Inhibition of radiation-induced DNA repair and prosurvival pathways contributes to vorinostat-mediated radiosensitization of pancreatic cancer cells

Abstract

Objective: The intrinsic radioresistance of pancreatic cancer (PaCa) is caused by multiple oncogenic signaling pathways. In contrast to combining radiation therapy (RT) with targeted therapeutic agent(s) whose blockade can be circumvented by redundant signaling pathways, we evaluated the combination of RT with a broad-spectrum histone deacetylase inhibitor, vorinostat.

Methods: Radiosensitization by vorinostat was analyzed using clonogenic survival assays. Apoptosis was evaluated using flow cytometry and immunoblotting. DNA repair was evaluated using immunofluorescence assessment of histone 2AX phosphorylation and immunoblotting for DNA repair proteins. Prosurvival pathway proteins were measured by immunoblotting and electrophoretic mobility shift assays.

Results: Vorinostat significantly sensitized PaCa cells to radiation, but vorinostat-induced apoptosis did not contribute significantly to the observed radiosensitization. However, vorinostat inhibited DNA damage repair by targeting key DNA repair proteins and also abrogated prosurvival pathways responsible for PaCa aggressiveness and radioresistance. Specifically, the constitutively overexpressed epidermal growth factor receptor and nuclear factor κB pathways were shown to be induced by radiation and inhibited by vorinostat.

Conclusions: Vorinostat augments the antitumor effects of RT by abrogating radioresistance responses of PaCa cells mediated by prosurvival and DNA repair pathways and promises to be a clinically relevant adjunct to RT for treatment of PaCa.

Figure 1. Vorinostat decreases the viability of pancreatic cancer cells

Colo357FG, AsPC-1 or MiaPaCa-2 cells (3 × 10⁴/mL) were exposed to different concentrations of vorinostat in a 96-well plate for 48 h and the viability was assessed by XTT assay (Roche). The percent viability was calculated with respect to DMSO-treated controls. Data points = mean ± SE of quadruplicates for each concentration.

Figure 2. Vorinostat pre-treatment sensitizes pancreatic cancer cells to ionizing radiation

AsPC-1 (a), MiaPaCa-2 (b) or Colo357FG (c) cells were exposed to 2 μM vorinostat for 48 h, following which they were irradiated to indicated doses and re-plated for colony formation. The enhancement in radiosensitivity by vorinostat was assessed on the basis of clonogenic cell survival assays in comparison with the controls (cells irradiated in the absence of vorinostat). Points, mean of the sextuplicates, bars, SE.

Figure 3. Apoptosis induction by vorinostat does not play a significant role in radiosensitization

(a) Assessment of apoptotic cell death by immunoblot analysis of caspase-3, caspase-9 and PARP cleavage. MiaPaCa-2 cells were treated with 2 μM vorinostat for 48 h, following which they were irradiated (6 Gy) and incubated for an additional 6 h. At the end of this treatment, cells were collected for immunoblot analysis. Quantification of the cleaved fragments for each protein was done by ImageQuant software. Fold cleavage with respect to the untreated control is indicated below each figure. (b) Assessment of vorinostat-induced apoptosis by TUNEL assay. Cells were treated with vorinostat for 48 hrs, irradiated (6 Gy) and DNA strand breaks were labeled with fluorescein-dUTP using TUNEL reaction (Roche) 24 h post-radiation. The apoptotic population was quantified by flow cytometry.

Figure 3. Apoptosis induction by vorinostat does not play a significant role in radiosensitization

(a) Assessment of apoptotic cell death by immunoblot analysis of caspase-3, caspase-9 and PARP cleavage. MiaPaCa-2 cells were treated with 2 μM vorinostat for 48 h, following which they were irradiated (6 Gy) and incubated for an additional 6 h. At the end of this treatment, cells were collected for immunoblot analysis. Quantification of the cleaved fragments for each protein was done by ImageQuant software. Fold cleavage with respect to the untreated control is indicated below each figure. (b) Assessment of vorinostat-induced apoptosis by TUNEL assay. Cells were treated with vorinostat for 48 hrs, irradiated (6 Gy) and DNA strand breaks were labeled with fluorescein-dUTP using TUNEL reaction (Roche) 24 h post-radiation. The apoptotic population was quantified by flow cytometry.

Figure 4. Vorinostat prolongs DNA repair

Vorinostat prolongs radiation-induced γ-H2AX foci. MiaPaCa-2 cells were treated with 2 μM vorinostat (48 h), irradiated (6 Gy), and fixed at indicated time intervals for immunofluorescence staining of nuclear γ-H2AX foci. (a) Representative images for each group; (b) Quantification of foci – columns = mean ±SE of nuclear foci counted in 50 cells.

Figure 4. Vorinostat prolongs DNA repair

Vorinostat prolongs radiation-induced γ-H2AX foci. MiaPaCa-2 cells were treated with 2 μM vorinostat (48 h), irradiated (6 Gy), and fixed at indicated time intervals for immunofluorescence staining of nuclear γ-H2AX foci. (a) Representative images for each group; (b) Quantification of foci – columns = mean ±SE of nuclear foci counted in 50 cells.

Figure 5. Effect of vorinostat on expression of DNA repair proteins

MiaPaCa-2 cells were exposed to indicated concentrations of vorinostat for 48 h, irradiated (6 Gy) and harvested at indicated times post-irradiation. Immunoblot analysis of DNA repair proteins was performed using the nuclear fraction (Lamin B) as loading control.

Figure 6. Vorinostat inhibits radiation-induced pro-survival signaling pathways

(a) Vorinostat inhibits radiation-induced EGFR phosphorylation. MiaPaCa-2 cells were exposed to vorinostat (2 μM; 48 hr), irradiated (6 Gy), and harvested at indicated time points post-irradiation for immunoblot analysis. (b) Vorinostat decreases radiation-induced expression of phospho (active) c-jun. Cells were treated with vorinostat (2 μM; 48 h), irradiated (6 Gy), and harvested at indicated time points post-irradiation for immunobloting. (c) Effect of vorinostat on NF-κB activation. Cells were treated with indicated concentrations of vorinostat for 48 h, irradiated (6 Gy), and harvested 3 h post-irradiation. NF-κB activity in the nuclear fraction was analyzed by EMSA.