wrwycr treatment with or without etoposide does not induce significant apoptosis.

<p><b>A:</b> Confocal microscopy images showing the transmitted channel (1^st column), cytochrome C-GFP (2^nd column), TMRE (3^rd column) and the overlay of GFP and TMRE channel (4^th column) followed by 24 h treatment. <b>B</b>: % Apoptotic cells scored by the release of cytochrome C (green bar), loss of TMRE (red bar), and typical morphological features of apoptosis such as spikes or blebs (grey bar).</p

Treatment with wrwycr interferes with the PC3 cell cycle over time.

<p>PC3 cells treated with 50, 100, 150, 200 µM wrwycr or 200 µM wrwyar for 24, 36 or 48 h. <b>A</b>: Phosphorylation of checkpoint proteins was analyzed using BDFacs Diva software. Significant increases in the fraction of pChk1⁺ cells were observed with 150 and 200 µM wrwycr, and in the fraction of pChk2⁺ cells with 100,150 and 200 µM wrwycr, after 36 h of treatment, as shown by two-way ANOVA using Bonferroni post test analysis. <b>B</b>: Cell cycle analysis of wrwycr or wrwyar- treated PC3 cells after 48 h. Graphical representation of the percentage of population in each of the phases of cell cycle done at 48 h are from 2 independent experiments, each with duplicate or triplicate independent samples. A significant increase in the fraction of S-phase cells was observed after treatment with 200 µM wrwycr compared to treatment with DMSO, as determined by one-way Anova using Bonferroni post-test analysis. <b>C</b>: Histograms of the DNA content in (<b>i</b>) wrwycr (<b>a–d</b>) or (<b>ii</b>) wrwyar- treated (<b>e–h</b>) cells at different peptide concentrations (0–200 µM). Cells in the G2 peak have twice the DNA content of G1. Cells in transition to G2 from G1 are considered to be in S-phase. <b>C</b>: Graphical representation of the percentage of population in each phase at 48 h from 2 independent experiments, each with duplicate or triplicate independent samples. A significant increase in the fraction of S-phase cells was observed after treatment with 200 µM wrwycr compared to treatment with DMSO, as determined by one-way ANOVA using Bonferroni post-test analysis.</p

MTT assays of PC3 cells treated with a combination of wrwycr and doxorubicin or docetaxel.

<p><b>A:</b> PC3 cells were treated with 10, 25, 50, 75 and 100 µM wrwycr and/or with 0.25, 0.5, 1 or 2.5 µM doxorubicin for 48 h. <b>B</b>: Time course of treatment with 75 or 100 µM of wrwycr and 1 µM doxorubicin either alone or in combination for 12–48 h. <b>C</b>: PC3 cells treated with 0, 25, 50 and 100 µM wrwycr and/or with 10, 25, or 50 nM of docetaxel. MTT activity was converted to % survival after normalizing to the activity of DMSO-treated cells.</p

wrwycr-induced γH2AX foci.

<p><b>A:</b> PC3 cells were treated with RPMI (<b>i–ii</b>), DMSO (<b>iii–iv</b>), 150 µM etoposide (<b>v–vi</b>), or wrwycr at 50 (<b>vii–viii</b>), 100 (<b>ix–x</b>), 150 (<b>xi–xii</b>) µM or wrwyrggrywrw at 1 (<b>xiii–xiv</b>), 10 (<b>xv–xvi</b>) or 50 (<b>xvii–xviii</b>) µM for 48 h. Etoposide was used as the positive control. The green γH2AX foci are visible within the nucleus. <b>B</b>: γH2AX foci in PC3 cells were determined over time (12, 24, 36, 48 h) and quantified using flow cytometry. The γH2AX positive population was gated with respect to FITC-conjugated isotype control antibody. The differences in % γH2AX positive cells are statistically significant for all wrwycr doses between 100–200 µM compared to DMSO after 36 and 48 h of treatment as determined by two-way ANOVA using Bonferroni post test analysis.</p

Tumor Cell Death Mediated by Peptides That Recognize Branched Intermediates of DNA Replication and Repair

<div><p>Effective treatments for cancer are still needed, both for cancers that do not respond well to current therapeutics and for cancers that become resistant to available treatments. Herein we investigated the effect of a structure-selective d-amino acid peptide wrwycr that binds replication fork mimics and Holliday Junction (HJs) intermediates of homologous recombination (HR) <i>in vitro</i>, and inhibits their resolution by HJ-processing enzymes. We predicted that treating cells with HJ-binding compounds would lead to accumulation of DNA damage. As cells repair endogenous or exogenous DNA damage, collapsed replication forks and HJ intermediates will accumulate and serve as targets for the HJ-binding peptides. Inhibiting junction resolution will lead to further accumulation of DNA breaks, eventually resulting in amplification of the damage and causing cell death. Both peptide wrwycr and the related wrwyrggrywrw entered cancer cells and reduced cell survival in a dose- and time-dependent manner. Early markers for DNA damage, γH2AX foci and 53BP1 foci, increased with dose and/or time exposure to the peptides. DNA breaks persisted at least 48 h, and both checkpoint proteins Chk1 and Chk2 were activated. The passage of the cells from S to G2/M was blocked even after 72 h. Apoptosis, however, was not induced in either HeLa or PC3 cells. Based on colony-forming assays, about 35% peptide-induced cytotoxicity was irreversible. Finally, sublethal doses of peptide wrwycr (50–100 µM) in conjunction with sublethal doses of several DNA damaging agents (etoposide, doxorubicin, and HU) reduced cell survival at least additively and sometimes synergistically. Taken together, the results suggest that the peptides merit further investigation as proof-of-principle molecules for a new class of anti-cancer therapeutics, in particular in combination with other DNA damaging therapies.</p></div

