M phase arrest and mitochondrial apoptosis pathway

Journal of Functional Foods 25 (2016) 523–536

Available online at www.sciencedirect.com

ScienceDirect j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff

Inhibition of allicin in Eca109 and EC9706 cells via G2/M phase arrest and mitochondrial apoptosis pathway Fenrong Chen a, Hong Li a, Yan Wang a, Meili Gao b, Yan Cheng a, Dong Liu a, Miao Jia a, Jun Zhang a,* a

Department of Gastroenterology, Second Affiliated Hospital of Xi’an Jiaotong University, The Key Laboratory of Gastrointestinal Motility Disorders of Shaanxi Province, Xi’an, Shaanxi Province 710004, China b Department of Biological Science and Engineering, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, Shaanxi Province 710049, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

The effects and underlying molecular mechanisms of allicin on oesophageal squamous cell

Received 5 December 2015

carcinoma (ESCC) are unclear. Herein, we investigated the effect and action mechanism of

Received in revised form 25 June

allicin in the ESCC cells of Eca-109 and EC9706. The cell viability and invasion ability of the

2016

two ESCC cells in allicin group were significantly decreased after allicin treatment. Allicin

Accepted 29 June 2016

reduced the percentage of G0/G1 and S phase and increased the population of G2/M phase

Available online 12 July 2016

in a dose-dependent manner. The expression levels were significantly increased for p53, p21, CHK1, and decreased for cyclinB after allicin treatment. The apoptotic rate was signifi-

Keywords:

cantly increased and accompanied by Cyt c release from the mitochondria to the cytosol,

Allicin

as well as Bax, caspase-3 and cleaved caspase-9 which were significantly increased. Fur-

Oesophageal squamous cell

thermore, suppression of tumour growth was evident in vivo. These results suggest that allicin

carcinoma

can serve as a novel and effective anti-oesophageal cancer agent for the treatment of ESCC.

Cell cycle

© 2016 Elsevier Ltd. All rights reserved.

Apoptosis

1.

Introduction

Oesophageal cancer (OC) is the sixth most common cause of cancer-related mortality worldwide, and is the third most common tumour in the digestive tracts, with an estimated 456,000 new cases per year. Oesophageal squamous cell carcinoma (ESCC) and oesophageal adenocarcinoma (EAC) are the two main histological subtypes (Du et al., 2013; Peng et al., 2015; Qia, Chaoa, & Chiu, 2012). In particular, ESCC accounts for 80% of cases of oesophageal cancer globes. Despite advances in

therapeutics such as endoscopic resection (ER), surgery, radiotherapy and chemotherapy, the overall 5-year survival rate of patients with ESCC is only 15–25% (Ohashi et al., 2015). This may be due to the fact that the majority of ESCC patients have advanced metastatic disease at initial diagnosis and curative resection would be inappropriate (Qia et al., 2012). Thus, development of a novel compound for its disease prevention effects on oesophageal cancer is worth investigating. Human consumption of foods containing phytochemicals is associated with a reduced risk of several different types of cancer (Braithwaite et al., 2014). Further, dietary organsulphides

* Corresponding author. Department of Gastroenterology, Second Affiliated Hospital of Xi’an Jiaotong University, The Key Laboratory of Gastrointestinal Motility Disorders of Shaanxi Province, Xi’an, Shaanxi Province 710004, China. Fax: +86 29 87679660. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.jff.2016.06.027 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

524

Journal of Functional Foods 25 (2016) 523–536

have attracted much attention for their health-related potential and chemoprevention of cancer (Chu, Ho, Chung, Rajasekaran, & Sheen, 2012). Garlic has been widely used as a food and flavouring agent, and is also a therapeutic remedy used for centuries around the world (Chu et al., 2013; Kubec et al., 2010). Allicin (2-propene-1-sulphinothioic acid S-2propenyl ester), as a garlic-derived sulphur compound, is one of the most bioactive compounds. It is formed from alliin via the enzyme of alliinase when the garlic is chopped or crushed (Ali, Thomson, & Afzal, 2000; Hirsch et al., 2000; Wang & Huang, 2015). Recent studies have indicated that allicin has a multiline of health beneficial effects, including antimicrobial, antifungal and anti-parasitic, anti-hypertensive, cardioprotective, anti-inflammatory and anti-cancer activities (Louis, Murphy, Thandapilly, Yu, & Netticadan, 2012; Wang, Liu, Cao, & Li, 2012; Zhang et al., 2010; Zhang, Zhu, Duan, Feng, & He, 2015). Among these effects, the reduction of the risk of certain cancers and cardiovascular diseases are two of the most important healthrelated properties for consumers (Wilde, Keppler, Palani, & Schwarz, 2016). Thus, allicin is an interesting functional ingredient. As for the effect of anti-cancer, garlic or allicin has been demonstrated in a range of cancers such as gastric cancer, colon cancer, liver cancer, lung cancer, breast cancer, cervical cancer, and lymph cancer (Chu et al., 2013; Xu et al., 2014). Based on these experiments, allicin is expected to be a potential anticancer or cancer-preventive agent of garlic. However, the effects and the underlying molecular mechanisms of this functional ingredient on ESCC are unclear. In this study, we examined the cell viability, cell cycle, cell apoptosis and related regulatory genes expression in ESCC cells of Eca109 and EC9706 following treatment with different doses of allicin. Further, Eca109 cell was injected subcutaneously in ICR mice, and the effect of allicin on Eca109 xenograft growth was assayed. These findings can potentially provide newer insights of allicin as a functional component of garlic or a potential therapeutic agent for the treatment of EC.

2.

Materials and methods

2.1.

Reagents and antibodies

Caspase-3, caspase-9, Bax, cyt C antibody were purchased from Cell Signaling Technology (Shanghai, China). CHK1, p53, p21, cyclinB were purchased from Abcam (Cambridge, England). Antiβ-actin was purchased from Bioworld, Minneapolis, MN, USA. Annexin V- FITC/PI and TUNEL assay kit was bought from Roche, New York, NY, USA. The 24-well plate assay for migration was purchased from BD Biosciences, New York, NY, USA. All cell culture agents and other assay reagents were obtained from Gibco (Shanghai, China).

