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Introduction

The dried fruits of S. chinensis Baill, a member of the Magnoliaceae family, are used extensively in the traditional medicinal systems of Korea, China and Japan (1). In particular, these fruits have been employed in the treatment and prevention of some chronic diseases, including hepatitis and cancer (2,3), which may result from damage to biomolecules by free radicals and reactive oxygen species. Additionally, the seeds and fruits of S. chinensis are enriched in lignans, and >40 lignans have been isolated from this plant (4).

αIC and α-iso-cubebene, which have very similar structures, were first purified from the dried fruits of S. chinensis using n-hexane (5). In an early study, nuclear magnetic resonance (NMR), mass spectrometry and circular dichroism results showed that the molecular formula of α-iso-cubebene was C15H24NH4, and harbors a cubebene sesquiterpene skeleton (5). α-iso-cubebene strongly stimulates intracellular calcium signaling and CXCL8 production in human neutrophils (5). This stimulation increases in a concentration-dependent manner, with maximal activity being evident at ~200 μg/ml. α-iso-cubebene also attenuates the activities of adhesion molecules, including vascular cell adhesion molecule-1, E-selectin, and intracellular adhesion molecule-1 in tumor necrosis factor-α-stimulated human vein endothelial cells (6). These features have revealed the potential of α-iso-cubebene as an effective novel anti-inflammatory agent for the prevention and treatment of vascular diseases. However, the principal biological function of αIC remains unclear, with the exception of the anti-inflammatory effect against bacterial infection or endotoxin recently reported by our group (7). The present study sought to clarify the function of αIC in the context of therapy for hepatocellular carcinoma.

Hepatocellular carcinoma is a primary malignancy of hepatocytes, and generally leads to death within 6–20 months. This disease is the fifth most common cancer in men and the eighth most common cancer in women worldwide (8). Cirrhosis of any etiology is a major risk factor for hepatocellular carcinoma (9). Approximately 80% of patients with newly diagnosed hepatocellular carcinomas have pre-existing cirrhosis in the liver, caused primarily by excessive alcohol use, as well as hepatitis B or C infection (10). The variety of therapeutic strategies for hepatocellular carcinoma include surgical resection and liver transplantation, although the treatment options available depend on the characteristics of the tumor (11,12). However, in many cases the drugs available for hepatocellular carcinoma therapy carry some disadvantages, such as toxicity, that hinder their clinical application.

Presently, in an effort to identify more efficacious therapeutic compounds, we investigated the anti-tumor functions of αIC newly isolated from S. chinensis using HepG2 hepatocellular carcinoma cells. Apoptosis in the αIC-treated cells was assessed using an established 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich)-based assay and western blotting. The present results indicate that high concentrations of αIC can induce significant cell death of hepatocellular carcinoma cells. Apoptosis induced by αIC is not correlated with Bax and p53 protein.

Materials and methods

Preparation of αIC compound

The αIC compound used in this study was prepared via the methods established in our laboratory (5). Lyophilized powders of αIC were dissolved in dimethylsulfoxide (DMSO, Sigma Aldrich, St. Louis, MO, USA) to a final concentration of 4 mM.

Cell culture and treatment

HepG2 human hepatoblastoma cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were grown in monolayers in minimum essential medium (MEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Gibco) during incubation at 37°C in a humidified incubator containing 5% CO2 in air. All other chemicals were purchased from Sigma-Aldrich.

In order to evaluate the dose-dependent effects, a stock solution (4 mM) of αIC was applied to the HepG2 cell line with different final concentrations (40, 80, 160 and 320 μM) of αIC for another 24 h. Additionally, the time-dependent effects were analyzed in the HepG2 cells treated for different times (3, 6, 12 and 24 h) with 320 μM of αIC.

Assay of cell proliferation

HepG2 cells were seeded at a density of 4×104 cells/200 μl in the wells of 96-well plates and were then grown for 24 h in a 37°C incubator. When the cells attained 70–80% confluence, they remained untreated (vehicle) or were exposed to 40, 80, 160 or 320 μM αIC dissolved in DMSO for another 24 h. Cell proliferation was determined using the tetrazolium compound MTT. After the supernatants in the αIC- or vehicle-treated wells were discarded, 200 μl of fresh MEM and 50 μl of MTT solution (2 mg/ml in phosphate buffered saline; PBS) were added to each well. The cells were then incubated in a 37°C incubator. The reduction of MTT to insoluble purple formazan dye crystals by viable cells was assessed in 220 μl recovered after 4 h. The formazan precipitate was dissolved in DMSO and the absorbance was directly read at 570 nm in the well using a SoftMax Pro5 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Additionally, the data were analyzed in terms of cell number versus absorbance, allowing for the quantification of changes in cell proliferation.

