Kang Yang

ALDH1A1 defines invasive cancer stem-like cells and predicts poor prognosis in patients with esophageal squamous cell carcinoma

Invasion and metastasis are the major cause of deaths in patients with esophageal cancer. In this study, we isolated cancer stem-like cells from an esophageal squamous cell carcinoma cell line EC109 based on aldehyde dehydrogenase 1A1 (ALDH1A1), and found that ALDH1A1high cells possessed the capacities of self-renewal, differentiation and tumor initiation, indications of stem cell properties. To support their stemness, ALDH1A1high cells exhibited increased potential of invasion and metastasis as compared with ALDH1A1low cells. ALDH1A1high esophageal squamous cell carcinoma cells expressed increased levels of mRNA for vimentin, matrix metalloproteinase 2, 7 and 9 (MMP2, MMP7 and MMP9), but decreased the level of E-cadherin mRNA, suggesting that epithelial–mesenchymal transition and secretary MMPs may be attributed to the high invasive and metastatic capabilities of ALDH1A1high cells. Furthermore, we examined esophageal squamous cell carcinoma specimens from 165 patients and found that ALDH1A1high cells were associated with esophageal squamous dysplasia and the grades, differentiation and invasion depth, lymph node metastasis and UICC stage of esophageal squamous cell carcinoma, as well as poor prognosis of patients. Our results provide the strong evidence that ALDH1A1high cancer stem-like cells contribute to the invasion, metastasis and poor outcome of human esophageal squamous cell carcinoma.

Nanosilver-doped DNA polyion complex membrane for electrochemical immunoassay of carcinoembryonic antigen using nanogold-labeled secondary antibodies

A simple and sensitive electrochemical immunoassay protocol was developed for the detection of carcinoembryonic antigen (CEA) using nanosilver-doped DNA polyion complex membrane (PIC) as sensing interface. To construct such an immunosensor, double-stranded DNA was initially assembled onto the surface of thionine/Nafion-modified screen-printed carbon electrode to adsorb silver ions with positive charges, then silver ions were reduced to nanosilver particles with the aid of NaBH(4), and then anti-CEA antibodies were immobilized on the nanosilver surface. Gold nanoparticles conjugated with horseradish peroxidase-labeled anti-CEA were employed as signal antibodies for the detection of CEA with a sandwich-type assay format. Under optimal conditions, the immunosensor exhibited a dynamic range of 0.03-32 ng mL(-1) with a low detection limit of 10 pg mL(-1) CEA. Intra- and inter-assay imprecision (CVs) were <9.5% and 6.5%, respectively. The response could remain 90.1% of the original current at 30th day. 50 real samples were evaluated using the immunosensor and the enzyme-linked immunosorbent assay, respectively, and received in accordance with those two methods.

Overexpression and Gender-specific Differences of SRC-3 (SRC-3/AIB1) Immunoreactivity in Human Non-Small Cell Lung Cancer: An In Vivo Study:

Steroid receptor coactivator-3 (SRC-3) has been reported to be overexpressed in the development and progression of many tumor types. SRC-3 has been detected in several lung cancer cell lines, but its expression and clinical significance in non-small cell lung cancer (NSCLC) remain unclear. In this study, 48 NSCLC tissues were collected and tissue microarrays were performed. The expression of SRC-3 was examined using nickel-intensified IHC. The results showed that of these 48 cases, 18 (37.5%) exhibited high levels of SRC-3 immunoreactivity, 23 (47.9%) exhibited moderate levels of SRC-3 immunoreactivity, and 7 (14.6%) were negative; thus, the total frequency of SRC-3 overexpression was 85.4% (41/48). This SRC-3 overexpression frequency was similar to the overexpression frequency observed for squamous cell carcinoma and adenocarcinoma (82.1% vs 90%) and for metastasis and non-metastasis patients (84.6% vs 85.7%). Data analysis demonstrated a significantly higher overexpression frequency in male patients compared with that in female patients (88.6% vs 76.9%). However, female patients tended to have higher expression levels of SRC-3, as measured by immunoreactivity, than male patients. These results demonstrate a high frequency of SRC-3 overexpression in NSCLC with a gender difference, suggesting that there is a specific role for SRC-3 in the pathogenesis of NSCLC. (J Histochem Cytochem 58:1121–1127, 2010)

