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. 2011 Sep;13(9):1000-10.
doi: 10.1093/neuonc/nor069. Epub 2011 Jul 9.

The microtubule stabilizer patupilone (epothilone B) is a potent radiosensitizer in medulloblastoma cells

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The microtubule stabilizer patupilone (epothilone B) is a potent radiosensitizer in medulloblastoma cells

Christoph Oehler et al. Neuro Oncol. 2011 Sep.

Abstract

Concurrent radiochemotherapy for medulloblastoma includes the microtubule disrupting agent vincristine; however, vincristine alone or as part of a combined treatment regimen is highly toxic. A major goal is therefore to replace vincristine with novel potent chemotherapeutic agents-in particular, with microtubule stabilizing and destabilizing compounds-with a larger therapeutic window. Here, we investigated the antiproliferative, cytotoxic and radiosensitizing effect of patupilone (epothilone B [EPO906]), a novel, non-taxane-related and nonneurotoxic microtubule-stabilizing agent in human medulloblastoma cell lines. The antiproliferative and cytotoxic effects of patupilone alone and in combination with ionizing radiation was determined in the 3 representative human medulloblastoma cell lines D341Med, D425Med, and DAOY. Patupilone alone effectively reduced the proliferative activity and clonogenicity of all medulloblastoma cell lines tested at picomolar concentrations (50-200 pM) and resulted in an at least additive anticlonogenic effect in combination with clinically relevant doses of ionizing radiation (2 or 5 Gy). Cell-cycle analysis revealed a sequential G2-M arrest and sub-G1 accumulation in a dose- and treatment-dependent manner after exposure to patupilone. In tumor xenografts derived from D425Med cells, a minimal treatment regimen with patupilone and fractionated irradiation (1 × 2 mg/kg plus 3 × 3 Gy) resulted in an extended tumor growth delay for the 2 single treatment modalities alone and a supra-additive treatment response for the combined treatment modality, with complete tumor regressions. These results demonstrate the potent efficacy of patupilone against medulloblastoma cell lines and indicate that patupilone represents a promising candidate to replace vincristine as part of a combined treatment strategy with ionizing radiation.

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Figures

Fig. 1.
Fig. 1.
Antiproliferative and cytotoxic effects of patupilone in human medulloblastoma cell lines. A–C, D341Med (A), D425Med (B), and DAOY (C) human medulloblastoma cells were treated with increasing doses of patupilone, and the antiproliferative activity was determined after 72 h of exposure. D–F, D341Med (D), D425Med (E), and DAOY (F) human medulloblastoma cells were treated with increasing doses of patupilone, and cell viability was determined by trypan blue exclusion after 72 h of exposure. (G) Clonogenicity of the D341Med cells after treatment with increasing doses of patupilone. (H) Clonogenicity of the D425Med and DAOY cells after treatment with increasing doses of patupilone. Results of a representative experiment are shown (n = 3).
Fig. 2.
Fig. 2.
Cell-cycle analysis after treatment with patupilone. Human medulloblastoma cell lines D341Med (top row), D425Med (middle row), and DAOY (bottom row) were treated with patupilone at low concentrations (D425Med and DAOY, 0.1 nM; D341Med, 0.25 nM) or high concentrations (D425Med, 0.5 nM; D341Med and DAOY, 1 nM) and subjected to cell-cycle determination by propidium iodide staining and FACS analysis at the indicated time points.
Fig. 3.
Fig. 3.
Caspase-3 activity and formation of acidic vesicular organelles (AVOs) after treatment with patupilone. Caspase-3 activity in patupilone-treated medulloblastoma cells (D341Med [A], D425Med [B], and DAOY [C]) was determined at the indicated time points. (D–F) AVO formation was determined in patupilone-treated medulloblastoma cells ([D] D341Med, 1 nM; [E] D425Med, 0.5 nM; [F] DAOY, 1 nM). Green (510–530 nm) and red (465 nm) fluorescence emission from 5 × 105 cells illuminated with blue (488 nm) excitation light was measured with a FACSCalibur at the indicated time points.
Fig. 4.
Fig. 4.
Antiproliferative and cytotoxic effect of patupilone in combination with ionizing radiation. (A–C) Human medulloblastoma cell lines D341Med (A), D425Med (B), and DAOY (C) were treated with patupilone alone or in combination with clinically relevant doses of ionizing radiation (2–5 Gy). Patupilone was added 30–60 min before irradiation. The antiproliferative activity was determined after 72 h of exposure. (D–F) Human medulloblastoma cell lines D341Med (D), D425Med (E) and DAOY (F) were treated with patupilone at low (D425Med and DAOY, 0.1 nM; D341Med, 0.25 nM) or high (D425Med, 0.5 nM; D341Med and DAOY, 1 nM) concentrations, alone or in combination with ionizing radiation (5 Gy), and cell viability was determined by trypan blue exclusion after 72 h of exposure. (G–H) Clonogenicity of human medulloblastoma cells after treatment with increasing doses of patupilone or in combination with ionizing radiation.
Fig. 5.
Fig. 5.
Apoptosis and autophagy after combined treatment with patupilone and ionizing radiation. (A–C) Human medulloblastoma cell lines D341Med (A), D425Med (B), and DAOY (C) were treated with patupilone at low (D425Med and DAOY, 0.1 nM; D341Med, 0.25 nM) or high (D425, 0.5 nM; D341Med and DAOY, 1 nM) concentrations, alone or in combination with ionizing radiation. Caspase-3 activity was determined after 48 h. (D–F) Human medulloblastoma cell lines D341Med (D), D425Med (E), and DAOY (F) were treated with patupilone at low (D425Med and DAOY, 0.1 nM; D341Med, 0.25 nM) or high (D425Med, 0.5 nM; D341Med and DAOY, 1 nM) concentrations, alone or in combination with ionizing radiation. Green (510–530 nm) and red (465 nm) fluorescence emission from 5 × 105 cells illuminated with blue (488 nm) excitation light was measured with a FACSCalibur at the indicated time points or after 48 h.
Fig. 6.
Fig. 6.
The effect of patupilone and ionizing radiation alone or in combination on the growth of D425Med-derived xenografts in nude mice. Mice were treated with patupilone (2 mg/kg once) and ionizing radiation (3 × 3 Gy on consecutive days), alone and in combination, with administration of patupilone or the vehicle 24 h prior to the first fraction of ionizing radiation. The horizontal bar indicates days of treatment. Each curve represents the mean tumor volume per group ± standard error.

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