BCL2-antagonist/killer 1 Research Articles

Anthocyanin-Mediated Autophagy in Hepatocellular Carcinoma: Gene Associations and Prognostic Implications.

Hepatocellular carcinoma (HCC) is a globally prevalent malignancy accompanied by high incidence, poor outcomes, and high mortality. Anthocyanins can inhibit tumor proliferation, migration, invasion, and promote apoptosis. Moreover, autophagy-related genes (ARGs) may play vital roles in HCC progression. This study aimed to decipher the mechanisms through which anthocyanins influence HCC via ARGs and to establish a novel prognostic model. Based on data from public databases, differential analysis and the Venn algorithm were employed to detect intersecting genes among differentially expressed genes (DEGs), anthocyanin- related targets, and ARGs. Consensus clustering was implemented to delineate molecular subtypes of HCC. The prognostic model was developed by Cox regression analyses. CIBIRSORT was engaged to assess the immune cell infiltration. Kaplan-Meier (KM) analysis and receiver operating characteristic (ROC) curve were utilized to evaluate the predictive efficiency of the prognostic signature. A total of 36 intersecting genes were identified from overlapping 1524 ARGs, 537 anthocyanin- related targets, and 5247 DEGs. Consensus clustering determined three molecular subtypes (cluster 1, cluster 2, and cluster 3). Cluster 1 showed worse outcomes and remarkably higher abundances of plasma cells and T follicular helper cells. Furthermore, four prognostic signatures [KDR (Kinase insert domain receptor), BAK1 (BCL2 antagonist/killer 1), HDAC1 (Histone deacetylase 1), and CDK2 (Cyclin-dependent kinase 2)] were identified and showing substantial predictive efficacy. This investigation identified three molecular subtypes of HCC patients and proposed a promising prognostic signature comprising KDR, BAK1, HDAC1, and CDK2, which could supply further robust evidence for additional clinical and functional studies.

Effects of insulin-like growth factor-1 on the proliferation and apoptosis of stallion testicular cells under normal and heat stress culture conditions

This study investigated the effect of heat stress on stallion testicular cells (TCs) and the effect of insulin-like growth factor (IGF)−1 on TC viability, proliferation, and apoptosis, including different stages of germ cells. TCs were divided into control or treatment groups with 0.01, 0.1, 1, 10, and 100 ng/mL of recombinant human IGF-1 (rhIGF-1) for 24 h at 34 °C and 37 °C. The population and viability were measured before and after treatment. The effects of rhIGF-1 on TC viability, proliferation, and apoptosis were determined using RT-qPCR. Proliferating cell nuclear antigen (PCNA) and marker of proliferation Ki-67 (MKI-67) were used as proliferation markers. Myeloid leukemia-1 (MCL-1) was used as an antiapoptotic marker. BCL2 antagonist/killer-1 (BAK-1) was used as a proapoptotic marker. The relative abundance of mRNA transcript of undifferentiated cell transcription factor 1 (UTF-1), protein gene product 9.5 (PGP9.5), and deleted in azoospermia-like (DAZL), was measured for spermatogenesis progression. TCs treated with 1 ng/mL rhIGF-1 at 34 °C exhibited the highest viability. Significant upregulation of the relative abundance of mRNA transcript of PCNA, MKI-67, and MCL-1 was observed in treated TCs compared with untreated TCs; however, BAK-1 was significantly downregulated in treated TCs. Germ cells treated with 1 ng/mL rhIGF-1 exhibited the highest relative abundance of mRNA transcript of UTF-1 and DAZL, whereas TCs exposed to 0.1 ng/mL showed the highest PGP9.5 level. These data confirm that heat stress in stallions decreases TC viability. These findings may help identify a basal IGF-1 level for TC proliferation and apoptosis during heat stress-induced testicular degeneration in stallions.

Decoding the concealed transcriptional signature of the apoptosis-related BCL2 antagonist/killer 1 (BAK1) gene in human malignancies.

BCL2 antagonist/killer (BAK) is a multidomain pro-apoptotic effector protein, encoded by the human BAK1 gene, which has emerged as a key checkpoint in the apoptotic machinery. Disassembly of BAK's tertiary structure, such as the truncation of the α1 helix, leads to deregulation of the pro-apoptotic functions and reduction of the protein's stability, thus being implicated in human malignancies. Although many studies have already clarified the vital role of BAK in cellular mechanisms, its pre-mRNA maturation process under cancerous and physiological human cells is neglected. In the present work, we developed and employed a custom multiplexed nanopore sequencing approach that enabled the identification and structural characterization of previously undescribed BAK1 mRNA transcripts (BAK1 v.2-v.11). The described novel mRNAs are derived from multiple types of alternative splicing events, including exon skipping and intron retentions. The implemented multiplexed long-read sequencing approach provided the detailed expression profile of the novel mRNAs in a wide panel of human malignancies and at the same time allowed their relative quantification as compared to the annotated BAK1 v.1. The validation of each novel transcript was carried out with qPCR-based assays. Our results strongly support that most of the novel BAK1 mRNAs harbor open reading frames with conserved BH domains that provide new insights into the correlated mechanisms of apoptosis suppression and cancer. The current study highlights for the first time the hidden aspects of BAK1's transcriptional landscape in both physiological and cancerous human cells and distinguishes the amino acid sequence of the putative BAK isoforms that may possess key apoptosis-related functions not only in diseases, but also under normal cellular conditions.

CircRNAPTK2 Promotes Cardiomyocyte Apoptosis in Septic Mice by Competitively Binding to miR-29b-3p with BAK1

Objective: Sepsis is a predominant reason for the growing morbidity and mortality in the world. The role of circular RNAs (CircRNAs) is actively researched in sepsis. In this study, we attempt to find out the effect of CircRNA protein tyrosine kinase 2 (circPTK2) on cardiomyocyte apoptosis in septic mice. Methods: Septic mouse model was established by cecal ligation and puncture. Then circPTK2 expression was detected and the role of circPTK2 in myocardial damage was assessed after circPTK2 expression was silenced using Ad-sh-circHIPK3. The subcellular localization of circPTK2 was analyzed. Besides, the binding relation between circPTK2 and microRNA (miR)-29b-3p and between miR-29b-3p and BCL2 antagonist/killer 1 (BAK1) was verified. The expression of miR-29b-3p and BAK1 in the myocardium was detected. Functional rescue was conducted to evaluate the role of miR-29b-3p and BAK1 in cardiomyocyte apoptosis in septic mice. Results: CircPTK2 was highly expressed in the myocardium of septic mice, while circPTK2 silencing relieved the cardiac function and reduced inflammatory reaction and cardiomyocyte apoptosis of septic mice. Mechanically, circPTK2 competitively bound to miR-29b-3p to upregulate BAK1 mRNA level. Inhibition of miR-29b-3p and BAK1 overexpression could counteract the protective role of circPTK2 silencing in the myocardium of septic mice. Conclusion: CircPTK2 is overexpressed in the myocardium of septic mice. CircPTK2 competitively bound to miR-29b-3p to upregulate BAK1 mRNA level, to promote cardiomyocyte apoptosis, inflammatory response, and myocardial damage of the myocardium of septic mice.

MiR-345-3p serves a protective role during gestational diabetes mellitus by targeting BAK1.

Recent studies have demonstrated that microRNAs (miRs) serve a crucial role during the development of gestational diabetes mellitus (GDM). However, the mechanisms underlying miR-345-3p and its protective role during GDM have not been previously reported. The present study investigated miR-345-3p expression and function in vitro, and the possible molecular mechanisms underlying GDM. Compared with healthy pregnant women, miR-345-3p was downregulated in the placental tissue and peripheral blood of patients with GDM. Further investigation revealed that BCL2-antagonist/killer 1 (BAK1) was a predicted target gene of miR-345-3p, and the expression of BAK1 was significantly increased in patients with GDM compared with healthy pregnant women. In vitro analysis revealed that miR-345-3p mimic significantly increased cell viability, migration and invasion, inhibited apoptosis, upregulated Bcl-2 and matrix metallopeptidase 9 expression, and decreased Bax expression compared with the control group. Furthermore, miR-245-3p mimic-induced alterations were reversed by BAK1 overexpression. The results suggested that miR-345-3p overexpression exhibited a protective role in patients with GDM by inhibiting HTR8-/SVneo cell apoptosis, and promoting cell proliferation and migration via targeting BAK1. The use of miR-345-3p for the diagnosis of GDM requires further investigation.

