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

Abstract

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 111 In-trastuzumab modified with nuclear-localization sequence peptides ( 111 In-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 trastuzumabresistant variants (trastuzumab-resistant-1 and -2 [TrR1 and TrR2]). Effects of coexposure of these cells to 111 In-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 gH2AX-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 111 In-NLStrastuzumab alone to that of cells treated concurrently with 111 In-NLS-trastuzumab and methotrexate, paclitaxel, or doxorubicin. Results: A reduction in the SF in HER2-positive 231-H2N (55.7% 6 1.3%) and TrR1 (62.6% 6 6.5%) cells was demonstrated after exposure to 111 In-NLS-trastuzumab (;0.2 MBq/mg, 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 111 In-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 111 In-NLS-trastuzumab, compared to treatment with 111 In-NLStrastuzumab alone, significantly increased residual gH2AX 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 111 In-NLS-trastuzumab. Cells exposed to low-dose methotrexate 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 111 In-NLS-trastuzumab through cell cycle distribution changes and in part through the inhibitory effects of these agents on DNA damage repair.

Changes in Lognormal Shape Parameter Guide Design of Patient-Specific Radiochemotherapy Cocktails

“…Although there is interest in using multimodal approaches that involve the concomitant delivery of chemotherapeutic and radiotherapeutic agents for cancer treatment (5,39), most efforts have not been directed at using agentspecific distribution profiles to target all malignant cells. To facilitate the design of cocktails that effectively target all cells of interest, an in-depth knowledge of the distribution profile of each agent is required.…”

Molecular and cellular effects of Auger emitters: 2008–2011

“…Use of the radiopharmaceutical, 111 In-DTPA-hEGF, proved to correlate well with the number of EGFR and γ H2AX foci formation so the authors suggest that γ H2AX foci formation be used as a biomarker for the eff ectiveness of 111 In-DTPA-hEGF therapy (Cai et al 2008). Costantini et al (2010a) also measured γ H2AX foci in cells treated with 111 In-NLS-trastuzumab in combination with methotrexate. Th e highest number of γ H2AX foci was found in cells treated with the 111 In-NLS-trastuzumab in combination with methotrexate and not when the radiopharmaceutical was combined with paclitaxel or doxorubicin (Costantini et al 2010a).…”

“…Costantini et al (2010a) also measured γ H2AX foci in cells treated with 111 In-NLS-trastuzumab in combination with methotrexate. Th e highest number of γ H2AX foci was found in cells treated with the 111 In-NLS-trastuzumab in combination with methotrexate and not when the radiopharmaceutical was combined with paclitaxel or doxorubicin (Costantini et al 2010a).…”

Nuclisome—targeting the tumor cell nucleus

“…Focus has been on delivery of 111 In utilizing nuclear-localization sequences (NLS) [5][6][7][8]. Proteins or peptides tagged with NLS can be transported form cytoplasmic compartments to the nucleus and if such proteins or peptides are radiolabeled and targeted to tumor markers a successful delivery of radionuclides to tumor-cell nuclei can potentially be achieved.…”