Trees of Liriodendron tulipifera L., yellow poplar (~0.9–1.4 meters in height), were collected from Kangjin, Chonnam Province, Korea. Air-dried trees (1.0 kg) were chopped off before extraction. The chopped plant materials were exhaustively extracted three times with either ethyl acetate or ethanol. Each extract was separately filtered and concentrated by a rotary evaporator. The resulting concentrates were completely dried via freeze dryer to yield brownish extract (11.3 g). In order to isolate the active ingredient, sesquiterpene lactones, including epi-Tulipinolide from ethyl acetate or ethanol extract, further reversed-phase high-performance liquid chromatography (HPLC) with a stepwise gradient solvent system from H₂O to methanol was performed.

The BaF3/WT and BaF3/T315I cells were provided by Dr M. Deininger (Huntsman Cancer Institute, Salt Lake City, UT, USA). The cells expressed wild-type Bcr-Abl and Bcr-Abl with the T315I mutation, respectively. BaF3/WT and BaF3/T315I cells were grown in Roswell Park Memorial Institute Medium-1640 (RPMI-1640), containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. RPMI-1640, FBS, and penicillin/streptomycin were purchased from Gibco (Grand Island, NY, USA). Imatinib was purchased from LC Laboratories (Woburn, MA, USA), and sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophe sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assays were purchased from WelGene Inc. (Daegu, Korea).

Cell viability of corresponding compounds was determined by XTT assay. The cells were seeded and treated onto 96-well plates at a density of 1×10³ cells per well and incubated at 37°C for 48 h. The cells were then treated with either CD-200 or Imatinib at the indicated concentrations (0.1–10 μg/ml) or (0.01–10 μM), respectively. Then, 10 μl of XTT labeling mixture [1 ml of XTT/20 μl of phenazinemethosulfate (PMS)] was added to each well. After incubation for 4 h, optical density (OD) was determined using a microplate reader by measuring the absorbance at wavelengths 540 nm and 620 nm. The absorbance rates for each well were calculated as OD₅₄₀–OD₆₂₀.

After the cells were treated with various concentrations of either CD-200 or Imatinib and incubated at 37°C for various times, they were collected and washed with cold phosphate-buffered saline (PBS). Then, the cells were lysed with a RIPA buffer (Biosesang, Seongnam, Korea) containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX, USA). The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto the nitrocellulose membranes. The blots were immunostained with appropriate primary antibodies followed by secondary antibodies conjugated to horseradish peroxidase. Antibody binding was detected with an enhanced chemiluminescence reagent (Bio-Rad, Hercules, CA, USA). Primary monoclonal antibodies against the following factors were used; p-Bcr-Abl (Tyr¹⁷⁷), p-Crkl (Tyr²⁰⁷), p-Stat5 (Tyr⁶⁹⁴), Bcr-Abl, Crkl, Stat5, cleave caspase-3, cleaved PARP, survivin, Mcl-1 and β-actin (Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

BaF3/T315I cells were plated in 48-well plates with RPMI-1640 medium and treated with CD-200 for various concentrations. The cells were then suspended on poly-l-lysine-coated slides, followed by Shandon Cytospin 3 (Akribis Scientific, Cheshire, WA, USA) for 3 min at 1000 rpm. Thereafter, the cells were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature and washed twice with PBS. The cells were blocked in 5% horse and goat serums in PBS for 1 h at room temperature, then incubated in a humidified chamber at 4°C overnight with p-Bcr-Abl (Tyr¹⁷⁷) antibody (Cell Signaling Technology). After washing twice with PBS, the cells were incubated with rabbit tetramethyl rhodamine isothiocyanate (TRITC) secondary antibody (Dianova, Germany) for 1 h at room temperature. They were also stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. The slides were then washed twice with PBS and covered with Dako (Carpinteria, CA, USA) before viewing with a confocal laser scanning microscope (Olympus, Tokyo, Japan).

Moreover, after deparaffinization, immunostaining was performed, using 8-μm-thick sections of the tumor samples. The tissue sections were then blocked with a normal goat or horse serum (Vector Laboratories, Burlingame, CA, USA) for 1 h, and were incubated at 4°C overnight with the primary antibody. After washing twice with PBS, the cells were incubated with the rabbit TRITC secondary antibody for 1 h at room temperature. They were also stained with DAPI to visualize the nuclei. The slides were then washed twice with PBS and covered with Dako before viewing with a confocal laser scanning microscope.

