Differential pro-apoptotic effect of allicin in oestrogen receptor-positive or -negative human breast cancer cells

Journal of Functional Foods 25 (2016) 341–353

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Differential pro-apoptotic effect of allicin in oestrogen receptor-positive or -negative human breast cancer cells Kyung-Ho Kim a,1, Seong-Jun Cho b,1, Byung-Oh Kim c, Suhkneung Pyo a,* a

School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Republic of Korea KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co., LTD, Seoul, Republic of Korea c School of Food Science & Biotechnology, College of Agriculture & Life Sciences, Kyungpook National University, Daegu, Republic of Korea b

A R T I C L E

I N F O

A B S T R A C T

Article history:

Allicin is known to induce apoptosis and inhibit tumourigenesis in various carcinoma cells.

Received 28 January 2016

However, the precise mechanism of allicin-induced apoptosis remains unclear in human

Received in revised form 7 June

breast cancer cells. Here we found that ERα−MDA-MB-231 cells were more sensitive to allicin-

2016

induced apoptosis as compared with ERα+MCF7 cells. We also found that allicin induced

Accepted 17 June 2016

reactive oxygens species (ROS)-mediated and caspase-dependent apoptosis in MDA-MB-

Available online

231 cells, but not in MCF7 cells. Additionally, we showed the p38 and JNK pathways were involved in allicin-induced apoptosis. Furthermore, we demonstrated that ERα-knockdown

Keywords:

increased cell growth suppression and apoptosis of MCF7 cells in response to allicin, whereas

Allicin

ERα-overexpression decreased cell growth suppression and apoptosis of MDA-MB-231 cells,

Apoptosis

implicating that ERα has a protective role during allicin-induced apoptosis. Collectively, these

Oestrogen receptor

findings suggest that allicin induces differential apoptotic pathways through MAPK acti-

Breast cancer

vated by ROS-dependent or -independent signalling pathway in breast cancer cells. © 2016 Elsevier Ltd. All rights reserved.

ER MAPK

1.

Introduction

Breast cancer is one of the most common types of cancer in women and is the leading cause of death in women. The di-

agnosis of breast cancer falls into two broad categories (oestrogen receptor α (ERα)-positive or ERα-negative) based on the presence or absence of ERα in the cancer cells (Allred, Brown, & Medina, 2004). ERα is expressed in about 70% of all breast cancers. ERα-positive breast cancer generally has a better

Chemical compounds: Allicin (PubChem CID: 65036); Penicillin (PubChem CID: 5904); Streptomycin (PubChem CID: 19649); HEPES (PubChem CID: 23831); CM-H2DCFDA (PubChem CID: 23847176); JC-1 (PubChem CID: 5492929); Cytochrome c (PubChem CID: 16057918); MTT (PubChem CID: 64965); Ethanol (PubChem CID: 702); Propidium iodide (PubChem CID: 104981); FITC (PubChem CID: 18730); Formaldehyde (PubChem CID: 62705); MgCl2 (PubChem CID: 5360315); KCl (PubChem CID: 4873); DTT (PubChem CID: 446094); PMSF (PubChem CID: 4784); SDS (PubChem CID: 3423265); Glycerol (PubChem CID: 753). * Corresponding author. School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Republic of Korea. Tel.: +82 31 290 7753; fax: +82 31 290 7733. E-mail address: [email protected] (S. Pyo). 1 Kyung-Ho Kim and Seong-Jun Cho contributed equally to this work. http://dx.doi.org/10.1016/j.jff.2016.06.019 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

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Journal of Functional Foods 25 (2016) 341–353

prognosis and is often responsive to anti-oestrogen therapy (Huang, Warner, & Gustafsson, 2014; Zhou et al., 2014). In contrast, ERα-negative breast cancers are more aggressive and are not responsive to anti-oestrogen treatment (Zhou et al., 2014). Despite advances in the diagnosis and treatment of breast cancer, breast cancer still has a poor outcome and lacks targeted therapy. There is a need for more effective chemopreventive agents for advanced breast cancer. Nutraceuticals and functional foods have attracted interest concerning their potential in disease prevention and health enhancement (Hussain, Panjagari, Singh, & Patil, 2015). The global growth has been driven mostly by functional foods. As dietary intake of fruits and vegetables provides health benefits beyond basic nutrition, perhaps including decreased incidence of chronic inflammatory disease, it might substitute for conventional medicine. Garlic has been widely used as a traditional medicine for thousands of years. It consists of complex compounds, including alliin (S-allyl-L-cysteine sulphoxide) and allinase (alliin lyase) (Borlinghaus, Albrecht, Gruhlke, Nwachukwu, & Slusarenko, 2014). Allicin (diallyl thiosulfinate) is produced by the action of allinase on alliin (Borlinghaus et al., 2014). Several studies have investigated the potential health benefits of allicin, including its anti-bacterial, anti-fungal, anti-parasitic, anti-atherosclerostic, anti-thrombotic and anti-tumour effects (Borlinghaus et al., 2014; Cho, Rhee, & Pyo, 2006). However, the relationship between allicininduced apoptosis and ERα in breast cancer cells is unclear. The aim of this study was to investigate the anti-cancer effects of allicin and molecular mechanism that regulate expression of ERα gene effects of allicin. Specifically, we investigated the apoptotic effect of allicin using ERα-positive MCF7 and ERα-negative MDA-MB-231 breast cancer cells. We also investigated allicin-mediated cellular and molecular mechanisms responsible for altering cell cycle arrest and inducing apoptosis. Although allicin decreased cell viability and induced cell cycle arrest and apoptosis in both breast cancer cell types, ERαnegative MDA-MB-231 cells appeared to be more sensitive to apoptosis than ERα-positive MCF7 cells. ERα significantly contributed to the different responses between the two breast cancer cells. The results bolster the evidence that allicin activates molecular mechanisms that reduce cell viability and induce a differential reactive oxygen species (ROS)-mediated mitochondrial apoptotic pathway in these breast cancer cell types.

2.

Materials and methods

2.1.

