Bcl-xL mRNA Research Articles

Abstract P6-13-06: Novel combination therapies for triple negative breast cancer identified by high-throughput screening

Abstract Background: Finding effective novel therapies for triple negative breast cancer (TNBC) remains an unmet challenge. Our objective was to assess the sensitivity of TNBC to single drugs and to combinations using a systematic screen of existing FDA approved drugs combined with other approved or experimental agents and to characterize the mechanism of action of the most promising combinations. Methods: We conducted a comprehensive high-throughput drug combination screen in six TNBC cell lines with different genetic backgrounds. We tested a panel of 128 agents with known targets for their growth inhibitory potential individually and in pairwise combinations with six FDA approved anticancer drugs in MDA-MB-231, MDA-MB-436, MDA-MB-468, BT-20, BT-549, and HCC-38 TNBC cell lines. High-throughput assays were performed in 384-well plates and dose response curves were generated using 5 concentrations of the secondary drug, while keeping the first drug at fixed dose. Cell viability was measured 72h after treatment exposure using CellTiter-Glo®. Drug combinations were assessed for overall inhibitory effect and also for superadditivity, assessed as deviations from non-interaction using Bliss model of synergy. Selected synergistic and effective drug combinations identified from the high-throughput screen were subsequently validated in a 96-well low-throughput format in all TNBC cell lines. Results: Cell cycle and apoptosis regulators were more inhibitory as single agents across TNBC cell lines relative to other drug classes. Bortezomib, carfilzomib, YM155, Flavopiridol, KP372-1, and dactinomycin were highly toxic to all TNBC lines as single agents. Combinations with either everolimus or erlotinib were particularly effective in BT-20 and MDA-MB-468, and combinations with crizotinib in MDA-MB-231 cells. Bay-11-7082/erlotinib and MK-1775/everolimus were few of the promising combinations that elicited potentiated response in all the cell lines. ABT-263/crizotinib was one of the top synergistic combination most effective in MDA-MB-231 cells that express highest relative mRNA and protein levels of respective drug targets, Bcl-xL, and AXL. The combination treatment with ABT-263 and crizotinib also resulted in apoptosis in MDA-MB-231 cells, indicated by marked PARP cleavage in these cells, while MDA-MB-436 cells with lowest expression of Bcl-xL were resistant to apoptosis or growth inhibition, indicating a potential efficacy of this combination in a subset of TNBCs. Conclusions: This study reveals novel combinations of cell cycle or apoptosis regulators and crizotinib, everolimus, or erlotinib with enhanced anticancer activity. Combinations of Bay-11-7082 and erlotinib; and MK-1775 and everolimus had mild synergistic activity in all TNBC lines, while ABT-263 and Crizotinib showed large synergistic antiproliferative activity in a subset of TNBCs. These promising drug combinations have great potential to improve cure rates in TNBC patients. Since crizotinib, everolimus, and erlotinib are US FDA-approved while ABT-263 and MK-1775 are in advanced clinical trials, there is a rapid path for clinical translation for these drug-combinations after validation in animal studies. Citation Format: Wali VB, Langdon CG, Held MA, Platt JT, Safonov A, Aktas B, Stern DF, Pusztai L, Hatzis C. Novel combination therapies for triple negative breast cancer identified by high-throughput screening. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-06.

Protective effects of retinoid x receptors on retina pigment epithelium cells

Age-related macular degeneration (AMD) is among the main pathologies leading to blindness in adults and has currently no cure or effective treatment. Selective apoptosis of retina pigment epithelial (RPE) cells results in the progressive loss of photoreceptor neurons, with the consequent gradual vision loss. Oxidative stress plays an important role in this process. We have previously determined that activation of RXRs protects rat photoreceptor neurons from oxidative stress-induced apoptosis. In this study we investigated whether RXR ligands prevented apoptosis in an RPE cell line, D407 cells, exposed to hydrogen peroxide (H2O2). H2O2 induced apoptosis of D407 cells, promoting p65NFκB nuclear translocation, increasing Bax mRNA expression, activating caspase-3 and altering cell morphology. We show, for the first time, that HX630, a RXR pan-agonist, protected D407 cells from H2O2-induced apoptosis, preventing p65NFκB nuclear translocation, increasing Bclxl and PPARγ mRNA levels and simultaneously decreasing Bax mRNA levels and caspase-3 activation. Pretreatment with a RXR antagonist blocked HX630 protection. LG100754, which binds RXRs but only activates heterodimers and is an antagonist of RXR homodimers, also had a protective effect. In addition, only agonists known to bind to RXR/PPARγ were protective. As a whole, our results suggest that RXR activation protects RPE cells from oxidative stress-induced apoptosis and this protection might involve signaling through a heterodimeric receptor, such as RXR/PPARγ. These data also imply that RXR agonists might provide potential pharmacological tools for treating retina degenerative diseases.

