Irinotecan and DNA-PKcs inhibitors synergize in killing of colon cancer cells

Abstract

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.

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

“…The end result of blocking DNA-PKcs activity is an inability to load Exo1 on DSB ends, consistent with the idea that autophosphorylation of DNA-PKcs helps to release it from ends and to allow access of other repair factors (21,55). In cells, the effects of DNA-PKcs inhibitors on DNA-PKcs protein levels and NHEJ efficiency, as well as the phosphorylation of DNAPKcs by ATM, likely mask the stimulatory effects of DNA-PKcs catalytic activity on resection and yield conflicting observations (28,29,31,56). Our results in vitro are consistent with previous observations that loss of DNA-PKcs catalytic activity (through truncation of the catalytic domain, chemical inhibition, or mutation of autophosphorylation sites) actually inhibits HR (29,30).…”

“…the effect of chemical inhibition of DNA-PK on HR remains controversial (28,29,31). We examined this by performing the DNA resection assay in the presence of a DNA-PK inhibitor NU-7441 (DNA-PKi) or vehicle (DMSO).…”

Chemical inhibition of DNA repair kinases as a promising tool in oncology

“…This inhibitor is believed to be the first specific DNA-PK inhibitor sensitizing breast cancer cells to doxorubicin (topoisomerase II inhibitor) (ref. 42,43 ). The IC86621 inhibitor (1-(2-hydroxy-4-morpholin-4-ylphenyl)-ethanone) is a methyl ketone derivative of the DNA-PK inhibitor IC60211 (2-Hydroxy-4-morpholin-4-ylbenzaldehyde) with IC 50 : 120 nM.…”

Studies of Malaysian Plants in Prevention and Treatment of Colorectal Cancer

“…For example, HT29 cells utilize glucose through the pentose phosphate pathway [53], whereas HCT116 cells have higher requirements for glutamine [52,54]. In terms of gene expression, HT29 is deficient in expression of p53 [55], while HCT116 cells possess mutations in PI3KCA and KRAS genes which confer constitutive activation of PI3K/AKT and KRAS pathways [56]. Since the two cells lines have different characteristics, the use of such cell lines in preliminary studies is substantial as it can set forth the mechanistic investigations on the effects of the plants against colon cancer.…”