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Epidermal Growth Factor Receptor Inhibition Modulates the Nuclear Localization and Cytotoxicity of the Auger Electron–Emitting Radiopharmaceutical ¹¹¹In-DTPA–Human Epidermal Growth Factor

¹ Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; ² Division of Applied Molecular Oncology, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada; ³ Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada; ⁴ Division of Nuclear Medicine, Toronto General Hospital Research Institute, Toronto, Ontario, Canada; and ⁵ Radiation Medicine Program, Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada

View larger version (39K): [in this window] [in a new window]   FIGURE 1.  EGF-induced EGFR phosphorylation and activation is blocked by 1 µM gefitinib in MDA-MB-468 cells. Western blot analyses of MDA-MB-468 cells without (–) or stimulated with 20 ng/mL EGF (+) for 15 min, following incubation with 0.05–1 µM gefitinib for 3 h at 37°C. (A) Total protein samples were probed with antiphospho-EGFR Tyr-1173 and anti-EGFR antibodies. (B) Anti-MAPK, antiphospho-MAPK (p42/p44), anti-SAPK/JNK, antiphospho-SAPK/JNK, anti-p38, and antiphospho-p38 antibodies were used to determine phosphorylation status of downstream proteins in EGFR signaling cascade. Phosphorylation of EGFR and activation of downstream proteins was completely blocked by 1 µM gefitinib.

View larger version (51K): [in this window] [in a new window]   FIGURE 2.  Visualization of nuclear fluorescein-labeled-hEGF in presence of gefitinib using confocal microscopy. (A) MDA-MB-468 cells treated with fluorescein-hEGF for 1 h at 37°C. (B) MDA-MB-468 cells pretreated with gefitinib (1 µM) for 3 h followed by fluorescein-hEGF and gefitinib for 1 h at 37°C. Cells were also incubated with DAPI to visualize the cell nucleus. Images represent a 1-µm slice through center of cell. Fluorescein-hEGF was observed in the nucleus in absence and presence of 1 µM gefitinib.

View larger version (13K): [in this window] [in a new window]   FIGURE 3.  Effect of gefitinib and 111In-DTPA-hEGF on induction of -H2AX foci in MDA-MB-468 cells. -H2AX assay was performed after exposure of MDA-MB-468 cells to DTPA-hEGF (250 ng/mL), gefitinib (1 µM), 111In-acetate (1.5 MBq/mL), 111In-DTPA-hEGF (250 ng/mL, 1.5 MBq/mL), gefitinib (1 µM) plus 111In-DTPA-hEGF (250 ng/mL, 1.5 MBq/mL), or DMEM alone (control) for 20 h. Optical sections (1.2 µm) through the cells were obtained using a confocal microscope and were processed using ImageJ (U.S. National Institutes of Health). Error bars represent SEM number of -H2AX foci from 3 separate experiments. Thirty cells were counted to generate each data point for each experiment. *P < 0.05 (compared with control).

View larger version (14K): [in this window] [in a new window]   FIGURE 4.  Gefitinib enhances 111In-DTPA-hEGF–mediated cytotoxicity. Clonogenic assay was performed using MDA-MB-468 cells treated with increasing concentrations of 111In-DTPA-hEGF (5–250 ng/mL, 6 MBq/µg) for 24 h at 37°C (black bars) or pretreated with gefitinib (1 µM) for 3 h followed by treatment with a range of concentrations of 111In-DTPA-hEGF plus gefitinib (1 µM) for 24 h at 37°C (white bars). At each concentration of 111In-DTPA-hEGF tested, addition of gefitinib resulted in enhanced cytotoxicity. The SF of cells exposed to DTPA-hEGF (250 ng/mL) in the absence or presence of gefitinib (1 µM) was also measured (lined and speckled bars, respectively). 111In-DTPA-hEGF (250 ng/mL) was 10-fold more cytotoxic than unlabeled DTPA-hEGF (SF, 6.1% ± 1.9% vs. 60.6% ± 9.9%, respectively; P < 0.01). The SF of cells exposed to DTPA-hEGF (250 ng/mL) plus gefitinib (1 µM) was not statistically significant from the SF of cells exposed to 1 µM gefitinib alone (39.9% ± 7.4% vs. 40.3% ± 6.1%, respectively; P = 0.5). Error bars represent SD of the mean SF calculated from 3 separate experiments. *P < 0.05 (compared with control).