Reagents

Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA). Foetal bovine serum, RPMI 1640, penicillin/streptomycin, and amphotericin B were obtained from Life Technologies. 5-(and-6)-chloromethyl-2′7′dichlorodihydrofluorescein diacetate (CM-H2DCFDA) and JC-1 were obtained from Molecular Probes (Eugene, OR, USA). ER, p53, extracellular signal-regulated kinase 1/2 (ERK1/2), phosphoERK1/2, p38, phospho-p38, c-Jun N-terminal kinase (JNK), phospho-JNK, Bcl-2, Bcl-xL, Bax, and apoptosis-inducing factor (AIF) antibodies were obtained from Cell Signaling Technol-

ogy (Danvers, MA, USA). Cytochrome c, caspase-3, and caspase-8 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Allicin was isolated and purified as described previously (Lee, Lee, Kim, Rhee, & Pyo, 2015).

2.2.

Cell culture

MDA-MB-231 and MCF7 human breast cancer cell lines (ATCC, Rockville, MD, USA) were propagated in Dulbecco’s modified Eagle’s Medium/F-12 (DMEM/F-12; Invitrogen Life Technologies Inc., Carlsbad, CA, USA) supplemented with 10% (v/v) foetal bovine serum (Invitrogen Life Technologies Inc.) and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin) (Invitrogen Life Technologies Inc.). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.

2.3.

Cell viability

Cell viability was measured by quantitative colorimetric assay with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylterazolium bromide (MTT) assay. Cells were seeded at a concentration of 1 × 105 cells/well in 96-well plates. The cells were washed once with Dulbecco’s phosphate-buffered saline (D-PBS) and 200 µl of MTT solution (25 µg/ml) and incubated for 4 hr at 37 °C and 5% CO2. The formazan blue crystals formed by the reduction of MTT were dissolved in 150 µl of dimethyl sulphoxide (DMSO). The cell viability was determined by absorbance at 540 nm using a microplate reader (Molecular Devices, Eugene, OR, USA).

2.4.

Cell cycle analysis

Cells were harvested, resuspended, and fixed with 70% (v/v) ice cold ethanol. Fixed cells were washed twice with ice cold PBS. Cells were incubated for 30 min at room temperature with staining solution [0.1 mg/ml RNAse A, 50 µg/ml propidium iodide (PI) in PBS]. Samples were analysed by flow cytometry using a FACS Calibur cytometer (BD Biosciences, San Jose, CA, BD, USA).

2.5.

Annexin V/PI analysis

Apoptosis was determined using an Annexin V/PI apoptosis kit. Briefly, cells were harvested by centrifuging at 300 × g, washed twice with ice-cold PBS, pelleted, and resuspended at 1 × 106 concentration in 100 µl of binding buffer. Five microlitres of Annexin V-FITC conjugate and 10 µl PI solution were added to the cells, and the cell solution was incubated in the dark for 15 min at room temperature. Three hundred microlitres of binding buffer were then added. At least 10,000 cells were subjected to flow cytometry analysis to identify viable, apoptotic, and necrotic populations, and quantified using CellQuest software (BD Biosciences) according to the manufacturer’s instructions.

2.6.

ROS production assay

CM-H2DCFDA, a redox-sensitive fluorescent dye, was used to evaluate the intracellular ROS level by flow cytometry. Cells were incubated in various conditions and then stained with 5 µM CM-H2DCFDA for 15 min at 37 °C. The cells were kept on ice

Journal of Functional Foods 25 (2016) 341–353

in the dark, and at least 10,000 cells from each sample were analysed by flow cytometry. The changes in the level of intracellular ROS were expressed as a percentage of cells not treated with allicin. Mitochondrial ROS production was also measured using the MitoSox probe. After treating cells with allicin for 4 hr, the medium was replaced by Hanks Buffered Salt Solution (HBSS) (Lonza, Walkersville, MD) containing 1 µM MitoSox for 10 min. The intensity of fluorescence signal was measured in a plate reader (BMG LABTECH, FLUOstar optima): excitation 510 nm, emission 580 nm.

2.7.

Analysis of mitochondrial membrane potential

For analysis of the mitochondrial membrane potential (ΔΨm) in cells, the membrane-permeable lipophilic cationic fluorochrome JC-1 was utilized in a Mitoscreen kit (BD Biosciences). JC-1 exhibits potential-dependent accumulation in mitochondria forming J-aggregates. An increase in green fluorescence indicates depolarization of the mitochondrial membrane potential. The ratio of red/green fluorescence was calculated and presented in arbitrary units. A decrease in this ratio indicates mitochondrial depolarization.

2.8.

Caspase activity assay

Caspase assay was performed using a caspase assay kit (BioSource International, Camarillo, CA, USA). Cell lysates were incubated with 25 µl of a specific substrate for caspase-3 (AcDEVD-pNA) or caspase-8 (Ac-IETD-pNA), in a 96-well plate. Following 2 hr incubation at room temperature, plates were read using a colorimetric plate reader (Titertek Instruments Inc., Huntsville, AL, USA) at 409 nm. The fold change in caspase activity was calculated compared to the untreated control.

2.9.

Plasmid and shRNA construction

To generate HA-tagged ERα, the coding region of ERα was amplified by PCR and cloned into the pcDNA3 vector. ERα was amplified with forward primer 5′-CCGGAATT CGCC ACCATGA CCATG ACCCTCCACACCAAAG-3′ and reverse primer 5′ATATCTCGAGTTAGCTCTGCAATGTTCC-3′. This ERα was then cloned between EcoR I and Xho I sites in the pcDNA3/HA-ERα vector. To generate ERα shRNA vector, oligonucleotides shERα 5′-GATCCCCTTCAGATAATCGACGCCAGTTCAA GAGACTGGC GTCGATTATCTGAATTTTTGGAAA-3′ and shATF3 5′-AGCT TTTCCAAAAATT CAGATAATCGACGCC AGTCTCTTGAACTGG CGTCGATTATCTGAAGGG-3′ were designed to target nucleotides of ERα mRNA. Oligonucleotides were annealed and cloned into the pSuper vector between BglII and HindIII sites (Oligoengine, Seattle, WA). Stable transfected clones expressing shRNA were selected using puromycin in MCF7 cells.

