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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.

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

    PubMed

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

    2006-02-01

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

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

    PubMed Central

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

    2000-01-01

    p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53 protein functionality in wild-type (wt) and mutant (mt) p53 cell lines. p53 mRNA and p53 protein expression were determined before and after exposure to equitoxic 5-FU concentration in six human carcinoma cell lines differing in p53 status and displaying marked differences in 5-FU sensitivity, with IC 50 values ranging from 0.2–22.6 mM. 5-FU induced a rise in p53 mRNA expression in mt p53 cell lines and in human papilloma virus positive wt p53 cell line, whereas significant decrease in p53 mRNA expression was found in wt p53 cell line. Whatever p53 status, 5-FU altered p53 transcriptional and translational regulation leading to up-regulation of p53 protein. In relation with p53 functionality, but independently of p53 mutational status, after exposure to 5-FU equitoxic concentration, all cell lines were able to arrest in G1. No relationship was evidenced between G1 accumulation ability and 5-FU sensitivity. Moreover, after 5-FU exposure, Bax and Bcl-2 proteins regulation was under p53 protein control and a statistically significant relationship (r= 0.880,P= 0.0097) was observed between Bcl-2/Bax ratio and 5-FU sensitivity. In conclusion, whatever p53 status, Bcl-2 or Bax induction and Bcl-2/Bax protein ratio were correlated to 5-FU sensitivity. © 2000 Cancer Research Campaign PMID:11044365

  4. 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.

  5. Bisdemethoxycurcumin enhances X-ray-induced apoptosis possibly through p53/Bcl-2 pathway.

    PubMed

    Enomoto, Atsushi; Yamada, Junko; Morita, Akinori; Miyagawa, Kiyoshi

    2017-03-01

    Bisdemethoxycurcumin (BDMC), which is isolated from the rhizomes of Curcuma longa, has anti-inflammatory and anti-carcinogenic activities. Here we found that BDMC enhanced X-ray-induced apoptosis in human T-cell leukemia MOLT-4 cells. Knockdown of p53 significantly attenuated the radiosensitizing effect of BDMC. However, BDMC did not enhance X-ray-mediated activation of the p53 signaling pathway via p53's transactivation or mitochondrial translocation. On the other hand, BDMC promoted the X-ray-induced dephosphorylation at Ser 70 in Bcl-2's flexible loop regulatory domain and Bcl-2 binding to p53. Overexpressing Bcl-2 completely blocked the BDMC's radiosensitization effect. Our results indicate that BDMC stimulates the dephosphorylation and p53-binding activity of Bcl-2 and suggest that BDMC may induce a neutralization of Bcl-2's anti-apoptotic function, thereby enhancing X-ray-induced apoptosis.

  6. Two Independent Positive Feedbacks and Bistability in the Bcl-2 Apoptotic Switch

    PubMed Central

    Lu, Haizhu; Sun, Tingzhe; Shen, Pingping

    2008-01-01

    Background The complex interplay between B-cell lymphoma 2 (Bcl-2) family proteins constitutes a crucial checkpoint in apoptosis. Its detailed molecular mechanism remains controversial. Our former modeling studies have selected the ‘Direct Activation Model’ as a better explanation for experimental observations. In this paper, we continue to extend this model by adding interactions according to updating experimental findings. Methodology/Principal Findings Through mathematical simulation we found bistability, a kind of switch, can arise from a positive (double negative) feedback in the Bcl-2 interaction network established by anti-apoptotic group of Bcl-2 family proteins. Moreover, Bax/Bak auto-activation as an independent positive feedback can enforce the bistability, and make it more robust to parameter variations. By ensemble stochastic modeling, we also elucidated how intrinsic noise can change ultrasensitive switches into gradual responses. Our modeling result agrees well with recent experimental data where bimodal Bax activation distributions in cell population were found. Conclusions/Significance Along with the growing experimental evidences, our studies successfully elucidate the switch mechanism embedded in the Bcl-2 interaction network and provide insights into pharmacological manipulation of Bcl-2 apoptotic switch as further cancer therapies. PMID:18213378

  7. 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

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

    PubMed

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

    2011-03-01

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

  10. BCL2 in breast cancer: a favourable prognostic marker across molecular subtypes and independent of adjuvant therapy received

    PubMed Central

    Dawson, S-J; Makretsov, N; Blows, F M; Driver, K E; Provenzano, E; Le Quesne, J; Baglietto, L; Severi, G; Giles, G G; McLean, C A; Callagy, G; Green, A R; Ellis, I; Gelmon, K; Turashvili, G; Leung, S; Aparicio, S; Huntsman, D; Caldas, C; Pharoah, P

    2010-01-01

    Background: Breast cancer is heterogeneous and the existing prognostic classifiers are limited in accuracy, leading to unnecessary treatment of numerous women. B-cell lymphoma 2 (BCL2), an antiapoptotic protein, has been proposed as a prognostic marker, but this effect is considered to relate to oestrogen receptor (ER) status. This study aimed to test the clinical validity of BCL2 as an independent prognostic marker. Methods: Five studies of 11 212 women with early-stage breast cancer were analysed. Individual patient data included tumour size, grade, lymph node status, endocrine therapy, chemotherapy and mortality. BCL2, ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) levels were determined in all tumours. A Cox model incorporating the time-dependent effects of each variable was used to explore the prognostic significance of BCL2. Results: In univariate analysis, ER, PR and BCL2 positivity was associated with improved survival and HER2 positivity with inferior survival. For ER and PR this effect was time dependent, whereas for BCL2 and HER2 the effect persisted over time. In multivariate analysis, BCL2 positivity retained independent prognostic significance (hazard ratio (HR) 0.76, 95% confidence interval (CI) 0.66–0.88, P<0.001). BCL2 was a powerful prognostic marker in ER− (HR 0.63, 95% CI 0.54–0.74, P<0.001) and ER+ disease (HR 0.56, 95% CI 0.48–0.65, P<0.001), and in HER2− (HR 0.55, 95% CI 0.49–0.61, P<0.001) and HER2+ disease (HR 0.70, 95% CI 0.57–0.85, P<0.001), irrespective of the type of adjuvant therapy received. Addition of BCL2 to the Adjuvant! Online prognostic model, for a subset of cases with a 10-year follow-up, improved the survival prediction (P=0.0039). Conclusions: BCL2 is an independent indicator of favourable prognosis for all types of early-stage breast cancer. This study establishes the rationale for introduction of BCL2 immunohistochemistry to improve prognostic stratification. Further work

  11. Contribution of apoptosis-associated signaling pathways to epileptogenesis: lessons from Bcl-2 family knockouts.

    PubMed

    Henshall, David C; Engel, Tobias

    2013-01-01

    Neuronal cell death is a pathophysiological consequence of many brain insults that trigger epilepsy and has been implicated as a causal factor in epileptogenesis. Seizure-induced neuronal death features excitotoxic necrosis and apoptosis-associated signaling pathways, including activation of multiple members of the Bcl-2 gene family. The availability of mice in which individual Bcl-2 family members have been deleted has provided the means to determine whether they have causal roles in neuronal death and epileptogenesis in vivo. Studies show that multiple members of the Bcl-2 family are activated following status epilepticus and the seizure and damage phenotypes of eight different knockouts of the Bcl-2 family have now been characterized. Loss of certain pro-apoptotic members, including Puma, protected against seizure-induced neuronal death whereas loss of anti-apoptotic Mcl-1 and Bcl-w enhanced hippocampal damage. Notably, loss of two putatively pro-apoptotic members, Bak and Bmf, resulted in more seizure-damage while deletion of Bid had no effect, indicating the role of certain Bcl-2 family proteins in epileptic brain injury is distinct from their contributions following other stressors or in non-CNS tissue. Notably, Puma-deficient mice develop fewer spontaneous seizures after status epilepticus suggesting neuroprotection may preserve functional inhibition, either directly by preserving neuronal networks or indirectly, for example by limiting reactive gliosis and pro-inflammatory responses to neuronal death. Together, these studies support apoptosis-associated molecular mechanisms controlling neuronal death as a component of epileptogenesis which might be targetable to protect against seizure-damage, cognitive deficits and mitigate the severity of syndrome following epilepsy-precipitating injuries to the brain.

  12. Latent membrane protein of Epstein-Barr virus induces cellular phenotypes independently of expression of Bcl-2.

    PubMed Central

    Martin, J M; Veis, D; Korsmeyer, S J; Sugden, B

    1993-01-01

    The stable expression of the Epstein-Barr virus (EBV) latent membrane protein (LMP) in certain EBV-negative Burkitt's lymphoma cell lines correlates with an increased expression of the oncogene Bcl-2 (S. Henderson, M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, and A. Rickinson, Cell 65:1107-1115, 1991). This finding is consistent with a model in which Bcl-2 contributes to the immortalization of B cells mediated by EBV. We therefore asked whether the expression of Bcl-2 protein correlates with the induction of three cellular phenotypes induced by or associated with LMP. The expression of Bcl-2 in primary B cells infected with the B95-8 strain of EBV varied between 1 and 1.8 times that in uninfected cells when 50% of the cells were infected, expressed LMP, and incorporated 20-fold more [3H]thymidine than did uninfected cells. This finding indicates that induced proliferation of these primary cells is not sufficient to induce Bcl-2. We found that BALB/c 3T3 cells and their derivatives transformed by LMP do not express Bcl-2 detectably. The expression of LMP at high levels in lymphoid cells is cytotoxic and correlates with an increased expression of Bcl-2 following stable selection for the introduced LMP gene; 2 days after transfection, control vector- and LMP-transfected populations, however, express equal levels of Bcl-2 protein. We also analyzed transient expression of LMP in an EBV-negative Burkitt's lymphoma cell line. Infection of BJAB cells with the B95-8 strain of EBV results in an increase in Bcl-2 expression with a time course similar to that of LMP expression, and LMP alone transiently induces an increase in Bcl-2 expression in these cells. We interpret these observations to indicate that increased expression of Bcl-2 is unlikely to contribute to the ability of EBV to immortalize primary B cells and that both the transformation of rodent cells and the cytotoxicity mediated by LMP are independent of Bcl-2. Images PMID:8394449

  13. 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."

  14. 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

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

    PubMed

    Xu, Wenyuan; Guo, Guifang; Li, Junjuan; Ding, Zhaolei; Sheng, Jianhui; Li, Juan; Tan, Wei

    2016-01-01

    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. 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. 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. 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 activation of the mitochondrial apoptosis

  16. Natural pyrethrins induces apoptosis in human hepatocyte cells via Bax- and Bcl-2-mediated mitochondrial pathway.

    PubMed

    Yang, Yun; Zong, Mimi; Xu, Wenping; Zhang, Yang; Wang, Bo; Yang, Mingjun; Tao, Liming

    2017-01-25

    Natural pyrethrins have been widely used for pest control in organic farming and for residential indoor pest managements. Although the specific mechanisms underlying their activity are incompletely understood, natural pesticides are considered the safest based on their target specificity and rapid degradation in the environment. Here, we used in vitro bioassays to characterize the cytotoxic effects of natural pyrethrins and attempted to delineate the cellular and molecular mechanisms of their cytotoxicity against human hepatocytes. The results demonstrate that natural pyrethrins reduce cell viability and enhance apoptosis in HepG2 cells. In addition, the current data indicate that natural pyrethrins cause a reduction in the mitochondrial membrane potential (Δψm), increase reactive oxygen species production, and up-regulate the Bax/Bcl-2 expression, leading to the release of cytochrome-c into the cytosol, activation of caspase-9 and caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). Taken together, the results indicate that natural pyrethrins has potentially exert adverse effects on human health by inducing caspase-dependent apoptosis in hepatocytes through Bax- and Bcl-2-mediated mitochondrial pathway.

  17. 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

  18. The Relationship between the Bcl-2/Bax Proteins and the Mitochondria-Mediated Apoptosis Pathway in the Differentiation of Adipose-Derived Stromal Cells into Neurons

    PubMed Central

    Wang, Quanquan; Zhang, Lili; Yuan, Xiaodong; Ou, Ya; Zhu, Xuhong; Cheng, Zanzan; Zhang, Pingshu; Wu, Xiaoying; Meng, Yan; Zhang, Liping

    2016-01-01

    Our objective is to study the relationship between the regulatory proteins Bcl-2/Bax and mitochondria-mediated apoptosis during the differentiation of adipose-derived stromal cells (ADSCs) into neurons. Immunocytochemistry and western blotting showed that the cells weakly expressed neuron-specific enolase (NSE) in the non-induced group and expressed NSE more strongly in the groups induced for 1 h, 3 h, 5 h and 8 h. NSE expression peaked at 5 h (P < 0.05), although there was no significant difference between 5 and 8 h (P > 0.05). Bcl-2 expression gradually decreased over time in the non-induced group (P < 0.05). However, Bax, caspase-9, Cyt-c and caspase-3 expression gradually increased and peaked at 8 h (P < 0.05). Transmission electron microscopy revealed karyopyknosis, chromatin edge setting, mitochondria swelling and cavitation in cells at 5 h, and the mitochondrial membrane potential decreased over time, as demonstrated by laser scanning confocal microscopy. After a 5 h induction, cells differentiated into typical neurons and expressed Bcl-2, which inhibited apoptosis. Bax showed a strong apoptosis-promoting capacity, leading to changes in the mitochondrial membrane potential and structure, and then triggered the caspase-independent apoptotic response through the mitochondrial pathway. At the same time, Cyt-c was directly or indirectly released from the mitochondria to the cytoplasm to trigger the caspase-dependent apoptotic response through the mitochondrial pathway. Therefore, Bcl-2/Bax play an important role in regulating caspase-dependent and caspase-independent apoptosis mediated by the mitochondrial pathway during the differentiation of ADSCs into neurons. PMID:27706181

  19. 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.

  20. 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

  1. 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.

  2. (-)-Epigallocatechingallate induces apoptosis in B lymphoma cells via caspase-dependent pathway and Bcl-2 family protein modulation.

    PubMed

    Wang, Jiangyan; Xie, Yu'an; Feng, Yan; Zhang, Litu; Huang, Xinping; Shen, Xiaoyun; Luo, Xiaoling

    2015-04-01

    (-)-Epigallocatechingallate (EGCG) as a representative polyphenol has attracted increasing attention due to its diversified effects, especially its potential as an agent for the prevention or treatment of certain cancers. However, the molecular mechanisms of EGCG-induced apoptosis in B lymphoma cells are unclear. The aim of this study was to investigate the effect of EGCG on proliferation and apoptosis in the B lymphoma cell lines Jeko-1 and Raji, and determine the underlying mechanisms. Cell proliferation and cytotoxicity were determined by the cell counting kit (CCK-8) assay; apoptosis was assessed by flow cytometry using the Annexin V-PE/7AAD double staining; Fas, Bcl-2 and Bax mRNA expression levels were determined by real-time PCR; caspase activity was measured by the caspase activity assay kit; the expression levels of apoptosis-associated proteins were determined by western blot analysis. We demonstrated that EGCG induced growth inhibition and apoptosis in a dose- and time-dependent manner. In agreement, EGCG upregulated the mRNA expression of Fas and Bax while downregulating Bcl-2. Protein expression levels of Bax, activated caspase-3, -7, -8, and -9, and PARP were increased, while Bcl-2 protein levels were reduced by EGCG treatment. Taken together, EGCG induces B lymphoma cell apoptosis by triggering caspase-dependent intrinsic (mitochondrial) and extrinsic (death receptor) pathways. These findings suggest that EGCG may be a potential agent for the treatment of B lymphoma.

  3. TESTOSTERONE-DOWNREGULATED AKT PATHWAY DURING CARDIAC ISCHEMIA/REPERFUSION: A MECHANISM INVOLVING BAD, BCL-2 AND FOXO3A

    PubMed Central

    Huang, Chunyan; Gu, Hongmei; Zhang, Wenjun; Herrmann, Jeremy L.; Wang, Meijing

    2010-01-01

    Background Lower levels of myocardial Akt activity in males are associated with a higher incidence of heart failure and worsened cardiac function after ischemia/reperfusion (I/R). While Akt activation by estrogen provides cardioprotection in females, no information exists regarding the effect of testosterone on the myocardial Akt pathway following I/R. We hypothesized that following I/R: 1) endogenous testosterone will decrease myocardial Akt activation in male hearts; 2) endogenous testosterone will mediate downstream signals of Akt, including Bad, Bcl-2 and FOXO3a; 3) administration of exogenous testosterone will recapitulate negative effects on the Akt pathway in castrated male hearts. Methods and Results Rat hearts from age-matched adult males, females, castrated males, males with androgen receptor blocker-flutamide, castrated males with chronic 5α-dihydrotestosterone (DHT) implantation or acute testosterone infusion (ATI) (n=9/group) were subjected to I/R (Langendorff). Castration or flutamide treatment significantly upregulated myocardial Akt activation, increased downstream apoptosis-regulatory molecules: p-Bad, Bcl-2, p-FOXO3a, but reduced Fas-L, consistent with decreased myocardial injury in male hearts following I/R. ATI administration, but not chronic DHT, reversed these effects on Akt signaling associated with further exacerbated cardiac dysfunction in castrated males. Notably, lower levels of MnSOD were observed in male hearts, and castration or flutamide treatment restored myocardial MnSOD expression to the levels of females in male hearts after I/R. Conclusion Our study represents the initial evidence of testosterone-induced downregulation of the Akt pathway in male hearts following I/R, thereby mediating cardiac injury through decreased p-Bad, reduced ratio of Bcl-2/Bax in the cytoplasm, and increased FOXO3a in the nucleus. PMID:20850791

  4. Triggering of Bcl-2-related pathway is associated with apoptosis of photoreceptors in Rpe65-/- mouse model of Leber's congenital amaurosis.

    PubMed

    Cottet, Sandra; Schorderet, Daniel F

    2008-03-01

    Mutations in RPE65 protein is characterized by the loss of photoreceptors, although the molecular pathways triggering retinal cell death remain largely unresolved. The role of the Bcl-2 family of proteins in retinal degeneration is still controversial. However, alteration in Bcl-2-related proteins has been observed in several models of retinal injury. In particular, Bax has been suggested to play a crucial role in apoptotic pathways in murine glaucoma model as well as in retinal detachment-associated cell death. We demonstrated that Bcl-2-related signaling pathway is involved in Rpe65-dependent apoptosis of photoreceptors during development of the disease. Pro-apoptotic Bax alpha and beta isoforms were upregulated in diseased retina. This was associated with a progressive reduction of anti-apoptotic Bcl-2, reflecting imbalanced Bcl-2/Bax ratio as the disease progresses. Moreover, specific translocation of Bax beta from cytosol to mitochondria was observed in Rpe65-deficient retina. This correlated with the initiation of photoreceptor cell loss at 4 months of age, and further increased during disease development. Altogether, these data suggest that Bcl-2-apoptotic pathway plays a crucial role in Leber's congenital amaurosis disease. They further highlight a new regulatory mechanism of Bax-dependent apoptosis based on regulated expression and activation of specific isoforms of this protein.

  5. Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells.

    PubMed

    Niu, Guomin; Yin, Songmei; Xie, Shuangfeng; Li, Yiqing; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavonol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

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

    PubMed

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

    2015-08-04

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

  7. Moclobemide upregulated Bcl-2 expression and induced neural stem cell differentiation into serotoninergic neuron via extracellular-regulated kinase pathway

    PubMed Central

    Chiou, Shih-Hwa; Ku, Hung-Hai; Tsai, Tung-Hu; Lin, Heng-Liang; Chen, Li-Hsin; Chien, Chan-Shiu; Ho, Larry L -T; Lee, Chen-Hsen; Chang, Yuh-Lih

    2006-01-01

    Moclobemide (MB) is an antidepressant drug that selectively and reversibly inhibits monoamine oxidase-A. Recent studies have revealed that antidepressant drugs possess the characters of potent growth-promoting factors for the development of neurogenesis and improve the survival rate of serotonin (5-hydroxytrytamine; 5-HT) neurons. However, whether MB comprises neuroprotection effects or modulates the proliferation of neural stem cells (NSCs) needs to be elucidated. In this study, firstly, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to demonstrate that 50 μM MB can increase the cell viability of NSCs. The result of real-time reverse transcription–polymerase chain reaction (RT–PCR) showed that the induction of MB can upregulate the gene expressions of Bcl-2 and Bcl-xL. By using caspases 8 and 3, ELISA and terminal dUTP nick-end labeling (TUNEL) assay, our data further confirmed that 50 μM MB-treated NSCs can prevent FasL-induced apoptosis. The morphological findings also supported the evidence that MB can facilitate the dendritic development and increase the neurite expansion of NSCs. Moreover, we found that MB treatment increased the expression of Bcl-2 in NSCs through activating the extracellular-regulated kinase (ERK) phosphorylation. By using the triple-staining immunofluorescent study, the percentages of serotonin- and MAP-2-positive cells in the day 7 culture of MB-treated NSCs were significantly increased (P<0.01). Furthermore, our data supported that MB treatment increased functional production of serotonin in NSCs via the modulation of ERK1/2. In sum, the study results support that MB can upregulate Bcl-2 expression and induce the differentiation of NSCs into serotoninergic neuron via ERK pathway. PMID:16702990

  8. The apoptotic pathway triggered by the Fhit protein in lung cancer cell lines is not affected by Bcl-2 or Bcl-x(L) overexpression.

    PubMed

    Roz, Luca; Andriani, Francesca; Ferreira, Carlos G; Giaccone, Giuseppe; Sozzi, Gabriella

    2004-12-02

    The expression of the tumour suppressor protein fragile histidine triad (Fhit) is often impaired in many human cancers and its restoration in Fhit-negative cancer cell lines suppresses tumorigenicity and induces apoptosis. Although the proapoptotic function of Fhit is well documented, little is known about its precise mechanism of action and further studies are needed in order to elucidate the putative therapeutic properties of this protein. To this end, we have engineered the lung cancer cell line NCI-H460 in order to express different molecules involved in the control of apoptotic pathways. Infection of these cells with an adenoviral vector transducing the Fhit gene (Ad-Fhit) revealed that complete protection from apoptosis was conferred by the inhibitor of caspases Cytokine response modifier A (CrmA) and by a dominant-negative form of the adapter protein Fas-associated death domain (FADD) and partial protection by a dominant-negative form of caspase-8, while cells over expressing mitochondrial mediators of the apoptotic response such as Bcl-2 or Bcl-x(L) that are resistant to treatment with cisplatin, remained highly susceptible to cell death triggered by Fhit gene transfer. In line to what was observed in H460 cells, Ad-Fhit efficacy was not affected by Bcl-2 overexpression also in two other lung cancer cell lines (A549 and Calu-1). Analysis of cytochrome c release also confirmed that in Bcl-2- or Bcl-x(L)-expressing cells apoptosis could be detected by terminal deoxynucleotidyl-transferase mediated dUTP nick-end labelling (TUNEL) assay before any evidence of mitochondrial membrane perturbation. In conclusion, our analysis indicates that the Fhit protein exerts its oncosuppressor activity through induction of an apoptotic mechanism that seems to be FADD dependent, caspase-8 mediated and independent from mitochondrial amplification.

  9. Insulin involved Akt/ERK and Bcl-2/Bax pathways against oxidative damages in C6 glial cells.

    PubMed

    Ramalingam, Mahesh; Kim, Sung-Jin

    2016-01-01

    Insulin, a hypoglycemic hormone, has multiple functions in the brain. The aim of this study to identify the mechanisms of insulin in hydrogen peroxide (H(2)O(2)-induced toxicity in the C6 glial cells. Cytotoxicity, lactate dehydrogenase, nitric oxide, reactive oxygen species and calcium ion, lipid peroxidation, protein oxidation and glutathione levels were determined. Signaling pathway molecules were assessed by western blotting and RT-PCR. The results showed that treatment with insulin reduced the cell death and cell membrane damages against H(2)O(2)-induced toxicity. Furthermore, insulin interfered H(2)O(2)-induced intracellular generation of reactive oxygen species and calcium-ion transport, apoptosis, including lipid and protein oxidation products. Cells treated with insulin reverted H(2)O(2)-induced suppression of reduced glutathione levels by blocking oxidized glutathione. Moreover, insulin treatment activates Akt, restores ERK1/2 and Bcl-2 by preventing Bax and Bax/Bcl-2 ratio. Our results suggest that treatment of insulin exerts potential role against 24 h of H(2)O(2)-induced toxicity in C6 cells.

  10. 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.

  11. Insulin resistance contributes to multidrug resistance in HepG2 cells via activation of the PERK signaling pathway and upregulation of Bcl-2 and P-gp.

    PubMed

    Liu, Xinyue; Li, Linjing; Li, Jing; Cheng, Yan; Chen, Jing; Shen, Minghui; Zhang, Shangdi; Wei, Hulai

    2016-05-01

    Liver tumorigenesis frequently causes insulin resistance which may be used as an independent risk factor for evaluation of survival and post-surgery relapse of liver cancer patients. In the present study, HepG2/IR, an insulin resistant HepG2 cell line, was established by exposing HepG2 cells to 0.5 µmol/l of insulin for 72 h, and comparison of HepG2/IR with the parental HepG2 cells indicated that the HepG2/IR cells showed significantly enhanced resistance to the most frequently used chemotherapeutics for solid tumors, such as cisplatin, 5-fluorouracil, vincristine and mitomycin. Flow cytometric analysis of cisplatin-treated HepG2/IR cells showed a significantly decreased hypodiploid peak and a significantly downregulated expression level of pro-apoptotic protein caspase-3 compared with the parental HepG2 cells. Our data further showed swollen endoplasmic reticulum (ER) in the cisplatin-treated HepG2/IR cells with significantly increased levels of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like ER kinase (p-PERK) and P-glycoprotein (P-gp). There was also an upregulated expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) whereas no significant change was observed for CCAAT-enhancer-binding protein homologous protein (CHOP), which is known to be induced by ER stress and to mediate apoptosis. Our results demonstrated that insulin resistance in HepG2 cells promoted a protective unfolded protein response and upregulated the expression of ER chaperone protein GRP78, which resulted in the phosphorylation of PERK kinase to activate the PERK-mediated ER stress signal transduction pathway and the upregulation of Bcl-2 and P-gp, leading to the inhibition of the caspase-3-dependent apoptosis pathway and to the survival of liver tumor cells.

  12. Defective cell death signalling along the Bcl-2 regulated apoptosis pathway compromises Treg cell development and limits their functionality in mice.

    PubMed

    Tischner, Denise; Gaggl, Irene; Peschel, Ines; Kaufmann, Manuel; Tuzlak, Selma; Drach, Mathias; Thuille, Nikolaus; Villunger, Andreas; Jan Wiegers, G

    2012-02-01

    The Bcl-2 regulated apoptosis pathway is critical for the elimination of autoreactive lymphocytes, thereby precluding autoimmunity. T cells escaping this process can be kept in check by regulatory T (Treg) cells expressing the transcription and lineage commitment factor Foxp3. Despite the well-established role of Bcl-2 family proteins in shaping the immune system and their frequent deregulation in autoimmune pathologies, it is poorly understood how these proteins affect Treg cell development and function. Here we compared the relative expression of a panel of 40 apoptosis-associated genes in Treg vs. conventional CD4(+) T cells. Physiological significance of key-changes was validated using gene-modified mice lacking or overexpressing pro- or anti-apoptotic Bcl-2 family members. We define a key role for the Bim/Bcl-2 axis in Treg cell development, homeostasis and function but exclude a role for apoptosis induction in responder T cells as relevant suppression mechanism. Notably, only lack of the pro-apoptotic BH3-only protein Bim or Bcl-2 overexpression led to accumulation of Treg cells while loss of pro-apoptotic Bad, Bmf, Puma or Noxa had no effect. Remarkably, apoptosis resistant Treg cells showed reduced suppressive capacity in a model of T cell-driven colitis, posing a caveat for the use of such long-lived cells in possible therapeutic settings. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    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

  14. Quercetin induces p53-independent apoptosis in human prostate cancer cells by modulating Bcl-2-related proteins: a possible mediation by IGFBP-3.

    PubMed

    Vijayababu, Marati R; Kanagaraj, P; Arunkumar, A; Ilangovan, R; Dharmarajan, A; Arunakaran, J

    2006-01-01

    Quercetin, a flavonoid found in onion, grapes, green vegetables, etc., has been shown to possess potent antiproliferative effects against various malignant cells. We report insulin-like growth factor-binding protein-3 (IGFBP-3) as an effector of quercetin-induced apoptosis in human prostate cancer cell lines in a p53-independent manner. We evaluated the production of IGFBP-3 in quercetin-treated cells. Apoptosis was studied in quercetin-treated cells to study the IGFBP-3-mediated role with flow cytometry and DNA fragmentation. Protein expressions of Bcl-2, Bcl-x(L), and Bax were studied by Western blot. Increased production of IGFBP-3 was associated with the increased ratio of proapoptotic to antiapoptotic members of the Bcl-2 family. In quercetin-treated PC-3 cells, an increase in Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein were observed. As PC-3 is a p53-negative cell line, these modulations of proapoptotic proteins and induction of apoptosis were independent of p53. The level of IGFBP-3 on the response of PC-3 cells to quercetin was examined. There was a twofold increase in IGFBP-3 level in conditioned media of 100 microM quercetin-treated cells. Quercetin also brought a peak at sub-G1 in PC-3 cells. Thus, increased level of IGFBP-3 was associated with increased proapoptotic proteins and apoptosis in response to quercetin, suggesting it may be a p53-independent effector of apoptosis in prostate cancer cells via its modulation of the Bax/Bcl-2 protein ratio.

  15. 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

  16. Training in Support of Research Project Entitled "Genetic Regulation of the Bcl-2/Bax Cell Death Pathway".

    DTIC Science & Technology

    1998-07-01

    programmed cell death in mammals, Bcl-2 family proteins can also induce or prevent cell death in the unicellular yeast; overexpression of Bax causes lethality...in both S. cerevisiae and S. pombe, and co-expression of either Bcl-2 or Bcl-XL can protect yeast against Bax-caused cell death . On the basis of these...observations, we have developed a functional screen and have identified two human genes, BI-1 and BI-2, which inhibit Bax-induced cell death in yeast

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

    PubMed

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

    2014-08-01

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

  18. Cannabinoid receptor type 2 agonist attenuates apoptosis by activation of phosphorylated CREB-Bcl-2 pathway after subarachnoid hemorrhage in rats.

    PubMed

    Fujii, Mutsumi; Sherchan, Prativa; Soejima, Yoshiteru; Hasegawa, Yu; Flores, Jerry; Doycheva, Desislava; Zhang, John H

    2014-11-01

    Early brain injury (EBI) which comprises of vasogenic edema and apoptotic cell death is an important component of subarachnoid hemorrhage (SAH) pathophysiology. This study evaluated whether cannabinoid receptor type 2 (CB2R) agonist, JWH133, attenuates EBI after SAH and whether CB2R stimulation reduces pro-apoptotic caspase-3 via up-regulation of cAMP response element-binding protein (CREB)-Bcl-2 signaling pathway. Male Sprague-Dawley rats (n=123) were subjected to SAH by endovascular perforation. Rats received vehicle or JWH133 at 1h after SAH. Neurological deficits and brain water content were evaluated at 24h after SAH. Western blot was performed to quantify phosphorylated CREB (pCREB), Bcl-2, and cleaved caspase-3 levels. Neuronal cell death was evaluated with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. Additionally, CREB siRNA was administered to manipulate the proposed pathway. JWH133 (1.0mg/kg) improved neurological deficits and reduced brain water content in left hemisphere 24h after SAH. JWH133 significantly increased activated CREB (pCREB) and Bcl-2 levels and significantly decreased cleaved caspase-3 levels in left hemisphere 24h after SAH. CREB siRNA reversed the effects of treatment. TUNEL positive neurons in the cortex were reduced with JWH133 treatment. Thus, CB2R stimulation attenuated EBI after SAH possibly through activation of pCREB-Bcl-2 pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. 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.

  20. Lewis y enhances CAM-DR in ovarian cancer cells by activating the FAK signaling pathway and upregulating Bcl-2/Bcl-XL expression.

    PubMed

    Yan, Limei; Wang, Changzhi; Lin, Bei; Liu, Juanjuan; Liu, Dawo; Hou, Rui; Wang, Yifei; Gao, Lili; Zhang, Shulan; Iwamori, Masao

    2015-06-01

    Oligosaccharides on the surface of adhesion molecules may contribute to the process of CAM-DR. To investigate the role of the Lewis y antigen in this process, we established a cell adhesion model mediated by the integrin α5β1-FN interaction in the ovarian cancer cell line, RMG-1-hFUT, which highly expresses Lewis y by transfection with α1,2-fucosyltransferase into RMG-1 cells. Our results indicate that the rates of carboplatin-induced apoptosis and necrosis are reduced in FN-adhered tumor cells, and carboplatin resistance is significantly decreased in the presence of anti-Lewis y antibody. CAM-DR in tumor cells has been correlated with elevated expression of the nuclear anti-apoptotic proteins Bcl-2 and Bcl-XL. Lewis y promotes the expression of the Bcl-2 and Bcl-XL genes by activating the focal adhesion kinase signaling pathway and accelerating their transcription. Thus, Lewis y leads to inhibition of apoptosis and enhancement of CAM-DR by activation of the FAK signaling pathway and upregulation of Bcl-2/Bcl-XL expression in ovarian cancer cell lines. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. P53 and MITF/Bcl-2 identified as key pathways in the acquired resistance of NRAS-mutant melanoma to MEK inhibition.

    PubMed

    Najem, Ahmad; Krayem, Mohammad; Salès, François; Hussein, Nader; Badran, Bassam; Robert, Caroline; Awada, Ahmad; Journe, Fabrice; Ghanem, Ghanem E

    2017-09-01

    Activating mutations in Neuroblastoma RAS viral oncogene homolog (NRAS) are found in 15-30% of melanomas and are associated with a poor prognosis. Although MAP kinase kinase (MEK) inhibitors used as single agents showed a limited clinical benefit in patients with NRAS-mutant melanoma due to their rather cytostatic effect and high toxicity, their combination with other inhibitors of pathways known to cooperate with MEK inhibition may maximise their antitumour activity. Similarly, in a context where p53 is largely inactivated in melanoma, hyperexpression of Microphthalmia associated transcription factor (MITF) and its downstream anti-apoptotic targets may be the cause of the restraint cytotoxic effects of MEK inhibitors. Indeed, drug combinations targeting both mutant BRAF and MITF or one of its important targets Bcl-2 were effective in mutant BRAF melanoma but had no effect on acquired resistance. Therefore, we aimed to further investigate the downstream MITF targets that can explain this anti-apoptotic effect and to evaluate in parallel the effect of p53 reactivation on the promotion of apoptosis under MEK inhibition in a panel of (Q61)NRAS-mutant melanoma cells. First, we showed that MEK inhibition (pimasertib) led to a significant inhibition of cell proliferation but with a limited effect on apoptosis that could be explained by the systematic MITF upregulation. Mimicking the MITF effect via cyclic adenosine monophosphate activation conferred resistance to MEK inhibition and upregulated Bcl-2 expression. In addition, acquired resistance to MEK inhibition was associated with a strong activation of the anti-apoptotic signalling MITF/Bcl-2. More importantly, selective Bcl-2 inhibition by ABT-199 or Bcl-2 knockout using CRISPR/Cas9 system annihilated the acquired resistance and restored the sensitivity of NRAS-mutant melanoma cells to MEK inhibition. Strikingly and similarly, direct p53 reactivation (PRIMA-1(Met), APR-246) also broke resistance and synergised with

  2. CACNA1C SNP rs1006737 associates with bipolar I disorder independent of the Bcl-2 SNP rs956572 variant and its associated effect on intracellular calcium homeostasis.

    PubMed

    Uemura, Takuji; Green, Marty; Warsh, Jerry J

    2016-10-01

    Intracellular calcium (Ca(2+)) dyshomeostasis (ICDH) has been implicated in bipolar disorder (BD) pathophysiology. We previously showed that SNP rs956572 in the B-cell CLL/lymphoma 2 (Bcl-2) gene associates with elevated B lymphoblast (BLCL) intracellular Ca(2+) concentrations ([Ca(2+)]B) differentially in BD-I. Genome-wide association studies strongly support the association between BD and the SNP rs1006737, located within the L-type voltage-dependent Ca(2+) channel α1C subunit gene (CACNA1C). Here we investigated whether this CACNA1C variant also associates with ICDH and interacts with SNP rs956572 on [Ca(2+)]B in BD-I. CACNA1C SNP rs1006737 was genotyped in 150 BD-I, 65 BD-II, 30 major depressive disorder patients, and 70 healthy subjects with available BLCL [Ca(2+)]B and Bcl-2 SNP rs956572 genotype measures. SNP rs1006737 was significantly associated with BD-I. The [Ca(2+)]B was significantly higher in BD-I rs1006737 A compared with healthy A allele carriers and also in healthy GG compared with A allele carriers. There was no significant interaction between SNP rs1006737 and SNP rs956572 on [Ca(2+)]B. Our study further supports the association of SNP rs1006737 with BD-I and suggests that CACNA1C SNP rs1006737 and Bcl-2 SNP rs956572, or specific causal variants in LD with these proxies, act independently to increase risk and ICDH in BD-I.

  3. Neuroprotective Effects of β-Asarone Against 6-Hydroxy Dopamine-Induced Parkinsonism via JNK/Bcl-2/Beclin-1 Pathway.

    PubMed

    Zhang, Sheng; Gui, Xue-Hong; Huang, Li-Ping; Deng, Min-Zhen; Fang, Ruo-Ming; Ke, Xue-Hong; He, Yu-Ping; Li, Ling; Fang, Yong-Qi

    2016-01-01

    β-asarone, a major component of Acorus tatarinowii Schott, has positive effects in neurodegeneration disease, however, its effect on the Parkinson's disease (PD) remains unclear. In this study, the effects of β-asarone on behavioral tests, neurotransmitters, tyrosine hydroxylase (TH), and α-synuclein (α-syn) were investigated in 6-hydroxydopamine (6-OHDA) induced rats. Furthermore, the JNK/Bcl-2/Beclin-1 autophagy pathway was also studied. The results showed that β-asarone improved the behavioral symptoms of rats in the open field, rotarod test, initiation time, and stepping time. And it increased the HVA, Dopacl, and 5-HIAA levels in striatum but not the DA and 5-HT levels. After administration of β-asarone, the TH level was elevated but the α-syn was declined in rats. It inhibited the expressions of LC3-II, but increased the p62 expression in SN4741 cells. Moreover, it affected the expressions of Beclin-1, Bcl-2, JNK, and p-JNK in vivo. We deduced that β-asarone may firstly downregulate expressions of JNK and p-JNK, and then indirectly increase the expression of Bcl-2. And the function of Beclin-1 could be inhibited, which could inhibit autophagy activation. Collectively, all data indicated that β-asarone may be explored as a potential therapeutic agent in PD therapy.

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

    PubMed Central

    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

  5. Intervention effects of QRZSLXF, a Chinese medicinal herb recipe, on the DOR-β-arrestin1-Bcl2 signal transduction pathway in a rat model of ulcerative colitis.

    PubMed

    Fan, Heng; Liu, Xing-xing; Zhang, Li-juan; Hu, Hui; Tang, Qing; Duan, Xue-yun; Zhong, Min; Shou, Zhe-xing

    2014-05-28

    Qingre Zaoshi Liangxue Fang (QRZSLXF) is a Chinese medicinal herb recipe that is commonly prescribed for the treatment of ulcerative colitis. It includes 5 quality assured herbs: Sophora flavescens Aiton., Baphicacanthus cusia (Nees) Bremek., Bletilla striata Rchb.f., Glycyrrhiza uralensis Fisch. and Coptis chinensis Franch. The main phytochemical ingredient of QRZSLXF includes ammothamnine, sophocarpidine, liquiritin, berberine and indirubin. QRZSLXF has been clinically proven for use in the treatment of ulcerative colitis for over twenty years. In the past ten years, research has confirmed the therapeutic effect of QRZSLXF in ulcerative colitis and partially revealed its mechanism of action. Here, we further reveal the therapeutic mechanism of QRZSLXF in ulcerative colitis. To investigate the role of the DOR-β-arrestin1-Bcl-2 signal transduction pathway in ulcerative colitis and to determine the effects of QRZSLXF on this signal transduction pathway. Eighty-four Sprague-Dawley rats were randomly divided into six groups: normal control group, model group, mesalazine group, and QRZSLXF high-dose, medium-dose group and low-dose groups (n=14). Experimental colitis was induced by trinitrobenzenesulfonic acid (TNBS) in each group, except the normal control group. After modeling, bloody stool, mental state and diarrhea were observed and recorded. Two rats were randomly selected from the model groups adfnd sacrificed on day 3 to observe pathological changes in the colon tissue by microscopy. The rats in the QRZSLXF-treated groups received intramuscular injections of different concentrations of QRZSLXF for 15 days. The rats in the mesalazine group were treated with mesalazine solution (0.5 g/kg/day) by gastric lavage for 15 days. The rats in the normal control group and the model group were treated with 3 mL water by gastric lavage for 15 days. On the 16th day, after fasting for 24 h, the remaining rats were sacrificed and their colon tissues were used to detect the m

  6. The C. elegans protein CEH-30 protects male-specific neurons from apoptosis independently of the Bcl-2 homolog CED-9.

    PubMed

    Schwartz, Hillel T; Horvitz, H Robert

    2007-12-01

    The developmental control of apoptosis is fundamental and important. We report that the Caenorhabditis elegans Bar homeodomain transcription factor CEH-30 is required for the sexually dimorphic survival of the male-specific CEM (cephalic male) sensory neurons; the homologous cells of hermaphrodites undergo programmed cell death. We propose that the cell-type-specific anti-apoptotic gene ceh-30 is transcriptionally repressed by the TRA-1 transcription factor, the terminal regulator of sexual identity in C. elegans, to cause hermaphrodite-specific CEM death. The established mechanism for the regulation of specific programmed cell deaths in C. elegans is the transcriptional control of the BH3-only gene egl-1, which inhibits the Bcl-2 homolog ced-9; similarly, most regulation of vertebrate apoptosis involves the Bcl-2 superfamily. In contrast, ceh-30 acts within the CEM neurons to promote their survival independently of both egl-1 and ced-9. Mammalian ceh-30 homologs can substitute for ceh-30 in C. elegans. Mice lacking the ceh-30 homolog Barhl1 show a progressive loss of sensory neurons and increased sensory-neuron cell death. Based on these observations, we suggest that the function of Bar homeodomain proteins as cell-type-specific inhibitors of apoptosis is evolutionarily conserved.

  7. 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

    2015-05-07

    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.

  8. Testosterone-down-regulated Akt pathway during cardiac ischemia/reperfusion: a mechanism involving BAD, Bcl-2 and FOXO3a.

    PubMed

    Huang, Chunyan; Gu, Hongmei; Zhang, Wenjun; Herrmann, Jeremy L; Wang, Meijing

    2010-11-01

    Lower levels of myocardial Akt activity in males are associated with a higher incidence of heart failure and worsened cardiac function after ischemia/reperfusion (I/R). While Akt activation by estrogen provides cardioprotection in females, no information exists regarding the effect of testosterone on the myocardial Akt pathway following I/R. We hypothesized that following I/R: (1) endogenous testosterone will decrease myocardial Akt activation in male hearts; (2) endogenous testosterone will mediate downstream signals of Akt, including Bad, Bcl-2, and FOXO3a; (3) administration of exogenous testosterone will recapitulate negative effects on the Akt pathway in castrated male hearts. Rat hearts from age-matched adult males, females, castrated males, males with androgen receptor blocker-flutamide, castrated males with chronic 5α-dihydrotestosterone (DHT) implantation, or acute testosterone infusion (ATI) (n = 9/group) were subjected to I/R (Langendorff). Castration or flutamide treatment significantly up-regulated myocardial Akt activation, increased downstream apoptosis-regulatory molecules p-Bad, Bcl-2, p-FOXO3a, but reduced Fas-L, consistent with decreased myocardial injury in male hearts following I/R. ATI administration, but not chronic DHT, reversed these effects on Akt signaling associated with further exacerbated cardiac dysfunction in castrated males. Notably, lower levels of MnSOD were observed in male hearts, and castration or flutamide treatment restored myocardial MnSOD expression to the levels of females in male hearts after I/R. Our study represents the initial evidence of testosterone-induced down-regulation of the Akt pathway in male hearts following I/R, thereby mediating cardiac injury through decreased p-Bad, reduced ratio of Bcl-2/Bax in the cytoplasm, and increased FOXO3a in the nucleus. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Anti-apoptotic BCL-2 family proteins in acute neural injury

    PubMed Central

    Anilkumar, Ujval; Prehn, Jochen H. M.

    2014-01-01

    Cells under stress activate cell survival and cell death signaling pathways. Cell death signaling frequently converges on mitochondria, a process that is controlled by the activities of pro- and anti-apoptotic B-cell lymphoma 2 (BCL-2) proteins. In this review, we summarize current knowledge on the control of neuronal survival, development and injury by anti-apoptotic BCL-2 family proteins. We discuss overlapping and differential effects of the individual family members BCL-2, BCL-extra long (BCL-XL), myeloid cell leukemia 1 (MCL-1), and BCL2-like 2 (BCL-W) in the control of survival during development and pathophysiological processes such as trophic factor withdrawal, ischemic injury, excitotoxicity, oxidative stress and energy stress. Finally we discuss recent evidence that several anti-apoptotic BCL-2 proteins influence mitochondrial bioenergetics and control neuronal Ca2+ homeostasis independent of their classical role in cell death signaling. PMID:25324720

  10. 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.

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

    PubMed

    Yang, Jiong; Yao, Shukun

    2015-10-27

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

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

    PubMed Central

    Yang, Jiong; Yao, Shukun

    2015-01-01

    Autophagy is associated with drug resistance which has been a threat in chemotherapy of hepatocellular carcinoma (HCC). The interconnected molecular regulators between autophagy and apoptosis serve as switching points critical to the ultimate outcome of the cell. Our study was performed to investigate the crosstalk between autophagy and apoptosis in HCC after the treatment of matrine. Flow cytometry and TUNEL (terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling) assay were used to detect apoptosis in vitro and in vivo, respectively. Bax oligomerization and Cytochrome c release assay were performed. Immunoprecipitation and siRNA transfection were used to detect the interplay between Bcl-2/Bcl-xL,Bax, and Beclin 1. Our results showed that: (1) matrine not only activated caspase and PARP (poly ADP-ribose polymerase) cleavage, but also triggered autophagy as shown by the increased levels of LC3II, Beclin 1, and PI3KC3, and the decreased level of p62; (2) matrine treatment promoted the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway; (3) Bax was oligomerized, the mitochondrial membrane potential altered, and Cytochrome c was released subsequently; (4) Bax interacts with Beclin 1 and inhibits autophagy, which may be a new crosstalk point; and (5) finally, we showed that matrine suppressed the growth of a MHCC97L xenograft in vivo for the first time. In conclusion, the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates the crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin 1. PMID:26516844

  13. 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

  14. The apoptotic effect of brucine from the seed of Strychnos nux-vomica on human hepatoma cells is mediated via Bcl-2 and Ca2+ involved mitochondrial pathway.

    PubMed

    Deng, Xukun; Yin, Fangzhou; Lu, Xiaoyu; Cai, Baochang; Yin, Wu

    2006-05-01

    In an attempt to dissect the mechanism of Strychnos nux-vomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, on human hepatoma cells (HepG2) were screened by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrasolium bromide (MTT) assay. Brucine, among the four alkaloids, exhibited the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucine-treated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular [Ca2+], which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of [Ca2+]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca2+ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis.

  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. 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

    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

    2015-03-11

    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.

  17. Interleukin 7 signaling prevents apoptosis by regulating bcl-2 and bax via the p53 pathway in human non-small cell lung cancer cells.

    PubMed

    Liu, Zi-Hui; Wang, Ming-Hui; Ren, Hong-Jiu; Qu, Wei; Sun, Li-Mei; Zhang, Qing-Fu; Qiu, Xue-Shan; Wang, En-Hua

    2014-01-01

    Interleukin 7/Interleukin 7 receptor (IL-7/IL-7R) signaling induces the upregulation of cyclin D1 to promote cell proliferation in lung cancer, but its role in preventing the apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. To study the role of IL-7 in lung cancer cell apoptosis, normal HBE cells as well as A549 and H1299 NSCLC cells were examined using flow cytometry. The results showed that the activation of IL-7R by its specific ligand, exogenous interleukin-7, was associated with a significant decline in apoptotic cells. Western blot and real-time PCR assays indicated that the activation of IL-7/IL-7R significantly upregulated anti-apoptotic bcl-2 and downregulated pro-apoptotic bax and p53 at both protein and mRNA levels. The knockdown of IL-7R through small interfering RNAs significantly attenuated these effects of exogenous IL-7. However, there was no significant anti-apoptotic effect in H1299 (p53-) cells. Furthermore, the inhibition of p53 significantly abolished the effects of IL-7/IL-7R on lung cancer cell apoptosis. These results strongly suggest that IL-7/IL-7R prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax, potentially via the p53 pathway in A549 and HBE cells.

  18. 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

  19. 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.

