Reduced Bcl-2 Levels Research Articles

Abstract 4836: Ly6-neurotoxin1 is a potential target in pancreatic ductal adenocarcinoma cells

Abstract Ly6/neurotoxin1 (Lynx1) is a cholinergic transmission modulator, which was described as a tumor suppressor in lung cancer. In addition, the expression of Lynx1 was downregulated in rat PDAC cells colonizing rat liver. As cholinergic signaling via the vagus nerve may slow pancreatic tumor progression, we investigated the role of Lynx1 in human PDAC cell lines. Transient gene knockdown (KD) by siRNA was used to modulate the expression of Lynx1 in BXPC3 and Miapaca cells and KD was confirmed by qRT-PCR and western blot (WB). Resulting effects were determined by respective assays for proliferation, migration and colony formation. Induction of apoptosis was assessed by Hoechst and Annexin V-FITC staining. Induction of autophagy was determined by acridine orange and immunofluorescence (IF) staining for LC3b. Finally, we performed microarrays for gene expression in cells with Lynx1 KD and analyzed by WB cancer pathways of related signaling molecules. Following successful KD of Lynx1, the response of Miapaca and BXPC3 cells was altered regarding proliferation (-60% and -10%), migration (+200% and -10%) at 72 h after transfection and colony formation (-50% and -40%) at 9 d after transfection. Hoechst staining revealed increased rates of apoptosis in both cell lines. This was confirmed by Annexin V assay in Miapaca cells and these changes were associated with significantly reduced BCL2 levels. Autophagy was increased in both cell lines as were LC3b levels detected by IF. Alterations in mRNA expression, as assessed by microarray, were analyzed by IPA software using a 1.5 fold cutoff. Miapaca cells showed significant activation of 43 pathways including phospholipase C signaling, cholecystokinin/gastrin mediated signaling, and cell cycle regulation. In addition, five pathways showed uniform downregulation, i.e estrogen mediated S phase entry, sirtuin signaling, small cell lung cancer signaling, cyclins and cell cycle regulation, and ataxia-telangiectasia-mutated (ATM) signaling. In contrast to the more sensitive Miapaca cells, BXPC3 cells showed modulation of few pathways only: activation of G protein beta gamma signaling and osteoarthritis pathways, as well as downregulation of the sirtuin signaling pathway. At protein level, the mTOR pathway was downregulated in both cell lines (including phosphorylated forms of mTOR, Rictor, Raptor, PRAS40), as were the upstream regulators PI3K and p-AKT. In conclusion, KD of Lynx1 caused changes in gene product levels, which are related to cholinergic signal transmission as well as to DNA damage and repair systems. The observed decreased colony formation in Lynx1 KD cells is also indicative of defective cholinergic signaling. Reduced mTOR pathway signaling caused induction of autophagy. These findings suggest that Lynx1 is beneficial for regular cellular functions and its lack will contribute to increased apoptosis, DNA damage and autophagy. Citation Format: Doaa M. Ali, Nevena Nedyalkova, Michael Zepp, Martin R. Berger. Ly6-neurotoxin1 is a potential target in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4836.

A phase I trial of palbociclib in combination with dexamethasone in relapsed or refractory adult B-cell acute lymphoblastic leukemia (ALL).

TPS7065 Background: c-Myb is a DNA-binding transcription factor that is highly expressed in immature hematopoietic cells. c-Myb and its products are essential in regulating normal hematopoiesis and influencing leukemogenesis. Knockdown of c-Myb causes cell cycle arrest and apoptosis in pre-B-ALL cells. The effects of c-Myb depend on transcriptional regulation of CDK6 and Bcl-2. c-Myb-silenced Ph+ ALL cells exhibit Rb-dependent cell cycle arrest and apoptosis, both of which are rescued by ectopic expression of cyclin D3, CDK6, and Bcl-2 expression. Preclinical studies suggest that the cytotoxic activity of dexamethasone in ALL cells may be due to decreased c-Myb expression and reduced Bcl-2 levels. Thus, the novel combination of palbociclib, a small molecule CDK4/6 inhibitor, and dexamethasone is a logical approach for the treatment of B-cell ALL. Methods: This is a single arm, phase I, dose escalation study with a traditional 3+3 design. Adult patients with relapsed or refractory B-cell ALL are eligible. Patients with Ph+ ALL must be refractory to or intolerant of standard tyrosine kinase inhibitor therapy. Patients receive a 1-week lead-in of palbociclib alone followed by induction with 4 weeks of palbociclib and dexamethasone. If an adequate response is seen, patients move to maintenance therapy, which consists of 1 week of palbociclib plus dexamethasone followed by 3 weeks of palbociclib alone. Treatment continues until disease progression, dose limiting toxicity, or availability of an alternative therapy. The primary endpoints are dose limiting toxicity and maximum tolerated dose of palbociclib and dexamethasone. Correlative studies, which are performed on pretreatment, day +1 and day +8 samples, include RB phosphorylation and FOXM1 expression as measures of palbociclib activity; CD19+ cell gene expression profiling of (1) p21 expression as an indicator of cell cycle activity, (2) S-Phase, Annexin V/Caspase 3 activation as indicators of proliferation and apoptosis and (3) Myb and Bcl-2 expression as indicators of dexamethasone sensitivity. Cohort 1 is currently enrolling. Once a maximum tolerated dose is established, an expansion cohort is planned. Clinical trial information: NCT03472573.

