Background
Cancer is one of the leading causes of death in the world and affects millions of patients worldwide despite significant advances have been made in the prevention, diagnosis, and treatment of cancers in recent years [
1]. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, ranging from 500,000 to 1 million new cases every year. Liver transplantation and surgical resection are the most efficient therapeutic strategies for early stage of HCC. However, current conventional therapies are still not effective to treat patients of advanced HCC [
2]. Hence, it is imperative to develop novel therapeutic drugs with high efficiency and low toxicity for HCC. In recent years, traditional Chinese medicine (TCM) has been increasingly applied in HCC prevention and treatment due to the advantages of safety and lower drug resistance [
3,
4]. Studies have shown that TCM possessed prophylactic and curative effects on human HCC with multiple actions and mechanisms [
4,
5].
There are two major types of cell death: necrosis and apoptosis. Necrotic cell death is commonly a result of pathological process and often stimulates local or systemic inflammation. In contrast, apoptosis, a programmed cell death, is usually a physiological event that does not provoke inflammation [
6]. Therefore, apoptosis was considered a potent therapeutic target for cancer treatment to reduce possible adverse side effects [
7]. Numerous studies have demonstrated that apoptosis can be triggered by diverse pro-apoptosis stimuli converging on mitochondria, causing mitochondrial depolarization, cytochrome
c release, caspase enzymes activation and eventually apoptotic cell death [
8]. A number of signaling pathways including caspase cascade and mitogen-activated protein kinase (MAPK) pathways play a vital role in the process of apoptosis. Additionally, tumor suppressor proteins, such as phosphatase and tensin homolog (PTEN) and p53, are also important in promoting apoptosis. PTEN is one of the most frequently mutated, deleted, or silenced tumor suppressor genes in cancers. It regulates p53 transcriptional activity and protein levels [
9]. Studies indicated that p53 induced apoptosis by either increasing transcriptional activity of pro-apoptotic genes or suppressing the activity of the anti-apoptotic genes [
10]. Many findings have demonstrated that PTEN and p53 played a critical role in DNA damage response and regulated cell cycle and apoptosis [
11,
12].
Recently, a number of natural steroidal saponins isolated from herbs display potential roles as apoptosis-promoting agents in a number of cancer cells [
13‐
15], which have received increased attentions due to their unique properties. Pro-oxidant-induced apoptosis results in an increase in intracellular reactive oxygen species (ROS) formation, which was closely coupled with a number of downstream events in apoptosis. It is well known that relatively high level of ROS induces redox imbalance, causes cell apoptosis or necrosis during physiological and pathological progress of many diseases. Tumor cells with higher level of ROS tend to be killed more easily than normal cells with lower level of ROS. Therefore, we could explore new anticancer drugs with high potential in promoting ROS production. Some steroidal saponins have been reported to exert pro-oxidant actions [
16,
17], which may be an important mechanism for their anticancer and apoptosis-inducing properties.
The rhizome of
Paris polyphylla var.
yunnanensis, or called
Rhizoma Paridis, a long been used herb in TCM, possesses multiple pharmacological activities [
18]. Notably, it has been used in combination with other herbs to treat cancer in clinical application of TCM [
19]. Several saponins with significantly biological activities have been isolated and identified from
Rhizoma Paridis and the steroidal saponins have been reported to have anticancer effects on many human cancer cell lines [
20,
21].
Polyphyllin VII (PP7), one of the steroidal saponins from
Paris polyphylla, has been found to show strong anticancer activity, as indicated by suppressing the growth of human cervical cancer HeLa cells [
22] and colorectal cancer cells [
23]. However, the underlying mechanisms of its anticancer activity are largely unknown. In this study, we aimed to investigate the anticancer effect of PP7 on human liver cancer HepG2 cells and the underlying molecular mechanisms. We found that PP7 induced apoptosis in HepG2 cells by inducing mitochondrial-mediated ROS generation and activating MAPK and PTEN/p53 pathways.
Methods
Reagents and antibodies
Polyphyllin II, VI and VII were kindly provided by Prof. Zhinan Mei (South-Central University for Nationalities). Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 were obtained from Gibco (Carlsbad, CA, USA). Fetal calf serum (FCS) was obtained from Tianhang Biotech (Hangzhou, Zhejiang, China). Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit, mitochondrial membrane potential assay kit 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetra-ethyl-benzimidazol-carbocyanine iodide (JC-1), Hoechst 33342 staining kit, SP600125, SB203580, and PD98059 were purchased from Beyotime (Nanjing, Jiangsu, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Molecular Probes (Eugene, OR, USA). 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich Co. LLC (Shanghai, China). Primary antibodies against Bcl-2, Bax, cytochrome c, p-BAD, BAD, p-stat3, stat3, p-PTEN, PTEN, extracellular signal-regulated kinase (ERK), p-ERK, p38 MAPK (p38), p-p38, c-Jun N-terminal kinase (JNK), p-JNK, p53, cleaved caspase-3, -8, -9 and poly (ADP-ribose) polymerase (PARP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and secondly antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) or Proteintech Group Inc (Wuhan, Hubei, China). The enhanced chemiluminescence (ECL) detection kit was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). All other chemicals of analytical grade were purchased from local sources.
