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Sample records for anti-apoptotic bcl-2 family

  1. The anti-apoptotic members of the Bcl-2 family are attractive tumor-associated antigens

    DEFF Research Database (Denmark)

    Straten, Per thor; Andersen, Mads Hald

    2010-01-01

    Anti-apoptotic members of the Bcl-2 family (Bcl-2, Bcl-X(L) and Mcl-2) are pivotal regulators of apoptotic cell death. They are all highly overexpressed in cancers of different origin in which they enhance the survival of the cancer cells. Consequently, they represent prime candidates for anti-ca...

  2. High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1.

    Science.gov (United States)

    Zhai, Dayong; Godoi, Paulo; Sergienko, Eduard; Dahl, Russell; Chan, Xochella; Brown, Brock; Rascon, Justin; Hurder, Andrew; Su, Ying; Chung, Thomas D Y; Jin, Chaofang; Diaz, Paul; Reed, John C

    2012-03-01

    Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

  3. Nitric oxide induces cell death by regulating anti-apoptotic BCL-2 family members.

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    Colleen M Snyder

    Full Text Available Nitric oxide (NO activates the intrinsic apoptotic pathway to induce cell death. However, the mechanism by which this pathway is activated in cells exposed to NO is not known. Here we report that BAX and BAK are activated by NO and that cytochrome c is released from the mitochondria. Cells deficient in Bax and Bak or Caspase-9 are completely protected from NO-induced cell death. The individual loss of the BH3-only proteins, Bim, Bid, Puma, Bad or Noxa, or Bid knockdown in Bim(-/-/Puma(-/- MEFs, does not prevent NO-induced cell death. Our data show that the anti-apoptotic protein MCL-1 undergoes ASK1-JNK1 mediated degradation upon exposure to NO, and that cells deficient in either Ask1 or Jnk1 are protected against NO-induced cell death. NO can inhibit the mitochondrial electron transport chain resulting in an increase in superoxide generation and peroxynitrite formation. However, scavengers of ROS or peroxynitrite do not prevent NO-induced cell death. Collectively, these data indicate that NO degrades MCL-1 through the ASK1-JNK1 axis to induce BAX/BAK-dependent cell death.

  4. A novel BH3 mimetic efficiently induces apoptosis in melanoma cells through direct binding to anti-apoptotic Bcl-2 family proteins, including phosphorylated Mcl-1.

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    Liu, Yubo; Xie, Mingzhou; Song, Ting; Sheng, Hongkun; Yu, Xiaoyan; Zhang, Zhichao

    2015-03-01

    The Bcl-2 family modulates sensitivity to chemotherapy in many cancers, including melanoma, in which the RAS/BRAF/MEK/ERK pathway is constitutively activated. Mcl-1, a major anti-apoptotic protein in the Bcl-2 family, is extensively expressed in melanoma and contributes to melanoma's well-documented chemoresistance. Here, we provide the first evidence that Mcl-1 phosphorylation at T163 by ERK1/2 and JNK is associated with the resistance of melanoma cell lines to the existing BH3 mimetics gossypol, S1 and ABT-737, and a novel anti-apoptotic mechanism of phosphorylated Mcl-1 (pMcl-1) is revealed. pMcl-1 antagonized the known BH3 mimetics by sequestering pro-apoptotic proteins that were released from Bcl-2/Mcl-1. Furthermore, an anthraquinone BH3 mimetic, compound 6, was identified to be the first small molecule to that induces endogenous apoptosis in melanoma cells by directly binding Bcl-2, Mcl-1, and pMcl-1 and disrupting the heterodimers of these proteins. Although compound 6 induced upregulation of the pro-apoptotic protein Noxa, its apoptotic induction was independent of Noxa. These data reveal the promising therapeutic potential of targeting pMcl-1 to treat melanoma. Compound 6 is therefore a potent drug that targets pMcl-1 in melanoma.

  5. The Bcl-2 homology domain 3 (BH3)-only proteins Bim and bid are functionally active and restrained by anti-apoptotic Bcl-2 family proteins in healthy liver.

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    Kodama, Takahiro; Hikita, Hayato; Kawaguchi, Tsukasa; Saito, Yoshinobu; Tanaka, Satoshi; Shigekawa, Minoru; Shimizu, Satoshi; Li, Wei; Miyagi, Takuya; Kanto, Tatsuya; Hiramatsu, Naoki; Tatsumi, Tomohide; Takehara, Tetsuo

    2013-10-18

    An intrinsic pathway of apoptosis is regulated by the B-cell lymphoma-2 (Bcl-2) family proteins. We previously reported that a fine rheostatic balance between the anti- and pro-apoptotic multidomain Bcl-2 family proteins controls hepatocyte apoptosis in the healthy liver. The Bcl-2 homology domain 3 (BH3)-only proteins set this rheostatic balance toward apoptosis upon activation in the diseased liver. However, their involvement in healthy Bcl-2 rheostasis remains unknown. In the present study, we focused on two BH3-only proteins, Bim and Bid, and we clarified the Bcl-2 network that governs hepatocyte life and death in the healthy liver. We generated hepatocyte-specific Bcl-xL- or Mcl-1-knock-out mice, with or without disrupting Bim and/or Bid, and we examined hepatocyte apoptosis under physiological conditions. We also examined the effect of both Bid and Bim disruption on the hepatocyte apoptosis caused by the inhibition of Bcl-xL and Mcl-1. Spontaneous hepatocyte apoptosis in Bcl-xL- or Mcl-1-knock-out mice was significantly ameliorated by Bim deletion. The disruption of both Bim and Bid completely prevented hepatocyte apoptosis in Bcl-xL-knock-out mice and weakened massive hepatocyte apoptosis via the additional in vivo knockdown of mcl-1 in these mice. Finally, the hepatocyte apoptosis caused by ABT-737, which is a Bcl-xL/Bcl-2/Bcl-w inhibitor, was completely prevented in Bim/Bid double knock-out mice. The BH3-only proteins Bim and Bid are functionally active but are restrained by the anti-apoptotic Bcl-2 family proteins under physiological conditions. Hepatocyte integrity is maintained by the dynamic and well orchestrated Bcl-2 network in the healthy liver.

  6. AT-101, a small molecule inhibitor of anti-apoptotic Bcl-2 family members, activates the SAPK/JNK pathway and enhances radiation-induced apoptosis

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    Rooswinkel Rogier

    2009-10-01

    Full Text Available Abstract Background Gossypol, a naturally occurring polyphenolic compound has been identified as a small molecule inhibitor of anti-apoptotic Bcl-2 family proteins. It induces apoptosis in a wide range of tumor cell lines and enhances chemotherapy- and radiation-induced cytotoxicity both in vitro and in vivo. Bcl-2 and related proteins are important inhibitors of apoptosis and frequently overexpressed in human tumors. Increased levels of these proteins confer radio- and chemoresistance and may be associated with poor prognosis. Consequently, inhibition of the anti-apoptotic functions of Bcl-2 family members represents a promising strategy to overcome resistance to anticancer therapies. Methods We tested the effect of (--gossypol, also denominated as AT-101, radiation and the combination of both on apoptosis induction in human leukemic cells, Jurkat T and U937. Because activation of the SAPK/JNK pathway is important for apoptosis induction by many different stress stimuli, and Bcl-XL is known to inhibit activation of SAPK/JNK, we also investigated the role of this signaling cascade in AT-101-induced apoptosis using a pharmacologic and genetic approach. Results AT-101 induced apoptosis in a time- and dose-dependent fashion, with ED50 values of 1.9 and 2.4 μM in Jurkat T and U937 cells, respectively. Isobolographic analysis revealed a synergistic interaction between AT-101 and radiation, which also appeared to be sequence-dependent. Like radiation, AT-101 activated SAPK/JNK which was blocked by the kinase inhibitor SP600125. In cells overexpressing a dominant-negative mutant of c-Jun, AT-101-induced apoptosis was significantly reduced. Conclusion Our data show that AT-101 strongly enhances radiation-induced apoptosis in human leukemic cells and indicate a requirement for the SAPK/JNK pathway in AT-101-induced apoptosis. This type of apoptosis modulation may overcome treatment resistance and lead to the development of new effective combination

  7. The molecular cell death machinery in the simple cnidarian Hydra includes an expanded caspase family and pro- and anti-apoptotic Bcl-2 proteins.

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    Lasi, Margherita; Pauly, Barbara; Schmidt, Nikola; Cikala, Mihai; Stiening, Beate; Käsbauer, Tina; Zenner, Gerhardt; Popp, Tanja; Wagner, Anita; Knapp, Regina T; Huber, Andreas H; Grunert, Michaela; Söding, Johannes; David, Charles N; Böttger, Angelika

    2010-07-01

    The fresh water polyp Hydra belongs to the phylum Cnidaria, which diverged from the metazoan lineage before the appearance of bilaterians. In order to understand the evolution of apoptosis in metazoans, we have begun to elucidate the molecular cell death machinery in this model organism. Based on ESTs and the whole Hydra genome assembly, we have identified 15 caspases. We show that one is activated during apoptosis, four have characteristics of initiator caspases with N-terminal DED, CARD or DD domain and two undergo autoprocessing in vitro. In addition, we describe seven Bcl-2-like and two Bak-like proteins. For most of the Bcl-2 family proteins, we have observed mitochondrial localization. When expressed in mammalian cells, HyBak-like 1 and 2 strongly induced apoptosis. Six of the Bcl-2 family members inhibited apoptosis induced by camptothecin in mammalian cells with HyBcl-2-like 4 showing an especially strong protective effect. This protein also interacted with HyBak-like 1 in a yeast two-hybrid assay. Mutation of the conserved leucine in its BH3 domain abolished both the interaction with HyBak-like 1 and the anti-apoptotic effect. Moreover, we describe novel Hydra BH-3-only proteins. One of these interacted with Bcl-2-like 4 and induced apoptosis in mammalian cells. Our data indicate that the evolution of a complex network for cell death regulation arose at the earliest and simplest level of multicellular organization, where it exhibited a substantially higher level of complexity than in the protostome model organisms Caenorhabditis and Drosophila.

  8. Increased ratio of anti-apoptotic to pro-apoptotic Bcl2 gene-family members in lithium-responders one month after treatment initiation

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    Lowthert Lori

    2012-09-01

    Full Text Available Abstract Background Lithium is considered by many as the gold standard medication in the management of bipolar disorder (BD. However, the clinical response to lithium is heterogeneous, and the molecular basis for this difference in response is unknown. In the present study, we sought to determine how the peripheral blood gene expression profiles of patients with bipolar disorder (BD changed over time following intitiation of treatment with lithium, and whether differences in those profiles over time were related to the clinical response. Methods Illumina Sentrix Beadchip (Human-6v2 microarrays containing > 48,000 transcript probes were used to measure levels of expression of gene-expression in peripheral blood from 20 depressed subjects with BD prior to and every two weeks during 8 weeks of open-label treatment with lithium. Changes in gene-expression were compared between treatment responders (defined as a decrease in the Hamilton Depression Rating Scale of 50% or more and non-responders. Pathway analysis was conducted using GeneGO Metacore software. Results 127 genes showed a differential response in responders vs. non-responders. Pathway analysis showed that regulation of apoptosis was the most significantly affected pathway among these genes. Closer examination of the time-course of changes among BCL2 related genes showed that in lithium-responders, one month after starting treatment with lithium, several anti-apoptotic genes including Bcl2 and insulin receptor substrate 2 (IRS2 were up-regulated, while pro-apoptotic genes, including BCL2-antagonist/killer 1 (BAK1 and BCL2-associated agonist of cell death (BAD, were down-regulated. In contrast, in lithium non-responders, BCL2 and IRS2 were down-regulated, while BAK1 and BAD up-regulated at the one-month time-point. Conclusions These results suggest that differential changes in the balance of pro- and anti- apoptotic gene-expression following treatment with lithium may explain some of

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

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    Marcus Wallgren

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

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

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

    2016-07-01

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

  11. SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes.

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    Yang, Yin; Wang, Zongdan; Sun, Luan; Shao, Lipei; Yang, Nan; Yu, Dawei; Zhang, Xin; Han, Xiao; Sun, Yujie

    2015-01-01

    Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3'-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways.

  12. Targeting anti-apoptotic BCL-2 proteins in cancer: the importance of intermolecular interactions and protein turnover

    NARCIS (Netherlands)

    Rooswinkel, R.W.

    2013-01-01

    Bcl-B, een van de anti-apoptotische Bcl-2-eiwitten, is instabiel. Dit beïnvloedt zijn capaciteit om apoptose te blokkeren negatief, toont Rogier Rooswinkel aan. Ook blijkt dat voor alle Bcl-2-familieleden eiwitstabiliteit de bepalende factor is voor hun anti-apoptotische capaciteit. Dit in tegenstel

  13. Apoptosis and oncogenesis: give and take in the BCL-2 family.

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    Llambi, Fabien; Green, Douglas R

    2011-02-01

    The mitochondrial pathway of apoptosis constitutes one of the main safeguards against tumorigenesis. The BCL-2 family includes the central players of this pathway that regulate cell fate through the control of mitochondrial outer membrane permeabilization (MOMP), and important progress has been made in understanding the dynamic interactions between pro-apoptotic and anti-apoptotic BCL-2 proteins. In particular, recent studies have delineated a stepwise model for the induction of MOMP. BCL-2 proteins are often dysregulated in cancer, leading to increased survival of abnormal cells; however, recent studies have paradoxically shown that apoptosis induction can under some circumstances drive tumor formation, perhaps by inducing compensatory proliferation under conditions of cellular stress. These observations underline the complexity of BCL-2 protein function in oncogenesis.

  14. Histone demethylase Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim.

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    Yang, Di; Okamura, Hirohiko; Teramachi, Jumpei; Haneji, Tatsuji

    2016-04-01

    Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down-regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation.

  15. The mystery of BCL2 family: Bcl-2 proteins and apoptosis: an update.

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    Siddiqui, Waseem Ahmad; Ahad, Amjid; Ahsan, Haseeb

    2015-03-01

    Apoptosis is a critically important biological process that plays an essential role in cell fate and homeostasis. An important component of the apoptotic pathway is the family of proteins commonly known as the B cell lymphoma-2 (Bcl-2). The primary role of Bcl-2 family members is the regulation of apoptosis. Although the structure of Bcl-2 family of proteins was reported nearly 10 years ago, however, it still surprises us with its structural and functional complexity and diversity. A number of studies have demonstrated that Bcl-2 family influences many other cellular processes beyond apoptosis which are generally independent of the regulation of apoptosis, suggesting additional roles for Bcl-2. The disruption of the regulation of apoptosis is a causative event in many diseases. Since the Bcl-2 family of proteins is the key regulator of apoptosis, the abnormalities in its function have been implicated in many diseases including cancer, neurodegenerative disorders, ischemia and autoimmune diseases. In the past few years, our understanding of the mechanism of action of Bcl-2 family of proteins and its implications in various pathological conditions has enhanced significantly. The focus of this review is to summarize the current knowledge on the structure and function of Bcl-2 family of proteins in apoptotic cellular processes. A number of drugs have been developed in the past few years that target different Bcl-2 members. The role of Bcl-2 proteins in the pathogenesis of various diseases and their pharmacological significance as effective molecular therapeutic targets is also discussed.

  16. Small Molecule Inhibitors of Bcl-2 Family Proteins for Pancreatic Cancer Therapy

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    Masood, Ashiq [Department of Internal Medicine/Pathology, Karmanos Cancer Institute, Wayne State University, 4100 John R, HWCRC 732, Detroit, MI 48201 (United States); Azmi, Asfar S. [Department of Pathology, Karmanos Cancer Institute, Wayne State University, 4100 John R, HWCRC 732, Detroit MI 48201 (United States); Mohammad, Ramzi M., E-mail: mohammar@karmanos.org [Department of Internal Medicine/Pathology, Karmanos Cancer Institute, Wayne State University, 4100 John R, HWCRC 732, Detroit, MI 48201 (United States); Department of Oncology, Karmanos Cancer Institute, 4100 John R, HWCRC 732, Detroit, MI 48201 (United States)

    2011-03-24

    Pancreatic cancer (PC) has a complex etiology and displays a wide range of cellular escape pathways that allow it to resist different treatment modalities. Crucial signaling molecules that function downstream of the survival pathways, particularly at points where several of these pathways crosstalk, provide valuable targets for the development of novel anti-cancer drugs. Bcl-2 family member proteins are anti-apoptotic molecules that are known to be overexpressed in most cancers including PC. The anti-apoptotic machinery has been linked to the observed resistance developed to chemotherapy and radiation and therefore is important from the targeted drug development point of view. Over the past ten years, our group has extensively studied a series of small molecule inhibitors of Bcl-2 against PC and provide solid preclinical platform for testing such novel drugs in the clinic. This review examines the efficacy, potency, and function of several small molecule inhibitor drugs targeted to the Bcl-2 family of proteins and their preclinical progress against PC. This article further focuses on compounds that have been studied the most and also discusses the anti-cancer potential of newer class of Bcl-2 drugs.

  17. IFPA Trophoblast Research Award Lecture: the dynamic role of Bcl-2 family members in trophoblast cell fate.

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    Ray, J; Jurisicova, A; Caniggia, I

    2009-03-01

    Bcl-2 family members are important regulators of cell fate in normal organ development and in disease status. Pro- and anti-apoptotic members of this family function through a complex network of homo- and hetero-dimers to determine whether a cell lives or dies. Members of the Bcl-2 family are classically recognized for their role in apoptosis, yet emerging evidence has highlighted their importance in the regulation of cell cycle. Cellular proliferation, differentiation and death accompany early placental development of the trophoblast lineage. We have recently reported on the expression and function of two Bcl-2 family members in normal placental development, namely the pro-apoptotic Mtd/Bok, and its anti-apoptotic partner Mcl-1 and have found that their expression is upregulated by low oxygen, a key mediator of trophoblast cell proliferation in early placentation. Interestingly, we have also reported that the expression of the Mtd/Mcl-1 system is altered in preeclampsia, a placental pathology associated with a status of oxidative stress and typically characterized by an immature proliferative trophoblast phenotype and excessive trophoblast cell death. In this pathology levels of pro-apototic Mtd-L and Mtd-P are increased and anti-apoptotic Mcl-1 is cleaved in to a pro-apoptotic isoform. Disruption in Mtd/Mcl-1 expression seen in preeclampsia may contribute to both the increased apoptosis and hyperproliferative nature of this disorder.

  18. Expression of the Bcl-2 family genes and complexes involved in the mitochondrial transport in prostate cancer cells.

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    Asmarinah, Asmarinah; Paradowska-Dogan, Agnieszka; Kodariah, Ria; Tanuhardja, Budiana; Waliszewski, Przemyslaw; Mochtar, Chaidir Arif; Weidner, Wolfgang; Hinsch, Elvira

    2014-10-01

    Alteration of molecular pathways triggering apoptosis gives raise to various pathological tissue processes, such as tumorigenesis. The mitochondrial pathway is regulated by both the genes of the Bcl-2 family and the genes encoding mitochondrial transport molecules. Those proteins allow a release of cyctochrome c through the outer mitochondrial membrane. This release activates the caspase cascade resulting in death of cells. There are at least two main transport systems associated with the family of Bcl-2 proteins that are involved in transport of molecules through the outer mitochondrial membrane, i.e., the voltage dependent anion channels (VDACs) and translocases of the outer mitochondrial membrane proteins (TOMs). We investigated the expression of genes of the Bcl-2 family, i.e., pro-apoptotic Bak and Bid, and anti-apoptotic Bcl-2; VDAC gene, i.e., VDAC1, VDAC2 and VDAC3; and TOMM genes, i.e., TOMM20, TOMM22 and TOMM40. This study was performed at the mRNA and the protein level. Fourteen paraffin embedded prostate cancer tissues and five normal prostate tissues were analyzed by the quantitative PCR array and immunohistochemistry. We found a significant increase in both mRNA expression of the anti-apoptotic Bcl-2 gene and VDAC1 gene in prostate cancer tissue in comparison with their normal counterparts. Translation of the anti-apoptotic Bcl-2 and VDAC1 genes in prostate cancer tissue was slightly increased. We observed no significant differences in the mRNA expression of the pro-apoptotic Bak and Bid genes, VDAC2 or VDAC3 genes or the three TOMM genes in these tissues. The pro-apoptotic Bax protein was downtranslated significantly in secretory cells of prostate cancer as compared to normal prostate. We suggest that this protein is a good candidate as biomarker for prostate cancer.

  19. Structural studies of Bcl-2-family regulators of apoptosis

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    Stevens, P.W. [Argonne National Lab., IL (United States). Center for Mechanistic Biology and Biotechnology]|[Northwestern Univ., Evanston, IL (United States). Dept. of Biomedical Engineering; Cai, X.; Schiffer, M. [Argonne National Lab., IL (United States). Center for Mechanistic Biology and Biotechnology

    1996-06-01

    The Bcl-2 family of proteins includes about a dozen different proteins which share two small regions of amino acid homology but otherwise exhibit rather modest sequence similarities. The members of this family function as molecular regulators of apoptosis, some as accelerators of cell death and others as inhibitors of apoptosis. The authors analyzed the predicted secondary structures of Bcl-2-family proteins and found that a series of four amphipathic helices, three short {beta}-strands, and a carboxyl-terminal transmembrane helix were conserved throughout the family. Since the Bcl-2-family proteins do not have homology with any proteins of known three-dimensional structure, it seems likely that the tertiary structure assumed by these conserved Bcl-2-family structural elements will represent a completely new protein fold. The authors have prepared recombinant versions of particular proteins of the Bcl-2-family so that the can analyze their molecular structures experimentally. In addition, since some of the Bcl-2-family members homodimerize, they are using small-zone size-exclusion chromatography to analyze the homodimerization of individual, purified Bcl-2-family proteins in order to determine the association and rate constants for these dimerization reactions using computer-simulation methods previously developed in the group. Since certain of these proteins also interest with each other to form heterodimers, the authors also hope to extend the analyses to similarly analyze the heterodimerization of pairs of purified Bcl-2-family proteins.

  20. Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

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    Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Cheah, Yew-Hoong [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur (Malaysia); Meenakshii, Nallappan [Biology Department, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

  1. Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström macroglobulinemia.

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    Gaudette, B T; Dwivedi, B; Chitta, K S; Poulain, S; Powell, D; Vertino, P; Leleu, X; Lonial, S; Chanan-Khan, A A; Kowalski, J; Boise, L H

    2016-01-28

    Waldenström macroglobulinemia (WM) is a proliferative disorder of IgM-secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels, which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for the inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing.

  2. The anti-apoptotic protein BCL2L1/Bcl-xL is neutralized by pro-apoptotic PMAIP1/Noxa in neuroblastoma, thereby determining bortezomib sensitivity independent of prosurvival MCL1 expression.

    Science.gov (United States)

    Hagenbuchner, Judith; Ausserlechner, Michael J; Porto, Verena; David, Reinhard; Meister, Bernhard; Bodner, Martin; Villunger, Andreas; Geiger, Kathrin; Obexer, Petra

    2010-03-05

    Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

  3. Staying alive: defensive strategies in the BCL-2 family playbook.

    Science.gov (United States)

    Cullen, Sean P; Henry, Conor M; Martin, Seamus J

    2011-11-18

    Much debate surrounds how prosurvival members of the BCL-2 family repress opening of the BAX/BAK channel to block apoptosis; in this issue Llambi et al. (2011) identify two modes of apoptosis inhibition that exhibit surprisingly different behavior upon repeat proapoptotic challenges by BH3-only proteins.

  4. The Role of Bcl-2 Family Proteins in Therapy Responses of Malignant Astrocytic Gliomas: Bcl2L12 and Beyond

    Directory of Open Access Journals (Sweden)

    Fotini M. Kouri

    2012-01-01

    Full Text Available Glioblastoma (GBM is a highly aggressive and lethal brain cancer with a median survival of less than two years after diagnosis. Hallmarks of GBM tumors include soaring proliferative indices, high levels of angiogenesis, diffuse invasion into normal brain parenchyma, resistance toward therapy-induced apoptosis, and pseudopallisading necrosis. Despite the recent advances in neurosurgery, radiation therapy, and the development of targeted chemotherapeutic regimes, GBM remains one of the deadliest types of cancer. Particularly, the alkylating agent temozolomide (TMZ in combination with radiation therapy prolonged patient survival only marginally, and clinical studies assessing efficacies of targeted therapies, foremost ATP mimetics inhibiting the activity of receptor tyrosine kinases (RTKs, revealed only few initial responders; tumor recurrence is nearly universal, and salvage therapies to combat such progression remain ineffective. Consequently, myriad preclinical and clinical studies began to define the molecular mechanisms underlying therapy resistance of GBM tumors, and pointed to the Bcl-2 protein family, in particular the atypical member Bcl2-Like 12 (Bcl2L12, as important regulators of therapy-induced cell death. This review will discuss the multi-faceted modi operandi of Bcl-2 family proteins, describe their roles in therapy resistance of malignant glioma, and outline current and future drug development efforts to therapeutically target Bcl-2 proteins.

  5. Clinical profiling of BCL-2 family members in the setting of BRAF inhibition offers a rationale for targeting de novo resistance using BH3 mimetics.

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    Dennie T Frederick

    Full Text Available While response rates to BRAF inhibitiors (BRAFi are high, disease progression emerges quickly. One strategy to delay the onset of resistance is to target anti-apoptotic proteins such as BCL-2, known to be associated with a poor prognosis. We analyzed BCL-2 family member expression levels of 34 samples from 17 patients collected before and 10 to 14 days after treatment initiation with either vemurafenib or dabrafenib/trametinib combination. The observed changes in mRNA and protein levels with BRAFi treatment led us to hypothesize that combining BRAFi with a BCL-2 inhibitor (the BH3-mimetic navitoclax would improve outcome. We tested this hypothesis in cell lines and in mice. Pretreatment mRNA levels of BCL-2 negatively correlated with maximal tumor regression. Early increases in mRNA levels were seen in BIM, BCL-XL, BID and BCL2-W, as were decreases in MCL-1 and BCL2A. No significant changes were observed with BCL-2. Using reverse phase protein array (RPPA, significant increases in protein levels were found in BIM and BID. No changes in mRNA or protein correlated with response. Concurrent BRAF (PLX4720 and BCL2 (navitoclax inhibition synergistically reduced viability in BRAF mutant cell lines and correlated with down-modulation of MCL-1 and BIM induction after PLX4720 treatment. In xenograft models, navitoclax enhanced the efficacy of PLX4720. The combination of a selective BRAF inhibitor with a BH3-mimetic promises to be an important therapeutic strategy capable of enhancing the clinical efficacy of BRAF inhibition in many patients that might otherwise succumb quickly to de novo resistance. Trial registrations: ClinicalTrials.gov NCT01006980; ClinicalTrials.gov NCT01107418; ClinicalTrials.gov NCT01264380; ClinicalTrials.gov NCT01248936; ClinicalTrials.gov NCT00949702; ClinicalTrials.gov NCT01072175.

  6. Noxa/Bcl-2 protein interactions contribute to bortezomib resistance in human lymphoid cells.

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    Smith, Alyson J; Dai, Haiming; Correia, Cristina; Takahashi, Rie; Lee, Sun-Hee; Schmitz, Ingo; Kaufmann, Scott H

    2011-05-20

    Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (K(D)) of 3.4 nm for Mcl-1, 70 nm for Bcl-x(L), and 250 nm for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-x(L) antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance.

  7. EXPRESSION AND SIGNIFICANCE OF bcl-2 FAMILY IN AMELOBLASTONA

    Institute of Scientific and Technical Information of China (English)

    WANG Jie; MA Jie; ZHONG Ming; LIU Jing-dong

    2006-01-01

    Objective: To study the expression of bcl-2 and bax in human ameloblastoma (AB), and investigate the role of apoptosis in genesis and development of AB and the relation of apoptosis with the clinic biological characteristics of AB. Methods:BCL-2 and BAX proteins were detected in 75 cases of AB (primary AB 31 cases, recurrent AB 37 cases, malignant AB 7cases) by S-P method. Oral normal mucosa (NOM) and Odontogenic kerotosyst (OKC) were used as controls. Bcl-2 and bax mRNA in 20 cases of AB, 12 cases of OKC were detected by in situ hybridization. Results: The positive ratio of BCL-2protein was 88.0% ( 66/75 ) in AB, 74.3% (26/35) in OKC and 44.4% (4/9) in NOM, respectively (P<0.001). BCL-2 protein was expressed in peripheral cells and a few scattered stellate-shape cells in AB. The positive ratio of BAX protein was 74.7%(56/75)in AB, 65.7%(23/35)in OKC and 77.8%(7/9) in NOM, respectively (P<0.001). BAX protein was expressed in peripheral cells and stellate-shape cells with similar intensity. BCL-2 expression increased in recurrent and AB canceration(P<0.01), while for BAX expression, the positive ratio was higher in recurrent AB, but lower than that of malignant AB. A moderate negative correlation between BCL-2 and BAX protein was found (rk=-0.331, P<0.001).Conclusion: AB has much more apoptosis-inhibiting protein than apoptosis- accelarating protein. Apoptosis plays an important role in genesis, development of AB. The fashion and intensity of bcl-2 and bax expression were different in various tissues and in benign or malignant AB.

  8. Prognostic Significance of Apoptosis Related Gene Family bcl-2 in Human Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To study the prognostic effect of bcl-2 oncogene and its gene family members bax, bcl-x expression in breast cancer patients. Methods: expression of bcl-2, bax proteins in 91 human breast cancer tissue sections were studied by immunohistochemical method. Bcl-x1 mRNA expression in frozen tissues from 16 breast cancer patients were detected using Northern blot method. Results: bcl-2 protein positivity was found in 60/91 (65.9%) patients, and bax positivity 59/91 (64.8%). Bcl-2 and bax expression levels were associated with apoptotic index(AI), histological grade, axillary lymph node metastasis, postoperative local recurrence and metastasis. Bcl-2 expression was related to ER positivity. In univariate analysis for disease free survival (DFS), bcl-2 and bax protein levels, and Al were all found to have prognostic value. The result of Cox's model multivariate analysis showed that bcl-2 protein level was an independent prognostic factor. In 16 frozen breast cancer tissues, 8/16(50%) had higher level of bcl-x1 mRNA, which showed correlation with bcl-2 protein expression and axillary lymph node metastasis. Conclusion: The findings indicate that dysregulated expressions of bcl-2, bax and bcl-x1 apoptosis-related genes, suggestive of serious deregulation of apoptotic process, may contribute to the biologic aggressiveness of breast cancer. Bcl-2 protein is an independent indicator of prognosis in breast cancer patients.

  9. Phenazine-1-carboxamide (PCN) from Pseudomonas sp. strain PUP6 selectively induced apoptosis in lung (A549) and breast (MDA MB-231) cancer cells by inhibition of antiapoptotic Bcl-2 family proteins.

    Science.gov (United States)

    Kennedy, R Kamaraj; Veena, V; Naik, P Ravindra; Lakshmi, Pragna; Krishna, R; Sudharani, S; Sakthivel, N

    2015-06-01

    Phenazine-1-carboxamide (PCN), a naturally occurring simple phenazine derivative isolated from Pseudomonas sp. strain PUP6, exhibited selective cytotoxic activity against lung (A549) and breast (MDA-MB-231) cancer cell lines in differential and dose-dependent manner compared to normal peripheral blood mononuclear cells. PCN-treated cancer cells showed the induction of apoptosis as evidenced by the release of low level of LDH, morphological characteristics, production of reactive oxygen species, loss of mitochondrial membrane potential (ΔΨm) and induction of caspase-3. At molecular level, PCN instigates apoptosis by mitochondrial intrinsic apoptotic pathway via the overexpression of p53, Bax, cytochrome C release and activation of caspase-3 with the inhibition of oncogenic anti-apoptotic proteins such as PARP and Bcl-2 family proteins (Bcl-2, Bcl-w and Bcl-xL). The in silico docking studies of PCN targeted against the anti-apoptotic members of Bcl-2 family proteins revealed the interaction of PCN with the BH3 domain, which might lead to the induction of apoptosis due to the inhibition of antiapoptotic proteins. Due to its innate inhibition potential of antiapoptotic Bcl-2 family proteins, PCN may be used as potent anticancer agent against both lung and breast cancer.

  10. IL-15 regulates Bcl-2 family members Bim and Mcl-1 through JAK/STAT and PI3K/AKT pathways in T cells.

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    Shenoy, Aparna R; Kirschnek, Susanne; Häcker, Georg

    2014-08-01

    Maintenance of T cells is determined by their survival capacity, which is regulated by Bcl-2 proteins. Cytokines signalling through the common gamma chains such as IL-2, IL-7 and IL-15 are important for T-cell survival but how these cytokines determine the expression of Bcl-2-family proteins is not clear. We report signalling events of cytokines that regulate expression of two key Bcl-2 proteins, pro-apoptotic Bim and anti-apoptotic Mcl-1, in resting C57BL/6 mouse T cells. IL-2, IL-7 and IL-15 inhibited apoptosis but paradoxically induced the expression of Bim, countered by concomitant induction of Mcl-1. Bim induction by IL-15 was found at the mRNA and protein levels and depended on both JAK/STAT and PI3K signals. A new STAT5-binding site was identified in the Bim promoter, which was occupied by STAT5 upon IL-15 stimulation. Although it also depended on JAK/STAT- and PI3K signalling, Mcl-1 regulation was independent of Mcl-1 mRNA levels and of regulation of protein stability, suggesting translational regulation. Concurrent CD3 signals inhibited some of the IL-7 effect but not the IL-15 effect on Bcl-2 proteins. The data suggest that cytokines induce Bim and prime T cells for apoptosis, but also inhibit apoptosis by stabilising Mcl-1. Later downregulation of short-lived Mcl-1 may induce efficient, Bim-dependent apoptosis.

  11. Inhibition of potentially anti-apoptotic proteins by antisense protein kinase C-alpha (Isis 3521) and antisense bcl-2 (G3139) phosphorothioate oligodeoxynucleotides: relationship to the decreased viability of T24 bladder and PC3 prostate cancer cells.

    Science.gov (United States)

    Benimetskaya, L; Miller, P; Benimetsky, S; Maciaszek, A; Guga, P; Beaucage, S L; Wilk, A; Grajkowski, A; Halperin, A L; Stein, C A

    2001-12-01

    Isis 3521 and G3139 are 20- and 18-mer phosphorothioate oligonucleotides, respectively, targeted to the protein kinase C (PKC)-alpha and bcl-2 mRNAs. Treatment of T24 bladder and PC3 prostate carcinoma cells with full-length and 3'-truncation mutants of Isis 3521 causes down-regulation of PKC-alpha protein and mRNA. However, at the level of a 15-mer and shorter, down-regulation of mRNA expression is no longer observed. Further, no diminution in cellular viability, as measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay, in response to increasing concentrations of paclitaxel, can be observed for these shorter oligomers. These observations not only indicate that PKC-alpha protein expression can be down-regulated by both RNase H-dependent and -independent mechanisms but also that down-regulation of PKC-alpha is insufficient by itself to "chemosensitize" cells. G3139, which down-regulates bcl-2 protein and mRNA expression, also down-regulates PKC-alpha protein and mRNA expression but not that of PKC-betaI, -epsilon, or -zeta. However, the down-regulation of PKC-alpha and bcl-2 are not linked. When the carrier Eufectin 5 is employed, only bcl-2 is down-regulated in both T24 and PC3 cells at 50 nM oligonucleotide concentration. At 100 nM, both bcl-2 and PKC-alpha expression are down-regulated, and only at this concentration can "chemosensitization" to paclitaxel and carboplatin be observed. In contrast, the down-regulation of bcl-2 seems to be linked with that of RelA (p65). However, this too is also not sufficient for chemosensitization, even though it leads to the loss of expression of genes under the putative control of nuclear factor-kappaB and to detachment of the cells from plastic surfaces. These results underscore the complexity of the intracellular requirements for the initiation of chemosensitization to anti-neoplastic agents.

  12. Bcl-2蛋白家族与运动%Bcl-2 Protein Family Members and Exercises

    Institute of Scientific and Technical Information of China (English)

    吴环成; 贺道远

    2004-01-01

    Bcl-2蛋白家族根据其在细胞凋亡中的作用,分为抑凋亡蛋白和促凋亡蛋白.在骨骼肌细胞和心肌细胞内,Bcl-2家族成员之间形成二聚体,调节细胞是否进入凋亡程序.不同强度的运动对骨骼肌、心肌和淋巴细胞凋亡的影响不尽相同.

  13. Bcl-2 gene family expression in the brain of rat offspring after gestational and lactational dioxin exposure.

    Science.gov (United States)

    Chang, Shwu-Fen; Sun, Yu-Yo; Yang, Liang-Yo; Hu, Ssu-Yao; Tsai, Shih-Ying; Lee, Wen-Sen; Lee, Yi-Hsuan

    2005-05-01

    Recent epidemiological studies have shown that dioxin, a persistent organic pollutant, is related to cognitive and behavioral abnormalities in the offspring of exposed cohort. In order to investigate the possible impact of dioxin in survival gene expression during brain development, we established an animal model of gestational and lactational dioxin-exposed rat offspring. The expressions of dioxin-responsive gene cytochrome P450 1A1 (CYP1A1), apoptotic gene Bax, and anti-apoptotic genes Bcl-2 and Bcl-xL were examined in rat liver and brains using Western blot analysis and RT-PCR. The results showed that treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (2 microg/kg body weight through oral delivery) at gestation day 15 resulted in an increase of Bcl-xL in offspring male liver and cerebral cortex, but a decrease in female offspring. In contrast, the expression of Bcl-xL in the cerebellum was decreased in male, but increased in female. Bcl-2, another anti-apoptotic gene, was also downregulated in P0 female liver, cerebral cortex, but was not observed in male. In the 4-month-old offspring, however, the Bcl-2 protein levels in the liver and cerebellum of both male and female pups were higher in the TCDD group as compared with the control group. However, the Bcl-2 level in the cerebral cortex of TCDD-treated groups was higher than the control group only in female but not male offspring at 4 months old. The expression of Bax showed no significant changes upon TCDD exposure at P0 stage, but was significantly reduced in the 4-month-old male cortex. These results indicate that early exposure of dioxin could affect the development of certain brain regions with gender difference, in terms of its differential effect on expressions of Bcl-xL, Bcl-2, and Bax.

  14. Human embryonic stem cells express elevated levels of multiple pro-apoptotic BCL-2 family members.

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    David T Madden

    Full Text Available Two of the greatest challenges in regenerative medicine today remain (1 the ability to culture human embryonic stem cells (hESCs at a scale sufficient to satisfy clinical demand and (2 the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms.

  15. Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor.

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    Giovanni Monaco

    Full Text Available The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R, the primary Ca(2+-release channel in the endoplasmic reticulum (ER. Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca(2+ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4 has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine "hinges" replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG. By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.

  16. Bcl-2 family members inhibit oxidative stress-induced programmed cell death in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chen, Shao-Rong; Dunigan, David D; Dickman, Martin B

    2003-05-15

    Selected antiapoptotic genes were expressed in baker's yeast (Saccharomyces cerevisiae) to evaluate cytoprotective effects during oxidative stress. When exposed to treatments resulting in the generation of reactive oxygen species (ROS), including H(2)O(2), menadione, or heat shock, wild-type yeast died and exhibited apoptotic-like characteristics, consistent with previous studies. Yeast strains were generated expressing nematode ced-9, human bcl-2, or chicken bcl-xl genes. These transformants tolerated a range of oxidative stresses, did not display features associated with apoptosis, and remained viable under conditions that were lethal to wild-type yeast. Yeast strains expressing a mutant antiapoptotic gene (bcl-2 deltaalpha 5-6), known to be nonfunctional in mammalian cells, were unable to tolerate any of the ROS-generating insults. These data are the first report showing CED-9 has cytoprotective effects against oxidative stress, and add CED-9 to the list of Bcl-2 protein family members that modulate ROS-mediated programmed cell death. In addition, these data indicate that Bcl-2 family members protect wild-type yeast from physiological stresses. Taken together, these data support the concept of the broad evolutionary conservation and functional similarity of the apoptotic processes in eukaryotic organisms.

  17. Expression of Bcl-2 Family Member Bid in Normal and Malignant Tissues

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    Maryla Krajewska

    2002-01-01

    Full Text Available Bid is the only known Bcl-2 family member that can function as an agonist of proapoptotic Bcl-2-related proteins such as Bax and Bak. Expression of the proapoptotic Bcl-2 family protein Bid was assessed by immunoblotting and immunohistochemical methods in normal murine and human tissues, and in several types of human cancers and tumor cell lines. Bid expression in normal tissues varied widely, with prominent Bid immunostaining occurring in several types of short-lived cells (e.g., germinal center B cells, peripheral blood granulocytes, differentiated keratinocytes and in apoptosissensitive cells (e.g., adult neurons. Analysis of Bid expression by immunostaining of 100 colon, 95 ovarian, and 254 prostate cancers, as well as 59 brain tumors and 50 lymphomas, revealed evidence of altered Bid regulation in sometypes of cancers. Correlations with clinical outcome data revealed association of higher levels of Bid with longer recurrence-free survival in men with locally advanced (T3 stage prostate cancer (P=0.04. Immunoblot analysis of Bid protein levels in the NCI's panel of 60 human tumor cell lines revealed a correlation between higher levels of Bid and sensitivity to ribonucleotide reductase (RR-inhibiting drugs (P<0.0005. Overexpression of Bid in a model tumor cell line by gene transfection resulted in increased sensitivity to apoptosis induction by a RR inhibitor. Taken together, these observations suggest a potential role for Bid in tumor responses to specific chemotherapeutic drugs, and lay a foundation for future investigations of this member of the Bcl-2 family in healthy and diseased tissues.

  18. Multimodal interaction with BCL-2 family proteins underlies the proapoptotic activity of PUMA BH3.

    Science.gov (United States)

    Edwards, Amanda L; Gavathiotis, Evripidis; LaBelle, James L; Braun, Craig R; Opoku-Nsiah, Kwadwo A; Bird, Gregory H; Walensky, Loren D

    2013-07-25

    PUMA is a proapoptotic BCL-2 family member that drives the apoptotic response to a diversity of cellular insults. Deciphering the spectrum of PUMA interactions that confer its context-dependent proapoptotic properties remains a high priority goal. Here, we report the synthesis of PUMA SAHBs, structurally stabilized PUMA BH3 helices that, in addition to broadly targeting antiapoptotic proteins, directly bind to proapoptotic BAX. NMR, photocrosslinking, and biochemical analyses revealed that PUMA SAHBs engage an α1/α6 trigger site on BAX to initiate its functional activation. We further demonstrated that a cell-permeable PUMA SAHB analog induces apoptosis in neuroblastoma cells and, like expressed PUMA protein, engages BCL-2, MCL-1, and BAX. Thus, we find that PUMA BH3 is a dual antiapoptotic inhibitor and proapoptotic direct activator, and its mimetics may serve as effective pharmacologic triggers of apoptosis in resistant human cancers.

  19. Boolean network-based model of the Bcl-2 family mediated MOMP regulation

    Science.gov (United States)

    2013-01-01

    Background Mitochondrial outer membrane permeabilization (MOMP) is one of the most important points in the majority of apoptotic signaling cascades and it is controlled by a network of interactions between the members of the Bcl-2 family. Methods To understand the role of individual members of this family within the MOMP regulation, we have constructed a Boolean network-based model of interactions between the Bcl-2 proteins. Results Computational simulations have revealed the existence of trapping states which, independently from the incoming stimuli, block the occurrence of MOMP. Our results emphasize the role of the antiapoptotic protein Mcl-1 in the majority of these configurations. We demonstrate here the importance of the Bid and Bim for activation of effectors Bax and Bak, and the irreversibility of this activation. The model further points to the antiapoptotic protein Bcl-w as a key factor preventing Bax activation. Conclusions In spite of relative simplicity, the Boolean network-based model provides useful insight into main functioning logic of the Bcl-2 switch, consistent with experimental findings. PMID:23767791

  20. Hypoxia-induced modulation of apoptosis and BCL-2 family proteins in different cancer cell types.

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    Audrey Sermeus

    Full Text Available Hypoxia plays an important role in the resistance of tumour cells to chemotherapy. However, the exact mechanisms underlying this process are not well understood. Moreover, according to the cell lines, hypoxia differently influences cell death. The study of the effects of hypoxia on the apoptosis induced by 5 chemotherapeutic drugs in 7 cancer cell types showed that hypoxia generally inhibited the drug-induced apoptosis. In most cases, the effect of hypoxia was the same for all the drugs in one cell type. The expression profile of 93 genes involved in apoptosis as well as the protein level of BCL-2 family proteins were then investigated. In HepG2 cells that are strongly protected against cell death by hypoxia, hypoxia decreased the abundance of nearly all the pro-apoptotic BCL-2 family proteins while none of them are decreased in A549 cells that are not protected against cell death by hypoxia. In HepG2 cells, hypoxia decreased NOXA and BAD abundance and modified the electrophoretic mobility of BIM(EL. BIM and NOXA are important mediators of etoposide-induced cell death in HepG2 cells and the hypoxia-induced modification of these proteins abundance or post-translational modifications partly account for chemoresistance. Finally, the modulation of the abundance and/or of the post-translational modifications of most proteins of the BCL-2 family by hypoxia involves p53-dependent and -independent pathways and is cell type-dependent. A better understanding of these cell-to-cell variations is crucial in order to overcome hypoxia-induced resistance and to ameliorate cancer therapy.

  1. Expression of bcl-2 gene family during resection induced liver regeneration:Comparison between hepatectomized and sham groups

    Institute of Scientific and Technical Information of China (English)

    Kamil Can Akcali; Aydin Dalgic; Ahmet Ucar; Khemaeis Ben Haj; Dilek Guvenc

    2004-01-01

    AIM: During liver regeneration cellular proliferation and apoptosis result in tissue remodeling to restore normal hepatic mass and structure. Main regulators of the apoptotic machinery are the Bcl-2 family proteins but their roles are not well defined throughout the liver regeneration. We aimed to analyze the expression levels of bcl-2gene family members during resection induced liver regeneration.METHODS: We performed semi-quantitative RT-PCR to examine the expression level of bak, bax, bcl-2 and bcl-xL in 70% hepatectomized rat livers during the whole regeneration process and compared to that of the sham and normal groups.RESULTS: The expression of bakand baxwas decreased whereas that of bcl-2and bcl-XL was increased in hepatectomized animals compared to normal liver at most time points. We also reported for the first time that sham group of animals had statistically significant higher expression of bakand bax than hepatectomized animals. In addition, the area under the curve (AUC) values of these genes was larger in sham groups than the hepatectomized groups.CONCLUSION: The expression changes of bak, bax, bcl-2 and bcl-,XL genes are altered not only due to regeneration,but also due to effects of surgical operations.

  2. Extracellular administration of BCL2 protein reduces apoptosis and improves survival in a murine model of sepsis.

    Directory of Open Access Journals (Sweden)

    Akiko Iwata

    Full Text Available BACKGROUND: Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2 family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP. We show that intraperitoneal injection of picomole range doses of recombinant human (rh BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo. CONCLUSIONS/SIGNIFICANCE: Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins.

  3. Crystal structure of a BCL-W domain-swapped dimer: implications for the function of BCL-2 family proteins.

    Science.gov (United States)

    Lee, Erinna F; Dewson, Grant; Smith, Brian J; Evangelista, Marco; Pettikiriarachchi, Anne; Dogovski, Con; Perugini, Matthew A; Colman, Peter M; Fairlie, W Douglas

    2011-10-12

    The prosurvival and proapoptotic proteins of the BCL-2 family share a similar three-dimensional fold despite their opposing functions. However, many biochemical studies highlight the requirement for conformational changes for the functioning of both types of proteins, although structural data to support such changes remain elusive. Here, we describe the X-ray structure of dimeric BCL-W that reveals a major conformational change involving helices α3 and α4 hinging away from the core of the protein. Biochemical and functional studies reveal that the α4-α5 hinge region is required for dimerization of BCL-W, and functioning of both pro- and antiapoptotic BCL-2 proteins. Hence, this structure reveals a conformational flexibility not seen in previous BCL-2 protein structures and provides insights into how these regulators of apoptosis can change conformation to exert their function.

  4. A forskolin derivative, FSK88, induces apoptosis in human gastric cancer BGC823 cells through caspase activation involving regulation of Bcl-2 family gene expression, dissipation of mitochondrial membrane potential and cytochrome c release.

    Science.gov (United States)

    Li, Zhonghai; Wang, Jingze

    2006-11-01

    FSK88, a forskolin derivative, was extracted and purified from cultured tropical plant roots, Coleus forskohlii. Our previous studies have demonstrated that FSK88 can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages. In this study, we showed that FSK88 can induce apoptotic death of human gastric cancer BGC823 cells in a dose- and time-dependent manner. Results showed that FSK88-induced apoptosis was accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in BGC823 cells. Furthermore, treatment with caspase-3 inhibitor (z-DEVD-fmk) was capable of preventing the FSK88-induced caspase-3 activity and apoptosis. FSK88-induced apoptosis in human gastric cancer BGC823 cells was also accompanied by the up-regulation of Bax, Bad and down-regulation of Bcl-2. Theses results clearly demonstrated that the induction of apoptosis by FSK88 involved multiple cellular and molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family genes, mitochondrial membrane potential (Deltapsi(m)), cytochrome c, and caspase-3, participate in the FSK88-induced apoptotic process in human gastric cancer BGC823 cells.

  5. Impact of conditional deletion of the pro-apoptotic BCL-2 family member BIM in mice.

    Science.gov (United States)

    Herold, M J; Stuchbery, R; Mérino, D; Willson, T; Strasser, A; Hildeman, D; Bouillet, P

    2014-10-09

    The pro-apoptotic BH3-only BCL-2 family member BIM is a critical determinant of hematopoietic cell development and homeostasis. It has been argued that the striking hematopoietic abnormalities of BIM-deficient mice (accumulation of lymphocytes and granulocytes) may be the result of the loss of the protein throughout the whole animal rather than a consequence intrinsic to the loss of BIM in hematopoietic cells. To address this issue and allow the deletion of BIM in specific cell types in future studies, we have developed a mouse strain with a conditional Bim allele as well as a new Cre transgenic strain, Vav-CreER, in which the tamoxifen-inducible CreER recombinase (fusion protein) is predominantly expressed in the hematopoietic system. We show that acute loss of BIM in the adult mouse rapidly results in the hematopoietic phenotypes previously observed in mice lacking BIM in all tissues. This includes changes in thymocyte subpopulations, increased white blood cell counts and resistance of lymphocytes to BIM-dependent apoptotic stimuli, such as cytokine deprivation. We have validated this novel conditional Bim knockout mouse model using established and newly developed CreER strains (Rosa26-CreER and Vav-CreER) and will make these exciting new tools for studies on cell death and cancer available.

  6. Bid, a Widely Expressed Proapoptotic Protein of the Bcl-2 Family, Displays Lipid Transfer Activity

    Science.gov (United States)

    Esposti, Mauro Degli; Erler, Janine T.; Hickman, John A.; Dive, Caroline

    2001-01-01

    Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis in primary tissues such as liver. Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria to facilitate the release of apoptogenic cytochrome c. The mechanism of Bid relocation to mitochondria was unclear. We report here novel biochemical evidence indicating that Bid has lipid transfer activity between mitochondria and other intracellular membranes, thereby explaining its dynamic relocation to mitochondria. First, physiological concentrations of phospholipids such as phosphatidic acid and phosphatidylgycerol induced an accumulation of full-length Bid in mitochondria when incubated with light membranes enriched in endoplasmic reticulum. Secondly, native and recombinant Bid, as well as tBid, displayed lipid transfer activity under the same conditions and at the same nanomolar concentrations leading to mitochondrial relocation and release of cytochrome c. Thus, Bid is likely to be involved in the transport and recycling of mitochondrial phospholipids. We discuss how this new role of Bid may relate to its proapoptotic action. PMID:11585909

  7. IRES-mediated translation of the pro-apoptotic Bcl2 family member PUMA.

    Science.gov (United States)

    Shaltouki, Atossa; Harford, Terri J; Komar, Anton A; Weyman, Crystal M

    2013-01-01

    The proapoptotic Bcl-2 family member PUMA is a critical regulator of apoptosis. We have previously shown that PUMA plays a pivotal role in the apoptosis associated with skeletal myoblast differentiation and that a MyoD-dependent mechanism is responsible for the increased expression of PUMA in these cells. Herein, we report that the increased expression of PUMA under these conditions involves regulation at the level of translation. Specifically, we have found that the increase in PUMA protein levels occurs under conditions of decreased total protein synthesis, eIF2-alpha phosphorylation and hypophosphorylation of eIF4E-BP, suggesting that PUMA translation is proceeding via an alternative initiation mechanism. Polyribosome analysis of PUMA mRNA further corroborated this suggestion. A combination of in vitro and ex vivo (cellular) approaches has provided evidence suggesting that PUMA mRNA 5'UTR harbors an Internal Ribosome Entry Site (IRES) element. Using mono- and bi-cistronic reporter constructs, we have delineated an mRNA fragment that allows for cap-independent translation in vitro and ex vivo (in skeletal myoblasts) in response to culture in differentiation media (DM), or in response to treatment with the DNA-damaging agent, etoposide. This mRNA fragment also supports translation in HeLa and 293T cells. Thus, our data has revealed a novel IRES-mediated regulation of PUMA expression in several cell types and in response to several stimuli. These findings contribute to our understanding and potential manipulation of any developmental or therapeutic scenario involving PUMA.

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

    Institute of Scientific and Technical Information of China (English)

    Hui Shen; Xiao-Dong Pan; Jing Zhang; Yu-Qi Zeng; Meng Zhou; Lu-Meng Yang; Bing Ye

    2016-01-01

    Background:Amyloid β (Aβ) deposits and the endoplasmic reticulum stress (ERS) are both well established in the development and progression ofAlzheimer's disease (AD).However,the mechanism and role of Aβ-induced ERS in AD-associated pathological progression remain to be elucidated.Methods:The five familial AD (5 ×FAD) mice and wild-type (WT) mice aged 2,7,and 12 months were used in the present study.Morris water maze test was used to evaluate their cognitive performance.Immunofluorescence and Western blot analyses were used to examine the dynamic changes of pro-apoptotic (CCAAT/enhancer-binding protein homologous protein [CHOP] and cleaved caspase-12) and anti-apoptotic factors (chaperone glucose-regulated protein [GRP] 78 and endoplasmic reticulum-associated protein degradation-associated ubiquitin ligase synovial apoptosis inhibitor 1 [SYVN1]) in the ERS-associated unfolded protein response (UPR) pathway.Results:Compared with age-matched WT mice,5 ×FAD mice showed higher cleaved caspase-3,lower neuron-positive staining at the age of 12 months,but earlier cognitive deficit at the age of 7 months (all P < 0.05).Interestingly,for 2-month-old 5×FAD mice,the related proteins involved in the ERS-associated UPR pathway,including CHOP,cleaved caspase-12,GRP 78,and SYVN1,were significantly increased when compared with those in age-matched WT mice (all P < 0.05).Moreover,ERS occurred mainly in neurons,not in astrocytes.Conclusions:These findings suggest that compared with those of age-matched WT mice,ERS-associated pro-apoptotic and anti-apoptoticproteins are upregulated in 2-month-old 5×FAD mice,consistent with intracellular Aβ aggregation in neurons.

  9. Alternative treatments for melanoma: targeting BCL-2 family members to de-bulk and kill cancer stem cells

    OpenAIRE

    Mukherjee, Nabanita; Schwan, Josianna V.; Fujita, Mayumi; David A Norris; Shellman, Yiqun G.

    2015-01-01

    For the first time new treatments in melanoma have produced significant responses in advanced diseases, but 30–90% of melanoma patients do not respond or eventually relapse after the initial response to the current treatments. The resistance of these melanomas is likely due to tumor heterogeneity, which may be explained by models such as the stochastic, hierarchical, and phenotype-switching models. This review will discuss the recent advancements in targeting BCL-2 family members for cancer t...

  10. Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis.

    Science.gov (United States)

    Srivastava, M; Ahmad, N; Gupta, S; Mukhtar, H

    2001-05-04

    Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.

  11. Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes

    DEFF Research Database (Denmark)

    Karlsson, Richard; Engström, Maria; Jönsson, Maria;

    2003-01-01

    not sustain their expression. Moreover, use of inhibitors implied that IL-3 was mainly exerting its effect on Bcl-2 at the level of transcription. The addition of LY294002 did not affect the expression of Bcl-2 and Bcl-XL, and thus, we conclude that expression of antiapoptotic Bcl-2 family member genes......Cytokines such as interleukin 3 (IL-3), kit ligand (KL), and flt3 ligand (FL) promote survival of hematopoietic stem cells and myeloid progenitor cells. In many cell types, members of the Bcl-2 gene family are major regulators of survival, but the mediating mechanisms are not fully understood...... is not dependent on PI-3 kinase activity. Our results indicate that cytokines exert distinct survival effects and that FL and IL-3 are capable of sustaining progenitor survival by up-regulating the expression of Bcl-2 and related genes....

  12. Regulatory Effect of Bcl-2 Family Proteins in CPB-induced Cardiomyocyte Apoptosis in Dog Hearts

    Institute of Scientific and Technical Information of China (English)

    孙宗全; 张顺业; 刘立新; 哈斯朝鲁

    2002-01-01

    Summary: Whether conventional hypothermic CPB induces myocyte apoptosis in dog hearts and modulation of bcl-2, bcl-xl, bax, bad, and caspase-3 pathways in this setting was investigated. Ten healthy adult dogs were randomized into sham-operated and CPB groups. Samples of left ventricle were obtained before, during and 3 h after CPB. In situ TUNEL was used to detect apoptotic my ocytes. Immunohistochemistry and flow cytometry were employed for detection of expressions of bcl 2, bcl-xl, bax and bad proteins. Z-DEVD-AMC substrate cleavage and TBARS methods were usedto measure the activity of caspase-3 and the content of lipid peroxide in LV myocardium, respective ly. After CPB, the number of apoptotic myocytes in CPB group was significantly increased. The re-sults of immunohistichemistry demonstrated that bcl-2, bcl-xl, bax and bad proteins were constitu tionally presnt on the sarcolemma of the LV myocytes. FACS results showed that, after CPB, ex-pressions of bax and bad in CPB group were significantly upregulated, while the expressions of bcl-2 and bcl-xl were not significantly changed in both groups. The activity of caspase-3 and the content of lipid peroxide in LV myocardium in CPB group were also significantly increased after CPB. The pre-sent study shows that there exists myocardiocyte apoptosis in dog hearts undergoing conventional hy-pothermic CPB and the myocyte apoptosis is initiated by ischemia and performed during reperfusion.Moreover, the CPB-induced myocyte apoptosis was associated with upregulation of expressions of bax and bad proteins, activation of caspase-3 and increase of oxidative stress.

  13. Regulatory effect of bcl-2 family proteins in CPB-induced cardiomyocyte apoptosis in dog hearts.

    Science.gov (United States)

    Sun, Zongquan; Zhang, Shunye; Liu, Lixin; Hasichaolu

    2002-01-01

    Whether conventional hypothermic CPB induces myocyte apoptosis in dog hearts and modulation of bcl-2, bcl-xl, bax, bad, and caspase-3 pathways in this setting was investigated. Ten healthy adult dogs were randomized into sham-operated and CPB groups. Samples of left ventricle were obtained before, during and 3 h after CPB. In situ TUNEL was used to detect apoptotic myocytes. Immunohistochemistry and flow cytometry were employed for detection of expressions of bcl-2, bcl-xl, bax and bad proteins. Z-DEVD-AMC substrate cleavage and TBARS methods were used to measure the activity of caspase-3 and the content of lipid peroxide in LV myocardium, respectively. After CPB, the number of apoptotic myocytes in CPB group was significantly increased. The results of immunohistochemistry demonstrated that bcl-2, bcl-xl, bax and bad proteins were constitutionally present on the sarcolemma of the LV myocytes. FACS results showed that, after CPB, expressions of bax and bad in CPB group were significantly upregulated, while the expressions of bcl-2 and bcl-xl were not significantly changed in both groups. The activity of caspase-3 and the content of lipid peroxide in LV myocardium in CPB group were also significantly increased after CPB. The present study shows that there exists myocardiocyte apoptosis in dog hearts undergoing conventional hypothermic CPB and the myocyte apoptosis is initiated by ischemia and performed during reperfusion. Moreover, the CPB-induced myocyte apoptosis was associated with upregulation of expressions of bax and bad proteins, activation of caspase-3 and increase of oxidative stress.

  14. Evaluation of Bcl-2 Family Gene Expression in Hippocampus of 3, 4-methylenedioxymethamphetamine Treated Rats

    Directory of Open Access Journals (Sweden)

    Hamed Hashemi-Nasl

    2012-01-01

    Full Text Available Objective: 3,4-methylenedioxymethamphetamine (MDMA is an illicit, recreational drugthat causes cellular death and neurotoxicity. This study evaluates the effects of differentdoses of MDMA on the expression of apoptosis–related proteins and genes in the hippocampusof adult rats.Materials and Methods: In this expremental study,a total of 20 male Sprague Dawley rats(200-250 g were treated with MDMA (0, 5, 10, 20 mg/kg i.p. twice daily for 7 days. Sevendays after the last administration of MDMA, the rats were killed. Bax and Bcl-2 genesin addition to protein expressions were detected by western blot and reverse transcriptionpolymerasechain reaction (RT-PCR.Results were analyzed using one-way ANOVA andp≤0.05 was considered statistically significant.Results: Our results showed that MDMA caused dose dependent up-regulation of Baxand down-regulation of Bcl-2 in the hippocampus. There was a significant alteration inbcl-2 and bax genes density.Conclusion: Changes in apoptosis-related proteins and respective genes relating to Baxand Bcl-2 might be involved in the molecular mechanism of MDMA-induced apoptosis.

  15. BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion.

    Science.gov (United States)

    Cekanova, Maria; Fernando, Romaine I; Siriwardhana, Nalin; Sukhthankar, Mugdha; De la Parra, Columba; Woraratphoka, Jirayus; Malone, Christine; Ström, Anders; Baek, Seung J; Wade, Paul A; Saxton, Arnold M; Donnell, Robert M; Pestell, Richard G; Dharmawardhane, Suranganie; Wimalasena, Jay

    2015-02-01

    We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.

  16. Selection of Aptamers for CED-9/Bcl-2 Family Cell Death Regulations and Their Application in Study of Apoptosis Regulation and Drug Design for Breast Cancer

    Science.gov (United States)

    2005-07-01

    Apoptosis Regulation and Drug Design for Breast Cancer PRINCIPAL INVESTIGATOR: Ding Xue, Ph.D. Chonglin Yang, Ph.D. Nathan Camp CONTRACTING ORGANIZATION...Aptamers for CED-9/Bcl-2 Family Cell Death Regulations and Their Application in Study of Apoptosis Regulation and Drug Design for Breast Cancer 5b. GRANT...aptamers for CED-9/Bcl-2 family cell death regulators and their application in study of apoptosis regulation and drug design for breast cancer. The 4th Era

  17. The role of the acidity of N-heteroaryl sulfonamides as inhibitors of bcl-2 family protein-protein interactions.

    Science.gov (United States)

    Touré, B Barry; Miller-Moslin, Karen; Yusuff, Naeem; Perez, Lawrence; Doré, Michael; Joud, Carol; Michael, Walter; DiPietro, Lucian; van der Plas, Simon; McEwan, Michael; Lenoir, Francois; Hoe, Madelene; Karki, Rajesh; Springer, Clayton; Sullivan, John; Levine, Kymberly; Fiorilla, Catherine; Xie, Xiaoling; Kulathila, Raviraj; Herlihy, Kara; Porter, Dale; Visser, Michael

    2013-02-14

    Overexpression of the antiapoptotic members of the Bcl-2 family of proteins is commonly associated with cancer cell survival and resistance to chemotherapeutics. Here, we describe the structure-based optimization of a series of N-heteroaryl sulfonamides that demonstrate potent mechanism-based cell death. The role of the acidic nature of the sulfonamide moiety as it relates to potency, solubility, and clearance is examined. This has led to the discovery of novel heterocyclic replacements for the acylsulfonamide core of ABT-737 and ABT-263.

