Bcl-2 Phosphorylation Research Articles

Cell Surface CD74–MIF Interactions Drive Melanoma Survival in Response to Interferon-γ

While melanoma is believed to be a highly immunogenic tumor and recent developments in immunotherapies are promising. Interferon-γ (IFN-γ) produced by immune cells plays a crucial role in tumor immune surveillance; however, it has also been reported to be pro-tumorigenic. In the current study, we found that IFN-γ enhances the expression of CD74, which interacts with its ligand, macrophage migration inhibitory factor (MIF), and thereby activates the PI3K/AKT pathway in melanoma, promoting tumor survival. IFN-γ increased phosphorylation of AKT Ser473 and upregulated total and cell surface expression of CD74 in human melanoma cell lines tested. CD74 was highly expressed in melanoma tissues. Moreover, the expression of CD74 on tumor cells correlated with plasma IFN-γ levels in melanoma patient samples. In our analysis of melanoma cell lines, all produced MIF constitutively. Blockade of CD74-MIF interaction reduced AKT phosphorylation and expression of pro-tumorigenic molecules, including interleukin-6, interleukin-8 and BCL-2. Inhibition of CD74-MIF interaction significantly suppressed tumor growth in the presence of IFN-γ in our xenograft mouse model. Thus, we conclude that IFN-γ promotes melanoma cell survival by regulating CD74-MIF signaling, suggesting that targeting the CD74-MIF interaction under IFN-γ–stimulatory conditions would be an effective therapeutic approach for melanoma.

Pharmaceutical screen identifies novel target processes for activation of autophagy with a broad translational potential.

Autophagy is a conserved homeostatic process active in all human cells and affecting a spectrum of diseases. Here we use a pharmaceutical screen to discover new mechanisms for activation of autophagy. We identify a subset of pharmaceuticals inducing autophagic flux with effects in diverse cellular systems modelling specific stages of several human diseases such as HIV transmission and hyperphosphorylated tau accumulation in Alzheimer's disease. One drug, flubendazole, is a potent inducer of autophagy initiation and flux by affecting acetylated and dynamic microtubules in a reciprocal way. Disruption of dynamic microtubules by flubendazole results in mTOR deactivation and dissociation from lysosomes leading to TFEB (transcription factor EB) nuclear translocation and activation of autophagy. By inducing microtubule acetylation, flubendazole activates JNK1 leading to Bcl-2 phosphorylation, causing release of Beclin1 from Bcl-2-Beclin1 complexes for autophagy induction, thus uncovering a new approach to inducing autophagic flux that may be applicable in disease treatment.

Overexpression of Bcl-2 induces STAT-3 activation via an increase in mitochondrial superoxide.

We recently reported a novel interaction between Bcl-2 and Rac1 and linked that to the ability of Bcl-2 to induce a pro-oxidant state in cancer cells. To gain further insight into the functional relevance of this interaction, we utilized computer simulation based on the protein pathway dynamic network created by Cellworks Group Inc. STAT3 was identified among targets that positively correlated with Rac1 and/or Bcl-2 expression levels. Validating this, the activation level of STAT3, as marked by p-Tyr705, particularly in the mitochondria, was significantly higher in Bcl-2-overexpressing cancer cells. Bcl-2-induced STAT3 activation was a function of GTP-loaded Rac1 and NADPH oxidase (Nox)-dependent increase in intracellular superoxide (O2•-). Furthermore, ABT199, a BH-3 specific inhibitor of Bcl-2, as well as silencing of Bcl-2 blocked STAT3 phosphorylation. Interestingly, while inhibiting intracellular O2•- blocked STAT3 phosphorylation, transient overexpression of wild type STAT3 resulted in a significant increase in mitochondrial O2•- production, which was rescued by the functional mutants of STAT3 (Y705F). Notably, a strong correlation between the expression and/or phosphorylation of STAT3 and Bcl-2 was observed in primary tissues derived from patients with different sub-sets of B cell lymphoma. These data demonstrate the presence of a functional crosstalk between Bcl-2, Rac1 and activated STAT3 in promoting a permissive redox milieu for cell survival. Results also highlight the potential utility of a signature involving Bcl-2 overexpression, Rac1 activation and STAT3 phosphorylation for stratifying clinical lymphomas based on disease severity and chemoresistance.