2.2.

Cell lines and culture

The human oesophageal cancer cell lines Eca109 and EC9706 were maintained in RPMI 1640 medium, which was supplemented with 10% heated inactivated foetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL). Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C.

2.3.

Cell viability assay

Cell viability was analysed using an MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] assay. Eca109 and EC9706 cells were plated in 96-well plates at 5 × 102 cells per well and indicated with various concentrations (0, 2.5, 5, 10, 20, 40, 80, 100 µg/mL) of allicin for 48h. In short, cells were washed after treatment with various conditions of allicin and 0.5 mg MTT/ mL in RPMI-1640 medium was added, and then cells were dissolved in dimethyl sulphoxide (DMSO) after 2 h at 37 °C. The absorbance at 550 nm was read and the results were expressed as percentage of cell viability compared with the controls.

2.4. cycle

Flow cytometry detection of effect of allicin on cell

Eca109 and EC9706 cells were collected and seeded in culture flasks at 5 × 104 ml/L, after culturing for 48 h; different concentrations of allicin were added, after culturing for another 48 h, and then culture medium was discarded, followed by digestion, centrifugation (800 g) for 8 min, to collect cells. The cells were washed with phosphate buffered saline (PBS) and fixed in 75% of ethanol at 20 °C overnight. After washing twice with cold PBS, cells were resuspended in PBS containing 50 mg/ mL PI and 100 mg/mL RNase A (Sigma, New York, NY, USA) at 37 °C for 30 min. Cells were then subjected to FACS Calibur flow cytometer (BD Biosciences). The data collection and analysis of the cell cycle distribution were analysed using cell Quest software. The experiment was repeated three times (Tao, Gao, Pan, & Wang, 2014).

2.5.

Annexin V-FITC/PI double staining assay

To analyse allicin induced apoptosis of Eca109 and EC9706 cells, we seeded cells at a concentration of 1 × 105 cells/well in a 12well plate. The Eca109 and EC9706 cells were treated with various concentrations (0, 2.5, 5, 10, 20, 40 µg/mL) of allicin for 48 h and then washed twice with PBS. Subsequently, the cells were resuspended in 1 × binding buffer with 1 × 106 cells/mL and transferred 100 µL of 1 × Annexin V binding buffer with 1 × 105 cells/mL to a 5 mL fluorescence activated cell sorting (FACS) tube. The cells were stained with Annexin V-FITC (5 µL) and PI (5 µL) for 15–30 min. Analysis was performed using a flow cytometer (BD Biosciences) within 1 h (Li et al., 2007).

2.6.

Transwell migration assay

Cancer cell migration was measured by Transwell assay with Millicell-24 Cell Culture Insert Plate (BD BioCoat Control Inserts from BD Biosciences). Eca109 and EC9706 cells were grown to 80% confluence and then serum starved overnight before the experiment was carried out. Briefly, cells were harvested and resuspended in starvation medium, plated at a density of 5 × 104 per well in 0.3 mL in the upper well. This upper well was immersed in a lower well containing complete medium which was added to various concentrations of allicin or 10% foetal calf serum (FBS). After incubation for 48 h at 37 °C with 5% CO2, the experiment was stopped. The membranes of the Transwell chambers were fixed with methanol for 15 min, stained with

Journal of Functional Foods 25 (2016) 523–536

Giemsa stain solution for 20 min, and dried at room temperature. Cell migration was examined by counting 10 randomly selected fields under microscope (Lee, Lee, Kim, Rhee, & Pyo, 2015; Liang et al., 2011).

2.7.

Western blot assay

Proteins were extracted with lysis buffer from Eca109 and EC9706 cells. A bicinchoninic acid (BCA) prorein assay was conducted to quantify the amount of protein. The proteins were then separated by SDS–PAGE and electrophoretically transferred onto nitrocellulose membranes. The membranes were incubated with a primary antibody overnight at 4 °C. After the membranes were washed with Tris-Buffered Saline and Tween (TBST), they were incubated with a rabbit radish peroxidasecoupled secondary antibody. The protein bands were visualized using a LumiGLO chemiluminescent substrate system (KPL, Guildford, UK). Band intensity was quantified by BandScan software (Glyko, Novato, CA, USA) (Li et al., 2010).

2.8.

In vivo assay

Male BALB/c mice (18 ± 2 g) were purchased from the Experimental Animal Center of Xi’an Jiaotong University (Shaanxi Province, China). The Eca109 cells were harvested, washed with PBS, and resuspended in PBS. Then mice were injected subcutaneously (s.c.) into each posterior hind flank region with approximately 6.0 × 106 cells, one site per mouse. When the average tumour diameter reached 5–7 mm, the mice were randomly divided into four groups of 15 mice each. The four groups were administrated orally by gavage with allicin at the doses of saline (0 mg/kg), 5, 10, 20 mg/kg.bw, respectively. The intragastrical administrations were conducted every 3 days for 28 days. At the end of the experiment, mice were sacrificed, blood was sampled, and the tumours were removed immediately, measured, and weighed. The volume (V) of each tumour was calculated as follows: V = Wid2 × Len/2 (Jiang, Huang, Wang, Yu, & Zhang, 2013). A TUNEL stain was conducted to determine the number of apoptotic tumour nuclei. Experiments on animals were performed according to the animal ethics guidelines of the institutional animal ethics committee.

2.9.

Statistical analysis

All experiments were performed at least three times. The significance of the differences between the two groups was determined using Student’s t test. All analyses were carried out using the SPSS 16.0 software package (SPSS, Chicago, IL, USA). All quantitative data were reported as “mean ± SD” and differences were considered significant at P values less than 0.05.

3.

