Abstract. Apart from their major function in the coordination of leukocyte recruitment, chemokines, in cooperation with their receptors, have been implicated in the progression of various diseases including different types of cancer, affecting survival, proliferation and metastasis. A complex network of chemokines and receptors exists in the tumor microenvironment and affects tumor development in various ways where chemokines activate typical signalling pathways by binding to the respective receptors. The identification and characterization of a group of atypical chemokine receptors [D6, Duffy antigen receptor for chemokines (DARC), ChemoCentryx chemokine receptor (CCX-CKR) and CXCR7] which appear to use unique biochemical properties to regulate the biological activities of these chemokines, is useful in the effort to therapeutically manipulate chemokines in a broad spectrum of diseases in which these chemokines play a critical role. The aim of this review was to investigate the combinatorial effect of two reported atypical chemokine receptors, D6 and DARC, on breast cancer cell invasion to understand their role and therapeutic potential in cancer treatment. In this regard, findings of the present review should be confirmed via the construction of recombinant D6 and DARC clones as well as the expression of the respective recombinant proteins using the Pichia pastoris (P. pastoris) expression system is to be performed in a future study in order to support findings of the current review.
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.
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