RPMI-1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from WelGENE Inc. (Daegu, Korea); 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI), phenol-chloroform, isoamyl alcohol, RNase A, dithiothreitol (DTT), propidium iodide (PI), paraformaldehyde, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl carbocyanine iodide (JC-1) and N-acetylcysteine (NAC), a ROS scavenger were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor and LY294002, a selective inhibitor of PI3K, were obtained from Calbiochem (San Diego, CA, USA) and Cell Signaling Technology, Inc. (Danvers, MA, USA), respectively. 2′,7′-Dichlorodihydrofluorescein diacetate (DCF-DA) and the enhanced chemiluminescence (ECL) kit were purchased from Molecular Probes (Leiden, The Netherlands) and Amersham Pharmacia Biotech (Arlington Heights, IL, USA), respectively. Primary antibodies (Table II) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Cell Signaling Technology, Inc., Bioworld Technology, Inc. (St. Louis Park, MN, USA) and Abcam (Cambridge, UK). Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin were purchased from Amersham Pharmacia Biotech. All other chemicals not mentioned in the present study were purchased from Sigma-Aldrich Chemical Co.

The four medicinal herbs forming HHS were obtained from Dongeui Oriental Hospital, Dongeui University College of Korean Medicine (Busan, Korea). The origin of the medicinal herbs was taxonomically confirmed by Professor Su Hyun Hong at the Dongeui University College of Korean Medicine (Busan). Each of the four herbs in HHS was cut into small pieces, which were mixed together to obtain a total amount of 76 g in the ratios shown in Table I. The mixture was boiled in distilled water (76 g/500 ml) for 3 h. The extract solution was filtered using a 0.45 μM filter to remove insoluble materials, and the blended supernatants were lyophilized and and then crushed into a thin powder (yield, 21% w/w, dried extract/crude herb). The dried extract (HHS) was dissolved in a 100 mg/ml concentration with distilled water, and the stock solution was then diluted with medium to the desired concentration prior to use.

CRC HCT116 cells were purchased from the American Type Culture Collection (ATCC; Manassas, MD, USA) and maintained at 37°C in an incubator with a humidified atmosphere of 5% CO₂ in air. The cells were cultured in RPMI-1640 medium containing 10% FBS, 100 U/ml of penicillin and 100 mg/ml of streptomycin in the presence or absence of HHS. Cell viability was determined using the MTT assay, which is based on the reduction of a tetrazolium salt by mitochondrial dehydrogenases in viable cells. In brief, HCT116 cells were seeded into a 96-well plate at a density of 1×10⁵ cells/well and treated with HHS or reagents. After incubating for 48 h, the medium was discarded, and the cells were washed with phosphate-buffered saline (PBS), followed by incubation with MTT (0.5 mg/ml) at 37°C. After incubating for a further 3 h, the supernatants were aspirated, and the formazan crystals in each well were dissolved in dimethyl sulfoxide (DMSO). Absorbance at 540 nm was read on an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA, USA).

For the assessment of apoptosis, the cells were washed with PBS, and the cell suspension was cytospun onto clean fat-free glass slides using a cytocentrifuge. The cells were fixed with 3.7% para-formaldehyde in PBS and then stained with 2.5 μg/ml of DAPI solution for 10 min at room temperature. The cells were washed twice with PBS, and the images were then captured using a fluorescence microscope (Carl Zeiss, Jena, Germany).

To analyze apoptotic sub-G1 hypodiploid cells, the cells were harvested, washed twice with ice-cold PBS, and fixed with 75% ethanol at 4°C for 30 min. The cells were then incubated with 1 U/ml of RNase A (DNase-free) and 10 μg/ml of PI overnight at room temperature in the dark. A flow cytometric analysis was performed using a flow cytometer (FACSCalibur; Becton-Dickinson, San Jose, CA, USA). The sub-G1 hypodiploid cells were assessed based on histograms generated by the CellQuest and ModFit computer programs (Becton-Dickinson). The level of apoptotic cells containing sub-G1 DNA content was determined as a percentage of the total number of cells. The cells were also stained with 5 μl of Annexin V-fluorescein isothiocyanate (FITC) (R&D Systems, Minneapolis, MN, USA) and 5 μl of PI. Following incubation for 15 min at room temperature in the dark, the degree of apoptosis was quantified as a percentage of the Annexin V-positive and PI-negative (Annexin V⁺/PI⁻ cells) cells using a flow cytometer (15).

The cells were harvested and lysed with lysis buffer [40 mM Tris (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40, 0.1 mM sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml leupeptin and 100 μg/ml phenymethylsulfonyl fluoride] for 30 min. In a parallel experiment, the mitochondrial and cytosolic fractions were isolated using a mitochondrial fractionation kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. The protein concentration was measured using a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). For the western blot analysis, equal amounts of protein (30–50 μg/lane) were fractionated in sodium dodecyl sulphate (SDS)-polyacrylamide gels prior to transfer to the nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). The membranes were blocked with Tris-buffered saline (10 mM of Tris-Cl, pH 7.4) containing 0.5% Tween-20 and 5% non-fat dry milk for 1 h at room temperature and then probed with the desired antibodies for 1 h. The membranes were then washed with PBS and incubated with the secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Immunoreactivity was detected using the ECL western blotting detection system according to the manufacturer's instructions.

