Bailey Kristy E, Costantini Danny L, Cai Zhongli, Scollard Deborah A, Chen Zhuo, Reilly Raymond M, Vallis Katherine A
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
J Nucl Med. 2007 Sep;48(9):1562-70. doi: 10.2967/jnumed.107.044073. Epub 2007 Aug 17.
(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.
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.
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).
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.
（111）铟-二乙三胺五乙酸-人表皮生长因子（（111）In-DTPA-hEGF [DTPA为二乙三胺五乙酸]）是一种靶向表皮生长因子受体（EGFR）阳性癌症的俄歇电子发射放射性药物。本研究的目的是确定吉非替尼抑制EGFR对过表达EGFR的MDA-MB-468人乳腺癌细胞中（111）In-DTPA-hEGF的内化、核转位和细胞毒性的影响。
采用蛋白质印迹分析确定消除EGFR激活的吉非替尼最佳浓度。通过共聚焦显微镜评估在存在或不存在1μM吉非替尼的情况下，异硫氰酸荧光素标记的hEGF在MDA-MB-468细胞（1.3×10⁶个EGFR/细胞）中的内化和核转位。在存在或不存在1μM吉非替尼的情况下，将MDA-MB-468细胞与（111）In-DTPA-hEGF在37℃孵育24小时后，通过细胞分级分离测量放射性分配到细胞质和细胞核中的比例。使用γ-H2AX检测对由（111）In引起的DNA双链断裂进行定量，并估计辐射吸收剂量。采用克隆形成存活试验测量单独使用（111）In-DTPA-hEGF或与吉非替尼联合使用时的细胞毒性。
吉非替尼（1μM）完全消除了MDA-MB-468细胞中的EGFR磷酸化。与对照相比，在吉非替尼处理的细胞中，异硫氰酸荧光素标记的EGF的内化和核转位没有减少。与单独使用（111）In-DTPA-hEGF相比，（111）In-DTPA-hEGF与吉非替尼联合使用时，定位于细胞核中的内化（111）In的比例在统计学上显著更高（平均值±标准差：分别为26.0%±5.5%和14.6%±4.0%；P<0.05）。与单独使用（111）In-DTPA-hEGF相比，用（111）In-DTPA-hEGF（250 ng/mL，1.5 MBq/mL）加吉非替尼（1μM）处理的MDA-MB-468细胞中γ-H2AX焦点的诱导更大（平均值±标准差：分别为每个细胞核35±4和24±5个焦点）。在克隆形成试验中，与单独使用（111）In-DTPA-hEGF相比，当（111）In-DTPA-hEGF（5 ng/mL，6 MBq/μg）与吉非替尼（1μM）联合使用时，观察到存活分数显著降低（分别为42.9%±5.7%和22.9%±3.6%；P<0.01）。
（111）In-DTPA-hEGF的疗效取决于放射性核素的内化和核摄取。当（111）In-DTPA-hEGF与吉非替尼联合使用时，核摄取、DNA损伤和细胞毒性增强。这些结果表明肽受体放射性核素治疗联合酪氨酸激酶抑制剂具有潜在的治疗作用。
