Mouse sarcoma 180 (S180) and human osteosarcoma U2OS cells were obtained from the Chinese Academy of Science (Shanghai, China) and were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin sodium and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. AMI-1 was synthesized in house, according to the method of Peng et al (21) and Ragno et al (22).

A total of 22 male Kunming mice (age, 6–7 weeks old; body weight, 18–22 g) were purchased from Lanzhou University (Gansu, China). The mice were acclimated to laboratory conditions (25°C, 12/12 h light/dark, 50% humidity and ad libitum access to food and water) for 3 days prior to experimentation. The present study was approved by the Institutional Animal Care and Treatment Committee of Lanzhou University (Gansu, China). On day 7, mice were euthanized prior to cervical dislocation with an intraperitoneal injection of 50 mg/kg pentobarbital sodium.

Briefly, S180 or U2OS cells were seeded at 2×10³ cells/well in 96-well plates. Following 24 h of culture, cells were treated with various concentrations of AMI-1 (0.6, 1.2 and 2.4 mM) and the control group was treated with the vehicle control (PBS). Cytotoxicity was evaluated using the Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer's protocol. S180 cells were seeded at 7.5×10⁴ cells/well in 24-well plates were incubated at 37°C at indicated timepoints (48, 72 or 96 h). The cell morphology and numbers were observed under a light inverted microscope (Olympus CK40; Olympus Corporation, Tokyo, Japan; magnification, ×100). Cytotoxicity was determined by measuring the absorbance at a wavelength of 450 nm using a plate reader. IC₅₀ values were evaluated using CurveExpert 1.3 software (Hyams Development, Mississippi, MS, USA).

U2OS cells were seeded at a density of 300 cells in 60 mm dishes and incubated for 24 h. S180 cells were not used due to them being suspended cells and not suitable for colony formation assay. RPMI medium with FBS was replaced with 5 ml fresh medium, containing AMI-1 (0.3 or 0.6 mM) or PBS (control) and incubated at 37°C for 18 days. Colonies were fixed with a 7:1 ratio of methanol to glacial acetic acid for 25 min at 25°C and then stained with 0.1% crystal violet (in 20% methanol and PBS) for 25 min at 25°C.

S180 cells were seeded at a density of 1.2×10⁵ cells/well in 6-well plates and treated with AMI-1 (1.2 and 2.4 mM) or vehicle (PBS) for 48 and 72 h. The cells were harvested, washed twice and resuspended in 1X binding buffer. A total of 500 µl S180 cells (1×10⁶ cells/ml) were incubated with 5 µl annexin V-fluorescein isothiocyanate and 5 µl propidium iodide for 15 min at room temperature in dark. The samples were then analyzed using a flow cytometer equipped with FCSDiva 6.2 software (LSR Fortessa™; BD Biosciences, Franklin Lakes, NJ, USA).

A total of 2×10⁶ S180 cells (in 0.2 ml 0.9% NaCl in PBS) were subcutaneously inoculated into the right axillary region of Kunming mice. Following 3 days of implantation with S180 cells, mice were divided into two groups (11 animals/group): AMI-1-treated (0.5 mg in 200 µl 0.9% NaCl) or vehicle treated (200 µl 0.9% NaCl). The treatments were administered intratumorally (200 µl per mouse, once daily for a total of 7 days). The weight of the mice was determined daily. On day 7, mice were sacrificed by cervical dislocation and tumors were removed and weighed. The inhibition rate of tumor viability (IR) was calculated as: (1-the average tumor weight of treated group/tumor weight of vehicle group) ×100%. The dose of AMI-1 was chosen in vivo experiments based on our preliminary experiments and previous literatures (23–26).