  18. Two independent positive feedbacks and bistability in the Bcl-2 apoptotic switch.

    Directory of Open Access Journals (Sweden)

    Jun Cui

    Full Text Available BACKGROUND: The complex interplay between B-cell lymphoma 2 (Bcl-2 family proteins constitutes a crucial checkpoint in apoptosis. Its detailed molecular mechanism remains controversial. Our former modeling studies have selected the 'Direct Activation Model' as a better explanation for experimental observations. In this paper, we continue to extend this model by adding interactions according to updating experimental findings. METHODOLOGY/PRINCIPAL FINDINGS: Through mathematical simulation we found bistability, a kind of switch, can arise from a positive (double negative feedback in the Bcl-2 interaction network established by anti-apoptotic group of Bcl-2 family proteins. Moreover, Bax/Bak auto-activation as an independent positive feedback can enforce the bistability, and make it more robust to parameter variations. By ensemble stochastic modeling, we also elucidated how intrinsic noise can change ultrasensitive switches into gradual responses. Our modeling result agrees well with recent experimental data where bimodal Bax activation distributions in cell population were found. CONCLUSIONS/SIGNIFICANCE: Along with the growing experimental evidences, our studies successfully elucidate the switch mechanism embedded in the Bcl-2 interaction network and provide insights into pharmacological manipulation of Bcl-2 apoptotic switch as further cancer therapies.

  19. BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion

    Energy Technology Data Exchange (ETDEWEB)

    Cekanova, Maria, E-mail: mcekanov@utk.edu [Department of Small Animal Clinical Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN (United States); Fernando, Romaine I. [Department of Obstetrics and Gynecology, Graduate School of Medicine, Medical Center, The University of Tennessee, Knoxville, TN (United States); Siriwardhana, Nalin [Department of Animal Science, The University of Tennessee, Knoxville, TN (United States); Sukhthankar, Mugdha [Department of Biomedical and Diagnostics Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN (United States); Parra, Columba de la [Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, PR (United States); Woraratphoka, Jirayus [Department of Obstetrics and Gynecology, Graduate School of Medicine, Medical Center, The University of Tennessee, Knoxville, TN (United States); Malone, Christine [Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Ström, Anders [Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Baek, Seung J. [Department of Biomedical and Diagnostics Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN (United States); Wade, Paul A. [Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Saxton, Arnold M. [Department of Animal Science, The University of Tennessee, Knoxville, TN (United States); Donnell, Robert M. [Department of Biomedical and Diagnostics Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN (United States); Pestell, Richard G. [Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA (United States); and others

    2015-02-01

    We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer. - Highlights: • BAD and p-BAD expressions are decreased in breast cancer compared with normal breast tissue. • BAD impedes breast cancer invasion and migration. • BAD inhibits the EMT and transcription factors that promote cancer cell migration. • Invasion and migration functions of BAD are distinct from the BAD's role in apoptosis.

  20. Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Two major apoptosis pathways have been defined in mammalian cells, the Fas/TNF-R1 death receptor pathway and the mitochondria pathway. The Bcl-2 family proteins consist of both anti-apoptosis and pro- apoptosis members that regulate apoptosis, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates downstream caspases. Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.

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

    Science.gov (United States)

    Schöneich, 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 cells 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.

  2. Anti-apoptotic BFL-1 is the major effector in activation-induced human mast cell survival.

    Directory of Open Access Journals (Sweden)

    Maria Ekoff

    Full Text Available Mast cells are best known for their role in allergic reactions, where aggregation of FcεRI leads to the release of mast cell mediators causing allergic symptoms. The activation also induces a survival program in the cells, i.e., activation-induced mast cell survival. The aim of the present study was to investigate how the activation-induced survival is mediated. Cord blood-derived mast cells and the mast cell line LAD-2 were activated through FcεRI crosslinking, with or without addition of chemicals that inhibit the activity or expression of selected Bcl-2 family members (ABT-737; roscovitine. Cell viability was assessed using staining and flow cytometry. The expression and function of Bcl-2 family members BFL-1 and MCL-1 were investigated using real-time quantitative PCR and siRNA treatment. The mast cell expression of Bfl-1 was investigated in skin biopsies. FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1. ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L, Bcl-2, Bcl-w and Mcl-1. Reducing BFL-1 but not MCL-1 levels by siRNA inhibited activation-induced mast cell survival. We also demonstrate that mast cell expression of Bfl-1 is elevated in birch-pollen-provocated skin and in lesions of atopic dermatitis and psoriasis patients. Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.

  3. Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation

    Directory of Open Access Journals (Sweden)

    Andrew N. Rouble

    2013-02-01

    Full Text Available In the context of normal cell turnover, apoptosis is a natural phenomenon involved in making essential life and death decisions. Apoptotic pathways balance signals which promote cell death (pro-apoptotic pathways or counteract these signals (anti-apoptotic pathways. We proposed that changes in anti-apoptotic proteins would occur during mammalian hibernation to aid cell preservation during prolonged torpor under cellular conditions that are highly injurious to most mammals (e.g. low body temperatures, ischemia. Immunoblotting was used to analyze the expression of proteins associated with pro-survival in six tissues of thirteen-lined ground squirrels, Ictidomys tridecemlineatus. The brain showed a concerted response to torpor with significant increases in the levels of all anti-apoptotic targets analyzed (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP as well as enhanced phosphorylation of Bcl-2 at S70 and T56. Heart responded similarly with most anti-apoptotic proteins elevated significantly during torpor except for Bcl-xL and xIAP that decreased and Mcl-1 that was unaltered. In liver, BI-1 increased whereas cIAP1/2 decreased. In kidney, there was an increase in BI-1, cIAP and xIAP but decreases in Bcl-xL and p-Bcl-2(T56 content. In brown adipose tissue, protein levels of BI-1, cIAP1/2, and xIAP decreased significantly during torpor (compared with euthermia whereas Bcl-2, Bcl-xL, Mcl-1 were unaltered; however, Bcl-2 showed enhanced phosphorylation at Thr56 but not at Ser70. In skeletal muscle, only xIAP levels changed significantly during torpor (an increase. The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart and brain where coordinated enhancement of anti-apoptotic proteins occurred in response to torpor.

  4. Evaluation of anti-apoptotic activity of different dietary antioxidants in renal cell carcinoma against hydrogen peroxide

    Institute of Scientific and Technical Information of China (English)

    Garg Neeraj K; Mangal Sharad; Sahu Tejram; Mehta Abhinav; Vyas Suresh P; Tyagi Rajeev K

    2011-01-01

    Objective: To evaluate the anti-apoptotic and radical scavenging activities of dietary phenolics, namely ascorbic acid, -tocopherol acetate, citric acid, salicylic acid, and estimate H2O2-induced apoptosis in renal cell carcinoma cells. Methods: The intracellular antioxidant potency of antioxidants was investigated. H2O2-induced apoptosis in RCC-26 was assayed with the following parameters: cell viability (% apoptosis), nucleosomal damage and DNA fragmentation, bcl-2 levels and flow cytometery analysis (ROS production evaluation). Results: The anticancer properties of antioxidants such as ascorbic acid, - tocopherol acetate, citric acid, salicylic acid with perdurable responses were investigated. It was observed that these antioxidants had protective effect (anti-apoptotic activity) against hydrogen peroxide (H2O2) in renal cell carcinoma (RCC-26) cell line. Conclusions: This study reveals and proves the anticancer properties. However, in cancer cell lines anti-apoptotic activity can indirectly reflect the cancer promoter activity through radicals scavenging, and significantly protect nucleus and bcl-2.

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

    Science.gov (United States)

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

    2007-01-01

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

  6. Assessment of expression of selected Bcl-2 family proteins in lymphoid infiltration in patients with B-cell chronic lymphocytic leukaemia treated with nucleoside analogues.

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    Janusz Kłoczko

    2008-12-01

    Full Text Available B-cell chronic lymphocytic leukaemia (B-CLL is characterized by clonal growth and accumulation of mature lymphoid cells due to disturbance in genetically regulated form of cell death called apoptosis. The intrinsic mechanism of apoptosis is controlled by Bcl-2 family proteins. Purine nucleoside analogues induce the apoptosis in cells in a state of quiescence. The aim of the study was to assess expression of selected Bcl-2 family proteins in neoplastic infiltration in bone marrow in patients with B-CLL treated with nucleoside analogues. The study comprised examination of bone marrow obtained routinely by trephine biopsy from 18 patients with B-CLL diagnosed before administration of purine nucleoside analogues treatment and after its completion. Expression of Bcl-2, Bcl-x and Bax proteins was examined. Lymphoid cells in bone marrow were present in all patients before administration of treatment. After treatment in two patients bone marrow was infiltrated in diffuse pattern, whereas other patients presented nodular pattern of infiltration. The difference between stage of infiltration before and after treatment was statistically significant (p<0.002. High percentage of infiltration cells with positive anti Bcl-2 reaction from 42.0% in one patient to 85.33+/-3.06% in four patients before treatment was observed. After treatment percentage of infiltration cells with positive anti Bcl-2 antibody reaction was from 33.0+/-18.38% in two patients to 99.0% in one patient. Positive correlation between stage of infiltration and expression of Bcl-2 protein was confirmed before and after treatment. Such correlations were not observed in case of Bax and Bcl-x. Strong staining of immunohistochemical reaction of cells in lymphoid infiltration with Bcl-2 antibody was confirmed. There was a difference between Bcl-/Bax ratio before and after treatment. Immunohistochemical assessment of expression of Bcl-2 family proteins in cells of lymphoid infiltration in bone

  7. Selective Impairment of TH17-Differentiation and Protection against Autoimmune Arthritis after Overexpression of BCL2A1 in T Lymphocytes

    Science.gov (United States)

    Iglesias, Marcos; Augustin, Juan Jesús; Alvarez, Pilar; Santiuste, Inés; Postigo, Jorge; Merino, Jesús; Merino, Ramón

    2016-01-01

    The inhibition of apoptotic cell death in T cells through the dysregulated expression of BCL2 family members has been associated with the protection against the development of different autoimmune diseases. However, multiple mechanisms were proposed to be responsible for such protective effect. The purpose of this study was to explore the effect of the T-cell overexpression of BCL2A1, an anti-apoptotic BCL2 family member without an effect on cell cycle progression, in the development of collagen-induced arthritis. Our results demonstrated an attenuated development of arthritis in these transgenic mice. The protective effect was unrelated to the suppressive activity of regulatory T cells but it was associated with a defective activation of p38 mitogen-activated protein kinase in CD4+ cells after in vitro TCR stimulation. In addition, the in vitro and in vivo TH17 differentiation were impaired in BCL2A1 transgenic mice. Taken together, we demonstrated here a previously unknown role for BCL2A1 controlling the activation of CD4+ cells and their differentiation into pathogenic proinflammatory TH17 cells and identified BCL2A1 as a potential target in the control of autoimmune/inflammatory diseases. PMID:27433938

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

    Science.gov (United States)

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

    2017-01-01

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

  9. p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum.

    Science.gov (United States)

    Ng, F W; Nguyen, M; Kwan, T; Branton, P E; Nicholson, D W; Cromlish, J A; Shore, G C

    1997-10-20

    We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

  10. Increased apoptosis in high-fat diet induced nonalcoholic steatohepatitis is associated with JNK activation and imbalance of pro- and anti-apoptotic proteins in Bcl-2 family in rats

    Science.gov (United States)

    Hepatocyte apoptosis in addition to increased oxidative stress could be a key component in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the cellular apoptotic response and its association with oxidative stress as well as the underlying mechanisms have not been investigated in hi...

  11. THE ROLES OF bcl-2 GENE FAMILY IN THE PULMONARY ARTERY REMODELING OF HYPOXIA PULMONARY HYPERTENSION IN RATS

    Institute of Scientific and Technical Information of China (English)

    杨成; 王胜发; 梁桃; 王巨; 王凯; 王柏春

    2001-01-01

    Objective. To investigate the roles of apoptosis in the pulmonary artery remodeling of pulmonary hypertension secondary to hypoxia and illustrate the relative genes expression.Methods. Thirty rats were divided into hypoxia group(10%O2, 8h/d) and normal control group. On the 15th day of hypoxia, pulmonary artery pressure and right ventricular hypertrophy index were measured and pulmonary artery vessels were studied by light microscope. Then terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)technique was used to detect nucleosomal DNA fragmentation of apoptotic cells.In situ hybridization and RT-PCR were used to detect the expression level of bcl-2 and bax.``Results. The pulmonary artery pressure and right ventricular hypertrophy index of hypoxia group were increased significantly, the pulmonary artery wall of hypoxic group become incrassate than control group. Apoptotic cells can be found in lung with hypoxia or without hypoxia. Compared with control group, apoptotic index of hypoxic group decreased significantly. Through the methods of in situ hybridization and RT-PCR, we found the expression of bcl-2 increased whereas bax decreased significantly in the hypoxic group.``Conclusion. The alternation in bcl-2 and bax expression induced by hypoxia play an important role in the pulmonary artery remodeling which is the main pathologic change of pulmonary hypertension secondary to hypoxia.

  12. Glutathione and Bcl-2 targeting facilitates elimination by chemoradiotherapy of human A375 melanoma xenografts overexpressing bcl-xl, bcl-2, and mcl-1

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    Mena Salvador

    2012-01-01

    Full Text Available Abstract Background Bcl-2 is believed to contribute to melanoma chemoresistance. However, expression of Bcl-2 proteins may be different among melanomas. Thus correlations among expression of Bcl-2-related proteins and in vivo melanoma progression, and resistance to combination therapies, was investigated. Methods Human A375 melanoma was injected s.c. into immunodeficient nude mice. Protein expression was studied in tumor samples obtained by laser microdisection. Transfection of siRNA or ectopic overexpression were applied to manipulate proteins which are up- or down-regulated, preferentially, during melanoma progression. Anti-bcl-2 antisense oligonucleotides and chemoradiotherapy (glutathione-depleting agents, paclitaxel protein-binding particles, daunorubicin, X rays were administered in combination. Results In vivo A375 cells down-regulated pro-apoptotic bax expression; and up-regulated anti-apoptotic bcl-2, bcl-xl, and mcl-1, however only Bcl-2 appeared critical for long-term tumor cell survival and progression in vivo. Reduction of Bcl-2, combined with partial therapies, decreased melanoma growth. But only Bcl-2 targeting plus the full combination of chemoradiotherapy eradicated A375 melanoma, and led to long-term survival (> 120 days without recurrence in 80% of mice. Tumor regression was not due to immune stimulation. Hematology and clinical chemistry data were within accepted clinical toxicities. Conclusion Strategies to target Bcl-2, may increase the effectiveness of antitumor therapies against melanomas overexpressing Bcl-2 and likely other Bcl-2-related antiapoptotic proteins.

  13. Chemoprevention of intestinal tumorigenesis by nabumetone: induction of apoptosis and Bcl-2 downregulation.

    Science.gov (United States)

    Roy, H K; Karoski, W J; Ratashak, A; Smyrk, T C

    2001-05-18

    Treatment of MIN mice with the nonsteroidal anti-inflammatory drug, nabumetone, resulted in a dose-dependent suppression of intestinal tumorigenesis. In both the uninvolved MIN mouse colonic epithelium and HT-29 colon cancer cells, nabumetone downregulated the anti-apoptotic protein, Bcl-2, with concomitant induction of apoptosis, suggesting a potential mechanism for colon cancer chemoprevention.

  14. Apoptosis induced by overall metabolic stress converges on the Bcl-2 family proteins Noxa and Mcl-1.

    Science.gov (United States)

    Wensveen, Felix M; Alves, Nuno L; Derks, Ingrid A M; Reedquist, Kris A; Eldering, Eric

    2011-07-01

    Apoptosis provoked by glucose shortage in dividing T cells is mediated via the BH3-only protein Noxa and inhibition of its binding partner Mcl-1. It is unknown how signals from cellular metabolism can affect the balance between Mcl-1 and Noxa and to what extent other Bcl-2 members are involved in this apoptosis cascade. Here, we defined the mechanism underlying apoptosis in relation to various types of metabolic stress. First, we established that the Noxa/Mcl-1 balance is regulated by glucose deprivation as well as by general metabolic stress, via changes in proteasome-mediated degradation of Mcl-1. Second, in contrast with cytokine-deprivation, no transcriptional modulation of Mcl-1, Puma, Bim or Noxa was observed during glucose deprivation. Third, no changes in PKB or GSK3 activity occurred and no clear role for AMPK was detected. Fourth, apoptosis triggered by nutrient deprivation was executed without signs of overt autophagy and independent of ROS production or p38 MAP kinase activity. Lastly, apoptosis under nutrient limitation could also be delayed by knock-down of Bim or overexpression of Bcl-2. In conclusion, Noxa functions in a specific apoptotic pathway that integrates overall nutrient stress, independent from attenuated PI3K/PKB signaling and without clear involvement of autophagy.

  15. SS-A/Ro52 promotes apoptosis by regulating Bcl-2 production

    Energy Technology Data Exchange (ETDEWEB)

    Jauharoh, Siti Nur Aisyah [Department of Clinical Pathology and Immunology, Kobe University Graduate School of Medicine, Hyogo 650-0017 (Japan); Faculty of Medicine and Health Science, Syarif Hidayatullah State Islamic University, Jakarta 15412 (Indonesia); Saegusa, Jun; Sugimoto, Takeshi [Department of Clinical Pathology and Immunology, Kobe University Graduate School of Medicine, Hyogo 650-0017 (Japan); Ardianto, Bambang [Department of Clinical Pathology and Immunology, Kobe University Graduate School of Medicine, Hyogo 650-0017 (Japan); Department of Child Health, Faculty of Medicine, Gadjah Mada University, Yogyakarta 55282 (Indonesia); Kasagi, Shimpei; Sugiyama, Daisuke; Kurimoto, Chiyo [Department of Clinical Pathology and Immunology, Kobe University Graduate School of Medicine, Hyogo 650-0017 (Japan); Tokuno, Osamu; Nakamachi, Yuji [Department of Laboratory Medicine, Kobe University Hospital, Hyogo 650-0017 (Japan); Kumagai, Shunichi [Department of Clinical Pathology and Immunology, Kobe University Graduate School of Medicine, Hyogo 650-0017 (Japan); Kawano, Seiji, E-mail: sjkawano@med.kobe-u.ac.jp [Department of Clinical Pathology and Immunology, Kobe University Graduate School of Medicine, Hyogo 650-0017 (Japan); Department of Laboratory Medicine, Kobe University Hospital, Hyogo 650-0017 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Ro52{sup low} HeLa cells are resistant to apoptosis upon various stimulations. Black-Right-Pointing-Pointer Ro52 is upregulated by IFN-{alpha}, etoposide, or IFN-{gamma} and anti-Fas Ab. Black-Right-Pointing-Pointer Ro52-mediated apoptosis is independent of p53. Black-Right-Pointing-Pointer Ro52 selectively regulates Bcl-2 expression. -- Abstract: SS-A/Ro52 (Ro52), an autoantigen in systemic autoimmune diseases such as systemic lupus erythematosus and Sjoegren's syndrome, has E3 ligase activity to ubiquitinate proteins that protect against viral infection. To investigate Ro52's role during stress, we transiently knocked it down in HeLa cells by siRo52 transfection. We found that Ro52{sup low} HeLa cells were significantly more resistant to apoptosis than wild-type HeLa cells when stimulated by H{sub 2}O{sub 2}- or diamide-induced oxidative stress, IFN-{alpha}, IFN-{gamma} and anti-Fas antibody, etoposide, or {gamma}-irradiation. Furthermore, Ro52-mediated apoptosis was not influenced by p53 protein level in HeLa cells. Depleting Ro52 in HeLa cells caused Bcl-2, but not other Bcl-2 family molecules, to be upregulated. Taken together, our data showed that Ro52 is a universal proapoptotic molecule, and that its proapoptotic effect does not depend on p53, but is exerted through negative regulation of the anti-apoptotic protein Bcl-2. These findings shed light on a new physiological role for Ro52 that is important to intracellular immunity.

  16. In situ hybridisation detects pro-apoptotic gene expression of a Bcl-2 family member in white syndrome-affected coral.

    Science.gov (United States)

    Ainsworth, T D; Knack, B; Ukani, L; Seneca, F; Weiss, Y; Leggat, W

    2015-12-09

    White syndrome has been described as one of the most prolific diseases on the Great Barrier Reef. Previously, apoptotic cell death has been described as the mechanism driving the characteristic rapid tissue loss associated with this disease, but the molecular mechanisms controlling apoptotic cell death in coral disease have yet to be investigated. In situ methods were used to study the expression patterns of 2 distinct regulators of apoptosis in Acropora hyacinthus tissues undergoing white syndrome and apoptotic cell death. Apoptotic genes within the Bcl-2 family were not localized in apparently healthy coral tissues. However, a Bcl-2 family member (bax-like) was found to localize to cells and tissues affected by white syndrome and those with morphological evidence for apoptosis. A potential up-regulation of pro-apoptotic or bax-like gene expression in tissues with apoptotic cell death adjacent to disease lesions is consistent with apoptosis being the primary cause of rapid tissue loss in coral affected by white syndrome. Pro-apoptotic (bax-like) expression in desmocytes and the basal tissue layer, the calicodermis, distant from the disease lesion suggests that apoptosis may also underlie the sloughing of healthy tissues associated with the characteristic, rapid spread of tissue loss, evident of this disease. This study also shows that in situ hybridisation is an effective tool for studying gene expression in adult corals, and wider application of these methods should allow a better understanding of many aspects of coral biology and disease pathology.

  17. Roscovitine-induced apoptosis in neutrophils and neutrophil progenitors is regulated by the Bcl-2-family members Bim, Puma, Noxa and Mcl-1.

    Directory of Open Access Journals (Sweden)

    Sanjivan Gautam

    Full Text Available Neutrophil granulocyte (neutrophil apoptosis plays a key role in determining inflammation in infectious and non-infectious settings. Recent work has shown that inhibitors of cyclin-dependent kinases (cdk such as roscovitine can potently induce neutrophil apoptosis and reduce inflammation. Using a conditional Hoxb8-expression system we tested the participation of Bcl-2-family proteins to roscovitine-induced apoptosis in mouse neutrophils and in neutrophil progenitor cells. Bcl-2 strongly protected against roscovitine-induced apoptosis in neutrophils. The isolated loss of either Bim or noxa provided significant, partial protection while protection through combined loss of Bim and noxa or Bim and Puma was only slightly greater than this individual loss. The only substantial change in protein levels observed was the loss of Mcl-1, which was not transcriptional and was inhibited by proteasome blockade. In progenitor cells there was no protection by the loss of Bim alone but substantial protection by the loss of both Bim and Puma; surprisingly, strongest protection was seen by the isolated loss of noxa. The pattern of protein expression and Mcl-1-regulation in progenitor cells was very similar to the one observed in differentiated neutrophils. In addition, roscovitine strongly inhibited proliferation in progenitor cells, associated with an accumulation of cells in G2/M-phase.

  18. MicroRNA-125b Induces Cancer Cell Apoptosis Through Suppression of Bcl-2 Expression

    Institute of Scientific and Technical Information of China (English)

    Aihua Zhao; Quan Zeng; Xiaoyan Xie; unnian Zhou; Wen Yue; Yali Li; Xuetao Pei

    2012-01-01

    MicroRNAs (miRNAs) are small,noncoding RNAs which can often act as an oncogene or a tumor suppressor.Several miRNAs are associated with the development of hepatocellular carcinoma (HCC).We demonstrated that miR-125b significantly suppresses HCC cell proliferation and promotes apoptosis by inhibiting the gene expression of the anti-apoptotic protein,Bcl-2.Bioinformatic analysis indicated that the 3'UTR of Bcl-2 has binding sites for miR-125b.Luciferase reporter assay confirmed the ability of miR-125b to dramatically suppress Bcl-2 transcription,suggesting that Bcl-2 is a target gene for miR-125b.We concluded that miR-125b acts as a tumor suppressor in hepatic tumor development by targeting Bcl-2 and inducing cancer cell apoptosis.

  19. FGF-2 inhibits TNF-α mediated apoptosis through upregulation of Bcl2-A1 and Bcl-xL in ATDC5 cells

    Directory of Open Access Journals (Sweden)

    Hey-Ryun Kim

    2012-05-01

    Full Text Available FGF-2 is involved in cell survival, proliferation, apoptosis, andangiogenesis in a wide variety of cells. FRGRs, PI3K and MAPkinases are well known mediators of FGF signaling. Despite itsknown roles during many developmental processes, includingosteogenesis, there are few known targets of FGF-2. In thepresent study, we identified Bcl2-A1 and Bcl-xL as two prominenttargets involved in promoting cell survival. Pretreatmentof ATDC5 cells with FGF-2 increased cell survival, whilesiRNAs specific for Bcl2-A1 and Bcl-xL compromised the anti-apoptotic effect of FGF-2, sensitized the cells to apoptosistriggered by TNF-α. Chemical inhibition of FGFR, NFkB, andPI3K activity by PD173074, pyrrolidine dithiocarbamate, andLY294002 respectively abrogated the FGF-2-mediated inductionof Bcl2-A1 and Bcl-xL expression. Taken together, our datademonstrate that a subset of Bcl2 family proteins are the targetsof FGF-2 signaling that promotes the survival of ATDC5cells. [BMB reports 2012; 45(5: 287-292

  20. Evaluation of anti-apoptotic activity of different dietary antioxidants in renal cell carcinoma against hydrogen peroxide

    Institute of Scientific and Technical Information of China (English)

    Garg; Neeraj; K; Mangal; Sharad; Sahu; Tejram; Mehta; Abhinav; Vyas; Suresh; P; Tyagi; Rajeev; K

    2011-01-01

    Objective:To evaluate the anti-apoptotic and radical scavenging activities of dietary phenolics, namely ascorbic acid,a-tocopherol acetate,citric acid,salicylic acid,and estimate H2O2induced apoptosis in renal cell carcinoma cells.Methods:The intracellular antioxidant potency of antioxidants was investigated.H2O-2-induced apoptosis in RCC-26 was assayed with the following parameters:cell viability(%apoptosis),nucleosomal damage and DNA fragmentation, bcl-2 levels and flow cytometery analysis(ROS production evaluation).Results:Ine anticancer properties of antioxidants such as ascorbic acid,a- tocopherol acetate,citric acid,salicylic acid with perdurable responses were investigated.It was observed that these antioxidants had protective effect(anti-apoptotic activity) against hydrogen peroxide(H2O2) in renal cell carcinoma(RCC-26) cell line.Conclusions:This study reveals and proves the anticancer properties.However,in cancer cell lines anti-apoptotic activity can indirectly reflect the cancer promoter activity through radicals scavenging,and significantly protect nucleus and bcl-2.

  1. Sesamin suppresses STZ induced INS-1 cell apoptosis through inhibition of NF-κB activation and regulation of Bcl-2 family protein expression.

    Science.gov (United States)

    Zheng, Shuguo; Zhao, Mengqiu; Ren, Younan; Wu, Yuanjie; Yang, Jieren

    2015-03-05

    Diverse risk factors for diabetes can induce oxidative stress, leading to pancreatic beta cell damage and insulin secretion dysfunction. In the present study, we evaluated the effect of sesamin on streptozotocin (STZ) induced apoptosis in INS-1 cells and the possible mechanisms implicated. After preincubation with indicated concentrations of sesamin (0.1, 1.0 and 10.0μmol/l) for 24h, INS-1 cells were exposed to STZ (3mmol/l) for 12h. Sesamin effectively improved STZ induced cell damage as determined by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] assay and insulin secretion capacity, and suppressed STZ induced cell apoptosis as evaluated by flow cytometry using annexin V and propidium iodide double staining. Western blot analysis demonstrated that sesamin markedly suppressed STZ induced nuclear factor kappa B (NF-κB) activation, with Bax protein down-regulated and Bcl-2 protein up-regulated significantly. Preincubation with sesamin resulted in an evident enhancement of total antioxidant capacity in INS-1 cells, accompanied by a significant reduction of intracellular reactive oxygen species and malondialdehyde, an end product of lipid peroxidation. Taken together, these findings suggested that sesamin was capable of suppressing STZ induced INS-1 cell apoptosis, which might be ascribed, at least partly, to the inhibition of NF-κB activation and subsequent regulation of Bcl-2 family protein expression. This study would provide a potential target for treatment of diabetes with sesamin as well as other antioxidants.

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

    Science.gov (United States)

    Aveic, Sanja; Pigazzi, Martina; Basso, Giuseppe

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sanja Aveic

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

  4. The Anti-Apoptotic Role of Berberine in Preimplantation Embryo In Vitro Development through Regulation of miRNA-21.

    Science.gov (United States)

    Zhang, Chao; Shi, Ya-Ran; Liu, Xiao-Ran; Cao, Yong-Chun; Zhen, Di; Jia, Zi-Ye; Jiang, Jin-Qi; Tian, Jian-Hui; Gao, Jian-Ming

    2015-01-01

    Traditional Chinese medicinal herbs containing berberine have been historically used to prevent miscarriage. Here, we investigated whether the anti-apoptotic effects of berberine on pre-implantation embryonic development are regulated by miRNA-21. Mouse pronuclear embryos were cultured in medium with or without berberine, and some were then microinjected with a miRNA-21 inhibitor. The in vitro developmental rates of 2- and 4-cell embryos and blastocysts, blastocyst cell numbers, apoptotic rates, and apoptotic cell numbers were measured in each group. Furthermore, we examined the transcription levels of miRNA-21 and its target genes (caspase-3, PTEN, and Bcl-2) and their translation levels. Comparisons were made with in vivo-developed and untreated embryos. We found that berberine significantly increased the developmental rates and cell numbers of mouse blastocysts and decreased apoptotic cell rates in vitro. Berberine also significantly increased miRNA-21 and Bcl-2 transcription levels and significantly decreased caspase-3 and PTEN transcription levels. In embryos treated with a miRNA-21 inhibitor, the results followed the opposite trend; PTEN and caspase-3 transcription levels increased significantly, while the transcription level of Bcl-2 decreased significantly. Additionally, berberine treatment significantly increased the Bcl-2 protein level and significantly decreased the caspase-3 and PTEN protein levels in blastocysts, but there were no significant differences observed in the levels of these proteins in 2- and 4-cell embryos. This study revealed that miRNA-21 is important for pre-implantation embryonic development, especially blastocyst development in vitro. Berberine elevates miRNA-21 expression, decreases PTEN and caspase-3 levels, increases Bcl-2 levels, and exerts anti-apoptotic and pro-growth effects.

  5. Expression of Bcl-2 in canine osteosarcoma

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    F. Piro

    2015-03-01

    Full Text Available Osteosarcoma (OS is the most common primary malignancy of bone. It is responsible for 80-85% of the primary bone tumors affecting dogs and it is characterized by aggressive and invasive behavior, with a high metastatic potential. Several studies on cancer and related tumorigenesis, show an involvement of the mechanisms of programmed cell death and cell survival. Many signals seem to be involved in the related mechanism of autophagy and in particular, our interest is focused on the expression of a family of Bcl-2 that seems to be involved either in the control of biomolecular mechanisms like autophagy and apoptosis. In this study we investigated the expression of Bcl-2 in different cases of spontaneous canine osteosarcoma and the related preliminary results are described. We found Bcl-2 activity was increased in OS tissue compared to normal bone tissue. These results suggested that Bcl-2 activity may play an important role in the formation of OS and as a diagnostic for neoplastic activity. However, further research is needed to confirm the role of Bcl-2 activity in OS in canines.

  6. Chemoprevention of intestinal tumorigenesis by nabumetone: induction of apoptosis and Bcl-2 downregulation

    OpenAIRE

    Roy, H K; Karoski, W J; Ratashak, A; Smyrk, T. C.

    2001-01-01

    Treatment of MIN mice with the nonsteroidal anti-inflammatory drug, nabumetone, resulted in a dose-dependent suppression of intestinal tumorigenesis. In both the uninvolved MIN mouse colonic epithelium and HT-29 colon cancer cells, nabumetone downregulated the anti-apoptotic protein, Bcl-2, with concomitant induction of apoptosis, suggesting a potential mechanism for colon cancer chemoprevention. © 2001 Cancer Research Campaign www.bjcancer.com

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

    Institute of Scientific and Technical Information of China (English)

    Qin FU; Chen HE; Zheng-rong MAO

    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.

  8. Serum Bcl-2 concentrations in overweight-obese subjects with nonalcoholic fatty liver disease

    Institute of Scientific and Technical Information of China (English)

    Giovanni Tarantino; Francesco Scopacasa; Annamaria Colao; Domenico Capone; Marianna Tarantino; Ernesto Grimaldi; Silvia Savastano

    2011-01-01

    AIM: To shed some light on the relationship between anti-apoptotic serum Bcl-2 concentrations and metabolic status, anthropometric parameters, inflammation indices, and non-alcoholic fatty liver disease severity were investigated in 43 young individuals with fatty liver (FL) and 41 with nonalcoholic steatohepatitis (NASH). METHODS: Circulating levels of Bcl-2 were detected in 84 patients with ultrasonographic findings of "bright liver" and/or hyper-transaminasemia of unknown origin and/or increase in γ-glutamyl-transpeptidase (γ-GT) strictly in the absence of other acute or chronic liver disease, whose age was not advanced, who gave consent to liver biopsy and were then divided on the basis of the histological results into two groups (43 with FL and 41 with NASH). Twenty lean subjects, apparently healthy and young, were chosen as controls.RESULTS: Serum Bcl-2 concentrations were significantly higher in the FL group than in the NASH group. Insulin resistance and γ-GT activity were significantly higher in NASH subjects. Apoptotic hepatocytes were significantly more numerous in NASH patients. NASH patients presented with larger spleens and augmented C-reactive protein (CRP) concentrations than healthy subjects. Steatosis grade at histology was similar in both NASH and FL populations. The number of apoptotic cells was significantly related to anti-apoptotic Bcl-2 protein values in FL patients. Bcl-2 serum levels positively correlated to body mass index (BMI) values (P ≤ 0.0001) but not to age of the population. Triglycerides/HDL ratio correlated well to waist circumference in males (P = 0.0008). γ-GT activity was associated with homeostatic metabolic assessment (HOMA) (P = 0.0003) and with serum ferritin (P = 0.02). Bcl-2 concentrations were not related to either spleen size or CRP values. NASH patients presented a weak negative correlation between lobular inflammation and Bcl-2 levels. A prediction by low values of serum Bcl-2 towards a greater presence of

  9. Destruxin B Isolated from Entomopathogenic Fungus Metarhizium anisopliae Induces Apoptosis via a Bcl-2 Family-Dependent Mitochondrial Pathway in Human Nonsmall Cell Lung Cancer Cells

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    Chun-Chi Wu

    2013-01-01

    Full Text Available Destruxin B, isolated from entomopathogenic fungus Metarhizium anisopliae, is one of the cyclodepsipeptides with insecticidal and anticancer activities. In this study, destruxin B was extracted and purified by ion-exchange chromatography, silica gel chromatography, and semipreparative high-performance liquid chromatography. The potential anticancer effects and molecular mechanisms of destruxin B in human nonsmall cell lung cancer cell lines were characterized. Our results showed that destruxin B induced apoptotic cell death in A549 cells. This event was accompanied by the activation of caspase-2, -3, and -9. Moreover, destruxin B increased the expression level of proapoptotic molecule, PUMA, while decreased antiapoptotic molecule Mcl-1. Additionally, the translocation of Bax from cytosol to mitochondrial membrane was observed upon destruxin B treatment. Knockdown of Bax by shRNA effectively attenuated destruxin-B-triggered apoptosis in A549 cells. Interestingly, similar toxic effects and underlying mechanisms including caspase activation, upregulation of PUMA, and downregulation of Mcl-1 were also observed in a p53-null lung cancer H1299 cell line upon destruxin B treatment. Taken together, our findings suggest that destruxin-B-induced apoptosis in human nonsmall cell lung cancer cells is via a Bcl-2 family-dependent mitochondrial pathway.

  10. Crizotinib (PF-2341066) induces apoptosis due to downregulation of pSTAT3 and BCL-2 family proteins in NPM-ALK(+) anaplastic large cell lymphoma.

    Science.gov (United States)

    Hamedani, Farid Saei; Cinar, Munevver; Mo, Zhicheng; Cervania, Melissa A; Amin, Hesham M; Alkan, Serhan

    2014-04-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is an aberrant fusion gene product with tyrosine kinase activity and is expressed in substantial subset of anaplastic large cell lymphomas (ALCL). It has been shown that NPM-ALK binds to and activates signal transducer and activator of transcription 3 (STAT3). Although NPM-ALK(+) ALCL overall shows a better prognosis, there is a sub-group of patients who relapses and is resistant to conventional chemotherapeutic regimens. NPM-ALK is a potential target for small molecule kinase inhibitors. Crizotinib (PF-2341066) is a small, orally bioavailable molecule that inhibits growth of tumors with ALK activity as shown in a subgroup of non-small lung cancer patients with EML4-ALK expression. In this study, we have investigated the in vitro effects of Crizotinib in ALCL cell line with NPM-ALK fusion. Crizotinib induced marked downregulation of STAT3 phosphorylation, which was associated with significant apoptotic cell death. Apoptosis induction was attributed to caspase-3 cleavage and marked downregulation of the Bcl-2 family of proteins including MCL-1. These findings implicate that Crizotinib has excellent potential to treat patients with NPM-ALK(+) ALCL through induction of apoptotic cell death and downregulation of major oncogenic proteins in this aggressive lymphoma.

  11. Phage display screen for peptides that bind Bcl-2 protein.

    Science.gov (United States)

    Park, Hye-Yeon; Kim, Joungmok; Cho, June-Haeng; Moon, Ji Young; Lee, Su-Jae; Yoon, Moon-Young

    2011-01-01

    Bcl-2 family proteins are key regulators of apoptosis associated with human disease, including cancer. Bcl-2 protein has been found to be overexpressed in many cancer cells. Therefore, Bcl-2 protein is a potential diagnostic target for cancer detection. In the present study, the authors have identified several Bcl-2 binding peptides with high affinity (picomolar range) from a 5-round M13 phage display library screening. These peptides can be used to develop novel diagnostic probes or potent inhibitors with diverse polyvalencies.

  12. c-Myc dependent expression of pro-apoptotic Bim renders HER2-overexpressing breast cancer cells dependent on anti-apoptotic Mcl-1

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    Jézéquel Pascal

    2011-09-01

    Full Text Available Abstract Background Anti-apoptotic signals induced downstream of HER2 are known to contribute to the resistance to current treatments of breast cancer cells that overexpress this member of the EGFR family. Whether or not some of these signals are also involved in tumor maintenance by counteracting constitutive death signals is much less understood. To address this, we investigated what role anti- and pro-apoptotic Bcl-2 family members, key regulators of cancer cell survival, might play in the viability of HER2 overexpressing breast cancer cells. Methods We used cell lines as an in vitro model of HER2-overexpressing cells in order to evaluate how anti-apoptotic Bcl-2, Bcl-xL and Mcl-1, and pro-apoptotic Puma and Bim impact on their survival, and to investigate how the constitutive expression of these proteins is regulated. Expression of the proteins of interest was confirmed using lysates from HER2-overexpressing tumors and through analysis of publicly available RNA expression data. Results We show that the depletion of Mcl-1 is sufficient to induce apoptosis in HER2-overexpressing breast cancer cells. This Mcl-1 dependence is due to Bim expression and it directly results from oncogenic signaling, as depletion of the oncoprotein c-Myc, which occupies regions of the Bim promoter as evaluated in ChIP assays, decreases Bim levels and mitigates Mcl-1 dependence. Consistently, a reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion. Western blot analysis confirms that naïve HER2-overexpressing tumors constitutively express detectable levels of Mcl-1 and Bim, while expression data hint on enrichment for Mcl-1 transcripts in these tumors. Conclusions This work establishes that, in HER2-overexpressing tumors, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of

  13. Silencing Bcl-2 Expression in Epithelial Cancer Cells Using “Smart” Particles

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    Yen-Ling Lin

    2014-09-01

    Full Text Available Short interfering RNA (siRNA targeted against anti-apoptotic Bcl-2 protein proved to knockdown its expression and trigger cancer cell death. We used degradable, pH-sensitive, comb-like [P(EAA-co-BMA-b-PNASI-g-P(HMA-co-TMAEMA] polymer to condense anti-Bcl-2 siRNA into “smart” particles, which proved to shuttle their cargo past the endosomal membrane and into the cytoplasm of HeLa and UM-SCC-17B cancer cells. HeLa and UM-SCC-17B cancer cells were treated with anti-Bcl-2 particles followed by quantifying Bcl-2 mRNA and protein levels using qRT-PCR and western blotting, respectively. “Smart” anti-Bcl-2 particles selectively suppress Bcl-2 mRNA and protein levels in HeLa cells by 50%–60% and 79%–81%, respectively. Similarly, “smart” anti-Bcl-2 particles inhibited Bcl-2 mRNA levels by 30%, 40%, and 20% upon incubation with UM-SCC-17B cancer cells for 48, 72, and 96 h, respectively. Bcl-2 protein expression in UM-SCC-17B cancer cells was inhibited by 30% after treatment for 72 h. Results show that pH-sensitive comb-like polymer complex anti-Bcl-2 siRNA forming “smart” nanoparticles that deliver their cargo into the cytoplasm of HeLa and UM-SCC-17B cancer cells causing Bcl-2 knockdown at the mRNA and protein levels.

  14. Study on the Regulation of Bcl-2 Gene on Rat Spermatogenic Cells Apoptosis in Transcription Level

    Institute of Scientific and Technical Information of China (English)

    董强; 杨宇如; 黄明孔; 李虹; 张卫东; 徐震波

    2000-01-01

    Objective To detect the change of Bcl-2 gene expression in the apopototic process of spermatogenic cells in rat with vasoligation and vasostomy, and to find out the relationship between the transcription of Bcl-2 and the apoptosis of spermatognic cells.Materials & Methods Sixty adult male Sprague-Dawley rats in 3 groups were operated with vasoligation and vasostomy. Then hybridization in situ with hypersensitive Bcl-2 RNA probe was used to detect the change of Bcl-2 mRNA.Results The transcription of Bcl-2 gene in spermatogenic cells was obviously inhibited in the vasoligation group compared with that in the control group (P<0. 05), and the transcription in the vasostomy group showed no difference from that of the control group.Conclusion Bcl-2 gene has an anti-apoptotic effect in rats with vasostomy, and there was a transcriptional regulation of Bcl-2 gene in rat spermatogenic cell during the period of pre-vasoligation to post-vasoligation and to post-vasosotomy.

  15. Bcl-2 family members in childhood acute lymphoblastic leukemia : relationships with features at presentation, in vitro and in vivo drug response and long-term clinical outcome

    NARCIS (Netherlands)

    Salomons, GS; Smets, LA; Verwijs-Janssen, M; Hart, AAM; Haarman, EG; Kaspers, GJL; Van Wering, ER; Van Der Does-Van Den Berg, A; Kamps, WA

    1999-01-01

    We have found that, in addition to Bcl-2 and Bar, the expression levels of apoptosis inducers (Bad, Bak) and inhibitors (Bcl-x(L), Mcl-1) were highly variable in blasts from 78 children with newly diagnosed acute lymphoblastic leukemia (ALL). The patients were enrolled in the national study ALL-7 of

  16. Expression of the Bcl-2 protein BAD promotes prostate cancer growth.

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    Adrienne J Smith

    Full Text Available BAD, a pro-apoptotic protein of the Bcl-2 family, has recently been identified as an integrator of several anti-apoptotic signaling pathways in prostate cancer cells. Thus, activation of EGFR, GPCRs or PI3K pathway leads to BAD phosphorylation and inhibition of apoptosis. Increased levels of BAD in prostate carcinomas have also been reported. It appears contradictory that instead of limiting expression of pro-apoptotic protein, prostate cancer cells choose to increase BAD levels while keeping it under tight phosphorylation control. Analysis of the effect of BAD on prostate cancer xenografts has shown that increased BAD expression enhances tumor growth, while knockdown of BAD expression by shRNA inhibits tumor growth. Tissue culture experiments demonstrated that increased BAD expression stimulates proliferation of prostate cancer cells. These results suggest that increased expression of BAD provides a proliferative advantage to prostate tumors, while BAD dephosphorylation increases sensitivity of prostate cancer cells to apoptosis. Combination of proliferative and apoptotic properties prompts prostate cancer cells to be "addicted" to increased levels of phosphorylated BAD. Thus, kinases that phosphorylate BAD are plausible therapeutic targets; while monitoring BAD phosphorylation could be used to predict tumor response to treatments.

  17. Anti-apoptotic effects of aspirin following cerebral ischemia-reperfusion injury in rats

    Institute of Scientific and Technical Information of China (English)

    Liying Qiu; Bin Du; Ying Li; Hongbin Fan; Zhiyong Yang

    2008-01-01

    /kg aspirin decreased MDA content and increased ATP levels. However, 6 mg/kg aspirin did not have the same effect. CONCLUSION: Aspirin reduced the number of apoptotic cells following CIRI. These results suggest that the neuroprotective mechanism of aspirin could be related to elevated Bcl-2 protein levels or decreased Bax protein expression. The increase in the ratio of Bcl-2 to Bax appears to be a common anti-apoptotic mechanism of aspirin.

  18. Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection.

    Science.gov (United States)

    Kim, H R; Luo, Y; Li, G; Kessel, D

    1999-07-15

    Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was approximately 2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (delta(psi)m), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage.

  19. MiR-122 increases sensitivity of drug-resistant BEL-7402/5-FU cells to 5-fluorouracil via down-regulation of bcl-2 family proteins.

    Science.gov (United States)

    Yin, Jie; Tang, Hui Fang; Xiang, Qiong; Yu, Jia; Yang, Xiao Yang; Hu, Nan; Lei, Xiao Yong

    2011-12-01

    To investigate the changes in drug sensitivity of miR-122 transfected BEL-7402/5-FU cells. MiR-122 and negative miRNA expression vectors were constructed and stably transfected into BEL-7402/5-FU cells. Real-time RT-PCR was used to detect the level of miR-122, Bcl-XL, Bcl-2 and P53 mRNA. Western Blotting was used to detect Bcl-2, Bcl-XL and P53 protein expression. Drug sensitivity of the cells to 5-fluorouracil (5-FU) was analyzed with MTT and flow cytometry. Compared with negative miRNA transfectants or untreated cells, mRNA and protein expression level of Bcl-2, Bcl-XL in stable miR-122 transfectants were decreased. Accordingly, P53 protein expression showed a significant up-regulation; MTT results showed that after incubation with 5-FU, miR-122 transfectants had higher cell inhibitory rates than negative miRNA or untreated cells; flow cytometry results demonstrated that apoptosis rate increased in miR-122 transfected cells, compared with negative miRNA or untreated cells. After addition of 5-FU (10 and 100 micromol/I), miR-122 transfected cells showed higher apoptosis rate than negative miRNA or untreated cells. MiR-122 can specifically down-regulate the expression of Bcl-2 and Bcl-XL, and increase P53 activity in BEL-7402/5-FU cells, which increased cells spontaneous apoptosis and sensitize cells to 5-FU. Therefore, MiR-122 can be used as a potential therapy agent against human hepatoblastoma.

  20. Induction of Apoptosis by Fucoidan in Human Leukemia U937 Cells through Activation of p38 MAPK and Modulation of Bcl-2 Family

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    Yung Hyun Choi

    2013-07-01

    Full Text Available The present study investigated possible mechanisms on the apoptosis induction of human leukemic cells by fucoidan, a sulfated polysaccharide found in marine algae. Fucoidan treatment of cells resulted in inhibition of growth and induction of apoptosis, as measured by 3-(4,5-dimetylthiazol-2-yl-2,5-diphenyl-tetrazolium (MTT assay, fluorescence microscopy, DNA fragmentation, and flow cytometry analysis. The increase in apoptosis was associated with the proteolytic activation of caspases, Bid cleavage, insertion of pro-apoptotic Bax into the mitochondria, release of cytochrome c from mitochondria to cytosol, and loss of mitochondria membrane potential (MMP in U937 cells. However, apoptosis induced by fucoidan was attenuated by caspase inhibitors, indicating that fucoidan-induced apoptosis was dependent on the activation of caspases. Furthermore, fucoidan treatment effectively activated the p38 mitogen-activated protein kinase (MAPK and p38 MAPK inhibitor, SB203580, and significantly reduced fucoidan-induced apoptosis through inhibition of Bax translocation and caspases activation, suggesting that the activation of p38 MAPK may play a key role in fucoidan-induced apoptosis. In addition, the authors found fucoidan-induced significantly attenuated in Bcl-2 overexpressing U937 cells, and pretreatment with fucoidan and HA 14-1, a small-molecule Bcl-2 inhibitor, markedly increased fucoidan-mediated apoptosis in Bcl-2 overexpressing U937 cells. Our findings imply that we may attribute some of the biological functions of p38 MAPK and Bcl-2 to their ability to inhibit fucoidan-induced apoptosis.

  1. A Urinary Bcl-2 Surface Acoustic Wave Biosensor for Early Ovarian Cancer Detection

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    Nathan D. Gallant

    2012-05-01

    Full Text Available In this study, the design, fabrication, surface functionalization and experimental characterization of an ultrasonic MEMS biosensor for urinary anti-apoptotic protein B-cell lymphoma 2 (Bcl-2 detection with sub ng/mL sensitivity is presented. It was previously shown that urinary Bcl-2 levels are reliably elevated during early and late stages of ovarian cancer. Our biosensor uses shear horizontal (SH surface acoustic waves (SAWs on surface functionalized ST-cut Quartz to quantify the mass loading change by protein adhesion to the delay path. SH-SAWs were generated and received by a pair of micro-fabricated interdigital transducers (IDTs separated by a judiciously designed delay path. The delay path was surface-functionalized with monoclonal antibodies, ODMS, Protein A/G and Pluronic F127 for optimal Bcl-2 capture with minimal non-specific adsorption. Bcl-2 concentrations were quantified by the resulting resonance frequency shift detected by a custom designed resonator circuit. The target sensitivity for diagnosis and identifying the stage of ovarian cancer was successfully achieved with demonstrated Bcl-2 detection capability of 500 pg/mL. It was also shown that resonance frequency shift increases linearly with increasing Bcl-2 concentration.

  2. Relationships Between Icariin and Anti-Apoptotic miRNA-21 in Mouse Blastocyst Development In vitro

    Institute of Scientific and Technical Information of China (English)

    SHI Ya-ran; WANG Zhan-he; CAO Yong-chun; LU Yan; TIAN Jin-ling; ZhANG Chao; JIA Zi-ye; CHEN Wu; GAO Jian-ming

    2013-01-01

    In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium (mCZB) as control group. The experimental group (Ica group) was supplemented with 0.6μg mL-1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21:pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group ((85.14±6.57)%vs. (72.04±11.58)%;(82.50±7.11)%vs. (66.80±11.70)%, respectively, P<0.01). The total cell number of blastocysts had extreme difference between Ica group and control group ((61.40±9.64) vs. (46.23±4.50), P<0.01). The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group ((1.47±0.51) vs. (2.94±0.66);(2.40±0.27)%vs. (6.25±0.62)%, respectively, P<0.01). Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 (P<0.01), down-regulated pro-apoptotic Caspase3 (P<0.05) and PTEN (P<0.01), up-regulated anti-apoptotic Bcl-2 (P<0.01). In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-apoptotic genes and down-regulated pro

  3. Downregulation of autophagy by Bcl-2 promotes MCF7 breast cancer cell growth independent of its inhibition of apoptosis.

    Science.gov (United States)

    Oh, S; Xiaofei, E; Ni, D; Pirooz, S D; Lee, J-Y; Lee, D; Zhao, Z; Lee, S; Lee, H; Ku, B; Kowalik, T; Martin, S E; Oh, B-H; Jung, J U; Liang, C

    2011-03-01

    The anti-apoptotic Bcl-2 protein, which confers oncogenic transformation and drug resistance in most human cancers, including breast cancer, has recently been shown to effectively counteract autophagy by directly targeting Beclin1, an essential autophagy mediator and tumor suppressor. However, it remains unknown whether autophagy inhibition contributes to Bcl-2-mediated oncogenesis. Here, by using a loss-of-function mutagenesis study, we show that Bcl-2-mediated antagonism of autophagy has a critical role in enhancing the tumorigenic properties of MCF7 breast cancer cells independent of its anti-apoptosis activity. A Bcl-2 mutant defective in apoptosis inhibition but competent for autophagy suppression promotes MCF7 breast cancer cell growth in vitro and in vivo as efficiently as wild-type Bcl-2. The growth-promoting activity of this Bcl-2 mutant is strongly correlated with its suppression of Beclin1-dependent autophagy, leading to sustained p62 expression and increased DNA damage in xenograft tumors, which may directly contribute to tumorigenesis. Thus, the anti-autophagic property of Bcl-2 is a key feature of Bcl-2-mediated oncogenesis and may in some contexts, serve as an attractive target for breast and other cancer therapies.

  4. The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis.

    Science.gov (United States)

    Eno, Colins O; Zhao, Guoping; Olberding, Kristen E; Li, Chi

    2012-05-15

    Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.

  5. Effect of Bcl-2 rs956572 polymorphism on age-related gray matter volume changes.

    Directory of Open Access Journals (Sweden)

    Mu-En Liu

    Full Text Available The anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2 gene is a major regulator of neural plasticity and cellular resilience. Recently, the Bcl-2 rs956572 single nucleotide polymorphism was proposed to be a functional allelic variant that modulates cellular vulnerability to apoptosis. Our cross-sectional study investigated the genetic effect of this Bcl-2 polymorphism on age-related decreases in gray matter (GM volume across the adult lifespan. Our sample comprised 330 healthy volunteers (191 male, 139 female with a mean age of 56.2±22.0 years (range: 21-92. Magnetic resonance imaging and genotyping of the Bcl-2 rs956572 were performed for each participant. The differences in regional GM volumes between G homozygotes and A-allele carriers were tested using optimized voxel-based morphometry. The association between the Bcl-2 rs956572 polymorphism and age was a predictor of regional GM volumes in the right cerebellum, bilateral lingual gyrus, right middle temporal gyrus, and right parahippocampal gyrus. We found that the volume of these five regions decreased with increasing age (all P<.001. Moreover, the downward slope was steeper among the Bcl-2 rs956572 A-allele carriers than in the G-homozygous participants. Our data provide convergent evidence for the genetic effect of the Bcl-2 functional allelic variant in brain aging. The rs956572 G-allele, which is associated with significantly higher Bcl-2 protein expression and diminished cellular sensitivity to stress-induced apoptosis, conferred a protective effect against age-related changes in brain GM volume, particularly in the cerebellum.

  6. Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

    Science.gov (United States)

    Petros, Andrew M; Swann, Steven L; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D; Kunzer, Aaron R; Souers, Andrew J; Sun, Chaohong

    2014-03-15

    Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts.

  7. Evidence for involvement of c-Src in the anti-apoptotic action of nitric oxide in serum-deprived RINm5F cells.

    Science.gov (United States)

    Tejedo, J R; Ramírez, R; Cahuana, G M; Rincón, P; Sobrino, F; Bedoya, F J

    2001-11-01

    The mechanism by which nitric oxide (NO) protects from apoptosis is a matter of debate. We have shown previously that phosphorylation of tyrosine residues participates in the protection from apoptosis in insulin-producing RINm5F cells (Inorg. Chem. Commun. 3 (2000) 32). Since NO has been reported to activate the tyrosine kinase c-Src and this kinase is involved in the activation of protein kinase G (PKG) in some cell systems, we aimed at studying the contribution of c-Src and PKG systems in anti-apoptotic actions of NO in serum-deprived RINm5F cells. Here we report that exposure of serum-deprived cells to 10 microM DETA/NO results in protection from degradation of the anti-apoptotic protein Bcl-2, together with a reduction of cytochrome c release from mitochondria and caspase-3 inhibition. Studies with the inhibitors ODQ and KT-5823 revealed that these actions are dependent on both activation of guanylate cyclase and PKG. DETA/NO was also able to induce autophosphorylation and activation c-Src protein both in vivo and in vitro and active c-Src was able to induce tyrosine phosphorylation of Bcl-2 in vitro. The c-Src kinase inhibitor PP1 abrogated the actions of DETA/NO on cGMP formation, PKG activation, caspase activation, cytochrome c release from mitochondria, and Bcl-2 phosphorylation and degradation in serum-deprived cells. We thus propose that activation of c-Src is an early step in the chain of events that signal cGMP-dependent anti-apoptotic actions of NO in mitocohondria.

  8. Amygdalin induces apoptosis through regulation of Bax and Bcl-2 expressions in human DU145 and LNCaP prostate cancer cells.

    Science.gov (United States)

    Chang, Hyun-Kyung; Shin, Mal-Soon; Yang, Hye-Young; Lee, Jin-Woo; Kim, Young-Sick; Lee, Myoung-Hwa; Kim, Jullia; Kim, Khae-Hawn; Kim, Chang-Ju

    2006-08-01

    Prostate cancer is one of the most common non-skin cancers in men. Amygdalin is one of the nitrilosides, natural cyanide-containing substances abundant in the seeds of plants of the prunasin family that have been used to treat cancers and relieve pain. In particular, D-amygdalin (D-mandelonitrile-beta-D-gentiobioside) is known to exhibit selective killing effect on cancer cells. Apoptosis, programmed cell death, is an important mechanism in cancer treatment. In the present study, we prepared the aqueous extract of the amygdalin from Armeniacae semen and investigated whether this extract induces apoptotic cell death in human DU145 and LNCaP prostate cancer cells. In the present results, DU145 and LNCaP cells treated with amygdalin exhibited several morphological characteristics of apoptosis. Treatment with amygdalin increased expression of Bax, a pro-apoptotic protein, decreased expression of Bcl-2, an anti-apoptotic protein, and increased caspase-3 enzyme activity in DU145 and LNCaP prostate cancer cells. Here, we have shown that amygdalin induces apoptotic cell death in human DU145 and LNCaP prostate cancer cells by caspase-3 activation through down-regulation of Bcl-2 and up-regulation of Bax. The present study reveals that amygdalin may offer a valuable option for the treatment of prostate cancers.

  9. ET-46ONCOLYTIC VIRAL THERAPY FOR MALIGNANT GLIOMAS USING MYXOMA VIRUS DELETED FOR ANTI-APOPTOTIC M11L GENE

    Science.gov (United States)

    Pisklakova, Alexandra; McKenzie, Brienne; Kenchappa, Rajappa; McFadden, Grant; Forsyth, Peter

    2014-01-01

    Brain Tumour Initiating Cells (BTICs) are stem-like cells hypothesized to mediate recurrence in high-grade gliomas. Myxoma virus (MyxV) is a promising oncolytic virus, which is highly effective in conventional long term resistant glioma cell lines and less effective in BTICs. We hypothesized that one possible factor limiting efficacy in BTICs is that cell death following infection with MyxV is inhibited by virally encoded anti-apoptotic proteins, such as the Bcl-2 structural homologue, M011L. To test this we evaluated and compared the efficacy of wtMYXV versus the viral construct MyxV-M011L-KO (in which the anti-apoptotic protein M11L has been deleted) in BTICs. We found that WT-MyxV does not induce significant level of apoptosis in infected BTICs, but that MyxV-M011L-KO induces dramatically more apoptosisas shown by caspase activation, PARP cleavage, and Cytochrome C release from the mitochondria M11L from the WT-MyxV localized to the mitochondrial membrane and prevented the association of Bax with the mitochondrial membrane. Finally, silencing of Bax using specific siRNAs significantly blocked the induction of apoptosis and cell death that occurs after infection with mutant MyxV-M011L-KO virus. Therefore MyxV-M011L-KO, which is has the anti-apoptotic virally derived gene M11L, dramatically improves the oncolytic efficacy in BTICs and this is dependent on the presence of the pro-apoptotic host protein, Bax. This is the first demonstration, that the MyxV mutant, genetically modified to promote apoptosis in tumor initiating cells, is significantly more efficacious than the wildtype virus. Strategies, such as this one, that promotes apoptosis in tumor initiating cells might be particularly effective.

  10. Anti-apoptotic response during anoxia and recovery in a freeze-tolerant wood frog (Rana sylvatica

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    Victoria E.M. Gerber

    2016-03-01

    Full Text Available The common wood frog, Rana sylvatica, utilizes freeze tolerance as a means of winter survival. Concealed beneath a layer of leaf litter and blanketed by snow, these frogs withstand subzero temperatures by allowing approximately 65–70% of total body water to freeze. Freezing is generally considered to be an ischemic event in which the blood oxygen supply is impeded and may lead to low levels of ATP production and exposure to oxidative stress. Therefore, it is as important to selectively upregulate cytoprotective mechanisms such as the heat shock protein (HSP response and expression of antioxidants as it is to shut down majority of ATP consuming processes in the cell. The objective of this study was to investigate another probable cytoprotective mechanism, anti-apoptosis during oxygen deprivation and recovery in the anoxia tolerant wood frog. In particular, relative protein expression levels of two important apoptotic regulator proteins, Bax and p-p53 (S46, and five anti-apoptotic/pro-survival proteins, Bcl-2, p-Bcl-2 (S70, Bcl-xL, x-IAP, and c-IAP in response to normoxic, 24 Hr anoxic exposure, and 4 Hr recovery stages were assessed in the liver and skeletal muscle using western immunoblotting. The results suggest a tissue-specific regulation of the anti-apoptotic pathway in the wood frog, where both liver and skeletal muscle shows an overall decrease in apoptosis and an increase in cell survival. This type of cytoprotective mechanism could be aimed at preserving the existing cellular components during long-term anoxia and oxygen recovery phases in the wood frog.

  11. Down-regulation of Bcl-2 in rat substantia nigra after focal cerebral ischemia.

    Science.gov (United States)

    Arango-Dávila, Cesar A; Cardona-Gomez, Gloria P; Gallego-Gomez, Juan C; Garcia-Segura, Luis M; Pimienta, Hernán J

    2004-06-28

    After occlusion of the middle cerebral artery in rats, a robust neuronal loss occurs in the ipsilateral substantia nigra reticulata. In this study we have assessed whether degeneration of the substantia nigra is accompanied by changes in the expression of the anti-apoptotic protein Bcl-2. Neuronal loss was assessed by neuronal nuclei (NeuN) immunoreactivity. A significant decrease of Bcl-2 expression was observed in the substantia nigra 12, 24 and 72 h after middle cerebral artery occlusion. These results suggest that the secondary neuronal loss in the substantia nigra could be related with the modification of proteins regulating programmed cell death. Exo-focal cell death may explain the appearance of neuropsychiatric symptoms that are not correlated with the primary site of lesion.

