Tetrastigma hemsleyanum (Sanyeqing) root tuber extracts induces apoptosis in human cervical carcinoma HeLa cells

Journal of Ethnopharmacology 165 (2015) 46–53

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Research Paper

Tetrastigma hemsleyanum (Sanyeqing) root tuber extracts induces apoptosis in human cervical carcinoma HeLa cells Yan Xiong a,b, Xuewen Wu b,n, Liqun Rao a,n a b

College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 411107, China College of Chemical Engineering, Xiangtan University, Xiangtan 411105, China

art ic l e i nf o

a b s t r a c t

Article history: Received 16 September 2014 Received in revised form 7 February 2015 Accepted 10 February 2015 Available online 18 February 2015

Aim of the study: Tetrastigma hemsleyanum (Sanyeqing) is traditionally used as a folk medicine for the treatment of cancer. However, the underlying mechanisms remain unclear. The purpose of this study was to investigate the possible mechanisms by which petroleum ether fraction (PEF) of Sanyeqing has antitumor activity on HeLa cells. Methods: The chemical components of PEF were analyzed by gas chromatography–mass spectrometry. The cytotoxicity of PEF on HeLa cells was measured by MTT assay. Apoptosis was evaluated by phosphatidylserine translocation, mitochondrial membrane potential (Δψm) changes and the activation of caspase-3, caspase-8 and caspase-9. The levels in T-SOD, CAT, GSH-PX and MDA were measured. Results: PEF of Sanyeqing inhibited the growth and induced apoptosis of HeLa cells in dose- and timedependent manner. PEF triggered intrinsic apoptotic pathway indicated by the loss of mitochondrial membrane potential and the activation of caspase-9 and caspase-3. In addition, PEF activated extrinsic apoptotic pathway indicated by the activation of caspase-8. Furthermore, PEF decreased T-SOD, CAT, GSH-PX activities and increased MDA level. Chemical analysis revealed the presence of fatty acids and phytosterol in PEF. Conclusions: PEF of Tetrastigma hemsleyanum Diels et. Gilg (Sanyeqing) exhibits cytotoxic effects, triggers both extrinsic and intrinsic apoptotic pathways, and augments oxidative stress in cervical carcinoma HeLa cells. Sanyeqing has strong potential to be developed as an agent for the treatment of cervical cancer. & 2015 Elsevier Ireland Ltd. All rights reserved.

Chemical compounds studied in this article: Oleic acid (PubChem CID: 445639) Linoleic acid (PubChem CID: 5280450) Hexadecanoic acid (PubChem CID: 985) β-Sitosterol (PubChem CID: 222284) Octadecanoic acid (PubChem CID: 5281). Keywords: Tetrastigma hemsleyanum Apoptosis HeLa cell GC–MS

1. Introduction Cervical cancer is one of the major causes of cancer death in women worldwide (Forouzanfar et al., 2011). The present treatment of cervical cancer possesses considerable side effects. Natural products or phytochemicals have gained an increasing interest due to their remarkable advantages, such as safety and lower drug resistance (Efferth et al., 2008). Great efforts have been made to develop effective anti-cancer drugs from plants. Over 60% anticancer drugs were derived from natural source (Cragg and Newman, 2005). Herbal medicines exhibit anticancer effects through inhibiting cell proliferation, inducing apoptosis, and modulating cell signaling pathway and oxidative stress-mediated mechanism (Hanahan and Weinberg, 2000; Li-Weber, 2013). Tetrastigma hemsleyanum Diels et. Gilg belongs to the grape family Vitaceae, known as “Sanyeqing” in China (Jiangsu New Medical

n

Corresponding authors. Tel./fax: þ86 731 58293549. E-mail addresses: [email protected] (X. Wu), [email protected] (L. Rao).

http://dx.doi.org/10.1016/j.jep.2015.02.030 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

College, 1977). Sanyeqing has been used in the treatment of tumors and several other diseases, such as high fever, infantile febrile convulsion, pneumonia, snake bite, and jaundice (Jiangsu New Medical College, 1977). Studies have examined the anticancer (Feng et al., 2014, 2006; Liu and Xia, 2010; Wang et al., 2012; Zhong et al., 2013), liver protection, antioxidant (Sun et al., 2013), anti-inflammatory, analgesic, antipyretic activities (Huang et al., 2005) and chemical components of the root tubers (Li et al., 2003; Liu et al., 2001; Liu and Yang, 1999). In addition, several studies investigated chemical components and biological activities of the leaves (Hossain et al., 2011; Shao et al., 2011; Sun et al., 2013). Petroleum ether fraction (PEF) is a major part that exhibits various biological activities, including antiinflammatory (Guo et al., 2011), anti-hypoxic activity (Ma et al., 2011), anti-nociceptive activity (Chen et al., 2008), anti-bacterial (Zhang et al., 2010) and anti-tumor (Cao et al., 2013) activities in Chinese folk medicine. Although Sanyeqing has long been used as a folk medicine in the treatment of tumors, the underlying mechanisms remain unclear. Therefore, in this study we aimed to investigate cytotoxic and apoptosis inducing activities and the possible

Y. Xiong et al. / Journal of Ethnopharmacology 165 (2015) 46–53

mechanisms of PEF of Sanyeqing, using HeLa cells as the experimental model.

2. Materials and methods 2.1. Plant materials Tetrastigama hemsleyanum Diels et. Gilg (Sanyeqing) root tuber was purchased (Yuanlin County, Hunan Province, China) and authenticated by Prof. Ribao Zhou (College of Pharmacy, Hunan University of Chinese Medicine, Hunan, China). A specimen was deposited at the Department of Pharmaceutical Engineering, College of Chemical Technology, Xiangtan University, Hunan. 2.2. Chemicals and reagents Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA 0.25%, streptomycin and penicillin were obtained from Hyclone (Logan, UT, USA). Dimethyl sulfoxide (DMSO), 3-(4,5dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) were purchased from Amresco (Cleveland, OH, USA). Annexin V-FITC/PI double staining detection kit, lactate dehydrogenase (LDH) cytotoxicity assay kit, mitochondrial membrane potential assay kit with JC-1, caspase-3, -8, -9 activity assay kit, propidium iodide (PI) were purchased from Beyotime (Beyotime Institute of Biotechnology, Shanghai, China). Catalase (CAT) assay kit, total superoxide dismutase (TSOD) assay kit, glutathione peroxidase (GSH-PX) assay kit and malondialdehyde (MDA) assay kit were purchased from Jiancheng (Jiancheng Biotechnology, Nanjing, China). All used chemicals and reagents were of analytical grade. 2.3. Preparation of extracts Sanyeqing was air dried for four days and dried in a hot air oven at 40 1C. The dried plant was ground to powder and refluxed in 80% ethanol for three times, each time for 2 h. Three extractions were combined, filtered and concentrated to dryness with a rotary evaporator under reduced pressure. The ethanol extract (yield: 5.21%) was suspended in water and sequentially partitioned with different solvents, including petroleum ether, chloroform, ethyl acetate and n-butanol. PEF was concentrated using a rotary evaporator, giving a yield of 8.87%. A stock solution of PEF was prepared in DMSO and stored at 4 1C, and diluted to the desired concentration with DMEM before each experiment. Maximum concentration of DMSO was maintained at 0.1% (v/v). 2.4. Gas chromatography–mass spectrometry (GC–MS) analysis GC–MS analysis was performed to analyze PEF composition with a Finnigan Voyager gas chromatograph fitted with a fused silica VF-5ms capillary column (29.5 m  0.25 mm; coating thickness 0.25 μm, VARIAN, USA) using the following temperature program. The initial temperature was 60 1C, raised to 315 1C at 6 1C/min and held for 3 min. The injector temperature was 230 1C and the MS source Helium was used as the carrier gas at a flow rate of 1.0 ml/min with a split ratio of 50:1 (v/v). The gas chromatograph was coupled to a Finnigan Voyager mass selective detector. The ionization source temperature was 250 1C. 2.5. Cell culture Human cervical carcinoma HeLa cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM containing 10% FBS, 100 μg/ml