Results

3.1. Allicin reduced cell viability and inhibited the migration of Eca109 and EC9706 cells The cell viability of allicin on the ESCC cells of Eca109 and EC9706 through MTT assay was first measured. Both cell lines were in-

525

cubated with various concentrations of allicin (0, 2.5, 5, 10, 20, 40, 80, 100 µg/mL) for 48 h. Cell survival was more than 90% in Eca109 and 80% in EC9706 cells, and no significant changes were observed when exposed to 20 µg/mL or lower concentrations of allicin (Fig. 1A). Cell viability in allicin-treated cells exhibited significant decreases (p < 0.01, p < 0.001, respectively) and indicated a dose-dependent manner at the concentrations of 40–100 µg/ mL for both cell lines (Fig. 1). Especially for EC9706 cells, the cell viability was below 40% at the concentration of 40 µg/mL. Thus, for the following experiment, we chose 40 µg/mL to be the maximum concentration group. To further confirm the effect of allicin on the migration of Eca109 and EC9706 cells, transwell migration assay was performed. Cells were treated with allicin at concentrations of 0, 2.5, 5, 10, 20, 40 µg/mL for 48 h. The representative photographs are shown in Figs 1B and 1C. The results showed that the migratory cells for Eca109 and EC9706 were decreased with increased concentration of allicin. Significant differences (p < 0.05, p < 0.01, p < 0.001, respectively) were observed in Eca109 and EC9706 cells as compared to the control group. No significant differences were observed between the two cell lines. Transwell assays indicated that allicin can suppress the migration of Eca109 and EC9706 cells in vitro.

3.2. Allicin induced G2/M phase arrest and disturbed the regulatory genes of G2/M in Eca109 and EC9706 cells In our study, we performed DNA cell cycle analysis using flow cytometry to assess the effect of allicin treatment on the distribution of Eca109 and EC9706 cells. As shown in Fig. 2, significant changes (p < 0.05, p < 0.01, p < 0.001, respectively) in cell cycle were noted in the two types of ESSC cells. As compared with the control group, allicin reduced the percentage of G0/G1 and S phase and increased the population of G2/M phase in a dose-dependent manner, which indicated that allicin mainly arrests cell cycle in G2/M phase, thus retarding cell growth. Based on the percentage distribution in cell cycle, we further assayed the protein expression of the associated regulatory genes of G2/M phase with Western blot. The p53, p21, cyclinB and CHK1 were analysed as shown in Fig. 3. The relative expression levels were significantly increased for p53, p21 and CHK1 (p < 0.05, p < 0.01, p < 0.001, respectively), except that in Eca109 cell at 2.5 µg/mL for p53, in EC9706 cell at 2.5 µg/mL for the three examined genes, at 5 µg/mL for p21 and CHK1, when compared with the corresponding controls. In contrast, allicin decreased the relative expression levels of cyclinB and showed significant differences at 20 and 40 µg/mL of allicin treatment. Also, no significant differences were observed between the two cell lines.

3.3. Allicin induced cell apoptosis of Eca109 and EC9706 cells In order to examine the effect of allicin on the cell apoptosis, the annexin V/propidium iodide (PI) double-staining assay was used to stain Eca109 and EC9706 cells, and the apoptosis rate was analysed by flow cytometry. As shown in Fig. 4, the basal early apoptotic rate of Eca109 was about 1.1% (Fig. 4a-A) and that of EC9706 cell was 0.4% (Fig. 4b-A). The representative histograms showed that the early apoptosis rates of Eca109 cells were

526

Journal of Functional Foods 25 (2016) 523–536

Fig. 1 – Effect of allicin on the cell viability of Eca109 and EC9706 (A) and on the migration of Eca109 and EC9706 cells (B and C). (A) The cell viability of Eca109 and Ec9706 cells as measured by the MTT assay. Cells were incubated with various concentrations of allicin for 48 h. Data are the averages of three independent experiments, each containing three replicates. (B) The representative photographs of migration of Ec109 and EC9706 cells at concentrations of allicin (0–40 µg/mL) for 48 h. (C) The statistic results of migration, significant differences were compared with the control groups. The results shown are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

5.6, 10.5, 18.2, 28.6, 39.4% (Fig. 4a-B-F) when they were treated with allicin at concentrations of 2.5, 5, 10, 20, 40 µg/mL, respectively, and that of EC9706 cells were 5.9, 9.9, 15.6, 24.9, 42.8% (Fig. 4b-B-F). The end stages of apoptotic rates of Eca109 were 0.3, 2.2, 7.3, 12.6, 18.7, 31.1% (Fig. 4a) when they were treated with

allicin at concentrations of 0, 2.5, 5, 10, 20, 40 µg/mL respectively, and that of EC9706 cells were 0.6, 3.2, 6.2, 10.7, 16.4, 32.5% (Fig. 4b). As shown in Fig. 4c, allicin treatment induced a significant apoptosis (p < 0.05, p < 0.01, p < 0.001, respectively) in Eca109 and EC9706 cells in a dose-dependent manner.

Journal of Functional Foods 25 (2016) 523–536

527

Fig. 2 – Effect of allicin on cell cycle of Eca109 (a and b) and EC9706 (c-d). (a) A–F indicated the representative histograms of allicin various concentrations (0, 2.5, 5, 10, 20, 40 µg/mL, respectively) for 48 h. (b) The percent of cells in G0/G1, S, and G2/M phases of the cell cycle are shown as mean ± SD (n = 3). (c) A–F indicated the representative histograms of allicin various concentrations (0, 2.5, 5, 10, 20, 40 µg/mL, respectively) for 48 h. (d) The percent of cells in G0/G1, S, and G2/M phases of the cell cycle are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

