http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(5): 544–550 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.850517
ORIGINAL ARTICLE
Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells Xu Chao1,2, Xiaojun Zhou2, Gang Zheng2, Changhu Dong2, Wei Zhang3, Xiaomei Song4, and Tianbo Jin5 The College of Preclinical Sciences, Shaanxi University of Chinese Medicine, Xianyang, P.R. China, 2The Second Affiliated Hospital of Shaanxi University of Chinese Medicine, Xianyang, P.R. China, 3Jilin Medical College, Jilin, P.R. China, 4The College of Pharmaceutical Sciences, Shaanxi University of Chinese Medicine, Xianyang, P.R. China, and 5The College of Chemistry and Materials Sciences, Northwest University, Xi’an, P.R. China
Abstract
Keywords
Context: Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong’s Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture. Objectives: This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells. Materials and methods: Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0 mmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot. Results: Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC50) values were 0.186, 0.158 and 0.123 mmol/ml at 24, 48 and 72 h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with downregulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of proapoptotic Bax and p53 proteins. Conclusion: Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.
Apoptosis, cell cycle, G2/M, HepG2 cells, osthole
Introduction Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] (Figure 1), a coumarin derivative (You et al., 2009) isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong’s Classic of Materia Medica in the Han Dynasty, exhibits many pharmacological and biological activities (Okamoto et al., 2007; Tang et al., 2008), including antidiabetic activity (Liang et al., 2009), anti-inflammatory activity (Zimecki et al., 2009) and antinociceptive effect (Riviere et al., 2006). Additionally, accumulating evidence indicates that osthole possesses antitumor effects by inhibiting tumor cell growth and induces apoptosis (Okamoto et al., 2005; Xu et al., 2011). Apoptosis, or programmed cell death, is implicated in cellular homeostasis and many physiological processes.
Correspondence: Dr Tianbo Jin, The College of Chemistry and Materials Sciences, Northwest University, Xi’an 710069, P.R. China. Tel: +86-2988303446. E-mail: [email protected]
History Received 12 November 2012 Revised 17 September 2013 Accepted 24 September 2013 Published online 15 November 2013
Most cancer cells block apoptosis, which allows them to survive despite undergoing genetic and morphologic transformations. It is often linked with carcinogenesis, which results in the abrogation of apoptotic processes (Blagosklonny, 2003; Rodriguez-Nieto & Zhivotovsky, 2006). Apoptotic cells are characterized by several unique features, including cell shrinkage, chromatin condensation, DNA fragmentation, cell surface expression of phosphatidylserine, and membrane blebbing (Han et al., 2008). It can be initiated either via extrinsic or intrinsic pathways. The extrinsic pathway is activated by cell surface receptors, and the intrinsic pathway is triggered by a mitochondria-mediated death-signaling cascade (Fulda & Debatin, 2006; Ghobrial et al., 2005; Harada & Grantet, 2003). These diverse routes ultimately converge and connect to each other via the caspase cascade. Recently, considerable attention has been devoted to the sequence of events referred to as apoptotic cell death and the role of this process in mediating the lethal effects of antineoplastic agents toward cancer cells (Cheng et al., 2003).
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Osthole induces apoptosis
DOI: 10.3109/13880209.2013.850517
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Figure 1. Chemical structure of osthole.
Recent studies have shown that many anticancer drugs or cancer chemopreventive agents act through the induction of apoptosis to prevent tumor promotion and progression (Chen et al., 2007). Reports indicated that osthole possesses antitumor effects by inhibiting tumor cell growth and inducing apoptosis (Chou et al., 2007; Riviere et al., 2006; Xu et al., 2011; Yang et al., 2003). It was reported that osthole induced G2/M arrest and apoptosis in lung cancer A549 cells by modulating the PI3K/Akt pathway (Xu et al., 2011). However, the anticancer effects of osthole on human hepatoma HepG2 cells and its mechanism have not been studied yet. In the present study, we demonstrated the antiproliferative activity of osthole toward human hepatocellular carcinoma HepG2 cells. Furthermore, we examined the mechanism by which osthole induces apoptosis. This study will help researchers evaluate the potential clinical use of osthole in liver cancer treatment.
Materials and methods
Cell viability was measured by the MTT assay (Cheng et al., 2003). Briefly, 180 ml of cell suspensions were seeded into 96well flat-bottom microplates (1 104 cells/well) and cultured in a humidified incubator to allow adhesion overnight. Cells were then treated with osthole at various concentrations of 0.004, 0.02, 0.1, 0.5 and 1.0 mmol/ml for 24 h. After incubation, 20 ml of MTT dye solution [5 mg/ml, in phosphate-buffered saline (PBS), pH 7.4] was added to each well and incubated at 37 C for 4 h. The medium was removed and 150 ml of DMSO was added to dissolve formazan. The optical density (OD) at 570 nm was measured by ELx-800 Universal Microplate Reader (Bio-Tek Instruments, Winooski, VT) and the cell viability (%) was calculated by the following formula: Cell viability ð%Þ ¼ 1 ½ðODcontrol ODtreated Þ =ðODcontrol ODblank Þ: Fluorescent morphologic assay Fluorescent morphological assays were used to detect apoptosis induced by osthole. Cells from exponentially growing cultures were seeded into 12-well culture plates and treated with 0.1 mmol/ml osthole for 24 h. The medium was then discarded and cells were washed twice with PBS, fixed in the mixture of methanol and acetic acid (3:1, v/v) for 10 min at 4 C, stained with Hoechst 33258 and PI for 15 min at 4 C, and examined using an Olympus Fluorescent Microscope (Olympus, Tokyo, Japan).
