Dried whole fruit of Torilis japonica was purchased from Na-num Pharmacy (Kyung-buk, Korea). Plant material (200 g) was extracted two times with 95% ethanol at room temperature for 3 day and was subsequently filtered. The combined filtrate was concentrated under vacuum at 60°C, and completely dried by freeze drying. The yield was 10% and TJE powder was dissolved in DMSO and filtrated by 0.2 μ pore size filter for in vitro studies.

N-acetyl cystein (NAC), 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DCFH-DA are purchased from Sigma-Aldrich (St. Louis, MO, USA). FITC-Annexin V apoptosis detection kit is obtained from BD Pharmingen (San Diego, CA, USA). Mitotracker was purchased from Molecular Probes (Eugene, OR, USA). Specific anti-bodies that recognized p-AMPKα1, AMPKα1, p38, p-p53, caspase-3, Bcl-2, Mcl-1, COX-4, β-actin are obtained from Cell Signaling Technology (Beverly, MA, USA) and Bax, Bak, PUMA, p-p38 MAPK, p53, cytochrome c are purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

HCT116 and HT-29, fibroblast cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) 3 month before experiment begun. HCT116, HT-29 cells were grown in RPMI-1640 medium and fibroblast cells were grown in DMEM medium containing 10% fetal bovine serum (both from HyClone, Waltham, MA, USA) and 1% antibiotics (100 mg/l streptomycin, 100 U/ml penicillin) at 37°C in a 5% CO₂ atmosphere. Cells were suspended by Trypsin-EDTA (HyClone) and separated 1.5×10⁵/ml at each plates, every 48 h. All the cell lines were authenticated by each 6 month repeat analysis at Korea Cell Line Bank (KCLB, Seoul, Korea)

Cells were seeded 1×10⁵/ml in 12-well plate with cover glasses. After treatment the indicated time and dose at 37°C in a 5% CO₂ atmosphere, the cells were incubated with 10 μM of DCFH-DA for 30 min and fixed with 3.7% formaldehyde for 20 min. Cells were washed with phosphate-buffered saline (PBS) twice and fluorescence was detected by fluorescence microscope (Carl Zeiss, Thornwood, NY, USA).

Cells were seeded 1×10⁶/ml in 100 mm plate and incubated for 24 h. After incubation, cells treated with test compound for 6 h at 37°C in a 5% CO₂ atmosphere. Cells were incubated with 40 μM of DCFH-DA for 30 min and harvested by trypsinization, collected by centrifugation, washed with PBS twice, and resuspended in PBS. Fluorescence intensity were analyzed by using flow cytometer (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA).

Cells were seeded at 4,000/ml each well in 96-well plate, and incubated 24 h. After the incubation, treated with test compound and incubate at 37°C in a 5% CO₂ atmosphere. After 24 h, cells were incubated with 20 μl MTT (5 mg/ml with PBS) solution for 1 h. Optical densities of solution, in each well, were determined by Microplate reader (Bio-Rad Laboratories, Inc., Tokyo, Japan) at 595 nm.

Cells were seeded at 1×10⁶/ml in 100 mm plate and incubated for 24 h. After incubation, cells treated with test compound for 24 h at 37°C in a 5% CO₂ atmosphere. Total cells were harvested by trypsinization, collected by centrifugation, washed with PBS, and resuspended in binding buffer. Cells were stained with Annexin V and PI for 15 min. Fluorescence intensity were analyzed by using flow cytometer (BD Biosciences).

Cells were seeded at 1×10⁶/ml in 100 mm plate and incubated for 24 h. After incubation, cells treated with test compound for 24 h at 37°C in a 5% CO₂ atmosphere. Total cells were harvested by trypsinization, collected by centrifugation, washed with PBS. Cell were incubated with JC-1 for 30 min 37°C in a 5% CO₂ atmosphere before the flow cytometer analysis (BD Biosciences).

We used caspase-3 activity assay kit (Abcam PLC, Cambridge, UK). Cells were seeded at 1×10⁶/ml in 100 mm plate and incubated for 24 h. After incubation, cells treated with test compound for 24 h at 37°C in a 5% CO₂ atmosphere. Total cells were harvested by trypsinization, collected by centrifugation, washed with PBS. Cells were resuspended in lysis buffer and mixed with 2X reaction buffer. Samples were reactivated with DEVD-p-NA for 2 h at 37°C. Optical densities of solution, in each well, were determined by Microplate reader (Bio-Rad Laboratories, Inc.) at 405 nm.

We used Mitochondria/Cytosol Fraction kit (Abcam PLC). Cells were seeded at 1×10⁶/ml in 100 mm plate and incubated for 24 h. After incubation, cells were treated with test compound for 24 h at 37°C in a 5% CO₂ atmosphere. Total cells were harvested by trypsinization, collected by centrifugation, washed with PBS, and homogenized in ice-cold cytosol extraction buffer mix containing DTT and protease inhibitor using a sonicator. The homogenates were centrifuged at 3,000 rpm for 10 min at 4°C and supernatants were collection. Supernatant were centrifuged at 13,000 rpm for 30 min at 4°C and collected supernatant for cytosol proteins and pellets were resuspended with ice cold mitochondria extraction buffer containing DTT and protease inhibitor for mitochondria proteins.

