A combination of p53-activating APR-246 and phosphatidylserine-targeting antibody potently inhibits tumor development in hormone-dependent mutant p53-expressing breast cancer xenografts

Figures & data

Figure 1 Protocol for treatment of tumor xenografts in nude mice with APR-246 and 2aG4.

Figure 2 APR-246 and PRIMA-1 reduce cell viability of mtp53-expressing breast cancer cells.

Notes: Cells (4–8×10³) were grown in DMEM/F12 medium with 5% FBS and incubated with vehicle (O) or increasing concentrations of APR-246 or PRIMA-1 for 24 or 48 hours. Cell viability was measured using SRB assays. Data are shown as the mean±SEM from six different determinations.
Abbreviations: FBS, fetal bovine serum; mtp53, mutated form of p53; SEM, standard error of the mean; SRB, sulforhodamine B.

Table 1 IC₅₀s (in μM) of APR-246 and PRIMA-1

Cell lines	Time (hours)	APR-246	PRIMA-1
BT-474	24	11.59±0.56	15.55±1.95
	48	5.69±0.31	9.43±0.53
T47-D	24	20.34±0.41	42.96±4.44
	48	15.00±0.65	31.77±3.34
MCF-7	24	–	–
	48	>50 μM	>50 μM

Figure 3 APR-246 increases DNA binding of mtp53 in BT-474 breast cancer cells.

Notes: Cells were grown overnight in DMEM/F12 medium supplemented with 5% FBS. Cells were then washed once with PBS and treated with 50 μM APR-246 for 1 hour, prior to harvest by scraping and subsequent preparation of nuclear extracts. TransAM assays were performed using nuclear extracts (2.5 μg), with each sample being analyzed in triplicate. Activation of DNA-binding ability was compared with the vehicle-treated group (control), whose value was set at 1. Nuclear extract of wtp53-expressing MCF-7 cells (provided with the TransAM kit) was used as a positive control (Positive-C). Data are shown as the mean±SEM from three different determinations. *Significantly different from untreated controls (P<0.05; ANOVA).
Abbreviations: ANOVA, analysis of variance; FBS, fetal bovine serum; mtp53, mutated form of p53; SEM, standard error of the mean.

Figure 4 APR-246 induces apoptosis in BT-474 breast cancer cells.

Notes: (A) Cells were grown for 24 hours in DMEM/F12 medium containing 5% FBS and then treated with 0 (vehicle control), 5, 10, or 20 μM APR-246 (A-5, A-10, A-20) for 24 hours. Cells were stained with Annexin V-FITC (BioVision) and PI, and the percentage of Annexin V+PI-positive cells (quadrant R3) was quantified by FACScan flow cytometry. Representative data are shown; inset values represent the percentage of Annexin V+PI-positive cells in the example shown. Bar graph presents the mean±SEM from three determinations. *Significantly different from control (C) (P<0.05; ANOVA). (B) Cells were treated with 0 (vehicle control; Con), 25, or 50 μM APR-246 for 3 or 12 hours. After cell harvest, whole-cell lysates (45 μg protein) were analyzed by Western blot for Bax and p21 protein expression and caspase-3 cleavage (caspase-3-Cle) from the caspase-3-Pro band. β-actin was used as a loading control.
Abbreviations: ANOVA, analysis of variance; Con, control; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PI, propidium iodide; SEM, standard error of the mean.

Figure 5 APR-246 and 2aG4 exert additive inhibitory effects on BT-474 tumor xenograft growth in nude mice.

Notes: Nude mice (n=8–10/group) were inoculated with an estrogen pellet 48 hours prior to injection of 5×10⁶ BT-474 cells into both flanks. Tumors were measured every 3 days with a digital caliper to determine volume. (A) Left panel, growth curves for tumors, with points representing mean tumor volumes±SEM in each group of mice. *Significantly different from the control group; **significantly different from the control and 2aG4 groups (P<0.05; ANOVA). Top right panel, number of tumors completely eradicated at the end of the experiment. The number of tumors eradicated was converted to a percentage of the tumor total and presented above each bar in the graph. Bottom right panel, animal weight during treatment period. Arrows indicate treatment start and end points. Values represent mean±SEM. (B) Representative images of tumor size from each group at the end of the experiment.
Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean.

Figure 6 APR-246 and 2aG4 exert additive inhibitory effects on T47-D tumor xenograft growth in nude mice.

Notes: Nude mice (n=8–10/group) were inoculated with an estrogen pellet 48 hours prior to injection of 5×10⁶ T47-D cells into both flanks. Tumors were measured every 3 days with a digital caliper to determine volume. (A) Left panel, growth curve for tumors, with points representing mean tumor volumes±SEM in each group of mice. *Significantly different from the control group; **significantly different from the control, APR-246, and 2aG4 groups (P<0.05; ANOVA). Top right panel, number of tumors eradicated at the end of the experiment. The number of tumors eradicated was converted to a percentage of the tumor total and presented above each bar in the graph. Right bottom panel, animal weight during the treatment period. Arrows indicate treatment start and end points. Values represent mean±SEM. (B) Representative images of tumor size from each group at the end of the experiment.
Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean.

Figure 7 APR-246 and 2aG4 combination treatment reduces the expression of Ki67 proliferation markers in breast tumor xenografts in nude mice.

Notes: At the termination of the experiments tumor tissue sections were immunostained for Ki67 and the signal was quantified. Left, representative images of immunohistochemical staining. Right, bar graph represents quantitated Ki67 signal shown as the mean±SEM. *Significantly different from the control group; **significantly different from APR-246 and 2aG4 groups (P<0.05; ANOVA). Inserts represent negative controls.
Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean.

Figure 8 APR-246 and 2aG4 combination treatment induces apoptosis in breast tumor xenografts in nude mice.

Notes: At the termination of the experiments tumor tissue sections were subjected to TUNEL assays and the signal was quantified. Left, representative images of TUNEL staining. Right, bar graph represents quantitated TUNEL signal shown as the mean±SEM. Inserts represent negative controls. *Significantly different from the control group; **significantly different from the control and 2aG4 groups (P<0.05; ANOVA).
Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean; TUNEL, terminal dUTP-mediated nick end labeling.

Figure 9 APR-246 and 2aG4 combination treatment reduces angiogenesis markers in breast tumor xenografts in nude mice.

Notes: At the termination of the experiments tumor tissue sections were immunostained for (A) VEGF and (B) CD31 and the signal was quantified. Left, representative images of immunostaining. Right, bar graph represents quantitated (A) VEGF signal or (B) blood vessel density shown as the mean±SEM. CD31 staining was used to count the blood vessels and determine blood vessel density. Inserts represent negative controls. *Significantly different from the control group; **significantly different from the control, 2aG4, and APR-246 groups (P<0.05; ANOVA).
Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean; VEGF, vascular endothelial growth factor.