MiR-23b targets cyclin G1 and suppresses ovarian cancer tumorigenesis and progression.

BackgroundIt has been proposed that cyclin G1 (CCNG1) participates in p53-dependent G1–S and G2 checkpoints and might function as an oncogenic protein in the initiation and metastasis of ovarian carcinoma. MicroRNA 23b (miR-23b) is a critical regulatory factor in the progression of many cancer cell types that targets the relevant genes.MethodsMiR-23b expression in ovarian tissues was quantified by quantitative reverse transcription–PCR. The ovarian cancer cell lines OVCAR3, HO8910-PM, and SKOV3/DDP were transfected with miR-23b, after we assayed the cell phenotype and expression of the relevant molecules. Dual-luciferase reporter assay and a xenograft mouse model were used to examine the expression of miR-23b and its target gene CCNG1.ResultsMIR23B mRNA expression was significantly lower in epithelial ovarian carcinoma and borderline tumors than in normal ovarian tissues and benign tumors, and miR-23b expression among ages and pathological subtypes was significantly different. CCNG1 mRNA expression was significantly lower in normal ovarian tissues than in benign tumors, borderline tumors, and ovarian carcinomas, and expression among pathological subtypes was significantly different. MiR-23b overexpression inhibited ovarian cancer cell proliferation, invasion, and migration, and induced apoptosis. Dual-luciferase reporter assay showed that miR-23b bound with the 3′ untranslated region of CCNG1. MiR-23b overexpression significantly downregulated CCNG1, urokinase, survivin, Bcl-xL, P70S6K, and matrix metallopeptidase-9 (MMP9) mRNA and protein expression. Furthermore, miR-23b inhibited tumor growth and suppressed CCNG1 expression in vitro.ConclusionsOur findings show that miR-23b may inhibit ovarian cancer tumorigenesis and progression by downregulating CCNG1 and the expression of the relevant genes. MiR-23b is a potentially novel application for regulating ovarian carcinoma progression.

Fucoidan from Acaudina molpadioides protects pancreatic islet against cell apoptosis via inhibition of inflammation in type 2 diabetic mice.

Inflammation induces pancreatic islet cell apoptosis. Effects of fucoidan from Acaudina molpadioides (Am-FUC) on inhibition of pancreatic islet cell apoptosis and inflammation in type 2 diabetic mice were investigated. Am-FUC repaired pancreatic islet cells, decreased serum C-reactive protein (CRP), macrophage inflammatory protein 1 (MIP-1), interleukin 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) levels, and increased the IL-10 level. Am-FUC also reduced TNF-α, CRP, MIP-1, IL-1β, and IL-6 mRNA expressions, and increased IL-10 mRNA expression in epididymal adipose tissues. Am-FUC reduced Bid, Bax, cytochrome c, caspase 9, and caspase 3 mRNA expressions, and increased Bcl-2 and Bcl-xL mRNA expressions. Am-FUC down-regulated t-Bid, Bax, cytochrome c, and caspase 9 activities, cleaved caspase 3 proteins, and up-regulated Bcl-2 and Bcl-xL proteins. Thus, an Am-FUCblocked mitochondrial pathway was the suppression mechanism in pancreatic islet cell apoptosis via regulation of inflammatory cytokines providing dietary intervention in type 2 diabetes and inflammation-induced pancreatic islet apoptosis.

P53-Derived peptides conjugation to PEI: an approach to producing versatile and highly efficient targeted gene delivery carriers into cancer cells

ABSTRACTObjectives: Targeted delivery of cytotoxic drugs or therapeutic antisense RNAs into specific cells is a major bottleneck in cancer therapy. To overcome this problem and improve the specificity for cancer cells, we describe a new-targeted delivery system using p53-derived peptides, namely PNC 27 and PNC 28. These peptides target HDM-2 on the surface of cancer cells. HDM-2 is overexpressed on the surface of cancerous cells, but not present on the untransformed cells.Methods: To determine HDM-2-expressing cells, we used immunocytochemistry and flow cytometry analysis on nine cell lines including MCF-7 and NIH-3t3. Conjugation of peptides to vectors was confirmed using reverse-phase high-pressure liquid chromatography (RP-HPLC). Physicochemical properties of vector/DNA complexes including particle size, surface charge and DNA condensation ability were determined. In transfection studies, three plasmids were used including luciferase, pEGFP and shRNA plasmid against Bcl-XL mRNA. The level of Bcl-XL expression was determined by real-time PCR and western blot techniques.Results: The results of gene delivery and shRNA-based gene silencing studies indicated that conjugation of PNC peptides could enhance gene delivery efficiently with high-targeted activity exclusively into cancer cells.Conclusion: Our results strongly indicated that this targeting system could be utilized as an efficient targeting method for most cancer cells.

Interleukin-22 ameliorates acute severe pancreatitis-associated lung injury in mice.

