Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 500 NMR spectrometer (Bruker Corporation, Billerica, MA, USA) with tetramethylsilane as an internal standard. High-resolution electrospray ionization mass spectrometry was conducted using a Micromass Autospec-Ultima TOF mass spectrophotometer (Micromass UK Ltd., Altrincham, UK). The melting point was acquired using micro melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China). The materials used for column chromatography (CC) were silica gel (SiO₂; 200–300 mesh; Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (18–111 μm; GE Healthcare, Amersham, UK). Thin-layer chromatography (TLC) was conducted using glass precoated with silica gel (GF254; 10–40 mm; Qingdao Marine Chemical Factory).

The roots of Euphorbia hylonoma were collected from Qinling Mountain, Shaanxi, China, in September 2010 and were identified by Professor Juxian Lu, Faculty of Pharmacy, Medical College of Xi’an Jiaotong University (Xi’an, China). The voucher specimen was retained at the Department of Pharmacy, Medical School of Xi’an Jiaotong University for future reference.

The HepG2 human hepatoma cell line was obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). HepG2 cells (5.0×10⁴ cells/ml) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), containing 2.0 mmol/l glutamine and 1% penicillin-streptomycin in 5% CO₂ at 37°C, and were allowed to adhere for 24 h. The experiments were divided into the following five groups in the cell proliferation assay (MTT assay): Negative control (dimethyl sulfoxide; DMSO); positive control (15 μmol/l oxaliplatin); low-dosage (22.5 μmol/l JNE2); middle-dosage (45 μmol/l JNE2); and high-dosage (90 μmol/l JNE2). However, the low-dosage group was cut-off in other assays due to its low efficiency in the MTT assay.

The dried and powdered roots (1 kg) of Euphorbia hylonoma were extracted with acetone three times (24 h per extraction) at room temperature to obtain 212-g extracts. A portion of the acetone extracts (20 g) was chromatographed on a silica gel (500-g) column. The column was eluted with a gradient of petroleum ether-ethyl acetate (100:1 to 1:100) and methanol. The volume of each elution was 250 ml and underwent TLC inspection. The fractions with the same TLC spectrum behavior were combined to obtain seven fractions, A–G. Subsequently, fraction D (4.3 g) was further isolated on a silica gel column and eluted with petroleum-ether acetate (7:3). Further CC of subfraction B (1.2 g) from fraction D was performed on a Sephadex LH-20 column that was eluted with methanol. Compound JNE2 (0.6 g) was obtained from subfraction B-6. The identification of this compound was performed through the analysis of the spectroscopic features and comparing the spectral data with those of previous studies (16,17). This compound was identified as JNE2 (Fig. 1) as follows as follows: White powder, m.p. 241–243°C; ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 7.48 (1H, d, J=5.3, H-5), 7.73 (1H, d, J=3.2, H-5), 4.05 (1H, s, 3-OCH₃), 4.08 (1H, s, 3′-OCH₃), 10.8 (1H, s, 4-OH); ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 111.07 (C-1), 140.94(C-2), 140.19(C-3), 151.26 (C-4), 111.65 (C-5), 111.82 (C-6), 158.37 (C-7), 114.19 (C-1′), 141.58 (C-2′), 141.98 (C-3′), 152.84 (C-4′), 112.02(C-5′), 112.72(C-6′), 152.40 (C-7′), 101.94(C-1″), 73.09(C-2″), 76.17(C-3″), 69.31(C-4″), 65.84 (C-5″), 61.48(3-OCH₃), 61.02 (3′-OCH₃).

HepG2 cells (2×10⁴ cells/well) were seeded in 96-well plates. Following overnight incubation, test substances were added and the incubation was continued at 37°C in an atmosphere containing 5% CO₂ for 3 days. Subsequently, 20 μl MTT (Sigma-Aldrich, St. Louis, MO, USA) solution (5 g/l) was added into each well and incubated for an additional 4 h. Supernatants were removed and formazan crystals were dissolved in 200 μl DMSO. The optical density was measured at 490 nm using a POLARstar Optima (BMG Labtech GmbH, Ortenberg, Germany).

Cell seeding and treatment were the same as MTT assay. After three days of treatment, cells were harvested by trypsinization and 1×10⁶ cells were counted and used for the analysis. Cells were fixed in ice-cold ethanol overnight at 4°C following washing with PBS. The cells were then washed in PBS again and incubated in 1 ml staining solution (20 μg/ml propidium iodide and 10 U/ml RNase A) for 30 min at room temperature. The cells were examined by fluorescence-activated cell sorting (FACS) using a flow cytometer (FACSort; Becton-Dickinson, Franklin Lakes, NJ, USA), and the cell cycle populations were determined using ModFit software (Verity Software House, Turramurra, Australia).

