Human promyelocytic leukemia HL-60 and plasma cell leukemia ARH-77 cells (Bioresource Collection and Research Centre) were cultured in RPMI-1640 medium plus 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO₂. Vero cells (a monkey kidney epithelial cell line) (Bioresource Collection and Research Centre) were cultivated in Eagle's Minimum Essential Medium (Thermo Fisher Scientific, Inc.) containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C, 5% CO₂.

Ethyl 2-anilino-4-oxo-4,5-dihydrofuran-3-carboxylate (compound 131) was manufactured and refined using high-performance liquid chromatography as described in a previous study (10). Briefly, a mixture of diethyl malonate (32.0 g, 0.2 mol) in 50 ml tetrahydrofuran (THF) with chloroacetyl chloride (11.3 g, 0.1 mol) in 100 ml THF was incubated at 10–12°C for 1 h followed by 40–45°C for 1 h; ethyl 2-ethoxy-4-oxo-4,5-dihydrofuran-3-carboxylate was produced post cooling. Finally, the ethoxy group in the compound was substituted with aniline after stirring at room temperature for 1 h and heating on a water bath at 80°C for 3 h to yield ethyl 2-anilino-4-oxo-4,5-dihydrofuran-3-carboxylate (compound 131). After the product of the reaction had been confirmed by performing via thin layer chromatography on silica gel-protected aluminum sheets (Type 60 F254; Merck KGaA) in which the spots were detected using a UV-lamp, the reaction was further mixed with 100 cc of ice water to form a precipitate; white crystals of compound 131 (18.29 g; yield, 74%; melting point, 115–116°C) were generated after the precipitate was recrystallized from 90% ethanol at room temperature °C for 1–2 days. The structure of compound 131 (Fig. 1) was confirmed via mass spectrometry (m/z) as follows: 3267.87 (-NH-), 1695.26 (C4=O), 1672.59 (C3-CO-OEt); UV λmax nm (MeOH) (log ε): 297 (4.523); 1H-NMR (200 MHz, CDCl₃) δ: 1.24 (3H, t, J=7 Hz, H-2″), 4.20 (2H, q, J=7 Hz, H-1″), 4.67 (2H, s, H-5), 7.25 (5H, m, H-2′, H-3′, H-4′, H-5′, H-6′), 10.264 (1H, s,-NH-); 13C-NMR (200 MHz, DMSO-d6) δ: 14.67 (C-2″), 59.37 (C-1″), 75.30 (C-5), 86.99 (C-3), 123.48 (C-2′, C-6′), 126.35 (C-4′), 129.27 (C-3′, C-5′), 135.24 (C-1′), 164.18 (C-2), 177.34 (C-3″), 188.84 (C-4).

HL-60, ARH-77 or Vero cells (1×10⁴/well) were cultured in 96-well plates in the presence or absence of compound 131 (0, 5, 25 and 50 µM) at 37°C for 2 days followed by the addition of 20 µl 0.5 mg/ml MTT (Sigma-Aldrich; Merck KGaA) per well for another 4-h incubation at 37°C. After removing the cultured media, the reduced form of MTT in the cells was solubilized using 150 µl dimethyl sulfoxide for 15 min. The survival rate of treated cells was measured as the ratio of the optical density (OD)_{(570–630 nm)} of treated cells to that of mock cells, similar to the calculation of the 50% cytotoxic concentration (CC₅₀).

A total of 2×10⁵ HL-60 cells were treated with compound 131 (0, 5, 25 or 50 µM) for 48 h at 37°C. Subsequently, cells were collected after centrifugation at 100 × g (Kubota Corporation) for 5 min at room temperature and cell cycle profile analysis was performed using propidium iodide (PI) staining and caspase-3 activity analysis using BD ApoAlert Caspase Fluorescent assay kit (cat. no. 51-6632AK and 51-6632BK; BD Biosciences) as described in our previous report (14,15). PI-stained cells were analyzed at a wavelength of 488 nm by flow cytometry (BD Biosciences). The supernatants of treated cell lysates were added to the wells of the caspase-3 assay with BD ApoAlert Caspase Fluorescent assay kit for a 2-h incubation at 37°C according to the manufacturer's protocol. Subsequently, caspase-3 activity was examined using a fluorescent substrate and a fluorescent plate reader (BioTek China) using an excitation wavelength of 380 nm and an emission wavelength of 460 nm.

