Cisplatin

Both B16 melanoma cell lines were seeded (5 × 10⁴ cells per well) in 12-well plates and imaged (from at least 10 different fields) after 24 and 48 h to examine differences in cell proliferation between the treatment and control groups. Additionally, B16 melanoma cells were seeded in 96-well plates (5 × 10³ cells per well), incubated for 24 h, and treated as indicated. Viability of co-transduced (DK + m-sgRNA-1) cells and untreated control cells was determined with a CellTiter-Glo Luminescent Cell Viability Assay kit (G7570, Promega, Madison, WI, USA) after 24 and 48 h in the presence or absence of two chemotherapeutic agents, cisplatin (13119, Cayman Chemicals, Ann Arbor, MI, USA) and tunicamycin (11445, Cayman Chemicals, Ann Arbor, MI, USA). Drugs were serially diluted from the stock solution (2 mM) and added to cell cultures in the range of 5 µM to 10 nM. At the end of 24 and 48 h of incubation, equal volumes (100 µL each) of assay solution and cell suspension were mixed and incubated at room temperature for 10 min. Relative luminescence was recorded with a spectrometer (Agilent BioTek, Winooski, VT, USA Gen5 Microplate reader), and the half-maximal inhibitory concentration (IC₅₀) value for each drug was plotted with GraphPad Prism, v7.0, Boston, MA, USA.

Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee of the Daegu Catholic University Medical Center Approval number: DCIAFCR-200626-13-Y, approval date: 26 June 2020). Seven-week-old male C57BL/6N mice were acquired from HyoSung Science Inc. (Daegu, Korea) and kept at 20–24 °C and 55% humidity for 1 week. The mice were divided into three groups (n = 8 per group): control (Con), cisplatin (CP), and cisplatin plus 6-shogaol (CP + 6-SHO). The CP group and the CP + 6-SHO group were given a single intraperitoneal injection of cisplatin (20 mg/kg; Sigma-Aldrich, St. Louis, MO, USA). The CP + 6-SHO group was also given an intraperitoneal injection of 6-shogaol [20 mg/kg; dissolved in dimethyl sulfoxide (DMSO); Cayman Chemical, Ann Arbor, MI] daily for 3 consecutive days, starting from 1 h after cisplatin injection. The Con group and the CP group received intraperitoneal injections of an equal volume of DMSO daily for 3 consecutive days. All mice were sacrificed 72 h after a single dose of cisplatin. The doses of cisplatin and 6-shogaol were selected based on the results of previous studies [25 (link),33 (link)].

4T1 and MCF7 cells were purchased from The American Type Culture Collection and cultured in Dulbecco's modified of Eagle's medium (DMEM) with 10% heat‐inactivated fetal calf serum, 2 mm l‐glutamine, and 100 μg·mL⁻¹ of both penicillin and streptomycin (Cellgro, Mediatech, Tewksbury, MA, USA) in a Heracell CO₂ incubator at 37 °C and 5% CO₂. For irradiation treatment, cells were plated in 100‐mm tissue culture plates overnight and irradiated at the required doses with a Cesium¹³⁷ source at a dose rate of 1.06 Gy·min⁻¹ or 2.29 Gy·min⁻¹. Cisplatin was purchased from Cayman Chemical Company (Ann Arbor, MI, USA) (L161,982), and ibuprofen, doxorubicin, and celecoxib were from Sigma, Franklin, MA, USA. MMT mammary tumor cells were from mice doubly transgenic for human mucin 1 Ag and polyomavirus middle T (PyMT) oncogene (Xia et al., 2003). MMT mice were a kind gift from Sandra J. Gendler, Mayo Clinic, Scottsdale, AZ. Such MMT mice carrying the PyMT oncogene driven by the mouse mammary tumor virus long terminal repeat developed mammary carcinoma at a 100% incidence (Guy et al., 1992).