2.10.

Immunofluorescence

Cells were washed with PBS, fixed with 3.7% formaldehyde in PBS for 15 min at room temperature, and washed again with PBS. Cells were permeabilized with 1% BSA/0.2% Triton X-100/ PBS for 1 hr. The cells were washed twice with permeabilization buffer, incubated for 5 min in a Hoechst 33342 (1 µg/ml)-

343

containing PBS solution, and washed with PBS. The cells were also labelled with mitotracker red (50 nM) to see the mitochondrial shape changes. Coverslips were mounted on glass slides and the cells were photographed with a LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany). To assure accuracy in morphology determination, the classification of morphology was judged by at least two individuals.

2.11.

Western blot analysis

Western blot analysis was performed by modification of the technique described previously (Lee et al., 2015). The cells were pretreated with allicin (0.1, 1, 10 ng/ml) for 2 h and were then incubated with fresh growth medium containing TNF-α (10 ng/ ml) for 4 hr. After treatment, cells were washed twice in phosphate-buffered saline (PBS) and suspended in 70 µl of buffer A [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and Protease Inhibitor Cocktail (Sigma)] and incubated on ice. After 15 min, 0.5% Nonidet P-40 was added to lyse the cells, which were then vortexed for 10 s. Then, cytosolic cell extracts were obtained through centrifugation at 1500 × g for 10 min at 4 °C. The pelleted nuclei were resuspended in 50 µl of buffer C [20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% (v/v) glycerol, 0.5 mM PMSF, and Protease Inhibitor Cocktail] and incubated on ice for 20 min with intermittent agitation. Nuclear cell extracts were recovered after centrifugation for 10 min at 13,000 × g and 4 °C. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Lab, Hercules, CA, USA) with BSA as the standard. The cytosolic and nuclear extracts (20 µg) were resolved in a 7.5% sodium dodecyl sulphate (SDS)polyacrylamide gel, respectively. The fractionated proteins were electrophoretically transferred to an immobilon polyvinylidene difluoride membrane (Amersham, Arlington Heights, IL, USA) and probed with the appropriate antibodies. The blots were developed using an enhanced chemiluminescence (ECL) kit (Amersham). In all immunoblotting experiments, the blots were reprobed with an anti-β-actin antibody to control for the protein loading.

2.12.

Statistical analyses

Results are reported as means ± SEM. One-way analysis of variance was used to determine significance among groups, after which a modified t-test with the Bonferroni correction was used for comparison between individual groups. Significant values (P < 0.05) are represented by an asterisk.

3.

Results

3.1. Differential inhibitory effect of allicin on the growth of MCF7 and MDA-MB-231 cells Since allicin can induce apoptosis in various cancer cells, we investigated how allicin regulates cell proliferation and apoptosis in two different types of breast cancer cells. To test allicin-induced cellular response in ERα-positive MCF7 and

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Fig. 1 – Effect of allicin on cell proliferation and apoptosis in breast cancer cells. (A) MDA-MB-231 and MCF7 cells were treated with various concentrations of allicin (1–10 µg/ml) for 12 or 24 hrs. Cell viability was determined by MTT assay. Data included mean ± S.E.M. of three independent experiments performed in duplicates. *P < 0.05 compared with untreated control. (B) MDA-MB-231 and MCF7 cells were treated with various concentrations of paclitaxel (10–1000 ng/ml) in the presence or absence of allicin (10 µg/ml) for 24 hrs. Cell viability was determined by MTT assay. Data included mean ± S.E.M. of three independent experiments performed in duplicates. *P < 0.05 compared with untreated control. # P < 0.05 compared with paclitaxel alone. (C and D) MDA-MB-231 and MCF7 cells were treated with various concentration of allicin (1–10 µg/ml) for 24 hrs. Cells were harvested, fixed with 70% ethanol and stained with a propidium iodide solution. DNA content was measured by cell cycle analysis, and apoptotic cells were determined by Annexin V/PI analysis using a FACS.

ERα-negative MDA-MB-231 cells, we examined cell proliferation and apoptosis after allicin treatment using the MTT assay, PI, and Annexin V/PI staining with flow cytometry. As shown in Fig. 1A, both 12 hr and 24 hr treatments of 10 µg/ml allicin were effective in reducing the cell viabilities of MCF7 and MDAMB-231 cells. In addition, even a low concentration of allicin significantly decreased cell viability in MDA-MB-231 cells. These data suggest that MDA-MB-231 cells are more sensitive to allicin-induced cytotoxicity than MCF7 cells. Our result also showed that co-treatment of allicin and paclitaxel significantly inhibited the cell viabilities of MCF7 and MDA-MB-231 cells, compared with the cells treated with allicin or paclitaxel alone (Fig. 1B). These results further indicated that ERαpositive MCF-7 cells were resistant to both compounds, whereas ER-negative MDA-MB-231 cells were sensitive to them. Next, we examined the effect of allicin on cell cycle arrest and apoptosis using flow cytometry analysis. Allicin increased the sub-G1 cell populations in both MCF7 and MDAMB-231 cells, with a greater effect evident in MDA-MB-231 cells (Fig. 1C). An Annexin V/FITC assay was done to confirm the proapoptotic effects of allicin in MDA-MB-231 than MCF7 cells. In MCF7 cells, apoptosis was only induced in response to 10 µg/ ml allicin, whereas in MDA-MB-231 cells allicin induced apoptosis in a dose-dependent manner (Fig. 1D). These results suggest that ERα-positive MCF7 cells are more resistant to allicin-induced apoptosis than ERα-negative MDA-MB-231 cells.

3.2.

