The propidium iodide (PI)/RNase staining buffer and Annexin-FITC kit for apoptosis were from BD Biosciences Pharmingen (San Diego, CA, USA). Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS, pH 7.4), 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Eagle’s minimum essential medium (EMEM), fetal bovine serum (FBS), penicillin-streptomycin and trypsin-EDTA were obtained from Hyclone Laboratories, Inc. (Logan, UT, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Osaka, Japan). MitoProbe™ JC-1 kit was obtained from Molecular Probes, Inc. (OR, USA). The primary antibodies used were polyclonal rabbit Bcl-2, Bax, cytochrome c, Apaf-1, procaspase-9, -3, -8 and (β-actin (Cell Signaling Technology, Inc., MA, USA). Polyclonal rabbit p53 and p21 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were of analytical reagent grade.

(3R,4S)-2,2-dimethyl-6-nitro-4-(4-(3-(trifluoromethyl)phenyl) piperazin-l-yl)chroman-3-ol (C110g), which appears as a light-yellow solid with the melting point 76–79°C, was synthesized from the laboratory of Dr D.S. Shin and identified according to the spectrometric data: [ a ] D 20 -143.6 (c 1.0, CH₂C1₂); ¹H NMR (400 MHz, CDC1₃) δ 1.17 (s, 3H), 1.46 (s, 3H), 2.53 (d, J= 2.0 Hz, 1H), 2.93–2.99 (m, 4H), 3.15–3.24 (m, 4H), 3.74 (d, J = 2.0 Hz, 1H), 3.80 (dd, J = 10.0,2.0 Hz, 1H), 6.79 (d, J = 9.2 Hz, 1H), 6.99–7.06 (m, 3H), 7.28 (t, J = 8.0 Hz, 1H), 7.97 (dd, J = 8.8, 2.4 Hz, 1H), 8.41 (d, J = 2.0 Hz, 1H); ¹³C NMR (100 MHz, CDC1₃) δ 18.9, 26.7, 49.7, 50.1, 63.1, 70.5,79.9,112.6,113.4, 116.2, 118.4, 119.2,121.2, 124.7,124.8 (d, J_CF = 3.1 Hz), 129.6, 136.2, 164.8, 167.7, 172.0; MS (EI) m/z: 451 (M⁺, 9%), 380 (22), 200 (100), 172 (17), 145 (8), 56 (8). Stock solution of C110g were prepared in DMSO and kept at 4°C. Further dilutions were made immediately prior to each experiment.

HeLa cells obtained from American Type Culture Collection (ATCC) were cultured in EMEM medium supplemented with 10% FBS at 37°C in an atmosphere of 5% CO₂.

Cell viability was determined by WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] colorimetric assay capable of detecting viable cells by the production of a yellow colored formazan, which is soluble in the culture medium (16). HeLa cells were seeded in 96-well plates at 5×10³ cells/well and allowed to attach overnight. Media were changed and the cells were incubated with C110g at various concentrations (2.5,5,10,20 and 40 μM) or DMSO as control for 24 and 48 h. After respective incubation period, the cells were treated with CCK-8 reagent for 2 h at 37°C. The viable cell number is directly proportional to the production of formazan which was read as absorbance values at 450 nm using the multimicroplate reader (Synergy HT, Biotek^®). For the cell proliferation assay, cells were seeded at 5×10³/ml media in 96-well plates and treated with 17 μM of C110g or vehicle alone as control for indicated time periods.

HeLa cells were seeded (1×10⁵ cells/well) into a 24-well plate and treated with 17 μM of C110g for 0, 12, 24 and 48 h. Cells were examined and photographed by Nikon Phase Contrast-2, ELWD 0.3 inverted microscope for the examination of morphological changes. For the Hoechst 33342 nuclear staining (17), HeLa cells were seeded into a 24-well plate containing 10-mm diameter sterile glass cover slips. After C110g treatment, cells were fixed with fixing solution (methanol:acetic acid = 3:1) and stained by Hoechst 33342. Observations were made using a fluorescence microscope with DAPI filter.

Cells treated with or without C110g were collected by trypsinization, washed with PBS 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 by FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA) with 10,000 events each time. The data were analyzed by the CellQuest software (Becton-Dickinson Instruments, Franklin Lakes, NJ, USA).

