Withanolide D Enhances Radiosensitivity of Human Cancer Cells by Inhibiting DNA Damage Non-homologous End Joining Repair Pathway

Abstract

Along with surgery and chemotherapy, radiation therapy (RT) is an important modality in cancer treatment, and the development of radiosensitizers is a current key challenge in radiobiology to maximize RT efficiency. In this study, the radiosensitizing effect of a natural compound from the withanolide family, withanolide D (WD), was assessed. Clonogenic assays showed that a 1 h WD pretreatment (0.7 μM) before irradiation decreased the surviving fraction of several cancer cell lines. To determine the mechanisms by which WD achieved its radiosensitizing effect, we then assessed whether WD could promote radiation-induced DNA damages and inhibit double-strand breaks (DSBs) repair in SKOV3 cells. Comet and γH2AX/53BP1 foci formation assays confirmed that DSBs were higher between 1 and 24 h after 2 Gy-irradiation in WD-treated cells compared to vehicle-treated cells, suggesting that WD induced the persistence of radiation-induced DNA damages. Immunoblotting was then performed to investigate protein expression involved in DNA repair pathways. Interestingly, DNA-PKc, ATM, and their phosphorylated forms appeared to be inhibited 24 h post-irradiation in WD-treated samples. XRCC4 expression was also down-regulated while RAD51 expression did not change compared to vehicle-treated cells suggesting that only non-homologous end joining (NHEJ) pathways was inhibited by WD. Mitotic catastrophe (MC) was then investigated in SKOV3, a p53-deficient cell line, to assess the consequence of such inhibition. MC was induced after irradiation and was predominant in WD-treated samples as shown by the few numbers of cells pursuing into anaphase and the increased amount of bipolar metaphasic cells. Together, these data demonstrated that WD could be a promising radiosensitizer candidate for RT by inhibiting NHEJ pathway and promoting MC. Additional studies are required to better understand its efficiency and mechanism of action in more relevant clinical models.

Keywords: DNA damage repair; cancer; mitotic catastrophe; radiation; radiosensitizer; withanolide D.

Figure 1

Structures of (A) withaferin A and (B) withanolide D.

Figure 2

WD sensitized several cancer cell lines to X-Ray irradiation. Cells in log phase were pretreated with 0.7 μM of WFA, WD or vehicle (DMSO) for 1 h and then irradiated at indicated doses. Data points represent colonies >50 cells as the mean ± SEM from three independent experiments with at least two replicates each. Significantly different values as determined by Wilcoxon signed-rank test (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

WD prolonged radiation-induced DNA damages in SKOV3 cells. (A) Representative images of alkaline comet assay of SKOV3 cells treated with vehicle (DMSO) or WD (0.7 μM) for 1 h and then irradiated at 2 Gy followed by 1 and 24 h incubation. Scale bar, 25 μm. (B) Average olive tail moment of at least 50 cells from two independent experiments. Data points represent the mean ± SEM. *Significantly different values as determined by Wilcoxon–Mann–Whitney test (p < 0.05). (C) Immunofluorescence showing γH2AX (red) and 53BP1 (green) foci formation in SKOV3 cells pre-treated with vehicle (DMSO) or WD (0.7 μM) for 1 h and then irradiated at 2 Gy followed by 1, 6, and 24 h incubation. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. (D) Ratio of radiation-induced γH2AX and (E) 53BP1 foci normalized to sham-irradiated cells, exposed to DMSO or WD. Data points represent the mean ± SEM of at least 60 nuclei from three independent experiments. Significantly different values as determined by Wilcoxon–Mann–Whitney test (*p < 0.05).

Figure 4

WD inhibited DNA repair proteins in irradiated SKOV3 cells. (A) Western blot is showing the expression level of ATM, ^S1981pATM, DNA-PKcs, ^S2056pDNA-PKcs, XRCC4, and RAD51 proteins in SKOV3 cells exposed to vehicle (DMSO) or WD (0.7 μM) for 1 h prior to sham- or 2 Gy-irradiation and incubated for 1, 6, or 24 h. NI, Non-Irradiated samples. (B) Bands intensities were quantified using ImageJ and normalized with GAPDH. Data points represent the mean ± SEM from three independent experiments. Significantly different values as determined by Wilcoxon–Mann–Whitney test (*p < 0.05).

Figure 5

WD induced MC in irradiated SKOV3 cells. (A) Representative images of immunostaining of mitotic spindle with anti α-tubulin (red) and pericentrin (green) in SKOV3 cells exposed to vehicle (DMSO) or WD (0.7 μM) for 1 h prior to sham- or 2 Gy-irradiation and incubated for 24 h. Nuclei were counterstained with DAPI (blue). Scale bar, 15 μm. Quantification of mitotic catastrophe as (B) the percentage of multipolar metaphases (in black on each bar chart, the number of metaphases scored is reported in white at the bottom of each bar chart) and (C) the percentage of cells in anaphase among cells in mitosis (from at least 200 mitotic events from three independent experiments). Significantly different values as determined by Welch's t-test (*p < 0.05).