Effect of allicin on ROS generation

To determine whether ROS is involved in allicin-induced apoptosis of MCF7 and MDA-MB-231 cells, ROS generation in allicintreated MCF7 and MDA-MB-231 cells was examined. Cells were treated with various concentrations of allicin for 24 hr and then flow cytometry analysis was performed. Allicin significantly increased the level of intracellular ROS in a concentrationdependent manner in MDA-MB-231 cells, but not in MCF7 cells (Fig. 2A). We also determined mitochondrial ROS production in allicin-treated cells. Allicin concentration dependently increased mitochondrial ROS production in MDA-MB-231 cells, but not in MCF7 cells (Fig. 2B). To further investigate the role of ROS in allicin-induced apoptosis, cells were pretreated with N-acetyl cysteine (NAC) antioxidant for 2 hr prior to treatment of allicin, followed by cell viability assay. NAC treatment restored the cell viability that had been decreased by allicin treatment in MDA-MB-231 cells, but not in MCF7 cells (Fig. 2C). To gain understanding of cell morphological alterations under allicin-induced apoptosis, we performed confocal microscopy analysis. In MDA-MB-231 cells, treatment with various

concentrations of allicin for 24 hr caused a significant increase in nuclear fragmentation and condensation, whereas no nuclear morphology changes were observed in MCF7 cells (Fig. 3A). In addition, allicin induced changes in mitochondrial shape (donut/blob shape) in MDA-MB-231 cells, but not in MCF7 cells (Fig. 3B). Thus, these data suggest that ROS generation mediates allicin-induced apoptosis of MCF7 cells, but not MDA-MB-231 cells.

3.3. Effect of allicin on loss of mitochondrial membrane potential (ΔΨm) regulating the expression of Bcl-2 family proteins in breast cancer cells Mitochondria play an important role in cellular functioning as the energy store of cells and are also critical to the cell death programme (Wang, 2001). The disruption of mitochondria induced by DNA damage and various cellular stimuli leads to an irreversible apoptotic cell death (Wang, 2001). Thus, ΔΨm is one parameter of mitochondrial function used as an indicator of cell apoptosis (Wang, 2001). The members of the Bcl2 family of proteins regulate outer mitochondrial membrane integrity and function. Once apoptotic signal transduces through the regulation of Bcl-2 family proteins, cytochrome c and AIF are released from the mitochondria into the cytosol and apoptosis is induced via caspase activation. To demonstrate this feature of mitochondria-mediated apoptosis in allicin-treated breast cancer cells, we examined the role of allicin on the expression of the Bcl-2 family of proteins, including antiapoptotic Bcl2 and Bcl-xL, and pro-apoptotic Bax. Allicin slightly decreased the level of Bcl-xL expression, significantly decreased levels of Bcl2 expression, and significantly increased the level of Bax expression in MCF7 cells (Fig. 4A). Unlike MCF7 cells, a different pattern of anti- or pro-apoptotic gene expression was observed in allicin-treated MDA-MB-231 cells. The expression of Bcl-xL was suppressed to a large extent and the increase of Bax expression induced by allicin was constant up to 10 µg/ml. Furthermore, we investigated whether allicin can cause the release of cytochrome c and AIF from the mitochondria to activate caspases in the breast cancer cells. Western blot analysis showed that allicin treatment induced the release of AIF and cytochrome c from the mitochondria to the cytosol in a dose-dependent manner in MDA-MB-231 cells, but to a lesser extent in MCF7 cells (Fig. 4B). Thus, we postulate that allicin induces apoptosis of breast cancer cells by regulating mitochondrial membrane permeability. ROS generation can potentially damage mitochondrial DNA and alter membrane potential (Circu & Aw, 2010). Therefore, we investigated whether allicin regulates the ΔΨm in breast

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Journal of Functional Foods 25 (2016) 341–353

Cell viability (% of control)

MCF7

12 hrs 24 hrs

120 100

*

80 60 40 20 0

0

1

3

5

MDA-MB-231 Cell viability (% of control)

A

10

100

*

80 60

Cell viability (% of control)

Cell viability (% of control)

100

* * *

*

80 60 40 20 0

-

C

+ -

-

-

-

+

20 0

0

1

3

5

10

+

+

MDA-MB-231

120 100

* *

80

*

60

#

#

#

+

+

+

40 20 0

-

10 100 1000 10 100 1000

+ -

-

-

-

Allicin (10 μg/ml)

10 100 1000 10 100 1000 Paclitaxel

Sub-G1 3.1% G1 52.15% S 22.75% G2/M 21.72%

Sub-G1 7.8% G1 56.94% S 16.12% G2/M 19.31%

Sub-G1 G1 S G2/M

13.35% 36.06% 29.12% 21.61%

Sub-G1 G1 S G2/M

17.22% 37.25% 19.53% 16.17%

MCF7

Sub-G1 3.28% G1 45.76% S 26.84% G2/M 19.04%

Sub-G1 9.46% G1 44.18% S 26.25% G2/M 15.69%

Sub-G1 G1 S G2/M

30.59% 22.69% 26.81% 10.25%

Sub-G1 G1 S G2/M

39.71% 19.62% 11.54% 13.75%

MDA-MB-231

control

D

1

5

10

MCF7

Allicin (10 μg/ml)

MDA-MB-231 Allicin (1 μg/ml)

control

PI

*

Allicin (μg/ml)

MCF7

120

*

40

Allicin (μg/ml)

B

12 hrs 24 hrs

120

Allicin (1 μg/ml)

control

2.73

0.32

3.33

1.32

2.44

0.82

4.21

3.03

96.66

0.09

95.20

0.15

96.70

0.04

92.27

0.49

Allicin (5 μg/ml)

Allicin (10 μg/ml)

Allicin (5 μg/ml)

Allicin (10 μg/ml)

4.49

2.14

5.06

7.64

4.25

8.11

7.39

31.15

93.00

0.37

86.72

0.54

86.58

1.06

59.29

2.17

Annexin V

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Journal of Functional Foods 25 (2016) 341–353

A

MCF7 control Allicin (1 μg/ml) Allicin (5 μg/ml) Allicin (10 μg/ml)

Cell count ROS (fold-change of fluorescence intensity)

MDA-MB-231 control Allicin (1 μg/ml) Allicin (5 μg/ml) Allicin (10 μg/ml)