  12. Autophagy Regulates the Post-Translational Cleavage of BCL-2 and Promotes Neuronal Survival

    Directory of Open Access Journals (Sweden)

    Laura Lossi

    2010-01-01

    Full Text Available B-cell lymphoma 2 protein (BCL-2 is one of the more widely investigated anti-apoptotic protein in mammals, and its levels are critical for protecting from programmed cell death. We report here that the cellular content of BCL-2 is regulated at post-translational level along the autophagy/lysosome pathways in organotypic cultures of post-natal mouse cerebellar cortex. Specifically this mechanism appears to be effective in the cerebellar granule cells (CGCs that are known to undergo massive programmed cell death (apoptosis during post-natal maturation. By the use of specific agonists/antagonist of calcium channels at the endoplasmic reticulum it was possible to understand the pivotal role of calcium release from intracellular stores in CGC neuroprotection. The more general significance of these findings is supported by a very recent study Niemann-Pick transgenic mice.

  13. Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

    Science.gov (United States)

    Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

    2015-03-01

    Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

  14. Cell Cycle Arrest and Apoptosis Induction via Modulation of Mitochondrial Integrity by Bcl-2 Family Members and Caspase Dependence in Dracaena cinnabari-Treated H400 Human Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Alabsi, Aied M; Lim, Kai Li; Paterson, Ian C; Ali-Saeed, Rola; Muharram, Bushra A

    2016-01-01

    Dracaena cinnabari Balf.f. is a red resin endemic to Socotra Island, Yemen. Although there have been several reports on its therapeutic properties, information on its cytotoxicity and anticancer effects is very limited. This study utilized a bioassay-guided fractionation approach to determine the cytotoxic and apoptosis-inducing effects of D. cinnabari on human oral squamous cell carcinoma (OSCC). The cytotoxic effects of D. cinnabari crude extract were observed in a panel of OSCC cell lines and were most pronounced in H400. Only fractions DCc and DCd were active on H400 cells; subfractions DCc15 and DCd16 exhibited the greatest cytotoxicity against H400 cells and D. cinnabari inhibited cells proliferation in a time-dependent manner. This was achieved primarily via apoptosis where externalization of phospholipid phosphatidylserine was observed using DAPI/Annexin V fluorescence double staining mechanism studied through mitochondrial membrane potential assay cytochrome c enzyme-linked immunosorbent and caspases activities revealed depolarization of mitochondrial membrane potential (MMP) and significant activation of caspases 9 and 3/7, concomitant with S phase arrest. Apoptotic proteins array suggested that MMP was regulated by Bcl-2 proteins family as results demonstrated an upregulation of Bax, Bad, and Bid as well as downregulation of Bcl-2. Hence, D. cinnabari has the potential to be developed as an anticancer agent.

  15. Cell Cycle Arrest and Apoptosis Induction via Modulation of Mitochondrial Integrity by Bcl-2 Family Members and Caspase Dependence in Dracaena cinnabari-Treated H400 Human Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Aied M. Alabsi

    2016-01-01

    Full Text Available Dracaena cinnabari Balf.f. is a red resin endemic to Socotra Island, Yemen. Although there have been several reports on its therapeutic properties, information on its cytotoxicity and anticancer effects is very limited. This study utilized a bioassay-guided fractionation approach to determine the cytotoxic and apoptosis-inducing effects of D. cinnabari on human oral squamous cell carcinoma (OSCC. The cytotoxic effects of D. cinnabari crude extract were observed in a panel of OSCC cell lines and were most pronounced in H400. Only fractions DCc and DCd were active on H400 cells; subfractions DCc15 and DCd16 exhibited the greatest cytotoxicity against H400 cells and D. cinnabari inhibited cells proliferation in a time-dependent manner. This was achieved primarily via apoptosis where externalization of phospholipid phosphatidylserine was observed using DAPI/Annexin V fluorescence double staining mechanism studied through mitochondrial membrane potential assay cytochrome c enzyme-linked immunosorbent and caspases activities revealed depolarization of mitochondrial membrane potential (MMP and significant activation of caspases 9 and 3/7, concomitant with S phase arrest. Apoptotic proteins array suggested that MMP was regulated by Bcl-2 proteins family as results demonstrated an upregulation of Bax, Bad, and Bid as well as downregulation of Bcl-2. Hence, D. cinnabari has the potential to be developed as an anticancer agent.

  16. Cyclin D1 inhibits whereas c-Myc enhances the cytotoxicity of cisplatin in mouse pancreatic cancer cells via regulation of several members of the NF-κB and Bcl-2 families

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    Ayman El-Kady

    2011-01-01

    Full Text Available Background: Cisplatin (CDDP is a drug used for treatment of many types of malignancy but pancreatic cancer is relatively resistant to it. This study aims to determine whether and how cyclin D1 (D1 and c-Myc influence the response of pancreatic cancer cells to CDDP. Materials and Methods: Ela-mycPT mouse pancreatic cancer cells were transfected with D1 or c-myc cDNA and treated with CDDP alone or together with NPCD, an inhibitor of cyclin dependent ckinase (CDK 4 and 6. Reverse transcription followed by polymerase chain reaction (RT-PCR and western blot assays were used to determine the mRNA and protein levels of interested genes. Cell viability was determined using 3-(4, 5-Dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT assay. Results: Treatment of Ela-mycPT1 cells with CDDP caused an increase in c-myc expression but a slightly latent decrease in D1 expression, whereas D1 and c-Myc proteins repressed each other. D1 or c-Myc rendered Ela-mycPT1 cells resistant or sensitive, respectively, to CDDP. D1 induced the expression of several members of the NF-κB family, including RelA, RelB, Nfκb1 and Nfκb2. D1 also induced BIRC5 and several pro-survival members of the Bcl-2 gene family, including Bcl-2, Mcl-1 and Bad while it decreased the level of the pro-apoptotic Noxa. Inhibition of CDK4 or CDK6 kinase activity by NPCD did not affect these effects of D1. In contrast, c-Myc in Ela-mycPT1 and Ela-mycPT4 cells has the opposite effects to D1 on the expression of most of these apoptosis regulating genes. Conclusion: Our results suggest that induction of c-Myc and inhibition of D1 may be mechanisms for CDDP to elicit cytotoxicity. On the other hand, D1 induces whereas c-Myc represses the expression of key NF-κB family members to induce and repress, respectively, the expression of BIRC5 and several Bcl-2 family members, in turn inhibiting or enhancing the response to CDDP.

  17. Growth factor deprivation induces an alternative non-apoptotic death mechanism that is inhibited by Bcl2 in cells derived from neural precursor cells.

    Science.gov (United States)

    Cárdenas-Aguayo, María del Carmen; Santa-Olalla, Jesús; Baizabal, José-Manuel; Salgado, Luis-Miguel; Covarrubias, Luis

    2003-12-01

    Although apoptosis has been considered the typical mechanism for physiological cell death, presently alternative mechanisms need to be considered. We previously showed that fibroblast growth factor-2 (FGF2) could act as a survival factor for neural precursor cells. To study the death mechanism activated by the absence of this growth factor, we followed the changes in cell morphology and determined cell viability by staining with several dyes after FGF2 removal from mesencephalic neural-progenitor-cell cultures. The changes observed did not correspond to those associated with apoptosis. After 48 h in the absence of FGF2, cells began to develop vacuoles in their cytoplasm, a phenotype that became very obvious 3-5 days later. Double-membrane vacuoles containing cell debris were observed. Vacuolated cells did not stain with either ethidium bromide or trypan Blue, and did not show chromatin condensations. Nonetheless, during the course of culture, vacuolated cells formed aggregates with highly condensed chromatin and detached from the plate. Neural progenitor cells grown in the presence of FGF2 did not display any of those characteristics. The vacuolated phenotype could be reversed by the addition of FGF2. Typical autophagy inhibitors such as 3-MA and LY294002 inhibited vacuole development, whereas a broad-spectrum caspase inhibitor did not. Interestingly, Bcl-2 overexpression retarded vacuole development. In conclusion, we identified a death autophagy-like mechanism activated by the lack of a specific survival factor that can be inhibited by Bcl2. We propose that anti-apoptotic Bcl2 family members are key molecules controlling death activation independently of the cell degeneration mechanism used.

  18. Methoxychlor induces atresia by altering Bcl2 factors and inducing caspase activity in mouse ovarian antral follicles in vitro.

    Science.gov (United States)

    Basavarajappa, Mallikarjuna S; Karman, Bethany N; Wang, Wei; Gupta, Rupesh K; Flaws, Jodi A

    2012-12-01

    Methoxychlor (MXC) is an organochlorine pesticide widely used in many countries against various species of insects that attack crops and domestic animals. MXC reduces fertility by increasing atresia (death) of antral follicles in vivo. MXC also induces atresia of antral follicles after 96 h in vitro. The current work tested the hypothesis that MXC induces morphological atresia at early time points (24 and 48 h) by altering pro-apoptotic (Bax, Bok, Casp3, and caspase activity) and anti-apoptotic (Bcl2 and Bcl-xL) factors in the follicles. The results indicate that at 24 h, MXC increased Bcl-xL and Bax mRNA levels and increased the ratio of Bax/Bcl2. At 48-96 h, MXC induced morphological atresia. At 24-96 h, MXC increased caspase activities. These data suggest that MXC may induce atresia by altering Bcl2 factors and inducing caspase activities in antral follicles.

  19. Chronic NMDA administration to rats increases brain pro-apoptotic factors while decreasing anti-Apoptotic factors and causes cell death

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    Rapoport Stanley I

    2009-09-01

    Full Text Available Abstract Background Chronic N-Methyl-d-aspartate (NMDA administration to rats is reported to increase arachidonic acid signaling and upregulate neuroinflammatory markers in rat brain. These changes may damage brain cells. In this study, we determined if chronic NMDA administration (25 mg/kg i.p., 21 days to rats would alter expression of pro- and anti-apoptotic factors in frontal cortex, compared with vehicle control. Results Using real time RT-PCR and Western blotting, chronic NMDA administration was shown to decrease mRNA and protein levels of anti-apoptotic markers Bcl-2 and BDNF, and of their transcription factor phospho-CREB in the cortex. Expression of pro-apoptotic Bax, Bad, and 14-3-3ζ was increased, as well as Fluoro-Jade B (FJB staining, a marker of neuronal loss. Conclusion This alteration in the balance between pro- and anti-apoptotic factors by chronic NMDA receptor activation in this animal model may contribute to neuronal loss, and further suggests that the model can be used to examine multiple processes involved in excitotoxicity.

  20. EFFECT OF HELICOBACTER PYLORI INFECTION ON GENE EXPRESSIONS OF Bcl-2 FAMILY MEMBERS IN GASTRIC ADENOCARCINOMA AND TISSUE ADJACENT TO THE TUMOR%幽门螺杆菌对胃癌及癌旁组织中Bcl-2家族基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    张浩; 房殿春; 刘海峰; 罗元辉

    2003-01-01

    目的研究幽门螺杆菌(H.pylori)感染对胃癌及癌旁组织中Bcl-2家族基因表达的影响.方法收集95例胃癌及癌旁组织标本,应用半定量RT-PCR法检测Bid、Bax和Bcl-2 mRNA的表达.结果胃癌组织中Bid和Bax mRNA表达按H.pylori阴性组、CagA- H.pylori感染组和CagA+ H.pylori感染组顺序依次增高(P<0.05),H.pylori阴性组的Bcl-2 mRNA表达低于H.pylori阳性组(P<0.05);癌旁组织中,Bid、Bax和Bcl-2表达按H.pylori阴性组、CagA- H.pylori感染组和CagA+ H.pylori感染组顺序均依次递增(P<0.05).H.pylori阴性组中,胃癌组织的Bid和Bax表达与Bcl-2表达呈负相关(P<0.05),而在H.pylori感染组中,胃癌组织的Bid和Bax表达与Bcl-2表达无相关性;癌旁组织的Bid和Bax表达与Bcl-2表达呈正相关(P<0.05).结论 H.pylori有促进Bid、Bax和Bcl-2表达的作用,并且能够诱发这些细胞凋亡调控基因的表达紊乱,这也许是H.pylori诱导胃癌发生的途径之一.

  1. Cannabinoids Regulate Bcl-2 and Cyclin D2 Expression in Pancreatic β Cells.

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    Jihye Kim

    Full Text Available Recent reports have shown that cannabinoid 1 receptors (CB1Rs are expressed in pancreatic β cells, where they induce cell death and cell cycle arrest by directly inhibiting insulin receptor activation. Here, we report that CB1Rs regulate the expression of the anti-apoptotic protein Bcl-2 and cell cycle regulator cyclin D2 in pancreatic β cells. Treatment of MIN6 and βTC6 cells with a synthetic CB1R agonist, WIN55,212-2, led to a decrease in the expression of Bcl-2 and cyclin D2, in turn inducing cell cycle arrest in G0/G1 phase and caspase-3-dependent apoptosis. Additionally, genetic deletion and pharmacological blockade of CB1Rs after injury in mice led to increased levels of Bcl-2 and cyclin D2 in pancreatic β cells. These findings provide evidence for the involvement of Bcl-2 and cyclin D2 mediated by CB1Rs in the regulation of β-cell survival and growth, and will serve as a basis for developing new therapeutic interventions to enhance β-cell function and growth in diabetes.

  2. Expression of Bcl-2 inhibited Fas-mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and"Death factor"family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of"Death factor" family, the transfection experiments with expression vectors pcDNA3-fland pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl2. The data showed that the expression of FasL in pcDNA3fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fltransient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hcpatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.

  3. Expression of bcl-2 protein in chronic hepatitis C: Effect of interferon alpha 2b with ribavirin therapy

    Institute of Scientific and Technical Information of China (English)

    Panasiuk Anatol; Prokopowicz Danuta; Dzieciol Janusz; Panasiuk Bozena

    2005-01-01

    AIM: Mechanisms responsible for persistence of HCV infection and liver damage in chronic hepatitis C are not clear. Apoptosis is an important form of host immune response against viral infections. Anti-apoptotic proteinbcl-2 expression on liver tissue as well as the influence of interferon alpha 2b (IFNα2b) and ribavirin (RBV) were analyzed in patients with chronic hepatitis C. METHODS: In 30 patients with chronic hepatitis C (responders - R and non-responders - NR) treated with IFNα2b+RBV, protein bcl-2 was determined in hepatocytes and in liver associated lymphocytes before and after the treatment.RESULTS: The treatment diminished bcl-2 protein accumulation in liver cells in_patients with hepatitis C (P<0.05). Before and after the therapy, we detected bcl-2 protein in R in 87±15% and 83±20% of hepatocytes andin 28± 18% and 26±10% of liver-associated lymphocytes, respectively. In NR, the values before treatment decreased from 94±32% to 88±21% of hepatocytes and 39±29% to 28±12% of lymphocytes with bcl-2 expression. There was no statistical correlation between bcl-2 expression on liver tissue with inflammatory activity, fibrosis and biochemical parameters before and after the treatment.CONCLUSION: IFNα2b+RBV treatment, by bcl-2 protein expression decrease, enables apoptosis of hepatocytes and associated liver lymphocytes, which in turn eliminate hepatitis C viruses.

  4. Association of BCL2-938C>A genetic polymorphism with glioma risk in Chinese Han population.

    Science.gov (United States)

    Li, Wei; Qian, Chunfa; Wang, Linxiong; Teng, Hong; Zhang, Li

    2014-03-01

    Glioma is the most common type of primary brain malignancy in adults. The anti-apoptotic protein B-cell lymphoma 2 (BCL2) has been implicated in the pathogenesis of glioma. This study aimed to evaluate the potential association between BCL2-938C>A genetic polymorphism and glioma susceptibility. This case-control study was conducted in Chinese Han populations consisting of 248 glioma cases and 252 cancer-free controls. The BCL2-938C>A genetic polymorphism was detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and verified using DNA sequencing methods. Our data suggested that the genotype/allele of BCL2-938C>A polymorphism were statistically associated with the increased risk of glioma where the risk of glioma for genotype AA or allele A is significantly higher than wild genotype CC (odds ratio (OR) = 2.23, 95% confidence interval (CI) 1.21-4.10, p = 0.009) or allele C (OR = 1.39, 95% CI 1.06-1.82, p = 0.016), respectively. In addition, the BCL2-938AA genotype was significantly more common in patients with glioblastoma and in patients with grade IV glioma. Our findings indicate that the BCL2-938C>A polymorphism is associated with the susceptibility to glioma in Chinese Han populations and might be used as molecular markers for evaluating glioma risk.

  5. Effects of Geldanamycin on Expression of Bcl-2 in Human Cervical Cancer HeLa Cells

    Institute of Scientific and Technical Information of China (English)

    Xue Du; Ruoran Mi; Quanxin Qu; Ye Qu; Tianfu Yue

    2008-01-01

    OBJECTIVE Geldanamycin, a natural product of Streptomyces geldanus, binds the heat shock protein 90 (Hsp90), a cell chaperone protein that interacts with Bcl-2. In this study, we investigated whether geldanamycin (GA) inhibits proliferation of HeLa cells through induction of apoptosis by decreasing the level of Bcl-2expression.METHODS HeLa cells, a human cervical cell line, were cultured in vitro and treated with different concentrations of GA (0, 0.02, 0.2,2, 10 μmol/L) for 24 h. Or were treated for different lengths of time at a GA concentration of 10 μmol/L. Proliferation of the cells was analyzed by an MTT assay, and cell apoptosis was determined by staining the cells with annexin V. In addition, cellular mRNA levels for Bcl-2 and Hsp90 were determined by the semi-quantitative polymerase chain reaction (PCR), and the levels of Bcl-2 and Hsp90 protein expression were determined by Western blots.RESULTS Treatment of cells with GA was found to inhibit HeLa cell proliferation in a concentration and time-dependent manner. The inhibition was a result of increased cellular apoptotic levels. Further analyses showed that while the mRNA and protein expression levels of Hsp90 were-not affected, GA treatment significantly reduced the level of Bcl-2 mRNA and protein expression in a concentration-dependent manner that correlated with the observed inhibition of cell proliferation.CONCLUSION GA can inhibit proliferation and increase apoptosis of HeLa cells by decreasing the transcription and expression of an anti-apoptotic gene bcl-2, probably through interaction and functional inhibition of Hsp90.

  6. Bcl-2 inhibits nuclear homologous recombination by localizing BRCA1 to the endomembranes.

    Science.gov (United States)

    Laulier, Corentin; Barascu, Aurélia; Guirouilh-Barbat, Josée; Pennarun, Gaëlle; Le Chalony, Catherine; Chevalier, François; Palierne, Gaëlle; Bertrand, Pascale; Verbavatz, Jean Marc; Lopez, Bernard S

    2011-05-15

    Genetic stability requires coordination of a network of pathways including DNA repair/recombination and apoptosis. In addition to its canonical anti-apoptotic role, Bcl-2 negatively impacts genome stability. In this study, we identified the breast cancer tumor suppressor BRCA1, which plays an essential role in homologous recombination (HR), as a target for Bcl-2 in the repression of HR. Indeed, ionizing radiation-induced BRCA1 foci assembly was repressed when Bcl-2 was expressed ectopically, in human SV40 fibroblasts, or spontaneously, in lymphoma t(14:18) cells and in HeLa and H460 cancer cell lines. Moreover, we showed that the transmembrane (TM) domain of Bcl-2 was required for both inhibition of BRCA1 foci assembly and the inhibition of HR induced by a double-strand break targeted into an intrachromosomal HR substrate by the meganuclease I-SceI. Fluorescence confocal microscopy, proximity ligation assay, and electron microscopy analyses as well as Western blot analysis of subcellular fractions showed that Bcl-2 and BRCA1 colocalized to mitochondria and endoplasmic reticulum in a process requiring the TM domain of Bcl-2. Targeting BRCA1 to the endomembranes depletes BRCA1 from the nucleus and, thus, accounts for the inhibition of HR. Furthermore, our findings support an apoptosis-stimulatory role for the cytosolic form of BRCA1, suggesting a new tumor suppressor function of BRCA1. Together, our results reveal a new mode of BRCA1 regulation and for HR in the maintenance of genome stability.

  7. Human bronchial epithelial cells exposed in vitro to diesel exhaust particles exhibit alterations in cell rheology and cytotoxicity associated with decrease in antioxidant defenses and imbalance in pro- and anti-apoptotic gene expression.

    Science.gov (United States)

    Seriani, Robson; de Souza, Claudia Emanuele Carvalho; Krempel, Paloma Gava; Frias, Daniela Perroni; Matsuda, Monique; Correia, Aristides Tadeu; Ferreira, Márcia Zotti Justo; Alencar, Adriano Mesquita; Negri, Elnara Marcia; Saldiva, Paulo Hilário Nascimento; Mauad, Thais; Macchione, Mariangela

    2016-05-01

    Diesel exhaust particles (DEPs) from diesel engines produce adverse alterations in cells of the airways by activating intracellular signaling pathways and apoptotic gene overexpression, and also by influencing metabolism and cytoskeleton changes. This study used human bronchial epithelium cells (BEAS-2B) in culture and evaluates their exposure to DEPs (15ug/mL for 1 and 2 h) in order to determine changes to cell rheology (viscoelasticity) and gene expression of the enzymes involved in oxidative stress, apoptosis, and cytotoxicity. BEAS-2B cells exposed to DEPs were found to have a significant loss in stiffness, membrane stability, and mitochondrial activity. The genes involved in apoptosis [B cell lymphoma 2 (BCL-2 and caspase-3)] presented inversely proportional expressions (p = 0.05, p = 0.01, respectively), low expression of the genes involved in antioxidant responses [SOD1 (superoxide dismutase 1); SOD2 (superoxide dismutase 2), and GPx (glutathione peroxidase) (p = 0.01)], along with an increase in cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) (p = 0.01). These results suggest that alterations in cell rheology and cytotoxicity could be associated with oxidative stress and imbalance between pro- and anti-apoptotic genes.

  8. Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage

    Institute of Scientific and Technical Information of China (English)

    Yuting Lin; Junichi Fukuchi; Richard A Hiipakka; John M Kokontis; Jialing Xiang

    2007-01-01

    Bcl-2 is an anti-apoptotic oncoprotein and its protein levels are inversely correlated with prognosis in many cancers.However, the role of Bcl-2 in the progression of prostate cancer is not clear. Here we report that Bcl-2 is required for the progression of LNCaP prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. The mRNA and protein levels of Bcl-2 are significantly increased in androgen-independent prostate cancer cells, shRNA-mediated gene silencing of Bcl-2 in androgen-independent prostate cancer cells promotes UV-induced apoptosis and suppresses the growth of prostate tumors in vivo. Growing androgen-dependent cells under androgen-deprivation conditions results in formation of androgen-independent colonies; and the transition from androgen-dependent to androgen-independent growth is blocked by ectopic expression of the Bcl-2 antagonist Bax or Bcl-2 shRNA. Thus, our results demonstrate that Bcl-2 is not only critical for the survival of androgen-independent prostate cancer cells, but is also required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage.

  9. Bcl-XL is qualitatively different from and ten times more effective than Bcl-2 when expressed in a breast cancer cell line

    Directory of Open Access Journals (Sweden)

    Leber Brian

    2006-08-01

    Full Text Available Abstract Background Bcl-2 and Bcl-XL are anti-apoptotic paralogues that inhibit apoptosis elicited by a wide variety of stimuli, and play critical roles in cancer development and resistance to treatment. Many clinical studies have indicated that expression of these anti-apoptotic proteins in tumours is associated with poor prognosis. It has therefore been assumed that in cells the essential difference between Bcl-2 and Bcl-XL involves regulation of expression and that they are otherwise functionally similar. To examine this issue, we have compared the function of the proteins and of mutants of Bcl-2 and Bcl-XL specifically targeted to different subcellular sites. Methods We generated clones of the human breast cancer line MCF-7 stably expressing known amounts of Bcl-2, or Bcl-XL as determined by quantitative immunoblotting. Clones expressing equivalent amounts of wild-type and mutants of Bcl-2 and Bcl-XL with subcellular localization restricted to the cytoplasm, endoplasmic reticulum or outer mitochondrial membrane were studied in both MCF-7 and Rat-1 fibroblasts. In MCF-7 cells we measured the functional activities of these proteins in preventing apoptosis induced by four different agents (doxorubicin, ceramide, thapsigargin, TNF-α. Etoposide and low serum were used to compare the effect of Bcl-2, Bcl-XL and mutants located at the endoplasmic reticulum on induction of apoptosis in fibroblasts. Results We noted both qualitative and quantitative differences in the functional activity of these two anti-apoptotic proteins in cells: Bcl-2 localized to the endoplasmic reticulum inhibits apoptosis induced by ceramide and thapsigargin but not by doxorubicin or TNFα, while Bcl-XL at the endoplasmic reticulum is active against all four drugs. In fibroblasts Bcl-2 localized to the ER did not prevent cell death due to etoposide whereas Bcl-XL in the same location did. Finally in MCF-7 cells, Bcl-XL is approximately ten times more active than Bcl-2 in

  10. Prognostic value of bcl-2 expression among women with breast cancer in Libya.

    Science.gov (United States)

    Ermiah, Eramah; Buhmeida, Abdelbaset; Khaled, Ben Romdhane; Abdalla, Fathi; Salem, Nada; Pyrhönen, Seppo; Collan, Yrjö

    2013-06-01

    We studied the association of the immunohistochemical bcl-2 expression in Libyan breast cancer with clinicopathological variables and patient outcome. Histological samples from 170 previously untreated primary Libyan breast carcinoma patients were examined. In immunohistochemistry, the NCL-L-bcl-2-486 monoclonal antibody was used. Positive expression of bcl-2 was found in 106 patients (62.4 %). The bcl-2 expression was significantly associated with estrogen receptor (p50 years; p=0.04). Histological subtypes and family history of breast cancer did not have significant relationship with bcl-2. Patients with positive expression of bcl-2 had lower recurrence rate than bcl-2-negative patients and better survival after median follow-up of 47 months. Patients with high bcl-2 staining were associated with the best survival. The role of bcl-2 as an independent predictor of disease-specific survival was assessed in a multivariate survival (Cox) analysis, including age, hormonal status, recurrence, histological grade, and clinical stage variables. Bcl-2 (p<0.0001) and clinical stage (p=0.016) were independent predicators of disease-specific survival. For analysis of disease-free survival, the same variables were entered to the model and only bcl-2 proved to be an independent predictor (p=0.002). Patients with positive expression of bcl-2 were associated with low grade of malignancy, with lower recurrence rate, with lower rate of death, and with longer survival time. Bcl-2 is an independent predictor of breast cancer outcome, and it provides useful prognostic information in Libyan breast cancer. Thus, it could be used with classical clinicopathological factors to improve patient selection for therapy.

  11. Anti-apoptotic treatment in mouse models of age-related hearing loss

    Institute of Scientific and Technical Information of China (English)

    Fengchan Han; Oumei Wang; Quanxiang Cai

    2016-01-01

    Age-related hearing loss (AHL), or presbycusis, is the most common neurodegenerative disorder and top communication deficit of the aged population. Genetic predisposition is one of the major factors in the development of AHL. Generally, AHL is associated with an age-dependent loss of sensory hair cells, spiral ganglion neurons and stria vascularis cells in the inner ear. Although the mechanisms leading to genetic hearing loss are not completely understood, caspase-family proteases function as important signals in the inner ear pathology. It is now accepted that mouse models are the best tools to study the mechanism of genetic hearing loss or AHL. Here, we provide a brief review of recent studies on hearing improvement in mouse models of AHL by anti-apoptotic treatment.

  12. The Bcl-2-associated death promoter (BAD) lowers the threshold at which the Bcl-2-interacting domain death agonist (BID) triggers mitochondria disintegration.

    Science.gov (United States)

    Howells, Christopher C; Baumann, William T; Samuels, David C; Finkielstein, Carla V

    2011-02-21

    The Bcl-2-associated death promoter (BAD) protein, like many other BH3-only proteins, is known to promote apoptosis through the intrinsic mitochondrial pathway. Unlike the BH3-interacting domain death agonist (BID) protein, BAD cannot directly trigger apoptosis but, instead, lowers the threshold at which apoptosis is induced. In many mathematical models of apoptosis, BAD is neglected or abstracted. The work presented here considers the incorporation of BAD and its various modifications in a model of the tBID-induction of BAK (Bcl-2 homologous antagonist killer) or the tBID-induction of BAX (Bcl-2-associated X protein). Steady state equations are used to develop an explicit formula describing the total concentration level of tBID, guaranteed to trigger apoptosis, as a bilinear function of the total BAD concentration level and the total anti-apoptotic protein concentration level (usually Bcl-2 or Bcl-xL). In particular, the formula explains how the pro-apoptotic protein BAD lowers the threshold at which tBID induces BAK/BAX activation-reducing the level of total Bcl-2/Bcl-xL available to inhibit tBID signaling in the mitochondria. Attention is then turned to the experimental data surrounding BAD phosphorylation, a process known to inhibit the pro-apoptotic effects of BAD. To address this data, the phosphorylation process is modeled following two separate kinetics in which either free unbound BAD is the assumed substrate or Bcl-xL/Bcl-2-bound BAD is the assumed substrate. Bifurcation analysis and further analysis of the bilinear equation validate experiments, which suggest that BAD phosphorylation prevents irreversible BAK/BAX-mediated apoptosis, even when phosphorylation-induced dissociation of Bcl-xL/Bcl-2-bound BAD is blocked. It is also shown that a cooperative, even synergistic, removal of mitochondrial BAD is seen when both types of phosphorylation are assumed possible. The presented work, however, reveals that the balance between BAD phosphorylation and

  13. Hypothermia enhances bcl-2 expression and protects against oxidative stress-induced cell death in Chinese hamster ovary cells.

    Science.gov (United States)

    Slikker, W; Desai, V G; Duhart, H; Feuers, R; Imam, S Z

    2001-08-01

    Oxidative stress is one of the major causes of cellular injury. Various reactive oxygen (ROS) and nitrogen (RNS) species such as superoxide, hydroxyl radical, peroxynitrite, and nitric oxide are involved in the manifestations of different types of organ toxicity and the resultant syndromes, symptoms, or diseases. Hypothermic conditions have been reported to reduce the oxidative stress in various in vitro and in vivo studies. In the present study, we sought to determine the effect of lowered temperatures on oxidative stress-induced cell death in Chinese hamster ovary (CHO) cells. We also investigated the oxidative stress-induced alterations in the expression of anti-apoptotic protein, bcl-2, in CHO cells at lowered temperatures. CHO cells were incubated at four different temperatures of 30, 32, 35, and 37 degrees C (control temperature) from 1 to 4 d. In another set, the cells were incubated with 100 microM hydrogen peroxide (H(2)O(2)) for 30 min before harvesting at different time points. The cells were harvested at 1, 2, 3, and 4 d. Cell survival was significantly higher at 30 degrees C as compared to 37 degrees C over 4 d of incubation. In cells incubated with H(2)O(2), significantly higher cell viability was observed at lower temperatures as compared to the cells incubated at 37 degrees C. The activity of glutathione peroxidase (GSH-Px) also increased significantly at lower temperatures. Lowered temperature also provided a significant increase in the expression of anti-apoptotic protein, bcl-2 after 4 d of incubation. These data suggest that hypothermic conditions lowers the risk of oxidative stress-induced cellular damage and programmed cell death by increasing the activity of GSH-Px and by the induction in the expression of the anti-apoptotic protein, bcl-2.

  14. Ginsenoside Rh2 Mitigates Pediatric Leukemia Through Suppression of Bcl-2 in Leukemia Cells

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    Xiaoru Wang

    2015-09-01

    Full Text Available Background/Aims: Acute myeloid leukemia (AML is a severe malignant cancer worldwide, in both adult and pediatric patients. Since bone marrow cell transplantation is seriously limited by the availability of the immune-paired donor sources, the therapy for pediatric leukemia remains challenging. Ginsenoside Rh2 (GRh2 is a well-characterized component in red ginseng, and has established therapeutic effects for different diseases, although whether GRh2 may have a therapeutic effect on pediatric leukemia has not been investigated. Methods: We examined the effects of GRh2 on the survival of mice in an acute leukemia model. We analyzed the effects of GRh2 on the cell viability of leukemia cell lines in vitro, using a CCK-8 assay and an MTT assay. We analyzed the effects of GRh2 on the apoptosis of leukemia cell lines in vitro, by flow cytometry. We analyzed the levels of Bcl-2 and microRNA-21 (miR-21 in GRh2-treated leukemia cells. Prediction of binding between miR-21 and 3'-UTR of Bcl-2 mRNA was performed by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. Results: GRh2 significantly prolonged the survival of mice with pediatric leukemia. GRh2 significantly decreased the viability of leukemia cells in vitro, through induction of apoptosis. GRh2 significantly decreased the levels of an anti-apoptotic protein Bcl-2 in leukemia cells, possibly through induction of miR-21, which suppressed the translation of Bcl-2 mRNA via 3'-UTR binding. Conclusion: GRh2 may be an effective treatment for pediatric leukemia, and GRh2 may induce apoptosis of leukemia cells through miR-21-modulated suppression of Bcl-2.

  15. µ-Calpain conversion of antiapoptotic Bfl-1 (BCL2A1 into a prodeath factor reveals two distinct alpha-helices inducing mitochondria-mediated apoptosis.

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    Juan García Valero

    Full Text Available Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.

  16. Apoptosis of brain cells and expression changes of bcl-2 gene family after traumatic brain injury in rats%大鼠脑创伤细胞凋亡和bcl-2基因家族表达的变化

    Institute of Scientific and Technical Information of China (English)

    杨云华; 张可成; 张艺梅

    2009-01-01

    目的:研究创伤性脑损伤(TBI)脑细胞凋亡发生及bcl-2基因家族表达变化,揭示TBI后继发性脑损伤的发生机制.方法:建立大鼠TBI模型,运用组织原位标记凋亡细胞检测、免疫组织化学等技术,观察TBI后1~14 d大脑伤侧皮层和海马等脑区细胞凋亡及bcl-2、bax基因表达的变化.结果:TBI后伤侧大脑广泛存在凋亡现象,以皮层及海马为甚;伤后1 d即出现凋亡细胞,3 d达高峰,7~14 d逐渐恢复正常;TBI后bcl-2、bax基因在伤脑表达增高,其增高的时相与凋亡规律相似,表达与细胞凋亡发生呈正相关.结论:细胞凋亡参与TBI后脑细胞的死亡机制,bcl-2基因在TBI后的脑组织中表达增加,对细胞凋亡发生有抑制作用;bax可促进细胞凋亡,TBI后表达明显增加,参与TBI后细胞凋亡的正向调节,可能是脑创伤后细胞死亡的重要机制.

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

    Science.gov (United States)

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

    2015-01-01

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

  18. HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

    Science.gov (United States)

    Hallgren, O; Gustafsson, L; Irjala, H; Selivanova, G; Orrenius, S; Svanborg, C

    2006-02-01

    HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

  19. New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum.

    Science.gov (United States)

    Rudner, J; Jendrossek, V; Belka, C

    2002-10-01

    The oncogenic protein Bcl-2 which is expressed in membranes of different subcellular organelles protects cells from apoptosis induced by endogenic stimuli. Most of the results published so far emphasise the importance of Bcl-2 at the mitochondria. Several recent observations suggest a role of Bcl-2 at the endoplasmic reticulum (ER). Bcl-2 located at the ER was shown to interfere with apoptosis induction by Bax, ceramides, ionising radiation, serum withdrawal and c-myc expression. Although the detailed functions of Bcl-2 at the ER remain elusive, several speculative mechanisms may be supposed. For instance, Bcl-2 at the ER may regulate calcium fluxes between the ER and the mitochondria. In addition, Bcl-2 is able to interact with the endoplasmic protein Bap31 thus avoiding caspase activation at the ER. Bcl-2 may also abrogate the function of ER located pro-apoptotic Bcl-2 like proteins by heterodimerization. Current data on the function of Bcl-2 at the ER, its role for the modulation of calcium fluxes and its influence on caspase activation at the ER are reviewed.

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

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    Bougras Gwenola

    2004-08-01

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

  1. MCL-1 is required throughout B-cell development and its loss sensitizes specific B-cell subsets to inhibition of BCL-2 or BCL-XL

    NARCIS (Netherlands)

    Vikström, Ingela B; Slomp, Anne; Carrington, Emma M; Moesbergen, Laura M; Chang, Catherine; Kelly, Gemma L; Glaser, Stefan P; Jansen, J H Marco; Leusen, Jeanette H W; Strasser, Andreas; Huang, David C S; Lew, Andrew M; Peperzak, Victor; Tarlinton, David M

    2016-01-01

    Pro-survival BCL-2 family members protect cells from programmed cell death that can be induced by multiple internal or external cues. Within the haematopoietic lineages, the BCL-2 family members BCL-2, BCL-XL and MCL-1 are known to support cell survival but the individual and overlapping roles of th

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

    Directory of Open Access Journals (Sweden)

    Mário Henrique Girão Faria

    2006-08-01

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

  3. MLN4924 induces Noxa upregulation in acute myelogenous leukemia and synergizes with Bcl-2 inhibitors.

    Science.gov (United States)

    Knorr, K L B; Schneider, P A; Meng, X W; Dai, H; Smith, B D; Hess, A D; Karp, J E; Kaufmann, S H

    2015-12-01

    MLN4924 (pevonedistat), an inhibitor of the Nedd8 activating enzyme (NAE), has exhibited promising clinical activity in acute myelogenous leukemia (AML). Here we demonstrate that MLN4924 induces apoptosis in AML cell lines and clinical samples via a mechanism distinct from those observed in other malignancies. Inactivation of E3 cullin ring ligases (CRLs) through NAE inhibition causes accumulation of the CRL substrate c-Myc, which transactivates the PMAIP1 gene encoding Noxa, leading to increased Noxa protein, Bax and Bak activation, and subsequent apoptotic changes. Importantly, c-Myc knockdown diminishes Noxa induction; and Noxa siRNA diminishes MLN4924-induced killing. Because Noxa also neutralizes Mcl-1, an anti-apoptotic Bcl-2 paralog often upregulated in resistant AML, further experiments have examined the effect of combining MLN4924 with BH3 mimetics that target other anti-apoptotic proteins. In combination with ABT-199 or ABT-263 (navitoclax), MLN4924 exerts a synergistic cytotoxic effect. Collectively, these results provide new insight into MLN4924-induced engagement of the apoptotic machinery that could help guide further exploration of MLN4924 for AML.

  4. Anti-apoptotic effect ofShudipingchan granule in the substantia nigra of rat models of Parkinson’s disease

    Institute of Scientific and Technical Information of China (English)

    Qing Ye; Xiao-lei Yuan; Jing He; Jie Zhou; Can-xing Yuan; Xu-ming Yang

    2016-01-01

    Levodopa is the gold-standard treatment for Parkinson’s disease. However, although it alleviates the clinical symptoms, it cannot delay the progressive apoptosis of dopaminergic neurons or prevent motor complications in the long term. In the present study, we investigated the effect ofShudipingchan granule on neuronal apoptosis in a rat model of Parkinson’s disease, established by injecting 6-hydroxydopamine into the substantia nigra pars compacta and ventral tegmental area. We then administered levodopa (20 mg/kg intraperitoneally, twice daily) with or withoutShudipingchan granule (7.5 mL/kg intragastrically, twice daily), for 4 weeks. hTe long-term use of levodopa accel-erated apoptosis of nigral cells and worsened behavioral symptoms by activating the extracellular signal-regulated kinase pathway and downstream apoptotic factors. However, administration ofShudipingchan granule with levodopa reduced expression of phosphorylated extracellular signal-regulated kinase 1/2 and Bax, increased tyrosine hydroxylase and Bcl-2, reduced apoptosis in the substantia nigra, and markedly improved dyskinesia. hTese ifndings suggest thatShudipingchan granule suppresses neuronal apoptosis by inhibiting the hyper-phosphorylation of extracellular signal-regulated kinase and downregulating expression of anti-apoptotic genes.Shudipingchan granule, used in combination with levodopa, can effectively reduce the symptoms of Parkinson’s disease.

  5. Herpesvirus pan encodes a functional homologue of BHRF1, the Epstein-Barr virus v-Bcl-2

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    Williams Tracey

    2005-02-01

    Full Text Available Abstract Background Epstein-Barr virus (EBV latently infects about 90% of the human population and is associated with benign and malignant diseases of lymphoid and epithelial origin. BHRF1, an early lytic cycle antigen, is an apoptosis suppressing member of the Bcl-2 family. In vitro studies imply that BHRF1 is dispensable for both virus replication and transformation. However, the fact that BHRF1 is highly conserved not only in all EBV isolates studied to date but also in the analogous viruses Herpesvirus papio and Herpesvirus pan that infect baboons and chimpanzees respectively, suggests BHRF1 may play an important role in vivo. Results Herpesvirus papio BHRF1 has been shown to function in an analogous manner to EBV BHRF1 in response to DNA damaging agents in human keratinocytes. In this study we show that the heterologous expression of the previously uncharacterised Herpesvirus pan BHRF1 in the human Burkitt's lymphoma cell line Ramos-BL provides similar anti-apoptotic functions to that of EBV BHRF1 in response to apoptosis triggered by serum withdrawal, etoposide treatment and ultraviolet (UV radiation. We also map the amino acid changes onto the recently solved structure of the EBV BHRF1 and reveal that these changes are unlikely to alter the 3D structure of the protein. Conclusions These findings show that the functional conservation of BHRF1 extends to a lymphoid background, suggesting that the primate virus proteins interact with cellular proteins that are themselves highly conserved across the higher primates. Further weight is added to this suggestion when we show that the difference in amino acid sequences map to regions on the 3D structure of EBV BHRF1 that are unlikely to change the conformation of the protein.

  6. Hydroalcoholic extract of cyperus rotundus ameliorates H2O2-induced human neuronal cell damage via its anti-oxidative and anti-apoptotic machinery.

    Science.gov (United States)

    Kumar, K Hemanth; Khanum, Farhath

    2013-01-01

    Hydrogen peroxide (H(2)O(2)), a major reactive oxygen species produced during oxidative stress, has been implicated in the pathophysiology of various neurodegenerative conditions. Cyperus rotundus is a traditional medicinal herb that has recently found applications in food and confectionary industries. In the current study, the neuroprotective effects of Cyperus rotundus rhizome extract (CRE) through its antioxidant and anti-apoptotic machinery to attenuate H(2)O(2)-induced cell damage on human neuroblastoma SH-SY5Y cells have been explored. The results obtained demonstrate that pretreatment of cells with CRE for 2 h before administration of H(2)O(2) for 24 h ameliorates the cytotoxicity induced by H(2)O(2) as evidenced by MTT and LDH assays. CRE exhibited potent antioxidant activity by regulating the enzymes/proteins levels such as SOD, CAT, GPx, GR, HSP-70, Caspase-3, and Bcl-2. The pretreatment restored H(2)O(2)-induced cellular, nuclear, and mitochondrial morphologies as well as increased the expression of Brain derived nerve growth factor (BDNF). The anti-oxidant and anti-apoptotic potentials of the plant extract may account for its high content of phenolics, flavonoids, and other active principles. Taken together, our findings suggest that CRE might be developed as an agent for neurodegeneration prevention or therapy.

  7. The Protective Properties of the Strawberry (Fragaria ananassa) against Carbon Tetrachloride-Induced Hepatotoxicity in Rats Mediated by Anti-Apoptotic and Upregulation of Antioxidant Genes Expression Effects.

    Science.gov (United States)

    Hamed, Sherifa S; Al-Yhya, Nouf A; El-Khadragy, Manal F; Al-Olayan, Ebtesam M; Alajmi, Reem A; Hassan, Zeinab K; Hassan, Salwa B; Abdel Moneim, Ahmed E

    2016-01-01

    The strawberry (Fragaria ananassa) has been extensively used to treat a wide range of ailments in many cultures. The present study was aimed at evaluating the hepatoprotective effect of strawberry juice on experimentally induced liver injury in rats. To this end, rats were introperitoneally injected with carbon tetrachloride (CCl4) with or without strawberry juice supplementation for 12 weeks and the hepatoprotective effect of strawberry was assessed by measuring serum liver enzyme markers, hepatic tissue redox status and apoptotic markers with various techniques including biochemistry, ELISA, quantitative PCR assays and histochemistry. The hepatoprotective effect of the strawberry was evident by preventing CCl4-induced increase in liver enzymes levels. Determination of oxidative balance showed that strawberry treatment significantly blunted CCl4-induced increase in oxidative stress markers and decrease in enzymatic and non-enzymatic molecules in hepatic tissue. Furthermore, strawberry supplementation enhanced the anti-apoptotic protein, Bcl-2, and restrained the pro-apoptotic proteins Bax and caspase-3 with a marked reduction in collagen areas in hepatic tissue. These findings demonstrated that strawberry (F. ananassa) juice possessed antioxidant, anti-apoptotic and anti-fibrotic properties, probably mediated by the presence of polyphenols and flavonoids compounds.

  8. High-quality NMR structure of human anti-apoptotic protein domain Mcl-1(171-327 for cancer drug design.

    Directory of Open Access Journals (Sweden)

    Gaohua Liu

    Full Text Available A high-quality NMR solution structure is presented for protein hMcl-1(171-327 which comprises residues 171-327 of the human anti-apoptotic protein Mcl-1 (hMcl-1. Since this construct contains the three Bcl-2 homology (BH sequence motifs which participate in forming a binding site for inhibitors of hMcl-1, it is deemed to be crucial for structure-based design of novel anti-cancer drugs blocking the Mcl1 related anti-apoptotic pathway. While the coordinates of an NMR solution structure for a corresponding construct of the mouse homologue (mMcl-1 are publicly available, our structure is the first atomic resolution structure reported for the 'apo form' of the human protein. Comparison of the two structures reveals that hMcl-1(171-327 exhibits a somewhat wider ligand/inhibitor binding groove as well as a different charge distribution within the BH3 binding groove. These findings strongly suggest that the availability of the human structure is of critical importance to support future design of cancer drugs.

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

    Science.gov (United States)

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

    2001-01-01

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

  10. Antiapoptotic Bcl-2 homolog CED-9 in Caenorhabditis elegans: dynamics of BH3 and CED-4 binding regions and comparison with mammalian antiapoptotic Bcl-2 proteins.

    Science.gov (United States)

    Modi, Vivek; Sankararamakrishnan, Ramasubbu

    2014-06-01

    Proteins belonging to Bcl-2 family regulate intrinsic cell death pathway. Although mammalian antiapoptotic Bcl-2 members interact with multiple proapoptotic proteins, the Caenorhabditis elegans Bcl-2 homolog CED-9 is known to have only two proapoptotic partners. The BH3-motif of proapoptotic proteins bind to the hydrophobic groove of prosurvival proteins formed by the Bcl-2 helical fold. CED-9 is also known to interact with CED-4, a homolog of the human cell death activator Apaf1. We have performed molecular dynamics simulations of CED-9 in two forms and compared the results with those of mammalian counterparts Bcl-XL, Bcl-w, and Bcl-2. Our studies demonstrate that the region forming the hydrophobic cleft is more flexible compared with the CED-4-binding region, and this is generally true for all antiapoptotic Bcl-2 proteins studied. CED-9 is the most stable protein during simulations and its hydrophobic pocket is relatively rigid explaining the absence of functional redundancy in CED-9. The BH3-binding region of Bcl-2 is less flexible among the mammalian proteins and this lends support to the studies that Bcl-2 binds to less number of BH3 peptides with high affinity. The C-terminal helix of CED-9 lost its helical character because of a large number of charged residues. We speculate that this region probably plays a role in intracellular localization of CED-9. The BH4-motif accessibility in CED-9 and Bcl-w is controlled by the loop connecting the first two helices. Although CED-9 adopts the same Bcl-2 fold, our studies highlight important differences in the dynamic behavior of CED-9 and mammalian antiapoptotic homologs.

  11. The CORM ALF-186 Mediates Anti-Apoptotic Signaling via an Activation of the p38 MAPK after Ischemia and Reperfusion Injury in Retinal Ganglion Cells

    Science.gov (United States)

    Ulbrich, Felix; Kaufmann, Kai B.; Meske, Alexander; Lagrèze, Wolf A.; Augustynik, Michael; Buerkle, Hartmut; Ramao, Carlos C.; Biermann, Julia

    2016-01-01

    Purpose Ischemia and reperfusion injury may induce apoptosis and lead to sustained tissue damage and loss of function, especially in neuronal organs. While carbon monoxide is known to exert protective effects after various harmful events, the mechanism of carbon monoxide releasing molecules in neuronal tissue has not been investigated yet. We hypothesize that the carbon monoxide releasing molecule (CORM) ALF-186, administered after neuronal ischemia-reperfusion injury (IRI), counteracts retinal apoptosis and its involved signaling pathways and consecutively reduces neuronal tissue damage. Methods IRI was performed in rat´s retinae for 1 hour. The water-soluble CORM ALF-186 (10 mg/kg) was administered intravenously via a tail vein after reperfusion. After 24 and 48 hours, retinal tissue was harvested to analyze mRNA and protein expression of Bcl-2, Bax, Caspase-3, ERK1/2, p38 and JNK. Densities of fluorogold pre-labeled retinal ganglion cells (RGC) were analyzed 7 days after IRI. Immunohistochemistry was performed on retinal cross sections. Results ALF-186 significantly reduced IRI mediated loss of RGC. ALF-186 treatment differentially affected mitogen-activated protein kinases (MAPK) phosphorylation: ALF-186 activated p38 and suppressed ERK1/2 phosphorylation, while JNK remained unchanged. Furthermore, ALF-186 treatment affected mitochondrial apoptosis, decreasing pro-apoptotic Bax and Caspase-3-cleavage, but increasing anti-apoptotic Bcl-2. Inhibition of p38-MAPK using SB203580 reduced ALF-186 mediated anti-apoptotic effects. Conclusion In this study, ALF-186 mediated substantial neuroprotection, affecting intracellular apoptotic signaling, mainly via MAPK p38. CORMs may thus represent a promising therapeutic alternative treating neuronal IRI. PMID:27764224

  12. Novel murine B-cell lymphoma/leukemia model to study BCL2-driven oncogenesis.

    Science.gov (United States)

    Meijerink, Jules P P; Van Lieshout, Esther M M; Beverloo, H Berna; Van Drunen, Ellen; Mensink, Ewald J B M; Macville, Merryn; Pieters, Rob

    2005-05-10

    The BCL-2 family has been implicated in the pathogenesis of various hematopoietic malignancies, including follicular non-Hodgkin lymphoma and B-cell chronic lymphocytic leukemia. To identify genes that act synergistically in BCL2-enforced leukemogenesis, we developed a murine B-cell lymphoma/leukemia model based on the IL-3-dependent Balb/C pro-B line (FL5.12). FL5.12 cells were stably transfected with antiapoptotic BCL-2 alone or in combination with proapoptotic BAX or nonfunctional mutant BAX, thereby creating various levels of imbalance within the BCL-2 family. Transfectants were intravenously injected into normal Balb/C mice. Whereas FL5.12 cells did not provoke leukemia, mice injected with stable transfectants died of leukemia over time. Disease incidence and latency time depended on the degree of imbalance in the BCL-2 family, supporting a model whereby BCL2 drives tumorigenesis. All mice presented with hepatosplenomegaly and leukemic FL5.12 cells in peripheral blood and bone marrow compartments. Leukemic conversion was accompanied by secondary genetic aberrations leading to clonal IL-3-responsive leukemia. Cellular transformation was independent of alterations in c-Myc or downstream apoptotic pathway. Leukemic clones retained a normal DNA damage response leading to elevated P53 and P21 levels and cell cycle arrest upon irradiation. In conclusion, our mouse model may prove a valuable tool to identify genes that cooperate in BCL2-enforced lymphoma/leukemogenesis.

  13. Targeting BCL-2 to enhance vulnerability to therapy in estrogen receptor-positive breast cancer.

    Science.gov (United States)

    Merino, D; Lok, S W; Visvader, J E; Lindeman, G J

    2016-04-14

    The last three decades have seen significant progress in our understanding of the role of the pro-survival protein BCL-2 and its family members in apoptosis and cancer. BCL-2 and other pro-survival family members including Mcl-1 and BCL-XL have been shown to have a key role in keeping pro-apoptotic 'effector' proteins BAK and BAX in check. They also neutralize a group of 'sensor' proteins (such as BIM), which are triggered by cytotoxic stimuli such as chemotherapy. BCL-2 proteins therefore have a central role as guardians against apoptosis, helping cancer cells to evade cell death. More recently, an increasing number of BH3 mimetics, which bind and neutralize BCL-2 and/or its pro-survival relatives, have been developed. The utility of targeting BCL-2 in hematological malignancies has become evident in early-phase studies, with remarkable clinical responses seen in heavily pretreated patients. As BCL-2 is overexpressed in ~75% of breast cancer, there has been growing interest in determining whether this new class of drug could show similar promise in breast cancer. This review summarizes our current understanding of the role of BCL-2 and its family members in mammary gland development and breast cancer, recent progress in the development of new BH3 mimetics as well as their potential for targeting estrogen receptor-positive breast cancer.

  14. Bcl-2/caspase 3 mucosal imbalance favors T cell resistance to apoptosis in dogs with inflammatory bowel disease.

    Science.gov (United States)

    Jergens, A; Young, J; Moore, D; Wang, C; Hostetter, J; Augustine, L; Allenspach, K; Schmitz, S; Mosher, C

    2014-04-15

    Canine idiopathic inflammatory bowel disease (IBD) is believed to result from complex interplay between genetic, microbial, and immunologic factors. Abnormal cell death by apoptosis may result in the persistence of activated intestinal T cells that contribute to mucosal inflammation and clinical severity. To test this hypothesis, we investigated the mucosal expression of pro- and anti-apoptotic proteins in different intestinal compartments and their association with inflammatory indices in dogs with IBD. Apoptosis of lamina propria (LP) T cells in duodenal, ileal, and colonic tissues in control and IBD dogs was analyzed by caspase 3/Bcl-2 immunohistochemistry and TUNEL assays. Densities and distributions of LP caspase 3 and Bcl-2 cells were correlated to histopathologic lesions and the clinical activity index (CIBDAI). Compared to control tissues, IBD dogs had significantly (Pdogs, there were significantly greater numbers of Bcl-2 cells at the apical and basilar villus in the duodenum as compared to the colon and to the apical and basilar villus in the ileum (Pdogs compared with controls (Pdogs and the CIBDAI (Pdogs with IBD. Mucosal imbalance of Bcl-2/caspase 3 expression favors T cell resistance to apoptosis which may contribute to T cell accumulation and chronic intestinal inflammation, similar to human IBD.

  15. Expanding the Cancer Arsenal with Targeted Therapies: Disarmament of the Antiapoptotic Bcl-2 Proteins by Small Molecules.

    Science.gov (United States)

    Yap, Jeremy L; Chen, Lijia; Lanning, Maryanna E; Fletcher, Steven

    2017-02-09

    A hallmark of cancer is the evasion of apoptosis, which is often associated with the upregulation of the antiapoptotic members of the Bcl-2 family of proteins. The prosurvival function of the antiapoptotic Bcl-2 proteins is manifested by capturing and neutralizing the proapoptotic Bcl-2 proteins via their BH3 death domains. Accordingly, strategies to antagonize the antiapoptotic Bcl-2 proteins have largely focused on the development of low-molecular-weight, synthetic BH3 mimetics ("magic bullets") to disrupt the protein-protein interactions between anti- and proapoptotic Bcl-2 proteins. In this way, apoptosis has been reactivated in malignant cells. Moreover, several such Bcl-2 family inhibitors are presently being evaluated for a range of cancers in clinical trials and show great promise as new additions to the cancer armamentarium. Indeed, the selective Bcl-2 inhibitor venetoclax (Venclexta) recently received FDA approval for the treatment of a specific subset of patients with chronic lymphocytic leukemia. This review focuses on the major developments in the field of Bcl-2 inhibitors over the past decade, with particular emphasis on binding modes and, thus, the origins of selectivity for specific Bcl-2 family members.

  16. Zebrafish bcl2l is a survival factor in thyroid development.

    Science.gov (United States)

    Porreca, Immacolata; De Felice, Elena; Fagman, Henrik; Di Lauro, Roberto; Sordino, Paolo

    2012-06-15

    Regulated cell death, defined in morphological terms as apoptosis, is crucial for organ morphogenesis. While differentiation of the thyroid gland has been extensively studied, nothing is yet known about the survival mechanisms involved in the development of this endocrine gland. Using the zebrafish model system, we aim to understand whether genes belonging to the Bcl-2 family that control apoptosis are implicated in regulation of cell survival during thyroid development. Evidence of strong Bcl-2 gene expression in mouse thyroid precursors prompted us to investigate the functions played by its zebrafish homologs during thyroid development. We show that the bcl2-like (bcl2l) gene is expressed in the zebrafish thyroid primordium. Morpholino-mediated knockdown and mutant analyses revealed that bcl2l is crucial for thyroid cell survival and that this function is tightly modulated by the transcription factors pax2a, nk2.1a and hhex. Also, the bcl2l gene appears to control a caspase-3-dependent apoptotic mechanism during thyroid development. Thyroid precursor cells require an actively maintained survival mechanism to properly proceed through development. The bcl2l gene operates in the inhibition of cell death under direct regulation of a thyroid specific set of transcription factors. This is the first demonstration of an active mechanism to ensure survival of the thyroid primordium during morphogenesis.

  17. Erythropoietin inhibits gamma-irradiation-induced apoptosis by upregulation of Bcl-2 and decreasing the activation of caspase 3 in human UT-7/erythropoietin cell line.

    Science.gov (United States)

    Liu, Yuan-Yuan; She, Zhen-Jue; Yao, Ming-Hui

    2010-05-01

    1. Erythropoietin (EPO) can reverse radiotherapy-induced anaemia by stimulating bone marrow cells to produce erythrocytes. However, there are limited studies that address the mechanisms by which EPO exerts its beneficial effects in radiotherapy-induced anaemia. In the present study, we used a human bone marrow-derived EPO-dependent leukaemia cell line UT-7/EPO that progressed further in erythroid development to evaluate the anti-apoptotic effects of EPO on irradiated human erythroid progenitor. 2. The UT-7/EPO cells exposed to gamma-irradiation were cultured in the presence or absence of EPO at a concentration of 7 U/mL. The cell viability, cell apoptosis and the expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 were examined. 3. The results showed that EPO protected the viability of human UT-7/EPO cells exposed to gamma-irradiation. EPO significantly inhibited gamma-irradiation-induced apoptosis in human UT-7/EPO cells: a significant decrease in the percentage of apoptotic cells was observed (62, 69 and 62% at 24, 48 and 72 h, respectively). Furthermore, EPO significantly increased the expression of Bcl-2 protein and the relative Bcl-2/Bax ratio, and decreased the activation of caspase 3 and formation of the p17 and p12 cleavage in similar conditions. 4. In conclusion, EPO exerts anti-apoptotic effects on irradiated human UT-7/EPO cells through upregulation of Bcl-2 protein and the relative Bcl-2/Bax ratio, and by decreasing the activation of caspase 3. These findings may contribute to our understanding of the beneficial function of EPO in radiotherapy-induced anaemia.

  18. MRP- and BCL-2-mediated drug resistance in human SCLC: effects of apoptotic sphingolipids in vitro.

    Science.gov (United States)

    Khodadadian, M; Leroux, M E; Auzenne, E; Ghosh, S C; Farquhar, D; Evans, R; Spohn, W; Zou, Y; Klostergaard, J

    2009-10-01

    Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

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

    Directory of Open Access Journals (Sweden)

    Azimahtol Hawariah LP

    2007-04-01

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

  20. Induction of leukemia cell apoptosis by cheliensisin A involves down-regulation of Bcl-2 expression

    Institute of Scientific and Technical Information of China (English)

    Li ZHONG; Chao-ming LI; Xiao-jiang HAO; Li-guang LOU

    2005-01-01

    Aim: To investigate the apoptosis-inducing effect of cheliensisin A (GC-51), a novel styryl-lactone isolated from Goniothalamus cheliensis, on human promyelocytic leukemia HL-60 cells and the mechanism of action involved.Methods: Apoptotic cell death was determined by morphological examination and DNA agarose gel electrophoresis. The activity of caspase-3 was assessed using Western blotting and the expression of Bcl-2 and Bax genes was analyzed using the reverse transcription-polymerase chain reaction (RT-PCR) method. Results:GC-51 significantly inhibited the proliferation of HL-60 cells with an IC50 of 2.4±0.2 μmol/L and effectively induced apoptosis in HL-60 cells. Exposure of HL-60cells to 10 μmol/L GC-51 for 8 h resulted in approximately 53% of the cells under going apoptosis. Caspase-3 was activated in GC-51-treated cells, which was manifested by the appearance of the 17 kDa active form of caspase-3 and the cleavage of poly(ADP-ribose) polymerase (PARP). Meanwhile, GC-51 markedly reduced the expression of the anti-apoptotic gene Bcl-2 and increased the expression of the pro-apoptotic gene Bax. The apoptosis-inducing effect of GC-51 was cAMP dependent protein kinase (PKA) dependent because PKA, but not the protein kinase C, specific inhibitor H-89, blocked the induction of apoptosis by GC-51 in HL-60 cells. Conclusion: The results demonstrate that GC-51 effectively induces apoptosis in HL-60 cells and that this effect is PKA-dependent and involves the downregulation of Bcl-2 expression and the activation of caspase-3.

  1. Efficient ferrocifen anticancer drug and Bcl-2 gene therapy using lipid nanocapsules on human melanoma xenograft in mouse.

    Science.gov (United States)

    Resnier, Pauline; Galopin, Natacha; Sibiril, Yann; Clavreul, Anne; Cayon, Jérôme; Briganti, Alessandro; Legras, Pierre; Vessières, Anne; Montier, Tristan; Jaouen, Gérard; Benoit, Jean-Pierre; Passirani, Catherine

    2017-01-31

    Metastatic melanoma has been described as a highly aggressive cancer with low sensibility to chemotherapeutic agents. New types of drug, such as metal-based drugs (ferrocifens) have emerged and could represent an alternative for melanoma treatment since they show interesting anticancer potential. Furthermore, molecular analysis has evidenced the role of apoptosis in the low sensibility of melanomas and especially of the key regulator, Bcl-2. The objective of this study was to combine two strategies in the same lipid nanocapsules (LNCs): i) gene therapy to modulate anti-apoptotic proteins by the use of Bcl-2 siRNA, and ii) ferrocifens as a new type of anticancer agent. The efficient gene silencing with LNCs was verified by the specific extinction of Bcl-2 in melanoma cells. The cellular toxicity of ferrocifens (ferrociphenol (FcDiOH) or Ansa-FcDiOH) was demonstrated, showing higher efficacy than dacarbazine. Interestingly, the association of siBcl-2 LNCs with Ansa-FcDiOH demonstrated a significant effect on melanoma cell viability. Moreover, the co-encapsulation of siRNA and ferrocifens was successfully performed into LNCs for animal experiments. A reduction of tumor volume and mass was proved after siBcl-2 LNC treatment and Ansa-FcDiOH LNC treatment, individually (around 25%). Finally, the association of both components into the same LNCs increased the reduction of tumor volume to about 50% compared to the control group. In conclusion, LNCs appeared to provide a promising tool for the co-encapsulation of a metal-based drug and siRNA.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  3. Thymosin beta 4 protects cardiomyocytes from oxidative stress by targeting anti-oxidative enzymes and anti-apoptotic genes.