Western blot analysis was performed as described previously (15). Briefly, tumor tissues were lysed using RIPA buffer (cat no. P0013B; Beyotime Institute of Biotechnology, Jiangsu, China) containing phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology) for 30 min at 4°C. The extract was centrifuged at 12,000 × g for 15 min at 4°C to clear insoluble debris. The protein concentration was assayed using Quick Start™ Bradford (cat no. 500-0205; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of protein (40 µg per lane) were separated by SDS-PAGE (12% gel) and then transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). The membranes were then blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 h at 25°C. Following blocking, membranes were incubated overnight at 4°C with the following primary antibodies: Anti-PRMT5 (1:500; cat no. sc-376937; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-PRMT7 (1:1,000; cat no. 14762; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-H4R3me2s (1:2,000; cat no. HW027; Signalway Antibody LLC, College Park, MD, USA), anti-H3R8me2s (1:1,000; cat no. HW015; Signalway Antibody LLC), anti-p53 (1:1,000; cat no. 9282; Cell Signaling Technology, Inc.) or anti-β-actin (1:2,000, cat no. 4970; Cell Signaling Technology, Inc.). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat nos. ZB-2301 and ZB-2305; OriGene Technologies, Inc., Beijing, China) for 1.5 h at 25°C. The protein bands were visualized by BeyoECL Plus kit (Beyotime Institute of Biotechnology). The densitometry was performed using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

The relevant data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). The difference between two groups was analyzed using Student's t-test. For comparison of multiple groups, one-way analysis of variance followed by Dunnett's post hoc test was used. P<0.05 was considered to indicate a statistically significant difference.

Cytotoxicity in response to AMI-1 treatment was determined using CCK-8 assay. At 72 h, IC₅₀ values for S180 and U2OS cells were 0.31±0.01 and 0.75±0.02 mM, respectively (data not shown). As presented in Fig. 1A-C, AMI-1 treatment inhibited the cell viability of sarcoma in S180 and U2OS cells in a time-dependent and dose-dependent manner in vitro. AMI-1 treatment significantly inhibited the viability of sarcoma S180 and U2OS cells in response to treatment of AMI-1 (0.6, 1.2 and 2.4 mM) for 48, 72 and 96 h (Fig. 1A and B).

To evaluate whether AMI-1 may inhibit cell viability by regulating cell apoptosis, S180 cells were treated with AMI-1 (1.2 and 2.4 mM) or vehicle for 48 and 72 h. Cellular apoptosis was evaluated using flow cytometry. The results demonstrated that AMI-1 may increase the percentages of cells undergoing apoptosis, compared with that in the vehicle group (Fig. 2). This indicated that AMI-1 reduces S180 cell viability through the induction of cell apoptosis.

To assess the antitumor activity of AMI-1 in vivo, S180 cells were subcutaneously inoculated into the right axillae of mice. Tumor weight and body weight of control and treated groups are presented in Table I. AMI-1 treatment significantly decreased tumor weight compared with that in control-treated mice (Fig. 3). Additionally, IR of tumor viability in response to AMI-1 was 41.42±6.34% (data not shown). Furthermore, there were no significant differences in body weight of mice treated with AMI-1, compared with the control (Table I).

A previous study demonstrated that AMI-1 inhibited the activity of type II arginine methyltransferase (PRMT5) (15). Therefore, in the present study, the expression PRMT5 in response to AMI-1 treatment was evaluated using a tumor xenograft model and western blot analysis. The results demonstrated that AMI-1 treatment significantly decreased the expression of PRMT5 but did not affect the expression of PRMT7 (Fig. 4). In addition, AMI-1 increased p53 protein levels, compared with control-treated tumors (Fig. 4).

PRMT5 is a major type II arginine methyltransferase that catalyzes ω-NG, N'G-symmetric dimethylarginine (H4R3me2s and H3R8me2s) (8,27,28). Western blot analysis was employed to investigate the molecular mechanism by which AMI-1 may inhibit the viability of S180 cells in vivo. The results demonstrated that AMI-1 treatment significantly decreased the levels of H4R3me2s and H3R8me2s compared with those in the control group (Fig. 4).