Materials Osthole was isolated from Cnidium monnieri as described previously (Renmin et al., 2004) in the College of Pharmaceutical Sciences, Shaanxi University of Chinese Medicine, Xianyang, P.R. China. Its structure was characterized by chemical and spectroscopic methods (1H NMR, 13C NMR and MS) and compared with the structure reported in the literature (Mao et al., 2011) (Figure 1). Its purity was above 99%, measured by high-performance liquid chromatography (HPLC). The chromatography was performed on an InertsilÕ ODS-SP analytical column (150 4.6 mm, 5 mm; Shimadzu Corporation, Tokyo, Japan). The mobile phase consisted of methanol and water (80:20, v/v) with detection wavelength at 320 nm. RPMI-1640 media, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), Hoechst 33258 stain, dimethylsulfoxide (DMSO) and antibodies (against Bax, Bcl-2, p-53, procaspase-3, -8, -9 and b-actin) were purchased from Sigma-Aldrich China, Inc. (Shanghai, China). Cell culture The human hepatoma cell line HepG2 was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured as monolayer with RPMI-1640 medium, supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 mg/ml streptomycin and 100 units/ml penicillin. The cultures were maintained at 37 C in a humidified atmosphere of 5% CO2. Cells in the exponential growth phase were used for all experiments.
Annexin V-FITC/PI staining experiments An annexin V-FITC (fluorescein isothiocyanate) Kit (BD Biosciences, San Jose, CA) was used to quantify the percentage of cells undergoing apoptosis. Briefly, HepG2 cells were treated with different concentrations of osthole for 24 h. Then cells were collected, washed twice with 400 ml of binding buffer, and incubated in 100 ml reagent mix containing 1 ml annexin V-FITC conjugate and 10 ml PI in the dark for 15 min at room temperature. The samples were subjected to FACSCaliber flow cytometry to quantify the percentage of cells at different stages. Cell cycle analysis HepG2 cells (1 104 cells/well) were seeded into 6-well plates to allow adhesion overnight and incubated with various concentrations of osthole. They were harvested after incubation, washed, and fixed in 70% ice-cold ethanol at 4 C for 2 h. Cells were then washed twice with PBS and re-suspended in 1 ml of PI stain solution (20 mg/ml PI, 1% Triton X-100, in PBS), containing 10 mg/ml RNaseA, at 37 C in the dark for 30 min. Fluorescence emitted from the PI-DNA complex was measured by FACSCaliber flow cytometry. Protein extraction and western blot assays HepG2 cells (1 106) were seeded in 10 cm dishes and incubated with various concentrations of osthole for 24 h. The total cell protein extracts were obtained according to the method of Levites et al. (2002). In brief, cells were harvested and washed with cold PBS, cell pellets were lysed in 50 ml
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lysis buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, protease inhibitor cocktail], and placed on ice for 30 min. Lysates were centrifuged at 14 000g for 20 min at 4 C. The proteins in the supernatants were collected and concentrations were determined using the bicinchoninic acid assay. The Western blotting assay was performed as described previously (Zhang et al., 2005). An aliquot of the denatured supernatant containing 30 mg of protein was mixed with loading dye and resolved by 10% SDS–polyacrylamide gel electrophoresis, and then electrophoretically transferred to a polyvinylidene fluoride membrane. The blot was blocked in blocking buffer (5% non-fat dry milk/1% Tween-20, in PBS) for 1 h at room temperature, then incubated with specific primary antibodies (rabbit monoclonal anti-human b-actin, anti-procaspase-3, anti-p53, anti-procaspase-8, anti-procaspase-9, anti-Bcl-2 and anti-Bax antibodies) overnight at 4 C, and further incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Detection was performed using an Enhanced Chemiluminescence Kit (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).
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Statistical analysis In all experiments, data were expressed as mean standard deviation. A significant difference from the respective controls for each experimental test condition was assessed using Student’s unpaired t-test and p50.05 was considered statistically significant.
Results Cytotoxicity of osthole on HepG2 As shown in Figure 2a, osthole exhibited a significant inhibition on the survival of HepG2 cells. The inhibitory effects of osthole on HepG2 cells showed concentrationdependent manner and statistically significant compared to the control group (p50.01). IC50 values for osthole treatment was 0.179 mmol/ml in HepG2 cell lines. Figure 2b shows the time course of the induction of cytotoxic effects on HepG2 cells by osthole. The IC50 values were 0.186, 0.158 and 0.123 mmol/ml for 24, 48 and 72 h, respectively. The cytotoxic effect of osthole at different concentrations (0.004, 0.02, 0.1, 0.5 and 1.0 mmol/ml) during 24 h of cultivation is illustrated in Figure 3. Osthole induced integrity
Figure 2. Cytotoxic activities of osthole measured by MTT assay. HepG2 cells were treated with different concentrations (0, 0.004, 0.02, 0.1, 0.5 and 1 mmol/ml) of osthole for 24 (unfilled rectangle), 48 (filled square) and 72 h (filled triangle) and cell viability was then determined by MTT assay.
Figure 3. Morphology of hepatocellular carcinoma HepG2 cells treated with osthole at different concentrations (0, 0.004, 0.02, 0.1, 0.5 and 1.0 mmol/ ml) for 24 h.
