Carboplatin

The animal studies were approved by the University of Oklahoma Animal Facility under the guidance of IACUC and were performed as described previously [32 (link)]. Briefly, female athymic nude mice (NCr-nu; 6–8 weeks old, ENVIGO Laboratories) were subcutaneously injected with CS-99 cells (1×10⁶/100 μL in Opti-MEM). Once the tumor volume reached approximately 100 mm³, mice were randomized to four different groups receiving vehicle, GLT (Eli-Lilly and company) or standard of care drugs carboplatin (Hospira, Inc.) and paclitaxel (Actavis Pharma, Inc.; CT) or GLT+ carboplatin and paclitaxel. GLT was administered orally at 75 mg/kg body weight twice daily for 14 days. carboplatin and paclitaxel were given once weekly by intraperitoneal injection at 50 and 15 mg/kg body weight, respectively [32 (link)]. After two cycles of treatment, mice were followed for tumor growth and euthanized according to IACUC limits (~1500 mm³). Tumor doubling time (DT) was calculated according to Mehrara and colleagues using the equation DT = LN (2)/SGR, and SGR (specific growth rate) = ln(V₂/V₁)/(t₂−t₁) [35 (link)]. In this experiment, we have utilized 7 mice in the vehicle-treated group, 9 mice in GLT group, 7 mice in C + T group and 9 mice in GLT+CT group, respectively.

MDA-MB-468 cells (ATCC HTB-132) were cultured in DMEM (Gibco), with 10% fetal bovine serum (FBS; Thermofisher), 100 µg/mL PenStrep (Gibco) and 2 mM L-glutamine (Gibco). Cells were tested with a mycoplasma testing kit (Lonza MycoAlert, LT07-318).
To induce resistance in MDA-MB-468 cells, the cells were exposed continuously to carboplatin (Hospira UK, Ltd), except for 48 h after splitting. The starting concentration of carboplatin was 0.4 µM and it was increased incrementally (nine times) until the cells grew comfortably at 2 µM carboplatin.
Lentiviral infections were performed as described by the RNAi Consortium (TRC). Infected cells were selected by puromycin selection (2 µg/mL; Thermofisher A11138-03).

The NSCLC cell lines A549, H460, H1703, and H358 were obtained directly from Dr. John Minna's laboratory (Dallas, TX) and maintained at 37°C and 5% CO₂ in RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Gibco) and 10% fetal bovine serum (Gibco). Human Bronchial Epithelial Cells (Cell Applications) were maintained in Bronchial/Tracheal Epithelial Growth Medium (Cell Applications) at 37°C and 5% CO₂. HBEpC cells beyond passage 3 were not used for experiments. PAPSS1 (1:1000) primary antibody was obtained from Abcam while β-Actin (1:50000), cleaved caspase-3 (1:1000), Cyclin A2 (1:2000), Cyclin E1 (1:1000), and cleaved PARP (1:1000) primary antibodies were purchased from Cell Signaling Technology. The chemotherapeutics cisplatin, carboplatin, irinotecan, topotecan, and docetaxel were obtained as ready-to-inject solutions from Hospira. Epirubicin and doxorubicin were obtained from Pfizer while oxaliplatin and mitomycin C were purchased from Sanofi Aventis and Novopharm respectively.

APR-246 (2-hydroxymethyl-2-methoxymethyl-1-azabicyclo [2,2,2] octan-3-one) and MQ (2-methylenequinuclidin-3-one) were from Aprea (Solna, Sweden). Cisplatin (Ebewe or Hospira), carboplatin (Hospira), docetaxel (Actavis) and doxorubicin (Teva) were purchased from the Pharmacy at Akademiska sjukhuset, Uppsala, Sweden. Cisplatin was also purchased from Sigma (St. Louis, MO, USA). Gemcitabine was from LC Laboratories (Woburn, MA, USA), and glutathione from Sigma-Aldrich (Steinheim, Germany).

Carboplatin was purchased from Hospira (Montreal, Canada) and Hande Tech Development Co (Houston, USA). 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol), 3ß-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2000 PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DSPG) were obtained from Avanti Polar Lipids Inc (Alabaster, USA).