis of infected host cells enabled the presentation of self antigens by major histocompatibility complex class II molecules in an inflammatory context. This was sufficient for the generation of an autoreactive TH17 subset of helper T cells, prominently associated with autoimmune disease. Once induced, the self-reactive TH17 cells promoted auto-inflammation and autoantibody generation. Our findings have implications for how infections precipitate autoimmunity. PMID:27455420

  6. Impact of simulated microgravity on microvascular endothelial cell apoptosis.

    PubMed

    Kang, Chun-Yan; Zou, Lin; Yuan, Ming; Wang, Yang; Li, Tian-Zhi; Zhang, Ye; Wang, Jun-Feng; Li, Yan; Deng, Xiao-Wei; Liu, Chang-Ting

    2011-09-01

    Cardiovascular deconditioning is known to occur in astronauts exposed to microgravity. Endothelial dysfunction at microcirculatory sites might contribute to cardiovascular deconditioning induced by weightlessness. Recent studies have reported changes in the morphology and gene expression of endothelial cells exposed to conditions of simulated microgravity. The present study was aimed at examining the effects of microgravity on the apoptosis of microvascular endothelial cells and the mechanism underlying these effects. We simulated a microgravity environment and found that microgravity induced microvascular endothelial cell apoptosis and that this effect was correlated with the downregulation of the PI3K/Akt pathway, increased expression of NF-κB, and depolymerization of F-actin. These findings may provide important insights into the origin of the adverse physiological changes occurring due to exposure to microgravity conditions. PMID:21287193

  7. Poliovirus protease 3C(pro) kills cells by apoptosis.

    PubMed

    Barco, A; Feduchi, E; Carrasco, L

    2000-01-20

    The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation.

  8. Evidence for Agrobacterium-induced apoptosis in maize cells.

    PubMed

    Hansen, G

    2000-06-01

    Agrobacterium spp. can genetically transform most dicotyledonous plant cells whereas many monocot species are recalcitrant to Agrobacterium-mediated transformation. One major obstacle is that co-cultivation of Agrobacterium spp. with plant tissues often results in cell death. Report here is that, in maize tissues, this process resembles apoptosis, with characteristic DNA cleavage into oligonucleosomal fragments and morphological changes. Two anti-apoptotic genes from baculovirus, p35 and iap, had the ability to prevent the onset of apoptosis triggered by Agrobacterium spp. in maize tissues. p35 is reported to act as a direct inhibitor of a certain class of proteases (caspase) whereas i.a.p. may act upstream to prevent their activation. This evidence raises the possibility that caspase-like proteases may also be involved in the apoptotic pathway in plant cells.

  9. Impact of simulated microgravity on microvascular endothelial cell apoptosis.

    PubMed

    Kang, Chun-Yan; Zou, Lin; Yuan, Ming; Wang, Yang; Li, Tian-Zhi; Zhang, Ye; Wang, Jun-Feng; Li, Yan; Deng, Xiao-Wei; Liu, Chang-Ting

    2011-09-01

    Cardiovascular deconditioning is known to occur in astronauts exposed to microgravity. Endothelial dysfunction at microcirculatory sites might contribute to cardiovascular deconditioning induced by weightlessness. Recent studies have reported changes in the morphology and gene expression of endothelial cells exposed to conditions of simulated microgravity. The present study was aimed at examining the effects of microgravity on the apoptosis of microvascular endothelial cells and the mechanism underlying these effects. We simulated a microgravity environment and found that microgravity induced microvascular endothelial cell apoptosis and that this effect was correlated with the downregulation of the PI3K/Akt pathway, increased expression of NF-κB, and depolymerization of F-actin. These findings may provide important insights into the origin of the adverse physiological changes occurring due to exposure to microgravity conditions.

  10. Intracellular GTP level determines cell's fate toward differentiation and apoptosis

    SciTech Connect

    Meshkini, Azadeh; Yazdanparast, Razieh Nouri, Kazem

    2011-06-15

    Since the adequate supply of guanine nucleotides is vital for cellular activities, limitation of their syntheses would certainly result in modulation of cellular fate toward differentiation and apoptosis. The aim of this study was to set a correlation between the intracellular level of GTP and the induction of relevant signaling pathways involved in the cell's fate toward life or death. In that regard, we measured the GTP level among human leukemia K562 cells exposed to mycophenolic acid (MPA) or 3-hydrogenkwadaphnin (3-HK) as two potent inosine monophosphate dehydrogenase inhibitors. Our results supported the maturation of the cells when the intracellular GTP level was reduced by almost 30-40%. Under these conditions, 3-HK and/or MPA caused up-regulation of PKC{alpha} and PI3K/AKT pathways. Furthermore, co-treatment of cells with hypoxanthine plus 3-HK or MPA, which caused a reduction of about 60% in the intracellular GTP levels, led to apoptosis and activation of mitochondrial pathways through inverse regulation of Bcl-2/Bax expression and activation of caspase-3. Moreover, our results demonstrated that attenuation of GTP by almost 60% augmented the intracellular ROS and nuclear localization of p21 and subsequently led to cell death. These results suggest that two different threshold levels of GTP are needed for induction of differentiation and/or ROS-associated apoptosis. - Graphical abstract: Display Omitted

  11. TGFβ Signaling Promotes Juvenile Granulosa Cell Tumorigenesis by Suppressing Apoptosis

    PubMed Central

    Mansouri-Attia, Nadéra; Tripurani, Swamy K.; Gokul, Nisha; Piard, Hermann; Anderson, Matthew L.; Eldin, Karen

    2014-01-01

    Molecular changes that give rise to granulosa cell tumors of the ovary are not well understood. Previously, we showed that deletion in granulosa cells of the bone morphogenetic protein receptor-signaling transcription factors, Smad1 and Smad5, causes development of metastatic granulosa cell tumors that phenocopy the juvenile form of granulosa cell tumors (JGCTs) in humans. The TGFβ-SMAD2/3 pathway is active in JGCTs, but its role is unknown. We tested the in vivo contribution of TGFβ-SMAD signaling to JGCT development by genetically deleting the common Smad4 from Smad1/5 double knockout mice. Smad1/5/4 triple knockout mice were sterile and had significantly increased survival and delayed tumor development compared to those for the Smad1/5 double knockout mice. The few tumors that did develop were smaller, showed no evidence of metastasis, and had increased apoptosis. In the human JGCT cell line COV434, TGFβ1 increased viability by inhibiting apoptosis through a TGFβ type I receptor–dependent repression of caspase activity and inhibition of poly(ADP-ribose) polymerase cleavage. These data support a tumor-promoting function of TGFβ in JGCTs through its ability to repress apoptosis. PMID:25243859

  12. Molecular mechanisms of asbestos-induced lung epithelial cell apoptosis.

    PubMed

    Liu, Gang; Beri, Rohinee; Mueller, Amanda; Kamp, David W

    2010-11-01

    Asbestos causes pulmonary fibrosis (asbestosis) and malignancies (bronchogenic lung cancer and mesothelioma) by mechanisms that are not fully elucidated. Accumulating evidence show that alveolar epithelial cell (AEC) apoptosis is a crucial initiating and perpetuating event in the development of pulmonary fibrosis following exposure to a wide variety of noxious stimuli, including asbestos. We review the important molecular mechanisms underlying asbestos-induced AEC apoptosis. Specifically, we focus on the role of asbestos in augmenting AEC apoptosis by the mitochondria- and p53-regulated death pathways that result from the production of iron-derived reactive oxygen species (ROS) and DNA damage. We summarize emerging evidence implicating the endoplasmic reticulum (ER) stress response in AEC apoptosis in patients with idiopathic pulmonary fibrosis (IPF), a disease with similarities to asbestosis. Finally, we discuss a recent finding that a mitochondrial oxidative DNA repair enzyme (8-oxoguanine DNA glycosylase; Ogg1) acts as a mitochondrial aconitase chaperone protein to prevent oxidant (asbestos and H(2)O(2))-induced AEC mitochondrial dysfunction and intrinsic apoptosis. The coupling of mitochondrial Ogg1 to mitochondrial aconitase is a novel mechanism linking metabolism to mitochondrial DNA that may be important in the pathophysiologic events resulting in oxidant-induced toxicity as seen in tumors, aging, and respiratory disorders (e.g. asbestosis, IPF). Collectively, these studies are illuminating the molecular basis of AEC apoptosis following asbestos exposure that may prove useful for developing novel therapeutic strategies. Importantly, the asbestos paradigm is elucidating pathophysiologic insights into other more common pulmonary diseases, such as IPF and lung cancer, for which better therapy is required. PMID:20380827

  13. A Potential Regulatory Role for Intronic microRNA-338-3p for Its Host Gene Encoding Apoptosis-Associated Tyrosine Kinase

    PubMed Central

    Kos, Aron; Olde Loohuis, Nikkie F. M.; Wieczorek, Martha L.; Glennon, Jeffrey C.; Martens, Gerard J. M.; Kolk, Sharon M.; Aschrafi, Armaz

    2012-01-01

    MicroRNAs (miRNAs) are important gene regulators that are abundantly expressed in both the developing and adult mammalian brain. These non-coding gene transcripts are involved in post-transcriptional regulatory processes by binding to specific target mRNAs. Approximately one third of known miRNA genes are located within intronic regions of protein coding and non-coding regions, and previous studies have suggested a role for intronic miRNAs as negative feedback regulators of their host genes. In the present study, we monitored the dynamic gene expression changes of the intronic miR-338-3p and miR-338-5p and their host gene Apoptosis-associated Tyrosine Kinase (AATK) during the maturation of rat hippocampal neurons. This revealed an uncorrelated expression pattern of mature miR-338 strands with their host gene. Sequence analysis of the 3′ untranslated region (UTR) of rat AATK mRNA revealed the presence of two putative binding sites for miR-338-3p. Thus, miR-338-3p may have the capacity to modulate AATK mRNA levels in neurons. Transfection of miR-338-3p mimics into rat B35 neuroblastoma cells resulted in a significant decrease of AATK mRNA levels, while the transfection of synthetic miR-338-5p mimics did not alter AATK levels. Our results point to a possible molecular mechanism by which miR-338-3p participates in the regulation of its host gene by modulating the levels of AATK mRNA, a kinase which plays a role during differentiation, apoptosis and possibly in neuronal degeneration. PMID:22363537

  14. Apoptosis inhibitor of macrophage (AIM) reduces cell number in canine histiocytic sarcoma cell lines

    PubMed Central

    UCHIDA, Mona; SAEKI, Kohei; MAEDA, Shingo; TAMAHARA, Satoshi; YONEZAWA, Tomohiro; MATSUKI, Naoaki

    2016-01-01

    Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines. PMID:27246397

  15. Viral Control of Mitochondrial Apoptosis

    PubMed Central

    Morselli, Eugenia; Touat, Zahia; Kroemer, Guido

    2008-01-01

    Throughout the process of pathogen–host co-evolution, viruses have developed a battery of distinct strategies to overcome biochemical and immunological defenses of the host. Thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. Since the elimination of infected cells via programmed cell death is one of the most ancestral defense mechanisms against infection, disabling host cell apoptosis might represent an almost obligate step in the viral life cycle. Conversely, viruses may take advantage of stimulating apoptosis, either to kill uninfected cells from the immune system, or to induce the breakdown of infected cells, thereby favoring viral dissemination. Several viral polypeptides are homologs of host-derived apoptosis-regulatory proteins, such as members of the Bcl-2 family. Moreover, viral factors with no homology to host proteins specifically target key components of the apoptotic machinery. Here, we summarize the current knowledge on the viral modulation of mitochondrial apoptosis, by focusing in particular on the mechanisms by which viral proteins control the host cell death apparatus. PMID:18516228

  16. Modulation of colony stimulating factor release and apoptosis in human colon cancer cells by anticancer drugs

    PubMed Central

    Calatayud, S; Warner, T D; Mitchell, J A

    2002-01-01

    Modulation of the immune response against tumour cells is emerging as a valuable approach for cancer treatment. Some experimental studies have shown that secretion of colony stimulating factors by cancer cells reduces their tumorigenicity and increases their immunogenicity probably by promoting the cytolitic and antigen presenting activities of leukocytes. We have observed that human colon cancer cells (HT-29) are able to secrete granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor when stimulated with cytokines (IL-1β and TNF-α). In this study we assessed, for the first time, the effects of several anticancer drugs on colony stimulating factor release or apoptosis in HT-29 cells. Cytokine-induced release of granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor was significantly increased by cisplatin and 6-mercaptopurine. Taxol only increased macrophage-colony stimulating factor release while reduced that of granulocyte-colony stimulating factor. No changes in colony stimulating factor secretion were observed after treatment with methotrexate. Only cisplatin and taxol induced apoptosis in these cells. Secretion of colony stimulating factors by colon cancer cells may contribute to the immune host response against them. Anticancer drugs such as cisplatin and 6-mercaptopurine increase colony stimulating factor secretion by cytokine stimulated cancer cells probably through mechanisms different to those leading to cell apoptosis, an effect that may contribute to their anti-neoplasic action. British Journal of Cancer (2002) 86, 1316–1321. DOI: 10.1038/sj/bjc/6600240 www.bjcancer.com © 2002 Cancer Research UK PMID:11953891

  17. The Type III Secretion Translocation Pore Senses Host Cell Contact

    PubMed Central

    Armentrout, Erin I.; Rietsch, Arne

    2016-01-01

    Type III secretion systems (T3SS) are nano-syringes used by a wide range of Gram-negative pathogens to promote infection by directly injecting effector proteins into targeted host cells. Translocation of effectors is triggered by host-cell contact and requires assembly of a pore in the host-cell plasma membrane, which consists of two translocator proteins. Our understanding of the translocation pore, how it is assembled in the host cell membrane and its precise role in effector translocation, is extremely limited. Here we use a genetic technique to identify protein-protein contacts between pore-forming translocator proteins, as well as the T3SS needle-tip, that are critical for translocon function. The data help establish the orientation of the translocator proteins in the host cell membrane. Analysis of translocon function in mutants that break these contacts demonstrates that an interaction between the pore-forming translocator PopD and the needle-tip is required for sensing host cell contact. Moreover, tethering PopD at a dimer interface also specifically prevents host-cell sensing, arguing that the translocation pore is actively involved in detecting host cell contact. The work presented here therefore establishes a signal transduction pathway for sensing host cell contact that is initiated by a conformational change in the translocation pore, and is subsequently transmitted to the base of the apparatus via a specific contact between the pore and the T3SS needle-tip. PMID:27022930

  18. The Salmonella effector SopB prevents ROS-induced apoptosis of epithelial cells by retarding TRAF6 recruitment to mitochondria.

    PubMed

    Ruan, Haihua; Zhang, Zhen; Tian, Li; Wang, Suying; Hu, Shuangyan; Qiao, Jian-Jun

    2016-09-16

    Microbial pathogens enter host cells by injecting effector proteins of the Type III secretion system (T3SS), which facilitate pathogen translocation across the host cell membrane. These effector proteins exert their effects by modulating a variety of host innate immune responses, thereby facilitating bacterial replication and systemic infection. Salmonella enterica serovar typhimurium (S.typhimurium) is a clinically important pathogen that causes food poisoning and gastroenteritis. The SopB effector protein of S. typhimurium, encoded by Salmonella pathogenicity islands (SPI)-1 T3SS, protects host epithelial cells from infection-induced apoptosis. However, how SopB influences apoptosis induction remains unclear. Here, we investigated the mechanism of SopB action in host cells. We found that SopB inhibits infection-induced apoptosis by attenuating the production of reactive oxygen species (ROS) in mitochondria, the crucial organelles for apoptosis initiation. Further investigation revealed that SopB binds to cytosolic tumor necrosis factor receptor associated factor 6 (TRAF6) and forms a trap preventing the mitochondrial recruitment of TRAF6, an essential event for ROS generation within mitochondria. By studying the response of Traf6(+/+) and Traf6(-/-)mouse embryonic fibroblasts to S. typhimurium infection, we found that TRAF6 promoted apoptosis by increasing ROS accumulation, which led to increased Bax/Bcl-2 ratio, Bax recruitment to mitochondrial membrane, and release of Cyt c into the cytoplasm. These findings show that SopB suppresses host cell apoptosis by binding to TRAF6 and preventing mitochondrial ROS generation. PMID:27473656

  19. Pharmacological induction of cell surface GRP78 contributes to apoptosis in triple negative breast cancer cells.

    PubMed

    Raiter, Annat; Yerushalmi, Rinat; Hardy, Britta

    2014-11-30

    Breast cancer tumor with triple-negative receptors (estrogen, progesterone and Her 2, receptors) is the most aggressive and deadly subtype, with high rates of disease recurrence and poor survival. Here, we show that induction in cell surface GRP78 by doxorubicin and tunicamycin was associated with CHOP/GADD153 upregulation and increase in apoptosis in triple negative breast cancer tumor cells. GRP78 is a major regulator of the stress induced unfolded protein response pathway and CHOP/GADD153 is a pro-apoptotic transcription factor associated exclusively with stress induced apoptosis. The blocking of cell surface GRP78 by anti-GRP78 antibody prevented apoptosis, suggesting that induction of cell surface GRP78 by doxorubicin and tunicamycin is required for apoptosis. A better understanding of stress induction of apoptotic signaling in triple negative breast cancer cells may help to define new therapeutic strategies.

  20. Poly I:C-induced tumor cell apoptosis mediated by pattern-recognition receptors.

    PubMed

    Zhao, Xiangzhong; Ai, Miao; Guo, Yuqi; Zhou, Xianbin; Wang, Li; Li, Xia; Yao, Chengfang

    2012-11-01

    Poly I:C is a synthetic dsRNA that can imitate a viral infection and elicit host immune responses by triggering specific pattern-recognition receptors (PRRs) such as toll-like receptor 3 and retinoic acid inducible gene I(RIG-I)-like receptors, including RIG-I and melanoma differentiation-associated gene 5. Activation of these PRRs by poly I:C triggers a signal transduction cascade that results in the activation of NF-κB and production of type I interferon. Poly I:C has been used as a vaccine adjuvant for cancer immunotherapy for several decades. Evidence from recent studies indicates that poly I:C can directly induce apoptosis in several types of tumor cells, thus providing a new therapeutic approach for cancer treatment. However, the molecular mechanism underlying the induction of apoptosis by poly I:C is still unclear. In this review, we summarize the current knowledge of poly I:C-induced tumor cell apoptosis, focusing on the key molecules and pathways involved in this process.

  1. Apoptosis of Endothelial Cells by 13-HPODE Contributes to Impairment of Endothelial Barrier Integrity

    PubMed Central

    Ryman, Valerie E.; Packiriswamy, Nandakumar

    2016-01-01

    Inflammation is an essential host response during bacterial infections such as bovine mastitis. Endothelial cells are critical for an appropriate inflammatory response and loss of vascular barrier integrity is implicated in the pathogenesis of Streptococcus uberis-induced mastitis. Previous studies suggested that accumulation of linoleic acid (LA) oxygenation products derived from 15-lipoxygenase-1 (15-LOX-1) metabolism could regulate vascular functions. The initial LA derivative from the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (HPODE), can induce endothelial death, whereas the reduced hydroxyl product, 13-hydroxyoctadecadienoic acid (HODE), is abundantly produced during vascular activation. However, the relative contribution of specific LA-derived metabolites on impairment of mammary endothelial integrity is unknown. Our hypothesis was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Exposure of bovine mammary endothelial cells (BMEC) to S. uberis did not increase 15-LOX-1 LA metabolism. However, S. uberis challenge of bovine monocytes demonstrated that monocytes may be a significant source of both 13-HPODE and 13-HODE during mastitis. Exposure of BMEC to 13-HPODE, but not 13-HODE, significantly reduced endothelial barrier integrity and increased apoptosis. Changing oxidant status by coexposure to an antioxidant during 13-HPODE treatment prevented adverse effects of 13-HPODE, including amelioration of apoptosis. A better understanding of how the oxidant status of the vascular microenvironment impacts endothelial barrier properties could lead to more efficacious treatments for S. uberis mastitis.

  2. 188Rhenium-induced cell death and apoptosis in a panel of tumor cell lines

    NASA Astrophysics Data System (ADS)

    Antoccia, Antonio; Banzato, Alessandra; Bello, Michele; Bollini, Dante; De Notaristefani, Francesco; Giron, Cecilia; Mazzi, Ulderico; Alafort, Laura Melendez; Moschini, Giuliano; Nadali, Anna; Navarria, Francesco; Perrotta, Andrea; Rosato, Antonio; Tanzarella, Caterina; Uzunov, Nikolay

    2007-02-01

    Assessment of "in vitro" tumor growth inhibition and radiobiological effects, such as apoptosis, have been evaluated in human neoplastic cells of different histotypes (H460 lung cancer cells, U87 glioblastoma, LnCaP prostate tumor cells) treated using solutions of 188Rhenium-perrhenate. The MTT assay, which measures mitochondrial metabolism in the entire cell culture is a recognized test for cytotoxicity and was used in cells exposed 48-72 h to specific activities ranged from 37 to 148 GBq/l. Whereas H460 and LnCaP were particularly sensitive to treatment, U87 glioblastoma cells behaved as radioresistant ones. However, evaluation of 188Re-induced apoptosis indicated that this kind of cell death contributed only marginally to the reduction in cell viability of H460 and LNCaP lines, suggesting the existence of protective mechanisms against apoptosis. In this respect, the membrane receptor, CD44, whose expression is dysregulated in most malignant cell types has proven to alter the response of cancer cells to apoptotic stimuli, including ionizing radiation. Cell samples decorated with a FITC-labelled CD44 antibody indicated, that in H460 and U87 cells the CD44(+) correlated well with an apoptosis-resistant response. Conversely, LnCap cells proven as CD44(-) did not display however sensitivity to radio-induced apoptosis.

  3. PDT-induced apoptosis in arterial smooth muscles cells

    NASA Astrophysics Data System (ADS)

    Nyamekye, Isaac; Renick, R.; Gilbert, C.; McEwan, Jean R.; Evan, G.; Bishop, Christopher C. R.; Bown, Stephen G.

    1995-03-01

    PDT kills smooth muscle cells (SMC) in vivo and thus prevents intimal hyperplasia after angioplasty. It causes little inflammation and structural integrity of the artery is not compromised. We have studied the process of the SMC death in vitro. Cultured rat SMC (cell line sv40 ATCC) were sensitized with aluminum disulphonated phthalocyanine (AlS2Pc), and then irradiated with 675 nm laser light (2.5 J/cm2). Controls were studied using only sensitizer or laser for treatment. The cells were incubated and the dying process observed with a time lapse video and microscope system. PDT caused a characteristic pattern of death. Cells lost contact with neighbors, shrank, and showed hyperactivity and membrane ruffling. The cells imploded into active and condensed membrane bound vesicles which were terminally reduced to residual bodies. These are the morphological changes of apoptosis. The control cells which were given AlS2Pc alone or laser alone showed no death. PDT induced cultured arterial SMC death by apoptosis rather than necrosis. An apoptotic mechanism of cell death in vivo would explain the relative lack of inflammation and local tissue destruction in the face of massive death.

  4. Signaling Perturbations Induced by Invading H. pylori Proteins in the Host Epithelial Cells

    PubMed Central

    Dampier, William; Tozeren, Aydin

    2007-01-01

    Helicobacter pylori (H. pylori), a gram-negative bacterium, infects the stomach of approximately 50% of the world population. H. pylori infection is a risk factor for developing chronic gastric ulcers and gastric cancer. The bacteria produce two main cytotoxic proteins: Vacuolating cytotoxin A (VacA) and Cytotoxin-Associated gene A (CagA). When these proteins enter the host cell they interfere with the host MAP Kinase and Apoptosis signaling pathways leading to aberrant cell growth and premature apoptosis. The present study expanded existing quantitative models of the MAP Kinase and Apoptosis signaling pathways to take into account the protein interactions across species using the CellDesigner tool. The resulting network contained hundreds of differential equations in which the coefficients for the biochemical rate constants were estimated from previously published studies. The effect of VacA and CagA on the function of this network were simulated by increasing levels of bacterial load. Simulations showed that increasing bacterial load affected the MAP Kinase signaling in a dose dependant manner. The introduction of CagA decreased the activation time of mapK signaling and extended activation indefinitely despite normal cellular activity to deactivate the protein. Introduction of VacA produced a similar response in the apoptosis pathway. Bacterial load activated both pathways even in the absence of external stimulation. Time course of emergence of transcription factors associated with cell division and cell death predicted by our simulation showed close agreement with that determined from a publicly accesible microarray dataset of H. pylori infected stomach epithelium. The quantitative model presented in this study lays the foundation for investigating the affects of single nucleotide polymorphisms (SNPs) on the efficiency of drug treatment. PMID:17559886

  5. Metformin impairs growth of endometrial cancer cells via cell cycle arrest and concomitant autophagy and apoptosis

    PubMed Central

    2014-01-01

    Background Effective therapies for early endometrial cancer usually involve surgical excision and consequent infertility Therefore, new treatment approaches that preserve fertility should be developed. Metformin, a well-tolerated anti-diabetic drug, can inhibit cancer cell growth. However, the mechanism of metformin action is not well understood. Here we investigate the roles of autophagy and apoptosis in the anti-cancer effects of metformin on endometrial cancer cells. Methods Ishikawa endometrial cancer cells were treated with metformin. WST-8 assays, colony formation assays, flow cytometry, caspase luminescence measurement, immunofluorescence, and western blots were used to assess the effects of metformin on cell viability, proliferation, cell cycle progression, apoptosis, and autophagy. Results Metformin-treated cells exhibited significantly lower viability and proliferation and significantly more cell cycle arrest in G1 and G2/M than control cells. These cells also exhibited significantly more apoptosis via both intrinsic and extrinsic pathways. In addition, metformin treatment induced autophagy. Inhibition of autophagy, either by Beclin1 knockdown or by 3-methyladenine-mediated inhibition of caspase-3/7, suppressed the anti-proliferative effects of metformin on endometrial cancer cells. These findings indicate that the anti-proliferative effects and apoptosis caused by metformin are partially or completely dependent on autophagy. Conclusions We showed that metformin suppresses endometrial cancer cell growth via cell cycle arrest and concomitant autophagy and apoptosis. PMID:24966801

  6. Safrole induces apoptosis in human oral cancer HSC-3 cells.

    PubMed

    Yu, F-S; Yang, J-S; Yu, C-S; Lu, C-C; Chiang, J-H; Lin, C-W; Chung, J-G

    2011-02-01

    Phytochemicals have been used as potential chemopreventive or chemotherapeutic agents. However, there are data suggesting a mutagenic effect of some phytochemicals. We hypothesized that safrole would have anticancer effects on human oral squamous cell carcinoma HSC-3 cells. Safrole decreased the percentage of viable HSC-3 cells via induction of apoptosis by an increased level of cytosolic Ca(2+) and a reduction in the mitochondrial membrane potential (ΔΨ(m)). Changes in the membrane potential were associated with changes in the Bax, release of cytochrome c from mitochondria, and activation of downstream caspases-9 and -3, resulting in apoptotic cell death. In vivo studies also showed that safrole reduced the size and volume of an HSC-3 solid tumor on a xenograft athymic nu/nu mouse model. Western blotting and flow cytometric analysis studies confirmed that safrole-mediated apoptotic cell death of HSC-3 cells is regulated by cytosolic Ca(2+) and by mitochondria- and Fas-dependent pathways.

  7. Metformin prevents methylglyoxal-induced apoptosis of mouse Schwann cells

    SciTech Connect

    Ota, Kimiko; Nakamura, Jiro; Li, Weiguo; Kozakae, Mika; Watarai, Atsuko; Nakamura, Nobuhisa; Yasuda, Yutaka; Nakashima, Eirtaro; Naruse, Keiko; Watabe, Kazuhiko; Kato, Koichi; Oiso, Yutaka; Hamada, Yoji . E-mail: yhama@med.nagoya-u.ac.jp

    2007-05-25

    Methylglyoxal (MG) is involved in the pathogenesis of diabetic complications via the formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS). To clarify whether the antidiabetic drug metformin prevents Schwann cell damage induced by MG, we cultured mouse Schwann cells in the presence of MG and metformin. Cell apoptosis was evaluated using Hoechst 33342 nuclear staining, caspase-3 activity, and c-Jun-N-terminal kinase (JNK) phosphorylation. Intracellular ROS formation was determined by flow cytometry, and AMP-activated kinase (AMPK) phosphorylation was also examined. MG treatment resulted in blunted cell proliferation, an increase in the number of apoptotic cells, and the activation of caspase-3 and JNK along with enhanced intracellular ROS formation. All of these changes were significantly inhibited by metformin. No significant activation of AMPK by MG or metformin was observed. Taken together, metformin likely prevents MG-induced apoptotic signals in mouse Schwann cells by inhibiting the formation of AGEs and ROS.

  8. Cord Blood Stem Cell-Mediated Induction of Apoptosis in Glioma Downregulates X-Linked Inhibitor of Apoptosis Protein (XIAP)

    PubMed Central

    Dasari, Venkata Ramesh; Velpula, Kiran Kumar; Kaur, Kiranpreet; Fassett, Daniel; Klopfenstein, Jeffrey D.; Dinh, Dzung H.; Gujrati, Meena; Rao, Jasti S.

    2010-01-01

    Background XIAP (X-linked inhibitor of apoptosis protein) is one of the most important members of the apoptosis inhibitor family. XIAP is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth and survival of malignant cells. We hypothesized that downregulation of XIAP by human umbilical cord blood mesenchymal stem cells (hUCBSC) in glioma cells would cause them to undergo apoptotic death. Methodology/Principal Findings We observed the effect of hUCBSC on two malignant glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310). In co-cultures of glioma cells with hUCBSC, proliferation of glioma cells was significantly inhibited. This is associated with increased cytotoxicity of glioma cells, which led to glioma cell death. Stem cells induced apoptosis in glioma cells, which was evaluated by TUNEL assay, FACS analyses and immunoblotting. The induction of apoptosis is associated with inhibition of XIAP in co-cultures of hUCBSC. Similar results were obtained by the treatment of glioma cells with shRNA to downregulate XIAP (siXIAP). Downregulation of XIAP resulted in activation of caspase-3 and caspase-9 to trigger apoptosis in glioma cells. Apoptosis is characterized by the loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic proteins Bax and Bad. Cell death of glioma cells was marked by downregulation of Akt and phospho-Akt molecules. We observed similar results under in vivo conditions in U251- and 5310-injected nude mice brains, which were treated with hUCBSC. Under in vivo conditions, Smac/DIABLO was found to be colocalized in the nucleus, showing that hUCBSC induced apoptosis is mediated by inhibition of XIAP and activation of Smac/DIABLO. Conclusions/Significance Our results indicate that downregulation of XIAP by hUCBSC treatment induces apoptosis, which led to the death of the glioma cells and xenograft cells. This study demonstrates the therapeutic

  9. Mechanisms of host cell invasion by Trypanosoma cruzi.

    PubMed

    Caradonna, Kacey L; Burleigh, Barbara A

    2011-01-01

    One of the more accepted concepts in our understanding of the biology of early Trypanosoma cruzi-host cell interactions is that the mammalian-infective trypomastigote forms of the parasite must transit the host cell lysosomal compartment in order to establish a productive intracellular infection. The acidic environment of the lysosome provides the appropriate conditions for parasite-mediated disruption of the parasitophorous vacuole and release of T. cruzi into the host cell cytosol, where replication of intracellular amastigotes occurs. Recent findings indicate a level of redundancy in the lysosome-targeting process where T. cruzi trypomastigotes exploit different cellular pathways to access host cell lysosomes in non-professional phagocytic cells. In addition, the reversible nature of the host cell penetration process was recently demonstrated when conditions for fusion of the nascent parasite vacuole with the host endosomal-lysosomal system were not met. Thus, the concept of parasite retention as a critical component of the T. cruzi invasion process was introduced. Although it is clear that host cell recognition, attachment and signalling are required to initiate invasion, integration of this knowledge with our understanding of the different routes of parasite entry is largely lacking. In this chapter, we focus on current knowledge of the cellular pathways exploited by T. cruzi trypomastigotes to invade non-professional phagocytic cells and to gain access to the host cell lysosome compartment. PMID:21884886

  10. Peripheral Red Blood Cell Split Chimerism as a Consequence of Intramedullary Selective Apoptosis of Recipient Red Blood Cells in a Case of Sickle Cell Disease

    PubMed Central

    Marziali, Marco; Isgrò, Antonella; Sodani, Pietro; Gaziev, Javid; Fraboni, Daniela; Paciaroni, Katia; Gallucci, Cristiano; Alfieri, Cecilia; Roveda, Andrea; De Angelis, Gioia; Cardarelli, Luisa; Ribersani, Michela; Andreani, Marco; Lucarelli, Guido

    2014-01-01

    Allogeneic cellular gene therapy through hematopoietic stem cell transplantation is the only radical cure for congenital hemoglobinopathies like thalassemia and sickle cell anemia. Persistent mixed hematopoietic chimerism (PMC) has been described in thalassemia and sickle cell anemia. Here, we describe the clinical course of a 6-year-old girl who had received bone marrow transplant for sickle cell anemia. After the transplant, the patient showed 36% donor hematopoietic stem cells in the bone marrow, whereas in the peripheral blood there was evidence of 80% circulating donor red blood cells (RBC). The analysis of apoptosis at the Bone Marrow level suggests that Fas might contribute to the cell death of host erythroid precursors. The increase in NK cells and the regulatory T cell population observed in this patient suggests that these cells might contribute to the condition of mixed chimerism. PMID:25408852

  11. Adipose stromal cells-secreted neuroprotective media against neuronal apoptosis.

    PubMed

    Wei, X; Zhao, L; Zhong, J; Gu, H; Feng, D; Johnstone, B H; March, K L; Farlow, M R; Du, Y

    2009-10-01

    Transplantation of pluripotent adipose stem/stromal cells (ASC) alleviates tissue damage and improves functional deficits in both stroke and cardiovascular disease animal models. Recent studies indicate that the primary mechanism of ASC-induced repair may not be directly related to tissue regeneration through differentiation, but rather through paracrine mechanisms provided by secreted pro-survival and repair-inducing trophic factors. In this study, we have found that ASC-conditioned medium (ASC-CM) potently protected cerebellar granule neurons (CGN) from apoptosis induced by serum and potassium deprivation. Neural cell protection was mostly attributable to activated caspase-3 and Akt-mediated neuroprotective pathway signaling. Specific neutralization of neurotrophic factor activity demonstrated that serum and potassium deprivation-induced Akt-mediated neuroprotection and caspase-3-dependent apoptosis were mainly modulated by IGF-1. These data suggest that of the many neuroprotective factors secreted by ASC, IGF-1 is the major factor that mediates protection against serum and potassium deprivation-induced CGN apoptosis. This study establishes a mechanistic basis supporting the therapeutic application of ASC for neurological disorders, specifically through paracrine support provided by trophic factor secretion. PMID:19549558

  12. Induction of apoptosis in a flounder gill cell line by lymphocystis disease virus infection.

    PubMed

    Hu, G-B; Cong, R-S; Fan, T-J; Mei, X-G

    2004-11-01

    Lymphocystis disease virus (LCDV), a large icosahedral DNA virus classified to the iridovirus family, is the causative agent of lymphocystis, a disease which occurs in marine and freshwater fish species and is characterized by formation of papilloma-like lesions on the surface of the skin. In vitro, LCDV infection causes flounder gill cells, an adherent cell line, to exhibit an obvious cytopathic effect (CPE). In order to test whether apoptosis is responsible for the observed CPE, cells infected with LCDV at a multiplicity of infection (m.o.i.) of 5 PFU per cell were examined at various time intervals for the appearance of apoptotic signs. Nuclear fragmentation, DNA laddering and caspase activation were observed in the infected cells at the time (i.e. 10 days post-infection) when an intensive CPE was observed. These findings demonstrate that LCDV is capable of inducing apoptosis in vitro, which is different from the result of LCDV infection in vivo, and consequently suggest an intricate LCDV-host interaction. PMID:15509260

  13. Interaction of chlamydiae and host cells in vitro.

    PubMed Central

    Moulder, J W

    1991-01-01

    The obligately intracellular bacteria of the genus Chlamydia, which is only remotely related to other eubacterial genera, cause many diseases of humans, nonhuman mammals, and birds. Interaction of chlamydiae with host cells in vitro has been studied as a model of infection in natural hosts and as an example of the adaptation of an organism to an unusual environment, the inside of another living cell. Among the novel adaptations made by chlamydiae have been the substitution of disulfide-bond-cross-linked polypeptides for peptidoglycans and the use of host-generated nucleotide triphosphates as sources of metabolic energy. The effect of contact between chlamydiae and host cells in culture varies from no effect at all to rapid destruction of either chlamydiae or host cells. When successful infection occurs, it is usually followed by production of large numbers of progeny and destruction of host cells. However, host cells containing chlamydiae sometimes continue to divide, with or without overt signs of infection, and chlamydiae may persist indefinitely in cell cultures. Some of the many factors that influence the outcome of chlamydia-host cell interaction are kind of chlamydiae, kind of host cells, mode of chlamydial entry, nutritional adequacy of the culture medium, presence of antimicrobial agents, and presence of immune cells and soluble immune factors. General characteristics of chlamydial multiplication in cells of their natural hosts are reproduced in established cell lines, but reproduction in vitro of the subtle differences in chlamydial behavior responsible for the individuality of the different chlamydial diseases will require better in vitro models. PMID:2030670

  14. Association between SET expression and glioblastoma cell apoptosis and proliferation

    PubMed Central

    He, Kunyan; Shi, Lihong; Jiang, Tingting; Li, Qiang; Chen, Yao; Meng, Chuan

    2016-01-01

    Glioblastoma multiforme (GBM) was one of the first cancer types systematically studied at a genomic and transcriptomic level due to its high incidence and aggressivity; however, the detailed mechanism remains unclear, even though it is known that numerous cytokines are involved in the occurrence and development of GBM. The present study aimed to determine whether the SET gene has a role in human glioblastoma carcinogenesis. A total of 32 samples, including 18 cases of glioma, 2 cases of meningioma and 12 normal brain tissue samples, were detected using the streptavidin-peroxidase method through immunohistochemistry. To reduce SET gene expression in U251 and U87MG cell lines, the RNA interference technique was used and transfection with small interfering (si)RNA of the SET gene was performed. Cell apoptosis was detected by flow cytometry, cell migration was examined by Transwell migration assay and cell proliferation was determined by Cell Counting Kit-8. SET, Bcl-2, Bax and caspase-3 mRNA and protein expression levels were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Positive protein expression of SET was observed in the cell nucleus, with the expression level of SET significantly higher in glioma tissues compared with normal brain tissue (P=0.001). Elevated expression of SET was significantly associated with gender (P=0.002), tumors classified as World Health Organization grade II (P=0.031), III (P=0.003) or IV (P=0.001), and moderately (P=0.031) or poorly differentiated (P=0.001) tumors. Compared with the negative and non-treatment (blank) control cells, SET gene expression was significantly inhibited (P=0.006 and P<0.001), cell apoptosis was significantly increased (P=0.001 and P<0.001), cell proliferation was significantly inhibited (P=0.002 and P=0.015), and cell migration was significantly decreased (P=0.001 and P=0.001) in siRNA-transfected U87MG−SET and U251−SET cells, respectively. In

  15. Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

    SciTech Connect

    Boudreau, Nancy; Sympson, C. J.; Werb, Zena; Bissell, Mina J.

    1994-12-01

    Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.

  16. microRNA-22 attenuates neuronal cell apoptosis in a cell model of traumatic brain injury

    PubMed Central

    Ma, Ji; Shui, Shaofeng; Han, Xinwei; Guo, Dong; Li, Tengfei; Yan, Lei

    2016-01-01

    Traumatic brain injury (TBI) is a major cause of injury-related deaths, and the mechanism of TBI has become a research focus, but little is known about the mechanism of microRNAs in TBI. The aim of this study is the role of microRNA-22 (miR-22) in TBI-induced neuronal cell apoptosis. Rat cortical neurons were cultured and the TBI model was induced by scratch injury in vitro, before which miR-22 level was altered by transfection of agomir or antagomir. Lactate dehydrogenase (LDH) release and TUNEL assays were performed to examine neuronal cell injury and apoptosis. The activity of caspase 3 (CASP3) and level changes of several apoptosis factors including B-cell lymphoma 2 (BCL2), BCL2-associated X protein (BAX), phosphatase and tensin homolog (PTEN) and v-AKT murine thymoma viral oncogene homolog 1 (AKT1) were detected. Results showed that TBI model cells possessed a downregulated miR-22 level (P < 0.001) and more LDH release and apoptotic cells indicating the aggravated neuronal cell injury and apoptosis induced by TBI. miR-22 agomir attenuated neuronal cell injury and apoptosis of the TBI model. It also caused the corresponding changes in CASP3 activity and other apoptosis factors, with cleaved CASP3, BAX and PTEN inhibited and BCL2 and phosphorylated AKT1 promoted, while miR-22 antagomir had the opposite effects. So miR-22 has neuroprotective roles of attenuating neuronal cell injury and apoptosis induced by TBI, which may be associated with its regulation on apoptosis factors. This study reveals miR-22 as a potential approach to TBI treatment and detailed mechanism remains to be uncovered. PMID:27186313

  17. Methods for production of proteins in host cells

    DOEpatents

    Donnelly, Mark; Joachimiak, Andrzej

    2004-01-13

    The present invention provides methods for the production of proteins, particularly toxic proteins, in host cells. The invention provides methods which use a fusion protein comprising a chaperonin binding domain in host cells induced or regulated to have increased levels of chaperonin which binds the chaperonin binding domain.

