AKT and MAPK are the two cell signaling pathways governing viability of colorectal cancer cell lines with different receptor expression patterns.

<p>Differences in cell viability in LoVo (A, B) and SW948 (C, D) cells following treatment with different agonists or inhibitors, as indicated. The data are presented as mean±SD, and **P<0.01 by unpaired Student’s t test.</p

Activation of MAPK or AKT signaling in colorectal cancer cells with different molecular patterns of expression.

<p>(A, B) Detection of AKT and MAPK signaling in SW948 and LoVo cells by western-blot, with the treatment indicated in the figure. (C, D) Detection of AKT and MAPK signaling by western-blot in LoVo and SW948 cells with HGF or HRG1-β1 treatment, with or without different agonists or antagonists, as indicated.</p

Expression of ErbB3, ErbB2 and c-MET in human colorectal cancer.

<p>(A, B, C) Expression of ErbB3, ErbB2 and c-MET in human colorectal cancer. Specimens of 105 paraffin-embedded colorectal cancer tissues were analyzed by IHC for ErbB3, ErbB2 and c-MET, and representative figures show strong and weak/negative staining for each marker with respective normal control samples. Average integrated optical density (IOD) was obtained by analyzing five fields for each slide, evaluated by Image-Pro Plus software (version 5.0) for IHC staining of ErbB3, ErbB2 and c-MET. The data are presented as mean ± SD, and *P<0.05, **P<0.01 by Mann-Whitney U test.</p

Proliferation of colorectal cancer cells is mediated by both ErbB3/ErbB2 and ErbB3/c-MET signaling Pathways.

<p>(A) Detection of ErbB3, ErbB2 and c-MET in the human colorectal cancer cell lines LoVo, sw948 and sw480 by western-blot. (B, C, D) Cell viability measured by MTS assay in the colorectal cancer cell lines LoVo, sw948 and sw480 treated as indicated in the figure. The data are presented as mean ± SD, and *P<0.05, **P<0.01 by unpaired Student’s t test.</p

Heterogeneous expression of ErbB3, ErbB2 and c-MET in human colorectal cancer.

<p>(A) Ratio of strong/moderate/weak/negative staining of ErbB3, ErbB2 and c-MET in human colorectal cancer. (B) Detection of ErbB3, ErbB2 and c-MET in protein extracted from tissue samples of 4 cases of colorectal cancer. (C) Venn diagram showing co-expression of ErbB3, ErbB2 and c-MET.</p

Quantitative Mass Spectrometry Combined with Separation and Enrichment of Phosphopeptides by Titania Coated Magnetic Mesoporous Silica Microspheres for Screening of Protein Kinase Inhibitors

We describe herein the development of a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) approach for screening of protein kinase inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed products of nonphosphorylated substrates, is a great challenge due to the ion suppression effect of highly abundant nonphosphorylated peptides in enzymatic reaction mixtures. To address this issue, a novel type of titania coated magnetic hollow mesoporous silica spheres (TiO₂/MHMSS) material was fabricated for capturing phosphopeptides from the enzymatic reaction mixtures prior to MS analysis. Under optimized conditions, even in the presence of 1000-fold of a substrate peptide of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated substrates at the femtomole level can be detected with high accuracy and reproducibility. With a synthetic nonisotopic labeled phosphopeptide, of which the sequence is similar to that of the phosphorylated substrate, as the internal standard, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides in peptide mixtures over the range of 0.1 to 4 with <i>r</i>² being 0.99. The IC₅₀ values of three EGFR inhibitors synthesized in our laboratory were then determined, and the results are consistent with those determined by an enzyme-linked immunosorbent assay (ELISA). The developed method is sensitive, cost/time-effective, and operationally simple and does not require isotope/radioative-labeling, providing an ideal alterative for screening of PKIs as therapeutic agents