Reduced expression of the chromatin remodeling gene ARID1A enhances gastric cancer cell migration and invasion via downregulation of E-cadherin transcription.

The chromatin remodeling gene AT-rich interactive domain-containing protein 1A (ARID1A) encodes the protein BAF250a, a subunit of human SWI/SNF-related complexes. Recent studies have identified ARID1A as a tumor suppressor. Here, we show that ARID1A expression is reduced in gastric cancer (GC) tissues, which are significantly associated with local lymph node metastasis, tumor infiltration and poor patient prognosis. ARID1A silencing enforces the migration and invasion of GC cells, whereas ectopic expression of ARID1A inhibits migration. The adhesive protein E-cadherin is remarkably downregulated in response to ARID1A silencing, but it is upregulated by ARID1A overexpression. E-cadherin overexpression significantly inhibits GC cell migration and invasion, whereas CDH1 (coded E-cadherin) silencing promotes migration. Restored expression of CDH1 in ARID1A-silenced cell lines restores the inhibition of cell migration. Luciferase reporter assays and chromatin immunoprecipitation indicate that the ARID1A-associated SWI/SNF complex binds to the CDH1 promoter and modulates CDH1 transcription. ARID1A knockdown induces evident morphological changes of GC cells with increased expression of mesenchymal markers, indicating an epithelial-mesenchymal transition. ARID1A silencing does not alter the level of ß-catenin but induces a subcellular redistribution of ß-catenin from the plasma membrane to the cytoplasm and nucleus. Immunohistochemical studies demonstrate that reduced expression of E-cadherin is associated with local lymph node metastasis, tumor infiltration and poor clinical prognosis. ARID1A and E-cadherin expression show a strong correlation in 75.4% of the analyzed GC tissues. They are synergistically downregulated in 23.5% of analyzed GC tissues. In conclusion, ARID1A targets E-cadherin during the modulation of GC cell migration and invasion.

Impact of glutathione-HbA1c on HbA1c measurement in diabetes diagnosis via array isoelectric focusing, liquid chromatography, mass spectrometry and ELISA.

Hemoglobin A1c (HbA1c) has been proven to be a key biomarker for diabetes screening, and glutathiolation of HbA1c (viz., GSS-HbA1c) has been identified. However, the impact of GSS-HbA1c on the measurement of HbA1c for diabetes screening has not been quantitatively assessed yet. To address the issue, the micropreparative capillary isoelectric focusing (cIEF) developed in our previous work was used for the high resolution separation and purification of hemoglobin (Hb) species. The main fractions of HbA0, HbA3 and HbA1c extracted from the developed cIEF were identified by validated Mono S method. The proposed GSS-HbA1c fractions in the cIEF were pooled and identified by electrospray ionization mass spectrometry (ESI-MS). The HbA1c enzyme-linked immunosorbent assay (ELISA) kit was employed for further quantitative analysis of GSS-HbA1c. A total of 34 blood samples with HbA1c levels from 4.2% to 13.4% were assessed via the above comprehensive strategy of IEF-HPLC-MS-ELISA. It was demonstrated that the HbA1c levels detected by cation exchange LC were considerably influenced by the glutathiolation of Hb and the range of detected GSS-HbA1c values was between 0.23% and 0.74%. The results and developed cIEF methods have considerable significances for investigation of diabetes and clinical diagnosis.

Novel moving reaction boundary-induced stacking and separation of human hemoglobins in slab polyacrylamide gel electrophoresis.

We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris-Gly), sample buffer (pH 6.78 Tris-Gly), and separation buffer (pH 8.52 Tris-Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below pI values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA1c, HbA0, and HbA2) in MRB-PAGE, in contrast to only one protein zone (HbA0) in ITP-PAGE for large-volume loading (≥15 µl), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory.

Comparative proteomics and molecular mechanical analysis in CDA-II induced therapy of LCI-D20 hepatocellular carcinoma model.

