Insect cell-derived human CD200-Fc increases zonula occludens-1 tight junction protein in urothelial carcinoma cells

Abstract

CD200 is a ligand that interacts with the CD200 receptor 1, with an anti-inflammatory effect. CD200 inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-& kappa;B) pathway, attenuating the inflammatory response and maintaining barrier function. In this study, human CD200 was fused with the fragment crystallizable (Fc) region of human immunoglobulin G (IgG) to generate the recombinant CD200-Fc protein, produced in insect cells using the baculovirus expression vector system. The CD200-Fc gene was cloned under the control of the polyhedrin promoter in the pFastBac-1 vector of the baculovirus expression vector system. Immunoblot analysis confirmed the expression of CD200-Fc in insect cells. The CD200-Fc protein was successfully purified using protein A affinity chromatography. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that purified CD200-Fc protein inhibited the proliferation of TCCSUP cells, a bladder epithelial cancer cell expressing CD200 receptor 1, at concentrations of 1, 10 and 100 ng/mL. Quantitative real-time reverse transcription PCR (qRT-PCR) and immunoblot analyses confirmed that the mRNA and protein levels of zonula occludens-1, a tight junction protein for barrier protection in epithelial tissues, were increased in TCCSUP cells treated with insect cell-derived CD200-Fc. These results suggest that insect cell-derived CD200-Fc could alter zonula occludens-1 expression in bladder epithelial cancer cells, enhancing the function of the cell barrier protein, which could inhibit cancer metastasis. Taken together, the baculovirus expression vector system can be applied to express antitumor therapeutic CD200-Fc protein for the inhibition of bladder cancer.