Powdered Curcuma longa (400 g) was extracted twice with hexane to remove curcumin and its derivatives. The extract (13.27 g) was then evaporated under vacuum. The residue was dissolved in MeOH. For the partial purification of ar-turmerone, the filtered solution was fractionated with a Buchi MPLC system (Buchi pump C-605, column 1.5×23 cm, fraction collector Buchi C-660) using LiChroprep C-18 (40–63 μm; Merck) as an adsorbent. The pooled fraction was extracted with ethyl acetate and water. The ethyl acetate layer was evaporated under reduced pressure, after which the residue was dissolved in MeOH. To achieve higher purity, the MeOH solution was injected into a semi-preparative liquid chromatography apparatus (Young Lin Instruments Co., Ltd., Korea) equipped with a Gemini C18 column (10×250 mm; Phenomenex Co., Ltd.). MeOH solution (70%) was maintained at a flow rate of 5 ml/min. The pooled fractions were evaporated under vacuum and extracted with ethyl acetate and water. The ethyl acetate layer was evaporated under reduced pressure. A viscous oil (131.3 mg) was subsequently generated, identified as ar-turmerone by GC-Mass analysis and by comparing with the NIST library data (23).

The viscous oil was analyzed by the GC-Mass electron impact ionization (EI) method on Agilent model 6890 GC interfaced to a 5973 mass selective detector. HP-5MS capillary columns (30 m×0.25 mm×0.25 μm film thickness) were used for GC. The oven temperature was maintained at 60°C for 6 min then programmed to 240°C at the rate of 5°C/min. The carrier gas was helium, at a flow rate of 0.9 ml/min, and the injection volume was 1 μl. In mass spectrometry EI was performed at an electron energy of 70 eV.

Mouse lymphoblast P388D1 cells from the Korean Cell Line Bank (KCLB) were cultured in RPMI-1640 media with 10% fetal bovine serum (FBS).

According to the protocol of the National Cancer Institute (NCI), USA (24), 6×10⁶ cells/0.1 ml/mouse of mouse lymphoblast cells (P388D1) were injected into subcutaneous tissues of BDF1 mice for 21 days. The animals commenced treatment on Day 2 of control (CMC vehicle, n=7), 200 mg/kg of ar-turmerone (n=7), and 10 mg/kg of Glivec (25) (n=7), a positive control by daily orogastric feeding daily. For the negative control, PBS was injected into subcutaneous tissues of BDF1 mice for 21 days and CMC vehicle were treated for 20 days orally. Then, changes of tumor volume were measured with Calipus (Mitutoyo Corp., Japan) twice a week, and the tumor inhibition rate of each group was calculated according to the following formula: inhibitory   rate   ( IR )   of   tumor   growth = C ( V 1 − V 0 ) − T ( V 1 − V 0 ) C ( V 1 − V 0 )where C is control group, T is treated group (ar-turmerone or Glivec), V₁ is the volume before treatment (mm³), V₀ is the volume after treatment (mm³).

This experiment was approved by the Ethics Committee for animal experiments at Dongseo University. When experimental animals are used, the NIH guidelines for laboratory animals (26) and the Korean revised law for animal protection, 8852, are followed. All precautions were taken to care for the animals and to minimize any pain or discomfort.

After 21 days of treatment, the mice were euthanized by cervical dislocation and thoroughly examined postmortem. Paraffin-embedded tumor samples were cut into 1 μm sections and attached on glass slides. Following deparaffination and dehydration, samples were stained with hematoxylin & eosin (H&E). H&E-stained ultra-thin sections were observed with a light microscope (BX41TF; Olympus, Japan) with magnification, ×1,000. Following cervical dislocation, blood was taken immediately, and analyzed by Hemavet 950 (Drew Scientific Inc.) without pretreatment.

Paraffin-embedded tumor samples were cut into 5-μm sections and attached on silane coated glass slides (Muto, Japan). Apoptosis in the tumor was measured with the In Situ Cell Death Detection kit, POD (Roche, Germany). The TUNEL assay was carried out following the manufacturer’s instructions. Tissue sections were deparaffinized in xylene, and hydrated through graded ethanol and then treated with proteinase K (20 μg/ml; Sigma, USA) for 20 min at RT. Endogenous peroxidase was blocked by 0.3% H₂O₂ in methanol for 15 min at RT. After the reaction, tissue sections were treated with Tris-HCl with 3% bovine serum albumin for 30 min and then incubated with TUNEL reaction mixture [containing terminal deoxynucleotidyl transferase from calf thymus (enzyme solution) and nucleotide mixture (label solution)] for 1 h at 37°C in a humidified chamber. Tissue samples were then combined with converter-POD (containing anti-fluorescein antibody Fab fragments from sheep, conjugated with horse-radish peroxidase), followed by washing and DAB (Sigma) color reaction. During the TUNEL procedure samples were washed in PBS. Tissue sections were counterstained by hematoxylin, dehydrated through graded ethanol, cleared in xylene, and mounted. Slides were observed with a light microscope (BX41TF; Olympus) for histological analysis. As a negative control, a section was incubated with label solution only instead of using the TUNEL reaction mixture. One thousand tumor cells were counted in five different sites of each tissue section at a magnification of ×200. The apoptotic index (AI) was calculated as follows: AI (%) = (number of apoptotic cells/total number of cells) ×100.

