Expression Levels Of B-cell Lymphoma-2 Research Articles

Characterization of Mechanisms of Acquired Venetoclax-Insensitivity in B-Cell Precursor Acute Lymphoblastic Leukemia

Deregulation of cell death pathways is a hallmark of many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Different pro- and anti-apoptotic molecules control apoptosis signaling. While pro-apoptotic BCL-2 homology domain 3 (BH3) proteins induce cellular death, apoptosis induction is counter-regulated by anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2). Therefore, inhibition of anti-apoptotic molecules has been developed as therapeutic strategy. Venetoclax (VEN) selectively binds to BCL-2, which leads to the release of pro-apoptotic molecules such as BIM resulting in apoptosis induction. In BCP-ALL, we and others showed preclinical activity of VEN and first clinical trials have started to evaluate VEN in ALL. Despite high activity, resistance can be acquired over time and acquisition of BCL2 mutations has been reported in CLL patients. In this study, we modeled VEN resistance in BCP-ALL and investigated underlying mechanisms in order to identify potential strategies to overcome VEN insensitivity. Starting from the BCP-ALL cell line RS4;11, five parallel VEN insensitive lines were generated by exposure to increasing concentrations of VEN over time (49 passages, over 8 months of continuous treatment). Simultaneously, five control lines were exposed to corresponding concentrations of solvent (DMSO). Measuring half maximal effective concentrations (EC50) over time showed increasing EC50 values from 4 nM to 26.2 µM in all VEN treated lines, reflecting acquired resistance in our model. Importantly, no mutations of the BCL2 gene were identified by sequencing at high coverage (Illumina AmpliSeq, 650- to 5951-fold), excluding acquisition of BCL2 mutations as mechanism of resistance. Next, we assessed expression levels of the mitochondrial apoptosis regulators BCL-2, MCL-1 and BCL-XL by western blot analysis. Comparing VEN insensitive to control cell lines, no differences in expression of the target molecule BCL-2 and similar levels of BCL-XL were observed. Most interestingly, all five VEN insensitive lines showed significant up-regulation of MCL-1 compared to all control lines. We next investigated the dependence of apoptosis signaling on different BCL-2 family members by exposing ALL cells to synthetic BH3-only peptides, which specifically bind to different regulators of mitochondrial apoptosis signaling (BH3-profiling), followed by analysis of apoptosis induction. Interestingly, dependence on BCL-2 was clearly reduced in all VEN insensitive lines indicated by almost lost mitochondrial priming. On the other hand, VEN insensitive ALL cells showed increased dependence on MCL-1. In order to analyze whether our modeled VEN resistance can be overcome by targeting MCL-1, we investigated sensitivity of these cells to the MCL-1 inhibitor S63845. High EC50 values for S63845 were found in all VEN insensitive and corresponding control lines, indicating low anti-MCL-1 activity. Importantly, the combination of S63845 with VEN synergistically induced cell death, showing that acquired VEN insensitivity in BCP-ALL can be overcome by co-targeting BCL-2 and MCL-1. To get insight into the molecular mechanisms underlying the synergism, we treated VEN insensitive ALL cells with VEN, S63845 or both and investigated binding of BIM to either BCL-2 or MCL-1 by immunoprecipitation. In the presence of VEN, clearly lower co-precipitation of BIM with BCL-2 but increased co-precipitation with MCL-1 was observed, indicating that pro-apoptotic BIM displaced from BCL-2 by VEN is sequestered by MCL-1, thereby counter-regulating VEN activity. Inversely, the MCL-1 inhibitor S63845 reduced binding of BIM to MCL-1 but increased BIM binding to BCL-2. Most interestingly, upon combination of both inhibitors, lower BIM binding to both BCL-2 and MCL-1 was found. This indicates that co-targeting MCL-1 can block the sequestration of pro-apoptotic BIM from BCL-2 to MCL-1, overcoming VEN resistance. Taken together, we show that acquired VEN resistance in BCP-ALL is characterized by up-regulated expression of counter-regulatory MCL-1 and can be overcome by simultaneous BCL-2 and MCL-1 inhibition, which prevents sequestration of BIM by MCL-1 after release from BCL-2. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Stilgenbauer:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau.

