Production of human embryonic kidney 293T cells stably expressing C-X-C chemokine receptor type 4 (CXCR4) as a screening tool for anticancer lead compound targeting CXCR4
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2022-08-15
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00243205
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18790631
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2-s2.0-85131138136
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35643380
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Life Sciences
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303
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SCOPUS
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Life Sciences Vol.303 (2022)
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Ha D.T.T., Glab-ampai K., Rojsitthisak P., Vajragupta O. Production of human embryonic kidney 293T cells stably expressing C-X-C chemokine receptor type 4 (CXCR4) as a screening tool for anticancer lead compound targeting CXCR4. Life Sciences Vol.303 (2022). doi:10.1016/j.lfs.2022.120661 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/83634
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Production of human embryonic kidney 293T cells stably expressing C-X-C chemokine receptor type 4 (CXCR4) as a screening tool for anticancer lead compound targeting CXCR4
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Aim: The C-X-C chemokine-receptor type 4 (CXCR4) is an emerging target for cancer drug discovery due to its high expression in cancer cells. The present study aimed to produce CXCR4 overexpressing HEK293T cells for a non-radioactive binding assay as a platform to identify drug candidates targeting CXCR4. Main methods: HEK293T cells stably expressing human CXCR4 were constructed by transfection of CXCR4 plasmids from the human CXCR4 gene. The CXCR4 overexpressing HEK293T cells were obtained by fluorescence-activated sorting and verified by conducting the competition binding assay of a known CXCR4 inhibitor, AMD3100 (plerixafor), to determine the IC50 value against monoclonal anti-human CD184 (hCD184) antibody tagged with fluorescence probe, phycoerythrin (PE). The non-radioactive binding assay using CXCR4 overexpressing HEK293T cells and PE-anti hCD184 was applied as a platform for identifying the target of natural compounds that exhibited cytotoxicity against cancer cell lines. Key findings: The CXCR4 overexpressing HEK293T cells were produced with high expression (99.8%). The IC50 value of plerixafor determined by fluorescence tagged antibody-based competition assay using our developed cells agree with previously reported values using a radioligand binding assay. We observed no significant displacement of bound PE-anti-hCD184 by the test natural compounds which could be due to non-specific binding to other functional targets or organelles, low potency of the natural compounds, or binding to CXCR4 at deeper pockets. Significance: The verified non-radioactive binding assay can serve as an alternative screening tool for anticancer lead compounds targeting CXCR4 and an essential tool for proof of mechanism study in the drug discovery.