β-Catenin gets an honorable discharge

Wnt signaling component is removed from active duty by exosomes

Proteomics profiling of cholangiocarcinoma exosomes: A potential role of oncogenic protein transferring in cancer progression

AbstractCholangiocarcinoma (CCA), a common primary malignant tumor of bile duct epithelia, is highly prevalent in Asian countries and unresponsive to chemotherapeutic drugs. Thus, a newly recognized biological entity for early diagnosis and treatment is highly needed. Exosomes are small membrane bound vesicles found in body fluids and released by most cell types including cancer cells. The vesicles contain specific subset of proteins and nucleic acids corresponding to cell types and play essential roles in pathophysiological processes. The present study aimed to assess the protein profiles of CCA-derived exosomes and their potential roles. We have isolated exosomes from CCA cells namely KKU-M213 and KKU-100 derived from Thai patients and their roles were investigated by incubation with normal human cholangiocyte (H69) cells. Exosomes were internalized into H69 cells and had no effects on viability or proliferation of the host cells. Interestingly, the exosomes from KKU-M213 cells only induced migration and invasion of H69 cells. Proteomic analysis of the exosomes from KKU-M213 cells disclosed multiple cancer related proteins that are not present in H69 exosomes. Consistent with the protein profile, treatment with KKU-M213 exosomes induced β-catenin and reduced E-cadherin expressions in H69 cells. Collectively, our results suggest that a direct cell-to-cell transfer of oncogenic proteins via exosomal pathway may be a novel mechanism for CCA progression and metastasis

Human chorion-derived mesenchymal stem cells suppress JAK2/STAT3 signaling and induce apoptosis of cholangiocarcinoma cell lines

Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the damaged epithelial cells of the biliary tract. Previous studies have reported that the multi-potent mesenchymal stem cells (MSCs) activate a series of tumor signaling pathways by releasing several cytokines to influence tumor cell development. However, the roles and mechanisms of human chorion-derived MSCs (CH-MSCs) in cholangiocarcinoma progression have not been fully addressed. This present study aims to examine the effects of conditioned media derived from CH-MSCs (CH-CM) on CCA cell lines and investigate the respective underlying mechanism of action. For this purpose, MSCs were isolated from chorion tissue, and three cholangiocarcinoma cell lines, namely KKU100, KKU213A, and KKU213B, were used. MTT assay, annexin V/PI analysis, and JC-1 staining were used to assess the effects of CH-CM on proliferation and apoptosis of CCA cells, respectively. Moreover, the effect of CH-CM on caspase-dependent apoptotic pathways was also evaluated. The western blotting assay was also used for measuring the expression of JAK2/STAT3 signaling pathway-associated proteins. The results showed that CH-CM suppressed proliferation and promoted apoptosis of CCA cell lines. CH-CM treatment-induced loss of mitochondrial membrane potential (∆Ψm) in CCA cell lines. The factors presented in the CH-CM also inhibited JAK2/STAT3 signaling, reduced the expression of BCL-2, and increased BAX expression in CCA cells. In conclusion, our study suggests that the CH-CM has a potent anti-cancer effect on cholangiocarcinoma cells and thus provides opportunities for use in alternative cell therapy or in combination with a conventional chemotherapeutic drug to increase the efficiency of CCA treatment

β-Catenin is a pH sensor with decreased stability at higher intracellular pH.

β-Catenin functions as an adherens junction protein for cell-cell adhesion and as a signaling protein. β-catenin function is dependent on its stability, which is regulated by protein-protein interactions that stabilize β-catenin or target it for proteasome-mediated degradation. In this study, we show that β-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster β-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase β-TrCP. While β-catenin phosphorylation was pH independent, higher pHi induced increased β-TrCP binding and decreased β-catenin stability. An evolutionarily conserved histidine in β-catenin (found in the β-TrCP DSGIHS destruction motif) is required for pH-dependent binding to β-TrCP. Expressing a cancer-associated H36R-β-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic β-catenin alleles. We identify pHi dynamics as a previously unrecognized regulator of β-catenin stability, functioning in coincidence with phosphorylation

