title: Epidermal growth factor receptor inhibition modulates the nuclear localization and cytotoxicity of the Auger electron emitting radiopharmaceutical 111In-DTPA human epidermal growth factor.
abstract: (111)In-DTPA-human epidermal growth factor ((111)In-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 (111)In-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 x 10(6) EGFRs/cell) in the presence or absence of 1 microM gefitinib. The proportion of radioactivity partitioning into the cytoplasm and nucleus of MDA-MB-468 cells after incubation with (111)In-DTPA-hEGF for 24 h at 37 degrees C in the presence or absence of 1 microM gefitinib was measured by cell fractionation. DNA double-strand breaks caused by (111)In were quantified using the gamma-H2AX assay, and radiation-absorbed doses were estimated. Clonogenic survival assays were used to measure the cytotoxicity of (111)In-DTPA-hEGF alone or in combination with gefitinib.
  
  
  RESULTS
  Gefitinib (1 microM) 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 (111)In that localized in the nucleus was statistically significantly greater when (111)In-DTPA-hEGF was combined with gefitinib compared with (111)In-DTPA-hEGF alone (mean +/- SD: 26.0% +/- 5.5% vs. 14.6% +/- 4.0%, respectively; P < 0.05). Induction of gamma-H2AX foci was greater in MDA-MB-468 cells that were treated with (111)In-DTPA-hEGF (250 ng/mL, 1.5 MBq/mL) plus gefitinib (1 microM ) compared with those treated with (111)In-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 (111)In-DTPA-hEGF (5 ng/mL, 6 MBq/microg) was combined with gefitinib (1 microM ) compared with (111)In-DTPA-hEGF alone (42.9% +/- 5.7% vs. 22.9% +/- 3.6%, respectively; P < 0.01).
  
  
  CONCLUSIONS
  The efficacy of (111)In-DTPA-hEGF depends on internalization and nuclear uptake of the radionuclide. Nuclear uptake, DNA damage, and cytotoxicity are enhanced when (111)In-DTPA-hEGF is combined with gefitinib. These results suggest a potential therapeutic role for peptide receptor radionuclide therapy in combination with tyrosine kinase inhibitors.
authors: Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors: Kristy E
authors: KE
authors: Bailey
authors: Danny L
authors: DL
authors: Costantini
authors: Zhongli
authors: Z
authors: Cai
authors: Deborah A
authors: DA
authors: Scollard
authors: Zhuo
authors: Z
authors: Chen
authors: Raymond M
authors: RM
authors: Reilly
authors: Katherine A
authors: KA
authors: Vallis
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D002451:Cell Compartmentation; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D004583:Electrons; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D000077156:Gefitinib; D006801:Humans; D007205:Indium Radioisotopes; D004369:Pentetic Acid / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D010766:Phosphorylation; D021381:Protein Transport; D011799:Quinazolines / Q000494:pharmacology; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics / Q000494:pharmacology*; D015398:Signal Transduction
chemical_list: C478252:111In-DTPA-human epidermal growth factor; D000970:Antineoplastic Agents; D005456:Fluorescent Dyes; D007205:Indium Radioisotopes; D011799:Quinazolines; D019275:Radiopharmaceuticals; D004815:Epidermal Growth Factor; D004369:Pentetic Acid; D066246:ErbB Receptors; D016650:Fluorescein-5-isothiocyanate; D000077156:Gefitinib