To investigate the potential protective effect of exogenous recombinant interleukin-22 (rIL-22) on L-arginine-induced acute severe pancreatitis (SAP)-associated lung injury and the possible signaling pathway involved. Balb/c mice were injected intraperitoneally with L-arginine to induce SAP. Recombinant mouse IL-22 was then administered subcutaneously to mice. Serum amylase levels and myeloperoxidase (MPO) activity in the lung tissue were measured after the L-arginine administration. Histopathology of the pancreas and lung was evaluated by hematoxylin and eosin (HE) staining. Expression of B cell lymphoma/leukemia-2 (Bcl-2), Bcl-xL and IL-22RA1 mRNAs in the lung tissue was detected by real-time PCR. Expression and phosphorylation of STAT3 were analyzed by Western blot. Serum amylase levels and MPO activity in the lung tissue in the SAP group were significantly higher than those in the normal control group (P < 0.05). In addition, the animals in the SAP group showed significant pancreatic and lung injuries. The expression of Bcl-2 and Bcl-xL mRNAs in the SAP group was decreased markedly, while the IL-22RA1 mRNA expression was increased significantly relative to the normal control group (P < 0.05). Pretreatment with PBS did not significantly affect the serum amylase levels, MPO activity or expression of Bcl-2, Bcl-xL or IL-22RA1 mRNA (P > 0.05). Moreover, no significant differences in the degrees of pancreatic and lung injuries were observed between the PBS and SAP groups. However, the serum amylase levels and lung tissue MPO activity in the rIL-22 group were significantly lower than those in the SAP group (P < 0.05), and the injuries in the pancreas and lung were also improved. Compared with the PBS group, rIL-22 stimulated the expression of Bcl-2, Bcl-xL and IL-22RA1 mRNAs in the lung (P < 0.05). In addition, the ratio of p-STAT3 to STAT3 protein in the rIL-22 group was significantly higher than that in the PBS group (P < 0.05). Exogenous recombinant IL-22 protects mice against L-arginine-induced SAP-associated lung injury by enhancing the expression of anti-apoptosis genes through the STAT3 signaling pathway.

Interleukin-6: A Constitutive Modulator of Glycoprotein 130, Neuroinflammatory and Cell Survival Signaling in Retina.

ObjectiveThe interleukin-6 (IL-6) family of cytokines and their signal transducer glycoprotein (gp130) are implicated in inflammatory and cell survival functions in glaucoma. There are several avenues for interdependent modulation of IL-6 family members and gp130 signaling. Here we investigated whether IL-6 modulates gp130 and related neuroinflammatory, cell survival and regulatory signaling in both healthy and glaucomatous retina.MethodsIn naïve and glaucomatous (Microbead Occlusion Model), wildtype (WT) and IL-6 knockout (IL-6−/−) mice, we examined gp130 protein expression and localization, using western blot and immunohistochemistry. Gene targets related to IL-6 and gp130 signaling and pertinent to neuroinflammation (TNFα, IL-1β), cell health (Bax, Bcl-xl) and STAT3 regulation (Socs3) were quantified using qRTPCR.ResultsIn the naïve retina, IL-6−/− retina contained significantly less gp130 compared to WT retina. This IL-6-related decrease in gp130 was accompanied by a reduction in mRNA expression of TNFα, Socs3 and Bax. After 4 weeks of microbead-induced ocular hypertension, both microbead- and saline-injected (control) eyes of IL-6−/− mice exhibited higher expression of TNFα, compared to WT mice. IL-1β expression was also reduced specifically in IL-6−/− retina with microbead-induced glaucoma. While saline and microbead injection increased Bcl-xl and Socs3 mRNA in both WT and IL-6−/− mice, IL-6−/− deficiency led to smaller increases for both Bcl-xl and Socs3.ConclusionsOur findings support a role for IL-6 in setting baseline parameters for neuroinflammatory, cell health and gp130 regulatory signaling that can impact the nature and magnitude of retinal responses to glaucoma-related stressors.

Influence of chronic alcohol treatment on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes in the mouse brain: Role of the C1473G polymorphism in the gene encoding tryptophan hydroxylase 2

Tryptophan hydroxylase 2 (Tph-2) is the key enzyme in serotonin biosynthesis. Serotonin is one of the main neurotransmitters involved in the regulation of various physiological functions and behavior patterns. The influence of chronic ethanol consumption on the expression of the Bdnf, Bax, Bcl-xL, and CASP3 genes was studied in the brain structures of B6-1473C (C/C) and B6-1473G (G/G) mice that had been obtained on the base of the C57BL/6 strain. The strains differed in the genotype for the C1473G single nucleotide polymorphism in the Tph-2 gene and in Tph-2 enzyme activity. It was found that chronic alcohol treatment led to a significant increase in the expression of the Bdnf gene in the midbrain of B6-1473G mice, but not in B6-1473С. Chronic alcohol treatment considerably decreased the expression of the ultimate brain apoptosis effector, caspase 3, in the frontal cortex, but increased it in the hippocampus of B6-1473G mice. At the same time, chronic ethanol administration reduced the level of the antiapoptotic Bcl-xL mRNA in the midbrain of B6-1473C mice. Thus, the C1473G polymorphism in the Tph-2 gene considerably influenced the changes in the expression patterns of genes involved in the regulation of neurogenesis and neural apoptosis induced by chronic ethanol treatment.