Osthole induces apoptosis
DOI: 10.3109/13880209.2013.850517
Effect of osthole on cell morphology
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The growth inhibitory effect of osthole was accompanied by cell shrinkage, as observed by phase-contrast microscopy. Cell shrinkage often indicates apoptotic cell death. We further examined the morphological changes in response to osthole treatment using fluorescence staining. Both control- and osthole-treated cells were stained with the fluorescent dyes Hoechst 33528 and PI, and were visualized by a fluorescent microscope. The nuclei of control cells were homogeneous (Figure 4a) while the cells treated with osthole exhibited the characteristics of apoptosis, with cell shrinkage, and condensation and fragmentation of nuclei (Figure 4b). Effects of osthole on apoptosis of HepG2 cells In an attempt to determine whether osthole induced apoptosis of HepG2 cells, the annexin V-FITC/PI staining experiment was performed. The proportion of annexin V-stained cells signified that both the early and late apoptotic cells increased with the concentration of osthole applied. The cells in the early phase of apoptosis were 3.15, 8.28, 15.32, 25.23 and 29.36%, at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ ml of the compound, respectively. The percentage of cells in the late phase of apoptosis were 2.29, 5.36, 18.08, 35.55 and 43.15% at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ ml of osthole, respectively (Table 1). These data show a shift in the cell population from normal to apoptotic/necrotic stages induced by osthole treatment, which may be the cause of its antitumor activity.
increasing concentrations of osthole compared to a vehicle control. These results indicate that osthole can induce a G2/M phase cell cycle arrest in HepG2 cells, which is associated with the inhibitory effects of osthole against HepG2 cells. Table 1. Percentage of HepG2 cells treated with osthole in each stage of apoptosis.
Concentration (mmol/ml) 0 0.004 0.02 0.1 0.5
Normal (%)
Early apoptosis (%)
Late apoptosis (%)
Others (%)
92.34 83.37 63.01 35.19 23.25
3.15 8.28 15.32 25.23 29.36
2.29 5.36 18.08 35.55 43.15
2.22 2.99 3.59 4.03 4.24
Annexin V-FITC/PI staining of HepG2 cells incubated with osthole for 24 h. HepG2 cells were exposed to different concentrations of osthole (0, 0.004, 0.02, 0.1 and 0.5 mmol/ml) for 24 h at 37 C in a humidified atmosphere of 5% CO2. Cells collected were subjected to annexin V-FITC/PI staining and analyzed by flow cytometry.
0 µmol/ml
100
0.004 µmol/ml 0.02 µmol/ml 0.1 µmol/ml
80 Cell number (%)
damage of cytoplasmic membranes, indicative of necrosis of the HepG2 cells.
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0.5 µmol/ml
60 40 20
Effect of osthole on cell cycle distribution To gain insights into whether osthole affects the cell cycle of HepG2 cells, cells treated with different concentrations of osthole for 24 h were subjected to flow cytometric analysis. After a 24-h exposure to osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ml, cells showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction (Figure 5). The percentage of cells in the G2/M fraction increased when treated with
0 sub-G1
G0/G1
S
G2/M
Cell cycle distribution Figure 5. Percentage of HepG2 cells in G1, S and G2/M phase after osthole treatment. HepG2 cells were exposed to different concentrations of osthole (0, 0.004, 0.02, 0.1 and 0.5 mmol/ml) for 24 h at 37 C in a humidified atmosphere of 5% CO2. Cells harvested were stained with PI and cell cycles were analyzed by flow cytometry.
Figure 4. Fluorescent staining of nuclei in both osthole-treated and untreated cells by Hoechst 33258 and PI. HepG2 cells were incubated with 0.1 mmol/ml of osthole for 24 h. Cells untreated (a) and cells treated with osthole (b) stained with the fluorescent dye Hoechst 33528 and PI were visualized by fluorescence microscope. Condensed and fragmented nuclei and apoptotic bodies were seen in the osthole-treated cells (b), but not in the control (a).
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Effects of osthole on the expression of apoptotic-related proteins To further understand the mechanism of the apoptotic phenomenon, we evaluated protein levels of various key extrinsic and intrinsic initiators and regulators in the apoptotic pathways by western blot analysis. The tumor suppressor p53 is known to be a member of the DNA damage-response pathway (Brune et al., 1998). It has been shown that the p53 protein increases at the early stages of cellular damage in response to a variety of stress inducing agents (Zimmermann et al., 2001). The p53 protein was analyzed by Western blot after treatment with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ml (Figure 6). The result showed osthole induced a slight increase of p53. Additionally, downregulation of Bcl-2 and up-regulation of Bax was also observed, suggesting that a decrease of Bcl-2/Bax ratios might be involved in apoptosis induced by osthole. Caspase-3 is one of the key proteases responsible for the cleavage of poly ADP-ribose polymerase (PARP) and subsequent apoptosis. It plays a necessary role in the morphological and biochemical changes during apoptosis (Wang et al., 2007). Therefore, the protein level of procaspase-3 in cells before and after osthole treatment was investigated by the Western blot analysis. As shown in Figure 6, the procaspase-3 and -9 decreased and their active cleaved forms increased when treated with osthole at concentrations from 0.004 to 0.5 mmol/ml. Meanwhile, a slight reduction of the protein
Figure 6. Western blot analysis of protein extracts obtained from HepG2 cells treated with 0, 0.004, 0.02, 0.1 and 0.5 mmol/ml of osthole, respectively. Total protein extracts were prepared after treatment for 24 h, and analyzed with antibodies to p53, Bcl-2, Bax, procaspase-3, -8 and -9.
