C57BL/6 and BALB/c mice were purchased from Samtaco Bio Korea and housed in the animal facility at Jeju National University. In this study, 7 to 12-week-old mice were used and all animal experiments in this study were performed in accordance with the Institutional Guidelines for Animal Use and Care of Jeju National University (No: 2021-0047). FBZ was purchased from Sigma-Aldrich (USA) and dissolved in dimethyl sulfoxide (Sigma-Aldrich). VC was purchased from REYON Pharmaceutical Co. (Korea) and was used as a positive control for anticancer drugs. Lipopolysaccharide (LPS) purified from Escherichia coli O26:B6 was purchased from Sigma-Aldrich and dissolved in sterile phosphate-buffered saline (PBS).

The EL4 (mouse lymphoma cell line) was purchased from Korean Cell Line Bank (Korea). The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin-streptomycin, and 2 mM L-glutamine.

The spleen was harvested and physically crushed to obtain cells. After hemolysis and removal of red blood cells using ammonium chloride potassium lysis buffer (Thermo Fisher Scientific, USA), spleen cells were filtered through a 70 µm cell strainer. RPMI 1640 medium containing 10% fetal bovine serum, L-glutamine, and penicillin-streptomycin was added and the cells were ready to be cultured.

To measure the metabolic activity of the cells, EL-4 and spleen cells were treated with FBZ or VC and cultured in 96-well plates for 3 days. EL-4 and spleen cells were cultured at concentrations of 1 × 10⁵ and 2 × 10⁶ cells/mL, respectively. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylltetrazolium bromide (MTT) solution was added at a concentration of 0.5 mg/mL and incubated at 37°C in 5% CO₂ for 4 hours. A 10% sodium dodecyl sulfate solution was added to dissolve the formazan product (crystal violet) produced by the cells and reacted for 2 hours. Optical density was measured at 570 nm using a microplate reader (Multiskan FC; Thermo Fisher Scientific).

Apoptosis, MMP, reactive oxygen species (ROS), and cell cycle analysis were performed using flow cytometry. The EL-4 and spleen cells were treated with FBZ or VC in 24-well plates and incubated for the required number of days, depending on the experiment. For VC, the IC50 was used as the representative concentration (25 ng/mL). EL-4 and spleen cells were cultured at concentrations of 1 × 10⁵ and 1 × 10⁶ cells/mL, respectively. To determine apoptosis, the cells were stained with annexin V-fluorescein isothiocyanate (FITC) (Thermo Fisher Scientific) and washed with annexin V-binding buffer. Subsequently, 1.5 µg/mL propidium iodide (PI; Sigma-Aldrich) were added at room temperature for 10 minutes in the dark. To measure MMP, cells were incubated for 48 hours and treated with 0.1 µg/mL rhodamine 123 (Sigma-Aldrich) for 30 minutes in the dark. The treated cells were washed twice with the fluorescence-activated cell sorting staining solution (FSS). For ROS detection, cells were treated for 72 hours, washed with pre-warmed PBS, and stained with 10 μM 2',7'-dichlorofluorescin diacetate (DCFDA, Sigma-Aldrich). After 1 hour incubation at 37°C, the stained cells were washed with FSS twice. Then, 2',7'-DCF, an oxidized form of DCFDA produced by ROS was measured. For cell cycle analysis, DNA content was evaluated by PI staining. Following treatment with FBZ or VC, the treated cells were washed with PBS and fixed with 70% cold ethanol for 20 minutes at -20℃. After the fixative was removed, the cells were stained with PI solution at a final concentration of 50 μg/mL, with 0.2 mg/mL RNase. All stained cells were analyzed using flow cytometry (CytoFLEX LX; Beckman Coulter, USA) and CytExpert software (Beckman Coulter).

To investigate whether FBZ-treated spleen cells can respond to LPS stimulation, spleen cells were pretreated with FBZ at a concentration of 2 × 10⁶ cells/mL. After 24 hours, the cells were harvested and washed with culture media, and the 2 × 10⁶ cells/mL viable cells were treated with 1 µg/mL LPS. The cells were incubated for another 48 hours, and the metabolic activity was measured using the MTT assay.

Statistical analyses were performed using Prism 9 (GraphPad Software, USA). Data are expressed as the mean ± standard deviation. All experiments were performed at least three times. Statistical significance was analyzed using ANOVA and an appropriate multiple comparison test.

Using the MTT assay, the metabolic activity of EL-4 cells and spleen cells was evaluated after treatment with FBZ or VC (Fig. 1). The metabolic activity of EL-4 decreased significantly at all concentrations, and significantly dropped at concentrations greater than 0.5 µM (p < 0.0001). The metabolic activity significantly decreased at some concentrations in the spleen cells, but was less than the effect observed in EL-4 cells.

Treatment of EL-4 cells with FBZ revealed a decrease in the percentage of live cells in a concentration-dependent manner (Fig. 2). We observed a 52% reduction in the ratio of viable cells following treatment of EL-4 cells with 1 µM FBZ, compared with the controls (0 µM FBZ). On the other hand, treatment of spleen cells with 1 µM FBZ revealed a 9% reduction in the ratio of viable cells compared to the controls. This result confirmed that FBZ markedly induced the death of EL-4 cells compared to spleen cells.

After excluding the estimated cell debris, ROS production in cells was measured using flow cytometry. In EL-4 cells, FBZ increased the mean fluorescence intensity (MFI) of DCFDA in a concentration-dependent manner (Fig. 3A). In particular, a 48% and 53% increase in the rate of ROS production at 1 µM and 4 µM FBZ compared to the control (0 µM) group was observed. In spleen cells, the tendency of ROS production was completely different; the MFI in spleen cells treated with DCFDA was not significantly different at all concentrations compared to the controls. VC (25 ng/mL) showed a similar trend of ROS production as FBZ in both cell types.

To investigate whether FBZ influences mitochondrial integrity of cells, we treated EL-4 cells with FBZ and stained them with rhodamine 123 solution. The MFI of rhodamine 123 was decreased by FBZ in a concentration-dependent manner in both EL-4 cells and spleen cells (Fig. 3B). At the final concentration of FBZ (4 µM), MMP was decreased by 43% and 33% in EL-4 cells and spleen cells, respectively. When treated with 25 ng/mL VC, the MMP decreased by 39% and 41% in EL-4 cells and spleen cells, respectively.

To determine whether FBZ affects the cell cycle, we treated EL-4 cells with FBZ and stained with PI solution. As shown in Fig. 4, the fraction of EL-4 cells in the G2/M phase increased at concentrations of 1 and 4 µM. The fraction of cells in the S phase was decreased, which was accompanied by a marked increase in the fraction of cells in the sub G0/G1 phase. In contrast, in spleen cells, each fraction of the cell cycle was not notably altered by FBZ treatment. This result confirmed that FBZ causes a distinct change in the G2/M phase of the cell cycle of EL-4 cells.

We investigated whether spleen cells treated with FBZ could maintain their activation status. First, spleen cells were pretreated with FBZ for 24 hours, and then viable cells were stimulated with LPS for another 48 hours (Fig. 5). The cells were significantly activated at all FBZ concentrations. Although the number of viable spleen cells was decreased by 21% at 1 µM FBZ (data not shown) compared to control (0 µM FBZ), the activation ability of the viable spleen cells to LPS stimulation did not decrease. These data reveal that FBZ had a minimal effect on the responding ability of viable spleen cells to LPS.