BET inhibitors reduce cell size and induce reversible cell cycle arrest in AML.

Inhibitors of the bromodomain and extraterminal domain family (BETi) offer a new approach to treat hematological malignancies, with leukemias containing mixed lineage leukemia rearrangements being especially sensitive due to a reliance on the regulation of transcription elongation. We explored the mechanism of action of BETi in cells expressing the t(8;21), and show that these compounds reduced the size of acute myeloid leukemia cells, triggered a rapid but reversible G0 /G1 arrest, and with time, cause cell death. Meta-analysis of PRO-seq data identified ribosomal genes, which are regulated by MYC, were downregulated within 3 hours of addition of the BETi. This reduction of MYC regulated metabolic genes coincided with the loss of mitochondrial respiration and large reductions in the glycolytic rate. In addition, gene expression analysis showed that transcription of BCL2 was rapidly affected by BETi but this did not cause dramatic increases in cell death. Cell cycle arrest, lowered metabolic activity, and reduced BCL2 levels suggested that a second compound was needed to push these cells over the apoptotic threshold. Indeed, low doses of the BCL2 inhibitor, venetoclax, in combination with the BETi was a potent combination in t(8;21) containing cells. Thus, BET inhibitors that affect MYC and BCL2 expression should be considered for combination therapy with venetoclax.

Cytotoxic, Antiproliferative and Apoptosis-inducing Activity of L-Amino Acid Oxidase from Malaysian Calloselasma rhodostoma on Human Colon Cancer Cells.

The aim of this study was to investigate the cytotoxic, antiproliferative activity and the induction of apoptosis by L-amino acid oxidase isolated from Calloselasma rhodostoma crude venom (CR-LAAO) on human colon cancer cells. CR-LAAO was purified using three chromatographic steps: molecular exclusion using G-50 gel filtration resin, ion-exchange by MonoQ column and desalted on a G25 column. The purity and identity of the isolated CR-LAAO was confirmed by SDS-PAGE and LC-MS/MS. CR-LAAO demonstrated time- and dose-dependent cytotoxic activity on SW480 (primary human colon cancer cells) and SW620 (metastatic human colon cancer cells) with an EC50 values of 6 μg/ml and 7 μg/ml at 48 hr, respectively. Quantification of apoptotic cells based on morphological features demonstrated significant increase in apoptotic cell population in both SW480 and SW620 cells which peaked at 48 hr. Significant increase in caspase-3 activity and reduction in Bcl-2 levels were demonstrated following CR-LAAO treatment. These data provide evidence on the potential anticancer activity of CR-LAAO from the venom of C. rhodostoma for therapeutic intervention of human colon cancer.

Silencing forkhead box M1 promotes apoptosis and autophagy through SIRT7/mTOR/IGF2 pathway in gastric cancer cells

Forkhead box M1 (FOXM1) was initially identified as an oncogenic transcription factor, and multiple lines of evidence have demonstrated that FOXM1 is abundantly expressed and plays an irreplaceable role in several types of human cancers. Also, evidence has shown the association of FOXM1 with gastric carcinoma metastasis and patients prognosis; however, the potential role and molecular mechanism of FOXM1 in gastric cancer cell apoptosis are still obscure. The current study indicates that FOXM1 is highly expressed in a variety of gastric carcinoma cell lines, such as BGC823, MGC803, AGS, and SGC-7901, compared with the normal gastric mucosal epithelial cell lines CES-1. FOXM1 silence markedly inhibits AGS and SGC-7901 cell survival and proliferation, increases their apoptosis, and modulates apoptosis-related protein expression, including reduced Bcl-2 level and increased Bax and caspase-3 levels. Further study showed that FOXM1 depletion induced cell autophagy through increasing the level of beclin-1 and decreasing the P62 expression. We next corroborated that FOXM1 silence abolished the expression of Sirtuin 7 (SIRT7) and increased the level of insulin-like growth factor 2 (IGF2) and mammalian target of rapamycin (mTOR). Finally, our data documented that the SIRT7/mTOR/IGF2 pathway was involved in the function of FOXM1 in AGS cell growth and apoptosis. In conclusion, these results confirmed that FOXM1 is involved in gastric carcinoma progression via the SIRT7/mTOR/IGF2 pathway.

Cytotoxic, Anti-Proliferative and Apoptosis Activity of l-Amino Acid Oxidase from Malaysian Cryptelytrops purpureomaculatus (CP-LAAO) Venom on Human Colon Cancer Cells.