  20. 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

  1. Perfluorocarbon reduces cell damage from blast injury by inhibiting signal paths of NF-κB, MAPK and Bcl-2/Bax signaling pathway in A549 cells

    PubMed Central

    Li, Huaidong; Li, Chunsun; Yang, Zhen; Li, Yanqin; She, Danyang; Cao, Lu; Wang, Wenjie; Liu, Changlin; Chen, Liangan

    2017-01-01

    Background and objective Blast lung injury is a common type of blast injury and has very high mortality. Therefore, research to identify medical therapies for blast injury is important. Perfluorocarbon (PFC) is used to improve gas exchange in diseased lungs and has anti-inflammatory functions in vitro and in vivo. The aim of this study was to determine whether PFC reduces damage to A549 cells caused by blast injury and to elucidate its possible mechanisms of action. Study design and methods A549 alveolar epithelial cells exposed to blast waves were treated with and without PFC. Morphological changes and apoptosis of A549 cells were recorded. PCR and enzyme-linked immunosorbent assay (ELISA) were used to measure the mRNA or protein levels of IL-1β, IL-6 and TNF-α. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity levels were detected. Western blot was used to quantify the expression of NF-κB, Bax, Bcl-2, cleaved caspase-3 and MAPK cell signaling proteins. Results A549 cells exposed to blast wave shrank, with less cell-cell contact. The morphological change of A549 cells exposed to blast waves were alleviated by PFC. PFC significantly inhibited the apoptosis of A549 cells exposed to blast waves. IL-1β, IL-6 and TNF-α cytokine and mRNA expression levels were significantly inhibited by PFC. PFC significantly increased MDA levels and decreased SOD activity levels. Further studies indicated that NF-κB, Bax, caspase-3, phospho-p38, phosphor-ERK and phosphor-JNK proteins were also suppressed by PFC. The quantity of Bcl-2 protein was increased by PFC. Conclusion Our research showed that PFC reduced A549 cell damage caused by blast injury. The potential mechanism may be associated with the following signaling pathways: 1) the signaling pathways of NF-κB and MAPK, which inhibit inflammation and reactive oxygen species (ROS); and 2) the signaling pathways of Bcl-2/Bax and caspase-3, which inhibit apoptosis. PMID:28323898

  2. Apigenin induces apoptosis via tumor necrosis factor receptor- and Bcl-2-mediated pathway and enhances susceptibility of head and neck squamous cell carcinoma to 5-fluorouracil and cisplatin.

    PubMed

    Chan, Leong-Perng; Chou, Tzung-Han; Ding, Hsiou-Yu; Chen, Pin-Ru; Chiang, Feng-Yu; Kuo, Po-Lin; Liang, Chia-Hua

    2012-07-01

    Apigenin, a natural plant flavone, may have chemopreventive and therapeutic potentials for anti-inflammatory, antioxidant, and anti-cancer. Nevertheless, the anti-tumor effect of apigenin on human head and neck squamous cell carcinoma (HNSCC) is not fully understood. The antioxidant capacity and protective effects of apigenin against oxidative stress in murine normal embryonic liver BNLCL2 cells are examined. Cell viability, morphologic change, clonogenic survival, cell cycle distribution, reactive oxygen species (ROS) production, glutathione formation, and death receptors- and Bcl-2-mediated caspase pathways of HNSCC SCC25 cells and A431 cells with apigenin are investigated. Apigenin inhibits the growth of SCC25 and A431 cells and induces cell cycle arrest in the G2/M phase. Apigenin has an antioxidant capacity as well as the ability to inhibit lipid peroxidation. It protects BNLCL2 cells against oxidative damage, and is potentially able to prevent cancer. Apigenin increases intracellular ROS levels and reduces levels of glutathione; it also induces cell apoptosis via tumor necrosis factor receptor (TNF-R)-, TNF-related apoptosis-inducing ligand receptor (TRAIL-R)-, and Bcl-2-mediated caspase-dependent cell death pathways in SCC25 cells. The combination of apigenin with 5-fluorouracil (5-Fu) or cisplatin induces the dramatic death of SCC25 cells. Apigenin induces SCC25 cell apoptosis via the up-regulation of both TNF-R and TRAIL-R signaling pathways, and has a synergistic effect on the inhibition of cell proliferation in combination with 5-Fu or cisplatin. These analytical findings suggest that apigenin may be a good therapeutic agent against HNSCC cells. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Granzyme H induces cell death primarily via a Bcl-2-sensitive mitochondrial cell death pathway that does not require direct Bid activation.

    PubMed

    Ewen, Catherine L; Kane, Kevin P; Bleackley, R Chris

    2013-07-01

    Natural killer and T cell-mediated cytotoxicity is important for the elimination of viruses and transformed cells. The granule lytic pathway utilizes perforin and granzymes to induce cell death, while receptor-mediated lytic pathways rely on molecules such as FasL. Pro-apoptotic activities of Granzyme B (GrB) and Fas are well-established, and many of their cellular targets have been identified. However, humans express additional related granzymes - GrA, GrM, GrK, and GrH. Neither the cytotoxic potential of GrH, nor the mechanism by which GrH may induce target cell death is currently understood. We proposed that GrH would have pro-apoptotic activity that would be distinct from that of GrB and FasL, which could be relevant when Fas/FasL or GrB activity or death pathways were impaired. Our results, using a purified recombinant form of GrH, revealed that GrH induced cell death via a Bcl-2-sensitive mitochondrial pathway without direct processing of Bid. Additionally, neither the apoptosome nor caspase-3 was essential to the induction of GrH-mediated cell death. However, GrH did directly process DFF45, potentially leading to DNA damage. Our findings support the idea that multiple, non-redundant death pathways may be initiated by cytotoxic cells to counteract various immune evasion strategies.

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

    PubMed Central

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

    2017-01-01

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

  5. Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension.

    PubMed

    Kataoka, T; Holler, N; Micheau, O; Martinon, F; Tinel, A; Hofmann, K; Tschopp, J

    2001-06-01

    The Bcl-2 family of proteins plays a central regulatory role in apoptosis. We have identified a novel, widely expressed Bcl-2 member which we have named Bcl-rambo. Bcl-rambo shows overall structural homology to the anti-apoptotic Bcl-2 members containing conserved Bcl-2 homology (BH) motifs 1, 2, 3, and 4. Unlike Bcl-2, however, the C-terminal membrane anchor region is preceded by a unique 250 amino acid insertion containing two tandem repeats. No interaction of Bcl-rambo with either anti-apoptotic (Bcl-2, Bcl-x(L), Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) or pro-apoptotic (Bax, Bak, Bik, Bid, Bim, and Bad) members of the Bcl-2 family was observed. In mammalian cells, Bcl-rambo was localized to mitochondria, and its overexpression induces apoptosis that is specifically blocked by the caspase inhibitors, IAPs, whereas inhibitors controlling upstream events of either the 'death receptor' (FLIP, FADD-DN) or the 'mitochondrial' pro-apoptotic pathway (Bcl-x(L)) had no effect. Surprisingly, the Bcl-rambo cell death activity was induced by its membrane-anchored C-terminal domain and not by the Bcl-2 homology region. Thus, Bcl-rambo constitutes a novel type of pro-apoptotic Bcl-2 member that triggers cell death independently of its BH motifs.

  6. 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.

  7. Cold-Inducible Protein RBM3 Protects UV Irradiation-Induced Apoptosis in Neuroblastoma Cells by Affecting p38 and JNK Pathways and Bcl2 Family Proteins.

    PubMed

    Zhuang, Rui-Juan; Ma, Jian; Shi, Xiang; Ju, Fei; Ma, Shuang-Ping; Wang, Lei; Cheng, Bin-Feng; Ma, Yan-Wen; Wang, Mian; Li, Tong; Feng, Zhi-Wei; Yang, Hai-Jie

    2017-08-22

    Induced by hypothermia, cold-inducible protein RBM3 (RNA-binding protein motif 3), has been implicated in neuroprotection against various toxic insults such as hypoxia and ischemia. However, whether mild hypothermia and RBM3 prevent neural cells from UV irradiation-elicited apoptosis is unclear. In the present study, human neuroblastoma cell line SH-SY5Y was used as a cell model for neural cell death, and it was demonstrated that mild hypothermia protects SH-SY5Y cells from UV irradiation-induced apoptosis. However, the protective effect of mild hypothermia was abrogated when RBM3 was silenced. Conversely, the overexpression of RBM3 rescued SH-SY5Y cells from UV-induced apoptosis, as indicated by the decreased levels of cleaved caspase-3 and PARP, and increased cell survival. The analysis on the mechanism underlying RBM3-mediated neuroprotection against UV insult showed that RBM3 could substantially block the activation of p38 and JNK signaling pathways. In addition, the overexpression of RBM3 reduced the expression of pro-apoptotic proteins Bax and Bad, leaving the pro-survival protein Bcl-2 unaffected. In conclusion, RBM3 is the key mediator of mild hypothermia-related protection against UV in neuroblastoma cells, and the neuroprotective effect might be exerted through interfering with pro-apoptotic signaling pathways p38 and JNK and regulating pro-apoptotic proteins Bax and Bad.

  8. Apigenin induces caspase-dependent apoptosis in human lung cancer A549 cells through Bax- and Bcl-2-triggered mitochondrial pathway.

    PubMed

    Lu, Hsu-Feng; Chie, Yu-Jie; Yang, Ming-Sung; Lee, Ching-Sung; Fu, Jene-John; Yang, Jai-Sing; Tan, Tzu-Wei; Wu, Shin-Hwar; Ma, Yi-Shih; Ip, Siu-Wan; Chung, Jing-Gung

    2010-06-01

    The molecular mechanism and possible signaling pathway of apigenin-induced cytotoxicity and apoptosis in human lung cancer cells has not been reported. We investigated the role of ROS, Ca2+, caspases and Bax proteins and mitochondria membrane potential in apigenin-induced apoptosis in A549 cells. Cells were incubated with different concentrations of apigenin then cell morphological changes, DNA damage, cell viability and apoptosis were determined by Comet assay, and flow cytometric analysis. Sub-G1 phase was also examined. Western blot analysis was used to determined the levels of Bax and Bcl-2 and apoptosis associated proteins, and confocal laser microscope for examining the translocation of associated protein after exposed to apigenin. The results indicated that apigenin induced morphological changes, decreased percentage of viable cells and induced apoptosis dose- and time-dependently. DAPI staining and Comet assay also confirmed that apigenin-induced DNA condensation and damage. The levels of caspase-3, -8 and -9 involved in apigenin-induced apoptosis indicating caspase-dependent pathway was induced by apigenin. Western blotting showed that apigenin promoted cytochrome c levels and also induced dysfunction of mitochondria leading to the release of cytochrome c, AIF and Endo G, causing the activation of caspase-9 and -3, then apoptosis in A549 cells.

  9. 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

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

    PubMed

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

    2015-03-12

    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.

  11. 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

  12. Effect of ginsenoside Rh-2 via activation of caspase-3 and Bcl-2-insensitive pathway in ovarian cancer cells.

    PubMed

    Kim, Jin Hee; Choi, Jae-Sun

    2016-12-13

    Ginsenoside has been reported to have therapeutic effects for some types of cancer, but its effect on ovarian cancer cells has not been evaluated. In this study, we monitored the effects of ginsenoside-Rh2 (Rh2) on the inhibition of cell proliferation and the apoptotic process in the ovarian cancer cell line SKOV3 using an MTT assay and TUNEL assay. We found that Rh2 inhibited cell proliferation and significantly induced apoptosis. We confirmed the apoptotic effects of Rh2 using western blot analysis of apoptosis-related proteins. Specifically, the levels of cleaved poly ADP ribose polymerase (PARP) and cleaved caspase-3 significantly increased in SKOV3 cells treated with Rh2. Therefore, Rh2 clearly suppressed the growth of SKOV3 cells in vitro, which was associated with induction of the apoptosis pathway. Moreover, the migration assay showed that Rh2 inhibited the invasive ability of SKOV3 cells. Taken together, our results suggest that Rh2 has anticancer effects in SKOV3 cells through inhibition of cell proliferation and induction of apoptosis. Considering the therapeutic potential of Rh2, more studies should be carried out to facilitate the future application of this natural product as a potential anti-cancer agent.

  13. 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

  14. 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.

  15. MicroRNA-497 Induces Apoptosis and Suppresses Proliferation via the Bcl-2/Bax-Caspase9-Caspase3 Pathway and Cyclin D2 Protein in HUVECs

    PubMed Central

    Wang, Mian; Xu, Xiangdong; Yao, Chen; Wang, Shenming

    2016-01-01

    Introduction MicroRNAs play crucial roles in various types of diseases. However, to date, no information about the role of miR-497 in the development of atherosclerosis has been reported. This study investigated the possible role of miR-497 in vascular endothelial cell injury during the early stage of atherosclerosis. Materials and Methods The expression level of miR-497 in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL was detected using qRT-PCR. To perform gain of function and loss of function analyses, miR-497 mimics were transfected into HUVECs, and miR-497 inhibitors were transfected into HUVECs stimulated with ox-LDL. Flow cytometry was used to analyze cell cycle progression and apoptosis. EdU and CCK-8 assays were employed to detect DNA synthesis and cell proliferation, respectively. After bioinformatics prediction, a dual Luciferase Reporter assay was used to analyze the direct target genes of miR-497. The mRNA and protein levels of the target genes were detected using qRT-PCR and western blot analyses, respectively. Caspase-9/3 activity was analyzed to determine the mechanism of endothelial dysfunction. Results We showed that miR-497 was significantly upregulated in HUVECs stimulated with ox-LDL. Ectopic expression of miR-497 suppressed cell proliferation, induced apoptosis and increased the activity of caspase-9/3. After verification, Bcl2 and CCND2 were shown to be direct target genes of miR-497 in HUVECs. MiR-497 significantly suppressed cell proliferation by arresting the cell cycle through the CCND2 protein and induced apoptosis through the Bcl2/Bax-caspase9-caspase3 pathway. Conclusion Overall, our study shows that miR-497 might play a role in the development of atherosclerosis by inducing apoptosis and suppressing the proliferation of vascular endothelial cells. Therefore, miR-497 could be a potential therapeutic target for the treatment of atherosclerosis. PMID:27918592

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

    PubMed Central

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

    2011-01-01

    A significant subset of gliomas arises after activation of the pro-proliferative platelet-derived growth factor (PDGF) pathway. The progression of low-grade gliomas to more malignant tumors may be due to oncogenic cellular programs combining with those suppressing apoptosis. Anti-apoptotic genes are overexpressed in a variety of cancers and the anti-apoptotic gene, BCL2, is associated with treatment resistance and tumor recurrence in gliomas. However, the impact of anti-apoptotic gene expression to tumor formation and progression is unclear. We overexpressed Bcl-2 in a PDGFB-dependent mouse model of oligodendroglioma, a common glioma subtype, to assess its effect in vivo. We hypothesized that the anti-apoptotic effect would complement the pro-proliferative effect of PDGFB to promote tumor formation and progression to anaplastic oligodendroglioma (AO). Here, we show that co-expression of PDGFB and Bcl-2 results in a higher overall tumor formation rate compared to PDGFB alone. Co-expression of PDGFB and Bcl-2 promotes progression to AO with prominent foci of necrosis, a feature of high-grade gliomas. Median tumor latency was shorter in mice injected with PDGFB and Bcl-2 compared to those injected with PDGFB alone. Although independent expression of Bcl-2 was insufficient to induce tumors, suppression of apoptosis (detected by cleaved caspase-3 expression) was more pronounced in AOs induced by PDGFB and Bcl-2 compared to those induced by PDGFB alone. Tumor cell proliferation (detected by phosphohistone H3 activity) was also more robust in high-grade tumors induced by PDGFB and Bcl-2. Our results indicate that suppressed apoptosis enhances oligodendroglioma formation and engenders a more malignant phenotype. PMID:21171016

  17. Ru(II)/diphenylphosphine/pyridine-6-thiolate complexes induce S-180 cell apoptosis through intrinsic mitochondrial pathway involving inhibition of Bcl-2 and p53/Bax activation.

    PubMed

    Pires, Wanessa Carvalho; Lima, Benedicto Augusto Vieira; de Castro Pereira, Flávia; Lima, Aliny Pereira; Mello-Andrade, Francyelli; Silva, Hugo Delleon; da Silva, Monize Martins; Colina-Vegas, Legna; Ellena, Javier; Batista, Alzir A; de Paul Silveira-Lacerda, Elisângela

    2017-08-09

    The aim of this work was the synthesis, characterization, and cytotoxicity evaluation of three new Ru(II) complexes with a general formula [Ru(Spy)(bipy)(P-P)]PF6 [Spy = pyridine-6-thiolate; bipy = 2,2'-bipyridine; P-P = 1,2-bis(diphenylphosphine)ethane (1); 1,3-bis(diphenylphosphine) propane (2); and 1,1'-bis(diphenylphosphino)ferrocene] (4). Complex (3) with the 1,4-bis(diphenylphosphine)butane ligand, already known from the literature, was also synthesized, to be better studied here. The cytotoxicities of the complexes toward two kinds of cancerous cells (K562 and S-180 cells) were evaluated and compared to normal cells (L-929 and PBMC) by MTT assay. The complex [Ru(Spy)(bipy)(dppb)]PF6 (3) was selected to study both the cellular and molecular mechanisms underlying its promising anticancer action in S-180 cells. The results obtained from this study indicated that complex (3) induces cell cycle arrest in the G0/G1 phase in S-180 cells associated with a decrease in the number of cells in S phase. After 24 and 48 h of exposure to complex (3), the cell viability decreased when compared to the negative control. Complex (3) does not appear to be involved in the DNA damage, but induced changes in the mitochondrial membrane potential in S-180 cells. Furthermore, there was also an increase in the gene expression of Bax, Caspase 9, and Tp53. According to our results, complex (3) induces cell apoptosis through p53/Bax-dependent intrinsic pathway and suppresses the expression of active antiapoptotic Bcl-2 protein.

  18. Bcl-2 delays cell cycle through mitochondrial ATP and ROS.

    PubMed

    Du, Xing; Fu, Xufeng; Yao, Kun; Lan, Zhenwei; Xu, Hui; Cui, Qinghua; Yang, Elizabeth

    2017-02-22

    Bcl-2 inhibits cell proliferation by delaying G0/G1 to S phase entry. We tested the hypothesis that Bcl-2 regulates S phase entry through mitochondrial pathways. Existing evidence indicates mitochondrial adenosine tri-phosphate (ATP) and reactive oxygen species (ROS) are important signals in cell survival and cell death, however, the molecular details of how these 2 processes are linked remain unknown. In this study, 2 cell lines stably expressing Bcl-2, 3T3Bcl-2 and C3HBcl-2, and vector-alone PB controls were arrested in G0/G1 phase by serum starvation and contact inhibition, and ATP and ROS were measured during re-stimulation of cell cycle entry. Both ATP and ROS levels were decreased in G0/G1 arrested cells compared with normal growing cells. In addition, ROS levels were significant lower in synchronized Bcl-2 cells than those in PB controls. After re-stimulation, ATP levels increased with time, reaching peak value 1-3 hours ahead of S phase entry for both Bcl-2 cells and PB controls. Consistent with 2 hours of S phase delay, Bcl-2 cells reached ATP peaks 2 hours later than PB control, which suggests a rise in ATP levels is required for S phase entry. To examine the role of ATP and ROS in cell cycle regulation, ATP and ROS level were changed. We observed that elevation of ATP accelerated cell cycle progression in both PB and Bcl-2 cells, and decrease of ATP and ROS to the level equivalent to Bcl-2 cells delayed S phase entry in PB cells. Our results support the hypothesis that Bcl-2 protein regulates mitochondrial metabolism to produce less ATP and ROS, which contributes to S phase entry delay in Bcl-2 cells. These findings reveal a novel mechanistic basis for understanding the link between mitochondrial metabolism and tumor-suppressive function of Bcl-2.

  19. 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

  20. Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival.

    PubMed

    Lossi, Laura; Gambino, Graziana; Ferrini, Francesco; Alasia, Silvia; Merighi, Adalberto

    2009-11-01

    Apoptosis can be modulated by K(+) and Ca(2+) inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca(2+) signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K(+)/Ca(2+) homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K(+)](e) and [Ca(2+)](i) to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K(+)](e) under conditions of immaturity, is independent of extracellular Ca(2+) and operates via IP3 channels. The second leads to influx of extracellular Ca(2+) following activation of ryanodine channels in the presence of physiological [K(+)](e), when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2.

  1. The miR-573/apoM/Bcl2A1-dependent signal transduction pathway is essential for hepatocyte apoptosis and hepatocarcinogenesis.

    PubMed

    Hu, Yan-Wei; Chen, Zhi-Ping; Hu, Xiu-Mei; Zhao, Jia-Yi; Huang, Jin-Lan; Ma, Xin; Li, Shu-Fen; Qiu, Yu-Rong; Wu, Xiao-Juan; Sha, Yan-Hua; Gao, Ji-Juan; Wang, Yan-Chao; Zheng, Lei; Wang, Qian

    2015-10-01

    Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with an increasing incidence worldwide. Apolipoprotein M (apoM) is a novel apolipoprotein that is mainly expressed in liver and kidney tissues. However, the anti-tumor properties of apoM remain largely unknown. We evaluated the anti-tumor activities and mechanisms of apoM in HCC both in vivo and in vitro. Bioinformatic analysis and luciferase reporter assay results showed that apoM was a potential target of hsa-miR-573 and was downregulated after transfection with hsa-miR-573 mimics. Overexpression of apoM suppressed migration, invasion, and proliferation of hepatoma cells in vitro. Overexpression of hsa-miR-573 in hepatoma cells reduced apoM expression, leading to promotion of the invasion, migration, and proliferation of hepatoma cells in vitro. In addition, hsa-miR-573 markedly promoted growth of xenograft tumors in nude mice with an accompanying reduction in cell apoptosis. ApoM markedly inhibited growth of xenograft tumors in nude mice and promoted cell apoptosis. Moreover, Bcl2A1 mRNA and protein levels were inhibited by apoM overexpression and an increase in apoptosis rate by apoM was markedly compensated by Bcl2A1 overexpression in HepG2 cells. These results provide evidence that hsa-miR-573 promoted tumor growth by inhibition of hepatocyte apoptosis and this pro-tumor effect might be mediated through Bcl2A1 in an apoM-dependent manner. Therefore, our findings may be useful to improve understanding of the critical effects of hsa-miR-573 and apoM in HCC pathogenesis.

  2. Cyclophilin D interacts with Bcl2 and exerts an anti-apoptotic effect.

    PubMed

    Eliseev, Roman A; Malecki, Jonathan; Lester, Tobias; Zhang, Yu; Humphrey, John; Gunter, Thomas E

    2009-04-10

    Cyclophilin D (CypD) is a mitochondrial immunophilin and a key positive regulator of the mitochondrial permeability transition (MPT). Several reports have shown that CypD is overexpressed in various tumors, where it has an anti-apoptotic effect. Because the MPT is a cell death-inducing phenomenon, we hypothesized that the anti-apoptotic effect of CypD is independent of the MPT but is due to its interaction with some key apoptosis regulator, such as Bcl2. Our data indicate that CypD indeed interacts with Bcl2 as confirmed with co-immunoprecipitation, pulldown, and mammalian two-hybrid assays. A cyclophilin D inhibitor, cyclosporine A, disrupts the CypD-Bcl2 interaction. CypD enhances the limiting effect of Bcl2 on the tBid-induced release of cytochrome c from mitochondria, which is not mediated via the MPT. Gain- and loss-of-function experiments confirm that CypD has a limiting effect on cytochrome c release from mitochondria and that such an effect of CypD is cyclosporine A- and Bcl2-dependent. On a cellular level, overexpression or knockdown of CypD respectively decreases or increases cytochrome c release from mitochondria and overall cell sensitivity to apoptosis progressing via the "intrinsic" pathway. Therefore, we here describe a novel function of CypD as a Bcl2 collaborator and an inhibitor of cytochrome c release from mitochondria independent of the MPT. This function of CypD may explain the anti-apoptotic effect of this protein observed in various cancer cells. The fact that some tumors overexpress CypD suggests that this may be an additional mechanism of suppression of apoptosis in cancer.

  3. Hyperoside induces apoptosis and inhibits growth in pancreatic cancer via Bcl-2 family and NF-κB signaling pathway both in vitro and in vivo.

    PubMed

    Li, Yilong; Wang, Yongwei; Li, Le; Kong, Rui; Pan, Shangha; Ji, Liang; Liu, Huan; Chen, Hua; Sun, Bei

    2016-06-01

    Although advanced surgical operation and chemotherapy have been under taken, pancreatic cancer remains one of the most aggressive and fatal human malignancies with a low 5-year survival rate of less than 5 %. Therefore, novel therapeutic strategies for prevention and remedy are urgently needed in pancreatic cancer. This present research aimed to investigate the anti-cancer effects of hyperoside in human pancreatic cancer cells. Our in vitro results showed that hyperoside suppressed the proliferation and promoted apoptosis of two different human pancreatic cancer cell lines, which correlated with up-regulation of the ratios of Bax/Bcl-2 and Bcl-xL and down-regulation of levels of nuclear factor-κB (NF-κB) and NF-κB's downstream gene products. What's more, using an orthotopic model of human pancreatic cancer, we found that hyperoside also inhibited the tumor growth significantly. Mechanically, these outcomes could also be associated with the up-regulation of the ratios of Bax/Bcl-2 and Bcl-xL and down-regulation of levels of NF-κB and NF-κB's downstream gene products. Collectively, our experiments indicate that hyperoside may be a promising candidate agent for the treatment of pancreatic cancer.

  4. Targeting the Bcl-2 Family for Cancer Therapy

    PubMed Central

    Thomas, Shibu; Quinn, Bridget A.; Das, Swadesh K.; Dash, Rupesh; Emdad, Luni; Dasgupta, Santanu; Wang, Xiang-Yang; Dent, Paul; Reed, John C.; Pellecchia, Maurizio; Sarkar, Devanand; Fisher, Paul B.

    2014-01-01

    Introduction Programmed cell death is well-orchestrated process regulated by multiple pro-apoptotic and anti-apoptotic genes, particularly those of the Bcl-2 gene family. These genes are well documented in cancer with aberrant expression being strongly associated with resistance to chemotherapy and radiation. Areas covered This review focuses on the resistance induced by the Bcl-2 family of anti-apoptotic proteins and current therapeutic interventions currently in preclinical or clinical trials that target this pathway. Major resistance mechanisms that are regulated by Bcl-2 family proteins and potential strategies to circumvent resistance are also examined. Although antisense and gene therapy strategies are used to nullify Bcl-2 family proteins, recent approaches use small molecule inhibitors and peptides. Structural similarity of the Bcl-2 family of proteins greatly favors development of inhibitors that target the BH3 domain, called BH3 mimetics. Expert opinion Strategies to specifically identify and inhibit critical determinants that promote therapy-resistance and tumor progression represent viable approaches for developing effective cancer therapies. From a clinical perspective, pretreatment with novel, potent Bcl-2 inhibitors either alone or in combination with conventional therapies hold significant promise for providing beneficial clinical outcomes. Identifying small molecule inhibitors with broader and higher affinities for inhibiting all of the Bcl-2 pro-survival proteins will facilitate development of superior cancer therapies. PMID:23173842

  5. Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway

    PubMed Central

    Xie, Zhengyuan; Xiao, Zhihua; Wang, Fenfen

    2017-01-01

    The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor (TNF)-α. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of NF-κB and miR-503. We found that overexpression of NS5A inhibited TNF-αinduced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the TNF-α induced Hep-mock cells was significantly less than the viability of the TNF-α induced Hep-NS5A cells, which demonstrates that NS5A inhibited TNF-α-induced HepG2 cell apoptosis. Under TNF-α treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited TNF-α-induced NF-κB activation and NF-κB regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse TNF-α-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits NF-κB activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis. PMID:28343379

  6. Clinical response after two cycles compared to HER2, Ki-67, p53, and bcl-2 in independently predicting a pathological complete response after preoperative chemotherapy in patients with operable carcinoma of the breast

    PubMed Central

    von Minckwitz, Gunter; Sinn, Hans-Peter; Raab, Günter; Loibl, Sibylle; Blohmer, Jens-Uwe; Eidtmann, Holger; Hilfrich, Jörn; Merkle, Elisabeth; Jackisch, Christian; Costa, Serban D; Caputo, Angelika; Kaufmann, Manfred

    2008-01-01

    Introduction To investigate the predictive value of clinical and biological markers for a pathological complete remission after a preoperative dose-dense regimen of doxorubicin and docetaxel, with or without tamoxifen, in primary operable breast cancer. Methods Patients with a histologically confirmed diagnosis of previously untreated, operable, and measurable primary breast cancer (tumour (T), nodes (N) and metastases (M) score: T2-3(≥ 3 cm) N0-2 M0) were treated in a prospectively randomised trial with four cycles of dose-dense (bi-weekly) doxorubicin and docetaxel (ddAT) chemotherapy, with or without tamoxifen, prior to surgery. Clinical and pathological parameters (menopausal status, clinical tumour size and nodal status, grade, and clinical response after two cycles) and a panel of biomarkers (oestrogen and progesterone receptors, Ki-67, human epidermal growth factor receptor 2 (HER2), p53, bcl-2, all detected by immunohistochemistry) were correlated with the detection of a pathological complete response (pCR). Results A pCR was observed in 9.7% in 248 patients randomised in the study and in 8.6% in the subset of 196 patients with available tumour tissue. Clinically negative axillary lymph nodes, poor tumour differentiation, negative oestrogen receptor status, negative progesterone receptor status, and loss of bcl-2 were significantly predictive for a pCR in a univariate logistic regression model, whereas in a multivariate analysis only the clinical nodal status and hormonal receptor status provided significantly independent information. Backward stepwise logistic regression revealed a response after two cycles, with hormone receptor status and lymph-node status as significant predictors. Patients with a low percentage of cells stained positive for Ki-67 showed a better response when treated with tamoxifen, whereas patients with a high percentage of Ki-67 positive cells did not have an additional benefit when treated with tamoxifen. Tumours overexpressing

  7. Sphallerocarpus gracilis polysaccharide protects pancreatic β-cells via regulation of the bax/bcl-2, caspase-3, pdx-1 and insulin signalling pathways.

    PubMed

    Guo, Jie; Wang, Junlong; Song, Shen; Liu, Qin; Huang, Yulong; Xu, Yunfei; Wei, YanXia; Zhang, Ji

    2016-12-01

    In this study, the structural characterization of Sphallerocarpus gracilis polysaccharide (SGP) and its hypoglycaemic activities are reported for the first time. SGP, which has a weight average molar mass (Mw) of 7.413×10(5), was isolated from Sphallerocarpus gracilis and purified by ion-exchange chromatography. The polysaccharide is composed of rhamnose, arabinose, mannose, glucose and galactose, with the molar ratio of 4.12: 8.99: 5.45: 65.94: 15.50. The mechanism underlying the hypoglycaemic effect of SGP was evaluated. Experimental results showed that SGP protected pancreatic β-cells from alloxan damage by several possible mechanisms, including: (1) repairing free radical damage; (2) reducing the apoptosis of pancreatic β-cells by inhibiting the activities of caspase-3 and bax, and enhancing the activity of bcl-2; (3) stimulating insulin secretion and upregulating the pancreatic and duodenal homeobox 1 gene and the insulin gene and the pancreatic in pancreatic β-cells. The results obtained in this study suggest that SGP may be a promising therapeutic agent in the treatment of diabetes mellitus.

  8. NHE9 induces chemoradiotherapy resistance in esophageal squamous cell carcinoma by upregulating the Src/Akt/β-catenin pathway and Bcl-2 expression

    PubMed Central

    Chen, Junying; Yang, Hong; Wen, Jing; Luo, Kongjia; Liu, Qianwen; Huang, Yijie; Zheng, Yuzhen; Tan, Zihui; Qingyuan Huang, Qinyuan; Fu, Jianhua

    2015-01-01

    Recently, we found that NHE9 mRNA was upregulated in chemoradiotherapy (CRT)-resistant esophageal squamous cell carcinoma (ESCC); however, the underlying mechanisms were unclear. Here, we aimed to clarify the functional contribution of NHE9 to CRT resistance, understand the molecular basis of NHE9-dependent resistance in ESCC, and identify potential therapeutic targets. Our results showed that NHE9 prevented CRT-induced apoptosis. Importantly, we found that RACK1 is a novel binding partner of NHE9 and that NHE9-dependent induction of CRT resistance requires the activation of RACK1-associated Src/Akt/β-catenin signaling. Moreover, upregulated Bcl-2 protein was also observed in cells exhibiting NHE9-induced CRT resistance. A higher NHE9 level was associated with a poor response to CRT and less decrease in T and N stage in ESCC patients. Furthermore, combining either Dasatinib or ABT-737 with CRT significantly reduced tumor volume, and the response to CRT was restored when these inhibitors were used together with CRT in a xenograft nude mouse model with NHE9 overexpression. Taken together, our findings demonstrate that NHE9 can be an effective predictor of CRT response and may be useful in the development of targeted therapies for CRT-resistant ESCC. PMID:25915159

  9. BET Inhibition Induces Apoptosis in Aggressive B-Cell Lymphoma via Epigenetic Regulation of BCL-2 Family Members.

    PubMed

    Hogg, Simon J; Newbold, Andrea; Vervoort, Stephin J; Cluse, Leonie A; Martin, Benjamin P; Gregory, Gareth P; Lefebure, Marcus; Vidacs, Eva; Tothill, Richard W; Bradner, James E; Shortt, Jake; Johnstone, Ricky W

    2016-09-01

    Targeting BET bromodomain proteins using small molecules is an emerging anticancer strategy with clinical evaluation of at least six inhibitors now underway. Although MYC downregulation was initially proposed as a key mechanistic property of BET inhibitors, recent evidence suggests that additional antitumor activities are important. Using the Eμ-Myc model of B-cell lymphoma, we demonstrate that BET inhibition with JQ1 is a potent inducer of p53-independent apoptosis that occurs in the absence of effects on Myc gene expression. JQ1 skews the expression of proapoptotic (Bim) and antiapoptotic (BCL-2/BCL-xL) BCL-2 family members to directly engage the mitochondrial apoptotic pathway. Consistent with this, Bim knockout or Bcl-2 overexpression inhibited apoptosis induction by JQ1. We identified lymphomas that were either intrinsically resistant to JQ1-mediated death or acquired resistance following in vivo exposure. Strikingly, in both instances BCL-2 was strongly upregulated and was concomitant with activation of RAS pathways. Eμ-Myc lymphomas engineered to express activated Nras upregulated BCL-2 and acquired a JQ1 resistance phenotype. These studies provide important information on mechanisms of apoptosis induction and resistance to BET-inhibition, while providing further rationale for the translation of BET inhibitors in aggressive B-cell lymphomas. Mol Cancer Ther; 15(9); 2030-41. ©2016 AACR.

  10. 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.

  11. 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

  12. 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

  13. Intranasal Administration of Interferon Beta Attenuates Neuronal Apoptosis via the JAK1/STAT3/BCL-2 Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy

    PubMed Central

    Dixon, Brandon J.; Chen, Di; Zhang, Yang; Flores, Jerry; Malaguit, Jay; Nowrangi, Derek; Zhang, John H.

    2016-01-01

    Neonatal hypoxic-ischemic encephalopathy (HIE) is an injury that often leads to detrimental neurological deficits. Currently, there are no established therapies for HIE and it is critical to develop treatments that provide protection after HIE. The objective of this study was to investigate the ability of interferon beta (IFNβ) to provide neuroprotection and reduce apoptosis after HIE. Postnatal Day 10 rat pups were subjected to unilateral carotid artery ligation followed by 2.5 hr of exposure to hypoxia (8% O2). Intranasal administration of human recombinant IFNβ occurred 2 hr after HIE and infarct volume, body weight, neurobehavioral tests, histology, immunohistochemistry, brain water content, blood–brain barrier permeability, enzyme-linked immunosorbent assay, and Western blot were all used to evaluate various parameters. The results showed that both IFNβ and the Type 1 interferon receptor expression decreases after HIE. Intranasal administration of human recombinant IFNβ was able to be detected in the central nervous system and was able to reduce brain infarction volumes and improve neurological behavior tests 24 hr after HIE. Western blot analysis also revealed that human recombinant IFNβ treatment stimulated Stat3 and Bcl-2 expression leading to a decrease in cleaved caspase-3 expression after HIE. Positive Fluoro-Jade C staining also demonstrated that IFNβ treatment was able to decrease neuronal apoptosis. Furthermore, the beneficial effects of IFNβ treatment were reversed when a Stat3 inhibitor was applied. Also an intraperitoneal administration of human recombinant IFNβ into the systemic compartment was unable to confer the same protective effects as intranasal IFNβ treatment. PMID:27683877

  14. Bcl-2–Mediated Drug Resistance

    PubMed Central

    Srivastava, Rakesh K.; Sasaki, Carl Y.; Hardwick, J. Marie; Longo, Dan L.

    1999-01-01

    Bcl-2 inhibits apoptosis induced by a variety of stimuli, including chemotherapy drugs and glucocorticoids. It is generally accepted that Bcl-2 exerts its antiapoptotic effects mainly by dimerizing with proapoptotic members of the Bcl-2 family such as Bax and Bad. However, the mechanism of the antiapoptotic effects is unclear. Paclitaxel and other drugs that disturb microtubule dynamics kill cells in a Fas/Fas ligand (FasL)-dependent manner; antibody to FasL inhibits paclitaxel-induced apoptosis. We have found that Bcl-2 overexpression leads to the prevention of chemotherapy (paclitaxel)-induced expression of FasL and blocks paclitaxel-induced apoptosis. The mechanism of this effect is that Bcl-2 prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes, a transcription factor activated by microtubule damage) by binding and sequestering calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT to move to the nucleus. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, it appears that paclitaxel and other drugs that disturb microtubule function kill cells at least in part through the induction of FasL. Furthermore, Bcl-2 antagonizes drug-induced apoptosis by inhibiting calcineurin activation, blocking NFAT nuclear translocation, and preventing FasL expression. The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation. PMID:10432288

  15. 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

  16. Structural insights into the transcription-independent apoptotic pathway of p53.

    PubMed

    Chi, Seung-Wook

    2014-03-01

    Reactivating the p53 pathway in tumors is an important strategy for anticancer therapy. In response to diverse cellular stresses, the tumor suppressor p53 mediates apoptosis in a transcription-independent and transcription-dependent manner. Although extensive studies have focused on the transcription-dependent apoptotic pathway of p53, the transcription-independent apoptotic pathway of p53 has only recently been discovered. Molecular interactions between p53 and Bcl-2 family proteins in the mitochondria play an essential role in the transcription-independent apoptosis of p53. This review describes the structural basis for the transcription-independent apoptotic pathway of p53 and discusses its potential application to anticancer therapy.

  17. Bcl-2 expression is a poor predictor for hepatocellular carcinoma prognosis of andropause-age patients

    PubMed Central

    Zhang, Xiao-Fei; Yang, Xin; Jia, Hu-Liang; Zhu, Wen-Wei; Lu, Lu; Shi, Wei; Zhang, Hao; Chen, Jin-Hong; Tao, Yi-Feng; Wang, Zheng-Xin; Yang, Jun; Wang, Lian-Xin; Lu, Ming; Zheng, Yan; Zhao, Jing; Dong, Qiong-Zhu; Qin, Lun-Xiu

    2016-01-01

    Objective: The expression of B-cell lymphoma 2 (Bcl-2) seems to be influenced by the endocrine environment. Numerous reports demonstrate the diverse expression of Bcl-2 family members under sex steroid regulation. With the exception of estrogen-related tumors, androgen-related tumors have shown their characteristics in Bcl-2 expression. In this study, the status of Bcl-2 expression in male hepatocellular carcinoma (HCC) patients was examined to verify the high incidence of HCC in males. Methods: Tumor tissue microarray was used to examine Bcl-2 expression levels in 374 HCC cases including 306 males and 68 females. Kaplan-Meier method, log-rank test, and Cox proportional hazards model were applied to investigate the predictive value of Bcl-2 in HCC patients. Results: Immunohistochemistry analysis showed that male patients with higher Bcl-2 levels had significantly longer median survival time and recurrence time than those with lower levels. However, no significant differences in outcomes were found between different Bcl-2 levels in female patients. When the male patients were stratified into several age points, the level of Bcl-2 expression showed poorer predictive efficiency in the 45–49 and 55–60 age groups in andropause-age patients compared with other age groups. Bcl-2 was an independent prognostic factor for both overall survival (P < 0.0001) and recurrence time (P = 0.0001) in male patients. After excluding male patients in the 45–60 age group, the predictive efficiency was enhanced (n = 147, OS, P = 0.0002, TTR, P < 0.0001). Conclusions: Bcl-2 expression is an independent predictor of survival and recurrence in male HCC. Bcl-2 levels may also be regulated by androgens or androgen receptors in male HCC patients. Bcl-2 levels change and exhibit poor predictive efficiency when androgen levels vary dramatically (andropause age). PMID:28154777

  18. Berberine protects against 6-OHDA-induced neurotoxicity in PC12 cells and zebrafish through hormetic mechanisms involving PI3K/AKT/Bcl-2 and Nrf2/HO-1 pathways.

    PubMed

    Zhang, Chao; Li, Chuwen; Chen, Shenghui; Li, Zhiping; Jia, Xuejing; Wang, Kai; Bao, Jiaolin; Liang, Yeer; Wang, Xiaotong; Chen, Meiwan; Li, Peng; Su, Huanxing; Wan, Jian-Bo; Lee, Simon Ming Yuen; Liu, Kechun; He, Chengwei

    2017-04-01

    Berberine (BBR) is a renowned natural compound that exhibits potent neuroprotective activities. However, the cellular and molecular mechanisms are still unclear. Hormesis is an adaptive mechanism generally activated by mild oxidative stress to protect the cells from further damage. Many phytochemicals have been shown to induce hormesis. This study aims to investigate whether the neuroprotective activity of BBR is mediated by hormesis and the related signaling pathways in 6-OHDA-induced PC12 cells and zebrafish neurotoxic models. Our results demonstrated that BBR induced a typical hormetic response in PC12 cells, i.e. low dose BBR significantly increased the cell viability, while high dose BBR inhibited the cell viability. Moreover, low dose BBR protected the PC12 cells from 6-OHDA-induced cytotoxicity and apoptosis, whereas relatively high dose BBR did not show neuroprotective activity. The hormetic and neuroprotective effects of BBR were confirmed to be mediated by up-regulated PI3K/AKT/Bcl-2 cell survival and Nrf2/HO-1 antioxidative signaling pathways. In addition, low dose BBR markedly mitigated the 6-OHDA-induced dopaminergic neuron loss and behavior movement deficiency in zebrafish, while high dose BBR only slightly exhibited neuroprotective activities. These results strongly suggested that the neuroprotection of BBR were attributable to the hormetic mechanisms via activating cell survival and antioxidative signaling pathways. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Evaluation of NF-κB subunit expression and signaling pathway activation demonstrates that p52 expression confers better outcome in germinal center B-cell-like diffuse large B-cell lymphoma in association with CD30 and BCL2 functions.

    PubMed

    Ok, Chi Young; Xu-Monette, Zijun Y; Li, Ling; Manyam, Ganiraju C; Montes-Moreno, Santiago; Tzankov, Alexandar; Visco, Carlo; Dybkær, Karen; Routbort, Mark J; Zhang, Li; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L; Hsi, Eric D; Choi, William W L; van Krieken, J Han; Huh, Jooryung; Ponzoni, Maurilio; Ferreri, Andrés J M; Parsons, Ben M; Rao, Huilan; Møller, Michael B; Winter, Jane N; Piris, Miguel A; Wang, Sa A; Medeiros, L Jeffrey; Young, Ken H

    2015-09-01

    Nuclear factor-κB (NF-κB) is a transcription factor with a well-described oncogenic role. Study for each of five NF-κB pathway subunits was only reported on small cohorts in diffuse large B-cell lymphoma (DLBCL). In this large cohort (n=533) of patients with de novo DLBCL, we evaluated the protein expression frequency, gene expression signature, and clinical implication for each of these five NF-κB subunits. Expression of p50, p52, p65, RELB, and c-Rel was 34%, 12%, 20%, 14%, and 23%, whereas p50/p65, p50/c-Rel, and p52/RELB expression was 11%, 11%, and 3%, respectively. NF-κB subunits were expressed in both germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL, but p50 and p50/c-Rel were associated with ABC-DLBCL. p52, RELB, and p52/RELB expressions were associated with CD30 expression. p52 expression was negatively associated with BCL2 (B-cell lymphoma 2) expression and BCL2 rearrangement. Although p52 expression was associated with better progression-free survival (PFS) (P=0.0170), singular expression of the remaining NF-κB subunits alone did not show significant prognostic impact in the overall DLBCL cohort. Expression of p52/RELB was associated with better overall survival (OS) and PFS (P=0.0307 and P=0.0247). When cases were stratified into GCB- and ABC-DLBCL, p52 or p52/RELB dimer expression status was associated with better OS and PFS (P=0.0134 and P=0.0124) only within the GCB subtype. However, multivariate analysis did not show p52 expression to be an independent prognostic factor. Beneficial effect of p52 in GCB-DLBC appears to be its positive correlation with CD30 and negative correlation with BCL2 expression. Gene expression profiling (GEP) showed that p52(+) GCB-DLBCL was distinct from p52(-) GCB-DLBCL. Collectively, our data suggest that DLBCL patients with p52 expression might not benefit from therapy targeting the NF-κB pathway.

  20. 17-DMAG Diminishes Hemorrhage-Induced Small Intestine Injury by Elevating Bcl-2 Protein and Inhibiting iNOS Pathway, TNF-alpha Increase, and Caspase-3 Activation

    DTIC Science & Technology

    2011-06-03

    Hemorrhagic shock has been shown to cause systemic inflammation response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), and multiple organ...available to tissues and results in an accumulation of carbon dioxide and metabolic waste , leading to activation of signal transduction pathways and...for each specimen was graded using a six-tiered scale [3]. Western blots. Jejunal tissue was minced in 100 µl Hanks’ balanced salt solution

  1. bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells.

    PubMed

    Delia, D; Aiello, A; Soligo, D; Fontanella, E; Melani, C; Pezzella, F; Pierotti, M A; Della Porta, G

    1992-03-01

    The present study provides immunobiochemical and molecular data on the differentiation-linked expression of the bcl-2 proto-oncogene in normal and neoplastic myeloid cells. Using a recently developed monoclonal antibody (MoAb) to the bcl-2 molecule, staining of normal bone marrow myeloblasts, promyelocytes, and myelocytes, but neither monocytes nor most polymorphonuclear cells, was demonstrated. By two-color flow cytometric analysis, bcl-2 was evidenced in CD33+ and CD33+/CD34+ myeloid cells as well as in the more primitive CD33-/CD34+ population. The leukemic cell lines HL-60, KG1, GM-1, and K562 were bcl-2 positive together with 11 of 14 acute myeloid leukemias (AML) and three of three chronic myeloid leukemias (CML) in blast crises; six of seven CML were negative. Among myelodysplastic cases, augmentation of the bcl-2 positive myeloblastic compartment was found in refractory anemia with excess of blasts (RAEB) and in transformation (RAEB-t). Western blots of myeloid leukemias and control lymphocytes extracts evidenced an anti-bcl-2 immunoreactive band of the expected size (26 Kd). Moreover, the HL-60 and KG1 cell lines, both positive for the bcl-2 protein, exhibited the appropriate size bcl-2 mRNA (7.5 Kb). These findings clearly indicate that the bcl-2 gene is operative in myeloid cells and that the anti-bcl-2 MoAb identifies its product and not a cross-reactive epitope. Induction of HL-60 differentiation toward the monocytic and granulocytic pathways was accompanied by a marked decrease in bcl-2 mRNA and protein levels; bivariate flow cytometric analysis showed that the fraction becoming bcl-2 negative was in the G1 phase of the cell cycle. These data establish that the bcl-2 proto-oncogene is expressed on myeloid cells and their progenitors and is regulated in a differentiation-linked manner.

  2. 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.

  3. BCL2L10 inhibits growth and metastasis of hepatocellular carcinoma both in vitro and in vivo.

    PubMed

    Bai, Yun; Wang, Jia; Han, Jing; Xie, Xiao-Li; Ji, Cheng-Guang; Yin, Jie; Chen, Lei; Wang, Cun-Kai; Jiang, Xiao-Yu; Qi, Wei; Jiang, Hui-Qing

    2017-03-01

    BCL2L10 is an apoptosis-related member of the BCL-2 protein family. The role of BCL2L10 in the pathogenesis of hepatocellular carcinoma (HCC) is poorly understood. This study was aimed to investigate the function and underlying mechanisms of BCL2L10 in HCC. BCL2L10 expression in human HCC and corresponding adjacent normal tissues was investigated by quantitative real-time PCR and Western blot. The biological functions of BCL2L10 in HCC cell lines were determined by cell viability, colony formation, cell apoptosis, cell cycle, and cell metastasis assays, and in vivo by tumorigenicity and lung metastasis assays in nude mice. Human cancer pathway PCR array was employed to explore the genes regulated by BCL2L10 in HCC. BCL2L10 was down-regulated in human HCC tissues compared to their adjacent non-tumor tissues. Ectopic expression of BCL2L10 in HepG2 and Huh7 cells suppressed cell growth as evidenced by cell viability and colony formation assay, and induced cell apoptosis. HCC cells transfected with BCL2L10 revealed an increased cell proportion arrested at G2/M phase, concomitant with a reduction in the cell proportion in S-phase as compared with control cells. Additional, BCL2L10 repressed cell migration and angiogenesis. Over-expression of BCL2L10 also restrained the tumorigenecity and lung metastasis capacity in nude mice. The activation of JAK-STAT3 signaling was suppressed by BCL2L10 in HCC. BCL2L10 was down-regulated in human HCC tissues compared to adjacent normal tissues. BCL2L10 suppressed HCC progression through inhibiting cell growth and metastasis. Thus, BCL2L10 functions as a tumor-suppressor in HCC. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Influence of p53 and bcl-2 on chemosensitivity in benign and malignant prostatic cell lines.

