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Cisplatin-Resistant Neuroblastoma Cells Express Enhanced Levels of Epidermal Growth Factor Receptor (EGFR) and Are Sensitive to Treatment with EGFR-Specific Toxins

Authors' Affiliations: ¹ Institut für Medizinische Virologie, Klinikum der J.W. Goethe Universität; ² Institut für Pharmazeutische Technologie, Johann Wolfgang Goethe-Universität; ³ Senckenbergisches Institut für Pathologie, Klinikum der J.W. Goethe Universität; ⁴ Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt am Main, Germany; and ⁵ Deutsches Krebsforschungszentrum (DKFZ), Klinische Kooperationseinheit Pädiatrische Onkologie (G340) and Pädiatrie III, Zentrum für Kinder- und Jugendmedizin, Heidelberg, Germany

Requests for reprints: Jindrich Cinatl, Jr., Institut für Medizinische Virologie, Paul Ehrlich-Str. 40, 60596 Frankfurt/Main, Germany. Phone: 49-69-6301-6409; Fax: 49-69-6301-4302; E-mail: Cinatl{at}em.uni-frankfurt.de.

Purpose: Neuroblastomas frequently show expression of the epidermal growth factor receptor (EGFR) and may therefore be susceptible to EGFR-targeted therapies. Here, EGFR expression and functionality was investigated in parental chemosensitive neuroblastoma cell lines (UKF-NB-3, IMR-32, NLF, SH-SY5Y) and their cisplatin-resistant sublines (UKF-NB-3^rCDDP¹⁰⁰⁰, IMR-32^rCDDP¹⁰⁰⁰, NLF^rCDDP¹⁰⁰⁰, and SH-SY5Y^rCDDP⁵⁰⁰). Moreover, the EGFR antibody cetuximab, the EGFR tyrosine kinase inhibitor Tyrphostin B46, and recombinant EGFR-targeted toxins were investigated for their influence on the viability and growth of neuroblastoma cells.

Experimental Design: EGFR expression and function was measured by flow cytometry or Western blot. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was examined by immunostaining for active caspase-3 or cleaved poly(ADP-ribose) polymerase. Cellular binding of FITC-labeled immunotoxins was studied by flow cytometry, and cellular uptake was studied by confocal laser scanning microscopy.

Results: The EGFR-targeted antibody and growth factor toxins scFv(14E1)- Pseudomonas exotoxin A (ETA) and TGF--ETA exerted anti-cancer effects in neuroblastoma cell lines that were insensitive to cetuximab or EGFR tyrosine kinase inhibitors. Furthermore, adaptation of chemosensitive neuroblastoma cells to cisplatin increased EGFR expression and sensitivity to both recombinant toxins. Treatment of chemosensitive neuroblastoma cells with cisplatin reversibly increased EGFR expression, whereas cisplatin-resistant cells showed enhanced EGFR expression independent of the presence of cisplatin. Combination treatment with scFv(14E1)-ETA or TGF--ETA and cisplatin exerted significantly improved anticancer effects compared with either single treatment in parental neuroblastoma cells, cisplatin-resistant sublines, and primary cultures.

Conclusions: EGFR-targeted cytotoxic reagents such as scFv(14E1)-ETA and TGF--ETA represent promising candidates for further development as antineuroblastoma agents, especially in combination with cisplatin.