The powdered roots of Pulsatilla koreana were extracted as described in our previous study (12). Briefly, they were extracted using 50% ethanol, while the final extracts were concentrated in vacuo to yield a light brown residue. The residue was suspended in acetone, then centrifuged, and the resulting supernatant was removed to obtain a brown precipitate. The precipitate was poured into water and subsequently filtered to remove the insoluble portion. The filtrate was concentrated into a brown mass.

Human ATC cell line 8505c was purchased from the Japanese Collection of Research Bioresources (JCRB, Shinjuku, Japan), while SNU-80 was purchased from the Korean Cell Line Bank (Seoul, Korea). 8505c cells were cultured in minimum essential medium Eagle (MEM) (Gibco-BRL, Carlsbad, CA, USA), whereas SNU-80 cells were cultured in RPMI-1640 (Gibco-BRL), supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) and 1% penicillin/streptomycin. Cultures were maintained at 37°C in a CO₂ incubator with a controlled humidified atmosphere composed of 95% air and 5% CO₂. Human umbilical vein endothelial cells (HUVECs) were grown in 0.2% gelatin-coated 75-cm² flasks in endothelial cell growth medium (ECGM) 2, containing its supplement mixture at 37°C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and proteinase K were purchased from Sigma-Aldrich (St. Louis, MO, USA). RNase A was purchased from Qiagen (Valencia, CA, USA).

Cell viability was performed through an MTT assay. Briefly, 8505c and SNU-80 cells were plated at a density of 7×10³ cells/well on 96-well plates overnight. The media were removed, and cells were treated with either saline as a control or various concentrations of PKE followed by incubation for 48 h. After that, MTT solutions were added to each well and incubated for 4 h at 37°C. The formazan crystals that formed were dissolved in dimethyl sulfoxide (DMSO). Absorbance was measured using a microplate reader at 540 nm. Three replicate wells were used for each analysis.

The cells were washed with ice-cold phosphate-buffered saline (PBS), then lysed with TNN buffer containing 1% Triton X-100, 1% Nonidet P-40, as well as the following protease and phosphatase inhibitors: aprotinin (10 mg/ml), leupeptin (10 mg/ml) (ICN Biomedicals, Inc., Asse-Relegem, Belgium), phenylmethylsulfonyl fluoride (1.72 mM), NaF (100 mM), NaVO₃ (500 mM) and Na₄P₂O₇ (500 mg/ml) (Sigma-Aldrich). Equal amounts of protein were separated by SDS-PAGE then transferred onto PVDF. Immunostaining of the blots was performed using the primary antibodies, followed by the secondary antibodies conjugated to horseradish peroxidase and detection by enhanced chemiluminescence reagent (ELPS, Seoul, Korea). The primary antibodies were monoclonal antibodies: anti-HIF-1α (BD Biosciences, San Jose, CA, USA), anti-vascular endothelial growth factor (VEGF) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-Bax, anti-Bcl-2 (Santa Cruz Biotechnology, Inc.), anti-cleaved caspase-3 and anti-cleaved poly ADP-ribose polymerase (PARP; Cell Signaling Technology, Inc., Danvers, MA, USA). The secondary antibodies were purchased from Amersham Biosciences, Inc., (Piscataway, NJ, USA) The bands were visualized with the ECL Plus system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

8505c cells were plated onto 18-mm cover glasses in MEM medium at ~70% confluence for 24 h. The cells were then treated with PKE at 100 μg/ml for 24 h. They were fixed in 2% ice-cold paraformaldehyde (PFA), washed with PBS, then stained with 2 μg/ml of 4,6-diamidino-2-phenylindole (DAPI) for 20 min at 37°C. The DAPI-stained cells were examined under a fluorescent microscope analyzing nuclear fragmentation. TUNEL was performed following the manufacturer’s instructions for TUNEL kit (Chemicon, Temecula, CA, USA).

Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was polymerized for 30 min at 37°C. HUVECs were suspended in ECGM2 medium, containing 50 ng/ml VEGF at a density of 3×10⁴ cells/ml, and 0.2 ml of cell suspension was added to each Matrigel-coated well, with or without the concentrations of PKE indicated for 10 h. The morphological changes of the tube formation were observed under a phase-contrast microscope and photographed at magnification, ×200.

HUVECs, plated on culture dishes of 60 mm diameter at 90% confluence, were wounded with a 2-mm razor blade and marked at the injury line. Subsequently, the cells that peeled off were removed with PBS and the wounded HUVECs were incubated in media with 50 ng/ml VEGF, 1 mM thymidine (Sigma-Aldrich) and/or PKE. HUVECs were allowed to migrate for 16 h and were then rinsed with PBS, followed by fixation with methanol.

8505c cells and HUVECs were seeded on 18-mm glass plates in growth medium at ~70% confluence for 24 h. The cells were treated with CoCl₂ for 1 h. Subsequently, PKE was added to the medium and incubated for 6 h. They were fixed in 2% ice-cold PFA, and washed with PBS. Immunostaining of the cells was performed using the primary antibodies, followed by the secondary antibodies conjugated to FITC (Vector Laboratories, Burlingame, CA, USA) or TRITC (Vector Laboratories) then stained with 2 μg/ml of DAPI for 20 min at 37°C. Each slide was observed using a confocal laser scanning microscope (Olympus, Tokyo, Japan).

