Cabozantinib

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.

LDL, HDL, and VLDL were obtained from Lee Biosolutions (Maryland Heights, MO, USA). Cabozantinib, sunitinib, axitinib, pazopanib, U18666A, and posaconazole were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Antibody against AR was obtained from Cell Signaling Technology (Danvers, MA, USA).

Vandetanib (VAN), and cabozantinib (CAB) were provided by Cayman Chemicals (Ann Arbor, MI, USA). Stock solutions (4 mM) were made in 100% dimethyl sulfoxide (DMSO) and diluted with culture media before use. Two human medullary thyroid carcinoma (MTC) cell lines, TT and MZ-CRC-1, were kindly provided by Prof. Lips (Utrecht, the Netherland). Cells were maintained at 37 °C in 5% CO₂ and cultured in T75 flasks filled with 10 mL of F-12K Kaighn’s modification medium (Gibco™ Thermo Fisher Scientific, Waltham, MA, USA). Media was supplemented with 10% heat-activated fetal bovine serum (FBS) (Invitrogen™ Thermo Fisher Scientific, Waltham, MA, USA) and 10⁵ U·L⁻¹ penicillin/streptomycin (EuroClone™, Milan, Italy). Cells were harvested by trypsinization (Trypsin 0.05% and EDTA 0.02%) (Sigma-Aldrich^® Merck KGaA, Darmstadt, Germany), resuspended in complete medium, then counted through an optical microscope using a standard haemocytometer before plating. Cells used in all experiments were below 5 passages. All in vitro experiments were monitored for up to 6 days of drug incubation. We performed long-term treatments due to the slow doubling time (about 4 days) of the MTC cell lines.