EFFECTS OF ANTIMICROBIAL PEPTIDES OF NEUTROPHILS ON TUMOR AND NORMAL CELLS IN CULTURE
O. V. Shamova,1, * G. A. Sakuta,2 D. S. Orlov,1
V. V. Zenin,2 G. I. Shtein,2 N. I. Kolodkin,3
I. V. Afonina,2 V. N. Kokryakov 1
1 Institute of Experimental Medicine RAMS, 2 Institute of Cytology RAS, and
3 Research Institute of Pure Biochemicals, St. Petersburg;
*e-mail: oshamova@yandex.ru
We performed a comparative study of effects of two structurally different cationic antimicrobial peptides of
cathelicidin family, porcine protegrin 1 (PG1) and caprine bactenecin 5 (Bac5) on selected tumor and normal
mammalian cells in vitro. Protegrins are amphiphilic β-hairpin molecules having broad-spectrum antimicrobial
activity due to their marked membranolytic properties. Bac5 belongs to the group of proline-rich peptides,
which adopt a polyproline type II extended helix and kill microorganisms rather by a non-lytic mechanism. We
have shown that while PG1 exerts distinct and fast cytotoxic effects on most of used tumor cells being slightly
less toxic for nontransformed host cell, the proline-rich Bac5 is much less cytotoxic for all the cells tested. The
toxic effects of PG1 were partially declined in the presence of 10 % fetal calf serum. It was revealed that PG1
was able to interact with proteins of serpin family (as had been previously established for human defensins by
Panyutich et al., 1995). Pre-incubation of PG1 with α1-antitrypsin caused the decrease of the cytotoxic activity
of the peptide and, on the other hand, the antiprotease activity of α1-antitrypsin was reduced after interaction of
the serpin with PG1 (not with Bac5). Confocal microscopy experiments allowed to monitor the internalization
of fluorescent labeled (by BODIPY FL) peptides into target cells and their intracellular distribution. Bac5-BODIPY
(at 5 μM) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations was also able to enter
the cells without significant damage to them. The comparative study of the kinetics of the peptides uptake into
the target cells and the influence of low temperature, energy-depletion and endocytosis inhibitors on the process
of the internalization of the peptides into the cells was carried out using flow cytometry.
Key words: antimicrobial peptides, caprine Bac5, protegrin 1, innate immunity, cytotoxicity,
serpins
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