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Introduction

Hepatocellular carcinoma (HCC) is a common tumor type with high metastatic ability and recurrence rate (1,2). HCC has a higher incidence in developing countries compared with that in developed countries and the highest incidence rates are found in China (3). Worlwide, HCC is the fifth most commonly diagnosed cancer, but the second most leading cause of cancer-related death (3). Some patients are deprived of the opportunity of curative therapy due to advanced stage disease, and systemic chemotherapy has shown little benefit on the survival rate of patients (4,5). Therefore, the underlying mechanisms of cancer cell migration, invasion and proliferation must be thoroughly elucidated to provide critical signaling effectors for effective molecularly targeted therapy.

Osthole (Fig. 1A), a herbal medicine, is a simple bioactive [7-methoxy-8-(3methy-2-butenyl)] coumarin derivative. It has been proven to exhibit various pharmacological functions. It is an anticonvulsant (6), prevents ischemia-reperfusion injury (7), has hepatocellular protective properties (8) and has anti-allergic function (9). Research on the effect of osthole on HCC is lacking, yet a few studies have reported that osthole possesses anticancer potential, and was found to inhibit the growth of human glioma (10), induce apoptosis in human lung cancer and human osteosarcoma (11,12), and inhibit metastasis in human glioblastoma and human lung cancer (13,14). In HCC, osthole was reported to play a crucial role in growth inhibition and induction of apoptosis (15), but it is unclear whether osthole has influence on HCC migration and invasion as well as critical signaling pathways.

Figure 1. Effect of osthole on the proliferation of hepatocellular carcinoma cells. (A) Chemical structure of osthole. (B) Cell viability was measured by MTT assay in hepatocellular carcinoma cell lines SMCC-7721, MHCC-97H, HCC-LM3 and BEL-7402 and normal cell line LO2 following treatment with osthole (0, 20, 40, 80, 120, 160 and 200 µM) for 24 and 48 h. *P<0.01 and **P<0.001, significant differences compared to the control group.

In the present study, we demonstrated for the first time the pharmacological function of osthole in inducing cell cycle arrest through downregulation of cycle B1 and Cdc2 levels and in triggering DNA damage through upregulation of ERCC1 and we further elucidated its effects on HCC cell migration associated with a decrease in MMP-9 and MMP-2 expression and modulating the epithelial-mesencheymal transition (EMT) signaling pathway.

Materials and methods

Materials, reagents and chemicals

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.

Cell lines and cell culture

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.

Drug preparation

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.

Cell viability assay

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 analysis by flow cytometry

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.

Wound healing assay

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.

Transwell migration assay

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.

Comet assay for the analysis of DNA damage

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.

Western blot analysis

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.

Statistical analysis

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.

Results

Effect of osthole on the proliferation of HCC cells

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.

Osthole induces cell G2/M phase arrest in HCC cell lines

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).

Figure 2. Analysis of cell cycle distribution by flow cytometry. (A) Cell cycle analysis of SMCC-7721 and MHCC-97H cells following treatment with 0, 80, 120 and 160 µM osthole for 24 h by flow cytometry. (B) The percentages of cells in the G1/G0, S, G2/M phases and apoptotic phase are shown in the histograms; *P<0.01 and **P<0.001 compared to the control group. Data are presented as means ± SD of three independent experiments. For each independent experiment, the assays were performed in duplicate.

DNA damage by osthole in HCC cell lines

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.

Figure 3. Osthole induces strand breaks in cellular DNA of hepatocellular carcinoma cells. (A) DNA damage was measured using comet assay in the SMCC-7721 and MHCC-97H cells following treatment with different concentrations of osthole (0, 80 and 120 µM). (B) The tail length of each comet was measured in each cell and tail length is expressed in µm as mean ± SD from at least 10 cells in each treatment group; *P<0.01 and **P<0.001 compared to the control group.

Osthole inhibits the migration of HCC cells

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.

Figure 4. Effect of osthole on the migration of hepatocellular carcinoma cells by Transwell assay. (A) Representative images of cell migration onto the underside of the Transwell membrane under a microscope at a magnification of ×200 field after cells were treated with osthole (0, 20 and 40 µM) for 24 h. (B) Average cell number of each field was counted. Migrated cells are expressed relative to that of the control group. The experiments were repeated three times; **P<0.001 compared to the control group.

Figure 5. Effects of osthole on the migration of hepatocellular carcinoma cells by wound healing assay. Cells were treated with osthole (0, 20 and 40 µM) for 24 h. (A) Representative images of cell migration in the wound healing assay under a microscope at a magnification of ×100 field before and after injury. (B) The migration of SMCC-7721 and MHCC-97H cells was quantified by measuring wound closure areas before and after injury. The experiments were repeated three times; *P<0.01 and **P<0.001 compared to the control group.