    PubMed

    Serafin, Antonio M; Bohm, Lothar

    2005-01-01

    The administration of cancer chemotherapeutic agents results in an increase in the apoptotic cells in the tumor: therefore, it has been assumed that anticancer drugs exhibit their cytotoxic effects via apoptotic signaling pathways. Characteristics that confer sensitivity to drug-induced apoptosis are, a functional p53 protein and expression of the apoptosis-promoting protein, bax. The role of p53 and bax/bcl-2 in drug-induced apoptosis was assessed in six prostate cell lines, 1532T, 1535T, 1542T, 1542N, BPH-1 and LNCaP using TD(50) concentrations of etoposide, vinblastine and estramustine. Cell death was monitored morphologically by fluorescent microscopy, and by flow cytometry (Annexin-V assay). Apoptotic morphology was rather low and ranged from 0.1% to 12.1%, 3.0% to 6.0% and 0.1% to 8.5% for etoposide, estramustine and vinblastine, respectively. Annexin-V binding and flow cytometry indicated apoptotic propensities of 0% to 4%, 0% to 3% and 0% to 5%, respectively. The percentage of cells responding to drug-induced apoptosis was, on average, higher in the tumor cell lines than in the normal cell lines, but showed no correlation with p53 status. The percentage of cells showing necrosis, assessed by Annexin binding and Propidium Iodide permeability in aqueous medium, tended to be much higher, and was found to be at the level of 5% to 30%. Immunoblotting demonstrated that bax and bcl-2 proteins were expressed at a basal level in all cell lines, but did not increase after exposure to TD(50) doses of the three drugs. The ratio of bax and bcl-2, measured by laser scanning densitometry, was not altered by the drug-induced DNA damage. The results suggest that apoptosis is not a major mechanism of drug-induced cell death in prostate cell lines and appears to be independent of p53 status and bax/bcl-2 expression.

  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. 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.

  7. 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.

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

    PubMed

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

    2015-01-01

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

  9. Yeast as a tool for studying proteins of the Bcl-2 family

    PubMed Central

    Polčic, Peter; Jaká, Petra; Mentel, Marek

    2015-01-01

    Permeabilization of the outer mitochondrial membrane that leads to the release of cytochrome c and several other apoptogenic proteins from mitochondria into cytosol represents a commitment point of apoptotic pathway in mammalian cells. This crucial event is governed by proteins of the Bcl-2 family. Molecular mechanisms, by which Bcl-2 family proteins permeabilize mitochondrial membrane, remain under dispute. Although yeast does not have apparent homologues of these proteins, when mammalian members of Bcl-2 family are expressed in yeast, they retain their activity, making yeast an attractive model system, in which to study their action. This review focuses on using yeast expressing mammalian proteins of the Bcl-2 family as a tool to investigate mechanisms, by which these proteins permeabilize mitochondrial membranes, mechanisms, by which pro- and antiapoptotic members of this family interact, and involvement of other cellular components in the regulation of programmed cell death by Bcl-2 family proteins. PMID:28357280

  10. Yeast as a tool for studying proteins of the Bcl-2 family.

    PubMed

    Polčic, Peter; Jaká, Petra; Mentel, Marek

    2015-03-02

    Permeabilization of the outer mitochondrial membrane that leads to the release of cytochrome c and several other apoptogenic proteins from mitochondria into cytosol represents a commitment point of apoptotic pathway in mammalian cells. This crucial event is governed by proteins of the Bcl-2 family. Molecular mechanisms, by which Bcl-2 family proteins permeabilize mitochondrial membrane, remain under dispute. Although yeast does not have apparent homologues of these proteins, when mammalian members of Bcl-2 family are expressed in yeast, they retain their activity, making yeast an attractive model system, in which to study their action. This review focuses on using yeast expressing mammalian proteins of the Bcl-2 family as a tool to investigate mechanisms, by which these proteins permeabilize mitochondrial membranes, mechanisms, by which pro- and antiapoptotic members of this family interact, and involvement of other cellular components in the regulation of programmed cell death by Bcl-2 family proteins.

  11. Structural biology of the Bcl-2 family and its mimicry by viral proteins

    PubMed Central

    Kvansakul, M; Hinds, M G

    2013-01-01

    lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways. PMID:24201808

  12. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak

    PubMed Central

    Lindqvist, Lisa M.; Heinlein, Melanie; Huang, David C. S.; Vaux, David L.

    2014-01-01

    Antiapoptotic B-cell lymphoma 2 (Bcl-2) family members such as Bcl-2, myeloid cell leukemia 1 (Mcl-1), and B-cell lymphoma-X large (Bcl-xL) are proposed to inhibit autophagy by directly binding to the BH3 domain of Beclin 1/Atg6. However, these Bcl-2 family proteins also block the proapoptotic activity of Bcl-2–associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), and many inducers of autophagy also cause cell death. Therefore, when the mitochondrial-mediated apoptosis pathway is functional, interpretation of such experiments is complicated. To directly test the impact of the endogenous antiapoptotic Bcl-2 family members on autophagy in the absence of apoptosis, we inhibited their activity in cells lacking the essential cell death mediators Bax and Bak. We also used inducible lentiviral vectors to overexpress Bcl-2, Bcl-xL, or Mcl-1 in cells and subjected them to treatments that promote autophagy. In the absence of Bax and Bak, Bcl-2, Bcl-xL, and Mcl-1 had no detectable effect on autophagy or cell death in myeloid or fibroblast cell lines. On the other hand, when Bax and Bak were present, inhibiting the prosurvival Bcl-2 family members stimulated autophagy, but this correlated with increased cell death. In addition, inhibition of autophagy induced by amino acid starvation, etoposide, or interleukin-3 withdrawal did not affect cell death in the absence of Bax and Bak. These results demonstrate that the antiapoptotic Bcl-2 family members do not directly inhibit components of the autophagic pathway but instead affect autophagy indirectly, owing to their inhibition of Bax and Bak. PMID:24912196

  13. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak.

    PubMed

    Lindqvist, Lisa M; Heinlein, Melanie; Huang, David C S; Vaux, David L

    2014-06-10

    Antiapoptotic B-cell lymphoma 2 (Bcl-2) family members such as Bcl-2, myeloid cell leukemia 1 (Mcl-1), and B-cell lymphoma-X large (Bcl-xL) are proposed to inhibit autophagy by directly binding to the BH3 domain of Beclin 1/Atg6. However, these Bcl-2 family proteins also block the proapoptotic activity of Bcl-2-associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), and many inducers of autophagy also cause cell death. Therefore, when the mitochondrial-mediated apoptosis pathway is functional, interpretation of such experiments is complicated. To directly test the impact of the endogenous antiapoptotic Bcl-2 family members on autophagy in the absence of apoptosis, we inhibited their activity in cells lacking the essential cell death mediators Bax and Bak. We also used inducible lentiviral vectors to overexpress Bcl-2, Bcl-xL, or Mcl-1 in cells and subjected them to treatments that promote autophagy. In the absence of Bax and Bak, Bcl-2, Bcl-xL, and Mcl-1 had no detectable effect on autophagy or cell death in myeloid or fibroblast cell lines. On the other hand, when Bax and Bak were present, inhibiting the prosurvival Bcl-2 family members stimulated autophagy, but this correlated with increased cell death. In addition, inhibition of autophagy induced by amino acid starvation, etoposide, or interleukin-3 withdrawal did not affect cell death in the absence of Bax and Bak. These results demonstrate that the antiapoptotic Bcl-2 family members do not directly inhibit components of the autophagic pathway but instead affect autophagy indirectly, owing to their inhibition of Bax and Bak.

  14. Expression of Bcl-2 in canine osteosarcoma

    PubMed Central

    Piro, F.; Leonardi, L.

    2015-01-01

    Osteosarcoma (OS) is the most common primary malignancy of bone. It is responsible for 80-85% of the primary bone tumors affecting dogs and it is characterized by aggressive and invasive behavior, with a high metastatic potential. Several studies on cancer and related tumorigenesis, show an involvement of the mechanisms of programmed cell death and cell survival. Many signals seem to be involved in the related mechanism of autophagy and in particular, our interest is focused on the expression of a family of Bcl-2 that seems to be involved either in the control of biomolecular mechanisms like autophagy and apoptosis. In this study we investigated the expression of Bcl-2 in different cases of spontaneous canine osteosarcoma and the related preliminary results are described. We found Bcl-2 activity was increased in OS tissue compared to normal bone tissue. These results suggested that Bcl-2 activity may play an important role in the formation of OS and as a diagnostic for neoplastic activity. However, further research is needed to confirm the role of Bcl-2 activity in OS in canines. PMID:26623359

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

    PubMed

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

    2013-06-01

    We studied the association of the immunohistochemical bcl-2 expression in Libyan breast cancer with clinicopathological variables and patient outcome. Histological samples from 170 previously untreated primary Libyan breast carcinoma patients were examined. In immunohistochemistry, the NCL-L-bcl-2-486 monoclonal antibody was used. Positive expression of bcl-2 was found in 106 patients (62.4 %). The bcl-2 expression was significantly associated with estrogen receptor (p<0.0001) and progesterone receptor positive tumors (p=0.002), small tumor size (p<0.0001), low tumor grade (p<0.0001), negative axillary lymph nodes (p<0.0001), early stages (p=0.001), and low risk of metastasis (p<0.0001). Positive expression was also associated with older patients (>50 years; p=0.04). Histological subtypes and family history of breast cancer did not have significant relationship with bcl-2. Patients with positive expression of bcl-2 had lower recurrence rate than bcl-2-negative patients and better survival after median follow-up of 47 months. Patients with high bcl-2 staining were associated with the best survival. The role of bcl-2 as an independent predictor of disease-specific survival was assessed in a multivariate survival (Cox) analysis, including age, hormonal status, recurrence, histological grade, and clinical stage variables. Bcl-2 (p<0.0001) and clinical stage (p=0.016) were independent predicators of disease-specific survival. For analysis of disease-free survival, the same variables were entered to the model and only bcl-2 proved to be an independent predictor (p=0.002). Patients with positive expression of bcl-2 were associated with low grade of malignancy, with lower recurrence rate, with lower rate of death, and with longer survival time. Bcl-2 is an independent predictor of breast cancer outcome, and it provides useful prognostic information in Libyan breast cancer. Thus, it could be used with classical clinicopathological factors to improve patient selection for

  16. Curcumin induces p53-independent necrosis in H1299 cells via a mitochondria-associated pathway.

    PubMed

    Li, Feie; Chen, Xi; Xu, Bing; Zhou, Hua

    2015-11-01

    Curcumin has been shown to have various therapeutic and/or adjuvant therapeutic effects on human cancers, as it inhibits cancer cell proliferation and induces apoptosis through p53-dependent molecular pathways. However, numerous cancer cell types bear a mutant p53 gene, and whether curcumin has any therapeutic effects on p53-deficient/mutant cancer cells has remained elusive. The present study sought to determine whether curcumin exerts any anti-proliferative and cytotoxic effects on the p53-deficient H1299 human lung cancer cell line via a p53-independent mechanism. An MTT assay and flow cytometric analysis indicated that curcumin significantly decreased cell proliferation and induced necrotic cell death. Western blot analysis of the cytosolic and mitochondrial fractions of H1299 cells as well as a fluorometric caspase assay indicated that curcumin-induced necrosis was mitochondria- and caspase-dependent, and resulted in cytochrome c release. Of note, this necrotic cell death was reduced following inhibition of B-cell lymphoma‑2 (Bcl-2)‑associated X protein (Bax) or Bcl‑2 homologous antagonist killer (Bak) as well as overexpression of Bcl-2. In conclusion, the present study suggested that curcumin-induced necrotic cell death was mediated via a p53-independent molecular pathway, which was associated with Bax and Bak translocation, caspase activation and cytochrome c release.

  17. 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. Copyright© Ferrata Storti Foundation.

  18. Immunohistochemically detectable bcl-2 expression in colorectal carcinoma: correlation with tumour stage and patient survival.

    PubMed Central

    Ofner, D.; Riehemann, K.; Maier, H.; Riedmann, B.; Nehoda, H.; Tötsch, M.; Böcker, W.; Jasani, B.; Schmid, K. W.

    1995-01-01

    The bcl-2 gene encodes for a mitochondrial membrane proto-oncoprotein, the expression of which is known to suppress programmed cell death (apoptosis). In the present study the prognostic value of bcl-2 proto-oncoprotein was immunohistochemically investigated in a series of 104 colorectal carcinomas. The bcl-2 staining patterns were semiquantitatively assessed and correlated with the pTNM stage, Dukes' classification, lymphocytic infiltration (Jass classification) and tumour grade as well as parameters not associated with prognosis (gender, age, tumour site, histological tumour type). Statistical analysis was carried out using the Kaplan-Meier method, the log-rank test, hazard ratios and their confidence intervals. Fifty-five out of 104 cases completely lacked immunohistochemical bcl-2 expression. Fewer than 5% of bcl-2-positive cells were found in 25, 5-50% in 17 and more than 50% in five cases. The extent of bcl-2 expression by tumour cells decreased significantly with respect to increasing tumour size (P < 0.05), decreasing lymphocytic infiltration (P < 0.05) and chance of poor clinical outcome (P < 0.05), but not with worsening of Dukes stages. In multivariate analysis (Cox regression model) bcl-2 expression remained as an independent prognostic parameter with Dukes' classification as stratification factor (P < 0.001). Although the biological functions of bcl-2 protein are not yet well understood, our results provide further evidence that bcl-2 oncoprotein appears to be associated with favourable clinical outcome. Thus immunohistochemical bcl-2 phenotyping of colorectal carcinoma may contribute in future to the clinical management of these patients. Images Figure 1 PMID:7547253

  19. 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

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

    PubMed

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

    2015-01-01

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

  1. Is Bax/Bcl-2 Ratio Considered as a Prognostic Marker with Age and Tumor Location in Colorectal Cancer?

    PubMed Central

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

    2015-01-01

    Background: Bax and Bcl-2 are the major members of Bcl-2 family whose play a key role in tumor progression or inhibition of intrinsic apoptotic pathway triggered by mitochondrial dysfunction. Therefore, the balance between pro- and anti-apoptotic members of this family can determine the cellular fate. Methods: In this study, the relative level of mRNA expression of Bax and Bcl-2 genes was determined using RNA extraction, cDNA synthesis and RT-qPCR technique from 22 tumoral tissues and adjacent non-tumoral tissues from adenocarcinoma colorectal cancer. Results: The potential prognostic and predictive significance of Bax and Bcl-2 gene expression and Bax/Bcl-2 ratio were demonstrated in colorectal cancer. The significant correlation between qPCR data and different clinicopathologic parameters of colorectal carcinoma, including age, gender, tumor size, tumor stage, tumor location, and tumor differentiation was also examined. Interestingly, no significant correlation was seen between Bax and Bcl-2 expressions and clinicopathological parameters of colorectal cancer. However, Bax/Bcl-2 ratio was statistically correlated with age and tumor location. Patients with age above 50 showed decreased levels of Bax/Bcl-2 ratio. Moreover, the Bax/Bcl-2 ratio was significantly lower in tumors resected from colon compared to sigmoid colon, rectosigmoid and rectum tumors. Conclusion: This study indicates a significant correlation between age and tumor location with Bax/Bcl-2 expression ratio, suggesting predictive value as a potential molecular marker of colorectal cancer. PMID:25864810

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

    PubMed

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

    2001-05-04

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

  3. Bcl-2△21 and Ac-DEVD-CHO Inhibit Death of Wheat Microspores

    PubMed Central

    Sinha, Rakesh K.; Pospíšil, Pavel; Maheshwari, Priti; Eudes, François

    2016-01-01

    Microspore cell death and low green plant production efficiency are an integral obstacle in the development of doubled haploid production in wheat. The aim of the current study was to determine the effect of anti-apoptotic recombinant human B-cell lymphoma-2 (Bcl-2△21) and caspase-3-inhibitor (Ac-DEVD-CHO) in microspore cell death in bread wheat cultivars AC Fielder and AC Andrew. Induction medium containing Bcl-2△21 and Ac-DEVD-CHO yielded a significantly higher number of viable microspores, embryo-like structures and total green plants in wheat cultivars AC Fielder and AC Andrew. Total peroxidase activity was lower in Bcl-2△21 treated microspore cultures at 96 h of treatment compared to control and Ac-DEVD-CHO. Electron paramagnetic resonance study of total microspore protein showed a different scavenging activity for Bcl-2△21 and Ac-DEVD-CHO. Bcl-2△21 scavenged approximately 50% hydroxyl radical (HO•) formed, whereas Ac-DEVD-CHO scavenged approximately 20% of HO•. Conversely, reduced caspase-3-like activities were detected in the presence of Bcl-2△21 and Ac-DEVD-CHO, supporting the involvement of Bcl-2△21 and Ac-DEVD-CHO in increasing microspore viability by reducing oxidative stress and caspase-3-like activity. Our results indicate that Bcl-2△21 and Ac-DEVD-CHO protects cells from cell death following different pathways. Bcl-2△21 prevents cell damage by detoxifying HO• and suppressing caspase-3-like activity, while Ac-DEVD-CHO inhibits the cell death pathways by modulating caspase-like activity. PMID:28082995

  4. Bcl-2△21 and Ac-DEVD-CHO Inhibit Death of Wheat Microspores.

    PubMed

    Sinha, Rakesh K; Pospíšil, Pavel; Maheshwari, Priti; Eudes, François

    2016-01-01

    Microspore cell death and low green plant production efficiency are an integral obstacle in the development of doubled haploid production in wheat. The aim of the current study was to determine the effect of anti-apoptotic recombinant human B-cell lymphoma-2 (Bcl-2△21) and caspase-3-inhibitor (Ac-DEVD-CHO) in microspore cell death in bread wheat cultivars AC Fielder and AC Andrew. Induction medium containing Bcl-2△21 and Ac-DEVD-CHO yielded a significantly higher number of viable microspores, embryo-like structures and total green plants in wheat cultivars AC Fielder and AC Andrew. Total peroxidase activity was lower in Bcl-2△21 treated microspore cultures at 96 h of treatment compared to control and Ac-DEVD-CHO. Electron paramagnetic resonance study of total microspore protein showed a different scavenging activity for Bcl-2△21 and Ac-DEVD-CHO. Bcl-2△21 scavenged approximately 50% hydroxyl radical (HO(•)) formed, whereas Ac-DEVD-CHO scavenged approximately 20% of HO(•). Conversely, reduced caspase-3-like activities were detected in the presence of Bcl-2△21 and Ac-DEVD-CHO, supporting the involvement of Bcl-2△21 and Ac-DEVD-CHO in increasing microspore viability by reducing oxidative stress and caspase-3-like activity. Our results indicate that Bcl-2△21 and Ac-DEVD-CHO protects cells from cell death following different pathways. Bcl-2△21 prevents cell damage by detoxifying HO(•) and suppressing caspase-3-like activity, while Ac-DEVD-CHO inhibits the cell death pathways by modulating caspase-like activity.

  5. Locating herpesvirus Bcl-2 homologs in the specificity landscape of anti-apoptotic Bcl-2 proteins

    PubMed Central

    Foight, Glenna Wink; Keating, Amy E.

    2015-01-01

    Viral homologs of the anti-apoptotic Bcl-2 proteins are highly diverged from their mammalian counterparts, yet they perform overlapping functions by binding and inhibiting BH3 motif-containing proteins. We investigated the BH3 binding properties of the herpesvirus Bcl-2 homologs KSBcl-2, BHRF1, and M11, as they relate to those of the human Bcl-2 homologs Mcl-1, Bfl-1, Bcl-w, Bcl-xL, and Bcl-2. Analysis of the sequence and structure of the BH3 binding grooves showed that, despite low sequence identity, M11 has structural similarities to Bcl-xL, Bcl-2, and Bcl-w. BHRF1 and KSBcl-2 are more structurally similar to Mcl-1 than to the other human proteins. Binding to human BH3-like peptides showed that KSBcl-2 has similar specificity to Mcl-1, and BHRF1 has a restricted binding profile; M11 binding preferences are distinct from those of Bcl-xL, Bcl-2 and Bcl-w. Because KSBcl-2 and BHRF1 are from human herpesviruses associated with malignancies, we screened computationally designed BH3 peptide libraries using bacterial surface display to identify selective binders of KSBcl-2 or BHRF1. The resulting peptides bound to KSBcl-2 and BHRF1 in preference to Bfl-1, Bcl-w, Bcl-xL, and Bcl-2, but showed only modest specificity over Mcl-1. Rational mutagenesis increased specificity against Mcl-1, resulting in a peptide with a dissociation constant of 2.9 nM for binding to KSBcl-2 and >1000-fold specificity over human Bcl-2 proteins, and a peptide with >70-fold specificity for BHRF1. In addition to providing new insights into viral Bcl-2 binding specificity, this study will inform future work analyzing the interaction properties of homologous binding domains and designing specific protein interaction partners. PMID:26009469

  6. Bcl-2-family proteins and hematologic malignancies: history and future prospects.

    PubMed

    Reed, John C

    2008-04-01

    BCL-2 was the first antideath gene discovered, a milestone that effectively launched a new era in cell death research. Since its discovery more than 2 decades ago, multiple members of the human Bcl-2 family of apoptosis-regulating proteins have been identified, including 6 antiapoptotic proteins, 3 structurally similar proapoptotic proteins, and several structurally diverse proapoptotic interacting proteins that operate as upstream agonists or antagonists. Bcl-2-family proteins regulate all major types of cell death, including apoptosis, necrosis, and autophagy. As such, they operate as nodal points at the convergence of multiple pathways with broad relevance to biology and medicine. Bcl-2 derives its name from its original discovery in the context of B-cell lymphomas, where chromosomal translocations commonly activate the BCL-2 protooncogene, endowing B cells with a selective survival advantage that promotes their neoplastic expansion. The concept that defective programmed cell death contributes to malignancy was established by studies of Bcl-2, representing a major step forward in current understanding of tumorigenesis. Experimental therapies targeting Bcl-2 family mRNAs or proteins are currently in clinical testing, raising hopes that a new class of anticancer drugs may be near.

  7. 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

  8. [Mechanism and clinical significance of anti-apoptotic gene bcl-2 expression in diffuse large B-cell lymphoma].

    PubMed

    Zeng, Li-ping; Wen, Yi-lei; Ma, Yun; Wang, Gui-qiu; Li, Ying; Wang, Jin; Xu, Li-li; Zhang, Xue-mei

    2011-06-01

    overall survival times, would face significant higher risk of death, these results suggested that bcl-2 could be a prognostic marker independent to clinical staging and immunophenotyping.

  9. Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane

    PubMed Central

    O'Neill, Katelyn L.; Huang, Kai; Zhang, Jingjing; Chen, Yi; Luo, Xu

    2016-01-01

    The mechanism of Bax/Bak activation remains a central question in mitochondria-dependent apoptotic signaling. While it is established that all proapoptotic Bcl-2 homology 3 (BH3)-only proteins bind and neutralize the anti-apoptotic Bcl-2 family proteins, how this neutralization leads to Bax/Bak activation has been actively debated. Here, genome editing was used to generate cells deficient for all eight proapoptotic BH3-only proteins (OctaKO) and those that lack the entire Bcl-2 family (Bcl-2 allKO). Although the OctaKO cells were resistant to most apoptotic stimuli tested, they underwent Bax/Bak-dependent and p53/Rb-independent apoptosis efficiently when both Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins, were inactivated or eliminated. Strikingly, when expressed in the Bcl-2 allKO cells, both Bax and Bak spontaneously associated with the outer mitochondrial membrane (OMM) through their respective helix 9, and this association triggered their homo-oligomerization/activation. Together, these results strongly suggest that the OMM, not BH3-only proteins or p53/Rb, is the long-sought-after direct activator of Bax/Bak following BH3-only-mediated neutralization of anti-apoptotic Bcl-2 proteins. PMID:27056669

  10. BCL-2 and BCL-XL restrict lineage choice during hematopoietic differentiation.

    PubMed

    Haughn, Loralee; Hawley, Robert G; Morrison, Deborah K; von Boehmer, Harald; Hockenbery, David M

    2003-07-04

    Differentiation of hematopoietic cells from multipotential progenitors is regulated by multiple growth factors and cytokines. A prominent feature of these soluble factors is promotion of cell survival, in part mediated by expression of either of the anti-apoptotic proteins, BCL-2 and BCL-XL. The complex expression pattern of these frequently redundant survival factors during hematopoiesis may indicate a role in lineage determination. To investigate the latter possibility, we analyzed factor-dependent cell-Patersen (FDCP)-Mix multipotent progenitor cells in which we stably expressed BCL-2 or BCL-XL. Each factor maintained complete survival of interleukin-3 (IL-3)-deprived FDCP-Mix cells but, unexpectedly, directed FDCP-Mix cells along restricted and divergent differentiation pathways. Thus, IL-3-deprived FDCP-Mix BCL-2 cells differentiated exclusively to granulocytes and monocytes/macrophages, whereas FDCP-Mix BCL-XL cells became erythroid. FDCP-Mix BCL-2 cells grown in IL-3 were distinguished from FDCP-Mix and FDCP-Mix BCL-XL cells by a striking reduction in cellular levels of Raf-1 protein. Replacement of the BCL-2 BH4 domain with the related BCL-XL BH4 sequence resulted in a switch of FDCP-Mix BCL-2 cells to erythroid fate accompanied by persistence of Raf-1 protein expression. Moreover, enforced expression of Raf-1 redirected FDCP-Mix BCL-2 cells to an erythroid fate, and prohibited generation of myeloid cells. These results identify novel roles for BCL-2 and BCL-XL in cell fate decisions beyond cell survival. These effects are associated with differential regulation of Raf-1 expression, perhaps involving the previously identified interaction between BCL-2-BH4 and the catalytic domain of Raf-1.

  11. 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.

  12. Expression of Bcl-2 and Bcl-2-Ig fusion transcripts in normal and neoplastic cells.

    PubMed Central

    Graninger, W B; Seto, M; Boutain, B; Goldman, P; Korsmeyer, S J

    1987-01-01

    We examined the expression of the Bcl-2 gene at chromosome segment 18q21, that is translocated into the Ig heavy chain gene locus in t(14;18) bearing lymphomas. Bcl-2, while B cell associated, is expressed in a variety of hematopoietic lineages including T cells. Bcl-2 mRNA levels are high during pre-B cell development, the time at which the t(14;18) translocation occurs, but are down regulated with maturation. Like certain other oncogenes, Bcl-2 is quiescent in resting B cells but up-regulated with B cell activation. Mature B cell lymphomas with a t(14;18) have log-folds more mRNA than matched counterparts without the translocation. A sensitive S1 protection assay revealed that all transcripts in t(14;18) B cells were Bcl-2-Ig fusion mRNAs and originated from the translocated allele. Thus, there is a marked deregulation of Bcl-2 when it is introduced into the Ig locus in t(14;18) lymphomas. Images PMID:3500184

  13. Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation.

    PubMed Central

    Linette, G P; Li, Y; Roth, K; Korsmeyer, S J

    1996-01-01

    BCL-2-deficient T cells demonstrate accelerated cell cycle progression and increased apoptosis following activation. Increasing the levels of BCL-2 retarded the G0-->S transition, sustained the levels of cyclin-dependent kinase inhibitor p27Kip1, and repressed postactivation death. Proximal signal transduction events and immediate early gene transcription were unaffected. However, the transcription and synthesis of interleukin 2 and other delayed early cytokines were markedly attenuated by BCL-2. In contrast, a cysteine protease inhibitor that also blocks apoptosis had no substantial affect upon cytokine production. InterleUkin 2 expression requires several transcription factors of which nuclear translocation of NFAT (nuclear factor of activated T cells) and NFAT-mediated transactivation were impaired by BCL-2. Thus, select genetic aberrations in the apoptotic pathway reveal a cell autonomous coregulation of activation. Images Fig. 3 Fig. 4 Fig. 7 PMID:8790367

  14. 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

  15. FBXO10 deficiency and BTK activation upregulate BCL2 expression in mantle cell lymphoma

    PubMed Central

    Li, Y; Bouchlaka, MN; Wolff, J; Grindle, KM; Lu, L; Qian, S; Zhong, X; Pflum, N; Jobin, P; Kahl, BS; Eickhoff, JC; Wuerzberger-Davis, SM; Miyamoto, S; Thomas, CJ; Yang, DT; Capitini, CM; Rui, L

    2016-01-01

    Targeting Bruton tyrosine kinase (BTK) by ibrutinib is an effective treatment for patients with relapsed/refractory mantle cell lymphoma (MCL). However, both primary and acquired resistance to ibrutinib have developed in a significant number of these patients. A combinatory strategy targeting multiple oncogenic pathways is critical to enhance the efficacy of ibrutinib. Here, we focus on the BCL2 anti-apoptotic pathway. In a tissue microarray of 62 MCL samples, BCL2 expression positively correlated with BTK expression. Increased levels of BCL2 were shown to be due to a defect in protein degradation because of no or little expression of the E3 ubiquitin ligase FBXO10, as well as transcriptional upregulation through BTK-mediated canonical nuclear factor-κB activation. RNA-seq analysis confirmed that a set of anti-apoptotic genes (for example, BCL2, BCL-XL and DAD1) was downregulated by BTK short hairpin RNA. The downregulated genes also included those that are critical for B-cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Targeting BCL2 by the specific inhibitor ABT-199 synergized with ibrutinib in inhibiting growth of both ibrutinib-sensitive and -resistant cancer cells in vitro and in vivo. These results suggest co-targeting of BTK and BCL2 as a new therapeutic strategy in MCL, especially for patients with primary resistance to ibrutinib. PMID:27157620

  16. The Anti-apoptotic Protein BCL2L1/Bcl-xL Is Neutralized by Pro-apoptotic PMAIP1/Noxa in Neuroblastoma, Thereby Determining Bortezomib Sensitivity Independent of Prosurvival MCL1 Expression*

    PubMed Central

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

    2010-01-01

    Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death. PMID:20051518

  17. 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

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

  19. Diosmetin triggers cell apoptosis by activation of the p53/Bcl-2 pathway and inactivation of the Notch3/NF-κB pathway in HepG2 cells

    PubMed Central

    Qiao, Jie; Liu, Jie; Jia, Kaiqiao; Li, Ning; Liu, Bin; Zhang, Qingyu; Zhu, Runzhi

    2016-01-01

    Diosmetin (DIOS), a flavonoid compound, is abundant in Citrus limon. Emerging studies have shown that DIOS is an effective compound implicated in multiple types of cancer. However, whether DIOS serves a role in hepatocellular carcinoma (HCC) is still obscure. HepG2 cells were used in the present study, and it was observed that DIOS exhibited antitumor activity against liver cancer cells. Western blotting was performed to evaluate cell apoptosis and survival-associated proteins, and the results demonstrated that DIOS treatment resulted in the activation of the p53-dependent apoptosis pathway. Our results revealed that DIOS caused inhibition of the nuclear factor (NF)-κB signaling pathway and downregulation of Notch3 receptor. Furthermore, by using small hairpin RNA-Notch3, it was confirmed that DIOS inhibited the NF-κB signaling pathway by inactivation of Notch3. In conclusion, the present results demonstrated that DIOS triggered cell apoptosis by activation of the p53 signaling pathway and inhibited the NF-κB cell survival pathway by downregulation of Notch3 receptor expression. DIOS is a potential agent for prevention of HCC. PMID:28101238

  20. 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

  1. 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.

  2. Predictive Bcl-2 Family Binding Models Rooted in Experiment or Structure

    PubMed Central

    DeBartolo, Joe; Dutta, Sanjib; Reich, Lothar; Keating, Amy E.

    2013-01-01

    Proteins of the Bcl-2 family either enhance or suppress programmed cell death and are centrally involved in cancer development and resistance to chemotherapy. BH3 (Bcl-2 homology 3)-only Bcl-2 proteins promote cell death by docking an α-helix into a hydrophobic groove on the surface of one or more of five pro-survival Bcl-2 receptor proteins. There is high structural homology within the pro-death and pro-survival families, yet a high degree of interaction specificity is nevertheless encoded, posing an interesting and important molecular recognition problem. Understanding protein features that dictate Bcl-2 interaction specificity is critical for designing peptide-based cancer therapeutics and diagnostics. In this study, we present peptide SPOT arrays and deep sequencing data from yeast display screening experiments that significantly expand the BH3 sequence space that has been experimentally tested for interaction with five human anti-apoptotic receptors. These data provide rich information about the determinants of Bcl-2 family specificity. To interpret and use the information, we constructed two simple data-based models that can predict affinity and specificity when evaluated on independent data sets within a limited sequence space. We also constructed a novel structure-based statistical potential, called STATIUM, which is remarkably good at predicting Bcl-2 affinity and specificity, especially considering it is not trained on experimental data. We compare the performance of our three models to each other and to alternative structure-based methods and discuss how such tools can guide prediction and design of new Bcl-2 family complexes. PMID:22617328

  3. 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.

  4. BCL-2 is dispensable for thrombopoiesis and platelet survival

    PubMed Central

    Debrincat, M A; Pleines, I; Lebois, M; Lane, R M; Holmes, M L; Corbin, J; Vandenberg, C J; Alexander, W S; Ng, A P; Strasser, A; Bouillet, P; Sola-Visner, M; Kile, B T; Josefsson, E C

    2015-01-01

    Navitoclax (ABT-263), an inhibitor of the pro-survival BCL-2 family proteins BCL-2, BCL-XL and BCL-W, has shown clinical efficacy in certain BCL-2-dependent haematological cancers, but causes dose-limiting thrombocytopaenia. The latter effect is caused by Navitoclax directly inducing the apoptotic death of platelets, which are dependent on BCL-XL for survival. Recently, ABT-199, a selective BCL-2 antagonist, was developed. It has shown promising anti-leukaemia activity in patients whilst sparing platelets, suggesting that the megakaryocyte lineage does not require BCL-2. In order to elucidate the role of BCL-2 in megakaryocyte and platelet survival, we generated mice with a lineage-specific deletion of Bcl2, alone or in combination with loss of Mcl1 or Bclx. Platelet production and platelet survival were analysed. Additionally, we made use of BH3 mimetics that selectively inhibit BCL-2 or BCL-XL. We show that the deletion of BCL-2, on its own or in concert with MCL-1, does not affect platelet production or platelet lifespan. Thrombocytopaenia in Bclx-deficient mice was not affected by additional genetic loss or pharmacological inhibition of BCL-2. Thus, BCL-2 is dispensable for thrombopoiesis and platelet survival in mice. PMID:25880088

  5. BCL-2 is dispensable for thrombopoiesis and platelet survival.

    PubMed

    Debrincat, M A; Pleines, I; Lebois, M; Lane, R M; Holmes, M L; Corbin, J; Vandenberg, C J; Alexander, W S; Ng, A P; Strasser, A; Bouillet, P; Sola-Visner, M; Kile, B T; Josefsson, E C

    2015-04-16

    Navitoclax (ABT-263), an inhibitor of the pro-survival BCL-2 family proteins BCL-2, BCL-XL and BCL-W, has shown clinical efficacy in certain BCL-2-dependent haematological cancers, but causes dose-limiting thrombocytopaenia. The latter effect is caused by Navitoclax directly inducing the apoptotic death of platelets, which are dependent on BCL-XL for survival. Recently, ABT-199, a selective BCL-2 antagonist, was developed. It has shown promising anti-leukaemia activity in patients whilst sparing platelets, suggesting that the megakaryocyte lineage does not require BCL-2. In order to elucidate the role of BCL-2 in megakaryocyte and platelet survival, we generated mice with a lineage-specific deletion of Bcl2, alone or in combination with loss of Mcl1 or Bclx. Platelet production and platelet survival were analysed. Additionally, we made use of BH3 mimetics that selectively inhibit BCL-2 or BCL-XL. We show that the deletion of BCL-2, on its own or in concert with MCL-1, does not affect platelet production or platelet lifespan. Thrombocytopaenia in Bclx-deficient mice was not affected by additional genetic loss or pharmacological inhibition of BCL-2. Thus, BCL-2 is dispensable for thrombopoiesis and platelet survival in mice.

  6. 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

  7. Algae: America’s Pathway to Independence

    DTIC Science & Technology

    2007-03-30

    Bioenergy, Biofuel, Energy Policy CLASSIFICATION: Unclassified The United States is dependent on foreign oil to meet 63% of its petroleum demand...source of bioenergy. ALGAE: AMERICA’S PATHWAY TO INDEPENDENCE Ensuring a secure supply of energy is a strategic challenge for...150 years,6 the U.S. will be competing with other nations to procure the 2 finite commodity. The Department of Energy (DOE) estimates that by the

  8. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyride neurotoxicity is attenuated in mice overexpressing Bcl-2.

    PubMed

    Yang, L; Matthews, R T; Schulz, J B; Klockgether, T; Liao, A W; Martinou, J C; Penney, J B; Hyman, B T; Beal, M F

    1998-10-15

    The proto-oncogene Bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that Bcl-2 protects against free radicals and that it increases mitochondrial calcium-buffering capacity. The neurotoxicity of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyride (MPTP) is thought to involve both mitochondrial dysfunction and free radical generation. We therefore investigated MPTP neurotoxicity in both Bcl-2 overexpressing mice and littermate controls. MPTP-induced depletion of dopamine and loss of [3H]mazindol binding were significantly attenuated in Bcl-2 overexpressing mice. Protection was more profound with an acute dosing regimen than with daily MPTP administration over 5 d. 1-Methyl-4-phenylpyridinium (MPP+) levels after MPTP administration were similar in Bcl-2 overexpressing mice and littermates. Bcl-2 blocked MPP+-induced activation of caspases. MPTP-induced increases in free 3-nitrotyrosine levels were blocked in Bcl-2 overexpressing mice. These results indicate that Bcl-2 overexpression protects against MPTP neurotoxicity by mechanisms that may involve both antioxidant activity and inhibition of apoptotic pathways.

  9. Molecular Basis for Bcl-2 Homology 3 Domain Recognition in the Bcl-2 Protein Family

    PubMed Central

    Moroy, Gautier; Martin, Elyette; Dejaegere, Annick; Stote, Roland H.

    2009-01-01

    The proteins of the Bcl-2 family are important regulators of apoptosis, or programmed cell death. These proteins regulate this fundamental biological process via the formation of heterodimers involving both pro- and anti-apoptotic family members. Disruption of the balance between anti- and pro-apoptotic Bcl-2 proteins is the cause of numerous pathologies. Bcl-xl, an anti-apoptotic protein of this family, is known to form heterodimers with multiple pro-apoptotic proteins, such as Bad, Bim, Bak, and Bid. To elucidate the molecular basis of this recognition process, we used molecular dynamics simulations coupled with the Molecular Mechanics/Poisson-Boltzmann Surface Area approach to identify the amino acids that make significant energetic contributions to the binding free energy of four complexes formed between Bcl-xl and pro-apoptotic Bcl-2 homology 3 peptides. A fifth protein-peptide complex composed of another anti-apoptotic protein, Bcl-w, in complex with the peptide from Bim was also studied. The results identified amino acids of both the anti-apoptotic proteins as well as the Bcl-2 homology 3 (BH3) domains of the pro-apoptotic proteins that make strong, recurrent interactions in the protein complexes. The calculations show that the two anti-apoptotic proteins, Bcl-xl and Bcl-w, share a similar recognition mechanism. Our results provide insight into the molecular basis for the promiscuous nature of this molecular recognition process by members of the Bcl-2 protein family. These amino acids could be targeted in the design of new mimetics that serve as scaffolds for new antitumoral molecules. PMID:19293158

  10. 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

  11. The Bcl-2 family: roles in cell survival and oncogenesis.

    PubMed

    Cory, Suzanne; Huang, David C S; Adams, Jerry M

    2003-11-24

    Apoptosis, the cell-suicide programme executed by caspases, is critical for maintaining tissue homeostasis, and impaired apoptosis is now recognized to be a key step in tumorigenesis. Whether a cell should live or die is largely determined by the Bcl-2 family of anti- and proapoptotic regulators. These proteins respond to cues from various forms of intracellular stress, such as DNA damage or cytokine deprivation, and interact with opposing family members to determine whether or not the caspase proteolytic cascade should be unleashed. This review summarizes current views of how these proteins sense stress, interact with their relatives, perturb organelles such as the mitochondrion and endoplasmic reticulum and govern pathways to caspase activation. It briefly explores how family members influence cell-cycle entry and outlines the evidence for their involvement in tumour development, both as oncoproteins and tumour suppressors. Finally, it discusses the promise of novel anticancer therapeutics that target these vital regulators.

  12. BCL2L12 Is a Novel Biomarker for the Prediction of Short-Term Relapse in Nasopharyngeal Carcinoma

    PubMed Central

    Fendri, Ali; Kontos, Christos K; Khabir, Abdelmajid; Mokdad-Gargouri, Raja; Scorilas, Andreas

    2011-01-01

    BCL2-like 12 (BCL2L12 ) is a new member of the apoptosis-related BCL2 gene family, members of which are implicated in various malignancies. Nasopharyngeal carcinoma is a highly metastatic, malignant epithelial tumor, with a high prevalence in Southeast Asia and North Africa. The purpose of the current study was to quantify and investigate the expression levels of the BCL2L12 gene in nasopharyngeal carcinoma biopsies and to assess its prognostic value. Total RNA was isolated from 89 malignant and hyperplastic nasopharyngeal biopsies from Tunisian patients. After testing the quality of the extracted RNA, cDNA was prepared by reverse transcription. A highly sensitive real-time polymerase chain reaction (PCR) method for BCL2L12 mRNA quantification was developed using SYBR® Green chemistry. GAPDH served as a reference gene. Relative quantification analysis was performed using the comparative CT (2−ΔΔCT) method. Higher BCL2L12 mRNA levels were detected in undifferentiated carcinomas of the nasopharynx, rather than in nonkeratinizing nasopharyngeal tumors (P = 0.045). BCL2L12 expression status was also found to be positively associated with the presence of distant metastases (P = 0.014). Kaplan-Meier survival analysis demonstrated that patients with BCL2L12-positive nasopharyngeal tumors have significantly shorter disease-free survival (P = 0.020). Cox regression analysis showed BCL2L12 expression to be an unfavorable and independent prognostic indicator of short-term relapse in nasopharyngeal carcinoma (P = 0.042). Our results suggest that mRNA expression of BCL2L12 may constitute a novel biomarker for the prediction of short-term relapse in nasopharyngeal carcinoma. PMID:21152697

  13. Nicotine increases the resistance of lung cancer cells to cisplatin through enhancing Bcl-2 stability

    PubMed Central

    Nishioka, T; Luo, L-Y; Shen, L; He, H; Mariyannis, A; Dai, W; Chen, C

    2014-01-01

    Background: Nicotine is able to activate mitogenic signalling pathways, which promote cell growth or survival as well as increase chemoresistance of cancer cells. However, the underlying mechanisms are not fully understood. Methods: In this study, we used immunoblotting and immunoprecipitation methods to test the ubiquitination and degradation of Bcl-2 affected by nicotine in lung cancer cells. Apoptotic assay was also used to measure the antagonising effect of nicotine on cisplatin-mediated cytotoxicity. Results: We demonstrated that the addition of nicotine greatly attenuated Bcl-2 ubiquitination and degradation, which further desensitised lung cancer cells to cisplatin-induced cytotoxicity. In this process, Bcl-2 was persistently phosphorylated in the cells cotreated with nicotine and cisplatin. Furthermore, Akt was proven to be responsible for sustained activation of Bcl-2 by nicotine, which further antagonised cisplatin-mediated apoptotic signalling. Conclusions: Our study suggested that nicotine activates its downstream signalling to interfere with the ubiquitination process and prevent Bcl-2 from being degraded in lung cancer cells, resulting in the increase of chemoresistance. PMID:24548862

  14. Preferential control of induced regulatory T cell homeostasis via a Bim/Bcl-2 axis.

    PubMed

    Wang, X; Szymczak-Workman, A L; Gravano, D M; Workman, C J; Green, D R; Vignali, D A A

    2012-02-09

    Apoptosis has an essential role in controlling T cell homeostasis, especially during the contraction phase of an immune response. However, its contribution to the balance between effector and regulatory populations remains unclear. We found that Rag1(-/-) hosts repopulated with Bim(-/-) conventional CD4(+) T cells (Tconv) resulted in a larger induced regulatory T cell (iTreg) population than mice given wild-type (WT) Tconv. This appears to be due to an increased survival advantage of iTregs compared with activated Tconv in the absence of Bim. Downregulation of Bcl-2 expression and upregulation of Bim expression were more dramatic in WT iTregs than activated Tconv in the absence of IL-2 in vitro. The iTregs generated following Tconv reconstitution of Rag1(-/-) hosts exhibited lower Bcl-2 expression and higher Bim/Bcl-2 ratio than Tconv, which indicates that iTregs were in an apoptosis-prone state in vivo. A significant proportion of the peripheral iTreg pool exhibits low Bcl-2 expression indicating increased sensitivity to apoptosis, which may be a general characteristic of certain Treg subpopulations. In summary, our data suggest that iTregs and Tconv differ in their sensitivity to apoptotic stimuli due to their altered ratio of Bim/Bcl-2 expression. Modulating the apoptosis pathway may provide novel therapeutic approaches to alter the balance between effector T cells and Tregs.

  15. Protect and serve: Bcl-2 proteins as guardians and rulers of cancer cell survival

    PubMed Central

    Braun, Frédérique; de Carné Trécesson, Sophie; Bertin-Ciftci, Joséphine; Juin, Philippe

    2013-01-01

    It is widely accepted that anti-apoptotic Bcl-2 family members promote cancer cell survival by binding to their pro-apoptotic counterparts, thereby preventing mitochondrial outer membrane permeabilization (MOMP) and cytotoxic caspase activation. Yet, these proteins do not only function as guardians of mitochondrial permeability, preserving it, and maintaining cell survival in the face of acute or chronic stress, they also regulate non-apoptotic functions of caspases and biological processes beyond MOMP from diverse subcellular localizations and in complex with numerous binding partners outside of the Bcl-2 family. In particular, some of the non-canonical effects and functions of Bcl-2 homologs lead to an interplay with E2F-1, NFκB, and Myc transcriptional pathways, which themselves influence cancer cell growth and survival. We thus propose that, by feedback loops that we currently have only hints of, Bcl-2 proteins may act as rulers of survival signaling, predetermining the apoptotic threshold that they also directly scaffold. This underscores the robustness of the control exerted by Bcl-2 homologs over cancer cell survival, and implies that small molecules compounds currently used in the clinic to inhibit their mitochondrial activity may be not always be fully efficient to override this control. PMID:23974114

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

    PubMed

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

    2011-01-01

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

  17. 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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. 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

  19. Conjugated linoleic acid induces apoptosis of murine mammary tumor cells via Bcl-2 loss

    PubMed Central

    Ou, Lihui; Ip, Clement; Lisafeld, Barbara; Ip, Margot M.

    2007-01-01

    Conjugated linoleic acid (CLA) is a powerful anticancer agent in a number of tumor model systems; however, its precise mechanism of action remains elusive. Here, we report that t10,c12 CLA, a component of synthetic CLA supplements, induced apoptosis and G1 arrest of p53 mutant TM4t murine mammary tumor cells. Furthermore, t10,c12-CLA induced a time- and concentration-dependent cleavage of caspases-3 and -9, and release of cytochrome c from mitochondria to cytosol. Levels of Bcl-2 protein were decreased both in total cellular lysates and in mitochondria after t10,c12-CLA treatment; however, there was no significant change in Bax or Bak. Overexpression of Bcl-2 attenuated apoptosis in response to t10,c12-CLA treatment. These results demonstrate that t10,c12-CLA triggers apoptosis of p53 mutant murine mammary tumor cells through the mitochondrial pathway by targeting Bcl-2. PMID:17400188

  20. Bcl-2 proteins and autophagy regulate mitochondrial dynamics during programmed cell death in the Drosophila ovary

    PubMed Central

    Tanner, Elizabeth A.; Blute, Todd A.; Brachmann, Carrie Baker; McCall, Kimberly

    2011-01-01

    The Bcl-2 family has been shown to regulate mitochondrial dynamics during cell death in mammals and C. elegans, but evidence for this in Drosophila has been elusive. Here, we investigate the regulation of mitochondrial dynamics during germline cell death in the Drosophila melanogaster ovary. We find that mitochondria undergo a series of events during the progression of cell death, with remodeling, cluster formation and uptake of clusters by somatic follicle cells. These mitochondrial dynamics are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery, and the autophagy machinery. Furthermore, Bcl-2 family mutants show a striking defect in cell death in the ovary. These data indicate that a mitochondrial pathway is a major mechanism for activation of cell death in Drosophila oogenesis. PMID:21177345

  1. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance?

    PubMed Central

    Williams, Michelle M.; Cook, Rebecca S.

