The Thioredoxin-1 Inhibitor, PX-12, Suppresses Local Osteosarcoma Progression

Materials and Methods

Bioinformatics. The Trx protein is encoded by TXN (6). We investigated the relationship between TXN (ILMN_1680314) expression and disease outcome in OS patients using public microarray data (GSE42352). TXN expression data were downloaded from the R2 microarray analysis and visualization platform, and the R2-based software was used to draw Kaplan–Meier survival curves. As described in a previous study (4), the best cut-off value for the survival analysis was determined as the expression value obtained when the statistic log-rank for the separation of the survival curves reached its highest level.

Antibodies and reagents. Antibodies and reagents were purchased from the following commercial sources, PX-12 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); N-Acetyl-L-cysteine (NAC) from Sigma-Aldrich (St. Louis, MO, USA); carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-FMK) from Adooq bioscience (Tokyo, Japan); MEM from Sigma-Aldrich; radioimmunoprecipitation acid (RIPA) lysis buffer from Santa Cruz Biotechnology; anti-Trx-1 (#2429), anti-p38 MAPK (#9212), anti-JNK MAPK (#9252), anti-phospho-p38 MAPK (Thr180/Tyr182) (#9211), anti-phospho-JNK MAPK (Thr183/Tyr185) (#9251), anti-beta-actin (#4967), and cleaved caspase-3 (Asp175) (#9661) from Cell Signaling Technology (Danvers, MA, USA); horseradish peroxidase-conjugated secondary antibodies and polyvinylidene difluoride (PVDF) western blotting membrane from GE Healthcare (Tokyo, Japan).

Cell culture. The LM8 cell line (RRID: CVCL_6669) was purchased from the RIKEN BioResource Center (Ibaraki, Japan). The cells were maintained in MEM supplemented with 10% fetal bovine serum (FBS) and 100 mg/ml penicillin/streptomycin in a humidified 5% CO₂ atmosphere at 37°C.

Cell viability assay. LM-8 cells were seeded at a concentration of 1×10⁶ cells/ml in a 96-well plate and stimulated with PX-12 (3.125, 6.25, 12.5, 25, and 50 μM) or dimethyl sulfoxide (DMSO) for 24 and 48 h. Subsequently, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (Cayman Chemical, Ann Arbor, MI, USA) was added to the cells at a volume equivalent to 10% of the culture volume under sterile conditions and incubated for 4 h in a 5% CO₂ incubator at 37°C. After incubation, a crystal-dissolving solution was added to the cultures and incubated for 4-18 h in a CO₂ incubator at 37°C. The absorbance of each sample at 570 nm was measured using a microplate reader (Multiskan Sky, Thermo Scientific, Tokyo, Japan). Cell viability (%) was calculated based on the following equation: (absorbance value for the experimental group/absorbance value for the control group) × 100.

IncuCyte^® cell proliferation assay. Cell proliferation was monitored for 96 h using the IncuCyte^® ZOOM system (Essen Bioscience, Ann Arbor, MI, USA), which uses an automated incubator to monitor live cells. Growth curves were generated using the algorithm in the ZOOM software and data points were acquired through six-hour-interval imaging. All samples were plated in quadruplicate.

RNA interference. For RNA interference, cells at 60% confluence were transfected with small interfering RNAs (siRNAs) (Life Technologies, Carlsbad, CA, USA) using the Lipofectamine RNAiMAX reagent (Life Technologies) according to the manufacturer’s instructions. Then, the cells were transfected with silencer siRNA targeting TXN (Catalog #4390824) and silencer select negative control siRNA. After knockdown with each siRNA for 48 h, siRNA-transfected cells were evaluated for cell viability or lysed for immunoblotting.

Immunoblotting. All wash buffers and extraction buffers contained a protease inhibitor cocktail (Roche, Tokyo, Japan), NaF (1 M), the PhosSTOP phosphatase inhibitor (Roche), and Na₃VO₄ (50 μM). Cell extracts were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. After the membranes were blocked with 2% bovine serum albumin in TBS-T (150 mM NaCl, 50 mM Tris HCl [pH 8.0], and 0.05% Tween-20), they were probed with antibodies. Antibody-antigen complexes were detected using an enhanced chemiluminescence system (GE Healthcare).

Caspase assay. DMSO- or PX-12-stimulated cells were lysed using a protease inhibitor-free RIPA lysis buffer. A substrate solution was prepared and mixed with phosphate-buffered saline (PBS), 2× reaction buffer (MBL, Tokyo, Japan), and fluorogenic caspase-3 substrate VII Calbiochem, Darmstadt, Germany). The supernatant of the cell solution was mixed with the substrate solution in the wells of a 96-well plate. The plates were incubated at 37°C for 2 h in the dark, and absorbance was measured using a microplate reader (Multiskan Sky, Thermo Scientific, Tokyo, Japan).

Wound healing assay. Cells were seeded in 6-well plates and cultured until a confluent monolayer was formed. A wound was created in the cell monolayer by scratching the plate with a sterile pipette tip, and the cells were washed with PBS. Then, the cells were pre-treated with 10 μg/ml mitomycin C (Abcam, Tokyo, Japan) at 37°C for 1 h to inhibit cell proliferation. Next, LM8 cells were incubated with DMSO or PX-12 (10 μM) in 10% FBS for 24 h. Finally, the cells were photographed at a magnification of ×100, 0, 12, and 24 h after incubation. The cell migration distance was measured between the 2 boundaries of the acellular area. The results obtained in the different treatment groups were expressed as the ratio of the original distance.

Animal studies. Five-week-old female C3H/HeSlc mice were purchased from Japan SLC (Shizuoka, Japan). The mice were maintained at a constant temperature (22±2°C) and humidity (50±10%) under a 12h light/12h dark cycle. All animal experiments were approved by the institutional animal care and use committee of the Chiba cancer center, Chiba, Japan, and were conducted in accordance with institutional guidelines. On day 0, 1×10⁷ LM8 cells in 300 μl PBS were subcutaneously injected into the backs of 12 syngeneic C3H/HeSlc mice. Primary tumor growth was allowed to proceed for 7 days prior to treatment until it reached a measurable size (measured using a caliper and determined as length × width²/2). The mice were then assigned to 2 groups of six mice each with similar tumor size distributions. The mice were treated with either 200 μl of vehicle control (40% polyethylene glycol 300+60% sterile PBS) or 12.5 mg/kg PX-12. Injections were administered when palpable tumors (100-150 mm³) were observed, and were provided intraperitoneally every other day till the end of the study. Tumor size measurements and animal weights were recorded once a week in a non-blinded manner. On day 35, the mice were euthanized under anesthesia, and the tumors were resected for tumor weight measurement.

Statistical analysis. Experimental data are expressed as the mean±standard deviation. The significance of differences between mean values was determined using Student’s t-test, with values of p<0.05 considered significant. All analyses were performed with SAS software, version 14.2 (SAS Institute, Inc.; Cary, NC, USA).