The aim of this study is to investigate the potential anti-cancer activity of l-amino acid oxidase (CP-LAAO) purified from the venom of Cryptelytrops purpureomaculatus on SW480 and SW620 human colon cancer cells. Mass spectrometry guided purification was able to identify and purify CP-LAAO. Amino acid variations identified from the partial protein sequence of CP-LAAO may suggest novel variants of these proteins. The activity of the purified CP-LAAO was confirmed with o-phenyldiamine (OPD)-based spectrophotometric assay. CP-LAAO demonstrated time- and dose-dependent cytotoxic activity and the EC50 value was determined at 13 µg/mL for both SW480 and SW620 cells. Significant increase of caspase-3 activity, reduction of Bcl-2 levels, as well as morphological changes consistent with apoptosis were demonstrated by CP-LAAO. Overall, these data provide evidence on the potential anti-cancer activity of CP-LAAO from the venom of Malaysian C. purpureomaculatus for therapeutic intervention of human colon cancer.

Alpha7 nicotinic acetylcholine receptor activation protects against myocardial reperfusion injury through modulation of autophagy

Alpha7 nicotinic acetylcholine receptor (α7nAChR) activation alleviates myocardial ischemia/reperfusion (MI/R) injury. However, the underlying mechanisms remain unclear. Here, we investigated the role of autophagy in α7nAChR-mediated cardioprotection and the molecular mechanisms involved. Activating α7nAChR with PNU-282987 at the initiation of reperfusion reduced myocardial infarct size in MI/R rats. PNU-282987 treatment also significantly inhibited MI/R-induced myocardial autophagy dysfunction as evidenced by the reduction of LC3-II/LC3-I ratio, Beclin-1 and p62 abundance. In addition, PNU-282987 treatment reduced hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury in vitro, accompanied with the inhibition of Beclin-1-associated autophagy and the restoration of autophagic flux. Interestingly, inhibiting autophagic flux attenuated α7nAChR-afforded improvement of mitochondrial function as well as inhibition of apoptosis in vitro. Mechanistically, co-administration of PNU-282987 with LY294002 (a PI3K inhibitor), AG490 (a JAK2 inhibitor) or Bcl-2 siRNA, but not compound C (an AMPK inhibitor), reduced Bcl-2 level and prevented the modulation of autophagy afforded by PNU-282987 in H/R cardiomyocytes. Collectively, these findings suggest that α7nAChR activation inhibits Beclin-1-associated autophagy dysfunction via the JAK2/Bcl-2 and PI3K/Bcl-2 cascades, leading to cardioprotection against MI/R injury.

Associations of Bcl-2 rs956572 genotype groups in the structural covariance network in early-stage Alzheimer\u2019s disease

BackgroundAlzheimer’s disease (AD) is a complex neurodegenerative disease, and genetic differences may mediate neuronal degeneration. In humans, a single-nucleotide polymorphism in the B-cell chronic lymphocytic leukemia/lymphoma-2 (Bcl-2) gene, rs956572, has been found to significantly modulate Bcl-2 protein expression in the brain. The Bcl-2 AA genotype has been associated with reduced Bcl-2 levels and lower gray matter volume in healthy populations. We hypothesized that different Bcl-2 genotype groups may modulate large-scale brain networks that determine neurobehavioral test scores.MethodsGray matter structural covariance networks (SCNs) were constructed in 104 patients with AD using T1-weighted magnetic resonance imaging with seed-based correlation analysis. The patients were stratified into two genotype groups on the basis of Bcl-2 expression (G carriers, n = 76; A homozygotes, n = 28). Four SCNs characteristic of AD were constructed from seeds in the default mode network, salience network, and executive control network, and cognitive test scores served as the major outcome factor.ResultsFor the G carriers, influences of the SCNs were observed mostly in the default mode network, of which the peak clusters anchored by the posterior cingulate cortex seed determined the cognitive test scores. In contrast, genetic influences in the A homozygotes were found mainly in the executive control network, and both the dorsolateral prefrontal cortex seed and the interconnected peak clusters were correlated with the clinical scores. Despite a small number of cases, the A homozygotes showed greater covariance strength than the G carriers among all four SCNs.ConclusionsOur results suggest that the Bcl-2 rs956572 polymorphism is associated with different strengths of structural covariance in AD that determine clinical outcomes. The greater covariance strength in the four SCNs shown in the A homozygotes suggests that different Bcl-2 polymorphisms play different modulatory roles.

Ablation of Akt2 prevents paraquat-induced myocardial mitochondrial injury and contractile dysfunction: Role of Nrf2

