Carboplatin

Cells were seeded into 96-well plates at a density of 5000 cells/well. After 24 h, the original medium was replaced with complete medium containing different concentrations of the following drugs: cisplatin (#AG-CR1-3590-M050, Funakoshi Frontiers In Life Science, Tokyo, Japan) at 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 μM; carboplatin (#033-25231, Fujifilm Wako Pure Chemical Corporation) at 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, and 0.98 μM; olaparib (#CS-0075, Funakoshi Frontiers In Life Science) at 200, 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 μM; docetaxel (#047-31281, Fujifilm Wako Pure Chemical Corporation) at 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02, and 0.01 nM. After 48 or 72 h, cell viability was determined using the Cell Counting Kit-8 (#CK04, Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s protocol. The absorbance at 450 nm was measured using a SpectraMax i3x Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). The half-maximal inhibitory concentration (IC₅₀) of the drug was calculated by fitting the dose-response curves to a four-parameter, variable slope sigmoid dose-response model using the ImageJ software (version 1.53f, NIH, Bethesda, MD, USA).

Two human invasive bladder cancer cell lines, T24 and T24PR, were used. T24 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). T24PR cells were established in our laboratory as an acquired platinum resistant cell line [11 (link)]. Briefly, T24 cells were grown and passaged upon reaching confluence in medium containing CDDP over a 6-month period to develop platinum resistance. All cells were routinely maintained in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Dainippon Pharmaceutical, Tokyo, Japan), at 37°C in a humidified 5% CO₂ atmosphere. DHMEQ, synthesized as described previously [10 (link),12 (link)], was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml and stored at −20°C. This stock solution was diluted in culture medium to a final concentration of <0.1%. CDDP and paclitaxel were kindly supplied by Nippon Kayaku Co. (Tokyo, Japan). Gemcitabine and carboplatin were obtained from Wako Pure Chemical Industries (Osaka, Japan).

OSCC cells were treated with 1 μm cisplatin (Wako Pure Chemical), carboplatin (Wako Pure Chemical), or nedaplatin (Wako Pure Chemical). Resistance to anticancer drugs was monitored using a MarkerGene Multiple Drug Resistance Microtiterplate Assay Kit (Marker Gene Technologies, Eugene, OR, USA) and was measured using a SpectraMax M2 multidetection microplate reader (Molecular Devices, Sunnyvale, CA, USA) at an emission wavelength of 504 nm and an excitation wavelength of 538 nm.

The drugs (1 wt%) were added to the solvents and stirred over 10 h at room temperature. When the drugs were not solubilised, the solutions were heated up to 80 °C and stirred for 2 h. We checked whether the drugs were solubilised by observing the particles of the drugs using an invert microscope (ECLIPSE Ts2, Nikon Corporation). Erythromycin, 3′-azido-3′-deoxythymidine, 5-iodo-2′-deoxyuridine, (+)-catechin hydrate, l-adrenaline, desloratadine, zoledronic acid monohydrate, testosterone, estriol, and oxaliplatin were purchased from Tokyo Chemical Industry Co., Ltd and used as received. Fluorouracil, carboplatin and cisplatin were purchased from Fujifilm Wako Pure Chemical Corporation and used as received. Adenosine 3′-phosphate and insulin were purchased from Nacalai Tesque Inc. and used as received. l-Thyroxine was purchased from Sigma-Aldrich Co., Llc. and used as received. Paclitaxel was purchased from Funakoshi Co., Ltd and used as received.