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. 2017 Feb 16;24(2):149-158.
doi: 10.1016/j.chembiol.2016.12.009. Epub 2017 Jan 12.

Glycan Alteration Imparts Cellular Resistance to a Membrane-Lytic Anticancer Peptide

Ken Ishikawa  1 , Scott H Medina  2 , Joel P Schneider  3 , Amar J S Klar  4
Affiliations

Glycan Alteration Imparts Cellular Resistance to a Membrane-Lytic Anticancer Peptide

Ken Ishikawa et al. Cell Chem Biol. .

Abstract

Although resistance toward small-molecule chemotherapeutics has been well studied, the potential of tumor cells to avoid destruction by membrane-lytic compounds remains unexplored. Anticancer peptides (ACPs) are a class of such agents that disrupt tumor cell membranes through rapid and non-stereospecific mechanisms, encouraging the perception that cellular resistance toward ACPs is unlikely to occur. We demonstrate that eukaryotic cells can, indeed, develop resistance to the model oncolytic peptide SVS-1, which preferentially disrupts the membranes of cancer cells. Utilizing fission yeast as a model organism, we show that ACP resistance is largely controlled through the loss of cell-surface anionic saccharides. A similar mechanism was discovered in mammalian cancer cells where removal of negatively charged sialic acid residues directly transformed SVS-1-sensitive cell lines into resistant phenotypes. These results demonstrate that changes in cell-surface glycosylation play a major role in tumor cell resistance toward oncolytic peptides.

Keywords: anticancer peptides; cancer cell resistance; genetics; glycosylation; membrane-lytic peptides.

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Conflict of interest statement

Competing information

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Identification of SVS-1 resistance in a model organism
(A) Left: Resistance mechanisms toward small molecule chemotherapeutics (red) have been widely explored, and include increased drug efflux, decreased cellular uptake and mutation of the target molecule. Right: Conversely, little is known about the potential of cells to gain resistance towards membrane-lytic anticancer peptides (green). (B) Resistance towards the oncolytic peptide SVS-1 was explored in yeast as a model organism. Colonies of wild-type yeast (wt; left) transferred to one-half of the plate without SVS-1 (-) grow as expected, while those replica plated onto a surface coated with SVS-1 (+) are killed. Displayed on the right, a spontaneous yeast mutant with loss of function of the pvg2 gene (labeled pvg2-1) exhibited strong resistance to the peptide. Colonies were grown from random spores of indicated strains. (C) Identified yeast mutations which lead to SVS-1 resistance. (D) Deletion mutants, constructed for pvg2, uge1 and gms1 genes, grew as well as the wt strain on regular yeast extract agar growth media, while only the deletion mutants grew on plates coated with the SVS-1 peptide.
Figure 2
Figure 2. Reduction in yeast cell-surface negative charge due to gene mutation
(A) Wild-type cells (wt) avidly bind to cationic Q-sepharose beads, while the pvg2 deletion mutant (pvg2∆) displayed weak binding interactions. Beads appear as large spheres, while cells look like short rods or ellipses. (B) Number of cells bound per bead for the wt yeast and the deletion mutants. (C) Cell-surface charge of each yeast strain as measured by zeta potential analysis. For panels B and C, results are shown as mean ± standard deviation for three replicates. Statistical significance compared to wt cells is shown as ** indicating p ≤ 0.01, and *** indicating p < 0.001.
Figure 3
Figure 3. Influence of yeast cell-surface charge on SVS-1 resistance
(A) Using an iterative enrichment protocol, yeast cell sub-populations with reduced negative charge were selected. (B) Representative images that show cells selected from each successive enrichment cycle displayed increased resistance to SVS-1 mediated lysis. (C) Correlation of the percentage of cells bound to Q-sepharose beads and their survival on SVS-1 coated plates, as a function of enrichment cycle number. Mean ± standard deviation from three independent experiments are shown.
Figure 4
Figure 4. Characterization of SVS-1 resistant A549 tumor cells
(A) Peptide cytotoxicity towards the resistant A549RES cell line derived through SVS-1 selection in culture. Parent cells used to initiate the resistant line (A549INT) or the same cells cultured over six months in blank media (A549CUL) were included as controls. (B) IC50 values from the viability data were calculated using non-linear regression. (C) Surface zeta potential values of the three cell lines in PBS buffer at pH7.4, 37°C. (D) Relative binding of fluorescently-labeled Annexin-V (A-V) to cell-surface phosphatidylserine was measured by flow cytometry. (E) Fluorescent microscopy images showing increased aggregation of A549RES cells (bottom) compared to the parent A549CUL cell line (top), after staining with a nuclear dye (10x magnification; scale bar = 400 μm). (F) Average cluster area of cell aggregates measured from the microscopy images for each cell line. Quantifications are shown as mean ± standard deviation. Statistical analysis was performed using the Student’s t-test, assuming unequal variance, with ** indicating p ≤ 0.01, and *** indicating p < 0.001.
Figure 5
Figure 5. Reduction of cell-surface sialic acid (SA) modulates resistance to peptide- sensitive tumor cells
(A) Percentage of SA measured at the surface of A549RES, A549INT and A549CUL cell lines. Treatment of A549CUL cells with the sialyltransferase inhibitior 3Fax-Peracetyl Neu5c, followed by enzymatic hydrolysis of residual carbohydrate, reduced surface SA content by ~80% compared to controls. All data is shown normalized to basal SA expression of untreated A549CUL cells. (B) Cytotoxicity of the SVS-1 peptide towards A549CUL cells before (pink) and after (red) removal of cell-surface SA. Peptide toxicity towards A549RES cells is included as a positive control (blue). Mean ± standard deviation from three independent experiments are shown. IC50 values were calculated using non-linear regression. (C) Model of cellular resistance towards the cationic ACP, SVS-1 (green). Electrostatic binding of SVS-1 with negatively charged saccharides at the cell surface (e.g. SA; red) promotes its partitioning to the membrane where it elicits its lytic function. Adaptation of cells through the loss of SA leads to reduced electrostatic engagement of the peptide with cell-surface glycans, thereby diminishing its potential to access the membrane and conferring resistance.
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