The Annexin V-FITC kit and propidium iodide (PI)/RNase staining buffer for apoptosis were obtained from BD Biosciences (Franklin Lakes, NJ, USA). Eagle's minimum essential medium (EMEM), penicillin-streptomycin and trypsin-EDTA were obtained from HyClone (HyClone: GE Healthcare Life Sciences, Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco-BRL (Gibco-BRL: Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell Counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich Chemical Co. (Sigma-Aldrich Chemical Co.: Merck KGaA, Darmstadt, Germany). All other chemicals were of analytical reagent grade.

HeLa cells obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) were cultured in EMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

1-[(3S,4R)-2,2-dimethyl-3-oxo-4-(2-piperidonyl)chroman-6-yl]-3-phenylurea (S32) and other chroman analogs (S10, S16, S18, S24, and S26) were obtained from the laboratory of Dr D.S. Shin (Changwon National University). The stock solutions of all chroman analogs were prepared in DMSO as 100 mM and maintained at 4°C. Further dilutions were made immediately prior to each experiment.

HeLa cells were seeded at 5×10³ cells into each well of a 96-well microplate. After 24 h, media were replaced with fresh media containing the various concentrations (20, 40 and 80 µM) of S32. The plate was incubated for a further 48 h. Then, CCK-8 reagent (10 µl) was added to each well of the plate and incubated for 2 h. The cell viability was assessed using WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] according to the manufacturer's recommendations (29). The optical density for living cells was read at 450 nm using a multi-microplate reader (Synergy HT; BioTek Instruments, Inc., Winooski, VT, USA). For determination of cell proliferation, cells were seeded at 5×10³ cells/ml media in 96-well microplates and treated with or without S32 (70 µM) for various times (0, 12, 24, 36, 48 and 60 h). Each experiment was repeated at least three times.

HeLa cells were distributed (1×10⁵ cells/well) into a 6-well plate and allowed to stand overnight. The cells were treated with S32 (70 µM) for 24 and 48 h. Wells that were not treated with S32 received an equivalent volume of DMSO (<0.1%) used as a control. Phase-contrast images were captured with a Nikon Phase Contrast-2, ELWD 0.3 inverted microscope.

Production of ROS was assessed using the fluorescent indicator 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), a cell-permeable indicator for ROS, shown to react with H₂O₂ (30). H₂DCF-DA is oxidized to highly green fluorescent 2′,7′-dichlorofluorescein (DCF) via the generation of ROS. HeLa cells (3×10⁵ cells in a 60-mm dish) treated with (70 µM) or without S32 were collected by trypsinization and centrifugation at 300 × g for 5 min. The pellets were washed with cold PBS and stained with 2 µl of H₂DCF-DA for 30 min at 37°C in a dark room. Relative fluorescence intensities were observed using the FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by CellQuest Pro software (Becton-Dickinson: BD Biosciences, Franklin Lakes, NJ, USA) with the FL-1 channel (green) set to 530 nm.

Changes in MMP were detected by using a fluorescent probe, rhodamine 123 (RH-123; Molecular Probes, Inc.; Thermo Fisher Scientific, Inc., Eugene, OR, USA). HeLa cells (3×10⁵ cells in a 60-mm dish) treated with (70 µM) or without S32 were collected by trypsinization and centrifugation at 300 × g for 5 min. The pellets were washed with cold PBS and stained with 5 µl of rhodamine and intensities were observed by the FACSCalibur flow cytometer (BD Biosciences) and analyzed by CellQuest software (Becton-Dickinson: BD Biosciences) with the FL-1 channel.

The [³H]-thymidine incorporation assay was performed as described in a previously study (31). HeLa cells were cultured in 6-well plates in growth media (EMEM + 10% FBS + 1% penicillin-streptomycin). After the cells were grown to 70–80% confluence, they were rendered quiescent by incubation for 24 h in EMEM containing 2% FBS. Cells were then treated with (70 µM) or without S32 in EMEM supplemented with 10% FBS. After incubation for 21 or 45 h, [³H]-thymidine was added at 1 µCi/ml (1 µCi = 37 kBq) and further incubated for 3 h. Incorporated [³H]-thymidine was measured by using a Liquid Scintillation Analyzer (Tris-Carb 2910TR; PerkinElmer, Inc., Waltham, MA, USA).

