Expression Levels Of B-cell Lymphoma-2 Research Articles

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.

The effect of sildenafil on right ventricular remodeling in a rat model of monocrotaline-induced right ventricular failure.

PurposePulmonary arterial hypertension (PAH) leads to right ventricular failure (RVF) as well as an increase in pulmonary vascular resistance. Our purpose was to study the effect of sildenafil on right ventricular remodeling in a rat model of monocrotaline (MCT)-induced RVF.MethodsThe rats were distributed randomly into 3 groups. The control (C) group, the monocrotaline (M) group (MCT 60 mg/kg) and the sildenafil (S) group (MCT 60 mg/kg+ sildenafil 30 mg/kg/day for 28 days). Masson Trichrome staining was used for heart tissues. Western blot analysis and immunohistochemical staining were performed.ResultsThe mean right ventricular pressure (RVP) was significantly lower in the S group at weeks 1, 2, and 4. The number of intra-acinar arteries and the medial wall thickness of the pulmonary arterioles significantly lessened in the S group at week 4. The collagen content also decreased in heart tissues in the S group at week 4. Protein expression levels of B-cell lymphoma-2 (Bcl-2)-associated X, caspase-3, Bcl-2, interleukin (IL)-6, matrix metalloproteinase (MMP)-2, endothelial nitric oxide synthase (eNOS), endothelin (ET)-1 and ET receptor A (ERA) in lung tissues greatly decreased in the S group at week 4 according to immunohistochemical staining. According to Western blotting, protein expression levels of troponin I, brain natriuretic peptide, caspase-3, Bcl-2, tumor necrosis factor-α, IL-6, MMP-2, eNOS, ET-1, and ERA in heart tissues greatly diminished in the S group at week 4.ConclusionSildenafil alleviated right ventricular hypertrophy and mean RVP. These data suggest that sildenafil improves right ventricular function.

Mangiferin inhibition of proliferation and induction of apoptosis in human prostate cancer cells is correlated with downregulation of B-cell lymphoma-2 and upregulation of microRNA-182.

Mangiferin, a flavonoid extracted from the mango tree, possesses anti-inflammatory, antibacterial, anti-herpes simplex and antitumor activity, and is able to affect immune function. The present study investigated the anticancer effects of mangiferin treatment on PC3 human prostate cancer cells, and the potential underlying mechanisms. In the present study, an MTT assay was used to analyze the proliferation of PC3 cells. Subsequently, flow cytometry and colorimetric assay kits were utilized to measure the PC3 cell apoptotic rate. The expression levels of B-cell lymphoma-2 (Bcl-2) and microRNA-182 (miR-182) were detected using western blot analysis and quantitative reverse transcription-polymerase chain reaction, respectively. Finally, miR-182 and anti-miR-182 were transfected into PC3 cells, which were used to investigate the effects of mangiferin. Mangiferin treatment reduced the proliferation of PC3 human prostate cancer cells in a concentration- and time-dependent manner. In addition, mangiferin was able to promote apoptosis and induce the caspase-3 activity of PC3 human prostate cancer cells. Mangiferin treatment was also able to significantly reduce Bcl-2 expression levels and enhance miR-182 expression in PC3 cells. Finally, it was observed that mangiferin inhibited proliferation and induced apoptosis in PC3 human prostate cancer cells, and this effect was correlated with downregulation of Bcl-2 and upregulation of miR-182.

Solamargine triggers hepatoma cell death through apoptosis.

Solamargine (SM), a steroidal alkaloid glycoside extracted from the traditional Chinese herb Solanum incanum, has been evidenced to inhibit the growth and induce apoptosis in a number of human cancer cell lines. In the present study, the anticancer effect of SM and underlying molecular mechanism of SM-induced apoptosis were investigated on the human hepatocellular carcinoma cells, SMMC7721 and HepG2. The proliferation effects of SM on the SMMC7721 and HepG2 cell lines were evaluated using MTT and colony formation assays. In addition, the percentage of apoptosis was measured using an Annexin V/propidium iodide staining method and the cell cycle distribution mediated by SM was analyzed using flow cytometry. The expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, caspase-9, proliferating cell nuclear antigen (pcna) and Ki67 proteins were examined to further demonstrate the proliferate and apoptosis effects of SM on the hepatoma cells. The results indicated that SM effectively inhibited hepatoma cell proliferation and promoted apoptosis. SM resulted in cell cycle arrest at the G2/M phase in the two cell lines. In addition, SM downregulated the levels of proliferation-associated (Ki67 and pcna) and anti-apoptotic (Bcl-2) proteins, and promoted the activity of apoptosis-associated proteins (Bax, caspase-3 and caspase-9). Therefore, the activation of the Bcl-2/Bax and caspase signaling pathways may be involved in the SM-induced apoptosis of hepatoma cells.

Curcumin induces the apoptosis of non-small cell lung cancer cells through a calcium signaling pathway.

