Abiraterone In Vitro Is Superior to Enzalutamide in Response to Ionizing Radiation

Abstract

Abiraterone acetate and Enzalutamide are novel anti-androgens that are key treatments to improve both progression-free survival and overall survival in patients with metastatic castration-resistant prostate cancer. In this study, we aimed to determine whether combinations of AR inhibitors with radiation are additive or synergistic, and investigated the underlying mechanisms governing this. This study also aimed to compare and investigate a biological rationale for the selection of Abiraterone versus Enzalutamide in combination with radiotherapy as currently selection is based on consideration of side effect profiles and clinical experience. We report that AR suppression with Enzalutamide produces a synergistic effect only in AR-sensitive prostate models. In contrast, Abiraterone displays synergistic effects in combination with radiation regardless of AR status, alluding to potential alternative mechanisms of action. The underlying mechanisms governing this AR-based synergy are based on the reduction of key AR linked DNA repair pathways such as NHEJ and HR, with changes in HR potentially the result of changes in cell cycle distribution, with these reductions ultimately resulting in increased cell death. These changes were also shown to be conserved in combination with radiation, with AR suppression 24 hours before radiation leading to the most significant differences. Comparison between Abiraterone and Enzalutamide highlighted Abiraterone from a mechanistic standpoint as being superior to Abiraterone for all endpoints measured. Therefore, this provides a potential rationale for the selection of Abiraterone over Enzalutamide.

Keywords: DNA damage; abiraterone; androgen receptor; enzalutamide; prostate cancer; radiotherapy.

Figure 1

Effect of Abiraterone and Enzalutamide treatment on cell viability of androgen-sensitive (LNCaP), androgen-insensitive (PC3) prostate cancer models and osteoblastic bone model (SJSA-1). LNCaP, PC3, and SJSA-1 cells were treated with a dose range of 10 nM to 100 μM of Abiraterone, Enzalutamide or DMSO. Cell viability was evaluated 72 hours post-treatment by MTT assay. Each value is the mean of three independent experiments performed in triplicate and error bars represent SEM.

Figure 2

Comparison of Abiraterone and Enzalutamide treatment on cell viability of androgen-sensitive (LNCaP), androgen-resistant (PC3) prostate cancer models and osteoblastic bone model (SJSA-1). LNCaP, PC3, SJSA-1 cells were treated with a dose range of 10 nM to 100 μM of Abiraterone and Enzalutamide or DMSO. Cell viability was evaluated 72 hours post-treatment by MTT assay. Each value is the mean of three independent experiments performed in triplicate (errors represent SEM).

Figure 3

Comparison of the combined effect of Abiraterone and Enzalutamide and DMSO as single agents or combined with 2 Gy radiation on survival fraction in androgen-sensitive LNCaPs and androgen insensitive PC3s prostate cancer models and osteoblastic bone model SJSA-1. PC3s and SJSA-1s were treated with 10 μM Abiraterone, Enzalutamide or DMSO 24 hours before radiation, while LNCaPs were treated with 200 nM due to their sensitivity. Cells were then left an appropriate amount of time to form sufficient colonies and any colonies of 50 cells or more counted. Each value is the mean of three independent experiments performed in triplicate (+/- SEM) and normalized to control. Unpaired students t-test was used for comparisons between two groups. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns= non significant.

Figure 4

Radiosensitization effects of Abiraterone, Enzalutamide and DMSO on radiation in androgen-insensitive PC3 prostate cancer model, androgen-sensitive LNCaP model and osteoblastic bone model SJSA-1 by colony formation assay. LNCaPs were treated with 200 nM, while PC3s and SJSA-1s were treated with 10 μM Abiraterone, Enzalutamide or DMSO 24 hours before X-Ray across a dose range of 0-8 Gy. Cells were then left to form appropriately sized colonies and survival fraction calculated using SF = (colonies counted) / (cells seeded x (PE/100) colonies counted). Error bars are standard error of the mean (+/- SEM) and for some points, the error bars are shorter than the height of the symbol (n=3).

Figure 5

Immunofluorescence of 53BP1 foci treated with Abiraterone and Enzalutamide on AR-insensitive PC3s and AR-sensitive LNCaP prostate models and osteoblastic bone model SJSA-1. All models were treated with 10 μM Abiraterone, Enzalutamide or DMSO and harvested 1-, 24 and 48-hours post-treatment before being fixed and stained with 53BP1 (n=3). Unpaired students t-test was used for comparisons between two groups *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and error bars represent SEM.