Synergistic induction of apoptosis by a polo-like kinase 1 inhibitor and microtubule-interfering drugs in Ewing sarcoma cells.

Since polo-like kinase 1 (PLK1) is highly expressed in Ewing sarcoma (ES), we evaluated the therapeutic potential of the PLK1 inhibitor BI 6727. Here, we identify a synergistic induction of apoptosis by BI 6727 and several microtubule-interfering drugs in ES cells, including vincristine (VCR), vinblastine (VBL), vinorelbine (VNR) and eribulin. Synergistic drug interaction is confirmed by calculation of combination index (CI). Also, BI 6727 and VCR act in concert to reduce long-term clonogenic survival. Mechanistically, BI 6727/VCR co-treatment cooperates to trigger mitotic arrest, phosphorylation of BCL-2 and BCL-XL and downregulation of MCL-1. This inactivation of anti-apoptotic BCL-2 family proteins in turn promotes activation of BAX and BAK, activation of caspase-9 and -3 and caspase-dependent apoptosis. Overexpression of BCL-2 or simultaneous knockdown of BAX and BAK significantly rescue BI 6727/VCR-induced apoptosis, indicating that engagement of the mitochondrial pathway is critical for BI 6727/VCR-mediated apoptosis. The clinical relevance of PLK1 inhibitor-based combination therapies is underscored by the fact that BI 6727 is currently evaluated in phase I clinical trials in childhood cancer. In conclusion, PLK1 inhibitors such as BI 6727 may provide a new strategy to chemosensitize ES.

Sphingosine kinase inhibitors decrease viability and induce cell death in natural killer-large granular lymphocyte leukemia

Sphingolipid metabolism has been identified as a potential therapeutic target in cancer. Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid metabolite produced by sphingosine kinases-1 and −2 (SPHK1 and SPHK2). Elevated SPHK1 has been found in numerous cancer types and been shown to contribute to survival, chemotherapeutic resistance and malignancy. However, its role in large granular Natural Killer (NK) large granular lymphocyte (LGL) leukemia has not been investigated. Here, we examine SPHK1 as a therapeutic target in LGL leukemia. We found that SPHK1 is overexpressed in peripheral blood mononuclear cells (PBMCs) from LGL leukemia patients which results in elevated S1P in the sera. The use of SPHK1 inhibitors, SKI-II or SKI-178, decreased leukemic NK cell viability and induced caspase-dependent apoptosis. SKI-II and SKI-178 restored the sphingolipid balance by increasing ceramide and decreasing S1P in leukemic NKL cells. SKI-II and SKI-178 also induced apoptosis in primary NK-LGLs from leukemia patients. Mechanistic studies in NK-LGL cell lines demonstrated that SKI-178 and SKI-II induced cell cycle arrest at G2/M. We found that SKI-178 induced phosphorylation of Bcl-2 at Ser70, and that this was dependent on CDK1. We further show that SPHK1 inhibition with SKI-178 leads to decreased JAK-STAT signaling. Our data demonstrate that SPHK1 represents a novel therapeutic target for the treatment of NK-LGL leukemia.

Zerumbone, a ginger sesquiterpene, induces apoptosis and autophagy in human hormone-refractory prostate cancers through tubulin binding and crosstalk between endoplasmic reticulum stress and mitochondrial insult.

Zerumbone, a natural monocyclic sesquiterpene, is the main component of the tropical plant Zingiber zerumbet Smith. Zerumbone induced antiproliferative and apoptotic effects against PC-3 and DU-145, two human hormone-refractory prostate cancer (HRPC) cell lines. Zerumbone inhibited microtubule assembly and induced an increase of MPM-2 expression (specific recognition of mitotic proteins). It also caused an increase of phosphorylation of Bcl-2 and Bcl-xL, two key events in tubulin-binding effect, indicating tubulin-binding capability and mitotic arrest to zerumbone action. Furthermore, zerumbone induced several cellular effects distinct from tubulin-binding properties. First, zerumbone significantly increased, while paclitaxel (as a tubulin-binding control) decreased, Mcl-1 protein expression. Second, paclitaxel but not zerumbone induced Cdk1 activity. Third, zerumbone other than paclitaxel induced Cdc25C downregulation. The data suggest that, in addition to targeting tubulin/microtubule, zerumbone may act on other targets for signaling transduction. Zerumbone induced mitochondrial damage and endoplasmic reticulum (ER) stress as evidenced by the loss of mitochondrial membrane potential and upregulation of GRP-78 and CHOP/GADD153 expression. Zerumbone induced an increase of intracellular Ca(2+) levels, a crosstalk marker between ER stress and mitochondrial insult, associated with the formation of active calpain I fragment. It induced apoptosis through a caspase-dependent way and caused autophagy as evidenced by dramatic LC3-II formation. In summary, the data suggest that zerumbone is a multiple targeting compound that inhibits tubulin assembly and induces a crosstalk between ER stress and mitochondrial insult, leading to apoptosis and autophagy in HRPCs.