Up-regulation of fas reverses cisplatin resistance of human small cell lung cancer cells

Background/AimFas/FasL system is a major regulator of apoptosis. The mechanisms by which Fas mediates cisplatin resistance remain unclear. The aim of this study is to explore the effect of Fas over-expression on cisplatin resistance of small cell lung cancer cells and its possible mechanisms.Materials and methodsFas was over-expressed in H446/CDDP cells by infection with the adenoviruses containing Fas. Sensitivity of Fas-overexpressed H446/CDDP cells to cisplatin was evaluated using MTT assay. Expressions of Fas, GST-π and ERCC1 were detected by RT-PCR and Western blot analysis. Apoptosis rate was examined by FACS.ResultsOver-expression of Fas in H446/CDDP cells significantly decreased the expressions of GST-π and ERCC1 at mRNA and protein levels, and increased the cell apoptosis. Furthermore, up-regulation of Fas significantly decreased the tolerance of H446/CDDP cells to cisplatin.ConclusionOver-expression of Fas reverses drug resistance of H446/CDDP cells, possibly due to the increased cell sensitivity to apoptosis and the decreased expressions of GST-π and ERCC1.

Why ligand cross-reactivity is high within peptide recognition domain families? A case study on human c-Src SH3 domain.

Many important protein-protein interactions in eukaryotic signaling networks are mediated by peptide recognition domains (PRDs), which bind short linear sequence motifs in other proteins. However, high ligand cross-reactivity is observed within most PRD families, rendering a broad specificity for the family members. In the present study, we attempt to explore the molecular mechanism and physicochemical origin of PRD cross-reactivity. In the procedure, a structure-based method called atomic cross-nonbonded interaction analysis (ACNIA) is described to extract atomic-level nonbonded interaction information at domain-peptide interface and to correlate the information with peptide affinity based on a set of structure-solved, affinity-known protein-peptide complex samples compiled from numerous literatures and databases. The ACNIA-derived affinity predictor is tested rigorously with statistical validation approach, which is also demonstrated to be capable of perceiving slight structural change in the interface using three distinct panels of SH3-binding peptide data. Subsequently, with help of the affinity predictor we adopt the human c-Src SH3 domain, one of the most sophisticated PRDs, as a paradigm to investigate the ligand cross-reactivity within SH3 family. It is found that most of the family members have only few non-essential residue differences in their peptide-binding pockets, and thus exhibit a similar peptide recognition profile and high cross-reactivity. The cross-reactivity is even shared by different subclasses of SH3 domains. The findings suggest that inherent binding specificity is not the only factor to select appropriate binders for specific SH3 domains, and other aspects such as cellular context and the rest of the SH3-containing proteins may play important roles in reducing their ligand cross-reactivity.

Rho Guanine Nucleotide Exchange Factor 5 Increases Lung Cancer Cell Tumorigenesis via MMP-2 and Cyclin D1 Upregulation