To further evaluate the mechanisms underlying allicin induced apoptosis in Eca109 and EC9706 cells, the changes in protein expression of the apoptosis-related genes Cyt c, Bax, caspase-3 and caspase-9 were examined by Western blot analysis using antibodies against the respective proteins. As shown in Fig. 5, for Cyt c and Bax, we assayed the changes in cytosolic and mitochondria. In cytosolic protein expression of Eca-109 and EC9706 cells, significant decreases were observed in Bax at the concentration of 10–40 µg/mL (p < 0.05) allicin and significant increases were observed in Cyt c at the concentration of 5–40 µg/mL (p < 0.05, p < 0.01, p < 0.001, respectively) allicin administration. In contrast, in mitochondria protein expression of Eca109 and EC9706 cells, Bax protein level increased significantly (p < 0.05, p < 0.01, respectively) and Cyt c expression level decreased significantly (p < 0.05, p < 0.01, respectively) after allicin treatment. The results indicated that allicin treatment resulted in Cyt c release from the mitochondria to the cytosol. As shown in Fig. 6, when Eca109 and EC9706 cells were treated with various concentrations of allicin for 48 h, allicin significantly decreased the protein expression levels of procaspase-3 and procaspase-9 (p < 0.05, p < 0.01, p < 0.001, respectively) except those at the concentration of 2.5 µg/mL of the expression of procaspase-3 and procaspase-9 in Eca109 cell, at the concentration of 2.5, 5 µg/mL of the expression of procaspase-3 in EC9706 cell, when compared with the corre-

sponding controls. Significant proteolytic cleavage of procaspase-9 and procaspase-3 was also detected in the experiment. Protein expression levels of cleaved caspase-3 and cleaved caspase-9 were significantly increased (p < 0.05, p < 0.01, p < 0.001, respectively), but not at the concentration of 2.5– 10 µg/mL of cleaved caspase-3, at the concentration of 2.5, 5 µg/mL of cleaved caspase-9 in EC9706 cell. Additionally, for the examined apoptosis-related genes Cyt c, Bax, caspase-3 and caspase-9, the effect of allicin was indicated in a concentration-dependent manner. No significant differences were observed between the two cell lines for induction of apoptosis of allicin.

3.4.

Allicin suppressed in vivo tumour growth

The antitumour effect of allicin was investigated in an ESCC of Eca109 cells bearing Male BALB/c mice. Thirty days after Eca109 cell transplantation, we chose 40 mice with tumour xenografts and randomly divided them into four groups. The results showed that Eca109 cell growth was significantly inhibited in different doses of allicin groups in tumour volume at the beginning of the fifth day (p < 0.05, p < 0.01, p < 0.001, respectively) and in tumour weight at the beginning of the fifth day compared with the control group (p < 0.05, p < 0.01) except that at the dose of 5 mg/kg body weight (Figs 7A and 7B).

528

Journal of Functional Foods 25 (2016) 523–536

Fig. 3 – Effect of allicin on p53, p21, cyclin B, CDK1 expression of Eca109 and EC9706 cells. (a) p53, p21, cyclin B, CDK1 expressions of Eca-109 and EC9706 cells detected by Western blotting, cells were treated with DMSO or different dose of allicin for 48 h. β-Actin was used as a loading control. (b) Expression level of each protein was estimated by densitometry and presented as a ratio to the loading control β-actin. Data are mean ± SD of three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

Fig. 4 – Effect of allicin on cell death in Eca109 (a) and EC9706 (b) cells. A–F indicated the representative histograms of allicin various concentrations (0, 2.5, 5, 10, 20, 40 µg/mL, respectively) for 48 h. Cell apoptosis was measured by annexinV-FITC and PI staining (flow cytometric analyses). Cells that stained positive for annexinV-FITC and negative for PI were at the early apoptotic stage (B4). Cells that stained positive for both annexinV-FITC and PI were at the end stages of apoptosis (B2). The data shown are representative of the best of three individual fluorescence activated cell sorter (FACS) acquisitions. (c) Allicin induced a significant apoptosis in Eca109 and EC9706 cells in a dose-dependent manner. Values represented mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

Journal of Functional Foods 25 (2016) 523–536

529

530

Journal of Functional Foods 25 (2016) 523–536

Fig. 5 – Effect of allicin on Cyt c and Bax expression of Eca109 and EC9706 cells. (a) The cytosolic and mitochondria protein Cyt c and Bax expressions of Eca109 and EC9706 cells detected by Western blotting, cells were treated with DMSO or different dose of allicin for 48 h. β-Actin was used as a loading control. (A) represents cytosolic and (B) represents mitochondria. (b) Expression level of each protein was estimated by densitometry and presented as a ratio to the loading control β-actin. Data are mean ± SD of three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

Journal of Functional Foods 25 (2016) 523–536

531

Fig. 6 – Effect of allicin on caspase-3 and caspase-9 expression of Eca109 and EC9706 cells. (a) procaspase-3, procaspase-9 and cleaved caspase-3, cleaved caspase-9 expressions of Eca109 and EC9706 cells detected by Western blotting, cells were treated with DMSO or different dose of allicin for 48 h. β-Actin was used as a loading control. (b) Expression level of each protein was estimated by densitometry and presented as a ratio to the loading control β-actin. Data are mean ± SD of three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

TUNEL staining was used to identify the apoptotic cells. A significant increase in the number of TUNEL-positive nuclei was observed in cells that had been incubated with allicin at the doses of 5, 10, 20 mg/kg body weight. The findings mirrored the results obtained in the in vitro experiments (Figs 7C and 7D).

4.

Discussion

In the present study, we determined the anti-cancer effect of allicin on ESCC with Eca109 and EC9706 cells. The molecular

532

Journal of Functional Foods 25 (2016) 523–536

Fig. 7 – Effect of allicin on Eca109 xenograft growth and on apoptosis in vivo. (A) Allicin suppresses the growth of the tumours compared with the control. (B) The tumour volume and weight of mouse model under different doses of allicin treatment. Statistically significant differences were compared with the control groups. For tumour volume, statistically significant differences were observed at the beginning of the fifth day (p < 0.05, p < 0.01, p < 0.001, respectively). For tumour weight, statistically significant differences were observed at the beginning of the fifth day (p < 0.05, p < 0.01). (C) Representative photographs of apoptotic cells detected with TUNEL and DAPI staining (200×) in the different groups. (D) The percentages of apoptotic cells in the different groups. The histogram was determined based on the average percentage of apoptotic cells in ten random visual fields of every group. Data are presented as the mean ± SD, *p < 0.05, **p < 0.01 versus control, ***p < 0.001 versus control.

