2298 ONCOLOGY REPORTS 40: 2298-2306, 2018 Lycorine inhibits cell proliferation and migration by inhibiting ROCK1/cofilin‑induced actin dynamics in HepG2 hepatoblastoma cells WUYI LIU*, QIAN ZHANG*, QIN TANG, CHANGPENG HU, JINGBIN HUANG, YALI LIU, YANYI LU, QING WANG, GUOBING LI and RONG ZHANG Department of Pharmacy, Xinqiao Hospital, Army Medical University, Chongqing 400037, P.R. China Received January 23, 2018; Accepted July 24, 2018 DOI: 10.3892/or.2018.6609 Abstract. Lycorine, a natural alkaloid extracted from the Introduction Amaryllidaceae plant family, has been reported to exhibit anti-cancer effects in various types of cancer cells. However, Hepatoblastoma, the most commonly diagnosed malignant the molecular mechanisms through which lycorine exhibits pediatric liver tumor, is frequently diagnosed in the first anti-hepatoblastoma activity are unclear. In the present 3 years of life. In recent years, the combination of surgery and study, the inhibitory effects of lycorine on the proliferation chemotherapy has improved the prognosis of patients with and migration of HepG2 hepatoblastoma cells were investi- hepatoblastoma (1). Furthermore, chemotherapeutic agents, gated. Lycorine inhibited the proliferation of HepG2 cells including cisplatin, have been applied in therapeutic strategies in a dose-dependent manner by inducing cell cycle arrest at for hepatoblastoma (2,3). However, conventional chemotherapy the G2/M phase, via downregulation of cyclin A, cyclin B1 agents frequently have limited clinical applications due to the and cyclin dependent kinase 1. Additionally, wound healing adverse side effects and drug resistance acquired following and Transwell assays revealed that treatment with lycorine long-term use. Consequently, it is vital to develop safe and resulted in a decrease in the migratory ability of HepG2 affordable alternative therapeutic agents for the treatment of cells. Also, treatment with lycorine decreased the expression hepatoblastoma. levels of matrix metalloproteinase (MMP)-9 and MMP-2. Recently, naturally occurring compounds have been valued Furthermore, lycorine induced the cleavage/activation of Rho as potential anticancer therapies due to their safety and effi- associated coiled-coil containing protein kinase 1 (ROCK1) cacy. Additionally, the majority of clinical chemotherapeutic and the downregulation of cofilin, accompanied by an increase drugs have an alkaloid structure, suggesting that alkaloids in polymerized filamentous actin and a loss of depolymerized are important antitumor agent candidates. Lycorine, a crude globular actin. Furthermore, pre-incubation of cells with alkaloid extracted from Amaryllidaceae genera, is reported Y‑27632, a specific ROCK1 inhibitor, markedly attenuated to have antimalarial, antiviral and anti‑inflammatory proper- lycorine-induced anti-proliferative and anti-migration effects. ties (4-7). Notably, lycorine is at least 15-fold more effective Taken together, the results demonstrated that lycorine inhibited against cancer cells compared with normal cells, suggesting the proliferation and migration of HepG2 cells by suppressing that lycorine is a selective anti-tumor compound (8). Multiple ROCK1/cofilin‑induced actin dynamics, which suggests that molecular mechanisms have been reported to be involved in lycorine has the potential to be developed into a novel drug for the anticancer effects of lycorine. Inducing apoptosis of cancer hepatoblastoma treatment. cells has a pivotal role among these mechanisms. For example, lycorine induces apoptosis of A549 cells via the adenosine monophosphate-activated protein kinase/serine/threo- nine-protein kinase mTOR/S6 kinase signaling pathway (9), and lycorine induces apoptosis of bladder cancer T24 cells by Correspondence to: Professor Rong Zhang or Dr Guobing Li, inhibiting protein kinase B phosphorylation and activating the Department of Pharmacy, Xinqiao Hospital, Army Medical University, intrinsic apoptotic cascade (10). However, increasing evidence 83 Xinqiao Road, Chongqing 400037, P.R. China has emphasized that lycorine also inhibits cancer cell prolif- E-mail: [email protected] E-mail: [email protected] eration and migration (11). Thus, it is important to examine the molecular mechanisms underlying the anti-proliferative and *Contributed equally anti-migration effects. In the present study, the HepG2 hepatoblastoma cell line Key words: lycorine, actin dynamics, Rho associated coiled-coil was used to investigate the inhibitory effects of lycorine on containing protein kinase 1, cofilin, hepatoblastoma cell proliferation and migration. Lycorine inhibited the prolif- eration of HepG2 cells and induced cell cycle arrest at the G2/M phase. Additionally, lycorine inhibited the migration LIU et al: ANTITUMOR ACTIVITY OF LYCORINE IN HEPATOBLASTOMA 2299 of HepG2 cells. Furthermore, mechanistic analyses revealed for 48 h, following which the culture medium was replaced that lycorine inhibited HepG2 cell proliferation and migration with fresh DMEM and cultured for 2 weeks; cells were through suppression of Rho associated coiled-coil