We used the human ARMS cell lines SJ-Rh30 (Rh30), SJ-Rh41 (Rh41), SJ-Rh3 (Rh3), SJ-Rh4 (Rh4), SJ-Rh18 (Rh18) and SJ-Rh28 (Rh28) that were kindly provided by Peter J. Houghton M, San Antonio, TX, USA), and the embryonal RMS (ERMS) cell lines RD that was obtained from JCRB (Japanese Collection of Research Bioresources) Cell Bank and RMS-YM that was kindly provided by Naoki Kakazu M.D. (Department of Environmental and Preventive Medicine, Shimane University School of Medicine, Shimane, Japan) (5,19). They were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ incubator. OBP-801 (Oncolys BioPharma, Inc., Tokyo, Japan) was dissolved in dimethyl sulfoxide (DMSO) and stored as a 1-mM stock solution in 50-µl aliquots at −20°C. The percentage of DMSO in all experiments was <0.01%.

Cells were lysed in RIPA buffer (08714-04; Nacalai Tesque, Kyoto, Japan). Protein concentrations in cell lysates were determined using BCA assay. A total of 20 µg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on NuPAGE Novex 4–12% Bis-Tris gels in NuPAGE MES SDS running buffer (Life Technologies; Thermo Fisher Scientific, Inc.). Proteins were subsequently transferred to Immobilon-P membranes in NuPAGE Transfer buffer (Life Technologies; Thermo Fisher Scientific, Inc). Membranes were blocked in phosphate-buffered saline with Tween-20 (PBST) containing 5% non-fat dry milk powder, and then incubated at 4°C overnight with primary antibodies against the following proteins: anti-histone-H4 (dilution 1:1,000; cat. no. 13919; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-acetyl histone-H4 (dilution 1:1,000; cat. no. 06–866; Merck KGaA, Darmstadt, Germany), anti-Rb (dilution 1:2,000; cat. no. 554136; BD Biosciences, Franklin Lakes, NJ, USA), anti-phospho-histone-H3 (dilution 1:2,000; cat. no. 3377; Cell Signaling Technology, Inc.), anti-survivin (dilution 1:2,000; cat. no. 2808; Cell Signaling Technology, Inc.) and anti-p21 Waf1/Cip1 (1:2,000; cat. no. 2946; Cell Signaling Technology, Inc.), anti-total caspase-3 (dilution 1:2,000; cat. no. 610322; BD Biosciences), anti-cleaved caspase-3 (dilution 1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.) and anti-β-actin (dilution 1:5,000; cat. no. A2228; Merck KGaA) for the reference protein. The membranes were then washed with PBST and incubated at room temperature for 1 h with sheep anti-mouse secondary antibody (dilution 1:10,000; cat. no. NA931; GE Healthcare, Little Chalfont, UK) or donkey anti-rabbit secondary antibody (dilution 1:10,000; cat. no. NA934; GE Healthcare). Antibody binding was detected using the enhanced chemiluminescence detection system (ECL and ECL prime; GE Healthcare) (20). The protein density of western blot was measured by ImageJ 1.52a (National Institutes of Health, Bethesda, MD, USA).

We performed WST-8 colorimetric assays with Cell Counting Kit-8 (Nacalai Tesque, Inc.) according to the manufacturer's instructions. We seeded the cells in 96-well plates in 100 µl culture medium for 24 h, then added various reagents. We determined cell viability by assessing the optical density (OD) at 450 nm with a microplate reader (Multiskan™ JX; Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan), as previously described (20).

To analyze their cell cycle distribution, we cultured the cells in the presence of OBP-801 or an equivalent volume of DMSO for 24 h. We then isolated the cells by scraping, washed them with phosphate-buffered saline (PBS), and incubated them with propidium iodide at a concentration of 50 µg/ml for 30 min to stain DNA. We determined their DNA content on a FACSCalibur™ flow cytometer (BD Biosciences). We analyzed their cell cycle status with FlowJo software 7.6.5 (Tree Star, Inc., Ashland, OR, USA), as previously described (21).

We analyzed cell death after Annexin V-FITC and propidium iodide staining with a TACS Annexin V-FITC Apoptosis Detection kit (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions. We analyzed the data with FlowJo software (Tree Star, Inc.) as previously described (21).

We plated the cells on Falcon^® 8-Well Culture Slides (cat. no. 354118; BD Falcon; Corning, Inc., Corning, NY, USA), then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton™ X-100, washed with PBS, and incubated with anti-survivin (dilution 1:1,000; cat. no. 2808), anti-α-tubulin (dilution 1:1,000; cat. no. 3873), anti-phospho-histone-H3 (dilution 1:1,000; cat. no. 3377), or anti-phospho-H2AX (dilution 1:1,000; cat. no. 9718; all from Cell Signaling Technology, Inc.) at 4°C overnight. We then rinsed the slides with PBS and incubated them with Alexa Fluor 488/555-conjugated anti-mouse/rabbit IgG (dilution 1:200; cat. nos. A11008, A11001 and A21422; Cell Signaling Technology, Inc.) at room temperature for 1 h. Finally, we examined the slides by fluorescence microscopy with a KEYENCE BZ-X700 instrument (Keyence Corp., Osaka, Japan).