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level of procaspase-8 and the increase of its active cleaved forms were also detected.
Discussion The present study using the MTT assay demonstrated that osthole could significantly inhibit the proliferation of HepG2 cells in vitro. The IC50 value after 24 h was 0.179 mmol/ml in HepG2 cells (Figure 2a). The antiproliferative effects of osthole on HepG2 cells treated at different time points were further studied. The IC50 values were 0.186, 0.158 and 0.123 mmol/ml at 24, 48 and 72 h, respectively (Figure 2b). These results indicated that osthole had potent cytotoxicity toward the experimental cells and can inhibit cellular proliferation in a time- and dose-dependent manner. Apoptosis is a fundamental mechanism of cell death that can be activated by a variety of cellular insults. It is characterized by a number of cellular and biochemical hallmarks, including DNA chromatin fragmentation, nuclear condensation, and externalization of phosphatidylserine. In our study, cells treated with osthole were significantly reduced in number with increasing concentrations of the compound compared to the control. The experiment also indicated that osthole was able to induce the integrity damage of cytoplasmic membranes, indicative of necrosis of the HepG2 cells. The morphological changes in response to osthole treatment were also determined by fluorescence staining. The results demonstrated apoptotic morphological changes including cell shrinkage, nuclear condensation, and bright and fragmented nuclei (Figure 4b). These apoptotic effects were further validated by the significant increase in the apoptotic cell population after osthole treatment. It is known that cell-cycle dysregulation is a hallmark of tumor cells. In our study, flow cytometric analysis was used to determine if osthole altered the cell cycle of HepG2 cells. The results showed that cells exposed to osthole for 24 h had a statistically significant increase in the G2/M fraction, accompanied by a decrease in the G0/G1 fraction (Table 1). These results show that osthole could induce a G2/M phase cell-cycle arrest in HepG2 cells that was associated with its inhibitory effects. The caspase family plays an important role in the regulation of apoptosis. Caspase activation is a feature of apoptosis induction in response to death-inducing signals originating from cell surface receptors, mitochondria, or the endoplasmic reticulum. In response to diverse stimuli, proapoptotic Bcl-2 family proteins such as Bax initiate the intrinsic apoptotic pathway by forming channels on assimilating into the mitochondria, thus increasing outer mitochondrial membrane permeability, and thereby facilitating the release of cytochrome c and other pro-apoptotic factors from the mitochondrial inter-membrane space. Released cytochrome c forms an apoptosome complex with Apaf-1, which activates caspase 9, and in turn, its downstream caspase-3, resulting in the morphological features of apoptosis (Cohen, 1997). After 24 h of osthole treatment, the expression of procaspase-3 and -9 decreased and their active cleaved forms increased remarkably. It has been well established that caspasemediated apoptosis in most cells is induced through the
Osthole induces apoptosis
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DOI: 10.3109/13880209.2013.850517
activation of either the mitochondrial (intrinsic) pathway or the death receptor (extrinsic) pathway. The mitochondrial pathway generally involves an induction of mitochondrial permeability transition and the subsequent release of cytochrome c, the CARD adaptor protein APAF-1, and procaspase-9 assembly in the cytosol into the apoptosome, leading to caspase-9 activation, which in turn cleaves and activates effector caspases such as caspase-3 (Yang et al., 2009). In this study, the shift of the expression of procaspase-3 and -9 and their active cleaved forms suggest that the mitochondrial (intrinsic) pathway involved in the apoptosis pathway were induced by osthole. The tumor suppressor gene p53 plays a crucial role in causing cell cycle arrest, in apoptosis and in cell repair. The role of p53 in both osthole-induced cell cycle arrest and apoptosis was further analyzed, and we found that the expression of p53 was increased in a dose-dependent manner in HepG2 cells. This result suggests that osthole induced G2/M phase arrest and the observed apoptosis was a p53 dependent process. Bcl-2 family proteins play an important role in mitochondrial apoptosis pathway, which contains pro-apoptosis (Bax and Bak) and anti-apoptosis proteins (Bcl-2, Bcl-xl, Bcl-w and Mcl-1) (Reyes-Zurita et al., 2009). The formation of heterodimers among the pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family controls apoptosis (Andrew, 1995; Antonsson & Martinou, 2000; Reed, 1997). The heterodimerization results in mutual neutralization of the bound pro- and anti-apoptotic proteins. Therefore, the balance between expression levels of the proteins (e.g., Bcl-2 and Bax) is critical for cell survival or cell death. It is reported that osthole increased proapoptotic Bax expression and decreased anti-apoptotic Bcl-2 expression, leading to up-regulation of the ratio of Bax/Bcl-2 (Xu et al., 2011). The results of this study show that osthole treatment led to up-regulation of Bax and concurrent down-regulation of Bcl2. The balance between the expression levels of these proteins changed, resulting in cell apoptosis.
Conclusion Osthole displays a marked cytotoxicity toward cells and induced G2/M growth arrest. The osthole-treated cells exhibited cell shrinkage and chromatin condensation. The increase of apoptosis induced by osthole was correlated with down-regulation of anti-apoptotic Bcl-2 expression, up-regulation of pro-apoptotic Bax and p53 proteins. Also the shift of the expression of procaspase-3 and -9 and their active cleaved forms suggest that the mitochondrial (intrinsic) pathway involved in the apoptosis pathway were induced by osthole. Taken together, these findings demonstrate that the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.
Declaration of interest This study was supported by the Education Department of Shaanxi Province Natural Science Project (11JK0682).
