Induction of apoptosis and modulation of homologous recombination DNA repair pathway in prostate cancer cells by the combination of AZD2461 and valproic acid

Cancer therapies using defects in homologous recombination (HR) DNA repair pathway of tumor cells are not yet approved to be applicable in patients with malignancies other than BRCA1/2-mutated tumors. This study was designed to determine the efficacy of combination therapy of a histone deacetylase inhibitor, valproic acid (VPA) and a novel PARP inhibitor AZD2461 in both PC-3 (PTEN-mutated) and DU145 (PTEN-unmutated) prostate cancer cell lines. The Trypan blue dye exclusion assay and the tetrazolium-based colorimetric (MTT) assay were performed to measure the cytotoxicity while combination effects were assessed based on Chou-Talalay's principles. Flow-cytometric assay determined the type of cell death. The real-time PCR analysis was used to evaluate the alterations in mRNA levels of HR-related genes while their protein levels were measured using the ELISA method. γ-H2AX levels were determined as a marker of DNA damage. We observed a synergistic relationship between VPA and AZD2461 in all affected fractions of PC-3 cells (CI<0.9), but not in DU145 cells (CI>1.1). Annexin-V staining analysis revealed a significant induction of apoptosis when PC-3 cells were treated with VPA+AZD2461 (p<0.05). Both mRNA and protein levels of Rad51 and Mre11 were significantly decreased in PC-3 cells co-treated with VPA+AZD2461 while enhanced H2AX phosphorylation was found in PC-3 cells after 12 and 24 hours of co-treatment (p<0.05). Our findings established a preclinical rationale for selective targeting of HR repair pathways by a combination of VPA and AZD2461 as a mechanism for reducing the HR pathway sufficiency in PTEN-mutated prostate cancer cells.