Effect of ukrain on ischemia/reperfusion-induced kidney injury in rats

BackgroundThe aim of this study was to investigate the potential protective effect of ukrain on an experimental kidney injury model induced by ischemia and reperfusion (IR) in rats. Material and methodsA total of 24 male Sprague–Dawley rats were equally and randomly separated into three groups as follows: group-1: controls (C; only laparotomy); group 2: renal ischemia–reperfusion (IR; occlusion of the renal artery for 30 min and 2 h of reperfusion); and group 3: ukrain treatment and IR applied group (U + IR; occlusion of the renal artery for 30 min and 2 h of reperfusion; ukrain was intraperitoneally administered 1 h before the IR process). ResultsSerum total oxidant status (TOS) and total antioxidant status (TAS) levels were measured. The oxidative stress index was determined by calculating the TOS/TAS ratio. TAS serum levels significantly increased, and TOS serum levels also prominently decreased in U + IR group, when compared with the IR group (P < 0.001). Mean NGAL level was remarkably higher in IR group, when compared with the U + IR group (P < 0.001). Caspase-3 messenger RNA (mRNA) expression level increased in IR and decreased in U + IR group (P < 0.001). Bcl-xL serum and mRNA expression levels increased in the U + IR group (P < 0.001). In addition, serum iNOS and mRNA expression levels increased in IR group and decreased in U + IR group (P < 0.001). ConclusionsData established from the present study suggest that ukrain may exhibit protective effect against IR-induced kidney injury and that antioxidant activity primarily modulates this effect.

Superior Activity of BET Protein Bromodomain Antagonist-Based Combination with JAK Kinase, PIM Kinase or Heat Shock Protein 90 Inhibitor Against Post-MPN-MF sAML Cells

Myeloproliferative Neoplasms with myelofibrosis (MPN-MF) demonstrate constitutive activation of the JAK-STAT signaling and often progress (~20%) to AML (sAML). As a single agent, JAK1&2 inhibitor ruxolitinib confers significant clinical benefit in MPN-MF, but exhibits modest activity in sAML, which is also incurable with standard anthracycline and Ara-C-based chemotherapy. This supports the rationale to develop and test novel combination therapies for post-MPN-MF sAML. Genetic alterations documented in sAML include those in JAK2, MPL or calreticulin (CALR) gene, as well as in TP53, TET2, ASXL1, IDH1&2, SRSF2, RUNX1, MYC, PTPN11, NRAS, and SETBP1 genes. Variant allelic frequency analyses demonstrated a common co-occurrence of JAK2V617F and mutant TP53 in the dominant clones of sAML. We and others have previously reported that treatment with BET (bromodomain and extra-terminal) protein bromodomain antagonists (BA) results in growth arrest, differentiation and apoptosis of AML cells (Mol Cancer Ther 2014,13:2315). In the present studies, we demonstrate that treatment with JQ1 but not its inactive enantiomer (R-JQ1) dose-dependently (100 to 2000 nM) mediates growth inhibition and apoptosis of the cultured (HEL92.1.7, SET2 and UKE1 cells) and primary (p) post-MPN-MF sAML cells. JQ1 treatment reduced the promoter occupancy of BRD4 and RNA polymerase II on MYC, BCL2 and IL7R promoters, attenuated the mRNA and protein expressions of BCL2, BCL-xL, MYC, CDK4/6 and PIM1, and repressed the pSTAT5 and pSTAT3 levels, while concomitantly inducing the levels of HEXIM1, p21, p27 and BIM in the sAML cells. Following engraftment of NOD/scid/IL-2Rγ null) (NSG) mice with HEL92.1.7 cell xenografts, treatment with JQ1 (50 mg/kg/day, administered IP daily x 5 days per week x 3 weeks) also significantly improved the median survival of the mice (p < 0.01). Compared to treatment with each agent alone, co-treatment with JQ1 and ruxolitinib (100 to 1000 nM) or pacritinib (250 to 1000 nM), which is also a clinically active JAK2, JAK2-V617F and Fms-like tyrosine kinase 3 inhibitor that does not inhibit JAK1, was synergistically lethal against the cultured sAML cells (Combination indices of < 1.0 on the isobologram analyses). Additionally, co-treatment with JQ1 and ruxolitinib caused a marked inhibition of pJAK2, pSTAT5, pSTAT3, MYC, CDK4/6, BCL-xL and PIM1 in the sAML cells. Co-treatment with JQ1 and the pan-PIM kinase inhibitor AZD1208 (500-3000 nM), which inhibits the PIM kinase substrates BAD, p70S6 kinase and 4EBP1, also synergistically induced apoptosis of the cultured sAML cells (CI < 1.0). HEL92.1.7 and SET2 cells are not only positive for the JAK2V617F mutation, they also express mutant TP53 (M133K in HEL92.1.7 and R248W in SET2 cells). Co-treatment with JQ1 and the heat shock protein (HSP) 90 inhibitor AUY922 (2.5-20 nM), which is known to down-regulate the levels of mutant-TP53, JAK2, c-RAF, pSTAT5, pSTAT3 and pAKT, is synergistically lethal against HEL92.1.7 and SET2 cells. We have isolated JAK inhibitor-resistant HEL92.1.7 cells (> 10-fold resistant to ruxolitinib; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAK inhibitor (Clin Cancer Res 2011;17:7347). Compared to the parental HEL92.1.7, HEL/JIR cells are highly and collaterally sensitive to AUY922. HEL/JIR cells also remain sensitive to JQ1-induced apoptosis. Importantly, co-treatment with JQ1 and AUY922 is also synergistically lethal against HEL/JIR cells (CI < 1.0). Taken together, these findings highlight the pre-clinical activity of BA-based combination with JAK inhibitors (ruxolitinib or pacritinib), PIM kinase inhibitor (AZD1208) or HSP90 inhibitor (AUY922) against JAK kinase inhibitor-sensitive sAML, as well as of the BA-based combination with HSP90 inhibitor or pan-PIM kinase inhibitor against JAK inhibitor-resistant sAML cells. Collectively, these findings also support further in vivo testing of these BA-based combinations against sAML cells. DisclosuresNo relevant conflicts of interest to declare.