A212 CHROMOFUNGIN PROTECTS AGAINST DSS-INDUCED COLITIS BY REGULATING P-53 APOPTITIC PATHWAY

Abstract Background Development of ulcerative colitis is associated with epithelial apoptosis mediated by p53-apoptotic pathway through the activation B-cell lymphoma 2 (Bcl-2), Bcl-2 associated-X protein (BAX) and Bcl-2 antagonist/killer-1 (BAK1) proteins. Chromofungin (CHR), a chromogranin-A derived peptide expressing a cell penetrating peptide motif, decreased the severity of colitis via the suppression of mucosal and pro-inflammatory macrophages-related p53-dependent apoptosis. Aims We aimed to investigate a) whether the gene profile expression of apoptosis could be extended to other p53-associated genes; b) whether the gene expression of some of the p53-apoptosis marker could be confirmed by protein analysis; and c) whether due to the cell penetrating peptide motif, CHR could enter into peritoneal macrophages. Methods UC-related colitis was induced in C57BL/6 mice (7 weeks) by administering dextran sulfate sodium (DSS) (5%, 5 days). Preventive CHR (2.5 mg/kg/day) or vehicle treatment started 1-day before colitis induction and lasted for 5-days. Profiler™ PCR Array was performed to screen a panel of 84 genes representative of the p53 signal pathway in colitic whole mucosa distal colonic samples treated or not with CHR. Western blot analysis was performed to confirm individual protein changes. Naïve macrophages were plated overnight and nonadherent cells were removed the next day. Cells were incubated with rhodamined CHR (4 ul) for 5, 10, 20, 30 min before washing and fixing them, detection was made via confocal microscopy. Results In colitic conditions, an up regulation of 26 genes associated to the p53-dependent apoptosis pathway were detected including Apaf1, Bax, Bbc3, Bcl2, Cradd, Fadd, Cul9, Pmaip1, Tnfrsf10b. In vivo CHR treatment decreased significantly the colitis and was associated with a significant downregulation of 19 genes including the 9 aforementioned when compared with biopsies from colitic groups. Compared to untreated groups, colitic mice treated with CHR demonstrated a significant decrease of BAX and BAK protein and the apoptotic ER stress inducer marker, X-Binding Protein 1. A large number of peritoneal macrophages displayed rhodamine within the all intracellular compartment. The presence of the peptide inside the cell can be visible as early as 5 min and the signal gradually increases. Conclusions CHR decreases the inflammatory process via the suppression of a large number of p53-related apoptotic proteins. CHR quickly enters the macrophage but the exact mechanism of entrance needs to be further defined. Targeting functional analysis of CHR may lead in the future to novel therapeutics for UC. Funding Agencies CCCNSERC

Inflammation-dependent downregulation of miR-532-3p mediates apoptotic signaling in human sarcopenia through targeting BAK1.

Inflammation and apoptosis are considered as two major pathological causes of human sarcopenia. The current understanding based on different models recognizes that apoptosis does not trigger inflammation, while emerging evidence indicates that inflammation can induce apoptosis. Here, we provide solid evidence to suggest that the inflammation-dependent downregulation of miR-532 causes apoptosis through targeting a proapoptotic gene BAK1 (BCL2 antagonist/killer 1). To identify miRNAs and genes that are aberrantly expressed in the muscle tissues of sarcopenia patients, we conducted two independent microarray analyses. In total, we identified 53 miRNAs and 69 genes with differential expression levels. Of these aberrantly expressed miRNAs, miR-532-3p showed the most obvious changes in sarcopenia tissues, and more importantly, it can be repressed by the well-known inflammatory inducer lipopolysaccharide (LPS) in vitro. According to gene-based microarray results and the predicted targets of miR-532-3p, we presumed that BAK1 was a putative target of miR-532-3p. Further in vitro and in vivo analyses verified that miR-532-3p could directly bind to the three prime untranslated region (3'-UTR) of BAK1 through the seed sequence CUCCCAC. In addition, we found that NFKB1 (also known as p50), a subunit of the transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), could specifically bind to the promoter region of miR-532-3p and repress its expression. Further analysis revealed that the activation of TLR4 (Toll-like receptor 4) signaling led to the translocation of p50 from the cytoplasm to the nucleus, where it repressed miR-532-3p expression and thus led to an increase of BAK1. The accumulated BAK1 activated its downstream apoptotic signaling pathways and resulted in apoptosis, eventually causing the pathogenesis underlying sarcopenia. Overall, our results uncovered a new mechanism by which the inflammation-dependent downregulation of miR-532-3p contributed to the pathogenesis of sarcopenia through mediating BAK1 expression.