47

streptomycin and 100 units/ml penicillin at 37 1C in a humidified atmosphere with 5% CO2. 2.6. Cytotoxicity assay Cytotoxicity effect of PEF on HeLa cells was determined by MTT assay as described previously with some modifications (Sreejith et al., 2012). HeLa cells were seeded in 96-well plates (at a density of 1  104 cells/well) and cultured overnight, next the cells were treated with different concentrations of PEF for 24, 48 and 72 h. After treatment, the cells were washed twice with phosphate buffered saline (PBS) and incubated with 20 μl MTT (5 mg/ml) and 80 μl DMEM for 4 h. After 4 h, MTT solution was removed and 100 μl DMSO was added to each well. The absorbance was measured at 570 nm by Microplate reader. The viability of HeLa cells was calculated comparing with the control group. The percentage of viability of HeLa cells was calculated according to the following equation: percentage of viability (%)¼(Mean OD of test sample Mean OD of the blank/Mean OD of the control Mean OD of the blank)  100 2.7. Lactate dehydrogenase (LDH) releases assay The release of lactate dehydrogenase was analyzed by LDH release assay kit. Briefly, 1  104 cells/well was seeded in 96-well plate and cultured overnight. Next, the cells were washed with PBS twice, and fresh culture media containing low concentrations of serum or no serum were added, and the cells were treated with PEF (5, 10, 20, 40, 80, 160 μg/ml) for 24 h. Then 120 μl supernatants were transferred to a new 96-well plate and the absorbance was immediately measured at 490 nm by Microplate reader. The percentage of LDH release was calculated according to the following equation: Percentage of LDH release (%)¼(Mean OD of test sample Mean OD of the control)/(Mean OD of the maximum enzyme activity Mean OD of the control)  100 2.8. Detection of apoptosis by flow cytometry (FCM) The Annexin V-FITC/PI staining kit was used to detect the phosphatidylserine translocation, an important characteristic at an early stage of cell apoptosis. Briefly, cells were seeded in 6 well plates at a density of 2  105 cells/ml and cultured overnight, and then the cells were treated with different concentrations of PEF for 24 h. The cells were collected and washed in PBS, then were resuspended in 195 μl binding buffer, and incubated with 10 μl Annexin V-FITC and 5 μl PI in the dark for 20 min. Thereafter, the solutions were immediately tested by FCM (Beckman, USA). 2.9. Cell cycle analysis Cells were seeded in 6-well plates at a density of 2  105 cells/ml and cultured overnight. The cells were treated with different concentrations of PEF for 24 h. Next the cells were washed with PBS twice and fixed with 2 ml 70% cold ethanol at 4 1C for 24 h. Then, the cells were stained with PI in the dark for 20 min and immediately analyzed by FCM. 2.10. Mitochondrial membrane potential (Δψm) loss The mitochondrial membrane potential loss was detected by using mitochondrial membrane potential assay kit. Briefly, cells were seeded in 6-well plates at a density of 2  105 cells/ml and cultured overnight, then treated with different concentrations of PEF for 24 h. The cells were washed with PBS twice then stained

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with JC-1 at 37 1C for 20 min. Then the cells were washed with JC-1 staining buffer twice and analyzed immediately by FCM.

2.11. Measurement of caspase-3, caspase-8 and caspase-9 activity Cells were seeded in 6 cm2 cell culture dishes at a density of 1  105 cells/ml and cultured overnight, then treated with different concentration of PEF for 24 h. Next, the cells were washed with cold PBS three times and lysed with 100 μl RIPA buffer at 4 1C for 30 min. The cell lysates were collected and centrifuged at 12,000  g at 4 1C for 10 min. Supernatants were transferred to precooled centrifuge tubes for immediate measurement of caspase -3, -8, -9 activities by using caspase-3, -8, -9 activity assay kits according to the manufacturer's protocols. The protein content was measured by BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China).

2.12. Catalase (CAT) assay Cell lysate was prepared as described in 2.11 and CAT activity was analyzed by using catalase assay kit. The absorbance of yellow production was measured at 405 nm by Microplate reader. The CAT activity unit was evaluated as decomposing (H2O2) per milligram protein per minute.

2.13. Total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) assay Cell lysate was prepared as described in 2.11. T-SOD activity in cell lysate was detected by using T-SOD assay kit according to the manufacturer's protocols. The absorbance of reaction product was measured at 550 nm by Microplate reader. One enzyme active unit was defined as the amount of SOD that achieves the reduction rate of nitrite by 50% in per milligram protein per milliliter. GSH-PX activity in cell lysate was detected by using Glutathione Peroxidase assay kit. The absorbance at 412 nm was measured by Microplate reader. One enzyme active unit was defined as reduction of 1 μmol/L of GSH per milligram protein. 2.14. Malondialdehyde (MDA) assay MDA content was detected by using MDA assay kit according to the manufacturer's protocols. The absorbance at 532 nm was measured by Microplate reader.