BCL2-Inhibitors Target a Major Group of Newly-Diagnosed and Relapsed/Refractory Acute Myeloid Leukemia Ex Vivo

BACKGROUND:BCL-2 family members play a critical role in the regulation of apoptosis. BCL-2 and BCL-XL promote cell survival by preventing mitochondrial apoptotic pore formation. BH3 mimetic drugs such as venetoclax (ABT-199) promote apoptosis by inhibiting BCL-2 while navitoclax (ABT-263) inhibits both BCL-2 and BCL-XL. In AML, the expression of anti-apoptotic proteins is highly variable. In a recent study venetoclax showed single-agent activity in 6/12 AML cell lines and 20/25 patient samples. (Pan et al. Cancer Disc 2014). The samples with complex cytogenetics were largely resistant. Sensitivity correlated with increased BCL-2 protein levels and negatively correlated with BCL-XL and MCL-1 protein levels. We aimed to expand these data to both newly diagnosed and relapsed AML patients and to isolate biomarkers for patient selection.METHODS:We assessed the ex vivo sensitivity of fresh leukemic cells from 16 diagnosed and 36relapsed/refractory AML patient samples to venetoclax (25 samples) and navitoclax (52 samples). Exome sequencing was performed on 32 samples and gene expression of BCL2 family members (BCL-2, BCL-XL, MCL-1, BIK, BAX, BAK1, BID, BCL2L12, BIM, BCL2A1, PUMA and BAD) was determined on 31 samples by qRT-PCR. Samples from primary cells of healthy individuals (n=10), and CMML (n=7) or CLL (n=2) patients were used as controls. Drug sensitivity was determined over a 10,000-fold concentration range (1-10 000 nM). A leukemia-specific drug sensitivity score (sDSS) derived from area under the dose response curve calculations was used as the efficacy variable by comparing leukemia results with those from normal bone marrow cells (Bhagwan et al., Sci Rep 2014, Pemovska et al., Cancer Disc 2014).RESULTS:Compared to healthy controls, CMML samples were largely non-sensitive, whereas CLL samples were highly sensitive to BCL-2 inhibitors ex vivo. The AML samples exhibited heterogeneous responses. 15/25 (60%) of AML samples were sensitive to venetoclax and 35/52 (67%) to navitoclax.Both diagnostic (12 of 16 samples, 75%) and relapsed/refractory samples (24 of 36 samples, 64%) were sensitive to navitoclax. Similarly, 6/7 (86%) of diagnostic samples and 9/18 (50%) of relapsed/refractory samples were sensitive to venetoclax. We observed responses to venetoclax and navitoclax in each patient to be similar, although navitoclax showed efficacy at lower concentrations: in 25 samples tested with both agents, mean sDSS values were lower in navitoclax-treated samples (paired t-test, p=0.02). All except one patient sample exhibited a difference in resistance between the two drugs showing sensitivity to navitoclax but not to venetoclax.We observed responses across all mutational profiles, including samples with mutations to FLT3 -ITD, NPM1, TP53, NRAS and IDH1 and IDH2, as well as in samples with complex karyotypes. Intriguingly, three of four samples with mutated TP53 exhibited sensitivity to BCL2 inhibition. No single mutation predicted sensitivity or resistance. At the RNA level, no statistical correlation between BCL2 or BCL-XL expression for BCL2 inhibitor response was observed. Instead we observed high levels of beta-2-microglobulin (B2M) mRNA expression in BCL2 inhibitor-resistant samples with a strong negative correlation to navitoclax sensitivity (r=-0.60, P=0.0008).DISCUSSION:We did not observe BCL2 and BCL-XL mRNA expression to be optimal predictors for BCL2 inhibitor response. On the other hand, we observed high expression of B2M mRNA expression in resistant samples suggesting that it could serve as a biomarker for sensitivity to BCL2 inhibitors. The high B2M expression has been previously linked to poor prognosis in solid tumors and in AML (Albitar et al., Leukemia 2007). In cell line models B2M leads to phosphorylation and inactivation of proapototic protein BAD (Nokura et al., J Urol 2007). This may affect the balance between pro- and antiapoptotic proteins and thus offer a escape route from BCL2 inhibition.To conclude, we observed BCL2 inhibition to be effective ex vivo in over half of all AML samples, tested both in primary and relapsed/refractory state as well as across different subgroups defined by AML driver mutations. Of the potential biomarkers that were assessed, B2M was the best mRNA-level indicator for anti-BCL-2 drug efficacy. DisclosuresOff Label Use: BCL2 inhibitors are not approved for the treatment of AML. Heckman:Celgene: Honoraria, Research Funding; Pfizer: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

