Expression Of Bcl-2-associated Athanogene 3 Research Articles

Expression of Anti-apoptotic Protein BAG3 in Human Sebaceous Gland Carcinoma of the Eyelid.

Bcl-2-associated athanogene 3 (BAG3), a co-chaperone of heat shock protein 70 (HSP70), has been shown to play a role in anti-apoptosis of various malignant tumors. In this study, the expression of BAG3 was examined in human sebaceous gland carcinoma of the eyelid. The expression of BAG3 was evaluated by immunohistochemistry of surgical samples from 5 patients with sebaceous gland carcinoma in the eyelid. BAG3 was positive diffusely in the cytoplasm in all patients. The average positive rate of BAG3 was 73.0±26.0% in tumor cells of all patients. BAG3 was highly expressed in sebaceous gland carcinoma of the eyelid. BAG3 may play an important role in the pathogenesis and progression of sebaceous gland carcinoma of the eyelid.

Overexpression of the anti-apoptotic protein BAG3 in human choroidal melanoma: A case report.

Bcl-2-associated athanogene 3 (BAG3), a co-chaperone of heat shock protein 70 (HSP70), exerts anti-apoptotic effects in various malignant tumors. However, relationships between choroidal melanoma and BAG3 are poorly studied. This study investigated the expression of BAG3 in a case of human choroidal melanoma. Funduscopy, computed tomography, and single-photon emission computed tomography with the intravenous injection of N-isopropyl-p-[123I] iodoamphetamine strongly indicated choroidal melanoma in a 68-year-old woman. Accordingly, we carried out an enucleation and pathological diagnosis. Proteins and total RNA were extracted from normal retinochoroidal and tumor tissues. Proteins were also extracted from ocular nevus tissues of other patients. We examined the expression of BAG3 protein and mRNA using Western blotting and the real-time quantitative polymerase chain reaction, respectively. Immunohistochemical stains were positive for melan-A, HMB-45, and S-100. Histopathology confirmed a choroidal melanoma. The expression of BAG3 protein and mRNA in the choroidal melanoma tissue was upregulated with respect to both normal retinochoroidal tissue and ocular nevus tissues from other patients. Because BAG3 may inhibit apoptosis of choroidal melanoma and facilitate its survival, overexpression of this gene product may be a prognostic marker and therapeutic target.

Advanced glycation end products promote the proliferation and migration of primary rat vascular smooth muscle cells via the upregulation of BAG3.

The present study was aimed to investigate the role of reactive oxygen species (ROS) on advanced glycation end product (AGE)-induced proliferation and migration of vascular smooth muscle cells (VSMCs) and whether Bcl-2-associated athanogene 3 (BAG3) is involved in the process. Primary rat VSMCs were extracted and cultured in vitro. Cell viability was detected by MTT assay and cell proliferation was detected by EdU incorporation assay. Cell migration was detected by wound healing and Transwell assays. BAG3 was detected using qPCR and western blot analysis. Transcriptional and translational inhibitors (actinomycin D and cycloheximide, respectively) were used to study the effect of AGEs on the expression of BAG3 in VSMCs. Lentiviral plasmids containing short hairpin RNA (shRNA) against rat BAG3 or control shRNA were transduced into VSMCs. Cellular ROS were detected by 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining. Mitochondrial membrane potential was detected by tetramethylrhodamine methyl ester (TMRE) staining. AGEs significantly increased the expression of BAG3 in a dose-and time-dependent manner. Furthermore, AGEs mainly increased the expression of BAG3 mRNA by increasing the RNA synthesis rather than inhibiting the RNA translation. BAG3 knockdown reduced the proliferation and migration of VSMCs induced by AGEs. BAG3 knockdown reduced the generation of ROS and sustained the mitochondrial membrane potential of VSMCs. Reduction of ROS production by N-acetylcysteine (NAC), a potent antioxidant, also reduced the proliferation and migration of VSMCs. On the whole, the present study demonstrated for the first time that AGEs could increase ROS production and promote the proliferation and migration of VSMCs by upregulating BAG3 expression. This study indicated that BAG3 should be considered as a potential target for the prevention and/or treatment of vascular complications of diabetes.

