Targeting the DNA double strand break repair machinery in prostate cancer

Abstract

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways.

Figure 1. Ku55933 and NU7441 inhibit ATM and DNA-PK, respectively.

Whole LNCaP cell extracts were prepared 1 h after 10 Gy and 6 h after 1 µM doxorubicin ±10 µM Ku55933 or 1 µM NU7441. Extracts were analyzed by western blotting and probed with the indicated antibodies. Data is a representative of three independent experiments.

Figure 2. Ku55933 and/or NU7441 sensitised CaP cells to IR or doxorubicin.

LNCaP (A&B) or PC3 (C&D) cells were seeded in different densities and left to attach before 1 h incubation with varying concentration of Ku55933 or NU7441 prior to irradiation 1 Gy or 10 nM doxorubicin treatment for 24 h (P<0.001). LNCaP (E&F) or PC3 (G&H) cells were seeded in different densities and left to attach before 1 h incubation with Ku55933 (10 µM) or NU7441 (1 µM) prior to treatment with varying IR doses or doxorubicin concentration for 24 h (P<0.001). All results are the mean of three independent experiments ± SEM.

Figure 3. Ku55933 and NU7441 altered CaP cell lines cell cycle and increased apoptosis.

LNCaP (A&C) and PC3 (B&D) cells were incubated with Ku55933 (10 µM) and/or NU7441 (1 µM) before being exposed to the indicated doses of IR or doxorubicin for 48 h and then stained with PI and analysed directly on FACscan. Data is a representative three independent experiments. LNCaP (E) and PC3 (F) cells were treated as described above and stained with FITC-conjugated anti caspase 3 antibody before direct analysis on FACScan. Results are the mean of three independent experiments ± SD.

Figure 4. Ku55933 and NU7441 increased DNA DSB in CaP cell lines.

LNCaP (A&C) and PC3 (B&D) cells were treated with 10 Gy or 1 µM doxorubicin following 1 h incubation with Ku55933 (10 µM) and/or NU7441 (1 µM) and stained with FITC-conjugated H2AX antibody before being analysed directly on FACscan. Results are the mean of at least 5 independent experiments±SEM. PC3 (E) cells were treated as above for 24 h and analysed using comet assay, 50 cells were counted per arm treatment. Results are the mean of 3 independent experiments ± SEM.