Science.gov

Sample records for bcl-2 independent pathways

  1. Bcl2 at the endoplasmic reticulum protects against a Bax/Bak-independent paraptosis-like cell death pathway initiated via p20Bap31.

    PubMed

    Heath-Engel, Hannah M; Wang, Bing; Shore, Gordon C

    2012-02-01

    Bap31 is an integral ER membrane protein which functions as an escort factor in the sorting of newly synthesized membrane proteins within the endoplasmic reticulum (ER). During apoptosis signaling, Bap31 is subject to early cleavage by initiator caspase-8. The resulting p20Bap31 (p20) fragment has been shown to initiate proapoptotic ER-mitochondria Ca2+ transmission, and to exert dominant negative (DN) effects on ER protein trafficking. We now report that ectopic expression of p20 in E1A/DNp53-transformed baby mouse kidney epithelial cells initiates a non-apoptotic form of cell death with paraptosis-like morphology. This pathway was characterized by an early rise in ER Ca2+ stores and massive dilation of the ER/nuclear envelope, dependent on intact ER Ca2+ stores. Ablation of the Bax/Bak genes had no effect on these ER/nuclear envelope transformations, and delayed but did not prevent cell death. ER-restricted expression of Bcl2 in the absence of Bax/Bak, however, delayed both ER/nuclear envelope dilation and cell death. This prosurvival role of Bcl2 at the ER thus extended beyond inhibition of Bax/Bak, and correlated with its ability to lower ER Ca2+ stores. Furthermore, these results indicate that ER restricted Bcl2 is capable of antagonizing not only apoptosis, but also a non-apoptotic, Bax/Bak independent, paraptosis-like form of cell death. PMID:22197342

  2. Bcl-2 and porin follow different pathways of TOM-dependent insertion into the mitochondrial outer membrane.

    PubMed

    Motz, Christian; Martin, Heiko; Krimmer, Thomas; Rassow, Joachim

    2002-11-01

    The bcl-2 gene encodes a 26kDa protein which functions as a central regulator of apoptosis. Here we investigated the pathway of Bcl-2alpha into the mitochondrial outer membrane using the yeast Saccharomyces cerevisiae as a model organism. We found that interactions of Bcl-2alpha with the mitochondrial import receptor Tom20 are dependent on two positively charged lysine residues in the immediate vicinity of the carboxy-terminal hydrophobic membrane anchor. The targeting function of these residues is independent of Tom22. Subsequent insertion of Bcl-2alpha into the mitochondrial outer membrane does not require Tom5 or Tom40, indicating that Bcl-2alpha bypasses the general import pore (GIP). Bcl-2alpha shows a unique pattern of interactions with the components of the mitochondrial TOM complex, demonstrating that at least two different pathways lead from the import receptor Tom20 into the mitochondrial outer membrane. PMID:12419260

  3. Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway

    SciTech Connect

    Zhang, Cui-Li; Song, Fei; Zhang, Jing; Song, Q.H.

    2010-04-16

    Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72 h. Peak expression occurred at 12 h (3- to 4-fold, p < 0.05). p38 inhibitor (SB203580) blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs' proliferation and migration. Over-expression of Bcl-2 increased HAECs' tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation. Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.

  4. TYK2-STAT1-BCL2 Pathway Dependence in T-Cell Acute Lymphoblastic Leukemia

    PubMed Central

    Sanda, Takaomi; Tyner, Jeffrey W.; Gutierrez, Alejandro; Ngo, Vu N.; Glover, Jason; Chang, Bill H.; Yost, Arla; Ma, Wenxue; Fleischman, Angela G.; Zhou, Wenjun; Yang, Yandan; Kleppe, Maria; Ahn, Yebin; Tatarek, Jessica; Kelliher, Michelle A.; Neuberg, Donna S.; Levine, Ross L.; Moriggl, Richard; Müller, Mathias; Gray, Nathanael S.; Jamieson, Catriona H. M.; Weng, Andrew P.; Staudt, Louis M.; Druker, Brian J.; Look, A. Thomas

    2013-01-01

    Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the JAK tyrosine kinase family, TYK2, and its downstream effector STAT1 in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently demonstrated TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK kinase activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway i n T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of IL-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the anti-apoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway. PMID:23471820

  5. MCL-1-independent mechanisms of synergy between dual PI3K/mTOR and BCL-2 inhibition in diffuse large B cell lymphoma

    PubMed Central

    Lee, J. Scott; Tang, Sarah S.; Ortiz, Veronica; Vo, Thanh-Trang; Fruman, David A.

    2015-01-01

    The PI3K/AKT/mTOR axis promotes survival and is a frequently mutated pathway in cancer. Yet, inhibitors targeting this pathway are insufficient to induce cancer cell death as single agents in some contexts, including diffuse large B cell lymphoma (DLBCL). In these situations, combinations with inhibitors targeting BCL-2 survival proteins (ABT-199 and ABT-263) may hold potential. Indeed, studies have demonstrated marked synergy in contexts where PI3K/mTOR inhibitors suppress expression of the pro-survival protein, MCL-1. In this study, we use BH3 profiling to confirm that BCL-2 and BCL-XL support survival following PI3K pathway inhibition, and that the dual PI3K/mTOR inhibitor BEZ235 strongly synergizes with BCL-2 antagonists in DLBCL. However, we identify an alternative mechanism of synergy between PI3K/mTOR and BCL-2 inhibitors, independent of MCL-1 down-regulation. Instead, we show that suppression of AKT activation by BEZ235 can induce the mitochondrial accumulation of pro-apoptotic BAD and BIM, and that expression of a constitutively active form of AKT prevents sensitization to BCL-2 antagonism. Thus, our work identifies an additional mechanism of synergy between PI3K pathway inhibitors and BCL-2 antagonists that strengthens the rationale for testing this combination in DLBCL. PMID:26460954

  6. N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins

    PubMed Central

    Zhao, Guoping; Neely, Aaron M.; Schwarzer, Christian; Lu, Huayi; Whitt, Aaron G.; Stivers, Nicole S.; Burlison, Joseph A.; White, Carl; Machen, Terry E.; Li, Chi

    2016-01-01

    Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells. PMID:26758417

  7. N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins.

    PubMed

    Zhao, Guoping; Neely, Aaron M; Schwarzer, Christian; Lu, Huayi; Whitt, Aaron G; Stivers, Nicole S; Burlison, Joseph A; White, Carl; Machen, Terry E; Li, Chi

    2016-02-01

    Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells. PMID:26758417

  8. Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway

    SciTech Connect

    Li, Yangling; Luo, Peihua; Wang, Jincheng; Dai, Jiabin; Yang, Xiaochun; Wu, Honghai; Yang, Bo He, Qiaojun

    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.

  9. BH3 domain-independent apolipoprotein L1 toxicity rescued by BCL2 prosurvival proteins.

    PubMed

    Heneghan, J F; Vandorpe, D H; Shmukler, B E; Giovinazzo, J A; Giovinnazo, J A; Raper, J; Friedman, D J; Pollak, M R; Alper, S L

    2015-09-01

    The potent trypanolytic properties of human apolipoprotein L1 (APOL1) can be neutralized by the trypanosome variant surface antigen gene product known as serum resistance-associated protein. However, two common APOL1 haplotypes present uniquely in individuals of West African ancestry each encode APOL1 variants resistant to serum resistance-associated protein, and each confers substantial resistance to human African sleeping sickness. In contrast to the dominantly inherited anti-trypanosomal activity of APOL1, recessive inheritance of these two trypanoprotective APOL1 alleles predisposes to kidney disease. Proposed mechanisms of APOL1 toxicity have included BH3 domain-dependent autophagy and/or ion channel activity. We probed these potential mechanisms by expressing APOL1 in Xenopus laevis oocytes. APOL1 expression in oocytes increased ion permeability and caused profound morphological deterioration (toxicity). Coexpression of BCL2 family members rescued APOL1-associated oocyte toxicity in the order MCL1 ∼ BCLW > BCLXL ∼ BCL2A1 ≫ BCL2. Deletion of nine nominal core BH3 domain residues abolished APOL1-associated toxicity, but missense substitution of the same residues abolished neither oocyte toxicity nor its rescue by coexpressed MCL1. The APOL1 BH3 domain was similarly dispensable for the ability of APOL1 to rescue intact mice from lethal trypanosome challenge. Replacement of most extracellular Na(+) by K(+) also reduced APOL1-associated oocyte toxicity, allowing demonstration of APOL1-associated increases in Ca(2+) and Cl(-) fluxes and oocyte ion currents, which were similarly reduced by MCL1 coexpression. Thus APOL1 toxicity in Xenopus oocytes is BH3-independent, but can nonetheless be rescued by some BCL2 family proteins. PMID:26108665

  10. Neuroprotective action of cycloheximide involves induction of bcl-2 and antioxidant pathways.

    PubMed

    Furukawa, K; Estus, S; Fu, W; Mark, R J; Mattson, M P

    1997-03-10

    The ability of the protein synthesis inhibitor cycloheximide (CHX) to prevent neuronal death in different paradigms has been interpreted to indicate that the cell death process requires synthesis of "killer" proteins. On the other hand, data indicate that neurotrophic factors protect neurons in the same death paradigms by inducing expression of neuroprotective gene products. We now provide evidence that in embryonic rat hippocampal cell cultures, CHX protects neurons against oxidative insults by a mechanism involving induction of neuroprotective gene products including the antiapoptotic gene bcl-2 and antioxidant enzymes. Neuronal survival after exposure to glutamate, FeSO4, and amyloid beta-peptide was increased in cultures pretreated with CHX at concentrations of 50-500 nM; higher and lower concentrations were ineffective. Neuroprotective concentrations of CHX caused only a moderate (20-40%) reduction in overall protein synthesis, and induced an increase in c-fos, c-jun, and bcl-2 mRNAs and protein levels as determined by reverse transcription-PCR analysis and immunocytochemistry, respectively. At neuroprotective CHX concentrations, levels of c-fos heteronuclear RNA increased in parallel with c-fos mRNA, indicating that CHX acts by inducing transcription. Neuroprotective concentrations of CHX suppressed accumulation of H2O2 induced by FeSO4, suggesting activation of antioxidant pathways. Treatment of cultures with an antisense oligodeoxynucleotide directed against bcl-2 mRNA decreased Bcl-2 protein levels and significantly reduced the neuroprotective action of CHX, suggesting that induction of Bcl-2 expression was mechanistically involved in the neuroprotective actions of CHX. In addition, activity levels of the antioxidant enzymes Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and catalase were significantly increased in cultures exposed to neuroprotective levels of CHX. Our data suggest that low concentrations of CHX can promote neuron survival by

  11. Bcl-2 protein expression is the strongest independent prognostic factor of survival in primary cutaneous large B-cell lymphomas.

    PubMed

    Grange, Florent; Petrella, Tony; Beylot-Barry, Marie; Joly, Pascal; D'Incan, Michel; Delaunay, Michele; Machet, Laurent; Avril, Marie-Francoise; Dalac, Sophie; Bernard, Philippe; Carlotti, Agnes; Esteve, Eric; Vergier, Beatrice; Dechelotte, Pierre; Cassagnau, Elisabeth; Courville, Philippe; Saiag, Philippe; Laroche, Liliane; Bagot, Martine; Wechsler, Janine

    2004-05-15

    Bcl-2 protein expression has been associated with poor prognosis in patients with noncutaneous diffuse large B-cell lymphomas. In primary cutaneous large B-cell lymphomas, the location on the leg, the round-cell morphology defined as the predominance of centroblasts and immunoblasts over large centrocytes, and multiple skin lesions were identified as adverse prognostic factors. The prognostic value of bcl-2 protein expression has not been studied in large series of patients. We evaluated 80 primary cutaneous large B-cell lymphomas collected by the French Study Group on Cutaneous Lymphomas. The prognostic value of age, sex, number of lesions, cutaneous extent, location, serum lactate dehydrogenase (LDH) level, B symptoms, morphology, and bcl-2 protein expression was studied. The overall 5-year specific survival rate was 65%. In univariate analysis, advanced age, multiple skin lesions (n = 48), location on the leg (n = 25), round-cell morphology (n = 32), and bcl-2 expression (n = 39) were significantly related to death from lymphoma. In multivariate analysis, bcl-2 expression (P =.0003), multiple skin lesions (P =.004), and age remained independent prognostic factors. The 5-year specific survival rates in bcl-2-positive and bcl-2-negative patients were 41% and 89%, respectively (P <.0001). A new prognostic classification of primary cutaneous B-cell lymphoma should be based primarily on bcl-2 protein expression rather than the location of skin lesions. PMID:14726400

  12. Docetaxel induces Bcl-2- and pro-apoptotic caspase-independent death of human prostate cancer DU145 cells

    PubMed Central

    OGURA, TAKEHARU; TANAKA, YOSHIYUKI; TAMAKI, HIROKI; HARADA, MAMORU

    2016-01-01

    Docetaxel is a useful chemotherapeutic agent for the first-line treatment of hormone-refractory prostate cancer. Abnormal expression of Bcl-2 is commonly found in cancer cells, which increases their anti-apoptotic potency and chemo-resistance. We investigated the effects of Bcl-2 expression status on the susceptibility of DU145 cells, an androgen-independent human prostate cancer cell line, to docetaxel and other anticancer agents. A panel of Bcl-2-expressing DU145 cell lines was established. Bcl-2 expression levels were unrelated to the susceptibility of DU145 cells to docetaxel. The sensitivity of DU145 cells to cisplatin fluctuated, and the sensitivity to tumor necrosis factor (TNF)-α was decreased by Bcl-2 overexpression. In a xenograft mouse model, overexpression of Bcl-2 drastically decreased the sensitivity of DU145 cells to cisplatin and TNF-α; however, there was no change in the response to docetaxel. Fluorescent microscopy revealed that Bcl-2-overexpression had no effect on the docetaxel-induced death of DU145 cells, but significantly decreased DU145 cell death induced by cisplatin or TNF-α. Interestingly, docetaxel hardly induced caspase-3/7 activation in control or Bcl-2-overexpressing DU145 cells, but did at a low level in LNCaP cells, another prostate cancer cell line. Moreover, in contrast to LNCaP cells, the reduced viabilities of docetaxel-treated control and Bcl-2-overexpressing DU145 cells were not restored by the addition of either a Bid inhibitor or a panel of pro-apoptotic caspase inhibitors. These findings indicate that the antitumor effects of docetaxel on DU145 cells are independent of both Bcl-2 and pro-apoptotic caspases. PMID:27082738

  13. Docetaxel induces Bcl-2- and pro-apoptotic caspase-independent death of human prostate cancer DU145 cells.

    PubMed

    Ogura, Takeharu; Tanaka, Yoshiyuki; Tamaki, Hiroki; Harada, Mamoru

    2016-06-01

    Docetaxel is a useful chemotherapeutic agent for the first-line treatment of hormone-refractory prostate cancer. Abnormal expression of Bcl-2 is commonly found in cancer cells, which increases their anti-apoptotic potency and chemoresistance. We investigated the effects of Bcl-2 expression status on the susceptibility of DU145 cells, an androgen-independent human prostate cancer cell line, to docetaxel and other anticancer agents. A panel of Bcl-2-expressing DU145 cell lines was established. Bcl-2 expression levels were unrelated to the susceptibility of DU145 cells to docetaxel. The sensitivity of DU145 cells to cisplatin fluctuated, and the sensitivity to tumor necrosis factor (TNF)-α was decreased by Bcl-2 overexpression. In a xenograft mouse model, overexpression of Bcl-2 drastically decreased the sensitivity of DU145 cells to cisplatin and TNF-α; however, there was no change in the response to docetaxel. Fluorescent microscopy revealed that Bcl-2-overexpression had no effect on the docetaxel-induced death of DU145 cells, but significantly decreased DU145 cell death induced by cisplatin or TNF-α. Interestingly, docetaxel hardly induced caspase-3/7 activation in control or Bcl-2-overexpressing DU145 cells, but did at a low level in LNCaP cells, another prostate cancer cell line. Moreover, in contrast to LNCaP cells, the reduced viabilities of docetaxel-treated control and Bcl-2-overexpressing DU145 cells were not restored by the addition of either a Bid inhibitor or a panel of pro-apoptotic caspase inhibitors. These findings indicate that the antitumor effects of docetaxel on DU145 cells are independent of both Bcl-2 and pro-apoptotic caspases. PMID:27082738

  14. Cell death and the mitochondria: therapeutic targeting of the BCL-2 family-driven pathway

    PubMed Central

    Roy, M J; Vom, A; Czabotar, P E; Lessene, G

    2014-01-01

    The principal biological role of mitochondria is to supply energy to cells; although intriguingly, evolution has bestowed another essential function upon these cellular organelles: under physiological stress, mitochondria become the cornerstone of apoptotic cell death. Specifically, mitochondrial outer membrane permeabilization (MOMP) allows cell death factors such as cytochrome c to be released into the cytoplasm, thus inducing caspase activation and the eventual destruction of essential cellular components. Proteins of the B-cell lymphoma 2 (BCL-2) family control the tightly regulated pathway that causes MOMP. The equilibrium between pro-survival and pro-apoptotic members of the BCL-2 family dictates the fate of cells, the homeostasis of organs and, by extension, the health of whole organisms. Dysregulation of this equilibrium is involved in a large number of diseases such as cancer, autoimmunity and neurodegenerative conditions. Modulating the activity of the BCL-2 family of proteins with small molecules or peptides is an attractive but challenging therapeutic goal. This review highlights the latest developments in this field and provides evidence that this strategy is likely to have a positive effect on the treatment of still poorly addressed medical conditions. LINKED ARTICLES This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8 PMID:24117105

  15. BCL-2 Antagonism to Target the Intrinsic Mitochondrial Pathway of Apoptosis.

    PubMed

    Gibson, Christopher J; Davids, Matthew S

    2015-11-15

    Despite significant improvements in treatment, cure rates for many cancers remain suboptimal. The rise of cytotoxic chemotherapy has led to curative therapy for a subset of cancers, though intrinsic treatment resistance is difficult to predict for individual patients. The recent wave of molecularly targeted therapies has focused on druggable-activating mutations, and is thus limited to specific subsets of patients. The lessons learned from these two disparate approaches suggest the need for therapies that borrow aspects of both, targeting biologic properties of cancer that are at once distinct from normal cells and yet common enough to make the drugs widely applicable across a range of cancer subtypes. The intrinsic mitochondrial pathway of apoptosis represents one such promising target for new therapies, and successfully targeting this pathway has the potential to alter the therapeutic landscape of therapy for a variety of cancers. Here, we discuss the biology of the intrinsic pathway of apoptosis, an assay known as BH3 profiling that can interrogate this pathway, early attempts to target BCL-2 clinically, and the recent promising results with the BCL-2 antagonist venetoclax (ABT-199) in clinical trials in hematologic malignancies. See all articles in this CCR Focus section, "Cell Death and Cancer Therapy." PMID:26567361

  16. Activation of Bcl-2-Caspase-9 Apoptosis Pathway in the Testis of Asthmatic Mice

    PubMed Central

    Li, Junjuan; Ding, Zhaolei; Sheng, Jianhui; Li, Juan; Tan, Wei

    2016-01-01

    Background Apoptosis plays a critical role in controlling the proliferation and differentiation of germ cells during spermatogenesis. Dysregulation of the fine-tuned balance may lead to the onset of testicular diseases. In this study, we investigated the activation status of apoptosis pathways in the testicular tissues under the background of an asthmatic mouse model. Methods Ten BALB/c mice were divided into two groups: the acute asthma group and the control group. In the acute asthma group, ovalbumin (OVA)-sensitized mice were challenged with aerosolized OVA for 7 days, while the control group was treated with physiological saline. After that, both epididymis and testis were collected to determine the sperm count and motility. Apoptosis in the testis was evaluated by DNA ladder, immunochemistry and further by PCR array of apoptosis-related genes. Finally, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP) was determined by western blot and the enzymatic activities of caspase-9 and 3/7 were assessed using Caspase-Glo kits. Results Compared with control mice, significant decreases in the body weight, testis weight, sperm count and motility were seen in the experimental group. DNA ladder and immunochemistry showed significant increase in apoptotic index of the asthmatic testis, whereas a decrease in mRNA expression of Bcl-2 and increases in Bax, BNIP3, caspase-9, and AIF were observed in the asthma group. Furthermore, protein levels of AIF were significantly upregulated, while the translational expression of Bcl-2 was downregulated markedly. Consistently, caspase-9 activity in the testis of asthma mice was significantly higher than that of the control group. Conclusion Collectively, these results showed that Bcl-2-caspase-9 apoptosis pathway was clearly activated in the testis of asthmatic mice with the increased expression of apoptosis-related genes and proteins. To our knowledge, this is the first report demonstrating that asthma could lead to the

  17. Di-(2-ethylhexyl) phthalate induces apoptosis of GC-2spd cells via TR4/Bcl-2 pathway.

    PubMed

    Zhu, Lishan; Lu, Jinchang; Tang, Xiao; Fu, Guoqing; Duan, Peng; Quan, Chao; Zhang, Ling; Zhang, Zhibing; Chang, Wei; Shi, Yuqin

    2016-06-01

    Di-(2-ethylhexyl) phthalate (DEHP) is a widely used environmental endocrine disruptor. Many studies have reported that DEHP exposure causes reproductive toxicity and cells apoptosis. However, the mechanism by which DEHP exposure causes male reproductive toxicity remains unknown. This study investigated the role of the testicular orphan nuclear receptor4 (TR4)/Bcl-2 pathway in apoptosis induced by DEHP, which resulted in reproductive damage. To elucidate the mechanism underpinning the male reproductive toxicity of DEHP, we sought to investigate apoptotic effects, expression levels of TR4/Bcl-2 pathway in GC-2spd cells, including TR4, Bcl-2 and caspase-3. GC-2spd cells were exposed to various concentrations of DEHP (0, 50, 100, or 200μM). The results indicated that, with the increase of the concentrations of DEHP, the survival rate of cell decreased gradually. DEHP exposure at over 100μM significantly induced apoptotic cell death. DEHP decreased SOD and GSH-Px activity in 200μM group. Compared to the control group, the mRNA levels of caspase-3 increased significantly, however, Bcl-2 mRNA decreased (P<0.05). In addition, there was a significant reduction in TR4, Bcl-2 and procaspase-3 protein levels. Taken together, these results lead us to speculate that in vitro exposure to DEHP might induce apoptosis in GC-2spd cells through the TR4/Bcl-2 pathway. PMID:27084994

  18. Bcl-2

    PubMed Central

    Putz, Eva Maria; Schuster, Christian; Sexl, Veronika

    2012-01-01

    Recent findings from our laboratory provide the first indication that overexpression of Bcl-2 in Eµ-myc transgenic cells enhances tumor immunosurveillance by inducing NKG2D ligands. Initial evidence suggests that this model is relevant to human patients. Thus, antitumor therapies that target Bcl-2 harbor the risk of reducing tumor immunogenicity. PMID:22934270

  19. Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2

    PubMed Central

    McCarthy, Aine; Marzec, Jacek; Clear, Andrew; Petty, Robert D.; Coutinho, Rita; Matthews, Janet; Wilson, Andrew; Iqbal, Sameena; Calaminici, Maria; Gribben, John G.; Jia, Li

    2014-01-01

    Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL-2+/BCL-2− lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the BCL-2+ Su-DHL4 cell line compared with BCL-2− Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression profiles were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease. PMID:25362242

  20. Co-targeting of Bcl-2 and mTOR pathway triggers synergistic apoptosis in BH3 mimetics resistant acute lymphoblastic leukemia

    PubMed Central

    Allegretti, Matteo; Mirabilii, Simone; Licchetta, Roberto; Bergamo, Paola; Rinaldo, Cinzia; Zeuner, Ann; Foà, Robin; Milella, Michele; McCubrey, James A.; Martelli, Alberto M.; Tafuri, Agostino

    2015-01-01

    Several chemo-resistance mechanisms including the Bcl-2 protein family overexpression and constitutive activation of the PI3K/Akt/mTOR signaling have been documented in acute lymphoblastic leukemia (ALL), encouraging targeted approaches to circumvent this clinical problem. Here we analyzed the activity of the BH3 mimetic ABT-737 in ALL, exploring the synergistic effects with the mTOR inhibitor CCI-779 on ABT-737 resistant cells. We showed that a low Mcl-1/Bcl-2 plus Bcl-xL protein ratio determined ABT-737 responsiveness. ABT-737 exposure further decreased Mcl-1, inducing apoptosis on sensitive models and primary samples, while not affecting resistant cells. Co-inhibition of Bcl-2 and the mTOR pathway resulted cytotoxic on ABT-737 resistant models, by downregulating mTORC1 activity and Mcl-1 in a proteasome-independent manner. Although Mcl-1 seemed to be critical, ectopic modulation did not correlate with apoptosis changes. Importantly, dual targeting proved effective on ABT-737 resistant samples, showing additive/synergistic effects. Together, our results show the efficacy of BH3 mimetics as single agent in the majority of the ALL samples and demonstrate that resistance to ABT-737 mostly correlated with Mcl-1 overexpression. Co-targeting of the Bcl-2 protein family and mTOR pathway enhanced drug-induced cytotoxicity by suppressing Mcl-1, providing a novel therapeutic approach to overcome BH3 mimetics resistance in ALL. PMID:26392332

  1. Lymphomas are sensitive to perforin-dependent cytotoxic pathways despite expression of PI-9 and overexpression of bcl-2.

    PubMed

    Godal, Robert; Keilholz, Ulrich; Uharek, Lutz; Letsch, Anne; Asemissen, Anne Marie; Busse, Antonia; Na, Il-Kang; Thiel, Eckhard; Scheibenbogen, Carmen

    2006-04-15

    There is considerable interest in immunotherapeutic approaches for lymphoma. The expression of proteinase inhibitor 9 (PI-9), a molecule that inactivates granzyme B, is considered an immune escape mechanism in lymphoma. Further, lymphomas frequently overexpress the antiapoptotic molecule bcl-2, which is able to inhibit perforin-dependent cytotoxic pathways. In this study, the impact of PI-9 and bcl-2 expression on the sensitivity of lymphomas to T- and natural killer (NK) cell-mediated cytotoxicity was analyzed. We found PI-9 expression in 10 of 18 lymphoma cell lines and in 9 of 14 primary lymphomas. Overexpression of bcl-2 was found in 8 of 18 cell lines and in 12 of 14 primary lymphomas. All lymphoma cells were sensitive to cytolysis by specific T cells and cytokine-activated NK cells, and no difference in sensitivity was observed with respect to PI-9 or bcl-2 expression. Cytolysis was mediated predominantly through perforin-dependent pathways despite expression of PI-9 and bcl-2. Interestingly, the majority of lymphoma cells were resistant to cytolysis by resting allogeneic NK cells. This was due to the failure of lymphomas to induce degranulation of resting NK cells. These results show that resistance to perforin-dependent pathways is not a relevant immune escape mechanism in lymphoma and therefore is unlikely to impair clinical outcome of immunotherapeutic approaches. PMID:16373664

  2. Chrysin induces cell apoptosis via activation of the p53/Bcl-2/caspase-9 pathway in hepatocellular carcinoma cells

    PubMed Central

    ZHANG, QINGYU; MA, SHENG; LIU, BIN; LIU, JIE; ZHU, RUNZHI; LI, MINGYI

    2016-01-01

    Chrysin is a major active ingredient of flavonoids, known to exhibit protective effects against various types of cancer. However, the anticancer role of chrysin against hepatocellular carcinoma (HCC) and the underlying molecular mechanisms remain unclear. In order to evaluate the effects of chrysin on cell viability and apoptosis in human HCC, HepG2 and QGY7701 cells were used in the present study. Cell viability was monitored using an MTT assay. In addition, an Annexin V-fluorescein isothiocyanate/propidium iodide kit was used for the labeling of the apoptotic cells, which were then measured using flow cytometry. Western blotting was used to examine the protein expression of p53, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), Bcl-2-associated death promoter (Bad), Bcl-2 homologous antagonist/killer (Bak), caspases-3 and −9, and cleaved-caspases-3 and −9. The results of the present study revealed that chrysin suppressed the cell viability of HepG2 and QGY7701 cells in a concentration-dependent manner. In addition, chrysin induced significant apoptosis in HepG2 and QGY7701 cells. Furthermore, it was demonstrated that chrysin treatment increased the expression of proapoptotic proteins, including p53, Bax, Bad and Bak, while it decreased the protein level of antiapoptotic protein Bcl-2. It was also demonstrated that chrysin induced apoptosis in the HCC cells by regulating the p53/Bcl-2/caspase-9 signaling pathway. In conclusion, the results of the present study suggested that chrysin may be a potential candidate agent for the induction of cell apoptosis in human HCC. PMID:27347080

  3. Involvement of Bcl-2 Signal Pathway in the Protective Effects of Apigenin on Anoxia/Reoxygenation-induced Myocardium Injury.

    PubMed

    Chen, Chuanjun; He, Huan; Luo, Yong; Zhou, Min; Yin, Dong; He, Ming

    2016-02-01

    Apigenin is a type of flavonoids, which has been demonstrated to protect myocardium against ischemia/reperfusion (I/R) injury. However, the mechanism is still unclear. We hypothesized that the mechanism of cardioprotective action of apigenin on the I/R-induced injury might be caused via B-cell lymphoma (Bcl) signaling pathway. In this study, an in vitro I/R model was replicated on Langendorff-perfused heart and H9c2 cardiomyocytes by anoxia/reoxygenation (A/R) treatment. The recovery of cardiac contractile function, infarct size, lactate dehydrogenase (LDH) and creatine kinase (CK) in the perfusate, the expression and activity of Bcl-2 and caspase-3, and cardiomyocyte apoptosis were measured in the Langendorff heart undergoing A/R injury. In addition, the cell viability, LDH release, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), expression of cytochrome c in the cytosol, and cell apoptosis were examined in the culture of H9c2 cardiomyocytes after the A/R. The results showed that apigenin significantly improved rat heart contractile function, reduced LDH release, infarct size and apoptotic rate, upregulated the expression of Bcl-2 and caspase-3, and downregulated the expression of cleaved caspase-3 after the A/R. Moreover, apigenin increased the cell viability and decreased the release of LDH, production of reactive oxygen species, release of mitochondrial cytochrome c into the cytosol, and cell apoptosis in the culture of H9c2 cardiomyocytes after the A/R. In addition, inhibition of Bcl-2 activity by ABT-737 markedly attenuated the protective effect of apigenin on the A/R-induced myocardium injury. Taken together, we firstly demonstrated that the effect of apigenin against A/R injury in cardiomyocytes involves Bcl-2 signal pathway and at least partly depends on its effect of upregulating the expression of Bcl-2. PMID:26466327

  4. Silibinin induces apoptosis through inhibition of the mTOR-GLI1-BCL2 pathway in renal cell carcinoma.

    PubMed

    Ma, Zhenkun; Liu, Wei; Zeng, Jin; Zhou, Jiancheng; Guo, Peng; Xie, Hongjun; Yang, Zhao; Zheng, Long; Xu, Shan; Wang, Xinyang; Chang, Luke S; He, Dalin; Li, Lei

    2015-11-01

    The downstream transcriptional factor of the hedgehog (Hh) pathway, GLI family zinc finger 1 (GLI1), plays a crucial role in regulating tumor progression. In the present study, we demonstrated that silibinin, a natural flavonoid antioxidant isolated from extracts of the milk thistle herb, exerts its anticancer capabilities by restraining GLI1 function in renal cell carcinoma (RCC) cells in vitro and in vivo. In the present study, we confirmed that silibinin induced growth inhibition of RCC through caspase-dependent apoptosis and downregulation of GLI1 and BCL2, which could be partially reversed by GLI1 overexpression. Moreover, we determined that the decreased GLI1 expression by silibinin was mediated by the mammalian target of rapamycin (mTOR) pathway. The in vivo mouse xenograft study also showed that silibinin significantly reduced RCC tumor growth and specifically targeted the mTOR-GLI1-BCL2 signaling pathway. In conclusion, our findings demonstrated for the first time that silibinin induces apoptosis of RCC cells through inhibition of the mTOR-GLI1‑BCL2 pathway. These findings also indicate that GLI1 is a novel regulator for the potential therapeutic application of silibinin against RCC. PMID:26323996

  5. The BCL2 selective inhibitor venetoclax induces rapid onset apoptosis of CLL cells in patients via a TP53-independent mechanism

    PubMed Central

    Anderson, Mary Ann; Deng, Jing; Seymour, John F.; Tam, Constantine; Kim, Su Young; Fein, Joshua; Yu, Lijian; Brown, Jennifer R.; Westerman, David; Si, Eric G.; Majewski, Ian J.; Segal, David; Heitner Enschede, Sari L.; Huang, David C. S.; Davids, Matthew S.; Letai, Anthony

    2016-01-01

    BCL2 blunts activation of the mitochondrial pathway to apoptosis, and high-level expression is required for chronic lymphocytic leukemia (CLL) survival. Venetoclax (ABT-199) is a small-molecule selective inhibitor of BCL2 currently in clinical trials for CLL and other malignancies. In conjunction with the phase 1 first-in-human clinical trial of venetoclax in patients with relapsed or refractory CLL (M12-175), we investigated the mechanism of action of venetoclax in vivo, explored whether in vitro sensitivity assays or BH3 profiling correlated with in vivo responses in patients, and determined whether loss of TP53 function affected responses in vitro and in vivo. In all samples tested, venetoclax induced death of CLL cells in vitro at concentrations achievable in vivo, with cell death evident within 4 hours. Apoptotic CLL cells were detected in vivo 6 or 24 hours after a single 20-mg or 50-mg dose in some patients. The extent of mitochondrial depolarization by a BIM BH3 peptide in vitro was correlated with percentage reduction of CLL in the blood and bone marrow in vivo, whereas the half lethal concentration derived from standard cytotoxicity assays was not. CLL cell death in vitro and the depth of clinical responses were independent of deletion of chromosome 17p, TP53 mutation, and TP53 function. These data provide direct evidence that venetoclax kills CLL cells in a TP53-independent fashion by inhibition of BCL2 in patients and support further assessment of BH3 profiling as a predictive biomarker for this drug. PMID:27069256

  6. The BCL2 selective inhibitor venetoclax induces rapid onset apoptosis of CLL cells in patients via a TP53-independent mechanism.

    PubMed

    Anderson, Mary Ann; Deng, Jing; Seymour, John F; Tam, Constantine; Kim, Su Young; Fein, Joshua; Yu, Lijian; Brown, Jennifer R; Westerman, David; Si, Eric G; Majewski, Ian J; Segal, David; Heitner Enschede, Sari L; Huang, David C S; Davids, Matthew S; Letai, Anthony; Roberts, Andrew W

    2016-06-23

    BCL2 blunts activation of the mitochondrial pathway to apoptosis, and high-level expression is required for chronic lymphocytic leukemia (CLL) survival. Venetoclax (ABT-199) is a small-molecule selective inhibitor of BCL2 currently in clinical trials for CLL and other malignancies. In conjunction with the phase 1 first-in-human clinical trial of venetoclax in patients with relapsed or refractory CLL (M12-175), we investigated the mechanism of action of venetoclax in vivo, explored whether in vitro sensitivity assays or BH3 profiling correlated with in vivo responses in patients, and determined whether loss of TP53 function affected responses in vitro and in vivo. In all samples tested, venetoclax induced death of CLL cells in vitro at concentrations achievable in vivo, with cell death evident within 4 hours. Apoptotic CLL cells were detected in vivo 6 or 24 hours after a single 20-mg or 50-mg dose in some patients. The extent of mitochondrial depolarization by a BIM BH3 peptide in vitro was correlated with percentage reduction of CLL in the blood and bone marrow in vivo, whereas the half lethal concentration derived from standard cytotoxicity assays was not. CLL cell death in vitro and the depth of clinical responses were independent of deletion of chromosome 17p, TP53 mutation, and TP53 function. These data provide direct evidence that venetoclax kills CLL cells in a TP53-independent fashion by inhibition of BCL2 in patients and support further assessment of BH3 profiling as a predictive biomarker for this drug. PMID:27069256

  7. Exploration of Bcl-2 family and caspases-dependent apoptotic signaling pathway in Zearalenone-treated mouse endometrial stromal cells.

    PubMed

    Hu, Jin; Xu, Minglong; Dai, Yujian; Ding, Xiaolin; Xiao, Cheng; Ji, Hongjun; Xu, Yinxue

    2016-08-01

    Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. Although the toxicity of ZEA have been widely studied in a number of cell types, the mechanistic role of ZEA on apoptosis of endometrial stromal cells (ESCs) remains poorly understood. The objective of this study was to determine the effects of ZEA on apoptosis of mouse ESCs and explore the signaling pathway underlying the cytotoxicity of ZEA. The results showed that ZEA treatment caused obvious apoptosis in ESCs as determined by the flow cytometry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR) revealed that ZEA treatment increased the ratio of Bax/Bcl-2. The enzymatic activity assays revealed that caspases-3 and caspase-9 were activated by ZEA treatment in a dose-dependent manner. In addition, flow cytometry show that the apoptotic percentages of cells pretreated with Z-VAD-FMK and Z-LEHD-FMK were markedly reduced compared to the ZEA-treated cells. Overall, the results suggested that ZEA induced obvious apoptosis in ESCs via a Bcl-2 family and caspases-dependent signaling pathway. PMID:27286704

  8. Arsenite induces apoptosis in human mesenchymal stem cells by altering Bcl-2 family proteins and by activating intrinsic pathway

    SciTech Connect

    Yadav, Santosh; Shi Yongli; Wang Feng; Wang He

    2010-05-01

    Purpose: Environmental exposure to arsenic is an important public health issue. The effects of arsenic on different tissues and organs have been intensively studied. However, the effects of arsenic on bone marrow mesenchymal stem cells (MSCs) have not been reported. This study is designed to investigate the cell death process caused by arsenite and its related underlying mechanisms on MSCs. The rationale is that absorbed arsenic in the blood circulation can reach to the bone marrow and may affect the cell survival of MSCs. Methods: MSCs of passage 1 were purchased from Tulane University, grown till 70% confluency level and plated according to the experimental requirements followed by treatment with arsenite at various concentrations and time points. Arsenite (iAs{sup III}) induced cytotoxic effects were confirmed by cell viability and cell cycle analysis. For the presence of canonic apoptosis markers; DNA damage, exposure of intramembrane phosphotidylserine, protein and m-RNA expression levels were analyzed. Results: iAs{sup III} induced growth inhibition, G2-M arrest and apoptotic cell death in MSCs, the apoptosis induced by iAs{sup III} in the cultured MSCs was, via altering Bcl-2 family proteins and by involving intrinsic pathway. Conclusion: iAs{sup III} can induce apoptosis in bone marrow-derived MSCs via Bcl-2 family proteins, regulating intrinsic apoptotic pathway. Due to the multipotency of MSC, acting as progenitor cells for a variety of connective tissues including bone, adipose, cartilage and muscle, these effects of arsenic may be important in assessing the health risk of the arsenic compounds and understanding the mechanisms of arsenic-induced harmful effects.

  9. Pristimerin Induces Apoptosis in Prostate Cancer Cells by Down-regulating Bcl-2 through ROS-dependent Ubiquitin-proteasomal Degradation Pathway

    PubMed Central

    Liu, Yong Bo; Gao, Xiaohua; Deeb, Dorrah; Arbab, Ali S; Gautam, Subhash C

    2014-01-01

    Pristimerin is a quinonemethide triterpenoid with the potential of a promising anticancer agent. Pristimerin (PM) has shown anticancer activity against a range of cancer cell lines, but its activity for prostate cancer has not been adequately investigated. In the present study we have examined the underlying mechanisms of the apoptotic response of the hormone-sensitive (LNCaP) and hormone-refractory (PC-3) prostate cancer cell lines to PM. Treatment with PM induced apoptosis in both cell lines as characterized by increased annexin V-binding and cleavage of PARP-1 and procaspases-3 and -9. It also induced mitochondrial depolarization, cytochrome c release from mitochondria and generation of reactive oxygen species (ROS). Response to PM is regulated by Bcl-2 since it down-regulated Bcl-2 expression and overexpression of Bcl-2 rendered prostate cancer cells resistant to PM. ROS plays a role in down-regulation of Bcl-2, since treatment with PM in the presence of various ROS modulators, e.g., n-acetylcysteine (NAC), a general purpose antioxidant; diphenylene iodonium (DPI), a NADPH inhibitor; rotenone (ROT), a mitochondrial electron transport chain interrupter rotenone or MnTBAP, a O2 scavenger, attenuated the down-regulation of Bcl-2. Furthermore, ROS is also involved in the ubiquitination and proteasomal degradation of Bcl-2 as both of these events were blocked by O 2− scavenger MnTBAP. Thus, pristimerin induces apoptosis in prostate cancer cells predominately through the mitochondrial apoptotic pathway by inhibiting antiapoptic Bcl-2 through a ROS-dependent ubiquitin-proteasomal degradation pathway. PMID:24877026

  10. B1-induced caspase-independent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box

    SciTech Connect

    Liang Xin; Xu Ke; Xu Yufang; Liu Jianwen Qian Xuhong

    2011-10-01

    The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P{sub 2} promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment. - Research Highlights: > B1 induced apoptosis in MCF-7 cells, following a transcriptional decrease in Bcl-2. > B1 treatment triggered p53 activation and leads to a p53-dependent down-regulation of Bcl-2. > B1 induced significant increase of p53 binding to Bcl-2 P{sub 2} promoter TATA box.

  11. Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: role of the Akt/mTOR survival pathway and Bcl-2 family proteins.

    PubMed

    Marin, Jose J G; Hernandez, Alicia; Revuelta, Isabel E; Gonzalez-Sanchez, Ester; Gonzalez-Buitrago, Jose M; Perez, Maria J

    2013-08-01

    Acute accumulation of bile acids in hepatocytes may cause cell death. However, during long-term exposure due to prolonged cholestasis, hepatocytes may develop a certain degree of chemoresistance to these compounds. Because mitochondrial adaptation to persistent oxidative stress may be involved in this process, here we have investigated the effects of complete mitochondrial genome depletion on the response to bile acid-induced hepatocellular injury. A subline (Rho) of human hepatoma SK-Hep-1 cells totally depleted of mitochondrial DNA (mtDNA) was obtained, and bile acid-induced concentration-dependent activation of apoptosis/necrosis and survival signaling pathways was studied. In the absence of changes in intracellular ATP content, Rho cells were highly resistant to bile acid-induced apoptosis and partially resistant to bile acid-induced necrosis. In Rho cells, both basal and bile acid-induced generation of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, was decreased. Bile acid-induced proapoptotic signals were also decreased, as evidenced by a reduction in the expression ratios Bax-α/Bcl-2, Bcl-xS/Bcl-2, and Bcl-xS/Bcl-xL. This was mainly due to a downregulation of Bax-α and Bcl-xS. Moreover, in these cells the Akt/mTOR pathway was constitutively activated in a ROS-independent manner and remained similarly activated in the presence of bile acid treatment. In contrast, ERK1/2 activation was constitutively reduced and was not activated by incubation with bile acids. In conclusion, these results suggest that impaired mitochondrial function associated with mtDNA alterations, which may occur in liver cells during prolonged cholestasis, may activate mechanisms of cell survival accounting for an enhanced resistance of hepatocytes to bile acid-induced apoptosis. PMID:23597504

  12. The Bax/Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the RU486-treated rat uterus during early pregnancy.

    PubMed

    Theron, Kathrine E; Penny, Clement B; Hosie, Margot J

    2013-12-01

    An increase in apoptotic activity has been observed in both the rabbit and the rat endometria following treatment with RU486. The aim of this study was to assess whether Bax and Bcl-2 signaling, in response to RU486, could be crucial role players mediating apoptosis in the rat uterus during early pregnancy. RU486 is a partial progesterone (P4) and estrogen receptor antagonist, functioning to actively silence P4 receptor gene-associated transcription. Although an increase in apoptosis as a result of RU486 administration has been previously reported in rabbits, the specific apoptotic factors and pathways involved in driving this process have not yet been established. Immunofluorescent techniques were used to determine protein expression levels of both Bax and Bcl-2 in RU486-treated endometria at days 4.5, 5.5 and 6.5 of pregnancy. The Bax/Bcl-2 index was used to determine the overall pro- or anti-apoptotic setting at each day of pregnancy, following RU486 administration. Changes in the Bax and Bcl-2 gene expression levels as a consequence of RU486 administration were evaluated using RT-qPCR. Both the protein and gene expression analyses suggest that RU486 induces a change toward an overall anti-apoptotic signal within the Bax/Bcl-2 pathway. These results suggest that the observed increase in apoptosis following RU486 administration is not driven by a shift in the Bax/Bcl-2 ratio toward cell death, when the P4 and estrogen receptors are partially inactivated by RU486, but is possibly regulated by another apoptotic pathway. PMID:24287037

  13. MPT64 Protein from Mycobacterium tuberculosis Inhibits Apoptosis of Macrophages through NF-kB-miRNA21-Bcl-2 Pathway

    PubMed Central

    Wang, Qingmin; Liu, Shupeng; Tang, Ying; Liu, Qiuhong; Yao, Yongjie

    2014-01-01

    MPT64 is one of the secreted proteins from Mycobacterium tuberculosis. Little is known about its role in infection by Mycobacterium tuberculosis. In this study, we demonstrated that MPT64 could dose-dependently inhibit the apoptosis of RAW264.7 macrophages induced by PPD-BCG. Quantitative real-time PCR results showed that the expression of bcl-2 increased in macrophages treated with MPT64 compared with PPD-treated cells. Furthermore, the results provided strong evidence that bcl-2 up-regulation was positively controlled by miRNA-21. Finally, NF-κB was identified as the transcription factor for miRNA-21 using a ChIP assay. It can be concluded from our study that MPT64 could inhibit the apoptosis of RAW264.7 macrophages through the NF-κB-miRNA21-Bcl-2 pathway. PMID:25000291

  14. Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types.

    PubMed Central

    Zhu, W; Cowie, A; Wasfy, G W; Penn, L Z; Leber, B; Andrews, D W

    1996-01-01

    Human Bcl-2 is located in multiple intracellular membranes when expressed in MDCK and Rat-1/myc cells. We restricted expression to the endoplasmic reticulum or mitochondria by exchanging the Bcl-2 carboxy-terminal insertion sequence for an equivalent sequence from cytochrome b5 or ActA, respectively. MDCK cells are protected from serum deprivation-induced apoptosis by both wild-type Bcl-2 and the mutant targeted to mitochondria but not by the mutant targeted to endoplasmic reticulum. In contrast, when expressed in Rat-1/myc cells, the Bcl-2 mutant located at the endoplasmic reticulum is more effective than that targeted to mitochondria. In MDCK cells both mutants bind Bax as effectively as wild-type, demonstrating that Bax binding is not sufficient to prevent apoptosis. Images PMID:8861942

  15. Pyrroloquinoline Quinone Induces Cancer Cell Apoptosis via Mitochondrial-Dependent Pathway and Down-Regulating Cellular Bcl-2 Protein Expression.

    PubMed

    Min, Zhihui; Wang, Lingyan; Jin, Jianjun; Wang, Xiangdong; Zhu, Bijun; Chen, Hao; Cheng, Yunfeng

    2014-01-01

    Pyrroloquinoline quinone (PQQ) has been reported as a promising agent that might contribute to tumor cell apoptosis and death, yet little is known on its mechanisms. In current study, the effect of PQQ on cell proliferation and mitochondrial-dependent apoptosis were examined in 3 solid tumor cell lines (A549, Neuro-2A and HCC-LM3). PQQ treatment at low to medium dosage exhibited potent anti-tumor activity on A549 and Neuro-2A cells, while had comparably minimal impact on the viabilities of 2 human normal cell lines (HRPTEpiC and HUVEC). The apoptosis of the 3 tumor cell lines induced by PQQ were increased in a concentration-dependent manner, which might be attributed to the accumulation of intracellular reactive oxygen species (ROS), decline in ATP levels and dissipation of mitochondrial membrane potential (MMP), in conjunction with down-regulation of Bcl-2 protein expression, up-regulation of activated caspase-3, and disturbed phosphorylated MAPK protein levels. PQQ induced tumor cells apoptosis was significantly alleviated by pan-caspase inhibitor Z-VAD-FMK. The present work highlights the potential capability of PQQ as an anti-tumor agent with low toxicity towards normal cells through activating mitochondrial-dependent apoptosis pathways, and warrants its development for cancer therapy. PMID:25161699

  16. Neohesperidin induces cellular apoptosis in human breast adenocarcinoma MDA-MB-231 cells via activating the Bcl-2/Bax-mediated signaling pathway.

    PubMed

    Xu, Fei; Zang, Jia; Chen, Daozhen; Zhang, Ting; Zhan, Huiying; Lu, Mudan; Zhuge, Hongxiang

    2012-11-01

    Neohesperidin, a flavonoid compound found in high amounts in Poncirus trifoliata, has free radical scavenging activity. For the first time, our study indicated that neohesperidin also induces cell apoptosis in human breast adenocarcinoma MDA-MB-231 cells, which was possibly mediated by regulating the P53/Bcl-2/Bax pathway. MDA-MB-231 cells were subjected to treatment with neohesperidin. MTT and Trypan blue exclusion assays were applied to assess the cell viability. The morphological changes of cells were observed using an inverted microscope, and cell apoptosis was detected by flow cytometric analysis. Immunoblot analysis was conducted to evaluate the protein expressions of apoptosis-related genes, including P53, Bcl-2 and Bax. Our results indicated that the proliferation of MDA-MB-231 cells was inhibited by the treatment with neohesperidin in a time- and dose-dependent manner. The IC50 values of neohesperidin at 24 and 48 h were 47.4 +/- 2.6 microM and 32.5 +/- 1.8 microM, respectively. The expressions of P53 and Bax in the neohesperidin-treated cells were significantly up-regulated, while that of Bcl-2 was down-regulated. Our study suggested that neohesperidin could induce apoptosis of MDA-MB-231 cells, a process which was associated with the activation of the Bcl-2/Bax-mediated signaling pathway. PMID:23285810

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

    PubMed Central

    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

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

    PubMed

    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

  19. Targeting the apoptotic pathway with BCL-2 inhibitors sensitizes primary chronic lymphocytic leukemia cells to vesicular stomatitis virus-induced oncolysis.

    PubMed

    Tumilasci, Vanessa Fonseca; Olière, Stephanie; Nguyên, Thi Lien-Ahn; Shamy, April; Bell, John; Hiscott, John

    2008-09-01

    Chronic lymphocytic leukemia (CLL) is characterized by clonal accumulation of CD5(+) CD19(+) B lymphocytes that are arrested in the G(0)/G(1) phase of the cell cycle and fail to undergo apoptosis because of overexpression of the antiapoptotic B-cell CLL/lymphoma 2 (BCL-2) protein. Oncolytic viruses, such as vesicular stomatitis virus (VSV), have emerged as potential anticancer agents that selectively target and kill malignant cells via the intrinsic mitochondrial pathway. Although primary CLL cells are largely resistant to VSV oncolysis, we postulated that targeting the apoptotic pathway via inhibition of BCL-2 may sensitize CLL cells to VSV oncolysis. In the present study, we examined the capacity of EM20-25--a small-molecule antagonist of the BCL-2 protein--to overcome CLL resistance to VSV oncolysis. We demonstrate a synergistic effect of the two agents in primary ex vivo CLL cells (combination index of 0.5; P < 0.0001). In a direct comparison of peripheral blood mononuclear cells from healthy volunteers with primary CLL, the two agents combined showed a therapeutic index of 19-fold; furthermore, the combination of VSV and EM20-25 increased apoptotic cell death in Karpas-422 and Granta-519 B-lymphoma cell lines (P < 0.005) via the intrinsic mitochondrial pathway. Mechanistically, EM20-25 blocked the ability of the BCL-2 protein to dimerize with proapoptotic BAX protein, thus sensitizing CLL to VSV oncolytic stress. Together, these data indicate that the use of BCL-2 inhibitors may improve VSV oncolysis in treatment-resistant hematological malignancies, such as CLL, with characterized defects in the apoptotic response. PMID:18579592

  20. Bcl-2 does not require Raf kinase activity for its death-protective function.

    PubMed Central

    Olivier, R; Otter, I; Monney, L; Wartmann, M; Borner, C

    1997-01-01

    It has been widely accepted that the oncogene product bcl-2 protects mammalian cells from programmed cell death (apoptosis). The molecules and signalling pathways upon which bcl-2 acts are, however, still ill-defined. Recently, bcl-2 was shown to interact with c-raf-1 in vitro. Furthermore, an active form of c-raf-1 delayed apoptosis induced by trophic factor deprivation and enhanced the death-suppressive function of bcl-2 when co-expressed. This has led to the hypothesis that bcl-2 communicates cell-death protection via a raf-dependent signal transduction pathway. Here we show, by various immunological and biochemical methods, that bcl-2 does not stably associate with c-raf-1 in cellular extracts prepared from fibroblasts before or after treatment with agents that induce apoptosis. Unexpectedly, bcl-2 function is entirely maintained, if not improved, when raf-dependent signalling is experimentally abrogated. In fact, bcl-2 allows the stable overexpression of a kinase-defective dominant-negative raf mutant that usually interferes with cell viability and/or proliferation. Our results indicate that bcl-2 does not require c-raf-1 kinase activity and an associated mitogen-activated protein kinase signalling pathway for its survival function. This property may be exploited to dissect cellular events that are dependent or independent of c-raf-1 kinase activity. PMID:9164843

  1. Adenosine induces cell cycle arrest and apoptosis via cyclinD1/Cdk4 and Bcl-2/Bax pathways in human ovarian cancer cell line OVCAR-3.

    PubMed

    Shirali, Saeid; Aghaei, Mahmoud; Shabani, Mahdi; Fathi, Mojtaba; Sohrabi, Majid; Moeinifard, Marzieh

    2013-04-01

    Adenosine is a regulatory molecule with widespread physiological effects in almost every cells and acts as a potent regulator of cell growth. Adenosine has been shown to inhibit cell growth and induce apoptosis in the several cancer cells via caspase activation and Bcl-2/Bax pathway. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis in the OVCAR-3 human ovarian cancer cells. MTT viability, BrdU and cell counting assays were used to study the cell proliferation effect of adenosine in presence of adenosine deaminase inhibitor and the nucleoside transporter inhibitor. Cell cycle analysis, propidium iodide and annexin V staining, caspase-3 activity assay, cyclinD1, Cdk4, Bcl-2 and Bax protein expressions were assessed to detect apoptosis. Adenosine significantly inhibited cell proliferation in a concentration-dependent manner in OVCAR-3 cell line. Adenosine induced cell cycle arrest in G0/G1 phase via Cdk4/cyclinD1-mediated pathway. Adenosine induced apoptosis, which was determined by Annexin V-FITC staining and increased sub-G1 population. Moreover, down-regulation of Bcl-2 protein expression, up-regulation of Bax protein expression and activation of caspase-3 were observed in response to adenosine treatment. The results of this study suggest that extracellular adenosine induced G1 cell cycle arrest and apoptosis in ovarian cancer cells via cyclinD1/ Cdk4 and Bcl-2/Bax pathways and caspase-3 activation. These data might suggest that adenosine could be used as an agent for the treatment of ovarian cancer. PMID:23345014

  2. RLIP76-dependent suppression of PI3K/AKT/Bcl-2 pathway by miR-101 induces apoptosis in prostate cancer

    SciTech Connect

    Yang, Jing; Song, Qi; Cai, Yi; Wang, Peng; Wang, Min; Zhang, Dong

    2015-08-07

    MicroRNA-101 (miR-101) participates in carcinogenesis and tumor progression in various cancers. However, its biological functions in prostate cancer are still unclear. Here, we demonstrate that miR-101 represents a critical role in regulating cell apoptosis in prostate cancer cells. We first demonstrated that miR-101 treatment promoted apoptosis in DU145 and PC3 cells by using flow cytometric analysis and transmission electron microscopy (TEM). To verify the mechanisms, we identified a novel miR-101 target, Ral binding protein 1 (RLIP76). We found miR-101 transfection significantly suppresses RLIP76 expression, which can transactivate phosphorylation of PI3K-Akt signaling, and resulted in an amplification of Bcl2-induced apoptosis. Furthermore, we demonstrated that RLIP76 overexpression could reverse the anti-tumor effects of miR-101 in DU145 and PC3 cells by using flow cytometry assay and MTT assay. Taken together, our results revealed that the effect of miR-101 on prostate cancer cell apoptosis was due to RLIP76 regulation of the PI3K/Akt/Bcl-2 signaling pathway. - Highlights: • miR-101 inhibited prostate cancer cell proliferation and enhanced apoptosis. • miR-101 directly targeted and regulated RLIP76 expression. • miR-101 suppressed PI3K/Akt/Bcl-2 signaling pathway by targeting RLIP76.

  3. BCL2DB: database of BCL-2 family members and BH3-only proteins.

    PubMed

    Rech de Laval, Valentine; Deléage, Gilbert; Aouacheria, Abdel; Combet, Christophe

    2014-01-01

    BCL2DB (http://bcl2db.ibcp.fr) is a database designed to integrate data on BCL-2 family members and BH3-only proteins. These proteins control the mitochondrial apoptotic pathway and probably many other cellular processes as well. This large protein group is formed by a family of pro-apoptotic and anti-apoptotic homologs that have phylogenetic relationships with BCL-2, and by a collection of evolutionarily and structurally unrelated proteins characterized by the presence of a region of local sequence similarity with BCL-2, termed the BH3 motif. BCL2DB is monthly built, thanks to an automated procedure relying on a set of homemade profile HMMs computed from seed reference sequences representative of the various BCL-2 homologs and BH3-only proteins. The BCL2DB entries integrate data from the Ensembl, Ensembl Genomes, European Nucleotide Archive and Protein Data Bank databases and are enriched with specific information like protein classification into orthology groups and distribution of BH motifs along the sequences. The Web interface allows for easy browsing of the site and fast access to data, as well as sequence analysis with generic and specific tools. BCL2DB provides a helpful and powerful tool to both 'BCL-2-ologists' and researchers working in the various fields of physiopathology. Database URL: http://bcl2db.ibcp.fr. PMID:24608034

  4. IPS-1 differentially induces TRAIL, BCL2, BIRC3 and PRKCE in type I interferons-dependent and -independent anticancer activity

    PubMed Central

    Kumar, S; Ingle, H; Mishra, S; Mahla, R S; Kumar, A; Kawai, T; Akira, S; Takaoka, A; Raut, A A; Kumar, H

    2015-01-01

    RIG-I-like receptors are the key cytosolic sensors for RNA viruses and induce the production of type I interferons (IFN) and pro-inflammatory cytokines through a sole adaptor IFN-β promoter stimulator-1 (IPS-1) (also known as Cardif, MAVS and VISA) in antiviral innate immunity. These sensors also have a pivotal role in anticancer activity through induction of apoptosis. However, the mechanism for their anticancer activity is poorly understood. Here, we show that anticancer vaccine adjuvant, PolyIC (primarily sensed by MDA5) and the oncolytic virus, Newcastle disease virus (NDV) (sensed by RIG-I), induce anticancer activity. The ectopic expression of IPS-1 into type I IFN-responsive and non-responsive cancer cells induces anticancer activity. PolyIC transfection and NDV infection upregulate pro-apoptotic gene TRAIL and downregulate the anti-apoptotic genes BCL2, BIRC3 and PRKCE. Furthermore, stable knockdown of IPS-1, IRF3 or IRF7 in IFN-non-responsive cancer cells show reduced anticancer activity by suppressing apoptosis via TRAIL and anti-apoptotic genes. Collectively, our study shows that IPS-1 induces anticancer activity through upregulation of pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE via IRF3 and IRF7 in type I IFN-dependent and -independent manners. PMID:25950488

  5. IPS-1 differentially induces TRAIL, BCL2, BIRC3 and PRKCE in type I interferons-dependent and -independent anticancer activity.

    PubMed

    Kumar, S; Ingle, H; Mishra, S; Mahla, R S; Kumar, A; Kawai, T; Akira, S; Takaoka, A; Raut, A A; Kumar, H

    2015-01-01

    RIG-I-like receptors are the key cytosolic sensors for RNA viruses and induce the production of type I interferons (IFN) and pro-inflammatory cytokines through a sole adaptor IFN-β promoter stimulator-1 (IPS-1) (also known as Cardif, MAVS and VISA) in antiviral innate immunity. These sensors also have a pivotal role in anticancer activity through induction of apoptosis. However, the mechanism for their anticancer activity is poorly understood. Here, we show that anticancer vaccine adjuvant, PolyIC (primarily sensed by MDA5) and the oncolytic virus, Newcastle disease virus (NDV) (sensed by RIG-I), induce anticancer activity. The ectopic expression of IPS-1 into type I IFN-responsive and non-responsive cancer cells induces anticancer activity. PolyIC transfection and NDV infection upregulate pro-apoptotic gene TRAIL and downregulate the anti-apoptotic genes BCL2, BIRC3 and PRKCE. Furthermore, stable knockdown of IPS-1, IRF3 or IRF7 in IFN-non-responsive cancer cells show reduced anticancer activity by suppressing apoptosis via TRAIL and anti-apoptotic genes. Collectively, our study shows that IPS-1 induces anticancer activity through upregulation of pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE via IRF3 and IRF7 in type I IFN-dependent and -independent manners. PMID:25950488

  6. The MUC1 oncomucin regulates pancreatic cancer cell biological properties and chemoresistance. Implication of p42–44 MAPK, Akt, Bcl-2 and MMP13 pathways

    SciTech Connect

    Tréhoux, Solange; Duchêne, Bélinda; Jonckheere, Nicolas; Van Seuningen, Isabelle

    2015-01-16

    Highlights: • Loss of MUC1 decreases proliferation and tumor growth via β-catenin and p42–44 MAPK. • Inhibition of MUC1 decreases cell migration and invasion through MMP13. • Loss of MUC1 decreases survival and increases apoptosis via Akt and Bcl-2 pathways. • Loss of MUC1 sensitizes cells to gemcitabine and 5-Fluorouracil chemotherapeutic drugs. - Abstract: MUC1 is an oncogenic mucin overexpressed in several epithelial cancers, including pancreatic ductal adenocarcinoma, and is considered as a potent target for cancer therapy. To this aim, we undertook to study MUC1 biological effects on pancreatic cancer cells and identify pathways mediating these effects. Our in vitro experiments indicate that inhibiting MUC1 expression decreases cell proliferation, cell migration and invasion, cell survival and increases cell apoptosis. Moreover, lack of MUC1 in these cells profoundly altered their sensitivity to gemcitabine and 5-Fluorouracil chemotherapeutic drugs. In vivo MUC1-KD cell xenografts in SCID mice grew slower. Altogether, we show that MUC1 oncogenic mucin alters proliferation, migration, and invasion properties of pancreatic cancer cells and that these effects are mediated by p42–44 MAPK, Akt, Bcl-2 and MMP13 pathways.

  7. Insulin exerts neuroprotective effects via Akt/Bcl-2 signaling pathways in differentiated SH-SY5Y cells.

    PubMed

    Ramalingam, Mahesh; Kim, Sung-Jin

    2015-02-01

    In the present study, the changes in the cell viability at different concentrations of hydrogen peroxide (H2O2) for 3 h used to establish a model of oxidative stress. Further assays with 200 μM H2O2 induces significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), reactive oxygen species (ROS) and calcium ion (Ca(2+)) in neuronal cells, but insulin can effectively diminish the oxidative damages. Moreover, cells treated with insulin increased the H2O2-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of Akt, Bcl-2, Bax, IRβ, IGF-1Rβ, IRS-1 and IRS-2 showed that insulin treatment had a protective effect on H2O2-induced oxidative stress in RA-differentiated SH-SY5Y neuroblastoma cells. PMID:24849496

  8. Cytotoxicity of carteolol to human corneal epithelial cells by inducing apoptosis via triggering the Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway.

    PubMed

    Shan, Ming; Fan, Ting-Jun

    2016-09-01

    Carteolol is a frequently used nonselective β-adrenoceptor antagonist for glaucoma and ocular hypertension treatment, and its repeated/prolonged usage might be cytotoxic to the cornea, especially the outmost human corneal epithelium (HCEP). The aim of the present study was to characterize the cytotoxicity of carteolol to HCEP and its underlying cellular and molecular mechanisms using an in vitro model of HCEP cells. After HCEP cells were treated with carteolol at concentrations varying from 2% to 0.015625%, the cytotoxicity, apoptosis-inducing effect and pro-apoptotic pathway was investigated, respectively. Our results showed that carteolol at concentrations above 0.03125% induced time- and dose-dependent growth retardation, cytopathic morphological changes and viability decline of HCEP cells. Moreover, carteolol induced G1 phase arrest, plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCEP cells. Furthermore, carteolol also induced activation of caspase-9 and -3, disruption of mitochondrial transmembrane potential, up-regulation the cytoplasmic amount of cytochrome c and apoptosis-inducing factor, and up-regulation of pro-apoptotic Bax and Bad, down-regulation of anti-apoptotic Bcl-2 and Bcl-xL. In conclusion, carteolol above 1/64 of its clinical therapeutic dosage has a time- and dose-dependent cytotoxicity to HCEP cells, which is achieved by inducing apoptosis via triggering Bcl-2 family protein-mediated mitochondrial pro-apoptotic pathway. PMID:27216471

  9. Alantolactone induces apoptosis of human cervical cancer cells via reactive oxygen species generation, glutathione depletion and inhibition of the Bcl-2/Bax signaling pathway

    PubMed Central

    JIANG, YAN; XU, HANJIE; WANG, JIAFEI

    2016-01-01

    Alantolactone is the active ingredient in frankincense, and is extracted from the dry root of elecampane. It has a wide variety of uses, including as an insect repellent, antibacterial, antidiuretic, analgesic and anticancer agent. In addition, alantolactone induces apoptosis of human cervical cancer cells, however, its mechanism of action remains to be elucidated. Therefore, the present study investigated whether alantolactone was able to induce apoptosis of human cervical cancer cells, and its potential mechanisms of action were analyzed. Treatment of HeLa cells with alantolactone (0, 10, 20, 30, 40, 50 and 60 µM) for 12 h significantly inhibited growth in a dose-dependent manner. Cells treated with 30 µM of alantolactone for 0, 3, 6 and 12 h demonstrated marked induction of apoptosis in a time-dependent manner. Treatment of HeLa cells with 30 µM of alantolactone for 0, 3, 6 and 12 h significantly induced the generation of reactive oxygen species (ROS) and inhibited glutathione (GSH) production in HeLa cells in a dose-dependent manner. Alantolactone additionally markedly inhibited the Bcl-2/Bax signaling pathway in HeLa cells. Therefore, administration of alantolactone induced apoptosis of human cervical cancer cells via ROS generation, GSH depletion and inhibition of the Bcl-2/Bax signaling pathway. PMID:27313767

  10. Tissue Kallikrein Alleviates Cerebral Ischemia-Reperfusion Injury by Activating the B2R-ERK1/2-CREB-Bcl-2 Signaling Pathway in Diabetic Rats

    PubMed Central

    Yuan, Kunxiong; Hu, Bin; Sang, Hongfei; Xie, Yi; Xu, Lili; Cao, Qinqin; Chen, Xin; Zhao, Lingling; Liu, Xinfeng; Liu, Ling; Zhang, Renliang

    2016-01-01

    Diabetes mellitus (DM) substantially increases the risk of ischemic stroke and reduces the tolerance to ischemic insults. Tissue kallikrein (TK) has been demonstrated to protect neurons from ischemia/reperfusion (I/R) injury in orthoglycemic model by activating the bradykinin B2 receptor (B2R). Considering the differential effects of B2R or bradykinin B1 receptor (B1R) on cardioprotection and neuroprotection in I/R with or without diabetes, this study was designed to investigate the role of TK during cerebral I/R injury in streptozotocin-induced diabetic rats. Intravenous injection of TK inhibited apoptosis in neurons, alleviated edema and inflammatory reactions after focal cerebral I/R, significantly reduced the infarct volume, and improved functional recovery. These beneficial effects were accompanied by activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), cAMP response element-binding (CREB), and Bcl-2 signal proteins. Inhibition of the B2R or ERK1/2 pathway abated the effects of TK, whereas an antagonist of B1R enhanced the effects. These findings reveal that the neuroprotective effect of TK against cerebral I/R injury in streptozotocin-induced diabetic rats mainly involves the enhancement of B2R and ERK1/2-CREB-Bcl-2 signaling pathway activity.

  11. Anti-apoptotic activity of caffeic acid, ellagic acid and ferulic acid in normal human peripheral blood mononuclear cells: a Bcl-2 independent mechanism.

