Dulbecco's modified Eagle's medium (DMEM), glucose-free DMEM, neomycin (G418) and fetal bovine serum (FBS) were obtained from Gibco-Invitrogen (Carlsbad, CA, USA). Propidium iodide (PI), 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). We obtained 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and antibodies recognizing the phospho-specific forms of AMPKα-Thr¹⁷² and ACC-Ser⁷⁹ from Cell Signaling Technology (Boston, MA, USA). Antibodies against α-actinin, poly (ADP-ribose) polymerase (PARP) and AMPKα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Compound C was purchased from Calbiochem (San Diego, CA, USA).

HT-29 colon cancer cells (ATCC, Manassas, VA, USA) were maintained in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco-Invitrogen) at 37°C in a 5% CO₂ humidified incubator.

The air-dried root bark of Broussonetia kazinoki (voucher specimen no. SPH 07002) (0.6 kg) was extracted with ethanol. The extract (51 g) was fractionated into n-hexane, EtOAc, CHCl₃ and BuOH soluble fractions. The EtOAc fraction (31 g) was subject to silica gel column chromatography followed by eluting with an n-hexane/acetone gradient system (20:1→1:10) and 11 fractions were collected as previously described (16). Fraction 7 was chromatographed using a silica gel column with CHCl₃:MeOH (100:1→10:1) to yield 6 subfractions. Sub-fraction 3 of fraction 7 was chromatographed on a RP-C18 column with a gradient MeOH (40→100%) elution to yield kazinol C (260 mg). The purity was confirmed by HPLC analyses and ¹H-NMR spectrum. The chemical structure of kazinol C was elucidated by spectroscopic analyses and confirmed by comparison with previous studies (17).

Cell apoptosis was assessed using a fluorescence-activated cell sorter (FACS). In brief, the cells were treated with kazinol C for 24 h. The cells were harvested by trypsinization and washed with phosphate-buffered saline (PBS). After fixation in 70% ethanol, the cells were resuspended in PBS containing 10 μg/ml PI. The fluorescence intensity was determined using a FACSCanto™ II flow cytometer (BD Biosciences, San Jose, CA, USA). Cell viability was assessed using the MTT assay. The cells were treated with 5 μg/ml MTT solution and incubated for 3 h, and then dissolved in DMSO. The absorbance was measured at 570 nm.

For the in vitro cell growth analysis, the cells were seeded in a 6-well plate and treated with 0–60 μM of kazinol C for 24–48 h. The cells were harvested and counted using the trypan blue exclusion test. To determine anchorage-independent cell growth, 1×10³ HT-29 cells were suspended in 1.5 ml of the medium containing 0.3% agar, 10% FBS and 15 μM kazinol C, and the mixture was applied onto pre-solidified 0.5% agar (1.5 ml) in 6-well plates. After incubation of two weeks, soft agar colonies were observed under a phase-contrast microscope and photographed.

The cells were seeded in 6-well plates and incubated for 12 h in starvation medium. The cellular monolayer was wounded with a sterile 200-μl pipette tip and washed with starvation medium to remove detached cells from the plates. The cells were pretreated with 0–15 μM kazinol C for 30 min, and incubated in the presence or absence of 40 ng/ml TPA for 36 h. The medium was replaced with PBS, and the cells were photographed using a phase-contrast microscope.

The in vitro migration and invasion assays were performed using a 24-well Transwell unit with polycarbonate filters with a 6.5 mm diameter and 8.0-μm pore size (Corning Costar, Cambridge, MA, USA). For the invasion assay, the lower chamber of the Transwell was filled with DMEM plus 10% FBS as a chemoattractant and the Transwell was assembled to serve as the intervening invasive barrier in a 24-well plate. The cells (5×10⁴) were suspended in serum-free DMEM with 15 μM kazinol C, added to the upper chamber of the Transwell and incubated for 48 h at 37°C. The cells that attached to the upper surface of the filter were completely removed by wiping with a cotton swab, and the filters were stained with a 0.2% crystal violet/20% methanol (w/v) solution.

pcDNA3-AMPK dominant-negative (DN) form was prepared as previously described (18). The plasmid was transfected into HT-29 cells using PolyFect Transfection Reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Transfected cells were selected using complete medium containing 1 mg/ml neomycin.

For the whole-cell lysate preparation, the treated cells were lysed on ice in the PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Seoul, Korea) for 30 min at 4°C. The supernatant fractions were recovered by centrifugation (14,000 × g for 20 min at 4°C), and the protein concentration was determined using the Bradford protein assay. The samples were prepared with 2-mercaptoethanol and denatured by heating at 95°C for 3 min. The proteins were separated on 8–12% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 1% bovine serum albumin or 5% skim milk and hybridized with the primary antibody. Following hybridization with the HRP-conjugated secondary antibody, the protein bands were visualized using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and a LAS-3000 or LAS-4000 imaging system (Fujifilm Corporation, Tokyo, Japan). The western blotting band intensity was analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

The results are presented as the means ± SD. The data were analyzed for statistical significance using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). Significant differences were analyzed using one-way ANOVA followed by the Newman-Keuls multiple comparison tests >3 groups or the unpaired t-test for two groups. P<0.05 was considered to indicate a statistically significant result.