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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ORIGINAL ARTICLE
Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells Xu Chao1,2, Xiaojun Zhou2, Gang Zheng2, Changhu Dong2, Wei Zhang3, Xiaomei Song4, and Tianbo Jin5 The College of Preclinical Sciences, Shaanxi University of Chinese Medicine, Xianyang, P.R. China, 2The Second Affiliated Hospital of Shaanxi University of Chinese Medicine, Xianyang, P.R. China, 3Jilin Medical College, Jilin, P.R. China, 4The College of Pharmaceutical Sciences, Shaanxi University of Chinese Medicine, Xianyang, P.R. China, and 5The College of Chemistry and Materials Sciences, Northwest University, Xi’an, P.R. China
Abstract
Keywords
Context: Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong’s Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture. Objectives: This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells. Materials and methods: Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0 mmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot. Results: Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC50) values were 0.186, 0.158 and 0.123 mmol/ml at 24, 48 and 72 h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with downregulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of proapoptotic Bax and p53 proteins. Conclusion: Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.
Apoptosis, cell cycle, G2/M, HepG2 cells, osthole
Introduction Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] (Figure 1), a coumarin derivative (You et al., 2009) isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong’s Classic of Materia Medica in the Han Dynasty, exhibits many pharmacological and biological activities (Okamoto et al., 2007; Tang et al., 2008), including antidiabetic activity (Liang et al., 2009), anti-inflammatory activity (Zimecki et al., 2009) and antinociceptive effect (Riviere et al., 2006). Additionally, accumulating evidence indicates that osthole possesses antitumor effects by inhibiting tumor cell growth and induces apoptosis (Okamoto et al., 2005; Xu et al., 2011). Apoptosis, or programmed cell death, is implicated in cellular homeostasis and many physiological processes.
Correspondence: Dr Tianbo Jin, The College of Chemistry and Materials Sciences, Northwest University, Xi’an 710069, P.R. China. Tel: +86-2988303446. E-mail: [email protected]
History Received 12 November 2012 Revised 17 September 2013 Accepted 24 September 2013 Published online 15 November 2013
Most cancer cells block apoptosis, which allows them to survive despite undergoing genetic and morphologic transformations. It is often linked with carcinogenesis, which results in the abrogation of apoptotic processes (Blagosklonny, 2003; Rodriguez-Nieto & Zhivotovsky, 2006). Apoptotic cells are characterized by several unique features, including cell shrinkage, chromatin condensation, DNA fragmentation, cell surface expression of phosphatidylserine, and membrane blebbing (Han et al., 2008). It can be initiated either via extrinsic or intrinsic pathways. The extrinsic pathway is activated by cell surface receptors, and the intrinsic pathway is triggered by a mitochondria-mediated death-signaling cascade (Fulda & Debatin, 2006; Ghobrial et al., 2005; Harada & Grantet, 2003). These diverse routes ultimately converge and connect to each other via the caspase cascade. Recently, considerable attention has been devoted to the sequence of events referred to as apoptotic cell death and the role of this process in mediating the lethal effects of antineoplastic agents toward cancer cells (Cheng et al., 2003).
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Osthole induces apoptosis
DOI: 10.3109/13880209.2013.850517
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Figure 1. Chemical structure of osthole.
Recent studies have shown that many anticancer drugs or cancer chemopreventive agents act through the induction of apoptosis to prevent tumor promotion and progression (Chen et al., 2007). Reports indicated that osthole possesses antitumor effects by inhibiting tumor cell growth and inducing apoptosis (Chou et al., 2007; Riviere et al., 2006; Xu et al., 2011; Yang et al., 2003). It was reported that osthole induced G2/M arrest and apoptosis in lung cancer A549 cells by modulating the PI3K/Akt pathway (Xu et al., 2011). However, the anticancer effects of osthole on human hepatoma HepG2 cells and its mechanism have not been studied yet. In the present study, we demonstrated the antiproliferative activity of osthole toward human hepatocellular carcinoma HepG2 cells. Furthermore, we examined the mechanism by which osthole induces apoptosis. This study will help researchers evaluate the potential clinical use of osthole in liver cancer treatment.
Materials and methods
Cell viability was measured by the MTT assay (Cheng et al., 2003). Briefly, 180 ml of cell suspensions were seeded into 96well flat-bottom microplates (1 104 cells/well) and cultured in a humidified incubator to allow adhesion overnight. Cells were then treated with osthole at various concentrations of 0.004, 0.02, 0.1, 0.5 and 1.0 mmol/ml for 24 h. After incubation, 20 ml of MTT dye solution [5 mg/ml, in phosphate-buffered saline (PBS), pH 7.4] was added to each well and incubated at 37 C for 4 h. The medium was removed and 150 ml of DMSO was added to dissolve formazan. The optical density (OD) at 570 nm was measured by ELx-800 Universal Microplate Reader (Bio-Tek Instruments, Winooski, VT) and the cell viability (%) was calculated by the following formula: Cell viability ð%Þ ¼ 1 ½ðODcontrol ODtreated Þ =ðODcontrol ODblank Þ: Fluorescent morphologic assay Fluorescent morphological assays were used to detect apoptosis induced by osthole. Cells from exponentially growing cultures were seeded into 12-well culture plates and treated with 0.1 mmol/ml osthole for 24 h. The medium was then discarded and cells were washed twice with PBS, fixed in the mixture of methanol and acetic acid (3:1, v/v) for 10 min at 4 C, stained with Hoechst 33258 and PI for 15 min at 4 C, and examined using an Olympus Fluorescent Microscope (Olympus, Tokyo, Japan).
