Differential responses to 223Ra and Alpha-particles exposure in prostate cancer driven by mitotic catastrophe

Abstract

Introduction: Radium-223 (²²³Ra) has been shown to have an overall survival benefit in metastatic castration-resistant prostate cancer (mCRPC) involving bone. Despite its increased clinical usage, relatively little is known regarding the mechanism of action of ²²³Ra at the cellular level.

Methods: We evaluated the effects of ²²³Ra irradiation in a panel of cell lines and then compared them with standard X-ray and external alpha-particle irradiation, with a particular focus on cell survival and DNA damage repair kinetics.

Results: ²²³Ra exposures had very high, cell-type-dependent RBE_50% ranging from 7 to 15. This was significantly greater than external alpha irradiations (RBE_50% from 1.4 to 2.1). These differences were shown to be partially related to the volume of ²²³Ra solution added, independent of the alpha-particle dose rate, suggesting a radiation-independent mechanism of effect. Both external alpha particles and ²²³Ra exposure were associated with delayed DNA repair, with similar kinetics. Additionally, the greater treatment efficacy of ²²³Ra was associated with increased levels of residual DNA damage and cell death by mitotic catastrophe.

Conclusions: These results suggest that ²²³Ra exposure may be associated with greater biological effects than would be expected by direct comparison with a similar dose of external alpha particles, highlighting important challenges for future therapeutic optimization.

Keywords: alpha particles; bone metastases; mitotic catastrophe; radiation effects; radium-223.

Figure 1

Survival curves of (A) PC-3, (B) U2OS, (C) RWPE, (D) SJSA-1, and (E) Saos-2 cells obtained after exposure at different doses of X-rays (blue) and external alpha source (red) or exposure to ²²³Ra for 24 h (green). (F) Comparison of X-ray survival curves for different cell lines. Data were fit to the linear quadratic model. Points represent the mean of at least three independent experiments with respective standard error.

Figure 2

(A) Different ionization patterns visualized by immunofluorescence staining for 53BP1 in the nucleus of U2OS cells 1 h after exposure to 1 Gy of X-rays (left) and ²²³Ra (right). The average number of 53BP1 foci per cell induced 1 h after exposure to X-rays or external alpha particles or 24-h exposure to ²²³Ra for (B) PC-3, (C) U2OS, and (D) RWPE. Points represent the mean of three independent experiments and the respective standard error; all values are background-corrected for the average of foci in control samples.

Figure 3

LET-dependent repair kinetics of radiation-induced foci. The repair of alpha-particle-induced foci is slower than those induced by X-rays (green) regardless of whether high LET was delivered by an external alpha source (blue) or 24-h exposure to ²²³Ra (red), for (A) PC-3, (B) U2OS, and (C) RWPE. (D) Percentage of residual damage at 24 h after irradiation normalized to the initial yield of damage. Points represent the mean number of foci per cell of three independent experiments and the respective standard error. Data were corrected for the baseline mean foci value and fit to an exponential decay equation. Analysis was performed using t-student method; A significant change when compared to the control group is represented by * (p < 0.05; ** (p < 0.01); *** (p < 0.001); **** (p < 0.0001).

Figure 4

Clonogenic survival data on PC-3 cells exposed to ²²³Ra for (A) 6 h and (B) 24 h using vials with different activities per milliliter at the time of the experiment [denoted in legend in (A, B)]. While all cells were treated with the same activity for a given dose and time, different volumes of ²²³Ra stock solution were needed to achieve a given dose, showing clear differences in survival. Here, the SFs are displayed as logarithm values. This can also be seen as a function of added volume when cells were exposed to alpha-particle doses of 0.05, 0.1, 0.25, and 0.5 Gy from ²²³Ra exposures for (C) 6 h and (D) 24 h.

Figure 5

²²³Ra induces mitotic catastrophe in a cell- and dose-dependent manner. Percentage of cells that have (A) a giant nucleus 24 h after irradiation or (B) an aberrant nucleus 96 h after irradiation as a function of absorbed dose. Data were obtained by microscopic evaluation of cell morphology after DAPI staining of three independent experiments with the respective standard error.

Figure 6

Typical nuclear morphologies observed after 1 Gy of ²²³Ra irradiation in 24 h and fixed at 96 h after exposure; cell nuclei were labeled with DAPI. (A) U2OS control cells. (B) Morphology of a giant nuclei cell after irradiation. (C) Aberrant nucleus representative of cells that undergo mitotic catastrophe after irradiation. (D–I) Mitotic catastrophe in response to ²²³Ra irradiation is cell-specific. In all cell lines [PC-3 (D, E), U2OS (F, G), and RWPE (H, I)], there is a peak of giant nuclei at 24 h after ²²³Ra exposure and a peak for aberrant nuclei at 96 h after 2 Gy of irradiation. Bars represent the mean of three independent experiments and the respective standard error. Analysis was performed using t-student method; A significant change when compared to the control group is represented by * (p < 0.05; ** (p < 0.01); *** (p < 0.001); **** (p < 0.0001).

Figure 7

Cell cycle analysis of PC-3, U2OS, and RWPE 1 h (A, C, E) or 24 h (B, D, F) after treatment with 8 Gy of X-ray, 2 Gy of external alpha particles, or 2 Gy of ²²³Ra delivered in 24 h. Bars represent the mean of two independent experiments and the respective standard error. A significant change when compared to the control group is represented by * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

Figure 8

Different exposure conditions induce PARP cleavage in PC-3 24 h after irradiation. (A) Western blot demonstrating the protein expression of full and cleaved PARP-1 and β actin at 24 h following irradiation with 8 Gy of X-ray, 2 Gy of ²²³Ra, or2 Gy of external alpha beam. (B) The ratio between cleaved PARP-1 and β actin (loading control). Bars represent the mean of two independent experiments and the respective standard error.

Figure 9

(A) The simulated cell as a semi-ellipsoid with 20-μm-side diameters and 7.8-μm thickness. The nucleus is simulated as a full ellipsoid with 6-μm-side diameters and 3-μm thickness, centered at the middle of the cell. (B) The simulated cylindrical volume of a treatment solution with alpha-particle emissions that resulted from ²²³Ra decays. A simulated geometry of a cell with its nucleus is placed at the bottom of the simulated well. The simulated well geometry has a 200-μm base diameter, having a 100-μm height.