Journal of Functional Foods 25 (2016) 523–536

mechanisms involved in the allicin-induced anti-cancer effect in Eca109 and EC9706 cells were examined. MTT assay, which is used to detect cell survival and growth, is performed to determine the potential cytotoxic properties of allicin on Eca109 and EC9706 cells. No significant changes below the concentration of 20 µg/mL indicated a relative safety for allicin on Eca109 and EC9706 cells. Allicin effectively reduces the cell viability of the two cells in a dose-dependent manner at the concentration of 40–100 µg/mL. This trend suggested the allicin-induced inhibitory effect on the growth of Eca109 and EC9706 cells as the effect of allicin on other cancer cells (Cha, Choi, Cha, Choi, & Cho, 2012; Chu et al., 2013; Jiang et al., 2013; Xu et al., 2014; Zhang et al., 2015). This further confirmed that allicin inhibited the migration of human Eca109 and EC9706 cells. The results of the migration suggested the metastasis inhibitory activity of allicin in oesophageal cancer cells. Our results were in agreement with other reports that the metastasis inhibitory activity of allicin was found in colorectal cancer cells (Liang et al., 2011) and in breast cancer cells (Lee et al., 2015). Cell cycle is a key process in regulation of cells. Towards this, we used flow cytometry to study the regulation of the cell cycle distribution in Eca109 and EC9706 cells after allicin treatment. We found that allicin reduced the percentage of G0/G1 and S phase and increased the population of G2/M phase in the experiment. This demonstrated that allicin disrupted the cell cycle progression and induced cell cycle arrest at G2/M phase in Eca109 and EC9706 cells. Actually, disruption of cell cycle progression in cancer cells is considered to be a potentially effective strategy for the control of tumour growth (Pavletich, 1999). As we know, G2/M phase can ensure complete and accurate replication of cells as well as preventing damaged and unreplicated DNAs from entering into mitosis (Tao et al., 2014). Thus, the results suggest that various concentrations of allicin could arrest the damaged cells in the G2/M phase of cell cycle, increase the repair of damaged DNA, reduce chromosomal aberrations (Cao, Yang, Wu, & Zheng, 1996; Sakamoto, Lawson, & Milner, 1997) and thereby selectively act on G2/M phase of Eca109 and EC9706 cells and participate in cell cycle regulation (Tao et al., 2014). In fact, compounds of garlic, including allicin, were observed to induce G2/M phase arrest in various cancer cells, such as HL-60 human myeloid leukaemia cells (Dirsch, Gerbes, & Vollmar, 1998), human mammary MCF-7 cancer cells (Hirsch et al., 2000), gastric SGC7901 cancer cells (Tao et al., 2014), and liver HepG2 cancer cells (Luo, Li, Deng, & Zhang, 2015). As an anticancer drug in clinic therapy, taxol also showed antiproliferation of cancer cells and arrested them at G2/M phase of cell cycle (Huang, Shu, Chao, Chen, & Chen, 2000). So, allicin can be used as a potential cancer-preventive agent for ESCC. Cell cycle checkpoint can be triggered at different stages of cell cycle to guarantee the accuracy of DNA synthesis, replication and repair. G2/M checkpoint plays a key role in DNA damage repair and cell survival promotion (Diaz-Moralli, Tarrado-Castellarnau, Miranda, & Cascante, 2013). Based on previous findings, we further assayed the related regulatory genes of G2/M checkpoint. The p53 is a key molecule in DNA damage repair and a tumour suppressor protein which is involved in many physiological functions, such as cell death, stress, cell cycle, cell differentiation, and immune response. In cell cycle, p53 mainly regulates G1 and G2/M checkpoint (Chu et al., 2013;

533

Fig. 8 – Schematic representation of the mechanism by which allicin induce G2/M arrest and apoptosis in Eca109 and EC9706 cells.

Yang, Lee, Ou, Chang, & Wang, 2012). As one of the most commonly induced genes, p21 is an important cyclin-dependent kinase inhibitor which plays a vital role in regulating cell cycle progression and inducing cell cycle arrest in G1 or G2 phase (Chhabria, Akbarsha, Li, Kharkar, & Desai, 2015; Su, Xiang, & Su, 2012). Cell cycle is regulated by the highly conserved cyclin dependent kinase (Cdks) and their regulatory subunits cyclins. Among cyclins, cyclins A and B are very important for enhancing the cell cycle from G2 into M phase. Especially, cyclin B plays a pivotal role as a regulatory subunit for Cdk1, which finally promotes the G2/M transition (Chou et al., 2009; Luo et al., 2015). CHK1 is a component of the DNA damage response pathway and is activated in response to intrinsic DNA damage induced by normal cellular process such as replication fork collapse or in response to DNA damage induced by cytotoxic chemotherapeutic agents (Massey et al., 2015). Thus, in this study, we assayed the effects of allicin of these related genes in G2/M phase. We found that the expression levels of p53, p21 and CHK1 genes were significantly increased and cyclin B were significantly decreased after relatively high concentrations of allicin treatment. Reports have indicated that p21 is one of the major transcription factors in the downstream of p53 and accumulation of p53 can activate p21, further to induce apoptosis (Chhabria et al., 2015; Stark & Taylor, 2006; Su et al., 2012; Yang et al., 2012). In addition, p21 and CHK1 have been reported to inhibit the activity of CDK1/cyclinB and strengthen the G2/M arrest of cell cycle distribution in the investigation of cantharidin induction G2/M phase arrest and apoptosis of human colorectal cancer colo 205 cells (Huang et al., 2011). Thus, we can conclude that allicin induced G2/M arrest via the signal pathway of p53-p21-CDK1/cyclinB (Fig. 8) in Eca109 and EC9706 cells. In this study, allicin induced apoptosis was firstly confirmed by the percentage of annexin-V-FITC cells. Apoptosis is a well-known process of programmed cell death which is characterized by apoptotic body formation, chromatin