We sought to elucidate the role of Rho guanine nucleotide exchange factor 5 (ARHGEF5) in tumorigenesis of lung adenocarcinoma cells. ARHGEF5 protein levels were assessed in 91 human lung adenocarcinoma specimens, and A549 and NCI-H1650 cells, by IHC and Western blotting. In addition, ARHGEF5 mRNA expression was evaluated by quantitative reverse transcriptase-PCR. Furthermore, ARHGEF5 long and short isoform coexpression was detected by immunofluorescence. Finally, flow cytometry; CCK8 and wound-healing assays; cell invasion, migration and adhesion; and xenografts were used to evaluate the biologic significance of ARHGEF5. ARHGEF5 was significantly increased in lung adenocarcinoma tissues and cell lines. Interestingly, ARHGEF5 levels were significantly associated with tumor grade and pathologic stage, but not age, gender, T stage, or lymph node metastasis status. ARHGEF5 knockdown by RNAi resulted in dramatically reduced proliferation, adhesion, invasion, and migratory capability of A549 and NCI-H1650 cells. Likewise, protein levels of p-Src, p-Akt, and NF-κB were significantly decreased after ARHGEF5 knockdown. In parallel, increased S-phase population and MMP-2/cyclin D1 expression were observed in the cancer cells, which were not apoptotic. In addition, ARHGEF5 knockdown A549 and NCI-H1650 cells injected s.c. and i.v. into nude mice exhibited decreased xenograft volume and overtly reduced metastasis. Conversely, ARHGEF5 overexpression in A549 and NCI-H1650 cells increased their tumorigenicity in vitro. ARHGEF5 acts as a proto-oncogene in human lung adenocarcinoma cell tumorigenesis. Mol Cancer Ther; 14(7); 1671–9. ©2015 AACR.

Exploring the activity space of peptides binding to diverse SH3 domains using principal property descriptors derived from amino acid rotamers.

Although there were intensive works addressed on multivariate extraction of the informative components from numerous physicochemical parameters of amino acids in isolated state, the various conformational behaviors of amino acids in complicated biological context have long been underappreciated in the field of quantitative structure-activity relationship (QSAR). In this work, the amino acid rotamers, which were derived from statistical survey of protein crystal structures, were used to reproduce the conformational variety of amino acid side-chains in real condition. In this procedure, these rotamers were superposed into a nx x ny x nz lattice and an artificial probe was employed to detect four kinds of nonbonding field potentials (i.e., electrostatic, steric, hydrophobic, and hydrogen bonds) at each lattice point using a Gaussian-type potential function; the generated massive data were then subjected to a principal component analysis (PCA) treatment to obtain a set of few, informative amino acid descriptors. We used this set of descriptors, that we named principal property descriptors derived from amino acid rotamers (PDAR), to characterize over 13,000 peptides with known binding affinities to 10 types of SH3 domains. Genetic algorithm/ partial least square regression (GA/PLS) modeling and Monte Carlo cross-validation (MCCV) demonstrated that the correlation between the PDAR descriptors and the binding affinities of peptides are comparable with or even better than previously published models. Furthermore, from the PDAR-based QSAR models we concluded that the core motif of peptides, particularly the electrostatic property, hydrophobicity, and hydrogen bond at residue positions P3, P2, and/or P0, contribute significantly to the hAmph SH3 domain-peptide binding, whereas two ends of the peptides, such as P6, P4, P-4, and P5, only play a secondary role in the binding.

Co-expression of Rho guanine nucleotide exchange factor 5 and Src associates with poor prognosis of patients with resected non-small cell lung cancer

Specific and sensitive enough molecular biomarkers are lacking to accurately predict the survival of non-small cell lung cancer (NSCLC) patients. ARHGEF5 and Src have been shown to play an important role in tumorigenesis. However, the involvement of ARHGEF5 and Src in NSCLC remains unknown. Therefore, we evaluated the expression of ARHGEF5 and Src in resected NSCLC tissues and the correlation of co-expression of ARHGEF5 and Src and the prognosis of patients with resected NSCLC. Positive expression of ARHGEF5 was detected in 133 cases of 193 patients (68.91%). A total of 193 NSCLC patients (male: 145; female: 48; average age: 61.84 years; age range: 31-84) were enrolled in this study, of which 99 cases were squamous cell carcinomas (SCCs) (51.30%) and 94 cases were adenocarcinomas (ADCs) (48.70%). The expression of ARHGEF5 was mainly located in the cytoplasm of tumor cells, but not in the corresponding adjacent lung tissues. The levels of ARHGEF5 were significantly associated with age, differentiation and tumor stage. ARHGEF5 protein expression was associated with Src protein expression in NSCLC (χ(2) = 11.874, P<0.01) and in ADC (χ(2) = 12.194, P<0.01), but not in SCC. Co-immunoprecipitation revealed that there was a physical interaction between Src and ARHGEF5 in lung cancer cells. The patients with ARHGEF5(+)/Src(+) had a shorter survival time compared with the other patients (29.37 months versus 39.90 months, P = 0.029). In conclusion, ARHGEF5/Src can be considered as a prognostic biomarker and a therapeutic target for patients with resected NSCLC.