    PubMed

    Khanduja, Krishan Lal; Avti, Pramod Kumar; Kumar, Surender; Mittal, Nidhi; Sohi, Kiranjit Kaur; Pathak, Chander Mohan

    2006-02-01

    Polyphenols have been shown to induce apoptosis in a variety of tumor cells including leukemia both in vitro and in vivo. However, their action on normal human peripheral blood mononuclear cells (PBMCs) during oxidative stress remains to be explored. In this study, we have evaluated the anti-apoptotic and radical scavenging activities of dietary phenolics, namely caffeic acid (CA), ellagic acid (EA) and ferulic acid (FA). H2O2-induced apoptosis in normal human PBMCs was assayed by phosphotidylserine externalization, nucleosomal damage and DNA fragmentation. Incubation of PBMCs with 5 mM H2O2 led to increased Annexin-V binding to externalized phosphatidyl serine (PS), an event of pre-apoptotic stage of the cell. Peripheral blood mononuclear cells pretreated with phenolics could resist H2O2-induced apoptotic damage. Caffeic acid (60 and 120 microM) and EA (100 and 200 microM) caused no change in externalization of PS, whereas FA (100 and 200 microM) increased externalization of PS in PBMCs treated with H2O2. The effects of phenolics were abolished to a large extent by culturing the PBMCs for 24 h after washing the phenolics from the medium. Inhibitory activities of these phenolics on lipid peroxidation were in the order of EABcl-2 expression in PBMCs. In conclusion, the anti-apoptotic effect of EA, CA and FA in PBMCs seems to be through the Bcl-2 independent mechanism. PMID:16459021

  12. Bilberry extract (Antho 50) selectively induces redox-sensitive caspase 3-related apoptosis in chronic lymphocytic leukemia cells by targeting the Bcl-2/Bad pathway

    PubMed Central

    Alhosin, Mahmoud; León-González, Antonio J.; Dandache, Israa; Lelay, Agnès; Rashid, Sherzad K.; Kevers, Claire; Pincemail, Joël; Fornecker, Luc-Matthieu; Mauvieux, Laurent; Herbrecht, Raoul; Schini-Kerth, Valérie B.

    2015-01-01

    Defect in apoptosis has been implicated as a major cause of resistance to chemotherapy observed in B cell chronic lymphocytic leukaemia (B CLL). This study evaluated the pro-apoptotic effect of an anthocyanin-rich dietary bilberry extract (Antho 50) on B CLL cells from 30 patients and on peripheral blood mononuclear cells (PBMCs) from healthy subjects, and determined the underlying mechanism. Antho 50 induced concentration- and time-dependent pro-apoptotic effects in B CLL cells but little or no effect in PBMCs. Among the main phenolic compounds of the bilberry extract, delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside induced a pro-apoptotic effect. Antho 50-induced apoptosis is associated with activation of caspase 3, down-regulation of UHRF1, a rapid dephosphorylation of Akt and Bad, and down-regulation of Bcl-2. Antho 50 significantly induced PEG-catalase-sensitive formation of reactive oxygen species in B CLL cells. PEG-catalase prevented the Antho 50-induced induction of apoptosis and related signaling. The present findings indicate that Antho 50 exhibits strong pro-apoptotic activity through redox-sensitive caspase 3 activation-related mechanism in B CLL cells involving dysregulation of the Bad/Bcl-2 pathway. This activity of Antho 50 involves the glucoside and rutinoside derivatives of delphinidin. They further suggest that Antho 50 has chemotherapeutic potential by targeting selectively B CLL cells. PMID:25757575

  13. Levofloxacin increases the effect of serum deprivation on anoikis of rat nucleus pulposus cells via Bax/Bcl-2/caspase-3 pathway.

    PubMed

    Yang, Si-Dong; Bai, Zhi-Long; Zhang, Feng; Ma, Lei; Yang, Da-Long; Ding, Wen-Yuan

    2014-12-01

    Levofloxacin, a fluoroquinolone, is a widely-used and effective antibiotic. However, various adverse side effects are associated with levofloxacin. The purpose of this study was to further explore the effects of levofloxacin on rat nucleus pulposus cells (NPCs). Inverted phase-contrast microscopy, flow cytometry and caspase-3 activity assays were used and revealed that serum deprivation induced apoptosis, which was markedly increased by levofloxacin in a dose-dependent manner. Simultaneously, levofloxacin decreased cell binding to type II collagen (COL2). Thus, levofloxacin-induced apoptosis exhibits characteristics of anoikis, the process by which cell death is triggered by separation from the extracellular matrix, which contains COL2. Furthermore, real-time quantitative RT-PCR was used to further confirm that levofloxacin downregulates COL2 expression in a dose-dependent manner. At last, western blot was used to find that levofloxacin increased the ratio of Bax/Bcl-2 and active caspase-3 in a dose-dependent manner. Levofloxacin therefore increases the effects of serum deprivation on anoikis by downregulating COL2 in rat NPCs in vitro via Bax/Bcl-2/caspase-3 pathway. This research provides a novel insight into the mechanisms of levofloxacin-induced toxicity and may potentially lead to a better understanding of the clinical effects of levofloxacin, especially in terms of intervertebral disc degeneration. PMID:25224805

  14. Protection of Tong-Sai-Mai Decoction against Apoptosis Induced by H2O2 in PC12 Cells: Mechanisms via Bcl-2-Mitochondria-ROS-INOS Pathway

    PubMed Central

    Lee, Maxwell Kim Kit; Lu, Yin; Di, Liu-qing; Xu, Hui-qin

    2014-01-01

    Tong-Sai-Mai decoction (TSM) is a Chinese materia medica polyherbal formulation that has been applied in treating brain ischemia for hundreds of years. Because it could repress the oxidative stress in in vivo studies, now we focus on the in vitro studies to investigate the mechanism by targeting the oxidative stress dependent signaling. The relation between the neurogenesis and the reactive oxygen species (ROS) production remains largely unexamined. PC12 cells are excitable cell types widely used as in vitro model for neuronal cells. Most marker genes that are related to neurotoxicity, apoptosis, and cell cycles are expressed at high levels in these cells. The aim of the present study is to explore the cytoprotection of TSM against hydrogen peroxide- (H2O2-) induced apoptosis and the molecular mechanisms underlying PC12 cells. Our findings revealed that TSM cotreatment with H2O2 restores the expression of bcl-2, inducible nitric oxide synthase (INOS), and mitochondria membrane potential. Meanwhile, it reduces intracellular [Ca2+] concentration, lactate dehydrogenase (LDH) release, and the expression of caspase-3 and bax. The results of the present study suggested that the cytoprotective effects of the TSM might be mediated, at least in part, by the bcl-2-mitochondria-ROS-INOS pathway. Due to its nontoxic characteristics, TSM could be further developed to treat the neurodegenerative diseases which are closely associated with the oxidative stress. PMID:25404948

  15. Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway

    PubMed Central

    Hao, Cui; Gao, Lixia; Zhang, Yiran; Wang, Wei; Yu, Guangli; Guan, Huashi; Zhang, Lijuan; Li, Chunxia

    2015-01-01

    Chitosan oligosaccharides (COSs), depolymerized products of chitosan composed of β-(1→4) d-glucosamine units, have broad range of biological activities such as antitumour, antifungal, and antioxidant activities. In this study, peracetylated chitosan oligosaccharides (PACOs) and N-acetylated chitosan oligosaccharides (NACOs) were prepared from the COSs by chemcal modification. The structures of these monomers were identified using NMR and ESI-MS spectra. Their antagonist effects against glutamate-induced PC12 cell death were investigated. The results showed that pretreatment of PC12 cells with the PACOs markedly inhibited glutamate-induced cell death in a concentration-dependent manner. The PACOs were better glutamate antagonists compared to the COSs and the NACOs, suggesting the peracetylation is essential for the neuroprotective effects of chitosan oligosaccharides. In addition, the PACOs pretreatment significantly reduced lactate dehydrogenase release and reactive oxygen species production. It also attenuated the loss of mitochondrial membrane potential. Further studies indicated that the PACOs inhibited glutamate-induced cell death by preventing apoptosis through depressing the elevation of Bax/Bcl-2 ratio and caspase-3 activation. These results suggest that PACOs might be promising antagonists against glutamate-induced neural cell death. PMID:25775423

  16. Antiapoptotic and Antioxidant Properties of Orthosiphon stamineus Benth (Cat's Whiskers): Intervention in the Bcl-2-Mediated Apoptotic Pathway

    PubMed Central

    Abdelwahab, Siddig Ibrahim; Mohan, Syam; Mohamed Elhassan, Manal; Al-Mekhlafi, Nabil; Mariod, Abdelbasit Adam; Abdul, Ahmad Bustamam; Abdulla, Mahmood Ameen; Alkharfy, Khalid M.

    2011-01-01

    Antiapoptotic and antioxidant activities of aqueous-methanolic extract (CAME) of Orthosiphonstamineus Benth(OS), and its hexane (HF), chloroform (CF), n-butanol (NBF), ethyl acetate (EAF) and water (WF) fractions were investigated. Antioxidant properties were evaluated using the assays of Folin-Ciocalteu, aluminiumtrichloride, β-carotene bleaching and DPPH. The role of OS against hydrogen peroxide induced apoptosis on MDA-M231 epithelial cells was examined using MTT assay, phase contrast microscope, colorimetric assay of caspase-3, western blot and quantitative real-time PCR. Results showed that EAF showed the highest total phenolic content followed by CAME, NBF, WF, CF and HF, respectively. Flavonoid content was in the order of the CF > EAF > HF > CAME > NBF > WF. The IC50 values on DPPH assay for different extract/fractions were 126.2 ± 23, 31.25 ± 1.2, 15.25 ± 2.3, 13.56 ± 1.9, 23.0 ± 3.2, and 16.66 ± 1.5 μg/ml for HF, CF, EAF, NBF, WF and CAME, respectively. OSreduced the oxidation of β-carotene by hydroperoxides. Cell death was dose-dependently inhibited by pretreatment with OS. Caspase-3 and distinct morphological features suggest the anti-apoptotic activities of OS. This plant not only increased the expression of Bcl-2, but also decreased Bax expression, and ultimately reduced H2O2-induced apoptosis. The current results showed that phenolics may provide health and nutritional benefits. PMID:21234328

  17. Functional Implications of the spectrum of BCL2 mutations in Lymphoma.

    PubMed

    Singh, Khushboo; Briggs, James M

    2016-01-01

    Mutations in the translocated BCL2 gene are often detected in diffuse large B-cell lymphomas (DLBCLs), indicating both their significance and pervasiveness. Large series genome sequencing of more than 200 DLBCLs has identified frequent BCL2 mutations clustered in the exons coding for the BH4 domain and the folded loop domain (FLD) of the protein. However, BCL2 mutations are mostly contemplated to represent bystander events with negligible functional impact on the pathogenesis of DLBCL. BCL2 arbitrates apoptosis through a classic interaction between its hydrophobic groove forming BH1-3 domains and the BH3 domain of pro-apoptotic members of the BCL2 family. The effects of mutations are mainly determined by the ability of the mutated BCL2 to mediate apoptosis by this inter-member protein binding. Nevertheless, BCL2 regulates diverse non-canonical pathways that are unlikely to be explained by canonical interactions. In this review, first, we identify recurrent missense mutations in the BH4 domain and the FLD reported in independent lymphoma sequencing studies. Second, we discuss the probable consequences of mutations on the binding ability of BCL2 to non-BCL2 family member proteins crucial for 1) maintaining mitochondrial energetics and calcium hemostasis such as VDAC, IP3R, and RyR and 2) oncogenic pathways implicated in the acquisition of the 'hallmarks of cancer' such as SOD, Raf-1, NFAT, p53, HIF-1α, and gelsolin. The study also highlights the likely ramifications of mutations on binding of BCL2 antagonists and BH3 profiling. Based on our analysis, we believe that an in-depth focus on BCL2 interactions mediated by these domains is warranted to elucidate the functional significance of missense mutations in DLBCL. In summary, we provide an extensive overview of the pleiotropic functions of BCL2 mediated by its physical binding interaction with other proteins and the various ways BCL2 mutations would affect the normal function of the cell leading to the development

  18. Baicalin Attenuates Ketamine-Induced Neurotoxicity in the Developing Rats: Involvement of PI3K/Akt and CREB/BDNF/Bcl-2 Pathways.

    PubMed

    Zuo, Daiying; Lin, Li; Liu, Yumiao; Wang, Chengna; Xu, Jingwen; Sun, Feng; Li, Lin; Li, Zengqiang; Wu, Yingliang

    2016-08-01

    Ketamine is widely used as an anesthetic in pediatric clinical practice. However, numerous studies have reported that exposure to ketamine during the developmental period induces neurotoxicity. Here we investigate the neuroprotective effects of baicalin, a natural flavonoid compound, against ketamine-induced apoptotic neurotoxicity in the cortex and hippocampus of the Sprague-Dawley postnatal day 7 (PND7) rat pups. Our results revealed that five continuous injections of ketamine (20 mg/kg) at 90-min intervals over 6 h induced obvious morphological damages of neuron by Nissl staining and apoptosis by TUNEL assays in the prefrontal cortex and hippocampus of PND7 rat pups. Baicalin (100 mg/kg) pretreatment alleviated ketamine-induced morphological change and apoptosis. Caspase-3 activity and caspase-3 mRNA expression increase induced by ketamine were also inhibited by baicalin treatment. LY294002, an inhibitor of PI3K, abrogated the effect of baicalin against ketamine-induced caspase-3 activity and caspase-3 mRNA expression increase. In addition, Western blot studies indicated that baicalin not only inhibited ketamine-induced p-Akt and p-GSK-3β decrease, but also relieved ketamine-induced p-CREB and BDNF expression decrease. Baicalin also attenuated ketamine-induced Bcl-2/Bax decrease and caspase-3 expression increase. Further in vitro experiments proved that baicalin mitigated ketamine-induced cell viability decrease in the MTT assay, morphological change by Rosenfeld's staining, and caspase-3 expression increase by Western blot in the primary neuron-glia mixed cultures. LY294002 abrogated the protective effect of baicalin. These data demonstrate that baicalin exerts neuroprotective effect against ketamine-induced neuronal apoptosis by activating the PI3K/Akt and its downstream CREB/BDNF/Bcl-2 signaling pathways. Therefore, baicalin appears to be a promising agent in preventing or reversing ketamine's apoptotic neurotoxicity at an early developmental stage. PMID

  19. Interferon-alpha and bortezomib overcome Bcl-2 and Mcl-1 over-expression in melanoma cells by stimulating the extrinsic pathway of apoptosis

    PubMed Central

    Lesinski, Gregory B.; Raig, Ene T.; Guenterberg, Kristan; Brown, Lloyd; Go, Michael R.; Shah, Nisha N.; Lewis, Adrian; Quimper, Megan; Hade, Erinn; Young, Gregory; Chaudhury, Abhik Ray; Ladner, Katherine J.; Guttridge, Denis C.; Bouchard, Page

    2008-01-01

    We hypothesized that interferon-alpha (IFN-α) would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-α induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspases-3, -7, -8, -9, and with cleavage of Bid and PARP. Bortezomib plus IFN-α was effective at inducing apoptosis in melanoma cells that over-expressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The pro-apoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD prior to the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or siRNA targeting Fas. These data suggest that bortezomib and IFN-α act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-α displayed statistically significant anti-tumor activity as compared to either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-α for malignant melanoma. PMID:18922907

  20. Chemosensitization of Prostate Cancer by Modulating Bcl-2 Family Proteins

    PubMed Central

    Karnak, David; Xu, Liang

    2010-01-01

    A major challenge in oncology is the development of chemoresistance. This often occurs as cancer progresses and malignant cells acquire mechanisms to resist insults that would normally induce apoptosis. The onset of androgen independence in advanced prostate cancer is a prime example of this phenomenon. Overexpression of the pro-survival/anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 are hallmarks of this transition. Here we outline the evolution of therapeutics designed to either limit the source or disrupt the interactions of these pro-survival proteins. By either lessening the stoichiometric abundance of Bcl-2/xL/Mcl-1 in reference to their pro-apoptotic foils or freeing these pro-apoptotic proteins from their grip, these treatments aim to sensitize cells to chemotherapy by priming cells for death. DNA anti-sense and RNA interference have been effectively employed to decrease Bcl-2 family mRNA and protein levels in cell culture models of advanced prostate cancer. However, clinical studies are lagging due to in vivo delivery challenges. The burgeoning field of nanoparticle delivery holds great promise in helping to overcome the challenge of administering highly labile nucleic acid based therapeutics. On another front, small molecule inhibitors that block the hetero-dimerization of pro-survival with pro-apoptotic proteins have significant clinical advantages and have advanced farther in clinical trials with promising early results. Most recently, a peptide has been discovered that can convert Bcl-2 from a pro-survival to a pro-apoptotic protein. The future may lie in targeting multiple steps of the apoptotic pathway, including Bcl-2/xL/Mcl-1, to debilitate the survival capacity of cancer cells and make chemotherapy induced death their only option. PMID:20298153

  1. Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim

    PubMed Central

    Marshall, B; Puthalakath, H; Caria, S; Chugh, S; Doerflinger, M; Colman, P M; Kvansakul, M

    2015-01-01

    Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism. PMID:25766319

  2. Molecular and Computational Studies on Apoptotic Pathway Regulator, Bcl-2 Gene from Breast Cancer Cell Line MCF-7.

    PubMed

    Tiwari, Pragya; Khan, M J

    2016-01-01

    Cancer is a dreadful disease constituting abnormal growth and proliferation of malignant cells in the body. Next to lung cancer, breast cancer is the most common form of cancer affecting women. The apoptotic pathway regulators, B cell lymphoma family of protein, play a key role in various malignancies defining cancer and their constitutive expression plays an integral role in breast cancer chemotherapy. The research work discusses the identification and molecular cloning of a B cell lymphoma like gene from human breast cancer cell line. The open reading frame of the gene consisted of 965 nucleotides, encoding a protein of 380 amino acids with a predicted molecular weight of 42.5 kilodalton. The predicted physiochemical properties of the gene were as follows: Isoelectric point - 9.49, molecular formula - C1893H3004N534O548S16, total number of negatively charged residues, (Aspartate+Glutamate) - 26, total number of positively charged residues, (Arginine+Lysine)-39, instability index-42.08 (unstable protein) and grand average of hydropathicity is -0.202. Additionally, phobius prediction suggested non-cytoplasmic localization of the putative protein. The presence of secondary structure in the protein was determined by Memsat program. A 3 dimensional protein homology model was generated using threading based method of protein modeling for structural and functional annotation of the putative protein. Future prospects accounts for the biochemical characterization of the enzyme including in vitro assays on breast cancer cell line would establish the functional characteristics of the protein and its physiological mechanisms in breast cancer development and its therapeutic-target role in future. PMID:27168686

  3. Dioxinodehydroeckol protects human keratinocyte cells from UVB-induced apoptosis modulated by related genes Bax/Bcl-2 and caspase pathway.

    PubMed

    Ryu, BoMi; Ahn, Byul-Nim; Kang, Kyong-Hwa; Kim, Young-Sang; Li, Yong-Xin; Kong, Chang-Suk; Kim, Se-Kwon; Kim, Dong Gyu

    2015-12-01

    Although ultraviolet B (UVB) has a low level of skin penetration, it readily results in epidermal sunburn of keratinocytes that are destined to apoptosis after sun expose, and leads to DNA damage. Dioxinodehydroeckol (DHE), a phlorotannin from Ecklonia cava has been explored for its preventive activity against UVB-induced apoptosis in human keratinocyte (HaCaT) cells; however, the protective effects of treatment with low doses of DHE on UVB-damaged cells post-UVB exposure and their underlying mechanisms still remain unclear. The HaCaT cells were exposed to 20 mJcm(-2) of UVB irradiation which is the minimal erythema dose (MED) for individuals to be able to tan, and the expression levels of Bax/Bcl-2 and caspase-3,-8, -9 which are associated genes with apoptosis were investigated when we either treated cells with DHE doses after UVB irradiation or exposed them to UVB only. Our results suggest insight into proposed mechanistic pathway of protective activity of DHE on the HaCaT cells from UVB-induced apoptosis, indicating the benefit of DHE as a repair agent for skin damage against UVB. PMID:26529485

  4. Dihydromyricetin Enhances the Chemo-Sensitivity of Nedaplatin via Regulation of the p53/Bcl-2 Pathway in Hepatocellular Carcinoma Cells

    PubMed Central

    Ren, Hao; Ma, Sheng; Lu, CaiJie; Liu, Bin; Liu, Jie; Liang, Jian; Li, Mingyi; Zhu, Runzhi

    2015-01-01

    Chemotherapy is an effective weapon in the battle against cancer. Nedaplatin (NDP) is an improved platinum-containing drug with lower cytotoxicity than other similar drugs. However, the repeated use of NDP results in substantial hepatocyte damage as well as drug resistance in hepatocellular carcinoma (HCC) cases. Therefore, the development of effective chemotherapy strategies that enhance tumor sensitivity to chemotherapeutics and reduce the secondary damage to liver cells is urgently needed. Dihydromyricetin (DHM), a natural flavonoid compound, has been shown to have antitumor activity with no obvious toxicity to normal cells in vitro and in vivo. In this study, DHM and NDP were combined to treat liver cancer cells; we found that DHM functions as a protector of normal cells compared with the use of NDP alone. In addition, the synergy of DHM with NDP enhanced the effect of NDP on the induction of HCC cell apoptosis. We found that the combination caused clear changes in the level of reactive oxygen species (ROS). Furthermore, we demonstrated that the combination of DHM and NDP activated the p53/Bcl-2 signaling pathway, which resulted in mitochondrial dysfunction and induced cell death and growth inhibition in HCC cells. PMID:25915649

  5. Estradiol-17beta protects against hypoxia-induced hepatocyte injury through ER-mediated upregulation of Bcl-2 as well as ER-independent antioxidant effects.

    PubMed

    Lee, Min Young; Jung, Sun Chul; Lee, Jang Hern; Han, Ho Jae

    2008-04-01

    Although many previous studies have suggested that estrogen functions as a cytoprotective agent under oxidative stress conditions, the underlying mechanism by which this effect is exerted remains to be elucidated. This study assessed the effects of estradiol-17beta (E(2)) (10(-8) M) on hypoxia-induced cell injury and its related signaling in primary cultured chicken hepatocytes. Hypoxic conditions were found to augment the level of DNA damage and to reduce cell viability and the level of [(3)H]-thymidine incorporation, and these phenomena were prevented through treatment with E(2). Hypoxia also increased caspase-3 expression, but showed no evidence of an influence on the expression of Bcl-2. However, E(2) induced an increase in the level of Bcl-2 expression under hypoxic conditions and reduced the level of caspase-3 expression. The effects of E(2) on Bcl-2 and caspase expression were blocked by ICI 182780 (E(2) receptor (ER) antagonist, 10(-7) M). In addition, hypoxia resulted in an increase in the intracellular reactive oxygen species (ROS) generated. These effects were blocked by E(2), but not by E(2)-BSA and ICI 182780. Hypoxia also activated p38 mitogen-activated protein kinase (MAPK), c-JUN N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and nuclear factor-kappaB (NF-kappaB). These effects were blocked by E(2), but not by ICI 182780. The inhibition of p38 MAPK and JNK/SAPK blocked NF-kappaB activation. In conclusion, E(2) was found to protect against hypoxia-induced cell injury in chicken hepatocytes through ER-mediated upregulation of Bcl-2 expression and through reducing the activity of ROS-dependent p38 MAPK, JNK/SAPK and NF-kappaB. PMID:18379592

  6. Activation of the p53 pathway by the MDM2 inhibitor nutlin-3a overcomes BCL2 overexpression in a preclinical model of diffuse large B-cell lymphoma associated with t(14;18)(q32;q21)

    PubMed Central

    Drakos, E; Singh, R R; Rassidakis, G Z; Schlette, E; Li, J; Claret, F X; Ford, R J; Vega, F; Medeiros, L J

    2011-01-01

    p53 is frequently wild type (wt) in diffuse large B-cell lymphoma (DLBCL) associated with t(14;18)(q32;q21) that overexpresses BCL2. Nutlin-3a is a small molecule that activates the p53 pathway by disrupting p53–MDM2 interaction. We show that nutlin-3a activates p53 in DLBCL cells associated with t(14;18)(q32;q21), BCL2 overexpression and wt p53, resulting in cell cycle arrest and apoptosis. Nutlin-3a treatment had similar effects on DLBCL cells of activated B-cell phenotype with wt p53. Cell cycle arrest was associated with upregulation of p21. Nutlin-3a-induced apoptosis was accompanied by BAX and PUMA upregulation, BCL-XL downregulation, serine-70 dephosphorylation of BCL2, direct binding of BCL2 by p53, caspase-9 upregulation and caspase-3 cleavage. Cell death was reduced when p53-dependent transactivation activity was inhibited by pifithrin-α (PFT-α), or PFT-μ inhibited direct p53 targeting of mitochondria. Nutlin-3a sensitized activation of the intrinsic apoptotic pathway by BCL2 inhibitors in t(14;18)-positive DLBCL cells with wt p53, and enhanced doxorubicin cytotoxicity against t(14;18)-positive DLBCL cells with wt or mutant p53, the latter in part via p73 upregulation. Nutlin-3a treatment in a xenograft animal lymphoma model inhibited growth of t(14;18)-positive DLBCL tumors, associated with increased apoptosis and decreased proliferation. These data suggest that disruption of the p53–MDM2 interaction by nutlin-3a offers a novel therapeutic approach for DLBCL associated with t(14;18)(q32;q21). PMID:21394100

  7. DNA Hypermethylation of CREB3L1 and Bcl-2 Associated with the Mitochondrial-Mediated Apoptosis via PI3K/Akt Pathway in Human BEAS-2B Cells Exposure to Silica Nanoparticles

    PubMed Central

    Zou, Yang; Li, Qiuling; Jiang, Lizhen; Guo, Caixia; Li, Yanbo; Yu, Yang; Li, Yang; Duan, Junchao; Sun, Zhiwei

    2016-01-01

    The toxic effects of silica nanoparticles (SiNPs) are raising concerns due to its widely applications in biomedicine. However, current information about the epigenetic toxicity of SiNPs is insufficient. In this study, the epigenetic regulation of low-dose exposure to SiNPs was evaluated in human bronchial epithelial BEAS-2B cells over 30 passages. Cell viability was decreased in a dose- and passage-dependent manner. The apoptotic rate, the expression of caspase-9 and caspase-3, were significantly increased induced by SiNPs. HumanMethylation450 BeadChip analysis identified that the PI3K/Akt as the primary apoptosis-related pathway among the 25 significant altered processes. The differentially methylated sites of PI3K/Akt pathway involved 32 differential genes promoters, in which the CREB3L1 and Bcl-2 were significant hypermethylated. The methyltransferase inhibitor, 5-aza, further verified that the DNA hypermethylation status of CREB3L1 and Bcl-2 were associated with downregulation of their mRNA levels. In addition, mitochondrial-mediated apoptosis was triggered by SiNPs via the downregulation of PI3K/Akt/CREB/Bcl-2 signaling pathway. Our findings suggest that long-term low-dose exposure to SiNPs could lead to epigenetic alterations. PMID:27362941

  8. The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I.

    PubMed

    Porcelli, A M; Ghelli, A; Iommarini, L; Mariani, E; Hoque, M; Zanna, C; Gasparre, G; Rugolo, M

    2008-09-01

    Human thyroid carcinoma XTC.UC1 cells harbor a homoplasmic frameshift mutation in the MT-ND1 subunit of respiratory complex I. When forced to use exclusively oxidative phosphorylation for energy production by inhibiting glycolysis, these cells triggered a caspase-independent cell death pathway, which was associated to a significant imbalance in glutathione homeostasis and a cleavage of the actin cytoskeleton. Overexpression of the anti-apoptotic Bcl-2 protein significantly increased the level of endogenous reduced glutathione, thus preventing its oxidation after the metabolic stress. Furthermore, Bcl-2 completely inhibited actin cleavage and increased cell adhesion, but was unable to improve cellular viability. Similar effects were obtained when XTC.UC1 cells were incubated with exogenous glutathione. We hence propose that Bcl-2 can safeguard cytoskeletal stability through an antioxidant function. PMID:18695940

  9. Protective Effects of Scutellarin on Type II Diabetes Mellitus-Induced Testicular Damages Related to Reactive Oxygen Species/Bcl-2/Bax and Reactive Oxygen Species/Microcirculation/Staving Pathway in Diabetic Rat

    PubMed Central

    Long, Lingli; Wang, Jingnan; Lu, Xiaofang; Xu, Yuxia; Zheng, Shuhui; Luo, Canqiao; Li, Yubin

    2015-01-01

    The goal of our study is to evaluate the effect of Scutellarin on type II diabetes-induced testicular disorder and show the mechanism of Scutellarin's action. We used streptozotocin and high-fat diet to establish type II diabetic rat model. TUNEL and haematoxylin and eosin staining were used to evaluate the testicular apoptotic cells and morphologic changes. Immunohistochemical staining was used to measure the expression level of vascular endothelial growth factor and blood vessel density in testes. Oxidative stress in testes and epididymis was tested by fluorescence spectrophotometer and ELISA. The expression of Bcl-2/Bax and blood flow rate in testicular vessels were measured by western blot and Doppler. Our results for the first time showed that hyperglycemia induced apoptotic cells and morphologic impairments in testes of rats, while administration of Scutellarin can significantly inhibit these damages. This effect of Scutellarin is controlled by two apoptotic triggers: ROS/Bcl-2/Bax and ROS/microcirculation/starving pathway. PMID:25861655

  10. Protective effects of scutellarin on type II diabetes mellitus-induced testicular damages related to reactive oxygen species/Bcl-2/Bax and reactive oxygen species/microcirculation/staving pathway in diabetic rat.

    PubMed

    Long, Lingli; Wang, Jingnan; Lu, Xiaofang; Xu, Yuxia; Zheng, Shuhui; Luo, Canqiao; Li, Yubin

    2015-01-01

    The goal of our study is to evaluate the effect of Scutellarin on type II diabetes-induced testicular disorder and show the mechanism of Scutellarin's action. We used streptozotocin and high-fat diet to establish type II diabetic rat model. TUNEL and haematoxylin and eosin staining were used to evaluate the testicular apoptotic cells and morphologic changes. Immunohistochemical staining was used to measure the expression level of vascular endothelial growth factor and blood vessel density in testes. Oxidative stress in testes and epididymis was tested by fluorescence spectrophotometer and ELISA. The expression of Bcl-2/Bax and blood flow rate in testicular vessels were measured by western blot and Doppler. Our results for the first time showed that hyperglycemia induced apoptotic cells and morphologic impairments in testes of rats, while administration of Scutellarin can significantly inhibit these damages. This effect of Scutellarin is controlled by two apoptotic triggers: ROS/Bcl-2/Bax and ROS/microcirculation/starving pathway. PMID:25861655

  11. Bcl-2 family proteins: master regulators of cell survival.

    PubMed

    Hatok, Jozef; Racay, Peter

    2016-08-01

    The most prominent function of proteins of the Bcl-2 family is regulation of the initiation of intrinsic (mitochondrial) pathways of apoptosis. However, recent research has revealed that in addition to regulation of mitochondrial apoptosis, proteins of the Bcl-2 family play important roles in regulating other cellular pathways with a strong impact on cell survival like autophagy, endoplasmic reticulum (ER) stress response, intracellular calcium dynamics, cell cycle progression, mitochondrial dynamics and energy metabolism. This review summarizes the recent knowledge about functions of Bcl-2 family proteins that are related to cell survival. PMID:27505095

  12. MicroRNA-204 targets JAK2 in breast cancer and induces cell apoptosis through the STAT3/BCl-2/survivin pathway

    PubMed Central

    Wang, Xilong; Qiu, Wenxiu; Zhang, Guoqiang; Xu, Shujian; Gao, Qiang; Yang, Zhenlin

    2015-01-01

    MicroRNAs (miRNAs) have emerged as important regulators that potentially play critical roles in cancer cell biological processes. Previous studies have shown that miR-204 plays an important role in various human cancers. However, the underlying mechanisms of this microRNA in breast cancer remain largely unknown. In the present study, we investigated that miR-204 expression level was markedly reduced in both the human breast cancer tissue and cultured breast cancer cell lines (MCF-7, MDA-MB-231). Overexpression of miR-204 inhibited the proliferation and promoted the apoptosis in breast cancer cells, which were reversed by co-transfection of miR-204 inhibitor. We validated that Janus kinase 2 (JAK2), as a direct target of miR-204, is overexpressed in breast cancer. Knockdown of JAK2 suppressed cell viability and induced apoptosis in breast cancer cells. Moreover, the level of miR-204 is negatively correlated with p-STAT3 and anti-apoptotic genes BCl-2 and surviving in breast cancer. In conclusions, miR-204 targets JAK2 and suppressed JAK2 and p-JAK2 expression in breast cancer, which further inhibit the activation of STAT3, BCl-2 and survivin. These findings indicate that manipulation of miR-204 expression may represent a novel therapeutic strategy in the treatment of breast cancer. PMID:26191195

  13. Polyphenols Isolated from Allium cepa L. Induces Apoptosis by Induction of p53 and Suppression of Bcl-2 through Inhibiting PI3K/Akt Signaling Pathway in AGS Human Cancer Cells

    PubMed Central

    Lee, Won Sup; Yi, Sang Mi; Yun, Jeong Won; Jung, Ji Hyun; Kim, Dong Hoon; Kim, Hye Jung; Chang, Seong-Hwan; Kim, GonSup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2014-01-01

    Background: The extract of Allium cepa Linn is commonly used as adjuvant food for cancer therapy. We assumed that it includes a potential source of anti-cancer properties. Methods: We investigated anti-cancer effects of polyphenols extracted from lyophilized A. cepa Linn (PEAL) in AGS human cancer cells. Results: PEAL inhibited cell growth in a dose-dependent manner. It was related to caspase-dependent apoptosis. We confirmed this finding with annexin V staining. PEAL up-regulated p53 expression, and subsequent Bax induction, down regulated Bcl-2 protein, anti-apoptotic protein. In addition, PEAL suppressed Akt activity and PEAL-induced apoptosis were significantly accentuated with Akt inhibitor (LY294002). Conclusions: Our data suggested that PEAL induce caspase-dependent apoptosis through mitochondrial pathway by up-regulating p53 protein, and subsequent Bax protein as well as by modulating Bcl-2 protein, and that PEAL induces caspase-dependent apoptosis at least in part through the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. This study provides evidence that PEAL might be useful for the treatment of cancer. PMID:25337568

  14. miR-204 targets Bcl-2 expression and enhances responsiveness of gastric cancer.

    PubMed

    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

  15. miR-204 targets Bcl-2 expression and enhances responsiveness of gastric cancer

    PubMed Central

    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

  16. BRCA1 involved in regulation of Bcl-2 expression and apoptosis susceptibility to ionizing radiation

    NASA Astrophysics Data System (ADS)

    Wang, YanLing; Wang, Bing; Zhang, Hong; Li, Ning; Tanaka, Kaoru; Zhou, Xin; Chen, RuPing; Zhang, Xin

    2011-05-01

    BRCA1 has been proposed to be tightly linked to the resistance of tumor cells to ionizing radiation. The pathway leading to this phenomenon is not yet clear. In this work, we investigated the role of BRCA1 in the apoptosis regulation in response to carbon ion irradiation. We utilized three different cancer cell lines with various states for BRCA1 and p53 to identify the relationship between endogenous BRCA1 and the apoptosis-related genes, and determine whether p53 function would affect the role of BRCA1 in apoptosis regulation. By Western blot analysis, we found that Bax expressions were not significantly changed after irradiation in all of three cell lines. However, Bcl-2 expression showed an up-regulation by endogenous BRCA1 regardless of p53 status. Moreover, the changes in Bcl-2 protein were due to the increase in the transcriptional levels of Bcl-2 mRNA, based on real-time PCR assay. At the same time, BRCA1-deficient cells showed a greater apoptosis susceptibility to irradiation when compared with BRCA1-proficient cells. The results suggest that BRCA1 might exert p53-independent regulative activities for Bcl-2, which seems account for the low apoptosis susceptibility in BRCA1-proficient carcinomas.

  17. Dodecyl gallate induces apoptosis by upregulating the caspase-dependent apoptotic pathway and inhibiting the expression of anti-apoptotic Bcl-2 family proteins in human osteosarcoma cells

    PubMed Central

    CHENG, CHUN-HSIANG; CHENG, YEN-PO; CHANG, ING-LIN; CHEN, HSIN-YAO; WU, CHIA-CHIEH; HSIEH, CHEN-PU

    2016-01-01

    Dodecyl gallate (DG) is a gallic acid ester that has been shown to inhibit tumor growth. The aim of this study was to investigate the mechanism by which DG induces antiproliferative and apoptotic effects in MG-63 human osteosarcoma cells. Dose- and time-dependent cytotoxic effects of DG were determined using an MTT assay. The results showed that the half-maximal inhibitory concentration (IC50) of DG in MG-63 cells was 31.15 µM at 24 h, 10.66 µM at 48 h, and 9.06 µM at 72 h. Flow cytometric analysis demonstrated that exposure to 20 and 40 µM DG resulted in an increase in the sub-G1 phase population and in S-phase cell cycle arrest. Furthermore, western blot analysis of apoptosis-related protein expression revealed an increase in the activation of caspases 8 and 3, cleavage of poly (ADPribose) polymerase (PARP), and disruption of mitochondrial membrane permeability was measured by flow cytometry. An increase in the Bax/Bcl-2 ratio and a decrease in the expression of inhibitor of apoptosis protein (IAP) family members, namely X-linked inhibitor of apoptosis protein and survivin, were also observed following DG treatment. These data provide insight into the molecular mechanisms governing the ability of DG to induce apoptosis in human osteosarcoma cells in vitro. PMID:26707422

  18. BCL2 mutations are associated with increased risk of transformation and shortened survival in follicular lymphoma

    PubMed Central

    Correia, Cristina; Schneider, Paula A.; Dai, Haiming; Dogan, Ahmet; Maurer, Matthew J.; Church, Amy K.; Novak, Anne J.; Feldman, Andrew L.; Wu, Xiaosheng; Ding, Husheng; Meng, X. Wei; Cerhan, James R.; Slager, Susan L.; Macon, William R.; Habermann, Thomas M.; Karp, Judith E.; Gore, Steven D.; Kay, Neil E.; Jelinek, Diane F.; Witzig, Thomas E.; Nowakowski, Grzegorz S.

    2015-01-01

    Follicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. Although BCL2 mutations have been previously described, their relationship to FL progression remains unclear. In this study, we evaluated the frequency and nature of BCL2 mutations in 2 independent cohorts of grade 1 and 2 FLs, along with the correlation between BCL2 mutations, transformation risk, and survival. The prevalence of BCL2 coding sequence mutations was 12% in FL at diagnosis and 53% at transformation (P < .0001). The presence of these BCL2 mutations at diagnosis correlated with an increased risk of transformation (hazard ratio 3.6; 95% CI, 2.0-6.2; P < .0001) and increased risk of death due to lymphoma (median survival of 9.5 years with BCL2 mutations vs 20.4 years without; P = .012). In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro. Accordingly, BCL2 mutations can affect antiapoptotic Bcl-2 function, are associated with increased activation-induced cytidine deaminase expression, and correlate with increased risk of transformation and death due to lymphoma. PMID:25452615

  19. Bcl-2 is a critical mediator of intestinal transformation.

    PubMed

    van der Heijden, Maartje; Zimberlin, Cheryl D; Nicholson, Anna M; Colak, Selcuk; Kemp, Richard; Meijer, Sybren L; Medema, Jan Paul; Greten, Florian R; Jansen, Marnix; Winton, Douglas J; Vermeulen, Louis

    2016-01-01

    Intestinal tumour formation is generally thought to occur following mutational events in the stem cell pool. However, active NF-κB signalling additionally facilitates malignant transformation of differentiated cells. We hypothesized that genes shared between NF-κB and intestinal stem cell (ISCs) signatures might identify common pathways that are required for malignant growth. Here, we find that the NF-κB target Bcl-2, an anti-apoptotic gene, is specifically expressed in ISCs in both mice and humans. Bcl-2 is dispensable in homeostasis and, although involved in protecting ISCs from radiation-induced damage, it is non-essential in tissue regeneration. Bcl-2 is upregulated in adenomas, and its loss or inhibition impairs outgrowth of oncogenic clones, because Bcl-2 alleviates apoptotic priming in epithelial cells following Apc loss. Furthermore, Bcl-2 expression in differentiated epithelial cells renders these cells amenable to clonogenic outgrowth. Collectively, our results indicate that Bcl-2 is required for efficient intestinal transformation following Apc-loss and constitutes a potential chemoprevention target. PMID:26956214

  20. Bcl-2 is a critical mediator of intestinal transformation

    PubMed Central

    van der Heijden, Maartje; Zimberlin, Cheryl D.; Nicholson, Anna M.; Colak, Selcuk; Kemp, Richard; Meijer, Sybren L.; Medema, Jan Paul; Greten, Florian R.; Jansen, Marnix; Winton, Douglas J.; Vermeulen, Louis

    2016-01-01

    Intestinal tumour formation is generally thought to occur following mutational events in the stem cell pool. However, active NF-κB signalling additionally facilitates malignant transformation of differentiated cells. We hypothesized that genes shared between NF-κB and intestinal stem cell (ISCs) signatures might identify common pathways that are required for malignant growth. Here, we find that the NF-κB target Bcl-2, an anti-apoptotic gene, is specifically expressed in ISCs in both mice and humans. Bcl-2 is dispensable in homeostasis and, although involved in protecting ISCs from radiation-induced damage, it is non-essential in tissue regeneration. Bcl-2 is upregulated in adenomas, and its loss or inhibition impairs outgrowth of oncogenic clones, because Bcl-2 alleviates apoptotic priming in epithelial cells following Apc loss. Furthermore, Bcl-2 expression in differentiated epithelial cells renders these cells amenable to clonogenic outgrowth. Collectively, our results indicate that Bcl-2 is required for efficient intestinal transformation following Apc-loss and constitutes a potential chemoprevention target. PMID:26956214

  1. Acidosis Promotes Bcl-2 Family-mediated Evasion of Apoptosis

    PubMed Central

    Ryder, Christopher; McColl, Karen; Zhong, Fei; Distelhorst, Clark W.

    2012-01-01

    Acidosis arises in solid and lymphoid malignancies secondary to altered nutrient supply and utilization. Tumor acidosis correlates with therapeutic resistance, although the mechanism behind this effect is not fully understood. Here we show that incubation of lymphoma cell lines in acidic conditions (pH 6.5) blocks apoptosis induced by multiple cytotoxic metabolic stresses, including deprivation of glucose or glutamine and treatment with dexamethasone. We sought to examine the role of the Bcl-2 family of apoptosis regulators in this process. Interestingly, we found that acidic culture causes elevation of both Bcl-2 and Bcl-xL, while also attenuating glutamine starvation-induced elevation of p53-up-regulated modulator of apoptosis (PUMA) and Bim. We confirmed with knockdown studies that these shifts direct survival decisions during starvation and acidosis. Importantly, the promotion of a high anti- to pro-apoptotic Bcl-2 family member ratio by acidosis renders cells exquisitely sensitive to the Bcl-2/Bcl-xL antagonist ABT-737, suggesting that acidosis causes Bcl-2 family dependence. This dependence appears to be mediated, in part, by the acid-sensing G protein-coupled receptor, GPR65, via a MEK/ERK pathway. PMID:22685289

  2. Intracellular insulin-like growth factor-1 induces Bcl-2 expression in airway epithelial cells.

    PubMed

    Chand, Hitendra S; Harris, Jennifer Foster; Mebratu, Yohannes; Chen, Yangde; Wright, Paul S; Randell, Scott H; Tesfaigzi, Yohannes

    2012-05-01

    Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and it can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and insulin-like growth factor-1 (IGF-1) coincided with induced Bcl-2 expression compared with controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using short hairpin RNA showed that intracellular IGF-1 (IC-IGF-1) was increasing Bcl-2 expression. Blocking epidermal growth factor receptor or IGF-1R activation also suppressed IC-IGF-1 and abolished the Bcl-2 induction. Induced expression and colocalization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and epidermal growth factor receptor pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases. PMID:22461702

  3. Bcl-2 inhibitors potentiate the cytotoxic effects of radiation in Bcl-2 overexpressing radioresistant tumor cells

    SciTech Connect

    Hara, Takamitsu; Omura-Minamisawa, Motoko . E-mail: momuram@med.yokohama-cu.ac.jp; Chao Cheng; Nakagami, Yoshihiro; Ito, Megumi; Inoue, Tomio

    2005-02-01

    Purpose: Bcl-2, an inhibitor of apoptosis frequently shows elevated expression in human tumors, thus resulting in resistance to radiation therapy. Therefore, inhibiting Bcl-2 function may enhance the radiosensitivity of tumor cells. Tetrocarcin A (TC-A) and bcl-2 antisense oligonucleotides exhibit antitumor activity by inhibiting Bcl-2 function and transcription, respectively. We investigated whether these antitumor agents would enhance the cytotoxic effects of radiation in tumor cells overexpressing Bcl-2. Methods and materials: We used HeLa/bcl-2 cells, a stable Bcl-2-expressing cell line derived from wild-type HeLa (HeLa/wt) cells. Cells were incubated with TC-A and bcl-2 antisense oligonucleotides for 24 h after irradiation, and cell viability was then determined. Apoptotic cells were quantified by flow cytometric assay. Results: The HeLa/bcl-2 cells were more resistant to radiation than HeLa/wt cells. At concentrations that are not inherently cytotoxic, both TC-A and bcl-2 antisense oligonucleotides increased the cytotoxic effects of radiation in HeLa/bcl-2 cells, but not in HeLa/wt cells. However, in HeLa/bcl-2 cells, additional treatment with TC-A in combination with radiation did not significantly increase apoptosis. Conclusions: The present results suggest that TC-A and bcl-2 antisense oligonucleotides reduce radioresistance of tumor cells overexpressing Bcl-2. Therefore, a combination of radiotherapy and Bcl-2 inhibitors may prove to be a useful therapeutic approach for treating tumors that overexpress Bcl-2.

  4. Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

    PubMed

    Han, Cho Rong; Jun, Do Youn; Kim, Yoon Hee; Lee, Ji Young; Kim, Young Ho

    2013-10-01

    In Jurkat T cell clone (JT/Neo), G2/M arrest, apoptotic sub-G1 peak, mitochondrial membrane potential (Δψm) loss, and TUNEL-positive DNA fragmentation were induced following exposure to 17α-estradiol (17α-E2), whereas none of these events (except for G2/M arrest) were induced in Jurkat cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, phosphorylation at Thr161 and dephosphorylation at Tyr15 of Cdk1, upregulation of cyclin B1 level, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation, and Bim phosphorylation were detected in the presence of Bcl-2 overexpression. However, the 17α-E2-induced upregulation of Bak levels, activation of Bak, activation of caspase-3, and PARP degradation were abrogated by Bcl-2 overexpression. In the presence of the G1/S blocking agent hydroxyurea, 17α-E2 failed to induce G2/M arrest and all apoptotic events including Cdk1 activation and phosphorylation of Bcl-2, Mcl-1 and Bim. The 17α-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor but not by aurora A and aurora B kinase inhibitors. Immunofluorescence microscopic analysis showed that an aberrant bipolar microtubule array, incomplete chromosome congression at the metaphase plate, and prometaphase arrest, which was reversible, were the underlying factors for 17α-E2-induced mitotic arrest. The in vitro microtubule polymerization assay showed that 17α-E2 could directly inhibit microtubule formation. These results show that the apoptogenic activity of 17α-E2 was due to the impaired mitotic spindle assembly causing prometaphase arrest and prolonged Cdk1 activation, the phosphorylation of Bcl-2, Mcl-1 and Bim, and the activation of Bak and mitochondria-dependent caspase cascade. PMID:23707954

  5. Bcl-2 apoptosis proteins, mitochondrial membrane curvature, and cancer

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Schmidt, Nathan; Sanders, Lori; Mishra, Abhijit; Wong, Gerard; Ivashyna, Olena; Christenson, Eric; Schlesinger, Paul; Akabori, Kiyotaka; Santangelo, Christian

    2012-02-01

    Critical interactions between Bcl-2 family proteins permeabilize the outer mitochondrial membrane, a common decision point early in the intrinsic apoptotic pathway that irreversibly commits the cell to death. However, a unified picture integrating the essential non-passive role of lipid membranes with the contested dynamics of Bcl-2 regulation remains unresolved. Correlating results between synchrotron x-ray diffraction and microscopy in cell-free assays, we report activation of pro-apoptotic Bax induces strong pure negative Gaussian membrane curvature topologically necessary for pore formation and membrane remodeling events. Strikingly, Bcl-xL suppresses not only Bax-induced pore formation, but also membrane remodeling by disparate systems including cell penetrating, antimicrobial or viral fusion peptides, and bacterial toxin, none of which have BH3 allosteric domains to mediate direct binding. We propose a parallel mode of Bcl-2 pore regulation in which Bax and Bcl-xL induce antagonistic and mutually interacting Gaussian membrane curvatures. The universal nature of curvature-mediated interactions allows synergy with direct binding mechanisms, and potentially accounts for the Bcl-2 family modulation of mitochondrial fission/fusion dynamics.

  6. Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.

    PubMed

    Koty, P P; Zhang, H; Levitt, M L

    1999-02-01

    Programmed cell death (PCD) is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the ratio of PCD inducers (Bax) or inhibitors (Bcl-2). An abnormally high ratio of Bcl-2 to Bax prevents PCD, thus contributing to resistance to chemotherapeutic agents, many of which are capable of inducing PCD. Non-small cell lung cancer (NSCLC) cells demonstrate resistance to these PCD-inducing agents. If Bcl-2 prevents NSCLC cells from entering the PCD pathway, then reducing the amount of endogenous Bcl-2 product may allow these cells to spontaneously enter the PCD pathway. Our purpose was to determine the effects of bcl-2 antisense treatment on the levels of programmed cell death in NSCLC cells. First, we determined whether bcl-2 and bax mRNA were expressed in three morphologically distinct NSCLC cell lines: NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Cells were then exposed to synthetic antisense bcl-2 oligonucleotide treatment, after which programmed cell death was determined, as evidenced by DNA fragmentation. Bcl-2 protein expression was detected immunohistochemically. All three NSCLC cell lines expressed both bcl-2 and bax mRNA and had functional PCD pathways. Synthetic antisense bcl-2 oligonucleotide treatment resulted in decreased Bcl-2 levels, reduced cell proliferation, decreased cell viability, and increased levels of spontaneous PCD. This represents the first evidence that decreasing Bcl-2 in three morphologically distinct NSCLC cell lines allows the cells to spontaneously enter a PCD pathway. It also indicates the potential therapeutic use of antisense bcl-2 in the treatment of NSCLC. PMID:10217615

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

    PubMed

    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. PMID:12207177

  8. Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis

    PubMed Central

    Iwata, Akiko; de Claro, R. Angelo; Morgan-Stevenson, Vicki L.; Tupper, Joan C.; Schwartz, Barbara R.; Liu, Li; Zhu, Xiaodong; Jordan, Katherine C.; Winn, Robert K.; Harlan, John M.

    2011-01-01

    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. PMID:21390214

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

    PubMed

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

    2016-01-01

    In chronic lymphocytic leukemia the balance between the pro-apoptotic and anti-apoptotic members of the bcl-2 family is involved in the pathogenesis, chemorefractoriness and clinical outcome. Moreover, the recently proposed anti-bcl-2 molecules, such as ABT-199, have emphasized the potential role of of bcl-2 family proteins in the context of target therapies. We investigated bax/bcl-2 ratio by flow cytometry in 502 patients and identified a cut off of 1.50 to correlate bax/bcl-2 ratio with well-established clinical and biological prognosticators. Bax/bcl-2 was 1.50 or over in 263 patients (52%) with chronic lymphocytic leukemia. Higher bax/bcl-2 was associated with low Rai stage, lymphocyte doubling time over 12 months, beta-2 microglobulin less than 2.2 mg/dL, soluble CD23 less than 70 U/mL and a low risk cytogenetic profile (P<0.0001). On the other hand, lower bax/bcl-2 was correlated with unmutated IGHV (P<0.0001), mutated NOTCH1 (P<0.0001) and mutated TP53 (P=0.00007). Significant shorter progression-free survival and overall survival were observed in patients with lower bax/bcl-2 (P<0.0001). Moreover, within IGHV unmutated (168 patients) and TP53 mutated (37 patients) subgroups, higher bax/bcl-2 identified cases with significant longer PFS (P=0.00002 and P=0.039). In multivariate analysis of progression-free survival and overall survival, bax/bcl-2 was an independent prognostic factor (P=0.0002 and P=0.002). In conclusion, we defined the prognostic power of bax/bcl-2 ratio, as determined by a flow cytometric approach, and highlighted a correlation with chemoresistance and outcome in chronic lymphocytic leukemia. Finally, the recently proposed new therapies employing bcl-2 inhibitors prompted the potential use of bax/bcl-2 ratio to identify patients putatively resistant to these molecules. PMID:26565002

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

    PubMed Central

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

    2016-01-01

    In chronic lymphocytic leukemia the balance between the pro-apoptotic and anti-apoptotic members of the bcl-2 family is involved in the pathogenesis, chemorefractoriness and clinical outcome. Moreover, the recently proposed anti-bcl-2 molecules, such as ABT-199, have emphasized the potential role of of bcl-2 family proteins in the context of target therapies. We investigated bax/bcl-2 ratio by flow cytometry in 502 patients and identified a cut off of 1.50 to correlate bax/bcl-2 ratio with well-established clinical and biological prognosticators. Bax/bcl-2 was 1.50 or over in 263 patients (52%) with chronic lymphocytic leukemia. Higher bax/bcl-2 was associated with low Rai stage, lymphocyte doubling time over 12 months, beta-2 microglobulin less than 2.2 mg/dL, soluble CD23 less than 70 U/mL and a low risk cytogenetic profile (P<0.0001). On the other hand, lower bax/bcl-2 was correlated with unmutated IGHV (P<0.0001), mutated NOTCH1 (P<0.0001) and mutated TP53 (P=0.00007). Significant shorter progression-free survival and overall survival were observed in patients with lower bax/bcl-2 (P<0.0001). Moreover, within IGHV unmutated (168 patients) and TP53 mutated (37 patients) subgroups, higher bax/bcl-2 identified cases with significant longer PFS (P=0.00002 and P=0.039). In multivariate analysis of progression-free survival and overall survival, bax/bcl-2 was an independent prognostic factor (P=0.0002 and P=0.002). In conclusion, we defined the prognostic power of bax/bcl-2 ratio, as determined by a flow cytometric approach, and highlighted a correlation with chemoresistance and outcome in chronic lymphocytic leukemia. Finally, the recently proposed new therapies employing bcl-2 inhibitors prompted the potential use of bax/bcl-2 ratio to identify patients putatively resistant to these molecules. PMID:26565002

  11. Involvement of PI3K and MAPK Signaling in bcl-2-induced Vascular Endothelial Growth Factor Expression in Melanoma Cells

    PubMed Central

    Trisciuoglio, Daniela; Iervolino, Angela; Zupi, Gabriella; Del Bufalo, Donatella

    2005-01-01

    We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1α expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways. PMID:15987743

  12. Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein

    PubMed Central

    Trisciuoglio, D; Desideri, M; Farini, V; De Luca, T; Di Martile, M; Tupone, M G; Urbani, A; D'Aguanno, S; Del Bufalo, D

    2016-01-01

    Members of the bcl-2 protein family share regions of sequence similarity, the bcl-2 homology (BH) domains. Bcl-2, the most studied member of this family, has four BH domains, BH1–4, and has a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2-interacting factors should provide a critical advance in understanding the different functions of bcl-2. Here, we characterized bcl-2 interactome by mass spectrometry in human lung adenocarcinoma cells. In silico functional analysis associated most part of the identified proteins to mitochondrial functions. Among them we identified SRA stem–loop interacting RNA-binding protein, SLIRP, a mitochondrial protein with a relevant role in regulating mitochondrial messenger RNA (mRNA) homeostasis. We validated bcl-2/SLIRP interaction by immunoprecipitation and immunofluorescence experiments in cancer cell lines from different histotypes. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein and regulates mitochondrial mRNA levels. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding. PMID:26866271

  13. Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein.

    PubMed

    Trisciuoglio, D; Desideri, M; Farini, V; De Luca, T; Di Martile, M; Tupone, M G; Urbani, A; D'Aguanno, S; Del Bufalo, D

    2016-01-01

    Members of the bcl-2 protein family share regions of sequence similarity, the bcl-2 homology (BH) domains. Bcl-2, the most studied member of this family, has four BH domains, BH1-4, and has a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2-interacting factors should provide a critical advance in understanding the different functions of bcl-2. Here, we characterized bcl-2 interactome by mass spectrometry in human lung adenocarcinoma cells. In silico functional analysis associated most part of the identified proteins to mitochondrial functions. Among them we identified SRA stem-loop interacting RNA-binding protein, SLIRP, a mitochondrial protein with a relevant role in regulating mitochondrial messenger RNA (mRNA) homeostasis. We validated bcl-2/SLIRP interaction by immunoprecipitation and immunofluorescence experiments in cancer cell lines from different histotypes. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein and regulates mitochondrial mRNA levels. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding. PMID:26866271

  14. Induction of apoptosis in melanoma A375 cells by a chloroform fraction of Centratherum anthelminticum (L.) seeds involves NF-kappaB, p53 and Bcl-2-controlled mitochondrial signaling pathways

    PubMed Central

    2013-01-01

    Background Centratherum anthelminticum (L.) Kuntze (scientific synonyms: Vernonia anthelmintica; black cumin) is one of the ingredients of an Ayurvedic preparation, called “Kayakalp”, commonly applied to treat skin disorders in India and Southeast Asia. Despite its well known anti-inflammatory property on skin diseases, the anti-cancer effect of C. anthelminticum seeds on skin cancer is less documented. The present study aims to investigate the anti-cancer effect of Centratherum anthelminticum (L.) seeds chloroform fraction (CACF) on human melanoma cells and to elucidate the molecular mechanism involved. Methods A chloroform fraction was extracted from C. anthelminticum (CACF). Bioactive compounds of the CACF were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human melanoma cell line A375 was treated with CACF in vitro. Effects of CACF on growth inhibition, morphology, stress and survival of the cell were examined with MTT, high content screening (HSC) array scan and flow cytometry analyses. Involvement of intrinsic or extrinsic pathways in the CACF-induced A375 cell death mechanism was examined using a caspase luminescence assay. The results were further verified with different caspase inhibitors. In addition, Western blot analysis was performed to elucidate the changes in apoptosis-associated molecules. Finally, the effect of CACF on the NF-κB nuclear translocation ability was assayed. Results The MTT assay showed that CACF dose-dependently inhibited cell growth of A375, while exerted less cytotoxic effect on normal primary epithelial melanocytes. We demonstrated that CACF induced cell growth inhibition through apoptosis, as evidenced by cell shrinkage, increased annexin V staining and formation of membrane blebs. CACF treatment also resulted in higher reactive oxygen species (ROS) production and lower Bcl-2 expression, leading to decrease mitochondrial membrane potential (MMP). Disruption of the MMP facilitated the release of

  15. Diminishing Apoptosis by Deletion of Bax or Overexpression of Bcl-2 Does Not Protect against Infectious Prion Toxicity In Vivo

    PubMed Central

    Steele, Andrew D.; King, Oliver D.; Jackson, Walker S.; Hetz, Claudio A.; Borkowski, Andrew W.; Thielen, Peter; Wollmann, Robert; Lindquist, Susan

    2008-01-01

    B-cell lymphoma protein 2 (Bcl-2) and Bcl-2-associated X protein (Bax), key antiapoptotic and proapoptotic proteins, respectively, have important roles in acute and chronic models of neurologic disease. Several studies have implicated Bax and Bcl-2 in mediating neurotoxicity in prion diseases. To determine whether diminishing apoptotic cell death is protective in an infectious prion disease model we inoculated mice that either were null for proapoptotic Bax or overexpressed antiapoptotic Bcl-2. Interestingly, genetic manipulation of apoptosis did not lessen the clinical severity of disease. Moreover, some disease parameters, such as behavioral alterations and death, occurred slightly earlier in mice that are null for Bax or overexpress Bcl-2. These results suggest that Bax and Bcl-2 mediated apoptotic pathways are not the major contributing factor to the clinical or pathological features of infectious prion disease. PMID:18032675

  16. The BCL2 rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia

    PubMed Central

    Ploner, C; Rainer, J; Niederegger, H; Eduardoff, M; Villunger, A; Geley, S; Kofler, R

    2016-01-01

    Glucocorticoid (GC)-induced apoptosis is essential in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Pro- and anti-apoptotic members of the BCL2 family control many forms of apoptotic cell death, but the extent to which this survival ‘rheostat’ is involved in the beneficial effects of GC therapy is not understood. We performed systematic analyses of expression, GC regulation and function of BCL2 molecules in primary ALL lymphoblasts and a corresponding in vitro model. Affymetrix-based expression profiling revealed that the response included regulations of pro-apoptotic and, surprisingly, anti-apoptotic BCL2 family members, and varied among patients, but was dominated by induction of the BH3-only molecules BMF and BCL2L11/Bim and repression of PMAIP1/Noxa. Conditional lentiviral gene overexpression and knock-down by RNA interference in the CCRF-CEM model revealed that induction of Bim, and to a lesser extent that of BMF, was required and sufficient for apoptosis. Although anti-apoptotic BCL2 members were not regulated consistently by GC in the various systems, their overexpression delayed, whereas their knock-down accelerated, GC-induced cell death. Thus, the combined clinical and experimental data suggest that GCs induce both pro- and anti-apoptotic BCL2 family member-dependent pathways, with the outcome depending on cellular context and additional signals feeding into the BCL2 rheostat. PMID:18046449

  17. Depletion of Bcl-2 by an antisense oligonucleotide induces apoptosis accompanied by oxidation and externalization of phosphatidylserine in NCI-H226 lung carcinoma cells.

    PubMed

    Koty, Patrick P; Tyurina, Yulia Y; Tyurin, Vladimir A; Li, Shang-Xi; Kagan, Valerian E

    2002-01-01

    Oxidant-induced apoptosis involves oxidation of many different and essential molecules including phospholipids. As a result of this non-specific oxidation, any signaling role of a particular phospholipid-class of molecules is difficult to elucidate. To determine whether preferential oxidation of phosphatidylserine (PS) is an early event in apoptotic signaling related to PS externalization and is independent of direct oxidant exposure, we chose a genetic-based induction of apoptosis. Apoptosis was induced in the lung cancer cell line NCI-H226 by decreasing the amount of Bcl-2 protein expression by preventing the translation of bcl-2 mRNA using an antisense bcl-2 oligonucleotide. Peroxidation of phospholipids was assayed using a fluorescent technique based on metabolic integration of an oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid (PnA), into cellular phospholipids and subsequent HPLC separation of cis-PnA-labeled phospholipids. We found a decrease in Bcl-2 was associated with a selective oxidation of PS in a sub-population of the cells with externalized PS. No significant difference in oxidation of cis-PnA-labeled phospholipids was observed in cells treated with medium alone or a nonsense oligonucleotide. Treatment with either nonsensc or antisense bcl-2 oligonucleotides was not associated with changes in the pattern of individual phospholipid classes as determined by HPTLC. These metabolic and topographical changes in PS arrangement in plasma membrane appear to be early responses to antisense bcl-2 exposure that trigger a PS-dependent apoptotic signaling pathway. This observed externalization of PS may facilitate the 'labeling' of apoptotic cells for recognition by macrophage scavenger receptors and subsequent phagocytic clearance. PMID:12162425

  18. Functional BCL-2 regulatory genetic variants contribute to susceptibility of esophageal squamous cell carcinoma.

    PubMed

    Pan, Wenting; Yang, Jinyun; Wei, Jinyu; Chen, Hongwei; Ge, Yunxia; Zhang, Jingfeng; Wang, Zhiqiong; Zhou, Changchun; Yuan, Qipeng; Zhou, Liqing; Yang, Ming

    2015-01-01

    B-cell lymphoma-2 (BCL-2) prevents apoptosis and its overexpression could promote cancer cell survival. Multiple functional BCL-2 genetic polymorphisms, such as rs2279115, rs1801018 and rs1564483, have been identified previously and might be involved in cancer development through deregulating BCL-2 expression. Therefore, we examined associations between these three polymorphisms and esophageal squamous cell carcinoma (ESCC) susceptibility as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1588 ESCC patients and 1600 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. The impact of the rs2279115 polymorphism on BCL-2 expression was detected using esophagus tissues. Our results demonstrated that the BCL-2 rs2279115 AA genotype was significantly associated with decreased ESCC risk compared with the CC genotype (OR = 0.72, 95% CI = 0.57-0.90, P = 0.005), especially in nonsmokers (OR = 0.42, 95% CI = 0.29-0.59, P = 0.001) or nondrinkers (OR = 0.44, 95% CI = 0.32-0.62, P =  .002). Genotype-phenotype correlation studies demonstrated that subjects with the rs2279115 CA and AA genotypes had a statistically significant decrease of BCL-2 mRNA expression compared to the CC genotype in both normal and cancerous esophagus tissues. Our results indicate that the BCL-2 rs2279115 polymorphism contributes to ESCC susceptibility in Chinese populations. PMID:26132559

  19. The BCL-2 protein family, BH3-mimetics and cancer therapy

    PubMed Central

    Delbridge, A R D; Strasser, A

    2015-01-01

    Escape from apoptosis is a key attribute of tumour cells and facilitates chemo-resistance. The ‘BCL-2-regulated' or ‘intrinsic' apoptotic pathway integrates stress and survival signalling to govern whether a cancer cell will live or die. Indeed, many pro-apoptotic members of the BCL-2 family have demonstrated tumour-suppression activity in mouse models of cancer and are lost or repressed in certain human cancers. Conversely, overexpression of pro-survival BCL-2 family members promotes tumorigenesis in humans and in mouse models. Many of the drugs currently used in the clinic mediate their therapeutic effects (at least in part) through the activation of the BCL-2-regulated apoptotic pathway. However, initiators of this apoptotic pathway, such as p53, are mutated, lost or silenced in many human cancers rendering them refractory to treatment. To counter such resistance mechanisms, a novel class of therapeutics, ‘BH3-mimetics', has been developed. These drugs directly activate apoptosis by binding and inhibiting select antiapoptotic BCL-2 family members and thereby bypass the requirement for upstream initiators, such as p53. In this review, we discuss the role of the BCL-2 protein family in the development and treatment of cancer, with an emphasis on mechanistic studies using well-established mouse models of cancer, before describing the development and already recognised potential of the BH3-mimetic compounds. PMID:25952548

  20. bcl-2 overexpression promotes myocyte proliferation

    PubMed Central

    Limana, Federica; Urbanek, Konrad; Chimenti, Stefano; Quaini, Federico; Leri, Annarosa; Kajstura, Jan; Nadal-Ginard, Bernardo; Izumo, Seigo; Anversa, Piero

    2002-01-01

    To determine the influence of Bcl-2 on the developmental biology of myocytes, we analyzed the population dynamics of this cell type in the heart of transgenic (TG) mice overexpressing Bcl-2 under the control of the α-myosin heavy chain promoter. TG mice and non-TG (wild type, WT) mice were studied at 24 days, 2 months, and 4 months after birth. Bcl-2 overexpression produced a significant increase in the percentage of cycling myocytes and their mitotic index. These effects were strictly connected to the expression of the transgene, as demonstrated in isolated myocytes. The formation of mitotic spindle and contractile ring was identified in replicating cells. These typical aspects of mitosis were complemented with the demonstration of karyokinesis and cytokinesis to provide structural evidence of cell division. Apoptosis was low at all ages and was not affected by Bcl-2. The higher cell replication rate in TG was conditioned by a decrease in the expression of the cell-cycle inhibitors, p21WAF1 and p16INK4a, and by an increase in Mdm2-p53 complexes. In comparison with WT, TG had 0.4 × 106, 0.74 × 106, and 1.2 × 106 more myocytes in the left ventricle at 24 days, 2 months, and 4 months, respectively. Binucleated myocytes were 12% and 25% larger in WT than in TG mice at 2 and 4 months of age. Taken together, these observations reveal a previously uncharacterized replication-enhancing function of Bcl-2 in myocytes in vivo in the absence of stressful conditions. PMID:11983915

  1. HUMAN HOMOLOGUE OF S. POMBE RAD9 INTERACTS WITH BCL-2/BCL-X{SUB L} AND PROMOTES APOPTOSIS

    EPA Science Inventory

    DNA damage induces apoptosis through a Bcl-2 suppressible signaling pathway, but the mechanism is unknown. The human cell cycle checkpoint control protein HRad9 was found to interact with Bcl-2 and Bcl-XL but not Bax or Bad, as demonstrated by yeast two-hybrid and coimmunoprecipi...

