Introduction
Liver cancer is a malignant tumour with high morbidity and fatality rates that is common worldwide (1). New cases of liver cancer account for 4% of all malignant tumours in the world each year, and the morbidity of liver cancer in China has been slowly increasing in recent years, seriously affecting the life and health of its citizens (2). With the morbidity of liver cancer in China now higher than that of gastric cancer, liver cancer currently has the highest fatality rate among malignant tumours of the digestive tract. The number of liver cancer patients in China accounts for 55% of all cases in the world, and the death toll has reached 45% of the world total (3,4). The occurrence of liver cancer is related to viral hepatitis, excessive drinking and non-alcoholic cirrhosis, all of which may increase the morbidity of liver cancer (5). Additionally, because the onset of liver cancer is hidden, most patients are already at the end-stage once a definite diagnosis is made (5,6), depriving them of the chance for radical treatment. Currently, the main clinical approaches to treating primary liver cancer include partial hepatectomy, systemic or local chemotherapy, radiotherapy, radiofrequency ablative surgery and liver transplantation (7–11). However, all of these approaches have shortcomings, including poor prognosis and many side-effects. Thus, there is an urgent need to search for new therapeutics with effective antitumour action for liver cancer patients.
Due to the extremely high costs for developing chemical synthetic drugs, it is quite difficult to discover an anticancer drug from chemical compounds. Additionally, chemical drugs can have many side-effects. Therefore, it has become an important part of research on anticancer drugs both at home and abroad to search for a single Chinese herb or effective ingredient from plants. As one of the key approaches to tumour treatment, traditional Chinese medicine plays an indispensable role. With decades of effort, traditional Chinese medicine has achieved breakthroughs in terms of enhancing the effect and reducing the toxicity of chemotherapy and radiotherapy, preventing tumour metastasis and relapse after surgery, alleviating the clinical symptoms of advanced tumours, improving the quality of life of patients with tumours and extending the patient’s long-term survival. Traditional Chinese medicine has become a ‘hot’ topic in cancer drug research due to its antitumour characteristics of multi-target, multi-step and multi-effect, its action in various steps in the occurrence and development of tumours, its lower toxicity and fewer toxic effects, its ability to improve immunity and its lower tendency toward drug resistance. The anticancer mechanism of Chinese medicine is also a focus in today’s medical research.
The occurrence of liver cancer is associated not only with abnormal cell proliferation and differentiation but also with abnormal apoptosis. The proliferation and apoptosis of tumour cells consist of a precise process regulated by multiple genes. Many studies both at home and abroad have demonstrated that flavonoid compounds can inhibit the proliferation of tumour cells, induce the apoptosis of tumour cells and regulate the expression of relevant genes to block the occurrence and development of tumour cells (12–15). Isoquercitrin, the effective monomer from Bidens bipinnata L. extract, was used in the present study. The molecular formula of isoquercitrin is C20H21O12, with a molecular weight of 464.38 (Fig. 1 illustrates its structural formula). Isoquercitrin was used to treat human liver cancer cells to study its role in regulating the proliferation, apoptosis and cell cycle of liver cancer cells and to explore a possible antitumour signalling pathway.
Materials and methods
Cell culture, antibodies and reagents
Human liver cancer cell lines HepG2 and Hep3B were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were inoculated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (both from Gibco, USA) in a 37°C incubator containing 5% CO2 and saturated humidity. Isoquercitrin (≥98% purity) was purchased from Sigma, USA. The Annexin V-FITC apoptosis analysis kit was purchased from BD, USA. Antibodies for phospho-JNK, phospho-ERK1/2, phospho-p38MAPK and PKC were purchased from Santa Cruz Biotechnology, USA. The RT-PCR kit was purchased from Takara Bio (Japan).
