Topotecan

The cytotoxicity of the compounds to HeLa (human cervical cancer) and HEK293A (human embryonic kidney) cell lines was examined using the EZ4U Cell Proliferation and Cytotoxicity Assay (Biomedica, Vienna, Austria), according to the manufacturer’s protocols. The cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium) with 50 IU/mL penicillin, 50 μg/mL streptomycin (MP Biomedicals, Santa Ana, CA, USA), and 10% of fetal bovine serum (Biolot, St. Petersburg, Russia) in a 5% CO₂ atmosphere. After formation of a 30–50%-monolayer, the tested compounds were added to the medium. The volume of the added reagents was 1/100 of the total volume of the culture medium, and the amount of DMSO (Sigma, St. Louis, MO, USA) was 1% of the final volume. The cell culture was monitored for 3 days. To assess the influence of the inhibitors on the cytotoxic effect of topotecan (Selleck Chemicals, Houston, TX, USA), 50% cytotoxic concentrations of topotecan and of each inhibitor were determined to attain a defined single-agent effect. Then, a minimum of two independent tests were performed with each inhibitor in combination with topotecan. When using a combination of drugs, TDP1 inhibitors were first added, then topotecan was added immediately (within 10–15 min).

The cell culture media RPMI, supplements and all other chemicals were purchased from Sigma Aldrich SRL (Milan, Italy). Quantitative real-time PCR reagents were acquired from Applied Biosystems (Foster City, CA, USA). Topotecan and pazopanib were bought from Selleckchem (DBA Italia, Milan, Italy), and prepared in a stock solution of 10 mM in 100% dimethylsulfoxide (DMSO) for cell's experiments. Control's medium wells were treated with the same DMSO concentration used to formulate the medium of the wells with the highest concentration of pazopanib and Topotecan in the same experiment.

LC-T2A-GFP lines were seeded at 1200 cells per well of a 384-well plate, adhered overnight and treated with chemotherapeutics olaparib, rucaparib, paclitaxel, topotecan, carboplatin, cyclophosphamide, cisplatin and doxorubicin (Selleck) at empirically determined clinically relevant doses (1 μM). Cells were maintained at 37 °C with 5% CO₂ in treatment for 72 h. Four focal areas per replicate well were imaged using 4X magnification on bright field, RFP and GFP using the Cytation™ 3 imaging system (Biotek) maintained at 37 °C and 5% CO₂. Images were analysed as detailed below (Cytation™ 3 Multimode Imaging).