    Directory of Open Access Journals (Sweden)

    Chuanyu Wei

    Full Text Available BACKGROUND: Thymosin beta-4 (Tβ4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2O(2 induced cardiac damage. METHODS: Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H(2O(2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H(2O(2-induced cardiac damage was evaluated. RESULTS: Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl(2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the

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

    Directory of Open Access Journals (Sweden)

    Maryam Ghoshcian

    2016-05-01

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

  5. Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression.

    Directory of Open Access Journals (Sweden)

    Nathalie Esber

    Full Text Available The progesterone receptor (PR with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4 and ulipristal acetate (UPA, a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL2-L1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required.

  6. TW-37, a Small-Molecule Inhibitor of Bcl-2, Inhibits Cell Growth and Induces Apoptosis in Pancreatic Cancer: Involvement of Notch-1 Signaling Pathway

    OpenAIRE

    2009-01-01

    Overexpression of Bcl-2 family proteins has been found in a variety of aggressive human carcinomas, including pancreatic cancer, suggesting that specific agents targeting Bcl-2 family proteins would be valuable for pancreatic cancer therapy. We have previously reported that TW-37, a small-molecule inhibitor of Bcl-2 family proteins, inhibited cell growth and induced apoptosis in pancreatic cancer. However, the precise role and the molecular mechanism of action of TW-37 have not been fully elu...

  7. Anti-apoptotic signaling and failure of apoptosis in the ischemic rat hippocampus

    DEFF Research Database (Denmark)

    Müller, Georg Johannes; Lassmann, Hans; Johansen, Flemming Fryd

    2007-01-01

    Several anti-apoptotic proteins are induced in CA1 neurons after transient forebrain ischemia (TFI), but fail to protect the majority of these cells from demise. Correlating cell death morphologies (apoptosis-like and necrosis-like death) with immunohistochemistry (IHC), we investigated whether a...

  8. Copper Induces Apoptosis of Neuroblastoma Cells Via Post-translational Regulation of the Expression of Bcl-2-family Proteins and the tx Mouse is a Better Model of Hepatic than Brain Cu Toxicity.

    Science.gov (United States)

    Chan, Hsien W; Liu, Tianbing; Verdile, Giuseppe; Bishop, Glenda; Haasl, Ryan J; Smith, Mark A; Perry, George; Martins, Ralph N; Atwood, Craig S

    2008-01-01

    The basic mechanism(s) by which altered Cu homeostasis is toxic to hepatocytes and neurons, the two major cell types affected in copper storage diseases such as Wilson's disease (WD), remain unclear. Using human M17 neuroblastoma cells as a model to examine Cu toxicity, we found that there was a time- and concentration-dependent induction of neuronal death, such that at 24 h there was a approximately 50 % reduction in viability with 25 muM Cu-glycine(2). Cu-glycine(2) (25:50 muM) treatment for 24 h significantly altered the expression of 296 genes, including 8 genes involved with apoptosis (BCL2-associated athanogene 3, BCL2/adenovirus E1B 19kDa interacting protein caspase 5, regulator of Fas-induced apoptosis, V-jun sarcoma virus 17 oncogene homolog, claudin 5, prostaglandin E receptor 3 and protein tyrosine phosphatase, non-receptor type 6). Surprisingly, changes in the expression of more 'traditional' apoptotic genes (Bcl-2, Bax, Bak and Bad) did not vary more than 20 %. To test whether the induction of apoptosis in neuroblastoma cells was via post-translational mechanisms, we measured the protein expression of these apoptotic markers in M17 neuroblastoma cells treated with Cu-glycine(2) (0-100 muM) for 24-48 h. Compared with glycine treated cells, Cu-glycine(2) reduced Bcl-2 expression by 50 %, but increased Bax and Bak expression by 130% and 400 %, respectively. To assess whether Cu also induced apoptotic cell death in a mouse model of WD, we measured the expression of these apoptotic markers in the liver and brain of mice expressing an ATP7b gene mutation (tx(J) mice) at 10 months of age (near the end of their lives when overt liver pathology is displayed). Changes in the liver expression of these apoptotic markers in tx(J) mice compared to background mice mirrored those of Cu treated neuroblastoma cells. In contrast, few changes in apoptotic protein expression were detected in the brain between tx(J) and background mice, indicating the tx(J) mouse is a good

  9. PESV对K562细胞BCR/ABL融合基因及凋亡调控因子Bcl-2、Bad表达的影响%The Effects of PESV on the Expression of BCR/ABL Fusion Gene and Bcl-2, Bad of Apoptosis Regulators on the K562 Cells

    Institute of Scientific and Technical Information of China (English)

    于文俊; 杨文华; 杨向东; 史哲新; 王兴丽; 郝征; 张佳

    2012-01-01

    Objective: To investigate the PESV of K562 cells BCR / ABL fusion gene and apoptosis regulators bcl-2 and bad expression. Methods: K562 cells were cultured in vitro, by PESV for different times, the apoptosis rate by flow cytometry, fluorescence quantitative RT-PCR detection of BCR / ABL, Bcl-2, Bad mRNA level changes. Results: Compared with the control group, PESV treated K562 cells, apoptosis increased, BCR / ABL fusion gene reduced expression, anti-apoptotic gene Bcl-2 mRNA expression decreased, pro-apoptotic gene Bad mRNA expression. Conclusion: PESV reduced in K562 cells can reduce the BCR / ABL fusion gene, may regulate the expression of Bcl-2 and Bad, inhibit proliferation of K562 cells and promote their apoptosis.%目的:探讨PESV对K562细胞BCR/ABL融合基因及凋亡调控因子bcl-2和bad表达的影响.方法:将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,荧光定量RT-PCR检测BCR/ABL、Bcl-2、Bad mRNA水平变化.结果:与对照组相比,PESV处理后K562细胞,凋亡率增加,BCR/ABL融合基因表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加.结论:PESV能降低降低K562细胞BCR/ABL融合基因的表达,可能通过调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡.

  10. Atherosclerosis-Associated Endothelial Cell Apoptosis by MiR-429-Mediated Down Regulation of Bcl-2

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    Tao Zhang

    2015-10-01

    Full Text Available Background/Aims: Endothelial cell injury and subsequent apoptosis play a key role in the development and pathogenesis of atherosclerosis, which is hallmarked by dysregulated lipid homeostasis, aberrant immunity and inflammation, and plaque-instability-associated coronary occlusion. Nevertheless, our understanding of the mechanisms underlying endothelial cell apoptosis is still limited. MicroRNA-429 (miR-29 is a known cancer suppressor that promotes cancer cell apoptosis. However, it is unknown whether miR-429 may be involved in the development of atherosclerosis through similar mechanisms. We addressed these questions in the current study. Methods: We examined the levels of endothelial cell apoptosis in ApoE (-/- mice suppled with high-fat diet (HFD, a mouse model for atherosclerosis (simplified as HFD mice. We analyzed the levels of anti-apoptotic protein Bcl-2 and the levels of miR-429 in the purified CD31+ endothelial cells from mouse aorta. Prediction of the binding between miR-429 and 3'-UTR of Bcl-2 mRNA was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. The effects of miR-429 were further analyzed in an in vitro model using oxidized low-density lipoprotein (ox-LDL-treated human aortic endothelial cells (HAECs. Results: HFD mice developed atherosclerosis in 12 weeks, while the control ApoE (-/- mice that had received normal diet (simplified as NOR mice did not. HFD mice had significantly lower percentage of endothelial cells and significantly higher percentage of mesenchymal cells in the aorta than NOR mice. Significantly higher levels of endothelial cell apoptosis were detected in HFD mice, resulting from decreases in Bcl-2 protein, but not mRNA. The decreases in Bcl-2 in endothelial cells were due to increased levels of miR-429, which suppressed the translation of Bcl-2 mRNA via 3'-UTR binding. These in vivo findings were reproduced in vitro on ox-LDL-treated HAECs. Conclusion: Atherosclerosis

  11. Immunogenicity of Bcl-2 in patients with cancer

    DEFF Research Database (Denmark)

    Andersen, Mads Hald; Svane, Inge Marie; Kvistborg, Pia

    2005-01-01

    activities in preclinical models and are currently in several clinical trials. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The overexpression of Bcl......-2 in cancer and the fact that immune escape by down-regulation or loss of expression of this protein would impair sustained tumor growth makes Bcl-2 a very attractive target for anticancer immunotherapy. Herein, we describe spontaneous T-cell reactivity against Bcl-2 in peripheral blood from......B-cell lymphoma 2 (Bcl-2) is a pivotal regulator of apoptotic cell death and it is overexpressed in many cancers. Consequently, the Bcl-2 protein is an attractive target for drug design, and Bcl-2-specific antisense oligonucleotides or small-molecule Bcl-2 inhibitors have shown broad anticancer...

  12. Dimer-dependent intrinsic/basal activity of the class B G protein-coupled receptor PAC1 promotes cellular anti-apoptotic activity through Wnt/β-catenin pathways that are associated with dimer endocytosis.

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    Rongjie Yu

    Full Text Available The high expression of PACAP (pituitary adenylate cyclase-activating polypeptide-preferring receptor PAC1 is associated with nerve injury and tumors. Our previous report (Yu R, et al. PLoS One 2012; 7: e51811 confirmed the dimerization of PAC1 and found that the M-PAC1 mutation in the N-terminal first Cys/Ala lost the ability to form dimers. In this study, Chinese hamster ovary (CHO-K1 cells overexpressing wild-type PAC1 (PAC1-CHO had significantly higher anti-apoptotic activities against serum withdrawal-induced apoptosis associated with a lower caspase 3 activity and a higher Bcl-2 level in a ligand-independent manner than those of CHO cells overexpressing the mutant M-PAC1 (M-PAC1-CHO. PAC1-CHO had significantly higher β-catenin, cyclin D1 and c-myc levels corresponding to the Wnt/β-catenin signal than did M-PAC1-CHO. In addition, the Wnt/β-catenin pathway inhibitor XAV939 significantly inhibited the anti-apoptotic activities of PAC1-CHO. Top-flash assays demonstrated that PAC1-CHO had a significantly stronger Wnt/β-catenin signal than did M-PAC1-CHO. Acetylcysteine (NAC as an inhibitor of the dimerization of PAC1 inhibited the anti-apoptotic activities that were endowed by PAC1 and decreased the Wnt/β-catenin signal in Top-flash assays. In the PAC1 Tet (tetracycline-on inducible gene expression system by doxycycline (Dox, higher expression levels of PAC1 resulted in higher anti-apoptotic activities that were associated with a stronger Wnt/β-catenin signal. A similar correlation was also found with the down-regulation of PAC1 in the Neuro2a neuroblastoma cell. BiFC combined with fluorescence confocal imaging indicated that during serum-withdrawal-induced apoptosis, PAC1 dimers displayed significant endocytosis. These findings indicate that PAC1 has ligand-independent and dimer-dependent intrinsic/basal activity, conferring cells with anti-apoptotic activities against serum withdrawal, which is involved in the Wnt/β-catenin signal and

  13. Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells

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    Unoki, Takamitsu; Abiko, Yumi; Toyama, Takashi; Uehara, Takashi; Tsuboi, Koji; Nishida, Motohiro; Kaji, Toshiyuki; Kumagai, Yoshito

    2016-01-01

    Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death. PMID:27357941

  14. MDA-7/IL-24 induces Bcl-2 denitrosylation and ubiquitin-degradation involved in cancer cell apoptosis.

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    Hui Tian

    Full Text Available MDA-7/IL-24 was involved in the specific cancer apoptosis through suppression of Bcl-2 expression, which is a key apoptosis regulatory protein of the mitochondrial death pathway. However, the underlying mechanisms of this regulation are unclear. We report here that tumor-selective replicating adenovirus ZD55-IL-24 leads to Bcl-2 S-denitrosylation and concomitant ubiquitination, which take part in the 26S proteasome degradation. IL-24-siRNA completely blocks Bcl-2 ubiquitination via reversion of Bcl-2 S-denitrosylation and protects it from proteasomal degradation which confirmed the significant role of MDA-7/IL-24 in regulating posttranslational modification of Bcl-2 in cancer cells. Nitric oxide (NO is a key regulator of protein S-nitrosylation and denitrosylation. The NO donor, sodium nitroprusside (SNP, down-regulates Bcl-2 S-denitrosylation, attenuates Bcl-2 ubiquitination and subsequently counteracts MDA-7/IL-24 induced cancer cell apoptosis, whereas NO inhibitor 2-(4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO shows the opposite effect. At the same time, these NO modulators fail to affect Bcl-2 phosphorylation, suggesting that NO regulates Bcl-2 stability in a phosphorylation-independent manner. In addition, Bcl-2 S-nitrosylation reduction induced by ZD55-IL-24 was attributed to both iNOS decrease and TrxR1 increase. iNOS-siRNA facilitates Bcl-2 S-denitrosylation and ubiquitin-degradation, whereas the TrxR1 inhibitor auranofin prevents Bcl-2 from denitrosylation and ubiquitination, thus restrains the caspase signal pathway activation and subsequent cancer cell apoptosis. Taken together, our studies reveal that MDA-7/IL-24 induces Bcl-2 S-denitrosylation via regulation of iNOS and TrxR1. Moreover, denitrosylation of Bcl-2 results in its ubiquitination and subsequent caspase protease family activation, as a consequence, apoptosis susceptibility. These findings provide a novel insight into MDA-7/IL-24 induced growth

  15. A gammaherpesvirus Bcl-2 ortholog blocks B cell receptor-mediated apoptosis and promotes the survival of developing B cells in vivo.

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    Carrie B Coleman

    2014-02-01

    Full Text Available Gammaherpesviruses such as Epstein-Barr virus (EBV and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8 establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68, suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in

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

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    Bitar Fadi F

    2006-07-01

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

  17. Correlation of Baseline BCL-2 mRNA Expression and Clinical Response to Neoadjuvant Chemotherapy in Breast Cancer

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    Prihantono Prihantono

    2017-01-01

    Full Text Available Impairment of apoptosis is a hallmark of cancer. Tumor resistance to apoptosis usually caused by deregulation of the expression of BCL-2 family protein or mutation of the tumor suppressor gene p53. Over expression of Bcl-2 is commonly found in various types of cancer including breast cancer. Studies mentioned that analysis of Bcl-2 might predict response to selected endocrine and chemotherapies. This study is conducted to evaluate the correlation of BCL-2 mRNA expression and clinical response to neoadjuvant chemotherapy in breast cancer patients. Longitudinal study is used in this research, 30 subjects of breast cancer tissue samples prechemotherapy using cyclophosphamide-adriamycin-5FU regiment. Detection of mRNA expression of BCL-2 using qRT-PCR techniques. Evaluation of clinical response to chemotherapy is using RECIST criteria. Mean value of BCL-2 mRNA expression in breast cancer patients was 9.917± 2.568. Mean value of BCL-2 mRNA expression of responsive group was 9.887± 2.731. Mean value of BCL-2 mRNA expression of nonresponsive group was 10.017±2.122. Mean value of responsive group were lower than nonresponsive group, but there was no significant correlation between baseline mRNA expression of BCL-2 with clinical response to chemotherapy, value of r=0.378, p=0.223 (p>0.05. this study shows that there was no significant correlation between baseline expression of mRNA BCL-2 with clinical response to chemotherapy.

  18. Immunogenicity of Bcl-2 in patients with cancer

    DEFF Research Database (Denmark)

    Andersen, Mads Hald; Svane, Inge Marie; Kvistborg, Pia

    2005-01-01

    activities in preclinical models and are currently in several clinical trials. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The overexpression of Bcl......B-cell lymphoma 2 (Bcl-2) is a pivotal regulator of apoptotic cell death and it is overexpressed in many cancers. Consequently, the Bcl-2 protein is an attractive target for drug design, and Bcl-2-specific antisense oligonucleotides or small-molecule Bcl-2 inhibitors have shown broad anticancer......-2 in cancer and the fact that immune escape by down-regulation or loss of expression of this protein would impair sustained tumor growth makes Bcl-2 a very attractive target for anticancer immunotherapy. Herein, we describe spontaneous T-cell reactivity against Bcl-2 in peripheral blood from...

  19. Characterization of a novel interaction between Bcl-2 members Diva and Harakiri.

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    Lorenzo Sborgi

    Full Text Available Interactions within proteins of the Bcl-2 family are key in the regulation of apoptosis. The death-inducing members control apoptotic mechanisms partly by antagonizing the prosurvival proteins through heterodimer formation. Structural and biophysical studies on these complexes are providing important clues to understand their function. To help improve our knowledge on protein-protein interactions within the Bcl-2 family we have studied the binding between two of its members: mouse Diva and human Harakiri. Diva has been shown to perform both prosurvival and killing activity. In contrast, Harakiri induces cell death by interacting with antiapoptotic Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Combining the NMR data with the previously reported three-dimensional structure of Diva we find that Harakiri binds to a specific region in Diva. This interacting surface is equivalent to the known binding area of prosurvival Bcl-2 members from the reported structures of the complexes, suggesting that Diva could function at the structural level similarly to the antiapoptotic proteins of the Bcl-2 family. We illustrate this result by building a structural model of the heterodimer using molecular docking and the NMR data as restraints. Moreover, combining circular dichroism and NMR we also show that Harakiri is largely unstructured with residual (13% α-helical conformation. This result agrees with intrinsic disorder previously observed in other Bcl-2 members. In addition, Harakiri constructs of different length were studied to identify the region critical for the interaction. Differential affinity for Diva of these constructs suggests that the amino acid sequence flanking the interacting region could play an important role in binding.

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

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    Kapucuoglu, N; Losi, L; Eusebi, V

    1997-01-01

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

  1. Anti-apoptotic, anti-oxidant, and anti-inflammatory effects of thalidomide on cerebral ischemia/reperfusion injury in rats.

    Science.gov (United States)

    Palencia, Guadalupe; Medrano, Juan Ángel Núñez-; Ortiz-Plata, Alma; Farfán, Dolores Jiménez; Sotelo, Julio; Sánchez, Aurora; Trejo-Solís, Cristina

    2015-04-15

    Thalidomide has shown protective effects in different models of ischemia/reperfusion damage. To elucidate the mechanisms of such protection, this study assessed the effects of thalidomide on the oxidative stress and inflammatory response induced by ischemia/reperfusion episodes in rats. Rats underwent middle cerebral artery occlusion (MCAO) for 2hours. All animals were sacrificed after different reperfusion times. Rats were administered either DMSO or thalidomide (20mg/kg (i.p.)) at different times before or during reperfusion: 1) 1h before reperfusion; the infarct area was measured 2h after reperfusion. 2) 10min before reperfusion and 80min after reperfusion; the infarct area was measured 24h after reperfusion; and 3) 10min before reperfusion and 1h, 24h, 48h, and 68h after reperfusion; the infarct area was measured 72h after reperfusion. Thalidomide reduced the infarct area 24h and 72h after MCAO, and decreased the neurological deficit in all groups with respect to controls. Thalidomide also lowered significantly the number of TUNEL-positive cells, levels of Bax, caspase-3, lipoperoxidation, and pro-inflammatory cytokines, and increased the levels of SOD1, Bcl-2 and pAkt. These results show that thalidomide has neuroprotective effects, apparently due to its anti-apoptotic, anti-oxidant, and anti-inflammatory effects.

  2. Fetal Kidney Cells Can Ameliorate Ischemic Acute Renal Failure in Rats through Their Anti-Inflammatory, Anti-Apoptotic and Anti-Oxidative Effects.

    Science.gov (United States)

    Gupta, Ashwani Kumar; Jadhav, Sachin H; Tripathy, Naresh Kumar; Nityanand, Soniya

    2015-01-01

    Fetal kidney cells may contain multiple populations of kidney stem cells and thus appear to be a suitable cellular therapy for the treatment of acute renal failure (ARF) but their biological characteristics and therapeutic potential have not been adequately explored. We have culture expanded fetal kidney cells derived from rat fetal kidneys, characterized them and evaluated their therapeutic effect in an ischemia reperfusion (IR) induced rat model of ARF. The fetal kidney cells grew in culture as adherent spindle shaped/polygonal cells and expressed CD29, CD44, CD73, CD90, CD105, CD24 and CD133 markers. Administration of PKH26 labeled fetal kidney cells in ARF rats resulted in a significant decrease in the levels of blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin and decreased tubular necrosis in the kidney tissues (pkidney cells were observed to engraft around injured tubular cells, and there was increased proliferation and decreased apoptosis of tubular cells in the kidneys (pkidney tissues of ARF rats treated with fetal kidney cells had a higher gene expression of renotropic growth factors (VEGF-A, IGF-1, BMP-7 and bFGF) and anti-inflammatory cytokine (IL10); up regulation of anti-oxidative markers (HO-1 and NQO-1); and a lower Bax/Bcl2 ratio as compared to saline treated rats (pkidney cells express mesenchymal and renal progenitor markers, and ameliorate ischemic ARF predominantly by their anti-apoptotic, anti-inflammatory and anti-oxidative effects.

  3. Anti-apoptotic effects of Sonic hedgehog signalling through oxidative stress reduction in astrocytes co-cultured with excretory-secretory products of larval Angiostrongylus cantonensis

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    Chen, Kuang-Yao; Chiu, Cheng-Hsun; Wang, Lian-Chen

    2017-01-01

    Angiostrongylus cantonensis, the rat lungworm, is an important aetiologic agent of eosinophilic meningitis and meningoencephalitis in humans. Co-culturing astrocytes with soluble antigens of A. cantonensis activated the Sonic hedgehog (Shh) signalling pathway and inhibited the apoptosis of astrocytes via the activation of Bcl-2. This study was conducted to determine the roles of the Shh signalling pathway, apoptosis, and oxidative stress in astrocytes after treatment with excretory-secretory products (ESP) from A. cantonensis fifth-stage larvae. Although astrocyte viability was significantly decreased after ESP treatment, the expression of Shh signalling pathway related proteins (Shh, Ptch-1 and Gli-1) was significantly increased. However, apoptosis in astrocytes was significantly decreased after activation of the Shh signalling pathway. Moreover, superoxide and hydrogen superoxide levels in astrocytes were significantly reduced after the activation of Shh pathway signalling due to increasing levels of the antioxidants catalase and superoxide dismutase. These findings indicate that the anti-apoptotic effects of the Shh signalling pathway in the astrocytes of mice infected with A. cantonensis are due to reduced levels of oxidative stress caused by the activation of antioxidants. PMID:28169282

  4. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Song, Shasha [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Wang, Shuang [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China); Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Zhu, Daling, E-mail: dalingz@yahoo.com [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China)

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  5. Bcl-2 proteins in development, health, and disease of the hematopoietic system.

    Science.gov (United States)

    Kollek, Matthias; Müller, Alexandra; Egle, Alexander; Erlacher, Miriam

    2016-08-01

    Members of the Bcl-2 protein family regulate cell fate decisions following a variety of developmental cues or stress signals, with the outcomes of cell death or survival, thus shaping multiple mammalian tissues. This review describes in detail how anti- and proapoptotic Bcl-2 proteins contribute to the development and functioning of the fetal and adult hematopoietic systems and how they influence the generation and maintenance of different hematopoietic lineages. An overview on how stress signals such as genotoxic stress or inflammation can compromise blood cell production, partially by engaging the intrinsic apoptosis pathway, is presented. Finally, the review describes how Bcl-2 protein deregulation-either leading to increased apoptosis resistance or excessive cell death-contributes to many hematological disorders, with specific focus on rare disorders of hematopoiesis and how this knowledge may be used therapeutically.

  6. Structural and biochemical bases for the inhibition of autophagy and apoptosis by viral BCL-2 of murine gamma-herpesvirus 68.

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    Bonsu Ku

    2008-02-01

    Full Text Available All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2 to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine gamma-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3 domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.

  7. The gastroprotective effect of menthol: involvement of anti-apoptotic, antioxidant and anti-inflammatory activities.

    Directory of Open Access Journals (Sweden)

    Ariane Leite Rozza

    Full Text Available The aim of this research was to investigate the anti-apoptotic, antioxidant and anti-inflammatory properties of menthol against ethanol-induced gastric ulcers in rats. Wistar rats were orally treated with vehicle, carbenoxolone (100 mg/kg or menthol (50 mg/kg and then treated with ethanol to induce gastric ulcers. After euthanasia, stomach samples were prepared for histological slides and biochemical analyses. Immunohistochemical analyses of the cytoprotective and anti-apoptotic heat-shock protein-70 (HSP-70 and the apoptotic Bax protein were performed. The neutrophils were manually counted. The activity of the myeloperoxidase (MPO was measured. To determine the level of antioxidant functions, the levels of glutathione (GSH, glutathione peroxidase (GSH-Px, glutathione reductase (GR and superoxide dismutase (SOD were measured using ELISA. The levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6 and the anti-inflammatory cytokine interleukin-10 (IL-10 were assessed using ELISA kits. The menthol treated group presented 92% gastroprotection compared to the vehicle-treated group. An increased immunolabeled area was observed for HSP-70, and a decreased immunolabeled area was observed for the Bax protein in the menthol treated group. Menthol treatment induced a decrease in the activity of MPO and SOD, and the protein levels of GSH, GSH-Px and GR were increased. There was also a decrease in the levels of TNF-α and IL-6 and an increase in the level of IL-10. In conclusion, oral treatment with menthol displayed a gastroprotective activity through anti-apoptotic, antixidant and anti-inflammatory mechanisms.

  8. The gastroprotective effect of menthol: involvement of anti-apoptotic, antioxidant and anti-inflammatory activities.

    Science.gov (United States)

    Rozza, Ariane Leite; Meira de Faria, Felipe; Souza Brito, Alba Regina; Pellizzon, Cláudia Helena

    2014-01-01

    The aim of this research was to investigate the anti-apoptotic, antioxidant and anti-inflammatory properties of menthol against ethanol-induced gastric ulcers in rats. Wistar rats were orally treated with vehicle, carbenoxolone (100 mg/kg) or menthol (50 mg/kg) and then treated with ethanol to induce gastric ulcers. After euthanasia, stomach samples were prepared for histological slides and biochemical analyses. Immunohistochemical analyses of the cytoprotective and anti-apoptotic heat-shock protein-70 (HSP-70) and the apoptotic Bax protein were performed. The neutrophils were manually counted. The activity of the myeloperoxidase (MPO) was measured. To determine the level of antioxidant functions, the levels of glutathione (GSH), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and superoxide dismutase (SOD) were measured using ELISA. The levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and the anti-inflammatory cytokine interleukin-10 (IL-10) were assessed using ELISA kits. The menthol treated group presented 92% gastroprotection compared to the vehicle-treated group. An increased immunolabeled area was observed for HSP-70, and a decreased immunolabeled area was observed for the Bax protein in the menthol treated group. Menthol treatment induced a decrease in the activity of MPO and SOD, and the protein levels of GSH, GSH-Px and GR were increased. There was also a decrease in the levels of TNF-α and IL-6 and an increase in the level of IL-10. In conclusion, oral treatment with menthol displayed a gastroprotective activity through anti-apoptotic, antixidant and anti-inflammatory mechanisms.

  9. Bcl-2 gene therapy for apoptosis following traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    YANG Xiao-feng; ZHENG Xue-sheng; LIU Wei-guo; FENG Jun-feng

    2006-01-01

    Objective: To investigate the therapeutic effect of Bcl- 2 fusion protein on apoptosis in brain following traumatic brain injury.Methods: Bcl-2 gene was cloned by RT-PCR. Bcl-2 and EGFP genes were linked together and inserted into pAdeno-X vector. This recombinant vector was packaged into infectious adenovirus in HEK293 cells. Ninety Wistar rats were assigned randomly into experimental group(n=45) and control group (n=45). All rats were subjected to traumatic brain injury. Then recombinant adenovirus (for experimental group) or saline (for control group) was injected into the traumatic brain. The expression of Bcl-2 fusion protein was investigated by Western blotting, immunohistochemistry and fluorescence microscopy. Apoptosis in the injured brain was studied by TUNEL. Animals' behavior capacity was evaluated by tiltboard test.Results: In the experimental group, many fluorescent cells were found around the traumatic locus,which were also proven to be Bcl-2-positive by immunohistochemistry. On the contrary, few Bcl-2-positive cells and no fluorescent cell were detected in the control group. Bcl-2 expression of experimental group was much higher than that of control group, which was illustrated by Western blotting. The apoptosis index of experimental group was 0.027 ± 0.005, and that of control group was 0.141±0.025 (P<0.01). Two weeks after injury, animals of the experimental group behaved better than those of the control group.Conclusions: A recombinant adenovirus vector expressing Bcl-2 fusion protein has been constructed. Bcl-2 fusion protein can suppress apoptosis and promote cell survival. Moreover, the behavior recovery of the injured animal is promoted. Bcl-2 fusion protein provides a way to track the target cells in vivo.

  10. Long noncoding RNA-mediated anti-apoptotic activity in murine erythroid terminal differentiation.

    Science.gov (United States)

    Hu, Wenqian; Yuan, Bingbing; Flygare, Johan; Lodish, Harvey F

    2011-12-15

    Long noncoding RNAs (lncRNAs) are differentially expressed under both normal and pathological conditions, implying that they may play important biological functions. Here we examined the expression of lncRNAs during erythropoiesis and identified an erythroid-specific lncRNA with anti-apoptotic activity. Inhibition of this lncRNA blocks erythroid differentiation and promotes apoptosis. Conversely, ectopic expression of this lncRNA can inhibit apoptosis in mouse erythroid cells. This lncRNA represses expression of Pycard, a proapoptotic gene, explaining in part the inhibition of programmed cell death. These findings reveal a novel layer of regulation of cell differentiation and apoptosis by a lncRNA.

  11. The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2.

    Science.gov (United States)

    Hsu, A L; Ching, T T; Wang, D S; Song, X; Rangnekar, V M; Chen, C S

    2000-04-14

    This study investigates the apoptotic activity of the cyclooxygenase-2 (COX-2) inhibitor celecoxib in prostate carcinoma cells. COX-2 is constitutively expressed in androgen-responsive LNCaP and androgen-nonresponsive PC-3 cells. Exposure of these cells to celecoxib induces characteristic features of apoptosis, including morphological changes, DNA laddering, and caspase-3 activation, whereas piroxicam, a COX-1-specific inhibitor, displays no appreciable effect on either cancer cell line even after prolonged exposure. Moreover, the potency of celecoxib in apoptosis induction is significantly higher than that of other COX-2 inhibitors examined despite the observation that these inhibitors exhibit similar IC(50) in COX-2 inhibition. It is noteworthy that normal human prostate epithelial cells, expressing a marginally detectable level of COX-2, are insensitive to the induction of apoptosis by celecoxib. These data suggest a correlation between COX-2 expression and sensitivity to the apoptotic effect of the COX-2 inhibitor. In an effort to delineate the underlying mechanism, we examined the effect of celecoxib on the expression of Bcl-2 as well as the activation of the key anti-apoptotic kinase Akt. In contrast to an earlier report that attributed the apoptotic activity of NS398 in LNCaP cells to Bcl-2 down-regulation, we provide evidence that the induction of apoptosis by celecoxib in LNCaP and PC-3 cells is independent of Bcl-2. First, treatment with celecoxib does not alter the cellular Bcl-2 level in both cell lines. Second, enforced Bcl-2 expression in PC-3 cells does not confer protection against the induction of apoptosis by celecoxib. Our data show that celecoxib treatment blocks the phosphorylation of Akt. This correlation is supported by studies showing that overexpression of constitutively active Akt protects PC-3 cells from celecoxib-induced apoptosis. Nevertheless, how celecoxib down-regulates Akt is not clear because the drug does not adversely affect

  12. The Participation of p53 and bcl-2 Proteins in Gastric Carcinomas Associated with Helicobacterpylori and/or Epstein-Barr Virus (EBV).

    Science.gov (United States)

    Szkaradkiewicz, Andrzej; Karpiński, Tomasz M; Majewski, Jan; Malinowska, Kamila; Goślińska-Kuźniarek, Olga; Linke, Krzysztof

    2015-01-01

    In the presented studies p53 and bcl-2 proteins expression were evaluated in samples of gastric carcinomas in patients with Helicobacter pylori or EBV or without H. pylori/EBV infection. The studies were conducted on 64 adult patients with gastric adenocarcinomas: 16 patients with H. pylori (cagA+)-positivity (group 1), 14 with EBV-positive tumours (group 2), 12 with H. pylori/EBV-positive tumours (group 3) and 22 patients with H. pylori/EBV-negative tumours (group 4). H. pylori presence in gastric tumour specimens was detected using Giemsa staining and bacterial culture technique. Moreover, cagA gene was detected using PCR. EBV infection was detected based on EBER presence in the tissue by RNA in situ hybridization. Expressions of p53 and bcl-2 proteins were analysed using immunohistochemistry. Expression of p53 was noted in 14 (84%) patients from group 1, 8 (57%) patients from group 2, 7 (58%) patients from group 3, and 19 (86%) patients from group 4, whereas expression of bcl-2 was noted in 12 (75%) patients from group 1, in 10 (71%) patients from group 2, 9 (75%) patients from group 3, and 6 (27%) patients from group 4. The obtained results allow the conclusion, that H. pylori (cagA+)-associated development of the gastric adenocarcinoma is determined by abnormalities in the p53 protein function and overexpression of anti-apoptotic bcl-2 protein, whereas EBV-associated adenocarcinomas seem to be related with apoptosis resistance associated with bcl-2 overexpression.

  13. Adaptation of HepG2 cells to silver nanoparticles-induced stress is based on the pro-proliferative and anti-apoptotic changes in gene expression.

    Science.gov (United States)

    Brzóska, Kamil; Męczyńska-Wielgosz, Sylwia; Stępkowski, Tomasz M; Kruszewski, Marcin

    2015-05-01

    Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials due to their antibacterial properties. Owing to the recent boost in the usage of AgNPs-containing products, human exposure to AgNPs is increasing, highlighting the need for careful evaluation of AgNPs toxicity in humans. We used two cellular models, hepatic HepG2 and epithelial A549 cell lines, to study the mechanism of AgNPs-induced toxicity at the cellular level. These two cell lines differ significantly in their response to AgNPs treatment. In the case of A549 cells, a minor decrease in viability and increase in the extent of DNA breakage were observed. A markedly different response to AgNPs was observed in HepG2 cells. In short term, a massive induction of DNA breakage was observed, suggesting that the basal activity of antioxidant defence in these cells was not sufficient to effectively protect them from the nanoparticle-induced oxidative stress. After prolonged exposure, the extent of DNA breakage decreased to the level observed in the control cells proving that a successful adaptation to the new conditions had taken place. The cells that were unable to adapt must have died, as revealed by the Neutral Red assay that indicated less than half viable cells after 24-h treatment with 100 µg/ml of 20nm AgNPs. The gene expression analysis revealed that the observed adaptation was underlain by a pro-proliferative, anti-apoptotic signal leading to up-regulation of the genes promoting proliferation and inflammatory response (EGR1, FOS, JUN, HK2, IL4, MMP10, VEGFA, WISP1, CEBPB, IL8, SELPLG), genes coding the anti-apoptotic proteins (BCL2A1, CCL2) and factors involved in the response to stress (HSPB1, GADD45A). Such a selection of highly resistant population of cells should be taken into account in the case of medical applications of nanoparticles since the sustained proliferative signalling and resistance to cell death are hallmarks of cancer, acquired by the cells in the process of

  14. BAG1: The Guardian of Anti-Apoptotic Proteins in Acute Myeloid Leukemia

    OpenAIRE

    Sanja Aveic; Martina Pigazzi; Giuseppe Basso

    2011-01-01

    BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, M...

  15. Selective peptide inhibitors of antiapoptotic cellular and viral Bcl-2 proteins lead to cytochrome c release during latent Kaposi's sarcoma-associated herpesvirus infection.

    Science.gov (United States)

    Burrer, Christine M; Foight, Glenna W; Keating, Amy E; Chan, Gary C

    2016-01-04

    Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with B-cell lymphomas including primary effusion lymphoma and multicentric Castleman's disease. KSHV establishes latency within B cells by modulating or mimicking the antiapoptotic Bcl-2 family of proteins to promote cell survival. Our previous BH3 profiling analysis, a functional assay that assesses the contribution of Bcl-2 proteins towards cellular survival, identified two Bcl-2 proteins, cellular Mcl-1 and viral KsBcl-2, as potential regulators of mitochondria polarization within a latently infected B-cell line, Bcbl-1. In this study, we used two novel peptide inhibitors identified in a peptide library screen that selectively bind KsBcl-2 (KL6-7_Y4eK) or KsBcl-2 and Mcl-1 (MS1) in order to decipher the relative contribution of Mcl-1 and KsBcl-2 in maintaining mitochondrial membrane potential. We found treatment with KL6-7_Y4eK and MS1 stimulated a similar amount of cytochrome c release from mitochondria isolated from Bcbl-1 cells, indicating that inhibition of KsBcl-2 alone is sufficient for mitochondrial outer membrane permiabilzation (MOMP) and thus apoptosis during a latent B cell infection. In turn, this study also identified and provides a proof-of-concept for the further development of novel KsBcl-2 inhibitors for the treatment of KSHV-associated B-cell lymphomas via the targeting of latently infected B cells.

  16. The effect of Ginkgo biloba extract treatment in the Bcl-2 expression by osteoblasts in the femoral trabecular bone of Wistar rats with glucocorticoid-induced osteoporosis

    Directory of Open Access Journals (Sweden)

    Leda M.F. Lucinda

    2014-06-01

    Full Text Available Evaluate the effect of the extract of Ginkgo biloba L., Ginkgoaceae (EGb in the Bcl-2 expression by osteoblasts in the femoral trabecular bone of Wistar rats with glucocorticoid-induced osteoporosis. Rats were divided into five groups: osteoporosis; EGb1 (28 mg/kg; EGb2 (56 mg/kg; alendronate (0.2 mg/animal and control. The treatments were conducted for 20 or 30 days. The Bcl-2 expression by osteoblasts cells was evaluated in the femoral trabecular bone. The control group was compared with the osteoporosis-induced group (Student's t-test. The other groups were analyzed by ANOVA test followed by Tukey's test (p < 0.05. The percentage of Bcl-2 expression was reduced, when the control group (17.95 ± 3.45 20 days; 21.11 ± 3.43 30 days was compared with the osteoporosis group (10.64 ± 3.30 20 days; 9.72 ± 2.84 30 days. Nevertheless, this percentage increased in the EGb2 group (18.58 ± 3.41 20 days; 16.51 ± 1.80 30 days when compared to the osteoporosis group. The EGb increased the expression of the anti-apoptotic protein, suggesting a decrease in osteoblast apoptosis.

  17. Inhibition of mitochondria responsible for the anti-apoptotic effects of melatonin during ischemia-reperfusion

    Institute of Scientific and Technical Information of China (English)

    HAN Yi-xiang; ZHANG Sheng-hui; WANG Xi-ming; WU Jian-bo

    2006-01-01

    Objective: To investigate a possible mechanism responsible for anti-apoptotic effects of melatonin and provide theoretical evidences for clinical therapy. Methods: Ischemia-reperfusion mediated neuronal cell injury model was constructed in cerebellar granule neurons (CGNs) by deprivation of glucose, serum and oxygen in media. After ischemia, melatonin was added to the test groups to reach differential concentration during reperfusion. DNA fragmentation, mitochondrial transmembrane potential,mitochondrial cytochrome c release and caspase-3 activity were observed after subjecting cerebellar granule neurons to oxygen-glucose deprivation (OGD). Results: The results showed that OGD induced typical cell apoptosis change, DNA ladder and apoptosis-related alterations in mitochondrial functions including depression of mitochondrial transmembrane potential (its maximal protection ratio was 73.26%) and release of cytochrome c (its maximal inhibition ratio was 42.52%) and the subsequent activation of caspase-3 (its maximal protection ratio was 59.32%) in cytoplasm. Melatonin reduced DNA damage and inhibited release of mitochondrial cytochrome c and activation of caspase-3. Melatonin can strongly prevent the OGD-induced loss of the mitochondria membrane potential. Conclusion: Our findings suggested that the direct inhibition of mitochondrial pathway might essentially contribute to its anti-apoptotic effects in neuronal ischemia-reperfusion.

  18. Anti-apoptotic signature in thymic squamous cell carcinomas - functional relevance of anti-apoptotic BIRC3 expression in the thymic carcinoma cell line 1889c

    Directory of Open Access Journals (Sweden)

    Bei eHuang

    2013-12-01

    Full Text Available The molecular pathogenesis of thymomas and thymic carcinomas (TCs is poorly understood and results of adjuvant therapy are unsatisfactory in case of metastatic disease and tumor recurrence. For these clinical settings, novel therapeutic strategies are urgently needed. Recently, limited sequencing efforts revealed that a broad spectrum of genes that play key roles in various common cancers are rarely affected in thymomas and thymic carcinomas, suggesting that other oncogenic principles might be important. This made us re-analyze historic expression data obtained in a spectrum of thymomas and thymic squamous cell carcinomas (TSCC with a custom made cDNA microarray. By cluster analysis, different anti-apoptotic signatures were detected in type B3 thymoma and TSCC, including overexpression of BIRC3 in TSCCs. This was confirmed by qRT-PCR in the original and an independent validation set of tumors. In contrast to several other cancer cell lines, the BIRC3-positive TSCC cell line, 1889c showed spontaneous apoptosis after BIRC3 knock-down. Targeting apoptosis genes is worth testing as therapeutic principle in TSCC.

  19. 吗啡成瘾时脑内Fas、Bcl-2和Caspase-3蛋白表达的改变%Changes of Fas, Bcl-2 and Caspase-3 protein in rat brain during morphine addiction

    Institute of Scientific and Technical Information of China (English)

    刘立伟; 王新华; 傅舒昆; 吴青华; 傅强

    2012-01-01

    neuronal apoptosis in rat brain by enhancing the expression of pro-apoptotic Fas, Caspase-3 and decreasing the expression of anti-apoptotic Bcl-2, which might be one of the mechanisms for opiate-induced neuronal damage.

  20. Pan-Bcl-2 inhibitor AT-101 enhances tumor cell killing by EGFR targeted T cells.

    Directory of Open Access Journals (Sweden)

    Archana Thakur

    Full Text Available Pancreatic cancer is a deadly disease and has the worst prognosis among almost all cancers and is in dire need of new and improved therapeutic strategies. Conditioning of tumor cells with chemotherapeutic drug has been shown to enhance the anti-tumor effects of cancer vaccines and adoptive cell therapy. In this study, we investigated the immunomodulatory effects of pan-Bcl-2 inhibitor AT-101 on pancreatic cancer (PC cell cytotoxicity by activated T cells (ATC. The effects of AT-101 on cytotoxicity, early apoptosis, and Granzyme B (GrzB and IFN-γ signaling pathways were evaluated during EGFR bispecific antibody armed ATC (aATC-mediated killing of L3.6pl and MiaPaCa-2 PC cells pre-sensitized with AT-101. We found that pretreatment of tumor cells with AT-101 enhanced susceptibility of L3.6pl and MiaPaCa-2 tumor cells to ATC and aATC-mediated cytotoxicity, which was in part mediated via enhanced release of cytolytic granule GrzB from ATC and aATC. AT-101-sensitized L3.6pl cells showed up-regulation of IFN-γ-mediated induction in the phosphorylation of Ser(727-Stat1 (pS(727-Stat1, and IFN-γ induced dephosphorylation of phospho-Tyr(705-Stat3 (pY(705-Stat3. Priming (conditioning of PC cells with AT-101 can significantly enhance the anti-tumor activity of EGFRBi armed ATC through increased IFN-γ induced activation of pS(727-Stat1 and inhibition of pY(705-Stat3 phosphorylation, and resulting in increased ratio of pro-apoptotic to anti-apoptotic proteins. Our results verify enhanced cytotoxicity after a novel chemotherapy conditioning strategy against PC that warrants further in vivo and clinical investigations.

  1. Increased expression of Bcl-2 during mucous cell metaplasia induced by endotoxin and ozone

    Energy Technology Data Exchange (ETDEWEB)

    Tesfaigzi, J.; Ray, L.M.; Hotchkiss, J.A. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1995-12-01

    Apoptosis or programmed cell death is accompanied by characteristic morphological changes that distinguish apoptosis from other forms of cell death. These changes include DNA fragmentation, chromatin condensation, cell shrinkage, cell surface pseudopodia, and finally the cellular collapse into membrane-enclosed apoptotic bodies which are rapidly engulfed by macrophages or neighboring cells. Although the morphological features of apoptotic cells are well studied, the biochemical events that control apoptosis are not understood. Programmed cell death is triggered by a variety of pathways that are initiated by different stimuli including noxious agents, DNA damage, the activation of TNF receptors, or the withdrawl of growth factors. The central process of programmed cell death involves a cascade of biochemical events that begins with the initiation of a family of cysteine proteases, including the interleukin-1-{Beta}-converting enzyme, CPP-32, and Apopain. The ratio of Bax, a death-inducer gene, to Bcl-2, an apoptosis suppressor gene, determines whether or not the main apoptotic pathyway is blocked. Apoptosis is suppressed if the ratio of Bcl-2/Bax is > 1, and cells undergo apoptosis if the ratio is < 1. The overexpression of Bcl-2 has been shown to block the apoptotic program triggered by a variety of agents. Therefore, Bcl-2 must be involved in blocking the central pathway of the cell death program. In conclusion, this study showed that high levels of Bcl-2 were detected in some mucous cells at specific time points during mucous cell metaplasia, and this expression was reduced at later time points or was absent after remodeling of this epithelium.

  2. Anti-Apoptotic and Pro-Survival Effect of Alpinate Oxyphyllae Fructus (AOF) in a d-Galactose-Induced Aging Heart.

    Science.gov (United States)

    Chang, Yung-Ming; Chang, Hen-Hong; Kuo, Wei-Wen; Lin, Hung-Jen; Yeh, Yu-Lan; Padma Viswanadha, Vijaya; Tsai, Chin-Chuan; Chen, Ray-Jade; Chang, Hsin-Nung; Huang, Chih-Yang

    2016-03-29

    Aging, a natural biological/physiological phenomenon, is accelerated by reactive oxygen species (ROS) accumulation and identified by a progressive decrease in physiological function. Several studies have shown a positive relationship between aging and chronic heart failure (HF). Cardiac apoptosis was found in age-related diseases. We used a traditional Chinese medicine, Alpinate Oxyphyllae Fructus (AOF), to evaluate its effect on cardiac anti-apoptosis and pro-survival. Male eight-week-old Sprague-Dawley (SD) rats were segregated into five groups: normal control group (NC), d-Galactose-Induced aging group (Aging), and AOF of 50 (AL (AOF low)), 100 (AM (AOF medium)), 150 (AH (AOF high)) mg/kg/day. After eight weeks, hearts were measured by an Hematoxylin-Eosin (H&E) stain, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-assays and Western blotting. The experimental results show that the cardiomyocyte apoptotic pathway protein expression increased in the d-Galactose-Induced aging groups, with dose-dependent inhibition in the AOF treatment group (AL, AM, and AH). Moreover, the expression of the pro-survival p-Akt (protein kinase B (Akt)), Bcl-2 (B-cell lymphoma 2), anti-apoptotic protein (Bcl-xL) protein decreased significantly in the d-Galactose-induced aging group, with increased performance in the AOF treatment group with levels of p-IGFIR and p-PI3K (Phosphatidylinositol-3' kinase (PI3K)) to increase by dosage and compensatory performance. On the other hand, the protein of the Sirtuin 1 (SIRT1) pathway expression decreased in the aging groups and showed improvement in the AOF treatment group. Our results suggest that AOF strongly works against ROS-induced aging heart problems.

  3. Anti-apoptotic action of API2-MALT1 fusion protein involved in t(11;18)(q21;q21) MALT lymphoma.

    Science.gov (United States)

    Hosokawa, Y

    2005-01-01

    At least three distinct chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21) involving the API2 (also known as c-IAP2)-MALT1 fusion protein, BCL10, and MALT1, respectively, have been implicated in the molecular pathogenesis of mucosa associated lymphoid tissue (MALT) lymphoma. Our findings showed that several variants of the API2-MALT1 fusion protein can occur in patients with t(11;18)(q21;q21), and that API2-MALT1 can potently enfance activation of nuclear factor (NF)-kappaB signaling, which may be relevant to the pathogenesis of MALT lymphomas. We also found that MALT1 is rapidly degraded via the ubiquitin-proteasome pathway, as is the case with API2, but upon the synthesis of fusion, API2-MALT1 becomes stable against this pathway. This stability of API2-MALT1 may thus result in inappropriate nuclear factor (NF)-kappaB activation, thereby contributing to the pathogenesis of MALT lymphoma. Recent biochemical and genetic studies have clearly shown that BCL10 and MALT1 form a physical and functional complex and are both required for NF-kappaB activation by antigen receptor stimulation in T and B lymphocytes. It has also been shown that CARMA1, a newly discovered member of the membrane-associated guanylate kinase (MAGUK) families, is critical for antigen receptor-stimulated NF-kappaB activation. It can be assumed that API2-MALT1 can bypass this normal BCL10/MALT1 cellular signaling pathway linked to NF-kappaB activation, thereby inducing antigen receptor-independent proliferation of lymphocytes. Furthermore, BCL10/MALT1- and API2-MALT1-induced NF-kappaB activation may contribute to anti-apoptotic action probably through NF-kappaB-mediated upregulation of apoptotic inhibitor genes. We recently provided direct evidence that API2-MALT1 indeed exerts anti-apoptotic action, in part, through its direct interaction with apoptotic regulators including Smac. Taken together, these findings prompt us to hypothesize that the anti-apoptotic action

  4. A balanced translocation disrupting BCL2L10 and PNLDC1 segregates with affective psychosis

    Science.gov (United States)

    Bouwkamp, Christian G.; Kievit, Anneke J. A.; Olgiati, Simone; Breedveld, Guido J.; Coesmans, Michiel; Bonifati, Vincenzo

    2016-01-01

    Affective psychoses are a group of severe psychiatric disorders, including schizoaffective disorder and bipolar I disorder, together affecting ∼1% of the population. Despite their high heritability, the molecular genetics and neurobiology of affective psychosis remain largely elusive. Here, we describe the identification of a structural genetic variant segregating with affective psychosis in a family with multiple members suffering from bipolar I disorder or schizoaffective disorder, bipolar type. A balanced translocation involving chromosomes 6 and 15 was detected by karyotyping and fluorescence in‐situ hybridization (FISH). Using whole‐genome sequencing, we rapidly delineated the translocation breakpoints as corresponding intragenic events disrupting BCL2L10 and PNLDC1. These data warrant further consideration for BCL2L10 and PNLDC1 as novel candidates for affective psychosis. © 2016 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc. PMID:27260655

  5. Exosomes derived from human platelet-rich plasma prevent apoptosis induced by glucocorticoid-associated endoplasmic reticulum stress in rat osteonecrosis of the femoral head via the Akt/Bad/Bcl-2 signal pathway

    Science.gov (United States)

    Tao, Shi-Cong; Yuan, Ting; Rui, Bi-Yu; Zhu, Zhen-Zhong; Guo, Shang-Chun; Zhang, Chang-Qing

    2017-01-01

    An excess of glucocorticoids (GCs) is reported to be one of the most common causes of osteonecrosis of the femoral head (ONFH). In addition, GCs can induce bone cell apoptosis through modulating endoplasmic reticulum (ER) stress. Among the three main signal pathways in ER stress, the PERK (protein kinase RNA-like ER kinase)/CHOP (CCAAT-enhancer-binding protein homologous protein) pathway has been considered to be closely associated with apoptosis. Platelet-rich plasma (PRP) has been referred to as a concentration of growth factors and the exosomes derived from PRP (PRP-Exos) have a similar effect to their parent material. The enriched growth factors can be encapsulated into PRP-Exos and activate Akt and Erk pathways to promote angiogenesis. Activation of the Akt pathway may promote the expression of anti-apoptotic proteins like Bcl-2, while CHOP can inhibit B-cell lymphoma 2 (Bcl-2) expression to increase the level of cleaved caspase-3 and lead to cell death. Consequently, we hypothesized that PRP-Exos prevent apoptosis induced by glucocorticoid-associated ER stress in rat ONFH via the Akt/Bad/Bcl-2 signal pathway. To verify this hypothesis, a dexamethasone (DEX)-treated in vitro cell model and methylprednisolone (MPS)-treated in vivo rat model were adopted. Characterization of PRP-Exos, and effects of PRP-Exos on proliferation, apoptosis, angiogenesis, and osteogenesis of cells treated with GCs in vitro and in vivo were examined. Furthermore, the mechanism by which PRP-Exos rescue the GC-induced apoptosis through the Akt/Bad/Bcl-2 pathway was also investigated. The results indicate that PRP-Exos have the capability to prevent GC-induced apoptosis in a rat model of ONFH by promoting Bcl-2 expression via the Akt/Bad/Bcl-2 signal pathway under ER stress. PMID:28255363

  6. Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing BCL-2.

    Science.gov (United States)

    Adams, M L; Pierce, R H; Vail, M E; White, C C; Tonge, R P; Kavanagh, T J; Fausto, N; Nelson, S D; Bruschi, S A

    2001-11-01

    Mitochondria play an important role in the cell death induced by many drugs, including hepatotoxicity from overdose of the popular analgesic, acetaminophen (APAP). To investigate mitochondrial alterations associated with APAP-induced hepatotoxicity, the subcellular distribution of proapoptotic BAX was determined. Based on the antiapoptotic characteristics of BCL-2, we further hypothesized that if a BAX component was evident then BCL-2 overexpression may be hepatoprotective. Mice, either with a human bcl-2 transgene (-/+) or wild-type mice (WT; -/-), were dosed with 500 or 600 mg/kg (i.p.) APAP or a nonhepatotoxic isomer, N-acetyl-m-aminophenol (AMAP). Immunoblot analyses indicated increased mitochondrial BAX-beta content very early after APAP or AMAP treatment. This was paralleled by disappearance of BAX-alpha from the cytosol of APAP treated animals and, to a lesser extent, with AMAP treatment. Early pathological evidence of APAP-induced zone 3 necrosis was seen in bcl-2 (-/+) mice, which progressed to massive panlobular necrosis with hemorrhage by 24 h. In contrast, WT mice dosed with APAP showed a more typical, and less severe, centrilobular necrosis. AMAP-treated bcl-2 (-/+) mice displayed only early microvesicular steatosis without progression to extensive necrosis. Decreased complex III activity, evident as early as 6 h after treatment, correlated well with plasma enzyme activities at 24 h (AST r(2) = 0.89, ALT r(2) = 0.87) thereby confirming a role for mitochondria in APAP-mediated hepatotoxicity. In conclusion, these data suggest for the first time that BAX may be an early determinant of APAP-mediated hepatotoxicity and that BCL-2 overexpression unexpectedly enhances APAP hepatotoxicity.

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Institute of Scientific and Technical Information of China (English)

    徐磊; 赵晏; 展淑琴; 王会生; 史文春

    2002-01-01

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

  9. An anti-apoptotic peptide improves survival in lethal total body irradiation.

    Science.gov (United States)

    McDunn, Jonathan E; Muenzer, Jared T; Dunne, Benjamin; Zhou, Anthony; Yuan, Kevin; Hoekzema, Andrew; Hilliard, Carolyn; Chang, Katherine C; Davis, Christopher G; McDonough, Jacquelyn; Hunt, Clayton; Grigsby, Perry; Piwnica-Worms, David; Hotchkiss, Richard S

    2009-05-15

    Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5-10 microM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.

  10. Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

    LENUS (Irish Health Repository)

    Greene, C M

    2010-05-01

    alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

  11. Simultaneous delivery of Paclitaxel and Bcl-2 siRNA via pH-Sensitive liposomal nanocarrier for the synergistic treatment of melanoma

    Science.gov (United States)

    Reddy, Teegala Lakshminarayan; Garikapati, Koteswara Rao; Reddy, S. Gopal; Reddy, B. V. Subba; Yadav, J. S.; Bhadra, Utpal; Bhadra, Manika Pal

    2016-10-01

    pH-sensitive drug carriers that are sensitive to the acidic (pH = ~6.5) microenvironments of tumor tissues have been primarily used as effective drug/gene/siRNA/microRNA carriers for releasing their payloads to tumor cells/tissues. Resistance to various drugs has become a big hurdle in systemic chemotherapy in cancer. Therefore delivery of chemotherapeutic agents and siRNA’s targeting anti apoptotic genes possess advantages to overcome the efflux pump mediated and anti apoptosis-related drug resistance. Here, we report the development of nanocarrier system prepared from kojic acid backbone-based cationic amphiphile containing endosomal pH-sensitive imidazole ring. This pH-sensitive liposomal nanocarrier effectively delivers anti-cancer drug (Paclitaxel; PTX) and siRNA (Bcl-2), and significantly inhibits cell proliferation and reduces tumor growth. Tumor inhibition response attributes to the synergistic effect of PTX potency and MDR reversing ability of Bcl-2 siRNA in the tumor supporting that kojic acid based liposomal pH-sensitive nanocarrier as efficient vehicle for systemic co-delivery of drugs and siRNA.

  12. D-pinitol promotes apoptosis in MCF-7 cells via induction of p53 and Bax and inhibition of Bcl-2 and NF-κB.

    Science.gov (United States)

    Rengarajan, Thamaraiselvan; Nandakumar, Natarajan; Rajendran, Peramaiyan; Haribabu, Lingaiah; Nishigaki, Ikuo; Balasubramanian, Maruthaiveeran Periyasamy

    2014-01-01

    Development of drugs from natural products has been undergoing a gradual evoluation. Many plant derived compounds have excellent therapeutic potential against various human ailments. They are important sources especially for anticancer agents. A number of promising new agents are in clinical development based on their selective molecular targets in the field of oncology. D-pinitol is a naturally occurring compound derived from soy which has significant pharmacological activitites. Therefore we selected D-pinitol in order to evaluate apoptotic potential in the MCF-7 cell line. Human breast cancer cells were treated with different concentrations of D-pinitol and cytotoxicity was measured by MTT and LDH assays. The mechanism of apoptosis was studied with reference to expression of p53, Bcl-2, Bax and NF-kB proteins. The results revealed that D-pinitol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner, while upregulating the expression of p53, Bax and down regulating Bcl-2 and NF-kB. Thus the results obtained in this study clearly vindicated that D-pinitol induces apotosis in MCF-7 cells through regulation of proteins of pro- and anti-apoptotic cascades.

  13. A novel small molecule inhibitor targeted at Bcl-2

    Institute of Scientific and Technical Information of China (English)

    JIN; LiJi; ZHANG; ZhiChao; WANG; YuanYuan; WEI; MeiJiao; XU; Qin

    2007-01-01

    Recently, the heterocyclic compound 8-oxo-3-thiomorpholino-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) was synthesized and shown to induce apoptosis in both (H22) hematoma and (MCF-7) adenocarcinoma cells. The IC50 values of S1 against the two cell lines were 0.17 and 0.09 μmol/L, respectively. Furthermore, the apoptosis-inducing activity of this compound was highlighted both in vivo and in vitro. Subsequent experiments identified Bcl-2 as the primary target of S1, as a significant reduction in Bcl-2 protein levels was observed in H22 cells following a two-hour treatment with 10 μmol/L S1. While rapid depolarization of mitochondrial membranes led immediately to caspase 9 activation, no changes were identified in either caspase 8 levels or levels in Bcl-2 mRNA. These data were consistent with the results of circular dichroism (CD) spectra analysis, revealing that S1 inactivated the Bcl-2 protein by destroying its critical alpha helices. Taken together, these results suggest the potential of S1 in the development of new therapeutic agents.

  14. MiR-16 modulate temozolomide resistance by regulating BCL-2 in human glioma cells.

    Science.gov (United States)

    Han, Jing; Chen, Qianxue

    2015-01-01

    Temozolomide (TMZ) with radiotherapy is the current standard of care for newly diagnosed glioma. However, glioma patients who are treated with the drug often develop resistance to it and some other drugs. Recently studies have shown that microRNAs (miRNAs) play an important role in drug resistance. In present study, we first examined the sensitivity to temozolomide in six glioma cell lines, and established a resistant variant, U251MG/TR cells from TMZ-sensitive glioma cell line, U251MG. We then performed a comprehensive analysis of miRNA expressions in U251MG/TR and parental cells using cancer microRNA PCR Array. Among the downregulated microRNAs was miR-16, members of miR-15/16 family, whose expression was further validated by qRT-PCR in U251MG/TR and U251MG cells. The selective microRNA, miR-16 mimics or inhibitor was respectively transfected into U251MG/TR cells and AM38 cell. We found that treatment with the mimics of miR-16 greatly decreased the sensitivity of U251MG/TR cells to temozolomide, while sensitivity to these drugs was increased by treatment with the miR-16 inhibitor. In addition, the downregulation of miR-16 in temozolomide-sensitive AM38 cells was concurrent with the upregulation of Bcl-2 protein. Conversely, overexpression of miR-16 in temozolomide-resistant cells inhibited Bcl-2 expression and decreased temozolomide resistance. In conclusion, MiR-16 mediated temozolomide-resistance in glioma cells by modulation of apoptosis via targeting Bcl-2, which suggesting that miR-16 and Bcl-2 would be potential therapeutic targets for glioma therapy.

