7-PEC was synthesized according to the procedure described in our previous report (17). 7-PEC (>95% purity) was dissolved in DMSO and added to the experimental media to give the final concentrations. Antibodies for detecting p-Akt, Akt, p53, Bcl-2, Bax, cyt-c, pro-caspase-9, pro-caspase-3, PARP1 and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Ac-DEVD-CHO and Rhodamine 123 were purchased from the Beyotime Institute of Biotechnology (Haimen, China). Hoechst 33258 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 was purchased from Gibco (Invitrogen, Carlsbad, CA, USA). Neonatal bovine serum (NBS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co. (China).

HCT-116 human colon cancer cells were kindly provided by Shanghai Jiao Tong University. The cells were routinely cultured in RPMI-1640 medium, supplemented with 10% NBS. The culture was maintained at 37°C with a gas mixture of 5% CO₂/95% air. All media were supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin.

The cells were seeded in 96-well microtiter plates (3×10⁴/ml). After 12 h of incubation in the appropriate medium, cells were treated with various concentrations (1, 10, 25 and 50 μM ) of 7-PEC for another 72 h (24 or 48 h). Subsequently, 10 μl of MTT stock solution was added to each well for an additional 4 h of incubation. Then, 100 μl of DMSO was added to each well and the absorbance at 570 nm was determined with a microplate reader. Using the MTT method, cell numbers were obtained as absorbance values. The results were expressed as viability compared with that of control cells. Each treatment and time-point had three independent wells. The representative data shown in this study are the results of three independent experiments.

Cell morphological changes were assessed by Hoechst 33258 staining. Briefly, following exposure to 7- PEC for 48 h, the cells were washed twice with PBS and fixed with 4% formaldehyde at 4°C for 10 min. The samples were then washed with PBS and stained with Hoechst 33258 solution (0.5 μg/ml) for 10 min at room temperature. Finally, the cells were observed under the fluorescence microscope (Nikon Eclipse Ti-s, Nikon Corp., Tokyo, Japan).

ΔΨm was measured using Rhodamine 123. Briefly, cells under different concentrations of 7-PEC treatment were incubated with Rhodamine 123 (5 μg/ml) at 37°C for 30 min, and washed with PBS. The cell pellet was collected by centrifugation (1,500 × g, 3 min), and resuspended in 1 ml of PBS. Fluorescence intensities of Rhodamine 123 in cells were analyzed by flow cytometric analysis.

For cell cycle analysis, HCT-116 cells (1×10⁵ cells/ml, 3 ml) were cultured in 6-well plates, with or without 7-PEC (1.25, 2.5 and 5 μM) for 48 h. Cells were collected and resuspended in 500 μl of PBS containing 0.025 mg of propidium iodide (PI) and 50 μg of RNase for 30 min at room temperature in the dark. Flow cytometry was performed on Quanta SC (Beckman Coulter, Fullerton, CA, USA).

Briefly, HCT-116 cells (1×10⁵ cells/ml) exposed to 7-PEC for 48 h were determined by flow cytometry (Quanta SC, Beckman Coulter) using a detection kit. Following 7-PEC treatment, cells were collected and washed twice in cold PBS and resuspended in 200 μl of binding buffer (1×10⁵ cells/ml). The samples were incubated with 5 μl of Annexin V-FITC and 5 μl PI in the dark for 15 min at room temperature. Finally, samples were analyzed by flow cytometry and evaluated based on the percentage of cells for Annexin V-positive.

HCT-116 cells were treated with 7-PEC (1.25, 2.5 and 5 μM) for 48 h. Proteins were extracted with cell lysis buffer for western and IP (Beyotime Institute of Biotechnology). Equal amounts (40 μg/lane) of protein were separated on 10 or 15% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Bedford, MA, USA) and blocked at room temperature for 1 h in 3% (w/v) non-fat milk in TBST. The blots were incubated overnight at 4°C with the primary antibodies diluted in TBST buffer. The membranes were incubated with anti-Akt, p-Akt, Bcl-2, Bax, p53, cyt-c, pro-casapse-3, pro-caspase-9, PARP1 and β-actin primary antibodies (1:1000). After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:5000), and visualized with the ECL detection kit (Thermo, USA), according to the manufacturer’s instructions.

The elevations of intracellular ROS induced by 7-PEC in HCT-116 cells were detected by DCFH-DA (2′,7′-dichlorofluorescein diacetate) using flow cytometry. This compound is a cell-permeant indicator for ROS that is non-fluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell. Briefly, cells were seeded at 1×10⁵ cells/well in 6-well plates, and treated with or without 7-PEC (5, 10 and 20 μM). At the indicated times, cells were harvested and washed with PBS, then resuspended in PBS containing DCFH-DA (10 μM) and incubated for 20 min at 37°C. After the inhibition, cells were washed twice by PBS and then analyzed by flow cytometry.

Results are expressed as the mean ± SD for three independent experiments. Statistical differences were evaluated using Student’s t-test or one-way analysis of variance (ANOVA). P<0.05 was considered to indicate statistically significant differences.

The cytotoxic effects of 7-PEC on five different cell lines were examined by MTT assay. The results showed that the cytotoxicity of 7-PEC on HCT-116 cells is most potent; it is comparable with that of 5-FU as a positive control (Table I). HCT-116 cells in exponential growth were treated with graded concentrations of 7-PEC (1, 10, 25 and 50 μM) for 24, 48 and 72 h. Under the experimental conditions, 7-PEC treatment exhibited strong inhibition on the survival of HCT-116 cells in a time- and dose-dependent manner as shown in Fig. 2. The IC₅₀ values were calculated as 16.25, 5.49 and 1.5 μM in cells treated for 24, 48 and 72 h, respectively.