Anti-Apoptotic Protein Bcl-xL Expression in the Midbrain Raphe Region Is Sensitive to Stress and Glucocorticoids.

Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with Bcl-xL, caused a significant increase in tph2 mRNA levels and slightly enhanced tryptophan hydroxylase immunoreactivity in the midbrain. The increasing effect on the bcl-xl expression was specific to the short-term stress. Forced swim repeated daily for 2 weeks led to a decrease in bcl-xl mRNA in the midbrain without any effects on the Bcl-xL protein expression in the 5-HT neurons. In chronically stressed animals, an increase in tph2 gene expression was not associated with any changes in tryptophan hydroxylase protein levels. Our findings are the first to demonstrate that both short-term stress and acute glucocorticoid exposures induce Bcl-xL protein expression in the midbrain 5-HT neurons concomitantly with the activation of the 5-HT synthesis pathway in these neurons.

Pro-apoptotic effects of splice-switching oligonucleotides targeting Bcl-x pre-mRNA in human glioma cell lines.

Alternative splicing is a near-ubiquitous phenomenon with important roles in human diseases, including cancers. Splice-switching oligonucleotides (SSOs) have emerged as a class of antisense therapeutics that modulate alternative splicing by hybridizing to the pre-mRNA splice site. The Bcl-x gene is alternatively spliced to express anti‑apoptotic Bcl-xL and pro-apoptotic Bcl-xS. Bcl-xL expression is upregulated in many cancers and is considered a general mechanism by which cancer cells evade apoptosis. By redirecting Bcl-x pre-mRNA splicing from Bcl-xL to Bcl-xS, SSO exerted pro-apoptotic and chemosensitizing effects in various cancer cell lines. In this study, we investigated the effects of SSO targeting Bcl-x pre-mRNA in human glioma cell lines. First, we performed reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine the mRNA and protein expression levels of Bcl-xL in glioma cell lines (U87 and U251) and a normal human astrocyte cell line (HA1800). Then, the Bcl-x SSO was designed to bind to the downstream 5' alternative splice site of exon 2 in Bcl-x pre-mRNA and was modified using 2'-O-methoxyethyl-phosphorothioate. An oligonucleotide targeting aberrantly spliced human β-globin intron was used as a negative control. The SSOs were delivered with a cationic lipid into glioma and astrocyte cell lines. The antitumor effects of the SSOs were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and flow cytometry, and the switch in production from Bcl-xL to Bcl-xS was analyzed by RT-PCR and western blotting. Bcl-xL mRNA and protein were highly expressed in both glioma cell lines. The Bcl-x SSO modified Bcl-x pre-mRNA splicing and had pro-apoptotic effects on the glioma cell lines. By contrast, the lipid alone and the control SSO did not affect Bcl-xL expression or induce apoptosis. Our study demonstrated the antitumor activity of an SSO that targets Bcl-x pre-mRNA splicing in glioma cell lines. Bcl-x SSO may be a potential strategy for treating gliomas.

The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism

In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response.