Cell viability analysis
Liver cancer cells in logarithmic growth phase were digested with 0.25% trypsin, rinsed with phosphate-buffered saline (PBS), and resuspended in DMEM culture medium containing 10% FBS as single cells to be inoculated into a 96-well plate (1×104 cells/well) for overnight culture in a 5% CO2 incubator at 37°C. The following day, the medium was replaced, and isoquercitrin was added to final concentrations of 0, 100, 200, 400 and 800 μM. A blank was established in one well with culture medium only. There were 6 double-wells for each group that was cultured for 24, 48 and 72 h. The medium was replaced 4 h prior to the analysis, and MTT was added (20 μl at 0.5 mg/ml) into each well. Cells were cultured for 4 h in the incubator at 37°C. DMSO (150 μl) was added, and the OD values of each well were measured with a microplate reader at the wavelength of 490 nm.
Annexin V-FITC/PI double stain flow cytometry for apoptosis analysis
Cells in the logarithmic growth phase were inoculated in a 6-well plate, and the cell density was adjusted to 1×106 cells/well. After adherence, cells were treated with different concentrations of isoquercitrin added to the culture medium. Cells were trypsinised 48 h after the isoquercitrin treatment, and then, Annexin V-FITC (5 μl) and propidium iodide (PI) (10 μl) were added to the cell suspension, which was mixed and incubated for 15 min at room temperature in the dark before the flow cytometric analysis. This procedure was repeated with each experimental group three times.
Caspase activity assay
HepG2 cells were treated with different concentrations of isoquercitrin (0, 100, 200, 400 and 800 μM) for 48 h, collected and tested for the viability of caspase-3, -8 and -9 using a caspase activity assay kit according to the manufacturer’s instructions (Beyotime, China). Fluorescence was measured using a spectrophotometer at an excitation wavelength of 400 nm and an emission wavelength of 505 nm.
Cell cycle analysis
Cells in the logarithmic growth phase were inoculated in a 6-well plate at a density of 1×106 cells/well. After adherence, cells were treated with different concentrations of isoquercitrin added to the culture medium. Cells were then trypsinised 48 h after the isoquercitrin treatment and resuspended in solution. Cold absolute ethyl alcohol (75%) was added, and cells were fixed for >18 h at 4°C. Cells were washed twice with PBS, treated with RNase A (50 mg/l) for 30 min at 37°C, and subsequently put in an ice-bath for 2 min. Cells were stained with PI dye (50 mg/l) for 30 min at 4°C, then analysed using FACS. CellQuest software was used to analyse the distribution of all cells.
Western blot assays
Cells from all treatment conditions were collected and washed twice with chilled PBS. Lysis buffer was added, and then cells were placed on ice for 20 min and subsequently centrifuged at 13,000 rpm for 20 min at 4°C. The supernatant was collected, and total cellular protein was extracted. Protein was quantified using the BCA method. Samples were subjected to SDS-PAGE gel electrophoresis at 80 V and 4°C. Samples were electrophoresed in the stacking gel for 1 h, and then electrophoresed at 150 V for ~1.5 h until the bromophenol blue dye front reached the bottom of the separation gel, and the power was turned off. Separated lysates were then transferred to a membrane for 2 h at 100 V. Membranes were stained with Ponceau S and blocked with 5% milk via shaking at room temperature for 1 h. Primary antibodies were diluted in Tris-buffered saline Tween-20 (TBST) and incubated overnight at 4°C. Antibody dilutions were as follows: phospho-ERK1/2, 1:500; phospho-JNK, 1:800; phospho-p38, 1:800 and PKC, 1:600. Secondary antibodies were incubated via shaking at 37°C for 1 h, washed three times with TBST for 10 min each, and developed using chemiluminescence. The target protein expression levels were normalised using a ratio to β-actin levels.
RT-PCR
Total RNA was extracted from all cells and was subjected to reverse transcription following determination of purity and integrity. RNA concentration was calculated and RT-PCR reactions were performed according to the manufacturer’s instructions (Takara Bio). β-actin and PKC primers were synthesised by Invitrogen using the following sequences: β-actin forward primer 5′-AAGGAAGGCTGGAAGAGTGC-3′ and reverse primer 5′-CTGGGACGACATGGAGAAAA-3′; PKC forward primer 5′-TGAATCCTCAGTGGAATGAGT-3′ and reverse primer 5′-GGTTGCTTTCTGTCTTCTGAA-3′. The PCR reaction volume was 50 μl using the following reaction conditions: 94°C for 2 min and a 94°C denaturation for 30 sec; a 60°C annealing for 30 sec; and a 72°C extension for 30 sec for a total of 32 cycles. PCR products were electrophoresed on a 1.0% agarose gel, then scanned and analysed with a gel imaging system.