  18. Host cells and methods for production of isobutanol

    DOEpatents

    Anthony, Larry Cameron; He, Hongxian; Huang, Lixuan Lisa; Okeefe, Daniel P.; Kruckeberg, Arthur Leo; Li, Yougen; Maggio-Hall, Lori Ann; McElvain, Jessica; Nelson, Mark J.; Patnaik, Ranjan; Rothman, Steven Cary

    2016-08-23

    Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

  19. Artemisinin reduces cell proliferation and induces apoptosis in neuroblastoma.

    PubMed

    Zhu, Shunqin; Liu, Wanhong; Ke, Xiaoxue; Li, Jifu; Hu, Renjian; Cui, Hongjuan; Song, Guanbin

    2014-09-01

    Artemisinin, a natural product from the Chinese medicinal plant, Artemisia annua L., is commonly used in the treatment of malaria, and has recently been reported to have potent anticancer activity in various types of human tumors. Yet, the effect of artemisinin on neuroblastoma is still unclear. In the present study, we aimed to investigate the effects of artemisinin on neuroblastoma cells. We observed that artemisinin significantly inhibited cell growth and proliferation, and caused cell cycle arrest in the G1 phase in neuroblastoma cell lines. Annexin V-FITC/PI staining assay revealed that artemisinin markedly induced apoptosis. Soft agar assays revealed that artemisinin suppressed the ability of clonogenic formation of neuroblastoma cells and a xenograft study in NOD/SCID mice showed that artemisinin inhibited tumor growth and development in vivo. Therefore, our results suggest that the Chinese medicine artemisinin could serve as a novel potential therapeutic agent in the treatment of neuroblastoma. PMID:25017372

  20. Artemisinin reduces cell proliferation and induces apoptosis in neuroblastoma.

    PubMed

    Zhu, Shunqin; Liu, Wanhong; Ke, Xiaoxue; Li, Jifu; Hu, Renjian; Cui, Hongjuan; Song, Guanbin

    2014-09-01

    Artemisinin, a natural product from the Chinese medicinal plant, Artemisia annua L., is commonly used in the treatment of malaria, and has recently been reported to have potent anticancer activity in various types of human tumors. Yet, the effect of artemisinin on neuroblastoma is still unclear. In the present study, we aimed to investigate the effects of artemisinin on neuroblastoma cells. We observed that artemisinin significantly inhibited cell growth and proliferation, and caused cell cycle arrest in the G1 phase in neuroblastoma cell lines. Annexin V-FITC/PI staining assay revealed that artemisinin markedly induced apoptosis. Soft agar assays revealed that artemisinin suppressed the ability of clonogenic formation of neuroblastoma cells and a xenograft study in NOD/SCID mice showed that artemisinin inhibited tumor growth and development in vivo. Therefore, our results suggest that the Chinese medicine artemisinin could serve as a novel potential therapeutic agent in the treatment of neuroblastoma.

  1. Radiation-Induced Autophagy Contributes to Cell Death and Induces Apoptosis Partly in Malignant Glioma Cells

    PubMed Central

    Jo, Guk Heui; Bögler, Oliver; Chwae, Yong-Joon; Yoo, Heon; Lee, Seung Hoon; Park, Jong Bae; Kim, Youn-Jae; Kim, Jong Heon; Gwak, Ho-Shin

    2015-01-01

    Purpose Radiation-induced autophagy has been shown to play two different roles, in malignant glioma (MG) cells, cytocidal or cytoprotective. However, neither the role of radiation-induced autophagy for cell death nor the existence of autophagy-induced apoptosis, a well-known cell-death pathway after irradiation, has been verified yet. Materials and Methods We observed both temporal and dose-dependent response patterns of autophagy and apoptosis to radiation in MG cell lines. Additionally, we investigated the role of autophagy in apoptosis through knockdown of autophagy-related proteins. Results Autophagic activity measured by staining of acidic vesicle organelles and Western blotting of LC-3 protein increased in proportion to radiation dose from day 1 to 5 after irradiation. Apoptosis measured by annexin-V staining and Western blotting of cleaved poly(ADP-ribose) polymerase demonstrated relatively late appearance 3 days after irradiation that increased for up to 7 days. Blocking of pan-caspase (Z-VAD-FMK) did not affect apoptosis after irradiation, but silencing of Atg5 effectively reduced radiation-induced autophagy, which decreased apoptosis significantly. Inhibition of autophagy in Atg5 knockdown cells was shown to be beneficial for cell survival. Stable transfection of GFP-LC3 cells was observed after irradiation. Annexin-V was localized in cells bearing GFP-LC3 punctuated spots, indicating autophagy in immunofluorescence. Some of these punctuated GFP-LC3 bearing cells formed conglomerated spots and died in final phase. Conclusion These findings suggest that autophagy appears earlier than apoptosis after irradiation and that a portion of the apoptotic population that appears later is autophagy-dependent. Thus, autophagy is a pathway to cell death after irradiation of MG cells. PMID:25410762

  2. Vanadium induced ultrastructural changes and apoptosis in male germ cells.

    PubMed

    Aragón, M A; Ayala, M E; Fortoul, T I; Bizarro, P; Altamirano-Lozano, M

    2005-01-01

    Vanadium is a transition metal that is emitted to the atmosphere during combustion of fossil fuels. In the environment, vanadium occurs in the (V) oxidized form, but in the body it is found exclusively in the (IV) oxidized form. Vanadium tetraoxide is an inorganic chemical species in the (IV) oxidized form that has been shown to induce toxic effects in vitro and in vivo. The reproductive toxicity of vanadium in males was studied through monitoring germ cell apoptosis during spermatogenesis. We analyzed ultrastructural damage, and testosterone and progesterone concentrations following vanadium tetraoxide administered to male mice for 60 days. Spermatogenesis stages I-III and X-XII frequently showed apoptotic germ cells in control and treated animals; vanadium tetraoxide treatment induced an increase in the number of germ cell apoptosis in stages I-III and XII at 9.4 and 18.8 mg/kg, respectively. Although spermatogenesis is regulated by testosterone, in our study this hormone level was not modified by vanadium administration; thus, germ cell death was not related with testosterone concentration. At the ultrastructural level, we observed inclusion structures that varied as to location and content in the Sertoli and germ cells. PMID:15808796

  3. Noninvasive Imaging of Natural Killer Cell-Mediated Apoptosis in a Mouse Tumor Model.

    PubMed

    Singh, Thoudam Debraj; Lee, Jaetae; Jeon, Yong Hyun

    2016-01-01

    Natural killer (NK) cells are cytotoxic lymphocytes that induce apoptosis in cancer cells infected with viruses and bacteria through a caspase-3-dependent pathway. Effective NK cell-based immunotherapy requires highly sensitive imaging tools for in vivo monitoring of the dynamic events involved in apoptosis. Here, we describe a noninvasive bioluminescence imaging approach to determine the antitumor effects of NK cell-based therapy by serial imaging of caspase-3-dependent apoptosis in a mouse model of human glioma. PMID:27177676

  4. Matrine induces the apoptosis of lung cancer cells through downregulation of inhibitor of apoptosis proteins and the Akt signaling pathway.

    PubMed

    Niu, Huiyan; Zhang, Yifei; Wu, Baogang; Zhang, Yi; Jiang, Hongfang; He, Ping

    2014-09-01

    Lung cancer is the leading cause of cancer‑related mortality in humans. The prognosis for advanced lung cancer patients is extremely poor. Current standard care is rather ineffective for prolonging patient life while preserving satisfactory quality of life due to adverse side-effects. Matrine extracted from the traditional Chinese herbal plant Sophora flavescens was shown to induce cancer cell death in vitro. The aim of this study was to investigate the effect of matrine on the proliferation and apoptosis of lung cancer cells and the molecular basis of matrine-induced apoptosis. The results showed that matrine inhibited cell proliferation and induced apoptosis in lung cancer A549 and 95D cells in a dose- and time-dependent manner. The apoptotic effects of matrine on lung cancer cells appeared to act via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathway and downregulation of the expression of the inhibitor of apoptosis protein (IAP) family proteins. Matrine exerts its cancer-killing effect via promoting apoptosis in lung cancer cells and may be a useful adjuvant therapeutic scheme for treating advanced lung cancer patients.

  5. β-Arrestin1 inhibits chemotherapy-induced intestinal stem cell apoptosis and mucositis

    PubMed Central

    Zhan, Y; Xu, C; Liu, Z; Yang, Y; Tan, S; Yang, Y; Jiang, J; Liu, H; Chen, J; Wu, B

    2016-01-01

    The mechanism of chemotherapy-induced gastrointestinal (GI) syndrome (CIGIS) is still controversial, and it is unclear whether chemotherapy induces intestinal stem cell (ISC) apoptosis. β-Arrestins are regulators and mediators of G protein-coupled receptor signaling in cell apoptosis, division and growth. In this study, we aimed to investigate whether chemotherapy induces ISC apoptosis to contribute to mucositis in CIGIS and whether β-arrestin1 (β-arr1) is involved in this apoptosis. Different chemotherapeutic agents were used to generate a CIGIS model. Lgr5-EGFP-IRES-creERT2+/− knock-in mice were used as a CIGIS model to investigate ISC apoptosis. β-arr1 knockout mice were used to determine whether β-arr1 is involved in the apoptosis in CIGIS. Intestinal histology was performed, the ISC apoptosis was analyzed and the mucosal barrier was examined. The effects of β-arr1 in apoptosis were investigated in the samples from humans and mice as well as in cell lines. Here, we demonstrate that chemotherapy induced intestinal mucositis by promoting crypt cell apoptosis, especially in Lgr5+ stem cells and Paneth cells but not in goblet cells, epithelial cells or vascular endothelial cells. Furthermore, β-arr1 deficiency exacerbated the Lgr5+ stem cell apoptosis, but not Paneth cell apoptosis, in CIGIS. In addition, the data showed that β-arr1 reduced the chemotherapy-induced Lgr5+ stem cell apoptosis by inhibiting endoplasmic reticulum stress-mediated mitochondrial apoptotic signaling. Our study indicates that β-arr1 inhibits chemotherapy-induced ISC apoptosis to alleviate intestinal mucositis in CIGIS. PMID:27195676

  6. Hedgehog regulates cerebellar progenitor cell and medulloblastoma apoptosis.

    PubMed

    Noguchi, Kevin Kiyoshi; Cabrera, Omar Hoseá; Swiney, Brant S; Salinas-Contreras, Patricia; Smith, Julie Kathryn; Farber, Nuri B

    2015-11-01

    The external granule layer (EGL) is a proliferative region that produces over 90% of the neurons in the cerebellum but can also malignantly transform into a cerebellar tumor called the medulloblastoma (the most common malignant brain tumor in children). Current dogma considers Hedgehog stimulation a potent proliferative signal for EGL neural progenitor cells (NPCs) and medulloblastomas. However, the Hedgehog pathway also acts as a survival signal in the neural tube where it regulates dorsoventral patterning by controlling NPC apoptosis. Here we show that Hedgehog stimulation is also a potent survival signal in the EGL and medulloblastomas that produces a massive apoptotic response within hours of signal loss in mice. This toxicity can be produced by numerous Hedgehog antagonists (vismodegib, cyclopamine, and jervine) and is Bax/Bak dependent but p53 independent. Finally, since glucocorticoids can also induce EGL and medulloblastoma apoptosis, we show that Hedgehog's effects on apoptosis can occur independent of glucocorticoid stimulation. This effect may play a major role in cerebellar development by directing where EGL proliferation occurs thereby morphologically sculpting growth. It may also be a previously unknown major therapeutic effect of Hedgehog antagonists during medulloblastoma therapy. Results are discussed in terms of their implications for both cerebellar development and medulloblastoma treatment. PMID:26319366

  7. Apoptosis during embryonic tissue remodeling is accompanied by cell senescence

    PubMed Central

    Lorda-Diez, Carlos I.; Garcia-Riart, Beatriz; Montero, Juan A.; Rodriguez-León, Joaquín; Garcia-Porrero, Juan A; Hurle, Juan M.

    2015-01-01

    This study re-examined the dying process in the interdigital tissue during the formation of free digits in the developing limbs. We demonstrated that the interdigital dying process was associated with cell senescence, as deduced by induction of β-gal activity, mitotic arrest, and transcriptional up-regulation of p21 together with many components of the senescence-associated secretory phenotype. We also found overlapping domains of expression of members of the Btg/Tob gene family of antiproliferative factors in the regressing interdigits. Notably, Btg2 was up-regulated during interdigit remodeling in species with free digits but not in the webbed foot of the duck. We also demonstrate that oxidative stress promoted the expression of Btg2, and that FGF2 and IGF1 which are survival signals for embryonic limb mesenchyme inhibited Btg2 expression. Btg2 overexpression in vivo and in vitro induced all the observed changes during interdigit regression, including oxidative stress, arrest of cell cycle progression, transcriptional regulation of senescence markers, and caspase-mediated apoptosis. Consistent with the central role of p21 on cell senescence, the transcriptional effects induced by overexpression of Btg2 are attenuated by silencing p21. Our findings indicate that cell senescence and apoptosis are complementary processes in the regression of embryonic tissues and share common regulatory signals. PMID:26568417

  8. Novel synthetic chalcones induces apoptosis in human glioblastoma cells.

    PubMed

    Bittencourt, Lucas Felipe Fernandes; Oliveira, Karen Andrinéia de; Cardoso, Carine Bropp; Lopes, Flávia Garcia; Dal-Cim, Tharine; Chiaradia-Delatorre, Louise Domeneghini; Mascarello, Alessandra; Maluf, Sharbel Weidner; Yunes, Rosendo Augusto; Garcez, Ricardo Castilho; Tasca, Carla Inês; Nedel, Cláudia Beatriz

    2016-05-25

    Glioblastoma multiforme is the main and most frequent tumor in adults' central nervous system. With a survival average of 5% two years after diagnosis, this type of cancer is a main health problem. Substances like the chalcones have been tested in order to develop new treatments. Here, we studied the effects of three synthetic chalcones (A23, C31 and J11) on A172 and surgery obtained-glioma cells. All chalcones showed a decrease in cell viability, mainly C31. An increase in apoptosis levels with no further increase of necrosis was observed. This augmentation may be linked to the high oxidative effect found, caused by the increased presence of reactive oxygen species and nitric oxide production. Cell cycle distribution showed an arrest at G0/G1 and S phases, suggesting that C31 interferes in cell cycle control. Our results shall aid in directing future research with this substance and its antitumor effect.

  9. Dolichyl phosphate, a potent inducer of apoptosis in rat glioma C6 cells.

    PubMed

    Yasugi, E; Yokoyama, Y; Seyama, Y; Kano, K; Hayashi, Y; Oshima, M

    1995-11-22

    Exposure of rat glioma C6 cells to dolichyl phosphate resulted in cell shrinkage followed by nuclear fragmentation and internucleosomal cleavage of genomic DNA, yielding ladder patterns of oligonucleosomal fragments, all characteristics of apoptosis. This phenomenon occurred in a dose and time dependent manner. Dolichol and prenol failed to induce apoptosis. The inhibitors of N-glycosylation, tunicamycin and swainsonine had no apparent effect on dolichyl phosphate-induced apoptosis. Apoptotic changes were also observed in HL-60 cells, SIRC cells and HeLa cells. Thus, dolichyl phosphate functions as a potential apoptosis inducer as well as an essential carrier lipid in the biosynthesis of N-linked glycoprotein.

  10. The Cytotoxic Role of Intermittent High Glucose on Apoptosis and Cell Viability in Pancreatic Beta Cells

    PubMed Central

    Zhang, Zhen; Li, Jing; Yang, Lei; Chen, Rongping; Yang, Rui; Zhang, Hua; Cai, Dehong; Chen, Hong

    2014-01-01

    Objectives. Glucose fluctuations are both strong predictor of diabetic complications and crucial factor for beta cell damages. Here we investigated the effect of intermittent high glucose (IHG) on both cell apoptosis and proliferation activity in INS-1 cells and the potential mechanisms. Methods. Cells were treated with normal glucose (5.5 mmol/L), constant high glucose (CHG) (25 mmol/L), and IHG (rotation per 24 h in 11.1 or 25 mmol/L) for 7 days. Reactive oxygen species (ROS), xanthine oxidase (XOD) level, apoptosis, cell viability, cell cycle, and expression of cyclinD1, p21, p27, and Skp2 were determined. Results. We found that IHG induced more significant apoptosis than CHG and normal glucose; intracellular ROS and XOD levels were more markedly increased in cells exposed to IHG. Cells treated with IHG showed significant decreased cell viability and increased cell proportion in G0/G1 phase. Cell cycle related proteins such as cyclinD1 and Skp2 were decreased significantly, but expressions of p27 and p21 were increased markedly. Conclusions. This study suggested that IHG plays a more toxic effect including both apoptosis-inducing and antiproliferative effects on INS-1 cells. Excessive activation of cellular stress and regulation of cyclins might be potential mechanism of impairment in INS-1 cells induced by IHG. PMID:24772447

  11. Salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells

    SciTech Connect

    Hu, Xiaolan; Zhang, Xianqi; Qiu, Shuifeng; Yu, Daihua; Lin, Shuxin

    2010-07-16

    Research highlights: {yields} Salidroside inhibits the growth of human breast cancer cells. {yields} Salidroside induces cell-cycle arrest of human breast cancer cells. {yields} Salidroside induces apoptosis of human breast cancer cell lines. -- Abstract: Recently, salidroside (p-hydroxyphenethyl-{beta}-D-glucoside) has been identified as one of the most potent compounds isolated from plants of the Rhodiola genus used widely in traditional Chinese medicine, but pharmacokinetic data on the compound are unavailable. We were the first to report the cytotoxic effects of salidroside on cancer cell lines derived from different tissues, and we found that human breast cancer MDA-MB-231 cells (estrogen receptor negative) were sensitive to the inhibitory action of low-concentration salidroside. To further investigate the cytotoxic effects of salidroside on breast cancer cells and reveal possible ER-related differences in response to salidroside, we used MDA-MB-231 cells and MCF-7 cells (estrogen receptor-positive) as models to study possible molecular mechanisms; we evaluated the effects of salidroside on cell growth characteristics, such as proliferation, cell cycle duration, and apoptosis, and on the expression of apoptosis-related molecules. Our results demonstrated for the first time that salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells and may be a promising candidate for breast cancer treatment.

  12. Mitophagy switches cell death from apoptosis to necrosis in NSCLC cells treated with oncolytic measles virus.

    PubMed

    Xia, Mao; Meng, Gang; Jiang, Aiqin; Chen, Aiping; Dahlhaus, Meike; Gonzalez, Patrick; Beltinger, Christian; Wei, Jiwu

    2014-06-15

    Although apoptotic phenomena have been observed in malignant cells infected by measles virus vaccine strain Edmonston B (MV-Edm), the precise oncolytic mechanisms are poorly defined. In this study we found that MV-Edm induced autophagy and sequestosome 1-mediated mitophagy leading to decreased cytochrome c release, which blocked the pro-apoptotic cascade in non-small cell lung cancer cells (NSCLCs). The decrease of apoptosis by mitophagy favored viral replication. Persistent viral replication sustained by autophagy ultimately resulted in necrotic cell death due to ATP depletion. Importantly, when autophagy was impaired in NSCLCs MV-Edm-induced cell death was significantly abrogated despite of increased apoptosis. Taken together, our results define a novel oncolytic mechanism by which mitophagy switches cell death from apoptosis to more efficient necrosis in NSCLCs following MV-Edm infection. This provides a foundation for future improvement of oncolytic virotherapy or antiviral therapy.

  13. Polydatin inhibits growth of lung cancer cells by inducing apoptosis and causing cell cycle arrest.

    PubMed

    Zhang, Yusong; Zhuang, Zhixiang; Meng, Qinghui; Jiao, Yang; Xu, Jiaying; Fan, Saijun

    2014-01-01

    Polydatin (PD), a small natural compound from Polygonum cuspidatum, has a number of biological functions. However, the anticancer activity of PD has been poorly investigated. In the present study, thiazolyl blue tetrazolium bromide assay was used to evaluate the inhibitory effect of PD on cell growth. Cell cycle distribution and apoptosis were investigated by flow cytometry. In addition, the expression of several proteins associated with apoptosis and cell cycle were analyzed by western blot analysis. The results demonstrated that PD significantly inhibits the proliferation of A549 and NCI-H1975 lung cancer cell lines and causes dose-dependent apoptosis. Cell cycle analysis revealed that PD induces S phase cell cycle arrest. Western blot analysis showed that the expression of Bcl-2 decreased as that of Bax increased, and the expression of cyclin D1 was also suppressed. The results suggest that PD has potential therapeutic applications in the treatment of lung cancer. PMID:24348867

  14. Anemone altaica Induces Apoptosis in Human Osteosarcoma Cells.

    PubMed

    Chang, I-Chang; Chiang, Tsay-I; Lo, Chun; Lai, Yi-Hua; Yue, Chia-Herng; Liu, Jer-Yuh; Hsu, Li-Sung; Lee, Chia-Jen

    2015-01-01

    In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS. PMID:26224029

  15. Dose- and time-dependent apoptosis induced by avian H9N2 influenza virus in human cells.

    PubMed

    Shahsavandi, Shahla; Ebrahimi, Mohammad Majid; Sadeghi, Kaveh; Mosavi, Seyedeh Zahra; Mohammadi, Ashraf

    2013-01-01

    To understand human response to avian H9N2 influenza, we investigated the effects of the viral infection on A549, HepG2, and HeLa cells at low and high MOIs. To identify virus-host interplay, expression of Mx and NP genes was measured in the cells supernatants. Cell viability and apoptosis were evaluated by MTT assay, DNA fragmentation, and florescent staining. The virus titration and NP gene transcript levels indicate lower susceptibility of HeLa cell to H9N2 replication than other cells. Although H9N2 did produce a faster CPE in HepG2, high dose of the virus induced apoptosis within early stage of A549 infection. The DNA laddering was enhanced in the cell correlated with increase in virus transcripts. The undetectable to different regulation levels of Mx gene were observed in response to H9N2 infection suggesting that an insufficient antiviral defense in the noncompetent-IFN HepG2 cell promotes efficient viral replication. These results showed that the permissivity of HepG2 for H9N2 is comparable with A549; however, liver cells are not target tissue respond to the infection. These data revealed that the H9N2 virus induced apoptosis signaling via mitochondrial pathway in human alveolar epithelial cells, indicating that the induction may be associated with a dose-dependent manner.

  16. Raman spectrum reveals the cell cycle arrest of Triptolide-induced leukemic T-lymphocytes apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Daosen; Feng, Yanyan; Zhang, Qinnan; Su, Xin; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

    2015-04-01

    Triptolide (TPL), a traditional Chinese medicine extract, possesses anti-inflammatory and anti-tumor properties. Though some research results have implicated that Triptolide (TPL) can be utilized in the treatment of leukemia, it remains controversial about the mechanism of TPL-induced leukemic T-lymphocytes apoptosis. In this study, combining Raman spectroscopic data, principal component analysis (PCA) and atomic force microscopy (AFM) imaging, both the biochemical changes and morphological changes during TPL-induced cell apoptosis were presented. In contrast, the corresponding data during Daunorubicin (DNR)-induced cell apoptosis was also exhibited. The obtained results showed that Raman spectral changes during TPL-induced cell apoptosis were greatly different from DNR-induced cell apoptosis in the early stage of apoptosis but revealed the high similarity in the late stage of apoptosis. Moreover, above Raman spectral changes were respectively consistent with the morphological changes of different stages during TPL-induced apoptosis or DNR-induced apoptosis, including membrane shrinkage and blebbing, chromatin condensation and the formation of apoptotic bodies. Importantly, it was found that Raman spectral changes with TPL-induced apoptosis or DNR-induced apoptosis were respectively related with the cell cycle G1 phase arrest or G1 and S phase arrest.

  17. In situ tissue regeneration through host stem cell recruitment

    PubMed Central

    Ko, In Kap; Lee, Sang Jin; Atala, Anthony; Yoo, James J

    2013-01-01

    The field of tissue engineering has made steady progress in translating various tissue applications. Although the classical tissue engineering strategy, which involves the use of culture-expanded cells and scaffolds to produce a tissue construct for implantation, has been validated, this approach involves extensive cell expansion steps, requiring a lot of time and laborious effort before implantation. To bypass this ex vivo process, a new approach has been introduced. In situ tissue regeneration utilizes the body's own regenerating capacity by mobilizing host endogenous stem cells or tissue-specific progenitor cells to the site of injury. This approach relies on development of a target-specific biomaterial scaffolding system that can effectively control the host microenvironment and mobilize host stem/progenitor cells to target tissues. An appropriate microenvironment provided by implanted scaffolds would facilitate recruitment of host cells that can be guided to regenerating structural and functional tissues. PMID:24232256

  18. Linalool Induces Cell Cycle Arrest and Apoptosis in Leukemia Cells and Cervical Cancer Cells through CDKIs

    PubMed Central

    Chang, Mei-Yin; Shieh, Den-En; Chen, Chung-Chi; Yeh, Ching-Sheng; Dong, Huei-Ping

    2015-01-01

    Plantaginaceae, a popular traditional Chinese medicine, has long been used for treating various diseases from common cold to cancer. Linalool is one of the biologically active compounds that can be isolated from Plantaginaceae. Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible tumor cells. However, the signaling pathway for apoptosis remains undefined. In this study, the cytotoxic effect of linalool on human cancer cell lines was investigated. Water-soluble tetrazolium salts (WST-1) based colorimetric cellular cytotoxicity assay, was used to test the cytotoxic ability of linalool against U937 and HeLa cells, and flow cytometry (FCM) and genechip analysis were used to investigate the possible mechanism of apoptosis. These results demonstrated that linalool exhibited a good cytotoxic effect on U937 and HeLa cells, with the IC50 value of 2.59 and 11.02 μM, respectively, compared with 5-FU with values of 4.86 and 12.31 μM, respectively. After treating U937 cells with linalool for 6 h, we found an increased sub-G1 peak and a dose-dependent phenomenon, whereby these cells were arrested at the G0/G1 phase. Furthermore, by using genechip analysis, we observed that linalool can promote p53, p21, p27, p16, and p18 gene expression. Therefore, this study verified that linalool can arrest the cell cycle of U937 cells at the G0/G1 phase and can arrest the cell cycle of HeLa cells at the G2/M phase. Its mechanism facilitates the expression of the cyclin-dependent kinases inhibitors (CDKIs) p53, p21, p27, p16, and p18, as well as the non-expression of cyclin-dependent kinases (CDKs) activity. PMID:26703569

  19. Linalool Induces Cell Cycle Arrest and Apoptosis in Leukemia Cells and Cervical Cancer Cells through CDKIs.

    PubMed

    Chang, Mei-Yin; Shieh, Den-En; Chen, Chung-Chi; Yeh, Ching-Sheng; Dong, Huei-Ping

    2015-01-01

    Plantaginaceae, a popular traditional Chinese medicine, has long been used for treating various diseases from common cold to cancer. Linalool is one of the biologically active compounds that can be isolated from Plantaginaceae. Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible tumor cells. However, the signaling pathway for apoptosis remains undefined. In this study, the cytotoxic effect of linalool on human cancer cell lines was investigated. Water-soluble tetrazolium salts (WST-1) based colorimetric cellular cytotoxicity assay, was used to test the cytotoxic ability of linalool against U937 and HeLa cells, and flow cytometry (FCM) and genechip analysis were used to investigate the possible mechanism of apoptosis. These results demonstrated that linalool exhibited a good cytotoxic effect on U937 and HeLa cells, with the IC50 value of 2.59 and 11.02 μM, respectively, compared with 5-FU with values of 4.86 and 12.31 μM, respectively. After treating U937 cells with linalool for 6 h, we found an increased sub-G1 peak and a dose-dependent phenomenon, whereby these cells were arrested at the G0/G1 phase. Furthermore, by using genechip analysis, we observed that linalool can promote p53, p21, p27, p16, and p18 gene expression. Therefore, this study verified that linalool can arrest the cell cycle of U937 cells at the G0/G1 phase and can arrest the cell cycle of HeLa cells at the G2/M phase. Its mechanism facilitates the expression of the cyclin-dependent kinases inhibitors (CDKIs) p53, p21, p27, p16, and p18, as well as the non-expression of cyclin-dependent kinases (CDKs) activity.

  20. Bone marrow cell transplantation is associated with fibrogenic cells apoptosis during hepatic regeneration in cholestatic rats.

    PubMed

    Nunes de Carvalho, Simone; da Cunha Lira, Dalvaci; Costa Cortez, Erika Afonso; de Andrade, Daniela Caldas; Thole, Alessandra Alves; Stumbo, Ana Carolina; de Carvalho, Lais

    2013-04-01

    Liver fibrosis is accompanied by hepatocyte death and proliferation of α-SMA(+) fibrogenic cells (activated hepatic stellate cells and myofibroblasts), which synthesize extracellular matrix components that contribute to disorganization of the hepatic parenchyma and loss of liver function. Therefore, apoptosis of these fibrogenic cells is important to hepatic regeneration. This study aimed to analyze the effect of cell therapy using bone marrow mononuclear cell (BMMNC) transplantation on α-SMA expression and on apoptosis of hepatic cells during liver fibrosis induced by bile duct ligation (BDL). Livers were collected from normal rats, fibrotic rats after 14 and 21 days of BDL, and rats that received BMMNC at 14 days of BDL and were analyzed after 7 days. Apoptosis in fibrogenic cells was analyzed by immunoperoxidase, confocal microscopy, and Western blotting, and liver regeneration was assessed by proliferating cell nuclear antigen staining. Results showed that caspase-3 and proliferating cell nuclear antigen expression were significantly increased in the BMMNC-treated group. Additionally, confocal microscopy analysis showed cells coexpressing α-SMA and caspase-3 in these animals, suggesting fibrogenic cell death. These results suggest a novel role for BMMNC in liver regeneration during fibrotic disease by stimulating fibrogenic cells apoptosis and hepatocyte proliferation, probably through secretion of specific cytokines that modulate the hepatic microenvironment toward an antifibrogenic balance.

  1. Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis

    PubMed Central

    Anglada, Teresa; Terradas, Mariona; Hernández, Laia; Genescà, Anna; Martín, Marta

    2016-01-01

    In order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs) and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated) defective cell line, as Ataxia-Telangiectasia (AT) cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70–85% of the AT viable cells (TUNEL-negative) carried ≥10 γH2AX foci/cell, while only 12–27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of the γH2AX-positive AT cells undergoing early apoptosis carried ≥10 γH2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-induced γH2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residual γH2AX foci might function as a predictive tool to assess radiation-induced apoptosis. PMID:27057549

  2. Inducible resistance to Fas-mediated apoptosis in B cells.

    PubMed

    Rothstein, T L

    2000-12-01

    Apoptosis produced in B cells through Fas (APO-1, CD95) triggering is regulated by signals derived from other surface receptors: CD40 engagement produces upregulation of Fas expression and marked susceptibility to Fas-induced cell death, whereas antigen receptor engagement, or IL-4R engagement, inhibits Fas killing and in so doing induces a state of Fas-resistance, even in otherwise sensitive, CD40-stimulated targets. Surface immunoglobulin and IL-4R utilize at least partially distinct pathways to produce Fas-resistance that differentially depend on PKC and STAT6, respectively. Further, surface immunoglobulin signaling for inducible Fas-resistance bypasses Btk, requires NF-kappaB, and entails new macromolecular synthesis. Terminal effectors of B cell Fas-resistance include the known anti-apoptotic gene products, Bcl-xL and FLIP, and a novel anti-apoptotic gene that encodes FAIM (Fas Apoptosis Inhibitory Molecule). faim was identified by differential display and exists in two alternatively spliced forms; faim-S is broadly expressed, but faim-L expression is tissue-specific. The FAIM sequence is highly evolu- tionarily conserved, suggesting an important role for this molecule throughout phylogeny. Inducible resistance to Fas killing is hypothesized to protect foreign antigen-specific B cells during potentially hazardous interactions with FasL-bearing T cells, whereas autoreactive B cells fail to become Fas-resistant and are deleted via Fas-dependent cytotoxicity. Inadvertent or aberrant acquisition of Fas-resistance may permit autoreactive B cells to escape Fas deletion, and malignant lymphocytes to impede anti-tumor immunity.

  3. Regulation of cell proliferation and apoptosis by bioactive lipid mediators.

    PubMed

    Clària, Joan

    2006-11-01

    Bioactive lipid mediators are increasingly being recognized as important endogenous regulators of cell activation, signaling, apoptosis and proliferation. Most of these lipid mediators are originated from cleavage of constituents of cellular membranes under the activity of phospholipases and sphingomyelinases. One of the major cascades of bioactive lipid mediator production involves the release of arachidonic acid from membrane phospholipids followed by the formation of eicosanoids (i.e. prostaglandins, leukotrienes and lipoxins). These biologically active metabolites of arachidonic acid are emerging as key regulators of cell proliferation and neo-angiogenesis and agents that specifically target these lipid mediators are being investigated as potential anticancer drugs. On the other hand, the lysophospholipid family, which includes members of the sphingomyelin-ceramide-sphingosine-1-phosphate and lysophosphatidic acid subfamilies, has evolved as an important group of lipid signaling molecules implicated in cellular differentiation, cell growth and apoptosis. This article reviews the most recent patents in this field of research, covering the following strategies based on the modulation of bioactive lipid mediators: (1) prostaglandin H synthase-2 inhibitors, (2) lipoxin analogs and aspirin-triggered lipid mediators, and (3) lysophosphatidic acid and other lysophospholipids. PMID:18221047

  4. Apoptosis and the Airway Epithelium

    PubMed Central

    White, Steven R.

    2011-01-01

    The airway epithelium functions as a barrier and front line of host defense in the lung. Apoptosis or programmed cell death can be elicited in the epithelium as a response to viral infection, exposure to allergen or to environmental toxins, or to drugs. While apoptosis can be induced via activation of death receptors on the cell surface or by disruption of mitochondrial polarity, epithelial cells compared to inflammatory cells are more resistant to apoptotic stimuli. This paper focuses on the response of airway epithelium to apoptosis in the normal state, apoptosis as a potential regulator of the number and types of epithelial cells in the airway, and the contribution of epithelial cell apoptosis in important airways diseases. PMID:22203854

  5. Induction of G1 cell cycle arrest and apoptosis by berberine in bladder cancer cells.

    PubMed

    Yan, Keqiang; Zhang, Cheng; Feng, Jinbo; Hou, Lifang; Yan, Lei; Zhou, Zunlin; Liu, Zhaoxu; Liu, Cheng; Fan, Yidon; Zheng, Baozhong; Xu, Zhonghua

    2011-07-01

    Bladder cancer is the ninth most common type of cancer, and its surgery is always followed by chemotherapy to prevent recurrence. Berberine is non-toxic to normal cells but has anti-cancer effects in many cancer cell lines. This study was aimed to determine whether berberine inhibits the cell proliferation and induces cell cycle arrest and apoptosis in BIU-87 and T24 bladder cancer cell line. The superficial bladder cancer cell line BIU-87 and invasive T24 bladder cancer cells were treated with different concentrations of berberine. MTT assay was used to determine the effects of berberine on the viability of these cells. The cell cycle arrest was detected through propidium iodide (PI) staining. The induction of apoptosis was determined through Annexin V-conjugated Alexa Fluor 488 (Alexa488) staining. Berberine inhibited the viability of BIU-87 and T24 cells in a dose- and time-dependent manner. It also promoted cell cycle arrest at G0/G1 in a dose-dependent manner and induced apoptosis. We observed that H-Ras and c-fos mRNA and protein expressionswere dose-dependently and time-dependently decreased by berberine treatment. Also, we investigated the cleaved caspase-3 and caspase-9 protein expressions increased in a dose-dependent manner. Berberine inhibits the cell proliferation and induces cell cycle arrest and apoptosis in BIU-87, bladder cancer cell line and T24, invasive bladder cancer cell line. Berberine can inhibit the oncogentic H-Ras and c-fos in T24 cells, and can induce the activation of the caspase-3 and caspase-9 apoptosis. Therefore, berberine has the potential to be a novel chemotherapy drug to treat the bladder cancer by suppressing tumor growth.

  6. VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

    SciTech Connect

    Qian, Qinyi; Zhou, Hao; Chen, Yan; Shen, Chenglong; He, Songbing; Zhao, Hua; Wang, Liang; Wan, Daiwei; Gu, Wen

    2014-01-17

    Highlights: •This research confirmed VMP1 as a regulator of autophagy in colorectal cancer cell lines. •We proved the pro-survival role of VMP1-mediated autophagy in colorectal cancer cell lines. •We found the interaction between VMP1 and BECLIN1 also existing in colorectal cancer cell lines. -- Abstract: Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.

  7. Platinum-zoledronate complex blocks gastric cancer cell proliferation by inducing cell cycle arrest and apoptosis.

    PubMed

    Yang, Hui; Qiu, Ling; Zhang, Li; Lv, Gaochao; Li, Ke; Yu, Huixin; Xie, Minhao; Lin, Jianguo

    2016-08-01

    A series of novel dinuclear platinum complexes based on the bisphosphonate ligands have been synthesized and characterized in our recent study. For the purpose of discovering the pharmacology and action mechanisms of this kind of compounds, the most potent compound [Pt(en)]2ZL was selected for systematic investigation. In the present study, the inhibition effect on the human gastric cancer cell lines SGC7901 and action mechanism of [Pt(en)]2ZL were investigated. The traditional 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay and colony formation assay were carried out to study the effect of [Pt(en)]2ZL on the cell viability and proliferation capacity, respectively. The senescence-associated β-galactosidase staining and immunofluorescence staining were also performed to assess the cell senescence and microtubule polymerization. Fluorescence staining and flow cytometry (FCM) were used to monitor the cell cycle distribution and apoptosis, and Western blot analysis was applied to examine the expression of several apoptosis-related proteins. The results demonstrated that [Pt(en)]2ZL exhibited remarkable cytotoxicity and anti-proliferative effects on the SGC7901 cells in a dose- and time-dependent manner, and it also induced cell senescence and abnormal microtubule assembly. The cell apoptosis and cell cycle arrest induced by [Pt(en)]2ZL were also observed with the fluorescence staining and FCM. The expressions of cell cycle regulators (p53, p21, cyclin D1, cyclin E, and cyclin-dependent kinase (CDK)2) and apoptosis-related proteins (Bcl-2, Bax, caspase-3, poly ADP ribose polymerase (PARP), and survivin) were regulated by the treatment of [Pt(en)]2ZL, resulting in the cell cycle arrest and apoptosis. Therefore, [Pt(en)]2ZL exerted anti-tumor effect on the gastric cancer via inducing cell cycle arrest at G1/S phase and apoptosis. PMID:26891667

  8. Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

    PubMed Central

    Chen, Xin; Gu, Ying; Singh, Karnika; Shang, Chaowei; Barzegar, Mansoureh; Jiang, Shanxiang; Huang, Shile

    2014-01-01

    Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death. PMID:25531367

  9. Smac mimetic sensitizes renal cell carcinoma cells to interferon-α-induced apoptosis.

    PubMed

    Reiter, Michael; Eckhardt, Ines; Haferkamp, Axel; Fulda, Simone

    2016-05-28

    The prognosis of metastatic or relapsed renal cell carcinoma (RCC) is still very poor, highlighting the need for new treatment strategies. Here, we identify a cooperative antitumor activity of interferon-α (IFNα) together with the Smac mimetic BV6 that antagonizes antiapoptotic IAP proteins. BV6 and IFNα act together to reduce cell viability and to induce apoptosis in various RCC cell lines. Molecular studies revealed that BV6/IFNα co-treatment triggers apoptosis independently of autocrine/paracrine Tumor Necrosis Factor (TNF)α signaling, since the TNFα-blocking antibody Enbrel fails to rescue cell death. Importantly, knockdown of Receptor-Interacting Protein (RIP)1 significantly decreases BV6/IFNα-mediated apoptosis, whereas the RIP1 kinase inhibitor necrostatin-1 (Nec-1) provides no protection. This demonstrates that RIP1 protein is critically required for BV6/IFNα-induced apoptosis, while RIP1 kinase activity is dispensable, pointing to a scaffold function of RIP1. Consistently, BV6 and IFNα cooperate to trigger the interaction of RIP1, Fas-Associated Death Domain protein (FADD) and caspase-8 to form a cytosolic cell death complex that drives caspase activation. Addition of the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) significantly protects RCC cells against BV6/IFNα-induced apoptosis, demonstrating that caspase activity is required for apoptosis. In conclusion, the combination approach of IFNα and BV6 represents a promising strategy for cooperative induction of apoptosis in RCC cells, which warrants further investigation. PMID:26912071

  10. Effects of LG268 on Cell Proliferation and Apoptosis of NB4 Cells

    PubMed Central

    Xu, Ting; Zhong, Liang; Gan, Liu-Gen; Xiao, Chun-Lan; Shan, Zhi-Ling; Yang, Rong; Song, Hao; Li, Liu; Liu, Bei-Zhong

    2016-01-01

    Aims: To investigate the effect of LG100268 (LG268) on cell proliferation and apoptosis in NB4 cells. Methods: NB4 cells were treated with LG268 for 24 h or 48 h. The effect of LG268 on cell proliferation was assessed by the CCK-8 assay and colony-forming assay. Apoptosis and cell cycle were evaluated by flow cytometry. The protein expression levels of Survivin, PARP, c-Myc, cyclin D1, ERK, p-ERK, p38 MAPK, and p- p38 MAPK were detected by western blot. Results: We found that LG268 inhibited the proliferation of NB4 cells in a dose-dependent manner. Flow cytometry analysis showed that LG268 accelerated apoptosis in NB4 cells in a time- dependent manner and that LG268 treatment led to cell cycle arrest at G0/G1 phase. Moreover, LG268 significantly decreased the protein levels of Survivin, c-Myc, and cyclinD1. Cleaved PARP was observed in the LG268 treatment group but not in the control group. In addition, LG268 increased the phosphorylation level of p38 MAPK and decreased the phosphorylation level of ERK. Conclusions: LG268 inhibited cell proliferation and promoted cell apoptosis in NB4 cells. PMID:27429588

  11. Anaplasma phagocytophilum inhibits apoptosis and promotes cytoskeleton rearrangement for infection of tick cells.

    PubMed

    Ayllón, Nieves; Villar, Margarita; Busby, Ann T; Kocan, Katherine M; Blouin, Edmour F; Bonzón-Kulichenko, Elena; Galindo, Ruth C; Mangold, Atilio J; Alberdi, Pilar; Pérez de la Lastra, José M; Vázquez, Jesús; de la Fuente, José

    2013-07-01

    Anaplasma phagocytophilum causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects gene expression in both the vertebrate host and the tick vector, Ixodes scapularis. Here, we identified new genes, including spectrin alpha chain or alpha-fodrin (CG8) and voltage-dependent anion-selective channel or mitochondrial porin (T2), that are involved in A. phagocytophilum infection/multiplication and the tick cell response to infection. The pathogen downregulated the expression of CG8 in tick salivary glands and T2 in both the gut and salivary glands to inhibit apoptosis as a mechanism to subvert host cell defenses and increase infection. In the gut, the tick response to infection through CG8 upregulation was used by the pathogen to increase infection due to the cytoskeleton rearrangement that is required for pathogen infection. These results increase our understanding of the role of tick genes during A. phagocytophilum infection and multiplication and demonstrate that the pathogen uses similar strategies to establish infection in both vertebrate and invertebrate hosts.