The protective effect and mechanism of catalpol on high glucose-induced podocyte injury

BackgroundCatalpol, a natural iridoid glycoside in Rehmannia glutinosa, can alleviate proteinuria associated with diabetic nephropathy (DN), however, whether catalpol has a protective effect against podocyte injury in DN remains unclear.MethodsIn this study, we used a high glucose (HG)-induced podocyte injury model to evaluate the protective effect and mechanism of catalpol against HG-induced podocyte injury. Cell viability was determined by the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by commercial assay kits. Cell apoptosis and reactive oxygen species (ROS) were determined by using flow cytometry. Tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) levels were determined by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl2-associated x (Bax), cleaved caspase-3, nicotinamide adenine dinucleotide phosphate oxidase enzyme 4 (NOX4), toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MyD88), p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 MAPK (p-p38 MAPK), nuclear factor kappa B inhibitor alpha (IκBα) and phosphorylated IκBα (p-IκBα) were measured by western blotting. In addition, Bcl-2, Bax, caspase-3 and nuclear factor kappa B (NF-κB) levels were determined by immunofluorescence staining.ResultsCatalpol significantly increased cell viability and decreased LDH release in HG-induced podocyte injury. Catalpol significantly decreased ROS generation, apoptosis, level of MDA, levels of inflammatory cytokine TNF-α, IL-1β, and IL-6 and increased SOD activity in HG-induced podocyte injury. Moreover, catalpol significantly decreased expression of cleaved caspase-3, Bax, NOX4, TLR4, MyD88, p-p38 MAPK, p-IκBα and NF-κB nuclear translocation, as well as increased Bcl-2 expression in HG-induced podocyte injury.ConclusionCatalpol can protect against podocyte injury by ameliorating apoptosis and inflammation. These protective effects may be attributed to the inhibition of NOX4, which alleviates ROS generation and suppression of the TLR4/MyD88 and p38 MAPK signaling pathways to prevent NF-κB activation. Therefore, catalpol could be a promising drug for the prevention of DN.

Influence of lncRNA-MALAT1 on neuronal apoptosis in rats with cerebral infarction through regulating the ERK/MAPK signaling pathway.

To investigate the regulatory effect of long non-coding ribonucleic acid (lncRNA)-metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway, and to explore its influence on neuronal apoptosis in rats with cerebral infarction. A total of 45 adult male Sprague-Dawley rats were randomly divided into sham group (n=15), model group (n=15) and MALAT1 low-expression group (n=15). The model of cerebral infarction was successfully established in the model group and MALAT1 low-expression group via middle cerebral artery occlusion (MCAO). After 3 d, the nerve injury in each group was evaluated using Zea-Longa score. Meanwhile, the area of cerebral infarction in each group was detected via 2,3,5-triphenyl tetrazolium chloride (TTC) staining. After the cortical tissues were separated, the expression level of lncRNA-MALAT1 was detected via quantitative Polymerase Chain Reaction (qPCR). The apoptotic level of neurons in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The expression levels of inflammatory factors were detected using enzyme-linked immunosorbent assay (ELISA) kits. Furthermore, the expression levels of apoptosis-related proteins and ERK/MAPK signaling pathway-related proteins were detected via Western blotting. Compared with the sham group, the behavioral score and area of cerebral infarction in the model group were significantly increased (p<0.01). The low expression of MALAT1 could effectively reduce the behavioral score and area of cerebral infarction in the model group (p<0.01). The expression level of lncRNA-MALAT1 in cortical tissues of the model group was markedly higher than that of the sham group and MALAT1 low-expression group (p<0.01). Compared with the sham group, the content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cortical tissues was significantly increased (p<0.01). However, the content of IL-10 was remarkably decreased in the model group (p<0.01). Low expressed MALAT1 could markedly reduce the content of TNF-α and IL-6 and increase the content of IL-10 in cortical tissues (p<0.01). The level of apoptosis in cortical tissues was increased in the model group when compared with that of the sham group (p<0.01). Meanwhile, low expression of MALAT1 could effectively reduce the apoptosis level in cortical tissues in model group (p<0.01). In the model group, the expression levels of B-cell lymphoma-2/Bcl-2 associated X protein (Bcl-2/Bax), p-ERK and matrix metalloproteinase-2 (MMP-2) in cortical tissues were significantly declined than the sham group (p<0.01). However, the protein expression level of cleaved caspase-3 was markedly increased (p<0.01). Furthermore, the low expression of MALAT1 could remarkably increase the expressions of Bcl-2/Bax, p-ERK and MMP-2 (p<0.01), as well as decrease the expression of cleaved caspase-3 (p<0.01). LncRNA-MALAT1 may increase the release of inflammatory cytokines by inhibiting the ERK/MAPK signaling pathway, thereby up-regulating the level of neuronal apoptosis and aggravating the cerebral injury in rats with cerebral infarction.