Apoptotic effects of insect tea in HepG2 human hepatoma cells

Insect tea is a traditional and special tea in China. After the HepG2 cancer cells were treated with insect tea ethanol extracts (ITEE) for 48 h, HepG2 cell proliferation was inhibited, with 40, 80 and 160 μg/mL of ITEE showing inhibitory rates of 35.1%, 59.7% and 86.3%, respectively. Compared to the lower concentration, 160 μg/mL ITEE significantly induced apoptosis as determined by flow cytometry analysis, which showed the content of sub-G1 cancer cells was 38.8%. By RT-PCR, ITEE induced apoptosis in HepG2 cancer cells by increasing caspase-3, caspase-8, caspase-9, Bax, p53, p21, E2F1, p73 and decreasing Bcl-2, Bcl-xL, HIAP-1 and HIAP-2 mRNA expression levels. Insect tea is rich in polyphenols, caffeine, flavones, vitamin C and amino acids. There were 17 kinds of amino acids in insect tea. The functional components of insect tea induce apoptosis in HepG2 cancer cells. Insect tea is a good functional food by its anticancer effects.

Propofol-induced rno-miR-665 targets BCL2L1 and influences apoptosis in rodent developing hippocampal astrocytes

Propofol exerts neurotoxic effects on the developing mammalian brains, but the underlying molecular mechanism remains unclear. MicroRNAs (miRNAs) are a class of small noncoding RNAs that modulate gene expression at the post-transcriptional level. However, in specific types of neurocytes, the detailed functions of miRNAs were not entirely understood. We investigated the potential role of miRNAs in astrocyte pathogenesis caused by propofol. We performed genome-wide microRNA expression profiling in immature cultured hippocampal astrocytes by microarray analysis and predicted their targets and functions using bioinformatics tools. The functional effects of one differentially expressed miRNA were examined experimentally in relation to astrocyte viability. The results showed that 13 miRNAs were significantly differentially expressed after both short-term exposure to high-concentration propofol (10μg/ml for 1h) and long-term exposure to low-concentration propofol (0.9μg/ml for 48h), including rno-miR-665, differing significantly between the 2. Bioinformatics predicted putative binding sites for rno-miR-665 existing in the 3′-untranslated region of Bcl-2-like protein 1 BCL2L1 (Bcl-xl) mRNA. Moreover, such relationship was assessed by luciferase reporter assay, qRT-PCR and western blot. Rno-miR-665 which was significantly up-regulated by propofol can suppress BCL2L1 and elevate cleaved caspase-3 expression in immature astrocytes in vitro. Apoptosis of developing hippocampal astrocytes was thus significantly influenced by propofol or rno-miR-665, or both. Taken together, rno-miR-665 is involved in the neurotoxicity induced by propofol via a caspase-3 mediated mechanism by negatively regulating BCL2L1. It might act as an alternative therapeutic target for treatment of neurological disorders in peadiatric prolonged anesthesia or sedation with propofol clinically.

Recombinant Human Erythropoietin Protects Myocardial Cells from Apoptosis via the Janus-Activated Kinase 2/Signal Transducer and Activator of Transcription 5 Pathway in Rats with Epilepsy

ObjectiveTo investigate the potential mechanisms underlying the protective effects of recombinant human erythropoietin (rhEPO) and carbamylated EPO (CEPO) against myocardial cell apoptosis in epilepsy. MethodsRats were given an intra-amygdala injection of kainic acid to induce epilepsy. Groups of rats were treated with rhEPO or CEPO before induction of epilepsy, whereas additional rats were given a caudal vein injection of AG490, a selective inhibitor of Janus kinase 2 (JAK2). At different time points after seizure onset, electroencephalogram changes were recorded, and myocardium samples were taken for the detection of myocardial cell apoptosis and expression of JAK2, signal transducer and activator of transcription 5 (STAT5), caspase-3, and bcl-xl mRNAs and proteins. ResultsInduction of epilepsy significantly enhanced myocardial cell apoptosis and upregulated the expression of caspase-3 and bcl-xl proteins and JAK2 and STAT5a at both the mRNA and protein levels. Pretreatment with either rhEPO or CEPO reduced the number of apoptotic cells, upregulated bcl-xl expression, and downregulated caspase-3 expression in the myocardium of epileptic rats. Both myocardial JAK2 and STAT5a mRNAs, as well as phosphorylated species of JAK2 and STAT5a, were upregulated in epileptic rats in response to rhEPO—but not to CEPO—pretreatment. AG490 treatment increased apoptosis, upregulated caspase-3 protein expression, and downregulated bcl-xl protein expression in the myocardium of epileptic rats. ConclusionsThese results indicate that myocardial cell apoptosis may contribute to myocardial injury in epilepsy. EPO protects myocardial cells from apoptosis via the JAK2/STAT5 pathway in rats with experimental epilepsy, whereas CEPO exerts antiapoptotic activity perhaps via a pathway independent of JAK2/STAT5 signaling.