  12. Anaplasma phagocytophilum Inhibits Apoptosis and Promotes Cytoskeleton Rearrangement for Infection of Tick Cells

    PubMed Central

    Ayllón, Nieves; Villar, Margarita; Busby, Ann T.; Kocan, Katherine M.; Blouin, Edmour F.; Bonzón-Kulichenko, Elena; Galindo, Ruth C.; Mangold, Atilio J.; Alberdi, Pilar; Pérez de la Lastra, José M.; Vázquez, Jesús

    2013-01-01

    Anaplasma phagocytophilum causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects gene expression in both the vertebrate host and the tick vector, Ixodes scapularis. Here, we identified new genes, including spectrin alpha chain or alpha-fodrin (CG8) and voltage-dependent anion-selective channel or mitochondrial porin (T2), that are involved in A. phagocytophilum infection/multiplication and the tick cell response to infection. The pathogen downregulated the expression of CG8 in tick salivary glands and T2 in both the gut and salivary glands to inhibit apoptosis as a mechanism to subvert host cell defenses and increase infection. In the gut, the tick response to infection through CG8 upregulation was used by the pathogen to increase infection due to the cytoskeleton rearrangement that is required for pathogen infection. These results increase our understanding of the role of tick genes during A. phagocytophilum infection and multiplication and demonstrate that the pathogen uses similar strategies to establish infection in both vertebrate and invertebrate hosts. PMID:23630955

  13. Bacterial Heat Shock Protein GroEL (Hsp64) Exerts Immunoregulatory Effects on T Cells by Utilizing Apoptosis

    PubMed Central

    Nalbant, Ayten; Kant, Melis

    2016-01-01

    Aggregatibacter actinomycetemcomitans (Aa) expresses a 64-kDa GroEL protein belonging to the heat shock family of proteins. This protein has been shown to influence human host cells, but the apoptotic capacity of the GroEL protein regarding T cells is not yet known. The purpose of this study was to investigate the ability of A. actinomycetemcomitans GroEL (AaGroEL) protein to induce human peripheral blood T-cell apoptosis. Endogenous, purified AaGroEL protein was used as an antigen. In AaGroEL-treated T cells, the data indicated that phosphatidylserine exposure, an early apoptotic event, was dose- and time-dependent. The AaGroEL-treated T cells were also positive for active caspase-3 in a dose-dependent manner. The rate of AaGroEL-induced apoptosis was suppressed by the addition of the general caspase inhibitor Z-VAD-FMK. Furthermore, cleaved caspase-8 bands (40/36 kDa and 23 kDa) were identified in cells responding to AaGroEL. DNA fragmentation was also detected in the AaGroEL-treated T cells. Overall, we demonstrated that the endogenous GroEL from A. actinomycetemcomitans has the capacity to induce T-cell apoptosis. PMID:27736933

  14. Activation of the IFN-inducible enzyme RNase L causes apoptosis of animal cells.

    PubMed

    Díaz-Guerra, M; Rivas, C; Esteban, M

    1997-09-29

    The interferon (IFN)-induced enzyme RNase L produced by a recombinant vaccinia virus (VV) causes death of mammalian cells with morphological and biochemical characteristics of apoptosis. Coexpression of 2-5A-synthetase enhances apoptosis induced by RNase L Activation of endogenous RNase L by infection with a VV ts mutant (ts22) or with wild-type virus in the presence of the antipoxvirus drug isatin-beta-thiosemicarbazone, a treatment known to significantly increase the amount of double-stranded RNA late during infection, also causes pronounced apoptosis of infected cells. The effects observed with recombinant virus-derived RNase L or with the endogenous enzyme are specific, since apoptosis also occurs in cells derived from mice lacking the IFN-induced protein kinase (PKR). The apoptosis antagonist Bcl-2 prevents induction of cell death by RNase L activation. Apoptosis of mammalian cells by RNase L activation could be a mechanism mediating anticellular actions of IFN.

  15. Cytotoxicity of Cyclodipeptides from Pseudomonas aeruginosa PAO1 Leads to Apoptosis in Human Cancer Cell Lines

    PubMed Central

    Vázquez-Rivera, Dolores; González, Omar; Guzmán-Rodríguez, Jaquelina; Díaz-Pérez, Alma L.; Ochoa-Zarzosa, Alejandra; López-Bucio, José; Meza-Carmen, Víctor; Campos-García, Jesús

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen of plants and animals, which produces virulence factors in order to infect or colonize its eukaryotic hosts. Cyclodipeptides (CDPs) produced by P. aeruginosa exhibit cytotoxic properties toward human tumor cells. In this study, we evaluated the effect of a CDP mix, comprised of cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) that were isolated from P. aeruginosa, on two human cancer cell lines. Our results demonstrated that the CDP mix promoted cell death in cultures of the HeLa cervical adenocarcinoma and Caco-2 colorectal adenocarcinoma cell lines in a dose-dependent manner, with a 50% inhibitory concentration (IC50) of 0.53 and 0.66 mg/mL, for HeLa and Caco-2 cells, respectively. Flow cytometric analysis, using annexin V and propidium iodide as apoptosis and necrosis indicators, respectively, clearly showed that HeLa and Caco-2 cells exhibited apoptotic characteristics when treated with the CDP mix at a concentration <0.001 mg/mL. IC50 values for apoptotic cells in HeLa and Caco-2 cells were 6.5 × 10−5 and 1.8 × 10−4 mg/mL, respectively. Our results indicate that an apoptotic pathway is involved in the inhibition of cell proliferation caused by the P. aeruginosa CDP mix. PMID:25821788

  16. Vanadate induces apoptosis in epidermal JB6 P+ cells via hydrogen peroxide-mediated reactions.

    PubMed

    Ye, J; Ding, M; Leonard, S S; Robinson, V A; Millecchia, L; Zhang, X; Castranova, V; Vallyathan, V; Shi, X

    1999-12-01

    Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P+ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 microM vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H2O2 as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H2O2 production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H2O2 induced by vanadate plays a major role in this process. PMID:10705990

  17. Hexachlorobenzene triggers apoptosis in rat thyroid follicular cells.

    PubMed

    Chiappini, Florencia; Alvarez, Laura; Lux-Lantos, Victoria; Randi, Andrea S; Kleiman de Pisarev, Diana L

    2009-04-01

    Hexachlorobenzene (HCB) is a widespread environmental pollutant. Chronic exposure of humans to HCB produces a number of effects, such as triggering of porphyria, increased synthesis of liver microsomal enzymes, neurological symptoms, immunological disorders and thyroid dysfunctions. In rats, HCB induced hepatic porphyria, neurotoxic effects, and toxic effects on the reproductive system, thyroid function, and immune system. HCB is also known to cause tumors of the liver, thyroid and mammary gland in laboratory animals. The aim of this study was to investigate parameters of thyroid growth regulation, mainly cell proliferation and apoptosis in thyroid tissue from HCB (0.1, 1, 10, 100, and 500 mg/kg body weight)-treated female Wistar rats. The current study demonstrates that only the exposure to the highest HCB dose for 30 days, has adverse effects on thyroid endpoints examined related to thyroid gland morphology, and 3,3'5,5'-tetraiodothyronine (T(4), thyroxine) serum levels, without changes in thyroid-stimulating hormone concentrations or in thyroid gland weight. Morphological changes, included flattened epithelium and increased colloid size compared with control tissue. Transforming growth factor (TGF-beta1) mRNA levels, evaluated by RT-PCR, revealed a significant upregulation after exposure to HCB (1, 10, 100 mg/kg body weight). Cell proliferation evaluated by 5'-Br deoxiuridine incorporation into DNA, was not altered at any dose. HCB (1, 10, 100 mg/kg body weight) induces apoptosis, evaluated by in situ end labeling of fragmented DNA, terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling, in rat thyroid glands. This process is associated with dose-dependent increases in cytochrome c release from the mitochondria and procaspase-9 processing to its active product. Caspase-8 was not activated. These studies indicate that doses of HCB that do not disrupt thyroid economy induce TGF-beta1 expression and apoptosis in the thyroid

  18. TRAIL-expressing T cells induce apoptosis of vascular smooth muscle cells in the atherosclerotic plaque

    PubMed Central

    Sato, Kayoko; Niessner, Alexander; Kopecky, Stephen L.; Frye, Robert L.; Goronzy, Jörg J.; Weyand, Cornelia M.

    2006-01-01

    Acute coronary syndromes (ACS) are precipitated by a rupture of the atherosclerotic plaque, often at the site of T cell and macrophage infiltration. Here, we show that plaque-infiltrating CD4 T cells effectively kill vascular smooth muscle cells (VSMC). VSMCs sensitive to T cell–mediated killing express the death receptor DR5 (TNF-related apoptosis-inducing ligand [TRAIL] receptor 2), and anti-TRAIL and anti-DR5 antibodies block T cell–mediated apoptosis. CD4 T cells that express TRAIL upon stimulation are expanded in patients with ACS and more effectively induce VSMC apoptosis. Adoptive transfer of plaque-derived CD4 T cells into immunodeficient mice that are engrafted with human atherosclerotic plaque results in apoptosis of VSMCs, which was prevented by coadministration of anti-TRAIL antibody. These data identify that the death pathway is triggered by TRAIL-producing CD4 T cells as a direct mechanism of VSMC apoptosis, a process which may lead to plaque destabilization. PMID:16418392

  19. Butylbenzyl phthalate induces spermatogenic cell apoptosis in prepubertal rats.

    PubMed

    Alam, Mohammad Shah; Kurohmaru, Masamichi

    2016-02-01

    Butylbenzyl phthalate (BBP), a suspected endocrine disruptor, adversely affects male reproductive function. In this study, morphological alterations of prepubertal rat testes caused by single administration of BBP, were examined by light microscopy. Three-week-old male rats were given a single dose of 500 mg/kg BBP by oral gavage and sacrificed at 3, 12, and 24 h after administration. Histopathological examination revealed progressive detachment and sloughing of spermatogenic cells into the lumen, and a significant increase in the number of TUNEL-positive (apoptotic) spermatogenic cells in the treated groups, compared to the control. Semithin sections confirmed the apoptotic cells by their prominent basophilia, condensed chromatin, and shrunken cytoplasm, hallmarks of apoptotic cell death. Immunohistochemistry identified disruption of Sertoli cell vimentin and actin filaments in the treated groups. To elucidate the recovery effects of BBP, rats were treated in the same way and were sacrificed at D1-12h after administration. The apoptotic index returned to normal at D9. While, the testes revealed lower weight gain until D12. These results show for the first time that BBP induces collapse of vimentin filaments in Sertoli cells which may lead to disruption of Sertoli-spermatogenic cell physical interaction and induces spermatogenic cell apoptosis. PMID:26747412

  20. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    SciTech Connect

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen

    2015-01-09

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

  1. Induction of apoptosis and cell-cycle arrest in human colon cancer cells by meclizine.

    PubMed

    Lin, Jiunn-Chang; Ho, Yuan-Soon; Lee, Jie-Jen; Liu, Chien-Liang; Yang, Tsen-Long; Wu, Chih-Hsiung

    2007-06-01

    Meclizine (MEC), a histamine H1 antagonist, is used for the treatment of motion sickness and vertigo. In this study, we demonstrate that MEC dose-dependently induced apoptosis in human colon cancer cell lines (COLO 205 and HT 29 cells). Results of a DNA ladder assay revealed that DNA ladders appeared with MEC treatment in COLO 205 cells at dosage of >50 microM. In addition, the total cell number decreased dose-dependently after treatment with MEC in COLO 205 and HT 29 cells. Using flow cytometry, the percentage of COLO 205 cells arrested at G0/G1 phase increased dose-dependently. Analysis of changes in cell-cycle arrest-associated proteins with Western blotting showed that p53 and p21 were upregulated after treatment with MEC. The kinase activities of cyclin-dependent kinase 2 (CDK2) and CDK4 were suppressed in MEC-treated cells. As for apoptosis, MEC may induce upregulation of p53 and downregulation of Bcl-2, thus causing the release of cytochrome C from mitochondria and the translocation of apoptosis-inducing factor (AIF) to the nucleus. This resulted in the activation of caspase 3, 8, and 9. Our results provide the molecular basis of MEC-induced apoptosis and cell-cycle arrest in human colon cancer cells. PMID:17222494

  2. Protective effect of trifluoperazine on hydrogen peroxide-induced apoptosis in PC12 cells.

    PubMed

    Liu, Shichang; Han, Yangguang; Zhang, Tao; Yang, Zhuo

    2011-02-01

    This study investigated effects of trifluoperazine (TFP) against the cytotoxicity induced by H₂O₂ in PC12 cells and the mechanisms thereof. Different concentrations of H₂O₂ (100-500 μM) induced a significant decrease in cell viability accompanied by increased oxidative stress and cell apoptosis. Pretreatment with TFP inhibited H₂O₂-induced cell viability loss. The flow cytometric assay showed that TFP can inhibit intracellular reactive oxygen species (ROS) generation and reduce the cell apoptosis. The electrophysiological recordings indicated that when treated with H₂O₂, the calcium current was significantly increased. Pretreatment with TFP increased mitochondrial membrane potential (MMP) in cells of oxidative injury. These results suggested that TFP can reduce apoptosis by inhibiting ROS generation and preventing loss of MMP in cells. Meanwhile, the protective effect of TFP on the cell apoptosis may be related to the calcium overload. TFP may inhibit the calcium overload process to achieve the protection against apoptosis.

  3. Control of Host Cell Phosphorylation by Legionella Pneumophila

    PubMed Central

    Haenssler, Eva; Isberg, Ralph R.

    2011-01-01

    Phosphorylation is one of the most frequent modifications in intracellular signaling and is implicated in many processes ranging from transcriptional control to signal transduction in innate immunity. Many pathogens modulate host cell phosphorylation pathways to promote growth and establish an infectious disease. The intracellular pathogen Legionella pneumophila targets and exploits the host phosphorylation system throughout the infection cycle as part of its strategy to establish an environment beneficial for replication. Key to this manipulation is the L. pneumophila Icm/Dot type IV secretion system, which translocates bacterial proteins into the host cytosol that can act directly on phosphorylation cascades. This review will focus on the different stages of L. pneumophila infection, in which host kinases and phosphatases contribute to infection of the host cell and promote intracellular survival of the pathogen. This includes the involvement of phosphatidylinositol 3-kinases during phagocytosis as well as the role of phosphoinositide metabolism during the establishment of the replication vacuole. Furthermore, L. pneumophila infection modulates the NF-κB and mitogen-activated protein kinase pathways, two signaling pathways that are central to the host innate immune response and involved in regulation of host cell survival. Therefore, L. pneumophila infection manipulates host cell signal transduction by phosphorylation at multiple levels. PMID:21747787

  4. Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

    SciTech Connect

    Geel, Tessa M.; Meiss, Gregor; Gun, Bernardina T. van der; Kroesen, Bart Jan; Leij, Lou F. de; Zaremba, Mindaugas; Silanskas, Arunas; Kokkinidis, Michael; Ruiters, Marcel H.; McLaughlin, Pamela M.; Rots, Marianne G.

    2009-09-10

    TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18{sup Registered-Sign }:DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

  5. A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells

    PubMed Central

    Lorenzo, Daniela; Duarte, Alejandra; Mundiñano, Juliana; Berguer, Paula; Nepomnaschy, Irene; Piazzon, Isabel

    2016-01-01

    B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed. PMID:27603942

  6. A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells.

    PubMed

    Lorenzo, Daniela; Duarte, Alejandra; Mundiñano, Juliana; Berguer, Paula; Nepomnaschy, Irene; Piazzon, Isabel

    2016-01-01

    B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed. PMID:27603942

  7. Astaxanthin Inhibits Proliferation and Induces Apoptosis and Cell Cycle Arrest of Mice H22 Hepatoma Cells

    PubMed Central

    Shao, Yiye; Ni, Yanbo; Yang, Jing; Lin, Xutao; Li, Jun; Zhang, Lixia

    2016-01-01

    Background It is widely recognized that astaxanthin (ASX), a member of the carotenoid family, has strong biological activities including antioxidant, anti-inflammation, and immune-modulation activities. Previous studies have confirmed that ASX can effectively inhibit hepatoma cells in vitro. Material/Methods MTT was used to assay proliferation of mice H22 cells, and flow cytometry was used to determine apoptosis and cell cycle arrest of H22 cells in vitro and in vivo. Moreover, anti-tumor activity of ASX was observed in mice. Results ASX inhibited the proliferation of H22 cells, promoted cell necrosis, and induced cell cycle arrest in G2 phase in vitro and in vivo. Conclusions This study indicated that ASX can inhibit proliferation and induce apoptosis and cell cycle arrest in mice H22 hepatoma cells in vitro and in vivo. PMID:27333866

  8. Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells

    PubMed Central

    1994-01-01

    When cultivated on substrates that prevent cell adhesion (the polymer polyhydroxyethylmethacrylate, bovine serum albumin, and Teflon), human endothelial cells (EC) rapidly lost viability with a half-life of approximately 10 h. Dying EC showed the morphological and biochemical characteristics of apoptosis. The apoptotic process of suspended EC was delayed by the protein synthesis inhibitor cycloheximide. To obtain information as to the mechanism involved in the apoptosis of suspended EC, we investigated whether adhesion to matrix proteins or integrin occupancy in EC retaining a round shape may affect EC suicide. EC bound to low coating concentration of either fibronectin or vitronectin, retaining a round shape and failing to organize actin microfilaments, underwent to rapid cell death; by contrast, cells on high substrate concentrations became flattened, showed actin microfilament organization, and retained viability. Addition of saturating amounts of soluble vitronectin to suspended round-shaped EC did not reduce the process of apoptosis. Finally, when suspended EC bound Gly-Arg-Gly-Asp- Ser-coated microbeads (approximately 10 microbeads/cell), yet retaining a round shape, the apoptotic process was not affected. Oncogene- transformed EC in suspension were less susceptible to cell death and apoptosis than normal EC. Overall, these data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive. Modulation of interaction with the extracellular matrix can, therefore, be an important target for the control of angiogenesis. PMID:7523422

  9. Gravisensing, apoptosis, and drug recovery in Taxus cell suspensions

    NASA Technical Reports Server (NTRS)

    Durzan, D. J.

    1999-01-01

    Haploid and diploid cell suspensions of Taxus spp. were examined for their adaptive plasticity in response to simulated microgravity, unit gravity, and hypergravity. Cell suspensions produced the taxane, paclitaxel, (TAXOL (R)), which is useful for the treatment of various cancers. Amyloplasts contributed to taxane ring biosynthesis and to drug release at the cell wall. Drug-producing cells reacted as gravisensing osmotic tensiometers. In stressed cells, amyloplasts docked and fused in clusters to sites on the plasmalemma before taxane discharge into the culture medium. In simulated microgravity and compared to all other treatments, taxane production was reduced nearly 100-fold. The percent paclitaxel of total taxanes remained 3-to 6-fold greater, and biomass doubled. When p53-independent programmed cell death was induced, taxanes were released into the culture medium as free molecules (soluble and insoluble) or bound to membranes, nuclear fragments, xylan residues, and other particulate materials. Unit gravity and especially hypergravity promoted xylogenesis and significant drug overproduction. A model relating families of >touch = (TCH), taxane early response (TER), nuclear cycling, and apoptosis-regulating genes to gravisensing, cell wall modifications, and to taxane recovery accounted for most but not all of the observations.

  10. Cell responses to FGFR3 signalling: growth, differentiation and apoptosis

    SciTech Connect

    L'Hote, Corine G.M. . E-mail: Corine.LHote@cancer.org.uk; Knowles, Margaret A.

    2005-04-01

    FGFR3 is a receptor tyrosine kinase (RTK) of the FGF receptor family, known to have a negative regulatory effect on long bone growth. Fgfr3 knockout mice display longer bones and, accordingly, most germline-activating mutations in man are associated with dwarfism. Somatically, some of the same activating mutations are associated with the human cancers multiple myeloma, cervical carcinoma and carcinoma of the bladder. How signalling through FGFR3 can lead to either chondrocyte apoptosis or cancer cell proliferation is not fully understood. Although FGFR3 can be expressed as two main splice isoforms (IIIb or IIIc), there is no apparent link with specific cell responses, which may rather be associated with the cell type or its differentiation status. Depending on cell type, differential activation of STAT proteins has been observed. STAT1 phosphorylation seems to be involved in inhibition of chondrocyte proliferation while activation of the ERK pathway inhibits chondrocyte differentiation and B-cell proliferation (as in multiple myeloma). The role of FGFR3 in epithelial cancers (bladder and cervix) is not known. Some of the cell specificity may arise via modulation of signalling by crosstalk with other signalling pathways. Recently, inhibition of the ERK pathway in achondroplastic mice has provided hope for an approach to the treatment of dwarfism. Further understanding of the ability of FGFR3 to trigger different responses depending on cell type and cellular context may lead to treatments for both skeletal dysplasias and cancer.

  11. Foodborne Cereulide Causes Beta-Cell Dysfunction and Apoptosis

    PubMed Central

    Vangoitsenhoven, Roman; Rondas, Dieter; Crèvecoeur, Inne; D'Hertog, Wannes; Baatsen, Pieter; Masini, Matilde; Andjelkovic, Mirjana; Van Loco, Joris; Matthys, Christophe; Mathieu, Chantal; Overbergh, Lut; Van der Schueren, Bart

    2014-01-01

    Aims/Hypothesis To study the effects of cereulide, a food toxin often found at low concentrations in take-away meals, on beta-cell survival and function. Methods Cell death was quantified by Hoechst/Propidium Iodide in mouse (MIN6) and rat (INS-1E) beta-cell lines, whole mouse islets and control cell lines (HepG2 and COS-1). Beta-cell function was studied by glucose-stimulated insulin secretion (GSIS). Mechanisms of toxicity were evaluated in MIN6 cells by mRNA profiling, electron microscopy and mitochondrial function tests. Results 24 h exposure to 5 ng/ml cereulide rendered almost all MIN6, INS-1E and pancreatic islets apoptotic, whereas cell death did not increase in the control cell lines. In MIN6 cells and murine islets, GSIS capacity was lost following 24 h exposure to 0.5 ng/ml cereulide (P<0.05). Cereulide exposure induced markers of mitochondrial stress including Puma (p53 up-regulated modulator of apoptosis, P<0.05) and general pro-apoptotic signals as Chop (CCAAT/-enhancer-binding protein homologous protein). Mitochondria appeared swollen upon transmission electron microscopy, basal respiration rate was reduced by 52% (P<0.05) and reactive oxygen species increased by more than twofold (P<0.05) following 24 h exposure to 0.25 and 0.50 ng/ml cereulide, respectively. Conclusions/Interpretation Cereulide causes apoptotic beta-cell death at low concentrations and impairs beta-cell function at even lower concentrations, with mitochondrial dysfunction underlying these defects. Thus, exposure to cereulide even at concentrations too low to cause systemic effects appears deleterious to the beta-cell. PMID:25119564

  12. Direct activation of the apoptosis machinery as a mechanism to target cancer cells.

    PubMed

    Nguyen, Jack T; Wells, James A

    2003-06-24

    Apoptosis plays a pivotal role in the cytotoxic activity of most chemotherapeutic drugs, and defects in this pathway provide a basis for drug resistance in many cancers. Thus the ability to restore apoptosis by using small molecules could have important therapeutic implications. Using a cell-free assay to simultaneously target multiple components of the apoptosis pathway, we identified a class of compounds that activate caspases in a cytochrome c-dependent manner and induce apoptosis in whole cells. By reconstituting the apoptosis pathway with purified proteins, we determined that these compounds promote the protein-protein association of Apaf-1 into the functional apoptosome. These compounds exert cytostatic and cytotoxic effects on a variety of cancer cell lines while having little or no activity against the normal cell lines tested. These findings suggest that direct activation of the basic apoptosis machinery may be a viable mechanism to selectively target cancer.

  13. Impact of apoptosis on the on-line measured dielectric properties of CHO cells.

    PubMed

    Zalai, Dénes; Tobak, Teodóra; Putics, Ákos

    2015-12-01

    Apoptosis is a common type of cell death in biopharmaceutical cell culture processes which causes decrease in viable cell density and product yield. The progression of apoptosis has been reported to influence the dielectric properties of mammalian cells; however, the on-line detection of these effects has been rarely described. This study provides a comprehensive analysis of the on-line detectability of dielectric changes upon apoptosis induction in an industrial fed-batch process of CHO cells expressing a recombinant monoclonal antibody. Using capacitance signals, measured at 25 frequencies, the impact of apoptosis on the dielectric spectra was investigated in eight bioreactor cultivations in which various process conditions were combined with two different apoptosis induction strategies (camptothecin treatment and glucose starvation). To differentiate the apoptosis-related information from the cell concentration-associated variance in the multivariate capacitance datasets, principal component analysis (PCA) was used. A second principal component, explaining an explicit proportion (>20%) of the variance, was identified to be related to dielectric changes induced by apoptosis. Furthermore, the analysis of caspase-3 and -7 activation and DNA fragmentation showed that the detected dielectric change occurred in the early phase of apoptosis. The presented results verify that apoptosis has a considerable impact on the dielectric features of CHO cells and it can be monitored on-line with the introduced tool-set combining capacitance measurement with multivariate data analysis. PMID:26440966

  14. Impact of apoptosis on the on-line measured dielectric properties of CHO cells.

    PubMed

    Zalai, Dénes; Tobak, Teodóra; Putics, Ákos

    2015-12-01

    Apoptosis is a common type of cell death in biopharmaceutical cell culture processes which causes decrease in viable cell density and product yield. The progression of apoptosis has been reported to influence the dielectric properties of mammalian cells; however, the on-line detection of these effects has been rarely described. This study provides a comprehensive analysis of the on-line detectability of dielectric changes upon apoptosis induction in an industrial fed-batch process of CHO cells expressing a recombinant monoclonal antibody. Using capacitance signals, measured at 25 frequencies, the impact of apoptosis on the dielectric spectra was investigated in eight bioreactor cultivations in which various process conditions were combined with two different apoptosis induction strategies (camptothecin treatment and glucose starvation). To differentiate the apoptosis-related information from the cell concentration-associated variance in the multivariate capacitance datasets, principal component analysis (PCA) was used. A second principal component, explaining an explicit proportion (>20%) of the variance, was identified to be related to dielectric changes induced by apoptosis. Furthermore, the analysis of caspase-3 and -7 activation and DNA fragmentation showed that the detected dielectric change occurred in the early phase of apoptosis. The presented results verify that apoptosis has a considerable impact on the dielectric features of CHO cells and it can be monitored on-line with the introduced tool-set combining capacitance measurement with multivariate data analysis.

  15. Malaria Sporozoites Traverse Host Cells within Transient Vacuoles.

    PubMed

    Risco-Castillo, Veronica; Topçu, Selma; Marinach, Carine; Manzoni, Giulia; Bigorgne, Amélie E; Briquet, Sylvie; Baudin, Xavier; Lebrun, Maryse; Dubremetz, Jean-François; Silvie, Olivier

    2015-11-11

    Plasmodium sporozoites are deposited in the host skin by Anopheles mosquitoes. The parasites migrate from the dermis to the liver, where they invade hepatocytes through a moving junction (MJ) to form a replicative parasitophorous vacuole (PV). Malaria sporozoites need to traverse cells during progression through host tissues, a process requiring parasite perforin-like protein 1 (PLP1). We find that sporozoites traverse cells inside transient vacuoles that precede PV formation. Sporozoites initially invade cells inside transient vacuoles by an active MJ-independent process that does not require vacuole membrane remodeling or release of parasite secretory organelles typically involved in invasion. Sporozoites use pH sensing and PLP1 to exit these vacuoles and avoid degradation by host lysosomes. Next, parasites enter the MJ-dependent PV, which has a different membrane composition, precluding lysosome fusion. The malaria parasite has thus evolved different strategies to evade host cell defense and establish an intracellular niche for replication.

  16. Efficacy of an AC sinusoidal electric field for apoptosis induction in lung carcinoma cells (A549)

    NASA Astrophysics Data System (ADS)

    Park, Hyoun-Hyang; Lee, Seung S.; Hoon Lee, Dae

    2012-08-01

    An AC sinusoidal electric field was applied to lung carcinoma cells for the induction of apoptosis. The occurrence of apoptosis was determined by analysis of Annexin V/PI and DNA fragmentation. Additional evidence of apoptosis was confirmed by caspase-3 cleavage and disruption of mitochondrial membrane potential. These results demonstrated that the expression of apoptosis can be controlled by varying the magnitude and the duration of the field, and that the application of an AC electric field can stimulate the apoptosis via mitochondria-mediated pathway.

  17. Skin-stage schistosomula of Schistosoma mansoni produce an apoptosis-inducing factor that can cause apoptosis of T cells.

    PubMed

    Chen, Lin; Rao, Kakuturu V N; He, Yi-Xun; Ramaswamy, Kalyanasundaram

    2002-09-13

    Skin-stage schistosomula of Schistosoma mansoni were found to secrete molecules that are pro-apoptotic for skin T lymphocytes as measured by annexin V staining, caspase-3 activity, caspase-8 activities, and DNA fragmentation. Caspase-8 activities in lymphocytes peaked approximately 8 h and caspase-3 activity peaked approximately 16 h after exposure to the parasite secretions. Subset analysis showed that mainly CD4(+) and CD8(+) cells (but not B cells) were susceptible to the parasite-induced pro-apoptotic effect. In situ staining confirmed the presence of apoptotic T cells around challenge parasites in the skin of naive or immunized animals. Analysis of T cells to identify the potential molecular pathway of the parasite-induced apoptosis showed increases in the expression of Fas, FasL, and the Fas-associated death domain. Blocking of FasL with a fusion protein reversed the parasite-induced apoptosis, suggesting a role for the Fas/FasL-mediated pathway in the parasite-induced T cell apoptosis. Subsequent analyses of the secretions of skin-stage schistosomula identified the pro-apoptotic activity as being associated with a protein of approximately 23 kDa. This protein was termed S. mansoni-derived apoptosis-inducing factor.

  18. Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis

    PubMed Central

    Barott, Katie L.; Venn, Alexander A.; Perez, Sidney O.; Tambutté, Sylvie; Tresguerres, Martin

    2015-01-01

    Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H+-ATPase (VHA), which acidifies the symbiosome space down to pH ∼4. Inhibition of VHA results in a significant decrease in average H+ activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts. PMID:25548188

  19. Identification and function analysis of the host cell protein that interacted with Orf virus Bcl-2-like protein ORFV125.

    PubMed

    Tian, Hong; Chen, Yan; Wu, Jinyan; Lin, Tong; Liu, Xiangtao

    2016-10-01

    Orf virus (ORFV) causes contagious ecthyma, a non-systemic skin disease in sheep and goat. Bioinformatics analysis showed that ORFV125 has Bcl-2-like homologous domain and 3D structurally, it is generally known that Bcl-2 protein is known to be a key protein to control cell apoptosis. Maybe ORFV125 act as a Bcl-2-like manner to control cell apoptosis, but its exact function isn't very clear. So in this study, we use yeast two-hybrid system to identity the putative host cell protein interacting partners of ORFV125, and meanwhile using the data obtained from the Gene Ontology, Uniprot, and Kyoto Encyclopedia of Genes and Genomes databases to analysis the functions and pathways associated with them. Finally, five host proteins were shown to be interacted with ORFV125, including cytochrome b (cytb) gene, GUCY2C, BIRC5, GTF3C6 and SERBP1, we also found that BIRC5 has complex biological functions, can inhibit apoptosis, promote cell transformation and are involved in mitosis, and the interaction network of BIRC5 and ORFV125 were constructed. These findings provide a foundation to better understand the biology of the interactions between ORFV125 and the host proteins with which it directly interacts with and resultant downstream events. PMID:27663376

  20. Apoptosis in ovarian granulosa cells of cattle: morphological features and clearance by homologous phagocytosis.

    PubMed

    Carou, María Clara; Cruzans, Paula Romina; Maruri, Alejandro; Stockert, Juan Carlos; Lombardo, Daniel Marcelo

    2015-01-01

    Apoptosis is involved in many physiological processes of the ovary, such as recruitment of prenatal germ cells, follicular atresia, ovulation, and luteolysis. Based on the need for the involvement of phagocytic cells to achieve apoptosis clearance and that follicular atresia is triggered by weak apoptotic stimuli, we postulate that granulosa cells engullng apoptotic corpses (ACs) must carry out this macrophagic process. Since apoptosis was early defined in terms of morphological aspects, here we describe apoptosis induced by a GnRH analog (leuprolide acetate, LA) at histological level on bovine granulosa cells (primary culture, CPGB, and an established cell line, BGC-1). We observed two main types of apoptosis. In type A, the whole cell or most of it is compacted into a single large AC that is then engulfed by neighboring cells or simply detached. In type B, small portions of cells, either with or without nuclear material, become ACs that are also phagocytosed. Apoptosis and homologous phagocytosis were confirmed by TUNEL and immunocytochemistry for Bax and active caspase 3. Induction of apoptosis was significant in BGC-1 cells treated for 24 h with 100 nM LA. CPGB cells showed two types of response with different doses of LA. Fetal calf serum was necessary to find apoptosis induced by LA.

  1. A tumour necrosis factor receptor-like protein encoded by Singapore grouper iridovirus modulates cell proliferation, apoptosis and viral replication.

    PubMed

    Yu, Yepin; Huang, Youhua; Wei, Shina; Li, Pengfei; Zhou, Lingli; Ni, Songwei; Huang, Xiaohong; Qin, Qiwei

    2016-03-01

    It has been demonstrated that tumour necrosis factor receptor (TNFR) homologues encoded by viruses are usually involved in virus immune evasion by regulating the host immune response or mediating apoptotic cell death. Here, a novel TNFR-like protein encoded by Singapore grouper iridovirus (SGIV VP51) was cloned and characterized. Amino acid analysis showed that VP51 contained three cysteine-rich domains (CRDs) and a transmembrane domain at its C terminus. The expression of VP51 in vitro enhanced cell proliferation, and affected cell cycle progression via altering the G1/S transition. Furthermore, VP51 overexpression improved cell viability during SGIV infection via inhibiting virus-induced apoptosis, evidenced by the reduction of apoptotic bodies and the decrease of caspase-3 activation. In addition, overexpression of VP51 increased viral titre and the expression of viral structural protein gene MCP and cell proliferation promoting gene ICP-18. In contrast, the expression of the viral apoptosis inducing gene, LITAF, was significantly decreased. Although all three CRDs were essential for the action of VP51, CRD2 and CRD3 exerted more crucial roles on virus-induced apoptosis, viral gene transcription and virus production, while CRD1 was more crucial for cell proliferation. Together, SGIV TNFR-like products not only affected cell cycle progression and enhanced cell growth by increasing the expression of the virus encoded cell proliferation gene, but also inhibited virus-induced apoptotic cell death by decreasing the expression of the viral apoptosis inducing gene. Our results provided new insights into understanding the underlying mechanism by which iridovirus regulated the apoptotic pathway to complete its life cycle.

  2. Host cells and methods for producing isoprenyl alkanoates

    SciTech Connect

    Lee, Taek Soon; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-12-01

    The invention provides for a method of producing an isoprenyl alkanoate in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses an enzyme capable of catalyzing the esterification of an isoprenol and a straight-chain fatty acid, such as an alcohol acetyltransferase (AAT), wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) or lipase, under a suitable condition so that the isoprenyl alkanoate is produced.

  3. CpG oligodeoxynucleotide induces apoptosis and cell cycle arrest in A20 lymphoma cells via TLR9-mediated pathways.

    PubMed

    Qi, Xu-Feng; Zheng, Li; Kim, Cheol-Su; Lee, Kyu-Jae; Kim, Dong-Heui; Cai, Dong-Qing; Qin, Jun-Wen; Yu, Yan-Hong; Wu, Zheng; Kim, Soo-Ki

    2013-07-01

    Recent studies have suggested that the anti-cancer activity of CpG-oligodeoxynucleotides (CpG-ODNs) is owing to their immunomodulatory effects in tumor-bearing host. The purpose of this study is to investigate the directly cytotoxic activity of KSK-CpG, a novel CpG-ODN with an alternative CpG motif, against A20 and EL4 lymphoma cells in comparison with previously used murine CpG motif (1826-CpG). To evaluate the potential cytotoxic effects of KSK-CpG on lymphoma cells, cell viability assay, confocal microscopy, flow cytometry, DNA fragmentation, Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR) analysis were used. We found that KSK-CpG induced direct cytotoxicity in A20 lymphoma cells, but not in EL4 lymphoma cells, at least in part via TLR9-mediated pathways. Apoptotic cell death was demonstrated to play an important role in CpG-ODNs-induced cytotoxicity. In addition, both mitochondrial membrane potential decrease and G1-phase arrest were involved in KSK-CpG-induced apoptosis in A20 cells. The activities of apoptotic molecules such as caspase-3, PARP, and Bax were increased, but the activation of p27 Kip1 and ERK were decreased in KSK-CpG-treated A20 cells. Furthermore, autocrine IFN-γ partially contributed to apoptotic cell death in KSK-CpG-treated A20 cells. Collectively, our findings suggest that KSK-CpG induces apoptotic cell death in A20 lymphoma cells at least in part by inducing G1-phase arrest and autocrine IFN-γ via increasing TLR9 expression, without the need for immune system of tumor-bearing host. This new understanding supports the development of TLR9-targeted therapy with CpG-ODN as a direct therapeutic agent for treating B lymphoma. PMID:23357786

  4. TFF3 knockout in human pituitary adenoma cell HP75 facilitates cell apoptosis via mitochondrial pathway

    PubMed Central

    Gao, Feng; Pan, Suxia; Liu, Bing; Zhang, Huanzhi

    2015-01-01

    Trefoil factor 3 (TFF3), a regulatory protein composed of 59 amino acids, has been suggested to be involved in pathogenesis, proliferation, differentiation, invasion, migration and apoptosis in multiple malignant tumors. This study thus investigated the effect of TFF3 knockout in human pituitary adenoma cell line HP75 on cell apoptosis and related pathways. RNA interference approach was used to knock down the expression of TFF3 protein. The gene silencing was validated by RNA denaturing gel electrophoresis and Western blotting. The effect of TFF3 knockout on cell apoptosis was analyzed by Western blotting and flow cytometry. TFF3 protein level in pituitary adenoma was about 3.61 ± 0.48 folds of that in normal tissues (P < 0.01). After transfecting with small interference RNA (siRNA) against TFF3, the apoptotic ration was significantly elevated (P < 0.01). Apoptosis related protein Bcl-2 and caspase-3 levels were remarkably depressed after siRNA transfection, while Bax and cleaved caspase-3 levels were elevated. TFF3 protein knockout can facilitate apoptosis of human pituitary adenoma HP75 cells via mitochondrial pathway. PMID:26823779

  5. TFF3 knockout in human pituitary adenoma cell HP75 facilitates cell apoptosis via mitochondrial pathway.

    PubMed

    Gao, Feng; Pan, Suxia; Liu, Bing; Zhang, Huanzhi

    2015-01-01

    Trefoil factor 3 (TFF3), a regulatory protein composed of 59 amino acids, has been suggested to be involved in pathogenesis, proliferation, differentiation, invasion, migration and apoptosis in multiple malignant tumors. This study thus investigated the effect of TFF3 knockout in human pituitary adenoma cell line HP75 on cell apoptosis and related pathways. RNA interference approach was used to knock down the expression of TFF3 protein. The gene silencing was validated by RNA denaturing gel electrophoresis and Western blotting. The effect of TFF3 knockout on cell apoptosis was analyzed by Western blotting and flow cytometry. TFF3 protein level in pituitary adenoma was about 3.61 ± 0.48 folds of that in normal tissues (P < 0.01). After transfecting with small interference RNA (siRNA) against TFF3, the apoptotic ration was significantly elevated (P < 0.01). Apoptosis related protein Bcl-2 and caspase-3 levels were remarkably depressed after siRNA transfection, while Bax and cleaved caspase-3 levels were elevated. TFF3 protein knockout can facilitate apoptosis of human pituitary adenoma HP75 cells via mitochondrial pathway.