ROS (Relative intensity of fluorescence) 6 5 4 3 2 1 0 1

0

10

5

0

1

Mitochondrial ROS production (% of control)

B

MCF7

160

10

MDA-MB-231

*

*

140

*

120

5

Allicin (μg/ml)

Allicin (μg/ml)

100 80 60 40 20 0

C

1

5

10

MCF7

Allicin (μg/ml)

MDA-MB-231

Allicin Allicin+NAC

120 Cell viability (% of control)

*

*

Allicin Allicin+NAC

*

100

*

80 60 40 20 0

0

1

5

10

0

1

5

10 Allicin (μg/ml)

Fig. 2 – ROS mediated allicin-induced apoptosis in breast cancer cells. (A) MCF7 and MDA-MB-231 cells were treated with the indicated concentrations of allicin (1–10 µg/ml) for 2 hr and then with 10 µM CMH2DCFDA for 30 min at 37 °C. Flow cytometry analysis was used to assess the intracellular accumulation of ROS. (B) Mitochondrial ROS generation was measured by MitoSox probe after 4 hr incubation with various concentrations of allicin (1–10 µg/ml). Fluorescence was measured in a plate reader. Values are reported as mean ± S.E.M. of three independent experiments performed in triplicates. *P < 0.05 compared with untreated control. (C) MCF7 and MDA-MB-231 cells were treated with various concentrations of allicin (1–10 µg/ml) for 24 hrs in the absence or presence of 10 mM NAC. Cell viability was determined by MTT assay. The data are mean ± S.E.M. of four independent experiments performed in duplicate. *P < 0.05 compared with breast cancer cells treated with allicin alone.

Journal of Functional Foods 25 (2016) 341–353

A

Control

Allicin (10 μg/ml) Allicin (10 μg/ml) + NAC

MCF7

MDA-MB-231

B 0

1

5

10

347

caspase-3 in MDA-MB-231 cells, but not MCF7 cells (Fig. 5A and 5B). To further clarify whether allicin-induced apoptosis is caspase-dependent in the breast cancer cells, cell viability was determined in cells treated with allicin with or without pretreatment with a caspase inhibitor (z-VAD-fmk). Consistent with the data in Fig. 1A, allicin decreased cell viability of MCF7 and MDA-MB-231 cells (Fig. 5C). Notably, the treatment of caspase inhibitor partially attenuated the growth inhibition activity of allicin in MDA-MB-231 cells, but not MCF7 cells (Fig. 5C). These findings suggest that allicin-mediated growth suppression is caspase-dependent in MDA-MB-231 cells, but not MCF7 cells.

Allicin (μg/ml)

MCF7

MDA-MB-231

Fig. 3 – Nuclear and mitochondria morphological change in breast cancer cells. (A) Nuclear morphological change in breast cancer cells treated with allicin in the presence of NAC. Cells were incubated with allicin (10 µg/ml) for 24 hrs in the absence or presence of NAC. The cells were then fixed with 4% paraformaldehyde, stained with Hoechst 33342, and viewed using confocal laser microscopy. The results illustrated are from a single experiment and are representative of three separate experiments. (B) Mitochondrial morphology in allicin-treated MCF7 and MDA-MB-231 cells. Cells were labelled with Mitotracker red to see the mitochondrial shape changes. The results illustrated are from a single experiment and are representative of three separate experiments.

cancer cells and whether ROS generation mediates this regulation. Aggregated JC-1 showed red fluorescence in healthy mitochondria, whereas the monomeric form is characterized by green fluorescence when mitochondria are depolarized during apoptotic cell death. The ΔΨm was measured with JC-1 green fluorescence (cytosol) as compared with JC-1 red fluorescence (mitochondria) in breast cancer cells pretreated with NAC for 2 hr and incubated with 10 µg/ml allicin for 12 hr. Allicin treatment resulted in a marked decrease in the red/green fluorescence ratio in both cell types compared to untreated cells, indicating that allicin induced depolarization of ΔΨm (Fig. 4C). Although ΔΨm was decreased in both MCF7 and MDA-MB231 cells by allicin treatment, NAC treatment significantly inhibited ΔΨm only in MDA-MB-231 cells. These data suggest that exposure to allicin resulted in ROS-dependent depolarization and ΔΨm collapse in MDA-MB-231 cells, but not MCF7 cells.

3.4. Effect of allicin on caspase activation in breast cancer cells Next, we examined whether activation of caspases was involved in allicin-induced apoptosis in breast cancer cells. Allicin treatment increased the cleavage and enzyme activity of

3.5. Effect of mitogen-activated protein kinases (MAPKs) on allicin-induced apoptosis in breast cancer cells MAPK pathways, including ERK1/2, p38 MAPK, and JNK (Pearson et al., 2001), are activated by various extracellular stimuli, such as oxidative stress and UV radiation, which induce apoptosis. Garlic compounds like ajoene and diallyl disulphide are important in the induction of apoptosis, particularly in cancer cells (Hassan, 2004; Powolny & Singh, 2008; Singh et al., 2008). Therefore, we examined the involvement of the aforementioned MAPK pathways in allicin-induced apoptosis of breast cancer cells. The present results showed that allicin led to increased levels of phosphorylated ERK1/2, p38, and JNK in MCF7 and MDA-MB-231 cells and this was confirmed by densitometric analysis (Fig. 6A). To confirm that MAPK pathways were involved in allicin-induced cell growth suppression in breast cancer cells, we examined the effect of specific inhibitors of ERK1/2 (PD98059), p38 (SB203580), and JNK (SP600125) on allicininduced cell growth suppression using the MTT assay. We showed that pretreatment with SB203580 and SP600125 significantly reduced the growth suppression effect induced by allicin in MCF7 and MDA-MB-231cells (Fig. 6B). However, pretreatment with PD98059 had no effect on allicin-induced growth suppression. ROS generation can regulate activation of MAPKs in some systems (McCubrey, Lahair, & Franklin, 2006; Usatyuk et al., 2003). To determine whether this activation is due to the generation of ROS in the two breast cancer cell types, we assessed the effect of NAC on activation of MAPKs. The levels of phosphorylated ERK1/2, p38 MAPK, and JNK proteins, which were increased by allicin, were decreased by pretreatment of NAC in MDA-MB-231 cells, but not in MCF7 cells (Fig. 6C). These results indicate that allicin induces apoptosis in MCF7 through ROS-independent MAPK activation and in MDA-MB-231 cells through ROS-dependent MAPK activation.

3.6. Effect of ERα on allicin-induced apoptosis in breast cancer cells The therapeutic efficacy of chemotherapeutic agents might vary in breast cancer patients based on whether a tumour is ERαpositive or ERα-negative (Berry et al., 2006; Colleoni et al., 2000; Jiang et al., 2012). As observed above, ERα-negative MDA-MB231 cells are more sensitive to allicin-induced apoptosis than are ERα-positive MCF7 cells. Therefore, we hypothesized that the lower sensitivity of MCF7 cells to allicin-induced apoptosis might be associated with the presence of ERα. To investigate the effect of ER status on the sensitivity to allicin-induced

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A

B

MCF7 0

MCF7 0

1

5

1

MDA-MB-231

5

10

0

1

5

10

MDA-MB-231 10

1

0

Allicin (μg/ml) Cyt c

10 Allicin (μg/ml)

5

Bcl2

AIF

MT

Actin Bcl-Xl Cyt c

Bax Actin

CT

AIF Actin

Allicin (10 μg/ml)

Control 98.58

1.48

84.65

0.62

87.83

1.14

1.21

12.66

1.28

10.27

0.03

99.80

0.42

71.92

1.18

91.34

0.03

0.14

0.87

26.79

1.26

6.22

MDA-MB-231

JC-1 Red

0.23

MCF7

35

MCF7

0.05

Allicin (10 μg/ml) + NAC (10 mM)

Red/Green fluorescence ratio

C

MDA-MB-231 #

30 25 20 #

15 10

*

5 0

-

+ -

+ +

Allicin NAC

JC-1 Green

Fig. 4 – Allicin induces apoptosis through a mitochondria-mediated pathway. (A) Cells were treated with the indicated concentrations of allicin (1–10 µg/ml) for 12 hr. The levels of Bcl2, Bcl-xL, and Bax proteins were determined by Western blot analysis using the indicated antibodies. (B) Cells were treated with the indicated concentrations of allicin (1–10 µg/ml) for 12 hr. Cytosolic (CT) and mitochondrial fractions (MT) were isolated from breast cancer cells, as described in Materials and methods. The levels of cytochrome c (Cyt c), AIF, and Actin proteins were determined by Western blot analysis using the indicated antibodies. (C) Cells were treated with 10 µg/ml allicin for 12 hr in the presence or absence of 10 mM NAC. Mitochondrial membrane potential was measured by the uptake of a membrane potential-sensitive fluorescence dye (JC-1). The fluorescence intensity was analysed with a flow cytometer. The graph shows the red/green fluorescence intensity ratio. Values are reported as mean ± S.E.M. of three independent experiments. #P < 0.05 compared with untreated control. *P < 0.05 compared with allicin alone.

apoptosis, shRNA transfection was done to knockdown ERα in ERα-positive MCF7 cells or to overexpress ERα in ERα-negative MDA-MB-231 cells. Western blots were performed to confirm the level of pro-apoptotic (Bax) and anti-apoptotic (Bcl2, BclxL) proteins in allicin-treated breast cancer cells. Interestingly, knockdown of ERα decreased the level of Bcl2 and Bcl-xL proteins, and increased the level of Bax protein regardless of allicin treatment in MCF7 cells (Fig. 7A). Moreover, overexpression of ERα increased the level of Bcl2 and Bcl-xL proteins, and decreased the level of Bax protein regardless of allicin treatment in MDA-MB-231 cells (Fig. 7A). To determine whether the presence of ERα mediates allicininduced apoptosis in breast cancer, we examined the cell viability, and Annexin V/PI analysis was carried out with ERα knocked-down MCF7 and ERα-overexpressing MDA-MB-231 cells. Allicin decreased the cell viability of MCF7 cells, with the effect decreased by ERα knock-down (Fig. 7B). Consistently,

allicin slightly increased the apoptosis of MCF7 cells, especially in ERα knock-down cells (Fig. 7C). Allicin also decreased the cell viability of MDA-MB-231 cells but it was reduced by ERα overexpression (Fig. 7B). Allicin markedly decreased the apoptosis of MDA-MB-231 cells, but the effect was reduced by ERα overexpression (Fig. 7C). To further determine whether the presence of ERα influences to ΔΨm during allicin-induced apoptosis in breast cancer, we determined ΔΨm with ERα knocked-down MCF7 and ERα overexpressing MDA-MB-231 cells. Allicin slightly decreased ΔΨm of MCF7 cells, with the effect decreased by ERα knockdown (Fig. 7D). Allicin also significantly decreased ΔΨm of MDAMB-231 cells but it was reduced by ERα overexpression (Fig. 7D). These results demonstrate that ERα has a protective effect in allicin-induced apoptosis in breast cancer cells through the regulation of mitochondrial membrane potential controlled by anti- or pro-apoptotic proteins such as Bcl2, Bcl-xL, and Bax.

Journal of Functional Foods 25 (2016) 341–353

A

MCF7 0

1

5

4.