Cell culture and treatments
Human hepatocellular carcinoma cell lines HepG2, Hep3B and Bel7402, human lung carcinoma cell line A549, human breast adenocarcinoma cell line MCF-7, human colorectal adenocarcinoma cell line Caco-2, and human ovarian carcinoma cell line SKOV-3 were obtained from American Type Culture Collection (Manassas, VA, USA). Human hepatocellular carcinoma cell line SMMC-7721 was obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were grown as monolayers in RPMI 1640 or DMEM. Cells were supplemented with 10 % heat-inactivated FCS, and 100 units/mL penicillin-streptomycin (Caco-2 cells plus 4 mM glutamine and 1 % nonessential amino acids), and incubated at 37 °C in a humidified atmosphere containing 5 % CO2. For all in vitro assays, PP7 was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution, sterilized using a sterile 0.22 μm membrane filter and stored at -80 °C, at a final DMSO concentration of less than 0.1 %. The working solutions were freshly diluted in the basal medium.
Cell viability assay
The cytotoxicity of PP7 was measured using MTT colorimetric assay. Briefly, cells (1 × 104 cells/well) were seeded in 96-well plates. After complete adhesion, different dilutions of PP7 were added and incubated further for 24, 48 and 72 h. The treated cells were then incubated in freshly DMEM medium containing MTT (0.2 mg/mL) at 37 °C. After 4 h, the supernatants were discarded carefully and added DMSO to dissolve the formazan crystals. The absorbance at 570 nm was measured with a microplate reader (BioTek, Winooski, VT, USA). The relative viability of treated cells was expressed as percentage of control untreated cells.
Determination of apoptosis and necrosis
Apoptosis was determined by visualizing surface exposure of phosphatidylserine using annexin V-FITC [
24]. HepG2 cells were either treated with different concentrations of PP7 or left untreated as control for 24 h. Cells were then resuspended in 500 μL binding buffer and incubated with FITC-conjugated annexin V (0.25 μg/mL), Hoechst 33342 (10 μg/mL) and propidium iodide (3 μg/mL)/RNase (30 μg/mL) for 15 min at 37 °C in the dark. After supravital staining, the cells were immediately visualized using the InCell 2000 confocal microscope (GE Biosciences, Piscataway, NJ, USA). Each well was taken at least six images (original magnification × 20). Images were captured by visualizing blue fluorescence (Hoechst 33342), green fluorescence (Annexin-V) and red fluorescence (propidium iodide). The number of positive cells per well was counted using the software modules supplied with the InCell 2000 and was expressed relative to the total number of nuclei present.
Measurement of intracellular reactive oxygen species (ROS)
The intracellular ROS generation was detected using an oxidant sensitive fluorescent probe DCFH-DA. To test the effect of time-dependent ROS generation, HepG2 cells seeded in 96-well plates (1 × 104/well) were treated with PP7 at the concentration of 1.32 μM for 3, 6, 12 and 24 h, respectively. To test the effect of concentration-dependent ROS generation, cells were treated with PP7 of 0.59, 0.88, 1.32 and 1.98 μM for 24 h. After treatment with DCFH-DA for 30 min at 37 °C in the dark, the cells were washed three times with phosphate buffered saline and fluorescence images of the cells were acquired using the InCell 2000 confocal microscope. For quantitative evaluation of intracellular ROS production efficacy, the fluorescence was measured by the software modules supplied with the InCell 2000.
Measurement of mitochondrial membrane potential (ΔΨm)
The mitochondrial membrane potential was measured using JC-1, which is a mitochondria-specific lipophilic cationic fluorescence dye and is capable of selectively entering the mitochondria. HepG2 cells were plated in a 96 well plate at a density of 1 × 104 cells/well and grown for 24 h. Different concentrations of PP7 were added and incubated further for 24 h. HepG2 cells were then stained with JC-1 working solution for 20 min at 37 °C in the dark, washed twice with 100 μL JC-1 binding buffer, and placed in growth media. Plates were then imaged live with the InCell 2000 confocal microscope. Quantitative image analysis was carried out using the software modules supplied with the InCell 2000.
