Regorafenib

The HCC cell lines SNU398, SNU387, Huh7, HepG2, and PLC/PRF/5 were obtained from ATCC. Cells were grown in Roswell Park Memorial Institute (RPMI) medium or Dulbecco’s Modified Eagle’s Medium (DMEM); supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin; and maintained at 37°C with a 5% CO2 atmosphere. Regorafenib (#CS-1205) and sorafenib (#CS-0164) were purchased from ChemScene. Lenvatinib (#19375) and cabozantinib (#18464) were purchased from Cayman Chemical. Trametinib (#S-2673) was purchased from Selleck Chemicals, and buparlisib (#HY-70063) was from MedChem Express. Recombinant human IFNγ (#300-02) was obtained from PeproTech. Cell viability was quantified with Cell Count Reagent SF (nacalai tesque). Absorbance at 450 nm was measured on a micro- plate reader. For quantification of cytotoxicity, we used LDH Cytotoxicity Assay Kit (nacalai tesque). The absorbance value at 490 nm was measured.

The CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Tokyo, Japan), which includes a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was used according to the manufacturer’s instructions. Cells in culture medium were incubated with one of the following anticancer drugs, 10 mM 5-fluorouracil (5-FU, Sigma-Aldrich), 250 nM docetaxel (Sigma-Aldrich), 2 μM doxorubicin (Sigma-Aldrich), 200 μM irinotecan hydrochloride (Sigma-Aldrich), 2.5 μM suberoylanilide hydroxamic acid (SAHA, Cosmo Bio), 75 μM sorafenib tosylate (ChemScene, Monmouth Junction, NJ), 25 μM lenvatinib mesylate (Carbosynth, Berkshire, UK), 50 μM regorafenib (ChemScene), or 20 μM cabozantinib S-malate (ChemScene), 24 h at 37 °C in an atmosphere of 5% CO₂ in air. Cells incubated in culture medium alone were used as control. The optical density of the culture medium at 492 and 650 nm was measured by using an EnVision plate reader (PerkinElmer). The viability of cells treated with the anticancer drugs was calculated as a ratio with respect to their viability in the absence of anticancer drugs. Triplicate wells were analyzed for each assay.

Regorafenib (ChemScene LLC, Monmouth Junction, NJ, USA) was purchased and prepared as a 10-mM stock solution in dimethyl sulfoxide (DMSO).