    2015-01-01

    Apoptosis, cell death executed by caspases, is essential to normal breast development and homeostasis. Pro-apoptotic and anti-apoptotic signals are tightly regulated in normal breast epithelial cells. Dysregulation of this balance is required for breast tumorigenesis and increases acquired resistance to treatments, including molecularly targeted therapies, radiation and chemotherapies. The pro-apoptotic or anti-apoptotic Bcl-2 family members interact with each other to maintain mitochondrial integrity and regulate cellular commitment to apoptosis. Among the anti-apoptotic Bcl-2 family members, Mcl-1 is uniquely regulated by numerous oncogenic signaling pathways. This review will focus on the role of Bcl-2 family proteins in normal breast development, breast tumorigenesis and acquired resistance to breast cancer treatment strategies, while highlighting Mcl-1 as a promising target to improve breast cancer tumor cell killing. PMID:25784482

  2. Regulation of mitochondrial nutrient and energy metabolism by BCL-2 family proteins

    PubMed Central

    Giménez-Cassina, Alfredo; Danial, Nika N.

    2015-01-01

    Cells have evolved a highly integrated network of mechanisms to coordinate cellular survival/death, proliferation, differentiation, and repair with metabolic states. It is, therefore, not surprising that proteins with canonical roles in cell death/survival also modulate nutrient and energy metabolism and vice versa. The finding that many BCL-2 (B cell lymphoma 2) proteins reside at mitochondria or can translocate to this organelle has long motivated investigation into their involvement in normal mitochondrial physiology and metabolism. These endeavors have led to the discovery of homeostatic roles for BCL-2 proteins beyond apoptosis. Here, we predominantly focus on recent findings that link select BCL-2 proteins to carbon substrate utilization at the level of mitochondrial fuel choice, electron transport, and metabolite import independent of their cell death regulatory function. PMID:25748272

  3. BCL-2 family member BOK promotes apoptosis in response to endoplasmic reticulum stress

    PubMed Central

    Carpio, Marcos A.; Michaud, Michael; Zhou, Wenping; Fisher, Jill K.; Walensky, Loren D.; Katz, Samuel G.

    2015-01-01

    B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) is a BCL-2 family protein with high homology to the multidomain proapoptotic proteins BAX and BAK, yet Bok−/− and even Bax−/−Bok−/− and Bak−/−Bok−/− mice were reported to have no overt phenotype or apoptotic defects in response to a host of classical stress stimuli. These surprising findings were interpreted to reflect functional compensation among the BAX, BAK, and BOK proteins. However, BOK cannot compensate for the severe apoptotic defects of Bax−/−Bak−/− mice despite its widespread expression. Here, we independently developed Bok−/− mice and found that Bok−/− cells are selectively defective in their response to endoplasmic reticulum (ER) stress stimuli, consistent with the predominant subcellular localization of BOK at the ER. Whereas Bok−/− mouse embryonic fibroblasts exposed to thapsigargin, A23187, brefeldin A, DTT, geldanamycin, or bortezomib manifested reduced activation of the mitochondrial apoptotic pathway, the death response to other stimuli such as etoposide, staurosporine, or UV remained fully intact. Multiple organs in Bok−/− mice exhibited resistance to thapsigargin-induced apoptosis in vivo. Although the ER stress agents activated the unfolded protein response, both ATF4 and CHOP activation were diminished in Bok−/− cells and mice. Importantly, BAX and BAK were unable to compensate for the defective apoptotic response to ER stress observed in SV40-transformed and primary Bok−/− cells, and in vivo. These findings support a selective and distinguishing role for BOK in regulating the apoptotic response to ER stress, revealing—to our knowledge—the first bona fide apoptotic defect linked to Bok deletion. PMID:26015568

  4. Clinical significance of Cox-2, Survivin and Bcl-2 expression in hepatocellular carcinoma (HCC).

    PubMed

    Yang, Yu; Zhu, Jiang; Gou, Hongfeng; Cao, Dan; Jiang, Ming; Hou, Mei

    2011-09-01

    Cox-2, Survivin and Bcl-2 are frequently overexpressed in numerous types of cancers. They are known to be the important regulators of apoptosis. This study was designed to investigate the correlation between the clinical characteristics and the expression of Cox-2, Survivin and Bcl-2 in hepatocellular carcinoma. A total of 63 postoperative hepatocellular carcinoma (HCC) samples, 10 adjacent non-tumor samples and 10 normal liver samples were immunochemically detected for the expression of Cox-2, Survivin and Bcl-2. A median follow-up of 4 years for the 63 HCC patients was conducted. Univariate tests and multivariate Cox regression were performed for statistical analysis. The Kaplan-Meier method was used to analyze the survival. Positive expression of Cox-2 (84.3%) and Survivin (77.8%) was detected significantly more frequently in the HCC samples than in the normal liver tissues (30% and 0, respectively). Bcl-2 was highly expressed in the adjacent non-tumor tissue. Cox-2 was positively correlative to Survivin. Survivin and Bcl-2 were significantly associated with the pathological grade of HCC (P<0.05). Expression of both Cox-2 and Survivin was significantly associated with the poor overall survival (OS) (P=0.0141, P=0.0039). Furthermore, multivariate analysis confirmed the independent prognostic value of Survivin expression, along with tumor size and hepatic function. Cox-2 and Survivin were highly expressed in the HCC tissue. Survivin and Bcl-2 were significantly associated with the pathological grade of HCC. The expression of Survivin was an independent prognostic factor for HCC after a hepatectomy. Treatment that inhibits Survivin may be a promising targeted approach in HCC.

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

    PubMed

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

    2011-02-21

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

  6. 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

  7. Bcl-2 Antagonists: A Proof of Concept for CLL Therapy

    PubMed Central

    Balakrishnan, Kumudha; Gandhi, Varsha

    2014-01-01

    Defective apoptosis is a fundamental hallmark feature of CLL biology and is a major target of cancer therapy development. High levels of Bcl-2 family anti-apoptotic proteins are considered primarily responsible for inhibiting apoptosis in CLL cells. While several approaches were considered to selectively inhibit Bcl-2 family anti-apoptotic proteins, the discovery that gossypol binds and antagonizes anti-apoptotic effect of Bcl-2 family proteins was a major breakthrough in identifying specific Bcl-2 antagonists. The concept of mimicking BH3 domain emphasized the importance of Bcl-2 family-targeted therapy that can modulate the function of anti-apoptotic proteins. Although parent compound gossypol did not sustain in the clinic, its structural modifications led to the development of additional analogues that demonstrated improved efficacy and reduced toxicity in preclinical and clinical investigations. Proof of concept of this hypothesis was demonstrated by structure based BH3 mimetic ABT-737 that has shown greater cytotoxicity towards CLL cells both in pre-clinical models and clinical trials. Its oral compound ABT-263 has demonstrated the substantial susceptibility of chronic lymphocytic leukemia cells through Bcl-2 inhibition. Collectively, results of a Phase I Study of Navitoclax (ABT-263) in patients with relapsed or refractory disease warrants Bcl-2 as a valid therapeutic target in CLL. Importantly, molecules that mimic pro-apoptotic BH3 domains represent a direct approach to overcoming the protective effects of anti-apoptotic proteins such as Mcl-1, Bcl-2 and Bcl-XL. PMID:23907405

  8. Initiation of premature senescence by Bcl-2 in hypoxic condition.

    PubMed

    Wang, Wei; Wang, Desheng; Li, Hong

    2014-01-01

    Senescence, a state of cell cycle arrest, has been regarded as an intrinsic barrier to malignance. Although being repressed in most immortal tumors, the genetic program of senescence can be reactivated by critical regulators, including the apoptosis regulator Bcl-2. We showed here that hypoxic condition resulted in an irreversible senescence-like phenotype with increased expression of Bcl-2 in mouse melanoma B16 cells. In CoCl2-simulating hypoxic condition, characteristic morphological alterations and increased activity of senescence-associated β-galactosidase (SA-β-gal) can be detected with high level of Bcl-2, which was confirmed by western blot and co-staining of SA-β-gal and Bcl-2 by immunocytochemistry. Accordingly, Bcl-2 silence by specific siRNA ahead of hypoxia treatment interrupted the senescent development. Moreover Bcl-2 overexpression led to early onset of senescence. We propose that Bcl-2 is required to initiate and maintain the senescent phenotype. In addition, p53 and p16 were not involved in hypoxia-induced senescence according to the expression levels during senescent process. These results suggest that when encountering harmful stress (hypoxia), melanoma cells overexpress Bcl-2 and turn to senescence, a permanent cell-cycle arrest, for prolonged survival.

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

    SciTech Connect

    Stevens, P.W. |; Cai, X.; Schiffer, M.

    1996-06-01

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

  10. Expression of the bcl-2 gene product in follicular lymphoma.

    PubMed Central

    Gaulard, P.; d'Agay, M. F.; Peuchmaur, M.; Brousse, N.; Gisselbrecht, C.; Solal-Celigny, P.; Diebold, J.; Mason, D. Y.

    1992-01-01

    Expression of bcl-2 protein was analyzed in 140 cases of follicular lymphoma by immunohistologic staining of paraffin-embedded tissue; 85% of cases were positive, the frequency being related to histologic grade (100% for the small-cleaved cell type, 86% for the mixed cell type, and 76% for the large cell group). There was striking heterogeneity of bcl-2 content in a number of cases and the smaller neoplastic cells (i.e., centrocytes) were usually the most strongly labeled. In most cases, bcl-2 protein staining was much weaker in normal lymphoid cells than in the neoplastic cells. In several cases, staining for bcl-2 revealed patterns of neoplastic cell spread into adjacent tissue (e.g., normal follicles, lymphoid sinuses), and bcl-2 protein expression tended to be highest in these migratory cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 PMID:1374590

  11. 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

  12. Unidirectional crosstalk between Bcl-xL and Bcl-2 enhances the angiogenic phenotype of endothelial cells.

    PubMed

    Karl, E; Zhang, Z; Dong, Z; Neiva, K G; Soengas, M S; Koch, A E; Polverini, P J; Núñez, G; Nör, J E

    2007-09-01

    Expression of Bcl-x(L) correlates with the clinical outcomes of patients with cancer. While the role of Bcl-2 in angiogenesis is becoming increasingly evident, the function of Bcl-x(L) in angiogenesis is unclear. Here, we showed that epidermal growth factor (EGF) induces in vitro capillary sprouting and Bcl-x(L) expression in primary endothelial cells. Bcl-x(L)-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-x(L)), but not empty vector control cells, spontaneously organize into capillary-like sprouts. Searching for a mechanism to explain these responses, we observed that Bcl-x(L) induced expression of the pro-angiogenic chemokines CXC ligand-1 (CXCL1) and CXC ligand-8 (CXCL8), and that blockade of CXC receptor-2 (CXCR2) signaling inhibited spontaneous sprouting of HDMEC-Bcl-x(L). Bcl-x(L) led to Bcl-2 upregulation, but Bcl-2 did not upregulate Bcl-x(L), suggesting the existence of a unidirectional crosstalk from Bcl-x(L) to Bcl-2. EGF and Bcl-x(L) activate the mitogen-activated protein kinase/ERK pathway resulting in upregulation of vascular endothelial growth factor (VEGF), a known inducer of Bcl-2 in endothelial cells. Inhibition of VEGF receptor signaling in HDMEC-Bcl-x(L) prevented Bcl-2 upregulation and demonstrated the function of a VEGF-mediated autocrine loop. Bcl-2 downregulation by RNAi blocked CXCL1 and CXCL8 expression downstream of Bcl-x(L), and markedly decreased angiogenesis in vivo. We conclude that Bcl-x(L) functions as a pro-angiogenic signaling molecule controlling Bcl-2 and VEGF expression. These results emphasize a complex interplay between Bcl-2 family members beyond their classical roles in apoptosis.

  13. Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins.

    PubMed

    Aouacheria, Abdel; Baghdiguian, Stephen; Lamb, Heather M; Huska, Jason D; Pineda, Fernando J; Hardwick, J Marie

    2017-04-28

    The morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl-2 protein family. Connecting mitochondrial dynamics with life-death pathway forks may arise from the intersection of Bcl-2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl-2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl-2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL-2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl-2 family proteins integrate into some of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Mechanisms of anti-leukemic activity of the Bcl-2 homology domain-3 mimetic S1.

    PubMed

    Liu, Yubo; Li, Zhiqiang; Song, Ting; Xue, Zuguang; Zhang, Zhichao

    2013-09-01

    Most of leukemia exhibits inherent overexpressed Bcl-2-like proteins. Small molecule S1 is a BH3 mimetic discovered by our previous studies. The aim of this study is to dissect the details of apoptosis signaling induced by S1 in acute myeloid leukaemia (AML) cells and to provide a molecular basis for the use of S1 in AML treatment. The anti-leukemic activity of S1 was evaluated in three cultured AML cell lines and eight patient samples. S1 induced apoptosis via an intrinsic apoptosis pathway by the disruption of protein-protein interactions of Bcl-2 family members and triggered the activation of Bax and Bak in AML cells. For the first time, we report that S1 can release pro-apoptotic protein from Bcl-XL and selectively inhibits colony formation of primary AML cells. Bak activation and release determined S1 sensitivity in AML cells. Furthermore, S1-induced apoptosis was largely reduced in cells with shRNA-mediated downregulation of Bak but not Bax. The combination of S1 with PD98059 can inhibit Bcl-2 phosphorylation and enhance Bak release from Bcl-2. Our study identified Bak as a key mediator of S1-induced intrinsic apoptosis in AML cells. Moreover, our data suggest that Bcl-2 phosphorylation plays an anti-apoptotic role in S1-induced apoptosis. This study could contribute not only to the future clinical development of S1, but also the rational use of other pan-Bcl-2 inhibitors, alone or in combination with kinase inhibitor-based strategies. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  15. The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses

    SciTech Connect

    Muscarella, Donna E. Bloom, Stephen E.

    2008-04-01

    The c-Jun N-terminal kinase (JNK) pathway can play paradoxical roles as either a pro-survival or a pro-cell death pathway depending on type of stress and cell type. The goal of the present study was to determine the role of JNK pathway signaling for regulating B-cell apoptosis in two important but contrasting situations-global proteotoxic damage, induced by arsenite and hyperthermia, versus specific microtubule inhibition, induced by the anti-cancer drug vincristine, using the EW36 B-cell line. This cell line over-expresses the Bcl-2 protein and is a useful model to identify treatments that can overcome multi-drug resistance in lymphoid cells. Exposure of EW36 B-cells to arsenite or lethal hyperthermia resulted in activation of the JNK pathway and induction of apoptosis. However, pharmacological inhibition of the JNK pathway did not inhibit apoptosis, indicating that JNK pathway activation is not required for apoptosis induction by these treatments. In contrast, vincristine treatment of EW36 B-cells resulted in JNK activation and apoptosis that was suppressed by JNK inhibition. A critical difference between the two types of stress treatments was that only vincristine-induced JNK activation resulted in phosphorylation of Bcl-2 at threonine-56, a modification that can block its anti-apoptotic function. Importantly, Bcl-2 phosphorylation was attenuated by JNK inhibition implicating JNK as the upstream kinase. Furthermore, arsenite and hyperthermia treatments activated a p53/p21 pathway associated with apoptosis induction, whereas vincristine did not activate this pathway. These results reveal two stress-activated pathways, one JNK-dependent and another JNK-independent, either of which can bypass Bcl-2 mediated resistance, resulting in cell death.

  16. Distinct regions of the interleukin-7 receptor regulate different Bcl2 family members.

    PubMed

    Jiang, Qiong; Li, Wen Qing; Hofmeister, Robert R; Young, Howard A; Hodge, David R; Keller, Jonathan R; Khaled, Annette R; Durum, Scott K

    2004-07-01

    The antiapoptotic function of the interleukin-7 (IL-7) receptor is related to regulation of three members of the Bcl2 family: synthesis of Bcl2, phosphorylation of Bad, and cytosolic retention of Bax. Here we show that, in an IL-7-dependent murine T-cell line, different regions of the IL-7 receptor initiate the signal transduction pathways that regulate these proteins. Both Box1 and Y449 are required to signal Bcl2 synthesis and Bax cytosolic retention. This suggests a sequential model in which Jak1, which binds to Box1, is first activated and then phosphorylates Y449, leading to Bcl2 and Bax regulation, accounting for approximately 90% of the survival function. Phosphorylation of Bad required Box1 but not Y449, suggesting that Jak1 also initiates an additional signaling cascade that accounts for approximately 10% of the survival function. Stat5 was activated from the Y449 site but only partially accounted for the survival signal. Proliferation required both Y449 and Box1. Thymocyte development in vivo showed that deletion of Y449 eliminated 90% of alphabeta T-cell development and completely eliminated gammadelta T-cell development, whereas deleting Box 1 completely eliminated both alphabeta and gammadelta T-cell development. Thus the IL-7 receptor controls at least two distinct pathways, in addition to Stat5, that are required for cell survival.

  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

    2013-09-23

    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.

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

    PubMed

    Wallgren, Marcus; Lidman, Martin; Pedersen, Anders; Brännström, Kristoffer; Karlsson, B Göran; Gröbner, Gerhard

    2013-01-01

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

  19. Sigma-1 Receptors Regulate Bcl-2 Expression by Reactive Oxygen Species-Dependent Transcriptional Regulation of Nuclear Factor κB

    PubMed Central

    Meunier, Johann

    2010-01-01

    The expression of Bcl-2, the major antiapoptotic member of the Bcl-2 family, is under complex controls of several factors, including reactive oxygen species (ROS). The σ-1 receptor (Sig-1R), which was recently identified as a novel molecular chaperone at the mitochondria-associated endoplasmic reticulum membrane (MAM), has been shown to exert robust cellular protective actions. However, mechanisms underlying the antiapoptotic action of the Sig-1R remain to be clarified. Here, we found that the Sig-1R promotes cellular survival by regulating the Bcl-2 expression in Chinese hamster ovary cells. Although both Sig-1Rs and Bcl-2 are highly enriched at the MAM, Sig-1Rs neither associate physically with Bcl-2 nor regulate stability of Bcl-2 proteins. However, Sig-1Rs tonically regulate the expression of Bcl-2 proteins. Knockdown of Sig-1Rs down-regulates whereas overexpression of Sig-1Rs up-regulates bcl-2 mRNA, indicating that the Sig-1R transcriptionally regulates the expression of Bcl-2. The effect of Sig-1R small interfering RNA down-regulating Bcl-2 was blocked by ROS scavengers and by the inhibitor of the ROS-inducible transcription factor nuclear factor κB (NF-κB). Knockdown of Sig-1Rs up-regulates p105, the precursor of NF-κB, while concomitantly decreasing inhibitor of nuclear factor-κBα. Sig-1R knockdown also accelerates the conversion of p105 to the active form p50. Lastly, we showed that knockdown of Sig-1Rs potentiates H2O2-induced apoptosis; the action is blocked by either the NF-κB inhibitor oridonin or overexpression of Bcl-2. Thus, these findings suggest that Sig-1Rs promote cell survival, at least in part, by transcriptionally regulating Bcl-2 expression via the ROS/NF-κB pathway. PMID:19855099

  20. The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2

    PubMed Central

    1995-01-01

    A family of genes related to the bcl-2 protooncogene has recently emerged. One member of this family, mcl-1, was cloned from a human myeloblastic leukemia cell line (ML-1) undergoing differentiation. The intracellular localization of mcl-1, as well as the kinetics of its expression during differentiation, have now been studied. These studies show that the intracellular distribution of mcl-1 overlaps with, but is not identical to, that of bcl-2: mcl-1 is similar to bcl-2 in that the mcl-1 protein has a prominent mitochondrial localization, and in that it associates with membranes through its carboxyl hydrophobic tail. mcl- 1 differs from bcl-2, however, in its relative distribution among other (nonmitochondrial/heavy membrane) compartments, mcl-1 also being abundant in the light membrane fraction of immature ML-1 cells while bcl-2 is abundant in the nuclear fraction. Similarly, in differentiating ML-1 cells, the timing of expression of mcl-1 overlaps with, but is not identical to, that of bcl-2: the mcl-1 protein increases rapidly as cells initiate differentiation, and mcl-1 is a labile protein. In contrast, bcl-2 decreases gradually as cells complete differentiation. Overall, the mcl-1 and bcl-2 proteins have some properties in common and others tht are distinct. A burst of expression of mcl-1, prominently associated with mitochondria, complements the continued expression of bcl-2 in ML-1 cells differentiating along the monocyte/macrophage pathway. PMID:7896880

  1. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease

    PubMed Central

    1996-01-01

    Bcl-2 belongs to a family of apoptosis-regulatory proteins which incorporate into the outer mitochondrial as well as nuclear membranes. The mechanism by which the proto-oncogene product Bcl-2 inhibits apoptosis is thus far elusive. We and others have shown previously that the first biochemical alteration detectable in cells undergoing apoptosis, well before nuclear changes become manifest, is a collapse of the mitochondrial inner membrane potential (delta psi m), suggesting the involvement of mitochondrial products in the apoptotic cascade. Here we show that mitochondria contain a pre-formed approximately 50-kD protein which is released upon delta psi m disruption and which, in a cell-free in vitro system, causes isolated nuclei to undergo apoptotic changes such as chromatin condensation and internucleosomal DNA fragmentation. This apoptosis-inducing factor (AIF) is blocked by N- benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk), an antagonist of interleukin-1 beta-converting enzyme (ICE)-like proteases that is also an efficient inhibitor of apoptosis in cells. We have tested the effect of Bcl-2 on the formation, release, and action of AIF. When preventing mitochondrial permeability transition (which accounts for the pre-apoptotic delta psi m disruption in cells), Bcl-2 hyperexpressed in the outer mitochondrial membrane also impedes the release of AIF from isolated mitochondria in vitro. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitochondria and in mitochondria from Bcl-2- hyperexpressing cells. Furthermore, the presence of Bcl-2 in the nuclear membrane does not interfere with the action of AIF on the nucleus, nor does Bcl-2 hyperexpression protect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protease in mitochondria. PMID:8879205

  2. ABT-199 (venetoclax) and BCL-2 inhibitors in clinical development.

    PubMed

    Cang, Shundong; Iragavarapu, Chaitanya; Savooji, John; Song, Yongping; Liu, Delong

    2015-11-20

    With the advent of new agents targeting CD20, Bruton's tyrosine kinase, and phosphoinositol-3 kinase for chronic lymphoid leukemia (CLL), more treatment options exist than ever before. B-cell lymphoma-2 (BCL-2) plays a major role in cellular apoptosis and is a druggable target. Small molecule inhibitors of BCL-2 are in active clinical studies. ABT-199 (venetoclax, RG7601, GDC-0199) has been granted breakthrough designation by FDA for relapsed or refractory CLL with 17p deletion. In this review, we summarized the latest clinical development of ABT-199/venetoclax and other novel agents targeting the BCL-2 proteins.

  3. [Bcl-2 expression following the brain concussion in rats].

    PubMed

    Zhu, Xu-yang; Wang, Feng; Fang, Wei-hua; Wu, Mao-wang

    2007-02-15

    To evaluate the expression of Bcl-2 protein after brain concussion. Expression levels of Bel-2 protein in cortex, pontine and cerebellum of rats were investigated using immunohistochemistry. There was no expression of Bcl-2 protein in control group seen. The expression of Bcl-2 protein in brain concussion groups was detected at l hour, and the expression level reached its peak 4 days after the concussion and then declined gradually. Our findings suggest that the detection of Bel-2 protein could be an indicator for diagnosis of brain concussion and for estimation of the post injury time interval.

  4. Role of the Bcl-2 gene family in prostate cancer progression and its implications for therapeutic intervention.

    PubMed Central

    Chaudhary, K S; Abel, P D; Lalani, E N

    1999-01-01

    Prostate cancer (PC) is an escalating health burden in the western world. A large number of patients still present with extraprostatic (i.e., T3/T4, N0, M0/M1 or any T category and M1 disease or involved lymph nodes) and therefore incurable disease. Since the work of Huggins in 1940, there have been no major therapeutic advances and androgen ablation remains the best treatment option for extraprostatic androgen-responsive PC. Eighty to ninety percent of PC patients respond well to this form of treatment initially. After a median time of approximately 2 years, however, relapse to an androgen-independent (AI) state occurs, followed by death after a further median 6 months. Androgen ablation is rarely curative. The major molecular defect in extraprostatic and AI PC is the inability of PC cells to initiate apoptosis in response to a variety of stimuli, including different forms of androgen ablation and cytotoxic agents. The balance between cellular proliferation and cell death is regulated by multiple genes or families of genes through the cell cycle. The exact mechanisms governing this intricate and complex process are as yet not fully understood. One family of genes involved in cell survival/death control is the Bcl-2 gene family, which consists of homologous proteins that function to regulate distal and crucial commitment steps of the apoptotic pathway. The Bcl-2 family constitutes both agonists and antagonists of apoptosis that function at least in part through protein-protein interactions between various members of the family. The final outcome depends on the relative ratio of death agonists and antagonists. Bcl-2 expression has been closely associated with the AI phenotype of PC. Cytotoxic chemotherapy may be used as palliative therapy in AI PC but has not been found effective. Most chemotherapeutic cytotoxic agents induce apoptosis in cancer cells by direct and indirect action on the cell cycle. In vitro and in vivo studies have established that Bcl-2 expression

  5. 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.

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

    PubMed

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

    2013-10-18

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

  7. Identification of expression signatures predictive of sensitivity to the Bcl-2 family member inhibitor ABT-263 in small cell lung carcinoma and leukemia/lymphoma cell lines.

    PubMed

    Tahir, Stephen K; Wass, John; Joseph, Mary K; Devanarayan, Viswanath; Hessler, Paul; Zhang, Haichao; Elmore, Steve W; Kroeger, Paul E; Tse, Christin; Rosenberg, Saul H; Anderson, Mark G

    2010-03-01

    ABT-263 inhibits the antiapoptotic proteins Bcl-2, Bcl-x(L), and Bcl-w and has single-agent efficacy in numerous small cell lung carcinoma (SCLC) and leukemia/lymphoma cell lines in vitro and in vivo. It is currently in clinical trials for treating patients with SCLC and various leukemia/lymphomas. Identification of predictive markers for response will benefit the clinical development of ABT-263. We identified the expression of Bcl-2 family genes that correlated best with sensitivity to ABT-263 in a panel of 36 SCLC and 31 leukemia/lymphoma cell lines. In cells sensitive to ABT-263, expression of Bcl-2 and Noxa is elevated, whereas expression of Mcl-1 is higher in resistant cells. We also examined global expression differences to identify gene signature sets that correlated with sensitivity to ABT-263 to generate optimal signature sets predictive of sensitivity to ABT-263. Independent cell lines were used to verify the predictive power of the gene sets and to refine the optimal gene signatures. When comparing normal lung tissue and SCLC primary tumors, the expression pattern of these genes in the tumor tissue is most similar to sensitive SCLC lines, whereas normal tissue is most similar to resistant SCLC lines. Most of the genes identified using global expression patterns are related to the apoptotic pathway; however, all but Bcl-rambo are distinct from the Bcl-2 family. This study leverages global expression data to identify key gene expression patterns for sensitivity to ABT-263 in SCLC and leukemia/lymphoma and may provide guidance in the selection of patients in future clinical trials.

  8. Zinc induces apoptosis on cervical carcinoma cells by p53-dependent and -independent pathway.

    PubMed

    Bae, Seog Nyeon; Lee, Keun Ho; Kim, Jin Hwi; Lee, Sung Jong; Park, Lae Ok

    2017-02-26

    There is evidence that the mineral zinc is involved in the apoptotic cell death of various carcinoma cells. In this study, we aim to determine whether zinc in the form of CIZAR induces apoptosis in cervical carcinoma cells by increasing intracellular zinc concentration. CaSki and HeLa cervical carcinoma cells and HPV-16 DNA-transformed keratinocyte (CRL2404) were treated with different concentrations of CIZAR. The cell viability test was carried out, the intracellular level of zinc was determined, and apoptosis was confirmed by flow cytometry after propidium iodide (PI) staining and fluorescence microscopy under DAPI staining. The expression of cell-cycle regulators was analyzed by Western blot, including the knock down of p53 and expression of HPV E6 and E7 genes by RT-PCR. Intracellular zinc accumulation induced the down-regulation of E6/E7 proteins through targeting of the specific transcriptional factors in the upstream regulatory region. p53 was induced after CIZAR treatment and p53-dependent apoptosis did not occur after knock down by p53 siRNA. In cervical carcinoma cells, regardless of HPV-infection, CIZAR induces apoptosis by the activation of the p53-independent pathways through the up-regulation of p21waf1, the down-regulation of c-Myc, and by decreasing the Bcl-2/Bax ratio. CIZAR induces apoptosis not only through the restoration of p53/Rb-dependent pathways in HPV-positive cells, but also through the activation of p53/Rb-independent pathways and the mitochondrial death-signal pathway in cervical carcinoma cells regardless of HPV-infection. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis.

    PubMed

    Meresman, G F; Vighi, S; Buquet, R A; Contreras-Ortiz, O; Tesone, M; Rumi, L S

    2000-10-01

    To evaluate and compare spontaneous apoptosis and Bcl-2 and Bax expression in eutopic endometrium from women with and without endometriosis. Apoptosis and Bcl-2 and Bax expression were examined in eutopic endometrium from women with and without endometriosis. Instituto de Biología y Medicina Experimental-CONICET, Department of Gynecology and Department of Gynecological Pathology, Clínicas University Hospital, Buenos Aires, Argentina. Women with untreated endometriosis (n = 14) and controls (n = 16). Collection of endometrial samples during diagnostic or therapeutic laparoscopy. Apoptotic cells were detected with use of the dUTP nick-end labeling (TUNEL) assay; Bcl-2 and Bax expressions were assessed with use of immunohistochemical techniques. Spontaneous apoptosis was significantly lower in eutopic endometrium from patients with endometriosis, compared with healthy controls (2.26 +/- 0.53 and 9.37 +/- 1.69 apoptotic cells/field, respectively) and was independent of cycle phase. An increased expression of Bcl-2 protein was found in proliferative eutopic endometrium from patients with endometriosis. Bax expression was absent in proliferative endometrium, whereas there was an increase in its expression in secretory endometrium from both patients and controls. Women with endometriosis show decreased number of apoptotic cells in eutopic endometrium. The abnormal survival of endometrial cells may result in their continuing growth into ectopic locations.

  10. Bruton's tyrosine kinase inhibition increases BCL-2 dependence and enhances sensitivity to venetoclax in chronic lymphocytic leukemia.

    PubMed

    Deng, J; Isik, E; Fernandes, S M; Brown, J R; Letai, A; Davids, M S

    2017-02-14

    Although the BTK inhibitor ibrutinib has transformed the management of patients with chronic lymphocytic leukemia (CLL), it does not induce substantial apoptosis in vitro, and as such the mechanisms underlying its ability to kill CLL cells are not well understood. Acalabrutinib, a more specific BTK inhibitor now in development, also appears to be highly effective in CLL, but the connection of its mechanism with CLL cell death is also unclear. Using dynamic BH3 profiling, we analyzed alterations in the function of the mitochondrial apoptotic pathway induced by ibrutinib and acalabrutinib. We studied CLL patient samples treated ex vivo with both drugs, as well as primary samples from CLL patients on clinical trials of both drugs. We found that BTK inhibition enhances mitochondrial BCL-2 dependence without significantly altering overall mitochondrial priming. Enhancement of BCL-2 dependence was accompanied by an increase in the pro-apoptotic protein BIM. In contrast, treatment with the selective BCL-2 inhibitor venetoclax enhanced overall mitochondrial priming without increasing BCL-2 dependence. Pre-treatment of CLL cells with either BTK inhibitor, whether ex vivo or in vivo in patients, enhanced killing by venetoclax. Our data suggest that BTK inhibition enhances mitochondrial BCL-2 dependence, supporting the ongoing development of clinical trials combining BTK and BCL-2 inhibition.Leukemia advance online publication, 14 February 2017; doi:10.1038/leu.2017.32.

  11. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death.

    PubMed

    Yang, E; Zha, J; Jockel, J; Boise, L H; Thompson, C B; Korsmeyer, S J

    1995-01-27

    To extend the mammalian cell death pathway, we screened for further Bcl-2 interacting proteins. Both yeast two-hybrid screening and lambda expression cloning identified a novel interacting protein, Bad, whose homology to Bcl-2 is limited to the BH1 and BH2 domains. Bad selectively dimerized with Bcl-xL as well as Bcl-2, but not with Bax, Bcl-xs, Mcl-1, A1, or itself. Bad binds more strongly to Bcl-xL than Bcl-2 in mammalian cells, and it reversed the death repressor activity of Bcl-xL, but not that of Bcl-2. When Bad dimerized with Bcl-xL, Bax was displaced and apoptosis was restored. When approximately half of Bax was heterodimerized, death was inhibited. The susceptibility of a cell to a death signal is determined by these competing dimerizations in which levels of Bad influence the effectiveness of Bcl-2 versus Bcl-xL in repressing death.

  12. Bcl-2-related protein family gene expression during oligodendroglial differentiation.

    PubMed

    Itoh, Takayuki; Itoh, Aki; Pleasure, David

    2003-06-01

    Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.

  13. In vivo expression of p53 and Bcl-2 and their role in programmed cell death in premalignant and malignant lung lesions.

    PubMed

    Koty, Patrick P; Zhang, Haifan; Franklin, Wilbur A; Yousem, Samuel A; Landreneau, Rodney; Levitt, Mark L

    2002-02-01

    Forty-four specimens of non-malignant and malignant human lung tissue, taken from patients with non-small cell lung cancer (NSCLC), were examined for the expression of wild-type p53, mutant p53, and bcl-2 and the occurrence of programmed cell death (apoptosis). Wild-type p53 expression peaked in peritumoral and metaplastic samples, whereas mutant p53, bcl-2 and apoptosis were first detected in metaplasia and increased with progression to carcinoma. Bcl-2 positive samples had lower levels of apoptosis than bcl-2 negative samples and was independent of wild-type or mutant p53 expression. These results suggest that the over-expression of wild-type p53 may be an early cellular response to an alteration in normal cellular homeostasis. The ensuing increase in apoptosis appears to be relatively independent of mutant or wild-type p53 expression, but does not occur in cells expressing bcl-2.

  14. Bcl2 induces DNA replication stress by inhibiting ribonucleotide reductase.

    PubMed

    Xie, Maohua; Yen, Yun; Owonikoko, Taofeek K; Ramalingam, Suresh S; Khuri, Fadlo R; Curran, Walter J; Doetsch, Paul W; Deng, Xingming

    2014-01-01

    DNA replication stress is an inefficient DNA synthesis process that leads replication forks to progress slowly or stall. Two main factors that cause replication stress are alterations in pools of deoxyribonucleotide (dNTP) precursors required for DNA synthesis and changes in the activity of proteins required for synthesis of dNTPs. Ribonucleotide reductase (RNR), containing regulatory hRRM1 and catalytic hRRM2 subunits, is the enzyme that catalyzes the conversion of ribonucleoside diphosphates (NDP) to deoxyribonucleoside diphosphates (dNDP) and thereby provides dNTP precursors needed for the synthesis of DNA. Here, we demonstrate that either endogenous or exogenous expression of Bcl2 results in decreases in RNR activity and intracellular dNTP, retardation of DNA replication fork progression, and increased rate of fork asymmetry leading to DNA replication stress. Bcl2 colocalizes with hRRM1 and hRRM2 in the cytoplasm and directly interacts via its BH4 domain with hRRM2 but not hRRM1. Removal of the BH4 domain of Bcl2 abrogates its inhibitory effects on RNR activity, dNTP pool level, and DNA replication. Intriguingly, Bcl2 directly inhibits RNR activity by disrupting the functional hRRM1/hRRM2 complex via its BH4 domain. Our findings argue that Bcl2 reduces intracellular dNTPs by inhibiting ribonucleotide reductase activity, thereby providing insight into how Bcl2 triggers DNA replication stress.

  15. Non-apoptotic functions of BCL-2 family proteins.

    PubMed

    Gross, Atan; Katz, Samuel G

    2017-02-24

    The BCL-2 family proteins are major regulators of the apoptosis process, but the mechanisms by which they regulate this process are only partially understood. It is now well documented that these proteins play additional non-apoptotic roles that are likely to be related to their apoptotic roles and to provide important clues to cracking their mechanisms of action. It seems that these non-apoptotic roles are largely related to the activation of cellular survival pathways designated to maintain or regain cellular survival, but, if unsuccessful, will switch over into a pro-apoptotic mode. These non-apoptotic roles span a wide range of processes that include the regulation of mitochondrial physiology (metabolism, electron transport chain, morphology, permeability transition), endoplasmic reticulum physiology (calcium homeostasis, unfolded protein response (UPR)), nuclear processes (cell cycle, DNA damage response (DDR)), whole-cell metabolism (glucose and lipid), and autophagy. Here we review all these different non-apoptotic roles, make an attempt to link them to the apoptotic roles, and present many open questions for future research directions in this fascinating field.Cell Death and Differentiation advance online publication, 24 February 2017; doi:10.1038/cdd.2017.22.

  16. Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.

    PubMed Central

    Wilson, B E; Mochon, E; Boxer, L M

    1996-01-01

    Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during

  17. Quercetin induces apoptosis via caspase activation, regulation of Bcl-2, and inhibition of PI-3-kinase/Akt and ERK pathways in a human hepatoma cell line (HepG2).

    PubMed

    Granado-Serrano, Ana Belén; Martín, María Angeles; Bravo, Laura; Goya, Luis; Ramos, Sonia

    2006-11-01

    Dietary polyphenols have been associated with the reduced risk of chronic diseases such as cancer, but the precise underlying mechanism of protection remains unclear. The aim of this study was to investigate the effect of quercetin on the activation of the apoptotic pathway in a human hepatoma cell line (HepG2). Treatment of cells for 18 h with quercetin induced cell death in a dose-dependent manner; however, a shorter treatment (4 h) had no effect on cell viability. Incubation of HepG2 cells with quercetin for 18 h induced apoptosis by the activation of caspase-3 and -9, but not caspase-8. Moreover, this flavonoid decreased the Bcl-xL:Bcl-xS ratio and increased translocation of Bax to the mitochondrial membrane. A sustained inhibition of the major survival signals, Akt and extracellular regulated kinase (ERK), also occurred in quercetin-treated cells. These data suggest that quercetin may induce apoptosis by direct activation of caspase cascade (mitochondrial pathway) and by inhibiting survival signaling in HepG2.

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

    PubMed

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

    2017-01-01

    To investigate the underlying mechanism of neurotoxicity of cadmium, we examined the effects of intraperitoneal injection of cadmium on messenger RNA (mRNA) expression of Bcl-2 (B-cell lymphoma 2) and Bax (Bcl2-associated x) genes and caspase-3/7 activation in rat hippocampus and frontal cortex. Twenty-eight male Wistar rats weighing 200-250 g were randomly divided into four groups. Control group received saline and three other groups received cadmium at doses of 1, 2 and 4 mg/kg (body weight) for 15 successive days. One day after the last injection, the hippocampus and frontal cortex were dissected and removed and then the expression of Bcl-2 and Bax genes was evaluated using real-time polymerase chain reaction and apoptotic studies was done using caspase-3/7 activation assay. Cadmium reduced the mRNA level of Bcl-2 in the control group at doses of 1 ( p < 0.01), 2 and 4 mg/kg ( p < 0.001) in rat hippocampus and cortex cells. The mRNA level of Bax increased significantly compared to the control group at the doses of 1 ( p < 0.05), 2 and 4 mg/kg ( p < 0.001) in rat hippocampus. The mRNA level of Bax was increased significantly compared to the control group at the doses of 2 and 4 mg/kg ( p < 0.001) in rat cortex cells. Cadmium increased caspase-3/7 activity at doses of 1, 2 and 4 mg/kg in rat hippocampus. Caspase-3/7 activity was increased significantly at dose of 4 mg/kg in rat cortex. This decreased Bcl-2/Bax mRNA ratio induces cell apoptosis. Apoptotic effect of cadmium may be through the mitochondrial pathway by the activation of caspase-3/7.

  19. Resistance to BH3 mimetic S1 in SCLC cells that up-regulate and phosphorylate Bcl-2 through ERK1/2.

    PubMed

    Liu, Yubo; Zhang, Zhichao; Song, Ting; Liang, Furong; Xie, Mingzhou; Sheng, Hongkun

    2013-08-01

    B cell lymphoma 2 (Bcl-2) is a central regulator of cell survival that is overexpressed in the majority of small-cell lung cancers (SCLC) and contributes to both malignant transformation and therapeutic resistance. The purpose of this work was to study the key factors that determine the sensitivity of SCLC cells to Bcl-2 homology domain-3 (BH3) mimetic S1 and the mechanism underlying the resistance of BH3 mimetics. Western blot was used to evaluate the contribution of Bcl-2 family members to the cellular response of SCLC cell lines to S1. Acquired resistant cells were derived from initially sensitive H1688 cells. Quantitative PCR and gene silencing were performed to investigate Bcl-2 up-regulation. A progressive increase in the relative levels of Bcl-2 and phosphorylated Bcl-2 (pBcl-2) characterized the increased de novo and acquired resistance of SCLC cell lines. Furthermore, acute treatment of S1 induced Bcl-2 expression and phosphorylation. We showed that BH3 mimetics, including S1 and ABT-737, induced endoplasmic reticulum (ER) stress and then activated MAPK/ERK pathway. The dual function of MAPK/ERK pathway in defining BH3 mimetics was illustrated; ERK1/2 activation leaded to Bcl-2 transcriptional up-regulation and sustained phosphorylation in naïve and acquired resistant SCLC cells. pBcl-2 played a key role in creating resistance of S1 and ABT-737 not only by sequestrating pro-apoptotic proteins, but also sequestrating a positive feedback to promote ERK1/2 activation. These results provide significant novel insights into the molecular mechanisms for crosstalk between ER stress and endogenously apoptotic pathways in SCLC following BH3 mimetics treatment. © 2013 The British Pharmacological Society.

  20. Resistance to BH3 mimetic S1 in SCLC cells that up-regulate and phosphorylate Bcl-2 through ERK1/2

    PubMed Central

    Liu, Yubo; Zhang, Zhichao; Song, Ting; Liang, Furong; Xie, Mingzhou; Sheng, Hongkun

    2013-01-01

    Background and Purpose B cell lymphoma 2 (Bcl-2) is a central regulator of cell survival that is overexpressed in the majority of small-cell lung cancers (SCLC) and contributes to both malignant transformation and therapeutic resistance. The purpose of this work was to study the key factors that determine the sensitivity of SCLC cells to Bcl-2 homology domain-3 (BH3) mimetic S1 and the mechanism underlying the resistance of BH3 mimetics. Experimental Approaches Western blot was used to evaluate the contribution of Bcl-2 family members to the cellular response of SCLC cell lines to S1. Acquired resistant cells were derived from initially sensitive H1688 cells. Quantitative PCR and gene silencing were performed to investigate Bcl-2 up-regulation. Key Results A progressive increase in the relative levels of Bcl-2 and phosphorylated Bcl-2 (pBcl-2) characterized the increased de novo and acquired resistance of SCLC cell lines. Furthermore, acute treatment of S1 induced Bcl-2 expression and phosphorylation. We showed that BH3 mimetics, including S1 and ABT-737, induced endoplasmic reticulum (ER) stress and then activated MAPK/ERK pathway. The dual function of MAPK/ERK pathway in defining BH3 mimetics was illustrated; ERK1/2 activation leaded to Bcl-2 transcriptional up-regulation and sustained phosphorylation in naïve and acquired resistant SCLC cells. pBcl-2 played a key role in creating resistance of S1 and ABT-737 not only by sequestrating pro-apoptotic proteins, but also sequestrating a positive feedback to promote ERK1/2 activation. Conclusions and Implications These results provide significant novel insights into the molecular mechanisms for crosstalk between ER stress and endogenously apoptotic pathways in SCLC following BH3 mimetics treatment. PMID:23651505

  1. Bcl-2 and caspase-3 are major regulators in Agaricus blazei-induced human leukemic U937 cell apoptosis through dephoshorylation of Akt.

    PubMed

    Jin, Cheng-Yun; Moon, Dong-Oh; Choi, Yung Hyun; Lee, Jae-Dong; Kim, Gi-Young

    2007-08-01

    Agaricus blazei is a medicinal mushroom that possesses antimetastatic, antitumor, antimutagenic, and immunostimulating effects. However, the molecular mechanisms involved in A. blazei-mediated apoptosis remain unclear. In the present study, to elucidate the role of the Bcl-2 in A. blazei-mediated apoptosis, U937 cells were transfected with either empty vector (U937/vec) or vector containing cDNA encoding full-length Bcl-2 (U937/Bcl-2). As compared with U937/vec, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment of U937/vec with 1.0-4.0 mg/ml of A. blazei extract (ABE) for 24 h resulted in a significant induction of morphologic features indicative of apoptosis. In contrast, U937/Bcl-2 exposed to the same ABE treatment only exhibited a slight induction of apoptotic features. ABE-induced apoptosis was accompanied by downregulation of antiapoptotic proteins such as X-linked inhibitor of apoptosis protein (XIAP), inhibitor of apoptosis protein (cIAP)-2 and Bcl-2, activation of caspase-3, and cleavage of poly(ADP-ribose)polymerase (PARP). Ectopic expression of Bcl-2 was associated with significantly induced expression of antiapoptotic proteins, such as cIAP-2 and Bcl-2, but not XIAP. Ectopic expression of Bcl-2 also reduced caspase-3 activation and PARP cleavage in ABE treated U937 cells. Furthermore, treatment with the caspase-3 inhibitor z-DEVD-fmk was sufficient to restore cell viability following ABE treatment. This increase in viability was ascribed to downregulation of caspase-3 and blockage of PARP and PLC-gamma cleavage. ABE also triggered the downregulation of Akt, and combined treatment with LY294002 (an inhibitor of Akt) significantly decreased cell viability. The results indicated that major regulators of ABE-induced apoptosis in human leukemic U937 cells are Bcl-2 and caspase-3, which are associated with dephosphorylation of the Akt signal pathway.

  2. Bcl-2 expression during T-cell development: early loss and late return occur at specific stages of commitment to differentiation and survival.

    PubMed Central

    Gratiot-Deans, J; Merino, R; Nuñez, G; Turka, L A

    1994-01-01

    During T-cell development CD3-CD4-CD8- (double-negative) thymocytes proliferative and produce an enormous number of CD3loCD4+CD8+ (double-positive) thymocytes which are destined to die intrathymically unless rescued by positive selection. Those which survive become mature CD3hiCD4/8+ (single-positive) cells and are the precursor of peripheral blood lymphocytes. The product of the bcl-2 protooncogene has been implicated in preventing programmed cell death and is required for prolonged lymphocyte survival following maturation. Previously we and others have reported that Bcl-2 protein expression is biphasic, being high in proliferating double-negative stem cells, low in all double-positive thymocytes except for 1-5% of these cells, and restored in mature, single-positive thymocytes. However, it remained unclear which signaling and selection events regulate Bcl-2 during T-cell maturation. Now we have utilized four-color flow cytometry in normal and genetically altered mice for a detailed analysis of Bcl-2 expression as it relates to T-cell receptor (TCR) expression and positive selection. These studies show that (i) expression of a transgenic TCR in double-negative thymocytes does not lead to premature loss of Bcl-2; thus, Bcl-2 downregulation is not solely due to TCR expression; (ii) Bcl-2 expression is lost at the early transitional CD3-/loCD4-CD8+ stage, prior to expression of CD4; (iii) the Bcl-2+ double-positive thymocytes are those which have undergone positive selection; and (iv) upregulation of Bcl-2 during positive selection requires participation of the CD4 or CD8 co-receptor. These results demonstrate that Bcl-2 and TCR expression are regulated independently during T-cell development, and suggest a role for the CD4 or CD8 co-receptor in Bcl-2 induction during positive selection. Images PMID:7938012

  3. Bcl-2:Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch.

    PubMed

    Marquez, Rebecca T; Xu, Liang

    2012-01-01

    Cancer cells have developed novel mechanisms for evading chemotherapy-induced apoptosis and autophagy-associated cell death pathways. Upon the discovery that chemotherapeutics could target these cell death pathways in a manner that was not mutually exclusive, new discoveries about the interrelationship between these two pathways are emerging. Key proteins originally thought to be "autophagy-related proteins" are now found to be involved in either inducing or inhibiting apoptosis. Similarly, apoptosis inhibiting proteins can also block autophagy-associated cell death. One example is the complex formed by the autophagy protein, Beclin 1, and anti-apoptotic protein Bcl-2, which leads to inhibition of autophagy-associated cell death. Researchers have been investigating additional mechanisms that form/disrupt this complex in order to better design chemotherapeutics. This review will highlight the role Bcl-2 and Beclin 1 play in cancer development and drug resistance, as well as the role the Bcl-2:Beclin 1 complex in the switch between autophagy and apoptosis.

  4. Inhibition of Mcl-1 with the pan–Bcl-2 family inhibitor (–)BI97D6 overcomes ABT-737 resistance in acute myeloid leukemia

    PubMed Central

    Pan, Rongqing; Ruvolo, Vivian R.; Wei, Jun; Konopleva, Marina; Reed, John C.; Pellecchia, Maurizio; Ruvolo, Peter P.

