Anticancer Potential of Euphorbia helioscopia L Extracts Against Human Cancer Cells
Version of Record online: 20 DEC 2011
Copyright © 2011 Wiley Periodicals, Inc.
The Anatomical Record
Volume 295, Issue 2, pages 223–233, February 2012
How to Cite
Wang, Z. Y., Liu, H. P., Zhang, Y. C., Guo, L. Q., Li, Z. X. and Shi, X. F. (2012), Anticancer Potential of Euphorbia helioscopia L Extracts Against Human Cancer Cells. Anat Rec, 295: 223–233. doi: 10.1002/ar.21517
- Issue online: 11 JAN 2012
- Version of Record online: 20 DEC 2011
- Manuscript Accepted: 22 SEP 2011
- Manuscript Received: 10 JUN 2011
- Gansu Province Science & Technology Pillar Program. Grant Number: QS061-C33-4
- Central Laboratory of Lanzhou University Second Hospital
- Euphorbia helioscopia L;
- cell cycle;
Euphorbia helioscopia L is considered a traditional Chinese herb which is widely distributed in China. The active anticancer fractions and anticancer mechanism of the herb are unclear. In this study, we evaluated the growth inhibitory effects of Euphorbia helioscopia L extracts on five different human cancer cell lines for screening the main active fraction with antitumor effect. In this regard, the ethyl acetate extract (EAE) was found to markedly inhibit the proliferation of SMMC-7721 cells in a time- and dose-dependent manner. EAE treatment arrested cell cycle in G-1 phase and EAE used at the concentration range of 100–200 μg/mL induced a marked increase of subdiploid peak. After EAE treatment at the concentrations of 150 and 200 μg/mL, the percentage of apoptotic cells was increased. At the EAE concentration of 200 μg/mL, the typical morphology of early apoptotic change was observed in SMMC-7721 cells. Since tumorigenesis is often defined by an uncontrolled proliferation and transplantability, we also determined the anti-invasive effects of EAE. The EAE treatment displayed a dose-dependent inhibitory effect on tumor cell invasion and MMP-9 expression. Also, the major active fraction was assayed using high-performance liquid chromatography (HPLC). The data showed that the flavonoids could be the main constituents of EAE. Based on the evidence from these data, we inferred that the EAE of Euphorbia helioscopia L could have chemopreventive potential against the human cancer. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.
Cancer is the second most common cause of morbidity and mortality in the world, and is expected to rise in the coming decades (Mignogna et al.,2004; WHO,2011). For solid tumors, the conventional treatments include chemotherapy, radiation therapy, and surgery. In recent years, the mechanism of cancer development strongly suggests that cancer is a systemic disease. Despite the tremendous progress made over the past few decades towards understanding the molecular biology of cancer, the disease has not been fully controlled. Relying solely on local treatment such as surgical excision or radiation therapy makes it difficult to cure or prevent tumor recurrence and metastasis. By far, chemotherapy is the most promising and commonly used treatment to reduce the risk of cancer (Surh and Chun,2007; Mou et al.,2011). Nevertheless, developing new synthetic drugs can be costly and it is also difficult to screen the effective anticancer drugs from large numbers of chemical compounds. Therefore, the quest for safe and effective anticancer drugs from natural plants has become an important aspect of anticancer research.