BAG3 is involved in neuronal differentiation and migration

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

High expression of BAG3 predicts a poor prognosis in human medulloblastoma.

Bcl2-associated athanogene 3 (BAG3), a co-chaperone of the heat shock protein (Hsp) 70, regulates various physiological and pathological processes. However, its role in human medulloblastoma has not been clarified. First of all, the expression of BAG3 was examined in formalin-fixed, paraffin-embedded specimens by immunohistochemical staining. And then, the prognostic role of BAG3 was analyzed in 51 medulloblastoma samples. Finally, the roles of BAG3 in the proliferation, migration, and invasion of Daoy medulloblastoma cell were investigated using a specific short hairpin RNA (shRNA). The expression of BAG3 in medulloblastoma tissues was higher than nontumorous samples. Furthermore, BAG3 overexpression significantly correlated with poor prognosis of patients with medulloblastoma. The overall survival and tumor-free survival in patients with BAG3 low expression were higher than high expression. Univariate and multivariate analysis showed that BAG3 overexpression was an independent prognostic marker for medulloblastoma. After the BAG3 knockdown, the Daoy cells exhibited decreased the ability to proliferate and form neurosphere. The preliminary mechanism study showed that overexpression of BAG3 might facilitate the cell cycle transition from G1 to S phase by modulating the cyclin-dependent kinase 2 (CDK2) and cyclin E expression. Additionally, we found that BAG3 might enhance the medulloblastoma cell migratory and invasive ability. In summary, BAG3 overexpression may regulate the survival and invasive properties of medulloblastoma and may serve as a potential therapy target for medulloblastoma.

Network analysis of genes involved in the enhancement of hyperthermia sensitivity by the knockdown of BAG3 in human oral squamous cell carcinoma cells

BCL2-associated athanogene 3(BAG3), a co-chaperone of the heat shock 70kDa protein(HSPA) family of proteins, is a cytoprotective protein that acts against various stresses, including heat stress. The aim of the present study was to identify gene networks involved in the enhancement of hyperthermia(HT) sensitivity by the knockdown(KD) of BAG3 in human oral squamous cell carcinoma(OSCC) cells. Although a marked elevation in the protein expression of BAG3 was detected in human the OSCC HSC-3cells exposed to HT at 44˚C for 90min, its expression was almost completely suppressed in the cells transfected with small interfering RNA against BAG3(siBAG) under normal and HTconditions. The silencing of BAG3 also enhanced the cell death that was increased in the HSC-3cells by exposure to HT. Global gene expression analysis revealed many genes that were differentially expressed by>2-fold in the cells exposed to HT and transfected with siBAG. Moreover, Ingenuity®pathways analysis demonstrated two unique gene networks, designated as Pro-cell death and Anti-cell death, which were obtained from upregulated genes and were mainly associated with the biological functions of induction and the prevention of cell death, respectively. Of note, the expression levels of genes in the Pro-cell death and Anti-cell death gene networks were significantly elevated and reduced in the HT+BAG3-KD group compared to those in the HTcontrol group, respectively. These results provide further insight into the molecular mechanisms involved in the enhancement of HTsensitivity by the silencing of BAG3 in human OSCCcells.

BAG3-mediated Mcl-1 stabilization contributes to drug resistance via interaction with USP9X in ovarian cancer.

Paclitaxel in combination with carboplatin improves survival among patients with susceptible ovarian cancers, but no strategy has been established against resistant ovarian cancers. BAG3 (Bcl-2-associated athanogene 3) is one of six BAG family proteins, which are involved in such cellular processes as proliferation, migration and apoptosis. In addition, expression of BAG3 with Mcl-1, a Bcl-2 family protein, reportedly associates with resistance to chemotherapy. Our aim in this study was to evaluate the functional role of BAG3 and Mcl-1 in ovarian cancer chemoresistance and explore possible new targets for treatment. We found that combined expression of BAG3 and Mcl-1 was significantly associated with a poor prognosis in ovarian cancer patients. Invitro, BAG3 knockdown in ES2 clear ovarian cancer cells significantly increased the efficacy of paclitaxel in combination with the Mcl-1 antagonist MIM1, with or without the Bcl-2 family antagonist ABT737. Moreover, BAG3 was found to positively regulate Mcl-1 levels by binding to and inhibiting USP9X. Our data show that BAG3 and Mcl-1 are key mediators of resistance to chemotherapy in ovarian cancer. In BAG3 knockdown ES2 clear ovarian cancer cells, combination with ABT737 and MIM1 enhanced the efficacy of paclitaxel. These results suggest that inhibiting BAG3 in addition to anti-apoptotic Bcl-2 family proteins may be a useful therapeutic strategy for the treatment of chemoresistant ovarian cancers.