In order to elucidate whether the loss of HCT-116 cell viability induced by 7-PEC was associated with apoptosis, the occurrence of apoptosis was identified with Hoechst 33258 staining. HCT-116 cells were incubated with various concentrations of 7-PEC (2.5 and 5 μM) for 48 h and stained by Hoechst 33258 for observation of the morphology. As is clearly shown in Fig. 3E, significant nuclear condensation and morphological changes for HCT-116 cells were observed, whereas in the control group, the cells without 7-PEC treatment demonstrated normal nuclear morphology. These data confirmed that 7-PEC could induce apoptosis in HCT-116 cells.

To investigate the effects of 7-PEC on apoptosis and the cell cycle of HCT-116 cells, sub-diploid DNA-content and phosphatidylserine (PS) externalization were measured by FACS after PI and Annexin V-FITC/PI staining. For the cell cycle study, HCT-116 cells were treated with 7-PEC (1.25, 2.5 and 5 μM) for 48 h, and the DNA content of 10, 000 events was analyzed by flow cytometry. Fig. 3A and B show a dose-dependent increase of apoptosis induction which is indicated by percentage of sub-diploid DNA content. Apoptotic cells reached ~8.53 and 22.27% when the cells were exposed to 2.5 and 5 μM of 7-PEC, respectively. For the apoptosis study, HCT-116 cells were incubated with different concentrations of 7-PEC (4, 6, 8 and 10 μM) for 48 h, and then the cells were subjected to Annexin V-FITC/PI staining and analyzed by flow cytometry. Significant apoptosis for HCT-116 cells is observed in Fig. 3C and D. Upon treatment with 2 and 10 μM of 7-PEC, the percentage of apoptotic cells increased from 3.09 to 50.03%. These results suggest that the Annexin-V-FITC assay is more sensitive than sub-diploid DNA-content measurement for the evaluation of apoptosis.

Caspase, a family of cysteine proteases, is known to form integral parts of the apoptotic pathway (18). Caspase-3 activation is considered the central and final apoptotic marker enzyme for both mitochondrial intrinsic and death-domain receptor-dependent extrinsic pathways. Poly(ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair, is a substrate for caspase-3 (19). Therefore, we investigated the protein levels and activity of caspase-3. As is shown in Fig. 4E, the pro-caspase-9 and -3 protein levels were significantly decreased and cleavage of PARP1 was detected in 7-PEC-treated HCT-116 cells.

To confirm whether 7-PEC specifically triggers caspase-3 expression, caspase-3 protein expression was investigated in HCT-116 cells by treating with 5 μM of 7-PEC for 48 h in the presence or absence of caspase-3 inhibitor, Ac-DEVD-CHO. As shown in Fig. 4C, activation of caspase-3 induced by 7-PEC is blocked in the presence of Ac-DEVD-CHO. MTT results demonstrated that the cell growth inhibition activity of 7-PEC was also weakened by Ac-DEVD-CHO (Fig. 4F). These data suggest that 7-PEC-induced apoptosis might engage caspase-3 dependent signaling cascades. Taken together, our results indicate that 7-PEC-induced apoptosis is possibly via the caspase-dependent apoptotic pathway in HCT-116 cells.

The expression of Akt, p-Akt, p53, pro-caspase-3, pro-caspase-9, PARP1 and cyt-c was measured in HCT-116 cells treated with 7-PEC (1.25, 2.5 and 5 μM). As is shown in Fig. 4, 7-PEC treatment resulted in the decrease of antiapoptotic protein Bcl-2 and increase of the Bax (Fig. 4A), with an increase in the Bax/Bcl-2 ratio (Fig. 4B). In addition, upregulation of p53, cyt-c, pro-caspase-3, pro-caspase-9 and subsequent cleavage of PARP1 were detected in 7-PEC-treated HCT-116 cells. The exposure to 7-PEC had no effects on steady-state levels of total Akt protein, whereas p-Akt levels were decreased significantly in a dose-dependent manner (Fig. 4E). These findings suggest the activation of the mitochondria-based intrinsic apoptosis in HCT-116 cells after 7-PEC treatment.

Early apoptosis is always accompanied by the disruption of the mitochondrial membrane, resulting in a rapid collapse in the electrochemical gradient (20). In this study, we explored the effects of 7-PEC on the loss of ΔΨm using a cationic dye Rhodamine 123, which can diffuse into the mitochondria matrix and reflect the change of ΔΨm (21). Thus, HCT-116 cells were incubated with different concentrations of 7-PEC (4, 6, 8 and 10 μM) for 24 h, and then incubated with Rhodamine 123 dye for another 30 min. Fluorescence emission was measured by flow cytometry. As shown in Fig. 5, the ΔΨm was significantly decreased by 7-PEC in a dose-dependent manner.

Increased production of reactive oxygen species (ROS) triggers cytotoxicity and cell death by increasing oxidative stress. The intracellular production of ROS in HCT-116 cells was measured while treating with 7-PEC (5, 10 and 20 μM) and using DCFH-DA staining. The 7-PEC treatment of HCT-116 cells induced a dose-dependent increase of ROS production. Fig. 6 shows an example of FACS analysis of DCFH-DA-stained HCT-116 cells after 7-PEC treatment. The experiments were triplicated and similar results were obtained.