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.