Bcl-xL Phosphorylation Research Articles

Repurposing Suan-Zao-Ren-Tang as anticancer agents in apoptotic sensitization against non-small cell lung cancer cell lines through amplification of spindle assembly checkpoint activation

Treatment of non-small cell lung cancer (NSCLC) is a high unmet medical need. We repurposed Suan-Zao-Ren-Tang (SZRT), a widely used traditional Chinese medicine in alleviating insomnia, to sensitize vinorelbine-induced anti-NSCLC effect and have unveiled the underlying mechanism. B7E2, the partial purified extract from SZRT, was used through bioactivity-guided fractionation based on anti-proliferative activity in NSCLC cell lines A549 and NCI-H460. Three batches of B7E2 and their individual components were prepared for the examination of components and bioactivity and mechanistic study. B7E2 alone had no cytotoxic effect but its combination with vinorelbine displayed a synergistic activity in killing NSCLC cells. Apoptotic sensitization was verified by a remarkable increase of caspase activation and phosphorylation of Bcl-2 and Bcl-xL. Combination treatment prolonged mitotic arrest and induced a substantial activation of spindle assembly checkpoint (SAC) characterized by BUBR1 phosphorylation at Ser670 and Thr680, and cyclin B1 upregulation. Combination treatment enhanced the interaction between mitotic checkpoint complex (MCC) components, including BUBR1, MAD2, BUB3 and CDC20. Further analysis revealed that SZRT components, including senkyunolide-A, trans-neocnidilide, 3-butylidenephthalide, betulinic acid and linoleic acid, were responsible for B7E2 activities. Three B7E2 batches showed a good chemical similarity and similar activities between batches. The data suggest that B7E2 is a potential chemosensitizer in potentiating vinorelbine-induced anti-NSCLC activity through amplification and prolongation of SAC activation.

O-Linked N-Acetylglucosamine Transferase Ensures Survival of Mouse Fetal Liver Hematopoietic Progenitors Partly by Regulating Bcl-xL and Oxidative Phosphorylation.

O-linked N-acetylglucosamine transferase (OGT) critically regulates wide variety of biological processes such as gene expression, metabolism, stress response, signaling and proteostasis. In adult hematopoiesis, OGT is crucial for differentiation of B and T cells and the maintenance of hematopoietic stem cells (HSCs). However, a role for OGT in fetal liver (FL) hematopoiesis remains unknown. To investigate a role for OGT in FL hematopoiesis, we conditionally disrupted OGT in hematopoietic cells in developing FLs. Hematopoietic specific disruption of OGT resulted in embryonic lethality in late stage of gestation due to severe anemia and growth retardation. OGT loss led to profound reduction of differentiating erythroid cells and erythroid progenitors in FLs due to massive apoptosis. In addition, clonogenic capacity of FL cells was severely impaired by OGT loss. Interestingly, expression of BCL-XL, a well-known inhibitor of apoptosis in FL cells, dramatically decreased, and the levels of reactive oxygen species (ROS) were increased in OGT-deficient FL cells. Overexpression of Bcl-xL and reduction of ROS significantly restored the colony formation of OGT-deficient FL cells. This study revealed a novel role for OGT during embryogenesis, which ensures survival of FL hematopoietic cells partly by regulating Bcl-xL and oxidative phosphorylation.

Ser14 phosphorylation of Bcl-xL mediates compensatory cardiac hypertrophy in male mice

The anti-apoptotic function of Bcl-xL in the heart during ischemia/reperfusion is diminished by K-Ras-Mst1-mediated phosphorylation of Ser14, which allows dissociation of Bcl-xL from Bax and promotes cardiomyocyte death. Here we show that Ser14 phosphorylation of Bcl-xL is also promoted by hemodynamic stress in the heart, through the H-Ras-ERK pathway. Bcl-xL Ser14 phosphorylation-resistant knock-in male mice develop less cardiac hypertrophy and exhibit contractile dysfunction and increased mortality during acute pressure overload. Bcl-xL Ser14 phosphorylation enhances the Ca2+ transient by blocking the inhibitory interaction between Bcl-xL and IP3Rs, thereby promoting Ca2+ release and activation of the calcineurin-NFAT pathway, a Ca2+-dependent mechanism that promotes cardiac hypertrophy. These results suggest that phosphorylation of Bcl-xL at Ser14 in response to acute pressure overload plays an essential role in mediating compensatory hypertrophy by inducing the release of Bcl-xL from IP3Rs, alleviating the negative constraint of Bcl-xL upon the IP3R-NFAT pathway.

