The human OS cell line MG-63 was purchased from the Cell Bank of China (Wuhan, China). The cells were maintained at 37°C in 5% CO₂ and Dulbecco's modified Eagle medium (DMEM), which contains 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 µg/ml streptomycin and 100 units/ml penicillin.

Capsaicin, DMEM, 3,3′-dihexyloxacarbocyanine iodide (DiOC₆)(3) and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit-anti-human CRT polyclonal antibody was purchased from Stressgen (Victoria, BC, Canada; cat. no. SPC-122B). Mouse anti-human phycoerythrin (PE)-conjugated CD11c monoclonal antibody was purchased from eBioscience (San Diego, CA, USA; cat. no. 12-0116-42). Rabbit anti-B-cell lymphoma 2 (Bcl-2) and rabbit anti-Bcl-2-associated X protein (Bax) monoclonal antibodies, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA; cat. nos. sc-492, sc-6236 and sc-516087, respectively). PE-conjugated goat anti-rabbit IgG polyclonal antibody was obtained from R&D Systems, Inc. (Minneapolis, MN, USA; cat. no. IC108P). Recombinant human interleukin (IL)-2, IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from PeproTech (Rocky Hill, NJ, USA). Lymphocyte separation medium was purchased from Tianjin Haoyang Biological Manufacture, Co., Ltd. (Tianjin, China). IL-4 and IFN-γ enzyme-linked immunosorbent assay (ELISA) kits were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

MG-63 cells were seeded onto 96-well tissue culture plates at a density of 2×10³ cells/100 µl per well, and incubated for 24 h at 37°C. The next day, the media were replaced with 100 µl fresh complete medium containing capsaicin (0, 12.5, 25, 50, 100, 200 and 400 µM) and cisplatin (0, 4, 8, 16, 32, 64 and 128 µg/ml). The cells treated with equal quantities of normal medium or solvent, instead of drugs, served as the control. After 24 h incubation, 100 µl MTT solution (0.5 mg/ml) in DMEM without fetal bovine serum was added to each well and cultured for 4 h at 37°C in a humidified atmosphere. The medium was removed and 150 µl DMSO was added into each well to dissolve the purple crystals, then the absorbance (A) at 570 nm was recorded. The cell proliferation inhibition rate was calculated according to the following formula: Cell proliferation inhibition rate = (A_control - A_drug)/A_control × 100%.

The lipophilic fluorescence dye DiOC₆ (3) (40 nM) was used to assay the mitochondrial membrane permeabilization. MG-63 cells (2×10⁶) treated with 200 µM capsaicin or 32 µg/ml cisplatin were stained with DiOC₆ (3) for 15 min at 37°C and analyzed immediately by flow cytometry (FCM) equipped with a standard 15 mW argon-ion laser (488 nm) to excite DiOC₆ (3). Then, a narrow band filter was used to collect emissions between 515 and 545 nm. A minimum of 10,000 cells were analyzed by FCM for each data point.

MG-63 cells (2×10⁶) were cultured in DMEM medium containing 200 µM capsaicin or 32 µg/ml cisplatin for 12 or 24 h and harvested. Cells were then lysed with cell lysis buffer (Tris 50 mM, NaCl 150 mM, SDS 0.1%, Sodium Deoxycholate 0.5% and 1% Triton X-100) for 20 min on ice. The protein concentrations were determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), after which the proteins in the lysate (45 µg) were separated by 10% SDS-PAGE and transferred onto a PVDF membranes. The membranes were blocked with fat-free milk solution (5%, w/v) for 12 h and then incubated with rabbit anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1,000) or anti-CRT polyclonal antibody (1:1,000) at 4°C for 12 h. After washing 3 times with TBST, the membrane was incubated with an HRP-conjugated anti-rabbit IgG antibody (1:4,000) at room temperature for 1 h and developed using electrochemiluminescence.

MG-63 cells were cultured in DMEM medium containing 200 µM capsaicin or 32 µg/ml cisplatin for 12 h and harvested. After washing once with phosphate-buffered saline (PBS), the cells were incubated with rabbit anti-CRT polyclonal antibody (1:1,000) at room temperature for 1 h, following by washing and incubation with PE-conjugated goat anti-rabbit IgG polyclonal antibody (1:500) at room temperature for 2 h (avoiding light). After washing with PBS, the cells were analyzed by FCM to identify CRT on the cell surface.

Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using lymphocyte separation medium (density, 1.077 g/ml) according to the manufacturer's instructions. Following the last wash, PBMCs (5×10⁶) were resuspended in RPMI-1640 and plated in 6-well culture plates. After 3 h incubation, the monocytes were purified by removing the non-adherent cells in the supernatant. Purified monocytes were cultured in a medium supplemented with 30 ng/ml recombinant human GM-CSF and 10 ng/ml recombinant human IL-4 in 6-well plates (1×10⁶ cells/well). After 7 days, the non-adherent and loosely adherent cells were harvested as the dendritic cells (DCs) and were used as the effector cells for the phagocytosis assay. The MG-63 cells (1×10⁶) incubated with 200 µM capsaicin or 32 µg/ml cisplatin for 12 h were labeled with the green dye carboxyfluorescein diacetate succinimidyl ester (1 µM) for 10 min and were used as the target cells. The effector and target cells were co-cultured at 37°C for 2 h at a 1:1 effector/target ratio. After 3 washes, anti-human CD11c PE was added to the cell mixture for 30 min at room temperature (in the dark) to label the effector cells. The cells were washed with PBS and analyzed by FCM. The phagocytotic efficiency was represented by the cell ratio of the double-positive cell number over the total cell number.