Vaccination of nude mice
The animal experimentation program was approved by the Medical Ethics Committee of Guilin Medical University. Twenty male nude mice were purchased from the Animal Experiment Center of Guilin Medical University at the age of 6 to 8 weeks and a weight of ~20 g. The mice were randomly divided into two groups of 10 mice each: the model group and isoquercitrin group. Liver cancer cells were collected in the logarithmic log phase. After washing with PBS, the cells were suspended in serum-free medium. Subsequently, 200 μl of the cell suspension (containing 2×107 cells) was injected subcutaneously into the right groin area of the mice. After tumours formed, the mice were gavaged with isoquercitrin every day and observed for the growth of tumours at 7, 14, 21 and 28 days. Mice were sacrificed by cervical vertebral dislocation 4 weeks later. Subcutaneous transplanted tumour tissue was surgically removed under aseptic conditions for index analysis.
Statistical analysis
All the experimental data were analysed with SPSS 15.0 statistical software. The measurement data are expressed as the means ± standard deviation, and the counting data are expressed as percentages. The comparison among various groups was carried out using one-way analysis of variance, and the comparison between pairs within a group was conducted using the Q test. p<0.05 was considered to indicate a statistically significant difference.
Results
Isoquercitrin inhibits the proliferation of liver cancer cells
The human liver cancer cell lines HepG2 and Hep3B were treated with various concentrations of isoquercitrin (0, 100, 200, 400 and 800 μM) for 24, 48 and 72 h. Cellular activity was assayed with MTT. Isoquercitrin inhibited the proliferation of HepG2 and Hep3B cells in a time- and concentration-dependent manner (Fig. 2). When the concentration of isoquercitrin increased from 0 to 800 μM, the A490 values in the HepG2 and Hep3B cells decreased gradually; the most significant decrease was observed at 400 μM.
Isoquercitrin induces apoptosis of liver cancer cells
To confirm whether isoquercitrin induces the apoptosis of liver cancer cells, Annexin V-FITC/PI double stain flow cytometry was used to assay for the apoptosis of HepG2 cells following treatment with various concentrations of isoquercitrin for 48 h. As the concentration of isoquercitrin gradually increased, the percentage of dead HepG2 cells increased in a concentration-dependent manner when compared with the control group (Fig. 3).
Isoquercitrin activates caspases in liver cancer cells
To confirm whether isoquercitrin induces the apoptosis of liver cancer cells, the change in the activity of caspase-3, -8 and -9 was analysed after HepG2 cells were treated with various concentrations of isoquercitrin for 48 h. The activity of caspase-3, -8 and -9 increased significantly after HepG2 cells were treated with isoquercitrin (Fig. 4). Our findings showed that isoquercitrin induced the apoptosis of HepG2 cells in a caspase family-dependent manner.
Isoquercitrin inhibits the proliferation of liver cancer cells via the MAPK signalling pathway
To explore the molecular mechanism of isoquercitrin-induced inhibition of liver cancer cell proliferation, HepG2 cells were treated with various concentrations of isoquercitrin for 48 h, and then the expression and phosphorylation levels of the MAPK pathway proteins ERK, JNK and p38MAPK were assayed by western blotting. We found that as the concentration of isoquercitrin gradually increased, the phosphorylation levels of both ERK and p38MAPK decreased, and the phosphorylation level of JNK increased (Fig. 5). Our findings showed that isoquercitrin promoted the phosphorylation of JNK, which in turn promoted the apoptosis of liver cancer cells.