Flow cytometry analysis

The percentage of cells undergoing apoptosis and dead cells were detected via staining with fluorescein isothiocyanate (FITC) Annexin V (BD Bioscience, Franklin Lakes, NJ, USA). HepG2 cells were seeded at a density of 2×106 cells in 100 mm-diameter dishes and grown for 24–48 h in an incubator at 37°C. When the cells attained 70–80% confluence, vehicle, 40, 80, 160 and 320 μM of αIC were added to each culture dish and the cells were incubated for an additional 24 h. The cells were harvested, washed twice in ice-cold PBS, and resuspended in 1× binding buffer at a concentration of 1×106 cells/ml. Cells (~1×105) in 100 μl of solution were transferred to round-bottomed culture tubes. FITC Annexin V (5 μl) was added to stain the cells. After 15 min of incubation at room temperature, 400 μl of 1× binding buffer was added to each tube and each sample was analyzed with a FACS Calibur apparatus (BD Biosciences) within 1 h.

Western blot analyses

HepG2 cells harvested from 100-mm diameter culture dishes were solubilized with 1% Nonidet P-40 in 150 mM NaCl, 10 mM Tris HCl (pH 7.5), and 1 mM EDTA, and supplemented with a protein inhibitor mixture (Roche, Basel, Switzerland). They were then centrifuged for 10 min at 10,000 × g at 4°C. The homogenized proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 3 h and transferred to nitrocellulose membranes over 2 h at 40 V. The membranes were then incubated with primary antibodies [anti-Bcl-2 (SC-7382), anti-Bax (SC-493), anti-p53 (SC-6243) and anti-α-tubulin (Sigma-Aldrich), anti-caspase-3 (Cell Signaling Technology Inc.)] were used to detect Bcl-2, Bax, p53 and anti-α-tubulin. Each antigen-antibody complex was visualized with a biotinylated secondary antibody (goat anti-rabbit)-conjugated horseradish peroxidase streptavidin Histostain-Plus Kit (Zymed, South San Francisco, CA, USA) diluted to 1:1,500 in PBS.

Statistical analysis

Tests for significance between the vehicle- and αIC-treated groups were performed via one-way ANOVA tests of variance (SPSS version 10.10, SPSS Inc., Chicago, IL, USA). If a statistically significant difference was observed, the Student Newman-Keuls post hoc test was used. All the values are expressed as the means ± standard deviation (SD). A p<0.05 was considered significant.

Results

Effects of αIC on cell proliferation

Several lignans isolated from S. chinensis induce anti-proliferative and anti-apoptotic effects in hepatic carcinoma cells (13). Thus, we initially attempted to determine whether αIC exhibited an anti-proliferative effect in HepG2 hepatocellular carcinoma cells by determining the cell viability using an established MTT-based assay. The assay results confirmed that αIC significantly induced cell death at 320 μM (Fig. 1A). However, 40–160 μM αIC did not appreciably alter cell death. Additionally, HepG2 cells were observed via phase-contrast microscopy after 24 h of treatment with the same concentrations of αIC to determine whether cell death was concurrent with cell morphological changes. αIC concentrations of 40–160 μM did not affect HepG2 cell morphology, and these results were similar to those obtained with vehicle-treated cells. However, morphologically-altered cells in populations treated with 320 μM αIC were markedly increased relative to other groups (Fig. 1B). Additionally, to investigate the anti-proliferative effects depending on the time, HepG2 viability was detected at various times after treatment with 320 μM αIC. Significant changes in the death of HepG2 cells were initially detected 12 h after αIC treatment. At 24 h after αIC treatment, more than 80% of HepG2 cells were detected as dead cells (Fig 2). Therefore, these results showed that high concentrations of αIC and incubation for 24 h could effectively induce the death of hepatic carcinoma cells.

