Cisplatin

Normal human lung bronchial epithelial cells (NHBE) and NSCLC cell lines (A549 and H1299) were purchased from the ATCC (Manassas, USA). The NHBE, A549 and H1299 cells were cultured in DMEM (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, USA), 100 mg/ml streptomycin and 100 U/ml penicillin in a humidified air at 37 °C with 5% CO₂. To establish cisplatin-resistant A549 (A549/DDP) and H1299 cells (H1299/DDP), A549 and H1299 cells were first treated with 0.1 mg/ml cisplatin (Tocris Bioscience, Minneapolis, USA) and then gradually exposed to cisplatin with step-wise increased concentrations till 1 mg/ml. The established A549/DDP and H1299/DDP cells were cultured in full medium containing 1 mg/ml until further use.

GC cell lines were exposed to different concentrations of cisplatin (0–40 µM) (Accord Healthcare Limited, Harrow, United Kingdom) for 48 h to assess their sensitivity. Similarly, to evaluate the effect of TRPV2 blockade by tranilast (Tocris Bioscience, Bristol, United Kingdom) on cisplatin resistance of KATO-III cells, cisplatin (10 µM) was added to cell culture after 10 min since the addition of TRPV2 inhibitor (250 µM). After 48 h of culture, KATO-III cells were recovered by trypsinization and subjected to flow cytometry. In brief, cells were washed twice with cold PBS and then, after centrifugation, resuspended in 100 µL of 1x binding buffer at a concentration of 1 × 10⁶ cells/ml. Subsequently, 5 µL of FITC Annexin V and 5 µL propidium iodide (PI) (BD Biosciences, San Jose, CA, United States) were added, and cells were incubated for 15 min at room temperature (RT) in the dark. For each tube, an adequate volume of 1x binding buffer was added. All samples were acquired, within 1 h, by using a NAVIOS flow cytometer and analyzed by Kaluza software (Beckman Coulter Diagnostics, Brea, CA, United States). A total of 10⁴ events were acquired for each sample. Data from treated samples were normalized as fold change of their untreated controls and were reported as mean ± SE of at least three independent experiments.

Eight-week-old male CBA/J mice purchased from the Jackson Laboratory were divided into two groups after passing a hearing test. In the control group, CBA/J mice were administered normal saline daily whereas the mice in the cisplatin treatment group were administered cisplatin (Tocris, 2251) diluted with normal saline (0.9% NaCl) by intraperitoneal injection at a dosage of 10 mg/kg. To prevent dehydration after cisplatin injection, we administered 1 mL of warm (37°C) normal saline by subcutaneous injection until the body weight recovered to that before cisplatin injection, and the cisplatin-treated mice were placed on the heating pad at all times. Auditory brainstem response (ABR) threshold shifts were measured after the recovery period of the second cycle.