BAG3 regulates cell proliferation, migration, and invasion in human colorectal cancer

Bcl2-associated athanogene 3 (BAG3) has been reported to be elevated in various tumors. However, it is unclear whether BAG3 has a functional role in the initiation and progression of colorectal cancer (CRC). Here, we collected CRC samples and cell lines to validate the pathway by using gene and protein assays. RT-PCR showed that the expression of BAG3 mRNA in CRC tissues was obviously higher than that in non-tumor tissues (p < 0.001). Immunohistochemical analysis showed that immunoreactivity of BAG3 was found in most CRC tissues and strongly correlated with TNM stage (p = 0.001), differentiation (p = 0.003), and metastasis (p = 0.010). Low expression of BAG3 in HCT-8 significantly reduced cellular proliferation, migration, and invasion. The analysis of in vitro cell showed that HCT-8 cells were exposed to si-BAG3, and its growth was inhibited depending on modulation of cell cycle G1/S checkpoints and cell cycle regulators, involving cyclin D1, cyclin A2, and cyclin B1. Furthermore, suppression of the epithelial-mesenchymal transition (EMT) by si-BAG3 is linked to the decreased expression of E-cadherin and the increased expression of N-cadherin, vimentin, and MMP9. In conclusion, in the present study, we demonstrated that BAG3 overexpression plays a critical role in cell proliferation, migration, and invasion of colorectal cancer. Our data suggests targeted inhibition of BAG3 may be useful for patients with CRC.

The Evaluation of WBP2NL-Related Genes Expression in Breast Cancer.

Breast cancer is the most frequent cause of mortality in women all around the world; therefore, study on molecular aspects of breast cancer is necessary for finding new biomarkers. Recent studies have shown that WW Binding Protein 2 (WBP2) is an important protein for the oncogenic property of cancer. We have previously evaluated the WW Binding Protein 2N-Terminal Like (WBP2NL) gene expression in cancerous cell line and breast tumor tissues, and reported changes in expression, which could increase tumorigenic cell growth. However, the molecular mechanisms of WBP2NL and its clinical relevance have not been investigated. In this study, the expression of WBP2NL-related genes in the invasive breast carcinoma and normal breast tissues was evaluated for the first time. Analysis of WBP2NL-related genes expression was performed with reverse transcription-PCR and real time-PCR detection method. The target genes studied were as follow: WW domain containing E3 ubiquitin protein ligase 1(WWP1), membrane associated guanylatekinase containing WW and PDZ domain-1 (MAGI1), neural precursor cell expressed developmentally down-regulated 4 (NEDD4), formin binding protein-4 (FNBP4), BCL2-associated athanogene-3 (BAG3), WW domain-containing oxidoreductase (WWOX), yes-associated protein-1 (YAP1), WW domain containing transcription regulator (WWTR1), member RAS oncogene family (RAB2A), and small G protein signaling modulator 3 (SGSM3). The expression of WWP1, BAG3, and WWTR1 was significantly increased in breast cancer. In contrast, the expression of WWOX, YAP1, RAB2A, and SGSM3 was significantly decreased. The MAGI1 and NEDD4 expression was increased, while the expression of FNBP4 was unchanged. These findings lead us to suggest that WBP2NL might play roles as an anti-apoptotic factor or co-activator to promote breast cancer cell survival and proliferation.

A.P.9: Elucidation of the mechanism of disease in BAG3-related myofibrillar myopathy

Mutations in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been shown to cause myofibrillar myopathy (MFM). MFM is characterised at the cellular level by the formation of protein aggregates and structural failure of the Z-disk. In contrast to the other MFM causing proteins BAG3 has no structural role in the Z-disk but is involved in the regulation of autophagy and the degradation of misfolded proteins. Protein aggregation in BAG3-related MFM has therefore been proposed to result from a reduction in autophagic activity. To investigate the mechanism of disease in BAG3-related MFM we expressed the human wildtype BAG3 or the dominant myofibrillar myopathy causing mutant form BAG3^P209L in zebrafish muscle. The expression of BAG3^P209L resulted in protein aggregation and we performed time-lapse imaging to examine aggregate formation in vivo and examined their composition. To investigate the role of impaired autophagy in BAG related MFM we stimulated or inhibited autophagy in BAG3^wt or BAG3^P209L expressing embryos and examined its effect on protein aggregation. Our studies revealed that autophagy is active in BAG3^P209L expressing fish and that inhibition of autophagy is not sufficient to induce protein aggregation, suggesting that the pathology is not due to impaired autophagic activity. We did not observe fibre failure in the BAG3^P209L model fish and therefore examined the effect of knocking down BAG3 expression. Loss of BAG3 function resulted in fibre failure but not the formation of protein aggregates. Together our knockdown and overexpressing models demonstrate contraction-dependent fiber failure and the formation of protein aggregates. Furthermore, our experimental analysis of the transgenic and knockdown embryos demonstrates that protein aggregation is not due to an inhibition of autophagy and suggests a novel mechanism of disease with the cellular pathology caused by a combination of toxic gain of function and BAG3 insufficiency.

The Expression of BAG3 Gene in Chronic Lymphocytic Leukemia and Its Clinical Significance

Abstract 4605 Objective:BAG3 (BCL2-associated athanogene 3) is a member of BAG family. Previously, many reports indicated that BAG3 was over-expressed in different human cancer tissues, such as thyroid carcinoma, pancreatic cancer, prostate cancer, and leukemic cells. However, there is no or low expression of BAG3 in normal tissues. These imply that BAG3 maybe have some important roles in the human cancers. This study was aimed to investigate the expression level of BAG3 mRNA in chronic lymphocytic leukemia (CLL) and its significance in evaluation of CLL prognosis. Methods:100 CLL human patients and 20 healthy controls were enrolled in this study. The BAG3 mRNA expressions were measured by real-time PCR with fluorescent dye SYBR Green I, the β-actin was used as internal control. The relative quantitative value of BAG3 expression was calculated by means of 2 (–ΔCt). We analyzed the results with established CLL prognostic factors, overall survival (OS) and treatment-free survival (TFS). Results:The expression of BAG3 mRNA in 100 CLL patients was significantly higher than 20 normal control (p = 0.038). Furthermore, The BAG3 expression was obviously increased in CD38 positive patients CD38 (n = 31) than CD38 negetive patients it (n = 61, p = 0.0238). We also found that the expression of BAG3 in the patients with unmutated immunoglobulin heavy-chain (n = 29) was markedly higher than those with mutated immunoglobulin heavy-chain (IgHV) (n = 62, p = 0.0326). It is also found that the patients with enlarged lymph nodes (n = 24) have higher BAG3 expression, compared with those without enlarged lymph nodes (n = 76, p = 0.0004). The increased BAG3 level can also be found in the patients with splenomegaly (n = 32),compared with those without splenomegaly (n=68, p = 0.0393). No association was found between BAG3 expression and patient clinical baseline information (gender and age), as well as other established prognostic factors (lymphocyte count, cytogenetics analysis and p53 mutation status). 25 of these patients were treated with fludarabine-based therapy, interestingly, BAG3 expression level was significantly increased in patients who were relapse and/or refractory to fludarabine-based treatment (n = 10, p = 0.019). However, there are no differences in OS and TFS between patients with low BAG3 expressions and high expression, which indicated BAG3 didn't affect the patients' OS and TFS. Conclusion:These results showed that the BAG3 expression in CLL patients is markedly higher than normal controls. CD38, mutation of IgHV, lymph nodes and splenomegaly are related to the expression of BAG3 in CLL. Furthermore, the patients, who were relapse and/or refractory to fludarabine, have a higher expression of BAG3. These imply that BAG3 may be involved in the pathogenesis of CLL and the roles of BAG3 in CLL need further investigation. Disclosures:No relevant conflicts of interest to declare.