The cells treated with C110g were collected by trypsinization, fixed with PBS and 70% ethanol (v/v), and stained by PI/RNase staining buffer for 15 min at room temperature. The percentage of cells at different phases of cell cycle was measured by a FACSCalibur flow cytometer and the data were analyzed using Modfit software (Becton-Dickinson Instruments). To further verify the effect of C110g on the cell cycle process, [³H]dTTP incorporation assay was conducted as previously described (18). Briefly, after exposure to 17 μM of C110g for different time periods, cells were supplied with [³H]dTTP at 1 μCi/ml (1 μCi = 37 kBq) and incubated for another 3 h. The incorporated [³H]dTTP was extracted in cell lysis buffer and measured in a liquid scintillation analyzer (Tri-Carb 2910TR, Perkin-Elmer Inc., USA).

The production of ROS was monitored using a fluorescence spectrometer with an oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCF-DA) (19). Briefly, after C110g treatment, cells were harvested and suspended in 0.5 ml PBS containing DCFH-DA (10 μM) for 30 min at 37°C in the dark, followed by quantitative analysis in a FACScan cytometer with the Cell Quest software. To further determine the effect of ROS on cytotoxicity induced by C110g, NAC, a general ROS scavenger, was added to the culture medium 1 h before treatment with C110g. The cytotoxic effect of C110g was measured using CCK-8 kit in the presence or absence of ROS scavenger.

Variations in ΔΨm were measured using JC-1 (5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetra-ethylbenzimidazolylcarbocyanine iodide) as ΔΨm indicator (20). Briefly, after C110g treatment, cells were harvested and suspended in 0.5 ml PBS containing JC-1 (2 μM) for 30 min at 37°C in the dark, followed by quantitative analysis in a FACScan cytometer with the CellQuest software. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial membrane potential disrupter, was used as the positive control for the study of ΔΨm.

Total cell lysates and cytosolic fractions were prepared according to our previous study (21). Briefly, equal amounts of protein (20–50 μg/well) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Schleicher & Schuell, USA) by western blotting. The results were quantified using ImageJ 1.43 C) (National Institutes of Health, Bethesda, MD, USA).

To examine the DNA damage in the single-cell suspensions, we performed an alkaline single-cell gel electrophoresis (comet assay). The comet assay was carried out as previously described (22). The data were analyzed using the Comet 5.5 software. Olive tail moment (OTM) approved by Olive was used to evaluate DNA damage. OTM, expressed in arbitrary units, is calculated by multiplying the percent of DNA (fluorescence) in the tail by the length of the tail in μm (23,24). The tail length is measured between the edge of the Comet head and the end of its tail. A major advantage of using the OTM as an index of DNA damage is that the amount of damaged DNA and the distance of migration of the genetic material in the tail are represented by a single number (25).

Data were collected and expressed as the mean ± standard deviation (SD) of three independent experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA) and differences from the respective controls for each experimental test condition were determined using t-tests. The criterion for significance was set at P<0.05. The 19th version of SPSS (SPSS, Chicago, IL, USA) and Microsoft Excel 2007 (Roselle, IL, USA) were used for the statistical and graphical evaluations.

The cytotoxic effect of C110g on HeLa cells was initially evaluated using WST-8 assay. Cells were treated for 24 and 48 h with a range of drug doses (2.5–40 μM) to determine the IC₅₀ value of C110g. Cell viability was determined and dose response curves were plotted (Fig. 1B). Longer incubation with the drug (48 h), significantly reduced the IC₅₀ value to 17 μM which was used in all subsequent time course experiments. The effect of C110g on proliferation of HeLa cells is illustrated in Fig. 1C. The number of C110g-treated cells gradually increased to 36 h and began to decrease at 48 h, whereas the untreated cells maintained an exponential proliferation rate. A significant difference in the absorbance from the control was observed at 36 h, showing that C110g inhibited HeLa cell proliferation after a 36-h incubation.

Many anticancer drugs act through the induction of apoptosis (26). Microscopic examination of C110g-treated HeLa cell cultures revealed a time-dependent cell death with characteristics of apoptosis, such as cell shrinkage, membrane blebbing and fragmentation of cell into apoptotic bodies (Fig. 2A). Hoechst 33342 staining confirmed apoptotic death by the appearance of nuclear fragmentation and chromatin condensation in C110g-treated cells (Fig. 2B). To quantify the apoptosis triggered by C110g, cells were stained with Annexin V-FITC/PI, and subsequently analyzed by flow cytometry. This double-staining method allows live non-apoptotic cells (Annexin V^negative/PI^negative) to be distinguished from early apoptotic cells (Annexin V^positlve/PI^negative) and late apoptotic cells (Annexin V^positive/PI^positive) (27,28). After treatment with C110g for 0, 12, 24, 36 and 48 h, the corresponding quantities of early and late apoptotic cells were 1.22 and 3.26%, 2.65 and 5.29%, 11.93 and 5.81%, 19.18 and 7.7%, and 17.96 and 15.11%, respectively (Fig. 2C), showing that early apoptosis occurred at 24 h and peaked at 36 h, while late apoptosis became evident at 36 h and continuously increased at tested time-points.