The cells treated with compound 131 were harvested and washed with ice-cold PBS by centrifuging at 100 × g (Kubota Corporation) for 5 min at 4°C, and then mixed with the RIPA lysis buffer (cat. no. R0278, Sigma-Aldrich; Merck KGaA) in microfuge tubes for 30 min at 4°C. The lysate of treated cells was collected after centrifugation at 12000 × g for 20 min at 4°C, and the protein concentration was determined using coomassie protein assay reagent (cat. no. 27813, Sigma-Aldrich; Merck KgaA) at an absorbance of 280 nm. A total of 20 µg of lysate per lane from the cells treated with compound 131 was dissolved in 2X SDS-PAGE sample buffer (Sigma-Aldrich; Merck KGaA), boiled at 95°C for 5 min, loaded in 12% SDS-PAGE gels and then analyzed using a vertical electrophoresis system (Bio-Rad Laboratories, Inc.). The separated proteins in the gels were transferred to nitrocellulose papers, and were blocked with 5% skim milk at room temperature overnight. The immune reaction on the blots was performed using a 1:2,000 dilution of anti-caspase-3 (cat. no. 9662), anti-Bax (cat. no. 2772), anti-Bcl-2 (cat. no. 3498) and anti-β-actin (cat. no. 4970) antibodies in at room temperature overnight, and 1:3,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (cat. no. 7076) (Cell Signaling Technology, Inc.) at room temperature for 2 h. The immune-reactive bands were developed using enhanced chemiluminescence solution (Amersham Pharmacia Biotech Ltd.), as previously described (14,15).

To detect intracellular calcium levels, treated cells were collected and incubated with 10 µM Fluo-3/AM for 30 min at 37°C in the dark and analyzed at 526 nm by flow cytometry (16). In the intracellular ROS assay, cells stained with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) were examined using flow cytometry as described in our previous study (17). To measure the ΔΨ_m, compound 131-treated cells were analyzed with DiOC₆ staining at 37°C for 30 min and examined using flow cytometry (17).

Data from three independent experiments of mock cells and compound 131-treated cells were analyzed by one-way ANOVA followed by Scheffe's post hoc test using SPSS 12.0 (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Initially, 23 synthesized intermediates of furoquinolone at 50 µM were screened for anti-proliferative activity against HL-60 and Vero cells; only compound 131 (ethyl 2-anilino-4-oxo-4,5-dihydrofuran-3-carboxylate) exhibited a significant inhibitory effect on the proliferation of HL-60 (but not Vero cells) (Fig. S1). Subsequently, the cells were treated with compound 131 at 0, 5, 25 and 50 µM for 48 h at 37°C; the cell viability was tested using the MTT assay to determine the CC₅₀ values of compound 131 on the proliferation of HL-60, ARH-77 and Vero cells. The survival rates of HL-60 and ARH-77 cells treated with compound 131 were significantly lower compared with treated Vero cells (Fig. 2). The CC₅₀ values of compound 131 were 23.5 µM for HL-60, 24.2 µM for ARH-77 and 87.0 µM for Vero cells. These results demonstrated that compound 131 exhibits significant anti-proliferative effects against HL60 and ARH-77 cells.

To determine whether compound 131 initiated apoptosis, the cell cycle and caspase-3 activity of treated cells were further analyzed using flow cytometry, western blotting and caspase-3 enzymatic activity assays (Figs. 3 and 4). Cell cycle analysis indicated that compound 131 increased the percentage of cells in the sub-G₁ phase (apoptotic fraction) in a concentration-dependent manner in HL-60 cells (Fig. 3A). The percentage of apoptotic cells (sub-G₁ fraction) was 0.2, 1.6, 75.4 and 80.0% in cells treated with compound 131 at 0, 5, 25 and 50 µM, respectively (Fig. 3B). Moreover, western blot analysis of cell lysates indicated that compound 131 treatment increased the protein levels of pro- and active forms of caspase-3, and also upregulated Bax and downregulated Bcl-2 in a concentration-dependent manner (Fig. 4A and B). Fluorescence assays of caspase-3 enzymatic activity revealed that caspase-3 enzymatic activity in compound 131-treated cells was higher compared with mock cells by 6-, 43- and 140-fold for 5, 25 and 50 µM, respectively (Fig. 4C). The current results indicate that compound 131 significantly promotes caspase-3 and Bax activation, but suppresses Bcl-2 expression, in apoptotic cells.

Intracellular Ca²⁺ accumulation and ROS generation serve a critical role in apoptosis (17–20). The effects of compound 131 on intracellular Ca²⁺ and ROS levels in HL-60 cells were examined (Figs. 5 and 6). Following treatment with compound 131 (0, 5, 25 or 50 µM) at 37°C for 48 h, cells were harvested, stained with Fluo-3/AM or DCFH-DA and analyzed using flow cytometry. Compound 131 treatment resulted in the increase of intracellular Ca²⁺ release from the ER in HL-60 cells. A 39.2% increase resulted from treatment with 5 µM, a 93.8% increase from treatment with 25 µM and an 80.4% increase following treatment with 50 µM compared with PBS-treated cells, respectively (Fig. 5). In addition, compound 131 significantly increased the production of intracellular ROS compared with mock cells (Fig. 6). The results indicate that compound 131 treatment significantly stimulates an increase in intracellular Ca²⁺ and ROS levels in HL-60 cells.

The disruption of mitochondrial membrane potential (MMP) has been reported to correlate with ROS generation (21–23). Treated cells were tested to determine the MMP levels by staining with DiOC₆ and analysis using flow cytometry (Fig. 7). Cells treated with compound 131 exhibited a significant decrease in DiOC₆ intensity by 19.6% for 5 µM, 32.5% for 25 µM and 27.45% for 50 µM. Thus, it was revealed that treatment with compound 131 also resulted in a decrease in MMP in HL-60 cells.