Casticin was purchased from Biopurify Phytochemicals Ltd. (Chengdu, China). The compound has a molecular weight of 374.3, appears as yellow crystals and has a purity of 98.0%. Casticin was prepared in dimethyl sulfoxide (DMSO) as a 10 mmol/l stock solution and diluted in medium to the indicated concentration prior to use. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes (Eugene, OR, USA). Propidium iodide (PI), ethidium bromide, N-acetylcysteine (NAC, an oxygen-free radical scavenger) and SP600125 (a JNK inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-JNK1 and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-phosphorylated JNK1/2, -phosphorylated ASK1, -ASK1, -phosphorylated Bim-extra long (EL) and -Bim-EL antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).

The human colon cancer cell lines HT-29, HCT-116, SW480 and Caco-2 were purchased from the China Center for Type Culture Collection (Wuhan, China). The study was approved by the ethics committee of Hunan Normal University (Changsha, China). Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, and incubated at 37°C in a humidified atmosphere of 5% CO₂.

Cells were seeded at a density of 4×10⁶ cells/well in 250-ml culture flasks for 24 h and then treated with the medium containing various concentrations of casticin or 0.1% DMSO for 24 h, or 10.0 μM casticin for the indicated time periods. PI staining for DNA content analysis was performed as described previously (21). All analyses were performed using a flow cytometer (Coulter Epics XL-MSL, Beckman Coulter, Fullerton, CA, USA) with CellQuest™ software (BD Pharmingen, San Diego, CA, USA).

The cell apoptosis ELISA detection kit (Roche, Basel, Switzerland) was used to detect apoptosis in cells treated with casticin according to the manufacturer’s instructions. Briefly, cells were seeded in a 96-well plate at a density of 1×10⁴ cells/well for 24 h, the tested agents were added and the cells were then cultured in RPMI-1640 medium containing 10% fetal bovine serum. After 24 h, the cytoplasm of the control and treatment groups was transferred to the 96-well plate peridiumed by the streptavidin and incubated with the biotinylated histone antibody and peroxidase-tagged mouse anti-human DNA (both from Roche, Palo Alto, CA, USA) for 2 h at room temperature. The absorbance at 405 nm was measured with an enzyme-linked immunosorbent apparatus (ELX-800 type; Bio-Tek, Shanghai, China).

Cells were seeded at a density of 4×10⁶ cells/well in 250-ml culture flasks for 24 h and treated with medium containing various concentrations of the test/control agents or vehicle and 10% fetal bovine serum for 24 h. This assay was performed as previously described (21).

ROS generation in the control and casticin-treated cells was measured by flow cytometry (FCM) following staining with the DCFH-DA. Briefly, cells were seeded in six-well plates (1×10⁵ cells per well), allowed to attach overnight and exposed to DMSO (control) or the desired concentrations of casticin for specified time periods. The cells were stained with 20 μM DCFH-DA for 30 min at 37°C, and the fluorescence intensity of the DCF in the cells was determined using the flow cytometer with winMDI software (Microsoft Corp., Redmond, WA, USA). As a rule, 10,000 cells were counted in each determination.

Control siRNA and siRNA targeting ASK1 were obtained from Santa Cruz Biotechnology, Inc. The sense sequences of the siRNA reagents were 5′-GACGCGATCAGAGAGTAAT-3′ (siRNA control) and 5′-GGTGGCACAAGCAAGTTCT-3′ (siRNA ASK1). For transient siRNA transfection, HT-29 cells were seeded at a density of 5×10⁵ cells/ml into six-well plates. Cells were transfected on the following day with the Lipofectamine^® LTX with Plus™ reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 100 ng/well siRNA (ASK1 or control). Cells were transfected with each siRNA and incubated for 48 h. The interference of ASK1 protein expression was confirmed by western blot analysis using the anti-ASK1 antibody.

Western blot analysis was performed as described previously (22). Anti-JNK1, -phosphorylated JNK1/2, -phosphorylated ASK1, -ASK1, -phosphorylated Bim-EL, -Bim-EL and -β-actin antibodies were used as the primary antibodies. The signals were detected using an ECL Advance western blot analysis system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

Data are presented as the mean ± standard deviation for triplicate experiments and were analyzed using the Student’s t-test. Differences from the controls were considered significant when P<0.05.

