Vinorelbine

Docetaxel (S1148), paclitaxel (S1150), vinorelbine (S4269), epothilone B (S1364), daunorubicin (S3035), and cytarabine (S1648) were obtained from Selleckchem. All drugs were solubilized in DMSO at a concentration of 50 mM and stored frozen until use. Drugs were generally diluted 4 times in DMSO before further dilution in HBSS or media.

Cisplatin, carboplatin, docetaxel, vinorelbine, gemcitabine, gefitinib and erlotinib were obtained from SellekChem.

HS578T, BT549, MDA-MB-231 and HCC3153 breast cancer cell lines were provided by Drs J Losada and A Balmain (originally from Dr. JW Gray's Laboratory, who in turn obtained them from the ATCC or from collection development in the laboratories of Drs S Ethier and A Gazdar to avoid errors occurring when obtained through ‘second-hand’ sources). In addition cells were analyzed by STR at the molecular biology unit at the Salamanca University Hospital. Cell lines were maintained in DMEM (HS578T, BT549, MDA-MB-231) and RPMI (HCC3153) containing 10% fetal bovine serum (FBS), with 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine, respectively. The cell culture medium and supplements were obtained from Sigma Aldrich (St. Louis, MO). The multi-kinase inhibitor EC-70124 was prepared via a proprietary process by Entrechem S.L. (Oviedo, Spain). Chemotherapeutic agents (Docetaxel, Carboplatin and Vinorelbine) were purchased from Selleckchem.

Rabusertib, docetaxel, vinorelbine, gemcitabine, topotecan and olaparib were purchased from Selleckchem (Munich, Germany). Carboplatin and Cisplatin were purchased from Accord Healthcare (Middlesex, UK). Eribulin was from Eisai Inc. (Tokyo, Japan) and doxorubicin from Pfizer GEP, SL (NY, NY, USA).
Dose-response and drug combination studies were performed using MTT screening assays. Thus, cells were seeded into 48-multiwell plates (1 × 10⁴ cells per well), and 24 h later, they were treated either with single agents or a combination of them (IC50 or lower for the synergy studies). MTT (0.5 mg/mL) was added to the wells 72 h later and then they were incubated for 1 h at 37 °C. Then, the MTT was removed and DMSO was added to solubilize the formed crystals. Last, absorbance values were measured at 555 nm (555–690) in a multiwell plate reader (BMG Labtech, Ortenberg, Germany).
Results were plotted as the mean values of three independent experiments. Drugs interactions were calculated by determining combinational index (CI), based on the algorithm reported by Chou and Talalay, using Calcusyn Version 2.0 software (Biosoft, Ferguson, MO, USA). CI < 1 = synergistic effect; CI = 1 = additive effect; CI >1 = antagonistic effect.