Paraquat is a quaternary nitrogen herbicide triggering oxidative stress, mitochondrial damage and multi-organ injuries including hearts. To date, effective measure to combat paraquat toxicity is still lacking. Recent evidence has revealed a role for Akt in cardiac homeostasis. To this end, this study was designed to examine the role of Akt2 in acute paraquat exposure-induced cardiac contractile and mitochondrial injury using a unique murine model of Akt2 knockout. Cardiac contractile and intracellular Ca2+ properties were evaluated. Mitochondrial integrity, ROS production, lipid peroxidation, ER stress and apoptosis were evaluated using aconitase assay, citrate synthase activity, DHE staining, mitochondrial permeation pore opening, 4-hydroxy-nonenal (4-HNE) and Western blot. Our results revealed compromised echocardiographic, contractile and intracellular Ca2+ handling properties along with overt mitochondrial damage (reduced levels of PGC-1α, aconitase, citrate synthase activity and NAD+) in mice challenged with paraquat (45mg/kg, single injection, i.p.), the effects of which were attenuated by Akt ablation. Paraquat triggered O2− production, lipid peroxidation and apoptosis as evidenced by increased DHE staining, 4-HNE, caspase-3 activity, Bax and reduced Bcl-2 levels in association with unchanged ER stress. The redox signaling molecule nuclear factor erythroid related factor 2 (Nrf2) was upregulated in response to paraquat challenge. Findings from in vitro study revealed that stimulation of Nrf2 using sulforaphane (10μM) negated Akt2 ablation-offered beneficial effect against paraquat whereas inhibition of Nrf2 using luteolin (20μM) mimicked Akt2 ablation-induced beneficial effect against paraquat challenge. Taken together, our data indicate that Akt2 ablation may protect against paraquat toxicity-induced cardiac contractile defect and apoptosis possibly via regulation of Nrf2 activation and mitochondrial homeostasis.

Expression patterns of sirtuin 1-AMPK-autophagy pathway in chronic colitis and inflammation-associated colon neoplasia in IL-10-deficient mice

BackgroundInterleukin-10-deficient (IL-10 (−/−)) mice spontaneously develop chronic colitis and adenocarcinoma through the dysplasia sequence. Autophagy malfunction is associated to inflammatory bowel disease (IBD) and colorectal cancer (CRC) pathogenesis. Autophagy is regulated by silent information regulator-1 (SIRT1), a NAD+-dependent histone deacetylase. Our aim was to investigate the expression changes of SIRT1-AMPK-autophagy pathway in the progression from chronic colitis to CRC. MethodsWe studied C57BL/6-IL-10-deficient mice between 6 and 18weeks of age. Macroscopic and histological analysis, and characterization of inflammatory and tumor biomarkers were performed. ResultsIL-10-deficient mice developed colitis from the age of 6weeks onward. The severity of inflammation and dysplasia, and the proliferative activity increased gradually with age. IL-10 (−/−) mice were characterized by improved levels of TNF-α and decreased expression of SIRT1. Moreover, our findings show an increase in p-AMPK expression and an activation of the autophagy in IL-10 (−/−) mice from all stages, evidenced by the accumulation of LC3-II protein, the increase in Beclin 1 expression and the reduction in Bcl-2 levels. ConclusionsSIRT1-AMPK-autophagy pathway may be involved in the maintenance of chronic inflammation and dysplasia development in the IL-10-deficient mice model. Modulation of this pathway could be a novel strategy for IBD and CRC treatment.

Abstract B25: BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia

Abstract Introduction: Despite the improved survival of patients with T-cell acute lymphoblastic leukemia (T-ALL), therapy-resistant or refractory T-ALL remains a major clinical challenge. Recently, we and others reported promising therapeutic activity for ABT-199, a highly specific inhibitor of the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), especially in immature T-ALL. Nevertheless, the occurrence of dose-limiting toxicities and the emergence of therapy resistance provides a rationale for the evaluation of ABT-199 as part of combination therapies. Indeed, previous studies have shown that ABT-199 can synergize with conventional chemotherapeutic agents in human T-ALL. BRD4 is an important bromodomain and extra-terminal (BET) protein that facilitates the recruitment of the transcriptional apparatus to acetylated chromatin. Small molecule inhibitors that target BET-bromodomain proteins, such as JQ1, can broadly affect oncogenic gene transcription and have recently emerged as powerful anti-cancer agents in a variety of hematological malignancies, including T-ALL. Here, we evaluated JQ1 and ABT-199 combination therapy and provide in vitro and in vivo evidence that combined BCL-2 and BRD4 inhibition synergistically affects leukemic tumor growth in T-ALL. Experimental Procedures: In vitro synergism between ABT-199 and JQ1 in a panel of T-ALL cell lines was determined by the combination index (CI) based on cell viability 48h after compound administration. For in vivo evaluation of the ABT-199/JQ1 combination therapy, luciferase-labeled T-ALL cells were administered by tail vein injection in immune compromised NOD scid gamma mice. Upon engraftment of leukemic cells, mice were divided into four groups and treated daily with 50mg ABT-199/kg body weight and/or 50mg JQ1/kg body weight for 13 days. The percentage of leukemic cells in the blood and bone marrow after treatment was analyzed using FACS analysis upon staining with an anti-human CD45 antibody. Results: In vitro evaluation of ABT-199 and JQ1 combination therapy revealed strong synergism (CI<0.5) in the T-ALL cell lines LOUCY, DND-41, ALL-SIL and TALL-1. In contrast, no or only slight synergistic effects (CI>0.5) were observed in other T-ALL lines, including KOPTK1, JURKAT, PEER and HPB-ALL. Notably, the degree of synergism was not defined by the IC50 values for ABT-199 or JQ1, but was significantly correlated with BCL-2 expression levels. Next, in vivo drug treatment experiments using luciferase-positive LOUCY T-ALL cells confirmed the synergistic activity of the JQ1/ABT-199 combination therapy as evaluated by bioluminescence, percentage of leukemic blasts in blood or bone marrow and spleen size. Interestingly, although ABT-199 strongly reduced the leukemic burden during the first days of treatment, the therapeutic response of single ABT-199 therapy was not durable. In contrast, JQ1/ABT-199 combination therapy resulted in a sustained therapeutic response associated with tumor regression. As expected, combination therapy resulted in enhanced levels of cleaved PARP and increased caspase activity in synergistic T-ALL cell lines, in line with induction of cellular apoptosis upon ABT-199/JQ1 treatment. To better understand the molecular mechanism by which JQ1 enhances the anti-leukemic properties of ABT-199, we defined its effect on gene expression in T-ALL cell lines. Notably, this analysis showed that the balance of pro- and anti-apoptotic BCL2 family factors was affected by JQ1 treatment. For example, JQ1 treatment caused a significant reduction in BCL2 levels, but also induced a concomitant increase in anti-apoptotic MCL1. In addition, pro-apoptotic factors, such as BIM, were also upregulated upon JQ1 treatment. Conclusions: Here, we show that BET-bromodomain inhibition can sensitize BCL2-positive human T-ALL to ABT-199 treatment by interfering with the balance of pro- and anti-apoptotic BCL2 family members. Citation Format: Sofie Peirs, Filip Matthijssens, Tim Pieters, Niels Vandamme, Geert Berx, Bruce Poppe, Pieter Van Vlierberghe. BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B25.