The antitumor activity of a novel GCN2 inhibitor in head and neck squamous cell carcinoma cell lines

BackgroundGeneral control nonderepressible 2 (GCN2) senses amino acid deprivation and activates activating transcription factor 4 (ATF4), which regulates many adaptive genes. We evaluated the impact of AST-0513, a novel GCN2 inhibitor, on the GCN2-ATF4 pathway. Additionally, we evaluated the antitumor effects of AST-0513 in amino acid deprivation in head and neck squamous cell carcinoma (HNSCC) cell lines. MethodsGCN2 expression in HNSCC patient tissues was measured by immunohistochemistry. Five HNSCC cell lines (SNU-1041, SNU-1066, SNU-1076, Detroit-562, FaDu) grown under amino acid deprivation conditions, were treated with AST-0513. After AST-0513 treatment, cell proliferation was measured by CCK-8 assay. Flow cytometry was used to evaluate apoptosis and cell cycle phase. In addition, immunoblotting was performed to evaluate the effect of AST-0513 on the GCN2-ATF4 pathway, cell cycle arrest, and apoptosis. ResultsWe demonstrated that GCN2 was highly expressed in HNSCC patient tissues. AST-0513 inhibited the GCN2-ATF4 pathway in all five HNSCC cell lines. Inhibiting the GCN2-ATF4 pathway during amino acid deprivation reduced HNSCC cell proliferation and prevented adaptation to nutrient stress. Moreover, AST-0513 treatment led to p21 and Cyclin B1 accumulation and G2/M phase cycle arrest. Also, apoptosis was increased, consistent with increased bax expression, increased bcl-xL phosphorylation, and decreased bcl-2 expression. ConclusionA novel GCN2 inhibitor, AST-0513, inhibited the GCN2-ATF4 pathway and has antitumor activity that inhibits proliferation and promotes cell cycle arrest and apoptosis. Considering the high expression of GCN2 in HNSCC patients, these results suggest the potential role of GCN2 inhibitor for the treatment of HNSCC.

PUMA overexpression dissociates thioredoxin from ASK1 to activate the JNK/BCL-2/BCL-XL pathway augmenting apoptosis in ovarian cancer

ASK1-JNK signaling promotes mitochondrial dysfunction-mediated apoptosis, but the bridge between JNK and apoptosis is not fully understood. PUMA induces apoptosis through BAX/BAK. Our previous study suggests a therapeutic potential of PUMA for ovarian cancer. However, whether and how PUMA activates ASK1 remains unclear. Here, we found for the first time that PUMA activated ASK1 by dissociating thioredoxin (TRX) from ASK1, however, it neither interacted with ASK1 nor TRX. Furthermore, PUMA overexpression caused ROS release from mitochondrial. H2O2 significantly impaired the interaction of ASK1 with TRX, whereas ROS scavenger NAC effectively abrogated the H2O2 effect, partly rescued PUMA-interfered interaction of ASK1 with TRX, and also abolished ASK1 phosphorylation. Interestingly, PUMA could not impair the association of ASK1 with TRX-C32S or TRX-C35S, two TRX mutants which are no longer oxidized in response to ROS. We further showed that PUMA activated ASK1-JNK axis to phosphorylate BCL-2 and BCL-XL, further augmenting apoptosis of ovarian cancer cells. In vivo, PUMA adenovirus combined with paclitaxel significantly inhibited intrinsically cisplatin-resistant ovarian cancer growth, and caused phosphorylation of BCL-2 and BCL-XL. Our results from human ovarian cancer TMA chips also revealed a positive correlation between PUMA expression and the phosphorylation of BCL-2 and BCL-XL. More importantly, all patients had no distal metastasis, implying a possibly clinical significance. Collectively, our results reveal a new pro-apoptotic signal amplification mechanism for PUMA by which PUMA overexpression first induces ROS-mediated dissociation of TRX from ASK1, and then causes JNK activation-triggering BCL-2/BCL-XL phosphorylation, ultimately augmenting apoptosis in ovarian cancer.

Protease-activated receptor 2 stabilizes Bcl-xL and regulates EGFR-targeted therapy response in colorectal cancer.

