Epidermal growth factor receptor inhibition modulates the nuclear localization and cytotoxicity of the Auger electron-emitting radiopharmaceutical 111In-DTPA-human epidermal growth factor

Abstract

111In-DTPA-human epidermal growth factor (111In-DTPA- hEGF [DTPA is diethylenetriaminepentaacetic acid]) is an Auger electron-emitting radiopharmaceutical that targets EGF receptor (EGFR)-positive cancer. The purpose of this study was to determine the effect of EGFR inhibition by gefitinib on the internalization, nuclear translocation, and cytotoxicity of 111In-DTPA-hEGF in EGFR-overexpressing MDA-MB-468 human breast cancer cells. Methods: Western blot analysis was used to determine the optimum concentration of gefitinib to abolish EGFR activation. Internalization and nuclear translocation of fluorescein isothiocyanate-labeled hEGF were evaluated by confocal microscopy in MDA-MB-468 cells (1.3 × 106 EGFRs/cell) in the presence or absence of 1 μM gefitinib. The proportion of radioactivity partitioning into the cytoplasm and nucleus of MDA-MB-468 cells after incubation with 111In-DTPA-hEGF for 24 h at 37°C in the presence or absence of 1 μM gefitinib was measured by cell fractionation. DNA double-strand breaks caused by 111In were quantified using the γ-H2AX assay, and radiation-absorbed doses were estimated. Clonogenic survival assays were used to measure the cytotoxicity of 111In-DTPA- hEGF alone or in combination with gefitinib. Results: Gefitinib (1 μM) completely abolished EGFR phosphorylation in MDA-MB-468 cells. Internalization and nuclear translocation of fluorescein isothiocyanate-labeled EGF were not diminished in gefitinib-treated cells compared with controls. The proportion of internalized 111In that localized in the nucleus was statistically significantly greater when 111In-DTPA-hEGF was combined with gefitinib compared with 111In-DTPA-hEGF alone (mean ± SD: 26.0% ± 5.5% vs. 14.6% ± 4.0%, respectively; P < 0.05). Induction of γ-H2AX foci was greater in MDA-MB-468 cells that were treated with 111In-DTPA-hEGF (250 ng/mL, 1.5 MBq/mL) plus gefitinib (1 μM) compared with those treated with 111In-DTPA-hEGF alone (mean ± SD: 35 ± 4 vs. 24 ± 5 foci per nucleus, respectively). In clonogenic assays, a significant reduction in the surviving fraction was observed when 111In-DTPA-hEGF (5 ng/mL, 6 MBq/μg) was combined with gefitinib (1 μM) compared with 111In-DTPA-hEGF alone (42.9% ± 5.7% vs. 22.9% ± 3.6%, respectively; P < 0.01). Conclusion: The efficacy of 111In-DTPA-hEGF depends on internalization and nuclear uptake of the radionuclide. Nuclear uptake, DNA damage, and cytotoxicity are enhanced when 111In-DTPA-hEGF is combined with gefitinib. These results suggest a potential therapeutic role for peptide receptor radionuclide therapy in combination with tyrosine kinase inhibitors. Copyright © 2007 by the Society of Nuclear Medicine, Inc.