  15. Caspase Induction and BCL2 Inhibition in Human Adipose Tissue

    Science.gov (United States)

    Tinahones, Francisco José; Coín Aragüez, Leticia; Murri, Mora; Oliva Olivera, Wilfredo; Mayas Torres, María Dolores; Barbarroja, Nuria; Gomez Huelgas, Ricardo; Malagón, Maria M.; El Bekay, Rajaa

    2013-01-01

    OBJECTIVE Cell death determines the onset of obesity and associated insulin resistance. Here, we analyze the relationship among obesity, adipose tissue apoptosis, and insulin signaling. RESEARCH DESIGN AND METHODS The expression levels of initiator (CASP8/9) and effector (CASP3/7) caspases as well as antiapoptotic B-cell lymphoma (BCL)2 and inflammatory markers were assessed in visceral (VAT) and subcutaneous (SAT) adipose tissue from patients with different degrees of obesity and without insulin resistance or diabetes. Adipose tissue explants from lean subjects were cultured with TNF-α or IL-6, and the expression of apoptotic and insulin signaling components was analyzed and compared with basal expression levels in morbidly obese subjects. RESULTS SAT and VAT exhibited increased CASP3/7 and CASP8/9 expression levels and decreased BCL2 expression with BMI increase. These changes were accompanied by increased inflammatory cytokine mRNA levels and macrophage infiltration markers. In obese subjects, CASP3/7 activation and BCL2 downregulation correlated with the IRS-1/2–expression levels. Expression levels of caspases, BCL2, p21, p53, IRS-1/2, GLUT4, protein tyrosine phosphatase 1B, and leukocyte antigen-related phosphatase in TNF-α– or IL-6–treated explants from lean subjects were comparable with those found in adipose tissue samples from morbidly obese subjects. These insulin component expression levels were reverted with CASP3/7 inhibition in these TNF-α– or IL-6–treated explants. CONCLUSIONS Body fat mass increase is associated with CASP3/7 and BCL2 expression in adipose tissue. Moreover, this proapoptotic state correlated with insulin signaling, suggesting its potential contribution to the development of insulin resistance. PMID:23193206

  16. Effects of mecobalamin on Bax and Bcl-2 in neurons after peripheral nerve injury%甲钴胺对周围神经损伤后神经元细胞Bax和Bcl-2蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    王东强; 张平平; 李志军; 刘颖

    2015-01-01

    and significantly reduced Bcl-2 protein expression in spinal cord anterior horn motor neurons and ganglion sensory neurons compared with the sham-operation group (P<0.05).Compared with the model group and sham-operation group,the mecobalamin group had significantly reduced Bax protein expression and significantly increased Bcl-2 protein expression in spinal cord anterior horn motor neurons and ganglion sensory neurons (P<0.05).Conclusion Mecobalamin has anti-apoptotic effect,and it contributes to neurological function recovery possibly by inhibiting the death of injured neurons.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  18. Dexamethasone protected human glioblastoma U87MG cells from temozolomide induced apoptosis by maintaining Bax:Bcl-2 ratio and preventing proteolytic activities

    Directory of Open Access Journals (Sweden)

    Patel Sunil J

    2004-12-01

    Full Text Available Abstract Background Glioblastoma is the deadliest and most prevalent brain tumor. Dexamethasone (DXM is a commonly used steroid for treating glioblastoma patients for alleviation of vasogenic edema and pain prior to treatment with chemotherapeutic drugs. Temozolomide (TMZ, an alkylating agent, has recently been introduced in clinical trials for treating glioblastoma. Here, we evaluated the modulatory effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells. Results Freshly grown cells were treated with different doses of DXM or TMZ for 6 h followed by incubation in a drug-free medium for 48 h. Wright staining and ApopTag assay showed no apoptosis in cells treated with 40 μM DXM but considerable amounts of apoptosis in cells treated with 100 μM TMZ. Apoptosis in TMZ treated cells was associated with an increase in intracellular free [Ca2+], as determined by fura-2 assay. Western blot analyses showed alternations in the levels of Bax (pro-apoptotic and Bcl-2 (anti-apoptotic proteins resulting in increased Bax:Bcl-2 ratio in TMZ treated cells. Western blot analyses also detected overexpression of calpain and caspase-3, which cleaved 270 kD α-spectrin at specific sites for generation of 145 and 120 kD spectrin break down products (SBDPs, respectively. However, 1-h pretreatment of cells with 40 μM DXM dramatically decreased TMZ induced apoptosis, decreasing Bax:Bcl-2 ratio and SBDPs. Conclusion Our results revealed an antagonistic effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells, implying that treatment of glioblastoma patients with DXM prior to chemotherapy with TMZ might result in an undesirable clinical outcome.

  19. Pan-Bcl-2 inhibitor obatoclax delays cell cycle progression and blocks migration of colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Bruno Christian Koehler

    Full Text Available Despite the fact that new treatment regimes have improved overall survival of patients challenged by colorectal cancer (CRC, prognosis in the metastatic situation is still restricted. The Bcl-2 family of proteins has been identified as promising anti cancer drug target. Even though small molecules targeting Bcl-2 proteins are in clinical trials, little is known regarding their effects on CRC. The aim of this study was to preclinically investigate the value of ABT-737 and Obatoclax as anticancer drugs for CRC treatment. The effects of the BH3-mimetics ABT-737 and Obatoclax on CRC cells were assessed using viability and apoptosis assays. Wound healing migration and boyden chamber invasion assays were applied. 3-dimensional cell cultures were used for long term assessment of invasion and proliferation. Clinically relevant concentrations of pan-Bcl-2 inhibitor Obatoclax did not induce cell death. In contrast, the BH3-mimetic ABT-737 induced apoptosis in a dose dependent manner. Obatoclax caused a cell line specific slowdown of CRC cell growth. Furthermore, Obatoclax, but not ABT-737, recovered E-Cadherin expression and led to impaired migration and invasion of CRC cells. The proliferative capacity and invasiveness of CRC cells was strikingly inhibited by low dose Obatoclax in long term 3-dimensional cell cultures. Obatoclax, but not ABT-737, caused a G1-phase arrest accompanied by a downregulation of Cyclin D1 and upregulation of p27 and p21. Overexpression of Mcl-1, Bcl-xL or Bcl-2 reversed the inhibitory effect of Obatoclax on migration but failed to restore the proliferative capacity of Obatoclax-treated CRC cells. The data presented indicate broad and multifaceted antitumor effects of the pan-Bcl-2 inhibitor Obatoclax on CRC cells. In contrast to ABT-737, Obatoclax inhibited migration, invasion and proliferation in sublethal doses. In summary, this study recommends pan-Bcl-2 inhibition as a promising approach for clinical trials in CRC.

  20. Effects of acupoint versus non-acupoint electroacupuncture on cerebral cortical neuronal Bcl-2,Bax and caspase-3 expression in a rat model of focal cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Jun Wang; Junming Fan; Yongshu Dong; Xia Huang; Hongxia Zhang

    2008-01-01

    BACKGROUND: Several studies have demonstrated that electroacupuncture by acupoint selection can inhibit cerebral cortical neuronal apoptosis following cerebral ischemia/reperfusion.OBJECTIVE: To validate the effects of electroacupuncture by acupoint selection on the expression level of cortical neuronal anti-apoptotic Bcl-2 protein and the apoptotic executive protein, caspase-3, in rat models of focal cerebral ischemia/reperfusion.DESIGN, TIME AND SETTING: This randomized grouping, neural cell and molecular biology animal experiment was performed at the Laboratory of Pharmacology of Traditional Chinese Medicine and the Laboratory Animal Center of Henan Institute of Traditional Chinese Medicine between November 2006 and May 2007.MATERIALS: Atotal of 40 healthy male adult Sprague-Dawley rats were randomly and evenly divided into four groups: sham-operated, model, electroacupuncture and non-acupoint control. G6895 electro-acupuncture instruments were purchased from Shanghai Huayi Instrument Factory, China. Caspase-3, Bcl-2 and Bax kits were provided by Wuhan Boster Bioengineering Co., Ltd., China.METHODS: Middle cerebral artery occlusion was induced in the model, electroacupuncture and non-acupoint groups. In the electroacupuncture group, the acupoints Jianyu (LI15), Waiguan (SJ5), Biguan (ST31), and Zusanli (ST36) were given electroacupuncture. In the non-acupoint control group, at each time point (immediately after ischemia and after reperfusion, or 2 hours after reperfusion), electroacupuncture was performed at the midpoints of Tianquan (PC2)-Quze (PC 3) line, Quze (PC 3)-Ximen (PC4) line, Zuwuli (LRlO)-Yinbao (LRg) line, and Xiguan (LR7)-Zhongdu (LR6) line. Electroacupuncture parameters were set with a continuous wave with a frequency of 10 Hz, wave width 0.6 ms, voltage 1.5-3.0 V, and a duration of 10 minutes. The sham-operated and model groups received only animal fixation without electroacupuncture procedure.MAIN OUTCOME MEASURES: Five rats were selected from

  1. Anti-inflammatory and anti-apoptotic effects of (RS)-glucoraphanin bioactivated with myrosinase in murine sub-acute and acute MPTP-induced Parkinson's disease.

    Science.gov (United States)

    Galuppo, Maria; Iori, Renato; De Nicola, Gina Rosalinda; Bramanti, Placido; Mazzon, Emanuela

    2013-09-01

    This study was focused on the possible neuroprotective role of (RS)-glucoraphanin, bioactivated with myrosinase enzyme (bioactive RS-GRA), in an experimental mouse model of Parkinson's disease (PD). RS-GRA is one of the most important glucosinolates, a thiosaccharidic compound found in Brassicaceae, notably in Tuscan black kale seeds. RS-GRA was extracted by one-step anion exchange chromatography, further purified by gel-filtration and analyzed by HPLC. Following, pure RS-GRA was characterized by (1)H and (13)C NMR spectrometry and the purity was assayed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method. The obtained purity has been of 99%. To evaluate the possible pharmacological efficacy of bioactive RS-GRA (administrated at the dose of 10mg/kg, ip +5μl/mouse myrosinase enzyme), C57BL/6 mice were used in two different sets of experiment (in order to evaluate the neuroprotective effects in different phases of the disease), according to an acute (2 injections·40mg/kg MPTP) and a sub-acute (5 injections·20mg/kg MPTP) model of PD. Behavioural test, body weight changes measures and immunohistochemical localization of the main PD markers were performed and post-hoc analysis has shown as bioactive RS-GRA is able to reduce dopamine transporter degradation, tyrosine hydroxylase expression, IL-1β release, as well as the triggering of neuronal apoptotic death pathway (data about Bax/Bcl-2 balance and dendrite spines loss) and the generation of radicalic species by oxidative stress (results focused on nitrotyrosine, Nrf2 and GFAP immunolocalization). These effects have been correlated with the release of neurotrophic factors, such as GAP-43, NGF and BDNF, that, probably, play a supporting role in the neuroprotective action of bioactive RS-GRA. Moreover, after PD-induction mice treated with bioactive RS-GRA are appeared more in health than animals that did not received the treatment both for phenotypic behaviour and for general condition

  2. Alteration of Bcl-2, Bcl-x and Bax protein expression following fluid per cussion brain injury in rats%大鼠液压脑损伤后Bcl-2、Bcl-x和Bax蛋白表达的改变

    Institute of Scientific and Technical Information of China (English)

    骆纯; 朱诚; 卢亦成; 江基尧

    2001-01-01

    目的:探讨液压脑损伤后凋亡相 关基因bcl-2、bcl-x和bax在蛋白水平的表达变化规律及神经细胞凋亡的分子生物学机制 。方法:应用免疫组化方法分别检测大鼠中型液压脑损伤后不同时程B cl-2、Bcl-x和Bax蛋白表达情况。结果:伤后6 h,打击侧海马CA 3区Bcl-2和Bcl-x蛋白表达显著下降,Bax的表达无明显变化,(Bcl-2+Bcl-x)/Bax比 率下降主要由于前者下降所致。伤后1~3 d,Bax蛋白表达显著增加,Bcl-2和Bcl-x的表 达下降相对缓慢,(Bcl-2+Bcl-x)/Bax比率同样减小。结论:bc l-2基因家族参与了液压脑损伤后神经细胞凋亡,该基因家族不同成员的表达变化与神经 细胞凋亡有关。%Objective: To investigate the alteration of bcl- 2 gene family in the rat brain and the molecular mechanism of neuronal apoptosis following traumatic brain injury. Methods: Male Sprague -Dawley rats were subjected to lateral fluid percussion brain injury(FPI) of mo derate severity. Bcl-2, Bcl-x and Bax protein expression was detected by immun ohistochemistry. Results: (1) The immunoreactivity of Bcl-2 and Bcl-x protein decreased in the hippocampus ipsilateral impact site as early as 6 h post-injury, and this was the main cause of down-regulation of the ratio of Bcl-2+Bcl-x to Bax. (2) During 1-3 d after injury, the Bax protein express i on increased significantly, while the Bcl-2 and Bcl-x protein expression decre ased relatively slow. The decreased ratio of Bcl-2+Bcl-x to Bax was mainly due to the Bax up-regulation. Conclusion: The bcl-2 gene family is involved in neuronal apoptosis after FBI, and the protein expression alteration of the family members leads the neuronal cell to apoptosis.

  3. Thyroid cancer imaging in vivo by targeting the anti-apoptotic molecule galectin-3.

    Directory of Open Access Journals (Sweden)

    Armando Bartolazzi

    Full Text Available BACKGROUND: The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67% when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules. The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3. METHODS: The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific (99mTc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 microCi of (99mTc-labeled mAb to galectin-3 (30 microg protein/in 100 microl saline solution. Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera. FINDINGS: Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared. CONCLUSIONS: These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better

  4. Targeting the Anti-Apoptotic Protein c-FLIP for Cancer Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Safa, Ahmad R., E-mail: asafa@iupui.edu [Department of Pharmacology and Toxicology, Indiana University School of Medicine, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States); Indiana University Simon Cancer Center, Indiana University School of Medicine, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States); Pollok, Karen E. [Department of Pharmacology and Toxicology, Indiana University School of Medicine, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States); Indiana University Simon Cancer Center, Indiana University School of Medicine, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States); Herman B. Wells Center for Pediatric Research, 980 W. Walnut Street, R3-C524, Indianapolis, IN 46202 (United States)

    2011-03-29

    Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor and critical anti-apoptotic regulator that inhibits tumor necrosis factor-alpha (TNF-alpha), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as well as chemotherapy-triggered apoptosis in malignant cells. c-FLIP is expressed as long (c-FLIP{sub L}), short (c-FLIP{sub S}), and c-FLIP{sub R} splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 in a ligand-dependent and-independent fashion, which in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. Moreover, c-FLIP{sub L} and c-FLIP{sub S} are known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective signaling molecules. Upregulation of c-FLIP has been found in various tumor types, and its downregulation has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. For example, small interfering RNAs (siRNAs) that specifically knockdown the expression of c-FLIP{sub L} in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents, thereby enhancing effector caspase stimulation and apoptosis. Moreover, small molecules causing degradation of c-FLIP as well as decreasing mRNA and protein levels of c-FLIP{sub L} and c-FLIP{sub S} splice variants have been found, and efforts are underway to develop other c-FLIP-targeted cancer therapies. This review focuses on (1) the functional role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and drug resistance; (2) the molecular mechanisms that regulate c-FLIP expression; and (3) strategies to inhibit c-FLIP expression and function.

  5. Onco-miR-24 regulates cell growth and apoptosis by targeting BCL2L11 in gastric cancer

    Directory of Open Access Journals (Sweden)

    Haiyang Zhang

    2016-01-01

    Full Text Available ABSTRACT Gastric cancer is one of the most common malignancies worldwide; however, the molecular mechanism in tumorigenesis still needs exploration. BCL2L11 belongs to the BCL-2 family, and acts as a central regulator of the intrinsic apoptotic cascade and mediates cell apoptosis. Although miRNAs have been reported to be involved in each stage of cancer development, the role of miR-24 in GC has not been reported yet. In the present study, miR-24 was found to be up-regulated while the expression of BCL2L11 was inhibited in tumor tissues of GC. Studies from both in vitro and in vivo shown that miR-24 regulates BCL2L11 expression by directly binding with 3′UTR of mRNA, thus promoting cell growth, migration while inhibiting cell apoptosis. Therefore, miR-24 is a novel onco-miRNA that can be potential drug targets for future clinical use.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  7. Novel anti-apoptotic mechanism of A20 through targeting ASK1 to suppress TNF-induced JNK activation.

    Science.gov (United States)

    Won, M; Park, K A; Byun, H S; Sohn, K-C; Kim, Y-R; Jeon, J; Hong, J H; Park, J; Seok, J H; Kim, J M; Yoon, W-H; Jang, I-S; Shen, H M; Liu, Z G; Hur, G M

    2010-12-01

    The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of nuclear factor-κB (NF-κB) activation and apoptosis in tumor necrosis factor (TNF) receptor 1 signaling pathway. Although the molecular basis for the anti-NF-κB function of A20 has been well elucidated, the anti-apoptotic function of A20 is largely unknown. Here, we report a novel mechanism underlying the anti-apoptotic function of A20: A20 blocks TNF-induced apoptosis through suppression of c-jun N-terminal kinase (JNK) by targeting apoptosis signal-regulating kinase1 (ASK1). First, the ectopic expression of A20 drastically inhibits TNF-induced JNK activation and apoptosis in multiple cell types including those deficient of NF-κB activation. Unexpectedly, the blunting effect of A20 on TNF-induced JNK activation is not mediated by affecting the TNFR1 signaling complex formation. Instead, A20 interacts with ASK1, an important MAPKK kinase in the JNK signaling cascade. More importantly, overexpression of wild-type A20, but not of mutant A20 (ZnF4; C624A, C627A), promotes degradation of the ASK1 through the ubiquitin-proteasome system. Taken together, the results from this study reveal a novel anti-apoptotic mechanism of A20 in TNF signaling pathway: A20 binds to ASK1 and mediates ASK1 degradation, leading to suppression of JNK activation and eventually blockage of apoptosis.

  8. Intrinsic order and disorder in the bcl-2 member harakiri: insights into its proapoptotic activity.

    Directory of Open Access Journals (Sweden)

    Susana Barrera-Vilarmau

    Full Text Available Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-x(L and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual α-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-x(L and Bcl-2. The structural data in water indicate that the α-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric α-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works.

  9. Anti-apoptotic Effects of PCP4/PEP19 in Human Breast Cancer Cell Lines: A Novel Oncotarget

    OpenAIRE

    HAMADA, Taiji; Souda, Masakazu; Yoshimura, Takuya; Sasaguri, Shoko; Hatanaka, Kazuhito; Tasaki, Takashi; Yoshioka, Takako; Ohi, Yasuyo; Yamada, Sohsuke; Tsutsui, Masato; Umekita, Yoshihisa; TANIMOTO, Akihide

    2014-01-01

    The PCP4/PEP19 is a calmodulin-binding anti-apoptotic peptide in neural cells but its potential role in human cancer has largely been unknown. We investigated the expression of PCP4/PEP19 in human breast cancer cell lines MCF-7, SK-BR-3, and MDA-MB-231 cells, and found that estrogen receptor (ER)-positive MCF-7 and ER-negative SK-BR-3 cells expressed PCP4/PEP19. In the MCF-7 cells, cell proliferation was estrogen-dependent, and PCP4/PEP19 expression was induced by estrogen. In both cell lines...

  10. Research Progress of Galectin-3,Bcl-2 and Embryo Development Termination%Galectin-3和Bcl-2与胚胎停育研究进展

    Institute of Scientific and Technical Information of China (English)

    徐耀辉

    2012-01-01

    Galectin-3 is a member of galectin family,which interacts with intracellular glycoprotein,cell surface molecules and extracellular matrix proteins by its carbohydrate recognition domains, participates the progress of embryo implantation,embryogenesis and placenta formation,and establishes and maintains a close relationship with pregnancy. Inhibitors of apoptosis protein Bcl-2 and Galectin-3 have significant sequence similarity,and may have the same apoptosis pathway,which participates in the growth and differentiation of villous trophoblast cells in people's earlier pregnancy and the process of decidualization of endometrium,in addition,playing a critical role in the villous production,growth,placenta formation and in its tissue structure rebuilding and functional perfection.%Galectin-3 是半乳糖凝集素家族中的一员,能通过其糖识别域与细胞内糖蛋白、细胞表面分子和细胞外基质蛋白相互作用,参与胚胎着床、胚胎发生和胎盘形成等过程,与妊娠成功建立和维持密切相关.凋亡抑制蛋白Bcl-2与Galectin-3有明显的序列相似性,可能存在共同细胞凋亡通路,在人早孕过程中参与了绒毛滋养层细胞的增殖和分化、子宫内膜蜕膜化的过程,在绒毛的发生、发育、胎盘形成和组织结构改建及功能完善等方面发挥着重要作用.

  11. Combination Anti-Apoptotic Effect of Erythropoietin and Melatonin on Ischemia Reperfusion-Induced Renal Injury in Rats

    Directory of Open Access Journals (Sweden)

    Shokofeh Banaei

    2016-11-01

    Full Text Available Renal ischemia-reperfusion (IR contributes to the development of acute renal failure (ARF. Oxygen free radicals are considered to be principal components involved in the pathophysiological tissue alterations observed during renal IR. The purpose of this study was to investigate the combination effect of melatonin (MEL and erythropoietin (EPO, which are a potent antioxidant and anti-apoptotic agents, in IR-induced renal injury in rats. Wistar Albino rats were unilaterally nephrectomized and subjected to 45 min of renal pedicle occlusion followed by 24 h reperfusion. MEL (10 mg/kg, i.p and EPO (5000 U/kg, i.p were administered prior to ischemia. After 24 h reperfusion, following decapitation, blood samples were collected for the determination of superoxide dismutase (SOD, glutathione peroxidase (GPx, and malondialdehyde (MDA levels. Also, renal samples were taken for histological evaluation and apoptosis assay. Ischemia-reperfusion increased SOD, GPx, MDA levels, and TUNEL positive cells. Histopathological findings of the IR group confirmed that there was renal impairment in the tubular epithelium. Treatment with EPO and MEL decreased SOD, GPx, and MDA levels, histopathological changes, and TUNEL positive cells. These results indicated that the combination of MEL and EPO could not exert more nephroprotective and anti-apoptotic effects than MEL treatment in renal ischemia-reperfusion injury.

  12. Neuroglobin in Breast Cancer Cells: Effect of Hypoxia and Oxidative Stress on Protein Level, Localization, and Anti-Apoptotic Function

    Science.gov (United States)

    Fiocchetti, Marco; Cipolletti, Manuela; Leone, Stefano; Naldini, Antonella; Carraro, Fabio; Giordano, Daniela; Verde, Cinzia; Ascenzi, Paolo; Marino, Maria

    2016-01-01

    The over-expression of human neuroglobin (NGB), a heme-protein preferentially expressed in the brain, displays anti-apoptotic effects against hypoxic/ischemic and oxidative stresses enhancing neuron survival. As hypoxic and oxidative stress injury frequently occurs in fast proliferating neoplastic tissues, here, the effect of these stressors on the level, localization, and anti-apoptotic function of NGB in wild type and NGB-stable-silenced MCF-7 breast cancer cells has been assessed. The well-known endogenous NGB inducer 17β-estradiol (E2) has been used as positive control. The median pO2 present in tumor microenvironment of breast cancer patients (i.e., 2% O2) does not affect the NGB level in breast cancer cells, whereas hydrogen peroxide and lead(IV) acetate, which increase intracellular reactive oxygen species (ROS) level, enhance the NGB levels outside the mitochondria and still activate apoptosis. However, E2-induced NGB up-regulation in mitochondria completely reverse lead(IV) acetate-induced PARP cleavage. These results indicate that the NGB level could represent a marker of oxidative-stress in MCF-7 breast cancer cells; however, the NGB ability to respond to injuring stimuli by preventing apoptosis requires its re-allocation into the mitochondria. As a whole, present data might lead to a new direction in understanding NGB function in cancer opening new avenues for the therapeutic intervention. PMID:27149623

  13. Effects of Apigenin on the Expressions of Apoptosisrelated Genes Bcl-2/Bax in Cardiomyocyte of Rats with Ischemia and Reperfusion%芹菜素对缺血再灌注大鼠心肌细胞凋亡相关基因Bcl-2/Bax的影响

    Institute of Scientific and Technical Information of China (English)

    史婷婷; 于肯明; 梁月琴; 张明升; 邸涛; 冯璟

    2011-01-01

    Objective To investigate the effects of apigenin on expressions of apoptosis - related genes Bcl - 2/Bax in cardiomyocyte of rats with myocardial ischemia and reperfusion. Methods Sixty - four rats were randomly divided into eight groups: Normal group, sham - operation group, physiological saline ischemia - reperfusion group ( NS group), solvent cont rol group (Sol group), metoprolol positive control group (Meto group), apigenin low, mid and high dose group (Apil, Api2, Api4). The rats were treated for 10 min before ischemia except rats in sham - operation group. The rat models with ischemia - reperfusion were established with 45 min of myocardial ischemia,2 h reperfusion. Then the heart was quickly removed. The expression levels of Bcl - 2 mRNA and Bax mRNA were detected by reverse transcriptase- polymerase chain reaction(RT - PCR). Heart rate (HR) ,mean arterial pressure (MAP) ,and electrocardiogram(ECG) ST segment were recorded and the Lambeth arrhythmia score was calculated. Results Compared with NS group, the expression of Bcl- 2 mRNA was dose- dependently increased,and the expression of Bax mRNA was dose- dependently reduced in apigenin groups(P<0.05). The Lambeth arrhythmia score in apigenin groups was significantly lower than that in control group(P<0. 05). The duration of reperfusion arrhythmia was shorten in apigenin groups(P<0.05). Conclusion Apigenin had a protective effect on rats with myocardial ischemia and reperfusion. The apigenin could up - regulate the expression of Bcl - 2 and downregulate the expression of Bax at transcription level, which could be related to anti- apoptotic effect.%目的 探讨芹菜素对缺血再灌注大鼠心肌细胞凋亡相关基因Bcl-2/Bax表达的影响.方法 心肌缺血45 min,再灌注2 h制备缺血再灌注模型.随机分为正常组、假手术组、生理盐水缺血再灌注组、溶剂对照组、美托洛尔阳性对照组及芹菜素低、中、高剂量组,每组8只.除假手

  14. Magnetic thermoablation stimuli alter BCL2 and FGF-R1 but not HSP70 expression profiles in BT474 breast tumors

    Directory of Open Access Journals (Sweden)

    Stapf M

    2015-03-01

    Full Text Available Marcus Stapf, Nadine Pömpner, Melanie Kettering, Ingrid Hilger Institute of Diagnostic and Interventional Radiology, Jena University Hospital, Jena, Germany Abstract: Magnetically induced heating of magnetic nanoparticles (MNP in an alternating magnetic field (AMF is a promising minimal invasive tool for localized tumor treatment that eradicates tumor cells by applying thermal stress. While temperatures between 42°C and 45°C induce apoptosis and sensitize the cells for chemo- and radiation therapies when applied for at least 30 minutes, temperatures above 50°C, so-called thermoablative temperatures, rapidly induce irreversible cell damage resulting in necrosis. Since only little is known concerning the protein expression of anti-apoptotic B-cell lymphoma 2 (BCL2, fibroblast growth factor receptor 1 (FGF-R1, and heat shock protein (HSP70 after short-time magnetic thermoablative tumor treatment, these relevant tumor proteins were investigated by immunohistochemistry (IHC in a human BT474 breast cancer mouse xenograft model. In the investigated sample groups, the application of thermoablative temperatures (<2 minutes led to a downregulation of BCL2 and FGF-R1 on the protein level while the level of HSP70 remained unchanged. Coincidently, the tumor tissue was damaged by heat, resulting in large apoptotic and necrotic areas in regions with high MNP concentration. Taken together, thermoablative heating induced via magnetic methods can reduce the expression of tumor-related proteins and locally inactivate tumor tissue, leading to a prospectively reduced tumorigenicity of cancerous tissues. The presented data allow a deeper insight into the molecular mechanisms in relation to magnetic thermoablative tumor treatments with the aim of further improvements. Keywords: magnetic nanoparticles (MNP, thermoablation, in vivo, mouse model, breast cancer tumor

  15. Bim/Bcl-2 balance is critical for maintaining naive and memory T cell homeostasis

    Science.gov (United States)

    Wojciechowski, Sara; Tripathi, Pulak; Bourdeau, Tristan; Acero, Luis; Grimes, H. Leighton; Katz, Jonathan D.; Finkelman, Fred D.; Hildeman, David A.

    2007-01-01

    We examined the role of the antiapoptotic molecule Bcl-2 in combating the proapoptotic molecule Bim in control of naive and memory T cell homeostasis using Bcl-2−/− mice that were additionally deficient in one or both alleles of Bim. Naive T cells were significantly decreased in Bim+/−Bcl-2−/− mice, but were largely restored in Bim−/−Bcl-2−/− mice. Similarly, a synthetic Bcl-2 inhibitor killed wild-type, but not Bim−/−, T cells. Further, T cells from Bim+/−Bcl-2−/− mice died rapidly ex vivo and were refractory to cytokine-driven survival in vitro. In vivo, naive CD8+ T cells required Bcl-2 to combat Bim to maintain peripheral survival, whereas naive CD4+ T cells did not. In contrast, Bim+/−Bcl-2−/− mice generated relatively normal numbers of memory T cells after lymphocytic choriomeningitis virus infection. Accumulation of memory T cells in Bim+/−Bcl-2−/− mice was likely caused by their increased proliferative renewal because of the lymphopenic environment of the mice. Collectively, these data demonstrate a critical role for a balance between Bim and Bcl-2 in controlling homeostasis of naive and memory T cells. PMID:17591857

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  17. The apoptotic members CD95, BclxL, and Bcl-2 cooperate to promote cell migration by inducing Ca2+ flux from the endoplasmic reticulum to mitochondria

    Science.gov (United States)

    Fouqué, A; Lepvrier, E; Debure, L; Gouriou, Y; Malleter, M; Delcroix, V; Ovize, M; Ducret, T; Li, C; Hammadi, M; Vacher, P; Legembre, P

    2016-01-01

    Metalloprotease-processed CD95L (cl-CD95L) is a soluble cytokine that implements a PI3K/Ca2+ signaling pathway in triple-negative breast cancer (TNBC) cells. Accordingly, high levels of cl-CD95L in TNBC women correlate with poor prognosis, and administration of this ligand in an orthotopic xenograft mouse model accelerates the metastatic dissemination of TNBC cells. The molecular mechanism underlying CD95-mediated cell migration remains unknown. Here, we present genetic and pharmacologic evidence that the anti-apoptotic molecules BclxL and Bcl-2 and the pro-apoptotic factors BAD and BID cooperate to promote migration of TNBC cells stimulated with cl-CD95L. BclxL was distributed in both endoplasmic reticulum (ER) and mitochondrion membranes. The mitochondrion-localized isoform promoted cell migration by interacting with voltage-dependent anion channel 1 to orchestrate Ca2+ transfer from the ER to mitochondria in a BH3-dependent manner. Mitochondrial Ca2+ uniporter contributed to this flux, which favored ATP production and cell migration. In conclusion, this study reveals a novel molecular mechanism controlled by BclxL to promote cancer cell migration and supports the use of BH3 mimetics as therapeutic options not only to kill tumor cells but also to prevent metastatic dissemination in TNBCs. PMID:27367565

  18. Lycopene modulates cholinergic dysfunction, Bcl-2/Bax balance, and antioxidant enzymes gene transcripts in monosodium glutamate (E621) induced neurotoxicity in a rat model.

    Science.gov (United States)

    Sadek, Kadry; Abouzed, Tarek; Nasr, Sherif

    2016-04-01

    The effect of monosodium glutamate (MSG) on brain tissue and the relative ability of lycopene to avert these neurotoxic effects were investigated. Thirty-two male Wistar rats were distributed into 4 groups: group I, untreated (placebo); group II, injected with MSG (5 mg·kg(-1)) s.c.; group III, gastrogavaged with lycopene (10 mg·kg(-1)) p.o.; and group IV received MSG with lycopene with the same mentioned doses for 30 days. The results showed that MSG induced elevation in lipid peroxidation marker and perturbation in the antioxidant homeostasis and increased the levels of brain and serum cholinesterase (ChE), total creatine phosphokinase (CPK), creatine phosphokinase isoenzymes BB (CPK-BB), and lactate dehydrogenase (LDH). Glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT) activities and gene expression were increased and glutathione content was reduced in the MSG-challenged rats, and these effects were ameliorated by lycopene. Furthermore, MSG induced apoptosis in brain tissues reflected in upregulation of pro-apoptotic Bax while lycopene upregulated the anti-apoptotic Bcl-2. Our results indicate that lycopene appears to be highly effective in relieving the toxic effects of MSG by inhibiting lipid peroxidation and inducing modifications in the activity of cholinesterase and antioxidant pathways. Interestingly, lycopene protects brain tissue by inhibiting apoptosis signaling induced by MSG.

  19. Comparision between indirect immunofluorescence assay and in situ hybridization assay in detecting the expression of Bcl-2 and Bcl-2/Bax in acute leukemia%间接免疫荧光法和原位杂交法检测急性白血病中Bcl-2Bcl-2/Bax表达的对比研究

    Institute of Scientific and Technical Information of China (English)

    陈瑢; 李玲; 温丙昭

    2005-01-01

    目的:比较两种不同方法检测急性白血病(AL)患者Bcl-2Bcl-2/Bax表达情况.方法:采用链亲和素-胶体金原位杂交法(ISH-SAG)和间接免疫荧光法对57例AL患者的Bcl-2Bcl-2/Bax进行检测.结果:(1) ISH-SAG法中,完全缓解(CR)组Bcl-2细胞表达率高于间接免疫荧光法,且差异有统计学意义(P<0.05).(2) Bcl-2Bcl-2/Bax检测均显示ISH-SAG法的特异度、准确度、阳性预测值、阴性预测值、阳性似然比、阴性似然比优于间接免疫荧光法;Bcl-2/Bax检测也显示ISH-SAG法较间接免疫荧光法高.(3)ISH-SAG法中Bcl-2细胞表达率和积分值的阳性检出率无差别.结论:(1) ISH-SAG法测定Bcl-2Bcl-2/Bax优于间接免疫荧光法.(2)ISH-SAG法中Bcl-2积分值临床应用价值不大.

  20. The Anti-Apoptotic and Cardioprotective Effects of Salvianolic Acid A on Rat Cardiomyocytes following Ischemia/Reperfusion by DUSP-Mediated Regulation of the ERK1/2/JNK Pathway

    Science.gov (United States)

    Chen, Qiuping; Zhu, Shasha; Liu, Yang; Pan, Defeng; Chen, Xiaohu; Li, Dongye

    2014-01-01

    The purpose of this study was to observe the effects of salvianolic acid A (SAA) pretreatment on the myocardium during ischemia/reperfusion (I/R) and to illuminate the interrelationships among dual specificity protein phosphatase (DUSP) 2/4/16, ERK1/2 and JNK pathways during myocardial I/R, with the ultimate goal of elucidating how SAA exerts cardioprotection against I/R injury (IRI). Wistar rats were divided into the following six groups: control group (CON), I/R group, SAA+I/R group, ERK1/2 inhibitor PD098059+I/R group (PD+I/R), PD+SAA+I/R group, and JNK inhibitor SP600125+I/R group (SP+I/R). The cardioprotective effects of SAA on the myocardium during I/R were investigated with a Langendorff device. Heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum rate of ventricular pressure rise and fall (±dp/dtmax), myocardial infarction areas (MIA), lactate dehydrogenase (LDH), and cardiomyocytes apoptosis were monitored. To determine the crosstalk betwee JNK and ERK1/2 via DUSP2/4/16 with SAA pretreatment, siRNA-DUSP2/4/16 were performed. The expression levels of Bcl-2, Bax, caspase 3, p-JNK, p-ERK1/2 and DUSP2/4/16 in cardiomyocytes were assayed by Western blot. Our results showed that LDH, MIA and cell apoptosis were decreased, and various parameters of heart function were improved by SAA pretreatment and SP application. In the I/R group, the expression levels of p-ERK1/2 and DUSP4/16 were not significantly different compared with the CON group, however, the protein expression levels of p-ERK1/2, Bcl-2 and DUSP4/16 were higher, while p-JNK, Bax, caspase 3 and DUSP2 levels were reduced among the SAA+I/R, PD+SAA+I/R and SP+I/R groups. The above indices were not significantly different between the SAA+I/R and SP+I/R groups. Compared with the SAA+I/R group, p-ERK1/2 was increased and p-JNK was decreased in the SAA+si-DUSP2+I/R, however, p-ERK was downregulated and p-JNK was upregulated in SAA+si-DUSP4+I

  1. The anti-apoptotic and cardioprotective effects of salvianolic acid a on rat cardiomyocytes following ischemia/reperfusion by DUSP-mediated regulation of the ERK1/2/JNK pathway.

    Directory of Open Access Journals (Sweden)

    Tongda Xu

    Full Text Available The purpose of this study was to observe the effects of salvianolic acid A (SAA pretreatment on the myocardium during ischemia/reperfusion (I/R and to illuminate the interrelationships among dual specificity protein phosphatase (DUSP 2/4/16, ERK1/2 and JNK pathways during myocardial I/R, with the ultimate goal of elucidating how SAA exerts cardioprotection against I/R injury (IRI. Wistar rats were divided into the following six groups: control group (CON, I/R group, SAA+I/R group, ERK1/2 inhibitor PD098059+I/R group (PD+I/R, PD+SAA+I/R group, and JNK inhibitor SP600125+I/R group (SP+I/R. The cardioprotective effects of SAA on the myocardium during I/R were investigated with a Langendorff device. Heart rate (HR, left ventricular systolic pressure (LVSP, left ventricular end-diastolic pressure (LVEDP, maximum rate of ventricular pressure rise and fall (±dp/dtmax, myocardial infarction areas (MIA, lactate dehydrogenase (LDH, and cardiomyocytes apoptosis were monitored. To determine the crosstalk betwee JNK and ERK1/2 via DUSP2/4/16 with SAA pretreatment, siRNA-DUSP2/4/16 were performed. The expression levels of Bcl-2, Bax, caspase 3, p-JNK, p-ERK1/2 and DUSP2/4/16 in cardiomyocytes were assayed by Western blot. Our results showed that LDH, MIA and cell apoptosis were decreased, and various parameters of heart function were improved by SAA pretreatment and SP application. In the I/R group, the expression levels of p-ERK1/2 and DUSP4/16 were not significantly different compared with the CON group, however, the protein expression levels of p-ERK1/2, Bcl-2 and DUSP4/16 were higher, while p-JNK, Bax, caspase 3 and DUSP2 levels were reduced among the SAA+I/R, PD+SAA+I/R and SP+I/R groups. The above indices were not significantly different between the SAA+I/R and SP+I/R groups. Compared with the SAA+I/R group, p-ERK1/2 was increased and p-JNK was decreased in the SAA+si-DUSP2+I/R, however, p-ERK was downregulated and p-JNK was upregulated in SAA+si-DUSP4

  2. Anti-apoptotic effects of recombinant human granulocyte colony-stimulating factor in focal cerebral ischemic rats

    Institute of Scientific and Technical Information of China (English)

    Xia Yuan; Shiming Zhang; Wanli Dong; Qi Fang

    2011-01-01

    The neuroprotective effects of granulocyte colony-stimulating factor in cerebral ischemia/reperfusion injury are currently contentious. The present study examined the effects of subcutaneous injection of recombinant human granulocyte colony-stimulating factor (50 μg/kg) over 5 days in a model of cerebral ischemia/reperfusion with intraluminal filament occlusion in rats. The results indicated that recombinant human granulocyte colony-stimulating factor reduced brain infarct volume following cerebral ischemia/reperfusion injury in rats, down-regulated the expression of caspase-3 mRNA (a key protease for apoptosis in the cerebral ischemia zone), lowered the rate of neuronal apoptosis in the cerebral ischemia zone, and notably ameliorated neurological function. These results indicate that recombinant human granulocyte colony-stimulating factor has anti-apoptotic effects on neurons following focal cerebral ischemia/reperfusion injury, and exerts neuroprotective effects.

  3. Cytotoxicity of calotropin is through caspase activation and downregulation of anti-apoptotic proteins in K562 cells.

    Science.gov (United States)

    Wang, Shih-Chung; Lu, Mei-Chin; Chen, Hsiu-Lin; Tseng, Hsing-I; Ke, Yu-Yuan; Wu, Yang-Chang; Yang, Pei-Yu

    2009-12-01

    Calotropin is one of cardenolides isolated from milkweed used for medicinal purposes in many Asian countries. Whereas calotropin possesses cytotoxicity against several cancer cells, the mechanisms of action remain unclear. We set out to evaluate the cytotoxic mechanism of calotropin on human chronic myeloid leukemia K562 cells. Calotropin inhibited the growth of K562 cells in a time- and dose-dependent manner by G(2)/M phase arrest. It upregulated the expression of p27 leading to this arrest by downregulating the G2/M regulatory proteins, cyclins A and B, and by upregulating the cdk inhibitor, p27. Furthermore, it downregulated anti-apoptotic signaling (XIAP and survivin) and survival pathways (p-Akt and NFkappaB), leading to caspase-3 activation which resulted in the induction of apoptosis. In all, calotropin exerted its anticancer activity on K562 cells by modulating the pro-survival signaling that leads to induction of apoptosis.

  4. Icariin Attenuates OGD/R-Induced Autophagy via Bcl-2-Dependent Cross Talk between Apoptosis and Autophagy in PC12 Cells

    Science.gov (United States)

    2016-01-01

    Icariin (ICA), an active component of Epimedium brevicornum Maxim, exerts a variety of neuroprotective effects such as antiapoptosis. However, the mechanisms underlying antiapoptosis of ICA in neurons exposed to oxygen-glucose deprivation and reperfusion (OGD/R) are unclear. The B-cell lymphoma-2 (Bcl-2) protein family plays an important role in the regulation of apoptosis and autophagy through Bcl-2-dependent cross talk. Bcl-2 suppresses apoptosis by binding to Bax and inhibits autophagy by binding to Beclin-1 which is an autophagy related protein. In the present study, MTT result showed that ICA increased cell viability significantly in OGD/R treated PC12 cells (P < 0.01). Results of western blotting analysis showed that ICA increased Bcl-2 expression significantly and decreased expressions of Bax, cleaved Caspase-3, Beclin-1, and LC3-II significantly in OGD/R treated PC12 cells (P < 0.01). These results suggest that ICA protects PC12 cells from OGD/R induced autophagy via Bcl-2-dependent cross talk between apoptosis and autophagy. PMID:27610184

  5. Down-Regulation of Bcl-2 Protein Sensitizes NCI 460 Cells to Radiotherapy-Induced Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Dongmei He; Yuan Zhang; Gexiu Liu

    2006-01-01

    OBJECTIVE To determine whether Bcl-2 protein down-regulation can render NCI-460 cells more susceptible to gamma radiation-induced apoptosis by treatment with antisense oligonucleotide (ASODN) against the coding region of Bcl-2 mRNA.METHODS Cell survival was determined using the trypan blue dye exclusion. Expression of the Bcl-2 protein was assayed using immunofluorescence labeling with fluoresce isothiocyanate. Apoptosis was determined by Giemsa staining and flow cytomertry.RESULTS It was found that Bcl-2 ASODN combined with radiation significantly reduced the number of viable cells (P<0.05). There was no difference in cell survival between a nonsense oligodeoxynucleotide/radiation combination and cells treated with radiation alone. Bcl-2 ASODN combined with radiation significantly inhibited expression of the Bcl-2protein in the NCI-H460 cells (P<0.05). Using Giemsa staining, cells treated with Bcl-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptotic rates of the NCI-H460 cells treated with Bcl-2 ASODN combined with radiation significantly increased (P<0.05), compared with either a nonsense oligodeoxynucleotide/radiation combination or radiation-treatment cells alone.CONCLUSION ASODN against the coding region of Bcl-2 mRNA increases radiation-induced apoptosis in NCI-H460 cells.

  6. Expressions of bcl-2 and P53 protein in Bowen's disease%Bowen病bcl-2及P53蛋白的表达

    Institute of Scientific and Technical Information of China (English)

    张士发; 王良明; 赵丽萍; 许静; 顾绍裘

    2004-01-01

    目的:探讨bcl-2及P53蛋白在Bowen病及Bowen样鳞癌中的表达及其意义.方法:应用免疫组织化学技术对11例Bowen病及3例Bowen样鳞癌bcl-2和/或P53蛋白的表达进行了检测.结果:11例Bowen病中bcl-2蛋白阳性2例(18%),P53蛋白阳性3例(27%);3例Bowen样鳞癌均见bcl-2蛋白表达.Bowen病中bcl-2与P53蛋白表达显著正相关(r=0.769,P<0.05).结论:Bowen病中bcl-2蛋白表达与P53基因突变有关,并参与了Bowen病的进展及向Bowen样鳞癌的演变.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  8. Introduction of the anti-apoptotic baculovirus p35 gene in passion fruit induces herbicide tolerance, reduced bacterial lesions, but does not inhibits passion fruit woodiness disease progress induced by cowpea aphid-borne mosaic virus (CABMV

    NARCIS (Netherlands)

    Freitas, D.S.; Coelho, M.C.F.; Souza, M.T.; Marques, A.; Ribeiro, B.M.

    2007-01-01

    The introduction of anti-apoptotic genes into plants leads to resistance to environmental stress and broad-spectrum disease resistance. The anti-apoptotic gene (p35) from a baculovirus was introduced into the genome of passion fruit plants by biobalistics. Eleven regenerated plants showed the presen

  9. Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint-flanking probes

    NARCIS (Netherlands)

    Vaandrager, J W; Schuuring, E; Raap, T; Philippo, K; Kleiverda, K; Kluin, P

    2000-01-01

    Rearrangement of the BCL2 gene is an important parameter for the differential diagnosis of non-Hodgkin lymphomas. Although a relatively large proportion of breakpoints is clustered, many are missed by standard PCR. A FISH assay is therefore desired. Up to now, a lack of probes flanking the BCL2 gene

  10. Apoptosis Mediated by HIV Protease is Preceded by Cleavage of Bcl-2

    Science.gov (United States)

    Strack, Peter R.; West Frey, Michelle; Rizzo, Christopher J.; Cordova, Beverly; George, Henry J.; Meade, Raymond; Ho, Siew Peng; Corman, Jeanne; Tritch, Radonna; Korant, Bruce D.

    1996-09-01

    Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor α . We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NFkappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.

  11. The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells.

    Science.gov (United States)

    Soundararajan, Sridharan; Chen, Weiwei; Spicer, Eleanor K; Courtenay-Luck, Nigel; Fernandes, Daniel J

    2008-04-01

    We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 micromol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 micromol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 micromol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death.

  12. Bcl-2 expression significantly correlates with thymidylate synthase expression in colorectal cancer patients

    Institute of Scientific and Technical Information of China (English)

    Riyad Bendardaf; Raija Ristamaki; Kari Syrjanen; Seppo Pyrhonen

    2008-01-01

    AIM: To examine the expression of thymidylate synthase (TS) and oncoprotein Bcl-2 in advanced colorectal cancer (CRC) patients, and to determine their mutual relationship, association to therapeutic response and impact on disease outcome. METHODS: Tumor samples from 67 patients with CRC, who were treated at advanced stage with either irinotecan alone or in combination with 5-fluorouracil/ leucovorin, were analyzed for expression of TS and BCl-2 using immunohistochemistry. RESULTS: A significant linear correlation between lower expression levels of Bcl-2 and lower levels of TS expression was found (P=0.033). Patients with high levels of both TS and Bcl-2 expression had a significantly longer disease-free survival (DFS) (42.6 mo vs 5.4 mo, n=25) than those with low TS/Bcl-2 index (P=0.001). Tumors with low levels of both TS and Bcl-2 were associated with a longer survival with metastasis (WMS) interval in the whole patients group (η = 67, P = 0.035). TS/Bcl-2 index was not significantly related to disease-specific survival. CONCLUSION: The present data suggest that CRC patients with low TS/Bcl-2 demonstrate a significantly shorter DFS and longer WMS. 2008 The WJG Press. All dghts reserved,Key words: Thymidylate synthase, Bcl-2; Colorectal cancer; Disease-free survival; Survival with metastaseis Peer reviewers: Shu Zheng, Professor, Scientific Director of Cancer Institute, Zhejiang University, Secondary AffiliatedHospital, Zhejiang University, 88# Jiefang Road, Hangzhou 310009, Zhejiang Province, China; Lars A Pahlman, Professor,Department of Surgery, Colorcctal Unit, University Hospital,SE 751 85, Uppsala, Sweden; Damian Casadesus, MD, PhD,Calixto Garcia University Hospital, J and University, Vedado,Havana City, Cuba Bendardaf R, Ristamaki R, Syrjanen K, Pyrhonen S. Bcl-2 expression significantly correlates with thymidylate synthase expression in coiorectal cancer patients. World J Gastroenterol.

  13. Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models

    Directory of Open Access Journals (Sweden)

    Christian Lehmann

    2016-06-01

    Full Text Available Abstract Background Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML. In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. Methods The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. Results Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that

  14. Synergistic antitumoral activity and induction of apoptosis by novel pan Bcl-2 proteins inhibitor apogossypolone with adriamycin in human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jin-xia MI; Guang-feng WANG; Heng-bang WANG; Xiao-qing SUN; Xin-yan NI; Xiong-wen ZHANG; Jia-ming TANG; Da-jun YANG

    2008-01-01

    Aim: To investigate the in vitro and in vivo activities and related mechanism of apogossypoione (ApoG2) alone or in combination with adriamycin (ADM) against human hepatocellular carcinoma (HCC). Methods: The IC50 of ApoG2 in vitro was tested by WST assay, and the synergistic effect was analyzed using the CalcuSyn method. Cell apoptosis was determined using 4',6-diamidino-2-phenylindole staining and flow cytometric analysis. Western blotting was used to determine the expression of apoptosis-related proteins. In vivo activity was evaluated in the xenograft model in nude mice, and apoptosis in tumor tissues was determined by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay. Results: The IC50 of ApoG2 in HCC cells was 17.28-30.63 μmol/L. When ApoG2 was combined with ADM, in-creased cytotoxicity and apoptosis were observed in SMMC-7721 cells compared to treatment with ApoG2 alone. The Western blotting results indicated that the ApoG2 induced apoptosis in SMMC-7721 cells by downregulating anti-apoptotic proteins Bcl-2, Mcl-1, and Bcl-XL, up-regulating pro-apoptotic protein Noxa, and promoting the activities of caspases-9 and -3. The tumor growth of xenograft SMMC-7721 was inhibited in nude mice when ApoG2 was administered orally without causing damage to the normal tissues. The in vivo study also indicated an increasing anti-tumoral effect when ApoG2 at 100 or 200 mg/kg dosages were used together with ADM at 5.5 mg/kg, with relative tumor proliferation rate (T/C) values of 0.456 and 0.323, respectively. Apoptosis induced in vivo by ApoG2 alone or combined with ADM was confirmed by TUNEL assay in tumor tissues. Conclusion: ApoG2 is a potential non-toxic target agent that induces apoptosis by upregulating Noxa, while inhibiting anti-apoptotic proteins and pro-moting the effect of chemotherapy agent ADM in HCC.

  15. The BH3 α-Helical Mimic BH3-M6 Disrupts Bcl-XL, Bcl-2, and MCL-1 Protein-Protein Interactions with Bax, Bak, Bad, or Bim and Induces Apoptosis in a Bax- and Bim-dependent Manner*

    Science.gov (United States)

    Kazi, Aslamuzzaman; Sun, Jiazhi; Doi, Kenichiro; Sung, Shen-Shu; Takahashi, Yoshinori; Yin, Hang; Rodriguez, Johanna M.; Becerril, Jorge; Berndt, Norbert; Hamilton, Andrew D.; Wang, Hong-Gang; Sebti, Saïd M.

    2011-01-01

    A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-XL and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-XL, Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-XL/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-XL, Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612. PMID:21148306

  16. Anti-apoptotic effects of PCP4/PEP19 in human breast cancer cell lines: a novel oncotarget.

    Science.gov (United States)

    Hamada, Taiji; Souda, Masakazu; Yoshimura, Takuya; Sasaguri, Shoko; Hatanaka, Kazuhito; Tasaki, Takashi; Yoshioka, Takako; Ohi, Yasuyo; Yamada, Sohsuke; Tsutsui, Masato; Umekita, Yoshihisa; Tanimoto, Akihide

    2014-08-15

    The PCP4/PEP19 is a calmodulin-binding anti-apoptotic peptide in neural cells but its potential role in human cancer has largely been unknown. We investigated the expression of PCP4/PEP19 in human breast cancer cell lines MCF-7, SK-BR-3, and MDA-MB-231 cells, and found that estrogen receptor (ER)-positive MCF-7 and ER-negative SK-BR-3 cells expressed PCP4/PEP19. In the MCF-7 cells, cell proliferation was estrogen-dependent, and PCP4/PEP19 expression was induced by estrogen. In both cell lines, PCP4/PEP19 knockdown induced apoptosis and slightly decreased Akt phosphorylation. Knockdown of calcium/calmodulin-dependent protein kinase kinase 1 (CaMKK1), resulting in decreased phospho-Akt(Thr308), enhanced apoptosis in SK-BR-3 but not in MCF-7 cells. CaMKK2 knockdown moderately decreased phospho-Akt(Thr308) and increased apoptosis in MCF-7 cells but not in SK-BR-3 cells. These data indicated that PCP4/PEP19 regulates apoptosis but exact mechanism is still unknown. PCP4/PEP19 can therefore potentially serve as independent oncotarget for therapy of PCP4/PEP19-positive breast cancers irrespective of ER expression.

  17. Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

    Science.gov (United States)

    Fuchs, Christiane; Rosner, Margit; Dolznig, Helmut; Mikula, Mario; Kramer, Nina; Hengstschläger, Markus

    2012-03-01

    Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

  18. Metformin combined with sodium dichloroacetate promotes B leukemic cell death by suppressing anti-apoptotic protein Mcl-1.

    Science.gov (United States)

    Voltan, Rebecca; Rimondi, Erika; Melloni, Elisabetta; Gilli, Paola; Bertolasi, Valerio; Casciano, Fabio; Rigolin, Gian Matteo; Zauli, Giorgio; Secchiero, Paola

    2016-04-05

    Metformin and the mitochondrial targeting dichloroacetate (DCA) have recently received attention due to their ability to inhibit anaerobic glycolysis, which renders most cancer cells resistant to apoptosis induction. We observed that Metformin alone exhibited a dose-dependent anti-leukemic activity in both B leukemic cell lines and primary B-chronic lymphocytic leukemia (B-CLL) patients' cells and its anti-leukemic activity was enhanced when used in combination with DCA. In order to overcome the problems of poor bioavailability and cellular uptake, which limit DCA efficacy, we have designed and synthetized cocrystals consisting of Metformin and DCA (Met-DCA) at different stoichiometric ratios. Of note, the MetH(2)(++)•2DCA(-) cocrystal exhibited enhanced in vitro anti-leukemic activity, with respect to the treatment with the mix consisting of Metformin plus DCA. In particular, the treatment with the cocrystal MetH(2)(++)•2DCA(-) induced a synergistic apoptotic cell death coupled to a marked down-modulation of the anti-apoptotic Mcl-1 protein. Taken together, our data emphasize that innovative compounds based on Metformin-DCA combination merit to be further evaluated as chemotherapeutic agents for the treatment of B-CLL.

  19. Neuroprotective and Anti-Apoptotic Effects of CSP-1103 in Primary Cortical Neurons Exposed to Oxygen and Glucose Deprivation

    Science.gov (United States)

    Porrini, Vanessa; Sarnico, Ilenia; Benarese, Marina; Branca, Caterina; Mota, Mariana; Lanzillotta, Annamaria; Bellucci, Arianna; Parrella, Edoardo; Faggi, Lara; Spano, Pierfranco; Imbimbo, Bruno Pietro; Pizzi, Marina

    2017-01-01

    CSP-1103 (formerly CHF5074) has been shown to reverse memory impairment and reduce amyloid plaque as well as inflammatory microglia activation in preclinical models of Alzheimer’s disease. Moreover, it was found to improve cognition and reduce brain inflammation in patients with mild cognitive impairment. Recent evidence suggests that CSP-1103 acts through a single molecular target, the amyloid precursor protein intracellular domain (AICD), a transcriptional regulator implicated in inflammation and apoptosis. We here tested the possible anti-apoptotic and neuroprotective activity of CSP-1103 in a cell-based model of post-ischemic injury, wherein the primary mouse cortical neurons were exposed to oxygen-glucose deprivation (OGD). When added after OGD, CSP-1103 prevented the apoptosis cascade by reducing cytochrome c release and caspase-3 activation and the secondary necrosis. Additionally, CSP-1103 limited earlier activation of p38 and nuclear factor κB (NF-κB) pathways. These results demonstrate that CSP-1103 is neuroprotective in a model of post-ischemic brain injury and provide further mechanistic insights as regards its ability to reduce apoptosis and potential production of pro-inflammatory cytokines. In conclusion, these findings suggest a potential use of CSP-1103 for the treatment of brain ischemia. PMID:28106772

  20. Significant reduction of fungal disease symptoms in transgenic lupin (Lupinus angustifolius) expressing the anti-apoptotic baculovirus gene p35.

    Science.gov (United States)

    Wijayanto, Teguh; Barker, Susan J; Wylie, Stephen J; Gilchrist, David G; Cowling, Wallace A

    2009-10-01

    Narrow-leafed lupin (NLL; Lupinus angustifolius) is a recently domesticated but anciently propagated crop with significant value in rotation with cereals in Mediterranean climates. However, several fungal pathogens, traditionally termed necrotrophs, severely affect broad-acre production and there is limited genetic resistance in the NLL germplasm pool. Symptoms of many of these diseases appear as localized areas of dead cells exhibiting markers of programmed cell death. Based on our previous research, we hypothesized that engineered expression of the baculovirus anti-apoptotic p35 gene might reduce symptoms of these diseases. Using Agrobacterium tumefaciens-mediated transformation of a cultivar highly susceptible to several pathogens, 14 independent NLL lines containing both the p35 and bar genes were obtained (p35-NLL). Integration and expression of the transgenes were confirmed by polymerase chain reaction (PCR), progeny testing, Southern blot, Northern blot and reverse transcriptase-PCR analyses. Fecundity and nodulation were not altered in these lines. Third or fourth generation p35-NLL lines were challenged with necrotrophic fungal pathogens (anthracnose in stem and leaf, and Pleiochaeta root rot and leaf brown spot) in controlled environment conditions. Several p35-NLL lines had significantly reduced disease symptoms. Interestingly, as with natural resistance, no single line was improved for all three diseases which possibly reflecting spatial variation of p35 expression in planta. These data support an alternative molecular definition for 'necrotrophic disease' in plants and suggest new routes for achieving resistance against a range of pathogens.

  1. The orphan adapter protein SLY1 as a novel anti-apoptotic protein required for thymocyte development

    Directory of Open Access Journals (Sweden)

    Beer-Hammer Sandra

    2009-07-01

    Full Text Available Abstract Background SH3 containing Lymphocyte Protein (SLY1 is a putative adapter protein exclusively expressed in lymphocytes which is involved in antigen receptor induced activation. We previously have generated SLY1Δ/Δ mice harbouring a partial deletion in the N-terminal region of SLY1 which revealed profound immunological defects in T and B cell functions. Results In this study, T cell development in SLY1-/- and SLY1Δ/Δ mice was analysed ex vivo and upon cultivation with the bone marrow stromal cell line OP9. SLY1-deficient thymocytes were compromised in inducing nutrient receptor expression and ribosomal protein S6 phosphorylation, indicating a defect in mTOR complex activation. Furthermore, SLY1 was identified as a novel anti-apoptotic protein required for developmental progression of T cell precursors to the CD4+CD8+ double-positive stage by protecting from premature programmed cell death initiation in developing CD4-CD8- double-negative thymocytes. In addition, SLY1 phosphorylation was differentially regulated upon Notch ligand-mediated stimulation and expression of the preTCR. Conclusion Thus, our results suggest a non-redundant role for SLY1 in integrating signals from both receptors in early T cell progenitors in the thymus.

  2. Piezometric biosensors for anti-apoptotic protein survivin based on buried positive-potential barrier and immobilized monoclonal antibodies.

    Science.gov (United States)

    Stobiecka, Magdalena; Chalupa, Agata; Dworakowska, Beata

    2016-10-15

    The anti-apoptotic protein survivin (Sur) plays an important role in the regulation of cell division and inducing the chemotherapeutic drug resistance. The Sur protein and its mRNA have recently been studied as cancer biomarkers and potential targets for cancer therapy. In this work, we have focused on the design of immunosensors for the detection of Sur based on buried positive-potential barrier layer structure and anti-survivin antibody. The modification of solid AuQC piezoelectrodes was monitored by recording the resonance frequency shift and electrochemical measurements during each step of the sensor preparation. Our results indicate that the immunosensor with covalently bound monoclonal anti-survivin antibody can detect Sur with the limit of detection, LOD=1.7nM (S/N=3σ). The immunosensor applicability for the analysis of real samples was assessed by testing samples of cell lysate solutions obtained from human astrocytoma (glioblastoma) U-87MG cell line, with the experiments performed using the standard addition method. The good linearity of the calibration curves for PBS and lysate solutions at low Sur concentrations confirm the high specificity of the proposed biosensor and good discrimination against nonspecific interactions with lysate components. The calculations indicate that there is still room to increase the Sur capture capacity for Sur while miniaturizing the sensor. The important advantage of the sensor is that it can be reused by a simple regeneration procedure.

  3. 听神经瘤BCL-2蛋白及bcl-2/JH融合基因的研究☆%Detection of BCL-2 protooncogene protein expression and the related bcl-2(mbr)/JH fusion gene from archival paraffin-embedded tissue from acoustic neuromas.

    Institute of Scientific and Technical Information of China (English)

    刘绍明; 李龄; 刘鹏翀

    2001-01-01

    目的评价石蜡包埋听神经瘤组织中BGL-2蛋白表达及相关的bcl-2(mbr)/JH融合基因改变,以探讨bcl-2癌基因在听神经瘤发病中的可能意义.方法免疫组化检测石蜡包埋组织中BCL-2蛋白的表达;提取石蜡包埋组织的DNA,PCR检测bcl-2(mbr)/JH融合基因.结果本组40例听神经瘤,BCL-2蛋白表达阳性27例(67.5%),bcl-2(mbr)/JH融合基因检出阳性19例(47.5%).结论听神经瘤中存在BCL-2蛋白的高表达及t(14;18)染色体易位,提示雪旺氏细胞凋亡抑制可能是听神经瘤发病的分子病理基础之一.

  4. Expression of Bcl-2 in adult human brain regions with special reference to neurodegenerative disorders.

    Science.gov (United States)

    Vyas, S; Javoy-Agid, F; Herrero, M T; Strada, O; Boissiere, F; Hibner, U; Agid, Y

    1997-07-01

    The expression of the protooncogene bcl-2, an inhibitor of apoptosis in various cells, was examined in the adult human brain. Several experimental criteria were used to verify its presence; mRNA was analyzed by northern blot with parallel experiments in mouse tissues, by RNase protection, and by in situ hybridization histochemistry. Bcl-2 protein was detected by western blot analysis and immunohistochemistry. Two bcl-2 mRNA species were identified in the human brain. The pattern of distribution of bcl-2 mRNA at the cellular level showed labeling in neurons but not glia. The in situ hybridization signal was stronger in the pyramidal neurons of the cerebral cortex and in the cholinergic neurons of the nucleus basalis of Meynert than in the Purkinje neurons of the cerebellum. Both melanized and nonmelanized neurons were labeled in the substantia nigra. In the striatum, bcl-2 mRNA was detected in some but not all neurons. In the regions examined for Bcl-2 protein, the expression pattern correlated with the mRNA results. In patients with Alzheimer's and Parkinson's diseases, quantification of bcl-2 mRNA in the nucleus basalis of Meynert and substantia nigra, respectively, showed that the expression was unaltered compared with controls, raising the possibility that the expression of other components of apoptosis is modulated.

  5. A plant Bcl-2-associated athanogene is proteolytically activated to confer fungal resistance

    Directory of Open Access Journals (Sweden)

    Mehdi Kabbage

    2016-04-01

    Full Text Available The Bcl-2-associated athanogene (BAG family is a multifunctional group of proteins involved in numerous cellular functions ranging from apoptosis to tumorigenesis. These proteins are evolutionarily conserved and encode a characteristic region known as the BAG domain. BAGs function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in tumor growth, HIV infection, and neurodegenerative diseases; as a result, the BAGs are attractive targets for therapeutic interventions, and their expression in cells may serve as a predictive tool for disease development. The Arabidopsis genome contains seven homologs of BAG family proteins (Figure 1, including four with a domain organization similar to animal BAGs (BAG1-4. The remaining three members (BAG5-7 contain a predicted calmodulin-binding motif near the BAG domain, a feature unique to plant BAG proteins that possibly reflects divergent mechanisms associated with plant-specific functions. As reported for animal BAGs, plant BAGs also regulate several stress and developmental processes (Figure 2. The recent article by Li et al. focuses on the role of BAG6 in plant innate immunity. This study shows that BAG6 plays a key role in basal plant defense against fungal pathogens. Importantly, this work further shows that BAG6 is proteolytically activated to induce autophagic cell death and resistance in plants. This finding underscores the importance of proteases in the execution of plant cell death, yet little is known about proteases and their substrates in plants.