The Bcl-2 homolog Bcl-xL is emerging as a key factor in tumorigenesis due to its prominent pro-survival and cell death-independent functions. However, the regulation of Bcl-xL by microenvironment and its implication in cancer therapy of colorectal carcinoma (CRC) are unclear. Here, we demonstrated that Bcl-xL expression was positively associated with protease-activated receptor 2 (PAR2) in CRC. Activation of PAR2 stabilized Bcl-xL protein in a proteasome-dependent manner, whereas E3 ligase RING finger protein 152 (RNF152) accelerated the ubiquitination and degradation of Bcl-xL. RNF152 silencing by specific siRNAs rescued the expression of Bcl-xL in PAR2-deficient cells. Moreover, RNF152 physically interacted with Bcl-xL, which was disturbed by PAR2 activation. Further studies with serial mutation of Bcl-xL revealed that phosphorylation of Bcl-xL at S145 reduced its binding affinity for RNF152 and stabilized Bcl-xL. Importantly, inhibition of PAR2 signaling by its gene silencing or specific chemical inhibitors increased apoptosis induced by different EGFR-targeted therapies. In patient-derived xenograft model, inhibition of PAR2 increased the response of CRC to different EGFR-targeted therapies. These results indicate that PAR2 stabilizes Bcl-xL by altering RNF152 signaling and that PAR2 inhibition sensitizes CRC to EGFR-targeted therapies in vivo.

Enhanced hepatocyte survival and anti-apoptosis via Akt by Diallyl trisulfide, augments hepatic regeneration through hydrogen sulfide in partially hepatectomized rats

BackgroundThe hepatic resecting is the final decision for advanced end-stage liver diseases. Live donor liver transplantation is employed in the medical scenario due to less donor availability. It is mandatory to understand the signaling and therapeutic strategy to enhance hepatic regeneration to avoid small-for-size syndrome. Hence this current study was employed to explore the importance of H2S signaling in hepatic regeneration. MethodsThe propargyl glycine (PAG) was used to inhibit H2S production, and garlic organosulfur, diallyl trisulfide (DATS), was an H2S donor in 70% partially hepatectomized male Wistar rats. The hepatectomized liver was allowed to regenerate for 48 h. The liver regeneration was scrutinized by analyzing the proliferative markers PCNA & Ki-67 and the apoptotic markers such as Bcl & Bcl-xL and hepatic antioxidants and serum liver markers. ResultsThe inhibition of H2S signaling displayed reduced hepatic regeneration rate and elevated liver index. The elevated serum liver marker enzymes such as AST, ALT & ALP further confirmed the hepatocyte damage. DATS administration to the regenerating liver demonstrated enhanced hepatic antioxidants (SOD, catalase, & GSH) and regenerative potential (PCNA & Ki-67). Histological findings supported the DATS enhanced liver regeneration. Western blot profiling of DATS supplied regenerating liver displayed increased expression of Bcl-xL and Akt phosphorylation with reduced Bad. ConclusionThis study concludes that the H2S signaling in the partial hepatectomized liver through DATS mediated Bcl-xL and Akt phosphorylation, enhancing hepatocyte survival and hepatic regeneration.

Naringin Induces Lysosomal Permeabilization and Autophagy Cell Death in AGS Gastric Cancer Cells.

Autophagy is a process of active programmed cell death, where a dying cell induces autophagosomes and subsequently regulated by degradative machinery. The aim of this study was to investigate the mechanism behind induction of autophagic cell death by Naringin flavonoid in AGS cancer cells. Growth inhibition of AGS cells showed downregulation of PI3K/Akt/mTOR signaling by Naringin treatment. Transmission electron microscopy observation showed swollen mitochondria and lysosome near peri-nuclear zone fused with autophagic vacuoles. Rapamycin pre-treatment with Naringin showed significant decrease in mTOR phosphorylation and increase in LC3B activation in AGS cells. Decrease in mTOR phosphorylation is associated with lysosomal function activation was observed by time-dependent treatment of Naringin. Induction of lysosomal membrane permeabilization (LMP) was observed by LAMP1 activation leading lysosomal cell death by releasing Cathepsin D from lysosomal lumen to cytosol. Naringin treated AGS cells showed up-regulating BH3 domain Bad, down-regulating Bcl-xL, and Bad phosphorylation and significant mitochondrial fluorescence intensity expression. Significant localization of mitochondria and LC3B activation was examined by person coefficient correlation. Activation of ERK1/2-p38 MAPKs and production of intracellular ROS has been observed over Naringin treatment. It has also been elucidated that pre-treatment with NAC inhibited mitochondria-LC3B colocalization, where ROS acted as upstream of ERK1/2-p38 MAPKs activation. Lysosomal cell death involvement has been evaluated by BAF A1 pre-treatment, inhibiting LAMP1, Cathepsin D, ROS, and blocking autophagolysosome in AGS cell death. Taken together, these findings show that, Naringin induced autophagy cell death involves LMP mediated lysosomal damage and BH3 protein Bad activation in AGS cancer cells.