2.15. Statistical analysis All experiments were performed in triplicate and all data were expressed as mean7SD. The difference between two groups (treated group and untreated group) was analyzed by using two tailed Student's t-test. The difference among three or more groups was analyzed by using one-way ANOVA. Po0.05 was considered to be statistically significant.

3. Results 3.1. Chemical analysis of PEF composition by GC–MS PEF composition was analyzed by GC–MS and the different components of PEF were identified by comparison of their retention times (RIs) with the literature and their mass spectrum with the data library (Fig. 1). As shown in Table 1, total 30 components were identified, representing 93.33% of the total compounds. The major constituents were hexadecanoic acid (15.67%), linoleic acid (16.54%), oleic acid (17.72%), and β-Sitosterol (10.29%).

3.2. PEF exhibits cytotoxicity on HeLa cells MTT assay was conducted to investigate the cytotoxicity of PEF on HeLa cells. HeLa cells were treated with different concentrations of PEF (0, 10, 20, 40, 80 and 160 μg/ml) for 24, 48 and 72 h. As shown in Fig. 2, a dose- and time-dependent growth inhibition of HeLa cells was observed. The cell viability rate after 24, 48, 72 h exposure to PEF significantly decreased from 88.8972.28% (Po0.05), 80.9471.23% (Po0.05) to 66.7872.13% (Po0.05) even at the lowest concentration (10 μg/ml).

3.3. PEF promotes LDH release in HeLa cells LDH release is an important index for evaluating cell membrane integrity. LDH is a stable enzyme and is released into the cellculture media from the cytoplasm upon cell apoptosis or necrosis. When HeLa cells were treated for 24 h with different concentrations (0–160 μg/ml) of PEF, a dose-dependent LDH release was observed at the range of 5–160 μg/ml (Fig. 3). Compared to untreated cells, LDH release was significantly increased at dose of 5 μg/ml and 10 μg/ml (Po0.05), and even more significantly at the dose from 20 to 160 μg/ml (Po0.01).

Fig. 1. GC–MS analysis of PEF composition with a Finnigan Voyager gas chromatograph fitted with a fused silica VF-5ms capillary column (29.5 m  0.25 mm; coating thickness 0.25 μm, VARIAN, USA).

Y. Xiong et al. / Journal of Ethnopharmacology 165 (2015) 46–53

3.4. PEF induces apoptosis of HeLa cells To understand whether the cytotoxicity of PEF on HeLa cells is related to the induction of apoptosis, apoptotic cells were detected by Annexin-V/PI double staining and FCM. After treatment with PEF (10, 20 and 40 μg/ml) for 24 h, the percentage of early apoptosis cells increased from 0.73% (untreated cells) to 4.32%, 18.69% and 33.34%, respectively (Fig. 4). Thus PEF induced apoptosis of HeLa cells in a dose-dependent manner. 3.5. PEF modulates cell cycle of HeLa cells To investigate whether PEF induces cell cycle arrest, cell-cycle distribution analysis was performed by FCM. There was a remarkable decrease in the number of cells in S phase after 24 h treatment of PEF (Po0.01), accompanied by an increase in both G0/G1 (Po0.05) and G1/G2 (Po0.01) phase (Fig. 5 and Table 2). These results indicated that PEF induces cell cycle arrest both in G0/G1 and G1/G2 phase. 3.6. PEF induces the depletion of mitochondrial membrane potential in HeLa cells Cell apoptosis is associated with the depletion of mitochondrial membrane potential (Mignotte and Vayssiere, 1998). HeLa cells were treated with PEF (10, 20 and 40 μg/ml) for 24 h and the changes in Δψm were determined by FCM. As shown in Fig. 6, PEF treatment caused the disruption of Δψm in a dose-dependent manner.

