Overexpression Of Bcl-2 Research Articles

Expression of apoptosis repressor with caspase recruitment domain (ARC) in familial adenomatous polyposis (FAP) adenomas and its correlation with DNA mismatch repair proteins, p53, Bcl-2, COX-2 and beta-catenin

BackgroundColorectal familial adenomatous polyposis (FAP) adenomas exhibit a uniform pathogenetic basis caused by a germline mutation in the adenomatous polyposis gene (APC), but the molecular changes leading to their development are incompletely understood. However, dysregulated apoptosis is known to substantially affect the development of colonic adenomas. One of the key regulatory proteins involved in apoptosis is apoptosis repressor with caspase recruitment domain (ARC).MethodsThe expression of nuclear and cytoplasmic ARC in 212 adenomas from 80 patients was analyzed by immunohistochemistry. We also compared expression levels of ARC with the expression levels of p53, Bcl-2, COX-2, and MMR proteins. Statistical analyses were performed by Spearman’s rank correlation and linear regression test.ResultsARC was overexpressed in the nuclei and cytoplasm of most FAP adenomas investigated. Cytoplasmic ARC staining was moderately stronger (score 2) in 49.1% (n = 104/212) and substantially stronger (score 3) in 32.5% (n = 69/212) of adenomas compared to non-tumorous colorectal mucosa. In 18.4% (n = 39/212) of adenomas, cytoplasmic ARC staining was equivalent to that in non-tumorous mucosa.Nuclear expression of ARC in over 75% of cells was present in 30.7% (n = 65/212) of investigated adenomas, and nuclear expression in 10–75% of cells was detected in 62.7% (n = 133/212). ARC expression in under 10% of nuclei was found in 6.6% (n = 14/212) of adenomas.The correlation between nuclear ARC expression and cytoplasmic ARC expression was highly significant (p = 0.001). Moreover, nuclear ARC expression correlated positively with overexpression of Bcl-2, COX-2 p53 and β-catenin. Cytoplasmic ARC also correlated with overexpression of Bcl-2. Sporadic MMR deficiency was detected in very few FAP adenomas and showed no correlation with nuclear or cytoplasmic ARC.ConclusionsOur results demonstrated that both cytoplasmic and nuclear ARC are overexpressed in FAP adenomas, thus in a homogenous collective. The highly significant correlation between nuclear ARC and nuclear β-catenin suggested that ARC might be regulated by β-catenin in FAP adenomas. Because of its further correlations with p53, Bcl-2, and COX-2, nuclear ARC might play a substantial role not only in carcinomas but also in precursor lesions.98uN_KvtZQTaRuojLZPqfUVideo

Full Activation of Kinase Protein Kinase B by Phosphoinositide-Dependent Protein Kinase-1 and Mammalian Target of Rapamycin Complex 2 Is Required for Early Natural Killer Cell Development and Survival.

The role of PI3K-mTOR pathway in regulating NK cell development has been widely reported. However, it remains unclear whether NK cell development depends on the protein kinase B (PKB), which links PI3K and mTOR, perhaps due to the potential redundancy of PKB. PKB has two phosphorylation sites, threonine 308 (T308) and serine 473 (S473), which can be phosphorylated by phosphoinositide-dependent protein kinase-1 (PDK1) and mTORC2, respectively. In this study, we established a mouse model in which PKB was inactivated through the deletion of PDK1 and Rictor, a key component of mTORC2, respectively. We found that the single deletion of PDK1 or Rictor could lead to a significant defect in NK cell development, while combined deletion of PDK1 and Rictor severely hindered NK cell development at the early stage. Notably, ectopic expression of myristoylated PKB significantly rescued this defect. In terms of mechanism, in PDK1/Rictor-deficient NK cells, E4BP4, a transcription factor for NK cell development, was less expressed, and the exogenous supply of E4BP4 could alleviate the developmental defect of NK cell in these mice. Besides, overexpression of Bcl-2 also helped the survival of PDK1/Rictor-deficient NK cells, suggesting an anti-apoptotic role of PKB in NK cells. In summary, complete phosphorylation of PKB at T308 and S473 by PDK1 and mTORC2 is necessary for optimal NK cell development, and PKB regulates NK cell development by promoting E4BP4 expression and preventing cell apoptosis.

Emergence of Enzalutamide Resistance in Prostate Cancer is Associated with BCL-2 and IKKB Dependencies.

Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance. We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer. ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased. Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

Ion Binding to the bcl-2 G-Quadruplex from Polarizable Simulations with the Drude Force Field

G-quadruplexes (GQs) are noncanonical nucleic acid structures that can form in guanine-rich sequences and can adopt many diverse conformations depending on their constituent nucleotides. GQs are commonly present in DNA promoter regions, so alterations to their structures can contribute to diseases such as cancer. The B-cell lymphoma 2 (bcl-2) proto-oncogene codes for a protein that inhibits cell apoptosis and overexpression of bcl-2 is associated with cancer progression. Transcription of bcl-2 is controlled by two promoters, one of which contains a GQ-forming sequence. Understanding the stabilizing forces in this structure may lead to novel chemotherapeutic design. There are gaps in our current understanding of under what conditions GQs fold, which is crucial information that can give insight into the equilibrium of GQ conformations. To advance our understanding of the dominant bcl-2 promoter GQ, we performed molecular dynamics (MD) simulations using the Drude-2017 polarizable force field. Our simulation outcomes highlight two distinct changes within the GQ that coincide with ion binding. The first is the recruitment of a bulk K+ ion to the solvent-exposed face of the tetrad stem, which led to changes in base dipole moments and the K+ interaction energies of the guanines within the tetrad core. The second is the emergence of an “electronegative pocket” between the tetrad core and the long loop below the tetrad core. Ion binding in the electronegative pocket induces a backbone conformational change that may be relevant for inducing local conformational changes at K+ concentrations similar to those found in the cell. These results suggest two different locations within the bcl-2 GQ that may be targeted by small molecules to induce conformational changes.

Modeling the neuro-protection of theaflavic acid from black tea and its synergy with nimodipine via mitochondria apoptotic pathway.

Ischemic stroke presents a leading cause of mortality and morbidity worldwide. Theaflavic acid (TFA) is a theaflavin isolated from black tea that exerts a potentially neuro-protective effect. However, the dynamic properties of TFA-mediated protection remain largely unknown. In the current study, we evaluated the function of TFA in the mitochondria apoptotic pathway using mathematical modeling. We found that TFA-enhanced B-cell lymphoma 2 (Bcl-2) overexpression can theoretically give rise to bistability. The bistability is highly robust against parametric stochasticity while also conferring considerable variability in survival threshold. Stochastic simulations faithfully match the TFA dose response pattern seen in experimental studies. In addition, we identified a dose- and time-dependent synergy between TFA and nimodipine, a clinically used neuro-protective drug. This synergistic effect was enhanced by bistability independent of temporal factors. Precise application of pulsed doses of TFA can also promote survival compared with sustained TFA treatment. These data collectively demonstrate that TFA treatment can give rise to bistability and that synergy between TFA and nimodipine may offer a promising strategy for developing therapeutic neuro-protection against ischemic stroke.

BCL-6 promotes the methylation of miR-34a by recruiting EZH2 and upregulating CTRP9 to protect ischemic myocardial injury.

Acute myocardial infarction (AMI) and the following heart failure are public health problems faced all over the globe. The current study set out to investigate the role of B-cell lymphoma 6 (BCL-6) in cardiac protection after AMI. Initially, AMI mouse models and H9c2 cell oxygen-glucose deprivation (OGD) models were established. The cell models were transfected with the vectors containing oe-BCL-6, oe-EZH2, sh-EZH2, miR-34a mimic, and miR-34a inhibitor. RT-qPCR and Western blot analysis were applied to detect the expression patterns of microRNA-34a (miR-34a), BCL-6, enhancer of zeste homolog 2 (EZH2), and C1q tumor necrosis factor-related protein 9 (CTRP9) in the treated cell models. ChIP-qPCR and co-immunoprecipitation assay were performed to detect EZH2 enrichment and H3K27me3 levels in the miR-34a promoter region and the interaction between BCL-2 and EZH2, respectively. EdU staining, TUNEL staining, and flow cytometry were performed to detect cell proliferation and apoptosis, while ELISA was conducted to detect the oxidative stress levels. It was found that miR-34a was highly expressed in heart tissues of AMI models, while BCL-6 and EZH2 were poorly expressed. BCL-2 overexpression increased the recruitment of EZH2, upregulated H3K27me3 level in the miR-34a promoter region, and inhibited the miR-34a expression. Ctrp9, the downstream negative-regulatory molecule of miR-34a, was upregulated. Besides, miR-34a/CTRP9 expression changes were found to affect cardiomyocyte apoptosis, oxidation stress, and proliferation, and prevent myocardial injury in AMI mice. Our findings indicate that BCL-6 increases the level of H3K27me3 in the promoter region of miR-34a via EZH2 recruitment and CTRP9 upregulation, which inhibits the apoptosis of myocardial cells.