Abstract 350: Bcl-xL-Ser14 Phosphorylation is Critical for Compensatory Cardiac Hypertrophy

Cardiac hypertrophy is an adaptive response at least initially since it reduces wall stress. Phosphorylation of Bcl-xL at Serine (Ser) 14, which dissociates Bcl-xL from Bax and promotes apoptosis, is increased in the heart within one hour after transverse aortic constriction (TAC)-induced pressure overload (PO). Here, we investigated how the increased Ser14-phosphorylation affects hypertrophy during PO. The Bcl-xL knock-in (KI) mice, in which Ser14 was replaced with Ala (Ser14Ala), exhibited a significantly greater mortality than wild-type (WT) mice (p=0.001) after TAC, with elevated end diastolic pressure (LVEDP, 34.6 vs 16.5 mmHg, p<0.05), impaired systolic function (EF, 38.2% vs 67.5%, p<0.001), and increased fibrosis (1.6-fold, p<0.001). The level of apoptosis was similar between the KI and WT mice one week after TAC, as assessed by TUNEL staining. The KI mice showed less cardiomyocyte and cardiac hypertrophy (cardiomyocyte size, 0.71-fold; heart weight/tibia length, 0.88-fold, both P<0.001). Adult cardiomyocytes isolated from the KI mice two days after TAC showed significantly lower contractility compared to those isolated from WT mice (0.32-fold, p<0.001). Mechanistically, gene set enrichment analysis using the RNA-seq data obtained from one-day TAC hearts showed that ion channel activity-related gene sets enriched in WT mice are downregulated in the KI mice. In line with this result, angiotensin II (Ang II) increased Ser14-phosphorylation and cytosolic Ca 2+ level in WT-MEFs in vitro , whereas MEFs isolated from the KI mice showed a significantly lower elevation of cytosolic Ca 2+ against Ang II. Proteomics analysis showed that Ankyrin, an anchoring protein that targets and stabilizes ion channels on the membrane, interacts with endogenous Bcl-xL in the heart. Taken together, these data suggest that phosphorylation of Bcl-xL at Ser14 is critical for augmenting Ca 2+ release from the sarcoplasmic reticulum by modulating the ion channel activity in part via Ankyrin against PO or AngII, thereby developing compensatory hypertrophy and maintaining contractile function. Our findings indicate that increasing the Bcl-xL-Ser14 phosphorylation during acute phase PO could be a potential therapeutic strategy for maintaining cardiac function.

Dynamic PGAM5 multimers dephosphorylate BCL-xL or FUNDC1 to regulate mitochondrial and cellular fate.

Mitochondria are highly dynamic organelles and respond to stress by changing their fission-fusion cycle, undergoing mitophagy, or releasing apoptotic proteins to initiate cell death. The molecular mechanisms that sense different stresses and coordinate distinct effectors still await full characterization. Here, we show that PGAM5, which exists in an equilibrium between dimeric and multimeric states, dephosphorylates BCL-xL to inhibit apoptosis or FUNDC1 to activate mitofission and mitophagy in response to distinct stresses. In vinblastine-treated cells, PGAM5 dephosphorylates BCL-xL at Ser62 to restore BCL-xL sequestration of BAX and BAK and thereby resistance to apoptosis. Selenite-induced oxidative stress increases the multimerization of PGAM5, resulting in its dissociation from BCL-xL, which causes increased BCL-xL phosphorylation and apoptosis. Once freed, the more multimeric and active PGAM5 dephosphorylates FUNDC1 to initiate mitofission and mitophagy. The reciprocal interaction of PGAM5 with FUNDC1 and BCL-xL, controlled by PGAM5 multimerization, serves as a molecular switch between mitofission/mitophagy and apoptosis.

