Open AccessPichia-Expressed Recombinant D6 and DARC Negatively Affect Cell Migration and Invasion of Breast Cancer Cells
Author(s) -
Wee Yee Tan,
Boon Yin Khoo,
Ai Lan Chew
Publication year - 2021
Publication title -
sains malaysiana
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.251
H-Index - 29
ISSN - 0126-6039
DOI - 10.17576/jsm-2021-5010-15
Subject(s) - recombinant dna , chemotaxis , chemokine , chemokine receptor , cell migration , biology , viability assay , cancer cell , pichia pastoris , receptor , chemotaxis assay , microbiology and biotechnology , cell , cancer research , cancer , biochemistry , gene , genetics
Atypical chemokine receptor proteins are termed ‘decoy proteins’ as their binding to the respective ligands does not lead to a typical signaling pathway but intercepts the action of chemokines. This method of chemokine activity regulation may also function in tumor suppression. D6 and DARC (Duffy Antigen Receptor for Chemokines) have been reported as decoy chemokine receptors in cancer studies. Purified Pichia-expressed D6 and DARC, produced in-house, were used in cell-based studies to test their biological activities. Cell viability tests showed that recombinant D6 and DARC did not affect cell viability significantly, suggesting that they were not involved in breast cancer cell death. Wound healing assays showed that the presence of recombinant D6 or DARC at 10 µg/mL optimally inhibited the migration of breast cancer cells. ELISA showed an inverse relationship between the recombinant proteins and CCL2 levels in the treated cells. Migration assay using Boyden chamber demonstrated the function of the recombinant proteins in inhibiting chemotaxis activity of treated cells. Invasion assay showed the ability of the recombinant proteins in inhibiting the invasion property of treated cells. Comparison of single and combinatorial effects of the recombinant proteins showed that the combination of D6 and DARC at a 1:1 ratio (10 µg/mL) is most effective in reducing CCL2 levels and inhibiting the migration and invasion of treated cells. It was shown that the purified Pichia-expressed recombinant D6 and DARC are the negative regulators of breast cancer cell migration and invasion, and the inhibition effects were greater when they were used in combination.