Michaelis Martin, Bliss Jennifer, Arnold Sonja C, Hinsch Nora, Rothweiler Florian, Deubzer Hedwig E, Witt Olaf, Langer Klaus, Doerr Hans W, Wels Winfried S, Cinatl Jindrich
Institut für Medizinische Virologie, Klinikum der J.W. Goethe Universität, Germany.
Clin Cancer Res. 2008 Oct 15;14(20):6531-7. doi: 10.1158/1078-0432.CCR-08-0821.
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(r)CDDP(1000), IMR-32(r)CDDP(1000), NLF(r)CDDP(1000), and SH-SY5Y(r)CDDP(500)). 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.
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.
The EGFR-targeted antibody and growth factor toxins scFv(14E1)- Pseudomonas exotoxin A (ETA) and TGF-alpha-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-alpha-ETA and cisplatin exerted significantly improved anticancer effects compared with either single treatment in parental neuroblastoma cells, cisplatin-resistant sublines, and primary cultures.
EGFR-targeted cytotoxic reagents such as scFv(14E1)-ETA and TGF-alpha-ETA represent promising candidates for further development as antineuroblastoma agents, especially in combination with cisplatin.
神经母细胞瘤常表现出表皮生长因子受体（EGFR）的表达，因此可能对EGFR靶向治疗敏感。在此，对亲代化疗敏感的神经母细胞瘤细胞系（UKF-NB-3、IMR-32、NLF、SH-SY5Y）及其顺铂耐药亚系（UKF-NB-3(r)CDDP(1000)、IMR-32(r)CDDP(1000)、NLF(r)CDDP(1000)和SH-SY5Y(r)CDDP(500)）中的EGFR表达和功能进行了研究。此外，还研究了EGFR抗体西妥昔单抗、EGFR酪氨酸激酶抑制剂酪氨酸磷酸化抑制剂B46以及重组EGFR靶向毒素对神经母细胞瘤细胞活力和生长的影响。
通过流式细胞术或蛋白质免疫印迹法检测EGFR表达和功能。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法检测细胞活力。通过活性半胱天冬酶-3或裂解的聚（ADP-核糖）聚合酶免疫染色检测细胞凋亡。通过流式细胞术研究异硫氰酸荧光素标记的免疫毒素的细胞结合，通过共聚焦激光扫描显微镜研究细胞摄取。
EGFR靶向抗体和生长因子毒素单链抗体片段(14E1)-绿脓杆菌外毒素A（ETA）和转化生长因子-α-ETA在对西妥昔单抗或EGFR酪氨酸激酶抑制剂不敏感的神经母细胞瘤细胞系中发挥抗癌作用。此外，化疗敏感的神经母细胞瘤细胞对顺铂的适应性增加了EGFR表达以及对两种重组毒素的敏感性。用顺铂处理化疗敏感的神经母细胞瘤细胞可使EGFR表达可逆性增加，而顺铂耐药细胞显示出与顺铂存在无关的EGFR表达增强。与亲代神经母细胞瘤细胞、顺铂耐药亚系和原代培养物中的单一治疗相比，单链抗体片段(14E1)-ETA或转化生长因子-α-ETA与顺铂联合治疗具有显著改善的抗癌效果。
EGFR靶向细胞毒性试剂如单链抗体片段(14E1)-ETA和转化生长因子-α-ETA是有前景的抗神经母细胞瘤药物进一步开发候选物，尤其是与顺铂联合使用时。