Euphorbia helioscopia L belongs to the plant family Euphorbiaceae and genus Euphorbia (Editorial Committee of the Administration Bureau of Traditional Chinese Medicine “ABTCM”,1998), and is widely distributed in China. As an herbaceous plant, the stem of Euphorbia helioscopia L produces a typical milky juice which may cause toxic reactions following contact with skin and mucous membranes (Wilken and Schempp,2005). As a traditional Chinese medicine, Euphorbia helioscopia L has been widely used for centuries to treat different disease conditions, such as ascites, edema, tuberculosis, dysentery, scabies, lung cancer, cervical carcinoma, and esophageal cancer (Editorial Committee of the ABTCM,1998; Pang and Lian,2007; Yang et al.,2007). It is also believed to have antifungal and antibacterial properties (Uzair et al.,2009). During the past decade, numerous studies reported the isolation of various secondary metabolites from Euphorbia helioscopia L., such as diterpenoids (Yamamura et al.,1981; Shizuri et al.,1983; Shizuri et al.,1984a,b; Kosemura et al.,1985; Yamamura et al.,1989; Zhang and Guo,2006; Barile et al.,2008; Tao et al.,2008), flavonoids (Kawase and Kutani,1968; Chen et al.,1979), triterpenoids (Nazir et al.,1998), polyphenols (Wei-Sheng et al.,2009), steroids and lipids (Kosemura et al.,1985). Previously, high contents of quercetin, a plant-derived flavonoid, have been detected in the leaves of Euphorbia helioscopia L (Liu et al.,2011) and its anticancer properties were demonstrated (Kang and Liang,1997; Caltagirone et al.,2000). Although the antitumor activity of aquatic extract of Euphorbia helioscopia L root was also studied (Cai et al.,1999a,b), the effects of the whole plant have not been evaluated and the anticancer active fractions and the precise anticancer mechanism of the herb remain unclear.
The purpose of this study was to identify the main antitumor active fractions of aqueous-ethanol extracts of Euphorbia helioscopia L against five cancer cell lines in vitro, and to analyze the possible constituents of the main antitumor active fractions by high performance liquid chromatography (HPLC) in order to evaluate the possible anticancer mechanisms involved.
MATERIALS AND METHODS
The whole plant of Euphorbia helioscopia L was collected in June 2008 from Dingxi City, Gansu Province, China and was identified in the Institute of Botany, School of Life Sciences, Lanzhou University and Gansu Institute for Drug Control. The voucher specimens were deposited in the Lanzhou University Second Hospital for future reference.
The dried Euphorbia helioscopia L whole plant (10 g) was ground and extracted with 70% ethanol for 5 hr and then filtered. The filtrate was concentrated by rotary evaporator (BÜCHI, Switzerland). The concentrated extract was fractionated using petroleum ether, chloroform, ethyl acetate, and n-butanol, individually. The residue from each fractionation step was used to obtain the subsequent fraction, as shown in Fig. 1. The extracts from each fractionation step were evaporated to dryness under vacuum. These extracts were dissolved in dimethylsulfoxide (DMSO) and further diluted with cell culture medium. The final DMSO concentration was below 1% of total volume of the medium in all treatments and controls.
Cell Lines and Cell Culture
The human hepatocellular carcinoma cell lines SMMC-7721, BEL-7402, HepG2, gastric carcinoma cell line SGC-7901 and colorectal cancer cell line SW-480 were purchased from the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). SMMC-7721, BEL-7402, SGC-7901, and SW-480 were cultured in RPMI-1640 medium (Gibco, USA), whereas HepG2 was cultured in DMEM medium (Gibco, USA). Both culture media were supplemented with 10% fetal bovine serum (FBS; Gibco, Australia), antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin) and maintained at 37°C in a humidified atmosphere of 5% CO2.
Cell Proliferation Assay
Cancer cells in logarithmic growth phase were prepared at a concentration of 2 × 104 cells/mL and inoculated into 96-well cell culture plates (200 μL per well) and incubated at 37°C for 24 hr. After 24 hr, cells were treated with various concentrations of extracts (50, 75, 100, 150, and 200 μg/mL) for 24, 48, and 72 hr. Cell proliferation was measured by MTT assay according to the manufacturer's instructions. Briefly, after 24-, 48-, and 72-hr treatments with the extracts, 20 μL of MTT reagent (5 mg/mL; Sigma, USA) was added to each well and incubated at 37°C for 4 hr. After incubation, the medium with MTT was removed and 150 μL of DMSO was added to each well to solubilize the formazan crystals by incubation for 15 min at room temperature. The absorbance value per well was determined at 490 nm using a microplate reader (Thermo Scientific 3001, USA). All assays were repeated three times.