GENE-32. SYNTHETIC LETHAL INTERACTIONS WITH IDH1R132H IN GLIOMA STEM-LIKE CELLS

Abstract Gliomas are the most frequently diagnosed human primary brain tumors. Mutations in Isocitrate Dehydrogenase (IDH) 1 occur in the vast majority of low grade gliomas and secondary high grade glioblastomas. A single amino acid missense mutation in IDH1 at arginine 132 (R132H) is an early event in tumor development. IDH1R132H leads to the production of the oncometabolite 2-R-2-hydroxyglutarate. However the exact roles played by IDH1R132H in the development and malignant transformation of the tumors remain unclear. Further studies are required to determine optimal therapeutic strategies to target the IDH1 mutated subsets of gliomas. New generation high-throughput genetic perturbation technologies make it possible to systematically identify the genes and pathways required for the survival and proliferation of mammalian cells. Herein, we present preliminary results from a CRISPR-dCas9 derived activation to drive the transcriptional activation of more than 23,000 coding genes in both wild type and mutant IDH1 patient-derived pediatric glioma cells. Based on an average of three viability screens per cell type, we analyzed the sgRNA library representation in the IDH1 mutated and non-mutated glioma cultures after the genome wide activation. We identified 1553 candidate genes that upon gain of function trigger the death of glioma cells harboring the IDH1 mutation. The analysis of these results further pinpoints the activation of the Cyclin E1 (CCNE1), the BCL2 Antagonist/Killer 1 (BAK1) and the Homeobox B13 (HOXB13) as the most significant synthetic lethal targets in IDH1R132H glioma cells. Interestingly, results from RNAseq showed a decreased expression of these genes in IDH1 mutated compared to non-mutated glioma cells. Thus, this viability screening aims to elucidate genes that interact with IDH1R123H and play a role in tumor cell fitness. The functional analysis of these candidate genes will allow us to uncover their contribution to the progression of IDH1 mutated gliomas.

Stable expression of infliximab in CRISPR/Cas9-mediated BAK1-deficient CHO cells.

To establish stable infliximab-expressing Chinese hamster ovary (CHO) cells with high tolerance to serum-free culture. Bcl-2 antagonist/killer 1 (BAK1), which is a key mediator of the apoptosis pathway, was disrupted, and infliximab, which is a broadly used monoclonal antibody for the treatment of rheumatoid arthritis and other autoimmune diseases, was incorporated into the BAK1 locus of the CHO chromosome using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome-editing technique. The activating effects of serum starvation on BAK1 and cytochrome C (CytC) were suppressed in the genome-edited cells, and the ability of the cells to resist the serum starvation-induced loss of mitochondrial membrane potential and apoptosis was increased, as indicated by the results of polymerase chain reaction (PCR), flow cytometry, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) analysis. In addition, during subsequent passages, infliximab could be stably produced in the genome-edited CHO cells, and the recombinant antibody could effectively antagonize the cytotoxic effect of tumor necrosis factor α (TNFα). A CHO cell line capable of stably expressing infliximab and adapting to serum-free culture was constructed. This work lays the foundation for the development of infliximab biosimilars.

Genes associated with testicular germ cell tumors and testicular dysgenesis in patients with testicular microlithiasis.

