Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair

Mol Cancer Ther. 2008 Jul;7(7):1772-81. doi: 10.1158/1535-7163.MCT-07-2200.

Abstract

We have already reported that epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT signaling is an important pathway in regulating radiation sensitivity and DNA double-strand break (DNA-dsb) repair of human tumor cells. In the present study, we investigated the effect of AKT1 on DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and DNA-dsb repair in irradiated non-small cell lung cancer cell lines A549 and H460. Treatment of cells with the specific AKT pathway inhibitor API-59 CJ-OH (API; 1-5 micromol/L) reduced clonogenic survival between 40% and 85% and enhanced radiation sensitivity of both cell lines significantly. As indicated by fluorescence-activated cell sorting analysis (sub-G(1) cells) and poly(ADP-ribose) polymerase cleavage, API treatment or transfection with AKT1-small interfering RNA (siRNA) induced apoptosis of H460 but not of A549 cells. However, in either apoptosis-resistant A549 or apoptosis-sensitive H460 cells, API and/or AKT1-siRNA did not enhance poly(ADP-ribose) polymerase cleavage and apoptosis following irradiation. Pretreatment of cells with API or transfection with AKT1-siRNA strongly inhibited radiation-induced phosphorylation of DNA-PKcs at T2609 and S2056 as well as repair of DNA-dsb as measured by the gamma-H2AX foci assay. Coimmunoprecipitation experiments showed a complex formation of activated AKT and DNA-PKcs, supporting the assumption that AKT plays an important regulatory role in the activation of DNA-PKcs in irradiated cells. Thus, targeting of AKT enhances radiation sensitivity of lung cancer cell lines A549 and H460 most likely through specific inhibition of DNA-PKcs-dependent DNA-dsb repair but not through enhancement of radiation-induced apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects
  • Apoptosis / radiation effects
  • Carcinoma, Non-Small-Cell Lung / enzymology
  • Carcinoma, Non-Small-Cell Lung / pathology
  • Caspases / metabolism
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cell Proliferation / radiation effects
  • DNA Breaks, Double-Stranded* / drug effects
  • DNA Breaks, Double-Stranded* / radiation effects
  • DNA Repair* / drug effects
  • DNA Repair* / radiation effects
  • DNA-Activated Protein Kinase / antagonists & inhibitors
  • DNA-Activated Protein Kinase / metabolism*
  • Humans
  • Lung Neoplasms / enzymology
  • Lung Neoplasms / pathology
  • Mutation / genetics
  • Phosphorylation / drug effects
  • Phosphorylation / radiation effects
  • Poly(ADP-ribose) Polymerases / metabolism
  • Protein Binding / drug effects
  • Protein Binding / radiation effects
  • Protein Kinase Inhibitors / pharmacology
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins c-akt / antagonists & inhibitors*
  • Proto-Oncogene Proteins p21(ras)
  • Radiation Tolerance* / drug effects
  • Radiation Tolerance* / radiation effects
  • Radiation, Ionizing
  • ras Proteins / genetics

Substances

  • KRAS protein, human
  • Protein Kinase Inhibitors
  • Proto-Oncogene Proteins
  • Poly(ADP-ribose) Polymerases
  • DNA-Activated Protein Kinase
  • Proto-Oncogene Proteins c-akt
  • Caspases
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins