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Development of a lytic peptide derived from BH3-only proteins

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Amphiphilicity and positive charge are required for cell-killing properties of ABH3 peptides. (a) CD spectra of ABH3um, ABH3 and ABH3E. 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 ABH3um, ABH3 and ABH3E (24 h); (c) Cellular uptake of ABH3um FITC, ABH3FITC and ABH3E FITC, (1 h). Values are expressed as mean (±S.D.) (n=3).
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fig2: Amphiphilicity and positive charge are required for cell-killing properties of ABH3 peptides. (a) CD spectra of ABH3um, ABH3 and ABH3E. 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 ABH3um, ABH3 and ABH3E (24 h); (c) Cellular uptake of ABH3um FITC, ABH3FITC and ABH3E FITC, (1 h). Values are expressed as mean (±S.D.) (n=3).

Mentions: ABH3um showed limited helicity in PBS (Figure 2a) according to the CD spectrum and moderate cell-killing activity (IC50~30 μM) against HCT116 according to the MTT results (Figure 2b). Conformation constraining has achieved great success in making peptides more effective. These reports prompted us to incorporate conformation constraining amino acids to achieve more helical peptides and test their effects. The Ala and Gly in ABH3um were replaced with a dihedral angle-constraining amino acid, aminoisobutyric acid, to enhance the peptide’s conformation constraint.22 The resulting peptide, ABH3 (Ac-WLRQIURRLRRIUDELNR-NH2, U=aminoisobutyric acid), showed enhanced helicity and cellular activity (Figures 2a and b). These results indicated that conformation constraining is favourable for enhancing the cytotoxicity of ABH3. However, a less positively charged control peptide, ABH3E, with two amino acid residues altered to Glu (Ac-WLREIURQLRRIUDELNR-NH2, U=aminoisobutyric acid) showed higher helicity than ABH3 but negligible effect on cell growth of HCT116 cells (Figures 2a and b). As a result, the combination of conformation constraint and a higher positive charge were favourable for cell-killing activities of ABH3. Besides, cell viability correlated well with the cellular uptake of these peptides as quantified by FACS (Figure 2c). ABH3 exhibited a roughly fivefold increase in permeability compared with ABH3um or ABH3E (Figure 2c). These results illustrated the impact of both of the peptide’s helicity and net charge to their effective penetration and cell-killing. Although ABH3um showed similar cellular penetration with ABH3E, their cellular activities are quite different. In summary, this experiment confirmed that the net charge of the engineered peptide has a central role in the cellular activity of lytic peptides and demonstrated that positive charge tuning and conformation constraining were efficient methods for enhancing the cell-killing properties of lytic peptides. These observations could be further applied in lytic peptide development using other peptide skeletons.


Development of a lytic peptide derived from BH3-only proteins
Amphiphilicity and positive charge are required for cell-killing properties of ABH3 peptides. (a) CD spectra of ABH3um, ABH3 and ABH3E. 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 ABH3um, ABH3 and ABH3E (24 h); (c) Cellular uptake of ABH3um FITC, ABH3FITC and ABH3E FITC, (1 h). Values are expressed as mean (±S.D.) (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979451&req=5

fig2: Amphiphilicity and positive charge are required for cell-killing properties of ABH3 peptides. (a) CD spectra of ABH3um, ABH3 and ABH3E. 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 ABH3um, ABH3 and ABH3E (24 h); (c) Cellular uptake of ABH3um FITC, ABH3FITC and ABH3E FITC, (1 h). Values are expressed as mean (±S.D.) (n=3).
Mentions: ABH3um showed limited helicity in PBS (Figure 2a) according to the CD spectrum and moderate cell-killing activity (IC50~30 μM) against HCT116 according to the MTT results (Figure 2b). Conformation constraining has achieved great success in making peptides more effective. These reports prompted us to incorporate conformation constraining amino acids to achieve more helical peptides and test their effects. The Ala and Gly in ABH3um were replaced with a dihedral angle-constraining amino acid, aminoisobutyric acid, to enhance the peptide’s conformation constraint.22 The resulting peptide, ABH3 (Ac-WLRQIURRLRRIUDELNR-NH2, U=aminoisobutyric acid), showed enhanced helicity and cellular activity (Figures 2a and b). These results indicated that conformation constraining is favourable for enhancing the cytotoxicity of ABH3. However, a less positively charged control peptide, ABH3E, with two amino acid residues altered to Glu (Ac-WLREIURQLRRIUDELNR-NH2, U=aminoisobutyric acid) showed higher helicity than ABH3 but negligible effect on cell growth of HCT116 cells (Figures 2a and b). As a result, the combination of conformation constraint and a higher positive charge were favourable for cell-killing activities of ABH3. Besides, cell viability correlated well with the cellular uptake of these peptides as quantified by FACS (Figure 2c). ABH3 exhibited a roughly fivefold increase in permeability compared with ABH3um or ABH3E (Figure 2c). These results illustrated the impact of both of the peptide’s helicity and net charge to their effective penetration and cell-killing. Although ABH3um showed similar cellular penetration with ABH3E, their cellular activities are quite different. In summary, this experiment confirmed that the net charge of the engineered peptide has a central role in the cellular activity of lytic peptides and demonstrated that positive charge tuning and conformation constraining were efficient methods for enhancing the cell-killing properties of lytic peptides. These observations could be further applied in lytic peptide development using other peptide skeletons.

View Article: PubMed Central - PubMed

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.

No MeSH data available.