Testicular microlithiasis (TM) is one of the symptoms of testicular dysgenesis syndrome (TDS). TM is particularly interesting as an informative marker of testicular germ cell tumors (TGCTs). KIT ligand gene (KITLG), BCL2 antagonist/killer 1 (BAK1), and sprouty RTK signaling antagonist 4 (SPRY4) genes are associated with a high risk of TGCTs, whereas bone morphogenetic protein 7 gene (BMP7), transforming growth factor beta receptor 3 gene (TGFBR3), and homeobox D cluster genes (HOXD) are related to TDS. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, we investigated allele and genotype frequencies for KITLG (rs995030, rs1508595), SPRY4 (rs4624820, rs6897876), BAK1 (rs210138), BMP7 (rs388286), TGFBR3 (rs12082710), and HOXD (rs17198432) in 142 TGCT patients, 137 TM patients, and 153 fertile men (control group). We found significant differences in the KITLG GG_rs995030 genotype in TM (P = 0.01) and TGCT patients (P = 0.0005) compared with the control. We also revealed strong associations between KITLG_rs1508595 and TM (G allele, P = 0.003; GG genotype, P = 0.01) and between KITLG_rs1508595 and TGCTs (G allele, P = 0.0001; GG genotype, P = 0.0007). Moreover, there was a significant difference in BMP7_rs388286 between the TGCT group and the control (T allele, P = 0.00004; TT genotype, P = 0.00006) and between the TM group and the control (T allele, P = 0.04). HOXD also demonstrated a strong association with TGCTs (rs17198432 A allele, P = 0.0001; AA genotype, P = 0.001). Furthermore, significant differences were found between the TGCT group and the control in the BAK1_rs210138 G allele (P = 0.03) and the GG genotype (P = 0.01). KITLG and BMP7 genes, associated with the development of TGCTs, may also be related to TM. In summary, the KITLG GG_rs995030, GG_rs1508595, BMP7 TT_rs388286, HOXD AA_rs17198432, and BAK1 GG_rs210138 genotypes were associated with a high risk of TGCT development.

The role of peptidylarginine deiminase 4 in ovarian cancer cell tumorigenesis and invasion.

Peptidylarginine deiminase 4 (PADI4) is an enzyme that converts both histone arginine and mono-methyl arginine residues to citrulline, and it has been detected in various subtypes of ovarian cancer. However, the mechanism of action of PADI4 in ovarian carcinogenesis remains unknown. To examine the function of PADI4, we transfected two ovarian cancer cell lines, wild-type p53 A2780 and p53-null SKOV3, with PADI4-siRNA and negative control siRNA. The proliferation of both A2780 and SKOV3 cells decreased significantly following PADI4-siRNA treatment (P A2780 < 0.01; P SKOV3 < 0.001). The invasion and migration ability of A2780 cells also significantly decreased in response to PADI4-siRNA treatment (P < 0.001), but SKOV3 cells showed no such decrease. The apoptotic rate of A2780 cells increased in the presence of PADI4-siRNA, but there was no such increase in SKOV3 cells (P > 0.05). PCR arrays of A2780 cells treated with PADI4-siRNA revealed the up-regulated expression of six genes, including cell death-inducing DFFA-like effector a (CIDEA) and tumor necrosis factor receptor superfamily member 9 (TNFRSF9), and the down-regulation of seven genes, including integrin beta 3 (ITGB3) and BCL2-antagonist/killer 1 (BAK1). These results suggest an important role for PADI4 in the p53 pathway and the regulation of the proliferation, apoptosis, invasion and migration of ovarian cancer cells. Our study also demonstrated that PADI4 contributes to tumor metastasis by regulating the gene expression of insulin-like growth factor 1 (IGF1) and WAS/WASL-interacting protein family member 1 (WIPF1).

The Roles of the Interaction of BCL2-Antagonist/Killer 1, Apoptotic Peptidase Activating Factor 1 and Selenium in the Pathogenesis of Kashin-Beck Disease.

