Development of a lytic peptide derived from BH3-only proteins

Abstract

Despite great advances in cancer therapy, drug resistance is a difficult hurdle to overcome that requires development of anticancer agents with novel and effective modes of action. In a number of studies, lytic peptides have shown remarkable ability to eliminate cancer cells through a different way from traditional treatments. Lytic peptides are positively charged, amphiphilic, and are efficient at binding and disrupting the negatively charged cell membrane of cancer cells. In this study, we described the anticancer properties of a lytic peptide that was developed on the basis of the alignment of amphiphilic BH3 peptides. Our results demonstrated that the positive charge and conformation constraint were favourable for efficient cancer cell elimination. Artificial BCL-2 homology 3 peptides (ABH3) exhibited effective anticancer effects against a series of cancer cell lines in vitro and in HeLa human cervical tumour xenografts in vivo. ABH3 induced cell death in an apoptosis-independent manner through the lytic properties of the peptide that caused disruption of cell membrane. Our results showed that charge tuning and conformation constraining in a lytic peptide could be applied to optimise the anticancer activity of lytic peptides. These results also suggest that ABH3 may be a promising beginning for the development of additional lytic peptides as anticancer reagents.

Figure 1

Design of a lytic peptide ABH3 based on BH3-only proteins. (Blue: basic amino acids, red: acidic amino acids; purple: polar amino acids, white: non-polar amino acids; black: Gly or Ala).

Figure 2

Amphiphilicity and positive charge are required for cell-killing properties of ABH3 peptides. (a) CD spectra of ABH3_um, ABH3 and ABH3_E. CD spectral measurements were performed in PBS buffer, 10 mM, pH 7.4, 298 K; (b) Cell viability of HCT116 cell lines after treatment with ABH3_um, ABH3 and ABH3_E (24 h); (c) Cellular uptake of ABH3_{um FITC}, ABH3_FITC and ABH3_{E FITC}, (1 h). Values are expressed as mean (±S.D.) (n=3).

Figure 3

ABH3 induced cell death in an apoptosis-independent way. (a) Action of ABH3 on different cancer cell lines (24 h); (b) Action of ABH3 lytic peptide and ABT-737 with or without Z-VAD-FMK on HCT116 and the Bak/Bax double knockout HCT116 cell line; (c) Caspase activation in HCT116 after treatment with ABH3 and ABT-737 (10 μM, 4 h); (d) ATP depletion in HCT116 after treatment with ABH3 and ABT-737 (10 μM, 4 h). Values are expressed as mean (±S.D.) (n=3).

Figure 4

ABH3 induced cell membrane destruction. (a) LDH release of HCT116 following treatment of ABH3 or ABT-737 (20 μM, 4 h); (b) PI staining of U937 cells following treatment with ABH3 and ABT-737 (10 μM, 0.5 h); (c) Scanning electron microscope image of HCT116 upon treatment with ABH3 (10 μM, 1 h), (i) control, (ii) treated by ABH3. Values are expressed as mean (±S.D.) (n=3).

Figure 5

Anticancer efficacy of ABH3 in vivo. (a) Tumour size of HeLa xenografts. Tumour size was measured by calliper measurements over a period of 2 weeks. (b) Tumour weights of HeLa xenografts. Mice were killed and tumours were resected after the final injection. Error bars represent maximum and minimum; boxes represent the upper and lower quartiles and median; (c) H&E staining of tumour cross-sections from mice treated with control and ABH3. Scale bar, 20 μm. mean±S.D. (n=5).