Effects of osthole on cell cycle-, DNA damage-, EMT- and migration-related protein expression

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.

Figure 6. Effects of osthole on cell cycle- and DNA damage-related protein expression. (A) Cdc2 and cyclin B1 protein expression was assessed using western blotting in SMCC-7721 cells following treatment with osthole (0, 80,120 and 160 µM) for 24 h. (B) ERCC1 protein expression was assessed using western blotting in SMCC-7721 cells following treatment with osthole (0, 80 and 120 µM) for 24 h; statistical analysis of quantification is shown; *P<0.01 and **P<0.001 compared to the control group.

Figure 7. Effects of osthole on EMT- and migration-related protein expression. SMCC-7721 cells were treated with osthole (0, 20 and 40 µM) for 24 h. (A) The expression levels of migration-related proteins MMP-2 and MMP-9 were assessed by western blotting. (B) Protein expression was analyzed by western blotting for epithelial markers E-cadherin and β-catenin and mesenchymal proteins N-cadherin and vimentin; statistical analysis of quantification is shown; *P<0.01 and **P<0.001 compared to the control group.

Discussion

Frequent patient diagnosis at advanced stage disease and limited effective treatment options contribute to the high mortality of HCC. In-depth knowledge of the multistep process leading to hepatocarcinogenesis, and development of novel chemotherapy targeting crucial signaling pathways may effectively improve the overall survival of patients with HCC across different stages (4). Osthole has been widely investigated due to its varied pharmacological functions (6,8,15–20). It has been reported that osthole inhibited ovarian cancer cells in vitro (19). We used concentrations from 0 to 200 µM to detect its effect on inhibition of cell proliferation. Our data confirmed that osthole induced cell cycle G2/M phase arrest and triggered DNA damage in HCC cell lines. Moreover, osthole significantly inhibited cell migration in HCC cells and we clarified the underlying mechanisms of these functions.

Dysregulated cell cycle and DNA damage response are currently two principal mechanisms of anticancer drugs (21,22). Cell cycle arrest as a critical target to inhibit cell proliferation has been widely investigated in recent years. Cdc2 and cyclin B1 are key regulators that modulate the G2/M checkpoint for cancer therapy (11,23–25). Furthermore, our results showed that osthole induced G2/M phase arrest in SMCC-7721 and MHCC-97H cells. Then, we demonstrated that treatment with osthole led to the downregulation of Cdc2 and cyclin B1 levels. Several studies have illustrated that osthole inhibited cancer cell proliferation and growth by cell cycle arrest (11,26). Suggesting that the underlying mechanism of osthole-induced HCC cell G2/M arrest may occur by attenuating the expression of Cdc2 and cyclin B1, this result is consistent with a previous study in lung cancer (11). In contrast, after incubation with osthole, DNA damage was significantly detected. Excision cross-complementation group 1 (ERCC1) has been found to be involved in DNA damage repair (27,28), and we found that ERCC1 was markedly upregulated in the experiment. Thus, we can conclude that osthole triggers DNA damage in HCC cell lines.

A great number of studies have focused on cancer cell metastasis due to its advanced threat to cancer patients. Increased expression of matrix metalloproteinases (MMPs) contributes to cancer cell migration, invasion and angiogenesis. MMP-2 and MMP-9 belong to the family of MMPs that have been widely studied (29–31). The EMT signaling pathway is also associated with cancer metastasis (32–34), and inhibition of EMT could be a potential therapeutic target to fight cancer metastasis. Previous research revealed that osthole inhibited cancer cell migration and invasion, in osteosarcoma, breast and lung cancer (26,35,36). In the present study, we demonstrated that osthole inhibited HCC cell migration in a dose-dependent manner, and we also further elucidated the underlying mechanisms by which osthole inhibits HCC cell migration by downregulating MMP-2 and MMP-9 expression and suppressing EMT by increasing the expression of epithelial markers, E-cadherin and β-catenin while decreasing mesenchymal markers, N-cadherin and vimentin.

Taken together, osthole possessed potential anticancer effects on HCC cells by inhibiting cell growth, inducing cell G2/M phase arrest, triggering DNA damage and suppressing migration in vitro. The underlying mechanisms of these functions were associated with the dysregulated expression of multiple proteins including Cdc2, cyclin B1, ERCC1, MMP-2, MMP-9, E-cadherin, β-catenin, N-cadherin and vimentin. We confirmed the potential pharmacological functions of osthole against HCC and elucidated the various mechanisms underlying these effects, suggesting that osthole may have high application value in HCC therapy.

Acknowledgements

The present study was supported by the National Natural Science Foundation of China Research grant (no. 30870719 to Z.W.).

References