The impact of cell-of-origin, MYC/Bcl-2 dual expression and MYC rearrangement on disease relapse among early stage diffuse large B-cell lymphoma patients treated with combined modality therapy

We addressed the prognostic impact of cell-of-origin (COO), MYC and Bcl-2 overexpression as well as isolated MYC rearrangement among 111 patients with limited stage diffuse large B-cell lymphoma (DLBCL) treated with consolidative radiation therapy (RT) after a metabolic complete response to immunochemotherapy. With a median follow-up of 31.1 months (95% CI 27.4 − 34.8), 4 relapses occurred. The 3-year progression free survival (PFS), overall survival (OS), and loco-regional relapse free survival (LRFS) for the cohort were 95%, 96%, and 100%, respectively. There were no differences in OS, PFS, or LRFS based on COO or MYC/Bcl-2 dual expression (DE). Similarly, patients with MYC translocations without BCL2 or BCL6 rearrangements did not have worse outcomes. Consolidative RT produced excellent local control, regardless of DLBCL biology, with one late in-field failure.

Prognostic significance of BIRC7/Livin, Bcl-2, p53, Annexin V, PD-L1, DARC, MSH2 and PMS2 in colorectal cancer treated with FOLFOX chemotherapy with or without aspirin.

Evasion of apoptosis is associated with treatment resistance and metastasis in colorectal cancer (CRC). Various cellular processes are associated with evasion of apoptosis. These include overexpression of pro-apoptotic proteins (including p53 and PD-L1), anti-apoptotic proteins (BIRC7/Livin and Bcl-2), chemokine receptors (including DARC), and dysregulation of DNA mismatch repair proteins (including MSH2 and PMS2). The aim of this study was to determine the effect of folinic acid, 5-FU and oxaliplatin (FOLFOX) as a single agent and aspirin plus FOLFOX in various combinations on the aforementioned proteins in human CRC, SW480 cell line and rat models of N-Methyl-N-Nitrosourea (NMU)-induced CRC. In addition, effects of the NMU-induced CRC and chemotherapeutic regimens on haematological and biochemical parameters in the rat models were studied. Immunohistochemistry, immunofluorescence and immunoblot techniques were used to study the expression pattern of the related proteins in the human CRC cells pre- and post-treatment. Double contrast barium enema, post-mortem examination and histological analyses were used to confirm tumour growth and the effect of the treatment in vivo in rat models. Notably, we found in human mucinous CRC, a significant increase in expression of the BIRC7/Livin post-FOLFOX treatment compared with pre-treatment (p = 0.0001). This increase provides new insights into the prognostic role of BIRC7/Livin in evasion of apoptosis and facilitation of treatment resistance, local recurrence and metastasis particularly among mucinous CRCs post-FOLFOX chemotherapy. These poor prognostic features in the CRC may be further compounded by the significant suppression of DARC, PD-L1, PMS2 and overexpression of MSH2 and anti-apoptotic Bcl-2 and p53 proteins observed in our study (p < 0.05). Importantly, we found a significant reduction in expression of BIRC7/Livin and reactivation of DARC and PD-L1 with a surge in Annexin V expression in rat models of CRC cells post-treatment with a sequential dose of aspirin plus FOLFOX compared with other treatments in vivo (p <0.05). The mechanistic rational of these effects underscores the importance of expanded concept of possible aspirin combination therapy with FOLFOX sequentially in future CRC management. Validation of our findings through randomized clinical trials of aspirin plus FOLFOX sequentially in patients with CRC is therefore warranted.

Generation and Surgical Analysis of Genetic Mouse Models to Study NF-κB-Driven Pathogenesis of Diffuse Large B Cell Lymphoma.

Enforced activation of NF-κB signaling can be achieved by constitutive NF-κB-inducing kinases, IKK2 and NIK, or via lymphoma-associated mutants of MYD88, CARD11, and CD79B. In order to model Diffuse Large B Cell Lymphoma (DLBCL) in mice, conditional alleles for these proteins are combined with alleles targeting Cre recombinase expression in mature B cells. However, unopposed NF-κB signaling promotes plasmablast differentiation, and as a consequence the model system must be complemented with further mutations that block differentiation, such as Prdm1/BLIMP1 inactivation or overexpression of BCL6. Here, we describe the currently available tools for DLBCL models in mice and their relative advantages and drawbacks. Furthermore, we describe methods to monitor lymphomagenesis, using ultrasound tomography of the spleen, and the technique of partial splenectomy surgery with recovery. These powerful techniques allow paired comparison of individual lymphoma cases before and after interventions, including therapies, and to study the evolution of lymphoma over time. NF-κB activation also promotes widespread nodal involvement with lymphoma and we describe the post-mortem dissection of major nodal groups.

Mangiferin Inhibits Inflammation and Cell Proliferation, and Activates Proapoptotic Events via NF-κB Inhibition in DMBA-Induced Mammary Carcinogenesis in Rats.

