Background
==========

Liver cancer is a major cause of cancer-associated mortality and morbidity across the globe \[[@b1-medscimonit-25-1864]\]. The incidence of liver cancer and its mortality rates tend to increase with age \[[@b2-medscimonit-25-1864]\]. Moreover, liver cancer is reported to be 3 times more common in men than in women. The main treatments for liver cancer are surgery, chemotherapy, and radiotherapy \[[@b2-medscimonit-25-1864]\]. In china, 12.9% of all new cancer cases and 17.4% of all cancer deaths in 2012 were due to liver cancer. The liver cancer mortality rates have increased by 68.41% in males and by 48.39% in females in china since 2005 \[[@b3-medscimonit-25-1864]\]. Liver cancer is often difficult to treat as it is mostly diagnosed at advanced stages and the chemotherapeutic drugs often cause adverse effects \[[@b4-medscimonit-25-1864]\]. It is believed that molecules of natural origin with minimal toxicity may prove beneficial in the treatment of liver cancer. Consistently, new molecules are being screened for their anticancer activity against liver cancer \[[@b5-medscimonit-25-1864]\]. Flavonoids are an important group of plant secondary metabolites that have been shown to exhibit a wide range of pharmacological properties \[[@b6-medscimonit-25-1864]\], such as inhibiting the growth of cancer cells, and are considered to be prospective anticancer drugs \[[@b7-medscimonit-25-1864]\]. In the present study, we investigated the anticancer effects of morusinol against SK-HEP-1 liver cancer cells and normal hepatocytes. Chemically, morusinol is an important flavone isolated from the plant *Morus alba* \[[@b8-medscimonit-25-1864]\]. Morusinol has been reported to have great pharmacological potential, and a number of bioactivities have been attributed to this flavone, such as inhibition of arterial thrombosis \[[@b9-medscimonit-25-1864],[@b10-medscimonit-25-1864]\]. However, the anticancer potential of morusinol has not been thoroughly explored. In this study, we for the first time report the anticancer activity of morusinol against liver cancer cells. Herein, we show that morusinol exerts dose-dependent anticancer effects on SK-HEP-1 liver cancer cells, with no or minor effects on the growth of normal hepatocytes. The Ras/MEK/ERK signalling pathway is an important pathway that has been reported to be activated in several types of cancer cells \[[@b11-medscimonit-25-1864]\]. Several anticancerous molecules have been reported to inhibit the growth of cancer cells by targeting the Ras/MEK/ERK pathway \[[@b12-medscimonit-25-1864]\]. In the present investigation we observed that morusinol inhibits this pathway, indicating that morusinol may be an important lead molecule for the treatment of liver cancer.

Material and Methods
====================

Chemicals and other reagents
----------------------------

Morusinol (purity \>98%; determined by high-performance liquid chromatography), 3-(4, 5-dimethyl-2-thiazolyl)-2, and 5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Propidium iodide was purchased from Wuhan Boster Biological Technology (Wuhan, China). Dulbecco's modified Eagle's medium (DMEM) was purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Tianjin HaoYang Biological Manufacture Co. (Tianjin, China). Horseradish peroxidase-labelled anti-mouse and anti-rabbit secondary antibodies and all other antibodies were purchased from Cell Signalling Technology (MA, USA). Cell culture plasticware was purchased from BD Biosciences (San Jose, CA, USA).

Cell lines and culture conditions
---------------------------------

Liver cancer SK-HEP-1 cells and FL83B normal hepatocytes were procured from American Type Culture Collection. Both these cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, antibodies (100 units/mL penicillin and 100 μg/mL streptomycin), and 2 mM glutamine. The cells were cultured in an incubator (Thermo Scientific) at 37°C with 98% humidity and 5% CO~2~.

Proliferation assay
-------------------

For assessment of cell viability, the SK-HEP-1 and FL83B cells were cultured in a 96-well plates at a density of 5×10^3^ cells/well. The cells were incubated for 1 night and then the medium was removed and replaced with new medium with morusinol separately at different concentrations (0--200 μM) for 24 h. Then, cells were subjected to 0.5 mg/ml MTT solution for 4 h of incubation, after which the absorbance was measured at 570 nm.

