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

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 In-111-trastuzumab modified with nuclear-localization sequence peptides (In-111-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 In-111-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 gamma 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 In-111-NLS-trastuzumab alone to that of cells treated concurrently with In-111-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 In-111-NLS-trastuzumab (similar to 0.2 MBq/mu g, 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 In-111-NLS-trastuzumab toward 231-H2N and TrR1 cells but not toward MDA-MB-231 or TrR2 cells. The radiationenhancement 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 In-111-NLS-trastuzumab, compared to treatment with In-111-NLStrastuzumab alone, significantly increased residual gamma 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 (InNLS)-In-111- trastuzumab. Cells exposed to low-dosemethotrexate accumulated in the G(1)/S phase of the cell cycle, whereas low-dose paclitaxel or doxorubicin caused cells to arrest in the G(2)/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 In-111-NLS-trastuzumab through cell cycle distribution changes and in part through the inhibitory effects of these agents on DNA damage repair.