Development of Hek-293t cells stably expressing the c-x-c chemokine receptor type 4 (cxcr4) for binding assay of potential anti-breast cancer agents

Abstract

The C-X-C chemokine receptor type 4 (CXCR4) has been recognized as a potential target in cancer drug discovery based on the high expression in cancer cells, particularly invasive breast cancer. This study aims to construct CXCR4-overexpressed cells to screen new anti-breast cancer agents targeting CXCR4 and identify the mediated target for anticancer effect. Firstly, five genetic isotypes of CXCR4, namely A, B, C, D and E, were amplified from Jurkat T cells. Amplicons of each CXCR4 isotype were ligated into cloning vectors and subcloned into expression vectors. They were transformed into competent Escherichia coli strain DH5-alpha cells. The presence of CXCR4 plasmids in the selective transformed cells was determined by colony PCR and gel electrophoresis. The recombinant plasmids of CXCR4-isotype B were selected for stable transfection in HEK293T cells. CXCR4 expression was confirmed by flow cytometry and the population of the highly CXCR4 expressing cells was isolated using the fluorescence-activated sorting cells technique to obtain 99.8% of CXCR4-expressing cells. The CXCR4-overexpressed HEK293T cells were verified by conducting the competitive binding assay of a known CXCR4 inhibitor, AMD3100 (plerixafor), using monoclonal anti-human CD184 (CXCR4) antibody tagged with fluorescence probe, phycoerythrin. The obtained IC50 of plerixafor (305.5 nM) against the binding of the antibody to CXCR4 was in accordance with those determined by conventional competitive radioligand binding assay. Moreover, the developed CXCR4-overexpressed HEK293T cells were used in the competitive binding assay to identify the mediated target of natural compounds showing high cytotoxicity against the invasive breast cancer cell line MDA-MB-231. The tested compounds were extracted from Stephania pierrei, which were methoxy-8-uvariopsine, crebanine and dehydrocrebanine. However, the application of the fluorescence tagged antibody competition binding assay to determine the binding affinity of these potential compounds has been limited due to the poor solubility of natural compounds, which required additional solvent and surfactant to improve the solubility leading to high fluorescence quenching. Further development of low fluorescence quenching solvent and solubilizer is needed to implement this assay in the screening platform.