Knockdown of BAG3 induces epithelial-mesenchymal transition in thyroid cancer cells through ZEB1 activation.

The process by which epithelial features are lost in favor of a mesenchymal phenotype is referred to as epithelial-mesenchymal transition (EMT). Most carcinomas use this mechanism to evade into neighboring tissues. Reduction or a loss of E-cadherin expression is a well-established hallmark of EMT. As a potent suppressor of E-cadherin, transcription factor ZEB1 is one of the key inducers of EMT, whose expression promotes tumorigenesis and metastasis of carcinomas. Bcl-2-associated athanogene 3 (BAG3) affects multifaceted cellular functions, including proliferation, apoptosis, cell adhesion and invasion, viral infection, and autophagy. Recently, we have reported a novel role of BAG3 implicated in EMT, while the mechanisms are poorly elucidated. The current study demonstrated that knockdown of BAG3 induced EMT, and increased cell migratory and invasiveness in thyroid cancer cells via transcriptional activation of ZEB1. We also found that BAG3 knockdown led to nuclear accumulation of ß-catenin, which was responsible for the transcriptional activation of ZEB1. These results indicate BAG3 as a regulator of ZEB1 expression in EMT and as a regulator of metastasis in thyroid cancer cells, providing potential targets to prevent and/or treat thyroid cancer cell invasion and metastasis.

PKCδ-mediated phosphorylation of BAG3 at Ser187 site induces epithelial-mesenchymal transition and enhances invasiveness in thyroid cancer FRO cells.

Protein kinase C delta (PKCδ) is a serine (Ser)/threonine kinase, which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. In the current study, Chinese hamster ovary cells were transfected with either a constitutively activated PKCδ or a dominant negative PKCδ, phosphoprotein enrichment, two-dimensional difference gel electrophoresis and mass spectrometry was combined to globally identified candidates of PKCδ cascade. We found that Bcl-2 associated athanogene 3 (BAG3) was one of the targets of PKCδ cascade, and BAG3 interacted with PKCδ in vivo. In addition, we clarified that BAG3 was phosphorylate at Ser187 site in a PKCδ-dependent manner in vivo. BAG3 has been implicated in multiple cellular functions, including proliferation, differentiation, apoptosis, migration, invasion, macroautophagy and so on. We generated wild-type (WT)-, Ser187Ala (S187A)- or Ser187Asp (S187D)-BAG3 stably expressing FRO cells, and noticed that phosphorylation state of BAG3 influenced FRO morphology. Finally, for the first time, we showed that BAG3 was implicated in epithelial-mesenchymal transition (EMT) procedure, and phosphorylation state at Ser187 site had a critical role in EMT regulation by BAG3. Collectively, the current study indicates that BAG3 is a novel substrate of PKCδ, and PKCδ-mediated phosphorylation of BAG3 is implicated in EMT and invasiveness of thyroid cancer cells.

Influence of proline-rich inositol polyphosphate 5-phosphatase, on early development of fertilized mouse eggs, via inhibition of phosphorylation of Akt.

OBJECTIVES: Proline-rich inositol polyphosphate 5-phosphatase (PIPP) is one of the signal-modifying enzymes that play pivotal regulatory roles in PI3K signalling pathway. The aim of this study was to determine the role of PIPP in early development of fertilized mouse eggs, via inhibition of Akt activity and subsequent downstream signalling events. MATERIALS AND METHODS: The mRNA transcript levels of endogenous PIPP and Akt1, Akt2, Akt3 were detected in G(1) , S, G(2) and M phases of fertilized mouse eggs by RT-PCR. Levels of exogenous PIPP, phosphorylated Akt at Ser473, dephosphorylated cdc2 at Tyr15 and levels of CCNB1, were detected respectively by immunoblotting. Changes in Akt localization were observed by fluoroimmunoassay; meanwhile, changes in activity of Akt and its downstream MPF were detected. Percentages of cells undergoing division were determined by counting, using a dissecting microscope. RESULTS: PIPP and Akt1 transcripts were detectable in G(1), S, G(2) and M phases of fertilized mouse eggs, but Akt2 and Akt3 were not. We also observed that overexpression of PIPP in fertilized eggs decreased expression of phosphorylated Akt at Ser473 and altered membrane localization of phosphorylated Akt at Ser473 specifically. Furthermore, overexpression of PIPP resulted in decreases in mitosis-phase promoting factor activity, level of dephosphorylated cdc2 at Tyr15 and cleavage rate of fertilized mouse eggs. CONCLUSIONS: Our data suggest, for the first time, that PIPP may affect development of fertilized mouse eggs by inhibition of level of phosphorylated Akt at Ser473 and subsequent inhibition of downstream signal cascades.

Inhibitory effects of c-erbB-2 antisense oligonucleotide transfection on uterine endometrial cancer Ishikawa cell lines.

BACKGROUND: C-erbB-2 is a chief proto-oncogene of endometrial cancer, which plays an important role in the biological behavior of endometrial cancer. Its overespression is an important factor of poor progrosis. The objective of this study was to investigate the treatment effects of transfecting c-erbB-2 antisense oligonucleotide (ASODN) on the uterine endometrial cancer Ishikawa cell line. MATERIALS AND METHODS: The c-erbB-2 expression on Ishikawa cell membranes was determined by immunohistochemistry and then the aim was to transfect ASODN into Ishikawa cells and assay the cellular growth inhibition by MTT, to observe the cellular ultrastructure changes under transmission electron microscope (TEM), and to assay the cellular apoptotic rate, c-erbB-2 mRNA and protein expression by flow cytometry, RT-PCR and Western blot, respectively. RESULTS: C-erbB-2 protein was positively expressed on Ishikawa cell membranes. MTT showed that when the concentration of transfecting ASODNs was 0.3 uM and 0.6 uM, cell growth inhibition rates were 55.43% and 76.12%, respectively. After transfecting 0.3 uM ASODN, the Ishikawa cell ultrastructure was obviously damaged, the cellular apoptotic rate was 72.21%, and the c-erbB-2 mRNA and protein expression were 45.71% and 34.52%, respectively, compared with those of the normal control cells. CONCLUSIONS: Transfecting c-erbB-2 ASODN can obviously suppress its mRNA and protein expression in Ishikawa cells, cause cellular apoptosis and inhibit cell growth. It may have an important role in the gene therapy of endometrial cancer.

Musclin inhibits insulin activation of Akt/protein kinase B in rat skeletal muscle.

Musclin is a muscle-derived secretory peptide that induces insulin resistance in vitro. We studied the effect of musclin (0.5 microg/ml) on insulin-stimulated glucose uptake in rat skeletal muscles and also the effect of rosiglitazone (0.4 microg/ml). Preincubation of muscles with musclin resulted in decreased insulin-stimulated glucose uptake. Musclin also reduced expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) mRNAs, although expression of glucose transporter 4 mRNA was unaltered. Rosiglitazone attenuated the effects of musclin on glucose uptake and PPARgamma and LXRalpha mRNA expression. Western blotting demonstrated that activation of protein kinase B (Akt/PKB) in the insulin-signalling cascade was decreased by musclin but corrected by rosiglitazone. These findings suggest that musclin-induced impairment of insulin-stimulated glucose uptake in skeletal muscle is related to Akt/PKB inhibition and might be modulated by PPARgamma/LXRalpha.