The purpose of this study was to investigate the anti-inflammatory, antiproliferatiive, and proapoptotic molecular mechanisms of mangiferin (MGN) against mammary carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA). Mammary cancer in rats was induced by single-dose subcutaneous injection of 0.5 ml DMBA (80 mg/kg in sesame oil) in the mammary gland. Increased tumor incidence and volume and other tumorigenic properties were observed. Further, we observed in these rats reduced antioxidant enzyme activity and elevated thiobarbituric acid reactive substance (TBARS) levels in plasma and tissues. DMBA-induced rats shows enhanced expression of the inflammatory markers NF-κBp65, COX-2, and iNOS and proliferation of PCNA and Cyclin D1, and overexpression of the antiapoptotic marker Bcl-2. Mangiferin (100 mg/kg body weight), administered orally once per day, significantly enhanced (p < 0.05) antioxidant levels and reduced TBARS levels. Moreover, MGN inhibited NF-κBp65 nucleus transcriptional activation, thereby suppressing inflammation and cell proliferation, and it increased proapoptotic proteins. Apoptosis was confirmed by TUNEL assay. In summary, MGN suppressed DMBA-induced mammary carcinogenesis through enhanced antioxidant levels, NF-κB inhibition, and positive regulation of apoptotic signals.

Upregulation of MicroRNA-34a Sensitizes Ovarian Cancer Cells to Resveratrol by Targeting Bcl-2.

PurposeResveratrol (REV), a natural compound found in red wine, exhibits antitumor activity in various cancers, including ovarian cancer (OC). However, its potential anti-tumor mechanisms in OC are not well characterized. Here, we tried to elucidate the underlying mechanisms of REV in OC cells.Materials and MethodsThe anti-proliferative effects of REV against OC cells were measured using CCK-8 assay. Apoptosis was measured using an Annexin V-FITC/PI apoptosis detection kit. The anti-metastasis effects of REV were evaluated by invasion assay and wound healing assay. The miRNA profiles in REV-treated cells were determined by microarray assay.ResultsOur results showed that REV treatment suppresses the proliferation, induces the apoptosis, and inhibits the invasion and migration of OV-90 and SKOV-3 cells. miR-34a was selected for further study due to its tumor suppressive roles in various human cancers. We found miR-34a overexpression enhanced the inhibitory effects of REV on OC cells, whereas miR-34a inhibition had the opposite effect in OC cells. In addition, we verified that BCL2, an anti-apoptotic gene, was found directly targeted by miR-34a. We also found that REV reduced the expression of Bcl-2 in OC cells. Further investigations revealed that overexpression of Bcl-2 significantly abolished the anti-tumor effects of REV on OC cells.ConclusionOverall, these results demonstrated that REV exerts anti-cancer effects on OC cells through an miR-34a/Bcl-2 axis, highlighting the therapeutic potential of REV for treatment of OC.

Eriodictyol corrects functional recovery and myelin loss in SCI rats.

BackgroundThis study investigated the therapeutic potential of eriodictyol (EDC) in spinal cord injury (SCI) rats and also the mechanism involved.MethodsThe SCI model was created in Sprague-Dawley rats by the weight drop method. The SCI rats were divided into four groups, namely, Sham operated group (submitted for laminectomy only), control rats (vehicle treated), rats treated with 10 mg/kg EDC and rats treated with 20 mg/kg EDC. EDC or vehicle was injected in The SCI rats via subarachnoid route at the lumbar level 4 just after inducing SCI. The open field and inclined plane tests were done for assessing the locomotor activity. Histopathological analysis of the injured site of the spinal cord was done. Western blot analysis and immunohistochemical analysis were done for the expression of Bcl-2, Bax, glial cell line–derived neurotrophic factor (GCDNF) and brain-derived neurotrophic factor (BDNF).ResultsThe outcomes suggested that EDC-treated rats showed significant improvement in the locomotor activity and also exhibited low myelin loss. The rats also showed overexpression of Bcl-2 and Bax. The treatment of EDC also increased the levels of GCDNF and BDNF after SCI. These outcomes suggested that EDC exerted the neuroprotective effect and also improved the locomotor activity by improving the levels of GCDNF and BDNF and blocking the apoptosis-related proteins.ConclusionThis study suggests that EDC could ameliorate the locomotor function, and the neuroprotective action may be attributed to modulation of GCDNF and BDNF and blockade of apoptosis-associated proteins.

The Roles of Beclin 1 Expression in Gastric Cancer: A Marker for Carcinogenesis, Aggressive Behaviors and Favorable Prognosis, and a Target of Gene Therapy.