Figure 1 Dose-dependent effect of αIC on anti-proliferative activity of HepG2 cells. Cells were cultured with αIC in DMSO at various concentrations for 24 h. Vehicle-treatment was with DMSO only. (A) Cell proliferation activity was measured via an MTT assay. The values of the data represent the means ± SD of three experiments. *P<0.05 is the significance level as compared to the vehicle-treated group. (B) Microscopic images of HepG2 cells after 24 h of treatment with αIC at various concentrations. Vehicle-treated cells were treated with DMSO. Cellular morphology was viewed at a magnification ×20.

Figure 2 Time-dependent effect of αIC on anti-proliferative activity of HepG2 cells. Cells were cultured with 320 μM of αIC for different time and cell proliferation activity was measured via an MTT assay. The values of data are expressed as the means ± SD of three experiments. *P<0.05 is the significance level as compared to the vehicle-treated group.

Effects of αIC on apoptosis

FITC Annexin V is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis (14,15). In order to evaluate the effects of αIC on apoptosis, HepG2 cells treated with various concentrations of αIC were stained with FITC Annexin V, and fluorescence was detected by flow cytometry. As shown in Fig. 3, αIC induced a significant increase in the number of cells undergoing apoptosis from 15 to 98–99% in 24 h in all treated groups. In particular, cells treated with 320 μM αIC evidenced the highest numbers of apoptotic cells. Therefore, these results indicated that early events such as the loss of plasma membrane asymmetry in the apoptotic process may be induced by αIC in the majority of treated cells, at all concentrations.

Figure 3 Identification of apoptotic cells. HepG2 cells were incubated with αIC at various concentrations for 24 h, then stained with FITC Annexin V to detect apoptotic cells. The fluorescence emitted from these cells was analyzed via flow cytometry. M1 denotes live cells and M2 denotes cells that are either undergoing apoptosis or are dead. The values are expressed as the means ± SD of three experiments. *P<0.05 is the significance level as compared to the vehicle-treated group.

Furthermore, in order to demonstrate the activation of the apoptosis pathway, the cleavage of caspase-3 was detected in HepG2 cells treated with various concentrations of αIC. Pro-caspase-3 levels were reduced significantly from the 160 μM-treated group to the 320 μM-treated group, whereas they were maintained in both the 40- and 80 μM-treated groups. In particular, the reduction of pro-caspase-3 levels induced increases in the levels of active caspase-3, because the apoptosis signal induced the cleavage of pro-caspase-3 (32 kDa) into two small fragments (12 and 17 kDa) (16). The highest levels of active caspase-3 were detected in the 320 μM-treated group (Fig. 4). Therefore, these results also confirmed that αIC can activate caspase-3, a key regulator of the apoptosis process, at high concentration.

Figure 4 Effects of αIC on the activation of caspase-3. Expression levels of the pro-caspase-3 and active caspase-3 proteins were analyzed via western blot analysis. The membranes were incubated with antibodies for caspase-3 and β-actin proteins from HepG2 cells treated with various concentrations of αIC. Expression levels were quantified via an imaging densitometer and the sizes of the products are indicated. The data are expressed as the means ± SD of three experiments. *P<0.05 is the significance level as compared to the vehicle-treated group.

Effects of αIC on the apoptotic pathway

Bcl-2 belongs to the family of proteins that includes both pro- and anti-apoptotic members. Among these members, Bcl-2 proteins stimulate the anti-apoptotic process and the Bax protein significantly inhibits the anti-apoptotic actions of the Bcl-2 protein (17,18). In order to assess the effects of αIC treatment on proteins associated with the apoptosis signaling pathway, the expression levels of Bcl-2 and Bax proteins were determined in the vehicle and αIC-treated groups using western blot analysis. The expression level of Bcl-2 protein was markedly increased only in the 40-μM αIC-treated group as compared to the vehicle-treated group, and the protein level gradually decreased in a dose-dependent pattern. Cells treated with 320 μM αIC exhibited the lowest level of Bcl-2 expression. Bax protein expression was increased slightly in a dose-dependent pattern. The highest level of this protein was observed in cells treated with 320 μM αIC, although their levels were quite similar to those of the vehicle-treated group (Fig. 5). These results indicated that αIC could simultaneously induce the reduction of the protein levels associated with anti-apoptotic and the increase in protein levels associated with the pro-apoptotic process at high concentrations.