    2015-01-01

    Overexpression of antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-xL, and Mcl-1 is widely associated with tumor initiation, progression, and chemoresistance. Furthermore, it has been demonstrated that Mcl-1 upregulation renders several types of cancers resistant to the Bcl-2/Bcl-xL inhibitors ABT-737 and ABT-263. The emerging importance of Mcl-1 in pathogenesis and drug resistance makes it a high-priority therapeutic target. In this study, we showed that inhibition of Mcl-1 with a novel pan–Bcl-2 inhibitor (–)BI97D6 potently induced apoptosis in acute myeloid leukemia (AML) cells. (–)BI97D6 induced hallmarks of mitochondrial apoptosis, disrupted Mcl-1/Bim and Bcl-2/Bax interactions, and stimulated cell death via the Bak/Bax-dependent mitochondrial apoptosis pathway, suggesting on-target mechanisms. As a single agent, this pan–Bcl-2 inhibitor effectively overcame AML cell apoptosis resistance mediated by Mcl-1 or by interactions with bone marrow mesenchymal stromal cells. (–)BI97D6 was also potent in killing refractory primary AML cells. Importantly, (–)BI97D6 killed AML leukemia stem/progenitor cells while largely sparing normal hematopoietic stem/progenitor cells. These findings demonstrate that pan–Bcl-2 inhibition by an Mcl-1–targeting inhibitor not only overcomes intrinsic drug resistance ensuing from functional redundancy of Bcl-2 proteins, but also abrogates extrinsic resistance caused by the protective tumor microenvironment. PMID:26045609

  5. Inhibition of Mcl-1 with the pan-Bcl-2 family inhibitor (-)BI97D6 overcomes ABT-737 resistance in acute myeloid leukemia.

    PubMed

    Pan, Rongqing; Ruvolo, Vivian R; Wei, Jun; Konopleva, Marina; Reed, John C; Pellecchia, Maurizio; Andreeff, Michael; Ruvolo, Peter P

    2015-07-16

    Overexpression of antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-xL, and Mcl-1 is widely associated with tumor initiation, progression, and chemoresistance. Furthermore, it has been demonstrated that Mcl-1 upregulation renders several types of cancers resistant to the Bcl-2/Bcl-xL inhibitors ABT-737 and ABT-263. The emerging importance of Mcl-1 in pathogenesis and drug resistance makes it a high-priority therapeutic target. In this study, we showed that inhibition of Mcl-1 with a novel pan-Bcl-2 inhibitor (-)BI97D6 potently induced apoptosis in acute myeloid leukemia (AML) cells. (-)BI97D6 induced hallmarks of mitochondrial apoptosis, disrupted Mcl-1/Bim and Bcl-2/Bax interactions, and stimulated cell death via the Bak/Bax-dependent mitochondrial apoptosis pathway, suggesting on-target mechanisms. As a single agent, this pan-Bcl-2 inhibitor effectively overcame AML cell apoptosis resistance mediated by Mcl-1 or by interactions with bone marrow mesenchymal stromal cells. (-)BI97D6 was also potent in killing refractory primary AML cells. Importantly, (-)BI97D6 killed AML leukemia stem/progenitor cells while largely sparing normal hematopoietic stem/progenitor cells. These findings demonstrate that pan-Bcl-2 inhibition by an Mcl-1-targeting inhibitor not only overcomes intrinsic drug resistance ensuing from functional redundancy of Bcl-2 proteins, but also abrogates extrinsic resistance caused by the protective tumor microenvironment.

  6. Targeting BCL-2-like Proteins to Kill Cancer Cells.

    PubMed

    Cory, Suzanne; Roberts, Andrew W; Colman, Peter M; Adams, Jerry M

    2016-08-01

    Mutations that impair apoptosis contribute to cancer development and reduce the effectiveness of conventional anti-cancer therapies. These insights and understanding of how the B cell lymphoma (BCL)-2 protein family governs apoptosis have galvanized the search for a new class of cancer drugs that target its pro-survival members by mimicking their natural antagonists, the BCL-2 homology (BH)3-only proteins. Successful initial clinical trials of the BH3 mimetic venetoclax/ABT-199, specific for BCL-2, have led to its recent licensing for refractory chronic lymphocytic leukemia and to multiple ongoing trials for other malignancies. Moreover, preclinical studies herald the potential of emerging BH3 mimetics targeting other BCL-2 pro-survival members, particularly myeloid cell leukemia (MCL)-1, for multiple cancer types. Thus, BH3 mimetics seem destined to become powerful new weapons in the arsenal against cancer. This review sketches the discovery of the BCL-2 family and its impact on cancer development and therapy; describes how interactions of family members trigger apoptosis; outlines the development of BH3 mimetic drugs; and discusses their potential to advance cancer therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. 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.

  8. Autophagy regulates the post-translational cleavage of BCL-2 and promotes neuronal survival.

    PubMed

    Lossi, Laura; Gambino, Graziana; Salio, Chiara; Merighi, Adalberto

    2010-05-18

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

  9. 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

  10. 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.

  11. An antiapoptotic Bcl-2 family protein index predicts the response of leukaemic cells to the pan-Bcl-2 inhibitor S1

    PubMed Central

    Zhang, Z; Liu, Y; Song, T; Xue, Z; Shen, X; Liang, F; Zhao, Y; Li, Z; Sheng, H

    2013-01-01

    Background: Bcl-2-like members have been found to be inherently overexpressed in many types of haematologic malignancies. The small-molecule S1 is a BH3 mimetic and a triple inhibitor of Bcl-2, Mcl-1 and Bcl-XL. Methods: The lethal dose 50 (LD50) values of S1 in five leukaemic cell lines and 41 newly diagnosed leukaemia samples were tested. The levels of Bcl-2 family members and phosphorylated Bcl-2 were semiquantitatively measured by western blotting. The interactions between Bcl-2 family members were tested by co-immunoprecipitation. The correlation between the LD50 and expression levels of Bcl-2 family members, alone or in combination, was analysed. Results: S1 exhibited variable sensitivity with LD50 values ranging >2 logs in both established and primary leukaemic cells. The ratio of pBcl-2/(Bcl-2+Mcl-1) could predict the S1 response. Furthermore, we demonstrated that pBcl-2 antagonised S1 by sequestering the Bak and Bim proteins that were released from Mcl-1, andpBcl-2/Bak, pBcl-2/Bax and pBcl-2/Bim complexes cannot be disrupted by S1. Conclusion: A predictive index was obtained for the novel BH3 mimetic S1. The shift of proapoptotic proteins from being complexed with Mcl-1 to being complexed with pBcl-2 was revealed for the first time, which is the mechanism underlying the index value described herein. PMID:23558901

  12. Computational study of the mechanism of Bcl-2 apoptotic switch

    NASA Astrophysics Data System (ADS)

    Tokár, Tomáš; Uličný, Jozef

    2012-12-01

    In spite of attention devoted to molecular mechanisms of apoptosis, the details of functioning of one crucial component-the Bcl-2 apoptotic switch-are not completely understood. There are two competing mechanisms of its internal working-the indirect activation and the direct activation. In the absence of conclusive experimental data, we have used computational modeling to assess the properties of both mechanisms and their suitability to act as a biological switch. Since the two mechanisms form opposite poles of continuum of Bcl-2 molecular interaction models, we have constructed more general models including these two models as extreme cases. By studying the relationship between model parameters and the steady-state response we have found optimal interaction patterns which reproduce the behavior of the Bcl-2 apoptotic switch. Our results show, that stimulus-response ultrasensitivity is negatively affected by spontaneous activation of Bcl-2 effectors. We found that ultrasensitivity requires effectors activation, mediated by another subgroup of Bcl-2 proteins-activators. We have shown that the auto-activation of monomeric effector forms provides an ultrasensitivity enhancing feedback loop. Thorough robustness analysis revealed that the interaction pattern postulated in the direct activation hypothesis is able to conserve stimulus-response switching characteristics for wide range changes of its internal parameters. The robustness of the switch against the variation of the reaction parameter is strongly reduced for the intermediate hybrid model and even more for the indirect part of the models. Computer simulations of the more general model presented here suggest, that stimulus-response ultrasensitivity is an emergent property of the direct activation model that is unlikely to occur in the model of indirect activation. Introduction of indirect-model-specific interactions does not provide a better explanation of the Bcl-2 switch functionality compared to the direct

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

    PubMed

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

    2016-06-01

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

  14. Low incidence of MYC/BCL2 double-hit in Burkitt lymphoma.

    PubMed

    Yoshida, Maki; Ichikawa, Ayako; Miyoshi, Hiroaki; Kiyasu, Junichi; Kimura, Yoshizo; Niino, Daisuke; Ohshima, Koichi

    2015-09-01

    Translocations involving MYC are highly characteristic for Burkitt lymphoma (BL). BCL2 expression has also been found previously in about 10 to 20% of BL cases, and BCL2 translocation is a major mechanism for the deregulation of BCL2 expression in non-Hodgkin lymphomas. However, we know little about the incidence of MYC/BCL2 double-hit (DH) in BL. We examined BL cases to determine how frequently they contained BCL2 translocations in combination with MYC translocations using fluorescence in situ hybridization. We also determined the effect of BCL2 expression on clinical outcomes of BL. BCL2 translocations were detected in 3.5% (2/57 cases) of the cases, and BCL2 expression was detected in 33%. Two cases with BCL2 translocation also showed BCL2 expression. The incidence of BCL2 expression was significantly higher in patients 16 years of age and older (46%) than in patients under 16 years of age (6%). Among patients 16 years of age and older, we did not detect significant differences in overall survival with respect to BCL2 expression status. In conclusion, BCL2 translocation is a rare cytogenetic abnormality in BL, and BL probably accounts for only a small fraction of MYC/BCL2 DH lymphomas. BCL2 expression in BL is probably not associated with BCL2 translocations.

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

    PubMed

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

    2007-01-01

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

  16. 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.

  17. Molecular interactions of prodiginines with the BH3 domain of anti-apoptotic Bcl-2 family members.

    PubMed

    Hosseini, Ali; Espona-Fiedler, Margarita; Soto-Cerrato, Vanessa; Quesada, Roberto; Pérez-Tomás, Ricardo; Guallar, Victor

    2013-01-01

    Prodigiosin and obatoclax, members of the prodiginines family, are small molecules with anti-cancer properties that are currently under preclinical and clinical trials. The molecular target(s) of these agents, however, is an open question. Combining experimental and computational techniques we find that prodigiosin binds to the BH3 domain in some BCL-2 protein families, which play an important role in the apoptotic programmed cell death. In particular, our results indicate a large affinity of prodigiosin for MCL-1, an anti-apoptotic member of the BCL-2 family. In melanoma cells, we demonstrate that prodigiosin activates the mitochondrial apoptotic pathway by disrupting MCL-1/BAK complexes. Computer simulations with the PELE software allow the description of the induced fit process, obtaining a detailed atomic view of the molecular interactions. These results provide new data to understand the mechanism of action of these molecules, and assist in the development of more specific inhibitors of anti-apoptotic BCL-2 proteins.

  18. Discovery of a small-molecule pBcl-2 inhibitor that overcomes pBcl-2-mediated resistance to apoptosis.

    PubMed

    Song, Ting; Yu, Xiaoyan; Liu, Yubo; Li, Xiangqian; Chai, Gaobo; Zhang, Zhichao

    2015-03-23

    Although the role of Bcl-2 phosphorylation is still under debate, it has been identified in a resistance mechanism to BH3 mimetics, for example ABT-737 and S1. We identified an S1 analogue, S1-16, as a small-molecule inhibitor of pBcl-2. S1-16 efficiently kills EEE-Bcl-2 (a T69E, S70E, and S87E mutant mimicking phosphorylation)-expressing HL-60 cells and high endogenously expressing pBcl-2 cells, by disrupting EEE-Bcl-2 or native pBcl-2 interactions with Bax and Bak, followed by apoptosis. In vitro binding assays showed that S1-16 binds to the BH3 binding groove of EEE-Bcl-2 (Kd =0.38 μM by ITC; IC50 =0.16 μM by ELISA), as well as nonphosphorylated Bcl-2 (npBcl-2; Kd =0.38 μM; IC50 =0.12 μM). However, ABT-737 and S1 had much weaker affinities to EEE-Bcl-2 (IC50 =1.43 and >10 μM, respectively), compared with npBcl-2 (IC50 =0.011 and 0.74 μM, respectively). The allosteric effect on BH3 binding groove by Bcl-2 phosphorylation in the loop region was illustrated for the first time.

  19. The BCL2 Family: Key Mediators of the Apoptotic Response to Targeted Anticancer Therapeutics.

    PubMed

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

    2015-05-01

    The ability of cancer cells to suppress apoptosis is critical for carcinogenesis. The BCL2 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 antiapoptotic BCL2 family proteins. In this review, we summarize the current knowledge of the role of BCL2 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. Apoptosis, long known to be important for response to conventional cytotoxic chemotherapy, has more recently been shown to be essential for the efficacy of targeted therapies. Approaches that increase the likelihood of a cancer to undergo apoptosis following therapy may help improve targeted treatment strategies. Cancer Discov; 5(5); 475-87. ©2015 AACR. ©2015 American Association for Cancer Research.

  20. Bcl-2 family genetic profiling reveals microenvironment-specific determinants of chemotherapeutic response.

    PubMed

    Pritchard, Justin R; Gilbert, Luke A; Meacham, Corbin E; Ricks, Jennifer L; Jiang, Hai; Lauffenburger, Douglas A; Hemann, Michael T

    2011-09-01

    The Bcl-2 family encompasses a diverse set of apoptotic regulators that are dynamically activated in response to various cell-intrinsic and -extrinsic stimuli. An extensive variety of cell culture experiments have identified effects of growth factors, cytokines, and drugs on Bcl-2 family functions, but in vivo studies have tended to focus on the role of one or two particular members in development and organ homeostasis. Thus, the ability of physiologically relevant contexts to modulate canonical dependencies that are likely to be more complex has yet to be investigated systematically. In this study, we report findings derived from a pool-based shRNA assay that systematically and comprehensively interrogated the functional dependence of leukemia and lymphoma cells upon various Bcl-2 family members across many diverse in vitro and in vivo settings. This approach permitted us to report the first in vivo loss of function screen for modifiers of the response to a front-line chemotherapeutic agent. Notably, our results reveal an unexpected role for the extrinsic death pathway as a tissue-specific modifier of therapeutic response. In particular, our findings show that particular tissue sites of tumor dissemination play critical roles in demarcating the nature and extent of cancer cell vulnerabilities and mechanisms of chemoresistance. ©2011 AACR.

  1. BCL2 and MYC dual-hit lymphoma/leukemia.

    PubMed

    Tomita, Naoto

    2011-01-01

    Translocation of the BCL2 gene on the chromosome band 18q21.3 results in consistent expression of the Bcl2 protein, an apoptosis inhibitor. BCL2 usually translocates to the immunoglobulin (IG) heavy chain (IGH) gene as t(14;18)(q32;q21.3) and rarely to IG light chain (IGK, IGL) loci as t(2;18)(p11;q21.3) or t(18;22)(q21.3;q11). The t(14;18) translocation is observed in 70-95% of follicular lymphoma cases and 20-30% of diffuse large B-cell lymphoma (DLBCL) cases. The MYC gene on chromosome band 8q24 acts as an accelerator of cell proliferation. MYC translocates to 14q32/IGH as t(8;14)(q24;q32) or less commonly to 2p11/IGK as t(2;8)(p11;q24) or 22q11/IGL as t(8;22)(q24;q11). The 8q24/MYC translocation is detected in nearly all Burkitt lymphoma (BL) and up to 10% of DLBCL cases. Both translocations rarely occur in an identical cell and this lymphoid malignancy is termed BCL2 and MYC dual-hit lymphoma/leukemia (DHL). The pathological diagnosis in most cases of DHL with BCL2-IG and MYC-IG translocation is B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL, although DLBCL is most common in DHL with BCL2-IG and MYC-nonIG translocation. The frequency of DHL with BCL2 and MYC translocation is estimated at around 2% of all B-cell malignancies. The condition is characterized by elevated serum lactate dehydrogenase levels, the presence of B symptoms, bone marrow involvement, advanced disease stage, extranodal involvement, and central nervous system (CNS) involvement at presentation or disease progression. Despite treatment strategies including CNS-targeted therapy, the prognosis for DHL is extremely poor. In this review, the current knowledge of the clinicopathological status of DHL is summarized and discussed.

  2. Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues.

    PubMed

    Droga-Mazovec, Gabriela; Bojic, Lea; Petelin, Ana; Ivanova, Saska; Romih, Rok; Repnik, Urska; Salvesen, Guy S; Stoka, Veronika; Turk, Vito; Turk, Boris

    2008-07-04

    As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.

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

    PubMed

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

    2014-10-01

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

  4. Caspase Induction and BCL2 Inhibition in Human Adipose Tissue

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    2013-01-01

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

  6. Response of yeast to the regulated expression of proteins in the Bcl-2 family.

    PubMed Central

    Polcic, Peter; Forte, Michael

    2003-01-01

    The mechanisms by which pro-apoptotic members of the Bcl-2 family of proteins promote the release of mitochondrial factors like cytochrome c, subsequently activating the apoptotic cascade, or by which anti-apoptotic family members block this release, are still not understood. When expressed in yeast, Bcl-2 family members act directly upon conserved mitochondrial components that correspond to their apoptotic substrates in mammalian cells. Here we describe a system in which the levels of representative pro- and anti-apoptotic members of the Bcl-2 family can be regulated independently in yeast. Using this system, we have focused on the action of the anti-apoptotic family member Bcl-x(L), and have defined the quantitative relationships that underlie the antagonistic action of this protein on the lethal consequences of expression of the pro-apoptotic family member Bax. This system has also allowed us to demonstrate biochemically that Bcl-x(L) has two actions at the level of the mitochondrion. Bcl-x(L) is able to inhibit the stable integration of Bax into mitochondrial membranes, as well as hinder the action of Bax that does become stably integrated into these membranes. Taken together, our results suggest that both the functional and biochemical actions of Bcl-x(L) may be based on the ability of this molecule to disrupt the interaction of Bax with a resident mitochondrial target that is required for Bax action. Finally, we confirm that VDAC (voltage-dependent anion channel) is not required for the functional responses observed following the expression of either pro- or anti-apoptotic members of the Bcl-2 family. PMID:12780347

  7. 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.

  8. Concurrent Expression of MYC and BCL2 in Diffuse Large B-Cell Lymphoma Treated With Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone

    PubMed Central

    Johnson, Nathalie A.; Slack, Graham W.; Savage, Kerry J.; Connors, Joseph M.; Ben-Neriah, Susana; Rogic, Sanja; Scott, David W.; Tan, King L.; Steidl, Christian; Sehn, Laurie H.; Chan, Wing C.; Iqbal, Javeed; Meyer, Paul N.; Lenz, Georg; Wright, George; Rimsza, Lisa M.; Valentino, Carlo; Brunhoeber, Patrick; Grogan, Thomas M.; Braziel, Rita M.; Cook, James R.; Tubbs, Raymond R.; Weisenburger, Dennis D.; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Holcroft, Christina; Jaffe, Elaine S.; Staudt, Louis M.; Gascoyne, Randy D.

    2012-01-01

    Purpose Diffuse large B-cell lymphoma (DLBCL) is curable in 60% of patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). MYC translocations, with or without BCL2 translocations, have been associated with inferior survival in DLBCL. We investigated whether expression of MYC protein, with or without BCL2 protein expression, could risk-stratify patients at diagnosis. Patients and Methods We determined the correlation between presence of MYC and BCL2 proteins by immunohistochemistry (IHC) with survival in two independent cohorts of patients with DLBCL treated with R-CHOP. We further determined if MYC protein expression correlated with high MYC mRNA and/or presence of MYC translocation. Results In the training cohort (n = 167), MYC and BCL2 proteins were detected in 29% and 44% of patients, respectively. Concurrent expression (MYC positive/BCL2 positive) was present in 21% of patients. MYC protein correlated with presence of high MYC mRNA and MYC translocation (both P < .001), but the latter was less frequent (both 11%). MYC protein expression was only associated with inferior overall and progression-free survival when BCL2 protein was coexpressed (P < .001). Importantly, the poor prognostic effect of MYC positive/BCL2 positive was validated in an independent cohort of 140 patients with DLBCL and remained significant (P < .05) after adjusting for presence of high-risk features in a multivariable model that included elevated international prognostic index score, activated B-cell molecular subtype, and presence of concurrent MYC and BCL2 translocations. Conclusion Assessment of MYC and BCL2 expression by IHC represents a robust, rapid, and inexpensive approach to risk-stratify patients with DLBCL at diagnosis. PMID:22851565

  9. Down-Regulation of MicroRNA-133b Suppresses Apoptosis of Lens Epithelial Cell by Up-Regulating BCL2L2 in Age-Related Cataracts

    PubMed Central

    Zhang, Feng; Meng, Weizhe; Tong, Bin

    2016-01-01

    Background MicroRNA-133b (miR-133b) has been reported to be involved in many diseases, including ovarian cancer and osteosarcoma. Accumulating evidence suggests that miR-133b plays important roles in human disease. In this study, we aimed to investigate the molecular mechanism, including the potential regulator and signaling pathways, of BCL2L2. Material/Methods We first searched the online miRNA database (www.mirdb.org) using the “seed sequence” located within the 3′-UTR of the target gene, and then performed luciferase assay to test the regulatory relationship between miR-133b and BCL2L2. Western blot and real-time PCR were used to determine the expression of BCL2L2 in human samples or cells treated with miRNA mimics or inhibitors. Flow cytometry was conducted to evaluate the apoptosis status of the cells. Results We validated BCL2L2 to be the direct gene using a luciferase reporter assay. We also conducted real-time PCR and Western blot analyses to study the mRNA and protein expression level of BCL2L2 among different groups (control: n=29, cataract: n=33) or cells treated with scramble control, miR-133b mimics, BCL2L2 siRNA, and miR-133b inhibitors, and identified the negative regulatory relationship between miR-133b and BCL2L2. We also conducted experiments to investigate the influence of miR-133b and BCL2L2 on the viability and apoptosis of cells. The results showed that miR-133b positively interfered with the viability of cells, while BCL2L2 negatively interfered with the viability of cells, and that miR-133b inhibited apoptosis while BCL2L2 accelerated apoptosis. Conclusions BCL2L2 was the virtual target of miR-133b, and we found a negative regulatory relationship between miR-133b and BCL2L2. MiR-133b and BCL2L2 interfered with the viability and apoptosis of cells. PMID:27802259

  10. Bcl-2 Retards Cell Cycle Entry through p27Kip1, pRB Relative p130, and Altered E2F Regulation

    PubMed Central

    Vairo, Gino; Soos, Timothy J.; Upton, Todd M.; Zalvide, Juan; DeCaprio, James A.; Ewen, Mark E.; Koff, Andrew; Adams, Jerry M.

    2000-01-01

    Independent of its antiapoptotic function, Bcl-2 can, through an undetermined mechanism, retard entry into the cell cycle. Cell cycle progression requires the phosphorylation by cyclin-dependent kinases (Cdks) of retinoblastoma protein (pRB) family members to free E2F transcription factors. We have explored whether retarded cycle entry is mediated by the Cdk inhibitor p27 or the pRB family. In quiescent fibroblasts, enforced Bcl-2 expression elevated levels of both p27 and the pRB relative p130. Bcl-2 still slowed G1 progression in cells deficient in pRB but not in those lacking p27 or p130. Hence, pRB is not required, but both p27 and p130 are essential mediators. The ability of p130 to form repressive complexes with E2F4 is implicated, because the retardation by Bcl-2 was accentuated by coexpressed E2F4. A plausible relevant target of p130/E2F4 is the E2F1 gene, because Bcl-2 expression delayed E2F1 accumulation during G1 progression and overexpression of E2F1 overrode the Bcl-2 inhibition. Hence, Bcl-2 appears to retard cell cycle entry by increasing p27 and p130 levels and maintaining repressive complexes of p130 with E2F4, perhaps to delay E2F1 expression. PMID:10848600

  11. MYC or BCL2 copy number aberration is a strong predictor of outcome in patients with diffuse large B-cell lymphoma

    PubMed Central

    Lu, Ting-Xun; Fan, Lei; Wang, Li; Wu, Jia-Zhu; Miao, Kou-Rong; Liang, Jin-Hua; Gong, Qi-Xing; Wang, Zhen; Young, Ken H.; Xu, Wei; Zhang, Zhi-Hong; Li, Jian-Yong

    2015-01-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL). Patients with DLBCL harboring MYC aberrations concurrent with BCL2 or/and BCL6 aberrations constitute a specific group with extremely poor outcome. In this study, we retrospectively investigated the incidence and prognosis of MYC, BCL2, and BCL6 aberrations with DLBCL patients in Chinese population. We applied fluorescence in situ hybridization and immunohistochemical analysis in 246 DLBCL patients. The results showed that patients with MYC or BCL2 copy number aberration (CNA) had significantly worse overall survival (OS) and progression-free survival (PFS) than negative cases (P < 0.0001). Patients with both MYC and BCL2 CNA had similar outcomes to those with classic double hit lymphoma or protein double expression lymphoma (MYC and BCL2/BCL6 coexpression). By multivariate analysis, MYC CNA, BCL2 CNA and double CNA were the independent worse prognostic factors. In conclusions, patients with MYC or BCL2 CNA constituted a unique group with extremely poor outcome and may require more aggressive treatment regimens. PMID:26158410

  12. Differential effects of Bcl-2 and caspases on mitochondrial permeabilization during endogenous or exogenous reactive oxygen species-induced cell death: a comparative study of H₂O₂, paraquat, t-BHP, etoposide and TNF-α-induced cell death.

    PubMed

    Rincheval, Vincent; Bergeaud, Marie; Mathieu, Lise; Leroy, Jacqueline; Guillaume, Arnaud; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc

    2012-08-01

    In this study, we have compared several features of cell death triggered by classical inducers of apoptotic pathways (etoposide and tumour necrosis factor (TNF)-α) versus exogenous reactive oxygen species (ROS; hydrogen peroxide (H₂O₂), tert-butyl hydroperoxide (t-BHP)) or a ROS generator (paraquat). Our aim was to characterize relationships that exist between ROS, mitochondrial perturbations, Bcl-2 and caspases, depending on source and identity of ROS. First, we have found that these five inducers trigger oxidative stress, mitochondrial membrane permeabilization (MMP), cytochrome c (cyt c) release from mitochondria and cell death. In each case, cell death could be inhibited by several antioxidants, showing that it is primarily ROS dependent. Second, we have highlighted that during etoposide or TNF-α treatments, intracellular ROS level, MMP and cell death are all regulated by caspases and Bcl-2, with caspases acting early in the process. Third, we have demonstrated that H₂O₂-induced cell death shares many of these characteristics with etoposide and TNF-α, whereas t-BHP induces both caspase-dependent and caspase-independent cell death. Surprisingly, paraquat-induced cell death, which harbours some characteristics of apoptosis such as cyt c release and caspase-3 activation, is not modulated by Bcl-2 and caspase inhibitors, suggesting that paraquat also triggers non-apoptotic cell death signals. On the one hand, these results show that endogenous or exogenous ROS can trigger multiple cell death pathways with Bcl-2 and caspases acting differentially. On the other hand, they suggest that H₂O₂ could be an important mediator of etoposide and TNF-α-dependent cell death since these inducers trigger similar phenotypes.

  13. Pseudonegative BCL2 protein expression in a t(14;18) translocation positive lymphoma cell line: a need for an alternative BCL2 antibody.

    PubMed

    Masir, Noraidah; Campbell, Lisa J; Jones, Margaret; Mason, David Y

    2010-04-01

    The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein expression in most follicular lymphomas. However, a small number of cases lack BCL2 expression despite carrying the t(14;18)(q32;q21) translocation. This study aims to explore the mechanism accounting for the lack of BCL2 protein expression when the t(14;18) translocation is present. BCL2 expression in the t(14;18) positive cell lines FL18, Karpas-422, SU-DHL-4 and SU-DHL-6, was analysed by Western blotting and by immunohistochemistry using two different antibodies. FISH analysis was performed to confirm the cytogenetic changes in the cell lines and real time quantitative PCR was used to evaluate the BCL2 mRNA level. Sequence analysis of translocated BCL2 was performed on FL18, Karpas-422, SU-DHL-4 and SU-DHL-6 cell lines. In FL18, Karpas-422, and SU-DHL-4, the BCL2 mRNA level correlated with the BCL2 protein expression. In contrast, BCL2 protein was not detected in SU-DHL-6 line using standard anti-BCL2 antibody (BCL2/124), despite the presence of the t(14;18) translocation and high level of mRNA. cDNA sequencing of translocated BCL2 showed three mutations in the SU-DHL-6 cell line, one of which resulted in an amino acid substitution (I48F) in the region recognised by the standard BCL2 antibody, whereas the other two were silent mutations at aa71 and aa72. Interestingly, when BCL2 expression was tested with an alternative antibody, E17, the protein was detected in SU-DHL-6, suggesting that the 'negativity' of SU-DHL-6 line for BCL2 using the standard antibody is spurious. Amino acid changes were found in Karpas-422 (G47D, P59L) and SU-DHL-4 (P59T, S117R) but these did not affect BCL2 detection. This study suggests that some somatic mutations of the translocated BCL2 gene may prevent epitope recognition by BCL2 antibodies, and hence cause false negative expression using the standard antibody. It is recommended that in practice all BCL2 negative cases should routinely be stained with an alternative

  14. 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

  15. Relationship between reduced BCL-2 expression in circulating mononuclear cells and early nephropathy in type 1 diabetes.

    PubMed

    Cipollone, F; Chiarelli, F; Iezzi, A; Fazia, M L; Cuccurullo, C; Pini, B; De Cesare, D; Torello, M; Tumini, S; Cuccurullo, F; Mezzetti, A

    2005-01-01

    Microalbuminuria is the earliest clinical evidence of diabetic nephropathy, but the mechanisms linking hyperglycemia and kidney complications are not clear. The aim of this study was to evaluate whether enhanced oxidative stress in patients with microalbuminuria can contribute to diabetic nephropathy development through downregulation of the antiapoptotic gene Bcl-2 that promotes in turn a pro-inflammatory status. We studied 30 patients with type 1 diabetes (15 with and 15 without microalbuminuria) compared to 15 matched healthy controls. Plasma oxidant status, and expression of Bcl-2, activated NF-kB, inducible Nitric Oxide synthase (iNOS), and monocyte chemoattractant protein (MCP)-1 in circulating monocytes were evaluated at baseline and after 8-week oral vitamin E treatment (600 mg b.i.d.). Bcl-2 expression was significantly reduced in microalbuminuric diabetic patients as a consequence of increased oxidant burden secondary to persistent hyperglycemia. Bcl-2 down-regulation was associated with enhanced expression of NF-kB, iNOS and MCP-1, and showed a strong correlation with the albumin excretion rate. Low Bcl-2 expression and high inflammatory status were normalized by vitamin E both in vivo and in vitro. Our study showed that Bcl-2 down-regulation in diabetic patients with poor glycemic control results in the activation of the NF-kB pathway leading to the development of nephropathy. Vitamin E might provide a novel form of therapy for prevention of nephropathy in diabetic patients in which an acceptable glycemic control is difficult to achieve despite insulin therapy.

  16. Prognostic significance of bcl-2 protein expression in aggressive non-Hodgkin's lymphoma. Groupe d'Etude des Lymphomes de l'Adulte (GELA).

    PubMed

    Hermine, O; Haioun, C; Lepage, E; d'Agay, M F; Briere, J; Lavignac, C; Fillet, G; Salles, G; Marolleau, J P; Diebold, J; Reyas, F; Gaulard, P

    1996-01-01

    Little is known about the expression of bcl-2 protein in intermediate and high grade non-Hodgkin's lymphoma (NHL) and its clinical and prognostic significance. We performed immunohistochemical analysis of bcl-2 expression in tumoral tissue sections of 348 patients with high or intermediate grade NHL. These patients were uniformly treated with adriamycin, cyclophosphamide, vindesine, bleomycin, and prednisone (ACVBP) in the induction phase of the LNH87 protocol. Fifty eight cases were excluded due to inadequate staining. Of the 290 remaining patients, 131 (45%) disclosed homogeneous positivity (high bcl-2 expression) in virtually all tumor cells, whereas 65 (23%) were negative and 94 (32%) exhibited intermediate staining. High bcl-2 expression was more frequent in B-cell NHL (109 of 214, 51%) than in T-cell NHL (6 of 35, 17%) (P = .0004), and was heterogeneously distributed among the different histological subtypes. Further analysis was performed on the 151 patients with diffuse large B-cell lymphoma (centroblastic and immunoblastic) to assess the clinical significance and potential prognostic value of bcl-2 expression in the most frequent and homogeneous immunohistological subgroup. High bcl-2 expression, found in 44% of these patients (67 of 151), was more frequently associated with III-IV stage disease (P = .002). Reduced disease-free survival (DFS) (P < .01) and overall survival (P < .05) were demonstrated in the patients with high bcl-2 expression. Indeed, the 3-year estimates of DFS and overall survival were 60% and 61%, respectively (high bcl-2 expression) versus 82% and 78%, respectively (negative/intermediate bcl-2 expression). A multivariate regression analysis confirmed the independent effect of bcl-2 protein expression on DFS. Thus bcl-2 protein expression, as demonstrated in routinely paraffin-embedded tissue, appears to be predictive of poor DFS, in agreement with the role of bcl-2 in chemotherapy-induced apoptosis. It might be considered as a new

  17. Genetic profiling of MYC and BCL2 in diffuse large B-cell lymphoma determines cell-of-origin-specific clinical impact.

    PubMed

    Ennishi, Daisuke; Mottok, Anja; Ben-Neriah, Susana; Shulha, Hennady P; Farinha, Pedro; Chan, Fong Chun; Meissner, Barbara; Boyle, Merrill; Hother, Christoffer; Kridel, Robert; Lai, Daniel; Saberi, Saeed; Bashashati, Ali; Shah, Sohrab P; Morin, Ryan D; Marra, Marco A; Savage, Kerry J; Sehn, Laurie H; Steidl, Christian; Connors, Joseph M; Gascoyne, Randy D; Scott, David W

    2017-05-18

    The clinical significance of MYC and BCL2 genetic alterations in diffuse large B-cell lymphoma (DLBCL), apart from translocations, has not been comprehensively investigated using high-resolution genetic assays. In this study, we profiled MYC and BCL2 genetic alterations using next-generation sequencing and high-resolution SNP array in 347 de novo DLBCL cases treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) at the British Columbia Cancer Agency. Cell-of-origin (COO) subtype was determined by Lymph2Cx digital gene expression profiling. We showed that the incidence of MYC/BCL2 genetic alterations and their clinical significance were largely dependent on COO subtypes. It is noteworthy that the presence of BCL2 gain/amplification is significantly associated with poor outcome in activated B-cell-like and BCL2 translocation with poor outcome in germinal center B-cell subtypes, respectively. Both have prognostic significance independent of MYC/BCL2 dual expression and the International Prognostic Index (IPI). Furthermore, the combination of BCL2 genetic alterations with IPI identifies markedly worse prognostic groups within individual COO subtypes. Thus, high-resolution genomic assays identify extremely poor prognostic groups within each COO subtype on the basis of BCL2 genetic status in this large, uniformly R-CHOP-treated population-based cohort of DLBCL. These results suggest COO subtype-specific biomarkers based on BCL2 genetic alterations can be used to risk-stratify patients with DLBCL treated with immunochemotherapy. © 2017 by The American Society of Hematology.

  18. The alpha1-adrenoceptor antagonist terazosin induces prostate cancer cell death through a p53 and Rb independent pathway.

    PubMed

    Xu, Kexin; Wang, Xianghong; Ling, Patrick M T; Tsao, S W; Wong, Y C

    2003-01-01

    Prostate cancer is the second leading cause of cancer-related death in men. Treatment failure in prostate cancer is usually due to the development of androgen independence and resistance to chemotherapeutic drugs at an advanced stage. Recently, it was reported that the alpha1-adrenoceptor antagonist terazosin was able to inhibit prostate cancer cell growth and indicated that it may have an implication in the treatment of prostate cancer. The aim of the present study was to investigate the mechanisms involved in terazosin-induced prostate cancer cell death using two androgen-independent cell lines, PC-3 and DU145. Our results showed that terazosin inhibited not only prostate cancer cell growth but also colony forming ability, which is the main target of chemotherapy. We also found that the sensitivity of these cells to terazosin was not affected by the presence of either functional p53 or Rb, suggesting that the terazosin-induced cell death was independent of p53 and Rb. However, the terazosin-induced cell death was associated with G1 phase cell cycle arrest and up-regulation of p27KIP1. In addition, up-regulation of Bax and down-regulation of Bcl-2 was also observed indicating that these two apoptotic regulators may play important roles in terazosin-mediated cell death pathway. Our results provide evidence for the first time that terazosin may have a therapeutic potential in the treatment of advanced prostate cancer.

  19. Dysregulation of Reelin and Bcl-2 proteins in autistic cerebellum.

    PubMed

    Fatemi, S H; Stary, J M; Halt, A R; Realmuto, G R

    2001-12-01

    Autism is a severe neurodevelopmental disorder with potential genetic and environmental causes. Cerebellar pathology including Purkinje cell atrophy has been demonstrated previously. We hypothesized that cell migration and apoptotic mechanisms may account for observed Purkinje cell abnormalities. Reelin is an important secretory glycoprotein responsible for normal layering of the brain. Bcl-2 is a regulatory protein responsible for control of programmed cell death in the brain. Autistic and normal control cerebellar corteces matched for age, sex, and post-mortem interval (PMI) were prepared for SDS-gel electrophoresis and Western blotting using specific anti-Reelin and anti-Bcl-2 antibodies. Quantification of Reelin bands showed 43%, 44%, and 44% reductions in autistic cerebellum (mean optical density +/- SD per 30 microg protein 4.05 +/- 4.0, 1.98 +/- 2.0, 13.88 +/- 11.9 for 410 kDa, 330 kDa, and 180 kDa bands, respectively; N = 5) compared with controls (mean optical density +/- SD per 30 microg protein, 7.1 +/- 1.6, 3.5 +/- 1.0, 24.7 +/- 5.0; N = 8, p < 0.0402 for 180 kDa band). Quantification of Bcl-2 levels showed a 34% to 51% reduction in autistic cerebellum (M +/- SD per 75 microg protein 0.29 +/- 0.08; N = 5) compared with controls (M +/- SD per 75 microg protein 0.59 +/- 0.31; N = 8, p < 0.0451). Measurement of beta-actin (M +/- SD for controls 7.3 +/- 2.9; for autistics 6.77 +/- 0.66) in the same homogenates did not differ significantly between groups. These results demonstrate for the first time that dysregulation of Reelin and Bcl-2 may be responsible for some of the brain structural and behavioral abnormalities observed in autism.

  20. Bcl-2 Conformational Change as an Indicator of Chemotherapy Response

    DTIC Science & Technology

    2005-09-01

    conformational Control change in MCF-7 breast cancer cells. Immunostaining of apoptotic Bcl-2. H460 - cells were treated with or Paclitaxel without...NuBCPs target mitochondria. GFP-NuBCP and mitochondria-targeted Red Fluorescent Protein (RFP-mito) were transfected into H460 cells for 16 h. Confocal...microscopy analysis showed that in H460 cells GFP-NuBCP colocalized extensively with that of RFP-Mito, a red fluorescence protein (RFP) fused with a

  1. Statins, Bcl-2, and apoptosis: cell death or cell protection?

    PubMed

    Wood, W Gibson; Igbavboa, Urule; Muller, Walter E; Eckert, Gunter P

    2013-10-01

    Statins have proven their effectiveness in the treatment of cardiovascular disease. This class of drugs has also attracted attention as a potential treatment for dissimilar diseases such as certain types of cancers and neurodegenerative diseases. What appears to be a contradiction is that, in the case of cancer, it has been suggested that statins increase apoptosis and alter levels of Bcl-2 family members (e.g., reduce Bcl-2 and increase Bax), whereas studies mainly using noncancerous cells report opposite effects. This review examined studies reporting on the effects of statins on Bcl-2 family members, apoptosis, cell death, and cell protection. Much, but not all, of the evidence supporting the pro-apoptotic effects of statins is based on data in cancer cell lines and the use of relatively high drug concentrations. Studies indicating an anti-apoptotic effect of statins are fewer in number and generally used much lower drug concentrations and normal cells. Those conclusions are not definitive, and certainly, there is a need for additional research to determine if statin repositioning is justified for noncardiovascular diseases.

  2. Patients with diffuse large B-cell lymphoma of germinal center origin with BCL2 translocations have poor outcome, irrespective of MYC status: a report from an International DLBCL rituximab-CHOP Consortium Program Study.

    PubMed

    Visco, Carlo; Tzankov, Alexander; Xu-Monette, Zijun Y; Miranda, Roberto N; Tai, Yu Chuan; Li, Yan; Liu, Wei-min; d'Amore, Emanuele S G; Li, Yong; Montes-Moreno, Santiago; Dybkær, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Wang, Huan-You; Dunphy, Cherie H; His, Eric D; Zhao, X Frank; Choi, William W L; Zhao, Xiaoying; van Krieken, J Han; Huang, Qin; Ai, Weiyun; O'Neill, Stacey; Ponzoni, Maurilio; Ferreri, Andres J M; Kahl, Brad S; Winter, Jane N; Go, Ronald S; Dirnhofer, Stephan; Piris, Miguel A; Møller, Michael B; Wu, Lin; Medeiros, L Jeffrey; Young, Ken H

    2013-02-01

    Diffuse large B-cell lymphoma can be classified by gene expression profiling into germinal center and activated B-cell subtypes with different prognoses after rituximab-CHOP. The importance of previously recognized prognostic markers, such as Bcl-2 protein expression and BCL2 gene abnormalities, has been questioned in the new therapeutic era. We analyzed Bcl-2 protein expression, and BCL2 and MYC gene abnormalities by interphase fluorescence in situ hybridization in 327 patients with de novo disease treated with rituximab-CHOP. Isolated BCL2 and MYC rearrangements were not predictive of outcome in our patients as a whole, but only in those with the germinal center subtype of lymphoma. The prognostic relevance of isolated MYC rearrangements was weaker than that of BCL2 isolated translocations, but was probably limited by the rarity of the rearrangements. Seven of eight patients with double hit lymphoma had the germinal center subtype with poor outcome. The germinal center subtype patients with isolated BCL2 translocations had significantly worse outcome than the patients without BCL2 rearrangements (P=0.0002), and their outcome was similar to that of patients with the activated B-cell subtype (P=0.30), but not as bad as the outcome of patients with double hit lymphoma (P<0.0001). Bcl-2 protein overexpression was associated with inferior outcome in patients with germinal center subtype lymphoma, but multivariate analysis showed that this was dependent on BCL2 translocations. The gene expression profiling of patients with BCL2 rearrangements was unique, showing activation of pathways that were silent in the negative counterpart. BCL2 translocated germinal center subtype patients have worse prognosis after rituximab-CHOP, irrespective of MYC status, but the presence of combined gene breaks significantly overcomes the prognostic relevance of isolated lesions.

  3. Overexpression of MYC and BCL2 Predicts Poor Prognosis in Patients with Extranodal NK/T-cell Lymphoma, Nasal Type

    PubMed Central

    Wang, Jing-hua; Bi, Xi-wen; Li, Peng-fei; Xia, Zhong-jun; Huang, Hui-qiang; Jiang, Wen-qi; Zhang, Yu-jing; Wang, Liang

    2017-01-01

    Background: Recently double-hit lymphoma or double protein expressor lymphoma has been identified as a distinct group of diffuse large B cell lymphoma with poor prognosis. However, the expression status, clinical and prognostic effect of combined overexpression of MYC and BCL2 in extranodal NK/T-cell lymphoma, nasal type (ENKTL) are not known. Materials and methods: Paraffin-embedded lymphoma samples from 53 patients with newly diagnosed ENKTL were studied using immunohistochemistry for MYC and BCL2, and fluorescent in situ hybridization (FISH) for MYC and BCL2 were done on 5 tissue sections with highest percentages of both MYC and BCL2 positive lymphoma cells. Results: The median percentage of MYC-positive lymphoma cells and BCL2-positive lymphoma cells were 20% (range, 5%-45%) and 70% (10%-95%), respectively. Using median scores as cutoffs, we assigned each patient an IHC double-hit score (DHS) that ranged from 0 to 2. Using this DHS, 15 patients (28.3%) had a DHS of 0, 24 patients (45.3%) had a DHS of 1, and the remaining 14 patients (26.4%) had a DHS of 2. FISH analysis was performed on 5 tissue sections with DHS of 2, and none of them had MYC or BCL2 rearrangement. The DHS was not associated with patients' age, gender, disease stage, LDH level, B symptoms, performance status, or local tumor invasiveness. However, patients with tumor localized in extranasal sites seemed to have higher expression of BCL2 and higher DHS than nasal lesions (p=0.014 and 0.042, respectively). In univariate survival analysis, either high expression of MYC or BCL2 was significantly correlated with inferior PFS and OS (p<0.05). According to the DHS, patients with ENKTL could be divided into three significantly different risk groups for PFS and OS (3-year PFS rate for DHS of 0, 1, and 2 was 60%, 41%, and 21%, respectively, p=0.008; 3-year OS rate for DHS of 0, 1, and 2 was 79%, 49%, and 33%, respectively, p=0.015). In multivariate survival analysis, it was found that DHS was an

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

    PubMed Central

    Sharifah Sakinah, SA; Tri Handayani, S; Azimahtol Hawariah, LP

    2007-01-01

    Background Zerumbone is a cytotoxic component isolated from Zingiber zerumbet Smith, a herbal plant which is also known as lempoyang. This new anticancer bioactive compound from Z. zerumbet was investigated for its activity and mechanism in human liver cancer cell lines. Results Zerumbone significantly showed an antiproliferative activity upon HepG2 cells with an IC50 of 3.45 ± 0.026 μg/ml. Zerumbone was also found to inhibit the proliferation of non-malignant Chang Liver and MDBK cell lines. However the IC50 obtained was higher compared to the IC50 for HepG2 cells (> 10 μg/ml). The extent of DNA fragmentation was evaluated by the Tdt-mediated dUTP nick end labelling assay which showed that, zerumbone significantly increased apoptosis in HepG2 cells in a time-course manner. In detail, the apoptotic process triggered by zerumbone involved the up-regulation of pro-apoptotic Bax protein and the suppression of anti-apoptotic Bcl-2 protein expression. The changes that occurred in the levels of this antagonistic proteins Bax/Bcl-2, was independent of p53 since zerumbone did not affect the levels of p53 although this protein exists in a functional form. Western blotting analysis for Bax protein was further confirmed qualitatively with an immunoassay that showed the distribution of Bax protein in zerumbone-treated cells. Conclusion Therefore, zerumbone was found to induce the apoptotic process in HepG2 cells through the up and down regulation of Bax/Bcl-2 protein independently of functional p53 activity. PMID:17407577

  5. Down-modulation of Bcl-2 sensitizes PTEN-mutated prostate cancer cells to starvation and taxanes.

    PubMed

    Calastretti, Angela; Gatti, Giuliana; Quaresmini, Carolina; Bevilacqua, Annamaria

    2014-10-01

    The critical role of PTEN in regulating the PI3K/Akt/mTOR signaling pathway raises the possibility that targeting downstream effectors of the PI3K pathway, such as Bcl-2, might be an effective anti-proliferative strategy for PTEN-deficient prostate cancer cells. Four prostate cancer cell lines (LNCaP, PC3, DU145, 22Rv1) were assayed for their levels of total Akt and Ser473 phosphorylated Akt (p-Akt) by Western Blotting; their growth rates and sensitivity to different doses of paclitaxel were determined by cell counts after Trypan Blue dye exclusion assay. Cells were subjected to different combinations of starvation (growth factors and/or aminoacids withdrawal), paclitaxel treatment and Bcl-2 silencing by siRNA. Cell viability was evaluated by Trypan Blue dye exclusion assay, Propidium Iodide (PI) and Annexin-V/PI staining. We assessed the sensitivity of different prostate cancer cell lines to starvation and we observed a differential response correlated to the levels of Akt activation. The four prostate cancer cell lines also showed different sensitivity to taxol treatments; LNCaP and 22Rv1 cells were more resistant to paclitaxel than DU145 and PC3 cells. Combining taxol with growth factors and aminoacids deprivation leaded to a more than additive reduction of cell viability compared to single treatments in PTEN-mutant LNCaP cells. Down-modulation of anti-apoptotic Bcl-2 protein by siRNA sensitized LNCaP cells to taxanes and starvation induced cell death. Silencing Bcl-2 in PTEN-mutated prostate cancer cells enhances the apoptotic effects of combined starvation and taxol treatments, indicating that inhibition of Bcl-2 may be of significant value in PTEN-mutant tumor therapy. © 2014 Wiley Periodicals, Inc.

  6. Viral Bcl-2 Encoded by the Kaposi's Sarcoma-Associated Herpesvirus Is Vital for Virus Reactivation

    PubMed Central

    Gelgor, Anastasia; Kalt, Inna; Bergson, Shir; Brulois, Kevin F.; Jung, Jae U.

