Methotrexate, paclitaxel, and doxorubicin radiosensitize HER2-amplified human breast cancer cells to the Auger electron-emitting radiotherapeutic agent (111)In-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 (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 trastuzumab-resistant 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 gammaH2AX-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-NLS-trastuzumab 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% +/- 1.3%) and TrR1 (62.6% +/- 6.5%) cells was demonstrated after exposure to (111)In-NLS-trastuzumab (approximately 0.2 MBq/microg, 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 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 (111)In-NLS-trastuzumab, compared to treatment with (111)In-NLS-trastuzumab alone, significantly increased residual gammaH2AX 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.


CONCLUSIONS
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.
Costantini
Danny L
DL
Department of Pharmaceutical Sciences, University of Toronto, Ontario, Canada.
Villani
Daniela F
DF
Vallis
Katherine A
KA
Reilly
Raymond M
RM
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology* / Q000532:radiotherapy; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000008:administration & dosage / Q000494:pharmacology; D004583:Electrons; D005434:Flow Cytometry; D006801:Humans; D007205:Indium Radioisotopes / Q000737:chemistry*; D008727:Methotrexate / Q000008:administration & dosage / Q000494:pharmacology; D019913:Nuclear Localization Signals / Q000737:chemistry*; D017239:Paclitaxel / Q000008:administration & dosage / Q000494:pharmacology; D011838:Radiation-Sensitizing Agents / Q000008:administration & dosage / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D000068878:Trastuzumab
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D004317:Doxorubicin; D007205:Indium Radioisotopes; D008727:Methotrexate; D019913:Nuclear Localization Signals; D017239:Paclitaxel; D011838:Radiation-Sensitizing Agents; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C508053:ERBB2 protein, human