Abstract 13: Biophysical evidence for the existence of a functional interaction between the small GTPase Rac-1 and the anti-apoptotic protein Bcl-2

Abstract We recently reported a novel mechanism of anti-apoptotic activity of Bcl-2 by demonstrating a link between an altered mitochondrial metabolism and apoptosis resistance in cells overexpressing Bcl-21. This increase in mitochondrial metabolism was linked to induction of complex IV activity (Cytochrome C oxidase) as well as oxygen consumption2. The pro-oxidant activity of Bcl-2 was a function of an interaction with the small GTPase Rac1. This interaction was validated in a small number of clinical tissues derived from patients with aggressive B cell lymphomas. Interestingly, interrupting the interaction between Bcl-2 and Rac1 restored apoptosis sensitivity of Bcl-2 overexpressing cell lines3. In the present study we set out to identify the domains within the two proteins responsible for the interaction. We show that the pro-oxidant activity of Bcl-2 and its interaction with Rac1 are dependent on the phosphorylation of Bcl-2 at serine 70 within the non-structured loop region adjacent to the BH3 domain. Also, the activation status of Rac1 appears important in this interaction as GDP loading almost completely inhibits, whereas GTP loading facilitates interaction. To further substantiate those findings, isothermal titration calorimetry (ITC) was carried out using Rac1-GTPγS and Bcl-2 BH3 peptides to gain insight into the energetics of binding of BH3 peptide to Rac1-GTPγS. Indeed, the thermodynamic parameters ΔH and ΔS in the ITC experiments of Rac1- GTPγS and BH3 peptide were -2.55 × 104 Joules/mol and +35.6 joules/mol/deg, respectively, and the ΔG values were negative, indicating that the binding of Rac1-GTPγS with BH3 peptide was spontaneous and exothermic and that hydrophobic interactions played a major role; however, hydrogen bonds could not be discounted due to the positive ΔS value and the presence of polar amino acids in the peptide. Similar results were obtained with the S70 phosphorylation mimetic peptide of Bcl-2 (S70E) but not with the S70A phosphorylation dead peptide. Furthermore, the fluorescence intensity of Rac1 decreased steadily with increasing Bcl-2 peptide concentration, indicating an interaction between Rac1 and Bcl-2 peptides. These data provide strong evidence for the existence of a functional interaction between Bcl-2 and Rac1 and for the translational relevance of this interaction in human cancers that are refractory to chemotherapy due to the overexpression of Bcl-2. 1. Chen, Z, X. and Pervaiz, S. Cell Death Diff. 14(9): 1617-27, 2007. 2. Chen ZX, and Pervaiz S. Cell Death Differ. 2010 Mar;17(3):408-20. 3. Velaithan R, et al. Blood. 2011 Jun 9;117(23):6214-26. Citation Format: Jia Kang, Shani Ajumal, Kunchithapadam Swaminathan, Shazib Pervaiz. Biophysical evidence for the existence of a functional interaction between the small GTPase Rac-1 and the anti-apoptotic protein Bcl-2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 13. doi:10.1158/1538-7445.AM2015-13

Repurposing of phentolamine as a potential anticancer agent against human castration-resistant prostate cancer: A central role on microtubule stabilization and mitochondrial apoptosis pathway.