It has been previously reported that casticin significantly inhibits the proliferation of human colon cancer cells (7,8). To further investigate its mechanisms, the hypodiploid cell populations were detected by FCM. Fig. 1A shows that casticin increased the percentage of the sub-G1 population in a concentration-dependent manner both in the p53 mutant cell line HT-29, and in the HCT116, SW480 and Caco-2 cell lines (P<0.05). The sub-G1 population of HT-29 cells treated with casticin was increased at 12 h and peaked at 24 h (Fig. 1B). The histone/DNA fragments of the HT-29, HCT-116, SW480 and Caco-2 cells, as measured by the cell apoptosis ELISA detection kit, were increased in a dose-dependent manner (P<0.05) following treatment with casticin (Fig. 1C). Furthermore, DNA fragmentation analysis by agarose gel electrophoresis revealed a typical ladder pattern of internucleosomal DNA fragments in the colon cancer cells that were treated with 10.0 μmol/l casticin for 24 h (Fig. 1D). These results suggest that casticin inhibits colon cancer cell proliferation by a mechanism involving the induction of apoptosis. The sequential experiments in the study explored the molecular mechanism by which casticin caused apoptosis using the p53-mutated human colon cancer HT-29 cells.

We have previously demonstrated that the casticin-induced apoptosis of human cervical cancer cells is associated with the induction of ROS generation (9,11). Therefore, the present study investigated whether casticin also caused ROS production in colon cancer cells. Following the treatment of HT-29 cells with 5.0, 10.0 and 20.0 μM casticin for 3 h, the levels of ROS increased in a dose-dependent manner (Fig. 2A). Time-course experiments revealed that the levels of ROS increased initially at 0.5 h, reached a peak at 3 h and persisted for up to 24 h after treatment with 10.0 μM casticin (Fig. 2B).

To explore the role of ROS in casticin-induced colon cancer cell apoptosis, the antioxidant NAC was used. As shown in Fig. 2C, pretreatment of HT-29 cells with NAC (2.5, 5.0 and 10.0 mM) markedly attenuated casticin-induced apoptosis in a concentration-dependent manner. Furthermore, casticin-induced ROS production was almost completely inhibited by treatment with 10.0 mM NAC (Fig. 2A). These results suggest that casticin causes intracellular ROS generation, and the oxidative stress further contributed to apoptosis of the human colon cancer HT-29 cells.

Our previous report indicated that ROS generation and sustained JNK activation play a role in the casticin-induced apoptosis of human cervical cancer cells (11). The present study examined whether casticin activates the JNK pathway in human colon cancer cells. Fig. 3A and B shows that casticin treatment increased the phosphorylation levels of the JNK1/2 protein and its downstream molecule Bim-EL, and these phosphorylation levels were attenuated in the presence of 10.0 mM NAC (Fig. 3C) in the HT-29 cells. These results suggest that casticin caused JNK activation through intracellular ROS generation in the colon cancer HT-29 cells.

ASK1 is a member of the mitogen-activated protein kinase kinase kinase (MAPKK) family that activates the JNK pathways by directly phosphorylating and thereby activating its respective MAPKKs, mitogen-activated protein kinase kinase (MKK) 4/7 and MKK3/6 (20). In the present study, we next examined whether the casticin-induced ROS activated ASK1. ASK1 is one of the upstream regulators of JNK, and is known to be associated with cell death (21,23). Fig. 4A and B shows that casticin induced the phosphorylation of ASK1 in a concentration- and time-dependent manner, and this was attenuated by pretreatment with 10 mM NAC in the HT-29 cells. These results suggest that, in the colon cancer HT-29 cells, the activation of ASK1 caused by casticin was dependent on intracellular ROS generation.

To investigate the role of ASK1 in regulating JNK and inducing apoptosis caused by casticin, siRNA that specifically targeted ASK1 was used. The results of the western blot analysis showed that ASK1 was downregulated following the transfection of ASK1 siRNA in HT-29 cells (Fig. 5A). The effect of ASK1 siRNA on the activation of JNK caused by casticin treatment was examined. Fig. 5A shows that ASK1 siRNA attenuated the increased phosphorylation levels of JNK1/2 that were induced by casticin, suggesting that ASK1 mediates casticin-induced JNK activation.

In order to assess whether ASK1 activation is negatively regulated by JNK, the cells were treated with a pharmacological compound named SP600125, a known JNK inhibitor. The results from the western blot analysis showed that activated ASK1 was not altered by pretreatment with SP600125 (Fig. 5B). These results suggest that ASK1 was activated prior to JNK in HT-29 cells treated with casticin. Fig. 5C and D shows that the ASK1 siRNA and JNK inhibitor decreased the casticin-induced apoptosis in the HT-29 cells. We can conclude that casticin may have caused apoptosis by activating the ASK1-JNK signaling pathway in the HT-29 cells.

We next investigated whether casticin induces apoptosis by the same modality in other colon cancer cell lines, including HCT-116, SW480 and Caco-2. As shown in Fig. 6A, FCM results obtained by monitoring the DCFH-DA probe indicated that casticin significantly induced ROS generation. Western blot analysis showed that casticin treatment also caused an increase in ASK1, JNK and Bim-EL phosphorylation levels in the HCT-116, SW480 and Caco-2 cells (Fig. 6B). Together, these findings suggest that casticin-induced apoptotic cell death, ROS generation and the activation of ASK1, JNK and Bim were not specific to human colon cancer cell types.