Following deparaffination, the tissue sections were heated at 100°C for 20 min in 10 mM sodium citrate buffer with 0.05% Tween-20 (pH 6.0) for antigen retrieval. Then, the sections were incubated with anti-Bax or anti-Bcl-2 antibodies (Imgenex Corp.) at a 1:200 dilution at 4°C overnight. After washing with PBS, the secondary antibody, Alexa Fluor^® 488 conjugated anti-rabbit IgG (Invitrogen), was added and cells were incubated at room temperature for 1 h. The cells were then counterstained with 2 μg/ml Hoechst dye for visualizing the nuclei. Finally, cells were observed with fluorescence microscopy and anti-Bax or anti-Bcl-2 positive cells were detected. The number of whole cells and positive cells was counted using image analysis software (ImageJ™) (NIH, Bethesda, MD, USA).

Tumor samples were cryopreserved in liquid nitrogen and total RNA was extracted as previously described (27). Concentration of RNA was determined by the absorption at 260 nm. The oligonucleotide primers were: Bcl-2 (716 bp), 5′-GGAAATATGGCGCACGCT-3′ (sense) and 5′-TCACTTGTGGCCCAGAT-3′ (antisense); Bax (508 bp), 5′-CCAGCTCTGAGCAGATCAT-3′ (sense) and 5′-TATCAGCCCATCTTCTTCC-3′ (antisense); caspase-1 (1,533 bp), 5′-GATGGCACATTTCCAGGACTGA-3′ (sense) and 5′-TGTTGCAGATAATGAGGGCAAGAC-3′ (antisense); caspase-3 (1,466 bp), 5′-CTGCCGGAGTCTGACTGGAA-3′ (sense) and 5′-ATCAGTCCCACTGTCTGTCTCAATG-3′ (antisense); caspase-6 (1,281 bp), 5′-ACGTTGACTGGCTTGTTCAAAGG-3′ (sense) and 5′-GCGTGTACACAGACGCAGCA-3′ (antisense); caspase-9 (3,896 bp), 5′-GCTGGAACACTGGGCATTGA-3′ (sense) and 5′-GGCCTGATACCCAAGCAG GA-3′ (antisense); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (sense) and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ (anti-sense). Polymerase chain reactions were performed in a 50 μl reaction volume. PCR products were electrophoresed on agarose gel, and photographed under UV light, after staining with ethidium bromide.

The effects of ar-turmerone on ex vivo lymphoproliferative responses in splenocyte cultures isolated from chronically ar-turmerone-exposed and untreated tumor-implanted BDF1 mice were determined, respectively. Spleen cell suspensions from the mice were prepared in Earle’s balanced salt solution (EBSS), washed and resuspended in RPMI-1640 medium containing 5% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μ/ml streptomycin and 50 μM mercaptoethanol (ME). The cell number was adjusted to 1.25×10⁶ cells/ml of culture media. Cells were distributed in 96-well plates (Costar, Cambridge, MA, USA) in 180 μl volume. The cultures were incubated in a humidified 5% CO₂ incubator at 37°C for 72 h with a given concentration of mitogen in 20 μl, either lipopolysaccharide (LPS) (50 μg/ml) or concanavalin A (Con A; 1 μg/ml). Following incubation, 20 μl of Cell Titer 96 aqueous nonradioactive cell proliferation kit (Promega) was added in each well, and then the plate was incubated for an additional 4 h. The plate was read on an ELISA reader (Bio-Rad) using a wavelength of 490 nM. Results were expressed as mean absorbance minus the negative control’s absorbance.

Prepared cell suspensions from drug-exposed and untreated BDF1 mice were homogenated in RPMI-1640 medium containing 5% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μM ME. The spleen cells were adjusted to 1×10⁶ cells/ml and 180 μl of cells were distributed in 24-well plates with 20 μl of Con A (2 μg/μl) and recombinant IL-2 (rIL-2; Roche). Following incubation at 37°C for 24 h, brefeldin A (10 μg/10⁶ cells) was added and postincubated for another 5 h. Then, the cells were washed with staining buffer and blocked nonspecific binding by adding anti-mouse CD16/CD32 Fc receptor (1 μg/10⁶ cell/tube) for 20 min on ice. The CD4 cells were identified using FITC-conjugated anti-mouse CD4 (0.5 μg/tube; clone, GK 1.5) mAb, which was suspended in staining buffer and incubated for 30 min on ice in a dark room. To fix and permeabilize the cells, Cytofix/Cytoperm solution (100 μl/tube) was added and postincubated on ice for 20 min in the dark. Then 1X Perm/Wash solution (1 ml/tube) was added for washing and centrifuged two times at 4°C 1,500 rpm. Subsequently, 100 μl of Perm/Wash solution with anticytokine mAbs (PE-conjugated anti-mouse IL-2) were added and the cells were postincubated for 30 min on ice in the dark. Finally, 1X Perm/Wash solution (1 ml/tube) was added for washing and centrifugation two times at 4°C 1,500 rpm, and analyzed with flow cytometry after mixing with staining buffer (300 μl/tube).