Drug repurposing of phentolamine, an α-adrenoceptor antagonist, as an anticancer agent has been studied in human castration-resistant prostate cancer (CRPC). Cell proliferation was examined by sulforhodamine B and CFSE staining assays. Cell cycle progression and mitochondrial membrane potential (ΔΨm) were detected by flow cytometric analysis. Protein expression was detected by Western blotting. Effect on tubulin/microtubule was determined using confocal immunofluorescence microscopic examination, microtubule assembly detection, tubulin turbidity assay, and binding assay. Several assessments were used to characterize apoptotic signaling pathways and combinatory effect. Phentolamine induced anti-proliferative effect in PC-3 and DU-145, two CRPC cell lines, and P-glycoprotein (P-gp) overexpressing cells. This effect was not significantly reduced in paclitaxel-resistant cells. Rhodamine 123 efflux assay showed that phentolamine was not a P-gp substrate. Phentolamine induced mitotic arrest of the cell cycle and formation of hyperdiploid cells, followed by an increase of apoptosis. Mitotic arrest was confirmed by cyclin B1 up-regulation, Cdk1 activation, and a dramatic increase of mitotic protein phosphorylation. Both in vitro and cellular identification demonstrated that phentolamine, similar to paclitaxel, induced tubulin polymerization and formation of multiple nuclei. Besides, it did not compete with paclitaxel binding on tubulin. Phentolamine induced the phosphorylation and degradation of Bcl-2 and Bcl-xL, two anti-apoptotic Bcl-2 family members, and the loss of ΔΨm indicating the induction of mitochondrial damage. It ultimately induced the activation of caspase-9, -8, and -3 and apoptotic cell death. Moreover, combination treatment with phentolamine and paclitaxel caused a synergistic apoptosis. The data suggest that phentolamine is a potential anticancer agent. In contrast to a wide variety of microtubule disrupting agents, phentolamine induces microtubule assembly, leading to mitotic arrest of the cell cycle which "in turn" induces subsequent mitochondrial damage and activation of related apoptotic signaling pathways in CRPC cells. Furthermore, combination between phentolamine and paclitaxel induces a synergistic apoptotic cell death. Phentolamine has a simple chemical structure and is not a P-gp substrate. Optimization of phentolamine structure may also be a potential approach for further development.

Polo-like kinase 1 inhibition sensitizes neuroblastoma cells for vinca alkaloid-induced apoptosis.

High polo-like kinase 1 (PLK1) expression has been linked to poor outcome in neuroblastoma (NB), indicating that it represents a relevant therapeutic target in this malignancy. Here, we identify a synergistic induction of apoptosis by the PLK1 inhibitor BI 2536 and vinca alkaloids in NB cells. Synergistic drug interaction of BI 2536 together with vincristine (VCR), vinblastine (VBL) or vinorelbine (VNR) is confirmed by calculation of combination index (CI). Also, BI 2536 and VCR act in concert to reduce long-term clonogenic survival. Importantly, BI 2536 significantly enhances the antitumor activity of VCR in an in vivo model of NB. Mechanistically, BI 2536/VCR co-treatment triggers prolonged mitotic arrest, which is necessary for BI 2536/VCR-mediated apoptosis, since pharmacological inhibition of mitotic arrest by the CDK1 inhibitor RO-3306 significantly reduces cell death. Prolonged mitotic arrest leads to phosphorylation-mediated inactivation of BCL-2 and BCL-XL as well as downregulation of MCL-1, since inhibition of mitotic arrest by RO-3306 also prevents phosphorylation of BCL-2 and BCL-XL and MCL-1 downregulation. This inactivation of antiapoptotic BCL-2 proteins promotes activation of BAX and BAK, cleavage of caspase-9 and -3 and caspase-dependent apoptosis. Engagement of the mitochondrial pathway of apoptosis is critically required for BI 2536/VCR-induced apoptosis, since ectopic expression of a non-degradable MCL-1 phospho-mutant, BCL-2 overexpression or BAK knockdown significantly reduce BI 2536/VCR-mediated apoptosis. Thus, PLK1 inhibitors may open new perspectives for chemosensitization of NB.

Gartanin induces autophagy through JNK activation which extenuates caspase-dependent apoptosis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Development of novel agents to eradicate liver cancer cells is required for treatment of HCC. Gartanin, a xanthone-type compound isolated from mangosteen, is known to possess potent antioxidant, anti-inflammatory, antifungal and antineoplastic properties. In the present study, we investigated the cytotoxic effect of gartanin on HCC and explored the cell death mechanism. We showed that gartanin induced both the extrinsic and intrinsic apoptotic pathways, which were interconnected by caspase-8, -9 and -3 activation. We also provided convincing evidence that gartanin induced autophagy in various cancer cells, as 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. Additionally, gartanin induced the formation of typical autophagosomes and autolysosomes and enhanced the degradation rate of intracellular granule(s), including mitochondria. Notably, gartanin-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). These findings suggested that gartanin-mediated autophagic response protected against eventual cell death induced by gartanin. Moreover, gartanin treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor (SP600125) inhibited autophagy yet promoted gartanin-induced apoptosis, indicating a key requirement of the JNK-Bcl-2 pathway in the activation of autophagy by gartanin. Taken together, our data suggested that the JNK-Bcl-2 pathway was the critical regulator of gartanin-induced protective autophagy and a potential drug target for chemotherapeutic combination.