Female BALB/c nu/nu nude mice (4-weeks old, total 14, 11–16 g) were purchased from Japan SLC, Inc. (Shizuoka, Japan). All experiments and procedures were conducted in accordance with the institutional animal care and use committee guidelines. The present study was also approved by the Committee for Animal Research of Kyoto Prefectural University of Medicine (permission no. M27-477). Mice were maintained at 23±2°C under a 12-h light/dark cycle (light period, 07:00-19:00 h). Food and water were available ad libitum. We subcutaneously injected 1×10⁷ luciferase-positive Rh30 cells into the dorsal area of BALB/c nu/nu nude mice (n=3 and 4/group). We monitored tumor growth in live mice by bioluminescent detection of the luciferase activity of the Rh30 cells at days 3 and 52, as previously described (22). We assessed the tumor sizes twice per week using calipers using the formula (a × b²)/2. The mice were euthanized by barbiturate overdose.

We performed in vivo bioluminescence imaging of live mice using a Xenogen IVIS^®-Illumina system (PerkinElmer, Inc., Waltham, MA, USA). The animals were maintained under inhaled anesthesia (2% isoflurane in 100% oxygen at the rate of 2.5 l/min). For imaging of the firefly luciferase reporter harbored by the tumor cells, we administered a single luciferin dose of 150 mg/kg (PerkinElmer, Inc.) via intraperitoneal injection 20 min prior to imaging. The data were acquired and analyzed using the manufacturer's proprietary Living Image software 4.4 (PerkinElmer, Inc.).

Average values were expressed as the mean ± standard deviation (SD). We used the 2-tailed Student's t-test for comparison of the means between groups. Differences with a P-value of <0.05 were considered to indicate a statistically significant difference.

We examined the effect of the HDAC inhibitor OBP-801 on the growth of ERMS and ARMS cell lines. The mean half maximal inhibitory concentration (IC₅₀) values were 2.6±0.2 nM for the ERMS cells and 1.8±0.8 nM for the ARMS cells (Table I). OBP-801 inhibited the growth of RD and Rh30 in a concentration- and time-dependent manner (Fig. 1A and B).

To evaluate the extent of HDAC inhibition in RMS cells, we performed immunoblot analyses using anti-acetylated histone antibodies. After 24 h, OBP-801 induced the accumulation of acetylated histone H4 in a concentration-dependent fashion (Fig. 2A). OBP-801 also induced p21waf1/Cip1 in a concentration-dependent manner, and hypophosphorylation of Rb in ARMS, but not in ERMS cells (Fig. 2A). We observed that OBP-801 (10 nM, 24 h) induced arrest in the G1 and G2/M-phases in ARMS cells and in the G2/M-phase in ERMS cells (Fig. 2B). In addition, OBP-801 induced cell death in RMS cells (Fig. 1B). OBP-801 (10 nM) induced early and late apoptosis in RMS cells 48 h after the treatment, as indicated by Annexin V staining assessed by flow cytometry (Fig. 2C). Treatment with OBP-801 also led to the expression of cleaved caspase-3 in a concentration-dependent manner (Fig. 2D).

To examine if OBP-801 caused G2- or M-phase arrest, we immunoblotted with an antibody against phospho-histone H3, a marker of mitosis. Cells treated with OBP-801 had higher phosphorylation levels of histone H3 than control cells 24 h after the treatment; the effect was concentration-dependent (Fig. 3A). We next analyzed the nuclear morphology of RMS cells with OBP-801 treatment. We found that control cells exhibited normal chromosome distribution in metaphase, with correctly formed mitotic spindles. However, upon treatment with OBP-801, dividing RMS cells exhibited aberrant metaphase morphologies: their chromosomes were not aligned at the metaphase plate, they had an abnormal mitotic spindle distribution, survivin was not recruited to the centromeres, and exhibited lower levels of survivin than that in control cells (Fig. 3B and C); OBP-801 affected the abundance of survivin in a dose-dependent manner (Fig. 3A). In addition, OBP-801-treated RMS cells were stained with the γH2AX antibody, which indicated that they entered mitosis with damaged DNA (Fig. 3C).

We measured the bioluminescence from Rh30 cells in nude mice at days 3 and 52 after injection and found that the mice that had received OBP-801 had lower tumor-related bioluminescence intensities at day 52 (Fig. 4A). The mice in the drug-treated group had smaller tumors and survived longer than those in the control group (Fig. 4C and D). We did not observe statistically significant differences between the body weights of the mice in the drug-treated and control groups, indicating that the drug did not cause toxicity (Fig. 4B).