Beclin 1 is encoded by Becn1, and plays a role in tumorigenesis, neurodegeneration, apoptosis and autophagy. Here, the aggressive phenotypes and relevant proteins were examined after Beclin 1 expression was altered in gastric cancer cells. We also observed the effects of Beclin 1 on gastric carcinogenesis using Becn1 knockout mice. Finally, clinicopathological significances of Beclin 1 expression were analyzed using meta- and bioinformatics analyses. Becn1 overexpression was found to inhibit proliferation, glucose metabolism, migration and invasion of gastric cancer cells, whereas its knockdown caused the opposite effects. Beclin 1 suppressed the tumor growth by decreasing proliferation and increasing apoptosis. The heterozygous abrogation of Becn1 in gastric pit, parietal and chief cells could not cause any epithelial lesion. Beclin 1-mediated chemoresistance was closely linked to the autophagy, Bax underexpression, and the overexpression of Bcl-2, LRP1, MDR1, and ING5. Bioinformatics analysis showed higher Becn1 mRNA expression in intestinal- than diffuse-type carcinomas (P<0.05), and in male than female gastric cancer patients (P<0.05). Becn1 hyperexpression was positively associated with both overall and progression-free survival rates of the cancer patients (P<0.05). Meta-analysis showed that down-regulated Beclin 1 expression in gastric cancer was positively with lymph node metastasis, TNM staging, dedifferentiation and poor prognosis (P<0.05). Becn1-related signal pathways in gastric cancer included prostate, lung, renal, colorectal, endometrial and thyroid cancers, glioma, and leukemia, the metabolism of amino acid, lipid and sugar, and some signal pathways of insulin, MAPK, TRL, VEGF, JAK-STAT, chemokine, p53, lysosome, peroxidome and ubiquitin-mediated protein degradation (P<0.05). These suggested that Beclin 1 might be considered as a potential marker of gastric carcinogenesis, aggressiveness and prognostic prediction, and as a target of gene therapy in gastric cancer.

BCL(X)L and BCL2 increase the metabolic fitness of breast cancer cells: a single-cell imaging study.

The BCL2 family of proteins regulate apoptosis by controlling mitochondrial outer membrane permeability. However, the effects on mitochondrial structure and bioenergetics have also been reported. Here we comprehensively characterized the effects of BCL2 and BCL(X)L on cellular energetics in MCF7 breast cancer cells using time-lapse confocal single-cell imaging and mitochondrial and cytosolic FRET reporters. We found that BCL2 and BCL(X)L increase the metabolic robustness of MCF7 cells, and that this was associated with increased mitochondrial NAD(P)H and ATP levels. Experiments with the F1F0 synthase inhibitor oligomycin demonstrated that BCL2 and in particular BCL(X)L, while not affecting ATP synthase activity, more efficiently coupled the mitochondrial proton motive force with ATP production. This metabolic advantage was associated with an increased resistance to nutrient deprivation and enhanced clonogenic survival in response to metabolic stress, in the absence of profound effects on cell death. Our data suggest that a primary function of BCL(X)L and BCL2 overexpression in tumor cells is to increase their resistance to metabolic stress in the tumor microenvironment, independent of cell death signaling.

Pivarubicin Is More Effective Than Doxorubicin Against Triple-Negative Breast Cancer In Vivo.

Triple-negative breast cancer (TNBC) is unresponsive to antiestrogen and anti-HER2 therapies, requiring the use of cytotoxic drug combinations of anthracyclines, taxanes, cyclophosphamide, and platinum compounds. Multidrug therapies achieve pathological cure rates of only 20–40%, a consequence of drug resistance and cumulative dose limitations necessitated by the reversible cardiotoxic effects of drug therapy. Safer and more effective treatments for TNBC are required to achieve durable therapeutic responses. This study describes the mechanistic analyses of the novel anthracycline, pivarubicin, and its in vivo efficacy against human primary TNBC. Pivarubicin directly activates PKCd, triggers rapid mitochondrial-dependent apoptosis, and circumvents resistance conferred by overexpression of P-glycoprotein, Bcl-2, Bcl-XL, and Bcr-Abl. As a consequence, pivarubicin is more cytotoxic than doxorubicin against MDA-MB-231, and SUM159 TNBC cell lines grown in both monolayer culture and tumorspheres. Comparative in vivo efficacy of pivarubicin and doxorubicin was performed in an orthotopic NSG mouse model implanted with MDA-MB-231 human TNBC cells and treated with the maximum tolerated doses (MTDs) of pivarubicin and doxorubicin. Tumor growth was monitored by digital caliper measurements and determination of endpoint tumor weight and volume. Endpoint cardiotoxicity was assessed histologically by identifying microvacuolization in ventricular cardiomyocytes. Primary tumors treated with multiple rounds of doxorubicin at MTD failed to inhibit tumor growth compared with vehicle-treated tumors. However, administration of a single MTD of pivarubicin produced significant inhibition of tumor growth and tumor regression relative to tumor volume prior to initiation of treatment. Histological analysis of hearts excised from drug- and vehicle-treated mice revealed that pivarubicin produced no evidence of myocardial damage at a therapeutic dose. These results support the development of pivarubicin as a safer and more effective replacement for doxorubicin against TNBC as well as other malignancies for which doxorubicin therapy is indicated.