Transmission electron microscopy (TEM)
--------------------------------------

For TEM, the untreated and Morusinol-treated (0, 10, 20, and 40 μM) SK-Hep-1 cells were subjected to fixation in glutaraldehyde (2.5%) in phosphate buffer for 35 min and post-fixed in 1% osmium tetraoxide in the same buffer for 35 min. This was followed by dehydration of cells in molecular grade ethanol and subsequent washing with propylene oxide, and then embedded in Epon. This was followed by sectioning on a Reichert-Jung ultramicrotome at 90-nm thickness. The sections were then stained with 5% uranyl acetate and 5% lead citrate and observed on a Hitachi H7100 transmission electron microscope at 75 kV.

Cell cycle analysis
-------------------

The dissemination of the SK-HEP-1 cells in various phases of the cell cycle was assessed by flow cytometry. Briefly, 0, 10, 20, and 40 μM morusinol-treated SK-HEP-1 cells were harvested after 24 h of culturing, then subjected to washing with PBS. The harvested SK-HEP-1 cells were subjected to fixation with ethanol (70%) for 1 h and then again washed with PBS. Thereafter, the cells were suspended in a solution of PI (50 μl/ml) and RNase1 (250 μg/ml). The cells were again subjected to incubation for 30 min at 25°C, and detected with a fluorescence-activated cell sorting cater-plus cytometer.

Cell migration and invasion assay
---------------------------------

The cell migration of the SK-HEP-1 liver cancer cells was determined by wound healing assay. After culturing for 24 h, the media and the cells were subjected to PBS washing. Then, a wound was scratched using a sterile pipette tip and a picture was captured. The cells were then grown for another 24 h and a photograph was taken again. Cell invasion of the morusinol-treated cells was performed by Transwell assay. In brief, the cells were cultured at 2×10^5^ cells/mL density, followed by the addition of 200 ml of these cultured cell suspensions to the upper chamber and whole medium was added into the bottom wells. The cells were cultured as such for 24 h. The SK-HEP-1 cells that migrated via the chambers were subjected to fixation with methyl alcohol, followed by crystal violet staining. Finally, an inverted microscope was used to count cells (200×, 10 different fields).

Western blotting
----------------

Exponentially growing HCT-116 cells were treated with different concentrations (0, 10, 20, and 40 μM) of morusinol for 48 h. Cells were centrifuged at 400 g for 5 min at 4°C, washed with PBS, and cell pellets were lysed in RIPA buffer (50 mM Tris--HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 5 mM EDTA, 30 mM Na2HPO4, 50 mM NaF, 0.5 mM NaVO4, 2 mM phenylmethylsulfonyl fluoride, and 10% protease cocktail inhibitor). Cells were incubated on ice for 30 min, vortexed, and supernatant containing proteins was collected by centrifuge at 13 000 g for 15 min. Protein concentration was determined by Bradford method and stored at -80°C. For Western blot analysis, equal amounts of proteins (50 μg) were loaded and resolved on SDS-polyacrylamide gel. Following gel electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in blocking buffer (10 mM Tris--HCl, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat milk for 1 h at room temperature and blotted with mouse anti-human primary antibodies (purchased from Santa Cruz Biotechnology) overnight at 4°C. Blots were washed in TBS, incubated with horseradish peroxidase-conjugated secondary antibody for 1 h, washed again 3 times with TBS, and chemiluminescence was captured on hyperfilm after incubating the blots in ECL plus solution.

Statistical analysis
--------------------

The experiments were carried out in triplicate and the values expressed are the mean of the 3 replicates ±SD. The *t* test was used for statistical analysis using GraphPad prism software 7. The values were considered significant at *p*\<0.05.