Cell Cycle Analysis
SMMC-7721 cells were inoculated into 6-well culture plates. After 24 hr, the culture medium was removed and the cells were treated with ethyl acetate extract (EAE) of Euphorbia helioscopia L. After 24, 48, and 72 hr, adherent and floating cells were harvested, washed with cold phosphate-buffered saline (PBS) and then fixed with 70% cold ethanol at 4°C for 24 hr. The cells were re-suspended in 300 μL of cold PBS solution containing 50 μg/mL propidium iodide (PI), 0.1% Triton-100, and 100 μg/mL RNase for 30 min at temperature room. The samples were processed using a Beckman Coulter Epics flow cytometer (Beckman Coulter Epics-XL, USA) and the data was analyzed using Expor-32 software.
Apoptosis was detected by annexin V-FITC/PI staining assay. SMMC-7721 cells were seeded in 6-well culture plates. After 24-, 48-, and 72-hr treatment with various concentrations (150 and 200 μg/mL) of EAE, the adherent and floating cells were harvested and stained with 5 μL annexin V-FITC and 10 μL PI according to manufacturer's instructions. The apoptotic cells were analyzed by a Beckman Coulter Epics flow cytometer and using Expor-32 software.
Scanning Electron Microscopy
SMMC-7721 cells were treated with EAE at the concentration of 200 μg/mL for 24 hr. The cultured cells were harvested, fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.2) for 1 hr at 4°C, and then fixed in 1% osmium tetraoxide in the same buffer for 2 hr. The cells were dehydrated using a graded series of acetone and propylene oxide and embedded in epoxy resin Epon-812. Embedded cells were sliced into ultrathin sections and stained with uranyl acetate and lead citrate. The sections were examined by scanning electron microscope (JEM-1230, Japan).
After incubation for 24 hr, culture medium of SMMC-7721 cells was removed and replaced with EAE, followed by reincubation for 48 hr before performing the invasion assay. The invasion assay was carried out using transwell chamber (BD-Biosciences, USA) method according to the manufacturer's instructions. Briefly, 8 μm pore inserts were coated with Matrigel before use. Cells (5 × 104) were suspended in 200 μL of serum-free medium and inoculated into the upper chamber. Then 600 μL of medium containing 10% FBS was added to the lower chamber. After 24 hr incubation, the inserts were fixed with 4% buffered formalin phosphate and washed with PBS. Noninvasion cells were removed from the interior of the chamber insert with a swab and the fixed cells were stained with 0.1% crystal violet solution. Invasion was analyzed by counting the number of cells in six randomly selected fields (×100 magnification) using an inverted microscope (Olympus IX-71, Japan).
Determination of MMP-9 Expression
After 48-hr treatment with EAE, cell supernatants were collected for analysis. The concentration of matrix metalloproteinase-9 (MMP-9) was detected by enzyme-linked immunosorbent assay (ELISA). To this end, commercial MMP-9 ELISA kits (Quantikine, R&D Systems, USA) were used for quantitative determination of human active (82 kDa) and Pro- (92 kDa) MMP-9 concentrations in cell culture supernatants following the manufacturer's instructions. The limit of detection for MMP-9 was 0.156 ng/mL.
EAE was analyzed by HPLC using Agilent 1100 HPLC XB-C18 column (150 mm × 4.6 mm; 5 μm, USA) with UV-detector system. The mobile phase of acetonitrile—0.4 % phosphoric acid (30:70) solution was used at the flow rate of 1.0 mL/min at 30°C and the detection was performed at 360 nm wavelength. All tested solutions used were of spectra analytical grade and were filtered through 0.45-μm filters before use. The standards of myricetin, quercetin, and kaempferol were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
Statistical analysis was performed with SPSS 16.0 software. All data were presented as mean ± standard deviation values. Statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) and Student's t-test was used for comparing two groups. P-values < 0.05 were considered as statistically significant.