Figure 5 Effects of αIC on the apoptotic pathway. Expression levels of the Bcl-2 and Bax proteins were analyzed via western blot analysis. The membranes were incubated with antibodies against Bcl-2, Bax and β-actin proteins from HepG2 cells treated with various concentrations of αIC. Expression levels were quantified using an imaging densitometer and the sizes of the products are indicated. The data are expressed as the mean ± SD of three experiments. *P<0.05 is the significance level as compared to the vehicle-treated group.

Effects of αIC on the expression of p53 tumor suppressor protein

The p53 protein is considered an important regulator in the mechanism of apoptosis (19). In particular, the upregulation of Bax is stimulated by activated p53 (20). Therefore, in order to determine whether the p53 tumor suppressor protein would be affected by αIC in HepG2 cells, the expression levels of p53 protein were assessed in the vehicle- and αIC-treated groups. The highest levels of p53 expression were detected in vehicle-treated cells. After αIC treatment, the levels of p53 expression decreased gradually in a dose-dependent pattern. In particular, 320 μM αIC-treated HepG2 cells evidenced the lowest levels of p53 expression (Fig. 6). These results indicated that αIC could induce a reduction in p53 associated with tumor suppression. Furthermore, the expression of p53 did not contribute to the upregulation of Bax protein in HepG2 cells.

Figure 6 Effects of αIC on the expression level of p53 tumor suppressor protein. The expression levels of p53 proteins were analyzed via western blot analysis. The membranes were incubated with antibodies for p53 proteins and the β-actin proteins from the HepG2 cells with various concentrations of αIC. Expression levels were quantified using an Imaging densitometer and the sizes of the products are indicated. The data are expressed as the means ± SD of three experiments. *P<0.05 is the significance level compared to the vehicle-treated group.

Discussion

S. chinensis has a well-recognized history in traditional Chinese medicine. Also referred to as ‘Chinese Magnolia’, the plant is widely distributed throughout the Eastern regions of Siberia, China, Japan, and Korea (3). Over the past 20 years, many active lignans including Gomisin A, B, C, D, E, F, G, K3, N and J, Schisandrol B, Schisandrin, and Schisandrin C were isolated from this plant (3,21). The ligands have four thrapeutic properties: adaptogenic action, hepatic protection, hepatic stabilization and hepatic regeneration. Adaptogenic action involves specific action mechanisms such as energizing, nervous system stimulation, oxygenation, immune-modulation, anti-oxidation and skin protection (3,22–25). Additionally, these lignans reduce hyperlipemia and significantly inhibit the development of arteriosclerosis (26). With regard to hepatic protection, the lignans are correlated tightly with the increase in the hepatic levels of ascorbic acid, as well as the inhibition of NADPH oxidization and lipid peroxidation, which occur as the consequence of the induction effect of the hepatic cytochrome P-450 enzymatic system of the microsome. These lignans also protect against the cholestasis induced by toxic substances (27–30). The aforementioned ligands contribute to the hepatic regeneration process via the induction of mRNA transcription for hepatocyte growth factor, as well as the stimulation of hepatocyte proliferation and increased blood flow to the liver. Additionally, in an effort to improve regenerative capacity via reduced glutation and the synthesis of hepatic glucogen, the addition of these lignans induces increases in the levels of mitochondrial hepatic glutation and mitochondrial reductase glutation activity (31,32). However, relatively few functional studies have been conducted to investigate in detail whether the novel lignans isolated from S. chinensis influence cell death of the hepatocellular carcinoma cells in vitro. In this study, we investigated the novel function of αIC on the apoptosis of hepatocellular carcinoma cells.

Lignans are the most common constituents of S. chinensis. The major lignans in European seeds are deoxyschisandrin (0.07–1.09%), gomisin N (0.24–1.49%), schisandrin (0.75–1.86%), wuweizisu C (0.01–0.34%) and gomisin A (0.13–0.90%) (33). Furthermore, several C18 dibenzocyclooctadiene lignans isolated from S. chinensis evidence a variety of therapeutic activities involving anti-tumorigenesis (34,35), anti-hepatocarcinogenesis (36,37), anti-hepatotoxic (38), anti-human immunodeficiency virus (39), anti-oxidant (2,40), anti-inflammatory (35) and vascular relaxation (41) properties. Our laboratory previously evaluated the anti-cancer ability of a hexane extract of S. chinensis fruit indigenous to Korea, Japan and China (2). As part of an effort to identify new compounds, we recently purified two novel compounds-αIC and α-iso-cubebene-which have been employed in a variety of human disease therapies (5,6). These compounds have profound potential for development as novel drugs in the treatment of infection and inflammation. However, only a relatively minimal body of literature regarding the pharmacological mechanisms of action of αIC exists.