  2. Clathrin-Independent Pathways of Endocytosis

    PubMed Central

    Mayor, Satyajit; Parton, Robert G.; Donaldson, Julie G.

    2014-01-01

    There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration. PMID:24890511

  3. Getting away with murder: how does the BCL-2 family of proteins kill with immunity?

    PubMed

    Renault, Thibaud T; Chipuk, Jerry E

    2013-05-01

    The adult human body produces approximately one million white blood cells every second. However, only a small fraction of the cells will survive because the majority is eliminated through a genetically controlled form of cell death known as apoptosis. This review places into perspective recent studies pertaining to the BCL-2 family of proteins as critical regulators of the development and function of the immune system, with particular attention on B cell and T cell biology. Here we discuss how elegant murine model systems have revealed the major contributions of the BCL-2 family in establishing an effective immune system. Moreover, we highlight some key regulatory pathways that influence the expression, function, and stability of individual BCL-2 family members, and discuss their role in immunity. From lethal mechanisms to more gentle ones, the final portion of the review discusses the nonapoptotic functions of the BCL-2 family and how they pertain to the control of immunity. PMID:23527542

  4. Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies.

    PubMed

    Delbridge, Alex R D; Grabow, Stephanie; Strasser, Andreas; Vaux, David L

    2016-02-01

    The 'hallmarks of cancer' are generally accepted as a set of genetic and epigenetic alterations that a normal cell must accrue to transform into a fully malignant cancer. It follows that therapies designed to counter these alterations might be effective as anti-cancer strategies. Over the past 30 years, research on the BCL-2-regulated apoptotic pathway has led to the development of small-molecule compounds, known as 'BH3-mimetics', that bind to pro-survival BCL-2 proteins to directly activate apoptosis of malignant cells. This Timeline article focuses on the discovery and study of BCL-2, the wider BCL-2 protein family and, specifically, its roles in cancer development and therapy. PMID:26822577

  5. Multifunctional Role of Bcl-2 in Malignant Transformation and Tumorigenesis of Cr(VI)-Transformed Lung Cells

    PubMed Central

    Azad, Neelam; Wang, Liying; Jiang, Bing-Hua; Davis, Mary E.; Barnett, John B.; Guo, Lan; Rojanasakul, Yon

    2012-01-01

    B-cell lymphoma-2 (Bcl-2) is an antiapoptotic protein known to be important in the regulation of apoptosis in various cell types. However, its role in malignant transformation and tumorigenesis of human lung cells is not well understood. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation; however, the role of Bcl-2 in the transformation is unclear. Using a gene silencing approach, we showed that Bcl-2 plays an important role in the malignant properties of Cr(VI)-transformed cells. Downregulation of Bcl-2 inhibited the invasive and proliferative properties of the cells as well as their colony forming and angiogenic activities, which are upregulated in the transformed cells as compared to control cells. Furthermore, animal studies showed the inhibitory effect of Bcl-2 knockdown on the tumorigenesis of Cr(VI)-transformed cells. The role of Bcl-2 in malignant transformation and tumorigenesis was confirmed by gene silencing experiments using human lung carcinoma NCI-H460 cells. These cells exhibited aggressive malignant phenotypes similar to those of Cr(VI)-transformed cells. Knockdown of Bcl-2 in the H460 cells inhibited malignant and tumorigenic properties of the cells, indicating the general role of Bcl-2 in human lung tumorigenesis. Ingenuity Pathways Analysis (IPA) revealed potential effectors of Bcl-2 in tumorigenesis regulation. Additionally, using IPA together with ectopic expression of p53, we show p53 as an upstream regulator of Bcl-2 in Cr(VI)-transformed cells. Together, our results indicate the novel and multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of human lung epithelial cells chronically exposed to Cr(VI). PMID:22666341

  6. Bcl2 is a critical regulator of bile acid homeostasis by dictating Shp and lncRNA H19 function

    PubMed Central

    Zhang, Yuxia; Liu, Chune; Barbier, Olivier; Smalling, Rana; Tsuchiya, Hiroyuki; Lee, Sangmin; Delker, Don; Zou, An; Hagedorn, Curt H.; Wang, Li

    2016-01-01

    Bile acid (BA) metabolism is tightly controlled by nuclear receptor signaling to coordinate regulation of BA synthetic enzymes and transporters. Here we reveal a molecular cascade consisting of the antiapoptotic protein BCL2, nuclear receptor Shp, and long non-coding RNA (lncRNA) H19 to maintain BA homeostasis. Bcl2 was overexpressed in liver of C57BL/6J mice using adenovirus mediated gene delivery for two weeks. Hepatic overexpression of Bcl2 caused drastic accumulation of serum BA and bilirubin levels and dysregulated BA synthetic enzymes and transporters. Bcl2 reactivation triggered severe liver injury, fibrosis and inflammation, which were accompanied by a significant induction of H19. Bcl2 induced rapid SHP protein degradation via the activation of caspase-8 pathway. The induction of H19 in Bcl2 overexpressed mice was contributed by a direct loss of Shp transcriptional repression. H19 knockdown or Shp re-expression largely rescued Bcl2-induced liver injury. Strikingly different than Shp, the expression of Bcl2 and H19 was hardly detectable in adult liver but was markedly increased in fibrotic/cirrhotic human and mouse liver. We demonstrated for the first time a detrimental effect of Bcl2 and H19 associated with cholestatic liver fibrosis and an indispensable role of Shp to maintain normal liver function. PMID:26838806

  7. Partial lack of BCL2 in follicular lymphoma: An unusual immunohistochemical staining pattern explained by ongoing BCL2 mutation.

    PubMed

    van den Brand, Michiel; Garcia-Garcia, Mar; Mathijssen, Janneke J M; Colomo, Lluis; Groenen, Patricia J T A; Serrano, Sergio; van Krieken, J Han J M

    2016-02-01

    Follicular lymphomas are characterized by overexpression of BCL2 which, in the large majority of cases, is due to a t(14;18) translocation which juxtaposes the BCL2 locus to the immunoglobulin heavy chain locus (IGH). Here, we report partial absence of BCL2 immunohistochemical staining in a case of FL, due to a mutation in the part of BCL2 that encodes the epitope for the most frequently used antibody against BCL2. This finding shows that mutations in BCL2 occur in an ongoing process in follicular which can give rise to unusual immunohistochemical staining patterns. PMID:26725534

  8. Interaction of Bcl-2 with the Autophagy-related GABAA Receptor-associated Protein (GABARAP)

    PubMed Central

    Ma, Peixiang; Schwarten, Melanie; Schneider, Lars; Boeske, Alexandra; Henke, Nadine; Lisak, Dmitrij; Weber, Stephan; Mohrlüder, Jeannine; Stoldt, Matthias; Strodel, Birgit; Methner, Axel; Hoffmann, Silke; Weiergräber, Oliver H.; Willbold, Dieter

    2013-01-01

    Apoptosis and autophagy are fundamental homeostatic processes in eukaryotic organisms fulfilling essential roles in development and adaptation. Recently, the anti-apoptotic factor Bcl-2 has been reported to also inhibit autophagy, thus establishing a potential link between these pathways, but the mechanistic details are only beginning to emerge. Here we show that Bcl-2 directly binds to the phagophore-associated protein GABARAP. NMR experiments revealed that the interaction critically depends on a three-residue segment (EWD) of Bcl-2 adjacent to the BH4 region, which is anchored to one of the two hydrophobic pockets on the GABARAP molecule. This is at variance with the majority of GABARAP interaction partners identified previously, which occupy both hydrophobic pockets simultaneously. Bcl-2 affinity could also be detected for GEC1, but not for other mammalian Atg8 homologs. Finally, we provide evidence that overexpression of Bcl-2 inhibits lipidation of GABARAP, a key step in autophagosome formation, possibly via competition with the lipid conjugation machinery. These results support the regulatory role of Bcl-2 in autophagy and define GABARAP as a novel interaction partner involved in this intricate connection. PMID:24240096

  9. Glioma oncoprotein Bcl2L12 inhibits the p53 tumor suppressor

    PubMed Central

    Stegh, Alexander H.; Brennan, Cameron; Mahoney, John A.; Forloney, Kristin L.; Jenq, Harry T.; Luciano, Janina P.; Protopopov, Alexei; Chin, Lynda; DePinho, Ronald A.

    2010-01-01

    Glioblastoma multiforme (GBM) is a lethal brain tumor characterized by intense apoptosis resistance and extensive necrosis. Bcl2L12 (for Bcl2-like 12) is a cytoplasmic and nuclear protein that is overexpressed in primary GBM and functions to inhibit post-mitochondrial apoptosis signaling. Here, we show that nuclear Bcl2L12 physically and functionally interacts with the p53 tumor suppressor, as evidenced by the capacity of Bcl2L12 to (1) enable bypass of replicative senescence without concomitant loss of p53 or p19Arf, (2) inhibit p53-dependent DNA damage-induced apoptosis, (3) impede the capacity of p53 to bind some of its target gene promoters, and (4) attenuate endogenous p53-directed transcriptomic changes following genotoxic stress. Correspondingly, The Cancer Genome Atlas profile and tissue protein analyses of human GBM specimens show significantly lower Bcl2L12 expression in the setting of genetic p53 pathway inactivation. Thus, Bcl2L12 is a multifunctional protein that contributes to intense therapeutic resistance of GBM through its ability to operate on two key nodes of cytoplasmic and nuclear signaling cascades. PMID:20837658

  10. Emerging Understanding of Bcl-2 Biology: Implications for Neoplastic Progression and Treatment

    PubMed Central

    Correia, Cristina; Lee, Sun-Hee; Meng, X. Wei; Vincelette, Nicole D.; Knorr, Katherine L.B.; Ding, Husheng; Nowakowski, Grzegorz S.; Dai, Haiming; Kaufmann, Scott H.

    2015-01-01

    Bcl-2, the founding member of a family of apoptotic regulators, was initially identified as the protein product of a gene that is translocated and overexpressed in greater than 85% of follicular lymphomas (FLs). Thirty years later we now understand that Bcl-2 modulates the intrinsic apoptotic pathway by binding and neutralizing the mitochondrial permeabilizers Bax and Bak as well as a variety of pro-apoptotic proteins, including the cellular stress sensors Bim, Bid, Puma, Bad, Bmf and, under some conditions, Noxa. Despite extensive investigation of all of these proteins, important questions remain. For example, how Bax and Bak breach the outer mitochondrial membrane remains poorly understood. Likewise, how the functions of anti-apoptotic Bcl-2 family members such as eponymous Bcl-2 are affected by phosphorylation or cancer-associated mutations has been incompletely defined. Finally, whether Bcl-2 family members can be successfully targeted for therapeutic advantage is only now being investigated in the clinic. Here we review recent advances in understanding Bcl-2 family biology and biochemistry that begin to address these questions. PMID:25827952

  11. Bcl-2high mantle cell lymphoma cells are sensitized to acadesine with ABT-199

    PubMed Central

    Montraveta, Arnau; Xargay-Torrent, Sílvia; Rosich, Laia; López-Guerra, Mònica; Roldán, Jocabed; Rodríguez, Vanina; Lee-Vergés, Eriong; de Frías, Mercè; Campàs, Clara; Campo, Elias; Roué, Gaël; Colomer, Dolors

    2015-01-01

    Acadesine is a nucleoside analogue with known activity against B-cell malignancies. Herein, we showed that in mantle cell lymphoma (MCL) cells acadesine induced caspase-dependent apoptosis through turning on the mitochondrial apoptotic machinery. At the molecular level, the compound triggered the activation of the AMPK pathway, consequently modulating known downstream targets, such as mTOR and the cell motility-related vasodilator-stimulated phosphoprotein (VASP). VASP phosphorylation by acadesine was concomitant with a blockade of CXCL12-induced migration. The inhibition of the mTOR cascade by acadesine, committed MCL cells to enter in apoptosis by a translational downregulation of the antiapoptotic Mcl-1 protein. In contrast, Bcl-2 protein levels were unaffected by acadesine and MCL samples expressing high levels of Bcl-2 tended to have a reduced response to the drug. Targeting Bcl-2 with the selective BH3-mimetic agent ABT-199 sensitized Bcl-2 high MCL cells to acadesine. This effect was validated in vivo, where the combination of both agents displayed a more marked inhibition of tumor outgrowth than each drug alone. These findings support the notions that antiapoptotic proteins of the Bcl-2 family regulate MCL cell sensitivity to acadesine and that the combination of this agent with Bcl-2 inhibitors might be an interesting therapeutic option to treat MCL patients. PMID:26110568

  12. Quantification of protein copy number in single mitochondria: The Bcl-2 family proteins.

    PubMed

    Chen, Chaoxiang; Zhang, Xiang; Zhang, Shuyue; Zhu, Shaobin; Xu, Jingyi; Zheng, Yan; Han, Jinyan; Zeng, Jin-Zhang; Yan, Xiaomei

    2015-12-15

    Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5-97.5%) for Bcl-2 and from 65 to 700 (2.5-97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria. PMID:26176207

  13. Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis.

    PubMed

    Fuenzalida, Karen; Quintanilla, Rodrigo; Ramos, Patricio; Piderit, Daniela; Fuentealba, Rodrigo A; Martinez, Gabriela; Inestrosa, Nibaldo C; Bronfman, Miguel

    2007-12-21

    Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2. PMID:17965419

  14. A component of green tea (-)-epigallocatechin-3-gallate, promotes apoptosis in T24 human bladder cancer cells via modulation of the PI3K/Akt pathway and Bcl-2 family proteins

    SciTech Connect

    Qin Jie; Xie Liping . E-mail: xielp@zjuem.zju.edu.cn; Zheng Xiangyi; Wang Yunbin; Bai Yu; Shen Huafeng; Li Longcheng; Dahiya, Rajvir

    2007-03-23

    Bladder cancer is the fourth most common cancer in men and ninth most common in women. It has a protracted course of progression and is thus an ideal candidate for chemoprevention strategies and trials. This study was conducted to evaluate the chemopreventive/antiproliferative potential of (-)-epigallocatechin gallate (EGCG, the major phytochemical in green tea) against bladder cancer and its mechanism of action. Using the T24 human bladder cancer cell line, we found that EGCG treatment caused dose- and time-dependent inhibition of cellular proliferation and cell viability, and induced apoptosis. Mechanistically, EGCG inhibits phosphatidylinositol 3'-kinase/Akt activation that, in turn, results in modulation of Bcl-2 family proteins, leading to enhanced apoptosis of T24 cells. These findings suggest that EGCG may be an important chemoprevention agent for the management of bladder cancer.

  15. MicroRNA-181c targets Bcl-2 and regulates mitochondrial morphology in myocardial cells

    PubMed Central

    Wang, Hongjiang; Li, Jing; Chi, Hongjie; Zhang, Fan; Zhu, Xiaoming; Cai, Jun; Yang, Xinchun

    2015-01-01

    Apoptosis is an important mechanism for the development of heart failure. Mitochondria are central to the execution of apoptosis in the intrinsic pathway. The main regulator of mitochondrial pathway of apoptosis is Bcl-2 family which includes pro- and anti-apoptotic proteins. MicroRNAs are small noncoding RNA molecules that regulate gene expression by inhibiting mRNA translation and/or inducing mRNA degradation. It has been proposed that microRNAs play critical roles in the cardiovascular physiology and pathogenesis of cardiovascular diseases. Our previous study has found that microRNA-181c, a miRNA expressed in the myocardial cells, plays an important role in the development of heart failure. With bioinformatics analysis, we predicted that miR-181c could target the 3′ untranslated region of Bcl-2, one of the anti-apoptotic members of the Bcl-2 family. Thus, we have suggested that miR-181c was involved in regulation of Bcl-2. In this study, we investigated this hypothesis using the Dual-Luciferase Reporter Assay System. Cultured myocardial cells were transfected with the mimic or inhibitor of miR-181c. We found that the level of miR-181c was inversely correlated with the Bcl-2 protein level and that transfection of myocardial cells with the mimic or inhibitor of miR-181c resulted in significant changes in the levels of caspases, Bcl-2 and cytochrome C in these cells. The increased level of Bcl-2 caused by the decrease in miR-181c protected mitochondrial morphology from the tumour necrosis factor alpha-induced apoptosis. PMID:25898913

  16. [Osteosarcoma--apoptosis and proliferation. Study of bcl-2 expression].

    PubMed

    Pösl, M; Amling, M; Werner, M; Bäsler, I; Salzer-Kuntschik, M; Winkler, K; Delling, G

    1994-12-01

    The relationship between the growth of tumors and the expression of the protooncogene Bcl-2 could be shown in epithelial tumors. A bcl-2 expression leads to a prolonged cell survival due to an inhibition of apoptosis. The potential meaning of bcl-2 expression in mesenchymal tumors remains still unknown. The fact, that the heterogenous group of osteosarcoma is not sufficiently characterized at present, suggested to investigate the bcl-2 expression in osteosarcoma. Thus, immunohistochemistry was used to analyze 47 specimens of different osteosarcomas of 36 patients. Sixteen cases (46%) showed a strong expression of bcl-2 and 13 cases (35%) were moderately positive for bcl-2. Seven cases (19%) were negative for bcl-2. The heterogenous, negative up to strong expression of bcl-2 yield clues, that the Bcl-2 controlled regulation of programmed cell death could be an important factor of cellular kinetics. Additionally the cellular proliferation rate was determined with the monoclonal antibody MIB 1, directed against the Ki-67 epitope. The data of bcl-2 expression and cellular proliferation rate lead to a classification correlating with the histological classification. To verify the importance of apoptosis in the genesis of mesenchymal tumors and whether Bcl-2 may play an important role as a predictive factor for the prognosis of osteosarcoma, further investigations will be needed. PMID:7855102

  17. Glutathione Binding to the Bcl-2 Homology-3 Domain Groove

    PubMed Central

    Zimmermann, Angela K.; Loucks, F. Alexandra; Schroeder, Emily K.; Bouchard, Ron J.; Tyler, Kenneth L.; Linseman, Daniel A.

    2008-01-01

    Bcl-2 protects cells against mitochondrial oxidative stress and subsequent apoptosis. However, the mechanism underlying the antioxidant function of Bcl-2 is currently unknown. Recently, Bax and several Bcl-2 homology-3 domain (BH3)-only proteins (Bid, Puma, and Noxa) have been shown to induce a pro-oxidant state at mitochondria (1-4). Given the opposing effects of Bcl-2 and Bax/BH3-only proteins on the redox state of mitochondria, we hypothesized that the antioxidant function of Bcl-2 is antagonized by its interaction with the BH3 domains of pro-apoptotic family members. Here, we show that BH3 mimetics that bind to a hydrophobic surface (the BH3 groove) of Bcl-2 induce GSH-sensitive mitochondrial dysfunction and apoptosis in cerebellar granule neurons. BH3 mimetics displace a discrete mitochondrial GSH pool in neurons and suppress GSH transport into isolated rat brain mitochondria. Moreover, BH3 mimetics and the BH3-only protein, Bim, inhibit a novel interaction between Bcl-2 and GSH in vitro. These results suggest that Bcl-2 regulates an essential pool of mitochondrial GSH and that this regulation may depend upon Bcl-2 directly interacting with GSH via the BH3 groove. We conclude that this novel GSH binding property of Bcl-2 likely plays a central role in its antioxidant function at mitochondria. PMID:17690097

  18. Actinomycin D upregulates proapoptotic protein Puma and downregulates Bcl-2 mRNA in normal peripheral blood lymphocytes.

    PubMed

    Kalousek, Ivan; Brodska, Barbora; Otevrelova, Petra; Röselova, Pavla

    2007-08-01

    We have examined the ability of actinomycin D to induce apoptosis in human peripheral blood lymphocytes. Run-On assays were performed to specify the primary molecular damage, reverse transcription-PCR, Western blots and flow cytometry studies were performed to ascertain which proteins of the apoptosis machinery were affected to cause actinomycin D-induced cell death. Expression of 23 apoptosis-related genes was investigated. The down-regulation of ribosomal RNA synthesis caused by actinomycin D induced a mitochondria-dependent apoptosis. Although the expression of the majority of examined genes remained indifferent against actinomycin D activity, the cellular level of p53 protein increased, subsequently upregulating both Puma mRNA and protein. Puma-mediated mitochondrial apoptosis was accompanied by nucleolin cleavage and Bcl-2 mRNA destabilization. The stability of the cellular level of Bcl-2 protein independent of a mRNA decrease suggests that protection of Bcl-2 protein against proteasomal degradation can moderate the apoptotic process. In peripheral blood lymphocytes cultured in vitro, the apoptosis induced by a low concentration of actinomycin D (10 nmol/l) is dependent on p53 and Puma activation. This apoptotic pathway is demonstrated in peripheral blood lymphocytes for the first time. A different apoptotic pathway induced in peripheral blood lymphocytes using this drug has, however, been previously revealed by other authors. The combination of cell specificity and dose-dependent effects can likely play a decisive role in apoptosis observed in peripheral blood lymphocytes after genotoxic drug application. PMID:17581298

  19. Methionine adenosyltransferase α2 sumoylation positively regulate Bcl-2 expression in human colon and liver cancer cells

    PubMed Central

    Tomasi, Maria Lauda; Ryoo, Minjung; Ramani, Komal; Tomasi, Ivan; Giordano, Pasquale; Mato, José M.; Lu, Shelly C.

    2015-01-01

    Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and inhibits apoptosis via Bcl-2 by unknown mechanism. Methionine adenosyltransferase 2A (MAT2A) encodes for MATα2, the catalytic subunit of the MATII isoenzyme that synthesizes S-adenosylmethionine (SAMe). Ubc9, Bcl-2 and MAT2A expression are up-regulated in several malignancies. Exogenous SAMe decreases Ubc9 and MAT2A expression and is pro-apoptotic in liver and colon cancer cells. Here we investigated whether there is interplay between Ubc9, MAT2A and Bcl-2. We used human colon and liver cancer cell lines RKO and HepG2, respectively, and confirmed key finding in colon cancer specimens. We found MATα2 can regulate Bcl-2 expression at multiple levels. MATα2 binds to Bcl-2 promoter to activate its transcription. This effect is independent of SAMe as MATα2 catalytic mutant was also effective. MATα2 also directly interacts with Bcl-2 to enhance its protein stability. MATα2's effect on Bcl-2 requires Ubc9 as MATα2's stability is influenced by sumoylation at K340, K372 and K394. Overexpressing wild type (but not less stable MATα2 sumoylation mutants) protected from 5-fluorouracil-induced apoptosis in both colon and liver cancer cells. Colon cancer have higher levels of sumoylated MATα2, total MATα2, Ubc9 and Bcl-2 and higher MATα2 binding to the Bcl-2 P2 promoter. Taken together, Ubc9's protective effect on apoptosis may be mediated at least in part by sumoylating and stabilizing MATα2 protein, which in turn positively maintains Bcl-2 expression. These interactions feed forward to further enhance growth and survival of the cancer cell. PMID:26416353

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

    PubMed Central

    Wong, Yi Li; Wong, Won Fen; Ali Mohd, Mustafa; Hadi, A. Hamid A.

    2014-01-01

    Persea declinata (Bl.) Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill), which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl.) Kosterm bark methanolic crude extract (PDM). PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48 h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH) release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development. PMID:24808916

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

    PubMed

    Narrima, Putri; Paydar, Mohammadjavad; Looi, Chung Yeng; Wong, Yi Li; Taha, Hairin; Wong, Won Fen; Ali Mohd, Mustafa; Hadi, A Hamid A

    2014-01-01

    Persea declinata (Bl.) Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill), which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl.) Kosterm bark methanolic crude extract (PDM). PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48 h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH) release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development. PMID:24808916

  2. Glutathione binding to the Bcl-2 homology-3 domain groove: a molecular basis for Bcl-2 antioxidant function at mitochondria.

    PubMed

    Zimmermann, Angela K; Loucks, F Alexandra; Schroeder, Emily K; Bouchard, Ron J; Tyler, Kenneth L; Linseman, Daniel A

    2007-10-01

    Bcl-2 protects cells against mitochondrial oxidative stress and subsequent apoptosis. However, the mechanism underlying the antioxidant function of Bcl-2 is currently unknown. Recently, Bax and several Bcl-2 homology-3 domain (BH3)-only proteins (Bid, Puma, and Noxa) have been shown to induce a pro-oxidant state at mitochondria (1-4). Given the opposing effects of Bcl-2 and Bax/BH3-only proteins on the redox state of mitochondria, we hypothesized that the antioxidant function of Bcl-2 is antagonized by its interaction with the BH3 domains of pro-apoptotic family members. Here, we show that BH3 mimetics that bind to a hydrophobic surface (the BH3 groove) of Bcl-2 induce GSH-sensitive mitochondrial dysfunction and apoptosis in cerebellar granule neurons. BH3 mimetics displace a discrete mitochondrial GSH pool in neurons and suppress GSH transport into isolated rat brain mitochondria. Moreover, BH3 mimetics and the BH3-only protein, Bim, inhibit a novel interaction between Bcl-2 and GSH in vitro. These results suggest that Bcl-2 regulates an essential pool of mitochondrial GSH and that this regulation may depend upon Bcl-2 directly interacting with GSH via the BH3 groove. We conclude that this novel GSH binding property of Bcl-2 likely plays a central role in its antioxidant function at mitochondria. PMID:17690097

  3. MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199

    PubMed Central

    Benito, Juliana M.; Godfrey, Laura; Kojima, Kensuke; Hogdal, Leah; Wunderlich, Mark; Geng, Huimin; Marzo, Isabel; Harutyunyan, Karine G.; Golfman, Leonard; North, Phillip; Kerry, Jon; Ballabio, Erica; Chonghaile, Triona Ní; Gonzalo, Oscar; Qiu, Yihua; Jeremias, Irmela; Debose, LaKiesha; O’Brien, Eric; Ma, Helen; Zhou, Ping; Jacamo, Rodrigo; Park, Eugene; Coombes, Kevin R.; Zhang, Nianxiang; Thomas, Deborah A.; O’Brien, Susan; Kantarjian, Hagop M.; Leverson, Joel D.; Kornblau, Steven M.; Andreeff, Michael; Müschen, Markus; Zweidler-McKay, Patrick A.; Mulloy, James C.; Letai, Anthony; Milne, Thomas A.; Konopleva, Marina

    2015-01-01

    Summary Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias. PMID:26711339

  4. MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199.

    PubMed

    Benito, Juliana M; Godfrey, Laura; Kojima, Kensuke; Hogdal, Leah; Wunderlich, Mark; Geng, Huimin; Marzo, Isabel; Harutyunyan, Karine G; Golfman, Leonard; North, Phillip; Kerry, Jon; Ballabio, Erica; Chonghaile, Triona Ní; Gonzalo, Oscar; Qiu, Yihua; Jeremias, Irmela; Debose, LaKiesha; O'Brien, Eric; Ma, Helen; Zhou, Ping; Jacamo, Rodrigo; Park, Eugene; Coombes, Kevin R; Zhang, Nianxiang; Thomas, Deborah A; O'Brien, Susan; Kantarjian, Hagop M; Leverson, Joel D; Kornblau, Steven M; Andreeff, Michael; Müschen, Markus; Zweidler-McKay, Patrick A; Mulloy, James C; Letai, Anthony; Milne, Thomas A; Konopleva, Marina

    2015-12-29

    Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias. PMID:26711339

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

    PubMed

    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. PMID:26881825

  6. The BCL-2 family: key mediators of the apoptotic response to targeted anti-cancer therapeutics

    PubMed Central

    Hata, Aaron N.; Engelman, Jeffrey A.; Faber, Anthony C.

    2016-01-01

    The ability of cancer cells to suppress apoptosis is critical for carcinogenesis. The BCL-2 family proteins comprise the sentinel network that regulates the mitochondrial or intrinsic apoptotic response. Recent advances in our understanding of apoptotic signaling pathways have enabled methods to identify cancers that are “primed” to undergo apoptosis, and have revealed potential biomarkers that may predict which cancers will undergo apoptosis in response to specific therapies. Complementary efforts have focused on developing novel drugs that directly target anti-apoptotic BCL-2 family proteins. In this review, we summarize the current knowledge of the role of BCL-2 family members in cancer development and response to therapy, focusing on targeted therapeutics, recent progress in the development of apoptotic biomarkers, and therapeutic strategies designed to overcome deficiencies in apoptosis. PMID:25895919

  7. Heterogeneity of Bcl-2 expression in metastatic breast carcinoma.

    PubMed

    Subhawong, Andrea Proctor; Nassar, Hind; Halushka, Marc K; Illei, Peter B; Vang, Russell; Argani, Pedram

    2010-08-01

    Bcl-2 is an antiapoptotic protein that promotes cell survival, but also may block proliferation. In breast cancer, bcl-2 expression correlates with favorable prognosis and estrogen receptor (ER) positivity. However, experimental data have paradoxically suggested that bcl-2 promotes chemoresistance and metastasis. A direct and comprehensive comparison of bcl-2 expression between primary breast carcinomas and paired distant metastases has not been performed. We completed rapid autopsies on 17 patients with archived primary tumors and metastatic breast carcinoma, and created single-patient tissue microarrays containing each patient's primary tumor and matched metastases. Expression of bcl-2, ER, progesterone receptor, and HER-2 in primary tumors and matched metastases were compared by immunohistochemistry. All 11 ER-positive cases showed bcl-2 labeling in the primary tumor, whereas only 3 of 6 ER-negative cases did (P=0.029). In 10 cases, bcl-2 labeling in metastases was similar to that of the primary, although 3 cases showed significant variation among metastases. In six other cases, bcl-2 labeling was lost or significantly diminished in metastases. Five of the latter cases were Luminal A (ER-positive, HER-2-negative) primaries, three of which lost hormone receptors in metastases. Only 1 of 17 cases showed an increase in bcl-2 labeling in metastases compared with the paired primary tumor. In conclusion, bcl-2 is infrequently upregulated in metastatic breast carcinoma. Instead, downregulation of bcl-2 expression may occur in the setting of hormone therapy resistance. Our findings call into question the potential utility of anti-bcl-2 therapy in metastatic breast cancer. PMID:20495533

  8. The targeted inhibition of mitochondrial Hsp90 overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis

    SciTech Connect

    Yan, Chunlan; Oh, Joon Seok; Yoo, Seung Hee; Lee, Jee Suk; Yoon, Young Geol; Oh, Yoo Jin; Jang, Min Seok; Lee, Sang Yeob; Yang, Jun; Lee, Sang Hwa; Kim, Hye Young; Yoo, Young Hyun

    2013-01-01

    Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity, thus inducing the apoptotic pathway in the cells. Accordingly, G-TPP shows a potential as a promising drug for the treatment of cancer. A cell can die from different types of cell death such as apoptosis, necrosis, necroptosis, and autophagic cell death. In this study, we further investigated the mechanisms and modes of cell death in the G-TPP-treated Hep3B and U937 cell lines. We discovered that G-TPP kills the U937 cells through the apoptotic pathway and the overexpression of Bcl-2 significantly inhibits U937 cell death to G-TPP. We further discovered that G-TPP kills the Hep3B cells by activating necroptosis in combination with the partial activation of caspase-dependent apoptosis. Importantly, G-TPP overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. We also observed that G-TPP induces compensatory autophagy in the Hep3B cell line. We further found that whereas there is a Bcl-2-Beclin 1 interaction in response to G-TPP, silencing the beclin 1 gene failed to block LC3-II accumulation in the Hep3B cells, indicating that G-TPP triggers Beclin 1-independent protective autophagy in Hep3B cells. Taken together, these data reveal that G-TPP induces cell death through a combination of death pathways, including necroptosis and apoptosis, and overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. These findings are important for the therapeutic exploitation of necroptosis as an alternative cell death program to bypass the resistance to apoptosis. Highlights: ► G-TPP binds to mitochondrial Hsp90. ► G-TPP induces apoptosis in U937 human leukemia cancer cells. ► G-TPP induces combination of death pathways in Hep3B cell. ► G-TPP overcomes the resistance conferred by Bcl-2 in Hep3B cells via necroptosis. ► G-TPP triggers Beclin 1-independent

  9. HA14-1, a small molecule inhibitor of Bcl-2, bypasses chemoresistance in leukaemia cells.

    PubMed

    Oliver, Lisa; Mahé, Béatrice; Gréé, René; Vallette, François M; Juin, Philippe

    2007-06-01

    We analyzed the biological activity of HA14-1, a small organic compound inhibitor of Bcl-2, against established leukaemia cell lines and blasts from acute myeloid leukaemia (AML) patients. HA14-1 had a potent killing activity against the leukaemia cell line that expressed endogenous or ectopic Bcl-2. This activity was mostly caspase-independent and was not altered by the expression of a multidrug-resistant phenotype. Moreover, HA14-1 efficiently induced cell death in a broad spectrum of AML blasts but not in normal peripheral blood lymphocytes. Thus, single-agent regimens using Bcl-2 inhibitors such as HA14-1 may be advantageous in overcoming some forms of chemoresistance in AML. PMID:17224180

  10. Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2

    PubMed Central

    Yue, Wen; Zheng, Xi; Lin, Yong; Yang, Chung S.; Xu, Qing; Carpizo, Darren; Huang, Huarong; DiPaola, Robert S.; Tan, Xiang-Lin

    2015-01-01

    Metformin and aspirin have been studied extensively as cancer preventive or therapeutic agents. However, the effects of their combination on pancreatic cancer cells have not been investigated. Herein, we evaluated the effects of metformin and aspirin, alone or in combination, on cell viability, migration, and apoptosis as well as the molecular changes in mTOR, STAT3 and apoptotic signaling pathways in PANC-1 and BxPC3 cells. Metformin and aspirin, at relatively low concentrations, demonstrated synergistically inhibitory effects on cell viability. Compared to the untreated control or individual drug, the combination of metformin and aspirin significantly inhibited cell migration and colony formation of both PANC-1 and BxPC-3 cells. Metformin combined with aspirin significantly inhibited the phosphorylation of mTOR and STAT3, and induced apoptosis as measured by caspase-3 and PARP cleavage. Remarkably, metformin combined with aspirin significantly downregulated the anti-apoptotic proteins Mcl-1 and Bcl-2, and upregulated the pro-apoptotic proteins Bim and Puma, as well as interrupted their interactions. The downregulation of Mcl-1 and Bcl-2 was independent of AMPK or STAT3 pathway but partially through mTOR signaling and proteasome degradation. In a PANC-1 xenograft mouse model, we demonstrated that the combination of metformin and aspirin significantly inhibited tumor growth and downregulated the protein expression of Mcl-1 and Bcl-2 in tumors. Taken together, the combination of metformin and aspirin significantly inhibited pancreatic cancer cell growth in vitro and in vivo by regulating the pro- and anti-apoptotic Bcl-2 family members, supporting the continued investigation of this two drug combination as chemopreventive or chemotherapeutic agents for pancreatic cancer. PMID:26056043

  11. Molecular mechanisms of nutlin-induced apoptosis in multiple myeloma: evidence for p53-transcription-dependent and -independent pathways.

    PubMed

    Saha, Manujendra N; Jiang, Hua; Chang, Hong

    2010-09-15

    Multiple myeloma (MM) is an incurable plasma cell malignancy in which p53 is rarely mutated. Thus, activation of the p53 pathway by a small molecule inhibitor of the p53-MDM2 interaction, nutlin, in MM cells retaining wild type p53 is an attractive therapeutic strategy. Recently we reported that nutlin plus velcade (a proteasome inhibitor) displayed a synergistic response in MM. However, the mechanism of the p53-mediated apoptosis in MM has not been fully understood. Our data show that nutlin-induced apoptosis correlated with reduction in cell viability, upregulation of p53, p21 and MDM2 protein levels with a simultaneous increase in pro-apoptotic targets PUMA, Bax and Bak and downregulation of anti-apoptotic targets Bcl2 and survivin and activation of caspase in MM cells harboring wild type p53. Nutlin-induced apoptosis was inhibited when activation of caspase was blocked by the caspase inhibitor. Nutlin caused mitochondrial translocation of p53 where it binds with Bcl2, leading to cytochrome C release. Moreover, blocking the transcriptional arm of p53 by the p53-specific transcriptional inhibitor, pifithrin-α, not only inhibited nutlin-induced upregulation of p53-transcriptional targets but also augmented apoptosis in MM cells, suggesting an association of transcription-independent pathway of apoptosis. However, inhibitor of mitochondrial translocation of p53, PFT-μ, did not prevent nutlin-induced apoptosis, suggesting that the p53 transcription-dependent pathway was also operational in nutlin-induced apoptosis in MM. Our study provides the evidence that nutlin-induced apoptosis in MM cells is mediated by transcription-dependent and -independent pathways and supports further clinical evaluation of nutlin as a novel therapeutic agent in MM. PMID:20595817

  12. Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2

    PubMed Central

    Katz, Chen; Benyamini, Hadar; Rotem, Shahar; Lebendiker, Mario; Danieli, Tsafi; Iosub, Anat; Refaely, Hadar; Dines, Monica; Bronner, Vered; Bravman, Tsafrir; Shalev, Deborah E.; Rüdiger, Stefan; Friedler, Assaf

    2008-01-01

    We have characterized the molecular basis of the interaction between ASPP2 and Bcl-2, which are key proteins in the apoptotic pathway. The C-terminal ankyrin repeats and SH3 domain of ASPP2 (ASPP2Ank-SH3) mediate its interactions with the antiapoptotic protein Bcl-2. We used biophysical and computational methods to identify the interaction sites of Bcl-2 and its homologues with ASPP2. Using peptide array screening, we found that ASPP2Ank-SH3 binds two homologous sites in all three Bcl proteins tested: (i) the conserved BH4 motif, and (ii) a binding site for proapoptotic regulators. Quantitative binding studies revealed that binding of ASPP2Ank-SH3 to the Bcl-2 family members is selective at two levels: (i) interaction with Bcl-2-derived peptides is the tightest compared to peptides from the other family members, and (ii) within Bcl-2, binding of ASPP2Ank-SH3 to the BH4 domain is tightest. Sequence alignment of the ASPP2-binding peptides combined with binding studies of mutated peptides revealed that two nonconserved positions where only Bcl-2 contains positively charged residues account for its tighter binding. The experimental binding results served as a basis for docking analysis, by which we modeled the complexes of ASPP2Ank-SH3 with the full-length Bcl proteins. Using peptide arrays and quantitative binding studies, we found that Bcl-2 binds three loops in ASPP2Ank-SH3 with similar affinity, in agreement with our predicted model. Based on our results, we propose a mechanism in which ASPP2 induces apoptosis by inhibiting functional sites of the antiapoptotic Bcl-2 proteins. PMID:18719108

  13. High incidence of MYC and BCL2 abnormalities in mantle cell lymphoma, although only MYC abnormality predicts poor survival

    PubMed Central

    Li, Chengwen; Zhong, Shizhen; Chen, Weiwei; Li, Zengjun; Xiong, Wenjie; Liu, Wei; Liu, Enbin; Cui, Rui; Ru, Kun; Zhang, Peihong; Xu, Yan; An, Gang; Lv, Rui; Qi, Junyuan; Wang, Jianxiang; Cheng, Tao; Qiu, Lugui

    2015-01-01

    The incidence and prognostic role of MYC and BCL2 rearrangements in mature B-cell lymphomas have been extensively studied, except the infrequent mantle cell lymphoma (MCL). Here, we analyzed the MYC and BCL2 abnormalities and other cytogenetic aberrations by fluorescence in situ hybridization (FISH) in 50 MCL patients with bone marrow involvement. Eighteen patients (36.0%) had MYC gains and/or amplifications, and twelve patients (24.0%) had BCL2 gains and/or amplifications. Among the 18 patients with MYC abnormality, four had simultaneous MYC translocations, but no BCL2 translocation was detected among patients with BCL2 abnormality. Only two patients (4.0%) had both MYC and BCL2 abnormalities. The patients with a MYC abnormality had a significantly higher tumor burden, a higher percentage of medium/high risk MIPI group and genomic instability compared to those without this abnormality. However, no significant difference was observed between patients with or without a BCL2 abnormality in terms of clinical and cytogenetic factors. Patients with a MYC abnormality had poorer progress-free survival (PFS) (9.0 vs. 48.0 months, p = .000) and overall survival (OS) (12.0 vs. 94.5 months, p = .000), but the presence of a BCL2 abnormality did not significantly influence either PFS or OS. In multivariate analysis, the MYC abnormality was the independent adverse factor for both PFS and OS, and intensive chemotherapy did not improve the outcome of these patients. Thus, the presence of a MYC but not BCL2 abnormality predicted the poor survival of MCL patients, and a new treatment strategy should be developed for these patients. PMID:26517511

  14. Induction of apoptosis and bcl-2 expression in acute lymphoblastic leukaemia and non-Hodgkin's lymphoma in children.

    PubMed

    Pituch-Noworolska, A; Hajto, B; Balwierz, W; Klus, K

    2001-01-01

    bcl-2 expression is associated with the expression of the multidrug resistance molecule (p-gp) and the resistance of leukaemia cells to the induction of apoptosis. The activity of p-gp is the main mechanism of resistance of leukaemia cells to chemotherapy. This study assessed the induction of apoptosis of acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) blastic cells following in vitro treatment with dexamethasone (DXM), vincristine (VCR), and tumour necrosis factor (TNF) in relation to the expression of bcl-2 and p-gp. Common ALL (cALL; n = 24 patients), common ALL with co-expression of myeloid antigens (cALL + My; n = 9), ALL-T (n = 9), and NHL [n = 6 (T type, n = 2; B type, n = 4)] were included. The expression of bcl-2 and p-gp and apoptosis were assayed by flow cytometry. Spontaneous apoptosis was low (< 5%) in cALL and ALL-T and higher (> 8%) in NHL and cALL + My. A high frequency of bcl-2 expression was noted in cALL and cALL + My. A high frequency of p-gp expression was observed in cALL + My, ALL-T, and NHL. There was a reverse association between bcl-2 expression and spontaneous apoptosis. DXM-induced apoptosis was observed in 52.63%, TNF-induced in 42.85%, VCR-induced in 36.36%, and GM-CSF-induced in 33.3% of leukaemia and lymphoma cases. DXM and GM-CSF-driven apoptosis was reversibly associated with bcl-2-expression (bcl-2-dependent mechanism). VCR and TNF-driven apoptosis was not associated with bcl-2 expression, suggesting a different, bcl-2-independent, mechanism(s) of its induction. The in vitro induction of apoptosis was not associated with expression of p-gp. PMID:11855781

  15. Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy

    PubMed Central

    Strappazzon, Flavie; Vietri-Rudan, Matteo; Campello, Silvia; Nazio, Francesca; Florenzano, Fulvio; Fimia, Gian Maria; Piacentini, Mauro; Levine, Beth; Cecconi, Francesco

    2011-01-01

    BECLIN 1 is a central player in macroautophagy. AMBRA1, a BECLIN 1-interacting protein, positively regulates the BECLIN 1-dependent programme of autophagy. In this study, we show that AMBRA1 binds preferentially the mitochondrial pool of the antiapoptotic factor BCL-2, and that this interaction is disrupted following autophagy induction. Further, AMBRA1 can compete with both mitochondrial and endoplasmic reticulum-resident BCL-2 (mito-BCL-2 and ER-BCL-2, respectively) to bind BECLIN 1. Moreover, after autophagy induction, AMBRA1 is recruited to BECLIN 1. Altogether, these results indicate that, in normal conditions, a pool of AMBRA1 binds preferentially mito-BCL-2; after autophagy induction, AMBRA1 is released from BCL-2, consistent with its ability to promote BECLIN 1 activity. In addition, we found that the binding between AMBRA1 and mito-BCL-2 is reduced during apoptosis. Thus, a dynamic interaction exists between AMBRA1 and BCL-2 at the mitochondria that could regulate both BECLIN 1-dependent autophagy and apoptosis. PMID:21358617

  16. PPARα induces cell apoptosis by destructing Bcl2

    PubMed Central

    Xu, Ying; Gong, Xin; Zhang, Runyun; Zhou, Chenglin; Su, Zhaoliang; Jin, Jianhua; Shi, Haifeng; Shi, Juanjuan; Hou, Yongzhong

    2015-01-01

    PPARα belongs to the peroxisome-proliferator-activated receptors (PPARs) family, which plays a critical role in inhibiting cell proliferation and tumorigenesis, while the molecular mechanism is still unclear. Here we report that PPARα serves as an E3 ubiquitin ligase to govern Bcl2 protein stability. PPARα physically bound to Bcl2 protein. In this process, PPARα/C102 was critical for PPARα binding to BH3 domain of Bcl2, subsequently, PPARα transferred K48-linked polyubiquitin to lysine-22 site of Bcl2 resulting in its ubiquitination and proteasome-dependent degradation. Importantly, overexpression of PPARα enhanced cancer cell chemotherapy sensitivity. In contrast, silenced PPARα decreased this event. These findings revealed a novel mechanism of PPARα governed endogenous Bcl2 protein stability leading to reduced cancer cell chemoresistance, which provides a potential drug target for cancer treatment. PMID:26556865

  17. Bovine herpesvirus type 5 infection regulates Bax/BCL-2 ratio.

    PubMed

    Garcia, A F; Novais, J B; Antello, T F; Silva-Frade, C; Ferrarezi, M C; Flores, E F; Cardoso, T C

    2013-01-01

    Bovine herpesvirus 5 (BoHV-5) is an α-herpesvirus that causes neurological disease in young cattle and is also occasionally involved in reproductive disorders. Although there have been many studies of the apoptotic pathways induced by viruses belonging to the family Herpesviridae, there is little information about the intrinsic programmed cell death pathway in host-BoHV-5 interactions. We found that BoHV-5 is able to replicate in both mesenchymal and epithelial cell lines, provoking cytopathology that is characterized by cellular swelling and cell fusion. Viral antigens were detected in infected cells by immunofluorescence assay at 48 to 96 h post-infection (p.i.). At 48 to 72 h p.i., anti-apoptotic BCL-2 antigens were found at higher levels than Bax antigens; the latter is considered a pro-apoptotic protein. Infected cells had increased BCL-2 phenotype cells from 48 to 96 h p.i., based on flow cytometric analysis. At 48 to 96 h p.i., Bax mRNA was not expressed in any of the infected cell monolayers. In contrast, BCL-2 mRNA was found at high levels at all p.i. in both types of cells. BoHV-5 replication apparently modulates BCL-2 expression and gene transcription, enhancing production of virus progeny. PMID:24085451

  18. Clinical and pathological features of Burkitt lymphoma showing expression of BCL2--an analysis including gene expression in formalin-fixed paraffin-embedded tissue.

    PubMed

    Masqué-Soler, Neus; Szczepanowski, Monika; Kohler, Christian W; Aukema, Sietse M; Nagel, Inga; Richter, Julia; Siebert, Reiner; Spang, Rainer; Burkhardt, Birgit; Klapper, Wolfram

    2015-11-01

    The differential diagnosis between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) can be challenging. BL has been reported to express less BCL2 than DLBCL, but this issue has not been analysed systematically. BL expressing BCL2 can be considered to be MYC/BCL2 co-expressors, a feature that is associated with poorer outcome in DLBCL but that has not been correlated with outcome in BL so far. We analysed the expression of BCL2 in 150 cases of conventionally diagnosed BL using two different BCL2 antibodies. BCL2 expression was detected in 23% of the cases, though the expression varied in intensity and number of positive cells. We did not detect any relevant differences in clinical presentation and outcome between BCL2-positive and BCL2-negative BL in a subgroup of 43 cases for which detailed clinical data were available. An independent cohort of 17 BL with expression of BCL2 were analysed molecularly, with 13 of 17 cases classified as molecularly defined BL (Burkitt Lymphoma) using gene expression profiling on formalin-fixed paraffin-embedded tissues. The four lymphomas diagnosed molecularly as intermediates did not differ in clinical presentation and outcome from molecularly defined BL. PMID:26218299

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

    SciTech Connect

    Tesfaigzi, J.; Ray, L.M.; Hotchkiss, J.A.

    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.

  20. Bcl-2 expression and triple negative profile in breast carcinoma.

    PubMed

    Kallel-Bayoudh, Imen; Hassen, Hanen Ben; Khabir, Abdelmajid; Boujelbene, Noureddine; Daoud, Jamel; Frikha, Mounir; Sallemi-Boudawara, Tahia; Aifa, Sami; Rebaï, Ahmed

    2011-12-01

    Many biomarkers for breast cancer prognosis have been proposed during the last two decades, among which HER2 and oestrogen receptors are of common use in routine clinical practice. However, in recent years, BCL2 has been recognized as an important prognostic parameter in human breast cancer, although its clinical utility is well established. The aim of this study was to examine the protein expression patterns of BCL2, HER2, oestrogen (ER) and progesterone receptors (PR) and to evaluate their correlation with survival and other prognostic parameters such as tumour size, histological grade and metastasis. We used a retrospective study including 84 Tunisian women with breast cancer. Immunohistochemistry was used to measure protein expression levels of several biomarkers. Association with conventional biopathological factors was analysed by SPSS (version13). The expression rates of BCL2, HER2, ER and PR were, respectively, 69, 62, 58.3 and 51.2%. In univariate analyses, BCL2 was highly correlated with both PR (P < 0.001) and ER (P = 0.006) and also with HER2 expression (P = 0.001). The triple negative profile showed a significant association with SBR (P = 0.016) and BCL2 expression (P = 0.02). In multivariate analyses, a significant association was maintained between BCL2 and both PR and ER (P = 0.02 and P = 0.004, respectively). Survival analysis showed that BCL2 expression was positively correlated with patients survival (P = 0.032). A Bayesian network analysis of all the variables confirmed the high value of BCL2 expression as a predictor of survival. As conclusion, BCL2 expression seems to be a very useful factor that should be in combination with HER2 and ER in breast cancer prognosis. PMID:20890735

  1. Getting away with murder: how do the BCL-2 family of proteins kill with immunity?

    PubMed Central

    Renault, Thibaud T.; Chipuk, Jerry E.

    2013-01-01

    About 1 million per second is the number of white blood cells the adult human body produces. However, only a small fraction of them will survive as the majority is eliminated through a genetically controlled form of cell death referred to as apoptosis. This review places into perspective recent studies pertaining to the BCL-2 family of proteins as critical regulators of the development and function of the immune system, with particular attention on B cell and T cell biology. Here we discuss how elegant murine model systems have revealed the major contributions of the BCL-2 family in establishing an effective immune system. Moreover, we highlight some key regulatory pathways that influence the expression, function, and stability of individual BCL-2 family members, and discuss their role in immunity. From deadly methods to more gentle manners, the final portion of the review discusses the non-apoptotic functions of the BCL-2 family and how they pertain to the control of immunity. PMID:23527542

  2. TRAIL activates JNK and NF-κB through RIP1-dependent and -independent pathways.

    PubMed

    Zhang, Laiqun; Dittmer, Martin R; Blackwell, Ken; Workman, Lauren M; Hostager, Bruce; Habelhah, Hasem

    2015-02-01

    The death receptor (DR) ligand TRAIL is being evaluated in clinical trials as an anti-cancer agent; however, many studies have found that TRAIL also enhances tumor progression by activating the NF-κB pathway in apoptosis-resistant cells. Although RIP1, cFLIP and caspase-8 have been implicated in TRAIL-induced JNK and NF-κB activation, underlying mechanisms are unclear. By examining the kinetics of pathway activation in TRAIL-sensitive lymphoma cells wild-type or deficient for RIP1, TRAF2, cIAP1/2 or HOIP, we report here that TRAIL induces two phases of JNK and NF-κB activation. The early phase is activated by TRAF2- and cIAP1-mediated ubiquitination of RIP1, whereas the delayed phase is induced by caspase-dependent activation of MEKK1 independent of RIP1 and TRAF2 expression. cFLIP overexpression promotes the early phase but completely suppresses the delayed phase of pathway activation in lymphoma cells, whereas Bcl-2 overexpression promotes both the early and delayed phases of the pathways. In addition, stable overexpression of cFLIP in RIP1- or TRAF2-deficient cells confers resistance to apoptosis, but fails to mediate NF-κB activation. HOIP is not essential for, but contributes to, TRAIL-induced NF-κB activation in cFLIP-overexpressing cells. These findings not only elucidate details of the mechanisms underlying TRAIL-induced JNK and NF-κB activation, but also clarify conflicting reports in the field. PMID:25446254

  3. Primary Cutaneous Follicle Center Lymphomas Expressing BCL2 Protein Frequently Harbor BCL2 Gene Break and May Present 1p36 Deletion: A Study of 20 Cases.

    PubMed

    Szablewski, Vanessa; Ingen-Housz-Oro, Saskia; Baia, Maryse; Delfau-Larue, Marie-Helene; Copie-Bergman, Christiane; Ortonne, Nicolas

    2016-01-01

    The classification of cutaneous follicular lymphoma (CFL) into primary cutaneous follicle center lymphoma (PCFCL) or secondary cutaneous follicular lymphoma (SCFL) is challenging. SCFL is suspected when tumor cells express BCL2 protein, reflecting a BCL2 translocation. However, BCL2 expression is difficult to assess in CFLs because of numerous BCL2+ reactive T cells. To investigate these issues and to further characterize PCFCL, we studied a series of 25 CFLs without any extracutaneous disease at diagnosis, selected on the basis of BCL2 protein expression using 2 BCL2 antibodies (clones 124 and E17) and BOB1/BCL2 double immunostaining. All cases were studied using interphase fluorescence in situ hybridization with BCL2, BCL6, IGH, IGK, IGL breakapart, IGH-BCL2 fusion, and 1p36/1q25 dual-color probes. Nineteen CFLs were BCL2 positive, and 6 were negative. After a medium follow-up of 24 (6 to 96) months, 5 cases were reclassified as SCFL and were excluded from a part of our analyses. Among BCL2+ PCFCLs, 60% (9/15) demonstrated a BCL2 break. BCL2-break-positive cases had a tendency to occur in the head and neck and showed the classical phenotype of nodal follicular lymphoma (CD10+, BCL6+, BCL2+, STMN+) compared with BCL2-break-negative PCFCLs. Del 1p36 was observed in 1 PCFCL. No significant clinical differences were observed between BCL2+ or BCL2- PCFCL. In conclusion, we show that a subset of PCFCLs harbor similar genetic alterations, as observed in nodal follicular lymphomas, including BCL2 breaks and 1p36 deletion. As BCL2 protein expression is usually associated with the presence of a BCL2 translocation, fluorescence in situ hybridization should be performed to confirm this hypothesis. PMID:26658664

  4. Ceramide and glutathione define two independently regulated pathways of cell death initiated by p53 in Molt-4 leukaemia cells.

    PubMed Central

    El-Assaad, Wissal; Kozhaya, Lina; Araysi, Sawsan; Panjarian, Shoghag; Bitar, Fadi F; Baz, Elizabeth; El-Sabban, Marwan E; Dbaibo, Ghassan S

    2003-01-01

    The tumour suppressor p53 induces cell death by launching several pathways that are either dependent on or independent of gene transcription. Accumulation of the sphingolipid ceramide and reactive oxygen species are among these pathways. Crossregulation of these two pathways is possible owing to the demonstrated inhibition of neutral sphingomyelinase by glutathione, the predominant cellular antioxidant, and has been observed in some cytokine-dependent cell-death models. In a model of irradiation-induced cell death of Molt-4 leukaemia cells, it was found that ceramide accumulation and glutathione depletion were dependent on p53 up-regulation. The loss of p53 owing to expression of the papilloma virus E6 protein inhibited both pathways after irradiation. However, in this model, these two pathways appeared to be independently regulated on the basis of the following observations: (1) glutathione supplementation or depletion did not alter irradiation-induced ceramide accumulation, (2) exogenous ceramide treatment did not induce glutathione depletion, (3) glutathione depletion was dependent on new protein synthesis, whereas ceramide accumulation was independent of it and (4) caspase activation was required for ceramide accumulation but not for glutathione depletion. Furthermore, caspase 9 activation, which is dependent on the release of mitochondrial cytochrome c, was not required for ceramide accumulation. This suggested that a caspase, other than caspase 9, was necessary for ceramide accumulation. Interestingly, Bcl-2 expression inhibited these pathways, indicating a possible role for mitochondria in regulating both pathways. These findings indicate that these two pathways exhibit cross-regulation in cytokine-dependent, but not in p53-dependent, cell-death models. PMID:12967322

  5. Hyperammonia induces specific liver injury through an intrinsic Ca2+-independent apoptosis pathway

    PubMed Central

    2014-01-01

    Background Numerous pathological processes that affect liver function in patients with liver failure have been identified. Among them, hyperammonia is one of the most common phenomena.The purpose of this study was to determine whether hyperammonia could induced specific liver injury. Methods Hyperammonemic cells were established using NH4Cl. The cells were assessed by MTT, ELISA, and flow cytometric analyses. The expression levels of selected genes and proteins were confirmed by quantitative RT-PCR and western blot analyses. Results The effects of 20 mM NH4Cl pretreatment on the cell proliferation and apoptosis of primary hepatocytes and other cells were performed by MTT assays and flow cytometric analyses. Significant increasing in cytotoxicity and apoptosis were only observed in hepatocytes. The cell damage was reduced after adding BAPTA-AM but unchanged after adding EGTA. The expression levels of caspase-3, cytochrome C, calmodulin, and inducible nitric oxide synthase were increased and that of bcl-2 was reduced. The Na+-K+-ATPase activities in hyperammonia liver cells was no signiaficant difference compaired with the control group, but was decreased in astrocytes. NH4Cl pretreatment of primary hepatocytes promoted the activation of mitochondrial permeability transition pores and the mitochondria swelled irregularly. Conclusions Hyperammonia induces specific liver injury through an intrinsic Ca2+-independent apoptosis pathway. PMID:25145683

  6. The rheostat in the membrane: BCL-2 family proteins and apoptosis

    PubMed Central

    Volkmann, N; Marassi, F M; Newmeyer, D D; Hanein, D

    2014-01-01

    Apoptosis, a mechanism for programmed cell death, has key roles in human health and disease. Many signals for cellular life and death are regulated by the BCL-2 family proteins and converge at mitochondria, where cell fate is ultimately decided. The BCL-2 family includes both pro-life (e.g. BCL-XL) and pro-death (e.g. BAX, BAK) proteins. Previously, it was thought that a balance between these opposing proteins, like a simple ‘rheostat', could control the sensitivity of cells to apoptotic stresses. Later, this rheostat concept had to be extended, when it became clear that BCL-2 family proteins regulate each other through a complex network of bimolecular interactions, some transient and some relatively stable. Now, studies have shown that the apoptotic circuitry is even more sophisticated, in that BCL-2 family interactions are spatially dynamic, even in nonapoptotic cells. For example, BAX and BCL-XL can shuttle between the cytoplasm and the mitochondrial outer membrane (MOM). Upstream signaling pathways can regulate the cytoplasmic–MOM equilibrium of BAX and thereby adjust the sensitivity of cells to apoptotic stimuli. Thus, we can view the MOM as the central locale of a dynamic life–death rheostat. BAX invariably forms extensive homo-oligomers after activation in membranes. However, recent studies, showing that activated BAX monomers determine the kinetics of MOM permeabilization (MOMP), perturb the lipid bilayer and form nanometer size pores, pose questions about the role of the oligomerization. Other lingering questions concern the molecular mechanisms of BAX redistribution between MOM and cytoplasm and the details of BAX/BAK–membrane assemblies. Future studies need to delineate how BCL-2 family proteins regulate MOMP, in concert with auxiliary MOM proteins, in a dynamic membrane environment. Technologies aimed at elucidating the structure and function of the full-length proteins in membranes are needed to illuminate some of these critical issues. PMID

  7. Photoreactive Stapled BH3 Peptides to Dissect the BCL-2 Family Interactome

    PubMed Central

    Braun, Craig R.; Mintseris, Julian; Gavathiotis, Evripidis; Bird, Gregory H.; Gygi, Steven P.; Walensky, Loren D.

    2010-01-01

    SUMMARY Defining protein interactions forms the basis for discovery of biological pathways, disease mechanisms, and opportunities for therapeutic intervention. To harness the robust binding affinity and selectivity of structured peptides for interactome discovery, we engineered photoreactive stapled BH3 peptide helices that covalently capture their physiologic BCL-2 family targets. The crosslinking α-helices covalently trap both static and dynamic protein interactors, and enable rapid identification of interaction sites, providing a critical link between interactome discovery and targeted drug design. PMID:21168768

  8. TIMP-1 Inhibits Apoptosis in Lung Adenocarcinoma Cells via Interaction with Bcl-2

    PubMed Central

    Kutiyanawalla, Ammar; Gayatri, Sitaram; Lee, Byung Rho; Jiwani, Shahanawaz; Rojiani, Amyn M.; Rojiani, Mumtaz V.

    2015-01-01

    Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target. PMID:26366732

  9. AT-101 simultaneously triggers apoptosis and a cytoprotective type of autophagy irrespective of expression levels and the subcellular localization of Bcl-xL and Bcl-2 in MCF7 cells.

    PubMed

    Antonietti, P; Gessler, F; Düssmann, H; Reimertz, C; Mittelbronn, M; Prehn, J H M; Kögel, D

    2016-04-01

    The effects of autophagy on cell death are highly contextual and either beneficial or deleterious. One prime example for this dual function of autophagy is evidenced by the cell responses to the BH3 mimetic AT-101 that is known to induce either apoptotic or autophagy-dependent cell death in different settings. Based on previous reports, we hypothesized that the expression levels of pro-survival Bcl-2 family members may be key determinants for the respective death mode induced by AT-101. Here we investigated the role of autophagy in the response of MCF7 breast cancer cells to AT-101. AT-101 treatment induced a prominent conversion of LC3-I to LC3-II and apoptotic cell death characterized by the appearance of Annexin-positive/PI-negative early apoptotic cells and PARP cleavage. Inhibition of the autophagy pathway, either through application of 3-MA or by lentiviral knockdown of ATG5, strongly potentiated cell death, indicating a pro-survival function of autophagy. Overexpression of wild type Bcl-xL significantly diminished the net amount of AT-101-induced cell death, but failed to alter the death-enhancing effects of the ATG5 knockdown. This was also observed with the organelle-specific variants Bcl-xL-ActA and Bcl-2-ActA (mitochondrial) as well as Bcl-xL-cb5 and Bcl-2-cb5 (ER) which all reduced AT-101-induced cell death, but did not affect the death-enhancing effects of 3-MA. Collectively, our data indicate that in apoptosis-proficient MCF7 cells, AT-101 triggers Bcl-2- and Bcl-xL-dependent apoptosis and a cytoprotective autophagy response that is independent of the expression and subcellular localization of Bcl-xL and Bcl-2. PMID:26721623

  10. Born to be Alive: A Role for the BCL-2 Family in Melanoma Tumor Cell Survival, Apoptosis, and Treatment

    PubMed Central

    Anvekar, Rina A.; Asciolla, James J.; Missert, Derek J.; Chipuk, Jerry E.

    2011-01-01

    The global incidence of melanoma has dramatically increased during the recent decades, yet the advancement of primary and adjuvant therapies has not kept a similar pace. The development of melanoma is often centered on cellular signaling that hyper-activates survival pathways, while inducing a concomitant blockade to cell death. Aberrations in cell death signaling not only promote tumor survival and enhanced metastatic potential, but also create resistance to anti-tumor strategies. Chemotherapeutic agents target melanoma tumor cells by inducing a form of cell death called apoptosis, which is governed by the BCL-2 family of proteins. The BCL-2 family is comprised of anti-apoptotic proteins (e.g., BCL-2, BCL-xL, and MCL-1) and pro-apoptotic proteins (e.g., BAK, BAX, and BIM), and their coordinated regulation and function are essential for optimal responses to chemotherapeutics. Here we will discuss what is currently known about the mechanisms of BCL-2 family function with a focus on the signaling pathways that maintain melanoma tumor cell survival. Importantly, we will critically evaluate the literature regarding how chemotherapeutic strategies directly impact on BCL-2 family function and offer several suggestions for future regimens to target melanoma and enhance patient survival. PMID:22268005

  11. Characterizing Bcl-2 Family Protein Conformation and Oligomerization Using Cross-Linking and Antibody Gel-Shift in Conjunction with Native PAGE.

    PubMed

    Dewson, Grant

    2016-01-01

    The Bcl-2 family of proteins tightly controls the intrinsic or mitochondrial pathway of apoptosis. This family is subdivided based on function into pro-survival proteins (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Bfl-1/A1) and pro-apoptotic proteins. The pro-apoptotic subset is further divided into those proteins that initiate the pathway, the BH3-only proteins (including Bim, Puma, Noxa, and Bid), and those that execute the pathway, Bak and Bax. Whether a cell lives or dies in response to apoptotic stress is determined by the interactions of the Bcl-2 family, which is in turn influenced by their conformation. We describe here a protocol to interrogate the interactions and conformation of the Bcl-2 family of proteins under native conditions. PMID:27108440

  12. Simultaneous Detection of Tumor Cell Apoptosis Regulators Bcl-2 and Bax through a Dual-Signal-Marked Electrochemical Immunosensor.