A novel missense mutation of SLC7A9 frequent in Japanese cystinuria cases affecting the C-terminus of the transporter

Cystinuria is caused by the inherited defect of apical membrane transport systems for cystine and dibasic amino acids in renal proximal tubules. Mutations in either SLC7A9 or SLC3A1 gene result in cystinuria. The mutations of SLC7A9 gene have been identified mainly from Italian, Libyan Jewish, North American, and Spanish patients. In the present study, we have analyzed cystinuria cases from oriental population (mostly Japanese). Mutation analyses of SLC7A9 and SLC3A1 genes were performed on 41 cystinuria patients. The uptake of 14C-labeled cystine in COS-7 cells was measured to determine the functional properties of mutants. The protein expression and localization were examined by Western blot and confocal laser-scanning microscopy. Among 41 patients analyzed, 35 were found to possess mutations in SLC7A9. The most frequent one was a novel missense mutation P482L that affects a residue near the C-terminus end of the protein and causes severe loss of function. In MDCK II and HEK293 cells, we found that P482L protein was expressed and sorted to the plasma membrane as well as wild type. The alteration of Pro482 with amino acids with bulky side chains reduced the transport function of b0,+AT/BAT1. Interestingly, the mutations of SLC7A9 for Japanese cystinuria patients are different from those reported for European and American population. The results of the present study contribute toward understanding the distribution and frequency of cystinuria-related mutations of SLC7A9

Polyketides From the Endophytic Fungus Cladosporium sp. Isolated From the Mangrove Plant Excoecaria agallocha

Five new polyketides (2–6) and ten known compounds (1 and 7–15) were obtained from the fermentation products of the endophytic fungus Cladosporium sp. OUCMDZ-302 with the mangrove plant, Excoecaria agallocha (Euphorbiaceae). The new structures of 2–6 were established on the basis of ECD, specific rotation and spectroscopic data as well as the chemical calculation. Compound 7 showed cytotoxicity against H1975 cell line with an IC50 value of 10.0 μM. Compounds 4 and 8–10 showed radical scavenging activity against DPPH with the IC50 values of 2.65, 0.24, 5.66, and 6.67 μM, respectively. In addition, the absolute configuration of compound 1 was solidly determined by X-ray and sugar analysis of the acidic hydrolysates for the first time as well as those of compounds 8–10 in this paper

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

Background: T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. Objective: We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. Methods: T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. Results: Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD81 suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)–150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH2/2 or miRNA-1502/2 mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. Conclusions: This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains

Nontemplated Nucleotide Additions Distinguish the Small RNA Composition in Cells from Exosomes

Functional biomolecules, including small noncoding RNAs (ncRNAs), are released and transmitted between mammalian cells via extracellular vesicles (EVs), including endosome-derived exosomes. The small RNA composition in cells differs from exosomes, but underlying mechanisms have not been established. We generated small RNA profiles by RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes. A comprehensive bioinformatics and statistical analysis revealed nonrandomly distributed subsets of microRNA (miRNA) species between B cells and exosomes. Unexpectedly, 3′ end adenylated miRNAs are relatively enriched in cells, whereas 3′ end uridylated isoforms appear overrepresented in exosomes, as validated in naturally occurring EVs isolated from human urine samples. Collectively, our findings suggest that posttranscriptional modifications, notably 3′ end adenylation and uridylation, exert opposing effects that may contribute, at least in part, to direct ncRNA sorting into EVs.T.W. is supported by VIDI 91711366. D.M.P. is supported by personal Dutch Cancer Society research award (KWF-5510). This work was funded by AICR grant 11-0157 and NWO-VENI 91696087 awarded to D.M.P

Exosome release of β-catenin: a novel mechanism that antagonizes Wnt signaling

The tetraspanins CD9 and CD82 suppress Wnt signaling by promoting the discharge of β-catenin from cells