The MMP values were determined using the lipophilic cationic probe JC-1, a radiometric dual-emission fluorescent dye that is absorbed and concentrated by respiring mitochondria and can reflect MMP changes in cells. Briefly, the cells were fixed and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature and then incubated with 10 μM of JC-1 for 30 min at 37°C in the dark. Subsequently, the cells were washed with PBS to remove unbound dye, and the amount of JC-1 retained by 10,000 cells/sample was measured at 488 and 575 nm using a flow cytometer (16).

The enzymatic activity of the caspases induced by HHS was assayed using colorimetric assay kits (R&D Systems). Briefly, the cells were lysed in the supplied lysis buffer according to the manufacturer's instructions. The supernatants were collected, and equal amounts of protein were incubated with the supplied reaction buffer containing colorimetric tetrapeptides, Asp-Glu-Val-Asp (DEAD)-p-nitroaniline (pNA) for caspase-3, Ile-Glu-Thr-Asp (IETD)-pNA for caspase-8 and Leu-Glu-His-Asp (LEHD)-pNA for caspase-9. The caspase activity was determined by measuring changes in absorbance at 405 nm using the ELISA reader.

The fluorescent probe DCF-DA method was used to measure the level of intracellular ROS in the cells as previously described (17). Twenty-four hours after plating, the cells were treated with 1 mM NAC for 1 h; HHS was then added, and the cells were incubated for another 48 h. After the media were changed, 10 μM DCF-DA was added to each well, and the cells were incubated for an additional 30 min at 37°C. After washing with PBS, the cells were trypsinized and resuspended in PBS. The fluorescence was measured at specific time intervals using a flow cytometer.

The data derived from at least three independent experiments are expressed as mean ± standard deviation (SD). Statistical comparisons between different groups were performed using a one-way ANOVA, which was followed by Student's t-tests after each treated group was compared to the negative control. Values of p<0.05 were considered to indicate a statistically significant result.

To evaluate the effect of HHS on cell viability, the cells were treated with various concentrations of HHS for 48 h, and an MTT assay was performed. As shown in Fig. 1A, the results indicated that HHS exhibited cytotoxic effects in a concentration-dependent manner. In addition, DAPI staining revealed that the apparent morphological changes, such as nuclear condensation and fragmentation, were concentration-dependently increased in the cells treated with HHS, indicating an increasing number of apoptotic cells, whereas these features were not observed in the control cells (Fig. 1B). To measure apoptotic cell death upon HHS treatment, we stained the cells with PI, and the cell cycle profiles were analyzed using a flow cytometer. The results showed that HHS increased the percentage of cells in the apoptotic hypodiploid sub-G1 phase in a dose-dependent manner (Fig. 1C). Annexin V-FITC/PI double staining was also performed to assess the rate of apoptotic cell death, and a significant degree of apoptosis was detected in the HHS-treated cells (Fig. 1D).

To elucidate the mechanism of HHS-induced apoptosis in the HCT116 cells, we determined whether HHS affects the expression of DR-related proteins. As shown in Fig. 2A, the treatment of HCT116 cells with HHS increased the expression of the Fas ligand (FasL), DR4 and DR5 in a concentration-dependent manner, whereas the levels of Fas and tumor necrosis factor- related apoptosis-inducing ligand (TRAIL) were relatively unchanged in response to the HHS treatment. The western blot analysis also revealed that the exposure of HCT116 cells to HHS caused the progressive accumulation of truncated Bid (tBid), a BH3 interacting domain death agonist, presumably resulting from truncation by the activated caspase-8, in the HHS-treated cells (Fig. 2D).

To confirm whether HHS induced intrinsic mitochondrial-mediated apoptosis, we determined the effect of HHS on MMP, a hallmark of intrinsic apoptosis, by staining with JC-1 dye. The flow cytometric results demonstrated that HHS induced a significant reduction in the MMP levels with increased concentrations of HHS (Fig. 2B), suggesting the depolarization of the mitochondria by HHS. Since the loss of MMP promotes the release of pro-apoptotic proteins, such as cytochrome c into the cytosol (18,19), we determined the levels of cytochrome c in the cytosolic and mitochondrial fractions of HCT116 cells that were treated with different concentrations of HHS. As shown in Fig. 2C, HHS promoted a concentration-dependent increase in the release of cytochrome c from the mitochondria into the cytosol. Since the Bcl-2 family proteins play a crucial role in mitochondrial homeostasis (20,21), the effect of HHS on the levels of Bcl-2 family proteins was also monitored. Our results showed that the incubation of the HCT116 cells with HHS caused a marked reduction in the level of the anti-apoptotic Bcl-2 protein and increased the level of the pro-apoptotic Bax protein (Fig. 2D).