The NOXA-MCL1-BIM axis defines lifespan on extended mitotic arrest.

Cell death on extended mitotic arrest is considered arguably most critical for the efficacy of microtubule-targeting agents (MTAs) in anticancer therapy. While the molecular machinery controlling mitotic arrest on MTA treatment, the spindle assembly checkpoint (SAC), appears well defined, the molecular components executing cell death, as well as factors connecting both networks remain poorly understood. Here we conduct a mini screen exploring systematically the contribution of individual BCL2 family proteins at single cell resolution to death on extended mitotic arrest, and demonstrate that the mitotic phosphorylation of BCL2 and BCLX represent a priming event for apoptosis that is ultimately triggered by NOXA-dependent MCL1 degradation, enabling BIM-dependent cell death. Our findings provide a comprehensive model for the initiation of apoptosis in cells stalled in mitosis and provide a molecular basis for the increased efficacy of combinatorial treatment of cancer cells using MTAs and BH3 mimetics.

A 2-methoxyestradiol bis-sulphamoylated derivative induces apoptosis in breast cell lines.

IntroductionResearch involving antimitotic compounds identified 2-methoxyestradiol (2ME2), as a promising anticancer endogenous metabolite. Owing to its low bioavailability, several in silico-designed 2ME2 analogues were synthesized. Structure-activity relationship studies indicated that an already existing 17-β-estradiol analogue, namely (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) to exert potential in vitro anticancer activity.MethodsThis study investigated the in vitro apoptotic influence of EMBS in an estrogen receptor-positive breast adenocarcinoma epithelial cell line (MCF-7); an estrogen receptor-negative breast epithelial cell line (MDA-MB-231) and a non-tumorigenic breast cell line (MCF-12A). Cell cycle progression, a phosphatidylserine flip, caspase 6-, 7- and 8 enzyme activity levels, Bcl-2 phosphorylation status at serine 70 and Bcl-2- and p53 protein levels were investigated to identify a possible action mechanism for apoptotic induction.ResultsThe xCELLigence real-time label-independent approach revealed that EMBS exerted antiproliferative activity in all three cell lines after 24 h of exposure. A G2M block was observed and apoptosis induction was verified by means of flow cytometry using propidium iodide and Annexin V-FITC respectively. EMBS-treated cells demonstrated a reduced mitochondrial membrane potential. EMBS exposure resulted in a statistically significant increase in p53 protein expression, decreased Bcl-2 protein expression and a decrease in pBcl-2(s70) phosphorylation status in all three cell lines. Results support the notion that EMBS induces apoptosis in all three cell lines.ConclusionThis study includes investigation into the apoptotic hallmarks exerted by EMBS after exposure of three cell lines namely MCF-7-, MDA-MDA-231- and MCF-12A cells. Increased caspase 6-, caspase 7- and caspase 8 activities, upregulation of p53 protein expression and a decrease in phosphorylation status of Bcl-2 at serine 70 in tumorigenic and non-tumorigenic lines were demonstrated.

Antiproliferative activity of the isoindigo 5′-Br in HL-60 cells is mediated by apoptosis, dysregulation of mitochondrial functions and arresting cell cycle at G0/G1 phase

