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

Author(s): Xue-Qing Zhang, Lu-Ting Yu, Pei Du, Tian-Qi Yin, Zhi-Yuan Zhang, Ying Xu, Xiang Li, You-Jie Li, Min Wang*, Chen Luo*.

Journal Name: Anti-Cancer Agents in Medicinal Chemistry
(Formerly Current Medicinal Chemistry - Anti-Cancer Agents)

Volume 19 , Issue 5 , 2019

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Graphical Abstract:


Abstract:

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-Fluorouracil (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 (KD) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetrazolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay.

Results: The KD of scFv-Reg4 to Reg4 was determined to be 1.91×10-8. 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 MKN45 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.

Keywords: Gastrointestinal cancers, Reg4 protein, 5-FU, cell proliferation, phage library, single-chain antibody.

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Article Details

VOLUME: 19
ISSUE: 5
Year: 2019
Page: [610 - 619]
Pages: 10
DOI: 10.2174/1871520619666181122104720
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