BaF3/T315I cells were plated in 10-cm dishes with an RPMI-1640 medium and treated with CD-200 at the indicated concentrations (0–5 μg/ml). The cells were collected and fixed in cold 70% ethanol at −20°C overnight. After washing with PBS, the cells were subsequently stained with 50 μg/ml propidium iodide (PI) and 100 μg/ml RNase A for 30 min at room temperature in the dark; then a flow cytometric analysis was performed to determine the percentage of cells in specific sub-G₁ phases, using a FACS Calibur flow cytometry (BD Biosciences, San Jose, CA, USA).

BaF3/T315I cells were plated in 48-well plates with RPMI-1640 medium and treated with various concentrations of CD-200. The cells were then suspended on poly-l-lysine-coated slides, followed by Shandon Cytospin 3 for 3 min at 1000 rpm. They were then fixed in 4% PFA for 15 min at room temperature and washed twice with PBS. The terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was subsequently performed using a TUNEL kit (Merck Millipore, Temecula, CA, USA) in accordance to the manufacturer’s instructions.

Mitochondrial membrane potential (MMP, Δψ) was assessed using the Mitochondrial Membrane Potential Detection kit (BD Biosciences). BaF3/T315I cells were then treated with various concentrations of CD-200 for 8 h. The cells were incubated with JC-1 working solution at 37°C. After washing two times, the cells were suspended in 0.5 ml of 1X assay buffer prior to flow cytometry. The results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

BaF3/T315I cells were treated with various concentrations for 8 h. To label the mitochondria, the cells were incubated with 500 nM mitochondrion-specific dye (MitoTracker^® Green FM; Molecular Probes Inc., Eugene, OR, USA) for 45 min at 37°C prior to fixation. The cells were then suspended on poly-l-lysine-coated slides, followed by Shandon Cytospin 3 for 3 min at 1000 rpm. They were then fixed in 4% PFA for 15 min at room temperature and washed with PBS. The cells were incubated at 4°C overnight with cytochrome c antibody (Santa Cruz Biotechnology). After washing twice with PBS, the cells were incubated with mouse TRITC secondary antibody (Dianova). The cells were also stained with DAPI to visualize the nuclei. The slides were then washed twice with PBS and covered with Dako before viewing with a confocal laser scanning microscope.

All animal experiments were performed in accordance to the guidelines of the INHA Institutional Animal Care and Use Committee (INHA IACUC) at Inha University Medical School. The cells were harvested and mixed in PBS (200 μl/mouse). Six weeks old male BALB/c nude mice (Orient Bio, Seoul, Korea) were inoculated with 1×10⁵ cells in the flank. When the tumor size reached approximately 50–100 mm³, they were randomly divided into 3 groups, with 5 mice in each. Then, these mice were given CD-200 (50 mg/kg) or Imatinib (50 mg/kg) intra-peritoneally once a day for 11 days, and the control group was fed vehicle. The tumor size was measured every 2 days, and it was calculated using the formula, 0.5 × length × width².

The tissue sections were blocked with normal goat or horse serum (Vector Laboratories) for 1 h, and incubated at 4°C overnight in 1:50 dilutions of p-Bcr-Abl, Ki-67, and cleaved caspase-3 (Cell Signaling Technology). The sections were then incubated with biotinylated secondary antibodies (1:100) for 1 h. The sections were visualized by an avidin-biotin peroxidase complex solution using an ABC kit (Vector Laboratories), which were then washed in PBS and developed with a diaminobenzidine tetrahydrochloride substrate for 15 min, then counterstained with hematoxylin. At least 3 random fields of each section were examined at ×400 magnification and analyzed using a computer image analysis system (Media Cybernetics, Rockville, MD, USA).

Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using ANOVA and unpaired Student’s t-tests. Statistical significance was set to p<0.05.