Our new compound, 5′-Br [(E)-1-(5′-bromo-2′-oxoindolin-3′-ylidene)-6-ethyl-2,3,6,9-tetrahydro-2,9-dioxo-1H-pyrrolo[3,2-f]quinoline-8-carboxylic acid], had shown strong, selective antiproliferative activity against different cancer cell lines. Here, we aim to comprehensively characterize the mechanisms associated with its cytotoxicity in the human promyelocytic leukemia HL-60 cells. We focused at studying the involvement of apoptotic pathway and cell cycle effects. 5′-Br significantly inhibited proliferation by inducing caspase-dependent apoptosis. Involvement of caspase independent mechanism is also possible due to observed inability of z-VAD-FMK to rescue apoptotic cells. 5′-Br was found to trigger intrinsic apoptotic pathway as indicated by depolarization of the mitochondrial inner membrane, decreased level of cellular ATP, modulated expression and phosphorylation of Bcl-2 leading to loss of its association with Bax, and increased release of cytochrome c. 5′-Br treated cells were found arrested at G0/G1 phase with modulation in protein levels of cyclins, dependent kinases and their inhibitors. Expression and enzymatic activity of CDK2 and CDK4 was found inhibited. Retinoblastoma protein (Rb) phosphorylation was also inhibited whereas p21 protein levels were increased. These results suggest that the antiproliferative mechanisms of action of 5′-Br could involve apoptotic pathways, dysregulation of mitochondrial functions and disruption of cell cycle checkpoint.

Paxillin promotes colorectal tumor invasion and poor patient outcomes via ERK-mediated stabilization of Bcl-2 protein by phosphorylation at Serine 87.

Stabilization of Bcl-2 protein by paxillin (PXN)-mediated ERK activation was recently reported to cause an unfavorable response to 5-Fluorouracil-based chemotherapy. Here, we present evidence from cell and animal models to demonstrate that stabilization of Bcl-2 protein by phosphorylation at Serine 87 (pBcl-2-S87) via PXN-mediated ERK activation is responsible for cancer cell invasiveness and occurs via upregulation of MMP2 expression. Immunostainings of 190 tumors resected from colorectal cancer patients indicated that PXN expression was positively correlated with Bcl-2, pBcl-2-S87, and MMP2 expression. A positive correlation of pBcl-2-S87 with Bcl-2 and MMP2 was also observed in this study population. Patients with high PXN, Bcl-2, pBcl-2-S87, and MMP2 had poor overall survival (OS) and shorter relapse free survival (RFS). In conclusion, PXN promotes Bcl-2 phosphorylation at Serine 87 via PXN-mediated ERK activation, and its stabilization associated with increased tumor formation efficacy in mice and poor patient outcome in colorectal cancer patients.

Protective effects of astaxanthin on ConA-induced autoimmune hepatitis by the JNK/p-JNK pathway-mediated inhibition of autophagy and apoptosis.

ObjectiveAstaxanthin, a potent antioxidant, exhibits a wide range of biological activities, including antioxidant, atherosclerosis and antitumor activities. However, its effect on concanavalin A (ConA)-induced autoimmune hepatitis remains unclear. The aim of this study was to investigate the protective effects of astaxanthin on ConA-induced hepatitis in mice, and to elucidate the mechanisms of regulation.Materials and MethodsAutoimmune hepatitis was induced in in Balb/C mice using ConA (25 mg/kg), and astaxanthin was orally administered daily at two doses (20 mg/kg and 40 mg/kg) for 14 days before ConA injection. Levels of serum liver enzymes and the histopathology of inflammatory cytokines and other maker proteins were determined at three time points (2, 8 and 24 h). Primary hepatocytes were pretreated with astaxanthin (80 μM) in vitro 24 h before stimulation with TNF-α (10 ng/ml). The apoptosis rate and related protein expression were determined 24 h after the administration of TNF-α.ResultsAstaxanthin attenuated serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It performed anti-apoptotic effects via the descending phosphorylation of Bcl-2 through the down-regulation of the JNK/p-JNK pathway.ConclusionThis research firstly expounded that astaxanthin reduced immune liver injury in ConA-induced autoimmune hepatitis. The mode of action appears to be downregulation of JNK/p-JNK-mediated apoptosis and autophagy.

Bcl-2 phosphorylation triggers autophagy switch and reduces mitochondrial damage in limb remote ischemic conditioned rats after ischemic stroke.

Autophagy, an important intracellular degradation pathway, has been reported to clear impaired mitochondria and reduce mitochondria-mediated injury in ischemic disease. Our study and other recent investigations have shown that AKT-dependent autophagy contributes to the neuroprotection afforded by limb remote ischemic conditioning (RIC) in experimental stroke. However, how AKT triggers RIC-based autophagy and whether RIC-afforded autophagy is beneficial for mitochondrial function after cerebral ischemia remains unclear. The disruption of the Bcl-2/Beclin1 complex has been reported to trigger autophagy formation in the condition of Bcl-2 phosphorylation at Ser70. We investigated whether Bcl-2 phosphorylation triggers RIC-based autophagy and thereby confers RIC-induced neuroprotection against mitochondrial injury, using a transient cerebral ischemic rat model. We demonstrated that rats undergoing RIC treatment 30 min after the onset of ischemia (I-30) and at reperfusion (R-0) significantly upregulated Bcl-2 phosphorylation. Immunoprecipitation revealed that RIC increased dissociation of the Bcl-2/Beclin1 complex, leading to a higher level of autophagy than in ischemia/reperfusion rats. Furthermore, AKT activation was shown to play a critical role in regulating Bcl-2-mediated autophagy, as an AKT inhibitor (LY294002, AKTi) administered 30 min prior to ischemia significantly suppressed Bcl-2 phosphorylation and Bcl-2/Beclin1 complex dissociation, thereby reducing autophagy in RIC rats. Blocking Bcl-2 phosphorylation-dependent autophagy with AKTi suppressed RIC-afforded protection on mitochondrial potential and mitochondrial-dependent cell death effector pathway. These findings indicate that Bcl-2 phosphorylation and thereby Bcl-2/Beclin1 complex disruption play a crucial role in triggering autophagy and reducing mitochondrial damage in RIC rats after cerebral ischemia and require the involvement of the AKT activation.

Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells

The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E2)-induced cell cycle arrest and 2-MeO-E2-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E2 (0.5–1.0μM) caused G2/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G2/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E2 and the G1/S blocking agent aphidicolin resulted in G1/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E2-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10–20μM 2-MeO-E2 reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5–5.0μM 2-MeO-E2 increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E2 (0.5–1.0μM) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.

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

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.

Rituximab-induced early and late signaling have opposite effects on dexamethasone-induced apoptosis in human follicular lymphoma cells

The addition of rituximab (RTX) to standard chemotherapy has improved the treatment of B-cell malignancies. We show here that RTX and dexamethasone (Dex) induced synergistic apoptosis in follicular lymphoma cell lines. However, apoptosis was delayed by RTX-induced early protective signaling. RTX-induced early signaling also decreased Dex-induced apoptosis and led to phosphorylation of ERK1/2, Bcl-2 (at serine 70) and phosphorylation/degradation of BimL/EL. All these events were prevented by the MEK inhibitor, UO126. Therefore, we suggest that RTX-induced ERK-mediated signaling events lead to protection from apoptosis during early signaling and that blocking of Bim and Bcl-2 phosphorylation might be used as a novel strategy for lymphoma treatment.

Relationship between BH3 mimetic S1 and expression of BCL-2 family members in acute myeloid leukemia

This study was to investigate the molecular biomarkers of apoptosis induced by BH3 mimetic S1 in human primary AML cells. Mononuclear cells were isolated from 27 newly diagnosed AML samples. Apoptosis was analyzed by flow cytometry. IC(50) value of S1 on these samples was determined by XTT assay. The expression level of BCL-2 family members and phosphorylated BCL-2 were assessed by Western blot with subsequent semi-quantitatively densitometric analysis. XTT assay was performed to determine the cell viability of the combined use of S1 and MEK/ERK inhibitor PD98059. The interactions between BCL-2 and pro-apoptosis proteins were tested by co-immunoprecipitation. The flow-cytometry detection showed that S1 induced the apoptosis of primary AML cells. Based on the responses, 27 primary samples could be classified into three groups: (1) a sensitive group (12 of 27 cases) with IC(50)<14 µmol/L, (2) an intermediate group (8 of 27 cases) with IC(50) of 14-30 µmol/L and (3) a resistant group (7 of 27 cases) with IC(50)>30 µmol/L. The ratio of pBCL-2/(BCL-2+MCL-1) showed a good linear correlation with the IC(50) values. (R(2) = 0.71, P < 0.0001). PD98059 suppressed BCL-2 phosphorylation. When PD98059 suppressed BCL-2 phosphorylation, the apoptotic rate of drug-resistant cells induced by S1 increased from 9.8% to 64.5% (combination index, CI = 0.4), accompanied by more dissociation of BCL-2 heterodimers. The combination of S1 with PD98059 decrease pBCL-2 level of AML patients and inhibits of the anti-apoptotic function of BCL-2 through enhancing the dissociation of BCL-2 heterodimers.