Upregulated cyclin B1/CDK1 mediates apoptosis following 2-methoxyestradiol-induced mitotic catastrophe: Role of Bcl-XL phosphorylation

2-Methoxyestradiol is an endogenous nonpolar metabolite of 17β-estradiol with a strong antitubulin activity. Earlier we showed that 2-methoxyestradiol increases the level and activity of cyclin B1/CDK1, which subsequently induces mitotic prometaphase arrest. In the present study, we demonstrate that upregulation of cyclin B1/CDK1 is responsible for the increased phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-XL in 2-methoxyestradiol-induced, mitotically-arrested cancer cells. Additional analysis shows that only the increase in phosphorylation of Bcl-XL, but not Bcl-2, is associated with activation of the mitochondrial cell death pathway. We find that MAD2 is an important upstream mediator of the antitubulin function of 2-methoxyestradiol, resulting in activation of the MKK4-JNK1 pathway. JNK1 activation then leads to cyclin B1/CDK1 upregulation, which further increases Bcl-2 and Bcl-XL phosphorylation. Together, these results indicate that cyclin B1/CDK1 upregulation in cancer cells undergoing 2-methoxyestradiol-induced mitotic catastrophe causes apoptosis via Bcl-XL phosphorylation.

The JNK signaling pathway plays a key role in methuosis (non-apoptotic cell death) induced by MOMIPP in glioblastoma

BackgroundSynthetic indolyl- pyridinyl- propenones (IPPs) induce methuosis, a form of non-apoptotic cell death, in glioblastoma and other cancer cell lines. Methuosis is characterized by accumulation of cytoplasmic vacuoles derived from macropinosomes and late endosomes, followed by metabolic failure and rupture of the plasma membrane. However, not all IPPs that cause vacuolization are cytotoxic. The main goals of the present study were to identify key signaling pathways that contribute to methuosis induced by cytotoxic IPPs and to evaluate the anti-tumor potential of a prototype IPP in vivo.MethodsWe utilized metabolic flux analysis, glucose uptake, immunoblotting, and selective pharmacological inhibitors to compare the effects of closely related cytotoxic and non-cytotoxic IPPs in cultured glioblastoma cells. To determine whether the use of methuosis-inducing IPPs might be feasible in a therapeutic context, we quantified the distribution of our lead IPP compound, MOMIPP, in mouse plasma and brain, and tested its ability to inhibit tumor growth in an intracerebral glioblastoma xenograft model.ResultsThe cytotoxic IPP compound, MOMIPP, causes early disruptions of glucose uptake and glycolytic metabolism. Coincident with these metabolic changes, MOMIPP selectively activates the JNK1/2 stress kinase pathway, resulting in phosphorylation of c-Jun, Bcl-2 and Bcl-xL. At the same concentration, the non-cytotoxic analog, MOPIPP, does not activate these pathways. Pharmacologic inhibition of JNK activity promotes survival, even when cells are extensively vacuolated, but suppression of c-Jun transcriptional activity offers no protection. MOMIPP readily penetrates the blood-brain barrier and is moderately effective in suppressing progression of intracerebral glioblastoma xenografts.ConclusionsThe results suggest that interference with glucose uptake and induction of JNK-mediated phosphorylation of pro-survival members of the Bcl-2 family represent key events in the methuosis death process. In addition to providing new insights into the underlying molecular mechanism of methuosis, the results indicate that compounds of the cytotoxic IPP class may have potential for further development as therapeutic agents for brain tumors.

6-MOMIPP, a novel brain-penetrant anti-mitotic indolyl-chalcone, inhibits glioblastoma growth and viability.

3-(6-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (6-MOMIPP) is a novel indole-based chalcone that disrupts microtubules. The present study aims to define the mechanism through which 6-MOMIPP induces cell death and to evaluate the efficacy of the compound in penetrating the blood-brain barrier and inhibiting growth of glioblastoma xenografts. The effects of 6-MOMIPP were evaluated in cultured U251 glioblastoma cells, using viability, flow cytometry, and tubulin polymerization assays. Scintillation proximity and tubulin crosslinking methods were used to identify the binding site of 6-MOMIPP on tubulin, and western blots were performed to define the signaling pathways that contribute to cell death. LC/MS assays were used to study the pharmacokinetic behavior of 6-MOMIPP in mice. Subcutaneous and intracerebral xenograft models were utilized to assess the effects of 6-MOMIPP on growth of U251 glioblastoma in vivo. The findings indicate that 6-MOMIPP targets the colchicine site on β-tubulin. At concentrations ≥ 250nm, 6-MOMIPP induces mitotic arrest, caspase activation and loss of cell viability. Cells are protected by caspase inhibitors, pointing to an apoptotic mechanism of cell death. Loss of cell viability is preceded by activation of Cdk1(Cdc2) and phosphorylation of Bcl-2 and Bcl-xL. Inhibition of both events with a Cdk1 inhibitor prevents cell death. 6-MOMIPP has broad activity against the viability of multiple glioblastoma, melanoma and lung carcinoma cell lines. Viability of normal cells, including differentiated neurons, is not significantly affected at a drug concentration (1µM) that reduces viability in most cancer lines. Pharmacokinetic studies in mice show that concentrations of 6-MOMIPP in the brain mirror those in the plasma, indicating that 6-MOMIPP readily penetrates the blood-brain barrier. Studies with mice bearing human U251 glioblastoma xenografts demonstrate that 6-MOMIPP is effective in suppressing growth of subcutaneous and intracerebral tumors without causing general toxicity. The results indicate that 6-MOMIPP is anovel microtubule disruptor that targets the colchicine binding site on β-tubulin to induce mitotic arrest and cell death. The ability of 6-MOMIPP to penetrate the blood-brain barrier and inhibit growth of glioblastoma xenografts suggests that it warrants further preclinical evaluation as potential small-molecule therapeutic that may have advantages in treating primary and metastatic brain tumors.