Antiproliferative Effects of Euphorbia helioscopia L Extracts on Different Cancer Cell Lines
Human hepatocellular carcinoma cell lines SMMC-7721, BEL-7402, HepG2, gastric carcinoma cell line SGC-7901 and colorectal cancer cell line SW480 were used to investigate the antiproliferative effects of petroleum ether extract (PEE), chloroform extract (CE), EAE, and n-butanol extract (NBE) of Euphorbia helioscopia L. As shown in Fig. 2, after treatment for 24 hr, a significant suppression in the proliferation of SMMC-7721 cells was observed with EAE and CE at the concentrations of 150 and 200 μg/mL, each. The antiproliferative rate of EAE and CE at the concentration of 200 μg/mL was 59.98 and 36.8%, respectively (P < 0.01). After treating with EAE at the concentrations of 150 and 200 μg/mL for 48 hr (Fig. 3), growth inhibition rates of SMMC-7721 cells were 72.59 and 77.43%, respectively (P < 0.01). However, with CE (Fig. 3), the growth inhibition rates at 150 and 200 μg/mL concentrations were 30.84% (P < 0.05) and 41.98% (P < 0.01), respectively. After treating with 150 and 200 μg/mL concentrations using SMMC-7721 cells for 72 hr (Fig. 4), EAE inhibited cell growth by 77.47 and 80.91% (P < 0.01), respectively; whereas CE inhibited cell growth by 40.91 and 51.76% (P < 0.01), respectively. Also, after treatment for 24 hr (Fig. 2), marked antiproliferation of BEL-7402 cells was observed with EAE and CE at the concentration range of 100–200 μg/mL. The inhibition rates at 200 μg/mL concentration were 48.53% and 34.33%, respectively (P < 0.01). In addition, the proliferation of HepG-2, SGC-7901, and SW-480 cells was also inhibited remarkably with EAE and CE at the concentrations of 150 and 200 μg/mL for 24 hr (Fig. 2), each. After treatment for 48 hr (Fig. 3) or 72 hr (Fig. 4), growth suppressive effects of EAE and CE were also observed for BEL-7402, HepG-2, SGC-7901, and SW480 cells. On the other hand, there were no obvious inhibitory effects of PEE and NBE observed on these cells during the same time periods.
Effects of the EAE of Euphorbia helioscopia L on Cell Cycle
As shown in Fig. 5, after 24-, 48-, and 72-hr treatment, the G1-phase population of control (mock treated) cells was 58.34%, 76.47%, and 77.90%, respectively. Compared with controls, the G1-phase population of the EAE-treated cells increased at 24, 48, and 72 hr, and the highest population was 90.02%. In addition, treatment with EAE also decreased the percentage of cells in S-phase in a dose- and time-dependent manner; S-phase cell population after 72 hr of EAE treatment at 200 μg/mL concentration was found to be 9.66%. Moreover, compared with controls, EAE treatment at 100–200 μg/mL concentration range induced a marked increase of subdiploid peak to varying degrees. Treatment with EAE at 200 μg/mL concentration for 72 hr resulted in the highest percentage of cells (11.18%) in Sub-G1 phase of cell cycle. The percentages of cells in different cell cycle phases after various treatments are summarized in Table 1.
|24 hr||0 (control)||0||58.34||38.41||3.25|
|48 hr||0 (control)||0||76.47||22.39||1.14|
|72 h||0 (control)||0||77.90||12.88||9.22|
Induction of Apoptosis by EAE
The cells in the lower left quadrant were viable and negative for both annexin V and PI. The cells in the upper left quadrant, early apoptotic cells, were positive for annexin V and negative for PI; the late apoptotic cells in the upper right quadrant were positive for annexin V and PI; necrotic cell population in the lower right quadrant was positive for PI and negative for annexin V (Fig. 6). After treatment with EAE (200 μg/ml) for 24, 48, and 72 hr, the amount of apoptotic cells (both early and late apoptosis) were 27.35%, 34.53%, and 47.53%, respectively. After treatment with EAE at 150 or 200 μg/mL concentrations, compared with the control group, the percentage of apoptotic cells was significantly increased.