    2015-01-01

    ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 16 (orf16) encodes a viral Bcl-2 (vBcl-2) protein which shares sequence and functional homology with the Bcl-2 family. Like its cellular homologs, vBcl-2 protects various cell types from apoptosis and can also negatively regulate autophagy. vBcl-2 is transcribed during lytic infection; however, its exact function has not been determined to date. By using bacterial artificial chromosome 16 (BAC16) clone carrying the full-length KSHV genome, we have generated recombinant KSHV mutants that fail to express vBcl-2 or express mCherry-tagged vBcl-2. We show that the vBcl-2 protein is expressed at relatively low levels during lytic induction and that a lack of vBcl-2 largely reduces the efficiency of KSHV reactivation in terms of lytic gene expression, viral DNA replication, and production of infectious particles. In contrast, the establishment of latency was not affected by the absence of vBcl-2. Our findings suggest an important role for vBcl-2 during initial phases of lytic reactivation and/or during subsequent viral propagation. Given the known functions of vBcl-2 in regulating apoptosis and autophagy, which involve its direct interaction with cellular proteins and thus require high levels of protein expression, it appears that vBcl-2 may have additional regulatory functions that do not depend on high levels of protein expression. IMPORTANCE The present study shows for the first time the expression of endogenous vBcl-2 protein in KSHV-infected cell lines and demonstrates the importance of vBcl-2 during the initial phases of lytic reactivation and/or during its subsequent propagation. It is suggested that vBcl-2 has additional regulatory functions beyond apoptosis and autophagy repression that do not depend on high levels of protein expression. PMID:25740992

  7. Reflection: The Early Career Surgeon-Scientist's Pathway to Independence.

    PubMed

    Smith, Stephanie Shintani

    2016-01-01

    The surgeon-scientist offers a unique perspective as one who can arguably best comprehend clinical needs, identify areas ripe for research, and translate discoveries from bench to bedside. However, the long transition from postdoc to independent investigator can prove to be quite challenging. Surgeons have long been described as having results-oriented personalities, and so the long road to independence can be fraught with frustration at times. It requires humility in seeking scientific direction and mentorship, institutional support, and ultimately extramural funding. This reflection piece examines some hallmark steps along the pathway to independence for one otolaryngology-head and neck surgeon-scientist in her early academic career.

  8. Glomerular expression and elevated serum Bcl-2 and Fas proteins in lupus nephritis: preliminary findings.

    PubMed

    Fathi, N A; Hussein, M R; Hassan, H I; Mosad, E; Galal, H; Afifi, N A

    2006-11-01

    Programmed cell death (apoptosis) is involved in glomerular injuries leading to glomerulonephritis. Bcl-2 and Fas are proteins that promote cell survival and death, respectively. This study tests the hypothesis that lupus nephritis is associated with alterations of Bcl-2 and Fas protein expression. Thirty-six patients with lupus nephritis and 10 controls (normal individuals) were included in this study. Bcl-2 and Fas positive cells were examined in kidney biopsies by immunohistochemistry. Bcl-2 and Fas serum levels were evaluated by enzyme-linked immunosorbent assay (ELISA). In the glomeruli of normal kidneys, Bcl-2 and Fas proteins were completely absent. In lupus nephritis patients, glomerular expression of Bcl-2 and Fas was seen in mesangial cells (1.3 +/- 0.1 and 2.0 +/- 0.1 for Bcl-2 and Fas, respectively). Similarly, a statistically significantly higher Bcl-2 (217.1 +/- 85.9) and Fas (767.9 +/- 271) serum levels were found in lupus patients compared to controls (148.6 +/- 87, 550.3 +/- 91 for Bcl-2 and Fas, P < 0.05). A direct correlation between serum Bcl-2 and Fas and chronicity index was also found. Compared to normal controls, lupus nephritis is associated with glomerular expression and elevated serum levels of Bcl-2 and Fas proteins. These findings suggest possible roles for Bcl-2 and Fas in glomerular injury during evolution of lupus nephritis. The diagnostic, prognostic and therapeutic ramifications of our findings are open to further investigation.

  9. Chemical parsing: Dissecting cell dependencies with a toolkit of selective BCL-2 family inhibitors.

    PubMed

    Leverson, Joel D

    2016-01-01

    The BCL-2/BCL-XL inhibitor navitoclax has shown promise for the treatment of cancer but on-target toxicities have limited its utility. Recently, the generation of selective BCL-2 family inhibitors has enabled a careful dissection of BCL-2 biology, and early work indicates that these molecules have improved therapeutic profiles for the treatment of cancer.

  10. Mesothelin regulates growth and apoptosis in pancreatic cancer cells through p53-dependent and -independent signal pathway.

    PubMed

    Zheng, Chunning; Jia, Wei; Tang, Yong; Zhao, HuiLiang; Jiang, Yingsheng; Sun, Shaochuan

    2012-10-03

    Mesothelin, a secreted protein, is overexpressed in some cancers, including pancreatic cancer. Rescent studies have shown that overexpression of mesothelin significantly increased tumor cell proliferation, and downregulation of mesothelin inhibited cell proliferation in pancreatic cancer cells, but its exact function and mechanism remains unclear. The aim of the present study was to evaluate the effects of mesothelin on proliferation and apoptosis in pancreatic cancer cells with different p53 status and to explore its signal pathway. Mesothelin levels were detected by western blot and RT-PCR assay in human pancreatic cancer AsPC-1, HPAC and Capan-2, Capan-1 and MIA PaCa-2 cell lines. Mesothelin was slienced by shRNA in AsPC-1, Capan-2 and Capan-1 cells with rich mesothelin level, and mesothelin was overexpressed in the HPAC and Capan-2 cells with less mesothelin level. We observed that in the AsPC-1 and Capan-1cells with mt-p53, and Capan-2 cells with wt-p53, shRNA mediated sliencing of the mesothelin significantly increased PUMA and Bax expression and caspase-3 activity, and decreased bcl-2 expression, followed by the reduced proliferation and colony forming capability and increased cell apoptosis. When PUMA was slienced by siRNA in the stable mesothelin shRNA transfected cells, proliferative capability was significantly increased, and apoptosis was decreased. However, in the Capan-2 cells with wt-p53, suppression of the mesothelin significantly increased wt-p53 levels. When p53 was blocked by siRNA in the stable mesothelin shRNA transfected Capan-2 cells, PUMA was inhibited, followed by increased proliferative capability and decreased cell apoptosis. In the HPAC and Capan-2 cells with wt-p53 and in the MIA PaCa-2 cells with mt-p53, overexpression of the mesothelin significantly decreased bax levels and increased bcl-2 levels, followed by increased proliferative and colony forming capability. Furthermore, mesothelin-shRNA-transfected cells exhibited a reduced rate

  11. 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

  12. PK11195 inhibits mitophagy targeting the F1Fo-ATPsynthase in Bcl-2 knock-down cells.

    PubMed

    Seneviratne, M S D; Faccenda, D; De Biase, V; Campanella, M

    2012-05-01

    The pharmacological agent 1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is the prototypical ligand of the 18-kDa Translocator Protein (TSPO) but at μM concentrations deactivates the oncoprotein Bcl-2 increasing the efficiency of chemotherapeutic agents and promoting the Ca2+-dependent macro-autophagy (or autophagy). In this paper, we report that PK11195, in HeLa cells, modifies the mitochondria-targeted type of autophagy--hereafter referred to as mitophagy--and the associated resizing of the mitochondrial network but does so exclusively in absence of the oncoprotein Bcl-2 (Bcl-2 Kd cells). This is consequence of a "side" targeting of the mitochondrial F1Fo-ATPsynthase enzyme, since identical outcome is mimicked by the antibiotic Oligomycin, of which PK11195 matches the effect on: i) mitochondrial membrane potential (ΔΨm), ii) ATP homeostasis and iii) Reactive Oxygen Species (ROS) generation. Taken together, these data highlight a novel TSPO-independent biological effect for PK11195 and provide evidences for a hitherto uncovered Bcl-2-dependent role of the F1Fo-ATPsynthase in mitochondrial quality control.

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

    PubMed

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

    2017-07-15

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

  14. Prognostic impact of Bcl-2 depends on tumor histology and expression of MALAT-1 lncRNA in non-small-cell lung cancer.

    PubMed

    Schmidt, Lars Henning; Görlich, Dennis; Spieker, Tilmann; Rohde, Christian; Schuler, Martin; Mohr, Michael; Humberg, Julia; Sauer, Tim; Thoenissen, Nils H; Huge, Andreas; Voss, Reinhard; Marra, Alessandro; Faldum, Andreas; Müller-Tidow, Carsten; Berdel, Wolfgang E; Wiewrodt, Rainer

    2014-09-01

    Apoptosis is a crucial pathway in tumor growth and metastatic development. Apoptotic proteins regulate the underlying molecular cascades and are thought to modulate the tumor response to chemotherapy and radiation. However, the prognostic value of the expression of apoptosis regulators in localized non-small-cell lung cancer (NSCLC) is still unclear. We investigated the protein expression of apoptosis regulators Bcl-2, Bcl-xl, Mcl-1, and pp32/PHAPI, and the expression of the lncRNA MALAT-1 in tumor samples from 383 NSCLC patients (median age: 65.6 years; 77.5% male; paraffin-embedded tissue microarrays). For statistical analysis correlation tests, Log rank tests and Cox proportional hazard models were applied. Tumor histology was significantly associated with the expression of Bcl-2, Bcl-xl and Mcl-1 (all p < 0.001). Among the tested apoptotic markers only Bcl-2 demonstrated prognostic impact (hazard ratio = 0.64, p = 0.012). For NSCLC patients with non-adenocarcinoma histology, Bcl-2 expression was associated with increased overall survival (p = 0.036). Besides tumor histology, prognostic impact of Bcl-2 was also found to depend on MALAT-1 lncRNA expression. Gene expression analysis of A549 adenocarcinoma cells with differential MALAT-1 lncRNA expression demonstrated an influence on the expression of Bcl-2 and its interacting proteins. Bcl-2 expression was specifically associated with superior prognosis in localized NSCLC. An interaction of Bcl-2 with MALAT-1 lncRNA expression was revealed, which merits further investigation for risk prediction in resectable NSCLC patients.

  15. Abnormal expression of bcl-2 and bax in rat tongue mucosa during the development of squamous cell carcinoma induced by 4-nitroquinoline 1-oxide

    PubMed Central

    Ribeiro, Daniel A; Salvadori, Daisy M F; Marques, Mariângela E A

    2005-01-01

    4-Nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis is a useful model for studying oral squamous cell carcinoma. The aim of this study was to investigate the expression of bcl-2 and bax during tongue carcinogenesis induced by 4NQO. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. Although no histological changes were induced in the epithelium after 4 weeks of carcinogen exposure, bcl-2 and bax were over-expressed (P < 0.01) in all layers of the ‘normal’ epithelium. The expression levels were the same in all layers of epithelium for both the antibodies used (bcl-2 or bax). In dysplastic lesions at 12 weeks following carcinogen administration, the levels of bcl-2 and bax expression did not increase when compared to negative control with the immunoreactivity for bcl-2 being restricted to the superficial layer of epithelium. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, bcl-2 was expressed in some cells of tumour islands. On the other hand, immunostaining for bax was widely observed at the tumour nests. The labelling index for bcl-2 and bax showed an increase (P < 0.05) after only 4 weeks of 4NQO administration. In conclusion, our results suggest that abnormalities in the apoptosis pathways are associated with the development of persistent clones of mutated-epithelial cells in the oral mucosa. Bcl-2 and bax expression appears to be associated with a risk factor in the progression of oral cancer. PMID:16309543

  16. Cif (Cytochrome c Efflux-Inducing Factor) Activity Is Regulated by Bcl-2 and Caspases and Correlates with the Activation of Bid

    PubMed Central

    Han, Zhiyong; Bhalla, Kapil; Pantazis, Panayotis; Hendrickson, Eric A.; Wyche, James H.

    1999-01-01

    The cytosolic factor Cif (cytochrome c-efflux inducing factor) was activated by the apoptosis inducers staurosporine and anti-Fas antibodies and rapidly induced the efflux of cytochrome c from purified human mitochondria. HL-60 cells that stably overexpressed a bcl-2 cDNA transgene (Bcl-2:HL-60 cells) contained mitochondria and a cytosol that were resistant to exogenous Cif and that lacked detectable endogenous Cif activity, respectively. Therefore, Bcl-2 overexpression negated Cif activity and suggested that the requirement for Cif resides upstream of Bcl-2 on the apoptotic signal transduction pathway. The addition of purified caspase 3, caspase 7, or caspase 8 to the cytosolic extract from Bcl-2:HL-60 cells, however, restored Cif activity, demonstrating that the inhibition of Cif by Bcl-2 overexpression could be overcome by activated caspases. Moreover, the addition of purified caspases to cytosolic extracts prepared from parental HL-60 cells was also sufficient to cause Cif activation, suggesting that caspases might be required for Cif activation. Consistent with these observations, Fas-induced apoptosis in Jurkat cells resulted in caspase 8 activation and subsequently in activation of Cif. Finally, we demonstrate that the activation of Cif correlated with the activation of the Bcl-2 family member Bid by caspases and that Cif activity was selectively neutralized by anti-Bid antibodies. Taken together, these results indicate that Cif is identical to Bid and that it can be inhibited by Bcl-2 and activated by caspases. Thus, Cif (Bid) is an important biological regulator for the transduction of apoptotic signals. PMID:9891071

  17. Cif (Cytochrome c efflux-inducing factor) activity is regulated by Bcl-2 and caspases and correlates with the activation of Bid.

    PubMed

    Han, Z; Bhalla, K; Pantazis, P; Hendrickson, E A; Wyche, J H

    1999-02-01

    The cytosolic factor Cif (cytochrome c-efflux inducing factor) was activated by the apoptosis inducers staurosporine and anti-Fas antibodies and rapidly induced the efflux of cytochrome c from purified human mitochondria. HL-60 cells that stably overexpressed a bcl-2 cDNA transgene (Bcl-2:HL-60 cells) contained mitochondria and a cytosol that were resistant to exogenous Cif and that lacked detectable endogenous Cif activity, respectively. Therefore, Bcl-2 overexpression negated Cif activity and suggested that the requirement for Cif resides upstream of Bcl-2 on the apoptotic signal transduction pathway. The addition of purified caspase 3, caspase 7, or caspase 8 to the cytosolic extract from Bcl-2:HL-60 cells, however, restored Cif activity, demonstrating that the inhibition of Cif by Bcl-2 overexpression could be overcome by activated caspases. Moreover, the addition of purified caspases to cytosolic extracts prepared from parental HL-60 cells was also sufficient to cause Cif activation, suggesting that caspases might be required for Cif activation. Consistent with these observations, Fas-induced apoptosis in Jurkat cells resulted in caspase 8 activation and subsequently in activation of Cif. Finally, we demonstrate that the activation of Cif correlated with the activation of the Bcl-2 family member Bid by caspases and that Cif activity was selectively neutralized by anti-Bid antibodies. Taken together, these results indicate that Cif is identical to Bid and that it can be inhibited by Bcl-2 and activated by caspases. Thus, Cif (Bid) is an important biological regulator for the transduction of apoptotic signals.

  18. Combining CAR T cells and the Bcl-2 family apoptosis inhibitor ABT-737 for treating B-cell malignancy.

    PubMed

    Karlsson, H; Karlsson, S C H; Lindqvist, A C; Fransson, M; Paul-Wetterberg, G; Nilsson, B; Essand, M; Nilsson, K; Frisk, P; Jernberg-Wiklund, H; Loskog, A; Loskog, S I A

    2013-07-01

    B-cell malignancies upregulate the B-cell lymphoma 2 (Bcl-2) family inhibitors of the intrinsic apoptosis pathway, making them therapy resistant. However, small-molecule inhibitors of Bcl-2 family members such as ABT-737 restore a functional apoptosis pathway in cancer cells, and its oral analog ABT-263 (Navitoclax) has entered clinical trials. Gene engineered chimeric antigen receptor (CAR) T cells also show promise in B-cell malignancy, and as they induce apoptosis via the extrinsic pathway, we hypothesized that small-molecule inhibitors of the Bcl-2 family may potentiate the efficacy of CAR T cells by engaging both apoptosis pathways. CAR T cells targeting CD19 were generated from healthy donors as well as from pre-B-ALL (precursor-B acute lymphoblastic leukemia) patients and tested together with ABT-737 to evaluate apoptosis induction in five B-cell tumor cell lines. The CAR T cells were effective even if the cell lines exhibited different apoptosis resistance profiles, as shown by analyzing the expression of apoptosis inhibitors by PCR and western blot. When combining T-cell and ABT-737 therapy simultaneously, or with ABT-737 as a presensitizer, tumor cell apoptosis was significantly increased. In conclusion, the apoptosis inducer ABT-737 enhanced the efficacy of CAR T cells and could be an interesting drug candidate to potentiate T-cell therapy.

  19. Leishmania donovani-Induced Increase in Macrophage Bcl-2 Favors Parasite Survival

    PubMed Central

    Pandey, Rajeev Kumar; Mehrotra, Sanjana; Sharma, Smriti; Gudde, Ramachandra Subbaraya; Sundar, Shyam; Shaha, Chandrima

    2016-01-01

    Members of the Bcl-2 family are major regulators of apoptosis in mammalian cells, and hence infection-induced perturbations in their expression could result into elimination of the parasites or creation of a niche favoring survival. In this investigation, we uncover a novel role of host Bcl-2 in sustaining Leishmania donovani infection. A rapid twofold increase in Bcl-2 expression occurred in response to parasite challenge. Downregulation of post infection Bcl-2 increase using siRNA or functional inhibition using Bcl-2 small molecule inhibitors interfered with intracellular parasite survival confirming the necessity of elevated Bcl-2 during infection. An increased nitric oxide (NO) response and reduced parasitic burden was observed upon Bcl-2 inhibition, where restitution of the NO response accounted for parasite mortality. Mechanistic insights revealed a major role of elevated Th2 cytokine IL-13 in parasite-induced Bcl-2 expression via the transcription factor STAT-3, where blocking at the level of IL-13 receptor or downstream kinase JAK-2 dampened Bcl-2 induction. Increase in Bcl-2 was orchestrated through Toll like receptor (TLR)-2-MEK-ERK signaling, and changes in TLR-2 levels affected parasite uptake. In a mouse model of visceral leishmaniasis (VL), Bcl-2 inhibitors partially restored the antimicrobial NO response by at least a twofold increase that resulted in significantly reduced parasite burden. Interestingly, monocytes derived from the peripheral blood of six out of nine human VL subjects demonstrated Bcl-2 expression at significantly higher levels, and sera from these patients showed only marginally quantifiable nitrites. Collectively, our study for the first time reveals a pro-parasitic role of host Bcl-2 and the capacity of host-derived IL-13 to modulate NO levels during infection via Bcl-2. Here, we propose Bcl-2 inhibition as a possible therapeutic intervention for VL. PMID:27826299

  20. Association of Genetic Markers in the BCL-2 Family of Apoptosis-Related Genes with Endometrial Cancer Risk in a Chinese Population

    PubMed Central

    Dorjgochoo, Tsogzolmaa; Xiang, Yong-Bing; Long, Jirong; Shi, Jiajun; Deming, Sandra; Xu, Wang-Hong; Cai, Hui; Cheng, Jiarong; Cai, Qiuyin; Zheng, Wei; Shu, Xiao-Ou

    2013-01-01

    Background In vitro studies have demonstrated the role of the BCL-2 family of genes in endometrial carcinogenesis. The role of genetic variants in BCL-2 genes and their interactions with non-genetic factors in the development of endometrial cancer has not been investigated in epidemiological studies. Patients and Methods We examined the relationship between BCL-2 gene family variants and endometrial cancer risk among 1,028 patients and 1,922 age-matched community controls from Shanghai, China. We also investigated possible interactions between genetic variants and established risk factors (demographic, lifestyle and clinical). Individuals were genotyped for 86 tagging single nucleotide polymorphisms (SNPs) in the BCL2, BAX, BAD and BAK1 genes. Results Significant associations with endometrial cancer risk were found for 9 SNPs in the BCL2 gene (P trend<0.05 for all). For SNPs rs17759659 and rs7243091 (minor allele for both: G), the associations were independent. The odds ratio was 1.27 (95% CI: 1.04–1.53) for women with AG genotype for the SNP rs17759659 and 1.82 (95% CI: 1.21–2.73) for women with the GG genotype for the SNP rs7243091. No interaction between these two SNPs and established non-genetic risk factors of endometrial cancer was noticed. Conclusion Genetic polymorphisms in the BCL2 gene may be associated with the risk of endometrial cancer in Chinese women. PMID:23637776

  1. Telomerase activity, estrogen receptors (α, β), Bcl-2 expression in human breast cancer and treatment response

    PubMed Central

    Murillo-Ortiz, Blanca; Astudillo-De la Vega, Horacio; Castillo-Medina, Sebastian; Malacara, JM; Benitez-Bribiesca, Luis

    2006-01-01

    Background The mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences, lost during replication by means of an intrinsic RNA component as a template for polymerization. Among the telomerase subunits, hTERT (human telomerase reverse transcriptase) is expressed concomitantly with the activation of telomerase. The role of estrogens and their receptors in the transcriptional regulation of hTERT has been demonstrated. The current study determines the possible association between telomerase activity, the expression of both molecular forms of estrogen receptor (ERα and ERβ) and the protein bcl-2, and their relative associations with clinical parameters. Methods Tissue samples from 44 patients with breast cancer were used to assess telomerase activity using the TRAP method and the expression of ERα, ERβ and bcl-2 by means of immunocytochemical techniques. Results Telomerase activity was detected in 59% of the 44 breast tumors examined. Telomerase activity ranged from 0 to 49.93 units of total product generated (TPG). A correlation was found between telomerase activity and differentiation grade (p = 0.03). The only significant independent marker of response to treatment was clinical stage. We found differences between the frequency of expression of ERα (88%) and ERβ (36%) (p = 0.007); bcl-2 was expressed in 79.5% of invasive breast carcinomas. We also found a significant correlation between low levels of telomerase activity and a lack of ERβ expression (p = 0.03). Conclusion Lower telomerase activity was found among tumors that did not express estrogen receptor beta. This is the first published study demonstrating that the absence of expression of ERβ is associated with low levels of telomerase activity. PMID:16911782

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

    PubMed

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

    2015-02-01

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

  3. Caspase-Dependent and Caspase-Independent Pathways Are Involved in Cadmium-Induced Apoptosis in Primary Rat Proximal Tubular Cell Culture

    PubMed Central

    Long, Mengfei; Bian, Jianchun; Liu, Xuezhong; Gu, Jianhong; Yuan, Yan; Song, Ruilong; Wang, Yi; Zhu, Jiaqiao; Liu, Zongping

    2016-01-01

    We designed this study to investigate whether cadmium induces caspase-independent apoptosis and to investigate the relationship between the caspase-dependent and caspase-independent apoptotic pathways. Cadmium (1.25–2.5 μM) induced oxidative stress in rat proximal tubular (rPT) cells, as seen in the reactive oxygen species levels; N-acetylcysteine prevented this. Cyclosporin A (CsA) prevented mitochondrial permeability transition pore opening and apoptosis; there was mitochondrial ultrastructural disruption, mitochondrial cytochrome c (cyt c) translocation to the cytoplasm, and subsequent caspase-9 and caspase-3 activation. Z-VAD-FMK prevented caspase-3 activation and apoptosis and decreased BNIP-3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) expression levels and apoptosis-inducing factor/endonuclease G (AIF/Endo G) translocation. Simultaneously, cadmium induced prominent BNIP-3 expression in the mitochondria and cytoplasmic AIF/Endo G translocation to the nucleus. BNIP-3 silencing significantly prevented AIF and Endo G translocation and decreased the apoptosis rate, cyt c release, and caspase-9 and caspase-3 activation. These results suggest that BNIP-3 is involved in the caspase-independent apoptotic pathway and is located upstream of AIF/Endo G; both the caspase-dependent and caspase-independent pathways are involved in cadmium-induced rPT cell apoptosis and act synergistically. PMID:27861627

  4. Expression Profile of BCL-2, BCL-XL, and MCL-1 Predicts Pharmacological Response to the BCL-2 Selective Antagonist Venetoclax in Multiple Myeloma Models.

    PubMed

    Punnoose, Elizabeth A; Leverson, Joel D; Peale, Franklin; Boghaert, Erwin R; Belmont, Lisa D; Tan, Nguyen; Young, Amy; Mitten, Michael; Ingalla, Ellen; Darbonne, Walter C; Oleksijew, Anatol; Tapang, Paul; Yue, Peng; Oeh, Jason; Lee, Leslie; Maiga, Sophie; Fairbrother, Wayne J; Amiot, Martine; Souers, Andrew J; Sampath, Deepak

    2016-05-01

    BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL-selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2(High)/BCL-XL (Low) In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132-44. ©2016 AACR.

  5. The involvement of Bcl-2 family proteins in AKT-regulated cell survival in cisplatin resistant epithelial ovarian cancer

    PubMed Central

    Dai, Yan; Jin, Shiguang; Li, Xueping; Wang, Daxin

    2017-01-01

    Many studies involving patients with cisplatin-resistant ovarian cancer have shown that AKT activation leads to inhibition of apoptosis. The aim of this study was to examine the potential involvement of the Bcl-2 family proteins in AKT-regulated cell survival in response to cisplatin treatment. Cisplatin-sensitive (PEO1) and cisplatin-resistant (PEO4) cells were taken from ascites of patients with ovarian cancer before cisplatin treatment and after development of chemoresistance. It was found that cisplatin treatment activated the AKT signaling pathway and promoted cell proliferation in cisplatin-resistant EOC cells. When AKT was transfected into nucleus of cisplatin-resistant ovarian cancer cells, DNA-PK was phosphorylated at S473. The activated AKT (pAKT-S473) in these cells inhibited the death signal induced by cisplatin thereby inhibiting cisplatin-mediated apoptosis. Results from this study showed that the combination of cisplatin, DNA-PK inhibitor NU7441, and AKT inhibitor TCN can overcome drug resistance, increase apoptosis, and re-sensitize PEO4 cells to cisplatin treatment. A decrease in apoptotic activity was seen in PEO4 cells when Bad was downregulated by siRNA, which indicated that Bad promotes apoptosis in PEO4 cells. Use of the Bcl-2 inhibitor ABT-737 showed that ABT-737 binds to Bcl-2 but not Mcl-1 and releases Bax/Bak which leads to cell apoptosis. The combination of ABT-737 and cisplatin leads to a significant increase in the death of PEO1 and PEO4 cells. All together, these results indicate that Bcl-2 family proteins are regulators of drug resistance. The combination of cisplatin and Bcl-2 family protein inhibitor could be a strategy for the treatment of cisplatin-resistant ovarian cancer. PMID:27935869

  6. DENSpm overcame Bcl-2 mediated resistance against Paclitaxel treatment in MCF-7 breast cancer cells via activating polyamine catabolic machinery.

    PubMed

    Akyol, Zeynep; Çoker-Gürkan, Ajda; Arisan, Elif Damla; Obakan-Yerlikaya, Pınar; Palavan-Ünsal, Narçin

    2016-12-01

    The Bcl-2 mediated resistance is one of the most critical obstacle in cancer therapy. Conventional chemotherapeutics such as Paclitaxel, a commonly used in the treatment of metastatic breast cancer, is not sufficient to overcome Bcl-2 mediated drug resistance mechanism. Thus, combinational drug regimes are favored by researchers to overcome resistance phenotype against drugs. N1,N11-diethylnorspermine (DENSpm), a polyamine analogue, which is a promising drug candidate induced-cell cycle arrest and apoptosis in various cancer cells such as prostate, melanoma, colon and breast cancer cells via activated polyamine catabolism and reactive oxygen generation. Recent studies indicated the potential therapeutic role of DENSpm in phase I and II trials in breast cancer cases. Although the molecular targets of Paclitaxel in apoptotic cell death mechanism is well documented, the therapeutic effect of DENSpm and Paclitaxel in breast cancer cells has not been investigated yet. In this study, our aim was to determine the time dependent effect of DENSpm and Paclitaxel on apoptotic cell death via determination of polyamine metabolism related targets in wt and Bcl-2 overexpressing MCF-7 breast cancer cells. In our experimental study, Paclitaxel decreased cell viability in dose-dependent manner within 24h. Co-treatment of Paclitaxel (30nM) with DENSpm (20μM) further increased the cytoxicity of Paclitaxel (30nM) compared to alone Paclitaxel (30nM) treatment in MCF-7 Bcl-2+ breast cancer cells. In addition, we determined that resistance against Paclitaxel-induced apoptotic cell death in Bcl-2 overexpressed MCF-7 cells was overcome due to activation of polyamine catabolic pathway, which caused depletion of polyamines. DENSpm combinational treatment might increase the effect of low cytotoxic paclitaxel in drug-resistant breast cancer cases. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  7. 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.

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

    PubMed

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

    2016-01-15

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

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

    PubMed Central

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

    2015-01-01

    Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca2+ signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca2+] ([Ca2+]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca2+ eliminated the plateau phase increase of [Ca2+]cyt in lung cancer cells, indicating that the plateau phase of [Ca2+]cyt increase is due to Ca2+ influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca2+ or chelating intracellular Ca2+ with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca2+]cyt through Ca2+ influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. PMID:26491047

  10. Differential expression of Fas family members and Bcl-2 family members in benign versus malignant epithelial ovarian cancer (EOC) in North Indian population.

    PubMed

    Chaudhry, Parvesh; Srinivasan, Radhika; Patel, Firuza D

    2012-09-01

    Epithelial ovarian cancer (EOC) represents the most challenging of gynecological malignancies. Defective apoptosis is a major causative factor in the development and progression of cancer. The two important pathways of apoptosis are extrinsic death receptor pathway (Fas family) and intrinsic mitochondrial pathway (Bcl-2 family). In this study, differential protein expression of the major Fas family members (Fas, FasL, and FAP-1) and Bcl-2 family members (Bax, Bcl-2, and Bcl-X(L)) in benign versus malignant surface epithelial ovarian tumors was evaluated at the protein level by immunohistochemistry. The expression of these molecules was compared in 30 benign versus 35 malignant surface epithelial ovarian tumors. The findings of the present study showed that there was no significant difference in the expression of the Fas family members in benign and malignant ovarian tumors. However, benign tumors showed higher levels of anti-apoptotic Bcl-2 protein levels (p < 0.009), whereas malignant tumors showed higher levels of pro-apoptotic Bax (p < 0.001). In general, there was no significant difference in Bcl-X(L) protein levels. The observations made in the present study suggest that alterations in expression of the Fas family and the Bcl-2 family members occur and play a key role in the deregulated growth of epithelial ovarian cancer.

  11. Identification of a novel regulatory domain in Bcl-X(L) and Bcl-2.

    PubMed Central

    Chang, B S; Minn, A J; Muchmore, S W; Fesik, S W; Thompson, C B

    1997-01-01

    Bcl-X(L), a member of the Bcl-2 family, can inhibit many forms of programed cell death. The three-dimensional structure of Bcl-X(L) identified a 60 amino acid loop lacking defined structure. Although amino acid sequence within this region is not conserved among Bcl-2 family members, structural modeling suggested that Bcl-2 also contains a large unstructured region. Compared with the full-length protein, loop deletion mutants of Bcl-X(L) and Bcl-2 displayed an enhanced ability to inhibit apoptosis. Despite enhanced function, the deletion mutants did not have significant alterations in the ability to bind pro-apoptotic proteins such as Bax. The loop deletion mutant of Bcl-2 also displayed a qualitative difference in its ability to inhibit apoptosis. Full-length Bcl-2 was unable to prevent anti-IgM-induced cell death of the immature B cell line WEHI-231. In contrast, the Bcl-2 deletion mutant protected WEHI-231 cells from death. Substantial differences were observed in the ability of WEHI-231 cells to phosphorylate the deletion mutant of Bcl-2 compared with full-length Bcl-2. Bcl-2 phosphorylation was found to be dependent on the presence of an intact loop domain. These results suggest that the loop domain in Bcl-X(L) and Bcl-2 can suppress the anti-apoptotic function of these genes and may be a target for regulatory post-translational modifications. PMID:9118958

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

    PubMed Central

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

    2017-01-01

    The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation. PMID:28028215

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

    PubMed

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

    2017-01-10

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

  14. MicroRNAs affect BCL-2 family proteins in the setting of cerebral ischemia.

    PubMed

    Ouyang, Yi-Bing; Giffard, Rona G

    2014-11-01

    The BCL-2 family is centrally involved in the mechanism of cell death after cerebral ischemia. It is well known that the proteins of the BCL-2 family are key regulators of apoptosis through controlling mitochondrial outer membrane permeabilization. Recent findings suggest that many BCL-2 family members are also directly involved in controlling transmission of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria through a specialization called the mitochondria-associated ER membrane (MAM). Increasing evidence supports the involvement of microRNAs (miRNAs), some of them targeting BCL-2 family proteins, in the regulation of cerebral ischemia. In this mini-review, after highlighting current knowledge about the multiple functions of BCL-2 family proteins and summarizing their relationship to outcome from cerebral ischemia, we focus on the regulation of BCL-2 family proteins by miRNAs, especially miR-29 which targets multiple BCL-2 family proteins. Copyright © 2013 Elsevier Ltd. All rights reserved.

  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

    2016-05-05

    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.

  16. The selective Bcl-2 inhibitor venetoclax, a BH3 mimetic, does not dysregulate intracellular Ca(2+) signaling.

    PubMed

    Vervloessem, Tamara; Ivanova, Hristina; Luyten, Tomas; Parys, Jan B; Bultynck, Geert

    2016-11-30

    Anti-apoptotic B cell-lymphoma-2 (Bcl-2) proteins are emerging as therapeutic targets in a variety of cancers for precision medicines, like the BH3-mimetic drug venetoclax (ABT-199), which antagonizes the hydrophobic cleft of Bcl-2. However, the impact of venetoclax on intracellular Ca(2+) homeostasis and dynamics in cell systems has not been characterized in detail. Here, we show that venetoclax did not affect Ca(2+)-transport systems from the endoplasmic reticulum (ER) in permeabilized cell systems. Venetoclax (1μM) did neither trigger Ca(2+) release by itself nor affect agonist-induced Ca(2+) release in a variety of intact cell models. Among the different cell types, we also studied two Bcl-2-dependent cancer cell models with a varying sensitivity towards venetoclax, namely SU-DHL-4 and OCI-LY-1, both diffuse large B-cell lymphoma cell lines. Acute application of venetoclax did also not dysregulate Ca(2+) signaling in these Bcl-2-dependent cancer cells. Moreover, venetoclax-induced cell death was independent of intracellular Ca(2+) overload, since Ca(2+) buffering using BAPTA-AM did not suppress venetoclax-induced cell death. This study therefore shows that venetoclax does not dysregulate the intracellular Ca(2+) homeostasis in a variety of cell types, which may underlie its limited toxicity in human patients. Furthermore, venetoclax-induced cell death in Bcl-2-dependent cancer cells is not mediated by intracellular Ca(2+) overload. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

  17. 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

  18. Sequence and partial functional analysis of canine Bcl-2 family proteins.

    PubMed

    de Brot, S; Schade, B; Croci, M; Dettwiler, M; Guscetti, F

    2016-02-01

    Dogs present with spontaneous neoplasms biologically similar to human cancers. Apoptotic pathways are deregulated during cancer genesis and progression and are important for therapy. We have assessed the degree of conservation of a set of canine Bcl-2 family members with the human and murine orthologs. To this end, seven complete canine open reading frames were cloned in this family, four of which are novel for the dog, their sequences were analyzed, and their functional interactions were studied in yeasts. We found a high degree of overall and domain sequence homology between canine and human proteins. It was slightly higher than between murine and human proteins. Functional interactions between canine pro-apoptotic Bax and Bak and anti-apoptotic Bcl-xL, Bcl-w, and Mcl-1 were recapitulated in yeasts. Our data provide support for the notion that systems based on canine-derived proteins might faithfully reproduce Bcl-2 family member interactions known from other species and establish the yeast as a useful tool for functional studies with canine proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells.

    PubMed

    Cao, Yongmei; Jiang, Zhen; Zeng, Zhen; Liu, Yujing; Gu, Yuchun; Ji, Yingying; Zhao, Yupeng; Li, Yingchuan

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5% O2) or normoxic (21% O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

  20. Mitochondrial PKM2 regulates oxidative stress-induced apoptosis by stabilizing Bcl2

    PubMed Central

    Liang, Ji; Cao, Ruixiu; Wang, Xiongjun; Zhang, Yajuan; Wang, Pan; Gao, Hong; Li, Chen; Yang, Fan; Zeng, Rong; Wei, Ping; Li, Dawei; Li, Wenfeng; Yang, Weiwei

    2017-01-01

    Pyruvate kinase M2 isoform (PKM2) catalyzes the last step of glycolysis and plays an important role in tumor cell proliferation. Recent studies have reported that PKM2 also regulates apoptosis. However, the mechanisms underlying such a role of PKM2 remain elusive. Here we show that PKM2 translocates to mitochondria under oxidative stress. In the mitochondria, PKM2 interacts with and phosphorylates Bcl2 at threonine (T) 69. This phosphorylation prevents the binding of Cul3-based E3 ligase to Bcl2 and subsequent degradation of Bcl2. A chaperone protein, HSP90α1, is required for this function of PKM2. HSP90α1's ATPase activity launches a conformational change of PKM2 and facilitates interaction between PKM2 and Bcl2. Replacement of wild-type Bcl2 with phosphorylation-deficient Bcl2 T69A mutant sensitizes glioma cells to oxidative stress-induced apoptosis and impairs brain tumor formation in an orthotopic xenograft model. Notably, a peptide that is composed of the amino acid residues from 389 to 405 of PKM2, through which PKM2 binds to Bcl2, disrupts PKM2-Bcl2 interaction, promotes Bcl2 degradation and impairs brain tumor growth. In addition, levels of Bcl2 T69 phosphorylation, conformation-altered PKM2 and Bcl2 protein correlate with one another in specimens of human glioblastoma patients. Moreover, levels of Bcl2 T69 phosphorylation and conformation-altered PKM2 correlate with both grades and prognosis of glioma malignancy. Our findings uncover a novel mechanism through which mitochondrial PKM2 phosphorylates Bcl2 and inhibits apoptosis directly, highlight the essential role of PKM2 in ROS adaptation of cancer cells, and implicate HSP90-PKM2-Bcl2 axis as a potential target for therapeutic intervention in glioblastoma. PMID:28035139

  1. Microtubule-damaging drugs triggered bcl2 phosphorylation-requirement of phosphorylation on both serine-70 and serine-87 residues of bcl2 protein.

    PubMed

    Basu, A; Haldar, S

    1998-10-01

    Specifically anti-microtubule agents such as taxol, vincristine, vinblastine and dolastatin can trigger Bcl2 phosphorylation at G2-M phase of the cell cycle in malignant cells derived from a variety of human cancers. In this study, the status of Bcl2 phosphorylation was investigated in response to more antimicrotubule agents such as colchicine, colcemid or podophyllotoxin. Although these agents are not currently used for cancer therapy, they were able to trigger Bcl2 phosphorylation with simultaneous apoptosis in cancer cells. Previously, by using extensive site-directed mutagenesis studies we determined that mutation of serine-70 to alanine could not completely abrogate taxol induced Bcl2 phosphorylation. Studies reported here clearly indicate that serine-87 residue along with serine-70 of Bcl2 protein are necessary for microtubule damaging drug induced phosphorylation.

  2. TIC10/ONC201 synergizes with Bcl-2/Bcl-xL inhibition in glioblastoma by suppression of Mcl-1 and its binding partners in vitro and in vivo.

    PubMed

    Karpel-Massler, Georg; Bâ, Maïmouna; Shu, Chang; Halatsch, Marc-Eric; Westhoff, Mike-Andrew; Bruce, Jeffrey N; Canoll, Peter; Siegelin, Markus D

    2015-11-03

    Glioblastoma is the most frequent primary brain tumor in adults. Current therapeutic options are sparse and the prognosis of patients suffering from this disease is grim. Abundance in intratumoral heterogeneity among different deregulated signaling pathways is a hallmark of glioblastoma and likely accounts for its recurrence and resistance to treatment. Glioblastomas harbor a plethora of deregulated pathways driving tumor formation and growth. In this study, we show that TIC10/ONC201, a promising compound that is currently in planned clinical development, along with Bcl-2/Bcl-xL inhibition by ABT263 yields a strong synergistic antiproliferative effect on pediatric, adult, proneural glioblastoma and glioma stem-like cells. On the molecular level, treatment with TIC10/ONC201 results in a posttranslational decrease of the anti-apoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), through modulation of the chaperone Bag3 and the deubiquitinase Usp9X. Consistently, the combination treatment of TIC10/ONC201 and ABT263 required the presence of functional BAX and BAK to drive intrinsic apoptosis, but is surprisingly independent of the extrinsic apoptotic pathway. Moreover, the expression of Noxa protein was required for efficient apoptosis induction by TIC10/ONC201 and ABT263. Importantly, the drug combination of TIC10/ONC201 and the BH3-mimetic, ABT263, led to a regression of tumors in vivo, without any notable toxicity and side effects. Overall, TIC10/ONC201 along with Bcl-2/Bcl-xL inhibition holds significant promise as a novel potential approach for the treatment of recalcitrant tumors such as glioblastoma.

  3. Platelet production proceeds independently of the intrinsic and extrinsic apoptosis pathways.

    PubMed

    Josefsson, Emma C; Burnett, Deborah L; Lebois, Marion; Debrincat, Marlyse A; White, Michael J; Henley, Katya J; Lane, Rachael M; Moujalled, Diane; Preston, Simon P; O'Reilly, Lorraine A; Pellegrini, Marc; Metcalf, Donald; Strasser, Andreas; Kile, Benjamin T

    2014-03-17

    BH3 mimetic drugs that target BCL-2 family pro-survival proteins to induce tumour cell apoptosis represent a new era in cancer therapy. Clinical trials of navitoclax (ABT-263, which targets BCL-2, BCL-XL and BCL-W) have shown great promise, but encountered dose-limiting thrombocytopenia. Recent work has demonstrated that this is due to the inhibition of BCL-XL, which is essential for platelet survival. These findings raise new questions about the established model of platelet shedding by megakaryocytes, which is thought to be an apoptotic process. Here we generate mice with megakaryocyte-specific deletions of the essential mediators of extrinsic (Caspase-8) and intrinsic (BAK/BAX) apoptosis. We show that megakaryocytes possess a Fas ligand-inducible extrinsic apoptosis pathway. However, Fas activation does not stimulate platelet production, rather, it triggers Caspase-8-mediated killing. Combined loss of Caspase-8/BAK/BAX does not impair thrombopoiesis, but can protect megakaryocytes from death in mice infected with lymphocytic choriomeningitis virus. Thus, apoptosis is dispensable for platelet biogenesis.

  4. 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

  5. MYC/BCL2 double-hit high-grade B-cell lymphoma.

    PubMed

    Li, Shaoying; Lin, Pei; Young, Ken H; Kanagal-Shamanna, Rashmi; Yin, C Cameron; Medeiros, L Jeffrey

    2013-09-01

    Double-hit lymphoma (DHL) has been defined by others as a B-cell lymphoma with MYC/8q24 rearrangement in combination with a translocation involving another gene, such as BCL2, BCL3, or BCL6. The most common form of DHL has translocations involving MYC and BCL2, also known as MYC/BCL2 DHL. In recent years, a number of case series of MYC/BCL2 DHL have been published. Most cases of MYC/BCL2 DHL morphologically resemble diffuse large B-cell lymphoma (DLBCL) or B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma. These tumors are of B-cell lineage, have a germinal center B-cell immunophenotype with a high proliferation rate, and a complex karyotype. Patients with these tumors have an aggressive clinical course and poor prognosis despite high-intensity chemotherapy. More recently, studies have suggested expanding the spectrum of MYC/BCL2 DHL to include cases that have concurrent MYC and BCL2 cytogenetic abnormalities, but not necessarily translocations. In addition, overexpression of MYC and BCL2 has been shown in an appreciable subset of DLBCL tumors. These tumors show overlap with MYC/BCL2 DHL, but are not equivalent. In this review, we discuss the clinicopathologic, immunophenotypic, cytogenetic, and prognostic features of MYC/BCL2 DHL.

  6. Bcl2L13 is a ceramide synthase inhibitor in glioblastoma

    PubMed Central

    Jensen, Samuel A.; Calvert, Andrea E.; Volpert, Giora; Kouri, Fotini M.; Hurley, Lisa A.; Luciano, Janina P.; Wu, Yongfei; Chalastanis, Alexandra; Futerman, Anthony H.; Stegh, Alexander H.

    2014-01-01

    Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13–CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use. PMID:24706805

  7. Topographic distribution of bcl-2 protein in feline tissues in health and neoplasia.

    PubMed

    Madewell, B R; Gandour-Edwards, R; Edwards, B F; Walls, J E; Griffey, S M

    1999-11-01

    The bcl-2 family of genes encodes proteins that influence apoptosis. In the present immunohistochemical study, the topographic distribution of bcl-2 protein was examined in healthy feline fetal, neonatal, and adult tissues, a feline renal cell line, and feline tumors obtained from a veterinary hospital. The topographic distribution of bcl-2 in healthy tissues was similar to that described in human tissues. In lymphoid tissues, follicular mantle cells strongly expressed bcl-2. In complex and differentiating epithelium, bcl-2 expression was detected in stem cell and proliferation zones. Bcl-2 expression was also detected in lower crypts of the intestine and in skin basal layers. The feline Crandell kidney cells expressed bcl-2 diffusely throughout the cytoplasm. Of 180 tumors examined, bcl-2 was expressed almost uniformly in cutaneous basal cell tumors, thyroid adenomas, and mammary carcinomas and in 50% of the lymphomas examined. Bcl-2 may play a role in blocking apoptotic cell death in a broad range of normal feline tissues, whereas dysregulated bcl-2 may extend the life of certain tumors or render certain tumors resistant to therapy because most chemotherapeutic and radiotherapeutic agents eliminate tumor cells by triggering apoptosis.

  8. Attenuation of both apoptotic and necrotic actions of cadmium by Bcl-2.

    PubMed Central

    Ishido, Masami; Ohtsubo, Rieko; Adachi, Tatsumi; Kunimoto, Manabu

    2002-01-01

    We examined the effects of cadmium on the bcl-2 family of proteins--bcl-2, bax, bad, and bcl-xS/L--in cadmium-induced cytotoxicity. Addition of 10 microM cadmium to cultured porcine kidney LLC-PK(1) cells caused apoptosis. Western blot analyses revealed that cadmium markedly increased endogenous bcl-2 protein (to 3-4 times the level in wild-type cells) earlier than metallothionein induction, but that the metal did not enhance the induction of bax, bad, or bcl-xS proteins. Cadmium also induced the transcript of bcl-2, with the amount of bcl-2 reaching a maximum at 1-2 hr of exposure; this increase occurred earlier than cadmium-induced increase in the protooncogene such as c-myc. A cadmium-induced increase in endogenous bcl-2 protein was also seen in rat primary thymocytes. Overexpression of the bcl-2 protein by gene transfection prevented cadmium-induced apoptosis. Following the detection of apoptosis, lactate dehydrogenase release in the culture medium (a marker of necrosis) was observed, and this release was also inhibited by overexpression of bcl-2. Electron microscopic observations also supported the fact that cadmium induced apoptotic chromatin condensation at an early stage of exposure, followed by necrotic features of the cells, both of which were also inhibited by overexpression of bcl-2 proteins. Thus, our data demonstrated that both apoptotic and necrotic actions of cadmium were attenuated by bcl-2. PMID:11781163

  9. Bcl2L13 is a ceramide synthase inhibitor in glioblastoma.

    PubMed

    Jensen, Samuel A; Calvert, Andrea E; Volpert, Giora; Kouri, Fotini M; Hurley, Lisa A; Luciano, Janina P; Wu, Yongfei; Chalastanis, Alexandra; Futerman, Anthony H; Stegh, Alexander H

    2014-04-15

    Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13-CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use.

  10. Bcl-2 associated with positive symptoms of schizophrenic patients in an acute phase.

    PubMed

    Tsai, Meng-Chang; Liou, Chia-Wei; Lin, Tsu-Kung; Lin, I-Mei; Huang, Tiao-Lai

    2013-12-30

    B cell lymphoma protein-2 (Bcl-2) may contribute to the pathophysiology of schizophrenia in the brain. The aim of this study was to investigate the serum levels of Bcl-2 in schizophrenic patients in an acute phase, and evaluate Bcl-2 level changes after antipsychotic treatment. We consecutively enrolled 41 schizophrenia patients in an acute phase; 28 were followed up with a 4-week antipsychotic treatment. Serum Bcl-2 levels were measured with assay kits. All patients were evaluated by examining the correlation between Bcl-2 levels and Positive and Negative Syndrome Scale (PANSS) scores, using Pearson correlation coefficients. In schizophrenic patients in an acute phase, positive PANSS subscores were significantly negatively correlated with Bcl-2 levels. In addition, we found Bcl-2 levels had a significantly negative correlation with PANSS total scores and positive subscores in male patients in an acute phase. Using the paired t-test, we found no significant changes in Bcl-2 levels in schizophrenia patients who had received the 4-week treatment with antipsychotic drugs (n=28). In conclusion, our results suggest that Bcl-2 might be an indicator of schizophrenia severity in the acute phase. In addition, Bcl-2 levels might be associated with positive symptoms in male patients with schizophrenia.