  6. The Influence of Matrine on Apoptosis and Expression of Bax and Bci-2 in Colorectal Cancer Cells%苦参碱对大肠癌细胞凋亡及Bax、Bcl-2表达的影响

    Institute of Scientific and Technical Information of China (English)

    王雷; 刘明

    2012-01-01

    [Purpose] To investigate the effect of Matrine on proliferation inhibition, apoptotic and Bax and Bcl-2 expression in human colorectal cancer cell line Lovo. [Methods] Lovo cells cultured in vitro were interfered with 0.05-1.6mg/ml different concentration of Matrine. The proliferation inhibition effect on Lovo cells was observed by MTT method. Apoptosis induction effect on Lovo cells was detected by DNA ladder, flow cytometer and TUNEL staining. The expression of Bcl-2 and Bax proteins correlated with apoptosis were detected by Western Blot assay. [Results] After being exposed to Matrine (0.05-1.6mg/ml) for 24 and 48h, the proliferation of Lovo cells was inhibited in a dose-time dependent manner. DNA ladder, Annexin V-PI method and TUNEL staining showed Matrine was obviously increased along with Matrine concentration increased. The expression of pro-apoptotic protein Bax was increased, while anti-apoptotic protein Bcl-2 was decreased as Matrine doses increased. [Conclusion] Matrine can inhibit proliferation and induction of apoptosis in colorectal cancer cells. Increased expression of Bax and decreased expression of Bcl-2 might involve in Matrine-induced apoptosis.%[目的]探讨苦参碱对人大肠癌Lovo细胞增殖抑制和凋亡诱导作用及其对Bax、Bcl-2表达的影响.[方法] 0.05~l.6mg/ml不同浓度苦参碱作用Lovo细胞,采用MTT法检测苦参碱对大肠癌Lovo细胞增殖抑制作用,DNA ladder、AnnexinV -PI法及TUNEL染色检测细胞凋亡,Western Blot法检测凋亡相关蛋白Bax、Bcl-2表达的变化.[结果]0.05~1.6mg/ml苦参碱处理Lovo细胞24h或48h后,细胞增殖均明显受抑制;DNA ladder、Annexin V-PI法及TUNEL染色检测结果显示苦参碱呈时间、剂量依赖性诱导细胞凋亡;促凋亡蛋白Bax随着苦参碱剂量增加表达增加,抗凋亡蛋白Bcl-2随着苦参碱剂量增加表达减少.[结论]苦参碱具有抑制大肠癌细胞增殖,诱导其凋亡的作用.苦参碱诱导大肠癌细胞凋

  7. SMI of Bcl-2 TW-37 is active across a spectrum of B-cell tumors irrespective of their proliferative and differentiation status.

    Science.gov (United States)

    Al-Katib, Ayad M; Sun, Yuan; Goustin, Anton Scott; Azmi, Asfar Sohail; Chen, Ben; Aboukameel, Amro; Mohammad, Ramzi M

    2009-02-16

    The Bcl-2 family of proteins is critical to the life and death of malignant B-lymphocytes. Interfering with their activity using small-molecule inhibitors (SMI) is being explored as a new therapeutic strategy for treating B-cell tumors. We evaluated the efficacy of TW-37, a non-peptidic SMI of Bcl-2 against a range spectrum of human B-cell lines, fresh patient samples and animal xenograft models. Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models. Nanomolar concentrations of TW-37 were able to induce apoptosis in both fresh samples and established cell lines with IC50 in most cases of 165-320 nM. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg x 3 days) via tail vein. TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy regardless of the stage of B-cell differentiation.

  8. No dramatic age-related loss of hair cells and spiral ganglion neurons in Bcl-2 over-expression mice or Bax null mice

    Directory of Open Access Journals (Sweden)

    Ohlemiller Kevin K

    2010-07-01

    Full Text Available Abstract Age-related decline of neuronal function is associated with age-related structural changes. In the central nervous system, age-related decline of cognitive performance is thought to be caused by synaptic loss instead of neuronal loss. However, in the cochlea, age-related loss of hair cells and spiral ganglion neurons (SGNs is consistently observed in a variety of species, including humans. Since age-related loss of these cells is a major contributing factor to presbycusis, it is important to study possible molecular mechanisms underlying this age-related cell death. Previous studies suggested that apoptotic pathways were involved in age-related loss of hair cells and SGNs. In the present study, we examined the role of Bcl-2 gene in age-related hearing loss. In one transgenic mouse line over-expressing human Bcl-2, there were no significant differences between transgenic mice and wild type littermate controls in their hearing thresholds during aging. Histological analysis of the hair cells and SGNs showed no significant conservation of these cells in transgenic animals compared to the wild type controls during aging. These data suggest that Bcl-2 overexpression has no significant effect on age-related loss of hair cells and SGNs. We also found no delay of age-related hearing loss in mice lacking Bax gene. These findings suggest that age-related hearing loss is not through an apoptotic pathway involving key members of Bcl-2 family.

  9. Combined Targeting of JAK2 and Bcl-2/Bcl-xL to Cure Mutant JAK2-Driven Malignancies and Overcome Acquired Resistance to JAK2 Inhibitors

    Directory of Open Access Journals (Sweden)

    Michaela Waibel

    2013-11-01

    Full Text Available To design rational therapies for JAK2-driven hematological malignancies, we functionally dissected the key survival pathways downstream of hyperactive JAK2. In tumors driven by mutant JAK2, Stat1, Stat3, Stat5, and the Pi3k and Mek/Erk pathways were constitutively active, and gene expression profiling of TEL-JAK2 T-ALL cells revealed the upregulation of prosurvival Bcl-2 family genes. Combining the Bcl-2/Bcl-xL inhibitor ABT-737 with JAK2 inhibitors mediated prolonged disease regressions and cures in mice bearing primary human and mouse JAK2 mutant tumors. Moreover, combined targeting of JAK2 and Bcl-2/Bcl-xL was able to circumvent and overcome acquired resistance to single-agent JAK2 inhibitor treatment. Thus, inhibiting the oncogenic JAK2 signaling network at two nodal points, at the initiating stage (JAK2 and the effector stage (Bcl-2/Bcl-xL, is highly effective and provides a clearly superior therapeutic benefit than targeting just one node. Therefore, we have defined a potentially curative treatment for hematological malignancies expressing constitutively active JAK2.

  10. A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes.

    Directory of Open Access Journals (Sweden)

    Moritz Eißmann

    Full Text Available Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.

  11. A PRELIMINARY STUDY ON SURVIVIN AND BCL-2 EXPRESSION IN CERVICAL CARCINOMAS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To study the expression of a novel inhibitor of apptosis and survivin in cervical carcinoma and its relationship to the expression of Bcl-2.Methods Using SP immunohistochemical technique, we examined the expression of survivin and Bcl-2 in 59 cervical invasive squamous cell carcinomas.Results Survivin was expressed in 41 of 59 cases(69.5%) of cervical carcinomas. In contrast, no expression of survivin in normal cervical tissues was observed. Overexpression of survivin was related to the tumor grade and clinical stage. Survivin positive cases were strongly associated with Bcl-2 expression(80% versus 35.7%;P<0.005).Conclusion Apoptosis inhibition by survivin abnormal expression, alone or in cooperation with Bcl-2, may participate in the onset and progression of cervical carcinoma. Survivin is a new diagnostic/therapeutic target in cervical cancer.

  12. Expression of Survivin in pancreatic cancer and its correlation to expression of Bcl-2

    Institute of Scientific and Technical Information of China (English)

    Jian-Guo Qiao; Yu-Qing Zhang; Yu-Chun Yin; Zui Tan

    2004-01-01

    AIM: To investigate the expression of Survivin in pancreatic cancer and its correlation to the expression of Bcl-2.METHODS: Survivin and Bcl-2 expressions were examined by immunohistochemistry in 42 tissue samples from pancreatic cancer and 10 from normal pancrease. RESULTS: No survivin expression was detected in the tissue samples from normal pancrease, while it was detected in 34 of 42 tissue samples from pancreatic cancer (81.95%).There was a correlation between survivin expression and differentiation and stages of pancreatic cancer. Survivin positive cases were strongly correlated to Bcl-2 expression (28/30 vs 6/12, P<0.05).CONCLUSION: Overexpression of survivin plays an important role in the development and progression of pancreatic cancer, and correlates to the expression of Bcl-2. Survivin expression can be used as a prognostic factor.

  13. Identification of an HLA-A*0201 restricted Bcl2-derived epitope expressed on tumors

    DEFF Research Database (Denmark)

    Wang, Mingjun; Johansen, Britta; Nissen, Mogens H

    2006-01-01

    A large number of human tumor-associated antigen-derived peptides have been identified that are recognized by CTLs in a MHC-I restricted fashion. The apoptosis inhibitory protein Bcl2 is overexpressed in many human cancers as part of their neoplastic phenotype. Since inhibition or loss of Bcl2...... expression might impair tumor growth and survival, this protein may serve as a rational target for vaccine-induced CTL responses. By Western blot technique, we screened a panel of established human tumor cell lines for proteins involved in the apoptotic process. Two of eight tumor cell lines, a B lymphoma...... (Loukes) and a colon carcinoma (CCL220) cell line showed increased Bcl2 protein expression whereas the majority of tumor cell lines expressed proapoptotic proteins. Neither fibroblasts nor peripheral blood mononuclear cells showed Bcl2 expression. An HLA-A*0201 restricted CTL epitope was deduced in silica...

  14. Increased expression of the anti-apoptotic protein Bcl-xL in the brain is associated with resilience to stress-induced depression-like behavior.

    Science.gov (United States)

    Dygalo, Nikolay N; Kalinina, Tatyana S; Bulygina, Veta V; Shishkina, Galina T

    2012-07-01

    Clinical observations and the results of animal studies have implicated changes in neuronal survival and plasticity in both the etiology of mood disorders, especially stress-induced depression, and anti-depressant drug action. Stress may predispose individuals toward depression through down-regulation of neurogenesis and an increase in apoptosis in the brain. Substantial individual differences in vulnerability to stress are evident in humans and were found in experimental animals. Recent studies revealed an association between the brain anti-apoptotic protein B cell lymphoma like X, long variant (Bcl-xL) expression and individual differences in behavioral vulnerability to stress. The ability to increase Bcl-xL gene expression in the hippocampus in response to stress may be an important factor for determining the resistance to the development of stress-induced depression. Treatment with anti-depressant drugs may change Bcl-xL response properties. In the rat brainstem, expression of this anti-apoptotic gene becomes sensitive to swim stress during the long-term fluoxetine treatment, an effect that appeared concomitantly with the anti-depressant-like action of the drug in the forced swim test, suggesting that Bcl-xL may be a new target for depression therapy. The processes and pathways linking stress stimuli to behavior via intracellular anti-apoptotic protein are discussed here in the context of Bcl-xL functions in the mechanisms of individual differences in behavioral resilience to stress and anti-depressant-induced effects on the behavioral despair.

  15. Bcl-2 promotes malignant progression in a PDGF-B-dependent murine model of oligodendroglioma.

    Science.gov (United States)

    Doucette, Tiffany; Yang, Yuhui; Zhang, Wei; Fuller, Gregory N; Suki, Dima; Fults, Daniel W; Rao, Ganesh

    2011-11-01

    A significant subset of gliomas arises after activation of the proproliferative platelet-derived growth factor (PDGF) pathway. The progression of low-grade gliomas to more malignant tumors may be due to oncogenic cellular programs combining with those suppressing apoptosis. Antiapoptotic genes are overexpressed in a variety of cancers, and the antiapoptotic gene, BCL2, is associated with treatment resistance and tumor recurrence in gliomas. However, the impact of antiapoptotic gene expression to tumor formation and progression is unclear. We overexpressed Bcl-2 in a PDGFB-dependent mouse model of oligodendroglioma, a common glioma subtype, to assess its effect in vivo. We hypothesized that the antiapoptotic effect would complement the proproliferative effect of PDGFB to promote tumor formation and progression to anaplastic oligodendroglioma (AO). Here, we show that coexpression of PDGFB and Bcl-2 results in a higher overall tumor formation rate compared to PDGFB alone. Coexpression of PDGFB and Bcl-2 promotes progression to AO with prominent foci of necrosis, a feature of high-grade gliomas. Median tumor latency was shorter in mice injected with PDGFB and Bcl-2 compared to those injected with PDGFB alone. Although independent expression of Bcl-2 was insufficient to induce tumors, suppression of apoptosis (detected by cleaved caspase-3 expression) was more pronounced in AOs induced by PDGFB and Bcl-2 compared to those induced by PDGFB alone. Tumor cell proliferation (detected by phosphohistone H3 activity) was also more robust in high-grade tumors induced by PDGFB and Bcl-2. Our results indicate that suppressed apoptosis enhances oligodendroglioma formation and engenders a more malignant phenotype.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  17. The anti-apoptotic factor Che-1/AATF links transcriptional regulation, cell cycle control, and DNA damage response

    Directory of Open Access Journals (Sweden)

    Fanciulli Maurizio

    2007-07-01

    Full Text Available Abstract Che-1 is a RNA polymerase II binding protein involved in the transcriptional regulation of E2F target genes and in cell proliferation. Recently, it has been shown that Che-1 accumulates in cells responding to genotoxic agents such as Doxorubicin and ionizing radiation. The DNA damage-activated checkpoint kinases ATM and Chk2 interact with and phosphorylate Che-1, enhancing its accumulation and stability, and promoting Che-1-mediated transcription of p53-responsive genes and of p53 itself, as evidenced by microarray analysis. This transcriptional response is suppressed by expression of a Che-1 mutant lacking ATM and Chk2 phosphorylation amino acid residues, or by depletion of Che-1 by RNA silencing. In addition, chromatin immunoprecipitation analysis has shown that Che-1 is released from E2F target genes and recruited to the p21 and p53 promoters after DNA damage. Che-1 contributes to the maintenance of the G2/M checkpoint in response to genotoxic stress. These findings identify a new mechanism by which the checkpoint kinases regulate, via the novel effector Che-1, the p53 pathway. Lastly, increasing evidence suggests that Che-1 may be involved in apoptotic signaling in neural tissues. In cortical neurons, Che-1 exhibits anti-apoptotic activity, protecting cells from neuronal damage induced by amyloid β-peptide. In cerebellar granule neurons, Che-1 interacts with Tau in the cytoplasmic compartment and this interaction is modulated during neuronal apoptosis. Finally, Che-1 directly interacts with the neuronal cell-death inducer "NRAGE" which downregulates endogenous Che-1 by targeting it for proteasome-dependent degradation. These findings identify Che-1 as a novel cytoprotective factor against apoptotic insults and suggest that Che-1 may represent a potential target for therapeutic application.

  18. Methanolic extract of Origanum vulgare ameliorates type 1 diabetes through antioxidant, anti-inflammatory and anti-apoptotic activity.

    Science.gov (United States)

    Vujicic, Milica; Nikolic, Ivana; Kontogianni, Vassiliki G; Saksida, Tamara; Charisiadis, Pantelis; Orescanin-Dusic, Zorana; Blagojevic, Dusko; Stosic-Grujicic, Stanislava; Tzakos, Andreas G; Stojanovic, Ivana

    2015-03-14

    Type 1 diabetes (T1D), an autoimmune inflammatory disorder, develops as a consequence of pancreatic β-cell destruction and results in hyperglycaemia. Since current T1D therapy mainly involves insulin replacement, the aim of the present study was to evaluate the therapeutic potential of Origanum vulgare L. ssp. hirtum (Greek oregano) leaf extract rich in biophenols for the treatment of T1D. The phytochemical profile of methanolic oregano extract (MOE) and aqueous oregano extract (AOE) was determined by liquid chromatography/electrospray ion-trap tandem MS (LC/DAD/ESI-MSn), while their main compounds were quantified by HPLC with diode array detection. After establishing their potent in vitro antioxidant activity, the extracts were administered to C57BL/6 mice treated with multiple low doses of streptozotocin for diabetes induction. While prophylactic AOE therapy had no impact on diabetes induction, MOE reduced diabetes incidence and preserved normal insulin secretion. In addition, MOE scavenged reactive oxygen and nitrogen species and, therefore, alleviated the need for the up-regulation of antioxidant enzymes. MOE treatment specifically attenuated the pro-inflammatory response mediated by T helper 17 cells and enhanced anti-inflammatory T helper 2 and T regulatory cells through the impact on specific signalling pathways and transcription factors. Importantly, MOE preserved β-cells from in vitro apoptosis via blockade of caspase 3. Finally, rosmarinic acid, a predominant compound in MOE, exhibited only partial protection from diabetes induction. In conclusion, acting as an antioxidant, immunomodulator and in an anti-apoptotic manner, MOE protected mice from diabetes development. Seemingly, there is more than one compound responsible for the beneficial effect of MOE.

  19. Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Woo Jin Jeong

    Full Text Available In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO can alter the function of the basement membrane of retinal pigment epithelial (RPE cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.

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

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jun; YAO Zhen-xiang; ZHANG Jing

    1999-01-01

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

  1. c-Fos enhances the survival of thymocytes during positive selection by upregulating Bcl-2

    Institute of Scientific and Technical Information of China (English)

    Xiaoming Wang; Yafeng Zhang; Gang Xiao; Xiang Gao; Xiaolong Liu

    2009-01-01

    T cells are derived from progenitor thymocytes, of which only a minority receive the appropriate TCR signal, undergo positive selection and mature. Owing to the very short lifespan of thymocytes, the prerequisite for posi-tive selection is survival. TCR signal-induced Bcl-2 expression is believed to play a dominant role in the survival of positively selecting thymocytes, but how Bcl-2 is directly regulated is unknown. Here we report that the immediate early gene (lEG) c-Fos can stimulate the expression of Bcl-2, depending on a specific AP-l-binding site in the Bcl-2 promoter. In c-Fos transgenic (Fos-Tg) mice, c-Fos binds to this site and promotes the expression of Bcl-2. As a result, Fos-Tg thymocytes exhibited enhanced survival, and more mature single-positive (SP) thymocytes were generated, even on a unique TCR background. The TCR repertoire remained normal in Fos-Tg mice. Our results identified e-Fos as the mediator of the stimulatory effect of TCR signaling on Bcl-2 expression. Therefore, c-Fos, as an IEG, because of its early response ability, can quickly rescue the survival of short-lived thymocytes during positive selection. Our results provide novel insight into the mechanism regulating the survival of positively selecting thymocytes.

  2. Expression of P16 protein and Bcl-2 protein in malignant eyelid tumors

    Institute of Scientific and Technical Information of China (English)

    牛膺筠; 周占宇; 刘夫玲; 王红云

    2002-01-01

    Objective To investigate the relationship between P16 gene (the tumor suppressor gene) and the bcl-2 gene (the apoptosis inhibitor gene) and the incidence and development of malignant eyelid tumors. Methods The streptavidin-biotin-peroxidase complex immunohistochemistry method was used to study the expression of P16 gene and the bcl-2 gene in 96 cases of malignant eyelid tumors. Results Among the 96 cases, there were 40 basal cell carcinomas (BCCs), 33 squamous carcinomas and 23 sebaceous carcinoma, with P16 protein positive (nuclear staining) rates 70%, 54.6% and 56.5%, respectively. The P16 positive rate was negatively correlated with the degree of tumor histological differentiation, and the rate difference between the high differentiated carcinomas was significant (P<0.05). Positive Bcl-2 protein expression was detected in the cytoplasm. All 40 BCC cases were Bcl-2 positive, and nearly all of the tumor cells showed positive cytoplasmic expression, while in the 33 squamous cell carcinoma cases only one showed positive focal reaction, and the staining in the other 32 cases was relatively faint. None of the 23 sebaceous carcinomas expressed Bcl-2. Conclusions The expression of the P16 protein was related to the occurrence and degree of differentiation of malignant eyelid tumors. The overexpression of the Bcl-2 protein suggests that suppression of apoptosis might play a role in the tumorigenesis of BCC.

  3. Studies of Liposomal bcl-2 Antisense Oligode-oxynucleofide Induction of Apoptosis in Raji Cells

    Institute of Scientific and Technical Information of China (English)

    DongmeiHe; HuanZhong

    2004-01-01

    OBJECTIVE To explore the effect of liposomal G3139 and transfected antisense phosphorothioate oligodeoxynucleotides directed against the coding region of the bcl-2 messenger RNA and the translation site on apoptosis in Raji cells.METHODS Cytotoxic effects were measured by use of the MTT method; The expression levels of Bcl-2 protein were assayed by immunofiuorescence using a fluoresce isothiocyanate label. Apoptosis was determined by morphological observation and flow cytometric analysis.RESULTS The 2 antisense oligonucleotides and G3139 can reduce Bcl-2 protein levels and Raji cell viability (IC50=4.54, 4.72 and 4.26 μmol/L, respectively), and induce apoptosis. A scrambled sequence control oligonucleotide and empty liposomes did not alter cell viability, Bcl-2 protein expression or apoptosis rates. There was no difference in reducing Bcl-2 protein levels and apoptosis rates found among the 3 antisense oligonucleotides.CONCLUSION The 2 antisense oligodeoxynucleotides of bcl-2 messenger RNA can effectively induce apoptosis of Raji cells. The 2 antisense sequences and G3139 have a similarity in their antisense effect.

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  5. Oxidative stress-mediated down-regulation of bcl-2 promoter in hippocampal neurons.

    Science.gov (United States)

    Pugazhenthi, Subbiah; Nesterova, Albina; Jambal, Purevsuren; Audesirk, Gerald; Kern, Marcey; Cabell, Leigh; Eves, Eva; Rosner, Marsha R; Boxer, Linda M; Reusch, Jane E-B

    2003-03-01

    Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.

  6. Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression

    Directory of Open Access Journals (Sweden)

    Braithwaite Antony W

    2011-06-01

    Full Text Available Abstract Background The aim of both classical (e.g. taxol and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs, which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown. Results We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells. Conclusion Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.

  7. THZ1 targeting CDK7 suppresses STAT transcriptional activity and sensitizes T-cell lymphomas to BCL2 inhibitors

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    Cayrol, Florencia; Praditsuktavorn, Pannee; Fernando, Tharu M.; Kwiatkowski, Nicholas; Marullo, Rosella; Calvo-Vidal, M. Nieves; Phillip, Jude; Pera, Benet; Yang, Shao Ning; Takpradit, Kaipol; Roman, Lidia; Gaudiano, Marcello; Crescenzo, Ramona; Ruan, Jia; Inghirami, Giorgio; Zhang, Tinghu; Cremaschi, Graciela; Gray, Nathanael S.; Cerchietti, Leandro

    2017-01-01

    Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL biology represents an urgent need. Here we report that PTCL are sensitive to transcription-targeting drugs, and, in particular, to THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7). The STAT-signalling pathway is highly vulnerable to THZ1 even in PTCL cells that carry the activating STAT3 mutation Y640F. In mutant cells, CDK7 inhibition decreases STAT3 chromatin binding and expression of highly transcribed target genes like MYC, PIM1, MCL1, CD30, IL2RA, CDC25A and IL4R. In surviving cells, THZ1 decreases the expression of STAT-regulated anti-apoptotic BH3 family members MCL1 and BCL-XL sensitizing PTCL cells to BH3 mimetic drugs. Accordingly, the combination of THZ1 and the BH3 mimetic obatoclax improves lymphoma growth control in a primary PTCL ex vivo culture and in two STAT3-mutant PTCL xenografts, delineating a potential targeted agent-based therapeutic option for these patients. PMID:28134252

  8. Apoptosis of HL-60 human leukemia cells induced by Asiatic acid through modulation of B-cell lymphoma 2 family proteins and the mitogen-activated protein kinase signaling pathway.

    Science.gov (United States)

    Wu, Qiuling; Lv, Tingting; Chen, Yan; Wen, Lu; Zhang, Junli; Jiang, Xudong; Liu, Fang

    2015-07-01

    The toxicities of conventional chemotherapeutic agents to normal cells restrict their dosage and clinical efficacy in acute leukemia; therefore, it is important to develop novel chemotherapeutics, including natural products, which selectively target cancer-specific pathways. The present study aimed to explore the effect of the chemopreventive agent asiatic acid (AA) on the proliferation and apoptotic rate of the leukemia cell line HL-60 and investigated the mechanisms underlying its anti-tumor activity. The effect of AA on the proliferation of HL-60 cells was evaluated using the MTT assay. Annexin V-fluorescein isothiocyanate/propidium iodide double staining followed by flow cytometric analysis as well as Hoechst 33258 staining were used to analyze the apoptotic rate of the cells. Furthermore, changes of survivin, B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 expressions were detected by western blot analysis. AA blocked the growth of HL-60 cells in a dose- and time-dependent manner. The IC50-value of AA on HL-60 cells was 46.67 ± 5.08 µmol/l for 24 h. AA induced apoptosis in a dose-dependent manner, which was inhibited in the presence of Z-DEVD-FMK, a specific inhibitor of caspase. The anti-apoptotic proteins Bcl-2, Mcl-1 and survivin were downregulated by AA in a dose-dependent manner. Concurrently, AA inhibited ERK and p38 phosphorylation in a dose-dependent manner, while JNK phosphorylation was not affected. In conclusion, the present study indicated that the p38 and ERK pathways, as well as modulation of Bcl-2 family and survivin proteins were key regulators of apoptosis induced in HL-60 cells in response to AA.

  9. The Significance and Correlation of SODD and Bcl-2 Protein Expression in Acute Leukemia of Children

    Institute of Scientific and Technical Information of China (English)

    Hongfang Tao; Qun Hu; Liuqing Zhang; Aiguo Liu; Shuangyou Liu; Ying Hu

    2006-01-01

    OBJECTIVE To explore the expression of SODD and bcl-2 proteins in bone marrow cells of children with acute leukemia (AL), and to examine the relationship of their expression with the classification, clinical features,therapeutic effect and prognosis for AL patients.METHODS Using the SABC immunohistochemical staining method, the expression of SODD and bcl-2 proteins in the bone marrow cells of 86 AL cases was determined. The patients were studied based on the following groups: 1) a first-visiting group; 2) a refractory-relapse group(some patients were sensitive to therapy but then suffered a recurrence);3) a complete-remission group (CR); 4) a high risk (HR) and 5) standard risk (SR) group; 6) a control group of patients with non-hematological diseases.RESULTS The positive rates of SODD and bcl-2 expression in the firstvisit, refractory-relapse and CR groups were significantly higher (P<0.05)compared to the control group. There was no significant difference in the expression of SODD or bcl-2 proteins between an acute lymphoblastic leukemia (ALL) group and acute nonlymphoblastic leukemia (ANLL)group (t=1.874, t=1.583, P>0.05). The positive rates of SODD and bcl-2expression in the patients who developed complete remission after chemotherapy were significantly lower (t=2.054, t=2.703, P<0.05) compared to the first-visit pediatric patients. The expression of the SODD protein in the refractory-relapse group was notably higher compared to the group treated initially (t=-1.081, P<0.05). A high expression of the bcl-2 protein was found in both the first-visit and refractory-relapse groups, with no significant difference found between the two groups (t=-1.196, P>0.05), whereas the percentage of bcl-2 positive cells in the refractory-relapse group (45%~87%) was significantly higher compared to the first-visit group (5%~62%). The positive expression of the SODD and bcl-2 proteins in the high-risk (HR) group were both significantly higher than the SR group (t=-3

  10. Prognostic significance of bcl-2 and p53 expression in colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    ZHAO Dan-ping; DING Xiao-wen; PENG Jia-ping; ZHENG Yi-xiong; ZHANG Su-zhan

    2005-01-01

    Objective: This study was designed to detect the expression ofbcl-2 and p53 proteins in colorectal carcinomas and to determine their association with the patient survival and stage of the diseases. Methods: Immunohistochemistry method was used to detect the expression ofbcl-2 and p53 proteins in 93 cases of colorectal carcinoma. The stain results were obtained by analyzing the clinic-pathological characteristics of patients. Results: Fifty-seven percent (53/93) of the colorectal carcinomas were bcl-2 protein positive. The positive rate of bcl-2 protein in lymph node involvement cases was lower (15/37) than the cases without node involvement (38/58, P<0.01). The positive rate of p53 protein was 43% (40/93) in colon-rectum carcinomas. No significant correlation was observed between p53 protein expression and clinic-pathological manifestations (P>0.05) but the survival was significantly worse (P=0.0001) in the p53 protein positive cases. Neither bcl-2 nor p53 alone was correlated with stage of the disease. When combined bcl-2/p53 status was analyzed, a group with bcl-2(+) and p53(-) had the best prognosis. This group was significantly associated with earlier Dukes' stages (P=0.1763). In multivariate Cox regression analysis, lymph node involvement and p53 protein expression were two independent factors correlated with survival time. Conclusion: The expression of bcl-2 and p53 represent biological characteristics of colorectal carcinomas. Assessment of both bcl-2 and p53 status may be valuable in predicting the prognosis of patients.

  11. Immunohistochemical expression of Bcl-2 in oral epithelial dysplasia and oral squamous cell carcinoma

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    S Juneja

    2015-01-01

    Full Text Available BACKGROUND: The B cell lymphoma-2 gene is a proto-oncogene whose protein product inhibits apoptosis. Its role is associated with keeping cells alive, but not by stimulating them to proliferation, as other proto-oncogenes do. Increased expression of protein product of Bcl-2 gene appears in the early phase of carcinogenesis leading to apoptosis impairment and in consequence to the progression of neoplastic changes. OBJECTIVE: To evaluate and compare the expression of Bcl-2 protein in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC. MATERIALS AND METHODS: Sixty cases of formalin-fixed paraffin-embedded archival specimens comprising of 30 cases of leukoplakia with oral epithelial dysplasia and 30 cases of OSCC were taken for immunohistochemical analysis using monoclonal antibody against anti-human Bcl-2 oncoprotein. RESULTS: Immunostaining for Bcl-2 protein was identified in basal and parabasal layers as granular cytoplasmic staining in oral epithelial dysplasia. In OSCC, Bcl-2 immunoreactivity was most prominent in the peripheral cells of the infiltrating tumor islands which diminished toward the center in well-differentiated and moderately differentiated OSCC, whereas stronger and more diffuse expression of Bcl-2 oncoprotein was seen in poorly differentiated OSCC. Overall positivity of 26.7% (8/30 was observed in oral epithelial dysplasia and 30% (9/30 in OSCC in this study. INTERPRETATION AND CONCLUSION: Altered expression of Bcl-2 oncoprotein may be an early molecular event which leads to prolonged cell survival, increased chances of accumulation of genetic alterations, and subsequent increase in malignant transformation potential.

  12. Anti-apoptotic effect of morphine-induced delayed preconditioning on pulmonary artery endothelial cells with anoxia/reoxygenation injury

    Institute of Scientific and Technical Information of China (English)

    DING Weng-ang; ZHOU Hua-cheng; CUI Xiao-guang; LI Wen-zhi; GUO Yue-ping; ZHANG Bing; LIU Wei

    2008-01-01

    Background Opioid preconditioning (PC) reduces anoxiaJreoxygenation (NR) injury to various cells. However, it remains unclear whether opioid-induced delayed PC would show anti-apoptotic effects on pulmonary artery endothelial cells (PAECs) suffering from A/R injury. The present study was conducted to elucidate this issue and to investigate the potential mechanism of opioid-induced delayed PC.Methods Cultured porcine PAECs underwent 16-hour anoxia followed by 1-hour reoxygenation 24 hours after pretreatment with saline (NaCI; 0.9%) or morphine (1 μmol/L). To determine the underlying mechanism, a non-selective KATP channel inhibitor glibenclamide (Glib; 10 μmol/L), a nitric oxide (NO) synthase blocker NG-nitro-L-arginine methyl ester (L-NAME; 100 μmol/L), and an opioid receptor antagonist naloxone (Nal; 10 pmol/L) were given 30 minutes before the A/R load. The percentage of apoptotic cells was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, eNOS mRNA level was measured by real-time polymerase chain reaction (PCR). NO content of PAECs supernatants was measured with the Griess reagent.Results Compared to the A/R PAECs, morphine-induced delayed PC significantly reduced PAECs apoptosis ((18.1±1.9)% vs (5.5±0.3)%; P <0.05), increased NO release ((11.4±1.3) μmol/L vs (20.5±2.1) μmol/L, P <0.05), and up-regulated eNOS gene expression nearly 9 times (P<0.05). The anti-apoptosis effect of morphine was abolished by pretreatment with Glib, L-NAME and Nal, but the three agent-selves did not aggravate the A/R injury. Furthermore, L-NAME and Nal offset the enhanced release of NO caused by pretreatment with morphine.Conclusions Morphine-induced delayed PC prevents A/R injury of PAECs. This effect may be mediated by activation of KATP channel via opioid receptor and NO signaling pathways.

  13. Thymosin beta 4 prevents oxidative stress by targeting antioxidant and anti-apoptotic genes in cardiac fibroblasts.

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    Sandeep Kumar

    Full Text Available RATIONALE: Thymosin beta-4 (Tβ4 is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress. METHODS: Rat neonatal fibroblasts cells were exposed to hydrogen peroxide (H(2O(2 in presence and absence of Tβ4 and expression of antioxidant, apoptotic and pro-fibrotic genes was evaluated by quantitative real-time PCR and western blotting. Reactive oxygen species (ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant and antiapoptotic genes were silenced by siRNA transfections in cardiac fibroblasts and the effect of Tβ4 on H(2O(2-induced profibrotic events was evaluated. RESULTS: Pre-treatment with Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in the cardiac fibroblasts. This was associated with an increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD and catalase and reduction of Bax/Bcl(2 ratio. Tβ4 treatment reduced the expression of pro-fibrotic genes [connective tissue growth factor (CTGF, collagen type-1 (Col-I and collagen type-3 (Col-III] in the cardiac fibroblasts. Silencing of Cu/Zn-SOD and catalase gene triggered apoptotic cell death in the cardiac fibroblasts, which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that exhibits the targeted molecules modulated by Tβ4 under oxidative stress utilizing the cardiac fibroblasts. Tβ4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that Tβ4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl(2, thereby, preventing H(2O(2-induced profibrotic changes in the myocardium. Further studies are

  14. Effect of Bcl-2 and caspase-3 on calcium distribution in apoptosis of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there's a change of intracellular calcium distribution, moving from cytoplast especially Golgi's apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi's apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi's apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspas-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor.

  15. THE OVEREXPRESSION OF APOPTOSIS -RELATED GENES OF P53 AND BCL-2 IN CERVICAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate the significance of overexpression of P53 and bcl-2 protein in carcinogenesis of cervix. Methods 10 cases of cervical intraepithelial neoplasis(CIN) and 57 cases of invasive cancer were investigated with immunohistochemistry technique. Results The overexpresion of P53 protein in CIN and cervical cancer was significantly higher than that of control, respectively (P<0.01). But there was no significant difference between CIN and cervical cancer(P>0. 05). The immunoreactivity of bcl-2 in CIN was much more higher than that of control (P<0.05). The positive rate and immunoreactivity of bcl-2 in cervical carcinoma were both remarkably higher than those of control (P<0. 01) ,but there was no significant difference between CIN and cervical carcinoma (P>0. 05). It was also found that there was a remarkably positive correlation between the overexpression of bcl-2 and P53 (P<0.01). Conclusion Because of the loss of wtP53 function,the expression of bcl-2 can not be down-reguated,which is associated with the pathogenesis and development of cervical carcinoma.

  16. Increase of bcl-2 Protein Expression in Aggressive Basal Cell Carcinoma of Head and Neck

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    Cláudia CAZAL

    2006-09-01

    Full Text Available Objective: The aim of this study was to verify the bcl-2 protein expression in 22 cutaneous basal cell carcinomas (BCC of the head and neck, and to compare it with its aggressive behavior. Method: Tumors were histologically classified in non-aggressive (BCC 1 and aggressive (BCC 2 and then submitted to the immunohistochemistry technique with the streptavidin-biotin peroxidase method using the anti-bcl-2 antibody. Results: After proceeding to morphological analysis, sixteen tumors (72.7% were considered aggressive and six (27.3% non-aggressive. Immunohistochemistry analysis showed that thirteen (59.1% lesions were positive staining and nine (40.9% were negative to the bcl-2 protein. Considering the positive lesions, 12 (92.3% were aggressive and one (7.7% non-aggressive. The relation between bcl-2 protein staining and the tumor aggressiveness was statistically significant (p<0.05 - Fisher's exact Test. Conclusion: The results suggest a relationship between the bcl-2 protein expression and the histological aggressiveness grade in the BCC of the head and neck group studied may exist.

  17. Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro

    Institute of Scientific and Technical Information of China (English)

    Liang ZHOU; Hong-feng WANG; Hai-gang REN; Dong CHEN; Feng GAO; Qing-song HU; Chen FU

    2013-01-01

    To investigate whether sequestosome 1/p62 (p62),a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system,could directly regulate autophagy in vitro.Methods:HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62.The cells or the cell lysates were subsequently subjected to immunofluorescence assay,immunoprecipitation assay,or immunoblot analysis.In vitro pulldown assay was used to study the interaction of p62 with Bcl-2.Results:Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells,whereas knockdown of p62 significantly decreased the basal level of autophagy.In vitro pulldown assay showed that p62 directly interacted with Bcl-2.It was observed in HeLa cells that p62 co-localized with Bcl-2.Furthermore,knockdown of p62 in HEK 293 cells significantly increased the amount of Beclin 1 that co-immunoprecipitated with Bcl-2.Conclusion:p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.

  18. Expression of COX-2 and bcl-2 in oral lichen planus lesions and lichenoid reactions

    Science.gov (United States)

    Arreaza, Alven J; Rivera, Helen; Correnti, María

    2014-01-01

    Oral lichen planus and lichenoid reactions are autoimmune type inflammatory conditions of the oral mucosa with similar clinical and histological characteristics. Recent data suggest that oral lichenoid reactions (OLR) present a greater percentage of malignant transformation than oral lichen planus (OLP). Objective To compare the expression of bcl-2 and COX-2 in OLP and OLR. Methods The study population consisted of 65 cases; 34 cases diagnosed as OLR and 31 as OLP. A retrospective study was done, and bcl-2 and COX-2 expression was semiquantitatively analysed. Results Fifty-three per cent (18/34) of the ORL samples tested positive for COX-2, whereas in the OLP group, 81% of the samples (25/31) immunostained positive for COX-2. The Fisher’s exact test for the expression of COX-2 revealed that there are significant differences between the two groups, P = 0.035. With respect to the expression of the bcl-2 protein, 76% (26/34) of the samples were positive in OLR, while 97% (30/31) were positive in the group with OLP. The Fisher’s exact test for the expression of bcl-2 revealed that there are significant statistical differences between the two groups, P = 0.028. Conclusions The expression of bcl-2 and COX-2 was more commonly expressed in OLP when compared with OLR. PMID:24834112

  19. Overcoming paclitaxel resistance in lung cancer cells via dual inhibition of stathmin and Bcl-2.

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    Han, Zheng-Xiang; Wang, Hong-Mei; Jiang, Guan; Du, Xiu-Ping; Gao, Xiang-Yang; Pei, Dong-Sheng

    2013-06-01

    Lung cancer is the leading cause of death from malignancy in people and over 85% of these patients eventually die from disseminated disease. Paclitaxel (TAX) is widely used as an antimicrotubule agent for the treatment of lung cancer. Unfortunately, the resistance to this antimicrotubule agent occurs frequently. Stathmin (STMN1) is a ubiquitous microtubule destabilizing protein linked to cancer and cell health and its expression level often correlates with cancer stage progression and prognosis for survival. Overexpression of the antiapoptotic protein Bcl-2 has been shown to prolong drug-induced growth arrest, potentially inducing resistance. In this study, we used a short hairpin RNA (shRNA) approach to evaluate the effect of STMN1 and Bcl-2 downregulation in the sensitivity to TAX in lung cancer cells. We achieved significant downregulation of STMN1 and Bcl-2 mRNA and protein expression by a combination of double shRNA treatment strategy. Our experimental data showed that inhibition of STMN1 and Bcl-2 expression with RNA interference can sensitize lung cancer cells to TAX. These findings suggest a novel approach to improve the efficacy of certain antimicrotubule agents against lung cancer by regulating the function of STMN1 and Bcl-2.

  20. Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas.

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    Ramarao Malla

    Full Text Available BACKGROUND: Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. CONCLUSIONS/SIGNIFICANCE: In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.

  1. Membrane microdomain-associated uroplakin IIIa contributes to Src-dependent mechanisms of anti-apoptotic proliferation in human bladder carcinoma cells

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    Shigeru Kihira

    2012-08-01

    Our previous study demonstrated that tyrosine phosphorylation of p145met/β-subunit of hepatocyte growth factor receptor by epidermal growth factor receptor and Src contributes to the anti-apoptotic growth of human bladder carcinoma cell 5637 under serum-starved conditions. Here, we show that some other cell lines of human bladder carcinoma, but not other types of human cancer cells, also exhibit Src-dependent, anti-apoptotic proliferation under serum-starved conditions, and that low-density, detergent-insoluble membrane microdomains (MD serve as a structural platform for signaling events involving p145met, EGFR, and Src. As an MD-associated molecule that may contribute to bladder carcinoma-specific cellular function, we identified uroplakin IIIa (UPIIIa, an urothelium-specific protein. Results obtained so far revealed: 1 UPIIIa undergoes partial proteolysis in serum-starved cells; 2 a specific antibody to the extracellular domain of UPIIIa inhibits the proteolysis of UPIIIa and the activation of Src, and promotes apoptosis in serum-starved cells; and 3 knockdown of UPIIIa by short interfering RNA also promotes apoptosis in serum-starved cells. GM6001, a potent inhibitor of matrix metalloproteinase (MMP, inhibits the proteolysis of UPIIIa and promotes apoptosis in serum-starved cells. Furthermore, serum starvation promotes expression and secretion of the heparin-binding EGF-like growth factor in a manner that depends on the functions of MMP, Src, and UPIIIa. These results highlight a hitherto unknown signaling network involving a subset of MD-associated molecules in the anti-apoptotic mechanisms of human bladder carcinoma cells.

  2. RBP2 Promotes Adult Acute Lymphoblastic Leukemia by Upregulating BCL2.

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    Xiaoming Wang

    Full Text Available Despite recent increases in the cure rate of acute lymphoblastic leukemia (ALL, adult ALL remains a high-risk disease that exhibits a high relapse rate. In this study, we found that the histone demethylase retinoblastoma binding protein-2 (RBP2 was overexpressed in both on-going and relapse cases of adult ALL, which revealed that RBP2 overexpression was not only involved in the pathogenesis of ALL but that its overexpression might also be related to relapse of the disease. RBP2 knockdown induced apoptosis and attenuated leukemic cell viability. Our results demonstrated that BCL2 is a novel target of RBP2 and supported the notion of RBP2 being a regulator of BCL2 expression via directly binding to its promoter. As the role of RBP2 in regulating apoptosis was confirmed, RBP2 overexpression and activation of BCL2 might play important roles in ALL development and progression.

  3. RBP2 Promotes Adult Acute Lymphoblastic Leukemia by Upregulating BCL2

    Science.gov (United States)

    Wang, Xiaoming; Zhou, Minran; Fu, Yue; Sun, Ting; Chen, Jin; Qin, Xuemei; Yu, Yuan; Jia, Jihui; Chen, Chunyan

    2016-01-01

    Despite recent increases in the cure rate of acute lymphoblastic leukemia (ALL), adult ALL remains a high-risk disease that exhibits a high relapse rate. In this study, we found that the histone demethylase retinoblastoma binding protein-2 (RBP2) was overexpressed in both on-going and relapse cases of adult ALL, which revealed that RBP2 overexpression was not only involved in the pathogenesis of ALL but that its overexpression might also be related to relapse of the disease. RBP2 knockdown induced apoptosis and attenuated leukemic cell viability. Our results demonstrated that BCL2 is a novel target of RBP2 and supported the notion of RBP2 being a regulator of BCL2 expression via directly binding to its promoter. As the role of RBP2 in regulating apoptosis was confirmed, RBP2 overexpression and activation of BCL2 might play important roles in ALL development and progression. PMID:27008505

  4. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

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    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Cheng, Tian-Lu [Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Lin, Shinne-Ren [Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Long-Sen, E-mail: lschang@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China)

    2015-04-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.

  5. Mutual regulation of Bcl-2 proteins independent of the BH3 domain as shown by the BH3-lacking protein Bcl-x(AK.

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    Michael Plötz

    Full Text Available The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-x(AK, a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-x(AK may trigger apoptosis.For efficient overexpression, Bcl-x(AK was subcloned in an adenoviral vector under Tet-OFF control. The construct resulted in significant apoptosis induction in melanoma and nonmelanoma cell lines with up to 50% apoptotic cells as well as decreased cell proliferation and survival. Disruption of mitochondrial membrane potential, and cytochrome c release clearly indicated activation of the mitochondrial apoptosis pathways. Both Bax and Bak were activated as shown by clustering and conformation analysis. Mitochondrial translocation of Bcl-x(AK appeared as an essential and initial step. Bcl-x(AK was critically dependent on either Bax or Bak, and apoptosis was abrogated in Bax/Bak double knockout conditions as well by overexpression of Bcl-2 or Bcl-x(L. A direct interaction with Bcl-2, Bax, Bad, Noxa or Puma was however not seen by immunoprecipitation. Thus besides BH3-mediated interactions, there exists an additional way for mutual regulation of Bcl-2 proteins, which is independent of the BH3. This pathway appears to play a supplementary role also for other proapoptotic family members, and its unraveling may help to overcome therapy resistance in cancer.

  6. Development of bcl-2 mRNA repressor of apoptosis in human fetal central nervous system%人胎儿中枢神经系统凋亡抑制因子bcl-2mRNA的发育

    Institute of Scientific and Technical Information of China (English)

    李泽桂; 蔡文琴

    2003-01-01

    目的研究人胎儿中枢神经系统凋亡抑制因子 bcl-2 mRNA 的发育表达.方法用地高辛标记的bcl-2 cRNA 探针原位杂交组织化学技术,检测了12~39周胎儿中枢神经系统内bcl-2 mRNA的表达情况. 结果①在所检测的各脑区均有bcl-2 mRNA表达.第12周,有强阳性的bcl-2 mRNA出现在脊髓、延髓的运动神经元、大脑额叶的皮质板; 小脑和大脑室层的bcl-2 mRNA表达较弱.bcl-2 mRNA的水平一般是随胎龄的增长而下降,至第39周表达最弱.②bcl-2 mRNA主要在神经元表达.结论在人胎儿神经系统发育中表达的bcl-2可能与编程性细胞死亡有关.

  7. Amelioration of apoptotic events in the skeletal muscle of intra-nigrally rotenone-infused Parkinsonian rats by Morinda citrifolia--up-regulation of Bcl-2 and blockage of cytochrome c release.

    Science.gov (United States)

    Narasimhan, Kishore Kumar S; Paul, Liya; Sathyamoorthy, Yogesh Kanna; Srinivasan, Ashokkumar; Chakrapani, Lakshmi Narasimhan; Singh, Abhilasha; Ravi, Divya Bhavani; Krishnan, Thulasi Raman; Velusamy, Prema; Kaliappan, Kathiravan; Radhakrishnan, Rameshkumar; Periandavan, Kalaiselvi

    2016-02-01

    Parkinson's disease is a progressive neurodegenerative movement disorder with the cardinal symptoms of bradykinesia, resting tremor, rigidity, and postural instability, which lead to abnormal movements and lack of activity, which in turn cause muscular damage. Even though studies have been carried out to elucidate the causative factors that lead to muscular damage in Parkinson's disease, apoptotic events that occur in the skeletal muscle and a therapeutical approach to culminate the muscular damage have not been extensively studied. Thus, this study evaluates the impact of rotenone-induced SNPc lesions on skeletal muscle apoptosis and the efficacy of an ethyl acetate extract of Morinda citrifolia in safeguarding the myocytes. Biochemical assays along with apoptotic markers studied by immunoblot and reverse transcription-polymerase chain reaction in the current study revealed that the supplementation of Morinda citrifolia significantly reverted alterations in both biochemical and histological parameters in rotenone-infused PD rats. Treatment with Morinda citrifolia also reduced the expression of pro-apoptotic proteins Bax, caspase-3 and caspase-9 and blocked the release of cytochrome c from mitochondria induced by rotenone. In addition, it augmented the expression of Bcl2 both transcriptionally and translationally. Thus, this preliminary study paves a way to show that the antioxidant and anti-apoptotic activities of Morinda citrifolia can be exploited to alleviate skeletal muscle damage induced by Parkinsonism.

  8. MicroRNA-146a Induced by Hypoxia Promotes Chondrocyte Autophagy through Bcl-2

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    Fei Zhang

    2015-10-01

    Full Text Available Background/Aims: There have been many studies on the etiology of osteoarthritis (OA with regard to the function of inflammatory cytokines, the process of cartilage degradation, the function of miR-146a, hypoxia stimulation and autophagy in OA chondrocytes, but there have been no reports on the relationship between miR-146a and autophagy in cartilage, especially under hypoxia. This study aimed to confirm the relationship of miR-146a and autophagy in cartilage under hypoxia. Methods: Chondrocytes were treated by hypoxia gradients, and the main factors including HIF-1α, HIF-2α, miR-146a and Bcl-2 and autophagy markers ULK-1, ATG-5 were detected by quantitative PCR (Q-PCR and western blotting. The autophagy marker LC-3 was detected by immunofluorescence. The reciprocal effects between miR-146a and Bcl-2 were confirmed by several combinations of shRNAs and adenovirus-gene systems followed by Q-PCR and western blot detection. Results: Hypoxia maintained the chondrocytes phenotype and promoted autophagy and miR-146a expression via HIF-1α, but not HIF-2α, while miR-146a did not reversely affect HIF-1α. The autophagy induced by hypoxia through HIF-1α, miR-146a and Bcl-2. Simply, hypoxia induced HIF-1α, and HIF-1α increased miR-146a, but miR-146a suppressed Bcl-2, an autophagy inhibitor. While Bcl-2 affected neither HIF-1α nor miR-146a. The absence of both HIF-1α and miR-146a or Bcl-2 over-expression inhibited hypoxia-induced autophagy. Conclusion: HIF-1α, miR-146a and Bcl-2 play crucial roles during hypoxia-induced autophagy, Hypoxia, HIF-1α and miR-146a promote chondrocytes autophagy via depressing Bcl-2. We conclude that miR-146a may serve as a novel therapeutic target for protecting cartilage from degeneration in OA.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  10. Suppression of bcl-2 Gene by RNA Interference Increases Chemosensitivity to Cisplatin in Nasopharyngeal Carcinoma Cell Line CNE1

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hua YIN; Cai-Ping REN; Feng LI; Xu-Yu YANG; Hui LI; Ming ZHAO; Kai-Tai YAO

    2004-01-01

    To explore the effect of suppressing BCL-2 expression using RNA interference (RNAi) technique in nasopharyngeal carcinoma cell line CNE1. CNE1 cell lines stably expressing shRNAs targeted bcl-2 and GL3 gene were established and gene expression inhibition was assessed by Western blotting analysis. The effect of suppressing bcl-2 by RNAi on cell growth was studied, the apoptosis induction and the sensitization of CNE 1 cells to cisplatin were quantified by MTT assay and flow cytometry. The results showed that: stable transfection of CNE 1 cells with vectors expressing shRNAs against bcl-2 decreased the expression of BCL-2 protein; suppression of BCL-2 expression did not affect cell proliferation but could increase the chemosensitivity to cisplatin in CNE1 cells. This will help physicians to make some clinical trials of gene therapy on nasopharyngeal carcinoma by RNAi.

  11. Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells

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    Shekhar, Tanmay M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Green, Maja M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Department of Anatomy & Neuroscience, The University of Melbourne, Parkville 3010 (Australia); Rayner, David M.; Miles, Mark A.; Cutts, Suzanne M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Hawkins, Christine J., E-mail: c.hawkins@latrobe.edu.au [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia)

    2015-07-15

    Graphical abstract: - Highlights: • Mutagenicities of anti-cancer drugs were tested using HPRT, γH2AX and comet assays. • TRAIL, doxorubicin and etoposide were more mutagenic than BH3- or Smac-mimetics. • Physiologically achievable levels of the BH3-mimetic ABT-737 were not mutagenic. • High concentrations of ABT-737 provoked mutations via an off-target mechanism. • Even very high concentrations of IAP antagonists were not mutagenic. - Abstract: Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict

  12. Bcl-2—Enhanced Efficacy of Microtubule-Targeting Chemotherapy through Bim Overexpression: Implications for Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Amandine Savry

    2013-01-01

    Full Text Available Bcl-2 is commonly overexpressed in tumors, where it is often associated with unfavorable outcome. However, it has also been linked to a favorable sensitivity to microtubule-targeting agents (MTAs. We show that Bcl-2– overexpressing lung and breast cancer cells were more sensitive to both paclitaxel and vinorelbine. Bcl-2 overexpression also significantly potentiated in vivo efficacy of paclitaxel, in terms of tumor volume decrease and survival benefits, in models of nude mice bearing lung cancer xenografts. To further investigate this favorable effect of Bcl-2, a genomic approach was taken. It revealed that Bcl-2 overexpression induced up-regulation of the proapoptotic protein Bim in lung cancer cells and that, conversely, Bcl-2 silencing decreased Bim expression level. A gene regulation study implicated the transcription factor Forkhead box-containing protein, class O3a in Bim up-regulation. Lastly, we show that Bim was responsible for MTA-triggered lung cancer cell death through a dynamin-related protein 1–mediated mitochondrial fragmentation. The Bcl-2–governed Bim induction evidence offers for the first time an explanation for the favorable higher sensitivity to treatment shown by Bcl-2–overexpressing cells. We suggest that Bim could be a powerful predictive factor for tumor response to MTA chemotherapy. Our data also give new insight into some failures in the efficacy of therapies targeted against Bcl-2.

  13. Antisense bcl-2 retrovirus vector increases the sensitivity of a human gastric adenocarcinoma cell line to photodynamic therapy.

    Science.gov (United States)

    Zhang, W G; Ma, L P; Wang, S W; Zhang, Z Y; Cao, G D

    1999-05-01

    The bcl-2 oncoprotein directly prolongs cellular survival by blocking apoptosis and its overexpression is associated with cellular resistance to killing by chemotherapeutic drugs and gamma-irradiation. Meanwhile, it has been shown that bcl-2 antisense oligonucleotide can induce apoptosis or increase toxicity of the treatment in tumors in vivo and in vitro. However, it is difficult to obtain stable transfection by this approach and there are no reports about the effect of an antisense bcl-2 on the sensitivity to oxidative stress induced by photodynamic therapy (PDT). Here we investigated the effect of an antisense bcl-2 RNA retrovirus vector transfer on the sensitivity of 2-butylamino-2-demethoxy-hypocrellin A (2-BA-2-DMHA) photosensitization in a human gastric adenocarcinoma MGC803 cell line. The results indicate that antisense bcl-2-infected MGC803 cells expressed exogenous antisense bcl-2 mRNA measured by reverse transcription polymerase chain reaction and significantly reduced bcl-2 protein determined by western blotting analysis. The decreased expression of bcl-2 protein was accompanied by increased phototoxicity and susceptibility to apoptosis induced by 2-BA-2-DMHA PDT. Our finding suggests that reduction of bcl-2 protein in gastric cancers, and possibly also in a variety of other tumors, may be a novel and rational approach to improve photosensitivity and the treatment outcome.

  14. Hepatitis E virus ORF2 protein activates the pro-apoptotic gene CHOP and anti-apoptotic heat shock proteins.

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    Lijo John

    Full Text Available BACKGROUND: Hepatitis E virus (HEV is a non-enveloped plus-strand RNA virus that causes acute hepatitis. The capsid protein open reading frame 2 (ORF2 is known to induce endoplasmic reticulum stress in ORF2 expressing cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study we found that HEV ORF2 activates the expression of the pro-apoptotic gene C/EBP homologous protein (CHOP. ORF2 stimulates the CHOP promoter mainly through AARE (amino acid response elements and to a minor extent the ERSE (endoplasmic reticulum stress response elements. Activating transcription factor 4 (ATF4 protein binds and activates the AARE regulatory sites of the CHOP promoter. ORF2 expression also leads to increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α that in turn initiates the translation of ATF4 mRNA. The pro-apoptotic gene CHOP is an important trigger to initiate endoplasmic reticulum stress induced apoptosis. However, the activation of CHOP by ORF2 in this study did not induce apoptosis, nor did BCL2-associated X protein (Bax translocate to mitochondria. Microarray analysis revealed an ORF2 specific increased expression of chaperones Hsp72, Hsp70B', and co-chaperone Hsp40. Co-immunoprecipitation (Co-IP and in silico molecular docking analysis suggests that HEV ORF2 interacts with Hsp72. In addition, Hsp72 shows nuclear accumulation in ORF2 expressing cells. CONCLUSIONS/SIGNIFICANCE: These data provide new insight into simultaneously occurring counter-acting effects of HEV ORF2 that may be part of a strategy to prevent host suicide before completion of the viral replication cycle.

  15. Neuroprotective effects of lycopene in spinal cord injury in rats via antioxidative and anti-apoptotic pathway.

    Science.gov (United States)

    Hu, Wei; Wang, Hongbo; Liu, Zhenfeng; Liu, Yanlu; Wang, Rong; Luo, Xiao; Huang, Yifei

    2017-03-06

    Oxidative damage induced-mitochondrial dysfunction and apoptosis has been widely studied in spinal cord injury (SCI). Lycopene, a polyunsaturated hydrocarbon, has the highest antioxidant capacity compared to the other carotenoids. However, the role of lycopene in SCI is unknown. In the present study, we evaluated the antioxidant effects of lycopene on mitochondrial dysfunction and apoptosis following T10 contusion SCI in rats. The rats were randomized into 5 groups: the sham group, the SCI group and the SCI pre-treated with lycopene (5, 10, or 20mg/kg) group. The SCI group showed increased malondialdehyde (MDA) content, decreased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) ability, which indicated that SCI could induce oxidative damage. What's more, the SCI group showed decreased mRNA expression of cytochrome b and mitochondrial transcription factor A (Tfam), and decreased mitochondrial membrane potential (ΔYm), which indicated that SCI could induce mitochondrial dysfunction. Besides, the SCI group showed decreased protein expression of bcl-2 and mitochondrial cytochrome C, increased protein expression of cytosolic cytochrome C, cleaved caspase-9, cleaved caspase-3 and bax, and increased TUNEL-positive cell numbers, which indicated that SCI could induce cell apoptosis. Fortunately, the lycopene treatment significantly ameliorated oxidative damage, mitochondrial dysfunction and cell apoptosis via the reversion of those parameters described above in the dose of lycopene of 10 and 20mg/kg. In addition, lycopene significantly ameliorated the hind limb motor disturbances in the SCI+lyco10 group and the SCI+lyco20 group compared with the SCI group. These results suggested that lycopene administration could improve total antioxidant status and might have neuroprotective effects on SCI.

  16. Hyperbaric Oxygen Prevents Cognitive Impairments in Mice Induced by D-Galactose by Improving Cholinergic and Anti-apoptotic Functions.

    Science.gov (United States)

    Chen, Chunxia; Huang, Luying; Nong, Zhihuan; Li, Yaoxuan; Chen, Wan; Huang, Jianping; Pan, Xiaorong; Wu, Guangwei; Lin, Yingzhong

    2017-01-11

    Our previous study demonstrated that hyperbaric oxygen (HBO) improved cognitive impairments mainly by regulating oxidative stress, inflammatory responses and aging-related gene expression. However, a method for preventing cognitive dysfunction has yet to be developed. In the present study, we explored the protective effects of HBO on the cholinergic system and apoptosis in D-galactose (D-gal)-treated mice. A model of aging was established via systemic intraperitoneal injection of D-gal daily for 8 weeks. HBO was administered during the last 2 weeks of D-gal injection. Our results showed that HBO in D-gal-treated mice significantly improved behavioral performance on the open field test and passive avoidance task. Studies on the potential mechanisms of this effect showed that HBO significantly reduced oxidative stress and blocked the nuclear factor-κB pathway. Moreover, HBO significantly increased the levels of choline acetyltransferase and acetylcholine and decreased the activity of acetylcholinesterase in the hippocampus. Furthermore, HBO markedly increased expression of the anti-apoptosis protein Bcl-2 and glial fibrillary acidic protein meanwhile decreased expression of the pro-apoptosis proteins Bax and caspase-3. Importantly, there was a significant reduction in expression of Aβ-related genes, such as amyloid precursor protein, β-site amyloid cleaving enzyme-1 and cathepsin B mRNA. These decreases were accompanied by significant increases in expression of neprilysin and insulin-degrading enzyme mRNA. Moreover, compared with the Vitamin E group, HBO combined with Vitamin E exhibited significant difference in part of the above mention parameters. These findings suggest that HBO may act as a neuroprotective agent in preventing cognitive impairments.

  17. A comparison of two strategies for affinity maturation of a BH3 peptide toward pro-survival Bcl-2 proteins.

    Science.gov (United States)

    Zhang, Siyan; Long, Angel; Link, A James

    2012-03-16

    The Bcl-2 family of proteins regulates apoptosis at the level of mitochondrial permeabilization. Pro-death members of the family, including Bak and Bax, initiate apoptosis, whereas pro-survival members such as Bcl-x(L) and Mcl-1 antagonize the function of Bak and Bax via heterodimeric interactions. These heterodimeric interactions are primarily mediated by the binding of the helical amphipathic BH3 domain from a pro-death protein to a hydrophobic cleft on the surface of the pro-survival protein. Since high levels of pro-survival Bcl-2 proteins are present in many cancers, peptides corresponding to pro-death BH3 domains hold promise as therapeutics. Here we apply a high-throughput flow cytometry assay to engineer the Bak BH3 domain for improved affinity toward the pro-survival proteins Bcl-x(L) and Mcl-1. Two strategies, engineering the hydrophobic face of the Bak BH3 peptide and increasing its overall helicity, are successful in identifying Bak BH3 variants with improved affinity to Bcl-x(L) and Mcl-1. Hydrophobic face engineering of the Bak BH3 peptide led to variants with up to a 15-fold increase in affinity for Bcl-x(L) and increased specificity toward Bcl-x(L). Engineering of the helicity of Bak BH3 led to modest (3- to 4-fold) improvements in affinity with retention of promiscuous binding to all pro-survival proteins. HeLa cell killing studies demonstrate that the affinity matured Bak BH3 variants retain their expected biological function.

  18. Anti-apoptotic mechanism of Bacoside rich extract against reactive nitrogen species induced activation of iNOS/Bax/caspase 3 mediated apoptosis in L132 cell line.

    Science.gov (United States)

    Anand, T; Pandareesh, M D; Bhat, Pratiksha V; Venkataramana, M

    2014-10-01

    Nitric oxide is a highly reactive free radical gas that reacts with a wide range of bio-molecules to produce reactive nitrogen species and exerts nitrative stress. Bacopa monniera is a traditional folk and ayurvedic medicine known to alleviate a variety of disorders. Aim of the present study is to evaluate the protective propensity of Bacopa monniera extract (BME) through its oxido-nitrosative and anti-apoptotic mechanism to attenuate sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132). Our results elucidate that pre-treatment of L132 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP as evidenced by MTT and LDH leakage assays. BME pre-treatment inhibited NO generation by down-regulating inducible nitric oxide synthase expression. BME exhibited potent antioxidant activity by up-regulating the antioxidant enzymes. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic biomarkers such as Bax, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. By considering all these findings, we report that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.

