Cisplatin

LUAD cell lines (A549, H1975, and CALU-3) were cultured in RPMI 1640 medium (Gibco, Waltham, MA, USA), and human bronchial epithelioid cell line (HBE) and HEK293T cell lines were cultured in Dulbecco’s modified eagle medium (Gibco). The cell culture medium was supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (New Cell & Molecular Biotech, Suzhou, China). All cells were obtained from the cell bank of the Cancer Research Institute of Central South University (Changsha, China) [21 (link),22 (link)] and incubated at 37 °C with 5% CO₂. The cisplatin-resistant A549 cell line (A549/CDDP) was derived from A549 cells using stepwise increased cisplatin (Qilu Pharmaceutical, Shandong, China) concentration incubation. All cells were passaged to the third generation to start the experiments.

Human HNSCC lines, CAL27, and Detroit‐562 (purchased from the American Type Culture Collection, ATCC) were used in the study and were cultured under standard condition as described.4 The 2 primary cisplatin sensitive HNSCC cell lines, oral tongue squamous cell carcinoma, CAL27, and pharyngeal squamous carcinoma, Detroit‐562 were cultured to acquire cisplatin resistance. Cells were treated with gradually increasing concentrations of cisplatin (Qilu Pharmaceutical Co., Ltd, Jinan, China) ranging from 3 to 10 μmol/L at weekly intervals for 8 months to establish cisplatin‐resistant CAL27 (CAL27cis) and Detroit‐562 (Detroit‐562cis) cell lines. The establishment of cisplatin‐resistant cells was selected by exposure to sequential cycles of cisplatin for 8 months, which mimics the way the drug is used in the clinic.4, 16 The resistance was unaltered after culturing the cisplatin‐resistant cells in the drug‐free medium for 2 months. CAL27, Detroit‐562, CAL27cis, and Detroit‐562cis were inoculated with LfcinB for test of drug resistance and the expression of PD‐L1 and IL‐6. LfcinB (FKCRRWQWRMKKLGAPSITCVRRAF), 25‐AA peptide fragment, was synthesized by GL Biochem (Shanghai, China) via a stepwise solid phase methodology. The peptide was purified by a Sephadex gel column and HPLC, and the homogeneity of the purified peptide was greater than 98%.

Patients were intravenously dripped with 25 mg/m² cisplatin (specification: 20 mg; Qilu Pharmaceutical Co., Ltd., China; batch No. 20121216] added in 500 mL of normal saline within 3 days before chemotherapy, and intravenously dripped with 35 mg/m² docetaxel (specification: 10 mg; Hainan Sinochem Joint Pharmaceutical Industry Co., Ltd., China; H20057065)/100 mg/m2 gemcitabine (specification: 2,000 mg; Jiangsu Haosen Pharmaceutical Group Co., Ltd., China; H20030104), 1 h each time, on the 1st, 8th and 15th days. Slow intravenous bolus injection of 3 mg of granisetron (Sinopharm Group Guorui Pharmaceutical Co., Ltd., China; H20041206) was performed prior to each initiation of chemotherapy to prevent vomiting. Twenty-one days of chemotherapy were used as a cycle, and 4 cycles of chemotherapy were considered as a course of treatment.

Human lung cancer cell line A549 was obtained from BeNa Culture Collection (BNCC; Beijing, China). The cell line was confirmed by short tandem repeat profiling and tested for mycoplasma contamination. Paclitaxel (PTX) was purchased from Beijing Pharmaceutical (Beijing, China) and cisplatin (DDP) was procured from Qilu Pharmaceutical (Jinan, Shandong, China). Cells were cultured in RPMI 1640 (Gibco, Gaithersburg, MD) containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin, subcultured every 3–4 days, and incubated at 37°C in a humidified environment. A549 cells were continuously cultured with gradient concentration of PTX and DDP for more than 12 months until the cells showed the drug resistance against 200 μg/mL of PTX or 1000 μg/mL of DDP. Thereafter, PTX‐resistant A549 (A549/PTX) cell line and DDP‐resistant A549 (A549/DDP) cell line were screened out and prepared for subsequent experiments.

Cisplatin was purchased from QiLu Pharmaceutical (Jinan, China). MiR-182 inhibitor, PDCD4 siRNA, and their negative control oligonucleotides were obtained from Shanghai GeneChem Co., Ltd (Shanghai, China). These were used to transfect A549 cells using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) according to the instructions provided by the manufacturer. Monoclonal rabbit anti-human PDCD4 antibody (Cell Signaling Technology, Inc., Beverly, MA, USA) was used for Western blot analysis.