LncRNA ANRIL knockdown ameliorates retinopathy in diabetic rats by inhibiting the NF-κB pathway.

To evaluate the impacts of long non-coding ribonucleic acid (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) on diabetic retinopathy (DR) in rats and the underlying mechanism. A total of 60 adult male Sprague Dawley (SD) rats were randomly divided into 3 groups: the Sham group (n=20), DR group (n=20) and DR + lncRNA ANRIL knockdown group [DR + lncRNA ANRIL small interfering RNA (siRNA) group, n=20]. DR model in rats was established by intraperitoneal injection of streptozocin (STZ; 60 mg/kg). Meanwhile, a certain dose of lncRNA ANRIL siRNA was added dropwise into rat eyes of DR + lncRNA ANRIL siRNA group during model induction to downregulate lncRNA ANRIL expression in the retina. 16 weeks later, rat retinal tissues were taken to extract total RNA and protein. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression levels of lncRNA ANRIL, interleukin-1 (IL-1), IL-6 and monocyte chemotactic protein-1 (MCP-1) in each group of the rat retina. Pathological structure of rat retinal tissues in each group was observed via hematoxylin and eosin (H&E) staining. Immunohistochemistry was adopted to measure the expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and P65 in each group of retinal tissues. In addition, the retinal vascular permeability in each group of rats was detected by fluorescent staining. Finally, Western blotting was utilized to determine the expressions of genes in the P65 signaling pathway. Compared with rats in the Sham group, lncRNA ANRIL was upregulated in rat retinal tissues harvested from the DR + lncRNA ANRIL siRNA group (p<0.05). After knockdown of lncRNA ANRIL in the retinal tissues of DR rats, pathologic damage was alleviated notably, and the levels of inflammatory markers (IL-1, IL-10 and MCP-1) were lowered markedly (p<0.05). The protein expressions of Bax and P65 decreased evidently, while the protein level of Bcl-2 increased markedly (p<0.05) in DR rats with ANRIL knockdown. Furthermore, Western blotting results revealed that inhibition of lncRNA ANRIL could prominently repress the phosphorylation level of P65 in the retinal tissues of DR rats (p<0.05). LncRNA ANRIL knockdown can significantly ameliorate the retinopathy in diabetic rats by blocking the nuclear factor-kappa B (NF-κB) signaling pathway.

Effect of IL-6-mediated STAT3 signaling pathway on myocardial apoptosis in mice with dilated cardiomyopathy.

To investigate the effect of interleukin-6 (IL-6) gene knockout on apoptosis of myocardial cells in mice with Coxsackievirus B3 (CVB3)-induced dilated cardiomyopathy (DCM) and its potential mechanism, so as to provide certain references for the clinical prevention and treatment of DCM. A total of 40 male C57 mice were randomly divided into Sham group (n=20) and DCM group (n=20) using a random number table. Another 20 mice with IL-6 gene knockout were enrolled into DCM+IL-6 KO group (n=20). The DCM model was established via CVB3 repeated incremental infection. After 9 months, the heart weight/body weight (HW/BW) ratio of mice in each group was detected. The ejection fraction [EF (%)] and fraction shortening [FS (%)] of mice in each group were detected via two-dimensional ultrasonography. The cross-sectional area and pathological changes in myocardial cells in the heart in each group were determined using hematoxylin-eosin (HE) staining. The collagen content in myocardial tissues in each group was detected via Masson staining and picrosirius red (PSR) staining, and the expressions of Collagen I and Collagen III in myocardial tissues in each group were detected via immunohistochemistry. In addition, the myocardial apoptosis in myocardial tissues in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Finally, the protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and total STAT3 (t-STAT3) were detected via Western blotting. The expression of IL-6 messenger ribonucleic acids (mRNAs) in myocardial tissues in DCM group was significantly increased compared with that in Sham group (p<0.05). After IL-6 knockout, the HW/BW ratio of DCM mice significantly declined (p<0.05), and the cross-sectional area of myocardial cells was significantly reduced (p<0.05). According to the results of echocardiography, the cardiac function of mice in DCM+IL-6 KO group was significantly superior to that in DCM group, manifested as the significant increase in FS (%) and EF (%) (p<0.05). The results of Masson staining, PSR staining, and immunohistochemical staining showed that IL-6 knockout could reduce the collagen content and Collagen I and Collagen III expressions in myocardial tissues of DCM mice (p<0.05). Furthermore, it was found via TUNEL staining that the number of apoptotic myocardial cells in DCM+IL-6 KO group was markedly smaller than that in DCM group (p<0.05). At the same time, the Bax/Bcl-2 ratio in myocardial tissues in DCM+IL-6 KO group was lower (p<0.05). Finally, the results of Western blotting revealed that DCM+IL-6 KO group had a lower phosphorylation level of STAT3 than DCM group (p<0.05). Inhibiting IL-6 gene may improve the DCM-induced myocardial remodeling through reducing myocardial apoptosis.

