This study sought to measure the degree of synergy induced by specific small molecule inhibitors of
DNA-PK [
NU7026 and
IC486241 (ICC)], a major component of the non-homologous end-joining (NHEJ) pathway, with SN38 or
oxaliplatin. Synergy between the
DNA damaging drugs and the
DNA-PK inhibitors was assessed using the
sulforhodamine-B assay (SRB). Effects of
drug combinations on cell cycle and
DNA-PK activity were determined using flow cytometry and western blot analysis. DNA damage was assessed via comet assay and quantification of γH2AX. The role of homologous recombination repair (HRR) was determined by nuclear
Rad51 protein levels and a GFP reporter recombination assay. Significant reductions in the IC(50) values of SN38 were observed at 5 and 10 μM of
DNA-PK inhibitors. Moreover, at 1-2 μM (attainable concentrations with ICC in mice) these
DNA-
PKcs inhibitors demonstrated synergistic reductions in the IC(50) of SN38. Flow cytometric data indicated that SN38 and SN38 in combination with
DNA-
PKcs inhibitors showed dramatic G2/M arrest at 24 h. Furthermore, reduced phosphorylation of
DNA-
PKcs and increased DNA damage were observed at this time point with SN38 in combination with
DNA-
PKcs inhibitors as compared to cells treated with SN38 alone. SN38 alone and in the presence of ICC increased nuclear
Rad51 protein levels. Furthermore, inhibition of
DNA-
PKcs increased HRR suggesting that NHEJ is a negative regulator of HRR. These data indicate that small molecule inhibitors of
DNA-
PKcs dramatically enhance the efficacy of SN38 in
colon cancer cell lines.