534

Journal of Functional Foods 25 (2016) 523–536

condensation, DNA fragmentation, cell cycle arrest, endoplasmic reticulum (ER) stress, mitochondrion dysfunction, and activated caspases such as caspase-3 (Chu et al., 2012; Zhang et al., 2015). Thus, we further investigated the mechanism of allicin-induced apoptosis of Eca109 and EC9706 cells. As a member of the Bcl-2 family, Bax was the first pro-apoptotic member of this family that was identified. In this study, allicin increased Bax protein expression in the two cells. Published papers have indicated that Bax expression is increased by a variety of well-characterized apoptotic agents (Chen et al., 2014). This is in agreement with other reports which have confirmed the apoptotic characterization of allicin, especially on cancer or cancer cells (Louis et al., 2012; Wang et al., 2012; Xu et al., 2014; Zhang et al., 2010, 2015). Bax is located in the mitochondrial membrane that can alter the permeability of the membrane and trigger mitochondrial apoptosis (Adams & Cory, 1998; Xu et al., 2014). The release of cytochrome c into the cytosol represents a pivotal step of mitochondrial apoptosis signalling (Xu et al., 2014). The changes in Cyt c expression levels in cytosolic and mitochondria suggested that allicin treatment results in Cyt c release from the mitochondria to the cytosol of Eca109 and EC9706 cells. Our results agree with those of recent studies demonstrating that allicin induces Cyt c release from the mitochondria in HL60 and U937 cells, colon cancer cells of HCT-116, LS174T, HT-29, Caco-2, human ovarian cancer SKOV3 cells (Bat-Chen, Golan, Peri, Ludmer, & Schwartz, 2010; Miron et al., 2008; Xu et al., 2014). The release of Cyt c from the mitochondria to the cytosol further interacts with procaspase-9, after which it switches on caspase-3 or caspase7, leading to apoptosis (Wang, Wang, Dai, & Grant, 2002; Xu et al., 2014). Allicin decreased the protein expression levels of procaspase-3 and procaspase-9, and increased cleaved caspase-3 and cleaved caspase-9 protein expression levels, suggesting that allicin treatment triggered the activation of procaspase-3 and procaspase-3 and caspase-dependent apoptosis in Eca-109 and EC9706 cells as in other reports (Chu et al., 2013; Wang et al., 2002; Xu et al., 2014). Thus, the changes of Bax, Cyt c, caspase-3 and caspase-9 demonstrated that allicin may induce mitochondrial apoptosis signalling pathways (Fig. 8) in EC cancer cells. To test the inhibitory role of allicin in ESCC, we assayed the effect of allicin on the formation tumour of Eca109 in BALB/c nude mice. We found that an oral gavage administration of allicin suppressed tumour growth in vivo via decreased tumour volume and weight (Jiang et al., 2013). In addition, TUNELpositive stained cells were increased by the allicin treatment. These data demonstrated that allicin plays a tumour suppressive role in ESCC. Similar to other reports (Louis et al., 2012; Wang et al., 2012; Xu et al., 2014; Zhang et al., 2010, 2015), the effect of allicin on Eca109 and EC9706 cells showed a dosedependent manner. In the experiment, the low differentiation of EC9706 and high differentiation of Eca109 cells were assayed. Except at the concentration of 40 µg/mL on EC9706 viability, no significantly different changes were indicated between the two cells and suggested the anticancer effect of allicin had no relation to the differentiation stage of ESCC cell. This needs to be further confirmed in the future. On the other hand, some reports have shown that allicin is a short-lived compound. It rapidly reacts with free intracellular thiol groups. Several such metabolites, especially

S-allylmercapto-glutathione and S-allylmercapto-cysteine, which are more stable, have been shown to possess beneficial health effects, including an anticancer activity (Block, 2010; Hirsch et al., 2000). Therefore, though the inhibitory effect of allicin was shown in the present study, the metabolites of allicin on ESCC cells should be further studied in the future.

5.

Conclusions

Our findings for the first time demonstrated that the functional ingredient of allicin of garlic inhibited the cell viability, induced G2/M phase arrest via the signal pathway of p53-p21CDK1/cyclinB and apoptosis via mitochondrial signal pathways as well as suppressed tumour growth in vivo. As we know, disruption of cell cycle progression in cancer cells is considered to be a potentially effective strategy for the control of tumour growth. Activation of apoptosis signalling pathways may be responsible for treatment of malignant diseases. Our data suggest the potential anti-cancer agent of allicin in the treatment of patients with EC, especially ESCC. Allicin was implicated to medicate the biological activity of garlic. Therefore, the findings also provide the evidence of garlic in preventing the advancement of EC.

Acknowledgments This work was supported by the key Project Funded by the Clinic Hospital of Ministry of Health of the People’s Republic China (Program No. 2007353).

REFERENCES

Adams, J. M., & Cory, S. (1998). The Bcl-2 protein family: Arbiters of cell survival. Science, 281, 1322–1326. Ali, M., Thomson, M., & Afzal, M. (2000). Garlic and onions: Their effect on eicosanoid metabolism and its clinical relevance. Prostaglandins, Leukotrienes, and Essential Fatty Acids, 62(2), 55– 73. Bat-Chen, W., Golan, T., Peri, I., Ludmer, Z., & Schwartz, B. (2010). Allicin purified from fresh garlic cloves induces apoptosis in colon cancer cells via Nrf2. Nutrition and Cancer, 62(7), 947–957. Block, E. (2010). Applications of direct analysis in real timemass spectrometry (DART-MS) in Allium chemistry. (Z)-Butanethial S-oxide and 1-butenyl thiosulfinates and their S-(E)-1butenylcysteine S-oxide precursor from Allium siculum. Journal of Agricultural and Food Chemistry, 58(2), 1121–1128. Braithwaite, M. C., Tyagi, C., Tomar, L. C., Kumar, P., Choonara, Y. E., & Pillay, V. (2014). Nutraceutical-based therapeutics and formulation strategies augmenting their efficiency to complement modern medicine: An overview. Journal of Functional Foods, 6, 82–99. Cao, H., Yang, H., Wu, W., & Zheng, S. (1996). Study of the effect of allicin on tumor cell cycle by flow cytometry. Chinese Journal of Cancer, 15, 401–404. Cha, J. H., Choi, Y. J., Cha, S. H., Choi, C. H., & Cho, W. H. (2012). Allicin inhibits cell growth and induces apoptosis in U87MG human glioblastoma cells through an ERK-dependent pathway. Oncology Reports, 28(1), 41–48.