Data were analyzed by one-way ANOVA, Student’s t-test or Tukey’s HSD test.

First, we treated 200–400 mg/kg of ar-turmerone on normal BDF1 mice. The highest dose of ar-turmerone (400 mg/kg/day) caused severe diarrhea in the afternoon and decreased body weight, so the treatment could not be prolonged to 10 days. Then, we used reduced doses (200 or 300 mg/kg/day) of ar-turmerone on BDF1 mice transplanted with P388D1 lymphoblast cells. A few of the mice treated with a higher dose of ar-turmerone (300 mg/kg) showed diarrhea in the afternoon, and significantly reduced body weight (data not shown), therefore, according to the NCI manual, we could not continue ar-turmerone treatment and lymphoblast cell injections.

Lymphoma was induced by 21 days of P388D1 administration following the NCI manual (24). Tumor inhibition rates of ar-turmerone and Glivec were 11.79 and 14.16% (Table I). One of each of the experimental groups is shown in Fig. 1, and necrotic morphology is hardly shown compared to the negative control PBS group (Fig. 1). Control group, or P388D1 + CMC group, showed increased number of white blood cells (WBCs) compared to the negative control group, or PBS group (P<0.001). The number of WBCs in the ar-turmerone-treated group and the Glivec-treated group dropped significantly compared to the control group, P388D1 + CMC (P<0.001).

Although the WBC number and neutrophile percentage of the ar-turmerone- or Glivec-treated group decreased compared to the control P388D1 + CMC group at P<0.001, the lymphocyte percentage increased compared to control at P<0.001 (Table II). The number of red blood cells (RBCs) of the ar-turmerone- and the Glivec-treated group was not different from that of the control group in tumor-implanted mice. The number of hemoglobins in the ar-turmerone-treated group increased compared to control (Table II).

Positive staining was clearly shown in nuclei (Fig. 2). The apoptosis index (AI) of the control group, the ar-turmerone group and the Glivec group were 4.22±1.02, 5.45±1.46 and 10.01±2.01, respectively. AI value of the ar-turmerone-treated group increased slightly, but it did not show a significance. Only the Glivec-treated group showed a significant difference at P<0.05 compared to the control group (Table III).

Bcl family protein level was measured only in implanted tumor cells. Positive staining was located in the cytoplasm. The positive rates of Bcl-2 protein expression in the control, ar-turmerone and Glivec groups were 13.20±1.32, 11.92±1.04 and 12.36±2.02%, respectively. The positive rates of Bax protein expression in the control, ar-turmerone and Glivec groups were 14.12±1.82, 18.34±3.62 and 22.33±2.82%, respectively, in which the Glivec-treated group showed a marked change at the 1% level (Table IV).

The main signaling gene expression level was measured only in implanted tumor cells. The density of caspase-1, -3, -6, -9, Bcl-2, Bax, p21 and p53 mRNA in the control, ar-turmerone and Glivec groups is shown in Fig. 3. Most signals in both the ar-turmerone- and Glivec-treated groups did not show differences, with the exception of the Bax mRNA of the Glivec-treated group, which increased compared to the control group (Fig. 3).

The lymphocyte proliferation activities of tumor cells decreased compared to the negative control PBS group. T-lymphocytes proliferated with the induction of Con A, and B-lymphocytes proliferated with the induction of LPS (Fig. 4). In LPS induced treatment, B-lymphocyte proliferation of the control, ar-turmerone- and Glivec-treated groups decreased compared to the negative control (normal), but the control group (P388D1 + CMC) only showed significance at P<0.01. B-lymphocyte proliferation of the ar-turmerone- and the Glivec-treated group increased compared to the control group. In Con A-induced treatment, T-lymphocyte proliferation of control significantly decreased compared to the negative control (P<0.01), and the T-lymphocyte proliferation of the ar-turmerone- and the Glivec-treated group also decreased (P<0.05). T-lymphocyte proliferation of the ar-turmerone- and the Glivec-treated group significantly increased compared to the control group (P388D1 + CMC) (P<0.01).

The change in cytokine production activity caused by drugs is very important, especially in IL-2 production activity that affects T-cell differentiation (28,29). IL-2 production activity of P388D1 in implanted tumor cells decreased significantly compared to the negative control group. IL-2 production in the ar-turmerone-treated group increased compared to the control group (P<0.05), but IL-2 production in the Glivec-treated group did not increase in implanted tumor cells (Fig. 5).