Article Figures & Data
Figures
- Figure 1.
CF10 has potent antitumor activity in PDAC cells. A, Representative graphs of 5-day survival assay with quantification of relative cell number via PicoGreen in the indicated PDAC cell lines. B, Colony area percentage quantification from 10-day colony growth inhibition assays in the indicated cell lines. C, Graph of individual MIA-PaCa 2 flank-tumor volumes (n = 8 mice/arm) after 35 days of CF10 treatment (200 mg/kg 3×/week) as compared with vehicle (saline) control with graph of changes in relative weight after initiation of CF10 treatment versus vehicle. D, Representative images of Ki67 and cleaved caspase-3 IHC staining of tumor tissues from CF10- and vehicle-treated mice with quantification of IHC staining. For each in vitro experiment, three independent experiments were conducted, and errors bars represent SEMs. Significance was tested via two-way ANOVA with Bonferroni correction for in vitro assays and Student t test with Bonferroni correction for in vivo assays denoted by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- Figure 2.
CF10 directly inhibits TS/Top1, disrupts replication fork dynamics. and is more DNA directed than 5-FU. A, Representative Western blots detecting the TS CC after treatment in indicated PDAC cell lines with F10, CF10, and 5-FU at indicated concentrations for 24 hours. B, Representative immunofluorescence images with an antibody specific for Top1cc in indicated PDAC cell lines treated at indicated concentrations of F10, CF10, 5-FU for 24 hours and Topotecan for 1 hour with quantification graphs to the right. C, Schematic depicting the dose and timing scheme for DNA fiber experiment, representative images from PANC1 cells used in DNA fiber analysis (yellow arrows indicate terminal replication forks), and graphs for quantification of fork velocity and percent terminal forks in PANC1 cells. D, Quantifications of median bliss synergy scores in the indicated cell lines after cotreatment of F10, CF10, and 5-FU with thymidine and uridine. For each experiment, three independent experiments were conducted, and errors bars represent SEMs. Significance was tested via two-way ANOVA with Bonferroni correction for Top1cc immunofluorescence and Student t test with Bonferroni correction for DNA fiber and thymidine/uridine supplementation assays denoted by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- Figure 3.
CF10 and PDDX-04 synergy produces unresolved DNA damage which triggers apoptosis. A, Representative Bliss synergy plots of indicated PDAC cell lines concurrently treated with serial dilutions of CF10 plus olaparib, CF10 plus PDDX-04 and F10 plus PDDX-04. B, Representative immunofluorescence images of MIA PaCa-2 cells from an alkaline comet assay after treatment with CF10, PDDX-04 or their combination for 16 hours at the indicated concentrations and quantification of DNA tail moments in indicated PDAC cell lines. C, Representative Western blot analysis for PAR, γH2AX, and cleaved caspase-3 with appropriate loading controls in indicated PDAC cell lines treated with CF10, PDDX-04 or their combination for 48 hours with quantifications below. For each experiment, three independent experiments were conducted, and errors bars represent SEM. Significance was tested via two-way ANOVA with Bonferroni correction and denoted by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Tables
- Table 1.
IC50 values with SD for F10, CF10, and 5-FU across all PDAC cell lines examined. P values indicate differences between CF10 IC50s compared with F10 and 5-FU.
IC50 (nmol/L) MIA PaCa-2 P PANC1 P ASPC1 P PDX139 P CF10 7.06 ± 2.89 — 46.01 ± 1.19 — 85.30 ± 4.62 — 46.26 ± 3.01 — F10 25.43 ± 4.00 0.003 345.10 ± 33.00 <0.0001 1,203.00 ± 308.70 0.0033 226.30 ± 22.53 0.0002 5FU 6,277.00 ± 426.00 <0.0001 12,340.00 ± 1,163.00 <0.0001 15,820.00 ± 529.90 <0.0001 5,451.00 ± 286.70 <0.0001 - Table 2.
IC50 values with SD from 5-day survival assay of MIA PaCa-2 cells treated with CF10 or F10 after 48 hours of siRNA knockdown of PARG compared with control scrambled siRNA. P values indicate differences between siCon and siPARG cells.