HeLa cells (3×10⁵ cells in a 60-mm Petri dish) treated with (70 µM) or without S32 were collected by trypsinization and washed with cold PBS by centrifugation at 412 × g for 6 min. After suspension in PBS and fixation with 70% ethanol (v/v), samples were washed with cold PBS and stained with PI/RNase staining buffer for 15 min at room temperature. The number of cells in the different cell cycle phases was analyzed using a FACSCalibur flow cytometer analysis system (BD Biosciences) and 20,000 events were analyzed for each sample. The percentage of cells in the different phases was determined using ModFit software (Verity Software House, Inc., Topsham, ME, USA).

HeLa cells (3×10⁵ cells in a 60-mm dish) treated with (70 µM) or without S32 were collected by trypsinization and washed with ice-cold PBS via centrifugation at 2,500 × g for 3 min. Subsequently, 1×10⁵ cells were resuspended in 100 µl of binding buffer and stained with 5 µl of Annexin V-FITC and 10 µl of PI (50 µg/ml) for 15 min at room temperature in the dark. Analysis was performed using FACSCalibur flow cytometer (BD Biosciences) with 10,000 events each time. The data were analyzed by CellQuest Pro software (Becton-Dickinson: BD Biosciences).

After the treatment of HeLa cells (1×10⁵ cells in a 150-mm dish) with (70 µM) or without S32, total cell lysates and cytosolic fractions were prepared as described in a previous study (32). Protein contents of the lysates were determined by the Bradford protein assay. Proteins (20 µg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes by western blot analysis. The following primary polyclonal antibodies were used: β-actin (cat. no. 4967), pro-caspase-8 (cat. no. 4790), pro-caspase-9 (cat. no. 9502), Bcl-2 (cat. no. 2872) (1:1,000 dilution; rabbit; Cell Signaling Technology, Inc., Danvers, MA, USA), pro-caspase-3 (1:300; mouse; cat. no. sc-7272; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and Bax (1:1,000; mouse; cat. no. 556467; BD Biosciences, San Diego, CA, USA). The results were quantified using ImageJ v.1.43 software (National Institutes of Health, Bethesda, MD, USA).

Each experiment was repeated at least three times. The results are expressed as the mean ± standard deviation (SD) values of three independent experiments. Statistical analysis was performed by one-way analysis of variation (ANOVA). The criterion for significance was set at P<0.05. For the statistical and graphical evaluations, Microsoft Excel 2007 was used.

In the present study, we evaluated the cytotoxic effect of chroman compounds, S26 and S32, at different concentrations (75, 150, and 300 µM) on HeLa cells. After incubation with the chroman compounds for 48 h, we observed a significant (P<0.05) concentration-dependent cytotoxicity of S32 compared to that observed with the control. In addition, we found that S32 inhibited cell viability to a greater extent than the mother compound S26 (Fig. 2). Therefore, we selected S32 for further study. As shown in Fig. 3A, the IC₅₀ value of S32 was 70 µM. Treatment of HeLa cells with 70 µM of S32 for various time periods (0, 12, 24, 36, 48 and 60 h) showed that their growth gradually increased until 12 h and started to decrease at 24 h, whereas untreated cells maintained exponential proliferation (Fig. 3B). These data demonstrated that S32 decreased HeLa cell viability in a concentration- and time-dependent manner.

Morphological changes are important characteristics of apoptotic cells. Microscopic analysis revealed the occurrence of apoptosis in cells treated with 70 µM of S32 for various durations (Fig. 4). As shown in Fig. 4A and C, non-treated HeLa cells proliferated regularly throughout the culture plate and grew to near confluence. After 24 h of treatment with S32, some cells were detached from the plate but the majority of the attached cells maintained a normal shape (Fig. 4B). After 48 h of treatment, the number of floating cells increased and the attached cells showed cell shrinkage and disruption, indicating apoptosis (Fig. 4D).