Apoptosis (programmed cell death) is characterized by certain morphological features involving the loss of plasma membrane asymmetry and attachment, the fragmentation and condensation of the nucleus, and the formation of cytoplasmic vesicles (42,43). The loss of plasma membrane asymmetry is one of the earliest detectable features, and can be distinguished by Annexin V conjugated FITC (44,45). We stained HepG2 cells with Annexin V to evaluate the novel functions of αIC on hepatocellular carcinoma. Although significant cell death was detected only at high concentrations using an MTT assay and morphological analysis, the results of flow cytometry suggested that the majority of the living cells were undergoing apoptosis. Therefore, our results indicate that αIC can markedly induce the apoptosis of HepG2 cells even after treatment at low concentrations.

Apoptosis plays a critical role in a variety of physiological processes during fetal development and in adult life. Defects in the apoptosis process can lead to the progression of many diseases involving progressive cell accumulation, as well as cancer (46). The apoptotic process involves many families of proteins. Among these, the Bcl-2 proteins are key to the induction of the anti-apoptotic process (46). This protein is overexpressed in many solid tumors, and contributes to chemotherapy resistance and radiation-induced apoptosis (46,47). Unlike many other known human oncogenes, Bcl-2 exerts its influence by enhancing cell survival rather than by stimulating cell division (47). In this study, we attempted to determine whether the expression level of Bcl-2 protein could affect αIC treatment in hepatocellular carcinoma cells. As shown in Fig. 4, the expression of the Bcl-2 protein was markedly reduced with increases in the concentration of αIC. These results bear substantial similarities to the alteration pattern of apoptosis induced by other compounds, including gomisin N (13).

In addition, the Bax protein, another member of the Bcl-2 family, inhibits the anti-apoptotic actions of Bcl-2. However, Bax may function as a classic tumor suppressor gene in vivo. Several human tumors involved the loss of functional mutations for this protein, and the knockout of the Bax gene induces tumorigenesis in mice (48–51). When we evaluated the effects of αIC treatment on the expression level of Bax protein via western blotting, their expression was increased slightly at a high αIC concentration. These results indicate that the effects of αIC may be so minimal as to have a negligible effect on Bax expression.

Genetic mutations of the p53 gene are the most common alteration noted in hepatocellular carcinoma (52). Generally, the p53 protein functions as a transcriptional activator, which induces the transcription of many genes associated with cell metabolism. Among this variety of genes, apoptosis-related genes are considered important factors of the regulation process of balance between death and survival (53). In particular, Bax protein is a transcriptional target that can be regulated in response to a variety of p53-dependent apoptosis triggers. Zakaria et al(20) previously determined that the upregulation of Bax is stimulated by activated p53 after eurycomanone treatment. Although p53 activation is an important step in the regulation of apoptosis, the effects of p53 on the sensitivity of HepG2 cells to αIC have not been hitherto rigorously assessed. In this study, we showed that the expression levels of p53 were reduced dramatically by treatment with high concentrations of αIC. However, the expression level of Bax upregulated by activated p53 was slightly increased in HepG2 cells, as αIC was increased from 40 to 320 μM. Therefore, these results indicate that the apoptosis of HepG2 cells induced by αIC is regulated via a p53-independent pathway.

All of the aforementioned results suggest that αIC may be an excellent novel candidate for the therapeutic treatment of hepatocellular carcinoma. Furthermore, these compounds regulated the apoptotic process via a p53-independent pathway, which includes a slight increase in Bax expression and reduction in p53 expression. However, intensive work remains necessary to define precisely the role of this compound in inducing cell death of hepatocellular carcinomas for future clinical applications.

Acknowledgements

This study was supported by Technology Development Program for Agriculture and Forestry (106048031CG00).

References