Paeoniflorin inhibits tributyltin chloride-induced apoptosis in hypothalamic neurons via inhibition of MKK4-JNK signaling pathway

Ethnopharmacological relevancePaeoniflorin (PF) exerts a significant protective effect against neurotoxicity and mitochondrial damage in neurons. However, the mechanisms underlying PF-mediated rescue remain elusive. Therefore, we endeavored to further research the molecular mechanisms underlying PF-mediated inhibition of tributyltin chloride (TBTC)-induced apoptosis of neurons.Aim of the study: To investigate the influence and possible mechanism of action of PF in TBTC-induced neurodegenerative disease. Materials and methodsFirst, primary hypothalamic neurons were treated with tributyltin chloride (150 μg/L) and PF (25, 50, and 100 μM). 17β-estradiol (1 nM) was used as a positive control. Subsequently, CCK-8 assay was performed. The level of apoptosis was examined by flow cytometry and the function of mitochondria was reflected by MMP levels. The mRNA expression levels of B-cell lymphoma-2 (Bcl-2), together with Bax, were examined using qRT-PCR. The protein levels of mitogen-activated protein kinase kinase 4 (MKK4), c-Jun N-terminal kinase (JNK), Bcl-2, Bax, and Caspase-3 were examined using western blotting. Finally, pretreatment with JNK agonist, anisomycin, was done to observe the change in expressions of MKK4 and JNK. ResultsPaeoniflorin treatment reduced TBTC-induced damage and neuron loss in a dose-dependent manner. Decrease in mitogen-activated protein kinase (MAPK) as well as JNK levels were reversed by treatment with paeoniflorin via inhibition of JNK activation. Furthermore, ratio of levels of Bcl-2/Bax increased while the activation of caspase-3 was suppressed. In addition, pretreatment with JNK agonist, anisomycin effectively suppressed TBTC-induced cytotoxicity in hypothalamic neuron. ConclusionsPF can potentially be used to prevent and/or treat neurodegenerative diseases and neural injury by inhibiting MKK4-JNK signaling pathway.

Digital Analysis of BCL2 Expression in Laryngeal Squamous Cell Carcinoma.

Deregulation of apoptosis is critical regarding the development and progression of malignancies, including laryngeal squamous cell carcinoma (LSCC). B-Cell lymphoma 2 (BCL2) (gene locus:18q21.33), located on the outer mitochondrial membrane, acts mainly as an anti-apoptotic factor suppressing and blocking apoptotic signal transduction. Fifty (n=50) primary LSCC tissue sections were used. Immunohistochemistry and digital image analysis were implemented for evaluating BCL2 protein expression levels. High BCL2 protein expression levels were observed in 21/50 (42%) LSCC tissue sections, whereas the remaining cases (n=29) demonstrated a low expression. Overall, BCL2 expression was associated with grade (p=0.046) and anatomical region of the examined malignancies (transglottic, p=0.047). Interestingly, high BCL2 expression levels were strongly associated with radiotherapy-based only regimens (p=0.01) in corresponding patients. BCL2 overexpression was found to be correlated with an aggressive phenotype (advanced grade of differentiation) in LSCC, also demonstrating a potential selective anatomic localization (transglotic region). Additionally, BCL2 overexpression appears to be a negative regulator for successful radiotherapy implementation by reducing the apoptotic process in patients.

LINC00152 promotes the proliferation of gastric cancer cells by regulating B-cell lymphoma-2.

LINC00152 has been considered to be associated with the tumorigenesis and the occurrence of gastric cancer; however, the mechanism of LINC00152 has yet to be fully elucidated. In the present study, the expression levels of LINC00152 in tissues, serum, and peripheral blood mononuclear cells (PBMCs) of patients with gastric cancer were determined using real-time polymerase chain reaction. The functions of LINC00152 with respect to the proliferation, apoptosis, migration, and invasive abilities of the gastric cancer cells were evaluated by cell proliferation analysis, flow cytometry, cell scratch wound assay, and transwell migration experiments. A mouse xenotransplant model of gastric tumors was established to detect the role of LINC00152 in vivo, and the expression levels of B-cell lymphoma-2 (Bcl-2) family proteins were investigated by Western blot analysis. The results revealed that LINC00152 was overexpressed in tissues, serum, and PBMCs of patients with gastric cancer. Moreover, LINC00152 could promote the migration and invasive abilities and suppress the apoptosis, of gastric cancer cells through regulating the Bcl-2 protein family. LINC00152 could bind with Bcl-2 directly to induce the activation of cell cycle signaling, and this may be a potential target for the therapy of gastric cancer in the future.

