APR-246 Enhances Colorectal Cancer Sensitivity to Radiotherapy

Abstract

p53 mutation is common and highly related to radiotherapy resistance in rectal cancer. APR-246, as a small molecule, can restore the tumor-suppressor function to mutant p53. As there is currently no existing study on combining APR-246 with radiation in rectal cancer, our objective was to investigate whether APR-246 could enhance the sensitivity of colorectal cancer cells, regardless of their p53 status, to radiation treatment. The combination treatment had synergistic effects on HCT116p53-R248W/- (p53Mut) cells, followed by HCT116p53+/+ [wild-type p53 (p53WT)] cells, and exhibited an additive effect on HCT116p53-/- (p53Null) cells through inhibiting proliferation, enhancing reactive oxygen species, and apoptosis. The results were confirmed in zebrafish xenografts. Mechanistically, p53Mut and p53WT cells shared more activated pathways and differentially expressed genes following the combination treatment, compared with p53Null cells, although the combination treatment regulated individual pathways in the different cell lines. APR-246 mediated radiosensitization effects through p53-dependent and -independent ways. The results may provide evidence for a clinical trial of the combination in patients with rectal cancer.

Figure 1.

Radiosensitization effects of APR-246 in p53WT, p53Mut, and p53Null HCT116 cells. A–D, p53WT, p53Mut, and p53Null cells were seeded in 96-well plates, after attachment pretreated with or without APR-246 (0, 2.5, 5, 7.5, or 10 μmol/L, respectively) for 12 hours and then exposed to a single dose of radiation (0, 2, 4, or 6 Gy, respectively). At 72 hours after radiation, inhibition of radiation and/or APR-246 were calculated for an IC₅₀ value of APR-246 (A) without radiation and IC₅₀ of radiation (B) under different concentrations of APR-246 in p53WT, p53Mut, and p53Null cells. C, The combinational inhibitory activity of APR-246 and radiation was analyzed using a ZIP model. This model proposes a delta score (δ) to characterize the synergy landscape over the full dose-response matrix. The interaction between two factors was defined as synergistic when the synergy score was larger than 10; as additive, from −10 to 10; and as antagonistic, less than −10. 3D synergy landscape of APR-246 and radiation in p53WT, p53Mut, and p53Null cells. The red area refers to synergy and the green area refers to antagonism. D, Synergy score (δ) of APR-246 and radiation in each cell line shown in histogram. Data represent the mean ± SD (n = 3) obtained from three independent experiments. E and F, Radiosensitization effects of APR-246 determined by colony-formation assay. Cells were seeded in 6-well plates, after attachment pretreated with APR-246 (2.5 μmol/L) for 12 hours and then irradiated at 2 Gy. The representative photos of p53WT, p53Mut, and p53Null cells after treatments and staining with crystal violet (E). The ratio of signal intensity of the plates treated with APR-246 and/or radiation versus control is displayed and compared in histogram and table (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001. n,s., not statistically significant. ^#, P < 0.05; versus p53Mut cells. All experiments were performed three times.

Figure 2.

APR-246 enhanced radiation-induced S-phase arrest, ROS accumulation, apoptosis, and p53 expression in p53WT, p53Mut, and p53Null HCT116 cells. Cells were seeded in 10-cm dishes and treated with control, APR-246 (20 μmol/L), radiation (6 Gy), or a combination. A, Percentages of cells at S-phase in each cell line after treatments based on PI staining. B, Ratio of increased S-phase arrest caused by combination treatment to radiation alone in the three cell lines. C, ROS-positive cells after treatments shown and compared in the histogram. D, Ratio of ROS-positive cells caused by combination treatment compared with radiation alone in p53WT, p53Mut, and p53Null cells. E, Apoptotic cells of p53WT, p53Mut, and p53Null cells under different conditions based on Annexin V-FITC and propidium iodide (PI) staining. F, Ratio of increased apoptosis caused by combination treatment compared with radiation alone in each cell line. G and H, P53 expression levels were examined by Western blotting. p53 protein in p53WT (G) and p53Mut (H) cells were compared in the histogram. GAPDH was used as an interval control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not statistically significant. Statistical analysis was carried out with one-way ANOVA and Dunnett multiple comparisons test. Mean ± SD. n = 3.