Abstract B39: BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia

Abstract Introduction: Despite the improved survival of patients with T-cell acute lymphoblastic leukemia (T-ALL), therapy-resistant or refractory T-ALL remains a major clinical challenge. Recently, we and others reported promising therapeutic activity for ABT-199, a highly specific inhibitor of the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), especially in immature T-ALL. Nevertheless, the occurrence of dose-limiting toxicities and the emergence of therapy resistance provides a rationale for the evaluation of ABT-199 as part of combination therapies. Indeed, previous studies have shown that ABT-199 can synergize with conventional chemotherapeutic agents in human T-ALL. BRD4 is an important bromodomain and extra-terminal (BET) protein that facilitates the recruitment of the transcriptional apparatus to acetylated chromatin. Small molecule inhibitors that target BET-bromodomain proteins, such as JQ1, can broadly affect oncogenic gene transcription and have recently emerged as powerful anti-cancer agents in a variety of hematological malignancies, including T-ALL. Here, we evaluated JQ1 and ABT-199 combination therapy and provide in vitro and in vivo evidence that combined BCL-2 and BRD4 inhibition synergistically affects leukemic tumor growth in T-ALL. Experimental Procedures: In vitro synergism between ABT-199 and JQ1 in a panel of T-ALL cell lines was determined by the combination index (CI) based on cell viability 48h after compound administration. For in vivo evaluation of the ABT-199/JQ1 combination therapy, luciferase-labeled T-ALL cells were administered by tail vein injection in immune compromised NOD scid gamma mice. Upon engraftment of leukemic cells, mice were divided into four groups and treated daily with 50mg ABT-199/kg body weight and/or 50mg JQ1/kg body weight for 13 days. The percentage of leukemic cells in the blood and bone marrow after treatment was analyzed using FACS analysis upon staining with an anti-human CD45 antibody. Results: In vitro evaluation of ABT-199 and JQ1 combination therapy revealed strong synergism (CI<0.5) in the T-ALL cell lines LOUCY, DND-41, ALL-SIL and TALL-1. In contrast, no or only slight synergistic effects (CI>0.5) were observed in other T-ALL lines, including KOPTK1, JURKAT, PEER and HPB-ALL. Notably, the degree of synergism was not defined by the IC50 values for ABT-199 or JQ1, but was significantly correlated with BCL-2 expression levels. Next, in vivo drug treatment experiments using luciferase-positive LOUCY T-ALL cells confirmed the synergistic activity of the JQ1/ABT-199 combination therapy as evaluated by bioluminescence, percentage of leukemic blasts in blood or bone marrow and spleen size. Interestingly, although ABT-199 strongly reduced the leukemic burden during the first days of treatment, the therapeutic response of single ABT-199 therapy was not durable. In contrast, JQ1/ABT-199 combination therapy resulted in a sustained therapeutic response associated with tumor regression. As expected, combination therapy resulted in enhanced levels of cleaved PARP and increased caspase activity in synergistic T-ALL cell lines, in line with induction of cellular apoptosis upon ABT-199/JQ1 treatment. To better understand the molecular mechanism by which JQ1 enhances the anti-leukemic properties of ABT-199, we defined its effect on gene expression in T-ALL cell lines. Notably, this analysis showed that the balance of pro- and anti-apoptotic BCL2 family factors was affected by JQ1 treatment. For example, JQ1 treatment caused a significant reduction in BCL2 levels, but also induced a concomitant increase in anti-apoptotic MCL1. In addition, pro-apoptotic factors, such as BIM, were also upregulated upon JQ1 treatment. Conclusions: Here, we show that BET-bromodomain inhibition can sensitize BCL2-positive human T-ALL to ABT-199 treatment by interfering with the balance of pro- and anti-apoptotic BCL2 family members. Citation Format: Sofie Peirs, Filip Matthijssens, Tim Pieters, Niels Vandamme, Geert Berx, Bruce Poppe, Pieter Van Vlierberghe. BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B39.