Indolyl-chalcone derivatives induce hepatocellular carcinoma cells apoptosis through oxidative stress related mitochondrial pathway in vitro and in vivo

A facile method of assembling oxindole and phthalide units through a Lewis based catalyzed allylic alkylation reaction of Morita–Baylis–Hillman carbonates of isatins and 3-cyanophthalides was recently developed. The method efficiently delivers a hybrid of 3,3′-disubstituted oxindole with a valuable phthalide pharmacophore. In the present study, we proved the deleterious effects of 5h2c, a screened synthesis compound, against hepatocellular carcinoma (HCC) in both in vitro and in vivo models. 5h2c strongly decreased cell viability, caused over-release of lactate dehydrogenase, inhibited cell migration, and enhanced the apoptosis rate in HepG2 and PLC/PRF/5 cells. 5h2c led to an increase in intracellular reactive oxygen species levels and a decrease in mitochondrial membrane potential. In HepG2-and PLC/PRF/5-xenograft tumor mouse models, treatment with 5h2c inhibited tumor growth without affecting the animals' bodyweight or organ functions. Proteome profiling of tumor tissues after 24-h exposure to 5h2c showed significantly enhanced expression levels of Bcl-2 associated X protein, cleaved caspase −3, −8, and −9, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), heme oxygenase-2, paraoxonase 2, catalase, and factor associated suicide ligand, and reduced the expression levels of B-cell lymphoma-2, B-cell lymphoma-extra large, heat shock protein 27, heat shock protein 60, and heat shock protein 70 in HepG2 and PLC/PRF/5 cells. All of our data confirmed that oxidative stress-mediated mitochondrial apoptosis (especially the Nrf-2/HO-1 pathway) is responsible for 5h2c-induced HCC damage.

Non-endometrioid and high-grade endometrioid endometrial cancers show DNA fragmentation factor 40 (DFF40) and B-cell lymphoma 2 protein (BCL2) underexpression, which predicts disease-free and overall survival, but not DNA fragmentation factor 45 (DFF45) underexpression

BackgroundThe expression of DNA fragmentation factor 45 (DFF45) and B-cell lymphoma 2 (BCL2) in glands of the normal human endometrium is related to phases of the menstrual cycle and decreases after menopause, whereas the expression of DNA fragmentation factor 40 (DFF40) is stable. Moreover, DF45, BCL2 and DFF40 underexpression has been reported in numerous malignancies, including uterine leiomyosarcomas. In this study, we aimed to investigate DFF45, BCL2 and DFF40 expression in endometrioid and non-endometrioid types of endometrial cancers (ECs). We also evaluated the correlations between DFF45, BCL2 and DFF40 expression levels and clinicopathological parameters and determined the value of these three proteins as prognostic markers of disease-free survival (DFS) and overall survival (OS).MethodsImmunohistochemistry was performed to evaluate DFF45, BCL2 and DFF40 expression in 342 cases of ECs. Student’s t-test, the Mann-Whitney U-test, and the chi-squared test were used for the statistical analyses as appropriate. The Cox-Mantel test, Cox’s proportional hazard model, and relative risk analyses were used to evaluate associations between DFF40, DFF45, and BCL2 expression and clinicopathological characteristics.ResultsDFF40 and BCL2, but not DFF45, were significantly underexpressed in non-endometrioid and high-grade endometrioid ECs compared with low- and moderate-grade endometrioid ECs. Women with DFF40- and BCL2-negative tumors had higher risks of disease recurrence, lymph node involvement, lympho-vascular space infiltration, and deep myometrial invasion compared with women with DFF40- and BCL2-positive tumors. Additionally, women with DFF40- and BCL2-negative tumors had significantly lower OS and DFS than women with DFF40- and BCL2-positive tumors. A multivariable analysis of the model, including the clinicopathological characteristics and immunohistochemical results, showed that negative BCL2 expression, lymph node involvement, and high-stage and high-grade disease were independent predictors of OS, whereas negative BCL2 expression, lymph node involvement, and high-stage disease were independent predictors of DFS.ConclusionsCompared with low- and moderate-grade endometrioid ECs, non-endometrioid and high-grade endometrioid ECs showed significant DFF40 and BCL2 underexpression. The absence of DFF40 and BCL2 expression negatively affects DFS and OS. Further prospective studies are warranted to assess the potential utility of DFF40 and BCL2 as targets in the diagnosis or treatment of ECs.