Determination of the expression levels of apoptotic- and MAPK-Related Proteins
For Western blotting analysis, HepG2 cells of different treatment groups were collected. Proteins were extracted with radio immunoprecipitation assay lysis buffer and separated using 8-12 % sodium dodecylsulfate polyacrylamide gel electrophoresis, and electrotransferred to polyvinylidene fluoride membranes. The membranes were blocked with 5 % non-fat milk and incubated overnight at 4 °C with primary antibodies followed by incubation with the corresponding secondary antibodies. Signals were developed using an ECL detection kit according to the manufacturer’s instructions. Densitometric measurement of band intensity was performed with Image Lab Software (Bio-Rad, Hercules, CA, USA). The expression of GAPDH was used as an internal standard.
Inhibitor treatment
To clarify the roles of signaling pathways in PP7-induced apoptosis in HepG2 cells, the cells were pretreated with the following inhibitors individually before the treatment of PP7 (1.32 μM): 20 μM SP600125 (JNK specific inhibitor), 5 μM PD98059 (ERK specific inhibitor), 20 μM SB203580 (p38 specific inhibitor), or 10 μM NAC (a free radical scavenger). The cells were then subjected to the determination of apoptosis by annexin V-FITC staining and measurement of apoptosis-related protein expression by Western blotting as described above.
Statistical analysis
All data were given as the means ± standard deviation (SD) for at least three independent experiments, and analyzed for statistical significance using one-way analysis of variance, followed by Tukey post hoc analysis using GraphPad Prism 5 software package (GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered to be statistically significant.
Discussion
The steroidal saponins isolated from herbs possess structural diversity and significant anticancer activities [
13‐
15]. In recent years, studies demonstrated that the main anticancer components of
Rhizoma Paridis are steroidal saponins [
20,
21]. Polyphyllin VII (PP7), a steroidal saponin purified from
P. polyphylla has been identified as a potential anticancer agent in many pharmacological studies [
22,
23]. However, the cellular and molecular mechanisms for the anticancer activity of PP7 are poorly understood. In the present study, we found that PP7 could significantly inhibit the proliferation of a panel of human cancer cell lines in a dose-dependent manner (Additional file
1). Among the tested cancer cell lines, HepG2 was the most sensitive to PP7 treatment (Table
1), indicating that PP7 exhibited relatively selective anticancer activity. Considerable studies have shown that natural compounds isolated from TCM were effective in inhibiting cancer cell proliferation of HCC [
4]. Oridonin, from
Isodon rubescens, inhibited the proliferation of HepG2 cells with an IC
50 of 41.77 μM for 24 h [
26]. The IC
50 value of oroxylin A, from
Scutellaria baicalensis, was 23.94 μM against HepG2 cells after 24 h treatment [
27]. The IC
50 of arsenic trioxide after exposure HepG2 cells for 72 h, was approximately 2.25 μM [
28]. In the present study, PP7 significantly inhibited the growth of HepG2 cells, with an IC
50 value as low as 1.32 μM for 24 h treatment, indicating that PP7 exhibits strong cytotoxicity against HepG2 cells. Induction of apoptosis is regarded as a novel therapeutic strategy for cancers [
6]. Many anticancer agents have been reported to induce cell death of tumor cells by triggering apoptosis [
29]. In our results, PP7 strongly induced apoptotic cell death in HepG2 cells in a dose-dependent manner as evidenced by Annexin V/Hoechst 33342 staining (Fig.
1), suggesting that the anticancer effect of PP7 on HepG2 cells was mediated through the induction of apoptosis.
Various chemotherapeutic agents exert their anticancer effects through inducing the generation of ROS, and the intrinsic apoptotic pathway is especially susceptible to ROS. In the present study, the production of intracellular ROS increased remarkably in HepG2 cells treated with PP7 (Fig.
2c and
d), while inhibition of ROS production by the antioxidant NAC significantly decreased the apoptosis (Fig.
5b), suggesting that PP7-induced apoptosis in HepG2 cells was closely associated with the production of ROS, which may act as upstream signaling molecules to initiate mitochondria-mediated cell apoptosis. ROS is involved in the opening of the mitochondrial permeability transition pore, depolarization of the mitochondrial membrane, and then the release of mitochondrial pro-apoptotic factors in the process of mitochondria-mediated apoptosis [
30]. In our results, PP7 increased the ratio of JC-1 green-to-red fluorescence intensity (Fig.
2a), indicating a decreased ΔΨm and depolarization of the mitochondrial membrane, further confirmed that PP7 induced an ROS-involved mitochondrial pathway-mediated apoptosis in HepG2 cells. Similar results were reported that berberine, an isoquinoline alkaloid isolated from TCM, exhibited cytotoxicity on L929 murine fibroblast cells was related to accumulation of intracellular ROS, reduction of mitochondrial membrane potential, and cell apoptosis [
31].