Effect of Altered WIG-1 Expression on DDP Sensitivity in a DDPResistant Esophageal Squamous Cancer Cell Line

Esophageal cancer (EC) is the most common esophageal malignancy and has a dismal prognosis. Developing novel strategies to reverse the resistance to chemotherapeutics in EC is currently of intense interest. The wide-type p53 induced gene 1 (WIG-1) is a p53-regulated transcription factor. The effect of WIG-1 on the regulation of cisplatin (DDP) sensitivity was evaluated in DDP-resistant EC cells both in vitro and in vivo. The DDP-resistant sub-line EC109/DDP was successfully selected following eight months of culture. Overexpression of WIG-1 in EC109/DDP cells significantly lowered the IC(50) of DDP to 1.11 ± 0.54 μg/ml when compared to Control cells (4.57 ± 0.98 μg/ml, P < 0.05). In addition, WIG-1 exerted a negative effect on cell proliferation and on the cloning efficiency of EC109/DDP cells. A significant increase in the apoptosis index and in TUNEL-positive nuclei was observed when the expression of WIG-1 was upregulated. Furthermore, WIG-1-overexpressing DDP-resistant EC cells exhibited suppressed xenograft tumor growth and a lower green fluorescent protein (GFP) fluorescence intensity following DDP injection. WIG-1 also reduced the expression of ERCC1 and increased the expression of Bax in DDP-resistant EC cells, while the expression of Bcl-2, P-gp and GST-π was not significantly altered after up- or down-regulation of WIG-1. In summary, these results show that WIG-1 may reverse the DDP resistance of EC cells by reducing ERCC1 expression and increasing Bax expression. This study will provide a framework for understanding the mechanism of DDP resistance by WIG-1 and will aid in the therapeutic use of DDP in ESCC.

Prolonged Cardiac Allograft Survival in Mouse Model After Complement Depletion with Yunnan Cobra Venom Factor

BACKGROUND Activation of the complement system is the leading mechanism that causes antibody-mediated acute rejection and hyperacute rejection after xenotransplantation. The major cause of acute rejection in allogeneic transplantation is the T cell-mediated specific immune response. We studied the effects of complement on acute rejection after cardiac allotransplantation using complement depletion with cobra venom factor (CVF) in the mouse. MATERIALS AND METHODS The Balb/c-C57 mouse model of heterotopic cardiac allograft was used. The mice were divided into 2 groups, a control group and a CVF-treated group. After intravenous injection of CVF, the experimental group was observed for allograft survival time. Twelve mice from the control and experimental groups were sacrificed on days 3, 5, and 7 after the operation. The pathologic grade of acute rejection, deposition of C3 in tissue, extent of infiltration by CD4+ and CD8+ T cells, and expression of MHC-II, B7-1, and B7-2 were compared between the 2 groups. RESULTS In the CVF-treated group, mean (SD) survival of the cardiac allograft was 26.2 (1.7) days, and in the control group was 8.4 (0.4) days (P < .01). Pathologic examination and immunohistochemistry demonstrated that the grade of acute rejection, deposition of C3 in tissue, extent of infiltration of CD4+ and CD8+ T cells, and expression of MHC-II, B7-1, and B7-2 were significantly decreased in the CVF-treated group. CONCLUSION Depletion of complement in the serum with CVF inhibits acute cardiac allograft rejection in the mouse.