49

caspase activation. Caspase family is the most prominent protease family in apoptosis (Budihardjo et al., 1999), and are divided into two functional groups, the apoptosis initiators (caspase-8, -9, -10) and the apoptosis executors (caspase-3, -6, -7) (Nicholson, 1999). To explore the mechanism underlying PEF induced apoptosis, we detected the activities of caspase-3, -8 and -9. As shown in Fig. 7, caspase-3, -8 and -9 activities of HeLa cells were significantly increased by treatment with 80 μg/ml (Po0.05) and 160 μg/ml (Po0.05) of PEF for 24 h, while the lowest dose of 40 μg/ml did not have any significant effects on caspase-3, -8 and -9 activities. These results suggested that PEF could activate the caspase and induce the apoptosis of HeLa cells. 3.8. PEF augments oxidative stress in HeLa cells Cell apoptosis is associated with oxidative stress triggered by reactive oxygen species (ROS) (Simon et al., 2000). Several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) protect the cells against the effects of ROS (Kachadourian and Day, 2006; Wood et al., 2001). Malondialdehyde (MDA) is an important index of cellular oxidative stress. Therefore, we evaluated the effects of PEF on T-SOD, CAT, GSH-PX, and MDA in HeLa cells. Compared to the control group, we observed decreased CAT, GSH-PX and T-SOD activities and increased MDA level in HeLa cells treated by PEF (Table 3). These data suggested that the promotion of oxidative stress seems to be one of the mechanisms by which PEF induces the apoptosis of HeLa cells.

3.7. PEF activates caspase-3, caspase-8 and caspase-9

4. Discussion

Apoptosis is executed via two major pathways (intrinsic and extrinsic pathways) (Fulda and Debatin, 2006). Both pathways lead to

Herbal medicine plays a significant role in the prevention and treatment of cancer. In this study, we investigated the anticancer

Table 1 Chemical constituents of PEF in Sanyeqing analyzed by GC–MS. Peak Rt (min)

Molecular formula

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

C3H6O2 C3H8O3 C4H6O4 C16H32 C5H8O4 C14H22O C5H10O6 C6H12O5 C6H10O7 C6H12O6 C6H12O6 C16H22O4 C17H34O2 C16H32O2 C18H32O2 C20H38O2 C18H32O2 C18H34O2 C18H36O2 C16H22O4 C19H38O4 C22H42O2 C24H48O2 C10H15N5O2 C28H48O C29H48O C29H50O C29H48 C30H48O C32H52O2

7.16 12.01 12.97 16.69 17.07 17.67 21.68 22.22 22.67 23.98 24.03 25.38 25.93 26.74 28.52 28.61 29.25 29.34 29.68 33.69 34.25 36.27 37.08 40.60 41.84 42.08 42.74 43.23 44.01 44.53

Molecular weight 74.08 92.09 118.09 224.43 132.11 206.32 166.13 164.07 194.13 180.16 180.16 278.34 270.45 256.42 280.44 310.51 280.45 282.46 278.34 330.50 338.57 368,64 237.25 400.68 412.69 414.71 396.69 424.70 468.39

Compound

Area (%)

Propanoic acid Glycerol Succinic acid n-Hexadecane D-erythro-Pentonicacid, 2-deoxy-, g-lactone 2,6-di-tert-butylphenol Arabinonic acid 2-Deoxy-galactopyranose 2-Keto-d-gluconic acid Inositol Glucopyranose Dibutyl phthalate Hexadecanoic acid, ethyl ester Hexadecanoic acid Ethyl 9,12-hexadecadienoate Ethyl Oleate Linoleic acid Oleic acid Octadecanoic acid 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester 1-Monopalmitoylglycerol cis-13-Docosenoic acid Docosanoic acid, ethyl ester 8-Dimethylamino-1,3,7-trimethyl-3,7-dihydropurine-2,6-dione Campesterol Stigmasterol β-Sitosterol Stigmastan-3,5-diene 4,4,6a,6b,8a,11,11,14b-Octamethyl-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-octadecahydro-2H-picen-3-one Acetic acid, 17-(1,5-dimethylhex-4-enyl)-4,4,8,10,14-pentamethyl-2,3,4,5,6,7,8,9,10,11,12,14,15,16-tetradecahydro1H-cyclopenta [a]phenanthrene

0.47 4.90 0.39 0.25 0.26 0.25 0.36 0.33 4.54 0.29 0.39 0.51 1.64 15.67 2.74 1.44 16.54 17.72 5.38 1.27 0.77 2.09 0.71 0.75 1.58 0.90 10.29 0.30 0.30 0.30

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activity of PEF from Sanyeqing root tuber. We demonstrated that PEF inhibited the growth of HeLa cells in dose-dependent and time-dependent manner. Furthermore, our data revealed that PEF

140 Control 48 h

Cell viability (%)

120 100

*

*

80

24 h 72 h

* ** **

**

** ** **

** ** **

**

60

**

40 **

20 0 10

20 40 80 Concentration (μg/ml)

160

Fig. 2. Effect of PEF on the viability of HeLa cells. HeLa cells were treated with different concentrations of PEF (10, 20, 40, 80, and 160 μg/ml) for 24, 48, and 72 h. Untreated cells were used as control. The data represent the mean 7 SD of three replicates and three independent experiments. n significant difference from the control (Po 0.05), nn very significant difference from control (Po 0.01).

80

Control PEF

**

60

% LDH release

** **

40 ** *

20 *

0 5

10

20 40 80 Concentration (µg/ml)

160

Fig. 3. PEF promotes LDH release in HeLa cells. HeLa cells were treated with different concentrations of PEF (5, 10, 20, 40, 80, and 160 μg/ml) for 24 h. Untreated cells were used as control. Data represent the mean 7 SD of three replicates and three independent experiments. n significant difference from the control (Po 0.05), nn very significant difference from control (Po 0.01).

induced an arrest in G0/G1 and S phase in HeLa cells. The arrest of S phase may be associated with DNA damage. Extrinsic and intrinsic pathways are well-known to be involved in apoptosis (Fulda and Debatin, 2006). Extrinsic pathway, also known as death-receptors mediated apoptotic pathway, is mediated by “death receptors” of Tumor Necrosis Factor (TNF) superfamily. “Death receptors” activate the recruitment and activation of initiator caspase-8. Activated caspase-8 can cleave and activate downstream executioner caspase-3, eventually resulting in the induction of morphological and biochemical changes associated with apoptosis (Nicholson, 1999). In this study, we found increased level of activated caspase-8 in PEF treated HeLa cells, suggesting that PEF induces apoptosis of HeLa cells via the death-receptors mediated apoptosis pathway. Intrinsic pathway is also referred as mitochondrial-mediated apoptosis pathway, which involves the loss of mitochondrial membrane potential (ΔΨm) due to the outer mitochondrial membrane permeability, and subsequent release of cytochrome C. Cytochrome C induces Apaf-1 oligomerization, leading to the activation of initiator caspase-9, eventually activates the executioner caspase-3 and initiates apoptotic cell death (Nicholson, 1999). In the present study, increased level of activated caspase-9 was observed in HeLa cells treated with PEF, indicating that intrinsic pathway is involved in the apoptosis of HeLa cells. Collectively, both extrinsic and intrinsic apoptotic pathways seem to be involved in the induction of apoptosis by PEF. Reactive oxygen species mainly include superoxide anion (O2  ), hydrogen peroxide (H2O2) and hydroxyl radicals (HO). Both hydrogen peroxide and superoxide radical may be involved in the induction of apoptosis (Gorman et al., 1997). H2O2 has been reported to induce apoptosis by disruption of mitochondrial membrane potential, release of cytochrome C, and subsequent activation of the caspase-3, eventually resulting in activation of the intrinsic pathway (Chen et al., 2008; Gorman et al., 1997). The intrinsic apoptotic pathway is especially susceptible to ROS (Chen et al., 2008). Here, we provide evidence that the increase in HeLa cell sensitivity to the PEF treatment may be due to the increase production of H2O2 and O2  . Several studies have demonstrated that phytosterol possesses anticancer activity (Awad et al., 2001; Woyengo et al., 2009). β-Sitosterol, one of the major constituent in phytosetrol, is a potential candidate for cancer therapy. β-Sitosterol exhibits anticancer properties through antiproliferation, induction of apoptosis, cell cycle arrest and modulation of antioxidant enzyme (Moon et al., 2008; Vivancos and Moreno, 2005; Zhao et al., 2009). Here, our results showed that PEF contained a high content of β-Sitosterol (10.29%) and thus may have potential in treating tumors. Fatty acids including oleic acid, linoleic acids and stearic acid have shown activity in cancer prevention and treatment (Das, 1989; Zhu et al., 1989). Coix seed, a wellknown Chinese herbal medicine, exhibited antitumor activities in vivo, which can be attributed to fatty acids including palmitic,

Fig. 4. Effect of PEF on apoptosis of HeLa cells. HeLa cells were treated with different concentration of PEF (10, 20 and 40 μg/ml) for 24 h. Untreated cells were used as control. Four quadrants represent necrotic cells (F1); late apoptotic cells (F2); live cells or unstained cells (F3); early apoptotic cells (F4), respectively.