Figure 3.

Radiosensitization effects of APR-246 in three primary colorectal cancer cell cultures. A, Morphology of primary cell cultures 1597D, 3117D, and 3431D cells. Bars = 200 μm. B, The tumorigenesis of primary cells in nude mice. The xenografts of primary cell cultures were pointed with red arrows. C and D, Calculation of synergistic effect between APR-246 and radiation in primary cell cultures. All primary cells were seeded in 96-well plates. In 1597D primary cells, APR-246 was at 0, 10, 25, 30, 35, or 40 μmol/L, respectively, and radiation was at 0, 2, 4, 6, or 8 Gy, respectively. In 3117D and 3431D primary cells, the concentration of APR-246 was 0, 2.5, 5, 15, or 20 μmol/L, while the dose of radiation was 0, 1, 2, 4, 6, or 8 Gy, respectively. The red areas refer to synergy and the green areas refer to antagonism in 3D synergy landscape of APR-246 and radiation (C). Synergy score (δ) of APR-246 and radiation shown in histogram (D). Data represent the mean ± SD (n = 3) obtained from three independent experiments. E and F, Flow cytometry assay for the S-phase distribution (E) and cell apoptosis (F) of 3 primary cell cultures after treatment with radiation alone or combination with APR-246. The dose of APR-246 and radiation was 25 μmol/L and 2 Gy separately in 1597D primary cells, while 20 μmol/L and 2 Gy separately both in 3117D and 3431D primary cells. *, P < 0.05; **, P < 0.01.

Figure 4.

Radiosensitization effects of APR-246 in p53WT, p53Mut, and p53Null HCT116 cells in vivo. A, A workflow of zebrafish xenograft experiment. Cells were labeled on 0 dpi (2 dpf), APR-246 (20 μmol/L) was added on 0.5 dpi, and radiation (6 Gy) was done on 1 dpi (3 dpf); the experiment ended on 3 dpi (5 dpf). B–D, Lateral views of live zebrafish embryos after transplantation of fluorescently labeled p53WT (B), p53Mut (C), and p53Null (D) cells. Dots indicated individual tumors. E, The ratio of inhibition (i.e., the inhibition found after combination treatment divided by the inhibition caused by radiation alone) in each cell line. Scale bar: 400 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student t test).

Figure 5.

The Venn diagrams of significantly enriched pathways and genes between combination and radiation-alone treatment in three HCT116 cell lines. Cells were seeded in 10-cm dishes and treated with 6-Gy radiation alone or in combination (20 μmol/L APR-246 + 6 Gy radiation). After cell harvest, RNA sequencing was performed as described in the Materials and Methods section. The potential biological pathways and their enriched genes involved in the combinational effect of APR-246 with radiation in each cell line were obtained by performing GSEA with the clusterProfile package version 3.18.1 within the R software based on the Reactome database. Gene sets with |NES| > 1, nominal (NOM) P value < 0.05, and FDR q-value < 0.25 were considered significantly enriched. A, The Venn diagrams of deregulated pathways were drawn for the three cell lines. B–E, The Venn diagrams of differentially expressed genes in cell cycle (B), mRNA splicing (C), apoptosis (D), and transcriptional regulation by TP53 (E) pathways between combination and radiation-alone treatment were further drawn for the three cell lines.

Figure 6.

Negative p53 expression correlated with better response to radiotherapy (RT) in tumors of patients with rectal cancer with RT. A and B, The relationship of p53 status with apoptosis (A) and Ki67 expression (B) in resected tumor samples from patients with rectal cancer with RT. C and D, The relationship of p53 status with apoptosis (C) and Ki67 expression (D) in resected tumor samples from patients without RT (NRT).