  6. FLIP switches Fas-mediated glucose signaling in human pancreatic cells from apoptosis to cell replication

    NASA Astrophysics Data System (ADS)

    Maedler, Kathrin; Fontana, Adriano; Ris, Frédéric; Sergeev, Pavel; Toso, Christian; Oberholzer, José; Lehmann, Roger; Bachmann, Felix; Tasinato, Andrea; Spinas, Giatgen A.; Halban, Philippe A.; Donath, Marc Y.

    2002-06-01

    Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of cell turnover. In human islets, elevated glucose concentrations impair cell proliferation and induce cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive cells; FLIP was no longer detectable in such TUNEL-positive cells. Up-regulation of FLIP, by incubation with transforming growth factor or by transfection with an expression vector coding for FLIP, protected cells from glucose-induced apoptosis, restored cell proliferation, and improved cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.

  7. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    PubMed

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  8. SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells.

    PubMed

    Bonifati, Serena; Daly, Michele B; St Gelais, Corine; Kim, Sun Hee; Hollenbaugh, Joseph A; Shepard, Caitlin; Kennedy, Edward M; Kim, Dong-Hyun; Schinazi, Raymond F; Kim, Baek; Wu, Li

    2016-08-01

    SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G1/G0 phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection. PMID:27183329

  9. Hyperthermia Induces Apoptosis of 786-O Cells through Suppressing Ku80 Expression

    PubMed Central

    Qi, Defeng; Hu, Yuan; Li, Jinhui; Peng, Tao; Su, Jialin; He, Yun; Ji, Weidong

    2015-01-01

    Hyperthermia as an anticancer method has been paid increasing attention in recent years. Several studies have shown that hyperthermia can kill tumor cells by inducing apoptosis. However, the underlying molecular mechanisms of hyperthermia-induced apoptosis are largely unknown. To investigate the effects and molecular mechanism of hyperthermia on the apoptosis in renal carcinoma 786-O cells, we firstly examined apoptosis and Ku expression in 786-O cell line treated with heat exposure (42°C for 0-4 h). The results showed that hyperthermia induced apoptosis of 786-O cells, and suppressed significantly Ku80 expression, but not Ku70 expression. Next, we knock-down Ku80 in 786-O cells, generating stable cell line 786-O-shKu80, and detected apoptosis, cell survival and cell cycle distribution. Our data showed higher apoptotic rate and lower surviving fraction in the stable cell line 786-O-shKu80 compared with those in control cells, exposed to the same heat stress (42°C for 0-4 h). Moreover, the results also showed suppression of Ku80 led to G2/M phase arrest in the stable cell line 786-O-shKu80 following heat treatment. Together, these findings indicate that Ku80 may play an important role in hyperthermia-induced apoptosis and heat-sensitivity of renal carcinoma cells through influencing the cell cycle distribution. PMID:25902193

  10. Cell Survival and Apoptosis Signaling as Therapeutic Target for Cancer: Marine Bioactive Compounds

    PubMed Central

    Kalimuthu, Senthilkumar; Se-Kwon, Kim

    2013-01-01

    Inhibition of apoptosis leads to activation of cell survival factors (e.g., AKT) causes continuous cell proliferation in cancer. Apoptosis, the major form of cellular suicide, is central to various physiological processes and the maintenance of homeostasis in multicellular organisms. A number of discoveries have clarified the molecular mechanism of apoptosis, thus clarifying the link between apoptosis and cell survival factors, which has a therapeutic outcome. Induction of apoptosis and inhibition of cell survival by anticancer agents has been shown to correlate with tumor response. Cellular damage induces growth arrest and tumor suppression by inducing apoptosis, necrosis and senescence; the mechanism of cell death depends on the magnitude of DNA damage following exposure to various anticancer agents. Apoptosis is mainly regulated by cell survival and proliferating signaling molecules. As a new therapeutic strategy, alternative types of cell death might be exploited to control and eradicate cancer cells. This review discusses the signaling of apoptosis and cell survival, as well as the potential contribution of marine bioactive compounds, suggesting that new therapeutic strategies might follow. PMID:23348928

  11. Prolonged insulin treatment sensitizes apoptosis pathways in pancreatic β cells.

    PubMed

    Bucris, E; Beck, A; Boura-Halfon, S; Isaac, R; Vinik, Y; Rosenzweig, T; Sampson, S R; Zick, Y

    2016-09-01

    Insulin resistance results from impaired insulin signaling in target tissues that leads to increased levels of insulin required to control plasma glucose levels. The cycle of hyperglycemia and hyperinsulinemia eventually leads to pancreatic cell deterioration and death by a mechanism that is yet unclear. Insulin induces ROS formation in several cell types. Furthermore, death of pancreatic cells induced by oxidative stress could be potentiated by insulin. Here, we investigated the mechanism underlying this phenomenon. Experiments were done on pancreatic cell lines (Min-6, RINm, INS-1), isolated mouse and human islets, and on cell lines derived from nonpancreatic sources. Insulin (100nM) for 24h selectively increased the production of ROS in pancreatic cells and isolated pancreatic islets, but only slightly affected the expression of antioxidant enzymes. This was accompanied by a time- and dose-dependent decrease in cellular reducing power of pancreatic cells induced by insulin and altered expression of several ER stress response elements including a significant increase in Trb3 and a slight increase in iNos The effect on iNos did not increase NO levels. Insulin also potentiated the decrease in cellular reducing power induced by H2O2 but not cytokines. Insulin decreased the expression of MCL-1, an antiapoptotic protein of the BCL family, and induced a modest yet significant increase in caspase 3/7 activity. In accord with these findings, inhibition of caspase activity eliminated the ability of insulin to increase cell death. We conclude that prolonged elevated levels of insulin may prime apoptosis and cell death-inducing mechanisms as a result of oxidative stress in pancreatic cells. PMID:27411561

  12. HTLV-1 Tax oncoprotein stimulates ROS production and apoptosis in T cells by interacting with USP10.

    PubMed

    Takahashi, Masahiko; Higuchi, Masaya; Makokha, Grace Naswa; Matsuki, Hideaki; Yoshita, Manami; Tanaka, Yuetsu; Fujii, Masahiro

    2013-08-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), and the viral oncoprotein Tax plays key roles in the immortalization of human T cells, lifelong persistent infection, and leukemogenesis. We herein identify the ubiquitin-specific protease 10 (USP10) as a Tax-interactor in HTLV-1-infected T cells. USP10 is an antistress factor against various environmental stresses, including viral infections and oxidative stress. On exposure to arsenic, an oxidative stress inducer, USP10 is recruited into stress granules (SGs), and USP10-containing SGs reduce reactive oxygen species (ROS) production and inhibit ROS-dependent apoptosis. We found that interaction of Tax with USP10 inhibits arsenic-induced SG formation, stimulates ROS production, and augments ROS-dependent apoptosis in HTLV-1-infected T cells. These findings suggest that USP10 is a host factor that inhibits stress-induced ROS production and apoptosis in HTLV-1-infected T cells; however, its activities are attenuated by Tax. A clinical study showed that combination therapy containing arsenic is effective against some forms of ATL. Therefore, these findings may be relevant to chemotherapy against ATL.

  13. Autophagy as a Survival Mechanism for Squamous Cell Carcinoma Cells in Endonuclease G-Mediated Apoptosis

    PubMed Central

    Masui, Atsushi; Hamada, Masakazu; Kameyama, Hiroyasu; Wakabayashi, Ken; Takasu, Ayako; Imai, Tomoaki; Iwai, Soichi; Yura, Yoshiaki

    2016-01-01

    Safingol, L- threo-dihydrosphingosine, induces cell death in human oral squamous cell carcinoma (SCC) cells through an endonuclease G (endoG) -mediated pathway. We herein determined whether safingol induced apoptosis and autophagy in oral SCC cells. Safingol induced apoptotic cell death in oral SCC cells in a dose-dependent manner. In safingol-treated cells, microtubule-associated protein 1 light chain 3 (LC3)-I was changed to LC3-II and the cytoplasmic expression of LC3, amount of acidic vesicular organelles (AVOs) stained by acridine orange and autophagic vacuoles were increased, indicating the occurrence of autophagy. An inhibitor of autophagy, 3-methyladenine (3-MA), enhanced the suppressive effects of safingol on cell viability, and this was accompanied by an increase in the number of apoptotic cells and extent of nuclear fragmentation. The nuclear translocation of endoG was minimal at a low concentration of safingol, but markedly increased when combined with 3-MA. The suppressive effects of safingol and 3-MA on cell viability were reduced in endoG siRNA- transfected cells. The scavenging of reactive oxygen species (ROS) prevented cell death induced by the combinational treatment, whereas a pretreatment with a pan-caspase inhibitor z-VAD-fmk did not. These results indicated that safingol induced apoptosis and autophagy in SCC cells and that the suppression of autophagy by 3-MA enhanced apoptosis. Autophagy supports cell survival, but not cell death in the SCC cell system in which apoptosis occurs in an endoG-mediated manner. PMID:27658240

  14. Tissue Inhibitor of Metalloproteinase-4 Triggers Apoptosis in Cervical Cancer Cells.

    PubMed

    Lizarraga, Floria; Ceballos-Cancino, Gisela; Espinosa, Magali; Vazquez-Santillan, Karla; Maldonado, Vilma; Melendez-Zajgla, Jorge

    2015-01-01

    Tissue inhibitor of metalloproteinase-4 (TIMP-4) is a member of extracellular matrix (ECM) metalloproteinases inhibitors that has pleiotropic functions. However, TIMP-4 roles in carcinogenesis are not well understood. Cell viability and flow cytometer assays were employed to evaluate cell death differences between H-Vector and H-TIMP-4 cell lines. Immunobloting and semi-quantitative RT-PCR were used to evaluate the expression of apoptosis regulators. We showed that TIMP-4 has apoptosis-sensitizing effects towards several death stimuli. Consistent with these findings, regulators of apoptosis from Inhibitors of Apoptosis Proteins (IAP), FLICE-like inhibitor proteins (FLIP) and Bcl-2 family members were modulated by TIMP-4. In addition, TIMP-4 knockdown resulted in cell survival increase after serum deprivation, as assessed by clonogenic cell analyses. This report shows that TIMP-4 regulates carcinogenesis through apoptosis activation in cervical cancer cells. Understanding TIMP-4 effects in tumorigenesis may provide clues for future therapies.

  15. The relevance of spontaneous- and chemically-induced alterations in testicular germ cell apoptosis to toxicology.

    PubMed

    Richburg, J H

    2000-03-15

    Elimination of germ cells via apoptosis occurs spontaneously under normal physiologic conditions and is often heightened after chemical-induced testicular injury. Though many different apoptosis-related elements have been identified in the testis, the molecular and cellular mechanisms that regulate germ cell apoptosis are not thoroughly understood. In this review, the role of germ cell apoptosis in spermatogenesis and possible key regulators of apoptosis is described. The involvement of the Fas-signaling pathway between Sertoli cells and germ cells is highlighted as a crucial paracrine-signaling mechanism that responds to both physiologic- or toxicant-induced declines in the supportive capacity of the testis and reduces the germ cell population accordingly.

  16. Morin, a flavonoid from moraceae, induces apoptosis by induction of BAD protein in human leukemic cells.

    PubMed

    Park, Cheol; Lee, Won Sup; Go, Se-Il; Nagappan, Arulkumar; Han, Min Ho; Hong, Su Hyun; Kim, Gon Sup; Kim, Gi Young; Kwon, Taeg Kyu; Ryu, Chung Ho; Shin, Sung Chul; Choi, Yung Hyun

    2014-12-30

    Evidence suggests that phytochemicals can safely modulate cancer cell biology and induce apoptosis. Here, we investigated the anti-cancer activity of morin, a flavone originally isolated from members of the Moraceae family in human leukemic cells, focusing on apoptosis. An anti-cancer effect of morin was screened with several human leukemic cell lines. U937 cells were most sensitive to morin, where it induced caspase-dependent apoptosis in a dose-dependent manner. It also induced loss of MMP (ΔΨm) along with cytochrome c release, down-regulated Bcl-2 protein, and up-regulated BAX proteins. The apoptotic activity of morin was significantly attenuated by Bcl-2 augmentation. In conclusion, morin induced caspase-dependent apoptosis through an intrinsic pathway by upregulating BAD proteins. In addition, Bcl-2 protein expression is also important in morin-induced apoptosis of U937 cells. This study provides evidence that morin might have anticancer properties in human leukemic cells.

  17. Apoptosis of human pancreatic carcinoma cell-1 cells induced by Yin Chen Hao Decoction

    PubMed Central

    Zhou, Hai-Bo; Chen, Jing-Ming; Shao, Li-Ming; Chen, Zhi-Gang

    2015-01-01

    AIM: To evaluate human pancreatic carcinoma cell line (PANC-1) cells apoptosis and Bcl-2 and Bax expression induced by Yin Chen Hao Decoction (YCHD). METHODS: The cell growth inhibitory rate was determined by MTT assay. Apoptosis of PANC-1 cells before and after treatment with YCHD was determined by TUNEL staining. Expression of the apoptosis-associated genes, Bcl-2 and Bax, was detected by immunohistochemical staining and reverse transcription -PCR. RESULTS: YCHD inhibited the growth of PANC-1 cells. Following treatment with YCHD for 24-96 h, the apoptotic rate of PANC-1 cells increased with time. In addition, the positive rate of Bcl-2 protein expression decreased in a time-dependent manner, whereas the positive rate of Bax protein expression increased in a time-dependent manner. Following treatment of with YCHD for 24-96h, expression of BAX mRNA increased gradually and BCL-2 mRNA reduced gradually with time. CONCLUSION: YCHD induces apoptosis of PANC-1 cells mediated in part via up-regulation of BAX and down-regulation of BCL-2. PMID:26217086

  18. Glycoprotein 5 of porcine reproductive and respiratory syndrome virus strain SD16 inhibits viral replication and causes G2/M cell cycle arrest, but does not induce cellular apoptosis in Marc-145 cells

    SciTech Connect

    Mu, Yang; Li, Liangliang; Zhang, Beibei; Huang, Baicheng; Gao, Jiming; and others

    2015-10-15

    Cell apoptosis is common after infection with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV GP5 has been reported to induce cell apoptosis. To further understand the role of GP5 in PRRSV induced cell apoptosis, we established Marc-145 cell lines stably expressing full-length GP5, GP5{sup Δ84-96} (aa 84-96 deletion), and GP5{sup Δ97-119} (aa 97-119 deletion). Cell proliferation, cell cycle progression, cell apoptosis and virus replication in these cell lines were evaluated. Neither truncated nor full-length GP5 induced cell apoptosis in Marc-145 cells. However, GP5{sup Δ97-119}, but not full-length or GP5{sup Δ84-96}, induced a cell cycle arrest at the G2/M phase resulting in a reduction in the growth of Marc-145 cells. Additionally, GP5{sup Δ84-96} inhibited the replication of PRRSV in Marc-145 cells through induction of IFN-β. These findings suggest that PRRSV GP5 is not responsible for inducing cell apoptosis in Marc-145 cells under these experimental conditions; however it has other important roles in virus/host cell biology. - Highlights: • Marc-145 cell lines stable expression PRRSV GP5 or truncated GP5 were constructed. • GP5{sup Δ97-119} expression in Marc-145 cell induced cell cycle arrest at G2/M phase. • Expression of GP5 and truncated GP5 could not induce Marc-145 cells apoptosis. • PRRSV replication in Marc-145-GP5{sup Δ84-96} was significantly inhibited.

  19. Induction of apoptosis by Citrus paradisi essential oil in human leukemic (HL-60) cells.

    PubMed

    Hata, Tomona; Sakaguchi, Ikuyo; Mori, Masahiro; Ikeda, Norikazu; Kato, Yoshiko; Minamino, Miki; Watabe, Kazuhito

    2003-01-01

    Limonene is a primary component of citrus essential oils (EOs) and has been reported to induce apoptosis on tumor cells. Little is known about induction of apoptosis by citrus EOs. In this study, we examined induction of apoptosis by Citrus aurantium var. dulcis (sweet orange) EO, Citrus paradisi (grapefruit) EO and Citrus limon (lemon) EO. These EOs induced apoptosis in HL-60 cells and the apoptosis activities were related to the limonene content of the EOs. Moreover, sweet orange EO and grapefruit EO may contain components besides limonene that have apoptotic activity. To identify the components with apoptotic activity, grapefruit EO was fractionated using silica gel columns, and the components were analyzed by GC-MS. The n-hexane fraction contained limonene, and the dichloromethane fraction (DF) contained aldehyde compounds and nootkatone. Decanal, octanal and citral in the DF showed strong apoptotic activity, suggesting that the aldehyde compounds induced apoptosis strongly in HL-60 cells. PMID:14758720

  20. A novel schiff base zinc coordination compound inhibits proliferation and induces apoptosis of human osteosarcoma cells.

    PubMed

    Yan, Ming; Pang, Li; Ma, Tan-tan; Zhao, Cheng-liang; Zhang, Nan; Yu, Bing-xin; Xia, Yan

    2015-10-01

    Various kinds of schiff base metal complexes have been proven to induce apoptosis of tumor cells. However, it remains largely unknown whether schiff base zinc complexes induce apoptosis in human cancer cells. Here, we synthesized a novel schiff base zinc coordination compound (SBZCC) and investigated its effects on the growth, proliferation and apoptosis of human osteosarcoma MG-63 cells. A novel SBZCC was synthesized by chemical processes and used to treat MG-63 cells. The cell viability was determined by CCK-8 assay. The cell cycle progression, mitochondrial membrane potential and apoptotic cells were analyzed by flow cytometry. The apoptosis-related proteins levels were determined by immunoblotting. Treatment of MG-63 cells with SBZCC resulted in inhibition of cell proliferation and cell cycle arrest at G1 phase. Moreover, SBZCC significantly reduced the mitochondrial membrane potential and induced apoptosis, accompanied with increased Bax/Bcl-2 and FlasL/Fas expression as well as caspase-3/8/9 cleavage. Our results demonstrated that the synthesized novel SBZCC could inhibit the proliferation and induce apoptosis of MG-63 cells via activating both the mitochondrial and cell death receptor apoptosis pathways, suggesting that SBZCC is a promising agent for the development as anticancer drugs.

  1. Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

    PubMed Central

    Leitão, Elsa; Costa, Ana Catarina; Brito, Cláudia; Costa, Lionel; Pombinho, Rita; Cabanes, Didier; Sousa, Sandra

    2014-01-01

    Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation. PMID:24552813

  2. Host-pathogen reorganisation during host cell entry by Chlamydia trachomatis.

    PubMed

    Nans, Andrea; Ford, Charlotte; Hayward, Richard D

    2015-01-01

    Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C. trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C. trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C. trachomatis entry and how this might relate to those of other bacteria and viruses.

  3. Ethyl acetate extracts of Fructus Ligustri Lucide induce cell apoptosis in human neuroglioma cell

    PubMed Central

    Guo, Yun-Bao; Shao, Yi-Meng; Chen, Jing; Xu, Song-Bai; Zhang, Xing-Dong; Liu, Hai-Yan

    2015-01-01

    Objective: Previous studies have shown that Fructus Ligustri Lucide (FLL) can be used to improve the tumor cells sensitivity to chemotherapeutics and promote cell death. However, the mechanism by which FLL mediate this effect is unclear. In the present study, ethyl acetate extracts of FLL induced cell apoptosis in human neuroglioma cell was investigated. Methods: The cell viability was detected by the CCK8 assay. The cell apoptosis was assessed by annexin V-PI double-labeling staining and hoechst 33342 staining. The protein expression of cell cycle regulators and tumor suppressors were analyzed by western blotting. Results: Treatment of human neuroglioma cell with FLL induced cell death in a dose-and time-dependent manner by using CCK8 assay. Consistent with the CCK8 assay, the flow cytometry results showed that the proportion of the early and terminal phase of apoptosis cells had gained after FLL treatment as compared to untreatment group. Moreover, human neuroglioma cells were exposed to the ethyl acetate extracts of FLL for 48 h, which resulted in an accumulation of cells in G2/Mphase. Apoptotic bodies were clearly observed in human neuroglioma cells that had been treated with FLL for 48 h and then stained with Hochest 33342. The expression of Cyclin B1, CDC2 and cdc25C were downregulated upon FLL treatment in human neuroglioma cells. The expression level of Cyclin B1, CDC2 and cdc25C was negatively correlated with the time of treatment by FLL. In contrast, p53, p21 and p16 were obviously upregulated by FLL treatment in a time-dependent manner. Conclusions: These results confirmed that FLL could induce apoptosis in human neuroglioma cells, the underlying molecular mechanisms, at least partially, through activation p21/p53 and suppression CDC2/cdc25C signaling in vitro. PMID:26064281

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Peroxiredoxin III protects pancreatic ß cells from apoptosis.

    PubMed

    Wolf, Gabriele; Aumann, Nicole; Michalska, Marta; Bast, Antje; Sonnemann, Jürgen; Beck, James F; Lendeckel, Uwe; Newsholme, Philip; Walther, Reinhard

    2010-11-01

    Type 1 diabetes mellitus is characterized by a progressive autoimmune destruction of insulin-producing β cells. Macrophages and T lymphocytes release cytokines, which induce the synthesis of oxygen and nitrogen radicals in the pancreatic islets. The resulting cellular and mitochondrial damage promotes β cell death. β cells are very sensitive to the autoimmune free radical-dependent attack due to their low content of antioxidant enzymes such as glutathione peroxidase and catalase. A focal point of β cell protection should be the control of the mitochondrial redox status, which will result in the preservation of metabolic stimulus-secretion coupling. For this reason, there is a considerable interest in the mitochondrial peroxiredoxin III (PRX III), a thioredoxin-dependent peroxide reductase, which was shown to be able to protect against both oxidative and nitrosative stress. Using the Tet-On-system, we generated stably transfected rat insulinoma cells over- or under-expressing PRX III in a doxycyclin-dependent manner to analyze the effect of increased or decreased amounts of cellular PRX III, following treatment with several stressors. We provide evidence that PRX III protects pancreatic β cells from cell stress induced by accumulation of hydrogen peroxide, or the induction of inducible nitric oxide synthase or caspase-9 and -3 by pro-inflammatory cytokines or streptozotocin. Basal insulin secretion was markedly decreased in cells expressing lower levels of PRX III. We suggest PRX III may be a suitable target for promoting deceleration or even prevention of stress-associated apoptosis in pancreatic β cells and the manifestation of insulin-dependent diabetes mellitus. PMID:20807727

  7. The emerging role of matrix metalloproteases of the ADAM family in male germ cell apoptosis

    PubMed Central

    Urriola-Muñoz, Paulina; Lagos-Cabré, Raúl

    2011-01-01

    Constitutive germ cell apoptosis during mammalian spermatogenesis is a key process for controlling sperm output and to eliminate damaged or unwanted cells. An increase or decrease in the apoptosis rate has deleterious consequences and leads to low sperm production. Apoptosis in spermatogenesis has been widely studied, but the mechanism by which it is induced under physiological or pathological conditions has not been clarified. We have recently identified the metalloprotease ADAM17 (TACE) as a putative physiological inducer of germ cell apoptosis. The mechanisms involved in regulating the shedding of the ADAM17 extracellular domain are still far from being understood, although they are important in order to understand cell-cell communications. Here, we review the available data regarding apoptosis during mammalian spermatogenesis and the localization of ADAM proteins in the male reproductive tract. We propose an integrative working model where ADAM17, p38 MAPK, protein kinase C (PKC) and the tyrosine kinase c-Abl participate in the physiological signalling cascade inducing apoptosis in germ cells. In our model, we also propose a role for the Sertoli cell in regulating the Fas/FasL system in order to induce the extrinsic pathway of apoptosis in germ cells. This working model could be applied to further understand constitutive apoptosis in spermatogenesis and in pathological conditions (e.g., varicocele) or following environmental toxicants exposure (e.g., genotoxicity or xenoestrogens). PMID:22319668

  8. α-Mangostin Induces Apoptosis and Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cell

    PubMed Central

    Kwak, Hyun-Ho; Park, Bong-Soo

    2016-01-01

    Mangosteen has long been used as a traditional medicine and is known to have antibacterial, antioxidant, and anticancer effects. Although the effects of α-mangostin, a natural compound extracted from the pericarp of mangosteen, have been investigated in many studies, there is limited data on the effects of the compound in human oral squamous cell carcinoma (OSCC). In this study, α-mangostin was assessed as a potential anticancer agent against human OSCC cells. α-Mangostin inhibited cell proliferation and induced cell death in OSCC cells in a dose- and time-dependent manner with little to no effect on normal human PDLF cells. α-Mangostin treatment clearly showed apoptotic evidences such as nuclear fragmentation and accumulation of annexin V and PI-positive cells on OSCC cells. α-Mangostin treatment also caused the collapse of mitochondrial membrane potential and the translocation of cytochrome c from the mitochondria into the cytosol. The expressions of the mitochondria-related proteins were activated by α-mangostin. Treatment with α-mangostin also induced G1 phase arrest and downregulated cell cycle-related proteins (CDK/cyclin). Hence, α-mangostin specifically induces cell death and inhibits proliferation in OSCC cells via the intrinsic apoptosis pathway and cell cycle arrest at the G1 phase, suggesting that α-mangostin may be an effective agent for the treatment of OSCC. PMID:27478478

  9. Tyrphostin AG 1296 induces glioblastoma cell apoptosis in vitro and in vivo

    PubMed Central

    LI, HONGWEI; ZHENG, JUNNING; GUAN, RUIYUN; ZHU, ZIFENG; YUAN, XIANHOU

    2015-01-01

    Glioblastoma is the most common type of malignant human brain tumor. Currently available chemotherapies for glioblastoma focus on targeting tyrosine kinases. However, the existing inhibitors of tyrosine kinases have not produced the therapeutic outcomes that were anticipated. In order to investigate the viability alternative chemotherapeutic agents in this disease, the present study examined the anticancer effects of tyrphostin AG 1296, focusing on its involvement in apoptosis in glioblastoma cells. The study aimed to identify whether tyrphostin AG 1296 affects glioblastoma cell growth by inducing cell apoptosis. To achieve this, cell viability, propidium iodide analysis and cell invasion assay were used to measure cell growth, cell apoptosis and cell migration of human glioblastoma cells. The results showed that tyrphostin AG 1296 treatment reduced cell viability and suppressed migration of human glioblastoma cells. It was also demonstrated that tyrphostin AG 1296 induced cell apoptosis in vitro. Finally, tyrphostin AG 1296 was also shown to significantly inhibit the growth of glioblastoma cells and to increase tumor cell apoptosis in vivo. These findings suggest that tyrphostin AG 1296 induces apoptosis, thereby reducing cell viability and capacity for migration of glioblastoma cells. PMID:26788146

  10. Effect of human umbilical cord mesenchymal stem cells on endometriotic cell proliferation and apoptosis.

    PubMed

    Xu, L N; Lin, N; Xu, B N; Li, J B; Chen, S Q

    2015-12-11

    The objective of this study was to observe the effects of human umbilical cord mesenchymal stem cells (UCMSCs) on the proliferation and apoptosis of endometriotic cells. Endometriotic cells and UCMSCs were primarily cultured in vitro. In the experimental group, a UCMSC and endometriotic cell non-contact co-culture system was established. The control group consisted of 1 x 10(5) endometriotic cells cultured alone. The proliferation and apoptosis of endometriotic cells were respectively detected using the MTT method and flow cytometry. The mRNA expression level of the tensin homologue gene (PTEN) in endometriotic cells was detected by reverse transcription-polymerase chain reaction amplification. Compared with the control group, the proliferation of endometriotic cells in the experimental group was clearly inhibited (P < 0.05) and time-dependent (P < 0.05). In addition, the number of apoptotic cells were significantly increased (P < 0.05), and the amount of cells, which entered S phase from G1 phase, decreased significantly. Furthermore, the mRNA expression level of the PTEN gene in the experimental group was significantly higher than in the control group (P < 0.05). These results suggest that UCMSCs might inhibit the proliferation of human endometriotic cells in vitro and promote their apoptosis by upregulating the expression of PTEN.

  11. IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells.

    PubMed

    Long, Jun; Zhang, Xulong; Wen, Mingjie; Kong, Qingli; Lv, Zhe; An, Yunqing; Wei, Xiao-Qing

    2013-01-01

    Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

  12. In situ apoptosis of adaptive immune cells and the cellular escape of rabies virus in CNS from patients with human rabies transmitted by Desmodus rotundus.

    PubMed

    Fernandes, Elaine Raniero; de Andrade, Heitor Franco; Lancellotti, Carmen Lúcia Penteado; Quaresma, Juarez Antônio Simões; Demachki, Samia; da Costa Vasconcelos, Pedro Fernando; Duarte, Maria Irma Seixas

    2011-03-01

    The aim of the current study was to investigate the apoptosis of neurons, astrocytes and immune cells from human patients that were infected with rabies virus by vampire bats bite. Apoptotic neurons were identified by their morphology and immune cells were identified using double immunostaining. There were very few apoptotic neurons present in infected tissue samples, but there was an increase of apoptotic infiltrating CD4+ and TCD8+ adaptive immune cells in the rabies infected tissue. No apoptosis was present in NK, macrophage and astrocytes. The dissemination of the human rabies virus within an infected host may be mediated by viral escape of the virus from an infected cell and may involve an anti-apoptotic mechanism, which does not kill the neuron or pro-apoptosis of TCD4+ and TCD8+ lymphocytes and which allows for increased proliferation of the virus within the CNS by attenuation of the adaptive immune response. PMID:21255623

  13. Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria.

    PubMed

    Gupta, Shelly; Prasad, G V R Krishna; Mukhopadhaya, Arunika

    2015-12-25

    Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.

  14. Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture.

    PubMed

    Bennett, M R

    1999-02-01

    Apoptosis (programmed cell death) of vascular smooth muscle cells (VSMCs) has recently been identified as an important process in a variety of human vascular diseases, including atherosclerosis, arterial injury, and restenosis after angioplasty. VSMC apoptosis is regulated by interactions between the local cell-cell and cytokine environment within the arterial wall, and the expression of pro- and anti-apoptotic proteins by the cell, including death receptors, proto-oncogenes and tumour suppressor genes. This review summarises our current knowledge of the occurrence and mechanisms underlying VSMC apoptosis in atherosclerosis and arterial remodelling.

  15. DNA damage and mitochondria dysfunction in cell apoptosis induced by nonthermal air plasma

    SciTech Connect

    Kim, G. J.; Lee, J. K.; Kim, W.; Kim, K. T.

    2010-01-11

    Nonthermal plasma is known to induce animal cell death but the mechanism is not yet clear. Here, cellular and biochemical regulation of cell apoptosis is demonstrated for plasma treated cells. Surface type nonthermal air plasma triggered apoptosis of B16F10 mouse melanoma cancer cells causing DNA damage and mitochondria dysfunction. Plasma treatment activated caspase-3, apoptosis executioner. The plasma treated cells also accumulated gamma-H2A.X, marker for DNA double strand breaks, and p53 tumor suppressor gene as a response to DNA damage. Interestingly, cytochrome C was released from mitochondria and its membrane potential was changed significantly.

  16. DNA damage and mitochondria dysfunction in cell apoptosis induced by nonthermal air plasma

    NASA Astrophysics Data System (ADS)

    Kim, G. J.; Kim, W.; Kim, K. T.; Lee, J. K.

    2010-01-01

    Nonthermal plasma is known to induce animal cell death but the mechanism is not yet clear. Here, cellular and biochemical regulation of cell apoptosis is demonstrated for plasma treated cells. Surface type nonthermal air plasma triggered apoptosis of B16F10 mouse melanoma cancer cells causing DNA damage and mitochondria dysfunction. Plasma treatment activated caspase-3, apoptosis executioner. The plasma treated cells also accumulated gamma-H2A.X, marker for DNA double strand breaks, and p53 tumor suppressor gene as a response to DNA damage. Interestingly, cytochrome C was released from mitochondria and its membrane potential was changed significantly.

  17. Gambogic acid induces apoptosis in diffuse large B-cell lymphoma cells via inducing proteasome inhibition

    PubMed Central

    Shi, Xianping; Lan, Xiaoying; Chen, Xin; Zhao, Chong; Li, Xiaofen; Liu, Shouting; Huang, Hongbiao; Liu, Ningning; Zang, Dan; Liao, Yuning; Zhang, Peiquan; Wang, Xuejun; Liu, Jinbao

    2015-01-01

    Resistance to chemotherapy is a great challenge to improving the survival of patients with diffuse large B-cell lymphoma (DLBCL), especially those with activated B-cell-like DLBCL (ABC-DLBCL). Therefore it is urgent to search for novel agents for the treatment of DLBCL. Gambogic acid (GA), a small molecule derived from Chinese herb gamboges, has been approved for Phase II clinical trial for cancer therapy by Chinese FDA. In the present study, we investigated the effect of GA on cell survival and apoptosis in DLBCL cells including both GCB- and ABC-DLBCL cells. We found that GA induced growth inhibition and apoptosis of both GCB- and ABC-DLBCL cells in vitro and in vivo, which is associated with proteasome malfunction. These findings provide significant pre-clinical evidence for potential usage of GA in DLBCL therapy particularly in ABC-DLBCL treatment. PMID:25853502

  18. Human Adipose Derived Stem Cells Induced Cell Apoptosis and S Phase Arrest in Bladder Tumor

    PubMed Central

    Yu, Xi; Su, Boxing; Ge, Peng; Wang, Zicheng; Li, Sen; Huang, Bingwei; Gong, Yanqing; Lin, Jian

    2015-01-01

    The aim of this study was to determine the effect of human adipose derived stem cells (ADSCs) on the viability and apoptosis of human bladder cancer cells. EJ and T24 cells were cocultured with ADSCs or cultured with conditioned medium of ADSCs (ADSC-CM), respectively. The cell counting and colony formation assay showed ADSCs inhibited the proliferation of EJ and T24 cells. Cell viability assessment revealed that the secretions of ADSCs, in the form of conditioned medium, were able to decrease cancer cell viability. Wound-healing assay suggested ADSC-CM suppressed migration of T24 and EJ cells. Moreover, the results of the flow cytometry indicated that ADSC-CM was capable of inducing apoptosis of T24 cells and inducing S phase cell cycle arrest. Western blot revealed ADSC-CM increased the expression of cleaved caspase-3 and cleaved PARP, indicating that ADSC-CM induced apoptosis in a caspase-dependent way. PTEN/PI3K/Akt pathway and Bcl-2 family proteins were involved in the mechanism of this reaction. Our study indicated that ADSCs may provide a promising and practicable manner for bladder tumor therapy. PMID:25691904

  19. miRNA143 Induces K562 Cell Apoptosis Through Downregulating BCR-ABL

    PubMed Central

    Liang, Bing; Song, Yanbin; Zheng, Wenling; Ma, Wenli

    2016-01-01

    Background Leukemia seriously threats human health and life. MicroRNA regulates cell growth, proliferation, apoptosis, and cell cycle. Whether microRNA could be treated as a target for leukemia is still unclear and the mechanism by which microRNA143 regulates K562 cells needs further investigation. Material/Methods miRNA143 and its scramble miRNA were synthesized and transfected to K562 cells. MTT assay was used to detect K562 cell proliferation. Flow cytometry and a caspase-3 activity detection kit were used to test K562 cell apoptosis. Western blot analysis was performed to determine breakpoint cluster region-Abelson (BCR-ABL) expression. BCR-ABL overexpression and siRNA were used to change BCR-ABL level, and cell apoptosis was detected again after lipofection transfection. Results miRNA143 transfection inhibited K562 cell growth and induced its apoptosis. miRNA143 transfection decreased BCR-ABL expression. BCR-ABL overexpression suppressed miRNA143-induced K562 cell apoptosis, while its reduction enhanced miRNA143-induced apoptosis. Conclusions miRNA143 induced K562 cell apoptosis through downregulating BCR-ABL. miRNA143 might be a target for a new leukemia therapy. PMID:27492780

  20. Ceramide-Induced Apoptosis in Renal Tubular Cells: A Role of Mitochondria and Sphingosine-1-Phoshate

    PubMed Central

    Ueda, Norishi

    2015-01-01

    Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed. PMID:25751724

  1. Hijacking Host Cell Highways: Manipulation of the Host Actin Cytoskeleton by Obligate Intracellular Bacterial Pathogens

    PubMed Central

    Colonne, Punsiri M.; Winchell, Caylin G.; Voth, Daniel E.

    2016-01-01

    Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions. PMID:27713866

  2. The Mitochondria-Mediate Apoptosis of Lepidopteran Cells Induced by Azadirachtin

    PubMed Central

    Huang, Jingfei; Lv, Chaojun; Hu, Meiying; Zhong, Guohua

    2013-01-01

    Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis. PMID:23516491

  3. Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

    SciTech Connect

    Takahara, Kiyoshi; Ii, Masaaki; Inamoto, Teruo; Komura, Kazumasa; Ibuki, Naokazu; Minami, Koichiro; Uehara, Hirofumi; Hirano, Hajime; Nomi, Hayahito; Kiyama, Satoshi; Asahi, Michio; Azuma, Haruhito

    2014-04-18

    Highlights: • AdSC transplantation exhibits inhibitory effect on tumor progressions of PCa cells. • AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway. • High expression of the TGF-β1 gene in AdSCs. - Abstract: Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.

  4. Apoptosis and inhibition of proliferation of cancer cells induced by cordycepin

    PubMed Central

    TIAN, XUEWEN; LI, YUJIAN; SHEN, YINYU; LI, QIAOQIAO; WANG, QINGLU; FENG, LIANSHI

    2015-01-01

    Cordycepin, a 3-deoxyadenosine, is the predominant functional component of the fungus Cordyceps militaris, a traditional Chinese medicine. Previous studies investigating the inhibition of cancer cells by cordycepin identified that it not only promotes cell apoptosis, but also controls cell proliferation. Furthermore, studies have elucidated the molecular mechanisms of inhibiting cell proliferation by cordycepin binding the A3 adenosine receptor, activating G protein, inhibiting cAMP formation, decreasing glycogen synthase kinase-3β/β-catenin activation and suppressing cyclin D1 and c-myc expression. The most significant signaling pathway in which cell apoptosis is induced by cordycepin is the caspase pathway. Cordycepin induces cell apoptosis via binding the DR3 receptor and consequently activating caspase-8/-3. Taken together, these studies demonstrate that cordycepin may be used as a natural medicine, as it can not only control tumor cell proliferation, but also induce cancer cell apoptosis. PMID:26622539

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

    PubMed

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

    2015-09-25

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

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

    PubMed Central

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

    2015-01-01

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

  7. Coordinated Regulation of Apoptosis and Cell Proliferation by Transforming Growth Factor β1 in Cultured Uterine Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Rotello, Rocco J.; Lieberman, Rita C.; Purchio, Anthony F.; Gerschenson, Lazaro E.

    1991-04-01

    Cell and tissue growth is regulated through a complex interplay of stimulatory and inhibitory signals. We describe two biological actions of transforming growth factor β 1 (TGF-β 1) in primary cultures of rabbit uterine epithelial cells: (i) inhibition of cell proliferation and (ii) a concomitant increase in cells undergoing apoptosis (programmed cell death). It is proposed that proliferation and apoptosis together comprise normal cell growth regulation.

  8. Host epithelial geometry regulates breast cancer cell invasiveness

    PubMed Central

    Boghaert, Eline; Gleghorn, Jason P.; Lee, KangAe; Gjorevski, Nikolce; Radisky, Derek C.; Nelson, Celeste M.

    2012-01-01

    Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment. PMID:23150585

  9. Herpesvirus Genome Integration into Telomeric Repeats of Host Cell Chromosomes.

    PubMed

    Osterrieder, Nikolaus; Wallaschek, Nina; Kaufer, Benedikt B

    2014-11-01

    It is well known that numerous viruses integrate their genetic material into host cell chromosomes. Human herpesvirus 6 (HHV-6) and oncogenic Marek's disease virus (MDV) have been shown to integrate their genomes into host telomeres of latently infected cells. This is unusual for herpesviruses as most maintain their genomes as circular episomes during the quiescent stage of infection. The genomic DNA of HHV-6, MDV, and several other herpesviruses harbors telomeric repeats (TMRs) that are identical to host telomere sequences (TTAGGG). At least in the case of MDV, viral TMRs facilitate integration into host telomeres. Integration of HHV-6 occurs not only in lymphocytes but also in the germline of some individuals, allowing vertical virus transmission. Although the molecular mechanism of telomere integration is poorly understood, the presence of TMRs in a number of herpesviruses suggests it is their default program for genome maintenance during latency and also allows efficient reactivation.

