The methanol extract (1 g) of S. delavayi Diels. was separated by chromatography on a Sephadex LH-20 column (GE Healthcare, Uppsala, Sweden, 40i.d.x860 mm, 25–100 μm, eluted with methanol). Fraction 3 (700 mg) was further purified by C18 high performance liquid chromatography [YMC-Pack Pro, YMC GmbH, Leicestershire, UK, S-5 μm, 20i.d.x250 mm) with 10–30% aqueous acetonitrile (0.05% trifluoroacetic acid, Sigma-Aldrich Co., St. Louis, MO, USA) for 90 min at 7 ml/min, yielding FK-3000 (76 mg; retention time, 82.14 min) as pale brown needles. The ¹H, ¹³C, and two-dimensional nuclear magnetic resonance (2D NMR) spectra of the isolate were in good agreement with those of FK-3000 isolated from S. cepharantha (22).

The human breast carcinoma cell lines MDA-MB-231 and MCF-7 were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultivated in RPMI-1640 (Gibco/BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco/BRL), 2 mg/ml sodium bicarbonate (Gibco/BRL), 100 U/ml penicillin (Gibco/BRL), and 100 μg/ml streptomycin (Gibco/BRL).

Cells were seeded in 96-well plates (1.5×10⁴ cells/well) and incubated at 37°C in a 5% CO₂ atmosphere. To determine the IC₅₀ of FK-3000, MDA-MB-231 and MCF-7 cells were treated with 0.1% dimethyl sulfoxide (DMSO; vehicle only control) (Sigma-Aldrich Co.) or FK-3000 (0–5 μg/ml) 24 h after seeding. Cell proliferation was analyzed after 24 and 48 h using the cell counting kit-8 (Dojindo Molecular Technologies, Rockville, MD, USA) according to the manufacturer’s instructions.

To evaluate cell viability, cells were treated with 0.1% DMSO, the 48 h IC₅₀ of FK-3000 (0.52 μg/ml for MDA-MB-231 cells; 0.77 μg/ml for MCF-7 cells), 5.0 μM trans-1-(4-hydroxy-cyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)-imidazole (p38 MAPK inhibitor SB 239063, Sigma-Aldrich Co.), or cotreated with the 48 h IC₅₀ of FK-3000 and 5.0 μM SB 239063. Cell viability was evaluated after 48 h using the cell counting kit-8 (Dojindo Molecular Technologies). All experiments were performed in quadruplicate on different days.

Cells were seeded in 100-mm culture dishes (1.0×10⁶ cells/well). After attachment, cells were synchronized by fetal bovine serum withdrawal for 6 h and then treated in quadruplicate with DMSO only, FK-3000 (MDA-MB-231 cells, 0.5 μg/ml; MCF-7 cells, 0.7 μg/ml), SB 239063 (both cell lines, 5.0 μM), or combination treatment (MDA-MB-231 cells, 0.5 μg/ml FK-3000 + 5.0 μM SB 239063; MCF-7 cells, 0.7 μg/ml FK-3000 + 5.0 μM SB 239063). Cells were harvested after 24 or 48 h of treatment and fixed with ice-cold 70% ethanol at 4°C. After 24 h, the fixed cells were centrifuged at 1,200 rpm using a Gyro 416 G (Gyrozen, Daejeon, Korea) for 6 min and the supernatant was discarded. The cell pellets were resuspended in binding buffer consisting of 0.01 M HEPES/NaOH (pH 7.4) (Sigma-Aldrich Co.) containing 0.14 M NaCl (Sigma-Aldrich Co.), 2.5 mM CaCl₂ (Sigma-Aldrich Co.), 5 μl propidium iodide (Sigma-Aldrich Co.), and 80 μl/ml ribonuclease A (Sigma-Aldrich Co.). After 20–30 min of incubation at room temperature in the dark, the DNA content of the cells was examined using a BD Model FACScan flow cytometer (Becton-Dickinson, San Jose, CA, USA).