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

    Institute of Scientific and Technical Information of China (English)

    林蓓; 张淑兰; 赵长清

    2004-01-01

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

  20. Effects of Nerve Growth Factor on Bcl-2 Protein after Spinal Cord Injury in Rats

    Institute of Scientific and Technical Information of China (English)

    汤长华; 曹晓建; 王道新

    2002-01-01

    Objective To explore the protective mechanisms of nerve growth factor( NGF) ou spinal cord injury(SCI) and provide theoretical basis for its clinical application. MethodsThe SCI of Wistar rats was done by Allens weight dropping way by a 10 g × 2.5 cm impact on theposterior of spinal cord T8 NGF ( 3 g/L, 20d) or normal saline was injected to treatment group ratsthrough catheter into subarachnoid space at 0,2,4,8,12 and 24 h after SCI. The expression of bcl-2 protein levels in rat spinal cord was detected by immunohistoclemistry. Results The strong expres-sion sequence of bcl-2 protein was found in spinal cord of normal rat group. The levels of bcl-2 pro-tein after SCI in NGF treatment group increased more significantly than those in normal saline treatmentgroup (P<0. 01). Conclusion NGF could protect injured spinal cord by stimulating bcl-2 pro-tein expression and suppressing apoptosis after SCI.

  1. Expression of caspase-3, p53 and Bcl-2 in generalized aggressive periodontitis

    Directory of Open Access Journals (Sweden)

    Özdemir B Handan

    2006-06-01

    Full Text Available Abstract Background Apoptosis, or programmed cell death is a form of physiological cell death. It is increased or decreased in the presence of infection, inflammation or tissue remodelling. Previous studies suggest that apoptosis is involved in the pathogenesis of inflammatory periodontal disease. The aim of the present study was to investigate the clinical features and known indicators of apoptosis (p53, Bcl-2, Caspase-3 in patients with generalized aggressive periodontitis (GAP Methods Eight patients with GAP, who had sites with probing depths (PD > 5 mm, and 10 periodontally-healthy persons were included in the study. Clinical examinations and PD were performed, and the plaque index and gingival index were recorded. Gingival tissues biopsies were obtained from active site of each patient and from healthy individuals. The expression of caspase-3, Bcl-2, and p53 was evaluated by immunohistochemistry Results There were no significant differences between GAP and control group with respect to levels of caspase-3 and p53 expression (P > 0.05. Contrary, the frequency of grade 3 expression of Bcl-2 was higher in GAP group than the control group. Conclusion The higher frequency of Bcl-2 expression in GAP group indicates and delayed apoptosis can lead to increasing resident inflammatory cells in periodontal tissues and resulting in progressive periodontal destruction.

  2. miR-204 targets Bcl-2 expression and enhances responsiveness of gastric cancer

    Science.gov (United States)

    Sacconi, A; Biagioni, F; Canu, V; Mori, F; Di Benedetto, A; Lorenzon, L; Ercolani, C; Di Agostino, S; Cambria, A M; Germoni, S; Grasso, G; Blandino, R; Panebianco, V; Ziparo, V; Federici, O; Muti, P; Strano, S; Carboni, F; Mottolese, M; Diodoro, M; Pescarmona, E; Garofalo, A; Blandino, G

    2012-01-01

    Micro RNAs (miRs) are small non-coding RNAs aberrantly expressed in human tumors. Here, we aim to identify miRs whose deregulated expression leads to the activation of oncogenic pathways in human gastric cancers (GCs). Thirty nine out of 123 tumoral and matched uninvolved peritumoral gastric specimens from three independent European subsets of patients were analyzed for the expression of 851 human miRs using Agilent Platform. The remaining 84 samples were used to validate miRs differentially expressed between tumoral and matched peritumoral specimens by qPCR. miR-204 falls into a group of eight miRs differentially expressed between tumoral and peritumoral samples. Downregulation of miR-204 has prognostic value and correlates with increased staining of Bcl-2 protein in tumoral specimens. Ectopic expression of miR-204 inhibited colony forming ability, migration and tumor engraftment of GC cells. miR-204 targeted Bcl-2 messenger RNA and increased responsiveness of GC cells to 5-fluorouracil and oxaliplatin treatment. Ectopic expression of Bcl-2 protein counteracted miR-204 pro-apoptotic activity in response to 5-fluorouracil. Altogether, these findings suggest that modulation of aberrant expression of miR-204, which in turn releases oncogenic Bcl-2 protein activity might hold promise for preventive and therapeutic strategies of GC. PMID:23152059

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

    Science.gov (United States)

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

    2013-06-01

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

  4. Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yangling; Luo, Peihua; Wang, Jincheng; Dai, Jiabin; Yang, Xiaochun; Wu, Honghai; Yang, Bo, E-mail: yang924@zju.edu.cn; He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn

    2014-01-15

    Combretastatin A-4 (CA-4) has already entered clinical trials of solid tumors over ten years. However, the limited anticancer activity and dose-dependent toxicity restrict its clinical application. Here, we offered convincing evidence that CA-4 induced autophagy in various cancer cells, which was demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Interestingly, CA-4-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5 and Beclin 1). The enhanced anticancer activity of CA-4 and 3-MA was further confirmed in the SGC-7901 xenograft tumor model. These findings suggested that CA-4-elicited autophagic response played a protective role that impeded the eventual cell death while autophagy inhibition was expected to improve chemotherapeutic efficacy of CA-4. Meanwhile, CA-4 treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor or JNK siRNA inhibited autophagy but promoted CA-4-induced apoptosis, indicating a key requirement of JNK-Bcl-2 pathway in the activation of autophagy by CA-4. We also identified that pretreatment of Bcl-2 inhibitor (ABT-737) could significantly enhance anticancer activity of CA-4 due to inhibition of autophagy. Taken together, our data suggested that the JNK-Bcl-2 pathway was considered as the critical regulator of CA-4-induced protective autophagy and a potential drug target for chemotherapeutic combination. - Highlights: • Autophagy inhibition could be a potential for combretastatin A-4 antitumor efficacy. • The JNK-Bcl-2 pathway plays a critical role in CA-4-induced autophagy. • ABT-737 enhances CA-4 anticancer activity due to inhibition of autophagy.

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

    Directory of Open Access Journals (Sweden)

    Ender DÜZCAN

    2010-01-01

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

  6. bcl-2在受损面神经运动神经元中的表达与定位%Expression of bcl-2 in facial motoneurons and its ultrastructural localization following facial nerve injury

    Institute of Scientific and Technical Information of China (English)

    戴春富; 黄新生; 王正敏; 李宽俨; 赵晖

    2001-01-01

    Objective To explore the expression of bcl-2 in facial motoneurons and its subcellular distribution. Methods Wistar rats were used in this study. Facial nerve transection was performed at stylomastoid foramen or internal acoustic meatus. Facial nerve crush was made at stylomastiod foramen. The animal survived for 1, 3, 7, 15, 30 and 60 days respectively. Facial nucleus was treated with bcl-2 monoclonal antibody or bcl-2 DIG-labelling probe and studied with immunohistochemstry and in situ hybridization. The bcl-2 positive motoneuron was investigated with immuno-electron microscope. Results It was demonstrated that bcl-2 protein level was corresponded with bcl-2 mRNA expression. The level of bcl-2 expression in facial motoneurons was high in normal facial nerve. It increased on the first day and declined on the third day post-transection in facial motoneuron. It reached the lowest level on the 15th days following facial nerve injury (P0.05). After facial nerve transected, the reduction of bcl-2 expression was more significant when facial nerve transected close to facial nucleus than that far from facial nucleus (P0.05)。bcl-2的表达同损伤程度和损伤部位有关(0.01bcl-2 mRNA和bcl-2蛋白表达一致。免疫电镜发现bcl-2蛋白主要定位于神经元胞体中的线粒体、内质网和核膜上。结论 bcl-2转录和翻译可能同面神经核团中运动神经元胞体的死亡相关。通过转基因技术调节bcl-2基因的表达,可能为临床面瘫的治疗提供新的手段。

  7. Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells.

    Science.gov (United States)

    Dispersyn, G; Nuydens, R; Connors, R; Borgers, M; Geerts, H

    1999-08-05

    This report addresses the relation between Bcl-2 and mitochondrial membrane potential (DeltaPsi(m)) in apoptotic cell death. Rat pheochromocytoma (PC12) cells are differentiated into neuron-like cells with nerve growth factor (NGF). It is known that Bcl-2 can attenuate apoptosis induced by deprivation of neurotrophic factor. The protective effect of Bcl-2 has been correlated with preservation of DeltaPsi(m). Protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), collapse the proton gradient across the mitochondrial inner membrane, resulting in a complete abolition of the mitochondrial membrane potential. Based on the analysis of morphology, of phosphatidylserine exposure and of nuclear fragmentation we conclude that FCCP induces apoptosis in PC12 cells, which can be prevented by overexpression of Bcl-2. To determine whether the cytoprotective effect of Bcl-2 is due to stabilization of DeltaPsi(m), we investigated the effect of Bcl-2 on changes in DeltaPsi(m), induced by FCCP in PC12 cells. We showed that treatment with FCCP induced a reduction in DeltaPsi(m), as assessed with the lipophilic cationic membrane potential-sensitive dye JC-1, and that Bcl-2 protects against FCCP-induced changes in NGF differentiated PC12 cells. Our data indicate that Bcl-2 protects against FCCP-induced cell death by stabilizing DeltaPsi(m).

  8. Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma

    NARCIS (Netherlands)

    Kramer, M.H.H.; Hermans, J; Wijburg, E; Philippo, K; Geelen, E; van Krieken, J.H.J.M.; de Jong, D; Maartense, E; Schuuring, E; Kluin, P M

    1998-01-01

    Diffuse large B-cell lymphoma (DLCL) is characterized by a marked degree of morphologic and clinical heterogeneity. We studied 156 patients with de novo DLCL for rearrangements of the BCL2, BCL6, and MYC oncogenes by Southern blot analysis and BCL2 protein expression. We related these data to the pr

  9. 应用bcl-2基因及GDNF治疗大鼠脑缺血的研究%The Effect of bcl-2 Gene and GDNF of Rats after Focal Cerebral Ischemia Reperfusion Injury

    Institute of Scientific and Technical Information of China (English)

    ZHOU Tie-zhu; FU Xia

    2009-01-01

    目的 观察胶质细胞源性神经营养因子(GDNF)与bcl-2基因对大鼠局灶性脑缺血再灌注损伤(CIRI)的影响.方法 取健康Wistar大鼠40只,采用Longa改良栓线法制成大脑中动脉闭塞(MCAO)模型,缺血2 h后再灌注,随机分4组:对照组、bcl-2组、GDNF组、bcl-2+GDNF组,于再灌注3 h后经侧脑室注射GDNF 10μl、经颈动脉注射pLXSN-bcl-2质粒10μg,MCAO后3d(n=5),14 d(n=5),通过免疫组化染色检测bcl-2蛋白和GDNF的表达情况,TUNEL法检测细胞凋亡情况.结果 bcl-2+GDNF组bcl-2蛋白表达与GDNF表达水平均较其他组表达明显增加(P<0.05),bcl-2组、GDNF组较对照组明显增加(P<0.05);脑内细胞凋亡GDNF+bcl-2组较其他组明显减少(P<0.05),bcl-2组、GDNF组较对照组明显减少(P<0.05).结论 bcl-2基因和GDNF可能通过减少神经细胞凋亡,促进bcl-2与GDNF蛋白表达,增强对局灶性缺血脑组织的保护能力,加速中枢神经损伤的修复;bcl-2基因与GDNF协同作用,促使神经细胞在脑内的凋亡进一步减少,从而避免脑缺血再灌注后脑细胞进一步损伤.

  10. Construction of Bcl-2 Antisense RNA Expressed by Defect Retroviral Vector%反义bcl-2基因逆转录病毒表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    林祥华; 陈志哲; 杨婷; 吕联煌

    2001-01-01

    目的克隆bcl-2基因,构建其不同反义核酸的逆转录病毒表达载体。方法设计一对两端带有EcoRⅠ酶切位点的引物,RT-PCR克隆含bcl-2全部编码区的848 bp片段,并连接到T载体,测序正确后正反向亚克隆到pLXSN中EcoRⅠ位点;从pBluescriptⅡSK(-)bcl-2中用BamHⅠ切下含有bcl-2阅读框起始部位的部分编码区亚克隆到pLXSN BamHⅠ位点。结果构建带有正反向含bcl-2阅读框的全部编码区和部分编码区的逆转录病毒表达载体。结论在已知基因序列克隆中,联合RT-PCR和T载体是一个简单、快速、有效的方法,为基因转移和表达研究提供基础。%Objective To clone bcl-2 gene and develop two different bcl-2 antisense RNA expressed by defect retroviral vector. Methods Both upper and down primers with the end of EcoRⅠ site were designed to amplify 848 bp cDNA fragement including the whole bcl-2 exon by RT-PCR. After confirmation of the sequences of cloned bcl-2 exon by T vector, both forward and backward of bcl-2 cDNA were subcloned into the EcoRⅠ site of retroviral vector pLXSN. In the other way, 600 bp cDNA fragement with the beginning of coding bcl-2 exon was obtained by digestion of pBluescriptⅡ SK(-) bcl-2 with BamHⅠ restriction enzyme and also subcloned into the BamHⅠ site of retroviral vector pLXSN. Results Retroviral vectors with both forward and backward, whole and part of bcl-2 exon were successfully constructed. Conclusion Under the knowledge of targeting cDNA sequences, the approach for clonning of targeting gene by combination of RT-PCR with T vector is simple, rapid, effective, and offering the underlying bases of gene transfer and expression.

  11. Exposure to 50 Hz electromagnetic field raises the levels of the anti-apoptotic protein BAG3 in melanoma cells.

    Science.gov (United States)

    Basile, Anna; Zeppa, Rosario; Pasquino, Nicola; Arra, Claudio; Ammirante, Massimo; Festa, Michelina; Barbieri, Antonio; Giudice, Aldo; Pascale, Maria; Turco, Maria Caterina; Rosati, Alessandra

    2011-11-01

    The expression of the anti-apoptotic protein BAG3 is induced in several cell types by exposure to high temperature, oxidants, and other stressful agents. We investigated whether exposure to 50 Hz electromagnetic fields raised BAG3 levels in the human melanoma cell line M14, in vitro and in orthotopic xenografts. Exposure of cultured cells or xenografts for 6 h or 4 weeks, respectively, produced a significant (P < 0.01) increase in BAG3 protein amounts. Interestingly, at the same times, we could not detect any significant variation in the levels of HSP70/72 protein or cell apoptosis. These results confirm the stressful effect of exposure to ELF in human cells, by identifying BAG3 protein as a marker of ELF-induced stress. Furthermore, they suggest that BAG3 induction by ELF may contribute to melanoma cell survival and/or resistance to therapy.

  12. 视网膜母细胞瘤Bcl-2和Bax基因蛋白质表达%Expression of Bcl-2 and Bax gene protein in retinoblastoma

    Institute of Scientific and Technical Information of China (English)

    张小猛; 庞利民; 张晓光

    2000-01-01

    目的:研究凋亡及凋亡调控基因Bcl-2/Bax和视网膜母细胞瘤(Retinoblastoma,RB)的发生、发展及退化的关系.方法:收集36例RB标本,对其分别进行Bcl-2和Bax基因的蛋白质免疫组织化学染色.对其表达情况和染色强度进行观察.结果:①Bcl-2在分化型RB中表达比较好;②Bax在未分化型和分化型中表达都比较好.结论:①分化型和未分化型RB中都有Bcl-2/Bax基因蛋白表达;②随RB恶性度的增加,Bcl-2的表达逐渐减弱;Bax的表达无明显改变.③分化型RB受Bcl-2和Bax基因共同控制;未分化型RB受Bax基因调控,Bcl-2基因发挥很少的作用.

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

    Institute of Scientific and Technical Information of China (English)

    Min-Er Tang

    2016-01-01

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

  14. Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

    Directory of Open Access Journals (Sweden)

    F.S. Lizarte Neto

    2013-01-01

    Full Text Available Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA. We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21 and apoptotic (Fas, caspase-3 and caspase-8 genes was analyzed by relative quantification (real-time PCR of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3 and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP. KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

  15. Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

    Energy Technology Data Exchange (ETDEWEB)

    Lizarte, F.S. Neto; Tirapelli, D.P.C. [Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Ambrosio, S.R. [Universidade de Franca, Núcleo de Pesquisa em Ciências e Tecnologia, Franca, SP (Brazil); Tirapelli, C.R. [Universidade de São Paulo, Laboratório de Farmacologia, Departamento de Enfermagem Psiquiátrica e Ciências Humanas, Escola de Enfermagem de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Oliveira, F.M. [Universidade de São Paulo, Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Novais, P.C. [Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Peria, F.M.; Oliveira, H.F. [Universidade de São Paulo, Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Carlotti, C.G. Junior; Tirapelli, L.F. [Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil)

    2013-01-11

    Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

  16. Mitogenic and Anti-apoptotic Effects of Insulin in En-dometrial Cancer Cells Are Phosphatidylinositol 3-ki-nase/Akt Dependent

    Institute of Scientific and Technical Information of China (English)

    Shaofang HUA; Fengxia XUE; Jing ZHAO

    2009-01-01

    Background and objective Endometrial carcinoma is the most common gynecologic malignancy in the world. Although the insulin-resistant state or hyperinsulinemia was recently suggested as a potent risk factor for endometrial carcinogenesis and progression, there is only limited supporting evidence and the mechanism is unclear. In this study, we explored the roles of phosphatidylinositol 3-k/nase (PI3K)/Akt signaling pathway in the response of a human endometrial cancer cell line, Ishikawa3-H-12 cells, to insulin.Methods The Ishikawa 3-H-12 cells were serum-starved and then stimulated by insulin at various concentrations and for different time periods. To identify the insnlin-mediated signal pathway in the cells, LY294002, a selective inhibitor of PI3K, was used. The proliferation and the apoptotic rates were determined with methyl thiazolyl tetrazolium (MTT) and flow cytometric assays, respectively.Results The insulin receptor positive Ishikawa 3-H-12 cells had enhanced proliferation upon insulin stimulation in a rinse-and time-dependent manner. The growth promoting effect of insulin was blocked when the cells were pre-incubated with LY294002 for 60 rains.Insulin was able to protect the cells from serum-starvation-induced apoptosis in a concentration-dependent manner, while the anti-apoptotic effects of insulin was reversed by adding LY294002. Treatment with insulin at 1 μM for 15 rain resulted in an increased level of activated Akt The insulin-induced Akt activation was inhibited by LY294002 in a dose-dependent manner.Conclusion Insulin activates PI3K/Akt signaling pathway and is a mitogenic and anti-apoptotic agent for Ishikewa 3-H-12 endometrial cancer cells.

  17. Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases.

    Science.gov (United States)

    Lizarte Neto, F S; Tirapelli, D P C; Ambrosio, S R; Tirapelli, C R; Oliveira, F M; Novais, P C; Peria, F M; Oliveira, H F; Carlotti Junior, C G; Tirapelli, L F

    2013-01-01

    Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

  18. Prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim reduces the association of Bcl-2 with Bak or Bim, provoking Bak activation and mitochondrial apoptosis in nocodazole-treated Jurkat T cells.

    Science.gov (United States)

    Han, Cho Rong; Jun, Do Youn; Lee, Ji Young; Kim, Young Ho

    2014-01-01

    Treatment of Jurkat T cells with the microtubule-depolymerizing agent nocodazole (NOC) caused prometaphase arrest and apoptosis. NOC-induced mitochondrial apoptotic events including Bak activation, Δψm loss, cytochrome c release, and caspase cascade activation were blocked by Bcl-2 overexpression. However, mitotic arrest, Cdc25C activation, upregulation of cyclin B1 levels, Cdk1 activation, Bcl-2 phosphorylation at Thr-56 and Ser-70, and Bim phosphorylation were retained. The treatment of Jurkat T cells concomitantly with NOC and the G1/S-blocking agent hydroxyurea resulted in G1/S arrest and complete abrogation of all apoptotic events. The association of Bcl-2 with Bim or Bak declined after the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, whereas the association of Bcl-2 with Bax remained relatively constant. Although Bax was redistributed from the cytosol to the mitochondria, resulting in an increase in the mitochondrial level of Bax following NOC treatment, the subcellular localization of Bcl-2, Bim, Bak and apoptosis-inducing factor was confined to the mitochondrial fraction irrespective of NOC treatment. Experiments using selective caspase inhibitors showed that mitochondria-dependent activation of caspase-9 and -3 was crucial for NOC-induced apoptosis. NOC-induced phosphorylation of Bcl-2 and Bim, Δψm loss, and mitochondria-dependent apoptotic events were significantly suppressed by a Cdk1 inhibitor roscovitine, but not by the JNK inhibitor SP600125 or the p38 MAPK inhibitor SB203580. These results show that the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, which was mediated by Cdk1, could reduce the association of Bcl-2 with Bak or Bim to allow Bak activation and mitochondrial apoptotic events in Jurkat T cells exposed to NOC.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  20. Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

    OpenAIRE

    2015-01-01

    We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC inc...

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  2. Evaluation of CD40, its ligand CD40L and Bcl-2 in psoriatic patients

    Directory of Open Access Journals (Sweden)

    Bożena Chodynicka

    2012-04-01

    Full Text Available Psoriasis is a chronic, recurrent, inflammatory disease. Recent investigations indicate an autoimmune pathogenesis of the disease. Apoptosis plays an important role in the regulation of immune mechanisms in many autoimmune diseases. Although CD40, CD40L, and Bcl-2 have already been studied in psoriatic skin lesions, little is known about their circulating forms. The aim of the present study was to evaluate the serum concentrations of Bcl-2, soluble CD40 and CD40L in psoriatic patients. The study was performed using ELISA kits in 39 psoriatic patients before treatment and after two weeks of topical ointment. Data was analyzed with respect to severity of psoriasis, duration of the disease, and coexisting psoriatic arthritis. Our results revealed that serum concentrations of soluble CD40 and CD40L before and after treatment were significantly higher (p < 0.01 and p < 0.001 in patients with psoriasis compared to the control group. Topical treatment of psoriatic lesions with dithranol ointment failed to decrease serum of CD40 and CD40L, which has not been described until now. There was no significant difference in serum Bcl-2 concentration between the compared groups. We did not find significant differences in serum concentrations of Bcl-2, CD40 or CD40L between patients with mild or severe psoriasis, nor any correlation between disease duration and the presence of psoriatic arthritis symptoms. Our data indicates upregulation of the CD40/CD40L system in psoriatic patients despite topical treatment and suggests their possible role in the pathogenesis of psoriasis.

  3. bcl-1, bcl-2, p53, c-myc, and lyt-10 analysis in cutaneous lymphomas.

    Science.gov (United States)

    Garatti, S A; Roscetti, E; Trecca, D; Fracchiolla, N S; Neri, A; Berti, E

    1995-01-01

    In the present study we investigated the pathogenetic role of c-myc, bcl-2, and lyt-10 oncogenes, bcl-1 locus, and p53 suppressor gene in a representative panel of cutaneous lymphomas, including 25 cases of cutaneous B cell lymphoma (CBCL) and 29 cases of cutaneous T cell lymphoma (CTCL). In our analysis four cases of CBCL were found rearranged for bcl-2 and two for the bcl-1 locus. Two cases of CTCL and one case of CBCL were found rearranged for lyt-10. No rearrangements of c-myc oncogene were found in CBCL. Analysis of p53 gene showed mutation only in one case of mycosis fungoides in tumoral stage, at codon 163 of p53 gene (TAC-->CAC; Tyr--> Asp). Our data suggest that in primary CBCL bcl-2 oncogenes and bcl-1 locus are rarely involved. Furthermore, in primary CTCL p53 gene is not affected at significant frequency. The occurrence of p53 mutation in a patient affected by mycosis fungoides in tumoral stage may represent an involvement of p53 gene in tumor progression of CTCL, a finding observed in several types of human cancer.

  4. Germinal center reentries of BCL2-overexpressing B cells drive follicular lymphoma progression

    Science.gov (United States)

    Sungalee, Stéphanie; Mamessier, Emilie; Morgado, Ester; Grégoire, Emilie; Brohawn, Philip Z.; Morehouse, Christopher A.; Jouve, Nathalie; Monvoisin, Céline; Menard, Cédric; Debroas, Guilhaume; Faroudi, Mustapha; Mechin, Violaine; Navarro, Jean-Marc; Drevet, Charlotte; Eberle, Franziska C.; Chasson, Lionel; Baudimont, Fannie; Mancini, Stéphane J.; Tellier, Julie; Picquenot, Jean-Michel; Kelly, Rachel; Vineis, Paolo; Ruminy, Philippe; Chetaille, Bruno; Jaffe, Elaine S.; Schiff, Claudine; Hardwigsen, Jean; Tice, David A.; Higgs, Brandon W.; Tarte, Karin; Nadel, Bertrand; Roulland, Sandrine

    2014-01-01

    It has recently been demonstrated that memory B cells can reenter and reengage germinal center (GC) reactions, opening the possibility that multi-hit lymphomagenesis gradually occurs throughout life during successive immunological challenges. Here, we investigated this scenario in follicular lymphoma (FL), an indolent GC-derived malignancy. We developed a mouse model that recapitulates the FL hallmark t(14;18) translocation, which results in constitutive activation of antiapoptotic protein B cell lymphoma 2 (BCL2) in a subset of B cells, and applied a combination of molecular and immunofluorescence approaches to track normal and t(14;18)+ memory B cells in human and BCL2-overexpressing B cells in murine lymphoid tissues. BCL2-overexpressing B cells required multiple GC transits before acquiring FL-associated developmental arrest and presenting as GC B cells with constitutive activation–induced cytidine deaminase (AID) mutator activity. Moreover, multiple reentries into the GC were necessary for the progression to advanced precursor stages of FL. Together, our results demonstrate that protracted subversion of immune dynamics contributes to early dissemination and progression of t(14;18)+ precursors and shapes the systemic presentation of FL patients. PMID:25384217

  5. Bcl-2/MDM2 Dual Inhibitors Based on Universal Pyramid-Like α-Helical Mimetics.

    Science.gov (United States)

    Wang, Ziqian; Song, Ting; Feng, Yingang; Guo, Zongwei; Fan, Yudan; Xu, Wenjie; Liu, Lu; Wang, Anhui; Zhang, Zhichao

    2016-04-14

    No α-helical mimetic that exhibits Bcl-2/MDM2 dual inhibition has been rationally designed due to the different helicities of the α-helixes at their binding interfaces. Herein, we extracted a one-turn α-helix-mimicking ortho-triarene unit from o-phenylene foldamers. Linking benzamide substrates with a rotatable C-N bond, we constructed a novel semirigid pyramid-like scaffold that could support its two-turn α-helix mimicry without aromatic stacking interactions and could adopt the different dihedral angles of the key residues of p53 and BH3-only peptides. On the basis of this universal scaffold, a series of substituent groups were installed to capture the key residues of both p53TAD and BimBH3 and balance the differences of the bulks between them. Identified by FP, ITC, and NMR spectroscopy, a compound 6e (zq-1) that directly binds to Mcl-1, Bcl-2, and MDM2 with balanced submicromolar affinities was obtained. Cell-based experiments demonstrated its antitumor ability through Bcl-2/MDM2 dual inhibition simultaneously.

  6. 生酮饮食对MPTP 小鼠帕金森病模型黑质TH、Bcl-2和caspase-3基因表达的影响%Ketogenic diet affects substantia nigra TH, Bcl-2 and caspase-3 gene expressions in mouse models with 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine-induced PD

    Institute of Scientific and Technical Information of China (English)

    陈鑫; 王国坤; 周晓平

    2010-01-01

    目的 研究生酮饮食对PD小鼠黑质多巴胺能神经元的抗凋亡作用. 方法 1-甲基-4-苯基-1、2、3、6-四氢吡啶(MPTP)腹腔注射方法 制备PD模型小鼠.实验分为正常饮食模型组(正常饮食喂养后造模)、实验组(生酮饮食喂养后造模)、正常饮食组(不造模,正常饮食喂养)和生酮饮食组(不造模,生酮饮食喂养).初次MPTP给药前及末次给药后的次日进行滚轴实验并采集血清样本检测血清酮体和血糖浓度.荧光定量PCR技术检测黑质酪氨酸羟化酶(TH)、Bcl-2及caspase-3基因水平的表达情况. 结果 经过生酮饮食喂养后的小鼠经MPTP给药后,死亡率较正常饮食模型组降低;滚轴实验中实验组在转盘上停留时间较正常饮食模型组延长,差异均有统计学意义(P<0.05).实验组和生酮饮食组的血清酮体浓度较其他2组明显升高,差异有统计学意义(P<0.05).荧光定量PCR检测发现,与正常饮食模型组相比,实验组黑质中TH基因的表达明显升高,抗凋亡作用的Bcl-2基因也明显升高,促凋亡作用的caspase-3基因的表达明显减少,差异均有统计学意义(P<0.05). 结论 生酮饮食逆转了MPTP给药后的黑质中Bcl-2基因表达的下调和caspase-3基因表达的上调,进而抑制了多巴胺能神经元的凋亡,起到了保护黑质多巴胺能神经元的作用.%Objective To investigate the anti-apoptotic effect of ketogenic diet (KD) on substantia nigra dopaminergic neurons in mouse models with PD.Methods Eight-week-old male C57BL/6 mice, weighting 24-26 g, were randomly divided into normal diet and model group (1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine [MPTP]), experimental group (KD+MPTP), normal diet group and KD group. Mouse models with PD were established by subjecting them to intraperitoneal injection of MPTP. All the mice in the normal diet group and MPTP group were fed with normal diet and were free to access food, while mice in the KD+MPTP and KD

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

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Aliparasti

    2015-06-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Combined expression of gastrointestinal hormone SP and anti-apoptosis geneBcl-2 in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Yan Ling Feng; Qin Xian Zhang; Sheng Lei Li

    2000-01-01

    AIM To study the combined expression of gastrointestinal hormone substance P and anti-apoptosis gene Bcl-2 in gastric carcinoma and its significance.METHODS Substance P and Bcl-2 protein expression was examined by the S-P immunohistochemicalmethod in 33 cases of gastric carcinoma, 17 adjacent the carcinoma and 13 normal gastric mucoma.RESULTS Positive expression of SP in gastric carcinoma was higher than that of both adjacent and normalmucosa (P 0.05). The expression of bcl-2 both in gastric carcinoma and adjacent tissues werehigher than that of normal gastric mucosa (P< 0.05-0.01). But the positive expression of Bcl-2 had nostatistical significance between gastric carcinoma and adjacent tissues.CONCLUSION Both gastrointestinal hormone SP and Bcl-2 gene have synergistic expression in gastriccarcinoma, indicating that they all take part in the occurrence of gastric carcinoma. Abnormal expression ofBcl-2 gene occurred in benign gastric pathological changes, once they become carcinoma, the positiveexpression of cell is no more increased, possibly because that there is no more increase of the intensity of Bcl-2 inhibition of cell apoptosis.

  10. The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Vogler, Meike, E-mail: mv62@le.ac.uk [MRC Toxicology Unit, University of Leicester, LE1 9HN Leicester (United Kingdom); Dickens, David, E-mail: David.Dickens@liverpool.ac.uk [Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, L69 3GL Liverpool (United Kingdom); Dyer, Martin J.S., E-mail: mjsd1@le.ac.uk [MRC Toxicology Unit, University of Leicester, LE1 9HN Leicester (United Kingdom); Owen, Andrew, E-mail: aowen@liverpool.ac.uk [Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, L69 3GL Liverpool (United Kingdom); Pirmohamed, Munir, E-mail: munirp@liv.ac.uk [Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, L69 3GL Liverpool (United Kingdom); Cohen, Gerald M., E-mail: gmc2@le.ac.uk [MRC Toxicology Unit, University of Leicester, LE1 9HN Leicester (United Kingdom)

    2011-05-06

    Highlights: {yields} The BCL2-inhibitor ABT-263 is a substrate for P-glycoprotein. {yields} Apoptosis is inhibited by P-glycoprotein expression. {yields} Overexpression of P-glycoprotein may contribute to resistance to ABT-263 or ABT-737. -- Abstract: Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using {sup 3}H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

  11. Expression and its significance of Beclin-1 and Bcl-2 in prostatic adenocarcinoma%前列腺腺癌中Beclin-1和Bcl-2的表达及意义

    Institute of Scientific and Technical Information of China (English)

    肖芹; 顾学文; 田秀春; 王翠梅

    2013-01-01

    目的:探讨 Beclin-1和 Bcl-2在前列腺腺癌中的表达及意义方法采用免疫组化 PV-6000法检测76例前列腺腺癌和40例前列腺结节性增生组织中Beclin-1和Bcl-2的表达。结果在前列腺腺癌组织中,Beclin-1、Bcl-2的阳性表达率分别为47.4%、39.5%;在前列腺结节性增生组织中Beclin-1、Bcl-2的阳性表达率分别为95%、12.5%。Beclin-1、Bcl-2的表达在前列腺腺癌中与前列腺结节性增生组织中有明显差异(P均<0.01);同时两者的表达与前列腺腺癌组织的Gleason评分分级有相关性,Gleason评分越高,Beclin-1的表达越弱,而Bcl-2的表达越强(P<0.05);Beclin-1在前列腺腺癌中的阳性表达与Bcl-2的阳性表达呈负相关性(r=-0.235,P<0.05)。结论 Beclin-1和Bcl-2与前列腺腺癌的发生、发展有关,联合检测有助于临床预测患者预后。%Objective To study the expression and its significance of Beclin-1 and Bcl-2 in prostatic adenocarcinoma. Methods The expression of Beclin-1 and Bcl-2 was detected in 76 cases of prostatic adenocarcinoma,40 cases of benign prostatic hypertrophy by using immunohistochemistry of PV-6000. Results The positive expression rate of Beclin-1 and Bcl-2 were 47.4%, 39.5%in prostatic adenocarcinoma, and were 95%, 12.5%in benign prostatic hypertrophy,respectively. There were statistic significant difference of the the positive expression of Beclin-1, Bcl-2 between prostatic adenocarcinoma and benign prostatic hypertrophy.The positive expression of Beclin-1 and Bcl-2 were correlated with Gleason histologic grade of prostatic adenocarcinoma .The score of Gleason histologic grade of prostatic adenocarcinoma was higher, the positive expression of Beclin-1 was weaker, and that of Bcl-2 was stronger. The expression of Beclin-1 was reversely correlated with the expression of Bcl-2 in prostatic adenocarcinoma. Conclusion Beclin-1 and Bcl-2 play a certain role in the development, progression and

  12. Effect of U-74389G on apoptosis and bcl-2 expression following traumatic brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    骆纯; 卢亦成; 朱诚; 江基尧

    2003-01-01

    Objective: To investigate the relationship between oxidative stress and apoptosis and bcl-2 expression following traumatic brain injury (TBI). Methods: Male Sprague-Dawley rats were subjected to lateral fluid percussion brain injury (FPBI) of moderate severity. U-74389G (20 mg/kg) were administered intravenously before FPBI. The neurological functions were measured by beam-walk task (BWT) and beam-balance task (BBT). In addition to morphological evidence of apoptosis, TUNEL histochemistry was used to identify DNA fragmentation in situ with both light and electron microscopic levels. The internucleosomal fragments of DNA in apoptotic cells were examined using agarose gel electrophoresis. Bcl-2 protein expression was detected by immunohistochemistry. Results: The scores of BWT and BBT were significantly improved (P<0.01) in the treated animals. The treatment significantly reduced the number of apoptotic cells that was counted in the areas of the injured hemisphere at various time points following TBI. No DNA ladder was detected in the treated rats. Bcl-2 expression was observed in the cerebral cortex, subcortical white matter, dentate gyrus, hippocampal CA1 and CA3 region ipsilateral to injured hemisphere. Bcl-2 positive cells displayed normal nuclear morphology; Little Bcl-2 positive cells revealed morphological feature of apoptosis or necrosis. The immunoreactivity of Bcl-2 protein decreased significantly in the hippocampus ipsilateral impact site as early as 6 h post-injury. During 1-3 d after injury, the bcl-2 protein expression decreased relatively slow. In the U-74389G treated groups, the downregulation of bcl-2 expression was halted. Conclusion: In this model, apoptosis is associated with an activation of lipid peroxidation. U-74389G may block oxidative stress and halt the downregulation of bcl-2 expression. These may be one of the molecular mechanisms of the neuro-protective effects by U-74389G.

  13. EXPRESSIONS OF P53, PROLIFERATING CELL NUCLEAR ANITIGEN, BCL-2 PROTEIN AND THEIR SIGNIFICANCE IN SALIVARY ADENOID CYSTIC CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To study the effects of P53, PCNA, Bcl-2 protein and their relationship in salivary adenoid cystic carcinoma(SACC). Methods These proteins were examined by immunohistochemistry. Results Overexpressions of P53 and PCNA were revealed in ACC samples, they were higher than those in (polymorphous adenomas) PA, but expression of Bcl-2 protein was not different between ACC and PA. In 3 subtypes of ACC, expressions of 3 proteins were different. Conclusion Mutations of P53, Bcl-2 may be involed in the occurrence of SACC, expression of PCNA and mutation of P53 may coexist in the development of the SACC.

  14. Notch pathway is involved in high glucose-induced apoptosis in podocytes via Bcl-2 and p53 pathways.

    Science.gov (United States)

    Gao, Feng; Yao, Min; Shi, Yonghong; Hao, Jun; Ren, Yunzhuo; Liu, Qingjuan; Wang, Xiaomeng; Duan, Huijun

    2013-05-01

    Recent studies have shown that Notch pathway plays a key role in the pathogenesis of diabetic nephropathy (DN), however, the exact mechanisms remain elusive. Here we demonstrated that high glucose (HG) upregulated Notch pathway in podocytes accompanied with the alteration of Bcl-2 and p53 pathways, subsequently leading to podocytes apoptosis. Inhibition of Notch pathway by chemical inhibitor or specific short hairpin RNA (shRNA) vector in podocytes prevented Bcl-2- and p53-dependent cell apoptosis. These findings suggest that Notch pathway mediates HG-induced podocytes apoptosis via Bcl-2 and p53 pathways.

  15. Behavior of solvent-exposed hydrophobic groove in the anti-apoptotic Bcl-XL protein: clues for its ability to bind diverse BH3 ligands from MD simulations.

    Directory of Open Access Journals (Sweden)

    Dilraj Lama

    Full Text Available Bcl-XL is a member of Bcl-2 family of proteins involved in the regulation of intrinsic pathway of apoptosis. Its overexpression in many human cancers makes it an important target for anti-cancer drugs. Bcl-XL interacts with the BH3 domain of several pro-apoptotic Bcl-2 partners. This helical bundle protein has a pronounced hydrophobic groove which acts as a binding region for the BH3 domains. Eight independent molecular dynamics simulations of the apo/holo forms of Bcl-XL were carried out to investigate the behavior of solvent-exposed hydrophobic groove. The simulations used either a twin-range cut-off or particle mesh Ewald (PME scheme to treat long-range interactions. Destabilization of the BH3 domain-containing helix H2 was observed in all four twin-range cut-off simulations. Most of the other major helices remained stable. The unwinding of H2 can be related to the ability of Bcl-XL to bind diverse BH3 ligands. The loss of helical character can also be linked to the formation of homo- or hetero-dimers in Bcl-2 proteins. Several experimental studies have suggested that exposure of BH3 domain is a crucial event before they form dimers. Thus unwinding of H2 seems to be functionally very important. The four PME simulations, however, revealed a stable helix H2. It is possible that the H2 unfolding might occur in PME simulations at longer time scales. Hydrophobic residues in the hydrophobic groove are involved in stable interactions among themselves. The solvent accessible surface areas of bulky hydrophobic residues in the groove are significantly buried by the loop LB connecting the helix H2 and subsequent helix. These observations help to understand how the hydrophobic patch in Bcl-XL remains stable in the solvent-exposed state. We suggest that both the destabilization of helix H2 and the conformational heterogeneity of loop LB are important factors for binding of diverse ligands in the hydrophobic groove of Bcl-XL.

  16. MDM-2、bcl-2 和Ki-67在子宫颈癌中的表达及其意义%Expression and Evaluation of MDM-2, bcl-2 and Ki-67 in Cervical Carcinoma

    Institute of Scientific and Technical Information of China (English)

    李宏艳; 张建中

    2008-01-01

    目的 探讨MDM-2、bcl-2和Ki-67在子宫颈癌的发生、发展过程中的作用.方法 采用免疫组化法检测MDM-2、bcl-2和Ki-67在31例子宫颈癌、13例子宫颈非典型性增生、15例正常子宫颈组织中的表达水平.结果 在31例子宫颈癌组织中MDM-2表达阳性11例,bcl-2阳性13例,Ki-67阳性15例.bcl-2、MDM-2和Ki-67三者共同阳性9例(29.03%).对31例子宫颈癌组织中MDM-2与bcl-2共同阳性者、MDM-2与Ki-67共同阳性者及Ki-67与bcl-2共同阳性者进行配对资料χ2检验,P<0.05.子宫颈癌组织中bcl-2、MDM-2和Ki-6阳性表达率彼此间存在着明显的正性相关关系.结论 联合检测MDM-2、bcl-2和Ki-67在子宫颈癌中的表达,显示三者具有明显相关性,与子宫颈癌的发生、发展有关.

  17. The actions of Bcl-2 on anti-ischemic neuron injury and the effects of anti-ischemic drugs on Bcl-2%Bcl-2在缺血性神经细胞损伤中的作用及药物的影响

    Institute of Scientific and Technical Information of China (English)

    张予阳; 史琳琳; 郝冬海; 潘丽红

    2010-01-01

    Bcl-2基因是线虫c-elegans的抗凋亡基因Ced-9的人类同源基因,可参与调控细胞凋亡过程,包括抑制脑缺血半影区的神经细胞凋亡.该文就Bcl-2抗缺血性神经细胞损伤的作用和可能机制,以及抗脑缺血药物对Bcl-2的影响作一综述.

  18. Regulation of Calcium Fluxes and Apoptosis by BCL-2 Family Proteins in Prostate Cancer Cells

    Science.gov (United States)

    2006-02-01

    Carcinogenesis 2004;25:1089–97. 51. Scaffidi C, Volkland J, Blomberg I, Hoffmann I, Krammer PH, Peter ME. Phosphorylation of FADD/ MORT1 at serine 194 and...association with a 70-kDa cell cycle-regulated protein kinase. J Immunol 2000;164: 1236–42. 52. Alappat EC, Volkland J, Peter ME. Cell cycle effects by C

  19. Brain-derived neurotrophic factor and Bcl-2 expression in rat brain areas following chronic morphine treatment

    Institute of Scientific and Technical Information of China (English)

    Huiping Yu; Hua Hu; Huaqing Meng; Wei Deng; Yixiao Fu; Qinghua Luo

    2011-01-01

    The ventral tegmental area and the locus coeruleus are associated with psychological and physical dependence of opioid addiction. To date, very little is known about brain-derived neurotrophic factor (BDNF) and Bcl-2 gene and protein changes following morphine addiction. The present study utilized immunohistochemistry and in situ hybridization techniques, which revealed that there were increased BDNF levels, but decreased Bcl-2 levels in the prefrontal cortex, locus coeruleus, hippocampus, and the ventral tegmental area during morphine-dependence formation and abstinence. However, the levels of BDNF remained unchanged, and Bcl-2 expression was increased in the nucleus accumbens. These results showed that BDNF and Bcl-2 are involved in the development of morphine dependence, and precipitation of abstinence syndrome.

  20. Bcl-2 GENE REARRANGEMENT DETERMINED BY PCR AS A MEAN TO DETECT MINIMAL RESIDUAL DISEASE IN MALIGNANT LYMPHOMAS

    Institute of Scientific and Technical Information of China (English)

    XIANG Zhi-fu; LU Yu-ying; LAI Yong-rong; CHEN Yan; LI Hui-yu; ZOU Ping

    1999-01-01

    Objective: To develop a sensitive method to detect minimal residual disease and to elucidate the significance of bcl-2 gene rearrangement in diagnosis and treatment of malignant lymphoma. Methods: Using polymerase chain reaction (PCR) to detect bcl-2 gene rearrangement and using serial dilution method to define the sensitivity of PCR. Results: In 9 different malignant lymphoma cell lines, Su-DHL-4 and Su-DHL-6 were shown bcl-2(MBR)/JH rearrangement, the sensitivity of PCR was 1:105. In 16 patients with follicular lymphoma, the peripheral blood and bone marrow were PCR positive in 4 cases both at initial diagnosis and after complete remission. Conclusion:Detection of bcl-2 gene rearrangement by PCR provides a sensitive and specific assay of minimal residual disease.It is helpful to improve staging of disease, prognosis and evaluation of the treatment results.

  1. Clinical significance of bcl-2 protein expression and classification algorithm in diffuse large B-cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    李敏

    2013-01-01

    Objective To investigate the clinical significance of bcl-2 protein expression and three classification algorithms including Hans model,Chan model and Muris model in patients with diffuse large B-cell lymphoma(DLBCL).

  2. [Dexamethasone affect on the expression of bcl-2 and mTOR genes in T-lymphocytes from healthy donors].

    Science.gov (United States)

    Fatkhullina, A R; Abramov, S N; Skibo, Iu V; Abramova, Z I

    2014-01-01

    Synthetic glucocorticoids are able to activate apoptosis in the cells by regulating the transcription of the respective genes. Effect of dexamethasone on apoptosis is an established fact. However, its influence on another program of cell death autophagy, is currently unproven. Therefore, in this paper we have analyzed the influence of dexamethasone on the expression of bcl-2 and mTOR genes in T-lymphocytes from healthy donors. The results showed that dexamethasone reduced the expression of bcl-2 and mTOR genes. However, the nature of the effect of dexamethasone on mTOR and bcl-2 expression was different: the expression of bcl-2 gene in the long-term cultivation was maintained at the same reduced level, while the expression of mTOR was first reduced and then increased.

  3. C-MYC and BCL2 translocation frequency in diffuse large B-cell lymphomas: A study of 97 patients

    Directory of Open Access Journals (Sweden)

    Bahar Akkaya

    2016-01-01

    Full Text Available Purpose: Diffuse large B-cell lymphoma (DLBCL is an aggressive non-Hodgkin lymphoma with marked biologic heterogeneity. MYC and BCL2 rearrangements have been reported in a proportion of DLBCLs, where they may be associated with an adverse clinical outcome. The aim of this study was to determine the frequency of MYC and BCL2 translocations in DLBCL and assess the prognostic impact in DLBCL patients. Materials and Methods:   In the present study, we evaluated the expression patterns of CD 10, BCL6, and MUM 1 by immunohistochemistry in 121 cases with DLBCL in tissue microarray (TMA: 62 cases in germinal center B-cells (GCBs; and 59 cases in activated B-cells (ABCs of which 60 were females and 61 were males. MYC and BCL2 rearrangements were investigated by interphase fluorescence in situ hybridization on TMAs in 97 DLBCLs. Result: MYC rearrangements were observed in 11 of 97 cases. There was no association with other clinical features, including age, sex, and nodal/extranodal disease. MYC rearrangement was associated with significantly worse overall survival (P < 0.01. BCL2 rearrangements were observed in 14 of 97 cases. There was no association with other clinical features including age and sex. BCL2 rearrangement had a worse outcome (P < 0.01. MYC and BCL2 rearrangements were observed in 3 of 97 cases with the age of  53 (female, 53, 63 years old, respectively, died in 24, 18, and 35 months after the diagnosis. Two cases had primary nodal and one case primary extranodal presentations. All these patients had stage IV disease. Conclusion: We concluded that C-MYC and BCL2 may contribute to aggressive transformation, and more mechanism-based therapy should be explored. Targeted therapies involving these rearrangements and its associated pathways may change the fate of DLBCLs. Analysis of MYC gene rearrangement along with BCL2 is critical in the identification of high-risk patients with poor prognosis.

  4. Perbedaan Ekspresi Bcl-2 Pada Hiperplasia Endometrium Non Atipik Simpleks Dan Kompleks Di RS.H.Adam Malik Medan

    OpenAIRE

    Rahim, Ricca Puspita

    2015-01-01

    Background : Endometrial hyperplasia is an excessive cellular proliferation which leads to the increase of endometrial tissue volume. Endometrial hyperplasia is a precursor of malignancy in women’s genitalia. Apoptosis may play a role in endometrial hyperplasia and regulated by several genes, including Bcl-2. Purpose : To find the difference between immunohistochemistry expression of Bcl-2 in simplex and complex non-atypical endometrial hyperplasia Method : This is an analyt...

  5. THE EXPERIMENTAL STUDY ON THE CELL APOPTOSIS AND EXPRESSION OF BCL-2 PROTEIN IN INTRACEREBRAL HEMORRHAGE IN MODEL OF RATS

    Institute of Scientific and Technical Information of China (English)

    Bao Gang; Guo Ning; Zhang Zhonglin; Chen Wei; Bao Dehu

    2006-01-01

    Otjective To study whether there is the apoptosis of neural cells and the expressionof Bcl-2 protein in intracerebral hemorrhage (ICH) in model of rats, for the further understanding the mechanism of the delayed damage of the neural cells around the hematoma after ICH. Methods Fifty SD rats were randomly divided into 5 groups, ten in each. With the Group A as the control, the rest 40 were used to set up intracerebral hemorrhage model. The brains were taken out at 12th, 24th, 48th and 72th hours, respectively. Apoptosis cells were detected with terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), and the expression of Bcl-2 protein was detected with immunochemical stainging methed (SP). Results In the control group, no apoptosis cells and Bcl-2protein were detected. In rest groups, the apoptosis cells and Bcl-2 protein were expressed in different degree.Apoptosis rates verified and corresponded with the time after ICH, with the peak at 48th -72th hour after hemorrhage.The peak rate of apoptosis cells was (24. 50± 2.69)% and Bcl-2 protein expression was (20. 76 ± 1.97)% . There was significant difference between the experimental groups and control (P<0.05), and no linear relationship between the apoptosis rate and the expression of Bcl-2 protein. Conclusion Apoptosis may be an important factor in the secondary trauma of ICH. There is a time leg after hemorrhage. All this is instructive to clinical treatment in time. Bcl-2 protein keeps increasing in a certain time after hemorrhage, but not synchronize with the cell apoptosis. This indicates that bcl-2 has the effect to reduce the apoptosis of neural cells.

  6. Direct regulation of BCL-2 by FLI-1 is involved in the survival of FLI-1-transformed erythroblasts

    OpenAIRE

    Lesault, Isabelle; Tran Quang, Christine; Frampton, Jon; Ghysdael, Jacques

    2002-01-01

    Rearrangement of the FLI-1 locus with ensuing overexpression of FLI-1 is an early event in Friend murine leukemia virus-induced disease. When overexpressed in primary erythroblasts, FLI-1 blocks erythropoeitin (Epo)-induced terminal differentiation and inhibits apoptosis normally induced in response to Epo withdrawal. We show here that the survival-inducing property of FLI-1 is associated with increased transcription of BCL-2. We further show that FLI-1 binds BCL-2 promoter sequences in trans...

  7. Folic acid prevents Bcl-2 hypomethylation in rats with hyperhomocysteinemia%叶酸拮抗高同型半胱氨酸血症大鼠Bcl-2基因低甲基化研究

    Institute of Scientific and Technical Information of China (English)

    丛广志; 贾绍斌; 罗彩琴; 王义勇

    2012-01-01

    Objective To investigate the effects of folic acid on Bcl-2 gene methylation status in rats with hyperhomocystinemia induced by ingestion of excess methionine. Methods 36 healthy 6-week-old wistar male rats, weighing(160 ± 10) g, after being fed adaptable for one week,were randomly divided into control group(n = 12) , hyperhomocysteinemia group (n = 12) , folic acid treatment group ( n = 12). The control group was fed with AIN-93G diet. The hyperhomocysteinemia group was fed with high-methionion diet, consisting of AIN-93G diet plus 1.7% methionion. The folic acid treatment group was fed with high-methionion plus folic acid-rich diet, consisting of AIN-93G diet plus 1.7% methionion and 0. 008% folic acid. After be maintained for 18 weekson the previously described diets, the concentrations in the plasma Hcy and folic acid and Vit B12 were measured with the IMX assays. The thoracic aorta was harvested for immunohist℃ hemical analysis. The methylation status of Bcl-2 gene was determined by nest touch-down PCR combined MSP( methylation specific PCR). Real-time RT PCR was used to detect mRNA expression of arotic Bcl-2. Results The study showed the following; (a) A high methionine diet for 18 weeks is sufficient to induce hyperhomt ystinemia; Folic acid supplementation to the rats fed the high-methionine diet prevented an elevation homocysteine (Hey) levels in the plasma ( P < 0. 01). (b) Compared with the control group, the Hhcy group had a elevating Bcl-2 expression by immunohistochemical analysis in aorta, along with Bcl-2 hypomethylation ( P < 0. 05 ) and increased Bcl-2 mRNA expression ( P < 0. 05 ). (c) Most important, after folic acid supplementation, the lowering of Hey levels was accompanied by a marked decreased Bcl-2 expression by immunohistochemical analysis and Bcl-2 hypermethylation ( P < 0. 05 ) and reduced Bcl-2 mRNA expression ( P < 0. 05 ). Conclusions Folic acid supplementation can prevents Bcl-2 hypomethylation in rats with hyperhomocysteinemia

  8. The pan-Bcl-2 blocker obatoclax promotes the expression of Puma, Noxa, and Bim mRNA and induces apoptosis in neoplastic mast cells.

    Science.gov (United States)

    Peter, Barbara; Cerny-Reiterer, Sabine; Hadzijusufovic, Emir; Schuch, Karina; Stefanzl, Gabriele; Eisenwort, Gregor; Gleixner, Karoline V; Hoermann, Gregor; Mayerhofer, Matthias; Kundi, Michael; Baumgartner, Sigrid; Sperr, Wolfgang R; Pickl, Winfried F; Willmann, Michael; Valent, Peter

    2014-01-01

    Advanced SM is an incurable neoplasm with short survival time. So far, no effective therapy is available for these patients. We and others have shown recently that neoplastic MC in ASM and MCL express antiapoptotic Mcl-1, Bcl-2, and Bcl-xL. In this study, we examined the effects of the pan-Bcl-2 family blocker obatoclax (GX015-070) on primary neoplastic MC, the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Obatoclax was found to inhibit proliferation in primary human neoplastic MC (IC₅₀: 0.057 μM), in HMC-1.2 cells expressing KIT D816V (IC₅₀: 0.72 μM), and in HMC-1.1 cells lacking KIT D816V (IC₅₀: 0.09 μM), as well as in C2 cells (IC₅₀: 0.74 μM). The growth-inhibitory effects of obatoclax in HMC-1 cells were accompanied by an increase in expression of Puma, Noxa, and Bim mRNA, as well as by apoptosis, as evidenced by microscopy, TUNEL assay, and caspase cleavage. Viral-mediated overexpression of Mcl-1, Bcl-xL, or Bcl-2 in HMC-1 cells was found to introduce partial resistance against apoptosis-inducing effects of obatoclax. We were also able to show that obatoclax synergizes with several other antineoplastic drugs, including dasatinib, midostaurin, and bortezomib, in producing apoptosis and/or growth arrest in neoplastic MC. Together, obatoclax exerts major growth-inhibitory effects on neoplastic MC and potentiates the antineoplastic activity of other targeted drugs. Whether these drug effects can be translated to application in patients with advanced SM remains to be determined.

  9. Induction of apoptosis in HepG2 cells by solanine and Bcl-2 protein.

    Science.gov (United States)

    Ji, Y B; Gao, S Y; Ji, C F; Zou, X

    2008-01-17

    The nightshade (Solanum nigrum Linn.) has been widely used in Chinese traditional medicine as a remedy for the treatment of digestive system cancer. The anti-tumor activity of solanine, a steroid alkaloid isolated from the nightshade has been demonstrated. To observe the effect of anti-tumor and mechanism of solanine. The MTT assay was used to evaluate the IC(50) on the three digestive system tumor cell lines. The effect on the morphology was observed with a laser confocal microscopy; the rate of apoptosis and the cell cycle were measured using flow cytometry (FCM); the expression of Bcl-2 protein was measured by Western blot. The results show that the IC(50) for HepG(2), SGC-7901, and LS-174 were 14.47, >50, and >50 microg/ml, respectively; the morphology of cells in the negative control was normal; for the treated groups, typical signs for apoptosis were found. The rate of apoptosis in HepG(2) cells induced by solanine was found to be 6.0, 14.4, 17.3, 18.9, and 32.2%, respectively. Observation of the cell cycle showed that cells in the G(2)/M phases disappeared while the number of cells in the S phase increased significantly for treated groups. Western blot showed that solanine decreased the expression of Bcl-2 protein. Therefore, the target of solanine in inducing apoptosis in HepG(2) cells seems to be mediated by the inhibition in the expression of Bcl-2 protein.

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

    Institute of Scientific and Technical Information of China (English)

    刘巍; 李为民; 孙宁玲; 陈源源; 任哲; 虞有智

    2002-01-01

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

  11. Telomerase activity, estrogen receptors (α, β), Bcl-2 expression in human breast cancer and treatment response

    Science.gov (United States)

    Murillo-Ortiz, Blanca; Astudillo-De la Vega, Horacio; Castillo-Medina, Sebastian; Malacara, JM; Benitez-Bribiesca, Luis

    2006-01-01

    Background The mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences, lost during replication by means of an intrinsic RNA component as a template for polymerization. Among the telomerase subunits, hTERT (human telomerase reverse transcriptase) is expressed concomitantly with the activation of telomerase. The role of estrogens and their receptors in the transcriptional regulation of hTERT has been demonstrated. The current study determines the possible association between telomerase activity, the expression of both molecular forms of estrogen receptor (ERα and ERβ) and the protein bcl-2, and their relative associations with clinical parameters. Methods Tissue samples from 44 patients with breast cancer were used to assess telomerase activity using the TRAP method and the expression of ERα, ERβ and bcl-2 by means of immunocytochemical techniques. Results Telomerase activity was detected in 59% of the 44 breast tumors examined. Telomerase activity ranged from 0 to 49.93 units of total product generated (TPG). A correlation was found between telomerase activity and differentiation grade (p = 0.03). The only significant independent marker of response to treatment was clinical stage. We found differences between the frequency of expression of ERα (88%) and ERβ (36%) (p = 0.007); bcl-2 was expressed in 79.5% of invasive breast carcinomas. We also found a significant correlation between low levels of telomerase activity and a lack of ERβ expression (p = 0.03). Conclusion Lower telomerase activity was found among tumors that did not express estrogen receptor beta. This is the first published study demonstrating that the absence of expression of ERβ is associated with low levels of telomerase activity. PMID:16911782

  12. Telomerase activity, estrogen receptors (α, β, Bcl-2 expression in human breast cancer and treatment response

    Directory of Open Access Journals (Sweden)

    Malacara JM

    2006-08-01

    Full Text Available Abstract Background The mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences, lost during replication by means of an intrinsic RNA component as a template for polymerization. Among the telomerase subunits, hTERT (human telomerase reverse transcriptase is expressed concomitantly with the activation of telomerase. The role of estrogens and their receptors in the transcriptional regulation of hTERT has been demonstrated. The current study determines the possible association between telomerase activity, the expression of both molecular forms of estrogen receptor (ERα and ERβ and the protein bcl-2, and their relative associations with clinical parameters. Methods Tissue samples from 44 patients with breast cancer were used to assess telomerase activity using the TRAP method and the expression of ERα, ERβ and bcl-2 by means of immunocytochemical techniques. Results Telomerase activity was detected in 59% of the 44 breast tumors examined. Telomerase activity ranged from 0 to 49.93 units of total product generated (TPG. A correlation was found between telomerase activity and differentiation grade (p = 0.03. The only significant independent marker of response to treatment was clinical stage. We found differences between the frequency of expression of ERα (88% and ERβ (36% (p = 0.007; bcl-2 was expressed in 79.5% of invasive breast carcinomas. We also found a significant correlation between low levels of telomerase activity and a lack of ERβ expression (p = 0.03. Conclusion Lower telomerase activity was found among tumors that did not express estrogen receptor beta. This is the first published study demonstrating that the absence of expression of ERβ is associated with low levels of telomerase activity.

  13. Rapamycin increases pCREB, Bcl-2, and VEGF-A through ERK under normoxia

    Institute of Scientific and Technical Information of China (English)

    Yudong Liu; Qixin Zheng; Hongbin Wu; Xiaodong Guo; Jingfeng Li; Shaofei Hao

    2013-01-01

    Rapamycin may serve as a new anti-osteosarcoma (OSA) agent due to its ability to inhibit the metastatic behavior of OSA.However,only limited benefit is observed in rodent studies and clinical trials using rapamycin as a single agent in the treatment of OSA.The target of rapamycin,mammalian target of rapamycin has multiple biological functions and may be linked with the kinases that mediate the phosphorylation of cyclic AMP-responsive element-binding (CREB) protein,an import factor in tumor progression.By employing an OSA cell line MG-63,we investigated how rapamycin regulates the phosphorylation of CREB (pCREB) at Ser133 and the expressions of two putative CREB targets,B-cell lymphoma 2 (Bcl-2)and vascular endothelial growth factor-A (VEGF-A).Under normoxia,we found that rapamycin (100nM)induced an increase of pCREB that was prevented by mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126 or cAMP-dependent protein kinase (PKA) inhibitor H89.However,H89 enhanced Akt phosphorylation and did not decrease the cell viability upon rapamycin treatment.In contrast,U0126 did not enhance Akt phosphorylation and decreased the cell viability upon rapamycin treatment.Moreover,U0126 prevented the rapamycin-induced increase of Bcl-2 and VEGF-A levels.Under hypoxia,rapamycin effectively prevented the hypoxia-induced increase of pCREB,Bcl-2,and VEGF-A.Our study demonstrated that rapamycin might be less effective in treating OSA cells under normoxia and provided the rationale for a combination of rapamycin and MEK/ERK inhibitor in the treatment of OSA.

  14. Bcl-2 and N-Myc Coexpression Increases IGF-IR and Features of Malignant Growth in Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Rama Jasty

    2001-01-01

    Full Text Available The bcl-2 and c-myc oncogenes cooperate to transform multiple cell types. In the pediatric malignancy NB2, Bcl2 is highly expressed. In tumors with a poor prognosis, N-Myc, a protein homologous to c-Myc, is overexpressed as a result of gene amplification. The present study was designed to determine whether Bcl-2 cooperates with N-Myc to bestow a tumorigenic phenotype to neuroblastoma (NB cells. NB cell lines that at baseline express neither Bcl-2 nor N-Myc were stably transfected to express these gene products. In this model, we found Bcl-2 rescues N-Myc-expressing cells from apoptosis induced by serum withdrawal. Coexpression of Bcl-2 and N-Myc supports growth in low serum conditions and anchorage-independent growth in soft agar. Similarly, in vivo tumorigenic and angiogenic activity was dependent on coexpression. Our data further suggests that the mechanism underlying these changes involves the receptor for insulin growth factor type I (IGF-IR.

