RESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína Quinasa Activada por ADN/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Tolerancia a Radiación , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Mutación/genética , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/efectos de la radiación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/efectos de la radiación , Radiación Ionizante , Proteínas ras/genéticaRESUMEN
BACKGROUND AND PURPOSE: 2-Methoxyestradiol (2-ME) is described as an inhibitor of the superoxide dismutase (SOD) enzyme activity. However, it attenuates PI3K/Akt pathway and induces radiosensitization in human tumor cells as well. Since the activation of catalytic subunit of DNA-protein kinase (DNA-PKcs) is partially regulated by Akt activity, in the present study we investigated whether 2-ME-induced radiosensitization is dependent on inhibition of Akt and DNA-PKcs activities or on SOD targeting. MATERIALS AND METHODS: This study was performed using the lung carcinoma cell line A549. Ionizing radiation-induced SOD activity was analyzed by superoxide dismutase activity assay. Applying Western blotting, the pattern of radiation-induced SOD expression and activation of Akt as well as DNA-PKcs was analyzed. Colony formation assay and gammaH2AX foci assay were performed to measure radiosensitization and DNA-double strand break (DNA-DSB) repair. To downregulate SOD expression small interfering RNA (siRNA) was used. RESULTS: Irradiation with 4Gy stimulated SOD enzyme activity as early as 1min after radiation exposure. Expression of Cu/Zn-SOD (SOD1) as well as Mn-SOD (SOD2) was increased by single doses of 1-4Gy within 24-36h. 2-ME blocked radiation-induced SOD enzyme activity but not protein expression and enhanced radiation sensitivity. Pretreatment with 2-ME blocked IR-induced Akt as well as DNA-PKcs phosphorylation and impaired the repair of DNA-DSB. SiRNA targeting of SOD1 and SOD2 affected neither DNA-PKcs phosphorylation nor post-irradiation survival while inhibition of Akt by specific inhibitor abrogated 2-ME-induced radiosensitization. CONCLUSION: These results may indicate that 2-ME-induced radiosensitization is independent of SOD inhibition but mainly depends on inhibition of Akt and DNA-PKcs activities.