Cytoprotective effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) is mediated by the inhibition of BAK-dependent mitochondrial apoptosis pathway.

The inhibitory mechanism of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) against apoptosis induced by the microtubule-damaging agents (MDAs), nocodazole (NOC) and 2-methoxyestradiol (2-MeO-E2), or a DNA-damaging agent (DDA), camptothecin (CPT) were investigated in human Jurkat T cell clones (J/Neo and J/BCL-XL cells). Treatment of J/Neo cells with NOC, 2-MeO-E2, or CPT caused cytotoxicity and apoptotic DNA fragmentation but these events were significantly attenuated in the presence of CMEP-NQ. Although not only MDA (NOC or 2-MeO-E2)-induced mitotic arrest, CDK1 activation, and BCL-2, BCL-XL and BIM phosphorylation, but also DDA (CPT)-induced S-phase arrest and ATM-CHK1/CHK2-p53 pathway activation were not or were barely affected in the presence of CMEP-NQ, the levels of anti-apoptotic BAG3 and MCL-1, which were markedly downregulated after MDA- or DDA-treatment, were rather elevated by CMEP-NQ. Under the same conditions, MDA- or DDA-induced mitochondrial apoptotic events including BAK activation, mitochondrial membrane potential (Δψm) loss, caspase-9 activation, and PARP cleavage were significantly inhibited by CMEP-NQ. While MDA- or DDA-induced sub-G1 peak and Δψm loss were abrogated in J/BCL-XL cells, MDA-induced mitotic arrest and DDA-induced S-arrest were more apparent in J/BCL-XL cells than in J/Neo cells. Simultaneously, the induced cell cycle arrest in J/BCL-XL cells was not significantly disturbed by CMEP-NQ. MDA- or DDA-treatment caused intracellular reactive oxygen species (ROS) production; however, MDA- or DDA-induced ROS production was almost completely abrogated in J/BCL-XL cells. MDA- or DDA-induced ROS production in J/Neo cells was significantly suppressed by CMEP-NQ, but the suppressive effect was hardly observed in J/BCL-XL cells. Together, these results show that CMEP-NQ efficiently protects Jurkat T cells from apoptotic cell death via the elevation of BAG3 and MCL-1 levels, which results in the inhibition of intrinsic BAK-dependent mitochondrial apoptosis pathway, as does the overexpression of BCL-XL.

Abstract 405: Bcl-xL Serine 14 Phosphorylation is Critical for Pressure Overload-induced Cardiac Hypertrophy