Abstract B58: BIM, a key player in apoptosis induction and acquired venetoclax resistance in follicular lymphoma cells

Abstract The chromosomal translocation t(14;18) in follicular lymphoma (FL) cells is a primary oncogenic event resulting in BCL-2 over-expression. BCL-2 is not directly responsible for growth of tumor cells, but it protects them against death by binding pro-apoptotic proteins. This study investigates in vitro and in vivo activity of the BH3 mimetic venetoclax (ABT-199) and mechanisms of acquiring resistance in FL. Primary FL cells with similar expression of anti-apoptotic proteins, such as BCL-2 and MCL-1, showed variable sensitivity to treatment, which correlated with expression of BIM. To further study this correlation we used two t(14;18)+ cell lines, WSU-FSCCL and FC-TxFL2. As with primary cells, FC-TxFL2 cells with higher expression of BIM protein were more sensitive to venetoclax-induced apoptosis. The treatment caused dissociation of BCL-2/ BIM complexes, decreased mitochondrial potential, activation of caspase-3 and caspase-7, and PARP cleavage. Interestingly the population of cells that survived venetoclax treatment showed an increase of phospho-ERK1/2 and phospho-BIM (S69), as well as a decrease of total BIM levels. Venetoclax treatment of murine xenografts of FC-TxFL2 cells resulted in significant delay in tumor growth. However, viable cells isolated from treated tumors showed a decrease of sensitivity to venetoclax exposure (IC50 increased almost 10 times) in comparison with the cells from the mice treated with vehicle only. These cells contained higher levels of MCL-1 and lower levels of BIM protein compared to control cells. BCL-2 levels remained unchanged. Venetoclax resistant cells prepared in vitro by continuous treatment with increasing concentrations of venetoclax initially showed elevated levels of p-AKT, p-FOX01/3a, MCL-1, dissociation of BIM/BCL-2/BECLIN1 complex, decrease of p62 level (a sign of an increased autophagy) together with a slight decline of BIM expression. After a stable resistant cell line was established, a significant reduction of BCL-2 levels and almost total absence of BIMEL, BIML and BIMS isoforms was observed. Detailed knowledge of venetoclax action and the mechanisms for acquired resistance in t(14;18)+ cells may help inform biomarker strategies and dosing schedules in future clinical trials. Inhibition of pathways activated in order to protect the FL cells (e.g. by down-regulating BIM (ERK or AKT pathway) or by autophagy) can significantly augment venetoclax potency and decrease incidence of acquired resistance. Citation Format: Juraj Bodo, Xiaoxian Zhao, Andrew J. Souers, Darren C. Phillips, Mitchell R. Smith, Eric D. Hsi. BIM, a key player in apoptosis induction and acquired venetoclax resistance in follicular lymphoma cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B58.

Mechanisms Underlying Taurine Protection Against Glutamate-Induced Neurotoxicity

Taurine appears to exert potent protections against glutamate (Glu)-induced injury to neurons, but the underlying molecular mechanisms are not fully understood. The possibly protected targets consist of the plasma membrane and the mitochondrial as well as endoplasmic reticulum (ER) membranes. Protection may be provided through a variety of effects, including the prevention of membrane depolarization, neuronal excitotoxicity and mitochondrial energy failure, increases in intracellular free calcium ([Ca2+]i), activation of calpain, and reduction of Bcl-2 levels. These activities are likely to be linked spatially and temporally in the neuroprotective functions of taurine. In addition, events that occur downstream of Glu stimulation, including altered enzymatic activities, apoptotic pathways, and necrosis triggered by the increased [Ca2+]i, can be inhibited by taurine. This review discusses the possible molecular mechanisms of taurine against Glu-induced neuronal injury, providing a better understanding of the protective processes, which might be helpful in the development of novel interventional strategies.

Effect of Chronic Melatonin Administration on Several Physiological Parameters from Old Wistar Rats and Samp8 Mice