XTT assays were performed to evaluate the effect of CD-200 on the growth of BaF3/WT and BaF3/T315I cells. The cells were treated with various concentrations of CD-200 and Imatinib for 48 h. As shown in Fig. 1, both CD-200 and Imatinib treatments reduced the cell viability of BaF3/WT cells; the IC₅₀ values were about 0.6 μg/ml for CD-200 and 0.4 μM for Imatinib. In addition, CD-200 significantly reduced the cell viability of BaF3/T315I cells (IC₅₀≅0.8 μg/ml), while Imatinib had little effect. These data suggest that CD-200 exhibits the potent inhibitory activity in BaF3 cells, expressing Bcr-Abl T315I mutation resistance to Imatinib, as well as wild- type Bcr-Abl leukemic cells.

In order to assess the effects of CD-200 on the inhibition of Bcr-Abl activity, phosphorylation of Bcr-Abl and its respective downstream signals, Crkl and Stat5, were measured by western blotting. As shown in Fig. 2A, CD-200 strongly inhibited the phosphorylation of Bcr-Abl (Tyr¹⁷⁷) in BaF3/T315I cells. Likewise, the phosphorylation levels of Crkl (Tyr²⁰⁷) and Stat5 (Tyr⁶⁹⁴) were effectively suppressed. In contrast, Imatinib did not alter the phosphorylation levels of Bcr-Abl, Crkl, and Stat5 in BaF3/T315I cells. The expression of p-Bcr-Abl was confirmed by confocal fluorescent microscopy (Fig. 2B).

In order to investigate whether the anticancer effect of CD-200 in BaF3/T315I cells was associated with the induction of apoptosis, we performed several cell-based apoptosis assays. We first identified the nuclear morphology on 12 h after treatment with CD-200 by using TUNEL apoptosis assay kits. As a result, CD-200-treated cells were presented with more prominent DNA fragmentation in comparison to the control group (Fig. 3A). Next, we assessed the cell cycle distribution by a flow cytometric analysis. After 24 h treatment with CD-200, the cells were collected and stained with PI, then analyzed by FACS. As shown in the Fig. 3B, CD-200 increased the number of cells in the sub-G₁ phase, associated with early apoptosis without changes of the cell cycle arrest (Fig. 3B). In support of apoptotic effect of CD-200, we observed that CD-200 also increased cleaved caspase-3 positive cells by FACS (Fig. 3C).

To gain further insight into the mechanism underling apoptosis induced by CD-200, we examined the mitochondrial potential change, which plays an important role in the regulation of apoptosis. Since a loss of MMP induces the transition of mitochondrial permeability and release of cytochrome c from the mitochondria to cytosol (23), we measured the MMP and cytochrome c release in CD-200-treated BaF3/T315I cells. As shown in Fig. 4A, CD-200 significantly reduced the fluorescence intensity reflecting MMP, while no changes were observed in the Imatinib-treated group. Moreover, we observed that the treatment of CD-200 increased the release of cytochrome c by immunostaining (Fig. 4B). In addition, CD-200 inhibited the expression of mitochondria-mediated protein families, such as Mcl-1, and survivin (Fig. 4C) along with that of cleaved PARP. These results indicated that CD-200 induced apoptosis through the mitochondria-mediated intrinsic pathways in BaF3/T315I cells.

We extended our study to an in vivo mouse xenograft model. After inoculation with BaF3/T315I cells, mice were intraperitoneally injected with CD-200, at doses of 50 mg/kg and Imatinib, once a day for 11 days. CD-200 potently inhibited the progression of tumor growth, which became more noticeable and significant on day 11 as compared to the control group, whereas Imatinib treatment did not show significant anticancer effect in this BaF3/T315I cell xenograft model (Fig. 5A). No significant changes in body weight or adverse effect were observed in any of the groups (data not shown). To further confirm whether CD-200 inhibits tumor growth through the induction of apoptosis and inhibition of proliferation, we identified the expression of cleaved caspase-3 and Ki-67 in tumor tissues. As expected, CD-200-treated tumor showed an increased expression of cleaved caspase-3 and decreased Ki-67 expression compared to the control and Imatinib groups (Fig. 5B). Moreover, the treatment with CD-200 decreased the phosphorylation of p-Bcr-Abl (Fig. 5B). Taken together, these results demonstrate that CD-200 has antitumor potency in the mouse xenograft model bearing BaF3/T315I cells.