Novel peptide dermaseptin-PS1 exhibits anticancer activity via induction of intrinsic apoptosis signalling

Abstract

Antimicrobial peptides (AMP) secreted by the granular glands of frog skin have been widely reported to exhibit strong bacteriostatic and bactericidal activities. Many of them have been documented with potent antiproliferative effects on multiple cancer cells, many studies also suggested that AMPs exert their functions via disrupting cell membranes. However, whether and how other cell death induction mechanism is involved in mammalian cancer cells has rarely been investigated. In this study, a novel AMP named Dermaseptin-PS1 was isolated and identified from Phyllomedusa sauvagei, it showed strong antimicrobial activities against three types of microorganisms. In vitro antiproliferative studies on human glioblastoma U-251 MG cells indicated that Dermaseptin-PS1 disrupted cell membranes at the concentrations of 10^-5 M and above, while the cell membrane integrity was not affected when concentrations were decreased to 10^-6 M or lower. Further examinations revealed that, at the relatively low concentration (10^-6 M), Dermaseptin-PS1 induced apoptosis through mitochondrial-related signal pathway in U-251 MG cells. Thus, for the first time, we report a novel frog skin derived AMP with anticancer property by distinct mechanisms, which largely depends on its concentration. Together, our study provides new insights into the mechanism-illustrated drug design and the optimisation of dose control for cancer treatment in clinic.

Keywords: antimicrobial peptides; apoptosis; concentration; dermaseptin; mitochondria.

Figure 1

Primary structure identification, determination and analysis of Dermaseptin‐PS1 from Phyllomedusa sauvagei (A) nucleotide and translated open‐reading frame amino acid sequences of cloned cDNA encoding the Dermaseptin‐PS1 precursor from P. sauvagei skin secretion (EMBL accession number: LT840240). The putative signal peptide is double‐underlined, the mature peptide is single‐underlined and the stop codon is indicated by an asterisk. (B) Alignment of cDNA deduced mature Dermaseptin‐PS1 sequence with dermaseptin peptides from P. sauvagei species. The identical and conservative residues are indicated by asterisks (*) and full stop (.)/colon (:), respectively. (C) rp‐HPLC chromatogram of P. sauvagei skin secretion with an arrow indicating the retention time (at 123 min) of Dermaseptin‐PS1. The detection wavelength was 214 nm with a flow rate of 1 mL/min in 240 min. (D) Predicted single‐ and double‐charged b‐ and y‐ion series arising from LCQ MS/MS fragmentation of Dermaseptin‐PS1. The truly observed fragment ions following actual fragmentation are shown in red (b‐ions) and blue (y‐ions) typefaces respectively. (E) The CD spectra recorded for the purified synthetic Dermaseptin‐PS1 in (a) 10 mmol/L ammonium acetate (NH₄Ac) water solution and (b) 50% 2,2,2‐trifluoroethanol (TFE)‐50% NH₄Ac water solution. The molar ellipticity was plotted against wavelength and the tested peptide concentration was 100 μmol/L

Figure 2

Antimicrobial activity and haemolytic effect of Dermaseptin‐PS1. The minimum inhibitory concentrations of Dermaseptin‐PS1 against (A) Staphylococcus aureus, (B) Escherichia coli and (C) Candida albicans. The minimum bactericidal concentration (MBC) values against S. aureus, E. coli were 10⁻⁴ M and no MBC value was detected against C. albicans. Data were analysed with unpaired Student's t test using GraphPad Prism 5 software. Values are the mean ± SEM for three independent experiments. Veh represents vehicle control, *P < 0.05, **P < 0.01 and ***P < 0.001 vs vehicle control. (D) Haemolytic activity of Dermaseptin‐PS1 following incubation with horse erythrocytes for 2 h. Data were analysed with unpaired Student's t test using GraphPad Prism 5 software. Values are the mean ± SEM for three independent experiments. P represents positive control, ***P < 0.001 vs positive control (10% Triton X‐100)

