Subjects
C2C12 cell lines (ATCC, Manassas, VA, USA) were used for In vitro study. C2C12 murine myoblasts were cultured in a growth medium consisting of low-glucose Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) in a 5% CO2 incubator at a temperature of 37 °C. After culturing until 70–80% confluence, C2C12 cells were used for the following analysis.
An in vivo study was performed on 68 male C3H mice aged at 5 weeks (SLC, Hamamatsu, Japan). The animals were housed in a temperature-controlled environment and maintained on a 12-hour light–dark cycle with the standard chow diet for mice and rats (Rodent Diet CA-1, CLEA Japan Inc., Tokyo, Japan); water was available ad libitum. The experimental protocol was approved by the Animal Ethics Research Committee at Mie University.
Collection of conditioned medium
LM8 osteosarcoma cells, which were derived from the murine osteosarcoma cell line Dunn osteosarcoma through a repetition of eight cycles of the procedure, were grown in 10% FBS at 37 °C in 5% CO2 (23). When the confluence reached 80–90%, the cells were washed with phosphate-buffered saline (PBS) and the medium was replaced with DMEM without phenol red or serum. After 48 h, the conditioned medium was collected by centrifugation at 12,000 rpm at 4 °C for 10 min and filtration with a 0.22 µm filter to remove debris (24). The conditioned medium was preserved at -80 °C for further experiments.
Antioxidant effect of myoblasts incubated in conditioned medium
C2C12 cells were seeded at 1.0 × 104 cells/well on a 96 well-plate in DMEM with 10% FBS. After 24 h, the medium was replaced with DMEM containing 2% FBS for differentiation into the myotubes. At the same time, febuxostat was added at a concentration of either 3 µM or 30 µM for 24 h as premedication. Febuxostat was gratuitously provided by Teijin Pharma Limited (Tokyo, Japan). Following incubation in the conditioned medium for 2 and 24 h, levels of ROS were examined using 2’,7’-dichlorofluorescein diacetate (DCF-DA), according to the manufacturer’s instructions (Abcam, Tokyo, Japan). Myotubes were washed with PBS and fresh DMEM without phenol red, and incubated with 10 µM DCF-DA for 30 min in darkness at room temperature. The cells were immediately analyzed, and ROS levels were measured by an increase in the DCF fluorescence. DCF fluorescence was measured at an excitation wavelength of 488 nm and an emission wavelength of 519 nm.
Animal models and febuxostat administration: Male C3H mice (5 weeks old; SLC, Hamamatsu, Japan) were inoculated with 1.0 × 107 LM8 osteosarcoma cells in 0.2 ml of PBS by subcutaneous injection into the backs of the mice. Control mice were injected with 0.2 ml of PBS. Febuxostat was administered in the drinking water, from the day of inoculation with the tumor cells. Four different groups were established: group F5, tumor-bearing mice with febuxostat 5 µg/ml; group F25, tumor-bearing mice with febuxostat 25 µg/ml; group TB, tumor-bearing mice without febuxostat; and group C, control mice. At 4 weeks after tumor inoculation, mice were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (0.05 mg/g body weight), followed by body weight measurement. Then, muscles of gastrocnemius were fully excised from the attachment site of the bone, and wet weights were measured. Furthermore, lung metastasis was macroscopically observed.
Immunohistochemical analysis: The harvested gastrocnemius muscles were immediately fixed in 4% paraformaldehyde at 4 °C overnight. Then, the specimens were embedded in paraffin, and were longitudinally cut at 4-mm thickness on a microtome. Specimens were dewaxed in xylene and rehydrated in graded ethanol (99–70% (v/v)) in distilled water. Endogenous peroxidase activity was quenched by 30-min incubation in 0.3% (v/v) hydrogen peroxide in 99% methanol. Heat induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) using a pressure cooker (Delicio 6L; T-FAL, Rumily, France). The sections were then left to cool at room temperature in the citrate buffer. Nonspecific staining was blocked by incubating the sections in a solution of 1% bovine serum albumin for 20 min at room temperature. The bovine serum albumin was then drained off, and specimens were incubated at room temperature overnight in each antibody (1: 50). Goat monoclonal 8OHdG antibodies (Japan Institute for Control of Aging, Tokyo, Japan) were used as primary antibodies. Bound primary antibodies were detected using secondary anti-goat Ig antibodies and anti-rabbit Ig antibodies conjugated to horseradish peroxidase (1:100; Dako Japan, Kyoto, Japan) for 1 h at room temperature. Bound antibodies were visualized by a reaction with 3, 30-diaminobenzidine. Between the incubation steps, sections were washed with PBS (3 × 5 min) to eliminate excess non-bound antibodies or reagents. Sections were counterstained with hematoxylin. We counted the number of 8OHdG labeled nuclei, and calculated the positive rate of 8OHdG expression by dividing this number by the total number of nuclei.
XO assay
XO activity was measured using a commercially available kit (Sigma-Aldrich, Darmstadt, Germany). The harvested gastrocnemius muscles were homogenized with an assay buffer. The homogenate was centrifuged at 15,000 rpm for 10 min, and the supernatant was used for the assay. After the addition of reaction buffer, initial (Tinitial) and final (Tfinal) measurements were performed at 570 nm on a microplate reader (FluoStar Galaxy®,BMG LABTECH GmbH, Ortenberg, Germany), and XO activity was calculated by the change in the measurements from Tinitial to Tfinal.
Measurements of inflammatory cytokine
The harvested gastrocnemius muscles were quickly frozen in liquid nitrogen, homogenized using Cryopress (Microtech, Chiba, Japan), and centrifuged, and the supernatant was preserved at -80 °C. Quantitative analysis of TNF-α and IL-6 was done using enzyme-linked immunosorbent assay (ELISA) kit (BD Bioscience, Franklin Lakes, NJ).
Statistical analyses
SPSS Statistics Software version 23.0 (IBM, Armonk, NY, USA) was used. Results are shown as mean ± standard deviation (SD). The comparisons among groups were assessed using the Kruskal-Wallis test. Further, overall survival was determined using the Kaplan-Meier survival analysis. Values of p < 0.05 were considered statistically significant.