BCL2-antagonist/killer 1 (BAK1) and apoptotic peptidase activating factor 1 (APAF1) are significant genes in apoptosis signalling pathway of Kashin-Beck disease (KBD). We aimed to verify the protein expression levels of BAK1 and APAF1 in the cartilage and chondrocytes of patients with KBD. Additionally, we explored the relationship between the levels of these proteins and selenium concentration. Chondrocytes was cultured and treated with sodium selenite in vitro. Immunohistochemistry and Western blotting were used to verify the expression levels of BAK1 and APAF1. Compared with the control samples, APAF1 was upregulated and BAK1 was downregulated in the cartilage and chondrocytes of KBD patients. APAF1 expression was higher in the middle and deep zone in the KBD cartilage. APAF1 levels decreased gradually with the increasing selenium concentration (0.05, 0.10 and 0.25mg/L). BAK1 expression in the 0.25mg/L selenium group was lower than that of the control group. Different selenium concentrations had varying effects on BAK1 and APAF1 levels. APAF1 may play an important role in the pathogenesis of KBD. APAF1-related apoptosis was more pronounced in the middle and deep zones of the KBD cartilage. APAF may represent a potentially novel molecular target, which may be a biomarker of the role of selenium on the prevention and treatment of KBD. The role of BAK1 in the pathogenesis of KBD requires further study.

A preliminary study of genes related to concomitant chemoradiotherapy resistance in advanced uterine cervical squamous cell carcinoma

Background Tumor intrinsic chemoradiotherapy resistance is the primary factor in concomitant chemoradiotherapy failure in advanced uterine cervical squamous cell carcinoma. This study aims to identify a set of genes and molecular pathways related to this condition. Methods Forty patients with uterine cervical squamous cell carcinoma in International Federation of Gynecology and Obstetrics stage IIb or IIIb, treated with platinum-based concomitant chemoradiotherapy between May 2007 and December 2012, were enrolled in this trial. Patients included chemoradiotherapy resistant (n=20) and sensitive (n=20) groups. Total RNA was extracted from fresh tumor tissues obtained by biopsy before treatment and microarray analysis was performed to identify genes differentially expressed between the two groups. Results Microarray analysis identified 108 genes differentially expressed between concomitant chemoradiotherapy resistant and sensitive patients. Functional pathway cluster analysis of these genes revealed that DNA damage repair, apoptosis, cell cycle, Map kinase signal transduction, anaerobic glycolysis and glutathione metabolism were the most relevant pathways. Platelet-derived growth factor receptor alpha (PDGFRA) and protein kinase A type 1A (PRKAR1A) were significantly upregulated in the chemoradiosensitive group, while lactate dehydrogenase A (LDHA), bcl2 antagonist/killer 1 (BAK1), bcl2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and cyclin-dependent kinase 7 (CDK7) were upregulated in the chemoradiotherapy resistant group. Conclusion We have identified seven genes that are differentially expressed in concomitant chemoradiotherapy resistant and sensitive uterine cervical squamous cell carcinomas, which may represent primary predictors for this condition.

Genes Related to Suppression of Malignant Phenotype Induced by Maitake D-Fraction in Breast Cancer Cells

It is already known that the Maitake (D-Fraction) mushroom is involved in stimulating the immune system and activating certain cells that attack cancer, including macrophages, T-cells, and natural killer cells. According to the U.S. National Cancer Institute, polysaccharide complexes present in Maitake mushrooms appear to have significant anticancer activity. However, the exact molecular mechanism of the Maitake antitumoral effect is still unclear. Previously, we have reported that Maitake (D-Fraction) induces apoptosis in breast cancer cells by activation of BCL2-antagonist/killer 1 (BAK1) gene expression. At the present work, we are identifying which genes are responsible for the suppression of the tumoral phenotype mechanism induced by Maitake (D-Fraction) in breast cancer cells. Human breast cancer MCF-7 cells were treated with and without increased concentrations of Maitake D-Fraction (36, 91, 183, 367 μg/mL) for 24 h. Total RNA were isolated and cDNA microarrays were hybridized containing 25,000 human genes. Employing the cDNA microarray analysis, we found that Maitake D-Fraction modified the expression of 4068 genes (2420 were upmodulated and 1648 were downmodulated) in MCF-7 breast cancer cells in a dose-dependent manner during 24 h of treatment. The present data shows that Maitake D-Fraction suppresses the breast tumoral phenotype through a putative molecular mechanism modifying the expression of certain genes (such as IGFBP-7, ITGA2, ICAM3, SOD2, CAV-1, Cul-3, NRF2, Cycline E, ST7, and SPARC) that are involved in apoptosis stimulation, inhibition of cell growth and proliferation, cell cycle arrest, blocking migration and metastasis of tumoral cells, and inducing multidrug sensitivity. Altogether, these results suggest that Maitake D-Fraction could be a potential new target for breast cancer chemoprevention and treatment.