Cells were seeded in 100-mm culture dishes (1.0×10⁶ cells/well), incubated for 24 h, and then treated in quadruplicate with DMSO, FK-3000 (MDA-MB-231 cells, 0.5, 2.5, or 5.0 μg/ml; MCF-7 cells, 0.7, 3.5 or 7.0 μg/ml), 5.0 μM SB 239063 (both cell lines), or combination treatment (MDA-MB-231 cells, 0.5 μg/ml FK-3000 + 5.0 μM SB 239063; MCF-7 cells, 0.7 μg/ml FK-3000 + 5.0 μM SB 239063). After incubation for 45 min to 48 h, cells were harvested by trypsinization and washed twice with cold phosphate-buffered saline (PBS, Sigma-Aldrich Co.). Total protein was prepared with Pro-Prep™ (iNtRON Biotechnology, Seongnam, Korea), and the protein content of each sample was determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane in Trans-Blot^® Transfer Medium (Bio-Rad). Membranes were incubated with anti-phospho-p38 MAPK monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA; cat no. 9215), anti-phospho-CDC25C antibody (Cell Signaling Technology, cat no. 9527), anti-phospho-CDC25B antibody (Abgent, San Diego, CA, USA; AP3053a), anti-cyclin B antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-245), anti-phospho-CDC-2 antibody (Cell Signaling Technology, cat no. 9112), anti-cyclin A antibody (Santa Cruz Biotechnology, SC-751), anti-phospho-retinoblastoma (RB) antibody (Cell Signaling Technology, cat no. 9308), and anti-β-actin monoclonal antibody (Sigma-Aldrich Co., cat no. A-5316). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Cayman, Ann Arbor, MI, USA; cat no. 10004301) was used as the secondary antibody. Stained bands were analyzed using the ECL detection kit (Amersham Biosciences, Buckinghamshire, UK).

Attached cells were treated with DMSO alone, FK-3000 (MDA-MB-231 cells, 0.5 μg/ml; MCF-7 cells, 0.7 μg/ml), 5.0 μM SB 239063 (both cell lines), or combination treatment (MDA-MB-231 cells, 0.5 μg/ml FK-3000 + 5.0 μM SB 239063; MCF-7 cells, 0.7 μg/ ml FK-3000 + 5.0 μM SB 239063), and incubated for 2 h in a confocal dish (SPL Life Science, Pochoen, Korea). Cells were washed three times in cold PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich Co.) at room temperature, treated with 0.5% Triton X-100, blocked with Animal-Free Blocker™ (Vector, Burlingame, CA, USA) for 1 h and incubated overnight at 4°C with anti-phospho-p38 MAPK monoclonal antibody. Cells were then incubated for 1 h with fluorescein isothio-cyanate (FITC)-conjugated goat anti-rabbit IgG (Cayman) followed by 7 μg/ml bisbenzimide H 33342 trihydrochloride (Sigma-Aldrich Co.) for nuclear staining, and photographed using an LSM510 Meta Fluorescent Microscope with Plan-Apochromat 100x/1.4 Oil DIC (Carl-Zeiss, Jena, Germany).

Attached cells were treated for 48 h with DMSO alone, FK-3000 (MDA-MB-231 cells, 0.5 μg/ml; MCF-7 cells, 0.3 μg/ml), 5.0 μM SB 239063 (both cell lines), or combination treatment (MDA-MB-231 cells, 0.5 μg/ml FK-3000 + 5.0 μM SB 239063; MCF-7 cells, 0.3 μg/ml FK-3000 + 5.0 μM SB 239063). Cells were harvested by trypsinization, washed in cold PBS, and resuspended in binding buffer consisting of 0.01 M HEPES/NaOH (pH 7.4) containing 0.14 M NaCl and 2.5 mM CaCl₂. FITC-conjugated Annexin V (BioVision, Milpitas, CA, USA) and propidium iodide (5 μl each) (Becton-Dickinson) were added to the cells, which were gently mixed and incubated for 15 min at room temperature in the dark. Binding buffer was then added, and the cells were analyzed with BD Model FACScan (Becton-Dickinson).

Results are expressed as mean ± standard deviation (SD). Groups were compared using Tukey’s studentized range (HSD) test with SPSS Statics (IBM, Armonk, NY, USA); p<0.01 was considered statistically significant.

We screened 509 natural products for anticancer activity and identified 14 candidates. The compound 6,7-di-O-acetylsinococuline (FK-3000) was isolated from S. delavayi Diels. (Fig. 1; molecular weight, 417.45), and its chemical structure was confirmed by ¹H, ¹³C, and ²D NMR. The chemical structure of FK-3000 isolated from S. delavayi Diels. was in good agreement with the compound previously isolated from S. cepharantha (22).

Antiproliferative effects of FK-3000 against cancer cells have not previously been reported, we found that FK-3000 inhibited cell proliferation in a dose-and time-dependent manner in two human breast cancer cell lines. The antiproliferative effect of FK-3000 against MDA-MB-231 cells (24 h IC₅₀, 0.89 μg/ml; 48 h IC₅₀, 0.52 μg/ml) was greater than its effect against MCF-7 cells (24 h IC₅₀, 2.53 μg/ml; 48 h IC₅₀, 0.77 μg/ml).

Carcinogenesis is caused by cell cycle deregulation, typically an increase in positive regulators such as CDKs and/or decrease in negative regulators such as cyclin D1, CDK4, cyclin E, cyclin A, and Wee1. Because the cell cycle is no longer controlled, cell proliferation is excessive (6). We therefore measured the effect of FK-3000 on cell cycle regulation in MDA-MB-231 and MCF-7 cells. Doses were based on the 48 h IC₅₀ of FK-3000 for each cell line, corresponding to 1× IC₅₀ to 10× IC₅₀ for each cell line (MDA-MB-231, 0.5–5.0 μg/ml; MCF-7, 0.7–7.0 μg/ml). As shown in Fig. 2A and B, FK-3000 treatment resulted in G₂/M phase arrest in a time- and dose-dependent manner. In MDA-MB-231 cells treated with 1× IC₅₀ FK-3000 for 24 h, the percentage of G₂/M phase arrested cells was 23.50%, increasing to 38.95% after 48-h treatment with 10× IC₅₀ FK-3000. In MCF-7 cells treated with 1× IC₅₀ FK-3000 for 24 h, the percentage of G₂/M phase arrested cells was 28.93%, increasing to 40.13% after 48-h treatment with 10× IC₅₀ FK-3000.

In cancer cells, levels of phosphorylated p38 MAPK proteins are low whereas phosphorylated CDC25B protein levels are high. CDC25B plays a key role in G₂/M phase transition and CDC2 activation (23); phosphorylation of CDC25B is an important step leading to proliferation and metastasis of neoplastic cells. P38 MAPK induces G₂/M arrest by inhibiting CDC25B phosphorylation and blocking participation of the CDC2/cyclin B complex in G₂/M transition (20,21).

As shown in Fig. 3A, FK-3000 increased phosphorylation of p38 MAPK and decreased phosphorylation of CDC25B in both MDA-MB-231 and MCF-7 cell lines. Levels of phosphorylated p38 MAPK increased in a dose- and time-dependent manner in MCF-7 cells, whereas the level of phosphorylated p38 MAPK at 90 min differed from that of other time points in MDA-MB-231 cells. In MDA-MB-231 cells, phosphorylation of CDC25B in cells treated with 1× IC₅₀ FK-3000 was similar to that of the 5× IC₅₀ group at 45 min, but was almost completely abolished by 90-min treatment with 10× IC₅₀ FK-3000 and 120-min treatment with 5× IC₅₀ FK-3000. In MCF-7 cells, FK-3000 significantly reduced phospho-CDC25B in a dose-and time-dependent manner, and 10× IC₅₀ FK-3000 almost completely suppressed phosphorylation of CDC25B at all time points. These results suggest that FK-3000 inhibits CDC25B through p38 MAPK activation.

We next determined the effect of FK-3000 on the G₂/M phase regulatory factors and related proteins CDC-2, cyclin A, cyclin B, and RB. With the exception of cyclin B and phospho-CDC-2, we did not observe changes in these proteins (data not shown). Cyclin B levels were not altered by 24 h FK-3000 treatment in either MDA-MB-231 or MCF-7 cells, except in cells treated with 10× IC₅₀ FK-3000; however, this increase was attenuated at 48 h, and cyclin B was barely detectable after 48-h treatment with 10× IC₅₀ FK-3000 in both cell lines (Fig. 3B). FK-3000 decreased phosphorylation of CDC2 in a dose- and time-dependent manner, and 48-h treatment with 10× IC₅₀ FK-3000 in MDA-MB-231 cells and 5× IC₅₀ or 10× IC₅₀ FK-3000 in MCF-7 cell completely abolished phosphorylation of CDC2 (Fig. 3B).

CDC25B phosphorylation is regulated by p38 MAPK, which also blocks participation of the CDC2/cyclin B complex in G₂/M transition (20,21). Phosphorylation of p38 MAPK plays a role in cell death, cell differentiation, and cell cycle progression. Following DNA damage, phospho-p38 MAPK translocates from the cytoplasm into the nucleus (24), where accumulation of phospho-p38 MAPK triggers G₂/M phase arrest and DNA repair.

We assumed that FK-3000 induced p38 MAPK phosphorylation and then suppressed CDC25B phosphorylation. Our results showed that a 90-min FK-3000 treatment stimulated p38 MAPK phosphorylation and nuclear translocation in MDA-MB-231 and MCF-7 cells (Fig. 4A and B), and this effect was suppressed by SB 239063, a potent and selective inhibitor of p38 MAPK (25). We compared phospho-p38 MAPK and phospho-CDC25B levels in FK-3000 treated cells with that of untreated cells at 90 min (Fig. 4C). Phosphorylation of CDC25B was abolished in cells treated with FK-3000 in the presence or absence of SB 239063. Together, these findings indicate that FK-3000 inhibits CDC25B phosphorylation directly as well as indirectly through p38 MAPK phosphorylation.

To evaluate the mechanism of cell cycle arrest by FK-3000, we analyzed the cell cycle distribution of treated cells. Although the distribution of cells treated with SB 239063 was similar to that of the vehicle control, SB 239063 could not completely reverse FK-3000-induced G₂/M phase arrest (Fig. 4D).

To confirm that FK-3000 inhibited cell proliferation through p38 MAPK activation, we evaluated whether the p38 MAPK inhibitor SB 239063 could rescue the antiproliferative effect of FK-3000. Our results showed that SB 239063 attenuated but could not completely block the antiproliferative action of FK-3000. SB 239063 increased viability from 52.93 to 62.52% in FK-3000-treated MDA-MB-231 cells and increased viability from 50.59 to 60.63% in FK-3000-treated MCF-7 cells (Fig. 4E). As shown in Fig. 4E, the viability of cells treated with both SB 239063 and FK-3000 (77.69% in MDA-MB-231, 60.63% in MCF-7) did not fully recover to the level of control cells, suggesting that FK-3000 inhibits cell proliferation by an additional mechanism besides G₂/M phase arrest through p38 MAPK phosphorylation and CDC25B dephosphorylation. We therefore analyzed the effect of SB 239063 on the rate of apoptosis in cells treated with FK-3000 (Fig. 4F). Apoptosis in cells treated with FK-3000 (SB 239063 + FK-3000 cotreatment or FK-3000 only) was significantly higher than that of cells treated with the vehicle control or SB 239063 only. Thus, FK-3000 appears to induce apoptosis by a pathway independent of the p38 MAPK-CDC25B pathway.

Taken together, these findings indicate that FK-3000 is a promising anticancer drug candidate that exerts its antiproliferative activity through two pathways: induction of G₂/M phase arrest by p38 MAPK-CDC25B-CDC2-cyclin B modulation and stimulation of apoptosis independent of the p38 MAPK-CDC25B pathway.