    PubMed

    Zhou, Shiwei; Wang, Yingying; Zhu, Jun-Jie

    2016-03-01

    B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) are often used to monitor the apoptosis of tumor cells and evaluate cancer drug effect. In this work, a novel sandwich-type dual-signal-marked electrochemical biosensor was fabricated for simultaneous detection of Bcl-2 and Bax proteins. Reduced graphene oxide (RGO) layers were used as substrate to immobilize Bcl-2 and Bax antibodies for further capturing target antigens. CdSeTe@CdS quantum dots (QDs) and Ag nanoclusters (NCs) with antibody modification and mesoporous silica amplification were used as signal probes, which were proportional to the amount of Bcl-2 and Bax antigens. Mesoporous SiO2 can provide a larger surface area, more effectively charged by ethylene imine polymer or poly(diallyldimethylammonium chloride) to adsorb more probes. The Bcl-2 and Bax proteins were determined indirectly by the detection of oxidation peak currents of Cd and Ag using anodic stripping voltammetry, showing a good linear relationship in the protein concentration range from 1 ng/mL to 250 ng/mL. The detection limit of trace protein level was ∼0.5 fmol. The biosensor was further introduced to investigate Bcl-2 and Bax expressions from nilotinib-treated chronic myeloid leukemia K562 cells. With the increase of drug dosage and incubation time, the up-regulation for Bax and down-regulation for Bcl-2 were observed, which indicated that the apoptosis level of K562 cells could be regulated by Bcl-2 family. The ratio of Bax/Bcl-2 was further calculated for evaluation of its drug effect and apoptosis level. The limited cell amount for detection reached less than 1 × 10(3) cells, much lower than traditional methods. Furthermore, completely independent detection step and stable acid solutions containing Ag(+) and Cd(2+) for long-time storage contribute to reducing the error from the sample differences and avoiding the potential errors from the photodegradation of fluorescent probes, enzymolysis of DNA, or inactivation of

  13. Photoreactive stapled peptides to identify and characterize BCL-2 family interaction sites by mass spectrometry.

    PubMed

    Lee, Susan; Braun, Craig R; Bird, Gregory H; Walensky, Loren D

    2014-01-01

    Protein interactions dictate a myriad of cellular activities that maintain health or cause disease. Dissecting these binding partnerships, and especially their sites of interaction, fuels the discovery of signaling pathways, disease mechanisms, and next-generation therapeutics. We previously applied all-hydrocarbon peptide stapling to chemically restore α-helical shape to bioactive motifs that become unfolded when taken out of context from native signaling proteins. For example, we developed stabilized alpha-helices of BCL-2 domains (SAHBs) to dissect and target protein interactions of the BCL-2 family, a critical network that regulates the apoptotic pathway. SAHBs are α-helical surrogates that bind both stable and transient physiologic interactors and have effectively uncovered novel sites of BCL-2 family protein interaction. To leverage stapled peptides for proteomic discovery, we describe our conversion of SAHBs into photoreactive agents that irreversibly capture their protein targets and facilitate rapid identification of the peptide helix binding sites. We envision that the development of photoreactive stapled peptides will accelerate the discovery of novel and unanticipated protein interactions and how they impact health and disease. PMID:24974285

  14. Bcl-2 family proteins as targets for anticancer drug design.

    PubMed

    Huang, Z

    2000-12-27

    Bcl-2 family proteins are key regulators of programmed cell death or apoptosis that is implicated in many human diseases, particularly cancer. In recent years, they have attracted intensive interest in both basic research to understand the fundamental principles of cell survival and cell death and drug discovery to develop a new class of anticancer agents. The Bcl-2 family includes both anti- and pro-apoptotic proteins with opposing biological functions in either inhibiting or promoting cell death. High expression of anti-apoptotic members such as Bcl-2 and Bcl-XL commonly found in human cancers contributes to neoplastic cell expansion and interferes with the therapeutic action of many chemotherapeutic drugs. The functional blockade of Bcl-2 or Bcl-XL could either restore the apoptotic process in tumor cells or sensitize these tumors for chemo- and radiotherapies. This article reviews the recent progress in the design and discovery of small molecules that block the anti-apoptotic function of Bcl-2 or Bcl-XL. These chemical inhibitors are effective modulators of apoptosis and promising leads for the further development of new anticancer agents. PMID:11426648

  15. A New G-Quadruplex with Hairpin Loop Immediately Upstream of the Human BCL2 P1 Promoter Modulates Transcription.

    PubMed

    Onel, Buket; Carver, Megan; Wu, Guanhui; Timonina, Daria; Kalarn, Salil; Larriva, Marti; Yang, Danzhou

    2016-03-01

    The abnormal overexpression of the BCL2 gene is associated with many human tumors. We found a new 28-mer G-quadruplex-forming sequence, P1G4, immediately upstream of the human BCL2 gene P1 promoter. The P1G4 is shown to be a transcription repressor using a promoter-driven luciferase assay; its inhibitory effect can be markedly enhanced by the G-quadruplex-interactive compound TMPyP4. G-quadruplex can readily form in the P1G4 sequence under physiological salt condition as shown by DMS footprinting. P1G4 and previously identified Pu39 G-quadruplexes appear to form independently in adjacent regions in the BCL2 P1 promoter. In the extended BCL2 P1 promoter region containing both Pu39 and P1G4, P1G4 appears to play a more dominant role in repressing the transcriptional activity. Using NMR spectroscopy, the P1G4 G-quadruplex appears to be a novel dynamic equilibrium of two parallel structures, one regular with two 1-nt loops and a 12-nt middle loop and another broken-strand with three 1-nt loops and a 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) conformation in the long loop. The dynamic equilibrium of two closely related structures and the unique hairpin loop conformation are specific to the P1G4 sequence and distinguish the P1G4 quadruplex from other parallel structures. The presence of P1G4 and Pu39 in adjacent regions of the BCL2 P1 promoter suggests a mechanism for precise regulation of BCL2 gene transcription. The unique P1G4 G-quadruplex may provide a specific target for small molecules to modulate BCL2 gene transcription. PMID:26841249

  16. Genomic alterations in BCL2L1 and DLC1 contribute to drug sensitivity in gastric cancer.

    PubMed

    Park, Hansoo; Cho, Sung-Yup; Kim, Hyerim; Na, Deukchae; Han, Jee Yun; Chae, Jeesoo; Park, Changho; Park, Ok-Kyoung; Min, Seoyeon; Kang, Jinjoo; Choi, Boram; Min, Jimin; Kwon, Jee Young; Suh, Yun-Suhk; Kong, Seong-Ho; Lee, Hyuk-Joon; Liu, Edison T; Kim, Jong-Il; Kim, Sunghoon; Yang, Han-Kwang; Lee, Charles

    2015-10-01

    Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. Recent high-throughput analyses of genomic alterations revealed several driver genes and altered pathways in GC. However, therapeutic applications from genomic data are limited, largely as a result of the lack of druggable molecular targets and preclinical models for drug selection. To identify new therapeutic targets for GC, we performed array comparative genomic hybridization (aCGH) of DNA from 103 patients with GC for copy number alteration (CNA) analysis, and whole-exome sequencing from 55 GCs from the same patients for mutation profiling. Pathway analysis showed recurrent alterations in the Wnt signaling [APC, CTNNB1, and DLC1 (deleted in liver cancer 1)], ErbB signaling (ERBB2, PIK3CA, and KRAS), and p53 signaling/apoptosis [TP53 and BCL2L1 (BCL2-like 1)] pathways. In 18.4% of GC cases (19/103), amplification of the antiapoptotic gene BCL2L1 was observed, and subsequently a BCL2L1 inhibitor was shown to markedly decrease cell viability in BCL2L1-amplified cell lines and in similarly altered patient-derived GC xenografts, especially when combined with other chemotherapeutic agents. In 10.9% of cases (6/55), mutations in DLC1 were found and were also shown to confer a growth advantage for these cells via activation of Rho-ROCK signaling, rendering these cells more susceptible to a ROCK inhibitor. Taken together, our study implicates BCL2L1 and DLC1 as potential druggable targets for specific subsets of GC cases. PMID:26401016

  17. BCL2 accelerates inflammation-induced BALB/c plasmacytomas and promotes novel tumors with coexisting T(12;15) and T(6;15) translocations.

    PubMed

    Silva, Santiago; Kovalchuk, Alexander L; Kim, Joong Su; Klein, George; Janz, Siegfried

    2003-12-15

    Previous studies on peritoneal plasmacytomas (PCTs) in BALB/c (C) mice suggested that the enforced expression of the death repressor BCL2 in B cells might facilitate the malignant transformation of aberrant B cells containing Myc-activating T(12;15) translocations, generating an improved model of plasmacytomagenesis. To investigate this hypothesis, we backcrossed a human BCL2 transgene onto strain C and performed a PCT induction study with pristane in the newly generated C.BCL2 congenics. In specific pathogen-free-maintained C.BCL2 mice, PCT incidence (19 of 34, 56%) was 24 times higher than in specific pathogen-free-maintained C mice (1 of 44, 2.3%), and tumor onset (113 days) was half that of conventionally maintained C mice (220 days). BCL2 transgenic PCT harbored T(12;15) translocations (12 of 12 tumors) with an unusual clustering of translocation breakpoints in the near 5' flank of Myc (4 of 5 tumors, 80%). Five tumors contained coexisting T(12;15) and T(6;15) translocations (not observed in >300 karyotyped PCTs from conventionally maintained C mice). BCL2 transgenic C57BL/6 mice exclusively developed B lymphomas (11 of 20, 55%) that also contained T(12;15) translocations (11 of 11 cases) with breakpoints in the near 5' flank of Myc (five of five tumors). We conclude that BCL2 accelerates PCT with novel Myc-activating translocations independently of environmental antigen stimulation. Accelerated plasmacytomagenesis in strain C.BCL2 may be useful for designing and testing BCL2 inhibition strategies in human plasma cell tumors overexpressing BCL2, such as Waldenström's macroglobulinemia and multiple myeloma. PMID:14695177

  18. Hormone-independent pathways of sexual differentiation.

    PubMed

    Renfree, Marilyn B; Chew, Keng Yih; Shaw, Geoffrey

    2014-01-01

    New observations over the last 25 years of hormone-independent sexual dimorphisms have gradually and unequivocally overturned the dogma, arising from Jost's elegant experiments in the mid-1900s, that all somatic sex dimorphisms in vertebrates arise from the action of gonadal hormones. Although we know that Sry, a Y-linked gene, is the primary gonadal sex determinant in mammals, more recent analysis in marsupials, mice, and finches has highlighted numerous sexual dimorphisms that are evident well before the differentiation of the testis and which cannot be explained by a sexually dimorphic hormonal environment. In marsupials, scrotal bulges and mammary primordia are visible before the testis has differentiated due to the expression of a gene(s) on the X chromosome. ZZ and ZW gynandromorph finches have brains that develop in a sexually dimorphic way dependent on their sex chromosome content. In genetically manipulated mice, it is the X chromosomes, not the gonads, that determine many characters including rate of early development, adiposity, and neural circuits. Even spotted hyenas have sexual dimorphisms that cannot be simply explained by hormonal exposure. This review discusses the recent findings that confirm that there are hormone-independent sexual dimorphisms well before the gonads begin to produce their hormones. PMID:24577198

  19. Bcl-2-regulated apoptosis: mechanism and therapeutic potential.

    PubMed

    Adams, Jerry M; Cory, Suzanne

    2007-10-01

    Apoptosis is essential for tissue homeostasis, particularly in the hematopoietic compartment, where its impairment can elicit neoplastic or autoimmune diseases. Whether stressed cells live or die is largely determined by interplay between opposing members of the Bcl-2 protein family. Bcl-2 and its closest homologs promote cell survival, but two other factions promote apoptosis. The BH3-only proteins sense and relay stress signals, but commitment to apoptosis requires Bax or Bak. The BH3-only proteins appear to activate Bax and Bak indirectly, by engaging and neutralizing their pro-survival relatives, which otherwise constrain Bax and Bak from permeabilizing mitochondria. The Bcl-2 family may also regulate autophagy and mitochondrial fission/fusion. Its pro-survival members are attractive therapeutic targets in cancer and perhaps autoimmunity and viral infections. PMID:17629468

  20. BCL-2 family proteins as regulators of mitochondria metabolism.

    PubMed

    Gross, Atan

    2016-08-01

    The BCL-2 family proteins are major regulators of apoptosis, and one of their major sites of action are the mitochondria. Mitochondria are the cellular hubs for metabolism and indeed selected BCL-2 family proteins also possess roles related to mitochondria metabolism and dynamics. Here we discuss the link between mitochondrial metabolism/dynamics and the fate of stem cells, with an emphasis on the role of the BID-MTCH2 pair in regulating this link. We also discuss the possibility that BCL-2 family proteins act as metabolic sensors/messengers coming on and off of mitochondria to "sample" the cytosol and provide the mitochondria with up-to-date metabolic information. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26827940

  1. The Bcl-2-regulated apoptosis switch: mechanism and therapeutic potential

    PubMed Central

    Adams, Jerry M; Cory, Suzanne

    2009-01-01

    Apoptosis is essential for tissue homeostasis, particularly in the hematopoietic compartment, where its impairment can elicit neoplastic or autoimmune diseases. Whether stressed cells live or die is largely determined by interplay between opposing members of the Bcl-2 protein family. Bcl-2 and its closest homologs promote cell survival, but two other factions promote apoptosis. The BH3-only proteins sense and relay stress signals, but commitment to apoptosis requires Bax or Bak. The BH3-only proteins appear to activate Bax and Bak indirectly, by engaging and neutralizing their pro-survival relatives, which otherwise constrain Bax and Bak from permeabilizing mitochondria. The Bcl-2 family may also regulate autophagy and mitochondrial fission/fusion. Its pro-survival members are attractive therapeutic targets in cancer and perhaps autoimmunity and viral infections. PMID:17629468

  2. Selective targeting of BCL6 induces oncogene addiction switching to BCL2 in B-cell lymphoma

    PubMed Central

    Patel, Jayeshkumar; Hatzi, Katerina; Malik, Alka; Tam, Wayne; Martin, Peter; Leonard, John; Melnick, Ari; Cerchietti, Leandro

    2016-01-01

    The BCL6 oncogene plays a crucial role in sustaining diffuse large B-cell lymphomas (DLBCL) through transcriptional repression of key checkpoint genes. BCL6-targeted therapy kills lymphoma cells by releasing these checkpoints. However BCL6 also directly represses several DLBCL oncogenes such as BCL2 and BCL-XL that promote lymphoma survival. Herein we show that DLBCL cells that survive BCL6-targeted therapy induce a phenomenon of “oncogene-addiction switching” by reactivating BCL2-family dependent anti-apoptotic pathways. Thus, most DLBCL cells require concomitant inhibition of BCL6 and BCL2-family members for effective lymphoma killing. Moreover, in DLBCL cells initially resistant to BH3 mimetic drugs, BCL6 inhibition induces a newly developed reliance on anti-apoptotic BCL2-family members for survival that translates in acquired susceptibility to BH3 mimetic drugs ABT-737 and obatoclax. In germinal center B cell-like (GCB)-DLBCL cells, the proteasome inhibitor bortezomib and the NEDD inhibitor MLN4924 post-transcriptionally activated the BH3-only sensitizer NOXA thus counteracting the oncogenic switch to BCL2 induced by BCL6-targeting. Hence our study indicates that BCL6 inhibition induces an on-target feedback mechanism based on the activation of anti-apoptotic BH3 members. This oncogene-addition switching mechanism was harnessed to develop rational combinatorial therapies for GCB-DLBCL. PMID:26657288

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

    PubMed Central

    Kouri, Fotini M.; Jensen, Samuel A.; Stegh, Alexander H.

    2012-01-01

    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. PMID:22431925

  4. Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of anti-apoptotic factors.

    PubMed

    Zhu, Yi; Tchkonia, Tamara; Fuhrmann-Stroissnigg, Heike; Dai, Haiming M; Ling, Yuanyuan Y; Stout, Michael B; Pirtskhalava, Tamar; Giorgadze, Nino; Johnson, Kurt O; Giles, Cory B; Wren, Jonathan D; Niedernhofer, Laura J; Robbins, Paul D; Kirkland, James L

    2016-06-01

    Clearing senescent cells extends healthspan in mice. Using a hypothesis-driven bioinformatics-based approach, we recently identified pro-survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro-survival regulators identified was Bcl-xl. Here, we tested whether the Bcl-2 family inhibitors, navitoclax (N) and TW-37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl-xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl-2, Bcl-xl, and Bcl-w, while T targets Bcl-2, Bcl-xl, and Mcl-1. The combination of Bcl-2, Bcl-xl, and Bcl-w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl-2, Bcl-xl, and Mcl-1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl-2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type-restricted manner. The hypothesis-driven, bioinformatics-based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type-specific senolytic agents. PMID:26711051

  5. Bcl-2 antagonists interact synergistically with bortezomib in DLBCL cells in association with JNK activation and induction of ER stress.

    PubMed

    Dasmahapatra, Girija; Lembersky, Dmitry; Rahmani, Mohamed; Kramer, Lora; Friedberg, Jonathan; Fisher, Richard I; Dent, Paul; Grant, Steven

    2009-05-01

    Mechanisms underlying interactions between the proteasome inhibitor bortezomib and small molecule Bcl-2 antagonists were examined in GC- and ABC-type human DLBCL (diffuse lymphocytic B-cell lymphoma) cells. Concomitant or sequential exposure to non- or minimally toxic concentrations of bortezomib or other proteasome inhibitors and either HA14-1 or gossypol resulted in a striking increase in Bax/Bak conformational change/translocation, cytochrome c release, caspase activation and synergistic induction of apoptosis in both GC- and ABC-type cells. These events were associated with a sharp increase in activation of the stress kinase JNK and evidence of ER stress induction (e.g., eIF2alpha phosphorylation, activation of caspases-2 and -4, and Grp78 upregulation). Pharmacologic or genetic (e.g., shRNA knockdown) interruption of JNK signaling attenuated HA14-1/bortezomib lethality and ER stress induction. Genetic disruption of the ER stress pathway (e.g., in cells expressing caspase-4 shRNA or DN-eIF2alpha) significantly attenuated lethality. The toxicity of this regimen was independent of ROS generation. Finally, HA14-1 significantly increased bortezomib-mediated JNK activation, ER stress induction, and lethality in bortezomib-resistant cells. Collectively these findings indicate that small molecule Bcl-2 antagonists promote bortezomib-mediated mitochondrial injury and lethality in DLBCL cells in association with enhanced JNK activation and ER stress induction. They also raise the possibility that such a strategy may be effective in different DLBCL sub-types (e.g., GC- or ABC), and in bortezomib-resistant disease. PMID:19270531

  6. Bcl-2 antagonists interact synergistically with bortezomib in DLBCL cells in association with JNK activation and induction of ER stress

    PubMed Central

    Dasmahapatra, Girija; Lembersky, Dmitry; Rahmani, Mohamed; Kramer, Lora; Friedberg, Jonathan; Fisher, Richard I.; Dent, Paul; Grant, Steven

    2010-01-01

    Mechanisms underlying interactions between the proteasome inhibitor bortezomib and small molecule Bcl-2 antagonists were examined in GC- and ABC-type human DLBCL (diffuse lymphocytic B-cell lymphoma) cells. Concomitant or sequential exposure to non- or minimally toxic concentrations of bortezomib or other proteasome inhibitors and either HA14-1 or gossypol resulted in a striking increase in Bax/Bak conformational change/translocation, cytochrome c release, caspase activation and synergistic induction of apoptosis in both GC- and ABC-type cells. These events were associated with a sharp increase in activation of the stress kinase JNK and evidence of ER stress induction (e.g., eIF2α phosphorylation, activation of caspases-2 and -4, and Grp78 upregulation). Pharmacologic or genetic (e.g., shRNA knockdown) interruption of JNK signaling attenuated HA14-1/bortezomib lethality and ER stress induction. Genetic disruption of the ER stress pathway (e.g., in cells expressing caspase-4 shRNA or DN-eIF2α) significantly attenuated lethality. The toxicity of this regimen was independent of ROS generation. Finally, HA14-1 significantly increased bortezomib-mediated JNK activation, ER stress induction, and lethality in bortezomib-resistant cells. Collectively these findings indicate that small molecule Bcl-2 antagonists promote bortezomib-mediated mitochondrial injury and lethality in DLBCL cells in association with enhanced JNK activation and ER stress induction. They also raise the possibility that such a strategy may be effective in different DLBCL sub-types (e.g., GC- or ABC), and in bortezomib-resistant disease. PMID:19270531

  7. Inhibition of PI3K signaling triggered apoptotic potential of curcumin which is hindered by Bcl-2 through activation of autophagy in MCF-7 cells.

    PubMed

    Akkoç, Yunus; Berrak, Özge; Arısan, Elif Damla; Obakan, Pınar; Çoker-Gürkan, Ajda; Palavan-Ünsal, Narçin

    2015-04-01

    Curcumin is a natural anti-cancer agent derived from turmeric (Curcuma longa). Curcumin triggers intrinsic apoptotic cell death by activating mitochondrial permeabilization due to the altered expression of pro- and anti-apoptotic Bcl-2 family members. Phosphoinositol-3-kinase (PI3K) and Akt, key molecular players in the survival mechanism, have been shown to be associated with the Bcl-2 signaling cascade; therefore, evaluating the therapeutic efficiency of drugs that target both survival and the apoptosis mechanism has gained importance in cancer therapy. We found that Bcl-2 overexpression is a limiting factor for curcumin-induced apoptosis in highly metastatic MCF-7 breast cancer cells. Forced overexpression of Bcl-2 also blocked curcumin-induced autophagy in MCF-7 cells, through its inhibitory interactions with Beclin-1. Pre-treatment of PI3K inhibitor LY294002 enhanced curcumin-induced cell death, apoptosis, and autophagy via modulating the expression of Bcl-2 family members and autophagosome formation in MCF-7 breast cancer cells. Atg7 silencing further increased apoptotic potential of curcumin in the presence or absence of LY294002 in wt and Bcl-2+ MCF-7 cells. The findings of this study support the hypothesis that blocking the PI3K/Akt pathway may further increased curcumin-induced apoptosis and overcome forced Bcl-2 expression level mediated autophagic responses against curcumin treatment in MCF-7 cells. PMID:25960232

  8. Artesunate induces apoptosis via a ROS-independent and Bax-mediated intrinsic pathway in HepG2 cells.

    PubMed

    Qin, Guiqi; Wu, Liping; Liu, Hongyu; Pang, Yilin; Zhao, Chubiao; Wu, Shengnan; Wang, Xiaoping; Chen, Tongsheng

    2015-08-15

    This study aims to explore the detail molecular mechanism by which artesunate (ARS), an artemisinin derivative, induces apoptosis in HepG2 cells. ARS induced a loss of mitochondrial transmemberane potential (ΔΨm), phosphatidylserine (PS) externalization, as well as activations of Bax/Bak and caspases indicative of apoptosis induction. Silencing Bax but not Bak significantly inhibited ARS-induced apoptosis, demonstrating the key role of the Bax-mediated intrinsic pathway. Although ARS increased intracellular reactive oxygen species (ROS), ARS-induced apoptosis was neither prevented by pretreatment with ROS scavengers nor potentiated by pretreatment with l-buthionine-sulfoximine (BSO) that enhanced the ARS-induced intracellular ROS generation, demonstrating that ROS was not involved in ARS-induced apoptosis. In addition, ARS did not induce Bid translocation to mitochondria, and the cytotoxicity of ARS was not prevented by silencing Bim, Puma or Mcl-1, but was significantly enhanced by HA14-1 pretreatment, demonstrating that Bcl-2/-xl instead of Bid and Bim as well as Puma may be the upstream factor to regulate the Bax-mediated intrinsic pathway. Collectively, our data demonstrate that ARS induces ROS-independent apoptosis via the Bax-mediated intrinsic pathway in HepG2 cells. PMID:26163896

  9. Endothelium Expression of Bcl-2 Is Essential for Normal and Pathological Ocular Vascularization.

    PubMed

    Zaitoun, Ismail S; Johnson, Ryan P; Jamali, Nasim; Almomani, Reem; Wang, Shoujian; Sheibani, Nader; Sorenson, Christine M

    2015-01-01

    Bcl-2 is an anti-apoptotic protein with important roles in vascular homeostasis and angiogenesis. Mice globally lacking Bcl-2 (Bcl-2 -/-) are small in stature and succumb to renal failure shortly after weaning as a result of renal hypoplasia/cystic dysplasia. We have shown that Bcl-2 -/- mice displayed attenuated retinal vascular development and neovascularization. In vitro studies indicated that in addition to modulating apoptosis, Bcl-2 expression also impacts endothelial and epithelial cell adhesion, migration and extracellular matrix production. However, studies delineating the cell autonomous role Bcl-2 expression plays in the endothelium during vascular development, pruning and remodeling, and neovascularization are lacking. Here we generated mice carrying a conditional Bcl-2 allele (Bcl-2Flox/Flox) and VE-cadherin-cre (Bcl-2EC mice). Bcl-2EC mice were of normal stature and lifespan and displayed some but not all of the retinal vascular defects previously observed in global Bcl-2 deficient mice. Bcl-2EC mice had decreased numbers of endothelial cells, decreased retinal arteries and premature primary branching of the retinal vasculature, but unlike the global knockout mice, spreading of the retinal superficial vascular layer proceeded normally. Choroidal neovascularization was attenuated in Bcl-2EC mice, although retinal neovascularization accompanying oxygen-induced ischemic retinopathy was not. Thus, Bcl-2 expression in the endothelium plays a significant role during postnatal retinal vascularization, and pathological choroidal but not retinal neovascularization, suggesting vascular bed specific Bcl-2 function in the endothelium. PMID:26444547

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

    SciTech Connect

    Jauharoh, Siti Nur Aisyah; Saegusa, Jun; Sugimoto, Takeshi; Ardianto, Bambang; Kasagi, Shimpei; Sugiyama, Daisuke; Kurimoto, Chiyo; Tokuno, Osamu; Nakamachi, Yuji; Kumagai, Shunichi; Kawano, Seiji

    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.

  11. Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells

    PubMed Central

    Trisciuoglio, D; Gabellini, C; Desideri, M; Ragazzoni, Y; De Luca, T; Ziparo, E; Del Bufalo, D

    2011-01-01

    In addition to act as an antiapoptotic protein, B-cell lymphoma (bcl)-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia-inducible factor-1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes, such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally, we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2. PMID:21233846

  12. Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells.

    PubMed

    Trisciuoglio, D; Gabellini, C; Desideri, M; Ragazzoni, Y; De Luca, T; Ziparo, E; Del Bufalo, D

    2011-06-01

    In addition to act as an antiapoptotic protein, B-cell lymphoma (bcl)-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia-inducible factor-1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes, such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally, we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2. PMID:21233846

  13. Imatinib enhances human melanoma cell susceptibility to TRAIL-induced cell death: Relationship to Bcl-2 family and caspase activation.

    PubMed

    Hamaï, A; Richon, C; Meslin, F; Faure, F; Kauffmann, A; Lecluse, Y; Jalil, A; Larue, L; Avril, M F; Chouaib, S; Mehrpour, M

    2006-12-01

    sensitivity in G1 cells, indicating that the expression level of c-FLIP(L) and its interaction with TRAIL receptor2 play a crucial role in determining TRAIL resistance in metastatic melanoma cells. Our results also show that imatinib enhances TRAIL-induced cell death independently of BH3-interacting domain death agonist translocation, in a process involving the Bax:Bcl-X(L) ratio, Bax:Bcl-X(L)/Bcl-2 translocation, cytochrome c release and caspase activation. Our data indicate that imatinib sensitizes T1 cells by directly downregulating c-FLIP(L), with the use of an alternative pathway for antitumor activity, because PDGFRalpha is not activated in T1 cells and these cells do not express c-kit, c-ABL or PDGFRbeta. Caspase cascade activation and mitochondria also play a key role in the imatinib-mediated sensitization of melanoma cells to the proapoptotic action of TRAIL. PMID:16983347

  14. Phosphorylation of paxillin confers cisplatin resistance in non-small cell lung cancer via activating ERK-mediated Bcl-2 expression.

    PubMed

    Wu, D-W; Wu, T-C; Wu, J-Y; Cheng, Y-W; Chen, Y-C; Lee, M-C; Chen, C-Y; Lee, H

    2014-08-28

    Paxillin (PXN) is required for receptor tyrosine kinase-mediated ERK activation, and the activation of the Raf/MEK/ERK cascade has been linked with Bcl-2 expression. We hypothesized that phosphorylation of PXN by the EGFR/Src pathway might contribute to cisplatin resistance via increased Bcl-2 expression. We show that cisplatin resistance was dependent on PXN expression, as evidenced by PXN overexpression in TL-13 and TL-10 cells and PXN knockdown in H23 and CL1-5 cells. Specific inhibitors of signaling pathways indicated that the phosphorylation of PXN at Y118 and Y31 via the Src pathway was responsible for cisplatin resistance. We further demonstrated that ERK activation was also dependent on this PXN phosphorylation. Bcl-2 transcription was upregulated by phosphorylated PXN-mediated ERK activation via increased binding of phosphorylated CREB to the Bcl-2 promoter. A subsequent increase in Bcl-2 levels by a PXN/ERK axis was responsible for the resistance to cisplatin. Animal models further confirmed the findings of in vitro cells indicating that xenograft tumors induced by TL-13-overexpressing cells were successfully suppressed by cisplatin combined with Src or ERK inhibitor compared with treatment of cisplatin, Src inhibitor or ERK inhibitor alone. A positive correlation of phosphorylated PXN with phosphorylated ERK and Bcl-2 was observed in lung tumors from NSCLC patients. Patients with tumors positive for PXN, phosphorylated PXN, phosphorylated ERK and Bcl-2 more commonly showed a poorer response to cisplatin-based chemotherapy than did patients with negative tumors. Collectively, PXN phosphorylation might contribute to cisplatin resistance via activating ERK-mediated Bcl-2 transcription. Therefore, we suggest that Src or ERK inhibitor might be helpful to improve the sensitivity for cisplatin-based chemotherapy in NSCLC patients with PXN-positive tumors. PMID:24096476

  15. In LNCaP cells enhanced expression of the androgen receptor compensates for Bcl-2 suppression by antisense oligonucleotides

    PubMed Central

    Rubenstein, Marvin; Hollowell, Courtney M.P.; Guinan, Patrick

    2011-01-01

    Background and methods: Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth stimulatory gene products. While most oligos have targeted growth factors or their receptors, others have been directed against inhibitors of apoptosis. In LNCaP cells we evaluated a set of oligos which targeted and comparably suppressed the expression of the apoptosis inhibitor protein Bcl-2. LNCaP cells adapted to this restoration of apoptosis with a compensatory suppression of caspase-3 expression, a nontargeted promoter of this process. In a continuation of this study we now evaluate the expression of the androgen receptor (AR) following oligo mediated regulation of apoptosis with suppression of Bcl-2. Results: Monospecific and bispecific oligos directed against Bcl-2 suppressed both the targeted Bcl-2 protein (an inhibitor of apoptosis) and the nontargeted caspase-3 (a promoter of apoptosis), potentially negating the effect on apoptosis produced by specific inhibition of Bcl-2. In contrast, the expression of the AR was significantly enhanced by each type of oligo. Conclusions: This suggests that when Bcl-2 expression is inhibited there are compensatory changes in the expression of additional proteins which regulate tumor growth, apoptosis and cell survival, and in this scenario might increase or re-establish hormonal sensitivity. If tumors variants are selected which evade gene therapy additional mechanisms of compensation must be identified and subsequently suppressed. These experiments identify pathways by which tumors can develop resistance to gene therapy and suggests additional targets for intervention. PMID:21869905

  16. Bcl-2 Regulates Reactive Oxygen Species Signaling and a Redox-Sensitive Mitochondrial Proton Leak in Mouse Pancreatic β-Cells.

    PubMed

    Aharoni-Simon, Michal; Shumiatcher, Rose; Yeung, Anthony; Shih, Alexis Z L; Dolinsky, Vernon W; Doucette, Christine A; Luciani, Dan S

    2016-06-01

    In pancreatic β-cells, controlling the levels of reactive oxygen species (ROS) is critical to counter oxidative stress, dysfunction and death under nutrient excess. Moreover, the fine-tuning of ROS and redox balance is important in the regulation of normal β-cell physiology. We recently demonstrated that Bcl-2 and Bcl-xL, in addition to promoting survival, suppress β-cell glucose metabolism and insulin secretion. Here, we tested the hypothesis that the nonapoptotic roles of endogenous Bcl-2 extend to the regulation of β-cell ROS and redox balance. We exposed mouse islet cells and MIN6 cells to the Bcl-2/Bcl-xL antagonist Compound 6 and the Bcl-2-specific antagonist ABT-199 and evaluated ROS levels, Ca(2+) responses, respiratory control, superoxide dismutase activity and cell death. Both acute glucose stimulation and the inhibition of endogenous Bcl-2 progressively increased peroxides and stimulated superoxide dismutase activity in mouse islets. Importantly, conditional β-cell knockout of Bcl-2 amplified glucose-induced formation of peroxides. Bcl-2 antagonism also induced a mitochondrial proton leak that was prevented by the antioxidant N-acetyl-L-cysteine and, therefore, secondary to redox changes. We further established that the proton leak was independent of uncoupling protein 2 but partly mediated by the mitochondrial permeability transition pore. Acutely, inhibitor-induced peroxides promoted Ca(2+) influx, whereas under prolonged Bcl inhibition, the elevated ROS was required for induction of β-cell apoptosis. In conclusion, our data reveal that endogenous Bcl-2 modulates moment-to-moment ROS signaling and suppresses a redox-regulated mitochondrial proton leak in β-cells. These noncanonical roles of Bcl-2 may be important for β-cell function and survival under conditions of high metabolic demand. PMID:27070098

  17. The tissue dependent interactions between p53 and Bcl-2 in vivo

    PubMed Central

    Wang, Hongshen; Xu, Zhixiang; Li, Bin

    2015-01-01

    To further investigate the role of p53 in apoptosis in vivo and the interaction between p53 and Bcl-2 in the regulation of cellular apoptosis in vivo, we depleted p53 in Bcl-2-null mice. We found that the interaction between p53 and Bcl-2 are tissue dependent. Specifically, loss of p53 in Bcl-2−/− mice inhibits apoptotic induction in spleen and subsequently inhibits the Bcl-2-null-induced spleen atrophy. Furthermore, p53 deficiency overcomes loss of melanocyte stem cell (MSC)-induced apoptosis and subsequently prevents hair graying in Bcl-2- null mice. In addition, p53 deletion partly inhibits apoptosis in hair follicle keratinocytes, leading to the alleviation of hair growth delay in Bcl-2-null mice. However, p53 absence in Bcl-2-null mice cannot restore other defects in Bcl-2-null mice, including retardation of growth, short ears and polycystic kidney disease. PMID:26452131

  18. BCL2 protein expression in follicular lymphomas with t(14;18) chromosomal translocations.

    PubMed

    Masir, Noraidah; Campbell, Lisa J; Goff, Lindsey K; Jones, Margaret; Marafioti, Teresa; Cordell, Jacqueline; Clear, Andrew J; Lister, T Andrew; Mason, David Y; Lee, Abigail M

    2009-03-01

    The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein overexpression in most follicular lymphomas. However the expression of BCL2 is not always homogeneous and may demonstrate a variable degree of heterogeneity. This study analysed BCL2 protein expression pattern in 33 cases of t(14;18)-positive follicular lymphomas using antibodies against two different epitopes (i.e. the widely used antibody BCL2/124 and an alternative antibody E17). 16/33 (49%) cases demonstrated strong BCL2 expression. In 10/33 (30%) cases, BCL2 expression was heterogeneous and in some of these, its loss appeared to be correlated with cell proliferation, as indicated by Ki67 expression. Double immunofluorescence labelling confirmed an inverse BCL2/Ki67 relationship, where in 24/28 (86%) cases cellular expression of BCL2 and Ki67 was mutually exclusive. In addition, seven BCL2 'pseudo-negative' cases were identified in which immunostaining was negative with antibody BCL2/124, but positive with antibody E17. Genomic DNA sequencing of these 'pseudo-negative' cases demonstrated eleven mutations in four cases and nine of these were missense mutations. It can be concluded that in follicular lymphomas, despite carrying the t(14;18) translocations, BCL2 protein expression may be heterogeneous and loss of BCL2 could be related to cell proliferation. Secondly, mutations in translocated BCL2 genes appear to be common and may cause BCL2 pseudo-negative immunostaining. PMID:19120369

  19. Tamoxifen-Induced Cell Death of Malignant Glioma Cells Is Brought About by Oxidative-Stress-Mediated Alterations in the Expression of BCL2 Family Members and Is Enhanced on miR-21 Inhibition.

    PubMed

    Harmalkar, Mugdha; Upraity, Shailendra; Kazi, Sadaf; Shirsat, Neelam Vishwanath

    2015-10-01

    High-grade gliomas are refractory to the current mode of treatment primarily due to their inherent resistance to cell death. Tamoxifen has been reported to inhibit growth and induce cell death of glioma cells in vitro, in an estrogen-receptor-independent manner. Delineating the molecular mechanism underlying tamoxifen-induced cell death of human glioma cells would help in identifying pathways/genes that could be targeted to induce tumor-cell-specific cell death. In the present study, tamoxifen was found to bring about autophagic cell death of human glioma cells that was accompanied by oxidative stress induction, JNK activation, downregulation of anti-autophagic BCL2 family members, viz. BCL2 and BCL-XL, and increased expression of the pro-autophagic members BCL-Xs and BAK. Oxidative stress induction appears to be primarily responsible for the tamoxifen-induced cell death since the cell death, JNK activation, and the alterations in the expression levels of BCL2 family members were abrogated on pretreatment with antioxidant vitamin E. MiR-21, an oncogenic miRNA, is known to be highly upregulated in malignant glioma. Inhibition of miR-21 activity was found to enhance tamoxifen-induced cell death of U87 MG malignant glioma cells. Tamoxifen treatment coupled with miR-21 inhibition could therefore be an effective strategy for the treatment of malignant gliomas. PMID:26109525

  20. Clusterin silencing sensitizes pancreatic cancer MIA-PaCa-2 cells to gmcitabine via regulation of NF-kB/Bcl-2 signaling.

    PubMed

    Xu, Miao; Chen, Xiumei; Han, Yanling; Ma, Chunqing; Ma, Lin; Li, Shirong

    2015-01-01

    Clusterin (CLU) is known as a multifunctional protein involved in a variety of physiological processes including lipid transport, epithelial cell differentiation, tumorigenesis, and apoptosis. Our recent study has demonstrated that knockdown of clusterin sensitizes pancreatic cancer cell lines to gmcitabine treatment. However the details of this survival mechanism remain undefined. Of the various downstream targets of CLU, we examined activation of the NF-kB transcription factor and subsequent transcriptional regulation of BCL-2 gene in pancreatic cancer cell MIA-PaCa-2. The MIA-PaCa-2 cells were transfected with an antisense oligonucleotide (ASO) against clusterin, which led to a decreased protein level of the antiapoptotic gene BCL-2. Furthermore, inhibition of CLU decreased the function of NF-kB, which is capable of transcriptional regulation of the BCL-2 gene. Inhibiting this pathway increased the apoptotic effect of gmcitabine chemotherapy. Re-activated NF-kB resulted in attenuation of ASO-induced effects, followed by the bcl-2 upregulation, and bcl-2 re-inhibition resulted in attenuation of Re-activated NF-kB -induced effects. Animals injected with ASO CLU in MIA-PaCa-2 cells combined with gmcitabine treatment had fewer tumors than gmcitabine or ASO CLU alone. These findings suggest that knockdown of CLU sensitized MIA-PaCa-2 cells to gmcitabine chemotherapy through modulating NF-Kb/bcl-2 pathway. PMID:26550158

  1. Clusterin silencing sensitizes pancreatic cancer MIA-PaCa-2 cells to gmcitabine via regulation of NF-kB/Bcl-2 signaling

    PubMed Central

    Xu, Miao; Chen, Xiumei; Han, Yanling; Ma, Chunqing; Ma, Lin; Li, Shirong

    2015-01-01

    Clusterin (CLU) is known as a multifunctional protein involved in a variety of physiological processes including lipid transport, epithelial cell differentiation, tumorigenesis, and apoptosis. Our recent study has demonstrated that knockdown of clusterin sensitizes pancreatic cancer cell lines to gmcitabine treatment. However the details of this survival mechanism remain undefined. Of the various downstream targets of CLU, we examined activation of the NF-kB transcription factor and subsequent transcriptional regulation of BCL-2 gene in pancreatic cancer cell MIA-PaCa-2. The MIA-PaCa-2 cells were transfected with an antisense oligonucleotide (ASO) against clusterin, which led to a decreased protein level of the antiapoptotic gene BCL-2. Furthermore, inhibition of CLU decreased the function of NF-kB, which is capable of transcriptional regulation of the BCL-2 gene. Inhibiting this pathway increased the apoptotic effect of gmcitabine chemotherapy. Re-activated NF-kB resulted in attenuation of ASO-induced effects, followed by the bcl-2 upregulation, and bcl-2 re-inhibition resulted in attenuation of Re-activated NF-kB -induced effects. Animals injected with ASO CLU in MIA-PaCa-2 cells combined with gmcitabine treatment had fewer tumors than gmcitabine or ASO CLU alone. These findings suggest that knockdown of CLU sensitized MIA-PaCa-2 cells to gmcitabine chemotherapy through modulating NF-Kb/bcl-2 pathway. PMID:26550158

  2. Dual expression of MYC and BCL2 proteins predicts worse outcomes in diffuse large B-cell lymphoma.

    PubMed

    Clark Schneider, Kelli M; Banks, Peter M; Collie, Angela M B; Lanigan, Christopher P; Manilich, Elena; Durkin, Lisa M; Hill, Brian T; Hsi, Eric D

    2016-07-01

    Recent studies suggested that MYC and BCL2 protein co-expression is an independent indicator of poor prognosis in diffuse large B-cell lymphoma. However, the immunohistochemistry protocols for dual-expression staining and the scoring cut-offs vary by study. Sixty-nine cases of diffuse large B-cell lymphoma were evaluated for MYC and BCL2 protein expression using various cut-offs that have been recommended in prior studies. Independent of the International Prognostic Index risk group, cases with dual protein expression of BCL2 and MYC using ≥50%/40% cut-offs and ≥70%/40% had significantly shorter overall survival than cases without. It was verified in this patient population that the use of BCL2 and MYC immunohistochemistry, performed with available in vitro diagnostic-cleared antibodies, provides rapid prognostic information in patients with de novo diffuse large B-cell lymphoma. This study has practical implications for diagnostic laboratories and serves as a guide for implementation in the setting of future clinical trials. PMID:26421520

  3. Apoptosis during an early stage of nephrogenesis induces renal hypoplasia in bcl-2-deficient mice.

    PubMed Central

    Nagata, M.; Nakauchi, H.; Nakayama, K.; Nakayama, K.; Loh, D.; Watanabe, T.

    1996-01-01

    Renal development in bcl-2-deficient mice was monitored to examine the temporal and spatial function of this gene during nephrogenesis in vivo. Extensive apoptosis occurred during abnormal nephrogenesis in bcl-2-deficient mice. In embryos and newborn mice, the sequence of morphological events was monitored by morphology in conjunction with morphometry, and bcl-2 -/-, bcl-2 +/-, and bcl-2 +/+ mice were compared. In bcl-2 -/- mice, initial induction of nephrons was detected by embryonic day 13 (E-13) as normal. Then, apoptotic cells became five times more frequent at E-13 to E-16 with a significant reduction (1/5) in nephron number at E-17 to E-19 in bcl-2 -/- mice compared with bcl-2 +/+ mice. No morphological difference was evident between bcl-2 +/- mice and bcl-2 +/+ mice by morphometry. Apoptotic cells were found mainly among the mesenchyme and less frequently in tubuli. Little apoptosis among ureteric buds was noted. In bcl-2 -/- mice at E-17 to E-19, inactive branching and insufficient convolution of ureteric buds were accompanied by fulminant apoptosis in the mesenchyme. Neonatal bcl-2 -/- mice lacked the nephrogenic zone, exhibiting renal hypoplasia. Thus, bcl-2 seems to inhibit apoptosis in renal stem cells during the induction of nephrons in vivo. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8623928

  4. Expression of Bcl-2, p53, and MDM2 in Localized Prostate Cancer With Respect to the Outcome of Radical Radiotherapy Dose Escalation

    SciTech Connect

    Vergis, Roy; Corbishley, Catherine M.; Thomas, Karen

    2010-09-01

    Purpose: Established prognostic factors in localized prostate cancer explain only a moderate proportion of variation in outcome. We analyzed tumor expression of apoptotic markers with respect to outcome in men with localized prostate cancer in two randomized controlled trials of radiotherapy dose escalation. Methods and Materials: Between 1995 and 2001, 308 patients with localized prostate cancer received neoadjuvant androgen deprivation and radical radiotherapy at our institution in one of two dose-escalation trials. The biopsy specimens in 201 cases were used to make a biopsy tissue microarray. We evaluated tumor expression of Bcl-2, p53, and MDM2 by immunohistochemistry with respect to outcome. Results: Median follow-up was 7 years, and 5-year freedom from biochemical failure (FFBF) was 70.4% (95% CI, 63.5-76.3%). On univariate analysis, expression of Bcl-2 (p < 0.001) and p53 (p = 0.017), but not MDM2 (p = 0.224), was significantly associated with FFBF. Expression of Bcl-2 remained significantly associated with FFBF (p = 0.001) on multivariate analysis, independently of T stage, Gleason score, initial prostate-specific antigen level, and radiotherapy dose. Seven-year biochemical control was 61% vs. 41% (p = 0.0122) for 74 Gy vs. 64 Gy, respectively, among patients with Bcl-2-positive tumors and 87% vs. 81% (p = 0.423) for 74 Gy vs. 64 Gy, respectively, among patients with Bcl-2-negative tumors. There was no statistically significant interaction between dose and Bcl-2 expression. Conclusions: Bcl-2 expression was a significant, independent determinant of biochemical control after neoadjuvant androgen deprivation and radical radiotherapy for prostate cancer. These data generate the hypothesis that Bcl-2 expression could be used to inform the choice of radiotherapy dose in individual patients.

  5. The Bcl-2 apoptotic switch in cancer development and therapy.

    PubMed

    Adams, J M; Cory, S

    2007-02-26

    Impaired apoptosis is both critical in cancer development and a major barrier to effective treatment. In response to diverse intracellular damage signals, including those evoked by cancer therapy, the cell's decision to undergo apoptosis is determined by interactions between three factions of the Bcl-2 protein family. The damage signals are transduced by the diverse 'BH3-only' proteins, distinguished by the BH3 domain used to engage their pro-survival relatives: Bcl-2, Bcl-x(L), Bcl-w, Mcl-1 and A1. This interaction ablates pro-survival function and allows activation of Bax and Bak, which commit the cell to apoptosis by permeabilizing the outer membrane of the mitochondrion. Certain BH3-only proteins (e.g. Bim, Puma) can engage all the pro-survival proteins, but others (e.g. Bad, Noxa) engage only subsets. Activation of Bax and Bak appears to require that the BH3-only proteins engage the multiple pro-survival proteins guarding Bax and Bak, rather than binding to the latter. The balance between the pro-survival proteins and their BH3 ligands regulates tissue homeostasis, and either overexpression of a pro-survival family member or loss of a proapoptotic relative can be oncogenic. Better understanding of the Bcl-2 family is clarifying its role in cancer development, revealing how conventional therapy works and stimulating the search for "BH3 mimetics" as a novel class of anticancer drugs. PMID:17322918

  6. Granzyme B-activated p53 interacts with Bcl-2 to promote cytotoxic lymphocyte-mediated apoptosis.

    PubMed

    Ben Safta, Thouraya; Ziani, Linda; Favre, Loetitia; Lamendour, Lucille; Gros, Gwendoline; Mami-Chouaib, Fathia; Martinvalet, Denis; Chouaib, Salem; Thiery, Jerome

    2015-01-01

    Granzyme B (GzmB) plays a major role in CTLs and NK cell-mediated elimination of virus-infected cells and tumors. Human GzmB preferentially induces target cell apoptosis by cleaving the proapoptotic Bcl-2 family member Bid, which, together with Bax, induces mitochondrial outer membrane permeabilization. We previously showed that GzmB also induces a rapid accumulation of the tumor-suppressor protein p53 within target cells, which seems to be involved in GzmB-induced apoptosis. In this article, we show that GzmB-activated p53 accumulates on target cell mitochondria and interacts with Bcl-2. This interaction prevents Bcl-2 inhibitory effect on both Bax and GzmB-truncated Bid, and promotes GzmB-induced mitochondrial outer membrane permeabilization. Consequently, blocking p53-Bcl-2 interaction decreases GzmB-induced Bax activation, cytochrome c release from mitochondria, and subsequent effector caspases activation leading to a decreased sensitivity of target cells to both GzmB and CTL/NK-mediated cell death. Together, our results define p53 as a new important player in the GzmB apoptotic signaling pathway and in CTL/NK-induced apoptosis. PMID:25404359

  7. BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

    PubMed

    Gabellini, Chiara; De Luca, Teresa; Trisciuoglio, Daniela; Desideri, Marianna; Di Martile, Marta; Passeri, Daniela; Candiloro, Antonio; Biffoni, Mauro; Rizzo, Maria Giulia; Orlandi, Augusto; Del Bufalo, Donatella

    2013-11-01

    Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis. PMID:23836782

  8. Influence of BCL2-938 C>A promoter polymorphism and BCL2 gene expression on the progression of breast cancer.

    PubMed

    Bhushann Meka, Phanni; Jarjapu, Sarika; Vishwakarma, Sandeep Kumar; Nanchari, Santhoshi Rani; Cingeetham, Anuradha; Annamaneni, Sandhya; Mukta, Srinivasulu; Triveni, B; Satti, Vishnupriya

    2016-05-01

    BCL2 (B-cell leukemia/lymphoma 2) gene functions as antiapoptotic regulatory element and known to be associated with tumorigenesis. The SNP-938 (C>A) (rs2279115), located in the inhibitory P2 promoter of the BCL2 gene, influences differential binding affinities of transcriptional factors thereby affecting BCL2 expression. The present study is an attempt to evaluate the association between BCL2(-938C>A) polymorphism and clinical characteristics of breast cancer patients as well as to analyze BCL2 expression and Ki67 proliferation index with respect to the genotypes. One hundred ten primary breast cancer tumor tissues were genotyped for -938 C>A polymorphism through PCR-RFLP method as well as evaluated for BCL2 expression and ki67 proliferation index by immunohistochemistry. Evaluation of apoptosis level was performed by flowcytometry. The results revealed that AA genotype was associated with an increased risk (AA Vs AC + CC) by 2.86-fold (p = 0.07) for breast cancer development which reflected in elevated A allele frequency also. AA genotype was found to be predominant among BCL2 positive tumors as compared to BCL2 negative tumors. Further, AA genotype was found to be associated with advanced stage tumors, node positive status, and high Ki67 proliferation index compared to CA and CC genotypes indicating that elevated expression of BCL2 gene in the presence of A allele might be associated with decreased apoptosis and enhanced proliferation rate. AA genotype of BCL2-938C>A polymorphism might influence BCL2 gene expression there by associated with elevated risk for breast cancer progression. Probably, failure of apoptosis due to enhanced expression and antiapoptotic protein BCL2 might promote malignant growth. PMID:26662799

  9. Regulation of mitochondrial ceramide distribution by members of the BCL-2 family[S

    PubMed Central

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

    2015-01-01

    Apoptosis is an intricately regulated cellular process that proceeds through different cell type- and signal-dependent pathways. In the mitochondrial apoptotic program, mitochondrial outer membrane permeabilization by BCL-2 proteins leads to the release of apoptogenic factors, caspase activation, and cell death. In addition to protein components of the mitochondrial apoptotic machinery, an interesting role for lipids and lipid metabolism in BCL-2 family-regulated apoptosis is also emerging. We used a comparative lipidomics approach to uncover alterations in lipid profile in the absence of the proapoptotic proteins BAX and BAK in mouse embryonic fibroblasts (MEFs). We detected over 1,000 ions in these experiments and found changes in an ion with an m/z of 534.49. Structural elucidation of this ion through tandem mass spectrometry revealed that this molecule is a ceramide with a 16-carbon N-acyl chain and sphingadiene backbone (d18:2/16:0 ceramide). Targeted LC/MS analysis revealed elevated levels of additional sphingadiene-containing ceramides (d18:2-Cers) in BAX, BAK-double knockout MEFs. Elevated d18:2-Cers are also found in immortalized baby mouse kidney epithelial cells lacking BAX and BAK. These results support the existence of a distinct biochemical pathway for regulating ceramides with different backbone structures and suggest that sphingadiene-containing ceramides may have functions that are distinct from the more common sphingosine-containing species. PMID:26059977

  10. Minocycline mechanism of neuroprotection involves the Bcl-2 gene family in optic nerve transection.

    PubMed

    Levkovitch-Verbin, Hani; Waserzoog, Yael; Vander, Shelly; Makarovsky, Daria; Ilia, Piven

    2014-10-01

    The second-generation tetracycline, minocycline, has been shown to exhibit neuroprotective therapeutic benefits in many neurodegenerative diseases including experimental glaucoma and optic nerve transection (ONT). This study investigated the mechanism underlying minocycline neuroprotection in a model of ONT. ONT was applied unilaterally in 36 Wistar rat eyes. The rats were randomly divided into a minocycline (22 mg/kg/d) treatment group and a saline treatment group (control). Treatment (minocycline or saline) was given by intraperitoneal injections initiated 3 d before ONT and continued daily until the end of the experiment. The involvement of pro-apoptotic, pro-survival and inflammatory pathways was analyzed by quantitative Real-Time Polymerase Chain Reaction at 4 h and 3 d after the transection in both treatment groups. The involvement of Bcl-2 protein was evaluated by immunohistochemistry. We found that Minocycline significantly increased the expression of the antiapoptotic gene bcl-2 4 h after transection (n = 8, p = 0.008) and decreased the expression of Bax at the same time point (n = 8, p = 0.03). Tumor Necrosis Factor α (TNFα), Inhibitor of Apoptosis Protein (IAP1) and Gadd45α were significantly upregulated in the retinas of eyes with ONTs compared to control (n = 10 for each gene, p = 0.02, p = 0.03, p = 0.04, respectively) but this effect was unaffected by minocycline. This study further support that the mechanism underlying minocycline neuroprotection involves the Bcl-2 gene family, suggesting that minocycline has antiapoptotic properties that support its value as a promising neuroprotective drug. PMID:24410139

  11. Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation.

    PubMed

    Xie, Maohua; Park, Dongkyoo; You, Shuo; Li, Rui; Owonikoko, Taofeek K; Wang, Ya; Doetsch, Paul W; Deng, Xingming

    2015-01-01

    High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. (56)iron and (28)silicon) by inhibiting Mre11 complex activity. Exposure of cells to (56)iron or (28)silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development. PMID:25567982

  12. MicroRNA-744 inhibited cervical cancer growth and progression through apoptosis induction by regulating Bcl-2.

    PubMed

    Chen, Xiao-Fang; Liu, Yun

    2016-07-01

    Growing evidence suggests that microRNA plays an essential role in the development and metastasis of many tumor progressions, including cervical cancer. Aberrant miR-744 expression has been indicated in many growth of tumor, the mechanism of miR-744 inhibits both the proliferation and metastatic ability for cervical cancer remains unclear. Accumulating evidences reported that Bcl-2 signal pathway plays an important role in the cellular process, such as apoptosis, cell growth and proliferation. The goal of this study was to identify miR-744 that could inhibit the growth, migration, invasion, proliferation and metastasis of gastric cancer through targeting Bcl-2 expression. Real-time PCR (RT-qPCR) was used to quantify miR-744 expression in vitro and vivo experiments. The biological functions of miR-744 were determined via cell proliferation. Our study indicated that miR-744 targeted on Bcl-2, which leads to the inactivation of apoptosis signaling and the cell proliferation of cervical cancer cells, ameliorating cervical cancer growth and progression. In addition, both up-regulation of miR-744 and down-regulation of Bcl-2 could stimulate Caspase-3 expression, promoting apoptosis of cervical cancer cells. Therefore, our research revealed the mechanistic links between miR-744 and Bcl-2 in the pathogenesis of cervical cancer through modulation of Caspase-3, leading to the inhibition of cervical cancer cell growth. And targeting miR-744 could be served as a novel strategy for future cervical cancer therapy clinically. PMID:27261616

  13. p53 and bcl-2 expression in high-grade B-cell lymphomas: correlation with survival time.

    PubMed

    Piris, M A; Pezzella, F; Martinez-Montero, J C; Orradre, J L; Villuendas, R; Sanchez-Beato, M; Cuena, R; Cruz, M A; Martinez, B; Pezella F [corrected to Pezzella, F

    1994-02-01

    B-cell high-grade lymphomas are heterogeneous in terms of histology, clinical presentation, treatment response and prognosis. As bcl-2 and p53 gene deregulations are frequently involved in several types of lymphoid malignancies, we aimed our investigation at the study of the relation between bcl-2 and p53 expression and survival probability in a group of 119 patients with B-cell high-grade lymphoma. These were obtained from the Virgen de la Salud Hospital, Toledo, Spain (73 cases), John Radcliffe Hospital, Oxford, UK (31 cases), and the Istituto Nazionale dei Tumori, Milan, Italy (15 cases). The relation between bcl-2 protein expression and survival was small, depending on the primary localisation of the tumour (in lymph node of mucosae), and lacked a significant correlation with overall survival. In contrast with this, p53 expression was related to survival probability in our series, this relation being both significant and independent of histological diagnosis. p53-positive patients showed a sudden decrease in life expectancy in the first months after diagnosis. Multivariant regression analysis confirmed that the only parameters significantly related with survival were extranodal origin, which is associated with a better prognosis, and p53 expression, which indicates a poor prognosis. Simultaneous expression of bcl-2 and p53 was associated with a poorer prognosis than p53 alone. This is particularly significant for large B-cell lymphomas presenting in lymph nodes. The cumulative poor effect of both p53 and bcl-2 in large B-cell lymphomas, which is more significant in nodal tumours, could confirm the existence of a multistep genetic deregulation in non-Hodgkin's lymphoma. This indicates that the genetic mechanisms controlling apoptosis and their disregulation are critical steps in the progression of lymphomas. PMID:8297731

  14. p53 and bcl-2 expression in high-grade B-cell lymphomas: correlation with survival time.

    PubMed Central

    Piris, M. A.; Pezzella, F.; Martinez-Montero, J. C.; Orradre, J. L.; Villuendas, R.; Sanchez-Beato, M.; Cuena, R.; Cruz, M. A.; Martinez, B.; Pezella F [corrected to Pezzella, F. ].

    1994-01-01

    B-cell high-grade lymphomas are heterogeneous in terms of histology, clinical presentation, treatment response and prognosis. As bcl-2 and p53 gene deregulations are frequently involved in several types of lymphoid malignancies, we aimed our investigation at the study of the relation between bcl-2 and p53 expression and survival probability in a group of 119 patients with B-cell high-grade lymphoma. These were obtained from the Virgen de la Salud Hospital, Toledo, Spain (73 cases), John Radcliffe Hospital, Oxford, UK (31 cases), and the Istituto Nazionale dei Tumori, Milan, Italy (15 cases). The relation between bcl-2 protein expression and survival was small, depending on the primary localisation of the tumour (in lymph node of mucosae), and lacked a significant correlation with overall survival. In contrast with this, p53 expression was related to survival probability in our series, this relation being both significant and independent of histological diagnosis. p53-positive patients showed a sudden decrease in life expectancy in the first months after diagnosis. Multivariant regression analysis confirmed that the only parameters significantly related with survival were extranodal origin, which is associated with a better prognosis, and p53 expression, which indicates a poor prognosis. Simultaneous expression of bcl-2 and p53 was associated with a poorer prognosis than p53 alone. This is particularly significant for large B-cell lymphomas presenting in lymph nodes. The cumulative poor effect of both p53 and bcl-2 in large B-cell lymphomas, which is more significant in nodal tumours, could confirm the existence of a multistep genetic deregulation in non-Hodgkin's lymphoma. This indicates that the genetic mechanisms controlling apoptosis and their disregulation are critical steps in the progression of lymphomas. PMID:8297731

  15. Impact of dual expression of MYC and BCL2 by immunohistochemistry on the risk of CNS relapse in DLBCL.

    PubMed

    Savage, Kerry J; Slack, Graham W; Mottok, Anja; Sehn, Laurie H; Villa, Diego; Kansara, Roopesh; Kridel, Robert; Steidl, Christian; Ennishi, Daisuke; Tan, King L; Ben-Neriah, Susana; Johnson, Nathalie A; Connors, Joseph M; Farinha, Pedro; Scott, David W; Gascoyne, Randy D

    2016-05-01

    Dual expression of MYC and BCL2 by immunohistochemistry (IHC) is associated with poor outcome in diffuse large B-cell lymphoma (DLBCL). Dual translocation of MYC and BCL2, so-called "double-hit lymphoma," has been associated with a high risk of central nervous system (CNS) relapse; however, the impact of dual expression of MYC and BCL2 (dual expressers) on the risk of CNS relapse remains unknown. Pretreatment formalin-fixed paraffin-embedded DLBCL biopsies derived from patients subsequently treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) were assembled on tissue microarrays from 2 studies and were evaluated for expression of MYC and BCL2 by IHC. In addition, cell of origin was determined by IHC and the Lymph2Cx gene expression assay in a subset of patients. We identified 428 patients who met the inclusion criteria. By the recently described CNS risk score (CNS-International Prognostic Index [CNS-IPI]), 34% were low risk (0 to 1), 45% were intermediate risk (2 to 3), and 21% were high risk (4 or greater). With a median follow-up of 6.8 years, the risk of CNS relapse was higher in dual expressers compared with non-dual expressers (2-year risk, 9.7% vs 2.2%; P = .001). Patients with activated B-cell or non-germinal center B-cell type DLBCL also had an increased risk of CNS relapse. However, in multivariate analysis, only dual expresser status and CNS-IPI were associated with CNS relapse. Dual expresser MYC(+) BCL2(+) DLBCL defines a group at high risk of CNS relapse, independent of CNS-IPI score and cell of origin. Dual expresser status may help to identify a high-risk group who should undergo CNS-directed evaluation and consideration of prophylactic strategies. PMID:26834242

  16. c-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine

    PubMed Central

    Chaudhary, Pankaj; Vishwanatha, Jamboor K.

    2014-01-01

    Tetrandrine, a constituent of Chinese herb Stephania tetrandra, causes cell death in prostate cancer, but the molecular mechanisms leading to apoptosis is not known. Here we demonstrated that tetrandrine selectively inhibits the growth of prostate cancer PC3 and DU145 cells compared to normal prostate epithelial PWR-1E cells. Tetrandrine-induced cell death in prostate cancer cells is caused by reactive oxygen species (ROS)-mediated activation of c-Jun NH2-terminal kinase (JNK1/2). JNK1/2-mediated proteasomal degradation of c-FLIPL/S and Bcl2 proteins are key events in the sensitization of prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine. Tetrandrine-induced JNK1/2 activation caused the translocation of Bax to mitochondria by disrupting its association with Bcl2 which was accompanied by collapse of mitochondrial membrane potential (MMP), cytosolic release of cytochrome c and Smac, and apoptotic cell death. Additionally, tetrandrine-induced JNK1/2 activation increased the phosphorylation of Bcl2 at Ser70 and facilitated its degradation via the ubiquitin-mediated proteasomal pathway. In parallel, tetrandrine-mediated ROS generation also caused the induction of ligand-independent Fas-mediated apoptosis by activating procaspase-8 and Bid cleavage. Inhibition of procaspase-8 activation attenuated the cleavage of Bid, loss of MMP and caspase-3 activation suggest that tetrandrine-induced Fas-mediated apoptosis is associated with the mitochondrial pathway. Furthermore, most of the signaling effects of tetrandrine on apoptosis were significantly attenuated in the presence of antioxidant N-acetyl-L-cysteine, thereby confirming the involvement of ROS in these events. In conclusion, the results of the present study indicate that tetrandrine-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic pathway contributes to cell death. PMID:25181458

  17. c-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine.

    PubMed

    Chaudhary, Pankaj; Vishwanatha, Jamboor K

    2014-10-15

    Tetrandrine, a constituent of Chinese herb Stephania tetrandra, causes cell death in prostate cancer, but the molecular mechanisms leading to apoptosis is not known. Here we demonstrated that tetrandrine selectively inhibits the growth of prostate cancer PC3 and DU145 cells compared to normal prostate epithelial PWR-1E cells. Tetrandrine-induced cell death in prostate cancer cells is caused by reactive oxygen species (ROS)-mediated activation of c-Jun NH2-terminal kinase (JNK1/2). JNK1/2-mediated proteasomal degradation of c-FLIPL/S and Bcl2 proteins are key events in the sensitization of prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine. Tetrandrine-induced JNK1/2 activation caused the translocation of Bax to mitochondria by disrupting its association with Bcl2 which was accompanied by collapse of mitochondrial membrane potential (MMP), cytosolic release of cytochrome c and Smac, and apoptotic cell death. Additionally, tetrandrine-induced JNK1/2 activation increased the phosphorylation of Bcl2 at Ser70 and facilitated its degradation via the ubiquitin-mediated proteasomal pathway. In parallel, tetrandrine-mediated ROS generation also caused the induction of ligand-independent Fas-mediated apoptosis by activating procaspase-8 and Bid cleavage. Inhibition of procaspase-8 activation attenuated the cleavage of Bid, loss of MMP and caspase-3 activation suggest that tetrandrine-induced Fas-mediated apoptosis is associated with the mitochondrial pathway. Furthermore, most of the signaling effects of tetrandrine on apoptosis were significantly attenuated in the presence of antioxidant N-acetyl-l-cysteine, thereby confirming the involvement of ROS in these events. In conclusion, the results of the present study indicate that tetrandrine-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic pathway contributes to cell death. PMID:25181458

  18. LIN-35/Rb Causes Starvation-Induced Germ Cell Apoptosis via CED-9/Bcl2 Downregulation in Caenorhabditis elegans

    PubMed Central

    Láscarez-Lagunas, L. I.; Silva-García, C. G.; Dinkova, T. D.

    2014-01-01

    Apoptosis is an important mechanism for maintaining germ line health. In Caenorhabditis elegans, germ cell apoptosis occurs under normal conditions to sustain gonad homeostasis and oocyte quality. Under stress, germ cell apoptosis can be triggered via different pathways, including the following: (i) the CEP-1/p53 pathway, which induces germ cell apoptosis when animals are exposed to DNA damage; (ii) the mitogen-activated protein kinase kinase (MAPKK) pathway, which triggers germ cell apoptosis when animals are exposed to heat shock, oxidative stress, or osmotic stress; and (iii) an unknown mechanism that triggers germ cell apoptosis during starvation. Here, we address how starvation induces germ cell apoptosis. Using polysomal profiling, we found that starvation for 6 h reduces the translationally active ribosomes, which differentially affect the mRNAs of the core apoptotic machinery and some of its regulators. During starvation, lin-35/Rb mRNA increases its expression, resulting in the accumulation of this protein. As a consequence, LIN-35 downregulates the expression of the antiapoptotic gene ced-9/Bcl-2. We observed that the reduced translation of ced-9/Bcl-2 mRNA during food deprivation together with its downregulation drastically affects its protein accumulation. We propose that CED-9/Bcl-2 downregulation via LIN-35/Rb triggers germ cell apoptosis in C. elegans in response to starvation. PMID:24752899

  19. Neuregulin Promotes Incomplete Autophagy of Prostate Cancer Cells That Is Independent of mTOR Pathway Inhibition

    PubMed Central

    Schmukler, Eran; Shai, Ben; Ehrlich, Marcelo; Pinkas-Kramarski, Ronit

    2012-01-01

    Background Growth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death. Methodology/Principal Findings In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited. Conclusions/Significance Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition. PMID:22606295

  20. Manual acupuncture at the SJ5 (Waiguan) acupoint shows neuroprotective effects by regulating expression of the anti-apoptotic gene Bcl-2.

