Hepg2

Five HCC cell lines were used in subsequent experimental investigations. SK-Hep-1 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). Huh-7 cells were a gift from Dr. Hui-Ling Chiou (Chung Shan Medical University, Taichung, Taiwan). HepG2, PLC/PRF/5, and HA22T/VGH cells were purchased from the Bioresource Collection and Research Center and the Food Industry Research and Development Institute (Hsinchu, Taiwan). The SK-Hep-1, HuH-7, and HA22T/VGH cells were cultured in Dulbecco's modified Eagle's medium (DMEM), whereas the PLC/PRF/5 and HepG2 cells were cultured in minimum essential medium. The PLC/PRF/5 and HepG2 cells were supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), 100 μg/mL NEAA, and 100 mg/mL penicillin-streptomycin (Sigma Chemicals, St. Louis, MO, USA). These cell lines were maintained in a humidified atmosphere containing 5% CO2 at 37 °C.

The starting materials and other reagents for synthesis were purchased from Aldrich Company and Tokyo Chemical Industry and used as received without further purification unless otherwise indicated. The melting points were measured using an SMP3 StuartTM digital melting point apparatus from Bibby Sterlin, Ltd (Stone, United Kingdom). All synthesized compounds were analyzed using nuclear magnetic resonance (NMR) and mass spectrometry to confirm their structures. Proton and carbon NMR spectra were accomplished using the Bruker Avance III HD 300 spectrometer at 75, 100, 300, and 400 MHz. High-resolution mass spectra were measured with a micrOTOF electrospray ionization time-of-flight mass spectrometer (Bruker Daltonics, Germany). HepG2 (hepatocarcinoma), MOLT-3 (acute lymphoblastic leukemia), HuCCA-1 (cholangiocarcinoma), A549 (lung carcinoma), MRC-5 (normal embryonic lung cell), MDA-MB-231 (hormone-independent breast cancer), S102 (Thai liver cancer), HeLA (cervical carcinoma), T47-D (hormone-dependent breast cancer), H69AR (lung cancer, multidrug resistant), and HL-60 cell lines (promyeloblast) were purchased from Hyclone Laboratories. Doxorubicin (purity ≥98%) and etoposide (purity 98%) were purchased from Aldrich Company.

The human hepatoma cell line HepG2 (American Type Culture Collection [ATCC], Manassas, VA, USA) was cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM; SH30021.01, HyClone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin in a 5% CO₂ incubator at 37 °C. Cells were incubated in a humidified atmosphere at 37 °C and 5% CO₂ and cultured for 3 days to achieve 70% confluence before treatment with palmitate or DPP4i (teneligliptin). HepG2 cells were treated with palmitate (Sigma-Aldrich, 0.6 mM) for 18 h with or without pre-treatment with DPP4i (teneligliptin, 3 μM) for 6 h and were then analyzed. The mRNA and protein expression levels of DPP4, ER stress, and TRAIL-R2-mediated apoptosis markers were assessed. The mRNA levels of collagen1α1 and αSMA, the typical mesenchymal cell markers corresponding to the activated hepatocyte state that involves the deposition of extracellular matrix during liver fibrosis^{66 (link)}, were also analyzed. To analyze the effect of DPP4i on TRAIL-mediated apoptosis, HepG2 cells were incubated with the recombinant human TRAIL/TNFSF10 protein (TRAIL; Cat# 375-TL-010, R&D Systems) at concentrations of 50 and 100 ng/mL for 18 h, with or without pre-treatment with DPP4i (teneligliptin, 3 μM) for 6 h.