Lycorine (cat. no. A0415) was purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China) and dissolved in PBS as a stock solution. Y-27632 (cat. no. S1049) was obtained from Selleck Chemicals (Houston, TX, USA). Antibodies against cyclin A (cat. no. sc-751; 1:500), cyclin B1 (cat. no. sc-752; 1:500), cyclin dependent kinase 1 (cdc2; cat. no. sc-8395; 1:1,000) and GAPDH (cat. no. sc-51905; 1:10,000) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); antibodies against cofilin (cat. no. ab42824; 1:2,000) and ROCK1 (cat. no. ab45171; 1:1,000) were from Abcam (Cambridge, MA, USA); and antibodies against matrix metalloproteinase (MMP)-9 (cat. no. 13667; 1:1,000) and MMP-2 (cat. no. 40994; 1:1,000) were from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The human HepG2 hepatoblastoma cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; cat. no. PM150212; Procell Life Science & Technology Co., Ltd., Wuhan, China) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and cultured in a 37°C incubator with a humidified atmosphere of 5% CO₂. Once cells were adhering to the flask, the medium was changed every 2 days and the cells were digested using 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.).

Cells were seeded in a 96-well culture plate at a density of 1×10⁴ cells/well overnight, and treated with various concentrations of lycorine (0.2, 0.5, 1, 2, 10, 20 and 100 µM) the following day. Following incubation in a 5% CO₂ incubator at 37°C for 24 h or 48 h, the medium was removed and 20 µl MTT solution (5 mg/ml) was added to each well. Following incubation at 37°C for an additional 4 h, 150 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to dissolve the dark blue crystals. The optical density value was determined at 570 nm and measured on a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Inc.). All these results are expressed as a percentage of the control, which was set at 100%. Each experiment was repeated three times individually.

Cells (200 cells/well) were seeded in a 6-well plate. Cells were allowed to attach overnight and exposed to different concentrations of lycorine (10 and 20 µM) for 48 h, following which the culture medium was replaced with fresh DMEM and cultured for 2 weeks; cells were subsequently fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 10 min at room temperature. The number of colonies was counted using Photoshop CS6 software (Adobe Systems, Inc., San Jose, CA, USA). Each group had three repeat wells and this experiment was repeated three times.

The cell cycle distribution was determined by flow cytometry. Cells were seeded in a 6-well culture plate at a density of 1×10⁶ cells/well. Lycorine (10 and 20 µM) were added the subsequent day. Following incubation for 48 h, cells were harvested and washed twice with PBS. Cells were fixed in cold 75% ethanol overnight in 4°C. Cells were washed twice with cold PBS and suspended in PBS with 200 µg/ml RNase and 50 µg/ml propidium iodide (cat. no. 556547; BD Biosciences, San Jose, CA, USA) in the dark for 30 min. The results were measured by flow cytometry (FACScan; BD Biosciences) and analyzed using ModFit LT 3.2 software (Verity Software House, Inc., Topsham, ME, USA).

A wound healing assay was used to assess the migration ability of HepG2 cells. Briefly, cells were seeded in a 6-well culture plate. When cells reached 90% confluence, a wound was scratched with a 200 µl pipette tip. Cells were washed with PBS three times to remove the scratched cells, and lycorine (10 and 20 µM) was added and incubated for 24 or 48 h. The cells were imaged following replacement of the medium. The wound width was measured using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA): Wound healing rate (%) = 100 × (0 h width - 24/48 h width)/2/0 h width.

HepG2 cells were adjusted to a density of 2×10⁴ cells/well, resuspended and seeded in the upper chamber of Transwell chambers with 200 µl serum-free medium, and 600 µl complete medium containing 30% FBS was added in the lower chamber. Lycorine (10 and 20 µM) was added to the two chambers. Following incubation for 48 h, non-migrated cells on the top surface of the upper chamber were gently scraped away with a cotton swab. The lower membrane containing migrated cells was fixed in 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 15 min at room temperature. A microscope (×4 magnification; Olympus IX51; Olympus Corporation, Tokyo, Japan) was used to image the migrated cells. The total cell numbers were calculated from three different fields and three independent experiments.

Cells were harvested and lysed in lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China). Bicinchoninic acid protein quantification kits (Beyotime Institute of Biotechnology) were used to measure protein concentrations. A total of 15 µg protein was separated via 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 h at room temperature, specific primary antibodies were added to the membranes and incubated at 4°C overnight on a shaker. Membranes were washed in TBS with Tween-20 three times and incubated with anti-rabbit or anti-mouse horseradish peroxidase secondary antibodies (cat. nos. 074-1516 and 074-1802, respectively; 1:100,000; Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD, USA) for a further 2 h at room temperature. Enhanced chemiluminescence reagent (EMD Millipore) was used to visualize the bands. Densitometric analysis was performed using Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was used as an internal control.

Following treatment with lycorine (10 and 20 µM) for 48 h, cells were washed twice with PBS and fixed with ice-cold 75% ethanol for 15 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 for 5 min, fluorescent staining of filamentous and globular actin was performed by staining with fluorescent deoxyribonuclease I conjugates and fluorescent phallotoxins (Molecular Probes; Thermo Fisher Scientific, Inc.) for 30 min in the dark, and slides were washed and stained with DAPI for 5 min at room temperature (cat. no. C1002; Beyotime Institute of Biotechnology). Images were captured using a Leica scanning confocal microscope (×40 magnification; TCS SP2 AOB; Leica Microsystems GmbH, Wetzlar, Germany).