Bak and Bax oligomerization were assessed by chemical crosslinking. Briefly, mitochondria protein fractions were resuspended in conjugation buffer. For disulphide-bond formation, mitochondria protein fractions were incubated with the bis(maleimido)hexane (Thermo Fisher Scientific, Rockford, IL, USA) for 1 h at room temperature and the samples analyzed by non-reducing SDS-PAGE.

We used to surebead protein G magnetic beads kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cells were seeded at 1×10⁶/ml in 100 mm plate and incubated for 24 h. After incubation, cells were treated with test compound for 24 h at 37°C in a 5% CO₂ atmosphere. Mitochondria/cytosol proteins were fraction and mitochondria proteins were incubated with specific antibody bound magnetic bead. The beads were washed using a magnet and PBS. Target proteins were elusion in 1X sample buffer and analyzed by western blotting.

We used Muse Bcl-2 activation dual detection kit (Merck Milipore, Darmstadt. Germany). Cells were seeded at 1×10⁵/ml in 6-well plate and incubated for 24 h. After incubation, cells were treated with test compound for 24 h at 37°C in a 5% CO₂ atmosphere. Total cells were harvested by trypsinization, collected by centrifugation, washed with PBS. Cell were fixed and permeabilized by reagent in the kit. The antibody, which was combined with fluorescence tag, was reacted for 1 h at room temperature with slow agitation, and it was washed once with the assay buffer. Cells were resuspended in assay buffer and analyzed by Muse cell analyzer.

Small interfering RNA (siRNA) was purchased by Dharmacon (Chicago, IL, USA). For transient transfection, cells were seeded at 5×10³/ml on a 6-well plate with antibiotics free medium. After incubation overnight, targeting siRNA was transfected using DharmaFECT2 transfection reagent (Dharmacon) according to manufacturer’s instructions. After incubation for 72 h, cells were treated with TJE with NAC at indicated doses.

Cells were seeded at 1×10⁵/ml in a 6-well plate and incubated for 24 h, and after the incubation, treated with test compound for 6 h at 37°C in a 5% CO₂ atmosphere. Cells were rinsed twice with ice-cold PBS and scraped with lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF) and subjected to western blot analysis. First antibody was reacted overnight at 4°C and second antibody for 75 min at room-temperature with slow agitation.

Cells were seeded 1×10⁵/ml in a 12-well plate with cover glasses. After treatment with the indicated time and dose, the cells were stained with mitotracker for 30 min at 37°C in a 5% CO₂ atmosphere. Cells were fixed with 3.7% formaldehyde for 20 min and pemeabilized with 0.2% Triton X-100 for 20 min. Cells were washed with PBS twice and reacted with cytochrome c antibody overnight at 4°C. Cells were washed with PBS twice and reacted with secondary antibody for 1 h. Fluorescence was detected by confocal (Olympus, Tokyo, Japan).

Five-week-old male Balb/c nu/nu mice were obtained from SLC (Tokyo, Japan) and housed in sterile filer-topped cages. For tumor induction, HCT116 human colon cancer cells (2.5×10⁵ cells/0.1 ml) were subcutaneously injected into the left flank of the mice (each group had 5 animals). One week after the injection of cells, they were co-treated with TJE 80 mg/kg/day for 21 days. Tumor size was measured using a caliper at 2 day intervals, and the volume was calculated by the modified formula V = 1/2 (length x width²). After the 3-week treatment, tumor was removed and frozen in liquid nitrogen for western blot analysis or fixed with formalin for immunohistochemistry and H&E staining. All animal experiments were approved by the Ethics Committee for Animal Experimentation, Hannam University.

Tumor specimens from mice were fixed in 10% formaldehyde, embedded in paraffin and sectioned into 5 μm thick slices. Consecutive thin cryosections (5 μm) of OCT compound (Sakura Finetek, Torrance, CA, USA) embedded tumor tissues were fixed in acetone at 4°C for 10 min. After washing in PBS, sections were treated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity, and the sections were blocked with normal rabbit serum. Then, the sections were blocked and washed in PBS and incubated with specific antibody overnight at 4°C. Negative controls were incubated with the primary normal serum IgG for the species from which the primary antibody was obtained.

Levels of apoptosis in distal colon tissue were determined using the TdT-mediated dUTP nick-end labeling (TUNEL) method. Tumor specimens from mice were fixed in 10% formaldehyde, embedded in paraffin and sectioned into 5 μm thick slices. Tissue sections were processed according to manufacturer’s instructions for the ApopTag peroxidase in situ apoptosis detection kit (Vector Laboratories, Burlingame, CA, USA).

Cell viability and caspase-3 activity was statistically analyzed using unpaired t-test (SPSS, Chicago, IL, USA). P<0.05 was considered statistically significant.