Apoptosis is executed through mitochondrial-mediated intrinsic and cell death receptor-mediated extrinsic pathways, both of which are converged to the cascade activation of caspase proteases [
32]. In the intrinsic pathway, cytochrome
c releases from damaged mitochondria and causes apoptosome-dependent activation of caspase 9. This leads to the activation of the executioner caspases 3 [
33], and eventually inactivation of PARP [
30]. In the extrinsic pathway, activated initiator caspase-8 by death-inducing signaling complex also cleaves and activates caspase-3 and results in apoptotic cell death [
34]. Our results demonstrated that PP7 increased the levels of cleaved caspases-3, -8, -9 and PARP in a dose- and time-dependent manner (Fig.
3), indicating that both intrinsic and extrinsic pathways were involved in the process of PP7-induced apoptosis in HepG2 cells. Our findings are consistent with the previous findings that TCM induced cancer cell apoptosis through intrinsic and extrinsic pathways [
35,
36].
Increased Bax/Bcl-2 ratio leads to ΔΨ
m collapse, cytochrome
c release, caspase-3 activation, and eventually apoptosis [
37]. In addition, pro-apoptotic proteins BAD and Bax are closely associated with the control of mitochondrial membrane permeability and release of cytochrome
c [
38]. Our results showed that PP7 increased the ratio of Bax/Bcl-2, decreased the level of p-BAD (an anti-apoptotic form), and caused the release of cytochrome
c from mitochondria in a dose- and time-dependent manner (Fig.
3), further confirmed that mitochondrial pathway was involved in PP7-induced apoptosis in HepG2 cells.
Numerous reports have demonstrated that expression of apoptosis-related proteins is critically regulated by tumor suppressor proteins PTEN and p53 in response to DNA damage [
11,
12]. Oxidative stress promotes PTEN nuclear accumulation, which leads to p53 stabilization and p53-mediated apoptosis [
39]. Furthermore, ROS could cause DNA damage and then directly activate p53 and initiate apoptosis [
40]. In the current study, PP7 significantly increased the expression levels of PTEN and p53 in HepG2 cells (Fig.
4), suggesting their potential roles in PP7-induced apoptosis in HepG2 cells. The upregulation of PTEN and p53 expression could be promoted by increased intracellular ROS production in HepG2 cells by PP7, which is in line with previous reports [
41‐
43].
MAPKs are also the major oxidative stress-sensitive signal transducing pathways [
44] and serve as upstream signals for the initiation of apoptosis. JNK and p38 MAPK were activated in response to ROS generation and mitochondrial dysfunction, which are frequently associated with the induction of apoptosis [
45]. Li et al. [
46] reported that the total flavonoids from
Arachniodes exilis induced apoptosis in HepG2 cells through ROS-mediated MAPK activation and mitochondrial dysfunction. A novel oleanolic acid derivative induces human hepatoma cell apoptosis via an ROS/MAPK-dependent mitochondrial pathway [
47]. Our results showed that PP7 significantly increased the levels of phosphorylated JNK, ERK and p38 in a dose-dependent manner without affecting the expression of total proteins (Fig.
4), indicating that these MAPK pathways were activated in the process of PP7-induced apoptosis in HepG2 cells. Moreover, MAPK inhibitors decreased the apoptosis of HepG2 cells (Fig.
5) induced by PP7. These results demonstrated that activation of ERK, JNK, and p38 pathways were involved in PP7-induced apoptosis in HepG2 cells.
The transcription factor STAT3 promotes tumorigenesis mainly through preventing apoptosis by up-regulating the expression of apoptosis inhibitors in several primary human cancers [
48], and blockage of constitutive STAT3 signaling results in the growth inhibition and apoptosis in cancer cells [
49]. Studies reported that the constitutive activation of STAT3 was often negatively regulated by p53, PTEN, and MAPK pathways [
25,
50]. Our data showed that PP7 significantly decreased the phosphorylated STAT3 level in HepG2 cells (Fig.
4). We supposed that STAT3 might be inhibited by the upregulated p53, PTEN, and MAPKs, in which the details need to be further elucidated.
Competing interests
The authors declare that they have no competing interests.
Authors’ contribution
Conceived and designed the study: CZ and CH. Performed the experiments: CZ, XJ, JB, SC, KW and YZ. Analyzed the data: CZ, CH, JB and KW. Contributed reagents/materials/analysis tools: ZM, PL, JBW, HS and YTW. Wrote the manuscript: CZ and CH. All authors have read and approved the final manuscript.