MDA-MB-231 10

1

0

5

cleaved-Caspase-8 pro-Caspase-3

cleaved-caspase-3 Actin

Caspase activity (fold change)

B MCF7

MDA-MB-231 caspase-3 caspase-8

caspase-3 caspase-8

3

* *

2 1 0

0

1

5

10

1

0

Allicin (µg/ml)

5

10

Allicin (µg/ml)

Cell viability (% of contorl)

C 120

MCF7 MDA-MB-231

100

*

80 60 40 20 0 -

+ -

Discussion

10 Allicin (μg/ml) pro-Caspase-8

4

349

+ +

Allicin (10 µg/ml) z-VAD-fmk (20 µM)

Fig. 5 – Effect of allicin on caspase activation in breast cancer cells. (A) Caspase-3 and -8 cleavage was analysed by Western blot analysis and the activity of caspase-3 and -8 was measured by colorimetric plate reader (409 nm) in both cells after allicin treatment (1–10 µg/ml) for 12 hr. (B) Cells were pretreated with the indicated concentration (20 µM) of the caspase inhibitor z-VAD-FMK for 2 hr, followed by incubation with allicin (10 µg/ml) for 12 hr. Cell viability was determined by MTT assay. The data are mean ± S.E.M. of four independent experiments performed in duplicate. *P < 0.05 compared with control. β-actin was used as an equal loading control. The results shown are from a single experiment, as representative of three separate experiments.

Breast cancer is a malignant tumour that predominantly occurs in women. Invasive breast cancer can be divided into two subtypes based on whether or not tumour cells express ERα. In this study, we examined the effect of allicin on cell proliferation, cell cycle, and induction of apoptosis using ERα-positive MCF7 breast cancer cells and ERα-negative MDA-MB-231 breast cancer cells. Allicin induced cell growth suppression and apoptosis in MCF7, with a more potent effect in MDA-MB-231 cells. It is interesting to note that co-treatment of allicin and paclitaxel significantly inhibited the cell viability of MDA-MB-231 cells, compared with the cells treated with allicin or paclitaxel alone. Collectively, these data demonstrate a differential sensitivity in human breast cancer cell lines in response to allicin, suggesting that ER status influences the efficacy of allicin. ROS, as the byproduct of normal cellular oxidative processes, regulates the process involved in the initiation of apoptotic signalling and induce mitochondrial damage in various physiological conditions (Circu & Aw, 2010). Therefore, we examined the effect of allicin on the ROS-mediated mitochondrial apoptotic pathway. ROS generation was not detected in MCF7 cells treated with any concentration of allicin. In contrast, ROS generation increased in a dose-dependent manner in response to allicin in MDA-MB-231 cells. In addition, treatment with NAC, a ROS inhibitor, attenuated cell growth suppression in MDA-MB-231 cells, but had no effect in MCF7 cells. These results suggest that allicin-induced cell growth suppression is ROS-dependent in MDA-MB-231 cells, but not in MCF7 cells. Further support for this conclusion comes from the observation that allicin could induce mitochondrial ROS production and change in mitochondrial shape in MDA-MB-231 cells, but not in MCF7 cells. Apoptosis leads to characteristic morphological changes, such as membrane blebbing, cell shrinkage, chromatin condensation, and nuclear and cytoplasmic fragmentation (Ziegler & Groscurth, 2004). In the present study, we observed morphological changes of apoptotic cells in allicininduced apoptosis of breast cancer cells. MDA-MB-231 cells showed chromatin condensation, loss of nuclear construction, and formation of apoptotic bodies in an allicin dosedependent response, whereas MCF7 cells showed only the formation of apoptotic bodies at a concentration of 10 µg/ml allicin. Taken together, these results suggest that allicininduced apoptosis was dependent on ROS generation in MDAMB-231 cells, but not in MCF7 cells. The mitochondrial apoptotic pathway has been also shown to play an important role as mediator of cell apoptosis in mammalian cells (Wang, 2001). There are two major apoptotic pathways, an extrinsic and an intrinsic pathway, which cause alterations of mitochondrial levels and trigger mitochondrial membrane permeabilization (Elmore, 2007). Moreover, an increase in ROS generation contributes to loss of mitochondria membrane potential (Δψm) (Fleury, Mignotte, & Vayssiere, 2002; Marchi et al., 2012). Presently, allicin increased the loss of Δψm in both MCF7 and MDA-MB-231 cells. However, pretreatment with NAC attenuated the loss of Δψm in allicin-treated MDAMB-231 cells, whereas it had no effect in MCF7 cells. The results indicate that allicin-induced apoptosis was dependent on ROS generation in MDA-MB-231 cells but not in MCF7 cells.

350

Journal of Functional Foods 25 (2016) 341–353

B A MCF7 0

15

30

MDA-MB-231 60

90

0

15

30

60

90

Allicin (min) p-ERK

ERK

Cell viability (% of contorl)

120

p-p38

MCF7 MDA-MB-231

100

*

80

Ralative intensity (Arbitory unit)

JNK

p-ERK p-p38 p-JNK

* **

*

**

* *

1.0 0.8 0.6

*

*

*

p-ERK p-p38 p-JNK

1.2

*

0.4

*

***

*

#

40 20 0

C

-

+

+

+

+ Allicin (10 μg/ml)

-

-

+

-

- PD98058 (10 μM)

-

-

-

+

- SB203580 (10 μM)

-

-

-

-

+ SP600125 (10 μM)

MCF7

MDA-MB-231

-

+

+

-

+

+

Allicin (10 μg/ml)

-

-

+

-

-

+

NAC (10 mM) p-ERK

* *

#

60

p38

p-JNK

*

* *

ERK p-p38

0.2

p38

0

0

15

30

60

90

0

15

30

60

90 Allicin (min)

p-JNK JNK

Fig. 6 – Role of MAPKs activation on allicin-induced apoptosis. (A) MCF7 and MDA-MB-231 cells were treated with 10 µg/ml allicin for 90 min. The levels of phospho-ERK1/2 (p-ERK), phospho-p38 (p-p38), and phospho-JNK (p-JNK) proteins were measured by Western blot analysis using specific antibodies. ERK1/2, p38, and JNK were employed as loading controls. MAPK densitometry values were adjusted to total MAPK intensity, and then normalized to expression from the control sample. Densitometric assay was showed including the mean ± S.E.M. of three independent experiments. *P < 0.05 compared with control. (B) Effects of MAPKs inhibition on allicin-induced apoptosis. Cells were treated with 10 µg/ml allicin in the absence or presence of specific inhibitors of each MAPK. Cell viability was determined by MTT assay. *P < 0.05 compared with MCF7 cells treated with allicin alone. #P < 0.05 compared with MDA-MB-231 cells treated with allicin alone. (C) Cells were pretreated with the indicated concentration of NAC (10 mM) for 2 hr, followed by incubation with 10 µg/ml allicin for 90 min. The levels of MAPKs phosphorylation were determined by Western blot analysis using the indicated antibodies. Total ERK, p38, and JNK were employed as loading controls. The results shown are from a single experiment, as representative of three separate experiments.