Abstract 2102: Investigational agent 5F 203 modulates apoptotic regulatory gene expression and induces lysosomal membrane permeabilization in breast cancer cells.

Abstract Despite advances in targeted therapeutic agents for women with breast cancer that either express estrogen receptor (ER) alpha or over-expresses human epidermal growth factor receptor 2 (Her2), nearly 40,000 women die each year due to this malignancy. An investigational anticancer agent 2-(4-amino-3-methylphenyl)-5-benzothiazole (5F 203) potently inhibits the growth of breast cancer cells irrespective of either ER or Her2 status. In our current studies we used the Annexin V/PI assay to determine that 5F 203 induced a dose-dependent increase in the percentage of ER/Her2-positive T47D cells and ER/Her2-negative MDA-MB-468 breast cancer cells in early apoptosis. Cell death was not entirely caspase-dependent as pretreatment with pan-caspase inhibitor z-VAD-fmk only partially suppressed apoptosis. A pathway specific PCR-array revealed the ability for 5F 203 to increase the mRNA expression of pro-apoptotic genes bcl-2-antagonist/killer 1 (BAK1) and lymphotoxin alpha (LTA). Using the Acridine Orange assay, we found that 5F 203-mediated cell death was associated with lysosomal membrane permeabilization in both T47D and MDA-MB-468 cells. Additionally, we determined using the Cathepsin B assay that 5F 203 promoted the release of cathepsin B from the lysosomes in both breast cancer cell types. These data indicate 5F 203 upregulates BAK1 and LTA expression and promotes lysosomal cell death triggering the release of cathepsin B in breast cancer cells to confer its anticancer action. Citation Format: Eileen J. Brantley, Gabriell Thorne, Devin Daly, Dain Zylstra, Lancelot McLean. Investigational agent 5F 203 modulates apoptotic regulatory gene expression and induces lysosomal membrane permeabilization in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2102. doi:10.1158/1538-7445.AM2013-2102

Apoptotic Vascular Smooth Muscle Cell Depletion via BCL2 Family of Proteins in Human Ascending Aortic Aneurysm and Dissection

This study investigates the expression patterns of BCL2 (B-cell CLL/lymphoma2) family of proteins and the extent of vascular smooth muscle cell (VSMC) apoptosis in thoracic aortic aneurysms (TAA), type-A aortic dissections (TAD), and nondilated ascending aortic samples. Aortic wall specimens were obtained from patients undergoing surgical repair for TAA (n = 24), TAD (n = 20), and normal aortic tissues from organ donors (n = 6). The expression pattern of BCL2, BCL2L1 (BCL2-like1), BAK1 (BCL2-antagonist/killer1), and BAX (BCL2-associated X protein) proteins was investigated by immunohistochemistry. Furthermore, colocalization of alpha smooth muscle actin (ACTA2) and caspase3 (CASP3) in aortic VSMCs was analyzed by double-immunofluorescence staining. Onset of DNA fragmentation was measured by TUNEL assay. Apoptotic index was significantly increased in both TAD group (31.3 ± 17.2, P < 0.001) and TAA group (21.1 ± 12.7, P = 0.001) relative to control aortas (2.0 ± 1.2). Anti-CASP3 and ACTA2 double-immunostaining confirmed apoptosis in VSMCs in TAA and TAD groups but not in controls. Proapoptotic BAX expression was significantly elevated in VSMCs of TAA patients, compared with that of controls (OR = 20; P = 0.02; 95% CI, 16-250). In contrast, antiapoptotic BCL2L1 expression was higher in controls compared with that of TAA group (OR = 11.2; P = 0.049; 95% CI, 1.0-123.9). Furthermore, BAX/BCL2 ratio was significantly increased in both TAA (1.2 ± 0.7, P < 0.001) and TAD (0.6 ± 0.4, P = 0.05) groups relative to controls (0.2 ± 0.1, P < 0.001). Apoptotic VSMC depletion in human TAA/TAD is associated with disturbance of the balance between proapoptotic and antiapoptotic members of the BCL2 family proteins, which may have a role in the pathogenesis of vascular remodelling in aortic disease. In light of the future studies, targeting apoptotic pathways in TAA and TAD pathogenesis may provide therapeutic benefits to patients by slowing down the progression and even possibly preventing the TAD.

