PMA and calcitriol (VD₃) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and dissolved in ethanol. Anti-ICAM-1 mouse monoclonal antibody (anti-ICAM-1 mAb) and casticin were purchased from Calbiochem (La Jolla, CA, USA) and ChromaDex (Irvine, CA, USA), respectively. 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). α-Naphthyl Acetate (Non-Specific Esterase) kit was purchased from Muto Pure Chemicals Co. (Tokyo, Japan). Phenazine methosulfate (PMS) and an RNA extraction kit, ISOGEN were obtained from Wako Pure Chemical Industries. Column chromatography was performed on silica gel 60 (Nacalai Tesque, 70-230 mesh) using the indicated solvents. TLC was performed using pre-coated silica gel 60 F₂₅₄ plates (Merck KGaA) using the indicated solvents. Melting point (mp) was determined with an AS ONE melting point apparatus ATM-02 without correction. ¹H NMR spectra were recorded on a Bruker Avance™ III 600 (600 MHz) with tetramethylsilane (0 ppm) as an internal standard. ¹³C NMR spectra were recorded on a Bruker Avance III 600 (150 MHz) with dimethylsulfoxide (DMSO) (39.7 ppm) as an internal standard.

Preparation of Vitex was carried out according to the methods previously described (6). Briefly, dried ripened V. agnus-castus fruit from Israel was gently triturated. The extract was prepared from 1 g of the triturate with 10 ml of ethanol under reflux for 2 h. The extract was then cooled, filtered, evaporated and dried in a vacuum desiccator, a product of which was designated as Vitex. The yield of Vitex was 0.08-0.1 g from 1 g of dried fruit.

Vitex (1 g) was subjected to flash column chromatography using silica gel 60 (51.6 g) eluted with chloroform-methanol (250:1). The obtained eleven fractions (F1-F11: 25 ml per one fraction) were analyzed by thin-layer chromatography (TLC) [eluents: chloroform-methanol (20:1)] using commercial casticin as a standard sample. Based on the analysis, five fractions (F5–F9) containing casticin were concentrated under reduced pressure to give a brown oil residue. Then, the residue was further purified by recrystallization from n-hexane-EtOAc (3:1) to afford casticin as pale yellow crystals. mp 185–188°C (n-hexane-EtOAc); ¹H NMR (600 MHz, CDCl₃) 3.88 (3H, s), 3.92 (3H, s), 3.96 (3H, s), 3.99 (3H, s), 5.71 (1H, s), 6.51 (1H, s), 6.97 (1H, d, J=8.4 Hz), 7.69 (1H, d, J=1.2 Hz), 7.73 (1H, dd, J=8.4, 1.2 Hz), 12.60 (1H, s); ¹³C NMR (150 MHz, DMSO-d6) δ 55.9, 56.7, 59.9, 60.2, 91.5, 105.8, 112.1, 115.3, 120.6, 122.4, 131.8, 138.2, 146.6, 150.5, 151.8, 152.0, 155.8, 158.9, 178.5. Melting point, ¹H and ¹³C NMR spectroscopic data of the crystal were comparable with the reported value of casticin (17).

Leukemic cell lines HL-60 and U-937 were purchased from the Health Science Research Resources Bank (Tokyo, Japan). Peripheral blood mononuclear cells (PBMNC) were isolated from three healthy volunteers (Vitex treatment; 26±1, and casticin treatment; 26.3±3.21 years of age), as previously described (18). Briefly, 10 ml of heparinized blood was loaded on 3.5 ml of Ficoll Hypaque (Nakalai Tesque, Kyoto, Japan) and centrifuged at 2,000 rpm for 20 min, and PBMNC were separated. Both the cell lines and PBMNC were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and antibiotics [100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen)] at 37°C in a humidified atmosphere (5% CO₂ in air). Vitex and casticin were dissolved in DMSO. As for unstimulated HL-60 and U-937 control cells, and PBMNC, 2X 10⁵ cells/ml were precultured for 12 h, followed by the treatment with Vitex or casticin at various concentrations indicated at 37°C for a designated time. Control samples were prepared by treating cells with culture medium containing vehicle reagent, DMSO alone (final concentration: 0.1%). This study was approved by the IRB committee of Tokyo University of Pharmacy and Life Sciences. An informed consent was obtained from all healthy volunteers.

To induce differentiation, cells (3×10⁵ cells/ml) were exposed to 10 ng/ml PMA for 48 h, or 100 nM VD₃ for 96 h with medium change at 48-h post-exposure, respectively. As for control groups, cells were exposed to vehicle reagent, ethanol alone, at a final concentration of 0.1%. Morphological changes of HL-60 cells were observed using phase-contrast microscope (CK2, Olympus, Japan). After exposure to PMA, non-adherent cells were discarded, and tightly adherent cells were designated as PMA-treated HL-60 cells and harvested for cell counting. The number of PMA-treated HL-60 cells was ∼3×10⁶ in 5 ml medium (i.e. at the density of 6×10⁵ cells/ml) each time. Moreover, after exposure to VD₃, loosely adherent cells were designated as VD₃-treated HL-60 cells. After the cell density of VD₃-treated HL-60 and control group cells were also adjusted to 6×10⁵ cells/ml in fresh medium, the three kinds of cells were treated with Vitex or casticin at various concentrations indicated at 37°C for a designated time. Furthermore, in order to elucidate the involvement of ICAM-1 in the cytotoxicity of Vitex against differentiated HL-60 cells, HL-60 cells were treated with 10 ng/ml of PMA in the presence or absence of 1 μg/ml of anti-ICAM-1 mAb for 48 h, followed by the treatment with 50 μg/ml Vitex for 24 h.

Cell viability was determined by XTT dye-reduction assay according to the method previously described (19). Briefly, after treatment with various concentrations of Vitex or casticin for a designated time, cells were washed with PBS twice and resuspended in appropriate volume of PBS. An aliquot (0.2 ml) of cell suspension was inoculated into 96-well micro-plates followed by the addition of 50 μl XTT/PMS mixed solution [1.5 mM XTT and 0.025 mM PMS]. After incubation at 37°C for 4 h, plates were mixed on a mechanical plate shaker, and absorbance at 450 nm was measured with a microplate reader (Safire, Tecan, Switzerland). The relative cell viability was expressed as the ratio of the absorbance of each treatment group against those of the corresponding untreated control group. The IC₅₀ value of Vitex and casticin was calculated from the cell proliferation inhibition curve after 24 h of treatment.

To identify nonspecific esterase (NSE) activity, a commercially available kit for NSE staining was used. Briefly, smear preparations of stimulated cells were fixed with formalin-acetone fixative solution for 30 sec at 4°C, then washed with running water, followed by incubation with reaction agent [10 mg of fast garnet GBC salt, 10 μl of naphthyl butyrate, 0.5 ml of ethylene glycol monomethyl ether (EGME) and 9.5 ml of phosphate buffer (1/15 M, pH 6.3)] for 30 min at 37°C. After washing with running water, these preparations were stained with Karachi’s hematoxylin for 10 min at 37°C, then washed with running water for achieving proper color intensity. After drying, smear preparations were enclosed with glycerol-gelatin, followed by observation with inverted microscope (Axiovert 200, Carl Zeiss, Germany) and photographed with the software of AxioVision 4.5 (Carl Zeiss). Furthermore, to conduct the sodium fluoride (NaF) inhibition test, the above-mentioned reaction agent was replaced with reaction agent containing 4.5 mg of NaF.

DNA preparation and agarose gel electrophoresis were carried out according to a method previously reported (19). Extracted DNA was dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). DNA samples (∼20 μg/20 μl) and a Tracklt™ 100 bp DNA Ladder (Invitrogen) as a DNA size marker were electrophoresed on a 2% agarose gel (Agarose X, Nippon Gene, Tokyo, Japan) using TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA). Gels were stained with ethidium bromide and viewed under printgraph (ATTO Corp., Tokyo, Japan).

After treatment with 30 and 70 μg/ml Vitex, respectively, for 24 h, cells were washed twice with PBS and fixed with 1% glutaraldehyde/PBS at 4°C for at least 2 h. Then, cell were washed twice with PBS and resuspended in 10 μl of 200 μM Hoechst 33342 (Calbiochem)/PBS for 15 min. The stained cells were enclosed in FluorSave™ reagent in a mounting medium and then viewed using a fluorescence microscopy, Axiovert 200.

Total RNA isolation and complementary DNA were prepared according to methods previously described with modifications (20). Total RNA was extracted from cells using an RNA extraction kit, ISOGEN. Complementary DNA was synthesized from 1 μg of RNA using 100 pmol random primer and 50 units of moloney murine leukemia virus reverse transcriptase (Invitrogen) in a total volume of 20 μl, according to the manufacturer’s instructions. PCR was performed according to the methods previously described (20) using a Takara Thermal Cycler MP (Takara Shuzo Co., Osaka, Japan). DNA sequence of PCR primers and optimal conditions for PCR are shown in Table I. PCR primers were purchased from Sigma-Aldrich (Hokkaido, Japan). PCR products and a Tracklt™ 100 bp DNA Ladder as a DNA size marker were electrophoresed on a 2% UltraPure™ agarose gel (Invitrogen), respectively, and visualized by ethidium bromide staining, followed by viewing under UV Light Printgraph.

Experiments were independently repeated three times, and the results are shown as the mean ± standard deviation (SD) of three assays. Student’s t-test was applied, and p<0.05 was considered as significant.

The above-mentioned F5–F9 fractions containing casticin obtained by chromatographic separation from Vitex (1 g) were concentrated under reduced pressure to give a brown oil residue (100.3 mg). ¹H NMR analysis of the residue using 1,1,2-trichloroethane (0.25 mmol, 46 μl) as an internal standard indicated that the amount of casticin in the residue was estimated to be 0.0275 mmol (10.3 mg) (Fig. 1). The analysis indicated that casticin accounted for approximate 1% weight of Vitex.

After 24 h of cultivation, the ratio of the absorbance at 450 nm of both HL-60 and U-937 cells compared to that at 0 h increased by ∼2.5-fold, indicating cell growth rate of the two cells was almost same. Although Vitex exhibited cytotoxic activities against both cells in a dose-dependent manner, a significant difference in the IC₅₀ value was observed between HL-60 and U-937 cells (22.2±0.92 μg/ml in HL-60; 32.0±1.33 μg/ml in U-937; p<0.01) (Fig. 2A). Furthermore, similar phenomena were observed in both cells when treated with casticin (0.29±0.02 μg/ml in HL-60; 0.39±0.02 μg/ml in U-937; p<0.01) (Fig. 2B), indicating HL-60 is more sensitive to the cytotoxicity of Vitex and casticin compared to U-937. Although a relatively high concentration of Vitex exhibited cytocidal effect against PBMNC, the IC₅₀ value was more than 50 μg/ml and was ∼2 times higher than that in both leukemic cell lines (Fig. 2C). Of note, no apparent cytotoxicity of casticin was observed in PBMNC when treated with concentrations showing significant cytotoxicity in both leukemic cell lines (Fig. 2D).

After exposure to 10 ng/ml PMA and 100 nM VD₃ for 48 h and 96 h, respectively, the mature phenotype was confirmed by cell attachment, NSE activity, and manifestation of a maturation surface marker, CD11b. Whereas unstimulated HL-60 cells grew as single cell suspension cultures, PMA-treated HL-60 cells adhered tightly to plastic culture plate and showed apparent cellular aggregation, and morphology became macrophage-like (Fig. 3A). On the other hand, VD₃-treated HL-60 cells adhered loosely to plastic culture plate and its morphology became monocytoid (Fig. 3A). The NSE is a well-known selective cytochemical marker for the monocyte/macrophage lineage (21). Consistent with these previous reports, NSE activity (brownish-red granulation) was observed in PMA- and VD₃-treated HL-60 cells, but not in untreated HL-60 cells (Fig. 3B). Furthermore, NaF abolished NSE activity as expected (Fig. 3B). A predominant increase in CD11b mRNA was coincidentally observed in both PMA- and VD₃-treated HL-60 cells (Fig. 3C).

Compared to unstimulated control HL-60 cells, PMA- and VD₃-treated HL-60 cells exhibited acquired resistance to both Vitex and casticin treatment (Table II), although the IC₅₀ value of unstimulated control HL-60 cells indicated in Table II was different from that in Fig. 2 due to different cell-density at the point of treatment. In the case of Vitex treatment, the IC₅₀ value was >100 μg/ml and 39.3±0.55 μg/ml in PMA- and VD₃-treated HL-60 cells, respectively, and significantly higher than that in unstimulated control HL-60 cells (28.3±0.17 μg/ml; p<0.05). As for casticin treatment, the IC₅₀ value increased more than two times in both PMA- and VD₃-treated HL-60 cells compared to that in unstimulated control cells. Furthermore, an evident distinct increase of DNA fragmentation ladder representing apoptosis induction was observed at concentrations starting from 30 μg/ml Vitex in unstimulated control HL-60 cells (Fig. 4A). However, the DNA fragmentation was clearly abrogated in both differentiated HL-60 cells (Fig. 4A). Hoechst 33342 fluorescent staining also demonstrated that DNA fragmentation was significantly abrogated in PMA-stimulated cells even when treated with as high as 70 μg/ml Vitex (Fig. 4B). On the other hand, we also observed that a certain degree of apoptotic population was observed in the flasks that were not treated with Vitex after 24 additional hours of culture (Fig. 4B), similar to a previous study showing that a population of PMA-treated HL-60 cells spontaneously detached and became dead cells (14,22).

As shown in Fig. 5A, PMA induced cellular aggregation and adhesion within 48 h after stimulation. However, the addition of 1 μg/ml anti-ICAM-1 mAb significantly abrogated the effects of PMA. In agreement with results presented in Table II, no cytotoxicity was observed in PMA-treated HL-60 when treated with 50 μg/ml Vitex exhibiting apparent cytocidal effect against unstimulated control HL-60 cells (Fig. 5B and C). Again, the addition of anti-ICAM-1 mAb abrogated the acquired resistance to Vitex (Fig. 5C).

The expression of ICAM-1 mRNA was observed in PMA-treated HL-60 cells, but not in unstimulated control cells (Fig. 6), indicating treatment with PMA induced ICAM-1 gene expression associated with cellular adhesion and aggregation. Consistent with apparent cytotoxicity induced by Vitex in unstimulated control cells, the expression level of β-actin was clearly downregulated when treated with >50 μg/ml Vitex. However, only a slight downregulation of β-actin expression level was observed in PMA-treated HL-60 cells even when treated with as high as 70 and 100 μg/ml Vitex. Noteworthy, a clear upregulation of ICAM-1 mRNA was observed in PMA-treated HL-60 cells treated with Vitex ranging from 10 to 50 μg/ml, which exhibited no cytocidal effect against the cells. Furthermore, a prominent decrease in the expression level of ICAM-1 mRNA was observed in PMA-treated HL-60 when treated with >70 μg/ml Vitex.