Isoquercitrin inhibits the proliferation of liver cancer cells via the PKC signalling pathway
To further explore the molecular mechanism of isoquercitrin-induced inhibition of liver cancer cell proliferation, HepG2 and Hep3B cells were treated with isoquercitrin for 48 h, and then changes in PKC mRNA and protein expression were measured using RT-PCR and western blot assays, respectively. We found that, following treatment with isoquercitrin, both PKC mRNA and protein expression levels dropped significantly (Fig. 6). Our findings indicate that isoquercitrin inhibits the proliferation of liver cancer cells by downregulating PKC.
Isoquercitrin blocks the liver cancer cell cycle in the G1 phase
To investigate whether isoquercitrin regulates changes in the liver cancer cell cycle, flow cytometry was used to analyse changes in the cell cycle of HepG2 cells after they were treated with various concentrations of isoquercitrin for 48 h. It was found that as the concentration of isoquercitrin gradually increased, the percentages of HepG2 cells entering the S and the G2/M phases gradually decreased, while most cells were blocked in the G1 phase (Fig. 7). Our findings showed that the anticancer role that isoquercitrin plays in the liver may be induced by the blockade of the cell cycle.
Isoquercitrin inhibits transplanted tumour growth in nude mice
We found that the tumour volume of the isoquercitrin-treated mouse group was consistently smaller than that of the control group at every time point examined. Additionally, the weight of the tumours obtained by surgery was also significantly less than that of the control group (Fig. 8). These findings strongly indicate that isoquercitrin obviously inhibits the progression of liver cancer.
Discussion
Bidens bipinnata L. is a whole dry herb of feverfew Bidens bipinnata L. that mainly grows in the warm and humid environment of south China and is widely distributed in most areas of Guangxi Province. It has historically been used as a basic drug in the local area of Guangxi to treat such diseases as malaria, diarrhoea, dysentery, hepatitis and acute nephritis, having the effects of diaphoresis, clearing heat and toxicity, and dissipating stasis. In recent years, Bidens bipinnata L. has often been used by local people to treat hypertension, hyperlipidaemia, diabetes, and anti-hepatic fibrosis and as an antitumour agent with positive effects. Many studies have proven that the flavonoid compounds of Bidens bipinnata L. have antitumour effects (15–20). Isoquercitrin is a flavonoid compound isolated from Bidens bipinnata L. Ma et al (21) and Zhong et al (22) demonstrated that the main components of the total flavone of Bidens bipinnata L. are isoquercitrin and hyperin via separation using macroporous adsorption resin and high performance liquid chromatography. Zhong et al (23) and Yuan et al (24) also studied the role that the Bidens bipinnata L. flavonoid plays in protecting mice against acute liver injury using a mouse model of acute liver injury induced by circulating tumour cells (CTCs). However, no report is available on its role in liver cancer cells. The formation of liver cancer is closely associated with abnormal proliferation and apoptosis of liver cells. To understand the effect of isoquercitrin on liver cancer cells, its role in liver cancer cell inhibition was preliminarily explored in in vivo and in vitro experiments. The possible signalling pathways involved in the inhibition of liver cancer cell growth by isoquercitrin were analysed by determining the expression levels of relevant proteins and genes to provide an experimental basis for its development and use.
There are many flavonoid drugs that can inhibit the proliferation of tumour cells. Various concentrations of isoquercitrin were administered to human liver cancer cells, and the MTT method was used to analyse its impacts on cell proliferation capacity. We found that when the concentration of isoquercitrin was higher than 100 μM, the inhibition of human liver cancer cell proliferation gradually increased as the concentration of isoquercitrin increased. The inhibition of human liver cancer cell proliferation was most obvious when the concentration of isoquercitrin was 400 μM. This means that isoquercitrin can inhibit the progression of human liver cancer in vitro in a concentration-dependent manner. In addition, we found in the in vivo experiment that the tumour formation rate of the nude mice decreased and that tumour growth was restrained following treatment with isoquercitrin. This means that isoquercitrin can inhibit the progression of human liver cancer in vivo as well.