    PubMed

    Lin, Dong; Lin, Li-Li; Sutherland, Kyle; Cao, Chuan-Hai

    2016-02-01

    Acupuncture at the SJ5 (Waiguan) acupoint has neuroprotective effects in cerebral infarction, but the underlying mechanism remains poorly understood. Here, we analyzed gene expression in healthy rat cerebellum using a pathway-focused DNA microarray to screen 113 genes associated with 18 signal transduction pathways. After 20 minutes of acupuncture at SJ5, the expression of Bcl-2 and Birc1b mRNA was markedly increased. This was confirmed by real-time reverse transcription PCR. Furthermore, western blot analysis showed that Bcl-2 protein expression remained high in the cerebellum until at least 2 hours after cessation of acupuncture. These findings indicate that acupuncture at SJ5 exerts neuroprotective effects by regulating the expression of anti-apoptotic gene Bcl-2. PMID:27073385

  1. Manual acupuncture at the SJ5 (Waiguan) acupoint shows neuroprotective effects by regulating expression of the anti-apoptotic gene Bcl-2

    PubMed Central

    Lin, Dong; Lin, Li-li; Sutherland, Kyle; Cao, Chuan-hai

    2016-01-01

    Acupuncture at the SJ5 (Waiguan) acupoint has neuroprotective effects in cerebral infarction, but the underlying mechanism remains poorly understood. Here, we analyzed gene expression in healthy rat cerebellum using a pathway-focused DNA microarray to screen 113 genes associated with 18 signal transduction pathways. After 20 minutes of acupuncture at SJ5, the expression of Bcl-2 and Birc1b mRNA was markedly increased. This was confirmed by real-time reverse transcription PCR. Furthermore, western blot analysis showed that Bcl-2 protein expression remained high in the cerebellum until at least 2 hours after cessation of acupuncture. These findings indicate that acupuncture at SJ5 exerts neuroprotective effects by regulating the expression of anti-apoptotic gene Bcl-2. PMID:27073385

  2. Involvement of Bcl-2-associated athanogene (BAG)-family proteins in the neuroprotection by rasagiline

    PubMed Central

    Guo, Ji-Feng; He, Shuang; Kang, Ji-Feng; Xu, Qian; Hu, Ya-Cen; Zhang, Hai-Nan; Wang, Chun-Yu; Yan, Xin-Xiang; Tang, Bei-Sha

    2015-01-01

    Rasagiline, a novel monoamine oxidase (MAO)-B inhibitor, has a mild to moderate effect in relieving Parkinson’s disease (PD) symptoms as well as unique neuroprotective effects. Previous studies demonstrated rasagiline protect neurons by regulating Bcl-2 family proteins. Our study aimed to study whether Bcl-2-associated athanogene (BAG)-family proteins, which were reported closely associated with neurodegenerative disease, were involved in the neuroprotective effect of rasagiline. We found that after the administration of 1-methy1-4-phenvl-1,2,3,6-tetrahvdropvridine (MPTP), BAG2 and BAG5 proteins were up-regulated in the substantia nigra dopaminergic neurons of PD mouse model. A further increase of BAG2 and BAG5 was detected after intragastric administration of rasagiline to post-MPTP lesioned mice. Thus, the current study proved the association of BAG family proteins with PD, and suggested the involvement and a positive role of BAG2, BAG5 in the neuroprotection of rasagiline. These preliminary results implicate a novel pathway for further study on neuroprotection of rasagiline. PMID:26770414

  3. Neuroprotection via pro-survival protein kinase C isoforms associated with Bcl-2 family members.

    PubMed

    Weinreb, Orly; Bar-Am, Orit; Amit, Tamar; Chillag-Talmor, Orly; Youdim, Moussa B H

    2004-09-01

    This study provides new insights into neuroprotection involving interaction of protein kinase C (PKC) pathway with Bcl-2 family proteins. Using a model of serum deprivation, we investigated the mechanism by which the anti-Parkinson/monoamine oxidase (MAO)-B inhibitor drug, rasagiline, exerts its neuroprotective effect in rat pheochromocytoma PC12 cells. Here, we report that rasagiline (0.1-10 microM) decreased apoptosis via multiple protection mechanisms, including the stimulation of PKC phosphorylation; up-regulation of PKCalpha and PKC mRNAs, induction of Bcl-xL, Bcl-w, and brain-derived neurotrophic factor (BDNF) mRNAs; and down-regulation of Bad and Bax mRNAs. Moreover, rasagiline inhibited the cleavage and activation of procaspase-3 and poly (ADP-ribose) polymerase (PARP), whereas the PKC inhibitor, GF109203X, reversed these actions. Similarly, rasagiline decreased serum-free-induced levels of the important regulator of cell death, Bad, which was also blocked by GF109203X, indicating the involvement of PKC in rasagiline-induced cell survival. Furthermore, these studies have established that PKC- and Bcl-2-dependent neuroprotective activity of rasagiline is dependent on its propargyl moiety, because propargylamine had similar effects with the same potency. PMID:15247150

  4. H3K27 demethylation by JMJD3 at a poised enhancer of anti-apoptotic gene BCL2 determines ERα ligand dependency

    PubMed Central

    Svotelis, Amy; Bianco, Stéphanie; Madore, Jason; Huppé, Gabrielle; Nordell-Markovits, Alexei; Mes-Masson, Anne-Marie; Gévry, Nicolas

    2011-01-01

    Chromatin represents a repressive barrier to the process of ligand-dependent transcriptional activity of nuclear receptors. Here, we show that H3K27 methylation imposes ligand-dependent regulation of the oestrogen receptor α (ERα)-dependent apoptotic response via Bcl-2 in breast cancer cells. The activation of BCL2 transcription is dependent on the simultaneous inactivation of the H3K27 methyltransferase, EZH2, and the demethylation of H3K27 at a poised enhancer by the ERα-dependent recruitment of JMJD3 in hormone-dependent breast cancer cells. We also provide evidence that this pathway is modified in cells resistant to anti-oestrogen (AE), which constitutively express BCL2. We show that the lack of H3K27 methylation at BCL2 regulatory elements due to the inactivation of EZH2 by the HER2 pathway leads to this constitutive activation of BCL2 in these AE-resistant cells. Our results describe a mechanism in which the epigenetic state of chromatin affects ligand dependency during ERα-regulated gene expression. PMID:21841772

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

    PubMed Central

    Esber, Nathalie; Le Billan, Florian; Resche-Rigon, Michèle; Loosfelt, Hugues; Lombès, Marc; Chabbert-Buffet, Nathalie

    2015-01-01

    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. PMID:26474308

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

    PubMed

    Esber, Nathalie; Le Billan, Florian; Resche-Rigon, Michèle; Loosfelt, Hugues; Lombès, Marc; Chabbert-Buffet, Nathalie

    2015-01-01

    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. PMID:26474308

  7. Mechanism of fragility at BCL2 gene minor breakpoint cluster region during t(14;18) chromosomal translocation.

    PubMed

    Nambiar, Mridula; Raghavan, Sathees C

    2012-03-16

    The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg(2+) and Mn(2+) are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans. PMID:22275374

  8. Epithelial cell expression of BCL-2 family proteins predicts mechanisms that regulate Helicobacter pylori-induced pathology in the mouse stomach

    PubMed Central

    Yang, David X; Tashima, Kimihito; Taylor, Nancy S; Fox, James G

    2009-01-01

    Corpus-predominant infection with Helicobacter pylori (HP) results in the activation of programmed cell death pathways in surface, parietal, and chief cells. At present, mechanisms that regulate these pathways to result in HP-associated pathology are not fully understood. Because it is not known which survival and death pathways are present in gastric epithelial cells, we used an antibody panel to evaluate the expression of BCL-2 family prosurvival proteins or multi-Bcl-2 homology (BH)-domains (group 1) or BH3-only (group-2) proapoptotic proteins in the stomachs of uninfected or HP-infected C57BL/6 mice. This strategy identified BCL-2, BAK, and BAD as the major prosurvival and proapoptotic proteins, in surface cells and BAD as the only BCL-2 family protein expressed in parietal cells. Chief cells express altogether different effectors, including BCL-XL/BCL-2, for survival but have no constitutively expressed proapoptotic proteins. In model chief cells, however, the group 1 proapoptotic protein BCL-XS was expressed after exposure to proinflammatory cytokines concomitant with reduced viability, demonstrating that chief cells can transcriptionally regulate the induction of proapoptotic proteins to execute apoptosis. During HP infection, no additional BCL-2 family proteins were expressed in epithelial cells, whereas those present either remained unchanged or were reduced as cell deletion occurred over time. Additional studies demonstrated that the posttranslational regulation of BAD in surface and parietal cells was negatively affected by HP infection, a result that may be directly related to an increase in apoptosis during infection. Thus, gastric epithelial cells express cell-specific prosurvival and proapoptotic pathways. From the results presented here, mechanisms that regulate HP-related changes in the survival and death profile of gastric epithelial cells can be predicted and then tested, with the ultimate goal of elucidating important therapeutic targets to

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

    PubMed

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

    2016-01-01

    Borax, a boron compound and a salt of boric acid, is known to inhibit the growth of tumor cells. HepG2 cells have been shown to be clearly susceptible to the anti-proliferative effects of borax. However, the specific mechanisms regulating this effect are poorly understood. This study aimed to investigate the pathways underlying the growth inhibition induced by borax in HepG2 cells. The effects of borax on HepG2 cell viability were characterized using MTT. Apoptosis was also verified by annexin V/propidium iodide staining. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and p53, Bax, and Bcl-2 protein expression, respectively. Relevant mRNA levels were measured by qRT-PCR. Borax inhibited the proliferation of HepG2 cells in a time- and dose-dependent manner in vitro. The apoptotic process triggered by borax involved the upregulation of p53 and Bax and the downregulation of Bcl-2, which was confirmed by a change in the mitochondrial membrane potential. These results elucidate a borax-induced apoptotic pathway in HepG2 cells that involves the upregulation of p53 and Bax and the downregulation of Bcl-2. PMID:27420953

  10. A transcriptional regulatory element in the coding sequence of the human Bcl-2 gene

    PubMed Central

    Lang, Georgina; Gombert, Wendy M; Gould, Hannah J

    2005-01-01

    We investigated the protein-binding sites in a DNAse I hypersensitive site associated with bcl-2 gene expression in human B cells. We mapped this hypersensitive site to the coding sequence of exon 2 of the bcl-2 gene in the bcl-2-expressing REH B-cell line. Electrophoretic mobility shift assays (EMSAs) with extracts from REH cells revealed three previously unrecognized B-Myb-binding sites in this sequence. The protein was identified as B-Myb by using a specific antibody and EMSAs. Accordingly, the levels of B-Myb and bcl-2 proteins, and of Myb EMSA activity, were correlated over a wide range of cell lines, representing different stages of B-cell development. Transfection of REH cells with antisense B-myb down-regulated EMSA activity and the level of bcl-2, and led to the apoptosis of REH cells. Transfection of the bcl-2-non-expressing RPMI 8226 cell line with a B-Myb expression vector induced B-Myb EMSA activity and the expression of bcl-2. Reporter assays indicated that the HSS8 sequence containing the three B-Myb sites may act as an enhancer when it is linked to the bcl-2 gene promoter. Interaction of B-Myb with HSS8 may enhance bcl-2 gene expression by co-operating with positive regulatory elements (e.g. previously identified B-Myb response elements) or silencing negative response elements in the bcl-2 gene promoter. PMID:15606792

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

    PubMed Central

    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

  12. Bcl-2 associated with severity of manic symptoms in bipolar patients in a manic phase.

    PubMed

    Chen, Wei-Ting; Huang, Tiao-Lai; Tsai, Meng-Chang

    2015-02-28

    B cell lymphoma protein-2 (Bcl-2) may contribute to the pathophysiology of bipolar disorder, and may be involved in the therapeutic action of anti-manic drugs. The aim of this study was to investigate serum levels of Bcl-2 in bipolar patients in a manic phase, and evaluate the Bcl-2 changes after treatment. We consecutively enrolled 23 bipolar inpatients in a manic phase and 40 healthy subjects; 20 bipolar patients were followed up with treatment. Serum Bcl-2 levels were measured with assay kits. All 20 patients were evaluated by examining the correlation between Bcl-2 levels and Young Mania Rating Scale (YMRS) scores, using Spearman׳s correlation coefficients. The serum Bcl-2 levels in bipolar patients in a manic phase were higher than in healthy subjects, but without a significant difference. The YMRS scores were significantly negatively associated with serum Bcl-2 levels (p=0.042). Bcl-2 levels of the 20 bipolar patients were measured at the end of treatment. Using the Wilcoxon Signed Rank test, we found no significant difference in the Bcl-2 levels of bipolar patients after treatment. Our results suggest that Bcl-2 levels might be an indicator of severity of manic symptoms in bipolar patients in a manic phase. PMID:25563670

  13. AT-101 downregulates BCL2 and MCL1 and potentiates the cytotoxic effects of lenalidomide and dexamethasone in preclinical models of multiple myeloma and Waldenström macroglobulinaemia

    PubMed Central

    Akhtar, Sharoon; Personett, David; Miller, Kena C.; Thompson, Kevin J.; Carr, Jennifer; Kumar, Shaji; Roy, Vivek; Ansell, Stephen M.; Mikhael, Joseph R.; Dispenzieri, Angela; Reeder, Craig B.; Rivera, Candido E.; Foran, James; Chanan-Khan, Asher

    2015-01-01

    Summary Multiple myeloma, the second most common haematological malignancy in the U.S., is currently incurable. Disruption of the intrinsic apoptotic pathway by BCL2 and MCL1 upregulation is observed in >80% of myeloma cases and is associated with an aggressive clinical course. Remarkably, there is no approved drug with the ability to target BCL2 or MCL1. Thus, we investigated the anti-tumour effects of a pan-BCL2 inhibitor, AT-101, which has high binding specificity for BCL2 and MCL1 in preclinical models of plasma cell cancers (Multiple myeloma and Waldenström macroglobulinaemia). Gene expression and immunoblot analysis of six plasma cell cancer models showed upregulation of BCL2 family members. AT-101 was able to downregulate BCL2 and MCL1 in all plasma cell cancer models and induced apoptotic cell death in a caspase-dependent manner by altering mitochondrial membrane permeability. This cytotoxic effect and BCL2 downregulation were further potentiated when AT-101 was combined with lenalidomide/dexamethasone (LDA). NanoString nCounter mRNA quantification and Ingenuity Pathways Analysis revealed differential changes in the CCNA2, FRZB, FYN, IRF1, PTPN11 genes in LDA-treated cells. In summary, we describe for the first time the cellular and molecular events associated with the use of AT-101 in combination with lenalidomide/dexamethasone in preclinical models of plasma cell malignancy. PMID:24236538

  14. Downregulation of miR-23a and miR-27a following Experimental Traumatic Brain Injury Induces Neuronal Cell Death through Activation of Proapoptotic Bcl-2 Proteins

    PubMed Central

    Zhao, Zaorui; Loane, David J.; Wu, Junfang; Borroto, Carlos; Dorsey, Susan G.; Faden, Alan I.

    2014-01-01

    MicroRNAs (miRs) are small noncoding RNAs that negatively regulate gene expression at the post-transcriptional level. To identify miRs that may regulate neuronal cell death after experimental traumatic brain injury (TBI), we profiled miR expression changes during the first several days after controlled cortical impact (CCI) in mice. miR-23a and miR-27a were rapidly downregulated in the injured cortex in the first hour after TBI. These changes coincided with increased expression of the proapoptotic Bcl-2 family members Noxa, Puma, and Bax. In an etoposide-induced in vitro model of apoptosis in primary cortical neurons, miR-23a and miR-27a were markedly downregulated as early as 1 h after exposure, before the upregulation of proapoptotic Bcl-2 family molecules. Administration of miR-23a and miR-27a mimics attenuated etoposide-induced changes in Noxa, Puma, and Bax, reduced downstream markers of caspase-dependent (cytochrome c release and caspase activation) and caspase-independent (apoptosis-inducing factor release) pathways, and limited neuronal cell death. In contrast, miRs hairpin inhibitors enhanced etoposide-induced neuronal apoptosis and caspase activation. Importantly, administration of miR-23a and miR-27a mimics significantly reduced activation of Puma, Noxa, and Bax as well as attenuated markers of caspase-dependent and -independent apoptosis after TBI. Furthermore, miR-23a and miR-27a mimics significantly attenuated cortical lesion volume and neuronal cell loss in the hippocampus after TBI. These findings indicate that post-traumatic decreases in miR-23a and miR-27a contribute to neuronal cell death after TBI by upregulating proapoptotic Bcl-2 family members, thus providing a novel therapeutic target. PMID:25057207

  15. Artesunate induces apoptosis through caspase-dependent and -independent mitochondrial pathways in human myelodysplastic syndrome SKM-1 cells.

    PubMed

    Wang, Ying; Yang, Jingci; Chen, Li; Wang, Jiamin; Wang, Yaqian; Luo, Jianmin; Pan, Ling; Zhang, Xuejun

    2014-08-01

    Artesunate (ART) is a semi-synthetic derivative of artemisinin extracted from Artemisia annua (sweet wormwood) that is conventionally used in anti-malarial drugs and more recently in medications that induce tumor cell apoptosis. Here, we investigated the effects and mechanistic pathways of ART in human myelodysplastic syndrome (MDS), a condition that commonly progresses to acute myeloid leukemia (AML). Human MDS SKM-1 cells, primary bone marrow (PBM) mononuclear cells from patients with refractory anemia with excess blasts (RAEB) or MDS-AML (MDS cell group), and PBM stromal cells from three patients without hematological diseases (non-MDS cell group) were cultured for 24, 48, or 72 h with or without various ART concentrations. CCK-8, western blot, JC-1 fluorescence, and Annexin-V/Propidium iodide (PI) labeling were used to assess cell proliferation, protein levels, mitochondrial membrane potentials (MMPs) and apoptosis, respectively. ART administration dose- and time-dependently inhibited SKM-1 proliferation. At 24, 48, and 72 h, ART IC₅₀ values were 89.92, 4.24, and 1.28 μmol/L, respectively. ART only significantly inhibited proliferation in the MDS cell group, but it has little impact on proliferation of non-MDS cells. ART decreased MMPs, and dose-dependently induced SKM-1 cell apoptosis, peaking at 82.9% when treated with 200 μmol/L ART for 24h. Caspase-3 and -9 activation, poly(ADP-ribose) polymerase cleavage, decreased Bcl-2/Bax ratio and apoptosis inducing factor nuclear localization were implicated in apoptosis. Our results indicate that ART effectively induces apoptosis in SKM-1 cells through both caspase-dependent and -independent mitochondrial pathways. PMID:24704559

  16. Bcl-2 stabilization by paxillin confers 5-fluorouracil resistance in colorectal cancer

    PubMed Central

    Wu, D-W; Huang, C-C; Chang, S-W; Chen, T-H; Lee, H

    2015-01-01

    5-Fluorouracil (5-FU) is chemotherapeutic agent widely used for the treatment of colorectal cancer. Unfortunately, advanced colorectal cancer is often resistance to such chemotherapy and poor outcome. An adaptor protein paxillin (PXN) is phosphorylated at Y31/Y118 (pPXN-Y31/Y118) by Src contributes to cell mobility and Ser (S)272 of PXN in LD4 domain is important to the interaction between PXN and Bcl-2. We thus hypothesized that pPXN-Y31/Y118 may be required for Bcl-2 protein stability via PXN interacting with Bcl-2 to confer 5-FU resistance in colorectal cancer. Mechanistically, pPXN-S272 is phosphorylated through pPXN-Y31/Y118-mediated p21 protein-activated kinase 1 (PAK1) activation and pPXN-S272 is required for PXN to interact with Bcl-2. The interaction between PXN and Bcl-2 is essential for Bcl-2 protein stability through phosphorylation of Bcl-2 at S87 (pBcl-2-S87) by pPXN-Y31/Y118-mediated ERK activation. An increase in Bcl-2 expression by PXN is responsible for resistance to 5-FU. The resistance to 5-FU can be abolished by inhibitor of Src and PAK1 or Bcl-2 antagonist in cell and animal models. Among patients, Bcl-2 expression is positively correlated with expression of PXN and pPXN-S272, respectively. Patients with high PXN/high Bcl-2 or high pPXN-S272/high Bcl-2 tumors are commonly to have an unfavorable response to 5-FU-based chemotherapy, compared with patients who have high PXN, high pPXN-S272 or high Bcl-2 tumors alone. Therefore, we suggest that Src, PAK1 or Bcl-2 inhibitor may potentially overcome the resistance of 5-FU-based chemotherapy and consequently to improve outcomes in patients with PXN/Bcl-2 and pPXN-S272/Bcl-2-positive tumors. PMID:25323586

  17. Acidosis promotes Bcl-2 family-mediated evasion of apoptosis: involvement of acid-sensing G protein-coupled receptor Gpr65 signaling to Mek/Erk.

    PubMed

    Ryder, Christopher; McColl, Karen; Zhong, Fei; Distelhorst, Clark W

    2012-08-10

    Acidosis arises in solid and lymphoid malignancies secondary to altered nutrient supply and utilization. Tumor acidosis correlates with therapeutic resistance, although the mechanism behind this effect is not fully understood. Here we show that incubation of lymphoma cell lines in acidic conditions (pH 6.5) blocks apoptosis induced by multiple cytotoxic metabolic stresses, including deprivation of glucose or glutamine and treatment with dexamethasone. We sought to examine the role of the Bcl-2 family of apoptosis regulators in this process. Interestingly, we found that acidic culture causes elevation of both Bcl-2 and Bcl-xL, while also attenuating glutamine starvation-induced elevation of p53-up-regulated modulator of apoptosis (PUMA) and Bim. We confirmed with knockdown studies that these shifts direct survival decisions during starvation and acidosis. Importantly, the promotion of a high anti- to pro-apoptotic Bcl-2 family member ratio by acidosis renders cells exquisitely sensitive to the Bcl-2/Bcl-xL antagonist ABT-737, suggesting that acidosis causes Bcl-2 family dependence. This dependence appears to be mediated, in part, by the acid-sensing G protein-coupled receptor, GPR65, via a MEK/ERK pathway. PMID:22685289

  18. Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-xL

    PubMed Central

    Huang, David C. S.; Hahne, Michael; Schroeter, Michael; Frei, Karl; Fontana, Adriano; Villunger, Andreas; Newton, Kim; Tschopp, Juerg; Strasser, Andreas

    1999-01-01

    Fas activation triggers apoptosis in many cell types. Studies with anti-Fas antibodies have produced conflicting results on Fas signaling, particularly the role of the Bcl-2 family in this process. Comparison between physiological ligand and anti-Fas antibodies revealed that only extensive Fas aggregation, by membrane bound FasL or aggregated soluble FasL consistently triggered apoptosis, whereas antibodies could act as death agonists or antagonists. Studies on Fas signaling in cell lines and primary cells from transgenic mice revealed that FADD/MORT1 and caspase-8 were required for apoptosis. In contrast, Bcl-2 or Bcl-xL did not block FasL-induced apoptosis in lymphocytes or hepatocytes, demonstrating that signaling for cell death induced by Fas and the pathways to apoptosis regulated by the Bcl-2 family are distinct. PMID:10611305

  19. Bcl-2/IgH expression in minimal bone marrow infiltration by follicular lymphoma cells.

    PubMed

    Che, Yi-Qun; Liu, Peng; Wang, Yue; Zhang, Chang-Gong; Han, Ya-Ling; Shen, Di; Zhang, Ying; Zheng, Cui-Ling; Qi, Jun; Wang, Qing-Tao

    2012-02-01

    The purpose of this study was to investigate the roles of bcl-2 chromosomal translocation and Bcl-2 protein expression in follicular lymphoma (FL) minimal bone marrow (BM) infiltration. We identified the same bcl-2/IgH fusion gene in paraffin-embedded lymph node (LN) samples and BM samples using immunohistochemistry (IHC), immunocytochemistry (ICC), cytologic morphology and fluorescence in situ hybridization (FISH). The presence of the Bcl-2/IgH fusion gene in the BM samples and paraffin-embedded LN samples from 56 patients with follicular lymphomas was detected using FISH. The Bcl-2 protein levels in BM and paraffin-embedded tissues were quantified using ICC and IHC, respectively. Approximately 78.6% (44/56) of the paraffin‑embedded LN tissue sections that underwent FISH analysis had a bcl-2/IgH translocation. The primary lesion was also positive for the bcl-2/IgH fusion gene, as were the BM minimal infiltrates. The bcl-2/IgH rearrangement occurred in 88.6% (39/44) of the BM specimens. The bcl-2/IgH recombination rate in stage III/IV cancers was significantly different to that observed in stage I/II cancers (p=0.041). In 59% (23/39) of the cases with t(14;18), Bcl-2 was found to be present as assessed by ICC. Positive Bcl-2 ICC staining and the t(14;18) translocation (as detected using FISH) were positively correlated (p=0.028). We then applied the FISH method to slides that had previously been morphologically evaluated using Wright-Giemsa staining; any slides with at least one abnormal cell were subjected to FISH analysis following staining. The assessment of bcl-2/IgH translocation status may contribute to the better detection of minimal BM infiltration by FL cells. Utilizing FISH and cytologic morphology techniques allows for earlier and more accessible BM examination. PMID:22052344

  20. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy.

    PubMed

    Czabotar, Peter E; Lessene, Guillaume; Strasser, Andreas; Adams, Jerry M

    2014-01-01

    The BCL-2 protein family determines the commitment of cells to apoptosis, an ancient cell suicide programme that is essential for development, tissue homeostasis and immunity. Too little apoptosis can promote cancer and autoimmune diseases; too much apoptosis can augment ischaemic conditions and drive neurodegeneration. We discuss the biochemical, structural and genetic studies that have clarified how the interplay between members of the BCL-2 family on mitochondria sets the apoptotic threshold. These mechanistic insights into the functions of the BCL-2 family are illuminating the physiological control of apoptosis, the pathological consequences of its dysregulation and the promising search for novel cancer therapies that target the BCL-2 family. PMID:24355989

  1. [Prognosis value of the immunohistochemical expresion of the bcl-2 in the larynx epidermoid cancer].

    PubMed

    García Lozano, M C; Orradre Romero, J L; Caro García, M; Sáez del Castillo, A I; Galán Morales, J T; Piris Pinilla, M A

    2005-01-01

    In this paper we carried out an immunohistochemical study of bcl-2 protein expression in a series of 195 patients with laryngeal carcinoma that were diagnosticated, treated and followed at the Department of Otolaryngology at "Virgen de la Salud" Hospital (Toledo, Spain). In the cases with lymphonode metastasis we also analysed bcl-2 protein expression at this level. Furthermore we have studied the value of bcl-2 protein expression as a prognostic factor (tumor recurrence, deads due to cancer and survival) and we analysed the relationship between bcl-2 protein expression and other clinic and pathologic parameters. PMID:15803918

  2. Stabilization of G-quadruplex DNA and inhibition of Bcl-2 expression by a pyridostatin analog.

    PubMed

    Feng, Yun; Yang, Dazhang; Chen, Hongbo; Cheng, Wenli; Wang, Lixia; Sun, Hongxia; Tang, Yalin

    2016-04-01

    The G-quadruplexes located in the P1 promoter of B-cell lymphoma-2 (Bcl-2) gene are implicated to regulate Bcl-2 expression. Here, we designed a new pyridostatin analog named PDF, which exhibited high specificity and stabilizing effect toward G-quadruplexes. The luciferase assay demonstrated that PDF could significantly suppress Bcl-2 transcriptional activation in human laryngeal squamous carcinoma cells (Hep-2) cells. Besides, PDF also induced cell apoptosis in vitro assays. These results provide an excellent G-quadruplex specific ligand as an efficient Bcl-2 inhibitor. These results also implicate that PDF may be a potential anticancer drug to head neck cancer. PMID:26923693

  3. Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

    PubMed Central

    Poliseno, Laura; Bianchi, Laura; Citti, Lorenzo; Liberatori, Sabrina; Mariani, Laura; Salvetti, Alessandra; Evangelista, Monica; Bini, Luca; Pallini, Vitaliano; Rainaldi, Giuseppe

    2004-01-01

    We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed. PMID:14748742

  4. Antiapoptotic Bcl-2 protein as a potential target for cancer therapy: A mini review.

    PubMed

    Jagani, Hitesh; Kasinathan, Narayanan; Meka, Sreenivasa Reddy; Josyula, Venkata Rao

    2016-08-01

    Bcl-2, an antiapoptotic protein, is considered as a potential target in cancer treatment since its oncogenic potential has been proven and is well documented. Antisense technology and RNA interference (RNAi) have been used to reduce the expression of the Bcl-2 gene in many types of cancer cells and are effective as adjuvant therapy along with the chemotherapeutic agents. The lack of appropriate delivery systems is considered to be the main hurdle associated with the RNAi. In this review, we discuss the antiapoptotic Bcl-2 protein, its oncogenic potential, and various approaches utilized to target Bcl-2 including suitable delivery systems employed for successful delivery of siRNA. PMID:25801037

  5. Role of Bcl-2 and its associated miRNAs in vasculogenic mimicry of hepatocellular carcinoma

    PubMed Central

    Zhao, Nan; Sun, Bao-Cun; Zhao, Xiu-Lan; Wang, Yong; Meng, Jie; Che, Na; Dong, Xu-Yi; Gu, Qiang

    2015-01-01

    Objective: An investigation of the role of the anti-apoptotic protein Bcl-2 and its associated miRNAs in vasculogenic mimicry (VM) in hepatocellular carcinoma. Methods: The Bcl-2 expression plasmid was constructed for transfection into the hepatocellular carcinoma cell line HepG2. Changes in the expression profiles of the miRNAs induced by Bcl-2 overexpression and their relationships with vasculogenic mimicry were analysed. Real-time PCR was performed in frozen tissue specimens from 42 cases of hepatocellular carcinoma to analyse the relationship between Bcl-2 and miR-27a; Immunohistochemical staining was performed in paraffin-embedded tissue samples from 97 cases of hepatocellular carcinoma to analyse the relationship between Bcl-2 expression and the expression of vasculogenic mimicry (VM) related molecules VEGF and HIF1A, which were target genes of the Bcl-2 related miRNAs. Results: Overexpression of Bcl-2 results in a significant change in the expression of a wide range of miRNAs, and the target genes of these miRNAs are composed of various vasculogenic mimicry related genes; Bcl-2 expression was positively correlated with the expression of the miRNA target genes VEGF and HIF1A. The expression of VEGF and HIF1A was significantly and positively correlated with VM and poor prognosis of patients. Conclusion: Bcl-2 may play a role in vasculogenic mimicry through miRNAs by targeting angiogenesis associated genes. PMID:26884845

  6. Nicotine mediates oxidative stress and apoptosis through cross talk between NOX1 and Bcl-2 in lung epithelial cells.

    PubMed

    Zanetti, Filippo; Giacomello, Marta; Donati, Yves; Carnesecchi, Stephanie; Frieden, Maud; Barazzone-Argiroffo, Constance

    2014-11-01

    Nicotine contributes to the onset and progression of several pulmonary diseases. Among the various pathophysiological mechanisms triggered by nicotine, oxidative stress and cell death are reported in several cell types. We found that chronic exposure to nicotine (48h) induced NOX1-dependent oxidative stress and apoptosis in primary pulmonary cells. In murine (MLE-12) and human (BEAS-2B) lung epithelial cell lines, nicotine acted as a sensitizer to cell death and synergistically enhanced apoptosis when cells were concomitantly exposed to hyperoxia. The precise signaling pathway was investigated in MLE-12 cells in which NOX1 was abrogated by a specific inhibitor or stably silenced by shRNA. In the early phase of exposure (1h), nicotine mediated intracellular Ca(2+) fluxes and activation of protein kinase C, which in its turn activated NOX1, leading to cellular and mitochondrial oxidative stress. The latter triggered the intrinsic apoptotic machinery by modulating the expression of Bcl-2 and Bax. Overexpression of Bcl-2 completely prevented nicotine's detrimental effects, suggesting Bcl-2as a downstream key regulator in nicotine/NOX1-induced cell damage. These results suggest that NOX1 is a major contributor to the generation of intracellular oxidative stress induced by nicotine and might be an important molecule to target in nicotine-related lung pathologies. PMID:25151121

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

    PubMed Central

    Gaudette, Brian T.; Dwivedi, Bhakti; Chitta, Kasyapa S.; Poulain, Stéphanie; Powell, Doris; Vertino, Paula; Leleu, Xavier; Lonial, Sagar; Chanan-Khan, Asher A.; Kowalski, Jeanne; Boise, Lawrence H.

    2015-01-01

    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 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. PMID:25893290

  8. MYBL2 guides autophagy suppressor VDAC2 in the developing ovary to inhibit autophagy through a complex of VDAC2-BECN1-BCL2L1 in mammals

    PubMed Central

    Yuan, Jia; Zhang, Ying; Sheng, Yue; Fu, Xiazhou; Cheng, Hanhua; Zhou, Rongjia

    2015-01-01

    Oogenesis is essential for female gamete production in mammals. The total number of ovarian follicles is determined early in life and production of ovarian oocytes is thought to stop during the lifetime. However, the molecular mechanisms underling oogenesis, particularly autophagy regulation in the ovary, remain largely unknown. Here, we reveal an important MYBL2-VDAC2-BECN1-BCL2L1 pathway linking autophagy suppression in the developing ovary. The transcription factors GATA1 and MYBL2 can bind to and activate the Vdac2 promoter. MYBL2 regulates the spatiotemporal expression of VDAC2 in the developing ovary. Strikingly, in the VDAC2 transgenic pigs (Sus scrofa/Ss), VDAC2 exerts its function by inhibiting autophagy in the ovary. In contrast, Vdac2 knockout promotes autophagy. Moreover, VDAC2-mediated autophagy suppression is dependent on its interactions with both BECN1 and BCL2L1 to stabilize the BECN1 and BCL2L1 complex, suggesting VDAC2 as an autophagy suppressor in the pathway. Our findings provide a functional connection among the VDAC2, MYBL2, the BECN1-BCL2L1 pathway and autophagy suppression in the developing ovary, which is implicated in improving female fecundity. PMID:26060891

  9. MYBL2 guides autophagy suppressor VDAC2 in the developing ovary to inhibit autophagy through a complex of VDAC2-BECN1-BCL2L1 in mammals.

    PubMed

    Yuan, Jia; Zhang, Ying; Sheng, Yue; Fu, Xiazhou; Cheng, Hanhua; Zhou, Rongjia

    2015-01-01

    Oogenesis is essential for female gamete production in mammals. The total number of ovarian follicles is determined early in life and production of ovarian oocytes is thought to stop during the lifetime. However, the molecular mechanisms underling oogenesis, particularly autophagy regulation in the ovary, remain largely unknown. Here, we reveal an important MYBL2-VDAC2-BECN1-BCL2L1 pathway linking autophagy suppression in the developing ovary. The transcription factors GATA1 and MYBL2 can bind to and activate the Vdac2 promoter. MYBL2 regulates the spatiotemporal expression of VDAC2 in the developing ovary. Strikingly, in the VDAC2 transgenic pigs (Sus scrofa/Ss), VDAC2 exerts its function by inhibiting autophagy in the ovary. In contrast, Vdac2 knockout promotes autophagy. Moreover, VDAC2-mediated autophagy suppression is dependent on its interactions with both BECN1 and BCL2L1 to stabilize the BECN1 and BCL2L1 complex, suggesting VDAC2 as an autophagy suppressor in the pathway. Our findings provide a functional connection among the VDAC2, MYBL2, the BECN1-BCL2L1 pathway and autophagy suppression in the developing ovary, which is implicated in improving female fecundity. PMID:26060891

  10. Protein folding: independent unrelated pathways or predetermined pathway with optional errors.

    PubMed

    Bédard, Sabrina; Krishna, Mallela M G; Mayne, Leland; Englander, S Walter

    2008-05-20

    The observation of heterogeneous protein folding kinetics has been widely interpreted in terms of multiple independent unrelated pathways (IUP model), both experimentally and in theoretical calculations. However, direct structural information on folding intermediates and their properties now indicates that all of a protein population folds through essentially the same stepwise pathway, determined by cooperative native-like foldon units and the way that the foldons fit together in the native protein. It is essential to decide between these fundamentally different folding mechanisms. This article shows, contrary to previous supposition, that the heterogeneous folding kinetics observed for the staphylococcal nuclease protein (SNase) does not require alternative parallel pathways. SNase folding kinetics can be fit equally well by a single predetermined pathway that allows for optional misfolding errors, which are known to occur ubiquitously in protein folding. Structural, kinetic, and thermodynamic information for the folding intermediates and pathways of many proteins is consistent with the predetermined pathway-optional error (PPOE) model but contrary to the properties implied in IUP models. PMID:18480257

  11. BCL2 suppresses PARP1 function and non-apoptotic cell death

    PubMed Central

    Dutta, Chaitali; Day, Tovah; Kopp, Nadja; van Bodegom, Diederik; Davids, Matthew S.; Ryan, Jeremy; Bird, Liat; Kommajosyula, Naveen; Weigert, Oliver; Yoda, Akinori; Fung, Hua; Brown, Jennifer R.; Shapiro, Geoffrey I.; Letai, Anthony; Weinstock, David M.

    2014-01-01

    BCL2 suppresses apoptosis by binding the BH3 domain of pro-apoptotic factors and thereby regulating outer mitochondrial membrane permeabilization. Many tumor types, including B-cell lymphomas and chronic lymphocytic leukemia, are dependent on BCL2 for survival, but become resistant to apoptosis after treatment. Here we identified a direct interaction between the anti-apoptotic protein BCL2 and the enzyme poly(ADP) ribose polymerase 1 (PARP1), which suppresses PARP1 enzymatic activity and inhibits PARP1-dependent DNA repair in diffuse large B cell lymphoma cells. The BH3 mimetic ABT-737 displaced PARP1 from BCL2 in a dose-dependent manner, re-establishing PARP1 activity and DNA repair and promoting non-apoptotic cell death. This form of cell death was unaffected by resistance to single-agent ABT-737 that results from upregulation of anti-apoptotic BCL2 family members. Based on the ability of BCL2 to suppress PARP1 function, we hypothesized that ectopic BCL2 expression would kill PARP inhibitor-sensitive cells. Strikingly, BCL2 expression reduced the survival of PARP inhibitor-sensitive breast cancer and lung cancer cells by 90-100%, and these effects were reversed by ABT-737. Taken together, our findings demonstrate that a novel interaction between BCL2 and PARP1 blocks PARP1 enzymatic activity and suppresses PARP1-dependent repair. Targeted disruption of the BCL2-PARP1 interaction therefore may represent a potential therapeutic approach for BCL2-expressing tumors resistant to apoptosis. PMID:22689920

  12. B Cell Lymphoma (Bcl)-2 Protein Is the Major Determinant in bcl-2 Adenine-Uridine-rich Element Turnover Overcoming HuR Activity*

    PubMed Central

    Ghisolfi, Laura; Calastretti, Angela; Franzi, Sara; Canti, Gianfranco; Donnini, Martino; Capaccioli, Sergio; Nicolin, Angelo; Bevilacqua, Annamaria

    2009-01-01

    In the 3′-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins. PMID:19520857

  13. B cell lymphoma (Bcl)-2 protein is the major determinant in bcl-2 adenine-uridine-rich element turnover overcoming HuR activity.

    PubMed

    Ghisolfi, Laura; Calastretti, Angela; Franzi, Sara; Canti, Gianfranco; Donnini, Martino; Capaccioli, Sergio; Nicolin, Angelo; Bevilacqua, Annamaria

    2009-07-31

    In the 3'-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins. PMID:19520857

  14. Cladribine induces apoptosis in human leukaemia cells by caspase-dependent and -independent pathways acting on mitochondria.

    PubMed Central

    Marzo, I; Pérez-Galán, P; Giraldo, P; Rubio-Félix, D; Anel, A; Naval, J

    2001-01-01

    We have studied the role of caspases and mitochondria in apoptosis induced by 2-chloro-2'-deoxyadenosine (cladribine) in several human leukaemic cell lines. Cladribine treatment induced mitochondrial transmembrane potential (DeltaPsi(m)) loss, phosphatidylserine exposure, caspase activation and development of typical apoptotic morphology in JM1 (pre-B), Jurkat (T) and U937 (promonocytic) cells. Western-blot analysis of cell extracts revealed the activation of at least caspases 3, 6, 8 and 9. Co-treatment with Z-VAD-fmk (benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone), a general caspase inhibitor, significantly prevented cladribine-induced death in JM1 and Jurkat cells for the first approximately 40 h, but not for longer times. Z-VAD-fmk also partly prevented some morphological and biochemical features of apoptosis in U937 cells, but not cell death. Co-incubation with selective caspase inhibitors Ac-DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde), Ac-LEHD-CHO (N-acetyl-Leu-Glu-His-Asp-aldehyde) or Z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone), inhibition of protein synthesis with cycloheximide or cell-cycle arrest with aphidicolin did not prevent cell death. Overexpression of Bcl-2, but not CrmA, efficiently prevented death in Jurkat cells. In all cell lines, death was always preceded by Delta Psi(m) loss and accompanied by the translocation of the protein apoptosis-inducing factor (AIF) from mitochondria to the nucleus. These results suggest that caspases are differentially involved in induction and execution of apoptosis depending on the leukaemic cell lineage. In any case, Delta Psi(m) loss marked the point of no return in apoptosis and may be caused by two different pathways, one caspase-dependent and the other caspase-independent. Execution of apoptosis was always performed after Delta Psi(m) loss by a caspase-9-triggered caspase cascade and the action of AIF. PMID:11672427

  15. Anti-apoptotic gene Bcl2 is required for stapes development and hearing.

    PubMed

    Carpinelli, M R; Wise, A K; Arhatari, B D; Bouillet, P; Manji, S S M; Manning, M G; Cooray, A A; Burt, R A

    2012-01-01

    In this paper we describe novel and specific roles for the apoptotic regulators Bcl2 and Bim in hearing and stapes development. Bcl2 is anti-apoptotic while Bim is pro-apoptotic. Characterization of the auditory systems of mice deficient for these molecules revealed that Bcl2⁻/⁻ mice suffered severe hearing loss. This was conductive in nature and did not affect sensory cells of the inner ear, with cochlear hair cells and neurons present and functional. Bcl2⁻/⁻ mice were found to have a malformed, often monocrural, porous stapes (the small stirrup-shaped bone of the middle ear), but a normally shaped malleus and incus. The deformed stapes was discontinuous with the incus and sometimes fused to the temporal bones. The defect was completely rescued in Bcl2⁻/⁻Bim⁻/⁻ mice and partially rescued in Bcl2⁻/⁻Bim⁺/⁻ mice, which displayed high-frequency hearing loss and thickening of the stapes anterior crus. The Bcl2⁻/⁻ defect arose in utero before or during the cartilage stage of stapes development. These results implicate Bcl2 and Bim in regulating survival of second pharyngeal arch or neural crest cells that give rise to the stapes during embryonic development. PMID:22874999

  16. The Role of BCL-2 Family Members in Acute Kidney Injury.

    PubMed

    Borkan, Steven C

    2016-05-01

    B-cell lymphoma 2 (BCL-2) family proteins gather at the biologic cross-roads of renal cell survival: the outer mitochondrial membrane. Despite shared sequence and structural features, members of this conserved protein family constantly antagonize each other in a life-and-death battle. BCL-2 members innocently reside within renal cells until activated or de-activated by physiologic stresses caused by common nephrotoxins, transient ischemia, or acute glomerulonephritis. Recent experimental data not only illuminate the intricate mechanisms of apoptosis, the most familiar form of BCL-2-mediated cell death, but emphasizes their newfound roles in necrosis, necroptosis, membrane pore transition regulated necrosis, and other forms of acute cell demise. A major paradigm shift in non-cell death roles of the BCL-2 family has occurred. BCL-2 proteins also regulate critical daily renal cell housekeeping functions including cell metabolism, autophagy (an effective means for recycling cell components), mitochondrial morphology (organelle fission and fusion), as well as mitochondrial biogenesis. This article considers new concepts in the biochemical and structural regulation of BCL-2 proteins that contribute to membrane pore permeabilization, a universal feature of cell death. Despite these advances, persistent BCL-2 family mysteries continue to challenge cell biologists. Given their interface with many intracellular functions, it is likely that BCL-2 proteins determine cell viability under many pathologic circumstances relevant to the nephrologist and, as a consequence, represent an ideal therapeutic target. PMID:27339388

  17. The coffee diterpene kahweol sensitizes TRAIL-induced apoptosis in renal carcinoma Caki cells through down-regulation of Bcl-2 and c-FLIP.

    PubMed

    Um, Hee Jung; Oh, Jung Hwa; Kim, Yoon-Nyun; Choi, Yung Hyun; Kim, Sang Hyun; Park, Jong-Wook; Kwon, Taeg Kyu

    2010-06-01

    Kahweol, a coffee-specific diterpene, found in the beans of Coffea arabica, has potent anti-carcinogenic, anti-tumor, and anti-inflammatory properties. TRAIL is a potential anti-cancer compound that induces apoptosis in a wide variety of cancer cells, but not in most normal human cell types. In the present study, we show that kahweol sensitizes human renal cancer cells, but not normal human mesangial cells, to TRAIL-mediated apoptosis. Moreover, treatment with a combination of kahweol and TRAIL induces significant apoptosis in various cancer cell types, thus presenting an attractive novel strategy for cancer treatment. Our experiments show that treatment with a combination of kahweol and TRAIL-induced apoptosis, and stimulated of DEVDase activity, DNA fragmentation, and cleavage of PARP, which was prevented by pretreatment with z-VAD, indicative of cell death via a caspase-dependent pathway. Kahweol-induced down-regulation of Bcl-2 and ectopic expression of Bcl-2 led to attenuation of kahweol plus TRAIL-mediated apoptosis, indicative of Bcl-2 involvement in the apoptotic process. In addition, the c-FLIP and caspase signal pathways seem to play a crucial role in apoptosis triggered by the combination of kahweol and TRAIL in Caki cells. Our results collectively demonstrate that down-regulation of Bcl-2 and c-FLIP contributes to the sensitizing effect of kahweol on TRAIL-mediated apoptosis in cancer cells. PMID:20403343

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

    PubMed Central

    Waibel, Michaela; Solomon, Vanessa S.; Knight, Deborah A.; Ralli, Rachael A.; Kim, Sang-Kyu; Banks, Kellie-Marie; Vidacs, Eva; Virely, Clemence; Sia, Keith C.S.; Bracken, Lauryn S.; Collins-Underwood, Racquel; Drenberg, Christina; Ramsey, Laura B.; Meyer, Sara C.; Takiguchi, Megumi; Dickins, Ross A.; Levine, Ross; Ghysdael, Jacques; Dawson, Mark A.; Lock, Richard B.; Mullighan, Charles G.; Johnstone, Ricky W.

    2013-01-01

    Summary 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. PMID:24268771

  19. Expression of Mitochondria-Associated Genes (PPARGC1A, NRF-1, BCL-2 and BAX) in Follicular Development and Atresia of Goat Ovaries.

    PubMed

    Zhang, G; Wan, Y; Zhang, Y; Lan, S; Jia, R; Wang, Z; Fan, Y; Wang, F

    2015-06-01

    Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via the mitochondria-dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondria-associated genes peroxisome proliferator-activated receptor-gamma, coactivator1-alpha (PPARGC1A), nuclear respiratory factor-1 (NRF-1), B-cell CLL/lymphoma 2 (BCL-2) and BCL2-associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria-associated proteins (PGC-1α, which are encoded by the PPARGC1A gene, NRF-1, BCL-2 and BAX) mainly expressed in granulosa cells. The mRNA and protein levels of PPARGC1A/PGC-1α and NRF-1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF-1, increased BAX expression and increased ratio of BAX to BCL-2 expression. These results suggested that changes of the mitochondria-associated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development. PMID:25779891

  20. Sec6/8 regulates Bcl-2 and Mcl-1, but not Bcl-xl, in malignant peripheral nerve sheath tumor cells.

    PubMed

    Tanaka, Toshiaki; Kikuchi, Noriaki; Goto, Kaoru; Iino, Mitsuyoshi

    2016-05-01

    Sec6 and Sec8, which are components of the exocyst complex, has been concerned with various roles independent of its role in secretion, such as cell migration, invadopodia formation, cytokinesis, glucose uptake, and neural development. Given the vital roles of the exocyst complex in cellular and developmental processes, the disruption of its function may be closely related to various diseases such as cancer, diabetes, and neuronal disorders. Malignant peripheral nerve sheath tumors (MPNSTs) have high malignant potential and poor prognosis because of aggressive progression and metastasis. To date, no chemotherapeutic agents have been validated for MPNSTs treatment because how MPNSTs are resistant to chemotherapeutic agents remains unknown. This study demonstrates that combination of doxorubicin and sorafenib induces apoptosis in MPNST cells through downregulation of B cell lymphoma protein 2 (Bcl-2), Bcl-2-related protein long form of Bcl-x (Bcl-xl), and myeloid cell leukemia 1 (Mcl-1). Moreover, both Sec6 and Sec8 levels decreased after treatment with doxorubicin and sorafenib and were found to be associated with Bcl-2 and Mcl-1 expressions, but not Bcl-xl. Although Sec8 was found to be involved in the regulation of both Bcl-2 and Mcl-1 at the mRNA level, Sec6 regulated Bcl-2 at the mRNA level and the binding affinity of F-box and WD repeat domain containing 7 and Mcl-1, thereby controlling Mcl-1 at the protein level. Bcl-2 or Mcl-1 mRNA suppression by Sec6 or Sec8 depletion resulted in significant changes in nuclear factor-kappa B, cAMP response element, and p53 transcriptional activity. These results suggest that Sec6 and Sec8 are therapeutic target molecules in MPNST. PMID:26892009

  1. The selective BH4-domain biology of Bcl-2-family members: IP3Rs and beyond.

    PubMed

    Monaco, Giovanni; Vervliet, Tim; Akl, Haidar; Bultynck, Geert

    2013-04-01

    Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca(2+) signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IP3R): Bcl-2 likely acts as an endogenous inhibitor of the IP3R, preventing pro-apoptotic Ca(2+) transients, while Bcl-XL likely acts as an endogenous IP3R-sensitizing protein promoting pro-survival Ca(2+) oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-XL may underlie the divergence in IP3R regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IP3R, is likely to be Bcl-2's determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca(2+)-channel tail and might be more crucial for Bcl-XL's function. Furthermore, one amino acid critically different in the sequence of Bcl-2's and Bcl-XL's BH4 domains underpins their selective effect on Ca(2+) signaling and distinct biological properties of Bcl-2 versus Bcl-XL. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-XL functions in normal cells. Thus, IP3R-derived molecules that mimic the BH4 domain's binding site on the IP3R may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2's proto-oncogenic activity. Finally, a more general role for the BH4 domain on IP3Rs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions. PMID:22955373

  2. Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes.

    PubMed Central

    Lam, M; Dubyak, G; Chen, L; Nuñez, G; Miesfeld, R L; Distelhorst, C W

    1994-01-01

    BCL-2 is a 26-kDa integral membrane protein that represses apoptosis by an unknown mechanism. Recent findings indicate that Ca2+ release from the endoplasmic reticulum (ER) mediates apoptosis in mouse lymphoma cells. In view of growing evidence that BCL-2 localizes to the ER, as well as mitochondria and the perinuclear membrane, we investigated the possibility that BCL-2 represses apoptosis by regulating Ca2+ fluxes through the ER membrane. A cDNA encoding BCL-2 was introduced into WEHI7.2 cells and two subclones, W.Hb12 and W.Hb13, which express high and low levels of BCL-2 mRNA and protein, respectively, were isolated. WEHI7.2 cells underwent apoptosis in response to treatment with the glucocorticoid hormone dexamethasone, whereas W.Hb12 and W.Hb13 cells were protected from apoptosis, revealing a direct relationship between the level of BCL-2 expression and the degree of protection. Significantly, BCL-2 also blocked induction of apoptosis by thapsigargin (TG), a highly specific inhibitor of the ER-associated Ca2+ pump. TG completely inhibited ER Ca2+ pumping in both WEHI7.2 and W.Hb12 cells, but the release of Ca2+ into the cytosol after inhibition of ER Ca2+ pumping was significantly less in W.Hb12 cells than in WEHI7.2 cells, indicating that BCL-2 reduces Ca2+ efflux through the ER membrane. By reducing ER Ca2+ efflux, BCL-2 interfered with a signal for "capacitative" entry of extracellular Ca2+, preventing a sustained increase of cytosolic Ca2+ in TG-treated cells. These findings suggest that BCL-2 either directly or indirectly regulates the flux of Ca2+ across the ER membrane, thereby abrogating Ca2+ signaling of apoptosis. Images PMID:8022822

  3. Post-transcriptional and post-translational regulation of Bcl2.

    PubMed

    Willimott, Shaun; Wagner, Simon D

    2010-12-01

    Bcl2 is an important pro-survival protein that has an essential function in normal immunity and whose constitutive expression leads to the development of lymphomas. Although transcriptional control of Bcl2 has been reported, increasing evidence suggests an important component of Bcl2 regulation is post-transcriptional. Phosphorylation of Bcl2 has been shown to enhance activity to allow response to extracellular growth-factor-mediated signals. Bcl2 mRNA contains regulatory elements in both its 5'- and 3'-UTRs (untranslated regions). An IRES (internal ribosome entry sequence) in the 5'-UTR permits continued translation in the presence of cellular stresses that reduce cap-dependent translation. The 3'-UTR of Bcl2 mRNA is 5.2 kb in length and contains multiple predicted miRNA (microRNA) and RNA-BP (RNA-binding protein)-binding sites. miR-15a and miR-16-1 have been found to inhibit Bcl2 expression in B-cells, whereas the RNA-BP nucleolin has been shown to increase Bcl2 expression by binding to the 3'-UTR and enhancing mRNA stability. Both decreased expression of miR-15a and miR-16-1 and increased nucleolin have been shown to be associated with increased Bcl2 expression and resistance to apoptosis in the common human disease, chronic lymphocytic leukaemia. miRNA-based therapeutic approaches to treat cancer are emerging. Bcl2 is highly regulated by miRNAs and is therefore an excellent candidate for such approaches. PMID:21118128

  4. B-cell lymphomas with concurrent MYC and BCL2 abnormalities other than translocations behave similarly to MYC/BCL2 double-hit lymphomas.

    PubMed

    Li, Shaoying; Seegmiller, Adam C; Lin, Pei; Wang, Xuan J; Miranda, Roberto N; Bhagavathi, Sharathkumar; Medeiros, L Jeffrey

    2015-02-01

    Large B-cell lymphomas with IGH@BCL2 and MYC rearrangement, known as double-hit lymphoma (DHL), are clinically aggressive neoplasms with a poor prognosis. Some large B-cell lymphomas have concurrent abnormalities of MYC and BCL2 other than coexistent translocations. Little is known about patients with these lymphomas designated here as atypical DHL. We studied 40 patients of atypical DHL including 21 men and 19 women, with a median age of 60 years. Nine (23%) patients had a history of B-cell non-Hodgkin lymphoma. There were 30 diffuse large B-cell lymphoma (DLBCL), 7 B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma, and 3 DLBCL with coexistent follicular lymphoma. CD10, BCL2, and MYC were expressed in 28/39 (72%), 33/35 (94%), and 14/20 (70%) cases, respectively. Patients were treated with standard (n=14) or more aggressive chemotherapy regimens (n=17). We compared the atypical DHL group with 76 patients with DHLand 35 patients with DLBCL lacking MYC and BCL2 abnormalities. The clinicopathologic features and therapies were similar between patients with atypical and typical DHL. The overall survival of patients with atypical double-hit lymphoma was similar to that of patients with double-hit lymphoma (P=0.47) and significantly worse than that of patients with DLBCL with normal MYC and BCL2 (P=0.02). There were some minor differences. Cases of atypical double-hit lymphoma more often have DLBCL morphology (P<0.01), less frequently expressed CD10 (P<0.01), and patients less often had an elevated serum lactate dehydrogenase level (P=0.01). In aggregate, these results support expanding the category of MYC/BCL2 DHL to include large B-cell lymphomas with coexistent MYC and BCL2 abnormalities other than concurrent translocations. PMID:25103070

  5. The inhibition of PI3K and NFκB promoted curcumin-induced cell cycle arrest at G2/M via altering polyamine metabolism in Bcl-2 overexpressing MCF-7 breast cancer cells.

    PubMed

    Berrak, Özge; Akkoç, Yunus; Arısan, Elif Damla; Çoker-Gürkan, Ajda; Obakan-Yerlikaya, Pınar; Palavan-Ünsal, Narçin

    2016-02-01

    Bcl-2 protein has been contributed with number of genes which are involved in oncogenesis. Among the many targets of Bcl-2, NFκB have potential role in induction of cell cycle arrest. Curcumin has potential therapeutic effects against breast cancer through multiple signaling pathways. In this study, we investigated the role of curcumin in induction of cell cycle arrest via regulating of NFκB and polyamine biosynthesis in wt and Bcl-2+ MCF-7 cells. To examine the effect of curcumin on cell cycle regulatory proteins, PI3K/Akt, NFκB pathways and polyamine catabolism, we performed immunoblotting assay. In addition, cell cycle analysis was performed by flow cytometry. The results indicated that curcumin induced cell cycle arrest at G2/M phase by downregulation of cyclin B1 and Cdc2 and inhibited colony formation in MCF-7wt cells. However, Bcl-2 overexpression prevented the inhibition of cell cycle associated proteins after curcumin treatment. The combination of LY294002, PI3K inhibitor, and curcumin induced cell cycle arrest by decreasing CDK4, CDK2 and cyclin E2 in Bcl-2+ MCF-7 cells. Moreover, LY294002 further inhibited the phosphorylation of Akt in Bcl-2+ MCF-7 cells. Curcumin could suppress the nuclear transport of NFκB through decreasing the interaction of P-IκB-NFκB. The combination of wedelolactone, NFκB inhibitor, and curcumin acted different on SSAT expression in wt MCF-7 and Bcl-2+ MCF-7 cells. NFκB inhibition increased the SSAT after curcumin treatment in Bcl-2 overexpressed MCF-7 cells. Inhibition of NFκB activity as well as suppression of ROS generation with NAC resulted in the partial relief of cells from G2/M checkpoint after curcumin treatment in wt MCF-7 cells. In conclusion, the potential role of curcumin in induction of cell cycle arrest is related with NFκB-regulated polyamine biosynthesis. PMID:26796279

  6. Protective Effect of Aliskiren in Experimental Ischemic Stroke: Up-Regulated p-PI3K, p-AKT, Bcl-2 Expression, Attenuated Bax Expression.

    PubMed

    Miao, Jiangyong; Wang, Lina; Zhang, Xiangjian; Zhu, Chunhua; Cui, Lili; Ji, Hui; Liu, Ying; Wang, Xiaolu

    2016-09-01

    Aliskiren (ALK), a pharmacological renin inhibitor, is an effective antihypertensive drug and has potent anti-apoptotic activity, but it is currently unknown whether ALK is able to attenuate brain damage caused by acute cerebral ischemia independent of its blood pressure-lowering effects. This study aimed to investigate the role of ALK and its potential mechanism in cerebral ischemia. C57/BL6 mice were subjected to transient middle cerebral artery occlusion (tMCAO) and treated for 5 days with Vehicle or ALK (10 or 25 mg/kg per day via intragastric administration), whereas Sham-operated animals served as controls. Treatment with ALK significantly improved neurological deficits, infarct volume, brain water content and Nissl bodies after stroke (P < 0.05), which did not affect systemic blood pressure. Furthermore, the protection of ALK was also related to decreased levels of apoptosis in mice by enhanced activation of phosphatidylinositol 3-kinase (PI3K)/AKT pathway, increased level of Bcl-2 and reduced Bax expression (P < 0.05). In addition, ALK's effects were reversed by PI3K inhibitors LY294002 (P < 0.05). Our data indicated that ALK protected the brain from reperfusion injuries without affecting blood pressure, and this effect may be through PI3K/AKT signaling pathway. PMID:27180190

  7. Necrosis-like death can engage multiple pro-apoptotic Bcl-2 protein family members

    PubMed Central

    Soratroi, Claudia; Villunger, Andreas

    2016-01-01

    Necroptosis is a physiologically relevant mode of cell death with some well-described initiating events, but largely unknown executioners. Here we investigated necrostatin-1 (Nec-1) sensitive death elicited by different necroptosis stimuli in L929 mouse fibrosarcoma cells, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages. We found that TNFα- or zVAD-induced necroptosis occurs independently of the recently implicated executioners Bmf or PARP-2, but can involve the Bcl-2 family proteins Bid and Bak. Furthermore, this type of necroptosis is associated with mitochondrial cytochrome c release and partly sensitive to cyclosporine A inhibition, suggesting a cross talk with the mitochondrial permeability transition pore. Necroptosis triggered by cadmium (Cd) exposure caused fully Nec-1-sensitive and caspase-independent death in L929 cells that was associated with autocrine TNFα-mediated feed-forward signalling. In MEF Cd-exposure elicited a mixed mode of cell death that was to some extent Nec-1-sensitive but also displayed features of apoptosis. It was partly dependent on Bmf and Bax/Bak, proteins typically considered to act pro-apoptotic, but ultimately insensitive to caspase inhibition. Overall, our study indicates that inducers of “extrinsic” and “intrinsic” necroptosis can both trigger TNF-receptor signalling. Further, necroptosis may depend on mitochondrial changes engaging proteins considered critical for MOMP during apoptosis that ultimately contribute to caspase-independent necrotic cell death. PMID:22971741

  8. The functional basis of c-myc and bcl-2 complementation during multistep lymphomagenesis in vivo.

    PubMed

    Marin, M C; Hsu, B; Stephens, L C; Brisbay, S; McDonnell, T J

    1995-04-01

    Oncogenes are known to be deregulated by chromosomal translocations occurring at high frequency in specific malignancies. Among the most well characterized of these are c-myc, associated with the t(8;14) in Burkitt's lymphomas, and bcl-2, associated with the t(14;18) in follicular lymphomas. In addition to their role in regulating rates of proliferation, it is known that oncogenes and tumor suppressor genes can also regulate rates of apoptotic cell death. The contribution of c-myc and bcl-2 to the regulation of cell death during lymphomagenesis in vivo is assessed using bcl-2-Ig and emu-myc trangenic mice and bcl-2/myc hybrid transgenic mice. Translocations between the endogenous c-myc gene and immunoglobulin loci, e.g., t(12;15), are common in lymphomas arising in the bcl-2-Ig mice. Furthermore, bcl-2/c-myc double transgenic mice exhibit accelerated lymphomagenesis, indicating cooperation between these two oncogenes. Genetic complementation of c-myc and bcl-2 during lymphomagenesis resulted from the suppression of c-myc-associated apoptosis. Other genes are likely involved in regulating cell death during multistep lymphomagenesis. PMID:7698223

  9. Immunihistochemical detection of Bcl-2 in AIDS-associated and classical Kaposi's sarcoma.

    PubMed Central

    Morris, C. B.; Gendelman, R.; Marrogi, A. J.; Lu, M.; Lockyer, J. M.; Alperin-Lea, W.; Ensoli, B.

    1996-01-01

    Kaposi's Sarcoma (KS) is an angioproliferative disease that is characterized by proliferation of spindle-shaped cells predominantly of vascular endothelial cell origin, neoangiogenesis, inflammatory cell infiltration, and edema. Although the lesions of classical KS and AIDS-associated KS (AIDS-KS) share common histological features, AIDS-KS occurs at a markedly higher frequency with a more aggressive clinical course. Immunohistochemical analyses of 26 evolutionarily staged AIDS-KS lesions derived from HIV-infected patients demonstrate significant cytoplasmic levels of Bcl-2, a protooncogene known to prolong cellular viability and to antagonize apoptosis. Bcl-2 expression increases as the pathological stage of KS advances. Immunohistochemical analyses of classical KS lesions demonstrate prevalent expression of Bcl-2 as well, indicating that upregulation of Bcl-2 may be important in the pathogenesis of both classical and AIDS-associated KS. Coexpression of Bcl-2 and factor VIII-related antigen in spindle-shaped cells present within KS lesions suggests that Bcl-2 is upregulated within the vascular endothelial spindle-shaped cells of KS. The consequences of upregulated Bcl-2 expression within KS lesions may be prolonged spindle cell viability which, when coupled with dysregulated cellular proliferation due in part to synergistic activities of inflammatory and angiogenic cytokines and HIV-1 Tat protein, may result in the maintenance, growth, and progression of KS. Images Figure 1 Figure 2 Figure 3 PMID:8644847

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

    PubMed

    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. PMID:26257067

  11. Skin-Derived Precursors against UVB-Induced Apoptosis via Bcl-2 and Nrf2 Upregulation

    PubMed Central

    Zhong, Jianqiao

    2016-01-01

    Bcl-2 and Nrf2 are critical factors in protecting cells against UVB-induced apoptosis. Hair-follicle-bulge stem cells could resist ionization through Bcl-2 upregulation. Skin-derived precursors (SKPs) dwelling on the bulge may be against UVB irradiation. Initially, SKPs were isolated and identified. Then, SKPs were exposed to UVB and grew in medium for 24 hours. CCK-8 assay, TUNEL, and Ki67 staining evaluated cells apoptosis/proliferation, while SA-βgal staining evaluated cells senescence and pH2AX immunostaining evaluated DNA damage. Meanwhile, Bcl-2, Nrf2, HO-1, Bax, and Bak expressions were assessed by qRT-PCR and western blot. Two weeks later, floating spheres appeared and were identified as SKPs. After UVB radiation, SKPs maintained spherical colonies and outnumbered unirradiated ones, showing high Ki67 expression and low TUNEL, SA-βgal, and pH2AX expression. Fibroblasts (FBs), however, displayed deformation, senescence, and reduction, with increased TUNEL, SA-βgal, and pH2AX expression. Moreover, Bcl-2 and Nrf2 mRNA expression were significantly higher than Bak and Bax in irradiated SKPs. Conversely, Bcl-2 and Nrf2 mRNA levels greatly decreased compared with Bax and Bak in irradiated FBs. Interestingly, SKPs showed higher protein levels of Bcl-2, Nrf2, and HO-1 than FBs. SKPs exert a beneficial effect on resisting UVB-induced apoptosis, which may be associated with Bcl-2 and Nrf2 upregulation.

  12. Mitochondrial Bcl-2 family dynamics define therapy response and resistance in neuroblastoma.

    PubMed

    Goldsmith, Kelly C; Gross, Michelle; Peirce, Susan; Luyindula, Dema; Liu, Xueyuan; Vu, Annette; Sliozberg, Michael; Guo, Rong; Zhao, Huaqing; Reynolds, C Patrick; Hogarty, Michael D

    2012-05-15

    Neuroblastoma is a childhood tumor in which transient therapeutic responses are typically followed by recurrence with lethal chemoresistant disease. In this study, we characterized the apoptotic responses in diverse neuroblastomas using an unbiased mitochondrial functional assay. We defined the apoptotic set point of neuroblastomas using responses to distinct BH3 death domains providing a BH3 response profile and directly confirmed survival dependencies. We found that viable neuroblastoma cells and primary tumors are primed for death with tonic sequestration of Bim, a direct activator of apoptosis, by either Bcl-2 or Mcl-1, providing a survival dependency that predicts the activity of Bcl-2 antagonists. The Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-737 showed single-agent activity against only Bim:Bcl-2 primed tumor xenografts. Durable complete regressions were achieved in combination with noncurative chemotherapy even for highest risk molecular subtypes with MYCN amplification and activating ALK mutations. Furthermore, the use of unique isogenic cell lines from patients at diagnosis and at the time of relapse showed that therapy resistance was not mediated by upregulation of Bcl-2 homologues or loss of Bim priming, but by repressed Bak/Bax activation. Together, our findings provide a classification system that identifies tumors with clinical responses to Bcl-2 antagonists, defines Mcl-1 as the principal mediator of Bcl-2 antagonist resistance at diagnosis, and isolates the therapy resistant phenotype to the mitochondria. PMID:22589275

  13. Mitochondrial Bcl-2 family dynamics define therapy response and resistance in neuroblastoma

    PubMed Central

    Goldsmith, Kelly C.; Gross, Michelle; Peirce, Susan; Luyindula, Dema; Liu, Xueyuan; Vu, Annette; Sliozberg, Michael; Guo, Rong; Zhao, Huaqing; Reynolds, C. Patrick; Hogarty, Michael D.

    2012-01-01

    Neuroblastoma is a childhood tumor in which transient therapeutic responses are typically followed by recurrence with lethal chemoresistant disease. In this study, we characterized the apoptotic responses in diverse neuroblastomas using an unbiased mitochondrial functional assay. We defined the apoptotic set-point of neuroblastomas using responses to distinct BH3 death domains providing a BH3 response profile, and directly confirmed survival dependencies. We found that viable neuroblastoma cells and primary tumors are primed for death with tonic sequestration of Bim, a direct activator of apoptosis, by either Bcl-2 or Mcl-1, providing a survival dependency that predicts the activity of Bcl-2 antagonists. The Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-737 showed single agent activity against only Bim:Bcl-2 primed tumor xenografts. Durable complete regressions were achieved in combination with non-curative chemotherapy even for highest-risk molecular subtypes with MYCN amplification and activating ALK mutations. Furthermore, the use of unique isogenic cell lines from patients at diagnosis and at the time of relapse showed that therapy resistance was not mediated by upregulation of Bcl-2 homologues or loss of Bim priming, but by repressed Bak/Bax activation. Together, our findings provide a classification system that identifies tumors with clinical responses to Bcl-2 antagonists, defines Mcl-1 as the principal mediator of Bcl-2 antagonist resistance at diagnosis, and isolates the therapy resistant phenotype to the mitochondria. PMID:22589275

  14. Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression.

    PubMed

    Bogner, Christian; Dechow, Tobias; Ringshausen, Ingo; Wagner, Michaela; Oelsner, Madlen; Lutzny, Gloria; Licht, Thomas; Peschel, Christian; Pastan, Ira; Kreitman, Robert J; Decker, Thomas

    2010-01-01

    Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti- CD22 antibody fused to a 38-kDa Pseudomonas exotoxin-A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl-2 expression (NCEB-1, JeKo-1 and JVM-2) compared to Granta-519 cells with high Bcl-2 expression. While the expression of the antiapoptotic protein Mcl-1 declined (NCEB-1, Granta-519), Bcl-2 levels remained unchanged in Granta-519 cells. However transfection of BCL2 cDNA into NCEB-1, JeKo-1 and JVM-2 cells significantly reduced BL22-mediated toxicity. Accordingly we examined the effects of Bcl-2 inactivation in Granta-519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta-519 cells with silenced Bcl-2. Our results were confirmed in freshly isolated MCL-cells from patients with leukaemic MCL. We conclude that Bcl-2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells. PMID:19821820

  15. ALS-linked mutant SOD1 damages mitochondria by promoting conformational changes in Bcl-2

    PubMed Central

    Pedrini, Steve; Sau, Daniela; Guareschi, Stefania; Bogush, Marina; Brown, Robert H.; Naniche, Nicole; Kia, Azadeh; Trotti, Davide; Pasinelli, Piera

    2010-01-01

    In mutant superoxide dismutase (SOD1)-linked amyotrophic lateral sclerosis (ALS), accumulation of misfolded mutant SOD1 in spinal cord mitochondria is thought to cause mitochondrial dysfunction. Whether mutant SOD1 is toxic per se or whether it damages the mitochondria through interactions with other mitochondrial proteins is not known. We previously identified Bcl-2 as an interacting partner of mutant SOD1 specifically in spinal cord, but not in liver, mitochondria of SOD1 mice and patients. We now show that mutant SOD1 toxicity relies on this interaction. Mutant SOD1 induces mitochondrial morphological changes and compromises mitochondrial membrane integrity leading to release of Cytochrome C only in the presence of Bcl-2. In cells, mouse and human spinal cord with SOD1 mutations, the binding to mutant SOD1 triggers a conformational change in Bcl-2 that results in the uncovering of its toxic BH3 domain and conversion of Bcl-2 into a toxic protein. Bcl-2 carrying a mutagenized, non-toxic BH3 domain fails to support mutant SOD1 mitochondrial toxicity. The identification of Bcl-2 as a specific target and active partner in mutant SOD1 mitochondrial toxicity suggests new therapeutic strategies to inhibit the formation of the toxic mutant SOD1/Bcl-2 complex and to prevent mitochondrial damage in ALS. PMID:20460269

  16. Evidence of reciprocity of bcl-2 and p53 expression in human colorectal adenomas and carcinomas.

    PubMed Central

    Watson, A. J.; Merritt, A. J.; Jones, L. S.; Askew, J. N.; Anderson, E.; Becciolini, A.; Balzi, M.; Potten, C. S.; Hickman, J. A.

