Sains Malaysiana 50(10)(2021): 3015-3033

http://doi.org/10.17576/jsm-2021-5010-15

Pichia-Expressed Recombinant D6 and DARC Negatively Affect Cell Migration and Invasion of Breast Cancer Cells

(Rekombinan D6 dan DARC Hasilan Pichia Mempengaruhi Migrasi Sel dan Serangan Sel Kanser Payudara secara Negatif)

TAN WEE YEE, KHOO BOON YIN & CHEW AI LAN*

Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 USM, Penang, Malaysia

Received: 6 August 2020/Accepted: 19 February 2021

ABSTRACT

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.

Keywords: Breast cancer cells; CCL2; cell migration and invasion; D6; DARC

ABSTRAK

Protein reseptor kemokin atipikal disebut ‘umpan protein’ kerana pengikatannya dengan ligan masing-masing tidak membawa kepada jalan isyarat yang khas tetapi memintas tindakan kemokin. Ia merupakan satu kawalan aktiviti kemokin dan boleh berfungsi dalam penyekatan tumor. D6 dan DARC telah dilaporkan sebagai reseptor kemokin umpan dalam kajian kanser. D6 dan DARC ekspresi Pichia yang dihasilkan di makmal telah digunakan dalam kajian berdasarkan sel untuk menguji aktiviti biologinya. Ujian daya hidup sel menunjukkan bahawa rekombinan D6 dan DARC tidak mempengaruhi daya maju sel secara signifikan, menunjukkan bahawa mereka tidak terlibat dalam kematian sel barah payudara. Ujian penyembuhan luka menunjukkan bahawa kehadiran D6 atau DARC rekombinan pada 10 µg/mL menghalang penghijrahan sel barah payudara secara optimum. ELISA menunjukkan hubungan terbalik antara protein rekombinan dan tahap CCL2 pada sel yang dirawat. Ujian migrasi menggunakan ruang Boyden menunjukkan fungsi protein rekombinan dalam menghalang aktiviti kemotaksis sel yang dirawat. Ujian penaklukan menunjukkan kemampuan protein rekombinan dalam merencat sifat penaklukan sel yang dirawat. Membandingkan kesan tunggal dan gabungan protein rekombinan, gabungan D6 dan DARC pada nisbah 1: 1 (10 µg/mL) didapati paling baik dalam mengurangkan tahap CCL2 dan seterusnya menghalang migrasi dan penaklukan sel yang dirawat. Hasil kajian menunjukkan bahawa rekombinan D6 dan DARC hasilanPichia bukan hanya pengawal negatif migrasi dan penaklukan sel barah payudara tetapi kesan perencatannya lebih besar ketika digunakan dalam gabungan.

Kata kunci: CCL2; D6; DARC; migrasi dan penaklukan sel; sel payudara

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*Corresponding author; email: chew@usm.my