Original Article
Therapeutic proton irradiation results in apoptosis and caspase-3 activation in human peripheral blood lymphocytes
Abstract
Background: Proton therapy is effective in controlling many cancer types, allowing to spare the normal tissues and limiting the risk of adverse effects. However, cellular and molecular mechanisms by which protons induce cell death are still not fully understood. The purpose of this study was to investigate apoptotic mode of cell killing in human peripheral blood lymphocytes (HPBL) exposed ex vivo to 60 MeV proton beam radiation.
Methods: HPBL obtained from 5 healthy donors were irradiated ex vivo with 60 MeV protons in spread out Bragg peak (SOBP), in the dose range 0.3–4.0 Gy. The average proton dose rate was 0.075 Gy/s. After irradiation, HPBL were stained with Annexin V fluorescein isothiocyanate (V-FITC) at different time-points post-radiation exposure: 1, 4 and 24 hours. To assess caspase-3 activation following irradiation with protons, caspase-3 DEVD-R1100 Fluorometric assay was used.
Results: The apoptotic cell fraction stained with Annexin V-FITC, analysed after 1 and 4 h post proton-irradiation showed a dose-response increase in cell death. After 24 h post radiation exposure, the apoptotic fraction of cells represented a similar trend as in 1 and 4 h but less pronounced. Caspase-3 activation measured after 6 h of proton irradiation was significantly higher (P<0.05) compared to 24 h post-irradiation for all donors.
Conclusions: The data clearly demonstrates that 60 MeV therapeutic proton beam induced cell-killing in HPBL via apoptotic cell mode of death appears to be mediated by caspase-3 activation.
Methods: HPBL obtained from 5 healthy donors were irradiated ex vivo with 60 MeV protons in spread out Bragg peak (SOBP), in the dose range 0.3–4.0 Gy. The average proton dose rate was 0.075 Gy/s. After irradiation, HPBL were stained with Annexin V fluorescein isothiocyanate (V-FITC) at different time-points post-radiation exposure: 1, 4 and 24 hours. To assess caspase-3 activation following irradiation with protons, caspase-3 DEVD-R1100 Fluorometric assay was used.
Results: The apoptotic cell fraction stained with Annexin V-FITC, analysed after 1 and 4 h post proton-irradiation showed a dose-response increase in cell death. After 24 h post radiation exposure, the apoptotic fraction of cells represented a similar trend as in 1 and 4 h but less pronounced. Caspase-3 activation measured after 6 h of proton irradiation was significantly higher (P<0.05) compared to 24 h post-irradiation for all donors.
Conclusions: The data clearly demonstrates that 60 MeV therapeutic proton beam induced cell-killing in HPBL via apoptotic cell mode of death appears to be mediated by caspase-3 activation.