  10. Alterations of host cell ubiquitination machinery by pathogenic bacteria

    PubMed Central

    Alomairi, Jaafar; Bonacci, Thomas; Ghigo, Eric; Soubeyran, Philippe

    2015-01-01

    Response of immune and non-immune cells to pathogens infections is a very dynamic process. It involves the activation/modulation of many pathways leading to actin remodeling, membrane engulfing, phagocytosis, vesicle trafficking, phagolysosome formation, aiming at the destruction of the intruder. These sophisticated and rapid mechanisms rely on post-translational modifications (PTMs) of key host cells' factors, and bacteria have developed various strategies to manipulate them to favor their survival. Among these important PTMs, ubiquitination has emerged as a major mediator/modulator/regulator of host cells response to infections that pathogens have also learned to use for their own benefit. In this mini-review, we summarize our current knowledge about the normal functions of ubiquitination during host cell infection, and we detail its hijacking by model pathogens to escape clearance and to proliferate. PMID:25774357

  11. Viroporins Customize Host Cells for Efficient Viral Propagation

    PubMed Central

    Giorda, Kristina M.

    2013-01-01

    Viruses are intracellular parasites that must access the host cell machinery to propagate. Viruses hijack the host cell machinery to help with entry, replication, packaging, and release of progeny to infect new cells. To carry out these diverse functions, viruses often transform the cellular environment using viroporins, a growing class of viral-encoded membrane proteins that promote viral proliferation. Viroporins modify the integrity of host membranes, thereby stimulating the maturation of viral infection, and are critical for virus production and dissemination. Significant advances in molecular and cell biological approaches have helped to uncover some of the roles that viroporins serve in the various stages of the viral life cycle. In this study, the ability of viroporins to modify the cellular environment will be discussed, with particular emphasis on their role in the stepwise progression of the viral life cycle. PMID:23945006

  12. Par-4/NF-κB Mediates the Apoptosis of Islet β Cells Induced by Glucolipotoxicity.

    PubMed

    QiNan, Wu; XiaGuang, Gan; XiaoTian, Lei; WuQuan, Deng; Ling, Zhang; Bing, Chen

    2016-01-01

    Apoptosis of islet β cells is a primary pathogenic feature of type 2 diabetes, and ER stress and mitochondrial dysfunction play important roles in this process. Previous research has shown that prostate apoptosis response-4 (Par-4)/NF-κB induces cancer cell apoptosis through endoplasmic reticulum (ER) stress and mitochondrial dysfunction. However, the mechanism by which Par-4/NF-κB induces islet β cell apoptosis remains unknown. We used a high glucose/palmitate intervention to mimic type 2 diabetes in vitro. We demonstrated that the high glucose/palmitate intervention induced the expression and secretion of Par-4. It also causes increased expression and activation of NF-κB, which induced NIT-1 cell apoptosis and dysfunction. Overexpression of Par-4 potentiates these effects, whereas downregulation of Par-4 attenuates them. Inhibition of NF-κB inhibited the Par-4-induced apoptosis. Furthermore, these effects occurred through the ER stress cell membrane and mitochondrial pathway of apoptosis. Our findings reveal a novel role for Par-4/NF-κB in islet β cell apoptosis and type 2 diabetes. PMID:27340675

  13. Actin reorganization as the molecular basis for the regulation of apoptosis in gastrointestinal epithelial cells

    PubMed Central

    Wang, Y; George, S P; Srinivasan, K; Patnaik, S; Khurana, S

    2012-01-01

    The gastrointestinal (GI) epithelium is a rapidly renewing tissue in which apoptosis represents part of the overall homeostatic process. Regulation of apoptosis in the GI epithelium is complex with a precise relationship between cell position and apoptosis. Apoptosis occurs spontaneously and in response to radiation and cytotoxic drugs at the base of the crypts. By contrast, the villus epithelial cells are extremely resistant to apoptosis. The molecular mechanism underlying this loss of function of villus epithelial cells to undergo apoptosis shortly after their exit from the crypt is unknown. In this study we demonstrate for the first time, that deletion of two homologous actin-binding proteins, villin and gelsolin renders villus epithelial cells extremely sensitive to apoptosis. Ultrastructural analysis of the villin-gelsolin−/− double-knockout mice shows an abnormal accumulation of damaged mitochondria demonstrating that villin and gelsolin function on an early step in the apoptotic signaling at the level of the mitochondria. A characterization of functional and ligand-binding mutants demonstrate that regulated changes in actin dynamics determined by the actin severing activities of villin and gelsolin are required to maintain cellular homeostasis. Our study provides a molecular basis for the regulation of apoptosis in the GI epithelium and identifies cell biological mechanisms that couple changes in actin dynamics to apoptotic cell death. PMID:22421965

  14. Lipopolysaccharide Stimulates Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kurita-Ochiai, Tomoko; Fukushima, Kazuo; Ochiai, Kuniyasu

    1999-01-01

    We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3+-T-cell apoptosis followed by similar levels of both CD4+- and CD8+-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3+ PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4+ and CD8+ cells. PMID:9864191

  15. PF-04691502 triggers cell cycle arrest, apoptosis and inhibits the angiogenesis in hepatocellular carcinoma cells.

    PubMed

    Wang, Feng-Ze; Peng-Jiao; Yang, Na-Na; Chuang-Yuan; Zhao, Ya-Li; Liu, Qiang-Qiang; Fei, Hong-Rong; Zhang, Ji-Guo

    2013-07-01

    Hepatocellular carcinoma (HCC) is a major cause of morbidity and mortality in the world. The aim of the present study is to determine the antitumor effect of PF-04691502, a potent inhibitor of PI3K and mTOR kinases, on the apoptosis and angiogenesis of the hepatoma cancer cells. Our results indicate that treatment of cancer cells with PF-04691502 reduces cell viability and inhibits cell growth in a dose-dependent manner. PF-04691502 triggers apoptosis via a mitochondrial pathway, accompanied by activation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). Pre-treatment of hepatoma cells with the caspase-3 inhibitor (z-DEVD-fmk) blocks the PF-04691502-induced death of these cells. In addition, growth factors-induced tube formation and the migration of HUVECs are markedly inhibited by PF-04691502 treatment. The mechanisms of anti-angiogenesis of PF-04691502 are associated with inhibiting the expression of VEGF and HIF-1α. Based on the overall results, we suggest that PF-04691502 reduces hepatocellular carcinoma cell viability, induces cell apoptosis, and inhibits cell growth and tumor angiogenesis, implicating its potential therapeutic value in the treatment of HCC.

  16. Solena amplexicaulis induces cell cycle arrest, apoptosis and inhibits angiogenesis in hepatocarcinoma cells and HUVECs.

    PubMed

    Ren, Jie; Xu, Yuan Yuan; Jiang, He Fei; Yang, Meng; Huang, Qian Hui; Yang, Jie; Hu, Kun; Wei, Kun

    2014-01-01

    Solena amplexicaulis (Lam.) Gandhi (SA) has been used as a traditional medicine for the treatment of dysentery, multiple abscess, gastralgia, urethritis, and eczema in the minority area of China. This study was aimed to examine the cell proliferation inhibitory activity of the SA extract (SACE) and its mechanism of action in human hepatoma cell line (HepG2) and evaluate its anti-angiogenesis activity in human umbilical vein endothelial cell line (HUVEC). SACE could inhibit the growth of HepG2 cells in a dose- and time-dependent manner. FCM analysis showed that SACE could induce G2/M phase arrest, cell apoptosis, the mitochondrial membrane potential loss (ΔΨm) and increase the production of intracellular ROS of HepG2 cells. After treatment with SACE, topical morphological changes of apoptotic body formation, obvious increase of apoptosis-related protein expressions, such as Bax, cytochrome c, caspase-3, PARP-1, and decrease of Bcl-2, procaspase-9 protein expressions were observed at the same time. Moreover, SACE caused the significant inhibition of endothelial cell migration and tube formation in HUVEC cells. The results suggested that SACE could act as an angiogenesis inhibitor and induce cell apoptosis via a caspase-dependent mitochondrial pathway. Therefore, SACE could be a potent candidate for the prevention and treatment of liver cancer. PMID:25547924

  17. Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

    SciTech Connect

    Neel, Benjamin D.; Gillet, Germain . E-mail: g.gillet@ibcp.fr

    2005-11-15

    Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60 {sup v-src} tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60 {sup v-src}, we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60 {sup v-src} inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60 {sup v-src} inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.

  18. Suppression of PAX6 promotes cell proliferation and inhibits apoptosis in human retinoblastoma cells.

    PubMed

    Meng, Bo; Wang, Yisong; Li, Bin

    2014-08-01

    The aim of this study was to investigate the role of the transcription factor, PAX6, in the development of retinoblastoma. The expression of endogenous PAX6 was knocked down using PAX6-specific lentivirus in two human retinoblastoma cell lines, SO-Rb50 and Y79. Cell proliferation functional assays and apoptotic assays were performed on the cells in which PAX6 was knocked down. The results revealed that PAX6 knockdown efficiency was significant (P<0.01, n=3) in the SO-Rb50 and Y79 cells. The inhibition of PAX6 reduced tumor cell apoptosis (P<0.05, n=3), but induced cell cycle S phase arrest (SO-Rb50; P<0.05, n=3) and G2/M phase arrest (Y79; P<0.05, n=3). Western blot analysis indicated that the inhibition of PAX6 increased the levels of the anti-apoptotic proteins, Bcl-2, proliferating cell nuclear antigen (PCNA) and CDK1, but reduced the levels of the pro-apoptotic proteins, BAX and p21. In conclusion, our data demonstrate that the suppression of PAX6 increases proliferation and decreases apoptosis in human retinoblastoma cells by regulating several cell cycle and apoptosis biomarkers. PMID:24939714

  19. Suppression of PAX6 promotes cell proliferation and inhibits apoptosis in human retinoblastoma cells

    PubMed Central

    MENG, BO; WANG, YISONG; LI, BIN

    2014-01-01

    The aim of this study was to investigate the role of the transcription factor, PAX6, in the development of retinoblastoma. The expression of endogenous PAX6 was knocked down using PAX6-specific lentivirus in two human retinoblastoma cell lines, SO-Rb50 and Y79. Cell proliferation functional assays and apoptotic assays were performed on the cells in which PAX6 was knocked down. The results revealed that PAX6 knockdown efficiency was significant (P<0.01, n=3) in the SO-Rb50 and Y79 cells. The inhibition of PAX6 reduced tumor cell apoptosis (P<0.05, n=3), but induced cell cycle S phase arrest (SO-Rb50; P<0.05, n=3) and G2/M phase arrest (Y79; P<0.05, n=3). Western blot analysis indicated that the inhibition of PAX6 increased the levels of the anti-apoptotic proteins, Bcl-2, proliferating cell nuclear antigen (PCNA) and CDK1, but reduced the levels of the pro-apoptotic proteins, BAX and p21. In conclusion, our data demonstrate that the suppression of PAX6 increases proliferation and decreases apoptosis in human retinoblastoma cells by regulating several cell cycle and apoptosis biomarkers. PMID:24939714

  20. Zinc Induces Apoptosis of Human Melanoma Cells, Increasing Reactive Oxygen Species, p53 and FAS Ligand.

    PubMed

    Provinciali, Mauro; Pierpaoli, Elisa; Bartozzi, Beatrice; Bernardini, Giovanni

    2015-10-01

    The aim of this study was to examine the in vitro effect of zinc on the apoptosis of human melanoma cells, by studying the zinc-dependent modulation of intracellular levels of reactive oxygen species (ROS) and of p53 and FAS ligand proteins. We showed that zinc concentrations ranging from 33.7 μM to 75 μM Zn(2+) induced apoptosis in the human melanoma cell line WM 266-4. This apoptosis was associated with an increased production of intracellular ROS, and of p53 and FAS ligand protein. Treatment of tumor cells with the antioxidant N-acetylcysteine was able to prevent Zn(2+)-induced apoptosis, as well as the increase of p53 and FAS ligand protein induced by zinc. Zinc induces apoptosis in melanoma cells by increasing ROS and this effect may be mediated by the ROS-dependent induction of p53 and FAS/FAS ligand.

  1. Wavelength-dependent backscattering measurements for quantitative monitoring of apoptosis, Part 1: early and late spectral changes are indicative of the presence of apoptosis in cell cultures

    NASA Astrophysics Data System (ADS)

    Mulvey, Christine S.; Zhang, Kexiong; Liu, Wei-Han Bobby; Waxman, David J.; Bigio, Irving J.

    2011-11-01

    Apoptosis, a form of programmed cell death with unique morphological and biochemical features, is dysregulated in cancer and is activated by many cancer chemotherapeutic drugs. Noninvasive assays for apoptosis in cell cultures can aid in screening of new anticancer agents. We have previously demonstrated that elastic scattering spectroscopy can monitor apoptosis in cell cultures. In this report we present data on monitoring the detailed time-course of scattering changes in a Chinese hamster ovary (CHO) and PC-3 prostate cancer cells treated with staurosporine to induce apoptosis. Changes in the backscattering spectrum are detectable within 10 min, and continue to progress up to 48 h after staurosporine treatment, with the magnitude and kinetics of scattering changes dependent on inducer concentration. Similar responses were observed in CHO cells treated with several other apoptosis-inducing protocols. Early and late scattering changes were observed under conditions shown to induce apoptosis via caspase activity assay and were absent under conditions where apoptosis was not induced. Finally, blocking caspase activity and downstream apoptotic morphology changes prevented late scattering changes. These observations demonstrate that early and late changes in wavelength-dependent backscattering correlate with the presence of apoptosis in cell cultures and that the late changes are specific to apoptosis.

  2. Absence of pRb facilitates E2F1-induced apoptosis in breast cancer cells.

    PubMed

    Sun, Baohua; Wingate, Hannah; Swisher, Stephen G; Keyomarsi, Khandan; Hunt, Kelly K

    2010-03-15

    The transcription factor E2F1 is known for its interaction with pRb, controlling cell proliferation; however, E2F1 also has a pivotal role in regulating apoptosis.  The relationship between pRb and E2F1 balances cell proliferation and apoptosis giving pRb tumor suppressive properties. The intricacies of the pRb/E2F1 relationship and thus the regulation of cell fate is cell context dependent. To explore the role of pRb in the E2F1-induced apoptosis of human breast cancer cells, we examined cell growth and apoptosis induction in isogenic cell systems of immortalized breast epithelial cells lacking either pRb (76NE7) or p53 (76NE6). We found that E2F1 caused accumulation of cells in G2 and S phases of the cell cycle along with apoptosis in 76NE7 but not 76NE6 cells.  Variants of 76NE6 cells with functional p53 did not rescue the apoptotic response in these cells, whereas knocking down pRb resulted in significant E2F1-induced apoptosis. We also determined that the effect of E2F1 overexpression in two breast cancer cell lines, MDA-MB-436 and MDA-MB-468, which lack pRb and functional p53, was accumulation of cells in G2/S phase and apoptosis. However, E2F did not cause apotosis  in MCF-7 cells which harbor a functional pRb. Therefore, we conclude that in the absence of Rb, E2F1 overexpression results in apoptosis, not proliferation, and that this effect is independent of p53.

  3. Mast Cell Stabilization Improves Survival by Preventing Apoptosis in Sepsis

    PubMed Central

    Ramos, Laura; Peña, Geber; Cai, Bolin; Deitch, E. A.; Ulloa, Luis

    2011-01-01

    Inhibiting single cytokines produced modest effects in clinical trials, in part because the cytokines werenot specific for sepsis, and sepsis may require cellular strategies. Previous studies reported that mast cells (MCs) fight infections in early sepsis. In this study, we report that MC stabilizers restrain serum TNF levels and improve survival in wild-type but not in MC-deficient mice. Yet, MC depletion in knockout mice attenuates serum TNF but does not improve survival in sepsis. Serum HMGB1 was the only factor correlating with survival. MC stabilizers inhibit systemic HMGB1 levels and rescue mice from established peritonitis. MC stabilizers fail to inhibit HMGB1 secretion from macrophages, but they prevent apoptosis and caspase-3 activation in sepsis. These results suggest that MC stabilization provides therapeutic benefits in sepsis by inhibiting extracellular release of HMGB1 from apoptotic cells. Our study provides the first evidence that MCs have major immunological implications regulating cell death in sepsis and represent a pharmacological target for infectious disorders in a clinically realistic time frame. PMID:20519642

  4. Calpeptin Attenuated Apoptosis and Intracellular Inflammatory Changes in Muscle Cells

    PubMed Central

    Nozaki, Kenkichi; Das, Arabinda; Ray, Swapan K.; Banik, Naren L.

    2011-01-01

    In idiopathic inflammatory myopathies (IIMs), extracellular inflammatory stimulation is considered to induce secondary intracellular inflammatory changes including expression of major histocompatibility complex class-I (MHC-I) and to produce self-sustaining loop of inflammation. We hypothesize that activation of calpain, a Ca2+-sensitive protease, bridges between these extracellular inflammatory stress and intracellular secondary inflammatory changes in muscle cells. In this study, we demonstrated that treatment of rat L6 myoblast cells with interferon-gamma (IFN-γ) caused expression of MHC-I and inflammation related transcription factors (phosphorylated-extracellular signal-regulated kinase 1/2 and nuclear factor-kappa B). We also demonstrated that treatment with tumor necrosis factor-alpha (TNF-α) induced apoptotic changes and activation of calpain and cyclooxygenase-2. Further, we found that post-treatment with calpeptin attenuated the intracellular changes induced by IFN-γ or TNF-α. Our results indicate that calpain inhibition attenuates apoptosis and secondary inflammatory changes induced by extracellular inflammatory stimulation in the muscle cells. These results suggest calpain as a potential therapeutic target for treatment of IIMs. PMID:21290412

  5. [Ribonuclease binase induces death in T-cell acute lymphoblastic leukemia cells by apoptosis].

    PubMed

    Burnysheva, K M; Petrushanko, I Yu; Spirin, P V; Prassolov, V S; Makarov, A A; Mitkevich, V A

    2016-01-01

    Bacterial ribonuclease binase is a potential anticancer agent. In the present study, we have determined the toxic effect of binase towards cell lines of T-cell acute lymphoblastic leukemia Jurkat and CEMss. We have shown that binase induces apoptosis in these cells. At the same time, binase does not cause toxic effects in leukocytes of healthy donors, which suggests that binase activity towards leukemic cells is selective. We have found that the treatment of cancer cells with binase leads to a reduction in reactive oxygen species and transcription factor NFκB levels, and demonstrated that these effects are a common feature of the action of RNases on cancer cells.

  6. Aquatic viruses induce host cell death pathways and its application.

    PubMed

    Reshi, Latif; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-01-01

    Virus infections of mammalian and animal cells consist of a series of events. As intracellular parasites, viruses rely on the use of host cellular machinery. Through the use of cell culture and molecular approaches over the past decade, our knowledge of the biology of aquatic viruses has grown exponentially. The increase in aquaculture operations worldwide has provided new approaches for the transmission of aquatic viruses that include RNA and DNA viruses. Therefore, the struggle between the virus and the host for control of the cell's death machinery is crucial for survival. Viruses are obligatory intracellular parasites and, as such, must modulate apoptotic pathways to control the lifespan of their host to complete their replication cycle. This paper updates the discussion on the detailed mechanisms of action that various aquatic viruses use to induce cell death pathways in the host, such as Bad-mediated, mitochondria-mediated, ROS-mediated and Fas-mediated cell death circuits. Understanding how viruses exploit the apoptotic pathways of their hosts may provide great opportunities for the development of future potential therapeutic strategies and pathogenic insights into different aquatic viral diseases.

  7. Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells

    PubMed Central

    Pant, Kishor; Yadav, Ajay K.; Gupta, Parul; Rathore, Abhishek Singh; Nayak, Baibaswata; Venugopal, Senthil K.

    2016-01-01

    Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 μM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and β-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 μM and 5-fold with 100 μM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells. PMID:27708347

  8. Curcumin-induced recovery from hepatic injury involves induction of apoptosis of activated hepatic stellate cells.

    PubMed

    Priya, S; Sudhakaran, P R

    2008-10-01

    Hepatic stellate cells (HSCs) undergo activation and transdifferentiation to myofibroblast like cells in liver injury, leading to liver fibrosis. During recovery from injury, activated HSCs may either revert back to quiescent state or undergo apoptosis or both. In the present study, we have examined whether recovery from hepatic injury involves apoptosis of activated HSCs and tested whether curcumin (the yellow pigment from Curcuma longa Linn.) promotes recovery from hepatic injury by inducing apoptosis of these cells. Hepatic injury was induced by CCl4 and apoptosis was studied in HSCs isolated from liver by MTT assay, DNA fragmentation, and DAPI and annexin staining. Hepatic recovery was assessed by measuring hepatic marker activities, such as serum GOT, GPT and protein. Hepatic recovery occurred within 4 weeks after inducing injury in untreated control, whereas curcumin treatment caused hepatic recovery within 2 weeks, as evidenced by the reduction of hepatic marker activities to near normal levels. HSCs isolated from liver of animals treated with curcumin showed maximum apoptotic marker activities in 2nd week, whereas in HSCs from untreated control recovering from injury, maximum apoptosis was observed in 4th week. Induction of apoptosis in vivo during hepatic recovery was also suggested by increase in caspase-3 activity. Treatment of isolated HSCs in culture with curcumin caused apoptosis during later stages confirming that curcumin induced apoptosis of activated HSCs and not in unactivated quiescent HSCs. These results suggested that hepatoprotective effect of curcumin causing recovery from injury involved apoptosis of activated HSCs. PMID:19069843

  9. Adhesion and host cell modulation: critical pathogenicity determinants of Bartonella henselae.

    PubMed

    Franz, Bettina; Kempf, Volkhard A J

    2011-04-13

    Bartonella henselae, the agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis, contains to date two groups of described pathogenicity factors: adhesins and type IV secretion systems. Bartonella adhesin A (BadA), the Trw system and possibly filamentous hemagglutinin act as promiscous or specific adhesins, whereas the virulence locus (Vir)B/VirD4 type IV secretion system modulates a variety of host cell functions. BadA mediates bacterial adherence to endothelial cells and extracellular matrix proteins and triggers the induction of angiogenic gene programming. The VirB/VirD4 type IV secretion system is responsible for, e.g., inhibition of host cell apoptosis, bacterial persistence in erythrocytes, and endothelial sprouting. The Trw-conjugation system of Bartonella spp. mediates host-specific adherence to erythrocytes. Filamentous hemagglutinins represent additional potential pathogenicity factors which are not yet characterized. The exact molecular functions of these pathogenicity factors and their contribution to an orchestral interplay need to be analyzed to understand B. henselae pathogenicity in detail.

  10. Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells

    PubMed Central

    Vellasamy, Kumutha Malar; Mariappan, Vanitha; Shankar, Esaki M.; Vadivelu, Jamuna

    2016-01-01

    Background Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. Methods We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). Results We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. Conclusion Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections. PMID:27367858

  11. Inhibition of the cell cycle and induction of apoptosis by Noclosan and Loxat in cell lines irrespective of their origin.

    PubMed

    Schutte, B; Harmsa, M; Costongs, G; Henfling, M; Ummelen, M; Dignef, W; Ramaekers, F

    2004-06-01

    The effects of Noclosan and Loxat, extracts from the potato tuber, on cancer cell lines and human endothelial cells in culture were examined with respect to cell cycle inhibition and apoptosis induction. Both components effect the cell cycle of the cancer cell lines, most likely by inhibition of the M to G1-phase transition. Furthermore, both compounds very effectively induced apoptosis in a dose-dependent manner. Strikingly, combination of both compounds revealed a synergistic effect, that can be explained by the observation that induction of apoptosis occurs through both the mitochondrial and the non-mitochondrial route. Preliminary studies suggest that the components affect the SAPK/JNK cell signaling pathway.

  12. Confocal Raman data analysis enables identifying apoptosis of MCF-7 cells caused by anticancer drug paclitaxel

    NASA Astrophysics Data System (ADS)

    Salehi, Hamideh; Middendorp, Elodie; Panayotov, Ivan; Dutilleul, Pierre-Yves Collard; Vegh, Attila-Gergely; Ramakrishnan, Sathish; Gergely, Csilla; Cuisinier, Frederic

    2013-05-01

    Confocal Raman microscopy is a noninvasive, label-free imaging technique used to study apoptosis of live MCF-7 cells. The images are based on Raman spectra of cells components, and their apoptosis is monitored through diffusion of cytochrome c in cytoplasm. K-mean clustering is used to identify mitochondria in cells, and correlation analysis provides the cytochrome c distribution inside the cells. Our results demonstrate that incubation of cells for 3 h with 10 μM of paclitaxel does not induce apoptosis in MCF-7 cells. On the contrary, incubation for 30 min at a higher concentration (100 μM) of paclitaxel induces gradual release of the cytochrome c into the cytoplasm, indicating cell apoptosis via a caspase independent pathway.

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

    PubMed Central

    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

  14. Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells.

    PubMed

    Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

    2015-01-09

    To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting.

  15. Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

    PubMed Central

    Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

    2015-01-01

    To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting. PMID:25571970

  16. Antiproliferative and cell apoptosis-inducing activities of compounds from Buddleja davidii in Mgc-803 cells

    PubMed Central

    2012-01-01

    Background Buddleja davidii is widely distributed in the southwestern region of China. We have undertaken a systematic analysis of B. davidii as a Chinese traditional medicine with anticancer activity by isolating natural products for their activity against the human gastric cancer cell line Mgc-803 and the human breast cancer cell line Bcap-37. Results Ten compounds were extracted and isolated from B. davidii, among which colchicine was identified in B. davidii for the first time. The inhibitory activities of these compounds were investigated in Mgc-803, Bcap-37 cells in vitro by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and the results showed that luteolin and colchicine had potent inhibitory activities against the growth of Mgc-803 cells. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in Mgc-803 cells. The results also showed that the percentages of early apoptotic cells (Annexin V+/PI-, where PI is propidium iodide) and late apoptotic cells (Annexin V+/PI+) increased in a dose- and time-dependent manner. After 36 h of incubation with luteolin at 20 μM, the percentages of cells were approximately 15.4% in early apoptosis and 43.7% in late apoptosis; after 36 h of incubation with colchicine at 20 μM, the corresponding values were 7.7% and 35.2%, respectively. Conclusions Colchicine and luteolin from B. davidii have potential applications as adjuvant therapies for treating human carcinoma cells. These compounds could also induce apoptosis in tumor cells. PMID:22938042

  17. Vincristine induces cell cycle arrest and apoptosis in SH-SY5Y human neuroblastoma cells.

    PubMed

    Tu, Yue; Cheng, Shixiang; Zhang, Sai; Sun, Hongtao; Xu, Zhongwei

    2013-01-01

    Neuroblastoma is a common childhood tumor. Vincristine (VCR), an alkaloid extracted from Catharanthus roseus, is commonly used in combination chemotherapy. However, the mechanisms of VCR-induced neuroblastoma cell death are not clear. The aim of this study was to investigate the impact of VCR on mitosis and apoptosis of SH-SY5Y neuroblastoma cells and the underlying mechanisms. SH-SY5Y cells were treated with increasing VCR doses for different time points. Cell proliferation was detected using the MTT assay. Mitotic rate was quantified by immunofluorescence. Cell cycle and apoptosis were analyzed by flow cytometry. The mRNA and protein expression of caspase-3 and -9 (apoptotic factors), as well as cyclin B and D (cell cycle factors), was evaluated by real-time (RT)-PCR and western blot analysis, respectively. VCR inhibited SH-SY5Y cell proliferation in a time- and dose-dependent manner (P<0.05). The IC50 of VCR in SH-SY5Y cells was determined as 0.1 µM. VCR at 0.1 µM induced mitotic arrest and apoptosis, promoted the expression of caspase-3 and -9 and cyclin B, while decreasing the expression of cyclin D at 6, 12, 18 and 24 h. Except for the mRNA expression of cyclin D at 6 h, these changes were significant at both the mRNA and protein levels (P<0.05). VCR induces mitotic arrest of SH-SY5Y cells by regulating cyclin B and D. It further induces apoptosis in these cells through the activation of caspase-3 and -9. This study provides fundamental evidence for the application of VCR in neuroblastoma chemotherapy.

  18. Apoptosis as a mechanism of cytolysis of tumor cells by a pathogenic free-living amoeba.

    PubMed

    Alizadeh, H; Pidherney, M S; McCulley, J P; Niederkorn, J Y

    1994-04-01

    Previous studies have shown that trophozoites of the pathogenic free-living amoeba Acanthamoeba castellanii rapidly lysed a variety of tumor cells in vitro. Tumor cells undergoing parasite-mediated lysis displayed characteristic cell membrane blebbing reminiscent of apoptosis. The present investigation examined the role of apoptosis (programmed cell death) in Acanthamoeba-mediated tumor cell lysis. The results showed that more than 70% of tumor cell DNA was fragmented following exposure to Acanthamoeba cell extracts. By contrast, only 7% of untreated control cells underwent DNA fragmentation. DNA fragmentation increased significantly in a dose-dependent fashion following concentration of the parasite extract. Apoptosis was also confirmed by DNA ladder formation. Characteristic DNA ladders, consisting of multimers of approximately 180 to 200 bp, were produced by tumor cells exposed to Acanthamoeba cell extracts. The morphology of tumor cell lysis was examined by light and scanning electron microscopy. Tumor cells exposed to parasite extract displayed morphological features characteristic of apoptosis including cell shrinkage, cell membrane blebbing, formation of apoptotic bodies, and nuclear condensation. By contrast, similar effects were not found in tumor cells exposed to extract similarly prepared from normal mammalian cells (i.e., human keratocytes). The results suggest that at least one species of pathogenic free-living amoeba is able to lyse tumor cells by a process that culminates in apoptosis. PMID:8132336

  19. Signaling between tumor cells and the host bone marrow microenvironment.

    PubMed

    Kovacic, Natasa; Croucher, Peter I; McDonald, Michelle M

    2014-01-01

    Tumor cells with high skeletal homing affinity express numerous cell surface receptors that bind ligands produced in bone. Upon arrival, these cells survive in the host environment, encompassed in close proximity to bone marrow cells. Interactions between tumor cells and cells of the host microenvironment are essential to not only tumor cell survival but also their activation and proliferation into environment-modifying tumors. Through the production of RANKL, PTHrP, cytokines, and integrins, activated tumor cells stimulate osteoclastogenesis, enhance bone resorption, and subsequently release matrix-bound proteins that further promote tumor growth and bone resorption. In addition, alterations in the TGF-β/BMP and Wnt signaling pathways via tumor cell growth can either stimulate or suppress osteoblastic bone formation and function, leading to sclerotic or lytic bone disease, respectively. Hence, the presence of tumor cells in bone dysregulates bone remodeling, dramatically impairing skeletal integrity. Furthermore, through complex mechanisms, cells of the immune system interact with tumor cells to further impact bone remodeling. Lastly, with alterations in bone cell activity, the environment is permissive to promoting tumor growth further, suggesting an interdependence between tumor cells and bone cells in metastatic bone disease and multiple myeloma.

  20. Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    PubMed Central

    Gan, Huachen; Hao, Qin; Idell, Steven; Tang, Hua

    2015-01-01

    Influenza A virus (IAV) targets airway epithelial cells and exploits the host cell machinery to replicate, causing respiratory illness in annual epidemics and pandemics of variable severity. The high rate of antigenic drift (viral mutation) and the putative antigenic shift (reassortant strains) have raised the need to find the host cell inducible factors modulating IAV replication and its pathogenesis to develop more effective antiviral treatment. In this study, we found for the first time that transcription factor Runx3, a developmental regulator and tumor suppressor, was induced by IAV H1N1 and H3N2, viral RNA, a synthetic analog of viral double-stranded RNA (dsRNA) polyinosinic-polycytidylic acid, and type-II interferon-γ (IFNγ) in human airway epithelial cells. Whereas Runx3 was essentially not induced by type-I IFNα and type-III IFNλ, we show that Runx3 induction by IAV infection and viral RNA is mediated through the innate immune receptor MDA5 and the IκB kinase-β−NF-κB pathway. Moreover, we provide substantial evidence indicating that Runx3 plays a crucial role in airway epithelial cell apoptosis induced by IAV infection and dsRNA through the activation of extrinsic and intrinsic apoptosis pathways. Thus, we have identified Runx3 as an inducible and important transcription factor modulating IAV-induced host epithelial cell apoptosis. PMID:26643317

  1. Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis.

    PubMed

    Gan, Huachen; Hao, Qin; Idell, Steven; Tang, Hua

    2015-01-01

    Influenza A virus (IAV) targets airway epithelial cells and exploits the host cell machinery to replicate, causing respiratory illness in annual epidemics and pandemics of variable severity. The high rate of antigenic drift (viral mutation) and the putative antigenic shift (reassortant strains) have raised the need to find the host cell inducible factors modulating IAV replication and its pathogenesis to develop more effective antiviral treatment. In this study, we found for the first time that transcription factor Runx3, a developmental regulator and tumor suppressor, was induced by IAV H1N1 and H3N2, viral RNA, a synthetic analog of viral double-stranded RNA (dsRNA) polyinosinic-polycytidylic acid, and type-II interferon-γ (IFNγ) in human airway epithelial cells. Whereas Runx3 was essentially not induced by type-I IFNα and type-III IFNλ, we show that Runx3 induction by IAV infection and viral RNA is mediated through the innate immune receptor MDA5 and the IκB kinase-β-NF-κB pathway. Moreover, we provide substantial evidence indicating that Runx3 plays a crucial role in airway epithelial cell apoptosis induced by IAV infection and dsRNA through the activation of extrinsic and intrinsic apoptosis pathways. Thus, we have identified Runx3 as an inducible and important transcription factor modulating IAV-induced host epithelial cell apoptosis. PMID:26643317

  2. Anti-hepatoma cells function of luteolin through inducing apoptosis and cell cycle arrest.

    PubMed

    Ding, Shixiong; Hu, Airong; Hu, Yaoren; Ma, Jianbo; Weng, Pengjian; Dai, Jinhua

    2014-04-01

    The aim of this study is to explore the apoptotic induction and cell cycle arrest function of luteolin on the liver cancer cells and the related mechanism. The liver cancer cell line SMMC-7721, BEL-7402, and normal liver cells HL-7702 were treated with different concentrations of luteolin. Cell proliferation ability was tested. Morphological changes of the apoptotic cells were observed under inverted fluorescence microscope after Hoechst33342 staining. We investigated the effect of luteolin on cell cycling and apoptosis with flow cytometry. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Caspases-3 and Bcl-2 family proteins expression were analyzed by real-time PCR. Cell proliferation of SMMC-7721 and BEL-7402 were inhibited by luteolin, and the inhibition was dose-time-dependent. Luteolin could arrest the cells at G1/S stage, reduce mitochondrial membrane potential, and induce higher apoptosis rate and the typical apoptotic morphological changes of the liver carcinoma cells. Q-RT-PCR results also showed that luteolin increased Bax and caspase-3 expression significantly and upregulated Bcl-2 expression in a dose-dependent manner in liver carcinoma cells. However, the normal liver cells HL-7702 was almost not affected by luteolin treatment. Luteolin can inhibit SMMC-7721 and BEL-7402 cell proliferation in a time- and dose-dependent manner. And the mechanism maybe through arresting cell cycle at phase G1/S, enhancing Bax level, reducing anti-apoptotic protein Bcl-2 level, resulting in activating caspase-3 enzyme and decrease of mitochondrial membrane potential, and finally leading to cell apoptosis.

  3. N-acetyl-L-cysteine inhibits bleomycin induced apoptosis in malignant testicular germ cell tumors.

    PubMed

    Kucuksayan, Ertan; Cort, Aysegul; Timur, Mujgan; Ozdemir, Evrim; Yucel, Suleyman Gultekin; Ozben, Tomris

    2013-07-01

    Antioxidants may prevent apoptosis of cancer cells via inhibiting reactive oxygen species (ROS). However, to date no study has been carried out to elucidate the effects of strong antioxidant N-acetylcysteine (NAC) on Bleomycin induced apoptosis in human testicular cancer (NTERA-2, NT2) cells. For this reason, we studied the effects of Bleomycin and NAC alone and in combination on apoptotic signaling pathways in NT2 cell line. We determined the cytotoxic effect of bleomycin on NT2 cells and measured apoptosis markers such as Caspase-3, -8, -9 activities and Bcl-2, Bax, Cyt-c, Annexin V-FTIC and PI levels in NT2 cells incubated with different agents for 24 h. Early apoptosis was determined using FACS assay. We found half of the lethal dose (LD50) of Bleomycin on NT2 cell viability as 400, 100, and 20 µg/ml after incubations for 24, 48, and 72 h, respectively. Incubation with bleomycin (LD50 ) and H2O2 for 24 h increased Caspase-3, -8, -9 activities, Cyt-c and Bax levels and decreased Bcl-2 levels. The concurrent incubation of NT2 cells with bleomycin/H2O2 and NAC (5 mM) for 24 h abolished bleomycin/H2O2-dependent increases in Caspase-3, -8, -9 activities, Bax and Cyt-c levels and bleomycin/H2O2-dependent decrease in Bcl-2 level. Our results indicate that bleomycin/H2O2 induce apoptosis in NT2 cells by activating mitochondrial pathway of apoptosis, while NAC diminishes bleomycin/H2O2 induced apoptosis. We conclude that NAC has antagonistic effects on Bleomycin-induced apoptosis in NT2 cells and causes resistance to apoptosis which is not a desired effect in eliminating cancer cells. PMID:23386420

  4. Wavelength-dependent backscattering measurements for quantitative real-time monitoring of apoptosis in living cells

    NASA Astrophysics Data System (ADS)

    Mulvey, Christine S.; Sherwood, Carly A.; Bigio, Irving J.

    2009-11-01

    Apoptosis-programmed cell death-is a cellular process exhibiting distinct biochemical and morphological changes. An understanding of the early morphological changes that a cell undergoes during apoptosis can provide the opportunity to monitor apoptosis in tissue, yielding diagnostic and prognostic information. There is avid interest regarding the involvement of apoptosis in cancer. The initial response of a tumor to successful cancer treatment is often massive apoptosis. Current apoptosis detection methods require cell culture disruption. Our aim is to develop a nondisruptive optical method to monitor apoptosis in living cells and tissues. This would allow for real-time evaluation of apoptotic progression of the same cell culture over time without alteration. Elastic scattering spectroscopy (ESS) is used to monitor changes in light-scattering properties of cells in vitro due to apoptotic morphology changes. We develop a simple instrument capable of wavelength-resolved ESS measurements from cell cultures in the backward direction. Using Mie theory, we also develop an algorithm that extracts the size distribution of scatterers in the sample. The instrument and algorithm are validated with microsphere suspensions. For cell studies, Chinese hamster ovary (CHO) cells are cultured to confluence on plates and are rendered apoptotic with staurosporine. Backscattering measurements are performed on pairs of treated and control samples at a sequence of times up to 6-h post-treatment. Initial results indicate that ESS is capable of discriminating between treated and control samples as early as 10- to 15-min post-treatment, much earlier than is sensed by standard assays for apoptosis. Extracted size distributions from treated and control samples show a decrease in Rayleigh and 150-nm scatterers, relative to control samples, with a corresponding increase in 200-nm particles. Work continues to correlate these size distributions with underlying morphology. To our knowledge, this

  5. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells

    PubMed Central

    Popa, Crina M.; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding. PMID:27489796

  6. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells.

    PubMed

    Popa, Crina M; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding.

  7. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum.

    PubMed

    Lampe, Elisabeth O; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C; Hagedorn, Monica

    2016-03-01

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism. PMID:26712555

  8. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

    PubMed Central

    Lampe, Elisabeth O.; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C.

    2015-01-01

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism. PMID:26712555

  9. Protection against apoptosis in chicken bursa and thymus cells by phorbol ester in vitro

    SciTech Connect

    Asakawa, J.; Thorbecke, G.J. )

    1991-03-15

    Programmed suicide or apoptosis, due to activation of endogenous nucleases, occurs in immature CD4{sup {minus}}85{sup {minus}} mammalian thymus cells. Like the thymus, the bursa of Fabricius is a site of massive lymphopoiesis accompanied by cell death in vivo. In the present study the authors have, therefore, examined whether chicken bursa and thymus cells exhibit apoptosis. Bursa and thymus cells from SC chickens, 4-10 weeks of age, were incubated for 8-24 hrs with various reagents. Genomic DNA was isolated, electrophoresed in 3% Nusieve agarose gels, and examined for patterns of DNA fragmentation. A laddering of DNA in multiples of 200 base pairs, indicative of apoptosis, was observed with both bursa and thymus cells. These patterns of DNA fragmentation from bursa cells could be prevented by adding phorbol myristic acetate during culture and, more effectively, by PMA plus ionomycin, but not by ionomycin alone or by anti-{mu}. PMA did not affect the patterns of DNA fragmentation seen with spleen cells. Addition of the protein kinase C inhibitor staurosporin inhibited the preventive effect of PMA on apoptosis. PMA also greatly promoted the survival of bursa cells in culture, as assayed by percentage cell death and by {sup 3}H-thymidine incorporation. It is concluded that bursa and thymus cells from the chicken exhibit apoptosis. The data further suggest that protein kinase C activation protects apoptosis in cultured bursa cells.