Cardiac hypertrophy is an adaptive response to various stresses, wherein Ca 2+ serves as a messenger to induce hypertrophy. We found that phosphorylation of Bcl-xL at Serine (Ser) 14 is significantly increased in the heart within one hour after transverse aortic constriction (TAC)-induced pressure overload. However, the functional significance of the increased Ser14-phosphorylation in hypertrophy is unknown. To address this question, wild-type (WT) and phosphorylation-resistant knock-in (KI) mice with an Alanine mutation at Ser14 in the Bcl-x gene were subjected to TAC. The KI mice had significantly higher mortality than WT mice (p=0.001), accompanied by heart failure (LVEDP, 34.6 vs 16.5 mmHg, p<0.05) with contractile dysfunction (EF, 38.2% vs 67.5%, p<0.001) and increased fibrosis (1.6-fold, p<0.001). Although the level of apoptosis was similar between the KI and WT mice one week after TAC, as assessed by TUNEL staining and cleaved caspase 3 and 9 expressions, cardiomyocyte and cardiac hypertrophy were inhibited in the KI mice (cardiomyocyte size, 0.32-fold; heart weight/tibia length, 0.61-fold, both P<0.001). Consistent with this finding, ANP and BNP gene expressions were significantly suppressed in the KI mice despite worse cardiac function (0.17-fold (p<0.001) and 0.69-fold (p<0.05), respectively). Adult cardiomyocytes isolated from the KI mice two days after TAC showed significantly reduced contractility compared to those isolated from WT mice (0.32-fold, p<0.001). Mechanistically, gene set enrichment analysis using the RNA-seq data obtained from the mouse heart one day after TAC revealed significantly altered channel activity in the KI mice. Although angiotensin II (AngII) increases Ser14-phosphorylation and cytosolic Ca 2+ level in WT-MEFs in vitro , MEFs isolated from the KI mice showed a significantly lower elevation of cytosolic Ca 2+ against AngII stimulation. Taken together, these data suggests that Bcl-xL-Ser14 phosphorylation promotes Ca 2+ release from the sarcoplasmic reticulum in response to pressure overload or AngII stimulation, leading to compensatory hypertrophy. Our findings indicate that targeting the Bcl-xL-Ser14 phosphorylation could be a potential therapeutic strategy for acute heart failure.

Eribulin alone or in combination with the PLK1 inhibitor BI 6727 triggers intrinsic apoptosis in Ewing sarcoma cell lines.

In this study, we investigated the molecular mechanisms of eribulin-induced cell death and its therapeutic potential in combination with the PLK1 inhibitor BI 6727 in Ewing sarcoma (ES). Here, we show that eribulin triggers cell death in a dose-dependent manner in a panel of ES cell lines. In addition, eribulin at subtoxic, low nanomolar concentrations acts in concert with BI 6727 to induce cell death and to suppress long-term clonogenic survival. Mechanistic studies reveal that eribulin monotherapy at cytotoxic concentrations and co-treatment with eribulin at subtoxic concentrations together with BI 6727 arrest cells in the M phase of the cell cycle prior to the onset of cell death. This mitotic arrest is followed by increased phosphorylation of BCL-2 and BCL-xL as well as downregulation of MCL-1, suggesting inactivation of these antiapoptotic BCL-2 family proteins. Consistently, eribulin monotherapy and eribulin/BI 6727 co-treatment trigger activation of BAX, a key proapoptotic BCL-2 family protein, and increase proteolytic activation of caspase-9 and -3. Importantly, overexpression of BCL-2 or addition of the broad-range caspase inhibitor zVAD.fmk significantly rescue eribulin- as well as eribulin/BI 6727-induced cell death. Together, these findings demonstrate that eribulin induces cell death via the intrinsic pathway of apoptosis in ES cells, both alone at cytotoxic concentrations and in combination with BI 6727 at subtoxic concentrations. Thus, our study highlights the therapeutic potential of eribulin for the treatment of ES alone or in rational combination therapies.

Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation during Mitosis Prevents Chromosome Instability and Aneuploidy in Normal Human Diploid Fibroblasts.

Bcl-xL proteins undergo dynamic phosphorylation/dephosphorylation on Ser49 and Ser62 residues during mitosis. The expression of Bcl-xL(S49A), (S62A) and dual (S49/62A) phosphorylation mutants in tumor cells lead to severe mitotic defects associated with multipolar spindle, chromosome lagging and bridging, and micro-, bi- and multi-nucleated cells. Because the above observations were made in tumor cells which already display genomic instability, we now address the question: will similar effects occur in normal human diploid cells? We studied normal human diploid BJ foreskin fibroblast cells expressing Bcl-xL (wild type), (S49A), (S49D), (S62A), (S62D) and the dual-site (S49/62A) and (S49/62D) mutants. Cells expressing S49 and/or S62 phosphorylation mutants showed reduced kinetics of cell population doubling. These effects on cell population doubling kinetics correlated with early outbreak of senescence with no impact on the cell death rate. Senescent cells displayed typical senescence-associated phenotypes including high-level of senescence-associated β-galactosidase activity, interleukin-6 (IL-6) secretion, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 activation as well as γH2A.X-associated nuclear chromatin foci. Fluorescence in situ hybridization analysis and Giemsa-banded karyotypes revealed that the expression of Bcl-xL phosphorylation mutants in normal diploid BJ cells provoked chromosome instability and aneuploidy. These findings suggest that dynamic Bcl-xL(S49) and (S62) phosphorylation/dephosphorylation cycles are important in the maintenance of chromosome integrity during mitosis in normal cells. They could impact future strategies aiming to develop and identify compounds that could target not only the anti-apoptotic domain of Bcl-xL protein, but also its mitotic domain for cancer therapy.