    1996-01-01

    Evidence of accumulating for the failure of apoptosis as an important factor in the evolution of colorectal cancer and its poor response to adjuvant therapy. The proto-oncogene bcl-2 suppresses apoptosis. Its expression could provide an important survival advantage permitting the development of colorectal cancer. The expression of bcl-2 and p53 was determined by immunohistochemistry in 47 samples of histologically normal colonic mucosa, 19 adenomas and 53 adenocarcinomas. Expression of bcl-2 in colonic crypts > 5 cm from the tumours was confined to crypt bases but was more extensive and intense in normal crypts < 5 mm from cancers. A higher proportion of adenomas (63.2%) than carcinomas (36.5%) expressed bcl-2 (P < 0.05). A lower proportion of adenomas (31.6%) than carcinomas (62.3%) expressed p53 (P < 0.02). A total of 26.3% of adenomas and 22% of carcinomas expressed both bcl-2 and p53. To determine whether these samples contained cells which expressed both proteins, a dual staining technique for bcl-2 and p53 was used. Only 1/19 adenomas and 2/53 carcinomas contained cells immunopositive for both bcl-2 and p53. Moreover there was evidence of reciprocity of expression of bcl-2 and p53 in these three double staining neoplasms. We suggest that bcl-2 provides a survival advantage in the proliferative compartment of normal crypts and colorectal neoplasms. However, its expression is lost during the evolution from adenoma to carcinoma, whereas p53 expression is increased, an event generally coincident with the expression of stabilised p53, which we presume to represent the mutant form. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8611422

  17. Nrf2 Protein Up-regulates Antiapoptotic Protein Bcl-2 and Prevents Cellular Apoptosis*

    PubMed Central

    Niture, Suryakant K.; Jaiswal, Anil K.

    2012-01-01

    Nuclear transcription factor Nrf2 regulates the expression and coordinated induction of a battery of genes encoding cytoprotective and drug transporter proteins in response to chemical and radiation stress. This leads to reduced apoptosis, enhanced cell survival, and increased drug resistance. In this study, we investigated the role of Nrf2 in up-regulation of antiapoptotic protein Bcl-2 and its contribution to stress-induced apoptosis and cell survival. Exposure of mouse hepatoma (Hepa-1) and human hepatoblastoma (HepG2) cells to antioxidant tert-butylhydroquinone led to induction of Bcl-2. Mutagenesis and transfection assays identified an antioxidant response element between nucleotides −3148 and −3140 on the reverse strand of the Bcl-2 gene promoter that was essential for activation of Bcl-2 gene expression. Band/supershift and ChIP assays demonstrated binding of Nrf2 to Bcl-2 antioxidant response element. Alterations in Nrf2 led to altered Bcl-2 induction and cellular apoptosis. Moreover, dysfunctional/mutant inhibitor of Nrf2 (INrf2) in human lung cancer cells failed to degrade Nrf2, resulting in an increased Bcl-2 level and decreased etoposide- and UV/γ radiation-mediated DNA fragmentation. In addition, siRNA-mediated down-regulation of Nrf2 also led to decreased apoptosis and increased cell survival. Furthermore, the specific knockdown of Bcl-2 in Nrf2-activated tumor cells led to increased etoposide-induced apoptosis and decreased cell survival and growth/proliferation. These data provide the first evidence of Nrf2 in control of Bcl-2 expression and apoptotic cell death with implications in antioxidant protection, survival of cancer cells, and drug resistance. PMID:22275372

  18. Immunohistochemical detection of p53 and Bcl-2 in colorectal carcinoma: no evidence for prognostic significance.

    PubMed Central

    Tollenaar, R. A.; van Krieken, J. H.; van Slooten, H. J.; Bruinvels, D. J.; Nelemans, K. M.; van den Broek, L. J.; Hermans, J.; van Dierendonck, J. H.

    1998-01-01

    To evaluate the prognostic significance of immunohistochemically detected p53 and Bcl-2 proteins in colorectal cancer, tissue sections from 238 paraffin-embedded colorectal carcinomas were immunostained for p53 (MAb DO-7 and CM-1 antiserum) and Bcl-2 (MAb Bcl-2:124). Staining patterns were assessed semiquantitatively and correlated with each other and with sex, age, tumour site, Dukes' classification, tumour differentiation, mucinous characteristics, lymphocyte and eosinophilic granulocyte infiltration, and patient survival. In our series, 35% of carcinomas showed no nuclear staining and 34% (DO-7) to 40% (CM-1) showed staining in over 30% of tumour cell nuclei. A majority of carcinomas that had been immunostained with CM-1 showed cytoplasmic staining, but this was not observed with DO-7. With respect to Bcl-2, 51% of tumours were completely negative, 32% displayed weak and 15% moderate staining; only 3% showed strong positive staining. No evidence was found for reciprocity between Bcl-2 expression and nuclear p53 accumulation. From 13 cases containing tumour-associated adenoma, four were Bcl-2 negative in premalignant and malignant cells, in another four cases these cells showed similar staining intensities and in the remaining cases only the malignant colorectal cells were Bcl-2 negative. Therefore, our data indicate that Bcl-2 is dispensable in the progression towards carcinoma. Except for an association between nuclear p53 accumulation and mucinous tumours (P = 0.01), no significant correlation was found between the clinicopathological parameters mentioned above and immunostaining pattern of (nuclear or cytoplasmic) p53 or Bcl-2. PMID:9667656

  19. Dioscin-induced apoptosis of human LNCaP prostate carcinoma cells through activation of caspase-3 and modulation of Bcl-2 protein family.

    PubMed

    Chen, Jing; Li, Hui-min; Zhang, Xue-nong; Xiong, Chao-mei; Ruan, Jin-lan

    2014-02-01

    Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin (1, 2 and 4 μmol/L) could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family. PMID:24496691

  20. MicroRNA-29a induces apoptosis via increasing the Bax:Bcl-2 ratio in dermal fibroblasts of patients with systemic sclerosis.

    PubMed

    Jafarinejad-Farsangi, Saeideh; Farazmand, Ali; Mahmoudi, Mahdi; Gharibdoost, Farhad; Karimizadeh, Elham; Noorbakhsh, Farshid; Faridani, Habibeh; Jamshidi, Ahmad Reza

    2015-01-01

    The most prominent feature of systemic sclerosis (SSc) and other diseases associated with fibrosis is the prolonged activation of fibroblasts not eliminated by apoptosis, hence characterized by accumulation of more extra cellular matrix (ECM). We tend to verify if microRNA-29a (miR-29a) as an anti-fibrotic factor could induce apoptosis in SSc fibroblasts. We did not detect apoptosis in SSc fibroblasts. We found that Bcl-2 expression was upregulated in SSc fibroblasts and the ratio of Bax:Bcl-2 in these cells was significantly lower (p = 0.02) compared to normal fibroblasts. Transfection of both SSc and transforming growth factor-β (TGF-β) stimulated fibroblasts by miR-29a mimic, significantly decreased the expression of two anti-apoptotic members of the Bcl-2 family, Bcl-2 (p = 0.0005, p = 0.01) and Bcl-XL (p = 0.0001, p = 0.006), resulted in enhanced Bax:Bcl-2 ratio and induced a high rate of apoptosis. Recently, miR-29 has been introduced as an anti-fibrotic factor with potential therapeutic effect on SSc. Until now, it has not been proposed whether there is a relationship between miR-29a and apoptosis in SSc. According to our results, it seems that miR-29a is a potent inducer of apoptosis in SSc fibroblasts and an attenuator of ECM production in these cells. MiR-29a disrupted the expression profiling of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-XL) which is the central point of dynamic life-death rheostat in many apoptotic pathways. Furthermore, dermal fibroblasts from patients with SSc showed elevation in TNF-α mRNA levels, while restoration of miR-29a decreases TNF-α production in these cells. Although further molecular studies are necessary to investigate the underlying apoptotic pathways, the present findings suggest that anti-fibrotic and pro-apoptotic properties of miR-29a could provide novel benefits toward the development of fibroblast-specific anti-fibrotic therapies. PMID:25857445

  1. Immunohistochemical analysis of Mcl-1 and Bcl-2 proteins in normal and neoplastic lymph nodes.

    PubMed Central

    Krajewski, S.; Bodrug, S.; Gascoyne, R.; Berean, K.; Krajewska, M.; Reed, J. C.

    1994-01-01

    The Bcl-2 protein blocks programmed cell death and becomes overproduced in many follicular non-Hodgkin's lymphomas as the result of t(14; 18) translocations involving the Bcl-2 gene. Mcl-1 is a recently discovered gene whose encoded protein has significant homology with Bcl-2 but whose function remains unknown. In this study, we compared the in vivo patterns of Bcl-2 and Mcl-1 protein production in normal and neoplastic lymph node biopsies by immunohistochemical means using specific polyclonal antisera. Intracellular Mcl-1 immunoreactivity was located primarily in the cytosol in a punctate pattern and was also seen in association with the nuclear envelope in many cases, similar to the results obtained for Bcl-2, which resides in the outer mitochondrial membrane, nuclear envelope, and endoplasmic reticulum. In 4 of 4 reactive tonsils and 28 of 28 nodes with reactive follicular hyperplasia, reciprocal patterns of Bcl-2 and Mcl-1 protein expression were observed. Bcl-2 immunostaining was highest in mantle zone lymphocytes and absent from most germinal center cells, whereas Mcl-1 immunoreactivity was highest in germinal center lymphocytes and absent from mantle zone lymphocytes. Mcl-1 was also expressed in some interfollicular lymphocytes, particularly those that had the appearance of activated lymphocytes. Similar to the patterns of Bcl-2 and mcl-1 expression seen in reactive nodes, Mcl-1 protein was largely absent from the malignant cells in 2 of 2 mantle cell lymphomas, whereas strong Bcl-2 immunostaining was found in these cells. In contrast to normal nodes, however, the neoplastic follicles of t(14;18) containing follicular non-Hodgkin's lymphomas immunostained positively for both Bcl-2 and Mcl-1 in 24 of 27 cases. Intense immunostaining for Mcl-1 was also observed in Reed-Sternberg cells in 2 of 2 cases of Hodgkin's disease but Bcl-2 immunoreactivity was present at much lower levels. These findings demonstrate that the levels of Mcl-1 and Bcl-2 proteins are

  2. Vaccinia Virus N1l Protein Resembles a B Cell Lymphoma-2 (Bcl-2) Family Protein

    SciTech Connect

    Aoyagi, M.; Zhai, D.; Jin, C.; Aleshin, A.E.; Stec, B.; Reed, J.C.; Liddington, R.C.; /Burnham Inst.

    2007-07-03

    Poxviruses encode immuno-modulatory proteins capable of subverting host defenses. The poxvirus vaccinia expresses a small 14-kDa protein, N1L, that is critical for virulence. We report the crystal structure of N1L, which reveals an unexpected but striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. Although N1L lacks detectable Bcl-2 homology (BH) motifs at the sequence level, we show that N1L binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 family proteins in vitro, consistent with a role for N1L in modulating host antiviral defenses.

  3. Flavopiridol induces BCL-2 expression and represses oncogenic transcription factors in leukemic blasts from adults with refractory acute myeloid leukemia

    PubMed Central

    Nelson, Dwella M.; Joseph, Biju; Hillion, Joelle; Segal, Jodi; Karp, Judith E.; Resar, Linda M. S.

    2011-01-01

    Flavopiridol is a cyclin-dependent kinase inhibitor that induces cell cycle arrest, apoptosis, and clinical responses in selected patients with acute myeloid leukemia (AML). A better understanding of the molecular pathways targeted by flavopiridol is needed to design optimal combinatorial therapy. Here, we report that in vivo administration of flavopiridol induced expression of the BCL-2 anti-apoptotic gene in leukemic blasts from adult patients with refractory AML. Moreover, flavopiridol repressed the expression of genes encoding oncogenic transcription factors (HMGA1, STAT3, E2F1) and the major subunit of RNA Polymerase II. Our results provide mechanistic insight into the cellular pathways targeted by flavopiridol and suggest that blocking anti-apoptotic pathways could enhance cytotoxicity and improve outcomes in patients treated with flavopiridol. PMID:21728742

  4. Stabilization of cellular mRNAs and up-regulation of proteins by oligoribonucleotides homologous to the Bcl2 adenine-uridine rich element motif.

    PubMed

    Bevilacqua, Annamaria; Ghisolfi, Laura; Franzi, Sara; Maresca, Giovanna; Gherzi, Roberto; Capaccioli, Sergio; Nicolin, Angelo; Canti, Gianfranco

    2007-02-01

    Adenine-uridine rich elements (AREs) play an important role in modulating mRNA stability, being the target site of many ARE-binding proteins (AUBPs) that are involved in the decay process. Three 26-mer 2'-O-methyl oligoribonucleotides (ORNs) homologous to the core region of ARE of bcl2 mRNA have been studied for decoy-aptamer activity in UV cross-linking assays. Sense-oriented ORNs competed with the ARE motif for the interaction with both destabilizing and stabilizing AUBPs in cell-free systems and in cell lines. Moreover, ORNs induced mRNA stabilization and up-regulated both Bcl2 mRNA and protein levels in the cells. Bcl2 ORNs stabilized other ARE-containing transcripts and up-regulated their expression. These results indicate that Bcl2 ORNs compete for AUBP-ARE interactions independently of ARE class and suggest that in the cell, the default labile status of ARE-containing mRNAs depends on the combined interaction of such transcripts with destabilizing AUBPs. PMID:17077270

  5. Double-strand break formation by the RAG complex at the bcl-2 major breakpoint region and at other non-B DNA structures in vitro.

    PubMed

    Raghavan, Sathees C; Swanson, Patrick C; Ma, Yunmei; Lieber, Michael R

    2005-07-01

    The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity. PMID:15988007

  6. Synergistic killing of human small cell lung cancer cells by the Bcl-2-inositol 1,4,5-trisphosphate receptor disruptor BIRD-2 and the BH3-mimetic ABT-263

    PubMed Central

    Greenberg, E F; McColl, K S; Zhong, F; Wildey, G; Dowlati, A; Distelhorst, C W

    2015-01-01

    Small cell lung cancer (SCLC) has an annual mortality approaching that of breast and prostate cancer. Although sensitive to initial chemotherapy, SCLC rapidly develops resistance, leading to less effective second-line therapies. SCLC cells often overexpress Bcl-2, which protects cells from apoptosis both by sequestering pro-apoptotic family members and by modulating inositol 1,4,5-trisphosphate receptor (IP3R)-mediated calcium signaling. BH3-mimetic agents such as ABT-263 disrupt the former activity but have limited activity in SCLC patients. Here we report for the first time that Bcl-2-IP3 receptor disruptor-2 (BIRD-2), a decoy peptide that binds to the BH4 domain of Bcl-2 and prevents Bcl-2 interaction with IP3Rs, induces cell death in a wide range of SCLC lines, including ABT-263-resistant lines. BIRD-2-induced death of SCLC cells appears to be a form of caspase-independent apoptosis mediated by calpain activation. By targeting different regions of the Bcl-2 protein and different mechanisms of action, BIRD-2 and ABT-263 induce cell death synergistically. Based on these findings, we propose that targeting the Bcl-2–IP3R interaction be pursued as a novel therapeutic strategy for SCLC, either by developing BIRD-2 itself as a therapeutic agent or by developing small-molecule inhibitors that mimic BIRD-2. PMID:26720343

  7. L-Glutamine enhances enterocyte growth via activation of the mTOR signaling pathway independently of AMPK.

    PubMed

    Yi, Dan; Hou, Yongqing; Wang, Lei; Ouyang, Wanjin; Long, Minhui; Zhao, Di; Ding, Binying; Liu, Yulan; Wu, Guoyao

    2015-01-01

    Neonates (including human infants) require L-glutamine (Gln) for optimal intestinal health. This study tested the hypothesis that Gln enhances enterocyte growth via both mammalian target of rapamycin (mTOR) and AMP-activated kinase (AMPK) signaling pathways. Intestinal porcine epithelial cells (IPEC-1) were cultured for 3 days in Gln-free Dulbecco's modified Eagle medium containing 0 or 2 mM Gln. To determine the role of mTOR and AMPK on cell growth, additional experiments were conducted where medium contained 2 mM Gln and 10 nM rapamycin (Rap, an inhibitor of mTOR) or 1 μM compound C (an inhibitor of AMPK). IPEC-1 cell growth increased with increasing concentrations of Gln from 0 to 2 mM. Compared with 0 mM Gln, 2 mM Gln increased (P < 0.05) the amounts of phosphorylated 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (p70S6 kinase) proteins but did not affect abundances of total or phosphorylated AMPK protein. Gln also increased mRNA levels for Bcl-2, mTOR, p70S6 kinase, 4E-BP1, COX7C, ASCT2, ODC, SGLT-1, CFTR, Na(+)/K(+)-ATPase, HSP70, and ZO-1. Similarly, cells cultured with Rap and Gln exhibited higher (P < 0.05) abundances of phosphorylated 4E-BP1 and p70S6 kinase proteins than the Rap-only group, whereas abundances of phosphorylated mTOR and 4E-BP1 proteins were increased when AMPK was inhibited by compound C. Conversely, the amount of phosphorylated AMPK increased when mTOR was inhibited by Rap, suggesting a negative cross-talk between mTOR and AMPK. Collectively, these results indicate that Gln stimulates enterocyte growth by activating the mTOR signaling pathway independently of AMPK. PMID:25280462

  8. Molecular analysis integrating different pathways associated with androgen-independent progression in LuCaP 23.1 xenograft.

    PubMed

    Rocchi, Palma; Muracciole, Xavier; Fina, Frederic; Mulholland, Dave J; Karsenty, Gilles; Palmari, Jacqueline; Ouafik, L'haucine; Bladou, Franck; Martin, Pierre-Marie

    2004-12-01

    After therapeutic hormone deprivation, most prostate cancer (PrCa) cells develop androgen-independent (AI) growth. PrCa is highly heterogeneous and multifocal, suggesting that several molecular processes or pathways may be contributing to AI. The human LuCaP 23.1 xenograft model retains clinical hallmarks of PrCa, including heterogeneous growth, PSA production, androgen-responsiveness and progression to AI. In this work, we studied the effect of androgen depletion (castration) on the growth of LuCaP 23.1 xenografts. A total of 100 nude mice were implanted and analysed for their growth profiles before and after castration. By 11 and 15 weeks, tumours were harvested and assessed for molecular marker expression specific for PrCa. Prior to castration we found 37 fast growing (FG) tumours (948.9+/-76.9 mm(3)) and 63 slow growing (SG) tumours (229.6+/-18.4 mm(3)), a previously undescribed result for this PrCa model. Quantitative RT-PCR showed that in comparison to SGs, FGs contained high HER1, uPA and thymidilate synthetase (TS) expression with low levels of 5alpha-reductase 2 mRNA. All FG tumours progressed rapidly to AI growth 5 weeks after castration (FG-P). In SG castrated tumours, 66% of tumours (SG-P) showed retarded progression (by 12 weeks) to AI, whereas 34% responded to castration (SG-R). Molecular analysis permitted us to define distinct molecular profiles integrating different pathways associated with AI progression. FG-P, and a subgroup of SG-P tumours, presented significantly high levels of peptidylglycine alpha-amidating monooxygenase (PAM), HER1, HER2, TS, and uPA mRNA, all of which correlated with AR expression. The second subgroup of SG-P tumours showed overexpression of the antiapoptotic gene Bcl-2. A third subgroup of SG-P tumours showed significant expression of hypoxia-related gene (adrenomedullin) after castration. This work permitted to define distinct molecular profiles related to different AI growth in the LuCaP 23.1 xenograft. PMID:15489889

  9. Cantharidin inhibits cell proliferation and promotes apoptosis in tongue squamous cell carcinoma through suppression of miR-214 and regulation of p53 and Bcl-2/Bax.

    PubMed

    Tian, Xiaoguang; Zeng, Guang; Li, Xi; Wu, Zizhong; Wang, Lei

    2015-06-01

    Cantharidin, a type of terpenoid, is a chemical compount secreted by the blister beetle or Mylabris phelarata pallas of the Meloidae family. Cantharidin is known to have good antitumor activity. The present study aimed to investigate the anticancer effect of cantharidin and its possible underlying mechanism using tongue squamous cell carcinoma (TSCC) TCA8113 cells. TCA8113 cells were treated with various concentrations of cantharidin, and the cell viability and cytotoxicity were assessed using MTT and LDH assays, respectively. Flow cytometry was conducted to examine cell apoptosis and colorimetric protease assay was performed to analyze caspase-9/3 activities in TCA8113 cells. qPCR and western blot analysis were used to investigate microRNA-214 (miR-214) expression, as well as the expression of p53, Bcl-2 and Bax proteins in TCA8113 cells. miR-214 and anti-miR-214 were transfected with mimics to examine whether miR-214 expression regulated the anticancer effect of cantharidin on TCA8113 cells and p53, Bcl-2 and Bax protein expression. The anticancer effect of cantharidin significantly inhibited cell proliferation and increased cytotoxicity of TSCC Tca8113 cells in a dose- and time-dependent manner. In addition, cantharidin induced cell apoptosis and activated caspase-9/3 activities of TSCC Tca8113 cells. Cantharidin markedly weakened miR-214 expression level, activated p53 protein expression, and suppressed the Bcl-2/Bax signaling pathway in Tca8113 cells. Downregulation of miR-214 increased p53 protein expression and decreased the Bcl-2/Bax signaling pathway of TSCC Tca8113 cells. However, the overexpression of miR-214 reduced the anticancer effect of cantharidin on the proliferation and apoptosis of TSCC Tca8113 cells, inhibited p53 protein expression, and increased the Bcl-2/Bax signaling pathway. The results suggested that cantharidin is a potential anticancer drug that can be used to regulate the proliferation and apoptosis of human TSCC Tca8113 cells

  10. A Potent and Highly Efficacious Bcl-2/Bcl-xL Inhibitor

    PubMed Central

    McEachern, Donna; Yang, Chao-Yie; Meagher, Jennifer; Stuckey, Jeanne; Wang, Shaomeng

    2013-01-01

    Our previously reported Bcl-2/Bcl-xL inhibitor, 4, effectively inhibited tumor growth but failed to achieve complete regression in vivo. We have now performed extensive modifications on its pyrrole core structure, which has culminated in the discovery of 32 (BM-1074). Compound 32 binds to Bcl-2 and Bcl-xL proteins with Ki values of < 1 nM and inhibits cancer cell growth with IC50 values of 1-2 nM in four small-cell lung cancer cell lines sensitive to potent and specific Bcl-2/Bcl-xL inhibitors. Compound 32 is capable of achieving rapid, complete and durable tumor regression in vivo at a well-tolerated dose-schedule. Compound 32 is the most potent and efficacious Bcl-2/Bcl-xL inhibitor reported to date. PMID:23448298

  11. Regulatory effect of Bcl-2 in ultraviolet radiation-induced apoptosis of the mouse crystalline lens

    PubMed Central

    DONG, YUCHEN; ZHENG, YAJUAN; XIAO, JUN; ZHU, CHAO; ZHAO, MEISHENG

    2016-01-01

    The aim of the present study was to analyze the role of Bcl-2 during the process of apoptosis in the mouse crystalline lens. In total, 12 normal mice served as the control group and 12 Bcl-2 knockout (K.O) mice served as the experimental group. The mouse crystalline lens was sampled for the detection of Bcl-2 and caspase-3 expression following exposure to ultraviolet (UV) radiation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine Bcl-2 expression in the groups of normal mice receiving UV radiation or not receiving UV radiation. Samples of the murine crystalline lens were microscopically harvested and analyzed using western blotting. Apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, caspase 3 activity was examined using enzyme-linked immunosorbent assay kits, and RT-qPCR was used to analyze caspase-3 expression levels. The results of the present study demonstrated that there was no statistically significant difference in the level of Bcl-2 gene transcription between the two groups. In addition, UV radiation did not change the macrostructure of the crystalline lens in the group of normal mice or the group of Bcl-2 K.O mice. The results of the TUNEL assay indicated that the normal-UV group exhibited a more significant apoptosis level compared with the Bcl-2 K.O-UV group. Furthermore, the mRNA expression level of caspase-3 in the normal-UV group was significantly higher compared with the normal-nonUV group (P<0.05), while the levels in the Bcl-2 K.O-UV group were significantly higher compared with the Bcl-2 K.O and normal-nonUV groups (P<0.05). In addition, the mRNA expression level of caspase-3 was significantly higher in the normal-UV, as compared with the Bcl-2 K.O-UV group (P<0.05), and the variation trends in caspase-3 activity were consistent. In conclusion, the results of the present study demonstrated that Bcl-2 may have an important role in the

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

    PubMed

    Liu, Mu-En; Huang, Chu-Chung; Yang, Albert C; Tu, Pei-Chi; Yeh, Heng-Liang; Hong, Chen-Jee; Chen, Jin-Fan; Liou, Ying-Jay; Lin, Ching-Po; Tsai, Shih-Jen

    2013-01-01

    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. PMID:23437205

  13. Intracellular localization of the BCL-2 family member BOK and functional implications

    PubMed Central

    Echeverry, N; Bachmann, D; Ke, F; Strasser, A; Simon, H U; Kaufmann, T

    2013-01-01

    The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a ‘tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK. PMID:23429263

  14. Mito-priming as a method to engineer Bcl-2 addiction

    PubMed Central

    Lopez, Jonathan; Bessou, Margaux; Riley, Joel S.; Giampazolias, Evangelos; Todt, Franziska; Rochegüe, Tony; Oberst, Andrew; Green, Douglas R.; Edlich, Frank; Ichim, Gabriel; Tait, Stephen W. G.

    2016-01-01

    Most apoptotic stimuli require mitochondrial outer membrane permeabilization (MOMP) in order to execute cell death. As such, MOMP is subject to tight control by Bcl-2 family proteins. We have developed a powerful new technique to investigate Bcl-2-mediated regulation of MOMP. This method, called mito-priming, uses co-expression of pro- and anti-apoptotic Bcl-2 proteins to engineer Bcl-2 addiction. On addition of Bcl-2 targeting BH3 mimetics, mito-primed cells undergo apoptosis in a rapid and synchronous manner. Using this method we have comprehensively surveyed the efficacy of BH3 mimetic compounds, identifying potent and specific MCL-1 inhibitors. Furthermore, by combining different pro- and anti-apoptotic Bcl-2 pairings together with CRISPR/Cas9-based genome editing, we find that tBID and PUMA can preferentially kill in a BAK-dependent manner. In summary, mito-priming represents a facile and robust means to trigger mitochondrial apoptosis. PMID:26833356

  15. BH3 Response Profiles From Neuroblastoma Mitochondria Predict Activity of Small Molecule Bcl-2 Family Antagonists

    PubMed Central

    Goldsmith, Kelly C.; Lestini, Brian J.; Gross, Michelle; Ip, Laura; Bhumbla, Ashish; Zhang, Xuemei; Zhao, Huaqing; Liu, Xueyuan; Hogarty, Michael D.

    2009-01-01

    Bcl-2 family proteins regulate mitochondrial apoptosis downstream of diverse stressors. Cancer cells frequently deregulate Bcl-2 proteins leading to chemoresistance. We have optimized a platform for solid tumors in which Bcl-2 family resistance patterns are inferred. Functional mitochondria were isolated from neuroblastoma cell lines, exposed to distinct BH3-domain peptides, and assayed for cytochrome c release. Such BH3 profiles revealed three patterns of cytochrome c response. A subset had a dominant NoxaBH3 response implying Mcl1-dependence. These cells were more sensitive to small molecules that antagonize Mcl1 (AT-101) than those that antagonize Bcl-2, Bcl-xL and Bcl-w (ABT-737). A second subset had a dominant BikBH3 response, implying a Bcl-xL/-w dependence, and was exquisitely sensitive to ABT-737 (IC50 <200 nM). Finally, most neuroblastoma cell lines derived at relapse were relatively resistant to pro-death BH3 peptides and Bcl-2 antagonists. Our findings define heterogeneity for apoptosis resistance in neuroblastoma, help triage emerging Bcl-2 antagonists for clinical use, and provide a platform for studies to characterize post-therapy resistance mechanisms for neuroblastoma and other solid tumors. PMID:19893570

  16. [Reversion of multidrug resistance in HL-60/VCR cells by down-regulation of bcl-2 with bcl-2 siRNA].

    PubMed

    Piao, Ying; Chen, Xie-Qun; Liu, Li-Mei; Hong, Liu; Liu, Jing-Hua; Zhou, Fan; Liu, Yan-Qin

    2005-12-01

    To evaluate the feasibility of gene therapy using bcl-2 as target in multiple drug resistance of leukemia, the small interfering RNA eukaryotic expression vector specific to human bcl-2 gene was constructed by gene recombination, then transfected into HL-60/VCR cells. Stable transfectants were obtained by G418 screening. The growth curve and drug sensitivity were detected by using MTT. The expression of Bax and ZNRD1 was analyzed by Western blot. The results showed that mU6pro-bcl-2 siRNA was successfully constructed and transfected into HL-60/VCR cells. The IC(50) of transfected cells to vincristine and adriamycin was significantly reduced as compared with that of the control. The expression of ZNRD1 in transfected cells was decreased as compared with that of the control, while Bax not. It is concluded that the bcl-2 siRNA restores the sensitivity of HL-60/VCR cells to conventional chemotherapeutic agents to a certain degree. PMID:16403269

  17. The downregulation of Bcl-xL/Bcl-2-associated death promoter indicates worse outcomes in patients with small cell lung carcinoma

    PubMed Central

    Yu, Yaoyang; Zhong, Zhaokui; Guan, Yaowu

    2015-01-01

    It is well known that lung cancer is the 1st leading cause of death worldwide. Many reports have demonstrated that Bad, the Bcl-xL/Bcl-2-associated death promoter plays a crucial role in the intrinsic apoptosis pathway. The aim of this study was to explore the expression of Bad and its clinical significance in small cell lung carcinoma (SCLC) By analyzing the expression of Bad in 147 SCLC patient specimen, we found that Bad expression was remarkably decreased in 55.8% (82/147) cases, compared with the neighboring non-tumor tissues. Further study showed that Bad expression was correlated with adverse clinical characters such as clinical stage (P = 0.001), tumor size (P = 0.036) and tumor recurrence (P = 0.030). Furthermore, the results of Kaplan-Meier analysis showed that low Bad expression was significantly correlated to overall survival (P < 0.0001) and disease-free survival (P = 0.017) of patients with SCLC. Moreover, multivariate analyses revealed that Bad was an independent indicator of overall survival in SCLC (hazard ration = 0.620, 95% confidence interval: 0.389-0.987, P < 0.001). In summary, we can conclude that patients with SCLC represent downregulation of Bad and the latter could be served as a useful biomarker for the outcomes of SCLC. PMID:26722503

  18. MicroRNAs expression in ox-LDL treated HUVECs: MiR-365 modulates apoptosis and Bcl-2 expression

    SciTech Connect

    Qin, Bing; Xiao, Bo; Liang, Desheng; Xia, Jian; Li, Ye; Yang, Huan

    2011-06-24

    Highlights: {yields} We evaluated the role of miRNAs in ox-LDL induced apoptosis in ECs. {yields} We found 4 up-regulated and 11 down-regulated miRNAs in apoptotic ECs. {yields} Target genes of the dysregulated miRNAs regulate ECs apoptosis and atherosclerosis. {yields} MiR-365 promotes ECs apoptosis via suppressing Bcl-2 expression. {yields} MiR-365 inhibitor alleviates ECs apoptosis induced by ox-LDL. -- Abstract: Endothelial cells (ECs) apoptosis induced by oxidized low-density lipoprotein (ox-LDL) is thought to play a critical role in atherosclerosis. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth, proliferation, and apoptosis. However, whether miRNAs are associated with ox-LDL induced apoptosis and their effect on ECs is still unknown. Therefore, this study evaluated potential miRNAs and their involvement in ECs apoptosis in response to ox-LDL stimulation. Microarray and qRT-PCR analysis performed on human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL identified 15 differentially expressed (4 up- and 11 down-regulated) miRNAs. Web-based query tools were utilized to predict the target genes of the differentially expressed miRNAs, and the potential target genes were classified into different function categories with the gene ontology (GO) term and KEGG pathway annotation. In particular, bioinformatics analysis suggested that anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2) is a target gene of miR-365, an apoptomir up-regulated by ox-LDL stimulation in HUVECs. We further showed that transfection of miR-365 inhibitor partly restored Bcl-2 expression at both mRNA and protein levels, leading to a reduction of ox-LDL-mediated apoptosis in HUVECs. Taken together, our findings indicate that miRNAs participate in ox-LDL-mediated apoptosis in HUVECs. MiR-365 potentiates ox-LDL-induced ECs apoptosis by regulating the

  19. Selective impairment of CD4+CD25+Foxp3+ regulatory T cells by paclitaxel is explained by Bcl-2/Bax mediated apoptosis.

    PubMed

    Liu, Nan; Zheng, Yijie; Zhu, Ying; Xiong, Shudao; Chu, Yiwei

    2011-02-01

    Paclitaxel has become one of the most effective and widely used chemotherapeutic agents over the past decades. Although it has shown promise to selectively deplete regulatory T (Treg) cells in our previous study, the underlying molecular mechanism remains to be further elucidated. The present study focused on the effect of paclitaxel on Treg cells in 3LL Lewis tumor model and explored the possible molecular pathways involved in this process. We found that paclitaxel significantly decreased the percentage of Treg cells in CD4(+) cells and impaired their suppressive functions, but effector T (Teff) cells remained unaffected. Compared with Teff cells, Treg cells exhibited a high sensitivity to paclitaxel-mediated apoptosis in vitro. Interestingly, though paclitaxel has been characterized as a mitotic inhibitor, tubulin was not involved in the selective function of paclitaxel. Treg cells exposed to paclitaxel displayed downregulation of Bcl-2 and upregulation of Bax. Blocking the Bcl-2 pathway eliminated the difference between Treg and Teff cells responding to paclitaxel. These results suggest that Bcl-2 rather than tubulin contributes to the distinctive effect of paclitaxel on Treg cells. Therefore, we here identify a molecular pathway through which paclitaxel selectively ablates Treg cells. PMID:21115120

  20. Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts*

    PubMed Central

    Nagase, Yuichi; Iwasawa, Mitsuyasu; Akiyama, Toru; Kadono, Yuho; Nakamura, Masaki; Oshima, Yasushi; Yasui, Tetsuro; Matsumoto, Takumi; Hirose, Jun; Nakamura, Hiroaki; Miyamoto, Takeshi; Bouillet, Philippe; Nakamura, Kozo; Tanaka, Sakae

    2009-01-01

    The anti-apoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Bcl-2−/− mice are growth-retarded and exhibit increased bone volume of the primary spongiosa, mainly due to the decreased number and dysfunction of osteoclasts. Osteoblast function is also impaired in Bcl-2−/− mice. Ex vivo studies on osteoblasts and osteoclasts showed that Bcl-2 promoted the differentiation, activation, and survival of both cell types. Because Bcl-2−/− mice die before 6 weeks of age due to renal failure and cannot be compared with adult wild type mice, we generated Bcl-2−/−Bim+/− mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2+/−Bim+/− littermates. Loss of a single Bim allele restored normal osteoclast function in Bcl-2−/− mice but did not restore the impaired function of osteoblasts, and the mice exhibited osteopenia. These data demonstrate that Bcl-2 promotes the differentiation, activity, and survival of both osteoblasts and osteoclasts. The balance between Bcl-2 and Bim regulates osteoclast apoptosis and function, whereas other pro-apoptotic members are important for osteoblasts. PMID:19846553

  1. Distinctive Expression of Bcl-2 Factors in Regulatory T Cells Determines a Pharmacological Target to Induce Immunological Tolerance

    PubMed Central

    Gabriel, Sarah Sharon; Bon, Nina; Chen, Jin; Wekerle, Thomas; Bushell, Andrew; Fehr, Thomas; Cippà, Pietro Ernesto

    2016-01-01

    Distinctive molecular characteristics of functionally diverse lymphocyte populations may represent novel pharmacological targets for immunotherapy. The intrinsic apoptosis pathway is differently regulated among conventional and regulatory T cells (Tregs). Targeted pharmacological modulation of this pathway with a small molecule Bcl-2/Bcl-xL inhibitor (ABT-737) caused a selective depletion of effector T cells and a relative enrichment of Tregs in vivo. Treatment with ABT-737 resulted in a tolerogenic milieu, which was exploited to alleviate graft-versus-host disease, to prevent allograft rejection in a stringent fully MHC-mismatched skin transplantation model and to induce immunological tolerance in combination with bone marrow transplantation. This concept has the potential to find various applications for immunotherapy, since it allows pharmacologic exploitation of the immunomodulatory properties of Tregs without the need for cell manipulation ex vivo. PMID:26973650

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

    PubMed

    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. PMID:18381443

  3. BIM mediates synergistic killing of B-cell acute lymphoblastic leukemia cells by BCL-2 and MEK inhibitors

    PubMed Central

    Korfi, K; Smith, M; Swan, J; Somervaille, T C P; Dhomen, N; Marais, R

    2016-01-01

    B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological disease that kills ~50% of adult patients. With the exception of some BCR-ABL1+ patients who benefit from tyrosine kinase inhibitors, there are no effective targeted therapies for adult B-ALL patients and chemotherapy remains first-line therapy despite adverse side effects and poor efficacy. We show that, although the MEK/ERK pathway is activated in B-ALL cells driven by different oncogenes, MEK inhibition does not suppress B-ALL cell growth. However, MEK inhibition synergized with BCL-2/BCL-XL family inhibitors to suppress proliferation and induce apoptosis in B-ALL cells. We show that this synergism is mediated by the pro-apoptotic factor BIM, which is dephosphorylated as a result of MEK inhibition, allowing it to bind to and neutralize MCL-1, thereby enhancing BCL-2/BCL-XL inhibitor-induced cell death. This cooperative effect is observed in B-ALL cells driven by a range of genetic abnormalities and therefore has significant therapeutic potential. PMID:27054332

  4. The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila

    PubMed Central

    Matsushita, Yuka; Watanabe, Megumi; Vo, Nicole; Yoshida, Hideki; Yamaguchi, Masamitsu; Kataoka, Takao

    2016-01-01

    Bcl-2 family proteins play a central role in regulating apoptosis. We previously reported that human Bcl-rambo, also termed BCL2L13, localized to mitochondria and induced apoptosis when overexpressed in human embryonic kidney 293T cells. However, the physiological function of Bcl-rambo currently remains unclear. In the present study, human Bcl-rambo was ectopically expressed in Drosophila melanogaster. Bcl-rambo mainly localized to the mitochondria of Drosophila Schneider 2 (S2) cells. The overexpression of Bcl-rambo, but not Bcl-rambo lacking a C-terminal transmembrane domain, induced apoptosis in S2 cells. Moreover, the ectopic expression of Bcl-rambo by a GAL4-UAS system induced aberrant morphological changes characterized by atrophied wing, split thorax, and rough eye phenotypes. Bcl-rambo induced the activation of effector caspases in eye imaginal discs. The rough eye phenotype induced by Bcl-rambo was partly rescued by the co-expression of p35, Diap1, and Diap2. By using this Drosophila model, we showed that human Bcl-rambo interacted genetically with Drosophila homologues of adenine nucleotide translocators and the autophagy-related 8 protein. The results of the present study demonstrated that human Bcl-rambo localized to mitochondria and at least regulated an apoptosis signaling pathway in Drosophila. PMID:27348811

  5. Apoptin-induced cell death is modulated by Bcl-2 family members and is Apaf-1 dependent

    PubMed Central

    Burek, M; Maddika, S; Burek, CJ; Daniel, PT; Schulze-Osthoff, K; Los, M

    2010-01-01

    Apoptin, a chicken anemia virus-derived protein, selectively induces apoptosis in transformed but not in normal cells, thus making it a promising candidate as a novel anticancer therapeutic. The mechanism of apoptin-induced apoptosis is largely unknown. Here, we report that contrary to previous assumptions, Bcl-2 and Bcl-xL inhibit apoptin-induced cell death in several tumor cell lines. In contrast, deficiency of Bax conferred resistance, whereas Bax expression sensitized cells to apoptin-induced death. Cell death induction by apoptin was associated with cytochrome c release from mitochondria as well as with caspase-3 and -7 activation. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad spectrum caspase inhibitor, was highly protective against apoptin-induced cell death. Apoptosis induced by apoptin required Apaf-1, as immortalized Apaf-1-deficient fibroblasts as well as tumor cells devoid of Apaf-1were strongly protected. Thus, our data indicate that apoptin-induced apoptosis is not only Bcl-2- and caspase dependent, but also engages an Apaf-1apoptosome-mediated mitochondrial death pathway. PMID:16288204

  6. The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the β-TrCP homologue slimb.

    PubMed

    Colin, Jessie; Garibal, Julie; Clavier, Amandine; Rincheval-Arnold, Aurore; Gaumer, Sébastien; Mignotte, Bernard; Guénal, Isabelle

    2014-10-01

    The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of β-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family. PMID:25208640

  7. BIM mediates synergistic killing of B-cell acute lymphoblastic leukemia cells by BCL-2 and MEK inhibitors.

    PubMed

    Korfi, K; Smith, M; Swan, J; Somervaille, T C P; Dhomen, N; Marais, R

    2016-01-01

    B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological disease that kills ~50% of adult patients. With the exception of some BCR-ABL1(+) patients who benefit from tyrosine kinase inhibitors, there are no effective targeted therapies for adult B-ALL patients and chemotherapy remains first-line therapy despite adverse side effects and poor efficacy. We show that, although the MEK/ERK pathway is activated in B-ALL cells driven by different oncogenes, MEK inhibition does not suppress B-ALL cell growth. However, MEK inhibition synergized with BCL-2/BCL-XL family inhibitors to suppress proliferation and induce apoptosis in B-ALL cells. We show that this synergism is mediated by the pro-apoptotic factor BIM, which is dephosphorylated as a result of MEK inhibition, allowing it to bind to and neutralize MCL-1, thereby enhancing BCL-2/BCL-XL inhibitor-induced cell death. This cooperative effect is observed in B-ALL cells driven by a range of genetic abnormalities and therefore has significant therapeutic potential. PMID:27054332

  8. Radiosensitization by a novel Bcl-2 and Bcl-XL inhibitor S44563 in small-cell lung cancer.

    PubMed

    Loriot, Y; Mordant, P; Dugue, D; Geneste, O; Gombos, A; Opolon, P; Guegan, J; Perfettini, J-L; Pierre, A; Berthier, L K; Kroemer, G; Soria, J C; Depil, S; Deutsch, E

    2014-01-01

    Radiotherapy has a critical role in the treatment of small-cell lung cancer (SCLC). The effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using the Bcl-2/Bcl-XL inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 caused SCLC cells to acquire hallmarks of apoptosis. S44563 markedly enhanced the sensitivity of SCLC cells to radiation, as determined by a clonogenic assay. The combination of S44563 and cisplatin-based chemo-radiation showed a significant tumor growth delay and increased overall survival in mouse xenograft models. This positive interaction was greater when S44563 was given after the completion of the radiation, which might be explained by the radiation-induced overexpression of anti-apoptotic proteins secondary to activation of the NF-κB pathway. These data underline the possibility of combining IR and Bcl-2/Bcl-XL inhibition in the treatment of SCLC as they underscore the importance of administering conventional and targeted therapies in an optimal sequence. PMID:25232677

  9. Nitric oxide and bcl-2 mediated the apoptosis induced by nickel(II) in human T hybridoma cells

    SciTech Connect

    Guan Fuqin; Zhang Dongmei; Wang Xinchang; Chen Junhui . E-mail: jhchen@nju.edu.cn

    2007-05-15

    Although effects of nickel(II) on the immune system have long been recognized, little is known about the effects of nickel(II) on the induction of apoptosis and related signaling events in T cells. In the present study, we investigated the roles and signaling pathways of nickel(II) in the induction of apoptosis in a human T cell line jurkat. The results showed that the cytotoxic effects of Ni involved significant morphological changes and chromosomal condensation (Hoechst 33258 staining). Analyses of hypodiploid cells and FITC-Annexin V and PI double staining showed significant increase of apoptosis in jurkat cells 6, 12 and 24 h after nickel(II) treatment. Flow cytometry analysis also revealed that the loss of mitochondrial membrane potential (MMP) occurred concomitantly with the onset of NiCl{sub 2}-induced apoptosis. Induction of apoptotic cell death by nickel was mediated by reduction of bcl-2 expression. Furthermore, nickel stimulated the generation of nitric oxide (NO). These results suggest that nickel(II) chloride induces jurkat cells apoptosis via nitric oxide generation, mitochondrial depolarization and bcl-2 suppression.

  10. The Bcl-2/Bcl-xL inhibitor BH3I-2' affects the dynamics and subcellular localization of sumoylated proteins.

    PubMed

    Plourde, Mélodie B; Morchid, Aïda; Iranezereza, Lolita; Berthoux, Lionel

    2013-04-01

    Sumoylation modulates many proteins implicated in apoptosis such as Fas, TNFR1, Daxx, p53 and its regulator MDM2. Some of these proteins, such as DRP-1, are involved in the intrinsic apoptosis pathway. The intrinsic pathway is regulated at the mitochondrial level by the Bcl-2 family of proteins. The small-molecule inhibitor BH3I-2' binds to the hydrophobic groove of the BH3 domain of anti-apoptotic proteins Bcl-xL and Bcl-2. Following treatment with this inhibitor in various experimental conditions, we observed decreased levels of detergent-soluble SUMO-1, an increase in the relative levels of detergent-insoluble sumoylated proteins, or both. Accordingly, immunofluorescence microscopy revealed that the relative numbers and intensities of endogenously or exogenously expressed SUMO-1 foci in the nucleus were increased following BH3I-2' treatment. MG132 caused a large increase in steady-state levels of SUMO-1 and of sumoylated proteins, and this was especially true for detergent-insoluble proteins. The conjugation-incompetent GG-to-AA SUMO-1 mutant, which did not form nuclear foci, was only present in the detergent-soluble lysate fraction and was insensitive to BH3I-2', implying that BH3I-2' specifically affects SUMO in its conjugated form. Finally, BH3I-2' had similar effects on SUMO-2 and SUMO-3 as it had on SUMO-1. In conclusion, BH3I-2' causes an intracellular redistribution of sumoylated proteins, more specifically their targeting to PML and non-PML nuclear bodies in which they may be degraded by the proteasome. Interestingly, knocking down Bcl-2 also altered levels of sumoylated proteins and their presence in detergent-insoluble compartments, confirming the role of Bcl-2 as a modulator of the sumoylation pathway. PMID:23375957

  11. Bcl-2 and Hsp27 act at different levels to suppress programmed cell death.

    PubMed

    Guénal, I; Sidoti-de Fraisse, C; Gaumer, S; Mignotte, B

    1997-07-17

    Apoptosis and necrosis, two morphologically distinct forms of cell death, can be induced by common stimuli depending on the doses and the cell type. This study compares the protective effect of oncoprotein Bcl-2 and of the small stress protein Hsp27 on these two types of cell death. We use rat embryo fibroblasts conditionally immortalized by the tsA58 mutant of SV40 large T antigen as parental cells to develop cell lines carrying inducible bcl-2 or hsp27 genes. Two apoptotic stimuli were used: shift to the restrictive temperature that induced p53-mediated apoptosis and treatment with low doses of hydrogen peroxide. Necrosis was induced by high doses of hydrogen peroxide. Although Bcl-2 and Hsp27 protect these cells from necrotic death, only Bcl-2 appears capable of preventing apoptotic death. Bcl-2 protection is not mediated by a negative effect on the induction of the p53 responsive genes bax or waf1 but it slows down at least two stages of apoptosis: decrease of mitochondrial membrane potential and subsequent morphological changes. In contrast, although Hsp27 has been recently shown to inhibit apoptosis induced by various stimuli, its overexpression has no effect on apoptosis in this cell system. It should be also noticed that the apoptotic stimuli (temperature shift or hydrogen peroxide treatment) induce Hsp27, but not Bcl-2 accumulation suggesting that, in parental cells, Hsp27 might already provide some protection. However, taken together these results suggest that Hsp27, as well as Bcl-2, acts at several levels to inhibit cell death, but that their protective functions only partially overlap. PMID:9233769

  12. Expression of Bcl-2 and Bax in Mouse Renal Tubules during Kidney Development

    PubMed Central

    Song, Xiao-Feng; Ren, Hao; Andreasen, Arne; Thomsen, Jesper Skovhus; Zhai, Xiao-Yue

    2012-01-01

    Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated. PMID:22389723

  13. Multimodal Interaction with BCL-2 Family Proteins Underlies the Pro-Apoptotic Activity of PUMA BH3

    PubMed Central

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

    2013-01-01

    SUMMARY PUMA is a pro-apoptotic BCL-2 family member that drives the apoptotic response to a diversity of p53-dependent and independent cellular insults. Deciphering the spectrum of PUMA interactions that confer its context-dependent pro-apoptotic 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 anti-apoptotic proteins, directly bind to 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 anti-apoptotic inhibitor and pro-apoptotic direct activator, and its mimetics may serve as effective pharmacologic triggers of apoptosis in resistant human cancers. PMID:23890007

  14. A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2

    PubMed Central

    Lavik, Andrew R.; Greenberg, Edward; Choudhary, Yuvraj; Smith, Mitchell R.; McColl, Karen S.; Pink, John; Reu, Frederic J.; Matsuyama, Shigemi; Distelhorst, Clark W.

    2015-01-01

    Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton’s tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction. PMID:26317541

  15. A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2.

    PubMed

    Lavik, Andrew R; Zhong, Fei; Chang, Ming-Jin; Greenberg, Edward; Choudhary, Yuvraj; Smith, Mitchell R; McColl, Karen S; Pink, John; Reu, Frederic J; Matsuyama, Shigemi; Distelhorst, Clark W

    2015-09-29

    Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton's tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction. PMID:26317541

  16. Bcl-2 maintains the mitochondrial membrane potential, but fails to affect production of reactive oxygen species and endoplasmic reticulum stress, in sodium palmitate-induced β-cell death

    PubMed Central

    Welsh, Nils

    2014-01-01

    Background Sodium palmitate causes apoptosis of β-cells, and the anti-apoptotic protein Bcl-2 has been shown to counteract this event. However, the exact mechanisms that underlie palmitate-induced pancreatic β-cell apoptosis and through which pathway Bcl-2 executes the protective effect are still unclear. Methods A stable Bcl-2-overexpressing RINm5F cell clone (BMG) and its negative control (B45) were exposed to palmitate for up to 8 h, and cell viability, mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, and NF-κB activation were studied in time course experiments. Results Palmitate exposure for 8 h resulted in increased cell death rates, and this event was partially counteracted by Bcl-2. Bcl-2 overexpression promoted in parallel also a delayed induction of GADD153/CHOP and a weaker phosphorylation of BimEL in palmitate-exposed cells. At earlier time points (2–4 h) palmitate exposure resulted in increased generation of ROS, a decrease in mitochondrial membrane potential (Δψm), and a modest increase in the phosphorylation of eIF2α and IRE1α. BMG cells produced similar amounts of ROS and displayed the same eIF2α and IRE1α phosphorylation rates as B45 cells. However, the palmitate-induced dissipation of Δψm was partially counteracted by Bcl-2. In addition, basal NF-κB activity was increased in BMG cells. Conclusions Our results indicate that Bcl-2 counteracts palmitate-induced β-cell death by maintaining mitochondrial membrane integrity and augmenting NF-κB activity, but not by affecting ROS production and ER stress. PMID:25266628

  17. Polydatin promotes apoptosis through upregulation the ratio of Bax/Bcl-2 and inhibits proliferation by attenuating the β-catenin signaling in human osteosarcoma cells

    PubMed Central

    Xu, Ge; Kuang, Ge; Jiang, Wengao; Jiang, Rong; Jiang, Dianming

    2016-01-01

    Osteosarcoma is the most prevalent primary malignant bone tumor mainly endangering young adults. In this study, we explore whether polydatin (PD), a glycoside form of resveratrol, is effective for osteosarcoma. Our results showed that PD dose-dependently inhibited proliferation and promoted apoptosis in 143B and MG63 osteosarcoma cells, examined by MTT assay and Annexin V-FITC apoptosis detection. Further, we found PD increased expression of Bax and attenuated expression of Bcl-2, and consequently augmented caspase-3 activity. Moreover, PD also dose-dependently inhibited β-catenin signaling pathway as indicated by decreased β-catenin expression and activity, while overexpression of β-catenin by adenoviruses system could abrogate the anti-tumor effect of PD. Our finding indicated that PD could inhibit the proliferation by inhibiting the β-catenin signaling and induce apoptosis via upregulation the ratio of Bax/Bcl-2 in human osteosarcoma cells. PMID:27158379

  18. Endogenous bcl-2 is not required for the development of Emu-myc-induced B-cell lymphoma.

    PubMed

    Kelly, Priscilla N; Puthalakath, Hamsa; Adams, Jerry M; Strasser, Andreas

    2007-06-01

    Although myc and bcl-2 synergize in tumor development, particularly lymphomagenesis, it is not known whether endogenous bcl-2 is required for myc-induced tumorigenesis. To investigate the role of endogenous Bcl-2 in myc-induced lymphomagenesis, we bypassed the early death of Bcl-2-deficient mice by reconstituting lethally irradiated wild-type (wt) mice with a hematopoietic system from fetal liver-derived stem cells of Emu-myc/bcl-2(-/-) or control Emu-myc transgenic embryos. In premalignant (healthy) recipients, loss of Bcl-2 caused a moderate decrease in pre-B and immature B cells, and a dramatic reduction of mature B lymphocytes expressing the Emu-myc transgene. Furthermore, cultured preneoplastic Emu-myc/bcl-2(-/-) mature B cells displayed accelerated apoptosis compared with Emu-myc B cells. However, despite the striking reduction in B-cell numbers in vivo, ablation of endogenous Bcl-2 did not prevent or even delay development of Emu-myc lymphoma. Moribund mice presented with similar degrees of splenomegaly, blood leukocyte numbers, and tumor dissemination at death. These findings demonstrate that the initiation, development, continued growth, and severity of Emu-myc lymphoma do not depend upon endogenous Bcl-2, nor upon the total number of B lymphoid cells driven by the Emu-myc transgene. These results have implications for the treatment of hematopoietic tumors, particularly those that are not caused by Bcl-2 overexpression. PMID:17317859

  19. Bcl-2 overexpression inhibits generation of intracellular reactive oxygen species and blocks adriamycin-induced apoptosis in bladder cancer cells.

    PubMed

    Kong, Chui-Ze; Zhang, Zhe

    2013-01-01

    Resistance to induction of apoptosis is a major obstacle for bladder cancer treatment. Bcl-2 is thought to be involved in anti-apoptotic signaling. In this study, we investigated the effect of Bcl-2 overexpression on apoptotic resistance and intracellular reactive oxygen species (ROS) generation in bladder cancer cells. A stable Bcl-2 overexpression cell line, BIU87-Bcl-2, was constructed from human bladder cancer cell line BIU87 by transfecting recombinant Bcl-2 [pcDNA3.1(+)-Bcl-2]. The sensitivity of transfected cells to adriamycin (ADR) was assessed by MTT assay. Apoptosis was examined by flow cytometry and acridine orange fluorescence staining. Intracellular ROS was determined using flow cytometry, and the activities of superoxide dismutase (SOD) and catalase (CAT) were also investigated by the xanthinoxidase and visible radiation methods using SOD and CAT detection kits. The susceptibility of BIU87-Bcl-2 cells to ADR treatment was significantly decreased as compared with control BIU87 cells. Enhanced expression of Bcl-2 inhibited intracellular ROS generation following ADR treatment. Moreover, the suppression of SOD and CAT activity induced by ADR treatment was blocked in the BIU87-Bcl-2 case but not in their parental cells. The overexpression of Bcl-2 renders human bladder cancer cells resistant to ADR-induced apoptosis and ROS might act as an important secondary messenger in this process. PMID:23621258

  20. Removal of the BH4 domain from Bcl-2 protein triggers an autophagic process that impairs tumor growth.

    PubMed

    Trisciuoglio, Daniela; De Luca, Teresa; Desideri, Marianna; Passeri, Daniela; Gabellini, Chiara; Scarpino, Stefania; Liang, Chengyu; Orlandi, Augusto; Del Bufalo, Donatella

    2013-03-01

    Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1) the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2) Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3) BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein. PMID:23479509

  1. Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth12

    PubMed Central

    Trisciuoglio, Daniela; De Luca, Teresa; Desideri, Marianna; Passeri, Daniela; Gabellini, Chiara; Scarpino, Stefania; Liang, Chengyu; Orlandi, Augusto; Del Bufalo, Donatella

    2013-01-01

    Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1) the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2) Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3) BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein. PMID:23479509

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

    PubMed Central

    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

  3. Bcl-2 activates a programme of premature senescence in human carcinoma cells.

    PubMed Central

    Crescenzi, Elvira; Palumbo, Giuseppe; Brady, Hugh J M

    2003-01-01

    The apoptosis regulator Bcl-2 has been shown to modulate cell-cycle progression, favouring a quiescent state over a proliferative state, in both normal and tumour cells. We show here that constitutive expression of Bcl-2 in human carcinoma cells results in a cell-cycle arrest that within a few days can become irreversible. Arrested cells acquire a senescent-like phenotype, which consists of several characteristic morphological alterations and increased activity of senescence-associated beta-galactosidase. The induction of the premature senescence programme is mediated by inhibition of Cdk2 kinase activity, and p27(KIP1) is required to maintain the senescent phenotype. We propose that the ability to activate an endogenous premature senescence programme allows Bcl-2 to suppress tumour growth. These results suggest that the down-regulation of Bcl-2 expression, which has been observed during the development and progression of human carcinoma, is related to the ability of Bcl-2 to severely hamper the growth of carcinoma cells and to induce a permanent cell-cycle arrest, with the features of senescence. PMID:12871207

  4. Two cases of histiocytic sarcoma with BCL2 translocations and occult or subsequent follicular lymphoma.

    PubMed

    Fernandez-Pol, Sebastian; Bangs, Charles D; Cherry, Athena; Arber, Daniel A; Gratzinger, Dita

    2016-09-01

    Histiocytic sarcoma is rare and difficult to distinguish from histologic mimics such as myeloid sarcoma due to its relatively nonspecific immunoprofile. A subset of histiocytic sarcomas are clonally related to synchronous or metachronous follicular lymphomas. Interestingly, the histiocytic tumor component has been shown to harbor BCL2 gene translocations that are identical to those found in the lymphoma. We present one case of histiocytic sarcoma and initially occult follicular lymphoma in which detection of a BCL2 gene translocation helped support the diagnosis. We also provide follow-up regarding a previously published case of histiocytic sarcoma with IGH/BCL2 fusion gene in which the patient subsequently developed follicular lymphoma and, later, diffuse large B-cell lymphoma. Our findings suggest that BCL2 gene translocations are a recurrent feature of a distinct subset of histiocytic sarcomas that are associated with follicular lymphoma; the follicular lymphoma component may be clinically occult at the time of diagnosis. Testing for an IGH/BCL2 translocation should be considered in the diagnostic workup of difficult-to-characterize neoplasms with histiocytic/monocytic morphology and immunoprofile. PMID:27134111

  5. Endothelium Expression of Bcl-2 Is Essential for Normal and Pathological Ocular Vascularization

    PubMed Central

    Zaitoun, Ismail S.; Johnson, Ryan P.; Jamali, Nasim; Almomani, Reem; Wang, Shoujian; Sheibani, Nader; Sorenson, Christine M.

    2015-01-01

    Bcl–2 is an anti-apoptotic protein with important roles in vascular homeostasis and angiogenesis. Mice globally lacking Bcl–2 (Bcl–2 -/-) are small in stature and succumb to renal failure shortly after weaning as a result of renal hypoplasia/cystic dysplasia. We have shown that Bcl–2 -/- mice displayed attenuated retinal vascular development and neovascularization. In vitro studies indicated that in addition to modulating apoptosis, Bcl–2 expression also impacts endothelial and epithelial cell adhesion, migration and extracellular matrix production. However, studies delineating the cell autonomous role Bcl–2 expression plays in the endothelium during vascular development, pruning and remodeling, and neovascularization are lacking. Here we generated mice carrying a conditional Bcl–2 allele (Bcl-2Flox/Flox) and VE-cadherin-cre (Bcl-2EC mice). Bcl-2EC mice were of normal stature and lifespan and displayed some but not all of the retinal vascular defects previously observed in global Bcl–2 deficient mice. Bcl-2EC mice had decreased numbers of endothelial cells, decreased retinal arteries and premature primary branching of the retinal vasculature, but unlike the global knockout mice, spreading of the retinal superficial vascular layer proceeded normally. Choroidal neovascularization was attenuated in Bcl-2EC mice, although retinal neovascularization accompanying oxygen-induced ischemic retinopathy was not. Thus, Bcl–2 expression in the endothelium plays a significant role during postnatal retinal vascularization, and pathological choroidal but not retinal neovascularization, suggesting vascular bed specific Bcl–2 function in the endothelium. PMID:26444547

  6. Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx.

    PubMed

    Jiang, Tianyu; Liu, Minhao; Wu, Jianping; Shi, Yigong

    2016-02-23

    HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins. PMID:26858413

  7. Apigenin inhibits glioma cell growth through promoting microRNA-16 and suppression of BCL-2 and nuclear factor-κB/MMP‑9.

    PubMed

    Chen, Xin-Jun; Wu, Mian-Yun; Li, Deng-Hui; You, Jin

    2016-09-01

    The present study aimed to investigate the effect of apigenin on glioma cells and to explore its potential mechanism. U87 human glioma cells treated with apigenin were used in the current study. Cell Counting Kit‑8 solution and Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection kit were used to analyze the effect of apigenin on U87 cell viability and apoptotic cell death. Reverse transcription‑quantitative polymerase chain reaction analysis was also used to determine microRNA‑16 (miR‑16) and MMP‑9 gene expression levels. Nuclear factor‑κB (NF‑κB) and B‑cell CLL/lymphoma 2 (BCL2) protein expression levels were determined using western blot analysis. An anti‑miR‑16 plasmid was constructed and transfected into U87 cells. The current study demonstrated that apigenin significantly decreased cell viability and induced apoptotic cell death of U87 cells in a dose‑dependent manner. Additionally, it was demonstrated that apigenin significantly increased miR‑16 levels, suppressed BCL2 protein expression and suppressed the NF‑κB/MMP9 signaling pathway in U87 cells. Furthermore, downregulation of miR‑16 using the anti‑miR‑16 plasmid reversed the effect of apigenin on cell viability, BCL2 protein expression and the NF‑κB/MMP‑9 pathway in U87 cells. The results of the present study suggested that apigenin inhibits glioma cell growth through promoting miR‑16 and suppression of BCL2 and NF-κB/MMP-9. In conclusion, the present study demonstrated the potential anticancer effects of apigenin on glioma cells. PMID:27430517

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

    SciTech Connect

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui; Cheng, Tian-Lu; Lin, Shinne-Ren; Chang, Long-Sen

    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.

  9. Regulation of Bcl-2 expression by HuR in HL60 leukemia cells and A431 carcinoma cells.

    PubMed

    Ishimaru, Daniella; Ramalingam, Sivakumar; Sengupta, Tapas K; Bandyopadhyay, Sumita; Dellis, Stephanie; Tholanikunnel, Baby G; Fernandes, Daniel J; Spicer, Eleanor K

    2009-08-01

    Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3'-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability. PMID:19671677

  10. RBP2 Promotes Adult Acute Lymphoblastic Leukemia by Upregulating BCL2

    PubMed Central

    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

  11. Observation of real-time interactions of Bcl-2 family members during apoptosis

    NASA Astrophysics Data System (ADS)

    Herman, Brian; Frohlich, Victoria; Qiu, Ming; Takahashi, Akiyuki

    2001-04-01

    Apoptosis is a physiological process of cell death resulting from an intricate cascade of sequential protein-protein interactions. Using donor and acceptor mutant GFP fusion constructs, we have monitored the interaction between specific pro- and anti-apoptotic members of the Bcl-2 family with each other as well as proteins located in the outer mitochondrial membrane, as current hypotheses regarding apoptosis suggest that interaction of Bcl-2 family members with each other, or with other mitochondrial membrane proteins, regulates apoptosis. Our data indicate that specific interactions between pro- and anti-apoptotic Bcl-2 family members do occur in situ in the mitochondrial membrane, are altered during apoptosis and regulate cellular sensitivity to apoptosis. These findings are the first to demonstrate real time protein-protein interactions in situ at the level of individual mitochondria.

  12. The role of Bax and Bcl-2 in gemcitabine-mediated cytotoxicity in uveal melanoma cells.

    PubMed

    Wang, Jing; Jia, Renbing; Zhang, Yidan; Xu, Xiaofang; Song, Xin; Zhou, Yixiong; Zhang, He; Ge, Shengfang; Fan, Xianqun

    2014-02-01

    Gemcitabine (GEM), a new cytotoxic agent, was shown to be effective against uveal melanoma (UM) which is noted for its resistance to chemotherapy. In this study, we found the different sensitivities to GEM in UM cell lines and identified apoptotic cell death as the cause of GEM cytotoxicity. Both UM cell lines showed an increase in Bax protein levels and activation of cleaved Caspase 3. Additionally, SP6.5 cells showed a gradual increase in Bcl-2 expression over time, whereas VUP cells showed almost none. After interfering in the expression of Bcl-2, the sensitivity to GEM was obviously enhanced in SP6.5 cells. These results suggest that an increase in Bax plays a crucial role in apoptotic cell death induced by GEM in the absence of p53. Moreover, inhibition of Bcl-2 expression can efficiently enhance the cytotoxic effect of, and sensitivity to, GEM in UM cells. PMID:24014050

  13. A comparison of pathway-independent and pathway-dependent methods in the calculation of conformational free enthalpy differences.

    PubMed

    Lin, Zhixiong; van Gunsteren, Wilfred F

    2016-01-01

    The multistep umbrella sampling method, which belongs to pathway-dependent methods to calculate conformational free enthalpy differences, is used to calculate the free enthalpy difference between a right-handed 2.710/12 -helix and a left-handed 314 -helix of a hexa-β-peptide in methanol solution. The same conformational free enthalpy difference was previously investigated using pathway-independent methods such as direct counting and enveloping distribution sampling. Our results show that the pathway-dependent simulations are sensitive to the choice of the pathway and its parameter values. A pathway based on restraining distances of hydrogen-bonding atom pairs shows poor sampling for two different values of the restraining force constant. Another pathway based on restraining backbone dihedral angles did smoothly sample the transition between the two helical conformations, but only with a proper choice of the restraining force constant. The results illustrate that if, and only if, a proper pathway and proper parameters are chosen, the multistep umbrella sampling can be almost 50 times more efficient than the pathway-independent methods in this case. The analysis illustrates the advantages and pitfalls of the much used multistep umbrella sampling methodology. PMID:25975696

  14. Gambogic acid is an antagonist of anti-apoptotic Bcl-2-family proteins

    PubMed Central

    Zhai, Dayong; Jin, Chaofang; Shiau, Chung-wai; Kitada, Shinichi; Satterthwait, Arnold C; Reed, John C.

    2008-01-01

    The natural product Gambogic acid (GA) has been reported to have cytotoxic activity against tumor cells in culture, and was identified as an active compound in a cell-based high-throughput screening (HTS) assay for activators of caspases, proteases involved in apoptosis. Using the anti-apoptotic Bcl-2-family protein, Bfl-1, as a target for screening of a library of natural products, we identified GA as a competitive inhibitor that displaced BH3 peptides from Bfl-1 in a fluorescent polarization assay (FPA). Analysis of competition for BH3 peptide binding revealed that GA inhibits all 6 human Bcl-2-family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited (concentrations required for 50% inhibition [IC50] <1 μM). Competition for BH3 peptide binding was also confirmed using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. GA functionally inhibited the anti-apoptotic Bcl-2-family proteins, as demonstrated by experiments using isolated mitochondria in which recombinant purified Bcl-2-family proteins suppress SMAC release in vitro, showing that GA neutralizes their suppressive effects on mitochondria in a concentration-dependent manner. GA killed tumor cell lines via an apoptotic mechanism, whereas analogs of GA with greatly reduced potency at BH3 peptide displacement showed little or no cytotoxic activity. However, GA retained cytotoxic activity against bax−/− bak−/− cells in which anti-apoptotic Bcl-2-family proteins lack a cytoprotective phenotype, implying that GA also has additional targets that contribute to its cytotoxic mechanism. Altogether, the findings suggest that suppression of anti-apoptotic Bcl-2-family proteins may be among the cytotoxic mechanisms by which GA kills tumor cells. PMID:18566235

  15. Effects of Ex Vivo Transduction of Mesencephalic Reaggregates with Bcl-2 on Grafted Dopamine Neuron Survival

    PubMed Central

    Sortwell, Caryl E.; Bowers, William J.; Counts, Scott E.; Pitzer, Mark R.; Fleming, Matthew F.; McGuire, Susan O.; Maguire-Zeiss, Kathleen A.; Federoff, Howard J.; Collier, Timothy J.