Loss of ARID1A Expression Correlates With Tumor Differentiation and Tumor Progression Stage in Pancreatic Ductal Adenocarcinoma.

Mutations in the AT-rich interactive domain 1A gene, which encodes a subunit of the Switch/Sucrose nonfermentable chromatin remodeling complex, can result in loss of protein expression and are associated with different cancers. Here, we used immunohistochemistry to investigate the significance of AT-rich interactive domain 1A loss in 73 pancreatic ductal adenocarcinoma cases with paired paracancerous normal pancreatic tissues. The relationship between levels of the AT-rich interactive domain 1A protein product, BAF250a, and clinicopathological parameters in the 73 pancreatic cancer specimens was also analyzed. We found that the expression of AT-rich interactive domain 1A in normal pancreatic tissue was higher than that in tumor tissue. Loss of AT-rich interactive domain 1A expression in pancreatic tumors was associated with tumor differentiation (P = .002) and tumor stage (P = .048). Meanwhile, BAF250a protein levels were not related to lymph node metastasis, distant metastasis, sex, or age and were not associated with survival. Transfection of the pancreatic cancer cell lines AsPC-1 and PANC-1 with small-interfering RNA specific for AT-rich interactive domain 1A resulted in elevated messenger RNA and protein expression levels of B-cell lymphoma-2 (Bcl-2), CyclinD1, and Kirsten rat sarcoma viral oncogene (KRAS). The AT-rich interactive domain 1A expression level in the cells was increased following microRNA-31 (miR-31) inhibitor transfection. Our data provide additional evidence that AT-rich interactive domain 1A might function as a tumor suppressor gene in pancreatic carcinogenesis.

Knockdown of microRNA-29a regulates the expression of apoptosis-related genes in MCF-7 breast carcinoma cells.

MicroRNA (miR), as non-coding small RNA, are key regulators of cancer-related biological cell processes and contribute to tumor growth through regulation of groups of pro- and anti-apoptotic genes. The present study aimed to investigate the effects of miR-29a on the expression of genes involved in apoptosis, including p21, B-cell lymphoma 2 (BCL-2), p53 and survivin. The MCF-7 breast cancer cell line was transfected with anti-miR-29a and treated with Taxol in subdivided treatment groups including: Scramble; anti-miR-29a; anti-miR-29a + Taxol; Taxol; and control. Expression levels of p21, BCL-2, p53 and survivin were evaluated using reverse transcription-quantitative polymerase chain reaction. miR-29a knockdown resulted in p21 and p53 upregulation and a decrease in survivin expression. These results indicated that miR-29a inhibition regulates apoptosis. The present data suggested that miR-29a inhibition may be a promising strategy for the induction of apoptosis of tumor cells.

Expression of PIM-1 in salivary gland adenoid cystic carcinoma: Association with tumor progression and patients' prognosis.

Pim-1 proto-oncogene, serine/threonine kinase (PIM-1) phosphorylates a series of substrates to exert its oncogenic function in numerous malignancies. The present study investigated the clinical significance of the PIM-1 protein, apoptosis status and apoptosis-associated proteins, including forkhead box O3a (FOXO3a), B cell lymphoma-2 (BCL-2) and BCL-2-associted agonist of cell death (BAD), were investigated in salivary gland adenoid cystic carcinoma (ACC) tissues. PIM-1 expression levels in 4 pairs of ACC tissues and corresponding normal salivary gland tissues were determined by western blot analysis. PIM-1, FOXO3a, BAD and BCL-2 expression levels in 60 ACC tissues were evaluated by immunohistochemistry (IHC). A terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay was performed to detect the apoptosis status of ACC tissues. PIM-1 was revealed to be highly expressed in ACC tissues compared with adjacent normal tissues. IHC staining results demonstrated high expression ratios of PIM-1, FOXO3a, BCL-2 and BAD [33.33% (20/60), 51.67% (31/60), 51.67% (31/60) and 55% (33/60)], respectively, and significant correlations between the expression of PIM-1 and FOXO3a and BCL-2 (P<0.05). Apoptotic rates were significantly associated with PIM-1, FOXO3a, BCL-2 and BAD expression levels (P<0.05). PIM-1 expression levels were significantly associated with tumor size, lymph node involvement, nerve invasion, distant metastasis and weakly associated with tumor node metastasis stage. Kaplan-Meier survival curves revealed that PIM-1 expression level was significantly associated with disease-free survival of patients with ACC (P=0.009). Cox regression multivariate analysis results revealed that histotype, distant metastasis and apoptotic rate were independent prognosis factors for ACC. Assessment of PIM-1 may be useful in investigating the malignant behaviors of ACC and predicting the outcome of patients with ACC.

