Daunorubicin

HCT116 p53+/+ and HCT116 p53-/- cells (a gift from Dr. Bert Vogelstein, Howard Hughes Medical Institute, Baltimore, MD) were cultured in a 5% CO₂-containing atmosphere in DMEM (Wisent) supplemented with 10% fetal bovine serum (Sigma), 0.2U/mL penicillin G, and 100 mg/mL streptomycin (Invitrogen). Unless specified otherwise, cells stress treatment consisted of 250 nM daunorubicin (Sigma) for 8 h. All results presented with SEM were obtained from independent biological triplicates. Statistics were calculated using Student’s t-test.

Cells were seeded at a concentration of 3 × 10⁵/ml in Roswell Park Memorial Institute medium complemented with 0.1, 1, 10, 50, 100, or 250 μM of Ara-C (Sigma-Aldrich) or 0.01, 0.05, 0.1, 0.5, 1, or 10 μM of daunorubicin (Sigma-Aldrich). Viability was measured 24 h later using the MTS assay from Promega following the manufacturer’s protocol. IC₅₀ was calculated using the GraphPad PRISM software.

Abemaciclib was purchased from Selleckchem (Houston, TX, USA), palbociclib and ribociclib from MedChem Express (Monmouth Junction, NJ, USA). Daunorubicin, mitoxantrone, propidium iodide, RNAse A, NADP+, glucose-6-phosphate, and HPLC grade solvents were bought from Sigma-Aldrich (St. Louis, MO, USA). ABCB1 inhibitor LY335979 and Daunorubicinol were obtained from Toronto Research Chemicals (North York, ON, Canada), while ABCG2 inhibitor Ko143 was from Enzo Life Sciences (Farmingdale, NY, USA). Opti-MEM was acquired from Gibco BRL Life Technologies (Rockville, MD, USA). All other cell culture media and reagents were purchased from Sigma-Aldrich, Lonza (Walkersville, MD, USA), and PAA Laboratories (Pashing, Austria).

The panel of drugs used were Daunorubicin (30450, Sigma-Aldrich), Doxorubicin (D1515, Sigma-Aldrich), Epirubicin (E9406, Sigma-Aldrich), Mitoxantrone (M6545, Sigma-Aldrich) and Trastuzumab (HYP9907, MedChem Express). All drugs were dissolved in molecular biology grade water to a concentration of 10 mM per stock. DOX, DNR, EPI, and MTX stocks were stored at -80°C and working stocks used at 4°C for up to one week. TRZ was stored at a 1 mM concentration at 4°C for up to one month.

For the MDR assays, daunorubicin (DNR, Sigma-Aldrich, St. Louis, MO) and cyclosporine A (CsA, Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to make stock solutions of 350 μM and 1 mM, respectively. Working solutions of 35-μM DNR and 5-μM and 10-μM CsA were prepared by diluting stock solutions in Hanks’ Balanced Salt Solution (HBSS, Invitrogen Corp., Grand Island, NY). Working solutions of 1-μM Oregon Green-labeled paclitaxel (OG-PTX) and 10-μM CsA were prepared by diluting stock solutions in a similar fashion. Post-assay cell viability was tested using 0.4% trypan blue solution (ThermoFisher Scientific, Waltham, MA).
The MDA-MB-231 breast cancer cell lines were obtained from cryopreserved storage. The cells were maintained in Hyclone Dulbecco’s Modified Eagle Medium (DMEM)/High Glucose (GE Life Science, Marlborough, MA) with 10% fetal bovine serum (FBS, Life Technologies, Grand Island, NY) and 1% penicillin (Stemcell Technologies, Vancouver, BC) at 5% CO₂ and 37 °C. The doubling time for the cell lines was ~38 h; hence, growth medium was changed three times a week and cells were passaged every 72 h.