Human osteosarcoma cell line MG63 (CRL-1427) and human NK cell line NK-92 (CRL-2407) were purchased from the American Type Culture Collection. MG63 cells were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM; cat. no. D6429; MilliporeSigma) supplemented with 10% fetal bovine serum (FBS; cat. no. F8192; MilliporeSigma) and 100 µg/ml penicillin/streptomycin (cat. no. P4333; MilliporeSigma) within a humidified atmosphere containing 5% CO₂ at 37˚C. Sesamin (cat. no. SMB00705; MilliporeSigma) was prepared in dimethyl sulfoxide at a concentration of 50 mM, stored as aliquots at -20˚C, and diluted with a cell culture medium for use. For treatments with sesamin, cells were treated with sesamin with serial concentrations in serum-free DMEM for 24 or 48 h. After treatments, resulting cells were washed with phosphate-buffered saline (PBS; pH 7.2) for following analyses. NK-92 cells were cultured and maintained in complete RPMI-1640 media (cat. no. R8758; MilliporeSigma) (10% FBS, 1% HEPES, 1% L-glutamine, 1% penicillin-streptomycin) and 300 U/ml recombinant human IL-2 (Proleukin; IOVANCE Biotherapeutics). NK-92 cells were activated by culturing in complete medium containing 500 IU/ml IL-2 and 50 ng/ml IL-15 (R&D Systems, Inc.) for 24 h. Activated NK-92 cells were harvested for following experiments.

Cell viability was evaluated using MTT assay. Cells (x10⁵) in culture media treated with sesamin were incubated with (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (0.5 mg/ml) at 37˚C for 4 h. MTT solution was aspirated and the formazan crystals were dissolved in 200 µl DMSO. Number of viable cells was directly proportional to the production of formazan determined by measuring the absorbance at 570 nm.

After treatment with sesamin, 5x10⁵ of MG-63 cells were digested by trypsin, washed with PBS, and fixed with 70% alcohol on ice for 30 min. Cells were subsequently washed with cold PBS, suspended in 200 µl staining solution (PBS with 1% BSA, 10 mg/ml propidium iodide, 0.25 mg/ml RNase A) and incubated at 37˚C in the dark for 30 min. Resulting cells were analyzed using population of each phase of cell cycle, determined using BD FACS Canto II with the FlowJo v.10 software package (BD Biosciences).

After treatments, cells were lysed using lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride and 1 mM NaF). Cell lysates were centrifuged at 4˚C, 15,000 x g for 20 min. Supernatants were collected and assessed for protein concentration using the Bradford assay (cat. no. B6916; MilliporeSigma). A total of 20 µg of total protein was subjected to a 12.5% SDS-polyacrylamide gel electrophoresis followed by transferring onto a nitrocellulose membrane (MilliporeSigma). The resulting membranes were blocked with 5% w/v skimmed milk in PBS for 30 min at room temperature and then incubated for 2 h at room temperature with primary antibodies at a ratio of 1:1,000, which were against p21 (cat. no. MA5-31479), cyclin B1 (cat. no. MA1-155), cyclin-dependent kinase 1 (CdK1; cat. no. MA5-15631) and β-actin (cat. no. MA5-15739; all from Invitrogen; Thermo Fisher Scientific, Inc.), respectively. Blots were subsequently incubated with 1:2,000 dilution of horseradish peroxidase-conjugated secondary antibodies (cat. no. 31430; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature and antigen-antibody complexes were displayed using ECL chemiluminescence (Millipore Sigma). The protein bands were quantified with the ImageJ software (version 1.54; National Institutes of Health).

Total RNA of cells after treatment was isolated using TRIzol^® (MilliporeSigma) according to the manufacturer's protocol. Reverse transcription was performed using SuperScript IV VILO Master Mix (cat. no. 11756050; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Quantification of mRNA levels of the genes of interest was performed using the ABI PRISM 7700 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with designated primer pairs. FastStart Universal SYBR Green Master (Roche Applied Science) was used for qPCR. The thermocycling conditions for qPCR included initial denaturation at 95˚C for 5 min, 40 cycles of amplification (at 95˚C for 30 sec, 58˚C for 30 sec and 72˚C for 30 sec) and at 72˚C for 5 min. The threshold cycle numbers were calculated using the 2^-ΔΔCq relative value method (18) and normalized to GAPDH. Pairs of primers used for genes of interest are listed in Table I. Each qPCR reaction was repeated at least three times.

A total of 5x10³ MG-63 cells were seeded into a well of the 96-well culture plates with complete DMEM media and incubated for 24 h, followed by treatments with or without sesamin for another 48 h. Resulting MG-63 cells were co-cultured with activated NK-92 cells at a range of effector: target (E:T) ratios (1:5, 1:3, 1:1, 3:1, 5:1 and 10:1) for 4 h at 37˚C with 5% CO₂. After 4 h of co-culture, NK-92 cells were removed and MTT assay was carried out to assess viable cells by measuring the absorbance at 570 nm.

Concentrations of IFN-γ secreted by activated NK-92 cells in cultures with MG-63 cells were measured using ELISA according to manufacturer's protocol. The levels of IFN-γ in the culture medium was detected using IFN-γ ELISA Kit (R&D systems, Inc.).

A total of 1x10⁶ MG-63 cells were cultured with different concentrations of sesamin. The expression of the NKG2DLs was analyzed by immunofluorescence staining using anti-MICA (1:50; cat. no. MA5-36026; Invitrogen; Thermo Fisher Scientific, Inc.), anti-MICB (1:50; cat. no. MA5-29422; Invitrogen; Thermo Fisher Scientific, Inc.), anti-ULBP-1 (1:50; cat. no. MA5-38655; Invitrogen; Thermo Fisher Scientific, Inc.) and phycoerythrin. In all experiments, cells were stained at room temperature for 15 min with propidium iodide (1 µg/µl) to assess cell viability. Data acquisition and flow cytometric analysis were carried out on a BD FACSCanto II using the FlowJo v10 software package (FlowJo LLC).