Evaluation of vitamin C protective effect on the cerebrocortical antioxidant defense, histopathological, pro-apoptotic p53 and anti-apoptotic Bcl2 expressions against tramadol neurotoxicity in rats

BackgroundReported tramadol toxicity emphasizes the necessity to recognize its mechanism of toxicity, particularly to the brain tissue. Aim: This study aimed to evaluate the protective effect of vitamin C (Vit C) in cerebrocortical toxicity mediated by tramadol in rats using biochemical and histological parameters. Material and methodsForty-eight albino rats were randomly divided into eight groups, (n = 6/group) as follow: the control group received normal saline and vitamin C group received vitamin C (200 mg/kg per oral). Tramadol 50, 100, 150 groups received tramadol in doses of (50, 100 and 150 mg/kg per oral, respectively); Tramadol 50+ Vit C, 100+ Vit C, 150+ Vit C groups received vitamin C (200 mg/kg per oral) plus tramadol in doses of (50, 100 and 150 mg/kg per oral, respectively). Rats had received vitamin C and tramadol daily for 30 days. Blood and brain tissues samples were harvested for biochemical, histopathological, immunohistochemical and electron microscopic examinations. ResultsTramadol administration leads to a significant elevation of MDA, NO levels and a significant decrease in antioxidants parameters (CAT, SOD and GSH) in the tissues of cerebral cortices in rats which were directly proportional to the dose of tramadol. In histological investigations, tramadol-treated groups showed pyknotic pyramidal cells, multiple red neurons and shrinking red neurons with hallows around it and apoptotic cells were detected. These biochemical abnormalities and histological impairment were ameliorated in groups with tramadol low doses by the co-treatment with vitamin C. Conclusionvitamin C has antioxidant and anti-apoptotic potentials against tramadol neurotoxicity via suppression of oxidative stress, lipid peroxidation, structural abnormalities, and down-regulation of p53 and overexpression of Bcl2 in the nervous tissues.

Targeted RNA-Sequencing Enables Detection of Relevant Translocations and Single Nucleotide Variants and Provides a Method for Classification of Hematological Malignancies-RANKING.

Patients with hematological malignancies (HMs) carry a wide range of chromosomal and molecular abnormalities that impact their prognosis and treatment. Since no current technique can detect all relevant abnormalities, technique(s) are chosen depending on the reason for referral, and abnormalities can be missed. We tested targeted transcriptome sequencing as a single platform to detect all relevant abnormalities and compared it to current techniques. We performed RNA-sequencing of 1385 genes (TruSight RNA Pan-Cancer, Illumina) in bone marrow from 136 patients with a primary diagnosis of HM. We then applied machine learning to expression profile data to perform leukemia classification, a method we named RANKING. Gene fusions for all the genes in the panel were detected, and overexpression of the genes EVI1, CCND1, and BCL2 was quantified. Single nucleotide variants/indels were analyzed in acute myeloid leukemia (AML), myelodysplastic syndrome and patients with acute lymphoblastic leukemia (ALL) using a virtual myeloid (54 genes) or lymphoid panel (72 genes). RANKING correctly predicted the leukemia classification of all AML and ALL samples and improved classification in 3 patients. Compared to current methods, only one variant was missed, c.2447A>T in KIT (RT-PCR at 10-4), and BCL2 overexpression was not seen due to a t(14; 18)(q32; q21) in 2% of the cells. Our RNA-sequencing method also identified 6 additional fusion genes and overexpression of CCND1 due to a t(11; 14)(q13; q32) in 2 samples. Our combination of targeted RNA-sequencing and data analysis workflow can improve the detection of relevant variants, and expression patterns can assist in establishing HM classification.

Inhibition of the BCL6/miR-31/PKD1 axis attenuates oxidative stress-induced neuronal damage

Ischemic stroke (IS) is one of the most common cerebrovascular diseases worldwide. The aberrant expression of BCL6 has been previously implicated in the pathogenesis of IS. Meanwhile, miR-31 is known as a target of BCL6, and has also been suggested to diminish cell damage by suppressing the PKD1 expression. Expanding on this relationship, the current study set out to investigate whether BCL6 participates in ischemic stroke by targeting PKD1. Firstly, IS models were established in vitro and in vivo. TUNEL staining and MTT assay were performed to examine the apoptosis and cell survival. In addition, qRT-PCR and Western blot analysis were applied to examine the expression patterns of the BCL6/miR-31/PKD1 axis and its downstream pathway. Bioinformatics analysis was used to predict the target of miR-31. It was found that BCL6 over-expression promoted ODG-induced increase of apoptosis and decreased the cell survival and miR-31 expression levels, whereas the opposite effects were noted in vitro and in vivo models of IS that were treated with shBCL6. Furthermore, miR-31 down-regulation blocked the effect of BCL6 on ODG-induced cell injury. It was also verified that miR-31 directly-targets PKD1. Also, OGD induced the PKD1 expression and activation of the JAK2/STAT3 pathway, while down-regulation of PKD1 inhibited the OGD-induced cell injury and JAK2/STAT3 pathway activation. Lastly, down-regulation of BCL6 in brain brought about a significant reduction in the size of cerebral infarction and oxidative stress levels in IS mice. Collectively, our findings suggest that inhibition of BCL6 may attenuate oxidative stress-induced neuronal damage by targeting the miR-31/PKD1 axis.

