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Methotrexate, Paclitaxel, and Doxorubicin Radiosensitize HER2-Amplified Human Breast Cancer Cells to the Auger Electron–Emitting Radiotherapeutic Agent ¹¹¹In-NLS-Trastuzumab

¹ Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada; ² Department of Diagnostic Imaging, Hospital for Sick Children, Toronto, Ontario, Canada; ³ Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada; ⁴ Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom; ⁵ Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada; and ⁶ Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada

Correspondence: For correspondence or reprints contact: Raymond M. Reilly, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, Ontario M5S 3M2, Canada. E-mail: raymond.reilly{at}utoronto.ca

Our goal in this study was to elucidate the mechanisms by which methotrexate radiosensitizes HER2-positive human breast cancer cells to the Auger electron emitter ¹¹¹In-trastuzumab modified with nuclear-localization sequence peptides (¹¹¹In-NLS-trastuzumab) and to compare these mechanisms with the potential sensitizing effects of paclitaxel and doxorubicin when combined with this radiopharmaceutical. Methods: Experiments were performed in MDA-MB-231 human breast cancer cells, their HER2-transfected subclones (231-H2N), and 2 trastuzumab-resistant variants (trastuzumab-resistant-1 and -2 [TrR1 and TrR2]). Effects of coexposure of these cells to ¹¹¹In-NLS-trastuzumab and low-dose, radiosensitizing methotrexate, paclitaxel, or doxorubicin were assessed by clonogenic cell-survival assay. Quantification of residual DNA damage was measured by the H2AX-immunofluorescence assay, and cell cycle distribution was measured by fluorescence-activated cell sorting analysis. The radiation-enhancement ratio was calculated as the ratio of the surviving fraction (SF) of cells treated with ¹¹¹In-NLS-trastuzumab alone to that of cells treated concurrently with ¹¹¹In-NLS-trastuzumab and methotrexate, paclitaxel, or doxorubicin. Results: A reduction in the SF in HER2-positive 231-H2N (55.7% ± 1.3%) and TrR1 (62.6% ± 6.5%) cells was demonstrated after exposure to ¹¹¹In-NLS-trastuzumab (0.2 MBq/µg, 100 nmol/L) but not in MDA-MB-231 or TrR2 cells expressing low levels of HER2 (SF > 90%, P > 0.05). Coadministration of methotrexate, paclitaxel, or doxorubicin enhanced the cytotoxicity of ¹¹¹In-NLS-trastuzumab toward 231-H2N and TrR1 cells but not toward MDA-MB-231 or TrR2 cells. The radiation-enhancement ratios for methotrexate, paclitaxel, and doxorubicin for 231-H2N or TrR1 cells were 2.0–2.2, 1.6–1.8, and 2.7–2.8, respectively. Methotrexate or doxorubicin combined with ¹¹¹In-NLS-trastuzumab, compared to treatment with ¹¹¹In-NLS-trastuzumab alone, significantly increased residual H2AX foci in 231-H2N and TrR1 cells but not in MDA-MB-231 or TrR2 cells or in any cell line treated concurrently with paclitaxel and ¹¹¹In-NLS-trastuzumab. Cells exposed to low-dose methotrexate accumulated in the G₁/S phase of the cell cycle, whereas low-dose paclitaxel or doxorubicin caused cells to arrest in the G₂/M phase. Conclusion: Low-dose methotrexate, paclitaxel, or doxorubicin potently sensitized HER2-overexpressing human breast cancer cells, with and without acquired trastuzumab-resistance, to the Auger electron emissions from ¹¹¹In-NLS-trastuzumab through cell cycle distribution changes and in part through the inhibitory effects of these agents on DNA damage repair.

Key Words: Auger electron • radiosensitizer • trastuzumab • breast cancer • radioimmunotherapy

^* Contributed equally to this work.

COPYRIGHT © 2010 by the Society of Nuclear Medicine, Inc.

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