Antibodies against MMP-2, MMP-9, Cdc2, cyclin B1, β-catenin, E-cadherin, vimentin and β-actin were obtained from ProteinTech Group, Inc. (Chicago, IL, USA), and the antibody for N-cadherin was obtained from Cell Signaling Technology, Boston, MA, USA). An enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Sciences, Inc. (Piscataway, NJ, USA). Cell cycle detection and comet assay kits were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Transwells were obtained from BD Biosciences (San Jose, CA, USA). 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Osthole powder was purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). It contained ~98% proanthocyanidins and is stable for at least two years at 4°C.

The HCC, SMCC-7721, MHCC-97H, HCC-LM3 and BEL-7402 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were cultured in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin (all from Gibco-BRL, Grand Island, NY, USA) at 37°C with 5% CO₂ in a humidified incubator.

Osthole was dissolved in 100% DMSO at a concentration of 1 M as a stock solution and stored at 4°C. It was diluted with DMEM before each experiment. The final concentrations of DMSO were <0.1% in all osthole treatment groups.

The effect of osthole on cell viability was detected using the MTT assay. The cells (1×10⁴/well) were seeded into a 96-well plate and incubated for 24 h. After treatment with osthole (20, 40, 80, 120, 160 or 200 µM) and the negative control for 24 and 48 h, the viability of the cancer cells was detected with MTT. Twenty microliters of MTT solution [5 mg/ml in phosphate-buffered saline (PBS)] was added to each well, and the mixtures were incubated for 4 h at 37°C. Then, the MTT solution was removed and 150 µl of DMSO was added to the wells. The absorbance was measured using a Multiskan Ascent plate reader at a 540 nm wavelength.

Cell cycle progression was assayed by measuring DNA content with propidium iodide (PI) staining. The cells were treated with osthole (0, 80, 120 or 160 µM) for 24 h, washed twice with PBS and fixed with 70% ethanol overnight at 4°C. Following fixation, the DNA fragments were stained in PBS containing PI and RNase for 1 h at 37°C. The DNA content was evaluated using an Accuri C6 flow cytometer (BD Biosciences). The data were analyzed using ModFit LT V4.1.

SMCC-7721 and MHCC-97H cells were seeded into 24-well plates and scraped with the end of a 200-µl pipette tip. The plates were washed with PBS to remove detached cells, and then the cells were incubated with complete growth medium containing 0, 20 and 40 µM osthole solution for 24 h. Cell migration was observed under a phase-contrast microscope at a magnification of ×100 field at 0 and 24 h post-induction of injury. Migrated cells into the denuded area in each of six random fields were measured and quantified with computer-assisted microscope.

Cell migration and invasion were quantified by the Transwell assay. HCC cells were treated with 0, 20 and 40 µM osthole for 24 h and harvested. Cells (2×10⁴) in serum-free DMEM were added to each upper chamber and DMEM with 10% FBS was added to the lower chamber as a chemoattractant. After a 24-h incubation at 37°C, the cells remaining on the upper surface of the membrane were removed, and the cells that had migrated through the membrane were stained with 0.1% crystal violet for 10 min. Six random fields of each Transwell membrane were assessed under a light microscope at a magnification of ×200.

DNA damage induced by osthole in the HCC cells was determined using a comet assay according to the manufacturer's protocol. Briefly, cells were treated with osthole (80 and 120 µM) for 48 h in complete medium, and then the cells were harvested and re-suspended in ice-cold PBS buffer. Approximately 1×10⁴ cells in a volume of 75 µl of 0.5% (w/v) low-melting-point agarose were pipetted into a frosted glass slide coated with a thin layer of 1.0% (w/v) agarose, covered with a coverslip, and allowed to set on ice for 10 min. Coverslips were removed, and the slides were immersed in ice-cold lysis buffer. After 2 h at 4°C, the slides were placed into a horizontal electrophoresis tank filled with electrophoresis buffer and subjected to electrophoresis for 30 min at 30 V at 4°C. Cells were stained with 2.5 µg/ml PI for 5 min and visualized under a microscope at a magnification of ×200 field. Tail lengths of a minimum of 10 cells were quantified as the distance from the center of the cell nucleus to the tip of the tail.

Protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) and protease inhibitor (Biocolors, Shanghai, China) was added in a proportion of 1:100. Equal amount of protein was loaded on a 10% SDS-PAGE gel. The lysates were resolved by electrophoresis (80 V for 30 min and 120 V for 1.5 h) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated with the primary antibody overnight at 4°C, followed by incubation with relevant secondary antibodies for 1 h at room temperature. The protein bands were visualized using the chemiluminescent ECL assay kit (Amersham Life Sciences, Inc.) and images were captured using Bio-Rad ChemiDoc XRS. Protein expression was quantitatively determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA). β-actin was used as an internal reference for protein expression in all cells.

Data were analyzed using SPSS 15.0 software and are presented as means ± SD of three independent experiments. Statistical differences between two groups were analyzed using a Student's t-test. A difference was considered to be statistically significant at P<0.05.

Human HCC cell lines SMCC-7721, BEL-7402, MHCC-97H and HCC-LM3, and a normal cell line LO2 were incubated with different concentrations (0, 20, 40, 80, 120, 160 and 200 µM) of osthole for 24 and 48 h (Fig. 1B), and then the cell viability was determined by MTT assay. As shown in Fig. 1B, except for the MHCC-97H cell line, incubation with osthole at 80 µM for 24 h significantly inhibited HCC cell proliferation, and following 48 h of treatment with osthole at 80 µM marked effects were noted on the inhibiton of cell growth. However, a concentration of 120 µM oshole had a marked significant effect on the inhibition of cell growth. Additionally, HCC cell lines following osthole treatment demonstrated a significant decrease in cell viability in a dose- and time-dependent manner (P<0.05), while the same concentrations did not significantly affect the viability of the normal LO2 cells.

To examine whether osthole contributes to the induction of cell cycle arrest, the effect on the cell cycle was measured by flow cytometry. HCC cell lines were treated with osthole at different concentrations (0, 80, 120 and 160 µM) for 24 h and the SMCC-7721 and MHCC-97H cell lines exhibited significantly increased accumulation of cells in the G2/M phase after osthole incubation for 24 h, whereas osthole did not significantly affect the S phase (Fig. 2A). Along with increasing concentrations, the percentages of cells in the G2/M phase were markedly increased from 16.8% (control group) to 64.32% in the SMCC-7721 cells, and from 4.52% (control group) to 41.48% in the MHCC-97H cells (Fig. 2B), indicating that osthole was able to induce cell cycle arrest in the G2/M phase in a dose-dependent manner. The proportion of apoptotic cells increased with increasing concentrations of osthole (Fig. 2B).

The degree of DNA damage was evaluated by the comet assay after the HCC SMCC-7721 and MHCC-97H cell lines were exposed to osthole at various concentrations (0, 80 and 120 µM) for 24 h, respectively. The representative images of DNA damage acquired from the comet assay are presented in Fig. 3, which shows that the comet tail was significantly extended compared to the control group and DNA damage was increased with the increasing concentration of osthole, suggesting that osthole triggered DNA damage in a dose-dependent manner.

The effect of osthole on HCC cell migration was evaluated by Transwell and wound healing assays in the SMCC-7721 and MHCC-97H cell lines following treatment with different concentrations (0, 20 and 40 µM) of osthole for 24 h, which were not apoptotic. As shown in Fig. 4A and B, the mobility ratio of HCC cells from the upper to the lower chamber gradually declined along with increased concentrations of osthole. Then, we performed a wound healing assay (Fig. 5). The wound closure rate of the osthole-incubated cells was lower than that of the control group (Fig. 5B). The results revealed that osthole significantly inhibited HCC cell migration in a dose-dependent manner, suggesting a critical role for osthole in the inhibition of HCC cell metastasis.

To further confirm the underlying molecular mechanisms of the induction of G2/M phase arrest by osthole, we examined the expression of cell cycle marker proteins Cdc2 and cyclin B1. As shown in Fig. 6A, the expression levels of Cdc2 and cyclin B1 were decreased following treatment with increasing concentrations of osthole. Then, we investigated the level of DNA damage marker protein ERCC1. As shown in Fig. 6B, the level of ERCC1 was significantly upregulated after treatment with osthole in a dose-dependent manner. Finally, we assessed the expression of migration-related proteins MMP-2 and MMP-9 after cells were exposed to osthole. As shown in Fig. 7A, along with increasing concentrations of osthole, the expression of MMP-2 and MMP-9 was gradually decreased. Osthole also increased the expression of epithelial markers, E-cadherin and β-catenin, and significantly decreased expression of mesenchymal markers, N-cadherin and vimentin (Fig. 7B). These results indicate the potential link between osthole and HCC cell G2/M phase arrest, DNA damage, EMT and migration-related protein expression, and also confirmed our previous results on the pharmacological functions of osthole in HCC cells.