Overexpression of the OCT-1 Gene Is a Biomarker Associated with Poor Outcomes in Diffuse Large B-Cell Lymphoma (DLBCL) - Data from a Retrospective Cohort from Latin America: Defining a Very High-Risk Clinical-Molecular Subgroup

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy, representing 30-40% of all non-Hodgkin's lymphomas (NHLs). They comprise a group of aggressive and heterogeneous neoplasms in terms of clinical presentation, response to therapy and prognosis. The OCT-1 gene is a member of the homodomain-POU family of transcriptional regulators of B-lymphoid differentiation. OCT-1 acts by controlling the expression of specific B-cell genes, such as BCL-2, a potent inhibitor of apoptosis that is essential for the differentiation of B-cells in the germinal center. These genes can be expressed in DLBCL, but the role of BCL-2 in its prognosis has been contradictory and the prognostic impact of the OCT-1 gene has not yet been tested in this lymphoma. Methods: In this observational, retrospective, single-center study, we investigated the prognostic impact of BCL-2 and OCT-1 gene expression in Brazilian patients with DLCBL treated with immunopolychemotherapy R-CHOP in a real-world context. The BCL-2 and OCT-1 genes were assessed in 78.5% (77/98) DLBCL patients, and the RNA for quantitative real-time PCR (qRT-PCR) was isolated from formalin-fixed and paraffin-embedded (FFPE) samples. The values obtained for gene expression were transformed into categorical variables according to their medians (6.27 for BCL-2 and 24.5 for OCT-1). The association between clinical and laboratory variables and results of gene expression was verified by the Fischer test. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. Univariate analysis was performed using Cox's bivariate regression method and multivariate analysis using Cox multiple regression methodology. Results: The median age of the cohort was 54.5 years (15-84), 50% (49/98) were male, 49.4% (38/77) and 51.4% (40/77) showed expression of OCT-1 and BCL- 2 ≥ median, respectively. The clinical characteristics of the 98 Brazilian patients with DLBCL that comprised our cohort are summarized in Table 1. The overall response rate (ORR) in all patients was 68.4% (67/98), 65.3% (64/98) showed a complete response (CR), and 3.1% (3/98) showed partial response (PR), while 6.1% (6/98) were primary refractory. With a median follow-up of 3.77 years (95% CI: 3.2-4.1), the median overall survival (OS) was 5.43 years (95% CI: 2.2-NR) and the median progression-free survival (PFS) was 5.15 years (95% CI: 2.9-NR). The 5-year OS and PFS was 54.2% (42.2% -64.8%) and 52.0% (40.1-62.6%), respectively. In the univariate analysis OCT-1 ≥ median was associated with shortened OS (HR: 2.45, 95% CI: 1.21-4.96, p = 0.013) and PFS (HR: 2.27, 95% CI: 1.14-4.51, p = 0.019). Overexpression of BCL-2 was associated with worse PFS (HR: 2.00, 95% CI: 1.02-3.95, p = 0.043). Subgroup analysis showed that OCT-1 overexpression predominated in elderly individuals (≥ 60 years) in a statistically significant mode (29/38 cases - 76.3%, p = 0.029). It was also observed that overexpression of OCT-1 was associated with worse OS in the high-risk adjusted International Prognostic Index (aIPI) subgroup (p = 0.048) - Figure 1, and worse PFS in patients ≥ 60 years old (p = 0.025) - Figure 2. In the multivariate analysis, overexpression of OCT-1 was associated with poor PFS (HR: 2.22, 95% CI: 1.06-4.76, p = 0.035). Conclusion: In this study, we demonstrated that overexpression of the OCT-1 gene was an independent prognostic factor associated with adverse outcomes in Brazilian patients with DLCBL. We also show that in patients with unfavorable risk, such as the elderly and those with intermediate-high and high-risk IPI, overexpression of OCT-1 contributed to the identification of a very high-risk clinical-molecular subgroup, where the results with standard R-CHOP therapy are unsatisfactory, and they may benefit from intensified therapeutic strategies. Our results are preliminary and need to be validated in subsequent studies of prospective nature and with an expanded sample. Disclosures No relevant conflicts of interest to declare.