Y. Xiong et al. / Journal of Ethnopharmacology 165 (2015) 46–53

P e r c e n ta g e o f c e lls in c e ll p h a s e

80

60

51

Control 10 μg/ml 20 μ g/ml 40 μg/ml

* * *

40

*

** ** **

20

**

** **

0 G0/G1(%)

S(%)

G2/M(%)

Fig. 5. Effect of PEF on cell cycle of HeLa cells. (a) Control group (untreated cells); (b)10 μg/ml of PEF;(c) 20 μg/ml of PEF; (d) 40 μg/ml of PEF; (e) quantitative analysis of the cells at different phases. Data represent the mean 7SD of three replicates and three independent experiments. n significant difference from the control (Po 0.05), nn very significant difference from control (Po 0.01).

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Table 2 Distribution of cell cycle phases of HeLa cells (means 7SD, n ¼3). Groups Concentrations (μg/ml) G0/G1 (%)

G2/M (%)

52.05 70.41 39.29 7 0.48 9.79 7 0.70 52.57 70.83 31.137 1.25n 16.93 7 0.46nn 47.20 70.96n 29.167 0.34nn 24.957 0.55nn 54.60 70.23n 12.707 0.41n 34.247 1.54nn

Control PEF 10 20 40 n

S (%)

Po 0.05. Po 0.01 vs. Control.

nn

5. Conclusion

Lost Δψm percentage (%)

50

This is the first study that demonstrated anti-cancer properties of PEF of Sanyeqing root tuber on HeLa cells. PEF exhibits multiple anticancer effects, including cell growth inhibition, cell cycle arrest, loss of mitochondrial membrane potential, activation of caspase-3, -8, -9 and oxidative stress-mediated apoptosis. Therefore, Sanyeqing appears to be a valuable natural source for the development of agents for the treatment of cervical cancer.

*

40 *

30 20 *

Acknowledgments

10 0

The authors would like to acknowledge financial support from Xiangtan University (kz08041).

Control

20 10 Concentration (μg/ml)

40 References

Fig. 6. PEF induced the depletion of Δψm in HeLa cells. HeLa cells treated with different concentrations of PEF (10, 20 and 40 μg/ml) for 24 h and Δψm were analyzed by flow cytometry. The number in each dot plot represented the percentage of cells that lose Δψm. np o 0.05 vs. control group.

Caspase Activity (Fold of Control)

5 4

Control 40μg/ml 80μg/ml 160μg/ml

*

3 *

2 *

*

*

*

1 0 PEF Caspase-3

PEF Caspase-8

PEF Caspase-9

Fig. 7. Dose dependent activation of caspase-3, -8 and -9 by PEF. HeLa cells were treated with different concentrations of PEF (40, 80 and 160 μg/ml) for 24 h and the activities of caspase-3, -8 and -9 were analyzed. The data represent the mean 7 SD of three replicates independent experiments. np o 0.05 vs. control group.

Table 3 Effect of PEF in Sanyeqing on CAT, GSH-PX, T-SOD and MDA of HeLa cells. Concentrations (μg/ml)

CAT (U/ mgprot)

40 80 160 Control

34.647 2.04n 106.27 70.21 24.257 0.94nn 90.50 70.08nn 3.23 7 1.71nn 72.54 70.12nn 43.34 7 0.97 109.94 70.23

n

Po 0.05. Po 0.01 vs. Control.

nn

stearic, oleic, and linoleic acids (Numata et al., 1994). Furthermore, synergistic effects occur when anticancer drugs are used together with fatty acids. The use of fatty acids enhanced paclitaxel cytotoxicity on tumor cells due to the mediation of tumor cell chemo-sensitivity in paclitaxel-based therapy (Menendez et al., 2001). In the present study, the synergistic effect of phytosterol compounds and fatty acids may contribute to the cytotoxicity of PEF on HeLa cells.

GSH-PX (U/ mgprot)

T-SOD (U/ mgprot)

MDA (nmol/ ml)

6.48 70.14nn 1.137 0.15nn 6.37 70.11nn 1.52 7 0.09nn 6.14 70.04nn 2.177 0.10nn 7.39 70.08 0.747 0.15

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