Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) to Analyze the Disruption of EGFR/HER2 Dimers
| Autor: | Caroline Bascoul-Mollevi, Christel Larbouret, Julie Vallaghe, Herve Bazin, David Azria, Nadège Gaborit, Thierry Chardès, Marie-Alix Poul, Gérard Mathis, Evelyne Crapez, Frédéric Peyrusson, André Pèlegrin |
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| Rok vydání: | 2011 |
| Předmět: |
0303 health sciences
biology Cetuximab Cell Biology Lapatinib Biochemistry Molecular biology 3. Good health 03 medical and health sciences 0302 clinical medicine Trastuzumab 030220 oncology & carcinogenesis medicine biology.protein Erlotinib Epidermal growth factor receptor Pertuzumab skin and connective tissue diseases Erlotinib Hydrochloride neoplasms Molecular Biology Tyrosine kinase 030304 developmental biology medicine.drug |
| Zdroj: | Journal of Biological Chemistry. 286:11337-11345 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m111.223503 |
| Popis: | In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation. |
| Databáze: | OpenAIRE |
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