Mst1-mediated phosphorylation of Bcl-xL is required for myocardial reperfusion injury.

Mst1 is a central Ser-Thr kinase in the Hippo pathway, which promotes apoptosis and inhibits cell proliferation. We have shown previously that, in cardiomyocytes, oxidative stress activates Mst1 at mitochondria, where Mst1 phosphorylates Bcl-xL at Ser14, inducing dissociation of Bcl-xL from Bax and thereby promoting apoptosis. However, the functional significance of Ser14 phosphorylation of endogenous Bcl-xL in vivo remains elusive. We generated knockin (KI) mice in which Ser14 of Bcl-xL is replaced with Ala. KI mice were born at the expected Mendelian ratio, and adult KI mice exhibited normal cardiac morphology and function at baseline. However, KI mice were protected from myocardial ischemia/reperfusion (I/R) injury and exhibited reduced cardiomyocyte apoptosis. Although suppression of endogenous Mst1 also reduced I/R injury, there was no additive protective effect when Mst1 was inhibited in KI mice. The development of dilated cardiomyopathy induced by cardiac-specific overexpression of Mst1 was also ameliorated in KI mice. Lats2 and YAP, two other key components of the Hippo pathway, were not affected in KI mice. These results suggest that Ser14 phosphorylation of Bcl-xL plays an essential role in mediating both cardiomyocyte apoptosis and myocardial injury by acting as a key downstream mediator of Mst1 independently of the canonical Hippo pathway.

Manipulating the Hippo-Yap signal cascade in stem cells for heart regeneration.

The Hippo-Yap pathway was originally recognized as a crucial signal cascade controlling organ size, and more recently identified as an important component involved in the regulation of cardiomyocyte survival, proliferation, and regeneration. Negative stress responses can activate mammalian sterile 20-like kinase 1 (Mst1) to suppress protective autophagy and promote cardiomyocyte apoptosis via phosphorylation and inhibition of Bcl-xL. Moreover, decreased Yap activity and nuclear entry will decrease upon Mst1 activation, ultimately suppressing cardiomyocytes proliferation and regeneration. Based on these observations, there are potential therapeutic opportunities in cardiac structural and functional regeneration post myocardium infarction to be gained by manipulation of the Hippo-Yap signal cascade. This review will summarize the main components of the Hippo-Yap pathway and their molecular biological functions. It will then highlight the role of these signal modules in the acquisition of stem cell pluripotency, cardiogenic differentiation, cardiomyocyte proliferation and maturation, and mitochondrial biogenesis in cardiac stem cells. Finally, it will discuss the potential for future studies of Hippo-Yap pathway using induced pluripotent stem cell (iPSC) technology.

Mimicking Cdk2 phosphorylation of Bcl-xL at Ser73 results in caspase activation and Bcl-xL cleavage.

Cisplatin is a widely used chemotherapeutic agent, yet its efficacy is limited by nephrotoxicity. The severity of nephrotoxicity is associated with the extent of kidney cell death. Previously, we found that cisplatin-induced kidney cell death was dependent on Cdk2 activation, and inhibition of Cdk2 protected cells from cisplatin-induced apoptosis. Using an in vitro kination assay, we showed that Cdk2 phosphorylated Bcl-xL, an anti-apoptotic member of Bcl-2 family proteins, at serine 73. We also found that this phosphorylated Bcl-xL participated in cell death, as a phosphomimetic mutant of Bcl-xL at the serine 73 site (S73D-Bcl-xL) activated caspases. We now find that S73D-Bcl-xL was cleaved at D61 and D76, which are putative caspase cleavage sites, to generate 15-kDa and 12-kDa fragments. Unlike full-length Bcl-xL, these cleavage products of Bcl-xL were previously reported to be pro-apoptotic. We sought to determine whether these Bcl-xL fragments were necessary for the induction of cell death by S73D-Bcl-xL. Mutation of these caspase cleavage sites prevented the formation of the 15-kDa and 12-kDa Bcl-xL cleavage products, but apoptosis still persisted in a S73D modified Bcl-xL. Our findings show that Cdk2 phosphorylation of Bcl-xL at Ser73, but not the Bcl-xL cleavage products, is necessary and sufficient to induce cell death.