Figure 3

Dermaseptin‐PS1 display antiproliferative activity against U‐251 MG cell line. A, The antiproliferative effect of human neuronal glioblastoma cells, U‐251 MG cell line after the treatment of Dermaseptin‐PS1 gradient (10⁻⁴ to 10⁻⁹ M) for 24 h. The calculated IC₅₀ value was 5.419 μmol/L. Data were analysed with unpaired Student's t test using GraphPad Prism 5 software. Values are the mean ± SEM for three independent experiments. Veh represents vehicle control, ***P < 0.001 vs vehicle control. B, The morphology changes of U‐251 MG cells after the treatment of Dermaseptin‐PS1 gradient (10⁻⁴ to 10⁻⁷ M) for 24 h. The images were observed by a magnification factor ×200. C, Lactate dehydrogenase (LDH) release from U‐251 MG cells after the treatment of Dermaseptin‐PS1 gradient (10⁻⁴ to 10⁻⁸ M) for 24 h. Data were analysed with unpaired Student's t test using GraphPad Prism 5 software. Values are the mean ± SEM for three independent experiments. ***P < 0.001 vs negative control

Figure 4

Dermaseptin‐PS1 induced U‐251 MG cell death at 10⁻⁶ M via apoptosis. Western blot analysis was performed on U‐251 MG cells to test the expression of cleaved caspase 3 and caspase 3 after the treatment of (A) NC, 20 μmol/L, 15 μmol/L and 10 μmol/L etoposide and 10⁻⁴ to 10⁻⁸ M Dermaseptin‐PS1 for 24 h; (B) 10⁻⁶ M Dermaseptin‐PS1 and 20 μmol/L etoposide for indicated times; (C) 20 μmol/L Z‐VAD‐FMK for 2 h subsequent to 20 μmol/L etoposide and 10⁻⁶ M Dermaseptin‐PS1 treatment for 16 h. The detection of GAPDH protein was used as an internal control. The signal intensity was quantified by Image Lab software and GraphPad Prism 5 software was used for statistical comparison. NC, negative control. *P < 0.05, **P < 0.01 and ***P < 0.001 vs negative control. (D) The fluorescent microscopy images showing FITC‐annexin V positive membranes, propidium iodide (PI)‐stained nuclei and a merge image. The images were captured from a population of U‐251 MG with the exposure to Dermaseptin‐PS1 gradient (10⁻⁴ to 10⁻⁷ M) or 20 μmol/L etoposide for 16 h. Scale bar = 25 μm. (E) The mRNA levels of apoptosis‐related genes were analysed by real‐time PCR. The qPCR analysis performed on U‐251 MG cells by the treatment of 10⁻⁶ M Dermaseptin‐PS1 for 16 h. The sequences of pro‐apoptotic genes are displayed in Table 1. The mRNA expression of 18S was used as a calibration standard. Data were analysed with one‐way anova using GraphPad Prism 5 software. Values are the mean ± SEM for three independent experiments. ***P < 0.001 vs negative control

Figure 5

The examinations of extrinsic apoptotic cascade mediated by Dermaseptin‐PS1 in U‐251 MG cells. Protein expression of caspase 8/cleaved caspase 8 and FADD were analysed by Western blot in U‐251 MG cells treated for 4‐24 h with 10⁻⁶ M Dermaseptin‐PS1 or 16‐24 h with 20 μmol/L etoposide. The detection of GAPDH protein was used as an internal control

Figure 6

Dermaseptin‐PS1 induced U‐251 MG cell death through intrinsic apoptosis signalling. A, Protein expression of caspase 9/cleaved caspase 9, Apaf‐1, Bcl‐2, Bax, Bak, p‐Bad, Bad, p‐p53 and p53 were analysed by Western blot in U‐251 MG cells treated for 4‐24 h with 10⁻⁶ M Dermaseptin‐PS1 or 16‐24 h with 20 μmol/L etoposide. The detection of GAPDH protein was used as an internal control. The signal intensity was quantified by Image Lab software and GraphPad Prism 5 software was used for statistical comparison. NC, negative control. *or ^# P < 0.01, ** or ^## P < 0.05 and *** or ^### P < 0.001 vs NC. B, Cytochrome c release from the mitochondria into the cytosol was measured via Western blot. COX IV was used as a loading control for mitochondrial fractions. The signal intensity was quantified by Image Lab software and GraphPad Prism 5 software was used for statistical comparison. NC, negative control; PS1, Dermaseptin‐PS1; Eto, etoposide. *P < 0.05 vs protein expression in cytosol after etoposide treatment; ^### P < 0.001 vs protein expression in cytosol after Dermaseptin‐PS1 treatment