  11. BCL2L2 — EDRN Public Portal

    Cancer.gov

    From NCBI Gene: This gene encodes a member of the BCL-2 protein family. The proteins of this family form hetero- or homodimers and act as anti- and pro-apoptotic regulators. Expression of this gene in cells has been shown to contribute to reduced cell apoptosis under cytotoxic conditions. Studies of the related gene in mice indicated a role in the survival of NGF- and BDNF-dependent neurons. Mutation and knockout studies of the mouse gene demonstrated an essential role in adult spermatogenesis. Alternative splicing results in multiple transcript variants. Read-through transcription also exists between this gene and the neighboring downstream PABPN1 (poly(A) binding protein, nuclear 1) gene. [provided by RefSeq, Dec 2010

  12. A Surface Groove Essential for Viral Bcl-2 Function During Chronic Infection In Vivo

    PubMed Central

    Petros, Andrew M; Nettesheim, David; van Dyk, Linda F.; Labrada, Lucia; Speck, Samuel H; Levine, Beth

    2005-01-01

    Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the γ-herpesvirus 68 (γHV68) Bcl-2 family protein (γHV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the γHV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type γHV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of γHV68 from latency and efficient persistent γHV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection. PMID:16201011

  13. 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

    2016-06-21

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

  14. Proposal for a modified grading system based on mitotic index and Bcl2 provides objective determination of clinical outcome for patients with breast cancer.

    PubMed

    Abdel-Fatah, Tarek M A; Powe, Desmond G; Ball, Graham; Lopez-Garcia, Maria A; Habashy, Hany O; Green, Andrew R; Reis-Filho, Jorge S; Ellis, Ian O

    2010-12-01

    We hypothesized that the interaction between mitotic index (M) and Bcl2 could accurately discriminate between low- and high-grade breast cancer (BC) and provide a more objective measure of clinical outcome than histological grade, especially for patients with intermediate histological grade (G2), small size or oestrogen receptor (ER)-negative cancers. A well-characterized series of 1650 BCs with long-term follow-up was subjected to immunohistochemical analysis for Bcl2. Mitotic index (M) was assessed according to Nottingham Grading System (NGS) guidelines: M1: < 10 mitoses; M2: 10-18 mitoses; M3: > 18 mitoses. Results were validated in an independent series of patients (n = 245) uniformly treated with adjuvant anthracycline-based chemotherapy. Subsequently, BCs were classified according to the combined M/Bcl2 profile and compared with NGS. Multivariate Cox regression models using validated prognostic factors demonstrated that the subgroups defined by M/Bcl2 profile remained significantly associated with patients' outcome but also performed better than lymph node status and tumour size. Incorporation of the M/Bcl2 profile into the Nottingham Prognostic Index (NPI) reclassified twice as many patients into the excellent prognosis group, potentially improving decision-making and sparing patients unneeded systemic adjuvant therapy. Patients with M2-3/Bcl2- and M3/Bcl2+ (high risk) had a two- to three-fold increased risk of recurrence when treated with either adjuvant hormone therapy or anthracycline-based chemotherapy compared with those with M1/Bcl2 ± and M2/Bcl2+ (low risk) [HR = 3.4 (2.8-5.6); p < 0.0001 and HR = 2.3 (1.2-4.3); p = 0.0009]. In conclusion, a grading system defined by mitotic counting and Bcl2 expression accurately reclassified patients with NGS-G2, small tumour size or ER-negative cancers into two groups: low risk (NGS-G1-like) versus high risk (NGS-G3-like) of BC mortality and recurrence, improving prognosis and therapeutic planning.

  15. Natural Diterpenoid Compound Elevates Expression of Bim Protein, Which Interacts with Antiapoptotic Protein Bcl-2, Converting It to Proapoptotic Bax-like Molecule*

    PubMed Central

    Zhao, Lixia; He, Feng; Liu, Haiyang; Zhu, Yushan; Tian, Weili; Gao, Ping; He, Hongping; Yue, Wen; Lei, Xiaobo; Ni, Biyun; Wang, Xiaohui; Jin, Haijing; Hao, Xiaojiang; Lin, Jialing; Chen, Quan

    2012-01-01

    Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics. PMID:22065578

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

    PubMed Central

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

    2015-01-01

    Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3’-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways. PMID:26422397

  17. Brain-derived neurotrophic factor prevents changes in Bcl-2 family members and caspase-3 activation induced by excitotoxicity in the striatum.

    PubMed

    Pérez-Navarro, Esther; Gavaldà, Núria; Gratacòs, Elena; Alberch, Jordi

    2005-02-01

    Brain-derived neurotrophic factor (BDNF) prevents the loss of striatal neurons caused by excitotoxicity. We examined whether these neuroprotective effects are mediated by changes in the regulation of Bcl-2 family members. We first analyzed the involvement of the phosphatidylinositol 3-kinase/Akt pathway in this regulation, showing a reduction in phosphorylated Akt (p-Akt) levels after both quinolinate (QUIN, an NMDA receptor agonist) and kainate (KA, a non-NMDA receptor agonist) intrastriatal injection. Our results also show that Bcl-2, Bcl-x(L) and Bax protein levels and heterodimerization are selectively regulated by NMDA and non-NMDA receptor stimulation. Striatal cell death induced by QUIN is mediated by an increase in Bax and a decrease in Bcl-2 protein levels, leading to reduced levels of Bax:Bcl-2 heterodimers. In contrast, changes in Bax protein levels are not required for KA-induced apoptotic cell death, but decreased levels of both Bax:Bcl-2 and Bax:Bcl-x(L) heterodimer levels are necessary. Furthermore, QUIN and KA injection activated caspase-3. Intrastriatal grafting of a BDNF-secreting cell line counter-regulated p-AKT, Bcl-2, Bcl-x(L) and Bax protein levels, prevented changes in the heterodimerization between Bax and pro-survival proteins, and blocked caspase-3 activation induced by excitotoxicity. These results provide a possible mechanism to explain the anti-apoptotic effect of BDNF against to excitotoxicity in the striatum through the regulation of Bcl-2 family members, which is probably mediated by Akt activation.

  18. Oxygen diffusion pathways in a cofactor-independent dioxygenase

    PubMed Central

    Di Russo, Natali V.; Condurso, Heather L.; Li, Kunhua; Bruner, Steven D.; Roitberg, Adrian E.

    2015-01-01

    Molecular oxygen plays an important role in a wide variety of enzymatic reactions. Through recent research efforts combining computational and experimental methods a new view of O2 diffusion is emerging, where specific channels guide O2 to the active site. The focus of this work is DpgC, a cofactor-independent oxygenase. Molecular dynamics simulations, together with mutagenesis experiments and xenon-binding data, reveal that O2 reaches the active site of this enzyme using three main pathways and four different access points. These pathways connect a series of dynamic hydrophobic pockets, concentrating O2 at a specific face of the enzyme substrate. Extensive molecular dynamics simulations provide information about which pathways are more frequently used. This data is consistent with the results of kinetic measurements on mutants and is difficult to obtain using computational cavity-location methods. Taken together, our results reveal that although DpgC is rare in its ability of activating O2 in the absence of cofactors or metals, the way O2 reaches the active site is similar to that reported for other O2-using proteins: multiple access channels are available, and the architecture of the pathway network can provide regio- and stereoselectivity. Our results point to the existence of common themes in O2 access that are conserved among very different types of proteins. PMID:26508997

  19. Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans

    PubMed Central

    Denning, Daniel P.; Hatch, Victoria; Horvitz, H. Robert

    2013-01-01

    Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to

  20. bcl-2 Overexpression Reduces Apoptotic Photoreceptor Cell Death in Three Different Retinal Degenerations

    NASA Astrophysics Data System (ADS)

    Chen, Jeannie; Flannery, John G.; Lavail, Matthew M.; Steinberg, Roy H.; Xu, Jun; Simon, Melvin I.

    1996-07-01

    Apoptosis of photoreceptors occurs infrequently in adult retina but can be triggered in inherited and environmentally induced retinal degenerations. The protooncogene bcl-2 is known to be a potent regulator of cell survival in neurons. We created lines of transgenic mice overexpressing bcl-2 to test for its ability to increase photoreceptor survival. Bcl-2 increased photoreceptor survival in mice with retinal degeneration caused by a defective opsin or cGMP phosphodiesterase. Overexpression of Bcl-2 in normal photoreceptors also decreased the damaging effects of constant light exposure. Apoptosis was induced in normal photoreceptors by very high levels of bcl-2. We conclude that bcl-2 is an important regulator of photoreceptor cell death in retinal degenerations.

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

    PubMed

    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

    2016-02-02

    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.

  2. miR-15 and miR-16 induce apoptosis by targeting BCL2.

    PubMed

    Cimmino, Amelia; Calin, George Adrian; Fabbri, Muller; Iorio, Marilena V; Ferracin, Manuela; Shimizu, Masayoshi; Wojcik, Sylwia E; Aqeilan, Rami I; Zupo, Simona; Dono, Mariella; Rassenti, Laura; Alder, Hansjuerg; Volinia, Stefano; Liu, Chang-Gong; Kipps, Thomas J; Negrini, Massimo; Croce, Carlo M

    2005-09-27

    Chronic lymphocytic leukemia (CLL) is the most common human leukemia and is characterized by predominantly nondividing malignant B cells overexpressing the antiapoptotic B cell lymphoma 2 (Bcl2) protein. miR-15a and miR-16-1 are deleted or down-regulated in the majority of CLLs. Here, we demonstrate that miR-15a and miR-16-1 expression is inversely correlated to Bcl2 expression in CLL and that both microRNAs negatively regulate Bcl2 at a posttranscriptional level. BCL2 repression by these microRNAs induces apoptopsis in a leukemic cell line model. Therefore, miR-15 and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing tumors.

  3. Bcl-2 upregulation by HIV-1 Tat during infection of primary human macrophages in culture.

    PubMed

    Zhang, Mingjie; Li, Xingxiang; Pang, Xiaowu; Ding, Lina; Wood, Owen; Clouse, Kathleen A; Hewlett, Indira; Dayton, Andrew I

    2002-01-01

    The ability of cells of the human monocyte/macrophage lineage to host HIV-1 replication while resisting cell death is believed to significantly contribute to their ability to serve as a reservoir for viral replication in the host. Although macrophages are generally resistant to apoptosis, interruption of anti-apoptotic pathways can render them susceptible to apoptosis. Here we report that HIV-1(BAL )infection of primary human monocyte-derived macrophages (MDM) upregulates the mRNA and protein levels of the anti-apoptic gene, Bcl-2. Furthermore, this upregulation can be quantitatively mimicked by treating MDM with soluble HIV-1 Tat-86 protein. These results suggest that in infecting cells of the monocyte/macrophage lineage, HIV-1 may be benefiting from additional protection against apoptosis caused by specific upregulation of cellular anti-apoptotic genes.

  4. Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice.

    PubMed

    Vandenberg, C J; Josefsson, E C; Campbell, K J; James, C; Lawlor, K E; Kile, B T; Cory, S

    2014-05-01

    Bax and Bak are critical effectors of apoptosis. Although both are widely expressed and usually functionally redundant, recent studies suggest that Bak has particular importance in certain cell types. Genetic and biochemical studies indicate that Bak activation is prevented primarily by Mcl-1 and Bcl-xL, whereas Bax is held in check by all pro-survival Bcl-2 homologues, including Bcl-2 itself. In this study, we have investigated whether loss of Bak or elevated Mcl-1 modulates haemopoietic abnormalities provoked by overexpression of Bcl-2. The Mcl-1 transgene had little impact, probably because the expression level was insufficient to effectively reduce Bak activation. However, loss of Bak enhanced lymphocytosis in vavP-BCL-2 transgenic mice and increased resistance of their thymocytes to some cytotoxic agents, implying that Bak-specific signals can be triggered in certain lymphoid populations. Nevertheless, lack of Bak had no significant impact on thymic abnormalities in vavP-BCL-2tg mice, which kinetic analysis suggested was due to accumulation of self-reactive thymocytes that resist deletion. Intriguingly, although Bak(-/-) mice have elevated platelet counts, Bak(-/-)vavP-BCL-2 mice, like vavP-BCL-2 littermates, were thrombocytopaenic. To clarify why, the vavP-BCL-2 platelet phenotype was scrutinised more closely. Platelet life span was found to be elevated in vavP-BCL-2 mice, which should have provoked thrombocytosis, as in Bak(-/-) mice. Analysis of bone marrow chimaeric mice suggested the low platelet phenotype was due principally to extrinsic factors. Following splenectomy, blood platelets remained lower in vavP-BCL-2 than wild-type mice. However, in Rag1(-/-) BCL-2tg mice, platelet levels were normal, implying that elevated lymphocytes are primarily responsible for BCL-2tg-induced thrombocytopaenia.

  5. Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

    PubMed Central

    Vandenberg, C J; Josefsson, E C; Campbell, K J; James, C; Lawlor, K E; Kile, B T; Cory, S

    2014-01-01

    Bax and Bak are critical effectors of apoptosis. Although both are widely expressed and usually functionally redundant, recent studies suggest that Bak has particular importance in certain cell types. Genetic and biochemical studies indicate that Bak activation is prevented primarily by Mcl-1 and Bcl-xL, whereas Bax is held in check by all pro-survival Bcl-2 homologues, including Bcl-2 itself. In this study, we have investigated whether loss of Bak or elevated Mcl-1 modulates haemopoietic abnormalities provoked by overexpression of Bcl-2. The Mcl-1 transgene had little impact, probably because the expression level was insufficient to effectively reduce Bak activation. However, loss of Bak enhanced lymphocytosis in vavP-BCL-2 transgenic mice and increased resistance of their thymocytes to some cytotoxic agents, implying that Bak-specific signals can be triggered in certain lymphoid populations. Nevertheless, lack of Bak had no significant impact on thymic abnormalities in vavP-BCL-2tg mice, which kinetic analysis suggested was due to accumulation of self-reactive thymocytes that resist deletion. Intriguingly, although Bak−/− mice have elevated platelet counts, Bak−/−vavP-BCL-2 mice, like vavP-BCL-2 littermates, were thrombocytopaenic. To clarify why, the vavP-BCL-2 platelet phenotype was scrutinised more closely. Platelet life span was found to be elevated in vavP-BCL-2 mice, which should have provoked thrombocytosis, as in Bak−/− mice. Analysis of bone marrow chimaeric mice suggested the low platelet phenotype was due principally to extrinsic factors. Following splenectomy, blood platelets remained lower in vavP-BCL-2 than wild-type mice. However, in Rag1−/− BCL-2tg mice, platelet levels were normal, implying that elevated lymphocytes are primarily responsible for BCL-2tg-induced thrombocytopaenia. PMID:24464220

  6. Glomerular expression and elevated serum Bcl-2 and Fas proteins in lupus nephritis: preliminary findings

    PubMed Central

    Fathi, N A; Hussein, M R; Hassan, H I; Mosad, E; Galal, H; Afifi, N A

    2006-01-01

    Programmed cell death (apoptosis) is involved in glomerular injuries leading to glomerulonephritis. Bcl-2 and Fas are proteins that promote cell survival and death, respectively. This study tests the hypothesis that lupus nephritis is associated with alterations of Bcl-2 and Fas protein expression. Thirty-six patients with lupus nephritis and 10 controls (normal individuals) were included in this study. Bcl-2 and Fas positive cells were examined in kidney biopsies by immunohistochemistry. Bcl-2 and Fas serum levels were evaluated by enzyme-linked immunosorbent assay (ELISA). In the glomeruli of normal kidneys, Bcl-2 and Fas proteins were completely absent. In lupus nephritis patients, glomerular expression of Bcl-2 and Fas was seen in mesangial cells (1·3 ± 0·1 and 2·0 ± 0·1 for Bcl-2 and Fas, respectively). Similarly, a statistically significantly higher Bcl-2 (217·1 ± 85·9) and Fas (767·9 ± 271) serum levels were found in lupus patients compared to controls (148·6 ± 87, 550·3 ± 91 for Bcl-2 and Fas, P < 0·05). A direct correlation between serum Bcl-2 and Fas and chronicity index was also found. Compared to normal controls, lupus nephritis is associated with glomerular expression and elevated serum levels of Bcl-2 and Fas proteins. These findings suggest possible roles for Bcl-2 and Fas in glomerular injury during evolution of lupus nephritis. The diagnostic, prognostic and therapeutic ramifications of our findings are open to further investigation. PMID:17034587

  7. Prognostic value of mitotic index and Bcl2 expression in male breast cancer.

    PubMed

    Lacle, Miangela M; van der Pol, Carmen; Witkamp, Arjen; van der Wall, Elsken; van Diest, Paul J

    2013-01-01

    The incidence of male breast cancer (MBC) is rising. Current treatment regimens for MBC are extrapolated from female breast cancer (FBC), based on the assumption that FBC prognostic features and therapeutic targets can be extrapolated to MBC. However, there is yet little evidence that prognostic features that have been developed and established in FBC are applicable to MBC as well. In a recent study on FBC, a combination of mitotic index and Bcl2 expression proved to be of strong prognostic value. Previous papers on Bcl2 expression in MBC were equivocal, and the prognostic value of Bcl2 combined with mitotic index has not been studied in MBC. The aim of the present study was therefore to investigate the prognostic value of Bcl2 in combination with mitotic index in MBC. Immunohistochemical staining for Bcl2 was performed on tissue microarrays of a total of 151 male breast cancer cases. Mitotic index was scored. The prognostic value of Bcl2 expression and Bcl2/mitotic index combinations was evaluated studying their correlations with clinicopathologic features and their prediction of survival. The vast majority of MBC (94%) showed Bcl2 expression, more frequently than previously described for FBC. Bcl2 expression had no significant associations with clinicopathologic features such as tumor size, mitotic count and grade. In univariate survival analysis, Bcl2 had no prognostic value, and showed no additional prognostic value to tumor size and histological grade in Cox regression. In addition, the Bcl2/mitotic index combination as opposed to FBC did not predict survival in MBC. In conclusion, Bcl2 expression is common in MBC, but is not associated with major clinicopathologic features and, in contrast to FBC, does not seem to have prognostic value, also when combined with mitotic index.

  8. The functional differences between pro-survival and pro-apoptotic B cell lymphoma 2 (Bcl-2) proteins depend on structural differences in their Bcl-2 homology 3 (BH3) domains.

    PubMed

    Lee, Erinna F; Dewson, Grant; Evangelista, Marco; Pettikiriarachchi, Anne; Gold, Grace J; Zhu, Haoran; Colman, Peter M; Fairlie, W Douglas

    2014-12-26

    Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity "BH3-in-groove" complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. The Functional Differences between Pro-survival and Pro-apoptotic B Cell Lymphoma 2 (Bcl-2) Proteins Depend on Structural Differences in Their Bcl-2 Homology 3 (BH3) Domains*

    PubMed Central

    Lee, Erinna F.; Dewson, Grant; Evangelista, Marco; Pettikiriarachchi, Anne; Gold, Grace J.; Zhu, Haoran; Colman, Peter M.; Fairlie, W. Douglas

    2014-01-01

    Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity “BH3-in-groove” complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins. PMID:25371206

  10. Targeting BCL2 family in human myeloid dendritic cells: a challenge to cure diseases with chronic inflammations associated with bone loss.

    PubMed

    Olsson Åkefeldt, Selma; Ismail, Mohamad Bachar; Valentin, Hélène; Aricò, Maurizio; Henter, Jan-Inge; Delprat, Christine

    2013-01-01

    Rheumatoid arthritis (RA) and Langerhans cell histiocytosis (LCH) are common and rare diseases, respectively. They associate myeloid cell recruitment and survival in inflammatory conditions with tissue destruction and bone resorption. Manipulating dendritic cell (DC), and, especially, regulating their half-life and fusion, is a challenge. Indeed, these myeloid cells display pathogenic roles in both diseases and may be an important source of precursors for differentiation of osteoclasts, the bone-resorbing multinucleated giant cells. We have recently documented that the proinflammatory cytokine IL-17A regulates long-term survival of DC by inducing BCL2A1 expression, in addition to the constitutive MCL1 expression. We summarize bibliography of the BCL2 family members and their therapeutic targeting, with a special emphasis on MCL1 and BCL2A1, discussing their potential impact on RA and LCH. Our recent knowledge in the survival pathway, which is activated to perform DC fusion in the presence of IL-17A, suggests that targeting MCL1 and BCL2A1 in infiltrating DC may affect the clinical outcomes in RA and LCH. The development of new therapies, interfering with MCL1 and BCL2A1 expression, to target long-term surviving inflammatory DC should be translated into preclinical studies with the aim to increase the well-being of patients with RA and LCH.

  11. 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.

  12. Protection against Fatal Sindbis Virus Encephalitis by Beclin, a Novel Bcl-2-Interacting Protein

    PubMed Central

    Liang, Xiao Huan; Kleeman, Linda K.; Jiang, Hui Hui; Gordon, Gerald; Goldman, James E.; Berry, Gail; Herman, Brian; Levine, Beth

    1998-01-01

    bcl-2, the prototypic cellular antiapoptotic gene, decreases Sindbis virus replication and Sindbis virus-induced apoptosis in mouse brains, resulting in protection against lethal encephalitis. To investigate potential mechanisms by which Bcl-2 protects against central nervous system Sindbis virus infection, we performed a yeast two-hybrid screen to identify Bcl-2-interacting gene products in an adult mouse brain library. We identified a novel 60-kDa coiled-coil protein, Beclin, which we confirmed interacts with Bcl-2 in mammalian cells, using fluorescence resonance energy transfer microscopy. To examine the role of Beclin in Sindbis virus pathogenesis, we constructed recombinant Sindbis virus chimeras that express full-length human Beclin (SIN/beclin), Beclin lacking the putative Bcl-2-binding domain (SIN/beclinΔBcl-2BD), or Beclin containing a premature stop codon near the 5′ terminus (SIN/beclinstop). The survival of mice infected with SIN/beclin was significantly higher (71%) than the survival of mice infected with SIN/beclinΔBcl-2BD (9%) or SIN/beclinstop (7%) (P < 0.001). The brains of mice infected with SIN/beclin had fewer Sindbis virus RNA-positive cells, fewer apoptotic cells, and lower viral titers than the brains of mice infected with SIN/beclinΔBcl-2BD or SIN/beclinstop. These findings demonstrate that Beclin is a novel Bcl-2-interacting cellular protein that may play a role in antiviral host defense. PMID:9765397

  13. 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

  14. A novel role for Bcl-2 in regulation of cellular calcium extrusion.

    PubMed

    Ferdek, Pawel E; Gerasimenko, Julia V; Peng, Shuang; Tepikin, Alexei V; Petersen, Ole H; Gerasimenko, Oleg V

    2012-07-10

    The antiapoptotic protein Bcl-2 plays important roles in Ca(2+) signaling by influencing inositol triphosphate receptors and regulating Ca(2+)-induced Ca(2+) release. Here we investigated whether Bcl-2 affects Ca(2+) extrusion in pancreatic acinar cells. We specifically blocked the Ca(2+) pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca(2+) concentration after a period of enhanced Ca(2+) entry. Because external Ca(2+) was removed and endoplasmic reticulum Ca(2+) pumps were blocked, Ca(2+) extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca(2+) level much faster than control cells. The enhanced Ca(2+) extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na(+)/Ca(2+) exchange activity, because removal of external Na(+) did not influence the Ca(2+) extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca(2+) extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca(2+) extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca(2+) pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca(2+) extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.

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

    PubMed

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

    2004-05-01

    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.

  16. Bax protein expression is increased in Alzheimer's brain: correlations with DNA damage, Bcl-2 expression, and brain pathology.

    PubMed

    Su, J H; Deng, G; Cotman, C W

    1997-01-01

    We have shown that many neurons in Alzheimer's disease (AD) exhibit terminal deoxynucleotidyl transferase (TdT) labeling for DNA strand breaks, and upregulation of Bcl-2 is associated with neurons exhibiting nuclear DNA fragmentation, while downregulation of Bcl-2 is associated with tangle-bearing neurons in AD brains. Consequently, we examined the expression of bcl-associated X (Bax) protein in AD brain. Immunoreactivity for Bax was seen in neurons and microglia of the hippocampal formation, and was elevated in the majority of AD cases as compared to control cases. Interestingly, 3 transitional cases, which had mild degeneration changes, exhibited relatively high levels of Bax immunoreactivity. Most Bax-positive neurons showed either TdT-labeled nuclei or Bcl-2 immunoreactivity. Although Bax immunoreactivity was detected within most early tangle-bearing neurons, many Bax-positive neurons did not colocalize with later-stage tangle-bearing neurons. In regions containing relatively few tangles in mild AD brains, many TdT-labeled neurons were immunolabeled with Bax antibody and most of them lacked evidence of neurofibrillary changes. These findings suggest that Bax may contribute to neuronal cell death in AD. Furthermore, DNA damage and the upregulation of Bax appear to precede tangle formation or may represent an alternative pathway of cell death in AD.

  17. Akt-Dependent Glucose Metabolism Promotes Mcl-1 Synthesis to Maintain Cell Survival and Resistance to Bcl-2 Inhibition

    PubMed Central

    Coloff, Jonathan L.; Macintyre, Andrew N.; Nichols, Amanda G.; Liu, Tingyu; Gallo, Catherine A.; Plas, David R.; Rathmell, Jeffrey C.

    2011-01-01

    Most cancer cells utilize aerobic glycolysis, and activation of the phosphatidyl-inositol 3-kinase (PI3K)/Akt/mTOR pathway can promote this metabolic program to render cells glucose-dependent. While manipulation of glucose metabolism may provide a means to specifically eliminate cancer cells, mechanistic links between cell metabolism and apoptosis remain poorly understood. Here we examine the role and metabolic regulation of the anti-apoptotic Bcl-2 family protein Mcl-1 in cell death upon inhibition of Akt-induced aerobic glycolysis. In the presence of adequate glucose, activated Akt prevented the loss of Mcl-1 expression and protected cells from growth factor-deprivation induced apoptosis. Mcl-1 associated with and inhibited the pro-apoptotic Bcl-2 family protein Bim, contributing to cell survival. However, suppression of glucose metabolism led to induction of Bim, decreased expression of Mcl-1, and apoptosis. The pro-apoptotic Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, shows clinical promise, but Mcl-1 upregulation can promote resistance. Importantly, inhibition of glucose metabolism or mTORC1 overcame Mcl-1-mediated resistance in diffuse large B cell leukemic cells. Together these data show that Mcl-1 protein synthesis is tightly controlled by metabolism and that manipulation of glucose metabolism may provide a mechanism to suppress Mcl-1 expression and sensitize cancer cells to apoptosis. PMID:21670080

  18. 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.

  19. Molecular Interactions of Prodiginines with the BH3 Domain of Anti-Apoptotic Bcl-2 Family Members

    PubMed Central

    Soto-Cerrato, Vanessa; Quesada, Roberto; Pérez-Tomás, Ricardo; Guallar, Victor

    2013-01-01

    Prodigiosin and obatoclax, members of the prodiginines family, are small molecules with anti-cancer properties that are currently under preclinical and clinical trials. The molecular target(s) of these agents, however, is an open question. Combining experimental and computational techniques we find that prodigiosin binds to the BH3 domain in some BCL-2 protein families, which play an important role in the apoptotic programmed cell death. In particular, our results indicate a large affinity of prodigiosin for MCL-1, an anti-apoptotic member of the BCL-2 family. In melanoma cells, we demonstrate that prodigiosin activates the mitochondrial apoptotic pathway by disrupting MCL-1/BAK complexes. Computer simulations with the PELE software allow the description of the induced fit process, obtaining a detailed atomic view of the molecular interactions. These results provide new data to understand the mechanism of action of these molecules, and assist in the development of more specific inhibitors of anti-apoptotic BCL-2 proteins. PMID:23460874

  20. 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

    2014-08-05

    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.

  1. Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.

    PubMed

    Uchime, Onyinyechukwu; Dai, Zhou; Biris, Nikolaos; Lee, David; Sidhu, Sachdev S; Li, Sheng; Lai, Jonathan R; Gavathiotis, Evripidis

    2016-01-01

    The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Involvement of proapoptotic Bcl-2 family members in terbinafine-induced mitochondrial dysfunction and apoptosis in HL60 cells.

    PubMed

    Yang, Kuo-Ching; Wu, Chi-Chen; Wu, Chih-Hsiung; Chen, Jur-Hao; Chu, Chien-Hwa; Chen, Chien-Ho; Chou, Yean-Hwei; Wang, Ying-Jan; Lee, Wen-Sen; Tseng, How; Lin, Shyr-Yi; Lee, Chia-Hwa; Ho, Yuan-Soon

    2006-02-01

    Terbinafine (TB, lamisil), a promising world widely used oral-anti-fungal agent, has been used in the treatment of superficial mycosis. In this study, we found that apoptosis but not cell growth arrest was induced by TB (1 microM, for 24 h) in human promyelocytic leukemia (HL60) cells. The apoptotic effect induced by TB in the HL60 cell was not through the general differentiation mechanisms evidenced by evaluation of three recognized markers, including CD11b, CD33, and morphological features. In addition, our results also revealed that TB-induced apoptosis was not through the cellular surface CD 95 receptor-mediated signaling pathway. We found that the mitochondria membrane in the TB-treated HL60 cells was dissipated by decreasing of the electrochemical gradient (DeltaPsi(m)) led to leakage of cytochrome c from mitochondria into cytosol. Such effects were completely blocked by in vitro transfection of the HL60 cells with Bcl-2 overexpression plasmid (HL60/Bcl-2). However, our data found that TB-mediated apoptosis could not be completely prevented in the Bcl-2 over expressed (HL60/Bcl-2) cells. Such results implied that additional mediators (such as caspase-9) other than mitochondria membrane permeability might contribute to the TB-induced cellular apoptosis signaling. This hypothesis was supported by the evidence that administration of caspases-9 specific inhibitor (z-LEHD-fmk) blocked the TB-induced apoptosis. Our studies highlight the molecular mechanisms of TB-induced apoptosis in human promyelocytic leukemia (HL60) cells.

  3. Red photon treatment inhibits apoptosis via regulation of bcl-2 proteins and ROS levels, alleviating hypoxic-ischemic brain damage.

    PubMed

    Jiang, W; Chen, L; Zhang, X J; Chen, J; Li, X C; Hou, W S; Xiao, N

    2014-05-30

    Therapeutic options for hypoxic-ischemic brain damage (HIBD) are scarce and inefficient. Recently, many studies have demonstrated that red photon plays an important role in anti-inflammatory processes as well as apoptosis, the main trait of HIBD. In this study, we investigated whether red photon can protect from HIBD in SD rats and oxygen-glucose deprivation (OGD) in PC12 cells. Apoptosis, mitochondrial transmembrane potential (MMP), and reactive oxygen species (ROS) rates were assessed in PC12 cells. We found that 6-h irradiation resulted in decreased MMP, ROS and apoptosis rates, although these changes were reversible with prolonged irradiation. Importantly, these effects were sustained for 2-8h upon quenching of the red photon. Similar trends were observed for protein and mRNA expression of bax and bcl-2, with short-term irradiation (6h) inhibiting apoptosis in PC12 Cells. However, long-term (>6h) irradiation caused cell damage. In vivo experiments, bax mRNA and protein levels were reduced after 7days in HIBD model rats treated with red photon, in contrast to bcl-2. Furthermore, we found that bax and bcl-2 were mainly expressed in pyramidal cells of the hippocampus CA1 and CA3. Importantly, Morris Water Maze test results revealed an improvement in learning ability and spatial memory in rats after irradiation. Overall, our data showed that short-term irradiation with red photon in the acute phase inhibits the mitochondrial apoptotic pathway via regulation of bcl-2-related proteins and reduction of ROS levels, thereby decreasing apoptosis in nerve cells and improving the neurological prognosis of HIBD.

  4. Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site*

    PubMed Central

    Uchime, Onyinyechukwu; Dai, Zhou; Biris, Nikolaos; Lee, David; Sidhu, Sachdev S.; Li, Sheng; Lai, Jonathan R.; Gavathiotis, Evripidis

    2016-01-01

    The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation. PMID:26565029

  5. 4-Nonylphenol induces disruption of spermatogenesis associated with oxidative stress-related apoptosis by targeting p53-Bcl-2/Bax-Fas/FasL signaling.

    PubMed

    Duan, Peng; Hu, Chunhui; Butler, Holly J; Quan, Chao; Chen, Wei; Huang, Wenting; Tang, Sha; Zhou, Wei; Yuan, Meng; Shi, Yuqin; Martin, Francis L; Yang, Kedi

    2017-03-01

    4-Nonylphenol (NP) is a ubiquitous environmental chemical with estrogenic activity. Our aim was to test the hypothesis that pubertal exposure to NP leads to testicular dysfunction. Herein, 24 7-week-old rats were randomly divided into four groups and treated with NP (0, 25, 50, or 100 mg/kg body weight every 2 days for 20 consecutive days) by intraperitoneal injection. Compared to untreated controls, the parameters of sperm activation rate, curvilinear velocity, average path velocity, and swimming velocity were significantly lower at doses of 100 mg/kg, while sperm morphological abnormalities were higher, indicating functional disruption and reduced fertilization potential. High exposure to NP (100 mg/kg) resulted in disordered arrangement of spermatoblasts and reduction of spermatocytes in seminiferous tubules, while tissues exhibited a marked decline in testicular fructose content and serum FSH, LH, and testosterone levels. Oxidative stress was induced by NP (50 or 100 mg/kg) as evidenced by elevated MDA, decreased SOD and GSH-Px, and inhibited antioxidant gene expression (CAT, GPx, SOD1, and CYP1B1). In addition, NP treatment decreased proportions of Ki-67-positive cells and increased apoptosis in a dose-dependent manner. Rats treated with 100 mg/kg NP exhibited significantly increased mRNA expression of caspase-1, -2, -9, and -11, decreased caspase-8 and PCNA1 mRNA expression, downregulation of Bcl-2/Bax ratios and upregulation of Fas, FasL, and p53 at the protein and mRNA levels. Taken together, NP-induced apoptosis, hormonal deficiencies, and depletion of fructose potentially impairs spermatogenesis and sperm function. p53-independent Fas/FasL-Bax/Bcl-2 pathways may be involved in NP-induced oxidative stress-related apoptosis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 739-753, 2017.

  6. BCL2 translocation frequency rises with age in humans

    SciTech Connect

    Liu, Y.; Hernandez, A.M.; Shibata, D.; Cortopassi, G.A.

    1994-09-13

    The background frequency of t(14;18) (q32;q21) chromosomal translocations at the locus associated with B-cell leukemia/lymphoma-2 (BCL2) was determined from a survey of the peripheral blood lymphocytes (PBLs) of 53 living individuals and from tissues of 31 autopsies by using a nested PCR assay. The translocation was detected in 55% of PBLs and 35% of autopsied spleens with a frequency of between less than 1 to 853 translocations per million cells. Translocations copurified with B lymphocytes. The frequency of translocations significantly increased with age in PBLs and spleens, as does human risk for lymphoma. Average translocation frequency was more than 40 times greater in the spleen and 13 times greater in the peripheral blood in the oldest individuals (61 yr and older) compared with the youngest individuals (20 yr or younger). Particular t(14;18)-bearing clones persisted over a period of 5 months in two individuals. These findings demonstrate that clones harboring the oncogenic t(14;18) chromosomal translocation are commonly present in normal humans, that such clones are long-lived, and that they rise in frequency with age. A multihit model of lymphomagenesis involving t(14;18) translocation followed by antigen stimulation is proposed. 49 refs., 8 figs., 1 tab.

  7. The BCL2 gene is regulated by a special AT-rich sequence binding protein 1-mediated long range chromosomal interaction between the promoter and the distal element located within the 3′-UTR

    PubMed Central

    Gong, Feiran; Sun, Luan; Wang, Zongdan; Shi, Junfeng; Li, Wei; Wang, Sumeng; Han, Xiao; Sun, Yujie

    2011-01-01

    The 279-bp major breakpoint region (mbr) within the 3′-untranslated region (3′-UTR) of the BCL2 gene is a binding site of special AT-rich sequence binding protein 1 (SATB1) that is well known to participate in the long-range regulation of gene transcription. Our previous studies have revealed that the mbr could regulate BCL2 transcription over a 200-kb distance and this regulatory function was closely related to SATB1. This study is to explore the underlying mechanism and its relevance to cellular apoptosis. With chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP) assays we demonstrated that the mbr could physically interact with BCL2 promoter through SATB1-mediated chromatin looping, which was required for epigenetic modifications of the promoter, CREB accessibility and high expression of the BCL2 gene. During early apoptosis, SATB1 was a key regulator of BCL2 expression. Inhibition of SATB1 cleavage by treatment of cells with a caspase-6 inhibitor or overexpression of mutant SATB1 that was resistant to caspase-6, inhibited disassembly of the SATB1-mediated chromatin loop and restored the BCL2 mRNA level in Jurkat cells. These data revealed a novel mechanism of BCL2 regulation and mechanistically link SATB1-mediated long-range interaction with the regulation of a gene controlling apoptosis pathway for the first time. PMID:21310710

  8. Reactive oxygen species mediate heat stress-induced apoptosis via ERK dephosphorylation and Bcl-2 ubiquitination in human umbilical vein endothelial cells.

    PubMed

    Li, Li; Tan, Hongping; Yang, Hong; Li, Feng; He, Xuan; Gu, Zhengtao; Zhao, Ming; Su, Lei

    2016-12-25

    Heat stress can induce the mitochondrial apoptotic pathway in HUVEC cells, indicating that apoptosis may be a prominent pathological feature of heat stroke, however, little is known about the precise mechani sms involved in it. In this study, we describe the apoptotic effect of intense heat stress on HUVEC cells and our investigation of its underlying mechanisms. Treatment of cells with intense heat stress induced production of reactive oxygen species (ROS) and a concomitant increase in activation of the mitochondrial apoptotic pathway. Furthermore, by over-expression of MnSOD and GPx in cells, we show that ROS, and especially superoxide, is the primary oxidative species induced by intense heat stress and responsible for cell death. In addition, we explored the mechanism by which superoxide regulates the apoptotic effect of intense heat stress, and found that it involved Bcl-2 down-regulation through ubiquitin - proteasomal degradation. Superoxide production also led to Bcl-2 dephosphorylation through inactivation of MAP kinase ERK1/2, which promoted Bcl-2 ubiquitination. Taken together, these findings describe a novel pathway downstream of heat stress-induced apoptosis in HUVEC cells, and provide new insight into the process of redox-mediated down-regulation of Bcl-2 and apoptosis induction. These results could be important in the understanding of pathogenesis of heat stroke and for the development of preventive and treatment measures, both of which are currently lacking.

  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.

  10. Metabolic reprogramming in cancer cells: glycolysis, glutaminolysis, and Bcl-2 proteins as novel therapeutic targets for cancer.

    PubMed

    Li, Chunxia; Zhang, Guifeng; Zhao, Lei; Ma, Zhijun; Chen, Hongbing

    2016-01-20

    Nearly a century ago, Otto Warburg made the ground-breaking observation that cancer cells, unlike normal cells, prefer a seemingly inefficient mechanism of glucose metabolism: aerobic glycolysis, a phenomenon now referred to as the Warburg effect. The finding that rapidly proliferating cancer cells favors incomplete metabolism of glucose, producing large amounts of lactate as opposed to synthesizing ATP to sustain cell growth, has confounded scientists for years. Further investigation into the metabolic phenotype of cancer has expanded our understanding of this puzzling conundrum, and has opened new avenues for the development of anti-cancer therapies. Enhanced glycolytic flux is now known to allow for increased synthesis of intermediates for sustaining anabolic pathways critical for cancer cell growth. Alongside the increase in glycolysis, cancer cells transform their mitochondria into synthesis machines supported by augmented glutaminolysis, supplying lipid production, amino acid synthesis, and the pentose phosphate pathways. Inhibition of several of the key enzymes involved in these pathways has been demonstrated to effectively obstruct cancer cell growth and multiplication, sensitizing them to apoptosis. The modulation of various regulatory proteins involved in metabolic processes is central to cancerous reprogramming of metabolism. The finding that members of one of the major protein families involved in cell death regulation also aberrantly regulated in cancers, the Bcl-2 family of proteins, are also critical mediators of metabolic pathways, provides strong evidence for the importance of the metabolic shift to cancer cell survival. Targeting the anti-apoptotic members of the Bcl-2 family of proteins is proving to be a successful way to selectively target cancer cells and induce apoptosis. Further understanding of how cancer cells modify metabolic regulation to increase channeling of substrates into biosynthesis will allow for the discovery of novel drug

  11. Bcl-2 in combination to myeloid antigen expression in adult acute lymphoblastic leukemia and prognostic outcome.

    PubMed

    Amirghofran, Zahra; Daneshbod, Yahya; Gholijani, Naser

    2009-01-01

    The present study was performed to find the importance of two myeloid (CD13 and CD33) antigens aberrantly expressed on the blasts of acute lymphoblastic leukemia (ALL) patients and Bcl-2 expression in relation to clinical and biological features and treatment outcome. Bone marrow or peripheral blood samples of 50 patients were assessed for the expression of markers by immunostaining methods. Twenty-one patients (42%) showed more than 20% positivity for Bcl-2. Aberrant expression of myeloid antigens was found in 14% of cases. The expression of Bcl-2 was associated with shorter survival (p = 0.009). A significant correlation between expression of myeloid antigens (MY) and survival and complete remission duration was found. The mean survival was 656 + 301 days for MY+ cases and 1009 +/- 230 days for MY- patients (p < 0.0001). Expression of Bcl-2 in combination to myeloid antigens was associated with a poorer outcome. Survival of MY+ patients expressing Bcl-2 was shorter than MY- Bcl-2+ and MY+ Bcl-2- ALL cases (p = 0.038). In conclusion, results of this study indicated the prognostic value of Bcl-2 and myeloid antigen expression in ALL patients. Presence of these markers together on the leukemic cells was associated with a poorer response to therapy and may implicate modified therapeutic strategies in the patients.

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

    PubMed

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

    2001-01-01

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

  13. Differential sensitivity of skeletal and fusimotor neurons to Bcl-2-mediated apoptosis during neuromuscular development.

    PubMed

    Hui, K; Kucera, J; Henderson, J T

    2008-04-01

    Proper development of the nervous system requires that a carefully controlled balance be maintained between both proliferation and neuronal survival. The process of programmed cell death is believed to play a key role in regulating levels of neuronal survival, in large part through the action of antiapoptotic proteins, such as Bcl-2. Consistent with this, Bcl-2 has been shown to be a key regulator of apoptotic signaling in post-mitotic neurons. However, we still know remarkably little regarding the role that Bcl-2 plays in regulating the survival of specific motor neuron populations. In the present study, we have examined somatic motor neurons of the lumbar spinal cord, and branchiomotor neurons of the facial nucleus in bcl-2-null mice to determine the differential dependence among motor neuron populations with respect to Bcl-2-mediated survival. Examination of neuronal and axon number, axonal area, and the distribution of axonal loss in bcl-2-null mice demonstrates that, in contrast to the great majority of alpha motor neurons, gamma motor neurons exhibit a unique dependence upon bcl-2 for survival. These results demonstrate, for the first time, the connection between Bcl-2 expression, motor neuron survival, and the establishment of different motor populations.

  14. Mechanism of apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins.

    PubMed

    Chipuk, Jerry E; Fisher, John C; Dillon, Christopher P; Kriwacki, Richard W; Kuwana, Tomomi; Green, Douglas R

    2008-12-23

    Normal cellular lifespan is contingent upon preserving outer mitochondrial membrane (OMM) integrity, as permeabilization promotes apoptosis. BCL-2 family proteins control mitochondrial outer membrane permeabilization (MOMP) by regulating the activation of the pro-apoptotic BCL-2 effector molecules, BAX and BAK. Sustainable cellular stress induces proteins (e.g., BID, BIM, and cytosolic p53) capable of directly activating BAX and/or BAK, but these direct activators are sequestered by the anti-apoptotic BCL-2 proteins (e.g., BCL-2, BCL-xL, and MCL-1). In the event of accumulated or marked cellular stress, a coordinated effort between previously sequestered and nascent BH3-only proteins inhibits the anti-apoptotic BCL-2 repertoire to promote direct activator protein-mediated MOMP. We examined the effect of ABT-737, a BCL-2 antagonist, and PUMA, a BH3-only protein that inhibits the entire anti-apoptotic BCL-2 repertoire, with cells and mitochondria that sequestered direct activator proteins. ABT-737 and PUMA cooperated with sequestered direct activator proteins to promote MOMP and apoptosis, which in the absence of ABT-737 or PUMA did not influence OMM integrity or cellular survival. Our data show that the induction of apoptosis by inhibition of the anti-apoptotic BCL-2 repertoire requires "covert" levels of direct activators of BAX and BAK at the OMM.

  15. Cooperation of bcl-2 and myc in the neoplastic transformation of normal rat liver epithelial cells is related to the down-regulation of gap junction-mediated intercellular communication.

    PubMed

    DeoCampo, N D; Wilson, M R; Trosko, J E

    2000-08-01

    The objectives of this study were to isolate several rat liver epithelial cell clones containing the human bcl-2 and myc/bcl-2 genes in order to study their potential cooperative effect on neoplastic transformation and gap junction-mediated intercellular communication (GJIC) and to test the hypothesis that the loss of GJIC leads to tumorigenesis. Using anchorage-independent growth as a surrogate marker for neoplastic transformation, we transfected both normal rat liver epithelial cells, WB-F344, and a WB-F344 cell line overexpressing v-myc with human bcl-2 cDNA. Those cell lines that only expressed v-myc or human bcl-2 were unable to form colonies in soft agar. However, those cell lines that overexpressed both v-myc and human bcl-2 showed varying ability to form colonies in soft agar, which did not correlate with their human bcl-2 expression level. In order to test if there was a correlation between cell line growth in soft agar and the ability to communicate through gap junctions, we performed scrape load dye transfer and fluorescence recovery after photobleaching assays. Our results show that v-myc and human bcl-2 can cooperate in the transformation of normal cells, but the degree to which the cells are transformed is dependent on the cells' ability to communicate through gap junctions.

  16. Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer

    PubMed Central

    Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

    2016-01-01

    Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes. DOI: http://dx.doi.org/10.7554/eLife.20352.001 PMID:27805565

  17. Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer.

    PubMed

    Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

    2016-11-02

    Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.

  18. 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.

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

    PubMed

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

    2015-04-01

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

  20. A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history

    PubMed Central

    2010-01-01

    Background Poxviruses evade the immune system of the host through the action of viral encoded inhibitors that block various signalling pathways. The exact number of viral inhibitors is not yet known. Several members of the vaccinia virus A46 and N1 families, with a Bcl-2-like structure, are involved in the regulation of the host innate immune response where they act non-redundantly at different levels of the Toll-like receptor signalling pathway. N1 also maintains an anti-apoptotic effect by acting similarly to cellular Bcl-2 proteins. Whether there are related families that could have similar functions is the main subject of this investigation. Results We describe the sequence similarity existing among poxvirus A46, N1, N2 and C1 protein families, which share a common domain of approximately 110-140 amino acids at their C-termini that spans the entire N1 sequence. Secondary structure and fold recognition predictions suggest that this domain presents an all-alpha-helical fold compatible with the Bcl-2-like structures of vaccinia virus proteins N1, A52, B15 and K7. We propose that these protein families should be merged into a single one. We describe the phylogenetic distribution of this family and reconstruct its evolutionary history, which indicates an extensive gene gain in ancestral viruses and a further stabilization of its gene content. Conclusions Based on the sequence/structure similarity, we propose that other members with unknown function, like vaccinia virus N2, C1, C6 and C16/B22, might have a similar role in the suppression of host immune response as A46, A52, B15 and K7, by antagonizing at different levels with the TLR signalling pathways. PMID:20230632

  1. Combined inhibition of NF-κB and Bcl-2 triggers synergistic reduction of viability and induces apoptosis in melanoma cells.

    PubMed

    Watanabe, Mariko; Umezawa, Kazuo; Higashihara, Masaaki; Horie, Ryouichi

    2013-01-01

    Constitutive activation of nuclear factor κB (NF-κB) characterizes melanoma cells. To explore the molecular mechanism of melanoma cell survival by constitutive NF-κB activation, we used the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ), which directly binds to NF-κB. DHMEQ abrogated constitutive NF-κB activity, which included RelA (p65)/p50 in melanoma cell lines G361 and HMV-II; however, the reduction of the viability was marginal. Expression of c-FLIP was not observed in the melanoma cell lines tested, and DHMEQ could not repress the expression of the Bcl-2 family proteins Bcl-2 and Bcl-xL. Concomitant treatment with DHMEQ and the inhibitor of antiapoptotic Bcl-2 family proteins, GX15-070, triggered synergistic reduction of the viability and induced apoptosis of G361 cells. These results indicate that abrogation of the NF-κB pathway alone is not sufficient to suppress the survival of melanoma cells. The NF-κB and the antiapoptotic Bcl-2 pathways cooperatively support the survival, and the dual targeting triggers synergistic reduction of the viability and induces apoptosis of melanoma cells.

  2. Thymoquinone decreases F-actin polymerization and the proliferation of human multiple myeloma cells by suppressing STAT3 phosphorylation and Bcl2/Bcl-XL expression

    PubMed Central

    2011-01-01

    Background Thymoquinone (TQ), the major active component of the medicinal herb Nigella sativa Linn., has been described as a chemopreventive and chemotherapeutic compound. Methods In this study, we investigated the effect of TQ on survival, actin cytoskeletal reorganization, proliferation and signal transduction in multiple myeloma (MM) cells. Results We found that TQ induces growth arrest in both MDN and XG2 cells in a dose- and time-dependent manner. TQ also inhibited CXC ligand-12 (CXCL-12)-mediated actin polymerization and cellular proliferation, as shown by flow cytometry. The signal transducer and activator of transcription (STAT) and B-cell lymphoma-2 (Bcl-2) signaling pathways may play important roles in the malignant transformation of a number of human malignancies. The constitutive activation of the STAT3 and Bcl-2 pathways is frequently observed in several cancer cell lines, including MM cells. Using flow cytometry, we found that TQ markedly decreased STAT3 phosphorylation and Bcl-2 and Bcl-XL expression without modulating STAT5 phosphorylation in MM cells. Using western blotting, we confirmed the inhibitory effect of TQ on STAT3 phosphorylation and Bcl-2 and Bcl-XL expression. Conclusions Taken together, our data suggests that TQ could potentially be applied toward the treatment of MM and other malignancies. PMID:22177381

  3. 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

  4. 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

  5. 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.