PBMCs were isolated as described above. Following the last wash, PBMCs (5×10⁶) were resuspended in RPMI-1640 and plated in 6-well culture plates. After 3 h incubation, the non-adherent cells were harvested as lymphocytes, and DCs were induced as described above. The DCs loaded with drug-treated MG-63 cells were co-cultured with lymphocytes at a ratio of 1:4. A volume of 200 U/ml IL-2 was added into the cell mixture during the culture period. After 24 h, the supernatant was collected, and the IFN-γ and IL-4 expression levels in the supernatant were measured by ELISA, according to the manufacturer's instructions.

Data are presented as the mean ± standard deviation of three independent experiments. Student's t-tests were performed for the comparison of results between different groups. All of the tests were performed using SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

An MTT assay was used to examine the growth inhibiting effects of capsaicin and cisplatin on MG-63 cells. The result showed that capsaicin and cisplatin significantly inhibit the growth of MG-63 cells in a dose-dependent manner (Fig. 1). After 24 h treatment, the inhibition percentage was 48.5% for 200 µM capsaicin and 58.3% for 32 µg/ml cisplatin. Since these drug concentrations were close to median lethal doses (capsaicin, 165.7 µM; cisplatin, 16.76 µg/ml), these concentrations were used to treat cells in the following experiments.

Decreased MMP is a feature of apoptotic cells. In order to determine whether capsaicin and cisplatin can induce apoptosis of MG-63 cells, the effect of the drugs on the MMP was evaluated by FCM. The cells treated by 200 µM capsaicin or 32 µg/ml cisplatin were stained with DiOC₆ (3), and the MMP was detected by FCM. The results showed that treatment with both drugs could decrease the ratio of cells with green fluorescence (Fig. 2). This result suggests that capsaicin and cisplatin can induce the alteration of MMP in MG-63 cells, and then initiate endogenous apoptosis.

To investigate the molecular mechanism underlying capsaicin- and cisplatin-induced apoptosis in human MG-63 OS cells, the expression levels of apoptosis-related proteins Bax and Bcl-2 were investigated using western blot analysis. As shown in Fig. 3, the expression level of Bax was markedly upregulated, and anti-apoptotic Bcl-2 was markedly downregulated, when the cells were treated with 200 µM capsaicin or 32 µg/ml cisplatin for 24 h. This suggests that Bcl-2 and Bax proteins are involved in capsaicin- and cisplatin-induced apoptosis.

To validate whether capsaicin and cisplatin could affect CRT expression in MG-63 cells, a western blot assay was used to analyze CRT protein expression in whole cell lysates. The result showed that treating cells with capsaicin or cisplatin for 12 h did not change the total cellular CRT expression level (Fig. 4).

To validate whether capsaicin and cisplatin could affect CRT subcellular localization, MG-63 cells were treated with 200 µM capsaicin or 32 µg/ml cisplatin, and FCM was used to assay CRT expression on the cell surface. The result (Fig. 5) showed that capsaicin treatment for 12 h significantly increased the expression of CRT on the cell surface, but no such effect was observed when the cells were treated with cisplatin. In this experiment, MG-63 cells have been dyed with fluorescence- labeled secondary antibody alone as a control, to exclude non-specific fluorescence binding to cells (Fig. 5).

In view of the established role of CRT as an ‘eat me’ signal, and the results from the experiments described above, the difference of phagocytotic rates among the two drug-treated MG-63 cells by DCs was investigated. The DCs (effector cells) and drug-treated MG-63 cells (target cells) were co-cultured for 2 h in a 1:1 effector/target ratio, and the phagocytotic rate was determined by FCM. The results (Fig. 6) showed that MG-63 cells treated with capsaicin were phagocytosed at a higher rate than the MG-63 cells treated with cisplatin, indicating that CRT on the cell surface enhanced the phagocytosis of the human OS cells by DCs.

To investigate whether MG-63 cells with high CRT expression levels on the cell surface can induce antitumor immunity, the ability of DCs loaded with MG-63 cells to activate tumor cell-specific T-cell responses was investigated. The drug-treated MG-63 cells (target cells) were co-cultured with DCs (effector cells), then MG-63-loaded DCs were further co-cultured with lymphocytes for 24 h. The IFN-γ and IL-4 expression levels in the supernatant were then analyzed by ELISA. The result (Fig. 7) showed that, compared with MG-63 cells treated with cisplatin, capsaicin-treated MG-63 cells expressed a higher level of IFN-γ (P<0.01) in the supernatant, but no significant change was observed in the IL-4 expression level.