    2007-01-01

    Survival rates of dopamine (DA) neurons grafted to the denervated striatum are extremely poor (5-20%). Gene transfer of survival promoting factors, such as the anti-apoptotic protein bcl-2, to mesencephalic DA neurons prior to transplantation (ex vivo transduction) offers a novel approach to increase graft survival. However, specific criteria to assess the efficacy of various vectors must be adhered to in order to reasonably predict successful gene transfer with appropriate timing and levels of protein expression. Cell culture results utilizing three different herpes simplex virus (HSV) vectors to deliver the reporter ß-galactosidase gene (lacZ) indicate that transduction of mesencephalic cells with a helper virus-free HSV amplicon (HF HSVTH9lac) that harbors the 9-kb tyrosine hydroxylase (TH) promoter to drive lacZ gene expression elicits the transduction of the highest percentage (≈50%) of TH-immunoreactive (THir) neurons without significant cytotoxic effects. This transduction efficiency and limited cytotoxicity was superior to that observed following transduction with helper virus-containing HSV (HC HSVlac) and helper virus-free HSV amplicons (HF HSVlac) expressing lacZ under the transcriptional control of the HSV immediate-early 4/5 gene promoter. Subsequently, we assessed the ability of HSV-TH9lac and the bcl-2 expressing HSV-TH9bcl-2 amplicon to transduce mesencephalic reaggregates. Although an increase in bcl-2 and ß-galactosidase protein was induced by transduction, amplicon-mediated overexpression of bcl-2 did not lead to an increase in grafted THir neuron number. Even with highly efficient viral vector-mediated transduction, our results demonstrate that ex vivo gene transfer of bcl-2 to mesencephalic reaggregates is ineffective in increasing grafted DA neuron survival. PMID:17196186

  16. Arctigenin, a dietary phytoestrogen, induces apoptosis of estrogen receptor-negative breast cancer cells through the ROS/p38 MAPK pathway and epigenetic regulation.

    PubMed

    Hsieh, Chia-Jung; Kuo, Po-Lin; Hsu, Ying-Chan; Huang, Ya-Fang; Tsai, Eing-Mei; Hsu, Ya-Ling

    2014-02-01

    This study investigates the anticancer effect of arctigenin (ATG), a natural lignan product of Arctium lappa L., in human breast cancer MDA-MB-231 cells. Results indicate that ATG inhibits MDA-MB-231 cell growth by inducing apoptosis in vitro and in vivo. ATG triggers the mitochondrial caspase-independent pathways, as indicated by changes in Bax/Bcl-2 ratio, resulting in AIF and EndoG nuclear translocation. ATG increased cellular reactive oxygen species (ROS) production by increasing p22(phox)/NADPH oxidase 1 interaction and decreasing glutathione level. ATG clearly increases the activation of p38 MAPK, but not JNK and ERK1/2. Antioxidant EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, significantly decreases ATG-mediated p38 activation and apoptosis. Blocking p38 with a specific inhibitor suppresses ATG-mediated Bcl-2 downregulation and apoptosis. Moreover, ATG activates ATF-2, a transcription factor activated by p38, and then upregulates histone H3K9 trimethylation in the Bcl-2 gene promoter region, resulting in Bcl-2 downregulation. Taken together, the results demonstrate that ATG induces apoptosis of MDA-MB-231 cells via the ROS/p38 MAPK pathway and epigenetic regulation of Bcl-2 by upregulation of histone H3K9 trimethylation. PMID:24140706

  17. Independent elaboration of steroid hormone signaling pathways in metazoans.

    PubMed

    Markov, Gabriel V; Tavares, Raquel; Dauphin-Villemant, Chantal; Demeneix, Barbara A; Baker, Michael E; Laudet, Vincent

    2009-07-21

    Steroid hormones regulate many physiological processes in vertebrates, nematodes, and arthropods through binding to nuclear receptors (NR), a metazoan-specific family of ligand-activated transcription factors. The main steps controlling the diversification of this family are now well-understood. In contrast, the origin and evolution of steroid ligands remain mysterious, although this is crucial for understanding the emergence of modern endocrine systems. Using a comparative genomic approach, we analyzed complete metazoan genomes to provide a comprehensive view of the evolution of major enzymatic players implicated in steroidogenesis at the whole metazoan scale. Our analysis reveals that steroidogenesis has been independently elaborated in the 3 main bilaterian lineages, and that steroidogenic cytochrome P450 enzymes descended from those that detoxify xenobiotics. PMID:19571007

  18. Therapeutic potential of a peptide targeting BCL-2 cell guardians in cancer.

    PubMed

    Adams, Jerry M

    2012-06-01

    A promising approach to cancer therapy is to elicit apoptosis with "BH3 mimetic" drugs, which target proteins of the BCL-2 family. As of yet, however, such drugs can target only certain BCL-2 family proteins. Hence, in this issue of the JCI, LaBelle et al. assess instead the therapeutic potential of a "stapled" BH3 peptide from the BIM protein, which inactivates all its prosurvival relatives. The peptide killed cultured hematologic tumor cells and abated growth of a leukemia xenograft, without perturbing the hematopoietic compartment. Hence, such peptides might eventually provide a new way to treat refractory leukemias. PMID:22622043

  19. Expression of p63 and Bcl-2 in Malignant Thyroid Tumors and their Correlation with other Diagnostic Immunocytochemical Markers

    PubMed Central

    Jain, Shyama; Khurana, Nita; Kakar, Arun Kumar

    2016-01-01

    Introduction Bcl-2 is a marker recently studied in thyroid tumours and proposed to have prognostic significance. p63 is expressed in a proportion of papillary thyroid carcinoma cases and may have a role in tumour progression. Aim To study expression of Bcl2 and p63 in thyroid tumours and correlation of Bcl-2 with diagnostic markers including Thyroglobulin, Calcitonin and Carcinoembryonic antigen. Materials and Methods Cytology smears of 35 cases of thyroid cancer were studied over a period of 18 months. In 20 cases histopathology was available. Immunocytochemistry for Bcl-2 and p63 was done, and diagnostic markers were applied as and when required. Results p63 showed focal nuclear expression in 46.1% of papillary thyroid carcinoma cases, and was negative in all other tumours. Bcl-2 was positive in 88.9% of follicular carcinomas, 100% of papillary carcinomas and 83.3% of medullary carcinoma cases, and showed focal weak expression in 40% of Anaplastic Carcinoma (ATC) cases, thereby signifying down regulation (p-value = 0.001). There was significant down regulation of Thyroglobulin (Tg) in ATC vs well differentiated follicular derived tumours (p-value ≤ 0.016). Positive correlation was noted between expression of Bcl-2 and Calcitonin (0.93) and Bcl-2 and Carcinoembryonic Antigen (CEA) (0.89), and weak positive correlation (0.65) between Tg and Bcl-2. Conclusion Bcl-2 is downregulated in anaplastic carcinomas as compared to well differentiated thyroid tumours, and shows correlation with differentiation associated tumour antigens. Thus, loss of Bcl-2 was associated with loss of differentiation in thyroid tumours. Anaplastic carcinoma as such is associated with worse prognosis and loss of Bcl-2 may be partly responsible for the same. p63 is specific but less sensitive marker for PTC. Further studies are required to determine the role of Bcl-2 and p63 in thyroid tumours.

  20. Coexistent rearrangements of c-MYC, BCL2, and BCL6 genes in a diffuse large B-cell lymphoma.

    PubMed

    Ueda, Chiyoko; Nishikori, Momoko; Kitawaki, Toshio; Uchiyama, Takashi; Ohno, Hitoshi

    2004-01-01

    We present a patient with stage III de novo diffuse large B-cell lymphoma. The lymphoma cells showed mature B-cell immunophenotype but lacked surface immunoglobulin (Ig) expression. Long-distance and long-distance inverse polymerase chain reaction assays to detect the oncogene/Ig gene rearrangement revealed that the cells carried 3 independent fusion genes, namely, c-MYC/Ig heavy chain gene (IgH), BCL2/IgH, and Ig lambda light chain gene/BCL6. Thus, the lymphoma cells concurrently carried t(8;14)(q24;q32), t(14;18)(q32;q21), and t(3;22)(q27;q11), which developed in association with class switching, V/D/J recombination, and somatic hypermutation, respectively. The lymphoma responded to chemoradiotherapy, and the patient has been well for 2 years, suggesting that multiple oncogene rearrangements may not necessarily be associated with poor clinical outcome. PMID:14979479

  1. Association of Bcl-2 with cyclin a/Cdk-2 complex and its effects on Cdk-2 activity.

    PubMed

    Crescenzi, Elvira; Sannino, Maria; Tonziello, Gilda; Palumbo, Giuseppe

    2002-11-01

    In the human endometrial carcinoma cell line HEC1B, the overexpression of Bcl-2 leads to a G2/M cell cycle arrest. The experiments presented in this work suggest a direct interaction between the antiapoptotic protein Bcl-2 and the cyclin-dependent kinase Cdk-2 and suggest that such interaction is cell cycle dependent. While further experiments are currently under way in our laboratory to elucidate the significance of Cdk-2/Bcl-2 complexes in PCD and cell cycle regulation, we demonstrate also a specific inhibitory function of Bcl-2 on the activity of the coprecipitated kinase. PMID:12485875

  2. Bcl-2 is a novel interacting partner for the 2-oxoglutarate carrier and a key regulator of mitochondrial glutathione.

    PubMed

    Wilkins, Heather M; Marquardt, Kristin; Lash, Lawrence H; Linseman, Daniel A

    2012-01-15

    Despite making up only a minor fraction of the total cellular glutathione, recent studies indicate that the mitochondrial glutathione pool is essential for cell survival. Selective depletion of mitochondrial glutathione is sufficient to sensitize cells to mitochondrial oxidative stress (MOS) and intrinsic apoptosis. Glutathione is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. Therefore, regulation of mitochondrial glutathione transport is a key factor in maintaining the antioxidant status of mitochondria. Bcl-2 resides in the outer mitochondrial membrane where it acts as a central regulator of the intrinsic apoptotic cascade. In addition, Bcl-2 displays an antioxidant-like function that has been linked experimentally to the regulation of cellular glutathione content. We have previously demonstrated a novel interaction between recombinant Bcl-2 and reduced glutathione (GSH), which was antagonized by either Bcl-2 homology-3 domain (BH3) mimetics or a BH3-only protein, recombinant Bim. These previous findings prompted us to investigate if this novel Bcl-2/GSH interaction might play a role in regulating mitochondrial glutathione transport. Incubation of primary cultures of cerebellar granule neurons (CGNs) with the BH3 mimetic HA14-1 induced MOS and caused specific depletion of the mitochondrial glutathione pool. Bcl-2 was coimmunoprecipitated with GSH after chemical cross-linking in CGNs and this Bcl-2/GSH interaction was antagonized by preincubation with HA14-1. Moreover, both HA14-1 and recombinant Bim inhibited GSH transport into isolated rat brain mitochondria. To further investigate a possible link between Bcl-2 function and mitochondrial glutathione transport, we next examined if Bcl-2 associated with the 2-oxoglutarate carrier (OGC), an inner mitochondrial membrane protein known to transport glutathione in liver and kidney. After cotransfection of CHO cells, Bcl-2 was coimmunoprecipitated with OGC and this novel

  3. Bcl-2 is a novel interacting partner for the 2-oxoglutarate carrier and a key regulator of mitochondrial glutathione

    PubMed Central

    Wilkins, Heather M.; Marquardt, Kristin; Lash, Lawrence H.; Linseman, Daniel A.

    2011-01-01

    Despite making up only a minor fraction of the total cellular glutathione, recent studies indicate that the mitochondrial glutathione pool is essential for cell survival. Selective depletion of mitochondrial glutathione is sufficient to sensitize cells to mitochondrial oxidative stress (MOS)1 and intrinsic apoptosis. Glutathione is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. Therefore, regulation of mitochondrial glutathione transport is a key factor in maintaining the antioxidant status of mitochondria. Bcl-2 is resident in the outer mitochondrial membrane where it acts as a central regulator of the intrinsic apoptotic cascade. In addition, Bcl-2 displays an antioxidant-like function that has been linked experimentally to the regulation of cellular glutathione content. We have previously demonstrated a novel interaction between recombinant Bcl-2 and reduced glutathione (GSH) which was antagonized by either Bcl-2 homology-3 domain (BH3) mimetics or a BH3-only protein, recombinant Bim. These previous findings prompted us to investigate if this novel Bcl-2/GSH interaction might play a role in regulating mitochondrial glutathione transport. Incubation of primary cultures of cerebellar granule neurons (CGNs) with the BH3 mimetic, HA14-1, induced MOS and caused specific depletion of the mitochondrial glutathione pool. Bcl-2 was co-immunoprecipitated with GSH following chemical cross-linking in CGNs and this Bcl-2/GSH interaction was antagonized by pre-incubation with HA14-1. Moreover, both HA14-1 and recombinant Bim inhibited GSH transport into isolated rat brain mitochondria. To further investigate a possible link between Bcl-2 function and mitochondrial glutathione transport, we next examined if Bcl-2 associated with the 2-oxoglutarate carrier (OGC), an inner mitochondrial membrane protein known to transport glutathione in liver and kidney. Following co-transfection of CHO cells, Bcl-2 was co-immunoprecipitated with OGC

  4. The Natively Disordered Loop of Bcl-2 Undergoes Phosphorylation-Dependent Conformational Change and Interacts with Pin1

    PubMed Central

    Kang, CongBao; Bharatham, Nagakumar; Chia, Joel; Mu, Yuguang; Baek, Kwanghee; Yoon, Ho Sup

    2012-01-01

    Bcl-2 plays a central role in the regulation of apoptosis. Structural studies of Bcl-2 revealed the presence of a flexible and natively disordered loop that bridges the Bcl-2 homology motifs, BH3 and BH4. This loop is phosphorylated on multiple sites in response to a variety of external stimuli, including the microtubule-targeting drugs, paclitaxel and colchicine. Currently, the underlying molecular mechanism of Bcl-2 phosphorylation and its biological significance remain elusive. In this study, we investigated the molecular characteristics of this anti-apoptotic protein. To this end, we generated synthetic peptides derived from the Bcl-2 loop, and multiple Bcl-2 loop truncation mutants that include the phosphorylation sites. Our results demonstrate that S87 in the flexible loop of Bcl-2 is the primary phosphorylation site for JNK and ERK2, suggesting some sequence or structural specificity for the phosphorylation by these kinases. Our NMR studies and molecular dynamics simulation studies support indicate that phosphorylation of S87 induces a conformational change in the peptide. Finally, we show that the phosphorylated peptides of the Bcl-2 loop can bind Pin1, further substantiating the phosphorylation-mediated conformation change of Bcl-2. PMID:23272207

  5. [Bcl-2 inhibits p53-induced apoptosis after genotoxic damage by inhibitors of nuclear import of p53].

    PubMed

    Beham, A; Schumacher, G; McDonnell, T J; Marin, M C; Jauch, K W

    1998-01-01

    The tumor suppressor gene p53 in overexpressed in 50% of colorectal carcinomas and is an interesting target for gene therapeutic approaches. Furthermore the protooncogen bcl-2 is known to inhibit p53 induced apoptosis and is expressed in some colorectal carcinomas. In this study mechanism of bcl-2 cell death inhibition after p53 induction were evaluated. The human colon carcinoma cell line RKO posses wild-type p53 and also expresses bcl-2 protein. RKO cells were treated with liposomal bcl-2 antisense oligonucleotides (AS), control oligonucleotides (CO) and empty liposomes (EL) resulting in decreased bcl-2 expression. After induction of p53 with gamma-irradiation p53 protein expression was induced in AS, CO and EL pretreated cells. Microscopy and immunoblotting was used to characterize subcellular localization of p53 protein. Further p53 subcellular localisation was examined after p53 transfer of wt p53 cDNA in three bcl-2 expressing cell lines. Most of the p53 protein remained localized in the cytosol and apoptosis was decreased in bcl-2 expressing cells assessed by flow cytometric analysis (Ao). Our data suggests that bcl-2 is able to modulate transmembrane trafficking of p53. This resulted in inhibition of cell death implicating that bcl-2 function is involved in regulation of transmembrane gradients. PMID:14518224

  6. Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation

    PubMed Central

    Murakawa, Tomokazu; Yamaguchi, Osamu; Hashimoto, Ayako; Hikoso, Shungo; Takeda, Toshihiro; Oka, Takafumi; Yasui, Hiroki; Ueda, Hiromichi; Akazawa, Yasuhiro; Nakayama, Hiroyuki; Taneike, Manabu; Misaka, Tomofumi; Omiya, Shigemiki; Shah, Ajay M.; Yamamoto, Akitsugu; Nishida, Kazuhiko; Ohsumi, Yoshinori; Okamoto, Koji; Sakata, Yasushi; Otsu, Kinya

    2015-01-01

    Damaged mitochondria are removed by mitophagy. Although Atg32 is essential for mitophagy in yeast, no Atg32 homologue has been identified in mammalian cells. Here, we show that Bcl-2-like protein 13 (Bcl2-L-13) induces mitochondrial fragmentation and mitophagy in mammalian cells. First, we hypothesized that unidentified mammalian mitophagy receptors would share molecular features of Atg32. By screening the public protein database for Atg32 homologues, we identify Bcl2-L-13. Bcl2-L-13 binds to LC3 through the WXXI motif and induces mitochondrial fragmentation and mitophagy in HEK293 cells. In Bcl2-L-13, the BH domains are important for the fragmentation, while the WXXI motif facilitates mitophagy. Bcl2-L-13 induces mitochondrial fragmentation in the absence of Drp1, while it induces mitophagy in Parkin-deficient cells. Knockdown of Bcl2-L-13 attenuates mitochondrial damage-induced fragmentation and mitophagy. Bcl2-L-13 induces mitophagy in Atg32-deficient yeast cells. Induction and/or phosphorylation of Bcl2-L-13 may regulate its activity. Our findings offer insights into mitochondrial quality control in mammalian cells. PMID:26146385

  7. Expression levels of the BAK1 and BCL2 genes highlight the role of apoptosis in age-related hearing impairment.

    PubMed

    Falah, Masoumeh; Najafi, Mohammad; Houshmand, Massoud; Farhadi, Mohammad

    2016-01-01

    Age-related hearing impairment (ARHI) is a progressive and a common sensory disorder in the elderly and will become an increasingly important clinical problem given the growing elderly population. Apoptosis of cochlear cells is an important factor in animal models of ARHI. As these cells cannot regenerate, their loss leads to irreversible hearing impairment. Identification of molecular mechanisms can facilitate disease prevention and effective treatment. In this study, we compared the expression of the genes BAK1 and BCL2 as two arms of the intrinsic apoptosis pathway between patients with ARHI and healthy subjects. ARHI and healthy subjects were selected after an ear nose throat examination, otoscopic investigation, and pure tone audiometry. RNA was extracted from peripheral blood samples, and relative gene expression levels were measured using quantitative real-time polymerase chain reaction. BAK1 and the BAK1/BCL2 ratio were statistically significantly upregulated in the ARHI subjects. The BAK1/BCL2 ratio was positively correlated with the results of the audiometric tests. Our results indicate that BAK-mediated apoptosis may be a core mechanism in the progression of ARHI in humans, similar to finding in animal models. Moreover, the gene expression changes in peripheral blood samples could be used as a rapid and simple biomarker for early detection of ARHI. PMID:27555755

  8. Loss of PUMA (BBC3) does not prevent thrombocytopenia caused by the loss of BCL-XL (BCL2L1).

    PubMed

    Delbridge, Alex R D; Chappaz, Stephane; Ritchie, Matthew E; Kile, Benjamin T; Strasser, Andreas; Grabow, Stephanie

    2016-09-01

    Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime example. Their brief life span in the circulation is regulated by the intrinsic apoptosis pathway. Pro-survival BCL-XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro-apoptotic BCL-2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL-XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis in vivo remains unclear. The pro-apoptotic BH3-only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3-only protein that can inhibit all pro-survival BCL-2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL-XL. It therefore remains to be established whether other BH3-only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL-XL with BAK and BAX. PMID:27221652

  9. Neuroprotective effects of a chromatin modifier on ischemia/reperfusion neurons: implication of its regulation of BCL2 transactivation by ERα signaling.

    PubMed

    Guo, Jun; Zhang, Tao; Yu, Jia; Li, Hong-Zeng; Zhao, Cong; Qiu, Jing; Zhao, Bo; Zhao, Jie; Li, Wei; Zhao, Tian-Zhi

    2016-06-01

    An understanding of the molecular mechanisms involved in the regulation of estrogen receptor alpha (ERα)-mediated neuroprotective effects is valuable for the development of therapeutic strategy against neuronal ischemic injury. Here, we report the upregulated expression of metastasis-associated protein 1 (MTA1), a master chromatin modifier and transcriptional regulator, in the murine middle cerebral artery occlusion (MCAO) model. Inhibition of MTA1 expression by in vivo short interfering RNA treatment potentiated neuronal apoptosis in a caspase-3-dependent manner and thereafter aggravated MCAO-induced neuronal damage. Mechanistically, the pro-survival effects of MTA1 required the participation of ERα signaling. We also provide in vitro evidence that MTA1 enhances the binding of ERα with the BCL2 promoter upon ischemic insults via recruitment of HDAC2 together with other unidentified coregulators, thus promoting the ERα-mediated transactivation of the BCL2 gene. Collectively, our results suggest that the augmentation of endogenous MTA1 expression during neuronal ischemic injury acts additionally to an endocrinous cascade orchestrating intimate interactions between ERα and BCL2 pathways and operates as an indispensable defensive mechanism in response to neuronal ischemia/reperfusion stress. Future studies in this field will shed light on the modulation of the complicated neuroprotective effects by estrogen signaling. PMID:26728277

  10. Gambogic acid induces mitochondria-dependent apoptosis by modulation of Bcl-2 and Bax in mantle cell lymphoma JeKo-1 cells

    PubMed Central

    Xu, Jingyan; Zhou, Min; Wang, Jing; Zhang, Qiguo; Xu, Yong; Xu, Yueyi; Zhang, Qian; Xu, Xihui; Zeng, Hui

    2013-01-01

    Objective To study the mechanisms in gambogic acid (GA) -induced JeKo-1 human Mantle Cell Lymphoma cell apoptosis in vitro. Methods The proliferation of GA-treated JeKo-1 cells was measured by CCK-8 assay and Ki-67 immunocytochemical detection. Apoptosis, cell cycle and mitochondrial membrane potential were measured by flow cytometric analysis. Caspase-3, -8 and -9 were detected by colorimetric assay. Bcl-2 and Bax were analyzed by Western blotting. Results GA inhibited cell growth in a time- and dose- dependent manner. GA induces apoptosis in JeKo-1 cells but not in normal bone marrow cells, which was involved in reducing the membrane potential of mitochondria, activating caspases-3, -8 and -9 and decreasing the ratio of Bcl-2 and Bax without cell cycle arresting. Conclusions GA induced apoptosis in human MCL JeKo-1 cells by regulating Bcl-2/Bax and activating caspase-3, -8 and -9 via mitochondrial pathway without affecting cell cycle. PMID:23592899

  11. Expression levels of the BAK1 and BCL2 genes highlight the role of apoptosis in age-related hearing impairment

    PubMed Central

    Falah, Masoumeh; Najafi, Mohammad; Houshmand, Massoud; Farhadi, Mohammad

    2016-01-01

    Age-related hearing impairment (ARHI) is a progressive and a common sensory disorder in the elderly and will become an increasingly important clinical problem given the growing elderly population. Apoptosis of cochlear cells is an important factor in animal models of ARHI. As these cells cannot regenerate, their loss leads to irreversible hearing impairment. Identification of molecular mechanisms can facilitate disease prevention and effective treatment. In this study, we compared the expression of the genes BAK1 and BCL2 as two arms of the intrinsic apoptosis pathway between patients with ARHI and healthy subjects. ARHI and healthy subjects were selected after an ear nose throat examination, otoscopic investigation, and pure tone audiometry. RNA was extracted from peripheral blood samples, and relative gene expression levels were measured using quantitative real-time polymerase chain reaction. BAK1 and the BAK1/BCL2 ratio were statistically significantly upregulated in the ARHI subjects. The BAK1/BCL2 ratio was positively correlated with the results of the audiometric tests. Our results indicate that BAK-mediated apoptosis may be a core mechanism in the progression of ARHI in humans, similar to finding in animal models. Moreover, the gene expression changes in peripheral blood samples could be used as a rapid and simple biomarker for early detection of ARHI. PMID:27555755

  12. Both V(D)J coding ends but neither signal end can recombine at the bcl-2 major breakpoint region, and the rejoining is ligase IV dependent.

    PubMed

    Raghavan, Sathees C; Hsieh, Chih-Lin; Lieber, Michael R

    2005-08-01

    The t(14;18) chromosomal translocation is the most common translocation in human cancer, and it occurs in all follicular lymphomas. The 150-bp bcl-2 major breakpoint region (Mbr) on chromosome 18 is a fragile site, because it adopts a non-B DNA conformation that can be cleaved by the RAG complex. The non-B DNA structure and the chromosomal translocation can be recapitulated on intracellular human minichromosomes where immunoglobulin 12- and 23-signals are positioned downstream of the bcl-2 Mbr. Here we show that either of the two coding ends in these V(D)J recombination reactions can recombine with either of the two broken ends of the bcl-2 Mbr but that neither signal end can recombine with the Mbr. Moreover, we show that the rejoining is fully dependent on DNA ligase IV, indicating that the rejoining phase relies on the nonhomologous DNA end-joining pathway. These results permit us to formulate a complete model for the order and types of cleavage and rejoining events in the t(14;18) translocation. PMID:16024785

  13. Transcriptional upregulation of HNF-1β by NF-κB in ovarian clear cell carcinoma modulates susceptibility to apoptosis through alteration in bcl-2 expression.

    PubMed

    Suzuki, Erina; Kajita, Sabine; Takahashi, Hiroyuki; Matsumoto, Toshihide; Tsuruta, Tomoko; Saegusa, Makoto

    2015-08-01

    Hepatocyte nuclear factor-1β (HNF-1β) is a transcriptional factor that has an important role in endometriosis-ovarian clear cell carcinoma (OCCC) sequence by modulating cell kinetics and glucose metabolism. However, little is known about the detailed molecular mechanisms that govern its regulation and function. Herein, we focus on upstream and downstream regulatory factors of HNF-1β in OCCCs. In clinical samples, HNF-1β expression was positively correlated with the active form of NF-κB/p65 in OCCCs, and closely linked with a low nuclear grade and non-solid architecture. In cell lines, transfection of p65 resulted in increased HNF-1β mRNA and protein expression in TOV-21G cells (OCCC cell line with endogenous HNF-1β expression), in line with activation of the promoter, probably through interacting with the basic transcriptional machinery. Suppression of endogenous HNF-1β expression by siRNA increased apoptosis in TOV-21G cells, while treatment of Hec251 cells (endometrial carcinoma cell line with extremely low endogenous HNF-1β expression) stably overexpressing exogenous HNF-1β with doxorubicin abrogated apoptosis of the cells, along with increased ratio of bcl-2 relative to bax. Moreover, overexpression of HNF-1β led to upregulation of bcl-2 expression at the transcriptional level in TOV-21G cells, which provided evidence for a positive correlation between HNF-1β and bcl-2 expression in OCCCs. These data, therefore, suggest that association between HNF-1β and NF-κB signaling may participate in cell survival by alteration of apoptotic events, particularly in mitochondria-mediated pathways, through upregulation of bcl-2 expression in OCCCs. PMID:26030369

  14. Genetic dissimilarity between primary colorectal carcinomas and their lymph node metastases: ploidy, p53, bcl-2, and c-myc expression--a pilot study.

    PubMed

    Zalata, Khaled Refaat; Elshal, Mohamed Farouk; Foda, Abd AlRahman Mohammad; Shoma, Ashraf

    2015-08-01

    The current paradigm of metastasis proposes that rare cells within primary tumors acquire metastatic capability via sequential mutations, suggesting that metastases are genetically dissimilar from their primary tumors. This study investigated the changes in the level of expression of a well-defined panel of cell proliferation, differentiation, and apoptosis markers between the primary colorectal cancer (CRC) and the corresponding synchronous lymph node (LN) metastasis from the same patients. DNA flow cytometry and immunostaining of p53, bcl-2, and c-myc were carried out on 36 cases of CRC radical resection specimens with their corresponding LN metastases. There was very low probability that the histological patterns of primary tumors and LN metastases are independent (p < 0.001). Metastatic tumors were significantly more diffusely positive for p53 than the primary tumors (p < 0.001). Conversely, primary tumors were significantly more diffusely positive for c-myc than metastatic tumors (p = 0.011). No significant difference was found between the LNs and the primary tumors in bcl-2 positivity (p = 0.538) and DNA aneuploidy (p = 0.35), with a tendency towards negative bcl-2 and less aneuploidy in LN metastases than primary tumors. In conclusion, LN metastatic colorectal carcinomas have a tendency of being less differentiated, with a higher incidence of diffuse p53 staining, lower incidence of bcl-2 staining, and less aneuploidy in comparison to their primary counterparts suggesting a more aggressive biological behavior, which could indicate the necessity for more aggressive adjuvant therapy. PMID:25840688

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

    PubMed

    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. PMID:11355956

  16. Subversion of the Bcl-2 life/death switch in cancer development and therapy.

    PubMed

    Adams, J M; Huang, D C S; Strasser, A; Willis, S; Chen, L; Wei, A; van Delft, M; Fletcher, J I; Puthalakath, H; Kuroda, J; Michalak, E M; Kelly, P N; Bouillet, P; Villunger, A; O'Reilly, L; Bath, M L; Smith, D P; Egle, A; Harris, A W; Hinds, M; Colman, P; Cory, S

    2005-01-01

    The Bcl-2 protein family, which largely determines commitment to apoptosis, has central roles in tumorigenesis and chemoresistance. Its three factions of interacting proteins include the BH3-only proteins (e.g., Bim, Puma, Bad, Noxa), which transduce diverse cytotoxic signals to the mammalian pro-survival proteins (Bcl-2, Bcl-x(L), Bcl-w, Mcl-1, A-1), whereas Bax and Bak, when freed from pro-survival constraint, provoke the mitochondrial permeabilization that triggers apoptosis. We have discovered unexpected specificity in their interactions. Only Bim and Puma, which mediate multiple cytotoxic signals, engage all the pro-survival proteins. Noxa and Bad instead bind subsets and cooperate in killing, indicating that apoptosis requires neutralization of different pro-survival subsets. Furthermore, Mcl-1 and Bcl-x(L), but not Bcl-2, directly sequester Bak in healthy cells, and Bak is freed only when BH3-only proteins neutralize both its guards. BH3-only proteins such as Bim are tumor suppressors and mediate many of the cytotoxic signals from anticancer agents. Hence, compounds mimicking them may prove valuable for therapy. Indeed, the recently described ABT-737 is a promising "BH3 mimetic" of Bad. We find that, like Bad, ABT-737 kills cells efficiently only if Mcl-1 is absent or down-regulated. Thus, manipulation of apoptosis by targeting the Bcl-2 family has exciting potential for cancer treatment. PMID:16869785

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

    PubMed Central

    Kim, Jung Seok; Rho, Jun Gi; Shin, Jung Jae; Song, Woo Keun; Lee, Eun Kyung; Egan, Josephine M.; Kim, Wook

    2016-01-01

    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. PMID:26967640

  18. WAVE1 regulates Bcl-2 localization and phosphorylation in leukemia cells

    PubMed Central

    Rui, Kang; Daolin, Tang; Yan, Yu; Zhuo, Wang; Ting, Hu; Haichao, Wang; Lizhi, Cao

    2010-01-01

    Bcl-2 proteins are over-expressed in many tumors, and are critically important for cell survival. Their anti-apoptotic activities are determined by intracellular localization and post-translational modifications (such as phosphorylation). Here we showed that WAVE1, a member of the Wiskott-Aldrich syndrome protein family, was over-expressed in blood cancer cell lines, and functioned as a negative regulator of apoptosis. Further enhanced expression of WAVE1 by gene transfection rendered leukemia cells more resistant to anti-cancer drug-induced apoptosis; whereas suppression of WAVE1 expression by RNA interference restored leukemia cells' sensitivity to anti-drug-induced apoptosis. WAVE1 was found to be associated with mitochondrial Bcl-2, and its depletion led to mitochondrial release of Bcl-2, and phosphorylation of ASK1/JNK and Bcl-2. Furthermore, depletion of WAVE1 expression increased anti-cancer drug-induced production of reactive oxygen species in leukemia cells. Taken together, these results suggest WAVE1 as a novel regulator of apoptosis, and potential drug target for therapeutic intervention of leukemia. PMID:19890377

  19. Therapeutic Modulation of Apoptosis: Targeting the BCL-2 Family at the Interface of the Mitochondrial Membrane

    PubMed Central

    Nemec, Kathleen N.

    2008-01-01

    A vast portion of human disease results when the process of apoptosis is defective. Disorders resulting from inappropriate cell death range from autoimmune and neurodegenerative conditions to heart disease. Conversely, prevention of apoptosis is the hallmark of cancer and confounds the efficacy of cancer therapeutics. In the search for optimal targets that would enable the control of apoptosis, members of the BCL-2 family of anti- and pro-apoptotic factors have figured prominently. Development of BCL-2 antisense approaches, small molecules, and BH3 peptidomimetics has met with both success and failure. Success-because BCL-2 proteins play essential roles in apoptosis. Failure-because single targets for drug development have limited scope. By examining the activity of the BCL-2 proteins in relation to the mitochondrial landscape and drawing attention to the significant mitochondrial membrane alterations that ensue during apoptosis, we demonstrate the need for a broader based multi-disciplinary approach for the design of novel apoptosis-modulating compounds in the treatment of human disease. PMID:18972587

  20. Transferrin Receptor-Targeted Lipid Nanoparticles for Delivery of an Antisense Oligodeoxyribonucleotide against Bcl-2

    PubMed Central

    Yang, Xiaojuan; Koh, Chee Guan; Liu, Shujun; Pan, Xiaogang; Santhanam, Ramasamy; Yu, Bo; Peng, Yong; Pang, Jiuxia; Golan, Sharon; Talmon, Yeshayahu; Jin, Yan; Muthusamy, Natarajan; Byrd, John C.; Chan, Kenneth K.; Lee, L. James; Marcucci, Guido; Lee, Robert J.

    2013-01-01

    Antisense oligonucleotide G3139-mediated down-regulation of Bcl-2 is a potential strategy for overcoming chemoresistance in leukemia. However, the limited efficacy shown in recent clinical trials calls attention to the need for further development of novel and more efficient delivery systems. In order to address this issue, transferrin receptor (TfR)-targeted, protamine-containing lipid nanoparticles (Tf-LNs) were synthesized as delivery vehicles for G3139. The LNs were produced by an ethanol dilution method and lipid-conjugated Tf ligand was then incorporated by a post-insertion method. The resulting Tf-LNs had a mean particle diameter of ~ 90 nm and G3139 loading efficiency of 90.4%. Antisense delivery efficiency of Tf-LNs was evaluated in K562, MV4-11 and Raji leukemia cell lines. The results showed that Tf-LNs were more effective than non-targeted LNs and free G3139 (p <0.05) in decreasing Bcl-2 expression (by up to 62% at the mRNA level in K562 cells) and in inducing caspase-dependent apoptosis. In addition, Bcl-2 down-regulation and apoptosis induced by Tf-LN G3139 were shown to be blocked by excess free Tf and thus were TfR-dependent. Cell lines with higher TfR expression also showed greater Bcl-2 down-regulation. Furthermore, upregulation of TfR expression in leukemia cells by iron chelator deferoxamine resulted in a further increase in antisense effect (up to 79% Bcl-2 reduction in K562 at the mRNA level) and in caspase-dependent apoptosis (by ~ 3-fold) by Tf-LN. Tf-LN mediated delivery combined with TfR up-regulation by deferoxamine appears to be a potentially promising strategy for enhancing the delivery efficiency and therapeutic efficacy of antisense oligonucleotides. PMID:19183107

  1. Effect of bcl-2 overexpression in mice on ovotoxicity caused by 4-vinylcyclohexene

    SciTech Connect

    Flaws, Jodi A.; Marion, Samuel L.; Miller, Kimberly P.; Christian, Patricia J.; Babus, Janice K.; Hoyer, Patricia B. . E-mail: hoyer@u.arizona.edu

    2006-08-15

    The occupational chemical 4-vinylcyclohexene (VCH) destroys small preantral ovarian follicles in mice following repeated daily dosing. The cell survival gene bcl-2 is thought to protect against follicular death during embryogenesis because primordial follicle numbers in newborn bcl-2 overexpressing (OE) mice are greater than in wild-type (WT) controls. Thus, this study was designed to determine if overexpression of bcl-2 protects against VCH-induced follicle loss during embryonic development. Pregnant bcl-2 OE or WT mice were dosed (p.o.) daily with VCH (500 mg/kg) or sesame oil (vehicle control) on days 8-18 of pregnancy. Ovaries were collected from moms and female pups on pup postnatal day (PND) 8. Nonpregnant OE and WT females were also treated with VCH (500 mg/kg p.o.) or vehicle and evaluated in the same manner. As previously reported, ovaries from PND8 OE female pups contained 50% more primordial follicles than WT pups (P < 0.05). Unlike WT pups, relative to vehicle controls, in utero exposure to VCH resulted in a reduction in primordial (25% of control), primary (38% of control), and secondary (33% of control) follicles in ovaries of OE pups (P < 0.05). VCH had no significant effect on follicle numbers in OE or WT moms. Conversely, in nonpregnant adults, VCH did not affect WT mice but caused loss of primordial (55% of control), primary (51% of control), and secondary (69% of control) follicles in OE mice (P < 0.05). These results demonstrate that bcl-2 overexpression does not protect against, but instead increases susceptibility to VCH-induced follicle loss in transplacentally exposed or in nonpregnant mice.

  2. Characterization of apoptosis and autophagy through Bcl-2 and Beclin-1 immunoexpression in gestational trophoblastic disease

    PubMed Central

    Wargasetia, Teresa Liliana; Shahib, Nurhalim; Martaadisoebrata, Djamhoer; Dhianawaty, Diah; Hernowo, Bethy

    2015-01-01

    Background: The pathogenesis of Gestational Trophoblastic Disease (GTD) is not clearly known. Objective: In this study, immunoexpression of proteins Bcl-2 and Beclin-1 in trophoblastic lesions and normal trophoblastic tissue was conducted to study the mechanism of apoptotic and autophagic cell death that is expected to complete the study of GTD pathogenesis. Materials and Methods: Bcl-2 and Beclin-1 immunoexpression were studied on complete hydatidiform mole, partial hydatidiform mole, invasive mole, choriocarcinoma and normal placenta slides. Results: The average total scores of Bcl-2 immunoexpression had a decreasing value, starting from partial hydatidiform mole (3.09), complete hydatidiform mole (2.36), invasive mole (1.18) to choriocarcinoma (0) when compared to normal placenta (6). The results showed no significant difference in Beclin-1 immunoexpression total score between complete hydatidiform mole, partial hydatidiform mole and invasive mole, namely that the value of the average total score of Beclin-1 was low (2.27, 2.45 and 2.36), but on the contrary choriocarcinoma showed an increasing strong Beclin-1 expression with the average total score of 4.57. Conclusion: Bcl-2 expression decreases in line with the excessive proliferation of trophoblast cells in hydatidiform mole and leads to malignancy in invasive mole and choriocarcinoma. The decreased expression of Beclin-1 that leads to autophagy defects in complete hydatidiform mole, partial hydatidiform mole and invasive mole shows the role of autophagy as tumor suppressor, whereas strong Beclin-1 expression shows the survival role of autophagy in choriocarcinoma. The change of Bcl-2 activity as antiapoptosis and Beclin-1 as proautophagy plays a role in pathogenesis of GTD. PMID:26494988

  3. Genome-Wide Prediction and Validation of Peptides That Bind Human Prosurvival Bcl-2 Proteins

    PubMed Central

    DeBartolo, Joe; Taipale, Mikko; Keating, Amy E.

    2014-01-01

    Programmed cell death is regulated by interactions between pro-apoptotic and prosurvival members of the Bcl-2 family. Pro-apoptotic family members contain a weakly conserved BH3 motif that can adopt an alpha-helical structure and bind to a groove on prosurvival partners Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. Peptides corresponding to roughly 13 reported BH3 motifs have been verified to bind in this manner. Due to their short lengths and low sequence conservation, BH3 motifs are not detected using standard sequence-based bioinformatics approaches. Thus, it is possible that many additional proteins harbor BH3-like sequences that can mediate interactions with the Bcl-2 family. In this work, we used structure-based and data-based Bcl-2 interaction models to find new BH3-like peptides in the human proteome. We used peptide SPOT arrays to test candidate peptides for interaction with one or more of the prosurvival proteins Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. For the 36 most promising array candidates, we quantified binding to all five human receptors using direct and competition binding assays in solution. All 36 peptides showed evidence of interaction with at least one prosurvival protein, and 22 peptides bound at least one prosurvival protein with a dissociation constant between 1 and 500 nM; many peptides had specificity profiles not previously observed. We also screened the full-length parent proteins of a subset of array-tested peptides for binding to Bcl-xL and Mcl-1. Finally, we used the peptide binding data, in conjunction with previously reported interactions, to assess the affinity and specificity prediction performance of different models. PMID:24967846

  4. Effect of bcl-2 overexpression in mice on ovotoxicity caused by 4-vinylcyclohexene.

    PubMed

    Flaws, Jodi A; Marion, Samuel L; Miller, Kimberly P; Christian, Patricia J; Babus, Janice K; Hoyer, Patricia B

    2006-08-15

    The occupational chemical 4-vinylcyclohexene (VCH) destroys small preantral ovarian follicles in mice following repeated daily dosing. The cell survival gene bcl-2 is thought to protect against follicular death during embryogenesis because primordial follicle numbers in newborn bcl-2 overexpressing (OE) mice are greater than in wild-type (WT) controls. Thus, this study was designed to determine if overexpression of bcl-2 protects against VCH-induced follicle loss during embryonic development. Pregnant bcl-2 OE or WT mice were dosed (p.o.) daily with VCH (500 mg/kg) or sesame oil (vehicle control) on days 8-18 of pregnancy. Ovaries were collected from moms and female pups on pup postnatal day (PND) 8. Nonpregnant OE and WT females were also treated with VCH (500 mg/kg p.o.) or vehicle and evaluated in the same manner. As previously reported, ovaries from PND8 OE female pups contained 50% more primordial follicles than WT pups (P < 0.05). Unlike WT pups, relative to vehicle controls, in utero exposure to VCH resulted in a reduction in primordial (25% of control), primary (38% of control), and secondary (33% of control) follicles in ovaries of OE pups (P < 0.05). VCH had no significant effect on follicle numbers in OE or WT moms. Conversely, in nonpregnant adults, VCH did not affect WT mice but caused loss of primordial (55% of control), primary (51% of control), and secondary (69% of control) follicles in OE mice (P < 0.05). These results demonstrate that bcl-2 overexpression does not protect against, but instead increases susceptibility to VCH-induced follicle loss in transplacentally exposed or in nonpregnant mice. PMID:16631218

  5. MicroRNA-184 Modulates Doxorubicin Resistance in Osteosarcoma Cells by Targeting BCL2L1

    PubMed Central

    Lin, Bo-chuan; Huang, Dong; Yu, Chao-qun; Mou, Yong; Liu, Yuan-hang; Zhang, Da-wei; Shi, Feng-jun

    2016-01-01

    Background Early metastasis of osteosarcoma (OS) is highly lethal and responds poorly to drug and radiation therapies. MicroRNAs (miRNAs) are a class of small noncoding RNAs that modulate gene expression at the post-transcriptional level. However, the detailed functions of specific miRNAs are not entirely understood. The aim of the present study was to investigate the role of miR-184 as a mediator of drug resistance in human osteosarcoma. Material/Methods qRT-PCR was used to analyze the expression level of miR-184 in OS cell line U-2 OS and MG-63 treated with doxorubicin. MiR-184 agomir or miR-184 antagomir was transferred into cells to regulated miR-184. The target of miR-184 was predicted by TargetScan and confirmed by luciferase reporter assay. Bcl-2-like protein 1 (BCL2L1) expression was detected by Western blot. Cell apoptosis was determined by Annexin V staining and analysis by flow cytometry. Results Doxorubicin induced time-dependent expression of miR-184 in OS cell line U-2 OS and MG-63. Luciferase reporter assay identified BCL2L1 as the direct target gene of miR-184. Furthermore, doxorubicin reduced BCL2L1 expression, which was reversed by miR-184 overexpression and further decreased by miR-184 inhibition in OS cells. In addition, miR-184 agomir reduced doxorubicin-induced cell apoptosis, whereas miR-184 antagomir enhanced apoptosis in OS cells, suggesting that up-regulation of miR-184 contributes to chemoresistance of the OS cell line. Conclusions Our data show that miR-184 was up-regulated in OS patients treated with doxorubicin therapy and leads to poor response to drug therapy by targeting BCL2L1. PMID:27222034

  6. Connexin 36 and rod bipolar cell independent rod pathways drive retinal ganglion cells and optokinetic reflexes.

    PubMed

    Cowan, Cameron S; Abd-El-Barr, Muhammad; van der Heijden, Meike; Lo, Eric M; Paul, David; Bramblett, Debra E; Lem, Janis; Simons, David L; Wu, Samuel M

    2016-02-01

    Rod pathways are a parallel set of synaptic connections which enable night vision by relaying and processing rod photoreceptor light responses. We use dim light stimuli to isolate rod pathway contributions to downstream light responses then characterize these contributions in knockout mice lacking rod transducin-α (Trα), or certain pathway components associated with subsets of rod pathways. These comparisons reveal that rod pathway driven light sensitivity in retinal ganglion cells (RGCs) is entirely dependent on Trα, but partially independent of connexin 36 (Cx36) and rod bipolar cells. Pharmacological experiments show that rod pathway-driven and Cx36-independent RGC ON responses are also metabotropic glutamate receptor 6-dependent. To validate the RGC findings in awake, behaving animals we measured optokinetic reflexes (OKRs), which are sensitive to changes in ON pathways. Scotopic OKR contrast sensitivity was lost in Trα(-/-) mice, but indistinguishable from controls in Cx36(-/-) and rod bipolar cell knockout mice. Mesopic OKRs were also altered in mutant mice: Trα(-/-) mice had decreased spatial acuity, rod BC knockouts had decreased sensitivity, and Cx36(-/-) mice had increased sensitivity. These results provide compelling evidence against the complete Cx36 or rod BC dependence of night vision's ON component. Further, the findings suggest the parallel nature of rod pathways provides considerable redundancy to scotopic light sensitivity but distinct contributions to mesopic responses through complicated interactions with cone pathways. PMID:26718442

  7. Bifurcation of the endocytic pathway into Rab5-dependent and -independent transport to the vacuole

    NASA Astrophysics Data System (ADS)

    Toshima, Junko Y.; Nishinoaki, Show; Sato, Yoshifumi; Yamamoto, Wataru; Furukawa, Daiki; Siekhaus, Daria Elisabeth; Sawaguchi, Akira; Toshima, Jiro

    2014-03-01

    The yeast Rab5 homologue, Vps21p, is known to be involved both in the vacuolar protein sorting (VPS) pathway from the trans-Golgi network to the vacuole, and in the endocytic pathway from the plasma membrane to the vacuole. However, the intracellular location at which these two pathways converge remains unclear. In addition, the endocytic pathway is not completely blocked in yeast cells lacking all Rab5 genes, suggesting the existence of an unidentified route that bypasses the Rab5-dependent endocytic pathway. Here we show that convergence of the endocytic and VPS pathways occurs upstream of the requirement for Vps21p in these pathways. We also identify a previously unidentified endocytic pathway mediated by the AP-3 complex. Importantly, the AP-3-mediated pathway appears mostly intact in Rab5-disrupted cells, and thus works as an alternative route to the vacuole/lysosome. We propose that the endocytic traffic branches into two routes to reach the vacuole: a Rab5-dependent VPS pathway and a Rab5-independent AP-3-mediated pathway.

  8. BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies.

    PubMed

    Bogenberger, J M; Kornblau, S M; Pierceall, W E; Lena, R; Chow, D; Shi, C-X; Mantei, J; Ahmann, G; Gonzales, I M; Choudhary, A; Valdez, R; Camoriano, J; Fauble, V; Tiedemann, R E; Qiu, Y H; Coombes, K R; Cardone, M; Braggio, E; Yin, H; Azorsa, D O; Mesa, R A; Stewart, A K; Tibes, R

    2014-08-01

    Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response. PMID:24451410

  9. BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

    PubMed Central

    Bogenberger, J M; Kornblau, S M; Pierceall, W E; Lena, R; Chow, D; Shi, C-X; Mantei, J; Ahmann, G; Gonzales, I M; Choudhary, A; Valdez, R; Camoriano, J; Fauble, V; Tiedemann, R E; Qiu, Y H; Coombes, K R; Cardone, M; Braggio, E; Yin, H; Azorsa, D O; Mesa, R A; Stewart, A K; Tibes, R

    2014-01-01

    Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response. PMID:24451410

  10. hnRNP L binds to CA repeats in the 3'UTR of bcl-2 mRNA

    SciTech Connect

    Lee, Dong-Hyoung; Lim, Mi-Hyun; Youn, Dong-Ye; Jung, Seung Eun; Ahn, Young Soo; Tsujimoto, Yoshihide; Lee, Jeong-Hwa

    2009-05-08

    We previously reported that the CA-repeat sequence in the 3'-untranslated region (3'UTR) of bcl-2 mRNA is involved in the decay of bcl-2 mRNA. However, the trans-acting factor for the CA element in bcl-2 mRNA remains unidentified. The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an intron splicing factor, has been reported to bind to CA repeats and CA clusters in the 3'UTR of several genes. We reported herein that the CA repeats of bcl-2 mRNA have the potential to form a distinct ribonuclear protein complex in cytoplasmic extracts of MCF-7 cells, as evidenced by RNA electrophoretic mobility shift assays (REMSA). A super-shift assay using the hnRNP L antibody completely shifted the complex. Immunoprecipitation with the hnRNP L antibody and MCF-7 cells followed by RT-PCR revealed that hnRNP L interacts with endogenous bcl-2 mRNA in vivo. Furthermore, the suppression of hnRNP L in MCF-7 cells by the transfection of siRNA for hnRNP L resulted in a delay in the degradation of RNA transcripts including CA repeats of bcl-2 mRNA in vitro, suggesting that the interaction between hnRNPL and CA repeats of bcl-2 mRNA participates in destabilizing bcl-2 mRNA.

  11. Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses

    PubMed Central

    Holm, Christian K.; Rahbek, Stine H.; Gad, Hans Henrik; Bak, Rasmus O.; Jakobsen, Martin R.; Jiang, Zhaozaho; Hansen, Anne Louise; Jensen, Simon K.; Sun, Chenglong; Thomsen, Martin K.; Laustsen, Anders; Nielsen, Camilla G.; Severinsen, Kasper; Xiong, Yingluo; Burdette, Dara L.; Hornung, Veit; Lebbink, Robert Jan; Duch, Mogens; Fitzgerald, Katherine A.; Bahrami, Shervin; Mikkelsen, Jakob Giehm; Hartmann, Rune; Paludan, Søren R.

    2016-01-01

    Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV. PMID:26893169

  12. Aiolos transcription factor controls cell death in T cells by regulating Bcl-2 expression and its cellular localization.

    PubMed Central

    Romero, F; Martínez-A, C; Camonis, J; Rebollo, A

    1999-01-01

    We searched for proteins that interact with Ras in interleukin (IL)-2-stimulated or IL-2-deprived cells, and found that the transcription factor Aiolos interacts with Ras. The Ras-Aiolos interaction was confirmed in vitro and in vivo by co-immunoprecipitation. Indirect immunofluorescence shows that IL-2 controls the cellular distribution of Aiolos and induces its tyrosine phosphorylation, required for dissociation from Ras. We also identified functional Aiolos-binding sites in the Bcl-2 promoter, which are able to activate the luciferase reporter gene. Mutation of Aiolos-binding sites within the Bcl-2 promoter inhibits transactivation of the reporter gene luciferase, suggesting direct control of Bcl-2 expression by Aiolos. Co-transfection experiments confirm that Aiolos induces Bcl-2 expression and prevents apoptosis in IL-2-deprived cells. We propose a model for the regulation of Bcl-2 expression via Aiolos. PMID:10369681

  13. DNA rearrangement in human follicular lymphoma can involve the 5' or the 3' region of the bcl-2 gene

    SciTech Connect

    Tsujimoto, Y.; Bashir, M.M.; Givol, I.; Cossman, J.; Jaffe, E.; Croce, C.M.

    1987-03-01

    In most human lymphomas, the chromosome translocation t(14;18) occurs within two breakpoint clustering regions on chromosome 18, the major one at the 3' untranslated region of the bcl-2 gene and the minor one at 3' of the gene. Analysis of a panel of follicular lymphoma DNAs using probes for the first exon of the bcl-2 gene indicates that DNA rearrangements may also occur 5' to the involved bcl-2 gene. In this case the IgH locus and the bcl-2 gene are found in an order suggesting that an inversion also occurred during the translocation process. The coding region of the bcl-2 gene, however, are left intact in all cases of follicular lymphoma studied to date.

  14. A chemoprotective fish oil/pectin diet enhances apoptosis via Bcl-2 promoter methylation in rat azoxymethane-induced carcinomas

    PubMed Central

    Cho, Youngmi; Turner, Nancy D; Davidson, Laurie A; Chapkin, Robert S; Carroll, Raymond J; Lupton, Joanne R

    2014-01-01

    We have demonstrated that diets containing fish oil and pectin (FO/P) reduce colon tumor incidence relative to control (corn oil and cellulose [CO/C]) in part by inducing apoptosis of DNA-damaged colon cells. Relative to FO/P, CO/C promotes colonocyte expression of the antiapoptotic modulator, Bcl-2, and Bcl-2 promoter methylation is altered in colon cancer. To determine if FO/P, compared with CO/C, limits Bcl-2 expression by enhancing promoter methylation in colon tumors, we examined Bcl-2 promoter methylation, mRNA levels, colonocyte apoptosis and colon tumor incidence in azoxymethane (AOM)-injected rats. Rats were provided diets containing FO/P or CO/C, and were terminated 16 and 34 weeks after AOM injection. DNA isolated from paraformaldehyde-fixed colon tumors and uninvolved tissue was bisulfite modified and amplified by quantitative reverese transcriptase-polymerase chain reaction to assess DNA methylation in Bcl-2 cytosine–guanosine islands. FO/P increased Bcl-2 promoter methylation (P = 0.009) in tumor tissues and colonocyte apoptosis (P = 0.020) relative to CO/C. An inverse correlation between Bcl-2 DNA methylation and Bcl-2 mRNA levels was observed in the tumors. We conclude that dietary FO/P promotes apoptosis in part by enhancing Bcl-2 promoter methylation. These Bcl-2 promoter methylation responses, measured in vivo, contribute to our understanding of the mechanisms involved in chemoprevention of colon cancer by diets containing FO/P. PMID:23354397

  15. Antibody-independent activation of the classical pathway of complement by Epstein-Barr virus.

    PubMed

    Martin, H; McConnell, I; Gorick, B; Hughes-Jones, N C

    1987-03-01

    A purified preparation of Epstein-Barr virus (EBV) has been shown to activate the classical complement pathway by direct interaction with the first component of complement, C1, without the intervention of antibody. No evidence was found for activation of the alternative pathway. Following classical pathway activation the specific affinity of EBV for B cells can be presumed to be lost since the virus will become opsonized for clearance by phagocytic cells bearing complement receptors, CR1 and CR3. This activation is further evidence that complement plays a role in defence mechanisms independently of antibody activity. PMID:3038440

  16. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2

    PubMed Central

    Goldmacher, Victor S.; Bartle, Laura M.; Skaletskaya, Anna; Dionne, Cheryl A.; Kedersha, Nancy L.; Vater, Carol A.; Han, Jia-wen; Lutz, Robert J.; Watanabe, Shinya; McFarland, Ellen D. Cahir; Kieff, Elliott D.; Mocarski, Edward S.; Chittenden, Thomas

    1999-01-01

    Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins. PMID:10535957

  17. Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak.

    PubMed

    Willis, Simon N; Fletcher, Jamie I; Kaufmann, Thomas; van Delft, Mark F; Chen, Lin; Czabotar, Peter E; Ierino, Helen; Lee, Erinna F; Fairlie, W Douglas; Bouillet, Philippe; Strasser, Andreas; Kluck, Ruth M; Adams, Jerry M; Huang, David C S

    2007-02-01

    A central issue in the regulation of apoptosis by the Bcl-2 family is whether its BH3-only members initiate apoptosis by directly binding to the essential cell-death mediators Bax and Bak, or whether they can act indirectly, by engaging their pro-survival Bcl-2-like relatives. Contrary to the direct-activation model, we show that Bax and Bak can mediate apoptosis without discernable association with the putative BH3-only activators (Bim, Bid, and Puma), even in cells with no Bim or Bid and reduced Puma. Our results indicate that BH3-only proteins induce apoptosis at least primarily by engaging the multiple pro-survival relatives guarding Bax and Bak. PMID:17289999

  18. Lantana camara Induces Apoptosis by Bcl-2 Family and Caspases Activation.

    PubMed

    Han, Eun Byeol; Chang, Bo Yoon; Jung, Young Suk; Kim, Sung Yeon

    2015-04-01

    Breast cancer is one of the most common cancers worldwide, and the second most fatal cancer in women after lung cancer. Because there are instances of cancer resistance to existing therapies, studies focused on the identification of novel therapeutic drugs are very important. In this study, we identified a natural anticancer agent from Lantana camara, a flowering plant species of the genus Verbena. The extract obtained from the L. camara exhibited cell death properties in the human breast cancer cell line, MCF-7. We found that the apoptosis induced by treatment with the L. camara extract was regulated by the Bcl-2 family. Bid and Bax was increased and Bcl-2 was decreased by L. camara extract. L. camara extract modulated cleavage of caspase-8, and caspase-9, as well as poly (ADP-ribose) polymerase (PARP). Our results support the potential use of the L. camara extract as an anti-breast cancer drug. PMID:25145450

  19. The flavonoid morin from Moraceae induces apoptosis by modulation of Bcl-2 family members and Fas receptor in HCT 116 cells.

    PubMed

    Hyun, Hwang-Bo; Lee, Won Sup; Go, Se-Il; Nagappan, Arulkumar; Park, Cheol; Han, Min Ho; Hong, Su Hyun; Kim, Gonsup; Kim, Gi Young; Cheong, Jaehun; Ryu, Chung Ho; Shin, Sung Chul; Choi, Yung Hyun

    2015-01-01

    It is evident based on literature that flavonoids from fruit can safely modulate cancer cell biology and induce apoptosis. Therefore, we investigated the anticancer activity of morin, a flavonoid which is plentiful in twigs of mulberry focusing on apoptosis, and its mechanisms. Morin upregulated the Fas receptor, and activates caspase-8, -9 and -3 in HCT-116 cells. Morin also activates Bid, and induced the loss of mitochondrial membrane potential (MMP, ∆Ψm) with Bax protein activation and cytochrome c release. In addition, morin induced ROS generation which was not blocked by N-acetylcysteine. Morin also suppressed Bcl-2 and cIAP-1, anti-apoptotic proteins, which may contribute to augmentation of morin-triggered apoptosis. As an upstream signaling pathway, suppressed Akt activity by morin was associated to apoptosis. This study suggests that morin induces caspase-dependent apoptosis through extrinsic pathway by upregulating Fas receptor as well as through the intrinsic pathway by modulating Bcl-2 and IAP family members, and ROS generation, and that Akt is the critical upstream signaling that regulates the apoptotic effect of morin in human colon cancer HCT-116 cells. PMID:25892545

  20. IMPORTANCE OF APOPTOSIS MARKERS (MDM2, BCL-2 AND Bax) IN CONVENTIONAL RENAL CELL CARCINOMA.

    PubMed

    Saker, Z; Tsintsadze, O; Jiqia, I; Managadze, L; Chkhotua, A

    2015-12-01

    The goal of the current study was to analyze the expression of Bcl-2, MDM2 and Bax in benign and malignant renal tissue samples and assess their possible association with different clinical parameters. Prognostic significance of the markers in recurrence-free and cancer-specific survivals has also been evaluated. Activity of MDM2, Bcl-2 and Bax was evaluated in: 24 normal human kidney tissues resected from the patients of different ages (range: 21-80 years), and in 52 conventional RCC samples. Intensity of the markers' expression was compared between the groups and correlation was analyzed with different clinical parameters. Activity of anti-apoptotic MDM2 and Bcl-2 was significantly elevated while activity of pro-apoptotic Bax was decreased in RCC as compared with normal kidney tissues. Bax expression was positively correlated with patient age. Significant association has been detected between the evaluated markers and cancer clinical parameters like: tumor stage, grade, lymph node and distant metastases. The markers' activity was associates with the tumor morphological features, in particular: presence of tumor necrosis and microvascular invasion. Disease recurrence and 5-year patient survival were associated with the markers' activity. Cox regression analyses have shown that tumor size, pathological stage and grade are the risk factors for disease recurrence and patient death. Expression of MDM2 and Bcl-2 is significantly up-regulated, while Bax is down-regulated in RCC as compared with normal kidney tissue. Intensity of the markers'activities is associated with the tumor pathological and clinical parameters (stage, grade, lymph node and distant metastases, tumor recurrence and patient survival). Further studies with more patients and longer follow-up will uncover the clinical importance of the evaluated markers in RCC. PMID:26719546

  1. Effect of curcumin on Bcl-2 and Bax expression in nude mice prostate cancer

    PubMed Central

    Yang, Jiayi; Ning, Jianping; Peng, Linlin; He, Dan

    2015-01-01

    Prostate cancer is a common malignant tumor in urinary system. Curcumin has curative effect on many kinds of cancers and can inhibit prostate cancer (PC)-3 cells proliferation. This study aimed to explore the curcumin induced prostate cancer cell apoptosis and apoptosis related proteins Bcl-2 and Bax expression. PC-3 cells were injected subcutaneously to the nude mice to establish the tumor model. The nude mice were randomly divided into group C (normal saline), group B (6% polyethylene glycol and 6% anhydrous ethanol), group H, M, L (100 mg/kg, 50 mg/kg, and 25 mg/kg curcumin). The tumor volume was measured every 6 days to draw the tumor growth curve. The mice were killed at the 30th day after injection to weight the tumor. TUNEL assay was applied to determine cell apoptosis. Immunohistochemistry was used to detect Bcl-2 and Bax expression. The tumor volume and weight in group H, M, L were significantly lower than the control group (C, B) (P<0.05), and the inhibitory rate increased following the curcumin dose increase. Compared with the control group, Bcl-2 expression in group H, M, L gradually decreased, while Bax protein expression increased (P<0.05). The cell apoptosis rate showed no statistical difference between group B and C, while it increased in curcumin group H, M, and L (P<0.05). Curcumin could inhibit PC-3 growth, decrease tumor volume, reduce tumor weight, and induce cell apoptosis under the skin of nude mice by up-regulating Bax and down-regulating Bcl-2. PMID:26464676

  2. MiR-15b mediates liver cancer cells proliferation through targeting BCL-2

    PubMed Central

    Zhang, Yuping; Huang, Feizhou; Wang, Jian; Peng, Lin; Luo, Hongwu

    2015-01-01

    The incidence and mortality of liver cancer increased year by year. Our country presents high incidence of liver cancer. MicroRNAs have tissue sensitivity as tumor biomarkers that play a role by promoting tumor growth as oncogenes or inhibit malignant cell growth as tumor suppressor genes. Studies showed that miR-15b abnormal expression in the tumor and can be treated as one of the tumor molecular markers. However, miR-15b expression and role in the liver cancer cells have not been elucidated. This study intended to explore the mechanism of miR-15b effect on liver cancer occurrence and development. Liver cancer cell line HepG2 was transfected with miR-15b mimic or inhibitor. Real-time PCR was applied to detect miR-15b expression. MTT was used to test cell proliferation. Transwell assay was performed to determine cell invasive ability. Real-time PCR and Western blot were used to detect BCL2 expression. MiR-15b mimic transfection promoted miR-15b overexpression and inhibited HepG2 cell proliferation significantly (P < 0.05). MiR-15b overexpression downregulated BCL2 mRNA and protein expression obviously (P < 0.05). On the contrary, miR-15b inhibitor transfection markedly reduced miR-15b expression in liver cancer cells (P < 0.05), promoted tumor cell proliferation, and increased BCL2 mRNA and protein expression. MiR-15b expression changes did not affect cell invasion (P > 0.05). MiR-15b can inhibit HepG2 cell proliferation and down-regulate BCL2 mRNA and protein expression. PMID:26884837

  3. Expression of URG4/URGCP, Cyclin D1, Bcl-2, and Bax genes in retinoic acid treated SH-SY5Y human neuroblastoma cells

    PubMed Central

    Gundogdu, Gulsah; Koc, Tugba; Yonguc, G. Nilufer; Kucukatay, Vural; Satiroglu-Tufan, N. Lale

    2013-01-01

    Retinoic acid (RA) plays important roles in development, growth, and differentiation by regulating the expression of its target genes. The pro-apoptotic Bax gene may form channels through oligomerization in the mitochondrial membrane and facilitate the cytosolic release of cytochrome c. The anti-apoptotic Bcl-2 gene can inhibit this process. Up-regulated gene 4/Upregulator of cell proliferation (URG4/URGCP) is a novel gene located on 7p13. URG4/URGCP also stimulates cyclin D1 (CCND1) mRNA expression, and RNAi-mediated URG4/URGCP silencing diminishes CCND1 mRNA expression in HepG2 cells. In this study, the effects of RA treatment on URG4/URGCP, CCND1, Bcl-2 and Bax gene expression changes in undifferentiated and differentiated SHSY5Y neuroblastoma cells was analyzed. SHSY5Y cells were cultured in the appropriate conditions. To induce differentiation, the cells were treated with 10 micromolar RA in the dark for 3-10 days. SHSY5Y cells possess small processes in an undifferentiated state, and after treatment with RA, the cells developed long neurites, resembling a neuronal phenotype. Total RNA was isolated with Tri-Reagent. Expression profiles of the target genes were determined by semi-quantitative RT-PCR. According to the results, Bcl-2 and CCND1 gene expression levels were increased, while URG4/URGCP and Bax gene expression was decreased in RA treated cells compared to the control cells. Our preliminary results suggest that RA may induce cell proliferation and escape apoptosis using a novel pathway by the URG4/URGCP gene. Further investigations are needed to clarify more direct transcriptional targets of RA signaling and the interaction of RA pathways with other pro-regenerative signals. PMID:24592121

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

    SciTech Connect

    Tee, Thiam-Tsui; Cheah, Yew-Hoong; Meenakshii, Nallappan; Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie

    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.

  5. The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins.

    PubMed

    Mérino, Delphine; Giam, Maybelline; Hughes, Peter D; Siggs, Owen M; Heger, Klaus; O'Reilly, Lorraine A; Adams, Jerry M; Strasser, Andreas; Lee, Erinna F; Fairlie, Walter D; Bouillet, Philippe

    2009-08-10

    Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim's proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models. PMID:19651893

  6. Dexamethasone treatment promotes Bcl-2 dependence in multiple myeloma resulting in sensitivity to venetoclax.

    PubMed

    Matulis, S M; Gupta, V A; Nooka, A K; Hollen, H V; Kaufman, J L; Lonial, S; Boise, L H

    2016-05-01

    Venetoclax (ABT-199), a specific inhibitor of the anti-apoptotic protein Bcl-2, is currently in phase I clinical trials for multiple myeloma. The results suggest that venetoclax is only active in a small cohort of patients therefore we wanted to determine its efficacy when used in combination. Combining venetoclax with melphalan or carfilzomib produced additive or better cell death in four of the five cell lines tested. The most striking results were seen with dexamethasone (Dex). Co-treatment of human myeloma cell lines and primary patient samples, with Dex and venetoclax, significantly increased cell death over venetoclax alone in four of the five cell lines, and in all patient samples tested. The mechanism by which this occurs is an increase in the expression of both Bcl-2 and Bim upon addition of Dex. This results in alterations in Bim binding to anti-apoptotic proteins. Dex shifts Bim binding towards Bcl-2 resulting in increased sensitivity to venetoclax. These data suggest that knowledge of drug-induced alterations of Bim-binding patterns may help inform better combination drug regimens. Furthermore, the data indicate combining this novel therapeutic with Dex could be an effective therapy for a broader range of patients than would be predicted by single-agent activity. PMID:26707935

  7. Germinal center reentries of BCL2-overexpressing B cells drive follicular lymphoma progression

    PubMed Central

    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

  8. Axotomized neonatal motoneurons overexpressing the bcl2 proto-oncogene retain functional electrophysiological properties.

