Costantini Danny L, Bateman Katherine, McLarty Kristin, Vallis Katherine A, Reilly Raymond M
Leslie Dan Faculty of Pharmacy, Department of Pharmaceutical Sciences, University of Toronto, Ontario, Canada.
J Nucl Med. 2008 Sep;49(9):1498-505. doi: 10.2967/jnumed.108.051771. Epub 2008 Aug 14.
UNLABELLED: Our goals in this study were to determine whether (111)In-trastuzumab coupled to peptides harboring nuclear localizing sequences (NLSs) could kill trastuzumab-resistant breast cancer cell lines through the emission of Auger electrons and whether the combination of radiosensitization with methotrexate (MTX) would augment the cytotoxicity of this radiopharmaceutical. METHODS: Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate for reaction with NLS peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with (111)In. HER2 expression was determined by Western blot and by radioligand binding assay using (111)In-trastuzumab in a panel of breast cancer cell lines, including SK-BR-3, MDA-MB-231 and its HER2-transfected subclone (231-H2N), and 2 trastuzumab-resistant variants (TrR1 and TrR2). Nuclear importation of (111)In-NLS-trastuzumab and (111)In-trastuzumab in breast cancer cells was measured by subcellular fractionation, and the clonogenic survival of these cells was determined after incubation with (111)In-NLS-trastuzumab, (111)In-trastuzumab, or trastuzumab (combined with or without MTX). Survival curves were analyzed according to the dose-response model, and the radiation-enhancement ratio was calculated from the survival curve parameters. RESULTS: The expression of HER2 was highest in SK-BR-3 cells (12.6 x 10(5) receptors/cell), compared with 231-H2N and TrR1 cells (6.1 x 10(5) and 5.1 x 10(5) receptors/cell, respectively), and lowest in MDA-MB-231 and TrR2 cells (0.4 x 10(5) and 0.6 x 10(5) receptors/cell, respectively). NLS peptides increased the nuclear uptake of (111)In-trastuzumab in MDA-MB-231, 231-H2N, TrR1, and TrR2 cells from 0.1%+/-0.01%, 2.5%+/-0.2%, 2.8%+/-0.7%, and 0.5%+/-0.1% to 0.5%+/-0.1%, 4.6%+/-0.1%, 5.2%+/-0.6%, and 1.5%+/-0.2%, respectively. The cytotoxicity of (111)In-NLS-trastuzumab on breast cancer cells was directly correlated with the HER2 expression densities of the cells. On a molar concentration basis, the effective concentration required to kill 50% of 231-H2N and TrR1 cells for (111)In-NLS-trastuzumab was 9- to 12-fold lower than for (111)In-trastuzumab and 16- to 77-fold lower than for trastuzumab. MDA-MB-231 and TrR2 cells were less sensitive to (111)In-NLS-trastuzumab or (111)In-trastuzumab, and both cell lines were completely insensitive to trastuzumab. The radiation-enhancement ratio induced by MTX for 231-H2N and TrR1 cells after exposure to (111)In-NLS-trastuzumab was 1.42 and 1.68, respectively. CONCLUSION: Targeted Auger electron radioimmunotherapy with (111)In-NLS-trastuzumab can overcome resistance to trastuzumab, and MTX can potently enhance the sensitivity of HER2-overexpressing breast cancer cells to the lethal Auger electrons emitted by this radiopharmaceutical.
未标记：本研究的目的是确定与携带核定位序列（NLSs）的肽偶联的（111）In-曲妥珠单抗是否可通过俄歇电子发射杀死曲妥珠单抗耐药的乳腺癌细胞系，以及放射增敏剂甲氨蝶呤（MTX）与该药物联合使用是否会增强其细胞毒性。 方法：用4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯对曲妥珠单抗进行衍生化，使其与NLS肽反应，然后与二乙烯三胺五乙酸偶联以用（111）In标记。通过蛋白质印迹法和使用（111）In-曲妥珠单抗的放射性配体结合试验，在一组乳腺癌细胞系中测定HER2表达，这些细胞系包括SK-BR-3、MDA-MB-231及其HER2转染亚克隆（231-H2N），以及2个曲妥珠单抗耐药变体（TrR1和TrR2）。通过亚细胞分级分离法测量乳腺癌细胞中（111）In-NLS-曲妥珠单抗和（111）In-曲妥珠单抗的核摄取，并在与（111）In-NLS-曲妥珠单抗、（111）In-曲妥珠单抗或曲妥珠单抗（联合或不联合MTX）孵育后，测定这些细胞的克隆形成存活率。根据剂量反应模型分析存活曲线，并从存活曲线参数计算放射增强率。 结果：与231-H2N和TrR1细胞（分别为6.1×10⁵受体/细胞和5.1×10⁵受体/细胞）相比，SK-BR-3细胞中HER2表达最高（12.6×10⁵受体/细胞），而在MDA-MB-231和TrR2细胞中最低（分别为0.4×10⁵受体/细胞和0.6×10⁵受体/细胞）。NLS肽使MDA-MB-231、231-H2N、TrR1和TrR2细胞中（111）In-曲妥珠单抗的核摄取分别从0.1%±0.01%、2.5%±0.2%、2.8%±0.7%和0.5%±0.1%增加到0.5%±0.1%、4.6%±0.1%、5.2%±0.6%和1.5%±0.2%。（111）In-NLS-曲妥珠单抗对乳腺癌细胞的细胞毒性与细胞的HER2表达密度直接相关。在摩尔浓度基础上，（111）In-NLS-曲妥珠单抗杀死50%的231-H2N和TrR1细胞所需的有效浓度比（111）In-曲妥珠单抗低9至12倍，比曲妥珠单抗低16至77倍。MDA-MB-231和TrR2细胞对（111）In-NLS-曲妥珠单抗或（111）In-曲妥珠单抗较不敏感，且两种细胞系对曲妥珠单抗完全不敏感。MTX对暴露于（111）In-NLS-曲妥珠单抗后的231-H2N和TrR1细胞诱导的放射增强率分别为1.42和1.68。 结论：用（111）In-NLS-曲妥珠单抗进行靶向俄歇电子放射免疫治疗可克服对曲妥珠单抗的耐药性，且MTX可有效增强HER2过表达乳腺癌细胞对该放射性药物发射的致死性俄歇电子的敏感性。
Biomacromolecules. 2012-1-17
Curr Radiopharm. 2024
Nucl Med Biol. 2020
Zotero 插件