Several studies have reported that ROS can activate the mitochondrial permeability transition and loss of MMP (33). By using the cell permeable dye, we showed that S32 has the capacity to induce the generation of intracellular ROS. Treatment of the HeLa cells with 70 µM of S32 for 1 h induced ROS generation compared to that observed with the control. As shown in Fig. 5, the mean H₂DCF-DA fluorescence increased from 109.26 to 341.67 after treatment with S32 for 1 h. Apoptosis induces mitochondrial membrane depolarization. A decrease in H₂DCF-DA fluorescence suggests the loss of MMP. We examined S32-induced MMP loss in HeLa cells. As shown in Fig. 6, the mean RH-123 fluorescence significantly (P<0.05) increased from 91.93 (0 h) to 99.73 (12 h) and then significantly (P<0.05) decreased to 66.75 after treatment with S32 for 24 h, respectively, suggesting that S32 induced apoptosis.

To identify that S32 affects cells at the DNA level, we analyzed DNA replication in cells treated with 70 µM of S32 by using a [³H]-thymidine incorporation assay. As shown in Fig. 7A, [³H]-thymidine incorporation was significantly (P<0.05) reduced in HeLa cells treated with S32, suggesting that DNA replication was inhibited in a time-dependent manner.

Cell proliferation and apoptosis are controlled by regulators of cell cycle progression and apoptotic impulses (34,35). The appearance of a sub-G₀/G₁ peak, also termed apoptotic peak, on flow cytometric DNA content histograms is thought to be one of the features of cells undergoing apoptosis (36). To examine the effect of S32 on cell cycle progression, HeLa cells were treated with 70 µM of S32 for 24 or 48 h, and analyzed by using flow cytometry. Treatment with S32 (Fig. 7Bb and d) increased the fraction of G2-phase cells compared to the untreated cells (Fig. 7Ba and d). In addition, treatment of S32 increased the percentage of sub-G1 phase (apoptotic) cells in a time-dependent manner. These data demonstrated that S32 induced G2 phase cell cycle arrest in HeLa cells.

To confirm that apoptosis was induced by S32, HeLa cells were treated with 70 µM of S32 for 24 or 48 h and were then analyzed using flow cytometry after staining with Annexin V-FITC and PI. The staining of cells with Annexin V-FITC and PI is used to distinguish and quantify non-apoptotic (Annexin V-FITC⁻/PI⁻), early apoptotic (Annexin V-FITC⁺/PI⁻), and late apoptotic (or necrotic) (Annexin V-FITC⁺/PI⁺ and Annexin V-FITC⁻/PI⁺) cells (37). Treatment with S32 increased the fraction of apoptotic cells (Fig. 8B and D) compared to the non-treated cells (Fig. 8A and C), confirming that S32 induced apoptosis in HeLa cells in a time-dependent manner.

The Bcl-2 family of proteins plays a crucial role in the regulation of cell life and death (38). The ratio between pro- (e.g., Bax) and anti-apoptotic (e.g., Bcl-2) proteins determines, in part, the susceptibility of cells to a death signal (39). We evaluated the effect of S32 treatment on the Bax/Bcl-2 ratio by using western blot analysis. As shown in Fig. 9, the Bcl-2 level decreased while the Bax level increased with time in cells treated with S32, indicating that the Bax/Bcl-2 ratio significantly (P<0.05) increased in a time-dependent manner (Fig. 9B).

In response to apoptotic stimuli, the outer mitochondrial membrane becomes permeable, resulting in the release of cytochrome c and other caspase activators (11). We evaluated whether the caspase-dependent mitochondrial-mediated pathway is involved in S32-induced apoptosis to determine the underlying molecular mechanism of this process. The pro-caspase-9 and −3 levels were markedly decreased in cells treated with S32, while the pro-caspase-8 level decreased slightly in a time-dependent manner (Fig. 9), demonstrating the activation of the caspase cascade. These results suggested that S32 induces apoptosis in HeLa cells primarily via the mitochondrial-mediated pathway.