MIA PaCa-2 IC50 (nmol/L) siCon siPARG P CF10 24.54 ± 7.32 2.41 ± 0.33 0.0064 F10 90.14 ± 8.73 5.61 ± 0.70 <0.0001
Supplementary Data
- Supplementary Data and Methods - S1. (A) Representative graph of 5-day picogreen survival assay with PDX139, a patient derived xenograft line. (B) Representative images of 10-day colony growth inhibition assays with crystal violet staining in indicated PDAC cell lines. (C) Graph of changes in MIA-PaCa 2 flank-tumor volume after initiation of CF10 treatment (200mg/kg 3x/week) as compared to vehicle (saline) control. (D) Graph of individual flank-tumor volumes (n= 4 mice per arm) after 21 days of F10 treatment (200mg/kg 3x/week) as compared to vehicle (saline), Graph of changes in tumor volume after initiation of F10 treatment as compared to vehicle control, and graph of changes in relative weight after initiation of F10 treatment vs vehicle. (E) Representative images of Ki67 and cleaved caspase-3 IHC staining of tumor tissues from F10 experiment with quantifications of IHC staining. For each in-vitro experiment, three independent experiments were conducted, and errors bars represent standard error of the means. S2. Representative immunofluorescence images with an antibody specific for Top1 cleavage complexes (Top1cc) in MIA-PaCa 2 cells with either (A) no treatment or treated with (B) 10nM F10 for 24 hours (C) 10nM CF10 for 24 hours (D) 10nM 5FU for 24 hours (E) 1uM topotecan for 1 hour. S3. Representative immunofluorescence images with an antibody specific for Top1 cleavage complexes (Top1cc) in PANC1 cells with either (A) no treatment or treated with (B) 10nM F10 for 24 hours (C) 10nM CF10 for 24 hours (D) 10nM 5FU for 24 hours (E) 1uM topotecan for 1 hour. S4. (A) Quantification of Top1cc images of MIA-PaCa 2 cells from supplementary figure 2 (B) Quantification of Top1cc images of PANC1 cells from supplementary figure 3 For each experiment, three independent experiments were conducted, and errors bars represent standard error of the means. Significance was tested via two-way ANOVA with Bonferroni correction denoted by *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. S5. (A) Representative Bliss synergy plots from MIA-PaCa 2 cells concurrently treated with serial dilutions of thymidine and F10, CF10, and 5-FU. (B) Representative Bliss synergy plots from ASPC1 cells concurrently treated with serial dilutions of thymidine and F10, CF10, and 5-FU. (C) Representative bliss synergy plots of indicated MIA-PaCA 2 cells concurrently treated with serial dilutions of uridine and F10, CF10, and 5-FU. (D) Representative Bliss synergy plots from ASPC1 cells concurrently treated with serial dilutions of uridine and F10, CF10, and 5-FU. S6. (A) Representative cell viability plots used for bliss synergy analysis from MIA-PaCa 2 cells concurrently treated with serial dilutions of thymidine and F10, CF10, and 5FU. (B) Representative cell viability plots used for bliss synergy analysis from ASPC1 cells concurrently treated with serial dilutions of thymidine and F10, CF10, and 5FU. (C) Representative cell viability plots used for Bliss synergy analysis from MIA-PaCa 2 cells concurrently treated with serial dilutions of uridine and F10, CF10, and 5FU. (D) Representative cell viability plots for Bliss synergy analysis from ASPC1 cells concurrently treated with serial dilutions of uridine and F10, CF10, and 5FU. S7. (A) Representative cell viability plots used for Bliss synergy analysis from MIA-PaCa 2 cells concurrently treated with serial dilutions of CF10 plus Olaparib, CF10 plus PDDX-04 and F10 plus PDDX-04. (B) Representative cell viability plots used for Bliss synergy analysis from PANC1 cells concurrently treated with serial dilutions of CF10 plus Olaparib, CF10 plus PDDX-04 and F10 plus PDDX-04. (C) Representative Bliss synergy plots from MIA-PaCa 2 cells concurrently treated with serial dilutions of AraC plus PDDX and F10 plus AraC. (D) Representative Bliss synergy plots from PANC1 cells concurrently treated with serial dilutions of AraC plus PDDX and F10 plus AraC. (E) Representative cell viability plots from MIA-PaCa 2 cells concurrently treated with serial dilutions of AraC plus PDDX and F10 plus AraC. (F) Representative cell viability plots from PANC1 cells concurrently treated with serial dilutions of AraC plus PDDX and F10 plus AraC. S8. (A) 5-day survival assay of MIA-PaCa 2 cells treated with CF10 or F10 after siRNA knockdown of PARG compared to control scrambled siRNA with western blot validation of knockdown. (B) Representative western blot for PARG, PAR, yH2AX, and cleaved capase-3 with appropriate loading controls in MIA-PaCa 2 cells treated with CF10 after 48 hours of either siControl or siPARG knockdown with quantification of three independent experiments. Supplementary Table S1: Commercially available chemical compounds Supplementary Table S2: Western blot antibodies
Volume 19, Issue 4