Journal of Functional Foods 25 (2016) 523–536

Chen, S., Tang, Y., Qian, Y., Chen, R., Zhang, L., Wo, L., & Chai, H. (2014). Allicin prevents H2O2-induced apoptosis of HUVECs by inhibiting an oxidative stress pathway. BMC Complementary and Alternative Medicine, 14, 321. Chhabria, S. V., Akbarsha, M. A., Li, A. P., Kharkar, P. S., & Desai, K. B. (2015). In situ allicin generation using targeted alliinase delivery for inhibition of MIA PaCa-2 cells via epigenetic changes, oxidative stress and cyclin-dependent kinase inhibitor (CDKI) expression. Apoptosis: An International Journal on Programmed Cell Death, 20(10), 1388–1409. Chou, L. C., Yang, J. S., Huang, L. J., Wu, H. C., Lu, C. C., Chiang, J. H., Chen, K. T., Kuo, S. C., & Cheng, J. G. (2009). The synthesized 2-(2-fluorophenyl)-6,7- methylenedioxyquinolin4-one (CHM-1) promoted G2/M arrest through inhibition of CDK1 and induced apoptosis through the mitochondrialdependent pathway in CT-26 murine colorectal adenocarcinoma cells. Journal of Gastroenterology, 44(10), 1055– 1063. Chu, Y. L., Ho, C. T., Chung, J. G., Raghu, R., Lo, Y. C., & Sheen, L. Y. (2013). Allicin induces anti-human liver cancer cells through the p53 gene modulating apoptosis and autophagy. Journal of Agricultural and Food Chemistry, 61(41), 9839–9848. Chu, Y. L., Ho, C. T., Chung, J. G., Rajasekaran, R., & Sheen, L. Y. (2012). Allicin induces p53-mediated autophagy in Hep G2 human liver cancer cells. Journal of Agricultural and Food Chemistry, 60(34), 8363–8371. Diaz-Moralli, S., Tarrado-Castellarnau, M., Miranda, A., & Cascante, M. (2013). Targeting cell cycle regulation in cancer therapy. Pharmacology Therapy, 138(2), 255–271. Dirsch, V. M., Gerbes, A. L., & Vollmar, A. M. (1998). A compound of garlic induces apoptosis in human promyeloleukemic cells, accompanied by generation of reactive oxygen species and activation of nuclear factor. Molecular Pharmacology, 53(3), 402– 407. Du, X. X., Li, Y. J., Wu, C. L., Zhou, J. H., Han, Y., Sui, H., Wei, X. L., Liu, L., Huang, P., Yuan, H. H., Zhang, T. T., Zhang, W. J., Xie, R., Lang, X. H., Jia, D. X., & Bai, Y. X. (2013). Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie, 67(5), 417–424. Hirsch, K., Danilenko, M., Giat, J., Miron, T., Rabinkov, A., Wilchek, M., Mirelman, D., Levy, J., & Sharoni, Y. (2000). Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation. Nutrition and Cancer, 38(2), 245–254. Huang, T. S., Shu, C. H., Chao, Y., Chen, S. N., & Chen, L. L. (2000). Activation of MAD2 check protein and persistence of cyclin B1/CDC2 activity associated with paclitaxel-induced apoptosis in human nasopharyngeal carcinoma cells. Apoptosis: An International Journal on Programmed Cell Death, 5, 235–241. Huang, W. W., Ko, S. W., Tsal, H. Y., Chung, J. G., Chiang, J. H., Chen, K. T., Chen, Y. C., Chen, H. Y., Chen, Y. F., & Yang, A. S. (2011). Cantharidin induces G2/M phase arrest and apoptosis in human colorectal cancer colo 205 cells through inhibition of CDK1 activity and caspase-dependent signaling pathways. International Journal of Oncology, 38(4), 1067–1073. Jiang, W., Huang, Y., Wang, J. P., Yu, X. Y., & Zhang, L. Y. (2013). The synergistic anticancer effect of artesunate combined with allicin in osteosarcoma cell line in vitro and in vivo. Asian Pacific Journal of Cancer Prevention, 14(8), 4615–4619. Kubec, R., Cody, R. B., Dane, A. J., Musah, R. A., Schraml, J., Vattekkatte, A., & Block, E. (2010). Applications of direct analysis in real time mass spectrometry (DART-MS) in Allium chemistry. (Z)-Butanethial S-oxide and 1-butenyl thiosulfinates and their S-(E)-1-butenylcysteine S-oxide precursor from Allium siculum. Journal of Agricultural and Food Chemistry, 58(2), 1121–1128.

