Article Figures & Data
Figures
- 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.
- 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.
- FIGURE 3.
Effect of gefitinib and 111In-DTPA-hEGF on induction of γ-H2AX foci in MDA-MB-468 cells. γ-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 γ-H2AX foci from 3 separate experiments. Thirty cells were counted to generate each data point for each experiment. *P < 0.05 (compared with control).
- 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).
Tables
- TABLE 1
Effect of Gefitinib on Binding, Internalization, and Nuclear Localization of 111In-DTPA-hEGF in MDA-MB-468 Human Breast Cancer Cells
Proportion (%) Proportion of cell-bound 111In-DTPA-hEGF (%) Treatment Radioactivity bound to cells Cell-bound radioactivity internalized by cells Radioactivity within cytoplasm Radioactivity within nucleus 111In-DTPA-hEGF 54.7 ± 8.6 76.9 ± 5.9 62.3 ± 9.5 14.6 ± 4.0 111In-DTPA-hEGF + gefitinib 58.1 ± 2.8 81.1 ± 1.0 55.0 ± 6.2 26.0 ± 5.5* ↵* P < 0.05.
Data are expressed as mean ± SD of 3 experiments.
- TABLE 2
Effect of Gefitinib on Radiation-Absorbed Dose to Cell Nucleus from 111In-DTPA-hEGF Localized at Cell Membrane, Cytoplasm, or Nucleus of MDA-MB-468 EGFR-Overexpressing Human Breast Cancer Cells*
Treatment Cell compartment Ā (Bq × s)† S ([Gy/Bq × s] × 10−4) Radiation dose to nucleus 
, (Gy)‡111In-DTPA-hEGF Membrane 4,240 1.78 0.75 Cytoplasm 11,307 3.18 3.4 Nucleus 2,651 60.3 16.0 Total 20.15 111In-DTPA-hEGF + gefitinib Membrane 3,432 1.78 0.61 Cytoplasm 9,894 3.18 3.15 Nucleus 4,735 60.3 28.55 Total 32.31 ↵* Radiation-absorbed dose (
) to cell nucleus was estimated using cellular radiation dosimetry model of Goddu et al. (22): = Ā × S, where S is radiation-absorbed dose in nucleus (Gy) per unit of cumulated radioactivity in source compartment, Ā (Bq × s).↵† Ā = A0/λ, where A0 is amount of radioactivity localized in the compartment at time 0, and λ is radioactive decay constant for 111In (2.83 × 10−6/s). Rapid localization of 111In-DTPA-hEGF in the compartment and rate of elimination corresponding to radioactive decay of radionuclide 111In are assumed.
↵‡ Based on each MDA-MB-468 cell having a diameter of 10 μm and a nuclear diameter of 6 μm. Assumes targeting of a single cell with 111In-DTPA-hEGF to receptor saturation. At receptor saturation, ∼48 mBq 111In-DTPA-hEGF would be bound to each MDA-MB-468 cell at a specific activity of 3.7 MBq/μg.
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