Growth suppression of colorectal cancer expressing S492R EGFR by monoclonal antibody CH12 / 医学前沿

Colorectal cancer (CRC) is a common malignant tumor in the digestive tract, and 30%-85% of CRCs express epidermal growth factor receptors (EGFRs). Recently, treatments using cetuximab, also named C225, an anti-EGFR monoclonal antibody, for CRC have been demonstrated to cause an S492R mutation in EGFR. However, little is known about the biological function of S492R EGFR. Therefore, we attempted to elucidate its biological function in CRC cells and explore new treatment strategies for this mutant form. Our study indicated that EGFR and S492R EGFR accelerate the growth of CRC cells in vitro and in vivo and monoclonal antibody CH12, which specifically recognizes an EGFR tumor-specific epitope, can bind efficiently to S492R EGFR. Furthermore, mAb CH12 showed significantly stronger growth suppression activities and induced a more potent antibody-dependent cellular cytotoxicity effect on CRC cells bearing S492R EGFR than mAb C225. mAb CH12 obviously suppressed the growth of CRC xenografts with S492R EGFR mutations in vivo. Thus, mAb CH12 may be a promising therapeutic agent in treating patients with CRC bearing an S492R EGFR mutation.

Differential Regulation of E-Cadherin Expression by the Soluble Ectodomain and Intracellular Domain of Jagged1.

Aberrant Jagged1-mediated Notch activation is linked to cancer and induces epithelial-tomesenchymal transition through the repression of E-cadherin transcription. All three proteins are subject to sequential proteolytic events referred to as regulated intramembrane proteolysis. This process releases soluble protein ectodomains from the cell and, concomitantly, generates intracellular domains capable of nuclear translocation and transcriptional regulation. Aim: To determine the cognate roles of the Jagged1 ectodomain and intracellular domain fragments in the regulation of E-cadherin expression. Methodology: Human embryonic kidney cells were stably transfected with coding DNA constructs analogous to full-length Jagged1, the soluble Jagged1 ectodomain, or the intracellular domain fragment of the protein. Correct construct expression and processing were confirmed by immunoblot analysis of transfectant cell lysates and conditioned culture medium. The effects of the various Jagged1 constructs on endogenous E-cadherin expression and processing were subsequently monitored by immunoblot and RT-qPCR analyses. Results: Both full-length Jagged1 and the soluble Jagged1 ectodomain construct downregulated E-cadherin expression at the protein and RNA level. In contrast, the Jagged1 intracellular domain fragment construct enhanced E-cadherin expression but only at the RNA level. Conclusion: The soluble Jagged1 ectodomain is sufficient for the down-regulation of Ecadherin expression whereas the intracellular domain of the protein does not exhibit such an effect and actually increases E-cadherin RNA expression. These results raise the interesting possibility of E-cadherin regulation in cells distal to the site of soluble Jagged1 ligand generation.

Screening of TACE Peptide Inhibitors from Phage Display Peptide Library / 华中科技大学学报（医学）（英德文版）

To obtain the recombinant tumor necrosis factor-α converting enzyme (TACE) ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RTPCR, and the expression plasmids were constructed by inserting T800 and T1300 into plasmid pET28a and pET-28c respectively. The recombinant T800 and T1300 were induced by IPTG, and SDSPAGE and Western blotting analysis results revealed that T800 and T1300 were highly expressed in the form of inclusion body. After Ni2+-NTA resin affinity chromatography, the recombinant proteins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence (TRWLVYFSRPYLVAT) was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3 %. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE.