Computational protocol: Evaluation of Activity and Combination Strategies with the Microtubule Targeting Drug Sagopilone in Breast Cancer Cell Lines

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Protocol publication

[…] The effect of sagopilone and other anticancer agents on the proliferation of breast cancer cell lines was assessed using a cell proliferation assay based on crystal violet cell staining as described before (Lichtner et al., ). IC50 values were calculated from three independent experiments using the SigmaPlot software (SPSS, Friedrichsdorf, Germany). The survival index was calculated as ratio of cell number surviving a 72-h treatment with 100 nM sagopilone divided by initial cell number (Shi et al., ). The combined effect of sagopilone and ispinesib was measured in cell proliferation assays. Here, the combination index (CI) was calculated according to Chou ().To visualize the effects of sagopilone on mitotic and interphase microtubules, MDA-MB-231 cells were incubated with vehicle (ethanol), 3 or 40 nM sagopilone for 20 h, fixed with 4% paraformaldehyde, and stained with a monoclonal mouse anti-α-tubulin antibody (1:1000; Sigma-Aldrich, Munich, Germany), Alexa Fluor® 488 goat anti-mouse IgG secondary antibody (1:250; Invitrogen Inc., Carlsbad, CA, USA), and DRAQ5™ (Biostatus, Leicestershire, UK), according to standard protocols. Fixed and stained cells were analyzed using a Zeiss LSM 510 META microscope (Carl Zeiss AG, Jena, Germany) equipped with a Plan-Apochromat® 63×/1.4 (oil DIC) objective. Zeiss LSM software (version 3.0 SP3) was employed for confocal imaging. Alternatively, cells were stained with mouse anti-α-Tubulin–FITC (1:200; SIGMA-Aldrich) and Hoechst33342 (Invitrogen Inc., Carlsbad, CA, USA). Images were taken on the OPERA automated microscope (Evotec, Hamburg, Germany; 10× objective, binning_2) and analyzed using MetaXpress Software (Molecular Devices, Sunnyvale, CA, USA).Fluorescence-activated cell sorter analysis was performed to determine the distribution of cells in the cell cycle phases. Cells were incubated with sagopilone for 20 h, fixed with 70% ethanol, and stained with 50 μg/mL propidium iodide (PI; Sigma-Aldrich, Munich, Germany). Cellular DNA content was determined by flow cytometry using the BD FACSCalibur™ and statistically analyzed using CellQuest™ software (Becton, Dickinson and Company, San Jose, CA, USA).The activity of the apical caspases 3 and 7 was measured using a commercial chemiluminescence-based caspase 3/7-Kit (Promega, Madison, WI, USA). Cells were seeded at a density of 1000 cells/well in 96-well plates. At 24 h after cell seeding, sagopilone or vehicle were added. After 48 h of treatment, cells were lysed, and caspase 3/7 activity was measured according to manufacturer’s instructions.To investigate loss of mitochondrial membrane potential and cellular vitality, breast cancer cells were incubated continuously for 72 h with vehicle or the indicated drug concentration of sagopilone, trypsinized, stained with 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3); Invitrogen Inc., Carlsbad, CA, USA) and propidium iodide as before (Castedo et al., ), measured by flow cytometry using the BD FACSCalibur™ and statistically analyzed using CellQuest™ software (Becton, Dickinson and Company, San Jose, CA, USA).The amount of cellular senescence was determined based on the expression of senescence-associated β-galactosidase (Dimri et al., ) using the Senescence Cells Histochemical Stain Kit (Sigma-Aldrich, Munich, Germany). At 24 h after cell seeding, sagopilone was added for 6 days. Doxorubicin was used as positive control (Eom et al., ). Cells were fixed and treated with X-gal for staining of β-galactosidase according to manufacturer’s instructions. Stained and unstained cells were counted and the percentage of β-galactosidase-positive cells was calculated. […]

Pipeline specifications

Software tools SigmaPlot, MetaXpress
Diseases Breast Neoplasms, Heart Arrest, Neoplasms, Polyploidy
Chemicals Paclitaxel, Epothilones