Topotecan hydrochloride hydrate

The human malignant rhabdoid tumor cell line JMU-RTK-2 was obtained from the JCRB cell bank and cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum. The rhabdomyosarcoma cell line RMS-YM was obtained from RIKEN BRC cell bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 20 mM-HEPES, and 0.1 mM None-essential amino acids (FUJIFILM Wako Pure Chemical Corporation). All the cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Topoisomerase inhibitors, etoposide, doxorubicin, and epirubicin were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), ellipticine was purchased from Merck (DS, Darmstadt, Germany), topotecan hydrochloride hydrate was purchased from Sigma Aldrich (St Louis, MO, USA), and siremadlin was purchased from Medchemexpress (Monmouth Junction, NJ, USA).

Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 0.25% Trypsin-EDTA, Hanks’s balanced salt solution (HBSS), and penicillin/streptomycin solution were purchased from GIBCO (Invitrogen, NY, USA). Trypan blue, Triton X-100, dimethyl sulfoxide (DMSO), and topotecan hydrochloride hydrate were purchased from Sigma-Aldrich (Madrid, Spain). The 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Phalloidin was purchased from VWR. CytoTox 96^® Non-Radioactive Cytotoxicity Assay was purchased from Promega Corporation (WI, USA). PrestoBlue™ Cell Viability reagent and Qubit™ 1X dsDNA HS Assay Kit for DNA quantification was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Caspase 3, Caspase 8, and Caspase 9 Multiplex Activity Assay Kit (Fluorometric) was purchased from Abcam (Cambridge, UK).

Cassettes, reagent kits (Prod No. PE-FSPG-047-R), the precursor (Prod No. 3193.0075), elution solution (Prod No. PE-FSPG-047-R-V1), and reference standard (PE-FSPG-047-H) were purchased from ABX (Radeberg, Germany). N-Desmethyl topotecan was purchased from Synfine Research (Toronto, Canada). Topotecan hydrochloride hydrate (>98%) standard and N,N-dimethylformamide (DMF, anhydrous 99.8%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Sodium hydroxide National Institute of Standards and Technology (NIST) traceable solution (0.5 N) was purchased from Aqua Solutions, Inc. (Deer Park, TX, USA) and used without further purification. Solid-phase extraction cartridges were purchased from Waters (Milford, MA, USA). Ultra-high purity N.O.S. gas (99% nitrogen/1% oxygen) was purchased from Airgas (White Plains, NY, USA). All other reagents not listed above were of the highest grade available from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Scientific (Pittsburgh, PA, USA). End of synthesis radioactivity was determined using a Biodex AtomLab 500 dose calibrator (Shirley, NY, USA).

Dulbecco’s modified eagle medium (DMEM), calcium chloride dehydrate (CaCl₂·2H₂O), sodium bicarbonate (NaHCO₃), dimethyl sulphoxide (DMSO), thiazolyl blue tetrazolium bromide (MTT), and ethylene diamine tetraacetic acid (EDTA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMEM powder, fetal bovine serum (FBS), trypsin-ethylenediamine tetraacetate (trypsin-EDTA), and penicillin-streptomycin were obtained from Gibco BRL (CA, USA). Anticancer drugs including paclitaxel (Pac), docetaxel (Doc), mitomycin C from streptomyces caespitosus (Mito) and topotecan hydrochloride hydrate (Topo) were purchased from Sigma. Topotecan is not approved for breast cancer and is used as a control drug in this study to observe cytotoxic effects of variously indicated medications against breast cancer cells. Acetonitrile (ACN) and triethylamine (TEA) were from Fisher Scientific (Loughborough, UK). All the chemicals used for HPLC were HPLC grade. MCF-7 and 4T1 cells were originally from ATCC.

Three L. infantum WT independent clones, which were obtained by plating the MHOM/MA/67/ITMAP-263 strain onto solid M199, were independently selected in 25 cm² flasks containing 5 ml M199 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 5 μg/ml hemin in the presence of increasing TPT concentrations, as described previously [23 (link)]. Briefly, the stepwise drug selection ranged from 1 × the EC₅₀ of TPT (24 μM as determined in the present study) up to 16 × the EC₅₀ of TPT (384 μM), with a twofold increase in drug concentration (24, 48, 96, 192, and 384 μM) every three sub-culturing passages. Topotecan hydrochloride hydrate (Sigma-Aldrich, St. Louis, MO, USA) was used as the source of TPT.