Materials Osthole was isolated from Cnidium monnieri as described previously (Renmin et al., 2004) in the College of Pharmaceutical Sciences, Shaanxi University of Chinese Medicine, Xianyang, P.R. China. Its structure was characterized by chemical and spectroscopic methods (1H NMR, 13C NMR and MS) and compared with the structure reported in the literature (Mao et al., 2011) (Figure 1). Its purity was above 99%, measured by high-performance liquid chromatography (HPLC). The chromatography was performed on an InertsilÕ ODS-SP analytical column (150 4.6 mm, 5 mm; Shimadzu Corporation, Tokyo, Japan). The mobile phase consisted of methanol and water (80:20, v/v) with detection wavelength at 320 nm. RPMI-1640 media, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), Hoechst 33258 stain, dimethylsulfoxide (DMSO) and antibodies (against Bax, Bcl-2, p-53, procaspase-3, -8, -9 and b-actin) were purchased from Sigma-Aldrich China, Inc. (Shanghai, China). Cell culture The human hepatoma cell line HepG2 was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured as monolayer with RPMI-1640 medium, supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 mg/ml streptomycin and 100 units/ml penicillin. The cultures were maintained at 37 C in a humidified atmosphere of 5% CO2. Cells in the exponential growth phase were used for all experiments.
Annexin V-FITC/PI staining experiments An annexin V-FITC (fluorescein isothiocyanate) Kit (BD Biosciences, San Jose, CA) was used to quantify the percentage of cells undergoing apoptosis. Briefly, HepG2 cells were treated with different concentrations of osthole for 24 h. Then cells were collected, washed twice with 400 ml of binding buffer, and incubated in 100 ml reagent mix containing 1 ml annexin V-FITC conjugate and 10 ml PI in the dark for 15 min at room temperature. The samples were subjected to FACSCaliber flow cytometry to quantify the percentage of cells at different stages. Cell cycle analysis HepG2 cells (1 104 cells/well) were seeded into 6-well plates to allow adhesion overnight and incubated with various concentrations of osthole. They were harvested after incubation, washed, and fixed in 70% ice-cold ethanol at 4 C for 2 h. Cells were then washed twice with PBS and re-suspended in 1 ml of PI stain solution (20 mg/ml PI, 1% Triton X-100, in PBS), containing 10 mg/ml RNaseA, at 37 C in the dark for 30 min. Fluorescence emitted from the PI-DNA complex was measured by FACSCaliber flow cytometry. Protein extraction and western blot assays HepG2 cells (1 106) were seeded in 10 cm dishes and incubated with various concentrations of osthole for 24 h. The total cell protein extracts were obtained according to the method of Levites et al. (2002). In brief, cells were harvested and washed with cold PBS, cell pellets were lysed in 50 ml
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lysis buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, protease inhibitor cocktail], and placed on ice for 30 min. Lysates were centrifuged at 14 000g for 20 min at 4 C. The proteins in the supernatants were collected and concentrations were determined using the bicinchoninic acid assay. The Western blotting assay was performed as described previously (Zhang et al., 2005). An aliquot of the denatured supernatant containing 30 mg of protein was mixed with loading dye and resolved by 10% SDS–polyacrylamide gel electrophoresis, and then electrophoretically transferred to a polyvinylidene fluoride membrane. The blot was blocked in blocking buffer (5% non-fat dry milk/1% Tween-20, in PBS) for 1 h at room temperature, then incubated with specific primary antibodies (rabbit monoclonal anti-human b-actin, anti-procaspase-3, anti-p53, anti-procaspase-8, anti-procaspase-9, anti-Bcl-2 and anti-Bax antibodies) overnight at 4 C, and further incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Detection was performed using an Enhanced Chemiluminescence Kit (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).
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Statistical analysis In all experiments, data were expressed as mean standard deviation. A significant difference from the respective controls for each experimental test condition was assessed using Student’s unpaired t-test and p50.05 was considered statistically significant.
Results Cytotoxicity of osthole on HepG2 As shown in Figure 2a, osthole exhibited a significant inhibition on the survival of HepG2 cells. The inhibitory effects of osthole on HepG2 cells showed concentrationdependent manner and statistically significant compared to the control group (p50.01). IC50 values for osthole treatment was 0.179 mmol/ml in HepG2 cell lines. Figure 2b shows the time course of the induction of cytotoxic effects on HepG2 cells by osthole. The IC50 values were 0.186, 0.158 and 0.123 mmol/ml for 24, 48 and 72 h, respectively. The cytotoxic effect of osthole at different concentrations (0.004, 0.02, 0.1, 0.5 and 1.0 mmol/ml) during 24 h of cultivation is illustrated in Figure 3. Osthole induced integrity
Figure 2. Cytotoxic activities of osthole measured by MTT assay. HepG2 cells were treated with different concentrations (0, 0.004, 0.02, 0.1, 0.5 and 1 mmol/ml) of osthole for 24 (unfilled rectangle), 48 (filled square) and 72 h (filled triangle) and cell viability was then determined by MTT assay.
Figure 3. Morphology of hepatocellular carcinoma HepG2 cells treated with osthole at different concentrations (0, 0.004, 0.02, 0.1, 0.5 and 1.0 mmol/ ml) for 24 h.