Results
=======

Morusinol inhibits the growth of liver cancer cells via autophagy induction
---------------------------------------------------------------------------

The antiproliferative activity of morusinol ([Figure 1](#f1-medscimonit-25-1864){ref-type="fig"}) was evaluated against the SK-HEP-1 liver cancer cell line and normal FL83B cells. Morusinol inhibited the proliferation of SK-HEP-1 liver cancer cell line, with an IC~50~ of 20 μM. However, morusinol exhibited mild antiproliferative effects on the normal FL83B cells, with an IC~50~of 80 μM ([Figure 2](#f2-medscimonit-25-1864){ref-type="fig"}). Flavonoids have been reported to induce autophagy in cancer cells \[[@b13-medscimonit-25-1864]\], so assessed whether morusinol could trigger autophagy against SK-HEP-1 liver cancer cells. Transmission electron microscopy revealed that morusinol triggered the formation of autophagic vesicles in the SK-HEP-1 cells, indicative of autophagy in the liver cancer cells ([Figure 3](#f3-medscimonit-25-1864){ref-type="fig"}). To further validate the autophagic potential of morusinol, we assessed the expression of autophagy-associated proteins in the untreated and morusinol-treated cells, finding that the expression of Beclin-1 and LC3-II proteins was significantly upregulated in the morusinol-treated SK-HEP-1 cells. Moreover, the expression of p62 was significantly decreased in the morusinol-treated SK-HEP-1 cancer cells. However, no visible alterations were observed in the expression of LC3-I and Vps34 ([Figure 4](#f4-medscimonit-25-1864){ref-type="fig"}).

Morusinol triggers G2/M cell cycle arrest in liver cancer cells
---------------------------------------------------------------

Since several flavones have been shown to trigger cell cycle arrest of cancer cells \[[@b15-medscimonit-25-1864]\], we investigated whether morusinol triggers G2/M cell cycle arrest in SK-HEP-1 liver cancer cells. The results showed that morusinol increased the proportion of cells in G2 phase of the cell cycle in a concentration-dependent manner ([Figure 5](#f5-medscimonit-25-1864){ref-type="fig"}).

Morusinol inhibits cell migration and invasion of liver cancer cells
--------------------------------------------------------------------

Wound healing and transwell assays were used to assess the effect of morusinol on the migration and invasion of SK-HEP-1 cancer cells. The results showed that at IC~50~, morusinol inhibited the migration and invasion of SK-HEP-1 liver cancer cells ([Figure 6](#f6-medscimonit-25-1864){ref-type="fig"}), and a similar trend was observed in case cell invasion ([Figure 7](#f7-medscimonit-25-1864){ref-type="fig"}).

Morusinol inhibits the Raf/MEK/ERK signalling pathway of the liver cancer cells
-------------------------------------------------------------------------------

The Raf/MEK/ERK signalling pathway is an important therapeutic target for treatment of cancer. Hence, the effect of morusinol on the Raf/MEK/ERK signalling pathway was also examined. The results showed that morusinol suppressed the expression of p-MEK and p-ERK dose-dependently, while no effect was found on MEK and ERK protein expression ([Figure 8](#f8-medscimonit-25-1864){ref-type="fig"}).

Discussion
==========

Liver cancer is a common cancer worldwide and causes many deaths \[[@b16-medscimonit-25-1864]\]. However, the chemotherapeutic drugs used for treatment of liver cancer are inefficient and associated with adverse effects \[[@b2-medscimonit-25-1864]\]. Therefore, identification and evaluation of novel and efficacious molecules for the treatment of liver cancer urgently needed.

Morusinol is an important flavone isolated from the plants *Morus alba* and has been reported to exhibit a number of bioactivities \[[@b8-medscimonit-25-1864]\]. However, its anticancer activity has not been explored so far. In this study, we for the first time report the anticancer activity morusinol against liver cancer cells. We found that morusinol suppressed the growth of liver cancer, and exhibited an IC~50~ of 20 μM against SK-HEP-1 liver cancer cells. Previous studies have indicated flavones inhibit the proliferation of cancer by triggering autophagy in cancer cells. For instance, quercetin, a plant-derived flavone, induces autophagy in gastric cancer cells \[[@b17-medscimonit-25-1864]\]. Similarly, silibinin, which is also a flavonoid, triggers autophagic cell death in breast cancer cells \[[@b18-medscimonit-25-1864]\]. Therefore, transmission electron microscopy was used to assess if morusinol induces autophagic cell death in SK-Hep-1 liver cancer cells. Interestingly, TEM showed that morusinol induces autophagy in liver cancer cells as it leads to formation of the autophagic vesicles in the liver cancer cells. We also assessed the expression of autophagy-related proteins LC3-I, LC3-II, Beclin-1, Vps34, and p62, and observed that morusinol treatment enhanced the expression of LC3-II and beclin-1, with concomitant downregulation of p62 expression. These changes in the expression of autophagy-related proteins, especially due to the upregulation of LC3-II, are characteristic of autophagy \[[@b19-medscimonit-25-1864]\]. In the present study we also observed that morusinol prompted the arrest of liver cancer cells at G2/M phase of the cell cycle and this could also be responsible for the anticancer activity of morusinol.

