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Peptides From the N-terminal End of Bovine Lactoferrin Induce Apoptosis in Human Leukemic (HL-60) Cells

M. K. Roy^*, Y. Kuwabara, K. Hara, Y. Watanabe^* and Y. Tamai^*

^* Department of Biological Chemistry, Faculty of Agriculture, Ehime University, Japan 790-8566
Shikoku Milk Product Co. Ltd. Kawauchi, Ehime, Japan. 791-0397

Corresponding author:
Y. Tamai; e-mail:
tamai{at}agr.ehime-u.ac.jp

To determine the effects of the multifunctional iron-binding glycoprotein, lactoferrin (LF) and related compounds on the growth of leukemic cells, human myeloid leukemic cells (HL-60) were exposed to bovine lactoferrin (bLF) and proteolytic hydrolysates of bLF. Pepsin hydrolysates of bLF showed a greater growth suppressive effect than tryptic hydrolysates or mature bLF. Four peptides with proliferation inhibition activity were purified from pepsin hydrolysates by ion-exchange chromatography, reverse-phase HPLC, and gel-filtration. All peptides were from the N-terminal end, in a region where lactoferricin B (Lfcin B), an antibacterial peptide, is located. Among the four peptides, peptide 1 (pep1) was found to exhibit highest activity and corresponded to residues 17 to 38 of bLF, with a molecular weight of 2753.88. The IC₅₀ value of this peptide was 6.3 µg/ml. Three other peptides were less active and corresponded to sequences 1 to 16 and 45 to 48, linked by disulfide-bridge (pep2, molecular mass of 2430.13), 1 to 15 and 45 to 46 linked by disulfide bridge (pep3, molecular mass of 2017,92) and from residues 1 to 13 (pep4, molecular mass of 1558.73). Cell proliferation inhibition activity of the peptides was thought to be due to induction of apoptosis, which was evaluated by DNA ladder formation, DNA fragmentation, enhanced expression of phosphatidyl serine, and morphological changes. The IC₅₀ values of the three peptides were confirmed using synthetic peptides and were consistent with those of purified peptides.

Abbreviation key: bLF = bovine lactoferrin, FITC = fluorescein isothiocyante, pep = peptide, PI = propidium iodide, RP-HPLC = reverse-phase HPLC, TBE = 89 m, M Tris, 89 m, M boric acid, and 2 m, M EDTA buffer, , TFA = trifluoroacetic acid

Key Words: bovine lactoferrin • apoptosis • leukemic cells