PURPOSE: To investigate the differential proteins and related molecular mechanism of CDA-II (cell differentiation agent-II) induced therapy on a human hepatocellular carcinoma model in nude mice with high metastatic potential (LCI-D20). METHODS: After tumors were transplanted 11 days, mice were intraperitoneally injected with CDA-II (1,800 mg/kg) for 20 days continuously. The tumor growth-inhibitory efficiency in CDA-II treated groups was calculated. Proteins extracted from tumor tissue were separated by two-dimensional gel electrophoresis (2DE) and the differential proteins were identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Western blotting (WB) was performed to verify the expression of certain candidate proteins. Reverse transcription-polymerase chain reaction (RT-PCR) was engaged to study the molecular mechanism of the therapy. RESULTS: CDA-II suppressed the growth and metastasis of tumor. The tumor growth-inhibitory efficiency was 41.8%. In total, 27 differentially expressed proteins were identified, including HSP27, UGDH, CK8, Hsp60, ENOA and AnxA5, with functions involved in oncogene expression and/or cell differentiation. In addition, apparent alternations of HSP60 and beta-actin expression levels and their different posttranslational modifications (PTMs) were investigated. RT-PCR analysis confirmed that the cancer related genes c-myc, N-ras and MMP-9 were significantly down-regulated. CONCLUSION: Our results demonstrate that CDA-II presence can change the proteome profiling and favors of the tumor suppression in LCI-D20 cell differentiation. Our results also suggest that the dynamic PTM of HSP60 expression levels could be used to predict HCC and might be a promising and useful biomarker to prognosticate CDA-II therapeutic efficacy.

[Studies on the simvastatin effect on the artery of atherosclerotic rabbit using proteomics approaches].

AIM: To evaluate the inhibitory effect of simvastatin via investigating the overall expression level of proteins in the artery of atherosclerotic rabbit. METHODS: Experimental model was established by feeding the rabbits a high fat diet (cholesterol 0.5 g.kg-1.d-1, lard 0.5 mL.kg-1.d-1) for 8 weeks. Then simvastatin (5 mg.kg-1) were fed for 4 weeks to the rabbits. The overall protein levels were measured using two-dimensional gel electrophoresis and a PDQUEST data processing. RESULTS: Twenty nine protein spots showed significant quantitative changes in comparison with the normal and the diseased rabbits. Furthermore, after the diseased rabbit having taken simvastatin, an obvious decay of symptom of fatty liver was observed, and the intensity of most spots has not been back-regulated. CONCLUSION: Simvastatin facilitates the metabolism of fat in the blood, but the lesion of the internal wall of the atherosclerotic artery cannot be restored.

Protein expressions in macrophage-derived foam cells: comparative analysis by two-dimensional gel electrophoresis.

AIM: To study the overall protein expression changes induced by oxidized low-density lipoprotein (ox-LDL) in the U937 foam cells. METHODS: Foam cell model was established by incubating the human monoblastic leukemia (U937) cells with ox-LDL. Each protein samples in U937 control cells and U937 foam cells were separated by two-dimensional gel electrophoresis (2-DE). After the gels were stained by silver staining method, the images were analyzed by PDquest 2D-image-analysis software (Bio-Rad). Some of the spots were available via the Internet with links to the U937 proteomic map provided from the ExPASy Proteomics server. RESULTS: Using 2-DE, the overall protein map was obtained, in which 150 spots were matched with the control gel (match ratio: 75 %). Compared with U937 cells, 37 spots significantly changed in the foam cells (P<0.05), among which the expression levels in 28 spots increased and those in 9 spots decreased. Especially, 8 spots in U937 cells were absent in the foam cells, while 11 spots in the foam cells were absent in the control cells. CONCLUSION: The changed protein profiles induced by ox-LDL in U937 foam cells were established to support the functional studies on the macrophage-derived foam cells in atherosclerotic pathological states.