Liver cancer metastasis is very challenging, and molecules that can inhibit metastasis of liver cancer cells could be very promising \[[@b20-medscimonit-25-1864]\]. In the present study, we observed that morusinol inhibits the migration and invasion of SK-HEP-1 cancer cells. Dysregulation of the RAS/extracellular signal-regulated kinase (ERK) pathway is common in cancer cells; therefore, targeting this pathway may provide a novel alternative to conventional therapy \[[@b21-medscimonit-25-1864]\]. Hence, we examined the effect of morusinol on the RAF/MEK/ERK pathway and found that morusinol prevents the phosphorylation of MEK and ERK, suggesting that morusinol may prove a potent molecule for use in targeting this pathway.

Conclusions
===========

We conclude that morusinol could an important molecule for treatment of liver cancer. Morusinol inhibits the proliferation of liver cancer cells via induction of autophagy and cell cycle arrest, and warrants further *in vivo* studies.

**Source of support:** Departmental sources

**Conflict of interest**

None.

![Chemical structure of morusinol.](medscimonit-25-1864-g001){#f1-medscimonit-25-1864}

![Morusinol exerts antiproliferative effects on SK-HEP-1 liver cancer cells (as determined by MTT assay). The experiments were repeated 3 times and results are expressed as mean ±SD (\* *p*\<0.05).](medscimonit-25-1864-g002){#f2-medscimonit-25-1864}

![Induction of autophagy in SK-HEP-1 liver cancer cells as indicated by transmission electron microscopy at (**A**) 0 μM, (**B**) 10 μM, (**C**) 20 μM, and (**D**) 40 μM concentrations of morusinol. The autophagic vesicles increase with increase in the concentration of morusinol. The experiments were repeated 3 times (arrows indicate autophagosomes).](medscimonit-25-1864-g003){#f3-medscimonit-25-1864}

![Effect of different concentrations of morusinol on the protein expression of autophagy-related proteins as determined by Western blotting. The experiments were repeated 3 times. The expression of L3II and Beclin I was increased, while that of p62 was decreased at IC~50~. The experiments were repeated 3 times.](medscimonit-25-1864-g004){#f4-medscimonit-25-1864}

![Morusinol triggers cell cycle arrest in SK-HEP-1 cells in G2/M phase, as indicated by flow cytometry at (**A**) 0 μM, (**B**) 10 μM, (**C**) 20 μM, and (**D**) 40 μM concentrations of morusinol. The G2/M cells increase with increase in the concentration of the cells from 0 to 40 μM. The experiments were repeated 3 times.](medscimonit-25-1864-g005){#f5-medscimonit-25-1864}

![Inhibition of cell migration of SK-HEP-1 cells treated with morusinol at IC~50~. The experiments were repeated 3 times.](medscimonit-25-1864-g006){#f6-medscimonit-25-1864}

![Inhibition of cell invasion of SK-HEP-1 cells treated with morusinol at IC~50~. The experiments were repeated 3 times.](medscimonit-25-1864-g007){#f7-medscimonit-25-1864}

![Concentration-dependent effect of morusinol on the Ras/MEK/ERK signalling pathway as determined by Western blot analysis. The experiments were repeated 3 times.](medscimonit-25-1864-g008){#f8-medscimonit-25-1864}

[^1]: Study Design

[^2]: Data Collection

[^3]: Statistical Analysis

[^4]: Data Interpretation

[^5]: Manuscript Preparation

[^6]: Literature Search

[^7]: Funds Collection

[^8]: Zhen Zhu and Tianyu Xiao contributed to this work equally