Data are presented as the mean ± SD of the three independent experiments with conditions set up in three or six replicates. Comparisons were made by unpaired Student's t-test between two groups. One-way and two-way ANOVA were performed to assess the statistical significance of differences in measured variables between multiple groups with Tukey's post hoc test. Correlations between the protein expression levels with NK cell-mediated cytotoxicity were examined using Pearson's correlation analysis. All statistical analyses were conducted using SigmaPlot 10 (Systat Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The effect of sesamin on growth of osteosarcoma cells was determined using MTT assay. MG-63 osteosarcoma cells were treated at various concentrations of sesamin (0,12.5,25,50 and 100 µM) for 24 h or 48 h and measured for cell viability. No significant inhibitory effects were observed at 24 h. The cells responded time-dependently to sesamin treatment within the concentrations tested. The viability of MG-63 cells was decreased to 80.7±2.9 and 73.3±1.9% of controls in response to the incubation with 50 and 100 µM of sesamin for 48 h, respectively (P<0.05) (Fig. 1). The doses of sesamin significantly inhibiting viability of MG-63 cells were chosen for subsequent experiments.

The mechanism underlying sesamin-induced proliferation inhibition was investigated. Changes in cell cycle distribution in MG-63 cells treated with sesamin at designated concentrations were determined using flow cytometry. MG-63 cells exposed to sesamin at different concentrations for 24 h exhibited G₂/M cell cycle arrest (Fig. 2A). Treatment of MG-63 cells with 0, 12.5, 25, 50 and 100 µM sesamin for 48 h resulted in an increase in proportion of G₂/M phase cells from 12.8±3.5, 15.6±3.4, 19.2±3.2, 36.1±1.4 and 45.2±1.2%, respectively (Fig. 2B). The results revealed that sesamin induced G₂/M cell cycle arrest in a dose- and time-dependent manner.

To determine the molecular events involved in sesamin-induced cell cycle arrest, changes in expression of proteins involved in G₂/M phase including p21, Cdk1 and cyclin B1 in sesamin-treated MG-63 cells were assessed. The protein levels of p21, cyclinB1 and Cdk1 were detected using western blotting in MG-63 cells treated with 0, 12.5, 25, 50 and 100 µM sesamin for 48 h (Fig. 3A). Sesamin induced an increase in expression of protein p21, which is an inhibitor of Cdk/cyclin complex in MG-6 cells. The results revealed that sesamin inhibited the expression of cyclin B1 and Cdk1 at 48 h, which are involved in the G₂/M transition. The mRNA levels of p21, Cyclin B1 and Cdk 1 gene in MG-63 cells treated with sesamin for 48 h were validated using RT-qPCR. The mRNA expression of p21 was significantly upregulated in MG-63 cells treated with 50 and 100 µM sesamin (Fig. 3B). The mRNA levels of cyclin B1 and Cdk1 were significantly reduced in comparison with that of 0 µM MG-63 cells (Fig. 3C and D). The changes in p21, Cyclin B1 and Cdk 1 mRNA expression were parallel to that of the results of western blotting.

As increased p21 expression has been revealed in association with expression of NKG2DLs, it was investigated whether sesamin has influences on the expression of the ligands for NKG2D in MG-63 cells. Expression of NKG2DLs in MG-63 cells treated with or without sesamin were examined by flow cytometry and RT-qPCR. MG-63 cells constitutively express MICA, MICB and ULBP1 at different levels (Fig. 4A-C). Treatment with sesamin for 48 h resulted in increased percentage of MICA positive MG-63 cells in a dose-dependent manner. The percentage of MICB positive MG-63 cells was significantly increased at sesamin concentrations of 50 and 100 µM. Sesamin treatment increased the percentage of ULBP1 positive MG-63 cells in a dose-independent manner. Afterwards, the changes in mRNA levels of MICA, MICB and ULBP1 in MG-63 cells treated sesamin were investigated. The results of RT-qPCR revealed that mRNA expression of MICA, MICB and ULBP1 was significantly upregulated in MG-63 cells treated with sesamin for 48 h (Fig. 4D-F).

Since sesamin upregulated the expression of NKG2DLs in MG-63 cells, it was next investigated whether sesamin primes MG-63-cells for NK cell-mediated antitumor activity by increasing NKG2DL expression. MG-63 cells were treated with sesamin at various concentration for 48 h and were subsequently subjected to co-culture with NK-92 cells. NK-92 cells inhibited the viability of untreated MG-63 cells up to 50% at an E:T ratio of 10:1 (Fig. 5). Treatment with sesamin enhanced MG-63 cell susceptibility to NK-92 cell elimination, resulting in a significant decrease in viability of MG-63 cells in a dose-dependent manner. Data obtained from cytotoxicity assay using E:T ratios of 1:1, 3:1 or 5:1 were used to calculate IC₅₀ values, resulting in IC₅₀ of 75.1, 51.1 or 16.8 µM, respectively.

The possible correlation between NKG2DL expression and NK cell-mediated cytotoxicity in MG-63 osteosarcoma cells was investigated. The correlation coefficient between MICA and NK cell-mediated cytotoxicity was significant at E:T ratios of 1:3 (P=0.04) and 1:1 (P=0.04) (Table II). The correlation between MICB levels in MG-63 cells and NK cell mediated elimination was significant at all E:T ratios. A significant correlation was detected between percentage of NKG2DL positive cells and NK-mediated cytotoxicity. There was no correlation between ULBP1 and NK-cell mediated killing at all E:T ratios tested.