  6. Restrained Terminal Differentiation and Sustained Stemness in Neonatal Skin by Ha-Ras and Bcl-2.

    PubMed

    Lee, Sangjun; Rodriguez-Villanueva, Julio; McDonnell, Timothy

    2017-03-01

    Nonmelanoma skin cancer is the most frequently diagnosed cancer in the United States. Deregulation of bcl-2 and ras family members is commonly observed in nonmelanoma skin cancer. It has been previously demonstrated that simultaneous bcl-2 and Ha-ras gene expression in keratinocytes results in resistance to cell death induced by ultraviolet radiation and enhanced multistep skin carcinogenesis. In this study, we aimed to elucidate the central roles of Ha-Ras and Bcl-2 in maintaining epidermal homeostasis. To assess the effect of deregulated Ha-Ras and Bcl-2 on skin differentiation, we have generated skin-specific transgenic mouse model constitutively expressing both oncogenic Ha-Ras and Bcl-2. Ectopic expression of Ha-Ras and Bcl-2 in newborn double transgenic epidermal keratinocytes induced abnormal epidermal differentiation accompanied by increased cell proliferation and suppressed apoptotic cell death, which resulted in thickened and wrinkled skin morphology in neonate skins. Expression of epidermal differentiation marker cytokeratin 1 was decreased. Expression of other differentiation markers loricrin and filaggrin was also decreased and delayed to be detected only in the upper stratum granulosum, whereas the proliferative markers cytokeratin 14 and cytokeratin 6, which are expressed in constitutively proliferative basal layer and stem cell niches such as hair follicles or neoplastic lesions, respectively, were highly expressed. The abnormal expression of epidermal cytokeratins suggests that Ha-Ras and Bcl-2 suppress the terminal differentiation and sustain the stem cell-like features in epidermal keratinocytes.

  7. S-Nitrosylation of Bcl-2 Negatively Affects Autophagy in Lung Epithelial Cells

    PubMed Central

    Wright, Clayton; Iyer, Anand Krishnan V.; Kulkarni, Yogesh; Azad, Neelam

    2015-01-01

    Autophagy is a catabolic cellular mechanism involving lysosomal degradation of unwanted cellular components. Interaction between Beclin-1 and Bcl-2 proteins is known to play a critical role in the initiation of autophagy. We report that malignantly transformed lung epithelial cells are resistant to autophagy and express lower basal levels of autophagic proteins, Beclin-1 and LC3-II as compared to non-tumorigenic cells. Additionally, increased levels of nitric oxide (NO) and Bcl-2 were observed in transformed cells. Nitric oxide was found to negatively regulate autophagy initiation and autophagic flux by nitrosylating Bcl-2 and stabilizing its interaction with Beclin-1, resulting in inhibition of Beclin-1 activity. An increase in the apoptotic initiator caspase-9 and the apoptosis and autophagy-associated kinase p38/MAPK in both cell types indicated possible autophagy–apoptosis crosstalk. Pre-treatments with ABT-737 (Bcl-2 inhibitor) and aminoguanidine (NO inhibitor), and transfection with a non-nitrosylable Bcl-2 cysteine double-mutant plasmid resulted in increased autophagic flux (LC3-II/p62 upregulation) corresponding with decreased S-nitrocysteine expression, thus corroborating the regulatory role of Bcl-2 S-nitrosylation in autophagy. In conclusion, our study reveals a novel mechanism of autophagy resistance via post-translational modification of Bcl-2 protein by NO, which may be critical in driving cellular tumorigenesis. PMID:26241894

  8. Solution structure of a Bcl-2 homolog from Kaposi sarcoma virus

    PubMed Central

    Huang, Qiulong; Petros, Andrew M.; Virgin, Herbert W.; Fesik, Stephen W.; Olejniczak, Edward T.

    2002-01-01

    Kaposi sarcoma-associated herpes virus (KSHV) contains a gene that has functional and sequence homology to the apoptotic Bcl-2 family of proteins [Sarid, R., Sato, T., Bohenzky, R. A., Russo, J. J. & Chang, Y. (1997) Nat. Med. 3, 293–298]. The viral Bcl-2 protein promotes survival of infected cells and may contribute to the development of Kaposi sarcoma tumors [Boshoff, C. & Chang, Y. (2001) Annu. Rev. Med. 52, 453–470]. Here we describe the solution structure of the viral Bcl-2 homolog from KSHV. Comparison of the KSHV Bcl-2 structure to that of Bcl-2 and Bcl-xL shows that although the overall fold is the same, there are key differences in the lengths of the helices and loops. Binding studies on peptides derived from the Bcl-2 homology region 3 of proapoptotic family members indicate that the specificity of the viral protein is very different from what was previously observed for Bcl-xL and Bcl-2, suggesting that the viral protein has evolved to have a different mechanism of action than the host proteins. PMID:11904405

  9. Effects of BCL-2 over-expression on B cells in transgenic rats and rat hybridomas.

    PubMed

    Iscache, Anne-Laure; Ménoret, Séverine; Tesson, Laurent; Rémy, Séverine; Usal, Claire; Pedros, Christophe; Saoudi, Abdelhadi; Buelow, Roland; Anegon, Ignacio

    2011-10-01

    The rat is an important biomedical experimental model that benefited from the recent development of new transgenic and knockout techniques. With the goal to optimize rat mAb production and to analyze the impact of Bcl-2 on B-cell development, we generated bcl-2 transgenic rats. Transgenic rats showed Bcl-2 over-expression in B cells, increased B cell numbers in lymphoid organs, elevated production of immunoglobulins (Igs) and prolonged B-cell survival in vitro. Transgenic rats remained healthy, reproduced normally and did not develop autoimmunity. Fusions with bcl-2 transgenic splenocytes did not result in increased hybridoma generation. A comparison of on- and off-rates of 39 mAbs generated with bcl-2 transgenic and wild-type animals revealed no significant differences. Over-expression of Bcl-2 in hybridomas did not change cell proliferation but resulted in increased Ig production. Bcl-2 transgenic rats will be a useful tool for the generation of rat mAbs, the analysis of B cells in different pathophysiological models, such as autoimmunity, cancer or organ transplantation, and the study of rat B-cell biology.

  10. Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival.

    PubMed

    Viant, Charlotte; Guia, Sophie; Hennessy, Robert J; Rautela, Jai; Pham, Kim; Bernat, Claire; Goh, Wilford; Jiao, Yuhao; Delconte, Rebecca; Roger, Michael; Simon, Vanina; Souza-Fonseca-Guimaraes, Fernando; Grabow, Stephanie; Belz, Gabrielle T; Kile, Benjamin T; Strasser, Andreas; Gray, Daniel; Hodgkin, Phillip D; Beutler, Bruce; Vivier, Eric; Ugolini, Sophie; Huntington, Nicholas D

    2017-02-01

    Natural killer (NK) cells are innate lymphoid cells with antitumor functions. Using an N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a strain with an NK cell deficiency caused by a hypomorphic mutation in the Bcl2 (B cell lymphoma 2) gene. Analysis of these mice and the conditional deletion of Bcl2 in NK cells revealed a nonredundant intrinsic requirement for BCL2 in NK cell survival. In these mice, NK cells in cycle were protected against apoptosis, and NK cell counts were restored in inflammatory conditions, suggesting a redundant role for BCL2 in proliferating NK cells. Consistent with this, cycling NK cells expressed higher MCL1 (myeloid cell leukemia 1) levels in both control and BCL2-null mice. Finally, we showed that deletion of BIM restored survival in BCL2-deficient but not MCL1-deficient NK cells. Overall, these data demonstrate an essential role for the binding of BCL2 to BIM in the survival of noncycling NK cells. They also favor a model in which MCL1 is the dominant survival protein in proliferating NK cells.

  11. Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival

    PubMed Central

    Viant, Charlotte; Guia, Sophie; Hennessy, Robert J.; Rautela, Jai; Pham, Kim; Bernat, Claire; Goh, Wilford; Jiao, Yuhao; Delconte, Rebecca; Roger, Michael; Simon, Vanina; Souza-Fonseca-Guimaraes, Fernando; Grabow, Stephanie; Belz, Gabrielle T.; Kile, Benjamin T.; Strasser, Andreas; Gray, Daniel; Hodgkin, Phillip D.; Beutler, Bruce; Vivier, Eric

    2017-01-01

    Natural killer (NK) cells are innate lymphoid cells with antitumor functions. Using an N-ethyl-N-nitrosourea (ENU)–induced mutagenesis screen in mice, we identified a strain with an NK cell deficiency caused by a hypomorphic mutation in the Bcl2 (B cell lymphoma 2) gene. Analysis of these mice and the conditional deletion of Bcl2 in NK cells revealed a nonredundant intrinsic requirement for BCL2 in NK cell survival. In these mice, NK cells in cycle were protected against apoptosis, and NK cell counts were restored in inflammatory conditions, suggesting a redundant role for BCL2 in proliferating NK cells. Consistent with this, cycling NK cells expressed higher MCL1 (myeloid cell leukemia 1) levels in both control and BCL2-null mice. Finally, we showed that deletion of BIM restored survival in BCL2-deficient but not MCL1-deficient NK cells. Overall, these data demonstrate an essential role for the binding of BCL2 to BIM in the survival of noncycling NK cells. They also favor a model in which MCL1 is the dominant survival protein in proliferating NK cells. PMID:28057804

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

    PubMed

    Juneja, S; Chaitanya, N Babu; Agarwal, M

    2015-01-01

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

  13. 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.

  14. ATMIN defines an NBS1-independent pathway of ATM signalling

    PubMed Central

    Kanu, Nnennaya; Behrens, Axel

    2007-01-01

    The checkpoint kinase ATM (ataxia telangiectasia mutated) transduces genomic stress signals to halt cell cycle progression and promote DNA repair in response to DNA damage. Here, we report the characterisation of an essential cofactor for ATM, ATMIN (ATM INteracting protein). ATMIN interacts with ATM through a C-terminal motif, which is also present in Nijmegen breakage syndrome (NBS)1. ATMIN and ATM colocalised in response to ATM activation by chloroquine and hypotonic stress, but not after induction of double-strand breaks by ionising radiation (IR). ATM/ATMIN complex disruption by IR was attenuated in cells with impaired NBS1 function, suggesting competition of NBS1 and ATMIN for ATM binding. ATMIN protein levels were reduced in ataxia telangiectasia cells and ATM protein levels were low in primary murine fibroblasts lacking ATMIN, indicating reciprocal stabilisation. Whereas phosphorylation of Smc1, Chk2 and p53 was normal after IR in ATMIN-deficient cells, basal ATM activity and ATM activation by hypotonic stress and inhibition of DNA replication was impaired. Thus, ATMIN defines a novel NBS1-independent pathway of ATM signalling. PMID:17525732

  15. Oligomerization process of Bcl-2 associated X protein revealed from intermediate structures in solution.

    PubMed

    Shih, Orion; Yeh, Yi-Qi; Liao, Kuei-Fen; Sung, Tai-Ching; Chiang, Yun-Wei; Jeng, U-Ser

    2017-03-15

    Upon apoptotic stress, Bcl-2 associated X (BAX) protein undergoes conformational changes and oligomerizes, leading to the mitochondrial membrane permeabilization and cell death. While structures of the resultant oligomer have been extensively studied, little is known about the intermediates that describe the reaction pathway from the inactive monomers to activated oligomers. Here we characterize the intermediate structures of BAX using combined small-angle X-ray scattering (SAXS) with on-line gel-filtration and electron spin resonance (ESR). The intermediates, including monomers, dimers, and tetramers, are reconstructed via integrating the SAXS-envelopes and ESR-determined skeleton structures. The hence revealed structures suggest a linear oligomerization of BAX utilizing the extended dimers with the two flexible α6 chains protruded out as ditopic ligands. The results of molecular dynamics simulation also support the ditopic dimer conformation with mobile α6. The ditopic dimers could further wind into a helical rod structure with three dimers in one helical turn. Our results not only reveal the on-pathway intermediates, but also suggest a ditopic oligomerization mechanism that may bridge the observed intermediate structures in solution to the large BAX assemblies lately observed on mitochondria.

  16. Multiple Bcl-2 family immunomodulators from vaccinia virus regulate MAPK/AP-1 activation.

    PubMed

    Torres, Alice A; Albarnaz, Jonas D; Bonjardim, Cláudio A; Smith, Geoffrey L

    2016-09-01

    Vaccinia virus (VACV) is a poxvirus and encodes many proteins that modify the host cell metabolism or inhibit the host response to infection. For instance, it is known that VACV infection can activate the mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) pathway and inhibit activation of the pro-inflammatory transcription factor NF-κB. Since NF-κB and MAPK/AP-1 share common upstream activators we investigated whether six different VACV Bcl-2-like NF-κB inhibitors can also influence MAPK/AP-1 activation. Data presented show that proteins A52, B14 and K7 each contribute to AP-1 activation during VACV infection, and when expressed individually outwith infection. B14 induced the greatest stimulation of AP-1 and further investigation showed B14 activated mainly the MAPKs ERK (extracellular signal-regulated kinase) and JNK (Jun N-terminal kinase), and their substrate c-Jun (a component of AP-1). These data indicate that the same viral protein can have different effects on distinct signalling pathways, in blocking NF-κB activation whilst leading to MAPK/AP-1 activation.

  17. Over-expression of BCL2 rescues muscle weakness in a mouse model of oculopharyngeal muscular dystrophy.

    PubMed

    Davies, Janet E; Rubinsztein, David C

    2011-03-15

    Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscular dystrophy caused by a polyalanine expansion mutation in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene. In unaffected individuals, (GCG)(6) encodes the first 6 alanines in a homopolymeric stretch of 10 alanines. In most patients, this (GCG)(6) repeat is expanded to (GCG)(8-13), leading to a stretch of 12-17 alanines in mutant PABPN1, which is thought to confer a toxic gain of function. Thus, OPMD has been modelled by expressing mutant PABPN1 transgenes in the presence of endogenous copies of the gene in cells and mice. In these models, increased apoptosis is seen, but it is unclear whether this process mediates OPMD. The role of apoptosis in the pathogenesis of different muscular dystrophies is unclear. Blocking apoptosis ameliorates muscle disease in some mouse models of muscular dystrophy such as laminin α-2-deficient mice, but not in others such as dystrophin-deficient (mdx) mice. Here we demonstrate that apoptosis is not only involved in the pathology of OPMD but also is a major contributor to the muscle weakness and dysfunction in this disease. Genetically blocking apoptosis by over-expressing BCL2 ameliorates muscle weakness in our mouse model of OPMD (A17 mice). The effect of BCL2 co-expression on muscle weakness is transient, since muscle weakness is apparent in mice expressing both A17 and BCL2 transgenes at late time points. Thus, while apoptosis is a major pathway that causes muscle weakness in OPMD, other cell death pathways may also contribute to the disease when apoptosis is inhibited.

  18. Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes.

    PubMed Central

    Merino, R; Ding, L; Veis, D J; Korsmeyer, S J; Nuñez, G

    1994-01-01

    Cell death is a prominent feature of B cell development. For example, a large population of B cells dies at the pre-B cell stage presumably due to the failure to express a functional immunoglobulin receptor. In addition, developing B cells expressing antigen receptors for self are selectively eliminated at the immature B cell stage. The molecular signals that control B cell survival are largely unknown. The product of the bcl-2 proto-oncogene may be involved as its overexpression inhibits apoptotic cell death in a variety of biological systems. However, the physiological role of the endogenous Bcl-2 protein during B cell development is undetermined. Here we show a striking developmental regulation of the Bcl-2 protein in B lymphocytes. Bcl-2 is highly expressed in CD43+ B cell precursors (pro-B cells) and mature B cells but downregulated at the pre-B and immature B cell stages of development. We found that Bcl-2 expressed by B cells is a long-lived protein with a half-life of approximately 10 h. Importantly, susceptibility to apoptosis mediated by the glucocorticoid hormone dexamethasone is stage-dependent in developing B cells and correlates with the levels of Bcl-2 protein. Furthermore, expression of a bcl-2 transgene rescued pre-B and immature B cells from dexamethasone-induced cell death, indicating that Bcl-2 can inhibit the apoptotic cell death of progenitors and early B cells. Taken together, these findings argue that Bcl-2 is a physiological signal controlling cell death during B cell development. Images PMID:8313913

  19. Prognostic significance of the bcl-2 apoptotic family of proteins in primary and recurrent cervical cancer.

    PubMed Central

    Crawford, R. A.; Caldwell, C.; Iles, R. K.; Lowe, D.; Shepherd, J. H.; Chard, T.

    1998-01-01

    bcl-2 is one of a family of genes that control the apoptotic threshold of a cell. bcl-2 protein and its anti-apoptotic homologue, mcl-1, with the pro-apoptotic protein, bax, are thought to function by forming homo- and heterotypic dimers that then control the progression to apoptosis. p53 is also involved as a down-regulator of bcl-2 and a promoter of bax. To determine the effect of these apoptotic mechanisms, we used immunohistochemistry to determine the prognostic significance of the expression of bcl-2, mcl-1, bax and p53 in primary and recurrent cervical cancer. Tissues from 46 patients with primary cervical cancer and 28 women with recurrent carcinoma were stained for bcl-2, mcl-1, bax and p53. Kaplan-Meier survival analysis was performed using the log-rank test for differences between groups. In the primary disease group, positive staining for bcl-2 was associated with a better 5-year survival (bcl-2 +ve, 84% vs bcl-2 -ve, 53%, P = 0.03). Positive staining for p53 was associated with a survival disadvantage (p53 +ve, 4-year survival 38% vs p53 -ve, 4-year survival 78%, P = 0.02). mcl-1 and bax staining were not useful as prognostic indicators in primary disease. No marker was prognostic in recurrent disease. Positive bcl-2 staining defines a group of patients with primary disease with a good prognosis. p53, an activator of the bax promoter, identifies a group with a worse outcome. In recurrent disease, none of the markers reflected prognosis. PMID:9683295

  20. 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

  1. 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.

  2. Bax expression remains unchanged following antisense treatment directed against BCL-2.

    PubMed

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

    2011-09-01

    Antisense oligonucleotides (oligos) have been evaluated in both in vivo and in vitro prostate cancer models. Although most contain a single mRNA binding site, our laboratory has also evaluated bispecific types directed toward two proteins. This study evaluates the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)]. Employing RT-PCR, the expression of two apoptosis regulating proteins, bcl-2 and non-targeted bax, was then evaluated. LNCaP prostate tumor cells were initially incubated for 24 h in the presence of oligos (6.25 μM) directed against bcl-2 and compared to lipofectin containing controls. Comparable and significant growth inhibition was produced by both mono- and bispecific forms. Employing RT-PCR to determine the expression of bcl-2, we found that the greatest amount of mRNA suppression approached 100% for each oligo type: monospecific MR4 (directed only against bcl-2), 100%; and bispecifics MR24 and MR42, 86 and 100%, respectively. We conclude, based upon both inhibition of in vitro growth and bcl-2 expression, that bispecific antisense oligos directed against EGFR and bcl-2 mRNAs are at least as effective as a monospecific directed solely toward bcl-2. In an effort to determine a compensatory response by cells evading apoptosis in the presence of bcl-2 suppression, the levels of mRNA encoding the non-targeted apoptosis activating protein bax were evaluated. Non-targeted protein suppression by these bispecifics has previously been demonstrated against prostate-specific membrane antigen (PSMA). However, in contrast to effects against bcl-2 and PSMA, no significant alteration in bax expression was produced by either oligo type. In LNCaP cells, bcl-2 suppression does not influence bax expression and, at least for this protein, there is no compensatory change in bax expression regulating apoptosis at this level

  3. High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition.

    PubMed

    Bate-Eya, Laurel T; den Hartog, Ilona J M; van der Ploeg, Ida; Schild, Linda; Koster, Jan; Santo, Evan E; Westerhout, Ellen M; Versteeg, Rogier; Caron, Huib N; Molenaar, Jan J; Dolman, M Emmy M

    2016-05-10

    The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression.

  4. High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition

    PubMed Central

    Bate-Eya, Laurel T.; den Hartog, Ilona J.M.; van der Ploeg, Ida; Schild, Linda; Koster, Jan; Santo, Evan E.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.; Molenaar, Jan J.; Dolman, M. Emmy M.

    2016-01-01

    The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression. PMID:27056887

  5. ABT-199, a new Bcl-2–specific BH3 mimetic, has in vivo efficacy against aggressive Myc-driven mouse lymphomas without provoking thrombocytopenia

    PubMed Central

    Vandenberg, Cassandra J.

    2013-01-01

    BH3-only proteins trigger the stress apoptosis pathway and chemical mimetics have great potential for cancer therapy. BH3-only proteins inhibit antiapoptotic members of the Bcl-2 family. Promising BH3 mimetic ABT-737 and the related orally available compound ABT-263 (navitoclax) bind avidly to antiapoptotic Bcl-2, Bcl-xL, and Bcl-w. However, their interaction with Bcl-xL provokes thrombocytopenia, which has proven to be the dose-limiting toxicity. We have tested the efficacy of ABT-199, a new Bcl-2–specific BH3 mimetic, against aggressive progenitor cell lymphomas derived from bitransgenic myc/bcl-2 mice. As a single agent, ABT-199 was as effective as ABT-737 in prolonging survival of immunocompetent tumor-bearing mice without causing thrombocytopenia. Both drugs acted rapidly but, contrary to prevailing models, their apoptotic activity did not rely upon the BH3-only protein Bim. When ABT-737 was combined with the proteosome inhibitor bortezomib or CDK inhibitor purvalanol, many treated animals achieved long-term remission. PMID:23341542

  6. Regulation of Bcl-2 and Bcl-xL anti-apoptotic protein expression by nuclear receptor PXR in primary cultures of human and rat hepatocytes.

    PubMed

    Zucchini, Nathalie; de Sousa, Georges; Bailly-Maitre, Béatrice; Gugenheim, Jean; Bars, Rémi; Lemaire, Géraldine; Rahmani, Roger

    2005-08-15

    The pregnane X receptor (PXR) plays a major role in the protection of the body by regulating the genes involved in the metabolism and elimination of potentially toxic xeno- and endobiotics. We previously described that PXR activator dexamethasone protects hepatocytes from spontaneous apoptosis. We hypothesise a PXR-dependent co-regulation process between detoxication and programmed cell death. Using primary cultured human and rat hepatocytes, we investigated to determine if PXR is implicated in the regulation of Bcl-2 and Bcl-xL, two crucial apoptosis inhibitors. In the present study we demonstrated that the treatment of primary cultured hepatocytes with PXR agonists increased hepatocyte viability and protects them from staurosporine-induced apoptosis. The anti-apoptotic capacity of PXR activation was correlated with Bcl-2 and Bcl-xL induction at both the transcriptional and protein levels in man and rats, respectively. The inhibition of PXR expression by antisense oligonucleotide abolished PXR activators Bcl-xL induction. Accordingly, PXR overexpression in HepG2 cells led to bcl-2 induction upon clotrimazole treatment and protects cells against Fas-induced apoptosis. Our results demonstrate that PXR expression is required for Bcl-2 and Bcl-xL up-regulation upon PXR activators treatment in human and rat hepatocytes. They also suggest that PXR may protect the liver against chemicals by simultaneously regulating detoxication and the apoptotic pathway.

  7. Expression of Bcl-2 and Bax protein in normal pineal gland in children and young adult.

    PubMed

    Marcol, Wiesław; Kotulska, Katarzyna; Larysz-Brysz, Magdalena; Malinowska-Kołodziej, Izabela; Mandera, Marek; Lewin-Kowalik, Joanna

    2006-01-01

    The Bcl family contains both pro and antiapoptotic proteins participating in the regulation of neuronal cell death in several pathological conditions. However, very little is known about physiological profiles of Bcl-2/Bax expression in normal brain. In this study, we examined expression profile of Bcl-2 and Bax proteins in normal pineal gland in children. The material for analysis was obtained by biopsy of pineal parenchyma during surgery of pineal cysts. All specimens were labeled immunohistochemically and analyzed by means of confocal laser scanning microscope. We found only few Bcl-2 expressing (0.7%) and no Bax-immunopositive (0.0%) pinealocytes. Bcl-2-positive cells were mature neurons, neither young ones nor glia.

  8. 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

  9. Bcl2 is not required for the development and maintenance of leukemia stem cells in mice

    PubMed Central

    González-Herrero, Inés; Vicente-Dueñas, Carolina; Orfao, Alberto; Flores, Teresa; Jiménez, Rafael; Cobaleda, César; Sánchez-García, Isidro

    2010-01-01

    The existence of leukemia stem cells (LSCs) responsible for tumor maintenance has been firmly established. Therefore, therapeutic targeting of these LSCs may have a profound impact on cancer eradication. The anti-apoptotic protein Bcl2 has been proposed as a therapeutic target, but its role in LSC biology has not been investigated. In order to understand the role of Bcl2 in LSC generation and maintenance, we have taken advantage of our Sca1-BCRABLp210 mouse model of human chronic myeloid leukemia and bcl2 gene-targeted mice. This study provides genetic evidence that the inhibition of Bcl2 is not critical for the generation, selection or maintenance of the tumor initiating and maintaining cells in mice. PMID:20299524

  10. Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease.

    PubMed Central

    Strasser, A; Whittingham, S; Vaux, D L; Bath, M L; Adams, J M; Cory, S; Harris, A W

    1991-01-01

    The biological functions of the BCL2 gene were investigated in transgenic mice harboring human BCL2 cDNA under the control of an immunoglobulin heavy chain enhancer (E mu). Mice of a representative transgenic strain, E mu-bcl-2-22, had a great excess of B lymphocytes, immunoglobulin-secreting cells, and serum immunoglobulins, attributable to increased longevity of B-lineage cells. Pre-B and plasma cells as well as B cells exhibited prolonged survival in culture. Immunized animals produced an amplified and protracted antibody response. Within the first year of life, most mice spontaneously produced antibodies to nuclear antigens, and 60% developed kidney disease, diagnosed as immune complex glomerulonephritis. Thus E mu-bcl-2-22 mice constitute a transgenic model for a systemic autoimmune disease resembling the human disorder systemic lupus erythematosus. Images PMID:1924327

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

    PubMed

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

    2012-02-01

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

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

    PubMed

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

    2017-07-29

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

  13. 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

  14. Prognostic Influence of BCL2 on Molecular Subtypes of Breast Cancer

    PubMed Central

    Han, Wonshik; Kim, Jongjin; Moon, Hyeong-Gon; Oh, Sohee; Song, Yun Seon; Kim, Young A; Chang, Mee Soo; Noh, Dong-Young

    2017-01-01

    Purpose We aimed to reveal the prognostic influence of B-cell CLL/lymphoma 2 (BCL2) on molecular subtypes of breast cancer. Methods We analyzed 9,468 patients with primary breast cancer. We classified molecular subtypes according to the National Comprehensive Cancer Network (NCCN) and St. Gallen guidelines, mainly on the basis of the expression of hormonal receptor (HR), human epidermal growth factor receptor 2 (HER2), and Ki-67. Results Regarding NCCN classification, BCL2 was a strong favorable prognostic factor in the HR(+)/HER2(–) subtype (p<0.001) and a marginally significant favorable prognosticator in the HR(+)/HER2(+) subtype (p=0.046). BCL2 had no prognostic impact on HR(–)/HER2(+) and HR(–)/HER2(–) subtypes. In relation to St. Gallen classification, BCL2 was a strong favorable prognosticator in luminal A and luminal B/HER2(–) subtypes (both p<0.001). BCL2 was a marginally significant prognosticator in the luminal B/HER2(+) subtype (p=0.046), and it was not a significant prognosticator in HER2 or triple negative (TN) subtypes. The prognostic effect of BCL2 was proportional to the stage of breast cancer in HR(+)/HER2(–), HR(+)/HER2(+), and HR(–)/HER2(–) subtypes, but not in HR(–)/HER2(+) subtype. BCL2 was not a prognostic factor in TN breast cancer regardless of epidermal growth factor receptor expression. Conclusion The prognostic influence of BCL2 was different across molecular subtypes of breast cancer, and it was largely dependent on HR, HER2, Ki-67, and the stage of cancer. BCL2 had a strong favorable prognostic impact only in HR(+)/HER2(–) or luminal A and luminal B/HER2(–) subtypes, particularly in advanced stages. Further investigations are needed to verify the prognostic influence of BCL2 on molecular subtypes of breast cancer and to develop clinical applications for prognostication using BCL2. PMID:28382095

  15. Methioninase gene therapy with selenomethionine induces apoptosis in bcl-2-overproducing lung cancer cells.

    PubMed

    Yamamoto, Norio; Gupta, Anshu; Xu, Mingxu; Miki, Kenji; Tsujimoto, Yoshihide; Tsuchiya, Hiroyuki; Tomita, Katsuro; Moossa, A R; Hoffman, R M

    2003-06-01

    We have previously shown that the toxic pro-oxidant methylselenol is released from selenomethionine (SeMET) by cancer cells transformed with the adenoviral methionine alpha,gamma-lyase (methioninase, MET) gene cloned from Pseudomonas putida. Methylselenol damaged the mitochondria via oxidative stress, and caused cytochrome c release into the cytosol thereby activating caspase enzymes and thereby apoptosis. However, gene therapy strategies are less effective if tumor cells overexpress the antiapoptotic mitochondrial protein bcl-2. In this study, we investigated whether rAdMET/SeMET was effective against bcl-2-overproducing A549 lung cancer cells. We established two clones of the human lung cancer A549 cell line that show moderate and high expression levels of bcl-2, respectively, compared to the parent cell line, which has very low bcl-2 expression. Staurosporine-induced apoptosis was inhibited in the bcl-2-overproducing clones as well as in the parental cell line. In contrast to staurosporine, apoptosis was induced in the bcl-2-overproducing clones as well as the parental cell line by AdMET/SeMET. Apoptosis in the rAdMET-SeMET-treated cells was determined by fragmentation of nuclei, and release of cytochrome c from mitochondria to the cytosol. A strong bystander effect of AdMET/SeMET was observed on A549 cells as well as the bcl-2-overproducing clones. rAdMET/SeMET prodrug gene therapy is therefore a promising novel strategy effective against bcl-2 overexpression, which has blocked other gene therapy strategies.

  16. Expression of COX-2 and Bcl-2 in primary fallopian tube carcinoma: correlations with clinicopathologic features.

    PubMed

    Wang, Fang; Sun, Guo-Ping; Zou, Yan-Feng; Wu, Qiang; Wu, Hong-Yang; Wu, Ji-Feng; Zhou, Jia-De; Chen, Ke; Zhang, Xiu-Shan

    2011-01-01

    The aim of this study was to evaluate the expression of COX-2 and Bcl-2 in primary fallopian tube carcinoma (PFTC), as well as their correlations with clinicopathologic features. We studied a cohort of 33 patients with a pathological diagnosis of PFTC. Thirty normal tubal tissues used for controls were obtained from patients diagnosed with uterine myomas. Expression analysis for COX-2 and Bcl-2 was performed using the immunohistochemical technique. The rate of preoperative diagnosis was 18.2%. With a median survival of 61.0 months (95% CI: 43.2 to 78.8 months), the estimated five-year overall survival rate in the 33 patients was 39.0%. Increased expression of COX-2 and Bcl-2 was observed in tumor specimens compared to normal controls (p = 0.026; p = 0.003). The expression rate of COX-2 in node-positive tumors was significantly higher than that of node-negative tumors (p = 0.024). Moreover, the expression rate of COX-2 was statistically significantly higher in patients with infiltration through the serosa (p = 0.019). Positive significant associations were observed between Bcl-2 staining index and FIGO stage (p = 0.015), and between Bcl-2 staining and lymph node metastasis (p = 0.010). There was a significant correlation between COX-2 expression and Bcl-2 staining index (r = 0.517, p = 0.002). We conclude that COX-2 and Bcl-2 may potentially be useful prognostic markers for PFTC. The exact molecular mechanism for correlations between COX-2 and Bcl-2 remains to be elucidated.

  17. Methylation of CpG sites in BCL2 major breakpoint region and the increase of BCL2/JH translocation with aging.

    PubMed

    Martin-Guerrero, Idoia; de Prado, Elena; Ardanaz, Maite; Martin-Arruti, Maialen; Garcia-Orad, Cristina; Guerra, Isabel; Ruiz, Irune; Zabalza, Iñaki; Garcia-Orad, Africa

    2015-10-01

    The BCL2 breakage mechanism has been shown to be highly dependent on DNA methylation at the major breakpoint region (MBR) CpG sites. We recently described an increased frequency of BCL2/ JH translocation with aging. It is known that methylation levels change with aging. The present study aimed to determine whether methylation alterations at CpG sites of BCL2 MBR were the cause of increased breakages with aging. We analyzed the methylation levels of three CpG sites on the region by pyrosequencing and studied if methylation levels and/or polymorphisms affecting CpG sites were associated with an increase of translocations. We observed that although the methylation levels of MBR CpG sites were higher in individuals with BCL2/JH translocation, in contrast to our expectations, these levels decreased with the age. Moreover, we show that polymorphisms at those CpG sites leading to absence of methylation seem to be a protective factor for the apparition of translocations.

  18. IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact

    PubMed Central

    Bonneau, Benjamin; Ando, Hideaki; Kawaai, Katsuhiro; Hirose, Matsumi; Takahashi-Iwanaga, Hiromi; Mikoshiba, Katsuhiko

    2016-01-01

    IRBIT is a molecule that interacts with the inositol 1,4,5-trisphosphate (IP3)-binding pocket of the IP3 receptor (IP3R), whereas the antiapoptotic protein, Bcl2l10, binds to another part of the IP3-binding domain. Here we show that Bcl2l10 and IRBIT interact and exert an additive inhibition of IP3R in the physiological state. Moreover, we found that these proteins associate in a complex in mitochondria-associated membranes (MAMs) and that their interplay is involved in apoptosis regulation. MAMs are a hotspot for Ca2+ transfer between endoplasmic reticulum (ER) and mitochondria, and massive Ca2+ release through IP3R in mitochondria induces cell death. We found that upon apoptotic stress, IRBIT is dephosphorylated, becoming an inhibitor of Bcl2l10. Moreover, IRBIT promotes ER mitochondria contact. Our results suggest that by inhibiting Bcl2l10 activity and promoting contact between ER and mitochondria, IRBIT facilitates massive Ca2+ transfer to mitochondria and promotes apoptosis. This work then describes IRBIT as a new regulator of cell death. DOI: http://dx.doi.org/10.7554/eLife.19896.001 PMID:27995898

  19. Bcl-2 Inhibits the Innate Immune Response during Early Pathogenesis of Murine Congenital Muscular Dystrophy

    PubMed Central

    Jeudy, Sheila; Wardrop, Katherine E.; Alessi, Amy; Dominov, Janice A.

    2011-01-01

    Laminin α2 (LAMA2)-deficient congenital muscular dystrophy is a severe, early-onset disease caused by abnormal levels of laminin 211 in the basal lamina leading to muscle weakness, transient inflammation, muscle degeneration and impaired mobility. In a Lama2-deficient mouse model for this disease, animal survival is improved by muscle-specific expression of the apoptosis inhibitor Bcl-2, conferred by a MyoD-hBcl-2 transgene. Here we investigated early disease stages in this model to determine initial pathological events and effects of Bcl-2 on their progression. Using quantitative immunohistological and mRNA analyses we show that inflammation occurs very early in Lama2-deficient muscle, some aspects of which are reduced or delayed by the MyoD-hBcl-2 transgene. mRNAs for innate immune response regulators, including multiple Toll-like receptors (TLRs) and the inflammasome component NLRP3, are elevated in diseased muscle compared with age-matched controls expressing Lama2. MyoD-hBcl-2 inhibits induction of TLR4, TLR6, TLR7, TLR8 and TLR9 in Lama2-deficient muscle compared with non-transgenic controls, and leads to reduced infiltration of eosinophils, which are key death effector cells. This congenital disease model provides a new paradigm for investigating cell death mechanisms during early stages of pathogenesis, demonstrating that interactions exist between Bcl-2, a multifunctional regulator of cell survival, and the innate immune response. PMID:21850221

  20. BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth

    PubMed Central

    Deng, Jiusheng; Park, Dongkyoo; Wang, Mengchang; Nooka, Ajay; Deng, Qiaoya; Matulis, Shannon; Kaufman, Jonathan; Lonial, Sagar; Boise, Lawrence H.; Galipeau, Jacques; Deng, Xingming

    2016-01-01

    Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy. PMID:27049723

  1. Inducible functional expression of Bcl-2 in human astrocytes derived from NTera-2 cells.

    PubMed

    Ozdener, Hakan

    2007-01-15

    Astrocytes provide structural support for neurons and may also play important roles in both neuroprotection and neurodegeneration. We, here report that human astrocytes derived from on NTera-2 (NT2) cell line expressing a functional anti-apoptotic protein bcl-2 under the control of a tetracycline responsive promoter using the Tet-On and Tet-Off expression systems. NT2 cells were transfected with the Tet On or Tet Off vectors followed by pTRE carrying human bcl-2. Drug resistant cells were differentiated into astrocytes under retinoic acid exposure. These astrocyte lines were found to express astrocyte specific markers such glial fibrillary acidic protein and chemokine receptors (CCR5, CXCR4) but not CCR3 and CD4. Furthermore, NT2 astrocytes expressing bcl-2 showed rapid response to doxycycline presence in the Tet On and Tet off system. The inducible expression of bcl-2 was found to be tightly regulated by doxycycline concentration in the NT2 astrocytes. We also showed that the induction of bcl-2 expression prevented NT2 astrocytes from camptothecin-induced cellular damage. These results indicate that this system may be useful for the study of specific effects of bcl-2 gene expression on astrocyte function(s) and cellular damage.

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

    PubMed

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

    2003-03-01

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

  3. Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2.

    PubMed

    Wang, Xiaobo; Belguise, Karine; Kersual, Nathalie; Kirsch, Kathrin H; Mineva, Nora D; Galtier, Florence; Chalbos, Dany; Sonenshein, Gail E

    2007-04-01

    Aberrant constitutive expression of c-Rel, p65 and p50 NF-kappaB subunits has been reported in over 90% of breast cancers. Recently, we characterized a de novo RelB NF-kappaB subunit synthesis pathway, induced by the cytomegalovirus (CMV) IE1 protein, in which binding of p50-p65 NF-kappaB and c-Jun-Fra-2 AP-1 complexes to the RELB promoter work in synergy to potently activate transcription. Although RelB complexes were observed in mouse mammary tumours induced by either ectopic c-Rel expression or carcinogen exposure, little is known about RelB in human breast disease. Here, we demonstrate constitutive de novo RelB synthesis is selectively active in invasive oestrogen receptor alpha (ERalpha)-negative breast cancer cells. ERalpha signalling reduced levels of functional NF-kappaB and Fra-2 AP-1 and inhibited de novo RelB synthesis, leading to an inverse correlation between RELB and ERalpha gene expression in human breast cancer tissues and cell lines. Induction of Bcl-2 by RelB promoted the more invasive phenotype of ERalpha-negative cancer cells. Thus, inhibition of de novo RelB synthesis represents a new mechanism whereby ERalpha controls epithelial to mesenchymal transition (EMT).

  4. Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2

    PubMed Central

    Wang, Xiaobo; Belguise, Karine; Kersual, Nathalie; Kirsch, Kathrin H.; Mineva, Nora D.; Galtier, Florence; Chalbos, Dany; Sonenshein, Gail E.

    2007-01-01

    Aberrant constitutive expression of c-Rel, p65 and p50 NF-κB subunits has been reported in over 90% of breast cancers1,2. Recently, we characterized a de novo RelB NF-κB subunit synthesis pathway, induced by the cytomegalovirus (CMV) IE1 protein, in which binding of p50/p65 NF-κB and c-Jun/Fra-2 AP-1 complexes to the RELB promoter work in synergy to potently activate transcription3. While RelB complexes were observed in mouse mammary tumors induced by either ectopic c-Rel expression4 or carcinogen exposure5, little is known about RelB in human breast disease. Here, we demonstrate constitutive de novo RelB synthesis is selectively active in invasive estrogen receptor (ER)α negative breast cancer cells. ERα signaling reduced levels of functional NF-κB and Fra-2 AP-1 and inhibited de novo RelB synthesis, leading to an inverse correlation between RELB and ERα gene expression in human breast cancer tissues and cell lines. Induction of Bcl-2 by RelB promoted the more invasive phenotype of ERα negative cancer cells. Thus, inhibition of de novo RelB synthesis represents a new mechanism whereby ERα controls epithelial to mesenchymal transition (EMT). PMID:17369819

  5. 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

  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. Bcl-2-like Protein 11 (BIM) Expression Is Associated with Favorable Prognosis for Patients with Cervical Cancer.

    PubMed

    Kim, Bo Wook; Cho, Hanbyoul; Ylaya, Kris; Kitano, Haruhisa; Chung, Joon-Yong; Hewitt, Stephen M; Kim, Jae-Hoon

    2017-09-01

    Bcl-2-like protein 11 (BIM) is a pro-apoptotic member of the Bcl-2 protein family. BIM elicits cell death by binding to pro-survival Bcl-2 proteins. Even though the association of BIM expression with cell death has been investigated, its clinical survival significance in cervical cancer has not. In the current study, the prognostic significance of BIM in cervical cancer was investigated. The study included normal cervical tissues (n=254), cervical intraepithelial neoplasia (CIN) tissues (n=275), and invasive cervical cancer (n=164). In order to identify BIM expression, immunohistochemistry (IHC) was performed, and IHC scoring by quantitative digital image analysis was determined. Then, the association of BIM with prognostic factors was investigated. BIM expression was higher in cervical cancer than normal cervical tissues (p<0.001). Well and moderate differentiation indicated higher BIM expression than did poor differentiation (p=0.001). Also, BIM expression was high in radiation-sensitive cervical cancer relative to radiation-resistant cancer (p=0.049). High BIM expression showed better 5-year disease-free survival (DFS) and overall survival (OS) rates (p=0.049 and π=0.030, respectively) than did low expression. In a multivariate analysis, BIM was shown to be an independent risk factor for DFS and OS in cervical cancer, with hazard ratios of 0.22 (p=0.006) and 0.46 (p=0.046), respectively. BIM is associated with favorable prognostic markers for prediction of DFS and OS in cervical cancer. High BIM expression is a potential prognostic marker as well as a chemotherapeutic target for cervical cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  8. Metabolism of cysteine by cyteinesulfinate-independent pathway(s) in rat hepatocytes

    SciTech Connect

    Stipanuk, M.H.; De La Rosa, J.; Drake, M.R.

    1986-05-01

    The metabolism of cysteine (CYS) and that of cysteinesulfinate (CSA) were studied in freshly isolated hepatocytes from fed rats. In incubations of rat hepatocytes with either 1 or 25 mM CSA, over 90% of the /sup 14/CO/sub 2/ formed from (1-/sup 14/C)CSA could be accounted for by production of hypotaurine plus taurine. In similar incubations with 1 or 25 mM CYS, only 4% of /sup 14/CO/sub 2/ evolution from (1-/sup 14/C)CYS could be accounted for by production of hypotaurine plus taurine. Addition of unlabeled CSA inhibited recovery of label from (1-/sup 14/C)CYS as /sup 14/CO/sub 2/ by 33%. Metabolism of CYS and of CSA were affected differently by addition of ..cap alpha..-ketoglutarate, a cosubstrate for transamination, or of propargylglycine, an inhibitor of cystathionase activity. These data suggest that a substantial proportion of CYS is catabolized by CSA-independent pathways in the rat hepatocyte. Although addition of ..cap alpha..-ketoglutarate to incubations of hepatocytes with CSA resulted in a marked increase in CSA catabolism via the transamination pathway, addition of keto acids to incubation systems had little or no effect on production of any metabolite from CYS. Thus, CYS transamination does not appear to be a major pathway of CYS metabolism in the hepatocyte. Inhibition of cystathionase with propargylglycine reduced both /sup 14/CO/sub 2/ production from (1-/sup 14/C)CYS and ammonia plus urea nitrogen production from CYS by about 50%; CSA catabolism was not affected. Thus, cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for CYS catabolism in the liver.

  9. BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells

    PubMed Central

    Beurlet, Stephanie; Omidvar, Nader; Gorombei, Petra; Krief, Patricia; Le Pogam, Carole; Setterblad, Niclas; de la Grange, Pierre; Leboeuf, Christophe; Janin, Anne; Noguera, Maria-Elena; Hervatin, Florence; Sarda-Mantel, Laure; Konopleva, Marina; Andreeff, Michael; Tu, Andrea W.; Fan, Alice C.; Felsher, Dean W.; Whetton, Anthony; Pla, Marika; West, Robert; Fenaux, Pierre; Chomienne, Christine

    2013-01-01

    Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1+/c-Kit+, and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS–AML transformation and survival of leukemic cells. PMID:23943652

  10. Bcl-2/Bcl-xL inhibition increases the efficacy of MEK inhibition alone and in combination with PI3 kinase inhibition in lung and pancreatic tumor models.

    PubMed

    Tan, Nguyen; Wong, Maureen; Nannini, Michelle A; Hong, Rebecca; Lee, Leslie B; Price, Stephen; Williams, Karen; Savy, Pierre Pascal; Yue, Peng; Sampath, Deepak; Settleman, Jeffrey; Fairbrother, Wayne J; Belmont, Lisa D

    2013-06-01

    Although mitogen-activated protein (MAP)-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the efficacy of a MEK inhibitor, we evaluated a panel of 53 non-small cell lung cancer and pancreatic cancer cell lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-xL (BCL2/BCL2L1) antagonist, and a novel MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment combination increases cell killing compared with double- or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with a Bcl-2 family inhibitor. ©2013 AACR

  11. Inhibition of NF-κB Activation in Combination with Bcl-2 Expression Allows for Persistence of First-Generation Adenovirus Vectors in the Mouse Liver

    PubMed Central

    Lieber, André; He, Chen-Yi; Meuse, Leonard; Himeda, Charis; Wilson, Christopher; Kay, Mark A.

    1998-01-01

    NF-κB is a key regulator of the innate antiviral immune response, due in part to its transcriptional activation of cytokines and adhesion molecules, which, in turn, function in chemotaxis and activation of inflammatory cells. We reported earlier that viral gene expression in hepatocytes transduced with first-generation (E1-deleted) adenoviruses induced NF-κB activation, elevation of serum cytokines, and hepatocellular apoptosis during the first days postinfusion. These events did not occur in mice infused with an adenovirus vector deleted for E1, E2, E3, and late gene expression. In the present study, we used an adenovirus expressing an IκBα supersuppressor (Ad.IκBM) and bcl-2 transgenic mice to unravel the role of virus-induced NF-κB activation and apoptosis in the clearance of recombinant adenovirus vectors from the liver. The combined action of IκBM and Bcl-2 allowed for vector persistence in livers of C57BL/6 × C3H mice. In the absence of Bcl-2, IκBM expression in mouse livers significantly reduced NF-κB activation, cytokine expression, leukocyte infiltration, and the humoral immune response against the transgene product; however, this was not sufficient to prevent the decline of vector DNA in transduced cells. Infusion of Ad.IκBM caused extended apoptosis predominantly in periportal liver regions, indicating that NF-κB activation may protect transduced hepatocytes from apoptosis induced by adenovirus gene products. To confer vector persistence, bcl-2 transgene expression was required to block virus-induced apoptosis if NF-κB protection was inactivated by IκBM. Expression of gene products involved in early stages of apoptotic pathways was up-regulated in response to virus infusion in bcl-2 transgenic mice, which may represent a compensatory effect. Our study supports the idea that the suppression of innate defense mechanisms improves vector persistence. PMID:9765474

  12. 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

  13. Aspirin-induced Bcl-2 translocation and its phosphorylation in the nucleus trigger apoptosis in breast cancer cells.

    PubMed

    Choi, Bo-Hwa; Chakraborty, Goutam; Baek, Kwanghee; Yoon, Ho Sup

    2013-10-11

    Here, we report that B-cell lymphoma 2 (Bcl-2) is a novel target molecule of aspirin in breast cancer cells. Aspirin influenced the formation of a complex by Bcl-2 and FKBP38 and induced the nuclear translocation of Bcl-2 and its phosphorylation. These events inhibited cancer cell proliferation and subsequently enhanced MCF-7 breast cancer cell apoptosis. Bcl-2 knockdown using small interfering RNA (siRNA) delayed apoptotic cell death, which correlated with increased proliferation following aspirin exposure. In contrast, Bcl-2 overexpression enhanced the onset of aspirin-induced apoptosis, which was also associated with a significant increase in Bcl-2 phosphorylation in the nucleus. Therefore, this study may provide novel insight into the molecular mechanism of aspirin, particularly its anticancer effects in Bcl-2- and estrogen receptor-positive breast cancer cells.

  14. Aspirin-induced Bcl-2 translocation and its phosphorylation in the nucleus trigger apoptosis in breast cancer cells