Osthole induces apoptosis
DOI: 10.3109/13880209.2013.850517
Effect of osthole on cell morphology
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The growth inhibitory effect of osthole was accompanied by cell shrinkage, as observed by phase-contrast microscopy. Cell shrinkage often indicates apoptotic cell death. We further examined the morphological changes in response to osthole treatment using fluorescence staining. Both control- and osthole-treated cells were stained with the fluorescent dyes Hoechst 33528 and PI, and were visualized by a fluorescent microscope. The nuclei of control cells were homogeneous (Figure 4a) while the cells treated with osthole exhibited the characteristics of apoptosis, with cell shrinkage, and condensation and fragmentation of nuclei (Figure 4b). Effects of osthole on apoptosis of HepG2 cells In an attempt to determine whether osthole induced apoptosis of HepG2 cells, the annexin V-FITC/PI staining experiment was performed. The proportion of annexin V-stained cells signified that both the early and late apoptotic cells increased with the concentration of osthole applied. The cells in the early phase of apoptosis were 3.15, 8.28, 15.32, 25.23 and 29.36%, at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ ml of the compound, respectively. The percentage of cells in the late phase of apoptosis were 2.29, 5.36, 18.08, 35.55 and 43.15% at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ ml of osthole, respectively (Table 1). These data show a shift in the cell population from normal to apoptotic/necrotic stages induced by osthole treatment, which may be the cause of its antitumor activity.
increasing concentrations of osthole compared to a vehicle control. These results indicate that osthole can induce a G2/M phase cell cycle arrest in HepG2 cells, which is associated with the inhibitory effects of osthole against HepG2 cells. Table 1. Percentage of HepG2 cells treated with osthole in each stage of apoptosis.
Concentration (mmol/ml) 0 0.004 0.02 0.1 0.5
Normal (%)
Early apoptosis (%)
Late apoptosis (%)
Others (%)
92.34 83.37 63.01 35.19 23.25
3.15 8.28 15.32 25.23 29.36
2.29 5.36 18.08 35.55 43.15
2.22 2.99 3.59 4.03 4.24
Annexin V-FITC/PI staining of HepG2 cells incubated with osthole for 24 h. HepG2 cells were exposed to different concentrations of osthole (0, 0.004, 0.02, 0.1 and 0.5 mmol/ml) for 24 h at 37 C in a humidified atmosphere of 5% CO2. Cells collected were subjected to annexin V-FITC/PI staining and analyzed by flow cytometry.
0 µmol/ml
100
0.004 µmol/ml 0.02 µmol/ml 0.1 µmol/ml
80 Cell number (%)
damage of cytoplasmic membranes, indicative of necrosis of the HepG2 cells.
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0.5 µmol/ml
60 40 20
Effect of osthole on cell cycle distribution To gain insights into whether osthole affects the cell cycle of HepG2 cells, cells treated with different concentrations of osthole for 24 h were subjected to flow cytometric analysis. After a 24-h exposure to osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ml, cells showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction (Figure 5). The percentage of cells in the G2/M fraction increased when treated with
0 sub-G1
G0/G1
S
G2/M
Cell cycle distribution Figure 5. Percentage of HepG2 cells in G1, S and G2/M phase after osthole treatment. HepG2 cells were exposed to different concentrations of osthole (0, 0.004, 0.02, 0.1 and 0.5 mmol/ml) for 24 h at 37 C in a humidified atmosphere of 5% CO2. Cells harvested were stained with PI and cell cycles were analyzed by flow cytometry.
Figure 4. Fluorescent staining of nuclei in both osthole-treated and untreated cells by Hoechst 33258 and PI. HepG2 cells were incubated with 0.1 mmol/ml of osthole for 24 h. Cells untreated (a) and cells treated with osthole (b) stained with the fluorescent dye Hoechst 33528 and PI were visualized by fluorescence microscope. Condensed and fragmented nuclei and apoptotic bodies were seen in the osthole-treated cells (b), but not in the control (a).
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Effects of osthole on the expression of apoptotic-related proteins To further understand the mechanism of the apoptotic phenomenon, we evaluated protein levels of various key extrinsic and intrinsic initiators and regulators in the apoptotic pathways by western blot analysis. The tumor suppressor p53 is known to be a member of the DNA damage-response pathway (Brune et al., 1998). It has been shown that the p53 protein increases at the early stages of cellular damage in response to a variety of stress inducing agents (Zimmermann et al., 2001). The p53 protein was analyzed by Western blot after treatment with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 mmol/ml (Figure 6). The result showed osthole induced a slight increase of p53. Additionally, downregulation of Bcl-2 and up-regulation of Bax was also observed, suggesting that a decrease of Bcl-2/Bax ratios might be involved in apoptosis induced by osthole. Caspase-3 is one of the key proteases responsible for the cleavage of poly ADP-ribose polymerase (PARP) and subsequent apoptosis. It plays a necessary role in the morphological and biochemical changes during apoptosis (Wang et al., 2007). Therefore, the protein level of procaspase-3 in cells before and after osthole treatment was investigated by the Western blot analysis. As shown in Figure 6, the procaspase-3 and -9 decreased and their active cleaved forms increased when treated with osthole at concentrations from 0.004 to 0.5 mmol/ml. Meanwhile, a slight reduction of the protein
Figure 6. Western blot analysis of protein extracts obtained from HepG2 cells treated with 0, 0.004, 0.02, 0.1 and 0.5 mmol/ml of osthole, respectively. Total protein extracts were prepared after treatment for 24 h, and analyzed with antibodies to p53, Bcl-2, Bax, procaspase-3, -8 and -9.
