Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor

Abstract

The dermaseptin antimicrobial peptide family contains members of 27-34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5'-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested.

Keywords: amphibian; anticancer; antimicrobial; molecular cloning; peptide.

Figure 1

Nucleotide sequences of precursor cDNAs encoding dermaseptin-PD-1 (a) and dermaseptin-PD-2 (b) cloned from a Pachymedusa dacnicolor skin secretion library and deduced amino acid sequences of precursors. The nucleotide sequences of mature peptides are single-underlined, the nucleotide sequences of putative signal peptides are double-underlined and the stop codons are indicated by asterisks.

Figure 2

Alignment of amino acid sequences of dermaseptin-B2, dermaseptin-PD-1 and dermaseptin-PD-2. The identical amino acid residues are indicated by asterisks.

Figure 3

Reversed-phase HPLC chromatogram of Pachymedusa dacnicolor skin secretion indicating elution positions/retention times of the two novel dermaseptin peptides (arrows). X-axis indicates retention time in min and the Y-axis indicates relative absorbance at 214 nm in arbitrary units.

Figure 4

Predicted secondary structures of (a) dermaseptin-PD-1 and (b) dermaseptin-PD-2 using I-TASSER online server.

Figure 4

Predicted secondary structures of (a) dermaseptin-PD-1 and (b) dermaseptin-PD-2 using I-TASSER online server.

Figure 5

Synergistic effect between dermaseptin-PD-1 (PD-1) and dermaseptin-PD-2 (PD-2) against the growth of the Gram-negative bacterium, E. coli. The best combination concentrations of dermaseptin-PD-1 and dermaseptin-PD-2 are 4.9 μM and 1.26 μM, respectively. The ΣFIC was calculated as 0.5.

Figure 6

The effect on proliferation of cancer cell lines, H157 (a), PC-3 (b) and U251-MG (c) after treatment with dermaseptin-PD-2 within the concentration range of 10⁻⁹ to 10⁻⁴ M. The effect on proliferation of cancer cell line U251 MG (d) after treatment with dermaseptin-PD-1 within the concentration range of 10⁻⁹ to 10⁻⁴ M. The effect on normal human cell line, human microvessel endothelial cell, HMEC-1 after treatment with dermaseptin-PD-1 (e) and dermaseptin-PD-2 (f) within the concentration range of 10⁻⁹ to 10⁻⁴ M. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

The effect on proliferation of cancer cell lines, H157 (a), PC-3 (b) and U251-MG (c) after treatment with dermaseptin-PD-2 within the concentration range of 10⁻⁹ to 10⁻⁴ M. The effect on proliferation of cancer cell line U251 MG (d) after treatment with dermaseptin-PD-1 within the concentration range of 10⁻⁹ to 10⁻⁴ M. The effect on normal human cell line, human microvessel endothelial cell, HMEC-1 after treatment with dermaseptin-PD-1 (e) and dermaseptin-PD-2 (f) within the concentration range of 10⁻⁹ to 10⁻⁴ M. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7

The effect on proliferation of human neuronal glioblastoma cell line, U251-MG after treatment with an array of combinations of dermaseptin-PD-1 (PD-1) and dermaseptin-PD-2 (PD-2). The arrow indicates the highest inhibitory rate of a combination of dermaseptin-PD-1 (10 μM) and dermaseptin-PD-2 (10 μM) and was used to calculate the combination index (Q), which is 1.086 (Q < 0.85, Q > 1.15 and 0.85 < Q < 1.15 indicate antagonism, synergy, and additive effect, respectively.)