Curcumin is known for its anti-proliferative effects in lung cancer cells. Studies have demonstrated that an increase in the levels of intracellular free calcium ([Ca2+]i) is involved in curcumin-induced apoptosis. In this study, we aimed to investigate the involvement of calcium overload in the anti-proliferative effects of curcumin on lung cancer cells and the possible mechanisms involved. A549 and H1299 lung cancer cells were incubated with serial diluted curcumin. MTT assay was used to assess the cytotoxic effects of curcumin on the lung cancer cells; the inositol 1,4,5-trisphosphate receptor (IP3R, a key regulator of [Ca2+]i signaling) was blocked by its specific inhibitor, xestospongin C (XSC). Hoechst 33342, Fura-2/AM and rhodamine 123 fluorescence staining was employed to detect the apoptosis, the [Ca2+]i level and mitochondrial potential in the lung cancer cells. The expression levels of B-cell lymphoma-2 (Bcl-2), cleaved caspase-3 and cleaved caspase-9, and the phosphorylation level of IP3R were evaluated by western blot analysis. Our results revealed that curcumin inhibited cell growth, increased the [Ca2+]i level and increased the apoptosis of the lung cancer cells in a concentration-dependent manner. However, XSC attenuated the increase in the [Ca2+]i level and apoptosis, and also reversed the curcumin-induced loss of mitochondrial potential potential. Treatment with curcumin downregulated the expression of Bcl-2, and elevated the phosphorylation level of IP3R in a concentration-dependent manner. However, this effect was not reversed by treatment with XSC. In conclusion, the cytotoxic effects of curcumin on lung cancer cells were induced by calcium overload, which involves Bcl-2 mediated IP3R phosphorylation.

FOXA1 expression after neoadjuvant chemotherapy is a prognostic marker in estrogen receptor-positive breast cancer

Recent studies have indicated that the response to chemotherapy and the prognostic impact of a pathological complete response (pCR) after neoadjuvant chemotherapy differ among breast cancer subtypes. Predictors of response to chemotherapy and prognostic factors for survival might be different in estrogen receptor (ER)-positive breast cancer. Women with Stage II to III ER-positive HER2-negative breast cancer treated with anthracycline and taxane-containing neoadjuvant chemotherapy between 2003 and 2011 were retrospectively analyzed. Expression of forkhead box A1 (FOXA1), B cell lymphoma 2 (BCL2) and microtubule-associated protein tau (MAPT) as well as ER, progesterone receptor, HER2 and Ki67 was examined by immunohistochemistry in pre- and post-treatment specimens. Factors predictive of response to neoadjuvant chemotherapy and distant disease-free survival were analyzed. Tumor grade was positively correlated with Ki67 expression. Expression levels of ER were positively correlated with expression levels of HER2, BCL2, FOXA1 and MAPT in pre-treatment tumors. The Ki67 labeling index was the only factor that was significantly associated with clinical response measured by the reduction of tumor volume and pCR. Lymph node status, expression of ER before neoadjuvant chemotherapy and expression of FOXA1 after neoadjuvant chemotherapy were significantly associated with distant disease-free survival, both by univariate and multivariate analyses. Patients with ER-positive HER2-negative breast cancer should be selected for neoadjuvant chemotherapy. FOXA1 expression could be a prognostic marker in ER-positive breast cancer.

Accelerated fibrosis and apoptosis with ageing and in atrial fibrillation: Adaptive responses with maladaptive consequences

The aim of this study was to investigate whether abnormal expression of matrix metalloproteinase (MMP)-9/tissue inhibitors of MMPs (TIMP)-1 and B cell lymphoma 2 (BCL-2)/BCL-2-associated X protein (BAX) are correlated with the characteristic accelerated fibrosis and apoptosis during ageing and in atrial fibrillation (AF). Four groups of dogs were studied: adult dogs in sinus rhythm (SR), aged dogs in SR, adult dogs with AF induced by rapid atrial pacing and aged dogs with AF induced by rapid atrial pacing. The mRNA and protein expression levels of the target gene in the left atrium were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Pathohistological and ultrastructural changes were assessed by light and electron microscopy. The apoptotic indices of myocytes were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). The mRNA and protein expression levels of MMP-9 and BAX and those of TIMP-1 and BCL-2 were significantly upregulated and down-regulated, respectively, in the aged groups compared with the adult groups. Compared with the control groups, the adult and aged groups with AF exhibited significantly increased mRNA and protein expression levels of MMP-9 and BAX and decreased expression levels of TIMP-1 and BCL-2. Samples of atrial tissue demonstrated abnormal pathohistological and ultrastructural changes, accelerated fibrosis and apoptosis. MMP-9/TIMP-1 and BCL-2/BAX hold potential for use as substrates conducive to AF and their abnormal expression plays a major role in structural remodeling of the atrium.