    PubMed Central

    Alberi, S; Raggenbass, M; de Bilbao, F; Dubois-Dauphin, M

    1996-01-01

    Bcl2 overexpression prevents axotomy-induced neuronal death of neonatal facial motoneurons, as defined by morphological criteria. However, the functional properties of these surviving lesioned transgenic neurons are unknown. Using transgenic mice overexpressing the protein Bcl2, we have investigated the bioelectrical properties of transgenic facial motoneurons from 7 to 20 days after neonatal unilateral axotomy using brain-stem slices and whole cell patch-clamp recording. Nonaxotomized facial motoneurons from wild-type and transgenic mice had similar properties; they had an input resistance of 38 +/- 6 M omega and fired repetitively after injection of positive current pulses. When cells were voltage-clamped at or near their resting membrane potential, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), or vasopressin generated sustained inward currents. In transgenic axotomized mice, facial motoneurons could be found located ipsilaterally to the lesion; they had an input resistance of 150 +/- 30 M omega, indicating that they were smaller in size, fired repetitively, and were also responsive to AMPA, NMDA, and vasopressin. Morphological measurements achieved 1 week after the lesion have shown that application of brain-derived neurotrophic factor prevented the reduction in size of axotomized transgenic motoneurons. These data indicate that Bcl2 not only prevents morphological apoptotic death of axotomized neonatal transgenic motoneurons but also permits motoneurons to conserve functional electrophysiological properties. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8633001

  9. Cooperation of ETV6/RUNX1 and BCL2 enhances immunoglobulin production and accelerates glomerulonephritis in transgenic mice.

    PubMed

    Bauer, Eva; Schlederer, Michaela; Scheicher, Ruth; Horvath, Jaqueline; Aigner, Petra; Schiefer, Ana-Iris; Kain, Renate; Regele, Heinz; Hoermann, Gregor; Steiner, Günter; Kenner, Lukas; Sexl, Veronika; Villunger, Andreas; Moriggl, Richard; Stoiber, Dagmar

    2016-03-15

    The t(12;21) translocation generating the ETV6/RUNX1 fusion gene represents the most frequent chromosomal rearrangement in childhood leukemia. Presence of ETV6/RUNX1 alone is usually not sufficient for leukemia onset, and additional genetic alterations have to occur in ETV6/RUNX1-positive cells to cause transformation. We have previously generated an ETV6/RUNX1 transgenic mouse model where the expression of the fusion gene is restricted to CD19-positive B cells. Since BCL2 family members have been proposed to play a role in leukemogenesis, we investigated combined effects of ETV6/RUNX1 with exogenous expression of the antiapoptotic protein BCL2 by crossing ETV6/RUNX1 transgenic animals with Vav-BCL2 transgenic mice. Strikingly, co-expression of ETV6/RUNX1 and BCL2 resulted in significantly shorter disease latency in mice, indicating oncogene cooperativity. This was associated with faster development of follicular B cell lymphoma and exacerbated immune complex glomerulonephritis. ETV6/RUNX1-BCL2 double transgenic animals displayed increased B cell numbers and immunoglobulin titers compared to Vav-BCL2 transgenic mice. This led to pronounced deposition of immune complexes in glomeruli followed by accelerated development of immune complex glomerulonephritis. Thus, our study reveals a previously unrecognized synergism between ETV6/RUNX1 and BCL2 impacting on malignant disease and autoimmunity. PMID:26919255

  10. The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

    SciTech Connect

    Vogler, Meike; Dickens, David; Dyer, Martin J.S.; Owen, Andrew; Pirmohamed, Munir; Cohen, Gerald M.

    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. Cooperation of ETV6/RUNX1 and BCL2 enhances immunoglobulin production and accelerates glomerulonephritis in transgenic mice

    PubMed Central

    Bauer, Eva; Schlederer, Michaela; Scheicher, Ruth; Horvath, Jaqueline; Aigner, Petra; Schiefer, Ana-Iris; Kain, Renate; Regele, Heinz; Hoermann, Gregor; Steiner, Günter; Kenner, Lukas; Sexl, Veronika; Villunger, Andreas; Moriggl, Richard; Stoiber, Dagmar

    2016-01-01

    The t(12;21) translocation generating the ETV6/RUNX1 fusion gene represents the most frequent chromosomal rearrangement in childhood leukemia. Presence of ETV6/RUNX1 alone is usually not sufficient for leukemia onset, and additional genetic alterations have to occur in ETV6/RUNX1-positive cells to cause transformation. We have previously generated an ETV6/RUNX1 transgenic mouse model where the expression of the fusion gene is restricted to CD19-positive B cells. Since BCL2 family members have been proposed to play a role in leukemogenesis, we investigated combined effects of ETV6/RUNX1 with exogenous expression of the antiapoptotic protein BCL2 by crossing ETV6/RUNX1 transgenic animals with Vav-BCL2 transgenic mice. Strikingly, co-expression of ETV6/RUNX1 and BCL2 resulted in significantly shorter disease latency in mice, indicating oncogene cooperativity. This was associated with faster development of follicular B cell lymphoma and exacerbated immune complex glomerulonephritis. ETV6/RUNX1-BCL2 double transgenic animals displayed increased B cell numbers and immunoglobulin titers compared to Vav-BCL2 transgenic mice. This led to pronounced deposition of immune complexes in glomeruli followed by accelerated development of immune complex glomerulonephritis. Thus, our study reveals a previously unrecognized synergism between ETV6/RUNX1 and BCL2 impacting on malignant disease and autoimmunity. PMID:26919255

  12. The BCL2L1 and PGAM5 axis defines hypoxia-induced receptor-mediated mitophagy

    PubMed Central

    Wu, Hao; Xue, Danfeng; Chen, Guo; Han, Zhe; Huang, Li; Zhu, Chongzhuo; Wang, Xiaohui; Jin, Haijing; Wang, Jun; Zhu, Yushan; Liu, Lei; Chen, Quan

    2014-01-01

    Receptor-mediated mitophagy is one of the major mechanisms of mitochondrial quality control essential for cell survival. We previously have identified FUNDC1 as a mitophagy receptor for selectively removing damaged mitochondria in mammalian systems. A critical unanswered question is how receptor-mediated mitophagy is regulated in response to cellular and environmental cues. Here, we report the striking finding that BCL2L1/Bcl-xL, but not BCL2, suppresses mitophagy mediated by FUNDC1 through its BH3 domain. Mechanistically, we demonstrate that BCL2L1, but not BCL2, interacts with and inhibits PGAM5, a mitochondrially localized phosphatase, to prevent the dephosphorylation of FUNDC1 at serine 13 (Ser13), which activates hypoxia-induced mitophagy. Our results showed that the BCL2L1-PGAM5-FUNDC1 axis is critical for receptor-mediated mitophagy in response to hypoxia and that BCL2L1 possesses unique functions distinct from BCL2. PMID:25126723

  13. Effect of sphingosine on Ca2+ entry and mitochondrial potential of Jurkat T cells--interaction with Bcl2.

    PubMed

    Dangel, Georg Richard; Lang, Florian; Lepple-Wienhues, Albrecht

    2005-01-01

    Triggers of Jurkat T cell apoptosis include sphingosine and ceramide. Sphingosine and ceramide further inhibit capacitative Ca2+ entry (ICRAC), an effect leading to inactivation but not death of Jurkat T cells. Mitochondria are key organelles in the machinery leading to apoptosis and on the other hand have been shown to participate in the regulation of Ca2+ entry. The present experiments were performed to explore whether treatment of Jurkat T cells with sphingosine leads to apoptosis and reduced Ca2+ entry and whether those effects are sensitive to expression of the antiapoptotic protein Bcl2, localized in the outer mitochondrial membrane. Exposure of Jurkat T cells to 10 microM spingosine was according to DiOC6 fluorescence followed by mitochondrial depolarization and according to Fura-red/Fluo-3 fluorescence followed by decreased capacitative Ca2+ entry. Mitochondrial depolarization was significantly delayed in cells overexpressing wild type Bcl2 or Bcl2 targeted to the mitochondrial membrane, whereas no significant influence on mitochondrial depolarization was observed in cells expressing Bcl2 lacking the membrane targeting motif or Bcl2 targeted to the endoplasmatic reticulum. In contrast to mitochondrial potential, the blunting of capacitative Ca2+ entry following sphingosine treatment was not sensitive to mitochondrial Bcl2 expression. In conclusion sphingosine exposure leads to both, mitochondrial depolarization and inhibition of capacitative Ca2+ entry. Mitochondrial Bcl2 reverses the effect on mitochondria but not on Ca2+ entry and thus leads to dissociation of those two sequelae of sphingosine treatment. PMID:16121028

  14. An ATM-independent S-phase checkpoint response involves CHK1 pathway

    NASA Technical Reports Server (NTRS)

    Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

    2002-01-01

    After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

  15. The Candida albicans ESCRT Pathway Makes Rim101-Dependent and -Independent Contributions to Pathogenesis ▿

    PubMed Central

    Wolf, Julie M.; Johnson, Diedre J.; Chmielewski, David; Davis, Dana A.

    2010-01-01

    Candida albicans is an opportunistic pathogen that colonizes diverse mucosal niches with distinct environmental characteristics. To adapt to these different sites, C. albicans must activate and attenuate a variety of signal transduction pathways. A mechanism of signal attenuation is through receptor endocytosis and subsequent vacuolar degradation, which requires the endosomal sorting complex required for transport (ESCRT) pathway. This pathway comprises several polyprotein complexes (ESCRT-0, -I, -II, -III, and -DS) that are sequentially recruited to the endosomal membrane. The ESCRT pathway also activates the Rim101 transcription factor, which governs expression of genes required for virulence. Here, we tested the hypothesis that the ESCRT pathway plays a Rim101-independent role(s) in pathogenesis. We generated deletion mutants in each ESCRT complex and determined that ESCRT-I, -II, and -III are required for Rim101 activation but that ESCRT-0 and ESCRT-DS are not. We found that the ESCRT-0 member Vps27 and ESCRT-DS components are required to promote epithelial cell damage and, using a murine model of oral candidiasis, found that the vps27Δ/Δ mutant had a decreased fungal burden compared to that of the wild type. We found that a high-dose inoculum can compensate for fungal burden defects but that mice colonized with the vps27Δ/Δ strain exhibit less morbidity than do mice infected with the wild-type strain. These results demonstrate that the ESCRT pathway has Rim101-independent functions for C. albicans virulence. PMID:20581294

  16. Ligand-independent pathway that controls stability of interferon alpha receptor

    SciTech Connect

    Liu Jianghuai; Plotnikov, Alexander; Banerjee, Anamika; Suresh Kumar, K.G.; Ragimbeau, Josiane; Marijanovic, Zrinka; Baker, Darren P.; Pellegrini, Sandra; Fuchs, Serge Y.

    2008-03-07

    Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance.

  17. Inhibitory heterotrimeric GTP-binding proteins inhibit hydrogen peroxide-induced apoptosis by up-regulation of Bcl-2 via NF-{kappa}B in H1299 human lung cancer cells

    SciTech Connect

    Seo, Mi Ran; Nam, Hyo-Jung; Kim, So-Young; Juhnn, Yong-Sung

    2009-04-03

    Inhibitory heterotrimeric GTP-binding proteins (Gi proteins) mediate a variety of signaling pathways by coupling receptors and effectors to regulate cellular proliferation, differentiation, and apoptosis. However, the role of Gi proteins in the modulation of hydrogen peroxide-induced apoptosis is not clearly understood. Thus, we investigated the effect of Gi proteins on hydrogen peroxide-induced apoptosis and the underlying mechanisms in H1299 human lung cancer cells. The stable expression of constitutively active alpha subunits of Gi1 (G{alpha}i1QL), Gi2, or Gi3 inhibited hydrogen peroxide-induced apoptosis. The expression of G{alpha}i1QL up-regulated Bcl-2 expression, and the knockdown of Bcl-2 with siRNA abolished the anti-apoptotic effect of G{alpha}i1QL. G{alpha}i1 induced the transcription of Bcl-2 by activation of NF-{kappa}B, which resulted from an increase in NF-{kappa}B p50 protein. We conclude that G{alpha}i1 inhibits hydrogen peroxide-induced apoptosis of H1299 lung cancer cells by up-regulating the transcription of Bcl-2 through a p50-mediated NF-{kappa}B activation.

  18. Salt-Induced Remodeling of Spatially Restricted Clathrin-Independent Endocytic Pathways in Arabidopsis Root

    PubMed Central

    Baral, Anirban; Irani, Niloufer G.; Fujimoto, Masaru; Nakano, Akihiko; Mayor, Satyajit; Mathew, M.K.

    2015-01-01

    Endocytosis is a ubiquitous cellular process that is characterized well in animal cells in culture but poorly across intact, functioning tissue. Here, we analyze endocytosis throughout the Arabidopsis thaliana root using three classes of probes: a lipophilic dye, tagged transmembrane proteins, and a lipid-anchored protein. We observe a stratified distribution of endocytic processes. A clathrin-dependent endocytic pathway that internalizes transmembrane proteins functions in all cell layers, while a sterol-dependent, clathrin-independent pathway that takes up lipid and lipid-anchored proteins but not transmembrane proteins is restricted to the epidermal layer. Saline stress induces a third pathway that is clathrin-independent, nondiscriminatory in its choice of cargo, and operates across all layers of the root. Concomitantly, small acidic compartments in inner cell layers expand to form larger vacuole-like structures. Plants lacking function of the Rab-GEF (guanine nucleotide exchange factor) VPS9a (vacuolar protein sorting 9A) neither induce the third endocytic pathway nor expand the vacuolar system in response to salt stress. The plants are also hypersensitive to salt. Thus, saline stress reconfigures clathrin-independent endocytosis and remodels endomembrane systems, forming large vacuoles in the inner cell layers, both processes correlated by the requirement of VPS9a activity. PMID:25901088

  19. Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile.

    PubMed

    Tor, Yin Sim; Yazan, Latifah Saiful; Foo, Jhi Biau; Wibowo, Agustono; Ismail, Norsharina; Cheah, Yoke Kqueen; Abdullah, Rasedee; Ismail, Maznah; Ismail, Intan Safinar; Yeap, Swee Keong

    2015-01-01

    Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic

  20. Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile

    PubMed Central

    Tor, Yin Sim; Yazan, Latifah Saiful; Foo, Jhi Biau; Wibowo, Agustono; Ismail, Norsharina; Cheah, Yoke Kqueen; Abdullah, Rasedee; Ismail, Maznah; Ismail, Intan Safinar; Yeap, Swee Keong

    2015-01-01

    Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic

  1. miR-139-5p Inhibits the Epithelial-Mesenchymal Transition and Enhances the Chemotherapeutic Sensitivity of Colorectal Cancer Cells by Downregulating BCL2

    PubMed Central

    Li, Qingguo; Liang, Xin; Wang, Yuwei; Meng, Xianke; Xu, Ye; Cai, Sanjun; Wang, Zhimin; Liu, Jianwen; Cai, Guoxiang

    2016-01-01

    MicroRNAs (miRNAs) are important regulators involved in various cancers, including colorectal cancer (CRC). The functions and mechanisms of the miRNAs involved in CRC progress and metastasis are largely unknown. In this study, miRNA microarray analysis was performed to screen crucial miRNAs involved in CRC progress, and miR-139-5p was chosen for further study. The functional roles of miR-139-5p in colon cancer were demonstrated by CCK-8 proliferation assay, cell invasion and migration, cell apoptosis and in a KO mouse study. miR-139-5p expression was significantly decreased in cancer tissues compared to normal tissues. The miR-139-5p expression level was associated with tumour stage (P < 0.01). Function studies revealed that miR-139-5p was significantly correlated with the metastasis potential and drug resistance of colon cancer cells by affecting the epithelial-mesenchymal transition (EMT). Then, we identified BCL2 as a direct target of miR-139-5p cells in vitro. The patient samples and KO mice model showed that BCL2 expression was inversely correlated with the expression of miR-139-5p. In conclusion, we found that miR-139-5p targeted the BCL2 pathway to reduce tumour metastasis and drug sensitivity in CRC. This axis provided insight into the mechanism underlying miRNA regulation of CRC metastasis and a novel therapeutic target for CRC therapy. PMID:27244080

  2. Isolated Follicles Enriched for Centroblasts and Lacking t(14;18)/BCL2 in Lymphoid Tissue: Diagnostic and Clinical Implications

    PubMed Central

    Gratzinger, Dita; Jones, Carol D.; Zehnder, James L.; Bangs, Charles D.; Cherry, Athena; Warnke, Roger A.; Natkunam, Yasodha

    2016-01-01

    We sought to address the significance of isolated follicles that exhibit atypical morphologic features that may be mistaken for lymphoma in a background of reactive lymphoid tissue. Seven cases that demonstrated centroblast-predominant isolated follicles and absent BCL2 staining in otherwise-normal lymph nodes were studied. Four of seven cases showed clonal B-cell proliferations amid a polyclonal B cell background; all cases lacked the IGH-BCL2 translocation and BCL2 protein expression. Although three patients had invasive breast carcinoma at other sites, none were associated with systemic lymphoma up to 44 months after diagnosis. The immunoarchitectural features of these highly unusual cases raise the question of whether a predominance of centroblasts and/or absence of BCL2 expression could represent a precursor lesion or atypical reactive phenomenon. Differentiating such cases from follicular lymphoma or another mimic is critical, lest patients with indolent proliferations be exposed to unnecessarily aggressive treatment. PMID:26991267

  3. Downregulation of Bcl-2 Expression by miR-34a Mediates Palmitate-Induced Min6 Cells Apoptosis

    PubMed Central

    Lin, Xiaojie; Huang, Zhimin; Liu, Juan; Li, Hai; Wei, Guohong; Cao, Xiaopei; Li, Yanbing

    2014-01-01

    Recent studies have demonstrated that the expression of miR-34a is significantly upregulated and associated with cell apoptosis in pancreatic β-cell treated with palmitate. Nevertheless, the underlying detailed mechanism is largely unknown. Here, we showed that miR-34a was significantly induced in Min6 pancreatic β-cell upon palmitate treatment. Elevated miR-34a promoted Min6 cell apoptosis. Intriguingly, ectopic expression of miR-34a lowered the expression of Bcl-2, an antiapoptotic protein. Luciferase reporter assay indicated the direct interaction of miR-34a with the Bcl-2 3′-UTR. Moreover, downregulated expression of Bcl-2 induced by palmitate could be restored by inhibition of miR-34a. We conclude that direct suppression of Bcl-2 by miR-34a accounts for palmitate-induced increased apoptosis rate in pancreatic β-cell. PMID:24829923

  4. Isolated Follicles Enriched for Centroblasts and Lacking t(14;18)/BCL2 in Lymphoid Tissue: Diagnostic and Clinical Implications.

    PubMed

    Nybakken, Grant E; Bala, Rajeev; Gratzinger, Dita; Jones, Carol D; Zehnder, James L; Bangs, Charles D; Cherry, Athena; Warnke, Roger A; Natkunam, Yasodha

    2016-01-01

    We sought to address the significance of isolated follicles that exhibit atypical morphologic features that may be mistaken for lymphoma in a background of reactive lymphoid tissue. Seven cases that demonstrated centroblast-predominant isolated follicles and absent BCL2 staining in otherwise-normal lymph nodes were studied. Four of seven cases showed clonal B-cell proliferations amid a polyclonal B cell background; all cases lacked the IGH-BCL2 translocation and BCL2 protein expression. Although three patients had invasive breast carcinoma at other sites, none were associated with systemic lymphoma up to 44 months after diagnosis. The immunoarchitectural features of these highly unusual cases raise the question of whether a predominance of centroblasts and/or absence of BCL2 expression could represent a precursor lesion or atypical reactive phenomenon. Differentiating such cases from follicular lymphoma or another mimic is critical, lest patients with indolent proliferations be exposed to unnecessarily aggressive treatment. PMID:26991267

  5. TR4 orphan nuclear receptor functions as an apoptosis modulator via regulation of Bcl-2 gene expression

    SciTech Connect

    Kim, Eungseok; Ma, Wen-Lung; Lin, Din-Lii; Inui, Shigeki; Chen, Yuh-Ling; Chang, Chawnshang . E-mail: chang@urmc.rochester.edu

    2007-09-21

    While Bcl-2 plays an important role in cell apoptosis, its relationship to the orphan nuclear receptors remains unclear. Here we report that mouse embryonic fibroblast (MEF) cells prepared from TR4-deficient (TR4{sup -} {sup /-}) mice are more susceptible to UV-irradiation mediated apoptosis compared to TR4-Wildtype (TR4 {sup +/+}) littermates. Substantial increasing TR4{sup -} {sup /-} MEF apoptosis to UV-irradiation was correlated to the down-regulation of Bcl-2 RNA and protein expression and collaterally increased caspase-3 activity. Furthermore, this TR4-induced Bcl-2 gene expression can be suppressed by co-transfection with TR4 coregulators, such as androgen receptor (AR) and receptor-interacting protein 140 (RIP140) in a dose-dependent manner. Together, our results demonstrate that TR4 might function as an apoptosis modulator through induction of Bcl-2 gene expression.

  6. Regulation of cancer cell survival by BCL2 family members upon prolonged mitotic arrest: opportunities for anticancer therapy.

    PubMed

    Barillé-Nion, Sophie; Bah, Nourdine; Véquaud, Eloïse; Juin, Philippe

    2012-10-01

    Attacking cancer cell survival defense by targeting B-Cell Lymphoma 2 (BCL2) family of anti-apoptotic proteins may provide a powerful means to improve chemotherapy efficiency. This could be particularly relevant to anti-mitotic-based therapy, where tumor response relates to a competing network between mitotic cell death signaling and mitotic slippage as an adaptative response to a leaky mitotic checkpoint. In this review, we focus on recent findings that point out the major role played by BCL2 family members in response to anti-mitotic agents, which reveal dependence of cancer cell survival on BCL2 homologs during mitotic arrest and after mitotic slippage. Finally, we discuss pre-clinical data combining anti-mitotic agents with BCL2 inhibitors. PMID:23060542

  7. Estrogen Affects Levels of Bcl-2 Protein and mRNA in Medial Amygdala of Ovariectomized Rats

    PubMed Central

    Fan, Lu; Pandey, Subhash C.; Cohen, Rochelle S.

    2013-01-01

    The survival factor Bcl-2 is a cyclic AMP response element-binding protein (CREB) gene product implicated in mediating some of estrogen’s effects on neuroprotection. Previously, we showed an effect of estradiol benzoate (E) on numbers of neuron-specific protein (NeuN)- and phosphorylated CREB (pCREB)-positive cells in medial (MeA), but not central (CeA), amygdala of ovariectomized rats. To determine whether these effects are accompanied by an E-induced increase in Bcl-2, we examined the effects of E on levels of Bcl-2 protein and mRNA in MeA and CeA of ovariectomized rats treated with E regimens resulting in moderate (2.5μg E for 4 or 14 days) or high (10μg E for 14 days) plasma estradiol levels. As a physiological control, we showed that all E treatments increased uterine wet weight relative to vehicle; 10μg E for 14 days also increased uterine weight compared to that seen with lower E levels. Western blot analysis revealed that all E groups displayed an increase in uterine Bcl-2 protein levels compared to vehicle. We found that 2.5μg and 10μg E for 14 days increased levels of Bcl-2 gold immunolabeling compared to vehicle and 2.5μg E for 4 days in MeA, but not CeA. We measured Bcl-2 mRNA levels in vehicle and 2.5μg E for 14 days groups. There was a significant increase in Bcl-2 mRNA levels in MeA, but not CeA, of E-treated ovariectomized rats compared with vehicle controls. The E-induced increase in protein and mRNA levels of Bcl-2 in MeA may be important for neuroprotection in this region. PMID:18655204

  8. Mechanism of regulation of bcl-2 mRNA by nucleolin and A+U-rich element-binding factor 1 (AUF1).

    PubMed

    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. PMID:20571027

  9. Disturbance of Bcl-2, Bax, Caspase-3, Ki-67 and C-myc expression in acute and subchronic exposure to benzo(a)pyrene in cervix.

    PubMed

    Gao, Meili; Li, Yongfei; Ji, Xiaoying; Xue, Xiaochang; Chen, Lan; Feng, Guodong; Zhang, Huqin; Wang, Huichun; Shah, Walayat; Hou, Zhanwu; Kong, Yu

    2016-03-01

    Epidemiological studies have demonstrated that cigarette smoking is an important cofactor or an independent risk factor for the development of cervical cancer. Benzo(a)pyrene (BaP) is one of the most potent tobacco smoke carcinogens in tobacco smoke. BaP induced DNA damage and over expression in p53 cervical tissue of mice as demonstrated in our previous study. Here we present the findings of exposure to BaP on the expression of Bcl-2, C-myc, Ki-67, Caspase-3 and Bax genes in mouse cervix. Acute intraperitoneal administration of BaP (12.5, 25, 50, 100mg/kg body weight) to ICR female mice induced a significant increase in Bcl-2, C-myc, Ki-67 mRNA and protein level till 72h except in Bcl-2 at 24h with 12.5, 25, 50mg/kg as well as at 48h with 12.5mg/kg body weight post treatment. A significant increase was also seen in Caspase-3 and Bax mRNA and protein level with peak level at 24h and gradual decrease till 72h, however, the expression of caspase-3 increased while that of Bax decreased with increasing dose of Bap after 24h. In sub chronic intraperitoneal and oral gavage administration of BaP (2.5, 5, 10mg/kg body weight), similar significant increase was observed for all the examined genes as compared to the control and vehicle groups, however the expression of Bax decreased in a dose dependent manner. The findings of this study will help in further understanding the molecular mechanism of BaP induced carcinogenesis of cervical cancer. PMID:26709117

  10. Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

    PubMed Central

    Faião-Flores, Fernanda; Coelho, Paulo Rogério Pinto; Toledo Arruda-Neto, João Dias; Maria-Engler, Silvya Stuchi; Tiago, Manoela; Capelozzi, Vera Luiza; Giorgi, Ricardo Rodrigues; Maria, Durvanei Augusto

    2013-01-01

    Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and 7Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT. PMID:23527236

  11. Down-regulation of Bcl-2-interacting protein BAG-1 confers resistance to anti-cancer drugs.

    PubMed

    Takahashi, Noriko; Yanagihara, Miyako; Ogawa, Yuzi; Yamanoha, Banri; Andoh, Toshiwo

    2003-02-14

    BAG-1 was originally identified as a binding partner of anti-apoptotic factor Bcl-2 [Takayama et al., Cell 80 (1995) 279-284]. Exogenous expression of BAG-1 was reported to confer cells resistance to several stresses [Chen et al., Oncogene 21 (2002) 7050]. We have obtained human cervical cancer HeLa cells with down-regulated BAG-1 levels by using a highly specific and efficient RNA interference approach. Surprisingly, cells with down-regulated BAG-1 exhibited significantly lower sensitivity against several anti-cancer drugs than parental cells expressing normal levels of the protein. Furthermore, growth rate of the cells was reduced when BAG-1 was down-regulated. Activity of ERK pathway appeared to be decreased in BAG-1 down-regulated cells, as shown by the reduced phosphorylation of ERK1/2 proteins. Taken together resistance against anti-cancer drugs acquired by BAG-1 down-regulated cells may well be accounted for by the retardation of cell cycle progression, implicating the importance of BAG-1 in cell growth regulation. PMID:12565851

  12. Synthesis and Characterization of Novel 2-Amino-Chromene-Nitriles that Target Bcl-2 in Acute Myeloid Leukemia Cell Lines

    PubMed Central

    Mohan, Chakrabhavi D.; Madan, Vikas; Kanojia, Deepika; Shobith, Rangappa; Nanjundaswamy, Shivananju; Mason, Daniel J.; Bender, Andreas; Basappa; Rangappa, Kanchugarakoppal S.; Koeffler, H. Phillip

    2014-01-01

    The anti-apoptotic protein Bcl-2 is a well-known and attractive therapeutic target for cancer. In the present study the solution-phase T3P-DMSO mediated efficient synthesis of 2-amino-chromene-3-carbonitriles from alcohols, malanonitrile and phenols is reported. These novel 2-amino-chromene-3-carbonitriles showed cytotoxicity in human acute myeloid leukemia (AML) cell lines. Compound 4g was found to be the most bioactive, decreasing growth and increasing apoptosis of AML cells. Moreover, compound 4g (at a concentration of 5 µM) increased the G2/M and sub-G1 (apoptosis) phases of AML cells. The AML cells treated with compound 4g exhibited decreased levels of Bcl-2 and increased levels of caspase-9. In silico molecular interaction analysis showed that compound 4g shared a similar global binding motif with navitoclax (another small molecule that binds Bcl-2), however compound 4g occupies a smaller volume within the P2 hot spot of Bcl-2. The intermolecular π-stacking interaction, direct electrostatic interactions, and docking energy predicted for 4g in complex with Bcl-2 suggest a strong affinity of the complex, rendering 4g as a promising Bcl-2 inhibitor for evaluation as a new anticancer agent. PMID:25268519

  13. Variant translocation of the bcl-2 gene to immunoglobulin. lambda. light chain gene in chronic lymphocytic leukemia

    SciTech Connect

    Adachi, M.; Cossman, J.; Longo, D.; Croce, C.M.; Tsujimoto, Y. )

    1989-04-01

    The bcl-2 gene has been identified as a gene directly involved in the consistent chromosome translocation t(14;18), which is found in {approx} 90% of human follicular lymphoma cases, and is a prime candidate for the oncogene playing a crucial role in follicular lymphomagenesis. In this paper, the authors describe a case of chronic lymphocytic leukemia showing the juxtaposition of the bcl-2 gene on chromosome 18 to immunoglobulin {lambda} light chain (Ig{lambda}) gene on chromosome 22 in a head-to-head configuration. Sequencing analysis of the joining site of the bcl-2 gene and Ig{lambda} gene has shown that the breakpoint is within the 5{prime} flanking region of the bcl-2 gene and about 2.2 kilobases 5{prime} to the joining segment of Ig{lambda} locus in a germ-line configuration. The extranucleotide, commonly appearing at the joining site of the t(14;18) translocation involving the IgH locus, is absent from the joining site of bcl-2 and Ig{lambda}. The lack of extranucleotide suggests that the juxtaposition of the bcl-2 and Ig{lambda} genes occurred during physiological rearrangement of the Ig{lambda} gene since it has been shown that the rearrangement of the Ig{lambda} locus is not accompanied by extranucleotides.

  14. Bcl-2 over-expression promotes genomic instability by inhibiting apoptosis of cells exposed to hydrogen peroxide.

    PubMed

    Cox, Andrew G; Hampton, Mark B

    2007-10-01

    The anti-apoptotic oncogene bcl-2 is hypothesized to increase the antioxidant status of cells, thereby protecting them from oxidative stress. In this study, we examined hydrogen peroxide (H2O2)-mediated oxidative stress in Jurkat T lymphoma cells. Over-expression of Bcl-2 did not inhibit cytotoxicity at doses of H2O2 that caused necrosis (>200 microM), but it did block cell death at apoptotic doses (<200 microM). However, these cells exhibited the same initial level of protein and lipid oxidation following exposure to H2O2 as the parental cells, indicating that the anti-apoptotic activity is not associated with general antioxidant properties. Bcl-2 expression was able to protect against secondary protein carbonyl formation, which was linked to lysosome stabilization. Assessment of micronuclei formation in cells over-expressing Bcl-2 showed evidence of increased genomic instability, consistent with the impairment of apoptosis in damaged cells. We conclude that while Bcl-2 can block cytotoxicity associated with apoptosis-inducing levels of oxidative stress, it does not protect the cells from the stress itself. Bcl-2 may promote tumourigenesis by preventing the removal of oxidatively damaged cells. PMID:17434928

  15. Phenylboronic acid-functionalized polyamidoamine-mediated Bcl-2 siRNA delivery for inhibiting the cell proliferation.

    PubMed

    Wu, Di; Yang, Jiebing; Xing, Zhen; Han, Haobo; Wang, Tingting; Zhang, Aijun; Yang, Yan; Li, Quanshun

    2016-10-01

    In this study, the conjugation of phenylboronic acid (PBA) to amine-terminated polyamidoamine (PAMAM) was successfully conducted to prepare a tumor-targeted gene carrier PBA-functionalized PAMAM (PPP) for Bcl-2 siRNA delivery, using a heterobifunctional crosslinker NHS-PEG5k-Mal. The carrier possessed favorable capacity for siRNA condensation and could protect siRNA from the degradation against RNase and serum. The introduction of PBA could facilitate the cellular uptake and further transfection of Bcl-2 siRNA demonstrated by confocal laser scanning microscopy and flow cytometry. Meanwhile, PPP-mediated transfection of Bcl-2 siRNA could significantly inhibit the expression of Bcl-2 gene at both mRNA and protein levels. Furthermore, owing to the knock-down of Bcl-2, PPP/siRNA could significantly inhibit the cell proliferation by inducing the cell apoptosis, and also enhance the antitumor efficiency of doxorubicin by suppressing the resistance of tumor cells to chemotherapeutics. In conclusion, the PPP-mediated Bcl-2 siRNA delivery could potentially be an effective platform for solving the drug resistance and further achieving the combined chemotherapy and gene therapy in tumor treatment. PMID:27371891

  16. Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways

    PubMed Central

    Berkowska, Magdalena A.; Driessen, Gertjan J. A.; Bikos, Vasilis; Grosserichter-Wagener, Christina; Stamatopoulos, Kostas; Cerutti, Andrea; He, Bing; Biermann, Katharina; Lange, Johan F.; van der Burg, Mirjam; van Dongen, Jacques J. M.

    2011-01-01

    Multiple distinct memory B-cell subsets have been identified in humans, but it remains unclear how their phenotypic diversity corresponds to the type of responses from which they originate. Especially, the contribution of germinal center-independent responses in humans remains controversial. We defined 6 memory B-cell subsets based on their antigen-experienced phenotype and differential expression of CD27 and IgH isotypes. Molecular characterization of their replication history, Ig somatic hypermutation, and class-switch profiles demonstrated their origin from 3 different pathways. CD27−IgG+ and CD27+IgM+ B cells are derived from primary germinal center reactions, and CD27+IgA+ and CD27+IgG+ B cells are from consecutive germinal center responses (pathway 1). In contrast, natural effector and CD27−IgA+ memory B cells have limited proliferation and are also present in CD40L-deficient patients, reflecting a germinal center-independent origin. Natural effector cells at least in part originate from systemic responses in the splenic marginal zone (pathway 2). CD27−IgA+ cells share low replication history and dominant Igλ and IgA2 use with gut lamina propria IgA+ B cells, suggesting their common origin from local germinal center-independent responses (pathway 3). Our findings shed light on human germinal center-dependent and -independent B-cell memory formation and provide new opportunities to study these processes in immunologic diseases. PMID:21690558

  17. Induction of apoptosis in HepG2 cells by solanine and Bcl-2 protein.

    PubMed

    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. PMID:18022776

  18. Aspirin inhibits growth of ovarian cancer by upregulating caspase-3 and downregulating bcl-2

    PubMed Central

    LI, LIN; MAO, XIAOGANG; QIN, XIAOMIN; ZHOU, MIN; XING, HUI; DONG, FAN; JIANG, XIAOYUAN; ZHUANG, WENHUI

    2016-01-01

    The aim of the present study was to investigate the effect and mechanism of different concentrations of aspirin in inhibiting the ovarian cancer of p53N236S gene knock-in mice. In total, 28 male p53S mice, with an age range of 4–6 weeks and weight of 20–25 g were selected. The animals were transplanted with SKOV3 cells to establish subdermal human ovarian cancer. The mice were randomly divided into different groups according to the aspirin concentrations (mmol/l) used, i.e., 0, 1, 2 and 3. Subsequently, intraperitoneal injection was performed once every two days for 3 weeks. The tumor volume, lifetime, tumor cell proliferation inhibition rates, caspase-3 protein and bcl-2 protein expression of the four groups were analyzed and compared. Following aspirin treatment for 1, 2 and 3 weeks, the tumor volume of the 3 mmol/l aspirin group was significantly smaller than the other groups (P<0.05). The higher concentration of aspirin led to a smaller tumor size (P<0.05). The cell proliferation inhibition rate of the 3 mmol/l aspirin group was significantly larger than that of other groups (P<0.05). The relative expression level of caspase-3, bcl-2 protein of the 3 mmol/l aspirin group was significantly improved and reduced, respectively. In conclusion, aspirin can inhibit the growth of ovarian cancer of p53S rats due to its upregulation of the expression of caspase-3 protein and downregulation of the expression of bcl-2 protein. PMID:27347106

  19. Plasmacytoma induction in specific pathogen-free (SPF) bcl-2 transgenic BALB/c mice.

    PubMed

    Silva, S; Klein, G

    1999-01-01

    Germ-free (GF) and specific pathogen free (SPF) BALB/c mice are refractory to plasmacytoma induction by pristane (McIntire and Princler, 1969, Byrd et al 1991). It was therefore suggested that MPC development may depend on antigenic stimulation. If so, it may conceivably act by preventing the apoptotic elimination of tumor precursor cells. We have tested this idea by elevating the apoptotic threshold by the introduction of a bcl-2 transgene. We have found that MPCs could be induced by pristane oil in transgene carrying SPF mice. An E mu activated bcl-2 transgene was introduced into SPF BALB/c mice. The mice were used after two backcrosses (BC-2). Pristane oil treatment was started at 4 to 6 weeks of age (3 x 0.3 ml via i.p. at monthly intervals). For each transgene carrier a transgene negative littermate was used as control. Fifteen of 24 (63%) transgene carriers developed plasmacytomas after latency periods between 67 and 146 days (mean = 112 +/- 30 days) after the first pristane injection. Five additional transgene carriers developed lymphoma (3 cases) or mixed MPC and lymphoma (2 cases). In contrast, no tumors developed in 16 transgene negative littermates that were kept > 300 days under observation. Karyotyping showed that 10/15 (66%) of the MPCs carried a T(12;15) translocation, 4/15 (27%) carried both T(12;15) and T(6;15) translocations in the same metaphase plate, and 1/15 (7%) was translocation free. A T(12;15) translocation was also detected in one of the 2 mice with mixed tumor type. Pristane treated bcl-2 transgenic C57B1/6 mice remained tumor free, although T(12;15) translocation carrying cells were found in the peritoneal fluid of 4/20 mice 176 days after pristane. PMID:10396078

  20. Asymmetrically dividing Drosophila neuroblasts utilize two spatially and temporally independent cytokinesis pathways

    PubMed Central

    Roth, Michaela; Roubinet, Chantal; Iffländer, Niklas; Ferrand, Alexia; Cabernard, Clemens

    2015-01-01

    Precise cleavage furrow positioning is required for faithful chromosome segregation and cell fate determinant distribution. In most metazoan cells, contractile ring placement is regulated by the mitotic spindle through the centralspindlin complex, and potentially also the chromosomal passenger complex (CPC). Drosophila neuroblasts, asymmetrically dividing neural stem cells, but also other cells utilize both spindle-dependent and spindle-independent cleavage furrow positioning pathways. However, the relative contribution of each pathway towards cytokinesis is currently unclear. Here we report that in Drosophila neuroblasts, the mitotic spindle, but not polarity cues, controls the localization of the CPC component Survivin. We also show that Survivin and the mitotic spindle are required to stabilize the position of the cleavage furrow in late anaphase and to complete furrow constriction. These results support the model that two spatially and temporally separate pathways control different key aspects during asymmetric cell division, ensuring correct cell fate determinant segregation and neuroblast self-renewal. PMID:25791062

  1. Evidence for a Novel, Caspase-8-Independent, Fas Death Domain-Mediated Apoptotic Pathway

    PubMed Central

    Katsanis, Emmanuel

    2004-01-01

    Certain caspase-8 null cell lines demonstrate resistance to Fas-induced apoptosis, indicating that the Fas/FasL apoptotic pathway may be caspase-8-dependent. Some reports, however, have shown that Fas induces cell death independent of caspase-8. Here we provide evidence for an alternative, caspase-8-independent, Fas death domain-mediated apoptotic pathway. Murine 12B1-D1 cells express procaspase-3, -8, and -9, which were activated upon the dimerization of Fas death domain. Bid was cleaved and mitochondrial transmembrane potential was disrupted in this apoptotic process. All apoptotic events were completely blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK, but not by other peptide caspase inhibitors. Cyclosporin A (CsA), which inhibits mitochondrial transition pore permeability, blocked neither pore permeability disruption nor caspase activation. However, CsA plus caspase-8 inhibitor blocked all apoptotic events of 12B1-D1 induced by Fas death domain dimerization. Our data therefore suggest that there is a novel, caspase-8-independent, Z-VAD-FMK-inhibitable, apoptotic pathway in 12B1-D1 cells that targets mitochondria directly. PMID:15123887

  2. Photocontrolled Exposure of Pro-apoptotic Peptide Sequences in LOV Proteins Modulates Bcl-2 Family Interactions.

    PubMed

    Mart, Robert J; Meah, Dilruba; Allemann, Rudolf K

    2016-04-15

    LOV domains act as biomolecular sensors for light, oxygen or the environment's redox potential. Conformational changes upon the formation of a covalent cysteinyl flavin adduct are propagated through hydrogen-bonding networks in the core of designed hybrid phototropin LOV2 domains that incorporate the Bcl homology region 3 (BH3) of the key pro-apoptotic protein BH3-interacting-domain death agonist (BID). The resulting change in conformation of a flanking amphiphilic α-helix creates a light-dependent optogenetic tool for the modulation of interactions with the anti-apoptotic B-cell leukaemia-2 (Bcl-2) family member Bcl-xL . PMID:26493687

  3. Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling.

    PubMed

    Quintana, Megan T; Parry, Traci L; He, Jun; Yates, Cecelia C; Sidorova, Tatiana N; Murray, Katherine T; Bain, James R; Newgard, Christopher B; Muehlbauer, Michael J; Eaton, Samuel C; Hishiya, Akinori; Takayama, Shin; Willis, Monte S

    2016-08-01

    The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype in vivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins. PMID:27321750

  4. Human Cytomegalovirus Promotes Survival of Infected Monocytes via a Distinct Temporal Regulation of Cellular Bcl-2 Family Proteins

    PubMed Central

    Collins-McMillen, Donna; Kim, Jung Heon; Nogalski, Maciej T.; Stevenson, Emily V.; Caskey, Joshua R.; Cieply, Stephen J.

    2015-01-01

    ABSTRACT Monocytes play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to target organ systems. To infect monocytes and reprogram them to deliver infectious virus, HCMV must overcome biological obstacles, including the short life span of monocytes and their antiviral proapoptotic response to infection. We have shown that virally induced upregulation of cellular Mcl-1 promotes early survival of HCMV-infected monocytes, allowing cells to overcome an early apoptotic checkpoint at around 48 h postinfection (hpi). Here, we demonstrate an HCMV-dependent shift from Mcl-1 as the primary antiapoptotic player to the related protein, Bcl-2, later during infection. Bcl-2 was upregulated in HCMV-infected monocytes beginning at 48 hpi. Treatment with the Bcl-2 antagonist ABT-199 only reduced the prosurvival effects of HCMV in target monocytes beginning at 48 hpi, suggesting that Mcl-1 controls survival prior to 48 hpi, while Bcl-2 promotes survival after 48 hpi. Although Bcl-2 was upregulated following viral binding/signaling through cellular integrins (compared to Mcl-1, which is upregulated through binding/activation of epidermal growth factor receptor [EGFR]), it functioned similarly to Mcl-1, adopting the early role of Mcl-1 in preventing caspase-3 cleavage/activation. This distinct, HCMV-induced shift from Mcl-1 to Bcl-2 occurs in response to a cellular upregulation of proapoptotic Bax, as small interfering RNA (siRNA)-mediated knockdown of Bax reduced the upregulation of Bcl-2 in infected monocytes and rescued the cells from the apoptotic effects of Bcl-2 inhibition. Our data demonstrate a distinct survival strategy whereby HCMV induces a biphasic regulation of cellular Bcl-2 proteins to promote host cell survival, leading to viral dissemination and the establishment of persistent HCMV infection. IMPORTANCE Hematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy and is required for the

  5. PB-1: The Relationship Between Anti Apoptotic Marker (BCL-2) and Biochemical Markers in Type 2 Diabetes Patients

    PubMed Central

    Damitri, TD; Faridah, AR; Imran, Y; Hasnan, J.

    2006-01-01

    Purpose : To investigate the expression of anti apoptotic marker (bcl-2) and the level of biochemical markers in type 2 diabetes patients. METHODS : A cross-sectional study was conducted from August 2003 to November 2005. Forty one type 2 diabetes patients and 36 non diabetes (control) subjects aged between 20 to 70 years were included in this study. Blood samples were collected for fasting plasma glucose (FPG), triglycerides (TG), Total cholesterol (TC), High density lipoprotein cholesterol (HDLC), Low density lipoprotein cholesterol (LDLC) and analyzed in the Chemical Pathology laboratory, while glycosylated hemoglobin A1c (A1C) was analyzed in the Endocrine laboratory. The skin biopsy tissue samples were stained with immunohistochemistry (IHC) stain for expression of bcl-2 in the Pathology laboratory. RESULTS : There was a significant difference (p<0.001) between both groups for mean FPG (diabetics=11.02±4.25, control=4.41±1.12 mmol/L), HDLC (diabetics=1.00±0.38, control=1.47±0.72 mmol/L) and A1C (diabetics=9.50±2.24%, control=5.00±0.67%). However, there was no significant difference for TG, TC, and LDLC between both groups. Interestingly, the difference of mean bcl-2 expression were very highly significant (p<0.001) when compared between both groups. Mean bcl-2 expression was dibetics= 1.88±0.33 and control= 1.47±0.51. Positive bcl-2 expression was found in only 5 (12.2%) diabetics while 36 (87.8%) diabetics showed negative expression. Positive bcl-2 expression was observed in 19 (52.8%) controls while 17 (47.2%) showed negative expression. CONCLUSION : The expression of anti apoptotic marker bcl-2 was increased in non diabetic subjects in order to prevent cell death. However, the reduced expression of bcl-2 in diabetic patients may be associated with programmed cell death. The detailed mechanism for the gene expression of bcl-2 may help us to understand how bcl-2 is involved in apoptosis in diabetic microvasculature complications.

  6. Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    PubMed Central

    Trisciuoglio, Daniela; Gabellini, Chiara; Desideri, Marianna; Ziparo, Elio; Zupi, Gabriella; Del Bufalo, Donatella

    2010-01-01

    Background Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis. Methodology/Principal Findings By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon. Conclusions/Significance We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the

  7. From molecular PDT damage to cellular PDT responses: attempts at bridging the gap on the role of Bcl-2

    NASA Astrophysics Data System (ADS)

    Usuda, Jitsuo; Xue, Liang-yan; Chiu, Song-mao; Azizuddin, Kashif; Morris, Rachel L.; Mulvihill, John; Oleinick, Nancy L.

    2003-06-01

    Expression of the anti-apoptotic proteins Bcl-2 and/or Bcl-xL is greatly elevated in many advanced cancers, especially those resistant to standard therapies, such as radiation or chemotherapy. It has been suggested that those two proteins would be attractive targets for the development of new cancer treatments. Photodynamic therapy (PDT) with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, specifically attack the anti-apoptotic protein Bcl-2, generating a variety of oxidized, complexed, and cleaved photoproducts. The closely related protein Bcl-xL is also a target of Pc 4-PDT. In a recent study employing transient transfection of an expression vector encoding deletion mutants of Bcl-2, we identified the membrane anchorage regions of the protein that are required to form the photosensitive target. In spite of the demonstrated photodamage to Bcl-2 (and Bcl-xL), how the photodamage translates into changes in the sensitivity of cells to PDT-induced apoptosis or other modes of cell death is not clear, and it also remains unclear how elevated amounts of anti-apoptotic proteins in tumors might make them more or less responsive to PDT. In the present study, we have studied the PDT response of MCF7 human breast cancer cells overexpressing wild-type Bcl-2 or certain deletion mutants either in a transient or stable mode. We show that cells expressing modestly elevated amounts (<10-fold increase) of Bcl-2 and in which the pro-apoptotic protein Bax is not upregulated do not differ from the parental cells with respect to PDT-induced cell killing. In contrast, cells expressing higher amounts (>50-fold increase) of Bcl-2 or certain mutants are made significantly more resistant to the induction of apoptosis and the loss of clonogenicity upon exposure to Pc 4-PDT. In the presence of high levels of Bcl-2, extensive photodamage requires higher PDT doses. We conclude that Pc 4-PDT targets Bcl-2 and Bcl-xL, eliminating one mechanism that

  8. Hydrogen Sulfide Attenuates the Recruitment of CD11b+Gr-1+ Myeloid Cells and Regulates Bax/Bcl-2 Signaling in Myocardial Ischemia Injury

    PubMed Central

    Zhang, Youen; Li, Hua; Zhao, Gang; Sun, Aijun; Zong, Nobel C.; Li, Zhaofeng; Zhu, Hongming; Zou, Yunzeng; Yang, Xiangdong; Ge, Junbo

    2014-01-01

    Hydrogen sulfide, an endogenous signaling molecule, plays an important role in the physiology and pathophysiology of the cardiovascular system. Using a mouse model of myocardial infarction, we investigated the anti-inflammatory and anti-apoptotic effects of the H2S donor sodium hydrosulfide (NaHS). The results demonstrated that the administration of NaHS improved survival, preserved left ventricular function, limited infarct size, and improved H2S levels in cardiac tissue to attenuate the recruitment of CD11b+Gr-1+ myeloid cells and to regulate the Bax/Bcl-2 pathway. Furthermore, the cardioprotective effects of NaHS were enhanced by inhibiting the migration of CD11b+Gr-1+ myeloid cells from the spleen into the blood and by attenuating post-infarction inflammation. These observations suggest that the novel mechanism underlying the cardioprotective function of H2S is secondary to a combination of attenuation the recruitment of CD11b+Gr-1+ myeloid cells and regulation of the Bax/Bcl-2 apoptotic signaling. PMID:24758901

  9. Biological rational for sequential targeting of Bruton tyrosine kinase and Bcl-2 to overcome CD40-induced ABT-199 resistance in mantle cell lymphoma

    PubMed Central

    Chiron, David; Touzeau, Cyrille; Maïga, Sophie; Moreau, Philippe; Pellat-Deceunynck, Catherine; Le Gouill, Steven; Amiot, Martine

    2015-01-01

    The aggressive biological behavior of mantle cell lymphoma (MCL) and its short response to current treatment highlight a great need for better rational therapy. Herein, we investigate the ability of ABT-199, the Bcl-2-selective BH3 mimetic, to kill MCL cells. Among MCL cell lines tested (n = 8), only three were sensitive (LD50 < 200 nM). In contrast, all primary MCL samples tested (n = 11) were highly sensitive to ABT-199 (LD50 < 10 nM). Mcl-1 and Bcl-xL both confer resistance to ABT-199-specific killing and BCL2/(BCLXL + MCL1) mRNA ratio is a strong predictor of sensitivity. By mimicking the microenvironment through CD40 stimulation, we show that ABT-199 sensitivity is impaired through activation of NF-kB pathway and Bcl-xL up-regulation. We further demonstrate that resistance is rapidly lost when MCL cells detach from CD40L-expressing fibroblasts. It has been reported that ibrutinib induces lymphocytosis in vivo holding off malignant cells from their protective microenvironment. We show here for two patients undergoing ibrutinib therapy that mobilized MCL cells are highly sensitive to ABT-199. These results provide evidence that in situ ABT-199 resistance can be overcome when MCL cells escape from the lymph nodes. Altogether, our data support the clinical application of ABT-199 therapy both as a single agent and in sequential combination with BTK inhibitors. PMID:25797245

  10. Hexavalent chromium-induced apoptosis of granulosa cells involves selective sub-cellular translocation of Bcl-2 members, ERK1/2 and p53

    SciTech Connect

    Banu, Sakhila K.; Stanley, Jone A.; Lee, JeHoon; Stephen, Sam D.; Arosh, Joe A.; Hoyer, Patricia B.; Burghardt, Robert C.

    2011-03-15

    Hexavalent chromium (CrVI) has been widely used in industries throughout the world. Increased usage of CrVI and atmospheric emission of CrVI from catalytic converters of automobiles, and its improper disposal causes various health hazards including female infertility. Recently we have reported that lactational exposure to CrVI induced a delay/arrest in follicular development at the secondary follicular stage. In order to investigate the underlying mechanism, primary cultures of rat granulosa cells were treated with 10 {mu}M potassium dichromate (CrVI) for 12 and 24 h, with or without vitamin C pre-treatment for 24 h. The effects of CrVI on intrinsic apoptotic pathway(s) were investigated. Our data indicated that CrVI: (i) induced DNA fragmentation and increased apoptosis, (ii) increased cytochrome c release from the mitochondria to cytosol, (iii) downregulated anti-apoptotic Bcl-2, Bcl-XL, HSP70 and HSP90; upregulated pro-apoptotic BAX and BAD, (iv) altered translocation of Bcl-2, Bcl-XL, BAX, BAD, HSP70 and HSP90 to the mitochondria, (v) upregulated p-ERK and p-JNK, and selectively translocated p-ERK to the mitochondria and nucleus, (vi) activated caspase-3 and PARP, and (vii) increased phosphorylation of p53 at ser-6, ser-9, ser-15, ser-20, ser-37, ser-46 and ser-392, increased p53 transcriptional activation, and downregulated MDM-2. Vitamin C pre-treatment mitigated CrVI effects on apoptosis and related pathways. Our study, for the first time provides a clear insight into the effect of CrVI on multiple pathways that lead to apoptosis of granulosa cells which could be mitigated by vitamin C.

  11. Lentiviral-mediated delivery of Bcl-2 or GDNF protects against excitotoxicity in the rat hippocampus.

    PubMed

    Wong, Liang-Fong; Ralph, G Scott; Walmsley, Lucy E; Bienemann, Alison S; Parham, Stephen; Kingsman, Susan M; Uney, James B; Mazarakis, Nicholas D

    2005-01-01

    Nutrient deprivation during ischemia leads to severe insult to neurons causing widespread excitotoxic damage in specific brain regions such as the hippocampus. One possible strategy for preventing neurodegeneration is to express therapeutic proteins in the brain to protect against excitotoxicity. We investigated the utility of equine infectious anemia virus (EIAV)-based vectors as genetic tools for delivery of therapeutic proteins in an in vivo excitotoxicity model. The efficacy of these vectors at preventing cellular loss in target brain areas following excitotoxic insult was also assessed. EIAV vectors generated to overexpress the human antiapoptotic Bcl-2 or growth factor glial-derived neurotrophic factor (GDNF) genes protected against glutamate-induced toxicity in cultured hippocampal neurons. In an in vivo excitotoxicity model, adult Wistar rats received a unilateral dose of the glutamate receptor agonist N-methyl-D-aspartate to the hippocampus that induced a large lesion in the CA1 region. Neuronal loss could not be protected by prior transduction of a control vector expressing beta-galactosidase. In contrast, EIAV-mediated expression of Bcl-2 and GDNF significantly reduced lesion size thus protecting the hippocampus from excitotoxic damage. These results demonstrate that EIAV vectors can be effectively used to deliver putative neuroprotective genes to target brain areas and prevent cellular loss in the event of a neurological insult. Therefore these lentiviral vectors provide potential therapeutic tools for use in cases of acute neurotrauma such as cerebral ischemia. PMID:15585409

  12. Mitochondrial permeabilization relies on BH3 ligands engaging multiple prosurvival Bcl-2 relatives, not Bak.

    PubMed

    Uren, Rachel T; Dewson, Grant; Chen, Lin; Coyne, Stephanie C; Huang, David C S; Adams, Jerry M; Kluck, Ruth M

    2007-04-23

    The Bcl-2 family regulates apoptosis by controlling mitochondrial integrity. To clarify whether its prosurvival members function by sequestering their Bcl-2 homology 3 (BH3)-only ligands or their multidomain relatives Bak and Bax, we analyzed whether four prosurvival proteins differing in their ability to bind specific BH3 peptides or Bak could protect isolated mitochondria. Most BH3 peptides could induce temperature-dependent cytochrome c release, but permeabilization was prevented by Bcl-x(L), Bcl-w, Mcl-1, or BHRF1. However, their protection correlated with the ability to bind Bak rather than the added BH3 peptide and could be overcome only by BH3 peptides that bind directly to the appropriate prosurvival member. Mitochondria protected by both Bcl-x(L)-like and Mcl-1 proteins were disrupted only by BH3 peptides that engage both. BH3-only reagents freed Bak from Bcl-x(L) and Mcl-1 in mitochondrial and cell lysates. The findings support a model for the control of apoptosis in which certain prosurvival proteins sequester Bak/Bax, and BH3-only proteins must neutralize all protective prosurvival proteins to allow Bak/Bax to induce mitochondrial disruption. PMID:17452531

  13. Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax.

    PubMed

    Fletcher, Jamie I; Meusburger, Sarina; Hawkins, Christine J; Riglar, David T; Lee, Erinna F; Fairlie, W Douglas; Huang, David C S; Adams, Jerry M

    2008-11-25

    A central issue in the control of apoptosis is whether its essential mediators Bax and Bak must be restrained by Bcl-2-like prosurvival relatives to prevent their damaging mitochondria and unleashing apoptosis. The issue is particularly vexed for Bax, which is largely a cytosolic monomer in unstressed cells. To determine whether Bax regulation requires its binding by prosurvival relatives, we replaced a conserved aspartate in its BH3 interaction domain with arginine. Bax D68R functioned and behaved like wild-type Bax in localization and activation but had greatly impaired binding to the prosurvival family members. Nevertheless, Bcl-x(L) remained able to block apoptosis induced by Bax D68R. Whereas cells with sufficient Bcl-x(L) tolerated expression of Bax D68R, it provoked apoptosis when Bcl-x(L) was absent, downregulated, or inactivated. Moreover, Bax D68R rendered membrane bound by a C-terminal anchor mutation overwhelmed endogenous Bcl-x(L) and killed cells. These unexpected results suggest that engagement of Bax by its prosurvival relatives is a major barrier to its full activation. We propose that the Bcl-2-like proteins must capture the small proportion of Bax molecules with an exposed BH3 domain, probably on the mitochondrial membrane, to prevent Bax-imposed cell death, but that Bcl-x(L) also controls Bax by other mechanisms. PMID:18981409

  14. BCL-2 and Bax Expression in Skin Flaps Treated with Finasteride or Azelaic Acid

    PubMed Central

    Ayatollahi, Seyyed Abdulmajid; Ajami, Marjan; Reyhanfard, Hamed; Asadi, Yasin; Nassiri-Kashani, Mansour; Rashighi Firoozabadi, Mehdi; Davoodi, Sayed Hossein; Habibi, Esmaeil; Pazoki-Toroudi, Hamidreza

    2012-01-01

    Despite all modern surgical techniques, skin flap that is considered as the main method in most reconstructive surgeries puts the skin tissue at danger of necrosis and apoptosis derived from ischemia. Therefore, finding a treatment for decreasing the apoptosis derived from flap ischemia will be useful in clinic. In present study, we evaluated the effect of azelaic acid 20% and finasteride on expression of BCL-2 and bax proteins after the skin flap surgery. For this purpose, 21 rats were entered in three groups including control, azelaic acid 20% and finasteride, all experienced skin flap surgery and then flap tissue was assessed for determining the expression of proteins in 5 slices prepared from each rat that were graded between – to +++ scales. Both azelaic acid and finasteride increased the expression of BCL-2 protein (p < 0.05) and decrease the expression of bax protein (p < 0.05). These results suggested an antiapoptotic role for finasteride and azelaic acid in preserving the flap after the ischemia reperfusion insult. PMID:24250563

  15. Antagonizing Bcl-2 family members sensitizes neuroblastoma and Ewing's sarcoma to an inhibitor of glutamine metabolism.

    PubMed

    Olsen, Rachelle R; Mary-Sinclair, Michelle N; Yin, Zhirong; Freeman, Kevin W

    2015-01-01

    Neuroblastomas (NBL) and Ewing's sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated metabolism of cancer as a therapeutic strategy, this approach has not been fully examined in NBL and EWS. In this study, we first tested a panel of metabolic inhibitors and identified the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) as the most potent chemotherapeutic across all NBL and EWS cell lines tested. Myc, a master regulator of metabolism, is commonly overexpressed in both of these pediatric malignancies and recent studies have established that Myc causes cancer cells to become "addicted" to glutamine. We found DON strongly inhibited tumor growth of multiple tumor lines in mouse xenograft models. In vitro, inhibition of caspases partially reversed the effects of DON in high Myc expressing cell lines, but not in low Myc expressing lines. We further showed that induction of apoptosis by DON in Myc-overexpressing cancers is via the pro-apoptotic factor Bax. To relieve inhibition of Bax, we tested DON in combination with the Bcl-2 family antagonist navitoclax (ABT-263). In vitro, this combination caused an increase in DON activity across the entire panel of cell lines tested, with synergistic effects in two of the N-Myc amplified neuroblastoma cell lines. Our study supports targeting glutamine metabolism to treat Myc overexpressing cancers, such as NBL and EWS, particularly in combination with Bcl-2 family antagonists. PMID:25615615

  16. Prognostic Factors Including Proliferation Markers Ki-67, bax, and bcl-2 in Temporal Bone Paraganglioma

    PubMed Central

    Gjuric, Mislav; Völker, Uwe; Katalinic, Alexander; Wolf, Stephan Rüdiger

    1997-01-01

    Valuable criteria with which to predict the clinical behavior of the temporal bone paraganglioma or the response to treatment are lacking. The analysis of markers of cell proliferation is a possibility to estimate the prognosis. Extensive patient data on 40 temporal bone paragangliomas were gathered over the years and correlated with the data obtained by staining histologic sections with bcl-2, bax, and MIB I markers of cellular proliferation. The immunohistochemistry was in all cases negative for bcl-2, positive for bax, and for Ki-67 positive in 20% of tumors. The scores for Ki-67 did not correlate with the majority of clinical parameters, except for treatment modality, preoperative hearing loss, and cranial nerve involvement. The tendency toward poorer hearing and a higher incidence of preoperative lower cranial nerve palsies was demonstrated in patients with higher Ki-67 scores. Furthermore, the higher rate of subtotal tumor removals in these patients reveals technical difficulties in accomplishing a radical removal, although the incidence of residual tumors was thus not affected. In view of the present information obtained with proliferation markers, the site of tumor origin still remains the most predictive variable for the course of the disease. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:17171028

  17. Heavy smokers have higher bcl-2 mutation frequency and risk for lymphoma than non-smokers

    SciTech Connect

    Liu, Y.; Cortopassi, G.A.; Bell, D.A.

    1994-09-01

    Early detection of cells carrying somatic mutations at oncogenic loci could prove useful for identifying individuals at high risk for cancer and permit intervention prior to the onset of clinically recognizable disease. We have determined the frequency of rare t(14;18)(q32;q21) translocations at the bcl-2 proto-oncogene locus in the peripheral blood of 85 smokers and 35 nonsmokers using a sensitive nested PCR assay. The identical translocation occurs in 85% of follicular lymphoma tumors, and about 50% of all non-Hodgkin`s Lymphoma. Smokers with the highest exposure had a 3.6-fold higher mutation frequency relative to the nonsmokers. Logistic regression analysis showed that of the variables tested (age, race, sex, current smoking, years of smoking, and pack-years), the cumulative smoking measure (pack-years) was the best predictor of t(14;18) frequency (p=0.004). These observations are consistent with two recent epidemiological studies showing 2.3-fold and 3.8-fold increased risk for Non-Hodgkins lymphoma among heavy smokers. The results support the hypothesis that smokers have an increased burden of lymphocytes bearing bcl-2 mutations which raises their individual risk for future lymphoid tumors. We speculate that the increased frequency of oncogenic translocations in smokers may result either from the mutagenic or antigenic activity of cigarette smoke.

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

    PubMed

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

    2012-06-01

    Breast cancer is a disease in which cancer cells form in the tissues of the breast. The present study aimed to explore the effect of the flavonoid compound quercetin on the growth and apoptosis of human breast cancer cells. Varying concentrations (12.5, 25, 50, 100, 200 µM) of quercetin were applied to cultured MCF-7 human breast cancer cells for defined lengths of time. At 50 to 200 µM doses, quercetin significantly inhibited the proliferation of MCF-7 cells assessed by MTT colorimetry, in both dose- and time-dependent manners (P<0.05). The compound also increased apoptosis after 48 h of exposure (P<0.05). Furthermore, following quercetin treatment Bcl-2 expression decreased significantly while Bax expression increased significantly (P<0.05). In brief, quercetin inhibits cell growth and induces apoptosis in MCF-7 human breast cancer cells. The mechanisms behind these effects may stem from the downregulation of Bcl-2 protein expression and upregulation of Bax expression. PMID:22447039

  19. Targeting BCL-2 and ABL/LYN in Philadelphia chromosome-positive acute lymphoblastic leukemia.

    PubMed

    Leonard, Jessica T; Rowley, Joelle S J; Eide, Christopher A; Traer, Elie; Hayes-Lattin, Brandon; Loriaux, Marc; Spurgeon, Stephen E; Druker, Brian J; Tyner, Jeffrey W; Chang, Bill H

    2016-08-31

    Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL) remains a challenge. Although the addition of targeted tyrosine kinase inhibitors (TKIs) to standard cytotoxic therapy has greatly improved upfront treatment, treatment-related morbidity and mortality remain high. TKI monotherapy provides only temporary responses and renders patients susceptible to the development of TKI resistance. Thus, identifying agents that could enhance the activity of TKIs is urgently needed. Recently, a selective inhibitor of B cell lymphoma 2 (BCL-2), ABT-199 (venetoclax), has shown impressive activity against hematologic malignancies. We demonstrate that the combination of TKIs with venetoclax is highly synergistic in vitro, decreasing cell viability and inducing apoptosis in Ph(+)ALL. Furthermore, the multikinase inhibitors dasatinib and ponatinib appear to have the added advantage of inducing Lck/Yes novel tyrosine kinase (LYN)-mediated proapoptotic BCL-2-like protein 11 (BIM) expression and inhibiting up-regulation of antiapoptotic myeloid cell leukemia 1 (MCL-1), thereby potentially overcoming the development of venetoclax resistance. Evaluation of the dasatinib-venetoclax combination for the treatment of primary Ph(+)ALL patient samples in xenografted immunodeficient mice confirmed the tolerability of this drug combination and demonstrated its superior antileukemic efficacy compared to either agent alone. These data suggest that the combination of dasatinib and venetoclax has the potential to improve the treatment of Ph(+)ALL and should be further evaluated for patient care. PMID:27582059

  20. Nature promises new anticancer agents: Interplay with the apoptosis-related BCL2 gene family.

    PubMed

    Christodoulou, Maria-Ioanna; Kontos, Christos K; Halabalaki, Maria; Skaltsounis, Alexios-Leandros; Scorilas, Andreas

    2014-03-01

    Natural products display special attributes in the treatment and prevention of a variety of human disorders including cancer. Their therapeutic capacities along with the fact that nature comprises a priceless pool of new compounds have attracted the interest of researchers worldwide. A significant number of organic compounds from terrestrial and marine organisms exhibit anticancer properties as attested by both in vitro and in vivo studies. Emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. As a result, numerous natural compounds or their derivatives have entered clinical practice and are currently in the forefront of chemotherapeutics, showing beneficial effects for cancer patients. Induction of apoptosis seems to be the major mechanism of action induced by these natural agents in the race against cancer. This is mainly achieved through modulations of the expression of B-cell CLL/lymphoma 2 (BCL2) family members. These molecules appear to be the pivotal players determining cellular fate. In the current review, we provide a comprehensive overview of the major alterations in the gene and/or protein levels of BCL2-family members evoked in cancer cells after treatment with a gamut of natural compounds. The data cited suggest the need for exploitation of newly discovered natural products that, along with the improvement of currently employed chemotherapeutics, will significantly enrich the anticancer armamentarium. PMID:23848203

  1. Effect of Bcl-2 rs956572 SNP on regional gray matter volumes and cognitive function in elderly males without dementia.

    PubMed

    Liu, Mu-En; Huang, Chu-Chung; Hwang, Jen-Ping; Yang, Albert C; Tu, Pei-Chi; Yeh, Heng-Liang; Hong, Chen-Jee; Liou, Ying-Jay; Chen, Jin-Fan; Lin, Ching-Po; Tsai, Shih-Jen

    2013-04-01

    The Bcl-2 gene is a major regulator of neural plasticity and cellular resilience. A single-nucleotide polymorphism (SNP) in the Bcl-2 gene, Bcl-2 rs956572, significantly modulates the expression of Bcl-2 protein and cellular vulnerability to apoptosis. This study investigated the association between the Bcl-2 rs956572 SNP and brain structural abnormalities in non-demented elders, and to test the relationship between neuropsychological performance and regional gray matter (GM) volumes. Our sample comprised 97 non-demented elderly men with a mean age of 80.6 ± 5.6 years