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level of procaspase-8 and the increase of its active cleaved forms were also detected.
Discussion The present study using the MTT assay demonstrated that osthole could significantly inhibit the proliferation of HepG2 cells in vitro. The IC50 value after 24 h was 0.179 mmol/ml in HepG2 cells (Figure 2a). The antiproliferative effects of osthole on HepG2 cells treated at different time points were further studied. The IC50 values were 0.186, 0.158 and 0.123 mmol/ml at 24, 48 and 72 h, respectively (Figure 2b). These results indicated that osthole had potent cytotoxicity toward the experimental cells and can inhibit cellular proliferation in a time- and dose-dependent manner. Apoptosis is a fundamental mechanism of cell death that can be activated by a variety of cellular insults. It is characterized by a number of cellular and biochemical hallmarks, including DNA chromatin fragmentation, nuclear condensation, and externalization of phosphatidylserine. In our study, cells treated with osthole were significantly reduced in number with increasing concentrations of the compound compared to the control. The experiment also indicated that osthole was able to induce the integrity damage of cytoplasmic membranes, indicative of necrosis of the HepG2 cells. The morphological changes in response to osthole treatment were also determined by fluorescence staining. The results demonstrated apoptotic morphological changes including cell shrinkage, nuclear condensation, and bright and fragmented nuclei (Figure 4b). These apoptotic effects were further validated by the significant increase in the apoptotic cell population after osthole treatment. It is known that cell-cycle dysregulation is a hallmark of tumor cells. In our study, flow cytometric analysis was used to determine if osthole altered the cell cycle of HepG2 cells. The results showed that cells exposed to osthole for 24 h had a statistically significant increase in the G2/M fraction, accompanied by a decrease in the G0/G1 fraction (Table 1). These results show that osthole could induce a G2/M phase cell-cycle arrest in HepG2 cells that was associated with its inhibitory effects. The caspase family plays an important role in the regulation of apoptosis. Caspase activation is a feature of apoptosis induction in response to death-inducing signals originating from cell surface receptors, mitochondria, or the endoplasmic reticulum. In response to diverse stimuli, proapoptotic Bcl-2 family proteins such as Bax initiate the intrinsic apoptotic pathway by forming channels on assimilating into the mitochondria, thus increasing outer mitochondrial membrane permeability, and thereby facilitating the release of cytochrome c and other pro-apoptotic factors from the mitochondrial inter-membrane space. Released cytochrome c forms an apoptosome complex with Apaf-1, which activates caspase 9, and in turn, its downstream caspase-3, resulting in the morphological features of apoptosis (Cohen, 1997). After 24 h of osthole treatment, the expression of procaspase-3 and -9 decreased and their active cleaved forms increased remarkably. It has been well established that caspasemediated apoptosis in most cells is induced through the
Osthole induces apoptosis
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DOI: 10.3109/13880209.2013.850517
activation of either the mitochondrial (intrinsic) pathway or the death receptor (extrinsic) pathway. The mitochondrial pathway generally involves an induction of mitochondrial permeability transition and the subsequent release of cytochrome c, the CARD adaptor protein APAF-1, and procaspase-9 assembly in the cytosol into the apoptosome, leading to caspase-9 activation, which in turn cleaves and activates effector caspases such as caspase-3 (Yang et al., 2009). In this study, the shift of the expression of procaspase-3 and -9 and their active cleaved forms suggest that the mitochondrial (intrinsic) pathway involved in the apoptosis pathway were induced by osthole. The tumor suppressor gene p53 plays a crucial role in causing cell cycle arrest, in apoptosis and in cell repair. The role of p53 in both osthole-induced cell cycle arrest and apoptosis was further analyzed, and we found that the expression of p53 was increased in a dose-dependent manner in HepG2 cells. This result suggests that osthole induced G2/M phase arrest and the observed apoptosis was a p53 dependent process. Bcl-2 family proteins play an important role in mitochondrial apoptosis pathway, which contains pro-apoptosis (Bax and Bak) and anti-apoptosis proteins (Bcl-2, Bcl-xl, Bcl-w and Mcl-1) (Reyes-Zurita et al., 2009). The formation of heterodimers among the pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family controls apoptosis (Andrew, 1995; Antonsson & Martinou, 2000; Reed, 1997). The heterodimerization results in mutual neutralization of the bound pro- and anti-apoptotic proteins. Therefore, the balance between expression levels of the proteins (e.g., Bcl-2 and Bax) is critical for cell survival or cell death. It is reported that osthole increased proapoptotic Bax expression and decreased anti-apoptotic Bcl-2 expression, leading to up-regulation of the ratio of Bax/Bcl-2 (Xu et al., 2011). The results of this study show that osthole treatment led to up-regulation of Bax and concurrent down-regulation of Bcl2. The balance between the expression levels of these proteins changed, resulting in cell apoptosis.
Conclusion Osthole displays a marked cytotoxicity toward cells and induced G2/M growth arrest. The osthole-treated cells exhibited cell shrinkage and chromatin condensation. The increase of apoptosis induced by osthole was correlated with down-regulation of anti-apoptotic Bcl-2 expression, up-regulation of pro-apoptotic Bax and p53 proteins. Also the shift of the expression of procaspase-3 and -9 and their active cleaved forms suggest that the mitochondrial (intrinsic) pathway involved in the apoptosis pathway were induced by osthole. Taken together, these findings demonstrate that the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.
Declaration of interest This study was supported by the Education Department of Shaanxi Province Natural Science Project (11JK0682).
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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