The effect of melatonin administration on age-induced alterations in hepatocytes, central nervous system, immune system, and skin are reviewed. Twenty-two-month-old Wistar rats and SAMP8 (senescence prone) mice of 10 months of age were used as experimental models. Wistar rats were analyzed untreated or after the chronic administration of melatonin at a dose of 1 mg/kg/day in the drinking water for 10 weeks. At the end of the treatment period, the various parameters were investigated. Results were compared with those of 2-month-old controls. In hepatocytes, aging induced a significant increase in oxidative stress, inflammation, and apoptosis when compared to young animals. Melatonin administration significantly ameliorated all these age-related changes. The impairment of the cardiovascular system with aging appears to contribute to the increased morbidity and mortality of the aged subjects. The process was investigated in SAMP8 mice of 10 months of age. Melatonin was provided for 30 days at two different dosages (1 mg/kg/day and 10 mg/kg/day), also in the drinking water. After treatment, the expression of inflammatory mediators (tumor necrosis factor-α, interleukin 1 and 10, NFκBp50 and NFκBp52), apoptosis markers (BAD, BAX, and Bcl2), and parameters related to oxidative stress (heme oxygenases 1 and 2, endothelial and inducible nitric oxide synthases) were determined in the heart. Inflammation as well as oxidative stress and apoptosis markers were increased in old SAMP8 males, as compared to young controls. After treatment with melatonin, these age-altered parameters were partially reversed. The results suggest that oxidative stress and inflammation increase with aging and that chronic treatment with melatonin is able to reduce these parameters. In the skin, a reduction of epidermal thickness together with a marked increase of the hypodermis with great fat accumulation was observed in old rats, together with an increase in caspase 3, 8 of nucleosomes and LPO and a reduction in Bcl2 levels in the cultured keratinocytes. Melatonin treatment was able to reduce the fat content of the hypodermis and to increase Bcl2 and reduce nucleosomes, caspases, and LPO in keratinocytes. Melatonin administration exerts a beneficial effect against age-induced changes in several physiological parameters in Wistar rats and SAMP 8 mice.

Apoptosis induced by berbamine in retinoblastoma HXO-RB44 cells

[Objective] To investigate the effect of berbamine (BBM) on the proliferation and apoptosis of retinoblastoma (RB) HXO-RB44 cells and its possible mechanism in vitro.[Methods] RB cells in logarithmic growth phase were divided into BBM treated group and control group.RB cells in BBM treated group were cultured with different concentrations of BBM (2,4,8,16 and 32 mg/L) for 24,48 and 72 hours,respectively.The proliferation was assayed by methyl Thiazolyl tetrazolium (MTT).RB cells were cultured with different concentrations of BBM (4,8 and 16 mg/L) for 24 hours.The early apoptotic rates were detected by flow cytometry; the expression of bcl-2 and Bax were measured by enzyme-linked immunosorbent assay (ELISA) and the activity of Caspase-3 was detected by colorimetric assay.[Results] BBM could obviously inhibit the proliferation of RB cells in a time-and dose-dependent manner (24 hours:F=70.547,P＜0.01; 48 hours:F=603.438,P＜0.01; 72 hours:F=577.521,P＜0.01).The 1C50value at 24,48 and 72 hours were 25.26,10.94 and 6.25 mg/L,respectively.Necrosis rates of control group andBBM treated group were (1.25±0.45)％,(4.10±2.95)％,(4.39±0.21)％ and (10.54±4.38) ％ respectively; the difference between two groups was statistically significant (F =6.527,P＜0.05).Apoptotic and necrosis rates in advanced stage of control group and BBM treated group were (2.13±0.71)％,(5.45 ± 2.31)％,(9.86 ± 3.18)％ and ( 11.10 ± 1.70)％,respectively.The difference between two groups was statistically significant (F =10.845,P＜0.05).Early apoptotic rates of control group andBBM treated group were (0.51±0.26)％,(2.68±0.35)％,(5.97±0.50)％ and (11.22±1.17) ％,respectively.The difference between two groups was statistically significant (F=144.976,P＜0.01).In addition,BBM dose-dependently reduced bcl-2 level and increased Bax expression,causing the reduction of the bcl-2/Bax protein ratio as well as increased the Caspase-3 activity in RB cells remarkably (bcl2:F=835.726,P＜0.01; bax:F=111.963,P＜0.01; Caspase-3:F=298.058,P＜0.01).[Conclusion]s BBM can inhibit the proliferation and induce apoptosis or necrosis of RB cells in vitro,downregulating the expression of bcl-2,up-regulating the expression of Bax.Along with increased Caspase-3activity these may be the apoptotic mechanisms. Key words: Retinoblastoma/drug therapy; Berbamine/therapeutic use; Apoptosis; Cell proliferation

MiR-143 Overexpression Impairs Growth of Human Colon Carcinoma Xenografts in Mice with Induction of Apoptosis and Inhibition of Proliferation