containing subsequently fixed with 4% paraformaldehyde for 15 min and protein kinase 1 (ROCK1)/cofilin‑induced actin dynamics. stained with 0.1% crystal violet for 10 min at room tempera- Furthermore, pre‑incubation of cells with Y‑27632, a specific ture. The number of colonies was counted using Photoshop ROCK1 inhibitor, significantly attenuated the anti-prolif- CS6 software (Adobe Systems, Inc., San Jose, CA, USA). Each erative and anti-migration effects of lycorine. Additionally, group had three repeat wells and this experiment was repeated Y‑27632 attenuated lycorine‑induced cofilin downregulation three times. and G2/M phase cell cycle arrest. These results suggested that lycorine may be useful as a promising agent for treating Flow cytometry. The cell cycle distribution was determined by hepatoblastoma. flow cytometry. Cells were seeded in a 6‑well culture plate at a density of 1x106 cells/well. Lycorine (10 and 20 µM) were added Materials and methods the subsequent day. Following incubation for 48 h, cells were harvested and washed twice with PBS. Cells were fixed in cold Cells and antibodies. Lycorine (cat. no. A0415) was 75% ethanol overnight in 4˚C. Cells were washed twice with purchased from Chengdu Must Biotechnology Co., Ltd. cold PBS and suspended in PBS with 200 µg/ml RNase and (Chengdu, China) and dissolved in PBS as a stock solu- 50 µg/ml propidium iodide (cat. no. 556547; BD Biosciences, tion. Y-27632 (cat. no. S1049) was obtained from Selleck San Jose, CA, USA) in the dark for 30 min. The results were Chemicals (Houston, TX, USA). Antibodies against cyclin A measured by flow cytometry (FACScan; BD Biosciences) (cat. no. sc-751; 1:500), cyclin B1 (cat. no. sc-752; 1:500), and analyzed using ModFit LT 3.2 software (Verity Software cyclin dependent kinase 1 (cdc2; cat. no. sc-8395; 1:1,000) House, Inc., Topsham, ME, USA). and GAPDH (cat. no. sc-51905; 1:10,000) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti- Wound healing assay. A wound healing assay was used to assess bodies against cofilin (cat. no. ab42824; 1:2,000) and ROCK1 the migration ability of HepG2 cells. Briefly, cells were seeded (cat. no. ab45171; 1:1,000) were from Abcam (Cambridge, in a 6‑well culture plate. When cells reached 90% confluence, MA, USA); and antibodies against matrix metalloproteinase a wound was scratched with a 200 µl pipette tip. Cells were (MMP)-9 (cat. no. 13667; 1:1,000) and MMP-2 (cat. no. 40994; washed with PBS three times to remove the scratched cells, 1:1,000) were from Cell Signaling Technology, Inc. (Danvers, and lycorine (10 and 20 µM) was added and incubated for MA, USA). 24 or 48 h. The cells were imaged following replacement of the medium. The wound width was measured using ImageJ Cell culture. The human HepG2 hepatoblastoma cell line software (version 1.48; National Institutes of Health, Bethesda, was obtained from the American Type Culture Collection MD, USA): Wound healing rate (%) = 100 x (0 h width - 24/48 h (Manassas, VA, USA). Cells were cultured in Dulbecco's modi- width)/2/0 h width. fied Eagle's medium (DMEM; cat. no. PM150212; Procell Life Science & Technology Co., Ltd., Wuhan, China) supplemented Transwell assay. HepG2 cells were adjusted to a density of with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher 2x104 cells/well, resuspended and seeded in the upper chamber Scientific, Inc., Waltham, MA, USA) and cultured in a 37˚C of Transwell chambers with 200 µl serum-free medium, and incubator with a humidified atmosphere of 5% CO2. Once cells 600 µl complete medium containing 30% FBS was added in were adhering to the flask, the medium was changed every the lower chamber. Lycorine (10 and 20 µM) was added to the 2 days and the cells were digested using 0.25% trypsin (Gibco; two chambers. Following incubation for 48 h, non-migrated Thermo Fisher Scientific, Inc.). cells on the top surface of the upper chamber were gently scraped away with a cotton swab. The lower membrane MTT assay. Cells were seeded in a 96-well culture plate at a containing migrated cells was fixed in 4% paraformaldehyde density of 1x104 cells/well overnight, and treated with various for 10 min and stained with 0.1% crystal violet for 15 min at concentrations of lycorine (0.2, 0.5, 1, 2, 10, 20 and 100 µM) room temperature. A microscope (x4 magnification; Olympus the following day. Following incubation in a 5% CO2 incubator IX51; Olympus Corporation, Tokyo, Japan) was used to image at 37˚C for 24 h or 48 h, the medium was removed and 20 µl the migrated cells. The total cell numbers were calculated MTT solution (5 mg/ml) was added to each well. Following from three different fields and three independent experiments. incubation at 37˚C for an additional 4 h, 150 µl dimethyl sulf- oxide (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) Western blot analysis. Cells were harvested and lysed in was used to dissolve the dark blue crystals. The optical density lysis buffer containing 1 mM phenylmethylsulfonyl fluoride value was determined at 570 nm and measured on a microplate (Beyotime Institute of Biotechnology, Haimen, China).