Changes in Ultrastructural Morphology of Cells
The control cells exhibited normal morphology of nucleolus, cytoplasm and organelles (Fig. 7A). However, the ultrastructural changes in EAE-treated cells included chromatin condensation, chromatin marginalization, organelle swelling, and cytoplasmic vacuolization. In addition, an abundance of autophagic vacuoles was observed in the cytoplasm and these autophagic vacuoles contained degraded organelles.
Effects of EAE on Cellular Invasion
The numbers of cellular invasions through the Matrigel-coated chamber are shown in Fig. 8. In the control group, SMMC-7721 cells displayed an invasive response to serum in the absence of EAE. After treatment with EAE at the concentrations of 100, 150, and 200 μg/mL for 48 hr, the cell invasion inhibition rates were 38.69%, 45.78%, and 53.37%, respectively (P < 0.05).
MMP-9 Expression in EAE-Treated SMMC-7721 Cells
As shown in Fig. 9, MMP-9 concentration in the supernatant of control cells was 45.2 ± 0.28 ng/mL. Following treatment with EAE at the concentrations of 100, 150, and 200 μg/mL for 48 hr, MMP-9 concentrations in the cell supernatants were 35.67 ± 2.19, 26.75 ± 2.90, and 20.38 ± 2.38 ng/mL, respectively (P < 0.05).
HPLC Analysis of EAE
We selected myricetin, quercetin and kaempferol among flavonoids as standards for qualitative analysis. The standard (reference) and sample peaks of myricetin, quercetin, and kaempferol are shown in Fig. 10A,B, respectively. From the reference chromatogram, the Peak 5 is identified as quercetin which is found to be the major component of EAE chromatogram.
In this study, our data show that EAE and CE could inhibit the proliferation of all five human cancer cell lines in a dose- and time-dependent manner at the concentration range of 50–200 μg/mL (Figs. 2, 3, and 4). In comparison with EAE and CE, less inhibitory effect was observed with PEE and NBE. The five tumor cell lines showed differential sensitivities to EAE and CE with SMMC-7721 cells being the most sensitive to EAE and CE treatment. The growth inhibitory effect of EAE and CE on SMMC-7721 cells was enhanced with the increase of drug concentrations. The highest growth inhibition rates following treatments with EAE and CE, each used at the concentration of 200 μg/mL for 72 hr, were 80.91% and 51.76%, respectively (Fig. 4). Of note, the antiproliferative effect of EAE was strongest among the four tested extracts at all observed time points. These results indicate that the active anticancer component(s) present in Euphorbia helioscopia L may be enriched in the EAE fraction.
The data of cell cycle show that EAE mainly arrested cells in G-1 phase in a dose- and time-dependent manner and reduced the percentage of cells in S-phase. Interestingly, the percentages of cells in G2/M-phase increased at 24 and 48 hr but decreased at 72 hr, as compared with controls. A remarkably high subdiploid peak was also observed in the treatment group (Fig. 5), suggesting that EAE-induced antiproliferation may be related to apoptosis. However, the sub-G1 percentages of 150 μg/mL treatments are smaller than those of 100 μg/mL treatments at 24 and 48 hr (Table 1). Moreover, the similar situation existed in early apoptosis whereby the proportion of early apoptotic cells treated with 150 μg/mL at 48 hr is smaller than that of the same concentration used at 24 hr (Fig. 6). This may be due to the reason that herbal extract is a complex mixture which is composed of many different pharmacologically active components. Interactions between different active ingredients may lead to such results.
The early apoptotic rate of SMMC-7721 cells treated with 200 μg/mL of EAE extract was higher than that of control, as well as the proportion of apoptotic cells (both early and late apoptosis) increased as the treatment time prolonged from 24 to 72 hr. The data suggest that apoptosis plays a crucial role in the antitumor mechanism of EAE from Euphorbia helioscopia L against SMMC-7721 cancer cells. Furthermore, we observed the typical morphological features of early apoptosis in SMMC-7721 cells treated with EAE (200 μg/mL) for 24 hr. Scanning electron microscopy also revealed the presence of autophagosomes in SMMC-7721 cells treated with 200 μg/mL of EAE and the autophagic vacuoles were filled with degraded organelles (Fig. 7D,E). These results suggest that autophagy might also be involved, in addition to apoptosis, as a mechanism of growth inhibition in cancer cells treated with EAE. On the basis of these observations, we suggest that EAE of Euphorbia helioscopia L may be a potent inducer of apoptosis in cancer cells.
