Recombinant human soluble trail

HCC cell lines (SNU-353, SNU-423, SNU-449, and SNU-475) derived from HCC tissues of patients (26 (link)) were purchased from the Korean Cell Line Bank. Cells were cultured in RPMI medium (Invitrogen, Carlsbad, CA, USA) containing 10% (v/v) fetal bovine serum (Gibco BRL, Life Technologies, Carlsbad, CA, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in an incubator (5% CO₂ and 95% O₂ at 37°C) as adherent monolayers. The recombinant human soluble TRAIL was obtained from R&D Systems (Minneapolis, MN, USA). Doxorubicin (DOX) and vinblastine (VBL) were purchased from Sigma-Aldrich.

Hepatocellular carcinoma (HCC) cell lines (SNU-354, SNU-423, SNU-449 and SNU-475) derived from HCC tissues of patients were purchased from the Korea Cell Line Bank (17 (link)). TRAIL-resistant SNU-475/TR subline was isolated from SNU-475 cells by stepwise increases in concentrations of TRAIL, starting with 10 ng/ml and reaching 200 ng/ml. After approximately 2 month of drug exposure, SNU-475/TR cells were able to grow in the presence of 200 ng/ml TRAIL, and this phenotype remained stable through passages. SNU-475/TR cells were at least 100-fold more resistant than SNU-475 cells to TRAIL. Cells were maintained in RPMI-medium (Welgene) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Welgene), 100 U/ml penicillin and 100 µg/ml streptomycin in a 5% CO₂ humidified incubator at 37°C. Recombinant human soluble TRAIL was obtained from R&D systems. Celecoxib (CCB), 2,5-dimethyl celecoxib (DMC), cycloheximide (CHX), 3-methyladenine (3-MA), chloroquine (CQ), LY294002 (LY), and 4-phenylbutyric acid (4-PBA) were purchased from Sigma-Aldrich; Merck KGaA.