Male nude mice were obtained from the Central Animal Laboratory, Inc. (Seoul, Korea). Animal care and experimental procedures were in accordance with the approval and guidelines of the Inha Institutional Animal Care and Use Committee (INHA IACUC) of the Medical School of Inha University (Incheon, Korea). The animals were fed standard rat chow and tap water ad libitum, and were kept under 12 h dark/light cycle at 21°C. Male nude mice (6 weeks; weight, 20–22 g) were randomized to three groups (control, PKE 125 and PKE 250 mg/kg). 8505c cells were harvested and mixed with PBS (200 μl/mouse), and then inoculated into one flank of each nude mouse (1×10⁷ of 8505c cells). When the tumors had reached a volume of ~50 mm³, the mice were given a daily intraperitoneal injection of PKE (125 and 250 mg/kg, treated group) or vehicle (200 μl PBS, control group) for 28 days. The tumor dimensions were measured twice a week using a digital caliper, while the tumor volume was calculated using the formula: V= length × width² × 0.5. At the end of the experiment, the mice were sacrificed and the tumors were excised and weighed.

Data were expressed as the mean ± SD, and statistical analysis was performed using ANOVA and an unpaired Student’s t-test. P≤0.05 was considered to indicate a statistically significant difference. Statistical calculations were performed using SPSS software for Windows operating system (version 10.0; SPSS, Chicago, IL, USA).

The effect of PKE on the viability of two ATC cell lines (8505c and SNU-80) was examined. ATC cells were incubated in media containing 50–300 μg/ml of PKE for 48 h. The results showed that cell growth was inhibited by PKE treatment in a dose-dependent manner (Fig. 1). The IC₅₀ for growth inhibition was 120 μg/ml on SNU-80 and 140 μg/ml on 8505c ATC cells.

To identify the apoptotic effect of PKE in 8505c ATC cells, the expression of Bax and Bcl-2, as well as the cleavage of PARP and caspase-3 was measured by western blot analysis with PKE for 48 h. PKE was found to lead to the upregulation of Bax and cleaved PARP and caspase-3 and a downregulation of Bcl-2 in 8505c ATC cells in a dose-dependent manner (Fig. 2A). These results showed that PKE induced cell apoptosis in 8505c ATC cells. When treated with PKE (100 μg/ml), the 8505c ATC cells presented the morphological features of apoptotic cells, such as DNA fragmentation and perinuclear apoptotic bodies by DAPI (Fig. 2B). The results of the TUNEL also exhibited PKE-induced apoptosis by causing DNA strand breaks.

HIF-1α and VEGF are important angiogenic factors in tumor progression. Thus, the effect of PKE on the hypoxia-induced HIF-1α and VEGF expressions was examined. The cells were treated with varying concentrations of PKE under hypoxia-like conditions induced by CoCl₂ (100 μM) for 18 h. As shown in Fig. 3A, the HIF-1α expression was increased under hypoxic conditions, whereas PKE treatment at 100–300 μg/ml inhibited the hypoxia-induced HIF-1α expression in a dose-dependent manner. Additionally, VEGF expression was increased under the hypoxia-like conditions, while PKE inhibited VEGF expression at a dose of 100–300 μg/ml. Consistent with the results of the western blot analysis, immunofluorescence using a confocal microscope also showed that PKE inhibited HIF-1α expression (Fig. 3B). In addition, the anti-angiogenic potential of PKE was examined using HUVECs. During an in vitro tube formation assay, PKE was observed to have inhibited the formation of vessel-like structures, comprising the elongation and alignment of the cells at the indicated concentrations (Fig. 4A). Cell migration is critical for endothelia cells to form blood vessels in angiogenesis and is necessary for tumor growth and metastasis. Thus, a wound migration assay was carried out to examine the effect of PKE on cell migration. Notably, when the endothelial cells were wounded and incubated in media with 50 ng/ml VEGF and 1 mM thymidine in the presence of PKE (100 μg/ml) for 16 h, subsequent to PKE treatment the wound was unable to heal (Fig. 4B). In addition, since VEGF is one of the critical factors of the tube formation and migration of HUVECs, we investigated whether PKE affects the expression of VEGF under hypoxic conditions in HUVECs using immunofluorescence. As expected, PKE decreased VEGF expression under hypoxia (Fig. 4C). These results showed that PKE prevented the tube formation and migration of endothelial cells while inhibiting the VEGF expression, suggesting that PKE had a potent anti-angiogenic property.

Based on these findings demonstrating a strong efficacy of PKE against 8505c ATC cells, the in vivo efficacy of PKE against the 8505c ATC cells was next examined in a nude mouse xenograft. As shown in Fig. 5A, PKE induced dose-dependent tumor growth inhibition at the doses of 125 or 250 mg/kg for 24 days, when compared to the control group. PKE administration at the doses of 125 and 250 mg/kg resulted in a significant reduction of the tumor volume (57 and 74%, respectively) in nude mice. Consistent with this finding, the tumor weight isolated from PKE-treated groups was decreased by 54 and 36% at the PKE doses of 125 and 250 mg/kg, respectively, compared to the control (Fig. 5B and C, P<0.05). No difference in the body weight of PKE-treatment groups was observed, compared to the control group (Fig. 5D), indicating that PKE had a low toxicity in mice at curative doses. These results demonstrated the in vivo antitumor efficacy of PKE against ATC without any apparent sign of toxicity.