Kazinol C is an active component of Broussonetia kazinoki. The kazinol C chemical structure used in the present study is shown in Fig. 1A. Kazinol C reportedly prevents cellular injury from reactive oxygen species (12). However, the anticancer effects of kazinol C remain unclear. In the present study, we first examined the effects of kazinol C on HT-29 colon cancer cell death. The cells maintained in complete medium were exposed to kazinol C for 24 h. Cells exposed to low concentrations (<30 μM) of kazinol C for 24 h exhibited no substantial increase in cell death, as assessed by the MTT assay (Fig. 1B). However, high concentrations (60–120 μM) of kazinol C significantly increased HT-29 cell death. Additionally, treatment with 30–60 μM kazinol C for 48 h significantly reduced HT-29 cell growth; however, cell growth was not affected by treatment with 30 μM kazinol C for 24 h (Fig. 1C). To verify whether kazinol C induces apoptosis, we performed FACS analysis of the sub-G1 DNA content. Low concentrations (<30 μM) of kazinol C did not significantly affect HT-29 cell apoptosis at 24 h, whereas 60 μM kazinol C significantly induced apoptosis (Fig. 1D). This result was supported by PARP cleavage following treatment with 60 μM kazinol C (Fig. 1E). Collectively, these results showed that kazinol C effectively induces HT-29 cell apoptosis.

AMPK activation results in apoptosis induction by modulating signaling pathways involved in cell proliferation and apoptosis (19). In our previous screening for natural compounds that act as AMPK activators, kazinol C was selected as a potent AMPK activator (data not shown). To investigate the effect of kazinol C on AMPK activity in HT-29 cells, we treated the cells with kazinol C in a time- and dose-dependent manner. Kazinol C markedly increased Thr¹⁷² phosphorylation in the catalytic subunit of AMPK and Ser⁷⁹ phosphorylation in acetyl-CoA carboxylase (ACC), which is a well-characterized AMPK cellular substrate (Fig. 2A and B). These results suggested that kazinol C activates AMPK.

To examine the causal relationship between AMPK activation and kazinol C-induced cell death, the MTT assay was performed after HT-29 cells were pretreated with the AMPK inhibitor compound C or the AMPK activator AICAR. Compound C-mediated inhibition of AMPK activity markedly abrogated the kazinol C-induced cell death in a dose-dependent manner (Fig. 3A). By contrast, the AMPK activator AICAR did not significantly affect kazinol C-induced cell death. Of note, although the AICAR concentration did not induce cell death, AICAR increased AMPK activity at this concentration (Fig. 3A). Moreover, compound C inhibited kazinol C-induced apoptosis, as assessed by FACS analysis of the sub-G1 DNA content (Fig. 3B) or PARP cleavage (Fig. 3C). To confirm these results, we used a molecular approach to regulate AMPK activity. Stable clones of HT-29 cells following transfection with pcDNA3 or AMPK-DN plasmids were established. We confirmed that AMPK activity was attenuated by the stable expression of the DN form of AMPK and that kazinol C-induced AMPK activation was markedly reduced by AMPK-DN expression (Fig. 4A). As demonstrated via FACS analysis of the sub-G1 DNA content, kazinol C effectively induced cell apoptosis and expression of the AMPK-DN form significantly abrogated kazinol C-induced apoptosis (Fig. 4B). These results suggested that kazinol C markedly increases HT-29 cell apoptosis through the activation of AMPK, which plays an important role in apoptosis induction.

Tumor metastasis is a multi-step process that includes migration, invasion, adhesion, proliferation and angiogenesis. To evaluate the effect of kazinol C on tumor metastatic activity, we performed in vitro migration and wound healing assays. As shown in Fig. 5A, 7.5–30 μM kazinol C did not significantly affect HT-29 cell growth at 24 h. In HT-29 cells, kazinol C-treatment for 24 h significantly reduced wound healing (Fig. 5B). Similar results were observed with an in vitro migration assay (Fig. 5C). Given that TPA treatment typically stimulates colon cancer cell migration, we determined the effects of TPA on the kazinol C-induced inhibition of cell migration. Notably, TPA treatment increased wound healing and HT-29 cell migration, whereas kazinol C markedly abrogated TPA-induced wound healing and migration (Fig. 5B and C). We tested the ability of cells to form colonies in a semisolid medium as an indicator of metastatic potential. Untreated and TPA-treated HT-29 cells formed sizable colonies and increased proliferation in soft agar (Fig. 5D). However, kazinol C treatment significantly decreased basal and TPA-induced cell proliferation in soft agar. To examine the causal relationship between AMPK and kazinol C-induced inhibition of migration, we used a molecular approach to inhibit AMPK activity. AMPK inhibition via stable transfection with the AMPK-DN form significantly abrogated kazinol C-mediated inhibition of cancer cell migration (Fig. 6). Collectively, these results suggested that kazinol C inhibits the migration and anchorage-independent growth of HT-29 cells through AMPK activation.