Since the aggressiveness of malignant tumors relates to uncontrolled proliferation and transplantability, we also studied the effects of Euphorbia helioscopia L EAE on the invasion and metastasis of cancer cells. Using transwell chamber approach, we demonstrated that EAE negatively impacted invasiveness of SMMC-7721 cells in a dose-dependent manner. Notably, there have been reports suggesting that tumor invasion could be related to the matrix metalloproteinases (MMPs) expression (Overall and Kleifeld,2006; Duffy et al.,2008). Of these MMPs, MMP-9 is involved in degradation of extracellular matrix and plays an important role in cancer cell migration and invasion (Westermarck and Kahari,1999; Overall and Kleifeld,2006; Duffy et al.,2008). Compared with controls, MMP-9 levels in the supernatants of EAE-treated tumor cells were significantly decreased and the effect was demonstrated in a dose-dependent manner. These results indicated that EAE down-regulated the MMP-9 expression in cancerous cells.
Flavonoids, from medicinal herbs and dietary plants, contribute to induce apoptosis by arresting the cell cycle and inhibiting migration and proliferation of human tumor cell lines in vitro (Huang et al.,2010). Euphorbia helioscopia L mainly contains flavonoids and diterpenoids as biologically active substances (Yang et al.,2007). However, the main flavonoids that may be associated with antitumor activity of Euphorbia helioscopia L are still unclear. Ethyl acetate solvent has good extracting ability which extracts alkaloid, flavonoid, organic acid, and diterpenoid from natural herbs. Thus, the EAE of Euphorbia helioscopia L is a complex mixture comprising of different biologically active compounds. Compared with the standard peaks of three types of reference flavonoids, the similar peaks were also detected in the EAE at 360 nm wavelength, and the quercetin peak was identified as one of the major constituents of EAE (Fig. 10). Furthermore, quercetin, myricetin and kaempferol are naturally occurring flavonoids that have been shown to exert multiple pharmacological effects, including those of anticancer agents or supplementary anticancer agents (Kang and Liang,1997; Lee et al,2007; Calderón-Montaño et al.,2011). On the basis of these studies and the above-mentioned results, we speculate that flavonoids may play a major role in the anticancer activity of EAE of Euphorbia helioscopia L.
However, we are also aware of the caveats involved in this study. First, our data show that the antitumor activity of EAE of Euphorbia helioscopia L against SMMC-7721 cancer cells involves apoptosis; it is still unclear which apoptotic pathway plays a key role in antitumor response. Second, the data of morphological changes suggest that several compounds, rather than a single component, present in Euphorbia helioscopia L may induce apoptosis in SMMC-7721 cancer cells since it is as yet, unclear whether the main constituent plays a key role in the anticancer effect observed. Therefore, further studies are needed to evaluate the molecular mechanism of EAE from Euphorbia helioscopia L against various cancer cells as well as the isolation of its active components in order to clarify these issues.
In summary, this study demonstrated that EAE of Euphorbia helioscopia L had a significant antiproliferation potential against different types of cancer cells and mediated G1-phase arrest of cell cycle. Besides, apoptosis was the dominant mechanism involved in the antitumor activity of EAE. Importantly, it was further revealed that the anti-invasive effects of EAE on the human hepatocellular carcinoma cell line SMMC-7721 could be correlated with down-regulated expression of MMP-9 induced by EAE. These results suggest that the EAE of Euphorbia helioscopia L has an inhibitory potential against the human cancer. Although the HPLC analysis in this study provided the initial information on the active constituent(s) present in EAE of Euphorbia helioscopia L, further studies are required to establish a comprehensive screening of the various active constituents of this extract.
The authors thank the Central Laboratory of Lanzhou University Second Hospital for providing experimental and technical support and Dr. Wen-Guang Li for manuscript review.
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