Single-chain Antibody Against Reg4 Suppresses Gastric Cancer Cell Growth and Enhances 5-FU-induced Cell Death in vitro

Background: Regenerating islet-derived gene family member 4 (Reg4), a well-investigated growth factor in the regenerative pancreas, has recently been reported to be highly associated with a majority of gastrointestinal cancers. Pathological hyper-expression or artificial over-expression of Reg4 causes acceleration of tumor growth, migration, and resistance to chemotherapeutic 5-Fluoroumcil (5-FU). Until now, no method has been successfully established for eliminating the effects of Reg4 protein. Methods: This study reports the production of an engineered immunoglobin, a single-chain variable fragment (scFv-Reg4), to specifically bind Reg4 and block the bioactivity. The complementary-determining regions (CDRs) against Reg4 were assigned using MOE and ZDOCK servers. The binding affinity (K D ) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetra-zolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay. Results: The K-D of scFv-Reg4 to Reg4 was determined to be 1.91x10(-8). In MKN45 and AGS cell lines, scFv-Reg4 depressed Reg4-stimulated cell proliferation and the inhibitory rates were 27.7 +/- 1.5% and 17.3 +/- 2.6%, respectively. Furthermore, scFv significantly enhanced 5-FU-induced cell death, from 23.0 +/- 1.0% to 28.4 +/- 1.2% in MK.N45 and 28.2 +/- 0.7% to 36.6 +/- 0.6% in AGS cells. Treatment with scFv alone could lyse cancer cells to a certain extent, but no significance has been observed. Conclusion: The single-chain antibody (scFv-Reg4) significantly inhibited gastric cancer cell proliferation and synergistically enhanced the lethal effect of 5-FU. Thus, traditional chemo-/radio- therapeutics supplemented with scFv-Reg4 may provide advances in the strategy for gastrointestinal cancer treatment.