  10. Huaier polysaccharide induces apoptosis in hepatocellular carcinoma cells through p38 MAPK

    PubMed Central

    Bao, Haidong; Liu, Peng; Jiang, Ke; Zhang, Xianbin; Xie, Long; Wang, Zhongyu; Gong, Peng

    2016-01-01

    The underlying mechanism of the antitumor activity of Huaier polysaccharide (HP) remains to be explored. The present study aimed to investigate the inhibitory effect of HP on hepatocellular carcinoma (HCC) cells, and to explore the possible mechanisms of its anticancer effect. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while apoptotic nuclear changes were observed using Hoechst 33258 staining. The distribution of cell cycle and apoptosis were analyzed by flow cytometry, and western blotting was used to test the apoptotic pathways. Apoptosis and mitogen-activated protein kinase (MAPK) inhibitors were used to investigate the mechanism of apoptosis. HP triggered cell cycle arrest and apoptosis in HepG2 and Huh7 cells. Both the extrinsic and intrinsic apoptotic pathways were activated after HP treatment. Furthermore, HP enhanced the three major MAPK pathways (extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 MAPK) and inhibited the AKT/mechanistic target of rapamycin signaling pathway in HCC cells. Notably, the inactivation of p38 MAPK impaired the HP-induced cell death. HP exerted its antitumor effect on HCC cells through the regulation of the expression of the apoptosis-related proteins B-cell lymphoma (Bcl)-2, Bcl-2-associated X protein and survivin. The present study provides evidence that HP induces apoptosis in HCC cells and demonstrated the role of p38 MAPK in HP-triggered cancer cell death. PMID:27446394

  11. Downregulation of SENP1 inhibits cell proliferation, migration and promotes apoptosis in human glioma cells

    PubMed Central

    ZHANG, QIU-SHENG; ZHANG, MENG; HUANG, XIAN-JIAN; LIU, XIAO-JIA; LI, WEI-PING

    2016-01-01

    Small ubiquitin-related modifier protein (SUMO) is an evolutionarily conserved protein in a broad range of eukaryotic organisms. De-SUMOylation, the reverse reaction of SUMOylation, is regulated by a family of SUMO-specific proteases (SENPs). SENP1 is a member of the de-SUMOylation protease family involved in the de-SUMOylation of a variety of SUMOylated proteins. The present study demonstrates that small hairpin RNA (shRNA)-mediated downregulation of SENP1 inhibits cell proliferation and migration, and promotes apoptosis in human glioma cells. Firstly, LN-299 cells were transfected with a plasmid expressing SENP1 shRNA (pGenesil-1-SENP1). The messenger RNA and protein expression of SENP1 was detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Cell proliferation in vitro was assessed using a methyl thiazolyl tetrazolium assay. Flow cytometry (FCM) was used to detect the apoptosis of LN-299 cells. The effect of the downregulation of SENP1 on cell migration was detected by a Transwell migration system. The present results showed that, compared with the control shRNA group, the expression of SENP1 was significantly reduced in the SENP1 shRNA group. The proliferation was markedly inhibited in the SENP1 shRNA group. FCM findings revealed that apoptosis increased significantly in the SENP1 shRNA group. In addition, it was found that downregulation of SENP1 evidently suppressed tumor cell migration. Downregulation of SENP1 expression inhibited the proliferation and migration and promoted apoptosis in LN-299 cells. These results indirectly demonstrate that SENP1 is likely to play a critical role in human glioma cells. PMID:27347128

  12. TAZ promotes cell growth and inhibits Celastrol-induced cell apoptosis

    PubMed Central

    Wang, Shuren; Ma, Kai; Chen, Lechuang; Zhu, Hongxia; Liang, Shufang; Liu, Mei; Xu, Ningzhi

    2016-01-01

    Hippo pathway is a highly conservative signalling pathway related to the development of organisms, which has been demonstrated to be strongly linked to the tumorigenesis and tumour progression. As the major downstream effector of Hippo pathway, yes-associated protein (YAP), is a transcriptional activator of target genes that are involved in cell proliferation and survival. As an oncogene, YAP can promote cell growth and inhibit cell apoptosis. Another major downstream effector of Hippo pathway, transcriptional co-activators with PDZ-binding motif (TAZ), is nearly 60% homologous with YAP. In the present study, we assume that TAZ probably has the similar function to YAP. To test this issue, we established an inducible and a stable expression system of TAZ in T-Rex-293 and HEK293 cells respectively. The results of cell growth curves, colony formation assay and tumour xenograft growth showed that overexpression of TAZ could promote cell growth in vitro and in vivo. Meanwhile, we found that up-regulated expression of TAZ could partially restore Celastrol-induced cell apoptosis. Induced overexpression of TAZ could up-regulate its target genes including ankyrin repeat domain-containing protein (ANKRD), cysteine-rich 61 (CYR61) and connective tissue growth factor (CTGF), increase the expression of B-cell lymphoma-2 (Bcl-2), decrease the expression of Bcl-2 associated X protein (Bax) and activate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which may be the mechanism underlying anti-apoptosis of TAZ. All these findings indicated that TAZ acts as an oncogene that could be a key regulator of cell proliferation and apoptosis. PMID:27515420

  13. HIV-1 Vpr-Induced Apoptosis Is Cell Cycle Dependent and Requires Bax but Not ANT

    PubMed Central

    Andersen, Joshua L; DeHart, Jason L; Zimmerman, Erik S; Ardon, Orly; Kim, Baek; Jacquot, Guillaume; Benichou, Serge; Planelles, Vicente

    2006-01-01

    The HIV-1 accessory protein viral protein R (Vpr) causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage–signaling protein ATR as the cellular factor that mediates Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45α was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4–3 vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked. PMID:17140287

  14. Apoptosis Cell Death Effect of Scrophularia Variegata on Breast Cancer Cells via Mitochondrial Intrinsic Pathway

    PubMed Central

    Azadmehr, Abbas; Hajiaghaee, Reza; Baradaran, Behzad; Haghdoost-Yazdi, Hashem

    2015-01-01

    Purpose: Scrophularia variegata M. Beib. (Scrophulariaceae) is an Iranian medicinal plant which is used for various inflammatory disorders in traditional medicine. In this study we evaluated the anti-cancer and cytotoxic effects of the Scrophularia variegata (S. variegata) ethanolic extract on the human breast cancer cell line. Methods: The cytotoxicity effect of the extract on MCF-7 cells was evaluated by MTT assay. In addition, Caspase activity, DNA ladder and Cell death were evaluated by ELISA, gel electrophoresis and Annexin V-FITC/PI staining, respectively. Results: The S. variegata extract showed significant effect cytotoxicity on MCF-7 human breast cancer cell line. Treatment with the extract induced apoptosis on the breast cancer cells by cell cycle arrest in G2/M phase. The results indicated that cytotoxicity activity was associated with an increase of apoptosis as demonstrated by DNA fragmentation as well as an increase of the amount of caspase 3 and caspase 9. In addition, the phytochemical assay showed that the extract had antioxidant capacity and also flavonoids, phenolic compounds and phenyl propanoids were presented in the extract. Conclusion: Our findings indicated that S. variegata extract induced apoptosis via mitochondrial intrinsic pathway on breast cancer by cell cycle arrest in G2/M phase and an increase of caspase 3 and caspase 9. However future studies are needed. PMID:26504768

  15. Bupivacaine induces apoptosis through caspase-dependent and -independent pathways in canine mammary tumor cells.

    PubMed

    Chiu, Yi-Shu; Cheng, Yeong-Hsiang; Lin, Sui-Wen; Chang, Te-Sheng; Liou, Chian-Jiun; Lai, Yu-Shen

    2015-06-01

    Local anesthetics have been reported to induce apoptosis in various cell lines. In this study, we showed that bupivacaine also induced apoptosis in DTK-SME cells, a vimentin(+)/AE1(+)/CK7(+)/HSP27(+), tumorigenic, immortalized, canine mammary tumor cell line. Bupivacaine induced apoptosis in DTK-SME cells in a time- and concentration-dependent manner. Apoptosis-associated morphological changes, including cell shrinkage and rounding, chromatin condensation, and formation of apoptotic bodies, were observed in the bupivacaine-treated DTK-SME cells. Apoptosis was further confirmed with annexin V staining, TUNEL staining, and DNA laddering assays. At the molecular level, the activation of caspases-3, -8, and -9 corresponded well to the degree of DNA fragmentation triggered by bupivacaine. We also demonstrated that the pan-caspase inhibitor, z-VAD-fmk, only partially inhibited the apoptosis induced by bupivacaine. Moreover, treated cells increased expression of endonuclease G, a death effector that acts independently of caspases. Our data suggested that bupivacaine-induced apoptosis occurs through both caspase-dependent and caspase-independent apoptotic pathways.

  16. Sodium butyrate-induced DAPK-mediated apoptosis in human gastric cancer cells.

    PubMed

    Shin, Hyunsoo; Lee, Yeo Song; Lee, Yong Chan

    2012-04-01

    Epigenetic mechanisms of histone acetylation/deacetylation play an important role in the regulation of gene expression associated with the cell cycle and apoptosis. Recently, sodium butyrate, a histone deacetylase (HDAC) inhibitor, has been shown to exhibit anticancer effects via differentiation and apoptosis of cancer cells. Sodium butyrate may be a potential anticancer chemotherapeutic drug; however, the precise mechanism underlying the anticancer effects of sodium butyrate has not been clearly elucidated. In the present study, we investigated the role of death-associated protein kinase (DAPK) on the apoptosis of human gastric cancer cells induced by sodium butyrate. We observed that sodium butyrate induced apoptosis in human gastric cancer cells. Treatment with the HDAC inhibitor sodium butyrate increased the expression of caspase-3 and DAPK1/2 genes but decreased the expression of Bcl-2 in human gastric cancer cells. The expression of DAPK3, p53 and p21 were not altered by sodium butyrate treatment. Analysis of the general expression patterns revealed that sodium butyrate increased the expression of DAPK1/2 but decreased the expression of FAK and induced changes in the proliferation of apoptosis-related genes in human gastric cancer cells. These data suggest that DAPK expression prompts apoptosis by reducing the FAK protein level in sodium butyrate-induced apoptosis of human gastric cancer cells.

  17. Dimethylarsinic acid causes apoptosis in HL-60 cells via interaction with glutathione.

    PubMed

    Ochi, T; Nakajima, F; Sakurai, T; Kaise, T; Oya-Ohta, Y

    1996-01-01

    Inducibility of apoptosis in cultured human HL-60 cells by arsenic compounds, such as arsenite, arsenate, methylarsonic acid (MAA), and dimethylarsinic acid (DMAA), was investigated, together with the role of glutathione (GSH) in the induction. Among the arsenic compounds DMAA was the most potent in terms of the ability to cause the morphological changes (formation of nuclear fragmentation and apoptotic bodies) characteristic of apoptosis. Furthermore, fragmentation of internucleosomal DNA was also induced by DMAA. Depletion of cell GSH by L-buthionine-SR-sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase, enhanced the cytotoxicity of arsenite, arsenate, and MAA, while such depletion suppressed the cytotoxicity of DMAA. The depletion of GSH also suppressed the morphological changes and the fragmentation of internucleosomal DNA caused by DMAA, both of which are characteristic features of apoptosis. The results suggest that the death of cells caused by DMAA is due to apoptosis and that GSH is involved in the induction of apoptosis by this arsenic compound.

  18. Matrine-induced autophagy counteracts cell apoptosis via the ERK signaling pathway in osteosarcoma cells

    PubMed Central

    Ma, Kun; Huang, Man-Yu; Guo, Yan-Xing; Hu, Guo-Qiang

    2016-01-01

    The aim of the present study was to observe whether autophagy was induced by matrine, and to investigate the role of autophagy in the antitumor effects of matrine on human osteosarcoma MG-63 cells and its underlying mechanism. MG-63 cells were cultured in vitro in matrine at a concentration of 0.6, 0.8, 1.0 and 1.2 g/l for 0, 24, 48 and 72 h. A MTT assay was used to evaluate the proliferation inhibition of MG-63 cells by matrine, and Annexin V-fluorescein isothiocyanate/propidum iodide (PI) staining flow cytometry was used to analyze the apoptotic rate. Alterations in cell morphology was assessed by PI and Hoechst 33258 cell staining. Matrine-induced autophagy in MG-63 cells was confirmed by green fluorescent protein-microtubule-associated protein 1-light chain 3 (LC3) b transfection and fluorescence microscopy, and cell viability was investigated by MTT assay following inhibition of autophagy by chloroquine (CQ) pretreatment. The expression level of apoptosis-associated proteins B-cell lymphoma-2 (Bcl-2) and Bcl-2-like protein 4 (Bax), autophagy-associated LC3II protein, and the activation of extracellular signal-regulated kinase (ERK) was detected by western blotting. Cell proliferation was clearly inhibited by matrine in a dose- and time-dependent manner. Flow cytometry and Hoechst 33258/PI staining verified that matrine induced apoptosis in a time-dependent manner when cells were exposed to 1.1 g/l matrine; fluorescence microscopy demonstrated that green fluorescence puncta were enhanced with prolonged time of matrine incubation. Western blotting confirmed that the expression of pro-apoptosis-associated proteins Bax and LC3II, and phosphorylated-ERK were upregulated, and anti-apoptosis protein Bcl-2 was downregulated in a time-dependent manner following treatment with matrine. The cell viability of the matrine + CQ group was increased compared with the matrine group alone, which revealed that matrine treatment alone induced protective autophagy in MG-63 cells

  19. p53 integrates host defense and cell fate during bacterial pneumonia

    PubMed Central

    Madenspacher, Jennifer H.; Azzam, Kathleen M.; Gowdy, Kymberly M.; Malcolm, Kenneth C.; Nick, Jerry A.; Dixon, Darlene; Aloor, Jim J.; Draper, David W.; Guardiola, John J.; Shatz, Maria; Menendez, Daniel; Lowe, Julie; Lu, Jun; Bushel, Pierre; Li, Leping; Merrick, B. Alex; Resnick, Michael A.

    2013-01-01

    Cancer and infection are predominant causes of human mortality and derive, respectively, from inadequate genomic and host defenses against environmental agents. The transcription factor p53 plays a central role in human tumor suppression. Despite its expression in immune cells and broad responsiveness to stressors, it is virtually unknown whether p53 regulates host defense against infection. We report that the lungs of naive p53−/− mice display genome-wide induction of NF-κB response element–enriched proinflammatory genes, suggestive of type 1 immune priming. p53-null and p53 inhibitor–treated mice clear Gram-negative and -positive bacteria more effectively than controls after intrapulmonary infection. This is caused, at least in part, by cytokines produced by an expanded population of apoptosis-resistant, TLR-hyperresponsive alveolar macrophages that enhance airway neutrophilia. p53−/− neutrophils, in turn, display heightened phagocytosis, Nox-dependent oxidant generation, degranulation, and bacterial killing. p53 inhibition boosts bacterial killing by mouse neutrophils and oxidant generation by human neutrophils. Despite enhanced bacterial clearance, infected p53−/− mice suffer increased mortality associated with aggravated lung injury. p53 thus modulates host defense through regulating microbicidal function and fate of phagocytes, revealing a fundamental link between defense of genome and host during environmental insult. PMID:23630228

  20. Host cell proteases: critical determinants of coronavirus tropism and pathogenesis

    PubMed Central

    Millet, Jean Kaoru; Whittaker, Gary R.

    2015-01-01

    Coronaviruses are a large group of enveloped, single-stranded positive-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a critical role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential first step in establishing infection, the proteolytic activation step is often critical for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large number of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the critical entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range and pathogenicity. PMID:25445340

  1. Binase induces apoptosis of transformed myeloid cells and does not induce T-cell immune response.

    PubMed

    Ilinskaya, Olga N; Zelenikhin, Pavel V; Petrushanko, Irina Yu; Mitkevich, Vladimir A; Prassolov, Vladimir S; Makarov, Alexander A

    2007-10-01

    Microbial RNases along with such animal RNases as onconase and BS-RNase are a promising basis for developing new antitumor drugs. We have shown that the Bacillus intermedius RNase (binase) induces selective apoptosis of transformed myeloid cells. It attacks artificially expressing activated c-Kit myeloid progenitor FDC cells and chronic myelogenous leukemia cells K562. Binase did not induce apoptosis in leukocytes of healthy donors and in normal myeloid progenitor cells. The inability of binase to initiate expression of activation markers CD69 and IFN-gamma in CD4+ and CD8+ T-lymphocytes testifies that enzyme is devoid of superantigenic properties. Altogether, these results demonstrate that binase possesses therapeutic opportunities for treatment of genotyped human neoplasms expressing activated kit.

  2. YopJ-Induced Caspase-1 Activation in Yersinia-Infected Macrophages: Independent of Apoptosis, Linked to Necrosis, Dispensable for Innate Host Defense

    PubMed Central

    Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B.

    2012-01-01

    Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic

  3. YopJ-induced caspase-1 activation in Yersinia-infected macrophages: independent of apoptosis, linked to necrosis, dispensable for innate host defense.

    PubMed

    Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B

    2012-01-01

    Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and

  4. MicroRNA-15a/16 Regulates Apoptosis of Lung Epithelial Cells After Oxidative Stress

    PubMed Central

    Cao, Yong; Zhang, Duo; Moon, Hyung-Geun; Lee, Heedoo; Haspel, Jeffrey A; Hu, Kebin; Xie, Lixin; Jin, Yang

    2016-01-01

    Lung epithelial cell apoptosis is an important feature of hyperoxia-induced lung injury. The death receptor–associated extrinsic pathway and mitochondria-associated intrinsic pathway both mediate the development of lung epithelial cell apoptosis. Despite decades of research, molecular mechanisms of hyperoxia-induced epithelial cell apoptosis remain incompletely understood. Here, we report a novel regulatory paradigm in response to hyperoxia-associated oxidative stress. Hyperoxia markedly upregulated microRNA (miR)-15a/16 levels in lung epithelial cells, bronchoalveolar lavage fluid (BALF) and lung tissue. This effect was mediated by hyperoxia-induced reactive oxygen species. Functionally, miR-15a/16 inhibitors induced caspase-3–mediated lung epithelial cell apoptosis, in the presence of hyperoxia. MiR-15a/16 inhibitors robustly enhanced FADD level and downregulated Bcl-2 expression. Consistently, cleaved caspase-8 and -9 were highly induced in the miR-15a/16–deficient cells, after hyperoxia. Using airway epithelial cell–specific, miR-15a/16–/– mice, we found that Bcl-2 was significantly reduced in lung epithelial cells in vivo after hyperoxia. In contrast, caspase-3, caspase-8 and Bcl-2–associated death promoter (BAD) were highly elevated in the miR-15a/16–/– epithelial cells in vivo. Interestingly, in lung epithelial malignant cells, rather than benign cells, deletion of miR-15a/16 prevented apoptosis. Furthermore, deletion of miR-15a/16 in macrophages also prohibited apoptosis, which is the opposite of what we have found in normal lung epithelial cells. Taken together, our data suggested that miR-15a/16 may exert differential roles in different cell types. MiR-15a/16 deficiency results in lung epithelial cell apoptosis in response to hyperoxia, via modulating both intrinsic and extrinsic apoptosis pathways. PMID:27257854

  5. Proteasome inhibitors induce p53-independent apoptosis in human cancer cells.

    PubMed

    Pandit, Bulbul; Gartel, Andrei L

    2011-01-01

    Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.

  6. Apoptosis of pancreatic adenocarcinoma cells induced by LED-activated pheophorbide a

    NASA Astrophysics Data System (ADS)

    Liu, L.; Leung, A. W. N.; Xia, X. S.; Bai, D. Q.; Lin, H. D.; Xu, C. S.

    2011-02-01

    Apoptosis is an important form of cell death. The present study investigated apoptotic cell death of pancreatic adenocarcinoma cells treated by LED-activated pheophorbide a (Pa) using nuclear staining and flow cytometric analysis with Annexin V/PI staining 8 h after LED-activated Pa. We further investigated the changes of mitochondrial membrane potential 3 h after LED-activated Pa. The results showed that apoptosis significantly occurred and mitochondrial membrane potential markedly decreased in pancreatic adenocarcinoma cells after LED-activated Pa. Our data demonstrated that LED-activated Pa could remarkably induce apoptosis of pancreatic adenocarcinoma cells, and the collapse of mitochondrial membrane potential might be an important event in the apoptosis of pancreatic adenocarcinoma cells after LED-activated Pa.

  7. Akt is translocated to the mitochondria during etoposide-induced apoptosis of HeLa cells.

    PubMed

    Park, Byoungduck; Je, Young-Tae; Chun, Kwang-Hoon

    2015-11-01

    Akt, or protein kinase B, is a key serine-threonine kinase, which exerts anti-apoptotic effects and promotes cell proliferation in response to various stimuli. Recently, however, it was demonstrated that Akt exhibits a proapoptotic role in certain contexts. During etoposide‑induced apoptosis of HeLa cells, Akt enhances the interaction of second mitochondria‑derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO) and X‑linked inhibitor of apoptosis protein by phosphorylating Smac at serine 67, and thus promotes apoptosis. However, the detailed mechanisms underlying Akt regulation in etoposide‑mediated apoptosis remain to be determined. The present study investigated whether etoposide triggers the translocation of Akt into the mitochondria. It was found that Akt activity was increased and sustained during apoptosis triggered by etoposide in HeLa cells. During apoptosis, Akt was translocated from the cytoplasm into the mitochondria in a phosphoinositide 3‑kinase-dependent manner at the early and late stages of apoptosis. Concomitantly, the depletion of Akt in the nuclear fraction was observed after etoposide treatment from analysis of confocal microscopy. The results suggest that etoposide‑stimulated Akt is translocated into the mitochondria, thereby possibly enhancing its interaction with Smac and promoting apoptosis in HeLa cells. These results indicate that Akt may be a promising candidate for a pro-apoptotic approach in cancer treatment.

  8. Early Bunyavirus-Host Cell Interactions

    PubMed Central

    Albornoz, Amelina; Hoffmann, Anja B.; Lozach, Pierre-Yves; Tischler, Nicole D.

    2016-01-01

    The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion. PMID:27213430

  9. Early Bunyavirus-Host Cell Interactions.

    PubMed

    Albornoz, Amelina; Hoffmann, Anja B; Lozach, Pierre-Yves; Tischler, Nicole D

    2016-01-01

    The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion. PMID:27213430

  10. Carbamate pesticide-induced apoptosis and necrosis in human natural killer cells.

    PubMed

    Li, Q; Kobayashi, M; Kawada, T

    2014-01-01

    We previously found that ziram, a carbamate fungicide, significantly induced apoptosis and necrosis in human NK-92MI, a natural killer cell line. To investigate whether other carbamate pesticides also induce apoptosis and necrosis in human natural killer cell, we conducted further experiments with NK-92CI, a human natural killer cell line using a more sensitive assay. NK-92CI cells were treated with ziram, thiram, maneb or carbaryl at 0.031-40 microM for 2-24 h in the present study. Apoptosis and necrosis were determined by FITC-Annexin-V/PI staining. To explore the mechanism of apoptosis, intracellular levels of active caspases 3 and mitochondrial cytochrome-c release were determined by flow cytometry. We found that ziram and thiram also induced apoptosis and necrosis in a time- and dose-dependent manner; however, maneb and carbaryl induced apoptosis and necrosis only at higher doses in NK-92CI cells. The strength of the apoptosis-inducing effect differed among the pesticides, and the order was as follows: thiram > ziram greater than maneb greater than carbaryl. NK-92CI was more sensitive to ziram than NK-92MI. Moreover, ziram and thiram significantly increased the intracellular level of active caspase 3 in NK-92CI and caspase inhibitor significantly inhibited the apoptosis. Ziram and thiram significantly caused mitochondrial cytochrome-c release in NK-92CI. These findings indicate that carbamate pesticides can induce apoptosis in natural killer cells, and the apoptosis is mediated by both the caspase-cascade and mitochondrial cytochrome-c pathways.

  11. Genome surveillance in pluripotent stem cells: Low apoptosis threshold and efficient antioxidant defense

    PubMed Central

    Dannenmann, Benjamin; Lehle, Simon; Essmann, Frank; Schulze-Osthoff, Klaus

    2016-01-01

    ABSTRACT Pluripotent stem cells must be endowed with efficient genome surveillance. Here we describe the multiple mechanisms that ensure their genome integrity, including high susceptibility to apoptosis and efficient prevention of DNA lesions. In induced pluripotent stem cells, apoptosis hypersensitivity is mediated by increased expression of proapoptotic BCL-2 protein, whereas DNA damage is prevented by the upregulation of several antioxidant enzymes. Antioxidants might be therefore employed for safer stem cell therapies. PMID:27308586

  12. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    SciTech Connect

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-16

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  13. TRAP1 regulates cell cycle and apoptosis in thyroid carcinoma cells.

    PubMed

    Palladino, Giuseppe; Notarangelo, Tiziana; Pannone, Giuseppe; Piscazzi, Annamaria; Lamacchia, Olga; Sisinni, Lorenza; Spagnoletti, Girolamo; Toti, Paolo; Santoro, Angela; Storto, Giovanni; Bufo, Pantaleo; Cignarelli, Mauro; Esposito, Franca; Landriscina, Matteo

    2016-09-01

    Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a heat shock protein 90 (HSP90) molecular chaperone upregulated in several human malignancies and involved in protection from apoptosis and drug resistance, cell cycle progression, cell metabolism and quality control of specific client proteins. TRAP1 role in thyroid carcinoma (TC), still unaddressed at present, was investigated by analyzing its expression in a cohort of 86 human TCs and evaluating its involvement in cancer cell survival and proliferation in vitro Indeed, TRAP1 levels progressively increased from normal peritumoral thyroid gland, to papillary TCs (PTCs), follicular variants of PTCs (FV-PTCs) and poorly differentiated TCs (PDTCs). By contrast, anaplastic thyroid tumors exhibited a dual pattern, the majority being characterized by high TRAP1 levels, while a small subgroup completely negative. Consistently with a potential involvement of TRAP1 in thyroid carcinogenesis, TRAP1 silencing resulted in increased sensitivity to paclitaxel-induced apoptosis, inhibition of cell cycle progression and attenuation of ERK signaling. Noteworthy, the inhibition of TRAP1 ATPase activity by pharmacological agents resulted in attenuation of cell proliferation, inhibition of ERK signaling and reversion of drug resistance. These data suggest that TRAP1 inhibition may be regarded as potential strategy to target specific features of human TCs, i.e., cell proliferation and resistance to apoptosis. PMID:27422900

  14. Apoptosis Induced by Ginkgo biloba (EGb761) in Melanoma Cells Is Mcl-1-Dependent

    PubMed Central

    Cheng, Yao; Du, Jipei; Chen, Degao; Li, Chengtao; Zhang, Ji

    2015-01-01

    Melanoma is an aggressive skin cancer. Unfortunately, there is currently no chemotherapeutic agent available to significantly prolong the survival of the most patients with metastatic melanomas. Here we report that the Ginkgo biloba extract (EGb761), one of the most widely sold herbal supplements in the world, potently induces apoptosis in human melanoma cells by disturbing the balance between pro- and anti-apoptosis Bcl-2 family proteins. Treatment with EGb761 induced varying degrees of apoptosis in melanoma cell lines but not in melanocytes. Induction of apoptosis was caspase-dependent and appeared to be mediated by the mitochondrial pathway, in that it was associated with reduction in mitochondrial membrane potential and activation of Bax and Bak. Although EGb761 did not cause significant change in the expression levels of the BH3-only Bcl-2 family proteins Bim, Puma, Noxa, and Bad, it significantly downregulated Mcl-1 in sensitive but not resistant melanoma cells, suggesting a major role of Mcl-1 in regulating apoptosis of melanoma cells induced by EGb761. Indeed, siRNA knockdown of Mcl-1 enhanced EGb761-induced apoptosis, which was associated with increased activation of Bax and Bak. Taken together, these results demonstrate that EGb761 kills melanoma cells through the mitochondrial apoptotic pathway, and that Mcl-1 is a major regulator of sensitivity of melanoma cells to apoptosis induced by EGb761. Therefore, EGb761 with or without in combination with targeting Mcl-1 may be a useful strategy in the treatment of melanoma. PMID:25860257

  15. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells.

    PubMed

    Silva, Julhiany de Fátima da; Vicentim, Juliana; Oliveira, Haroldo Cesar de; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; Silva, Juliana Leal Monteiro da; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-06-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis. PMID:26038961

  16. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

    PubMed Central

    da Silva, Julhiany de Fátima; Vicentim, Juliana; de Oliveira, Haroldo Cesar; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; da Silva, Juliana Leal Monteiro; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-01-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis. PMID:26038961

  17. Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells

    NASA Technical Reports Server (NTRS)

    Plymale, D. R.; Tang, D. S.; Comardelle, A. M.; Fermin, C. D.; Lewis, D. E.; Garry, R. F.

    1999-01-01

    BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (<12%) had condensed chromatin and fragmented nuclei, the morphological features of apoptosis. The predominant alterations in acutely infected cells were distended endoplasmic reticulum and abnormal mitochondria; these ultrastructural changes are consistent with necrosis, although some infected cells simultaneously displayed features of both necrosis and apoptosis. Viability of cells persistently infected by HIV-1 was only minimally reduced from that of uninfected cells. This reduction was accounted for by an increased propensity of the persistently infected cells to die by apoptosis. Alterations in [Ca2+]i and deltapsi(m) occurred in both acutely and persistently infected cells. CONCLUSION: Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

  18. Exogenous nitric oxide inhibits apoptosis in guinea pig gastric mucous cells

    PubMed Central

    Potter, C; Hanson, P

    2000-01-01

    BACKGROUND—Increased nitric oxide (NO) synthase activity and enhanced apoptosis are features of gastric mucosa infected with Helicobacter pylori and a causative relation has been suggested. However, although NO can promote apoptosis, its actions vary with cell type.
AIMS—To determine whether exogenous NO, derived from an NO donor, might promote or counteract apoptosis in gastric mucous epithelial cells.
METHODS—Primary cultures of guinea pig gastric mucosal cells were exposed to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) for 24 hours. Apoptosis was detected from nuclear staining with Hoechst 33258, in situ nick end labelling of DNA, and the presence of DNA "ladders" in cell extracts. Cyclic GMP content and caspase activity were determined by immunoassay and fluorimetric assay respectively.
RESULTS—SNAP 1 mM did not alter the small proportion of cells on the culture plate (3-6%) which exhibited features of apoptosis. However, SNAP produced an inhibition of apoptosis, and of caspase 3 like activity, when enhanced by 25 µM N-hexanoyl-D-sphingosine (C6-ceramide), or by detachment of cells from the culture plate. The guanylate cyclase inhibitor, 1H-1, 2, 4-oxadiazole-4, 3-a-quinoxaline-1-one (ODQ), prevented the stimulation of cyclic GMP by SNAP, but not the anti- apoptotic effects of the NO donor. The cyclic GMP analogues 8-bromo-cyclic GMP and 8-(4-chlorophenylthio) guanosine-3',5'- cyclic monophosphate did not significantly inhibit apoptosis in the mucosal cells.
CONCLUSIONS—Exogenous NO inhibited apoptosis in guinea pig gastric mucous cells by a mechanism which did not involve elevation of cyclic GMP. NO, if produced from NO synthase during infection with H pylori, may therefore counter the proapoptotic effects of this pathogen.


Keywords: nitric oxide; gastric mucosa; stomach; apoptosis PMID:10644307

  19. Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality.

    PubMed

    Saha, Asim; O'Connor, Roddy S; Thangavelu, Govindarajan; Lovitch, Scott B; Dandamudi, Durga Bhavani; Wilson, Caleph B; Vincent, Benjamin G; Tkachev, Victor; Pawlicki, Jan M; Furlan, Scott N; Kean, Leslie S; Aoyama, Kazutoshi; Taylor, Patricia A; Panoskaltsis-Mortari, Angela; Foncea, Rocio; Ranganathan, Parvathi; Devine, Steven M; Burrill, Joel S; Guo, Lili; Sacristan, Catarina; Snyder, Nathaniel W; Blair, Ian A; Milone, Michael C; Dustin, Michael L; Riley, James L; Bernlohr, David A; Murphy, William J; Fife, Brian T; Munn, David H; Miller, Jeffrey S; Serody, Jonathan S; Freeman, Gordon J; Sharpe, Arlene H; Turka, Laurence A; Blazar, Bruce R

    2016-07-01

    Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

  20. Overcoming Hypoxic-Resistance of Tumor Cells to TRAIL-Induced Apoptosis through Melatonin

    PubMed Central

    Lee, You-Jin; Lee, Ju-Hee; Moon, Ji-Hong; Park, Sang-Youel

    2014-01-01

    A solid tumor is often exposed to hypoxic or anoxic conditions; thus, tumor cell responses to hypoxia are important for tumor progression as well as tumor therapy. Our previous studies indicated that tumor cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell apoptosis under hypoxic conditions. Melatonin inhibits cell proliferation in many cancer types and induces apoptosis in some particular cancer types. Here, we examined the effects of melatonin on hypoxic resistant cells against TRAIL-induced apoptosis and the possible mechanisms of melatonin in the hypoxic response. Melatonin treatment increased TRAIL-induced A549 cell death under hypoxic conditions, although hypoxia inhibited TRAIL-mediated cell apoptosis. In a mechanistic study, hypoxia inducible factor-1α and prolyl-hydroxylase 2 proteins, which increase following exposure to hypoxia, were dose-dependently down-regulated by melatonin treatment. Melatonin also blocked the hypoxic responses that reduced pro-apoptotic proteins and increased anti-apoptotic proteins including Bcl-2 and Bcl-xL. Furthermore, melatonin treatment reduced TRAIL resistance by regulating the mitochondrial transmembrane potential and Bax translocation. Our results first demonstrated that melatonin treatment induces apoptosis in TRAIL-resistant hypoxic tumor cells by diminishing the anti-apoptotic signals mediated by hypoxia and also suggest that melatonin could be a tumor therapeutic tool by combining with other apoptotic ligands including TRAIL, particularly in solid tumor cells exposed to hypoxia. PMID:25000265

  1. Overcoming hypoxic-resistance of tumor cells to TRAIL-induced apoptosis through melatonin.

    PubMed

    Lee, You-Jin; Lee, Ju-Hee; Moon, Ji-Hong; Park, Sang-Youel

    2014-07-04

    A solid tumor is often exposed to hypoxic or anoxic conditions; thus, tumor cell responses to hypoxia are important for tumor progression as well as tumor therapy. Our previous studies indicated that tumor cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell apoptosis under hypoxic conditions. Melatonin inhibits cell proliferation in many cancer types and induces apoptosis in some particular cancer types. Here, we examined the effects of melatonin on hypoxic resistant cells against TRAIL-induced apoptosis and the possible mechanisms of melatonin in the hypoxic response. Melatonin treatment increased TRAIL-induced A549 cell death under hypoxic conditions, although hypoxia inhibited TRAIL-mediated cell apoptosis. In a mechanistic study, hypoxia inducible factor-1α and prolyl-hydroxylase 2 proteins, which increase following exposure to hypoxia, were dose-dependently down-regulated by melatonin treatment. Melatonin also blocked the hypoxic responses that reduced pro-apoptotic proteins and increased anti-apoptotic proteins including Bcl-2 and Bcl-xL. Furthermore, melatonin treatment reduced TRAIL resistance by regulating the mitochondrial transmembrane potential and Bax translocation. Our results first demonstrated that melatonin treatment induces apoptosis in TRAIL-resistant hypoxic tumor cells by diminishing the anti-apoptotic signals mediated by hypoxia and also suggest that melatonin could be a tumor therapeutic tool by combining with other apoptotic ligands including TRAIL, particularly in solid tumor cells exposed to hypoxia.

  2. Apoptosis induction by aluminum phthalocyanine tetrasulfonate-based sonodynamic therapy in HL-60 cells

    NASA Astrophysics Data System (ADS)

    Iwase, Yumiko; Yumita, Nagahiko; Nishi, Koji; Kuwahara, Hiroyuki; Fukai, Toshio; Ikeda, Toshihiko; Chen, Fu-shih; Momose, Yasunori; Umemura, Shin-ichiro

    2015-07-01

    The present study aims to investigate sonodynamically-induced apoptosis using the phthalocyanine, chloroaluminum phthalocyanine tetrasulfonate (AlPcTS). HL-60 cells were exposed to ultrasound for up to 3 min in the absence and presence of AlPcTS. Apoptosis was analyzed by cell morphology, DNA fragmentation, and caspase-3 activity. Electron spin resonance was used to measure reactive oxygen species. The number of apoptotic cells showing membrane blebbing and cell shrinkage after combined treatment (ultrasound and AlPcTS) was significantly higher than following other treatments, including ultrasound alone and AlPcTS alone. Furthermore, DNA ladder formation, caspase-3 activation and enhanced nitroxide generation were observed in cells treated with ultrasound and AlPcTS. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. The significant reduction by histidine indicated that ultrasonically generated reactive oxygen species, such as singlet oxygen, is an important mediator of sonodynamically-induced apoptosis.

  3. Plasmodium species: master renovators of their host cells.

    PubMed

    de Koning-Ward, Tania F; Dixon, Matthew W A; Tilley, Leann; Gilson, Paul R

    2016-08-01

    Plasmodium parasites, the causative agents of malaria, have developed elaborate strategies that they use to survive and thrive within different intracellular environments. During the blood stage of infection, the parasite is a master renovator of its erythrocyte host cell, and the changes in cell morphology and function that are induced by the parasite promote survival and contribute to the pathogenesis of severe malaria. In this Review, we discuss how Plasmodium parasites use the protein trafficking motif Plasmodium export element (PEXEL), protease-mediated polypeptide processing, a novel translocon termed the Plasmodium translocon of exported proteins (PTEX) and exomembranous structures to export hundreds of proteins to discrete subcellular locations in the host erythrocytes, which enables the parasite to gain access to vital nutrients and to evade the immune defence mechanisms of the host.

  4. Research on effect of minor bupleurum decoction of proliferation and apoptosis of esophageal cancer cell strain eca-109 cell.

    PubMed

    Li, Xiaofang; Sun, Miaomiao; Zhao, Zhihua; Yang, Jianping; Chen, Kuisheng

    2014-09-01

    The research protocol is MTT (Methyl Thiazolyl Tetrazolium) method, Hoechst33342 staining method and flow cytometry detection to observe the effect of minor bupleurum decoction on proliferation inhibition and apoptosis-inducing of esophageal cancer cell strain Eca-109 cell and its purpose is to discuss the effect. The result of MTT method shows that minor buplerum decoction can obviously inhibit proliferation of esophageal cancer cell strain Eca-109 cell. Apoptosis number of esophageal cancer cell increased with the increase of concentration of tetrandrine by the Hoechst 35528 staining experiment of cancer cell in three different concentrations. Flow cytometry detection result showed that cells in cell cycle G0/G1 of esophageal cancer cell strain Eca-109 cell increased obviously and cell in s period decreased significantly. This research proved that minor bupleurum decoction had anti-tumor effect and can influent proliferation and apoptosis of esophageal cancer cell strain Eca-109 cell. PMID:25262517

  5. THE ROLE OF GSH EFFLUX IN STAUROSPORINE-INDUCED APOPTOSIS IN COLONIC EPITHELIAL CELLS

    PubMed Central

    Circu, Magdalena L.; Stringer, Sarah; Rhoads, Carol Ann; Moyer, Mary Pat; Aw, Tak Yee

    2008-01-01

    Staurosporine (STP) was shown to induce cell apoptosis through formation of reactive oxygen species, but a role for cellular redox has not been defined. In this study, we report that STP (2μM) caused apoptosis (24±3% at 24h) of human colon adenocarcinoma epithelial cell line HT29 that was preceded by significant GSH and GSSG efflux (6h), but independent of changes in cellular GSH/GSSG redox status. The blockade of GSH efflux by γ-glutamyl glutamate (γ-GG) or ophthalmic acid was associated with apoptosis attenuation; however, γ-GG administration after peak GSH efflux (8h) did not confer cytoprotection. Moreover, lowering cellular GSH through inhibition of its synthesis prevented extracellular GSH accumulation and cell apoptosis, thus validating a link between cellular GSH export and the trigger of cell apoptosis. Inhibition of γ-glutamyl transferase (GGT1 EC 2.3.2.2)-catalyzed extracellular GSH degradation with acivicin significantly blocked GSH efflux, suggesting that GSH breakdown is a driving force for GSH export. Interestingly, acivicin treatment enhanced extracellular GSSG accumulation, consistent with GSH oxidation. STP-induced HT29 cell apoptosis was associated with caspase-3 activation independent of caspase-8 or caspase-9 activity; accordingly, inhibitors of the latter caspases were without effect on STP-induced apoptosis. STP similarly induced GSH efflux and apoptosis in a nonmalignant human NCM460 colonic cell line in association with caspase-3 activation. Collectively, our results demonstrate that STP induction of apoptosis in malignant and non-malignant colonic cells is temporally linked to the export of cellular GSH and the activation of caspase-3 without caspase-8 or -9 involvement. PMID:18840413

  6. Apoptosis in yeast: a new model system with applications in cell biology and medicine.

    PubMed

    Madeo, Frank; Engelhardt, Silvia; Herker, Eva; Lehmann, Nina; Maldener, Corinna; Proksch, Astrid; Wissing, Silke; Fröhlich, Kai-Uwe

    2002-07-01

    Apoptosis is a highly coordinated cellular suicide program crucial for metazoan health and diseases. Although its increasing importance in cancer, neurodegenerative disorders and AIDS led to intense research and a better understanding of apoptosis, many details of its regulation or the apoptotic phenotypes are poorly understood. The complex regulatory network and the often contradictory results obtained with human cell lines made application of an easier model system desirable. Apoptosis in yeast promises to provide a better understanding of the genetics of apoptosis. During the past 2 years, scientists were successful in identifying new cell-death regulators of humans, plants and fungi using Saccharomyces cerevisiae. The finding of apoptotic phenotypes, even in protists, suggests that apoptosis developed in unicellular organisms long before the evolutionary separation between fungi, plants and metazoan animals occurred.

  7. Boron neutron capture therapy induces cell cycle arrest and cell apoptosis of glioma stem/progenitor cells in vitro

    PubMed Central

    2013-01-01

    Background Glioma stem cells in the quiescent state are resistant to clinical radiation therapy. An almost inevitable glioma recurrence is due to the persistence of these cells. The high linear energy transfer associated with boron neutron capture therapy (BNCT) could kill quiescent and proliferative cells. Methods The present study aimed to evaluate the effects of BNCT on glioma stem/progenitor cells in vitro. The damage induced by BNCT was assessed using cell cycle progression, apoptotic cell ratio and apoptosis-associated proteins expression. Results The surviving fraction and cell viability of glioma stem/progenitor cells were decreased compared with differentiated glioma cells using the same boronophenylalanine pretreatment and the same dose of neutron flux. BNCT induced cell cycle arrest in the G2/M phase and cell apoptosis via the mitochondrial pathway, with changes in the expression of associated proteins. Conclusions Glioma stem/progenitor cells, which are resistant to current clinical radiotherapy, could be effectively killed by BNCT in vitro via cell cycle arrest and apoptosis using a prolonged neutron irradiation, although radiosensitivity of glioma stem/progenitor cells was decreased compared with differentiated glioma cells when using the same dose of thermal neutron exposure and boronophenylalanine pretreatment. Thus, BNCT could offer an appreciable therapeutic advantage to prevent tumor recurrence, and may become a promising treatment in recurrent glioma. PMID:23915425

  8. Stereospecific induction of apoptosis in tumor cells via endogenous C16-ceramide and distinct transcripts

    PubMed Central

    Blaess, M; Le, HP; Claus, RA; Kohl, M; Deigner, H-P

    2015-01-01

    Concentration and distribution of individual endogenous ceramide species is crucial for apoptosis induction in response to various stimuli. Exogenous ceramide analogs induce apoptosis and can in turn modify the composition/concentrations of endogenous ceramide species and associated signaling. In this study, we show here that the elevation of endogenous C16-ceramide levels is a common feature of several known apoptosis-inducing triggers like mmLDL, TNF-alpha, H2O2 and exogenous C6-ceramide. Vice versa apoptosis requires elevation of endogenous C16-ceramide levels in cells. Enantiomers of a synthetic ceramide analog HPL-1RS36N have been developed as probes and vary in their capacity to inducing apoptosis in macrophages and HT-29 cells. Apoptosis induction by the two synthetic ceramide analogs HPL-39N and HPL-1R36N correlates with generation of cellular C16-ceramide concentration. In contrast to the S-enantiomer HPL-1S36N, the R-enantiomer HPL-1R36N shows significant effects on the expression of distinct genes known to be involved in cell cycle, cell growth and cell death (CXCL10, CCL5 and TNF-alpha), similarly on apoptosis induction. Enantioselective effects on transcription induced by metabolically stable synthetic probes provide clues on molecular mechanisms of ceramide-induced signaling, as well as leads for future anti-cancer agents. PMID:27551447

  9. Effect of arsenic, cadmium and lead on the induction of apoptosis of normal human mononuclear cells

    PubMed Central

    DE LA FUENTE, H; PORTALES-PÉREZ, D; BARANDA, L; DÍAZ-BARRIGA, F; SAAVEDRA-ALANÍS, V; LAYSECA, E; GONZÁLEZ-AMARO, R

    2002-01-01

    The aim of this work was to investigate the effect of cadmium, lead and arsenic on the apoptosis of human immune cells. Peripheral blood mononuclear cells (MNC) were incubated with increasing concentrations of these metals and then cellular apoptosis was determined by flow cytometry and by DNA electrophoresis. We found that arsenic induced a significant level of apoptosis at 15 μm after 48h of incubation. Cadmium had a similar effect, but at higher concentrations (65 μm). In addition, cadmium exerted a cytotoxic effect on MNC that seemed to be independent of the induction of apoptosis. In contrast, concentrations of lead as high as 500 μm were nontoxic and did not induce a significant degree of apoptosis. Additional experiments showed that arsenic at concentrations as low as 1·0 μm had a significant pro-apoptotic effect when cells were cultured in the presence of this pollutant for more than 72. Non-T cells were more susceptible than T lymphocytes to the effect of arsenic and cadmium. Interestingly, MNC from children chronically exposed to arsenic showed a high basal rate of apoptosis and a diminished in vitro sensibility to this metalloid. Our results indicate that both arsenic and cadmium are able to induce apoptosis of lymphoid cells, and suggest that this phenomenon may contribute to their immunotoxic effect in vivo. PMID:12100024

  10. Okadaic acid inhibits cell multiplication and induces apoptosis in a549 cells, a human lung adenocarcinoma cell line

    PubMed Central

    Wang, Renjun; Lv, Lili; Zhao, Yunfeng; Yang, Nana

    2014-01-01

    This essay aims to research the effect of okadaic acid (OA) on A549 cell multiplication, and cell apoptosis induced by OA was observed by cell morphology. MTT assay, trypan blue exclusion test (TBET), Giemsa staining method and acridine orange (AO) fluorescence staining assay were applied. The results of cell survival evaluated by TBET and colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) showed: The number of A549 cells was decreased in a dose-dependent manner. Cytomorphology observation of okadaic acid-treated cells showed that cells became shrinkage and turned round, some cells floated in the nutrient medium with nucleus agglutination broken, resulting in apoptotic bodies. Above-mentioned results indicated that OA exerted significantly inhibitory effect on A549 cell multiplication due to the apoptosis induced by OA. PMID:25232383

  11. Th9 Cells Drive Host Immunity against Gastrointestinal Worm Infection.

    PubMed

    Licona-Limón, Paula; Henao-Mejia, Jorge; Temann, Angela U; Gagliani, Nicola; Licona-Limón, Ileana; Ishigame, Harumichi; Hao, Liming; Herbert, De'broski R; Flavell, Richard A

    2013-10-17

    Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4⁺ effector T cells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection.

  12. Tocilizumab inhibits neuronal cell apoptosis and activates STAT3 in cerebral infarction rat model

    PubMed Central

    Wang, Shaojun; Zhou, Jun; Kang, Weijie; Dong, Zhaoni; Wang, Hezuo

    2016-01-01

    Cerebral infarction is a severe hypoxic ischemic necrosis with accelerated neuronal cell apoptosis in the brain. As a monoclonal antibody against interleukin 6, tocilizumab (TCZ) is widely used in immune diseases, whose function in cerebral infarction has not been studied. This study aims to reveal the role of TCZ in regulating neuronal cell apoptosis in cerebral infarction. The cerebral infarction rat model was constructed by middle cerebral artery occlusion and treated with TCZ. Cell apoptosis in hippocampus and cortex of the brain was examined with TUNEL method. Rat neuronal cells cultured in oxygen-glucose deprivation (OGD) conditions and treated with TCZ were used to compare cell viability and apoptosis. Apoptosis-related factors including B-cell lymphoma extra large (Bcl-xL) and Caspase 3, as well as the phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in brain cortex were analyzed from the protein level. Results indicated that TCZ treatment could significantly prevent the promoted cell apoptosis caused by cerebral infarction or OGD (P < 0.05 or P < 0.01). In brain cortex of the rat model, TCZ up-regulated Bcl-xL and down-regulated Caspase 3, consistent with the inhibited cell apoptosis. It also promoted tyrosine 705 phosphorylation of STAT3, which might be the potential regulatory mechanism of TCZ in neuronal cells. This study provided evidence for the protective role of TCZ against neuronal cell apoptosis in cerebral infarction. Based on these fundamental data, TCZ is a promising option for treating cerebral infarction, but further investigations on related mechanisms are still necessary. PMID:26773188

  13. Apoptosis and proliferation of oligodendrocyte progenitor cells in the irradiated rodent spinal cord

    SciTech Connect

    Atkinson, Shelley L.; Li Yuqing; Wong, C. Shun . E-mail: shun.wong@sw.ca

    2005-06-01

    Purpose: Oligodendrocytes undergo early apoptosis after irradiation. The aim of this study was to determine the relationship between oligodendroglial apoptosis and proliferation of oligodendrocyte progenitor cells (OPC) in the irradiated central nervous system. Methods and Materials: Adult rats and p53 transgenic mice were given single doses of 2 Gy, 8 Gy, or 22 Gy to the cervical spinal cord. Apoptosis was assessed using TUNEL (Tdt-mediated dUTP terminal nick-end labeling) staining or by examining nuclear morphology. Oligodendrocyte progenitor cells were identified with an NG2 antibody or by in situ hybridization for platelet-derived growth factor receptor {alpha}. Proliferation of OPC was assessed by in vivo bromodeoxyuridine (BrdU) labeling and subsequent immunohistochemistry. Because radiation-induced apoptosis of oligodendroglial cells is p53 dependent, p53 transgenic mice were used to study the relationship between apoptosis and cell proliferation. Results: Oligodendrocyte progenitor cells underwent apoptosis within 24 h of irradiation in the rat. That did not result in a change in OPC density at 24 h. Oligodendrocyte progenitor cell density was significantly reduced by 2-4 weeks, but showed recovery by 6 weeks after irradiation. An increase in BrdU-labeled cells was observed at 2 weeks after 8 Gy or 22 Gy, and proliferating cells in the rat spinal cord were immunoreactive for NG2. The mouse spinal cord showed a similar early cell proliferation after irradiation. No difference was observed in the proliferation response in the spinal cord of p53 -/- mice compared with wild type animals. Conclusions: Oligodendroglial cells undergo early apoptosis and OPC undergo early proliferation after ionizing radiation. However, apoptosis is not likely to be the trigger for early proliferation of OPC in the irradiated central nervous system.

  14. Cell-permeable intrinsic cellular inhibitors of apoptosis protect and rescue intestinal epithelial cells from radiation-induced cell death.

    PubMed

    Matsuzaki-Horibuchi, Shiori; Yasuda, Takeshi; Sakaguchi, Nagako; Yamaguchi, Yoshihiro; Akashi, Makoto

    2015-01-01

    One of the important mechanisms for gastrointestinal (GI) injury following high-dose radiation exposure is apoptosis of epithelial cells. X-linked inhibitor of apoptosis (XIAP) and cellular IAP2 (cIAP2) are intrinsic cellular inhibitors of apoptosis. In order to study the effects of exogenously added IAPs on apoptosis in intestinal epithelial cells, we constructed bacterial expression plasmids containing genes of XIAP (full-length, BIR2 domain and BIR3-RING domain with and without mutations of auto-ubiquitylation sites) and cIAP2 proteins fused to a protein-transduction domain (PTD) derived from HIV-1 Tat protein (TAT) and purified these cell-permeable recombinant proteins. When the TAT-conjugated IAPs were added to rat intestinal epithelial cells IEC6, these proteins were effectively delivered into the cells and inhibited apoptosis, even when added after irradiation. Our results suggest that PTD-mediated delivery of IAPs may have clinical potential, not only for radioprotection but also for rescuing the GI system from radiation injuries.

  15. Phosphoribosylpyrophosphate Synthetase 1 Knockdown Suppresses Tumor Formation of Glioma CD133+ Cells Through Upregulating Cell Apoptosis.

    PubMed

    Li, Chen; Yan, Zhongjie; Cao, Xuhua; Zhang, Xiaowei; Yang, Liang

    2016-10-01

    Relapse is the main cause of mortality in patients with glioblastoma multiforme (GBM). Treatment options involve surgical resection followed by a combination of radiotheraphy and chemotherapy with temozolomide. Several genes and genetic pathways have been identified to contribute to therapeutic resistance, giving rise to recurrence of the malignancy. In the last decades, glioma stem cells (GSCs) with the capacity of self-renewal have been demonstrated to maintain tumor propagation and treatment resistance. Here, we isolated CD133-positive (CD133+) and CD133-negative (CD133-) cells from glioblastoma U98G and U87MG cell lines. The role of phosphoribosylpyrophosphate synthetase 1 (PRPS1), which catalyzes the first step of the synthesis of nucleotide, in proliferation and apoptosis was investigated. We found that PRPS1 had a remarkable effect on cell proliferation and sphere formation in both CD133+ and CD133- cells. Compared to CD133- cells, CD133+ cells exhibited more significant results in cell apoptosis assay. CD133+ T98G and U87MG cells were used in xenograft mouse model of tumor formation. Interestingly, the mice implanted with PRPS1 knockdown T98G or U87MG stem cells exhibited prolonged survival time and reduced tumor volume. By immunostaining caspase-3 in tumor tissues of these mice, we demonstrated that the apoptotic activities in tumor cells were positively correlated to the survival time but negatively correlated to PRPS1 expression. Our results indicate that PRPS1 plays an important role in proliferation and apoptosis in GSCs and provide new clues for potential PRPS1-targeted therapy in GBM treatment. PMID:27343059

  16. THE PROS AND CONS OF APOPTOSIS ASSAYS FOR USE IN THE STUDY OF CELLS, TISSUES AND ORGANS

    EPA Science Inventory

    Abstract
    Programmed cell death or apoptosis occurs in many tissues during normal development and in the normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and disease. Increased interest in apoptosis and cell death in general...

  17. Profiling of Host Cell Response to Successive Canine Parvovirus Infection Based on Kinetic Proteomic Change Identification.

    PubMed

    Zhao, Hang; Cheng, Yuening; Wang, Jianke; Lin, Peng; Yi, Li; Sun, Yaru; Ren, Jingqiang; Tong, Mingwei; Cao, Zhigang; Li, Jiawei; Deng, Jinliang; Cheng, Shipeng

    2016-01-01

    Canine parvovirus (CPV) reproduces by co-opting the resources of host cells, inevitably causing cytotoxic effects to the host cells. Feline kidney F81 cells are sensitive to CPV infection and show disparate growing statuses at different time points post-infection. This study analysed the response of F81 cells to CPV infection at successive infection time points by iTRAQ-based quantitative proteomics. Differentially expressed proteins (DEPs) during 60 h of infection and at selected time points post-infection were identified by an analysis of variance test and a two-tailed unpaired t test, respectively. DEPs with similar quantitative changes were clustered by hierarchical clustering and analysed by gene ontology enrichment, revealing that 12 h and 60 h post-infection were the optimal times to analyse the autonomous parvovirus replication and apoptosis processes, respectively. Using the Metacore(TM) database, 29 DEPs were enriched in a network involved in p53 regulation. Besides, a significantly enriched pathway suggests that the CPV-induced cytopathic effect was probably due to the deficiency of functional CFTR caused by CPV infection. This study uncovered the systemic changes in key cellular factors involved in CPV infection and help to understand the molecular mechanisms of the anti-cancer activity of CPV and the cytopathic effects induced by CPV infection. PMID:27406444

  18. Profiling of Host Cell Response to Successive Canine Parvovirus Infection Based on Kinetic Proteomic Change Identification

    PubMed Central

    Zhao, Hang; Cheng, Yuening; Wang, Jianke; Lin, Peng; Yi, Li; Sun, Yaru; Ren, Jingqiang; Tong, Mingwei; Cao, Zhigang; Li, Jiawei; Deng, Jinliang; Cheng, Shipeng

    2016-01-01

    Canine parvovirus (CPV) reproduces by co-opting the resources of host cells, inevitably causing cytotoxic effects to the host cells. Feline kidney F81 cells are sensitive to CPV infection and show disparate growing statuses at different time points post-infection. This study analysed the response of F81 cells to CPV infection at successive infection time points by iTRAQ-based quantitative proteomics. Differentially expressed proteins (DEPs) during 60 h of infection and at selected time points post-infection were identified by an analysis of variance test and a two-tailed unpaired t test, respectively. DEPs with similar quantitative changes were clustered by hierarchical clustering and analysed by gene ontology enrichment, revealing that 12 h and 60 h post-infection were the optimal times to analyse the autonomous parvovirus replication and apoptosis processes, respectively. Using the MetacoreTM database, 29 DEPs were enriched in a network involved in p53 regulation. Besides, a significantly enriched pathway suggests that the CPV-induced cytopathic effect was probably due to the deficiency of functional CFTR caused by CPV infection. This study uncovered the systemic changes in key cellular factors involved in CPV infection and help to understand the molecular mechanisms of the anti-cancer activity of CPV and the cytopathic effects induced by CPV infection. PMID:27406444

  19. Profiling of Host Cell Response to Successive Canine Parvovirus Infection Based on Kinetic Proteomic Change Identification.

    PubMed

    Zhao, Hang; Cheng, Yuening; Wang, Jianke; Lin, Peng; Yi, Li; Sun, Yaru; Ren, Jingqiang; Tong, Mingwei; Cao, Zhigang; Li, Jiawei; Deng, Jinliang; Cheng, Shipeng

    2016-01-01

    Canine parvovirus (CPV) reproduces by co-opting the resources of host cells, inevitably causing cytotoxic effects to the host cells. Feline kidney F81 cells are sensitive to CPV infection and show disparate growing statuses at different time points post-infection. This study analysed the response of F81 cells to CPV infection at successive infection time points by iTRAQ-based quantitative proteomics. Differentially expressed proteins (DEPs) during 60 h of infection and at selected time points post-infection were identified by an analysis of variance test and a two-tailed unpaired t test, respectively. DEPs with similar quantitative changes were clustered by hierarchical clustering and analysed by gene ontology enrichment, revealing that 12 h and 60 h post-infection were the optimal times to analyse the autonomous parvovirus replication and apoptosis processes, respectively. Using the Metacore(TM) database, 29 DEPs were enriched in a network involved in p53 regulation. Besides, a significantly enriched pathway suggests that the CPV-induced cytopathic effect was probably due to the deficiency of functional CFTR caused by CPV infection. This study uncovered the systemic changes in key cellular factors involved in CPV infection and help to understand the molecular mechanisms of the anti-cancer activity of CPV and the cytopathic effects induced by CPV infection.

  20. Clustered Intracellular Salmonella enterica Serovar Typhimurium Blocks Host Cell Cytokinesis

    PubMed Central

    Durkin, Charlotte H.; Helaine, Sophie; Boucrot, Emmanuel

    2016-01-01

    Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonize the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonization. We found that intracellular Salmonella enterica serovar Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 h postinvasion and that the effect was dependent on an intact Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organizing center of infected epithelial cells. During host cell division, these clustered microcolonies prevented the correct localization of members of the chromosomal passenger complex and mitotic kinesin-like protein 1 and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, resulting in either chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S. Typhimurium delays epithelial cell turnover in the intestine. PMID:27185791

  1. Clustered Intracellular Salmonella enterica Serovar Typhimurium Blocks Host Cell Cytokinesis.

    PubMed

    Santos, António J M; Durkin, Charlotte H; Helaine, Sophie; Boucrot, Emmanuel; Holden, David W

    2016-07-01

    Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonize the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonization. We found that intracellular Salmonella enterica serovar Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 h postinvasion and that the effect was dependent on an intact Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organizing center of infected epithelial cells. During host cell division, these clustered microcolonies prevented the correct localization of members of the chromosomal passenger complex and mitotic kinesin-like protein 1 and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, resulting in either chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S Typhimurium delays epithelial cell turnover in the intestine.

  2. A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9.

    PubMed

    Shu, Benshui; Wang, Wenxiang; Hu, Qingbo; Huang, Jingfei; Hu, Meiying; Zhong, Guohua

    2015-07-01

    The induction of apoptosis by azadirachtin, a well-known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC50 at 48 and 72 h was 2.727 × 10(-6) and 6.348 × 10(-9) μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase-1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase-dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds. PMID:25828604

  3. Low intensity-pulsed ultrasound induced apoptosis of human hepatocellular carcinoma cells in vitro.

    PubMed

    Shi, Mingfang; Liu, Bangzhong; Liu, Guanghua; Wang, Ping; Yang, Mingzhen; Li, Yun; Zhou, Jian

    2016-01-01

    The present study was conducted to determine whether low intensity-pulsed ultrasound (LIPUS) could induce apoptosis of human hepatocellular carcinoma cells, SMMC-7721, and to define the mechanism of ultrasound-induced apoptosis, in vitro. MTT assay was used to measure cell proliferation. Apoptosis was investigated by multiple methods such as flow cytometry, DNA fragmentation, Ca(2+) mobilizations, pro- and anti-apoptotic protein expression, and light as well as ultramicroscopic morphology. The results provide evidence that LIPUS induced a dose-dependent effect on cell viability and apoptosis of SMMC-7721 cells. Specifically, exposure of cells to >0.5 W/cm(2) intensity significantly increased cell apoptosis, caused shifts in cell cycle phase, and induced structural changes. Ultrasound significantly increased intracellular Ca(2+) concentrations and modulated expression of caspase-3, Bcl-2 and Bax. The findings suggest that this novel technology can be used to induce SMMC-7721 apoptosis via the Ca(2+)/mitochondrial pathway and could potentially be of clinical use for the treatment of hepatocellular carcinoma (SMMC-7721 cell line) and other cancers.

  4. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro.

    PubMed

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-05-11

    Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 μM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer. PMID:22546556

  5. Low-power laser irradiation inhibits amyloid beta-induced cell apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Heng; Wu, Shengnan

    2011-03-01

    The deposition and accumulation of amyloid-β-peptide (Aβ) in the brain are considered a pathological hallmark of Alzheimer's disease(AD). Apoptosis is a contributing pathophysiological mechanism of AD. Low-power laser irradiation (LPLI), a non-damage physical therapy, which has been used clinically for decades of years, is shown to promote cell proliferation and prevent apoptosis. Recently, low-power laser irradiation (LPLI) has been applied to moderate AD. In this study, Rat pheochromocytoma (PC12) cells were treated with amyloid beta 25-35 (Aβ25-35) for induction of apoptosis before LPLI treatment. We measured cell viability with CCK-8 according to the manufacture's protocol, the cell viability assays show that low fluence of LPLI (2 J/cm2 ) could inhibit the cells apoptosis. Then using statistical analysis of proportion of apoptotic cells by flow cytometry based on Annexin V-FITC/PI, the assays also reveal that low fluence of LPLI (2 J/cm2 ) could inhibit the Aβ-induced cell apoptosis. Taken together, we demonstrated that low fluence of LPLI (2 J/cm2 ) could inhibit the Aβ-induced cell apoptosis, these results directly point to a therapeutic strategy for the treatment of AD through LPLI.

  6. Asymmetric dimethylarginine (ADMA) treatment induces apoptosis in cultured rat mesangial cells via endoplasmic reticulum stress activation.

    PubMed

    Park, Min-Jung; Oh, Ki-Seok; Nho, Jong-Hyun; Kim, Gye-Yeop; Kim, Dong-Il

    2016-06-01

    Asymmetric dimethylarginine (ADMA), a high risk factor for endothelial dysfunction and cardiovascular disease (CVD), has been reported to promote cellular dysfunction via endoplasmic reticulum (ER) stress activation in various cells. Additionally, increased serum ADMA levels have been observed in incipient kidney diseases. Previously, we reported that activated ER stress is associated with mesangial cell apoptosis, observed mainly in overt nephropathy or chronic kidney disease (CKD). However, the effect of ADMA on mesangial cell apoptosis is unknown. Thus, we investigated the effects of ADMA on mesangial cell apoptosis and ER stress signaling. ADMA treatment increased caspase-3 activity and activated three branches of ER stress signaling (PERK, IRE1, and ATF6) that induce mesangial cell apoptosis. Pharmacological inhibitors of ER stress (inhibitors of PERK, IRE1, and S1P) attenuated ADMA-induced cleavage of caspase-3 and induced a decrease in the mitochondrial membrane potential. Furthermore, these inhibitors diminished the number of apoptotic cells induced by ADMA treatment. Taken together, our results indicated that ADMA treatment induces mesangial cell apoptosis via ER stress signaling. These results suggest that ADMA-induced mesangial cell apoptosis could contribute to the progression of overt nephropathy and CKD.

  7. Aminomethylphosphonic acid and methoxyacetic acid induce apoptosis in prostate cancer cells.

    PubMed

    Parajuli, Keshab R; Zhang, Qiuyang; Liu, Sen; You, Zongbing

    2015-01-01

    Aminomethylphosphonic acid (AMPA) and its parent compound herbicide glyphosate are analogs to glycine, which have been reported to inhibit proliferation and promote apoptosis of cancer cells, but not normal cells. Methoxyacetic acid (MAA) is the active metabolite of ester phthalates widely used in industry as gelling, viscosity and stabilizer; its exposure is associated with developmental and reproductive toxicities in both rodents and humans. MAA has been reported to suppress prostate cancer cell growth by inducing growth arrest and apoptosis. However, it is unknown whether AMPA and MAA can inhibit cancer cell growth. In this study, we found that AMPA and MAA inhibited cell growth in prostate cancer cell lines (LNCaP, C4-2B, PC-3 and DU-145) through induction of apoptosis and cell cycle arrest at the G1 phase. Importantly, the AMPA-induced apoptosis was potentiated with the addition of MAA, which was due to downregulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2), leading to activation of caspases 7 and 3. These results demonstrate that the combination of AMPA and MAA can promote the apoptosis of prostate cancer cells, suggesting that they can be used as potential therapeutic drugs in the treatment of prostate cancer.

  8. Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality

    PubMed Central

    O’Connor, Roddy S.; Thangavelu, Govindarajan; Lovitch, Scott B.; Dandamudi, Durga Bhavani; Vincent, Benjamin G.; Tkachev, Victor; Pawlicki, Jan M.; Furlan, Scott N.; Kean, Leslie S.; Aoyama, Kazutoshi; Taylor, Patricia A.; Panoskaltsis-Mortari, Angela; Foncea, Rocio; Ranganathan, Parvathi; Devine, Steven M.; Burrill, Joel S.; Guo, Lili; Sacristan, Catarina; Snyder, Nathaniel W.; Blair, Ian A.; Milone, Michael C.; Dustin, Michael L.; Riley, James L.; Bernlohr, David A.; Murphy, William J.; Fife, Brian T.; Munn, David H.; Miller, Jeffrey S.; Serody, Jonathan S.; Freeman, Gordon J.; Sharpe, Arlene H.; Turka, Laurence A.

    2016-01-01

    Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1–/– donors. PD-L1–deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1–/– donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell–mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD. PMID:27294527

  9. Clinostat rotation induces apoptosis in luteal cells of the pregnant rat

    NASA Technical Reports Server (NTRS)

    Yang, Hyunwon; Bhat, Ganapathy K.; Sridaran, Rajagopala

    2002-01-01

    Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.

  10. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    SciTech Connect

    Sun, Bin; Cai, Yingyue; Li, Yongshu; Li, Jingjing; Liu, Kaiyu; Li, Yi; Yang, Yongbo

    2013-05-25

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization.

  11. Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

    PubMed Central

    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-apoptosis

  12. Perfluorooctanesulfonate Mediates Renal Tubular Cell Apoptosis through PPARgamma Inactivation.

    PubMed

    Wen, Li-Li; Lin, Chien-Yu; Chou, Hsiu-Chu; Chang, Chih-Cheng; Lo, Hau-Yin; Juan, Shu-Hui

    2016-01-01

    Perfluorinated chemicals (PFCs) are ubiquitously distributed in the environments including stainless pan-coating, raincoat, fire extinguisher, and semiconductor products. The PPAR family has been shown to contribute to the toxic effects of PFCs in thymus, immune and excretory systems. Herein, we demonstrated that perfluorooctanesulfonate (PFOS) caused cell apoptosis through increasing ratio of Bcl-xS/xL, cytosolic cytochrome C, and caspase 3 activation in renal tubular cells (RTCs). In addition, PFOS increased transcription of inflammatory cytokines (i.e., TNFα, ICAM1, and MCP1) by NFκB activation. Conversely, PFOS reduced the mRNA levels of antioxidative enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase, as a result of reduced PPARγ transactivational activity by using reporter and chromatin immuoprecipitation (ChIP) assays. PFOS reduced the protein interaction between PPARγ and PPARγ coactivator-1 alpha (PGC1α) by PPARγ deacetylation through Sirt1 upregulation, of which the binding of PPARγ and PGC1α to a peroxisome proliferator response element (PPRE) in the promoter regions of these antioxidative enzymes was alleviated in the ChIP assay. Furthermore, Sirt1 also deacetylated p53 and then increased the binding of p53 to Bax, resulting in increased cytosolic cytochrome C. The effect of PPARγ inactivation by PFOS was validated using the PPARγ antagonist GW9662, whereas the adverse effects of PFOS were prevented by PPARγ overexpression and activators, rosiglitozone and L-carnitine, in RTCs. The in vitro finding of protective effect of L-carnitine was substantiated in vivo using Balb/c mice model subjected to PFOS challenge. Altogether, we provide in vivo and in vitro evidence for the protective mechanism of L-carnitine in eliminating PFOS-mediated renal injury, at least partially, through PPARγ activation. PMID:27171144

  13. Perfluorooctanesulfonate Mediates Renal Tubular Cell Apoptosis through PPARgamma Inactivation

    PubMed Central

    Chou, Hsiu-Chu; Chang, Chih-Cheng; Lo, Hau-Yin; Juan, Shu-Hui

    2016-01-01

    Perfluorinated chemicals (PFCs) are ubiquitously distributed in the environments including stainless pan-coating, raincoat, fire extinguisher, and semiconductor products. The PPAR family has been shown to contribute to the toxic effects of PFCs in thymus, immune and excretory systems. Herein, we demonstrated that perfluorooctanesulfonate (PFOS) caused cell apoptosis through increasing ratio of Bcl-xS/xL, cytosolic cytochrome C, and caspase 3 activation in renal tubular cells (RTCs). In addition, PFOS increased transcription of inflammatory cytokines (i.e., TNFα, ICAM1, and MCP1) by NFκB activation. Conversely, PFOS reduced the mRNA levels of antioxidative enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase, as a result of reduced PPARγ transactivational activity by using reporter and chromatin immuoprecipitation (ChIP) assays. PFOS reduced the protein interaction between PPARγ and PPARγ coactivator-1 alpha (PGC1α) by PPARγ deacetylation through Sirt1 upregulation, of which the binding of PPARγ and PGC1α to a peroxisome proliferator response element (PPRE) in the promoter regions of these antioxidative enzymes was alleviated in the ChIP assay. Furthermore, Sirt1 also deacetylated p53 and then increased the binding of p53 to Bax, resulting in increased cytosolic cytochrome C. The effect of PPARγ inactivation by PFOS was validated using the PPARγ antagonist GW9662, whereas the adverse effects of PFOS were prevented by PPARγ overexpression and activators, rosiglitozone and L-carnitine, in RTCs. The in vitro finding of protective effect of L-carnitine was substantiated in vivo using Balb/c mice model subjected to PFOS challenge. Altogether, we provide in vivo and in vitro evidence for the protective mechanism of L-carnitine in eliminating PFOS-mediated renal injury, at least partially, through PPARγ activation. PMID:27171144

  14. An insulin-like growth factor homologue of Singapore grouper iridovirus modulates cell proliferation, apoptosis and enhances viral replication.

    PubMed

    Yan, Yang; Cui, Huachun; Guo, Chuanyu; Li, Jun; Huang, Xiaohong; Wei, Jingguang; Qin, Qiwei

    2013-12-01

    Insulin-like growth factors (IGFs) play crucial roles in regulating cell differentiation, proliferation and apoptosis. In this study, a novel IGF homologue gene (IGF-like) encoded by Singapore grouper iridovirus (SGIV) ORF062R (termed SGIV-IGF), was cloned and characterized. The coding region of SGIV-IGF is 771 bp in length, with a variable number of tandem repeats (VNTR) locus at the 3'-end. We cloned one isoform of this novel gene, 582 bp in length, containing the predicted IGF domain and 3.6 copy numbers of the 27 bp repeat unit. SGIV-IGF was an early transcribed gene during viral infection, and SGIV-IGF was distributed predominantly in the cytoplasm with a diffused granular appearance. Intriguingly, overexpression of SGIV-IGF was able to promote the growth of grouper embryonic cells (GP cells) by promoting G1/S phase transition, which was at least partially dependent on its 3'-end VNTR locus. Furthermore, viral titre assay and real-time quantitative PCR (RT-qPCR) analysis proved that SGIV-IGF could promote SGIV replication in grouper cells. In addition, overexpression of SGIV-IGF mildly facilitated apoptosis in SGIV-infected non-host fathead minnow (FHM) cells. Together, our study demonstrated a novel functional gene of SGIV which may regulate viral replication and cellular processes through multiple mechanisms that appear to be cell type-dependent.

  15. Receptor-binding cancer antigen expressed on SiSo cells induces apoptosis via ectodomain shedding.

    PubMed

    Sonoda, Kenzo; Miyamoto, Shingo; Nakashima, Manabu; Wake, Norio

    2010-07-01

    Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a secreted antigen that induces apoptosis in putative receptor-expressing cells, including peripheral lymphocytes and natural killer (NK) cells. RCAS1 expression is associated with aggressive characteristics and poor overall survival for 15 different human malignancies. The putative RCAS1 receptor has not been isolated and the mechanism of RCAS1 apoptosis induction remains unclear. This study explores how RCAS1 is involved in apoptosis initiation. The cell lines SiSo and MCF-7, human uterine carcinoma and breast adenocarcinoma, respectively, both express RCAS1, but RCAS1 secretion is undetectable in MCF-7 cells. SiSo and MCF-7 cells were stimulated to induce RCAS1 ectodomain shedding followed by assessment of RCAS1 expression and secretion. Additionally, the RCAS1 putative receptor-expressing human chronic myelogenous leukemia cell line K562 was co-cultured with SiSo, MCF-7, or soluble RCAS1 to follow RCAS1 secretion in apoptosis initiation. RCAS1 secretion was strongly suppressed by inhibitors of metalloproteases, protein kinase C (PKC)-delta, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK), epidermal growth factor (EGF), and G-protein-coupled receptor (GPCR). K562 apoptosis could be induced only by co-culturing with SiSo or soluble RCAS1. RCAS1 is thus secreted by ectodomain shedding, which may represent a pivotal step in RCAS1-induced apoptosis initiation.

  16. Apoptosis of human gastric carcinoma cells induced by Euphorbia esula latex

    PubMed Central

    Fu, Zhao-Ying; Han, Xiao-Dong; Wang, Ai-Hong; Liu, Xiao-Bin

    2016-01-01

    AIM: To investigate the effect of Euphorbia esula (E. esula) extract in inhibiting proliferation and inducing apoptosis in SGC-7901 cells. METHODS: E. esula extract at different concentrations was used to inhibit proliferation and induce apoptosis of human gastric carcinoma SGC-7901 cells. Inhibition of proliferation was detected with thiazolyl blue assay, and apoptosis was detected with fluorescence microscopy, transmission electron microscopy, and flow cytometry. The mechanisms were studied by measurement of caspase-3 and caspase-8 activities and Bax and Bcl2 mRNA expression. RESULTS: The thiazolyl blue assay showed that SGC-7901 cell viability and proliferation were inhibited significantly by E. esula extract in a time- and concentration-dependent manner. Fluorescence microscopy revealed that the cell nuclei showed the characteristic changes of apoptosis, such as uneven staining and chromatin marginalization. Some key features of apoptosis were also observed under transmission electron microscopy, which included cellular shrinkage and the foaming or bubbling phenomenon. When the cells were analyzed by flow cytometry, a sub-G1 peak could be seen clearly. Spectrophotometric assay of caspase-3 and caspase-8 activities in the treated cells showed an approximately two-fold increase. Reverse transcription polymerase chain reaction showed that Bax mRNA expression was upregulated, while Bcl2 mRNA expression was downregulated. CONCLUSION: E. esula extract inhibited proliferation and induced apoptosis in SGC-7901 cells, in a caspase-dependent manner, involving upregulation of Bax and downregulation of Bcl2. PMID:27053848

  17. Depletion of mitochondrial fission factor DRP1 causes increased apoptosis in human colon cancer cells

    SciTech Connect

    Inoue-Yamauchi, Akane; Oda, Hideaki

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer DRP1 is required for mitochondrial fission in colon cancer cells. Black-Right-Pointing-Pointer DRP1 participates in inhibition of colon cancer cell apoptosis. Black-Right-Pointing-Pointer DRP1 can inhibit apoptosis through the regulation of cytochrome c release. -- Abstract: Mitochondria play a critical role in regulation of apoptosis, a form of programmed cell death, by releasing apoptogenic factors including cytochrome c. Growing evidence suggests that dynamic changes in mitochondrial morphology are involved in cellular apoptotic response. However, whether DRP1-mediated mitochondrial fission is required for induction of apoptosis remains speculative. Here, we show that siRNA-mediated DRP1 knockdown promoted accumulation of elongated mitochondria in HCT116 and SW480 human colon cancer cells. Surprisingly, DRP1 down-regulation led to decreased proliferation and increased apoptosis of these cells. A higher rate of cytochrome c release and reductions in mitochondrial membrane potential were also revealed in DRP1-depleted cells. Taken together, our present findings suggest that mitochondrial fission factor DRP1 inhibits colon cancer cell apoptosis through the regulation of cytochrome c release and mitochondrial membrane integrity.

  18. Chloride channel protein 2 prevents glutamate-induced apoptosis in retinal ganglion cells

    PubMed Central

    Bi, Miao-Miao; Hong, Sen; Ma, Ling-Jun; Zhou, Hong-Yan; Lu, Jia; Zhao, Jing; Zheng, Ya-Juan

    2016-01-01

    Objective(s): The purpose of this study was to investigate the role of chloride channel protein 2 (ClC-2) in glutamate-induced apoptosis in the retinal ganglion cell line (RGC-5). Materials and Methods: RGC-5 cells were treated with 1 mM glutamate for 24 hr. The expression of ClC-2, Bax, and Bcl-2 was detected by western blot analysis. Cell survival and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Caspase-3 and -9 activities were determined by a colorimetric assay. The roles of ClC-2 in glutamate-induced apoptosis were examined by using ClC-2 complementary deoxyribonucleic acid (cDNA) and small inference ribonucleic acid (RNA) transfection technology. Results: Overexpression of ClC-2 in RGC-5 cells significantly decreased glutamate-induced apoptosis and increased cell viability, whereas silencing of ClC-2 with short hairpin (sh) RNA produced opposite effects. ClC-2 overexpression increased the expression of Bcl-2, decreased the expression of Bax, and decreased caspase-3 and -9 activation in RGC-5 cells treated with glutamate, but silencing of ClC-2 produced opposite effects. Conclusion: Our data suggest that ClC-2 chloride channels might play a protective role in glutamate-induced apoptosis in retinal ganglion cells via the mitochondria-dependent apoptosis pathway.

  19. Promyelocytic leukemia protein enhances apoptosis of gastric cancer cells through Yes-associated protein.

    PubMed

    Xu, Zhipeng; Chen, Jiamin; Shao, Liming; Ma, Wangqian; Xu, Dingting

    2015-09-01

    It has been shown that Yes-associated protein (YAP) acts as a transcriptional co-activator to regulate p73-dependent apoptosis in response to DNA damage in some cell types, and promyelocytic leukemia (PML) protein is involved in the regulation loop through stabilization of YAP through sumoylation. Although YAP has been shown to be significantly upregulated in gastric cancer, whether the YAP/PML/p73 regulation loop also functions in gastric cancer is unknown. Here, we show significantly higher levels of YAP and significantly lower levels of PML in the gastric cancer specimen. Overexpression of YAP in gastric cancer cells significantly increased cell growth, but did not affect apoptosis. However, overexpression of PML in gastric cancer cells significantly increased cell apoptosis, resulting in decreases in cell growth, which seemed to require the presence of YAP. The effect of PML on apoptosis appeared to be conducted through p73-mediated modulation of apoptosis-associated genes, Bcl-2, Bak, and caspase9. Thus, our study suggests the presence of a YAP/PML/p73 regulatory loop in gastric cancer, and highlights PML as a promising tumor suppressor in gastric cancer through YAP-coordinated cancer cell apoptosis.

  20. Chloride channel protein 2 prevents glutamate-induced apoptosis in retinal ganglion cells

    PubMed Central

    Bi, Miao-Miao; Hong, Sen; Ma, Ling-Jun; Zhou, Hong-Yan; Lu, Jia; Zhao, Jing; Zheng, Ya-Juan

    2016-01-01

    Objective(s): The purpose of this study was to investigate the role of chloride channel protein 2 (ClC-2) in glutamate-induced apoptosis in the retinal ganglion cell line (RGC-5). Materials and Methods: RGC-5 cells were treated with 1 mM glutamate for 24 hr. The expression of ClC-2, Bax, and Bcl-2 was detected by western blot analysis. Cell survival and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Caspase-3 and -9 activities were determined by a colorimetric assay. The roles of ClC-2 in glutamate-induced apoptosis were examined by using ClC-2 complementary deoxyribonucleic acid (cDNA) and small inference ribonucleic acid (RNA) transfection technology. Results: Overexpression of ClC-2 in RGC-5 cells significantly decreased glutamate-induced apoptosis and increased cell viability, whereas silencing of ClC-2 with short hairpin (sh) RNA produced opposite effects. ClC-2 overexpression increased the expression of Bcl-2, decreased the expression of Bax, and decreased caspase-3 and -9 activation in RGC-5 cells treated with glutamate, but silencing of ClC-2 produced opposite effects. Conclusion: Our data suggest that ClC-2 chloride channels might play a protective role in glutamate-induced apoptosis in retinal ganglion cells via the mitochondria-dependent apoptosis pathway. PMID:27635193

  1. Programmed cell death and apoptosis: origins of the theory.

    PubMed

    Lockshin, R A; Zakeri, Z

    2001-07-01

    Interest in the study of apoptosis grew with the recognition that it is a highly regulated process. Such a change in attitude allowed the intellectual and technical breakthroughs that led to the explosive development of this subject.