BackgroundMicroRNAs (miRNAs) are aberrantly expressed in human cancer and involved in the (dys)regulation of cell survival, proliferation, differentiation and death. Specifically, miRNA-143 (miR-143) is down-regulated in human colon cancer. In the present study, we evaluated the role of miR-143 overexpression on the growth of human colon carcinoma cells xenografted in nude mice (immunodeficient mouse strain: N: NIH(s) II-nu/nu).Methodology/Principal FindingsHCT116 cells with stable miR-143 overexpression (Over-143) and control (Empty) cells were subcutaneously injected into the flanks of nude mice, and tumor growth was evaluated over time. Tumors arose ∼ 14 days after tumor cell implantation, and the experiment was ended at 40 days after implantation. miR-143 was confirmed to be significantly overexpressed in Over-143 versus Empty xenografts, by TaqMan® Real-time PCR (p<0.05). Importantly, Over-143 xenografts displayed slower tumor growth compared to Empty xenografts from 23 until 40 days in vivo (p<0.05), with final volumes of 928±338 and 2512±387 mm3, respectively. Evaluation of apoptotic proteins showed that Over-143 versus Empty xenografts displayed reduced Bcl-2 levels, and increased caspase-3 activation and PARP cleavage (p<0.05). In addition, the incidence of apoptotic tumor cells, assessed by TUNEL, was increased in Over-143 versus Empty xenografts (p<0.01). Finally, Over-143 versus Empty xenografts displayed significantly reduced NF-κB activation and ERK5 levels and activation (p<0.05), as well as reduced proliferative index, evaluated by Ki-67 immunohistochemistry (p<0.01).ConclusionsOur results suggest that reduced tumor volume in Over-143 versus Empty xenografts may result from increased apoptosis and decreased proliferation induced by miR-143. This reinforces the relevance of miR-143 in colon cancer, indicating an important role in the control of in vivo tumor progression, and suggesting that miR-143 may constitute a putative novel therapeutic tool for colon cancer treatment that warrants further investigation.

Aristolochic acid induces apoptosis of human umbilical vein endothelial cells in vitro by suppressing PI3K/Akt signaling pathway

To investigate whether aristolochic acid (AA) induced the apoptosis of human umbilical vein endothelial cells (HUVECs) in vitro and the underlying mechanism. HUVECs were treated with AA (5, 10 or 20 μg/mL) for 12, 24 and 48 h. Cell viabilities were determined with MTT assay. Hoechst 33258 staining and flow cytometry were used to examine the apoptosis of HUVECs. Western blotting was used to evaluate Akt phosphorylation. Bcl-2 and Bax levels were measured using Western blotting and RT-PCR assays. Treatment of HUVECs with AA significantly decreased the cell viabilities in dose- and time-dependent manners. Morphological changes of apoptosis were observed in AA-treated cells. AA inhibited Akt activation, which was attenuated by pretreatment of the cells with LY294002 (20 μmol/L) or wortmannin (50 nmol/L). Furthermore, AA reduced Bcl-2 levels and increased Bax levels. AA induces apoptosis of HUVECs in vitro via the PI3K/Akt signaling pathway and by modulating the ratio Bcl-2 and Bax.

Arsenic‐trioxide‐induced apoptosis of chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) cells are characterized by defective apoptosis which leads to their extended survival. Arsenic trioxide (As(2)O(3)) was reported to induce cell death in many malignant cells, but the specific pathway of As(2)O(3)-induced apoptosis/necrosis remains controversial. Our aim was to determine if As(2)O(3) kills CLL cells through apoptosis and whether this is accompanied by reduction in Bcl-2 levels. Cells from nine patients with CLL were incubated with increasing concentrations of As(2)O(3) (0.5-2 microM) for 2, 7, or 14 days. Cells viability was measured using Alamar Blue assay and apoptosis using human Annexin V-FITC and propidium iodine (PI) kit (BMS306FI; Bender MedSystems, Vienna, Austria). Intracellular Bcl-2, Bax, and caspase-3 levels were measured by flow cytometry. As(2)O(3) significantly reduced CLL cell viability (P < 0.01) and induced apoptotic cell death in a time- and dose-dependent manner. After 7 days, CLL cells showed a significant decrease in mean fluorescence intensity (MFI) of Bcl-2 on flow cytometry study. Bax and caspase-3 levels showed significant decrease in MFI only after prolonged incubations (7 and 14 days) and mostly at higher concentrations of As(2)O(3). The mechanism underlying the reduction in viability of CLL cells incubated with As(2)O(3) is mediated by induction of apoptosis maybe through the down-regulation of Bcl-2. Further studies are needed to elucidate the potential therapeutic role of As(2)O(3) in CLL.

Cytosolic prion protein induces apoptosis in human neuronal cell SH-SY5Y via mitochondrial disruption pathway

Different neurodegenerative disorders like prion disease, is caused by protein misfolding conformers. Reverse-transfected cytosolic prion protein (PrP) and PrP expressed in the cytosol have been shown to be neurotoxic. To investigate the possible mechanism of neurotoxicity due to accumulation of PrP in cytosol, a PrP mutant lacking the signal and GPI (CytoPrP) was introduced into the SH-SY5Y cell. MTT and trypan blue assays indicated that the viability of cells expressing CytoPrP was remarkably reduced after treatment of MG-132. Obvious apoptosis phenomena were detected in the cells accumulated with CytoPrP, including loss of mitochondrial transmembrane potential, increase of caspase-3 activity, more annexin V/PI-double positive-stained cells and reduced Bcl-2 level. Moreover, DNA fragmentation and TUNEL assays also revealed clear evidences of late apoptosis in the cells accumulated CytoPrP. These data suggest that the accumulation of CytoPrP in cytoplasm may trigger cell apoptosis, in which mitochondrial relative apoptosis pathway seems to play critical role.