Therapeutic Activity of Combining BCL-2 and HMG-CoA Reductase Inhibition in Systemic Light-Chain Amyloidosis

Introduction: Overexpression of BCL-2 in association with t(11;14) in multiple myeloma (MM) and systemic light-chain amyloidosis (AL) makes it a therapeutic target for the BCL-2 inhibitor venetoclax; response rates of 60-80% in t(11;14) have been reported in MM (Kumar S, Blood 2017 &amp; Vaxman I, Expert Rev Hematol 2018). Addition of HMG-CoA reductase inhibitors (statins) may augment venetoclax activity (Lee JS, Sci Transl Med 2018). We now report ex-vivo functional activity of venetoclax with AL patient CD138-selected plasma cells and provide clinical outcomes of 8 patients with relapsed/refractory AL who were treated with venetoclax and a statin. Methods: To construct a functional assay NCI-H929 and KMS-12-PE cells were used as controls and incubated with venetoclax to assess the IC50. Cell viability was measured with CellTiter-Glo and caspase activity with Caspase-Glo 3/7 (Promega, Madison, WI). H929 cells were the negative and KMS cells the positive control. From patient marrows CD138+ cells were isolated (Miltenyi Biotec, Auburn, CA) as previously described (Ma X, Gene Ther 2016) and incubated with 100 nM of venetoclax for 18 hours with controls. In AL patients, Venetoclax was started at 200 mg daily and escalated to 400 mg daily after 2 weeks if tolerated. Statin (atorvastatin, 10-40 mg daily or simvastatin 40 mg daily) was started concurrently or for lack of response to venetoclax alone. Bone marrow assessment was performed prior to starting venetoclax and response assessment was performed monthly. Results: Venetoclax induced apoptosis in a dose escalated manner with KMS-12-PE cells (t(11;14) positive) when compared with H929 cells (Figure 1A-B). CD138-selected plasma cells from 20 patients (17 AL, 2 MM, 1 MGUS) were incubated with venetoclax (100 nM) for 18 hours and had a median caspase 3/7 activity level that was significantly higher in patients with t(11;14) (Figure 1C). Eight patients were treated with venetoclax in combination with a statin. Baseline characteristics are provided in Table 1. Median age of the cohort was 70 (range, 59 - 77), of which five (63%) were male, and 6 were λ-type (1-kappa, 1-heavy chain). At diagnosis, four (50%) patients had involvement of two or more organ systems (cardiac, renal, gastrointestinal, vascular, and/or neurological). Cardiac involvement was the most common (88%). Seven patients had t(11;14) and/or positive cyclin D1 staining on pre-treatment marrow studies. The median number of prior therapies was 2 (1 - 5), and all except one had been previously treated with daratumumab. Venetoclax-statin combination was started due to hematologic progression (4), organ progression (1), or suboptimal response to prior therapy (3). At a median follow-up of 8 weeks (5 - 25), overall hematologic response rate is 63% (1 CR, 3 VGPR, 1 PR and 1 progression). Cardiac response was seen in 2 patients. One patient who lacked the t(11;14) mutation had early disease progression. One patient with stage-3 cardiac AL amyloidosis experienced cardiac progression without hematologic response on venetoclax alone but responded promptly with both hematologic and cardiac response to addition of simvastatin, 40 mg daily. Statin dose was reduced in 1 patient due to grade 1 myalgia. All responders continue on treatment at their most recent follow-up. Conclusion: In this cohort of 8 patients with AL amyloidosis treated with a combination of venetoclax and statin, hematologic response rate was 63% and &amp;gt;VGPR was seen in 50%. The combination was well tolerated. Consistent with the preclinical activity of venetoclax in MM, functional activity of venetoclax was similarly higher in plasma cells from AL patients harboring t(11;14). As t(11;14) is the most common cytogenetic abnormality in systemic AL amyloidosis, venetoclax and statin combination may provide a potent therapeutic alternative for relapsed/refractory AL patients and requires validation in clinical trials. Disclosures Chaulagain: Sanofi Genzyme: Honoraria. Comenzo:Takeda: Consultancy, Research Funding; Prothena: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Amgen: Consultancy; Karyopharm: Consultancy, Research Funding; Caleum: Consultancy; Unum: Consultancy; Sanofi: Consultancy. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor and currently approved for non-Hodgkin's lymphoma and Acute myeloid leukemia. Venetoclax has shown clinical activity in clinical trials with multiple myeloma, especially patients who harbor t(11;14). Given the preclinical and clinical evidence of its efficacy, we treated 8 relapsed/refractory patients with systemic light-chain amyloidosis with a combination of venetoclax and a statin.