Increased ratio of anti-apoptotic to pro-apoptotic Bcl2 gene-family members in lithium-responders one month after treatment initiation

BackgroundLithium is considered by many as the gold standard medication in the management of bipolar disorder (BD). However, the clinical response to lithium is heterogeneous, and the molecular basis for this difference in response is unknown. In the present study, we sought to determine how the peripheral blood gene expression profiles of patients with bipolar disorder (BD) changed over time following intitiation of treatment with lithium, and whether differences in those profiles over time were related to the clinical response.MethodsIllumina Sentrix Beadchip (Human-6v2) microarrays containing > 48,000 transcript probes were used to measure levels of expression of gene-expression in peripheral blood from 20 depressed subjects with BD prior to and every two weeks during 8 weeks of open-label treatment with lithium.Changes in gene-expression were compared between treatment responders (defined as a decrease in the Hamilton Depression Rating Scale of 50% or more) and non-responders. Pathway analysis was conducted using GeneGO Metacore software.Results127 genes showed a differential response in responders vs. non-responders. Pathway analysis showed that regulation of apoptosis was the most significantly affected pathway among these genes. Closer examination of the time-course of changes among BCL2 related genes showed that in lithium-responders, one month after starting treatment with lithium, several anti-apoptotic genes including Bcl2 and insulin receptor substrate 2 (IRS2) were up-regulated, while pro-apoptotic genes, including BCL2-antagonist/killer 1 (BAK1) and BCL2-associated agonist of cell death (BAD), were down-regulated. In contrast, in lithium non-responders, BCL2 and IRS2 were down-regulated, while BAK1 and BAD up-regulated at the one-month time-point.ConclusionsThese results suggest that differential changes in the balance of pro- and anti- apoptotic gene-expression following treatment with lithium may explain some of the heterogeneity in clinical response in BD patients.

BAK1 (BCL2-antagonist/killer 1)

Review on BAK1 (BCL2-antagonist/killer 1), with data on DNA, on the protein encoded, and where the gene is implicated.

BCL2-associated X protein (BAX); BCL2-antagonist/killer 1 (BAK1)

BCL2-associated X protein (BAX); BCL2-antagonist/killer 1 (BAK1)

Haplotype of BAK1 (BCL2 antagonist killer 1) polymorphisms associated with the risk of developing Kawasaki disease in Taiwanese children

Kawasaki disease (KD) is a pediatric systemic vasculitis of unknown etiology for which a genetic influence has been suggested. BCL2 antagonist killer 1 (BAK1) has been considered to play a critical role in the development of autoimmune disease. The aim of this study was to examine the association of BAK1 polymorphisms with KD risk in Taiwanese children. Five single nucleotide polymorphisms (SNPs)—rs210132, rs210135, rs210139, rs210145 and rs396746—in the BAK1 gene were analysed in a case-control study comprising 93 KD patients and 680 genderand agematched healthy controls. The results showed that the frequencies of the SNP rs210132 TT genotypes were significantly higher in KD patients without coronary artery aneurysm than in control subjects (OR, 1.93 [95% CI: 1.08–3.46]; p = 0.037). The estimated frequency of the GAGC haplotype (rs210132-rs210139-rs210145-rs396746) was significantly lower in KD patients than in controls (OR, 0.60 [95% CI: 0.36–1.00]; p = 0.047). In addition, the frequency of the TAGC haplotype (rs210132-rs210139-rs210145-rs396746) was significantly higher in KD patients than in control subjects (OR, 9.97 [95% CI: 3.72–26.7]; p < 0.0001). In conclusion, the results suggest that the BAK1 gene polymorphisms are associated with the risk of KD in the Taiwanese population.