Effect of ADAM10 Inhibitor GI254023X on Proliferation and Apoptosis of Acute T-Lymphoblastic Leukemia Jurkat Cells In Vitro and Its Possible Mechanisms

To investigate the effect of ADAM10 inhibitor GI254023X on the proliferation and apoptosis of acute T-lymphoblastic leukemia Jurkat cells and its mechanisms. Jurkat cells were treated with different concentrations of GI254023X, the proliferation-inhibition curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis was detected by flow cytometry with Annexin V and 7-AAD staining, the cleavage of Notch1 protein was determined by Western blot, the transcripts of anti-apoptotic genes BCL-2, MCL-1, BCL-xl and Notch1 target gene Hes-1 were detected by real-time PCR. The GI254023X obviously inhibited the proliferation of Jurkat cells in concentration-dependent manner. As compared with the control group, the apoptosis of cells increased along with increment of GI254023X concentration. Compared with control group, the expression of Cleaved Notch1 was down-regulated while the expression of Notch1 was up-regulated in a time-dependent manner after the treatment with GI254023X. The levels of MCL-1 and Hes-1 mRNA transcripts in Jurkat cells were reduced in GI254023X treated group, but did not show obvious effect on the level of BCL-2 and BCL-xl mRNA transcripts. GI254023X can remarkably inhibit proliferation and induce apoptosis of Jurkat cells. The inhibition of Notch1 activation and the down-regulation of apoptosis-related gene MCL-1 may be involved in the process of apoptosis.

The effects of the glial cell line-derived neurotrophic factor (GDNF) on the levels of mRNA of apoptotic genes Bax and Bcl-xl in the brain of mice genetically predisposed to pathological behavior

Similar to the other neurotrophic factors, the glial cell line-derived neurotrophic factor (GDNF) may inhibit apoptosis in vitro. However, the antiapoptotic GDNF effects in vivo are not known. We studied the effect of GDNF central administration of on the mRNA levels of genes encoding the proapoptotic protein Bax and the antiapoptotic protein Bcl-xl in the brain of ASC mice genetically predisposed to depressivelike behavior and mice of their nondepressive parental CBA strain. We found that the GDNF injection increased the Bcl-xl mRNA content in the hippocampi of mice of both strains (ρ < 0.05) and Bax mRNA (ρ < 0.05) in the hippocampus of ASC mice. Thus, we revealed both the antiand proapoptotic effects of GDNF in vivo. These effects substantially depended on the genotype of the animals. We also observed significant interstrain differences in the Bax and Bcl-xl mRNA levels. In ASC mice, the Bax mRNA levels were significantly higher (ρ < 0.001 and ρ < 0.01) in all investigated structures and the content of Bcl-xl mRNA was elevated in the midbrain. Our data demonstrate the activation of apoptotic processes in ASC mice and the substantial compensatory changes, probably directed to the rise of the threshold for neuronal apoptosis.

Myeloid cell leukemia-1 is an important apoptotic survival factor in triple-negative breast cancer

Breast cancer is the second-most frequently diagnosed malignancy in US women. The triple-negative breast cancer (TNBC) subtype, which lacks expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, afflicts 15% of patients and is refractory to current targeted therapies. Like many cancers, TNBC cells often deregulate programmed cell death by upregulating anti-apoptotic proteins of the B-cell CLL/lymphoma 2 (Bcl-2) family. One family member, myeloid cell leukemia-1 (Mcl-1), is commonly amplified in TNBC and correlates with a poor clinical prognosis. Here we show the effect of silencing Mcl-1 and Bcl-2-like protein 1 isoform 1 (Bcl-xL) expression on viability in a panel of seventeen TNBC cell lines. Cell death was observed in a subset upon Mcl-1 knockdown. In contrast, Bcl-xL knockdown only modestly reduced viability, indicating that Mcl-1 is a more important survival factor. However, dual silencing of both Mcl-1 and Bcl-xL reduced viability in most cell lines tested. These proliferation results were recapitulated by BH3 profiling experiments. Treatment with a Bcl-xL and Bcl-2 peptide had only a moderate effect on any of the TNBC cell lines, however, co-dosing an Mcl-1-selective peptide with a peptide that inhibits Bcl-xL and Bcl-2 was effective in each line tested. Similarly, the selective Bcl-xL inhibitor WEHI-539 was only weakly cytotoxic across the panel, but sensitization by Mcl-1 knockdown markedly improved its EC50. ABT-199, which selectively inhibits Bcl-2, did not synergize with Mcl-1 knockdown, indicating the relatively low importance of Bcl-2 in these lines. Mcl-1 sensitivity is not predicted by mRNA or protein levels of a single Bcl-2 family member, except for only a weak correlation for Bak and Bax protein expression. However, a more comprehensive index composed of Mcl-1, Bcl-xL, Bim, Bak and Noxa protein or mRNA expression correlates well with Mcl-1 sensitivity in TNBC and can also predict Mcl-1 dependency in non-small cell lung cancer cell lines.