All data were analyzed using GraphPad Prism 6.0 software (GraphPad Software, Inc., San Diego, CA, USA). Data presented are expressed as the mean ± standard deviation at least three independent experiments. Differences between groups were analyzed by one-way analysis of variance (ANOVA) with Dunnett's or Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

The chemical structure and molecular weight of lycorine is presented in Fig. 1A. The cytotoxicity of lycorine in HepG2 cells was determined by MTT assay. Cells were treated with various concentrations of lycorine (0.2, 0.5, 1, 2, 10, 20 and 100 µM) for 24 or 48 h, which resulted in significant decreases in cell viability in a dose-dependent manner (P<0.01, P<0.001; Fig. 1B). Exposure of cells to 2 µM lycorine resulted in a modest decrease in cell viability, and these events became significant following exposure of cells to ≥10 µM lycorine (Fig. 1B). To further confirm the anti-proliferative effect of lycorine, a clone formation assay was performed. In accordance with the results of the MTT assay, lycorine significantly inhibited the clone formation of HepG2 cells compared with the control cells (P<0.01, 10 µM; P<0.001, 20 µM; Fig. 1C). Taken together, these results demonstrated that lycorine inhibited the proliferation of HepG2 cells.

Cell cycle arrest is an important mechanism involved in the inhibition of cell growth (12). To examine the effect of lycorine on the cell cycle of HepG2 cells, cell cycle dynamics were assessed by flow cytometry. Following exposure of cells to lycorine (10 and 20 µM) for 48 h, the proportion of cells in the G2/M phase increased from 10.81% (without lycorine) to 14.64% (in the presence of 10 µM lycorine) and 20.15% (in the presence of 20 µM lycorine; P<0.01 vs. control; Fig. 2A). These results indicated that lycorine induced HepG2 cell cycle arrest at the G2/M phase. To further examine the molecular mechanisms under lycorine-induced G2/M phase arrest, western blot analysis was performed to assess the expression of cyclin A, cyclin B1 and cdc2. The results revealed that exposure of cells to lycorine resulted in a marked decrease in the expression of cyclin A, cyclin B1 and cdc2 in HepG2 cells (P<0.001; Fig. 2B). These data suggested that lycorine has an inhibitory effect on HepG2 cell cycle progression, which may cause the inhibition of HepG2 cell proliferation.

The wound healing assay indicated that lycorine induced a marked decrease in cell migration in HepG2 cells in a dose and time-dependent manner (P<0.01 and P<0.001; Fig. 3A). Furthermore, the Transwell assay revealed that lycorine significantly inhibited the migratory ability of HepG2 cells (P<0.001; Fig. 3B). Evidence has confirmed that MMPs, particularly MMP-9 and MMP-2, are important regulators in the process of cancer migration (13,14). To investigate whether lycorine alters the expression of MMP-9 and MMP-2, western blot analysis was performed. The results demonstrated that compared with the control group, lycorine decreased the expression levels of MMP-9 and MMP-2 in a concentration-dependent manner (P<0.05, P<0.01 and P<0.001; Fig. 3C). These data indicated that lycorine inhibits HepG2 cell migration.

Actin, an essential component of the cytoskeleton, has a critical role in a wide range of cellular processes, including cell migration and cell division (15). In the current study, exposure of cells to lycorine (10 and 20 µM) resulted in an increase in polymerized filamentous actin (F-actin) and a decrease in depolymerized globular actin (G-actin; Fig. 4A). It has been reported that cofilin regulates actin dynamics by severing actin filaments (16). Therefore, western blot analysis was performed to determine whether cofilin is involved in the lycorine-induced altering of actin cytoskeletal dynamics. As presented in Fig. 4B, HepG2 cells were treated with lycorine (1, 2.5, 5, 10 and 20 µM) for 48 h, which resulted in a decrease in the expression of cofilin (P<0.01 and P<0.001). Collectively, these results suggested that lycorine suppresses the expression of cofilin and alters actin cytoskeletal dynamics.

ROCK1 has been confirmed to have an important role in regulating cell polarity and migration (17,18). In the present study, it was investigated whether ROCK1 activation is involved in lycorine-induced anticancer effects. Treatment of HepG2 cells with lycorine (1, 2.5, 5, 10 and 20 µM) for 48 h induced cleavage/activation of ROCK1 in a dose-dependent manner (Fig. 5A). To further confirm the role of ROCK1 in lycorine-induced cell proliferation and migration inhibition, Y-27632, a ROCK1 specific inhibitor, was used. Western blot analysis indicated that pre-incubation of cells with Y-27632 inhibited lycorine-induced ROCK1 cleavage/activation (Fig. 5B). Pre-incubation of cells with Y-27632 attenuated the lycorine-induced cofilin decrease (Fig. 5C). Furthermore, pre-incubation with Y-27632 also attenuated the decreases in cyclin A, cyclin B1, cdc2, MMP-9 and MMP-2, which indicated that Y-27632 attenuated lycorine-induced G2/M cell cycle arrest and migration ability (Fig. 5D and E). A clone formation assay demonstrated that combined treatment with Y-27632 and lycorine significantly increased the colony number compared with treatment with lycorine alone (P<0.01; Fig. 5F). The wound healing and Transwell assays revealed that co-administration of Y-27632 and lycorine markedly attenuated the lycorine-induced inhibitory effect on cell migration (P<0.01, P<0.001; Fig. 5G and H). Collectively, these results demonstrated that ROCK1 activation has a critical role in the lycorine-induced effects on actin cytoskeletal dynamics, and anti-proliferative and anti-migration activity in HepG2 cells.