Figure 6

Immunofluorescence of 53BP1 foci treated with Abiraterone and Enzalutamide in combination with 2 Gy X-ray in AR-insensitive PC3s and AR-sensitive LNCaP prostate models and osteoblastic bone model SJSA-1. All models were treated with 10 μM Abiraterone, Enzalutamide or DMSO 24 hours before being administered 2 Gy radiation. Samples were then harvested 1, 24- and 48-hours post-radiation before being fixed and stained with 53BP1 (n=3). Unpaired students t-test was used for comparisons between two groups *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 ****p ≤ 0.0001, and error bars represent SEM.

Figure 7

Impact of Abiraterone and Enzalutamide on RAD51 and PSA protein expression in AR-sensitive LNCaP prostate model, AR-insensitive PC3 prostate model and osteoblastic bone model SJSA-1. All models were treated with 10 μM Abiraterone, Enzalutamide or DMSO. Samples were then harvested 1, 24- and 48-hours post-treatment and expression levels measured via Western blot. β-Actin was used as a loading control. (n=3).

Figure 8

Impact of Abiraterone and Enzalutamide in combination with 2 Gy X-ray on RAD51 and PSA expression in AR-insensitive PC3s and AR-sensitive LNCaP prostate models and osteoblastic bone model SJSA-1. All models were treated with 10 μM Abiraterone, Enzalutamide or DMSO 1 or 24 hours before radiation. Samples were then harvested 1, 24 and 48 hours post-radiation and expression levels measured via Western blot. β-Actin was used as a loading control. (n=3).

Figure 9

Impact of Abiraterone and Enzalutamide on DNA-PK and PSA protein expression in AR-sensitive LNCaP and AR-insensitive PC3 prostate models and osteoblastic bone model SJSA-1. All models were treated with 10 μM Abiraterone, Enzalutamide or DMSO. Samples were then harvested 1, 24 and 48 hours post-treatment and expression levels measured via Western blot (A) and densitometric analysis (B). β -Actin was used as a loading control. (n=3). Unpaired students t-test was used for comparisons between two groups *p < 0.05 and error bars represent SEM.

Figure 10

Impact of Abiraterone and Enzalutamide in combination with 2 Gy X-ray on DNA-PK and PSA expression in AR-insensitive PC3s, AR-sensitive LNCaP prostate models and osteoblastic bone model SJSA-1. All models were treated with 10 μM Abiraterone, Enzalutamide or DMSO for 1 or 24 hours before being administered 2 Gy radiation. Samples were then harvested 1, 24 and 48 hours post-radiation and expression levels measured via Western blot (A) and densitometric analysis (B). β -Actin was used as a loading control. (n=3). Unpaired students t-test was used for comparisons between two groups *p < 0.05 and error bars represent SEM.

Figure 11

Cell-cycle analysis of AR-sensitive LNCaP prostate model, AR- insensitive PC3 prostate model and osteoblastic bone model SJSA-1. Cells were treated with 10 μM Abiraterone, Enzalutamide or DMSO, fixed in ice cold ethanol 1h, 24h and 48h post-treatment and stained with PI/RNaseA for 30 minutes before the cell-cycle profile was determined by flow cytometry. Error bars are standard error of the mean (SEM) (n=3).

Figure 12

Impact of Abiraterone and Enzalutamide on PARP cleavage in AR-insensitive PC3s, AR-sensitive LNCaP prostate models and osteoblastic bone model SJSA-1. Models were treated with 10 μM Abiraterone, Enzalutamide or DMSO. Samples were then harvested 1, 24 and 48 hours post-treatment and expression levels measured via Western blot. β-Actin was used as a loading control. (n=3).

Figure 13

Cell-cycle analysis of AR-sensitive LNCaP prostate model, AR- insensitive PC3 prostate model and osteoblastic bone model SJSA-1. Cells were treated with 10 μM Abiraterone, Enzalutamide or DMSO 1 or 24 hours before radiation with 2 Gy. Post radiation cells were fixed in ice cold ethanol 1h, 24h and stained with PI/RNaseA for 30 minutes before the cell-cycle profile was determined by flow cytometry. Error bars are standard error of the mean (SEM) (n=3).

Figure 14

Impact of Abiraterone and Enzalutamide and 2Gy radiation on PARP cleavage in AR-insensitive PC3s and AR-sensitive LNCaP prostate models and osteoblastic bone model SJSA-1. Models were treated with 10 μM Abiraterone, Enzalutamide or DMSO 1 or 24 hours before radiation with 2Gy. Samples were then harvested 1,24 and 48 hours post-radiation and expression levels measured via Western blot. β -Actin was used as a loading control. (n=3).