We investigated the anti-proliferative and apoptotic effects of TJE. We treated cells with TJE (10–100 μg/ml) for 12 and 24 h, and then assayed for cellular viability and apoptosis. Cells treated with TJE showed a decrease in viability and an increase in the number of Annexin V-positive cells in a dose-dependent manner. In normal human fibroblasts, however, TJE had no effect on cellular viability (Fig. 1A–C).

To understand the mechanism by which TJE induces apoptosis, we measured the mitochondrial membrane potential after treatment with different concentrations of TJE via JC-1 staining (Fig. 1D). Our results showed that TJE reduced the membrane potential dose-dependently.

We analyzed the changes in p-AMPKα1, p-p38, p53 and p-p53 levels and the apoptosis-related proteins Bax, Bak, PUMA, cleaved caspase-3, Mcl-1 and Bcl-2 after treatment with different concentrations of TJE by western blotting. Our results showed that TJE strongly activated AMPK, p38 and p53 dose-dependently (Fig. 2A). Moreover, TJE reduced the expression of Mcl-1 and Bcl-2 and induced the expression and mitochondrial translocation of Bax, Bak and PUMA. The latter three proteins became localized to the outer membrane of the mitochondria, which led to secretion of cytochrome c from the mitochondria to the cytosol by a reduction in the mitochondrial membrane potential (Fig. 2B and C).

We examined whether TJE promotes the generation of ROS in HCT116 colon cancer cells. We measured intracellular ROS levels following treatment of cells with TJE (20–80 μg/ml) for 6 h. As shown in Fig. 3A (left panel), TJE increased ROS levels at the indicated concentrations. These effects were completely blocked by co-treatment with NAC, a ROS scavenger (right panel).

To make sure that the increase in intracellular ROS levels was related to TJE-regulated signaling proteins and the induction of apoptosis, we co-treated cells with NAC and then analyzed protein levels and the concentration of Annexin V-positive cells. The cells co-treated with NAC and TJE did not undergo increases in AMPK, p38 or p53 phosphorylation and showed decreased expression, oligomerization, and binding of apoptosis-related proteins and increases in the expression of anti-apoptotic proteins in comparison to cells treated with TJE alone (Figs. 3E and 4). In addition, cells co-treated with TJE and NAC did not undergo cell death and did not exhibit a reduction in the mitochondrial membrane potential (Fig. 3F). By contrast, TJE-treated cells displayed increased levels of apoptotic cell death through changes in the mitochondrial membrane potential which led to secretion of cytochrome c from the mitochondria to the cytosol (Fig. 3G). Generally, cytochrome c in the cytosol initiates caspase cleavage and activates effector caspases such as caspase-3, which in turn induce apoptosis. We demonstrated that TJE induced caspase-3 activation dose-dependently. Co-treatment with NAC abolished this effect (Fig. 3H).

To show that TJE treatment induced cytochrome c release from the mitochondria to the cytosol, we co-treated cells with TJE and NAC for 24 h and stained cells in order to visualize the mitochondria and cytochrome c. In TJE-treated cells, cytochrome c translocated from the mitochondria to the cytosol; in contrast, in control cells and those co-treated with both TJE and NAC, cytochrome c remained in the mitochondria (Fig. 5).

In order to identify the intracellular signaling pathway that mediates the effects of TJE, we transfected cells with siRNAs against AMPK, p38 and p53. The cells in which AMPK and p38 were knocked down did not undergo apoptosis following TJE treatment, but the cells treated with p53 siRNA still showed an increase in apoptotic cell death (Fig. 6A). In addition, TJE did not affect caspase-3 activity or cytochrome c translocation in the cells treated with AMPK or p38 siRNA, but did affect those activities in the p53 siRNA-treated group (Fig. 6B).

To confirm that apoptotic cell death following TJE treatment is p53-independent, we analyzed apoptosis-related protein expression and translocation to the mitochondria in HCT116 cells transfected with siRNAs against apoptosis-related proteins. Our results showed that TJE did not induce pro-apoptotic protein expression or downregulation of anti-apoptotic proteins in AMPK siRNA- or p38 siRNA-transfected cells, but the effects of TJE were still seen in p53 siRNA-transfected cells, except those treated with PUMA; PUMA cannot act independently of p53 (Fig. 6C and D). Moreover, TJE induced apoptotic cell death in HT-29 colon cancer cells, which have a mutation in the p53 gene (Fig. 6F).

To analyze the consequences of TJE treatment in an HCT116 xenograft model of tumor growth, we performed histological analysis from control- and TJE (80 mg/kg/day)-treated tumor tissue stained with H&E using the TUNEL assay. The amount of tumor tissue in TJE-treated samples was considerably less than that of control samples (Fig. 7A). The number of TUNEL-positive cells was significantly increased and cancer tissue was degraded in TJE-treated samples (Fig. 7E). In addition, in a similar in vitro experiment, the AMPK/p38 MAPK and p53 signaling pathways were activated, and apoptosis-related proteins showed an increase in expression and translocation to the cytosol (Fig. 7B–D and F).