Effects of wrwycr on DNA breaks in HeLa cells.

<p>HeLa cells were incubated with the indicated treatments for 24-VAD-fmk (V) and a mock inhibitor z-FA-fmk (m) were used in conjunction with various treatments to determine if apoptosis contributes significantly to the accumulation of DNA breaks. The p-values reported are the results of Wilcoxon exact two-tailed test.</p

Viability assessment of cancer cell lines treated with wrwycr.

<p><b>A:</b> Morphological changes in wrwycr-treated PC3 cells. PC3 cells were treated with 50–200 µM of wrwycr (iii–vi) for 48 h. (i): Untreated (RPMI), (ii): 2% DMSO were kept as negative controls. The cells were viewed under 25× objective using phase-contrast microscopy. The photographs were processed to greyscale using Canvas software. <b>B</b>: MTT assay using different tumor cells (PC3, Du145, LnCAP, PPC-1, DuPro-1,HeLa and A549) treated with (i): wrwycr at doses 50, 75, 100, 150, 200 µM; (ii): wrwyrggrywrw at doses 1, 5, 10, 25, or 50 µM for 48 h, except for Du145 which was treated for 24 h. MTT activity was normalized to 100% metabolic activity of cells treated with DMSO alone. <b>C</b>: Representative flow cytometry dot plots of the live-dead assay with wrwycr-treated PC3 cells using cell permeant calcein AM and cell impermeable propidium iodide dyes. Live cells are capable of converting calcein AM to fluorescent calcein (green) via nonspecific esterases. Dead cells are permeable to propidium iodide, indicating membrane disruption. <b>D–E</b>: wrwycr treatment exerts permanent damage and reduces HeLa cell viability. <b>D</b>: Representative figures of HeLa cells in colony forming assay. HeLa cells were treated for 24 h and allowed to recover and form colonies in the absence of treatment for 5–6 days. Shown here are representative colonies after treatment with (i) DMEM, (ii) 1.5% DMSO and (iii) 150 µM wrwycr. <b>E</b>: Graph summarizing the colony-forming assay results of 2 independent experiments. Reduced numbers of surviving colonies were found with 100 and 150 µM of wrwycr compared to the corresponding vehicle controls, as determined by one-way ANOVA using Bonferroni post-test analysis.</p

Determination of the intracellular concentration of wrwycr.

<p><b>A:</b> PC3 cells were independently treated with 50, 100, 150, 200 µM of wrwycr for 48 h. (i): Intracellular concentration was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078751#s2" target="_blank">Materials and Methods</a>. Final intracellular peptide concentrations (in mM) were calculated taking into account two reported volumes for the cells: the minimum concentration (blue line) denotes wrwyrggrywrw concentration based on a cell volume of 2.4 pl and the maximum concentration (red line) denotes the concentration based on a cell volume of 1.6 pl. (ii): Extracellular concentration was calculated from the peptide left in the media and graph against input concentration of wrwyrggrywrw in X-axis. <b>B</b>: HeLa cells were independently treated with 50, 100, 150, 200 µM of wrwycr for 24 h, cell lysates were fractionated using HPLC, and the area under the curve of the peptide-dependent peaks was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078751#s2" target="_blank">Materials and Methods</a>.</p

Treatment with wrwycr elicits 53BP1 focus formation in the PC3 nucleus.

<p>PC3 cells were treated as specified for 48<b>A</b>: Fluorescence microscopy images of 53BP1 foci and merged images with the nuclear stain DAPI and 53BP1 foci in cells treated with 50–200 µM wrwycr. <b>B</b>: Foci formed in cells treated as specified were counted and the average number of foci per cell was calculated for 35–79 cells from 2 independent experiments. Significant accumulation of 53BP1 was observed with 200 µM wrwycr vs. DMSO, as determined by one-way ANOVA using Bonferroni post test analysis.</p

Co-treatment of PC3 cells with doxorubicin and wrwycr significantly increases the population of pChk2⁺ cells compared to corresponding individual treatments.

<p>PC3 cells were treated with 50, 100 µM wrwycr and 1 µM doxorubicin for 48 h either alone or in combination. Significance was determined with one-way ANOVA using Bonferroni post-test analysis. *** indicates p<0.001, ** indicates p<0.01.</p