Flow cytometry data exhibited accumulation of cells in the G1 phase with reduction in the S-phase after 24-h treatment with C110g, whereas only modest alteration in the G2/M phases (Fig. 3A). Consequently, the G1/S ratio, which was used as an index of G1 arrest, increased significantly in a time-dependent manner in treated groups (P<0.05), while kept equivalent level in control group through 12–48 h. Moreover, Fig. 3B shows a significant reduction of the [³H]dTTP incorporation in HeLa cells during S-phase of cell cycle, suggesting that there was an inhibitory effect of C110g on the process of DNA synthesis after 24 h. The data reveal that the actions of C110g blocked HeLa cells in the transition from the G1 to the S-phase of cell cycle, suggesting that the observed apoptosis induced by C110g might be due to an arrest of DNA synthesis, thereby, inhibiting further progress in the cell cycle.

To further investigate the molecular mechanisms by which C110g induced G1 cell cycle arrest in HeLa cell, the expression level of G1/S-phase regulatory proteins, p53 and its effector p21 was examined using western blot analysis. As shown in Fig. 3C, p53 and p21 protein levels were markedly increased in early time-points and further decreased with time, peaking at 12 and 24 h respectively. These results suggest that C110 induces cell cycle arrest and apoptosis in a p53-dependent manner in HeLa cells.

To determine the involvement of ROS in C110g-induced cytotoxicity of HeLa cells, ROS production was measured using DCF-DA. Fig. 4A shows that ROS levels were reduced in treated groups, compared with untreated control. Moreover, treatment of NAC, a general ROS scavenger, did not affect cell death induced by C110g in HeLa cells (Fig. 4B). These results indicate that C110-induced apoptotic cell death in HeLa cells did not require the generation of ROS.

It is known that the loss of ΔΨm is associated with apoptotic cell death induced by various stimuli (29). In this study, JC-1, a mitochondria-specific and voltage-dependent dye, was employed to determine whether the mitochondrial metabolism played a role in the apoptosis of HeLa cell by C110g. The mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. As shown in Fig. 4C, treatment of cells with C110g (17 μM) after 6 h or positive control CCCP resulted in significant depolarization of ΔΨm as revealed by a dramatic change in fluorescence from red to green (P<0.05). Marked collapse of ΔΨm demonstrate that dysfunction of mitochondria may be involved in the apoptosis induced by C110g.

Bcl-2 family of proteins, which comprises both pro-apoptotic and anti-apoptotic members, play an important role in p53-dependent apoptosis (30). In our experiments, after treatment with C110g, the pro-apoptotic protein Bax was pronouncedly elevated (P<0.05), while the anti-apoptotic member Bcl-2 was kept in equivalent level (Fig. 5A). Correspondingly, the ratio of Bax/Bcl-2, which is crucial for activation of mitochondrial apoptotic pathway, increased dramatically (P<0.05). Furthermore, cytochrome c was released from the mitochondria to the cytosol and Apaf-1 protein expression was increased. Caspases are another important family of proteins involved in the downstream events of p53-mediated apoptosis (7). Decreases in procaspase -3, -8 and -9 expression levels were observed (P<0.05), demonstrating the activation of caspase cascade (Fig. 5B).

Damage in DNA initiates the DNA damage response, which cooperates with the effector proteins such as p53 inducing cell cycle arrest and DNA repair or apoptosis (31). To determine directly if C110g induces DNA damage, we performed an alkaline comet assay, a sensitive method that detects DNA single- and double-strand breaks, alkali-labile sites, and incomplete excision repair sites at the single-cell level based on the increased tail of DNA migration (32). As Fig. 6 shows, the DNA from C110g treated cells exhibited typical formation of comet, while control cells showed a well defined circular nucleus. DNA damage, as indicated by OTM, started to be evident as early as 12 h after treatment (P<0.05), and further increased in a time-dependent manner. After 48 h, almost the entire population of HeLa cells contained damaged DNA.