  15. EFFECT OF BcL-2 ANTISENSE DRUG WITH DIFFERENT STRUCTURE ON THE BIOLOGICAL FUNCTION OF K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    雷小勇; 张洹; 何冬梅

    2004-01-01

    Objective: To study the differences and similarities of the antisense drugs with different structures on the biological functions of K562 cells. Methods: Cytotoxic effects were measured by use of a cell viability assay. Flow cytometric analysis and agarose gel electrophoresis of DNA fragmentation were also performed. The expression level of protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Results: PNA targeting the coding region of the Bcl-2 messenger RNA could effectively inhibit K562 cell viability, down-regulate the synthesis of the Bcl-2 protein and increase cell apoptosis. By 72 h after the Bcl-2 antisense PNA treatment, K562 cells showed more reduction in the level of Bcl-2 protein compared with cells treated with the antisense ODN. After treatment with 10μmol/L of Bcl-2 antisense PNA or antisense ODN for 72 h, apoptotic rates of K562 cells were 13.15±1.13 and 11.72±1.12, respectively. Furthermore, there was significant difference in the percentage of apoptotic cells between antisense PNA group and antisense ODN group. Conclusion: The results suggest that antisense PNA targeting the coding region of Bcl-2 mRNA has better antisense effects than the antisense oligonucleotides on inducing apoptosis of K562 cells.

  16. Mechanism of regulation of bcl-2 mRNA by nucleolin and A+U-rich element-binding factor 1 (AUF1).

    Science.gov (United States)

    Ishimaru, Daniella; Zuraw, Lisa; Ramalingam, Sivakumar; Sengupta, Tapas K; Bandyopadhyay, Sumita; Reuben, Adrian; Fernandes, Daniel J; Spicer, Eleanor K

    2010-08-27

    The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3'-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3'-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5' end of the 136-nucleotide bcl-2 AU-rich element (ARE(bcl-2)). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to ARE(bcl-2). In RNA decay assays, ARE(bcl-2) transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of ARE(bcl-2) transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.

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

    OpenAIRE

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

    2000-01-01

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

  18. 雌激素对大鼠胸腺细胞凋亡及Bcl-2、Bax表达的影响%Effects of estrogen on apoptosis and expression of Bcl-2 and Bax in rat thymus

    Institute of Scientific and Technical Information of China (English)

    李雅娜; 孙研; 崔春红; 殷彦君

    2011-01-01

    目的:探讨苯甲酸雌二醇对大鼠胸腺Bcl-2和Bax表达及细胞凋亡的影响及其机制.方法:雌性大鼠行卵巢切除术,给予苯甲酸雌二醇后,观察胸腺指数的变化,Hochest33342荧光染色及透射电镜标本观察胸腺细胞凋亡情况,免疫组织化学检测胸腺组织中Bcl-2和Bax的表达情况,原位杂交技术检测Bcl-2、Bax m RNA的表达情况.结果:双侧卵巢切除组大鼠胸腺指数较假手术组增加,双侧卵巢切除+雌激素组大鼠胸腺指数较双侧卵巢切除组减小;假手术组和双侧卵巢切除组大鼠胸腺组织中以正常胸腺细胞为主,偶见凋亡细胞或凋亡小体,双侧卵巢切除+雌激素组可见较多凋亡细胞和凋亡小体;双侧卵巢切除+雌激素组大鼠胸腺组织中Bcl-2表达较双侧卵巢切除组和假手术组增高明显降低,而Bax表达呈现相反趋势;Bcl-2 mRNA、Bax mRNA的表达与Bcl-2、Bax的表达呈一致性.结论:雌激素可以降低大鼠胸腺指数,抑制胸腺组织中Bcl-2的表达,促进Bax的表达,从而诱导大鼠胸腺细胞凋亡,促进雌性大鼠胸腺退化.%Objective-. To explore the effects of estrogen on the apoptosis and the expression of Bcl-2 and Bax in rat thymus. Methods-. The rats performed with ovariectomy were injected with estradiol benzoate. Thymus was obtained 7 days after the injection. Thymic indexes were measured. Apoptosis of the thymus was detected after Hochest 33342 staining and examined under an electron microscope. The expression of Bcl-2 and Bax in the thymus was detected with a immunohistochemical method. The expression of Bcl-2 mRNA and Bax mRNA in the thymus was detected by in situ hybridization. The test was taken in statistical treatment. Results: The thymus quality index in ovariectomy group was higher than that in the model control group. The thymus quality index of rats injected with estradiol benzoate was reduced respectively. Apoptotic cells and apoptotic bodies were found in the

  19. Functional expression of CD95/Fas Antigen and Bcl-2 on Cord blood Hematopoietic Progenitor Cells

    Institute of Scientific and Technical Information of China (English)

    马艳萍; 邹萍

    2002-01-01

    The cell-surface expression and functional status of the CD95/Fas antigen on primitive hematopoietic progenitors isolated from human cord blood (CB) were studied. The CD34+ cells freshly isolated from CB displayed low CD95 expression. The combinations of cytokines such as SCF+FL could up-regulate the expression of CD95 in vitro culture and tumor necrosis factor-e (TNF-α) and interon-γ (IFN-γ) further increased the CD95 expression induced by positive cytokines. The functional status of CD95-mediated apoptosis were analyzed by incubation of CD34+CB cells in the presence of anti-CD95 monoclonal antibodies (McAbs). The effects of anti-CD95 McAbs were measured by viable cell counting, flow cytometry, LTIC and CFU-C assays. A decrease of viable cells, CFUC and LTIC numbers were observed in the presence of anti-CD95 McAbs and TNF-α or IFN-γ.However, growth factor deprivation or the early-acting cytokine such as SCF and FL cross-linking to CD95 caused low apoptosis of CD34+ cells. The correlation of increased intracytoplasmic levels of bcl2 and the presence of CD95 on fresh CB CD34+ cells suggested that bcl-2 might be involved in protecting against CD95-mediated apoptosis of CB CD34+ cells.

  20. Nesfatin-1 protects dopaminergic neurons against MPP+/MPTP-induced neurotoxicity through the C-Raf–ERK1/2-dependent anti-apoptotic pathway

    Science.gov (United States)

    Shen, Xiao-Li; Song, Ning; Du, Xi-Xun; Li, Yong; Xie, Jun-Xia; Jiang, Hong

    2017-01-01

    Several brain-gut peptides have been reported to have a close relationship with the central dopaminergic system; one such brain-gut peptide is nesfatin-1. Nesfatin-1 is a satiety peptide that is predominantly secreted by X/A-like endocrine cells in the gastric glands, where ghrelin is also secreted. We previously reported that ghrelin exerted neuroprotective effects on nigral dopaminergic neurons, which implied a role for ghrelin in Parkinson’s disease (PD). In the present study, we aim to clarify whether nesfatin-1 has similar effects on dopaminergic neurons both in vivo and in vitro. We show that nesfatin-1 attenuates the loss of nigral dopaminergic neurons in the 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. In addition, nesfatin-1 antagonized 1-methyl-4-phenylpyridillium ion (MPP+)-induced toxicity by restoring mitochondrial function, inhibiting cytochrome C release and preventing caspase-3 activation in MPP+-treated MES23.5 dopaminergic cells. These neuroprotective effects could be abolished by selective inhibition of C-Raf and the extracellular signal-regulated protein kinase 1/2 (ERK1/2). Our data suggest that C-Raf-ERK1/2, which is involved in an anti-apoptotic pathway, is responsible for the neuroprotective effects of nesfatin-1 in the context of MPTP-induced toxicity. These results imply that nesfatin-1 might have therapeutic potential for PD. PMID:28106099

  1. Lactobacillus acidophilus Increases the Anti-apoptotic Micro RNA-21 and Decreases the Pro-inflammatory Micro RNA-155 in the LPS-Treated Human Endothelial Cells.

    Science.gov (United States)

    Kalani, Mehdi; Hodjati, Hossein; Sajedi Khanian, Mahdi; Doroudchi, Mehrnoosh

    2016-06-01

    Given the anti-inflammatory and protective role of probiotics in atherosclerosis and the regulatory role of micro RNA (miRNA) in endothelial cell (dys) functions, this study aimed to investigate the effect of Lactobacillus acidophilus (La) on cellular death and the expression of miRNA-21, 92a, 155, and 663 in human umbilical vein endothelial cell (HUVEC) induced by Escherichia coli lipopolysaccharide (Ec-LPS). LPS-treated and untreated HUVECs were cultured in the presence of different La conditions such as La-conditioned media (LaCM), La water extract (LaWE), La culture-filtered (LaFS) and unfiltered supernatants (LaUFS). After 24 h, apoptosis, necrosis and the levels of the mentioned miRNAs were measured using flow cytometry and real-time PCR methods, respectively. LaCM decreased apoptosis, necrosis and inflammatory miR-155 and conversely increased anti-apoptotic miR-21 in Ec-LPS-treated HUVECs. Association analysis revealed negative correlations between necrosis and the levels of miR-21, miR-92a, and miR-155. The beneficial effects of L. acidophilus on the ECs death and expression of atherosclerosis related miRNAs in these cells imply a new aspect of its regulation in cardiovascular diseases rather than previously described ones and suggest this probiotic bacterium as a candidate in the preventative therapy of atherosclerosis.

  2. Anti-apoptotic and hepatoprotective effects of gomisin A on fulminant hepatic failure induced by D-galactosamine and lipopolysaccharide in mice.

    Science.gov (United States)

    Kim, Sung-Hwa; Kim, Yeong Shik; Kang, Sam Sik; Bae, Kihwan; Hung, Tran Manh; Lee, Sun-Mee

    2008-02-01

    This study examined the effects of gomisin A, a lignan compound from Schisandra fructus, on D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic apoptosis and liver failure. Mice were given an intraperitoneal injection of GalN (700 mg/kg) / LPS (10 microg/kg). Gomisin A (25, 50, 100, and 200 mg/kg) was administered intraperitoneally 1 h before the GalN/LPS injection. The liver injury was assessed biochemically and histologically. GalN/LPS increased the serum aminotransferase levels and lipid peroxidation but decreased the reduced glutathione level. The pretreatment with gomisin A attenuated these changes in a dose-dependent manner. The survival rate of the gomisin A group was significantly higher than that of the control. The mitochondria isolated after the mice had been injected with GalN/LPS were swollen, which was attenuated by the gomisin A pretreatment. The elevation of serum tumor necrosis factor-alpha and activation of caspase-3 were observed in the GalN/LPS group, which was attenuated by gomisin A. The gomisin A-pretreated groups showed significantly fewer apoptotic (TUNEL-positive) cells and DNA fragmentation as compared with the GalN/LPS mice. The liver protection afforded by gomisin A is the result of the reduced oxidative stress and its anti-apoptotic activity.

  3. Bcl-2 Expression in the Aortas of Model Rats with Aldosterone-Producing Adenomas%Bcl-2在醛固酮腺瘤模型大鼠主动脉中的表达

    Institute of Scientific and Technical Information of China (English)

    闫永吉; 陈戬; 刘建和; 张劲松; 姜永明; 王光; 张海燕

    2011-01-01

    目的:观察醛固酮对主动脉Bcl-2表达的影响.方法:32只SD大鼠随机均分为4组:①空白溶剂设为对照(60%丙二醇+10%乙醇+30%双蒸水);②醛固酮腺瘤:泵入醛固酮(1μg/h);③醛固酮腺瘤+依普利酮组:依普利酮(100 mg·kg-1·d-1);④醛固酮腺瘤+肼苯哒嗪组:肼苯哒嗪(25 mg·kg-1·d-1).渗透泵内分别注入醛固酮或空白溶剂,然后将其埋植于大鼠背部皮下.8周后,免疫组织化学和免疫印迹检测主动脉Bcl-2的表达.结果:与对照组相比,腺瘤组大鼠主动脉Bcl-2表达显著减少(P<0.05);依普利酮能够拮抗醛固酮对Bcl-2表达的抑制作用(P<0.05).结论:醛固酮通过抑制Bcl-2表达,调节细胞凋亡状态,可能是醛固酮诱导主动脉重构的机制之一.%Objective: To observe the effect of aldosterone on Bcl-2 expression in the aorta. Methods: Thirtytwo male rats were randomly divided into 4 groups; vehicle (control), aldosterone-producing adenoma, aldosterone-producing adenoma plus eplerenone or hydralazine. They were then implanted with an osmotic mini-pump that infused either aldosterone or the vehicle. After 8 weeks, Bcl-2 was examined by immunohistochemistry and Western blotting. Results:Compared with controls, the model rats with aldosterone-producing adenoma exhibited decreased aortic expression of Bcl-2. This effect of aldosterone was significantly reversed after co-treatment with eplerenone but not with hydralazine. Conclusions:This study's findings suggest that aldosterone directly suppresses aortic expression of Bcl-2 that may play an important role in aldosterone-induced aortic remodeling.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  5. Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

    Science.gov (United States)

    Chen, Ying-Jung; Liu, Wen-Hsin; Kao, Pei-Hsiu; Wang, Jeh-Jeng; Chang, Long-Sen

    2010-06-15

    CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation.

  6. Anti-tumour activity of a novel coumarin-chalcone hybrid is mediated through intrinsic apoptotic pathway by inducing PUMA and altering Bax/Bcl-2 ratio.

    Science.gov (United States)

    Singh, Neetu; Sarkar, Jayanta; Sashidhara, Koneni V; Ali, Shakir; Sinha, Sudhir

    2014-06-01

    Coumarins and chalcones are secondary plant metabolites which have shown an array of pharmacological properties including anti-tumour activity. We have previously reported on the synthesis and anti-proliferative activity of a series of novel coumarin-chalcone hybrids. Now we report on the in vivo efficacy as well as mechanism of action of the most potent molecule of the series, S009-131. Oral administration of this molecule resulted in regression of tumours induced by HeLa cell xenografts in nod SCID mice. The molecule inhibited proliferation of cervical cancer cells (HeLa and C33A) by inducing apoptosis and arresting cell cycle at G2/M phase. Apoptosis was induced through induction of caspase-dependent intrinsic pathway and alterations in the cellular levels of Bcl-2 family proteins. The mitochondrial transmembrane potential got highly depleted in S009-131 treated cells due to an increase in Bax/Bcl-2 ratio and intracellular ROS. The molecule induced release of cytochrome c into the cytosol and activation of initiator caspase-9 and executioner caspases-3/7. Tumour suppressor protein p53 and its transcriptional target PUMA were up regulated, suggesting their role in mediating the cell death. These results suggest that S009-131 is a potent candidate for the chemotherapy of cervical carcinoma.

  7. Bcl-2基因与神经生长因子(NGF)抑制神经细胞凋亡的研究%Abaissement role of bcl-2 gene and NGF on the apoptosis of nerve cel s

    Institute of Scientific and Technical Information of China (English)

    韩贵和; 魏威; 顾军

    2013-01-01

    目的探讨原癌基因bcl-2与神经生长因子(NGF)联合应用对抑制神经细胞凋亡的协同作用。方法培养PC12细胞至对数生长期,用100感染复数(multiplicity of infection,MOI)的携带bcl-2基因的慢病毒质粒及未携带bcl-2基因的慢病毒质粒感染PC12细胞。再将其分为A、B、C、D、E、F六组,A组为bcl-2-PC12细胞培养液中不加H2O2及NGF(bcl-2-PC12组);B组为bcl-2-PC12细胞培养液中加H2O2(bcl-2-PC12+ H2O2组);C组为bcl-2-PC12细胞培养液中加H2O2及NGF(bcl-2-PC12+ H2O2+NGF组)。D组为NC-PC12细胞培养液中不加H2O2及NGF(NC-PC12组);E组为NC-PC12细胞培养液中加H2O2(NC-PC12+H2O2组);F组为NC-PC12细胞培养液中加H2O2及NGF(NC-PC12+ H2O2+NGF组)。应用流式细胞仪检测各组细胞的凋亡率,采用BCA(bicinchoninic acid)法检测bcl-2基因表达蛋白浓度,数据进行统计学分析。结果 A组细胞凋亡率较D组低(u=2.16,0.02bcl-2基因表达蛋白浓度高于D组(t=2.87,0.005bcl-2基因及NGF均对正常神经细胞的凋亡有抑制作用,能够增强神经细胞的抗损伤能力,二者联合应用时具有协同作用。%Objective To approach synergia abaissement role of the proto-oncogene bcl-2 and NGF on the apoptosis of nerve cells.Methods PC12 cells were cultured to the logarithmic growth phase,slow virus plasmid carrying the bcl-2 gene and slow virus plasmid infected respectively PC12 cells by 100 MOI.Then they were divided into six groups(groupA,groupB,groupC ,groupD,groupE and groupF ). Group A:PC12 cells with slow virus plasmid carrying the bcl-2 gene.Group B: PC

  8. 胃癌变过程中Hp感染与凋亡基因Bcl-2表达相关性研究%Correlation between Helicobacter pylori infection and expression of Bcl - 2 gene in carcinogenesis of gastric muco

    Institute of Scientific and Technical Information of China (English)

    刘爱群; 葛莲英; 罗元; 林思彤; 韦宗萍

    2011-01-01

    Objective To study the correlation among Helicobacter pylori ( Hp ) infection, expression and clinical significance of Bcl -2 in carcinogenesis of gastric mucosa. Methods Hp infection was detected by rapid urease Test, methylene blue and Warthin -Starry staining, while the expression of Bcl -2 and cell apoptosis were evaluated with immunohistochemical staining and TUNEL staining, respectively, in 62 cases of chronic superficial gastris ( CSG ), 55 cases of chronic atrophic gastric ( CAG ), 52 cases of intestinal melaplasia ( IM ), 46 cases of atypical hyperplasia ( AH ) and 65 cases of gastric carcinoma ( GC ). Results The infection of Hp and the positive Bcl - 2 expression increased with the carcinogenesis of gastric mucosa. The infection rates of Hp in CAG, IM, AH and GC were significantly higher than that in CSG ( P < 0. 05 ), with higher rate in GC than that in CAG ( P < 0. 05 ). Significantly higher expression of Bcl - 2 was revealed in IM, AH and GC than that in CSG ( P < 0. 05 ), so was in GC than that in IM ( P < 0. 01 ). Significantly higher positive rates of Bcl - 2 expression were observed in AH and GC with positive Hp infection than those with negative Hp infection ( P < 0. 05 ). The apoptosis indexes with positive Bcl - 2 in IM, AH and GC were significantly lower than those with negative Bcl - 2 ( P < 0. 05 ). Furthermore, the expression of Bcl - 2 protein in GC with Hp infection was closely correlated with tissue differentiation. The expression of Bcl - 2 with undifferentiation or poorly - differentiation was significantly higher than that with well - differentiation ( P < 0. 05 ). Conclusion Hp infection up - regulates the expression of Bcl -2 gene during the procession of gastric cancer, and inhibits cell apoptosis and differentiation in carcinogenesis.%目的 研究胃癌变过程中幽门螺杆菌(Hp)感染与凋亡基因Bcl-2表达、细胞凋亡和临床意义.方法 用快速尿素酶法、W-S银染法和美蓝法联合检测62

  9. Heterogeneous breakpoints on the immunoglobulin genes are involved in fusion with the 5' region of BCL2 in B-cell tumors.

    Science.gov (United States)

    Yonetani, N; Ueda, C; Akasaka, T; Nishikori, M; Uchiyama, T; Ohno, H

    2001-09-01

    The 5' flanking region of the BCL2 gene (5'-BCL2) is a breakpoint cluster of rearrangements with immunoglobulin genes (IGs). In contrast to t(14;18)(q32;q21) affecting the 3' region of BCL2, 5'-BCL2 can fuse to not only the heavy chain gene (IGH), but also two light chain gene (IGL) loci. We report here cloning and sequencing of a total of eleven 5'-BCL2 / IGs junctional areas of B-cell tumors, which were amplified by long-distance polymerase chain reaction-based assays. The breakpoints on 5'-BCL2 were distributed from 378 to 2312 bp upstream of the translational initiation site and, reflecting the alteration of regulatory sequences of BCL2, 5'-BCL2 / IGs-positive cells showed markedly higher levels of BCL2 expression than those of t(14;18)-positive cells. In contrast, the breakpoints on the IGs were variable. Two 5'-BCL2 / IGH and two 5'-BCL2 / IGLkappa junctions occurred 5' of the joining (J) segments, suggesting operation of an erroneous variable (V) / diversity (D) / J and V / J rearrangement mechanism. However, two other 5'-BCL2 / IGH junctions affected switch regions, and the kappa-deleting element, which is located 24 kb downstream of the constant region of IGLkappa, followed the 5'-BCL2 in another case. One 5'-BCL2 / IGLkappa and two 5'-BCL2 / IGLlambda junctions involved intronic regions where the normal recombination process does not occur. In the remaining one case, the 5'-BCL2 fused 3' of a Vlambda gene that was upstream of another Vlambda / Jlambda complex carrying a non-producing configuration, indicating that the receptor editing mechanism was likely involved in this rearrangement. Our study revealed heterogeneous anatomy of the 5'-BCL2 / IGs fusion gene leading to transcriptional activation of BCL2, and suggested that the mechanisms underlying the formation of this particular oncogene / IGs recombination are not identical to those of t(14;18).

  10. Combination of erlotinib and EGCG induces apoptosis of head and neck cancers through posttranscriptional regulation of Bim and Bcl-2.

    Science.gov (United States)

    Haque, Abedul; Rahman, Mohammad Aminur; Chen, Zhuo Georgia; Saba, Nabil F; Khuri, Fadlo R; Shin, Dong M; Ruhul Amin, A R M

    2015-07-01

    Combinatorial approaches using two or more compounds are gaining increasing attention for cancer therapy. We have previously reported that the combination of the EGFR-TKI erlotinib and epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and by inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis. shRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p = 0.005). On the other hand, overexpression of Bcl-2 markedly protected cells from apoptosis (p = 0.003), whereas overexpression of constitutively active AKT only minimally protected cells from apoptosis induced by the combination of the two compounds. Analysis of mRNA expression by RT-PCR revealed that erlotinib, EGCG and their combination had no significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2 suggesting the post-transcriptional regulation of these molecules. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim and stabilized Bim after inhibition of protein translation by cycloheximide. Taken together, our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the posttranscriptional level.

  11. TNF-α induces apoptosis of Molt-4 cells and cell cycle specificity of Bcl-2 phosphyrylation

    Institute of Scientific and Technical Information of China (English)

    Changyong Yang; Huijie Zhao; Jianping Gong

    2010-01-01

    Objective:The aim of the study was to observe the expression of Bcl-2 and its phosphorylation in Molt-4 cells induced by tumor necrosis factor-α(TNF-α),and to investigate the possible mechanism of cell cycle specificity of apoptosis.Methods:Exponentially growing Molt-4 cells were treated with TNF-α.Apoptosis was detected by DNA fragmentation assay.API method was applied to illustrate the cell cycle specificity of apoptotic cells.Cells of sub-phases were sorted by FACSvantage flow cytometer and then submitted to immunoblot.Results:Molt-4 cells which were treated with TNF-α went to apoptosis and showed a DNA ladder pattern.Most apoptosis happened in G1-phase of cell cycle.Bcl-2 expression increased for the Molt-4 cells treated with TNF-α.The phosphorylation state of Bcl-2 was only presented in G1-phase cells,in accordance with the specified time and cell cycle phase of apoptosis.Conclusion:The phosphorylation of Bcl-2 in the Molt-4 cells treated with TNF-α happened with the same cell cycle specificity as cell apoptosis.The cell cycle specificity of Bcl-2 phosphorylation was one of the mechanisms of receptor-mediated apoptosis.The cell cycle machine can trigger the apoptosis program.

  12. Expression of the Bcl-2 apoptotic marker in cats diagnosed with inflammatory bowel disease and gastrointestinal lymphoma.

    Science.gov (United States)

    Swanson, Christine M; Smedley, Rebecca C; Saavedra, Paulo Vilar; Kiupel, Matti; Kitchell, Barbara E

    2012-10-01

    Immunolabeling for the critical lymphocyte survival factor, Bcl-2, of intestinal biopsies from cats with histologic evidence of inflammatory bowel disease (IBD) or gastrointestinal (GI) lymphoma was evaluated to determine if expression differed significantly between these two disease processes. Immunolabeling for Bcl-2 was performed on small intestinal endoscopic or full thickness biopsy sections from 55 cats. Diagnosis of IBD, T-cell lymphoma or B-cell lymphoma was established previously. The percentage of infiltrating lymphocytes that were positively labeled for Bcl-2 was subjectively determined for each case. Eight cats were diagnosed with IBD and 47 cats with lymphoma. A significantly higher percentage of cells were positively immunolabeled for Bcl-2 in cats with GI lymphoma [median (range); 90 (5-95)%] compared with cats with IBD [60 (15-95)%] (P = 0.029). However, the overall degree of positive immunolabeling in both groups tended to be high. This over-expression of Bcl-2 may prove useful as a therapeutic target for IBD and GI lymphoma in cats.

  13. Bcl-2和Bax在皮肤血管瘤组织中的表达及意义%Expression of Bcl-2 and Bax in different phases of human dermal hemagniomas

    Institute of Scientific and Technical Information of China (English)

    陕声国; 张端莲; 刘昱; 余瑛; 杨勇; 熊彦娥; 李红

    2008-01-01

    Objective To investigate the function of Bcl-2 and Bax in the pathogenesis,development and regression of human hemangiomas.Methods We examined the expression of Bcl-2 and Bax in proliferating versus involuting human hemangioma tissues and normal skin tissues using immunohistochemical technique.Results The expression of Bcl-2 in proliferating hemangiomas was significantly higher than that in involuting hemangiomas and normal skin tissues(P<0.01).No significant difference was found between the expression of Bcl-2 in involuting hemangiomas and that in normal skin tissues(P>0.05).The expression of Bax in involuting hemangiomas was significantly higher than that in proliferating hemangiomas and normal skin tissues(P<0.01);the expression of Bax in proliferating hemangiomas was significantly higher than that in normal skin tissues(P<0.05).Conclusion Bcl-2 and Bax participate in the development and involution of hemangioma,Bcl-2 plays a role in accelerating the proliferation of hemangioma by inhibiting the apoptosis of endothelial cells,and Bax promotes the switching from proliferation to involution in hemangiomas through inducing the apoptosis of endothelial cells.%目的 探讨Bcl-2和Bax在皮肤血管瘤发生、发展及退化过程中的作用及意义.方法 采用免疫组织化学方法(S-P法)检测人皮肤血管瘤增生期、退化期及正常组织中Bcl-2和Bax的表达水平.结果 Bcl-2在增生期血管瘤内皮细胞的表达明显高于退化期血管瘤内皮细胞和正常皮肤组织血管内皮细胞(P<0.01);Bcl-2在退化期血管瘤内皮细胞的表达与正常皮肤组织血管内皮细胞相比,差异无统计学意义(P>0.05).Bax在退化期血管瘤内皮细胞的表达明显高于增生期血管瘤内皮细胞和正常皮肤组织血管内皮细胞(P<0.01);Bax在增生期血管瘤内皮细胞的表达高于正常皮肤组织血管内皮细胞(P<0.05).结论 Bcl-2和Bax参与了血管瘤的发生、发展和退化.Bcl-2通过

  14. The role of the expression of bcl-2, p53 gene in tamoxifen-induced apoptosis of breast cancer cells and its relationship with hormone receptor status

    Energy Technology Data Exchange (ETDEWEB)

    Noh, Woo Chul; Ham, Yong Ho [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1998-01-01

    To investigate the relationship of bcl-2, p53, ER and tamoxifen-induced apoptosis of breast cancer cells, MCF-7 (ER+/bcl-2+/p53-) and MB MDA 468 (ER-/bcl-2-/p53+) cell line were cultured in estrogen-free condition. E2(10`-`9M) and tamoxifen (10`-`5M) were added to the media. The changes of bcl-2 and mutant p53 protein were checked by Western blot and apoptosis were measured by flowcytometry. In MCF-7 cells, we found that treatment with tamoxifen resulted in a decrease in bcl-2 protein level, but produced no change in mutant p53. In MB MDA 468 cell however, there were no changes of bcl-2 and mutant p53 protein level when E2 or tamoxifen were added. Apoptotic cells increased with time-dependent pattern when tamoxifen was added to MCF-7 cells. According to these result, ER+/blc-2+/mutant p53- cells, when treated with tamoxifen, were converted into bcl-2/mutant p53- cells which were more prone to apoptosis than bcl-2-/mutant p53+ cells. The paradoxical correlation of bcl-2 and ER which had been observed in clinical studies might be explained with this results and bcl-2 protein seems to be one of important factors that can predict the effect of hormone therapy. (author). 26 refs., 5 figs

  15. Tumor necrosis factor suppresses interleukin 10 in peripheral B cells via upregulating Bcl2-like protein 12 in patients with inflammatory bowel disease.

    Science.gov (United States)

    Guo, Xiutian; Li, Mao-Gang; Li, Shan-Shan; Liu, Feng-Hua; Liu, Zhan-Ju; Yang, Ping-Chang

    2017-03-01

    The pathogenesis of the immune regulation dysfunction is unclear. Bcl2-like protein 12 (Bcl2L12) has immune suppression function. This study tests a hypothesis that tumor necrosis factor (TNF) increases Bcl2L12 to suppress the expression of interleukin (IL) 10 in peripheral B cells of patients with inflammatory bowel disease (IBD). In this study, peripheral blood samples were collected from IBD patients and healthy controls. B cells were isolated from the blood samples. The expression of IL-10 and Bcl2L12 in B cells was analyzed by quantitative reverse transcription polymerase chain reaction and Western blotting. We observed that the expression of Bcl2L12 in the peripheral B cells was higher in IBD patients than that in healthy controls. The IL-10 levels in B cells were negatively correlated with the expression of Bcl2L12. Exposure of B cells to TNF in the culture enhanced the expression of Bcl2L12. The Bcl2L12 mediated the effects of TNF on suppression of IL-10 in B cells. In conclusion, Bcl2L12 mediates the effects of TNF to suppress the expression of IL-10 in B cells. The data suggest that Bcl2L12 may be a therapeutic target for the treatment of IBD.

  16. Significance of Fas and bcl-2 gene in the pathogenesis and treatment of myasthenia gravis%Fas和bcl-2基因与重症肌无力的发生与治疗

    Institute of Scientific and Technical Information of China (English)

    赖成虹; 李作孝

    2005-01-01

    OBJECTIVE: At present the function of Fas and Bcl-2 gene in the pathogenesis of myasthenia gravis has gained more attention and more studies have been extensively made inside and outside, to investigate the modulation of Fas and Bcl-2 gene expression is of extremely significance for exploring the pathogenesis and treatment of myasthenia gravis.DATA SOURCE: The related articles published in English from January 1990 to December 2004 were computer searched in Medline database using the terms of "Fas", "Bcl-2", "Myasthenia Gravis" and "gene".STUDY SELECTION: The data were identified preliminarily and literatures on the relationship between Fas and Bcl-2 gene and myasthenia gravis, as well as on Fas and Bcl-2 gene and genotherapy were remained for searching the full content.DATA EXTRACTION: A great deal of literatures about Fas and Bcl-2 gene were searched, 21 of which were studies on Fas and Bcl-2 gene and myasthenia gravis, and 32 on myasthenia gravis related genotherapy. A total of 20 representative articles were selected for analysis in this study.DATA SYNTHESIS: Eight out of 20 literatures were about the relationship between Fas gene and myasthenia gravis, 5 about the Bcl-2 gene and myasthenia gravis, 7 about the genotherapy in the treatment of myasthenia gravis and immune diseases.CONCLUSION: Abnormal Fas and Bcl-2 gene modulation and uncontrollable expression were closely associated with apoptosis defect of T lymphocyte and thymocytes, and even closely related to the pathogenesis of myasthenia gravis. To explore pathogenesis of myasthenia gravis at gene level by inducing cell apoptosis and depressing self-antibody production, would provide new means for the treatment of myasthenia gravis.%背景:目前Fas和Bcl-2基因在重症肌无力发病机制中的作用越来越受到重视,国内外已做了较多的研究,阐明调控Fas和Bcl-2基因的表达对重症肌无力发生及其疾病过程中的治疗均具有密切的关系.资料来源:

  17. TIEG1对HL-60细胞凋亡及Bcl-2/Bax表达的影响%Influence of TIEG1 on Apoptosis of HL-60 Cells and Expression of Bcl-2/Bax

    Institute of Scientific and Technical Information of China (English)

    姚鲲; 杨莹; 胡荣; 苗苗; 廖爱军; 杨威; 刘卓刚

    2013-01-01

    This study was aimed to investigate the influence of TIEG1 on apoptosis of HL-60 cells and the expression of Bcl-2/Bax.Different concentration of TIEG1 were used to treat HL-60 cells,the cell growth inhibition rate was detected by MTT method.After treating HL-60 cells with 12.03 ng/ml TIEG1,cell apoptosis was detected with flow cytometry.Bcl-2 and Bax was detected with RT-PCR.The results showed that TIEG1 had inhibitory effect on HL-60 cell proliferation,and in time-and dose-dependent manners.The more obvious inhibitory effect was observed in HL-60 cells treated with TIEG1 of 12.03 ng/ml.During the course of cell apoptosis,Bax expression increasied,but Bcl-2 expression decreased(P < 0.05).It is concluded that TIEG1 inhibits HL-60 cell proliferation and induces apoptosis in time and dose-dependent manners.During the course of HL-60 cells apoptosis induced by TIEG1,Bcl-2/Bax are assosciated with HL-60 cell apoptosis induced by TIEG1.%本研究旨在观察TIEG1对HL-60细胞凋亡及Bcl-2/Bax表达的影响.用不同浓度的TIEG1处理HL-60细胞,采用MTT法检测细胞生长抑制率.用12.03 ng/ml TIEG1处理HL-60细胞,以流式细胞术检测细胞凋亡,以RT-PCR方法检测Bcl-2及Bax的表达变化.结果表明,TIEG1对HL-60细胞具有增殖抑制作用,其抑制效应呈时间及剂量依赖性.TIEG1 12.03 ng/ml增殖抑制作用较为明显.在细胞凋亡过程中,Bax表达呈升高趋势,而Bcl-2呈下降趋势(P<0.05).结论:TIEG1可以抑制HL-60细胞增殖,进而诱导其凋亡,且呈时间、剂量依赖性.在TIEG1诱导HL-60细胞凋亡的过程中,Bcl-2/Bax与TIEG1诱导HL-60细胞凋亡相关.

  18. Examination of a Viral Infection Mimetic Model in Human iPS Cell-Derived Insulin-Producing Cells and the Anti-Apoptotic Effect of GLP-1 Analogue.

    Directory of Open Access Journals (Sweden)

    Megu Yamaguchi Baden

    Full Text Available Viral infection is associated with pancreatic beta cell destruction in fulminant type 1 diabetes mellitus. The aim of this study was to investigate the acceleration and protective mechanisms of beta cell destruction by establishing a model of viral infection in pancreatic beta cells.Polyinosinic:polycytidylic acid was transfected into MIN6 cells and insulin-producing cells differentiated from human induced pluripotent stem cells via small molecule applications. Gene expression was analyzed by real-time PCR, and apoptosis was evaluated by caspase-3 activity and TUNEL staining. The anti-apoptotic effect of Exendin-4 was also evaluated.Polyinosinic:polycytidylic acid transfection led to elevated expression of the genes encoding IFNα, IFNβ, CXCL10, Fas, viral receptors, and IFN-inducible antiviral effectors in MIN6 cells. Exendin-4 treatment suppressed the elevated gene expression levels and reduced polyinosinic:polycytidylic acid-induced apoptosis both in MIN6 cells and in insulin-producing cells from human induced pluripotent stem cells. Glucagon-like peptide-1 receptor, protein kinase A, and phosphatidylinositol-3 kinase inhibitors counteracted the anti-apoptotic effect of Exendin-4.Polyinosinic:polycytidylic acid transfection can mimic viral infection, and Exendin-4 exerted an anti-apoptotic effect both in MIN6 and insulin-producing cells from human induced pluripotent stem cells.

  19. Follicle-stimulating hormone potentiates the steroidogenic activity of chorionic gonadotropin and the anti-apoptotic activity of luteinizing hormone in human granulosa-lutein cells in vitro.

    Science.gov (United States)

    Casarini, Livio; Riccetti, Laura; De Pascali, Francesco; Nicoli, Alessia; Tagliavini, Simonetta; Trenti, Tommaso; La Sala, Giovanni Battista; Simoni, Manuela

    2016-02-15

    Luteinizing hormone (LH) and choriogonadotropin (hCG) are glycoprotein hormones regulating ovarian function and pregnancy, respectively. Since these molecules act on the same receptor (LHCGR), they were traditionally assumed as equivalent in assisted reproduction techniques (ART), although differences between LH and hCG were demonstrated at molecular and physiological level. In this study, we demonstrated for the first time that co-treatment with a follicle-stimulating hormone (FSH) dose in the ART therapeutic range potentiates different LH- and hCG-dependent responses in vitro, measured in terms of cAMP, phospho-CREB, -ERK1/2 and -AKT activation, gene expression, progesterone and estradiol production in human granulosa-lutein cells (hGLC). We show that in the presence of FSH, hCG biopotency is about 5-fold increased, in the presence of FSH, in terms of cAMP activation. Accordingly, CREB phosphorylation and steroid production is increased under hCG and FSH co-treatment. LH effects, evaluated as steroidogenic cAMP/PKA pathway activation, do not change in the presence of FSH, which, however, increases LH-dependent ERK1/2 and AKT, but not CREB phosphorylation, resulting in anti-apoptotic effects. The different modulatory activity of FSH on LH and hCG action in vitro corresponds to their different physiological functions, reflecting proliferative effects exerted by LH during the follicular phase and before trophoblast development, and the high steroidogenic potential of hCG requested to sustain pregnancy from the luteal phase onwards.

  20. Human Noxin is an anti-apoptotic protein in response to DNA damage of A549 non-small cell lung carcinoma.

    Science.gov (United States)

    Won, Kyoung-Jae; Im, Joo-Young; Yun, Chae-Ok; Chung, Kyung-Sook; Kim, Young Joo; Lee, Jung-Sun; Jung, Young-Jin; Kim, Bo-Kyung; Song, Kyung Bin; Kim, Young-Ho; Chun, Ho-Kyung; Jung, Kyeong Eun; Kim, Moon-Hee; Won, Misun

    2014-06-01

    Human Noxin (hNoxin, C11Orf82), a homolog of mouse noxin, is highly expressed in colorectal and lung cancer tissues. hNoxin contains a DNA-binding C-domain in RPA1, which mediates DNA metabolic processes, such as DNA replication and DNA repair. Expression of hNoxin is associated with S phase in cancer cells and in normal cells. Expression of hNoxin was induced by ultraviolet (UV) irradiation. Knockdown of hNoxin caused growth inhibition of colorectal and lung cancer cells. The comet assay and western blot analysis revealed that hNoxin knockdown induced apoptosis through activation of p38 mitogen-activated protein kinase (MAPK)/p53 in non-small cell lung carcinoma A549 cells. Furthermore, simultaneous hNoxin knockdown and treatment with DNA-damaging agents, such as camptothecin (CPT) and UV irradiation, enhanced apoptosis, whereas Trichostatin A (TSA) did not. However, transient overexpression of hNoxin rescued cells from DNA damage-induced apoptosis but did not block apoptosis in the absence of DNA damage. These results suggest that hNoxin may be associated with inhibition of apoptosis in response to DNA damage. An adenovirus expressing a short hairpin RNA against hNoxin transcripts significantly suppressed the growth of A549 tumor xenografts, indicating that hNoxin knockdown has in vivo anti-tumor efficacy. Thus, hNoxin is a DNA damage-induced anti-apoptotic protein and potential therapeutic target in cancer.

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

    Directory of Open Access Journals (Sweden)

    W. Y. Liu

    2013-01-01

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

  2. Evaluation of Bcl-2, Bcl-x and Cleaved Caspase-3 in Malignant Peripheral Nerve Sheath Tumors and Neurofibromas

    Directory of Open Access Journals (Sweden)

    KARIN S. CUNHA

    2013-11-01

    Full Text Available AIMS: To study the expression of Bcl-2, Bcl-x, as well the presence of cleaved caspase-3 in neurofibromas and malignant peripheral nerve sheath tumors. The expression of Bcl-2 and Bcl-x and the presence of cleaved caspase 3 were compared to clinicopathological features of malignant peripheral nerve sheath tumors and their impact on survival rates were also investigated. MATERIALS AND METHODS: The evaluation of Bcl-2, Bcl-x and cleaved caspase-3 was performed by immunohistochemistry using tissue microarrays in 28 malignant peripheral nerve sheath tumors and 38 neurofibromas. Immunoquantification was performed by computerized digital image analysis. CONCLUSIONS: Apoptosis is altered in neurofibromas and mainly in malignant peripheral nerve sheath tumors. High levels of cleaved caspase-3 are more common in tumors with more aggressive histological features and it is associated with lower disease free survival of patients with malignant peripheral nerve sheath tumors.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  4. Expression of bcl-2 oncogene in gastric precancerous lesions and its correlation with syndromes in traditional Chinese medicine

    Institute of Scientific and Technical Information of China (English)

    Ling Hu; Shao-Xian Lao; Chun-Zhi Tang

    2005-01-01

    AIM: To observe the protein and mRNA expression of bcl-2 oncogene in gastric precancerous lesions (GPL) and to analyze its correlation with syndromes in traditional Chinese medicine (TCM).METHODS: Sixty-seven patients with GPL confirmed by gastroscopy and pathology were studied, including 39 cases of moderate gastric mucosal dysplasia, 19 casesof severe gastric mucosa dysplasia, g cases of incompletecolon metaplasia. In syndrome differentiation of TCM, 17 cases belonged to the syndrome of qi and yin deficiency of the spleen and stomach complicated by qi stagnation, 21 cases belonged to the syndrome of qi and yin deficiency of the spleen and stomach complicated by stomach heat, 29 cases belonged to the syndrome of qi and yin deficiency of the spleen and stomach complicated by blood stasis. Protein and mRNA expression of bcl-2 oncogene weredetected by labeled streptavidin biotin (LSAB) immunohistochemistry and in situ hybridization respectively. RESULTS: Abnormal expression of protein and mRNA on bcl-2 oncogene was found in GPL, which increased gradually with the course of lesions. In moderate and severe gastric mucosal dysplasia and incomplete colon metaplasia, there was no difference in the expression of bcl-2 oncogene (P>0.05). In different accompanying syndromes, the expression of protein and mRNA on bcl-2 oncogene increased gradually in the following order: deficiency of both qi and yin of the spleen and stomach accompanying qi stagnation → stomach heat → blood stasis. In GPL, compared with accompanying blood stasis, there was an obvious difference in the expression of bd-2 oncogene between the syndrome of qi and yin deficiency of the spleen and stomach and accompanying stomach heat, so did accompanying qi stagnation (the level of protein: χ2 = 8.45, P<0.05; the level of mRNA: χ2 = 7.35,P<0.05).CONCLUSION: Apoptosis-associated bcl-2 oncogene is abnormally expressed in GPL, which correlates with different accompanying syndromes in TCM.

  5. Analysis of the Expression of Fas, FasL and Bcl-2 in the Pathogenesis of Autoimmune Thyroid Disorders

    Institute of Scientific and Technical Information of China (English)

    Shenren Chen; S.M.Fazle Akbar; Zhichao Zhen; Yiping Luo; Lijuan Deng; Haihua Huang; Linxin Chen; Wei Li

    2004-01-01

    To investigate the expression of apoptosis-related protein (Fas, FasL, and Bcl-2) in the pathogenesis of autoimmune thyroid disorders (ATDs), immunohistochemical staining was performed on 20 Hashimoto's thyroiditis (HT), 20 Graves' disease (GD), and 20 thyroid follicular adenoma (TFA, as control). All the cases expressed Fas, mainly on the cell surface and cytoplasm. FasL was found in 17 cases of the TFA. Bcl-2 was detected in 15 cases of HT, 19 of GD and 17 of TFA. In TFA, a moderate Fas expression and a minimal or no FasL expression was detected on follicular cells. In HT, the follicles adjacent to infiltrating lymphocytes showed increased levels of Fas and FasL expression. A weaker staining of Fas and FasL was exhibited on infiltrating lymphocytes than on thyrocytes. In a comparison of GD with HT, thyrocytes and lymphocytes showed similar Fas staining, but for FasL the staining was rather weaker in HT. The expression of Bcl-2 was nearly identical in GD and TFA, but much weaker on the follicular cells in vicinity of lymphocytes and on the lymphocytes located in germinal centers of HT tissues. The expression of Fas, FasL, Bcl-2 in Hashimoto's thyroiditis and Graves' disease were almost same. FasL strong expression and Bcl-2 weak expression on the follicles in HT may induce apoptosis. These results provided evidence for expression of Fas, FasL and Bcl-2 in the pathogenesis of autoimmune thyroid disease. The lymphocytes seem not to be directly engaged in the process via their own FasL, but they may provide some cytokines that, in turn, upregulate Fas and/or FasL expression to induce apoptosis.

  6. Analysis of the Expression of Fas, FasL and Bcl-2 in the Pathogenesis of Autoimmune Thyroid Disorders

    Institute of Scientific and Technical Information of China (English)

    ShenrenChen; S.M.FazleAkbar; ZhichaoZhen; YipingLuo; LijuanDeng; HaihuaHuang; LinxinChen; WeiLi

    2004-01-01

    To investigate the expression of apoptosis-related protein (Fas, FasL, and Bcl-2) in the pathogenesis of autoimmune thyroid disorders (ATDs), immunohistochemical staining was performed on 20 Hashimoto's thyroiditis (HT), 20 Graves' disease (GD), and 20 thyroid follicular adenoma (TFA, as control). All the cases expressed Fas, mainly on the cell surface and cytoplasm. FasL was found in 17 cases of the TFA. Bcl-2 was detected in 15 cases of HT, 19 of GD and 17 of TFA. In T FA, a moderate Fas expression and a minimal or no FasL expression was detected on follicular cells. In HT, the follicles adjacent to infiltrating lymphocytes showed increased levels of Fas and FasL expression. A weaker staining of Fas and FasL was exhibited on infiltrating lymphocytes than on thyrocytes. In a comparison of GD with HT, thyrocytes and lymphocytes showed similar Fas staining, but for FasL the staining was rather weaker in HT. The expression of Bcl-2 was nearly identical in GD and TFA, but much weaker on the follicular cells in vicinity of lymphocytes and on the lymphocytes located in germinal centers of HT tissues. The expression of Fas, FasL, Bcl-2 in Hashimoto's thyroiditis and Graves' disease were almost same. FasL strong expression and Bcl-2 weak expression on the follicles in HT may induce apoptosis. These results provided evidence for expression of Fas, FasL and Bcl-2 in the pathogenesis of autoimmune thyroid disease. The lymphocytes seem not to be directly engaged in the process v/a their own FasL, but they may provide some cytokines that, in turn, upregulate Fas and/or FasL expression to induce apoptosis.

  7. Relationship between reduced BCL-2 expression in circulating mononuclear cells and early nephropathy in type 1 diabetes.

    Science.gov (United States)

    Cipollone, F; Chiarelli, F; Iezzi, A; Fazia, M L; Cuccurullo, C; Pini, B; De Cesare, D; Torello, M; Tumini, S; Cuccurullo, F; Mezzetti, A

    2005-01-01

    Microalbuminuria is the earliest clinical evidence of diabetic nephropathy, but the mechanisms linking hyperglycemia and kidney complications are not clear. The aim of this study was to evaluate whether enhanced oxidative stress in patients with microalbuminuria can contribute to diabetic nephropathy development through downregulation of the antiapoptotic gene Bcl-2 that promotes in turn a pro-inflammatory status. We studied 30 patients with type 1 diabetes (15 with and 15 without microalbuminuria) compared to 15 matched healthy controls. Plasma oxidant status, and expression of Bcl-2, activated NF-kB, inducible Nitric Oxide synthase (iNOS), and monocyte chemoattractant protein (MCP)-1 in circulating monocytes were evaluated at baseline and after 8-week oral vitamin E treatment (600 mg b.i.d.). Bcl-2 expression was significantly reduced in microalbuminuric diabetic patients as a consequence of increased oxidant burden secondary to persistent hyperglycemia. Bcl-2 down-regulation was associated with enhanced expression of NF-kB, iNOS and MCP-1, and showed a strong correlation with the albumin excretion rate. Low Bcl-2 expression and high inflammatory status were normalized by vitamin E both in vivo and in vitro. Our study showed that Bcl-2 down-regulation in diabetic patients with poor glycemic control results in the activation of the NF-kB pathway leading to the development of nephropathy. Vitamin E might provide a novel form of therapy for prevention of nephropathy in diabetic patients in which an acceptable glycemic control is difficult to achieve despite insulin therapy.

  8. Expression of Bcl-2 and Bax in Cerebral Cortex of Sodium Valproate Induced Autism Rats%丙戊酸钠诱导孤独症模型大鼠皮层Bcl-2、Bax的表达①

    Institute of Scientific and Technical Information of China (English)

    姜志梅; 崔利军; 郭津; 张士岭; 郭岚敏

    2013-01-01

    Objective To explore the role of Bcl-2 and Bax in pathogenesis of the autism. Methods Female Sprague-Dawley rats were given a single intraperitoneal injection of sodium valproate (VPA, 600 mg/kg) on 12.5 d after pregnancy, their offspring were as the model group;while the other pregnancy rats were given normal saline, their offspring were as the control group. Both groups were observed with the Nissl staining, immunohistochemistry of Bcl-2 and Bax and image analysis 1 d, 7 d, 14 d, 28 d, 56 d after birth. Results Compared with the control group, Nissl staining showed the number of cortical neurons decreased on 1 d and 7 d after birth in the model group, rapidly in-creased on 14 d after birth, and maintained in high level on 28d , 56 d after birth. For immunohistochemistry, the integrated optical density (IOD) of Bcl-2 and Bax decreased in cortex on 1~14 d after birth (P0.05). Com-pared with the control group, the IOD of Bcl-2 decreased much more at every time point (P0.05). The ratio of Bcl-2/Bax was the most on 1 d af-ter birth, and then decreased to approximately 1 in the control group, while it was the least on 7 d, most on 14 d, and decrease to less than 1 28 d after birth. Conclusion Apoptosis of cerebral cortex neurons increases in the autism model rats, especially in the early time.%  目的探讨Bcl-2、Bax在孤独症发病中的作用。方法孕12.5 d Sprague-Dawley孕鼠腹腔注射丙戊酸钠600 mg/kg建立子代孤独症模型大鼠,对照组注射同等剂量生理盐水。利用尼氏染色、免疫组化和图像分析技术观察比较出生后1 d、7 d、14 d、28 d和56 d两组大鼠脑部Bcl-2、Bax表达。结果尼氏染色:出生后1 d、7 d模型组神经元数量较少,出生14 d后剧增,出生后28 d、56 d仍高于对照组。免疫组化:出生后1~14 d两组Bcl-2、Bax表达均显著升高(P0.05);与对照组相比,模型组各日龄大鼠Bcl-2表达水平均显著降低(P0.05)。对照组Bcl-2/Bax值出生后1 d

  9. P53和Bcl-2蛋白在基底细胞癌中的表达及临床意义%Expression of protein P53 and Bcl-2 in basal cell carcinoma and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    魏荣

    2014-01-01

    目的:探讨P53、B细胞淋巴瘤/白血病-2基因(bcl-2)蛋白在基底细胞癌(BCC)组织中的表达及临床意义。方法用免疫组化链霉素抗生素蛋白-过氧化物酶连结(SP)法检测P53、Bcl-2蛋白在40例BCC(BCC组)组织中的表达,以20例正常皮肤组织作为对照组。结果 P53、Bcl-2蛋白在BCC组表达阳性率分别为55%(22/40)、85%(34/40),在对照组表达阳性率分别为15%(3/20)、10%(2/20)。P53、Bcl-2蛋白在BCC组中的表达明显强于对照组,差异有统计学意义(χ2=6.35、7.45,P<0.01)。结论 P53、Bcl-2蛋白的异常表达与BCC相关,说明其可能在BCC的发生及发展过程中起重要作用。%Objective To investigate the expression and clinical significance of protein P53 and B cell lymphom/leukemia-2 gene(Bcl-2) in basal cell carcinoma(BCC). Methods The expression of protein P53 and Bcl-2 was measured by streptavidin-peroxidase(SP) immunohistochemical method in 40 cases of BCC(BCC group),and 20 cases of normal skin tissue were selected as control group. Results The positive rates of protein P53 and Bcl-2 in the BCC group were 55%(22/40) and 85%(34/40) respectively,while they were 15%(3/20) and 10%(2/20) respectively in the control group. The expression of pro-tein P53 and Bcl-2 in the BCC group was significantly stronger than that in the control group with statistically significant differ ence(χ2=6.35,7.45;P<0.01). Conclusion The abnormal expression of protein P53 and Bcl-2 is related with BCC,which shows that protein P53 and Bcl-2 might play an important role in the occurrence and development of BCC .

  10. Bcl-2 expression during amelogenesis in mouse molars%Bcl-2在成釉细胞分化、分泌过程中的表达研究

    Institute of Scientific and Technical Information of China (English)

    于西佼; 唐开亮; 杜毅; 李纾

    2012-01-01

    目的:检测凋亡调控抑制蛋白Bcl-2在成釉细胞分化、分泌过程中的表达,观察细胞超微结构的变化,探讨Bcl-2和细胞凋亡在该过程的可能作用.方法:制备出生后2、5、7、9、14 d不同发育阶段的BALB/C小鼠下颌第一磨牙牙胚标本,采用原位末端标记法(TUNEL法)和PV免疫组织化学技术观察成釉细胞分化、分泌和釉质发育完成各阶段细胞凋亡以及Bcl-2的表达情况;透射电镜观察细胞超微结构的变化.结果:出生后第2~5天,小鼠下颌第一磨牙成釉细胞处于分化期,超微结构可见胞浆内有高尔基复合体和线粒体,并有细胞增生的核分裂;免疫组化结果显示Bcl-2阳性表达,TUNEL检测结果发现部分细胞胞核阳性表达,提示细胞凋亡的存在;出生后第7天,成釉细胞已开始分泌,可见新形成的釉质,胞核远基底排列,胞核附近可见大量线粒体,胞质内可见大量高尔基复合体和粗面内质网,胞浆呈Bcl-2强阳性表达,TUNEL检测结果发现少量细胞胞核阳性表达;出生后14 d,釉质发育完成,成釉细胞变短,间隙变大,细胞器数量减少,核膜逐渐不清,核糖体脱颗粒水肿,呈现凋亡征象,胞浆未见Bcl-2阳性表达.结论:细胞凋亡在釉质发育的各期皆有表达,Bcl-2作为凋亡抑制基因可能参与了成釉细胞的分化、分泌的调控.%AIM: To observe Bcl-2 expresion during amelogenesis in developing mouse molars. METH- ODS : First molar germs of postnatal 2 - 14 d BALB/C mice were extracted. The morphology and distribution of amelo-blasts in the tooth germ were examined by light and transmission electron microscopy. PV two-step immunohistochemi-cal method was used to detect the expression of Bcl-2 protein. Apoptosis was identified by the terminal deoxy-transfer-ase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method. RESULTS: Bcl-2 positive cells was detected in the proliferating pre-ameloblasts and secretory stage ameloblasts

  11. Expression of Bcl-2 and Ki-67 in the endometria diseases%Bcl-2和Ki-67对子宫内膜病变价值的探讨

    Institute of Scientific and Technical Information of China (English)

    文华清; 耿源源; 胡红文; 王海涛; 尹伟; 罗应彪

    2009-01-01

    Objective:To explore the expression of the Bcl-2 and Ki-67 in the endometrial. Methods:Detecting the expression of Bcl-2 and Ki-67 in the endometrial, including 25 cases of normal endometrial tissues, 150 cases of hyperplasia endometrial tissues, 75 cases of endometrial polyps tissues and 50 cases of endometrial cancer by the technology of immunohistochemistry. Results:In normal endometria, hyperplasia endometrial, endometrial polyps, endometrial cancer the posive expression rates of Bcl-2 was 84%, 62.67%, 70.67%, 38%; while those of Ki-67 was 12%, 37.33%, 17.33%, 82%. Both the expression of Bcl-2 and Ki-67 were correlated with histologic differentiation(P0.05). Conclusion:The positive expression in endometrial cancers were correlated with histologic differentiation, and it concluded that Bcl-2 and Ki-67 could be related with the endometrial cancers and normal endometrial. The expression of Bcl-2 and Ki-67 in endometrial carcinoma,in the existence of negative correlation,suggesting the application of immunohistochemical SP method for simultaneous detection of endometrial tissue in the Bcl-2,Ki-67 in abnormal protein expression, may contribute to the early detection of endometrial cancer for the early prediction of endometrial cancer and found a reference of the indicators.%目的:通过检测子宫内膜病变组织中Bcl-2和Ki-67蛋白的表达,探讨应用Bcl-2和Ki-67蛋白测定在鉴别子宫内膜病变方面的价值.方法:应用免疫组化技术SP法检测25例正常子宫内膜组织、150例子宫内膜增生症组织、75例子宫内膜息肉组织、50例子宫内膜癌组织中Bcl-2和Ki-67蛋白的表达情况.结果:Bcl-2蛋白在正常子宫内膜组织、子宫内膜增生症(单纯型、复杂型、不典型)组织、子宫内膜息肉组织、子宫内膜癌组织的阳性表达率呈递减趋势,而Ki-67的阳性表达率呈递增趋势.子宫内膜癌组织与正常子宫内膜组织、子宫内膜增生症组织、子宫内膜息肉组织的Bcl

  12. Co-overexpression of bcl-2 and c-myc in uterine cervix carcinomas and premalignant lesions

    Directory of Open Access Journals (Sweden)

    Z. Protrka

    2011-03-01

    Full Text Available To establish the role of co-overexpression of bcl-2 and c-myc protooncogenes in uterine cervix carcinogenesis, we examined 138 tissue samples of low grade cervical squamous intraepithelial lesions (SIL, high grade SIL, portio vaginalis uteri (PVU carcinoma in situ and PVU carcinoma invasive, stage IA-IIA (study group and 36 samples without SIL or malignancy (control group. The expression of bcl-2 and c-myc was detected immunohistochemically using a monoclonal antibody. Fisher’s exact test (P<0.05 was used to assess statistical significance. Overexpression of bcl-2 was found to increase in direct relation to the grade of the cervical lesions. High sensitivity was of great diagnostic significance for the detection of these types of changes in the uterine cervix. On the basis of high predictive values it can be said that in patients with bcl-2 overexpression there is a great possibility that they have premalignant or malignant changes in the uterine cervix. Co-overexpression of bcl-2 and c-myc oncogenes was found only in patients with PVU invasive carcinoma (6/26-23.0%. Statistically significant difference was not found in the frequency of co-overexpression in patients with PVU invasive carcinoma in relation to the control group (Fisher’s test; P=0.064. The method's sensitivity of determining these oncogenes with the aim of detecting PVU invasive carcinoma was 23%, while specificity was 72.2%. On the basis of high predictive values (100%, speaking in statistical terms, it can be concluded that all patients with co-overexpression of bcl-2 and c-myc oncogenes will have PVU invasive carcinoma. We confirmed in our research that co-overexpression of bcl-2 and c-myc oncogenes was increased only in PVU invasive carcinoma. However, a more extensive series of samples and additional tests are required to establish the prognostic significance of bcl-2 and c-myc co-overexpression in cervical carcinogenesis.

  13. Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

    Science.gov (United States)

    Phatak, Nitasha R.; Stankowska, Dorota L.

    2016-01-01

    Purpose Brn3b is a class IV POU domain transcription factor that plays an important role in the development of retinal ganglion cells (RGCs), RGC survival, and particularly axon growth and pathfinding. Our previous study demonstrated that recombinant adenoassociated virus serotype 2 (rAAV-2)–mediated overexpression of Brn3b in RGCs promoted neuroprotection in a rodent model of glaucoma. However, the mechanisms underlying neuroprotection of RGCs in rats overexpressing Brn3b in animal models of glaucoma remain largely unknown. The goal of this study was to understand some of the mechanisms underlying the neuroprotection of RGCs overexpressing Brn3b during intraocular pressure (IOP) elevation in Brown Norway rats. Methods One eye of Brown Norway rats (Rattus norvegicus) was injected with an AAV construct encoding either green fluorescent protein (GFP; recombinant adenoassociated virus–green fluorescent protein, rAAV-hSyn-GFP) or Brn3b (rAAV-hSyn-Brn3b). Expression of antiapoptotic proteins, including B cell lymphoma/leukemia-2 (Bcl-2) family proteins (Bcl-2 and Bcl-xL), and p-AKT, was observed following immunostaining of rat retinas that overexpress Brn3b. In a different set of experiments, intraocular pressure was elevated in one eye of Brown Norway rats, which was followed by intravitreal injection with AAV constructs encoding either GFP (rAAV-CMV-GFP) or Brn3b (rAAV-CMV-Brn3b). Retinal sections were stained for prosurvival factors, including Bcl-2, Bcl-XL, and p-AKT. Results AAV-mediated expression of transcription factor Brn3b promoted statistically significant upregulation of the Bcl-2 protein and increased expression of p-AKT in RGCs of Brown Norway rats. In addition, following IOP elevation, AAV-mediated Brn3b expression also statistically significantly increased levels of Bcl-2 in the RGC layer in Brown Norway rats. Conclusions Adenoassociated virus–mediated Brn3b protein overexpression may promote neuroprotection by upregulating key antiapoptotic

  14. The inhibition of malignant epithelial cells in mucosal injury in the oral cavity of strains by pomegranate fruit extract (Punica granatum linn through Bcl-2 expression

    Directory of Open Access Journals (Sweden)

    Sri Hernawati

    2013-03-01

    Full Text Available Background: Squamous cell carcinoma of oral cavity is a malignant neoplasms derived from epithelia. The malignant neoplasms are cells that have changed their structure and function, and their number becomes increasing abnormally, invasive, and metastatic. Carcinoma can be caused by the resistance of malignant cell apoptosis. Bcl-2 is a proto-oncogene of Bcl family that inhibits the process of cell apoptosis and suppresses Bax protein (pro-apoptotic. The management efforts of cancer diseases, however, still have many obstacles. Thus, the researcher was triggered to explore more herbal plants, namely pomegranate. Pomegranate as a medicinal plant is accessible and cheap. Ellagic acid (EA is a single active compound derived from whole pomegranate fruit extract (PGL, which has anti-cancer activity as in vitro, but EA is low concentration in plasma, low water solubility, and insoluble in intestinal. These facts prompted the researcher to compare between pomegranate extract, which consists of several active compounds, and that, which only consists of ellagic acid. Thus, this research is expected to know how some active compounds can work synergistically in the PGL, so the effect can be more potent. Purpose: The purpose of this research, therefore, was to compare EA with PGL in reducing the expression of Bcl-2. Methods: This laboratory experimental research was used 32 male mice (Balb/c in the age of 5 months. They were randomly divided into 4 groups: 2 control groups (K0: which was not exposed with benzopirene and also untreated and K1: which was exposed with benzopirene and also untreated, 2 treatment groups (P1: which was exposed with benzopirene and also treated with EA and P2: which was exposed with benzopirene and also treated with the PGL. Next, an examination was conducted by using immunohistochemical techniques. results: The results then showed that the provision of the PGL could decrease the expression of Bcl-2 significantly higher than that

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