  2. Lentiviral Delivery of HIV-1 Vpr Protein Induces Apoptosis in Transformed Cells

    NASA Astrophysics Data System (ADS)

    Stewart, Sheila A.; Poon, Betty; Jowett, Jeremy B. M.; Xie, Yiming; Chen, Irvin S. Y.

    1999-10-01

    Most current anticancer therapies act by inducing tumor cell stasis followed by apoptosis. HIV-1 Vpr effectively induces apoptosis of T cells after arrest of cells at a G2/M checkpoint. Here, we investigated whether this property of Vpr could be exploited for use as a potential anticancer agent. As a potentially safer alternative to transfer of genes encoding Vpr, we developed a method to efficiently introduce Vpr protein directly into cells. Vpr packaged into HIV-1 virions lacking a genome induced efficient cell cycle arrest and apoptosis. Introduction of Vpr into tumor cell lines of various tissue origin, including those bearing predisposing mutations in p53, XPA, and hMLH1, induced cell cycle arrest and apoptosis with high efficiency. Significantly, apoptosis mediated by virion-associated Vpr was more effective on rapidly dividing cells compared with slow-growing cells, thus, in concept, providing a potential differential effect between some types of tumor cells and surrounding normal cells. This model system provides a rationale and proof of concept for the development of potential cancer therapeutic agents based on the growth-arresting and apoptotic properties of Vpr.

  3. Is Hydrogen Peroxide a Suitable Apoptosis Inducer for All Cell Types?

    PubMed Central

    Xiang, Jinmei; Wan, Chunyun; Guo, Rui

    2016-01-01

    Hydrogen peroxide is currently the most widely used apoptosis inducer due to its broad cytotoxic efficacy against nearly all cell types. However, equivalent cytotoxicity is achieved over a wide range of doses, although the reasons for this differential sensitivity are not always clear. In this study, three kinds of cells, the 293T cell line, primary fibroblasts, and terminally differentiated myocardial cells, were treated with a wide range of H2O2 doses. Times to apoptosis initiation and end were measured cytochemically and the changes in expression of caspase-9, P53, NF-κB, and RIP were determined by RT-PCR. The 293T cell line was the most sensitive to H2O2, undergoing necroptosis and/or apoptosis at all concentrations from 0.1 to 1.6 mM. At > 0.4 mM, H2O2 also caused necroptosis in primary cells. At < 0.4 mM, however, primary cells exhibited classic signs of apoptosis, although they tended to survive for 36 hours in < 0.2 mM H2O2. Thus, H2O2 is a broadly effective apoptosis inducer, but the dose range differs by cell type. For cell lines, a low dose is required and the exposure time must be reduced compared to primary cells to avoid cell death primarily by necroptosis or necrosis. PMID:27595106

  4. Is Hydrogen Peroxide a Suitable Apoptosis Inducer for All Cell Types?

    PubMed Central

    Xiang, Jinmei; Wan, Chunyun; Guo, Rui

    2016-01-01

    Hydrogen peroxide is currently the most widely used apoptosis inducer due to its broad cytotoxic efficacy against nearly all cell types. However, equivalent cytotoxicity is achieved over a wide range of doses, although the reasons for this differential sensitivity are not always clear. In this study, three kinds of cells, the 293T cell line, primary fibroblasts, and terminally differentiated myocardial cells, were treated with a wide range of H2O2 doses. Times to apoptosis initiation and end were measured cytochemically and the changes in expression of caspase-9, P53, NF-κB, and RIP were determined by RT-PCR. The 293T cell line was the most sensitive to H2O2, undergoing necroptosis and/or apoptosis at all concentrations from 0.1 to 1.6 mM. At > 0.4 mM, H2O2 also caused necroptosis in primary cells. At < 0.4 mM, however, primary cells exhibited classic signs of apoptosis, although they tended to survive for 36 hours in < 0.2 mM H2O2. Thus, H2O2 is a broadly effective apoptosis inducer, but the dose range differs by cell type. For cell lines, a low dose is required and the exposure time must be reduced compared to primary cells to avoid cell death primarily by necroptosis or necrosis.

  5. Heat-shock protein expression on the membrane of T cells undergoing apoptosis.

    PubMed Central

    Poccia, F; Piselli, P; Vendetti, S; Bach, S; Amendola, A; Placido, R; Colizzi, V

    1996-01-01

    Heat-shock proteins (hsp) represent a highly conserved family of proteins, normally localized in the cytoplasm and nucleus, whose expression is induced in situations involving cell stress. This paper reports the unusual translocation of hsp to the cell membrane of T cells undergoing apoptosis. We observed that glucocorticosteroid-induced thymocyte death is associated to the surface expression of hsp 60 and hsp 70 in a discrete fraction of apoptotic cells. hsp surface expression is closely related to a thymic subset of immature CD3low/- T cells. The expression of surface hsp 60 appears early after treatment with dexamethasone (3 hr) whereas the membrane expression of hsp 70 follows different kinetics and peaks later. Morphological analysis of the hsp+ apoptotic cells suggest that this subset represents late-stage apoptotic cells at their minimal volume before fragmentation into apoptotic bodies. Membrane expression of hsp is also associated with apoptosis in peripheral blood mononuclear cells from AIDS patients cultured in vitro. Altogether, we show that a discrete fraction of cells undergoing apoptosis expresses membrane hsp 60 and hsp 70, supporting the hypothesis that apoptosis causes a radical alteration in the expression of cell surface molecules. Surface hsp expressed during apoptosis may constitute a novel immune-context able to generate packages of self- and exogenous antigens, originating from degradation of altered cells. Images Figure 1 Figure 3 PMID:8707351

  6. Positive Feedback Cycle of TNFα Promotes Staphylococcal Enterotoxin B-Induced THP-1 Cell Apoptosis

    PubMed Central

    Zhang, Xiaopeng; Shang, Weilong; Yuan, Jizhen; Hu, Zhen; Peng, Huagang; Zhu, Junmin; Hu, Qiwen; Yang, Yi; Liu, Hui; Jiang, Bei; Wang, Yinan; Li, Shu; Hu, Xiaomei; Rao, Xiancai

    2016-01-01

    Staphylococcal enterotoxin B (SEB) has been demonstrated to be of importance in Staphylococcus aureus related diseases, such as atopic dermatitis (AD). Dysregulated apoptosis in AD is remarkable, and SEB can induce apoptosis of various cell types. However, the mechanisms by which SEB induces apoptosis and influences disease processes remain unclear. In this study, the recombinant SEB-induced THP-1 monocyte apoptosis was demonstrated in the absence of preliminary cell activation in a time- and dose-dependent manner. SEB could up-regulate the expression of tumor necrosis factor alpha (TNFα) in THP-1 cells and induce apoptosis via an extrinsic pathway. TNFα could in turn increase the expression of HLA-DRa, the SEB receptor on the cell surface. As a result, a positive feedback cycle of TNFα was established. TNFα expression and SEB-induced apoptosis were decreased by knocking down the expression of either HLA-DRa or TNFR1. Therefore, the feedback cycle of TNFα is crucial for SEB functions. This work provides insights into the mechanisms of SEB-induced monocyte apoptosis and emphasizes the major role of TNFα in future related studies. PMID:27709104

  7. Influence of Artemisia annua extract derivatives on proliferation, apoptosis and metastasis of osteosarcoma cells.

    PubMed

    Tang, Chao; Zhao, Yongqiang; Huang, Suizhu; Jin, Yi; Liu, Jijun; Luo, Jianping; Zheng, Jia; Shi, Dapeng

    2015-03-01

    Regarding the Artemisia annua extract derivatives called dihydroarteminin (DHA) as the object, we studied about its influence to the proliferation, apoptosis and metastasis of human osteosarcoma cells. First, we cultured in vitro the osteosarcoma cell strain and divided them into groups, then detected the cell proliferation, apoptosis and cell metastasis, etc by multiple measurement technique. Finally, we observed the influence of DHA to human osteosarcoma cells. Osteosarcoma cells were all sensitive to DHA, and the appropriate concentration range was 10~40μM. DHA could effectively restrain its protein expression, and there was a significant difference between experimental group and control group. These finding suggest that, the Artemisia annua extract derivatives (DHA) has a biological effect of observably restraining the proliferation and metastasis of human osteosarcoma cells and promoting the tumour cell apoptosis. PMID:25796153

  8. Glycyrrhizin induces apoptosis in human stomach cancer KATO III and human promyelotic leukemia HL-60 cells.

    PubMed

    Hibasami, Hiroshige; Iwase, Hiroshi; Yoshioka, Kazumi; Takahashi, Hidehisa

    2005-08-01

    We have investigated the effects of glycyrrhizin (GL) on cell proliferations of human stomach cancer KATO III and promyelotic leukemia HL-60 cells, and on DNA of those cell lines. GL displayed growth inhibitory effect against KATO III and HL-60 cells. Morphological change showing apoptotic bodies was observed in the KATO III and HL-60 cells treated with GL. The fragmentation of DNA by GL to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in both cell lines. Caspase inhibitors such as Z-VAD-FMK and Z-Asp-CH2-DCB suppressed the DNA fragmentation induced by GL. The data of the present study show that the suppression of KATO III and HL-60 cell-growth by GL results from the induction of apoptosis by GL, and that caspase is involved in the induction of apoptosis by GL in these cells. PMID:16012754

  9. Diosgenin induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma cells.

    PubMed

    Li, Yongjian; Wang, Xiaorong; Cheng, Silu; Du, Juan; Deng, Zhengting; Zhang, Yani; Liu, Qun; Gao, Jingdong; Cheng, Binbin; Ling, Changquan

    2015-02-01

    Diosgenin is a major compound of Dioscoreaceae plants such as yam, which is used as a drug in Traditional Chinese Medicine, and a common vegetable worldwide. The anticancer effect of diosgenin has been reported in various tumor cells, including leukemia, gastric, colorectal, and breast cancer. However, the activity of diosgenin on hepatocellular carcinoma (HCC) and the underlying mechanism have not been completely investigated. Therefore, we investigated the efficacy and associated mechanisms of diosgenin in HCC cells. Flow cytometric analysis was performed to determine the presence of cell cycle arrest and apopotic cells. Diosgenin significantly inhibited the growth of Bel-7402, SMMC-7721 and HepG2 HCC cells in a concentration-dependent manner. Diosgenin treatment for 24 h induced G2/M cell cycle arrest and apoptosis of hepatoma cells. Diosgenin inhibited Akt phosphorylation and upregulated p21 and p27 expression, but did not alter the expression of p53, suggesting diosgenin-induced upregulation of p21 and p57 is p53-independent in HCC cells. Diosgenin induced HCC cell apoptosis by activating caspase cascades -3, -8 and -9. However, diosgenin did not affect Bcl-2 and Bax levels. In conclusion, results of the present study suggest that diosgenin may be an active anti-HCC agent obtained from natural plants and provide new insights in understanding the mechanisms of diosgenin. PMID:25434486

  10. A Novel Isoform of the B Cell Tyrosine Kinase BTK Protects Breast Cancer Cells from Apoptosis

    PubMed Central

    Eifert, Cheryl; Wang, Xianhui; Kokabee, Leila; Kourtidis, Antonis; Jain, Ritu; Gerdes, Michael J.; Conklin, Douglas S.

    2016-01-01

    Tyrosine kinases orchestrate key cellular signaling pathways and their dysregulation is often associated with cellular transformation. Several recent cases in which inhibitors of tyrosine kinases have been successfully used as anticancer agents have underscored the importance of this class of proteins in the development of targeted cancer therapies. We have carried out a large-scale loss-of-function analysis of the human tyrosine kinases using RNA interference to identify novel survival factors for breast cancer cells. In addition to kinases with known roles in breast and other cancers, we identified several kinases that were previously unknown to be required for breast cancer cell survival. The most surprising of these was the cytosolic, nonreceptor tyrosine kinase, Bruton’s tyrosine kinase (BTK), which has been extensively studied in B cell development. Down regulation of this protein with RNAi or inhibition with pharmacological inhibitors causes apoptosis; overexpression inhibits apoptosis induced by Doxorubicin in breast cancer cells. Our results surprisingly show that BTK is expressed in several breast cancer cell lines and tumors. The predominant form of BTK found in tumor cells is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. This alternate form of BTK is expressed at significantly higher levels in tumorigenic breast cells than in normal breast cells. Since this protein is a survival factor for these cells, it represents both a potential marker and novel therapeutic target for breast cancer. PMID:23913792

  11. Acitretin induces apoptosis through CD95 signalling pathway in human cutaneous squamous cell carcinoma cell line SCL-1

    PubMed Central

    Lin, Xiu-Ying; He, Chun-Di; Xiao, Ting; Jin, Xin; Chen, Jiang; Wang, Ya-Kun; Liu, Mei; Wang, Kai-Bo; Jiang, Yi; Wei, Hua-Chen; Chen, Hong-Duo

    2009-01-01

    Skin cancers are by far the most common human malignancies. Retinoids have shown promising preventive and therapeutic effects against a variety of human malignancies. The aim of this study was to investigate the apoptosis-inducing effect of acitretin on human skin squamous cell carcinoma (SCC) SCL-1 cells. We found that acitretin preferentially inhibited the growth of SCL-1 cells in a dose- and time-dependent manner, but not of non-malignant keratinocyte HaCaT cells. This inhibition appeared to be due to induction of apoptosis as revealed by enzyme-linked immunosorbent assay. AnnexinV/propidium iodide assay and morphological observation confirmed the pro-apoptotic effect of acitretin on SCL-1 cells. We further demonstrated that apoptosis was induced within 1–2 days and involved activation of caspases-8, -9, -3 and poly (ADP-ribose) polymerase (PARP). Caspase-8 inhibitor effectively suppressed acitretin-induced apoptosis whereas caspase-9 inhibitor did not. Acitretin increased the levels of CD95 (Fas), CD95-ligand and Fas-associated death domain. Neutralizing ZB4 anti-Fas antibody significantly inhibited the apoptosis in SCL-1 cells induced by acitretin. These results suggest that acitretin is able to induce apoptosis in skin cancer cells possibly via death receptor CD95 apoptosis pathway without affecting the viability of normal keratinocyte. PMID:18624760

  12. Induction of Apoptosis in TNF-Treated L929 Cells in the Presence of Necrostatin-1

    PubMed Central

    Sawai, Hirofumi

    2016-01-01

    It has been shown that necroptosis—caspase-independent programmed necrotic cell death—can be induced by treatment with tumor necrosis factor (TNF) in the L929 murine fibrosarcoma cell line, even in the absence of a caspase inhibitor. Although it was reported that necrostatin-1—a specific inhibitor of necroptosis—inhibited TNF-induced necroptosis in L929 cells, it has not been elucidated whether the cells eventually die by apoptosis in the presence of necrostatin-1. In this paper, induction of apoptosis was demonstrated in TNF-treated L929 cells in the presence of necrostatin-1. Co-treatment with cycloheximide expedited apoptosis induction in necrostatin-1/TNF-treated L929 cells: typical apoptotic morphological changes, including membrane blebbing and nuclear fragmentation, induction of caspase-3 activity, proteolytic activation of caspases-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase (PARP) (a well-known substrate of caspase-3) were observed. Moreover, co-treatment with Z-VAD-fmk (a pan-caspase inhibitor) inhibited apoptosis by completely inhibiting caspases, resulting in a shift from apoptosis to necroptosis. In contrast, co-treatment with Z-Asp-CH2-DCB (a caspase inhibitor preferential to caspase-3) inhibited apoptosis without expediting necroptosis. These results indicate that apoptosis can be induced in TNF-treated L929 cells when the cells are protected from necroptosis, and support the notion that partial activation of caspase-8 in the presence of a caspase inhibitor preferential to caspase-3 suppresses both apoptosis and necroptosis. PMID:27739412

  13. Fluid Shear Stress Sensitizes Cancer Cells to Receptor-Mediated Apoptosis via Trimeric Death Receptors

    PubMed Central

    Mitchell, Michael J.

    2013-01-01

    Cancer metastasis, the process of cancer cell migration from a primary to distal location, typically leads to a poor patient prognosis. Hematogenous metastasis is initiated by intravasation of circulating tumor cells (CTCs) into the bloodstream, which are then believed to adhere to the luminal surface of the endothelium and extravasate into distal locations. Apoptotic agents such as tumor necrosis factor (TNF) apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer (NK) cells, have shown promise in treating CTCs to reduce the probability of metastasis. The role of hemodynamic shear forces in altering the cancer cell response to receptor-mediated apoptosis has not been previously investigated. Here, we report that human colon cancer COLO 205 and prostate cancer PC-3 cells exposed to a uniform fluid shear stress in a cone-and-plate viscometer become sensitized to TRAIL-induced apoptosis. Shear-induced sensitization directly correlated with the application of fluid shear stress, and TRAIL-induced apoptosis increased in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis was not affected by the application fluid shear stress. Interestingly, fluid shear stress did not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments revealed that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear force can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents. PMID:25110459

  14. Antiaging Gene Klotho Attenuates Pancreatic β-Cell Apoptosis in Type 1 Diabetes.

    PubMed

    Lin, Yi; Sun, Zhongjie

    2015-12-01

    Apoptosis is the major cause of death of insulin-producing β-cells in type 1 diabetes mellitus (T1DM). Klotho is a recently discovered antiaging gene. We found that the Klotho gene is expressed in pancreatic β-cells. Interestingly, halplodeficiency of Klotho (KL(+/-)) exacerbated streptozotocin (STZ)-induced diabetes (a model of T1DM), including hyperglycemia, glucose intolerance, diminished islet insulin storage, and increased apoptotic β-cells. Conversely, in vivo β-cell-specific expression of mouse Klotho gene (mKL) attenuated β-cell apoptosis and prevented STZ-induced diabetes. mKL promoted cell adhesion to collagen IV, increased FAK and Akt phosphorylation, and inhibited caspase 3 cleavage in cultured MIN6 β-cells. mKL abolished STZ- and TNFα-induced inhibition of FAK and Akt phosphorylation, caspase 3 cleavage, and β-cell apoptosis. These promoting effects of Klotho can be abolished by blocking integrin β1. Therefore, these cell-based studies indicated that Klotho protected β-cells by inhibiting β-cell apoptosis through activation of the integrin β1-FAK/Akt pathway, leading to inhibition of caspase 3 cleavage. In an autoimmune T1DM model (NOD), we showed that in vivo β-cell-specific expression of mKL improved glucose tolerance, attenuated β-cell apoptosis, enhanced insulin storage in β-cells, and increased plasma insulin levels. The beneficial effect of Klotho gene delivery is likely due to attenuation of T-cell infiltration in pancreatic islets in NOD mice. Overall, our results demonstrate for the first time that Klotho protected β-cells in T1DM via attenuating apoptosis. PMID:26340932

  15. p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

    SciTech Connect

    Huang, Shi-Wei; Wu, Chun-Ying; Wang, Yen-Ting; Kao, Jun-Kai; Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu; Chiu, Husan-Wen; Chang, Chuan-Hsun; Liang, Shu-Mei; Chen, Yi-Ju; Huang, Jau-Ling; Shieh, Jeng-Jer

    2013-02-15

    Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.

  16. Columbianadin Inhibits Cell Proliferation by Inducing Apoptosis and Necroptosis in HCT116 Colon Cancer Cells.

    PubMed

    Kang, Ji In; Hong, Ji-Young; Choi, Jae Sue; Lee, Sang Kook

    2016-05-01

    Columbianadin (CBN), a natural coumarin from Angelica decursiva (Umbelliferae), is known to have various biological activities including anti-inflammatory and anti-cancer effects. In this study, the anti-proliferative mechanism of actions mediated by CBN was investigated in HCT-116 human colon cancer cells. CBN effectively suppressed the growth of colon cancer cells. Low concentration (up to 25 µM) of CBN induced apoptosis, and high concentration (50 µM) of CBN induced necroptosis. The induction of apoptosis by CBN was correlated with the modulation of caspase-9, caspase-3, Bax, Bcl-2, Bim and Bid, and the induction of necroptosis was related with RIP-3, and caspase-8. In addition, CBN induced the accumulation of ROS and imbalance in the intracellular antioxidant enzymes such as SOD-1, SOD-2, catalase and GPx-1. These findings demonstrate that CBN has the potential to be a candidate in the development of anti-cancer agent derived from natural products.

  17. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    SciTech Connect

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  18. Apoptosis in Living Animals Is Assisted by Scavenger Cells and Thus May Not Mainly Go through the Cytochrome C-Caspase Pathway

    PubMed Central

    Liu, Bingya; Xu, Ningzhi; Man, Yangao; Shen, Haihong; Avital, Itzhak; Stojadinovic, Alexander; Liao, D. Joshua

    2013-01-01

    Because billions of cells die every day in their bodies, animals have evolutionarily developed apoptosis to preserve the tissue environment from adverse effects of dead cells, a process achieved via phagocytosis of the cell corpses by professional or amateur phagocytes that are collectively referred to as scavengers. Hence, apoptosis is a merger of two procedures separately occurring inside the dying and the scavenger cells, respectively. The task of apoptosis research is to study how these death procedures occur without hurting the host tissues, and recruitment of in vitro system into the study must be justified for this purpose. Cells in culture have no motivation to preserve the environment, and their death does not involve corpse clearance by scavengers. Therefore, programmed cell death in culture should be redefined, for example as stress-induced cell death, to avoid many sources of confusions, since the word “apoptosis” had already been defined, prior to the era of cell culture, as a silent and beneficial cell suicide with corpse clearance as a distinctive hallmark. We should start over again on apoptosis research by determining whether different physiological apoptotic procedures in animals involve the cytochrome c-caspase pathway, since it has been established from cultured cells as a central mechanism of “apoptosis” but whether it overarches any physiological apoptotic procedure in animals is still unclear. Probably, cells in living animals are programmed to use scavengers to assist their apoptosis but cells in culture have no scavengers to help and thus need to go mainly through the cytochrome c-caspase pathway. PMID:24312141

  19. Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis

    PubMed Central

    Vétillard, Alexandra; Jonchère, Barbara; Moreau, Marie; Toutain, Bertrand; Henry, Cécile; Fontanel, Simon; Bernard, Anne-Charlotte; Campone, Mario; Guette, Catherine; Coqueret, Olivier

    2015-01-01

    Activated in response to chemotherapy, senescence is a tumor suppressive mechanism that induces a permanent loss of proliferation. However, in response to treatment, it is not really known how cells can escape senescence and how irreversible or incomplete this pathway is. We have recently described that cells that escape senescence are more transformed than non-treated parental cells, they resist anoikis and rely on Mcl-1. In this study, we further characterize this emergence in response to irinotecan, a first line treatment used in colorectal cancer. Our results indicate that Akt was activated as a feedback pathway during the early step of senescence. The inhibition of the kinase prevented cell emergence and improved treatment efficacy, both in vitro and in vivo. This improvement was correlated with senescence inhibition, p21waf1 downregulation and a concomitant activation of apoptosis due to Noxa upregulation and Mcl-1 inactivation. The inactivation of Noxa prevented apoptosis and increased the number of emergent cells. Using either RNA interference or p21waf1-deficient cells, we further confirmed that an intact p53-p21-senescence pathway favored cell emergence and that its downregulation improved treatment efficacy through apoptosis induction. Therefore, although senescence is an efficient suppressive mechanism, it also generates more aggressive cells as a consequence of apoptosis inhibition. We therefore propose that senescence-inducing therapies should be used sequentially with drugs favoring cell death such as Akt inhibitors. This should reduce cell emergence and tumor relapse through a combined induction of senescence and apoptosis. PMID:26485768

  20. Bromelain-induced apoptosis in GI-101A breast cancer cells.

    PubMed

    Dhandayuthapani, Sivanesan; Perez, Honey Diaz; Paroulek, Alexandra; Chinnakkannu, Panneerselvam; Kandalam, Umadevi; Jaffe, Mark; Rathinavelu, Appu

    2012-04-01

    Bromelain is a proteolytic enzyme extracted from the stems and the immature fruits of pineapple that was found to be antitumorigenic in different in vitro models. Bromelain has been reported to promote apoptosis, particularly in breast cancer cells, with the up-regulation of c-Jun N-terminal kinase and p38 kinase. Our study was designed to determine if bromelain could induce apoptosis in GI-101A breast cancer cells. GI-101A cells were treated with increasing concentrations of bromelain for 24 hours. The effect of bromelain for inducing cell death via activation of the apoptosis mechanism in GI-101A cells was further determined by using caspase-9 and caspase-3 assays along with the M30-Apoptosense assay to measure cytokeratin 18 (CK18) levels in the cytoplasm of the cultured cancer cells. A dose-dependent increase in the activities of caspase-9 and caspase-3 coinciding with elevation of CK18 levels was found in bromelain-treated cells compared with control cells. Furthermore, the apoptosis induction by bromelain was confirmed by DNA fragmentation analysis and 4,6'-diamino-2-phenylindole dihydrochloride fluorescence staining of the nucleus. Our results indicate an increase in apoptosis-related cell death in breast cancer cells with increasing concentrations of bromelain.

  1. Role of Long Noncoding RNA HOTAIR in the Growth and Apoptosis of Osteosarcoma Cell MG-63

    PubMed Central

    Zheng, Hua

    2016-01-01

    This study investigated the function of HOTAIR in the growth and apoptosis of OS MG-63 cell line in vitro and further clarified its mechanism. The expression levels of HOTAIR in OS MG-63 cell line and normal osteoblast hFOB1.19 cell line were determined by RT-PCR, respectively. The growth and apoptosis of MG-63 cells in vitro were investigated by MTT assay and flow cytometry assay after HOTAIR was knocked down with retroviral vector construction. And the expression levels of cell growth and apoptosis related factors TGF-β, p53, Bcl-2, and TNF-α were determined to clarify the mechanism. We found that HOTAIR was highly expressed in osteosarcoma MG-63 cell line compared with normal osteoblast hFOB1.19 cell line. The proliferation rate was lower and the apoptosis rate was higher significantly in shHOTAIR MG-63 cells than those in EV MG-63 cells. TGF-β and Bcl-2 were downregulated significantly when HOTAIR was knocked down. p53 and TNF-α were upregulated significantly when HOTAIR was knocked down. These results indicated that HOTAIR functioned as a carcinogenic lncRNA, which could promote the proliferation and inhibit the apoptosis of MG-63 cells in vitro. HOTAIR could be a potential target for the treatment of osteosarcoma.

  2. Role of Long Noncoding RNA HOTAIR in the Growth and Apoptosis of Osteosarcoma Cell MG-63

    PubMed Central

    Zheng, Hua

    2016-01-01

    This study investigated the function of HOTAIR in the growth and apoptosis of OS MG-63 cell line in vitro and further clarified its mechanism. The expression levels of HOTAIR in OS MG-63 cell line and normal osteoblast hFOB1.19 cell line were determined by RT-PCR, respectively. The growth and apoptosis of MG-63 cells in vitro were investigated by MTT assay and flow cytometry assay after HOTAIR was knocked down with retroviral vector construction. And the expression levels of cell growth and apoptosis related factors TGF-β, p53, Bcl-2, and TNF-α were determined to clarify the mechanism. We found that HOTAIR was highly expressed in osteosarcoma MG-63 cell line compared with normal osteoblast hFOB1.19 cell line. The proliferation rate was lower and the apoptosis rate was higher significantly in shHOTAIR MG-63 cells than those in EV MG-63 cells. TGF-β and Bcl-2 were downregulated significantly when HOTAIR was knocked down. p53 and TNF-α were upregulated significantly when HOTAIR was knocked down. These results indicated that HOTAIR functioned as a carcinogenic lncRNA, which could promote the proliferation and inhibit the apoptosis of MG-63 cells in vitro. HOTAIR could be a potential target for the treatment of osteosarcoma. PMID:27660759

  3. Actinobacillus actinomycetemcomitans toxin induces both cell cycle arrest in the G2/M phase and apoptosis.

    PubMed

    Ohguchi, M; Ishisaki, A; Okahashi, N; Koide, M; Koseki, T; Yamato, K; Noguchi, T; Nishihara, T

    1998-12-01

    We found that the culture supernatant of the periodontopathic bacterium Actinobacillus actinomycetemcomitans had a cytotoxic effect on several cell lines. In this study, we purified the toxin from the culture supernatant of A. actinomycetemcomitans Y4 by a four-step procedure: ammonium sulfate precipitation, POROS HQ/M column chromatography, polymyxin B matrix column chromatography, and Mono-Q column chromatography. The purified toxin gave two major bands of protein with molecular masses of 80 and 85 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death of the B-cell hybridoma cell line HS-72 was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis. Overexpression of human Bcl-2 suppressed apoptosis in HS-72 cells, indicating that the toxin from A. actinomycetemcomitans induces apoptosis by a Bcl-2-inhibitable mechanism. Flow cytometric analysis revealed that the toxin caused cell cycle arrest in the G2/M phase and apoptosis in HS-72 cells. In addition, aurintricarboxylic acid, a DNA endonuclease inhibitor, markedly decreased the percentage of apoptotic cells but had no effect on cell cycle arrest in the G2/M phase. Taken together, these findings suggest that the toxin from A. actinomycetemcomitans could mediate the development of periodontal diseases through cell cycle arrest in the G2/M phase and apoptosis in B lymphocytes of periodontal tissue. PMID:9826381

  4. Multiple effects of TRAIL in human carcinoma cells: Induction of apoptosis, senescence, proliferation, and cytokine production

    SciTech Connect

    Levina, Vera; Marrangoni, Adele M.; DeMarco, Richard; Gorelik, Elieser; Lokshin, Anna E.

    2008-04-15

    TRAIL is a death ligand that induces apoptosis in malignant but not normal cells. Recently the ability of TRAIL to induce proliferation in apoptosis-resistant normal and malignant cells was reported. In this study, we analyzed TRAIL effects in apoptosis sensitive MCF7, OVCAR3 and H460 human tumor cell lines. TRAIL at low concentrations preferentially induced cell proliferation. At 100 ng/ml, apoptotic death was readily observed, however surviving cells acquired higher proliferative capacity. TRAIL-stimulated production of several cytokines, IL-8, RANTES, MCP-1 and bFGF, and activation of caspases 1 and 8 was essential for this effect. Antibodies to IL-8, RANTES, and bFGF blocked TRAIL-induced cell proliferation and further stimulated apoptosis. For the first time, we report that high TRAIL concentrations induced cell senescence as determined by the altered morphology and expression of several senescence markers: SA-{beta}-gal, p21{sup Waf1/Cip1}, p16{sup INK4a}, and HMGA. Caspase 9 inhibition protected TRAIL-treated cells from senescence, whereas inhibition of caspases 1 and 8 increased the yield of SLP cells. In conclusion, in cultured human carcinoma cells, TRAIL therapy results in three functional outcomes, apoptosis, proliferation and senescence. TRAIL-induced proapoptotic and prosurvival responses correlate with the strength of signaling. TRAIL-induced cytokine production is responsible for its proliferative and prosurvival effects.

  5. Baicalein induces apoptosis of human cervical cancer HeLa cells in vitro.

    PubMed

    Peng, Yong; Guo, Congshan; Yang, Yanhong; Li, Fenglin; Zhang, Yanxia; Jiang, Bin; Li, Qingwang

    2015-03-01

    A number of studies have shown that baicalein shows high antitumor activity in vitro and in vivo. In this study, the inhibitory effect of baicalein on human cervical cancer HeLa cells was studied in vitro. HeLa cells were treated with high (100 µg/ml) and low (50 µg/ml) doses of baicalein, and cell growth inhibition rates were examined by the MTT assay. The morphological changes of apoptotic cells were observed under the light and electron microscope, while the rate of cell apoptosis was examined by flow cytometry. The expression of apoptosis-related proteins was analyzed by western blot, and caspase-3 activation was examined by a caspase-3 activity assay and spectrophotometry. The results demonstrated that baicalein inhibits the proliferation of HeLa cells and induces apoptosis in a caspase-3-dependent pathway, through downregulation of the B-cell lymphoma 2 (Bcl-2) protein and upregulation of the Bcl-2-associated X protein (Bax), Fas, Fas ligand (FasL) and caspase-8. Thus, we conclude that baicalein induces apoptosis of HeLa cells via the mitochondrial and the death receptor pathways. Cell apoptosis in HeLa cells was most likely promoted by the activation of the proteolytic enzyme caspase-3 in both pathways.

  6. C/EBP homologous protein (CHOP) deficiency aggravates hippocampal cell apoptosis and impairs memory performance.

    PubMed

    Chen, Chang-Mu; Wu, Cheng-Tien; Chiang, Chih-Kang; Liao, Bor-Wu; Liu, Shing-Hwa

    2012-01-01

    Neurodegenerative disorders are growing burdens in modern societies because of increased life expectancy. Most neurodegenerative disorders commonly possess a similar neuropathological feature--the accumulation of abnormal protein aggregates or inclusions (misfolded proteins) in the brain. One of the main functions of endoplasmic reticulum (ER) is to initiate proper protein folding to facilitate protein secretion through the induction of unfolded protein response (UPR). C/EBP homologous protein (CHOP) induction has been demonstrated to be a signaling event underlying ER stress-induced cell apoptosis. In this study, we explored the role of CHOP in the hippocampal cell apoptosis and memory performance injury under an induced ER stress condition. Adult male wild type (C57BL/6J) and CHOP knockout (CHOP-/-) mice were intracerebroventricularly injected with tunicamycin. Tunicamycin can induce ER stress and cell apoptosis in mouse hippocampus. Compared with wild type mice, CHOP-/- mice showed an enhanced hippocampal cell apoptosis, worse performance in memory-related behavioral tests, and attenuated IRE-1 expression under tunicamycin treatment. The aggravated cell apoptosis and worse memory performance in CHOP-/- mice might be due to the deficiency of CHOP protein resulted in the impaired adaptive/pathological transcriptional response, the decreased IRE-1 and XBP-1 expressions, and the increased JNK phosphorylation to cope with ER stress. Taken together, these results suggest that CHOP may play a protective role in the hippocampal cell apoptosis and impairment of memory performance.

  7. Targeting Proliferating Cell Nuclear Antigen and Its Protein Interactions Induces Apoptosis in Multiple Myeloma Cells

    PubMed Central

    Müller, Rebekka; Bachke, Siri; Gilljam, Karin M.; Våtsveen, Thea K.; Rø, Torstein B.; Bellacchio, Emanuele; Sundan, Anders; Otterlei, Marit

    2013-01-01

    Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA’s protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells’ sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment. PMID:23936203

  8. Cancer cell(s) cycle sequencing reveals universal mechanisms of apoptosis.

    PubMed

    Marretta, R M Ardito; Ales, F

    2010-12-01

    In this paper, cell cycle in higher eukaryotes and their molecular networks signals both in G1/S and G2/M transitions are replicated in silico. Biochemical kinetics, converted into a set of differential equations, and system control theory are employed to design multi-nested digital layers to simulate protein-to-protein activation and inhibition for cell cycle dynamics in the presence of damaged genomes. Sequencing and controlling the digital process of four micro-scale species networks (p53/Mdm2/DNA damage, p21mRNA/cyclin-CDK complex, CDK/CDC25/weel/SKP2/APC/CKI and apoptosis target genes system) not only allows the comprehension of the mechanisms of these molecule interactions but paves the way for unraveling the participants and their by-products, until now quite unclear, which have the task of carrying out (or not) cell death. Whatever the running simulations (e.g., different species signals, mutant cells and different DNA damage levels), the results of the proposed cell digital multi-layers give reason to believe in the existence of a universal apoptotic mechanism. As a consequence, we identified and selected cell check points, sizers, timers and specific target genes dynamic both for influencing mitotic process and avoiding cancer proliferation as much as for leading the cancer cell(s) to collapse into a steady stable apoptosis phase. PMID:21141676

  9. Metformin Induces Cell Cycle Arrest and Apoptosis in Drug-Resistant Leukemia Cells

    PubMed Central

    Rodríguez-Lirio, A.; Pérez-Yarza, G.; Fernández-Suárez, M. R.; Alonso-Tejerina, E.; Boyano, M. D.; Asumendi, A.

    2015-01-01

    Recent epidemiological studies indicate that the antidiabetic drug metformin has chemosensitizing and chemopreventive effects against carcinogenesis. Here, we demonstrate that metformin exerts varying degrees of antitumor activity against human leukemia cells, as reflected by differences in growth inhibition, apoptosis, and alterations to metabolic enzymes. In metformin-sensitive cells, autophagy was not induced but rather it blocked proliferation by means of arresting cells in the S and G2/M phases which was associated with the downregulation of cyclin A, cyclin B1, and cdc2, but not that of cyclin E. In 10E1-CEM cells that overexpress Bcl-2 and are drug-resistant, the effect of metformin on proliferation was more pronounced, also inducing the activation of the caspases 3/7 and hence apoptosis. In all sensitive cells, metformin decreased the Δψm and it modified the expression of enzymes involved in energy metabolism: PKCε (PKCepsilon) and PKCδ (PKCdelta). In sensitive cells, metformin altered PKCε and PKCδ expression leading to a predominance of PKCε over PKCδ which implies a more glycolytic state. The opposite occurs in the nonresponsive cells. In conclusion, we provide new insights into the activity of metformin as an antitumoral agent in leukemia cells that could be related to its capability to modulate energy metabolism. PMID:26688757

  10. Saikosaponin-d, a novel SERCA inhibitor, induces autophagic cell death in apoptosis-defective cells

    PubMed Central

    Wong, V KW; Li, T; Law, B YK; Ma, E DL; Yip, N C; Michelangeli, F; Law, C KM; Zhang, M M; Lam, K YC; Chan, P L; Liu, L

    2013-01-01

    Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump, leading to autophagy induction through the activation of the Ca2+/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells. PMID:23846222

  11. Host Cell Factors as Antiviral Targets in Arenavirus Infection

    PubMed Central

    Linero, Florencia N.; Sepúlveda, Claudia S.; Giovannoni, Federico; Castilla, Viviana; García, Cybele C.; Scolaro, Luis A.; Damonte, Elsa B.

    2012-01-01

    Among the members of the Arenaviridae family, Lassa virus and Junin virus generate periodic annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of West Africa and Argentina, respectively. Given the human health threat that arenaviruses represent and the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. Since diverse host cell pathways and enzymes are used by RNA viruses to fulfill their replicative cycle, the targeting of a host process has turned an attractive antiviral approach in the last years for many unrelated virus types. This strategy has the additional benefit to reduce the serious challenge for therapy of RNA viruses to escape from drug effects through selection of resistant variants triggered by their high mutation rate. This article focuses on novel strategies to identify inhibitors for arenavirus therapy, analyzing the potential for antiviral developments of diverse host factors essential for virus infection. PMID:23170173

  12. Apoptosis-like cell death induced by Salmonella in Acanthamoeba rhysodes.

    PubMed

    Feng, Ye; Hsiao, Yi-Hsing; Chen, Hsiu-Ling; Chu, Chishih; Tang, Petrus; Chiu, Cheng-Hsun

    2009-08-01

    Free-living amoebae act as environmental hosts of several intracellular pathogens. We examined the interaction between Acanthamoeba rhysodes and Salmonella, a human intracellular pathogen. There was no difference among three different serovars of Salmonella in terms of their growth within A. rhysodes over time. The number of intracellular bacteria increased at 6 h post-infection, and the viability of A. rhysodes was significantly reduced at 24 h post-infection. Amoebic cell death was characterized by TUNEL and Annexin V assay, without DNA ladder identified, indicating an apoptosis-like cell death in Salmonella-infected A. rhysodes. Global gene expression screening between intracellular and extracellular Salmonella by microarray and quantitative PCR showed that genes from Salmonella pathogenicity islands and virulence plasmid were up-regulated within A. rhysodes. The phase-dependent expression pattern suggests their distinct roles in the pathogenesis. A. rhysodes and Salmonella provide a model to study transient symbiosis between bacterial pathogens and protozoa in an aquatic ecosystem. PMID:19446019

  13. Apoptosis-like cell death induced by Salmonella in Acanthamoeba rhysodes.

    PubMed

    Feng, Ye; Hsiao, Yi-Hsing; Chen, Hsiu-Ling; Chu, Chishih; Tang, Petrus; Chiu, Cheng-Hsun

    2009-08-01

    Free-living amoebae act as environmental hosts of several intracellular pathogens. We examined the interaction between Acanthamoeba rhysodes and Salmonella, a human intracellular pathogen. There was no difference among three different serovars of Salmonella in terms of their growth within A. rhysodes over time. The number of intracellular bacteria increased at 6 h post-infection, and the viability of A. rhysodes was significantly reduced at 24 h post-infection. Amoebic cell death was characterized by TUNEL and Annexin V assay, without DNA ladder identified, indicating an apoptosis-like cell death in Salmonella-infected A. rhysodes. Global gene expression screening between intracellular and extracellular Salmonella by microarray and quantitative PCR showed that genes from Salmonella pathogenicity islands and virulence plasmid were up-regulated within A. rhysodes. The phase-dependent expression pattern suggests their distinct roles in the pathogenesis. A. rhysodes and Salmonella provide a model to study transient symbiosis between bacterial pathogens and protozoa in an aquatic ecosystem.