We then examined the expression and activity of caspases in the HHS-treated HCT116 cells to determine whether HHS-induced apoptosis was associated with the activation of the caspases. The results showed that HHS treatment downregulated the levels of pro-caspase-8 and -9, which are the initiator caspases of the extrinsic and intrinsic pathway, respectively, in a concentration-dependent manner (Fig. 3A). The subsequent immunoblotting analysis showed that the levels of pro-caspase-3, an effector caspase, were also downregulated in the HHS-treated cells. Correspondingly, the HHS treatment led to the progressive proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP, 113-kDa), a known caspase-3 substrate (22), which yielded an 85-kDa cleaved fragment. For the further quantification of the proteolytic activation of caspases, the lysates equalized by the protein from the cells treated without or with HHS were assayed for their enzymatic activities. As shown in Fig. 3B, the HHS treatment resulted in the significantly increased activity of caspase-8, -9 and -3. In addition, we examined the expression of inhibitor of the apoptosis protein (IAP) family proteins, which bind to caspases and lead to their inactivation (23,24). As shown in Fig. 3C, HHS treatment reduced the expression of the IAP family proteins, such as XIAP, cIAP-1, cIAP-2 and survivin, indicating that the HHS-induced activation of caspases was associated with the reduction in the IAP family proteins.

To confirm the involvement of caspase activation in the apoptosis induced by HHS, we conducted a test to determine whether the caspase inhibitor could attenuate the HHS-induced apoptosis and viability reduction. As shown in Fig. 4A and B, pre-treatment with z-VAD-fmk, a pan-caspase inhibitor, resulted in the significant prevention of apoptotic features and the accumulation of hypodiploid cells. The MTT assay also revealed that cells that were co-treated with HHS and z-VAD-fmk had significantly greater cell viability than those that were treated with HHS only (Fig. 4C).

The PI3K/Akt pathway has been known to transduce anti-apoptotic signals that confer a survival advantage and resistance of cancer cells against various chemotherapeutic agents (7,9). We therefore sought to determine the involvement of the PI3K/Akt signaling pathway in the HHS-induced apoptosis in HCT116 cells. The constitutive activation of both PI3K and AKT, one of the main downstream effectors of PI3K, was observed in the control cells, whereas HHS treatment significantly suppressed their phosphorylation in a concentration-dependent manner (Fig. 5). The levels of total PI3K and Akt were unaffected by HHS treatment. To determine whether the mechanism in PI3K/Akt signaling is involved in HHS-induced apoptosis, the cells were treated with HHS in the presence or absence of LY294002, a representative PI3K inhibitor. As shown in Fig. 6A and B, the treatment of LY294002 markedly increased the HHS-induced apoptosis, which was demonstrated by nuclear morphological changes and the flow cytometric analysis. In addition, in combination with LY294002, HHS strongly inhibited cell viability (Fig. 6C), indicating that the inactivation of the PI3K/Akt signaling pathway is involved in HHS-induced HCT116 cell apoptosis.

Since the increase of intracellular ROS generation is proposed to be one of the early events in the activation of apoptotic signaling, which alters the cellular redox state and MMP (8,25), we determined whether the generation of ROS is responsible for the apoptosis induced by HHS in HCT116 cells. The flow cytometric analysis revealed that the generation of ROS was observed at 0.5 h, and the levels continued to increase up to 5 h in response to the HHS treatment. In contrast, the pretreatment of cells with NAC, a commonly used reactive oxygen intermediate scavenger, significantly blocked the HHS-induced increase in ROS generation (Fig. 7A).

In order to show that the generation of ROS is a key step in HHS-induced mitochondrial dysfunction, we compared the levels of cytochrome c in the cytosolic and mitochondrial fractions. As shown in Fig. 7B, the blocking of ROS generation by the pretreatment of cells with NAC effectively prevented the HHS-induced cytochrome c release into the cytosol, suggesting that the increment in the intracellular ROS level by HHS was linked to mitochondrial dysfunction. NAC pretreatment also markedly prevented the HHS-induced cleavage of caspases (caspase-8, -9 and -3) and the degradation of PARP (Fig. 7C). Next, we confirmed whether the HCC-induced ROS generation had any functional role in the inactivation of the PI3K/Akt pathway. The results indicated that the pretreatment of HCT116 cells with NAC effectively attenuated the HHS-induced phosphorylation of both PI3K and Akt (Fig. 7D). Furthermore, the apoptotic events in the HHS-treated HCT116 cells were prevented to a considerable extent by the blocking of ROS generation (Fig. 8A and B), which was associated with recovered cell viability, which was revealed by the MTT assay (Fig. 8C), indicating that the HHS-induced inactivation of the PI3K/Akt pathway was mediated by ROS generation.