Glycan Alteration Imparts Cellular Resistance to a Membrane-Lytic Anticancer Peptide  Ken Ishikawa, Scott H. Medina, Joel P. Schneider, Amar J.S. Klar  Cell Chemical Biology  Volume 24, Issue 2, Pages 149-158 (February 2017) DOI: 10.1016/j.chembiol.2016.12.009 Copyright © 2017 Terms and Conditions

Cell Chemical Biology 2017 24, 149-158DOI: (10. 1016/j. chembiol. 2016 Copyright © 2017 Terms and Conditions

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 toward membrane-lytic ACPs (green). (B) Resistance toward the oncolytic peptide SVS-1 was explored in yeast as a model organism. Colonies of wild-type (WT) yeast (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. Cell Chemical Biology 2017 24, 149-158DOI: (10.1016/j.chembiol.2016.12.009) Copyright © 2017 Terms and Conditions

Figure 2 Reduction in Yeast Cell-Surface Negative Charge due to Gene Mutation (A) WT avidly bind to cationic Q Sepharose beads, while the pvg2 deletion mutant (pvg2Δ) displayed weak binding interactions. Beads appear as large spheres, while cells appear as 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 (B) and (C), results are shown as mean ± SD for three replicates. Statistical significance compared with WT cells is shown as **p ≤ 0.01 and ***p < 0.001. Cell Chemical Biology 2017 24, 149-158DOI: (10.1016/j.chembiol.2016.12.009) Copyright © 2017 Terms and Conditions

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 ± SD from three independent experiments are shown. Cell Chemical Biology 2017 24, 149-158DOI: (10.1016/j.chembiol.2016.12.009) Copyright © 2017 Terms and Conditions

Figure 4 Characterization of SVS-1-Resistant A549 Tumor Cells (A) Peptide cytotoxicity toward 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 6 months in blank medium (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 pH 7.4 and 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 with the parent A549CUL cell line (top), after staining with a nuclear dye (10× magnification; scale bar, 400 μm). (F) Average cluster area of cell aggregates measured from the microscopy images for each cell line. For panels A-D and F, values are shown as mean ± SD. Statistical analysis was performed using the Student's t test, assuming unequal variance, with **p ≤ 0.01 and ***p < 0.001. Cell Chemical Biology 2017 24, 149-158DOI: (10.1016/j.chembiol.2016.12.009) Copyright © 2017 Terms and Conditions

Figure 5 Reduction of Cell-Surface 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 inhibitor 3Fax-Peracetyl Neu5c, followed by enzymatic hydrolysis of residual carbohydrate, reduced surface SA content by ∼80% compared with controls. All data are shown normalized to basal SA expression of untreated A549CUL cells. (B) Cytotoxicity of the SVS-1 peptide toward A549CUL cells before (pink) and after (red) removal of cell-surface SA. Peptide toxicity toward A549RES cells is included as a positive control (blue). Mean ± SD from three independent experiments are shown. IC50 values were calculated using non-linear regression. (C) Model of cellular resistance toward 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. Cell Chemical Biology 2017 24, 149-158DOI: (10.1016/j.chembiol.2016.12.009) Copyright © 2017 Terms and Conditions