Pink1 attenuates propofol-induced apoptosis and oxidative stress in developing neurons.

The underlying mechanisms of propofol-induced neurotoxicity in developing neurons are still not completely understood. We examined the role of PTEN-induced kinase 1 (Pink1), an antioxidant protein, in propofol-induced apoptosis in developing neurons. Primary hippocampal neurons isolated from neonatal Sprague-Dawley rats were exposed to propofol 20μM for 2, 4, 6 and 12h. Subsequently, neurons underwent overexpression and knockdown of Pink1, followed by propofol exposure (20μM, 6h). Neuron apoptosis was detected by terminal transferase deoxyuridine triphosphate-biotin nick-end labeling (TUNEL). Reactive oxygen species (ROS) production in neurons was detected by using a 2,7-dichlorodihydro-fluorescein diacetate probe and target protein or mRNA levels were analyzed by Western blotting or real-time polymerase chain reaction. Propofol treatment time-dependently increased the number of TUNEL-positive neurons and the expression levels of cleaved caspase-3 and B-cell lymphoma 2 (BcL-2) associated X protein, but decreased expression levels of BcL-2. Furthermore, propofol treatment time-dependently reduced the expression levels of Pink1 mRNA and protein. ROS production and the markers of oxidative stress, 2,4-dinitrophenol and 4-hydroxynonenal, were increased by propofol treatment. However, these propofol-induced changes were significantly restored by Pink1 overexpression. Pink1 plays an important role in neuronal apoptosis induced by propofol. Our results may provide some new insights in propofol-induced neurotoxicity in developing neurons.

Synergy of BCL2 and histone deacetylase inhibition against leukemic cells from cutaneous T-cell lymphoma patients

AbstractThe presence and degree of peripheral blood involvement in patients with cutaneous T-cell lymphoma (CTCL) portend a worse clinical outcome. Available systemic therapies for CTCL may variably decrease tumor burden and improve quality of life, but offer limited effects on survival; thus, novel approaches to the treatment of advanced stages of this non-Hodgkin lymphoma are clearly warranted. Mutational analyses of CTCL patient peripheral blood malignant cell samples suggested the antiapoptotic mediator B-cell lymphoma 2 (BCL2) as a potential therapeutic target. To test this, we developed a screening assay for evaluating the sensitivity of CTCL cells to targeted molecular agents, and compared a novel BCL2 inhibitor, venetoclax, alone and in combination with a histone deacetylase (HDAC) inhibitor, vorinostat or romidepsin. Peripheral blood CTCL malignant cells were isolated from 25 patients and exposed ex vivo to the 3 drugs alone and in combination, and comparisons were made to 4 CTCL cell lines (Hut78, Sez4, HH, MyLa). The majority of CTCL patient samples were sensitive to venetoclax, and BCL2 expression levels were negatively correlated (r = −0.52; P = .018) to 50% inhibitory concentration values. Furthermore, this anti-BCL2 effect was markedly potentiated by concurrent HDAC inhibition with 93% of samples treated with venetoclax and vorinostat and 73% of samples treated with venetoclax and romidepsin showing synergistic effects. These data strongly suggest that concurrent BCL2 and HDAC inhibition may offer synergy in the treatment of patients with advanced CTCL. By using combination therapies and correlating response to gene expression in this way, we hope to achieve more effective and personalized treatments for CTCL.

Atractylenolide II Inhibits Proliferation, Motility and Induces Apoptosis in Human Gastric Carcinoma Cell Lines HGC-27 and AGS.

