Cell lines
HepG2 and Hep3B human hepatocellular carcinoma cell lines were purchased from Bioresource Collection and Resource Center (BCRC numbers RM60025 and 60434), Taiwan and were grown in DMEM medium supplemented with 10% FBS (Invitrogen), 100 μg/mL penicillin, and 100 U/mL streptomycin.
Animal
Mouse experiments were conducted with the ethics approval from Animal Care and Use Committee in Chang Gung Memorial Hospital, Keelung. Male nude mice CAnN.Cg-Foxn1nu/CrlNarl (4–6 weeks old, 20 g) were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All mice were sacrificed by using CO2 for 20 min. All animal procedures were conducted in Abnova Co., and were performed according to approved protocols and by recommendations for the proper use and care of laboratory animals.
Chemicals
RA190 and RA190B were synthesized in-house and purified to 99% as previously described [11]. Sorafenib was purchased from L.C. laboratories.
qRT-PCR
Total RNA was isolated from cells using the RNeasy mini kit (Qiagen). Extracted RNA was normalized for concentration and reverse transcribed using iScript cDNA synthesis kit (Bio-Rad). Taqman gene expression assays measured Bip-1, ATF-4, CHOP10, RPN13, iNOS, GAPDH expression levels utilizing Taqman gene expression master mix (Applied Biosystems) per the manufacturer’s instructions. Spliced XBP1 mRNA was assayed with SsoFast EvaGreen Supermix (Bio-rad) following the recommendations for the iCycler System. Forward and reverse primer was: 5'-TGCTGAGTCCGCAGCAGGTG and 5'-TGGGTCCAAGTTGTCCAG AATGCC. Calculations were done according to the Livak method and normalized to reference gene GAPDH. Each condition was replicated three times; each sample was run in triplicate. 2 μL cDNA sample was used for PCR amplification with iQ™ SYBR® Green Supermix (Bio-Rad Laboratories) according to the manufacturer's protocol.
Biotin labeling assay
HepG2 cells (5X106) were lysed using MPER (Pierce) lysis buffer (1 mL) according to the manufacturer protocol. Cell lysates were centrifuged at 10,000 rpm briefly (2 min) at 4 °C to remove cell debris. Lysate supernatant (100 µL) was pre-cleared with streptavidin dyna beads (20 µL) for one hour at 4 °C to remove non-specific biotin binding and incubated with compounds (indicated concentrations) at 4 °C for 1 hr. An equal amount of each sample (20 µL of lysate) was mixed with the same volume of Laemmli sample buffer (20 µL) (BioRad) and was boiled for 5 min. The proteins were separated using 4-15% Bio-Rad Mini-PROTEAN SDS-PAGE gel (1 hr at 100 V) and transferred to the membrane overnight at 4 °C (24 V). The membrane was blocked with 5% BSA in PBST (phosphate buffered saline containing 0.1% Tween 20) for 1 hr at room temperature and washed for 20 min (3X in PBST). Then the membrane was probed with HRP-streptavidin (1:10,000 in PBST) for 1 hr at room temperature and soaked for 30 min (3X in PBST) and developed using HyGLO chemiluminescent detection reagent (Denville) for biotin recognition.
Western blot analysis
50 μg/well of protein from the HepG2 cell lysate was separate using SDS-PAGE and transferred to a nitrocellulose membrane (G.E. Bioscience). After blocking with 5% skim milk in PBST for 1 hr at room temperature, membranes were incubated overnight with primary antibody at 4°C. Layers were then washed with PBST and incubated with horseradish peroxidase (HRP)–conjugated secondary antibody before visualization with ECL plus (G.E. Bioscience). All antibodies, including NF-κB (Cell signaling D14E12), IκBα (Cell signaling L35A5), poly-Ub (Enzo FK2), P21 (Cell Signaling 12D1), Actin (Abcam EPR16769), lamin A (Abcam EPR4068), and tubulin (Abcam EPR13796) were diluted in 5% BSA buffer. The dilution ratio of the antibody followed the manufacturer's recommendation.
Clonogenicity Assay
The clonogenicity assay was performed as previously described[16]. HepG2 cells (500 cells/well) were plated in a 6-well plate at day 1. Cells were treated on day 2 with RA190 at the indicated concentrations. After 14-days incubation, colonies were stained with crystal violet (0.5% w/v) and imaged.
Immunofluorescence stain
1.5 x104 cells of HEpG2 per well were seeded in the 8-chamber slide in 500 µL of culture medium, incubated at 37°C under humidified 95% air/5% CO2 for 18 hours. After treatment for 30 minutes, the slides were fixed with 4% paraformaldehyde (in PBS pH 7.4) for 10 min, and permeabilized with 400 µL of 0.1% Triton-X-100 in 1X PBS at room temperature for 5 min. After blocking with 3 mL 10 % donkey serum in PBS pH 7.4 at R.T. 30 min, the slides were incubated overnight with the primary antibody, IκBα (Cell Signaling 4812S), NF-κB (B.D. 558393) and proteasome (19S, NOVUS NB100-1483) at 4°C. After washing slides with PBST pH 7.4, 2° Ab was added (anti-Rabbit—DyLight 550 (Bethyl), Proteasome-Goat—DyLight650 (Bethyl)) in 1% donkey serum in PBS pH7.4 for 2 hr. Finally, NF-κB—FITC (Biorbyt) in 1% donkey serum in PBS pH7.4 was added for 2 hr. The nucleus was stained with DAPI (Thermo) in 1% donkey serum in PBS pH7.4 for 10 min, and the slides mounted using antifade. The immunofluorescence signal was visualized by confocal microscopy using a Leica 2000 and edited by Leica 2000 software.
Flow cytometry analysis
Cell were stained with Annexin V (P.E., B.D. 560930), propidium iodide (B.D. 556463), or Active Caspase 3 (P.E., B.D. 561011) and flow cytometric analyses performed on a Becton-Dickinson FACScan with CELLQuest software (Becton-Dickinson Immunocytometry System, Mountain View, CA) and Flowjo 10 software.
Orthotopic tumor implantation model
Male nude mice, 4–6 weeks old, were anesthetized via i.p. administration of 80 mg/kg ketamine and 10 mg/kg xylazine. While under demonstrable anesthesia, an upper midline incision of the abdomen was made and 5x105 HepG2-Luc cells mixed with Matrigel (1:1) in 20 µL was injected into the left lobe of the liver in male nude mice through a 23-gauge syringe by laparotomy, six in each group. To avoid the leakage of the tumor and seeding into the peritoneum, the injection site was compressed by cotton swab for 2 minutes until no bleeding was evident from the liver surface [17]. After tumor inject, the abdominal wound was closed by interrupted stitches. To monitor the tumor growth in mice, the tumor was imaged by the IVIS system as previously described[18].
Statistical analysis
All data are expressed as mean ± S.E. where indicated and are representative of at least two separate experiments. In the tumor treatment experiments, the outcome of interest was the duration of survival until euthanasia based on the animal protocol (in stress, weight change greater than 20%). The event-time distributions for different mice were compared using the Kaplan–Meier method and the log-rank statistic by Prism 6 software. All p-values < 0.05 were considered significant. The statistics were calculated by Prism 6 software.