535

Lee, C. G., Lee, H. W., Kim, B. O., Rhee, D. K., & Pyo, S. (2015). Allicin inhibits invasion and migration of breast cancer cells through the suppression of VCAM-1: Regulation of association between p65 and ER-α. Journal of Function Foods, 15, 172–185. Li, L., Han, W., Gu, Y., Qiu, S., Lu, Q., Jin, J., Luo, J., & Hu, X. (2007). Honokiol induces a necrotic cell death through the mitochondrial permeability transition pore. Cancer Research, 67(10), 4894–4903. Li, S., Dong, P., Wang, J., Zhang, J., Gu, J., Wu, X., Wu, W., Fei, X., Zhang, Z., Wang, Y., Quan, Z., & Liu, Y. (2010). Icariin, a natural flavonol glycoside, induces apoptosis in human hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway. Cancer Letters, 298(2), 222–230. Liang, D., Qin, Y., Zhao, W., Zhai, X., Guo, Z., Wang, R., Tong, L., Lin, L., Chen, H., Wong, Y. C., & Zhong, Z. (2011). S-allylmercaptocysteine effectively inhibits the proliferation of colorectal cancer cells under in vitro and in vivo conditions. Cancer Letter, 310(1), 69–76. Louis, X. L., Murphy, R., Thandapilly, S. J., Yu, L., & Netticadan, T. (2012). Garlic extracts prevent oxidative stress, hypertrophy and apoptosis in cardiomyocytes: A role for nitric oxide and hydrogen sulfide. BMC Complementary and Alternative Medicine, 12, 140. Luo, Q., Li, Y., Deng, J., & Zhang, Z. (2015). PARP-1 inhibitor sensitizes arsenic trioxide in hepatocellular carcinoma cells via abrogation of G2/M checkpoint and suppression of DNA damage repair. Chemico- Biological Interaction, 226, 12–22. Massey, A. J., Stokes, S., Browne, H., Foloppe, N., Fiumana, A., Scrace, S., Fallowfield, M., Bedford, S., Webb, P., Baker, L., Christie, M., Drysdale, M. J., & Wood, M. (2015). Identification of novel, in vivo active Chk1 inhibitors utilizing structure guided drug design. Oncotarget, 6(34), 35797–35812. PMID: 26437226. Miron, T., Wilchek, M., Sharp, A., Nakagawa, Y., Naoi, M., Nozawa, Y., & Akao, Y. (2008). Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells. Journal of Nutrition Biochemistry, 19(8), 524–535. Ohashi, S., Miyamoto, S., Kikuchi, O., Goto, T., Amanuma, Y., & Muto, M. (2015). Recent advances from basic and clinical Studies of esophageal squamous cell carcinoma. Gastroenterology, doi:10.1053/j.gastro.2015.08.054. Pavletich, N. (1999). Mechanisms of cyclin-dependent kinase regulation: Structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. Journal of Molecular Biology, 287(5), 821–828. Peng, Y., Zhou, Y., Cheng, L., Hu, D., Zhou, X., Wang, Z., Xie, C., & Zhou, F. (2015). The anti-esophageal cancer cell activity by a novel tyrosine/ phosphoinositide kinase inhibitor PP121. Biochemical and Biophysical Research Communications, 465(1), 137–144. Qia, Y. J., Chaoa, W. X., & Chiu, J. F. (2012). An overview of esophageal squamous cell carcinoma proteomics. Journal of Proteomics, 75(11), 3129–3137. Sakamoto, K., Lawson, L. D., & Milner, J. A. (1997). Allyl sulfides from garlic suppress the in vitro proliferation of human A549 lung tumor cells. Nutrition and Cancer, 29(2), 152–156. Stark, G. R., & Taylor, W. R. (2006). Control of the G2/M transition. Molecular Biotechnology, 32(3), 227–248. Su, B., Xiang, L., & Su, J. (2012). Diallyl disulfide increases histone acetylation and P21/WAF1 expression in human gastric cancer cells in vivo and in vitro. Biochemical Pharmacology, 1, 1–10. Tao, M., Gao, L., Pan, J., & Wang, X. (2014). Study on the inhibitory effect of allicin on human gastric cancer cell line SGC-7901 and its mechanism. African Journal of Traditional Complementary and Alternative Medicine, 11(1), 176–179.

536

Journal of Functional Foods 25 (2016) 523–536

Wang, H., & Huang, D. (2015). Dietary organosulfur compounds from garlic and cruciferous vegetables as potent hypochlorite scavengers. Journal of Functional Foods, 18, 986–993. Wang, Z., Liu, Z., Cao, Z., & Li, L. (2012). Allicin induces apoptosis in EL-4 cells in vitro by activation of expression of caspase-3 and -12 and up-regulation of the ratio of Bax/Bcl-2. Naturnal Product Research, 26(11), 1033–1037. Wang, Z., Wang, S., Dai, Y., & Grant, S. (2002). Bryostatin 1 increases 1-β-D-arabinofuranosylcytosine-induced cytochrome c release and apoptosis in human leukemia cells ectopically expressing Bcl-xL. Journal of Pharmacology and Experimental Therapecutics, 301, 568–577. Wilde, S. C., Keppler, J. K., Palani, K., & Schwarz, K. (2016). β-Lactoglobulin as nanotransporter for allicin: Sensory properties and applicability in food. Food Chemistry, 199, 667– 674. Xu, L., Yu, J., Zhai, D., Zhang, D., Shen, W., Bai, L., Cai, Z., & Yu, C. (2014). Role of JNK activation and mitochondrial Bax translocation in allicin-induced apoptosis in human ovarian

cancer SKOV3 cells. Evidence-based complementary and alternative medicine : eCAM, 2014, 378684. Yang, T. P., Lee, H. J., Ou, T. T., Chang, Y. J., & Wang, C. J. (2012). Mulberry leaf polyphenol extract induced apoptosis involving regulation of adenosine monophosphate-activated protein kinase/ fatty acid synthase in a p53-negative hepatocellular carcinoma cell. Journal of Agricultural and Food Chemistry, 60(27), 6891–6897. Zhang, W., Ha, M., Gong, Y., Xu, Y., Dong, N., & Yuan, Y. (2010). Allicin induces apoptosis in gastric cancer cells through activation of both extrinsic and intrinsic pathways. Oncology Reports, 24(6), 1585–1592. Zhang, X., Zhu, Y., Duan, W., Feng, C., & He, X. (2015). Allicin induces apoptosis of the MGC-803 human gastric carcinoma cell line through the p38 mitogen-activated protein kinase/ caspase-3 signaling pathway. Molecular Medicine Reports, 11(4), 2755–2760.