Fig. 7 – Role of ERα in allicin-induced apoptosis. (A) MCF7 cells and MDA-MB-231 cells were transfected with a vector control or vector carrying shRNA against ERα (shERα) or HA-tagged ERα cDNA (HAERα) and then treated with 5 µg/ml allicin for 24 hr. Total cells lysates were subject to Western blot assay using ERα, Bcl2, Bcl-xL or Bax antibodies. β-actin was used as an equal loading control. (B and C) Cells were transfected with vector control, HAERα, or shERα and then treated with 5 µg/ml allicin for 24 hr. Cell viability and apoptosis were determined by MTT assay and Annexin V/PI staining, respectively. *P < 0.05 as compared to allicin-treated MCF7 cells transfected with control under the same conditions. #P < 0.05 as compared to allicin-treated MDA-MB-231cells transfected with HA control under the same conditions. (D) Cells were transfected with vector control, HAERα, or shERα and then treated with 5 µg/ml allicin for 24 hr. Mitochondrial membrane potential was evaluated by the uptake of a membrane potential-sensitive fluorescence dye (JC-1). Fluorescence intensity was analysed by flow cytometry. A typical result from three independent experiments is shown.

351

Journal of Functional Foods 25 (2016) 341–353

A shControl -

shERα -

+

B

MDA-MB-231 HAControl -

+

+

shERα -

+

Allicin (5 μg/ml) ERα Bcl2 Bcl-xL Bax

MCF7

120 Cell viability (% of contorl)

MCF7

100 80

40 20 0 -

+

shControl

2.73

Allicin (5 μg/ml) 0.39

-

Allicin + + HAControl shERα (10 μg/ml)

+

shERα

MDA-MB-231

MCF7 untreated

#

*

60

Actin

C

MDA-MB-231

3.79

Allicin (5 μg/ml)

untreated 3.91

3.03

1.83

8.59

24.52

96.79 3.23

0.09

96.70

0.54

7.29

0.04

HAControl

PI

PI

shControl

15.22

93.95 3.40

0.31 1.00

64.98 3.95

1.91 5.22

shERα

0.20

96.03

71.17

HAERα

6.32

0.12

95.48

Annexin V

D

0.00

MDA-MB-231 Allicin (5 μg/ml)

95.94

0.64

Annexin V

MCF7 untreated

90.19

0.01

Allicin (5 μg/ml)

untreated 0.02

90.42

92.94

0.00

77.85

0.03

3.03

0.03

9.54

0.01

94.71

0.00

83.28

HAControl

JC-1 Red

JC-1 Red

shControl

0.02

7.02

0.00

22.15

0.00

90.84

0.03

83.68

shERα

0.05

5.23

0.09

16.63

JC-1 Green

Allicin increased caspase-3 activation in MDA-MB-231 cells, but not MCF7 cells. In addition, the inhibition of caspase also reduced allicin-induced growth suppression in MDA-MB-231 cells. Our results indicate that allicin regulates levels of expressed apoptosis-related proteins in both types of cells and causes caspase-dependent apoptosis in MDA-MB-231 cells and caspase-independent apoptosis in MCF7 cells.

HAERα

0.23

9.14

0.06

13.23

JC-1 Green

The MAPK family including ERK1/2, p38, and JNK is an important mediator of signal transduction for cell survival and apoptosis in response to a variety of extracellular signals (Pearson et al., 2001). Therefore, we examined the effect of allicin on each MAPK pathway. Allicin significantly increased phosphorylation of ERK1/2, p38, and JNK in MCF7 and MDA-MB231 cells. However, the ERK inhibitor PD98059 failed to attenuate

352

Journal of Functional Foods 25 (2016) 341–353

the cell apoptosis induced by allicin, indicating that ERK1/2 was not responsible for allicin-induced apoptosis in MCF7 and MDA-MB-231 cells. On the contrary, p38 inhibitor (SB203580) and JNK inhibitor (SP600125) inhibited allicin-induced apoptosis. ROS can induce the activation of the MAPK pathways (McCubrey et al., 2006). Antioxidant-mediated inhibition of ROS accumulation blocks the activation of MAPKs in response to extracellular signals. We confirmed that NAC treatment decreased activation of the MAPK pathway in MDAMB231 cells, but not in MCF7 cells. These results suggest that allicin only induced the activation of a MAPK signalling pathway, through ROS generation, in MDA-MB-231 cells. Several studies have reported the relationship between ERα and resistance to chemotherapeutic agents in breast cancer cells (Colleoni et al., 2000; Zhou et al., 2014). We investigated the mechanism of allicin-induced differential apoptosis in the two human breast cancer cell types. Interestingly, knock-down of ERα decreased the level of anti-apoptotic genes and increased pro-apoptotic gene, consequently inducing cell growth suppression and apoptosis in ERα-positive MCF7 cells. Overexpression of ERα induced cell growth suppression and apoptosis in ERα-negative MDA-MB-231 cells. These results suggest that regulation of ERα expression in response to allicin is an important event for controlling two different breast cancer cells and the presence of ERα in breast cancer cells contributes to their resistance to allicin-induced apoptosis.

5.

Conclusions

Allicin differentially induces growth suppression and apoptosis of breast cancer cells as the presence of oestrogen receptor. And the sensitivity to allicin-induced cytotoxicity is caused by the activation state of caspase pathway and MAPK pathway through ROS generation. The present data provide novel insights into the mechanisms of allicin and suggest the therapeutic potential of allicin in non-invasive or invasive cancer such as breast cancer because of its anti-proliferative effect.

Conflict of interest On behalf of all the authors, there are no potential conflicts of interest.

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