Involvement of JNK and NF-κB pathways in lipopolysaccharide (LPS)-induced BAG3 expression in human monocytic cells

Lipopolysaccharide (LPS) is an outer-membrane glycolipid component of Gram-negative bacteria known for its fervent ability to activate monocytic cells and for its potent proinflammatory capabilities. Bcl-2-associated athanogene 3 (BAG3) is a survival protein that has been shown to be stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, proteasome inhibition, and human immunodeficiency virus 1 (HIV-1) infection. In addition, BAG3 regulates replication of Varicella–Zoster Virus (VZV) and Herpes Simplex Virus (HSV) replication, suggesting that BAG3 could participate in the host response to infection. In the current study, we found that LPS increased the expression of BAG3 in a dose- and time-dependent manner. Actinomycin D completely blocked the LPS-induced BAG3 accumulation, as well as LPS activated the proximal promoter of BAG3 gene, supported that the induction by LPS occurred at the level of gene transcription. LPS-induced BAG3 expression was blocked by JNK or NF-κB inhibition, suggesting that JNK and NF-κB pathways participated in BAG3 induction by LPS. In addition, we also found that induction of BAG3 was implicated in monocytic cell adhesion to extracellular matrix induced by LPS. Overall, the data support that BAG3 is induced by LPS via JNK and NF-κB-dependent signals, and involved in monocytic cell-extracellular matrix interaction, suggesting that BAG3 may have a role in the host response to LPS stimulation.

4.5 Expression of the BAG3 Gene in Chronic Lymphocytic Leukemia and Its Clinical Significance

Abstract Abstract 4605 Objective: BAG3 (BCL2-associated athanogene 3) is a member of BAG family. Previously, many reports indicated that BAG3 was over-expressed in different human cancer tissues, such as thyroid carcinoma, pancreatic cancer, prostate cancer, and leukemic cells. However, there is no or low expression of BAG3 in normal tissues. These imply that BAG3 maybe have some important roles in the human cancers. This study was aimed to investigate the expression level of BAG3 mRNA in chronic lymphocytic leukemia (CLL) and its significance in evaluation of CLL prognosis. Methods: 100 CLL human patients and 20 healthy controls were enrolled in this study. The BAG3 mRNA expressions were measured by real-time PCR with fluorescent dye SYBR Green I, the β-actin was used as internal control. The relative quantitative value of BAG3 expression was calculated by means of 2 (–ΔCt). We analyzed the results with established CLL prognostic factors, overall survival (OS) and treatment-free survival (TFS). Results: The expression of BAG3 mRNA in 100 CLL patients was significantly higher than 20 normal control (p = 0.038). Furthermore, The BAG3 expression was obviously increased in CD38 positive patients CD38 (n = 31) than CD38 negetive patients it (n = 61, p = 0.0238). We also found that the expression of BAG3 in the patients with unmutated immunoglobulin heavy-chain (n = 29) was markedly higher than those with mutated immunoglobulin heavy-chain (IgHV) (n = 62, p = 0.0326). It is also found that the patients with enlarged lymph nodes (n = 24) have higher BAG3 expression, compared with those without enlarged lymph nodes (n = 76, p = 0.0004). The increased BAG3 level can also be found in the patients with splenomegaly (n = 32),compared with those without splenomegaly (n=68, p = 0.0393). No association was found between BAG3 expression and patient clinical baseline information (gender and age), as well as other established prognostic factors (lymphocyte count, cytogenetics analysis and p53 mutation status). 25 of these patients were treated with fludarabine-based therapy, interestingly, BAG3 expression level was significantly increased in patients who were relapse and/or refractory to fludarabine-based treatment (n = 10, p = 0.019). However, there are no differences in OS and TFS between patients with low BAG3 expressions and high expression, which indicated BAG3 didn't affect the patients' OS and TFS. Conclusion: These results showed that the BAG3 expression in CLL patients is markedly higher than normal controls. CD38, mutation of IgHV, lymph nodes and splenomegaly are related to the expression of BAG3 in CLL. Furthermore, the patients, who were relapse and/or refractory to fludarabine, have a higher expression of BAG3. These imply that BAG3 may be involved in the pathogenesis of CLL and the roles of BAG3 in CLL need further investigation. Disclosures: No relevant conflicts of interest to declare.