For the analysis of apoptotic cell populations, cells were trypsinized and washed in PBS. Staining with Alexa-Fluor 647 Annexin V (Invitrogen Life Technologies, Carlsbad, CA, USA) and propidium iodide was performed in 20 mmol/l HEPES buffer (pH 7.4), containing 150 mM NaCl and 2.5 mmol/l CaCl_2, for 15 min at room temperature. The cells were examined by FACS using a flow cytometer (FACSort; Becton-Dickinson), and the apoptotic populations were determined using ModFit software (Verity Software House).

Cell invasion was evaluated using the Chemicon QCM™ 24-well collagen-based cell invasion assay (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. In brief, 0.3 ml serum-free medium was added to the interior of each insert to rehydrate the collagen layer for 30 min at room temperature. The medium was then replaced with 0.3 ml prepared serum-free cell suspension containing 3.0×10⁵ cells and the corresponding test substances. Medium (500 μl) containing 10% FBS was added to the lower chamber and the cells were incubated for 24 h at 37°C. Following this, all non-invaded cells were removed from the interior of the insert and the invaded cells were stained with crystal violet. The stained cells were analyzed on an Olympus fluorescence microscope (BX43; Olympus Corporation, Tokyo, Japan).

Cell seeding and treatment were the same as MTT assay. After three days of treatment, cells were harvested and washed in PBS. Cell protein lysates were separated in 10% SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes (Roche Diagnostics, Mannheim, Germany). The lysates were then detected using rabbit polyclonal antibodies that were specific for Bcl-2, BAX, caspase-3 and CCND1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and a commercial ECL kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Protein loading was estimated by human anti-β-actin monoclonal antibody (Santa Cruz Biotechnology, Inc.).

Statistical analysis was performed by one-way ANOVA test followed by Fisher’s protected least significant difference post hoc test for multiple comparisons using the StatView program (Abacus Concepts, Berkeley, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To evaluate the antitumor role of JNE2 on human hepatoma cells, MTT and colony formation assays were employed to detect the growth of HepG2 cells at various time points following treatment with JNE2 at various concentrations. The results showed that JNE2 exhibited a growth inhibitory effect on HepG2 cells in a dose- and time-dependent manner. Following 24 h of treatment, the OD value of the high-dosage group (JNE2, 90 μmol/l) was significantly lower than that of the negative control group (P<0.05) and also lower than that of the positive control group, but without significant difference (Fig. 2A). The time-effect curve also demonstrated the antiproliferation ability of JNE2 (Fig. 2B). Furthermore, the colony formation results shown in Fig. 3 confirmed that a high dosage of JNE2 may inhibit the growth of HepG2 cells. These results suggested that JNE2 exhibits an inhibitory effect on the proliferation of hepatoma cells.

To explore the mechanism underlying JNE2-suppressed cell proliferation, the impact of JNE2 on cell cycle progression was investigated by FACS and the results are presented in Fig. 4A. Following treatment with high and middle dosages of JNE2, the cell cycle shifted from a high S phase population to a high G1 phase population, together with an accumulation of a G2/M phase population. Whereas, little effect on the cell cycle was observed in the control groups. These results indicated that JNE2 blocks the G1/S transition.

To examine the effect of JNE2 on apoptosis, hepatoma cells were treated with various concentrations of JNE2. Compared with the cells that were treated with the negative control, the cells that were treated with a high dosage of JNE2 exhibited a similar apoptotic rate to those treated with oxaliplatin, whereas the cells that were treated with 45 μmol/l JNE2 exhibited slightly lower apoptotic rates. In the positive and JNE2-treated groups, the number of cells at the early and late stages of apoptosis marginally increased. However, no significant differences were identified (Fig. 4B) between the groups. These results showed that JNE2 induces apoptosis in human hepatoma cells in a dose-dependent manner.

Cancer invasion is the process by which cells break away from the primary tumor and migrate through the surrounding tissue. This enables the cancer cells to move into blood vessels and travel through the body, possibly establishing a secondary tumor at an additional site (18). To determine whether JNE2 inhibits invasion, HepG2 cells were treated with various concentrations of JNE2 and the control groups were treated separately. Evidently, the invasion was inhibited by a high concentration of JNE2, whereas, migration was not altered in the cells that were treated with the negative control (Fig. 5). These results markedly suggest that JNE2 inhibits the invasion of HepG2 cells.

To clarify the apoptotic mechanism of JNE2 on HepG2 cells, western blot analysis was performed to examine the protein expression levels of Bcl-2, Bax, caspase-3 and CCND1 (cyclin D1). The cells that were treated with JNE2 exhibited upregulated levels of Bax and caspase-3 expression when compared with the negative control cells. This result may explain why JNE2 inhibited the growth of HepG2 cells, and this function may be associated with mitochondrial pathway-induced apoptosis. Conversely, the protein levels of Bcl-2 and CCND1 were decreased with JNE2 treatment (Fig. 6). The apoptotic effect of JNE2 was confirmed by the upregulation of Bax and downregulation of Bcl-2. Based on the G0/G1 arresting capability, the downregulation of CCND1 may provide an explanation. These results suggested that JNE2 may exhibit its apoptotic effect through the upregulation of Bax and caspase-3, and the downregulation of Bcl-2 and CCND1.