Atractylenolide II (AT-II) exhibits several biological and pharmacological functions, especially anti-cancer activity as the major sesquiterpene lactones isolated from Atractylodes macrocephala (also named Baizhu in Chinese). However, the effects and mechanisms of AT-II on human gastric cancer remain unclear. Cell Counting Kit-8 (CCK-8) assay, morphological changes, flow cytometry, wound healing assay and Western blot analysis were used to investigate the effects of AT-II on cell proliferation, apoptosis and motility of human gastric carcinoma cell lines HGC-27 and AGS. Our results indicated that AT-II could significantly inhibit cell proliferation, motility and induce apoptosis in a dose and time-dependent manner. Western blot analysis showed that the expression level of Bax was upregulated and the expression levels of B-cell lymphoma-2 (Bcl-2), phosphorylated-protein kinase B (p-Akt) and phosphorylated-ERK (p-ERK) were downregulated compared to control group. In conclusion, the findings suggested that AT-II exerted significant anti-tumor effects on gastric carcinoma cells by modulating Akt/ERK signaling pathway, which might shed light on therapy of gastric carcinoma.

Downregulation of transcription factor GATA4 sensitizes human hepatoblastoma cells to doxorubicin-induced apoptosis.

Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.

Effect of Polygonatum odoratum extract on human breast cancer MDA-MB-231 cell proliferation and apoptosis.

Traditional Chinese medicine (TCM) is important in the provision of anti-tumor drugs. Recently, studies have shown that certain types of TCM agents are able to control the growth of tumors, enhance the body's immune function and enhance the therapeutic effect of chemotherapeutic drugs. In women, breast carcinoma is the most common tumor type and the second most common cause of death from cancer. Polygonatum odoratum (P. odoratum) is commonly used in TCM. The aim of the present study was to investigate the effects of P. odoratum extract on the proliferation and apoptosis of MDA-MB-231 breast cancer cells. Cell proliferation was assessed using MTT and colony formation assays. In addition, propidium iodide (PI)/Annexin V-FITC staining was used to investigate the apoptosis of MDA-MB-231 cells following treatment with P. odoratum extract. The protein expression levels of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were also detected using western blot analysis, while a JC-1 staining assay was used to assess the mitochondrial membrane potential (ΔΨm). The results of the MTT assay showed that the proliferation and colony formation of MDA-MB-231 cells were inhibited following treatment with the extract. Furthermore, the PI/Annexin-V staining showed that the apoptosis of MDA-MB-231 cells was enhanced by the extract, in a concentration-dependent manner. The extract also lowered the ΔΨm of MDA-MB-231 cells, upregulated the expression of Bax and inhibited the expression of Bcl-2. In conclusion, these results showed that the P. odoratum extract inhibited the proliferation and induced apoptosis of breast cancer MDA-MB-231 cells.

Effect of API-1 and FR180204 on cell proliferation and apoptosis in human DLD-1 and LoVo colorectal cancer cells.

The activation of the phosphatidylinositol-3 kinase/v-akt murine thymoma viral oncogene homolog (Akt) and mitogen activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathways are implicated in the majority of cancers. Selective inhibition of Akt and ERK represents a potential approach for cancer therapy. Therefore, the present study aimed to investigate the apoptotic and anti-proliferative effects of the novel and selective Akt inhibitor 4-amino-5,8-dihydro-5-oxo-8-β-D-ribofuranosyl-pyrido[2,3-d]pyrimidine-6-carboxamide (API-1) and selective ERK1/2 inhibitor FR180204 (FR) alone and in combination on colorectal cancer (CRC) cells (DLD-1 and LoVo). In addition, the effects of API-1 and FR on Akt and ERK signaling pathways were also investigated. The effects of the agents on DLD-1 and LoVo cells were evaluated in terms of cell viability, cytotoxicity, DNA synthesis rate, DNA fragmentation and caspase-3 activity levels. In addition, quantitative reverse transcription-polymerase chain reaction and western blot analysis were performed to examine relevant mRNA and protein levels. The present study observed that the combination of FR with API-1 resulted in significant apoptosis and cytotoxicity compared with any single agent alone in a time-dependent manner in these cells. Also, treatment with FR and API-1 in combination decreased the expression levels of B-cell lymphoma-2 (BCL2), Bcl-2-like1, cyclin D1 and cMYC, and increased the expression levels of BCL2-associated X protein and BCL2 antagonist/killer via phosphorylated Akt and phosphorylated ERK1/2 downregulation. The combination of Akt and ERK1/2 inhibitors resulted in enhanced apoptotic and anti-proliferative effects against CRC cells. The present study hypothesizes that the combination of FR and API-1 in CRC cells may contribute toward potential anti-carcinogenic effects. Additional analyses using other cancer cell lines and animal models are required to confirm these findings in vitro and in vivo.

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&amp;lt;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&amp;gt;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.