Liver cancer is caused by the proliferation of tumour cells and the unbalanced regulation of apoptosis due to the abnormal activation of cell proliferation signals and the abnormal inhibition of cell apoptotic signals (25,26). In these experiments, the apoptosis of liver cancer cells was tested using the flow cytometry Annexin V/PI double stain method following treatment with isoquercitrin for 48 h. We found that isoquercitrin induced the apoptosis of liver cancer cells in a concentration-dependent manner. The apoptosis of cells is achieved mainly through two pathways, the death receptor pathway and the mitochondrial pathway (27–29). Caspases are specific cysteine aspartic acid proteases, and they are present in an inactive zymogen state in normal cells. Once a caspase is activated, it participates in initiating and executing cell apoptosis. Caspase-9 is at a comparatively upstream stage of the cell apoptotic signal transduction process. If caspase-9 is activated, it activates the downstream effector cysteine proteases caspase-3 and -8 to trigger a caspase cascade reaction, which in turn promotes subsequent cell apoptosis signals and the initiation of apoptosis. We found that the expression of caspase-3, -8 and -9 all increased following treatment with isoquercitrin. This means that isoquercitrin induces cell apoptosis by activating caspase family proteins inside human liver cancer cells.
Tumour growth is a disorder of cell cycle regulation due to changes in multiple genes. Two key points in the cell cycle regulatory mechanism are the G1 and the S phases. The blockade of tumour cells in the G1 or the S phase is an important mechanism to resist the growth of tumour cells (30,31). In the present study, changes in the cell cycle of human liver cells were analysed by flow cytometry after they were treated with isoquercitrin to determine the mechanism by which isoquercitrin inhibits the proliferation of human liver cancer cells. We found that the percentage of tumour cells in the G1 phase increased after these cells were treated with isoquercitrin for 48 h, meaning that cells were blocked in the G1 phase. Therefore, isoquercitrin had the effect of preventing human liver cancer cells from transitioning from the G1 to the S phase and the G2/M phase.
The MAPK signalling transduction pathway is a key information transfer pathway for signals to enter the cell nucleus from the cell surface and an intersection for signal transfer between cell proliferation and differentiation. It mainly consists of extracellular regulating protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38MAPK pathways. Three pathways, ERK, JNK and p38MAPK, were found to be closely associated with many malignant tumours, including breast, ovarian cancer and non-small cell lung cancer. ERK mainly takes part in the proliferation and differentiation of cells (32–35), while JNK and p38MAPK mainly take part in the stress reaction and induction of apoptosis. Additionally, JNK plays a role as a proto-oncogene in hepatocellular carcinoma (36). When JNK is activated through phosphorylation, it regulates the expression of downstream target genes and the activity of target proteins to induce apoptosis (37). When Nateri et al knocked down the c-Jun gene or changed the locus of JNK phosphorylation, intestine tumours in mice became smaller, the number of tumour cells decreased, and the lifespan of the mice was extended (38). The activation of the p38MAPK pathway plays a key role in inflammation, apoptosis, cell cycle regulation and cell differentiation. The abnormal activation of the p38MAPK pathway is closely correlated to the occurrence of tumours. Some studies indicate that p38MAPK is extensively and continuously activated in many human tumours. In addition, the p38MAPK pathway participates in all processes of tumour development and metastasis. One study proved that the use of a p38MAPK inhibitor inhibited the occurrence of some types of tumours (39). Therefore, further study concerning the key genes and proteins of the MAPK signalling pathway is warranted. We found that isoquercitrin strongly inhibited the phosphorylation of ERK and p38MAPK proteins while promoting the phosphorylation of JNK, which indicates that isoquercitrin may play a role in regulating the proliferation and apoptosis of human liver cancer cells via the MAPK signalling pathway.
Protein kinase C (PKC) plays an important role in the transmembrane signal transfer process. By catalysing Ser/Thr phosphorylation of various proteins, PKC regulates the metabolism, growth, proliferation and differentiation of various types of cells. Current studies indicate that PKC is closely related to the proliferation and attack of liver cancer cells (40). We found that isoquercitrin significantly lowered the expression level of phosphorylated PKC; thus, isoquercitrin may affect the progression of liver cancer via the PKC signalling pathway.
In summary, we found that isoquercitrin inhibited the progression of human liver cancer cells. Therefore, isoquercitrin may be a potential new antitumour drug for the treatment of liver cancer.